Vasoactive Intestinal Polypeptide mRNA Research Articles

Chronic constipation in Parkinson's disease: clinical features and molecular insights on the intestinal epithelial barrier.

Chronic constipation (CC) is a severe symptom in Parkinson's disease (PD), with an unclear pathogenesis. Abnormalities of the enteric nervous system (ENS) and/or intestinal epithelial barrier (IEB) may be pathophysiologically relevant in PD patients with CC. We investigated possible molecular changes of the IEB in PD/CCs compared with CCs and controls. Twelve PD/CCs (2 female, age range 51-80 years), 20 CCs (15 female, age range 27-78 years), and 23 controls (11 female, age range 32-74 years) were enrolled. Ten PD/CCs and 10 CCs were functionally characterized by anorectal manometry (AM) and transit time (TT). Colon biopsies were obtained and assessed for gene and protein expression, and localization of IEB tight junction markers claudin-4 (CLDN4), occludin-1 (OCCL-1), and zonula occludens-1 (ZO-1) by RT-qPCR, immunoblot and immunofluorescence labeling. PD/CCs were clustered in 2 functional categories: patients with delayed TT and altered AM (60%), and a second group showing only modifications in AM pattern (40%). Gene expression of CLDN4, OCCL-1 and ZO-1 was higher in PD/CCs than controls (P<0.05). Conversely, PD/CCs showed a trend to decrease (P>0.05) in CLDN4 and OCCL-1 protein levels than controls, whereas ZO-1 protein was comparable. In PD/CCs compared with controls, decreasing tendency of vasoactive intestinal polypeptide mRNA, protein and immunoreactive fiber density were observed, although the difference was not statistically significant. Transit and anorectal dysfunctions in PD/CCs are associated with difference in ZO-1, OCCL-1 and CLDN4 expression, thus supporting the role of an altered IEB as a contributory mechanism to possible neuronal abnormalities.

Downregulation of neuronal vasoactive intestinal polypeptide in Parkinson's disease and chronic constipation.

Chronic constipation (CC) is a common and severe gastrointestinal complaint in Parkinson's disease (PD), but its pathogenesis remains poorly understood. This study evaluated functionally distinct submucosal neurons in relation to colonic motility and anorectal function in PD patients with constipation (PD/CC) vs both CC and controls. Twenty-nine PD/CC and 10 Rome III-defined CC patients were enrolled. Twenty asymptomatic age-sex matched subjects served as controls. Colonic transit time measurement and conventional anorectal manometry were evaluated in PD/CC and CC patients. Colonoscopy was performed in all three groups. Colonic submucosal whole mounts from PD/CC, CC, and controls were processed for immunohistochemistry with antibodies for vasoactive intestinal polypeptide (VIP) and peripheral choline acetyltransferase, markers for functionally distinct submucosal neurons. The mRNA expression of VIP and its receptors were also assessed. Four subgroups of PD/CC patients were identified: delayed colonic transit plus altered anorectal manometry (65%); delayed colonic transit (13%); altered manometric pattern (13%); and no transit and manometric impairment (9%). There were no differences in the number of neurons/ganglion between PD/CC vs CC or vs controls. A reduced number of submucosal neurons containing VIP immunoreactivity was found in PD/CC vs controls (P<.05). VIP, VIPR1, and VIPR2 mRNA expression was significantly reduced in PD/CC vs CC and controls (P<.05). Colonic motor and rectal sensory functions are impaired in most PD/CC patients. These abnormalities are associated with a decreased VIP expression in submucosal neurons. Both sensory-motor abnormalities and neurally mediated motor and secretory mechanisms are likely to contribute to PD/CC pathophysiology.

