Ontogeny of PAC1-R and VPAC1-R in the frog, Rana esculenta

The distribution of pituitary adenylate cyclase-activating polypeptide (PACAP) and PACAP receptors in the brain of amphibians has been previously described. In the present study, we have investigated the ontogeny of the selective PACAP receptor, PAC1-R, and the PACAP-vasoactive intestinal polypeptide (VIP) mutual receptor, VPAC1-R, in frog embryos by whole-mount in situ hybridization histochemistry. At stage 20, expression of PAC1-R and/or VPAC1-R mRNAs was detected in the brain, the auditory vesicles, the external gills, the buds of the lateral lines and the coelomatic cavity. At stage 25, PAC1-R and/or VPAC1-R mRNAs were observed in the buds of the orbital lateral line, the pancreas and heart. At stage 30, PAC1-R and VPAC1-R mRNAs were widely distributed in the telencephalon and diencephalon as well as in the bud of the lateral line, the heart and the pancreas. The anatomical distribution of PAC1-R and VPAC1-R mRNAs, although similar, did not totally overlap, indicating that PACAP and VIP may exert differential effects in frog during development.     

Cytosolic Ca2+ responses to sub-picomolar and nanomolar PACAP in pancreatic beta-cells are mediated by VPAC2 and PAC1 receptors

Pituitary adenylate cyclase-activating polypeptide (PACAP) potentiates glucose-induced insulin release and increases cytosolic Ca2+ concentration ([Ca2+]i) in islet beta-cells in a concentration-dependent manner with two peaks at 10(-13) and 10(-9) M. PAC1 receptor (PAC1-R) and VPAC2 receptor (VPAC2-R) are expressed in pancreatic beta-cells and thought to be involved in insulin release. We aimed to determine the receptor types involved in the [Ca2+]i responses to 10(-13) and 10(-9) M PACAP. We measured [Ca2+]i in beta-cells and examined comparative effects of PAC1-R-selective agonist maxadilan, its antagonist M65, VPAC2-R-selective agonist Ro25-1553, and native ligands of PACAP and VIP. In the presence of 8.3 mM glucose, maxadilan, Ro25-1553, PACAP, and VIP at 10(-13) and 10(-9) M all increased [Ca2+]i. PACAP and maxadilan elicited greater effects at 10(-9) M than at 10(-13) M both in the incidence and amplitude of [Ca2+]i responses. For VIP and Ro25-1553, in contrast, the effects at 10(-9) and 10(-13) M were comparable. Furthermore, the amplitude of [Ca2+]i responses to 10(-9) M PACAP, but not 10(-13) M PACAP, was suppressed by M65. The results suggest that VPAC2-R and PAC1-R contribute equally to [Ca2+]i responses to sub-picomolar concentrations of PACAP, while PAC1-R has greater contribution to [Ca2+]i responses to nanomolar concentrations of this peptide.     

VIP and PACAP receptors coupled to adenylyl cyclase in human lung cancer: a study in biopsy specimens

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are important neuropeptides in the control of lung physiology. Both of these commonly bind to specific G protein coupled receptors named VPAC(1)-R and VPAC(2)-R, and PAC(1)-R (with higher affinity for PACAP). VIP and PACAP have been implicated in the control of cell proliferation and tumor growth. This study examined the presence of VIP and PACAP receptors in human lung cancer samples, as well as the functionality of adenylyl cyclase (AC) stimulated by both peptides. Results from RT-PCR and immunoblot experiments showed the expression of VPAC(1)-, VPAC(2)- and PAC(1)-R in lung cancer samples. Immunohistochemical studies showed the expression of VPAC(1) and VPAC(2) receptors. These receptors were positively coupled to AC, but the enzyme activity was impaired as compared to normal lung. There were no changes in Galpha(s) or Galpha(i) levels. Present results contribute to a better knowledge of VIP/PACAP actions in lung cancer and support the interest for the development of VIP/PACAP analogues with therapeutic roles.     

