[Clinical importance of adenylate cyclase-activating polypeptide (PACAP)]. / Az adenilát-cikláz-aktiváló polipeptid (PACAP) klinikai jelentosége.

The pituitary adenylate cyclase-activating polypeptide (PACAP) was isolated and characterized from sheep hypothalami. Its amino acid sequence, gene, receptors and receptor genes and its distribution in the mammalian organism were soon described. PACAP is a member of the secretin peptide family. Its closest relative is the vasoactive intestinal polypeptide (VIP). Its widespread occurrence suggests that it plays a significant role in physiological processes. With the aim of animal models, the role of PACAP was intensively investigated worldwide in a possible treatment of various diseases. The first part of this work contains the most important experimental data regarding the structure, genes and occurrence of the peptide and its receptors in mammalian body. In the second part, we overviewed the ever-increasing data on human material according to organ systems. The review contains the data where there is a chance to use PACAP for therapeutic purposes in the clinical practice. Determining the concentration of PACAP in the blood would help in establishing a clinical and differential diagnosis. In the future, there may be a possibility for non-invasive therapy of tumors expressing PACAP receptors. PACAP regulates the pituitary functions, stimulates vasopressin release, adrenalin secretion, insulin secretion. It is smooth muscle relaxant, immunosuppressant. PACAP is a neurotransmitter, it is neuroprotective in various diseases of the nervous system and cytoprotective in peripheral organs. PACAP inhibits apoptosis and the formation of pro-inflammatory factors and stimulates the anti-inflammatory factors and development of tumor cells. PACAP participates in regulating the daily rhythm of physiological functions. Orv Hetil. 2023; 164(33): 1300-1310.

Vitamin D Deficiency Reduces Vascular Reactivity of Coronary Arterioles in Male Rats.

BACKGROUND: Vitamin D deficiency (VDD) may be considered an independent cardiovascular (CV) risk factor, and it is well known that CV risk is higher in males. Our goal was to investigate the pharmacological reactivity and receptor expression of intramural coronary artery segments of male rats in cases of different vitamin D supply. METHODS: Four-week-old male Wistar rats were divided into a control group (n = 11) with optimal vitamin D supply (300 IU/kgbw/day) and a VDD group (n = 11, <0.5 IU/kgbw/day). After 8 weeks of treatment, intramural coronary artery segments were microprepared, their pharmacological reactivity was examined by in vitro microangiometry, and their receptor expression was investigated by immunohistochemistry. RESULTS: Thromboxane A2 (TXA2)-agonist induced reduced vasoconstriction, testosterone (T) and 17-ß-estradiol (E2) relaxations were significantly decreased, a significant decrease in thromboxane receptor (TP) expression was shown, and the reduction in estrogen receptor-α (ERα) expression was on the border of significance in the VDD group. CONCLUSIONS: VD-deficient male coronary arteries showed deteriorated pharmacological reactivity to TXA2 and sexual steroids (E2, T). Insufficient vasoconstrictor capacity was accompanied by decreased TP receptor expression, and vasodilator impairments were mainly functional. The decrease in vasoconstrictor and vasodilator responses results in narrowed adaptational range of coronaries, causing inadequate coronary perfusion that might contribute to the increased CV risk in VDD.

Vitamin D Deficiency Induces Elevated Oxidative and Biomechanical Damage in Coronary Arterioles in Male Rats.

BACKGROUND: Several reports prove interconnection between vitamin D (VD) deficiency and increased cardiovascular risk. Our aim was to investigate the effects of VD status on biomechanical and oxidative-nitrative (O-N) stress parameters of coronary arterioles in rats. METHODS: 4-week-old male Wistar rats were divided into a control group (11 animals) with optimal VD supply (300 IU/kgbw/day) and a VD-deficient group (11 animals, <5 IU/kg/day). After 8 weeks, coronary arteriole segments were prepared. Geometrical, elastic, and biomechanical characteristics were measured by in vitro arteriography. O-N stress markers were investigated by immunohistochemistry. RESULTS: Inner radius decreased; wall thickness and wall-thickness/lumen diameter ratio increased; tangential wall stress and elastic modulus were reduced in VD-deficient group. No difference could be found in wall-cross-sectional area, intima-media area %. While the elastic elements of the vessel wall decreased, the α-smooth muscle actin (α-SMA) immunostaining intensity showed no changes. Significant elevation was found in the lipid peroxidation marker of 4-hidroxy-2-nonenal (HNE), while other O-N stress markers staining intensity (poly(ADP)ribose, 3-nitrotyrosine) did not change. CONCLUSIONS: Inward eutrophic remodeling has developed. The potential background of these impairments may involve the initial change in oxidative damage markers (HNE). These mechanisms can contribute to the increased incidence of the cardiovascular diseases in VD deficiency.

