EC:2.7.11.11 - FACTA Search

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Query: EC:2.7.11.11 (AMPK)
12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Pituitary adenylate-cyclase-activating peptide (PA-CAP) and PACAP-27 are novel hypothalamic peptides that can stimulate adenylate cyclase in cultured anterior pituitary cells. Because these peptides are present in the gut and are homologous with vasoactive intestinal peptide (VIP), itself known to stimulate intestinal ion transport, we examined the effects of these peptides on the T84 colonocyte cell line. Using cells grown on semipermeable supports and mounted in Ussing chambers, we showed that PACAP and PACAP-27 potently activate intestinal secretion. The half-maximal secretory response was produced with 0.5 nmol/L PA-CAP and 0.1 nmol/L PACAP-27. PACAP resembled VIP in that it stimulated a secretory response potentiated by carbachol, inhibited by bumetanide and barium chloride, and not further stimulated by the subsequent addition of VIP. Like VIP, PACAP also stimulated 5' cyclic adenosine monophosphate (cAMP) production and the phosphorylation of cellular proteins known to be substrates for cAMP-dependent protein kinase. In addition, PACAP inhibited 125I-VIP binding to T84 cells, and the secretion it stimulated was reduced by the VIP receptor antagonist, L-8-K. Thus PACAP and PACAP-27 potently stimulate colonocyte ion transport via mechanisms mediated by the VIP receptor and cAMP-dependent signaling.
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PMID:Pituitary adenylate cyclase-activating polypeptide stimulates secretion in T84 cells. 132 72

Two novel polypeptides known as pituitary adenylate cyclase activating polypeptide with 38 residues (PACAP38) and a shorter form of the peptide corresponding to the N-terminal 27 residues (PACAP27) were isolated from ovine hypothalamus. The N-terminal 28 residues of PACAP show 68% homology with vasoactive intestinal peptide (VIP). VIP has been reported to have specific binding sites in lymphocytes and inhibit mitogen-stimulated lymphocyte proliferation through a receptor-mediated stimulation of cAMP-dependent protein kinase. Using concanavalin A-induced proliferation of murine splenocytes as a model system, we now report that both PACAP38 and PACAP 27 can inhibit the proliferation of these cells in the same dose-dependent manner as VIP. The minimal effective concentration of the PACAPs was 10(-10)-10(-9) M. However, neither PACAP affected lipopolysaccharide-induced proliferation of murine splenocytes. The binding of [125I]PACAP27 to these splenocytes was rapid, time dependent, reversible, and proportional to the numbers of murine splenocytes. Scatchard analysis of displacement of the bound tracer by unlabeled PACAP27 indicated the existence of two classes of binding sites. The dissociation constant (Kd) was 0.86 +/- 0.24 nM and the maximal binding capacity (Bmax) was 1.13 +/- 0.39 fmol/10(6) cells for the high affinity binding site. The low affinity binding site had a Kd of 0.13 +/- 0.03 microM with a Bmax of 73.5 +/- 9.5 fmol/10(6) cells. PACAP38 and VIP displaced the binding of [125I]PACAP27 in the same manner as PACAP27 and Scatchard analyses indicated the presence of two classes of binding sites with Kd and Bmax similar to those for PACAP27. Furthermore, when [125I]VIP was used as a radiolabeled ligand, PACAP27 and PACAP38 displaced the [125I]VIP binding to the same degree as unlabeled VIP. Scatchard analysis indicated that there was no significant difference of the Kd or Bmax between PACAP and VIP. Taken together, these data suggest that PACAPs bind to a site similar or identical to that used by VIP which inhibit the proliferation of murine splenocytes induced by concanavalin A.
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PMID:Inhibition of mitogen-stimulated proliferation of murine splenocytes by a novel neuropeptide, pituitary adenylate cyclase activating polypeptide: a comparative study with vasoactive intestinal peptide. 198 59

