Somatostatin Acts Via a Pertussis Toxin-Sensitive Mechanism on Calcitonin Secretion in C-Cells

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Henry Ford Hospital Medical Journal

Volume 40 Number 3 Article 35

9-1992

Angela Zink

Hans Scherubl

Friedhelm Raue

Reinhard Ziegler

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Recommended Citation Zink, Angela; Scherubl, Hans; Raue, Friedhelm; and Ziegler, Reinhard (1992) "Somatostatin Acts Via a Pertussis Toxin-Sensitive Mechanism on Calcitonin Secretion in C-Cells," Henry Ford Hospital Medical Journal : Vol. 40 : No. 3 , 289-292. Available at: https://scholarlycommons.henryford.com/hfhmedjournal/vol40/iss3/35

This Article is brought to you for free and open access by Henry Ford Health System Scholarly Commons. It has been accepted for inclusion in Henry Ford Hospital Medical Journal by an authorized editor of Henry Ford Health System Scholarly Commons. Somatostatin Acts Via a Pertussis Toxin-Sensitive Mechanism on Calcitonin Secretion in C-Cells

Angela Zink,* Hans Scherubl,^ Friedhelm Raue,* and Reinhard Ziegler'

The effect ofthe somatostatin analog octreotide on cAMP-mediated calcitonin (CT) secretion and cAMP accumulation in C-cells was investigated. Glucagon stimulated cAMP accumulation and CT secretion with a maximal eff'ecl at a concentration oflQ-^M. The cAMP antagonist RpcAMPs blocked the glucagon-induced CT secretion down to control levels. Therefore, no other second messengers seem to he involved in glucagon-stimulated CT secretion. Octreotide in increasing doses (70'' to 10'^ M) inhibited cAMP accumulation and CT secretion with a maximal effect at a concentration of 10'^ (40% and 29% of control values, respectively). Pretreatment ofthe cells with 100 ng/mL pertussis toxin for 24 hours abolished the inhibitory effect of octreotide on cAMP accumulation and CT secretion (82% and 58% of control values, respectively). Similar results were obtained under the inftuence ofthe phosphodiesterase inhibitor IBMX. Therefore, we conclude that somatostatin modu lates adenylate cyclase-coupled CT secretion in C-cells via a pertussis toxin-sensitive G-protein possibly in an autocrine/paracrine way. (Henry Ford Hosp MedJ 1992:40:289-92)

omatostatin is a physiologically important inhibitor of Determination of cAMP S growth hormone release and is also known to inhibit Confluent cells on 35 mm dishes were washed twice with hormone secretion in a variety of extrapituitary tissues includ PBS-buffer and further incubated with medium containing test ing brain, thyroid, pancreas, and gut (1,2). Concerning C-cells in agents or vehicle alone at 37 °C. After 15 minutes, medium was vivo, somatostatin inhibits calcitonin (CT) secretion (3,4), but removed, cells were washed twice with PBS-buffer, and denatu- the physiological role of somatostatin and the mechanism of ac rated with ice-cold ethanol (100% pH 3). After 2 hours at 4 °C, tion in CT-secreting cells is still unclear. the supematant was evaporated at 37 °C under Nj and the result Hormone secretion by C-cells is mainly regulated through ing pellet was resuspended in the cAMP-assay buffer. cAMP changes in the extracellular calcium concentration via voltage- was determined by competitive protein binding assay as re dependent calcium channels, but various peptides such as glu ported previously (8). Total cell protein was determined by the cagon or growth hormone-releasing peptide influence CT secre method of Bradford (9). tion in C-cells via the adenylate cyclase pathway (5). It has been shown that in pituitary eeds somatostatin acts independently on Secretion experiments both second messenger systems, the cAMP-mediated and the To determine CT secretion, confluent cells on replicate 35 calcium-mediated pathway (6). In C-cells, the effect of somato mm dishes were preincubated with serum-free DMEM for 2 statin on adenylate cyclase-mediated CT secretion is still un hours. Subsequently, ceUs were washed twice with PBS-buffer clear. Therefore, we studied the effect of somatostatin on the and further incubated with medium containing test agents or ve glucagon-stimulated CT secretion in an established rat medul hicle alone. After 2 hours, medium from each dish was collected lary carcinoma cell line (rMTC 6-23) (7) in order to elucidate the and stored at -20 °C until assayed. CT was measured by radio role of somatostatin on cAMP-mediated CT secretion and to immunoassay. Total cell protein was determined after die study the possible involvement of a pertussis toxin-sensitive method of Bradford (9). Viability as tested by trypan blue exclu mechanism in cAMP-mediated CT secretion. sion was > 90% in each experiment.

