Pituitary Adenylate Cyclase-activating Polypeptide Treatment Research Articles

Effects of pituitary adenylate cyclase activating polypeptide in retinal degeneration induced by monosodium-glutamate

Pituitary adenylate cyclase activating polypeptide (PACAP) is a pleiotropic neuropeptide with a wide range of effects in the central and peripheral nervous systems. PACAP has well-documented neurotrophic and neuroprotective actions in both in vitro and in vivo models of different neuronal injuries. The aim of the present study was to investigate the possible neuroprotective effect of PACAP in retinal degeneration induced by monosodium-glutamate (MSG) in neonatal rats. Preceding the MSG treatment, PACAP (1 or 100 pmol/5 μl) was injected unilaterally into the vitreous body on postnatal days 1, 5 and 9. Immediately after the PACAP treatment, pups were treated with 2 mg/g body weight MSG subcutaneously. At 3 weeks of age, rats were sacrificed and retinas were removed and processed for histological examination. Our results show that MSG treatment caused severe degeneration, primarily of the inner retinal layers. The thickness of the entire retina was only approximately half of that of the normal retinas, and the inner nuclear layer seemed to be fused with the ganglionic cell layer, with no discernible inner plexiform layer. Retinas of animals treated with 1 pmol PACAP showed a similar degree of degeneration. However, retinas of rats treated with 100 pmol PACAP showed significantly less damage, with clearly distinguishable inner retinal layers. In summary, our present study shows that local PACAP treatment could attenuate the retinal degeneration induced by the excitotoxic effects of glutamate.

Effect of PACAP on LH release studied by cell immunoblot assay depends on the gender, on the time of day and in female rats on the day of the estrous cycle

We have previously demonstrated that pituitary adenylate cyclase activating polypeptide (PACAP) can be released from cultured rat anterior pituitary cells and when added to the medium in physiological concentration it releases LH from individual gonadotropes. In the present work, we studied whether the release of PACAP and the responsiveness of LH cells to PACAP depend on the gender, on the time of day when the animals were sacrificed, and in females on the stage of the estrous cycle. Anterior pituitary cells were cultured on nitrocellulose membrane. We found that the number of PACAP releasing cells was higher in proestrous than in diestrous female or in male rats and their number was always higher in the evening than at the other times. The effect of PACAP on LH cells was stimulatory in the morning of proestrus and diestrus. In proestrous rats, PACAP did not influence LH release in the afternoon or the evening, but in diestrous rats it decreased it in the afternoon and the evening. In males, there was a decrease of LH due to PACAP treatment at 10 and 20 h; however, PACAP did not influence LH at 16 h. It was concluded that in vivo PACAP might be involved in the circadian and episodic release of LH at pituitary level.

Morphological and functional effects of PACAP in 6-hydroxydopamine-induced lesion of the substantia nigra in rats

Pituitary adenylate cyclase activating polypeptide (PACAP) has several different actions in the nervous system, including neuroprotective effects. In the present study, we investigated the effects of different doses of PACAP on the functional and morphological outcome in a rat model of Parkinson's disease. Rats were given unilateral injections of 6-hydroxydopamine (6-OHDA) into the substantia nigra. PACAP-treated animals received 1, 0.1 or 0.01 μg PACAP as a pretreatment. Control animals without PACAP treatment displayed severe hypokinesia at 1 and 10 days post-lesion when compared to normal animals or those receiving saline only. PACAP treatment resulted in less severe acute hypokinesia, and complete recovery by 10 days. Asymmetrical signs were observed in all lesioned animals 1 day post-lesion. PACAP-treated animals, however, showed better recovery as they ceased to display asymmetrical signs 10 days later and showed markedly less apomorphine-induced rotations. Best behavioral outcome was observed in animals treated with 0.1 μg PACAP. Tyrosine-hydroxylase (TH) immunohistochemistry revealed increased number of dopaminergic neurons in the substantia nigra pars compacta and in the ventral tegmental area in all PACAP-treated rats in contrast to the severe cell loss in control animals. These results indicate that PACAP may be a promising therapeutic agent in Parkinson's disease.

