Injection Of Pituitary Adenylate Cyclase-activating Polypeptide Research Articles

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

BackgroundTrigeminal 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. ObjectiveWe 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 designSprague-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. ResultsThe 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. ConclusionAfter 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.

Intravitreal Injection of PACAP Attenuates Acute Ocular Hypertension-Induced Retinal Injury Via Anti-Apoptosis and Anti-Inflammation in Mice.

PurposePituitary adenylate cyclase-activating polypeptide (PACAP) has shown potent neuroprotective effects in central nervous system and retina disorders. However, whether PACAP can attenuate retinal neurodegeneration induced by acute ocular hypertension (AOH) and the underlying mechanisms remain unknown. In this study, we aimed to investigate the effects of PACAP on the survival and function of retinal ganglion cells (RGCs), apoptosis, and inflammation in a mouse model of AOH injury.MethodsPACAP was injected into the vitreous body immediately after inducing AOH injury. Hematoxylin and eosin staining and optical coherence tomography were used to evaluate the loss of retina tissue. Pattern electroretinogram was used to evaluate the function of RGCs. TUNEL assay was used to detect apoptosis. Immunofluorescence and western blot were employed to evaluate protein expression levels.ResultsPACAP treatment significantly reduced the losses of whole retina and inner retina thicknesses, Tuj1-positive RGCs, and the amplitudes of pattern electroretinograms induced by AOH injury. Additionally, PACAP treatment remarkably reduced the number of TUNEL-positive cells and inhibited the upregulation of Bim, Bax, and cleaved caspase-3 and downregulation of Bcl-xL after AOH injury. Moreover, PACAP markedly inhibited retinal reactive gliosis and vascular inflammation, as demonstrated by the downregulation of GFAP, Iba1, CD68, and CD45 in PACAP-treated mice. Furthermore, upregulated expression of NF-κB and phosphorylated NF-κB induced by AOH injury was attenuated by PACAP treatment.ConclusionsPACAP could prevent the loss of retinal tissue and improve the survival and function of RGCs. The neuroprotective effect of PACAP is probably associated with its potent anti-apoptotic and anti-inflammatory effects.

Identification of PACAP-Expressing Neurons in the Hypothalamic Origins of Sympathetic Outflow Tracts Terminating in Brown Adipose Tissue of Mice

Abstract Adaptive thermogenesis in brown adipose tissue is stimulated by the sympathetic nervous system (SNS) in response to cold stress. Using retrograde viral transneuronal tract tracers, previous studies have identified that the paraventricular nucleus (PVN), ventromedial hypothalamus (VMH), and median preoptic nucleus (MnPO) contain neurons that are part of sympathetic outflow tracts to brown adipose tissue, presumptively involved in SNS stimulation of interscapular brown adipose tissue (iBAT). Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) is a peptide hormone known to regulate energy homeostasis, acting in both the central (CNS) and peripheral nervous system (PNS). Mice lacking PACAP have impaired adrenergic-induced thermogenesis and a cold-sensitive phenotype. In the CNS, PACAP is highly expressed in the VMH, MnPO, and PVN of the hypothalamus. Injection of PACAP into the VMN increased core body temperature and sympathetic nerve activity to brown adipose tissue. While these studies show exogenous PACAP can activate sympathetic outflow tracts to brown adipose tissue, they do not confirm that endogenously expressed PACAP induces sympathetic nerve activity as an adaptive mechanism to cold stress, or if sympathetic outflow tracts originating in the hypothalamus express PACAP. We hypothesize that PACAP is expressed in neurons of sympathetic outflow tracts originating in the hypothalamus. To test this hypothesis, PACAP-eGFP transgenic mice were injected with the retrograde neural tracer, pseudorabies virus tagged with β-galactosidase (β-gal, PRV-BaBlu), in iBAT where postganglionic nerves innervate the tissue. Five-days post-infection, animals were culled, brains removed and cryosectioned. Neurons positive for green fluorescent protein (eGFP) and/or β-gal immunoreactivity (ir) were identified by immunohistochemistry in serial coronal and sagittal brain cryo-sections. Co-occurrence of eGFP-ir and β-gal-ir, inferred PACAP expressing neurons present in sympathetic outflow tracts (ImageJ). Co-occurrence was identified in several structures in the hypothalamus and thalamus. In conclusion, this study presents neuroanatomical evidence for populations of PACAPinergic neurons in the hypothalamus that are part of sympathetic outflow tracts to brown adipose tissue, providing further evidence of a central role for PACAP in regulating energy homeostasis.