PACAP-27 tyrosine phosphorylates mitogen activated protein kinase and increases VEGF mRNAs in human lung cancer cells

The effects of pituitary adenylate cyclase activating polypeptide (PACAP) on human lung cancer cell line NCI-1299 mitogen activated protein kinase (MAPK) tyrosine phosphorylation and vascular endothelial cell growth factor (VEGF) expression were investigated. PACAP-27 (100 nM) increased MAPK tyrosine phosphorylation 3-fold, 5 min after addition to NCI-H1299 cells. PACAP caused tyrosine phosphorylation in a concentration-dependent manner being half-maximal at 10 nM PACAP-27. PACAP-27 or PACAP-38 (100 nM) but not PACAP28-38 or VIP caused increased MAPK tyrosine phosphorylation using NCI-H1299 cells. Also, the increase in MAPK tyrosine phosphorylation caused by PACAP-27 was totally inhibited by 10 microM PACAP(6-38), a PAC(1) receptor antagonist or 10 microM PD98059, a MAPKK inhibitor. These results suggest that PAC(1) receptors regulate tyrosine phosphorylation of MAPK in a MAPKK-dependent manner. PACAP-27 (100 nM) caused increased VEGF mRNA in NCI-H1299 cells after 8 h. The increase in VEGF mRNA caused by PACAP-27 was partially inhibited by PACAP(6-38), PD98059 and H-89. Addition of VIP to NCI-H1299 cells caused increased VEGF mRNA, which was totally inhibited by H89, a PKA inhibitor. These results suggest that PAC(1) and VPAC(1) receptors regulate VEGF expression in lung cancer cells.     

Identification of key residues that cause differential gallbladder response to PACAP and VIP in the guinea pig

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) have opposite actions on the gallbladder; PACAP induces contraction, whereas VIP induces relaxation. Here, we have attempted to identify key residues responsible for their interactions with PACAP (PAC1) and VIP (VPAC) receptors in the guinea pig gallbladder. We synthesized PACAP-27/VIP hybrid peptides and compared their actions on isolated guinea pig gallbladder smooth muscle strips using isotonic transducers. [Ala4]- and [Val5]PACAP-27 were more potent than PACAP-27 in stimulating the gallbladder. In contrast, [Ala4, Val5]- and [Ala4, Val5, Asn9]PACAP-27 induced relaxation similarly to VIP. [Asn9]-, [Thr11]-, or [Leu13]PACAP-27 had 20-70% contractile activity of PACAP-27, whereas [Asn24,Ser25,Ile26]PACAP-27 showed no change in the activity. All VIP analogs, including [Gly4,Ile5,Ser9]VIP, induced relaxation. In the presence of a PAC1 receptor antagonist, PACAP(6-38), the contractile response to PACAP-27 was inhibited and relaxation became evident. RT-PCR analysis revealed abundant expressions of PAC1 receptor, "hop" splice variant, and VPAC1 and VPAC2 receptor mRNAs in the guinea pig gallbladder. In conclusion, PACAP-27 induces contraction of the gallbladder via PAC1/hop receptors. Gly4 and Ile5 are the key NH2-terminal residues of PACAP-27 that distinguish PAC1/hop receptors from VPAC1/VPAC2 receptors. However, both the NH2-terminal and alpha-helical regions of PACAP-27 are required for initiating gallbladder contraction.     

Structural motifs of pituitary adenylate cyclase-activating polypeptide (PACAP) defining PAC1-receptor selectivity

Pituitary adenylate cyclase-activating polypeptide (PACAP) interacts with three types of PACAP/VIP-receptors. The PAC1-receptor accepts PACAP as a high affinity ligand but not vasoactive intestinal peptide (VIP) similarly binding to VPAC1- and VPAC2-receptors. To identify those amino acids not present in VIP defining PAC1-receptor selectivity of PACAP, radio receptor binding assays on AR4-2J cells were performed. It could be shown that PACAP(1-27) exhibited a distinct and much higher susceptibility to VIP-amino acid substitutions, compared to PACAP(1-38). Positions 4 and 5 seem to be most important for receptor binding of PACAP(1-27), whereas position 13 was identified to be crucial for maximal affinity of PACAP(1-38). PACAP(29-38) extension analogues of VIP revealed a stabilizing effect of the C-terminus of PACAP(1-38) on the optimal peptide conformation. The substitution analogues were also checked for their capacity to stimulate IP3 and cAMP formation in AR4-2J cells. Compared to PACAP(1-27) and PACAP(1-38), most analogues revealed potencies reduced congruously to their lower binding affinities. However, one of the analogues, PACAP(1-27) substituted in position 5, may represent a weak antagonist since this peptide was less potent in inducing second messengers than in label displacement. Our findings indicate that PACAP(1-27) and PACAP(1-38) differ in terms of their requirement of the amino acids in positions 4, 5, 9, 11 and 13 for maximal interaction with the PAC1-receptor.     

