Interaction Between Adrenergic and Peptide Stimulation in the Rat Pineal: Pituitary Adenylate Cyclase-Activating Peptide

Abstract: The 27 amino acid peptide, pituitary adenylate cyclase-activating polypeptide (PACAP-27), and its 38 amino acid analogue, PACAP-38, stimulate serotonin- N -acetyltransferase (NAT) activity and N -acetylserotonin (NAS) and melatonin content of pineal glands from adult rats. Maximal stimulation of rat pineal NAT by PACAP-38 is not increased further significantly by concurrent stimulation with the two related peptides, vasoactive intestinal polypeptide (VIP) and/or peptide N-terminal histidine C-terminal isoleucine (PHI). Isoproterenol was a more potent inducer of NAT activity than any of these peptides alone or in combination. PACAP-38 also stimulates melatonin production by chicken pineal cells in culture as does VIP. Stimulation by both was not greater than after either alone. Prior stimulation of rat pineal NAT activity with VIP, PHI, or PACAP-38 reduces the magnitude of subsequent stimulation with PACAP-38 or forskolin. Concurrent stimulation of α-receptors or treatment with active phorbol ester augments rat pineal response to PACAP-38 stimulation just as it increases the response to VIP, PHI, and β-receptor stimulation. Pineals from newborn rats respond to PACAP-38 with an increase in NAT activity and the increase is augmented by concomitant α₁-adrenergic stimulation. The putative PACAP inhibitor PACAP (6–38) and the putative VIP inhibitor (Ac-Tyr, d -Phe)-GRF 1–29 amide, in 100–1,000-fold excess, did not affect the stimulatory activity of any of the peptides. Pineal melatonin concentration parallels changes in pineal NAT activity.     

Pituitary Adenylate Cyclase-Activating Polypeptide Causes Ca2+ Release from Ryanodine/Caffeine Stores Through a Novel Pathway Independent of Both Inositol Trisphosphates and Cyclic AMP in Bovine Adrenal Medullary Cells

Abstract: Pituitary adenylate cyclase-activating polypeptide (PACAP) causes both Ca²⁺ release and Ca²⁺ influx in bovine adrenal chromaffin cells. To elucidate the mechanisms of PACAP-induced Ca²⁺ release, we investigated expression of PACAP receptors and measured inositol trisphosphates (IP₃), cyclic AMP, and the intracellular Ca²⁺ concentration in bovine adrenal medullary cells maintained in primary culture. RT-PCR analysis revealed that bovine adrenal medullary cells express the PACAP receptor hop, which is known to couple with both IP₃ and cyclic AMP pathways. The two naturally occurring forms of PACAP, PACAP38 and PACAP27, both increased cyclic AMP and IP₃, and PACAP38 was more potent than PACAP27 in both effects. Despite the effects of PACAP on IP₃ production, the Ca²⁺ release induced by PACAP38 or by PACAP27 was unaffected by cinnarizine, a blocker of IP₃ channels. The potencies of the peptides to cause Ca²⁺ release in the presence of cinnarizine were similar. The Ca²⁺ release induced by PACAP38 or by PACAP27 was strongly inhibited by ryanodine and caffeine. In the presence of ryanodine and caffeine, PACAP38 was more potent than PACAP27. PACAP-induced Ca²⁺ release was unaffected by Rp-adenosine 3′,5′-cyclic monophosphothioate, an inhibitor of protein kinase A. Ca²⁺ release induced by bradykinin and angiotensin II was also inhibited by ryanodine and caffeine, but unaffected by cinnarizine. Although IP₃ production stimulated by PACAP38 or bradykinin was abolished by the phospholipase C inhibitor, U-73122, Ca²⁺ release in response to the peptides was unaffected by U-73122. These results suggest that PACAP induces Ca²⁺ release from ryanodine/caffeine stores through a novel intracellular mechanism independent of both IP₃ and cyclic AMP and that the mechanism may be the common pathway through which peptides release Ca²⁺ in adrenal chromaffin cells.     

