The allosteric modulation effects of doxycycline, minocycline, and their derivatives on the neuropeptide receptor PAC1-R.

Class B G-protein coupled receptors (GPCR) PAC1-R is a neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP)-preferring receptor that mediates the effective neuroprotective activity. Based on our previous data showing that doxycycline and minocycline work as the positive allosteric modulator (PAM) of PAC1-R, we used computer molecular docking and isothermal titration calorimetry assay to further determine the bindings of doxycycline/minocycline's derivatives including tetracycline/tigecycline with the N-terminal extracellular domain of PAC1-R (PAC1-EC1). Then the cAMP assay combined with the PAC1-R natural agonist PACAP27 was used to confirm the possible PAM roles of the small-molecule antibiotics. The results showed that tetracycline/tigecycline had significant lower affinity to PAC1-EC1 than doxycycline/minocycline, which was consistent with their non-positive allosteric modulation activity on PAC1-R. Furthermore, by comparing the key residues contributing to the PAM binding with the predicted allosteric site in PAC1-EC1, we characterized four motifs contributing to PAM binding in PAC1-EC1. The site-directed mutation results showed that ASN60 played the most important role in the PAM binding of the small-molecule antibiotics, while ASP116 played a sensitive marginal role in the PAM binding. These results not only help to explain the clinical and experimental neuroprotective effects of doxycycline/minocycline, but also help to characterize the PAM binding site in PAC1-EC1, which will promote the screening and characterization of novel small-molecule PAMs targeting PAC1-EC1 with drug development potency in nerve system disease.

Src family kinase inhibitors blunt PACAP-induced PAC1 receptor endocytosis, phosphorylation of ERK, and the increase in cardiac neuron excitability.

Pituitary adenylate cyclase activating polypeptide (PACAP, Adcyap1) activation of PAC1 receptors ( Adcyap1r1) significantly increases excitability of guinea pig cardiac neurons. This modulation of excitability is mediated in part by plasma membrane G protein-dependent activation of adenylyl cyclase and downstream signaling cascades. However, additional mechanisms responsible for the enhanced excitability are activated following internalization of the PAC1 receptor and endosomal signaling. Src family kinases play critical roles mediating endocytosis of many trophic factor and G protein-coupled receptors. The present study investigated whether Src family kinases also support the PACAP-induced PAC1 receptor internalization, phosphorylation of ERK, and enhanced neuronal excitability. Using human embryonic kidney cells stably expressing a green fluorescent protein-tagged PAC1 receptor, treatment with the Src family kinase inhibitor PP2 (10 µM) markedly reduced the PACAP-induced PAC1 receptor internalization, and in parallel, both PP2 and Src inhibitor 1 (Src-1, 2 µM) reduced ERK activation determined by Western blot analysis. In contrast, Src family kinase inhibitors did not eliminate a PACAP-induced rise in global calcium generated by inositol (1,4,5)-trisphosphate-induced release of calcium from endoplasmic reticulum stores. From confocal analysis of phosphorylated ERK immunostaining, PP2 treatment significantly attenuated PACAP activation of ERK in neurons within cardiac ganglia whole mount preparations. Intracellular recordings demonstrated that PP2 also significantly blunted a PACAP-induced increase in cardiac neuron excitability. These studies demonstrate Src-related kinase activity in PAC1 receptor internalization, activation of MEK/ERK signaling, and regulation of neuronal excitability. The present results provide further support for the importance of PAC1 receptor endosomal signaling as a key mechanism regulating cellular function.

Rearrangement of a polar core provides a conserved mechanism for constitutive activation of class B G protein-coupled receptors.