The hypothalamic factor pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates an increase in the cytoplasmic free Ca2+ ion concentration ([Ca2+]i) in GH-secreting somatotropes and LH-secreting gonadotropes of the rat anterior pituitary gland. The dynamics of the PACAP-induced Ca2+ responses and their dependence on extracellular Ca2+ are markedly different in the two cell types, suggesting separate mechanisms of action of PACAP in somatotropes and gonadotropes. The present study reports a full characterization of the Ca2+ responses seen in the two cell types over a wide range of PACAP concentrations. In addition, the involvement of the cAMP-dependent protein kinase (PKA) system in the mediation of PACAP-stimulated Ca2+ was tested using the R-isomer of cAMP (RpcAMPs) as a specific inhibitor of PKA. In identified somatotropes, PACAP (10(-11)-10(-6) M) stimulated Ca2+ responses in 49% of the cells tested, with two types of Ca2+ response profile observed. The first was a slow rise in [Ca2+]i to a new level (Ca2+ step; 28% of somatotropes); the second was characterized by repetitive transient rises in [Ca2+]i (Ca2+ transients; 21% of somatotropes). The range of PACAP concentrations tested (10(-11)-10(-6) M) did not markedly alter the number of cells responding or the type of response observed. In some gonadotropes, PACAP stimulated Ca2+ step responses similar to those seen in somatotropes; however, the most common response observed was a rapid, high amplitude, but transient spike of [Ca2+]i, which was often accompanied by rapid oscillations in [Ca2+]i. This response profile was termed a Ca2+ spike-oscillations response, and the proportion of cells exhibiting this response increased from 25% at a PACAP concentration of 10(-11) M to 73% at 10(-6) M PACAP. PACAP-induced Ca2+ responses in somatotropes were blocked by pretreatment with the cAMP antagonist RpcAMPs (10(-3) M). In addition, other factors known to increase cAMP in somatotropes (GH-releasing factor and 8-bromo-cAMP) stimulated Ca2+ responses in these cells that were qualitatively similar to those induced by PACAP. In contrast, the Ca2+ responses in gonadotropes were insensitive to the cAMP antagonist RpcAMPs, and the membrane-permeable cAMP analog 8-bromo-cAMP failed to stimulate responses in this cell type. These results suggest that the intracellular mechanisms of PACAP action in the two cell types are markedly different. In somatotropes, the rise in [Ca2+]i stimulated by PACAP is probably through the production of cAMP and the activation of protein kinase-A.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Pituitary adenylate cyclase-activating polypeptide regulates cytosolic Ca2+ in rat gonadotropes and somatotropes through different intracellular mechanisms. 838 87

The effect of pituitary adenylate cyclase activating polypeptide (PACAP) on the L-type Ca2+ channel current (L-channel current) was studied in smooth muscle cells prepared from the rat tail artery. PACAP caused an increase in the amplitude of the L-channel current. The maximal increase (56%) occurred at a PACAP concentration of 1 x 10(-8) M; higher concentrations resulted in a smaller increase. Investigation into the intracellular mechanisms of PACAP action revealed that the increase in L-channel currents was blocked by calphostin C and bisindolylmaleimide IV [protein kinase C (PKC) inhibitors] and mimicked by 4 beta-phorbol 12-myristate 13-acetate (PMA), an activator of PKC. PACAP was also found to cause translocation of PKC, suggesting that the increase in the current by PACAP was due to PKC. In contrast, activation of cAMP-dependent protein kinase (PKA) by 8-bromo-cAMP caused an inhibition of the L-channel current. A high concentration of PACAP (1 x 10(-6) M) had no effect on the L-channel current. The null effect of PACAP on the L-channel current could be converted to an increase by Rp-cAMPs, a cAMP antagonist, and a decrease by calphostin C. PACAP also increased cAMP accumulation. These observations indicate the effect of PACAP on the L-channel current represents the integration of two signaling mechanisms that involve the activation of PKA and PKC.
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PMID:PACAP modulates L-type Ca2+ channel currents in vascular smooth muscle cells: involvement of PKC and PKA. 883 45

