Pleural mesothelioma from fluoro-edenite exposure: PACAP and PAC1 receptor. A preliminary report.

Pleural mesothelioma is a devastating malignancy primarily associated with asbestos exposure. However, emerging evidence suggests that exposure to fluoro-edenite fibers, a naturally occurring mineral fiber, can also lead to the development of pleural mesothelioma. In this study, based on the hypothesis that pituitary adenylate cyclase-activating polypeptide (PACAP) and PACAP-preferring receptor (PAC1R) expressions could be dysregulated in pleural mesothelioma samples and that they could potentially act as diagnostic or prognostic biomarkers, we aimed to investigate the immunohistochemical expression of PACAP and PAC1R in pleural biopsies from patients with pleural mesothelioma exposed to fluoro-edenite fibers. A total of 12 patients were included in this study, and their biopsies were processed for immunohistochemical analysis to evaluate the expression of PACAP and its receptor. The study revealed a correlation between the overexpression of PACAP and PAC1R and shorter overall survival in patients with malignant mesothelioma. These findings suggest that PACAP and PAC1R expression levels could serve as potential prognostic biomarkers for malignant mesothelioma. Furthermore, the immunohistochemical analysis of PACAP and PAC1R may provide valuable information for clinicians to guide therapeutic decisions and identify patients with poorer prognosis.

Dynamic, sex- and diet-specific pleiotropism in the PAC1 receptor-mediated regulation of arcuate proopiomelanocortin and Neuropeptide Y/Agouti related peptide neuronal excitability by anorexigenic ventromedial nucleus PACAP neurons.

This study furthers the investigation of how pituitary adenylate cyclase activating polypeptide (PACAP) and the PAC1 receptor (PAC1R) regulate the homeostatic energy balance circuitry. We hypothesized that apoptotic ablation of PACAP neurones in the hypothalamic ventromedial nucleus (VMN) would affect both energy intake and energy expenditure. We also hypothesized that selective PAC1R knockdown would impair the PACAP-induced excitation in anorexigenic proopiomelanocortin (POMC) neurones and inhibition of orexigenic neuropeptide Y (NPY)/agouti-related peptide (AgRP) neurones in the hypothalamic arcuate nucleus (ARC). The results show CASPASE-3-induced ablation of VMN PACAP neurones leads to increased energy intake and meal frequency as well as decreased energy expenditure in lean animals. The effects were more robust in obese males, whereas we saw the opposite effects in obese females. We then utilized visualized whole-cell patch clamp recordings in hypothalamic slices. PAC1R knockdown in POMC neurones diminishes the PACAP-induced depolarization, increase in firing, decreases in energy intake and meal size, as well as increases in CO2 production and O2 consumption. Similarly, the lack of expression of the PAC1R in NPY/AgRP neurones greatly attenuates the PACAP-induced hyperpolarization, suppression of firing, decreases in energy intake and meal frequency, as well as increases in energy expenditure. The PACAP response in NPY/AgRP neurones switched from predominantly inhibitory to excitatory in fasted animals. Finally, the anorexigenic effect of PACAP was potentiated when oestradiol was injected into the ARC in ovariectomized females. This study demonstrates the critical role of anorexigenic VMN PACAP neurones and the PAC1R in exciting POMC and inhibiting NPY/AgRP neurons to control homeostatic feeding.

Impact Assessment of Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) and Hemostatic Sponge on Vascular Anastomosis Regeneration in Rats.

