Brain Research, 205 (1981) 299-311 © Elsevier/North-Holland Biomedical Press
299
TIME COURSE OF APPEARANCE AND RELEASE OF [asS]CYSTEINE LABELLED NEUROPHYSINS AND PEPTIDES IN THE NEUROHYPOPHYSIS
JAMES T. RUSSELL, M I C H A E L J. BROWNSTEIN and H A R O L D G A I N E R
Section on Functional Neurochemistry, Laboratory of Developmental Neurobiology, National Institutes of Child Health and Human Development and Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, Md. 20205 (U.S.A.) (Accepted July 10th, 1980)
Key words: axonal transport - - precursor processing - - neurohypophysial release
SUMMARY
[35S]cysteine injected adjacent to the supraoptic nuclei (SON) of rats is rapidly incorporated into two macromolecular (both about 20,000 Daltons) common precursors of arginine vasopressin (AVP) and its associated neurophysin, and oxytoxin (OT) and its neurophysin2, xl. Conversion of the larger precursor proteins to the smaller peptides appears to occur intragranularly during axonal transport to the neurohypophysis 4-6. The labelled products of this conversion (neurophysin, AVP, and OT) are released, in a Ca2+-dependent manner, from the posterior pituitary in response to depolarization by veratridine. Both the rates of biosynthesis and of processing of the precursors are greatly increased by increased functional activity (i.e. secretion) of the hypothalamo-neurohypophysial system.
INTRODUCTION
The neurohypophysial peptides vasopressin (VP) and oxytocin (OT) and their respective neurophysins (Np) are synthesized by neurons in the supraoptic (SON) and paraventricular nuclei (PVN) of the hypothalamus, and transported intra-axonally to the neurohypophysis where they are stored in nerve terminals for release12, lz. These peptidergic neurons of the hypothalamo-neurohypophysial system are ideal for studies of the biosynthesis, axonal transport, and release of neuronal peptides. The neurohypophysial peptides and proteins are synthesized as parts of macromolecular precursors by a ribosomal mechanism4-6,12,14. We have recently isolated two [zSS]cysteine labelled precursor proteins from the SON of rats and have
300 demonstrated, by limited proteolysis and immunological techniques, that one is the common precursor for vasopressin and its associated neurophysin (Np-Vp) and the other is the common precursor for oxytocin and its neurophysin (Np-Ot) 5,10-12. In this paper, the mechanisms of axonal transport and release are used to study the regulation of the rate of post-translational modifications by functional activity (e.g. dehydration), and to evaluate the nature of the secretory products derived from the precursors. MATERIALS AND METHODS
Labellingprocedure Female Osborn-Mendel rats weighing 225-250 g were used in these experiments. The animals were anaesthetized with ether and injected bilaterally adjacent to the SONs with 20 #Ci of[35S]cysteine (New England Nuclear, spec. act. diluted to give 100 Ci/mmol) in 0.9 ~ NaC1 and 10 mM dithiothreitol in a vol. of 1 /d/site as described earlier2, 4. At different times after injection, (see Results) the animals were killed, their neurohypophyses were removed and homogenized in 0.1 N HC1, and the acid insoluble material was removed by centrifugation in a microfuge (Beckman Instruments) for 1 min. The HC1 soluble fraction was analyzed for labelled peptides. Some of the animals (as noted below) were given 2 ~ NaC1 to drink for one week before injection of [35S]cysteine. Others were treated with colchicine (100/~g in 0.9 ~ NaCI) intraventricularly 24 h prior to injection of [35S]cysteine.
Analysis of labelledpolypeptides The 0.1 N HC1 soluble fraction was applied on a Sephadex G- 50 column (0.9 × 60 cm) equilibrated with 0.1 N formic acid. Forty-five 1.0 ml fractions were collected and aliquots were counted for [zSS]cysteine radioactivity.
