Pituitary Adenylate Cyclase-activating Peptide Expression Research Articles

Pharmacology of PACAP and VIP receptors in the spinal cord highlights the importance of the PAC1 receptor.

The spinal cord is a key structure involved in the transmission and modulation of pain. Pituitary adenylate cyclase-activating peptide (PACAP) and vasoactive intestinal peptide (VIP), are expressed in the spinal cord. These peptides activate G protein-coupled receptors (PAC1, VPAC1 and VPAC2) that could provide targets for the development of novel pain treatments. However, it is not clear which of these receptors are expressed within the spinal cord and how these receptors signal. Dissociated rat spinal cord cultures were used to examine agonist and antagonist receptor pharmacology. Signalling profiles were determined for five signalling pathways. The expression of different PACAP and VIP receptors was then investigated in mouse, rat and human spinal cords using immunoblotting and immunofluorescence. PACAP, but not VIP, potently stimulated cAMP, IP1 accumulation and ERK and cAMP response element-binding protein (CREB) but not Akt phosphorylation in spinal cord cultures. Signalling was antagonised by M65 and PACAP6-38. PACAP-27 was more effectively antagonised than either PACAP-38 or VIP. The patterns of PAC1 and VPAC2 receptor-like immunoreactivity appeared to be distinct in the spinal cord. The pharmacological profile in the spinal cord suggested that a PAC1 receptor is the major functional receptor subtype present and thus likely mediates the nociceptive effects of the PACAP family of peptides in the spinal cord. However, the potential expression of both PAC1 and VPAC2 receptors in the spinal cord highlights that these receptors may play differential roles and are both possible therapeutic targets.

Increased pituitary adenylate cyclase-activating peptide genes expression in the prefrontal cortex in schizophrenia in relation to suicide.

Pituitary adenylate cyclase-activating peptide (PACAP) is a stress-related neuropeptide that is produced in several brain areas. It acts by 3 receptors: PACAP type-1 (PAC1), vasoactive intestinal peptide (VIP) -1 and -2 (VPAC1 and 2). Data on polymorphisms in PACAP and PAC1 indicate a relationship of the PACAP system with schizophrenia (SCZ). The prefrontal cortex was chosen to measure PACAP-gene related expression changes, since this is a central structure in the symptoms of schizophrenia (SCZ). We investigated alterations in the expression of the PACAP-related genes by qPCR in the human dorsolateral prefrontal cortex (DLPFC) and anterior cingulate cortex (ACC) of 35 SCZ patients and 34 matched controls in relation to SCZ, suicide, gender and medication. The ACC revealed an upregulation in PACAP, PAC1, VPAC1 and VPAC2 in SCZ suicide (S) completers compared to controls. An increase in PACAP, VPAC1 and VPAC2 expression was also present in the ACC in SCZ-S compared to SCZ patients who died naturally (SCZ-N). In the DLPFC, an increase in PAC1 was found in SCZ-N patients compared to SCZ-S and controls. Moreover, an increase in all PACAP-related genes was present in SCZ-N male patients compared to SCZ-N females. Concluding, expression changes were found in PACAP-related genes in relation to SCZ, suicide and gender. In particular, there was a higher PACAP-related gene expression in SCZ patients in the ACC in relation to suicide and in DLPFC in relation to SCZ. These findings suggest a potential link between PACAP and the pathophysiology of SCZ and suicide. Further research is needed to understand the functional significance and potential clinical applications of these changes.

Constitutive and conditional deletion reveals distinct phenotypes driven by developmental versus neurotransmitter actions of the neuropeptide PACAP.

