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.     

Effects of a novel hypothalamic peptide, pituitary adenylate cyclase-activating polypeptide, on pituitary hormone release in rats.

We have demonstrated that the novel hypothalamic peptide pituitary adenylate cyclase-activating polypeptide (PACAP-38; 0.1-100 nmol/l) caused an increase in the release of GH, ACTH, LH and alpha-subunit and accumulation of intracellular cyclic AMP from dispersed rat anterior pituitary cells in static culture for 24 h. There were no significant effects on TSH or prolactin release over the same time-period. PACAP-38 (10 nmol/l) increased the release of GH by 1.3-fold (P less than 0.05), ACTH by 1.9-fold (P less than 0.05), LH by 3.5-fold (P less than 0.001) and alpha-subunit by 2.0-fold (P less than 0.005) and the accumulation of intracellular cyclic AMP by greater than 2-fold (P less than 0.001) after 24 h. However, the time-course for the effect of PACAP-38 (1 mmol/l) on hormone release and intracellular cyclic AMP levels showed a temporal dissociation. The effect of PACAP-38 on GH and ACTH levels did not reach significance until 24 h whereas the effect of PACAP-38 on LH and alpha-subunit release reached significance after 4 h implying a different mechanism of action for their release. To investigate the PACAP-induced secretion of LH and alpha-subunit further, we examined the effects of PACAP after down-regulation of protein kinase C (PKC). PACAP-38 at a dose maximal for the stimulation of LH and alpha-subunit release (10 nmol/l) added together with the PKC activator, 12-O-tetradecanoyl-phorbol-13-acetate (TPA; 0.1 mumol/l) had no greater effect on LH and alpha-subunit release than TPA alone over a 4 h incubation period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Atrial natriuretic factor does not affect basal, forskolin- and CRF-stimulated adenylate cyclase activity, cAMP formation or ACTH secretion, but does stimulate cGMP synthesis in anterior pituitary

The report that ANF inhibits basal and CRF-stimulated adenylate cyclase activity in anterior pituitary homogenates suggested that the atrial peptide could inhibit ACTH secretion. This possibility was investigated in the ACTH-secreting AtT-20 mouse pituitary tumor cell line as well as homogenates or primary cell cultures from rat anterior hypophysis. ANF (up to 5 X 10(-7) M) was found to be completely ineffective in stimulating basal, CRF- and/or forskolin-stimulated adenylate cyclase activity, cAMP accumulation and ACTH secretion. Similarly, ANF had no effect on spontaneous or GRF-induced GH release from cells in primary culture. ANF receptors, however, are present in AtT-20 cells and anterior pituitary cells as evidenced by the ability of the peptide to stimulate intracellular cGMP accumulation. The data, therefore, suggests that ANF does not have a negative modulatory action on the secretory function of anterior pituitary. The role of cGMP in any other action(s) of ANF remains unknown.     

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.     

Pituitary adenylate cyclase-activating polypeptide (PACAP) potentiates the gonadotropin-releasing activity of luteinizing hormone-releasing hormone

In order to determine if the newly discovered neuropeptide, pituitary adenylate cyclase activating polypeptide (PACAP), interacts with the known hypothalamic releasing factors to modulate pituitary hormone secretion, the effect of PACAP, either alone or in combination with either LHRH, TRH, CRF or GHRH, was examined in rat anterior pituitary cell cultures. While PACAP alone weakly stimulated LH and FSH release, PACAP and LHRH, in combination, interacted synergistically to stimulate gonadotropin secretion. No significant changes in the secretion of either TSH, ACTH, or GH were observed in response to PACAP, either alone or in combination with the other releasing factors. Addition of an LHRH antagonist demonstrated that the PACAP effect on gonadotropin release was neither mediated by the LHRH receptor nor the result of LHRH contamination of the PACAP preparation. Because of the sequence homology (68%) between the N-terminal 28 amino acids of PACAP and VIP, the addition of a VIP antagonist was used to demonstrate that the PACAP effect is not mediated through the VIP receptor. The observation that PACAP interacts synergistically with LHRH in stimulating gonadotropin release suggests intriguing possibilities for PACAP in regulating gonadotropin secretion and reproductive function.     

