Pituitary adenylate cyclase-activating polypeptide specifically increases cytosolic calcium ion concentration in rat gonadotropes and somatotropes.

The hypothalamic peptide, pituitary adenylate cyclase-activating polypeptide (PACAP), is a potent stimulator of cAMP accumulation in the anterior pituitary gland, though its physiological function has yet to be defined. To establish the target cells of PACAP action we have measured PACAP-induced changes in cytosolic free calcium ion concentration ([Ca2+]i) in single identified anterior pituitary cells. This was achieved by combining fura-2 videomicroscopy, to measure [Ca2+]i, and reverse hemolytic plaque assays, to identify the secreted hormone. PACAP (100 nM) increased [Ca2+]i in 32% of all pituitary cells. These responses were predominantly seen in identified gonadotropes and somatotropes, but rarely in corticotropes or lactotropes. PACAP induced two forms of Ca2+ response in gonadotropes; a "Ca2+ spike" (independent of extracellular Ca2+) in 72% of responding gonadotropes, and an extracellular Ca(2+)-dependent "Ca2+ plateau" (28% of cells). In somatotropes, PACAP stimulated either Ca2+ plateau responses (58% of responding somatotropes) or repetitive "Ca2+ transients" (42% of cells), both of which were dependent upon extracellular Ca2+. PACAP, therefore, produces distinct changes in [Ca2+]i in gonadotropes and somatotropes, which may be related to distinct intracellular messenger pathways. The identification of these cell types as targets of PACAP action suggests a role in the regulation of reproduction and growth.     

Goldfish brain somatostatin-28 differentially affects dopamine- and pituitary adenylate cyclase-activating polypeptide-induced GH release and Ca(2+) and cAMP signals

Dopamine (DA) and pituitary adenylate cyclase-activating polypeptide (PACAP) stimulate goldfish growth hormone (GH) release via cAMP- and Ca(2+)-dependent pathways while DA also utilizes NO. In this study, identified goldfish somatotropes responded to sequential applications of PACAP and the DA D1 agonist SKF38393 with increased intracellular Ca(2+) levels ([Ca(2+)](i)), indicating that PACAP and DA D1 receptors were present on the same cell. A native goldfish brain somatostatin (gbSS-28) reduced SKF38393-stimulated cAMP production and PACAP- and NO donor-elicited GH and [Ca(2+)](i) increases, but not PACAP-induced cAMP production nor the GH and [Ca(2+)](i) responses to forskolin, 8-bromo-cAMP and SKF38393. gbSS-28 might inhibit PACAP-induced GH release by interfering with PACAP's ability to increase [Ca(2+)](i) in a non-cAMP-dependent manner. However, DA D1 receptor activation bypassed gbSS-28 inhibitory effects on cAMP production and NO actions via unknown mechanisms to maintain a normal [Ca(2+)](i) response leading to unhampered GH release.     

Thyrotropin-releasing hormone and epidermal growth factor stimulate prolactin synthesis by a pathway(s) that differs from that used by phorbol esters: dissociation of actions by calcium dependency and additivity