Neuropeptides may exert trophic effects during development, and then neurotransmitter roles in the developed nervous system. One way to associate peptide-deficiency phenotypes with either role is first to assess potential phenotypes in so-called constitutive knockout mice, and then proceed to specify, regionally and temporally, where and when neuropeptide expression is required to prevent these phenotypes. We have previously demonstrated that the well-known constellation of behavioral and metabolic phenotypes associated with constitutive pituitary adenylate cyclase-activating peptide (PACAP) knockout mice are accompanied by transcriptomic alterations of two types: those that distinguish the PACAP-null phenotype from wild-type (WT) in otherwise quiescent mice (cPRGs), and gene induction that occurs in response to acute environmental perturbation in WT mice that do not occur in knockout mice (aPRGs). Comparing constitutive PACAP knockout mice to a variety of temporally and regionally specific PACAP knockouts, we show that the prominent hyperlocomotor phenotype is a consequence of early loss of PACAP expression, is associated with Fos overexpression in hippocampus and basal ganglia, and that a thermoregulatory effect previously shown to be mediated by PACAP-expressing neurons of medial preoptic hypothalamus is independent of PACAP expression in those neurons in adult mice. In contrast, PACAP dependence of weight loss/hypophagia triggered by restraint stress, seen in constitutive PACAP knockout mice, is phenocopied in mice in which PACAP is deleted after neuronal differentiation. Our results imply that PACAP has a prominent role as a trophic factor early in development determining global central nervous system characteristics, and in addition a second, discrete set of functions as a neurotransmitter in the fully developed nervous system that support physiological and psychological responses to stress.

PACAP6-38 improves nitroglycerin-induced central sensitization by modulating synaptic plasticity at the trigeminal nucleus caudalis in a male rat model of chronic migraine

AimsChronic migraine (CM) is a common neurological disorder with complex pathogenesis. Evidence suggests that pituitary adenylate cyclase-activating peptide (PACAP) induces migraine-like attacks and may be potential a new target for migraine treatment, but the therapeutic results of targeting PACAP and its receptors are not uniform. Therefore, the aim of this study was to investigate the regulatory effect of PACAP type I receptor (PAC1R) antagonist, PACAP6-38, on nitroglycerin (NTG)-induced central sensitization in a CM model.MethodsSprague–Dawley (SD) rats received repeated injections of NTG to construct a CM model. Mechanical and thermal thresholds were measured using Von Frey filaments and hot plate tests. C-Fos expression was measured by western blotting and immunofluorescence staining to assess the central sensitization. PACAP6-38 was intracerebrally injected into the trigeminal nucleus caudalis (TNC), and then the changes in c-Fos, the synaptic-associated proteins, phospho-ERK1/2 (p-ERK1/2), phosphorylation of cyclic adenosine monophosphate response element-binding protein (p-CREB) and brain-derived neurotrophic factor (BDNF) were detected. Transmission electron microscopy (TEM) and Golgi-Cox staining were used to observe the ultrastructure of synapses and dendritic structures of TNC neurons.ResultsThe results showed that PACAP and PAC1R expression were significantly raised in the TNC after repeated NTG injections. Additionally, PACAP6-38 treatment alleviated nociceptive sensitization, inhibited NTG-induced overexpression of c-Fos and synaptic-associated proteins in the TNC of CM rat, restored aberrant synaptic structures. Furthermore, the expression of ERK/CREB/BDNF pathway was depressed by PACAP6-38.ConclusionsOur results demonstrated that abnormal synaptic structure in the TNC of CM, which could be reversed by inhibition of PAC1R via down-regulating the ERK/CREB/BDNF signaling pathway. PACAP6-38 improves NTG-induced central sensitization by regulating synaptic plasticity in the TNC of CM rat, which may provide new insights into the treatments targeting PACAP/PAC1R in migraine.

A brainstem peptide system activated at birth protects postnatal breathing.

Among numerous challenges encountered at the beginning of extrauterine life, the most celebrated is the first breath that initiates a life-sustaining motor activity 1. The neural systems that regulate breathing are fragile early in development, and how they adjust to support breathing at the time of birth is not well understood. Here, we identify a neuropeptide system that becomes activated immediately upon birth and supports breathing. Mice lacking pituitary adenylate cyclase-activating peptide (PACAP) selectively in retrotrapezoid nucleus (RTN) neurons displayed increased apneas and blunted CO2-stimulated breathing; re-expression of PACAP in RTN neurons corrected these breathing deficits. Deletion of the PACAP receptor, PAC1, from the pre-Bötzinger Complex (preBötC), an RTN target region responsible for respiratory rhythm generation, phenocopied breathing deficits observed with RTN deletion of PACAP, and suppressed PACAP-evoked respiratory stimulation in the preBötC. Notably, a striking postnatal burst of PACAP expression occurred in RTN neurons precisely at the time of birth, coinciding with exposure to the external environment. Neonatal mice with deletion of PACAP in RTN neurons displayed increased apneas that were further exacerbated by changes in ambient temperature. Our findings demonstrate that well-timed PACAP expression by RTN neurons provides an important supplementary respiratory drive immediately after birth, and reveal key molecular components of a peptidergic neural circuit that supports breathing at a particularly vulnerable period in life.

