Effects of the Neuropeptides Pituitary Adenylate Cyclase Activating Polypeptide and Vasoactive Intestinal Peptide in Male Fertility.

Background and Objectives: The neuroendocrine system plays a crucial role in regulating various bodily functions, including reproduction, with evidence suggesting its significant involvement in male fertility and sperm development. Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP) are expressed in both male and female reproductive tissues, influencing penile erection and regulating steroidogenesis in males. Therefore, our study aimed to compare the protein levels of VIP and PACAP in seminal plasma between healthy controls and sub-fertile patients. Additionally, we sought to correlate the levels of these biomarkers with clinical, functional, and laboratory findings in the participants. Materials and Methods: The study included a total of 163 male participants for analysis. The participants were further stratified into subgroups of fertile and sub-fertile men of four subgroups according to the 2021 WHO guidelines. Seminal plasma concentrations of the neuropeptides VIP and PACAP were measured using human enzyme-linked immunosorbent assay technique. Results: The findings showed statistically significant differences in total sperm count, sperm concentration, total motility, and vitality (p < 0.001) between the fertile group and the sub-fertile group. Specifically, significant differences found between healthy males and oligoasthenospermic patients (p = 0.002), and between asthenospermic and oligoasthenospermic patients (p = 0.039). An ROC analysis showed associated sensitivity and specificity values of 62.2% and 55.6%, respectively, to PACAP seminal levels differentiated between sub-fertile patients from fertile males (p = 0.028). No significant difference in seminal levels of VIP was found between the sub-fertile and fertile groups. Conclusions: Previous research leads to the point of PACAP active involvement in spermatogenesis. In accordance to our study, in human semen samples, we have seen a significance change in PACAP levels amongst patients with low sperm count or with both low sperm count and low motility, hinting at its contribution and acting as a possible factor in this complex process. Thus, alterations in the levels or actions of these neuropeptides have been associated with certain reproductive disorders in males.

Investigation of pituitary adenylate cyclase activating polypeptide (PACAP) in human amniotic fluid samples.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide acting as a hormone, a neuromodulator, a neurotransmitter, a trophic factor and is involved in a variety of developmental and regenerative processes. PACAP is present in several human tissues and biological fluids. In many pathological conditions, changes in PACAP levels have been described to reflect disease progression, therefore PACAP has diagnostic value as a potential biomarker. Since PACAP has been shown to play an important role in reproductive physiology and development, it was of interest to examine whether this neuropeptide occurs in the human amniotic fluid. Amniotic fluid samples were collected between the 15-19th weeks of gestation from volunteering pregnant women undergoing amniocentesis as a prenatal diagnostic tool due to maternal age. Pathological cases were excluded after prenatal karyotype analysis. PACAP-like immunoreactivity was measured by radioimmunoassay and could be detected in all samples. The present study provides evidence for the presence of PACAP in human amniotic fluid, but determination of the exact physiological or pathological significance awaits further investigation.

Pituitary adenylate cyclase activating polypeptide concentrations in the sheep mammary gland, milk, and in the lamb blood plasma after suckling.

Pituitary adenylate cyclase activating polypeptide (PACAP) is involved in development and reproduction. We previously described elevated PACAP levels in the milk compared to the plasma, and the presence of its specific PAC1 receptor in the mammary gland. This study aimed to determine PACAP and vasoactive intestinal peptide (VIP) levels in female suckling lambs compared to ewe plasma and mammary gland, as well as their age-dependent alterations. mRNA expressions of PACAP, VIP, PAC1 receptor and brain-derived neurotrophic factor (BDNF) were quantified in the milk whey and mammary gland. PACAP38-like immunoreactivity (PACAP38-LI) was measured in plasma, milk whey and mammary gland by radioimmunoassay, VIP-LI by enzyme-linked immunoassay. PACAP38-LI was 5, 6 times higher in the milk compared to the plasma of lactating sheep. It significantly increased in the lamb plasma 1 h, but returned to basal level 2 h after suckling. However, VIP mRNA was not present in the mammary gland, we detected the VIP protein in the milk whey. BDNF mRNA significantly decreased with age to approximately 60% and 25% in the 3- and 10-year-old sheep respectively, compared to the 3-month-old lambs. No differences were found between mammary and jugular vein plasma PACAP and VIP concentrations, or during the daily cycle. We propose a rapid absorption of PACAP38 from the milk and/or its release in suckling lambs. PACAP accumulated in the milk might be synthesized in the mammary gland or secreted from the plasma of the mothers. PACAP is suggested to have differentiation/proliferation promoting and immunomodulatory effects in the newborns and/or a local function in the mammary gland.

