Pituitary Adenylate Cyclase-activating Polypeptide mRNA Expression Research Articles

Changes in expression of PACAP in rat sensory neurons in response to sciatic nerve compression.

In the present study, expression of pituitary adenylate cyclase-activating polypeptide (PACAP) in rat dorsal root ganglion (DRG) neurons and sciatic nerve following experimental sciatic nerve compression was studied with the use of quantitative immunohistochemistry and in situ hybridization. Previously, we have investigated changes in PACAP expression after nerve transection and, here, the far more frequently encountered condition of nerve compression injury is examined. Nerve compression was performed unilaterally on the rat sciatic nerve, at mid-thigh level, by application of a narrow silicone tube around the nerve for 3, 7, 14 or 28 days, respectively. We detect a statistically significant upregulation in the number and density of PACAP mRNA expression in both small and large DRG neurons in response to nerve compression. An increased number of PACAP-immunoreactive neurons is also found in the ipsilateral DRG. In addition, PACAP immunoreactivity is observed in the compressed sciatic nerve segment and adjacent nerve tissue after nerve compression. The present findings can be compared with previous studies where we have shown that PACAP expression is upregulated in DRG; in response to peripheral inflammation (primarily in small-medium neurons), and after axotomy (dramatic upregulation in medium-large neurons). In view of the recent findings of an increased PACAP expression in DRG after nerve compression, as well as the previous findings of a modulation of PACAP expression in response to axotomy and inflammation, it is likely that PACAP is also involved in the modulation of the response to peripheral nerve compression.

Expression of mRNAs for PACAP and its receptor in human neuroblastomas and their relationship to catecholamine synthesis

Purpose: Pituitary adenylate cyclase-activating polypeptide (PACAP), a member of the secretin/glucagon/vasoactive intestinal peptide family, induces the expression of catecholamine-synthesizing enzymes in adrenal medullary cells. In addition, PACAP and its receptor have been detected in human neuroblastoma tissues and cell lines, though it is not yet known whether PACAP enhances the expression of genes encoding catecholamine-synthesizing enzymes. To address this question, we analyzed PACAP, PACAP receptor and tyrosine hydroxylase (TH) mRNAs in neuroblastomas. Methods: The levels of mRNA for PACAP and vasoactive intestinal peptide (VIP), as well as their receptors and the mRNA for TH were measured by RT-PCR or real-time PCR analysis. Results: VPAC1R mRNA was detected in all of 16 tissues and 3 cell lines that were examined, while VPAC2R mRNA was detected in 5 of 16 (31%) tissue and 2 of 3 cell lines. PAC1R mRNA was detected in 6 out of 16 (38%) tissues and none of 3 cell lines. mRNA expression of PACAP and TH were detected in many tissues (10/16 and 16/16, respectively). However, neither in tissues nor cell lines did PACAP mRNA expression correlate with TH mRNA expression. Conclusion: Our findings suggest that PACAP is not involved in the regulation of expression of TH in neuroblastomas.

PACAP mRNA is expressed in rat spinal cord neurons

This study examines the expression of pituitary adenylate cyclase activating polypeptide (PACAP) mRNA in the rat spinal cord during normal conditions and in response to sciatic nerve transection. Previously, PACAP immunoreactivity has been found in fibers in the spinal cord dorsal horn and around the central canal and in neurons in the intermediolateral column (IML). Furthermore, in the dorsal root ganglia, PACAP immunoreactivity and PACAP mRNA expression have been observed preferentially in nerve cell bodies of smaller diameter terminating in the superficial laminae of the dorsal horn. However, neuronal expression of PACAP mRNA in adult rat spinal cord appeared limited to neurons of the IML. By using a refined in situ hybridization protocol, we now detect PACAP mRNA expression in neurons primarily in laminae I and II, but also in deeper laminae of the spinal cord dorsal horn and around the central canal. In addition, PACAP mRNA expression is observed in a few neurons in the ventral horn. PACAP expression in the ventral horn is increased in a population of large neurons, most likely motor neurons, both after distal and proximal sciatic nerve transection. The proposed role of PACAP in nociception is strengthened by our findings of PACAP mRNA-expressing neurons in the superficial laminae of the dorsal horn. Furthermore, increased expression of PACAP in ventral horn neurons, in response to nerve transection, suggests a role for PACAP in repair/regeneration of motor neurons.

