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, interleukin-6 and glucocorticoids regulate the release of vascular endothelial growth factor in pituitary folliculostellate cells

There is increasing evidence that hormones play an important role in the control of endothelial cell function and growth by regulating the production of vascular endothelial growth factor (VEGF). VEGF regulates vascular permeability and represents the most powerful growth factor for endothelial cells. In the normal anterior pituitary, VEGF has been detected only in folliculostellate (FS) cells. In the present study, the regulation of the release of VEGF from FS-like mouse TtT/GF cells, and from FS cells of rat pituitary monolayer cell cultures was investigated using a specific VEGF ELISA. Basal release of VEGF was demonstrated in cultures of both TtT/GF cells and rat pituitary cells. Interestingly, the VEGF secretion was stimulated by both forms of pituitary adenylate cyclase-activating polypeptide (PACAP-38 and PACAP-27), indicating that this hypothalamic peptide regulates endothelial cell function and growth within the pituitary. VEGF secretion was also stimulated by interleukin-6 (IL-6) whereas basal, IL-6- and PACAP-stimulated secretion was inhibited by the synthetic glucocorticoid dexamethasone. The inhibitory action of dexamethasone was reversed by the glucocorticoid receptor antagonist RU486, suggesting that in FS cells functional glucocorticoid receptors mediate the inhibitory action of glucocorticoids on the VEGF secretion. The endocrine and auto-/paracrine control of VEGF production in pituitary FS cells by PACAP, IL-6 and glucocorticoids may play an important role both in angiogenesis and vascular permeability regulation within the pituitary under physiological and pathophysiological conditions.     

Interactions of ovarian steroids with pituitary adenylate cyclase-activating polypeptide and GnRH in anterior pituitary cells

Pituitary adenylate cyclase-activating polypeptide (PACAP) releases LH and FSH from anterior pituitary cells. Although this effect is relatively weak, it has a strong sensitizing action on GnRH-induced gonadotropin secretion. Here we investigated the possibility that ovarian steroids, which are well-known modulators of LH secretion, interact with PACAP and GnRH in pituitary gonadotrophs. Rat pituitary cells were treated for 48 h with vehicle, 1 nmol/l estradiol, 1 nmol/l estradiol + 100 nmol/l progesterone or 48 h with 1 nmol/l estradiol and 4 h with 100 nmol/l progesterone. The cells were stimulated for 3 h with 1 nmol/l GnRH or 100 nmol/l PACAP. Estradiol treatment alone enhanced basal as well as GnRH- or PACAP-stimulated LH secretion. LH release was facilitated by additional short-term progesterone treatment. Long-term treatment with estradiol and progesterone led to reduced LH responses to GnRH and PACAP. Neither treatment paradigms affected cAMP production. However, estradiol treatment led to enhanced cAMP accumulation in quiescent or GnRH-stimulated cells. PACAP-induced increases of cAMP production were inhibited by estradiol treatment. After 7-h preincubation with 10 nmol/l PACAP, cells responded with enhanced LH secretion to GnRH stimulation. When steroid pretreatment was performed the responsiveness of gonadotrophs to low concentrations of GnRH was still increased. In contrast, at high concentrations of GnRH the sensitizing action of PACAP on agonist-induced LH secretion was lost in steroid-treated cells. There were no significant differences between the steroid treatment paradigms. It is concluded that estradiol but not progesterone acts as a modulator of adenylyl cyclase in gonadotrophs. The stimulatory effect of estradiol is thought to be involved in its sensitizing action on agonist-induced LH secretion. The inhibitory effect of estradiol on PACAP-stimulated adenylyl cyclase activities seems to be responsible for the loss of its action to sensitize LH secretory responses to GnRH.     