The proper regeneration of vessel anastomoses in microvascular surgery is crucial for surgical safety. Pituitary adenylate cyclase-activating polypeptide (PACAP) can aid healing by decreasing inflammation, apoptosis and oxidative stress. In addition to hematological and hemorheological tests, we examined the biomechanical and histological features of vascular anastomoses with or without PACAP addition and/or using a hemostatic sponge (HS). End-to-end anastomoses were established on the right femoral arteries of rats. On the 21st postoperative day, femoral arteries were surgically removed for evaluation of tensile strength and for histological and molecular biological examination. Effects of PACAP were also investigated in tissue culture in vitro to avoid the effects of PACAP degrading enzymes. Surgical trauma and PACAP absorption altered laboratory parameters; most notably, the erythrocyte deformability decreased. Arterial wall thickness showed a reduction in the presence of HS, which was compensated by PACAP in both the tunica media and adventitia in vivo. The administration of PACAP elevated these parameters in vitro. In conclusion, the application of the neuropeptide augmented elastin expression while HS reduced it, but no significant alterations were detected in collagen type I expression. Elasticity and tensile strength increased in the PACAP group, while it decreased in the HS decreased. Their combined use was beneficial for vascular regeneration.

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) of the Bed Nucleus of the Stria Terminalis Mediates Heavy Alcohol Drinking in Mice.

Alcohol use disorder (AUD) is a complex psychiatric disease characterized by periods of heavy drinking and periods of withdrawal. Chronic exposure to ethanol causes profound neuroadaptations in the extended amygdala, which cause allostatic changes promoting excessive drinking. The bed nucleus of the stria terminalis (BNST), a brain region involved in both excessive drinking and anxiety-like behavior, shows particularly high levels of pituitary adenylate cyclase-activating polypeptide (PACAP), a key mediator of the stress response. Recently, a role for PACAP in withdrawal-induced alcohol drinking and anxiety-like behavior in alcohol-dependent rats has been proposed; whether the PACAP system of the BNST is also recruited in other models of alcohol addiction and whether it is of local or nonlocal origin is currently unknown. Here, we show that PACAP immunoreactivity is increased selectively in the BNST of C57BL/6J mice exposed to a chronic, intermittent access to ethanol. While pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 receptor-expressing cells were unchanged by chronic alcohol, the levels of a peptide closely related to PACAP, the calcitonin gene-related neuropeptide, were found to also be increased in the BNST. Finally, using a retrograde chemogenetic approach in PACAP-ires-Cre mice, we found that the inhibition of PACAP neuronal afferents to the BNST reduced heavy ethanol drinking. Our data suggest that the PACAP system of the BNST is recruited by chronic, voluntary alcohol drinking in mice and that nonlocally originating PACAP projections to the BNST regulate heavy alcohol intake, indicating that this system may represent a promising target for novel AUD therapies.

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) and Sudden Infant Death Syndrome: A Potential Model for Investigation.

Sudden infant death syndrome (SIDS) represents a significant cause of post-neonatal mortality, yet its underlying mechanisms remain unclear. The triple-risk model of SIDS proposes that intrinsic vulnerability, exogenous triggers, and a critical developmental period are required for SIDS to occur. Although case-control studies have identified potential risk factors, no in vivo model fully reflects the complexities observed in human studies. Pituitary adenylate cyclase-activating polypeptide (PACAP), a highly conserved neuropeptide with diverse physiological functions, including metabolic and thermal regulation, cardiovascular adaptation, breathing control, stress responses, sleep-wake regulation and immunohomeostasis, has been subject to early animal studies, which revealed that the absence of PACAP or its specific receptor (PAC1 receptor: PAC1R) correlates with increased neonatal mortality similar to the susceptible period for SIDS in humans. Recent human investigations have further implicated PACAP and PAC1R genes as plausible contributors to the pathomechanism of SIDS. This mini-review comprehensively synthesizes all PACAP-related research from the perspective of SIDS and proposes that PACAP deficiency might offer a promising avenue for studying SIDS.

Protective effect of pituitary adenylate cyclase activating polypeptide in diabetic keratopathy.

