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Different patterns of molecular forms of somatostatin are released by the rat median eminence and hypothalamus
Neuroscience Letters, 57 (1985) 215--220
215
Elsevier Scientific Publishers Ireland Ltd. NSL 03356
DIFFERENT P A T T E R N S OF M O L E C U L A R F O R M S OF S O M A T O S T A T I N ARE R E L E A S E D BY THE RAT M E D I A N E M I N E N C E AND HYPOTHALAMUS*
ADRIAN R. PIEROTTI, ANTHONY J. HARMAR**, LESLEY A. TANNAHILL and GORDON W. ARBUTHNOTT MRC Brain Metabolism Unit, University Department of Pharmacology, 1 George Square, Edinburgh EH8 9JZ (U.K.)
(Received February 19th, 1985; Revised version received and accepted March 15th, 1985)
Key words" somatostatin-14 - somatostatin-28 - hypothalamus - median eminence - release - high-per-
formance liquid chromatography - rat
The molecular forms of somatostatin (SOM) released from hypothalamic slices and from the isolated median eminence (ME) in vitro were compared by high-performance liquid chromatography and radioimmunoassay. SOM-14 was the predominant form of the peptide released from hypothalamic slices, although small amounts of SOM-28 were detected. Perifusates of ME tissue contained a larger proportion of SOM-28 and higher molecular weight peptides were present; depolarization increased the rates of release of all molecular forms of SOM. These results suggest that the capacity to release SOM-28 and high-molecular-weight forms of SOM may be a specialized function of nerve terminals in the ME.
S o m a t o s t a t i n - l i k e i m m u n o r e a c t i v i t y ( S O M - L I ) has been shown to exist in tissues in several m o l e c u l a r forms, all o f which are derived f r o m a c o m m o n p o l y p e p t i d e prec u r s o r ( p r o s o m a t o s t a t i n , mol.wt. 10,400) [10]. T h e first o f these forms to be described was SOM-14, a t e t r a d e c a p e p t i d e m e d i a t i n g the h y p o t h a l a m i c c o n t r o l o f g r o w t h horm o n e ( G H ) secretion [5]. SOM-28, an o c t a c o s a p e p t i d e c o n t a i n i n g the sequence o f S O M - 1 4 with an N - t e r m i n a l extension o f 14 a m i n o acids [7, 19], is m o r e p o t e n t t h a n - S O M - 1 4 in i n h i b i t n g the release o f insulin from the p a n c r e a s [15] a n d the secretion o f G H f r o m the a n t e r i o r p i t u i t a r y g l a n d [4], a n d m a y act selectively at a subclass o f S O M receptors with a higher affinity for S O M - 2 8 t h a n for S O M - 1 4 [20]. T w o forms o f S O M - L I with higher m o l e c u l a r weights (6000 a n d 8000-10,000) have also been described in extracts o f n e r v o u s tissue [3]; the a m i n o acid sequences a n d possible biological functions o f these larger peptides have yet to be established. The m e d i a n eminence ( M E ) , which c o n t a i n s S O M - 1 4 a n d S O M - 2 8 in a l m o s t e q u i m o l a r concent r a t i o n s [18], differs f r o m the rest o f the h y p o t h a l a m u s , in which S O M - 1 4 p r e d o m i n a t e s . A l t h o u g h the c a l c i u m - d e p e n d e n t , p o t a s s i u m - e v o k e d release o f S O M *Some of the observations reported here have been published in preliminary form [1, 11]. **Author for correspondence. 0304-3940,'85/$ 03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd.
