Zhi-wei Yang

Mechanisms of hydrogen peroxide-induced contraction of rat aorta

It has been suggested that reactive oxygen species may be involved in the regulation of vascular tone. However, the underlying mechanisms remain to be elucidated. The present studies were designed to investigate the contractile effects of hydrogen peroxide (H2O2), one of the reactive oxygen species, on isolated ring segments of rat aorta with and without endothelium. H2O2 induced an endothelium-independent contraction in isolated rat aorta ring segments in a concentration-dependent manner at concentrations from 5 x 10(-6) to 5 x 10(-3) M. H2O2-induced contractions of denuded rat aorta rings were stronger than those on intact rat aorta segments. The contractile effects of H2O2 were inhibited completely by 1200 u/ml catalase. The presence of 1.0 microM Fe2+ or 10 microM proadifen, a cytochrome P450 monooxygenase inhibitor, potentiated the contractile effect of H2O2 on isolated rat aorta segments. 1 mM deferoxamine (a Fe2+ chelator) or 100 microM dimethyl sulfoxide (a hydroxyl radical scavenger) significantly attenuated the vessel contractions induced by hydrogen peroxide plus Fe2+ or hydrogen peroxide itself. Removal of extracellular Ca2+ ([Ca2+]0), addition of 5 microM verapamil, administration of a protein kinase C inhibitor (staurosporine), treatment with an inhibitor of protein tyrosine phosphorylation (genistein) or employment of 5.0 microM indomethacin resulted in a significant attenuation of the contractile responses of the vessels to H2O2. Pharmacological antagonists (e.g. a muscarinic acetylcholine receptor antagonist (atropine), an antagonist of histamine H1 receptors (diphenhydramine), an antagonist of histamine H2 receptors (cimetidine), an alpha-adrenoceptor antagonist (phentolamine), a beta-adrenoceptor antagonist (propranolol) and an antagonist of serotonin receptor (methysergide)) did not inhibit or attenuate the contractions induced by H2O2. Exposure of primary aortic smooth muscle cells to H2O2 (5 x 10(-6) to 5 x 10(-3) M) produced significant rises of intracellular Ca2+ ([Ca2+]i) within 20 s. Employment of 1.0 microM Fe2+ markedly enhanced the increment in [Ca2+]i in the smooth muscle cells. 10 microM proadifen treatment failed to alter the hydrogen peroxide-induced increment in [Ca2+]i of the smooth muscle cells. However, the presence of 5 microM indomethacin significantly attenuated the rise in [Ca2+]i in smooth muscle cells. The present results suggest that H2O2 can induce contractions of rat aorta segments, at pathophysiological concentrations, which are Ca2+-dependent. Hydroxyl radicals (.OH), cyclooxygenase products, protein kinase C and products of protein tyrosine phosphorylation appear to play some role in hydrogen peroxide-induced contractions. Metabolites catalyzed by cytochrome P450-dependent enzymes (upon treatment with hydrogen peroxide) appear to exert a vasodilator effect on rat aorta segments. Lastly, some unidentified mediators, produced by a cytochrome P450 inhibitor (proadifen), during hydrogen peroxide treatment, appear to play some role in contraction of vascular smooth muscle of rat aorta segments in vitro.

Ethanol-Induced Contractions in Cerebral Arteries

Background and Purpose— The relationship between alcohol consumption and stroke appears complex; moderate ingestion is associated with reduced stroke risk, while heavy intake is associated with increased stroke risk. Ethanol has been shown both experimentally and epidemiologically to induce hemorrhagic and ischemic strokes, which are associated with cerebral vasoconstriction. Ethanol is known to induce contraction in isolated cerebral arteries and intact microvessels from diverse mammalian animals. The relationships between ethanol-induced contractions in cerebral arteries, intracellular free Ca2+ ([Ca2+]i), tyrosine kinases (including the src family), and mitogen-activated protein kinases (MAPK) were investigated in the present study. Methods— Cerebral arterial muscle tension and [Ca2+]i were quantified by an isometric contraction technique and direct visualization of Ca2+ in single cells. Results— Ethanol induces concentration-dependent contractions in intact canine basilar arteries, which are attenuated significantly by pretreatment of the arteries with low concentrations of an antagonist of protein tyrosine kinases (genistein); an src homology 2 (SH2) domain inhibitor peptide; a highly specific antagonist of p38 MAPK (SB-203580); a potent, selective antagonist of MEK1/MEK2 (U0126); and a selective antagonist of mitogen-activated protein kinase kinase (MAPKK) (PD-98059). IC50 levels obtained for these 5 antagonists are consistent with reported Ki values for these tyrosine kinase, MAPK, and MAPKK antagonists. Ethanol induces transient and sustained increases in [Ca2+]i in primary single smooth muscle cells from canine basilar arteries, which are markedly attenuated in the presence of genistein, an SH2 domain inhibitor peptide, SB-203580, U0126, and PD-98059. Several specific antagonists of known endogenously formed vasoconstrictors do not inhibit or attenuate either the ethanol-induced contractions or the elevation of [Ca2+]i. Conclusions— The present study suggests that activation of protein tyrosine kinases (including the src family) and MAPK appear to play important roles in the ethanol-induced contractions and the elevation of [Ca2+]i in smooth muscle cells from canine basilar arteries. The results could be used to suggest that selective antagonists of protein tyrosine kinases and MAPK may be useful both prophylactically and therapeutically in alcohol-induced strokes.

