Storage of Serotonin and Serotonin Binding Protein in Synaptic Vesicles

Journal o/ Newor hemi<rr j Vol. 33. pp 35 to 44 Pergamon Preys Ltd 197') Prmtcd in Great Britain 0 lniernational Society fur Neurochemistry Ltd

0022-3042/79/070 I-0035502 W/O

STORAGE OF SEROTONIN AND SEROTONIN BINDING PROTEIN IN SYNAPTIC VESICLES

HADASSAH TAMIR' and MICHAEL D. GERSHON Departments of Psychiatry and Anatomy, Columbia University College of Physicians &

Surgeons and Division of Neuroscience, New York State Psychiatric Institute, New York, NY 10032, U.S.A.

(Received 29 Deceniher 1978. Accepted 29 January 1979)

Abstract-We have used the newly introduced method of DELORENZO & FREEDMAN (1978) for isolating synaptic vesicles to determine if such vesicles contain both serotonin (5-HT) and serotonin binding protein (SBP). Two fractions were obtained. A 55,000 g fraction was morphologically heterogeneous and contained coated vesicles. A 135,000g vesicle (dia. 51.3 nm) fraction was homogeneous in ultrastructure and contained no coated vesicles. The specific activity of SBP in this fraction was much higher than that in the supernatant. Unlike SBP, very little lactic dehydrogenase activity appeared in the 135,OOOg fraction. Qualitative and quantitative differences were observed between the polypeptide profiles of soluble proteins extracted from the vesicles and supernatant proteins on SDS gels. Therefore, entrapment of cytosol in the vesicles of the 135,OOOg fraction was minimal. The 5-HT concentration of the 135,000g vesicles was 5.5 ng/mg protein and in the supernatant, 11.3 ng/mg protein. The ATP concentration in the 135,000 g vesicle fraction was only 0.8 ng/mg Pr. Rabbit spinal cords were transected in order to determine if SBP is moved proximo-distally in axons by rapid axonal transport as would be predicted for a constituent of synaptic vesicles. SBP accumulated above the cut at a rate consistent with fast transport (78 mm/day). SBP activity fell caudal to the point of transection and there was no evidence, such as an accumulation below the lesion, that might indicate retrograde transport of SBP. These experiments indicate that SBP is probably synthesized in the cell bodies of serotonergic neurons and some is rapidly transported down axons to be stored in terminals in vesicles.

A SPECIFIC serotonin binding protein (SBP) has been found in serotonergic neurons in brain (TAMIR & HUANG, 1974; TAMIR & KUHAR, 1975; TAMIR et a/., 1977) and gut (JONAKAIT et al., 1977) as well as in non-neuronal cells that store serotonin (5-HT). These non-neuronal cells include parafollicular cells of the thyroid gland (JONAKAIT et ul., 1977; BERND et a/., 1979) and blood platelets (PIGNATTI & CAVALLI- SFORZA, 1975). Since reserpine inhibits the binding of 5-HT to this protein and newly synthesized 5-HT is bound to it (TAMIR & RAPPORT, 1978), a role of SBP in intracellular storage of 5-HT has been suggested.

The subcellular localization of SBP, and indeed the subcellular site or sites of 5-HT storage in neurons have been difficult to ascertain. Both SBP and 5-HT have been associated with synaptosomes. After disruption of synaptosomes both substances are largely recovered in the 100,OOOg supernatant and very little

' To whom correspondence should be addressed, at the Division of Neuroscience, New York State Psychiatric In- stitute, 722 West 168th Street, New York, NY 10032, U.S.A.

Abbreuiations used: 5-HT, 5-hydroxytryptamine; LDH, lactic dehydrogenase; MAO, monoamine oxidase; MOPS, morpholino-propane sulfonic acid buffer; Pr, protein; SBP, serotonin binding protein.

of either appears in synaptic vesicles conventionally prepared from the disrupted synaptosomes (TAMIR & HUANG, 1974). These observations might be taken to suggest that both SBP and 5-HT are stored in the cytosol of synaptosomes. If so, binding of 5-HT to SBP might serve to protect the amine from degrada- tion by the monoamine oxidase (MAO) of synaptic mitochondria. However, these experiments could be interpreted differently. SBP and 5-HT might be stored within vesicles in oiuo but be released to the supernatant fraction during the processes of synaptosomal disruption and vesicle isolation. The possibility that vesicles which store 5-HT are more fragile than other vesicles must be considered.

Indirect evidence has recently been obtained indicating that at least some SBP and 5-HT are stored in vesicles in enteric serotonergic neurons (JONAKAIT et al., in press). Both SBP and 5-HT are released from these neurons by a Caz+-dependent mechanism upon electrical stimulation. The cytosol marker protein, lactic dehydrogenase (LDH; EC 1.1.127) is not similarly released. While one could conceive of a C a 2 + - dependent transmembrane transport mechanism for the release of 5-HT, it is difficult to envision such a mechanism operating in the release of SBP. There- fore, exocytosis is probably the mechanism of transmitter and protein release, and this implies a vesicular mode of storage.