Expression of functional PACAP/VIP receptors in human prostate cancer and healthy tissue

Vasoactive intestinal peptide (VIP) is involved in prostate cell proliferation and function. VIP and pituitary adenylate cyclase-activating peptide (PACAP) are similarly recognized by VPAC(1)/VPAC(2) receptors whereas PACAP binds with higher affinity than VIP to PAC(1) receptor. Here we systematically studied the presence and distribution of functional PAC(1), VPAC(1) and VPAC(2) receptors in human normal and malignant prostate tissue. Functional PACAP/VIP receptors were detected in normal and malignant prostate by adenylyl cyclase stimulation with PACAP-27/38 and VIP. RT-PCR experiments showed PAC(1) (various isoforms due to alternative splicing), VPAC(1) and VPAC(2) receptor expression at the mRNA level, whereas Western blots found the three receptor protein classes in normal and pathological conditions. No conclusive differences could be established when comparing control and cancer tissue samples. Immunohistochemistry showed a weaker immunostaining in tumoral than in normal epithelial cells for the three receptor subtypes. In conclusion, we demonstrate the expression of functional PAC(1), VPAC(1) and VPAC(2) receptors in human prostate as well as its maintenance after malignant transformation.     

A structure–function study of PACAP using conformationally restricted analogs: Identification of PAC1 receptor-selective PACAP agonists

Pituitary adenylate cyclase-activating polypeptide (PACAP) has widespread physiological/pathophysiological actions and there is increased interest for its use therapeutically, especially in the CNS (neuroprotection). Unfortunately, no selective PACAP-analogs exist for PACAP-preferring PAC1-receptors, primarily because of its high sequence identity to VIP and particularly, because of the inability of structure–function studies to separate the pharmacophore of PAC1-R from VPAC1-R, which has high affinity for PACAP and VIP. The present study attempted to develop PAC1-R-selective agonists primarily by making conformationally restricted PACAP-analogs in positions important for receptor-selectivity/affinity. Forty-six PACAP-related-analogs were synthesized with substitutions in positions 1–4, 14–17, 20–22, 28, 34, 38 and receptor-selectivity determined in PAC1-R,VPAC1-R,VPAC2-R-transfected or native cells from binding or cAMP-generation experiments. Fifteen PACAP-analogs had 6–78-fold higher affinities for PAC1-R than VPAC1-R and 13 were agonists. Although binding-affinities correlated significantly with agonist potency, the degree of receptor-spareness varied markedly for the different PACAP-analogs, resulting in selective potencies for activating the PAC1 receptor over the VPAC1 receptor from 0- to 103-fold. In addition, a number of PACAP-analogs were identified that had high selectivity for PAC1-R over VPAC2-R as well as PACAP-analogs that could prove more useful therapeutically because of substitutions known to extend their half-lives (substitutions at potential sites of proteolysis and attachment of long-chain fatty acids). This study provides for the first time a separation of the pharmacophores for PAC1-R and VPAC1-R, resulting in PACAP-related analogs that are PAC1-R-preferring. Some of these analogs, or their modifications, could prove useful as therapeutic agents for various diseases.     

GI side-effects of a possible therapeutic GRF analogue in monkeys are likely due to VIP receptor agonist activity.