Effect of anti-nerve growth factor treatment on pituitary adenylate cyclase activating polypeptide expression in adult sensory neurons exposed to adjuvant induced inflammation

Expression of pituitary adenylate cyclase activating polypeptide (PACAP) is increased in sensory neurons exposed to adjuvant induced peripheral inflammation. Local elevation in expression of the neurotrophin nerve growth factor (NGF) is a main factor contributing to the neuronal response to inflammation. This study examines the role of endogenous NGF in inflammation-associated increases in PACAP expression using the adjuvant-induced peripheral inflammation model with or without systemic administration of antibodies against NGF. Quantitative in situ hybridization was used to detect changes in neuronal PACAP mRNA expression and to correlate this expression with neuronal mRNA expression of the NGF receptor tyrosine kinase (trk) A. The results from this study show that inflammation triggered increases in PACAP expression occurs in small- to medium-sized dorsal root ganglion (DRG) neurons that also express trkA, and that this elevation in PACAP expression is prevented by systemic injection of anti-NGF. This supports a role for NGF as a positive regulator of PACAP expression during inflammation.

Differential expression of the pituitary gonadotropin subunit genes during male rat sexual maturation: reciprocal relationship between hypothalamic pituitary adenylate cyclase-activating polypeptide and follicle-stimulating hormone beta expression.

The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) has been shown to differentially regulate the expression of the gonadotropin subunit genes in cultures of rat pituitary cells. PACAP is expressed within the hypothalamus, and concentrations of PACAP are 2- to 4-fold higher in the portal circulation than in the general circulation. Therefore, PACAP is a candidate regulator of pituitary function. In the present study, we examined the expression of PACAP mRNA within the paraventricular nucleus (PVN) during maturation (Days 20-60) in the male rat and compared this expression to the levels of the gonadotropin subunits, follistatin, and GnRH-receptor mRNAs within the anterior pituitary. Serum concentrations of FSH and LH confirm the established maturational pattern of divergent secretion of LH and FSH. Northern analysis of the gonadotropin subunit mRNAs revealed that FSHbeta expression parallels FSH secretion whereas LHbeta mRNA concentrations do not change during development. Expression of the GnRH receptor in the pituitary parallels that of FSHbeta. In situ hybridization revealed a developmental pattern of PACAP mRNA within the PVN that is reciprocal to that of FSHbeta. Competitive reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of total pituitary follistatin mRNA revealed no significant changes; however, semiquantitative RT-PCR analyses revealed the presence of two follistatin mRNA species, one of which, corresponding to follistatin-288, was developmentally regulated. These studies identified a reciprocal relationship between PVN PACAP and FSHbeta gene expression in maturing rats. We propose that PACAP contributes to the selective regulation of FSHbeta expression during maturation in the male rat, perhaps via regulation of follistatin.

Expression of mRNA for PACAP and its receptors in intra- and extra-adrenal human pheochromocytomas and their relationship to catecholamine synthesis

Purpose: Pituitary adenylate cyclase-activating polypeptide (PACAP), a member of the secretin/glucagons/vasoactive intestinal peptide family, induces the expression of catecholamine-synthesizing enzymes in adrenal medullary cells. In addition, PACAP and its receptor have been detected in human pheochromocytoma tissues, though it is not yet known whether PACAP enhances the expression of genes encoding catecholamine-synthesizing enzymes. To address this question, we analyzed PACAP, PACAP receptor, and tyrosine hydroxylase (TH) and phenylethanolamine- N-methyltransferase (PNMT) mRNAs in pheochromocytomas. Methods: The levels of the mRNA for PACAP and vasoactive intestinal peptide (VIP), and their receptors, and for TH and PNMT were measured by RT-PCR or real-time PCR analysis, and the concentrations of catecholamines were measured by HPLC in 24 intra-adrenal and six extra-adrenal pheochromocytomas. Results: mRNA expression of PACAP and its receptor VPAC1R were detected in many pheochromocytomas (24/30 and 29/30, respectively), but mRNA expression of the PAC1R and VPAC2R receptor subtypes were detected in only one of six extra-adrenal pheochromocytomas. PACAP mRNA expression correlated with TH ( p=0.0018) and PNMT ( p=0.05) mRNA expression, as well as epinephrine ( p=0.0342) levels in 16 intra-adrenal pheochromocytomas. Conclusion: Our findings support a possible role for PACAP in the regulation of expression of genes encoding catecholamine-synthesizing enzymes in intra-adrenal pheochromocytomas.