35 N.C. 3311- c

36 HADASSAH TAMIR and MICHAEL D. GERSHON

The present experiments were designed t o re- evaluate the subcellular s torage of SBP a n d 5-HT. In particular, we sought t o determine if 5-HT and SBP could be recovered in vesicles if synaptosomes were rapidly disrupted and vesicles were prepared by the newly introduced technique of DELORENZO & FREEDMAN (1978). In addition, we studied the axonal t ransport of SBP t o determine if tha t protein is rapidly transported, as a r e other particulate components of neurons destined for the synaptic appar- atus.

MATERIALS AND METHODS

Preparation of wsicles. Highly purified synaptic vesicles were prepared from brains of male Sprague-Dawley rats according to DELORENZO & FREEDMAN (1978). This method has two principal features; it shortens the time of osmotic shock of synaptosomes, and the medium to which the released vesicles are exposed is kept similar to intracellular conditions with respect to K’, and is free of Ca2 + (1 60 mM-KCI, 5 mM-NaCI, 10 mM-Tris-maleate, pH 6.5, 5 mM-MgCl,).

The purification procedure used to isolate vesicles utilizes differential centrifugation. Briefly, a crude inter- mediate fraction was first obtained by centrifuging the shocked synaptosomal preparation at 55,OOOg for 1 h. A second step, involving centrifugation of the 55,000 g supernatant at 135,OOOg for 45 min was then used to sediment the purified vesicles. The final purified vesicle fraction was then resuspended in 1 ml of 0.02 M-potassium phosphate buffer at pH 7.5 unless otherwise stated. A O.2ml aliquot was set aside for determination of protein, 5-HT and ATP. The remainder was then frozen and thawed to release the soluble contents of the vesicles. The vesicle mentbranes were obtained by centrifuging the disrupted vesicle suspension at 100,000 g for 60 min. For comparison, vesicles were also prepared by the method of WHITTAKER et al. (1964).

5-HT determination. 5-HT concentrations were measured according to the method of SAAVEDRA et a/. (1973). Extracts were incubated with serotonin-N-acetyl transferase (EC 2.3.1.5). and then hydroxyindole-0-methyl transferase (S-adenosyl-L-methionine: N-aceytl serotonin-0-methyl transferase, EC 2.1. I .4), in the presence of [ ’Hlmethyl- S-adenosyl-methionine (Amersham/Searle, Arlington Heights, IL). The tritiated melatonin produced from 5-HT by this treatment was isolated and counted. Standards containing known amounts of 5-HT were simultaneously analyzed.

Assay of serotonin binding protein. The assay for SBP has been described previously (TAMIR et al., 1976). The binding assay involves incubation of partially purified protein for 15 min with [G-3H]-5-HT (0.2 jim) in the presence of Fez+ (0.1 mM). The resulting protein-iron-[G-’H]-5-HT complex is then separated from the free 5-HT on Sephadex G-50. [G-’H]-5-HT was obtained from Amersham/Searle. Non-specific binding, measured in the presence of a 1000-fold excess of nonradioactive 5-HT, was subtracted from the total binding to provide an estimate of the specific binding of [G3H]-5-HT to SBP.

Assay of A T P in vesicles. The purified vesicle preparation was suspended in morpholino-propane sulfonic acid (MOPS) buffer at pH 7.4 and kept a t -60°C overnight. The ATP content was then determined using the

firefly luciferin-luciferase assay (STREHLER, 1965), run with a DuPont Biometer. The incubation mixture (purchased from E.I. DuPont & Co.; 0.5 ml) contained: MOPS buffer (0.01 M, pH 7.4) MgSO, (0.01 M), crystalline luciferin (0.71 mM), and luciferase (100units). The reaction was started by adding 0.01 ml of the vesicle suspension containing 60pg protein. This method is sensitive to 1 pg/ml of ATP.

Other assays. LDH activity was measured as described by KORNBERG (1955). Protein was determined by the method of LOWRY et a/. (1951) after alkaline digestion with 1.0 N-NaOH for 30min at room temperature. Electro- phoresis on 10% polyacrylamide tube gels was carried out using the SDS buffer system of LAEMMLI (1970) as modified by MAHADIK et al. (1976). For electrophoretic analysis, sedimented membrane proteins and the 100.000 g supernatant protein were directly dissolved in a buffer containing 0.125 M-Tris-HCI (pH 6 4 , 1.2Sx SDS, I:<, mercaptoethanol, 12.551 glycerol and 2 mM-EDTA, at a concentration of 1-2 mg/ml of protein (Pr). The SDS concentration was adjusted to give a weight ratio of SDS to protein of 5. After heating at 80°C for 10min and cooling, additional 1% mercaptoethanol was added to replace that which was lost. Gels were run at a constant current of 1 mA/gel and were cooled with circulating tap water. Gels were stained with Coomassie Blue, destained as described by FAIRBANKS et a/. (1971) and scanned at 550nm in a Gilford scanner.