Current State of Understanding of the Role of PACAP in the Hypothalamo-Hypophyseal Gonadotropin Functions of Mammals.

PACAP was discovered 30 years ago in Dr. Akira Arimura's laboratory. In the past three decades since then, it has become evident that this peptide plays numerous crucial roles in mammalian organisms. The most important functions of PACAP are the following: 1. neurotransmitter, 2. neuromodulator, 3. hypophysiotropic hormone, 4. neuroprotector. This paper reviews the accumulated data regarding the distribution of PACAP and its receptors in the mammalian hypothalamus and pituitary gland, the role of PACAP in the gonadotropin hormone secretion of females and males. The review also summarizes the interaction between PACAP, GnRH, and sex steroids as well as hypothalamic peptides including kisspeptin. The possible role of PACAP in reproductive functions through the biological clock is also discussed. Finally, the significance of PACAP in the hypothalamo-hypophysial system is considered and the facts missing, that would help better understand the function of PACAP in this system, are also highlighted.

Intranasal application of PACAP and ß-cyclodextrin before the "critical period of proestrous stage" can block ovulation.

INTRODUCTION: It was previously shown that intracerebroventricular administration of pituitary adenylate cyclase-activating polypeptide (PACAP) prior to GnRH mobilization in proestrus prevents ovulation in rats. In this study, we examined whether PACAP given intranasally could influence luteinizing hormone (LH) and prolactin (PRL) surges and ovulation. METHODS: On the day of proestrus PACAP, p-cyclodextrin (modifier of blood-brain barrier) or PACAP + p-cyclodextrin was applied intranasally between 12:30 and 13:00. Blood samples were taken at 16:00, 18:00, and 20:00 for measuring plasma hormone levels. In the next morning, the expelled ova were counted. p-Cyclodextrin was also administered to male and diestrous female rats between 12:30 and 13:00 and blood was taken at 18:00. RESULTS: PACAP prevented LH and PRL surges and ovulation in about half of the rats, p-cyclodextrin alone more effectively prevented ovulation. When PACAP and p-cyclodextrin were administered together, more rats ovulated like when PACAP given alone. p-Cyclodextrin did not influence LH and PRL levels in diestrous females; however, in males, it significantly enhanced PRL level. DISCUSSION: Not only the intracerebroventricular, but the intranasal application of PACAP prevented ovulation. p-Cyclodextrin alone is more effective than PACAP and enhances PRL levels in male rats. PACAP and p-cyclodextrin given together weaken each other's effect. p-Cyclodextrin, as excipient of various drugs, has to be used carefully in human medications.

Geometric, elastic and contractile-relaxation changes in coronary arterioles induced by Vitamin D deficiency in normal and hyperandrogenic female rats.

Vitamin D (VitD) hypovitaminosis and androgen excess (AE) are both risk factors for cardiovascular diseases in fertile women. However, the possible early interaction between AE and VitD status is not clear. Our goal was to describe how VitD status influences early changes in the biomechanical reactivity of small coronary arterioles in adult female rats after transdermal testosterone treatment. Forty-six adolescent, 90-110-gram-weighed female Wistar rats were randomly grouped into 4 groups. Twenty-four animals received an optimal VitD-supplemented diet, from which 12 animals underwent transdermal testosterone treatment. Twenty-two animals received a VitD-deficient diet, from which 11 were treated with testosterone. At 8 weeks of treatment, invasive arterial blood pressure was registered after in vivo cannulation of carotid artery. Arteriolar end and side branches (200 µm diameter) of the left anterior descendent coronary artery (LAD) were obtained and examined with pressure arteriography in vitro. Similar segments were removed for histological examination. The inner and outer radii of the arterioles were measured using video-microscopy. Normal myogenic tone, maximal passive vasorelaxation and vasoconstriction of the arterioles were measured and statistically analyzed. The vessels' maximal smooth muscle relaxant potential, thromboxane-induced contraction capacity and normal myogenic tone were significantly influenced by actual VitD status. A lower relaxation capacity and increased wall thickness were observed in VitD-deficient groups, which could cause rigidity of the coronary arterioles and elevate cardiovascular risk. Supplementation of VitD could improve myogenic tone and relaxation and hold cardiovascular benefits.