The glucagon receptor (GCGR) belongs to the secretin-like (class B) family of G protein-coupled receptors (GPCRs) and is activated by the peptide hormone glucagon. The structures of an activated class B GPCR have remained unsolved, preventing a mechanistic understanding of how these receptors are activated. Using a combination of structural modeling and mutagenesis studies, we present here two modes of ligand-independent activation of GCGR. First, we identified a GCGR-specific hydrophobic lock comprising Met-338 and Phe-345 within the IC3 loop and transmembrane helix 6 (TM6) and found that this lock stabilizes the TM6 helix in the inactive conformation. Disruption of this hydrophobic lock led to constitutive G protein and arrestin signaling. Second, we discovered a polar core comprising conserved residues in TM2, TM3, TM6, and TM7, and mutations that disrupt this polar core led to constitutive GCGR activity. On the basis of these results, we propose a mechanistic model of GCGR activation in which TM6 is held in an inactive conformation by the conserved polar core and the hydrophobic lock. Mutations that disrupt these inhibitory elements allow TM6 to swing outward to adopt an active TM6 conformation similar to that of the canonical ß2-adrenergic receptor complexed with G protein and to that of rhodopsin complexed with arrestin. Importantly, mutations in the corresponding polar core of several other members of class B GPCRs, including PTH1R, PAC1R, VIP1R, and CRFR1, also induce constitutive G protein signaling, suggesting that the rearrangement of the polar core is a conserved mechanism for class B GPCR activation.

Backup Mechanisms Maintain PACAP/VIP-Induced Arterial Relaxations in Pituitary Adenylate Cyclase-Activating Polypeptide-Deficient Mice.

BACKGROUND: Pituitary adenylate cyclase-activating polypeptide (PACAP) is a multifunctional neuropeptide in the VIP/secretin/glucagon peptide superfamily. Two active forms, PACAP1-38 and PACAP1-27, act through G protein-coupled receptors, the PAC1 and VPAC1/2 receptors. Effects of PACAP include potent vasomotor activity. Vasomotor activity and organ-specific vasomotor effects of PACAP-deficient mice have not yet been investigated; thus, the assessment of its physiological importance in vasomotor functions is still missing. We hypothesized that backup mechanisms exist to maintain PACAP pathway activity in PACAP knockout (KO) mice. Thus, we investigated the vasomotor effects of exogenous vasoactive intestinal peptide (VIP) and PACAP polypeptides in PACAP wild-type (WT) and PACAP-deficient (KO) male mice. METHODS: Carotid and femoral arteries were isolated from 8- to 12-week-old male WT and PACAP-KO mice. Vasomotor responses were measured with isometric myography. RESULTS: In the arteries of WT mice the peptides induced relaxations, which were significantly greater to PACAP1-38 than to PACAP1-27 and VIP. In KO mice, PACAP1-38 did not elicit relaxation, whereas PACAP1-27 and VIP elicited significantly greater relaxation in KO mice than in WT mice. The specific PAC1R and VPAC1R antagonist completely blocked the PACAP-induced relaxations. CONCLUSION: Our data suggest that in PACAP deficiency, backup mechanisms maintain arterial relaxations to polypeptides, indicating an important physiological role for the PACAP pathway in the regulation of vascular tone.

PAC1R agonist maxadilan enhances hADSC viability and neural differentiation potential.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a structurally endogenous peptide with many biological roles. However, little is known about its presence or effects in human adipose-derived stem cells (hADSCs). In this study, the expression of PACAP type I receptor (PAC1R) was first confirmed in hADSCs. Maxadilan, a specific agonist of PAC1R, could increase hADSC proliferation as determined by Cell Counting Kit-8 and cell cycle analysis and promote migration as shown in wound-healing assays. Maxadilan also showed anti-apoptotic activity in hADSCs against serum withdrawal-induced apoptosis based on Annexin V/propidium iodide analysis and mitochondrial membrane potential assays. The anti-apoptotic effects of maxadilan correlated with the down-regulation of Cleaved Caspase 3 and Caspase 9 as well as up-regulation of Bcl-2. The chemical neural differentiation potential could be enhanced by maxadilan as indicated through quantitative PCR, Western blot and cell morphology analysis. Moreover, cytokine neural redifferentiation of hADSCs treated with maxadilan acquired stronger neuron-like functions with higher voltage-dependent tetrodotoxin-sensitive sodium currents, higher outward potassium currents and partial electrical impulses as determined using whole-cell patch clamp recordings. Maxadilan up-regulated the Wnt/ß-catenin signalling pathway associated with dimer-dependent activity of PAC1R, promoting cell viability that was inhibited by XAV939, and it also activated the protein kinase A (PKA) signalling pathway associated with ligand-dependent activity of PAC1R, enhancing cell viability and neural differentiation potential that was inhibited by H-89. In summary, these results demonstrated that PAC1R is present in hADSCs, and maxadilan could enhance hADSC viability and neural differentiation potential in neural differentiation medium.