SUN-116 The Endogenous Pituitary Adenylate Cyclase-Activating Polypeptide Increases Food Intake by Modulating the Expression of Neuropeptides in the Hypothalamus

Pituitary adenylate cyclase activating polypeptide (PACAP) is a pleiotropic neuropeptide which is involved in a variety physiological function. Regarding the appetite control, there are several reports that intracerebroventricular (ICV) injection of PACAP to mice reduced food intake (Resch et al, 2011, 2013, 2014). On the other hand, food intake is decreased in PACAP knockout (KO) mice (Nakata et al, 2004). Thus, there is some discrepancy in the functional role of PACAP in energy homeostasis including appetite control. Since the mechanism how PACAP regulates appetite remains unknown, we attempted to elucidate how PACAP is involved in the regulation of appetite in terms of the relationship with other neuropeptides regulating appetite.To address this issue, we assessed the expression of several neuropeptide which is involved in appetite control in PACAP knockout (KO) mice, and by administration of PACAP antagonist, PACAP[6-38] which is specific for PAC1R and VPAC2R. At first, we confirmed that nocturnal and daily food intake of PACAP KO mice was significantly lower than those of wild-type (WT) mice. Then, we observed that in food consumption at 4, 8, or 24 hours of refeeding after fasting for 2 days, the food intake at 8 hours of the refeeding was significantly lower in PACAP KO mice than in the WT mice, that is, PACAP KO mice showed a decrease in food intake in the fasting/refeeding paradigm. On the other hand, PACAP mRNA level was increased significantly by fasting in the whole hypothalamus of WT mice. The ICV administration of a PACAP antagonist PACAP[6-38] (1 nmol) reduced the diurnal food intake of WT mice after 1, 2, 4 and, 24 hours of injection. Furthermore we also observed that the expression level of agouti-related peptide (AgRP) was decreased in PACAP KO mice but that of proopiomelanocortin (POMC) was increased in the hypothalamus of PACAP KO mice after refeeding for 4 hours. On the other hand, the expression of AgRP was down-regulated by the injection of PACAP[6-38] at 4 hours after refeeding in WT mice. Taken together, our study demonstrated that food intake in mice was enhanced by the endogenous PACAP not only in daily but also in fasting-refeeding paradigms via the modulation of neuropeptides AgRP and/or POMC in the mouse hypothalamus, indicating endogenous PACAP might upregulate appetite in concert with other neuropeptides controlling appetite.

Expression Patterns of PACAP and PAC1R Genes and Anorexigenic Action of PACAP1 and PACAP2 in Zebrafish.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide with potent suppressive effects on feeding behavior in rodents, chicken, and goldfish. Teleost fish express two PACAPs (PACAP1, encoded by the adcyap1a gene, and PACAP2, encoded by the adcyap1b gene) and two PACAP receptors (PAC1Rs; PAC1Ra, encoded by the adcyap1r1a gene, and PAC1Rb, encoded by the adcyap1r1b gene). However, the mRNA expression patterns of the two PACAPs and PAC1Rs, and the influence and relationship of the two PACAPs on feeding behavior in teleost fish remains unclear. Therefore, we first examined mRNA expression patterns of PACAP and PAC1R in tissue and brain. All PACAP and PAC1Rs mRNAs were dominantly expressed in the zebrafish brain. However, adcyap1a mRNA was also detected in the gut and testis. In the brain, adcyap1b and adcyap1r1a mRNA levels were greater than that of adcyap1a and adcyap1r1b, respectively. Moreover, adcyap1b and adcyap1r1a mRNA were dominantly expressed in telencephalon and diencephalon. The highest adcyap1a mRNA levels were detected in the brain stem and diencephalon, while the highest levels of adcyap1r1b were detected in the cerebellum. To clarify the relationship between PACAP and feeding behavior in the zebrafish, the effects of zebrafish (zf) PACAP1 or zfPACAP2 intracerebroventricular (ICV) injection were examined on food intake, and changes in PACAP mRNA levels were assessed against feeding status. Food intake was significantly decreased by ICV injection of zfPACAP1 (2 pmol/g body weight), zfPACAP2 (2 or 20 pmol/g body weight), or mammalian PACAP (2 or 20 pmol/g). Meanwhile, the PACAP injection group did not change locomotor activity. Real-time PCR showed adcyap1 mRNA levels were significantly increased at 2 and 3 h after feeding compared with the pre-feeding level, but adcyap1b, adcyap1r1a, and adcyap1r1b mRNA levels did not change after feeding. These results suggest that the expression levels and distribution of duplicated PACAP and PAC1R genes are different in zebrafish, but the anorexigenic effects of PACAP are similar to those seen in other vertebrates.

