thapsigargin and 9-(tetrahydro-2-furyl)-adenine

We tested the hypothesis that adrenomedullin reduces calcium influx independent of potassium channels in depolarized endothelium-denuded mesenteric artery from pregnant rats.. Adrenomedullin reduced the CaCl(2)-induced contraction, while the receptor antagonist calcitonin gene-related peptide (CGRP)(8-37), but not adrenomedullin(22-52), reversed these effects. Adenylate cyclase inhibition by SQ22536 did not prevent adrenomedullin effects on CaCl(2)-induced contraction. Adrenomedullin did not inhibit depolarization-induced calcium entry to isolated vascular smooth muscle. Inhibition of myosin light-chain (MLC) phosphatase by calyculin A reversed the effects of adrenomedullin on contraction caused by submillimolar concentrations of CaCl(2), while adrenomedullin still inhibited contraction caused by higher concentrations of CaCl(2). However, the ratio of phosphorylated to total myosin phosphatase target 1, the regulatory subunit of MLC phosphatase, did not change with adrenomedullin, indicating a lack of MLC phosphatase activation. Interestingly, sodium fluoride, a nonspecific protein phosphatase inhibitor, completely blocked the effect of adrenomedullin on CaCl(2)-induced contraction. Adrenomedullin inhibited calcium mobilization from intracellular stores induced by thapsigargin.. Adrenomedullin inhibits CaCl(2)-induced contraction, without affecting calcium influx, through a CGRP(8-37)-sensitive receptor, but not using the cyclic adenosine monophosphate pathway, probably through activation of protein phosphatases. Inhibition of intracellular calcium release is an additional role played by adrenomedullin in calcium homeostasis in vascular smooth muscle.

Topics: Adenine; Adenylyl Cyclase Inhibitors; Adenylyl Cyclases; Adrenomedullin; Animals; Calcitonin Gene-Related Peptide; Calcium Chloride; Calcium Signaling; Calcium-Transporting ATPases; Cells, Cultured; Cyclic AMP; Dose-Response Relationship, Drug; Enzyme Inhibitors; Female; Marine Toxins; Membrane Potentials; Mesenteric Arteries; Muscle, Smooth, Vascular; Myosin-Light-Chain Phosphatase; Oxazoles; Peptide Fragments; Phosphorylation; Potassium; Pregnancy; Protein Phosphatase 1; Rats; Receptors, Adrenomedullin; Receptors, G-Protein-Coupled; Sodium Fluoride; Thapsigargin; Time Factors; Vasoconstriction; Vasoconstrictor Agents

The aim of this study was to investigate the signal transduction mechanisms underlying the inhibitory effect induced by pituitary adenylate cyclase activating peptide (PACAP-27) on the spontaneous contractile activity of longitudinal muscle of mouse ileum. Mechanical activity of ileal segments was recorded isometrically in vitro. PACAP-27 produced apamin-sensitive reduction of the amplitude of the spontaneous contractions. 9-(Tetrahydro-2-furanyl)-9H-purin-6-amine (SQ 22,536), adenylate cyclase inhibitor, or genistein and tyrphostin 25, tyrosine kinase inhibitors, had negligible effects on PACAP-27-induced inhibition. PACAP-27 effects were significantly inhibited by U-73122, phopholipase C (PLC) inhibitor, by 2-aminoethoxy-diphenylborate (2-APB), permeable blocker of inositol 1,4,5-triphosphate (IP3) receptors and by depletion of Ca2+ stores with cyclopiazonic acid or thapsigargin. Ryanodine did not reduce PACAP-27-inhibitory responses. We suggest that, in mouse ileum, the inhibitory responses to PACAP-27 involve stimulation of PLC, increased production of IP3 and localised Ca2+ release from intracellular stores, which could provide the opening of apamin-sensitive Ca2+-dependent K+ channels.