Differences in pituitary adenylate cyclase-activating peptide and calcitonin gene-related peptide release in the trigeminovascular system.

Several neurotransmitters are expressed in the neurons of the trigeminal ganglion. One such signalling molecule is the pituitary adenylate cyclase-activating peptide (PACAP). PACAP signalling has been suggested to have a possible role in the pathophysiology of primary headaches. The present study was designed to investigate the relationship between PACAP and calcitonin gene-related peptide, currently the two most relevant migraine peptides. In the current study, we used ELISA to investigate PACAP and calcitonin gene-related peptide release in response to 60 mM K+ or capsaicin using a rat hemi-skull model. We combined this analysis with qPCR and immunohistochemistry to study the expression of PACAP and calcitonin gene-related peptide receptors and ligands. Calcitonin gene-related peptide (CGRP) is released from the trigeminal ganglion and dura mater. In contrast, PACAP is only released from the trigeminal ganglion. We observed a weak correlation between the stimulated release of the two neuropeptides. PACAP-38 immunoreactivity was expressed alone and in a subpopulation of neurons in the trigeminal ganglion that also store calcitonin gene-related peptide. The receptor subtype PAC1 was mainly expressed in the satellite glial cells (SGCs), which envelop the neurons in the trigeminal ganglion, in some neuronal processes, inside the Aδ-fibres and in the outermost layer of the myelin sheath that envelopes the Aδ-fibres. Unlike CGRP, PACAP is only released within the trigeminal ganglion. This raises the question of whether a migraine therapy aimed at preventing peripheral PACAP signalling would be as successful as the CGRP signalling targeted treatments.

Activation of PACAP expression in mouse RTN neurons at birth protects postnatal breathing

The brainstem systems that regulate breathing are established prenatally but they remain immature and vulnerable to failure at the time of birth; this reflects, in part, underdevelopment of the respiratory chemosensory systems that typically monitor blood gases (O2, CO2) to regulate breathing. In the mouse retrotrapezoid nucleus (RTN), Phox2b‐expressing respiratory chemoreceptor neurons are a nexus for integration of multiple respiratory‐related inputs, homeostatically regulating blood gases by providing CO2‐modulated drive to brainstem respiratory rhythm generator and pre‐motor circuits. In this work, we demonstrate that postnatal expression of a neuropeptide in chemosensitive RTN neurons is precisely tuned to influence breathing at the time of birth. First, by using single cell qRT‐PCR and multiplex in situ hybridization, we define a striking postnatal burst of pituitary adenylate cyclase‐activating peptide (PACAP) expression in RTN neurons that is activated in neonates within minutes of exposure to the external environment. Further, by using whole body plethysmography, we show that mouse pups (P2‐P12) lacking PACAP in Phox2b neurons (Phox2b::Cre;PACAPfl/fl) have severely blunted CO2‐stimulated ventilation (reduced by >50%) and a striking ~3‐fold greater incidence of apneic episodes. PACAP expression in RTN neurons persists into adulthood, albeit at apparently lower levels. PACAP deletion/depletion in adult RTN neurons (in Phox2b::Cre;PACAPfl/fl mice or by shRNA‐mediated knockdown) also depresses CO2‐regulated breathing (by ~30–40%) and increases apneas (~3‐fold), while viral‐mediated re‐expression of PACAP in RTN neurons corrects these deficits. After PACAP knockdown in RTN, CO2‐dependent Fos expression was retained in RTN neurons but decreased in cells of the preBötzinger complex (preBötC), which contains respiratory rhythm‐generating neurons targeted by the RTN. Consistent with the preBötC as a site of action for RTN‐derived PACAP, we show that: 1) the PACAP receptor, PAC1, is expressed in preBötC; 2) PACAP injection directly into the preBötC stimulated central respiratory output, both in vitro and in vivo; and 3) shRNA‐mediated depletion of PAC1 in the preBötC phenocopied breathing deficits observed with RTN deletion of PACAP. Notably, PACAP variants are associated with sudden infant death syndrome (SIDS), and with a SIDS‐like phenotype in PACAP‐null mice. Collectively, these data demonstrate that well‐timed PACAP expression by RTN neurons protects the developmentally immature respiratory control system from hypoventilation and potentially dangerous apneas at birth, and identifies key molecular components of a circuit that supports breathing at this particularly vulnerable period in life.Support or Funding InformationR01 HL108609; R01 HL074011