Diabetic keratopathy (DK) is the major complication of the cornea characterizing diabetes-affected patients. This ocular pathology is correlated with the hyperglycemic state leading to delayed corneal wound healing and recurrent corneal ulcers. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide with widespread distribution throughout the body, and exerting cytoprotective effects in the neural and non-neuronal parts of the eye, including the cornea. The purpose of the present study was to investigate whether changes in PACAP expression can concur for delayed epithelial wound healing in diabetic cornea and whether the protective effect of the peptide could be mediated through the activation of the EGFR signaling pathway, which has been reported to be impaired in DK. Expression and distribution of PACAP, PAC1R, and EGFR were investigated through immunohistochemistry analysis in the cornea of normal and diabetic rats. The role of the peptide on wound healing during DK was evaluated in an in vitro model represented by rabbit corneal epithelial cells grown in high glucose conditions. Western blotting and immunofluorescence analysis were used to examine the ability of PACAP to induce the activation of the EGFR/ERK1/2 signaling pathway. Our results showed that in diabetic cornea the expression of PACAP, PAC1R, and EGFR is drastically reduced. The treatment with PACAP via PAC1R activation enhanced cell viability and corneal epithelium wound healing in cells grown under high glucose conditions. Furthermore, both EGFR and ERK1/2 signaling was induced upon the peptide treatment. Overall, our results showed the trophic efficiency of PACAP for enhancing the corneal epithelium re-epithelialization suggesting that the peptide could be beneficially valuable as a treatment for DK.

PACAP/PAC1-R activation contributes to hyperalgesia in 6-OHDA-induced Parkinson's disease model rats via promoting excitatory synaptic transmission of spinal dorsal horn neurons.

Pain is a common annoying non-motor symptom in Parkinson's disease (PD) that causes distress to patients. Treatment for PD pain remains a big challenge, as its underlying mechanisms are elusive. Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptor PAC1-R play important roles in regulating a variety of pathophysiological processes. In this study, we investigated whether PACAP/PAC1-R signaling was involved in the mechanisms of PD pain. 6-hydroxydopamine (6-OHDA)-induced PD model was established in rats. Behavioral tests, electrophysiological and Western blotting analysis were conducted 3 weeks later. We found that 6-OHDA rats had significantly lower mechanical paw withdrawal 50% threshold in von Frey filament test and shorter tail flick latency, while mRNA levels of Pacap and Adcyap1r1 (gene encoding PAC1-R) in the spinal dorsal horn were significantly upregulated. Whole-cell recordings from coronal spinal cord slices at L4-L6 revealed that the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) in dorsal horn neurons was significantly increased, which was reversed by application of a PAC1-R antagonist PACAP 6-38 (250 nM). Furthermore, we demonstrated that intrathecal microinjection of PACAP 6-38 (0.125, 0.5, 2 µg) dose-dependently ameliorated the mechanical and thermal hyperalgesia in 6-OHDA rats. Inhibition of PACAP/PAC1-R signaling significantly suppressed the activation of Ca2+/calmodulin-dependent protein kinase II and extracellular signal-regulated kinase (ERK) in spinal dorsal horn of 6-OHDA rats. Microinjection of pAAV-Adcyap1r1 into L4-L6 spinal dorsal horn alleviated hyperalgesia in 6-OHDA rats. Intrathecal microinjection of ERK antagonist PD98059 (10 µg) significantly alleviated hyperalgesia in 6-OHDA rats associated with the inhibition of sEPSCs in dorsal horn neurons. In addition, we found that serum PACAP-38 concentration was significantly increased in PD patients with pain, and positively correlated with numerical rating scale score. In conclusion, activation of PACAP/PAC1-R induces the development of PD pain and targeting PACAP/PAC1-R is an alternative strategy for treating PD pain.

Calcitonin/PAC1 receptor splice variants: a blind spot in migraine research.

The neuropeptides calcitonin gene-related peptide (CGRP) and pituitary adenylate cyclase-activating polypeptide (PACAP) and their receptors are linked to migraine neurobiology. Recent antimigraine therapeutics targeting the signaling of these neuropeptides are effective; however, some patients respond suboptimally, indicating an incomplete understanding of migraine pathophysiology. The CGRP- and PACAP-responsive receptors can be differentially spliced. It is known that receptor splice variants can have different pathophysiological effects in other receptor-mediated pain pathways. Despite considerable knowledge on the structural and pharmacological differences of the CGRP- and PACAP-responsive receptor splice variants and their expression in migraine-relevant tissues, their role in migraine is rarely considered. Here we shine a spotlight on the calcitonin and PACAP (PAC1) receptor splice variants and examine what implications they may have for drug activity and design.