Production of interleukin-6 by anterior pituitary cells is stimulated by increased intracellular adenosine 3',5'-monophosphate and vasoactive intestinal peptide

Interleukin-6 (IL-6) is an inflammatory cytokine that is produced by a variety of cells and tissues. We recently demonstrated that IL-6 is produced by anterior pituitary cells in response to the bacterial endotoxin lipopolysaccharide and phorbol diester in vitro. Lipopolysaccharide (0.01-100 ng/ml) increased, whereas dexamethasone (0.1-100 nM) decreased, IL-6 production by anterior pituitary cells in vitro as measured by the 7TD1 cell growth factor assay. In addition, we now report that IL-6 production by anterior pituitary cells is stimulated by agents that elevate intracellular cAMP concentrations. Exposure of anterior pituitary cells to (Bu)2cAMP (0.01-10 mM), prostaglandin E2 (1.0-1000 nM), forskolin (50-1000 nM), or cholera toxin (0.25-250 ng/ml) for 6 h resulted in concentration-related increases in the production of IL-6, which, in the cases of forskolin and cholera toxin, correlated well with increased intracellular cAMP concentrations. Vasoactive intestinal peptide (1-1000 nM), which stimulates adenylate cyclase activity in the anterior pituitary, caused a concentration-related enhancement of IL-6 production that was unaffected in the presence of 10-100 nM somatostatin. In contrast, GH-releasing factor had no effect on IL-6 production. These data suggest that anterior pituitary cells produce IL-6 in response to increased intracellular cAMP, and that the neuropeptide vasoactive intestinal peptide may act to regulate IL-6 production.     

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.     

Adenosine-sensitive adenylate cyclase in rat anterior pituitary

An adenosine-sensitive adenylate cyclase has been demonstrated in anterior pituitary cultured cells in the present studies. N-ethylcarboxamideadenosine (NECA), L-N6-phenylisopropyladenosine (PIA), and 5'-N-methylcarboxamideadenosine (MECA), all stimulated adenylate cyclase in a concentration-dependent manner in the order of potency NECA greater than PIA greater than MECA. Adenosine showed a biphasic effect on adenylate cyclase: stimulation at lower and inhibition at higher concentrations, whereas 2'-deoxyadenosine only inhibited adenylate cyclase in a concentration-dependent manner. The stimulatory effect of NECA on adenylate cyclase was dependent on metal ion concentrations and was blocked by 3-isobutyl-1-methylxanthine and 8-phenyltheophylline. Various agonists such as isoproterenol, prostaglandins (PGE1), vasoactive intestinal peptide, corticotropin-releasing factor, NaF, and forskolin, all stimulated adenylate cyclase to various degrees. The stimulatory effect of vasoactive intestinal peptide and corticotropin-releasing factor on adenylate cyclase was found to be almost additive with the stimulation exerted by NECA. These data indicate the presence of adenosine stimulatory receptors ('Ra') in anterior pituitary which are coupled to adenylate cyclase. It is possible that adenosine may act as one of the important regulators to regulate and/or modulate the effects of agents/factors in the release of pituitary hormones.     

Pituitary adenylate cyclase polypeptide (PACAP) stimulates cyclic AMP formation in pituitary fibroblasts and 3T3 tumor fibroblasts: lack of enhancement by protein kinase C activation.

A number of neuropeptides were shown to produce potent mitogenic effects on Swiss 3T3 fibroblasts by activating the phospholipase C pathway. Here we provide evidence for the activation by PACAP of the adenylate cyclase pathway in 3T3, as well as in non-tumoral pituitary fibroblasts, similarly to what was seen in pituitary endocrine cells. In these cells, PACAP triggered elevation of both intracellular and extracellular contents of cAMP and the effect was time- and dose-dependent, with half-maximal stimulations being induced with about 0.1 nM. Following activation of protein kinase C (PKC) by the phorbol ester phorbol 12-myristate 13-acetate (PMA), PACAP-induced cAMP production was amplified in pituitary endocrine cells, but was either unchanged or dampened in 3T3 and pituitary fibroblasts, respectively. Pretreatment of cells with pertussis toxin (PT) failed to change the effect of PMA on PACAP-stimulated adenylate cyclase activity, irrespective of the cell type being used. However, PT dramatically reduced the potentiation by PMA of cAMP production enhanced by forskolin in 3T3 cells. These results provide new evidence pointing to the presence in fibroblasts of receptors for PACAP, coupled to cAMP production, which may play a role in the modulation of the mitogenic signal. They also indicate that, compared with pituitary endocrine cells, PKC activation in fibroblasts differentially affected PACAP-induced cAMP formation and that these effects were unaltered upon inhibition by PT of Gi-like proteins.     