Regulation of neurotrophic peptide expression in sympathetic neurons: quantitative analysis using radioimmunoassay and real-time quantitative polymerase chain reaction.

The regulated expression of the peptide and transcript levels of the neurotrophic peptides, pituitary adenylate cyclase-activating polypeptide (PACAP), galanin and vasoactive intestinal peptide (VIP) were examined in sympathetic neurons of the rat superior cervical ganglion (SCG). Real-time quantitative PCR methods were developed to assess modulation of neuronal peptide precursor protein transcript levels following experimental paradigms of neuropeptidergic plasticity. Oligonucleotide primer, fluorogenic probe and amplification conditions were optimized for maximal assay sensitivity. Depolarization of primary cultured sympathetic neurons stimulated PACAP, galanin, and VIP peptide contents and releases with differing magnitudes and temporal profiles. The rank order of increased neuronal peptide content paralleled the augmented peptide release (VIP>galanin>PACAP). Maximal cellular PACAP and VIP levels were achieved by 72 and 96 h, respectively; galanin levels did not plateau during the treatment period. PACAP transcript elevation was rapid and transient; PACAP mRNA expression diminished at longer depolarization times, which diverged markedly from the sustained high peptide production levels. By contrast, VIP and galanin mRNAs reached maximal levels at later times, and appeared to correlate more closely with peptide production. We previously described multiple proPACAP mRNA variants resulting from alternative 3' untranslated region cleavage and polyadenylation. The shorter depolarization-induced PACAP transcripts exhibit longer half-lives, suggesting that the short proPACAP mRNA variant may function to impart PACAP translational efficiency and sustain PACAP peptide production.

Involvement of intracellular Ca2+ elevation but not cyclic AMP in PACAP-induced p38 MAP kinase activation in PC12 cells.

We have recently shown that in PC12 cells, pituitary adenylate cyclase-activating polypeptide (PACAP) and NGF synergistically stimulate PACAP mRNA expression primarily via a mechanism involving a p38 mitogen-activated protein kinase (MAPK)-dependent pathway. Here we have analyzed p38 MAPK activation by PACAP and the mechanism underlying this action of PACAP in PC12 cells. PACAP increased phosphorylation of p38 MAPK with a bell-shaped dose-response relationship and a maximal effect was obtained at 10(-8) M. PACAP (10(-8) M)-induced p38 MAPK phosphorylation was already evident at 2.5 min, maximal at 5 min, and rapidly declined thereafter. PACAP-induced p38 MAPK phosphorylation was potently inhibited by depletion of Ca(2+) stores with thapsigargin and partially inhibited by the phospholipase C inhibitor U-73122, L-type voltage-dependent calcium channel inhibitors nifedipine and nimodipine, and the Ca(2+) chelator EGTA, whereas the protein kinase C inhibitor calphostin C, the protein kinase A inhibitor H-89, the cAMP antagonist Rp-cAMP, and the nonselective cation channel blocker SKF96365 had no effect. These results indicate that PACAP activates p38 MAPK in PC12 cells through activation of a phospholipase C, mobilization of intracellular Ca(2+) stores, and Ca(2+) influx through voltage-dependent Ca(2+) channels, but not cyclic AMP-dependent mechanisms.

