PACAP and PAC1 Receptor Expression in Human Insulinomas

International Journal of Peptide Research and Therapeutics - Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide with widespread occurrence and diverse functions. PACAP...     

Effects of in ovo treatment with PACAP antagonist on general activity, motor and social behavior of chickens

Pituitary adenylate cyclase activating polypeptide (PACAP) has been shown to influence nervous system development. The aim of the present study was to investigate the effects of in ovo treatment with the PACAP antagonist PACAP6-38 during embryonic life (E8 and E16) on motor activity and social behavior in chicken. Our results showed that a single injection of PACAP6-38 during the first half of embryonic life caused subtle transient changes in general behavior and motor control when compared to saline-treated controls. Increased activity and reduced anxiety were observed also in a novel environment at 2 days after hatching. However, most of these behavioral differences disappeared by 2 weeks. PACAP6-38-treatment during the first half of embryonic life resulted in markedly reduced social behavior, which was still present at 2 weeks of age. Treatment during the second half of embryonic life resulted in no behavioral differences between control and PACAP6-38-treated chicken. PACAP content in different brain areas was not different between control and PACAP6-38-treated chicken at 5 days or 3 weeks of age, but it decreased significantly with age in both groups. In summary, our results show that PACAP6-38 treatment at E8 caused transient changes in motor behavior, and long-lasting reduction in social behavior.     

Comparative distribution of VIP in the central nervous system of various species measured by a new radioimmunoassay

Vasoactive intestinal polypeptide (VIP) occurs in high concentrations throughout the gut and the nervous system. The presence of VIP has been shown in a number of species, mainly by immunohistochemistry. The aim of the present study was to develop a new, highly specific VIP radioimmunoassay to investigate the distribution of VIP in the central nervous system of various vertebrate and invertebrate species. Different areas of the brain and spinal cord were removed from rats, chickens, turtles, frogs and fishes. The cerebral ganglia and the ventral ganglionic chain were investigated in the earthworm. The tissue samples were processed for VIP radioimmunoassay. Our results show that the antiserum used in the radioimmunoassay turned to be C-terminal specific, without significant affinity to other members of the VIP peptide family. Detection limit of the assay was 0.1 fmol/ml. Highest concentrations were found in the turtle diencephalon, followed by other brain areas in the turtle and rat. All other brain areas in the examined species contained significant levels of VIP. Immunoreactivity was also shown in the cerebral and ventral ganglia of the earthworm. In summary, our results show comparative quantitative distribution in representative species of the phylogenetic line, using the same experimental conditions.     

Influence of pinealectomy on levels of PACAP and cAMP in the chicken brain

One of the recently found functions of pituitary adenylate cyclase activating polypeptide (PACAP) is the modulation of circadian rhythms. Widespread distribution of PACAP-containing neurons and receptors has been shown in the chicken. Recently, we have demonstrated that PACAP levels oscillate in a circadian manner in the chicken brain. Daily variation in PACAP levels might be influenced by several regulatory mechanisms. Among the structures that may regulate PACAP levels, one candidate is the pineal gland. Therefore, in the present study, we investigated the effect of pinealectomy on the levels of PACAP in the chicken brain. Animals were kept under 12:12-h light-dark schedule. Pinealectomy was performed at 3 weeks of age; sham-operated animals were used as controls. The animals were sacrificed at 15 and 24 h 1 week after pinealectomy. The brainstem and diencephalon were removed, and tissue samples were processed for PACAP and cAMP radioimmunoassay (RIA).PACAP and cAMP levels showed nighttime elevations in both the sham-operated and pinealectomized animals, except for the PACAP content in the diencephalon of pinealectomized chicken. PACAP levels of pinealectomized animals were significantly higher in the diencephalon and brainstem as compared to the control animals at both time-points. Levels of cAMP correlated well with levels of PACAP. The present results provide evidence that the pineal gland has an inhibitory impact on PACAP-neurons in the chicken brainstem and diencephalon.     

Effects of pretreatment with PACAP on the infarct size and functional outcome in rat permanent focal cerebral ischemia

PACAP exerts neuroprotective effects under various neurotoxic conditions in vitro. In vivo, it reduces brain damage after global and transient focal ischemia. The present study investigated whether PACAP has neuroprotective effects when applied before the onset of permanent ischemia. Rats were given bolus injections of PACAP38 intracerebroventricularly, and then underwent permanent middle cerebral artery occlusion. The results show that 2 μg of PACAP significantly reduced the infarct size measured 12 and 24 h after the onset of ischemia. No further reduction was obtained by a 7-day pretreatment. PACAP also ameliorated certain sensorimotor deficits. Our present study provides further evidence for the neuroprotective effects of PACAP, and implies that it might be a promising preventive therapeutic agent in ameliorating ischemic brain damage.     

Pituitary adenylate cyclase-activating polypeptide type 1 (PAC1) receptor is expressed during embryonic development of the earthworm

Pituitary adenylate cyclase activating polypeptide (PACAP)-like molecules have been shown to be present in cocoon albumin and in Eisenia fetida embryos at an early developmental stage (E1) by immunocytochemistry and radioimmunoassay. Here, we focus on detecting the stage at which PAC1 receptor (PAC1R)-like immunoreactivity first appears in germinal layers and structures, e.g., various parts of the central nervous system (CNS), in developing earthworm embryos. PAC1R-like immunoreactivity was revealed by Western blot and Far Western blot as early as the E2 developmental stage, occurring in the ectoderm and later in specific neurons of the developing CNS. Labeled CNS neurons were first seen in the supraesophageal ganglion (brain) and subsequently in the subesophageal and ventral nerve cord ganglia. Ultrastructurally, PAC1Rs were located mainly on plasma membranes and intracellular membranes, especially on cisternae of the endoplasmic reticulum. Therefore, PACAP-like compounds probably influence the differentiation of germinal layers (at least the ectoderm) and of some neurons and might act as signaling molecules during earthworm embryonic development.     