216 from hypothalamic slices has been demonstrated [12], there is little information concerning the molecular forms of the peptide released. Accordingly, we have used highperformance liquid chromatography (HPLC) to compare the molecular forms of SOM released from hypothalamic slices with those released from the isolated ME in vitro. Male COB Wistar rats (180-230 g body wt.) were killed by decapitation and the brains removed. Hypothalamic tissue was dissected by the procedure of Glowinski and Iversen [8] and sliced in the sagittal plane at 250/~m intervals using a McIIwain tissue chopper. ME tissue was removed from the intact brain by dissection with iridectomy scissors. Hypothalamic slices or MEs were placed in a modified Krebsbicarbonate medium (identical to that described by Iversen et al. [12] except that Trasylol, 400 Kallikrein inactivator units/ml, was added) and transferred to small plastic chambers [13]. Tissues were perifused at 37'~C with Krebs-bicarbonate medium at a flow-rate of 0.3 ml/min. After an initial wash period of 30 rain, samples were collected for 3-min periods into plastic tubes containing 0.5 ml aqueous trifluoroacetic acid (TFA: 3"~, v/v). Total SOM-LI released was determined after extraction of samples on disposable columns of octadecylsilica (Baker-10 ODS, 1 ml; J.T. Baker). Columns were preconditioned by washing with 2 ml of T F A - m e t h a n o l (l~,, v/v) followed by 2 ml of aqueous T F A (1",,;, v/v). After application of the sample, columns were washed with 1 ml aqueous T F A and peptides eluted with I ml of methanolTFA. Samples were evaporated to dryness in a vacuum oven prior to determination of SOM-LI by radioimmunoassay (RIA) [18]. The antiserum used (final dilution 1:750,000) was directed towards the central or C-terminal region of the SOM-14 sequence; SOM-14 and SOM-28 were equally active on a molar basis in displacing tracer from the antiserum whereas 13 other neuropeptides were inactive. [1251]Tyr°SOM-14 was used as tracer and the limit of sensitivity of the assay was 2.2 fmol SOM-14 per tube. In normal Krebs medium, SOM-LI was released from hypothalamic slices with a rate constant of less than 0.02',!Jo/min (Fig. lb). Addition of 50-raM KCI to the medium caused an immediate increase of 15-20-fold in the rate of release of SOM-LI. Basal release of SOM-LI was diminished, and K ' - e v o k e d release dramatically reduced, when Mg 2+ was substituted for Ca 2+ in the perifusion medium (Fig. la). The basal rate of release of SOM-LI from the isolated ME was 0.005°J~,/min, and depolarization with 50 mM KCI stimulated the release of SOM-LI by 6.9-fold (Fig. l c). K ~-evoked release was abolished in calcium-free medium. To characterize the pattern of molecular forms of SOM-LI released from the hypothalamus and ME, perifusate samples from 8 hypothalami or 60 MEs were combined. Chromatographic analysis was performed using an Altex Model 421 H P L C system with an analytical column of Ultrasphere 5 ~m ODS (Altex). Samples were loaded directly onto a disposable precolumn (Spheri-5 cyano; Brownlee Labs.) which replaced the sample loop of an Altex 210 injection valve. After the precolumn had been placed in the solvent stream, peptides were eluted at a flow-rate of 2 ml/min with a gradient from solvent A [(0.2o/o, v/v) T F A in water, pH 2.1] to solvent B [(0.31~,,, v/v) T F A in acetonitrile] in 3 linear segments (0.0"i, to 31.0°;, B over 5 rain. 31.0°i,
217
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Fig. 1. Release of SOM-LI from hypothalamic slices and from the isolated ME in vitro. Hypothalamic slices (a, b) or M E s (c) were perifused with modified Krebs-bicarbonate medium, and SOM-LI released was measured by RIA as described in the text: Tissues were exposed to a depolarizing pulse of KCI (50 m M ) for the periods indicated, a: hypothalamic slices, Mg z+ substituted for Ca 2+ in the medium throughout the experiment, b: hypothalamic slices, normal Krebs medium, c: MEs, Mg 2+ substituted for Ca 2+ for the period indicated. Results are expressed as percentage total tissue SOM-LI released per minute, based on measurements of the tissue content at the end of the perifusion (mean + S.E.M. for 4 experiments).