Characterization of Pituitary Adenylate Cyclase-Activating Polypeptide 38 (PACAP38)-, PACAP27-, and Vasoactive Intestinal Peptide-Stimulated Responses in N1E-115 Neuroblastoma Cells

Abstract: In this study, the effects of three related peptides, pituitary adenylate cyclase-activating polypeptide 38 (PACAP38), PACAP27, and vasoactive intestinal peptide (VIP), on cyclic AMP (cAMP) accumulation and intracellular Ca²⁺ concentration ([Ca²⁺]_i) were compared in N1E-115 cells. PACAP38 and PACAP27 stimulated cAMP accumulation up to 60-fold with EC₅₀ values of 0.54 and 0.067 n M , respectively. The effect of VIP on cAMP accumulation was less potent. The binding of ¹²⁵I-PACAP27 to intact cells was inhibited by PACAP38 and PACAP27 (IC₅₀ values of 0.44 and 0.55 n M , respectively) but not by VIP. In fura-2-loaded cells, both PACAP38 and PACAP27 increased [Ca²⁺]_i with EC₅₀ values around 10 n M . The interactions of these three peptides with ionomycin, a Ca²⁺ ionophore, and 4β-phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, were also determined. Ionomycin increased the cAMP accumulation caused by all three peptides. With low concentrations of PACAP38 or PACAP27, the effect of PMA was inhibitory, whereas at higher concentrations of PACAP (>1 n M ), the effect of PMA was stimulatory. Similar to other agents that elevate cAMP, PACAP38 was an effective stimulator of neurite outgrowth. These results show that (a) PACAP27 and PACAP38 stimulate cAMP accumulation and increase [Ca²⁺]_i through the type I PACAP receptors in N1E-115 cells, (b) ionomycin enhances cAMP accumulation by all three peptides, and (c) activation of protein kinase C has a dose-dependent stimulatory or inhibitory effect on the PACAP38- or PACAP27-stimulated cAMP accumulation.     

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Stimulates Adenylyl Cyclase and Phospholipase C Activity in Rat Cerebellar Neuroblasts

Abstract: The presence of receptors for the novel neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) has been recently demonstrated in the external granule cell layer of the cerebellum, a germinative matrix that generates the majority of cerebellar interneurons. In the present study, we have taken advantage of the possibility of obtaining a culture preparation that is greatly enriched in immature cerebellar granule cells to investigate the effect of PACAP on the adenylyl cyclase and phospholipase C transduction pathways. The two molecular forms of PACAP, i.e., 27-(PACAP27) and 38-(PACAP38) amino-acid forms of PACAP, induced a dose-dependent stimulation of cyclic AMP production in granule cells. The potencies of PACAP27 and PACAP38 were similar (ED₅₀ = 0.12 ± 0.01 and 0.23 ± 0.07 n M , respectively), whereas vasoactive intestinal polypeptide (VIP) was ∼100 times less potent. PACAP27 and PACAP38 also induced a dose-dependent stimulation of polyphosphoinositide breakdown (ED₅₀ = 19.1 ± 6.3 and 13.4 ± 6.0 n M , respectively), whereas VIP had no effect on polyphosphoinositide metabolism. The effect of PACAP38 on inositol phosphate formation was significantly reduced by U-73122 and by pertussis toxin, indicating that activation of PACAP receptors causes stimulation of a phospholipase C through a pertussis toxin-sensitive G protein. In contrast, forskolin and dibutyryl cyclic AMP did not affect PACAP-induced stimulation of inositol phosphates. Taken together, the present results demonstrate that PACAP stimulates independently the adenylyl cyclase and the phospholipase C transduction pathways in immature cerebellar granule cells. These data favor the concept that PACAP may play important roles in the control of proliferation and/or differentiation of cerebellar neuroblasts.     

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Regulation of Sympathetic Neuron Neuropeptide Y and Catecholamine Expression

