Effects Of PACAP Research Articles

Modulation of nociceptive transmission by pituitary adenylate cyclase activating polypeptide in the spinal cord of the mouse.

Superficial layers of the dorsal horn receive a dense plexus of nerve fibers immunoreactive to pituitary adenylate cyclase activating polypeptide (PACAP). In vivo experiments were conducted in the mice to evaluate the effects of PACAP-38, herein referred to as PACAP, PACAP receptor antagonist PACAP(6-38) and PACAP-antiserum on nociceptive behaviors induced by radiant heat, intrathecally administered N-methyl-D-aspartate (NMDA) or intraplantarly administered formalin. PACAP (0.05-0.5 microg) dose-dependently decreased the paw-withdrawal latencies induced by thermal stimulation and enhanced the aversive licking and biting behaviors induced by intrathecally injected NMDA. Pretreatment with the PACAP receptor antagonist PACAP(6-38) (0.5-2 microg) or PACAP-antiserum (1:500-2,000 dilution) dose-dependently attenuated the second phase, but not the first phase, of nociceptive responses to formalin. Next, the effects of PACAP on NMDA- and kainate-induced currents evoked in single dorsal horn neurons were studied. Whole-cell patch recordings were made from superficial dorsal horn neurons of spinal cord slices from 14- to 20-day-old mice. PACAP at the concentrations of 100 and 200 nM, which caused no significant change of holding currents, increased NMDA-but not kainate-induced currents in superficial dorsal horn neurons. Our results suggest that exogenously applied PACAP sensitizes the dorsal horn neurons to formalin stimulation, and facilitates NMDA receptor-mediated nociceptive response. As a corollary, PACAP, which may be released from primary afferent fibers potentiates nociceptive transmission to the dorsal horn by interacting primarily with NMDA receptors.

Plasticity of pheochromocytoma cell lines from neurofibromatosis knockout mice.

Adrenergic mouse pheochromocytoma (MPC) cells from heterozygous neurofibromatosis knockout mice show little or no expression of the NGF receptor trk A and do not undergo neuronal differentiation in response to NGF. However, they express high levels of receptor tyrosine kinase, Ret, and GDNF family receptor alpha(1) (GFRalpha(1)) in vivo and in vitro and respond to glial cell line-derived neurotrophic factor (GDNF). In addition, they form short processes in response to PACAP or cyclic AMP. Morphological effects of GDNF, PACAP, or cyclic AMP are similar to those of NGF, PACAP, or cyclic AMP on PC12 cells, and all three agents cause downregulation of PNMT mRNA. The MAP kinase kinase inhibitor U0126 inhibits both baseline proliferation and stimulated process outgrowth, consistent with a model in which sustained low-level ERK activation drives proliferation, and more intense activation drives neuronal differentiation. The sensitivity of MPC cells to U0126 both may reflect mechanisms that cause pheochromocytomas in neurofibromatosis and aid in their clarification.

Possible role of a neuropeptide PACAP (pituitary adenylate cyclase-activating polypeptide) on stimulus-secretion coupling in catecholamine neuron

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide first isolated from ovine hypothalamic tissue. This peptide stimulates adenylate cyclase activation. However, few details were known of the function of this peptide on stimulus-secretion coupling in neuronal cells. The authors have investigated the role of PACAP on catecholamine biosynthesis and secretion using cultured bovine adrenal chromaffin cells as a model for catecholamine-containing neurons. PACAP38, the 38-amino acid form of PACAP, increased cAMP formation in bovine adrenal chromaffin cells. In addition, PACAP38 increased [Ca2+]i associated with PI turnover and Ca2+ influx into the cells. The synthesis of catecholamine and the phosphorylation of tyrosine hydroxylase, a rate-limiting enzyme of catecholamine biosynthesis, stimulated by the maximal effective concentration of dibutyryl cAMP or a high concentration (56 mM) of K+ were further enhanced by PACAP38. Thus PACAP38 stimulated the pathway of catecholamine biosynthesis mainly by both activation of cAMP- and Ca2(+)-dependent protein kinases. On catecholamine secretion from the cells, the effect of PACAP38 was markedly potentiated by addition of ouabain, an inhibitor of Na+/K+ ATPase. This markedly potentiated secretion was greatly reduced with Na+ omitted-sucrose medium. PACAP38 increased 22Na+ influx into the cells treated with ouabain. Thus PACAP38 with ouabain stimulated catecholamine secretion by accumulation of intracellular Na+, resulting in an increase in Ca2+ influx. These results indicate that the neuropeptide PACAP has an important role in stimulus-secretion coupling in adrenal chromaffin cells.