Methods Cell culture rMTC 6-23 cells were purchased from the American Type Submitted for publication: Oclober 14, I99I. Culture Collection and grown as monolayers in Dulbecco's Accepted for publication: November 27, 1991. *Department of Intemal Medicine 1, Endocrinology and Metabolism, University of Hei modified Eagle's medium (DMEM) supplemented with 15% delberg, Heidelberg. Germany, horse seram in a humidified atmosphere with 5% COj and 95% -j-Pharmakologischesln.stitul derFreien Universitat Berlin, Beriin, Germany. Address correspondence to Dr. Zink, Departnient of Intemal Medicine 1, Endocrinology, air. Luisenstrabe 5, D-6900 Heidelberg, Germany.

Henry Ford Hosp Med 1—Vol 40, Nos 3 & 4, 1992 Effect of Somatostatin on CT Secretion in C-cells—Zink et al 289 Table Effect ofthe cAMP Antagonist RpcAMPs on 600 Glucagon-induced Calcitonin Secretion in rMTC Cells* ^ 500 Calcitonin Secretion 400 (pg CT/mg Protein/2 hrs)

300 - Control 60 + 4.8 £ Glucagon (10-'' M) 124 ±9 a 200 RpcAMPs (1 O't M) 59 ±7 < Glucagon (IQ-^ M) 4- RpcAMPs (10"* M) 61 ±9 100 *Points show mean ± SEM of four representative experiments.

300

and reached levels 300% and 150% over control levels for

OJ cAMP and CT secretion, respectively. The cAMP antagonist

^ 200 RpcAMPs blocked the glucagon-induced CT secretion down to 9) control values (Table). Increasing doses of octreotide (10"^ to O 10"* M) inhibited the glucagon-induced cAMP accumulation o. and CT secretion maximally to 40% and 29%, respectively, by a concentration of 10"^ M octreotide (Fig 2). Similar results were 100 I- seen under the influence of the phosphodiesterase inhibitor 2 IBMX (10'"* M, data not shown). Fig 3 shows the effect of pre treatment of the cells for 24 hours with pertussis toxin. This pre treatment partially blocked the inhibitory effect of octreotide on cAMP accumulation and CT secretion to 82% and 58% of con Glucagon 6 -lg(M) trol values, respectively (Fig 3). A maximal effect of pertussis toxin was reached by a concentration of 100 ng/mL. Similar re sults were obtained under the influence of IBMX (10M, data Fig 1—Effect of increasing doses of glucagon on cAMP accu not shown). mulation (upper panel) and CT secretion (lower panel) in rMTC cells. Cells were grown and experiments performed as de scrihed in the text. Points represent mean ± SEM of four repre Discussion sentative experiments. The glucagon-induced CT secretion in our experiments paral leled the glucagon-induced cAMP accumulation, which is con sistent with previous findings (5). Generation of other second Materials messengers such as IP3, which influences intracellular calcium DMEM and trypsin/EDTA were obtained from Biochrom by glucagon, is unlikely in C-cells, as it has been shown eartier (Berlin, Germany) and L-glutamine, HEPES-buffer, PBS- that glucagon had no ef fect on intracellular calcium concentra buffer, and horse seram from Gibco (Paisley, UK). Glucagon tion in C-cells measured with fura-2-loaded cells (10). Further and pertussis toxin were purchased by Sigma (Deisenhofen, more, addition of the cAMP antagonist RpcAMPS completely Germany). The cyclic somatostatin analog octreotide was a gift suppressed glucagon-stimulated CT secretion. Therefore, we from Sandoz (Nuraberg, Germany). Plastic tissue culture ware considered activation of adenylate cyclase the sole signal- was purchased from Falcon (Los Angeles, CA). transducing pathway mediating the glucagon-induced CT secre tion in our cell system and used it as a model to study the effect of somatostatin on cAMP-mediated CT secretion. Statistics Somatostatin is able to inhibit cAMP accumulation as well as Data are represented as means ± SEM. Statistical analysis was cAMP-mediated CT-secretion in C-cells. This is consistent with performed using the computer program STATVIEW (Abacus findings in pituitary or adrenal glomerulosa cells, where so Concepts, Inc., Berkeley, CA). Statistical significance was as matostatin inhibits cAMP-mediated hormone secretion in a sim sessed by Wilcoxon rank sum test or the Mann-Whitney- ilar way (11,12). In the rMTC 6-23 cells as well as in parafolli Wilcoxon test; P < 0.05 was considered significant. cular and GH-pituitary cells, the inhibitory effect of somato statin was not complete (13-15). The reason for this is unclear. Results Binding studies with different somatostatin analogs revealed Glucagon stimulated cAMP accumulation and CT secretion similar or even higher binding affinities or biological effects of in rMTC 6-23 cells in a dose-dependent manner as shown in Fig the analogs in pituitary cells (16). Thus stmctural differences are 1. Maximal stimulation occurred at a concentration of 10"^ M not likely to be the reason for this partial inhibitory effect. As C-

