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Calcium-dependence of somatostatin binding to receptors
Peptides, Vol. 6, pp. 831-833, 1985. © A n k h o International Inc. Printed in the U.S.A.
0196-9781/85 $3.00 + .00
Calcium-Dependence of Somatostatin Binding to Receptors C H . S U S I N I , J. P. ESTEVE, N. V A Y S S E A N D A . R I B E T
l n s e r m U 151, C H U Rangueil Ls, 31054 Toulouse Cedex, France R e c e i v e d 8 F e b r u a r y 1985 CH. SUSINI, J. P. ESTEVE, N. VAYSSE AND A. RIBET. Calcium-dependence ofsomatostatin binding to receptors. PEPTIDES 6(5)831-833, 1985.--Binding of 12~I-[Tyr"]somatostatin (S14) and '25I-[TyrqS14 has been studied in pancreatic acini and cerebral cortex. Ca2+-dependence of somatostatin binding to receptors was observed only with the highly non degradable iodinated analog '~I-[Tyr' q somatostatin but not with 125I-[Tyrqsomatostatin. The inhibitory activity of S 14 on secretin-stimulated cAMP cellular content was decreased when Ca 2+ concentration in the medium was reduced to 30 nM, indicating that the Ca 2+ dependence of somatostatin binding to receptors is also present with the native peptide. Somatostatin receptors
Pancreatic membranes
Cerebro-cortical membranes
WE have previously reported that Ca .'+ regulates the binding of 12S[Tyrlt] somatostatin (S14) to intact cells [7] and membranes [9] from guinea pig pancreatic acini. Such a Ca .'+ dependence of somatostatin binding to receptors, for Ca .'+ concentrations lower than 0.1 raM, has not been described by others [1, 2, 4, 5, 8]. The purpose of this study was to define in which conditions the effect of Ca .'+ on somatostatin binding is observable. We compared the binding of two different ligands '25I[TyrqS14 and ~SI[Tyrn]S14 to membranes from pancreatic acini at different free Ca 2+ concentrations. In order to examine whether the effect of Ca .'+ is a more general phenomenon, we have also studied the effect of Ca .'+ on the binding of these two ligands to membranes from cerebral cortex. In pancreatic acinar cells, somatostatin is known to inhibit the formation of secretin-stimulated cellular c A M P [6]. Thus, we also investigated the possibility that Ca .'+ might affect the biological activity of somatostatin by studying the ability of the peptide to reduce secretinstimulated cellular c A M P levels in low Ca .'+ medium.
Cyclic AMP
in triethylammonium phosphate (V/V), pH 3.0. The recovery was 93-98% of total injected radioactivity. ~25I-[Tyr"]S14 was eluted as a main peak (95% of total radioactivity) in fractions 30-36. A small amount of radioactivity appeared in fractions 8-10 (<5%), representing iodinated tyrosine [6]. The same findings were observed with '25I-[TyrqS 14. Cyclic A M P cellular content was measured on intact icini incubated with 5 mM theophylline as described [6]. RESULTS
Influence of Ca 2+ on Somatostatin Binding to Membranes (Fig. 1) The same pattern of time course of binding was observed both with pancreatic membranes and cortical membranes (Fig. 1 A and B). The binding of ~25I-[Tyrn]S14 to membranes incubated in standard medium (0.2 mM Ca ~+) reached equilibrium at 90 rain. In standard medium plus 0.6 mM EGTA (30 nM free CaZ+), the amount of specifically bound ligand was strongly decreased, reached a plateau value at 30 rain and remained constant up to 90 rain. When membranes were incubated at 37°C with ~25I-[TyrqS14 the amount of specifically bound ligand was not depending on free-Ca 2+ concentration. The specific binding reached a maximal value at 20-30 min, lower than that observed with lzSI-Tyr~qS14 and then decreased. In pancreatic membranes this decrease was faster than in cerebrocortical preparations. Non specific binding was similar in the two preparations and was higher with '25I-[Tyr~]S14 (30% of total binding) than with lZSI[Tyr~qS14 (10% of total binding). Some experiments with ~25I-[TyrqS 14 were performed at 25°C. The rate of association was slower with steady-state binding being reached at 70 min; maximum binding was slightly increased and the period of equilibrium was prolonged up to 140 min (not shown). Relative potency of unlabeled somatostatin in inhibiting the binding of the two differently labeled somatostatin molecules to pancreatic membranes were; EC50=1.2_0.2 nM with 125I-[Tyrl]S14; EC50=0.5-+0.3 nM with 12~I[ T y r ' ] S 1 4 (not shown). The same correlation between free