Growth hormone (GH) is used or is being evaluated for efficacy in treatment of short stature, aspects of aging, cardiac disorders, Crohn's disease, and short bowel syndrome. Therefore, we synthesized several stable growth hormone-releasing factor (GRF) analogues that could be therapeutically useful. One potent analog, [D-Ala(2),Aib(8, 18,)Ala(9, 15, 16, 22, 24-26,)Gab(27)]hGRF(1-27)NH(2) (GRF-6), with prolonged infusion caused severe diarrhea in monkeys; however, it had no side-effects in rats. Because GRF has similarity to VIP/PACAP and VIPomas cause diarrhea, this study investigated the ability of this and other GRF analogues to interact with the VIP/PACAP receptors. Rat VPAC(1)-R (rVPAC(1)-R), human VPAC(1)-R (hVPAC(1)-R), rVPAC(2)-R and hVPAC(2)-R stably transfected CHO and PANC 1 cells were made and T47D breast cancer cells containing native human VPAC(1)-R and AR4-2J cells containing PAC(1)-R were used. hGRF(1-29)NH(2) had low affinity for both rVPAC(1)-R and rVPAC(2)-R while VIP had a high affinity for both receptors. GRF-6 had a low affinity for both rVPAC(1)-R and rVPAC(2)-R and very low affinity for the rPAC(1)-R. VIP had a high affinity, whereas hGRF(1-29)NH(2) had a low affinity for both hVPAC(1)-R and hVPAC(2)-R. In contrast GRF-6, while having a low affinity for hVPAC(2)-R, had relatively higher affinity for the hVPAC(1)-R. In guinea pig pancreatic acini, all GRF analogues were full agonists at the VPAC(1)-R causing enzyme secretion. These results demonstrate that in contrast to native hGRF(1-29)NH(2,) GRF-6 has a relatively high affinity for the human VPAC(1)-R but not for the human VPAC(2)-R, rat VPAC(1)-R, rat VPAC(2)-R or rat PAC(1)-R. These results suggest that the substituted GRF analog, GRF-6, likely causes the diarrheal side-effects in monkeys by interacting with the VPAC(1)-R. Furthermore, they demonstrate significant species differences can exist for possible therapeutic peptide agonists of the VIP/PACAP/GRF receptor family and that it is essential that receptor affinity assessments be performed in human cells or from a closely related species.     

Signaling involved in pituitary adenylate cyclase-activating polypeptide-stimulated ADNP expression

Activity-dependent neurotrophic protein (ADNP) was discovered as a novel response gene for VIP and has neuroprotective potential. When the VIP paralog, PACAP38 was added to mouse neuron-glia co-cultures, it induced ADNP mRNA expression in a bimodal fashion at subpico- and nanomolar concentrations with greater response at subpicomolar level. The response was attenuated by a PAC1-R antagonist at both concentrations and by a VPAC1-R antagonist at nanomolar concentration only. An IP3/PLC inhibitor attenuated the response at both concentrations of PACAP38, but a MAPK inhibitor had no effect. A PKA inhibitor suppressed the response at nanomolar concentration only. These findings suggest that ADNP expression is mediated through multiple receptors and signaling pathways that are regulated by different concentrations of PACAP.     

Identification and functional properties of the pituitary adenylate cyclase activating peptide (PAC1) receptor in human benign hyperplastic prostate

Pituitary adenylate cyclase activating peptide (PACAP) is a novel neuropeptide with regulatory and trophic functions that is related to vasoactive intestinal peptide (VIP). Here we investigate the expression of specific PACAP receptors (PAC1) and common VIP/PACAP receptors (VPAC1 and VPAC2) in the human hyperplastic prostate by immunological methods. The PAC1 receptor corresponded to a 60-KDa protein whereas the already known VPAC1 and VPAC2 receptors possessed molecular masses of 58 and 68 KDa, respectively. The heterogeneity of VIP/PACAP receptors in this tissue was confirmed by radioligand binding studies using [125I]PACAP-27 by means of stoichiometric and pharmacological experiments. At least two classes of PACAP binding sites showing different affinities could be resolved, with Kd values of 0.81 and 51.4 nM, respectively. The order of potency in displacing [125I]PACAP-27 binding was PACAP-27 approximately equal to PACAP-38 > VIP. PACAP-27 and VIP stimulated similarly adenylate cyclase activity, presumably through common VIP/PACAP receptors. The PAC1 receptor was not coupled to activation of either adenylate cyclase, nitric oxide synthase, or phospholipase C. It appears to be a novel subtype of PAC1 receptor because PACAP-27 (but not PACAP-38 or VIP) led to increased phosphoinositide synthesis, an interesting feature because phosphoinositides are involved via receptor mechanisms in the regulation of cell proliferation.     

