Pituitary Adenylate Cyclase-activating Polypeptide Immunoreactivity Research Articles

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) of the Bed Nucleus of the Stria Terminalis Mediates Heavy Alcohol Drinking in Mice.

Alcohol use disorder (AUD) is a complex psychiatric disease characterized by periods of heavy drinking and periods of withdrawal. Chronic exposure to ethanol causes profound neuroadaptations in the extended amygdala, which cause allostatic changes promoting excessive drinking. The bed nucleus of the stria terminalis (BNST), a brain region involved in both excessive drinking and anxiety-like behavior, shows particularly high levels of pituitary adenylate cyclase-activating polypeptide (PACAP), a key mediator of the stress response. Recently, a role for PACAP in withdrawal-induced alcohol drinking and anxiety-like behavior in alcohol-dependent rats has been proposed; whether the PACAP system of the BNST is also recruited in other models of alcohol addiction and whether it is of local or nonlocal origin is currently unknown. Here, we show that PACAP immunoreactivity is increased selectively in the BNST of C57BL/6J mice exposed to a chronic, intermittent access to ethanol. While pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 receptor-expressing cells were unchanged by chronic alcohol, the levels of a peptide closely related to PACAP, the calcitonin gene-related neuropeptide, were found to also be increased in the BNST. Finally, using a retrograde chemogenetic approach in PACAP-ires-Cre mice, we found that the inhibition of PACAP neuronal afferents to the BNST reduced heavy ethanol drinking. Our data suggest that the PACAP system of the BNST is recruited by chronic, voluntary alcohol drinking in mice and that nonlocally originating PACAP projections to the BNST regulate heavy alcohol intake, indicating that this system may represent a promising target for novel AUD therapies.

Pituitary adenylate cyclase-activating polypeptide: Postnatal development in multiple brain stem respiratory-related nuclei in the rat

The pituitary adenylate cyclase-activating polypeptide (PACAP) plays an important role in anterior pituitary hormone secretion, neurotransmission, and the control of breathing. Mice lacking PACAP die suddenly mainly in the 2nd postnatal week, coinciding temporally with a critical period of respiratory development uncovered by our laboratory in the rat. The goal of the current study was to test our hypothesis that PACAP expression is reduced during the critical period in normal rats. We undertook immunohistochemistry and optical densitometry of PACAP (specifically PACAP38) in several brain stem respiratory-related nuclei of postnatal days P2-21 rats, and found that PACAP immunoreactivity was significantly reduced at P12 in the pre-Bötzinger complex, nucleus ambiguus, hypoglossal nucleus, and the ventrolateral subnucleus of the nucleus tractus solitarius. No changes were observed in the control, non-respiratory cuneate nucleus at P12. Results imply that the down-regulation of PACAP during normal postnatal development may contribute to the critical period of vulnerability, when the animals’ response to hypoxia is at its weakest.

Pituitary adenylate cyclase-activating polypeptide promotes eccrine gland sweat secretion.

Sweat secretion is the major function of eccrine sweat glands; when this process is disturbed (paridrosis), serious skin problems can arise. To elucidate the causes of paridrosis, an improved understanding of the regulation, mechanisms and factors underlying sweat production is required. Pituitary adenylate cyclase-activating polypeptide (PACAP) exhibits pleiotropic functions that are mediated via its receptors [PACAP-specific receptor (PAC1R), vasoactive intestinal peptide (VIP) receptor type 1 (VPAC1R) and VPAC2R]. Although some studies have suggested a role for PACAP in the skin and several exocrine glands, the effects of PACAP on the process of eccrine sweat secretion have not been examined. To investigate the effect of PACAP on eccrine sweat secretion. Reverse transcriptase-polymerase chain reaction and immunostaining were used to determine the expression and localization of PACAP and its receptors in mouse and human eccrine sweat glands. We injected PACAP subcutaneously into the footpads of mice and used the starch-iodine test to visualize sweat-secreting glands. Immunostaining showed PACAP and PAC1R expression by secretory cells from mouse and human sweat glands. PACAP immunoreactivity was also localized in nerve fibres around eccrine sweat glands. PACAP significantly promoted sweat secretion at the injection site, and this could be blocked by the PAC1R-antagonist PACAP6-38. VIP, an agonist of VPAC1R and VPAC2R, failed to induce sweat secretion. This is the first report demonstrating that PACAP may play a crucial role in sweat secretion via its action on PAC1R located in eccrine sweat glands. The mechanisms underlying the role of PACAP in sweat secretion may provide new therapeutic options to combat sweating disorders.