Stimulatory effect of pituitary adenylate-cyclase activating polypeptide (PACAP) and its PACAP type I receptor (PAC1R) on prolactin synthesis in rat pituitary somatolactotroph GH3 cells

In this present study, we investigated the role of pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptor, PACAP type I receptor (PAC1R) on prolactin synthesis in pituitary somatolactotroph GH3 cells. PACAP increased prolactin promoter activity up to 1.3 ± 0.1-fold. This increase, while significant, was less than the increase resulting from thyrotropin-releasing hormone (TRH) stimulation. By transfection of a PAC1R expression vector to the cells, the response to PACAP on prolactin promoter activity was dramatically potentiated to a degree proportional to the amount of PAC1R transfected. In the PAC1R expressing GH3 cells, TRH and PACAP alone increased prolactin promoter up to 3.3 ± 0.3-fold and 4.9 ± 0.2-fold, respectively, and combined treatment with TRH and PACAP further increased prolactin promoters up to 6.8 ± 0.6-fold. PACAP binds both Gs- and Gq-coupled receptors and stimulates adenylate cyclase/cAMP and protein kinase C/extracellular signal-regulated kinase (ERK) signaling pathways. PACAP increased ERK phosphorylation in PAC1R expressing cells to the same degree as TRH. Combined treatment with TRH and PACAP had a synergistic effect on ERK activation. GH3 cells produce both prolactin and growth hormone. Stimulation of GH3 cells with TRH significantly increased the mRNA level of prolactin and attenuated growth hormone mRNA expression. PACAP increased both prolactin and growth hormone mRNA levels, particularly in PAC1R expressing cells. In addition, increasing amount of PAC1R in GH3 cells potentiated the action of TRH on prolactin promoter activity, as well as on ERK phosphorylation. PAC1R was induced by PACAP itself, but not by TRH. Our current study demonstrates that PACAP and its PAC1R, functions as a stimulator of prolactin alone or with TRH in prolactin producing cells.     

Somatostatin inhibits multireceptor stimulation of cyclic AMP formation and corticotropin secretion in mouse pituitary tumor cells.

The AtT-20/D16-16 mouse pituitary tumor cell secretes corticotropin (ACTH) in response to corticotropin-releasing factor (CRF), (-)-isoproterenol, and vasoactive intestinal peptide (VIP). These responses are associated with a rapid increase in cyclic AMP formation. Somatostatin (SRIF) markedly decreases the stimulatory effect of CRF, (-)-isoproterenol, and VIP on both cyclic AMP formation and immunoreactive ACTH secretion. Forskolin and cholera toxin, adenylate cyclase activators, also stimulate cyclic AMP formation and ACTH secretion in AtT-20 cells and these responses are all inhibited by SRIF. The ACTH secretory responses to melittin and to the calcium ionophore A23187, neither of which increases cyclic AMP in AtT-20 cells, were not inhibited by SRIF. SRIF did not affect the binding of a tritiated beta-adrenergic receptor antagonist to AtT-20 membranes nor did it decrease basal cyclic AMP formation even in the presence of excess phosphodiesterase inhibitor, indicating that the reduction of cyclic AMP levels by SRIF did not involve either an interference with beta-adrenergic agonist binding to receptors or stimulation of cyclic AMP degradation. These results indicate that the inhibition of CRF-, (-)-isoproterenol-, and VIP-stimulated ACTH secretion by SRIF may be regulated by its inhibitory action on adenylate cyclase.     

Prolonged stimulation with thyrotropin-releasing hormone and pituitary adenylate cyclase-activating polypeptide desensitize their receptor functions in prolactin-producing GH3 cells

We used somatolactotroph GH3 cells to examine changes in response to stimulation with thyrotropin-releasing hormone (TRH) and pituitary adenylate cyclase-activating polypeptide (PACAP) after sustained treatment with these peptides. TRH and PACAP increased prolactin promoter activity in mock- and PACAP type 1 receptor (PAC1R)-transfected cells. When the cells were pretreated with TRH for 48 h, the response of the prolactin promoter to both TRH and PACAP was diminished. Similarly, in PAC1R-transfected GH3 cells pretreated with PACAP, the effects of TRH and PACAP on the prolactin promoter were eliminated. The stimulation of prolactin mRNA expression by TRH and PACAP was eliminated by prolonged pretreatment with these peptides in PAC1R-transfected cells. Both the serum response element (SRE) promoters and cAMP response element (CRE) promoters were activated by TRH and PACAP in either mock- or PAC1R-transfected cells. Pretreatment for 48 h with TRH also eliminated the effects of TRH and PACAP on the SRE and CRE promoters, and pretreatment of PAC1R-transfected cells with PACAP for 48 h reduced the responses of the SRE and CRE promoters to TRH and PACAP. These observations demonstrated that sustained stimulation with TRH and PACAP desensitizes their own and each other’s receptors.     