Pituitary Adenylate Cyclase-Activating Polypeptide-27 (PACAP-27) in the Thalamic Paraventricular Nucleus Is Stimulated by Ethanol Drinking.

BACKGROUND: The paraventricular nucleus of the thalamus (PVT) is a limbic brain structure that affects ethanol (EtOH) drinking, but the neurochemicals transcribed in this nucleus that may participate in this behavior have yet to be fully characterized. The neuropeptide, pituitary adenylate cyclase-activating polypeptide (PACAP), is known to be transcribed in other limbic areas and to be involved in many of the same behaviors as the PVT itself, possibly including EtOH drinking. It exists in 2 isoforms, PACAP-38 and PACAP-27, with the former expressed at higher levels in most brain regions. The purpose of this study was to characterize PACAP in the PVT and to assess its response to EtOH drinking. METHODS: First, EtOH-naïve, Sprague Dawley rats were examined using quantitative real-time polymerase chain reaction (qPCR) and immunohistochemistry, to characterize PACAP mRNA and peptide throughout the rostrocaudal axis of the PVT. Next, EtOH-naïve, vGLUT2-GFP transgenic mice were examined using immunohistochemistry, to identify the neurochemical phenotype of the PACAPergic cells in the PVT. Finally, Long Evans rats were trained to drink 20% EtOH under the intermittent-access paradigm and then examined with PCR and immunohistochemistry, to determine the effects of EtOH on endogenous PACAP in the PVT. RESULTS: Gene expression of PACAP was detected across the entire PVT, denser in the posterior than the anterior portion of this nucleus. The protein isoform, PACAP-27, was present in a high percentage of cell bodies in the PVT, again particularly in the posterior portion, while PACAP-38 was instead dense in fibers. All PACAP-27+ cells colabeled with glutamate, which itself was identified in the majority of PVT cells. EtOH drinking led to an increase in PACAP gene expression and in levels of PACAP-27 in individual cells of the PVT. CONCLUSIONS: This study characterizes the PVT neuropeptide, PACAP, and its understudied protein isoform, PACAP-27, and demonstrates that it is involved in pharmacologically relevant EtOH drinking. This indicates that PACAP-27 should be further investigated for its possible role in EtOH drinking.

Migraine, Neurogenic Inflammation, Drug Development - Pharmacochemical Aspects.

BACKGROUND: Migraine is a primary headache disorder. Despite numerous studies conducted with the aim to understand the pathophysiology of migraine, several aspects are still unclear. The trigeminovascular system plays a key role. Neurogenic inflammation is presumed to be an important factor in migraine pathophysiology, mediated by the activation of primary neurons, leading to the release of various pro-inflammatory neuropeptides and neurotransmitters such as Calcitonin Gene-Related Peptide (CGRP), substance P (SP), and vasoactive intestinal peptide (VIP). Nitric oxide (NO), Pituitary adenylate cyclase-activating polypeptide (PACAP) and Glutamate (Glu) also play an important role in the modulation of inflammatory mechanisms. OBJECTIVE: To review the literature focusing on novel therapeutic targets in migraine, related to neurogenic inflammation. METHOD: A systematic literature search in the database of PUBMED was conducted regarding therapeutic strategies in migraine, focusing on substances and cytokines released during neurogenic inflammation, published until January 2017. RESULTS: Ongoing phase III clinical studies with monoclonal antibodies against CGRP and CGRP receptors offer promising novel aspects for migraine treatment. Preclinical and clinical studies targeting SP and nitric oxide synthase (NOS) were all terminated with no significant results compared to placebo. New promising therapeutic goal could be PACAP and its receptor (PAC1), and kynurenic acid (KYNA) analogues. CONCLUSION: Current migraine treatment offers pain relief only for a small proportion of migraine patients and might not be adequate for patients with cardiovascular comorbidity due to side effects. Better understanding of migraine pathophysiology might, therefore, lead to novel therapeutic lines both in migraine attack treatment and prophylaxis.