TRH, epidermal growth factor (EGF), and 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulate PRL synthesis by GH4C1 rat pituitary cells. Recent evidence suggests that TPA activates directly phospholipid- and calcium-dependent protein kinase C in other cell types and that TRH might act analogously by altering phospholipid metabolism in GH4C1 cells. To examine the pathways by which these three agents stimulate PRL synthesis, we determined their calcium dependencies as well as their combined effects on PRL production. By equilibration of GH4C1 cells in a protein-free medium for 24 h, the free cytosolic calcium concentration ([Ca2+]i) was found to increase (from 90 to 360 nM) when the extracellular calcium concentration ([Ca2+]e) was varied from 15 to 800 microM. Basal PRL production increased in parallel (from 1 to 4 micrograms/ml X 24 h). TPA-stimulated PRL production was highly calcium dependent and required 180 nM [Ca2+]i for maximal enhancement. TRH-stimulated PRL production was constant between 10 and 660 microM [Ca2+]e, whereas EGF stimulated PRL production to a similar extent as TRH at 10 microM [Ca2+]e, but continued to enhance production with increasing [Ca2+]e. TRH elevated [Ca2+]i acutely, and at [Ca2+]e greater than 100 microM caused both a burst and a plateau phase in elevated [Ca2+]i. At lower [Ca2+]e, at which TRH still caused a maximal stimulation of PRL production, only the burst phase of [Ca2+]i occurred. When cultures were treated with any combination of maximally effective concentrations of TPA, TRH, or EGF, PRL production was increased by additive increments. The additive actions of TPA and TRH could not be explained by a calcium-promoted increase in TPA-stimulated PRL production. We conclude that TPA stimulates PRL production by a highly calcium-dependent pathway and that TRH and EGF stimulate PRL production by a different pathway(s) requiring lower [Ca2+]i.     

Pituitary adenylate cyclase activating polypeptide and vasoactive intestinal peptide increase cytosolic free calcium concentration in cultured rat hippocampal neurons.

Recently, pituitary adenylate cyclase activating polypeptide (PACAP) was isolated from ovine hypothalamus and it was shown to stimulate adenylate cyclase in rat pituitary cells, neurons, and astrocytes. PACAP exhibits a 68% amino acid sequence homology with vasoactive intestinal peptide (VIP); however, it is 1000 times more potent than VIP in stimulating adenylate cyclase. In view of the wide distribution of PACAP and its receptor in the central nervous system, PACAP is likely to act as a neurotransmitter or neuromodulator as well. In the present study, we investigated the effects of PACAP38 on cytosolic-free calcium concentrations ([Ca2+]i) and compared these effects with those of VIP in cultured rat hippocampal neurons. Calcium concentrations, at the single cell level, were measured using fura-2, a calcium sensitive fluorescent dye, and fura-2-loaded neurons were continuously superfused at 37 C and viewed under an inverted microscope. Images of these neurons were recorded at 10-sec intervals by a video camera equipped with an Argus-50/CA system which controls the image acquisition and display. [Ca2+]i was quantitated from the intensities of fluorescence of the cells at two excitation wavelengths of 340 and 380 nm. The ratio of the intensities of emitted fluorescence (340/380 nm) was calibrated to determine [Ca2+]i. PACAP38 (0.1 nM) increased [Ca2+]i in some hippocampal neurons. As the concentration of peptide was increased from 0.1 to 10 nM, the accumulated number of hippocampal neurons responding to PACAP38 progressively increased and reached a plateau at 10 nM. Total neurons (33.0 +/- 5.3%, n = 4; 502 neurons) were found to respond to 100 nM PACAP38. The half-maximal concentration (ED50) of PACAP38 was 2.60 +/- 0.77 nM. Typically, 60-90 sec after the addition of PACAP38 (10 nM), [Ca2+]i increased from basal levels of 50-100 to 150-300 nM. VIP also increased [Ca2+]i, but required 1 microM or higher concentration for a considerable number of cells to respond. The number of hippocampal neurons responding to VIP at 1 microM was 28.9 +/- 9.8% (n = 4; 442 cells) which was comparable to the population of neurons responding to 10 nM PACAP38. The ED50 for VIP was 0.68 +/- 0.38 microM which was approximately 260 times higher than the ED50 for PACAP38. Neither 1-10 microM nitrendipine, a L-type voltage-dependent Ca2+ channel blocker, or 1 microM omega-conotoxin GVIA, a N-type voltage-dependent Ca2+ channel blocker, altered the PACAP-induced Ca2+ increment. Removal of Ca2+ from the superfusion media did not influence the PACAP38-induced increase of [Ca2+]i.(ABSTRACT TRUNCATED AT 400 WORDS)     

Role of Ca2+ stores in dopamine- and PACAP-evoked growth hormone release in goldfish

