[35S]cysteine labeled peptides transported to the neurohypophyses of adrenalectomized, lactating, and Brattleboro rats

Brain Research, 201 (1980) 227-234 © Elsevier/North-Holland Biomedical Press

227

psS]cysteine labeled peptides transported to the neurohypophyses of adrenalectomized, lactating, and Brattleboro rats

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 Institute 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: neurohypophysis - - adrenalectomy - - lactation

Arginine-(8)-vasopressin (AVP) oxytocin (OT) and neurophysin (Np) are synthesized in the supraoptic (SON) and the paraventricular nuclei (PVN) of the hypothalamus and transported intra-axonally to the neurohypophysisa,la. Since both the neurophysins and the nonapeptides are cysteine rich, they can be labeled with [35S]cysteine administered near the nuclei; and the selective fast axonal transport of the secretory products provides an ideal tool by which to study the labeled products at the terminala,4,10,11. In this study, we have used affinity chromatography and high performance liquid chromatography to analyze the [3sS]cysteine labeled peptides in the neurohypophysis under conditions of enhanced secretory activity (e.g. lactation and adrenalectomy). We found that the nature and amount of labeled peptides which were transported to the pituitary were influenced by the site of injection and the state of secretory activity of the system. [a5]Cysteine (New England Nuclear, specific activity diluted to 100 Ci/mmol) was injected bilaterally adjacent to the SONs or PVNs of rats (Osborn Mendel, 225-250 g), as described previously2. In one set of experiments Brattleboro rats (homozygous and heterozygous) were used. The adrenalectomized animals were studied 10 days postoperatively and the lactating animals were injected 3 days post partum. Twentyfour hours post injection of the [aSS]cysteine the posterior pituitaries and median eminencies (M.E.) were removed. Three posterior pituitaries or median eminences were pooled in each sample and homogenized in 0.1 N HCI. The HC1 soluble material was chromatographed on a Sephadex G-50 column. The labeled peptide fraction, which appeared in the Vt of the column (see Figs. 1A, 2A and 3A), was lyophilized and then applied on a neurophysin-Sepharose affinity column. The Np-bound fraction after lyophihzation, was analyzed by high performance liquid chromatography. The experimental methods are described in detail in the legend to Fig. 1. The labeled material in the neurohypophysis and median eminence 24 h after injection of [aSS]cysteine was separated by Sephadex G-50 into 3 molecular weight

228

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Fig. 1. Analysis of [asS]cysteine-labeled peptides In the neurohypophysls of Brattleboro rats. A: Brattleboro rats, homozygotes (solid symbols) and heterozygotes (open symbols) were injected bilaterally adjacent to the SONs with [asS]cystelne in 0.9 % NaCl in a volume of 1/,1/site. The pituitaries were collected 24 h later and homogenized in 0.1 N HCI. The HCI soluble material was applied on a Sephadex G-50 column (0 9 ~, 60 cm) and eluted with 0.1 N formic acid, 1.0 mt fractions being collected and 100 ffl aliquots were counted. The radioactivity was separated into 3 distinct peaks. B: Np-Sepharose affinity chromatography of [asS]cysteine-labeled peptides. The peptide peak (panel A) was collected lyophilized and redissolved in 0.1 M ammonium acetate (pH 5.7) and chromatographed on a NpSepharose column (0.5 × 3 cm). The bound fraction was etuted from the column with 0.1 N formic acid. Fractions of 0.5 ffl were collected and aliquots were counted. The fraction of labeled peptldes bound in the heterozygote (54.4 %) is larger than the homozygous Brattleboro rats (t6.1%). C: high performance liquid chromatography analysis of the Np-bound peptides. The chromatographic system (Waters Associates, Millford, Mass.) consisted of two pumps (model 6000), a solvent programmer (model 660) and an injector (model UGK). The Np-bound peptides were dissolved in 35 m M ammonium acetate buffer (pH 6.0) and a 2 0 0 / 4 aliquot was injected on a reverse phase (uCls) column and eluted with 35 % methanol in 35 mM ammonium acetate buffer (pH 6 0). Two step changes in methanol concentration of the mobile phase were made as indicated in the figure The flow rate was 1.5 ml/mm at room temperature and 1.0 ml fractions were collected and counted. The arrows indicate the elution positions for A V P and OT Note that in the homozygous Brattleboro animals the A V P peak is absent.