Np-Sepharose affinity chromatography Neurophysin-Sepharose affinity chromatography was used to separate the labelled peptides with affinity for Np. Bovine neurophysin was coupled to cyanogen bromide activated Sepharose 4B as described previously 11. The [z~S]cysteine labelled neurohypophysial peptides were taken up in 0.1 M ammonium acetate (pH 5.7), applied on a Np-Sepharose column, (0.5 × 3 cm) and eluted with the same buffer solution. Seventeen fractions of 0.5 ml each were collected, the eluent was changed to 0.1 N formic acid, and another 15 fractions were collected.
High performance liquid chromatography of Np-boundpeptides The Np-bound peptides were analyzed on a HPLC system which separated [8]arginine-vasopressin (AVP) and OT. The HPLC instrumentation (Waters, Millford, Mass.) consisted of two pumps (model 6000 A), a programmer (model 660) and an injector (model UGK), The Np-bound peptides were taken up in 0.1% acetic acid, a 200 #1 aliquot was injected on a reverse phase column (/~C 18 P/N 27324, Waters), and the column was eluted with stepping and linear gradients (see Fig. 3). The mobile
301 phase was increasing methanol concentrations in 1 ~o acetic acid and the flow rate was 1.5 ml/min at room temperature. Fractions of 1.0 ml (42 sec) were collected and counted.
Release of [35S]cysteine labelledpolypeptides from isolated neurohypophyses Ten rats were injected with [35S]cysteine adjacent to their SONs (see above) and 24 h later the neural lobes were removed and placed in a medium of the following composition (mM) NaC1, 130; KC1, 4.8; CaC12, 2.8; MgSO4, 1.3; dextrose, 10; HEPES (N-2-hydroxyethyl piperazine-N-2-ethane sulfonic acid, 10 (pH 7.32). The lobes were incubated in this medium at 37 °C with constant oxygenation for 30 rain. At this time the medium was replaced with 1.0 ml of fresh warm medium. Every 5 min the incubate was collected and bathing medium was replaced. The lobes were stimulated by changing the medium to one containing veratridine (60/~M) with or without Ca 2+. The Ca 2+ deficient medium was prepared simply by omitting CaCI2. The incubation protocol is shown in Fig. 4.
L VP-Sepharose affinity chromatography [8-Lysine]-vasopressin (LVP) was covalently coupled to cyanogen bromide activated Sepharose 4-B as described earlier 9. The radioactive peak at the 10,000 Mr region of the Sephadex G-50 elution was taken up in 0.1 M ammonium acetate (pH 5.7), applied on a LVP-Sepharose column, and eluted with the same buffer. The bound peptides were eluted with 0.1 M ammonium formate (pH 8.5). Fractions of 0.5 ml were collected and aliquots were counted.
Polyacrylamide gel isoelectricfocusing The LVP-bound fraction of the 10,000 Dalton posterior pituitary proteins was dissolved in sample buffer containing 8 M urea, 1 ~ (v/v) Triton X-100 and 2 ~o (w/v) ampholytes (Bio Rad.). The sample was loaded at the anodal end of the gel and focused as described previously2. The gels were fixed in TCA, and 3 mm slices were cut and counted in an ammonium hydroxide-NCS counting solution. All column eluent aliquots were counted using a Beckman Liquid scintillation spectrometer (model LS 330) in a Triton X-100:toluene (1:2 v/v) counting solution. The aqueous sample content was maintained at 10 ~. RESULTS
Analysis of the [zaS]cysteine labelledpeptides transported to the neurohypophysis Twenty-four hours after injection of [zsS]cysteine adjacent to the supraoptic nuclei of rats, the posterior pituitaries contained labelled material which separated into 3 peaks on Sephadex G-50 (Fig. 1A, open symbols). The small peak at the V0 of the column contained relatively large proteins. The heavily labelled peak at the 10,000 Mr (10 kDalton) region was composed primarily of neurophysin as shown by binding to a LVP-Sepharose column (more than 98 ~ of the radioactivity was bound) and by isoelectric focusing (data not shown). Fifty to sixty per cent of the radioactivity in the
302 35S-CYSTEINE LABELLED MATERIAL TRANSPORTED TO POSTERIOR PITUITARY
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Fig. 1. A: Sephadex G-50 chromatography of posterior pituitary homogenates from control (open symbols) and colchicine-treated animals (solid symbols). The separation was carried out as described in legend to Fig. 2. In the colchicine-treated animals 100 Hg colchicine in 0.9 ~ NaCl was injected intraventricularly 24 h prior to [35S]cysteine injections into the SON, and the posterior pituitaries were harvested another 24 h later. Note that in colchicine-treated animals none of the radioactivity is present in the 10k Dalton region of the column, but significant labelled peptides are found (see B). B: N p Sepharose affinity chromatography of the peptides from the posterior pituitaries (see A) of normal (open symbols) and colchicine-treated animals (solid symbols), (see legend to Fig. 5A for methods). Note that in colchicine-treated animals none of the labelled peptides were bound by Np-Sepharose.