Electron microscopy. Sedimented vesicles were fixed overnight in 4% glutaraldehyde in 0.2 M-pOtaSSiUm phosphate buffer at pH 7.5. Following fixation tissues were rinsed in 0.2 M-potassium phosphate buffer and post fixed with ly< OsO, in the same buffer. Tissue was then dehy- drated with ethanol, cleared in propylene oxide and embedded in Epon 812. Silver sections were cut with a diamond knife, stained en bloc with uranyl acetate and lead citrate and examined in a JEM 100 C electron micro- scope. Electron micrographs were analyzed quantitatively to provide an estimate of the relative volumes occupied by the various subcellular constituents found in the fractions. Point count planimetry was used as described by ELIAS et ul. (1971).

Determination ofrate of protein trunsport. Rabbits (aver- aging 3 kg) were anesthetized intravenously with a mixture of pentobarbital and chloral hydrate (Chloropent, Fort Dodge Laboratories, Fort Dodge, IA). A laminectomy was performed and the spinal cord was exposed at the first lumbar vertebra. The spinal cord was cut and the bleeding was stopped by inserting gelfoam into the space between the rostra1 and caudal ends of the severed cord. Control animals were operated upon but their spinal cords were not severed. Following spinal cord section, the lesion was closed and the animals were allowed to survive for additional periods ranging from 1 to 48 h. After the recovery period, animals were stunned by a blow on the head and rapidly exsanguinated. The bodies of the animals were cooled and brain and spinal cords were then transferred to a glass plate on ice. Meningi were removed and the cord was divided into consecutive 2cm segments which were kept in order. The segments were cut into small pieces with a razor blade and homogenized in tight fitting glass homogenizers in a hypotonic potassium phosphate buffer (0.02 M; pH 7.5; 3 x 0.5 ml). The resulting suspension was centrifuged (lO0,OOO g. 60 min) to remove al l particulate material. The supernatant was dialyzed for 3 h to remove endogenous 5-HT and the protein and binding capacity of SBP were assayed as described above.

FIG. 1. An electron micrograph of the 55.000y vesicular fraction. dcrived from disrupted rat brain synaptosornes. Note the abundance of irregularly shaped pleornorphic membranous profiles. This fraction also contains coated vcsicles (arrows) and smcoth round to elliptical vesicles. The inset shows

a coated vesicle at higher magnification. The markers indicate 100 nm.

37

Fici. 2. An electron micrograph of the 135,OOOg vesicular fraction. Note the relative homogeneity of vesicular contours and diameters. Although small agranular vesicles are most abundant, occasional

larger vesicles may also be seen. The marker indicates 100 nm.

38

Serotonin binding protein in synaptic vesicles 39

RESULTS

Electron microscopic studies

The nature of the two vesicle fractions, obtained by centrifuging at 55,000 g and 135,000 g respectively, was assessed by electron microscopy. The 55,000 g fraction (Fig. 1) was relatively crude. It contained a wide variety of irregularly shaped membrane enclosed structures which were much larger than synaptic vesicles and appeared empty. In addition, the fraction also contained smaller membrane enclosed vesicles whose size (4g100 nm) and regular round or elliptical profiles were consistent with their identification as small or large synaptic vesicles. Granular debris and coated vesicles could also be seen. Electron micrographs of the fraction were analyzed stereologically to estimate the relative volumes of the fractions occupied by the various organelles. Random micrographs, cut at different levels in the tissue blocs, were overlain with a regular grid of dots and the relative volumes were determined by point count planimetry. The relative volumes are given in Table 1.

In contrast to the crude 55,000g fraction, the 135,000 g fraction appeared quite homogeneous (Fig. 2). Most of the fraction was made up of small vesicles which were slightly elliptical in profile. The smooth regular contour of the trilaminar limiting membrane of these vesicles was consistent with their being synaptic vesicles. The vesicles were remarkably uniform in size. The mean vesicular diameter was 5 1.3 nm and the standard error was only 1.9 nm. Most of the vesicles had relatively electron-lucent contents which appeared somewhat more dense than the extravesicular medium and were sometimes faintly granular. Rarely, larger vesicles (approx 94 nm in diameter) with electron dense cores were observed. Coated vesicles which were present in the 55,000g fraction were never encountered in this 135,000 g preparation. There was some contamination of the purified vesicle fraction with irregularly shaped membrane enclosed structures. Granular extravesicular debris was also seen. This granular material, present in both subfractions, probably represented, at least in part, mem-

branes cut tangentially. It is sometimes difficult to distinguish these in electron micrographs. Therefore, the proportion of the fractional volume occupied by granular debris shown in Table 1 is likely to be an overestimate.