Ameliorative effects of PACAP against cartilage degeneration. Morphological, immunohistochemical and biochemical evidence from in vivo and in vitro models of rat osteoarthritis.

Osteoarthritis (OA); the most common form of degenerative joint disease, is associated with variations in pro-inflammatory growth factor levels, inflammation and hypocellularity resulting from chondrocyte apoptosis. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide endowed with a range of trophic effects in several cell types; including chondrocytes. However; its role in OA has not been studied. To address this issue, we investigated whether PACAP expression is affected in OA cartilage obtained from experimentally-induced OA rat models, and then studied the effects of PACAP in isolated chondrocytes exposed to IL-1ß in vitro to mimic the inflammatory milieu of OA cartilage. OA induction was established by histomorphometric and histochemical analyses. Changes in PACAP distribution in cartilage, or its concentration in synovial fluid (SF), were assessed by immunohistochemistry and ELISA. Results showed that PACAP abundance in cartilage tissue and SF was high in healthy controls. OA induction decreased PACAP levels both in affected cartilage and SF. In vitro, PACAP prevented IL-1ß-induced chondrocyte apoptosis, as determined by MTT assay; Hoechst staining and western blots of apoptotic-related proteins. These changes were also accompanied by decreased i-NOS and COX-2 levels, suggesting an anti-inflammatory effect. Altogether, these findings support a potential role for PACAP as a chondroprotective agent for the treatment of OA.

Localization of CGRP, CGRP receptor, PACAP and glutamate in trigeminal ganglion. Relation to the blood-brain barrier.

Calcitonin gene-related peptide (CGRP) receptor antagonists have demonstrated anti-migraine efficacy. One remaining question is where do these blockers act? We hypothesized that the trigeminal ganglion could be one possible site. We examined the binding sites of a CGRP receptor antagonist (MK-3207) and related this to the expression of CGRP and its receptor in rhesus trigeminal ganglion. Pituitary adenylate cyclase-activating polypeptide (PACAP) and glutamate were examined and related to the CGRP system. Furthermore, we examined if the trigeminal ganglion is protected by the blood-brain barrier (BBB). Autoradiography was performed with [(3)H]MK-3207 to demonstrate receptor binding sites in rhesus trigeminal ganglion (TG). Immunofluorescence was used to correlate binding and the presence of CGRP and its receptor components, calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1), and the distribution of PACAP and glutamate in rhesus and rat TG. Evans blue was used to examine large molecule penetration into the rat TG. High receptor binding densities were found in rhesus TG. Immunofluorescence revealed expression of CGRP, CLR and RAMP1 in trigeminal cells. CGRP positive neurons expressed PACAP but not glutamate. Some neurons expressing CLR and RAMP1 co-localized with glutamate. Evans blue revealed that the TG is not protected by BBB. This study demonstrates CGRP receptor binding sites and expression of the CGRP receptor in rhesus and rat TG. The expression pattern of PACAP and glutamate suggests a possible interaction between the glutamatergic and CGRP system. In rat the TG is outside the BBB, suggesting that molecules do not need to be CNS-penetrant to block these receptors.

Expression of neuropeptides and anoctamin 1 in the embryonic and adult zebrafish intestine, revealing neuronal subpopulations and ICC-like cells.

This immunohistochemical study in zebrafish aims to extend the neurochemical characterization of enteric neuronal subpopulations and to validate a marker for identification of interstitial cells of Cajal (ICC). The expression of neuropeptides and anoctamin 1 (Ano1), a selective ICC marker in mammals, was analyzed in both embryonic and adult intestine. Neuropeptides were present from 3 days postfertilization (dpf). At 3 dpf, galanin-positive nerve fibers were found in the proximal intestine, while calcitonin gene-related peptide (CGRP)- and substance P-expressing fibers appeared in the distal intestine. At 5 dpf, immunoreactive fibers were present along the entire intestinal length, indicating a well-developed peptidergic innervation at the onset of feeding. In the adult intestine, vasoactive intestinal peptide (VIP), pituitary adenylate cyclase-activating peptide (PACAP), galanin, CGRP and substance P were detected in nerve fibers. Colchicine pretreatment enhanced only VIP and PACAP immunoreactivity. VIP and PACAP were coexpressed in enteric neurons. Colocalization stainings revealed three neuronal subpopulations expressing VIP and PACAP: a nitrergic noncholinergic subpopulation, a serotonergic subpopulation and a subpopulation expressing no other markers. Ano1-immunostaining revealed a 3-dimensional network in the adult intestine containing multipolar cells at the myenteric plexus and bipolar cells interspersed between circular smooth muscle cells. Ano1 immunoreactivity first appeared at 3 dpf, indicative of the onset of proliferation of ICC-like cells. It is shown that the Ano1 antiserum is a selective marker of ICC-like cells in the zebrafish intestine. Finally, it is hypothesized that ICC-like cells mediate the spontaneous regular activity of the embryonic intestine.