peptide fraction bound to a Np-Sepharose affinity column (Fig. 1B, open symbols), in a pH-dependent manner. In theory, labelled Np-unbound peptides could be peptides other than AVP and OT derived from the precursors during post-translational cleavage or extragranular non-neurosecretory peptides. Alternatively, because of the anatomical proximity of the injection site (the SON) to the neurohypophysis some label could diffuse to the neurohypophysis and be incorporated into proteins and peptides there. In order to assess these possibilities we injected colchicine (100 #g which blocks axonal transport) intraventricularly 24 h prior to the injection of [35S]cysteine. Twenty-four hours after cysteine administration the posterior pituitaries were harvested. The analysis of the labelled 0.1 N HCI soluble polypeptides from these pituitaries is presented in Fig. 1A (solid symbols). We found that all the radioactivity was associated with a single peak appearing at the Vt of the Sephadex G-50 column (<2500 Daltons, Fig. IA, solid symbols). These peptides were not bound by the Np-Sepharose affinity support (Fig. 1B, solid symbols) showing that the transport of labelled secretory proteins and Np-bound peptides had been blocked by colchicine. The labelled peptides which did not bind to Np were similar to the unbound peptides from untreated animals which were found after two dimensional cellulose plate thin layer chromatography (data not shown). Thus the Np-unbound peptides seem to be non-neuronal and to result from local incorporation of label by the posterior pituitary.
303 NORMALRATS
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Fig. 2. Sephadex G-50 separation of [35S]cysteine labelled materials transported to the posterior pituitary. At different times (indicated in the figure) after injection of [asS]cysteine near the SONs, the rats were killed and their posterior pituitaries removed and homogenized in 0.1 N HCI. The homogenate was applied on a Sephadex G-50 column (0.9 × 60 cm). The column was eluted with 0.1 N formic acid, 1.0 ml fractions being collected and 100 pl aliquots were counted. In normal rats, at 2 h after injection, the labelled large molecular weight proteins (in V0) make up a significant proportion of the total label. The label in V0 decreases with time, whereas the total amount of label increases (mainly in the 10k Dalton peak). In contrast, in the salt-treated rats, the relative proportion of the 3 peaks changes only slightly with time. This indicates that the rate of pro-hormone conversion to neurophysin (10 kDalton) and peptide products is increased by salt treatment of the rats (see Table I for analysis of peptides).
F o r this r e a s o n w e c o n f i n e d our s u b s e q u e n t a n a l y s e s to the N p - b o u n d p e p t i d e s . W e e x a m i n e d the t i m e course o f a p p e a r a n c e a n d the release o f N p - b o u n d labelled peptides.
