Advent and Recent Advances in Research on the Role of Pituitary Adenylate Cyclase-activating Polypeptide (PACAP) in the Regulation of Gonadotropic Hormone Secretion of Female Rats

PACAP (ADCYAP1) was isolated from ovine hypothalami. PACAP activates three distinct receptor types: G-protein coupled PAC1, VPAC1, and VPAC2 with seven transmembrane domains. Eight splice variants of PAC1 receptor are described. A part of the hypothalamic PACAP is released into the hypophyseal portal circulation. Both hypothalamic and pituitary PACAP are involved in the dynamic control of gonadotropic hormone secretion. In female rats, PACAP in the paraventricular nucleus is upregulated in the morning and pituitary PACAP is upregulated in the late evening of the proestrus stage of the reproductive cycle. PACAP mRNA peak in the hypothalamic PVN precedes the LHRH release into the portal circulation. It is supposed that PACAP peak is evoked by the elevated estrogen on proestrous morning. At the beginning of the so-called critical period of the same day, PACAP level starts to decline allowing LHRH release into the portal circulation, resulting in the LH surge that evokes ovulation. Just before the critical period, icv-administered exogenous PACAP blocks the LH surge and ovulation. The blocking effect of PACAP is mediated through CRF and endogenous opioids. The effect of the pituitary-born PACAP depends on the intracellular cross-talk between PACAP and LHRH.     

PACAP Stimulates Functional Recovery after Spinal Cord Injury through Axonal Regeneration

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuroprotective peptide expressed in the central nervous system. Although many studies have shown a neuroprotective effect of PACAP, the mechanism of PACAP in the treatment of spinal cord injury (SCI) is yet to be elucidated. The purpose of this study was to examine the efficacy and underlying mechanism of PACAP in a mouse SCI model where PACAP was delivered via a biodegradable hydrogel. When PACAP or saline was delivered immediately after SCI, the functional motor recovery 14 days after SCI was significantly improved in the PACAP group compared with that in the saline group. Expression levels of messenger RNA (mRNA) for collapsin response mediator protein 2 (CRMP2), a factor related to axonal regeneration, were increased in the PACAP group 14 days after SCI compared with those in the saline group. A significantly increased number of CRMP2-positive cells were observed around the injury lesion in the PACAP group, while CRMP2 co-labeling with neuronal and oligodendrocyte markers was detected in intact spinal cord. Fourteen days after SCI, anterograde tracing revealed that a significantly increased number of neuronal fibers extended caudally from the lesion epicenter in the PACAP group. These results suggest that PACAP stimulates functional motor recovery after SCI through axonal regeneration mediated by CRMP2.     

Structural and Morphometric Comparison of the Molar Teeth in Pre-eruptive Developmental Stage of PACAP-Deficient and Wild-Type Mice

Pituitary adenylate cyclase activating polypeptide (PACAP) is a pleiotropic neuropeptide with widespread distribution. It plays pivotal role in neuronal development. PACAP-immunoreactive fibers have been found in the tooth pulp, and recently, it has been shown that PACAP may also play a role in the regeneration of the periodontium after luxation injuries. However, there is no data about the effect of endogenous PACAP on tooth development. Ectodermal organogenesis including tooth development is regulated by different members of bone morphogenetic protein (BMP), fibroblast growth factor (FGF), hedgehog (HH), and Wnt families. There is also a growing evidence to support the hypothesis that PACAP interacts with sonic hedgehog (SHH) receptor (PTCH1) and its downstream target (Gli1) suggesting its role in tooth development. Therefore, our aim was to study molar tooth development in mice lacking endogenous PACAP. In this study morphometric, immunohistochemical and structural comparison of molar teeth in pre-eruptive developmental stage was performed on histological sections of 7-day-old wild-type and PACAP-deficient mice. Further structural analysis was carried out with Raman microscope. The morphometric comparison of the 7-day-old samples revealed that the dentin was significantly thinner in the molars of PACAP-deficient mice compared to wild-type animals. Raman spectra of the enamel in wild-type mice demonstrated higher diversity in secondary structure of enamel proteins. In the dentin of PACAP-deficient mice higher intracrystalline disordering in the hydroxyapatite molecular structure was found. We also obtained altered SHH, PTCH1 and Gli1 expression level in secretory ameloblasts of PACAP-deficient mice compared to wild-type littermates suggesting that PACAP might play an important role in molar tooth development and matrix mineralization involving influence on SHH signaling cascade.     