Early Alterations of PACAP and VIP Expression in the Female Rat Brain Following Spinal Cord Injury.

Previous evidence shows that rapid changes occur in the brain following spinal cord injury (SCI). Here, we interrogated the expression of the neuropeptides pituitary adenylyl cyclase-activating peptide (PACAP), vasoactive intestinal peptides (VIP), and their binding receptors in the rat brain 24 h following SCI. Female Sprague-Dawley rats underwent thoracic laminectomy; half of the rats received a mild contusion injury at the level of the T10 vertebrate (SCI group); the other half underwent sham surgery (sham group). Twenty-four hours post-surgery, the hypothalamus, thalamus, amygdala, hippocampus (dorsal and ventral), prefrontal cortex, and periaqueductal gray were collected. PACAP, VIP, PAC1, VPAC1, and VPAC2 mRNA and protein levels were measured by real-time quantitative polymerase chain reaction and Western blot. In SCI rats, PACAP expression was increased in the hypothalamus (104-141% vs sham) and amygdala (138-350%), but downregulated in the thalamus (35-95%) and periaqueductal gray (58-68%). VIP expression was increased only in the thalamus (175-385%), with a reduction in the amygdala (51-68%), hippocampus (40-75%), and periaqueductal gray (74-76%). The expression of the PAC1 receptor was the least disturbed by SCI, with decrease expression in the ventral hippocampus (63-68%) only. The expression levels of VPAC1 and VPAC2 receptors were globally reduced, with more prominent reductions of VPAC1 vs VPAC2 in the amygdala (21-70%) and ventral hippocampus (72-75%). In addition, VPAC1 downregulation also extended to the dorsal hippocampus (69-70%). These findings demonstrate that as early as 24 h post-SCI, there are region-specific disruptions of PACAP, VIP, and related receptor transcript and protein levels in supraspinal regions controlling higher cognitive functions.

Altered Hippocampal and Striatal Expression of Endothelial Markers and VIP/PACAP Neuropeptides in a Mouse Model of Systemic Lupus Erythematosus.

Neuropsychiatric systemic lupus erythematosus (NPSLE) is one of the most common and severe manifestations of lupus; however, its pathogenesis is still poorly understood. While there is sparse evidence suggesting that the ongoing autoimmunity may trigger pathogenic changes to the central nervous system (CNS) microvasculature, culminating in inflammatory/ischemic damage, further evidence is still needed. In this study, we used the spontaneous mouse model of SLE (NZBWF1 mice) to investigate the expression of genes and proteins associated with endothelial (dys)function: tissue and urokinase plasminogen activators (tPA and uPA), intercellular and vascular adhesion molecules 1 (ICAM-1 and VCAM-1), brain derived neurotrophic factor (BDNF), endothelial nitric oxide synthase (eNOS) and Krüppel-like factor 4 (KLF4) and neuroprotection/immune modulation: pituitary adenylate cyclase-activating peptide (PACAP), vasoactive intestinal peptide (VIP), PACAP receptor (PAC1), VIP receptors 1 and 2 (VPAC1 and VPAC2). Analyses were carried out both in the hippocampus and striatum of SLE mice of two different age groups (2 and 7 months old), since age correlates with disease severity. In the hippocampus, we identified a gene/protein expression profile indicative of mild endothelial dysfunction, which increased in severity in aged SLE mice. These alterations were paralleled by moderate alterations in the expression of VIP, PACAP and related receptors. In contrast, we report a robust upregulation of endothelial activation markers in the striatum of both young and aged mice, concurrent with significant induction of the VIP/PACAP system. These data identify molecular signatures of endothelial alterations in the hippocampus and striatum of NZBWF1 mice, which are accompanied by a heightened expression of endogenous protective/immune-modulatory neuropeptides. Collectively, our results support the idea that NPSLE may cause alterations of the CNS micro-vascular compartment that cannot be effectively counteracted by the endogenous activity of the neuropeptides PACAP and VIP.

