Effects of pituitary adenylate cyclase-activating polypeptide and vasoactive intestinal polypeptide on cyclic AMP accumulation in sheep pituitary cells in vitro

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are known to stimulate adenylate cyclase activity in rat pituitary cells but no direct effects have been reported on sheep pituitary cells. In this study we determined whether either peptide could stimulate intracellular cAMP accumulation in dispersed sheep pituitary cells in primary culture. Time course studies with PACAP showed that tachyphylaxis developed rapidly and so a short incubation time (5 min) was used to define the dose-response relationship. PACAP dose-dependently stimulated intracellular cAMP levels with a half-maximum response at 2.9 +/- 0.2 nmol/l (n = 4). In contrast, VIP only caused a small increase in intracellular cAMP levels at the highest dose tested (1 mumol/l). The VIP antagonist [4Cl-D-Phe6,Leu17]VIP had no effect on the cAMP response to either PACAP or VIP while the peptide PACAP(6-38), a putative PACAP antagonist, blocked the cAMP response to PACAP. The desensitisation to PACAP was further investigated by pretreating cells with PACAP for 30 min. After a further 15 min in culture medium alone, these cells showed no cAMP response to subsequent treatment with PACAP but could respond to forskolin. When a longer incubation period of 240 min was used between the first and second treatment with PACAP, a partial return in responsiveness to PACAP was observed. In summary, these results show that PACAP activates adenylate cyclase in sheep pituitary cells but that there is rapid development of tachyphylaxis. Experiments with the antagonists suggest that the response to PACAP is via the PACAP type I receptor. In contrast, physiological doses of VIP do not stimulate cAMP accumulation in sheep pituitary cells.     

Multiple splice variants of the pituitary adenylate cyclase-activating polypeptide type 1 receptor detected by RT-PCR in single rat pituitary cells

The urodynamic profiles of 97 patients with benign prostatic hyperplasia undergoing low-energy transurethral microwave thermotherapy (TUMT) for lower urinary tract symptoms were analysed using the Abrams/Griffiths nomogram, the urethral resistance algorithm, the linPURR, Sch?fer nomogram, and the CHESS classification. A significant clinical response was seen for the whole group, as shown by changes in symptom score, free flow rate, and residual urine. The best symptomatic response was identified in patients in whom obstruction was present, whatever the classification used. Only the two-dimensional CHESS classification was found to predict a group of patients with a better response in both symptoms and objective variables. Obviously, a better response from TUMT can only be predicted by a classification system that identifies the independent variables of footpoint and slope of the PURR. The CHESS classification was the only one of those studied that satisfactorily identified these two parameters and could be used as a system of case selection for this minimally invasive treatment.     

Gonadotropin-releasing hormone and pituitary adenylate cyclase-activating polypeptide affect levels of cyclic adenosine 3',5'-monophosphate-dependent protein kinase A (PKA) subunits in the clonal gonadotrope alphaT3-1 cells: evidence for cross-talk between PKA and protein kinase C pathways

We have shown previously that protein kinase A (PKA) subunit levels are regulated by activation of PKA or protein kinase C (PKC) in anterior pituitary cells. GnRH also influenced PKA subunit levels, suggesting that hormonal regulation occurs in gonadotrophs, and therefore, we have reexamined this question using the clonal gonadotrope-derived cell line (alphaT3-1 cells). Western blot analysis, using specific immunoaffinity purified immunoglobulins, revealed expression of catalytic (Cat) and regulatory type I (RI) and type II (RII) subunits of PKA in these cells. Activation of adenylyl cyclase (AC) with forskolin, or of PKC with tetradecanoyl phorbol acetate (TPA), caused a rapid (detectable at 0.5-1 h) and concentration-dependent loss of all PKA subunits. Forskolin (10-100 microM) reduced Cat and RI by 60% and RII by 30%, whereas TPA (0.1-1 microM) reduced Cat and RII by 50% and RI by 40%. Simultaneous activation of PKA and PKC caused the expected dose-dependent reductions in Cat, and the effects of forskolin or TPA were nearly additive. RI and RII were reduced similarly by 10 nM TPA, whereas 100 nM TPA tended to prevent the reduction of RI or RII caused by forskolin. GnRH, which activates phosphoinositidase C and not AC in these cells, caused a clear loss of Cat or RII at all concentrations tested and of RI at 0.1 nM. Pituitary adenylate cyclase-activating polypeptide 38, which acts via PVR-1 receptors to stimulate both phosphoinositidase C and AC in these cells, also caused a clear dose-dependent decrease in Cat, RI, and RII, although higher concentrations were needed for the latter effects. Together, the data demonstrate that catalytic and regulatory subunits of PKA are subject to both hormonal and receptor-independent regulation in alphaT3-1 cells, reinforcing the possibility that such effects occur in nonimmortalized gonadotropes. Whereas the effects of PKA activators very likely involve proteolytic degradation of the dissociated PKA holoenzyme, the effects of TPA and GnRH occur in the absence of cAMP elevation by unknown mechanisms. Whatever the mechanisms involved, the data reveal a mechanism for cross-talk between phosphoinositidase C and AC-mediated hormonal signals, in which PKC activation seems to play a pivotal role.     