Expression of PACAP and PAC1 Receptor in Normal Human Thyroid Gland and in Thyroid Papillary Carcinoma.

Pituitary adenylate cyclase activating polypeptide (PACAP) belongs to the vasoactive intestinal peptide-secretin-glucagon peptide family, isolated first from ovine hypothalamus. The diverse physiological effects of PACAP are known mainly from animal experiments, including several actions in endocrine glands. Alteration of PACAP expression has been shown in several tumors, but changes in expression of PACAP and its specific PAC1 receptor in human thyroid gland pathologies have not yet been investigated. Therefore, the aim of the present study was to investigate expression of PACAP and its PAC1 receptor in human thyroid papillary carcinoma, the most common endocrine malignant tumor. PACAP and PAC1 receptor expressions were investigated from thyroid gland samples of patients with papillary carcinomas. The staining intensity of follicular epithelial cells and thyroid colloid of tumor tissue was compared to that of tumor-free tissue in the same thyroid glands in a semi-quantitative way. Our results reveal that both PACAP(-like) and PAC1 receptor(-like) immunoreactivities are altered in papillary carcinoma. Stronger PACAP immunoreactivity was observed in active follicles. Colloidal PACAP immunostaining was either lacking or very weak, and more tumorous cells displayed strong apical immunoreactivity. Regarding PAC1 receptor, cells of the normal thyroid tissue showed strong granular expression, which was lacking in the tumor cells. The cytoplasm of tumor cells displayed weak, minimal staining, while in a few tumor cells we observed strong PAC1 receptor expression. This pattern was similar to that observed in the PACAP expression, but fewer in number. In summary, we showed alteration of PACAP and PAC1 receptor expression in human thyroid papillary carcinoma, indicating that PACAP regulation is disturbed in tumorous tissue of the thyroid gland. The exact role of PACAP in thyroid tumor growth should be further explored.

PACAP suppresses dry eye signs by stimulating tear secretion.

Dry eye syndrome is caused by a reduction in the volume or quality of tears. Here, we show that pituitary adenylate cyclase-activating polypeptide (PACAP)-null mice develop dry eye-like symptoms such as corneal keratinization and tear reduction. PACAP immunoreactivity is co-localized with a neuronal marker, and PACAP receptor (PAC1-R) immunoreactivity is observed in mouse infraorbital lacrimal gland acinar cells. PACAP eye drops stimulate tear secretion and increase cAMP and phosphorylated (p)-protein kinase A levels in the infraorbital lacrimal glands that could be inhibited by pre-treatment with a PAC1-R antagonist or an adenylate cyclase inhibitor. Moreover, these eye drops suppress corneal keratinization in PACAP-null mice. PACAP eye drops increase aquaporin 5 (AQP5) levels in the membrane and pAQP5 levels in the infraorbital lacrimal glands. AQP5 siRNA treatment of the infraorbital lacrimal gland attenuates PACAP-induced tear secretion. Based on these results, PACAP might be clinically useful to treat dry eye disorder.

Co-expression of PACAP with VIP, SP and CGRP in the Porcine Nodose Ganglion Sensory Neurons

Our previous study revealed the expression of substance P (SP) and calcitonin gene-related peptide (CGRP) in sensory distal ganglion of the vagus (nodose ganglion) neurons in the pig. As these neuropeptides may be involved in nociception, the goal of these investigations was to determine possible expression of vasoactive intestinal polypeptide (VIP), SP and CGRP in the pituitary adenylate cyclase-activating polypeptide-immunoreactive (PACAP-IR) porcine nodose perikarya. Co-expression of these substances was examined using a double-labelling immunofluorescence technique. To reveal the ganglionic cell bodies, the pan-neuronal marker protein gene product 9.5 (PGP 9.5) was used. Quantitative analysis of the neurons revealed that 67.25% of the PGP 9.5+ somata in the right-side ganglion and 66.5% in the left side, respectively, co-expressed PACAP-IR. Moreover, 60.6% of the PACAP-IR cells in the right-side ganglion and 62.1% in the left, respectively, co-expressed VIP. SP-IR was observed in 52.2 and 39.9% of the right and left ganglia, respectively. CGRP was found in 27.7 and 34.1% of the right and left distal ganglion of the vagus, respectively. High level of co-expression of PACAP with VIP, SP and CGRP in the distal ganglia of the vagus sensory perikarya directly implicates studied peptides in their functional interaction during nociceptive vagal transduction.