Growth hormone (GH) secretion, evoked by either pituitary adenylate cyclase-activating polypeptide (PACAP) or dopamine (DA), is dependent on both voltage-sensitive calcium channels (VSCC) and cAMP signaling in goldfish. We further characterized the involvement of Ca2+ in evoked release by PACAP and DA, by examining the sensitivity of evoked GH release to perturbations of Ca2+ signaling. Both VSCC and calmodulin/calmodulin-dependent kinase are involved in PACAP signaling as had been shown for DA. In spite of this apparent dependence on VSCC, blockade of TMB-8 but not ryanodine-sensitive intracellular Ca2+ stores inhibited both PACAP- and DA-evoked GH release. Using sarcoplasmic/endoplasmic reticulum Ca-ATPases (SERCA) inhibitors, we found BHQ blocked, whereas thapsigargin (Tg) enhanced stimulated GH release, suggesting that Tg-sensitive SERCA may counteract these cAMP-mobilizing neuroendocrine regulators by sequestering [Ca2+]i. As GH secretion stimulated by two endogenous gonadotropin-releasing hormones is not affected by Tg, it appears that distinct multiple Ca2+ stores mediate the hormone releasing response to different neuroendocrine regulators.     

Lowering of cytosolic free Ca2+ by carbachol, a muscarinic cholinergic agonist, in clonal pituitary cells (GH3 cells)

Muscarinic cholinergic agonists have been shown to inhibit PRL secretion in normal and tumor-derived pituitary cells. Evidence from experiments with the fluorescent Ca2+ probe quin 2 shows that carbachol, acting through muscarinic acetylcholine receptors, lowers the cytosolic free Ca2+ concentration ([Ca2+]i), in GH3 cells. A decrease in [Ca2+]i is observed rapidly after carbachol addition, the lowered steady state [Ca2+]i is maintained, and upon the addition of atropine [Ca2+]i returns to the initial basal value. The lowering from a basal [Ca2+]i, averaging 110 +/- 2 nM (+/- SEM, n = 9), to a steady state [Ca2+]i of 63 +/- 4 nM (+/- SEM, n = 5) at 10 micron carbachol is dose dependent, a significant decrease from basal [Ca2+]i being observed at 0.1 micron. Carbachol does not prevent TRH-induced mobilization of Ca2+ but attenuates the resulting rise in [Ca2+]i. The lowering of steady state [Ca2+]i and the attenuation of the rise in [Ca2+]i provoked by stimulators of PRL secretion could explain the inhibition of both basal and stimulated PRL secretion. Concomitantly with the action on [Ca2+]i, carbachol causes hyperpolarization of GH3 cells. Together with the established inhibition of adenylate cyclase by muscarinic cholinergic agonists, these findings suggest a relation between changes in trans-membrane Ca2+ fluxes and cAMP generation.     

Regulation of growth hormone release in common carp pituitary cells by pituitary adenylate cyclase-activating polypeptide: signal transduction involves cAMP- and calcium-dependent mechanisms