Pituitary adenylate cyclase-activating polypeptide, helospectin, and vasoactive intestinal polypeptide in human corpus cavernosum

1. The distribution and effects of pituitary adenylate cyclase-activating polypeptide (PACAP-27 and -38), helospectin (Hel-1 and Hel-2), and vasoactive intestinal polypeptide (VIP), were investigated in isolated preparations of human corpus cavernosum (CC). 2. Immunohistochemistry revealed coinciding profiles of nerve structures that showed immunoreactivities for VIP and PACAP, and VIP and Hel. Confocal microscopy showed the co-existence of VIP- and PACAP-immunoreactivities, and VIP- and Hel-immunoreactivities in most (90%) varicose nerve structures. 3. As determined by radioimmunoassay, the amounts of VIP, PACAP-27, and PACAP-38 in the preparations were 61.7 +/- 11.6, 0.1 +/- 0.05, and 3.7 +/- 0.5 pmol g-1 wet weight of tissue (pmol g-1 wet wt.), respectively. In tissue from patients with diabetes, the content of VIP was lower (13.7 +/- 0.5 pmol g-1 wet wt.), whereas that of PACAP (-27 and -38) was unchanged. 4. Cyclic nucleotide levels were determined in preparations exposed to PACAP-27, PACAP-38, Hel-1, Hel-2, and VIP. All the peptides, but Hel-2, significantly increased the concentrations of cyclic AMP, whereas the levels of cyclic GMP were unchanged. 5. The peptides concentration-dependently relaxed noradrenaline-contracted preparations. The order of potency was VIP > PACAP 27 > Hel-1 > Hel-2 > PACAP-38. 6. Hel-1, VIP and PACAP-27 effectively counteracted electrically induced contractions. At 10(-6) M, the highest peptide concentration used, the inhibitory effects obtained reached 96 +/- 3%, 87 +/- 6%, and 80 +/- 3%, respectively. 7. The results suggest that PACAP and Hel-1 are co-localized with VIP in nerve structures within the human cavernous tissue, and that the peptides are effective relaxants of CC preparations in vitro. The role of the investigated peptides for penile erection remains to be established.     

Involvement of pituitary adenylate cyclase-activating polypeptide II vasoactive intestinal peptide 2 receptor in mouse neocortical astrocytogenesis

At the end of neuronal migration, the neopallial germinative zone produces glial cells destined to colonize the upper layers of neocortex. High densities of binding sites for vasoactive intestinal peptide (VIP) have been found in the rodent germinative zone just after completion of neuronal migration, suggesting a possible role of VIP in neocortical astrocytogenesis. In the present study, administration of a VIP antagonist at embryonic days 17 and 18 to pregnant mice was followed by a dramatic depletion of astrocytes in the upper cortical layer of the offspring. The depletion of astrocytes was dose-dependent, with a 42% reduction in the density of astrocytes observed with 50 microg of antagonist. The antagonist effect was reversed by cotreatment with VIP or pituitary adenylate cyclase-activating polypeptide (PACAP), suggesting the involvement of a receptor common to these two neuropeptides. VIP antagonist-induced inhibition of astrocytogenesis was also blocked by Ro 25-1553, a long-acting cyclic VIP analogue selective for the PACAP II VIP2 receptor subclass. Our results demonstrate that VIP and/or PACAP play a crucial physiological role in neocortical astrocytogenesis, possibly through interaction with PACAP II VIP2 receptors.     