Comparison of Expression and Proliferative Effect of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) and its Receptors on Human Astrocytoma Cell Lines

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide considered to be a potent regulator of astrocytes. It has been reported that PACAP also affects astrocytoma cell properties, but the proliferative effects of this peptide in previous reports were inconsistent. The purpose of this study was to search for correlations between malignant potential, PACAP/PACAP receptor expression, and the proliferative potential of four astrocytoma cell lines (KNS-81, KINGS-1, SF-126, and YH-13). Immunohistochemical observations were performed using astrocyte lineage markers with a view to establishing malignant potential, which is inversely correlated to differentiation status in astrocytoma cells. YH-13 showed the most undifferentiated astrocyte-like status, and was immunopositive to a cancer stem cell marker, CD44. These observations suggest that YH-13 is the most malignant of the astrocytoma cell lines tested. Moreover, the strongest PAC1-R immunoreactivity was observed in YH-13 cells. Using real-time PCR analysis, no significant differences among cell lines were detected with respect to PACAP mRNA, but PAC1-R and VPAC1-R mRNA levels were significantly increased in YH-13 cells compared with the other cell lines. Furthermore, when cell lines were treated with PACAP (10^?11 M) for 3 days, the YH-13 cell line, but not of the other cell lines, exhibited a significantly increased cell number. These results suggest that PACAP receptor expression is correlated with the malignant and proliferative potential of astrocytoma cell lines.     

Investigation of the Possible Functions of PACAP in Human Trophoblast Cells

Pituitary adenylate cyclase activating polypeptide (PACAP) is an endogenous neuropeptide having a widespread distribution both in the nervous system and peripheral organs including the female reproductive system. Both the peptide and its receptors have been shown in the placenta but its role in placental growth, especially its human aspects, remains unknown. The aim of the present study was to investigate the effects of PACAP on invasion, proliferation, cell survival, and angiogenesis of trophoblast cells. Furthermore, cytokine production was investigated in human decidual and peripheral blood mononuclear cells. For in vitro studies, human invasive proliferative extravillous cytotrophoblast (HIPEC) cells and HTR-8/SVneo human trophoblast cells were used. Both cell types were used for testing the effects of PACAP on invasion and cell survival in order to investigate whether the effects of PACAP in trophoblasts depend on the examined cell type. Invasion was studied by standardized invasion assay. PACAP increased proliferation in HIPEC cells, but not in HTR-8 cells. Cell viability was examined using MTT test, WST-1 assay, and annexin V/propidium iodide flow cytometry assay. Survival of HTR-8/SVneo cells was studied under oxidative stress conditions induced by hydrogen peroxide. PACAP as pretreatment, but not as co-treatment, significantly increased the number of surviving HTR-8 cells. Viability of HIPEC cells was investigated using methotrexate (MTX) toxicity, but PACAP1-38 could not counteract its toxic effect. Angiogenic molecules were determined both in the supernatant and the cell lysate by angiogenesis array. In the supernatant, we found that PACAP decreased the secretion of various angiogenic markers, such as angiopoietin, angiogenin, activin, endoglin, ADAMTS-1, and VEGF. For the cytokine assay, human decidual and peripheral blood lymphocytes were separated and treated with PACAP1-38. Th1 and Th2 cytokines were analyzed with CBA assay and the results showed that there were no significant differences in control and PACAP-treated cells. In summary, PACAP seems to play various roles in human trophoblast cells, depending on the cell type and microenvironmental influences.     

Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) Dilates Cerebellar Arteries Through Activation of Large-Conductance Ca2+-Activated (BK) and ATP-Sensitive (KATP) K+ Channels

Pituitary adenylate cyclase activating polypeptide (PACAP) is a potent vasodilator of numerous vascular beds, including cerebral arteries. Although PACAP-induced cerebral artery dilation is suggested to be cyclic AMP (cAMP)-dependent, the downstream intracellular signaling pathways are still not fully understood. In this study, we examined the role of smooth muscle K⁺ channels and hypothesized that PACAP-mediated increases in cAMP levels and protein kinase A (PKA) activity result in the coordinate activation of ATP-sensitive K⁺ (K_ATP) and large-conductance Ca²⁺-activated K⁺ (BK) channels for cerebral artery dilation. Using patch-clamp electrophysiology, we observed that PACAP enhanced whole-cell K_ATP channel activity and transient BK channel currents in freshly isolated rat cerebellar artery myocytes. The increased frequency of transient BK currents following PACAP treatment is indicative of increased intracellular Ca²⁺ release events termed Ca²⁺ sparks. Consistent with the electrophysiology data, the PACAP-induced vasodilations of cannulated cerebellar artery preparations were attenuated by approximately 50 % in the presence of glibenclamide (a K_ATP channel blocker) or paxilline (a BK channel blocker). Further, in the presence of both blockers, PACAP failed to cause vasodilation. In conclusion, our results indicate that PACAP causes cerebellar artery dilation through two mechanisms: (1) K_ATP channel activation and (2) enhanced BK channel activity, likely through increased Ca²⁺ spark frequency.     

PACAP Application Improves Functional Outcome of Chronic Retinal Ischemic Injury in Rats-Evidence From Electroretinographic Measurements

Retinoprotective effects of pituitary adenylate cyclase activating polypeptide (PACAP) are well-known and have been demonstrated in various pathological conditions, such as diabetic retinopathy, excitotoxic retinal injury, UV light-induced degeneration, and ischemic retinal lesion. The neuronal degeneration observed in the different retinal layers under the above pathological conditions can be successfully decreased by PACAP; however, whether this morphological improvement is also reflected in functional amelioration remains unknown. Therefore, our purpose was to investigate the protective effect of PACAP on the rat retina after bilateral common carotid artery occlusion (BCCAO) with electroretinography (ERG) to parallel the functional data with the previous morphological and neurochemical observations. Control eyes received saline treatment while PACAP was injected into the vitreous space of the other eye immediately after the induction of ischemia. Retinal damage and protective effects of PACAP were quantified by the changes in the wave forms and amplitudes. On postoperative days 2 and 14, several parameters were assessed with special attention to the changes of b wave. The results confirm that the previously described morphological protection induced by PACAP treatment is reflected in functional improvement in ischemic retinal lesions.     

PACAP Enhances Barrier Properties of Cerebral Microvessels

Cerebral microvascular endothelial cells—coming in contact with pericytes and astrocytes—constitute the structural basis of the blood-brain barrier (BBB). The continuous belt of interendothelial tight junctions (TJs) and the presence of specific transport systems, enzymes, and receptors in the brain endothelium regulate the molecular and cellular traffic into the central nervous system. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide having several cellular protective effects. However, little is known about the effects of PACAP on the cerebral endothelium and BBB functions. Here, we show that PACAP has no significant pro-survival role in cerebral microvascular endothelial cells; however, it improves the barrier properties of the brain endothelium. PACAP induces an increase in the transendothelial electrical resistance, which is the most important marker of the tightness of the TJs. Moreover, PACAP has a protective role against glucose deprivation- and oxidative stress-induced junctional damage in microvascular brain endothelial cells.     

