41
Electrophysiology of Vascular Smooth Muscle ~OLLIE E. I-IOLMAN * Table of Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137 Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 $ Large elastic arteries . . . . . . . . . . . . . . . . . . . . . . . . . . 139 Muscular arteries . . . . . . . . . . . . . . . . . . . . . . . . . . . 140 Small arteries and arterioles . . . . . . . . . . . . . . . . . . . . . . . 141 Capillaries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 Small veins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 Larger veins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 Electrical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . 144 1. Resting membrane potential . . . . . . . . . . . . . . . . . . . . . . . 144 2. Passive electrical properties . . . . . . . . . . . . . . . . . . . . . . . t45 3. Action potentials . . . . . . . . . . . . . . . . . . . . . . . . . . . t49 4. Origin of spontaneous activity . . . . . . . . . . . . . . . . . . . . . . 154 5. Effects of stimulation of the nerve supply to vascular smoth muscle ..... 156 6. Action of catecholamines . . . . . . . . . . . . . . . . . . . . . . . . 161 7. Action of acetylcholine . . . . . . . . . . . . . . . . . . . . . . . . . 165 8. Responses to changes in length . . . . . . . . . . . . . . . . . . . . . 166 9. Ionic basis of the action potential . . . . . . . . . . . . . . . . . . . . 167 10. Activation of contraction . . . . . . . . . . . . . . . . . . . . . . . . 170 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173 Introduction This review deals with the electrical activity of vascular smooth muscle and, as far as possible, with data concerned with cell membrane potentials. Recent work in this field will be viewed in the light of present knowledge of the membrane phenomena of other smooth muscles which have been studied extensively during the last ten years (e.g. guinea-pig taenia coli, vas deferens of various small laboratory animals, cat longitudinal intestinal muscle, etc., see reviews by BOHR, t 964; SCHATZMANN, t 964 ; BURNSTOCK and HOLMAN, t 966). Evidence is steadily accumulating which suggests that under normal con- ditions, the contractile apparatus of vascular smooth muscle may be activated by a change in cell membrane potential. This does not mean that changes in membrane potential are the only mechanisms by which contractile activity can be controlled. In cardiac muscle, there is no doubt that action potentials * Department of Physiology, Monash University, Clayton, Victoria, Australia.

[Reviews of Physiology, Biochemistry and Pharmacology] Reviews of Physiology, Biochemistry and Experimental Pharmacology Volume 61 || Electrophysiology of vascular smooth muscle

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Page 1: [Reviews of Physiology, Biochemistry and Pharmacology] Reviews of Physiology, Biochemistry and Experimental Pharmacology Volume 61 || Electrophysiology of vascular smooth muscle

Electrophysiology of Vascular Smooth Muscle ~OLLIE E. I-IOLMAN *

T a b l e of C o n t e n t s

Introduct ion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 3 7

Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 $ Large elastic arteries . . . . . . . . . . . . . . . . . . . . . . . . . . 139 Muscular arteries . . . . . . . . . . . . . . . . . . . . . . . . . . . 140 Small arteries and arterioles . . . . . . . . . . . . . . . . . . . . . . . 141 Capillaries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 Small veins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 Larger veins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143

Electrical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . 144

1. Resting membrane potential . . . . . . . . . . . . . . . . . . . . . . . 144 2. Passive electrical properties . . . . . . . . . . . . . . . . . . . . . . . t45 3. Action potentials . . . . . . . . . . . . . . . . . . . . . . . . . . . t49 4. Origin of spontaneous ac t iv i ty . . . . . . . . . . . . . . . . . . . . . . 154 5. Effects of s t imulat ion of the nerve supply to vascular smoth muscle . . . . . 156 6. Action of catecholamines . . . . . . . . . . . . . . . . . . . . . . . . 161 7. Action of acetylcholine . . . . . . . . . . . . . . . . . . . . . . . . . 165 8. Responses to changes in length . . . . . . . . . . . . . . . . . . . . . 166 9. Ionic basis of the action potential . . . . . . . . . . . . . . . . . . . . 167

10. Activat ion of contraction . . . . . . . . . . . . . . . . . . . . . . . . 170

Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173

R e f e r e n c e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173

I n t r o d u c t i o n

T h i s r e v i e w d e a l s w i t h t h e e l e c t r i c a l a c t i v i t y of v a s c u l a r s m o o t h m u s c l e

a n d , as f a r as poss ib le , w i t h d a t a c o n c e r n e d w i t h cel l m e m b r a n e p o t e n t i a l s .

R e c e n t w o r k in t h i s f ie ld wi l l be v i e w e d in t h e l i g h t of p r e s e n t k n o w l e d g e of

t h e m e m b r a n e p h e n o m e n a of o t h e r s m o o t h m u s c l e s w h i c h h a v e b e e n s t u d i e d

e x t e n s i v e l y d u r i n g t h e l a s t t e n y e a r s (e .g . g u i n e a - p i g t a e n i a coli , va s de fe r ens

of v a r i o u s s m a l l l a b o r a t o r y a n i m a l s , c a t l o n g i t u d i n a l i n t e s t i n a l musc le , e t c . , see

r e v i e w s b y BOHR, t 964; SCHATZMANN, t 964 ; BURNSTOCK a n d HOLMAN, t 966).

E v i d e n c e is s t e a d i l y a c c u m u l a t i n g w h i c h s u g g e s t s t h a t u n d e r n o r m a l con-

d i t i o n s , t h e c o n t r a c t i l e a p p a r a t u s of v a s c u l a r s m o o t h m u s c l e m a y b e a c t i v a t e d

b y a c h a n g e in cel l m e m b r a n e p o t e n t i a l . T h i s does n o t m e a n t h a t c h a n g e s in

m e m b r a n e p o t e n t i a l a re t h e o n l y m e c h a n i s m s b y w h i c h c o n t r a c t i l e a c t i v i t y

c a n b e c o n t r o l l e d . I n c a r d i a c musc l e , t h e r e is no d o u b t t h a t a c t i o n p o t e n t i a l s

* Depar tment of Physiology, Monash Universi ty, Clayton, Victoria, Australia.

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138 IVI. E. HOLMAN: Electrophysiology of v a s c u l a r s m o o t h muscle

initiate contractions; however there is a vast literature concerned with the variation in contractile performance which cannot be correlated with changes

in action potential configuration. In smooth muscle, contraction and relaxa- tion may be produced by the appropriate drugs or neurohormones in com- pletely depolarized cells. Nevertheless, the first step in analysing the mecha-

nisms which control the contractile behaviour of smooth muscle, whether

vascular or visceral, would seem to be a study of the electrical activity gener-

ated by the smooth muscle cell membrane.

In order to be able to interpret the results of electrophysiological experi-

ments on vascular smooth muscle, it is essential to know its morphology --

particularly the relationship between individual smooth muscle cells and the

pattern of their innervation. A brief account of the structure of those muscles

whose electrical activity has been analysed in some detail will be given. The

architecture, fine structure and innervation of blood vessels varies greatly

from one region of the body to another, and it must be emphasized that the

observations summarised below should not be extrapolated too far.

Morphology The walls of blood vessels are generally considered to be made up of three

coats: tunica intima, tunica media and tunica adventitia. The tunica intima consists of a single, continuous layer of endothelial cells

with overlapping end to end contacts (PEASE, t962). The endothelium of larger blood vessels is separated from the tunica media by an acellular layer of connective tissue elements with a large proportion of elastin - - the internal elastic lamina. In small vessels (less than 100 ~m diameter) the internal elastic lamina breaks up and eventually disappears. Endothelial cell processes come into close relationship with the smooth muscle cells of the media, forming characteristic myo-endothelial junctions (see below).

The tunica media may be defined from the functional point of view, as that region of the blood vessel wall which contains cells which have the ap- pearance of smooth muscle cells elsewhere in the body. Some text books con- sider the outer layer of longitudinal smooth muscle characteristic of large veins to be a component of the adventi t ia rather than the media. I t would seem to be less confusing to consider all smooth muscle components of blood vessels as being a part of the tunica media.

Since no cellular elements, other than those of the endothelium and the smooth muscle cells of the media, have been observed within these layers it is likely that the connective tissue skeleton of the vessel wall is maintained as a result of the secretory activity of the endothelial and muscle cells. This idea was put forward by PEASE (1962) who proposed that the neonatal development

of walls of elastic arteries was "accomplished b y the activities and regulation of tile muscle cells, perhaps aided in the intima by the endothelium". The

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Larger elastic arteries 139

absence of cells from the media, other than those with the characteristics of smooth muscle, is of special interest in relation to the genesis of arterial disease (KARRER, 1961 ; WlSSLER, 1967).

Tile tunica adventitia Call be defined as the outermost layer of the vessel wall where there is a transition from tissue elements functionally associated with the vessel to the perivascular connective tissue space. It is difficult to establish ally clear boundary for the adventit ia under the electronmicroscope - - especially for veins. The adventitia, so defined, consists of loosely packed bundles of collagen, fibroblast cells and bundles of nerve fibres with their associated Schwann cells.

Those aspects of the structure of the tunica media and its innervation, which are relevant to current electrophysiological studies will now be sum- marized.

Large elastic arteries

The connective tissue skeleton of large elastic arteries consists of con- centric cylinders (laminae) of elastin and collagen, with strands of collagen and sometimes elastin linking them together. Tile detailed architecture of the connective tissue skeleton varies greatly for different elastic arteries from different species (PEASE, 1962). KEATINGE (t966) described the arrangement of this structure in the sheep carotid artery. The thickness of the laminae is greatest (10 am) towards the outer part of the media, diminishing to t to 2 am near the intima. Laminae consist of collagen fibres surrounding bands of elastin. The strands of collagen passing between adjacent lamina interweave in bands, about t am thick, between single or small groups of smooth muscle cells, t~EATINGE'S observations with the light microscope suggest that there may be 4 to 6 smooth muscle cells sandwiched between each lamina. At the adventitial border these cells are arranged in circular bands. Near the intima, tile orientation of the smooth muscle cells is more variable.

The classical electronmicroscope s tudy of elastic arteries is that of PEASE

and PAULE (t 960) on rat aorta. Here every smooth muscle cell extends from one elastic lamina to the next - - "in essence, binding together adiacent lamellae". Neighbouring smooth muscle cells lie in close proximity. CLIFF (1967) found numerous examples of tight junctions (regions of membrane fusion) between them.

PEASE and PAULE (t960) emphasized the atypical appearance of the smooth muscle cells of the rat aorta. Although myofibrils and dense bodies character- istic of other smooth muscles are found within the cytoplasm, cell membranes lack a well developed basement membrane, and micropinocytotic vesicles are rarely seen.

Innervalion. PEASE and PAULE (1960) found no evidence for innervation of the media of the rat aorta. Subsequently, the use of the fluorescent method

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140 M.E. I-IoLMAN: Electrophysiology of vascular smooth muscle

of FALCK and HILLARI" (FALcK, t962) enabled KEATIXGE (t966) to demon- strate the presence of a fairly dense but thin layer of noradrenergic fibres at

the medio-adventitiai border of the sheep carotid artery from which bundles

of varicose axons penetrate within the connective tissue laminae of the media

and occasionally within the larger strands of collagen between adjacent

laminae. Fluorescent fibres were absent from the intima and innermost part

of the media (70 to t 50 am outwards from the lumen of the vessel). Similar

observations for other elastic arteries have been summarised by EHINGER,

FALCK and SPORRONG (t967) and FILLENZ (t967). EHINGER et al. suggest that those noradrenergic fibres penetrating the media of elastic arteries accompany

the vaso vasorum. VERITY, HUGHES and BEVAN (t965) used the electron- microscope to study the neuromuscular relationships of the outer border of

the media of the rabbit pulmonary artery. They found a minimum separation of 0.4 ~m between axon and muscle membranes with a mean separation of

t .9 ~m (see also VERITY and BEVAN, 1967).

Muscular arteries

Most text books agree that there is no sharp discontinuity between the

structure of elastic arteries and large muscular (distributing) arteries (e.g.

HAM, t965). In general, as the diameter of the vessel decreases, the elastic

components of the connective tissue skeleton gradually disappear from the bulk of the media and are limited to the internal and external elastic laminae.