Stage-specific expression of pituitary adenylate cyclase-activating polypeptide (PACAP) mRNA in the rat seminiferous tubules

We have used in situ hybridization and Northern blot analysis with oligonucleotide probe to characterize the site of pituitary adenylate cyclase-activating polypeptide (PACAP) synthesis in the rat testis. We observed strong hybridization signal in one third of the cross-sections of the seminiferous tubules, whereas some tubules were devoid of hybridization signal, thus suggesting that PACAP mRNA is expressed in a stage-specific manner. More detailed analysis showed that PACAP mRNA was present in round spermatids at stages III-VII of the cycle. Northern blot hybridization to RNAs extracted from samples of seminiferous tubules at different stages of the epithelial cycle confirmed that expression of PACAP mRNA is restricted to specific stages of the cycle. The highest amount of PACAP mRNA was detected at stages V to early -VII of the cycle, whereas very low levels of mRNA were present at stages I-II and IX-XIV. The present results demonstrate that PACAP mRNA is expressed in the developing germ cells. This suggests that PACAP may function as a paracrine or autocrine regulatory factor for the Sertoli and germ cells, with a specific function during early spermiogenesis, shortly before the onset of nuclear elongation, at the last period of haploid gene activity.     

Pharmacological, molecular and functional characterization of vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating polypeptide receptors in the rat pineal gland

Melatonin secretion from the mammalian pineal gland is strongly stimulated by noradrenaline and also by vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP). Three types of receptors for VIP and PACAP have been characterized so far: VIP1/PACAP receptors and VIP2/PACAP receptors, which possess similar high affinities for VIP and PACAP, and PACAP1 receptors which exhibit a 100-1000-fold higher affinity for PACAP. The aim of the present study was to characterize the receptor subtype(s) mediating the stimulatory effects of VIP and PACAP on melatonin synthesis in the rat pineal gland. Autoradiographic studies showed that PACAP and VIP were equally potent in displacing binding of radioiodinated PACAP27 from pineal sections. Amplification of pineal complementary DNAs by polymerase chain reaction using specific primers for the different receptor subtypes revealed that all three receptor messenger RNAs are expressed and that VIP1/PACAP receptor messenger RNA was predominant over VIP2/PACAP receptor messenger RNA. In vitro, VIP and PACAP stimulated melatonin synthesis with similar high potency and the effect of the two peptides were not additive. The selective VIP1/PACAP receptor agonists [R16]chicken secretin (1-25) and [K15, R16, L27]VIP(1-7)/growth hormone releasing factor(8-27) were significantly more potent than the selective VIP2/PACAP receptor agonist RO 25-1553 in stimulating melatonin secretion. The stimulatory effects of VIP and PACAP were similarly inhibited by the VIP1/PACAP antagonist [acetyl-His1, D-Phe2, K15, R16, L27]VIP(3-7)/growth hormone releasing factor(8-27). These data strongly suggest that VIP and PACAP exert a stimulatory effect on melatonin synthesis mainly through activation of a pineal VIP1/PACAP receptor subtype.     

Embryonic expression of pituitary adenylate cyclase-activating polypeptide in sensory and autonomic ganglia and in spinal cord of the rat

Sequential changes in small separated texture elements can produce perception of a moving form with continuous boundaries. This process of spatiotemporal boundary formation may exist to provide a robust means of detecting moving objects that occlude more distant textured surfaces. Whereas most research on spatiotemporal boundary formation has been focused on boundary and shape perception, two experiments are reported here on the perception of surface qualities in spatiotemporal boundary formation. In experiment 1 a free-report procedure was used to investigate whether surface perception can be determined by dynamic information alone, apart from static spatial differences. Results showed that dynamic information was sufficient to determine the appearance of a surface. This dynamic information may play an important role in other aspects of perception. In experiment 2, it was shown that dynamically specifying an extended, opaque surface facilitated edge perception. Implications for the relation of boundary and surface perception and for theories of perceptual transparency are discussed.     

Control of human luteal steroidogenesis: role of growth hormone-releasing hormone, vasoactive intestinal peptide, and pituitary adenylate cyclase-activating peptide

OBJECTIVE: To examine the possible effect of growth hormone-releasing hormone (GHRH), vasoactive intestinal peptide, and pituitary adenylate cyclase-activating peptide on basal and hCG-stimulated P production by human luteal cells. DESIGN: Cultures of human luteal cells from the early and midluteal phase. SETTING: All corpora lutea were obtained from the Obstetrics and Gynecology Department of the Università Cattolica, a public care center. PATIENT(S): Ten nonpregnant women between 35 and 47 years of age underwent surgery for various nonendocrine disorders, such as leiomyomatosis. INTERVENTION(S): Corpora lutea were obtained at the time of hysterectomy. MAIN OUTCOME MEASURE(S): Luteal cells were incubated with GHRH, vasoactive intestinal peptide, and pituitary adenylate cyclase-activating peptide with or without hCG at different concentrations. RESULT(S): Pituitary adenylate cyclase-activating peptide stimulated P production in a dose- and time-dependent manner, whereas GHRH and vasoactive intestinal peptide did not affect luteal steroidogenesis. None of the three peptides were found to synergize with hCG. CONCLUSION(S): Pituitary adenylate cyclase-activating peptide can influence human luteal steroidogenesis.     