Spinal astrocytic activation contributes to both induction and maintenance of pituitary adenylate cyclase-activating polypeptide type 1 receptor-induced long-lasting mechanical allodynia in mice.

BACKGROUND: Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors are present in the spinal dorsal horn and dorsal root ganglia, suggesting an important role of PACAP-PACAP receptors signaling system in the modulation of spinal nociceptive transmission. We have previously reported that a single intrathecal injection of PACAP or a PACAP specific (PAC1) receptor selective agonist, maxadilan, in mice induced dose-dependent aversive behaviors, which lasted more than 30 min, and suggested that the maintenance of the nociceptive behaviors was associated with the spinal astrocytic activation. RESULTS: We found that a single intrathecal administration of PACAP or maxadilan also produced long-lasting hind paw mechanical allodynia, which persisted at least 84 days without affecting thermal nociceptive threshold. In contrast, intrathecal application of vasoactive intestinal polypeptide did not change mechanical threshold, and substance P, calcitonin gene-related peptide, or N-methyl-D-aspartate induced only transient mechanical allodynia, which disappeared within 21 days. Western blot and immunohistochemical analyses with an astrocytic marker, glial fibrillary acidic protein, revealed that the spinal PAC1 receptor stimulation caused sustained astrocytic activation, which also lasted more than 84 days. Intrathecal co-administration of L-α-aminoadipate, an astroglial toxin, with PACAP or maxadilan almost completely prevented the induction of the mechanical allodynia. Furthermore, intrathecal treatment of L-α-aminoadipate at 84 days after the PAC1 stimulation transiently reversed the mechanical allodynia accompanied by the reduction of glial fibrillary acidic protein expression level. CONCLUSION: Our data suggest that spinal astrocytic activation triggered by the PAC1 receptor stimulation contributes to both induction and maintenance of the long-term mechanical allodynia.

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

BACKGROUND AND PURPOSE: 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. EXPERIMENTAL APPROACH: 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. KEY RESULTS: 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. CONCLUSIONS AND IMPLICATIONS: 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.

Pharmacological characterization of VIP and PACAP receptors in the human meningeal and coronary artery.

OBJECTIVE: We pharmacologically characterized pituitary adenylate cyclase-activating polypeptides (PACAPs), vasoactive intestinal peptide (VIP) and the VPAC(1), VPAC(2) and PAC(1) receptors in human meningeal (for their role in migraine) and coronary (for potential side effects) arteries. METHODS: Concentration response curves to PACAP38, PACAP27, VIP and the VPAC(1) receptor agonist ([Lys15,Arg16,Leu27]-VIP[1-7]-GRF[8-27]) were constructed in the absence or presence of the PAC(1) receptor antagonist PACAP6-38 or the VPAC(1) receptor antagonist, PG97269. mRNA expression was measured using qPCR. RESULTS: PACAP38 was less potent than VIP in both arteries. Both peptides had lower potency and efficacy in meningeal than in coronary arteries, while mRNA expression of VPAC(1) receptor was more pronounced in meningeal arteries. PACAP6-38 reduced the E(max) of PACAP27, while PG97269 right-shifted the VIP-induced relaxation curve only in the coronary arteries. CONCLUSION: The direct vasodilatory effect of VIP and PACAP might be less relevant than the central effect of this compound in migraine pathogenesis.

VPAC and PAC receptors: From ligands to function.

Vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase activating polypeptides (PACAPs) share 68% identity at the amino acid level and belong to the secretin peptide family. Following the initial discovery of VIP almost four decades ago a substantial amount of knowledge has been presented describing the mechanisms of action, distribution and pleiotropic functions of these related peptides. It is now known that the physiological actions of these widely distributed peptides are produced through activation of three common G-protein coupled receptors (VPAC(1), VPAC(2) and PAC(1)R) which preferentially stimulate adenylate cyclase and increase intracellular cAMP, although stimulation of other intracellular messengers, including calcium and phospholipase D, has been reported. Using a range of in vitro and in vivo approaches, including cell-based functional assays, transgenic animals and rodent models of disease, VPAC/PAC receptor activation has been associated with numerous physiological processes (e.g. control of circadian rhythms) and clinical conditions (e.g. pulmonary hypertension), which underlies on-going research efforts and makes these peptides and their cognate receptors attractive targets for the pharmaceutical industry. However, despite the considerable interest in VPAC/PAC receptors and the processes which they mediate, there is still a paucity of selective and available, non-peptide ligands, which has hindered further advances in this field both at the basic research and clinical level. This review summarises the current knowledge of VIP/PACAP and the VPAC/PAC receptors with regard to their distribution, pharmacology, signalling pathways, splice variants and finally, the utility of animal models in exploring their physiological roles.

Differential activation of guinea pig intrinsic cardiac neurons by the PAC1 agonists maxadilan and pituitary adenylate cyclase-activating polypeptide 27 (PACAP27).

Pituitary adenylate cyclase-activating polypeptide (PACAP) evokes tachycardia followed by a larger cholinergic bradycardia in isolated guinea pig hearts. We used the selective PAC1 receptor agonist maxadilan and vasoactive intestinal polypeptide (VIP) to test the hypothesis that PACAP27-evoked tachycardia and bradycardia are mediated by VPAC and PAC1 receptors, respectively. Chronotropic actions of these peptides were evaluated in isolated perfused hearts. Direct neuronal actions were determined by intracellular voltage recordings from cholinergic neurons in atrial ganglion whole mounts. Administration of 1 nmol of PACAP27 to isolated hearts evoked typical biphasic rate responses, whereas 1 nmol of maxadilan caused only a minor rate decrease. Desensitization with VIP eliminated the positive chronotropic effect of PACAP27 selectively. Local application of PACAP27 to cardiac neurons frequently evoked slow depolarization and caused prolonged increase of neuronal excitability. Maxadilan rarely affected membrane potential but consistently increased excitability. VIP had no effect on excitability and evoked depolarization in only a few neurons. Because maxadilan increased neuronal excitability but did not trigger action potentials as PACAP often does, we evaluated the interaction of maxadilan with substance P (SP) in isolated hearts. SP depolarizes cardiac neurons more consistently than PACAP, often triggers neuronal action potentials, and causes bradycardia but does not increase neuronal excitability. Maxadilan had a persistent effect to augment negative chronotropic responses to SP. These findings support our hypothesis that PACAP evokes tachycardia and bradycardia through VPAC and PAC1 receptors, respectively. They also suggest that maxadilan and PACAP27 differ in activating PAC1 receptors on cardiac neurons and/or stimulating downstream signaling mechanisms.

Long-term administration of maxadilan improves glucose tolerance and insulin sensitivity in mice.

Maxadilan and its truncated variant, M65, are agonist and antagonist specific, respectively, for the PAC1 receptor. PAC1 is the specific receptor for the pituitary adenylate cyclase-activating peptide (PACAP), which is not shared by vasoactive intestinal peptide (VIP). PACAP is a ubiquitous peptide of the glucagon superfamily that is involved in glucose homeostasis and regulation of insulin secretion. This study employed the recombinant maxadilan and M65 to evaluate the PAC1 receptor-mediated effects on energy metabolism using NIH mice. First, the acute effect of maxadilan-induced hyperglycemia was blocked by M65. In long-term studies, NIH mice were given daily intraperitoneal injections with maxadilan, M65, or vehicle for 21 days. Maxadilan suppressed feeding and enhanced water intake significantly for the first several days. After that period, maxadilan treatment continued to promote food and water intake. Long-term administration of maxadilan led to an increase in body weight (P<0.01), decrease in body fat (P<0.01), down-regulation of basal plasma glucose (P<0.01), upregulation of basal plasma insulin (P<0.01) and improved glucose tolerance (P<0.01) and insulin sensitivity (P<0.01). An elevation in plasma LDL (P<0.01) was also observed in the maxadilan group. However, M65 displayed no significant adverse effects on the aforementioned parameters except basal plasma glucose (P<0.05). The significant changes induced by maxadilan indicate that the PAC1 receptor plays multiple key roles in carbohydrate metabolism, lipid metabolism and energy homeostasis in mice.