This study examines the neural lobe of the pituitary gland for the presence of receptors for pituitary adenylate cyclase-activating polypeptide (PACAP) and their possible involvement in the regulation of neurosecretion. The presence of PACAP receptors of type I was revealed in the neural lobe, as well as in anterior and intermediate lobes, by means of RT-PCR amplification using selective oligonucleotide pairs of primers. They appeared to be expressed in the tissues as a short form together with an isoform of heavier molecular weight. Activation of receptors in the presence of PACAP stimulated both formation of cyclic AMP (cAMP) and secretion of arginine vasopressin (AVP) in neural lobes, in a dose-related fashion, with half-maximum (EC50) values of 1.0 +/- 0.2 x 10(-9) M and 1.4 +/- 0.3 x 10(-8) M, respectively. Parallel with AVP, PACAP also stimulated oxytocin (OXT) output, with an EC50 value of 0.6 +/- 0.1 x 10(-8) M. In an attempt to localize receptors on cells (mainly astrocyte-like glials or pituicytes) and/or on nerve fibers of the gland, we used cultures of neural lobe cells and explants (in which nerve fibers undergo degeneration), as well as isolated nerve endings. In both cells and nerve terminals, PACAP enhanced accumulation of cAMP, while it triggered AVP secretion from the latter. The stimulatory effect of PACAP on both AVP and OXT release was mimicked by dbcAMP and blocked by H89, an inhibitor of cAMP-dependent protein kinase. We conclude that in the neural lobe, PACAP receptors are localized on both nerve terminals and pituicytes, which participate in the modulation of secretion of neurohypophyseal hormones in an interactive way and mainly through the cAMP signalling route.
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PMID:Evidence for the presence of receptors for pituitary adenylate cyclase-activating polypeptide in the neurohypophysis that are positively coupled to cyclic AMP formation and neurohypophyseal hormone secretion. 885 10

Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates catecholamine release and biosynthesis in sympathetic postganglionic cells. Moreover, PACAP receptor activation in cultured adrenal chromaffin and superior cervical ganglion cells has been reported to increase the expression of the gene coding for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis. However, the relative contribution of transcriptional and posttranscriptional mechanisms to the effects of PACAP on TH gene expression has not been evaluated. Therefore, in this study we compared the temporal effects of PACAP on TH gene transcription with the duration of its effects on TH mRNA levels. We had previously shown that vasoactive intestinal polypeptide, peptide histidine isoleucine, and secretin, peptides closely related to PACAP, induce TH gene expression through a cyclic AMP (cAMP)-dependent pathway. Therefore, using a mutant PC12 cell line deficient in cAMP-dependent protein kinase II (PKA), we also evaluated the role of the cAMP pathway in the effect of PACAP on TH gene expression. Continuous treatment of wild-type PC12 cells with PACAP (1 nM) increased TH mRNA levels maximally by 12 h and maintained TH mRNA at near maximal levels for at least 2 days. In contrast, the rate of TH gene transcription, as measured by a nuclear run-on assay, was maximal by 1 h and returned to basal levels by 3 h. The fact that a new steady-state level of TH mRNA was achieved and maintained for days in the absence of a sustained increase in TH gene transcription supports the involvement of posttranscriptional mechanisms. Removal of PACAP after 12 h, a time at which TH gene transcription was at basal levels, resulted in a subsequent return of TH mRNA to unstimulated levels within 36 h. Thus, continuous PACAP stimulation is required to maintain sustained increases in TH mRNA levels in the absence of a sustained elevation of transcription. To examine the role of the cAMP pathway in these effects, we compared the effects of PACAP in wild-type PC12 cells and in a mutant PC12 cell line (A126-1B2) that is deficient in PKA. PACAP failed to stimulate either TH mRNA levels or TH gene transcription in the mutant cells. In contrast to the effects of PACAP, dexamethasone increased TH mRNA levels by the same magnitude in both cell lines. It is noteworthy that stimulation of the PKA-deficient mutant cells with a combination of PACAP and dexamethasone (1 microM) produced a synergistic increase in TH mRNA levels, which was nearly twice that induced by dexamethasone stimulation alone. This synergistic effect was not transcriptionally mediated. The effect of the combined treatment on TH gene transcription was identical to the effect of dexamethasone alone. Taken together, these data indicate that PACAP regulates TH gene expression through a transcriptional mechanism requiring an intact cAMP pathway and through posttranscriptional mechanisms under the control of a cAMP-independent pathway(s).
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PMID:Transcriptional and posttranscriptional control of tyrosine hydroxylase gene expression during persistent stimulation of pituitary adenylate cyclase-activating polypeptide receptors on PC12 cells: regulation by protein kinase A-dependent and protein kinase A-independent pathways. 968 37

