PACAP sequence modifications modulate the peptide antimicrobial activity against bacterial pathogens affecting aquaculture.

The global aquaculture industry has significant losses each year due to disease outbreaks. Antibiotics are one of the common methods to treat fish infections, but prolonged use can lead to the emergence of resistant strains. Aeromonas spp. Infections are a common and problematic disease in fish, and members of this genera can produce antibiotic resistant strains. Antimicrobial peptides (AMPs) have emerged as an alternative method to treat and prevent infections and pituitary adenylate cyclase activating polypeptide (PACAP) is a prominent member of this family. The objective of this research was to study PACAP's direct antimicrobial activity and its toxicity in fish cells. Four synthetic variants of the natural PACAP from Clarias gariepinus were tested in addition to the natural variant. The experimental results show a different antimicrobial activity against A. salmonicida and A. hydrophila of each PACAP variant, and for the first time show dependence on the culture broth used. Furthermore, the results suggest that the underlying mechanism of PACAP antimicrobial activity includes a bacterial membrane permeabilizing effect, classifying PACAP as a membrane disruptive AMP. This study also demonstrated that the five PACAP variants evaluated showed low toxicity in vitro, at concentrations relevant for in vivo applications. Therefore, PACAP could be a promising alternative to antibiotics in the aquaculture sector.

Targeting the PACAP-38 pathway is an emerging therapeutic strategy for migraine prevention.

INTRODUCTION: The pituitary adenylate cyclase-activating polypeptide-38 (PACAP-38) has emerged as a key mediator of migraine pathogenesis. PACAP-38 and its receptors are predominantly distributed in arteries, sensory and parasympathetic neurons of the trigeminovascular system. Phase 2 trials have tested human monoclonal antibodies designed to bind and inhibit PACAP-38 and the pituitary adenylate cyclase-activating polypeptide type I (PAC1) receptor for migraine prevention. AREAS COVERED: This review focuses on the significance of the PACAP-38 pathway as a target in migraine prevention. English peer-reviewed articles were searched in PubMed, Scopus and ClinicalTrials.gov electronic databases. EXPERT OPINION: A PAC1 receptor monoclonal antibody was not effective for preventing migraine in a proof-of-concept trial, paving the way for alternative strategies to be considered. Lu AG09222 is a humanized monoclonal antibody targeting PACAP-38 that was effective in preventing physiological responses of PACAP38 and reducing monthly migraine days in individuals with migraine. Further studies are necessary to elucidate the clinical utility, long-term safety and cost-effectiveness of therapies targeting the PACAP pathway.

Immunostimulant effects of Pituitary Adenylate Cyclase-Activating Polypeptide and double-stranded (ds)RNA in Orconectes propinquus.

Disease outbreaks in crustacean aquaculture caused by opportunistic and obligate pathogens cause severe economic losses to the industry. Antibiotics are frequently used as prophylactic treatments worldwide, although its overuse and misuse has led to microbial resistance, which has driven the search for novel molecules with immunostimulant and antibacterial activities. Antimicrobial peptides (AMP) and double-stranded (ds)RNAs constitute promising immunostimulants in the fight against infectious diseases in aquaculture. Scientists have made significant progress in testing these molecules in aquatic organisms as potential candidates for replacing conventional antibiotics. However, most studies have been conducted in teleost fish, thus little is known about the immunostimulatory effects in crustaceans, especially in freshwater crayfishes. Consequently, in the present work, we evaluate the immunomodulatory effects of the AMP Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) and high molecular weight (HMW) Poly (I:C) in the northern clearwater crayfish Orconectes propinquus. Two bioassays were conducted to evaluate the effects of different doses of PACAP and Poly (I:C) HMW, different administration routes, as well as the effects of the combined treatment on the crayfish immune system. Results showed the immunostimulatory role of PACAP and Poly (I:C) HMW with effects depending on the dose, the site of injection and the treatment assessed. These findings offer new insights into the crayfish immune system and contribute to the development of effective broad-spectrum immune therapies in aquaculture.

