Molecular und Cellular Endocrinology, 0 1992 Elsevier Scientific Publishers

MOLCEL

213

86 (1992) 213-219 Ireland, Ltd. 0303.7207/92/$05.00

02800

Inhibitory effect of somatostatin on CAMP accumulation and calcitonin secretion in C-cells: involvement of pertussis toxin-sensitive G-proteins A. Zink ‘, H. Scheriibl ‘, D. Kliemann a, M. Hijflich ‘, R. Ziegler a and F. Raw ” and ’ Abteilung

” Abteilung Innere Medizin I, ~In~i~ers~t~tHeidelberg, D-6900 ~~eideiberg, Germany, Innere Medizi~ - ~ch~~erpunkt Gastroe~teroiogie, Unir.er.~it~tskli,likum Stegiitz, FU Berlin, Beriirz 45, Germany (Received

Kq words: Calcitonin

secretion;

C-cell: Pertussis

28 February

1992; accepted

toxin; Somatostatin;

29 April 1992)

Adenylate

cyclase;

Cytosolic

calcium;

G-protein

Summary The effect of somatostatin on CAMP accumulation and calcitonin secretion in C-cells of the rat medullary thyroid carcinoma cell line rMTC 6-23 was investigated. Intracellular CAMP accumulation as well as calcitonin secretion could be dose-dependently stimulated by rat growth hormone releasing factor (rGRF). The long-acting somatostatin analogue octreotide inhibited rGRF-stimulated CAMP accumulation and calcitonin secretion dose dependently but failed to block 8-bromo-CAMP-stimulated calcitonin secretion. The inhibitory effect of octreotide on rGRF-induced calcitonin secretion was partially abolished by pretreating the cells with pertussis toxin. The octreotide effect was not due to changes in the degradation of CAMP, as it was similarily seen in the presence of isobutylmethylxanthine. Thus we conclude that pertussis toxin-sensitive G-proteins are involved in the CAMP-mediated regulation of calcitonin secretion in C-cells.

Introduction Somatostatin inhibits hormone secretion from a variety of tissues including various neuroendocrine cells. In pituitary and islet cells of the pancreas two mechanisms of somatostatin action on hormone secretion have been proposed (Dorflinger and Schonbrunn, 1983; Wollheim et al., 1990): first, inhibition of adenylate cyclase, which lowers cellular CAMP levels, and second, interference with intracellular calcium ([Ca2’],>. Increases in CAMP levels and/or [Ca’+], are criti-

Correspondence to: Prof. F. Raue, Department of Internal Medicine I, University of Heidelberg. Luisenstrasse 5, 6900 Heidelberg, Germany. Tel. 06221-568613; Fax 06221-563101.

cal triggering events in the secretion of many hormones and neurotransmitters. In both mechanisms of transmembrane signalling of somatostatin, G-proteins are involved (Pate1 et al., 1990). In the first instance the somatostatin receptor is negatively coupled to adenylate cyclase via a pertussis toxin (PT)-sensitive G,-protein (Birnbaumer et al., 1990). In the second instance interference of somatostatin with [Ca2+li either occurs through a subset of K+ channels, which are directly coupled to the receptor via a PT-sensitive G, (Yatani et al., 19901, or through a direct effect of somatostatin receptors on voltage-dependent calcium channels (Dolphin et al., 1991). Here again there is evidence for the involvement of PT-sensitive G-proteins (Rosenthal et al., 1990; Scheriibl et al., 1992).

214

Calcitonin (CT) secretion by C-cells is mainly regulated through changes in the extracellular calcium concentration. These changes are mediated through corresponding changes in [Ca*‘], through voltage-dependent calcium channels (Hishikawa et al., 1985; Raue et al., 1989; Scheriibl et al., 1990). Recently it has been shown that somatostatin inhibits calcium-induced CT secretion by reducing calcium influx in C-cells (Scheriibl et al., 1991). These effects were due to a direct effect of somatostatin on voltage-dependent calcium channels and were mediated through PT-sensitive G-proteins independent of changes in intracellular CAMP. Neurotransmitters such as norepinephrine and gastrointestinal hormones such as glucagon and growth hormone-releasing factor also stimulate CT secretion in C-cells (Zeytin and Brazeau, 1984; Scheriibl et al., 1989; Raue et al., 1991). These substances are known to activate the CAMP second messenger pathway but recent reports suggest that norepinephrine and growth hormone-releasing factor additionally lead to an increase of ICaZili in C-cells (Fried and Tashijan, 1987). Details about signal transduction in response to norepinephrine or growth hormone-releasing factor are not well known so far. Therefore we used the established rat medullary carcinoma cell line rMTC 6-23 (Gage1 et al., 1980) to characterize the signal transduction pathway of rat growth hormone-releasing factor (rGRF) in rMTC cells, to study the influence of the long-acting somatostatin analogue octreotide on CAMP-mediated CT secretion, and to investigate the possible invoIvement of PT-sensitive Gproteins in the signal transduction of rGRF. Materials