In Silico Screening Identified Novel Small-molecule Antagonists of PAC1 Receptor.

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 signaling systems in the modulation of spinal nociceptive transmission. Previously, we found that intrathecal injection of PACAP or maxadilan, a selective PACAP type I (PAC1) receptor agonist, induced transient aversive responses followed by a long-lasting mechanical allodynia in mice, suggesting that PACAP-PAC1 receptor systems are involved in chronic pain and that selective PAC1 antagonists may become a new class of analgesics. Although several PAC1 antagonists, such as PACAP 6-38, have been reported, all of them are peptide compounds. In the present study, we identified new small-molecule antagonists of the PAC1 receptor using in silico screening and in vitro/vivo pharmacological assays. The identified small-molecule compounds, named PA-8 and PA-9, dose dependently inhibited the phosphorylation of CREB induced by PACAP in PAC1-, but not VPAC1- or VPAC2-receptor-expressing CHO cells. PA-8 and PA-9 also dose dependently inhibited PACAP-induced cAMP elevation with an IC50 of 2.0 and 5.6 nM, respectively. In vivo pharmacological assays showed that intrathecal injection of these compounds blocked the induction of PACAP-induced aversive responses and mechanical allodynia in mice. In contrast, the compounds when administered alone exerted neither agonistic nor algesic actions in the in vitro/vivo assays. The compounds identified in the present study are new and the first small-molecule antagonists of the PAC1 receptor; they may become seed compounds for developing novel analgesics.

PACAP Is Protective in a Rat Model of Retinopathy of Prematurity.

The oxygen-induced retinopathy (OIR) is a well-established rodent model of retinopathy of prematurity (ROP), which is one of the most common causes of childhood visual impairment affecting preterm babies. Pituitary adenylate cyclase-activating polypeptide (PACAP) is known to have neuroprotective effects. Several studies have revealed the presence of PACAP and its receptors in the retina and reported its protective effects in ischemic and diabetic retinopathy. In this study, we investigated whether PACAP administration can influence the vascular changes in the rat OIR model. OIR was generated by placing the animals in daily alternating 10/50 oxygen concentrations from postnatal day (PD) 0 to PD14 then returned them to room air. Meanwhile, animals received PACAP or saline intraperitoneally or intravitreally from PD1 to PD8 or on PD11, PD14, and PD17, respectively. On PD19±1, the retinas were isolated and the vessels were visualized by isolectin staining. The percentage of avascular to whole retinal areas and the number of branching points were measured. Change in cytokine expression was also determined. Intravitreal treatment with PACAP remarkably reduced the extent of avascular area compared to the non- and saline-treated OIR groups. Intraperitoneal PACAP injection did not influence the vascular extent. Retinal images of room-air controls did not show vascular alterations. No changes in the number of vessel branching were observed after treatments. Alterations in cytokine profile after local PACAP injection further supported the protective role of the peptide. This is the first study to examine the effects of PACAP in ROP. Although the exact mechanism is still not revealed, the present results show that PACAP treatment can ameliorate the vascular changes in the animal model of ROP.

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Is Involved in Adult Mouse Hippocampal Neurogenesis After Stroke.

In the subgranular zone (SGZ) of the hippocampus, neurogenesis persists throughout life and is upregulated following ischemia. Accumulating evidence suggests that enhanced neurogenesis stimulated by ischemic injury contributes to recovery after stroke. However, the mechanisms underlying the upregulation of neurogenesis are unclear. We have demonstrated that a neuropeptide, pituitary adenylate cyclase-activating polypeptide (PACAP), exerts a wide range of effects on neural stem cells (NSCs) during neural development. Here, we examined the effects of endogenous and exogenous PACAP in adult NSCs of the SGZ. Immunostaining showed expression of the PACAP receptor PAC1R in nestin-positive NSCs of adult naive mice. PACAP injection into the lateral ventricle increased bromodeoxyuridine (BrdU)-positive proliferative cells in the SGZ. These data suggest that PACAP promoted the proliferation of NSCs. In global ischemia model mice, the number of BrdU-positive cells was increased in wild-type mice but not in PACAP heterozygous knockout mice. The BrdU-positive cells that increased in number after ischemia were immunopositive for SOX2, a marker of NSCs, and differentiated into NeuN-positive mature neurons at 4weeks after ischemia. These findings suggest that PACAP contributes to the proliferation of NSCs and may be associated with recovery after brain injury.

