Pituitary adenylate cyclase-activating polypeptide (PACAP) is important for embryo implantation in mice

Mice lacking pituitary adenylate cyclase-activating polypeptide (PACAP) show high mortality during the postnatal period, as well as impaired reproduction in females. This study characterizes the reproductive phenotype in female mice lacking PACAP due to targeted disruption (knockout) of the single copy pacap gene (Adcyap1) to determine the site(s) of action of PACAP in the cascade of reproductive events. PACAP null females showed normal puberty onset, estrous cycles, and seminal plugs when paired with a male of proven fertility. However, significantly fewer PACAP null females (21%) than wild-type females (100%) gave birth following mating. Although a defect was not detected in ovulation, ovarian histology or fertilization of released eggs in PACAP null females, only 13% had implanted embryos 6.5 days after mating. Associated with the decrease in implantation, prolactin and progesterone levels were significantly lower in females lacking PACAP than in wild types on day 6.5 after mating. Our evidence suggests that impaired implantation is the defect responsible for decreased fertility in PACAP null female mice.     

Growth hormone-releasing hormone and pituitary adenylate cyclase-activating polypeptide in the reproductive system

Growth hormone-releasing hormone (GHRH) and pituitary adenylate cyclase-activating polypeptide (PACAP) are both members of the glucagon superfamily that, with gonadotropins, act at central and peripheral levels as paracrine and autocrine coregulators of reproductive function. GHRH and PACAP are ancient peptides. Their original forms (both 27 amino acids long) were encoded by a single ancestral gene, several duplications of which led to the genes that encode the neuropeptides of the glucagon superfamily. In the male and female reproductive tracts, GHRH and PACAP interact with a subset of G protein-coupled receptors that are structurally similar to the PACAP receptor and variants of the vasoactive intestinal peptide receptor, and share several biological actions. These are related mainly to the modulation of cAMP-dependent and other signal transduction pathways in several cells of the pituitary–gonadal axis. The recent discovery that antagonists of GHRH and PACAP suppress the growth of human cancer cell lines that are derived from reproductive tissues indicates the potential importance of these peptides as local regulators of cell division, cell cycle arrest, differentiation and cell death.     

Altered psychomotor behaviors in mice lacking pituitary adenylate cyclase-activating polypeptide (PACAP)

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been conserved remarkably during evolution and is widely expressed in the mammalian brain. In Drosophila, mutation of the PACAP homologue results in behavioral defects, including impaired olfaction-associated learning and changes in ethanol sensitivity. Here, we report the generation of mice lacking the PACAP gene (PACAP(-/-)). PACAP(-/-) mice were born in the expected Mendelian ratios but had a high early-mortality rate. The surviving adult PACAP(-/-) mice displayed remarkable behavioral changes; they exhibited hyperactive and explosive jumping behaviors in an open field, increased exploratory behavior, and less anxiety in the elevated plus maze, emergence, and novel-object tests. Analysis of PACAP(-/-) mice brains revealed that the serotonin metabolite 5-hydroxyindoleacetic acid was slightly decreased in the cortex and striatum compared with wild-type mice. The present study provides evidence that PACAP plays a previously uncharacterized role in the regulation of psychomotor behaviors.     

Roles and mechanisms of action of pituitary adenylate cyclase-activating polypeptide (PACAP) in growth hormone and prolactin secretion.

It appears that PACAP has a direct action on somatotrophs to release GH. The intracellular signal transduction mechanisms for PACAP might be similar to but partly distinct from those for GRH. PACAP might play a role in GH secretion induced by serotoninergic mechanisms but not in ultradian rhythm of GH secretion in the rat. PACAP can stimulate PRL release from the pituitary in rats possibly through indirect paracrine effect. In addition, PACAP might participate in regulation of PRL secretion via hypothalamic VIP.     