PACAP-38 related modulation of the cranial parasympathetic projection: A novel mechanism and therapeutic target in severe primary headache.

BACKGROUND AND PURPOSE: Little is known of how cranial autonomic symptoms (CAS) in cluster headache and migraine may contribute to their severe headache phenotype. This strong association suggests the involvement of the cranial parasympathetic efferent pathway. To investigate its contribution, we studied the role of pituitary adenylate cyclase activating polypeptide-38 (PACAP-38), a potent sensory and parasympathetic neuropeptide, in modulating pre- and post-ganglionic cranial parasympathetic projection neurons, and their influence on headache-related trigeminal-autonomic responses. EXPERIMENTAL APPROACH: Using PACAP-38 and PACAP-38 responsive receptor antagonists, electrophysiological, behavioural and facial neurovascular-blood flow was measured in rats to probe trigeminal- and parasympathetic-neuronal, periorbital thresholds and cranial-autonomic outcomes, as they relate to primary headaches. KEY RESULTS: Sumatriptan attenuated the development of PACAP-38 mediated activation and sensitization of trigeminocervical neurons and related periorbital allodynia. PACAP-38 also caused activation and enhanced responses of dural-responsive pre-ganglionic pontine-superior salivatory parasympathetic neurons. Further, the PACAP-38 responsive receptor antagonists dissected a role of VPAC1 and PAC1 receptors in attenuating cranial-autonomic and trigeminal-neuronal responses to activation of the cranial parasympathetic projection, which requires post-ganglionic parasympathetic neurotransmission. CONCLUSION AND IMPLICATIONS: Given the prevailing view that sumatriptan acts to some degree via a peripheral mechanism, our data support that PACAP-38 mediated receptor activation modulates headache-related cranial-autonomic and trigeminovascular responses via peripheral and central components of the cranial parasympathetic projection. This provides a mechanistic rationale for the association of CAS with more severe headache phenotypes in cluster headache and migraine, and supports the cranial parasympathetic projection as a potential novel locus for treatment by selectively targeting PACAP-38 or PACAP-38 responsive VPAC1 /PAC1 receptors.

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.

Changes in the Phenotype of Intramural Inhibitory Neurons of the Porcine Descending Colon Resulting from Glyphosate Administration.

Environmental contamination and the resulting food contamination represent a serious problem and pose a major threat to animal and human health. The gastrointestinal tract is directly exposed to a variety of substances. One is glyphosate, whose presence in the soil is commonly observed. This study demonstrates the effects of low and high glyphosate doses on the populations of intramural neurons of the porcine descending colon. An analysis was performed on neurons ex-pressing the vasoactive intestinal peptide, pituitary adenylate cyclase-activating peptide, a neuronal isoform of nitrogen oxide synthase, and galanin. Even a low dose of glyphosate increased the number of neurons immunoreactive against the studied substances. However, the changes depended on both the plexus analysed and the substance tested. Meanwhile, a high glyphosate dose resulted in quantitative changes (an increase in the number) within neurons immunoreactive against all the studied neuropeptides/enzymes in the myenteric plexus and both submucosal plexuses. The response of the enteric nervous system in the form of an increase in the number of neurons immunoreactive against neuroprotective substances may suggest that glyphosate has a toxic effect on enteric neurons which attempt to increase their survivability through the released neuroprotective substances.

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.

PAC1 receptor modulation of freezing and flight behavior in periaqueductal gray.