Insulin resistance in an animal model of polycystic ovary disease is aggravated by vitamin D deficiency: Vascular consequences.

Hyperandrogenic state in females is accompanied with metabolic syndrome, insulin resistance and vascular pathologies. A total of 67%-85% of hyperandrogenic women suffer also from vitamin D deficiency. We aimed to check a potential interplay between hyperandrogenism and vitamin D deficiency in producing insulin resistance and effects on coronary resistance arteries. Adolescent female rats were divided into four groups, 11-12 animals in each. Transdermal testosterone-treated and vehicle-treated animals were kept either on vitamin D-deficient or on vitamin D-supplemented diet for 8 weeks. Plasma sexual steroid, insulin, leptin and vitamin D plasma levels were measured, and oral glucose tolerance test was performed. In coronary arterioles, insulin receptor and vitamin D receptor expressions were tested by immunohistochemistry, and insulin-induced relaxation was measured in vitro on isolated coronary resistance artery segments. Testosterone impaired glucose tolerance, and it diminished insulin relaxation but did not affect the expression of insulin and vitamin D receptors in vascular tissue. Vitamin D deficiency elevated postprandial insulin levels and homeostatic model assessment insulin resistance. It also diminished insulin-induced coronary arteriole relaxation, while it raised the expression of vitamin D and insulin receptors in the endothelial and medial layers. Our conclusion is that both hyperandrogenism and vitamin D deficiency reduce sensitivity of coronary vascular tissue to insulin, but they do it with different mechanisms.

Vitamin D deficiency causes inward hypertrophic remodeling and alters vascular reactivity of rat cerebral arterioles.

BACKGROUND AND PURPOSE: Vitamin D deficiency (VDD) is a global health problem, which can lead to several pathophysiological consequences including cardiovascular diseases. Its impact on the cerebrovascular system is not well understood. The goal of the present work was to examine the effects of VDD on the morphological, biomechanical and functional properties of cerebral arterioles. METHODS: Four-week-old male Wistar rats (n = 11 per group) were either fed with vitamin D deficient diet or received conventional rat chow with per os vitamin D supplementation. Cardiovascular parameters and hormone levels (testosterone, androstenedione, progesterone and 25-hydroxyvitamin D) were measured during the study. After 8 weeks of treatment anterior cerebral artery segments were prepared and their morphological, biomechanical and functional properties were examined using pressure microangiometry. Resorcin-fuchsin and smooth muscle actin staining were used to detect elastic fiber density and smooth muscle cell counts in the vessel wall, respectively. Sections were immunostained for eNOS and COX-2 as well. RESULTS: VDD markedly increased the wall thickness, the wall-to-lumen ratio and the wall cross-sectional area of arterioles as well as the number of smooth muscle cells in the tunica media. As a consequence, tangential wall stress was significantly lower in the VDD group. In addition, VDD increased the myogenic as well as the uridine 5'-triphosphate-induced tone and impaired bradykinin-induced relaxation. Decreased eNOS and increased COX-2 expression were also observed in the endothelium of VDD animals. CONCLUSIONS: VDD causes inward hypertrophic remodeling due to vascular smooth muscle cell proliferation and enhances the vessel tone probably because of increased vasoconstrictor prostanoid levels in young adult rats. In addition, the decreased eNOS expression results in endothelial dysfunction. These morphological and functional alterations can potentially compromise the cerebral circulation and lead to cerebrovascular disorders in VDD.

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.

Distribution of secretin receptors in the rat central nervous system: an in situ hybridization study.

Secretin shows a wide distribution in the brain. Functional significance of central secretin is stressed since it has been associated with autism and schizophrenia. The presence of the secretin receptor was previously demonstrated in the brain by different methods. Neurons in the cerebellum, hypothalamic paraventricular and supraoptic nuclei, and in the vascular organ of lamina terminalis were shown to express secretin receptor mRNA by using in situ hybridization with digoxigenin-labeled probe. In this work, we used a very sensitive radioactive in situ hybridization technique and systematically mapped the expression of secretin receptor mRNA in the brain. The densest labeling was observed in the nucleus of solitary tract and in the laterodorsal thalamic nucleus, where decreasing number of receptors was seen in the vascular organ of lamina terminalis, and the lateral habenular complex, and then in the supraoptic nucleus. Only a few scattered labeled cells were observed in the median frontal gyrus, entorhinal cortex, hypothalamic paraventricular nucleus, perifornical region, lateral hypothalamic area, head of the caudate nucleus, spinal trigeminal nucleus, and cerebellum. Secretin receptor mRNA showed a far wider distribution than was known before, suggesting a more significant functional relevance than thought earlier.