to 32.2~o B over 12 min, 32.2~o to 100~ B over 15 min). This chromatographic system has been shown to resolve SOM-LI in hypothalamic extracts into 3 components corresponding to SOM-14, SOM-28 and a composite peak of high-molecular-weight SOM (HMW-SOM) containing 6000 and 10,000 mol.wt, species of SOM-LI [18]. Perifusates of hypothalamic slices contained two molecular forms of SOM-LI corresponding to SOM-14 and SOM-28 (Fig. 2a-c). 76~ of the SOM-LI released during a pulse of K ÷ depolarization corresponded to SOM-14, and SOM-28 represented a further 17~o. K + depolarization increased by 12-fold the rate of release of SOM-14, but did not significantly influence the rate of release of SOM-28. In contrast, perifusates of isolated MEs contained SOM-14, SOM-28 and HMW-SOM (Fig. 2d-f) and K + depolarization increased the rates of release of the 3 peptides by 4.3-, 5.5- and 1.6-fold, respectively. The proportions of SOM-14, SOM-28 and HMW-SOM in the perifusate during K÷-depolarization were 58~, 25~o and 17~o. In experiments to investigate the possible degradation of SOM-28 and SOM-14 by hypothalamic slices, 20 ng of each peptide was added to the superfusion medium and passed through a perifusion chamber containing slices derived from 8 hypothalami. Analysis of the perifusates by HPLC followed by RIA indicated that the recoveries of both peptides were in excess of 80~o. The techniques described above provide a powerful method for the quantitation of SOM-LI released from nervous tissue in vitro and for the analysis of the multiple forms of the peptide released. Concentration and desalting of tissue perifusates on ODS columns enabled us to determine basal and stimulated levels of SOM release at concentrations falling well within the limits of sensitivity of our RIA. The use of
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Fig. 2. HPLC of SOM-LI released from (a-c) hypothalamic slices and (d-f) MEs in vitro. Perifusate samples from 8 hypothalami or 60 MEs were collected for 9-min periods before (a, d), during (b, e) and after (c, f) exposure of tissues to a depolarizing pulse of KCI. HPLC analysis and RIA were performed as described in the text. Results are expressed as femtomoles SOM-LI per milliliter of HPLC eluate; the elution positions of synthetic SOM-28 and SOM-14, and of HMW-SOM, are indicated.
a cartridge precolumn for the concentration of the SOM present in large volumes of tissue perifusates permits HPLC analysis to be performed without elaborate sample preparation. These methods may be of general application in the quantitation of neuropeptide release in vitro. We have confirmed that the K +-evoked release of SOM from hypothalamic slices and from the isolated ME is Ca2+-dependent. The predominant form of SOM-LI released from hypothalamic slices was SOM-14. Although a small amount of SOM28 was released, the rate of release of peptide was not increased by K +-depolarization. Perifusates of ME tissue contained a strikingly different pattern of SOM-LI from perifusates of the whole hypothalamus; in addition to SOM-14, significant amounts of SOM-28 and HMW-SOM were present, and depolarization increased the rates of release of all 3 forms of SOM-LI. Evidence is now accumulating to suggest that, in different tissues, prosomatostatin may be processed to generate different patterns of biologically active products. In most regions, SOM-14 is the predominant end-product of biosynthesis, but in some tissues SOM-28 is synthesized in comparable or greater amounts.The submucosa and muscle layer of the small intestine contain almost exclusively SOM-14 of neuronal origin, whereas in the endocrine cells of the mucosa SOM-28 predominates [2, 17]. SOM-14 is the most abundant form of SOM in the hypothalamus, but the ME [18] and neurohypophysis [9] contain almost equimolar amounts of SOM-14 and SOM-
219 28. SOM-14 a n d SOM-28 m a y play separate roles in the regulation of endocrine function; SOM-28 is more p o t e n t t h a n SOM-14 in i n h i b i t i n g the secretion o f G H from the pituitary gland [4] a n d is secreted into hypophysial portal b l o o d in vivo [16]. In the pancreas, SOM-28 inhibits insulin release with a p o t e n c y some 10 times greater t h a n that o f SOM-14 [15], whereas SOM-14 is more p o t e n t in the i n h i b i t i o n of glucagon release. There is evidence that pituitary S O M receptors m a y be of a type exhibiting a selective affinity for SOM-28, whereas those in the rest of the h y p o t h a l a m u s are more sensitive to SOM-14 [20]. These results provide supportive evidence for the existence o f two p o p u l a t i o n s of S O M - c o n