Nesting behavior is associated with VIP expression and VIP–Fos colocalization in a network-wide manner

Many species, including humans, engage in a series of behaviors that are preparatory to the arrival of offspring. Such “nesting behaviors” are of obvious importance, but relevant neuroendocrine mechanisms remain little studied. We here focus on the potential roles of vasoactive intestinal polypeptide (VIP) in the performance of appetitive and consummatory nesting behaviors in male and female zebra finches (Taeniopygia guttata). Using combined immunocytochemistry for Fos and in situ hybridization for VIP, we now show that many VIP cell groups show increased transcriptional activity in response to nest building in male and female zebra finches. Particularly strong data come from the preoptic area (medial preoptic area and medial preoptic nucleus), where VIP–Fos co-expression correlates positively with three different measures of nesting behavior, as does the number of VIP-expressing cells. Remarkably, we find that VIP mRNA and/or VIP–Fos co-expression is correlated with nesting behavior in virtually every brain area that we examined, including the medial amygdala (anterior and posterior), medial bed nucleus of the stria terminalis, medial preoptic area, medial preoptic nucleus, anterior hypothalamus, ventromedial hypothalamus, periaqueductal gray complex (central gray and nucleus intercollicularis), and ventral tegmental area. Near-significant effects are also obtained in the tuberoinfundibular hypothalamus. Although most correlations are positive, negative correlations are observed for the VIP cell group of the anterior hypothalamus, a population that selectively promotes aggression, and also the periaqueductal gray complex. These data demonstrate a network-wide relationship between peptide production and social behavior that is, to our knowledge, unparalleled by other peptidergic modulators.

Effects of neonatal alcohol exposure on vasoactive intestinal polypeptide neurons in the rat suprachiasmatic nucleus

Neonatal alcohol exposure produces long-term changes in the suprachiasmatic nucleus (SCN) that are presumably responsible for disturbances in the light–dark regulation of circadian behavior in adult rats, including the pattern of photoentrainment, rate of re-entrainment to shifted light–dark cycles, and phase-shifting responses to light. Because SCN neurons containing vasoactive intestinal polypeptide (VIP) receive direct photic input via the retinohypothalamic tract and thus play an important role in the circadian regulation of the SCN clock mechanism by light, the present study examined the long-term effects of neonatal alcohol exposure on VIP neuronal populations within the SCN of adult rats. Male Sprague-Dawley rat pups were exposed to alcohol (EtOH; 3.0, 4.5, or 6.0 g/kg/day) or isocaloric milk formula (gastrostomy control; GC) on postnatal days 4–9 using artificial-rearing methods. At 2–3 months of age, animals from the suckle control (SC), GC, and EtOH groups were exposed to constant darkness (DD) and SCN tissue was harvested for subsequent analysis of either VIP mRNA expression by quantitative polymerase chain reaction (PCR) and in situ hybridization or of VIP-immunoreactive (ir) neurons using stereological methods. Neonatal alcohol exposure had no impact on VIP mRNA expression but dramatically altered immunostaining of neurons containing this peptide within the SCN of adult rats. The relative abundance of VIP mRNA and anatomical distribution of neurons expressing this transcript were similar among all control- and EtOH-treated groups. However, the total number and density of VIP-ir neurons within the SCN were significantly decreased by about 35% in rats exposed to alcohol at a dose of 6.0 g/kg/day relative to that observed in both control groups. These results demonstrate that VIP neuronal populations in the SCN are vulnerable to EtOH-induced insult during brain development. The observed alterations in SCN neurons containing VIP may have an impact upon clock responses to light input and thus contribute to the long-term effects of neonatal alcohol exposure on the photic regulation of circadian behavior.

PACAP-cytokine interactions govern adrenal neuropeptide biosynthesis after systemic administration of LPS

We have examined induction of neuropeptide expression in adrenal medulla after treatment of mice with lipopolysaccharide (LPS), a model for septic shock, which activates both immune and stress responses in vivo. Messenger RNAs encoding vasoactive intestinal polypeptide (VIP) and galanin, both modulators of steroidogenesis in neighboring adrenal cortex, are up-regulated at 24 h (eight-fold for VIP and two-fold for galanin) after LPS injection, and remain elevated for the following 24 h. Up-regulation of VIP and galanin by LPS is abrogated in pituitary adenylate cyclase-activating polypeptide (PACAP)-deficient mice, suggesting an interaction between LPS, or LPS-induced cytokines, and PACAP released in adrenal medulla from the splanchnic nerve. Treatment of cultured chromaffin cells with 100 nM PACAP and 10 nM tumor necrosis factor-alpha (TNF-α), a cytokine whose production is elevated by LPS, results in long-term synergistic up-regulation of VIP and galanin mRNA. PACAP blocks the earlier induction by TNF-α of mRNA encoding inhibitor of NF-κB alpha (IκBα), normally a negative autoregulator of TNF-α signaling through nuclear factor-κB (NF-κB), without affecting the induction of TNF-α-induced protein 3 (TNFAIP3), another NF-κB-dependent gene induced by TNF-α in chromaffin cells. By acting downstream of NF-κB to inhibit IκBα gene induction by TNF-α, PACAP may block IκBα-dependent negative autoregulation of TNF-α signaling through NF-κB, prolonging TNF-α-dependent signaling to neuropeptide-encoding genes in chromaffin cells. This mechanism may also underlie PACAP-dependent neuropeptide gene induction by LPS in vivo.