The 38-amino-acid isoform of pituitary adenylate cyclase-activating polypeptide (PACAP38) elicits a robust outgrowth of neurites in cultured PC12 cells. Initiation of neurite outgrowth occurs within 4-8 hr after the addition of PACAP38. Treatment with PACAP38 does not elicit collateral activation of p140(trk) nerve growth factor receptor tyrosine kinase activity, nor is it associated with tyrosine phosphorylation of suc1-associated neurotrophic factor target, a selective target of neurotrophin tyrosine kinase receptors. Coadministration of epidermal growth factor with PACAP38 elicits an enhanced response. Induction of neurites is also observed on the addition of PACAP38 to dominant negative Src and Ras PC12 cell variants. PACAP38 stimulates extracellular signal-regulated kinase (Erk) activity >10-fold within 5 min, and the effect is augmented by cotreatment with epidermal growth factor. Pretreatment with the cAMP-dependent protein kinase-selective inhibitor, H-89, is ineffective as an antagonist of PACAP38-induced neurite outgrowth, whereas down-regulation of protein kinase C (PKC) by phorbol ester or incubation with PKC-selective inhibitors GF109203X and calphostin C effectively blocks PACAP38-stimulated neurite formation. Stimulation of Erk activity is inhibited by incubation with PD90859, a pharmacological antagonist of the threonine/tyrosine dual-specificity Erk. Inhibition of ligand-stimulated Erk activation prevents PACAP38-induced neurite outgrowth. Collectively, these findings indicate that PACAP38-stimulated neuritogenesis requires PKC and Erk activation but is independent of cAMP-dependent protein kinase, nerve growth factor receptor tyrosine kinase, p21(ras) G protein, and pp60(c-src) cytoplasmic tyrosine kinase.
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PMID:The 38-amino-acid form of pituitary adenylate cyclase-activating polypeptide induces neurite outgrowth in PC12 cells that is dependent on protein kinase C and extracellular signal-regulated kinase but not on protein kinase A, nerve growth factor receptor tyrosine kinase, p21(ras) G protein, and pp60(c-src) cytoplasmic tyrosine kinase. 973 Sep 14