t a i n i n g n e u r o n s in the h y p o t h a l a m u s , already suggested by n e u r o a n a t o m i cal [14] a n d ontogenic [6] studies. One p o p u l a t i o n , with cell bodies in the rostral periventricular area which send projections to the ME, releases both SOM-14 a n d S O M 28 as h o r m o n a l regulators of pituitary G H secretion. The second p o p u l a t i o n consists o f i n t e r n e u r o n s in the arcuate, ventromedial a n d other nuclei, which release SOM- 14. We t h a n k Professor G. F i n k for advice a n d e n c o u r a g e m e n t a n d N o r m a Brearley for the careful p r e p a r a t i o n o f the manuscript. A.R.P. a n d L.T. are M R C Research Students. 1 Arbuthnott, G.W., Harmar, A.J., Pierotti, A.R. and Tannahill, L., Release of two molecular forms of somatostatin from the rat hypothalamus in vitro, J. Physiol. (Lond.), 349 (1984) 34P. 2 Baskin, D.G. and Ensinck, J.W., Somatostatin in epithelial cells of intestinal mucosa is present primarily as somatostatin 28, Peptides, 5 (1984) 615-621. 3 Benoit, R., Ling, N., Alford, B. and Guillemin, R., Seven peptides derived from pro-somatostatin in rat brain, Biochem. Biophys. Res. Commun., 107 (1982) 944-950. 4 Brazeau, P., Ling, N., Esch, F., Bohlen, P., Benoit R. and Guillemin, R., High biological activity of the synthetic replicates of somatostatin-28 and somatostatin-25, Regulat. Pept., 1 (1981) 255-264. 5 Brazeau, P., Vale, W., Burgus, R., Ling, N., Butcher, M., River, J. and Guillemin, R., Hypothalamic polypeptide that inhibits the secretion of immunoreactive pituitary growth hormone, Science, 179 (1973) 77-79. 6 Daikoku, S., Hisano, S., Kawano, H., Okamura, Y. and Tsuruo, Y., Ontogenetic studies on the topographical heterogeneity of somatostatin-containing neurones in rat hypothalamus, Cell Tiss. Res., 233 (1983) 347-354. 7 Esch, F., Bohlen, P., Ling, N., Benoit, R., Brazeau, P. and Guillemin, R., Primary structure of ovine hypothalamic somatostatin-28 and somatostatin-25, Proc. Natl. Acad. Sci. USA, 77 (1980) 6827-6831. 8 Glowinski, J. and Iversen, L.L., Regional studies of the distribution of catecholamines in rat brain. I. The distribution of 3H-norepinephrine,3H-dopamineand 3H-DOPA in various regions of the brain, J. Neurochem., 13 (1966) 655-699. 9 Gomez, S.,.Morel, A., Nicolas, P. and Cohen, P., Regional distribution of the Mr 15,000 somatostatin precursor somatostatin-28 and somatostatin-14 in the rat brain suggests a differential intracellular processing of the high molecular weight species, Biochem. Biophys. Res. Commun., 112 (1983) 297-305. I0 Goodman, R.H., Aron, D.C. and Roos, B.A., Rat prepro-somatostatin, structure and processing by microsomal membranes, J. Biol. Chem., 258 (1983) 5570-5573. 11 Harmar, A.J. and Pierotti, A.R., The pattern of molecular forms of somatostatin released by the rat median eminence differs from that released by the hypothalamus as a whole, J. Physiol. (Lond.), 357 (1984) 95P. 12 Iversen, L.L., Iversen, S.D., Bloom, F., Douglas, C., Brown, M. and Vale, W., Calcium-dependent release of somatostatin and neurotensin from rat brain in vitro, Nature (Lond.), 273 (1978) 161-163. 13 Jessell, T.M., Iversen, L.L. and Kanazawa, I., Release and metabolism of substance P in rat hypothalamus, Nature (Lond.), 264 (1976) 81-83.
220 14 Kawano, H., Diakoku, S. and Sailo, S., Immunohislochemical studies of inlrahypoikalamic ~;olnalo stalin-containing neurones in rat, Brain Res., 242 ~ 1982) 227 232. 15 Mandarino, L., Stenner. D., Blanchard, W., Nissem S., (icrich, J., Ling, N., Brazcam P., Bohlen, P., Esch, F., and Guillemin, R., Selective effects o1" somalostatin-14, 25, 28 on m viiro insulin and glucagon secretion, Nature (Lond.), 291 119811 76 77. 16 Millar, R.P., Sheward, W.J., Wegener, I. and Fink, G., Somalostalin-28 is a hormonally aciivc pepiidc released into hypophysial portal vessel blood, Brain Res., 260 (1983) 334 337. 17 Penman, E., Wass, J.A.H., Butler, M.G., Penny, E.S., Price, J., W m P. and Rees, L.H., Distribution and characterisaiion of immunoreactive somalostalin in h u m a n gastrointestinal tract, Regulat. Pept. 7(1983) 53 65. 18 Pierotti, A,R. and Harmar, A.J., Multiple forms of somatostatin-like immunoreactivity in the hypothalamus and amygdala of the rat: selective Iocalisalion of somatostatin-28 in the median eminence, J. Endocrinol., in press. 19 Pradayrol, L., Jornvall, H., Mutt, V. and Ribet, A., N-Terminally exlended somatostatin: the primary structure of somatostalin-28, FEBS Lett., 109 (1980) 55 58. 20 Srikant, C.B. and Patel, Y.C., Receptor binding of somatostatin-28 is lissue specific, Nature (Lond.), 294(1981) 259 260.