Pituitary Adenylyl Cyclase-activating Polypeptide Prevents Induced Cell Death in Retinal Tissue through Activation of Cyclic AMP-dependent Protein Kinase

Multiple neuroactive substances are secreted by neurons and/or glial cells and modulate the sensitivity to cell death. In the developing retina, it has been shown that increased intracellular levels of cAMP protect cells from degeneration. We tested the hypothesis that the neuroactive peptide pituitary adenylyl cyclase-activating polypeptide (PACAP) has neuroprotective effects upon the developing rat retina. PACAP38 prevented anisomycin-induced cell death in the neuroblastic layer (NBL) of retinal explants, and complete inhibition of induced cell death was obtained with 1 nm. A similar protective effect was observed with PACAP27 and with the specific PAC1 receptor agonist maxadilan but not with glucagon. Photoreceptor cell death induced by thapsigargin was also prevented by PACAP38. The neuroprotective effect of PACAP38 upon the NBL could be reverted by the competitive PACAP receptor antagonist PACAP6-38 and by the specific PAC1 receptor antagonist Maxd.4. Molecular and immunohistochemical analysis demonstrated PAC1 receptors, and treatment with PACAP38 induced phospho-cAMP-response element-binding protein immunoreactivity in the anisomycin-sensitive undifferentiated postmitotic cells within the NBL. PACAP38 produced an increase in cAMP but not inositol triphosphate, and treatment with the cAMP-dependent protein kinase inhibitor R(p)-cAMPS blocked the protective effect of PACAP38. The results indicate that activation of PAC1 receptors by PACAP38 modulates cell death in the developing retina through the intracellular cAMP/cAMP-dependent protein kinase pathway.

Transcriptional and post-transcriptional regulation of Tyrosine Hydroxylase messenger RNA in PC12 cells during persistent stimulation by VIP and PACAP38: differential regulation by protein kinase A and protein kinase C -dependent pathways

VIP and PACAP38 are closely related peptides that are released in the adrenal gland and sympathetic ganglia and regulate catecholamine synthesis and release. We used PC12 cells as a model system to examine receptor and second messenger pathways by which each peptide stimulates transcriptional and post-transcriptional mechanisms that regulate the level of the mRNA for tyrosine hydroxylase (TH), the rate-limiting enzymatic step in catecholamine synthesis. Concentration–response studies revealed that PACAP38 had both greater efficacy and potency than VIP. The specific PAC1 receptor antagonist PACAP[6–38] blocked the effects of each peptide on THmRNA content while the PACAP/VIP type II receptor antagonist (N-AC-Tyr1-D-Phe2)-GRF-(1–29)-NH2 was without effect. At equipotent concentrations, each peptide stimulated a transient increase in TH gene transcription lasting less than 3h. Continuous VIP treatment stimulated a transient increase in TH mRNA lasting less than 24h. In contrast, continuous exposure to PACAP38 stimulated a stable increase in TH mRNA that persisted for 2 days in the absenceofelevated transcription, pointing to different post-transcriptional effects of the two peptides. PACAP38 alone had no effect on the magnitude of TH gene transcription or TH mRNA in A126-1B2 PKA-deficient PC12 cells. However, when combined with dexamethasone, PACAP38 produced a synergistic increase in TH mRNA in the absence of PACAP38-stimulated TH gene transcription. In contrast, VIP had no effect on either TH mRNA content or TH gene transcription in this model. PACAP38, but not VIP, stimulated PKC activity. Calphostin C antagonized the effect of PACAP38 on the persistent post-transcriptional elevation in TH mRNA. Thus, the results support the conclusion thatVIP and PACAP38 each stimulate PAC1 receptors to increase TH gene transcription through a PKA-controlled pathway, but their divergent post-transcriptional effects result at least partly from differing abilities to stimulate PKC.

