Calcitonin peptide inhibition of TRH-stimulated prolactin secretion

JOURNAL CLUB Calcitonin Peptide Inhibition of TRH-Stimulated Prolactin Secretion Additional Evidence for Inhibitory Regulation of Phospholipase C Thomas

F.J. Martin

Calcitonin peptides have been reported to exert direct inhibitory effects on stimulated prolactin secretion from lactotrophs. Several studies indicate that calcitonin peptide inhibition is rather selective for the stimulator-y effects of thyrotropin-releasing hormone (TRH), but not those of other secretagogues. Recent reports demonstrate inhibitory effects of calcitonin peptides on TRWinduced calcium mobilization and inositol phosphate generation. The possibility is discussed that calcitonin peptides act at pituitary receptors that are coupled to Metab phospholipase C in an inhibitory manner. (Trends Endocrlnol 1992;3:82-85)

l

Articles Reviewed

Judd AM, Kubota T, Kuan SI, Jarvis WD, Spangelo BL, MacLeod RM: 1990. Calcitonin decreases thyrotropin-releasing hormone-stimulated prolactin release through a mechanism that involves inhibition of inosit01 phosphate production. Endocrinology 127:191-199. Shah GV, Kennedy D, Dockter ME, Crowley WR: 1990a. CaIcitonin inhibits thymtmpin-releasing hormoneinduced increases in cytosolic Ca2+in isolated rat anterior pituitary cells. Endocrinology 127:6 13-620. Shah GV, Wang W, Grosvenor CE, Crowley WR: 1990b. Calcitonin inhibits basal and thyrotropin-releasing hormone-induced release of prolactin from anterior pituitary cells: evidence for a selective action exerted proximal to secretagogue-induced increases in cytosolic Ca2+. Endocrinology 127:621-628.

Thomas F.J. Martin is at the Department of Zoology, University of Wisconsin, Madison, WI 53706, USA. 82

Pituitary lactotrophs and tumorous cell lines derived therefrom (for example, GH, cells) have long been regarded as favored model systems for investigating mechanisms underlying the regulation of secretion in neuroendocrine cells. Prolactin (PIU) secretion in vivo and in vitro can be stimulated by a variety of hormones, including thyrotropin-releasing hormone (TRH), angiotensin II (AD), neurotensin (NT.), and vasoactive intestinal peptide (VIP). Studies with lactotrophs have been instrumental in elucidating the postreceptor mechanism of action of TRH, the best studied of these secretagogues. Of particular importance for in situ regulation of the lactotroph, however, are the inhibitory regulators of PRL secretion, foremost of which is dopamine (DA). Studies of DA action, as well as that of other inhibitory hormones such as somatostatin (SIUH), have provided additional insights into cellular mechanisms that regulate secretory exocytosis. Several recent reports describe an additional inhibitory regulator of PFU secretion, sahnon calcitonin (sCT)like peptides. sCT, a peptide of 32 amino acids, differs ii-om human and rat CT at 01992,

14 residues

of 32 and from

calcitonin

gene-related peptide (CGRP, an alternative splicing product of the CT gene) at 21 of 32 amino acids. The presence of sCT-like immunoreactive peptides in the human posterior hypothalamus, median eminence and pituitary has previously been noted (Fischer et al. 1981). In addition, direct inhibitory effects of sCT on pituitary PRL release in vitro have been described (Shah et al. 1988). The three reports reviewed here (Shah et al. 1990a and b; Judd et al. 1990) are noteworthy because the results of recent studies indicate that sCT exerts inhibitory effects on PRL secretion by a mechanism distinct from that utilized by DA or SRIH; hence, further studies should provide new information on inhibitory mechanisms. Stimulatory hormonal secretagogues acting on lactotrophs are known to activate transduction mechanisms involving either phospholipase C or adenylyl cyclase and probably Cal+ channels (Figure 1). TRH stimulation of PRL secretion occurs in two phases, both of which are mediated by signals derived from TRH receptor-triggered phospholipase C activation (Drummond 1986; Gershengorn 1986). The initial transient response, during which rates of PRL secretion are markedly elevated, involves an inositol-1,4,5_trisphosphate (IP,)induced cytoplasmic Ca2+ rise, the effects of which may be potentiated by the concomitant rapid 1,2-diacylglycerol (DAG)-mediated activation of protein kinase C (Ronning and Martin 1986a; Martin et al. 1990). The sustained secondary secretory response, during which rates of PRL secretion are increased twoto fourfold, appears to be driven by the increased entry of Ca2+ through voltagedependent Ca2+ channels and the enhanced sensitivity of the secretory apparatus to Ca2+, triggered by the preceding period of protein kinase C activation (Ronning and Martin 1986b; Dufy et al. 1987). Stimulatory mechanisms in lactotrophs mediated through adenylyl cyclase activation are less well understood than those for phospholipase C activation. VIP stimulation of PRL secre-