further incubated in medium with or without the phosphodiesterase inhibitor isobu~lmethylxanthine (IBMX, lo-” M) and containing test agents or vehicle alone at 37°C. After 15 min medium was removed, cells were washed twice with PBS and then denaturated with ice-cold ethanol (loo%, pH 3). After 2 h at 4°C supernatant was evaporated at 37°C under N, and the resulting pellet was resuspended in CAMP assay buffer. CAMP was determined by competitive protein binding assay as reported previously (Armbruster et al., 1986). Total cell protein was determined by the method of Bradford (Bradford et al., 1976). Secretion experiments To determine CT secretion confluent cells on replicate 35 mm dishes were preincubated with serum-free DMEM for 2 h. Subsequently, cells were washed twice with PBS and further incubated with medium containing test agents or ve-

and methods

Cell culture Rat MTC 6-23 cells (rMTC 6-23) were purchased from the American Type Culture Collection. The cells were grown as monolayers in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 15% horse serum in a humidified atmosphere with 5% CO, and 95% air. Determination of CAMP Confluent cells on 35 mm dishes were washed twice with phosphate-buffered saline (PBS) and

&RF

0

10

9

8

7

6

-MM)

Fig. 1. Stimulation of adenylate cyclase and calcitonin (CT) secretion by increasing doses of rGRF. Upper panel shows the accumulation of CAMP, lower pane! secretion. For experimental details see Materials and methods. The mean of six representative experiments + SD is shown. * p < 0.05 was considered significant.

21.5

bovine serum albumin and 20 mM Hepes (pH 7.4). Fluorescence was measured as previously described using a microspectrofluorimeter (Nobiling et al., 1989; Eckert et al., 1989). Fura 2 signals were taken from single cells which were alternately excited at 340 and 380 nm wavelengths. Fluorescence trackings corresponding to the appropriate wavelength were measured by a photomultiplier and simultaneously digitally recorded.

hicle alone. After 2 h, medium from each dish was collected and kept at -20°C until assayed. CT was measured by radioimmunoassay. Again total cell protein was determined by the method of Bradford (Bradford et al., 1976). Cell viability as tested by trypan blue exclusion was > 90% in each experiment.

Cytosolic calcium measurements

rMTC 6-23 cells were loaded with the calcium indicator fura 2-AM at 37°C for 30 min in PBS. Then the PBS/furs 2-AM solution was replaced by a buffer containing 0.5 mM CaCl,, 137 mM NaCI, 5.6 mM KCI, 0.8 mM Na,HPO,, 1 mM MgSO,, 5.6 mM glucose, 10 FM EGTA, 1 mg/ml

Statistics

Data are represented as means f SEM. Statistical analysis was performed using the computer program STATVIEW (Abacus Concepts, Berkeley, CA, USA). Statistical significance was as-

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0 1 0

t

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Ca

I

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I 6U Time

I 120

(set)

Fig. 2. Effect of &RF and calcium on the intracellular calcium concentration in rat C-cells. A single cell was loaded with furaand excited with 340 and 380 nm wavelength as described in Materials and methods. A representative experiment out of six is shown. The arrow indicates the addition of rGRF (lo-’ MI and/or calcium (3 mM>.