Pituitary adenylate cyclase-activating polypeptide and vasoactive intestinal peptide-stimulated cyclic AMP synthesis in rat cerebral cortical slices: interaction with noradrenaline, adrenaline, and forskolin.

Pituitary adenylate cyclase-activating polypeptide (PACAP; 0.001-1 microM) and vasoactive intestinal peptide (VIP; 0.01-1 microM) produced a concentration-dependent stimulation of cyclic AMP (cAMP) formation in rat cerebral cortical slices prelabeled with [3H]adenine. The effects of PACAP38 and PACAP27 were similar, and more efficacious (at 0.1 and 1 microM) than those of VIP. Adrenaline and noradrenaline (each at 100 microM) also stimulated cAMP formation, with the latter compound being more effective. Combination of PACAP38, PACAP27 (each at 0.1 microM) and VIP (1 microM) with adrenaline or noradrenaline resulted in most cases in additive effects, with some supraadditive (PACAP27 plus adrenaline) or subadditive (PACAP38 or VIP plus noradrenaline) fluctuations. In contrast, combination of each of the three peptides with 3 microM forskolin resulted in synergistic effects. These results indicate that in rat cerebral cortex there is no synergism between PACAP or VIP with noradrenaline or adrenaline; however, based on the forskolin data, it seems likely that synergistic effects may take place with VIP or PACAP and other cAMP-stimulating neuroregulators.

VIP and PACAP inhibit L-, N- and P/Q-type Ca2+ channels of parasympathetic neurons in a voltage independent manner.

In this study, we investigated the effects of vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide 1-38 (PACAP) on the voltage-gated calcium currents in hamster submandibular ganglion neurons. VIP and PACAP inhibited the high threshold voltage-gated calcium current in a voltage-independent and a concentration-dependent manner via the G protein-mediated pathway. L-, N- and P/Q-type components of the total maximum voltage-gated calcium current accounted for 48.0 +/- 3.1% (n = 4), 35.1 +/- 4.7% (n = 4), and 13.5 +/- 2.3% (n = 3) of the total peak amplitude, respectively. VIP at a concentration of 1 microM inhibited the L-type calcium current by 33.2% +/- 1.4% (n = 4), the N-type current by 31.0 +/- 3.6%, and the P/Q-type current by 3.2 +/- 1.1% (n = 3). PACAP at a concentration of 1 microM inhibited the L-type current by 35.6 +/- 5.7%, the N-type current by 34.4 +/- 3.1% (n = 4), and the P/Q-type current by 6.4 +/- 2.1% (n = 2). However, VIP and PACAP did not inhibit the low threshold voltage-gated (T-type) calcium current. The rank order of potency was PACAP > VIP. In experiments replacing GTP with GDP-beta-S, the inhibitory effects of VIP and PACAP were prevented. In experiments of double-pulse protocol, depolarizing conditioning pulses could not relieve the inhibition of total high threshold voltage-gated calcium currents produced by VIP and PACAP. Therefore, the inhibition of the high threshold voltage-gated calcium channels produced by VIP and PACAP in hamster parasympathetic neurons differed in its mechanisms from that of N-type calcium channels in rat sympathetic neurons.