Distribution, characterization, and growth hormone-releasing activity of pituitary adenylate cyclase-activating polypeptide in the European eel, Anguilla anguilla

The complementary DNA encoding pituitary adenylate cyclase-activating polypeptide (PACAP) has been cloned from two species of teleost fishes, the Sockeye salmon and the Thai catfish, and the amino acid sequence of PACAP has been determined in another teleost, the stargazer. However, to date, the detailed distribution of PACAP immunoreactivity has never been investigated in the fish brain. In the present study, we have determined the localization of PACAP-immunoreactive neurons in the central nervous system of a primitive teleost fish, the European eel Anguilla anguilla, using an antiserum raised against PACAP27. PACAP-positive perikarya were exclusively observed in the diencephalon, i.e. in the preoptic nucleus of the hypothalamus and in the dorsal and ventral nuclei of the thalamus. PACAP-immunoreactive fibers were detected in various areas of the brain, notably in the ventral telencephalon, the diencephalon, the mesencephalon, the cerebellar valvula, and the medulla oblongata. In addition, a dense accumulation of PACAP-containing nerve terminals was found in the pars distalis of the pituitary. The PACAP-like immunoreactivity contained in the eel brain was characterized by HPLC analysis combined with RIA quantification. The major form of PACAP-immunoreactive material coeluted with mammalian PACAP38. Molecular cloning of the PACAP precursor has previously shown that in fish, PACAP and GH-releasing hormone (GHRH) originate from the same precursor. We have thus investigated the effects of PACAP and GHRH on GH secretion from eel pituitary cells in primary culture. Dose-response experiments revealed that PACAP27 and PACAP38 possessed the same efficacy, but PACAP38 was 12 times more potent than PACAP27 in stimulating GH release (ED50 = 4.3 x 10(-10) and 3.5 x 10(-9) M, respectively). In contrast, GHRH, even at a high concentration (10(-6) M), had no effect on GH release. Taken together, these data indicate that in the eel, PACAP may play a significant role in the regulation of somatotrope cells: 1) PACAP-immunoreactive neurons are exclusively located in the diencephalon and send numerous projections in the pars distalis; and 2) PACAP, but not GHRH, dose dependently stimulates GH secretion from cultured eel pituitary cells.     

Distribution and effects of pituitary adenylate cyclase-activating peptide in the rabbit eye

Pituitary adenylate cyclase-activating peptide (PACAP)-like immunoreactivity was demonstrated by immunocytochemistry together with calcitonin gene-related peptide (CGRP)-like immunoreactivity in small to medium-sized neurons in the trigeminal ganglion and in nerve fibers in the iris, ciliary body, cornea, choroid and sclera of the rabbit eye. The regional distribution of PACAP-27- and PACAP-38-like immunoreactivity in the eye was studied by radioimmunoassay: the highest concentrations were found in the iris sphincter and ciliary body. The distribution pattern resembled that of CGRP-like immunoreactivity, which is a well-known constituent of sensory C-fibre neurons. Intravitreal injection of PACAP-27 or PACAP-38 induced conjunctival hyperemia, swelling of the anterior segment of the eye, miosis and breakdown of the blood-aqueous barrier, manifested as a marked aqueous flare response. Tetrodotoxin pretreatment inhibited the conjunctival hyperemia, the swelling of the anterior segment of the eye, and the miosis but not the aqueous flare response. The concentration of PACAP-like immunoreactivity in the aqueous humor was increased greatly following infrared irradiation of the iris, topical application of formaldehyde to the cornea, or intravitreal injection of endotoxin or bovine serum albumin. Also the concentration of CGRP-like immunoreactivity in the aqueous humor was increased greatly. Both in vivo and in vitro studies showed that capsaicin caused a parallel release of PACAP-like immunoreactivity and CGRP-like immunoreactivity from the uvea. Injection of PACAP-27 and PACAP-38 resulted in the release of CGRP-like immunoreactivity (and PACAP-like immunoreactivity) into the aqueous humor and PACAP-27 and PACAP-38 were also found to evoke tachykinin-mediated contractions of the isolated iris sphincter muscle, indicating that PACAP induces positive feedback on C-fibres. Thus, PACAP is a sensory neuropeptide in the eye. Since the PACAP-induced ocular responses mimicked the symptoms of inflammation, and since the PACAP-like immunoreactivity concentration in the aqueous humor was greatly increased following noxious stimulation, we suggest that it takes part in the inflammatory responses of the rabbit eye.     