216

sessed by the Wilcoxon Mann-Whitney-Wilcoxon sidered significant.

rank sum test or the test; p < 0.05 was con-

Materials DMEM and trypsin/EDTA were obtained from Biochrom (Berlin, Germany), r_-glutamine, Hepes, PBS and horse serum from Gibco (Pailsey, UK). 8-Bromo-CAMP, rGRF, IBMX and PT were purchased from Sigma (Deisenhofen, FRG), the CAMP antagonist RpcAMPs from Biologic Life Science Institute (Bremen). The cyclic somatostatin analogue octreotide was a gift from Sandoz, Niirnberg, Germany. Plastic tissue culture ware was purchased from Falcon (Los Angeles, CA, USA). Results

In order to characterize the second messenger pathways of rGRF in C-cells, we measured CAMP and [Ca”],, two important mediators of CT secretion. Incubation with rGRF in increasing doses (lo-“’ to lo-’ M) led to increases in CAMP accumulation and CT secretion (Fig. 1). The increase in CT secretion paralleled the increase in intracellular CAMP accumulation. Measurements of [Ca2+li did not show any influence of rGRF (lo-’ M) on [Ca’+], in rMTC 6-23 cells (Fig. 2),

140 .-gz 5N b= ki ‘5 rnz Ch ._ Em .z g ic$ ov

120 100 80 60 40 20 0

rGRF RpcAMPs

0 0

7 0

0 4

7 4

-wJv -kl(M)

Fig. 3. Influence of the CAMP antagonist RpcAMPs (10m4 M) on basal and rGRF (lo-’ M)-stimulated calcitonin secretion. RpcAMPs was incubated 20 min at 37°C before stimulation of the cells with rGRF was performed, as described in Materials and methods. The mean of six representative experiments+ SD is shown. * p < 0.05 was considered significant.

rGRF Octreotide

0 0

0 6

77 0

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7 8

7 7

7 6

-km -WV

Fig. 4. Effects of increasing doses of the somatostatin analogue octreotide on &RF-induced CAMP accumulation (upper panel) and calcitonin secretion (lower panel). The mean of six representative experiments + SD is shown. p < 0.05 was considered significant.

whereas an increase of the extracellular calcium concentration from 1 to 3 mM promptly resulted in an increase of [Ca2+li. The recently developed CAMP antagonist RpcAMPs blocks signal transduction via CAMP by binding to the CAMP-dependent proteins kinase without activating the enzyme. Thereby RpcAMPs sufficiently inhibits CAMP-mediated hormone secretion in multiple cell types (Van Haastert et al., 1984; Pereia et al., 1987; Lynne et al., 1988). Basal CT secretion in rMTC 6-23 cells was not influenced by RpCAMPS. rGRF (lo-’ M&stimulated CT secretion, however, was completely blocked by RpCAMPS (10m4 M) (Fig. 3). We studied the effect of the long-acting somatostatin analogue octreotide on rGRF-induced CAMP accumulation and CT secretion. Addition of increasing doses of octreotide (lo-” to 10ph M) inhibited rGRF-induced CAMP accumulation and CT secretion nearly completely, with a maximal effect at lop6 M octreotide (Fig. 4). Basal

TABLE 1 EFFECT OF INCREASiNG DOSES OF PERTUSSIS TOXIN ON BASAL AND rGRF-INDUCED CAMP ACCUMULATION IN rMTC 6-23 CELLS Intracellular CAMP accumulation was measured in pmol cAMP/mg protein/l0 min. The data were obtained in the presence of IBMX (2x 10m4 M). For experimental details see Materials and methods. The mean of six representative experiments + SD is shown. rGRF

0

lo-‘M

S-bromo-CAMP octreotide

Pertussis toxin (ng/mi) 0

1

10

100

2.2 + 0.5 49.5.6+38

3.9+0.7 498529

4.5 rt 1.2 * 577*35 *

5.422 * 624f54 *

* p < 0.05 was considered significant.

intracellular CAMP concentration was not influenced by octreotide. Beside its effect on ion channels, somatostatin is known to inhibit adenylate cyclase through PT-sensitive G-proteins (Jakobs et al., 1983; Koch et al., 1984; Pate1 et al., 1990). Therefore we tested the influence of PT

rGRF

‘Octfeotide PT

077777 0 0

0

0

7 7 0

0

to

7

10

0

100

7 100

-@l(M) -IQ(M) nglml

Fig. 5. Effect of pertussis toxin (PT) on octreotide-induced inhibition of rGRF-stimulated CAMP accumulation (upper panel) and calcitonin secretion (lower panel). The mean of six representative experiments+ SD is shown. * p < 0.05 was considered significant.