215
Elsevier Scientific Publishers Ireland Ltd. NSL 03356
DIFFERENT P A T T E R N S OF M O L E C U L A R F O R M S OF S O M A T O S T A T I N ARE R E L E A S E D BY THE RAT M E D I A N E M I N E N C E AND HYPOTHALAMUS*
ADRIAN R. PIEROTTI, ANTHONY J. HARMAR**, LESLEY A. TANNAHILL and GORDON W. ARBUTHNOTT MRC Brain Metabolism Unit, University Department of Pharmacology, 1 George Square, Edinburgh EH8 9JZ (U.K.)
(Received February 19th, 1985; Revised version received and accepted March 15th, 1985)
Key words" somatostatin-14 - somatostatin-28 - hypothalamus - median eminence - release - high-per-
formance liquid chromatography - rat
The molecular forms of somatostatin (SOM) released from hypothalamic slices and from the isolated median eminence (ME) in vitro were compared by high-performance liquid chromatography and radioimmunoassay. SOM-14 was the predominant form of the peptide released from hypothalamic slices, although small amounts of SOM-28 were detected. Perifusates of ME tissue contained a larger proportion of SOM-28 and higher molecular weight peptides were present; depolarization increased the rates of release of all molecular forms of SOM. These results suggest that the capacity to release SOM-28 and high-molecular-weight forms of SOM may be a specialized function of nerve terminals in the ME.
S o m a t o s t a t i n - l i k e i m m u n o r e a c t i v i t y ( S O M - L I ) has been shown to exist in tissues in several m o l e c u l a r forms, all o f which are derived f r o m a c o m m o n p o l y p e p t i d e prec u r s o r ( p r o s o m a t o s t a t i n , mol.wt. 10,400) [10]. T h e first o f these forms to be described was SOM-14, a t e t r a d e c a p e p t i d e m e d i a t i n g the h y p o t h a l a m i c c o n t r o l o f g r o w t h horm o n e ( G H ) secretion [5]. SOM-28, an o c t a c o s a p e p t i d e c o n t a i n i n g the sequence o f S O M - 1 4 with an N - t e r m i n a l extension o f 14 a m i n o acids [7, 19], is m o r e p o t e n t t h a n - S O M - 1 4 in i n h i b i t n g the release o f insulin from the p a n c r e a s [15] a n d the secretion o f G H f r o m the a n t e r i o r p i t u i t a r y g l a n d [4], a n d m a y act selectively at a subclass o f S O M receptors with a higher affinity for S O M - 2 8 t h a n for S O M - 1 4 [20]. T w o forms o f S O M - L I with higher m o l e c u l a r weights (6000 a n d 8000-10,000) have also been described in extracts o f n e r v o u s tissue [3]; the a m i n o acid sequences a n d possible biological functions o f these larger peptides have yet to be established. The m e d i a n eminence ( M E ) , which c o n t a i n s S O M - 1 4 a n d S O M - 2 8 in a l m o s t e q u i m o l a r concent r a t i o n s [18], differs f r o m the rest o f the h y p o t h a l a m u s , in which S O M - 1 4 p r e d o m i n a t e s . A l t h o u g h the c a l c i u m - d e p e n d e n t , p o t a s s i u m - e v o k e d release o f S O M *Some of the observations reported here have been published in preliminary form [1, 11]. **Author for correspondence. 0304-3940,'85/$ 03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd.