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a member of the glucagon/secretin peptide family and its molecular structure is highly conserved among vertebrates. In this study, the role of PACAP in regulating growth hormone (GH) secretion in fish was examined in vitro using common carp pituitary cells under column perifusion. A dose-dependent increase in GH release was observed after exposing pituitary cells to increasing doses of ovine PACAP38 (oPACAP38) and PACAP27 (oPACAP27), but not vasoactive intestinal polypeptide (VIP). A lack of GH response to VIP stimulation is consistent with the pharmacological properties of PAC-1 receptors, suggesting that this receptor subtype may be involved in PACAP-induced GH secretion in carp species. Although the maximal GH responses induced by oPACAP38 and oPACAP27 were similar, the minimal effective dose and ED50 value for oPACAP38 were significantly lower than that for oPACAP27. These results may indicate that common carp PAC-1 receptors are more sensitive to stimulation by oPACAP38 than by oPACAP27. In parallel studies, oPACAP38 and oPACAP27 were also effective in increasing cAMP release, cellular cAMP content, total cAMP production, and intracellular Ca(2+) ([Ca(2+)](i)) levels in common carp pituitary cells. Besides, the rise in [Ca(2+)](i) induced by oPACAP38 was blocked by removing extracellular Ca(2+) ([Ca(2+)](e)) or by treatment with nifedipine, an inhibitor of voltage-sensitive Ca(2+) channels (VSCC). The dose dependence of PACAP-stimulated GH release in common carp pituitary cells was mimicked by activating adenylate cyclase using forskolin, inhibiting cAMP degradation using IBMX, increasing functional levels of intracellular cAMP using CPT-cAMP, or inducing [Ca(2+)](e) entry using the Ca(2+) ionophore A23187. In contrast, the GH-releasing effect of oPACAP38 was suppressed by treatment with the adenylate cyclase inhibitor MDL12330A, protein kinase A inhibitor H89, and VSCC blocker nifedipine, or by perifusion with a Ca(2+)-free culture medium. These results, as a whole, suggest that PACAP functions as a GH-releasing factor in common carp by activating pituitary receptors resembling mammalian PAC-1 receptors. Apparently, the GH-releasing action of PACAP is mediated through the adenylate cyclase/cAMP/protein kinase A pathway and [Ca(2+)](e) influx through VSCC.     

Growth hormone-releasing factor stimulates calcium entry in the GH3 pituitary cell line

The GH3 pituitary cell line has been extensively used to study various aspects of the stimulus secretion coupling process. It is known that GH3 cells release PRL and GH in the basal state and in response to various secretagogues. However, this cell line was considered unsuitable as a model for studying the effects of GHRF since the neuropeptide did not affect GH secretion or gene expression. This suggested that the GH3 cells may lack GHRF receptors. The present study investigates the effect of GHRF on free intracellular Ca2+ concentrations in GH3 cells. Cytosolic free calcium concentrations ([Ca2+]i) were monitored in individual cells by microspectrofluorimetry using the fluorescent dye indo 1. When the cells were challenged with a brief application of GHRF (100 nM; 15 sec), 36 out of 59 of these cells responded within a few seconds by a marked increase in [Ca2+]i. GHRF enhanced the frequency of [Ca2+]i oscillations in spontaneously active cells or triggered [Ca2+]i oscillations in inactive cells. The response to GHRF was totally blocked by external Ca2+ free solutions and Ca2+ channel blockers. Combined electrophysiological and fluorescent experiments were carried out in 16 cells. Eleven responded to GHRF. In all cases, the Ca2+ transients triggered by GHRF were associated with action potentials. The Ca2+ responses observed in our experiments clearly show that GH3 cells possess membrane receptors to GHRF. Thus, it is likely that the lack of secretory response observed in GH3 cells does not result from the absence of binding sites to the peptide. It is more likely to be related to alterations of transduction mechanisms resulting in uncoupling between stimulation and secretion.     

Subpopulations of somatotropes with differing intracellular calcium concentration responses to secretagogues

Multiple secretagogues stimulate the release of growth hormone (GH). The present studies examined the ability of chicken somatotropes to respond to GH secretagogues with increased intracellular calcium concentrations ([Ca2+]i). It was hypothesized that there are subsets of the somatotrope population with different responsiveness to the various secretagogues. Somatotropes were identified and distinguished from other adenohypophyseal cells, by their unique ability to respond to GH-releasing hormone with increased [Ca2+]i with immunocytochemistry used as a post-hoc confirmatory test. Large increases in [Ca2+]i (222 +/- 16 nM) were evoked by thyrotropin-releasing hormone in only 73% of the somatotropes. Similarly, [Ca2+]i was increased by perifusion with pituitary adenylate cyclase-activating peptide in 85% and by leptin but only in 51% of somatotropes. Ghrelin acutely increased [Ca2+]i in only 21% of somatotropes. Perfusion with gonadotropin-releasing hormone elevated [Ca2+]i, but in only 40% of somatotropes. The kinetics of calcium transients and the magnitude of the response differed from those observed in the presumptive gonadotropes. It is concluded that there are subsets of the somatotrope population in the anterior pituitary gland with differences in their ability to respond to various secretagogues.     