Abstract: Two forms of pituitary adenylate cyclase-activating polypeptide (PACAP), the 38- and 27-amino-acid forms (PACAP38 and PACAP27, respectively), which share amino acid sequence homology with vasoactive intestinal peptide (VIP), were evaluated for their abilities to regulate sympathetic neuron catecholamine and neuropeptide Y (NPY) expression. PACAP38 and PACAP27 potently and efficaciously stimulated NPY and catecholamine secretion in primary cultured superior cervical ganglion (SCG) neurons; 100- to 1,000-fold higher concentrations of VIP were required to modulate secretion, suggesting that SCG neurons express the PACAP-selective type I receptor. PACAP38 elicited a sustained seven- to ninefold increase in the rate of NPY secretion and three-fold stimulation in the rate of catecholamine release. PACAP38 and PACAP27 produced parallel neuronal NPY and catecholamine release, but cellular levels of NPY and catecholamines were differentially regulated. Sympathetic neuron NPY content was decreased, whereas cellular total catecholamine levels were elevated by the PACAP peptides; total NPY and catecholamine levels (secreted plus cellular content) were increased. In concert with the increased total peptide and transmitter production, pro-NPY and tyrosine hydroxylase mRNA levels were elevated. Furthermore, PACAP38 was more efficacious than PACAP27 in regulating pro-NPY and tyrosine hydroxylase mRNA. SCG neuronal expression of mRNA encoding the type I PACAP receptor further supported the studies demonstrating that sympathetic neuronal levels of NPY and catecholamine content and secretion and mRNA are differentially regulated by the PACAP peptides.     

Vasoactive Intestinal Peptide and Pituitary Adenylate Cyclase-Activating Polypeptide Potentiate c-fos Expression Induced by Glutamate in Cultured Cortical Neurons

Abstract: Previous reports have demonstrated that glutamate stimulates c- fos mRNA expression in primary cultures of mouse cerebral cortical neurons. We show here that vasoactive intestinal peptide (VIP) induces c- fos mRNA expression; however, this effect of VIP is completely inhibited by the noncompetitive NMDA receptor antagonist MK-801, therefore indicating that VIP stimulates c- fos expression in a glutamate-dependent manner. A similar effect was observed with pituitary adenylate cyclase-activating polypeptide27 (PACAP27). At the intracellular level, coactivation of protein kinases A and C mediates the glutamate-dependent stimulation of c- fos expression evoked by VIP, because either H-89 or staurosporin inhibits the effect of VIP as well as that of glutamate. These results point to a "biochemical AND gate" mechanism, which implies the obligatory activation of both protein kinases A and C in the transduction of c- fos expression. In summary, this article provides evidence that VIP and PACAP27 potentiate the effect of glutamate, the principal effector on c- fos expression, suggesting that both peptides can increase the "throughput" or "strength" of glutamate-containing circuits in the cerebral cortex.     

Capsaicin-Induced Ion Fluxes Increase Cyclic GMP but Not Cyclic AMP Levels in Rat Sensory Neurones in Culture

Capsaicin, which induces fluxes of sodium, calcium, and potassium ions in a subset of both neonatal and adult rat dorsal root ganglion neurones, increased cyclic GMP (cGMP) levels by a factor of 20 (EC50 0.07 microM) to 10-20 pmol cGMP/mg protein in these cells. Cyclic AMP (cAMP) levels were unaffected. Nonneuronal cells derived from rat ganglia, and both neurones and nonneuronal cells from chick were unresponsive to capsaicin. Capsaicin-induced cGMP elevation in rat dorsal root ganglion (DRG) neurones was unaffected by pertussis toxin, lowered by compounds that block voltage-sensitive calcium channels, and was abolished by the removal of extracellular calcium. Calcium, guanidine, and rubidium fluxes were unaffected by treatment of DRG cells with sodium nitroprusside or dibutyryl cGMP. The cGMP response to capsaicin is thus a function of capsaicin-evoked calcium uptake through voltage-sensitive calcium channels. Elevated cGMP levels do not, however, contribute to capsaicin-evoked ion fluxes or to their desensitisation.     

Intracellular pH on Protein Kinase C and Ionomycin Potentiation of Isoproterenol-Stimulated Cyclic AMP and Cyclic GMP Production in Rat Pinealocytes