Comparative Protein Composition of the Brains of PACAP-Deficient Mice Using Mass Spectrometry-Based Proteomic Analysis

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a widespread neuropeptide acting as a neurotransmitter, neuromodulator, or neurotrophic factor. The diverse biological actions provide the background for the variety of deficits observed in mice lacking endogenous PACAP. PACAP-deficient mice display several abnormalities, such as sudden infant death syndrome (SIDS)-like phenotype, decreased cell protection, and increased risk of Parkinson’s disease. However, the molecular and proteomic background is still unclear. Therefore, our aim was to investigate the differences in peptide and protein composition in the brains of PACAP-deficient and wild-type mice using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometric (MS)-based proteomic analysis. Brains from PACAP-deficient mice were removed, and different brain areas (cortex, hippocampus, diencephalon, mesencephalon, brainstem, and cerebellum) were separated. Brain pieces were weighed, homogenized, and further processed for electrophoretic analysis. Our results revealed several differences in diencephalon and mesencephalon. The protein bands of interest were cut from the gel, samples were digested with trypsin, and the tryptic peptides were measured by matrix-assisted laser desorption ionization time of flight (MALDI TOF) MS. Results were analyzed by MASCOT Search Engine. Among the altered proteins, several are involved in metabolic processes, energy homeostasis, and structural integrity. ATP-synthase and tubulin beta-2A were expressed more strongly in PACAP-knockout mice. In contrast, the expression of more peptides/proteins markedly decreased in knockout mice, like pyruvate kinase, fructose biphosphate aldolase-A, glutathione S-transferase, peptidyl propyl cis-trans isomerase-A, gamma enolase, and aspartate amino transferase. The altered expression of these enzymes might partially account for the decreased antioxidant and detoxifying capacity of PACAP-deficient mice accompanying the increased vulnerability of these animals. Our results provide novel insight into the altered biochemical processes in mice lacking endogenous PACAP.     

Calcium Influx,But Not Intracellular Calcium Release,Supports PACAP-Mediated ERK Activation in HEK PAC1 Receptor Cells

In HEK cells expressing GFP-tagged PAC1Hop1 receptors, PACAP augments ERK phosphorylation through two parallel pathways: one through PACAP/PAC1 receptor internalization/endosome MEK/ERK signaling and the other through PLC/DAG/PKC activation. We examined whether elevation of intracellular calcium ([Ca²⁺]_i) was required for either of the PACAP/PAC1 receptor-mediated ERK activation mechanisms. The PACAP (25 nM)-induced elevation of [Ca²⁺]_i was greater with cells maintained in Ca²⁺-containing than in Ca²⁺-deficient solution, suggesting that both calcium release from internal stores and calcium influx contributed to the rise in [Ca²⁺]_i. A thapsigargin-induced increase in [Ca²⁺]_i also was greater with calcium in the external solution. OAG, the cell permeable analogue of DAG, increased [Ca²⁺]_i, but only in Ca²⁺-containing solution. Decreasing external calcium or depleting internal calcium stores did not block PACAP-induced PAC1 receptor internalization. Omission of calcium from the external solution, but not thapsigargin pretreatment, significantly blunted PACAP-stimulated ERK phosphorylation. The PKC inhibitor BimI decreased PACAP-mediated ERK activation in both Ca²⁺-containing or Ca²⁺-deficient solutions. In contrast, following Pitstop 2 pretreatment to block endocytic mechanisms, PACAP activated ERK only when calcium was present in the external solution. We conclude that the endosome signaling pathway is largely calcium-independent whereas calcium influx appears necessary for the PLC/DAG/PKC component of PACAP-induced ERK activation.     

