Differential effects of peroxynitrite on the function of arginine vasopressin V(1a) receptors and alpha(1)-adrenoceptors in vivo.

OBJECTIVE: The aim of this study was to provide evidence that peroxynitrite may differentially affect the function of arginine vasopressin (AVP) V(1a) receptors and alpha(1)-adrenoceptors in vascular smooth muscle of the rat METHODS: The vasoconstrictor responses elicited by AVP, or the alpha(1)-adrenoceptor agonist, phenylephrine, were determined in anesthetized rats before and after injections of (i) peroxynitrite, the thiol chelator, para-hydroxymercurobenzoic acid (PHMBA), or the electron acceptor, nitroblue tetrazolium (NBT). The ability of the reducing agent, glutathione, to reverse the loss of response to phenylephrine and AVP in peroxynitrite-treated rats was also examined. RESULTS: The AVP-induced responses were suppressed 10-20 min but not 60-70 min after the administration of peroxynitrite. Glutathione reversed the above loss of response to AVP at 10-20 min. The responses elicited by phenylephrine were suppressed 10-20 min and 60-70 min after administration of peroxynitrite. Glutathione did not reverse the above losses of response to phenylephrine. In addition, the vasoconstrictor actions of AVP and phenylephrine were markedly suppressed after administration of PHMBA or nitroblue tetrazolium. CONCLUSIONS: The above findings provide evidence that exogenously administered peroxynitrite may differentially affect the function of AVP V(1a) receptors and alpha(1)-adrenoceptors in vascular smooth muscle of the rat. The possibility that peroxynitrite impairs AVP V(1a) receptor function by transient oxidation events whereas peroxynitrite impairs alpha(1)-adrenoceptor function by transient oxidation and permanent nitration events will be discussed.

L-beta,beta-dimethylcysteine attenuates the haemodynamic responses elicited by systemic injections of peroxynitrite in anaesthetized rats.

1 There is direct chemical evidence that L-beta,beta-dimethylcysteine (L-penicillamine (L-PEN)) is a scavenger of peroxynitrite. The aim of this study was to determine whether L-PEN attenuates the haemodynamic responses elicited by peroxynitrite in pentobarbital-anaesthetized rats. 2 Peroxynitrite (1-20 micromol kg(-1), i.v.) elicited dose-dependent reductions in mean arterial blood pressure (MAP) and mesenteric and hindquarter vascular resistances. 3 L-PEN (2 mmol kg(-1), i.v.) elicited relatively minor but significant increases in MAP and vascular resistances. The initial reductions in MAP and vascular resistances elicited by peroxynitrite were not diminished after administration of L-PEN whereas they were much shorter in duration. As such, the total reductions in MAP and vascular resistances were markedly reduced by L-PEN. 4 The finding that L-PEN (2 mmol kg(-1), i.v.) did not affect the hypotensive or vasodilator responses elicited of the ATP-dependent potassium-channel agonist, cromakalim (3-18 microg kg(-1), i.v.), suggests that this dose of L-PEN is not a nonselective inhibitor of vasodilation. 5 These findings suggest that L-PEN may effectively scavenge peroxynitrite in vivo and/or interfere with the mechanisms by which peroxynitrite elicits its vasodilator responses.

Peroxynitrite elicits dysfunction of stereoselective s-nitrosocysteine recognition sites.

The aim of this study was to determine whether induction of tachyphylaxis to peroxynitrite (induced by giving 10 intravenous injections of a 10-micromol/kg dose) differentially affects the vasodilator responses elicited by systemic injections of the L- and D-isomers of S-nitrosocysteine (L-SNC and D-SNC), in pentobarbital-anesthetized rats. L- and D-SNC (12.5-200 nmol/kg, iv) elicited dose-dependent reductions in hindquarter, mesenteric, and renal vascular resistances. The L-SNC-induced vasodilator responses in the hindquarter and renal vascular beds were virtually abolished whereas the vasodilator responses in mesenteric bed were markedly diminished after administration of peroxynitrite. The D-SNC-induced vasodilator responses in the hindquarter and renal beds were slightly attenuated whereas the vasodilator responses in the mesenteric bed were not diminished after administration of peroxynitrite. The vasodilator responses elicited by the nitric oxide donor, MAHMA NONOate (5-50 nmol/kg, iv), were not attenuated by peroxynitrite. The finding that induction of tachyphylaxis to peroxynitrite diminishes the effects of L- and D-SNC but not MAHMA NONOate suggests that the stereoisomers exert their vasodilator effects by mechanisms other than their decomposition to nitric oxide. Moreover, the finding that induction of tachyphylaxis to peroxynitrite causes a more pronounced attenuation of the vasodilator effects of L- than D-SNC supports evidence that the stereoisomers differentially interact with stereoselective S-nitrosothiol recognition sites in the vasculature. Taken together, these novel results support the possibility that peroxynitrite diminishes the vasodilator potencies of L- and D-SNC by oxidation and/or nitration of amino acids in these recognition sites.