In rat pinealocytes, alpha 1-adrenergic activation, which leads to cytoplasmic alkalinization, also potentiates the beta-adrenergic stimulated cyclic AMP (cAMP) and cyclic GMP (cGMP) responses. Both elevation of intracellular calcium ([Ca2+]i) and activation of protein kinase C are involved in the potentiation mechanism. Recently, intracellular pH has also been found to modulate the adrenergic-stimulated cyclic nucleotide responses, suggesting intracellular pH may also affect the potentiation mechanism. This possibility was examined in the present study. Cytoplasmic alkalinization by ammonium chloride had an enhancing effect on the isoproterenol and ionomycin-stimulated cAMP and cGMP accumulation. In comparison, cytoplasmic acidification by sodium propionate reduced the isoproterenol and ionomycin-stimulated cAMP and cGMP responses. Direct measurement of [Ca2+]i indicated that neither ammonium chloride nor sodium propionate had an effect on the ionomycin-stimulated elevation of [Ca2+]i, suggesting their effects on cyclic nucleotide responses may be independent of [Ca2+]i. In cells stimulated by isoproterenol and an activator of protein kinase C, ammonium chloride had an enhancing effect on both cAMP and cGMP responses, whereas sodium propionate had no effect. Taken together, these results suggest that a site distal to elevation of [Ca2+]i and activation of protein kinase C, of importance to the potentiation mechanism, is modulated by intracellular pH.     

The Mammalian Pineal Expresses the Cone but Not the Rod Cyclic GMP Phosphodiesterase

Abstract: To determine the presence of cone or rod cyclic GMP phosphodiesterase (EC 3.1.4.17) in the mammalian pineal, extracts from adult rat and bovine pineals were injected onto a Mono Q anion-exchange HPLC column and eluted with an NaCl linear gradient. Fractions were immunoadsorbed with monoclonal antibodies specific to rod and cone phosphodiesterases (ROS-1) and to calmodulin-phosphodiesterase complexes (ACC). Profiles were assayed with 10 µmol/L [³H]cyclic GMP in the presence of calcium-calmodulin, histone, or trypsin. Rat and bovine pineals displayed a single peak of activity recognized by ROS-1, which corresponded to the activity of the cone but, not to the rod in bovine retina. ROS-1 immunoadsorbed ∼80% of the activity in the 60-day-old rat pineal but only 26% of the activity in bovine pineal. ACC immunoadsorbed the remaining activity in both species. Western blot analysis of rat pineal extracts revealed three polypeptides of ∼87, 15, and 10 kDa when probed with a rod/cone phosphodiesterase-specific antiserum. The specific activity of the cone-like phosphodiesterase in 10-day-old rat pineals was twice that of this isozyme in the bovine retina and 150 times that in the bovine pineal. The specific activity of phosphodiesterase in rat pineals decreased with age. We conclude that an enzyme with biochemical and antigenic characteristics similar to cone, but distinct from rod phosphodiesterase, is present in bovine and rat pineals.     

Calcium Potentiates Cyclic AMP Stimulation of Pineal Arylalkylamine N-Acetyltransferase

Abstract: Pineal arylalkylamine N -acetyltransferase ( N -acetyltransferase) controls large daily changes in melatonin production. It is generally thought that the activity of this enzyme is controlled by norepinephrine acting exclusively via elevation of cyclic AMP. However, norepinephrine also elevates pineal intracellular Ca²⁺ concentration ([Ca²⁺]_i), and it is not known whether Ca²⁺ is involved in regulating N -acetyltransferase activity other than through its established role in cyclic AMP production. In this study, the issue of whether Ca²⁺ enhances the effects of cyclic AMP on N -acetyltransferase activity was investigated. The effects of cyclic AMP protagonists (isobutylmethylxanthine, N ⁶, 2'- O -dibutyryladenosine 3',5'-cyclic monophosphate, 8-bromoadenosine 3',5'-cyclic monophosphate, and adenosine 3',5'-cyclic monophosphothioate, Sp-diastereomer) were examined in combination with [Ca²⁺]_i protagonists (A23187, ionomycin, and phenylephrine). All [Ca²⁺]_i protagonists potentiated the effects of cyclic AMP protagonists. For example, ionomycin potentiated the effects of low concentrations of 8-bromoadenosine 3',5'-cyclic monophosphate, and A23187 potentiated the effects of isobutylmethylxanthine without altering cyclic AMP accumulation. These findings indicate that Ca²⁺ and cyclic AMP probably act physiologically in a coordinated manner to stimulate N -acetyltransferase activity; these second messengers could act directly at one or more sites or through indirect actions mediated by kinases.     