5-H T in synaptosomal suhjiructions

The total amount of 5-HT found in the final 135,000 g supernatant, obtained after differential centrifugation of the disrupted synaptosomal suspension, was far greater than the total amount of 5-HT found in the combined 55,000 g and 135,000 g vesicular subfractions (Table 2). Only 12% of the 5-HT, originally present in the synaptosomal fraction prior to its disruption, was recovered in the vesicular subfractions. Therefore, after synaptosomal disruption, the bulk of 5-HT continues to be associated with the high speed supernatant even when the milder conditions of DELORENZO & FREEDMAN (1978) are used to try to avoid leakage of vesicular contents. Moreover, the 5-HT concentration in the purified 135,000 g vesicular fraction was only approximately half that of the 135,000 g supernatant. Nevertheless, 5-HT was found in the 135,000 g vesicular fraction. This indicates that if the vesicular fraction is not contaminated by supernatant material then some 5-HT is stored in vesicles. In contrast, essentially no 5-HT (less than 1.0 ng/mg Pr) was found in vesicles prepared by the method of WHITTAKER et al. (1964).

Serotonin binding protein activity

The presence of 5-HT in the 135,000g vesicular subfraction raised the possibility that SBP might be associated with this fraction as well. We therefore further disrupted the 135,000 g vesicular fraction by freezing and thawing in order to assay the SBP activity of vesicular contents and vesicular membranes separately. The disrupted vesicular suspension was centrifuged at 100,000g for 60 min. The resulting supernatant was then dialyzed for 3 h to remove endogenous 5-HT and assayed for SBP activity (Table 2). The pellet was similarly analyzed. Note that

TABLE 1. RELATIVE VOLUMES OF SUBCELLULAR CONSTITUENTS IN SUBFRACTIONS OF DISRUPTED SYNAPTOSOMES

55,OOOg pellet 135,000 g pellet Per cent Per cent Per cent Per cent

of fraction* particulate? of fraction particulate

31.8 Extraparticulate space 26.1 ~

40-60 nm Vesicles 5.1 6.9 41.3 69.4 80-1 10 nm Vesicles 3.1 4.1 3.3 4.9 Coated vesicles 4.1 6.4 0 0 Irregular membranous profiles 46.1 62.4 6.3 9.2 Granular material 11.9 16.1 10.3 15.1 Large round bodies 3.0 4.1 1 .o I .4

-

* The per cent of fraction gives the number of dots striking the constituent divided by

?The dots falling over space have been subtracted from the total to give the per cent the total number of dots analyzed (3000) x 100 for the entire fraction.

particulate volume.


TABLE 2. SUBSYNAPTOSOMAL DISTRIBUTION OF 5-HT, SEROTONIN BINDING PROTEIN AND LACTATE DEHYDROGENASE

Specific binding Total Pr 5-HT Total* 5-HT capacity (SBP) LDH

Fraction (mg) (ngimg Pr) (ng) (c.p.m./mg Pr) (m/min/mg Pr)

Supernatant 74.5 f 3.5 11.3 f 0.02 842 15,150 & 40861- 0. I2 f 0.021:

Vesicles (55,000 y) 8.95 rf: 1.20 7.40 f 1.35 66 - 0.02 & 0.01 Vesicles (1 35,000 y) 3.44 f 1.99 5.52 f 1.08 19 22,500 k 750 - Membrane derived from 2.67 f 0.30 -

Soluble fraction derived 1.09 f 0.07 - - 61,160 f 11,540t 0.04 f O.Ol$,b

(135,000~)

- - -~

vesicles ( I 35,000 y) + from vesicles (135,000 y)

to Whittaker’s technique - 4285 k 15 - Vesicles prepared according 12.7 & 2.3 < 1

* ng/mg Pr of 5-HT x mg protein. t P < 0.01. $ P < 0.05. $ In some experiments no LDH activity is detectable

considerable SBP activity appeared in the purified 135,000 g vesicular fraction (Table 2). The specific ac- tivities of both vesicular membrane and especially vesicular contents were higher than that of the 135,000 g vesicular supernatant. Therefore, there is an enrichment of SBP in the purified 135,OOOg vesicular fraction. This enrichment of SBP activity suggests that most of the contents of the vesicles were not derived from synaptosomal cytosol entrapped within membranes that re-sealed when the synaptosomes were disrupted to release the vesicles. This conclusion is further substantiated by the distribution of the cytosol marker protein LDH (Table 2). Unlike SBP, LDH is concentrated in the 135,000 g vesicular supernatant and very little or none appears in the purified 135,000 g vesicular fraction or its subfractions. There- fore, the contamination of the 135,000 g purified vesicular fraction with cytosol must be minimal and the SBP activity (as well as the 5-HT) of this fraction

probably represents material contained within actual vesicles.

We have demonstrated recently that detergent (lo/(, Triton) will enhance several-fold the binding capacity of SBP present in the 100,OOOg supernatant of brain homogenates (DEN & TAMIR, 1978). The detergent enhanced the binding capacity of the membranous material derived from vesicles, as well as from the 135,000 g supernatant, almost 20-fold.