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TEM Vol. 3, No. 3, 1992

1. Schematic summary of signal transduction pathways and their relation to the stimulation of secretion. Three receptor-regulated pathways are ilhtstrated, representing examples of G-protein-linked receptors coupled to distinct effecters. G proteins are indicated as heterotrimers of a&r subunits. (Middle) Receptor-G-protein-regulated ion channel function that may directly, or indirectly through membrane potential, regulate Caz+influx. This pathway may be utilized by LHRH and AII in pituitary cells. (Top) Receptor-G-protein-regulated adenylyl cyclase (AC) and CAMP generation result in the activation of the CAMP-dependent protein kinase (PKA) that may alter ion channel (Ca2+ channel) function or modulate a component of the exocytotic apparatus. VIP and forskolin stimulation of PRL secretion are mediated, at least in part, through this pathway. (Bottom) Receptor-G-protein-regulated phospholipase C (PLC) that hydrolyzes phosphoinositides and generates two second messengers, diacylglycerol (DAG) and inositol-1,4,Mrisphosphate (IP,). DAGis an activator of protein kinase C (PKC) that may regulate ion channel (Ca 2+ channel) function or modulate a component of the exocytotic apparatus. IP, triggers Caz+ mobilization from an intracellular, nonmitochondrial pool. TRH, AII, and NT are believed to stimulate PRL secretion via this pathway. Each of these signal transduction pathways can promote an increase in cytoplasmic Ca2+, which is the principal activator of secretion in neuroendocrine cells. PKA- and PKC-mediated pathways can enhance Ca2+-activatedsecretory responses. Numbers correspond to sites discussed in the text for potential inhibition of stimulated secretion by inhibitory agonists. Figure

tion is slower in onset than that for TRH, corresponding to the relatively slow onset of cyclic (c) AMP accumulation. CAMP stimulation of secretion may be mediated through a combination of mechanisms involving stimulated influx of Ca2+ (Koch et al. 1988) and an increased responsiveness of the secretory apparatus to Caz+ (Guild et al. 1988). Small increases in cAMP that have been observed in lactotrophs in response to TRH TEM Vol. 3, No. 3, 1992

may result from effects of protein kinase C in activating adenylyl cyclase (Quilliam et al. 1989) and may provide synergy with pathways involving Ca2+ entry or Caz+-activated exocytosis (Delbeke et al. 1984). In general, cytoplasmic Ca2+ appears to be the principal direct regulator of secretion in neuroendocrine cells, with cAMP-dependent or protein kinase C-mediated pathways serving to modulate Caz+-dependent steps (see Figure 1). 01992,