216 from hypothalamic slices has been demonstrated [12], there is little information concerning the molecular forms of the peptide released. Accordingly, we have used highperformance liquid chromatography (HPLC) to compare the molecular forms of SOM released from hypothalamic slices with those released from the isolated ME in vitro. Male COB Wistar rats (180-230 g body wt.) were killed by decapitation and the brains removed. Hypothalamic tissue was dissected by the procedure of Glowinski and Iversen [8] and sliced in the sagittal plane at 250/~m intervals using a McIIwain tissue chopper. ME tissue was removed from the intact brain by dissection with iridectomy scissors. Hypothalamic slices or MEs were placed in a modified Krebsbicarbonate medium (identical to that described by Iversen et al. [12] except that Trasylol, 400 Kallikrein inactivator units/ml, was added) and transferred to small plastic chambers [13]. Tissues were perifused at 37'~C with Krebs-bicarbonate medium at a flow-rate of 0.3 ml/min. After an initial wash period of 30 rain, samples were collected for 3-min periods into plastic tubes containing 0.5 ml aqueous trifluoroacetic acid (TFA: 3"~, v/v). Total SOM-LI released was determined after extraction of samples on disposable columns of octadecylsilica (Baker-10 ODS, 1 ml; J.T. Baker). Columns were preconditioned by washing with 2 ml of T F A - m e t h a n o l (l~,, v/v) followed by 2 ml of aqueous T F A (1",,;, v/v). After application of the sample, columns were washed with 1 ml aqueous T F A and peptides eluted with I ml of methanolTFA. Samples were evaporated to dryness in a vacuum oven prior to determination of SOM-LI by radioimmunoassay (RIA) [18]. The antiserum used (final dilution 1:750,000) was directed towards the central or C-terminal region of the SOM-14 sequence; SOM-14 and SOM-28 were equally active on a molar basis in displacing tracer from the antiserum whereas 13 other neuropeptides were inactive. [1251]Tyr°SOM-14 was used as tracer and the limit of sensitivity of the assay was 2.2 fmol SOM-14 per tube. In normal Krebs medium, SOM-LI was released from hypothalamic slices with a rate constant of less than 0.02',!Jo/min (Fig. lb). Addition of 50-raM KCI to the medium caused an immediate increase of 15-20-fold in the rate of release of SOM-LI. Basal release of SOM-LI was diminished, and K ' - e v o k e d release dramatically reduced, when Mg 2+ was substituted for Ca 2+ in the perifusion medium (Fig. la). The basal rate of release of SOM-LI from the isolated ME was 0.005°J~,/min, and depolarization with 50 mM KCI stimulated the release of SOM-LI by 6.9-fold (Fig. l c). K ~-evoked release was abolished in calcium-free medium. To characterize the pattern of molecular forms of SOM-LI released from the hypothalamus and ME, perifusate samples from 8 hypothalami or 60 MEs were combined. Chromatographic analysis was performed using an Altex Model 421 H P L C system with an analytical column of Ultrasphere 5 ~m ODS (Altex). Samples were loaded directly onto a disposable precolumn (Spheri-5 cyano; Brownlee Labs.) which replaced the sample loop of an Altex 210 injection valve. After the precolumn had been placed in the solvent stream, peptides were eluted at a flow-rate of 2 ml/min with a gradient from solvent A [(0.2o/o, v/v) T F A in water, pH 2.1] to solvent B [(0.31~,,, v/v) T F A in acetonitrile] in 3 linear segments (0.0"i, to 31.0°;, B over 5 rain. 31.0°i,
217
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Fig. 1. Release of SOM-LI from hypothalamic slices and from the isolated ME in vitro. Hypothalamic slices (a, b) or M E s (c) were perifused with modified Krebs-bicarbonate medium, and SOM-LI released was measured by RIA as described in the text: Tissues were exposed to a depolarizing pulse of KCI (50 m M ) for the periods indicated, a: hypothalamic slices, Mg z+ substituted for Ca 2+ in the medium throughout the experiment, b: hypothalamic slices, normal Krebs medium, c: MEs, Mg 2+ substituted for Ca 2+ for the period indicated. Results are expressed as percentage total tissue SOM-LI released per minute, based on measurements of the tissue content at the end of the perifusion (mean + S.E.M. for 4 experiments).
to 32.2~o B over 12 min, 32.2~o to 100~ B over 15 min). This chromatographic system has been shown to resolve SOM-LI in hypothalamic extracts into 3 components corresponding to SOM-14, SOM-28 and a composite peak of high-molecular-weight SOM (HMW-SOM) containing 6000 and 10,000 mol.wt, species of SOM-LI [18]. Perifusates of hypothalamic slices contained two molecular forms of SOM-LI corresponding to SOM-14 and SOM-28 (Fig. 2a-c). 76~ of the SOM-LI released during a pulse of K ÷ depolarization corresponded to SOM-14, and SOM-28 represented a further 17~o. K + depolarization increased by 12-fold the rate of release of SOM-14, but did not significantly influence the rate of release of SOM-28. In contrast, perifusates of isolated MEs contained SOM-14, SOM-28 and HMW-SOM (Fig. 2d-f) and K + depolarization increased the rates of release of the 3 peptides by 4.3-, 5.5- and 1.6-fold, respectively. The proportions of SOM-14, SOM-28 and HMW-SOM in the perifusate during K÷-depolarization were 58~, 25~o and 17~o. In experiments to investigate the possible degradation of SOM-28 and SOM-14 by hypothalamic slices, 20 ng of each peptide was added to the superfusion medium and passed through a perifusion chamber containing slices derived from 8 hypothalami. Analysis of the perifusates by HPLC followed by RIA indicated that the recoveries of both peptides were in excess of 80~o. The techniques described above provide a powerful method for the quantitation of SOM-LI released from nervous tissue in vitro and for the analysis of the multiple forms of the peptide released. Concentration and desalting of tissue perifusates on ODS columns enabled us to determine basal and stimulated levels of SOM release at concentrations falling well within the limits of sensitivity of our RIA. The use of
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Fig. 2. HPLC of SOM-LI released from (a-c) hypothalamic slices and (d-f) MEs in vitro. Perifusate samples from 8 hypothalami or 60 MEs were collected for 9-min periods before (a, d), during (b, e) and after (c, f) exposure of tissues to a depolarizing pulse of KCI. HPLC analysis and RIA were performed as described in the text. Results are expressed as femtomoles SOM-LI per milliliter of HPLC eluate; the elution positions of synthetic SOM-28 and SOM-14, and of HMW-SOM, are indicated.
