Maxadilan interacts with receptors for pituitary adenylyl cyclase activating peptide in human SH-SY5Y and SK-N-MC neuroblastoma cells

Receptors for pituitary adenylyl cyclase activating peptide (PACAP) have been identified in human SH-SY5Y neuroblastoma cells with PACAP being 1000-fold more potent than vasoactive intestinal peptide (VIP) in [(125)I]PACAP binding inhibition and stimulation of cAMP accumulation. Maxadilan, a vasodilator peptide from the salivary gland of the sand fly Lutzomyia longipalpis also specifically bound to SH-SY5Y cells, and was equipotent to PACAP in [(125)I]PACAP and [(125)I]maxadilan binding inhibition, and stimulation of cAMP accumulation. Maxadilan and PACAP also increased the cytosolic free calcium concentration. In human SK-N-MC neuroblastoma cells PACAP, VIP and maxadilan equipotently stimulated cAMP accumulation. The maximal effects of VIP and maxadilan were additive and reached those of PACAP alone. In human T47D breast carcinoma cells PACAP and VIP were also equipotent in the stimulation of cAMP accumulation, but maxadilan was inactive. The results are consistent with the interaction of maxadilan with PACAP specific PAC(1)receptors in SH-SY5Y cells, but not with VPAC receptors, not differentiating between VIP and PACAP in T47D cells. Moreover, maxadilan is a PAC(1)receptor specific agonist which allows discrimination of co-expressed PAC(1)and VPAC receptors in SK-N-MC cells.     

PACAP activates calcium influx-dependent and -independent pathways to couple met-enkephalin secretion and biosynthesis in chromaffin cells

Pituitary adenylate cyclase activating polypeptide-27 (PACAP-27) caused a dose-dependent increase in met-enkephalin secretion and increased production of met-enkephalin peptide and proenkephalin A (PEnk) mRNA in bovine chromaffin cells, at concentrations as low as 300 pM. PACAP-38 was less potent than PACAP-27, but had similar effects. Vasoactive intestinal polypeptide (VIP) (1–100 nM) was without appreciable effect on either enkephalin secretion or biosynthesis, implicating PACAP type I receptors in PACAP-stimulated enkephalin secretion and synthesis. PACAP type I receptors can activate adenylate cyclase and stimulate phospholipase C through heterotrimeric G protein interactions, leading to increased intracellular cyclic AMP (cAMP), inositol triphosphate (IP³)-mediated calcium mobilization, and calcium- and diacylglycerol (DAG)-mediated protein kinase C (PKC) activation. Enkephalin secretion evoked by 10–100 nM PACAP-27 was not inhibited by 1 μM (-)-202-791, an L-type specific dihydropyridine calcium channel blocker, but was inhibited 65–80% by the arylalkylamine calcium channel blocker D600. Forty mM potassium-evoked secretion was inhibited >90% by both D600 and (-)-202-791, 25 μM forskolin-induced secretion was blocked <50% by D600 and was unaffected by (-)-202-791, and 100 nM phorbol myristate acetate (PMA)-induced secretion was unaffected by either D600 or (-)-202-791. Enkephalin biosynthesis was increased by 10 nM PACAP-27, as measured by increased met-enkephalin pentapeptide content and PEnk A mRNA levels. PACAP-, forskolin-, and PMA-stimulated enkephalin synthesis were not blocked by D600 or (-)-202-791. Elevated potassium-induced enkephalin biosynthesis upregulation was completely blocked by either D600 or (-)-202-791 at the same concentrations. PACAP acting through type I PACAP receptors couples calcium influx-dependent enkephalin secretion and calcium influx-independent enkephalin biosynthesis in chromaffin cells. Restriction of the effects of enhanced calcium influx to stimulation of secretion, but not of biosynthesis, is unique to PACAP. By contrast, potassium-induced enkephalin biosynthesis upregulation is completely calcium influx dependent, specifically via calcium influx through L-type calcium channels. We propose that subpopulations of voltage-dependent calcium channels are differentially linked to intracellular signal transduction pathways that control neuropeptide gene expression and secretion in chromaffin cells.     