Increased expression of CART, nNOS, VIP, PACAP, SP and GAL in enteric neurons of the porcine stomach prepyloric region following hydrochloric acid infusion.

Stomach hyperacidity leads to damage of the mucus/bicarbonate barrier, ulcerations and the development of stomach cancer. Key regulators of the mucosal barrier/luminal acid balance are neurotransmitters secreted by intramural neurons. The aim of the current study was to determine the expression of gastric neuropeptides and nNOS in the porcine stomach following hydrochloric acid instillation. We report on increased expression of enteric neurotransmitters involved in adaptive reaction to an experimentally-induced hyperacidity state. The investigation was conducted on eight 12-18 kg pigs. The influence of intragastric infusion of hydrochloric acid on the expression of cocaine- and amphetamine-regulated transcript peptide (CART), neuronal nitric oxide synthase (nNOS), vasoactive intestinal polypeptide (VIP), pituitary adenylate cyclase-activating peptide (PACAP), substance P (SP) and galanin (GAL) in the submucous and myenteric gastric neurons of the pig has been studied with double immunofluorescence. A mimicked hyperacidity state significantly increased the proportion of enteric neurons immunoreactive to CART, nNOS, VIP, PACAP, SP and GAL in the submucous gastric neurons. In the myenteric plexus, a significant increase of the number of VIP-, CART- and GAL-immunoreactive (IR) neurons was found. Similarly, the percentage of myenteric nNOS-IR and PACAP-IR neurons tended to increase, while the fraction of SP-IR cells did not change. Stomach hyperacidity modifies the expression of the studied neurotransmitters in a specific way depending on the location of the neurons in particular plexuses of the stomach. Increased numbers of neurons expressing CART, nNOS, VIP, PACAP, SP and GAL clearly indicate their regulatory engagement in the restoration of the physiological gastric balance following hyperacidity.

Pituitary adenylate cyclase-activating peptide (PACAP) and its receptors in Mus musculus testis

To enlighten the involvement of PACAP/receptors system in the control of mammal testis, we investigated the expression of PACAP and the localization of PACAP and its receptors PAC1, VPAC1, and VPAC2 in the testis of Mus musculus. By molecular and immunohistochemical investigations, we highlighted that PACAP and its receptors are widely represented in germ cells of Mus testis, particularly in spermatocytes I, spermatids, and spermatozoa, strongly suggesting their involvement in spermatogenesis process. Moreover, for the first time in the adult mouse testis we highlighted that PACAP is present within Leydig cells, as PACAP receptors, confirming its involvement in the control of steroidogenesis in mouse.