Differentiation of PC12 Cells Results in Enhanced VIP Expression and Prolonged Rhythmic Expression of Clock Genes

To examine for circadian rhythmicity, the messenger RNA (mRNA) amount of the clock genes Per1 and Per2 was measured in undifferentiated and nerve-growth-factor-differentiated PC12 cells harvested every fourth hour. Serum shock was needed to induce circadian oscillations, which in undifferentiated PC12 cultures lasted only one 24-h period, while in differentiated cultures, the rhythms continued for at least 3 days. Thus, neuronal differentiation provided PC12 cells the ability to maintain rhythmicity for an extended period. Both vasoactive intestinal polypeptide (VIP) and its receptor VPAC2 are expressed in the suprachiasmatic nucleus (SCN), and in agreement with VIP signaling being crucial for maintenance of rhythmicity, we found both VIP and VPAC2 mRNA increased after differentiation of PC12 cells. Pituitary adenylate cyclase activating polypeptide (PACAP) exerts time- and concentration-dependent effects on Per gene expression in the SCN. We added 1 nM and 1 microM PACAP to oscillating PC12 cells at times corresponding to midday and early and late night to evaluate whether the effects were similar as in SCN. Induction of Per1 mRNA was found at all three times, which differs from results in SCN. Thus, PC12 cells seem more useful for studying mechanisms behind acquirement of rhythmicity of cell cultures than for resetting of circadian rhythm.

Expression localisation and functional activity of pituitary adenylate cyclase-activating polypeptide, vasoactive intestinal polypeptide and their receptors in mouse ovary

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) positively affect several parameters correlated with the ovulatory process. PACAP is transiently expressed in rat preovulatory follicles, while VIP is present in nerve fibres at all stages of development. These two peptides act by interacting with three types of receptors: PACAP type I receptor (PAC1-R), which binds with higher affinity to PACAP, and two VIP receptors (VPAC1-R and VPAC2-R), which bind to PACAP and VIP with equal affinity. The aim of the present study was to characterise the PACAP/VIP/receptor system in the mouse ovary. Results obtained by RT-PCR, immunohistochemistry and in situ hybridisation showed that PACAP was transiently expressed in granulosa cells of preovulatory follicles after human chorionic gonadotrophin (hCG) stimulation, while VIP mRNA was never observed. All the receptors were present in 22-day-old untreated mice. In preovulatory follicles, PAC1-R was expressed both in granulosa cells and in residual ovarian tissue but was stimulated by hCG mainly in granulosa cells; VPAC2-R was present in both the cell compartments and was only mildly stimulated; VPAC1-R was present mainly in the residual ovarian tissue and was downregulated by hCG. PACAP and VIP were equipotent in inhibiting apoptosis in granulosa cells, confirming the presence of functional PACAP/VIP receptors. The contemporary induction by hCG of PACAP and PAC1-R in granulosa cells of preovulatory follicles suggests that, also in mouse ovary, PACAP may play a significant role around the time of ovulation. Moreover, the presence of PACAP/VIP receptors in the untreated ovary suggests a possible role for PACAP and VIP during follicle development.