304 TABLE I Np-Sepharose binding of i 35S]cysteine labelled peptides arriving at the posterior pituitary
Np-Sepharose affinity chromatography of 135S]cysteinelabelled posterior pituitary peptides. The peptides were separated on a Sephadex G-50 column (see Fig. 2) and then applied on a column of NpSepharose (0.5 × 3 cm) in 0.1 M ammonium acetate buffer (pH 5.7). These were eluted as described in legend to Fig. 5A. The percentage of the total radioactivity bound to Np-Sepharose is tabulated. Note that 2 h after SON injection of the [zsS]cysteine,the pituitaries of control (unstimulated) rats contained considerably less [3~S]cysteinelabelled bound peptides than the pituitaries of the salt-treated rats (see text for discussion). Time after :~sS/cysteine injection in the S O N ( h )
olo Total peptides bound to Np-Sepharose ........................................ Control Salt-treated
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T i m e course o f appearance o f [ 3 5 S j c y s t e i n e labelled peptides at the neurohypophysis
The elution profiles of [35S]cysteine labelled material in the neurohypophysis of normal and salt-treated rats 2, 4, 6, 24 and 48 h after injection of [35S]cysteine near both SONs, are shown in Fig. 2. This gel filtration analysis illustrates the quantitative differences in the material transported to the neurohypophysis with time and with stimulation of the system. In control animals (Fig. 2A-D), 2 h after injection the radioactivity is low and the Vo peak which contains the precursors of N p - V p and N p - O t as well as other proteins4, 5 makes up to 31 ~ of the radioactivity reaching the posterior pituitary (Fig. 2A). With time, however, the amount of radioactivity in the posterior pituitary increases dramatically. Furthermore, at the later times the Vo peak accounts for only 5-7 ~ of the total label. In the salt-treated animals (Fig. 2E-H), vasopressin release has been augmented several fold 4,8. Even 2 h after injection the amount of labelled peptides arriving at the posterior pituitary (Fig. 2E) is 7 times higher than that in control animals (Fig. 2A). This increased labelling is maintained up to 24 h after injection of [35S]cysteine. At longer times however, the difference is not observed, presumably because of increased release of the newly synthesized material in the salt-treated animals (Fig. 2D vs Fig. 2H). At all times the Vo peak proteins contain only about 6.5 ~ of the total radioactivity. Furthermore, the amount of label reaching the neurohypophysis increases only slightly with time. The labelled peptides in the Vt of the Sephadex G-50 column were separated by their affinity to neurophysin. Table I shows the increase in the proportion of Npbound labelled peptides in the neurohypophysis with time. Note that at early times after injection (Table I, 2 h) the percentage of Np-bound peptides was approximately 3-fold greater in the salt-treated in comparison to the control rats, indicating that the conversion rates of the precursors are enhanced by salt-treatment. The Np-bound peptides were fractionated on a reverse phase H P L C column
305
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Fig. 3. HPLC analysis of the [a~S]cysteine labelled, Np-bound peptides transported to posterior pituitaries. The Np-Sepharose-bound fractions of the posterior pituitary peptides were lyophilized and taken up in 1 ~ acetic acid, and a 200/~!aliquot was applied on a reverse phase ~Cls) column. Elution conditions are described in Methods. Note that particularly in earlier time periods, the labelled peptides are heterogeneous. Note also that in salt-treated rats (F-J) the proportion of label associated with AVP is larger compared with the normal animals (A-E). (Fig. 3). The labelled Np-bound peptides in the posterior pituitary mainly co-ran with AVP and oxytocin. However, at earlier time points (2-6 h Fig. 3A-C) in the control animals, there were two significant unidentified peaks with retention times close to AVP. These two small peptide peaks were absent after longer chase times. In salttreated animals, the laeptide profile is less heterogeneous f r o m 4 h post-injection on. One consistent difference between the labelled peptides transported to the pituitaries of salt-treated vs normal rats is that more label was associated with the AVP peak relative to the O T peak at all times in the salt-treated animals (Fig. 3). When the Npunbound fractions of the neurohypophysial labelled peptides were analyzed in the same H P L C system, we found that they consisted of a complex class of very hydrophilic peptides excluded by the column (data not shown).