Presence of pituitary adenylate cyclase-activating polypeptide (PACAP) in the plasma and milk of ruminant animals

Milk contains a variety of proteins and peptides that possess biological activity. Growth factors, such as growth hormone, insulin-like, epidermal and nerve growth factors are important milk components which may regulate growth and differentiation in various neonatal tissues and also those of the mammary gland itself. We have recently shown that pituitary adenylate cyclase-activating polypeptide (PACAP), an important neuropeptide with neurotrophic actions, is present in the human milk in much higher concentration than in the plasma of lactating women. Investigation of growth factors in the milk of domestic animals is of utmost importance for their nutritional values and agricultural significance. Therefore, the aim of the present study was to determine the presence and concentration of PACAP in the plasma and milk of three ruminant animal species. Furthermore, the presence of PACAP and its specific PAC1 receptor were investigated in the mammary glands. Radioimmunoassay measurements revealed that PACAP was present in the plasma and the milk of the sheep, goat and the cow in a similar concentration to that measured previously in humans. PACAP38-like immunoreactivity (PACAP38-LI) was 5-20-fold higher in the milk than in the plasma samples of the respective animals, a similar serum/milk ratio was found in all the three species. The levels did not show significant changes within the examined 3-month-period of lactation after delivery. Similar PACAP38-LI was measured in the homogenates of the sheep mammary gland samples taken 7 and 30 days after delivery. PAC1 receptor expression was detected in these udder biopsies by fluorescent immunohistochemistry suggesting that this peptide might have an effect on the mammary glands themselves.These data show that PACAP is present in the milk of various ruminant domestic animal species at high concentrations, the physiological implications of which awaits further investigation.     

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.     

Pituitary adenylate cyclase-activating polypeptide relaxes rat gastrointestinal smooth muscle.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a new member of the secretin/glucagon peptides family, being most homologous to vasoactive intestinal peptide (VIP). The present study was designed to investigate a possible effect of PACAP on the rat gastrointestinal smooth muscle in vitro. We demonstrated that 1) PACAP reduced basal smooth-muscle contractions in all portions of the gastrointestinal tract, but the effect of VIP was region-specific. The inhibitory effect of PACAP in midcolon was approximately 100 times greater than that of VIP. 2) PACAP significantly inhibited smooth-muscle contractions induced by acetylcholine or carbachol. The inhibitory effect of PACAP was not affected by hexamethonium and was additive to the inhibitory effect of atropine and pirenzepine. 3) PACAP inhibited smooth-muscle contractions induced by substance P, cholecystokinin, and galanin, even after atropine treatment. Although the exact mechanism of the inhibitory action of PACAP remains to be clarified, PACAP appears to exert its effect in the rat at a site other than muscarinic receptors, probably through a direct effect on gastrointestinal smooth muscle in vitro.     

The origin and function of the pituitary adenylate cyclase-activating polypeptide (PACAP)/glucagon superfamily

The pituitary adenylate cyclase-activating polypeptide (PACAP)/ glucagon superfamily includes nine hormones in humans that are related by structure, distribution (especially the brain and gut), function (often by activation of cAMP), and receptors (a subset of seven-transmembrane receptors). The nine hormones include glucagon, glucagon-like peptide-1 (GLP-1), GLP-2, glucose-dependent insulinotropic polypeptide (GIP), GH-releasing hormone (GRF), peptide histidine-methionine (PHM), PACAP, secretin, and vasoactive intestinal polypeptide (VIP). The origin of the ancestral superfamily members is at least as old as the invertebrates; the most ancient and tightly conserved members are PACAP and glucagon. Evidence to date suggests the superfamily began with a gene or exon duplication and then continued to diverge with some gene duplications in vertebrates. The function of PACAP is considered in detail because it is newly (1989) discovered; it is tightly conserved (96% over 700 million years); and it is probably the ancestral molecule. The diverse functions of PACAP include regulation of proliferation, differentiation, and apoptosis in some cell populations. In addition, PACAP regulates metabolism and the cardiovascular, endocrine, and immune systems, although the physiological event(s) that coordinates PACAP responses remains to be identified.     