a cartridge precolumn for the concentration of the SOM present in large volumes of tissue perifusates permits HPLC analysis to be performed without elaborate sample preparation. These methods may be of general application in the quantitation of neuropeptide release in vitro. We have confirmed that the K +-evoked release of SOM from hypothalamic slices and from the isolated ME is Ca2+-dependent. The predominant form of SOM-LI released from hypothalamic slices was SOM-14. Although a small amount of SOM28 was released, the rate of release of peptide was not increased by K +-depolarization. Perifusates of ME tissue contained a strikingly different pattern of SOM-LI from perifusates of the whole hypothalamus; in addition to SOM-14, significant amounts of SOM-28 and HMW-SOM were present, and depolarization increased the rates of release of all 3 forms of SOM-LI. Evidence is now accumulating to suggest that, in different tissues, prosomatostatin may be processed to generate different patterns of biologically active products. In most regions, SOM-14 is the predominant end-product of biosynthesis, but in some tissues SOM-28 is synthesized in comparable or greater amounts.The submucosa and muscle layer of the small intestine contain almost exclusively SOM-14 of neuronal origin, whereas in the endocrine cells of the mucosa SOM-28 predominates [2, 17]. SOM-14 is the most abundant form of SOM in the hypothalamus, but the ME [18] and neurohypophysis [9] contain almost equimolar amounts of SOM-14 and SOM-
219 28. SOM-14 a n d SOM-28 m a y play separate roles in the regulation of endocrine function; SOM-28 is more p o t e n t t h a n SOM-14 in i n h i b i t i n g the secretion o f G H from the pituitary gland [4] a n d is secreted into hypophysial portal b l o o d in vivo [16]. In the pancreas, SOM-28 inhibits insulin release with a p o t e n c y some 10 times greater t h a n that o f SOM-14 [15], whereas SOM-14 is more p o t e n t in the i n h i b i t i o n of glucagon release. There is evidence that pituitary S O M receptors m a y be of a type exhibiting a selective affinity for SOM-28, whereas those in the rest of the h y p o t h a l a m u s are more sensitive to SOM-14 [20]. These results provide supportive evidence for the existence o f two p o p u l a t i o n s of S O M - c o n t a i n i n g n e u r o n s in the h y p o t h a l a m u s , already suggested by n e u r o a n a t o m i cal [14] a n d ontogenic [6] studies. One p o p u l a t i o n , with cell bodies in the rostral periventricular area which send projections to the ME, releases both SOM-14 a n d S O M 28 as h o r m o n a l regulators of pituitary G H secretion. The second p o p u l a t i o n consists o f i n t e r n e u r o n s in the arcuate, ventromedial a n d other nuclei, which release SOM- 14. We t h a n k Professor G. F i n k for advice a n d e n c o u r a g e m e n t a n d N o r m a Brearley for the careful p r e p a r a t i o n o f the manuscript. A.R.P. a n d L.T. are M R C Research Students. 1 Arbuthnott, G.W., Harmar, A.J., Pierotti, A.R. and Tannahill, L., Release of two molecular forms of somatostatin from the rat hypothalamus in vitro, J. Physiol. (Lond.), 349 (1984) 34P. 2 Baskin, D.G. and Ensinck, J.W., Somatostatin in epithelial cells of intestinal mucosa is present primarily as somatostatin 28, Peptides, 5 (1984) 615-621. 3 Benoit, R., Ling, N., Alford, B. and Guillemin, R., Seven peptides derived from pro-somatostatin in rat brain, Biochem. Biophys. Res. Commun., 107 (1982) 944-950. 