Release of [35S]cysteine labelledpeptidesfrom the neurohypophysis Twenty-four hours after injection of [35S]cysteine adjacent to the SONs of 10
306 RELEASEOF 35S-CYSTEINELABELLEDMATERIAL FROM POSTERIORPITUITARY
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Fig. 4. A: release of [35S]cysteine labelled material from isolated neurohypophyses. Twenty-four hours after microinjection of [asS]cysteine near the SONs of rats, the posterior pituitaries were removed and placed in a medium containing (mM) NaCl, 130; KCI, 4.8; CaCl2, 2.8; MgSO_~, 1.3; Dextrose, 10; HEPES, 10 (pH 7.32). Ca 2+ was removed in the O-Ca medium. After a 30 rain equilibration period the medium was replaced and the incubation was begun in 1 ml fresh medium at 37 °C under constant oxygenation. The medium was exchanged every 5 min as indicated and aliquots of the incubate were counted. Note the 10-fold increase in released radioactivity upon veratridine depolarization only in the presence of Ca 2+. B: analysis of the released radioactivity on Sephadex G-50. The incubation medium during the first 3 periods (in the presence of 2.8 mM Ca 2+, see A; control, open symbols) and the two periods containing Ca 2+ and veratridine (veratridine, solid symbols) were separately pooled and lyophilized. This material was separated on a Sephadex G-50 column (0.9 × 60 cm) equilibrated with 0.1 N formic acid (see legend to Fig. 2 for methods). Note the marked increase in the 10,000 Mr region peak and the increase in the peptide peak with veratridine depolarization.
n o r m a l rats, their n e u r o h y p o p h y s e s were r e m o v e d a n d incubated at 37 °C (see Fig. 4). Fig. 4 A shows the r a d i o a c t i v i t y in the b a t h i n g m e d i u m at the end o f each 5 min collection period. R e m o v a l o f Ca 2÷ did n o t alter the release o f r a d i o a c t i v i t y in n o r m a l or v e r a t r i d i n e - c o n t a i n i n g media. However, veratridine in the presence o f C a 2+ caused a 10-fold increase in the release. The material released u n d e r c o n t r o l c o n d i t i o n s (the first 3 p e r i o d s ) a n d the two 5 min p e r i o d s with 6 0 / ~ M veratridine a n d 2.8 m M Ca 2÷ were p o o l e d separately a n d analyzed by Sephadex G-50 c h r o m a t o g r a p h y followed by N p - S e p h a r o s e a n d L V P - S e p h a r o s e affinity c h r o m a t o g r a p h y . Fig. 4B shows t h a t the released r a d i o a c t i v i t y separated into two m a j o r peaks on Sephadex G-50; one at the 10,000 M r region a n d a n o t h e r at the Vt o f the c o l u m n (peptides). N p - S e p h a r o s e affinity c h r o m a t o g r a p h y o f the released peptides revealed that under c o n t r o l conditions a very small fraction o f the [35S]cysteine labelled peptides were b o u n d by the c o l u m n (Fig. 5A). On the other h a n d the veratridine d e p o l a r i z a t i o n in the presence o f C a 2+ increased the N p - b o u n d fraction several fold w i t h o u t a
307 HPLCOFNp-BOUNDRELEASEDPEPTIDES
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Fig. 5. Analysis of the released [asS]cysteinelabelled peptides. A: affinity chromatography of the released peptides on Np-Sepharose. The peptide fraction separated by Sephadex G-50 chromatography (see Fig. 4B) was lyophilized and redissolved in 0.1 M ammonium acetate (pH 5.7) and applied on a Np-Sepharose column (0.5 × 3 cm). The column was eluted initially with the same buffer and the bound fraction was collected by changing the eluent to 0.1 N formic acid at the arrow. Note the marked increase only of the bound fraction during veratridine depolarization. B: HPLC separation of the Npbound released peptides. The Np-bound peptides were separated by a HPLC system described in the legend to Fig. 3. Note that veratridine stimulation increased the release of AVP and OT. Under both conditions more label was associated with the oxytocin peak than with the AVP peak.
