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488 BIOCHIMICA ET BIOPHYSICA ACTA BBA 12216 THE INDUCTION OF A TRANSFER ADENOSINE TRIPHOSPHATE PHOSPHOHYDROLASE IN EMBRYONIC CHICK HEART RICHARD L. KLEIN Department of Pharmacology, University of Mississippi Medical Center, Jackson, Miss. (U.S.A.) (Received December 5th, 1962 ) SUMMARY The ATP phosphohydrolase of isolated mitochondria and microsomes from em- bryonic chick ventricles was studied at different stages of development. At 12 days of embryonic age the particulate fractions contribute at least 85 9 ° % of the total Mg-activated ATP phosphohydrolase activity of the whole ventricle homogenate, the myofibrils contribute the remainder. A typical myocardial cation transfer ATP phosphohydrolase is demonstrated in the microsomal fraction: a Mg-activated ATP phosphohydrolase stimulated by Na, K and Na + K and inhibited by ouabain at concentrations which also inhibit active transport of Na and K. The induction of this transfer ATP phosphohydrolase is believed to occur between 4 and 7 days of embryonic development. INTRODUCTION In an earlier study of the ontogenesis of ATP phosphohydrolase (ATPase) in whole homogenates of embryonic chick ventricle, a marked increase in Mg-activated ATPase was noted between 4 and 7 days of development 1. On the basis of a number of other findings 2-4, it was hypothesized that this increase in Mg-activated ATPase may denote the induction of an enzyme for the active extrusion of Na from the myo- cardium. However, the question was raised as to whether this increase in enzyme activity might not represent myofibrillar (actomyosin) Mg-activated ATPase rather than particulate (mitochondria and microsomes) ATPase, even though the particulates had been shown to possess a high Mg activity and myofibrils are known to be relatively sparse during early myocardial development. From studies on other systems one would expect to find the so-called "transfer" ATPase associated with the microsomal or membrane fraction 5-1~. The general features of the transfer ATPase are a stimulation by Na +, K + and/or Na + plus K +, the latter increment of which is inhibited by digitalis glycosides and similar acting compounds at concentrations which also inhibit active cation transport (see refs. 3, 8, I I, 12 and 16, and this paper). In this study we present data concerning the relative contribution of the myo- Biochim, Biophys. Acta, 73 (I963) 488 498

The induction of a transfer adenosine triphosphate phosphohydrolase in embryonic chick heart

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488 BIOCHIMICA ET BIOPHYSICA ACTA

BBA 12216

T H E I N D U C T I O N OF A T R A N S F E R A D E N O S I N E T R I P H O S P H A T E P H O S P H O H Y D R O L A S E I N E M B R Y O N I C C H I C K H E A R T

R I C H A R D L. K L E I N

Department of Pharmacology, University of Mississippi Medical Center, Jackson, Miss. (U.S.A.)

(Received December 5th, 1962 )

SUMMAR Y

The ATP phosphohydrolase of isolated mitochondria and microsomes from em- bryonic chick ventricles was studied at different stages of development. At 12 days of embryonic age the particulate fractions contribute at least 85 9 ° % of the total Mg-activated ATP phosphohydrolase activity of the whole ventricle homogenate, the myofibrils contribute the remainder. A typical myocardial cation transfer ATP phosphohydrolase is demonstrated in the microsomal fraction: a Mg-activated ATP phosphohydrolase stimulated by Na, K and Na + K and inhibited by ouabain at concentrations which also inhibit active transport of Na and K. The induction of this transfer ATP phosphohydrolase is believed to occur between 4 and 7 days of embryonic development.

INTRODUCTION

In an earlier s tudy of the ontogenesis of ATP phosphohydrolase (ATPase) in whole homogenates of embryonic chick ventricle, a marked increase in Mg-activated ATPase was noted between 4 and 7 days of development 1. On the basis of a number of other findings 2-4, it was hypothesized that this increase in Mg-activated ATPase may denote the induction of an enzyme for the active extrusion of Na from the myo- cardium.