The amount of connective tissue within the media decreases as the diameter

of the vessel decreases. The orientation of the smooth muscle cells within the vessel wall of the

larger distributing arteries may vary from a circular arrangement close to the

intima ("ring" muscles) to that of a spiral whose angle of pitch increases to-

wards the adventitia ("tension" muscles). STRONG ( t938), was able to unravel

the media of muscular arteries from various species after they had been partial- ly digested in HC1. He wrote that " the media was found without exception

to lack a fascicular arrangement and to be composed of compact, concentric

laminae of obliquely directed, fusiform smooth muscle cells united so closely

as to support suggestions of the syncytial character of the muscle".

Attempts to confirm STRONCr'S findings in this laboratory, for the larger muscular arteries of the rabbit, have so far been unsuccessful. Our impression is that the organi- zation of the media is predominantly circular. Microdissection of "fresh" specimens of these vessels in our hands produces tings, linked by strands of tissue to each other, rather than continuous spirals.

PEASE and MOLINARI (t960) studied the smooth muscle of cat pial arteries

and the characteristics they describe are typical of muscular arteries elsewhere.

The most conspicuous feature of the smooth muscle cells of these vessels are

the bands of dense cytoplasm (dense bodies) which appear to be connected to

the cell membrane. The smooth muscle cells have well defined basement

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Smal l arteries and ar ter ioles 14 t

membranes and micropinocytotic vesicles are evident. Regions of close contact between neighbouring cells were observed in cat pial arteries from which basement membrane material was excluded. Tight junctions of the variety which indicate membrane fusion have yet to be found for these or any

other muscular arteries. Znnervation. The innervation of muscular arteries has been studied with

the fluorescence method (e.g. EHINGER et al., t 967; FILLENZ, t 967; DE LA LAN- DE and WATEI~SON, t967). All authors agree that noradrenergic axons occur in a compact sheath just outside the media and that they only extend for a very

short distance into the media - - if at all. EHINGER et al. claim that the den- sity of this network decreases with the diameter of the vessel.

Small arteries and arterioles

Ill contrast with the limited amount of work that has been done at the ultrastructure level on the large muscular arteries there have been many stu- dies on the structure and innervation of small arteries and arterioles [see RHODIN (t967) for earlier references]. On the basis of electronmicroscopic studies on the microcirculation of the fascia covering the medial thigh muscles of the rabbit, RHODIN (t967) classified the small vessels on the arterial side of

the circulation as follows: Arterioles: vessels 100 to 50 am in diameter (dilated) with a fragmented

sheath of connective tissue elements replacing the internal elastic lamina and

2 to 4 layers of smooth muscle in the media. Terminal arterioles: less than 50 am in diameter, with few connective tissue

elements and no internal elastic lamina, but myoendothelial connections between the intima and media. These vessels have a single layer of helically

arranged smooth muscle cells. Pre-capillary sphincters and metarterioles: side branches coming off at

right angles from terminal arterioles; t 0 to t 5 am diameter with a single layer of circularly arranged smooth muscle cells at their junction with the terminal arteriole (pre-capfllary sphincter) and irregularly placed smooth muscle cells extending for 20 to 30 am from this junction (metarterioles) ; scanty connective tissue elements and many myoendothelial junctions.

Several points arise from RHODIN'S elegant s tudy which are of interest in relation to the electrophysiological studies that have been made on small vessels. Regions of close contact between neighbouring smooth muscle cells, presumed to be tight junctions, were observed in all these small vessels, but they were especially prominent in sections of terminal arterioles and pre- capillary sphincters. Lateral contacts between neighbouring cells, in which a process from one cell appeared to invaginate into the wall of a neighbouring cell occurred frequently and it is likely that these may be associated with

extensive electrical coupling between cells.

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42 M.E. HOLMA•: Electrophysiology of vascular smooth muscle

RHODIN observed filamentous material within the endothelial cells, especial- ly those of terminal arterioles. Although he was unable to differentiate between the filaments of the endothelial cells and those of neighbouring smooth muscle

cells, RHODIN did not feel that this could be taken to indicate that the endo- thelial cells showed active contractile activity.

The myoendothelial junctions, characteristic of terminal arterioles and pre-capillary sphincters, are of special interest. RHODIN'S suggestion that they may provide a means by which metabolites could be exchanged between the lumen of the vessel and the smooth muscle cells is an attractive one.

The smooth muscle cells of these vessels resemble those of muscular arte- ries. They contMn prominent dense bodies usually at tached to cell membranes and often organized into bar-like structures associated with microtubules.

Innervation. There have been a number of studies on the innervation of small arteries and arterioles. RHODIN (t967) describes bundles of terminal axons partially surrounded by Schwann cells running with arterioles towards their terminal branches. These are usually at least I ~m distant from the smooth muscle cells but occasionally axons lacking a Schwann cell sheath, approach the membrane of a smooth muscle cell more closely. RHODIN emphasized that terminal axons are especially abundant in the region of the pre-capillary sphincter. He describes apparent neuromuscular contacts of 200 A or less, with no basement membrane material between axon and muscle membranes for terminal axons and pre-capillary sphincters but points out that it is often difficult to identify a terminal axon under these conditions.

The morphologic relationships between the membranes of terminal axons and the membranes of the nearest smooth muscle cells are of special interest from the functional point of view. There is general agreement that the dis- tribution of axons is limited to the medioadventitial border. LEVER, GRAHAM and SPRIGGS (1967), for example, found no evidence for nervous penetration of the media of small arteries and arterioles of the pancreas. The closest neuro- muscular contacts observed in their experiments were within the range

800--4,000 A. DEVINE and SIMPSON (1967) compared the separation of neuro- muscular contacts in small arteries and arterioles of rat mesentry. A wide range of separation, from less than 0.t to 1 ~m or more was observed. Their results suggested that arterioles were more abundant ly supplied with contacts of less than 0.5 vtm separation than small arteries.

In summary, varicose terminal axons, often running together in bundles accompanied by Schwann cells, follow small arteries, arterioles, and terminal arterioles towards their peripheral extremities. Although serial sections have yet to be studied, it is likely that these axons resemble those described else-

where (MALMFORS, 1965; FILLENZ, t967; LEVER et al., 1967; ~'~ERRILLEES, t 968). Varicose regions, about I ~m diameter and 1--2 ~m in length probably alternate with narrow (0.t ~m) intervaricosities. The contents of these varico-

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Larger veins 143

sities resemble those described for autonomic terminal axons elsewhere. Neuro- tubules and mitochondria occur, together with a variety of vesicles. Small, 500 ~ unit vesicles with an electron dense core have been described by several

authors (e.g. SIMPSON and DEVINE, t966; LEVER et al., 1967; VERITY and BEVAN, t967). These are probably sites for storage and synthesis of nor- adrenaline (IVERSON, f 967). Partially exposed profiles of axon varicosities come into various neuromuscular relationships with the cells of the outer layer of the media. Close contacts (less than 200 A) in which basement membrane material disappears are very rare, though they may occur at the level of the terminal arterioles and precapillary sphincter (RHODIN, 1967). More usually it would seem that transmitter is released into wider extracellular spaces and would probably reach the smooth muscle membrane after considerable dilution by the extracellular fluid.

Capillaries

Capillaries may be conveniently defined as small vessels whose walls lack any smooth muscle elements. The chances of impaling the thin endothelial cell wall of most capillaries with a microelectrode are somewhat remote. Scanty electrophysiological data are available for these vessels (FUNAKI, t958). It seems unlikely that capillaries receive any innervation.

Small veins

Small veins are distinguishable from arterioles and small arteries by the large diameter of their lumen, compared with the thickness of their walls. In general their connective tissue skeleton is less well developed than that of arteries and arterioles of similar wall thickness. An internal elastic lamina is evident, even in small veins. The external elastic lamina may be absent or

rudimentary. Innervation. The innervation of small veins resembles that of small arteries

though they are usually less well supplied with terminal axons (EHINGER et at.,

1967). Since the external elastic lamina is missing, closer contact between terminal axons and smooth muscle membranes might be expected to occur but this point has not been studied at the ultrastructure level.

Larger veins

The morphology of veins varies greatly from one region of the body to another (HAM, t965). The structure of the mesenteric and portal veins of small laboratory animals is of interest since there have been a number of electro- physiological studies on these preparations. The internal elastic lamina is well developed (HoLMAN, KASBY, SUTHERS and WILSON, t968). The media contains two layers of smooth muscle separated from each other b y a layer of connective tissue. The inner layer is of smooth muscle cells arranged in a near circular

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144 .'-~1. ]~. I'{OLMA.N.': Electrophysiology of vascular smooth muscle

direction around the lumen ("ring" muscle). In the rabbit portal vein this layer is about 30 am thick. The outer layer of smooth muscle (about 80 am thick) is orientated in a more or less longitudinal direction - - probably forming a spiral of large angle of pitch. Contraction of this component of the vessel wall might be expected to lead to shortening and to an increase in diameter of the

vessel (RHoDIN, 1967). The smooth muscle cells of the outer layer are grouped together into bundles with quite large connective tissue spaces surrounding the bundles but narrow spaces between the smooth muscle cells within the bundle. The membranes of neighbouring cells within such a bundle frequently come

into close contact (200 A separation). Regions of membrane fusion (tight junctions) have been observed in rabbit portal vein (HOLMAN etal. , t968). Tile ultrastructure of the smooth muscle cells themselves resembles that of visceral smooth muscle. As mentioned previously, there is no distinct layer of connective tissue separating the outer layer of media from the adventitia or from the perivascular connective tissue space.

Innervation. Fluorescent fibres can be found along the outer border of the

longitudinal layer of smooth muscle of the portal vein, in the region between the outer layer and the inner circular layer and between the bundles of smooth muscle cells in the outer layer (HoLMAN et al., 1968). A similar distribution of nerve fibres has been observed in the electronmicroscope. No axons have been found within the inner layer of smooth muscle and few, if any, appear to penetrate the bundles of cells of the outer layer. Varicose terminal axons occur in the larger connective tissue spaces between these bundles - - occasionally a naked axon varicosity comes into contact with a smooth muscle cell. I t must be emphasized that the density of innervation of the portal vein is very sparce compared with smooth muscles such as the nictitating membrane or vas deferens. Furthermore, the dense layer of noradrenergic axons typical of the medio-adventitial border of muscular arteries is not seen in these or other abdominal veins.

Larger muscular veins, saphenous, facial, penile, etc. (EHINGER et al., i967) contain many noradrenergic fibres which are especially well demonstrated in longitudinal sections. These authors suggest that tile dense innervation of these vessels might be associated with a lack of muscle cell to cell interaction.

E l e c t r i c a l properties 1. Resting membrane potential

The maximum level of membrane potential, recorded in the absence of electrical activity (slow waves or action potentials) is within the range quoted for visceral smooth muscle (BuRNSTOCK, HOLMAN and PROSSER, 1963). Su, BEVAN and URStLLO (1964) recorded values of up to 75 mV from cells near the intimal border of rabbit pulmonary artery and similar values were obtained

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Passive electrical properties ~45

by SPEDEN (t967) for small arteries, in vitro. Somewhat lower values have been found for vascular smooth muscle undergoing myogenic or neurally induced activity. For example, STEEDMAN (1966) recorded values of up to 60 mV for small mesenteric arteries and arterioles, in vivo. NAKAJIMA and HOl~N (1967) found that the resting potential of the anterior mesenteric vein of the guinea- pig, in vitro, reached values of up to 62 mV between bursts of action potentials and similar values were reported by FUNAKI and BOHR (t964) for the spon- taneously active rat portal vein.

The resting membrane potential of visceral smooth muscle depends on the equilibrium potentials for Na, K and C1 ions and the relative values of their membrane permeability, ill much the same way as that of skeletal muscle and nerve (HoLMAN, t 968 a). In visceral smooth muscle there is evidence that the permeability for K ions may be lower than is the case for skeletal muscle. AXELSSON, WAHLSTROM, JOHANSSON and JONSSON (1967) studied the effect of changes in external K concentration on the resting potential of the rat portal vein. They also concluded that K permeability was lower in this smooth muscle than in skeletal muscle or nerve. A decrease in the value of the ratio of K to Na permeability, PK]PNa, may account for the divergence of the resting poten- tial from the K equilibrium potential which is probably not much lower in smooth muscle than in nerve and skeletal muscle (BuI~NSTOCK and HOLMAN,

1966).