Stress-specific changes of galanin and PACAP expression in the rat hypothalamus and adrenal gland

Galanin and pituitary adenylate cyclase-activating peptide (PACAP) are involved in the stress response, and both produce multiple effects in the central and peripheral nervous systems. In rodents, galanin brain expression is altered by different stressors, while PACAP has a critical role in the circuits mediating the stress-response. The aim of this study was to investigate whether acute and/or repeated immobilization (IMO) and restraint (R) differently affect galanin and PACAP expression in the rat hypothalamus and adrenal gland. Relative expression of mRNAs was examined by qPCR and relative changes in the protein content were analyzed using Western blotting. The distribution of galanin and PACAP peptides in the hypothalamus was analyzed by immunofluorescence. Relative hypothalamic expression of both galanin and PACAP mRNAs was increased only after repeated restraint. Hypothalamic galanin protein levels were increased after all applied stressors, whereas there were no changes in the PACAP content. In the adrenal glands, galanin and PACAP mRNAs were elevated only after acute IMO, whereas the protein levels were not changed, regardless of the applied stressor or its duration. Immunofluorescence staining demonstrated the presence of galanin in the paraventricular nuclei (PVN) of the hypothalamus in all groups. Galanin immunoreactivity was stronger in the PVN of all stressed groups in comparison to the control. We concluded that the patterns of transcriptional activation and translational control of galanin and PACAP in the examined tissues depended on the type (IMO vs. R) and duration (acute vs. repeated) of the applied stressor.

Genetic blockade of the dopamine D3 receptor enhances hippocampal expression of PACAP and receptors and alters their cortical distribution

Dopamine D3 receptors (D3Rs) are implicated in several aspects of cognition, but their role in aversive conditioning has only been marginally uncovered. Investigations have reported that blockade of D3Rs enhances the acquisition of fear memories, a phenomenon tightly linked to the neuropeptide pituitary adenylate cyclase-activating peptide (PACAP). However, the impact of D3R ablation on the PACAPergic system in regions critical for the formation of new memories remains unexplored. To address this issue, levels of PACAP and its receptors were compared in the hippocampus and cerebral cortex (CX) of mice devoid of functional D3Rs (D3R−/−) and wild-types (WTs) using a series of comparative immunohistochemical and biochemical analyses. Morphometric and stereological data revealed increased hippocampal area and volume in D3R−/− mice, and augmented neuronal density in CA1 and CA2/3 subfields. PACAP levels were increased in the hippocampus of D3R−/− mice. Expression of PACAP receptors was also heightened in mutant mice. In the CX, PACAP immunoreactivity (IR), was restricted to cortical layer V in WTs, but was distributed throughout layers IV–VI in D3R−/− mice, along with increased mRNAs, protein concentration and staining scores. Consistently, PAC1, VPAC1 and VPAC2 IRs were variably redistributed in CX, with a general upregulation in cortical layers II–IV in knockout animals. Our interpretation of these findings is that disturbed dopamine neurotransmission due to genetic D3R blockade may enhance the PACAP/PAC1–VPAC axis, a key endogenous system for the processing of fear memories. This could explain, at least in part, the facilitated acquisition and consolidation of aversive memories in D3R−/− mice.

Pituitary adenylate cyclase-activating peptide (PACAP) and PAC1 receptor in the testis of cartilaginous fish Torpedo marmorata: A molecular and phylogenetic study.

The role of PACAP in spermatogenesis and steroidogenesis has been largely investigated in last years in mammals; conversely, a few studies have been performed in non mammalian vertebrates. In this paper we investigated the sequence, expression and localization of PACAP and its PAC1 receptor in the testis of the benthic elasmobranch Torpedo marmorata, the marbled electric ray. Cloning a partial PACAP cDNA, we demonstrated for the first time in elasmobranches that PACAP shows a highly conserved sequence, compared with the PACAP of other chordates (tunicates and vertebrates). Moreover, the phylogenetic analysis revealed that PACAP has been well preserved during evolution and that a negative selection acts on PACAP sequence, leading to the conservation of the coding sites. The phylogenetic consensus tree showed also that Torpedo PACAP is more related with the amphibian PACAP than with the teleost one. Finally, we demonstrated that in T. marmorata PACAP and its PAC1 receptor are synthesized directly in the testis, where they show a wider localization than mammals, suggesting that this neuropeptide is involved in the control of Torpedo spermatogenesis.

The effects of sex and neonatal stress on pituitary adenylate cyclase-activating peptide expression.