concomitant increase in the release of unbound peptides. The H P L C profiles of the Np-bound peptides are shown in Fig. 5B. Under both control and stimulated conditions the released [35S]cysteine labelled peptides were primarily made up of vasopressin and oxytocin. Furthermore, there was 2.5 times as much radioactivity associated with the oxytocin peak as with the AVP peak (Fig. 5B). The major increase in the released radioactivity on veratridine depolarization was associated with the 10,000 Mr region on Sephadex G-50 chromatography (Fig. 4B). More than 95 ~ of this peak was bound to LVP-Sepharose affinity column thus identifying it as neurophysin (Fig. 6A). The LVP-bound fraction focused as a single peak (Fig. 6B). The peak of label seen in Fig. 6B can actually be resolved into two separate neurophysins ( N p - V P and N P - O T ) using more expanded p H gradients in the acid range during electrofocusing2, 5. However, we chose this wider p H gradient in order to detect whether more basic proteins were also released. DISCUSSION Several lines of evidence suggest that processing of the precursor occurs intragranularly during axonal transport. Sachs found 12,13 that the vasopressin content of neurohypophysial neurosecretory granules is 5 times higher than that of hypothalamic granules. In previous studies, we have shown that the Np-precursors are transported into the axons and terminals 4-6, where the precursors (Mr 20,000) are processed into smaller neurophysins (Mr 10,000). Electron microscopic autoradiography shows that most of the cysteine-labelled material transported to the pituitary is
308 ISOELECTRIC FOCUSING OF LVP-BOUND RELEASED 10K PEAK
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Fig. 6. Analysis of the released [35S]cysteine labelled l0 kDalton peak. A: LVP-Sepharose affinity chromatography of the released 10 kDalton peak. The 10,000 Mr labelled proteins obtained by Sephadex G-50 chromatography of the veratridine released material (see Fig. 4B) were redissolved in 0.1 M ammonium acetate pH 5.7. This material was applied on a LVP-Sepharose column and eluted with the same buffer and 0.5 ml fractions were collected. The bound fraction was eluted by the affinity support in a pH-dependent manner. B: polyacrylamide gel isoelectric focusing of LVP-bound released proteins. The LVP-bound fraction of the 10 kDalton proteins released by veratridine was dissolved in sample buffer and focused on a 7.5 % acrylamide gel (see Methods). The gel was fixed in TCA and 3 mm slices were cut and counted for radioactivity. Note that virtually all the radioactivity was found in a single peak corresponding to the migration position of rat neurophysin (rat Np).
in the neurosecretory granules 7. Therefore, we have concluded that the conversion process is normally occurring intragranularly during axonal transport. This is the only neuronal system in which evidence for an anatomical compartment (i.e. the secretory granules) for post-translational cleavage of a pro-peptide has been reported. However, processing can also occur at the level of the hypothalamic nuclei. Under conditions where axonal transport of the granules is blocked with colchicine (100 /zg, intraventricular), accumulation of [35S]cysteine labelled neurophysin and AVP and OT can be observed in the SON, 6 h after injection of labelled cysteine (unpublished observations). In this study, we have shown that the rate of processing is regulated by the functional activity of the system. During dehydration, induced either by water deprivation or salt-loading, the rats release large quantities of neurohypophysial hormones 8, and biosynthesis of the hormones increases between 5- and 10-fold (see ref. 4 and Fig. 2). The time course study illustrated in Fig. 2 shows that in normal (nondehydrated) rats, 2 h post-injection of [35S]cysteine in the SON, about 31% of the labelled material transported to the pituitary is still in the precursor form (i.e. in the Vo peak). In contrast, at 2 h, the labelled material in the salt-treated rats is completely converted to neurophysin (i.e. the 10 kDalton peak). The data shown in Table I also illustrates this point with regard to the peptides.