AXELSSON et al. found that the resting potential of the rat portal vein decreased in K-free solution. This may have been due to a decrease of K perme- ability with decreasing extracellular K concentrations - - as suggested for cardiac muscle (HoFFMAN and CRANEFIELD, t 960). Alternatively, it is possible that the " N a - p u m p " in smooth muscle is electrogenic (see HOLMAN, 1968a). The absence of K ions from the perfusing solution might have inhibited the "Na-pump" and this effect could also have contributed to the decrease in resting potential.

The resting potential of the rat portal vein decreased in Ca-free solution and increased in Ca-rich solutions - - as reported for visceral smooth muscle (BENXETT, t967). Curiously enough, CUTHBERT (t967) reported that Ca-lack caused hyperpolarization of the rabbit anterior mesenteric artery.

2. Passive electrical properties

During the last few years significant advances have been made toward a solution of the problem of the way in which a large population of individual smooth muscle cells can act together as a "single-unit" (BozLER, t 948). Intra- cellular recording techniques, combined with the use of large external stimu- lating electrodes, have confirmed the work of BOZLER, BOLBRING, BURNSTOCK, HOLMAN, PROSSER and others who showed that it was necessary to activate

t0 Ergebnisse der Physiologie, Bd. 61

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t46 M.E . HOLMAN: Electrophysiology of vascular smooth muscle

a sufficiently large number of cells, more or less simultaneously, if excitation was to spread throughout the muscle without decrement. It is now clear that

a bundle of smooth muscle cells, t 00 am or more in diameter, can behave elec- trically as if it were a single "core-conductor" with a length constant of I to 3 mm and a time constant of about t 00 msec (ToMITA, t966). The characteristics of the stimulus required to elicit a conducted action potential in such a bundle and the velocity of conduction of the action potential can be predicted from the core-conductor properties of the bundle. Calculated values agree well with

those observed experimentally (ABE and TOMITA, t 968). It seems likely that the individual smooth muscle cells of such a bundle are

coupled together electrically by regions of membrane fusion (tight junctions) (BARR, DEWEY and BERGER, 1968). The electrical resistance of similar tight junctions observed in various epithelial tissues is very low (LOEwENSTEIN,

t966). In visceral smooth muscle (e.g. the taenia coli of the guinea-pig) neigh- bouring cells establish quite extensive regions of contact with each other. Sometimes a bulging lateral process from one cell pushes into the side of a neighbouring cell and contiguous cell membranes are fused for distances of several micrometers (BENNETr and ROGERS, 1967; BARR et al., 1968). Similar regions of contact occur between the smooth muscle cells of some elastic arteries, small arteries, arterioles and the longitudinal muscle of the portal vein

(see above). It has been suggested that the cells of some visceral smooth muscles are

coupled together so tightly that the whole tissue forms a 3-dimensional elec- trical network resembling that of cardiac muscle (ToMITA, t966). This model for smooth muscle has important implications in relation to the pat tern of innervation which is required for effective transmission (BENNETT and ROGERS,

t 967). If only a single point within such a syncytium is depolarized by excitatory transmitter action or by the injection of depolarizing current from an intra- cellular electrode, it is difficult to initiate an action potential. The threshold membrane potential at which the response to depolarization becomes self- regenerative is very much higher than the threshold at which an action poten- tial may be initiated by stimulation with external electrodes. It follows that a whole series of smooth muscle cells needs to be depolarized more or less simultaneously if an action potential is to be initiated at a reasonably low

threshold. Some smooth muscles may be less extensively coupled than is apparent ly the case

for the guinea-pig taenia coli. In this case, only a small fraction of the current injected into a cell by an intracellular stimulating electrode would flow across the membranes of neighbouring cells. Thus, although it would be relatively easy to excite an action potential in the impaled cell, it would be unlikely tha t an action potential, so initiated, would be able to excite neighbouring cells. Once again, if only a single smooth muscle cell was influenced by transmitter action, there would be little chance that the excitation would spread very far. Thus the innervation of a group of cells rather than a single cell is also required for effective transmission (HOLMAN, t968b).

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Passive electrical properties 147

The degree of coupling between the cells of vascular smooth muscle has yet to be analysed. As pointed out by FURCHGOTT (1955) and BOHR (t964) the ability of many different blood vessels to show rhythmic coordinated contrac- tions implies that excitation can spread to some degree from cell to cell. BOHR'S group studied the spontaneous contractile activity of a number of different blood vessel in vitro. In normal physiological saline solution the portal vein of rat, rabbit and dog all showed regular phasic contractile activity. SUTTER'S (t965) observations on the mechanical activity of mammalian veins suggest that coordinated myogenic activity is a property of the longitudinal smooth muscle component, rather than the inner circular layer of these vessels. He was unable to find any evidence for conduction of excitation in the vena cava or jugular vein.

Spontaneous mechanical activity, in vitro, was also recorded from strips cut from the cutaneous arteries of the dog's paw (JOHANSSON and BOHR, 1966}. Coordinated phasic contractions were superimposed on a maintained tonic contraction.

Manipulation of the ionic composition of the bathing solution (high K in the presence of high Ca) was needed to induce such activity in most prepa- rations studied by BOHR (t 964). The degree of automaticity of these muscles decreased in the following order:

t . portal vein of rat, rabbit and dog,

2. human umbilical arteries,

3. rat aorta,

4. dog cerebral resistance vessels,

5. dog coronary vessels,

6. dog mesenteric resistance vessels,

7. dog carotid,

8. rabbit aorta.

The finding that coordinated contractions could be obtained from all these vessels suggested to BOHR that most vascular smooth muscles probably have the capacity to behave as a single unit. (It must be emphasized that a corre- lation between the degree of coupling between smooth muscle cells and a tendency to automaticity cannot be assumed at this time.)

Some degree of electrical coupling is indicated by the observation of quite large potentials in the sucrose-gap apparatus. The sucrose-gap measures the potential difference between two regions of a bundle or strip of muscle which are separated by a compartment several mm in length perfused with an iso- tonic sucrose solution of high electrical resistance. If one end of the strip is perfused with isotonic KISO4, so that its membrane potential is reduced to 0 mV, the sucrose-gap could, theoretically, measure the full value of the resting potential. However, "short-circuiting" usually occurs, depending on the

tO*

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148 I~¢1. E. HOLMAN: Electrophysiology of vascular smooth muscle

resistance of the preparation compared with the resistance of the sucrose solution. I t is difficult to see how values of 50 mV or so could be found for the resting potential of vascular smooth muscles with this technique, if there were no low resistance connections between cells.

Sucrose-gap records have been obtained from strips of sheep carotid artery

(KEATINGE, 1964, 1966, 1967, t968), longitudinal muscle of the mesenteric veins of several species (CUTHBERT and SUTTER, 1964, 1965; CUTHBERT, MATTHEWS and SUTTER, t965 ; CUTHBERT, 1967; HOLMAN and MCLEAN, 1967) and the portal vein of rat, rabbit and guinea-pig (AxELSSON, JOHANSSON and JoNssoN, 1966; AXELSSON, JOHANSSON, JONSSON and WAHLSTROM, 1967;

JOHANSSON and LJUNG, t967b; JOHANSSON, JONSSON, AXELSSON and WAHL- STROM, t967; AXEI, SSON, WAHLSTROM, j'OHANSSON and JONSSON, f967; HOL- MAN, t967; HOLMAN et al., t968).

Interaction between cells is also a necessary property of smooth muscles from which records can be obtained with "pressure" electrodes. These elec- trodes are usually constructed in the same way as the micropipettes used for intracellular recording. Their diameter may range from 0.5 am up to t0 am. When they are pushed against visceral smooth muscle they record the electrical acti- vi ty of the muscle in a monophasic manner with the same polarity as that of an intracellular electrode. The operation of "pressure" electrodes has been dis- cussed by GILLESPIE (1962) and BORTOFF (t967). STEEDMAN'S (4966) obser- vation of action potentials and slow waves in small mesenteric arteries with "pressure" electrodes may be taken as indirect evidence for electrical coupling between the smooth muscle cells of these vessels.

Very convincing evidence for electrical interaction, and hence the "single uni t" nature of the longitudinal muscle of the rat portal vein has been put

forward by JOHANSSON and LJUNG (1967b). They recorded tile mechanical activity of two neighbouring segments of vein which were separated by a compartment containing a number of different test solutions. When conduction through this compartment was normal both segments showed synchronous contractions. If conduction was blocked by hypertonic solutions or by local anaesthetics contractions were asynchronous.

RODDIE (I 967) reported that the conducted action potentials of the arteries and veins of turtles could be picked up by a microelectrode before contraction occurred at the site of the recording electrode. Ganglion-blocking agents (e.g. pentolinium) and cocaine had no effect on conduction. RODDIE concluded that conduction was "due to spread of current via low resistance pathways from cell to cell".

Much work needs to be done to establish the characteristics of the passive electrical properties of vascular smooth muscles, following the experimental procedures of BULBRING, KURIYAMA and TOMITA (see TOMITA, t966). This should be a relatively simple mat ter for strip preparations from the larger arteries and veins. I t is more difficult to see how these techniques could be applied to smaller vessels.

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Action potentials 149

3. Action potentials It has become increasingly difficult to find an adequate definition for the

term "action potential" which can be usefully applied to the electrical records

from smooth muscle (HoLMAN, t968a). Perhaps this term should be reserved for those changes in membrane potential which have the following properties.

1. They are initiated by a decrease in membrane potential. 2. They are self-regenerative and may be all-or-nothing in amplitude. 3. They may lead to activation of the contractile apparatus. It has become customary to refer to changes in membrane potential of

much smaller amplitude (5--30 mV), which have a t ime course which is similar to that of an established action potential, as "abortive action potentials" or simply, as action potentials. Furthermore, ill some papers, this term has been used without reservation for any detectable depolarization of rapid time course. Although such presumptive action potentials may represent regenerative activity somewhere in the neighbourhood of the recording electrode, one must be cautious in drawing analogies between these potentials and action poten-

tials observed in other excitable tissues. Elastic Arteries. Extracellular wick electrodes were used by TAKENAKA

(t964) to s tudy the electrical activity of strips, prepared from a number of blood vessels, in vitro. Spontaneous activity was rare in all preparations tested, but this could be induced by Ba ions or by noradrenaline. The aorta of rabbit and guinea-pig showed bursts of "spikes" at intervals of 3--5 sec which pre- ceded the development of tension. TAKENAKA'S records show diphasic poten- tials which may indicate that at least some of these spikes were conducted.

The strips of common carotid artery studied by KEATINGE (t964) in the sucrose-gap were not spontaneously active. Perfusion with noradrenaline caused depolarization and contraction. Only in occasional preparations did tile form of the depolarization resemble an action potential. In contrast, carotid artery strips which had been exposed to inhibitors of oxidative metabolism regularly responded to noradrenaline with a series of action potentials of the plateau form (about 20 sec in duration). It is difficult to understand why the presence of metabolic inhibitors should predispose the cell membrane to generate a series of action potentials rather than a maintained depolarization preceded by what might correspond to the rising phase of a single action potential. The action of inhibitors on the state or distribution of membrane Ca may be involved here (Section t0). When strips of carotid artery were ex- posed to Ca-free solutions for 20 min or more they became spontaneously active (KEATINGE, 1968). A small contraction was associated with the onset of action potentials but this disappeared during the next 40--60 min although electrical activity was maintained for up to t 80 rain after removing Ca. Action potentials occurred at frequencies of up to 7t/min. They were of the spike form with a total duration of about t sec.

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150 2"~i. E , HOLMAN: Electrophysiology of v a s c u l a r s m o o t h m u s c l e

It may be tentatively concluded that the smooth muscle cells of some elastic arteries of mammals are capable of generating changes in membrane potential which may have the properties of action potentials as defined above. Although intracellular records are obviously desirable to check this point, the technical difficulties involved in their use with large elastic arteries may be insurmountable. The sucrose-gap might turn out to be the most suitable method for studying the electrical activity of these vessels.