Pituitary Adenylate Cyclase Activating Polypeptide, A Potential Therapeutic Agent for Diabetic Retinopathy in Rats: Focus on the Vertical Information Processing Pathway.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neurotrophic and neuroprotective peptide that has been shown to exert protective effects in different neuronal injuries, such as retinal degenerations. Diabetic retinopathy (DR), the most common complication of diabetes, affects the microvasculature and neuronal architecture of the retina. We have proven earlier that PACAP is also protective in a rat model of DR. In this study, streptozotocin-induced DR was treated with intravitreal PACAP administration in order to further analyze the synaptic structure and proteins of PACAP-treated diabetic retinas, primarily in the vertical information processing pathway. Streptozotocin-treated Wistar rats received intravitreal PACAP injection three times into the right eye 2weeks after the induction of diabetes. Morphological and molecular biological (qRT-PCR; Western blot) methods were used to analyze retinal synapses (ribbons, conventional) and related structures. Electron microscopic analysis revealed that retinal pigment epithelium, the ribbon synapses and other synaptic profiles suffered alterations in diabetes. However, in PACAP-treated diabetic retinas more bipolar ribbon synapses were found intact in the inner plexiform layer than in DR animals. The ribbon synapse was marked with C-terminal binding protein 2/Bassoon and formed horseshoe-shape ribbons, which were more retained in PACAP-treated diabetic retinas than in DR rats. These results are supported by molecular biological data. The selective degeneration of related structures such as bipolar and ganglion cells could be ameliorated by PACAP treatment. In summary, intravitreal administration of PACAP may have therapeutic potential in streptozotocin-induced DR through maintaining synapse integrity in the vertical pathway.

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.

BackgroundPituitary 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.ResultsWe 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.ConclusionOur data suggest that spinal astrocytic activation triggered by the PAC1 receptor stimulation contributes to both induction and maintenance of the long-term mechanical allodynia.

Neuromodulatory effect of Gαs- or Gαq-coupled G-protein-coupled receptor on NMDA receptor selectively activates the NMDA receptor/Ca2+/calcineurin/cAMP response element-binding protein-regulated transcriptional coactivator 1 pathway to effectively induce brain-derived neurotrophic factor expression in neurons.

Although coordinated molecular signaling through excitatory and modulatory neurotransmissions is critical for the induction of immediate early genes (IEGs), which lead to effective changes in synaptic plasticity, the intracellular mechanisms responsible remain obscure. Here we measured the expression of IEGs and used bioluminescence imaging to visualize the expression of Bdnf when GPCRs, major neuromodulator receptors, were stimulated. Stimulation of pituitary adenylate cyclase-activating polypeptide (PACAP)-specific receptor (PAC1), a Gαs/q-protein-coupled GPCR, with PACAP selectively activated the calcineurin (CN) pathway that is controlled by calcium signals evoked via NMDAR. This signaling pathway then induced the expression of Bdnf and CN-dependent IEGs through the nuclear translocation of CREB-regulated transcriptional coactivator 1 (CRTC1). Intracerebroventricular injection of PACAP and intraperitoneal administration of MK801 in mice demonstrated that functional interactions between PAC1 and NMDAR induced the expression of Bdnf in the brain. Coactivation of NMDAR and PAC1 synergistically induced the expression of Bdnf attributable to selective activation of the CN pathway. This CN pathway-controlled expression of Bdnf was also induced by stimulating other Gαs- or Gαq-coupled GPCRs, such as dopamine D1, adrenaline β, CRF, and neurotensin receptors, either with their cognate agonists or by direct stimulation of the protein kinase A (PKA)/PKC pathway with chemical activators. Thus, the GPCR-induced expression of IEGs in coordination with NMDAR might occur via the selective activation of the CN/CRTC1/CREB pathway under simultaneous excitatory and modulatory synaptic transmissions in neurons if either the Gαs/adenylate cyclase/PKA or Gαq/PLC/PKC-mediated pathway is activated.