229 TABLE I [asSJcysteine labeled neurophysin distribution 24 h after injection o f label in the S O N or P V N o f rats

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0.042 0.39

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2.1 4.5

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(--) (65)

15.7 14.3

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A. SON injection Control Lactation Adrenalectomy B. PVN injection Control Adrenalectomy Brattleboro rats

C. SON injection Heterozygote(control) 1.01 Homozygote 0.66

classes: >20,000 (V0); 10,000; and a peptide fraction (Vt, < 2500 mw) (Figs. 1A, 2A and 3A). The Mr 10,000 peaks from the Sephadex G-50 column were identified as neurophysins (96-98 ~o) as shown by their affinity to a lysine vasopressin-Sepharose affinity column (not illustrated). Using this experimental paradigm we examined how much labeled neurophysin arrived at the median eminence and neurohypophysis 24 h after injection. Table I summarizes these data for a variety of experimental conditions. As can be seen in Table I, SON injections in normal (Osborn-Mendel) rats were about 8-fold more effective than PVN injections in labeling neurophysin in control pituitaries. Using the SON injection no change was found between the pituitaries of control and adrenalectomized rats, but there was a 1.9-fold increase in labeled neurophysin in lactating rat pituitaries (Table IA). PVN injections revealed a more than 2-fold increase in labeled neurophysin in adrenalectomized over control rat pituitaries. These observations are consistent with suggestions of a relationship between the regulation of corticotropic function and the activity of neurosecretory neurons specifically in the PVN 6. In this connection it is interesting to note that extracts of neurohypophyses have been shown to contain corticotropin releasing activity (CRF) 14. The major site of increased neurophysin in response to adrenalectomy is known to be the zona externa of the M.E. which receives projections principally from the PVN (see ref. 6 for review). The data in Table I are also consistent with this view. Comparison of the differences in labeled neurophysin between control and adrenalectomized rats shows that there was a 9-fold increase in the latter after PVN injection (Table IB), and in contrast, a decrease in labeled neurophysin in the M.E. of adrenalectomized rats after SON injection (Table IA). The above data show that the increase in newly synthesized neurophysin in

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Fig. 2. Analysis of [asS]cysteine labeled material from the neurohypophysls of normal (solid circles), adrenalectomized (open circles), and lactating (open squares) rats injected near the SONs. The adrenalectomlzed animals were injected 10 days after surgery. During this time, they were given 0.9 % NaC1 to drink. The lactating animals were injected 3 days postpartum. The pups were removed during the injection time and were allowed to suckle immediately after the mother was awake, thus ensuring almost constant stimulation. A: gel filtration of Sephadex G-50 of 0.1 N HCI radioactivity from posterior pituitaries. The chromatography was carried out as described in legend to Fig. 1A. Note that while the amount of labeling is unchanged m adrenalectomized animals, in lactating rats it is higher than the controls. B: Np-Sepharose affinity chromatography of peptides (see Fig. 2A). The methods are described in Fig. lB. The percentages of bound labeled pepttdes in the control, lactating, and adrenalectomized rats were 48.3 %, 62.8 % and 51.3 %, respectively. C: HPLC analysis of Np-bound peptides (see Legend to Fig. 1C for methods). Note the label associated with new peaks m lactating animals. pituitary and M.E. after adrenalectomy is largely derived from P V N neurons, and that this change is greatest in the median eminence. N o comparisons between P V N and S O N injections were done for lactating animals, nor for Brattleboro rats. In the latter, the heterozygote (control) appeared to contain more labeled neurophysin m

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Fig• 3. Analysis of labeled neurohypophysial peptides after injection of [asS]cysteine adjacent to the PVNs of rats. Solid symbols, adrenalectomized rats; open symbols, normal rats. Adrenalectomized animals were handled as described in legend to Fig. 2. A: Sephadex G-50 chromatography of 0.1 N HCI soluble label• Note the marked increase in labeling in the adrenalectomized animals unlike when injected near SONs (Fig• 2A)• The chromatographic technique ]s described in legend to Fig. 1A. B: Np-Sepharose affimty chromatography of the peptide fraction from gel filtration (see F]g. 3A). The percentages of bound labeled peptides in the control and adrenalectomized rats were 33.1% and 48.6 ~ , respectively. C: HPLC analyms of the Np-bound peptldes. Radioactivity associated with both the AVP and OT peaks are increased 2-3 fold.

the M . E . relative to the posterior pituitary (after S O N injection, T a b l e IC) t h a n in c o n t r o l O s b o r n - M e n d e l rats (Table IA).