309 We have used Np-bindingl,ll, 14 and HPLC chromatography as identifying criteria for analyzing the [35S]cysteine labelled peptides. As can be seen in Fig. 1, many of the labelled Np-unbound peptides in the neurohypophyses do not derive from the hypothalamus via axonal transport. At present we cannot distinguish between those Np-unbound peptides which are in the pituitary through axonal transport and those which are produced locally. For this purpose, further development of the HPLC system for better resolution of these hydrophilic peptides (Mr 700-1000) is still necessary. The data in Table I indicate that 2 h after injection, the conversion of the labelled peptides to Np-binding peptides is almost complete in salt-treated rats, but is considerably slower in control (non-dehydrated) rats. Even when the peptides bind to Np, there are subtle differences in the HPLC patterns (Fig. 3) of Np-bound peptides from salt-treated vs normal rats, which suggest a slower rate of (or less complete) processing in the latter. In normal (unstimulated) animals at the earliest times, the Np-bound peptides show more heterogeneity after HPLC analysis (Fig. 3). It is possible that the extra peptide peaks observed may be intermediates in the intragranular processing of the precursors. Recently we found that mild trypsinization of the isolated precursor proteins over different periods of time produces Np-binding peptides which showed precursor product relationships (i.e. converting to peptides with different chromatographic (HPLC) properties with time) 11. Since the NH2-terminal sequence of the peptide (Cys-Tyr) is essential for binding to Npl, 14, the subsequent alterations of the Np-bound peptides must be occurring at the C-terminal. The release paradigm illustrated in Figs. 4-6 was used in order to provide additional evidence that the labelled Np and Np-bound peptides were indeed intragranular. The Ca2+-dependent ~ veratridine-induced release consisted exclusively of Np (Figs. 4 and 6), and Np-bound peptides (Fig. 5). Analysis of the HPLC patterns of the Np-bound released peptides showed that there was a preferential release of OT using this stimulation paradigm (compare Figs. 3D and 5B). It is not clear at present why, 24 h after transport to the neurohypophysis, the labelled OT is released more readily than the labelled AVP. Presumably, this reflects differences in the granule storage characteristics of the OT- and AVP-containing terminals. Another possibility is that there are other cysteine-containing, Np-bound peptides transported to and released by the neurohypophysis, and that these peptides co-migrated with the OT on our HPLC system. Separation of these labelled peptides by other HPLC eluents and protocols, in the future, may help to resolve this issue. Since there are approximately 12-14 cysteine residues in each neurophysin molecule and 2 cysteine residues in each nonapeptide (OT or AVP), and if the Np and OT (or AVP) are stored and released in a 1:1 M ratio, then one might expect that the ratio of Np cpm and Np-bound peptide cpm would be 6-7. Analysis of this ratio for [sS]cysteine material transported to the neurohypophysis gives a ratio of about 6 (from data in Fig. 1). However, analysis of this ratio for the veratridine-released labelled material (Figs. 4-6) gives a ratio of 2.5-3. Two explanations might account for this discrepancy: (1) in our release paradigm, the recovery of the larger Np molecule which is released may be less than the smaller and more rapidly diffusible peptides; and/or (2)
310 as discussed above, there may be other cysteine containing peptides that migrate near OT (the O T peak is large and asymmetrical on H PLC; Figs. 3 and 5B). The peptides thus unresolved by H P L C could account for the larger than expected radioactivity associated with the peptide fraction. I f the latter explanation is correct, then we would expect that the other cysteine-containing peptides would derive from the OT precursor, since we have shown that the A V P -+- N p - v p precursor contains a non-cysteine containing glycoprotein at its N-terminal region10,11. Indeed, trypsinization o f isolated OT + N p - o t precursor suggests that more than one OT-like peptide may be derived from each precursor molecule, unlike in the case o f the A V P + N p - v p c o m m o n precursor (see ref. 10). The data presented here show that the increase in h o r m o n e biosynthesis in the hypothalamic neurohypophysial system, induced by an increased functional activity, is accompanied by an increase in rate o f post-translational cleavage of the p r o - h o r m o n e within the granules. While this is understandable in a teleological dense (i.e. the terminals in the neurohypophysis are depleted of granules in the salt-treated animal 8, and release of hormones under these conditions may then depend upon 'on-line' delivery from the hypothalamus), we do not yet know either the enzymes involved in the post-translational cleavage, or the mechanisms which underly their regulation. In addition, it will be of considerable interest to determine whether the increased electrical (or synaptic) activity of the cell, or the depletion of the contents at its terminals acts as the intracellular signal to regulate the biosynthesis and conversion rates of the neurosecretory cell. ACKNOWLEDGEMENTS We wish to thank Miss Sharon Holmes for excellent technical assistance and Mrs. N a n c y Garvey for typing the manuscript. We also would like to acknowledge the contributions of Dr. Marina Mata to the early stages o f this work.