Downregulation of PACAP and the PAC1 Receptor in the Basal Ganglia, Substantia Nigra and Centrally Projecting Edinger-Westphal Nucleus in the Rotenone model of Parkinson's Disease.

Numerous in vitro and in vivo models of Parkinson's disease (PD) demonstrate that pituitary adenylate cyclase-activating polypeptide (PACAP) conveys its strong neuroprotective actions mainly via its specific PAC1 receptor (PAC1R) in models of PD. We recently described the decrease in PAC1R protein content in the basal ganglia of macaques in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of PD that was partially reversed by levodopa therapy. In this work, we tested whether these observations occur also in the rotenone model of PD in the rat. The rotarod test revealed motor skill deterioration upon rotenone administration, which was reversed by benserazide/levodopa (B/L) treatment. The sucrose preference test suggested increased depression level while the open field test showed increased anxiety in rats rendered parkinsonian, regardless of the received B/L therapy. Reduced dopaminergic cell count in the substantia nigra pars compacta (SNpc) diminished the dopaminergic fiber density in the caudate-putamen (CPu) and decreased the peptidergic cell count in the centrally projecting Edinger-Westphal nucleus (EWcp), supporting the efficacy of rotenone treatment. RNAscope in situ hybridization revealed decreased PACAP mRNA (Adcyap1) and PAC1R mRNA (Adcyap1r1) expression in the CPu, globus pallidus, dopaminergic SNpc and peptidergic EWcp of rotenone-treated rats, but no remarkable downregulation occurred in the insular cortex. In the entopeduncular nucleus, only the Adcyap1r1 mRNA was downregulated in parkinsonian animals. B/L therapy attenuated the downregulation of Adcyap1 in the CPu only. Our current results further support the evolutionarily conserved role of the PACAP/PAC1R system in neuroprotection and its recruitment in the development/progression of neurodegenerative states such as PD.

The functional heterogeneity of PACAP: Stress, learning, and pathology.

Pituitary adenylate cyclase-activating peptide (PACAP) is a highly conserved and widely expressed neuropeptide that has emerged as a key regulator of multiple neural and behavioral processes. PACAP systems, including the various PACAP receptor subtypes, have been implicated in neural circuits of learning and memory, stress, emotion, feeding, and pain. Dysregulation within these PACAP systems may play key roles in the etiology of pathological states associated with these circuits, and PACAP function has been implicated in stress-related psychopathology, feeding and metabolic disorders, and migraine. Accordingly, central PACAP systems may represent important therapeutic targets; however, substantial heterogeneity in PACAP systems related to the distribution of multiple PACAP isoforms across multiple brain regions, as well as multiple receptor subtypes with several isoforms, signaling pathways, and brain distributions, provides both challenges and opportunities for the development of new clinically-relevant strategies to target the PACAP system in health and disease. Here we review the heterogeneity of central PACAP systems, as well as the data implicating PACAP systems in clinically-relevant behavioral processes, with a particular focus on the considerable evidence implicating a role of PACAP in stress responding and learning and memory. We also review data suggesting that there are sex differences in PACAP function and its interactions with sex hormones. Finally, we discuss both the challenges and promise of harnessing the PACAP system in the development of new therapeutic avenues and highlight PACAP systems for their critical role in health and disease.

Shared and independent roles of CGRP and PACAP in migraine pathophysiology.

