Peroxynitrite resistance of sarco/endoplasmic reticulum Ca2+ pump in pig coronary artery endothelium and smooth muscle.

We examined the effects of peroxynitrite pre-treatment on sarco/endoplasmic reticulum Ca(2+) (SERCA) pump in pig coronary artery smooth muscle and endothelium. In saponin-permeabilized cells, smooth muscle showed much greater rates of the SERCA Ca(2+) pump-dependent (45)Ca(2+) uptake/mg protein than did the endothelial cells. Peroxynitrite treatment of cells inhibited the SERCA pump more severely in smooth muscle cells than in endothelial cells. To determine implications of this observation, we next examined the effect of the SERCA pump inhibitor cyclopiazonic acid (CPA) on intracellular Ca(2+) concentration of intact cultured cells. CPA produced cytosolic Ca(2+) transients in cultured endothelial and smooth muscle cells. Pre-treatment with peroxynitrite (200 microM) inhibited the Ca(2+) transients in the smooth muscle but not in the endothelial cells. CPA contracts de-endothelialized artery rings and relaxes precontracted arteries with intact endothelium. Peroxynitrite (250 microM) pre-treatment inhibited contraction in the de-endothelialized artery rings, but not the endothelium-dependent relaxation. Thus, endothelial cells appear to be more resistant than smooth muscle to the effects of peroxynitrite at the levels of SERCA pump activity, CPA-induced Ca(2+) transients in cultured cells, and the effects of CPA on contractility. The greater resistance of endothelium to peroxynitrite may play a protective role in pathological conditions such as ischemia-reperfusion when excess free radicals are produced.

Effects of peroxynitrite on pig coronary artery smooth muscle.

We examined the effects of peroxynitrite pretreatment of pig coronary arteries on their sarcoplasmic reticulum (SR) Ca(2+) pump function. Pretreating rings from de-endothelialized arteries with peroxynitrite, followed by a wash to remove this agent, led to a decrease in the force of contraction produced in response to the SR Ca(2+) pump inhibitor cyclopiazonic acid (CPA, IC(50) = 87 +/- 6 microM). Inclusion of catalase and superoxide dismutase with the peroxynitrite did not alter its effect indicating that the inhibition was produced by peroxynitrite. Contractions produced by 30 mM KCl were not affected by up to 250 microM peroxynitrite. Smooth muscle cells cultured from this artery gave a transient increase in cytosolic Ca(2+) in response to CPA. Treating the cells with peroxynitrite inhibited this increase. Treating the SR-enriched isolated subcellular membrane fraction with peroxynitrite produced an inhibition of the ATP-dependent azide-insensitive oxalate-stimulated Ca(2+) uptake. Thus, peroxynitrite damages the SR Ca(2+)pump in the coronary artery, and this inhibition appears to lead to an inability of the arteries to respond to CPA. Thus, peroxynitrite produced from superoxide and NO in the arteries may compromise regulation of coronary tone which requires mobilization of Ca(2+) from the SR.

Peroxynitrite and nitric oxide differ in their effects on pig coronary artery smooth muscle.

Peroxynitrite generated in arteries from superoxide and nitric oxide (NO) may damage their function. Here, we compare the effects of peroxynitrite and peroxynitrite/NO-generating agents SIN-1 (3-morpholinosydnonimine hydrochloride), SNAP (S-nitroso-N-acetyl-penicillamine), SNP (sodium nitroprusside), and NONOate (spermine NONOate) on pig coronary artery. Deendothelialized artery rings were pretreated with these agents and then washed before examining their contractility. Pretreatment with all agents (200 microM) results in a decrease in the force of contraction in response to the sarco(endo)plasmic Ca(2+) (SERCA) pump inhibitor cyclopiazonic acid (CPA): SNAP > NONOate > or = peroxynitrite > or = SIN-1 > SNP. Pretreatment with SNAP, NONOate, or SIN-1 also inhibits the force of contraction produced with 30 mM KCl, with SNAP being the most potent. Including catalase plus superoxide dismutase (SOD) during the preincubation has no effect. Including an NO scavenger [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide] or a guanylate cyclase inhibitor (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) partially protects against SNAP. Pretreatment of cultured cells with peroxynitrite, but not with SNAP, inhibits the Ca(2+) transients produced in response to CPA. Pretreating isolated membrane vesicles with peroxynitrite inhibits the Ca(2+) uptake due to the SERCA pump, with all the other agents being less effective. Thus peroxynitrite and NO both inhibit the CPA-induced contractions in deendothelialized artery rings, peroxynitrite by damage to the SERCA pump and NO possibly by a step downstream from the increase in cytosolic Ca(2+).