Molecular Mechanisms Underlying the Nephroprotective Effects of PACAP in Diabetes

Diabetic nephropathy is the leading cause of end-stage renal failure and accounts for 30–40 % of patients entering renal transplant programmes. The nephroprotective effects of the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP38) against diabetes have been shown previously, but the molecular mechanisms responsible for these effects remain unknown. In the present study, we showed that PACAP treatment counteracted the diabetes-induced increase in the level of the proapoptotic pp38MAPK and cleaved caspase-3 and also decreased the p60 subunit of NFκB. The examined antiapoptotic factors, including pAkt and pERK1/2, showed a slight increase in the diabetic kidneys, while PACAP treatment resulted in a notable elevation of these proteins. PCR and Western blot revealed the downregulation of fibrotic markers, like collagen IV and TGF-β1 in the kidney. PACAP treatment resulted in increased expression of the antioxidant glutathione. We conclude that the nephroprotective effect of PACAP in diabetes is, at least partly, due to its antiapoptotic, antifibrotic and antioxidative effect in addition to the previously described antiinflammatory effect.     

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Induces Relaxations of Peripheral and Cerebral Arteries,which are Differentially Impaired by Aging

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a well-known neuropeptide, which also has vasomotor effects. However, little is known regarding its age-related and organ-specific vasomotor effects. We hypothesized that the vasomotor effects of PACAP depend on the tissue origin of the vessels and aging substantially modulates its actions. Thus, carotid (CA) and basilar arteries (BA) were isolated from young (2 months old), middle age (12 months old), and old (30 months old) rats. Their vasomotor responses were measured with an isometric myograph (DMT610M) in response to cumulative concentrations of PACAP1-38 (10^?9–10^?6 M). PACAP1-38 induced (1) significantly greater concentration-dependent relaxations in CA compared to that of BA of young, middle age, and old rats; (2) relaxations of CA significantly decreased, whereas they did not change substantially in BA, as a function of age; (3) sodium nitroprusside (SNP)-induced relaxation did not change after PACAP1-38 administration in any conditions; and (4) inhibition of PAC1 receptors by selective PAC1 receptor blocker (PACAP6-38) completely diminished the responses to PACAP in all age groups of BA and CA. In conclusion, these findings suggest that PACAP1-38 has greater vasomotor effect in CA than that in BA, whereas aging has less effect on PACAP-induced relaxation of cerebral arteries and BA than that in peripheral arteries and CA suggesting that the relaxation to PACAP is maintained in cerebral arteries even in old age.     

PACAP Protects Against Inflammatory-Mediated Toxicity in Dopaminergic SH-SY5Y Cells: Implication for Parkinson’s Disease

There has been a growing recognition of the role of neuroinflammation caused by microglia-exaggerated release of inflammatory mediators in the pathogenesis of Parkinson’s disease (PD). Pituitary adenylate cyclase activating polypeptide (PACAP) is an endogenous 38 amino acid containing neuropeptide that has been shown to possess neurotrophic as well as neuroprotective properties. In this study, we sought to determine whether PACAP could protect SH-SY5Y dopaminergic cells against toxicity induced by inflammatory mediators. For this purpose, THP-1 cells which possess microglia-like property were stimulated by a combination of lipopolysaccharide (LPS) and interferon gamma (IFN-γ), and the media containing inflammatory mediators were isolated and applied to SH-SY5Y cells. Such treatment resulted in approximately 54 % cell death as well as a reduction in brain-derived neurotrophic factor (BDNF) and phosphorylated cyclic AMP response element-binding protein (p-CREB). Pretreatment of the SH-SY5Y cells with PACAP (1-38) dose-dependently attenuated toxicity induced by the inflammatory mediators. PACAP effects, in turn, were dose-dependently blocked by the PACAP receptor antagonist (PACAP 6-38). These results suggest protective effects of PACAP against inflammatory-induced toxicity in a cellular model of PD that is likely mediated by enhancement of cell survival markers through activation of PACAP receptors. Hence, PACAP or its agonists could be of therapeutic benefit in inflammatory-mediated PD.     