Decreased VIP and VPAC2 receptor expression in the biological clock of the R6/2 Huntington's disease mouse

Huntington's disease (HD) is a fatal genetic neurodegenerative disorder caused by a CAG triplet repeat expansion in the gene encoding the protein huntingtin. The most studied model of HD, the R6/2 transgenic mouse, replicates many features of the disease. In addition to motor, cognitive, and endocrine dysfunctions, these mice exhibit a progressive disruption of circadian rhythms. This is accompanied by an altered expression of the circadian clock genes in the suprachiasmatic nucleus/nuclei (SCN), the principal circadian pacemaker in the brain. The neuropeptide vasoactive intestinal polypeptide (VIP) and its receptor VPAC2 are highly expressed in the SCN, and VIPergic signaling plays an essential role in maintenance of ongoing circadian rhythmicity. We found a marked reduction in both VIP mRNA and VPAC2 receptor mRNA, quantified by RT-PCR, as well as a decrease in VIP immunostaining in the SCN of R6/2 mice. These changes were coupled to a disruption of circadian rhythm. We observed no loss of neurons in the SCN and therefore suggest that the changes in VIP and VPAC2 receptor are due to their decreased expression. In conclusion, we propose that impaired VIPergic signaling is an additional candidate mechanism for disruption of circadian rhythms in R6/2 mice.

The tissue specifier element (TSE) functions as a Ca2+ response element for Ca2+ and cAMP synergistic signaling to the human vasoactive intestinal polypeptide (VIP) gene

Vasoactive intestinal polypeptide (VIP) is a pleiotropic neurotransmitter with roles in development, circadian function, immune regulation, and pain sensation. VIP gene expression is synergistically activated by elevation of cAMP and Ca2+ (Hamelink et al., J. Neurosci. 22:, 2002). The effects of these two signaling pathways on VIP gene regulation were examined in NBFL human neuroblastoma cells, which endogenously express VIP. Treatment with forskolin or the Ca2+ionophore A23187, alone modestly (10-fold) increased VIP mRNA, but together caused a synergistic (75-fold) increase in NBFL cells. Although stimulation of neuroendocrine genes by both cAMP and Ca2+ via convergence on a common cis-active element, the cyclic AMP-response element (CRE) is common, we found, unexpectedly, that the CRE of a full-length VIP luciferase reporter gene, while essential for cAMP-stimulated transcription and synergistic stimulation with cAMP and Ca2+, was dispensable for activation by Ca2+ alone. Instead, Ca2+ activation required a single element of 280 bases located 4 kb upstream from the VIP proximal promoter that was previously identified as a tissue specifier element (TSE; Hahm et al., JBC 273:,1998). The TSE of the VIP gene is a novel calcium response element in a neuron-specific gene that is independent of the CRE, but acts together with it to enable synergistic cAMP and Ca2+signaling.

Short‐term constant light potentiation of large‐magnitude circadian phase shifts induced by 8‐OH‐DPAT: effects on serotonin receptors and gene expression in the hamster suprachiasmatic nucleus

Nonphotic phase-shifting of mammalian circadian rhythms is thought to be mediated in part by serotonin (5-HT) acting in the suprachiasmatic nucleus (SCN) circadian clock. Previously we showed that brief (1-3 days) exposure to constant light (LL) greatly potentiates nonphotic phase-shifting induced by the 5-HT agonist, (+/-)2-dipropyl-amino-8-hydroxyl-1,2,3,4-tetrahydronapthalene (8-OH-DPAT). Here we investigated potential mechanisms for this action of LL, including 5-HT receptor upregulation and SCN clock gene and neuropeptide gene expression. Autoradiographic analysis of ritanserin inhibition of [3H]8-OH-DPAT binding indicated that LL (approximately 2 days) did not affect 5-HT7 receptor binding in the SCN or dorsal raphe. Measurement of 5-HT1A autoreceptors in the median raphe and 5-HT1B receptors in the SCN also showed no effect of LL. In experiment 2, hamsters held under a 14-h light : 10-h dark photocycle (LD) or exposed to LL for approximately 2 days received an intraperitoneal injection of 8-OH-DPAT or vehicle at zeitgeber time (ZT) 6 or 0 and were killed after 2 h of dark exposure. 8-OH-DPAT suppressed SCN Per1 and Per2 mRNAs at both ZTs, as assessed by in situ hybridization. Per1 mRNA was also suppressed by LL alone. In addition, in situ hybridization of arginine vasopressin (AVP) mRNA and vasoactive intestinal polypeptide mRNA showed that LL significantly suppressed the former but not the latter. The LL-induced suppression of SCN Per1 mRNA and AVP mRNA may be involved in LL-induced potentiation of pacemaker resetting, especially as these data provide additional evidence that LL suppresses circadian pacemaker amplitude, thus rendering the clock more susceptible to phase-shifting stimuli.