Alpha-adrenergic inhibition of thyrotropin-releasing hormone-induced prolactin secretion in GH4C1 cells is associated with a depressed rise in intracellular Ca2+.

alpha-Adrenergic receptors are present on the plasma membrane of normal anterior pituitary cells and alpha-adrenergic agonists may play a role in the secretion of corticotropin (ACTH) and thyrotropin (TSH). However, alpha-adrenergic involvement in prolactin (PRL) secretion is uncertain. We have therefore examined this question in the PRL-secreting clonal rat pituitary tumor-derived GH4C1 cells. Norepinephrine (NE), an alpha-adrenergic agonist, had no effect on basal PRL secretion but abolished thyrotropin-releasing hormone (TRH)-induced PRL secretion in a dose-dependent manner (EC50 100 nM). NE also significantly suppressed the TRH-stimulated rise in [Ca2+]i. Phentolamine (PA), a non-selective alpha-adrenergic antagonist, reversed the inhibitory effect of NE on both the TRH-stimulated PRL secretion and [Ca2+]i rise. NE did not inhibit the rise in PRL secretion or [Ca2+]i induced by depolarizing 30 mM K+, 30% hyposmolarity or BAY K-8644, a specific L-type Ca2+ channel agonist. The inhibitory effect of NE on TRH-induced PRL and [Ca2+]i changes was also present when Ca2+ influx was prevented by removing medium Ca2+ or by blocking L-type Ca2+ channels with 2 microM nifedipine. The TRH-stimulated first-phase rise in [Ca2+]i in GH4C1 cells is believed to result primarily from release of sequestered Ca2+ from an intracellular pool through the activation of inositol 1,4,5-trisphosphate (IP3) and this [Ca2+]i spike stimulates PRL secretion. Our data thus suggest that GH4C1 cells have alpha-adrenergic receptors and that alpha-adrenergic agonists either suppress IP3 generation or block IP3 release of sequestered intracellular Ca2+.     

1,25-Dihydroxycholecalciferol enhances both the bombesin-induced transient in intracellular free Ca2+ and the bombesin-induced secretion of prolactin in GH4C1 pituitary cells

In GH4C1 rat pituitary cells, 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] enhances both the synthesis of PRL and the TRH-induced transient increase in cytosolic free calcium ( [Ca2+]i). In the present report we investigated whether 1,25-(OH)2D3 could enhance the effect of the tetradecapeptide bombesin (BBS) in GH4C1 cells. Pretreatment of the cells with 1 nM 1,25-(OH)2D3 for 24 h enhanced the BBS-induced transient increase in [Ca2+]i compared to that in control cells, while having no significant effect on the plateau phase of [Ca2+]i. Addition of the Ca2+ channel blocker nimodipine or chelating extracellular Ca2+ with EGTA did not abolish the enhancement of the BBS response in 1,25-(OH)2D3-pretreated cells. Furthermore, the BBS-induced efflux of 45Ca2+ from cells preequilibrated with 45Ca2+ was larger in cells treated with 1,25-(OH)2D3. Incubating GH4C1 cells with 1,25-(OH)2D3 alone or in combination with BBS for up to 72 h did not stimulate synthesis of PRL. However, the BBS-induced secretion of PRL was enhanced in cells pretreated with 1,25-(OH)2D3 for 24 h compared with that in vehicle-treated control cells. The effect of 1,25-(OH)2D3 on BBS-induced secretion was dose dependent, with 10(-11) M 1,25-(OH)2D3 enhancing the stimulated secretion of PRL. We conclude that in GH4C1 cells, pretreatment with 1,25-(OH)2D3 enhances the BBS-induced transient increase in [Ca2+]i. This effect may be due to a modulation of the availability of sequestered intracellular Ca2+ and/or membrane Ca2+ conductance. Furthermore, pretreatment with 1,25-(OH)2D3 enhanced secretion of PRL stimulated by BBS. The enhanced transient increase in [Ca2+]i may be the factor inducing the enhanced BBS-induced secretion of PRL.     