Ethanol-Induced Contractions in Cerebral Arteries: Role of Tyrosine and Mitogen-Activated Protein Kinases

Background and Purpose— The relationship between alcohol consumption and stroke appears complex; moderate ingestion is associated with reduced stroke risk, while heavy intake is associated with increased stroke risk. Ethanol has been shown both experimentally and epidemiologically to induce hemorrhagic and ischemic strokes, which are associated with cerebral vasoconstriction. Ethanol is known to induce contraction in isolated cerebral arteries and intact microvessels from diverse mammalian animals. The relationships between ethanol-induced contractions in cerebral arteries, intracellular free Ca2+ ([Ca2+]i), tyrosine kinases (including the src family), and mitogen-activated protein kinases (MAPK) were investigated in the present study. Methods— Cerebral arterial muscle tension and [Ca2+]i were quantified by an isometric contraction technique and direct visualization of Ca2+ in single cells. Results— Ethanol induces concentration-dependent contractions in intact canine basilar arteries, which are attenuated significantly by pretreatment of the arteries with low concentrations of an antagonist of protein tyrosine kinases (genistein); an src homology 2 (SH2) domain inhibitor peptide; a highly specific antagonist of p38 MAPK (SB-203580); a potent, selective antagonist of MEK1/MEK2 (U0126); and a selective antagonist of mitogen-activated protein kinase kinase (MAPKK) (PD-98059). IC50 levels obtained for these 5 antagonists are consistent with reported Ki values for these tyrosine kinase, MAPK, and MAPKK antagonists. Ethanol induces transient and sustained increases in [Ca2+]i in primary single smooth muscle cells from canine basilar arteries, which are markedly attenuated in the presence of genistein, an SH2 domain inhibitor peptide, SB-203580, U0126, and PD-98059. Several specific antagonists of known endogenously formed vasoconstrictors do not inhibit or attenuate either the ethanol-induced contractions or the elevation of [Ca2+]i. Conclusions— The present study suggests that activation of protein tyrosine kinases (including the src family) and MAPK appear to play important roles in the ethanol-induced contractions and the elevation of [Ca2+]i in smooth muscle cells from canine basilar arteries. The results could be used to suggest that selective antagonists of protein tyrosine kinases and MAPK may be useful both prophylactically and therapeutically in alcohol-induced strokes.

Importance of extracellular Ca2+ and intracellular Ca2+ release in ethanol-induced contraction of cerebral arterial smooth muscle

The present study was designed to investigate the roles of extracellular Ca2+ ([Ca2+]0) influx and intracellular free Ca2+ ([Ca2+]i) release in ethanol-induced contractions of isolated canine cerebral arteries and primary cultured, cerebral vascular smooth muscle cells. Ethanol (20-200 mM) produced significant contractions in isolated canine basilar arterial rings in a concentration-dependent manner. Removal of [Ca2+]0 and pretreatment of canine basilar arterial rings with verapamil (an antagonist of voltage-gated Ca2+ channels), thapsigargin (a selective antagonist of the sarcoplasmic reticulum Ca2+ pump), caffeine plus ryanodine (a specific antagonist of ryanodine-sensitive Ca2+ release), or heparin (an inositol 1,4,5,-trisphosphate [InsP3]-mediated Ca2+ release antagonist) markedly attenuated (approximately 50%-80%) ethanol-induced contractions. The absence of [Ca2+]0 and preincubation of primary single smooth muscle cells obtained from canine basilar arteries with verapamil, thapsigargin, heparin, or caffeine plus ryanodine markedly attenuated (approximately 50%-80%) the transient and sustained elevations in [Ca2+]i induced by ethanol. Results of the present study suggest to us that both Ca2+ influx through voltage-gated Ca2+ channels and Ca2+ release from intracellular stores (both InsP3 sensitive and ryanodine sensitive) are required for ethanol-induced contractions of isolated canine basilar arteries.

Roles of tyrosine kinase-, 1-phosphatidylinositol 3-kinase-, and mitogen- activated protein kinase-signaling pathways in ethanol-induced contractions of rat aortic smooth muscle: possible relation to alcohol-induced hypertension