RODDIE (1962) used intracellular electrodes to record from the aorta and inferior vena cava of turtles. He chose these preparations because " they ex- hibit spontaneous activity in vitro and the muscle cells in their walls are surrounded by a relatively small amount of connective tissue" (RODDIE, 1967). The action potentials of the turtle aorta were of the plateau type and of ex- tremely long duration (up to 30 sec). Individual action potentials gave rise to long, slow contractions lasting for more than 30 sec. RODDIE emphasized that contractions were always associated with discrete action potentials and were never triggered by gradual changes in membrane potential. In this context, it is interesting that KF-ATINGE (t964) also observed discrete contractions in association with the action potentials recorded in metabolically "poisoned" strips of carotid artery, in response to noradrenaline.

Microelectrode studies on rabbit pulmonary arteries by Su, BEVAN and URSlLLO (t964) failed to reveal any changes in membrane potential of the action potential type. S u e t al. suggest that "electrical quiescence during sympathetic nerve and (catechol) amine stimulation is a characteristic of this tissue". In contrast with the action of noradrenaline and sympathetic nerve stimulation, KC1 caused depolarization but again, there were no changes in membrane potential resembling action potentials.

Although there is no basis for the assumption that all vertebrate smooth muscles are capable of generating action potentials, the assertion that the intimal layer of the pulmonary artery is electrically quiescent does raise some difficulties in understanding how the contractile activity of this smooth muscle can be controlled (see Section 5 below). It is reasonable to ask whether there might be an alternative explanation for this finding. The following suggestions have been made.

1. The microelectrodes of S u e t al. were not recording from smooth muscle cells. High resistance electrodes of small tip diameter develop various DC potentials when they are pushed into the midst of a tissue. However, such potentials are usually of positive polarity. It is just conceivable that, when the preparation was exposed to the action of KC1, the observed negative po- tentials could decay to zero as a result of movement artifacts, but this explana- tion seems most unlikely.

2. The smooth muscle cells close to the intima, which were studied by S u e t al. (t964), may have been insensitive to noradrenaline. One of the in-

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Action potentials 15 !

herent disadvantages of the microelectrode technique is the difficulty in corre- lating the mechanical activity of the whole preparation with the electrical activity recorded from one or a few cells. The contractions observed by Su et al. might have reflected excitation of smooth muscle cells nearer the

adventitia.

3. The smooth muscle of the pulmonary artery may be of the "mult iuni t" type (BozLER, t948). If SO, it would be extremely difficult to record electrical activity with microelectrodes. The impaled cell would have to be in perfect physiological condition. In single unit smooth muscles, current flow from cell to ceil can alter the membrane potential of an impaled cell #assively. Such changes in membrane potential could be recorded with an electrode which injured the cell to a degree which prevented it from generating its own acti- vity. On the other hand, if the individual cells are electrically isolated from each other, an intracellular electrode would only record active changes in membrane potential generated by the impaled cell.

Muscular distributing arteries. The only report of electrical activity which may represent action potentials is that of TAKENAKA (t964) who recorded complex, diphasic, spike-like potentials from dog renal artery and pig coronary arteries in the presence of a low concentration of Ba ions. SPEDEN (1967) used microelectrodes to record from rabbit arteries, in vitro (ear artery, a branch of the posterior mesenteric artery and a caecal artery). No action potentials were recorded from unstimulated preparations nor were they seen in response to sympathetic nerve stimulation.

Small arteries and arterioles, pre-capillary sphincters and venules. Several workers have been successful in recording changes in membrane potential from these vessels which may well be classified as action potentials. FUNAKI (1958, t960, 1961) must be acknowledged as the pioneer in this field. His initial studies were carried out on small arteries and venules (20--I 00 ~m dia- meter) of the frog's tongue. Stimulating pulses were applied by a capillary electrode of 5 M~ resistance placed close to the recording electrode. Action potentials of up to 60 mV in amplitude and 200 msec duration were recorded in response to brief stimuli. Like the spikes of the taenia coil (]3fJLBRING, BURN- STOCK and HOLMAN, t958) these were not always all-or-none. Responses to stimulation occasionally showed a complex shape suggesting that they may have been recorded from cells which were partially depolarized by the spread of activity from neighbouring cells.

FUNAKI was able to record action potentials from regions which he con- sidered to be pre-capillary sphincters. These cells may correspond with the circular layer of smooth muscle at the junction of a metarteriole with a ter- minal arteriole as described by RHODIN (1967). Such action potentials were of the spike form, with a duration of about 20 msec. FUNAKI (1958) also recorded

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152 M.E. HOLSIAN: Electrophysiology of vascular smooth muscle

resting membrane potentials of about 50 mV from the endothelial cells of ca- pillaries but was unable to elicit any electrical activity from these vessels.

In t 960, he reported similar results for the small vessels of tile mesentery

of mice. Further studies were carried out by FUNAKI (t96t) on the small blood

vessels of the skin of the lateral region of the abdomen of frogs in vivo. Some of these vessels were spontaneously active, discharging action potentials of around 200 msec with prominent pre-potentials. The pattern of activity recorded from these vessels resembled that of a cardiac pacemaker cell. Bursts of action potentials lasted for several seconds. FUNAI<I'S work on the small

vessels of frogs has been confirmed recently (STEEDMAN, t966; SIGGINS and BER~AN, t 967). It should be noted that his method of stimulation may have excited motor nerve fibres in preference to direct stimulation of the smooth

muscle ceils themselves. STEEDMAN (1966, t967) studied small arteries and arterioles of rat mesen-

t ry with intracellular electrodes. She found that the smooth muscle of these vessels was spontaneously electrically active, in vivo. Fluctuations in membrane potential of the "slow wave" type were observed. Action potentials arose from the peaks of slow waves and were of the spike form, their half duration being of the order of 20 msec. Their repolarization phase was often faster than their depolarization phase. Many showed a prominent phase of hyperpolarization. Action potentials of the spike form were also recorded by SPEDEN (t 964) from small blood vessels of the mesentery of guinea-pigs in vivo. Both authors noted that the "foot" of the spike was rounded and that spikes showed a slow initial rate of depolarization. This feature is characteristic of the action potentials of visceral smooth muscle, recorded during spontaneous activity or in response to direct stimulation with large external electrodes. In contrast, action poten- tials in response to intracellular stimulation of visceral smooth muscle have a much faster initial rate of rise. It has been suggested that the slow "foot" of the conducted action potential or of action potentials recorded during spon- taneous activity is determined by the time course of the passive spread of depolarization through bundles of smooth muscle cells (ToMITA, 1966). The slow "foot" of the spikes recorded from arteriolar smooth muscle may indi- cate that the time course of the spread of electrotonus in this smooth muscle is similar to that of visceral smooth muscle.

Veins. In t 964, two accounts of the electrical activity of mammalian veins appeared almost simultaneously. CUTHBERT and SUTTER (1964) used tile suc- rose-gap apparatus to record from strips of rabbit anterior mesenteric veins. They reported that one preparation showed electrical activity consisting of a "plateau of depolarization on which spike-like action potentials were super- imposed". Phasic contractions were associated with these bursts of action potentials. FUNAKI and BOI~R (t964) used intracellular electrodes to record

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Action potentials ! 53

from isolated strips of rat portal vein. Electrical activity again consisted of irregular bursts of spike-like potentials. Individual spikes ranged from 8 to 30 mV in amplitude and 50 to t00 msec in duration.

Since these initial reports the longitudinal muscle of the anterior mesenteric and portal veins of common laboratory animals has been widely used as a model for studies on the electrical activity of vascular smooth muscle. In spite of an earlier report to the contrary (CUTHBERT and SUTTER, t964) this has turned out to be a simple and convenient preparation for sucrose-gap studies and it is not very difficult to obtain intracellular records - - at least for short periods. Microelectrodes have been used to record from the guinea-pig portal vein (NAKAJIMA and HORN, 1967), rabbit mesenteric veins (CUTHBERT et al., t965 ; MATTHEWS and SUTTER, t967) and rabbit portal vein (K. YONE- MURA - - unpublished work). Several different patterns of activity were ob- served in these preparations:

I. Long, continuous trains of action potentials of the spike form resembling those recorded from the guinea-pig taenia coll.

2. Regular bursts of short trains of spikes alternating with silent periods.

3. A mixture of single spikes and bursts of spikes.

Individual spikes had amplitudes of up to 60 mV and were of about t 00 msec total duration.

The patterns of activity recorded with the sucrose-gap apparatus from longitudinal muscle strips from the portal vein of rats (AxELSSON, JOHANSSON, JONSSON and WAHLSTROM, t967; AXELSSON, WAHLSTROM, JOHANSSON and JONSSON, t 967; JOHANSSON and LJUNG, 1967b) and rabbits (HoLMAN, KASBY, SUTHERS and WILSON, t 968) were similar to those observed with microelectro- des. In the rat, electrical activity appears to consist only of short bursts of spikes closely correlated with phasic contractions, alternating with periods of inactivity. In the rabbit, similar bursts of spikes (multi-spike complexes) can be recorded from most preparations but some undergo continuous spike acti- vity. The observed pattern of activity appears to be characteristic of the pre- paration under study. Variation between the two extreme forms, continuous "spiking" and bursts of spikes, occurred from one preparation to another, in spite of every precaution that could be taken to ensure that muscles were not stretched while being set up in the sucrose-gap apparatus (R. MILLS, unpub- lished work).

Individual spikes resemble those of visceral smooth muscle, having a slow initial rate of rise and a rapid decay phase which often leads to an apparent after-hyperpolarization.

In summary, changes in membrane potential resembling the action poten- tials of other excitable cells have been recorded from a limited number of examples of most types of vascular smooth muscle. Action potentials have

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154 M.E. HOLMAN: Electrophysiology of vascular smooth muscle

yet to be observed in muscular (distributing) arteries. No information is available for the circular muscle of veins.

4. Origin of s p o n t a n e o u s ac t iv i ty

FURCHGOTT (1955) wrote that "vascular smooth muscle, like most other smooth muscle, can exhibit inherent or spontaneous tone (an active sustained contraction not demonstrably dependent on stimuli from outside the muscle)". Reports of both tonic and phasic contractions of small vessels in vivo (vasomo- tion) and of strips of larger vessels in vitro are summarized in his article. Al- though FURCHGOTT (t955) felt that the mechanism of the development of this spontaneous contractile activity was not understood at that time, he quotes PETERSON'S (1936a and b) results which suggested that this might be initiated by the rhythmic firing of action potentials.

Recent evidence that the spontaneous electrical activity of some blood vessels is of myogenic origin has come from studies on the action of tetrodo- toxin. This drug is known to block the excitation and conduction of nerve impulses but is without effect on the initiation of spontaneous act ivi ty of visceral smooth muscle (NONOMURA, HOTTA and OHASHI, 1966; KURIYAMA, OSA and TOIDA, t 966; HASHIMOTO, HOLMAN and McLEAN, 1967). Tetrodotoxin likewise had no effect on the patterns of electrical or mechanical activity recorded from sheep mesenteric veins (HoLMAN and MCLEAN, 1967), rabbit portal vein (HoLMAN et at., t968) or from the sheep carotid artery exposed

to Ca-free solution 1 (KEATINGE, t 968). In contrast, there have been numerous reports that the spontaneous acti-

v i ty of small vessels, in vivo, is markedly affected by sympathetic denervation or by the action of sympathetic blocking drugs (see FURCHGOTT, 1955). STEED- MAN (1966) found that the spikes and slow waves of rat mesenteric arteries were depressed by section of the greater splanchnic nerves and blocked by intravenous injections of dihydroergotamine. However, she did not feel that her experiments ruled out the possibility that the electrical activity of these vessels was basically of myogenic origin.

STEEDMAN'S records are surprisingly reminiscent of those obtained with similar electrodes from the longitudinal muscle of the small intestine of many mammalian species (see HOLMAN, t968a). Although similar changes in mem- brane potential may be the ultimate result of a variety of different changes in membrane properties, it is reasonable to draw an analogy between the myo- genic slow waves of the gut and those of small blood vessels - - at least as a basis for subsequent experimentation. Both types of slow waves resemble sine

waves which have been "rectified" at a variable level.

1 The expression 'Ca-free' will be used to describe solutions from which Ca salts were omitted. If chelating agents were added to ensure that the solution was entirely free of Ca ions, this is noted in the text.