Pituitary adenylate cyclase activating polypeptide protects dopaminergic neurons and improves behavioral deficits in a rat model of Parkinson’s disease

Pituitary adenylate cyclase activating polypeptide (PACAP) is a pleiotropic neuropeptide, exerting different actions in the central and peripheral nervous systems. Among others, it has neurotrophic and neuroprotective effects. In the present study, we investigated the effects of PACAP in a rat model of Parkinson’s disease. Rats were given unilateral injections of 6-hydroxydopamine (6-OHDA) into the substantia nigra. PACAP-treated animals received 0.1 μg PACAP as a pretreatment. Control animals without PACAP treatment displayed severe hypokinesia at 1 and 10 days postlesion when compared to animals receiving saline only. In only 1 day postlesion, by contrast, PACAP-treated rats showed no hypokinesia. Asymmetrical signs, such as turning, rearing and biased thigmotaxic scanning were observed in all lesioned animals 1 day postlesion. PACAP-treated animals, however, showed better recovery as they ceased to display asymmetrical signs 10 days later and showed markedly less apomorphine-induced rotations. Tyrosine-hydroxylase immunohistochemistry revealed that control animals had more than 95% loss of the dopaminergic cells in the ipsilateral substantia nigra, while PACAP-treated animals had only approximately 50% loss of dopaminergic cells. In summary, the present results show the neuroprotective effect of PACAP in 6-OHDA-induced lesion of substantia nigra, with less severe acute neurological symptoms and a more rapid amelioration of behavioral deficits.

The effects of PACAP and PACAP antagonist on the neurobehavioral development of newborn rats

Recent studies show that pituitary adenylate cyclase activating polypeptide (PACAP) plays an important role in the development of the nervous system. The aim of the present study was to investigate the effects of PACAP38 and the PACAP antagonist PACAP6-38 on the development of neonatal behavior in rats. Pups were treated subcutaneously until day 14, a period during which the blood–brain barrier is not yet complete. Rats were tested daily for the appearance of physical features, sensory and motor neurological signs, and for exploratory behavior on days 14 and 21. Facial development and most neurological signs were accelerated by PACAP treatment, while anti-PACAP retarded ear unfolding, eye opening, hindlimb placing and righting reflex. PACAP-treated animals also showed altered behavior in the open-field, in particular at 3 weeks of age. The number of areas entered and rearings were much higher than in the vehicle-treated group, and they spent less time along the walls and in corners. Anti-PACAP had little effect in the exploratory behavior of the pups. In summary, these data provide additional evidence for the neurotrophic effects of both endogenously present and exogenously administered PACAP-38.

Pituitary adenylate cyclase-activating polypeptide acts as a paracrine regulator of melatonin-responsive cells of the ovine pars tuberalis.

The pars tuberalis (PT) region of the anterior pituitary plays a physiological role in seasonal animals. The primary signal transduction mechanism of the melatonin receptor in this tissue is an inhibition of cAMP signaling. However, nothing is known about the endocrine signals that activate cAMP synthesis in the cells of the PT, as previous studies relied on the pharmacological tool, forskolin, to stimulate cAMP synthesis. Here we show that pituitary adenylate cyclase-activating polypeptide (PACAP) activates cAMP synthesis in the cells of the PT. The pharmacology of cAMP activation by PACAP peptides suggests that cAMP activation is mediated by the type I PACAP receptor. PACAP treatment of PT cells results in cellular responses that are consistent with cAMP activation in these cells, including activation of MAPK and elevation of melatonin receptor mt1 mRNA expression. These responses can be inhibited by melatonin, demonstrating that activation of cAMP occurs within the melatonin-responsive cells. However, although PACAP activates cAMP in the cells of the PT, the effect of PACAP may not be direct, as colocalization in situ hybridization studies demonstrates that the type I PACAP receptor and the melatonin mt1 receptor do not colocalize on the cells of the PT.