Insights into the relations between and among ethanol-induced contractions in rat aorta, tyrosine kinases (including src family of cytoplasmic tyrosine kinases), 1-phosphatidylinositol 3-kinases (PI-3Ks), mitogen-activated protein kinases (MAPKs), and regulation of intracellular free Ca(2+) ([Ca(2+)](i)) were investigated in the present study. Ethanol-induced concentration-dependent contractions in isolated rat aortic rings were attenuated greatly by pretreatment of the arteries with low concentrations of an antagonist of protein tyrosine kinases (genistein), an src homology domain 2 (SH2) inhibitor peptide, a highly specific antagonist of p38 MAPK (SB-203580), a potent, selective antagonist of two specific MAPK kinases-MEK1/MEK2 (U0126)-and a selective antagonist of mitogen-activated protein kinase kinase (MAPKK) (PD-98059), as well as by treatment with wortmannin or LY-294002 (both are selective antagonists of PI-3Ks). Inhibitory concentration 50 (IC(50)) levels obtained for these seven antagonists were consistent with reported inhibition constant (Ki) values for these tyrosine kinase, MAPK, and MAPKK antagonists. Ethanol-induced transient and sustained increases in [Ca(2+)](i) in primary single smooth muscle cells from rat aorta were markedly attenuated in the presence of genistein, an SH2 domain inhibitor peptide, SB-203580, U0126, PD-98059, wortmannin, and LY-294002. A variety of specific antagonists of known endogenously formed vasoconstrictors did not inhibit or attenuate either the ethanol-induced contractions or the elevations of [Ca(2+)](i). Results of the present study support the suggestion that activation of tyrosine kinases (including the src family of cytoplasmic tyrosine kinases), PI-3Ks, and MAPK seems to play an important role in ethanol-induced contractions and the elevation of [Ca(2+)](i) in smooth muscle cells from rat aorta. These signaling pathways thus may be important in hypertension in human beings associated with chronic alcohol consumption.

Low extracellular Mg2+ contraction of arterial muscle: role of protein kinase C and protein tyrosine phosphorylation.

The effects of extracellular Mg2+ ion ([Mg2+]0) deficiency on basal tension of isolated rat aortae and rat aortic smooth muscle cell Ca2+ metabolism were investigated in the present study. The contractions of rat aortae induced by diverse concentrations of low [Mg2+]0 were potentiated, greatly, by removal of the endothelium or pre-incubation of intact rat aortic rings with L-N(G)-monomethyl-arginine (L-NMMA). [Mg2+]0 deficiency-induced contractions were inhibited, to different degrees, by pre-treatment of the vessels with low concentrations of Gö6976, bisindolymaleimide I, genistein or a combination of bisindolymaleimide I with genistein. IC50 levels found for these three agents were found to be not too different from Ki values for these drugs. Pre-treatment of rat aortic smooth muscle cells with Gö6976, bisindolymaleimide I, genistein or a combination of bisindolymaleimide I with genistein suppressed, significantly, or almost eliminated both the rapid and stable increments in [Ca2+]i induced by Mg2+-free medium. The present findings suggest that both protein kinase C and protein tyrosine phosphorylation appear to play important roles in Mg2+ deficiency-induced contractions of isolated rat aortic smooth muscle, most likely via phosphorylation of L-type Ca2+ channels.

Ethanol-Induced Contractions in Cerebral Arteries : Role of Tyrosine and Mitogen-Activated Protein Kinases Editorial Comment: Role of Tyrosine and Mitogen-Activated Protein Kinases

Background and Purpose— The relationship between alcohol consumption and stroke appears complex; moderate ingestion is associated with reduced stroke risk, while heavy intake is associated with increased stroke risk. Ethanol has been shown both experimentally and epidemiologically to induce hemorrhagic and ischemic strokes, which are associated with cerebral vasoconstriction. Ethanol is known to induce contraction in isolated cerebral arteries and intact microvessels from diverse mammalian animals. The relationships between ethanol-induced contractions in cerebral arteries, intracellular free Ca2+ ([Ca2+]i), tyrosine kinases (including the src family), and mitogen-activated protein kinases (MAPK) were investigated in the present study. Methods— Cerebral arterial muscle tension and [Ca2+]i were quantified by an isometric contraction technique and direct visualization of Ca2+ in single cells. Results— Ethanol induces concentration-dependent contractions in intact canine basilar arteries, which are attenuated significantly by pretreatment of the arteries with low concentrations of an antagonist of protein tyrosine kinases (genistein); an src homology 2 (SH2) domain inhibitor peptide; a highly specific antagonist of p38 MAPK (SB-203580); a potent, selective antagonist of MEK1/MEK2 (U0126); and a selective antagonist of mitogen-activated protein kinase kinase (MAPKK) (PD-98059). IC50 levels obtained for these 5 antagonists are consistent with reported Ki values for these tyrosine kinase, MAPK, and MAPKK antagonists. Ethanol induces transient and sustained increases in [Ca2+]i in primary single smooth muscle cells from canine basilar arteries, which are markedly attenuated in the presence of genistein, an SH2 domain inhibitor peptide, SB-203580, U0126, and PD-98059. Several specific antagonists of known endogenously formed vasoconstrictors do not inhibit or attenuate either the ethanol-induced contractions or the elevation of [Ca2+]i. Conclusions— The present study suggests that activation of protein tyrosine kinases (including the src family) and MAPK appear to play important roles in the ethanol-induced contractions and the elevation of [Ca2+]i in smooth muscle cells from canine basilar arteries. The results could be used to suggest that selective antagonists of protein tyrosine kinases and MAPK may be useful both prophylactically and therapeutically in alcohol-induced strokes.