Progesterone Receptor Activation Mediates LH-Induced Type-I Pituitary Adenylate Cyclase Activating Polypeptide Receptor (PAC1) Gene Expression in Rat Granulosa Cells

We have previously reported that the pituitary adenylate cyclase activating polypeptide (PACAP) gene is regulated in ovarian granulosa cells by the autocrine and/or paracrine interaction between progesterone and its nuclear receptor progesterone receptor (PR). To initiate studies on the functional significance of the progesterone-induced PACAP production in luteinizing granulosa cells, we sought to determine the expression and hormonal regulation of PACAP receptors in the rat ovary. The relative mRNA levels of three known PACAP receptor subtypes (PAC1, VPAC1, and VPAC2) were determined in ovaries of immature rats treated with gonadotropins, by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) assays. Results show that all PAC1, VPAC1, and VPAC2 transcripts are expressed at a detectable level in immature rat ovaries. Importantly, the ovarian level of PAC1, but not VPAC1 or VPAC2, mRNA notably changes during gonadotropin challenges. Ovarian PAC1 mRNA expression decreases during the pregnant mare's serum gonadotropin (PMSG)-induced follicular phase but substantially increases during the human chorionic gonadotropin (hCG)-induced periovulatory period. Because the hCG-induced increase in ovarian PAC1 mRNA expression is attributable to the hormone-induced PAC1 mRNA expression in granulosa cells of the preovulatory follicles, we next examined whether hCG regulates PAC1 mRNA expression by directly acting on granulosa cells. When granulosa cells isolated from PMSG (40 h)-primed immature rats were challenged with hCG (or forskolin), PAC1, but not VPAC1 or VPAC2, mRNA expression significantly increased within 6 h. Because the LH-induced PAC1 mRNA expression (6 h) proceeds PR activation (3 h) in granulosa cells as the LH-induced PACAP mRNA expression (6 h) does, we further determined the cause–effect relationship among LH, PR activation and PAC1 receptor gene expression, by examining the effect of PR antagonist, ZK98299, on the ability of LH to increase PAC1 mRNA levels in luteinizing granulosa cells. Results show that ZK98299 inhibited the stimulatory effect of hCG (or forskolin) on PAC1 mRNA expression, at the level of all known splice variants of PAC1 mRNA in granulosa cells. In summary, our results demonstrating that PR activation is critical for the LH-induced PAC1 gene expression in luteinizing granulosa cells suggest that PR activation regulates the finely tuned expression of the PACAP/PACAP receptor genes in luteinizing granulosa cells and thus dictates the timing of the autocrine and/or paracrine function of PACAP in preovulatory follicles.

Presence of pituitary adenylate cyclase-activating polypeptide (PACAP) and its relaxant activity in the rectum of a teleost, the stargazer, Uranoscopus japonicus

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide and a member of the secretin/glucagon superfamily of peptides that include vasoactive intestinal polypeptide. PACAP is not only present in the central nervous system but also in peripheral organs, such as the gastrointestinal tract, gonads and adrenal glands, and plays various roles in mammals. Recently, we isolated and characterized PACAP, which is very similar to PACAP of mammalian origin, from the brain of a teleost, the stargazer, Uranoscopus japonicus. In the present study, the expression of PACAP mRNA was detected in the stargazer rectum using the reverse transcriptase/polymerase chain reaction (RT-PCR) method. The distribution of PACAP-like immunoreactivity in the rectum was also examined immunohistochemically, using an antiserum raised against PACAP 27, and PACAP-like immunoreactive neuronal cell bodies and fibers were found in the myenteric plexuses and the smooth muscle layers of the rectum. The present study also investigated the relaxant activity of synthesized homologous PACAP on rectal contraction. Stargazer PACAP, like that of mammalian origin, inhibited contractions stimulated by acetylcholine or potassium chloride. PACAP-induced inhibition was not affected by preincubation with atropine, propranolol, or phentolamine. These results suggest that PACAP may act directly as an inhibitory neuropeptide in the stargazer rectum.