The neuropeptides calcitonin gene-related peptide (CGRP) and pituitary adenylate cyclase-activating polypeptide (PACAP) have emerged as mediators of migraine pathogenesis. Both are vasodilatory peptides that can cause migraine-like attacks when infused into people and migraine-like symptoms when injected into rodents. In this narrative review, we compare the similarities and differences between the peptides in both their clinical and preclinical migraine actions. A notable clinical difference is that PACAP, but not CGRP, causes premonitory-like symptoms in patients. Both peptides are found in distinct, but overlapping areas relevant to migraine, most notably with the prevalence of CGRP in trigeminal ganglia and PACAP in sphenopalatine ganglia. In rodents, the two peptides share activities, including vasodilation, neurogenic inflammation, and nociception. Most strikingly, CGRP and PACAP cause similar migraine-like symptoms in rodents that are manifested as light aversion and tactile allodynia. Yet, the peptides appear to act by independent mechanisms possibly by distinct intracellular signaling pathways. The complexity of these signaling pathways is magnified by the existence of multiple CGRP and PACAP receptors that may contribute to migraine pathogenesis. Based on these differences, we suggest PACAP and its receptors provide a rich set of targets to complement and augment the current CGRP-based migraine therapeutics.

Activation of the PACAP/PAC1 Signaling Pathway Accelerates the Repair of Impaired Spatial Memory Caused by an Ultradian Light Cycle.

The mechanism of light-induced spatial memory deficits, as well as whether rhythmic expression of the pituitary adenylyl cyclase-activating polypeptides (PACAP)-PAC1 pathway influenced by light is related to this process, remains unclear. Here, we aimed to investigate the role of the PACAP-PAC1 pathway in light-mediated spatial memory deficits. Animals were first housed under a T24 cycle (12 h light:12 h dark), and then light conditions were transformed to a T7 cycle (3.5 h light:3.5 h dark) for at least 4 weeks. The spatial memory function was assessed using the Morris water maze (MWM). In line with behavioral studies, rhythmic expression of the PAC1 receptor and glutamate receptors in the hippocampal CA1 region was assessed by western blotting, and electrophysiology experiments were performed to determine the influence of the PACAP-PAC1 pathway on neuronal excitability and synaptic signaling transmission. Spatial memory was deficient after mice were exposed to the T7 light cycle. Rhythmic expression of the PAC1 receptor was dramatically decreased, and the excitability of CA1 pyramidal cells was decreased in T7 cycle-housed mice. Compensation with PACAP1-38, a PAC1 receptor agonist, helped T7 cycle-housed mouse CA1 pyramidal cells recover neuronal excitability to normal levels, and cannulas injected with PACAP1-38 shortened the time to find the platform in MWM. Importantly, the T7 cycle decreased the frequency of AMPA receptor-mediated excitatory postsynaptic currents. In conclusion, the PACAP-PAC1 pathway is an important protective factor modulating light-induced spatial memory function deficits, affecting CA1 pyramidal cell excitability and excitatory synaptic signaling transmission.

Sensory Neurons, PIEZO Channels and PAC1 Receptors Regulate the Mechanosensitive Release of Soluble Ectonucleotidases in the Murine Urinary Bladder Lamina Propria.

The urinary bladder requires adequate concentrations of extracellular adenosine 5'-triphosphate (ATP) and other purines at receptor sites to function properly. Sequential dephosphorylation of ATP to ADP, AMP and adenosine (ADO) by membrane-bound and soluble ectonucleotidases (s-ENTDs) is essential for achieving suitable extracellular levels of purine mediators. S-ENTDs, in particular, are released in the bladder suburothelium/lamina propria (LP) in a mechanosensitive manner. Using 1,N6-etheno-ATP (eATP) as substrate and sensitive HPLC-FLD methodology, we evaluated the degradation of eATP to eADP, eAMP and eADO in solutions that were in contact with the LP of ex vivo mouse detrusor-free bladders during filling prior to substrate addition. The inhibition of neural activity with tetrodotoxin and ω-conotoxin GVIA, of PIEZO channels with GsMTx4 and D-GsMTx4 and of the pituitary adenylate cyclase-activating polypeptide type I receptor (PAC1) with PACAP6-38 all increased the distention-induced but not spontaneous release of s-ENTDs in LP. It is conceivable, therefore, that the activation of these mechanisms in response to distention restricts the further release of s-ENTDs and prevents excessive hydrolysis of ATP. Together, these data suggest that afferent neurons, PIEZO channels, PAC1 receptors and s-ENTDs form a system that operates a highly regulated homeostatic mechanism to maintain proper extracellular purine concentrations in the LP and ensure normal bladder excitability during bladder filling.