Calcium signal-mediated expression of the vasoactive intestinal polypeptide gene and its small contribution to activity-dependent survival of mouse cerebellar granule cells.

We have demonstrated previously in primary cultures of mouse cerebellar granule cells (CGCs) that endogenously synthesized pituitary adenylate cyclase-activating polypeptide (PACAP) contributes at least in part to the activity-dependent survival of CGCs (Tabuchi et al. [2001] Neurosci. Res. 39:85-93). In this study, we have demonstrated that expression of vasoactive intestinal polypeptide (VIP), a member of the same VIP/secretin/glucagon family as PACAP, was activated markedly by Ca(2+) influx through L-type voltage-dependent Ca(2+) channels (L-VDCCs), which could be induced under the depolarizing condition induced by high concentration of potassium (K(+)) in the medium. The activation of VIP mRNA expression, different from that of PACAP, was dependent partly on de novo protein synthesis. On the other hand, mRNA expression of secretin and PACAP/VIP receptors (PAC(1), VPAC(1), and VPAC(2)) was not activated by the Ca(2+) influx; rather, PAC(1) mRNA expression was reduced. Exogenously added VIP prevented apoptosis of CGCs under nondepolarizing conditions, the effect of which was mediated specifically through the VPAC(1) receptor. Furthermore, the survival of CGCs under depolarizing conditions could be mediated partly through VPAC(1), the contribution of which was much less than that of PAC(1). These findings indicate that PACAP and VIP genes are coordinately activated by the Ca(2+) signals in CGCs, but the contribution of VIP to the activity-dependent survival of CGCs is quite small.

Daily and circadian expression of neuropeptides in the suprachiasmatic nuclei of nocturnal and diurnal rodents

The suprachiasmatic nuclei (SCN) of the hypothalamus are necessary for coordination of major aspects of circadian rhythmicity in mammals. Although the molecular clock mechanism of the SCN has been a field of intense research during the last decade, the role of the neuropeptides in the SCN, including arginine–vasopressin (AVP), vasoactive intestinal polypeptide (VIP) and gastrin-releasing peptide (GRP), in the clock itself or in circadian organization is still largely unknown. Previous studies mainly performed in the rat have examined the profiles of AVP, VIP and GRP mRNA and peptide levels and suggested that the AVP rhythm is controlled by the circadian clock, whereas those of VIP and GRP are directly dependent on lighting conditions. Here, both daily (i.e., under light–dark cycle [LD]) and circadian (i.e., in constant darkness [DD]) profiles of neuropeptide mRNA were investigated in the SCN of the nocturnal mouse Mus musculus and the diurnal rodent Arvicanthis ansorgei to gain insight into a possible role in circadian organization. Our data show that AVP mRNA exhibits a clear circadian rhythm in the SCN peaking by the end of the subjective day in both species. Contrary to what has been observed in rats, oscillations of VIP and GRP mRNA in the SCN are found to be clock-controlled in mice and A. ansorgei, but with different phases for peak expression. While both VIP and GRP mRNA peak during the middle of the subjective night (i.e., with a 6-h lag compared to AVP mRNA) in mice, they peak almost in phase with AVP mRNA in A. ansorgei. Contrary to what has been reported in the rat, mean levels of VIP and GRP peptide mRNA levels tended to be increased by light in the mice. The different circadian organization of SCN neuropeptides mRNA profiles in both light/dark and constant darkness conditions between mice and A. ansorgei could be related with diurnality.

Autocrine/paracrine action of pituitary vasoactive intestinal peptide on lactotroph hyperplasia induced by estrogen.