38-Amino acid form of pituitary adenylate cyclase activating peptide induces process outgrowth in human neuroblastoma cells

Permanent cell lines from human neuroblastoma, a sympathoadrenal malignancy, are known to exhibit a more neuronal phenotype characterized by outgrowth of long processes in response to multiple second messenger analogs. In this report we demonstrate that the 38-amino acid form of a peptide homologous to vasoactive intestinal peptide (VIP), pituitary adenylate cyclase activating peptide (PACAP), as well as the 27-amino acid form of PACAP, induce NB-OK human neuroblastoma cells to extrude cellular processes within 5 hr of treatment with either peptide at 10^?8 M. Treatment of NB-OK cells with PACAP38 or PACAP27 at 10^?8 M for 1 hr also elevates cAMP content greater than 100-fold and inositol lipid turnover 11- to 12-fold. VIP acutely induces process outgrowth and elevates intracellular second messenger levels in NB-OK cells only at higher concentrations, 10^?6 M or greater. In contrast to the equipotency of PACAP27 and PACAP38 in stimulating the outgrowth of processes observed after 5 hr of treatment, PACAP38 is much more potent than PACAP27 when NB-OK cells are scored for process outgrowth after 72 hr of treatment. Correlating with the extended time course over which morphologic changes are seen with PACAP38, cAMP levels remain elevated for a more prolonged time span during treatment with PACAP38 than PACAP27. After 72 hr of treatment with PACAP38 versus treatment with PACAP27, cAMP levels are elevated 10-fold versus 3-fold, respectively. PACAP38 at 10^?8 M also induces process outgrowth in two additional human neuroblastoma lines tested, SMS-KAN and LA-N-1, whereas PACAP27 and VIP at the same concentration are less effective. The effects of PACAP38 on neuroblastoma cells may be a model for neurotrophic activities of PACAP38 via G protein-linked intracellular messengers in human sympathoadrenal cells. © 1993 Wiley-Liss, Inc.     

Neuroanatomical and Physiological Evidence for the Involvement of Pituitary Adenylate Cyclase-Activating Polypeptide in the Regulation of the Distal Lobe of the Frog Pituitary

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 38 amino-acid peptide which belongs to the glucagon/secretin/ vasoactive intestinal peptide superfamily. The sequence of PACAP is identical in all mammalian species studied so far but frog PACAP differs by one amino-acid from mammalian PACAP. The aim of the present study was to investigate the presence of PACAP in the hypothalamo-pituitary complex of the frog Rana ridibunda and to determine the biological activity of frog PACAP on homologous pituitary cells. The distribution of PACAP-containing neurons and fibers was examined by the indirect immunofluores-cence method using an antiserum raised against the N-terminal region of the peptide. In the hypothalamus, PACAP-immunoreactive perikarya were localized in the preoptic nucleus and the dorsal and ventral infundibular nuclei. Beaded nerve fibers were observed coursing from the ventral infundibular nucleus to the external vascular layer of the median eminence. A dense network of immunoreactive axons terminated in the vicinity of the capillaries of the hypophysial portal system. The neurointermediate lobe and the distal lobe of the pituitary were devoid of immunoreactive elements. The amount of PACAP-like immunoreactive material in hypothalamus extracts was measured by radioimmunoassay; the apparent concentration of PACAP was 4.5 ng/mg protein. Synthetic frog PACAP38 and PACAP27 induced a similar dose-dependent stimulation of cAMP production in isolated frog distal lobe pituitary fragments (ED₅₀= 2 × 10^?8 M). At the maximum dose tested (5 × 10^?6 M), both frog PACAP38 and PACAP27 produced a 4-fold increase in cAMP production. In contrast, the truncated form [Des-His¹frog PACAP38 did not affect adenylate cyclase activity demonstrating therefore that the N-terminal histidyl residue is essential for the biological activity of the peptide. [Des-His¹]frog PACAP38 did not antagonize the stimulatory effect of frog PACAP38 or PACAP27 on cAMP production. Taken together, these data support the concept that, in amphibians as in mammals, PACAP may act as a hypophysiotropic neuropeptide.     

Desensitization of the human D1 dopamine receptor: Evidence for Involvement of both cyclic AMP-dependent and receptor-specific protein kinases

Human SK-N-MC neurotumor cells express D₁ dopamine receptors coupled to adenylyl cyclase. Following exposure of the cells to dopamine, there was a time- and dose-dependent loss of dopamine responsiveness that involved both a reduction in the maximum response (V_max) and a shift to less sensitivity in the dose response (K_act). Although the shift in K_act occurred more rapidly than the reduction in V_max, both effects were completed within a 20-min exposure of the cells to dopamine. During this rapid period of desensitization, there was no loss of D₁ receptors from membranes prepared from dopamine-treated cells, but there was a reduction in the proportion of agonist high-affinity binding sites. More prolonged exposure to D₁ agonists led to a progressive loss of binding activity. The desensitization was homologous as exposure of the cells, which have β₁-adrenergic receptors, to isoproterenol did not alter their response to dopamine. A shift in K_act but not in V_max was observed when membranes from control cells were incubated with dopamine, ATP, and the catalytic subunit of cyclic AMP-dependent protein kinase (PKA). To pursue the role of protein kinases in the desensitization process, cells were transiently made permeable, loaded with a PKA inhibitor or with heparin, an inhibitor of the β-adrenergic receptor kinase, and exposed to dopamine. The PKA inhibitor blocked the shift in K_act, whereas heparin inhibited the reduction in V_max. Our results suggest that desensitization of human D₁ dopamine receptors involves both PKA and a receptor-specific kinase and that the action of both kinases in intact cells requires agonist-occupied receptors.     