The midbrain periaqueductal gray (PAG) region is a critical anatomical regulator of fear-related species-specific defensive reactions (SSDRs). Pituitary adenylate-cyclase-activating polypeptide (PACAP), and its main receptor PAC1, play an important role in fear-related behavior and anxiety disorders. However, the function of the PACAP-PAC1 system within the PAG with regards to SSDRs has received little attention. To address this gap, we used transgenic PAC1flox/flox mice to examine both conditional and unconditional defensive reactions. We performed conditional PAC1 gene deletion within the ventrolateral(vl)PAG of PAC1flox/flox mice using an adeno-associated virus (AAV) coding for Cre recombinase. Following viral expression, we used a white noise fear conditioning preparation that produces both an unconditional activity burst to the onset of noise that is followed by conditional freezing. On Day 1, mice received five white noise foot-shock pairings, whereas on Day 2, they were exposed to white noise five times without shock and we scored the activity burst and freezing to the white noise. Following behavioral testing, histology for immunofluorescent analysis was conducted in order to identify PACAP positive cells and stress-induced c-fos activity respectively. We found that PAC1 deletion in vlPAG increased the unconditional activity burst response but disrupted conditional freezing. PAC1 deletion was accompanied by higher c-fos activity following the behavioral experiments. Furthermore, a significant portion of PACAP-EGFP positive cells showed overlapping expression with VGAT, indicating their association with inhibitory neurons. The findings suggested that intact PACAP-PAC1 mechanisms are essential for SSDRs in vlPAG. Therefore, midbrain PACAP contributes to the underlying molecular mechanisms regulating fear responses.

Neuroprotective effect of the PACAP-ADNP axis on SOD1G93A mutant motor neuron death induced by trophic factors deprivation.

Amyotrophic lateral Sclerosis (ALS) is a neurodegenerative disease characterized by progressive degeneration of motor neurons in the central nervous system. Mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1) account for approximately in 20% of familial ALS cases. The pathological mechanisms underlying the toxicity induced by mutated SOD1 are still unknown. However, it has been hypothesized that oxidative stress (OS) has a crucial role in motor neuron degeneration in ALS patients. Moreover, it has been described that SOD1 mutation interferes expression of nuclear factor erythroid 2-related factor 2 (Nrf2), a protective key modulator against OS and reactive oxygen species (ROS) formation. The protective effect of pituitary adenylate cyclase-activating peptide (PACAP) has been demonstrated in various neurological disorders, including ALS. Some of its effects are mediated by the stimulation of an intracellular factor known as activity-dependent protein (ADNP). The role of PACAP-ADNP axis on mutated SOD1 motor neuron degeneration has not been explored, yet. The present study aimed to investigate whether PACAP prevented apoptotic cell death induced by growth factor deprivation through ADNP activation and whether the peptidergic axis can counteract the OS insult. By using an in vitro model of ALS, we demonstrated that PACAP by binding to PAC1 receptor (PAC1R) prevented motor neuron death induced by serum deprivation through induction of the ADNP expression via PKC stimulation. Furthermore, we have also demonstrated that the PACAP/ADNP axis counteracted ROS formation by inducing translocation of the Nfr2 from the cytoplasm to the nucleus. In conclusion, our study provides new insights regarding the protective role of PACAP-ADNP in ALS.

PACAP-Sirtuin3 alleviates cognitive impairment through autophagy in Alzheimer's disease.

BACKGROUND: Autophagy is vital in the pathogenesis of neurodegeneration. Thus far, no studies have specifically investigated the relationship between pituitary adenylate cyclase-activating polypeptide (PACAP) and autophagy, particularly in the context of Alzheimer's disease (AD). This study used in vitro and in vivo models, along with clinical samples, to explore interactions between PACAP and autophagy in AD. METHODS: AD model mice were administered 6 µl of 0.1 mg/ml PACAP liquid intranasally for 4 weeks, then subjected to behavioral analyses to assess the benefits of PACAP treatment. The underlying mechanisms of PACAP-induced effects were investigated by methods including real-time quantitative polymerase chain reaction, RNA sequencing, immunofluorescence, and western blotting. Exosomes were extracted from human serum and subjected to enzyme-linked immunosorbent assays to examine autophagy pathways. The clinical and therapeutic implications of PACAP and autophagy were extensively investigated throughout the experiment. RESULTS: Impaired autophagy was a critical step in amyloid ß (Aß) and Tau deposition; PACAP enhanced autophagy and attenuated cognitive impairment. RNA sequencing revealed three pathways that may be involved in AD progression: PI3K-AKT, mTOR, and AMPK. In vivo and in vitro studies showed that sirtuin3 knockdown diminished the ability of PACAP to restore normal autophagy function, resulting in phagocytosis dysregulation and the accumulation of pTau, Tau, and Aß. Additionally, the autophagic biomarker MAP1LC3 demonstrated a positive association with PACAP in human serum. CONCLUSIONS: PACAP reverses AD-induced cognitive impairment through autophagy, using sirtuin3 as a key mediator. MAP1LC3 has a positive relationship with PACAP in humans. These findings provide insights regarding potential uses of intranasal PACAP and sirtuin3 agonists in AD treatment. TRIAL REGISTRATION: NCT04320368.