Quantitative peptidomics for discovery of circadian-related peptides from the rat suprachiasmatic nucleus.

In mammals the suprachiasmatic nucleus (SCN), the master circadian clock, is sensitive to light input via the optic chiasm and synchronizes many daily biological rhythms. Here we explore variations in the expression levels of neuropeptides present in the SCN of rats using a label-free quantification approach that is based on integrating peak intensities between daytime, Zeitgeber time (ZT) 6, and nighttime, ZT 18. From nine analyses comparing the levels between these two time points, 10 endogenous peptides derived from eight prohormones exhibited significant differences in their expression levels (adjusted p-value <0.05). Of these, seven peptides derived from six prohormones, including GRP, PACAP, and CART, exhibited ≥ 30% increases at ZT 18, and the VGRPEWWMDYQ peptide derived from proenkephalin A showed a >50% increase at nighttime. Several endogenous peptides showing statistically significant changes in this study have not been previously reported to alter their levels as a function of time of day, nor have they been implicated in prior functional SCN studies. This information on peptide expression changes serves as a resource for discovering unknown peptide regulators that affect circadian rhythms in the SCN.

Changes of PACAP immunoreactivities and cytokine levels after PACAP-38 containing intestinal preservation and autotransplantation.

Small bowel is one of the most sensitive organs to ischemia-reperfusion injury, which is a significant problem during transplantation. Pituitary adenylate cyclase-activating polypeptide (PACAP) has cytoprotective effect in ischemic injuries of various tissues. The aim of our study was to measure changes of PACAP-38 and PACAP-27 immunoreactivities and cytokine levels in intestinal grafts stored in PACAP-38-containing preservation solution. Small bowel autotransplantation was performed on male Wistar rats. Grafts were stored in University of Wisconsin (UW) solution at 4 °C for 1 h (group (G)I), for 3 h (GII), and for 6 h (GIII) and in PACAP-38-containing UW solution for 1 h (GIV), for 3 h (GV), and for 6 h (GVI). After preservation, performing vessel anastomosis reperfusion began, which lasted 3 h in each group. Tissue biopsies were collected after laparotomy (control) and at the end of the reperfusion periods. Intestinal PACAP-38 and PACAP-27 immunoreactivities were measured by radioimmunoassay. To measure cytokines from tissue homogenates, we used rat cytokine array and Luminex Multiplex Immunoassay. Levels of PACAP-38 and PACAP-27 immunoreactivity decreased after 1 and 3 h preservation compared to control levels. This decrease was significant following 6 h cold storage (p < 0.05). Values remained significantly higher in grafts stored in PACAP-38-containing UW. Cytokine array revealed that expression of the soluble intercellular adhesion molecule-1 (CD54) and L-selectin (CD62L/LECAM-1) was increased in GIII. Both 6 h cold storage in PACAP-38-containing UW solution and 3 h reperfusion caused strong reduction in these cytokines activation in GVI. RANTES (CCL5) levels were increased in all groups. Strong activation of the tissue inhibitor of metalloproteinase-1 was in GIII. However, PACAP-38-containing cold storage could decrease its activation in GVI. Furthermore, strong activation of the tissue inhibitor of metalloproteinase-1 was detected in 6 h preserved grafts without PACAP-38 (GIII). PACAP-38-containing cold storage could decrease its activation in GVI. Our present study showed that PACAP-38 and PACAP-27 immunoreactivities decreased in a time-dependent manner during intestinal cold preservation, which could be ameliorated by administration of exogenous PACAP-38 to the preservation solution. Moreover, PACAP-38 could attenuate tissue cold ischemic injury-induced changes in cytokine expression.