Pituitary adenylyl cyclase‐activating polypeptide controls the proliferation of retinal progenitor cells through downregulation of cyclin D1

During retinal development, cell proliferation and exit from the cell cycle must be precisely regulated to ensure the generation of the appropriate numbers and proportions of the various retinal cell types. Previously, we showed that pituitary adenylyl cyclase‐activating polypeptide (PACAP) exerts a neuroprotective effect in the developing retina of rats, through the cAMP–cAMP‐dependent protein kinase (protein kinase A) (PKA) pathway. Here, we show that PACAP also regulates the proliferation of retinal progenitor cells. PACAP, PACAP‐specific receptor (PAC1), and the receptors activated by both PACAP and vasoactive intestinal peptide (VIP), VPAC1 and VPAC2, are expressed during embryonic and postnatal development of the rat retina. Treatment of retinal explants with PACAP38 reduced the incorporation of [³H]thymidine as well as the number of 5‐bromo‐2′‐deoxyuridine‐positive and cyclin D1‐positive cells. Pharmacological experiments indicated that PACAP triggers this antiproliferative effect through the activation of both PAC1 and VPACs, and the cAMP–PKA pathway. In addition, PACAP receptor activation decreased both cyclin D1 mRNA and protein content. Altogether, the data support the hypothesis that PACAP is a cell‐extrinsic regulator with multiple roles during retinal development, including the regulation of proliferation in a subpopulation of retinal progenitor cells.     

Pituitary adenylate cyclase-activating polypeptide (PACAP) can act as determinant of the tyrosine hydroxylase phenotype of dopaminergic cells during retina development

In the chick retina, dopaminergic cells are generated between embryonic days 3 and 7 (E3/E7). However, the expression of tyrosine hydroxylase (TH), the first enzyme in the catecholamine synthetic pathway, is only detected after E11/E12. During the interval comprising E7 to E12, signals conveyed by cAMP are important to determine the TH phenotype. The present study shows that pituitary adenylyl cyclase-activating polypeptide (PACAP), via cAMP, is a major endogenous component in defining the TH phenotype of retina dopaminergic cells during development. PACAP type 1 receptor and its mRNA were detected in retinas since E6. PACAP was also immunodetected in cells localized in the inner nuclear layer of retinas since E8. This peptide promoted greater than 10-fold increase in cAMP accumulation of retinas obtained from embryos since E8, an effect that was blocked by PACAP6-38 (PAC1 receptor antagonist). In cultured retina cells from E8 and E9, maintained for 6 days in vitro with 10 nM PACAP (for 5 days), the number of dopaminergic cells expressing tyrosine hydroxylase increased 2.4-fold. The cAMP analog, 8-Br-cAMP and 3-isobutyl-1-methylxanthine (IBMX, a phosphodiesterase inhibitor) also increased the number of tyrosine hydroxylase-positive cells by 4- to 6-fold. IBMX plus PACAP treatment resulted in 17-fold increase in the number of cells positive for tyrosine hydroxylase. Under this condition the amount of tyrosine hydroxylase expression, as detected by western blot analysis, was also increased. The protein kinase-A inhibitor, rp-cAMPS, significantly reduced the effect of PACAP. Our data show that this peptide is an important factor influencing the definition of the tyrosine hydroxylase phenotype of retina dopaminergic cells within a narrow window of development.     

Alpha 1-adrenoceptor augmentation of beta-stimulated cAMP formation is enhanced by estrogen and reduced by progesterone in rat hypothalamic slices