Topics: Adenine; Adenylyl Cyclase Inhibitors; Animals; Apamin; Boron Compounds; Calcium; Calcium-Transporting ATPases; Dose-Response Relationship, Drug; Enzyme Inhibitors; Estrenes; Genistein; Ileum; In Vitro Techniques; Indoles; Male; Mice; Mice, Inbred C57BL; Muscle Contraction; Muscle, Smooth; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Phosphodiesterase Inhibitors; Pituitary Adenylate Cyclase-Activating Polypeptide; Protein-Tyrosine Kinases; Pyrrolidinones; Ryanodine; Thapsigargin; Tyrphostins; Vasodilator Agents

The species-specific sound production of acoustically communicating grasshoppers can be stimulated by pressure injection of both nicotinic and muscarinic agonists into the central body complex and a small neuropil situated posterior and dorsal to it. To determine the role of muscarinic acetylcholine receptors (mAChRs) in the control of acoustic communication behavior and to identify the second-messenger pathways affected by mAChR-activation, muscarinic agonists and membrane-permeable drugs known to interfere with specific mechanisms of intracellular signaling pathways were pressure injected to identical sites in male grasshopper brains. Repeated injections of small volumes of muscarine elicited stridulation of increasing duration associated with decreased latencies. This suggested an accumulation of excitation over time that is consistent with the suggested role of mAChRs in controlling courtship behavior: to provide increasing arousal leading to higher intensity of stridulation and finally initiating a mating attempt. At sites in the brain where muscarine stimulation was effective, stridulation could be evoked by forskolin, an activator of adenylate cyclase (AC); 8-Br-cAMP-activating protein kinase A (PKA); and 3-isobuty-1-methylxanthine, leading to the accumulation of endogenously generated cAMP through inhibition of phosphodiesterases. This suggested that mAChRs mediate excitation by stimulating the AC/cAMP/PKA pathway. In addition, muscarine-stimulated stridulation was inhibited by 2'-5'-dideoxyadenonsine and SQ 22536, two inhibitors of AC; H-89 and Rp-cAMPS, two inhibitors of PKA; and by U-73122 and neomycin, two agents that inhibit phospholipase C (PLC) by independent mechanisms. Because the inhibition of AC, PKA, or PLC by various individually applied substances entirely suppressed muscarine-evoked stridulation in a number of experiments, activation of both pathways, AC/cAMP/PKA and PLC/IP(3)/diacylglycerine, appeared to be necessary to mediate the excitatory effects of mAChRs. With these studies on an intact "behaving" grasshopper preparation, we present physiological relevance for mAChR-evoked excitation mediated by sequential activation of the AC- and PLC-initiated signaling pathways that has been reported in earlier in vitro studies.

Topics: Acetylcholine; Adenine; Adenylyl Cyclases; Animal Communication; Animals; Brain; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Dideoxyadenosine; Diglycerides; Enzyme Inhibitors; Estrenes; Grasshoppers; Inositol 1,4,5-Trisphosphate; Isoquinolines; Muscarine; Muscarinic Agonists; Phosphodiesterase Inhibitors; Purinones; Pyrrolidinones; Receptors, Muscarinic; Second Messenger Systems; Sphingosine; Sulfonamides; Thapsigargin; Thionucleotides; Type C Phospholipases

Bitter substances are a structurally diverse group of compounds that appear to act via several transduction mechanisms. The bitter-tasting denatonium ion has been proposed to act via two different G-protein-regulated pathways, one involving inositol 1,4, 5-trisphosphate and raised intracellular calcium levels, the other involving phosphodiesterase and membrane depolarization via a cyclic nucleotide-suppressible cation channel. The aim of the present study was to examine these transduction mechanisms in taste cells of the mudpuppy Necturus maculosus by calcium-imaging and whole-cell recording. Denatonium benzoate increased intracellular calcium levels and induced an outward current independently of extracellular calcium. The denatonium-induced increase in intracellular calcium was inhibited by U73122, an inhibitor of phospholipase C, and by thapsigargin, an inhibitor of calcium transport into intracellular stores. The denatonium-induced outward current was blocked by GDP-beta-S, a blocker of G-protein activation. Neither resting nor denatonium-induced intracellular calcium levels were affected by inhibition of phosphodiesterase (with IBMX) or adenylate cyclase (with SQ22536) or by raising intracellular cyclic nucleotides directly (with cell permeant analogs). Our results support the hypothesis that denatonium is transduced via a G-protein cascade involving phospholipase C, inositol 1,4,5-trisphosphate, and raised intracellular calcium levels. Our results do not support the hypothesis that denatonium is transduced via phosphodiesterase and cAMP.