Vasoactive intestinal polypeptide (VIP) content is increased in the hyperplastic pituitaries of estrogen (E)-treated rats, thus suggesting that this neuropeptide could mediate the E effect on lactotrophs. E also decreases pituitary TGF-beta1 content, an autocrine/paracrine inhibitor of lactotroph proliferation, and induces pituitary angiogenesis. To elucidate the role of VIP in this context, lactotroph hyperplasia was induced in female Fisher 344 rats by implanting sc pellets of diethylstilbestrol (DES). Twenty-five days later, the rats were treated with three different increasing doses of a VIP receptor antagonist or the vehicle for 5 d. DES treatment resulted in a marked increase of serum prolactin (PRL), pituitary PRL content, PRL mRNA expression, pituitary weight, and pituitary proliferating cell nuclear antigen. DES treatment also increased pituitary VIP content and VIP mRNA levels, but not in the hypothalamus and cerebral cortex. Simultaneously, DES treatment decreased the pituitary TGF-beta1 content and increased the pituitary content of vascular endothelial growth factor. VIP receptor antagonist partially reverted the effect of DES on serum PRL and pituitary PRL, proliferating cell nuclear antigen, TGF-beta1, and vascular endothelial growth factor contents, as well as on pituitary weight, in a dose-dependent relation. These data suggest that pituitary VIP mediates the effect of E on lactotroph hyperplasia, pituitary TGF-beta1, and angiogenesis.

Increased expression of vasoactive intestinal polypeptide in cultured myenteric neurons from adult rat small intestine

Adult neurons possess the ability to adapt to a changing environment. Loss of target-derived neurotrophic factors due to axotomy or isolation by culturing is known to induce changes in neuropeptide expression in several types of peripheral neurons. The aim of the present study was to investigate changes in the expression of vasoactive intestinal polypeptide (VIP) and nitric oxide synthase (NOS) in cultured myenteric ganglia and dissociated neurons. Myenteric ganglia and neurons from rat small intestine were dissociated and cultured for up to 21 days. Immunocytochemistry was used to determine the total number of neurons and the proportions of subpopulations containing VIP or NOS or both in preparations of whole mounts (controls used to determine the conditions in vivo), myenteric ganglion culture and dissociated myenteric neuronal culture. In situ hybridization was used to determine changes in the expressions of NOS and VIP mRNA. The relative number of VIP-expressing neurons increased significantly during culturing. The percentage of all neurons expressing VIP was 3.6±0.3% in whole mounts, 22–24% in cultured myenteric ganglia, and up to 35% in cultured dissociated neurons. NOS-expressing neurons constituted approximately 30–40% of all neurons in whole mounts as well as in cultured ganglia or dissociated neurons. A dramatic increase in NOS/VIP-containing neurons were detected in cultured neurons irrespective of whether they were arranged in ganglia or dissociated, as compared to whole mount preparations. This suggests that the NOS-containing neurons are the ones that increase their VIP expression. The induced expression of VIP in cultured adult myenteric neurons indicates that VIP is important for neuronal adaptation, maintenance and survival.

Coincident Elevation of cAMP and Calcium Influx by PACAP-27 Synergistically Regulates Vasoactive Intestinal Polypeptide Gene Transcription through a Novel PKA-Independent Signaling Pathway

Pituitary adenylate cyclase-activating polypeptide (PACAP) causes calcium influx, intracellular calcium release, and elevation of cAMP in chromaffin cells. Calcium influx is required for PACAP-stimulated secretion of catecholamines and neuropeptides. The role of cAMP elevation in the action of PACAP at either sympathetic or adrenomedullary synapses, however, is unknown. Here, we show that PACAP-27-induced calcium influx through voltage-sensitive calcium channels (VSCCs), together with elevation of intracellular cAMP, was sufficient to stimulate vasoactive intestinal polypeptide (VIP) biosynthesis at least 40-fold. Combined treatment of chromaffin cells with 40 mm KCl, which elevates intracellular calcium, and 25 micrometer forskolin, which elevates intracellular cAMP, caused an increase in VIP peptide and mRNA much greater than that elicited by either agent alone, and comparable to the increase caused by 10-100 nm PACAP-27. Elevation of VIP mRNA by either KCl plus forskolin, or PACAP, (1) was independent of new protein synthesis, (2) was blocked by inhibition of calcium influx through voltage-sensitive calcium channels, (3) was calcineurin dependent, and (4) was dependent on MAP kinase activation but not activation of protein kinase A. The degree of activation of two different second-messenger pathways, calcium influx and cAMP elevation, appears to determine the magnitude of transcriptional activation of the VIP gene in chromaffin cells. Maximal stimulation of VIP biosynthesis by PACAP appears to require the coincident activation of both of these pathways.

Differential expression of vasoactive intestinal polypeptide receptor 1 and 2 mRNA in murine intestinal T lymphocyte subtypes.