Synergistic induction of pituitary adenylate cyclase-activating polypeptide (PACAP) gene expression by nerve growth factor and PACAP in PC12 cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP) gene expression was analyzed in PC12 cells. PC12 cells transfected with a PACAP promoter-luciferase reporter construct were utilized to investigate the effects of PACAP, either alone or in combination with nerve growth factor (NGF), on PACAP transcriptional response. PACAP induced transcription from the PACAP promoter through PACAP type I receptor (PAC1 receptor). PACAP gene transcription was also induced by NGF. Simultaneous treatment with PACAP and NGF resulted in a synergistic transcriptional response that was more than three times the predicted response, based on a simple additive effect of both agents. This synergism in transcriptional response paralleled the PACAP mRNA levels, as determined by RT-PCR and northern blotting. The level of PACAP mRNA peaked 3 h after stimulation and gradually returned to basal levels by 48 h. PC12 cells are known to express predominantly the hop isoform of the PAC1 receptor, which positively couples to both adenylate cyclase and phospholipase C. To determine the role of the cyclic AMP and protein kinase C pathways in PACAP gene expression, the effects of forskolin and phorbol 12-myristate 13-acetate (PMA) were then examined. PMA did not alter PACAP mRNA levels but enhanced forskolin-induced PACAP mRNA expression. Down-regulation of protein kinase C blocked the ability of PACAP to stimulate PACAP mRNA expression. The mitogen-activated protein kinase extracellular signal-regulated kinase (ERK) kinase 1/2 (MEK1/2) inhibitor PD98059 also blocked the PACAP mRNA expression induced by either PACAP or NGF but not that induced by a combination of PACAP and NGF. These results suggest that PACAP stimulates the PACAP gene expression in PC12 cells at least in part through activation of adenylate cyclase and protein kinase C signaling pathways and that the ERK1/2 cascade is involved in PACAP and NGF-induced PACAP gene expression, although redundant signaling pathways may also be involved. The present finding showing that PACAP in combination with NGF causes a synergistic increase in PACAP gene expression in PC12 cells supports the idea that PACAP acts as an autocrine regulatory factor.

Role of Progesterone Receptor Activation in Pituitary Adenylate Cyclase Activating Polypeptide Gene Expression in Rat Ovary1

It is well known that the pituitary gonadotropin surge induces progesterone receptor (PR) gene expression in luteinizing granulosa cells and that PR activation is critical for successful ovulation. To further understand the molecular mechanism(s) by which PR plays a role critical for granulosa cell functions, we wanted to identify progesterone-induced genes in granulosa cells. We employed a PCR-based subtraction cloning strategy to screen for genes expressed differentially in granulosa cells that were challenged with forskolin in the presence of progesterone or ZK98299. One such differentially expressed clone was identified as the pituitary adenylate cyclase activating polypeptide (PACAP). To begin to understand the relationship between PR activation and PACAP gene expression in luteinizing granulosa cells, we examined whether PR and PACAP messenger RNA (mRNA) expression is temporally correlated. In cultured granulosa cells, both human CG and forskolin induced PR and PACAP mRNA levels in a dose-dependent manner, as determined by semi-quantitative RT-PCR assays. However, the peak expression for PR and PACAP mRNAs was observed at 3 h and 6 h after hormone treatment, respectively. This time difference in cAMP-responsive expression of the PR and PACAP genes is due, at least in part, to the requirement of ongoing protein synthesis for PACAP expression, as demonstrated by the inhibitory effect of cycloheximide on cAMP-induced PACAP, but not PR, mRNA levels. To determine whether PR synthesis is prerequisite for PACAP expression, we examined the effect of ZK98299, a specific PR antagonist, on cAMP-induced PACAP mRNA expression. This compound blocked cAMP-induced PACAP mRNA expression in a dose-dependent manner, indicating that PR activation is required for PACAP gene expression in granulosa cells. We then compared cellular localization and hormonal regulation of ovarian PR and PACAP gene expression in immature rats treated with gonadotropins as well as in adult rats during the preovulatory period by using in situ hybridization and semiquantitative RT-PCR assays. Results show that both PR and PACAP mRNAs are induced in granulosa cells of preovulatory follicles by human CG, but that the PR gene is expressed before the PACAP gene. Taken together, these results demonstrate that PRs mediate the LH-induced PACAP gene expression in rat granulosa cells.