垂体腺苷酸环化酶激活多肽对胰腺癌细胞的生长调控

目的 观察垂体腺苷酸环化酶激活多肽（pituitary adenylate cyclase activating polypeptide,PACAP)对人胰腺癌细胞株的生长调控作用;并确定神经鞘磷脂是否作为第二信使参与受体后信息传导。方法 人胰腺癌细胞株JF305,HS766T,ASPC－1进行细胞培养,传代。分别加入不同浓度的PACAP1－38（10^-6-10^-12mol/L)于三种癌细胞中。应用MTT法观察细胞增程度。薄层层析法测定细胞神经鞘磷脂。放射免疫法测定细胞内cAMP含量。Fura-2/AM测定细胞内游离Ca^2 浓度。结果 PACAP1－38促进JF305,HS766T,ASPC－1细胞的生长;PACAP1－38增加细胞内神经鞘磷脂、cAMP、Ca^2 的生长;生长抑素可明显抑制PACAP1－38诱导的JF305细胞的生长等作用。结论 PACAP1－38促进人胰腺癌细胞株的增殖。PACAP受体后信息传递途径;（1）腺苷 酸环化酶途径;（2）钙－钙调素途径。神经鞘磷脂作为第二信使也参与此过程。     

Adenosine and its analogue (-)-N6-R-phenyl-isopropyladenosine modulate anterior pituitary adenylate cyclase activity and prolactin secretion in the rat

The effect of adenosine and its analogue (-)-N6-R-phenylisopropyladenosine (PIA) on both anterior pituitary adenylate cyclase activity and prolactin secretion was examined in the rat. Adenosine inhibited basal adenylate cyclase activity in a dose-dependent manner and also reduced the stimulation of the enzyme by vasoactive intestinal peptide (VIP). Likewise, in primary cultures of anterior pituitary cells, adenosine decreased prolactin secretion in both basal and VIP-stimulated conditions. In perifusion experiments, adenosine also inhibited prolactin release in both basal and TRH-stimulated conditions. PIA produced a biphasic pattern of response of basal adenylate cyclase activity, being inhibitory at low and stimulatory at high concentrations. In VIP-stimulated conditions, low concentrations of PIA inhibited both adenylate cyclase activity and prolactin release from primary cultures of pituitary cells, while no additive stimulatory effect was seen at high concentrations. Similarly, low concentrations of PIA reduced both basal and TRH-stimulated prolactin release from perifused pituitaries, while increasing PIA concentrations restored prolactin release. These data show that adenosine affects basal and stimulated prolactin secretion from anterior pituitary cells. Adenosine receptors seem to be coupled to the adenylate cyclase system in the anterior pituitary gland, suggesting a possible relationship between the effect of adenosine on adenylate cyclase activity and prolactin secretion.     

Inhibition of mitogen-stimulated proliferation of murine splenocytes by a novel neuropeptide, pituitary adenylate cyclase activating polypeptide: a comparative study with vasoactive intestinal peptide

Two novel polypeptides known as pituitary adenylate cyclase activating polypeptide with 38 residues (PACAP38) and a shorter form of the peptide corresponding to the N-terminal 27 residues (PACAP27) were isolated from ovine hypothalamus. The N-terminal 28 residues of PACAP show 68% homology with vasoactive intestinal peptide (VIP). VIP has been reported to have specific binding sites in lymphocytes and inhibit mitogen-stimulated lymphocyte proliferation through a receptor-mediated stimulation of cAMP-dependent protein kinase. Using concanavalin A-induced proliferation of murine splenocytes as a model system, we now report that both PACAP38 and PACAP 27 can inhibit the proliferation of these cells in the same dose-dependent manner as VIP. The minimal effective concentration of the PACAPs was 10(-10)-10(-9) M. However, neither PACAP affected lipopolysaccharide-induced proliferation of murine splenocytes. The binding of [125I]PACAP27 to these splenocytes was rapid, time dependent, reversible, and proportional to the numbers of murine splenocytes. Scatchard analysis of displacement of the bound tracer by unlabeled PACAP27 indicated the existence of two classes of binding sites. The dissociation constant (Kd) was 0.86 +/- 0.24 nM and the maximal binding capacity (Bmax) was 1.13 +/- 0.39 fmol/10(6) cells for the high affinity binding site. The low affinity binding site had a Kd of 0.13 +/- 0.03 microM with a Bmax of 73.5 +/- 9.5 fmol/10(6) cells. PACAP38 and VIP displaced the binding of [125I]PACAP27 in the same manner as PACAP27 and Scatchard analyses indicated the presence of two classes of binding sites with Kd and Bmax similar to those for PACAP27. Furthermore, when [125I]VIP was used as a radiolabeled ligand, PACAP27 and PACAP38 displaced the [125I]VIP binding to the same degree as unlabeled VIP. Scatchard analysis indicated that there was no significant difference of the Kd or Bmax between PACAP and VIP. Taken together, these data suggest that PACAPs bind to a site similar or identical to that used by VIP which inhibit the proliferation of murine splenocytes induced by concanavalin A.     