Ca2+ plays an essential role in pituitary adenylate cyclase-activating polypeptide (PACAP)-stimulated growth hormone (GH) secretion from porcine somatotropes. Here, Indo-1 microfluorimetry was used to investigate the dynamics of free cytosolic Ca2+ concentration ([Ca2+]i) in single porcine somatotropes in response to PACAP38 and PACAP27. We also evaluated the relative contributions of extra- and intracellular Ca2+ sources and of cAMP-dependent protein kinase (PKA) and phospholipase C (PLC). A high proportion of somatotropes responded to PACAP38 (79.4%) and PACAP27 (68.4%) with [Ca2+]i rises that could be followed by a refractory plateau (type 1 response), or by a decrease in [Ca2+]i during which somatotropes were responsive to a subsequent PACAP pulse (type II response). Although Ca2+ profiles in response to both peptides were similar, PACAP38-induced [Ca2+]i rises were higher. Somatotrope response to PACAP38 or PACAP27 was markedly reduced by removing extracellular Ca2+, blocking Ca2+ entry through L-type voltage sensitive Ca2+ channels (VSCC), or inhibiting PKA. Conversely, Ca2+ depletion from intracellular stores or PLC inactivation did not affect the response to PACAP27 but considerably reduced maximal [Ca2+]i induced by PACAP38. We conclude that both peptides stimulate extracellular Ca2+ influx through L-type VSCC by a PKA-dependent mechanism. However, PACAP38 also triggers a PLC-mediated Ca2+ mobilization from intracellular stores, thereby indicating that the two molecular forms of PACAP activate common and distinct second messenger pathways within porcine somatotropes.
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PMID:Pituitary adenylate cyclase-activating polypeptides 38 and 27 increase cytosolic free Ca2+ concentration in porcine somatotropes through common and distinct mechanisms. 992 28

The neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) suppress monocyte/macrophage production of proinflammatory agents. The transcription factor NF-kappa B regulates the transcription of most agents. VIP/PACAP inhibit NF-kappa B transactivation in the lipopolysaccharide-stimulated human monocytic cell line THP-1 at multiple levels. First, VIP/PACAP inhibit p65 nuclear translocation and NF-kappa B DNA binding by stabilizing the inhibitor I kappa B alpha. Second, VIP/PACAP induce phosphorylation of the CRE-binding protein (CREB) and its binding to the CREB-binding protein (CBP). This results in a decrease in p65.CBP complexes, which further reduces NF-kappa B transactivation. Third, VIP and PACAP reduce the phosphorylation of the TATA box-binding protein (TBP), resulting in a reduction in TBP binding to both p65 and the TATA box. All these effects are mediated through the specific receptor VPAC1. The cAMP/cAMP-dependent protein kinase pathway mediates the effects on CBP and TBP, whereas a cAMP-independent pathway is the major transducer for the effects on p65 nuclear translocation. Since NF-kappaB represents a focal point for various stimuli and induces the expression of many proinflammatory genes, its targeting by VIP and PACAP positions them as important anti-inflammatory agents. The VIP/PACAP inhibition of NF-kappa B at various levels and through different transduction pathways could offer a significant advantage over other anti-inflammatory agents.
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PMID:Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide inhibit nuclear factor-kappa B-dependent gene activation at multiple levels in the human monocytic cell line THP-1. 1102 67

We examined whether mitogen-activated protein (MAP) kinase was activated by stimulation of the cAMP pathway and whether MAP kinase activation was involved in synthesis of PRL and GH in GH(3) cells. Treatment of the cells with a cAMP analog, 8-(4-chlorophenylthio)cAMP (CPT-cAMP), activated MAP kinase and increased PRL at both the protein and messenger RNA levels. The protein and messenger RNA of GH were decreased by the treatment. We constructed the luciferase reporter genes after the promoters of PRL and GH and found the activation of both promoters by the CPT-cAMP treatment. We confirmed that overexpression of the catalytic subunit of cAMP-dependent protein kinase had essentially the same effects on MAP kinase activation and synthesis of PRL and GH as the CPT-cAMP treatment. Furthermore, treatment of the cells with pituitary adenylate cyclase-activating polypeptide 27 activated MAP kinase. The activation of PRL promoter by CPT-cAMP and pituitary adenylate cyclase-activating polypeptide 27 was abolished by pretreatment with PD098059 and H89. Although the increase in PRL and GH secretion by CPT-cAMP was inhibited by H89, PD098059 had no effect on secretion. These results suggest that cAMP-induced MAP kinase activation is essential for PRL gene expression, but not for secretion of PRL and GH.
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PMID:Involvement of mitogen-activated protein kinase in cyclic adenosine 3',5'-monophosphate-induced hormone gene expression in rat pituitary GH(3) cells. 1141

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