However, the question was raised as to whether this increase in enzyme activity might not represent myofibrillar (actomyosin) Mg-activated ATPase rather than particulate (mitochondria and microsomes) ATPase, even though the particulates had been shown to possess a high Mg activity and myofibrils are known to be relatively sparse during early myocardial development.

From studies on other systems one would expect to find the so-called "transfer" ATPase associated with the microsomal or membrane fraction 5-1~. The general features of the transfer ATPase are a stimulation by Na +, K + and/or Na + plus K +, the latter increment of which is inhibited by digitalis glycosides and similar acting compounds at concentrations which also inhibit active cation transport (see refs. 3, 8, I I, 12 and 16, and this paper).

In this s tudy we present data concerning the relative contribution of the myo-

Biochim, Biophys. Acta, 73 (I963) 488 498

A T P A s E IN EMBRYONIC HEART 489

fibrils to the overall ventricular Mg-activated ATPase activity during development, and the effects of Mg ~+, Ca *+, Na + and K +, and ouabain on the ATPase of mito- chondria and microsomes isolated from embryonic-chick ventricles at various ages.

METHODS

Handling of embryos

Embryona ted chicken eggs were incubated and ventricles dissected as previously described2, 3.

Tissue fractionation

Isolated ventricles were homogenized in 0.308 M sucrose containing o.ooi M sodium EDTA. In an effort to prevent undue fragmentation of myofibrils, a teflon pestle and smooth glass tube were used with gentle homogenization. The younger embryonic tissues are very soft and easy to homogenize. The extent of cell breakage was confirmed microscopically. Three fractions were obtained: myofibril, mito- chondrial and microsomal. The fibril fraction was the fraction tha t sedimented at 800 × g for 5 min, which was resuspended and centrifuged 3 times to remove mito- chondria and fragmented sarcoplasmic reticulum. The mitochondrial and microsomal fractions were prepared as previously described 1. Several criteria were employed to enhance and check the relative puri ty of mitochondria and microsomes including gross and microscopic appearance and succinate dehydrogenase (EC 1-3.99. I) activity. In mitochondrial fractions the light-colored fluffy layer (presumably microsomes) was removed from the tan-colored mitochondrial sediment. In microsomal sediments any remaining tan mitochondrial pellet (at bottom) was left behind when washing off the upper layer of microsomes. Spot checks of the mitochondrial enzyme succinate- dehydrogenase on typical mitochondrial and microsomal suspensions from I2-day embryonic hearts revealed but 0-5 % of this enzyme associated with the microsomes at particulate concentrations used to measure ATPase.

A ctomyosin extraction

Actomyosin was extracted from embryonic ventricles according to the method described by MOMMAERTS 17 using Gubba-Straub solution. Dissolved actomyosin was cleared and reprecipitated one time before experimentation.

Enzyme studies

ATPase and Pi determinations were performed as previously described 1. One additional precaution was taken to minimize acid hydrolysis of ATP ; solutions were maintained at ice-bath temperature during the 6-8-min when trichloroacetic acid was added to stop the ATPase reaction and protein was precipitated, filtered out, and the filtrate neutralized to pH 5-7 with NaOH.

Succinate dehydrogenase was determined by Warburg manometry according to the method of SCHNEIDER AND POTTER 18. Enzyme activity was measured in

Biochim. Biophys. Acta, 73 (1963) 488-498

49 ° R.L. KLEIN

3 .oml of m e d i u m w i t h final concentrations of o .o33Mphospha te buffer at pH7.4 , o .ooooI3M cytochrome c, o.ooo4M CaC12 and A1Cla, and o .osM sodium succinate at 38 ° .

Protein determinations

Due to the small samples available in a number of these experiments, the method of LOWRY et al. ~9 for protein determination was employed which is about IOO times as sensitive as the previously used biuret test. Five times crystallized albumin (Nutr. Biochem. Corp.) was used as a standard.