Pituitary adenylate cyclase activating polypeptide protects cardiomyocytes against oxidative stress-induced apoptosis

Pituitary adenylate cyclase activating polypeptide (PACAP) has well-known neuroprotective effects, and one of the main factors leading to neuroprotection seems to be its anti-apoptotic effects. The peptide and its receptors are present also in the heart, but whether PACAP can be protective in cardiomyocytes, is not known. Therefore, the aim of the present study was to investigate the effects of PACAP on oxidative stress-induced apoptosis in cardiomyocytes. Our results show that PACAP increased cell viability by attenuating H2O2-induced apoptosis in a cardiac myocyte culture. PACAP also decreased caspase-3 activity and increased the expression of the anti-apoptotic markers Bcl-2 and phospho-Bad. These effects of PACAP were counteracted by the PACAP antagonist PACAP6-38. In summary, our results show that PACAP is able to attenuate oxidative stress-induced cardiomyocyte apoptosis.     

PKA-Bad-14-3-3 and Akt-Bad-14-3-3 signaling pathways are involved in the protective effects of PACAP against ischemia/reperfusion-induced cardiomyocyte apoptosis

The neuropeptide PACAP (pituitary adenylate cyclase activating polypeptide) and its receptors are widely expressed in the nervous system and various other tissues. PACAP has well-known anti-apoptotic effects in neuronal cell lines. Recent data suggest that PACAP exerts anti-apoptotic effects also in non-neuronal cells. The peptide is present in the cardiovascular system, and has various distinct effects. The aim of the present study was to investigate whether PACAP is protective against in vitro ischemia/reperfusion-induced apoptosis in cardiomyocytes. Cultured cardiomyocytes were exposed to 60 min ischemia followed by 120 min reperfusion. The addition of PACAP1-38 significantly increased cell viability and decreased the ratio of apoptotic cells as measured by MTT test and flow cytometry. PACAP induced the phosphorylation of Akt and protein kinase A. In the present study we also examined the possible involvement of Akt- and protein kinase A-induced phosphorylation and thus inactivation of Bad, a pro-apoptotic member of the Bcl-2 family. It was found that ischemia significantly decreased the levels of phosphorylated Bad, which was counteracted by PACAP. Furthermore, PACAP increased the levels of Bcl-xL and 14-3-3 protein, both of which promote cell survival, and decreased the apoptosis executor caspase-3 cleavage. All effects of PACAP1-38 were inhibited by the PACAP antagonist PACAP6-38. In summary, our results show that PACAP has protective effects against ischemia/reperfusion-induced cardiomyocyte apoptosis and provides new insights into the signaling mechanisms involved in the PACAP-mediated anti-apoptotic effects.     

Pituitary Adenylate Cyclase-Activating Polypeptide Ameliorates Experimental Acute Ileitis and Extra-Intestinal Sequelae

Background

The neuropeptide Pituitary adenylate cyclase-activating polypeptide (PACAP) plays pivotal roles in immunity and inflammation. So far, potential immune-modulatory properties of PACAP have not been investigated in experimental ileitis.

Methodology/Principal Findings

Mice were perorally infected with Toxoplasma (T.) gondii to induce acute ileitis (day 0) and treated daily with synthetic PACAP38 from day 1 to 6 post infection (p.i.; prophylaxis) or from day 4 to 6 p.i. (therapy). Whereas placebo-treated control mice suffered from acute ileitis at day 7 p.i. and succumbed to infection, intestinal immunopathology was ameliorated following PACAP prophylaxis. PACAP-treated mice exhibited increased abundance of small intestinal FOXP3+ cells, but lower numbers of ileal T lymphocytes, neutrophils, monocytes and macrophages, which was accompanied by less ileal expression of pro-inflammatory cytokines such as IL-23p19, IL-22, IFN-γ, and MCP-1. Furthermore, PACAP-treated mice displayed higher anti-inflammatory IL-4 concentrations in mesenteric lymph nodes and liver and higher systemic anti-inflammatory IL-10 levels in spleen and serum as compared to control animals at day 7 p.i. Remarkably, PACAP-mediated anti-inflammatory effects could also be observed in extra-intestinal compartments as indicated by reduced pro-inflammatory mediator levels in spleen (TNF-α, nitric oxide) and liver (TNF-α, IFN-γ, MCP-1, IL-6) and less severe histopathological sequelae in lungs and kidneys following prophylactic PACAP treatment. Strikingly, PACAP prolonged survival of T. gondii infected mice in a time-of-treatment dependent manner.

Conclusion/Significance

Synthetic PACAP ameliorates acute small intestinal inflammation and extra-intestinal sequelae by down-regulating Th1-type immunopathology, reducing oxidative stress and up-regulating anti-inflammatory cytokine responses. These findings provide novel potential treatment options of inflammatory bowel diseases.