calyculin-a and 9-(tetrahydro-2-furyl)-adenine

The relaxant effect of hydrogen sulfide (H(2)S) in the vascular tree is well established but its influence and mechanism of action in gastrointestinal smooth muscle was hardly investigated. The influence of H(2)S on contractility in mouse gastric fundus was therefore examined. Sodium hydrogen sulfide (NaHS; H(2)S donor) was administered to prostaglandin F(2alpha) (PGF(2alpha))-contracted circular muscle strips of mouse gastric fundus, before and after incubation with interfering drugs. NaHS caused a concentration-dependent relaxation of the pre-contracted mouse gastric fundus strips. The K(+) channels blockers glibenclamide, apamin, charybdotoxin, 4-aminopyridin and barium chloride had no influence on the NaHS-induced relaxation. The relaxation by NaHS was also not influenced by L-NAME, ODQ and SQ 22536, inhibitors of the cGMP and cAMP pathway, by nerve blockers capsazepine, omega-conotoxin and tetrodotoxin or by several channel and receptor blockers (ouabain, nifedipine, 2-aminoethyl diphenylborinate, ryanodine and thapsigargin). The myosin light chain phosphatase (MLCP) inhibitor calyculin-A reduced the NaHS-induced relaxation, but the Rho-kinase inhibitor Y-27632 had no influence. We show that NaHS is able to relax PGF(2alpha)-contracted mouse gastric fundus strips. The results suggest that in the mouse gastric fundus, H(2)S causes relaxation at least partially via activation of MLCP.

Topics: Adenine; Amides; Animals; Enzyme Activation; Gastric Fundus; Glyburide; Hydrogen Sulfide; In Vitro Techniques; Male; Marine Toxins; Mice; Muscle Relaxation; Myosin-Light-Chain Phosphatase; NG-Nitroarginine Methyl Ester; omega-Conotoxins; Oxazoles; Pyridines; rho-Associated Kinases; rhoA GTP-Binding Protein; Signal Transduction; Tetrodotoxin

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

Reactive oxygen species (ROS) are believed to cause vascular injury in the pathophysiology of atherosclerosis, diabetes, and vasoocclusion in sickle cell disease. Studies have shown that ROS causes increased adhesion of monocytes and neutrophils to the endothelium. We investigated the effects of tert-butylhydroperoxide (t-BuOOH), an inducer of oxidant stress, to determine the cellular signaling pathway leading to the transendothelial migration of polymorphonuclear leukocytes. Our studies revealed that signaling by t-BuOOH in human umbilical vein endothelial cells (HUVECs) causes a twofold increase in the transendothelial migration of monocyte-like HL-60 cells and a fivefold increase in platelet endothelial cell adhesion molecule-1 (PECAM-1) phosphorylation. The transmigration induced by t-BuOOH was inhibited by an antibody to PECAM-1. These events were inhibited by antioxidants and inhibitors of protein kinase C, p21ras and glutathione synthesis. However, treatment of HUVECs with the phosphatase inhibitor calyculin A augmented the t-BuOOH-mediated transendothelial migration of monocytes and PECAM-1 phosphorylation. Our results suggest that oxidative stress can induce the transendothelial migration of monocytes as a result of phosphorylation of PECAM-1, a crucial event in the diapedesis of leukocytes during pathophysiology of vascular diseases.

Topics: Adenine; Antibodies, Monoclonal; Antioxidants; Cells, Cultured; Chemotaxis, Leukocyte; Endothelium, Vascular; Enzyme Inhibitors; HL-60 Cells; Humans; Indoles; Kainic Acid; Kinetics; Maleimides; Marine Toxins; Monocytes; Oxazoles; Oxidative Stress; Peroxides; Phosphorylation; Platelet Endothelial Cell Adhesion Molecule-1; Probucol; Reactive Oxygen Species; Signal Transduction; tert-Butylhydroperoxide; Thiobarbituric Acid Reactive Substances; Umbilical Veins; Vitamin E