METHOD
Radioiodinated ligands were prepared by high pressure liquid chromatography (H.P.L.C.) [3]. Membranes from cerebral cortex and plasma membranes from guinea-pig pancreatic acini were prepared as described [5,9]. F o r binding assays, membranes were suspended in Tris 50 mM medium supplemented with 0.2% albumin, 0.2 mM calcium, 0.30 mg/ml soybean trypsin inhibitor, I mg/ml bacitracin in a total volume of 150-p.l and incubated with 0.08 nM iodinated somatostatin. Binding procedure has been previously described [9]. Binding data were specific binding defined as the difference between total binding and binding observed in the presence of 1/~M S 14. Free Ca 2+ concentration in the incubation medium was maintained by Ca.'+/EGTA buffers and calculated by computer simulation [9]. Ligand degradation in the incubation medium was evaluated by HPLC. The column was eluted with 25% (l.'5I-[Tyr~]S 14) or 27% (~.'5I-[Tyr']S14) acetonitrile
831
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time (rnin] free calcium concentration (IogM) FIG. 1. Influence of calcium on somatostatin binding. (A,B): Time-course of binding of m I - [ T y r q S l 4 ( & , & ) or nsI[ T y r " ] S l 4 ( © , e ) to membranes from pancreatic acini or cerebral cortex at 37°C, in 0.2 m M Ca ~+ medium ( & , e ) , or in 30 nM Ca 2+ medium (/\,©). (C): Effect of free Ca 2+ concentration on binding of '2'~]-[Tyr t qS 14 to membranes from pancreatic
acini or cerebral cortex. In each experiment each value was determined in duplicate, and results given are representative of at least 3 separate experiments.
TABLE 1 HIGH PRESSURELIQUID CHROMATOGRAPHICANALYSISOF DEGRADATIONOF RADIOIODINATEDSOMATOSTATININ INCUBATIONMEDIUMAT VARIOUSTIMES OF INCUBATIONAND TEMPERATURES:PERCENTAGEOF RADIOACTIVITYELUTING IN FRACTIONS30-36 (INTACTLIGAND) 12'~I-[Tyr]qS 14
l~I-[Tyr']S 14
37°C Incubation period Pancreatic membranes Cortical membranes
20 min 95-+ 3 94 _+ 4
37°C
70 min 92_+ 3 93 _+ 2
20 min 51 + 4 54 _+ 10
70 min 27_+ 5 43 _+ 10
25°C 20 min 55-+ 8 81 _+ 8
70 min 42_+ 5 60 _+ 6
Results are means -+ SE of 3 separate experiments.
Ca 2+ medium concentration and the amount of specific binding of 12'~I-[TyrV]S14 was found with membranes from pancreas or from cerebral cortex (Fig. 1 C).
Stability of ~2'~l-Labeled Somatostatin Analogs During Incubation with Membranes (Table 1) When t2'~I-[Tyr']S14 was incubated with membranes, the same elution profile as that of standard was obtained, up to 70 min incubation. When ~25I-[TyrqS14 was incubated with membranes, the percentage of total radioactivity eluting in fractions 30-36 (intact ligand) decreased strongly especially at 70 min and in supernatants from pancreatic membranes. The remainder was mostly found in fractions 8-10 (iodotyrosine). Decreasing the temperature of incubation, with 125I-[Tyr!]S14,10wered the amount of degradation but in this condition, longer period of incubation was required to reach the equilibrium. Therefore, the rate of degradation at the equilibrium at 25°C (70 min) was the same as that found at 37°C, when the equilibrium was reached (20 min). The presence of E G T A in the incubation medium did not modify the extent of degradation of the ligands (not shown).
Influence of Low Ca 2+Medium on the Biological Activity of Somatostatin (Fig. 2) In acini incubated in 30 nM Ca 2+ medium, 1 nM secretin caused a 24_+3.9 fold increase in cellular cAMP content which was the same as the 23.3_+3.3 fold increase measured in acini incubated in 0.2 mM Ca 2+ medium. Nevertheless, lowering extracellular Ca 2+ produced an impairment in the ability of somatostatin to inhibit secretin-stimulated cellular c A M P content. The inhibition caused by S14 in secretinstimulated cellular cAMP was significantly' greater when acini were incubated with 0.2 mM Ca 2+ than with 30 nM Ca 2+ up to 10-9 M somatostatin. DISCUSSION The present study demonstrates that the Ca 2+ dependence of somatostatin binding previously observed with pancreatic membranes [9] and intact acini [7] is only found with ~25I[Tyr"]S14 but not with '25I-[TyrqS14. In pancreatic membranes, Ca 2+ concentration was found to regulate both the number of binding sites and the receptor affinity for somatostatin [9].