PACAP promotes neuron survival in early experimental diabetic retinopathy

Metabolic changes induced by diabetes lead to a multifactorial progressive disease of the retina with an extremely complex pathogenesis. One of the mechanisms of retinal cell death in diabetes is via apoptosis. Our previous results show that pituitary adenylate cyclase activating polypeptide (PACAP) attenuates the morphological and neurochemical changes in a rat model of diabetic retinopathy. The aim of this study was to investigate the mechanisms of this protective effect.Retinas of streptozotocin-induced diabetic rats were analyzed using apoptosis detection combined with immunolabeling. Western blot was used to measure levels of pro- and anti-apoptotic pathways.Intraocular PACAP injection markedly attenuated diabetic retinal injury: increased levels of the anti-apoptotic p-Akt, p-ERK1, p-ERK2, PKC, Bcl-2, while decreased levels of the pro-apoptotic p-p38MAPK and activated caspases (8, 3, 12) were detected. The number of apoptotic cells increased in all nuclear layers of diabetic retinas, but significantly decreased after PACAP treatment. Our results clearly demonstrate that the protective effects of PACAP are mediated, at least partly, by attenuating apoptosis, including also that of the dopaminergic amacrine cells. Inhibition of apoptosis is one of the PACAP-induced pathways with therapeutic potential in early experimental diabetic retinopathy.

Intrahypothalamic pituitary adenylate cyclase-activating polypeptide regulates energy balance via site-specific actions on feeding and metabolism

Numerous studies have demonstrated that both the hypothalamic paraventricular nuclei (PVN) and ventromedial nuclei (VMN) regulate energy homeostasis through behavioral and metabolic mechanisms. Receptors for pituitary adenylate cyclase-activating polypeptide (PACAP) are abundantly expressed in these nuclei, suggesting PACAP may be critical for the regulation of feeding behavior and body weight. To characterize the unique behavioral and physiological responses attributed to select hypothalamic cell groups, PACAP was site-specifically injected into the PVN or VMN. Overall food intake was significantly reduced by PACAP at both sites; however, meal pattern analysis revealed that only injections into the PVN produced significant reductions in meal size, duration, and total time spent eating. PACAP-mediated hypophagia in both the PVN and VMN was abolished by PAC1R antagonism, whereas pretreatment with a VPACR antagonist had no effect. PACAP injections into the VMN produced unique changes in metabolic parameters, including significant increases in core body temperature and spontaneous locomotor activity that was PAC1R dependent whereas, PVN injections of PACAP had no effect. Finally, PACAP-containing afferents were identified using the neuronal tracer cholera toxin subunit B (CTB) injected unilaterally into the PVN or VMN. CTB signal from PVN injections was colocalized with PACAP mRNA in the medial anterior bed nucleus of the stria terminalis, VMN, and lateral parabrachial nucleus (LPB), whereas CTB signal from VMN injections was highly colocalized with PACAP mRNA in the medial amygdala and LPB. These brain regions are known to influence energy homeostasis perhaps, in part, through PACAP projections to the PVN and VMN.

PACAP Attenuates NMDA-Induced Retinal Damage in Association with Modulation of the Microglia/Macrophage Status into an Acquired Deactivation Subtype

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been known as a neuroprotectant agent in several retinal injury models. However, a detailed mechanism of this effect is still not well understood. In this study, we examined the retinoprotective effects and associated underlying mechanisms of action of PACAP in the mouse N-methyl-D-aspartic acid (NMDA)-induced retinal injury model, focusing on the relationship between PACAP and retinal microglia/macrophage (MG/MΦ) status. Adult male C57BL/6 mice received an intravitreal injection of NMDA to induce retinal injury. Three days after NMDA injection, the number of MG/MΦ increased significantly in the retinas. The concomitant intravitreal injection of PACAP suppressed NMDA-induced cell loss in the ganglion cell layer (GCL) and significantly increased the number of MG/MΦ. These outcomes associated with PACAP were attenuated by cotreatment with PACAP6-38, while the beneficial effects of PACAP were not seen in interleukin-10 (IL-10) knockout mice. PACAP significantly elevated the messenger RNA levels of anti-inflammatory cytokines such as transforming growth factor beta 1 and IL-10 in the injured retina, with the immunoreactivities seen to overlap with markers of MG/MΦ. These results suggest that PACAP enhances the proliferation and/or infiltration of retinal MG/MΦ and modulates their status into an acquired deactivation subtype to favor conditions for neuroprotection.