Gel electrophoresis studies

Contamination of the 135,OOOg vesicles by supernatant proteins was further evaluated by electrophoresis on SDS gels. Soluble proteins derived from both vesicles and the supernatant fraction were sub- jected to SDS gel electrophoresis. The absorbance profiles (550nm) of the stained gels are shown in Fig. 3. The range of polypeptides with molecular

I 94.000

,..l,. 68,000 , 111 j j i j I

!__- I -

~ __-___-, I I I I I I 1 1 I I

loo 80 90 10 20 30 40 50 60 70 DISTANCE (mm)

25,000

1

A u

FIG. 3. A densitometric scan of electropherograms of soluble proteins extracted from vesicles (-) and supernatant (----). Electrophoresis was performed in SDS on 10% polyacrylamide gels. Gels were stained with Coomassie Blue. The migration of standard proteins is shown by arrows with numbcrs

indicating the molecular weight of the standards.


SEGMENT NUMBER FIG. 4. SBP activity along the length of the control rabbit spinal cord. SBP activity is shown on the ordinate and segment numbers are indicated in ascending order from foramen magnum to coccyx on the abscissa. Each segment was 2cm in length. The open circles indicate the SBP activity in the pons and medulla (stem). Brackets represent the S.E. The arrow indicates the position where the

spinal cord was transected in subsequent experiments.

weights between 25,000 and 94,000 is very extensive. However, both qualitative and quantitative differences can be observed between the electrophoreto- grams of the two fractions. The peptides derived from vesicles are enriched in the region of smaller molecular weight (below 25,000), around the 45,000 region and at the 50,000 region. On the other hand, peptides of the supernatant fraction are enriched in the region between 68,000 and 94,000 and are impoverished in the region of molecular weight below 40,000.

The distinctive pattern of the low molecular weight peptides derived from vesicles and the significant quantitative differences observed in the high molecular weight region strongly support the conclusion that the soluble material derived from the 135,000 g vesicles does not represent contamination by supernatant.

A T P content in synaptic vesicles

The ATP concentration of three vesicle preparations was found to be 0.83 f 0.05 ng/mg Pr or 1.42pmol/mg Pr. As shown in Table 2 the 5-HT level is 5.52 ng/mg Pr or 31.4 pmol/mg Pr. Thus the molar ratio of 5-HT to ATP in the vesicle preparation, 37.8, is very high, suggesting that the amine may not form a complex with the nucleotide.

Determination of rute of niiyrution of SBP in spinal cord

SBP binding activity and protein were measured in the pons and medulla in consecutive 2 cm segments of spinal cord from the foramen magnum to the coccyx (Fig. 4). The caudal segments included cauda equina and filum terminale. The SBP binding activity was much lower in the cervical spinal cord than in the brain stem but increased caudally as a function of distance from the foramen magnum. However, a plateau is reached in the thoracic and lumbar regions

of the cord and the approximate center of this plateau was selected for lesioning. The spinal cord was transected and the SBP activity and protein concentration were again measured in consecutive 2cm segments rostra1 and caudal to the lesion of the cord as a function of time after the transection. There was no significant change in concentration of total protein in the segments above or below the cut. However, there was a significant accumulation of SBP binding activity in the first segment proximal to the cut. SBP activity appeared to increase as a linear function of time up to 4 h (Fig. 5), after which the rate of increase was no longer linear. Forty-eight hours after severing the cord the binding capacity of SBP in the 2cm

2 4 20 48 T I M E (hrs)

FIG. 5. The increase in SBP activity in the spinal cord segment immediately proximal to a transection of the cord is plotted as a function of elapsed time after the lesion. The increase is linear for the first 4 h (continuous line; y = 2 . 0 9 ~ + 0.27; r = 0.96) and remains elevated for at

least 48 h.


segment above the lesion was 200% of control. SBP activity below the lesion was 50% of control

The rate of SBP transport in the cord was calcu- lated from the formula, transport (mm/day) = SBP units per day/SBP units per mm. The units per day represent the increase over control in binding capacity in the segment above the cut normalized to 24 h. The units per mm represent the binding capacity in the segment above the segment just proximal to the cut, divided by the length of that segment (20mm). The initial accumulation rate was determined to be 78mm/day. This rate is probably a gross underestimate of the actual rate because, for technical reasons, the accumulation had to be measured in 2cm segments. The increase in SBP activity probably occurred only in the first few mm of the segment above the cut. The segment distal to the cut showed no accumulation of SBP; in fact there was a decrease in the binding capacity of this segment as compared to control ( P < 0.01).

(P < 0.01).

DISCUSSlON

These experiments were designed to test the hy- pothesis that 5-HT and SBP are stored in the terminals of serotonergic neurons in synaptic vesicles.

We prepared synaptic vesicles by a method which was reported to decrease the leakage or release of vesicular contents. The method yielded a purified vesicular subfraction that was remarkably homogeneous when its ultrastructure was analyzed stereologically. The fraction was largely composed of small agranular vesicles which looked like synaptic vesicles. The relative uniformity of vesicular diameters (51.3 1.9 nm) indicates that the purified fraction contained only a subpopulation of synaptic vesicles. Large granular vesicles, for example, were not included in the purified fraction. Since no coated vesicles were seen in the purified vesicular fraction, it seems likely that the vesicles were primarily synaptic and not endocytotic (HEUSER & REESE, 1977). More- over, the low level of LDH activity in the vesicular fraction, and the difference in electrophoretic profile between the vesicular contents and the supernatant protein, indicates that the purified vesicular fraction was minimally contaminated with supernatant or trapped cytosol. It is thus likely that materials found in the fraction are actually components of synaptic vesicles. However, as the fraction does not include all of the vesicles originally present in the tissue, it cannot be said that material found in other fractions did not also arise from synaptic vesicles. DELORENZO & FREEDMAN (1978) reported that 30% of the vesicles in their purified vesicular fraction were coated. How- ever, in our study all of the coated vesicles appeared in the 55,000 g subfraction. The discrepancy in results probably indicates that coated vesicles are more dense than the synaptic vesicles of our purified fraction but that 55,000 g may be just barely adequate to sediment the coated vesicles.