IL-6 knockout mice are protected from cocaine-induced kindling behaviors; possible involvement of JAK2/STAT3 and PACAP signalings.

IL-6 has been recognized as an anticonvulsant against certain neuroexcitotoxicities. We aimed to investigate on the interactive role between IL-6 and PACAP in cocaine-induced kindling behaviors. Although we found that cocaine (45 mg/kg, i.p./day x 5) significantly increased IL-6 and TNF-α expression, it resulted in a decrease in IFN-γ expression. We observed that the cocaine-induced increase in IL-6 expression was more pronounced than that in TNF-α expression. Genetic depletion of IL-6 significantly activated cocaine kindling behaviors. This phenomenon was also consistently observed in WT mice that received a neutralizing IL-6 receptor antibody. Cocaine-treated IL-6 knockout mice exhibited significantly decreased PACAP and PACAP receptor (PAC1R) mRNA levels and significantly increased TNF-α gene expression. TNF-α knockout mice were protected from cocaine kindling via an up-regulation of IL-6, phospho-JAK2/STAT3, PACAP, and PAC1R levels, which produced anti-apoptotic effects. Recombinant IL-6 protein (rIL-6, 2 µg, i.v./mouse/day x 5) also up-regulated phospho-JAK2/STAT3, PACAP, and PAC1R mRNA levels, leading to anti-apoptotic effects in IL-6 knockout mice. Consistently, AG490, a JAK2/STAT3 inhibitor, and PACAP 6-38, a PAC1 receptor antagonist, counteracted rIL-6-mediated protection. Combined, our results suggest that IL-6 gene requires up-regulation of phospho-JAK2/STAT3, PACAP, and PAC1R and down-regulation of the TNF-α gene to modulate its anticonvulsive/neuroprotective potential.

The neuropeptide PACAP promotes the alpha-secretase pathway for processing the Alzheimer amyloid precursor protein.

The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) has neurotrophic as well as anti-apoptotic properties and is involved in learning and memory processes. Its specific G protein-coupled receptor PAC1 is expressed in several central nervous system (CNS) regions, including the hippocampal formation. Here we examined the effect of PAC1 receptor activation on alpha-secretase cleavage of the amyloid precursor protein (APP) and the production of secreted APP (APPsalpha). Stimulation of endogenously expressed PAC1 receptors with PACAP in human neuroblastoma cells increased APPsalpha secretion, which was completely inhibited by the PAC1 receptor specific antagonist PACAP-(6-38). In HEK cells stably overexpressing functional PAC1 receptors, PACAP-27 and PACAP-38 strongly stimulated alpha-secretase cleavage of APP. The PACAP-induced APPsalpha production was dose dependent and saturable. This increase of alpha-secretase activity was completely abolished by hydroxamate-based metalloproteinase inhibitors, including a preferential ADAM 10 inhibitor. By using several specific protein kinase inhibitors, we show that the MAP-kinase pathway [including extracellular-regulated kinase (ERK) 1 and ERK2] and phosphatidylinositol 3-kinase mediate the PACAP-induced alpha-secretase activation. Our findings provide evidence for a role of the neuropeptide PACAP in stimulation of the nonamyloidogenic pathway, which might be related to its neuroprotective properties.

Maxadilan, a PAC1 receptor agonist from sand flies.

In 1991, a potent 61 amino acid vasodilator peptide, named maxadilan, was isolated from the salivary glands of the sand fly. Subsequently, it was shown that this peptide specifically and potently activated the mammalian PAC1 receptor, one of the three receptors for PACAP. These studies and the link between maxadilan and leishmaniasis are discussed.