Primary structure of frog pituitary adenylate cyclase-activating polypeptide (PACAP) and effects of ovine PACAP on frog pituitary.

Pituitary adenylate cyclase-activating polypeptide (PACAP), a peptide of the glucagon-secretin-vasoactive intestinal polypeptide superfamily, was isolated in pure form from the brain of the European green frog, Rana ridibunda. The primary structure of the peptide indicates that evolutionary pressure to conserve the complete amino acid sequence has been very strong. Frog PACAP comprises 38 amino acid residues and contains only 1 substitution (isoleucine for valine at position 35) compared with human/ovine/rat PACAP. In the presence of the phosphodiesterase inhibitor isobutylmethylxanthine, synthetic ovine PACAP-(1-38) produced a dose-dependent increase in the concentration of cAMP in isolated frog anterior pituitary fragments (ED50 = 2.1 +/- 0.6 x 10(-7) M; mean +/- SE; n = 6). Maximum stimulation (an approximately 8-fold increase in concentration over basal values) was produced by 10(-6) M peptide. The truncated form of PACAP [PACAP-(1-27)] also produced a dose-dependent increase in cAMP in frog anterior pituitary fragments, and the potency of the peptide (ED50 = 5.9 +/- 0.6 x 10(-8) M) was comparable to that of PACAP-(1-38). The data suggest, therefore, that the function as well as the structure of PACAP have been conserved during the evolution of amphibia to mammals.     

Interleukin 1 potentiates the secretion of beta-endorphin induced by secretagogues in a mouse pituitary cell line (AtT-20).

Previous work has shown that corticotropin releasing factor, vasoactive intestinal peptide, phorbol ester, and forskolin cause the secretion of adrenocorticotropic hormone and beta-endorphin from the AtT-20 mouse pituitary cell line. Human recombinant interleukin 1 alpha and 1 beta also stimulated adrenocorticotropic hormone and beta-endorphin secretion from AtT-20 cells in a time- and dose-related manner. The effect appeared only after pretreatment with interleukin 1 (IL-1) for at least 18 hr and was maximum at 24 hr. After pretreatment of the cells over a period of time with IL-1, the secretion induced by corticotropin releasing factor and vasoactive intestinal peptide was increased in more than an additive manner. The enhancement of corticotropin releasing factor-induced beta-endorphin release produced by IL-1 was apparent after 12 hr and reached a maximum at 24 hr. IL-1 did not affect forskolin-induced cAMP generation but enhanced the effect of forskolin on beta-endorphin secretion. This suggests that IL-1 does not induce adenylate cyclase and that forskolin causes the secretion of beta-endorphin by a mechanism independent of cAMP. IL-1 enhanced phorbol ester-induced beta-endorphin secretion. After prolonged treatment with phorbol ester (an activator of protein kinase C), the secretion induced by phorbol ester was abolished as well as the enhancement induced by IL-1. However, prolonged treatment with phorbol ester had no effect on IL-1-induced beta-endorphin secretion. These observations suggest that IL-1 enhances peptide-generated secretion of beta-endorphin by inducing protein kinase C.     