Neuropeptides may exert a variety of effects on the immune cells at both systemic and mucosal immune sites. The immunoregulatory properties refer to the ability of physiological signals and pathways to influence various immune functions. Vasoactive intestinal polypeptide (VIP), a neuropeptide present in high concentration in gut, was studied for its production and receptor expression in intraepithelial and lamina propria T lymphocytes of mouse intestine. Using reverse transcription-polymerase chain reaction (RT-PCR) analysis, it was demonstrated that VIP receptor 1 (VIPR1) was constantly expressed in intraepithelial and lamina propria T lymphocytes from both small and large intestine. In contrast, VIPR2 was identified only in T cells from small intestine. Further studies on purified subpopulations of T lymphocytes indicated the existence of VIPR2 in CD8(+) T cells, but not CD4(+) and CD4CD8 double negative T cells, although all these three subpopulations displayed VIPR1. In addition, VIPR1 mRNA was detected in splenic T lymphocytes, but no signal was obtained for VIPR2 mRNA, even after stimulation of the cells with anti-CD3(epsilon)-chain mAb, phorbol 12-myristate 13-acetate (PMA) and/or VIP. The presence of VIP receptor(s) on intestinal T lymphocytes was supported by the detection of VIP on the cell surface using dual colour immunoflowcytometry. In-vitro treatment with VIP resulted in a tendency towards an increased size of the VIP immunoreactive T cell population and significantly enhanced the average immunofluorescence intensity of the surface labelling. This indicates that the receptors are partially occupied by locally produced VIP in vivo and that more peptide molecules can be bound on the lymphocytes when needed, released and accumulated in higher concentration at the action sites. We failed to detect the expression of VIP mRNA in T lymphocytes, from either intestine or spleen. These observations support that VIP may be an important immune modulator in gut acting through specific receptors on T lymphocytes. The differential mRNA expression of VIP receptor subtypes in cells with different phenotypes and in different immune compartments may suggest diverse regulatory roles of the neuropeptide in immune responses.

Nerve growth factor restores mRNA levels and the expression of neuropeptides in the suprachiasmatic nucleus of rats submitted to chronic ethanol treatment and withdrawal.

Chronic ethanol treatment and withdrawal from alcohol decrease the synthesis and expression of neuropeptides in the hypothalamic suprachiasmatic nucleus. Given the existing evidence that neurotrophins modulate the synthesis and expression of neurotransmitters/neuromodulators in the mature brain, we have hypothesized that such alterations might result from the reduced biological activity or brain content of neurotrophic factors. To test this possibility, nerve growth factor (NGF) was delivered intraventricularly, over a 4-week period, to rats submitted to ethanol treatment for 6 months and to withdrawn rats. Vasopressin (AVP) and vasoactive intestinal polypeptide (VIP), and the respective mRNAs were detected by immunocytochemistry and in situ hybridization histochemistry, and their levels estimated using stereological methods and densitometry. In ethanol-treated and withdrawn rats, NGF produced increases in the number of AVP- and VIP-immunostained neurons to values identical to those of controls. Corresponding variations were detected in AVP and VIP mRNA levels, which indicates that NGF restored the expression of AVP and VIP by enhancing neuropeptide synthesis. These findings show that NGF can correct the changes induced by chronic ethanol treatment and withdrawal in the gene expression and protein content of the neuropeptides synthesized by suprachiasmatic neurons. They also reveal that NGF plays an important role in the maintenance of the neurochemical phenotype of the suprachiasmatic nucleus in the adult rat. Because suprachiasmatic neurons do not express trkA, NGF might have exerted its effects either through direct signalling of suprachiasmatic neurons via p75(NTR) activation or, indirectly, by enhancing the activity of the cholinergic and/or glutamatergic afferents to the suprachiasmatic nucleus, or both.

Effects of androgen and ageing on gene expression of vasoactive intestinal polypeptide in rat corpus cavernosum.

