icatibant and chelerythrine

Transient receptor potential melastatin-7 (TRPM7) channels have recently been identified to be regulated by vasoactive agents acting through G protein-coupled receptors in vascular smooth muscle cells (VSMC). However, downstream targets and functional responses remain unclear. We investigated the subcellular localization of TRPM7 in VSMCs and questioned the role of TRPM7 in proinflammatory signaling by bradykinin. VSMCs from Wistar-Kyoto rats were studied. Cell fractionation by sucrose gradient and differential centrifugation demonstrated that in bradykinin-stimulated cells, TRPM7 localized in fractions corresponding to caveolae. Immunofluorescence confocal microscopy revealed that TRPM7 distributes along the cell membrane, that it has a reticular-type intracellular distribution, and that it colocalizes with flotillin-2, a marker of lipid rafts. Bradykinin increased expression of calpain, a TRPM7 target, and stimulated its cytosol/membrane translocation, an effect blocked by 2-APB (TRPM7 inhibitor) and U-73122 (phospholipase C inhibitor), but not by chelerythrine (PKC inhibitor). Expression of proinflammatory mediators VCAM-1 and cyclooxygenase-2 (COX-2) was time-dependently increased by bradykinin. This effect was blocked by Hoe-140 (B2 receptor blocker) and 2-APB. Our data demonstrate that in bradykinin-stimulated VSMCs: 1) TRPM7 is upregulated, 2) TRPM7 associates with cholesterol-rich microdomains, and 3) calpain and proinflammatory mediators VCAM-1 and COX2 are regulated, in part, via TRPM7- and phospholipase C-dependent pathways through B2 receptors. These findings identify a novel signaling pathway for bradykinin, which involves TRPM7. Such phenomena may play a role in bradykinin/B2 receptor-mediated inflammatory responses in vascular cells.

Topics: Animals; Benzophenanthridines; Boron Compounds; Bradykinin; Bradykinin B2 Receptor Antagonists; Calpain; Caveolae; Cells, Cultured; Cyclooxygenase 2; Enzyme Inhibitors; Estrenes; Inflammation Mediators; Magnesium; Mesenteric Arteries; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Protein Kinase C; Protein Transport; Pyrrolidinones; Rats; Rats, Inbred WKY; Receptor, Bradykinin B2; Signal Transduction; TRPM Cation Channels; Type C Phospholipases; Up-Regulation; Vascular Cell Adhesion Molecule-1

We examined the involvement of the oxidative stress in high glucose-induced suppression of human aortic endothelial cell proliferation. Chronic glucose treatment for 72 h concentration-dependently (5.6-22.2 mol/l) inhibited human coronary endothelial cell proliferation. Temocaprilat, an angiotensin-converting enzyme inhibitor, at 10 nmol/l to 1 micromol/l inhibited high glucose (22.2 mmol/l)-mediated suppression of human aortic endothelial cell proliferation. Temocaprilat at 1 micromol/l inhibited high glucose-induced membrane-bound protein kinase C activity in human aortic endothelial cells. The protein kinase C inhibitors calphostin C 100 nmol/l or chelerythrine 1 micromol/l inhibited high glucose-mediated suppression of human aortic endothelial cell proliferation. Chronic high glucose treatment for 72 h increased intracellular oxidative stress, directly measured by flow cytometry using carboxydichlorofluorescein diacetate bis-acetoxymethyl ester, and this increase was significantly suppressed by temocaprilat 10 nmol/l to 1 micromol/l. Bradykinin B2 receptor antagonist icatibant 100 nmol/l significantly reduced the action of temocaprilat; whereas bradykinin B1 receptor antagonist des-Arg9-Leu8-bradykinin 100 nmol/l had no effect. These findings suggest that high glucose inhibits human aortic endothelial cell proliferation and that the angiotensin-converting enzyme inhibitor temocaprilat inhibits high glucose-mediated suppression of human aortic endothelial cell proliferation, possibly through suppression of protein kinase C, bradykinin B2 receptors and oxidative stress.

Topics: Alkaloids; Angiotensin-Converting Enzyme Inhibitors; Aorta; Benzophenanthridines; Bradykinin; Bradykinin B2 Receptor Antagonists; Cell Division; Cell Membrane; Cells, Cultured; Coronary Vessels; Dose-Response Relationship, Drug; Endothelial Cells; Glucose; Humans; Naphthalenes; Oxidative Stress; Phenanthridines; Protein Kinase C; Receptor, Bradykinin B1; Receptor, Bradykinin B2; Thiazepines; Time Factors

Myocardial protection can be achieved by brief ischemia-reperfusion of remote organs, a phenomenon described as remote preconditioning (RPC). Since the intracellular mechanisms of RPC are not known, we tested the hypothesis that RPC might activate myocardial PKCepsilon, an essential mediator of classical ischemic preconditioning. Furthermore, we tried to delineate the mechanisms by which RPC is transduced to the heart with respect to the possible contribution of kinins and neuronal reflexes.. Anesthetized rats were randomised to undergo either 30 min of waiting (controls) or RPC (brief mesenteric artery occlusion followed by reperfusion) in the absence or presence of chelerythrine (5 mg kg(-1)), a specific PKC inhibitor. Myocardial infarct size was measured by TTC staining after 30 min of coronary artery occlusion followed by 150 min of reperfusion. In separate sets of experiments RPC was performed with or without pretreatment with HOE140, a selective B(2)-antagonist or hexamethonium was used to explore the influence of ganglion blockade on RPC. Translocation of PKCepsilon from cytosol to the particulate fraction was measured by quantitative immunoblotting.. RPC significantly reduced infarct size which was completely blocked by the PKC inhibitor. RPC shifted the ratio between cytosolic and particulate PKCepsilon, an indicator for PKC-activation, from 0.95+/-0.06 in controls to 0.41+/-0.09 (P<0.05), and this effect was abolished by HOE140. Activation of PKCepsilon could not be achieved after pretreatment with HEX (0.69+/-0.06 in HEX vs. 0.78+/-0.06 in HEX+RPC).. RPC activates myocardial PKCepsilon through a neuronal and bradykinin-dependent pathway. We assume that activation of PKCepsilon is an important step in cardioprotection induced by remote preconditioning.

Topics: Adrenergic beta-Antagonists; Alkaloids; Animals; Benzophenanthridines; Bradykinin; Enzyme Activation; Enzyme Inhibitors; Ganglionic Blockers; Hexamethonium; Intestines; Ischemic Preconditioning; Ischemic Preconditioning, Myocardial; Isoenzymes; Male; Myocardial Infarction; Myocardium; Phenanthridines; Protein Kinase C; Random Allocation; Rats; Rats, Wistar