Activation of PAC1 and VPAC receptor subtypes elicits differential physiological responses from sympathetic preganglionic neurons in the anaesthetized rat

Pituitary adenylate cyclase-activating polypeptide (PACAP) is an excitatory neuropeptide with central and peripheral cardiovascular actions. Intrathecal PACAP increases splanchnic sympathetic nerve activity and heart rate, but not mean arterial pressure (MAP). We hypothesize that the three PACAP receptors (PAC(1) , VPAC(1) and VPAC(2) ) have different actions in central cardiovascular control, and that their summed effect results in the lack of MAP response observed following intrathecal PACAP injection. The effects of the PACAP receptors on baseline cardiovascular parameters were investigated using selective agonists and antagonists administered into the intrathecal space of urethane-anaesthetized, vagotomized and artificially ventilated male Sprague-Dawley rats. Selective activation of the PACAP receptors had different effects on MAP. When activated by maxadilan, PAC(1) receptors increased MAP. The VPAC receptors decreased MAP when both were activated with vasoactive intestinal polypeptide or when only VPAC(1) receptors were activated. The PAC(1) and VPAC(2) receptor antagonist PACAP(6-38) had no cardiovascular effects, suggesting that PACAP is not tonically released. PACAP neurotransmission was not responsible for the moment-to-moment tonic regulation of central cardiovascular control mechanisms. Nevertheless, PACAP release within the spinal cord may have pleiotropic effects on sympathetic outflow depending on the postsynaptic receptor type. PAC(1) and VPAC receptor subtypes produced opposing changes in blood pressure when activated by intrathecal PACAP-38 in the anaesthetized Sprague-Dawley rat, resulting in no net change in MAP.

Immunohistochemical localization of PACAP and VIP receptors in major salivary glands and the effect of PACAP on saliva secretion in mice

The control of saliva secretion is mainly under the autonomic nervous control. Pituitary adenylate cyclase‐activating polypeptide (PACAP) is now recognized as the multi‐functional neuropeptide in various organs. Vasoactive intestinal polypeptide (VIP) is similar function to PACAP. In this study, we compared the distribution of PACAP receptor, PAC1R and VIP receptor, VPAC1, in major salivary glands; parotid, submandibular and sublingual glands, and examined the effect of PACAP on saliva secretion in 8 weeks old C57BL/6 mice. In parotid gland, PAC1R and VPAC1 were detected in the cells of striated duct. In the case of submandibular gland, PAC1R and VPAC1 were detected in the tall columnar epithelial cells, called pillar cells, in granular ducts and some of the cells in the striated ducts. In sublingual gland, PAC1R and VPAC1 were expressed mainly in the epithelial cells of striated ducts. Intranasal injection of PACAP enhanced the secretion of saliva. Taken together, these results indicated that the distribution of PAC1R and VPAC1 were similar in three major salivary glands and that PACAP and VIP might be the useful agents for the future dry mouth therapuutics.

Stimulation of the hypothalamic ventromedial nuclei by pituitary adenylate cyclase-activating polypeptide induces hypophagia and thermogenesis

Numerous studies have demonstrated that the hypothalamic ventromedial nuclei (VMN) regulate energy homeostasis by integrating and utilizing behavioral and metabolic mechanisms. The VMN heavily express pituitary adenylate cyclase-activating polypeptide (PACAP) type I receptors (PAC1R). Despite the receptor distribution, most PACAP experiments investigating affects on feeding have focused on intracerebroventricular administration or global knockout mice. To identify the specific contribution of PACAP signaling in the VMN, we injected PACAP directly into the VMN and measured feeding behavior and indices of energy expenditure. Following an acute injection of PACAP, nocturnal food intake was significantly reduced for 6 h after injections without evidence of malaise. In addition, PACAP-induced suppression of feeding also occurred following an overnight fast and could be blocked by a specific PAC1R antagonist. Metabolically, VMN-specific injections of PACAP significantly increased both core body temperature and spontaneous locomotor activity with a concurrent increase in brown adipose uncoupling protein 1 mRNA expression. To determine which signaling pathways were responsive to PACAP administration into the VMN, we measured mRNA expression of well-characterized hypothalamic neuropeptide regulators of feeding. One hour after PACAP administration, expression of pro-opiomelanocortin mRNA was significantly increased in the arcuate nuclei (ARC), with no changes in neuropeptide Y and agouti-related polypeptide mRNA levels. This suggests that PAC1R expressing VMN neurons projecting to pro-opiomelanocortin neurons contribute to hypophagia by involving melanocortin signaling. While the VMN also abundantly express PACAP protein, the present study demonstrates that PACAP input to the VMN can influence the control of energy homeostasis.