Calcium currents and fura-2 signals in fluorescence-activated cell sorted lactotrophs and somatotrophs of rat anterior pituitary

Optical and electrical recording techniques were applied to single primary pituitary cells to characterize the types of voltage-dependent calcium currents (ICa) and levels of intracellular calcium ([Ca2+]i). GH-containing somatotrophs and PRL-containing lactotrophs were isolated from adult female rats using fluorescence-activated cell-sorting techniques and were maintained in culture for 1-4 days. Whole cell patch-clamp recordings were made to analyze the ICa, and [Ca2+]i was measured with fura-2. Cell type was verified after each recording by indirect immunocytochemistry. GH and PRL cells could be divided into two groups: silent and spontaneously active. Silent cells had stable membrane potentials and stable levels of [Ca2+]i. Spontaneously active cells exhibited spontaneous action potentials and large fluctuations in [Ca2+]i. Two types of ICa were found: a low threshold, transient current which was insensitive to the dihydropyridine -Bay 5417 (the negative isomer of Bay K 8644), and a high threshold, sustained current which was enhanced by -Bay 5417. Both types of ICa were present in PRL and GH cells, but each cell type differed quantitatively in the proportion of each current type. While the GH cells had a more prominent, low threshold, transient ICa, the PRL cells had a more prominent, high threshold, sustained ICa. The enhancement of ICa by -Bay 5417 was greater in the PRL cells, which have a larger dihydropyridine-sensitive ICa. Parallel fura-2 measurements showed an increase in [Ca2+]i in response to 50 mM KCl and -Bay 5417 for both lactotrophs and somatotrophs.     

Pituitary adenylate cyclase-activating polypeptide releases 7B2, adrenocorticotrophin, growth hormone and prolactin from the mouse and rat clonal pituitary cell lines AtT-20 and GH3.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide originally isolated from ovine hypothalami and so called because of its ability to stimulate pituitary adenylate cyclase activity. Alternative amidation and proteolytic processing of prepro-PACAP gives rise to two bioactive-amidated forms, PACAP-NH2(1-38) (PACAP-38) and PACAP-NH2(1-27) (PACAP-27). 7B2 is a polypeptide of 185 amino acids which is predominantly found in secretory granules and is widely distributed in rat and human tissues. We investigated the ability of the two forms of PACAP to stimulate GH, prolactin and 7B2 release by the rat pituitary clonal cell line GH3, and ACTH and 7B2 by the mouse pituitary clonal cell line AtT-20. PACAP-38 and PACAP-27 stimulated 7B2 and GH/prolactin or ACTH secretion with a similar efficacy over the 2-h incubation period from GH3 and AtT-20 cells respectively. 7B2 secretion was also stimulated by corticotrophin-releasing factor (CRF-41) and vasoactive intestinal polypeptide (VIP) in AtT-20 cells, and thyrotrophin-releasing hormone (TRH) and VIP in GH3 cells. Addition of PACAP to CRF-41 resulted in an additive effect on ACTH secretion and a synergistic effect on 7B2 secretion in AtT-20 cells. No synergism was observed when PACAP was added together with TRH, either on GH and prolactin secretion or on 7B2 release from GH3 cells. PACAP-mediated 7B2 secretion from both cell lines and PACAP-stimulated ACTH release from AtT-20 cells were reduced by 5 mg octapeptide synthetic somatostatin analogue/l (5 mg SMS 201-995/l).     