Loss of K+ATP-channel-mediated vasodilation after induction of tachyphylaxis to peroxynitrite.

Systemic injections of peroxynitrite elicit pronounced vasodilator responses in rats by activation of ATP-dependent K+ channels (K+ATP-channels). The aim of this study was to determine whether development of tachyphylaxis to the vasodilator actions of peroxynitrite involves the loss of K+ATP-channel function. The falls in mean arterial blood pressure (MAP) and mesenteric and hindquarter vascular resistances produced by the K+ATP-channel agonist, cromakalim (3-18 microg/kg, iv), and the nitric oxide (NO) donor, sodium nitroprusside (SNP; 1-4 microg/kg, iv), were determined in pentobarbital-anesthetized rats before and after induction of tachyphylaxis to peroxynitrite induced by the administration of 10 injections of peroxynitrite (10 micromol/kg, iv). The first dose of peroxynitrite elicited pronounced falls in MAP and vascular resistances whereas the tenth injection elicited much smaller responses that were equivalent to those of decomposed peroxynitrite. Before induction of tachyphylaxis to peroxynitrite, cromakalim and SNP produced dose-dependent reductions in MAP and vascular resistances. The hemodynamic actions of cromakalim were markedly attenuated after induction of tachyphylaxis to peroxynitrite whereas the SNP-induced responses were only slightly attenuated. These results suggest that tachyphylaxis to the vasodilator actions of peroxynitrite involves the loss of K+ATP-channel function whereas tachyphylaxis to peroxynitrite minimally affects NO-mediated vasodilation. Taken together, these findings raise the possibility that peroxynitrite inhibits K+ATP-channel function by oxidation and/or nitration of amino acids in these channels.

Role of ATP-sensitive K+ -channels in hemodynamic effects of peroxynitrite in anesthetized rats.

The aim of this study was to determine whether the hypotensive and vasodilator actions of peroxynitrite in pentobarbital-anesthetized rats involve the activation of ATP-sensitive K+-channels (K+ATP-channels). The effects of the K+ATP-channel agonist, cromakalim (9-36 microg/kg, iv), peroxynitrite (0.5-10 micromol/kg iv), and L-S-nitrosocysteine (12.5-200 nmol/kg, iv) on mean arterial blood pressure (MAP) and mesenteric (MR) and hindquarter (HQR) vascular resistances were determined before and after injection of the K+ATP-channel blocker, glibenclamide (40 micromol/kg, iv). Cromakalim, peroxynitrite, and L-S-nitrosocysteine produced dose-dependent reductions in MAP, MR, and HQR. Administration of glibenclamide did not affect resting hemodynamic parameters but markedly attenuated the hemodynamic actions of cromakalim. The maximal falls in MAP and HQR produced by peroxynitrite were attenuated by glibenclamide whereas the maximal falls in MR were not affected. In addition, the duration of the hypotensive and vasodilator effects of peroxynitrite in the mesenteric and hindquarter beds were markedly diminished by glibenclamide. In contrast, glibenclamide did not affect the maximal hypotensive or vasodilator effects of L-S-nitrosocysteine or the duration of these responses. These results suggest that the hypotensive and vasodilator actions of peroxynitrite in anesthetized rats involve the activation of K+ATP-channels whereas the hemodynamic actions of L-S-nitrosocysteine do not.

Potential role of nitration and oxidation reactions in the effects of peroxynitrite on the function of beta-adrenoceptor sub-types in the rat.

This study examined the hemodynamic responses elicited by the beta-adrenoceptor agonist, isoproterenol (1 and 10 microg/kg, i.v.) before and after administration of (i) peroxynitrite (10 x 10 micromol/kg, i.v.), (ii) the thiol chelator, para-hydroxymercurobenzoic acid (pHMBA, 75 micromol/kg, i.v.), and (iii) the electron acceptor, nitroblue tetrazolium (NBT, 10 micromol/kg, i.v.) in pentobarbital-anesthetized rats. The tachycardia elicited by the lower dose of isoproterenol was diminished whereas the tachycardia elicited by the higher dose was not attenuated after administration of peroxynitrite. The falls in hindquarter and renal vascular resistances elicited by both doses of isoproterenol were substantially diminished whereas the isoproterenol-induced falls in mesenteric vascular resistance were not changed after administration of peroxynitrite. All of the isoproterenol-induced responses were markedly attenuated after administration of pHMBA or NBT. These findings suggest that the oxidation and/or nitration of beta-adrenoceptors impair the ability of isoproterenol to bind to and/or activate these G protein-coupled receptors. beta1-, beta2- and beta3-adrenoceptors contain extracellular cysteine residues susceptible to oxidation (i.e., disulfide-bridge formation) whereas only the beta1- and beta2-adrenoceptors contain extracellular tyrosine residues susceptible to nitration. These findings also suggest that sustained impairment of beta1- and beta2-adrenoceptor function by peroxynitrite is due to nitration of extracellular tyrosine residues in these receptors. By analogy, beta3-adrenoceptors may not be permanently affected by peroxynitrite because these receptors are devoid of extracellular tyrosine residues.