Strategies to Convert PACAP from a Hypophysiotropic Neurohormone Into a Neuroprotective Drug

In neurological insults, such as cerebral ischemia and traumatic brain injury, complex molecular mechanisms involving inflammation and apoptosis are known to cause severe neuronal cell loss, emphasizing the necessity of developing therapeutic strategies targeting simultaneously these two processes. Over the last decade, numerous in vitro and in vivo studies have demonstrated the unique therapeutical potential of pituitary adenylate cyclase-activating polypeptide (PACAP) for the treatment of neuronal disorders involving apoptotic cell death and neuroinflammation. The neuroprotective activity of PACAP is based on its capacity to reduce the production of deleterious cytokines from activated microglia, to stimulate the release of neuroprotective agents from astrocytes and to inhibit pro-apoptotic intracellular pathways. However, the use of PACAP as a clinically applicable drug is hindered by its peptidic nature. As most natural peptides, native PACAP shows poor metabolic stability, low bioavailability, inadequate distribution and rapid blood clearance. Moreover, injection of PACAP to human can induce peripheral adverse side effects. Therefore, targeted chemical modifications and/or conjugation of PACAP to different macromolecules are required to improve the pharmacokinetic and pharmacological properties of PACAP. This review presents the chemical, biochemical and pharmacological strategies that are currently under development to convert PACAP from a hypophysiotropic neurohormone into a clinically relevant neuroprotective drug.

The melanocortin system is involved in regulating autonomic nerve activity through central pituitary adenylate cyclase-activating polypeptide

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a peptidergic neurotransmitter that is highly expressed in the nervous system. We have previously reported that a central injection of PACAP leads to changes in the autonomic nervous system tones including sympathetic excitation and parasympathetic inhibition. An anatomical study revealed that melanocortin and PACAP are colocalized in some hypothalamic nuclei. Here, we investigated the possible role of the melanocortin system in autonomic control by PACAP using SHU9119, an antagonist of the melanocortin receptors (MC3-R/MC4-R). Pretreatment with SHU-9119 did not affect the activating neural responses of adrenal, renal, and lumbar sympathetic nerves following a PACAP injection However, SHU9119 significantly eliminated the suppressing effect of a PACAP injection on gastric vagal nerve activity and excitation effects on liver and brown adipose tissue sympathetic nerve activities. These results suggest that the brain melanocortin system might play a key role in the control of thermogenic sympathetic outflows and digestive parasympathetic outflow by PACAP, but this system does not participate in the central effects of PACAP on cardiovascular function and neural activities of renal, adrenal, and lumbar sympathetic nerves.

Regulation of autonomic nerve activities by central pituitary adenylate cyclase-activating polypeptide

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a peptidergic neurotransmitter that is expressed in high levels in nervous systems. Here, we investigated the roles of PACAP in autonomic system regulation by evaluating the changes caused in the autonomic nerve activities after injecting PACAP into the central nervous system (CNS) and examining stress-induced blood glucose changes in PACAP-deficient (PACAP−/−) mice. Renal sympathetic nerve activity (RSNA), blood pressure, and heart rate were elevated after injecting PACAP into the third cerebral ventricle (3CV). Similarly, other sympathetic nerve activities, including adrenal sympathetic nerve activity (ASNA), celiac sympathetic nerve activity (CSNA), and brown adipose tissue sympathetic nerve activity (BAT-SNA), were accelerated by PACAP injection. In contrast, injecting PACAP into 3CV significantly suppressed parasympathetic nerve activities, including gastric vagal nerve activity (GVNA) and celiac vagal nerve activity (CVNA). In addition, blood glucose elevations induced by stress, such as immobilization or ether exposure, were disrupted in PACAP−/− mice, although basal glucose levels in mutants were comparable to that in wild-type mice. These results suggest that CNS PACAP regulates autonomic function by maintaining a sympathetic–parasympathetic balance and contributes to peripheral homeostatic maintenance, especially under conditions of stress.