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Origin of s p o n t a n e o u s a c t i v i t y 155

The slow waves of rat mesenteric arterioles varied in frequency from one

animal to another (period 4.5--7.5 sec) but were relatively constant for any one preparation. Their duration was "only occasionally affected significantly

by various stimulating agents; in most cases their mean value remained un- changed" (STEEDMAN, t966). In contrast with the stability of their duration, the amplitude of the slow waves varied with depth of anaesthesia, during stimulation of sympathetic nerves and applied neurohormones (see below). Stability of frequency as opposed to amplitude is also characteristic of the

slow waves of the longitudinal intestinal muscle of most species. U n d e r n o r m a l condi t ions t h e s m o o t h musc le cells of t he l ong i tud ina l l aye r of the

i n t e s t i ne are p r o b a b l y in a s t a t e of exc i t ab i l i t y w h i c h is v e r y close to t h a t a t w h i c h ac t ion p o t e n t i a l s arise spon taneous ly . A n y process w h i c h t ends to increase t h e i r exc i t ab i l i ty , to lower t he i r r e s t ing m e m b r a n e po ten t i a l , or bo th , will cause a n ac t ion p o t e n t i a l or a series of ac t ion po t en t i a l s to develop. T h u s t h e depo l a r i za t i on of t he slow w a v e is able to i n i t i a t e one or a b u r s t of ac t ion po ten t i a l s . I f t h e muscle is hype rexc i t ab l e , showing a c o n t i n u o u s d i scharge of ac t ion po ten t i a l s , s low w a v e f l uc tua t ions in m e m b r a n e p o t e n t i a l c an m o d u l a t e t h e i r f requency. I f t he muscle is hypoexc i t ab le , depo la r i za t ion b y slow waves m a y n o t r each th re sho ld for the i n i t i a t i o n of a n ac t ion po ten t ia l . U n d e r these cond i t ions t he re is no c o n t r a c t i o n b u t slow waves can st i l l be recorded w i t h su i t ab le e lec t rodes (HoLMAN, 1968a).

The regular phasic contractions which have been observed in the longi- tudinal muscle of portal and mesenteric veins are usually associated with bursts of action potentials superimposed on a wave of depolarization (e. g. NAKAJI~A and HoRx, t967). It is interesting that the mechanical activity of strips of rabbit portal vein from which a continouus discharge of spikes was recorded in the sucrose-gap, occasionally showed phasic variations in tension in spite of an apparent lack of modulation of spike frequency, at least in that region of the muscle from which the recording was made. The longitudinal muscle of these veins may possess an inherent "slow wave" rhythm but this may be less effective in modulating spike frequency than the slow waves of small arteries and arterioles. Slow waves in the absence of spikes have yet to be observed in vein preparations - - in contrast with those of small arteries and arterioles.

The frequency of the bursts of spikes and phasic contractions characteristic of most preparations of portal veins appears, at first sight, to be more labile than that of the slow waves of small vessels or of intestinal muscle. Threshold stimulation of sympathetic nerves and low concentrations of sympathomimetic amines may cause an increase in the frequency of "bu r s t s " and phasic con- tractions (Sections 5 and 6). It is possible that this effect may be due to the development of new pacemaker regions rather than an increase in frequency of those which were driving the activity of the preparation before the onset

of excitatory stimulation ( JoHANSSON and L J U N G , t967a). Records obtained from isolated strips of the portal vein of rabbit and guinea-pig during just supra-threshold stimulation of sympathetic nerve and low concentrations of noradrenaline suggest that this might be the case (KASB'Z, MILLS and SUTHERS,

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156 M . E . HOLMAN : Electrophysiology of vascular smooth muscle

unpublished work). Small "ec topic" depolarizations sometimes appeared dur- ing the intervals between successive spike complexes. They increased in ampli- tude and eventually led to their own phasic contractions so that the frequency of both electrical and mechanical activity was twice that of the control.

It is clearly important to establish whether or not the spontaneous activity

of the longitudinal muscles of these veins is due to a slow wave mechanism which is similar to that of the small vessels studied by STEEDMAN (1966). The vein preparations have many advantages for in vitro studies. It is to be hoped that data concerned with their physiology, which are rapidly accumulating at the present time, may also be applicable to small arteries and arterioles.

The ionic basis of the slow waves of both vascular and intestinal smooth muscle remains a mystery. It has been suggested that they may be due to rhythmic fluctuations in the activity of an electrogenic Na pump (DANIEl, and CHAPMAN, 1963) but conclusive evidence for this view has yet to be found.

5. Ef fec ts of s t imu la t i on of the ne rve supply to v a s c u l a r s m o o t h m u s c l e

Several different methods have been used to stimulate the nerve supply to blood vessels in vivo and in vitro.

1. Electrical stimulation of sympathetic nerves:

a) by stimulation of anatomically distinct nerve trunks (e.g. the greater splanchnic nerves) ;

b) by stimulation of peri-arterial nerves; c) by stimulation of nerve fibres within the wall of the vessel by "trans-

mural" or "f ie ld" stimulating electrodes (PATON, 1955; KURIYAMA, 1963).

2. Pharmacological stimulation of sympathetic nerve terminals with acetyl- choline or related drugs (see Section 8).

Most authors have given pharmacological evidence confirming that the changes in electrical activity observed during nerve stimulation were due to the release of noradrenaline from post-ganglionic sympathetic nerves.

Elastic arteries. The apparent electrical quiescence of the rabbit pulmonary

artery during stimulation of its sympathetic nerve supply in the right recurrent cardiac nerve has already been described. Since S u e t al. (1964) were recording from the intimal surface of the vessel and the noradrenergic terminal axons are limited to the medio-adventitial border this is not an unexpected finding. How- ever these authors were unable to detect any changes in membrane potential during the direct application of noradrenaline. Since they were recording changes in tension from the whole arterial ring it cannot be assumed that the muscle cells on the intimal side of the artery were undergoing contraction during nerve stimulation or during exposure to noradrenaline. In fact, the simplest explanation for their findings might be that tile intimal smooth

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Effects of stimulation of the nerve supply to vascular smooth muscle 1 5 7

muscle has no receptors for this neurohormone. If the " in t imal" smooth muscle of this vessel is unreactive to noradrenaline then the question arises as to the way in which its contractile activity is controlled. There appear to be at least two possible explanations. Firstly, the muscle of the intimal layers may be excited, in vivo, by the spread of excitation which is initiated by neuro- muscular transmission at the medio-adventitial border. It is possible that the excitability of the smooth muscle is so markedly depressed by perfusion with physiological saline in vitro that conduction can no longer occur (see below, in relation to muscular arteries). Alternatively, the contractile activity of the smooth muscle of the intimal side of the vessel wall may be controlled by a completely different mechanism which is unrelated to the sympathetic system. Tile possibility that the " r ing" muscle cells of muscular arteries are controlled by vasodllator nerves, local hormones or metabolites, whereas the outer " tens ion" muscle cells are controlled by sympathetic constrictor nerves has been discussed by FOLKOW (t964a). However it is difficult to imagine how FOLKOW'S model might operate for large elastic arteries. It must be emphasized that no nerve fibres have been found near the intima in any artery - - elastic or muscular.

KEATINGE (1966) stimulated the noradrenergic nerve supply to sheep carotid artery strip preparation with acetylcholine. His observations suggest that the release of noradrenaline from sympathetic nerve terminals can initiate action potentials in this elastic artery. Irregular bursts of small spike potentials occurred which will be described in Section 8, below.

Small muscular arteries and arterioles. SPEDEN (t 964) recorded the electrical activity of small mesenteric arteries of the guinea-pig, in vivo, during stimu- lation of splanchnic nerves. Single stimuli gave rise to discrete waves of de- polarization which resembled the excitatory junction potentials (EJPs) re- corded from other smooth muscles in response to stimulation of excitatory nerves (HOLMAN, 1968b). E J P s in mesenteric arteries arose after a latency of about t50 msec. Their time course was similar to that of the E J P s of the guinea-pig vas deferens in response to stimulation of the hypogastric nerve (about 1 sec total duration). If they were of sufficient amplitude they appeared to give rise to action potentials.

Several conclusions can be drawn from SPEDEN'S results:

t . The invasion of the terminal axons at the medio-adventitial border by action potentials in response to a single stimulus gives rise to the release of sufficient noradrenaline to cause a large suprathreshold depolarization of the smooth muscle of the media. Although the distance separating axon terminals from smooth muscle cell membranes is greater than that described for some of the heavily innervated organs of the pelvic viscera (e.g. the vas deferens) it is clear that dilution of noradrenaline during diffusion does not prevent effec- tive transmission.

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158 M.E. HOLMAN: Electrophysiology of vascular smooth muscle

2. The noradrenaline released during transmission is probably "inacti- va t ed" fairly quickly - - within t sec of its release. This time is, of course, very long compared with the duration of transmitter action at the skeletal neuro-

muscular junction where acetylcholine is destroyed by cholinesterase within a few msec. However it may be quite short compared with the apparent du- ration of transmitter action in other blood vessels (e.g. veins, see below).

It is known that sympathetic terminal axons are capable of taking up nor- adrenaline from their extracellular fluid against a large concentration gradient (IVERSON, t967). This uptake process may be important in determining the duration of transmitter action at sympathetic nerve terminals. Alternatively, the concentration of noradrenaline arising from a single nerve stimulus could be expected to decrease rapidly as the result of simple diffusion. These questions have been discussed in detail elsewhere (HOLMAN, 1968b).

3- The smooth muscle of these vessels is capable of generating action potentials, either spontaneously, or when the membrane is depolarized by transmitter action. Apart from their probable role in initiating contraction such action potentials may be able to propagate for some distance, both proximally and distally, along the wall of the vessel so extending the effects of the release of transmitter from a limited number of axon varicosities to a considerable length of the vessel. However it must be emphasized that direct evidence for the conduction of action potentials from one cell to another has yet to be found for arteriolar smooth muscle (see Section 2 above).

More recently, SPEDEN (1967) studied the effects of peri-arterial nerve stimulation of isolated arterial segments taken from rabbits (ear artery, posterior mesenteric and caecal arteries). E J P s of up to 10 mV amplitude and 0.5 to t .0 sec duration were observed. However, in contrast with his previous finding for small guinea-pig vessels in vivo, these E J P s did not give rise to action potentials.

E J P s in response to repetitive stimulation showed marked facilitation. At frequencies greater than 2/sec successive E J P s summed with each other and at high rates of stimulation (5/sec or more) gave rise to a steady depolarization. The time course of the decay of this depolarization, after the cessation of stimulation, was similar to that of the E J P in response to a single stimulus.

Although no action potentials were observed during these experiments the vessels contracted in response to frequencies of 2/sec or more. This would seem to indicate that the contractile apparatus was activated by either a relatively small decrease in membrane potential (less than 20 mV) or a more direct action of noradrenaline on the contractile mechanism, or both. This point will be taken up again in Section 10.

Although action potentials have yet to be observed in normal solution in these or any other muscular arteries, in vivo or in vitro, SPEDEN suggested that

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Effects of s t imula t ion of the nerve supply to vascular smoo th muscle 159

this lack of excitability might be due to removal of the arteries from their natural site and their perfusion at low pressures (15--35 mm Hg).

Since the innervation of muscular arteries is limited to the medio-adventitial border, some mechanism must exist for activation of the smooth muscle cells of the media other than those in the immediate neighbourhood of the terminal axons. (The media is of the order of t00 am thick.) Three possibilities may be considered:

t. Diffusion of noradrenaline.

2. Electrotonic spread of the E J P which is generated by the action of nor- adrenaline on the smooth muscle cells at the medio-adventitial border.

3. The initiation of an action potential by such E J P s and conduction of the action potential throughout the media.

The brief duration (500 msec) of some of the E J P s recorded by SPEDEN might be taken as evidence against the diffusion theory. Both the other alter- natives imply that the smooth muscle of these arteries is of the single-unit type and that ions can flow from one cell to another via low resistance pathways. Evidence for the spread of electrotonus has yet to be found but this may be due to technical difficulties. If the passive electrical properties of arterial smooth muscle should turn out to be similar to those of visceral smooth muscle one might expect the E J P to spread rapidly throughout the media of quite large vessels with little decrement. (The length constant for the spread of electro- tonus in bundles of longitudinal intestinal muscle is t mm or more.) If the E J P exceeded threshold for initiation of an action potential, it is likely that the entire media would undergo excitation in an "all-or-none" way.