Molecular and Physiological Functions of PACAP in Sweat Secretion.

Sweat plays a critical role in human body, including thermoregulation and the maintenance of the skin environment and health. Hyperhidrosis and anhidrosis are caused by abnormalities in sweat secretion, resulting in severe skin conditions (pruritus and erythema). Bioactive peptide and pituitary adenylate cyclase-activating polypeptide (PACAP) was isolated and identified to activate adenylate cyclase in pituitary cells. Recently, it was reported that PACAP increases sweat secretion via PAC1R in mice and promotes the translocation of AQP5 to the cell membrane through increasing intracellular [Ca2+] via PAC1R in NCL-SG3 cells. However, intracellular signaling mechanisms by PACAP are poorly clarified. Here, we used PAC1R knockout (KO) mice and wild-type (WT) mice to observe changes in AQP5 localization and gene expression in sweat glands by PACAP treatment. Immunohistochemistry revealed that PACAP promoted the translocation of AQP5 to the lumen side in the eccrine gland via PAC1R. Furthermore, PACAP up-regulated the expression of genes (Ptgs2, Kcnn2, Cacna1s) involved in sweat secretion in WT mice. Moreover, PACAP treatment was found to down-regulate the Chrna1 gene expression in PAC1R KO mice. These genes were found to be involved in multiple pathways related to sweating. Our data provide a solid basis for future research initiatives in order to develop new therapies to treat sweating disorders.

Hypothalamic PACAP/PAC1R Involvement in Feeding and Body Weight Regulation.

Pituitary adenylate cyclase-activating polypeptide (PACAP) and its cognate receptor PAC1R play key roles in energy balance. Central neuropeptide systems like PACAP are critical to the neuroendocrine system that regulates energy homeostasis in regions of the hypothalamus. A thorough investigation into central PACAP's influence on energy balance presents an opportunity to reveal putative causes of energy imbalance that could lead to obesity. In this review, we provide a brief overview of preclinical studies that have examined hypothalamic PACAP's influence on feeding behavior and metabolic regulation. Notably, due to the complexity and pleiotropic nature of the PACAP system, we highlight the need for a nuanced examination of PACAP signaling that utilizes a complex intersection of signaling circuitry in energy regulation that could ultimately offer insights to future therapeutic targets relevant for treating obesity.

Inhibiting PAC1 receptor internalization and endosomal ERK pathway activation may ameliorate hyperalgesia in a chronic migraine rat model.

BACKGROUND: Pituitary adenylate cyclase-activating polypeptide (PACAP) is a multipotent neuropeptide widely distributed in the trigeminovascular system (TVS) and higher brain regions. At present, the underlying mechanism of PACAP/PACAP type1 (PAC1) receptor in migraine generation remains unclear. METHODS: The rat model of chronic migraine (CM) was established by repeated intraperitoneal injection of nitroglycerin (NTG). Von Frey filaments and hot plate tests were used to measure the mechanical and thermal thresholds. The expression levels of c-Fos, calcitonin gene-related peptide (CGRP), PACAP, PAC1, protein kinase A (PKA) and phosphorylated extracellular signal-regulated kinase (ERK) were assessed by western blotting or immunofluorescence staining. The internalization of PAC1 receptor was visualized by fluorescence microscope and laser scanning confocal microscope. RESULTS: The results showed that c-Fos and CGRP expression significantly increased after repeated administrations of NTG or PACAP. Pitstop2 notably improved hyperalgesia in CM rats, while PACAP6-38 offered no benefit. In addition, PACAP-induced PAC1 receptor internalization, PKA and ERK pathways activation were blocked by Pitstop2 instead of PACAP6-38. CONCLUSIONS: Our results demonstrate that inhibition of PAC1 receptor internalization could effectively improve allodynia in CM rats by restraining ERK signaling pathway activation in a chronic migraine rat model. Modulation of receptor internalization may be a novel perspective to explore specific mechanisms of PACAP signaling activation in the trigeminal vascular system.