Pituitary Adenylate Cyclase-Activating Polypeptide Acts as a Paracrine Regulator of Melatonin-Responsive Cells of the Ovine Pars Tuberalis

The pars tuberalis (PT) region of the anterior pituitary plays a physiological role in seasonal animals. The primary signal transduction mechanism of the melatonin receptor in this tissue is an inhibition of cAMP signaling. However, nothing is known about the endocrine signals that activate cAMP synthesis in the cells of the PT, as previous studies relied on the pharmacological tool, forskolin, to stimulate cAMP synthesis. Here we show that pituitary adenylate cyclase-activating polypeptide (PACAP) activates cAMP synthesis in the cells of the PT. The pharmacology of cAMP activation by PACAP peptides suggests that cAMP activation is mediated by the type I PACAP receptor. PACAP treatment of PT cells results in cellular responses that are consistent with cAMP activation in these cells, including activation of MAPK and elevation of melatonin receptor mt1 mRNA expression. These responses can be inhibited by melatonin, demonstrating that activation of cAMP occurs within the melatonin-responsive cells. However, although PACAP activates cAMP in the cells of the PT, the effect of PACAP may not be direct, as colocalization in situ hybridization studies demonstrates that the type I PACAP receptor and the melatonin mt1 receptor do not colocalize on the cells of the PT.

Activation of Trk Neurotrophin Receptor Signaling by Pituitary Adenylate Cyclase-activating Polypeptides

Pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide that acts through G protein-coupled receptors, exerts neuroprotective effects upon many neuronal populations. However, the intracellular signaling mechanisms that account for PACAP's trophic effects are not well characterized. Here we have tested the possibility that PACAP uses neurotrophin signaling pathways. We have found that PACAP treatment resulted in an increase in TrkA tyrosine kinase activity in PC12 cells and TrkB activity in hippocampal neurons. The activation of TrkA receptors by PACAP required at least 1 h of treatment and did not involve binding to nerve growth factor. Moreover, PACAP induced an increase in activated Akt through a Trk-dependent mechanism that resulted in increased cell survival after trophic factor withdrawal. The increases in Trk and Akt were blocked by K252a, an inhibitor of Trk receptor activity. In addition, transactivation of TrkA receptors by PACAP could be inhibited with PP1, an inhibitor of Src family kinases or BAPTA/AM, (1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid acetoxymethyl ester), an intracellular calcium chelator. Therefore, PACAP can exert trophic effects through a mechanism involving Trk receptors and utilization of tyrosine kinase signaling. This ability may explain several neuroprotective actions of PACAP upon neuronal populations after injury, nerve lesion, or neurotrophin deprivation.

Control of CCK gene transcription by PACAP in STC-1 cells.

The mechanisms by which neuroendocrine stimulants regulate CCK gene transcription are unclear. We examined promoter activation by pituitary adenylate cyclase-activating polypeptide (PACAP), a known CCK secretagogue, in the enteroendocrine cell line STC-1. The promoter region from -70 to -87 bp, relative to the transcriptional start site, contains a composite calcium/cyclic AMP response element (CRE)/activator protein 1 (AP1) site that may bind CRE binding protein (CREB) and AP1. PACAP (with IBMX) stimulated expression of an 87-bp construct 3.35+/-0.36-fold but had no effect on a -70 construct. The effect was blocked by the protein kinase A inhibitor H-89 and by a dominant-negative CREB plasmid. Mutation of the CRE/AP1 site to a canonical CRE site did not affect the response to PACAP, but mutation to a canonical AP1 site prevented it. CREB phosphorylation was increased after PACAP treatment. Electrophoretic mobility shift assay and supershift analysis revealed that CREB and not AP1 bound to the CRE/AP1 site and that PACAP increased the proportion of phosphorylated CREB that was bound. We conclude that PACAP increases CCK gene expression via a cAMP-mediated pathway involving CREB phosphorylation by protein kinase A and activation of a composite CRE/AP1 site.

A role of pituitary adenylate cyclase activating polypeptide (PACAP) as a regulator of paracrine interactions between folliculo-stellate cells and gonadotropes through the control of activin-follistatin interactions.

Pituitary folliculo-stellate (FS) cells were able to modify the effect of activin-A on gonadotropes through the paracrine factor, follistatin. The present study was aimed to examine whether a hypothalamic peptide, pituitary adenylate cyclase activating polypeptide (PACAP), could be a regulator of this paracrine interaction. Co-culture of FS cell-originated cell line TtT/GF cells with rat anterior pituitary cells showed faint inhibitory effect on the stimulatory action of activin-A on FSH secretion. When PACAP was added to the culture during the co-culture period, however, the presence of TtT/GF cells caused significant suppression of the effect of activin-A on FSH secretion. Conditioned-media (CM) from TtT/GF cells, obtained by incubation of TtT/GF cells in the presence or absence of PACAP, were next added to the cultures of anterior pituitary cells alone. CM from TtT/GF cells without PACAP treatment revealed slight, but not significant, suppressive effect on activin-induced increases in FSH secretion and the percentage of FSH cells. Meanwhile, CM from PACAP-treated TtT/GF cells attenuated both effects of activin-A. Furthermore, the inhibitory effect of the CM was neutralized when follistatin antibody was present in the culture. These results suggest that PACAP is able to regulate the paracrine action of FS cells on pituitary gonadotropes. Besides expressing direct actions on pituitary endocrine cells, PACAP may have roles as a regulator of cell-to-cell interactions within the pituitary gland.