The consequences of this chain of events in relation to the contractile behaviour of the vessel wall must depend on the characteristics of excitation- contraction coupling. If this were such that depolarization in the form of an E J P could initiate contraction it would not be necessary to propose that this smooth muscle was capable of generating action potentials. Furthermore the absence of action potentials might lead to a smoother grading of the response to nerve stimulation than that associated with "all-or-none" action potentials. However, there is little to be gained by further speculation along these lines until more is known about the electrical and mechanical properties of arterial smooth muscle.

The effect of repetitive nerve stimulation on small arteries and arterioles (vessels ranging from 80 to 150 ~xm diameter) was studied by STEEDMAN (1966) who used rat mesentery, in vivo. The cut peripheral end of the right splanchnic nerve was stimulated with short trains (3--5 sec duration) of pulses whose frequency (5 to t0/sec) and intensity were sufficient to cause marked vaso- constriction. She observed an increase in amplitude of the slow waves and the appearance of one or a burst of spikes on their crests. Extracellular "pressure"

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t 60 M.E. HOLMAN: Electrophysiology of vascular smooth muscle

electrodes were used in this s tudy together with high frequencies of stimulation and E J P s similar to those described by SPEDEN were not observed.

In contrast with SPEDEN'S finding for rabbit arteries, STEEDMAN reported that repetitive stimulation caused a prolonged increase in electrical activity of

the small vessels of the rat mesentery. The total number of spikes recorded during successive 50 sec intervals was increased to a level well above that of the control for more than 300 sec after a 5 sec period of stimulation. It is rather surprising that the effects of 25 to 50 stimuli should last more than 300 times longer than the effects of a single stimulus to blood vessels of comparable size in the guinea-pig mesentery (SPEDEN, 1964). However, as STEEDMAN pointed out, the prolonged response to splanchnic nerve stimulation observed ill her experiments could have been due to the "organization of the ganglia", the influence of the intact left splanchnic nerves, or to the release of catecholamines

from the adrenal medulla. Portal vein. No E J P s in response to a single stimulus have been observed so

far in the rabbit portal (HOLMAN, KASBY, MILLS, SUTHERS and YONEMURA, unpublished work). Furthermore, we have been unable to detect any changes in the pattern of electrical or mechanical activity in response to a single stimulus (HOLMAN et ah, t968). Repetitive stimulation appears to be an essential pre- requisite for effective transmission, as is also the case for the transmission of inhibition from sympathetic nerves to the smooth muscle of the taenia coli (BENNETT, BURNSTOCK and HOLMAN, 1966). In both tissues, this may be related to the pattern of innervation. Terminal axons are limited to the larger spaces between bundles of smooth muscle cells and rarely (if at all in the taenia coli) make close contact with muscle cell membranes. Under these conditions it would seem that the amount of noradrenaline released by a single maximal stimulus is insufficient to modulate the pattern of spontaneous activity of the preparation. This may be due to any or all of the following:

t . The concentration of noradrenaline is too low to cause a change in mem- brane properties as a consequence of its dilution by extracellular fluid.

2. The time during which an effective concentration of noradrenaline is present in the vicinity of the smooth muscle membrane is too short to bring

about a significant change in electrical activity. 3. The area of smooth muscle membrane which is exposed to a supra-thresh-

old concentration of noradrenaline is too small to be effective in changing the pat tern of activity of the majori ty of smooth muscle cells of the preparation.

I t must be emphasized that relatively few terminal axons can be found within the media of these veins. A dense noradrenergic plexus like that at the medio-adventitial border of arteries is absent from these vessels.

The mechanical effects of repetitive stimulation of the right splanchnic nerve were recorded by JOHANSSON and LJUNG (t967a) from portal veins of rabbits and cats in vivo and in vitro. Tonic contractions occurred in response to

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Action of catecholamines 161

frequencies of I/sec or more on which phasic contractions, sometimes at an increased frequency, were superimposed. The amplitude of the response in- creased rapidly as frequency was increased from t to 8/sec.

HOLMAN et al. (t 968) used '" field" electrodes to stimulate intramural nerve fibres in strips of rabbit portal vein, i n vi tro, and obtained mechanical responses

similar to those described by JOHANSSON and LJUNG (t967a). They found that maximal nerve stimulation gave a smooth uninterrupted rise in tension which reached a maximum within t0 sec of the onset of stimulation. (5 to t0 sec trains of pulses of variable frequency and intensity were used ill these experi- ments.) Phasic contractions could not be distinguished at the peak of these responses but returned as the level of tension fell back to normal during the next 30 to 50 sec.

Both JOHANSSON et al. and HOLMAN et al. found that the response to maximal nerve stimulation was smaller than that in response to a large dose of noradrenaline (see also HOLMAN and MCLEAN, 1967).

The electrical events occurring during nerve stimulation have been de- scribed (HOLMAN et al., t 968). Threshold stimuli caused an increase in frequency of spike complexes which could be correlated with phasic contractions. Stimuli of greater intensity or frequency, which were associated with an abrupt rise in tension and the absence of phasic contractions, caused a continuous dis- charge of low amplitude action potentials which were superimposed upon a maintained depolarization. It is interesting that the time course of this re- sponse was similar to that of the small mesenteric vessels studied by STEEDMAN

(t966).

6. Act ion of catecholamines

Noradrenaline caused a maintained depolarization of the carotid artery strip which was accompanied by contraction (KEATINGE, t 964). Depolarization was abrupt in onset and was sometimes preceded by a single spike with an incomplete repolarization phase. BARR (t 961) used large " w i c k " electrodes to record from rings of dog carotid artery. He found that contractions in response to noradrenaline were associated with maintained electrical quiescence. In contrast, responses to direct electrical stimulation consisted of low amplitude spike activity. His records of the action of noradrenaline, however, do not rule out the possibility that contraction was preceded by a brief burst of action potantials (compare Fig. 3 A with Fig. 2 of BARR, t961). I t is unlikely that his methods of recording would have detected a phase of maintained depolarization.

These responses may be comparable with those of visceral smooth muscle to large doses of excitatory neurohormones (BURNSTOCK and PROSSER, t960). The taenia coli of the guinea-pig, for example, responds to acetylcholine with an initial burst of spikes which rapidly reverts to an oscillation and finally

results in a maintained depolarization (BuRNSTOCK and HOLMAN, t 966).

I I Ergebnisse der Physiologie, Bd. 61

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162 M.E. HOLMAI~: Electrophysiology of vascular smooth muscle

A maintained depolarization response to acetylcholine contrasts with the action of this neurohormone on skeletal muscles of the twitch type. Prolonged exposure of the end- plate region to acetylcholine and related drugs causes rapid receptor desensitisation and depolarization is not maintained. Neither "slow" skleletal muscle fibres nor smooth muscle appear to possess such a fast desensitisation mechanism.

The application of noradrenaline and adrenaline to the small arteries of the rat, in vivo, caused a marked increase in the frequency of firing of action potentials (STEEDMAN, t966). Thus t0 -s g/ml adrenaline caused a 7-fold in- crease in the number of spikes which occurred in successive 50 sec intervals. The threshold concentration required for a detectable effect on electrical acti- vi ty was lower for noradrenMine (t0 -1° to 10 -8 g/ml) than for adrenaline (t 0 -9 to 10 -s g/ml). In spite of the increase in spike frequency the duration of the slow waves was unchanged, as described previously for sympathetic nerve stimulation. Higher doses of catecholamines prolonged these effects.

STEEDMAi% showed that it was possible to mimic the action of the noradren- aline released upon nerve stimulation, with the direct application of low con- centrations of noradrenaline (t 0 -s g/ml) to the outer (adventitial) layer of the vessel. This suggests that a high local concentration of noradrenaline in the junctional gap between axon terminals and muscle membrane is not necessarily

required for effective transmission. There have been several descriptions of the excitatory effect of noradren-

aline on the electrical activity of the anterior mesenteric and portM veins of various species (e.g. FUNAKI, t 9 6 7 ; JOHANSSON, JONSSON, AXELSSON and WAHLSTROM, 1967; NAKAJIMA and HORN, t967; HOLMAN et al., t968; CUTH- BERT, 1967; CUTHBERT and SUTTER, t965). All authors have reported that "threshold" concentrations of noradrenaline (as low as 10 -l° g/ml) caused an overall increase in the frequency of action potentials. This increase was related to an increase in the number of spikes per burst, to an increase in the frequency of bursts, or to both. The electrical effect was associated with an increase in amplitude and, or frequency of phasic contractions. JOHANSSON, JONSSON,

AXELSSON and WAHLSTROM (t967) did not feel that the increase in electrical activity could entirely account for the observed increase in contractile activity. They proposed that noradrenaline may have had a direct excitatory action on the contractile mechanism or on excitation-contraction coupling (see below).

Higher concentrations of noradrenaline caused a continuous discharge of action potentials and eventually maintained depolarization. NAKAJIMA and HORN (t967), for example, recorded a depolarization of 20 mV during pro- longed exposure to 10 -~ g/ml noradrenaline.

CUTHBERT and SUTTER (1965) argued that the maintained depolarization observed in the rabbit vein was not of sufficient intensity to account for the absence of action potentials. When this muscle was depolarized to a similar degree by K-rich solutions a continuous discharge of action potentials was observed. CUTHBERT and SUTTER also concluded that the maintained tension

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Action of catecholamines 163

observed in the presence of noradrenaline was due, in part, to the direct action of this neurohormone on the contractile mechanism. It is difficult to measure absolute levels of membrane polarization in the sucrose-gap and a microelec- trode study would be useful to check this point.

The behaviour of the smooth muscle membrane in response to high con- centrations of noradrenaline may provide important clues as to the ionic basis of its action. However, it seems unlikely that the smooth muscle of these vessels would ever encounter such concentrations of catecholamines, in vivo. It is perhaps of greater interest to analyse the electrical effects of noradrenaline which cause contractions of similar magnitude to those in response to sym- pathetic nerve stimulation.

In the rabbit portal vein it was also possible, in some preparations, to ex- actly mimic the effects of a short period (5 sec) of sympathetic nerve stimulation by perfusing the muscle with a small dose of noradrenaline (less than 0,t a g - - see HOLMAN et al., t 968). Similar observations for the mechanical activity of rat portal vein, in vivo, have been reported by JOHANSSON and LJUNG (1967a). These responses are presumably mediated by ~-adrenergic receptors since they could be blocked by phenoxybenzamine and other s-receptor antagonists.

There is evidence for the presence of r-receptors in the portal vein. Isopren- aline abolished electrical spontaneous activity and associated phasic contrac- tions (JOHANSSON, JONSSON, AXELSSON and WAHLSTROM, 1967; CUTHBERT, 1967; HOLMAN, 1967; HOLMAN et al., 1968). This effect was blocked by r-re- ceptor antagonists (e.g. propranolol). Occasionally preparations of rabbit portal vein gave a diphasic response to just supra-threshold concentrations of noradrenaline. An initial increase in activity was followed by inhibition. This inhibition may have been due to the combination of noradrenaline with r- receptors (MILLS, unpublished work).

The inhibitory action of isoprenaline on the rabbit portal vein was asso- ciated with a decrease in frequency of spike complexes. In preparations show- ing a continuous discharge of action potentials or in those where there was evidence for depolarization of the "pace-maker" type between bursts of action potentials, isoprenaline caused hyperpolarization. There was no evidence for dissociation of electrical and mechanical activity (HOLMAN et al., t968). In contrast, on the rat portal vein, isoprenaline caused "membrane depolarization, a shortening of the bursts of action potentials, a shortening of the interval between bursts and a reduction in tension development that could not be fully accounted for by a change in spike activity" (JoHANSSON, JONSSON, AXELSSON and WAHLSTR6M, 1967). In order to check whether the decrease in amplitude of phasic contractions of the rat vein was due to a "desynchronising" effect of isoprenaline, JOHANSSON et al. used the preparation of JOHANSSON and LJUNG (1967) described above. However, the mechanical activity of both segments

i 1 "

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t64 Y[. E. HOLMAN: Electrophysiology of vascular smooth muscle

of vein remained in phase, in spite of the changes in frequency induced by the isoprenaline.