Vasodilator actions of the endothelium-derived relaxing factor L-S-nitrosocysteine in anaesthetized rats are markedly diminished by peroxynitrite.

The aim of the present study was to assess the effects of the potent oxidant and nitrating agent peroxynitrite on the haemodynamic actions of the endothelium-derived S-nitrosothiol L-S-nitrosocysteine. The haemodynamic actions of L-S-nitrosocysteine (12.5-100 nmol/kg, i.v.) were determined in pentobarbital-anaesthetized rats before and after the induction of tachyphylaxis to peroxynitrite achieved by giving 10 intravenous injections of a 10 micromol/kg dose. L-S-Nitrosocysteine elicited dose-dependent reductions in mean arterial blood pressure and in hindquarter and mesenteric vascular resistance. The L-S-nitrosocysteine-induced responses were substantially attenuated after administration of peroxynitrite. We have reported previously that nitric oxide-mediated vasodilation is not diminished after the induction of tachyphylaxis to peroxynitrite. Taken together, these findings support the concept that peroxynitrite reduces the vasodilator actions of L-S-nitrosocysteine via oxidation and/or nitration of putative membrane-bound S-nitrosothiol recognition sites.

A formidable challenge: the diagnosis and treatment of viral myocarditis in children.

It is generally well accepted that one third of patients with viral myocarditis experience a complete recovery of normal cardiac function, one third improve clinically but show residual cardiac dysfunction, and one third experience chronic heart failure and die or require heart transplantation. It is hoped that a better understanding of the underlying cause and pathogenesis of this disease will increase the number of patients who experience a complete recovery. New advances in both the diagnosis and treatment of viral myocarditis continue to enter clinical practice at a rapid pace, and it is likely that a genomic approach to the diagnostic evaluation and treatment of this disease will become possible in the near future. Viral myocarditis, however, will remain a significant diagnosticand therapeutic challenge to both physicians and scientists alike.

Peroxynitrite inhibits epidermal growth factor receptor signaling in Caco-2 cells.

Intestinal mucosa serves as an important barrier that may be disrupted by inflammation. A complex system of cellular and humoral factors, including epidermal growth factor (EGF), maintains the integrity of this barrier. We hypothesized that peroxynitrite, generated in inflamed intestinal epithelium, can alter the EGF receptor function by nitrating tyrosine residues and blocking ligand-activated tyrosine autophosphorylation. Caco-2 cells or A431 cell membranes were treated with peroxynitrite or its decomposed form. Cell proliferation was measured by [3H]thymidine uptake. Immunoblot and immunoprecipitation were used to assess the tyrosine phosphorylation and nitration. Binding of epidermal growth factor to its receptor was detected by affinity labeling with 125I-EGF. Peroxynitrite inhibited EGF-induced Caco-2 cell proliferation and binding of EGF to its receptor in a concentration-dependent manner. Peroxynitrite abolished EGF-stimulated receptor autophosphorylation and nitrated EGF receptor tyrosine residues. Peroxynitrite generated during inflammation may disrupt the EGF-induced signaling in intestinal epithelial cells.

Ablation of the heat shock factor-1 increases susceptibility to hyperoxia-mediated cellular injury.

High concentrations of oxygen (hyperoxia) are known to cause cellular injury and death. The heat shock response is a highly conserved cellular defense mechanism that protects cells against various environmental stressors, including hyperoxia. Herein we determined the role of heat shock factor-1 (HSF-1), a major component of the heat shock response, in protecting cells against hyperoxia. Embryonic fibroblasts from HSF-1-null mutant mice (HSF-1 -/- cells) were compared to wild-type embryonic fibroblasts (HSF-1 +/+ cells) following 24 hours' exposure to room air or hyperoxia (95% O(2)). Acute survival in hyperoxia was decreased in HSF-1 -/- cells as compared to HSF-1 +/+ cells. Intracellular ATP levels were significantly lower in the HSF-1 -/- cells as compared to the HSF-1 +/+ cells exposed to hyperoxia. Isoprostane levels, a marker of membrane lipid peroxidation, were significantly higher in the HSF-1 -/- cells as compared to the HSF-1 +/+ cells exposed to hyperoxia. Restoration of HSF-1 in the HSF-1 -/- cells by stable transfection with a HSF-1 expression plasmid improved survival in hyperoxia when compared to HSF-1 -/- cells stably transfected with the empty expression vector. Hyperoxia increased activation of HSF-1 in HSF-1 +/+ cells and in HSF-1 -/- cells stably transfected with the HSF-1 expression plasmid. These data demonstrate that HSF-1 plays an important role in conferring resistance to hyperoxia in vitro.