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Hormones with inhibitory effects on stimulated PRL secretion may counteract any of the cellular events responsible for stimulation (see Figure 1 legend). Modes of inhibition might include (1) antagonism of receptor binding by stimulator-y hormones; (2) activation of inhibitory G proteins (for example, Gi antagonism of G,-mediated activation of adenylyl cyclase); (3) inhibition of adenylyl cyclase or phospholipase C by other means (for instance, inhibition of Ca2+-dependent phospholipase C by decreasing Cal+); (4) increased dissipation of second-messenger molecules (for example, enhanced hydrolysis of CAMP, decreased influx or enhanced efflux of Ca2+, and increased metabolism of IP, or DAG); (5) inhibition of second-messengeractivated pathways (for instance, inhibition of IP,-mediated Ca2+ mobilization, inhibition of protein kinases, and activation of protein phosphatases); and (6) distal inhibitory influences on components of the exocytotic apparatus. DA and SRIH share certain similarities in the mechanisms by which they inhibit secretion. These inhibitory agonists counteract the stimulatory effects of multiple secretagogues, including hormones that activate the adenylyl cyclase cascade (VIP) or the phosphoinositide cascade (TRH). Inhibitory coupling of DA and SRIH receptors to adenylyl cyclase mediated through Gi proteins has been demonstrated (Vallar and Meldolesi 1989; Schonbnmn 1990). DA and SIUH also lower cytoplasmic Ca2+ levels in pituitary cells by decreasing Ca2+ influx (Schofield 1983; Schlegel et al. 1984). For both DA and SRIH, receptor-G-protein-mediated activation of K+ channels affects the probability of action potential generation and action potential duration, and results in reduced Ca2+ entry (Vallar and Meldolesi 1989; Schonbrunn 1990). Direct receptorG-protein-mediated inhibition of Ca2+ channel activity may also occur (Mollard et al. 1988; Brown and Birnbaumer 1990). The inhibitory effects of DA on PRL secretion are reversed by combined, but not individual, treatment with agents that increase CAMP and cytoplasmic Ca2+ (Delbeke and Dannies 1985), indicating dual inhibitory effects of DA exerted through reductions in CAMP and Ca2+levels. A similar model may account for inhibitory effects of SRIH, although there is also evidence in pituitary cells 83

for distal sites of SRIH action, indicated by reports of SRIH inhibition in permeable cells where CAMP, K+, and Ca2+ levels are clamped (Luini and DeMatteis 1990). DA (but not SRIH) treatment has also been reported to inhibit TRH-stimulated IP, generation (Vallar et al. 1988; Enjalbert et al. 1990). Although it was initially suggested that this effect of DA was proximal, it now appears that most of the inhibition is secondary to a lowering of Ca2+ levels and resultant inhibition of TRH-regulated phospholipase C (Vallar et al. 1988; Enjalbert et al. 1990). A second component of inhibition, possibly involving proximal inhibitory effects of DA receptors on phospholipase C activity, has also been suggested (Enjalbert et al. 1990). In one recent study, DA was found to decrease resting cytoplasmic Ca2+ levels without reducing TRHstimulated intracellular Ca2+ mobilization, consistent with a primary mode of action of inhibiting Ca2+ influx (Law et al. 1988). The three articles reviewed here (Shah

secretion, it was argued that direct receptor antagonism can be excluded (Shah et al. 1990b). This point needs to be more carefully examined in future studies, since single doses of TR.H and sCT were examined in TSH release studies, and there may be differences between the efficacy of antagonism at thyrotropic and lactotropic TRH receptors based on differences in receptor occupancy required to trigger secretory responses. Assuming that receptor antagonism is not responsible for the inhibition of TRH effects by sCT, the results provided by Shah et al. (1990a) provide a suggestion of the underlying mechanism. These authors reported that sCT was very effective in blocking TRH-stimulated increases in cytoplasmic Ca2+. Both firstphase and second-phase TRH-induced elevations of Ca2+ were blocked by sCT, corresponding to mobilization of intracellular and influx of extracellular Ca2+, respectively. Similar results have been reported for sCT inhibition in GH, cells (Epand et al. 1989). Removal and chela-

et al. 1990a and b; Judd et al. 1990) extend the earlier report (Shah et al. 1988) that sCT inhibits TRH-stimulated PRL release. sCT inhibition developed rapidly (within several minutes) and was exerted at low concentrations of the peptide (0.1-100 nM). The evidence presented indicates that the mechanism of sCT action may differ from that of DA or SRIH described above. The inhibition of stimulated PRL secretion by sCT was restricted to the stimulatory effects of TRH, whereas stimulation by VIP, AII, and NT was not affected by sCT (Judd et al. 1990). Although it might be argued that sCT and TRH receptors were limited to a subset of lactotrophs distinct from those that bear VIP, AII, or NT receptors, it was also reported that sCT failed to inhibit PRL release stimulated by pharmacologic treatments intended to mimic intracellular regulators (ionophores and Ca2+ channel activators, protein kinase C activators, and the adenylyl cyclase activator forskolin)(Shah et al. 1990b; Judd et al. 1990). Such results suggest that sCT inhibition is exerted proximal to the sites of second-messenger generation and is somehow restricted to TRH receptorassociated events. The obvious possibility that sCT is an antagonist of the TRH receptor has not been directly examined. Since sCT failed to inhibit (but possibly enhanced) TRH-stimulated TSH