JOHANSSON, JONSSON, AXELSSON and WAHLSTR6M, t967 drew attention to the similarity of the response of the rat portal vein to isoprenaline with that in response to Ca-free solutions. They quote a suggestion of BOHR (1961) that fl inhibition of vascular smooth muscle may be due to a mechanism which reduces the concentration of Ca ions available for contraction.

High concentrations of isoprenaline (greater than 10 -~ g/ml) gave a diphasic response. There was initial inhibition of spike complexes and phasic contrac- tions which was followed by an excitatory phase resembling that in response to noradrenaline. This latter response was blocked by phenoxybenzamine.

Studies on the effect of catecholamines in the presence of an altered ionic environment may provide important clues as to the ionic mechanisms involved in their action. A great deal of work along these lines has been carried out on visceral smooth muscle (BURNSTOCK and HOLMAN, 1966). AXELSSON, JOHANS- SON, JONSSOl~ and WAHLSTR6M (t967) studied the action of isoprenaline on rat preparations exposed to K-rich solutions. A 4-fold increase in K caused a continuous discharge of action potentials which was unaffected by iso- prenaline, although some mechanical relaxation was observed. In the rabbit vein, K-rich solutions reduced the hyperpolarization observed in response to isoprenaline in those preparations which did not show steady resting mem- brane potentials.

These findings may be taken to indicate that the action of isoprenaline is due, at least in part, to an increase in K and, or C1 conductance. The K per- meability and conductance of the portal vein may be relatively low under normal conditions (see above). An increase in extracellular K concentration probably causes an increase in K conductance and the membrane potential may approach the K equilibrium potential given by the Nernst equation. A further increase in K conductance would have no effect on the membrane potential. Furthermore, if K conductance was already high, a further increase, due to the action of isoprenaline, might be less effective in stabilizing spon- taneous activity, than under normal conditions.

The effect of catecholamines on preparations of rat portal vein exposed to nominally Ca-free solutions is of special interest (AxELSSON, WAHLSTR(~M,

JOI~ANSSON and Jo~sso~, t967; JOI-IANSSON, JONSSON, AXELSSON and WAHL- STR6M, 1967). Prolonged exposure to Ca-free solution caused depolarization and the complete cessation of electrical and mechanical activity. At this time both spike discharge and phasic contractions could be restored by the appli- cation of noradrenaline. High concentrations of isoprenaline also had this effect which was abolished by phenoxybenzamine.

In Ca-free solution the ratio of K conductance to Na conductance is re- duced in many different excitable cells, probably as the result of an increase

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Action of acetylcholine 165

in Na conductance (BENNETT, t967). This generally leads to a decrease in resting membrane potential. It is likely that the effect of the combination of noradrenaline with ~-receptors in vascular smooth muscle is to increase con- ductance for cations (HoL~AN, t967). If Na conductance is already high in Ca-free solution, the effects of an increase in K conductance may dominate the response to noradrenaline. This might lead to an increase in membrane poten- tial and, possibly, to the restoration of activity. It must be pointed out that JO~IANSSON, JONSSON, AXELSSON and WAHLSTR6~ (1967) did not r e p o r t a n increase in membrane potential upon exposure to noradrenaline in the absence of Ca. Nevertheless, the relatively large amplitude of tile spikes shown in their Fig. 4 d suggests that the membrane potential must have been reasonably high at this time. JO~ANSSON et aI. suggest that an increase in membrane permeability for Ca ions or mobilization of bound Ca may also be involved in "~-receptor stimulation". This point wilt be discussed below in Section t0.

It is clear that many more experiments will need to be carried out before it is possible to say whether or not the excitatory and inhibitory actions of catecholamines on vascular smooth muscle are due to the same ionic mecha- nisms as those proposed for visceral smooth muscle. It should be possible to measure both changes in membrane potential and conductance during amine action (e.g. BOLBRING and TOmTA, t968). I t is probably safe to predict rapid developments in this area of research into the physiology of vascular smooth muscle in the near future.

7. Ac t ion of ace ty lcho l ine

The effects of this neurohormone on vascular smooth muscle are complex, probably due to its action on sympathetic nerve terminals (FERRY, t966). There is much evidence that acetylcholine and related nicotinic agonists can excite noradrenergic terminal axons, causing the release of transmitter. This action of acetylcholine is blocked by hexamethonium. KEATINGE (1966) used this method to stimulate intramural nerve fibres in carotid artery strips. The electrical activity induced by acetylcholine (25 Ezg/ml) and nicotine (25 ~zg/ml) differed rather markedly from that ill response to added noradrenaline (25 ~g/t00 ml). In contrast to the prolonged depolarization in response to noradrenaline, acetylcholine caused an irregular discharge of small spikes. KEATINGE suggested that this pattern of activity could be due to the asyn- chronous firing of different groups of smooth muscle cells. He felt that only some of the cells within such group would be excited directly by released noradrenaline, the remainder of the cells being activated "indirectly by trans- mission of action potentials from one smooth muscle cell to another" (KEA- TINGE, 1967).

In KEATINGE'S experiments, nicotine did not completely abolish contrac- tions in response to acetylcholine. Although ~-adrenergic antagonists and

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166 M.E . HOLMAN: Electrophysiology of vascular smooth muscle

hexamethonium blocked the action of nicotine, they did not block contrac- tions in response to acetylcholine, in the presence o/ nicotine. Thus it seems that the presence of receptors for acetylcholine mediating on excitatory action cannot be excluded for this smooth muscle. HOLMAN and MCLEAN (1967) came to a similar conclusion as a result of their pharmacological studies on sheep mesenteric veins.

SIJ and BEVAN (1967) found that acetylcholine caused depolarization of the intimal layer of the pulmonary artery. This is an especially interesting finding, in view of the lack of effect of noradrenaline on the membrane poten- tial of this preparation. Again this indicates that there are receptors for acetylcholine which lead to excitation rather than inhibition.

The direct application of high concentrations of acetylcholine (10 -4 to t0 -3 gm/ml) to rat mesenteric arterioles caused abolition of action potentials and a decrease in slow wave amplitude (STEEDMAN, t966). In contrast with the work described above, STEEDMAN did not observe any excitatory effects which might have been due to the direct action of acetylcholine or to the release of noradrenaline from terminal axons.

Acetylcholine had complex effects on the portal vein. FUNAKI and BOI4R (1964) reported a diphasic action in which an initial period of hyperpolarization was followed by a secondary depolarization. The initial period of hyperpolari- zation did not block spontaneous activity but action potentials of increased magnitude were observed at this time. Their frequency was increased during the later phase of depolarization. Similar results were obtained for the guinea- pig preparation by NAKAJIMA and HORN (t967).

These responses probably arise from a combination of a direct hyper- polarizing (inhibitory) action of acetylcholine on the smooth muscle membrane, followed by an excitatory effect due to the release of noradrenaline or to a secondary excitatory action of acetylcholine on the smooth muscle membrane. It is questionable whether excitatory responses to acetylcholine have any physiological significance; at least in those blood vessels studies so far.

8. Responses to changes in length Many types of vascular smooth muscle are sensitive to mechanical stimuli.

The contractile responses of small vessels may be important for haemostasis. I t is also possible that their response to a mechanical stimulus in terms of in- creased intraluminal pressure may be significant in relation to the "auto- regulation" of blood flow with variation in perfusion pressure such as that which occurs in various vascular beds, notably the kidney (FURCHGOTT, 1955; FOLKOW, 1964a and b).

Electrophysiological studies on this important aspect of the physiology of vascular smooth muscle have been limited so far to the effect of changes in length on the electrical activity of the portal vein of several species. These

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Ionic basis of the action potential 167

preparations are extremely sensitive to "s t re tch" , responding to a small in- crease in length (a few %) with an inital phasic contraction followed by a main-

tained increase in tension (CUTHBERT, t967; I-IOLMAN, KASBY and SUTI~ERS, t968). The initial phasic response of the rabbit portal vein was associated with depolarization and a high frequency burst of low amplitude spikes which lasted for about t0 sec (HOLMAN, KASBY and SUTHERS, 1968). Activity gradually returned to a pattern which resembled that of the control though, in many preparations, the frequency of the bursts of spikes increased. In other prepa- rations contractions (and bursts of spikes) eventually returned to the same frequency as that of the control period but the number of spikes per complex and the amplitude of the phasic contractions were increased.

The relevance of these findings to the problem of autoregulation in small

vessels has yet to be established.

9. Ionic basis of the action potential

Several lines of evidence suggest that the ionic basis of the action potentials of visceral smooth muscle may be different from that of nerve and skeletal muscle (BENNETT, 1967; HOLlVIAN, t968a). The nerve impulse is due to a voltage-dependent increase in conductance for Na ions which permits a net influx of Na ions and a rapid, regenerative change in membrane potential of the order of several hundred V/sec. The action potentials of many visceral smooth muscles differ from classical " N a " action potentials in the following

ways: t . They have a slow rate of rise, often less than 20 V/sec. 2. They are relatively independent of a reduction in Na concentration of the

external medium to less than 20 meq/1. (In general, completely Na-free solutions depress or abolish action potentials. This may be part ly due to the decrease in resting membrane potential which occurs in visceral smooth muscles exposed to Na-free solution for 20 rain or more.)

3. They are unaffected by tetrodotoxin (which blocks " N a " action po- tentials).

The action potentials of visceral smooth muscle resemble Na action poten- tials in the following ways:

1. They are associated with an increase in membrane conductance. 2. They are dependent on the Ca concentration of the external medium. 3. They are dependent on the value of the membrane potential at which

they are initiated. Studies on the ionic basis of the action potentials of crustacean striated

muscle have established an alternative mechanism for the generation of an all- or-none, regenerative response to depolarization which does not depend on an influx of Na ions. In these preparations the action potential is due to the inward movement of Ca ions which are probably derived from a "store" of Ca ions

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168 M.E. HOLMAN: Electrophysiology of vascular smooth muscle

bound to sites on or near the outside of the cell membrane (HAGIWARA and TAKAHASHI, t967). Similarities between the action potentials of smooth and crustacean muscle suggest that they may both depend on the same ionic mechanism. However, the possibility still exists that Na ions might make some contribution to the rising phase of the smooth muscle action potential. An in- flux of both Ca and Na probably occurs during the action potentials of cardiac muscle (HAGIWARA and NAKAJIMA, t965; DUDEL, PEPER, RUDEL and TRAUT- WEIr, t967).

Since tetrodotoxin does not block the action potentials of vascular smooth muscle it seemed likely that their ionic basis might be similar to that of the action potentials of visceral smooth muscle. However, KEATINGE (t968) has put forward the view that the action potentials of the sheep carotid artery are Na rather than Ca dependent. As already noted, his preparations were spontane- ously active during long periods of exposure in Ca-free solution. If EDTA was added to the solution there was rapid depolarization and action potentials were abolished. KEATINGE concluded that this was due to the fall in resting potential caused by the EDTA rather than the absence of Ca ions in the external solution. If the resting potential was increased by the use of low Na solutions (Tris re- placing Na) spontaneous electrical activity returned, even in the presence of EDTA. KEATINGE stated that "when spikes had ceased after the addition of EDTA, 50 % of the sodium in the perfusion fluid was replaced by Tris to in- crease the membrane potential. This restored spikes in every case (three experi- ments)"

Further evidence for dependence of action potentials on Na rather than Ca came from experiments in which arteries were exposed ini t ial ly to Ca-free, low Na solutions. These preparations had high membrane potentials and were not spontaneously active. EDTA caused depolarization and spontaneous activity. This only occurred if some Na was present in the bathing solution.

Although KEATINGE'S analysis of these data, in terms of a tetrodotoxin- independent, Na action potential is attractive, an alternative explanation, in terms of a Ca action potential is still possible.