Vasoactive intestinal peptide and peptide with N-terminal histidine and C-terminal isoleucine increase prolactin secretion in cultured rat pituitary cells (GH4C1) via a cAMP-dependent mechanism which involves transient elevation of intracellular Ca2+

Vasoactive intestinal peptide (VIP) and peptide (P) with N-terminal histidine and C-terminal isoleucine (PHI) stimulated prolactin (PRL) secretion from GH4C1 cells equipotent with ED50 values of 30-50 nM. In a parafusion system optimized to give high time resolution both VIP and PHI increased PRL secretion with a delay of about 60 s and subsequent to the activation of the adenylate cyclase. Thyroliberin (TRH) increased PRL secretion within 4 s. The dose-response curves for VIP- and PHI-stimulated cAMP accumulation were superimposable on those for PRL secretion. At submaximal concentrations the effects of VIP and PHI on both cAMP accumulation and PRL secretion were additive, whereas the effects were not additive at concentrations giving maximal effects. VIP and PHI increased [Ca2+]i measured by quin-2 in a different way than TRH, without inducing changes in the electrophysiological membrane properties of the GH4C1 cells. We conclude that both VIP and PHI stimulate PRL secretion via a cAMP-dependent process involving an increase in [Ca2+]i.     

U-73122, an aminosteroid phospholipase C antagonist, noncompetitively inhibits thyrotropin-releasing hormone effects in GH3 rat pituitary cells.

TRH increases cytosolic-free calcium ([Ca2+]i) by activating phospholipase C(PL-C), which induces phosphoinositol hydrolysis, leading to Ca2+ mobilization from inositol trisphosphate (IP3) sensitive stores, and by increasing Ca2+ influx. Increases in [Ca2+]i stimulate PRL secretion. We investigated the effects of U-73122, an aminosteroid inhibitor of PL-C dependent processes, on TRH-stimulated second messenger pathways and on PRL secretion in GH3 rat pituitary cells. [Ca2+]i was monitored by Indo-1 fluorescence, and IP3 and metabolites separated on ion exchange columns. In Ca(2+)-free buffer, [Ca2+]i was 96 +/- 6 nM and increased to 323 +/- 23 nM (P less than 0.001) after TRH (100 nM). U-73122 dose dependently inhibited the TRH effect (IC50 = 967 nM; complete inhibition at 3-5 microM). Subsequent addition of monensin (100 microM) increased [Ca2+]i from 107 +/- 4 to 142 +/- 4 nM (P < 0.001), confirming our previous findings of a non-TRH regulated Ca2+ pool in GH3 cells. Pretreatment (15 sec) with U-73122 partly inhibited the TRH effect on [Ca2+]i; complete suppression occurred with 70 sec of pretreatment. An inactive analog (U-73343) had no inhibitory effect at 5 microM. U-73122 acted noncompetitively, as the mean maximum velocity (expressed as percent increase in [Ca2+]i after TRH) was reduced from 225 to 91 while the Michaelis-Menten constant for TRH was unchanged (15.4 vs. 13.8 nM, n = 3). Of note, U-73122, at 3-5 microM, increased basal [Ca2+]i from 109 +/- 5 to 120 +/- 5 nM (P less than 0.001). In 1.3 mM Ca2+ buffer containing nifedipine (1 microM) and verapamil (50 microM), similar effects of U-73122 (5 microM) were observed on basal and TRH-stimulated [Ca2+]i. IP3, IP2, and IP1 increased to 241 +/- 12%, 148 +/- 23%, and 167 +/- 39% of control, 30 sec after TRH (100 nM); these responses were prevented by 1 microM U-73122. At 5 microM, U-73122 also significantly increased IP3 levels. TRH (100 nM) increased 4-h PRL secretion from 16.3 +/- 1.4 to 27.6 +/- 3.2 ng/well (P less than 0.05). U-73122 (5 microM) increased basal PRL secretion to 35.9 +/- 3.2 ng/well (P less than 0.05), but abolished the TRH effect. In contrast, U-73343 (with Ca2+ channel blockers) did not inhibit the TRH effect on PRL (control: 24.3 +/- 2.1; TRH: 51.0 +/- 6.3 ng/well).(ABSTRACT TRUNCATED AT 400 WORDS)     