Solutions

Because of the known effects of tonicity on mitochondrial and microsomal ATPase activity, all media regardless of salt content were made isotonic (0.308 M in non-electrolyte equivalents) by addition of sucrose. To avoid the introduction of Na or K into ATPase reaction media without intent, Tris-HCl buffer (pH 7.2) and the Tris salt of ATP was used throughout. The lat ter was made at ice-bath temperatures by passing sodium ATP through Dowex-5o WX8 (4 ml Dowex/g ATP) and subsequent neutralization with Tris. ATP hydrolysis was negligible by this method.

In ouabain experiments, the particulates were incubated with the drug 45 min - i h previous to the determination of ATPase activity; controls were treated in the same manner without the drug.

RESULTS

Contribution of myofibril and particulate fractions to Mg-activated A TPase of whole- ventricle homogenate

The possibility existed that actomyosin being Mg- as well as Ca-activated might be largely responsible for the increase in Mg-activated ATPase demonstrated in the whole-ventricle homogenate between 4 and 7 day sl. This raised a critical point con- cerning our hypothesis of an induced Mg-activated ATPase for active cation transport at this time, as the latter would most likely involve an enzyme associated with the particulate fraction of the cells.

To clarify this point, pooled embryonic ventricles at different ages were homo- genized and divided into myofibril, mitochondrial and microsomal fractions by fractional centrifugation. The production of relatively pure fractions was hampered by the necessity for complete recovery of ATPase in the whole homogenate from small amounts of tissue, especially at the early ages. Even more difficulty was ex- perienced due to the fact that it was virtually impossible to produce a fibril fraction not highly contaminated with mitochondria regardless of how many washings were performed. The lat ter has been experienced by others using skeletal muscle S° .

From phase-contrast examination we felt that the mitochondrial fractions were relatively pure and free of myofibrillar fragments. As succinate dehydrogenase is localized in the mitochondria ~1, it was assumed tha t from the ratio of Mg-activated ATPase: succinate dehydrogenase act ivi ty in the mitochondria, one could estimate

Biochim. Biophys. Acta, 73 (1963) 488-498

ATPAsE IN EMBRYONIC HEART 491

what portion of the Mg-activated ATPase of the fibril fraction was due to mitochon- drial contamination, if one also determined the succinate dehydrogenase associated with the myofibrils.

In Fig. I we have plotted the ratio of succinate dehydrogenase and Mg-activated ATPase associated with the myofibril fraction compared to that in the myofibril plus mitochondrial fractions. This ratio was plotted for two reasons. (a) An increase in the Mg-activated ATPase curve compared to the succinate dehydrogenase curve should be a measure of the Mg-activated ATPase contributed by the myofibrils. (b) An indication as to the amount of mitochondrial contamination of the fibril fraction could be shown at different ages.

O.B"

T I

s l

0.6 ~ II

o ' K 0 4 [ L . O "

~'~ , ~--g 0.2- 1 o r ~ a .

. ~ I ~ ~) ( 4 )

0 4

(9)

I "~' I i I 8 12 16 Embryonic age(doys)

O'~<3Succ -dehyd~+ F

~ - - - -~ Mg- ATPase

I T I I ! I

1"t (8) (18)

' ~o

Fig. I. S-D = succ ina te dehydrogenase , F = myofibr i l lar fract ion, M = mi tochondr i a l f ract ion. F igu res in p a r e n t h e s e s show n u m b e r o f e x p e r i m e n t s ave r aged a t each age. Vert ical l ines ind ica te va r i ab i l i ty in s e p a r a t i n g m i t o c h o n d r i a f rom myofibr i l s a t each age (see text ) . Pooled 4 dozen

hea r t s per e x p e r i m e n t a t 5 days .

The averaged values show that we were unable to demonstrate any significant contribution by the myofibrils to the overall Mg-activated ATPase activi ty by this method. The range of values (vertical lines in Fig. I) give an indication only of how consistent was our separation of enzymes into myofibril and mitochondrial fractions from experiment to experiment. The actual agreement between points on the two curves in any one experiment was relatively good, an average of + lO%. The lower ratio at the oldest age attr ibutes to the fact that the separation of mito- chondria from myofibrils was best accomplished in the older heart homogenates under the conditions employed.