232 In the following section we consider the nature of the labeled peptldes which were found in the posterior pituitary under the above experimental conditions. No such analyses were performed on the median eminence samples since they contained too little radioactivity. When the peptide fraction obtained from Sephadex G-50 separation of labeled material from the posterior pituitary is applied on a Np-Sepharose affinity column, peptides such as oxytocin and vasopressin, containing an NH2-terminal sequence ot Cys-Tyr-hydrophobic amino acid (Leu or Phe) are specifically bound to the Np on the column, and are eluted at acid pH (eg. Figs. IB, 2B, and 3B). The nature of this interaction between the Np and the peptldes is well characterized, and involves both ionic and hydrophobic interactions 1,9,a6. The Np-bound peptldes labeled with [.3.~S]cystelne are synthesized in the hypothalamus and transported intra-axonally to the posterior lobe3, tz. In colchicine injected rats, where fast axonal transport is inhibited ~, no Np-bound, labeled peptldes can be detected in the neurohypophysis i2. In a previous paper 3, we suggested that [35S]cysteine-labeled peptldes, other than OT and AVP, may be transported to the neurohypophysis. However, ~t was not known whether any of those labeled peptides bound to Np. The analysis of the Np-bound fraction by HPLC using elution at pH 6.0 (Figs. 1C, 2C, and 3C) in contrast to elution at p H 3.0 tz, suggests that these labeled peptides are qmte heterogeneous. Analysis of the Np-bound peptldes by HPLC is illustrated in Figs. 1C, 2C, and 3C. Homozygous rats with hereditary diabetes mslpldus (Brattleboro strain) do not synthesize the precursor for AVP 2, or store AVP m their hypothalamo-neurohypophysial system15, aT. The heterozygote does synthesize and contain AVP. We found that the neurohypophyses of heterozygous rats contained labeled peptides which were similar to those m normal animals (Fig. 1C, open circles and Fig. 2C, solid circles). In the homozygote, however, labeled AVP was completely absent (Fig. t C solid circles). As expected, since the AVP was absent, the fraction of labeled peptides in the pituitary which bound to Np-Sepharose was much smaller in the homozygous than in the heterozygous Brattleboro rats (Fig. 1B). It ~s of interest to note here, that the labeled peaks other than OT and AVP seen m F~g. 1C (particularly the large peak elutmg m 8 0 ~ methanol), are still present in the homozygous rat pituitary. Therefore, these peaks cannot be associated with AVP synthesis They may be associated with OT synthesis, i.e. they may be co-ordmately synthesized with OT on a common precursor3.11 . The data in Fig. 2 compare the [zsS[cysteine labeled peptides transported to the neurohypophysls following injection in the SON, in normal (control), adrenalectomized and lactating rats. We studied lactating rats with the expectation that there would be a preferential increase in biosynthesis of OT (as opposed to AVP) in these animals, since suckling induces selective stimulation of OT neurons 7 and depletes OT in the neurohypophysis s. When we analyzed the [3sS]tabeled peptides in the neurohypophysis of lactating rats, we found that as with neurophysm (Table l) the peptide labeling was 2-fold higher in lactating than m control ammals (Fig. 2 open squares). However, to our surprise, the analysis of the peptides on HPLC showed (Fig. 2C open squares) that the labeling was increased for both OT and AVP. The increase in labeling found was

233 very similar to t h a t seen in rats given 2 ~ NaC1 to d r i n k l L T h e latter t r e a t m e n t a u g m e n t s b o t h O T a n d A V P release 5. Thus, we c o n c l u d e t h a t in the lactating animal, the biosynthesis o f b o t h A V P a n d O T is enhanced. A l s o n o t a b l e in Fig. 2C is the presence o f a new p e a k (fraction no. 10) in the pituitaries o f lactating rats, n o t seen with a n y o t h e r e x p e r i m e n t a l c o n d i t i o n . T h e d a t a in Fig. 3C show t h a t the increased biosynthesis o f n e u r o p h y s i n after a d r e n a l e c t o m y (see T a b l e I) observed with P V N injected animals, was also a c c o m p a n i e d by an increased p e p t i d e labeling in the p o s t e r i o r p i t u i t a r y (in c o n t r a s t to S O N rejection, Fig. 2C, o p e n circles). There was no qualitative change in the H P L C p a t t e r n . It will be i m p o r t a n t in the future to examine whether any qualitative changes in H P L C p a t t e r n s o c c u r r e d at the M . E . level. W e wish to t h a n k Mrs. N a n c y G a r v e y for t y p i n g this manuscript.