SOMATOSTATIN BINDING
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sornatostatin c ~ t i o n [Iogl~ Fig. 2. Effect of-c~ciu-m-on--somatostatin inhibition of secretinstimulated cellular cAMP in pancreatic acini. Acini were incubated with 1 nM secretin plus different concentrations of S14 in 0.2 mM Ca2+ (0) or 30 nM Ca2÷ (,t) medium. Values are expressed as percentages of increase with secretin alone. Results given are means -+SE for duplicate determinations in 4 separate experiments. *Significantly lower (.0<0.05) than values with secretin alone by Student's test for paired data.
Our parallel use of '25I-[Tyr~]S14 and r'5I-[Tyr~qS14 in studies of time-course of the binding in 0.2 mM Ca 2÷ showed also striking differences in the rate of reaching steady-state
binding and the amount of bound radioactivity. Concentration of native peptide required to decrease the binding by 50% was slightly different. No attempt was made to analyze further other differences in binding constants such as maximal binding capacity or dissociation rates, since high non specific binding and extensive degradation of l.'5I-[Tyrl]S14 prevent accurate determination of these constants with this probe. Therefore, it could not be decided whether the observed differences might be due to selectivity in the binding of the radiolabeled peptides to different somatostatin receptor populations related to structural differences among the two peptides. Nevertheless the high extent of degradation observed with ~.'5I-[TyrqS14 might account at least in part for these differences. This study also provides evidence that 1."5I-[TyrH]S14 binding to cerebro-cortical membranes is Ca .'+ dependent, indicating that Ca .'+ dependence might be a more general phenomenon. In addition, our finding that the inhibitory activity of somatostatin on secretin-stimulated cAMP cellular content was decreased in 30 nM Ca 2÷ medium suggests that the Ca .'÷ dependence of somatostatin binding is also present with the native peptide and is not conferred by iodination of Tyr ~l. It also indicates that somatostatin binding sites are receptors that mediate the action of somatostatin on cAMP accumulation.
ACKNOWLEDGEMENTS The authors wish to thank Dr. G. Krejs, Dallas, TX and Dr. A. Estival, Toulouse, France for carefully reading their manuscript.
REFERENCES 1. Aguilera, G. and D. S. Parker. Pituitary somatostatin receptors. J Biol Chem 257:1134-1137, 1982. 2. Aguilera, G., D. S. Parker and K. J. Catt. Characterization of somatostatin receptors in the rat adrenal glomerulosa zone. Endocrinology 111: 1376--1384, 1982. 3. Antoniotti, H., D. Fourmy, J. P. Esteve, P. Fagot-Revurat, L. Pradayrol and A. Ribet. Purification and radioiodinated somatostatin derivatives by RP-HPLC. J Chromatogr 296: 181-188, 1984. 4. Czernik, A. J. and B. Petrack. Somatostatin receptor binding in rat cerebral cortex. J Biol Chem 5525-5530, 1983. 5. Epelbaum, J., L. Tapia-Arancibia, C. Kordon and A. Enjalbert. Characterization, regional distribution, and subcellular distribution of J~I-TyrLsomatostatin binding sites in rat brain. J Neurochern 38: 1515-1523, 1982.
258:
6. Esteve, J. P., N. Vaysse, C. Susini, J. M. Kunsch, D. Fourmy, L. Pradayrol, E. Wiinsch, E. Moroder and A. Ribet. Bimodal regulation of pancreatic exocrine function in vitro by somatostatin-28. Am J Physiol 245: G208-G216, 1983. 7. Esteve, J. P., C. Susini, N. Vaysse, H. Antoniotti, E. Wfinsch, G. Berthon and A. Ribet. Binding of somatostatin to pancreatic acinar cells. Am J Physiol 247: G62-G69, 1984. 8. Srikant, C. B. and Y. C. Patel. Somatostatin receptors: identification and characterization in rat brain membranes. Proc Natl Acad Sci USA 78: 3930-3934, 1981. 9. Taparel, D., J. P. Esteve, C. Susini, N. Vaysse, D. Balas, G. Berthon, E. Wiinsch and A. Ribet. Binding of somatostatin to guinea pig pancreatic membranes: regulation by ions. Biochem Biophys Res Commun 115: 827-833, 1983.