Distribution of pituitary adenylate cyclase-activating polypeptide (PACAP) in the human testis and in testicular germ cell tumors

Pituitary adenylate cyclase-activating peptide (PACAP) is a neuropeptide expressed in the central nervous system and peripheral organs. Previous studies revealed the role and distribution of PACAP in the rodent testis, however, its presence in the human testis and in testicular germ cell tumors is not known. We used RT-PCR and immunohistological observations to investigate whether human testicular tissue and testicular germ cell tumors contain PACAP. The mRNAs for PACAP and its receptors were detected in total RNA extracted from human testes. PACAP immunoreactivity was observed in spermatogonia and spermatids from normal testes. In contrast, diffuse PACAP immunopositivity was observed in seminoma tumor cells, while only faint immunoreactivity was observed in embryonal carcinoma cells. Our data suggest that PACAP may play a role in human spermatogenesis and in testicular germ cell tumor development.

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.

Pituitary adenylatecyclase-activating polypeptide-immunoreactive nerve fibers in the rat epiglottis and pharynx

The distribution of pituitary adenylatecyclase-activating polypeptide-immunoreactive (PACAP-IR) nerve fibers was studied in the rat epiglottis and pharynx. PACAP-IR nerve fibers were located beneath the mucous epithelium, and occasionally penetrated the epithelium. These nerve fibers were abundant on the laryngeal side of the epiglottis and in the dorsal and lateral border region between naso-oral and laryngeal parts of the pharynx. PACAP-IR nerve fibers were also detected in taste buds within the epiglottis and pharynx. In addition, many PACAP-IR nerve fibers were found around acinar cells and blood vessels. The double immunofluorescence method demonstrated that distribution of PACAP-IR nerve fibers was similar to that in CGRP-IR nerve fibers in the epithelium and taste bud. However, distributions of PACAP-IR and CGRP-IR nerve fibers innervating mucous glands and blood vessels were different. The retrograde tracing method also demonstrated that PACAP and CGRP were co-expressed by vagal and glossopharyngeal sensory neurons innervating the pharynx. These findings suggest that PACAP-IR nerve fibers in the epithelium and taste bud of the epiglottis and pharynx which originate from the vagal and glossopharyngeal sensory ganglia include nociceptors and chemoreceptors. The origin of PACAP-IR nerve fibers which innervate mucous glands and blood vessels may be the autonomic ganglion.

Pituitary Adenylate Cyclase-Activating Polypeptide-Immunoreactive Cells in the Ageing Gerbil Hippocampus

In the present study, we investigated age-related changes in pituitary adenylate cyclase-activating polypeptide (PACAP) immunoreactivity and its protein levels in the gerbil hippocampus at various ages using immunohistochemistry and western blot analysis. In the post-natal month 1 (PM 1) group, PACAP-immunoreactive cells were found in all hippocampal subregions. The number of PACAP-immunoreactive cells was decreased in the PM 3 group and was still more decreased in the PM 6 and 12 groups. Thereafter, in the PM 18 and 24 groups, PACAP-immunoreactive cells were significantly increased again. However, in the mossy fibre zone, PACAP immunostaining was very strong in the adult group, especially in the PM 6 group. In addition, PACAP protein level was highest at PM 6, showing a slight decrease at PM 24. These results indicate that PACAP-immunoreactive cells are lowest in the adult stage and highest in the aged stage. However, PACAP immunoreactivity in the mossy fibre zone and PACAP protein level in the hippocampus are highest in the adult stage.

Pituitary adenylate cyclase-activating polypeptide (PACAP): A regulator of the innate and acquired immune functions in juvenile fish

To date, published in-vivo studies on the action of the PACAP in fish are few and these are concerned with reproduction, brain development and feeding behavior. Recently, we demonstrated for the first time that PACAP, apart from its neuroendocrine role, influences immune functions in fish larvae. In this work, we have evaluated the effects of recombinant Clarias gariepinus PACAP administration by intraperitoneal injection on important immune parameters in juvenile fish. We observed that a single injection of the recombinant peptide (0.1 μg per g of body weight) was able to increase the nitric oxide synthase-derived metabolites (NOS) and total immunoglobulin M (IgM) concentration in serum of juvenile catfish C. gariepinus and tilapia Orechromis niloticus respectively, after 24 h of its administration. In addition, our results showed that recombinant PACAP increases IgM, NOS and lysozyme in serum correlated with its ability to enhance growth performance in juvenile fish. Finally, the PACAP mRNA expression and PACAP immunoreactivity detected in peripheral blood leucocytes from juvenile catfish suggest a direct autocrine or/and paracrine mechanism of regulation of this peptide to mediate immune functions in fish.