These experiments examined the influence of estradiol and progesterone given in vivo on norepinephrine (NE) regulation of cAMP synthesis in hypothalamic and preoptic area slices in vitro. Administration of progesterone to estrogen-primed female rats attenuated NE-induced slice cAMP accumulation. This hormone-dependent reduction in NE-stimulated cAMP synthesis was observed in slices incubated with TTX and in slices prepared from hypophysectomized rats, suggesting that progesterone effects on NE receptor activation of cAMP-generating systems are not secondary to the release of neurotransmitters that inhibit adenylyl cyclase or to changes in pituitary hormone secretion. Progesterone suppression of NE-induced cAMP formation could be prevented by incubating slices in the presence of a phorbol ester. In additional studies, the activity of beta-NE receptors was assessed by measuring isoproterenol (ISO)-stimulated cAMP accumulation in the presence of the phosphodiesterase inhibitor RO-20-1724, and the activity of alpha 1 receptors was evaluated by measuring phenylephrine (PHE) augmentation of the ISO response. Estradiol reduced the cAMP response to ISO in both hypothalamic and preoptic area slices, and this effect was not reversed by subsequent progesterone treatment. Estradiol also enhanced PHE augmentation of ISO-stimulated cAMP synthesis. Moreover, administration of progesterone subsequent to estradiol eliminated alpha 1-receptor augmentation of the ISO response. An alpha 1 enhancement of the ISO response is observed if the progestin receptor antagonist RU 38486 is administered before progesterone. Progesterone also abolished PHE potentiation of vasoactive intestinal polypeptide-stimulated cAMP accumulation. In contrast, neither phorbol ester nor muscarinic (carbachol) potentiation of the cAMP response to ISO was affected by progesterone. The data suggest that ovarian steroids regulate the coupling of both alpha 1 and beta receptors to the membrane effector systems that generate intracellular cAMP.     

Mechanism of Action of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) in Human Nonfunctioning Pituitary Tumors

Several evidence suggest that pituitary adenylate cyclase activating polypeptides (PACAP-38 and -27) could function as hypophysiotropic factors. Both peptides interact with either the type I receptor, which preferentially binds the two PACAPs and has a much lower affinity for vasoactive intestinal polypeptide (VIP) or the type II receptor, which binds the two PACAPs and VIP with a nearly equal affinity. In addition to the stimulation of adenylyl cyclase (AC) activity, in different cell types PACAP causes an increase of cytosolic calcium levels ([Ca²⁺]i), consequent to phospholipase-C activation. In the present study, we investigated the effect of PACAP on cAMP formation and [Ca²⁺]i levels in 16 human nonfunctioning pituitary adenomas (NFPA). PACAP-38 increased cAMP formation in all tumors; the peptide stimulated either AC activity in membrane preparations from 26 ± 10 to 214 ± 179 pmol/mg prot/min (P < 0.01) or cAMP efflux from 12 ± 5.4 to 73.2 ± 32 pmol/well (P < 0.01) in cultured cells. The effect, detectable at concentrations higher than 1-10 pM, was maximal at 0.1-10 nM. While PACAP-38 and PACAP-27 were nearly equally effective and potent, 100-fold higher concentrations of VIP were required to obtain similar AC activation. GHRH and CRH were ineffective in any NFPA. The PACAP effect was not antagonized by a VIP antagonist, while PACAP fragment 6–27 amide partially reduced the stimulatory effects of both PACAP-27 and VIP in 2 out of 3 tumors tested. PACAP-38 caused a [Ca²⁺]i rise in cells obtained from 7 NFPA (from 110 ± 34 to 151 ± 40 nM [Ca²⁺]i, P < 0.05) while in the remaining 7 the peptide was ineffective at any concentrations tested (from 1 nM to 10 μM). In the responsive tumors, PACAP-38 effect was not consequence of phospholipase-C activation since removal of extracellular Ca²⁺ as well as blockade of L-type Ca²⁺ channels by dihydropyridine antagonists abolished [Ca²⁺]i increase triggered by the peptide. These data indicate that PACAP is by far the most potent activator of cAMP formation in NFPA and suggest a possible modulatory action of this peptide on cell growth.     

Involvement of metabotropic glutamate receptors in Gi- and Gs-dependent modulation of adenylate cyclase activity induced by a novel cognition enhancer NS-105 in rat brain

The effect of a novel cognition enhancer [(+)-5-oxo-

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-prolinepiperidinamide monohydrate] (NS-105) on cAMP formation was investigated in both slices and membranes of the rat cerebral cortex. NS-105 (10⁻⁸–10⁻⁶ M) inhibited forskolin-stimulated cAMP formation in membranes, however, the compound significantly enhanced the cAMP formation in pertussis toxin-pre-treated membranes, an action that was abolished by cholera toxin. In contrast, in digitonin-permeabilized membranes, NS-105 had no influence on Mn²⁺-stimulated cAMP formation. Both of the inhibitory and facilitatory actions of NS-105 on cAMP formation were mimicked by a metabotropic glutamate receptor (mGluR) agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) and an adrenergic α₂ agonist UK-14,304, and blocked by a mGluR antagonist 2-amino-3-phosphonopropanoate but not by an α₂ antagonist yohimbine. In cortical slices, NS-105 (10⁻⁸–10⁻⁷ M) inhibited forskolin-stimulated cAMP accumulation but enhanced isoproterenol-stimulated cAMP accumulation, as did by a GABA_B agonist (−)baclofen. On the other hand, (−)baclofen, while it significantly inhibited cAMP accumulation in slices, did no longer inhibit cAMP accumulation, when treated with NS-105 (10⁻⁸–10⁻⁵ M). Similarly, (−)baclofen-induced inhibition of the cAMP accumulation was reversed by 1S,3R-ACPD and UK-14,304. NS-105 (10⁻⁶) increased [³⁵S]GTPγS binding in the intact but not digitonin-permeabilized cortical membranes, as produced by UK-14,304, although the compound (10⁻⁹–10⁻³ M) had no influence on various neurotransmitter receptor bindings, including α₂ receptors. These results suggest that NS-105 modulates adenylate cyclase activity by stimulating mGluRs which might coupled to both G_i/G_o and G_s. © 1997 Elsevier Science B.V. All rights reserved.     