Vasoactive intestinal polypeptide and pituitary adenylate cyclase-activating polypeptides stimulate mitogen-activated protein kinase in the pituitary cell line GH4C1 by a 3',5'-cyclic adenosine monophosphate pathway

Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP38) regulate anterior pituitary cell secretion and proliferation. In the somatolactotrope GH4C1 cell line, these effects are mediated through the type-II-like PACAP receptor (VPAC2) coupled to the cAMP pathway. In this study, the control of the extracellularly responsive kinases (ERKs) by VIP and PACAP38 was investigated in GH4C1 cells. VIP and PACAP38 increased ERK1 and ERK2 phosphorylation and were equipotent stimulators of both kinases. ERK activation was mimicked by cholera toxin, forskolin and 8bromo-cAMP. VIP and PACAP38 activation of ERK2 was blocked by the protein kinase A inhibitor H89, whereas the protein kinase C inhibitor GF109203X, or prior PMA-induced depletion of the protein kinases C, failed to inhibit VIP and PACAP38 activation of ERK2. In contrast, thyrotropin-releasing hormone (TRH) elicited ERK activation by a PKC-dependent process. ERK activation by VIP or PACAP38 and TRH were additive and both sensitive to the MEK inhibitors PD98059 and U0126. In parallel, U0126 reduced prolactin (PRL) mRNA levels induced by VIP. These results demonstrate for the first time that VIP and PACAP38 activate ERK in GH4C1 cells. Cyclic AMP increase is sufficient to elicit ERK activation in these cells and thus likely to represent the transduction pathway underlying VIP- and PACAP38-dependent ERK activation. This mechanism seems to be involved in VIP-induced PRL gene regulation.     

Differential involvement of nitric oxide signaling in dopamine and PACAP stimulation of growth hormone release in goldfish

Previous studies in goldfish pituitary cells have shown that nitric oxide synthase (NOS)/nitric oxide (NO) signaling is involved in mediating the growth hormone (GH) release response to gonadotropin-releasing hormones. In this study, the involvement of this signaling pathway in mediating the action of two cAMP-mobilizing neuroendocrine stimulators of GH release, pituitary adenylate cyclase-activating polypeptide (PACAP) and dopamine (DA), was investigated in cell column perifusion experiments with primary cultures of dispersed pituitary cells. GH responses to PACAP were unaffected by three NOS inhibitors, aminoguanidine hemisulfate, 1400W and 7-nitroindazole (7-Ni). PACAP-stimulated GH release was also not reduced by two NO scavengers, rutin hydrate and PTIO, but NO-donor sodium nitroprusside (SNP)-elicited GH release was additive to the GH response to PACAP. In contrast, DA-induced GH secretion was reduced by 7-Ni, rutin hydrate and PTIO while not being additive to the GH response induced by SNP. These results indicate that although both PACAP and DA stimulation of acute GH release involve activation of adenylate cyclase/cAMP, DA- but not PACAP-signaling also utilizes the NOS/NO second messenger system.     

Effects of calcitonin on basal and thyrotropin-releasing hormone-stimulated prolactin secretion in man

Plasma PRL fell in nine healthy subjects and four patients with hyperprolactinemia after iv administration of salmon calcitonin (CT). The maximum fall was observed 30--60 min after the infusion. There was no change in the plasma concentrations of the other anterior pituitary hormones tested (GH, FSH, LH, and TSH). In five healthy subjects, TRH was injected 60 min after the CT infusion. This protocol was repeated in the same subjects at 3-day intervals, except CT was not administered. Plasma PRL before TRH injection was clearly lower when CT had been administered. Plasma concentrations of the other anterior pituitary hormones did not change. PRL and TSH responses to TRH were markedly inhibited when CT had been previously infused. These observations are in agreement with preceeding studies showing a similar effect of CT on the plasma concentration of various other polypeptide hormones. This general effect of CT could be attributed to a change in intracellular calcium of the secreting cells.