What is the central question of this study? Does sex or neonatal stress affect the expression of pituitary adenylate cyclase-activating peptide or its receptors? What is the main finding and its importance? Neonatal-maternal separation stress has little long-lasting effect on the expression of pituitary adenylate cyclase-activating peptide or its receptors, but sex differences exist in these genes between males and females at baseline. Sex differences in classic stress hormones have been studied in depth, but pituitary adenylate cyclase-activating peptide (PACAP), recently identified as playing a critical role in the stress axes, has not. Here we studied whether baseline levels of PACAP differ between sexes in various stress-related tissues and whether neonatal-maternal separation stress has a sex-dependent effect on PACAP gene expression in stress pathways. Using quantitative RT-PCR, we found sex differences in PACAP and PACAP receptor gene expression in several respiratory and/or stress-related tissues, while neonatal-maternal separation stress did little to affect PACAP signalling in adult animals. We propose that sex differences in PACAP expression are likely to contribute to differences between males and females in responses to stress.

PACAP in the BNST Produces Anorexia and Weight Loss in Male and Female Rats

Recent gene association studies have implicated pituitary adenylate cyclase-activating peptide (PACAP) systems in several psychiatric disorders associated with stressor exposure, and we have argued that many of the behavioral consequences of repeated stressor exposure may depend on the expression of PACAP in the bed nucleus of the stria terminalis (BNST). One behavioral consequence of the activation of stress systems can be anorexia and subsequent weight loss, and both the activation of central PACAP systems as well as neuronal activity in the BNST have also been associated with anorexic states in rodents. Hence, we investigated the regulation of food and water intake and weight loss following BNST PACAP infusion. BNST PACAP38 dose-dependently decreased body weight, as well as food and water intake in the first 24 h following infusion. Because different BNST subregions differentially regulate stress responding, we further examined the effects of PACAP38 in either the anterior or posterior BNST. Anterior BNST PACAP38 infusion did not alter weight gain, whereas posterior PACAP38 infusion resulted in weight loss. PACAP38 infused into the lateral ventricles did not alter weight, suggesting that the effects of BNST-infused PACAP were not mediated by leakage into the ventricular system. These data suggest that PACAP receptor activation in posterior BNST subregions can produce anorexia and weight loss, and corroborate growing data implicating central PACAP activation in mediating the consequences of stressor exposure.

Age-related decrease in cerebrovascular-derived neuroprotective proteins: Effect of acetaminophen

As the population ages, the need for effective methods to maintain brain function in older adults is increasingly pressing. Vascular disease and neurodegenerative disorders commonly co-occur in older persons. Cerebrovascular products contribute to the neuronal milieu and have important consequences for neuronal viability. In this regard vascular derived neuroprotective proteins, Such as vascular endothelial growth factor (VEGF), pigment epithelium-derived factor (PEDF), and pituitary adenylate cyclase activating peptide (PACAP) are important for maintaining neuronal viability, especially in the face of injury and disease. The objective of this study is to measure and compare levels of VEGF, PEDF and PACAP released from isolated brain microvessels of Fischer 344 rats at 6, 12, 18, and 24months of age. Addition of acetaminophen to isolated brain microvessels is employed to determine whether this drug affects vascular expression of these neuroprotective proteins. Experiments on cultured brain endothelial cells are performed to explore the mechanisms/mediators that regulate the effect of acetaminophen on endothelial cells. The data indicate cerebrovascular expression of VEGF, PEDF and PACAP significantly decreases with age. The age-associated decrease in VEGF and PEDF is ameliorated by addition of acetaminophen to isolated brain microvessels. Also, release of VEGF, PEDF, and PACAP from cultured brain endothelial cells decreases with exposure to the oxidant stressor menadione. Acetaminophen treatment upregulates VEGF, PEDF and PACAP in brain endothelial cells exposed to oxidative stress. The effect of acetaminophen on cultured endothelial cells is in part inhibited by the selective thrombin inhibitor hirudin. The results of this study suggest that acetaminophen may be a useful agent for preserving cerebrovascular function. If a low dose of acetaminophen can counteract the decrease in vascular-derived neurotrophic factors evoked by age and oxidative stress, this drug might be useful for improving brain function in the elderly.