I f extracted actomyosin ATPase can be considered representative of myo- fibrillar ATPase, one can get an estimate of how much Mg-activated ATPase is contributed by the contractile protein from a different approach. Actomyosin was extracted from I2-day embryonic ventricles and its Mg- and Ca-activated ATPase activi ty determined under the same conditions as above. Mg activity varied between 20 and 40% of the Ca activity in isotonic sucrose media with 0.0025 M ATP and 0.005 M Ca or Mg. Assuming tha t IOO% of the Ca activity of the whole homogenate z

Biochim. Biophys. Acta, 73 (1963) 488-498

492 R.L. KLEIN

represents myofibri l lar ATPase, one can calculate from the ratio of Mg- to Ca-act ivated ATPase of actomyosin tha t the myofibrils m a y contr ibute at most lO-15 % of the tota l Mg-act ivated ATPase in the whole homogenate at 12 days. This est imate is most likely high, as both mi tochondr ia and microsomes also cont r ibute some to the Ca ac t iv i ty of the whole homogenate (Fig. 2).

Effects of Mg and Ca concentrations

The effects of [Mg] and ECa] at 0.0025 M ATP on isolated mitochondria l and microsomal ATPase are shown in Fig. 2. Wi th either par t icula te Mg act ivat ion is greatest when the Mg : ATP ratio is u n i t y or a little less. Ca ac t iva t ion is greatest when the Ca : A T P is at least 4 : I. The la t ter ac t iv i ty is much more sensitive to [Ca], t han is Mg ac t iv i ty to [Mg].

In earlier ATPase studies on whole-ventricle homogenates the overall enzyme ac t iv i ty over the first IO or 3o rain was not great ly affected by using either o.oo5 or

;y 8o -

"~ 60-

g " o 4 0 "

~- 20" o

n

0 , , , J , i i , i f i J , , , ,

0 2.5 5.0 7.5 10 0 2.5 5.0 7.5 10 mM Mg** or Ca"' mM Mg** op Ca**

Fig. 2. A, mitochondrial fraction and B, microsomal fraction. O - - O , Mg; A - - ~ , Ca. Particulates suspended in 0.02 M Tris-HC1 buffer (pH 7.2), 0.0025 M Tris-ATP, plus MgC12 or CaC] 2 and made isotonic with sucrose. Maximal ATPase activity with Mg or Ca is set equal to ioo %. Absolute ATPase activity: mitochondria at 2.5 mM Mg = 2.98 and Ca = 0.57 #moles Pi/mg protein N/min, microsomes at 2. 5 mM Mg = 3.33 and Ca z o.98ktmoles Pl/mg protein N/min. An

average of 6 experiments using each particulate from i2-day embryonic ventricles.

0.0025 M A T P at 0.005 M MgCI~ or CaC12 (see ref. I). Therefore, the lower [ATP] was used after the pre l iminary experiments. Ex t rac ted actomyosin from I2-day embryonic ventricles is also best ac t ivated at this Mg : A T P ratio of 2 : I (to be reported elsewhere). For compara t ive purposes the la t ter rat io was employed in the present s tudy.

Effects of KCl and NaCl concentrations

The effects of [KC1] and/or [NaC1] on the Mg-act ivated ATPase of isolated mi tochondr ia and microsomes are shown in Fig. 3. The Mg-act ivated ATPase of mi tochondr ia is max imal at about 0.03 M salt or a l i t t le less, and for microsomes at 0.05 M salt or a l i t t le higher. In media where no sucrose was added to ma in t a in isotonicity, microsomal ac t iv i ty was no t great ly changed, however, mi tochondr ia l ac t iv i ty was considerably increased at the lower salt concentra t ions a t tes t ing to a tonic i ty effect here.