Protective Effects of Pituitary Adenylate-Cyclase-Activating Polypeptide on Retinal Vasculature and Molecular Responses in a Rat Model of Moderate Glaucoma.

Despite the high probability of glaucoma-related blindness, its cause is not fully understood and there is no efficient therapeutic strategy for neuroprotection. Vascular factors have been suggested to play an important role in glaucoma development and progression. Previously, we have proven the neuroprotective effects of pituitary adenylate-cyclase-activating polypeptide (PACAP) eye drops in an inducible, microbeads model in rats that is able to reproduce many clinically relevant features of human glaucoma. In the present study, we examined the potential protective effects of PACAP1-38 on the retinal vasculature and the molecular changes in hypoxia. Ocular hypertension was induced by injection of microbeads into the anterior chamber, while control rats received PBS. PACAP dissolved in vehicle (1 µg/drop) or vehicle treatment was started one day after the injections for four weeks three times a day. Retinal degeneration was assessed with optical coherence tomography (OCT), and vascular and molecular changes were assessed by immunofluorescence labeling. HIF1-α and VEGF-A protein levels were measured by Western blot. OCT images proved severe retinal degeneration in the glaucomatous group, while PACAP1-38 eye drops had a retinoprotective effect. Vascular parameters were deteriorated and molecular analysis suggested hypoxic conditions in glaucoma. PACAP treatment exerted a positive effect against these alterations. In summary, PACAP could prevent the severe damage to the retina and its vasculature induced by ocular hypertension in a microbeads model.

Effect of pituitary adenylate cyclase-activating polypeptide supplementation, applied during or after vitrification on mouse embryo.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide with widespread occurrence and diverse functions. It occurs in high levels in the gonads suggesting a potential central role in reproduction. The aim of our study was to assess the effect of PACAP treatment during embryo vitrification on the developmental rate and the expression of the heparin-binding EGF-like growth factor gene (Hbegf). Mouse embryos, obtained from superovulated females were allocated into the four treatment groups. In EM1 and EM2, the embryos were prepared for vitrification in an Equilibration Solution that was supplemented with 1 or 2 µM PACAP1-38, respectively. The embyos in groups CM1 and CM2 were not treated prior to vitrification but were cultured in a medium supplemented with 1 or 2 µM PACAP1-38 after thawing. The Vitrified Control group consisted of embryos vitrified and thawed then cultured without PACAP1-38 treatment. A non-vitrified, non-treated Fresh Control group was also used. After 24 h of culture, the developmental rate of the embryos, as well as the relative expression level of the Hbegf gene, as determined by qPCR, were compared among groups. Higher developmental rate and Hbegf gene expression level were found in the embryos treated with a higher concentration of PACAP. These results indicate that PACAP treatment has a beneficial effect on the survival and development of vitrified/thawed mouse embryos.

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.

PACAP-ADNP axis prevents outer retinal barrier breakdown and choroidal neovascularization by interfering with VEGF secreted from retinal pigmented epitelium cells.