84

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SRIH, sCT was not found to inhibit basal or stimulated CAMP levels. Inhibitory regulation of phospholipase C has been suggested to account for the actions of a variety of agonists (Linden and Delahunty 1989). It will be important to determine whether artificial elevation of cytoplasmic Ca2+ is capable of reversing the inhibitory effects of sCT on TRH-stimulatedphosphoinositide hydrolysis, since sCT-induced reductions in stimulated inositol phosphate generation might be secondary to a lowering of Ca2+, as appears to be largely the case for DA. In one study (Epand et al. 1989), but not two others (Shah et al. 1990a; Judd et al. 1990), sCT alone slightly decreased resting Ca2+ levels in pituitary cells. However, because of leakage of fluorescent Ca2+ reporter dyes and extensive cellular heterogeneity, possible inhibitory effects of sCT on Ca2+ influx cannot be entirely excluded based on the available data. In three reports (Shah et al. 1988 and 1990b; Epand et al. 1989), but not in a fourth (Judd et al. 1990), sCT inhibited basal rates of PRL. release. The basis for this effect remains to be clarified, although an effect of sCT on resting cytoplasmic Ca2+ would be a likely explanation. It is unlikely that a reduction in resting cytoplasmic Ca2+ would result from inhibition of basal Ip, generation, since IP, probably does not contribute to the maintenance of resting Ca2+ levels. It is difficult to envision a selective action of sCT on TRH-activated phosphoinositide hydrolysis without similar effects on AII- or NT-stimulated inositol phosphate generation, although distinct receptor-bearing populations of lactotrophs could be responsible for this selectivity. The alternative possibility that TRH and AI1 or NT activate distinct phospholipase C isoenzymes cannot be entirely excluded. TRH receptors stimulate phospholipase C activity by a mechanism that employs a pertussis-toxin-insensitive G protein (G,) shown recently to be related or identical to Gg,,, (Martin et al. 1989; Hsieh and Martin, submitted). In contrast, recent indirect evidence has indicated possible inhibitory G protein regulation of phospholipase C activity by a variety of agonists such as adenosine, possibly DA, opioid peptides, muscarinic agonists, and excitatory amino acids. These studies have been reviewed re-

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cently (Linden and Delahunty 1989) and have generated the hypothesis of dual regulation of phospholipase C via G . . . (i mhrbrto$ (s, st’lm ulatory) and G proteins, by analogy wi%thd established dual mode of adenylyl cyclase regulation by G, and Gi proteins. The evidence provided for sCT similarly suggests the possibility of inhibitory regulation through sCT receptor coupling to a Gpcij protein. The most compelling and direct evidence for this type of regulation would be the demonstration of receptormediated, GTP-dependent inhibition of phospholipase C activity in permeable cells or isolated membranes, as has recently been reported for inhibitory effects of carbachol in FRTLS cells (Bizzarri et al. 1990). Such studies eliminate indirect, secondary effects of an agonist on Ca2+ or other second messengers. For understanding sCT actions in lactotrophs, similar studies represent the next logical step. References Brown AM, Bimbaumer L: 1990. Ionic channels and their regulation by G protein subunits. Armu Rev Physiol52:197-213. Bizzarri C, DiGirolamo MD, D’Orazio MC, Corda D: 1990. Evidence that a guanine nucleotide-binding protein linked to a muscarinic receptor inhibits directly phospholipase C. Proc Natl Acad Sci USA 87:48894893. Delbeke D, Dannies PS: 1985. Stimulation of the adenosine 3’, 5’-monophosphate and the Ca2+ messenger systems together reverse dopaminergic inhibition of prolactin release. Endocrinology 117:439-446. Delbeke D, Kojima I, Darmies PS, Rasmussen H: 1984. Synergistic stimulation of prolactin release by phorbol ester, A23 187 and forskolin. Biochem Biophys Res Commun 123:735-741. Drummond AI-L 1986. Inositol lipid metabolism and signal transduction in clonal pituitary cells. J Exp Biol 124:337-358. Dufy B, Jaken S, Barker JL: 1987. Intracellular Ca2+-dependent protein kinase C activation mimics delayed effects of thyrotropinreleasing hormone on clonal pituitary cell excitability. Endocrinology 121:793-802.