0 0

0 6

3 0

3 6

Fig. 6. Effect of octreotide on 8-bromo-CAMP-stimulated calcitonin secretion. Cells were stimulated with either S-bromoCAMP (lo-’ M) or octreotide (lOmh M) or both as described in Materials and methods. The mean of six representative experiments*SD is shown. p < 0.05 was considered significant.

on rGRF-stimulated/octreotide-inhibited rMTC 6-23 cells. Stimulation with PT alone (10-100 ng/ml) resulted in a small but significant increase of intracelIular CAMP. Simultaneous incubation with rGRF and PT had an additive effect on intracellular CAMP (Table 1). Parallel to the increase in either basal or rGRF-stimulated CAMP accumulation, PT stimulated basal and rGRF-induced CT secretion. Pretreatment of the cells with PT for 24 h partially abolished the inhibitory effect of octreotide on the rGRF-induced stimulation of intracellular CAMP production (Fig. 5). Similar results were found in secretion experiments. Pretreatment with PT blocked the inhibitory effect of octreotide on rGRF-stimulated CT secretion (Fig. 5). Recent reports suggest that, in addition to its plasma membrane effects, somatostatin interferes with more distal sites in hormone secretion (Wollheim et al., 1990). In order to evaluate distal effects of somatostatin, we bypassed the adenylate cyclase by stimulating CT secretion with S-bromo-CAMP. CT secretion was stimulated by B-bromo-CAMP (Fig. 6). This stimulatory effect could not be blocked by octreotide (Fig. 6). Discussion Consistent with previous reports, rGRF induced CT secretion in rat C-cells which paralleled rGRF-induced CAMP accumulation (Reyl-

218

Desmares and Zeytin, 1985). Fried and Tashijan (1987) showed that rGRF additionally increased [Ca2’Ji in rat C-cells in a biphasic manner and the initial rise in fCa2’li was interpreted to be due to an influx from the extracellular space. A similar increase of [Ca2+li due to rGRF has been described in somatotrophs (Lussier et al., 1991). Lussier et al. postulated that this increase of [Ca2’]; was mediated through CAMP-mediated changes in Na” conductance, which would lead to a depolarization of the cell and thus to an opening of voltage-dependent calcium channels. Concerning the release of growth hormone in somatotrophs, the GRF-induced increase of [Ca’+], did not play any role, however. In contrast to the findings of Fried and Tashijan (19871, we could not find any influence of rGRF on [Ca2+li, whereas an increase of extracellular calcium induced a sudden increase of [Ca’+]i. The reason for this discrepancy is unclear but might be due to the different rat C-cell line we used. Additionally, the CAMP antagonist RpcAMPs was able to block rGRF-induced CT secretion eompletely. This antagonist is an intracellular inhibitor of CAMP-dependent systems and its antagonistic action has been shown - among others - in gIucagon-induced glucose production in hepatocytes, in the CAMP activation of Dycty~st~~iu~~ discoides and in hormone-induced steroidogenesis in cultured granulosa and Leydig cells (Van Haastert et al., 1984; Pereia et al., 1987; Lynne et al., 19881. The fact that RpcAMPs bfocked rGRF-induced CT secretion completely points to a major roIe of CAMP as a second messenger for rGRF-induced CT secretion in rMTC 6-23 cells. [CaZili or other second messengers do not seem to be important for rGRF-induced CT secretion in C-cells. The somatostatin analogue octreotide was able to block both rGRF-induced cAMP accumulation and CT secretion. The decrease in CAMP levels produced by octreotide appeared not to result from changes in degradation as the phosphodiesterase inhibitor ‘IBMX did not reduce octreotide inhibition of rGRF-stimulated CAMP accumulation. This is consistent with previous findings in pituitary and adrenal cells (Hausdorff et al., 1989; Narayan et aI., 1989; Lussier et al., 19911, in which the somatostatin receptor has