VIP and PACAP Effects on Mouse Major Pelvic Ganglia Neurons

Major pelvic ganglia (MPG) neurons innervate urogenital organs and components of the lower bowel. Immunoreactivity for vasoactive intestinal polypeptide (VIP) has previously been observed in the MPG, and VIP knockout animals have impaired micturition reflexes suggesting a role for this neuropeptide in urogenital function. Here, we investigate the presence and action of VIP and a related neuropeptide, pituitary adenylate cyclase activating polypeptide (PACAP), in the pelvic ganglia of male mice. An abundance of VIP-immunoreactive (IR) neurons and nerve fibers were observed in the ganglion, whereas PACAP immunoreactivity was not seen. Extracts from acutely isolated MPG contained transcripts for the VPAC1, VPAC2, and PAC1 receptors. Local application of VIP, PACAP, or maxadilan to isolated pelvic ganglion neurons shortened the duration of the afterhyperpolarization (AHP) of action potentials elicited by brief intracellular depolarization. All three peptides also increased neuronal excitability within a subpopulation of the sampled neurons. Bath application of apamin, a peptide antagonist of SK channels, shortened the duration of the AHP indicating that AHP duration in pelvic neurons is determined principally by SK-channel activity. The results suggest that VIP has a role in the neural control of pelvic organ function and activation of VPAC and/or PAC1 receptors can modulate the activity of the autonomic neurons innervating pelvic organs.

PACAP Expression in Explant Cultured Mouse Major Pelvic Ganglia

The major pelvic ganglia (MPG) contain both parasympathetic and sympathetic postganglionic neurons and provide much of the autonomic innervation to urogenital organs and components of the lower bowel. Whereas many parasympathetic neurons were found to express vasoactive intestinal polypeptide (VIP), no MPG neurons exhibited immunoreactivity for pituitary adenylate cyclase-activating polypeptide (PACAP). However, in 3-day cultured MPGs, numerous PACAP-IR cells and nerve fibers were present, and transcript levels for PACAP increased significantly. In 3-day cultured MPGs, PACAP immunoreactivity was seen in cells that were also immunoreactive for VIP or neuronal nitric oxide synthase, but not tyrosine hydroxylase, indicating that PACAP expression occurred preferentially in MPG parasympathetic postganglionic neurons. Transcript levels for the VPAC2, but not VPAC1 or PAC1 receptor, also increased significantly following 3days in culture. Transcript levels of activating transcription factor 3 (ATF-3), a marker of cellular injury, were increased 64-fold in 3-day explants, and ATF-3-IR nuclei were evident in both TH-IR and nNOS-IR neurons as well as in non-neuronal cells. In sum, these results demonstrate that, although only the parasympathetic neurons in explant cultured MPGs increase expression of PACAP, both sympathetic and parasympathetic postganglionic neurons in the cultured MPG whole-mount increase expression of ATF-3.

Presence of pituitary adenylate cyclase-activating polypeptide (PACAP) defines a subpopulation of hypothalamic POMC neurons

Several lines of evidence support a role for pituitary adenylate cyclase-activating polypeptide (PACAP) in the regulation of energy balance. In the present study, we have used fluorescent in situ hybridization and immunohistochemistry to investigate in detail the cellular localization and chemical content of PACAP mRNA- and peptide-containing neuronal cell bodies in the mediobasal hypothalamus of the rat. PACAP mRNA-containing cell bodies were demonstrated in high numbers in the ventromedial hypothalamic nucleus (VMH) and in lower numbers in the arcuate nucleus (Arc). In colchicine-treated rats, PACAP immunoreactivity was demonstrated in many cell bodies of the VMH and several cell bodies of the ARC. Double-labeling revealed that PACAP immunoreactivity was present in approximately 20% of pro-opiomelanocortin (POMC) neurons in the ventrolateral Arc as shown by presence of α-melanocyte-stimulating hormone (α-MSH), but not in agouti-related peptide (AgRP)-containing neurons in the ventromedial aspect of the Arc. PACAP immunoreactivity was also colocalized with the vesicular acetylcholine transporter (VAChT; a marker for cholinergic neurons) in Arc POMC neurons. Brainstem POMC neurons in the commissural part of the solitary tract nucleus were devoid of PACAP immunoreactivity. However, several VAChT-positive neurons in the dorsal motor nucleus of the vagus nerve were also PACAP immunoreactive, whereas VAChT-positive neurons of the motor nucleus of the hypoglossal nerve were PACAP-negative. The results show presence of PACAP with α-MSH in a subpopulation of hypothalamic POMC neurons and point further to the neurochemical heterogeneity of hypothalamic, but not brainstem, POMC neurons.