Intranasal application of secretin, similarly to intracerebroventricular administration, influences the motor behavior of mice probably through specific receptors.

Secretin and its receptors show wide distribution in the central nervous system. It was demonstrated previously that intravenous (i.v.) and intracerebroventricular (i.c.v.) application of secretin influenced the behavior of rat, mouse, and human. In our previous experiment, we used a special animal model, Japanese waltzing mice (JWM). These animals run around without stopping (the ambulation distance is very limited) and they do not bother with their environment. The i.c.v. secretin attenuated this hyperactive repetitive movement. In the present work, the effect of i.c.v. and intranasal (i.n.) application of secretin was compared. We have also looked for the presence of secretin receptors in the brain structures related to motor functions. Two micrograms of i.c.v. secretin improved the horizontal movement of JWM, enhancing the ambulation distance. It was nearly threefold higher in treated than in control animals. The i.n. application of secretin to the left nostril once or twice a day or once for 3 days more effectively enhanced the ambulation distance than i.c.v. administration. When secretin was given twice a day for 3 days it had no effect. Secretin did not improve the explorative behavior (the rearing), of JWM. With the use of in situ hybridization, we have found very dense secretin receptor labeling in the cerebellum. In the primary motor cortex and in the striatum, only a few labeled cells were seen. It was supposed that secretin exerted its effect through specific receptors, mainly present in the cerebellum.

Sexual dimorphism in the effect of concomitant progesterone administration on changes caused by long-term estrogen treatment in pituitary hormone immunoreactivities of rats.

BACKGROUND: Since in clinical practice long-term estrogen (E) treatment is frequently applied, our aim was to study the effect of concomitant progesterone (P) administration on changes caused by long-term estrogen treatment in the secretion of LH, FSH, PRL and GH. MATERIAL/METHODS: Diethylstilbestrol (DES), P or both in silastic capsules were implanted under the skin of prepubertal Sprague-Dawley male and female rats. Animals survived for two or five months. We have also studied whether the changed hormone secretion caused by DES can return to normal level 1 or 2 months after removing DES capsule. RESULTS: 1.) The males more rapidly responded than females with decreasing basal LH release upon treatments. The basal FSH release was decreased only in males. The effect of DES persisted in males; however, in females basal LH and FSH levels were upregulated after removal of DES capsule. 2.) The basal GH levels were low in each group. The body weight and length were depressed by DES in both genders and P little blunted this effect. The body weight and length in males remained low after removal of DES capsule, in females it was nearly similar to intact rats. 3.) There was no sexual dimorphism in the effect of steroids on PRL secretion. In both genders DES extremely enhanced the PRL levels, P prevented the effect of DES. PRL levels returned to intact value after removal of DES influence. 4.) Removal of DES capsule reversed the changes in the immunohistochemical appearance of hormone immunoreactivities. CONCLUSIONS: There was sexual dimorphism in the change of basal gonadotropic hormone and GH secretion but not of PRL upon DES and DES+P treatments. P was basically protective and this role may be mediated by P receptors locally in the pituitary gland.

Secretin attenuates the hereditary repetitive hyperactive movements in a mouse model.

It was previously demonstrated that secretin influenced the behavior of rats investigated by open-field test. In the present experiment, we have compared the effect of intracerebroventricular administration of 2 µg of secretin on the behavior of CFLP white and Japanese waltzing mice. These latter animals exhibit stereotypic circular movements. The effect of secretin on the horizontal (ambulation) and vertical movements (rearing and jumping) was investigated in open-field test. The ambulation time and distance were shorter, and the number of rearing and jumping were much lower in Japanese waltzing mice than in CFLP white mice during 30 min-experimental period. In white mice, 2 µg of secretin had no effect on the above-mentioned parameters; however, in Japanese waltzing mice, secretin enhanced the ambulation time and distance to the level of CFLP white mice, but did not influence the rearing and jumping. On the basis of the results, it was concluded that intracerebroventricularly administered secretin attenuated the stereotypic (circulating) movement and improved the horizontal movement indicated by the normalization of the ambulation time and distance; however, it did not influence the explorative behavior (rearing and jumping) in our special animal model.

Secretin mRNA in the subdivision of primary sensory neurons in the trigeminal ganglion of rats.