4 Brazeau, P., Ling, N., Esch, F., Bohlen, P., Benoit R. and Guillemin, R., High biological activity of the synthetic replicates of somatostatin-28 and somatostatin-25, Regulat. Pept., 1 (1981) 255-264. 5 Brazeau, P., Vale, W., Burgus, R., Ling, N., Butcher, M., River, J. and Guillemin, R., Hypothalamic polypeptide that inhibits the secretion of immunoreactive pituitary growth hormone, Science, 179 (1973) 77-79. 6 Daikoku, S., Hisano, S., Kawano, H., Okamura, Y. and Tsuruo, Y., Ontogenetic studies on the topographical heterogeneity of somatostatin-containing neurones in rat hypothalamus, Cell Tiss. Res., 233 (1983) 347-354. 7 Esch, F., Bohlen, P., Ling, N., Benoit, R., Brazeau, P. and Guillemin, R., Primary structure of ovine hypothalamic somatostatin-28 and somatostatin-25, Proc. Natl. Acad. Sci. USA, 77 (1980) 6827-6831. 8 Glowinski, J. and Iversen, L.L., Regional studies of the distribution of catecholamines in rat brain. I. The distribution of 3H-norepinephrine,3H-dopamineand 3H-DOPA in various regions of the brain, J. Neurochem., 13 (1966) 655-699. 9 Gomez, S.,.Morel, A., Nicolas, P. and Cohen, P., Regional distribution of the Mr 15,000 somatostatin precursor somatostatin-28 and somatostatin-14 in the rat brain suggests a differential intracellular processing of the high molecular weight species, Biochem. Biophys. Res. Commun., 112 (1983) 297-305. I0 Goodman, R.H., Aron, D.C. and Roos, B.A., Rat prepro-somatostatin, structure and processing by microsomal membranes, J. Biol. Chem., 258 (1983) 5570-5573. 11 Harmar, A.J. and Pierotti, A.R., The pattern of molecular forms of somatostatin released by the rat median eminence differs from that released by the hypothalamus as a whole, J. Physiol. (Lond.), 357 (1984) 95P. 12 Iversen, L.L., Iversen, S.D., Bloom, F., Douglas, C., Brown, M. and Vale, W., Calcium-dependent release of somatostatin and neurotensin from rat brain in vitro, Nature (Lond.), 273 (1978) 161-163. 13 Jessell, T.M., Iversen, L.L. and Kanazawa, I., Release and metabolism of substance P in rat hypothalamus, Nature (Lond.), 264 (1976) 81-83.
220 14 Kawano, H., Diakoku, S. and Sailo, S., Immunohislochemical studies of inlrahypoikalamic ~;olnalo stalin-containing neurones in rat, Brain Res., 242 ~ 1982) 227 232. 15 Mandarino, L., Stenner. D., Blanchard, W., Nissem S., (icrich, J., Ling, N., Brazcam P., Bohlen, P., Esch, F., and Guillemin, R., Selective effects o1" somalostatin-14, 25, 28 on m viiro insulin and glucagon secretion, Nature (Lond.), 291 119811 76 77. 16 Millar, R.P., Sheward, W.J., Wegener, I. and Fink, G., Somalostalin-28 is a hormonally aciivc pepiidc released into hypophysial portal vessel blood, Brain Res., 260 (1983) 334 337. 17 Penman, E., Wass, J.A.H., Butler, M.G., Penny, E.S., Price, J., W m P. and Rees, L.H., Distribution and characterisaiion of immunoreactive somalostalin in h u m a n gastrointestinal tract, Regulat. Pept. 7(1983) 53 65. 18 Pierotti, A,R. and Harmar, A.J., Multiple forms of somatostatin-like immunoreactivity in the hypothalamus and amygdala of the rat: selective Iocalisalion of somatostatin-28 in the median eminence, J. Endocrinol., in press. 19 Pradayrol, L., Jornvall, H., Mutt, V. and Ribet, A., N-Terminally exlended somatostatin: the primary structure of somatostalin-28, FEBS Lett., 109 (1980) 55 58. 20 Srikant, C.B. and Patel, Y.C., Receptor binding of somatostatin-28 is lissue specific, Nature (Lond.), 294(1981) 259 260.