Regulation of neuronal pituitary adenylate cyclase-activating polypeptide expression during culture of guinea-pig cardiac ganglia

The trophic neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) increases in many different neuron types following injury; a response postulated to support cell survival and regeneration. In acutely isolated cardiac ganglia, approximately 1% of the cardiac neurons exhibited PACAP immunoreactivity whereas after 72 h in culture, ∼25% of the neurons were PACAP immunoreactive. In contrast, there was no increase in vasoactive intestinal polypeptide (VIP)-immunoreactive (IR) cells. Using a combination of immunocytochemical and molecular techniques, we have quantified PACAP expression, during explant culture of guinea-pig cardiac ganglia. Using real time polymerase chain reaction, PACAP transcript levels increased progressively up to 48 h in culture with no further increase after 72 h. PACAP transcript levels were reduced by neurturin at 48 h in culture but not after 24 or 72 h in culture. In addition, neurturin partially suppressed the percentage of PACAP-IR neurons after 72 h in culture, an effect mediated by activation of the phosphatidylinositol 3-kinase and mitogen-activated protein kinase signaling pathways. The addition of different known regulatory molecules, including ciliary neurotrophic factor (CNTF), interleukin-1 beta (Il-1β), tumor necrosis factor-alpha (TNFα), fibroblast growth factor basic (bFGF), transforming growth factor-beta (TGF-β) and nerve growth factor (NGF) did not increase the percentage of PACAP-IR neurons after 24 h in culture; a result indicating that the generation and secretion of these factors did not stimulate PACAP expression. The presence of 20 nM PACAP or 10 μM forskolin increased the percentage of PACAP-IR cardiac neurons in 24 h cultures, but not in 72 h cultures. Neither treatment enhanced the number of VIP-IR neurons. The addition of the PACAP selective receptor (PAC1) receptor antagonist, M65 (100 nM) suppressed the 20 nM PACAP-induced increase in percentage of PACAP-IR cells in 24 h cultures indicating the effect of PACAP was mediated through the PAC1 receptor. However, 100 nM M65 had no effect on the percentage of PACAP-IR cells in either 24 or 48 h cultures not treated with exogenous PACAP, suggesting that endogenous release of PACAP likely did not contribute to the enhanced peptide expression. We postulate that the enhanced PACAP expression, which occurs in response to injury is facilitated in the explant cultured cardiac ganglia by the loss of a target-derived inhibitory factor, very likely neurturin. In intact tissues the presence of neurturin would normally suppress PACAP expression. Lastly, our results indicate that many common trophic factors do not enhance PACAP expression in the cultured cardiac neurons. However, the stimulatory role of an, as yet, unidentified factor cannot be excluded.

PACAP Enhances Mouse Urinary Bladder Contractility and Is Upregulated in Micturition Reflex Pathways after Cystitis

Pituitary adenylate cyclase-activating polypeptide (PACAP) elicits a transient contraction, sustained increase in the amplitude of spontaneous phasic contractions, and significantly increases the amplitude of nerve-mediated contractions in mouse urinary bladder smooth muscle (UBSM) strips. PACAP immunoreactivity (IR) is increased in micturition reflex pathways following cystitis. PACAP may contribute to altered sensation and bladder overactivity in the chronic bladder inflammatory syndrome, interstitial cystitis.