been shown to be coupled to adenyIate cyclase via inhibitory PT-sensitive G-proteins. To evaluate the role of G-proteins in CAMP-mediated CT secretion, we used the islet-activating protein PT. PT is known to uncouple G-proteins from their associated receptors on the plasma membrane by ADP-ribosylating the GTP-binding a-subunit (Milligan, 1988). There is further evidence that PT also acts via G-proteins on intracellular organelles such as chromaffin secretory granules (Sontag et al., 1991). In C-cells, PT increased basal and rGRF-stimulated secretion of CT. The fact that PT augmented rGRF-induced CAMP accumulation and that similar results were seen in the presence of IBMX clearly points to adenylate cyclase as the site of action of PT. Pretreatment of the cells with PT abolished the inhibitory effect of octreotide on CT secretion. Similar results were obtained with IBMX. These results confirm that PT-sensitive G-proteins are involved in the inhibitory effect of octreotide on CT secretion. It is remarkable, however, that PT only partially abolishes the inhibitor effect of octreotide. Recent findings provide evidence for an additional more distal effect of somatostatin on hormone secretion. Wollheim et al. (1990) showed in permeabilized insuIin-secreting cells in which the membrane channels are short circuited that somatostatin inhibited calcium-stimuiated insulin secretion - an effect which was abolished in PT-pretreated ceils. They interpreted these results as suggesting a possible direct action of somatostatin on the secretory processes of hormones distal to the membrane and postulated the involvement of PT-sensitive G-proteins. In order to investigate a similar action of somatostatin on sites distal to the membrane we used 8-bromoCAMP to bypass the adenylate cyclase and therefore to bypass the inhibitory effect of somatostatin on the membrane. Gctreotide was not able to block 8-bromo-CAMP-mediated CT secretion. Thus an effect of somatostatin on more distal sites of the secretory process is not likely to play a role in CT secretion in C-cells. We conclude from our results that somatostatin exerts its inhibitor effect in C-cells through a receptor coupled to the adenylate cycIase by an inhibitory PT-sensitive G-protein. However, effects of PT more distal to the plasma membrane and independent

219

from somatostatin are not excluded from our experiments, especially as the inhibitory effect of octreotide was not completely abolished by pretreatment with PT. Recent reports about PT-insensitive G,-proteins, which inhibit cAMP accumulation independent from PT (Wang et al., 1992) stimulate speculations about a similar mechanism in C-cells, which could explain the partial effect of PT, too. The greater expression of G, in neural tissues compared to other tissues supports this hypothesis, but further studies are required to confirm the existence of such a mechanism in C-cells. The physiological role of somatostatin in Ccells and CT secretion remains unclear. Somatostatin blocks CT secretion in primary cultures of rat C-cells (Endo et al., 1988) and clinically it has been shown that somatostatin lowers the levels of plasma CT in patients with medulla~ carcinoma of the thyroid (Modigliani et al., 1988). Our studies, together with recently reported findings of a direct effect of somatostatin on voltage-dependent calcium channels in C-cells via G-proteins (Scheriibl et al., 19911, implicate that somatostatin acts independently on two important second messenger pathways which regulate CT secretion. Since somatostatin is secreted from Ccells themselves (Aaron et al., 1981; Gage1 et al., 19861, it is an attractive speculation that somatostatin modulates CT secretion in a tonic inhibitory way, possibly in an autocrine or paracrine fashion. References Armbruster, F.P., Scharfenstein, H., Hitzler, W. and Schmidt-Gayk, H. (1983) Artzl. Lab. 32, 115-120. Aron. DC, Musziyuski, M., Birnbaum, R.S., Sabo, SW. and Roos, B.A. (1983) Endocrinology 118, 1643-1651. Birnbaumer, L., Abranowitz, J. and Broen, A.M. (1990) Biochim. Biophys. Acta 1031, 163-224. Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. Dolphin, A.C. (1991) Biochim. Biophys. Acta 1091, 68-80. Dorflinger. L.J. and Schonbrunn, A. (1983) Endocrinology, 113, 1551-1558. Eckert, R.W., Scheriibl, H., Petzelt, Ch., Raue, F. and Ziegler, R. (1989) Mol. Ceil. Endocrinol. 64, 267-270. Endo, T., Saito, T., Uchida, T. and Onaya, T. (19883 Acta Endocrinol. 117, 214-218. Fried, R.M. and Tashijan, A.H. (1986) J. Biol. Chem. 261, 7669-7674. Fried, R.M. and Tashijan, A.H. (1987) J. Bone Min. Res. 2, 579-585.