During diabetic retinopathy (DR) progression, the retina undergoes various metabolic changes, including hypoxia-signalling cascade induction in the cells of retinal pigmented epithelium (RPE). The overexpression of hypoxic inducible factors causes transcription of many target genes including vascular endothelial growth factor (VEGF). The RPE cells form the outer blood retinal barrier (oBRB), a specialized structure that regulates ions and metabolites flux into the retina to maintain a suitable quality of its extracellular microenvironment. VEGF worsens retinal condition since its secretion from the basolateral compartment of RPE cells compromises the barrier's integrity and induces choroidal neovascularization. In this work, we hypothesized that PACAP prevents the damage to oBRB and controls choroidal neovascularization through the induction of ADNP. Firstly, we demonstrated that ADNP is expressed in Streptozotocin (STZ)-induced diabetic animals. To validate our hypothesis, we cultured endothelial cells (H5V) forming vessels-like structures, in a conditioned medium (CM) derived from ARPE-19 cells exposed to hyperglycaemic/hypoxic insult, containing a known VEGF concentration. The involvement of PACAP-ADNP axis on oBRB integrity was evaluated through the measurement of trans-epithelial-electrical resistance and permeability assay performed on ARPE cell monolayer cultured in CM and by analysing the expression of two tight junction forming proteins, ZO1 and occludin. By culturing H5V in CM, we demonstrated that PACAP-ADNP axis counteracted vessels-like structures formation promoted by VEGF. In conclusion, the results suggested a primary role of PACAP/ADNP axis in preventing oBRB damage and in controlling aberrant choroidal neovascularization induced by VEGF secreted from RPE cells exposed to hyperglycaemia/hypoxic insult in DR.

Long-term Effects of the pituitary-adenylate cyclase-activating Polypeptide (PACAP38) in the Adult Mouse Retina: Microglial Activation and Induction of Neural Proliferation.

The degenerative retinal disorders characterized by progressive cell death and exacerbating inflammation lead ultimately to blindness. The ubiquitous neuropeptide, PACAP38 is a promising therapeutic agent as its proliferative potential and suppressive effect on microglia might enable cell replacement and attenuate inflammation, respectively. Our previous finding that PACAP38 caused a marked increase of the amacrine cells in the adult (1-year-old) mouse retina, served as a rationale of the current study. We aimed to determine the proliferating elements and the inflammatory status of the PACAP38-treated retina. Three months old mice were intravitreally injected with 100 pmol PACAP38 at 3 months intervals (3X). Retinas of 1-year-old animals were dissected and effects on cell proliferation, and expression of inflammatory regulators were analyzed. Interestingly, both mitogenic and anti-mitogenic actions were detected after PACAP38-treatment. Further analysis of the mitogenic effect revealed that proliferating cells include microglia, endothelial cells, and neurons of the ganglion cell layer but not amacrine cells. Furthermore, PACAP38 stimulated retinal microglia to polarize dominantly into M2-phenotype but also might cause subsequent angiogenesis. According to our results, PACAP38 might dampen pro-inflammatory responses and help tissue repair by reprogramming microglia into an M2 phenotype, nonetheless, with angiogenesis as a warning side effect.

Discovery and In Vitro Characterization of BAY 2686013, an Allosteric Small Molecule Antagonist of the Human Pituitary Adenylate Cyclase-Activating Polypeptide Receptor.

The human pituitary adenylate cyclase-activating polypeptide receptor (hPAC1-R), a class B G-protein-coupled receptor (GPCR) identified almost 30 years ago, represents an important pharmacological target in the areas of neuroscience, oncology, and immunology. Despite interest in this target, only a very limited number of small molecule modulators have been reported for this receptor. We herein describe the results of a drug discovery program aiming for the identification of a potent and selective hPAC1-R antagonist. An initial high-throughput screening (HTS) screen of 3.05 million compounds originating from the Bayer screening library failed to identify any tractable hits. A second, completely revised screen using native human embryonic kidney (HEK)293 cells yielded a small number of hits exhibiting antagonistic properties (4.2 million compounds screened). BAY 2686013 (1) emerged as a promising compound showing selective antagonistic activity in the submicromolar potency range. In-depth characterization supported the hypothesis that BAY 2686013 blocks receptor activity in a noncompetitive manner. Preclinical, pharmacokinetic profiling indicates that BAY 2686013 is a valuable tool compound for better understanding the signaling and function of hPAC1-R. SIGNIFICANCE STATEMENT: Although the human pituitary adenylate cyclase-activating polypeptide receptor (hPAC1-R) is of major significance as a therapeutic target with a well documented role in pain signaling, only a very limited number of small-molecule (SMOL) compounds are known to modulate its activity. We identified and thoroughly characterized a novel, potent, and selective SMOL antagonist of hPAC1-R (acting in an allosteric manner). These characteristics make BAY 2686013 an ideal tool for further studies.