REFERENCES l Breslow, E., Aanning, H. L., Abrash, L. and Schmir, M., Physical properties of the bovine neurophysins, J. biol. Chem., 246 (1971) 5179-5188. 2 Brownstein, M. J. and Gainer, H., Neurophysin biosynthesis in normal rats and in rats with hereditary diabetes insipidus, Proc. nat. Acad. Sci. (Wash.), 74 (1977) 4046-4049. 3 Douglas, W. W., Mechanism ofrelease of neurohypophysial hormones : stimulus secretion coupling. In E. Knobil and W. H. Sawyer (Eds.), Handbook of Physiology, Section 7, Vol. 4, American Physiological Society, Washington, D.C., 1974, pp. 191-224. 4 Gainer, H., Sarne, Y. and Brownstein, M. J., Biosynthesis and axonal transport of rat neurohypophysial proteins and peptides, J. Cell Biol., 73 (1977) 366-381. 5 Gainer, H. and Brownstein, M. J., Identification of the precursors of the rat neurophysins. In J.-D. Vincent and C. Kordon (Eds.), Cell Biology ofHypothalamic Neurosecretion, Centre National de la Recherche Scientifique, Paris, 1978, pp. 526-541. 6 Gainer, H., Loh, Y. and Sarne, Y., Biosynthesis of neuronal peptides. In H. Gainer (Ed.), Peptides in Neurobiology, Plenum Press, New York, 1977, pp. 183 219. 7 Kent, C. and Williams, M. A., The nature ofhypothalamo-neurohypophysial neurosecretion in rat. A study by light- and electron-microscope autoradiography, J. Cell Biol., 60 (1974) 554-570.
311 8 Jones, C. W. and Pickering, B. T., Comparison of the effects of water deprivation and sodium chloride inhibition on the hormones content of the neurohypophysis of the rat, J. PhysioL (Lond.), 203 (1969) 458--499. 9 Robinson, I. C. A. F., Edgar, D. H. and Hope, D. B., A new method of coupling (8-1ysine) vasopressin to agarose; the purification of neurophysins by affinity chromatography, Neuroscience, 1 (1976) 35-39. 10 Russell, J. T., Brownstein, M. J. and Gainer, H., Biosynthesis of vasopressin, oxytocin and neurophysins: isolation and characterization of the two common precursors (propressophysin and prooxyphysin), Endocrinology, in press. 11 Russell, J. T., Brownstein, M. J. and Gainer, H., Liberation by trypsin of an arginine vasopressin like peptide and neurophysin from a Mr 20,000 putative common precursor, Proc. nat. Acad. Sci. (Wash.), 76 (1979) 6086-6090. 12 Sachs, H., Neurosecretion. In A. Lajtha (Ed.), Handbook of Neurochemistry, Plenum Press, New York, 1970, pp. 373428. 13 Sachs, H., Studies on intracellular distribution of vasopressin, J. Neurochern., 10 (1960) 297-303. 14 Stoufer, J. E., Hope, D. B. and Du Vigneaud, V., Neurophysin, oxytocin, and desamino oxytocin. In C. F. Cori, V. G. Foglia, L. F. Leloir and S. Ochoa, (Eds.), Perspectives in Biology, Elsevier, Amsterdam, 1963, pp. 75-80.