Biochim. Biophys. Acta, 73 (1963) 488-498

ATPAsE IN EMBRYONIC HEART 493

100-

.~ 90- >

'~ 80-

iJ 7 0 -

60 I

?_ 5 0 -

40 6 2;~ 4'0 6'o ~;o 1~o ' ' ' 2~o

mM KCIorNflCI

Fig. 3. O - - Q , mic rosomes ; & - - A , mi tochondr ia . Pa r t i cu l a t e s s u s p e n d e d in 0.02 M Tr i s -HCI buffer (pH 7.2), o.oo 5 M MgC] v 0.0025 M T t i s - A T P p lus KC1 or NaC1 and m a d e isotonic wi th sucrose. M a x i m a l Mg-ac t iva t ed A T P a s e ac t iv i ty set equa l to lOO%. Media wi th 200 m M sa l t added is correc ted for t he h y p e r t o n i c i t y effect ( inhibit ion) by c o m p a r i n g wi th ac t iv i ty in IOO m M sa l t + sucrose, wh ich was equa l ly hype r ton ic . A t i o o % ac t iv i ty , mic rosomes = 3.56 and mi to- chondr i a = 3 .55/*moles P i / m g p ro te in N /min . An average of 19 KC1 and 24 NaC1 e x p e r i m e n t s u s ing microsomes , a n d 9 KC1 and 9 NaC1 e x p e r i m e n t s us ing m i t o c h o n d r i a f rom i 2 - d a y embryon ic

ventr ic les .

Effects of ouabain

The bar graph in Fig. 4 shows the additional stimulation of Mg-activated ATPase by adding KCI to I2-day embryonic heart microsomes suspended in a me- dium already containing o.I M NaC1 (open bars). The baseline activity (zero) is that

,~+25" I

£ +151

oi

' ~o ' ,;o 6'o' 8 ~ ' ~)o mM KCI

((added to 0.1M NtmCI medium)

Fig. 4. Microsomes f rom i 2 - d a y e m b r y o n i c ventr ic les . Open bars, increased Mg-ac t iva t ed A T P a s e ac t i v i t y due to add i t ion of KC1 to m e d i a a l r eady con ta in ing o. i M NaC1. Shaded bars, degree o f inh ib i t ion w h e n lO -4 M o u a b a i n is added. T he per c en t changes in ac t iv i ty are expressed re la t ive to t h a t a c t i v i t y in t he presence of a n equal ionic s t r e n g t h of NaC1 only (see text) . Media wi th o . i M NaC1 + o. i M KC1 are correc ted for t he h y p e r t o n i c i t y effect (inhibition) b y compar ing w i th a c t i v i t y in o . i M NaC1 + sucrose wh ich was equa l ly hyper ton ic . An average of 12 ex-

pe r iments .

in a medium of equal ionic strength using NaC1 only (e.g.o.I M NaC1 + 0.03 M KC1 is compared with o.13 M NaC1). The latter is necessary to account for the effect due solely to changes in ionic strength. Under the present conditions the sum of the stimulations by NaC1 (Fig. 3) + KCI (Fig. 4) is up to 35 % above the ATPase activity with Mg alone.

The shaded bars show the degree of inhibition of Mg-activated ATPase activity

Biochim. Biophys. Acta, 73 (1963) 488-498

494 R.L. KLEIN

when lO -4 M ouabain is also present in these NaC1-KC1 mixtures. Whereas ouabain has little effect on microsomal Mg-activated ATPase in o.I M NaC1 alone (KC1 = o), the drug completely prevents the stimulatory effect of added KC1. In fact, at lower [KCll the Mg-activated ATPase activity is lower than at an equal ionic strength using NaC1 alone, therefore, ouabain can inhibit some of the NaC1 stimulation when KC1 is also present. The effects of ouabain at lO -3 M are quantitatively similar to those at lO -4 M; lower concentrations tend to stimulate the Mg-activated ATPase.