Vasoactive intestinal polypeptide- and pituitary adenylate cyclase activating polypeptide-mediated control of catecholamine release from chromaffin tissue in the rainbow trout, Oncorhynchus mykiss

The aim of the present investigation was to assess the relative contributions of cholinergic (acetylcholine) and non-cholinergic vasoactive intestinal polypeptide (VIP), and pituitary adenylate cyclase activating polypeptide (PACAP) neurotransmitters in the neuronal control of catecholamine secretion from the chromaffin tissue lining the posterior cardinal vein of the rainbow trout (Oncorhynchus mykiss). Using an in situ saline-perfused posterior cardinal vein preparation, it was demonstrated that exogenous administration of chicken VIP or human PACAP-27 caused a dose-dependent increase in adrenaline secretion; noradrenaline secretion was unaffected. Analysis of dose-response curves indicated that VIP and PACAP stimulated the secretion of adrenaline with a similar degree of potency (ED(50) for VIP=1.90x10(-11) mol/kg; ED(50) for PACAP=1.03x10(-11) mol/kg). The VIP/PACAP-elicited secretion was diminished in the presence of the VIP receptor antagonist, VIP 6-28, but was unaffected by the PACAP receptor antagonist, PACAP 6-27, or the cholinergic antagonists, hexamethonium and atropine. Thus, this is the first study to demonstrate a direct stimulatory role for VIP or PACAP in catecholamine secretion from piscine chromaffin cells. The relative contribution of cholinergic and non-cholinergic neurotransmitters in the neuronal control of catecholamine secretion from the chromaffin tissue was evaluated using an in situ nerve-stimulating technique previously validated by us in the rainbow trout. This was accomplished by comparing catecholamine secretion in the presence or absence of cholinergic and the VIP and PACAP receptor antagonists during different levels of electrical stimulation. The results demonstrated that cholinergic stimulation predominated during high frequency of electrical stimulation (20 Hz) while the non-cholinergic component prevailed at low frequency (1 Hz). Overall, the results of the present investigation demonstrate that VIP and/or PACAP may directly stimulate adrenaline secretion from trout chromaffin cells at low levels of neuronal activity. Therefore, the neuronal control of catecholamine secretion in teleosts may not be confined to cholinergic-evoked events.     

Pituitary adenylate cyclase-activating polypeptide stimulates secretion in T84 cells.

Pituitary adenylate-cyclase-activating peptide (PA-CAP) and PACAP-27 are novel hypothalamic peptides that can stimulate adenylate cyclase in cultured anterior pituitary cells. Because these peptides are present in the gut and are homologous with vasoactive intestinal peptide (VIP), itself known to stimulate intestinal ion transport, we examined the effects of these peptides on the T84 colonocyte cell line. Using cells grown on semipermeable supports and mounted in Ussing chambers, we showed that PACAP and PACAP-27 potently activate intestinal secretion. The half-maximal secretory response was produced with 0.5 nmol/L PA-CAP and 0.1 nmol/L PACAP-27. PACAP resembled VIP in that it stimulated a secretory response potentiated by carbachol, inhibited by bumetanide and barium chloride, and not further stimulated by the subsequent addition of VIP. Like VIP, PACAP also stimulated 5' cyclic adenosine monophosphate (cAMP) production and the phosphorylation of cellular proteins known to be substrates for cAMP-dependent protein kinase. In addition, PACAP inhibited 125I-VIP binding to T84 cells, and the secretion it stimulated was reduced by the VIP receptor antagonist, L-8-K. Thus PACAP and PACAP-27 potently stimulate colonocyte ion transport via mechanisms mediated by the VIP receptor and cAMP-dependent signaling.     

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.     

Possible key residues that determine left gastric artery blood flow response to PACAP in dogs

AIM:To determine the effect of pituitary adenylate cy-clase-activating polypeptide (PACAP) on left gastric artery (LGA) flow and to unveil the structural or functional important sites that may be critical for discrimination of different receptor subtypes. METHODS: Peptides, including PACAP-27, PACAP-38, amino acid substituted PACAP-27 and C-terminus truncated analogues PACAP (27-38), were synthesized by a simultaneous multiple solid-phase peptide synthesizer. Flow probes of an ultrasound transit-time blood ...     