Class II G protein-coupled receptors for VIP and PACAP: structure, models of activation and pharmacology.

VIP and PACAP impact strongly on human pathophysiology. Their receptors are very promising targets for developing new drugs in the treatment of inflammatory and neurodegenerative diseases. This article reviews the present knowledge regarding VIP and PACAP receptors, i.e. VPAC1, VPAC2 and PAC1. This includes: (I) a critical review of instrumental peptide agonists and antagonists; (II) a survey of recent data regarding the structure of VPAC1 receptor and the docking of VIP in the receptor binding domain. Structural models for the VPAC2 and PAC1 receptor N-terminal ectodomains are also described; (III) A critical description of the two models of VPAC1 receptor activation in the general context of class II/family B G protein-coupled receptors.

Role of transglutaminase 2 in PAC1 receptor mediated protection against hypoxia-induced cell death and neurite outgrowth in differentiating N2a neuroblastoma cells.

The PAC1 receptor and tissue transglutaminase (TG2) play important roles in neurite outgrowth and modulation of neuronal cell survival. In this study, we investigated the regulation of TG2 activity by the PAC1 receptor in retinoic acid-induced differentiating N2a neuroblastoma cells. TG2 transamidase activity was determined using an amine incorporation and a peptide cross linking assay. In situ TG2 activity was assessed by visualising the incorporation of biotin-X-cadaverine using confocal microscopy. TG2 phosphorylation was monitored via immunoprecipitation and Western blotting. The role of TG2 in PAC1 receptor-induced cytoprotection and neurite outgrowth was investigated by monitoring hypoxia-induced cell death and appearance of axonal-like processes, respectively. The amine incorporation and protein crosslinking activity of TG2 increased in a time and concentration-dependent manner following stimulation with pituitary adenylate cyclase-activating polypeptide-27 (PACAP-27). PACAP-27 mediated increases in TG2 activity were abolished by the TG2 inhibitors Z-DON and R283 and by pharmacological inhibition of protein kinase A (KT 5720 and Rp-cAMPs), protein kinase C (Ro 31-8220), MEK1/2 (PD 98059), and removal of extracellular Ca2+. Fluorescence microscopy demonstrated PACAP-27 induced in situ TG2 activity. TG2 inhibition blocked PACAP-27 induced attenuation of hypoxia-induced cell death and outgrowth of axon-like processes. TG2 activation and cytoprotection were also observed in human SH-SY5Y cells. Together, these results demonstrate that TG2 activity was stimulated downstream of the PAC1 receptor via a multi protein kinase dependent pathway. Furthermore, PAC1 receptor-induced cytoprotection and neurite outgrowth are dependent upon TG2. These results highlight the importance of TG2 in the cellular functions of the PAC1 receptor.

Neuroendocrine Underpinnings of Increased Risk for Posttraumatic Stress Disorder in Women.

Women are particularly vulnerable to the effects of psychological trauma and the development of trauma-, stressor-, and anxiety-related mental illnesses such as posttraumatic stress disorder (PTSD). In the current chapter, we examine the female hormonal systems that interact with psychobiological stress response systems to elicit maladaptive behavior and mental disease states in traumatized female populations. In addition, we provide a contemporary translational example of a stress vulnerability genomic profile (coding for pituitary adenylate cyclase-activating polypeptide) that may underlie the specific susceptibilities observed in women. Translational scientific investigations such as those described herein may lead to the identification of risk and resilience factors for PTSD as well as enhanced clinical interventions for treating excessive fear and anxiety.

Role of pituitary adenylyl cyclase-activating polypeptide in intracellular calcium dynamics of neurons and satellite cells in rat superior cervical ganglia.