Pituitary adenylate cyclase-activating peptide inhibits neutrophil chemotaxis

Pituitary adenylate cyclase-activating peptide 38 (PACAP 38) is a neuropeptide that displays several biological effects of interest in the context of airway diseases such as asthma and chronic obstructive pulmonary disease. These effects include inhibition of airway and vascular smooth muscle tone as well as modulation of inflammatory cell activity. However, little is known about the effect of PACAP on granulocytes. The present study was designed to investigate if PACAP and the closely related peptide vasoactive intestinal peptide (VIP) could affect neutrophil migration. A standard 48 well chemotaxis chamber was used to assess the effects of PACAP on N-Formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-induced neutrophil chemotaxis and spontaneous random migration. PACAP 38 and VIP inhibited fMLP-induced human neutrophil chemotaxis. Furthermore, both peptides also exhibited a dose-related trend toward inhibiting the spontaneous, unstimulated migration of neutrophils. Since enhanced cell migration in cell chamber systems is reported to correlate with increased invasive properties in vivo, the presented inhibitory effects of PACAP 38 on neutrophil chemotaxis, supports the idea of an anti-inflammatory role for PACAP. This together with the well documented bronchodilatory capacity of PACAP might indicate a role for PACAP-agonists in future treatment of asthma and other inflammatory airway diseases.

Study on the hypothalamic factors mediating the inhibitory effect of PACAP38 on ovulation

1. IntroductionThe pituitary adenylate cyclase activating polypeptide(PACAP) was first isolated from ovine hypothalami in anattempt to discover new hypothalamic factors [25]. It ispresent in two bioactive amidated forms: PACAP38 andPACAP27 with 38 and 27 amino acid residues, respectively[25,26]. In the brain, PACAP38 is the predominant form[4]. Immunohistochemical studies have shown PACAP im-munoreactive cell bodies in the hypothalamus and fibers inthe median eminence and in the posterior pituitary [16,17].Gradually accumulating observations suggest that PACAPis a hypophysiotropic factor:1. PACAP immunoreactive fibers can be found in theexternal zone of the median eminence around theportal capillaries [15,16].2. PACAP is released into the portal blood where itsconcentration is higher than in the general circulation[9].3. PACAP receptors are present in the anterior pituitary[12].4. PACAP influences the anterior pituitary functions (seeRawlings and Hezareh for review [29]).Although a lot of data suggests the role of PACAP as ahypophysiotropic factor, there is also evidence which showsthat PACAP may act as a neurotransmitter or neuromodu-lator, influencing other hypothalamic factors which are in-volved in the control of anterior pituitary functions. Ourprevious experiments have shown that PACAP38 givenintracerebroventricularly (ICV) before the critical period ofthe proestrous afternoon was able to block the ovulation andthe preovulatory LH surge, while the i.v. administration hadno effect [19]. It was concluded that PACAP38 does notexert its inhibitory effect at the pituitary level, rather it ismediated by other hypothalamic factors. This hypothesis issupported by in vitro experiments. In a superfusion systemPACAP38 stimulated rather than inhibited the LH release[25]. With the use of a special cell culture and immunohis-tochemistry (cell immunoblot assay, CIBA) it was alsofound that PACAP38 stimulated the LH release from ante-rior pituitary cells taken from proestrous rats [20]. On thebasis of the above mentioned results, it is obvious that IVadministered PACAP38 has no inhibitory effect on the LHrelease in proestrous female rats. The in vitro results furthersupport the hypothesis that PACAP38 exerts its inhibitoryeffect on the gonadotrop hormone secretion through hypo-thalamic factors. Our candidates were the endogenous opi-oids, melatonin, somatostatin, CRF, and GABA, all of themwith well known inhibitory effect on LH secretion.1.1. Opiates and endogenous opioidsThe inhibitory action of opiates on LH secretion wasshown by Barraclough and Sawyer nearly five decades ago[5]. The major site of action seems to be the hypothalamus,since morphine inhibits the firing frequency of the LHRHneurons [14] and the LHRH release into the hypophysialportal blood [7]. (D-Met

PACAP-(6–38) inhibits the effects of vasoactive intestinal polypeptide, but not PACAP, on the small intestinal circular muscle

Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating peptide-(1–38) (PACAP) have been found to stimulate distension-induced peristaltic motility in the guinea-pig isolated small intestine. In this study, we tested whether the putative VIP/PACAP receptor antagonist PACAP-(6–38) counteracts the properistaltic effect of VIP and PACAP in isolated segments of the guinea-pig small intestine. VIP (100 nM) and PACAP (30 nM) had a stimulatory effect, i.e., lowered the peristaltic pressure threshold at which peristaltic waves were triggered and enhanced the frequency of peristaltic waves. PACAP-(6–38) (3 μM) was per se without effect on peristalsis but prevented or reversed the peristaltic motor stimulation caused by VIP, when it was given before or after the agonist, respectively. PACAP-(6–38), however, failed to antagonize the properistaltic effect of PACAP. In ileal circular strips treated with tetrodotoxin (1 μM) and indomethacin (3 μM), spontaneous myogenic activity was inhibited by VIP (5–30 nM). This effect was significantly reduced by a pretreatment with PACAP-(6–38) (3 μM). A similar inhibition by PACAP-(1–38) (10–500 nM) was not influenced by the antagonist. It is concluded that PACAP-(6–38) is a VIP receptor antagonist, both in the peristaltic motor pathways and at the level of the circular muscle of the guinea-pig small intestine. The lack of a motor effect of PACAP-(6–38) on its own indicates that VIP acting on PACAP-(6–38)-sensitive receptors (located on neurons and/or the smooth muscle) is unlikely to participate in peristaltic motor regulation.

PACAP stimulates gastric acid secretion in the rat by inducing histamine release.

Previous studies have shown that pituitary adenylate cyclase-activating peptide (PACAP) stimulates enterochromaffin-like (ECL) cell histamine release, but its role in the regulation of gastric acid secretion is disputed. This work examines the effect of PACAP-38 on aminopyrine uptake in enriched rat parietal cells and on histamine release and acid secretion in the isolated vascularly perfused rat stomach and the role of PACAP in vagally (2-deoxyglucose) stimulated acid secretion in the awake rat. PACAP has no direct effect on the isolated parietal cell as assessed by aminopyrine uptake. PACAP induces a concentration-dependent histamine release and acid secretion in the isolated stomach, and its effect on histamine release is additive to gastrin. The histamine H2 antagonist ranitidine potently inhibits PACAP-stimulated acid secretion without affecting histamine release. Vagally stimulated acid secretion is partially inhibited by a PACAP antagonist. The results from the present study strongly suggest that PACAP plays an important role in the neurohumoral regulation of gastric acid secretion. Its effect seems to be mediated by the release of ECL cell histamine.

Regulation of olfactory neurogenesis by amidated neuropeptides.

The existence of stem cells in the CNS raises issues concerning the ability of nervous tissues to regenerate in the adult mammal and provides new perspectives on the treatment of degenerative disease and traumatic injury of the nervous system. These cells have a relatively limited range of locations within the nervous system and include cells of the rostral migratory stream, hippocampus, retina, and olfactory epithelium. The olfactory epithelium has been studied as a model of adult neuronal regeneration, with neuronal precursor/basal cells serving as the olfactory "stem cells." The identification of factors that promote neuronal proliferation or regeneration within the olfactory epithelium can provide clues to the process of adult mammalian nervous system repair and treatment. Multiple factors have been examined that appear to influence the proliferation and subsequent maturation of basal cells. These factors include nerve growth factor, fibroblast growth factor-2, epidermal growth factor, and insulin/insulin-like growth factor-1. Recently, two amidated neuropeptides, neuropeptide Y (NPY) and pituitary adenylate cyclase-activating polypeptide (PACAP38), identified in the olfactory epithelium have been shown to promote dramatically neuronal proliferation. The effects of NPY and PACAP suggest that amidated neuropeptides may serve a broad developmental and regenerative role in the mammalian olfactory epithelium.

Differential effects of PACAP-38 on synaptic responses in rat hippocampal CA1 region.