The primary sensory neurons use glutamate as a major neurotransmitter. Several neuropeptides are also found in these neurons. In our laboratory we demonstrated secretin-like immunoreactivity in primary sensory neurons of several species including human, rat and cat. In the present experiment utilizing in situ hybridization, we have demonstrated for the first time that secretin is not only immunostained but is also expressed in the primary sensory neurons of the trigeminal ganglion of male rats. In intact rats, secretin mRNA was not observed; we had to use intracerebroventricular colchicine administration to induce the expression of secretin. Secretin was expressed in about 5% of the cells in all the three subdivisions of the trigeminal ganglion. The secretin-synthetizing cells were large and medium sized, and their mean diameter was about 50 µm. When we compared the percentage and the size of secretin to that of calcitonin gene-related peptide (CGRP), substance-P (SP) and vasoactive intestinal polypeptide (VIP) cells, it was found that CGRP, SP and VIP are present in about 15-20% of the cells and their mean diameter is about 20-25 µm. The morphometric data indicate that secretin is present in a subdivision of neurons that is different from the subdivision of the CGRP, SP and VIP cells. It is suggested that secretin may modulate the function of the primary neurotransmitter.

Effect of concomitant progesterone administration or the effect of removal of estrogen capsule on changes caused by long-term estrogen treatment in pituitary VIP immunoreactivities.

In the anterior pituitary besides the classical tropic hormones, peptides of a small molecular weight are also synthesized. One of them is the vasoactive intestinal polypeptide (VIP). VIP immunoreactivity is readily detected in human and monkey pituitaries; however, in the rat VIP immunoreactive cells were observed in about 50% of intact rats. In estrogen treated rats VIP immunoreactive cells were observed in the anterior pituitary of all animals. In this work, we have examined the effect of long-term sexual steroid treatments on the VIP immunoreactivity of the anterior pituitary using diethylstilbestrol (DES) or progesterone (P) filled capsules. The effectiveness of steroid treatments was tested by the measurement of plasma prolactin (PRL) level and by the appearance of prolactinoma. DES enhanced the plasma PRL level and 5 months later it induced prolactinomas, the concomitant P treatment prevented both the elevation of plasma PRL level and the formation of prolactinomas. These results indicated that there was enough steroid in the capsules. There was a positive correlation between the duration of DES influence and the number of VIP immunoreactive cells. Two months after the implantation of DES there was a considerable number of VIP cells in the anterior pituitary, and 5 months after implantation the number of VIP cells was greatly increased so as to form a VIP-oma. Concomitant implantation of P prevented the formation of VIP-oma. Two months after the implantation, the DES capsule was removed. Already 2 months after removal the number of VIP cells approximated to the control level. It has been concluded that P can prevent the undesired effect of DES not only on the PRL, but on the VIP immunoreactivity as well.

The characteristic change in the distribution of S-100 immunoreactive folliculostellate cells in rat anterior pituitary upon long-term estrogen treatment is prevented by concomitant progesterone treatment.

The presence of folliculostellate cells in the anterior pituitary was described 49 years ago. These cells give about 10% of the whole cell population and through their long processes they provide intrahypophyseal communication. The folliculostellate cells contain S-100 protein. Its immunostaining was used to identify these cells. It was previously found that the diethylstilbestrol treatment basically influences the morphology and function of the trophic hormone secreting as well as the folliculostellate cells. In the present experiment, we have studied whether a concomitant progesterone treatment can prevent or attenuate changes caused by diethylstilbestrol treatment in the distribution of folliculostellate, prolactin, and GH cells. Diethylstilbestrol alone induced the appearance of prolactinomas. Inside the prolactinomas, folliculostellate cells were scattered but outside the prolactinomas they formed a demarcation line. Inside the prolactinomas, there were only a few growth hormone immunoreactive cells but they surrounded the prolactinomas in a ring-like pattern. When diethylstilbestrol was implanted with progesterone, the changes being characteristic for diethylstilbestrol treatment, could not develop. Concomitant progesterone influence prevented morphological changes in the anterior pituitary. Progesterone alone had no effect. In accordance with the formation of prolactinomas, the plasma prolactin level was very high in diethylstilbestrol treated rats. Concomitant progesterone treatment prevented the effect of diethylstilbestrol. Progesterone alone did not influence the prolactin level. GH levels did not significantly differ in any groups.

PACAP is transiently expressed in anterior pituitary gland of rats: in situ hybridization and cell immunoblot assay studies.