To clarify the effects of androgens (testosterone and dihydrotestosterone, DHT) and ageing on gene expression of vasoactive intestinal polypeptide (VIP), assessed as the expression of VIP mRNA, in rat corpus cavernosum. Materials and methods The study comprised 160 male Sprague Dawley rats divided into group A (56 rats, 5 weeks old), group B (50 rats, 10 weeks old) and group C (54 rats, 58 weeks old). Groups A-C were subdivided, respectively, into subgroups 1 (intact controls), 2 (castrated), 3 (castrated but given testosterone undecanoate 25 mg/kg per month by intramuscular injection), 4 (castrated with testosterone undecanoate 50 mg/kg per month) and 5 (treated with finasteride 4.5 mg/kg per day, orally). At 4 and 10 weeks after these treatments half the rats were killed. Serum samples were taken for the measurement of total and free testosterone and DHT, using a radioimmunoassay. Penile samples (corpus cavernosum) were frozen in liquid nitrogen and stored at -80 degrees C. VIP mRNA was estimated using a semi-quantitative reverse-transcription polymerase chain reaction. There was no significant difference in VIP mRNA in rat corpus cavernosum between intact control and castrated subgroups, or subgroups treated with finasteride, in groups A-C, including both the 4- and 10-week old animals (P > 0.05). Penile VIP mRNA was unchanged at any dose of testosterone in the castrated subgroups in all groups (P > 0.05). There was no significant relationship between penile VIP mRNA and ageing (P > 0.05). The gene expression of VIP in rat corpus cavernosum is independent of androgens (testosterone and DHT) and ageing. Androgens probably induce penile erection by pathways other than VIP; ageing may have little effect on penile VIP mRNA.

Pituitary adelylate cyclase-activating peptide is an activator of vasoactive intestinal polypeptide gene transcription in human neuroblastoma cells

In many ganglia, the neuropeptide pituitary adenylate cyclase-activating peptide (PACAP) innervates nerve cell bodies containing the homologous neuropeptide vasoactive intestinal polypeptide (VIP). We therefore investigated whether PACAP affected the VIP gene expression and elucidated the molecular mechanisms using the human neuroblastoma cell line NB-1. A concentration dependent induction of the VIP mRNA level was found upon PACAP stimulation. Five nM PACAP mediated transient elevation of the VIP mRNA being evident after 2 h, the maximal 65-fold induction was reached after 6–8 h and hereafter the level decreased rapidly. In cell extracts, the concentration of immunoreactive VIP was elevated four-fold upon PACAP stimulation for 8 h, and it remained elevated during the next 40 h. In conditioned medium, a stable 20-fold VIP increase was seen after 8–24 h. Experiments with the translational inhibitor cycloheximide showed a direct effect of PACAP on the VIP mRNA level, and nuclear run-on assays revealed a three- to four-fold enhancement of the VIP gene transcription rate after PACAP stimulation. The VIP mRNA induction was abolished by transcriptional inhibition with the actinomycin D, and PACAP did not seem to mediate any changes in the VIP mRNA half-life. However, the VIP mRNA level seemed very stable during the transcriptional cessation. Reporter gene constructs were used to evaluate involvement of the VIP CRE site in the PACAP mediated induction of the VIP gene transcription. Mutation of the CRE site did not abolish the induction suggesting it to be of minor if any importance for the induction. In conclusion, the PACAP mediated induction of the VIP gene expression suggests that PACAP released from nerve terminals could influence the function of VIP’ergic neurons in target tissues.

Temporal profiles of vasoactive intestinal polypeptide precursor mRNA and its receptor mRNA in the rat suprachiasmatic nucleus

Many vasoactive intestinal polypeptide (VIP) neurons are distributed in the suprachiasmatic nucleus (SCN) and most of them arborize throughout the nuclei. We determined temporal patterns of VIP receptor (VPAC2 receptor) mRNA in the rat SCN in constant darkness and light–dark cycles, by means of in situ hybridization histochemistry. Amounts of VIP precursor mRNA in the SCN were also quantified in adjacent sections to compare its temporal profile with that of VPAC2 receptor mRNA. In constant darkness no circadian rhythms in VIP precursor mRNA or VPAC2 receptor mRNA in the SCN were detected. In light–dark cycles VIP precursor mRNA and VPAC2 receptor mRNA in the SCN showed robust but different daily rhythms. VPAC2 receptor mRNA decreased over the course of the light period and then gradually recovered during the dark period. VIP precursor mRNA was also reduced in response to light but the reduction was transient. This difference in the nature of light responsiveness between VIP mRNA and VPAC2 receptor mRNA suggests that pre- and postsynaptic events of the VIP transduction system represent different stages of light information processing in the SCN.