0196-9781/85 $3.00 + .00
Calcium-Dependence of Somatostatin Binding to Receptors C H . S U S I N I , J. P. ESTEVE, N. V A Y S S E A N D A . R I B E T
l n s e r m U 151, C H U Rangueil Ls, 31054 Toulouse Cedex, France R e c e i v e d 8 F e b r u a r y 1985 CH. SUSINI, J. P. ESTEVE, N. VAYSSE AND A. RIBET. Calcium-dependence ofsomatostatin binding to receptors. PEPTIDES 6(5)831-833, 1985.--Binding of 12~I-[Tyr"]somatostatin (S14) and '25I-[TyrqS14 has been studied in pancreatic acini and cerebral cortex. Ca2+-dependence of somatostatin binding to receptors was observed only with the highly non degradable iodinated analog '~I-[Tyr' q somatostatin but not with 125I-[Tyrqsomatostatin. The inhibitory activity of S 14 on secretin-stimulated cAMP cellular content was decreased when Ca 2+ concentration in the medium was reduced to 30 nM, indicating that the Ca 2+ dependence of somatostatin binding to receptors is also present with the native peptide. Somatostatin receptors
Pancreatic membranes
Cerebro-cortical membranes
WE have previously reported that Ca .'+ regulates the binding of 12S[Tyrlt] somatostatin (S14) to intact cells [7] and membranes [9] from guinea pig pancreatic acini. Such a Ca .'+ dependence of somatostatin binding to receptors, for Ca .'+ concentrations lower than 0.1 raM, has not been described by others [1, 2, 4, 5, 8]. The purpose of this study was to define in which conditions the effect of Ca .'+ on somatostatin binding is observable. We compared the binding of two different ligands '25I[TyrqS14 and ~SI[Tyrn]S14 to membranes from pancreatic acini at different free Ca 2+ concentrations. In order to examine whether the effect of Ca .'+ is a more general phenomenon, we have also studied the effect of Ca .'+ on the binding of these two ligands to membranes from cerebral cortex. In pancreatic acinar cells, somatostatin is known to inhibit the formation of secretin-stimulated cellular c A M P [6]. Thus, we also investigated the possibility that Ca .'+ might affect the biological activity of somatostatin by studying the ability of the peptide to reduce secretinstimulated cellular c A M P levels in low Ca .'+ medium.
Cyclic AMP
in triethylammonium phosphate (V/V), pH 3.0. The recovery was 93-98% of total injected radioactivity. ~25I-[Tyr"]S14 was eluted as a main peak (95% of total radioactivity) in fractions 30-36. A small amount of radioactivity appeared in fractions 8-10 (<5%), representing iodinated tyrosine [6]. The same findings were observed with '25I-[TyrqS 14. Cyclic A M P cellular content was measured on intact icini incubated with 5 mM theophylline as described [6]. RESULTS
Influence of Ca 2+ on Somatostatin Binding to Membranes (Fig. 1) The same pattern of time course of binding was observed both with pancreatic membranes and cortical membranes (Fig. 1 A and B). The binding of ~25I-[Tyrn]S14 to membranes incubated in standard medium (0.2 mM Ca ~+) reached equilibrium at 90 rain. In standard medium plus 0.6 mM EGTA (30 nM free CaZ+), the amount of specifically bound ligand was strongly decreased, reached a plateau value at 30 rain and remained constant up to 90 rain. When membranes were incubated at 37°C with ~25I-[TyrqS14 the amount of specifically bound ligand was not depending on free-Ca 2+ concentration. The specific binding reached a maximal value at 20-30 min, lower than that observed with lzSI-Tyr~qS14 and then decreased. In pancreatic membranes this decrease was faster than in cerebrocortical preparations. Non specific binding was similar in the two preparations and was higher with '25I-[Tyr~]S14 (30% of total binding) than with lZSI[Tyr~qS14 (10% of total binding). Some experiments with ~25I-[TyrqS 14 were performed at 25°C. The rate of association was slower with steady-state binding being reached at 70 min; maximum binding was slightly increased and the period of equilibrium was prolonged up to 140 min (not shown). Relative potency of unlabeled somatostatin in inhibiting the binding of the two differently labeled somatostatin molecules to pancreatic membranes were; EC50=1.2_0.2 nM with 125I-[Tyrl]S14; EC50=0.5-+0.3 nM with 12~I[ T y r ' ] S 1 4 (not shown). The same correlation between free
METHOD
