Recent studies from our laboratory have indicated that renal vascular smooth muscle cells metabolize arachidonic acid via a P4504A dependent pathway to 20-HETE, and that this system serves as a novel signal transduction pathway that plays a central role in the regulation of renal vascular tone by regulate the open state probability of Ca++-activated K+ channels. This proposal will examine the hypothesis that nitric oxide (NO) inhibits the production of 20-HETE in the kidney and that this contributes to its inhibitory effects on renal vascular tone and tubuloglomerular feedback. The influence of NO on the renal metabolism of arachidonic acid will be evaluated using renal microsomes, intact renal microvessels and tubules. Visible light spectroscopy will be utilized to study the binding of NO to recombinant P4504A isoforms expressed with a baculovirus and Sf9 insect cells. The effects of NO on K+ channel activity in renal vascular smooth muscle cells will be characterized using patch clamp techniques., and the relative contribution of 20-HETE versus cGMGP to these responses will be determined by altering intracellular levels of 20-HETE in the presence and absence of inhibitors of guanylyl cyclase and cGMP-dependent protein kinase. Parallel studies will determine the contribution of changes in the production of 20-HETE versus cGMP to the effects of NO on membrane potential and vascular tone in isolated perfused renal arterioles. The effects of P4504A inhibitors on the changes in arterial pressure and renal blood flow produced by NO donors and synthase inhibitors will be studied in rats to evaluate the significance of NO-20- HETE interactions in the regulation of renal and peripheral vascular tone in vivo, and tubular perfusion studies will be performed to determine the contribution of 20-HETE to the inhibitory effects of NO on tubuloglomerular feedback. Finally, the effects of changes in salt intake and circulating levels of AII on the distribution of P4504A mRNA, protein and enzyme activity along the nephron will be measured using competitive RT-PCR, Western blots and radio-chemical assays to determine whether changes in the expression of this system might contribute to the modulation of renal vascular tone and tubuloglomerular feedback associated with changes in salt intake.
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