Topics: 1-Methyl-3-isobutylxanthine; Adenine; Adenylate Cyclase Toxin; Animals; Calcium; Calcium Channels; Cyclic AMP; Cyclic GMP; Enzyme Inhibitors; Estrenes; Fluorescent Dyes; Fura-2; GTP-Binding Proteins; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Membrane Potentials; Necturus; Patch-Clamp Techniques; Phosphodiesterase Inhibitors; Pyrrolidinones; Quaternary Ammonium Compounds; Receptors, Cytoplasmic and Nuclear; Ryanodine; Signal Transduction; Taste; Taste Buds; Thapsigargin; Virulence Factors, Bordetella

The expression by T lymphocytes (T cells) of more than one of the functionally distinct subtypes of prostaglandin E2 (PGE2) receptors (Rs), designated EP1, EP2, EP3, and EP4 Rs, is a principal determinant of specificity and diversity of the immune effects of PGE2. The cultured line of human leukemic T cells, termed HSB.2, co-expresses a total of 7282 +/- 1805 EP3, EP4, and EP2 Rs per cell with a Kd of 3.7 +/- 1.4 nM (mean +/- S.E., n = 9). The EP3/EP1 R-selective agonist sulprostone, EP3/EP2/EP4 R-selective agonists M&B 28767 and misoprostol, and EP2 R-selective agonist butaprost but not the EP1 R-selective antagonist SC-19220 competitively inhibited the binding of [3H]PGE2 to HSB.2 cells. Stimulation of increases in the intracellular concentration of cyclic AMP ([cAMP]i) by PGE2, misoprostol, and butaprost and of increases in the intracellular concentration of calcium ([Ca2+]i) by PGE2 and sulprostone demonstrated the respective involvement of EP2/EP4 Rs and EP3 Rs in transduction of biochemical signals. Matrix metalloproteinase (MMP)-9 was identified by zymography and Western blots as the principal MMP secreted by HSB.2 cells. The cytosolic level and secretion of MMP-9 were increased maximally after 24 h of incubation of HSB.2 cells with 10(-8)-10(-6) M PGE2, sulprostone, M&B 28767, and misoprostol but not with 10(-6) M PGF2alpha, PGD2, PGI2, or butaprost, suggesting a principal dependence on EP3 Rs. That stimulation of MMP-9 secretion by PGE2 was not diminished in Ca2+-free medium but was suppressed significantly and dose-dependently by thapsigargin, an inhibitor of endomembrane Ca2+-ATPase, suggested that MMP-9 expression by HSB.2 cells is mediated by increases in [Ca2+]i attributable to release of Ca2+ from intracellular stores. The lack of effect of dibutyryl cAMP, forskolin, and SQ 22536, an adenylyl cyclase inhibitor, on MMP-9 secretion by HSB.2 cells argued against any role for cAMP-dependent mechanisms linked to EP2/EP4 Rs. Cycloheximide and actinomycin D, which respectively inhibited protein and RNA synthesis, suppressed basal and PGE2 induction of MMP-9 production by HSB.2 cells. Northern analysis indicated that PGE2 and sulprostone time-dependently increased expression of MMP-9 mRNA. Thus, stimulation of MMP-9 in HSB.2 T cells by PGE2 is attributable to [Ca2+]i-dependent EP3 R-mediation of increases in message transcription.

Topics: Adenine; Alprostadil; Blotting, Northern; Bucladesine; Calcium; Cell Line; Colforsin; Collagenases; Cyclic AMP; Cycloheximide; Dactinomycin; Dinoprostone; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Kinetics; Leukemia, T-Cell; Matrix Metalloproteinase 9; Misoprostol; Prostaglandins E, Synthetic; Receptors, Prostaglandin E; RNA, Messenger; T-Lymphocytes; Thapsigargin; Transcription, Genetic; Tumor Cells, Cultured