(Applicant?s abstract) The Na+:Ca2+ exchanger is a major regulator of intracellular calcium ([Ca2+]I) in renal contractile cells including afferent arteriole smooth muscle cells and mesangial cells. Dysregulation of [Ca2+]I in these contractile cells can lead to deleterious pathogenesis of salt sensitive hypertension through dysregulation of [Ca2+]I is oxidative stress, a condition characterized by excessive levels of reactive oxygen metabolites (ROM). The mechanism by which oxidative stress effects dysregulation of renal contractile cell [Ca2+]I in salt-sensitive hypertension is not known. However, studies have shown that under oxidative stress conditions, levels of the cells, is attenuated. This occurs, in part, through inaction of NO with the superoxide anion, O2, forming peroxynitrite (ONOO-). Peroxynitrite has been implicated in cancer, neurodegenerative diseases, and salt-sensitive hypertension. The formation of this product leads to nitration of tyrosine residues in a number of proteins and inactivation of these proteins, in part, through protein aggregation and degradation. Because of the critical role that the Na+-Ca2+ exchanger plays in regulating [Ca2+], in renal contractile cells, we propose and will test the hypothesis that dysregulation of [Ca2+}I under oxidative stress conditions occurs through attenuation of Na+:Ca2+ exchanger activity in these cells. We will examine the effects of oxidative stress on Na+-Ca2+ exchanger activity in primary cultures of mesangial cells of Salt Sensitive (S) and Salt Resistant Dahl/Rapp rats, a genetic model of hypertension that mimics this disorder in humans. We will also examine the effects of oxidative stress on exchanger activity in opossum proximal tubule (OK-PTH) cells which have been stably transfected with either the salt resistant (RNCX1) or the salt sensitive (SNCX1) Na+:Ca2+ exchanger isoform that we have recently cloned from mesangial cells of S and R rats. We will examine exchanger nitration, phosphorylation, and translocation under oxidative stress conditions and determine how they relate to the attenuation of exchanger activity.