Synergystic relationship between GnRH AND GATA on the activity of pituitary adenylate cyclase-activating peptide (PACAP) gene promoter

PACAP has widespread distribution and function in the reproductive system. In the pituitary, PACAP is both secreted by and acts on gonadotrope cells. Previously, we have demonstrated induction of pituitary PACAP gene expression by either GnRH or GATA. The objective of these studies was to investigate their relationship in promoting PACAP expression.

Expression of pituitary adenylate cyclase-activating peptide, vasoactive intestinal peptide and their receptors in diabetic rat retinas

Pituitary adenylate cyclase-activating peptide (PACAP), vasoactive intestinal peptide (VIP) and their related receptors have already been described in the retina of different mammalian species during development. Based on relative affinity studies, at least three different binding sites have been identified for PACAP and VIP: the PACAPpreferring PAC1 receptors and, VPAC1 and VPAC2 receptors which bind with equal affinity both PACAP and VIP. However, peptides and receptors expression in early diabetic retinopathy remains unknown. In the present study we investigated PACAP, VIP and relative receptors expression and distribution in the retina of streptozotocin (STZ)induced diabetic rats. Diabetes was induced in Wistar rats by a single STZ intraperitoneal injection. The expression of PACAP, VIP and receptors in healthy and early diabetic retinas were assayed both after 1 and 3 weeks by quantitative real-time PCR, Western blot and immunohistochemical analyses. VPAC1 and VPAC2 receptors, as well as PACAP and VIP peptides mRNA levels were transiently induced by STZ-treatment within 1 week. After 3 weeks, PAC1, VPAC receptors and their related peptides expression levels were remarkably reduced in diabetic as compared to healthy rat retinas. These findings were confirmed at the protein level by immunoblot. Interestingly, immunohistochemical analyses revealed some peculiar changes in PACAP, VIP and receptors distribution between the two animal groups. In fact, after 1 week treatment, PACAP was more expressed in the retinal pigmented epithelium (RPE) and in the inner segment of diabetic rats. Similarly, VIP reactivity was remarkably increased in the RPE, but also in the inner plexiform layer of STZ-treated rats. No apparent changes were observed in PAC1 immunoreactivity between healthy and diabetic rats, although the RPE was slightly less reactive in the latter group. VPAC2 receptors seemed to be more expressed in the inner nuclear layer but less evident in the outer nuclear layer of STZ-treated rats. VPAC1 receptor retinal distribution was not affected by STZ-treatment. These differences were no longer observable 3 weeks after the STZ injection, although an overall reduction of both peptides and receptors expression occurred. In conclusion, this study provides evidence of an involvement of both PACAP and VIP peptides in early diabetic retinopathy. Furthermore, the overall downregulation of both peptides/receptors expression in retinas of STZ-treated rats after 3 weeks suggests that some deregulatory mechanisms might be activated in the later stages of retinal degeneration.

Differential regulation of CXCL12 and PACAP mRNA expression after focal and global ischemia

Pituitary adenylate cyclase activating peptide (PACAP) and the chemokine stromal cell-derived factor (SDF-1) have been implicated in neuroprotection, neurogenesis, and regeneration. Focal ischemia is associated with rapid upregulation of PACAP in perifocal neurons and delayed induction of SDF-1 in hypoxic/ischemic tissues, the latter process being involved in the recruitment of stem cells and inflammatory cells. Here, we studied mRNA patterns of PACAP, SDF-1 and the cognate receptors PAC1 and CXCR4 by in situ hybridization in the rat hippocampus after transient global ischemia, a rat model for programmed death of CA1 pyramidal neurons. Cell death in CA1 was not associated with local induction of PACAP and SDF-1 expression or recruitment of CXCR4-expressing infiltrates. However, there was a transient, almost complete loss of SDF-1 expression in microvessels in all hippocampal regions. Granule cells transiently showed a decrease of SDF-1 and an increase of PACAP expression. While PAC1 mRNA was moderately decreased throughout the hippocampus, CXCR4 expression was selectively increased in the subgranular layer. We propose that altered PACAP and SDF-1 gene expression in granule cells plays a role in regulated neurogenesis after global ischemia. The finding that programmed neuronal death after global ischemia was not associated with SDF-1 upregulation or recruitment of CXCR4-expressing cells is in sharp contrast to SDF-1/CXCR4-mediated infiltration of infarct tissue after focal ischemia. Hence, the different modes of neuronal death after focal and global ischemia are associated with distinct SDF-1 and PACAP gene regulation patterns and distinct reorganization mechanisms.