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) 38 and PACAP27 Activate Common and Distinct Intracellular Signaling Pathways to Stimulate Growth Hormone Secretion from Porcine Somatotropes1

We have recently shown that the two bioactive forms of pituitary adenylate cyclase-activating polypeptide, PACAP38 and PACAP27, stimulate GH release and GH messenger RNA (mRNA) accumulation in cultured porcine pituitary cells. However, dose- and time-related differences in the response to both peptides suggested that the signaling mechanisms activated by PACAP38 and PACAP27 in this cell type could differ. To test this hypothesis, we have evaluated hormone release and GH mRNA content after PACAP treatment in combination with selective activators and inhibitors of the adenylate cyclase/cAMP/protein kinase A and the phospholipase C/inositol phosphate (IP)/protein kinase C pathways, and with blockers of intra- and extracellular Ca2+. Our results show that activation of the adenylate cyclase/cAMP/protein kinase A system, and extracellular Ca2+ entry through L-type Ca2+-channels are prevailing and requisite signals for the transduction of the stimulatory effects of both PACAP38 and PACAP27 on GH release and transcription in porcine somatotropes. However, phospholipase C and intracellular Ca2+ also contribute, although partially, to PACAP38-induced, but not to PACAP27-induced increase in porcine GH secretion and mRNA levels. These findings demonstrate that in normal somatotropes, PACAP38 can activate multiple transduction pathways that differ from those employed by PACAP27. Moreover, these differences could account for the previously described divergences in the actions of either peptide in porcine somatotropes.

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.

Continuous activation of pituitary adenylate cyclase-activating polypeptide receptors elicits antipodal effects on cyclic AMP and inositol phospholipid signaling pathways in CATH.a cells: role of protein synthesis and protein kinases.

Continuous exposure of cells to agonists develops a process that determines the extent to which the cells eventually respond to further stimuli. Here we used CATH.a cells (a catecholaminergic neuron-like cell line), which express pituitary adenylate cyclase-activating polypeptide (PACAP) receptors linked to both adenylyl cyclase and phospholipase C-beta pathways, to investigate the influence of prolonged hormonal treatment on dual signaling and gene transcription. Prolonged incubation of cells with PACAP failed to down-regulate the density and affinity of membrane binding sites and caused opposite changes in messenger systems: PACAP-stimulated cyclic AMP accumulation was attenuated in a time- and dose-dependent fashion (t(1/2) = 6.7 h and IC50 = 0.1 nM), whereas phosphoinositide turnover was overstimulated. Both effects were insensitive to pertussis toxin, whereas the drop in cyclic AMP concentration was also unchanged in the presence of 3-isobutyl-1-methylxanthine, indicating that neither Gi-like proteins nor cyclic nucleotide phosphodiesterases play a critical role in these processes. Blockade of protein synthesis with cycloheximide, as well as inhibition by H89 of cyclic AMP-dependent protein kinase (but not by bisindolylmaleimide of protein kinase C) antagonized the influences exerted by PACAP on adenylyl cyclase activity and inositol phosphate formation. Transcription of the chimeric GAL4-CREB construct, transiently transfected into CATH.a cells, was stimulated by PACAP, and this effect was potentiated as a result of chronic PACAP treatment. The results of the present investigation provide new insight into the possible differential regulation and cross-talks of transduction signals of receptors linked to multiplex signaling. They demonstrate that prolonged exposure of CATH.a cells to PACAP results in the desensitization of the cyclic AMP pathway and superinduction of the inositol phosphate signal, through protein neosynthesis and cyclic AMP-dependent protein kinase activation. At the same time, they show that desensitization of cyclic AMP signaling not only fails to hamper, but actually amplifies PACAP-stimulated CREB-regulated transcription.