Effects of peroxynitrite on sarcoplasmic reticulum Ca²⁺ pump in pig coronary artery smooth muscle

Abstract

Peroxynitrite generated in arteries from superoxide and NO may damage Ca(2+) pumps. Here, we report the effects of peroxynitrite on ATP-dependent azide-insensitive uptake of Ca(2+) into pig coronary artery vesicular membrane fractions F2 [enriched in plasma membrane (PM)] and F3 [enriched in sarcoplasmic reticulum (SR)]. Membranes were pretreated with peroxynitrite and then with DTT to quench this agent. This pretreatment inhibited Ca(2+) uptake in a peroxynitrite concentration-dependent manner, but the effect was more severe in F3 than in F2. The inhibition was thus not overcome by excess DTT used to quench peroxynitrite and was not affected if catalase, SOD, or mannitol was added along with peroxynitrite. Such damage to the pump protein would be difficult to repair if produced during ischemia-reperfusion. The acylphosphates formed with ATP in F3 corresponded mainly to the SR Ca(2+) pump (110 kDa), but in F2 both PM (140 kDa) and 110-kDa bands were observed. Peroxynitrite treatment of F2 inhibited only the 110-kDa band. Inhibition of Ca(2+) uptake and acylphosphate formation from ATP correlated well in peroxynitrite-treated F3 samples. However, inhibition of acylphosphates from orthophosphate (reverse reaction of the pump) was slightly poorer. Peroxynitrite treatment also covalently cross-linked the pump protein, yielding no dimers but only larger oligomers. In contrast, cross-linking of the SR Ca(2+) pump in skeletal and cardiac muscles gives dimers as the first oligomers. Therefore, we speculate that SERCA2 has a different quaternary structure in the coronary artery smooth muscle.

Effects of peroxynitrite on sarco/endoplasmic reticulum Ca2+ pump isoforms SERCA2b and SERCA3a

“…Thus SERCA2b and SERCA3a differed in their oligomerization patterns and the inhibition of the acylphosphate formation by the monomers. The pattern of oligomerization observed with SERCA2b differs from that observed previously in the pig coronary artery smooth muscle and is closer to that observed upon peroxynitrite treatment of SERCA2a in slow-twitch muscle (24,40). Thus it is likely that the oligomerization pattern may also be tissue dependent.…”

“…It is inhibited by peroxynitrite and its decomposition product hydrogen peroxide. To avoid such interference from peroxynitrite in the subsequent assays, after incubation for 10 min at 37°C, the peroxynitrite and any hydrogen peroxide produced from its decomposition was quenched by adding dithiothreitol (DTT) to a final concentration of 1 mM and placing the samples on ice (24,41,43). Ca 2ϩ uptake.…”

“…Peroxynitrite differs from peroxide and superoxide in that it can act directly on proteins by S-nitrosation or tyrosine nitration rather than by simply oxidizing cysteine residues in proteins. In cardiac muscle, slowtwitch skeletal muscle, and pig coronary artery smooth muscle, the peroxynitrite treatment produces a loss of the SERCA pump ATPase activity (24,39,40,42). All of these tissues express SERCA2 gene products.…”

Platelet Sarcoplasmic Endoplasmic Reticulum Ca 2+ -ATPase and μ-Calpain Activity Are Altered in Type 2 Diabetes Mellitus and Restored by Rosiglitazone

“…20 Higher concentrations of ONOO Ϫ , on the other hand, enhanced the tyrosine nitration of SERCA-2 in platelets from nondiabetic individuals and decreased platelet Ca 2ϩ -ATPase activity, findings that are consistent with reports in overexpressing systems 21 and in skeletal and smooth muscle. 22,23 At this point, we can only speculate about the in vivo source of the ONOO Ϫ that targets platelets and other plasma proteins and results in their increased tyrosine nitration, 24 but it is certainly possible that it is derived from an uncoupled NOS in either the vascular wall or other circulating cells. 25 Over the last few years, several reports have highlighted the importance of calpain in platelet granule secretion and aggregation.…”

Effects of Free Radicals on Coronary Artery

“…In general, the SERCA pumps are more susceptible to ROS than are the PM Ca 2+ pumps [46][47][48][49]. The resistance is important as it allows the cells to survive as excessive extracellular Ca 2+ would cause cell death.…”