Pituitary adenylate cyclase-activating polypeptide (PACAP-38) is a member of the vasointestinal polypeptide (VIP)/secretin/glucagon family of neuropeptides for which neuroregulatory functions have been postulated. PACAP-38 receptors are expressed in different brain regions, including hippocampus. In this study, we examined the dose-dependent effects of PACAP-38 on the excitatory postsynaptic field potential (fEPSP) evoked at the Schaffer collateral-CA1 synapse in rat hippocampal slices. Bath application of low dose (0.05 nM) of PACAP-38 induced long-lasting facilitation of the fEPSP. This enhancement was blocked by the cholinergic receptor antagonist atropine and partially by the NMDA receptor antagonist 2-amino-5-phosphonovalerate (APV) and therefore, shares a common mechanism with LTP. In contrast, a high dose (1 microM) of PACAP-38 induced a persistent depression of the fEPSP that was not blocked by antagonists of cholinergic receptors (i.e., atropine and mecamylamine), adenosine receptors (i.e., DCPCX), or glutamatergic NMDA receptors (APV). Intermediate doses (0.1-0.5 microM) of PACAP-38 produced an initial decrease of the fEPSP followed by an enhancement. This decrease was not blocked by atropine whereas the facilitation was. These results show that PACAP-38 modulates CA1 synaptic transmission in a dose-dependent manner and that the peptide interacts with cholinergic and glutamatergic systems.

Pituitary adenylate cyclase-activating polypeptide stimulates secretoneurin release and secretogranin II gene transcription in bovine adrenochromaffin cells through multiple signaling pathways and increased binding of pre-existing activator protein-1-like transcription factors.

Secretoneurin (SN) is a novel bioactive peptide that derives from the neuroendocrine protein secretogranin II (SgII) by proteolytic processing and participates in neuro-immune communication. The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP-38) dose-dependently stimulates (EC(50) approximately 3 nM) SN release (up to 4-fold) and SgII gene expression (up to 60-fold) in cultured bovine adrenochromaffin cells. The effect of PACAP on both SN secretion and SgII mRNA levels is rapid and long lasting. We analyzed in this neuroendocrine cell model the transduction pathways involved in both SN secretion and SgII gene transcription in response to PACAP. The cytosolic calcium chelator BAPTA-AM and the nonselective calcium channel antagonist NiCl(2) equally inhibited both secretion of the peptide and transcription of the SgII gene, indicating a major contribution of calcium influx in PACAP-induced SN biosynthesis and release in chromaffin cells. Inhibition of protein kinase A (PKA) or C (PKC) also reduced PACAP-evoked SN release but did not alter the stimulatory effect of PACAP on SgII mRNA levels. Conversely, application of mitogen-activated protein kinase inhibitors suppressed PACAP-induced SgII gene expression. The effect of PACAP on SgII mRNA levels, like the effect of the PKC stimulator 12-O-tetradecanoylphorbol-13-acetate (TPA), was not affected by cycloheximide, whereas the effects of the PKA stimulator forskolin or cell-depolarization by high K(+) were significantly reduced by the protein synthesis inhibitor. PACAP and TPA both increased the binding activity of the SgII cAMP response element to trans-acting factors present in chromaffin cell nuclear extracts, which are recognized by antibodies to activator protein-1-related proteins. These data indicate that SN biosynthesis is regulated by PACAP in chromaffin cells through complex signaling cascades, suggesting that SN may play a function during trans-synaptic stimulation of the adrenal medulla.

Regulation of neuroblast mitosis is determined by PACAP receptor isoform expression.

Although neurogenesis in the embryo proceeds in a region- or lineage-specific fashion coincident with neuropeptide expression, a regulatory role for G protein-coupled receptors (GPCR) remains undefined. Pituitary adenylate cyclase activating polypeptide (PACAP) stimulates sympathetic neuroblast proliferation, whereas the peptide inhibits embryonic cortical precursor mitosis. Here, by using ectopic expression strategies, we show that the opposing mitogenic effects of PACAP are determined by expression of PACAP receptor splice isoforms and differential coupling to the phospholipase C (PLC) pathway, as opposed to differences in cellular context. In embryonic day 14 (E14) cortical precursors transfected with the hop receptor variant, but not cells transfected with the short variant, PACAP activates the PLC pathway, increasing intracellular calcium and eliciting translocation of protein kinase C. Ectopic expression of the hop variant in cortical neuroblasts transforms the antimitotic effect of PACAP into a promitogenic signal. Furthermore, PACAP promitogenic effects required PLC pathway function indicated by antagonist U-73122 studies in hop-transfected cortical cells and native sympathetic neuroblasts. These observations highlight the critical role of lineage-specific expression of GPCR variants in determining mitogenic signaling in neural precursors.