290 Henry Ford Hosp Med J—Vol 40, Nos 3 & 4, 1992 Effeci of Somatostatin on CT Secretion in C-cell.s—Zink et al = 100

c •fii o a1— o> E

< o a

200

c 1 •fij o o u. 100 o> £ E la: o Gl Cl Glucagon 0 7 -lg(M) Ilili Octreotide 0 9 -lg(M) Glucagon 07707707 7 -lg(M) Octreotide 00700700 7 -lg(M) Fig 2—Effect of increasing doses of octreotide on glucagon- PT 0 0 0 10 10 10 100 100 100 ng/ml stimulated cAMP accumulation (upper panel) and CT secretion (lower panel) in rMTC cells. Cells were grown and experiments Fig 3—Effect of diff'ereni doses of pertussis toxin on the inhib performed as described in the text. Points show mean ± SEM of itory effect of octreotide on glucagon-stimulated cAMP accu four representative experiments. mulation and CT secretion in rMTC cells. Cells were pretreated with pertussis toxin for 24 hours before stimulation was per formed. For details, see the text. Points show mean ± SEM of cells have been shown to secrete somatostatin (17,18), an auto four representative experiments. crine or paracrine desensitization of the cells might explain the incomplete effect of somatostatin. A similar desensitization by somatostatin has been described in pituitary cells (19). The octreotide-induced inhibition of cAMP accumulation as Acknowledgment well as the inhibition of CT secretion could be prevented by pre This work was supported by a grant of the DFG, Ra 327/1-4. treatment of the cells with pertussis toxin. This indicates that a pertussis toxin-sensitive mechanism is involved in the cAMP- mediated CT secretion in C-cells. As the effect of pertussis toxin References occurred with or without IBMX, it is not due to changes in deg 1. Reichiin S. Somatostatin. N Engl J Med I983;309:1495-501,1556-63. radation of cAMP. More likely, octreotide acts via somatostatin 2. Patel YC, Srikant CB, Somatostatin mediation of adenohypophysial secre receptors which are coupled to adenylate cyclase via inhibitory tion. Annu Rev Physiol 1986;48:552-67. pertussis toxin-sensitive G-proteins, as reported in other cell 3. Linehan WM, Cooper CW, Bolman RM III, Wells SA Jr. Inhibition of in systems (20,21). vivo secretion of calcitonin in the pig by somatostatin. Endocrinology 1979; 104:1602-7. The physiological role of the somatostatin effect on C-ceds 4. Laurberg P. Somatostatin and calcitonin release from perfused dog thyroid remains unclear. In pituitary eeds an effect of somatostatin on lobes: Studies with calcium and pentaga.strin. Endocrinology 1984; 114:2234- Ca+"^- and voltage-gated K*-channels has been described (22), 41. and there is evidence that somatostatin may influence secretory 5. Raue F, Zink A, Scherubl H. Regulation of calcitonin secretion and calci tonin gene expression. In: Medullary thyroid carcinoma. Recent Res Cancer Res processes via more distal events (23). In C-cells, somatostatin 1992;125:1-18. has been shown to inhibit calcium-induced CT secretion via G- 6. Schonbrunn A. Somatostatin action in pituitary cells involves two proteins direcdy coupled to the voltage-gated calcium channels independent transduction mechanisms. Metabolism 1990;39(suppl 2):96- (24) and chronic exposure of C-cells to somatostatin inhibits 100. proliferation of the cells (Mekonnen Y, et al, personal communi 7. Gagel RF, Zeytinoglu FN, Voelkel EF, Tashjian AH Jr Establishment of a calcitonin-producing rat medullary carcinoma cell line, II. Secretory studies of cation, 1991). Together with the finding that somatostatin itself the tun-iorand cells in culture. Endocrinology 1980;107:516-23. is secreted by C-cells, a modulatory role of somatostatin on pro 8. Armbruster FP. Scharfenstein H. Hitzler W. Schmidt-Gayk H. cAMP-proliferation and CT secretion in an autocrine or paracrine way tein-bindungs-assay: Proteinabhangigkeit der cAMP-bindung. Arztl Lab 1986; seems to be evident. 32:115-20.