When NaC1 is added to o.i M KC1 media, microsomal Mg-activated ATPase is also stimulated, however, the stimulation is less than shown in Fig. 4, and the inhibition by ouabain is seen only at the higher concentrations of added NaC1.

Similar experiments were performed with isolated mitochondria (data not presented). One finds a questionable stimulation of Mg-activated ATPase upon the addition of KC1 to o.I M NaC1 media. A small but definite stimulation of Mg-activated ATPase is produced when ouabain at lO -4 or IO -~ M is added.

Statistical analyses of the effects of ouabain at 4, 7 and z2 days

The characterization of a heart microsomal Mg-activated ATPase at 12 days of embryonic development prepared us for a more direct approach to test the possibility that the marked increase in Mg-activated ATPase of whole-ventricle homogenates between 4 and 7 days of development may represent the induction of an enzyme for active cation transport.

Microsomes were isolated from pooled embryonic ventricles at 4 and 7 days. The difficulties in cell fractionation procedures using the small amounts of heart tissue are obvious. However, microsomal pellets although small could be obtained with easily measurable ATPase activity. The microsomal suspensions were diluted such that the levels of enzyme activity were reasonably equal at 4 and 7 days. The effects of ouabain on microsomes isolated from 4-, 7- and I2-day embryonic ventricles are

T A B L E I

STATISTICAL SIGNIFICANCE OF THE INHIBITION OF N a + K-DEPENDENT Mg-ACTIVATED A T P A s E BY OUABAIN

M g - a c t i v a t e d A T P a s e a c t i v i t y o f m i c r o s o m a l f r a c t i o n s u s p e n d e d i n o .o2 M T r i s - H C 1 b u f f e r ( p H 7.2) , o . i o M NaC1 + o .o 3 M KC1, 0 . 0 0 5 M MgC12, 0 . 0 0 2 5 M T r i s - A T P . O u a b a i n a t lO -4 M. d - - a r i t h m a t i c m e a n , S ,E . = s t a n d a r d e r r o r o f t h e d i f f e r e n c e , t = v a l u e f o r u n p a i r e d v a r i a t e s , -- t0. ,~ e t c . a r e v a l u e s t a k e n f r o m F i s h e r ' s t d i s t r i b u t i o n t a b l e s . T h e F t e s t s h o w e d e q u a l i t y o f v a r i a n c e s a t e a c h o f t h e a g e s . P o o l e d 4 d o z e n h e a r t s p e r e x p e r i m e n t a t 4 d a y s . R e s u l t s e x p r e s s e d

a s / , m o l e s P l / m g p r o t e i n N ] m i n .

4-days 7 -days i2-days

Control Ouabain Control Ouabain Control Ouabain

N u m b e r o f o b s e r - v a t i o n s 23 23 36 36 18 18

d 2 .o2 i .92 3 .78 3 .28 3.81 3.31 D i f f e r e n c e o. I o o. 5 o o. 5 ° S . E . o .14 o. i o 0 .02 t o .71 4 .77 29 .7 L e v e l o f s i g n i f i c a n c e --/0.25 - - 0 .68 --t0.00o5 = 3 .43 --t0.0001 = 3 .61

Biochim. Biophys. Acta, 73 (1963) 4 8 8 - 4 9 8

ATPASE IN EMBRYONIC HEART 495

shown in Table I. Statistical analyses were performed to determine the significance of the differences between means of control and ouabain experiments. At 4 days in 23 pairs of samples, 14 showed inhibition, 5 stimulation and 4 were unchanged by ouabain. Although the mean for ouabain experiments was 5 ~/o less than the controls, the t value permits one to accept the null hypothesis, indicating that the means are not significantly different at the usual critical level of --t0.05 to --t0.0v The in- hibition by ouabain of some 13% at 7 days is highly significant, as is that also of 13% at 12 days.

From the values of the differences due to ouabain inhibition, an estimate of the actual increase in the specific activity of the Na + K-dependent Mg-activated ATPase between 4 and 7 days is at least 500.%, i .e.o.5o/o.lo = 5 times.