Expression of pituitary adenylate cyclase‐activating polypeptide in the primary lymphoid organs of the duck Anas platyrhynchos

The expression of pituitary adenylate cyclase-activating polypeptide (PACAP) was studied in the thymus and bursa of Fabricius of the duck Anas platyrhynchos, at different ages, using immunohistochemistry, Western blotting, RT-PCR and sequencing. In the thymus, PACAP immunoreactivity (-ir) was found in lymphoid cells. CD68/ and PGP 9.5/PACAP38 double labelling showed that PACAP was not expressed either in macrophages or in epithelial cells, suggesting that the PACAP-positive cells observed were lymphoid cells. Immunoreactive lymphocytes were observed in the interlobular septa. They increased in number with ageing. In the bursa, PACAP-ir was found in nerve fibres and in a few lymphoid cells. RT-PCR revealed PACAP mRNA expression in the thymus but not in the bursa. These results suggest that PACAP plays a role in the functions of the immune system in birds.

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) as a Growth Hormone (GH)-Releasing Factor in Grass Carp. I. Functional Coupling of Cyclic Adenosine 3′,5′-Monophosphate and Ca2+/Calmodulin-Dependent Signaling Pathways in PACAP-Induced GH Secretion and GH Gene Expression in Grass Carp Pituitary Cells

Pituitary adenylate cyclase-activating polypeptide (PACAP), a member of the glucagon/secretin peptide family, has been recently proposed to be the ancestral GH-releasing factor. Using grass carp as a model for bony fish, we examined the mechanisms for PACAP regulation of GH synthesis and secretion at the pituitary level. Nerve fibers with PACAP immunoreactivity were identified in the grass carp pituitary overlapping with the distribution of somatotrophs. At the somatotroph level, PACAP was shown to induce cAMP synthesis and Ca(2+) entry through voltage-sensitive Ca(2+) channels (VSCC). In carp pituitary cells, PACAP but not vasoactive intestinal polypeptide increased GH release, GH content, total GH production, and steady-state GH mRNA levels. PACAP also enhanced GH mRNA stability, GH promoter activity, and nuclear expression of GH primary transcripts. Increasing cAMP levels, induction of Ca(2+) entry, and activation of VSCC were all effective in elevating GH secretion and GH mRNA levels. PACAP-induced GH secretion and GH mRNA expression, however, were abolished by inhibiting adenylate cyclase and protein kinase A, removing extracellular Ca(2+) or VSCC blockade, or inactivating calmodulin (CaM)-dependent protein kinase II (CaM kinase II). Similar sensitivity to VSCC and CaM kinase II blockade was also observed by activating cAMP production as a trigger for GH release and GH gene expression. These results suggest that PACAP stimulates GH synthesis and secretion in grass carp pituitary cells through PAC(1) receptors. These stimulatory actions probably are mediated by the adenylate cyclase/cAMP/protein kinase A pathway coupled to Ca(2+) entry via VSCC and subsequent activation of CaM/CaM kinase II cascades.

Changes in pituitary adenylate cyclase activating polypeptide expression in urinary bladder pathways after spinal cord injury

These studies examined changes in the pituitary adenylate cyclase activating polypeptide (PACAP) expression in micturition reflex pathways after spinal cord injury (SCI) of various durations. In spinal-intact animals, PACAP immunoreactivity (IR) was expressed in fibers in the superficial dorsal horn in all segmental levels examined (L1, L2, L4–S1). Bladder-afferent cells (35–45%) in the dorsal root ganglia (DRG; L1, L2, L6, S1) from spinal-intact animals also exhibited PACAP-IR. After SCI (6 weeks), PACAP-IR was dramatically increased in spinal segments and DRG (L1, L2, L6, S1) involved in micturition reflexes. The density of PACAP-IR was increased in the superficial laminae (I–II) of the L1, L2, L6, and S1 spinal segments. No changes in PACAP-IR were observed in the L4–L5 segments. Staining was also dramatically increased in a fiber bundle extending ventrally from Lissauer's tract (LT) in lamina I along the lateral edge of the dorsal horn to the sacral parasympathetic nucleus (SPN) in the L6–S1 spinal segments (lateral collateral pathway of Lissauer, LCP). After SCI (range 48 h to 6 weeks), PACAP-IR in cells in the L1, L2, L6, and S1 DRG significantly ( P ≤ 0.001) increased and the percentage of bladder-afferent cells expressing PACAP-IR also significantly ( P ≤ 0.001) increased (70–92%). No changes were observed in the L4–L5 DRG. PACAP-IR was reduced throughout the urothelium and detrusor smooth muscle whole mounts after SCI. These studies demonstrate changes in PACAP expression in micturition reflex pathways after SCI that may contribute to urinary bladder dysfunction or reemergence of primitive voiding reflexes after SCI.