Pituitary adenylate cyclase-activating polypeptide stimulates calcium mobilization in amphibian pituitary cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 38-amino acid peptide of the glucagon-secretin-vasoactive intestinal polypeptide superfamily. Although PACAP is a potent stimulator of adenylate cyclase activity in the adenohypophysis, the precise target cells for PACAP in the anterior pituitary remain unknown. The aim of the present study was to investigate whether PACAP could stimulate calcium mobilization in individual cells of the pituitary and to determine the type of cells that responded to PACAP. Enzymatically dispersed frog distal pituitary cells were plated on photoetched coverslips and cultured for 3-7 days. The cells were loaded with the fluorescent calcium indicator indo-1, and changes in intracellular calcium concentrations ([Ca2+]i) were monitored using dual wavelength microfluorimetry. The individual cells were localized with the aid of the alpha/numeric grid of the coverslips and identified retrospectively by immunofluorescence. Approximately 45% of GH and PRL cells and 25% of ACTH and TSH cells responded to PACAP (10(-5) M) ejection by an elevation of [Ca2+]i. Only 16% of gonadotropes were stimulated by PACAP. The time course of [Ca2+]i variations showed three different patterns: transient spikes, sustained stimulations, and oscillatory responses. In addition, heterogenous responses were observed within each cell type. These data provide evidence for the involvement of calcium mobilization in the mechanism of action of PACAP on pituitary cells. The results also indicate that in frogs, PACAP may stimulate the secretory activity of GH and PRL cells and, to a lesser extent, ACTH, TSH, and gonadotrope cells.     

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.     

Dopaminergic inhibition of anterior pituitary adenylate cyclase activity and prolactin release: the effects of perturbing calcium on catalytic adenylate cyclase activity

The dopaminergic inhibition of anterior pituitary adenylate cyclase activity, cAMP accumulation, and prolactin release was studied in the presence of the Ca2+ channel activator, maitotoxin. In isobutylmethylxanthine (IBMX)-treated cells, maitotoxin stimulated prolactin secretion within 30 s and cAMP accumulation within 1 min. Although dopamine reduced cAMP accumulation and prolactin release, the effectiveness of the catecholamine was reduced in the presence of maitotoxin. When hemipituitary glands were exposed for 10 min to 100 ng maitotoxin/ml, their membranes showed increased adenylate cyclase activity. The hypothesis that maitotoxin stimulates adenylate cyclase activity by increasing Ca2+ availability was supported by the observation that, at concentrations up to 100 microM, Ca2+-stimulated anterior pituitary adenylate cyclase activity. Although dopamine decreased basal and maitotoxin-stimulated pituitary cAMP accumulation, via changes in adenylate cyclase activity, the decrement in cyclic nucleotide production, but not prolactin release, can be ascribed to the effect of the catecholamine on the basal activities of these parameters. These data provide additional evidence that an increased Ca2+ flux is stimulating to cAMP generation and prolactin release, whereas dopamine is inhibitory to these processes.     

Bronchodilation by pituitary adenylate cyclase-activating peptide and related peptides.

Pituitary adenylate cyclase-activating peptide (PACAP) is present in nerves in the vicinity of bronchial and vascular smooth muscle in the airways. At least one endogenous form of PACAP, PACAP 1-27, has high affinity binding sites in the lung, probably including cholinergic nerve terminals, bronchial smooth muscle, epithelial and mononuclear inflammatory cells. The mechanism of action for PACAP 1-27 and 1-38 in vivo involves endogenous catecholamines, peptidases and nitric oxide, depending on tissue type. Intracellularly, cyclic adenosine monophosphate (cAMP) as well as calcium and sodium mobilization is probably involved. PACAP 1-27 and 1-38 inhibit airway smooth muscle tone in vitro and in vivo. The inhibitory effect of PACAP 1-38 is more sustained than that of PACAP 1-27, in vitro as well as in vivo. PACAP 1-38 also causes more sustained inhibition of bronchoconstriction after inhalation in vivo, than does vasoactive intestinal peptide (VIP). PACAP 1-27 given intravenously virtually abolishes allergen-induced bronchoconstriction in vivo. Novel synthetic analogues of PACAP 1-27 cause more sustained inhibition of airway smooth muscle tone in vitro and in vivo than do PACAP 1-27 or 1-38. Both PACAP 1-27 and 1-38 inhibit arterial smooth muscle tone but, administration of PACAP 1-27, 1-38 or a structural analogue of PACAP 1-27 in the airways, induces no cardiovascular side effects at doses inhibiting bronchoconstriction. PACAP 1-38 enhances phagocytosis in macrophages and inhibits the release of the pro-inflammatory cytokine interleukin-2 in lymphocytes, suggesting antiinflammatory effects. It is concluded that pituitary adenylate cyclase-activating peptide 1-27 and 1-38, or structurally related molecules, may be useful as bronchodilators but their effect on human bronchial smooth muscle and on human inflammatory cells is in need of evaluation.     