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Quilham LA, Dobson PRM, Brown BL: 1989. Regulation of GH, pituitary tumour-cell adenylate cyclase activity by activators of protein kinase C. Biochem J 262:829-834. Ronning SA, Martin TFJ: 1986a. Characterization of Cal+-stimulated secretion in permeable GH, pituitary cells. J Biol Chem 261:7834-7839. Ronning SA, Martin TFJ: 1986b. Characterization of phorbol ester- and diacylglycerolstimulated secretion in permeable GH, cells. J Biol Chem 261:7840-7845. Schlegel W, Wuarin F, Wollheim CB, Zahnd GR: 1984. Somatostatin lowers the cytosolic free Caz+concentration in clonal rat pituitary cells (GH, cells). Cell Calcium 5:223-236.

Judd AM, Kubota T, Kuan SI, Jarvis WD, Spangelo BL, MacLeod RM: 1990. Calcitonin decreases thyrotropin-releasing hormone-stimulated prolactin release through a mechanism that involves inhibition of inositol phosphate production. Endocrinology 127:191-199.

Schofield JG: 1983. Use of a trapped fluorescent indicator to demonstrate effects of thyroliberin and dopamine on cytoplasmic calcium concentrations in bovine anterior pituitary cells. FEBS Lett 159379-82.

Koch BD, Blalock JB, Schonbnmn A: 1988. Characterization of the cyclic AMP-independent actions of somatostatin in GH cells. J Biol Chem 263:216-225.

Schonbrunn A: 1990. Somatostatin action in pituitary cells involves two independent transduction mechanisms. Metabolism 39:96-l 00.

Law GJ, Pachter JA, Dannies PS: 1988. Dopamine has no effect on thyrotropinreleasing hormone mobilization of calcium from intracellular stores in rat anterior pituitary cells. Mol Endocrinol2:966-972.

Shah GV, Epand RM, Orlowski RC: 1988. Calcitonin inhibition of prolactin secretion in isolated rat pituitary cells. J Endocrinol 116:279-286.

Linden J, Delahunty TM: 1989. Receptors that inhibit phosphoinositide breakdown. Trends Pharmacol Sci 10: 114-l 20. Luini A, DeMatteis MA: 1990. Evidence that receptor-linked G protein inhibits exocytosis by a post-second-messenger mechanism in AtT-20 cells. J Neurochem 54:30-38. Martin TFJ, Hsieh K-P, Porter BW: 1990. The sustained second phase of hormonestimulated diacylglycerol accumulation does not activate protein kinase C in GH, cells. J Biol Chem 265:7623-7631. Martin TFJ, Lewis JE, Kowalchyk JA: 1991. Phospholipase C-8 is regulated by a pertussis toxin-insensitive G-protein. Biochem J 280:753-760. Mollard P, Vacher P, Dufy B, Barker JL: 1988. Somatostatin blocks Ca2+ action potential activity in prolactin-secreting pituitary tumor

Enjalbert A, Guillon G, Mouillac B, et al.: 1990. Dual mechanisms of inhibition by dopamine of basal and thyrotropin-releasing hormone-stimulated inositol phosphate production in anterior pituitary cells. J Biol Chem 265:18,816-18,822.

Shah GV, Kennedy D, Dockter ME, Crowley WR: 1990a. Calcitonin inhibits thyrotropinreleasing hormone-induced increases in cytosolic Caz+in isolated rat anterior pituitary cells. Endocrinology 127:613-620. Shah GV, Wang W, Grosvenor CE, Crowley WR: 1990b. Calcitonininhibitsbasal andthyrotropinreleasing hormone-induced release of prolactin from anterior pituitary cells: evidence for a selective action exerted proximal to secretagogue-induced increases in cytosolic Ca2+.Endocrinology 127:621428. Vallar L, Meldolesi J: 1989. Mechanisms of signal transduction at the dopamine D, receptor. Trends Pharmacol Sci 10:74--77. Vallar L, Vicentini LM, Meldolesi J: 1988. Inhibition of inositol phosphate production is a late, Caz+-dependent effect of D, dopaminergic receptor activation in rat lactotroph cells. JBiol Chem 263:10,127-10,134. TEM

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Epand RM, Stafford AR, Orlowski RC: 1989. Calcitonin inhibits the rise of intracellular

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cells through coordinate actions on K+ and Ca2+conductances. Endocrinology 123:72 l732.

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