BDNF mediates the neuroprotective effect of PACAP-38 on rat cortical neurons.

In primary cultures from rat cerebral cortex, pituitary adenylate cyclase-activating polypeptide (PACAP-38) exerted a protective effect on cell death induced by the excitotoxin NMDA in neuron-enriched cultures and also on apoptotic cell death induced by serum deprivation in mixed neuronal-glial cultures. The neuroprotective effect was already observed at subnanomolar concentrations of PACAP and was slightly more pronounced against excitotoxic cell death. BDNF protein expression was reduced by NMDA and much more markedly by serum deprivation (approximately 28 and 93% reduction respectively). In both cellular injury conditions, the diminished BDNF expression was significantly prevented by PACAP. When purified neuronal cultures were preincubated with an antiserum anti-BDNF, at a concentration without any intrinsic effect on cell viability, the neuprotective effect of PACAP was no longer observed. The results suggest that the neuroprotective effect of PACAP-38 is mediated, at least in part, by preventing the suppressed expression of a neurotrophin essential for cortical neuron survival.

Effects of pituitary adenylate cyclase activating polypeptide on lumbosacral preganglionic neurons in the neonatal rat spinal cord

The effects of PACAP-38 on phasic and tonic preganglionic neurons (PGN) in L6 and S1 spinal cord slices from neonatal rats (5–11 days old) were studied using the whole-cell patch clamp technique. PGN were identified by retrograde axonal transport of a fluorescent dye (Fast Blue, 5 μl of 4% solution) injected into the intraperitoneal space 3–7 days prior to the study. Bath application of pituitary adenylate cyclase activating polypeptide (PACAP) (20 nM) increased the frequency of spontaneous excitatory postsynaptic potentials (EPSPs) and spontaneous firing in both types of PGN. PACAP markedly increased the number (200–800%) and frequency of action potentials elicited by depolarizing current pulses in phasic PGN, but had a smaller effect on tonic PGN. PACAP decreased the threshold for action potential generation by approximately 25% in both types of neurons (e.g. −34.0±1.5 to −38.4±1.7 mV from a holding potential of −50 mV in phasic PGN, P<0.005). PACAP did not affect the duration of the action potential. The amplitude of the spike after hyperpolarization was not changed but the duration was significantly reduced by PACAP from 204.4±12.2 to 106.2±8.1 ms in tonic but not in phasic PGN. PACAP suppressed a transient outward current that was also suppressed by 4-aminopyridine (0.5 mM). These results coupled with the immunohistochemical identification of a dense collection of PACAP fibers in the region of the PGN, raises the possibility that PACAP may function as an excitatory transmitter in lumbosacral parasympathetic reflex pathways in the neonatal rat.

G-protein-coupled receptor kinase 3- and protein kinase C-mediated desensitization of the PACAP receptor type 1 in human Y-79 retinoblastoma cells

Pituitary adenylyl cyclase-activating polypeptide (PACAP) receptor type 1 (PAC 1) signaling and desensitization were investigated in human retinoblastoma Y-79 cells. Concentration-dependent stimulation of cAMP accumulation was observed in Y-79 cells incubated for 30 min with PACAP38, PACAP27, or VIP (10 −12 to 10 −6 M). The following EC 50 values were calculated: PACAP38, 24±3 pM; PACAP27, 99±8 pM; and VIP, 29±3 nM. Homologous desensitization of PAC 1 receptors in Y-79 cells pretreated with 10 nM PACAP38 or PACAP27 for 60 min was characterized by a 30–50% reduction in PACAP-stimulated cAMP accumulation ( p<0.0001) and a two- to fivefold rightward shift in EC 50 values ( p<0.0001). PAC 1 receptor desensitization was not accompanied by a reduction in PAC 1 mRNA expression. We concluded that the desensitizing effect of PACAP38 was homologous because neither corticotropin-releasing factor- nor (−)-isoproterenol-stimulated cAMP accumulation was altered by PACAP38 preincubation. Pretreating Y-79 cells with the protein kinase A (PKA) inhibitor H89 failed to inhibit homologous PAC 1 receptor desensitization. Similarly, pretreating Y-79 cells with the protein kinase C (PKC) inhibitors staurosporine or bisindolylmaleimide failed to alter homologous PAC 1 receptor desensitization. Although activation of PKA by dibutyryl cAMP or forskolin did not desensitize PAC 1 receptors, direct activation of PKC by PMA heterologously desensitized PAC 1 receptors, reducing cAMP accumulation 34.2±2.2% ( p<0.001). Using RT–PCR, mRNA levels for G-protein-coupled receptor kinase 3 (GRK3), but not GRK2, were found to increase 2.2- to 4.8-fold in Y-79 cells exposed to PACAP38 for 10 min to 24 h ( p<0.001). PAC 1 receptor desensitization decreased 72.5±4.3% ( p<0.001) in Y-79 cells transfected with a GRK3 antisense cDNA construct that also reduced GRK3 protein expression 48.5±7.9% ( p<0.0005). These experiments demonstrate that GRK3 plays an important role in the homologous desensitization of retinoblastoma PAC 1 receptors, whereas PKC, but not PKA, contributes to the heterologous desensitization of retinoblastoma PAC 1 receptors.

