nitroarginine and ebselen

Heme is a co-factor required for the stimulation of soluble guanylate cyclase (sGC) by nitric oxide (NO) and carbon monoxide, and sGC activation by these agents is inhibited by superoxide. Because heme promotes oxidant generation, we examined the influence of rat pulmonary microvascular endothelial cells (PMECs) with a stable human heme oxygenase-1 (HO-1) transfection and heme on oxidant generation and cGMP. Culture of PMEC with low serum heme decreased cGMP and the detection of peroxide with 10 microM 2',7'-dichlorofluorescin diacetate and increased HO-1 further decreased cGMP without altering the peroxide detection under these conditions. Under conditions where heme (30 microM) has been shown to stimulate cGMP production in PMECsby mechanisms involving NO and CO, heme increased the detection of peroxide in a PMEC-dependent manner and HO-1 transfection did not markedly alter the effects heme on peroxide detection. The addition of 1 microM catalase markedly inhibited the effects of heme on peroxide detection whereas increasing (0.1 mM ebselen) or decreasing (depleting glutathione with 7 mM diethylmaleate) rates of intracellular peroxide metabolism or inhibiting the biosynthesis of oxidants (with 10 microM diphenyliodonium or 0.1 mM nitro-L-arginine) had only modest effects. The detection of superoxide by 10 microM dihydroethidium from PMECs was not increased by exposure to heme. These actions of oxidant probes suggest that intracellular oxidants have a minimal influence on the response to heme. Thus, exposure of PMECs to heme causes a complex response involving an extracellular generation of peroxide-derived oxidant species, which do not appear to originate from increases in intracellular superoxide or peroxide. This enables heme and HO to regulate sGC through mechanisms involving NO and CO, which are normally inhibited by superoxide.

Topics: Animals; Antioxidants; Azoles; Catalase; Cells, Cultured; Cyclic GMP; Endothelium, Vascular; Enzyme Inhibitors; Heme; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; Humans; Isoindoles; Lung; Maleates; Membrane Proteins; Nitric Oxide Synthase; Nitroarginine; Organoselenium Compounds; Oxidants; Rats; Reactive Oxygen Species; Transfection

Growing evidence indicates that reactive oxygen species (ROS) as well as nitric oxide (NO) have a profound influence on contractile function of skeletal muscle possibly through modulation of excitation-contraction coupling. We hypothesized that if NO and xanthine oxidase (XO) interact at key sites in excitation-contraction coupling, the effects of XO with nitric oxide synthase (NOS) inhibitors and NO donors on contractile function of the unfatigued diaphragm would not be additive. Diaphragm fibre bundles were extracted from 4-month Fischer-344 rats and placed in Krebs solution bubbled with 95% O2, 5% CO2. Baseline twitch tension, tension at 20 Hz (low-frequency), and maximal tetanic tension (Po) at 120 Hz were then measured (PRE). In Experiment 1 diaphragm fibre bundles were exposed to Krebs with 200 microM hypoxanthine as a control (CON); 0.02 U mL-1 XO + 200 microM hypoxanthine; 1 mM of the NOS inhibitor N-nitro-L-arginine (L-NNA) or L-NNA + XO. Five minutes were allowed for equilibration, and a second set of contractile measures was taken (POST). In Experiment 2 we exposed diaphragm fibre bundles to one of the following four solutions: CON, XO, 100 microM of the NO donor sodium nitroprusside (SNP) and XO + SNP, and evaluated contractile function as described above. In Experiment 3 we tested to determine if peroxynitrite production from the reaction of superoxide anion and NO affected the above results for SNP using 30 microM ebselen as a peroxynitrite quencher. Xanthine oxidase resulted in a significant potentiation of diaphragm twitch tension and tension at 20 Hz (+29%) without affecting Po. L-NNA also significantly increased 20 Hz tension but did not alter Po. However, the combination of XO + L-NNA did not further increase low-frequency contractility. Sodium nitroprusside alone did not affect diaphragm contractility, but did attenuate XO-induced potentiation in the XO + SNP group. Ebselen did not alter the impact of SNP on XO in the diaphragm. These data support the hypothesis that XO and NO interact or compete at similar sites of action that modulate contractility of the unfatigued diaphragm.

Topics: Analysis of Variance; Animals; Antioxidants; Azoles; Diaphragm; Enzyme Inhibitors; Hypoxanthine; In Vitro Techniques; Isoindoles; Muscle Contraction; Nitrates; Nitric Oxide; Nitroarginine; Nitroprusside; Organoselenium Compounds; Rats; Rats, Inbred F344; Reactive Oxygen Species; Vasodilator Agents; Xanthine Oxidase