1 Breslow, E., Aannmg, H. L., Abrash, L. and Schmir, M., Physical and chemical properties of the bovine neurophysins, J. bzoL Chem., 246 ( 1971) 5179-5188. 2 Brownstein, M. J. and Gainer, H., Neurophysln biosynthesis in normal rats and in rats with hereditary diabetes insipidus, Proc. nat. Acad. Sci. (Wash.), 74 (1977) 4046-4049. 3 Gainer, H., Same, Y. and Brownstein, M. J., Biosynthesis and axonal transport of rat neurohypophyslal proteins and peptides, J. Cell BioL, 73 (1977) 366-381. 4 Gainer, H. and Brownstein, M. J., Identification of the precursors of the rat neurophyslns. In J. P. Vincent and C. Kordon (Eds.), Cell Biology ofHypothalamic Neurosecretion, Centre National de la Recherche Scmntifique, Paris, 1978, pp. 525-541. 5 Jones, C. W. and Pickering, B. T., Comparison of the effects of water deprivation and sodium chloride inhibition on the hormone content of the neurohypophysis of the rat, J. PhysioL (Lond.), 203 (1969)448--458. 6 Krieger, D. T. and Zimmerman, E. A., The nature of CRF and its relationship to vasopressin. In L. Martini, and G. M. Besser (Eds.), Clinical Neuroendocrinology, Academic Press, New York, 1977, pp. 363-391. 7 Lincoln, D. W. and Wakerley, J. B., Factors governing the periodic activation of supraoptic and paraventrtcular neurosecretory cells during suckling in the rat, J. Physiol. (Lond.), 250 (1975) 443-461. 8 0 k a , T., Blockade by chohnerglc and adrenergic blocking agents of the suckling reduced depletion of oxytocin from the neurohypophysis of the lactating rat, Acta Endocr. (Kbh.), 68 (1971) 707-714. 9 Pickering, B. T. and Jones, C. W., The neurophysins. In Hormonal Proteins andPeptides Vol. 5. Academic press, New York, 1978, pp. 103-158. 10 Russell, J. T., Brownstein, M. J. and Gainer, H., Liberation by trypsin ofa vasopressin-like peptide and neurophysm from a Mr 20,000 putative common precursor, Proc. nat. Acad. Sci. ( Wash ), 76 (1979) 6086-6090. 11 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), submitted for publication. 12 Russell, J. T., Brownstein, M. J. and Gainer, H., Time course of appearance and release of [ass]cysteme labeled neurophyslns and peptides in the neurohypophysis, submitted for publication. 13 Sachs, H., Neurosecretion. In A. Lajtha (Ed.), Handbook of Neurochemistry, Plenum press, New York, 1970, pp. 373-428. 14 Saffran, M., Schally, A. V. and Benfry, B. G., Stimulation of the release of corticotropin from the adenohypophyszs by a neurohypophysial factor, Endocrinology, 57 (1955) 439 444. 15 Sokol, H. W., Zlmmerman, E. A., Sawyer, W. N. and Robmson, A. G., The hypothalamo-neurohypophyslal system of the rat" localization and quantification of neurophysin by light microscopic immunocytochemlstry in normal rats and in Brattleboro rats deficient in vasopressin and a neurophysin, Endocrinology, 98 (1976) 1176-1185.

234 16 Stouffer, J. E., Hope, D. B. and Du V~gneaud,V., Neurophysin, oxytocin and des amino oxytocm In C. F. Cori, V. G. Foglia, L. F. Leloir and S. Ochoa (Eds.), Perspectives in Biology, Elsevier, Amsterdam, 1963, pp 75-80. 17 Valtln, H., Stewart, J. and Sokol, H. W , Genetic control of the production of posterior p~tu~tary principles. In E. Knobll and W. H. Sawyer (Eds), Handbook of Physiology, Section 7, Fol 4, American Physiology Society, Washington, D.C., 1974, pp. 131-171