Orexins/Hypocretins Acting at G<Subscript>i</Subscript> Protein-Coupled OX<Subscript>2</Subscript> Receptors Inhibit Cyclic AMP Synthesis in the Primary Neuronal Cultures

Orexins A and B are newly discovered neuropeptides with pleiotropic activity. They signal through two G protein-coupled receptors: OX₁ and OX₂. In this study, we examined the expression of orexin receptors and effects of the receptors’ activation on cyclic AMP formation in the primary neuronal cell cultures from rat cerebral cortex. Both types of orexin receptors were expressed in rat cortical neurons; the level of OX₂R was markedly higher compared to OX₁R. Orexin A (an agonist of OX₁R and OX₂R) and [Ala¹¹-D-Leu¹⁵]orexin B (a selective agonist of OX₂R) did not affect basal cyclic AMP formation in the primary neuronal cell cultures. Both peptides (0.001–1 μM) inhibited, in a concentration-dependent manner and IC₅₀ values in low nanomolar range, the increase in the nucleotide production evoked by forskolin (1 μM; a direct activator of adenylyl cyclase), pituitary adenylate cyclase-activating polypeptide (PACAP27; 0.1 μM), and vasoactive intestinal peptide (VIP; 3 μM). Effects of orexin A on forskolin-, PACAP27-, and VIP-stimulated cyclic AMP synthesis were blocked by TCS OX2 29 (a selective antagonist of OX₂R), and unaffected by SB 408124 (a selective antagonist of OX₁R). Pretreatment of neuronal cell cultures with pertussis toxin (PTX) abolished the inhibitory action of orexin A on forskolin- and PACAP-stimulated cyclic AMP accumulation. It is suggested that in cultured rat cortical neurons orexins, acting at OX₂ receptors coupled to PTX-sensitive G_i protein, inhibit cyclic AMP synthesis.     

Identification of Intracellular Signaling Cascades Mediating the PACAP-induced Increase in Guinea Pig Cardiac Neuron Excitability

The pituitary adenylate cyclase-activating polypeptide (PACAP) increases excitability of guinea pig cardiac neurons, an effect mediated through activation of PAC1 receptors. The signaling cascades that couple activation of the PAC1 receptor to alterations in membrane ionic conductances responsible for the PACAP effect are unknown. Intracellular recordings were made from neurons in kinase inhibitor-treated cardiac ganglia preparations to determine which of the intracellular cascades activated by PAC1 receptor stimulation mediate the PACAP effect. In control cells, long depolarizing-current steps elicited one to three action potentials. In contrast, during the application of 10 nM PACAP, depolarizing-current pulses elicited multiple action potential firing (greater than or equal to five action potentials) in 79% of the neurons. Pretreatment with an adenylyl cyclase inhibitor, SQ 22536 (100 microM), suppressed the PACAP-induced increase in excitability, whereas the presence of U-73122 (10 microM), a potent phospholipase C (PLC) inhibitor, had no effect. Thus, the activation of adenylyl cyclase, but not PLC, was a critical step mediating the PACAP effect. Pretreatment with H-89 (1 microM), a protein kinase A inhibitor, and PD 98059 (50 microM), a MEK kinase inhibitor, also significantly blunted the PACAP-induced increase in excitability. Furthermore, treatment with forskolin (5 microM), an activator of adenylyl cyclase, or exposure to the cell-permeable cyclic adenosine monophosphate (cAMP) analogue, 8-bromo-cAMP (1 mM), partially recapitulated the effect of PACAP on excitability. We conclude that the activation of signaling cascades downstream of cAMP mediate the PACAP-induced increase in cardiac neuron excitability.     

Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) and Its Receptors Are Present and Biochemically Active in the Central Nervous System of the Pond Snail Lymnaea stagnalis

PACAP is a highly conserved adenylate cyclase (AC) activating polypeptide, which, along with its receptors (PAC1-R, VPAC1, and VPAC2), is expressed in both vertebrate and invertebrate nervous systems. In vertebrates, PACAP has been shown to be involved in associative learning, but it is not known if it plays a similar role in invertebrates. To prepare the way for a detailed investigation into the possible role of PACAP and its receptors in a suitable invertebrate model of learning and memory, here, we undertook a study of their expression and biochemical role in the central nervous system of the pond snail Lymnaea stagnalis. Lymnaea is one of the best established invertebrate model systems to study the molecular mechanisms of learning and memory, including the role of cyclic AMP-activated signaling mechanisms, which crucially depend on the learning-induced activation of AC. However, there was no information available on the expression of PACAP and its receptors in sensory structures and central ganglia of the Lymnaea nervous system known to be involved in associative learning or whether or not PACAP can actually activate AC in these ganglia. Here, using matrix-assisted laser desorption ionization time of flight (MALDI-TOF) and immunohistochemistry, we established the presence of PACAP-like peptides in the cerebral ganglia and the lip region of Lymnaea. The MALDI-TOF data indicated an identity with mammalian PACAP-27 and the presence of a squid-like PACAP-38 highly homologous to vertebrate PACAP-38. We also showed that PACAP, VIP, and maxadilan stimulated the synthesis of cAMP in Lymnaea cerebral ganglion homogenates and that this effect was blocked by the appropriate general and selective PACAP receptor antagonists.     

Molecular, cellular, and functional characterizations of pituitary adenylate cyclase-activating polypeptide and its receptors in the cerebellum of New and Old World monkeys

The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) exerts trophic activities during cerebellar development, and a neuroprotective effect of PACAP has been demonstrated in pathological conditions such as stroke. However, all these data have been obtained in rodents, and neuroprotective effects of PACAP in primates remain unknown. Because of their evolutionary relationships with humans, monkeys represent powerful models for validating the therapeutic interest in PACAP. The objective of the present study was to characterize PACAP and its receptors in the cerebellum of two nonhuman primates. RT-PCR and in situ hybridization experiments revealed that PACAP is expressed in the cerebellum by Purkinje cells. Via immunohistochemistry, PACAP was detected in Purkinje cells and radial glial fibers. With regard to PACAP receptors, PAC1-R and VPAC1-R were detected by RT-PCR. In situ hybridization revealed a strong expression of PAC1-R and VPAC1-R in the granule cell layer (GCL), and VPAC1-R was also expressed in the Purkinje cell layer. A high density of PACAP binding sites was visualized in the GCL and the Purkinje cell layer. Competition studies indicated that, in the GCL, PACAP induced complete displacement of [(125)I]PACAP27 binding, whereas vasoactive intestinal polypeptide (VIP) was a weak competitor. In contrast, in the Purkinje cell layer, both PACAP and VIP displaced [(125)I]PACAP27 binding. Measurement of cAMP levels showed that PACAP is a powerful activator of adenylyl cyclase, whereas VIP is about 100-fold less potent. Altogether, these observations constitute the first demonstration of a functional PACAPergic system in monkey cerebellum. They strongly suggest that neuroprotective effects of PACAP can be transposed to primates, including human.     

PACAP Stimulation of Gonadotropin-II Secretion in Goldfish Pituitary Cells: Mechanisms of Action and Interaction with Gonadotropin Releasing Hormone Signalling

Pituitary adenylate cyclase-activating polypeptide (PACAP) has recently been shown to be a hypophysiotropic factor in the goldfish. In this study, we examined the mechanisms of PACAP action on goldfish maturational gonadotropin (GTH-II) release using primary cultures of pituitary cells. The GTH-II response to mammalian PACAP1-38 (mPACAP) was inhibited by a PACAP receptor antagonist suggesting a receptor-mediated action. Addition of either an adenylate cyclase inhibitor or a protein kinase A (PKA) inhibitor reduced the mPACAP-induced GTH-II release. In addition, when GTH-II release was already stimulated by either forskolin or 8-bromo-cAMP (8Br-cAMP), mPACAP did not further increase GTH-II secretion. These results strongly implicated the involvement of an adenylate cyclase/cAMP/PKA pathway in PACAP-stimulated GTH-II release. Although mPACAP induced a rise in intracellular Ca2+ level in identified gonadotropes, results with voltage-sensitive Ca2+ channel inhibitors indicated that the GTH-II responses to mPACAP, forskolin and 8Br-cAMP did not depend upon Ca2+ entry through these channels. Two protein kinase C (PKC) inhibitors did not affect mPACAP-elicited GTH-II release, and mPACAP further increased GTH-II secretion in the presence of PKC activators. These results indicate that PKC-dependent elements are not essential for the stimulatory action of mPACAP in gonadotropes. Interestingly, while GTH-II responses to a stimulatory concentration of mPACAP were additive to responses elicited by maximal effective concentrations of two endogenous gonadotropin releasing hormones (GnRHs), a subthreshold concentration of mPACAP potentiated GnRH and PKC activator stimulation of GTH-II secretion. Similarly, submaximal concentrations of forskolin potentiated the GTH-II response to the PKC activator, tetradecanoyl phorbol acetate. These data suggest that PACAP and its cAMP-dependent signalling mechanisms provide an alternate stimulatory input to goldfish gonadotropes and may influence the effectiveness of the major neuroendocrine control exerted by the PKC-dependent GnRH signalling pathway.     