DISCUSSION

The microsomal Mg-activated ATPase of ventricles from the embryonic chick is quali tatively similar to tha t from adult hearts of other species x~-14, and from other sourcesS-lX, xS. Characteristic of heart microsomes, it is st imulated by Na or K alone, and by Na + K, and is inhibited by ouabain. The latter stimulation and inhibition of Mg=activated ATPase is relatively small compared to some of the other systems, however, this may be simply a reflection of the puri ty of the enzyme, the method of preparat ion and ageingX~, 14.

Thus, we have shown that ouabain at IO -* M inhibits both the transfer ATPase and active Na extrusion and K entry 8 in embryonic ventricles. The degree of inhibi- tion of each remains the same at 7 and 12 days of embryonic age. At IO -e M, ouabain causes visible positive inotropic effects, but could not be shown to inhibit active cation transport, and if anything, this concentration tends to stimulate the microsomal Mg-activated ATPase.

I t was shown (Fig. 4) tha t the inhibition of microsomal Mg-activated ATPase by ouabain was dependent on the presence of added KC1 to o.I M NaC1 media. At lower concentrations of added KC1 ouabain also inhibited some of the NaC1 stimula- tion ; the inhibition of NaC1 stimulation did not occur in the absence of KC1. I f this is t ruly an ATPase involved in active transport of Na and K, the above evidence lends support to the contention tha t active Na extrusion is dependent on the presence of K, and tha t ouabain should reduce Na efflux to the lowest value when K is present in low concentrations. The latter interpretation is in keeping with the findings of GLYNN 22 on the ability of digoxin to inhibit Na efflux at different K concentrations using red blood cells.

In a previous s tudy of the unidirectional fluxes of Na and K in embryonic chick heart s, it was found that an increase of 8 times or a decrease to 1/s the normal con- centration of Ca in the suspending Ringer's medium had relatively little effect on the ability of ouabain to decrease the transfer coefficients for Na effiux and K influx, both of which involve active movements. I t did have rather pronounced effects on the passive Na influx and K efflux. Although the transfer Mg-activated ATPase can be markedly inhibited by Ca (see refs. 5, 6, I I , 13, 23), in the present and other studies the addition of Ca to the medium is not necessary to demonstrate the inhibi- tion of Na + K stimulation by ouabain 6-8,1z-~4,9..

Thus, the inhibition of both active transport and transfer ATPase by ouabain

Biochim. Biophys. Acta, 73 (1963) 488-498

496 R . L . K L E I N

appears to be relatively insensitive to or does not require Ca in the medium. In contrast, the therapeutic effect of the d r u g (positive inotropism) in cardiac muscle is sensitive to the Ca concentration and will not occur if Ca is omitted from the medium. This is additional evidence in favor of the dissociation of the therapeutic effects of ouabain on the contractile mechanism and the suggested toxic effects on active transport, which we made earlier s.

I t should be pointed out, however, that although the microsomes were prepared in sucrose medium containing EDTA, which would be expected to eliminate any traces of free or loosely bound Ca from the suspension, the possibility remains that ouabain may still mobilize firmly bound Ca from the microsomal membranes which is needed for the inhibition of the Na + K dependent Mg-activated ATPase. The presence of firmly bound Ca and the ability to concentrate Ca in the microsomal fraction of muscle is well established 24-~7.

In an earlier s tudy of the ontogenesis of ATPase in whole-ventricle homogenates of the embryonic chick, a marked increase in Mg-activated ATPase was noted between 4 and 7 days of developmenO. This Mg-activated ATPase is associated primarily with the particulate fraction of the embryonic myocardium. Present data from fractional centrifugation and actomyosin extraction studies suggest that the myofibrils contribute at most lO-15% of the Mg-activated ATPase of the whole ventricle homogenate. The nature of embryonic heart actomyosin ATPase will be reported in detail elsewhere.