Immunohistochemical demonstration of a novel hypothalamic peptide, pituitary adenylate cyclase-activating polypeptide, in the ovine hypothalamus

We recently reported isolation, characterization and synthesis of a novel ovine hypothalamic peptide with 38 residues which stimulates accumulation of cAMP in rat anterior pituitary cell cultures. The peptide was named PACAP38 (pituitary adenylate cyclase-activating polypeptide with 38 residues). The presence of another peptide corresponding to the N-terminal 1-27 residues (PACAP27) was also demonstrated. Both PACAP38 and PACAP27 have an amidated C-terminus. Antisera against synthetic PACAP27 were generated in rabbits. These antisera were tested for titer and specificity in enzyme-linked immunosorbent assay. One of the antisera (no. 88121-3) exhibited a high titer of antibody, which was specific to PACAP27 and PACAP38 with exception of slight cross-reactivity with ovine CRF (oCRF). Therefore, the antibodies against oCRF were removed from the antiserum using a solid phase method. Removal of oCRF antibodies was confirmed by enzyme-linked immunosorbent assay. A dense immunoreactive fiber network was found in both external and internal zones of the median eminence and pituitary stalk. The fibers were demonstrated to be in close contact with the hypophysial portal capillaries. The preabsorption of antiserum with vasoactive intestinal polypeptide or with the mixture containing TRH, LHRH, oCRF, ovine GH-releasing factor, somatostatin, and bovine thyroglobulin did not affect the immunostaining. On the other hand, the preabsorption of antiserum with an excess of PACAP27 or PACAP38 abolished the immunostaining. Therefore, the staining is considered specific for PACAP27 and PACAP38. Stained fibers were also present in the posterior pituitary. A dense fiber network was observed and the lateral hypothalamus the fibers appeared to cling to unstained neuronal cell bodies and their dendrites. In the lateral septum the fibers surrounded some blood vessels. Immunolabeled cell bodies were found in the paraventricular and supraoptic nuclei. These findings support the view that PACAP may play a multifunctional role, including that of a hypophysiotropic hormone, neurotransmitter, neuromodulator, and vasoregulator.     

Antagonist of pituitary adenylate cyclase activating polypeptide suppresses prolactin secretion without changing the activity of dopamine neurons in lactating rats

Pituitary adenylate cyclase activating polypeptide (PACAP) is a relatively new neuropeptide, and it has a potent stimulatory effect on adenylate cyclase activity in rat pituitary cells. However, the role of PACAP in the physiological control of prolactin (PRL) secretion is still unclear. In the present study, we investigated the physiological significance of endogenous PACAP on PRL secretion in lactating rats. On lactation days 7-8, pups were separated from their mother rats for 5 h before the onset of suckling and PACAP6-38 (16 microg), a receptor antagonist, was injected through the lateral ventricle cannula just after the removal of pups. The effects of PACAP6-38 on PRL and oxytocin secretion, and on the activity of tyrosine hydroxylase (TH), were examined after the onset of suckling. Administration of PACAP6-38 inhibited PRL levels in response to suckling, but it did not affect the activity of TH, as measured by DOPA accumulation at 15 min after administration of NSD 1015 (25.0 mg/kg), an L-aromatic amino acid decarboxylase inhibitor, or the plasma concentrations of oxytocin in lactating rats. Injection of alpha-methyl-p-tyrosine (alpha-MT; 50 mg/kg), an inhibitor of dopamine synthesis, increased PRL levels, and suckling caused a further increase in the plasma concentrations of PRL. An injection of PACAP6-38 (i.c.v.) also inhibited the PRL response to suckling under dopamine depletion. These results suggest that endogenous PACAP acts as a neurotransmitter or neuromodulator within the hypothalamus and plays an important role for PRL secretion in lactating rats. Endogenous PACAP may regulate PRL secretion, possibly mediated by PRL-releasing factors such as vasoactive intestinal polypeptide or vasopressin.