Henry Ford Hosp Med J—Vol 40. Nos 3 & 4. 1992 Effect of Somatostatin on CT Secretion in C-cells—Zink el al 291 9. Bradford MM. A rapid sensitive method for the quantitation of microgram 17. Aron DC, Muszynski M, Birnbaum RS, Sabo SW, Roos BA. Somatostatin quantities of protein utilizing the principle of protein-dye binding. Anal Bio elaboration by monolayer cell cultures derived from transplantable rat medullary chem 1976;72:248-54. thyroid carcinoma: Synergistic stimulatory effects of glucagon and calcium. En 10. Eckert RW, Scherubl H, Petzelt C, Raue F, Ziegler R, Rhythmic oscilla docrinology 1981;109:1830-4. tions of cytosolic free calcium in rat C-cells. Mol Cell Endocrinol 1989;64:267- 18. Gagel RF, Palmer WN, Leonhart K, Chan L, Leong SS, Somatostatin pro 70. duction by a human medullary thyroid carcinoma cell line. Endocrinology 11. Reyl-Desmars F. Zeytin F. Somatostatin inhibits growth-hormone-releas 1986;118:1643-51, ing factor-stimulated adenylate cyclase activity in GH cells. Biochim Biophys 19. Smith MA. Yamamoto G, Vale WW, Somatostatin desensitization in rat Res Commun 1985;127:986-91. anterior pituitary cells. Mol Cell Endocrinol 1984;37:311-8. 12. Hausdorff WP. AguileraG, Catt KJ. Inhibitory actions of somatostatin on 20. Koch BD, Schonbrunn A. The somatostatin receptor is directly coupled to cyclic AMP and aldosterone production in agonist-stimulated adrenal glomeru adenylate cyclase in GH^C, pituitary cell membranes. Endocrinology 1984; losa cells. Cell Signal 1989;l;377-86. 114:1784-90. 13. Dorflinger LJ, Schonbrunn A. Somatostatin inhibits basal and vasoactive 21. Bimbaumer L. Abramowitz J. Brown AM. Receptor-effector coupling by intestinal peptide-stimulated hormone release by different mechanisms in GH G proteins. Biochim Biophys Acta I990;1031;163-224. pituitary cells. Endocrinology 1983; 113:1551-8. 22. White RE. Schonbrunn A. Armstrong DL. Somatostatin stimulates Ca^-^- 14. Dorflinger LJ. Schonbrunn A. Somatostatin inhibits vasoactive intestinal activated K* channels through protein depho.sphorylation. Nature 1991:351: peptide-.stimulated cyclic adenosine monophosphate accumulation in GH pitu 570-3. itary cells. Endocrinology 1983;113:1541-50. 23. Wollheim CB, Winiger BP, Ullrich S, Wuarin F, Schlegel W, So 15. Endo T, Saito T, Uchida T, Onaya T. Effects of somatostatin and serotonin matostatin inhibition of hormone release: Effects on cytosolic Ca-^-^ and on calcitonin secretion from cultured rat parafollicular cells. Acta Endocrinol interference with distal secretory events. Metabolism 1990;39(suppl): 101- 1988;117:214-8. 4. 16. Schonbrunn A, Rorstad OP, Westendorf JM, Martin JB, Somatostatin an 24. Scherubl H, Hescheler J, Schultz G, et al. Inhibition of Ca--^-induced calci alogs: Correlation between receptor binding affinity and biological potency in tonin secretion by somatostatin: Roles of voltage dependent Ca--^ channels and GH pituitary cells. Endocrinology 1983;113:1559-67. G-proleins, Cell Signal 1992;4:77-85,

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