PACAP-Mediated ATP Release from Rat Urothelium and Regulation of PACAP/VIP and Receptor mRNA in Micturition Pathways after Cyclophosphamide (CYP)-Induced Cystitis

Pituitary adenylate cyclase-activating peptide (PACAP) peptides are expressed in micturition pathways, and PACAP expression is regulated by urinary bladder inflammation. Previous physiological studies have demonstrated roles for PACAP27 and PACAP38 in detrusor smooth muscle (DSM) contraction and a PAC1 receptor antagonist reduced cyclophosphamide (CYP)-induced bladder hyperreflexia. To gain insight into PACAP signaling in micturition and regulation with cystitis, receptor characterization by real-time quantitative polymerase chain reaction and physiological assays were performed. PACAP receptors were identified in tissues of rat micturition pathway, including DSM, urothelium (U), and dorsal root ganglia (DRG) after acute (4 h), intermediate (48 h) or chronic (8 days) CYP-induced cystitis. PAC1 messenger RNA expression significantly (p < or = 0.05) increased in U and DSM after 48 h and chronic CYP-induced cystitis after an initial decrease at 4 h. VPAC1 and VPAC2 transcripts increased in U and DSM after acute and intermediate CYP-induced cystitis followed by a decrease in VPAC2 expression with chronic cystitis. Application of PACAP27 (100 nM) to cultured urothelial cells evoked adenosine triphosphate (ATP) release that was blocked by the PAC1 specific antagonist, M65 (1 microM). PACAP38 (100 nM) also evoked ATP release from cultured urothelial cells, but ATP release was less than that observed with PACAP27. PACAP transcripts were increased in the U with intermediate and chronic cystitis, whereas vasoactive intestinal polypeptide (VIP) expression in both tissues was very low and showed no regulation with cystitis. Regulation of PACAP, galanin, and substance P transcripts expression was observed in lumbosacral DRG, but no regulation for VIP was observed. The current data demonstrate PACAP and PAC1 regulation in micturition pathways with inflammation and PACAP-mediated ATP release from urothelium.

Enhanced expression of CGRP in rat trigeminal ganglion neurons during cell and organ culture

The sensory innervation of intracranial vessels originates in the trigeminal ganglion with calcitonin gene-related peptide (CGRP), substance P (SP) and pituitary adenylate cyclase activating peptide (PACAP) as frequent neuronal messengers. The present study was designed to study the expression of these neuropeptides (a) in primary culture of adult rat trigeminal ganglion neuronal cells and (b) in organ culture of sections of the trigeminal ganglion. In cell culture, axons grow in the peripheral direction for up to 48 h. Immunocytochemistry revealed that the cell bodies showed increased expression of CGRP at 24 h and SP at 24–48 h ( p < 0.05), whereas cell culture did not increase the expression of PACAP at 24 h ( p > 0.05), but at 48 h ( p < 0.05). A significant elevation of CGRP mRNA was seen at 12 h, i.e. before the increased CGRP immunoreaction was observed. In organ culture of sections of trigeminal ganglia, the number of CGRP immunoreactive (-ir) cells and the mRNA expression were significantly increased at 24 and 48 h of incubation as compared to control ( p < 0.05), while the number of SP-ir cells was not altered ( p > 0.05). In conclusion, neurons of rat trigeminal ganglia alter their expression of neuropeptides during cell and organ culture differently, but it is mainly the CGRP system that is up-regulated. We have compared two methods for future studies of underlying molecular mechanisms responsible for regulation of neuropeptide expression in the trigeminal system.