Melatonin Synthesis in the Bovine Pineal Gland Is Regulated by Type II Cyclic AMP-Dependent Protein Kinase

Abstract: We investigated the expression of regulatory (R) and catalytic (C) subunits of cyclic AMP-dependent protein kinase (cAK; ATP:protein phosphotransferase; EC 2.7.1.37) in the bovine pineal gland. In total RNA extracts of bovine pineal glands moderate levels of RIα/RIIβ and high levels of Cα and Cβ mRNA were found. We were able to detect a strong signal for RII and C subunit at the protein level, whereas RI was apparently absent. Probing sections of the intact bovine pineal gland with RI and RII antibodies stained only RII in pinealocytes. Pairs of cyclic AMP analogues complementing each other in activation of type II cAK, but not cAKI-directed analogue pairs, showed synergistic stimulation of melatonin synthesis. Moreover, melatonin synthesis stimulated by the physiological activator norepinephrine in pineal cell cultures was inhibited by cAK antagonists. Taken together these results show the presence of RII regulatory and both Cα and Cβ catalytic subunits and thus cAKII holoenzyme in the bovine pineal gland. The almost complete inhibition of norepinephrine-mediated melatonin synthesis by the cAK antagonists emphasizes the dominant role of cyclic AMP as the second messenger and cAK as the transducer in bovine pineal signal transduction.     

Differential Effects of Two Protein Kinase C Inhibitors, Calphostin C and Gö6976, on Pineal Cyclic Nucleotide Accumulation

Abstract: In rat pinealocytes, protein kinase C (PKC) is involved in the α₁-adrenergic-mediated potentiation of β-adrenergic-stimulated cyclic nucleotide responses; however, the specific PKC isozyme(s) involved in the potentiation mechanism remain unknown. In the present study, we compared the effects of two PKC inhibitors, calphostin C, a specific inhibitor of PKC, and Gö6976, a selective inhibitor of PKCα and PKCβ₁, on the adrenergic-stimulated cyclic nucleotide accumulation in rat pinealocytes. Surprisingly, Gö6976 was found to have an enhancing effect on basal cyclic GMP and isoproterenol-stimulated cyclic AMP and cyclic GMP accumulation, an effect not shared by calphostin C. Gö6976 also increased the norepinephrine- and ionomycin-induced potentiation of isoproterenol-stimulated cyclic AMP and cyclic GMP accumulation, whereas the effect of calphostin C was inhibitory. The enhancing effect of Gö6976 was abolished in the presence of isobutylmethylxanthine or zaprinast, but not rolipram, suggesting that this effect of Gö6976 may be mediated through type V or the retinal type of phosphodiesterase. Based on these observations, we propose that some of the PKC isozyme(s) inhibited by calphostin C are involved in the potentiation of β-adrenergic-stimulated cyclic nucleotide responses and that they act by enhancing synthesis. However, PKC isozymes inhibited by Gö6976 appear to be basally active and tonically inhibit cyclic nucleotide accumulation through their stimulatory action on phosphodiesterase.     

Potentiation of Agonist-Stimulated Cyclic AMP Accumulation by Tyrosine Kinase Inhibitors in Rat Pinealocytes

Abstract: To study cross-talk mechanisms in rat pinealocytes, the role of tyrosine kinase or kinases in the regulation of adrenergic-stimulated cyclic AMP production was investigated. Both norepinephrine- and isoproterenol-stimulated cyclic AMP accumulation were increased by two distinct tyrosine kinase inhibitors, genistein or erbstatin, in a concentration-dependent manner. A similar increase was observed with two other inhibitors, tyrphostin B44 and herbimycin. In contrast, daidzein, an inactive analogue of genistein, was ineffective; whereas vanadate, a phosphotyrosine phosphatase inhibitor, reduced the adrenergic-stimulated cyclic AMP accumulation. The tyrosine kinase inhibitors were effective in potentiating the cholera toxin-or forskolin-stimulated cyclic AMP accumulation, indicating that their sites of action are at the postreceptor level. Neither an activator nor inhibitors of protein kinase C influenced the potentiation of the cyclic AMP responses by genistein, suggesting that the potentiation effect by tyrosine kinase inhibitors does not involve the phospholipase C/protein kinase C pathway. However, when the phosphodiesterase was inhibited by isobutylmethylxanthine, genistein failed to potentiate and vanadate did not inhibit the adrenergic-stimulated cyclic AMP accumulation, indicating that the phosphodiesterase is a probable site of action for these inhibitors. These results suggest that cyclic AMP metabolism in the pinealocytes is tonically inhibited by tyrosine kinase acting on the cyclic AMP phosphodiesterase.     