Gag& R.F., Zextinoglu, F., Voelkel, E.F. and Tashijan, AH. (1980) Endocrinology 107, 516-523. Gage], R.F., Palmer, W.N., Leonhardt, K., Chan, L. and Leon& S.S. (1986) Endocrinology 118, 1643-1651. Hausdorff, W.P., Aguilers, G. and Catt, K.J. (1989) Ceil, Signal. l(4), 377-386. Jakobs, K.H., Aktorios, K. and Schultz, G. (1983) Nature 303, 177-178. Koch, B.D. and Schonbrunn, A. (1984) Endocrinology 114, 1784-1790. Lussier, B.T., French, M.B., Moor, B.C. and Kraicer, J. (1991) Endocrinology 128, 592-603. Lynne, H., Botelho, P., Rothermel, J.D., Coombs, R.V. and Jastorff, B. (1988) Methods Enzymoi. 159, 159-172. Milligan, G. (1988) Biochem. J. 255, I-13. Modigliani. E., Chayvialle, J.A., Cohen, R., Perret, G., Guliana, J.M., Vassy, R., Roger, P., Siame Mourot, C., Bennet, M., Bentata Pessayre, M., Baulieu, J.L., Charpantier, G.. Ruznieswki, P., Deidier. A. and Calmettes, C. (1988) Harm. Metab. Res. 20. 773-776. Narayanan, N., Lussier, B., French M., Moor, B. and Kraicer, J. (1989) Endocrinology 124, 484-495. Nobiling, R. and Biihrle, C.P. (1989) J. Microsc. 156, 149-161. Patel, Y.C., Murthy, K.K., Escher, E.E., Banville, D., Spiess, J. and Srikant, C.B. (1990) Metabolism 39, 63-69. Pereia, M.E., Segaloff, D., Ascoli, M. and Eckstein, F. (1987) J. Biol. Chem. 262(13), 6091-6100. Raue, F., Serve, H., Grauer, A., Rix, E., Scheriihl, H.. Schneider, H.G. and Ziegler, R. (1989) Khn. Wochenschr. 67, 635-639. Raue, F., Zink, A. and Scheriibf, H. (1991) in Medultary Thyroid Carcinoma (Calmettes, C. and Guliana, J.M., eds.), Vol. 211, pp, 31-38. Colloque INSERM/John Libbey Eurotest, Paris. Reyl-Desmares, F. and Zeytin. F. (1985) Biochim. Biophys. Res. Commun. 127,986-991. Rosenthal, W., Hescheler, J., Eckert, R., Gffermanns, S.. Schmidt, A., Hinsch, K.D., Spicher, K., Trautwein, W. and Schultz, C. (1990) Adv. Second Messenger Phosphoprotein Res. 24, 89-94. Scheriibl, H., Raue, F., Zopf, G. and Ziegler, R. (1989) Worm. Metab. Res. Suppl. 21, 18-21. Scheriibl, H., Schultz, G. and Hescheler, J. (1990) FEBS Lett. 27X51-54 [Errata 278, 287 t1991)]. Scheriibl, H. Hescheler, J., Schultz, G., Khemann, D., Zink. A., Ziegler, R. and Raue, F. (1992) Cell. Signal. 4, 77-85. Schonbrunn, A. (1990) Metabolism 39(9), 96-100. Sontag, J.M., Thierse, D., Rouot, B., Aunis. D. and Bader, M.F. (1991) Biochem. J. 274, 339-347, VanHaastert, P.J.M., Van Driel, R., Jastorff, B., Baraniak, J., Stec, W.J. and DeWit, R.J.W. (1984) J. Biol. Chem. 259(16), 10020-10024. Wollheim, C.B., Winiger, B.P., Ulrich, S., Wuarin, F. and Schlegel, W. (1990) Metabolism 39, 101-104. Wong, Y.H., Conklin, B.R. and Bourne, H.R. (1992) Science 255, 339-342. Yatani. A., Birnbaumer, L. and Brown, A.M. (1990) Metabolism 39(9) Suppl. 2, 91-95. Zeytin. F. and Brazeau, P. (1984) Biochem. Biophys. Res. Commun. 123(2), 497-506.

Inhibitory effect of somatostatin on cAMP accumulation and calcitonin secretion in C-cells: involvement of pertussis toxin-sensitive G-proteins.

The effect of somatostatin on cAMP accumulation and calcitonin secretion in C-cells of the rat medullary thyroid carcinoma cell line rMTC 6-23 was inv...
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