Neuropeptides of the pituitary adenylate cyclase-activating polypeptide/vasoactive intestinal polypeptide/growth hormone-releasing hormone/secretin family in testis

Mammalian testicular development and the maintenance of spermatogenesis are hormone-dependent processes that are controlled by the pituitary gonadotropins and testosterone. Recent studies have demonstrated the presence of many neuropeptides and their receptors in the testis, suggesting that these peptides operate as local regulators of testicular germ cell development and function. Among these testicular neuropeptides, the peptides that belong to the pituitary adenylate cyclase-activating polypeptide (PACAP) family, particularly growth hormone-releasing hormone and secretin, appear to show some unique common features in terms of intratesticular localization and the time of expression during the spermatogenic cycle. However, their precise physiologic roles and mechanisms of action remain unknown. This review analyzes the available information on the functional interactions among the testicular cells that appear to be mediated by locally produced neuropeptides, with a special emphasis on the peptides of the PACAP family.     

Pituitary response to luteinizing hormone-releasing hormone in women with variant luteinizing hormone

OBJECTIVE: To assess the LH response of the pituitary gland to GnRH stimulation in healthy women with a mutant beta-subunit (Trp8 to Arg8 and Ile15 to Thr15). DESIGN: Clinical study. PATIENTS: We studied 40 healthy non-pregnant Japanese women of known zygosity for the LH beta-subunit gene (3 homozygotes for the mutant gene, 17 heterozygotes, and 20 homozygotes for the wild type). All women had normal ovulatory cycles. MEASUREMENTS: Serum LH status was determined by comparing LH immunoassays results using a monoclonal antibody recognizing only wild-type LH with those from a polyclonal antibody assay recognizing both variant and wild-type LH. The ratio of monoclonal to polyclonal immunoassay results determined the serum LH status. LH secretion in response to a GnRH stimulation test was measured. RESULTS: All women with the wild-type LH showed a normal response of LH to GnRH according to both assays. Over the time course of the response, the ratios in women with wild-type LH showed no remarkable changes. The response curves in women heterozygous for the mutant peaked 15-30min after GnRH injection; their response patterns included a statistically significant decrease in the rates of response at 15min after injection. CONCLUSIONS: There are the differences in circulatory kinetics between the two LH forms and in regulation of the two types of LHbeta genes. The maximal response of the variant LH to pituitary stimulation with GnRH appears to be greater than that of wild-type LH.     

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.     

Central pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) decrease the baroreflex sensitivity in trout

Although PACAP and VIP exert diverse actions on heart and blood vessels along the vertebrate phylum, no information is currently available concerning the potential role of these peptides on the regulation of the baroreflex response, a major mechanism for blood pressure homeostasis. Consequently, the goal of this study was to examine in our experimental model, the unanesthetized rainbow trout Oncorhynchus mykiss, whether PACAP and VIP are involved in the regulation of the cardiac baroreflex sensitivity (BRS). Cross-spectral analysis techniques using a fast Fourier transform algorithm were employed to calculate the coherence, phase and gain of the transfer function between spontaneous fluctuations of systolic arterial blood pressure and R-R intervals of the electrocardiogram. The BRS was estimated as the mean of the gain of the transfer function when the coherence between the two signals was high and the phase negative. Compared with vehicle, intracerebroventricular (ICV) injections of trout PACAP-27 and trout VIP (25-100 pmol) dose-dependently reduced the cardiac BRS to the same extent with a threshold dose of 50 pmol for a significant effect. When injected intra-arterially at the same doses as for ICV injections, only the highest dose of VIP (100 pmol) significantly attenuated the BRS. These results suggest that the endogenous peptides PACAP and VIP might be implicated in the central control of cardiac baroreflex functions in trout.     

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.     

Pituitary apoplexy caused by luteinizing hormone-releasing hormone in prolactin-producing adenoma

We report a case in which pituitary apoplexy developed shortly after an intravenous (i.v.) injection of luteinizing hormone-releasing hormone (LH-RH). A 56-year-old man with prolactin-producing pituitary tumor complained of severe headache, visual field loss and facial nerve palsy shortly after LH-RH test. Magnetic resonance image (MRI) revealed a hemorrhage in the pituitary adenoma. He showed dramatic improvement in his symptoms after decompression surgery. These findings suggest a causal relationship between the i.v. injection of LH-RH and pituitary apoplexy. Possible pituitary apoplexy should be kept in mind during pituitary testing.