Changes in pituitary adenylate cyclase-activating Peptide 27-like immunoreactive nervous structures in the porcine descending colon during selected pathological processes.

This study reports on changes in the pituitary adenylate cyclase-activating peptide 27-like immunoreactive (PACAP-27-LI) nerve structures of the enteric nervous system (ENS) in the porcine descending colon, caused by chemically induced inflammation, nerve injury, and proliferative enteropathy (PE), which is a "natural" inflammation of the porcine digestive tract. The distribution pattern of PACAP-27-LI structures was studied using the immunofluorescence technique in the circular muscle layer, enteric plexuses (i.e., myenteric plexus (MP), outer submucous plexus (OSP), and inner submucous plexus (ISP)), and in the mucosal layer. Under physiological conditions, PACAP-27-LI perikarya have been shown to constitute 4.04 ± 0.66, 6.66 ± 0.77, and 11.19 ± 0.74 % in the MP, OSP, and ISP, respectively. Changes in PACAP-27 immunoreactivity depended on the pathological factor studied. The numbers of the PACAP-27-LI perikarya amounted to 12.26 ± 1.43, 12.28 ± 0.79, and 21.13 ± 1.19 % in chemically induced colitis, 17.83 ± 0.88, 9.03 ± 1.05, and 20.72 ± 1.35 % during PE and 10.65 ± 0.82, 6.88 ± 1.04, and 14.04 ± 1.09 % after axotomy in MP, OSP, and ISP, respectively. All of the studied processes generally resulted in an increase in the number of PACAP-27-LI nerve fibers in the circular muscle and mucosal layers. The obtained results suggest that PACAP-27-LI nerve structures of ENS may participate in various pathological states within the porcine descending colon, and their functions probably depend on the type of pathological factor.

PACAP immunoreactivity in human malignant tumor samples and cardiac diseases.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a pleiotropic and multifunctional neuropeptide having important roles in various physiological processes. Recent trends in PACAP research point to the clinical introduction of PACAP or its analogs/fragments possibly in the near future. Recently, we have shown the presence of PACAP in human plasma, milk, placenta, and follicular fluid samples. However, relatively few data are available on PACAP in human tissues from patients with different disorders. The aim of the present study was to determine, by radioimmunoassay, the tissue level of PACAP38-like immunoreactivity (LI) and PACAP27-LI in different primary non-small cell lung cancer, colon tumor samples, and in cardiac muscle samples from patients suffering from ischemic heart disease and valvular disorders. We also labeled the PAC1 receptors in human cardiac cells. All samples showed significantly higher PACAP38-LI compared with PACAP27-LI. We found significantly lower levels of PACAP38-LI and PACAP27-LI in tumoral and peripheral samples compared with normal healthy tissue in both lung and colon cancers. Further investigations are necessary to describe the exact function of PACAP in oncogenesis. We showed that PACAP38-LI and PACAP27-LI are significantly higher in ischemic heart diseases compared with valvular abnormalities, suggesting that PACAP might play a role in ischemic heart disorders.

Effects of pituitary adenylate cyclase activating polypeptide on human sperm motility.

Pituitary adenylate cyclase activating polypeptide (PACAP), a neuropeptide with diverse effects, was originally isolated as a hypothalamo-hypophyseal peptide. Subsequent studies showed highest levels of PACAP in the testis after the brain, suggesting that it influences the development and functioning of spermatozoa. Indeed, it has been proven that PACAP has an effect on spermatogenesis, both locally and via influencing the hypothalamo-hypophyseal-gonadal axis. The aim of the present study was to determine whether PACAP has an effect on human sperm motility and whether it is present in the human seminal fluid. Furthermore, the sperm head morphology was studied in mice lacking endogenous PACAP. Human samples were obtained from healthy adult volunteers and andrological patients. The effects of PACAP on the motility of human sperm cells were investigated using a computer aided sperm analysis system. In cases where the motility was lower, addition of PACAP to the samples increased the motility and the ratio of rapid progressive and medium progressive sperm motility groups. The presence of PACAP could not be detected in human seminal fluid samples by means of mass spectrometry. Investigating sperm head morphology with routine histology in PACAP deficient mice revealed that both the longitudinal and transverse diameters were significantly lower in PACAP deficient mice, without marked difference in the shape, as revealed by scanning electron microscopy.