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.     

Pituitary vasoactive intestinal peptide regulates prolactin secretion in the hypothyroid rat

In this study, we demonstrated that the cell content and basal secretion of vasoactive intestinal peptide (VIP) in primary rat pituitary cell cultures were increased in hypothyroidism. VIP release from hypothyroid pituitary cells in vitro was stimulated by thyrotropin releasing hormone (TRH 10(-8) to 10(-6) M) and growth hormone (GH)-releasing hormone (GHRH 10(-9) to 10(-8) M) but not by corticotropin-releasing hormone or luteinizing hormone-releasing hormone in concentrations up to 10(-6) M. In the presence of anti-VIP antisera, there was a significant decrease in basal prolactin secretion from cultured hypothyroid pituitary cells (p less than 0.005) indicating that VIP exerts a tonic stimulatory effect on prolactin (PRL) secretion. The increment in PRL secretion following TRH was not affected by exposure to anti-VIP indicating that PRL release after TRH is not mediated by VIP at the pituitary level. In contrast to changes in PRL, exposure to anti-VIP had no effect on basal GH secretion, indicating that the PRL changes are hormone specific. Similarly, GHRH-induced GH release was unaffected by VIP immunoneutralization.     

Over-expression of NCS-1 in AtT-20 cells affects ACTH secretion and storage.

The effect of over-expressing neuronal calcium sensor 1 (NCS-1) upon stimulated adrenocorticotrophin (ACTH) secretion was studied in AtT-20 cells. Stably-transfected AtT-20 cell lines over-expressing NCS-1 were obtained and compared to wild type AtT-20 cells. Corticotrophin releasing factor (CRF-41)-stimulated ACTH secretion from NCS-1 over-expressing cells was significantly reduced from that obtained in wild type AtT-20 cells. The effects of other stimulants of ACTH secretion from wild type AtT-20 cells were not attenuated in NCS-1 over-expressing cells. Calcium, guanosine 5'-O-(3'-thiotriphosphate) (GTP-gamma-S) and mastoparan stimulated ACTH secretion from permeabilised wild type AtT-20 and NCS-1 over-expressing AtT-20 cells with significantly greater ACTH secretion obtained in NCS-1 over-expressing cells. This study shows that in intact cells over-expression of NCS-1 reduces exocytotic ACTH release, while in permeabilised cells increases ACTH release. NCS-1 has multiple cellular targets and that directly and indirectly via these targets acts to increase the releasable ACTH pool while inhibiting CRF-41 stimulus-secretion coupling.     

Characterization and distribution of binding sites for the hypothalamic peptide, pituitary adenylate cyclase-activating polypeptide

A novel bioactive peptide was recently isolated from ovine hypothalamus and was named PACAP (pituitary adenylate cyclase-activating polypeptide). PACAP was present in two bioactive, amidated forms, PACAP27 and PACAP38 (27 and 38 amino acids, respectively), and showed a 68% sequence homology with vasoactive intestinal peptide (VIP) in the N-terminal 28 residues. PACAP38 was at least 1000 times more potent than VIP in stimulating adenylate cyclase in pituitary cells, but both peptides exhibited comparable vasodepressor activity. Thus, we sought to determine whether PACAP acts on specific binding sites in the anterior pituitary or other tissues and whether these binding sites are different from those of VIP. Binding of [125I] PACAP27 to freshly prepared rat anterior pituitary membranes in the presence and absence of 212 nM unlabeled PACAP27 was specific, saturable, and more rapid at 22 C than at 4 C. Scatchard analysis of this binding site using increasing doses of unlabeled PACAP27 revealed a single high affinity site with a Kd of 446 +/- 141 pM and a maximum number of sites of 1312 +/- 182 fmol/mg protein. These results do not exclude the possibility of a second pituitary binding site with significantly lower affinity. Unlabeled PACAP38 and PACAP38OH exhibited significantly higher affinity binding (3- to 5-fold) than PACAP27 with a similar number of pituitary sites. A variable distribution of binding sites was observed between PACAP27 and VIP when binding to different tissue membranes was measured with 125I-labeled peptides. Very high specific binding of both PACAP27 and VIP was observed in lung membranes. An almost identical relative magnitude of binding was observed between PACAP27 and VIP in lung, liver, duodenum, ovary, and thymus. However, whereas PACAP27 binding to hypothalamic and pituitary membranes was great, VIP binding to these tissues was almost absent. To determine if VIP and PACAP might share a binding site in peripheral tissues, displacement curves were generated using [125I]PACAP27 binding to lung membranes and VIP, PACAP27, and PACAP38 as unlabeled ligands. VIP was highly potent in displacing [125I] PACAP27 binding in lung membrane, and the IC50 values for all three of these peptides were between 1-10 nM. These results suggest that 1) a saturable, high affinity binding site for PACAP is present on anterior pituitary membranes; 2) PACAP27 and PACAP38, but not VIP, share this binding site in the anterior pituitary and possibly the hypothalamus; and 3) PACAP27, PACAP38, and VIP share a similar or identical binding site on lung membranes and possibly other peripheral tissues.     