Radioiodinated ligands were prepared by high pressure liquid chromatography (H.P.L.C.) [3]. Membranes from cerebral cortex and plasma membranes from guinea-pig pancreatic acini were prepared as described [5,9]. F o r binding assays, membranes were suspended in Tris 50 mM medium supplemented with 0.2% albumin, 0.2 mM calcium, 0.30 mg/ml soybean trypsin inhibitor, I mg/ml bacitracin in a total volume of 150-p.l and incubated with 0.08 nM iodinated somatostatin. Binding procedure has been previously described [9]. Binding data were specific binding defined as the difference between total binding and binding observed in the presence of 1/~M S 14. Free Ca 2+ concentration in the incubation medium was maintained by Ca.'+/EGTA buffers and calculated by computer simulation [9]. Ligand degradation in the incubation medium was evaluated by HPLC. The column was eluted with 25% (l.'5I-[Tyr~]S 14) or 27% (~.'5I-[Tyr']S14) acetonitrile
831
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SUSINI, ESTEVE, VAYSSE AND RIBET
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time (rnin] free calcium concentration (IogM) FIG. 1. Influence of calcium on somatostatin binding. (A,B): Time-course of binding of m I - [ T y r q S l 4 ( & , & ) or nsI[ T y r " ] S l 4 ( © , e ) to membranes from pancreatic acini or cerebral cortex at 37°C, in 0.2 m M Ca ~+ medium ( & , e ) , or in 30 nM Ca 2+ medium (/\,©). (C): Effect of free Ca 2+ concentration on binding of '2'~]-[Tyr t qS 14 to membranes from pancreatic
acini or cerebral cortex. In each experiment each value was determined in duplicate, and results given are representative of at least 3 separate experiments.
TABLE 1 HIGH PRESSURELIQUID CHROMATOGRAPHICANALYSISOF DEGRADATIONOF RADIOIODINATEDSOMATOSTATININ INCUBATIONMEDIUMAT VARIOUSTIMES OF INCUBATIONAND TEMPERATURES:PERCENTAGEOF RADIOACTIVITYELUTING IN FRACTIONS30-36 (INTACTLIGAND) 12'~I-[Tyr]qS 14
l~I-[Tyr']S 14
37°C Incubation period Pancreatic membranes Cortical membranes
20 min 95-+ 3 94 _+ 4
37°C
70 min 92_+ 3 93 _+ 2
20 min 51 + 4 54 _+ 10
70 min 27_+ 5 43 _+ 10
25°C 20 min 55-+ 8 81 _+ 8
70 min 42_+ 5 60 _+ 6
Results are means -+ SE of 3 separate experiments.
Ca 2+ medium concentration and the amount of specific binding of 12'~I-[TyrV]S14 was found with membranes from pancreas or from cerebral cortex (Fig. 1 C).
Stability of ~2'~l-Labeled Somatostatin Analogs During Incubation with Membranes (Table 1) When t2'~I-[Tyr']S14 was incubated with membranes, the same elution profile as that of standard was obtained, up to 70 min incubation. When ~25I-[TyrqS14 was incubated with membranes, the percentage of total radioactivity eluting in fractions 30-36 (intact ligand) decreased strongly especially at 70 min and in supernatants from pancreatic membranes. The remainder was mostly found in fractions 8-10 (iodotyrosine). Decreasing the temperature of incubation, with 125I-[Tyr!]S14,10wered the amount of degradation but in this condition, longer period of incubation was required to reach the equilibrium. Therefore, the rate of degradation at the equilibrium at 25°C (70 min) was the same as that found at 37°C, when the equilibrium was reached (20 min). The presence of E G T A in the incubation medium did not modify the extent of degradation of the ligands (not shown).
Influence of Low Ca 2+Medium on the Biological Activity of Somatostatin (Fig. 2) In acini incubated in 30 nM Ca 2+ medium, 1 nM secretin caused a 24_+3.9 fold increase in cellular cAMP content which was the same as the 23.3_+3.3 fold increase measured in acini incubated in 0.2 mM Ca 2+ medium. Nevertheless, lowering extracellular Ca 2+ produced an impairment in the ability of somatostatin to inhibit secretin-stimulated cellular c A M P content. The inhibition caused by S14 in secretinstimulated cellular cAMP was significantly' greater when acini were incubated with 0.2 mM Ca 2+ than with 30 nM Ca 2+ up to 10-9 M somatostatin. DISCUSSION The present study demonstrates that the Ca 2+ dependence of somatostatin binding previously observed with pancreatic membranes [9] and intact acini [7] is only found with ~25I[Tyr"]S14 but not with '25I-[TyrqS14. In pancreatic membranes, Ca 2+ concentration was found to regulate both the number of binding sites and the receptor affinity for somatostatin [9].