Changes in PACAP immunoreactivity in human milk and presence of PAC1 receptor in mammary gland during lactation.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide with widespread occurrence in the nervous system and peripheral organs, including the mammary gland. Previously, we have shown that PACAP38 is present in the human milk at higher levels than in respective blood samples. However, it is not known how PACAP levels and the expression of PAC1 receptor change during lactation. Therefore, the aim of our study was to investigate PACAP38-like immunoreactivity (PACAP38-LI) in human colostrums and transitional and mature milk during lactation and to compare the expression of PAC1 receptors in lactating and non-lactating mammary glands. We found that PACAP38-LI was significantly higher in human colostrum samples than in the transitional and mature milk. PACAP38-LI did not show any significant changes within the first 10-month period of lactation, but a significant increase was observed thereafter, up to the examined 17th month. Weak expression of PAC1 receptors was detected in non-lactating sheep and human mammary glands, but a significant increase was observed in the lactating sheep samples. In summary, the present study is the first to show changes of PACAP levels in human milk during lactation. The presence of PACAP in the milk suggests a potential role in the development of newborn, while the increased expressions of PAC1 receptors on lactating breast may indicate a PACAP38/PAC1 interaction in the mammary gland during lactation.

Correlation between oocyte number and follicular fluid concentration of pituitary adenylate cyclase-activating polypeptide (PACAP) in women after superovulation treatment.

Follicular growth, ovulation, and luteinization are influenced by interactions of peptide and steroid hormone-signaling cascades in the ovary. Pituitary adenylate cyclase-activating polypeptide (PACAP) plays an important role in the regulation of several endocrine processes and is present in ovarian follicular fluid (FF). However, little is known about PACAP in FF with regard to maturation, ovulation, fertilization, and successful pregnancy. The aim of this pilot study was to investigate whether there is a correlation between PACAP concentration in FF and ovarian response to superovulation treatment in infertile women, performed in volunteers (n = 132; aged between 20 and 35). After treatment, the number of harvested oocytes was recorded and PACAP immunoreactivity in FF was measured by radioimmunoassay. All the corresponding PACAP concentrations were below 290 fmol/ml in cases when the number of harvested oocytes exceeded 14 per patient, while in all cases above 290 fmol/ml, the number of oocytes was below 14. Using these cutoff values, we determined three study groups: high-PACAP concentration, high-oocyte number, and low-PACAP concentration-low-oocyte number groups. Median values of PACAP concentration in these groups were 411.2, 106.5, and 101.0 fmol/ml, respectively, while the median values of harvested oocytes were 5.5, 19.0, and 5.0, respectively. Differences were significant, indicating a correlation between concentration of PACAP in FF and the number of recruited oocytes. Higher concentrations of PACAP in FF might be associated with lower number of developing oocytes, while low concentrations of PACAP might correlate with a markedly higher number of ova retrieved, thus predicting a higher chance for ovarian hyperstimulation. Our present study is among the first few human clinical studies with direct conclusions drawn for possible clinical impact of PACAP.

Immunohistochemical characterisation of dorsal root ganglia neurons supplying the porcine mammary gland.

The present study investigated the chemical coding of mammary gland-projecting dorsal root ganglia (DRG) neurons using double-labelling immunohistochemistry. Earlier investigations revealed the presence of Fast blue - positive (FB+) neurons in Th9-Th12 DRG after injection of the tracer into the second, right thoracic mamma. Neurons projecting to the last right abdominal mamma were found in L1-L3 DRG. In the present study, the cryostat sections from these ganglia were stained for calcitonin gene-related peptide (CGRP), substance P (SP), nitric oxide synthase (NOS), galanin (GAL) and pituitary adenylate cyclase activating polypeptide (PACAP). Immunohistochemistry revealed that the vast majority of FB+ mammary gland-projecting neurons contained immunoreactivity to CGRP (68.87±0.7%), SP (63.4±0.9%), NOS (32.47±0.9%), GAL (16.28±0.8%) and less numerous nerve cells stained for PACAP (5.87±0.5%). The present results largely correspond with findings dealing with immunohistochemical characterization of nerve fibres supplying porcine mammary gland structures described earlier.