It is unlikely that exposure to a nominally Ca-free solution would seriously deplete the Ca store in or near the cell membrane. Thus JOHANSSON, JONSSON, AXELSSON and WAHLSTR/JM (1967) were able to restore both electrical and mechanical activity of preparations exposed to Ca-free solution by catechol- amines. In an example of visceral smooth muscle (mouse vas deferens) Ca-free solution abolished the action potentials in response to intracellular stimulation but this appeared to be due primarily to a fall in resting potential (HAsHIMOTO and HOLMAN, 1969). If this was increased by prolonged hyperpolarizing cur- rents action potentials could again be elicited. The carotid artery is not nor- mally spontaneously active and has a high resting membrane potential. If it is accepted that the membrane potential must be within a critical "f i r ing"

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Ionic basis of the action potential t 69

zone for spontaneous activity to occur, then Ca-free solutions may have induced activity by causing a decrease in membrane potential (BURNSTOCK, HOLMAN

and PROSSER, 1963). The addition of EDTA to the Ca-free solution probably depolarized the membrane beyond the firing zone.

The depolarization induced by Ca-free solution, which is accelerated by EDTA, is probably due to an increase in Na conductance (BENNETt, 1967). This could explain why KEATINGE observed little or no depolarization when EDTA was added to preparations exposed to Na-free, Tris solution and hence, no electrical activity. If Na was only partially replaced by Tfis, the resting membrane potential should be high, owing to a decrease in the value of the Na equilibrium potential. An increase in Na conductance by EDTA would be still expected to decrease the resting potential and cause a transient restoration of activity.

I t is more difficult to explain the results of experiments in which this sequence of events was reversed, i.e. spontaneous activity was first blocked by EDTA and this effect was subsequently reversed by solutions containing

50% Na and 50% Tris. Prolonged exposure to EDTA would be expected to cause a loss of Ca from the membrane store and a loss of excitability. Pro- vided some Ca remained in the store, the increase in membrane potential due to the low Na solution might reverse the EDTA depolarization and so put the membrane potential back into the firing zone. It would be interesting to know whether or not KEATINGE was able to obtain this effect after prolonged exposure to EDTA.

One possible test for the Na hypothesis would be to check whether action potentials can occur in Na-free solution. KEATINGE did, in fact, f ind that action potentials (of relatively small amplitude) could be elicited by an increase in K concentration in Na-free solution. One could invoke a store of bound Na ions to explain this result. The final analysis of this problem must await the appli- cation of voltage clamp techniques to smooth muscle and the direct identifi- cation of the ions which carry the inward current during the rising phase of the action potential.

I t is of interest that carotid arteries exposed to Ca-free solution behave in many ways like visceral smooth muscle in equilibrium with solutions contain- ing about 2.5 mM CaCI~. I t may well be incorrect to assume that the activity of the Ca ions in the immediate vicinity of the cell membrane of smooth muscle, in vivo, is equal to the concentration of the Ca ions in the solution used for in vitro studies. Little is known of the physico-chemical properties of t h e " base- ment membrane material" which surrounds the unit cell membranes of most smooth muscle cells. I t is possible that this may have a significant effect on the activity of Ca ions and that this may differ from one smooth muscle to another. Interestingly enough, PEASE (t962) noted that basement membrane material was absent from the smooth muscle cells of another elastic ar tery (rat aorta).

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170 M.E. HOLMAN : Electrophysiology of vascular smooth muscle

There seems no reason to doubt that the repolarization phase of action

potentials of the spike form, whether these are from vascular or visceral smooth muscle, is due to an increase in conductance for K ions (BuRNSTOCK and HOLMAN, t966). For example, the action of Ba ions, which decrease the K conductance of both resting and active membranes, is similar for both types of smooth muscle (a fall in resting membrane potential and a decrease in rate of repolarization during the action potential; I-IOLMAN, unpublished work).

10. Ac t iva t i on of c o n t r a c t i o n

No at tempt will be made here to summarize the vast amount of literature

dealing with this question in relation to vascular smooth muscle. This aspect of the physiology of smooth muscle in general has been the subject of a number of recent reviews (BOHR, t964; DANIEL, t964, t965; SCHATZMANN, t964). The following is a highly speculative account of some recent work which is of

interest in relation to the electrical activity of vascular smooth muscle. It seems likely that the short term mechanical activity of smooth muscle is

regulated by the concentration of ionized Ca in the neighbourhood of the myo- filaments (BOHR, 1964). In the relaxed state, the concentration of ionized Ca in the cytoplasm is probably maintained at less than t EzM, as is the case for striated muscle (SANDOW, 1965). I t has not yet been possible to obtain sub- cellular fractions from smooth muscles which have biochemical properties similar to those described for the "relaxing factor" of skeletal muscle. How- ever, there is evidence for an active process which transfers ionized Ca from the intracellular space 'of smooth muscle to the cell membrane (VAN BREEMEN, DANIEL and VAN BREEMEN, t966). I t may turn out that the cell membranes of smooth muscle have very similar properties to the membranes of the sarco- plasmic reticulum of striated muscle. In this case smooth muscle may prove to be convenient tissue for more detailed studies on the mechanisms involved in the removal of ionized Ca from the vicinity of the contractile apparatus.

There is a number of possible ways in which the intracellular ionized Ca concentration might be increased to a level at which contractile activity is turned on. The action potential itself is one such mechanism. If the rising phase of the action potential is due partly, or entirely, to an increase in membrane permeability for Ca and to a net inward movement of these ions down their electrochemical gradient is it possible that this might lead to a sufficiently

high concentration of ionized Ca to initiate contraction ? GOODFORD (1967) calculated that the Ca influx during a single action poten-

tial (taenia coli) would be barely adequate to raise the intracellular concen- tration of Ca ions to a level which would activate contraction although he felt

that "five or ten such events (action potentials) would produce full act ivat ion". Similar calculations from this laboratory which were based on a rate of de- polarization of 20 V/sec and a membrane capacitance of t ~F/cm 2 suggested

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Activation of contraction 171

that, if the faster spikes recorded from visceral smooth muscle were entirely due to Ca current, a single action potential could lead to an increase in Ca concentration of the order of 2 to 5 × t0 -6 M. SCHADLER (1967) observed a graded increase in tension and ATPase activity of glycerol extracted smooth muscles (guinea-pig taenia coli and cow carotid artery) over a range of Ca

concentrations from 10-8--t0 -~ to 10 -~ to I0 -5 M. Since the concentration of actomyosin in smooth muscle is probably only 1/10 that of skeletal muscle (NEEDHAM and WILLIAMS, 1963) it seems just possible that a " C a " action potential might lead directly to the partial activation of contraction.

If the Ca current of the action potential is sufficient to trigger contraction it follows that, provided that contractile apparatus is functional, action poten- tials should always trigger contractions. There have been a number of reports of the dissociation of action potentials and contractions which would appear, at first sight, to rule out this hypothesis. In Ca-free solution, electrical activity continued for several minutes after the cessation of contractions of tile rabbit anterior mesenteric vein (CUTHBERT and SUTTER, 1965). In the sheep carotid artery, spontaneous electrical activity continued for more than 100 rain in the absence of detectable mechanical activity. In contrast with these results, how- ever, is the finding of JOHANSSON, JONSSON, AXELSSON and WAHLSTR6M (1967) who showed that noradrenaline was able to restore both action potentials and contractions of the rat portal vein which had been rendered both electrically and mechanically quiescent by prolonged exposure to Ca-free solution.

Dissociation of action potentials from contractions of the taenia coli, during perfusion with solutions in which Na had been replaced with Li, hydrazine and choline ions, was reported by AXELSSON (1961). However, as AXELSSON pointed out, it is possible that changes in spike configuration might have occurred during exposure to these solutions which could be responsible for the abolition of tension.

It will be interesting to discover if any or all of the above examples of dis- sociation can be explained in terms of a decrease in the Ca current associated with the action potential. It is certainly possible that a regenerative change in membrane potential might still occur even though the inward Ca current was too small to initiate contraction.

There is no doubt that the contractile apparatus of smooth muscle can be activated by means other than action potentials. Depolarization by K-rich solutions causes an initial phasic contraction in many smooth muscles which is associated with a brief burst of action potentials. The initial phasic contraction is followed by a tonic contraction whose amplitude is related to the concen- tration of K and, therefore, to the degree of depolarization of the membrane (CHAPMAN and HOLMAN, t968). Many smooth muscles relax completely during prolonged exposure to high K solutions, although the membrane remains in a depolarized state (EVANS, SCHILD and THESLEFF, t958). It is well known that

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172 M . E . HOLMAN : E l e c t r o p h y s i o l o g y of v a s c u l a r s m o o t h musc le

excitatory drugs and neurohormones are still able to elicit contractions at this

time although there is little likelihood that they would cause a significant change in membrane potential. Since excitatory agents (e.g. noradrenaline, for vascular smooth muscle) can cause contraction of preparations which have partially or completely relaxed during prolonged K depolarization it would seem that the source of Ca ions which is utilized during the tonic response to high K may be different from that utilized by noradrenaline. Alternatively, if

the relaxation in high K solutions is due to depletion of a " s to re" of Ca ions, noradrenaline may act by reloading Ca ions into this store. However, until the source of the Ca ions which are involved in the tonic response to K depolari- zation has been identified, and the cause of the subsequent relaxation has been found, it is unwise to speculate any further along these lines.

HINKE'S (1964) studies on arterial smooth muscle led him to propose that noradrenaline released Ca ions from a " t ight ly bound" fraction of membrane Ca whereas contractions in response to high K solution were due to the release of Ca from a more labile store (a "loosely bound" membrane fraction). I t is interesting that many of the characteristics of HINKE'S "tightly bound" frac- tion of membrane Ca might well apply to the membrane bound Ca which is probably involved in the generation of action potentials. Thus both hypo- thetical Ca stores are stable in Ca-free media bu t can be depleted by EDTA and the "binding sites" of both stores are probably saturated at low external

Ca concentrations. It is likely that the action of noradrenaline on muscles with normal resting

membrane potentials is associated with a fall in membrane potential due to an increase in cation permeability. If this depolarization is large enough, action potentials and contractions are initiated (arterial smooth muscle may be an

exception). The initial step in this sequence of events may well be the release of Ca ions

from the " t ight ly bound" membrane store. If the membrane is incapable of producing an action potential, the release of Ca ions from the membrane may still be of sufficient magnitude to trigger contraction (e.g. arterial smooth

muscle). According to HINKE (t964), the tonic response to depolarization by K-rich

solutions involves the release of Ca ions other than those of t h e " tightly bound" fraction. Useful clues as to the nature of this source of Ca may come from comparative studies on the time course of the relaxation which eventually

occurs in high K solutions. The " t igh t ly" bound fraction of membrane Ca may be associated with

negative charges on the outer layer of the unit cell membrane (see GOODFORD, t967, for further references). The "loosely" bound fraction may be related to the nature of the basement membrane material, probably consisting of muco- polysaccharides, which surrounds most smooth muscle cells. (It has already

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References t 73

been suggested that the activity and exchangeability of the Ca ions in the immediate vicinity of the cell membrane may differ from one smooth muscle to another, perhaps due to specific differences ill the physical chemistry of this layer.) It must be emphasized that many authors would not agree with such a simplified scheme, but have found it necessary to postulate a more complicated partititioning of membrane Ca (DANIEL, 1965). However, this working hypo- thesis suggests a number of experiments which may eventually help to resolve

this question.

Conclusion Evidence has been presented which suggests that a number of different

vascular smooth muscles may have many characteristics in common with visceral smooth muscle (e.g. some elastic arteries, small arteries and arterioles, the longitudinal smooth muscle of mesenteric and portal veins). It would seem that the longitudinal muscle of veins might turn out to be a useful model for other spontaneously active vascular smooth muscles which are less accessible to experimental work in vitro. However, it remains to be shown, whether or not the physiology and pharmacology of these preparations is similar to that of smaller resistance vessels.

Little is known of the cellular physiology of the smooth muscle of muscular arteries, in spite of the considerable amount of work that has been carried out on their innervation. It is to be hoped that this obvious gap in our present understanding of the physiology of smooth muscle will be filled by the time this review is published.

At this t ime it would seem that Ca plays a key role in the physiology of both vascular and visceral smooth muscle. It is tempting to speculate that the " individual i ty" of vascular smooth muscles (BOHR, 1965) may be related to:

1. the Ca binding properties of the cell membrane, 2. the ionic envirement of the cell membrane. The latter might depend on the

physico-chemical properties of vascular extra-cellular space - - in particular, the nature of the mucopolysaccharides which constitute the so-called basement membrane material.

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