Helospectin I and II evoke vasodilation in the intact peripheral microcirculation

Helospectin I and II, two closely related mammalian neuropeptides of the secretin/glucagons/vasoactive intestinal peptide (VIP) superfamily of peptides, are co-localized with VIP in nerve fibers surrounding vascular smooth muscle. However, the role if any, VIP receptors play in transducing the vasorelaxant effects of helospectin I and II in the intact peripheral microcirculation is uncertain. The purpose of this study was to determine whether helospectin I and II elicit vasodilation in the intact peripheral microcirculation and, if so, whether this response is mediated, in part, by VIP or pituitary adenylate cyclase activating peptide (PACAP) receptor engagement, and through local elaboration of cyclooxygenase products of arachidonic acid metabolism. Using intravital microscopy, we found that suffusion of helospectin I and II (each, 1.0 nmol) evoked potent vasodilation and of similar magnitude in the intact hamster cheek pouch microcirculation (P < 0.05). Suffusion of 0.1 nmol helospectin I and II had no significant effects on arteriolar diameter. Pretreatment with VIP(10-28), a VPAC1/VPAC2 receptor antagonist, or PACAP(6-38), a PAC1/VPAC2 receptor antagonist, had no significant effects on helospectin I- and II-induced responses. In addition, pretreatment with indomethacin had no significant effects on helospectin I- and II-induced vasodilation. Collectively, these data indicate that helospectin I and II evoke potent vasodilation in the intact peripheral microcirculation that is not transduced by VIP or PACAP receptors nor through cyclooxygenase products of arachidonic acid metabolism.     

Generation of highly selective VPAC2 receptor agonists by high throughput mutagenesis of vasoactive intestinal peptide and pituitary adenylate cyclase-activating peptide

Pituitary adenylate cyclase-activating peptide (PACAP) has a specific receptor PAC1 and shares two receptors VPAC1 and VPAC2 with vasoactive intestinal peptide (VIP). VPAC2 activation enhances glucose-induced insulin release while VPAC1 activation elevates glucose output. To generate a large pool of VPAC2 selective agonists for the treatment of type 2 diabetes, structure-activity relationship studies were performed on PACAP, VIP, and a VPAC2 selective VIP analog. Chemical modifications on this analog that prevent recombinant expression were sequentially removed to show that a recombinant peptide would retain VPAC2 selectivity. An efficient recombinant expression system was then developed to produce and screen hundreds of mutant peptides. The 11 mutations found on the VIP analog were systematically replaced with VIP or PACAP sequences. Three of these mutations, V19A, L27K, and N28K, were sufficient to provide most of the VPAC2 selectivity. C-terminal extension with the KRY sequence from PACAP38 led to potent VPAC2 agonists with improved selectivity (100-1000-fold). Saturation mutagenesis at positions 19, 27, 29, and 30 of VIP and charge-scanning mutagenesis of PACAP27 generated additional VPAC2 selective agonists. We have generated the first set of recombinant VPAC2 selective agonists described, which exhibit activity profiles that suggest therapeutic utility in the treatment of diabetes.     

Mutual dependence of VIP/PACAP and CCK receptor signaling for a physiological role in duck exocrine pancreatic secretion

Unlike in rodents, CCK has not been established as a physiological regulator in avian exocrine pancreatic secretion. In the isolated duck pancreatic acini, 1 nM CCK was required for stimulation of amylase secretion, maximal effect being achieved at 10 nM; picomolar CCK was without effect. Vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase activating peptide (PACAP) receptor (VPAC) agonists PACAP-38 and PACAP-27 (10(-12)-10(-7) M) alone had no effect, but made picomolar CCK effective. VPAC agonist VIP 10(-10)-10(-7) M stimulated amylase secretion marginally, but made CCK 10(-12)-10(-10) M effective also. PACAP-27 and VIP both shifted the maximal CCK concentration from 10(-8) to 10(-9) M. This sensitizing effect was mimicked by forskolin. CCK dose dependently induced intracellular Ca2+ concentration ([Ca2+]i) oscillations. PACAP-38 (1 nM), PACAP-27 (1 nM), VIP (10 nM), or forskolin (10 microM) alone did not stimulate [Ca2+]i increase, neither did they modulate CCK (1 nM)-induced oscillations; but when they were added to cells simultaneously exposed to subthreshold CCK (10 pM), calcium spikes emerged. Amylase secretion induced by the simultaneous presence of 10 pM CCK and VPAC agonists was completely blocked by removing extracellular calcium, but the protein kinase C inhibitor staurosporine (1 microM) was without effect. CCK (10 nM)-induced secretion was inhibited by CCK1 receptor antagonist FK480 (1 microM). Gastrin from 10(-12) to 10(-6) M did not stimulate amylase secretion nor did it (100 nM) induce [Ca2+]i increase. The above data suggest that duck pancreatic acini possess both CCK1 and VPAC receptors; simultaneous activation of both is required for each to play a physiological role.     