Pituitary adenylyl cyclase-activating polypeptide (PACAP) is a bioactive peptide with diverse effects in the nervous system. The present study investigated whether stimulation of PACAP receptors (PACAPRs) induces responses in neurons and satellite cells of the superior cervical ganglia (SCG), with special reference to intracellular Ca2+ ([Ca2+]i) changes. The expression of PACAPRs in SCG was detected by reverse transcription-PCR. PACAP type 1 receptor (PAC1R), vasoactive intestinal peptide receptor type (VPAC)1R, and VPAC2R transcripts were expressed in SCG, with PAC1R showing the highest levels. Confocal microscopy analysis revealed that PACAP38 and PACAP27 induced an increase in [Ca2+]i in SCG, first in satellite cells and subsequently in neurons. Neither extracellular Ca2+ removal nor Ca2+ channel blockade affected the PACAP38-induced increase in [Ca2+]i in satellite cells; however, this was partly inhibited in neurons. U73122 or xestospongin C treatment completely and partly abrogated [Ca2+]i changes in satellite cells and in neurons, respectively, whereas VPAC1R and VPAC2R agonists increased [Ca2+]i in satellite cells only. This is the first report demonstrating the expression of PACAPRs specifically, VPAC1 and VPAC2 in SCG and providing evidence for PACAP38-induced [Ca2+]i changes in both satellite cells and neurons via Ca2+ mobilization.

Neuropeptide mimetics and antagonists in the treatment of inflammatory disease: focus on VIP and PACAP.

Corticosteroids are the mainstay treatment for most severe inflammatory disorders. Due to the considerable toxicity associated with their long-term use, there is a great need for alternative treatments. Recently, two closely related neuropeptides with potent neuromodulatory activities, vasoactive intestinal peptide (VIP) and pituitary adenylyl cyclase activating peptide (PACAP) have emerged as candidate molecules for the treatment of such pathologies. These peptides act primarily on three high affinity receptor subtypes expressed on multiple immune cell types, and orchestrate a cytokine response that is primarily anti-inflammatory. In this regard, systemic treatment with these peptides has been shown to greatly reduce the clinical symptoms and alter the pathogenic and cytokine profiles in animal models of rheumatoid arthritis, Crohn's disease, septic shock, and multiple sclerosis. Likewise, VIP and PACAP receptor knockout and overexpressing mice show altered immune responses in different models. We review here data demonstrating the potential effectiveness of these peptides in immune disorders, discuss receptor pharmacology and signaling pathways, describe the development of receptor specific agonists and antagonists, and discuss pharmaceutical considerations relevant to the specific delivery of analogs to the appropriate targets.

The Protective Role of PAC1-Receptor Agonist Maxadilan in BCCAO-Induced Retinal Degeneration.

A number of studies have proven that pituitary adenylate cyclase activating polypeptide (PACAP) is protective in neurodegenerative diseases. Permanent bilateral common carotid artery occlusion (BCCAO) causes severe degeneration in the rat retina. In our previous studies, protective effects were observed with PACAP1-38, PACAP1-27, and VIP but not with their related peptides, glucagon, or secretin in BCCAO. All three PACAP receptors (PAC1, VPAC1, VPAC2) appear in the retina. Molecular and immunohistochemical analysis demonstrated that the retinoprotective effects are most probably mainly mediated by the PAC1 receptor. The aim of the present study was to investigate the retinoprotective effects of a selective PAC1-receptor agonist maxadilan in BCCAO-induced retinopathy. Wistar rats were used in the experiment. After performing BCCAO, the right eye was treated with intravitreal maxadilan (0.1 or 1 µM), while the left eye was injected with vehicle. Sham-operated rats received the same treatment. Two weeks after the operation, retinas were processed for standard morphometric and molecular analysis. Intravitreal injection of 0.1 or 1 µM maxadilan caused significant protection in the thickness of most retinal layers and the number of cells in the GCL compared to the BCCAO-operated eyes. In addition, 1 µM maxadilan application was more effective than 0.1 µM maxadilan treatment in the ONL, INL, IPL, and the entire retina (OLM-ILM). Maxadilan treatment significantly decreased cytokine expression (CINC-1, IL-1α, and L-selectin) in ischemia. In summary, our histological and molecular analysis showed that maxadilan, a selective PAC1 receptor agonist, has a protective role in BCCAO-induced retinal degeneration, further supporting the role of PAC1 receptor conveying the retinoprotective effects of PACAP.