In this work the expression of PACAP (pituitary adenylate cyclase activating polypeptide) in rat anterior pituitary was demonstrated for the first time using in situ hybridization. The number of cells showing PACAP signal in intact male rats was negligible similarly to that of diestrous rats. In proestrous rats sacrificed at 10h there was a moderate increase in the expression and after a decrease at 16 h and 18 h, there was a transient peak at 20 h and then the number of labeled cells was declined again (22 h). In the cell immunoblot assay study it was observed that the number of PACAP blot forming (PACAP releasing) cells in an anterior pituitary cell culture changed according to a similar pattern as the number of PACAP expressing cells. The number of blots was also the highest when the animals were sacrificed in the evening of proestrus at 20h. The results obtained by in situ hybridization and cell immunoblot assay well correlate with each other. The above-mentioned results support our hypothesis that the enhanced expression and secretion of PACAP in the pituitary gland may be involved in ceasing the LH surge.

Secretin and autism: a basic morphological study about the distribution of secretin in the nervous system.

For the first time, the relationship between secretin and autism has been demonstrated by one of us. Intravenous administration of secretin in autistic children caused a fivefold higher pancreaticobiliary fluid secretion than in healthy ones and, at least in some of the patients, better mental functions were reported after the secretin test. Because the precise localization of secretin in the brain is still not completely known, the abovementioned observation led us to map secretin immunoreactivity in the nervous system of several mammalian species. In the present work, the distribution of secretin immunoreactivity in cat and human nervous systems was compared with that of rats using an immunohistochemical approach. Secretin immunoreactivity was observed in the following brain structures of both humans and in colchicine-treated rats: (1) Purkinje cells in the cerebellar cortex; (2) central cerebellar nuclei; (3) pyramidal cells in the motor cortex; and (4) primary sensory neurons. Additionally, secretin immnoreactive cells were observed in the human hippocampus and amygdala and in third-order sensory neurons of the rat auditory system. In cats, secretin was only observed in the spinal ganglia. Our findings support the view that secretin is not only a gastrointestinal peptide but that it is also a neuropeptide. Its presence or the lack of its presence may have a role in the development of behavioral disorders.

The role of PACAP in gonadotropic hormone secretion at hypothalamic and pituitary levels.

The presence of pituitary adenylate cyclase-activating polypeptide (PACAP) and its mRNAin the three levels of the hypothalamo-hypophyseal-ovarian axis was previously demonstrated using immunohistochemistry, in situ hybridization, and reverse transcriptase polymerase chain reaction (RT-PCR). In the hypothalamus, PACAP is present in neuroendocrine effector cells and in the median eminence. In the anterior pituitary and ovary, PACAP is transiently present during the proestrous stage of the estrous cycle. In the pituitary, PACAP was observed in gonadotropes. In the ovary, PACAP was demonstrated in the granulosa cells of the preovulatory ovarian follicles. The effect of PACAP on luteinizing hormone (LH) secretion was demonstrated in in vivo and in vitro models. In our work we have studied the role of PACAP in gonadotropic hormone secretion at hypothalamic and pituitary levels. At the hypothalamic level, PACAP, administered intracerebroventricularly to female rats before the critical period of the proestrus stage, can inhibit LH release and ovulation. Its inhibiting effect is mediated through corticotropin-releasing factor (CRF) and endogenous opioids. PACAP administered to neonatal female rats delayed the onset of puberty by influencing the luteinizing hormone-releasing hormone (LHRH) neuronal system. In the pituitary gland, the release of PACAP depended on the stage of the estrous cycle and on the time of day the animals were sacrificed. On the day of proestrus, the number of PACAP-releasing cells showed a diurnal change with two peaks (in the morning and in the evening). The peak was much higher in the evening at the end of the LH surge than in the morning.

Distribution of PACAP and its mRNA in several nonneural tissues of rats demonstrated by sandwich enzyme immunoassay and RT-PCR technique.

The presence of PACAP in various organs was previously demonstrated using immunohistochemistry and radioimmunoassay. The aim of our work was to get information whether the presence of immunoreactive PACAP in various organs, mainly in the gastric mucosa, also indicates the place of its synthesis. The immunoreactive PACAP and its mRNA were measured in parallel assays using sandwich enzyme immunoassay (S-EIA) and RT-PCR technique. PACAP and its mRNA were demonstrated in the pancreas, testes, adrenal glands, ovaries, and in the oxyntic mucosa of the stomach. These results support our previous observation that PACAP is present not only in the nervous system and endocrine glands, but might be synthetized in the oxyntic mucosa of the stomach as well.