I t was hypothesized that the increase in Mg-activated ATPase between 4 and 7 days of development may denote the induction of an enzyme for the active extru- sion of Na from the myocardial cells. A number of facts support this reasoning. (a) The initially high 600-700 mM Na associated with the 2-day tubular embryonic chick heart decreases rapidly to about 80 mM by 7 days; this is believed to represent a decrease in extracellular Na associated with the mucopolysaccharide cardiac jelly 1,4. The 4-7-day period marks the beginning of the phase of more gradual decrease of ventricular Na from 80 mM at 7 days to 30 mM shortly after hatching, and the inverse change in Na exchangeability from only 5-1o% at 7 days to over 70% at hatching 2. The presence of a transfer ATPase at this time could cause a gradual shift in the equilibrium between intracellular-bound and free Na toward the adult level with the consequent net loss of tissue Na from the bound state. (b) As far as one can tell, the electron micrographic studies of the embryonic myocardium of the chick 2s,29 and the mouse s° suggest no marked increase in myofibrillar, mitochondrial or microsomal components of this tissue between 4 and 7 days. We would like to investigate this further. (c) Analyses of ventricles during development indicate an actual decrease in protein nitrogen during early development up to 7 or 8 days, after which an increase ensues. A decrease in protein has been associated by some with enzyme induction. (d) The growth curve for the embryonic heart does not parallel the increase in Mg-activated ATPase between 4 and 7 day ssl. (e) The ouabain- inhibited Na + K-dependent Mg-activated ATPase activity of isolated microsomes increases at least 5-fold between 4 and 7 days (Table I).

I f the increase in Mg-activated ATPase of the whole-ventricle homogenate at least in part represents an induction of a transfer ATPase or of a much higher level of its activity, one might expect to find that at 4 days ouabain should have no or low inhibitory effects, whereas at 7 days characteristic inhibition by ouabain should

Biochim. Biophys. Acta, 73 (1963) 488-498

ATPASE IN EMBRYONIC HEART 497

be evident. A typical inhibition by ouabain was found at 7 days similar to tha t at 12 days. At 4 days there was an average inhibition of 5% by ouabain, however, statistical analyses indicated this was not a significant difference at the usually accepted t levels (Table I). Thus, one can not state with assurance whether an active cation transport system is entirely absent or is operable at a low level in hearts younger than 4 days.

I t is suggested tha t in the early embryonic myocardium, before the establish- ment of a fully operable cation transport system, free intracellular Na is maintained at a low level by some type of binding or occlusion mechanism at least in part de- pendent on the structural integrity of the myocardial cell 4.

Although it is felt there is good evidence for the induction of a myocardial transfer ATPase between 4 and 7 days of embryonic development in the chick, it is not claimed that this is the sole result or purpose of the increase in Mg-activated ATPase activity. On the contrary, the magnitude of the increase may very well signify greater myocardial metabolic activity and ATP production.

I t was also pointed out in an earlier communication 1 that HUGHES '32 estimate of the work of the embryonic heart between 4 and 7 days parallels the increase in Mg-activated ATPase activi ty very closely. That this coincidence is fortuitous is questionable. A first assumption might correlate the increased work with increased myofibrillar Mg-activated ATPase activity. However, the Mg-activated ATPase even at 12 days is estimated to be a max imum of only lO-15% myofibrillar in origin. The Ca activity of the whole-ventricle homogenate, which may be a bet ter measure of the relative change in actomyosin ATPase, shows a much smaller and more gradual increase between 4 and 7 days. Possibly the utilization of ATP by the contractile proteins becomes more efficient, as might occur if structural orientation of the myo- fibrils was greatly improved during this period. I t is at this t ime tha t the first signs of myofibrillar orientation are noted; fibrils lie parallel, striations line up transversely and Z bands fuse 28.

ACKNOWLEDGEMENTS

The author is indebted to Mrs. A. P. BRELAND, A . BEESON, M. PFAFFMAN and S . OUTLAW for their technical assistance.

This research was supported by Public Health Service Research Grants H-4289 and H-7o56, and Career Development Award HE-K3-5892.

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