VIP and PACAP Enhance the In Vivo Generation of Memory TH2 Cells by Inhibiting Peripheral Deletion of Antigen-Specific Effectors

In an immune response, antigen-specific CD4 T cells proliferate and differentiate into effector cells capable to produce large amounts of cytokines upon restimulation. Most effector T cells are later eliminated through antigen-induced cell death (AICD), mediated through FasL/Fas interactions. A low percentage of effector T cells survive and differentiate into long-lived memory cells. Mechanisms must operate not only to destroy no longer needed and even potentially damaging T cells, but also to allow the survival of a small number of activated T cells. Little is known about the factors and mechanisms that regulate the shift from an apoptosis-sensitive to an apoptosis-resistant phenotype. VIP and the structurally related peptide, PACAP, synthesized and/or released in the immune organs act on both innate and adaptive immunity. Recently, VIP and PACAP were shown to inhibit AICD in peripheral CD4 T cells by down-regulating FasL expression. In view of these findings, VIP and PACAP are reasonable candidates for the generation of memory T cells. To test this hypothesis, we analyzed the effects of VIP and PACAP in various models for effector and memory T cells. Our data demonstrate that both neuropeptides promote the in vivo effector function and memory phenotype of Th2, but not Th1 cells, by preferentially inhibiting the clonal deletion of Th2 cells. To our knowledge, this is the first report describing the role of a neuropeptide present in the lymphoid microenvironment on the generation and maintenance of long-lived memory T cells.

The induction of carbon monoxide-mediated airway relaxation by PACAP 38 in isolated guinea pig airways.

Pituitary adenylate cyclase--activating peptide 38 (PACAP 38) displays several biologic activities relevant to obstructive airway disease. Carbon monoxide (CO) has recently emerged as a potent, endogenously produced mediator of bronchodilation. In this study, we have analyzed the occurrence of PACAP 38 and the corresponding occurrence of heme oxygenase (HO), the rate-limiting enzyme for CO production, in guinea pig trachea, using immunocytochemistry. We have also investigated whether the dilatory effects of PACAP 38 are dependent on CO, using an in vitro setup for tracheal studies. A moderate supply of PACAP-like immunoreactive nerve fibers was seen in association with tracheal smooth muscle. HO-like immunoreactivity was observed in the respiratory epithelium and in association with smooth muscle bundles. PACAP 38 induced a concentration-dependent relaxation of precontracted tracheal segments. This dilation was nearly abolished after pretreatment with zincprotoporphyrine, an inhibitor of heme oxygenase. The same effect was accomplished with Rp-8Br-cyclicGMPS, an inhibitor of cyclicGMP, whereas the nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine had no effect on the PACAP 38--induced dilation. The presented data suggest that PACAP 38 can induce bronchodilation by means of a CO-dependent, cyclicGMP-related mechanism, thereby providing a link between neurotransmission and local CO release in the airway smooth muscle.