Pyridoxal phosphate inhibits pituitary cell proliferation and hormone secretion

Pyridoxal phosphate (PLP), a bioactive form of pyridoxine, dose-dependently (10-1000 microm) inhibited cell proliferation in rat pituitary MMQ and GH3 cells and in mouse AtT-20 cells. After 4 d, MMQ cell numbers were reduced by up to 81%, GH3 cell numbers were reduced by up to 64% (P < 0.05), and AtT-20 cell numbers were reduced by up to 90%. Cell proliferation rates recovered and dose-dependently reverted to control levels after PLP withdrawal. After 4 d, PLP (400 and 1000 microm) decreased [3H]thymidine incorporation by up to 71% (P < 0.05). PLP (400-1000 microm) reduced GH3 cell GH and prolactin secretion and AtT-20 cell ACTH secretion (adjusted for cell number) by approximately 70% after 2 d. The 100 microm PLP also inhibited prolactin secretion (65%, P < 0.05) in primary rat pituitary cells treated for 2 d. PLP decreased the percentage of AtT-20 and GH3 cells in S phase and increased those in G0-G1 phase. Furthermore, PLP induced AtT-20 and GH3 cell apoptosis (28 vs. 6, P < 0.05; 26 vs. 3, P < 0.05, respectively) and dose-dependently reduced content of the antiapoptosis gene Bcl-2. These results indicate that pharmacological doses of PLP inhibit pituitary cell proliferation and hormone secretion, in part mediated through PLP-induced cell-cycle arrest and apoptosis. Pyridoxine may therefore be appropriate for testing as a relatively safe drug for adjuvant treatment of hormone-secreting pituitary adenomas.     

A novel role for endogenous pituitary adenylate cyclase activating polypeptide in the magnocellular neuroendocrine system

Central release of vasopressin (VP) by the magnocellular neuroendocrine cells (MNCs) responsible for systemic VP release is believed to be important in modulating the activity of these neurons during dehydration. Central VP release from MNC somata and dendrites is stimulated by both dehydration and pituitary adenylate cyclase activating polypeptide (PACAP). Although PACAP is expressed in MNCs, its potential role in the magnocellular response to dehydration is unexplored. The current study demonstrates that prolonged dehydration increases immunoreactivity for PACAP-27, PACAP-38, and the type I PACAP receptor in the supraoptic nucleus (SON) of the rat. In addition, PACAP stimulates local VP release in the euhydrated rat SON in vitro, and this effect is reduced by the PACAP receptor antagonist PAC(6-27) (100 nm), suggesting the participation of PACAP receptors. Concomitant with its effects on local VP release, PACAP also reduces basal glutamate and aspartate release in the euhydrated rat SON. Furthermore, somatodendritic VP release elicited by acute dehydration is blocked by PAC(6-27), suggesting that endogenous PACAP participates in this response. Consistent with this, RIA revealed that local PACAP-38 release within the SON is significantly elevated during acute dehydration. These results suggest that prolonged activation of hypothalamic MNCs is accompanied by up-regulation of PACAP and the type I PACAP receptor in these cells and that somatodendritic VP release in response to acute dehydration is mediated by activation of PACAP receptors by endogenous PACAP released within the SON. A potential role for PACAP in promoting efficient, but not exhaustive, systemic release of VP from MNCs during physiological challenge is discussed.