The 5-HT concentration of the purified vesicles, 5.52ng/ml Pr, is very similar t o the concentration of NE found in similarly prepared vesicles by DELOR- ENZO & FREEDMAN (1978). Only a small fraction of the total 5-HT present in the crude synaptosomal starting material (about 2%) was recovered in the purified vesicular fraction. The crude vesicles contained 10% of the 5-HT and the rest was recovered in the high speed supernatant. Therefore, although vesicles d o appear to contain 5-HT, the possibility that there is also a 5-HT pool in the cytosol cannot be ruled out. However, a pool in the cytosol has not been established either. The procedure of osmotic shock used to liberate synaptic vesicles might have led to leakage from them. Loss of 5-HT from synaptic vesicles does appear to occur because conventionally prepared vesicles contained far less 5-HT than those prepared by the method of DELORENZO & FREEDMAN (1978; see also ZIEHER & DEROBERTIS, 1963). There- fore, this method decreases leakage of 5-HT from vesicles but we have no evidence that the method eliminates it. It is even possible that the vesicular storage mechanism may be linked to the structural integ- rity of the terminal serotonergic varicosity.

In contrast to 5-HT, SBP was enriched in the purified vesicular fraction and was more concentrated in the vesicles than in the high speed supernatant. There- fore, SBP is probably a constitutent of synaptic vesicles. However, since substantial amounts of SBP were still found in the supernatant it is possible that SBP exists in the cytosol as well as in vesicles. It is of interest that all of the varicosities identified by DES- CARRIES et a!. (1975) as serotonergic using electron microscopic radioautography contained agranular vesicles similar in size to those of our 135,ooOg fraction They postulated that these vesicles are 5-HT storage vesicles. Our data are consistent with this pos- tulate.

This conclusion with regard to the localization of SBP was supported by the experiments involving spinal cord section. The serotonergic cells bodies of the CNS are mainly situated in the nuclei of the median raphe (DAHLSTROM & FUXE, 1964). The caudal raphe nuclei project to the spinal cord and the lesions we made in the cord would have cut the axons of these neurons. There are no serotonergic perikarya in the rabbit spinal cord and thus all of the transected serotonergic axons would have been descending. SBP accumulated on the cephalic side of the lesion; there was no accumulation of SBP on the caudal side of the cut and, in fact, SBP activity eventually decreased below the level of the transection. Moreover, the accumulation of SBP above the lesion was rapid. These data support the view that SBP is an intracellular protein found in serotonergic neurons (TAMIR & KUHAR, 1975). The data also indicate that the protein is synthesized in the cell bodies and is transported proximo-distally down the axons of these neurons. No evidence, such as accumulation of SBP on the caudal side of the cut, was obtained in favor of the


counter process of retrograde transport. The observed rate of anterograde translocation of SBP, 78 mm/day, is compatible with movement by fast transport (GRAFSTEIN, 1977). Actually, this measured rate is probably an underestimate of the true rate of transport. The segments we had to use for assay were large, 2 cm, and our calculations are based on the probably unjustified assumption that all of the SBP in the axons above the lesion is free to move. If only a por- tion of the SBP in axons is available for transport then the true transport rate would be higher than our estimate. Nevertheless, the rate of transport of SBP that we did measure, 78mm/day, is not too much different from the rate of fast transport measured by AZMITIA & SECAL (1978), 108mm/day, for the ascending projections of raphe neurons following injection of [G-3H]proline into the perikarya. Clearly, the rate of transport, even at 78mm/day, is much faster than the 1-2 mm/day that characterizes the process of slow axonal transport (GRAFSTEIN, 1977). Fast transport has been found to be associated with movement of particulate material destined for the synaptic terminal (MCEWEN et a/., 1971). There- fore, the fast transport of SBP is consistent with its being a constituent of serotonergic synaptic vesicles.

We also found a rostro-caudal gradient in SBP activity along the spinal cord. This gradient probably reflects the density of the serotonergic innervation of the cord. A similar gradient has also been reported for the concentration of 5-HT in the spinal cord (ANDEN et al., 1969; ANDERSON, 1972; CARLSSON et a/ . , 1973). The large rise that we found in the SBP activity in the most caudal segments most probably reflects the intense serotonergic innervation of the filum terminale (OLSON & NYGREN, 1972).