Receptors for VIP and PACAP in guinea pig cerebral cortex

Receptors for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) in guinea pig cerebral cortex were characterized by (1) radioreceptor binding of ¹²⁵I-labeled VIP (human/rat/porcine), and (2) cyclic AMP (cAMP) formation. Saturation analysis of ¹²⁵I-VIP binding to membranes of guinea pig cerebral cortex resulted in a linear Scatchard plot, suggesting the presence of a single class of high-affinity receptor-binding sites, with a Kd of 0.63 nM and a Bmax of 77 fmol/mg protein. Various peptides from the PACAP/VIP/secretin family displaced the specific binding of ¹²⁵I-VIP to guinea pig cerebrum with the relative rank order of potency: chicken VIP (cVIP) ≥ PACP38 ∼ PACAP27 ∼ guinea pig VIP (gpVIP) ≥ mammalian (human/rat/porcine) VIP (mVIP) > peptide histidine-methionine (PHM) > peptide histidine-isoleucine (PHI) > secretin. Analysis of the competition curves revealed displacement of ¹²⁵I-VIP from high- and lower-affinity binding sites, with IC50 values in the picomolar and the nanomolar range, respectively. About 70% of the specific ¹²⁵I-VIP-binding sites in guinea pig cerebral cortex were sensitive to Gpp(NH)p, a nonhydrolyzable analog of GTP. Pituitary adenylate cyclase-activating polypeptide 38 (PACAP38), PACAP27, cVIP, gpVIP, mVIP, PHM, and PHI stimulated cAMP production in [³H]adenine-prelabeled slices of guinea pig cerebral cortex in a concentration-dependent manner. Of the tested peptides, the most effective were PACAP38 and PACAP27, which at a 1 μM concentration produced a 17- to 19-fold rise in cAMP synthesis, increasing the nucleotide production to approx 11% conversion above the control value. The three forms of VIP (cVIP, mVIP, and gpVIP) at the highest concentration used, i.e., 3 μM, produced net increases in cAMP production in the range of 8–9% conversion, whereas 5 μM PHM and PHI, by, respectively, 6.7% and 4.9% conversion. It is concluded that cerebral cortex of guinea pig contains VPAC- type receptors positively linked to cAMP formation. In addition, the observed stronger action of PACAP (both PACAP38 and PACAP27), when compared to any form of VIP, on cAMP production in this tissue, suggests its interaction with both PAC1 and VPAC receptors.     

(N-stearyl, norleucine17) VIPhybrid is a broad spectrum vasoactive intestinal peptide receptor antagonist

The effects of a (N-stearyl, Norleucine17) vasoactive intestinal peptide hybrid ((SN)VIPhybrid) on cells stably transfected with VPAC,, VPAC2, or PAC1 receptors were investigated. (SN)VIPhybrid inhibited specific 125I-VIP binding to membranes derived from CHO cells transfected with VPAC, or VPAC2 receptors with high affinity (IC50 = 30 and 50 nM). (SN)VIPhyb inhibited specific 125I-PACAP-27 binding to membranes derived from NIH/3T3 cells transfected with PAC1 receptors with high affinity (IC50 = 65 nM). PACAP-27 caused cAMP elevation in NIH/3T3 cells transfected with PAC1 receptors and the increase cAMP caused by pituitary adenylated cyclase (PACAP) was inhibited by (SN)VIPhyb. Also, the increase in cAMP caused by VIP using CHO cells transfected with VPAC1 or VPAC2 receptors was antagonized by (SN)VIPhyb. These results indicate that (SN)VIPhyb is an antagonist for VPAC1, VPAC2, and PAC1 receptors.     