Pituitary adenylate cyclase-activating polypeptide activates K(ATP) current in rat atrial myocytes

Because the electrophysiological effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on the heart are little known, we studied the regulation of the atrial ATP-sensitive K(+) (K(ATP)) current by PACAP on primary cultured neonatal rat atrial myocytes. PACAP-38 stimulates cAMP production with EC(50) = 0.28 nmol/l (r = 0.92, P < 0.02). PACAP-38 and PACAP-27 (10 nmol/l) have similar maximal effects, whereas 100 nmol/l vasoactive intestinal polypeptide (VIP) is 2.7 times less effective (P < 0.05). RT-PCR shows the presence of cloned PACAP receptors PAC(1) (> or =2 isoforms), VPAC(1), and VPAC(2). PACAP-38 dose dependently activates the whole cell atrial K(ATP) current with EC(50) = 1-3 nmol/l (n = 44). Maximal effects occur at 10 nmol/l (91 +/- 15 pA/pF, n = 18). Diazoxide further increases the PACAP-activated current by 78% (P < 0.05; n = 6). H(89) (500 nmol/l), a protein kinase A (PKA) inhibitor, reduces the PACAP-activated K(ATP) current to 17.8 +/- 9.6% (n = 5) of the maximal diazoxide-induced current and totally inhibits the cAMP-induced K(ATP) current. A protein kinase C (PKC) inhibitor peptide (50 micromol/l) in the pipette reduces the PACAP-38-induced K(ATP) current to 33 +/- 17 pA/pF (P < 0.05, n = 6) without significantly affecting the currents induced by cAMP or VIP. The results suggest that: 1) PAC(1), VPAC(1), and VPAC(2) are present in atrial myocytes; and 2) PACAP-38 activates the atrial K(ATP) channels through both PKA and PKC pathways.     

PACAP-(1-38) as neurotransmitter in the porcine adrenal glands

The concentration of pituitary adenylyl cyclase-activating polypeptide [PACAP-(1-38)] in porcine adrenal glands amounted to 14 +/- 3 pmol/g tissue. PACAP immunoreactive (PACAP-IR) fibers innervated adrenal chromaffin cells (often co-localized with choline acetyltransferase). Subcapsular fibers traversed the cortex-innervating endocrine cells and blood vessels [some co-storing mainly calcitonin gene-related peptide but also vasoactive intestinal polypeptide (VIP)]. PACAP-IR fibers were demonstrated in the splanchnic nerves, whereas IR adrenal nerve cell bodies were absent. In isolated, vascularly perfused adrenal gland, splanchnic nerve stimulation (16 Hz) and capsaicin (10(-5) M) increased PACAP-(1-38) release (1.6-fold and 6-fold respectively, P = 0.02). PACAP-(1-38) dose-dependently stimulated cortisol (2 x 10(-10) M; 24-fold increase, P = 0.02) and chromogranin A fragment (2 x 10(-9) M; 15-fold increase, P = 0.05) secretion. Both were strongly inhibited by the PAC(1)/VPAC(2) receptor antagonist PACAP-(6-38) (10(-7) M). PACAP-(6-38) also inhibited splanchnic nerve (10 Hz)-induced cortisol secretion but lacked any effect on splanchnic nerve-induced pancreastatin secretion. PACAP-(1-38) (2 x 10(-10) M) decreased vascular resistance from 5.5 +/- 0.6 to 4.6 +/- 0.4 mmHg. min. ml(-1). PACAP-(6-38) had no effect on this response. We conclude that PACAP-(1-38) may play a role in splanchnic nerve-induced adrenal secretion and in afferent reflex pathways.     

The 38-amino acid form of pituitary adenylate cyclase-activating polypeptide stimulates dual signaling cascades in PC12 cells and promotes neurite outgrowth.