If SBP is a soluble consituent of serotonergic synaptic vesicles, its release from stimulated axon terminals by exocytosis along with the transmitter, 5-HT, would be predicted. Such a release has, in fact, been demonstrated from enteric serotonergic neurons (JONAKAIT et a[., in press). There is a spontaneous release of 5-HT and SBP from these neurons which is greatly increased by nerve stimulation. The stimulated increased release, but not the resting release, is Ca2+-dependent. The present results also support the view that the SBP associated with synaptic vesicles is, at least in part, soluble. Maximum SBP activity could only be measured from the preparations after disruption by freeze-thawing. However, the membranes derived from the purified vesicles also showed some specific SBP activity (22,000 c.p.m./mg Pr). This activity could be endogenous to the membrane. It is interesting to note that detergent increased the binding capacity of both soluble and membranous protein considerably. Whereas one expects the detergent to solubilize the membranous fraction and enhance its binding, it is not clear by what mechanism the detergent enhances the SBP activity of the soluble fraction. Therefore, SBP may be found both in the contents and membranes of synaptic vesicles.

Some amine storage vesicles such as chromaffin granules (HILLARP, 1958), blood platelets (DA PRADA & PLETSCHER. 1968), dense cored vesicles from sym- pathetic neurons (SCHUMANN, 1958), and cholinergic vesicles (DOWDALL et d., 1979, have been shown to contain high concentrations of ATP in association with protein and biogenic amines. More recently, DELORENZO & FREEDMAN (1978) demonstrated a cor- relation between Ca’+-dependent neurotransmitter release and phosphorylation of vesicular proteins. We therefore assayed the ATP content in our 135,000 g vesicular fraction but found it very low. Less than 2 pmol ATP/mg protein were detected. The serotonin plus norepinephrine content of the fraction is over 60 pmol/mg Pr. Thus, if ATP is really present in these vesicles its molar ratio to the biogenic amines is 1/30 or less. Even this low value of ATP is questionable since the concentration of ATP in whole brain is 20 nmol/mg Pr and thus 0.01% contamination by cytosol could contribute the amount of ATP found in vesicles. It is interesting to note that ATP and to a lesser extent ADP and AMP inhibit the binding of 5-HT to SBP (TAMIR et al., 1976). Thus. it is con- cluded that unlike platelet granules, the storage of 5-HT in synaptic vesicles may not involve complexing with ATP.

The observations made in this study support the conclusion that SBP and 5-HT are stored in synaptic vesicles. However, these observations do not rule out the additional possibility that either or both of these substances may also be stored in the cytosol.

Acknowledgements-Excellent technical assistance was pro- vided by DIANE SHERMAN and FRANCES MULLER. The authors wish to thank Dr. M. BANAY-SCHWARTZ for help- ing with the ATP assay and Dr. MAURICE M. R ~ P P O R T for advice and encouragement. This work was supported by NIH grants NS 12506 and NS 12969.

REFERENCES

ANDEN N. E., FUXE H . & HENNIG M. (1969) Mechanisms of noradrenaline and 5-hydroxytryptamine disappear- ance induced by -methyl-metatyrosine. Eur. J , Pharmac.

ANDERSON E. G. (1972) Bulbospinal serotonin containing neurons and motor neurons. Fedn Proc. Fedn Am. Socs. exp. Bid 31, 107-1 12.

AZMITIA E. C . & SEGAL M. (1978) An autoradiographic analysis of the differential ascending projections of the dorsal and median raphe nuclei in the rat. J . comp. Neurol. 179, 64-659.

BERND P., GERSHON M. D., N~JNEZ E. A. & TAMIR H. (1979) Localization of highly specific neuronal protein, serotonin binding protein, in thyroid parafollicular cells. Anat. Rec. 193, 257--268.

CARLSSON A,. LINDQVIST M., MAGNUSSON T. & ATACK C. (1973) Effect of acute transection on the synthesis and turnover of 5-HT in rat spinal cord. Naunyn-Schmiede- berg’s Arch. exp. path. Pharmak. 217, 1-12.

DAHLSTROM A. & FUXE R. (1964) Evidence for the existence of monoarnine containing neurons in central nervous

8, 302-309.


system 1. Demonstration of monoamines in the cell bodies of brain stem neurons. Actu physiol. scand. 62, Suppl. 262, 2-55, 1965.

DA PRAVA M. & PLETSCHER A. (1968) Isolated 5-hydroxytryptamine organelles of rabbit blood platelets: physiological properties and drug induced. Br. J . Phurmuc. 34,

DELORENZO R. J. & FREEDMAN S. D. (1978) Calcium dependent neurotransmitter release and protein phosphorylation in synaptic vesicles. Biochem. biophys. Res. Commun. 80, 182-192.

DEN H. & TAMIR H. (1978) Serotonin binding protein: effects of Triton X-100. Trans. Am. Soc. Neurochem. 9, 196.

DESCARRIES L., BEA~JDET A. & WATKINS K. C. (1975) Sero- tonin nerve terminals in adult rat neocortex. Brain Res.