Characterization of the equine glycoprotein hormone alpha-subunit gene reveals divergence in the mechanism of pituitary and placental expression

The equine glycoprotein hormone alpha-subunit gene is expressed in both pituitary and placenta, unlike that of all other nonprimate mammals studied, in which expression is limited to pituitary. Previous studies of the 5'-flanking region of the equine alpha-subunit promoter have revealed unique characteristics as well as similarities with the human alpha-subunit promoter, which demonstrates a similar pattern of tissue-specific expression. We have cloned and sequenced the equine alpha-subunit gene and have used tissue culture systems and transgenic mice to characterize its expression. Unlike the human promoter, the cloned equine alpha-subunit promoter failed to direct trophoblast-specific expression in either tissue culture or transgenic mouse models, suggesting an entirely different mechanism for expression. In contrast, the equine alpha-subunit promoter was able to direct gonadotroph expression in both tissue culture and transgenic mouse models. In alphaT3-1 cells, 550 base pair (bp) was sufficient for expression. This expression involves promoter elements identified in other species as playing a role in gonadotroph expression, but mutation of these elements reveals differences in their relative contributions to promoter activity. In mice, 2800 bp of 5'-flanking sequence allowed specific expression in gonadotrophs but not in thyrotrophs or placenta. The pattern of estrogen regulation observed in transgenic mice matched neither the repression that has been observed with human and bovine alpha-subunit promoters in transgenic mice nor the stimulation in mRNA levels reported in mares, suggesting a unique mechanism that is not recapitulated in the transgenic model. Thus the equine alpha-subunit promoter uses a combination of conserved and unique features of gene regulation to direct its pattern of tissue-specific expression.     

Production of alpha-subunit of glycoprotein hormones by pituitary somatotroph adenomas in vitro

Somatotroph adenomas of the pituitary secrete growth hormone in excess and are associated with acromegaly. Morphologically, they can be separated into two entities, densely and sparsely granulated variants. It has been shown that a number of somatotroph adenomas produce alpha-subunit of glycoprotein hormones; however, it is not clear whether alpha-subunit production correlates with tumor cell morphology. We studied 32 surgically removed pituitary somatotroph adenomas in tissue culture to determine structure-function correlations of growth hormone and alpha-subunit production. All tumors were classified on the basis of detailed histological, immunocytochemical and electron-microscopic studies. Fifteen tumors were densely granulated and 17 were sparsely granulated. In addition to growth hormone, all 15 densely granulated tumors released alpha-subunit in vitro, whereas of the 17 sparsely granulated tumors only 4 released alpha-subunit; moreover, the mean baseline levels of alpha-subunit were significantly higher in densely granulated adenomas than in sparsely granulated adenomas. Parallel response of release of both hormones was found during stimulation with growth hormone-releasing hormone or thyrotropin-releasing hormone and during suppression with somatostatin or bromocriptine in densely granulated tumors. alpha-subunit response to stimulation or suppression could not be determined with significance in sparsely granulated tumors because of low basal levels. The results indicate that alpha-subunit production and release is characteristic of densely granulated somatotroph adenomas and that alpha-subunit is coregulated with growth hormone by adenohypophysiotropic substances; in contrast, alpha-subunit production, by sparsely granulated somatotroph adenomas is rare and, when present, much lower in quantity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pituitary adenylate cyclase-activating polypeptide (PACAP) 38 and PACAP27 differentially stimulate growth hormone release and mRNA accumulation in porcine somatotropes

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been suggested to regulate growth hormone (GH) secretion in several species. Here, we analyzed the in vitro effects of PACAP38 and PACAP27 on the secretory activity of porcine somatotropes. Cultures of porcine pituitary cells were treated with PACAP38 and PACAP27, and GH release, intracellular GH content, and GH mRNA levels were evaluated. Also, the time course of changes in the somatotrope content of GH and its mRNA in response to PACAPs were measured. Both PACAPs stimulated GH release from porcine somatotropes in a broad range of doses (10(-10)-10(-6) M), yet only PACAP27 elicited a dose-dependent response. GH cell content remained essentially unchanged after PACAP treatment. In contrast, both PACAPs induced significant and sustained increases in GH mRNA cell content, although the response to PACAP27 appeared faster (8 h) than to PACAP38 (16 h). These results demonstrate that PACAP stimulates GH production in porcine somatotropes. Furthermore, the differential responses induced by PACAP38 and PACAP27 suggest that distinct mechanisms mediate their effects on this cell type.