Pituitary adenylate cyclase activating polypepetide is expressed by developing rat Purkinje cells and decreases the number of cerebellar γ-amino butyric acid positive neurons in culture

The neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) is expressed in various parts of the developing and adult rat brain, including the cerebellum. In situ hybridization was employed to localize the precise site of mRNA expression for PACAP and PACAP receptor I (PRI). During prenatal cerebellar development, PACAP mRNA was present in developing Purkinje cells and some deep cerebellar nuclei, whilst PRI mRNA was expressed by adjacent cells in the Purkinje cell layer (PCL). There was a shift in PRI mRNA expression to the external germinal cell layer around birth. PACAP decreased the number of neurons positive for the inhibitory neurotransmitter γ-amino butyric acid (GABA) in cultures from embryonic cerebellum, but did not affect overall cell survival. In conclusion, our results show the pattern of PACAP mRNA expression in embryonic cerebellum and suggest a physiological role for PACAP on GABAergic cerebellar neurons.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is an islet substance serving as an intra-islet amplifier of glucose-induced insulin secretion in rats.

1. We examined whether pituitary adenylate cyclase-activating polypeptide with 38 or 27 residues (PACAP-38 or PACAP-27) serves as an intra-islet regulator of glucose-induced insulin secretion in rats. PACAP antiserum specific for PACAP-38 and PACAP-27 was used to neutralize the effect of endogenous PACAP in islets. PACAP release from islets was bioassayed using the response of cytosolic Ca2+ concentration ([Ca2+]i) in single beta-cells, monitored by dual-wavelength fura-2 microfluorometry. Expression of PACAP mRNA was studied by reverse transcription-polymerase chain reaction (RT-PCR), while expression of PACAP was studied by metabolic labelling and immunoblotting. Localization of PACAP receptors was studied immunohistochemically. 2. High glucose-stimulated insulin release from isolated islets was attenuated by PACAP antiserum but not by non-immune sera. 3. The islet incubation medium with high glucose (Med) possessed a capacity, which was neutralized by PACAP antiserum, to increase [Ca2+]i in beta-cells. PACAP antiserum also neutralized the [Ca2+]i-increasing action of synthetic PACAP-38 and PACAP-27, but not that of vasoactive intestinal polypeptide (VIP) and glucagon. 4. Both Med and synthetic PACAP increased [Ca2+]i in beta-cells only in the presence of stimulatory, but not basal, glucose concentrations. In contrast, ATP, a substance that is known to be released from beta-cells, increased [Ca2+]i in beta-cells at both and stimulatory glucose concentrations. 5. Expression of PACAP mRNA and biosynthesis of PACAP-38 were detected in islets and a beta-cell line, MIN6. 6. Immunoreactivity for PACAP-selective type-I receptor was observed in islets. 7. [Ca2+]i measurements combined with immunocytochemistry with insulin antiserum revealed a substantial population of glucose-unresponsive beta-cells, many of which were recruited by PACAP-38 into [Ca2+]i responses. 8. These results indicate that PACAP-38 is a novel islet substance that is synthesized and released by islet cells and then, in an autocrine and/or paracrine manner, potentiates and arouses beta-cell responses to glucose, thereby amplifying glucose-induced insulin secretion in islets.

Expression of pituitary adenylate cyclase-activating polypeptide (PACAP) receptors and PACAP in human fetal retina.