DOWDALL M. J., BOYNE A. & WATKINS K. C. (1975) Sero- tonin nerve terminals in adult rat neocortex. Brain Res.

ELIAS H., HENNING A. & SCHWARTZ D. E. (1971) Stere- ology application to biomedical research. Physiol. Reo.

EULER U. S. VON & HILLARP N. A. (1956) Evidence for the presence of noradrenaline in submicroscopic structures of adrenergic axons. Nurure, Lond. 177, 44-45.

FAIRBANKS G., STECK T. L. & WALLACH D. F. H. (1971) Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry 10, 2607 -261 7.

GRAFSTEIN B. (1977) Axonal transport: the intracellular traffic of the neuron, in Handbook of Physiology (BROOK- HART J. M., MOUNTCASTLE V. B., KANDEL E. R. & GEIGER S. R., eds.) pp. 691-717. American Physiological Society Press, Bethesda, MD.

HEUSER J . E. 6t REESE T . (1977) Structure of the synapse, in Handbooh of Physiology (BROOKHART J. M., MOUNT- CASTLE V. B., KANDEL E. R . & GEIGER S. R., eds.) pp. 261 -264. American Physiological Society Press, Beth- esda MD.

HILLARP N. A. (1958) Adenosine phosphate and inorganic phosphate in adrenaline and noradrenaline containing granules or the adrenal medulla. Acta physiol. scand. 42,

591-597.

100, 563-588.

100, 5 6 3 ~ 588.

51 ( I ) , 158-200.

321-332. JONAKAIT 1. M., TAMIR H., RAPPORT M. M. & GERSHON

M. D. (1977) Detection of a soluble serotonin-binding protein in the mammalian myenteric plexus and other peripheral sites of serotonin storage, J . Neurochem. 28,

JONAKAIT J . M., TAMIR H., GINTZLER A. R. & GERSHON M. D. Release of serotonin and its binding protein from

277-284.

KORNBERC A. (1955) Lactic dehydrogenase assay, in Methods in Enzymology (COLOWICK S. P. & KAPLAN N. O., eds.) Vol. 1, pp. 441, Academic Press, New York.

LAEMML~ U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T,. Nature, Lond. 227, 680-685.

LOWRY 0. H., ROSEBROUCH N. J., FARR A. L. & RANDALL R. J. (1951) Protein measurement with the Folin phenol reagent. J . biol. Cheni. 193, 265-275.

MAHADIK S. P., KORENOVSKY A. & RAPPORT M. M. (1976) Slab gel analysis of the polypeptide components of rat brain subcellular organelles. Anulyt. Biochem. 76,

MCEWEN B. S. D., FORMAN S. & GRAFSTEIN B. (1971) Components of fast and slow axonal transport in gold- fish optic nerve J . Neurobiol. 2, 361-377.

OLSON L. & NYGREN L. B. (1972) A new model for fluor- escent histochemical study for central 5-hydroxytryptamine and noradrenaline nerve fibers. The filum terminale spread preparation. Histochemie 20, 265-273.

PIGNATTI P. F. & CAVALLI-SFORZA L. L. (1975) Serotonin binding proteins from human blood platelets. Neuro-

SAAVEDRA J. M., BROWNSTEIN M. & AXELROD J. (1973) A specific and sensitive enzymatic-isotopic micro-assay for serotonin in tissues. J . Pharmuc. exp. Ther. 186,

SCHUMANN H. J. (1958) Uber der noradrenalin und ATP gehalt synaptisher nerven. Naanyn-Schniiedeberg's Arch. exp. Path. Pharmuk. 233, 296-300.

STREHLER B. L. (1965) Determination of adenosine-5-tri- phosphate, in Methods of Enzymutic Analysis (BERG- MEYER H. V., ed.) pp. 563-568. Academic Press, New York.

TAMIR H. & HUANG Y. L. (1974) Binding of serotonin to soluble binding protein from synaptosomes, Life Sci. 14,

TAMIR H. & KUHAR M. J. (1975) Association of serotonin binding protein with projections of the midbrain raphe nuclei. Bruin Res. 83, 169-172.

TAMIR H., KLEIN A. & RAPPORT M. M. (1976) Serotonin binding protein: enhancement of binding by F e + * and inhibition of binding by drugs. J . Neurorhem. 26,

TAMIR H. & RAPPORT M. M. (1978) Effects of neurotoxins in oitro on the binding of serotonin to serotonin binding protein. Ann. N.Y. Acad. Sci. 305, 85-95.

WHITTAKER V. P., MICHAELSON I. A. & KIRKLAND R. J . A. (1964) The separation of synaptic vesicles from nerve ending particles (synaptosomes). Biochem. J . 90,293-305.

ZIEHER L. M. & DE ROBERTIS E. (1963) Subcellular localization of 5-hydroxytryptamine in rat brain. Biochem.

615-633.

bioiogy 5, 65-74.

508-5 12.

83-93.

871-878.

enteric neurons. Brain Reg. In press. Phurmac. 12, 596-598.

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Storage of Serotonin and Serotonin Binding Protein in Synaptic Vesicles