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Articles in PresS, published online ahead of print November 6, 2002
Am J Physiol Cell Physiol, 10.1152/ajpcell.00297.2002
Submitted on June 26, 2002
Accepted on September 6, 2002
1 Medicine, McMaster University, Hamilton, ON, Canada; Biology, McMaster University, Hamilton, ON, Canada
2 Medicine, McMaster University, Hamilton, ON, Canada
* To whom correspondence should be addressed. E-mail: groverak{at}mcmaster.ca.
Peroxynitrite generated in the arteries from superoxide and NO may damage Ca2+ pumps. Here, we report the effects of peroxynitrite on the ATP-dependent azide insensitive uptake of Ca2+ 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 dithiothreitol to quench this agent. This pretreatment inhibited the Ca2+ 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 nor was it affected if catalase, superoxide dismutase plus mannitol were added along with peroxynitrite. Such damage to the pump protein would be difficult to repair if produced during ischaemia-reperfusion. The acylphosphates formed with ATP in F3 corresponded mainly to the SR Ca2+ pump (110 kDa) but in F2 both PM (140 kDa) and 110 kDa bands were observed. Peroxynitrite treatment of F2 inhibited only the 110 band. The inhibition of Ca2+ 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 covalent cross linked the pump protein yielding no dimers but only larger oligomers. In contrast, cross linking of the SR Ca2+ pump in skeletal and cardiac muscles gives dimers as the first oligomers. Therefore, we suggest that SERCA2 has a different quaternary structure in the coronary artery smooth muscle.
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