Vasoactive intestinal peptide (VIP) activates adenylylcyclase in sympathoadrenal cells at concentrations greater than 10(-6) M. We demonstrate here that two forms of a newly discovered peptide with homology to VIP named pituitary adenylate cyclase-activating polypeptide (PACAP) are much more potent activators of signal transduction in PC12 cells. Both the 27- and 38-amino acid forms of PACAP elevate cAMP levels in PC12 cells and stimulate adenylylcyclase in PC12 membranes, with an EC50 near 10(-9) M. PACAP38 additionally is a potent activator of the inositol lipid cascade in PC12 cells, elevating the content of inositol phosphates by 8-fold at 10(-8) M (EC50 = 7 x 10(-9) M). PACAP38 and PACAP27 have been thought to have essentially identical actions, but PACAP27 is 2-3 logs less potent in increasing inositol lipid levels. Moreover, PACAP38 at 10(-8) M is an effective inducer of neuronal morphology in PC12 cells, whereas PACAP27 is much less active in promoting neurite outgrowth. In contrast to the PACAP-preferring receptors on PC12 cells, another class of PACAP-binding sites with equal high affinities for VIP, PACAP38, and PACAP27 has been identified on several other cell types. We find that the cAMP content of rat CH3 pituitary cells, known to have high affinity VIP receptors, is in fact potently elevated by PACAP27 and PACAP38 as well as by VIP. However, PACAP38, even at 10(-6) M, is not capable of significant activation of inositol lipid turnover via these VIP/PACAP nondiscriminating sites.     

Pharmacological evidence for both neuronal and smooth muscular PAC1 receptors and a VIP-specific receptor in rat colon

The receptors for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide (PACAP) were characterised in vitro on rat colon longitudinal smooth muscle with adherent myenteric ganglia. VIP, PACAP-38 and PACAP-27 all caused concentration-dependent relaxations. PACAP-27 and PACAP-38 were equipotent, while VIP was less potent. Tetrodotoxin (10(-6) M), L-NAME (10(-4) M), 7-NINA (10(-5) M) and ODQ (3 x 10(-6) M) reduced the amplitude of the relaxatory responses to PACAP-38 but did not affect relaxations induced by VIP or PACAP-27. Apamin (10(-6) M) almost totally abolished the PACAP-27- and PACAP-38-induced relaxations, while VIP-induced relaxations were only slightly reduced. Tetraethylammonium (TEA) reduced VIP- but not PACAP-27-induced relaxations, while charybdotoxin was ineffective. Cross-desensitisation between PACAP-27, PACAP-38 and VIP could be revealed to some extent. In conclusion: VIP, PACAP-27 and PACAP-38 are effective relaxants in rat colon longitudinal muscle. The receptors involved are classified as: (1) a neuronal PAC1 receptor localised on NO-synthesising neurones, the preferred ligand being PACAP-38. Activation of this receptor leads to an increased NO production. (2) A smooth muscle PAC1 receptor, the preferred ligand being PACAP-27. However, also PACAP-38 and, to a less extent, VIP activate this receptor. The relaxatory responses elicited by both PACAP-27 and PACAP-38 are abolished by apamin and thus mediated through small conductance Ca2+-activated K+ channels. (3) A VIP-specific receptor localised on smooth muscle cells. The mechanisms whereby this receptor elicits a relaxatory response involve, at least to some extent, TEA-sensitive K+ channels.     

Ontogeny of PACAP receptors in the human cerebellum: perspectives of therapeutic applications

It is now well established that pituitary adenylate cyclase-activating polypeptide (PACAP) exerts anti-apoptotic and pro-differentiating actions during development of the rodent cerebellum. Cell signaling involved in the neurotrophic effects of PACAP has been precisely investigated. In particular, PACAP is a potent inhibitor of the mitochondrial apoptotic pathway through an ERK- and PKA-dependent mechanism. However, transposition of the neurodevelopmental activities of PACAP to the human cerebellum remains speculative, essentially because of the lack of data concerning the PACAP-ergic system. The present review is based on recent results that provide the first molecular, pharmacological and anatomical characterizations of PACAP receptors in the developing human cerebellum. It is now clearly established that the distribution pattern of PAC1-R and VPAC1-R mRNA in the human cerebellum is very similar to that already described in rodents. [(125)I]PACAP27 binding sites are closely associated with germinative neuroepithelia in fetal stages and with mature granule cells in infants and adults. Pharmacological characterization revealed that, in fetuses, PACAP binding sites exhibit a PAC1-R profile while, in adult patients, they correspond to a heterogeneous population of PAC1-R and VPAC(1/2)-R. Altogether, these data provide the first evidence that PACAP may exert neurodevelopmental functions in the human cerebellum.