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.

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.

Inhibition of the retinal orexin receptors affects the hypothalamic-pituitary-gonadal axis through retinal pituitary adenylate cyclase activating polypeptide (PACAP) in male Wistar rats.

Orexins A and B (OXA and OXB) and their receptors are expressed in the retina of both human and rodents and play a vital role in regulating signal transmission circuits in the retina. There is an anatomical-physiological relationship between the retinal ganglion cells and suprachiasmatic nucleus (SCN) through glutamate as a neurotransmitter and retinal pituitary adenylate cyclase-activating polypeptide (PACAP) as a co-transmitter. SCN is the main brain center for regulating the circadian rhythm, which governs the reproductive axis. The impact of retinal orexin receptors on the hypothalamic-pituitary-gonadal axis has not been investigated. Retinal OX1R or/and OX2R in adult male rats by 3 µl of SB-334867 (1 µg) or/and 3 µl of JNJ-10397049 (2 µg) were antagonized via intravitreal injection (IVI). Four time-periods were considered (3, 6, 12, and 24 h) for the controls without any treatment, SB-334867, JNJ-10397049, and SB-334867 + JNJ-10397049 groups. Antagonizing retinal OX1R or/and OX2R resulted in a significant elevation of retinal PACAP expression compared to control animals. In addition, expression of GnRH increased non-significantly in the hypothalamus over the 6 h of the study, and the serum concentration of LH decreased significantly in the SB-334867 group after 3 h of injection. Furthermore, testosterone serum levels declined significantly, especially within 3 h of injection; serum levels of progesterone were also exposed to a significant rise at least within 3 h of injection. However, the retinal PACAP expression changes were mediated by OX1R more effectively than by OX2R. In this study, we report the retinal orexins and their receptors as light-independent factors by which the retina affects the hypothalamic-pituitary-gonadal axis.

High plasma calcitonin gene-related peptide and serum pituitary adenylate cyclase-activating polypeptide levels in patients with neuropathic pain.

INTRODUCTION: Based on animal studies, calcitonin gene-related peptide (CGRP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are thought to play a role in neurobiological events such as neuropathic pain, neuroprotection, neurotransmission, neural plasticity, and neurotrophic effects. The aim of the study is to investigate whether there is a change in the blood level of CGRP and PACAP in patients with neuropathic pain and to look for clues about the utility of these peptides as pharmacological targets in the treatment of neuropathic pain in humans. METHODS: The study included 60 polyneuropathy patients with neuropathic pain, 30 polyneuropathy patients without neuropathic pain (NNP) and 29 healthy subjects as control group. Polyneuropathy patients with neuropathic pain were divided into two groups as diabetic (D-PNP) and non-diabetic polyneuropathy (ND-PNP) patients. Plasma CGRP and serum PACAP levels were measured from venous blood samples of the patients and healthy controls. RESULTS: The CGRP level was significantly higher in the D-PNP and ND-PNP groups compared to the control and NNP groups (P<0.05). PACAP levels were significantly higher in the D-PNP and ND-PNP groups compared to the control and NNP groups (P<0.05). There was no significant correlation between CGRP and PACAP levels and neuropathic pain scale (NPS). CONCLUSIONS: This study is the first to demonstrate elevated plasma CGRP and serum PACAP levels in polyneuropathy patients with neuropathic pain. The results of this study are important in terms of showing that both CGRP and PACAP can be new pharmacological targets in the treatment of neuropathic pain and polyneuropathy in humans.