PACAP及其衍生物治疗糖尿病及其并发症的研究进展

垂体腺苷酸环化酶激活肽(PACAP)是近年新发现的神经多肽,属于促胰液素/胰高血糖素/血管活性肽(VIP)家族中的新成员,广泛分布于脑和外周组织器官,尤其在内分泌胰腺、性腺、呼吸和生殖系统,在能量代谢、神经保护、免疫系统等发挥重要生理学功能。糖尿病是一种常见的主要以高血糖为特征的慢性代谢性疾病,糖尿病并发症日益严重威胁着人们的身体健康,已成为导致糖尿病患者致死、致残的主要原因。主要对PACAP治疗糖尿病及其并发症国内外研究的最新进展进行论述。     

Stimulatory Effect of Histamine on Cyclic AMP Formation in Chick Pineal Gland

Abstract: Histamine (HA) potently stimulated cyclic AMP accumulation in intact pineal glands taken from light-exposed chicks. The action of HA was stronger in the presence of forskolin and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). The effect of HA was mimicked by HA H₁- and H₂-receptor-selective agonists in the following order of potency: HA > 4-methylhistamine (H₂) > 2-methylhistamine (H₁) > 2-thiazolylethylamine (H₁) ≫ dimaprit (H₂). The HA H₃-receptor-selective agonist (R)α-methylhistamine was poorly active. The effect of HA was antagonized by selective H₂-receptor blockers (tiotidine > oxmetidine > cimetidine = ranitidine) and was not significantly affected by the selective H₁- and H₃-receptor blockers mepyramine and thioperamide. A detailed analysis of an antagonistic action of ranitidine (versus HA) revealed a noncompetitive mode of action of the H₂ blocker. The stimulatory action of the H₁ agonist 2-thiazolylethylamine (both under basal conditions and in the presence of forskolin or IBMX) was not significantly influenced by three H₁-receptor-selective blockers (mepyramine, triprolidine, and diphenhydramine), but it was totally counteracted by ranitidine. Using accepted selective agonists and antagonists of the HA H₁, H₂, and H₃ receptor we were unable to identify clearly the receptor subtype mediating the HA action on the cyclic AMP-generating system of the chick pineal. It is suggested that the receptor under consideration may represent either an H₂-like (in terms of mammalian criteria) or avian-specific HA receptor. The data suggest that HA may be considered a modulator of the pineal activity in chicks.     

Presynaptic and Postsynaptic Effects of Neuropeptide Y in the Rat Pineal Gland

Abstract: Neuropeptide Y is colocalized with noradrena-line in sympathetic fibers innervating the rat pineal gland. In this article we present a study of the effects and mechanisms of action of neuropeptide Y on the pineal noradrenergic transmission, the main input leading to the rhythmic secretion of melatonin. At the presynaptic level, neuropeptide Y inhibits by 45%, with an EC₅₀ of 50 n M , the potassium-evoked noradrenaline release from pineal nerve endings. This neuropeptide Y inhibition occurs via the activation of pertussis toxin-sensitive G protein-coupled neuropeptide Y-Y2 receptors and is independent from, but additive to, the α₂-adrenergic inhibition of noradrenaline release. At the postsynaptic level, neuropeptide Y decreases by a maximum of 35%, with an EC₅₀ of 5 n M , the β-adrenergic induction of cyclic AMP elevation via the activation of neuropeptide Y-Y1 receptors. This moderate neuropeptide Y-induced inhibition of cyclic AMP accumulation, however, has no effect on the melatonin secretion induced by a β-adrenergic stimulation. On the contrary, in the presence of 1 m M ascorbic acid, neuropeptide Y potentiates (up to threefold) the melatonin secretion. In conclusion, this study has demonstrated that neuropeptide Y modulates the noradrenergic transmission in the rat pineal gland at both presynaptic and postsynaptic levels, using different receptor subtypes and transduction pathways.