Paternal influences on treatment outcome of behavioral parent training in children with attention-deficit/hyperactivity disorder

This study aims to explore the influence of paternal variables on outcome of behavioral parent training (BPT) in children with attention-deficit/hyperactivity disorder (ADHD). 83 referred, school-aged children with ADHD were randomly assigned to BPT plus ongoing routine clinical care (RCC) or RCC alone. Treatment outcome was based on parent-reported ADHD symptoms and behavioral problems. Moderator variables included paternal ADHD symptoms, depressive symptoms, and parenting self-efficacy. We conducted repeated measures analyses of variance (ANOVA) for all variables, and then analyzed the direction of interaction effects by repeated measures ANOVA in high and low scoring subgroups. Paternal ADHD symptoms and parenting self-efficacy played a moderating role in decreasing behavioral problems, but not in decreasing ADHD symptoms. Paternal depressive symptoms did not moderate either treatment outcome. BPT is most beneficial in reducing children’s behavioral problems when their fathers have high levels of ADHD symptoms or high-parenting self-efficacy.     

The effect of serotonin 1A receptor polymorphism on the cognitive function of premenstrual dysphoric disorder

Estrogen and serotonin play vital roles in the mechanism of premenstrual dysphoric disorder (PMDD). Cognitive deficit in the premenstrual phase contributes to impaired life function among women with PMDD. The aim of this study was to evaluate the difficulties in cognitive control and working memory (WM) in PMDD and to explore the effects of gonadotropic hormone and polymorphism of serotonin 1A receptor (HTR1A; rs6295) on cognitive deficit in PMDD. Women with PMDD completed diagnostic interviewing, questionnaire assessment, the Go/Nogo task, 2-back and 3-back tasks, and gonadotropic hormone analysis in the premenstrual and follicular phases. Further, they were followed up for two menstrual cycles to confirm two consecutive symptomatic cycles. A total of 59 subjects with PMDD and 74 controls completed all evaluation, fulfilled the criteria, and entered into the final analysis. The results demonstrated cognitive control and WM decline in the premenstrual among women with PMDD. The G/G genotype of HTR1A (rs6295) was found to be associated with impaired WM in the premenstrual phase and premenstrual decline of cognitive function. It also contributed to the vulnerability of cognitive function to the menstrual cycle effect and PMDD effect. As the G/G genotype of HTR1A (rs6295) involves in reducing serotonin neurotransmission, our results provide insight into the serotonin mechanism of cognitive function among women with PMDD.     

Theiler’s virus infection provokes the overexpression of genes coding for the chemokine Ip10 (CXCL10) in SJL/J murine astrocytes,which can be inhibited by modulators of estrogen receptors

Theiler’s murine encephalomyelitis virus (TMEV) induces demyelination in susceptible strains of mice (SJL/J) through an immunopathological process that is mediated by CD4⁺ Th1 T cell. These T cells are chemoattracted to the central nervous system by chemokines. Hence, in this study, we focused on the production of the chemokine “interferon-gamma-inducible protein 10 kDa,” or IP-10/CXCL10, by cultured SJL/J mouse astrocytes infected with the BeAn strain of TMEV and its capacity to attract activated T cells. The analysis of the whole murine genome by DNA hybridization with cRNAs from mock- and TMEV-infected cultures revealed the upregulation of six sequences that potentially encode for CXCL10. This increased CXCL10 expression was validated by PCR and qPCR. The presence of this chemokine was further demonstrated by enzyme-linked immunoassay (ELISA). Significantly, astrocytes from BALB/c mice, a strain resistant to demyelination, did not produce CXCL10. The secreted CXCL10 was biologically active, inducing chemoattraction of activated lymphocytes. The inflammatory cytokines, IL-1α, IFN-γ, and TNF-α, were strong inducers of CXCL10 in astrocytes. Serum from TMEV-infected SJL/J but not BALB/c mice contains CXCL10, the levels of which peak at the onset of the clinical disease. Finally, this in vitro inflammation model was fully inhibited by 17β-estradiol and four selective estrogen receptor modulators, as demonstrated by ELISA and qPCR.