Specific receptors for pituitary adenylate cyclase-activating polypeptide (PACAP), a novel peptide with neuroregulatory and neurotrophic functions, have recently been identified in the retinas of different mammalian species. In the present study, expression of PACAP receptors and PACAP was investigated in the retinas of 12-18-week human embryos. Radioligand binding studies showed that the two forms of PACAP with 38 and 27 amino acids (PACAP 38 and PACAP 27, respectively) displaced the binding of 125I-PACAP 27 with IC50 values in the picomolar range, whereas functional receptor assays demonstrated that the two peptides were potent and effective stimulators of adenylyl cyclase activity. In contrast, vasoactive intestinal peptide (VIP) and human peptide histidine-isoleucine, which are homologous to PACAP, displayed lower affinities for the 125I-PACAP 27 binding site and were much less potent stimulators of cyclic AMP formation. Glucagon and secretin were inactive in both receptor assays. The expression of specific PACAP receptors was further investigated by reverse transcription-polymerase chain reaction technique, which showed the presence of mRNAs coding for PACAP type I and for nonselective PACAP type II (both VIP1 and VIP2) receptors. By the same technique, expression of PACAP mRNA was also detected. These data indicate that the developing human retina synthesizes PACAP and that the peptide may act on retinal cells by predominantly stimulating PACAP type I receptors coupled to cyclic AMP formation.

Expression of pituitary adenylate cyclase-activating polypeptide (PACAP) in the mesencephalic trigeminal nucleus of the rat after transsection of the masseteric nerve.

Pituitary adenylate cyclase-activating polypeptide (PACAP), a member of the VIP (vasoactive intestinal polypeptide) family of peptides, has been demonstrated in neurons of the sensory system. PACAP expression of these neurons is sensitive to nerve damages such as nerve crush or axotomy. In the present study, PACAP expression in the mesencephalic trigeminal nucleus of the rat was examined after transsection of the main trunk of the masseteric nerve. The primary sensory neurons of the nucleus are considered to have purely proprioceptive functions. By quantitative in situ hybridization using a PACAP [35S]cRNA probe, an increase in PACAP mRNA was observed on the side ipsilateral to transsection already after 3 h and the expression reached a peak 24 h after surgery after which the levels gradually decreased during the next 14 days. A low and constant expression of PACAP mRNA could be seen on the side contralateral to transsection. PACAP immunoreactivity was demonstrated on the ipsilateral side after 18 h, using a specific monoclonal PACAP antibody. Co-existence of PACAP with NPY and galanin was demonstrated 7 days after transsection. Analysis of the masseteric nerve by radioimmunoassay on transsected and normal nerve stumps revealed an increase of PACAP-38 immunoreactivity in the nerve proximal to the transsection compared to the normal side (15.3 vs. 6.1 pmol/g wt). The results suggest that PACAP has a role in the early phase of adaptation to nerve injury in the proprioceptive neurons.

The expression of pituitary adenylate cyclase-activating polypeptide (PACAP) mRNA in rat brain: possible role of endogenous PACAP in vasopressin release

We investigated the expression of pituitary adenylate cyclase-activating polypeptide (PACAP) mRNA in rat brain by in situ hybridization. PACAP mRNA was prominently expressed in arcuate nucleus (ARC). Three days of water deprivation significantly increased plasma arginine vasopressin and markedly potentiated the expression of PACAP mRNA in ARC. These results suggest that PACAP in ARC may play some physiological role, possibly one of which may be the control of vasopressin release.

Stage-specific expression of pituitary adenylate cyclase-activating polypeptide (PACAP) mRNA in the rat seminiferous tubules.

We have used in situ hybridization and Northern blot analysis with oligonucleotide probe to characterize the site of pituitary adenylate cyclase-activating polypeptide (PACAP) synthesis in the rat testis. We observed strong hybridization signal in one third of the cross-sections of the seminiferous tubules, whereas some tubules were devoid of hybridization signal, thus suggesting that PACAP mRNA is expressed in a stage-specific manner. More detailed analysis showed that PACAP mRNA was present in round spermatids at stages III-VII of the cycle. Northern blot hybridization to RNAs extracted from samples of seminiferous tubules at different stages of the epithelial cycle confirmed that expression of PACAP mRNA is restricted to specific stages of the cycle. The highest amount of PACAP mRNA was detected at stages V to early -VII of the cycle, whereas very low levels of mRNA were present at stages I-II and IX-XIV. The present results demonstrate that PACAP mRNA is expressed in the developing germ cells. This suggests that PACAP may function as a paracrine or autocrine regulatory factor for the Sertoli and germ cells, with a specific function during early spermiogenesis, shortly before the onset of nuclear elongation, at the last period of haploid gene activity.