OMICS Analyses Unraveling Related Gene and Protein-Driven Molecular Mechanisms Underlying PACAP 38-Induced Neurite Outgrowth in PC12 Cells.

The study aimed to understand mechanism/s of neuronal outgrowth in the rat adrenal-derived pheochromocytoma cell line (PC12) under pituitary adenylate cyclase-activating polypeptide (PACAP) treatment. Neurite projection elongation was suggested to be mediated via Pac1 receptor-mediated dephosphorylation of CRMP2, where GSK-3ß, CDK5, and Rho/ROCK dephosphorylated CRMP2 within 3 h after addition of PACAP, but the dephosphorylation of CRMP2 by PACAP remained unclear. Thus, we attempted to identify the early factors in PACAP-induced neurite projection elongation via omics-based transcriptomic (whole genome DNA microarray) and proteomic (TMT-labeled liquid chromatography-tandem mass spectrometry) analyses of gene and protein expression profiles from 5-120 min after PACAP addition. The results revealed a number of key regulators involved in neurite outgrowth, including known ones, called 'Initial Early Factors', e.g., genes Inhba, Fst, Nr4a1,2,3, FAT4, Axin2, and proteins Mis12, Cdk13, Bcl91, CDC42, including categories of 'serotonergic synapse, neuropeptide and neurogenesis, and axon guidance'. cAMP signaling and PI3K-Akt signaling pathways and a calcium signaling pathway might be involved in CRMP2 dephosphorylation. Cross-referencing previous research, we tried to map these molecular components onto potential pathways, and we may provide important new information on molecular mechanisms of neuronal differentiation induced by PACAP. Gene and protein expression data are publicly available at NCBI GSE223333 and ProteomeXchange, identifier PXD039992.

PACAP inhibition alleviates neuropathic pain by modulating Nav1.7 through the MAPK/ERK signaling pathway in a rat model of chronic constriction injury.

BACKGROUND: Trigeminal neuralgia is a common chronic maxillofacial neuropathic pain disorder, and voltage-gated sodium channels (VSGCs) are likely involved in its pathology. Prior studies report that pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide highly expressed in the trigeminal ganglion, may contribute to dorsal root ganglion neuron excitability by modulating the Nav1.7. OBJECTIVE: We investigated whether PACAP can regulate Nav1.7 through the mitogen-activated protein kinase/ERK kinase/extracellular-signal-regulated kinase (MEK/ERK) pathway in the trigeminal ganglion after chronic constriction injury of the infraorbital nerve (ION-CCI) in rats. STUDY DESIGN: Sprague-Dawley rats underwent ION-CCI, followed by intrathecal injection of PACAP 6-38 (PAC1 receptor antagonist) and PD98059 (MEK/ERK antagonist). Quantitative real-time PCR and western blot were used to quantify ATF3, PACAP, ERK, p-ERK, and Nav1.7 expression. RESULTS: The mechanical pain threshold decreased from day 3 to day 21 after ION-CCI and reached the lowest testing value by day 14; however, it increased after PACAP 6-38 and PD98059 injections. Additionally, ION-CCI surgery increased ATF3, PACAP, and p-ERK expression in the rat trigeminal ganglion and decreased Nav1.7 and PAC1 receptor expression; however, there was no difference in ERK expression. PACAP 6-38 injection significantly decreased PACAP, p-ERK, and Nav1.7 expression and increased the PAC1 receptor expression, with no change in ERK expression. Moreover, PD98059 injection decreased PACAP, p-ERK, and Nav1.7 expression and increased the expression of PAC1 receptor. CONCLUSION: After ION-CCI, PACAP in the rat trigeminal ganglion can modulate Nav1.7 through the MEK/ERK pathway via the PAC1 receptor. Further, PACAP inhibition alleviates allodynia in ION-CCI rats.