l-DOPA-induced dyskinesia in hemiparkinsonian rats is associated with up-regulation of adenylyl cyclase type V/VI and increased GABA release in the substantia nigra reticulata

l-DOPA treatment induces abnormal involuntary movements (AIMs) in Parkinson's patients and experimental animals. We examined the relationship between the development of AIMs (dyskinesia) and changes in [³H]-GABA release and cAMP signaling in striatonigral terminals of rats with unilateral 6-OHDA lesions. Analysis of AIMs scores in hemiparkinsonian rats treated with l-DOPA for 20 days was fitted by the sum of two Gaussian distributions showing the presence of two populations: one with mild and the other with severe dyskinesia. cAMP signaling was evaluated in the two populations by determining changes in cAMP formation, Gα_olf and adenylyl cyclase type V/VI levels. In animals that were not treated with l-DOPA, all the parameters were significantly increased in the denervated side. In the animals that had mild dyskinesia, l-DOPA treatment normalized these parameters. In contrast, in the animals in which l-DOPA treatment induced severe dyskinesia all the parameters, except for Gα_olf levels, were significantly higher in the denervated side. Similarly, D1-stimulated [³H]-GABA release was not elevated in l-DOPA-treated animals with mild dyskinesia but was increased in animals with severe dyskinesia. Changes in Gα_olf and adenylyl cyclase type V/VI levels in the striatum paralleled the response in the SNr. The linkage between the changes in [³H]-GABA release and cAMP activity was further evaluated with the selective adenylyl cyclase V/VI antagonist NKY80. This inhibitor blocked the increases of both [³H]-GABA release and cAMP production. These results indicate that increased expression of adenylyl cyclase V/VI is a major determinant of increased GABAergic transmission in the substantia nigra pars reticulata of animals in which l-DOPA induces severe dyskinesia.     

PACAP and VIP Stimulate Ca2+ Oscillations in Rat Gonadotrophs through the PACAP/VIP Type 1 Receptor (PVR1) Linked to a Pertussis Toxin-Insensitive G-Protein and the Activation of Phospholipase C-β

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are hypothalamic factors that play roles in the regulation of anterior pituitary cell activity. PACAP exists in 2 forms physiologically, a 38 amino acid form (PACAP38) and a form possessing the N-terminal 27 amino acids of PACAP38 (PACAP27). We have previously shown that PACAP38 stimulates an increase in [Ca²⁺]_i in rat gonadotrophs. In an attempt to identify the PACAP receptor type underlying this effect, we compared the potency of PACAP38, PACAP27 and VIP to stimulate Ca²⁺ changes in identified single rat gonadotrophs. All 3 peptides at 100  nM were capable of stimulating high amplitude Ca²⁺ oscillations, which were also observed in the absence of extracellular Ca²⁺. The order of potency of these peptides was PACAP38>PACAP27>VIP, and a potent antagonist of the PACAP/VIP type II binding site ([4-Cl-D-Phe⁶, Leu¹⁷]-VIP) failed to block these responses, suggesting that these effects are mediated through a PACAP/VIP type 1 receptor (PVR1). The Ca²⁺ responses to PACAP38 and VIP were unaffected by overnight treatment of the cells with pertussis toxin (PTX; 250  ng/ml) indicating that these responses are mediated by a PTX-insensitive G-protein. Finally, the Ca²⁺ responses stimulated by PACAP38 and VIP were blocked by the phospholipase C- β blocker U73122 (5  μM). In summary, PACAP stimulates Ca²⁺ oscillations in rat gonadotrophs through the activation of the PVR1 linked to a PTX-insensitive G-protein and the activation of phospholipase C- β . VIP can stimulate the same pathway in rat gonadotrophs, although it is at least 100 fold less potent than PACAP38.     

垂体腺苷酸环化酶激活肽增加新生大鼠小脑颗粒细胞钙通道电流

应用膜片箝"全细胞"电流记录方法，研究了垂体腺苷酸环化酶激活肽（PACAP）对新生大鼠小脑颗粒细胞高电压激活（HVA）的钙电流作用。实验结果发现：PACAP－38、PACAP－27能明显增加小脑颗粒细胞钙电流的幅值，两者增加钙内流的效应无显著差异，并且都具有脱敏现象；而出于同一家族的血管活性肠肽（VIP）对此电流却没有任何影响。当记录电极内加入GTPγS后，PACAP增加钙电流的效应成为不可逆；而细胞内液加入GDPβS或预先用百日咳毒素孵育细胞12h后，PACAP的钙通道激活作用完全消失。此外，细胞内应用cAMP和磷酸二酯酶抑制剂3－isobutyl－1－methylxanthine（IBMX），既不影响钙通道本身的活动，也不修饰PACAP增加颗粒细胞钙通道电流的效应。我们的结果提示：新生大鼠小脑颗粒细胞上存在着具有功能意义的PACAP受体，该受体被PACAP－38和PACAP－27激活后可增加细胞膜上钙通道的开放，促进钙内流。PACAP的这种作用与百日咳毒素敏感的G蛋白偶联，但并不依赖腺苷酸环化酶参与的第二信使系统。