SOMATOSTATIN BINDING
833
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,
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,
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sornatostatin c ~ t i o n [Iogl~ Fig. 2. Effect of-c~ciu-m-on--somatostatin inhibition of secretinstimulated cellular cAMP in pancreatic acini. Acini were incubated with 1 nM secretin plus different concentrations of S14 in 0.2 mM Ca2+ (0) or 30 nM Ca2÷ (,t) medium. Values are expressed as percentages of increase with secretin alone. Results given are means -+SE for duplicate determinations in 4 separate experiments. *Significantly lower (.0<0.05) than values with secretin alone by Student's test for paired data.
Our parallel use of '25I-[Tyr~]S14 and r'5I-[Tyr~qS14 in studies of time-course of the binding in 0.2 mM Ca 2÷ showed also striking differences in the rate of reaching steady-state
binding and the amount of bound radioactivity. Concentration of native peptide required to decrease the binding by 50% was slightly different. No attempt was made to analyze further other differences in binding constants such as maximal binding capacity or dissociation rates, since high non specific binding and extensive degradation of l.'5I-[Tyrl]S14 prevent accurate determination of these constants with this probe. Therefore, it could not be decided whether the observed differences might be due to selectivity in the binding of the radiolabeled peptides to different somatostatin receptor populations related to structural differences among the two peptides. Nevertheless the high extent of degradation observed with ~.'5I-[TyrqS14 might account at least in part for these differences. This study also provides evidence that 1."5I-[TyrH]S14 binding to cerebro-cortical membranes is Ca .'+ dependent, indicating that Ca .'+ dependence might be a more general phenomenon. In addition, our finding that the inhibitory activity of somatostatin on secretin-stimulated cAMP cellular content was decreased in 30 nM Ca 2÷ medium suggests that the Ca .'÷ dependence of somatostatin binding is also present with the native peptide and is not conferred by iodination of Tyr ~l. It also indicates that somatostatin binding sites are receptors that mediate the action of somatostatin on cAMP accumulation.
ACKNOWLEDGEMENTS The authors wish to thank Dr. G. Krejs, Dallas, TX and Dr. A. Estival, Toulouse, France for carefully reading their manuscript.
REFERENCES 1. Aguilera, G. and D. S. Parker. Pituitary somatostatin receptors. J Biol Chem 257:1134-1137, 1982. 2. Aguilera, G., D. S. Parker and K. J. Catt. Characterization of somatostatin receptors in the rat adrenal glomerulosa zone. Endocrinology 111: 1376--1384, 1982. 3. Antoniotti, H., D. Fourmy, J. P. Esteve, P. Fagot-Revurat, L. Pradayrol and A. Ribet. Purification and radioiodinated somatostatin derivatives by RP-HPLC. J Chromatogr 296: 181-188, 1984. 4. Czernik, A. J. and B. Petrack. Somatostatin receptor binding in rat cerebral cortex. J Biol Chem 5525-5530, 1983. 5. Epelbaum, J., L. Tapia-Arancibia, C. Kordon and A. Enjalbert. Characterization, regional distribution, and subcellular distribution of J~I-TyrLsomatostatin binding sites in rat brain. J Neurochern 38: 1515-1523, 1982.
258:
6. Esteve, J. P., N. Vaysse, C. Susini, J. M. Kunsch, D. Fourmy, L. Pradayrol, E. Wiinsch, E. Moroder and A. Ribet. Bimodal regulation of pancreatic exocrine function in vitro by somatostatin-28. Am J Physiol 245: G208-G216, 1983. 7. Esteve, J. P., C. Susini, N. Vaysse, H. Antoniotti, E. Wfinsch, G. Berthon and A. Ribet. Binding of somatostatin to pancreatic acinar cells. Am J Physiol 247: G62-G69, 1984. 8. Srikant, C. B. and Y. C. Patel. Somatostatin receptors: identification and characterization in rat brain membranes. Proc Natl Acad Sci USA 78: 3930-3934, 1981. 9. Taparel, D., J. P. Esteve, C. Susini, N. Vaysse, D. Balas, G. Berthon, E. Wiinsch and A. Ribet. Binding of somatostatin to guinea pig pancreatic membranes: regulation by ions. Biochem Biophys Res Commun 115: 827-833, 1983.