Presence of pituitary adenylate cyclase activating polypeptide and its type I receptor in the rat kidney.

Pituitary adenylate cyclase activating polypeptide (PACAP), a multifunctional neuropeptide, has 2 active forms, PACAP38 and PACAP27. It is now well-established that PACAP has several actions also in peripheral organs, including renoprotective effects. The peptide itself has not been previously identified in the rat kidney. The first aim of our study was to identify PACAP in the rat kidney using mass spectrometry and radioimmunoassay (RIA). Receptor mRNA and binding studies revealed the existence of all 3 PACAP receptors (PAC1, VPAC1, and VPAC2) in the kidney, but their exact localization in histologic sections was not evident. Because most of the cytoprotective effects of PACAP relate to its specific PAC1 receptor, our second aim was to identify the cell types wherein the PAC1 receptor is expressed in the rat kidney. Mass spectrometry revealed the presence of PACAP38 in the kidney. RIA measurements showed both PACAP38- and PACAP27-like immunoreactivities in kidney homogenates, with PACAP38 being dominant. Immunohistochemistry revealed PAC1 receptor-like immunoreactivity in kidney sections, mainly expressed in cortical tubular epithelial cells. These results showed PACAP to be endogenously present in the kidney. The tubular localization of the PAC1 receptor provides the basis for the renal effects of the peptide under physiologic and pathologic conditions.

Investigation of pituitary adenylate cyclase activating polypeptide in human gynecological and other biological fluids by using MALDI TOF mass spectrometry.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a multifunctional and pleiotropic neuropeptide. PACAP has diverse effects in the endocrine system, among others, it plays important roles in oogenesis, implantation and development of the nervous system. However, it is not known whether PACAP is present in the fluids of the human reproductive organs. The aim of the present study was to determine, by means of mass spectrometry and radioimmunoassay, whether PACAP is present in human amniotic fluid, ovarian follicular fluid and cervico-vaginal fluid. Samples were obtained from healthy adult volunteers. Our MALDI TOF and MALDI TOF/TOF spectrometry results show that PACAP38 is present in all of the follicular fluid samples, and PACAP-like immunoreactivity was also measured by radioimmunoassay. However, we did not find the characteristic peak representing the unmodified 38 amino acid form of the peptide in normal cervico-vaginal smear and amniotic fluid samples. Furthermore, we analyzed other body fluids for comparison, such as human nasal fluid, saliva and aqueous humor. PACAP was not found in these latter samples. In summary, the present study provides evidence for the presence of PACAP in human follicular fluid, suggesting a role in oocyte function, but determination of the exact physiological significance awaits further investigation.

Immunolocalization of neurotransmitter-synthesizing enzymes and neuropeptides with associated receptors in the photophores of the hatchetfish, Argyropelecus hemigymnus Cocco, 1829.

Anatomical and functional studies of the autonomic innervation of the photophores of luminescent fishes are scarce. The present immunohistochemical study demonstrated the presence of nerve fibers in the luminous epithelium and lens epithelium of the photophores of the hatchet fish, Argyropelecus hemigymnus and identified the immunoreactive elements of this innervation. Phenylethanolanine N-methyltransferase (PNMT) and catecholamine (CA)-synthesizing enzymes were detected in nerve varicosities inside the two epithelia. Neuropeptides were localized in neuropeptide Y (NPY) and substance P (SP)- and its NK11 receptor-immunopositive nerves in the lens epithelium. Neuropeptides were also localized in non-neural cell types such as the lens cells, which displayed immunoreactivities for pituitary adenylate cyclase activating peptide (PACAP) and their receptors R-12 and 93093-3. This reflects the ability of the neuropeptide-containing nerves and lens cells to turn on and off the expression of selected messengers. It appears that the neuropeptide-containing nerves demonstrated in this study may be sensory. Furthermore, neuronal nitric oxide synthase-immunopositive axons associated with photocytes in the luminous epithelium have previously been described in this species. Whereas it is clear that the photophores receive efferent (motor) fibers of spinal sympathetic origin, the origin of the neuropeptide sensory innervation remains to be determined. The functional roles of the above neuropeptides or their effects on the bioluminescence or the chemical nature of the terminals, either sensory or postganglionic neurons innervating the photophores, are still not known.