Hypokalemia impairs the transport function of the thick ascending limb (TAL) and diminishes urinary concentrating ability. Since apical K channels play an important role in K recycling across the apical membrane in the TAL, inhibition of the apical K channels is expected to block the function of the Na/K/Cl cotransporter and to attenuate the urinary concentrating ability. Preliminary results have shown that the activity of apical K channels in the mTAL from animals on a K-deficient diet is significantly lower than those of a high K diet. Moreover, K- depletion increases the production of 20-hydroxyeicosatetraenoic acid and inhibition of cytochrome P450 monoxygenase of arachidonic acid (AA) increases the activity of the apical 70 pS K channels which contribute 80% of K conductance to the apical membrane. Thus, we will test the hypothesis that an increase in cytochrome p450-dependent metabolites of AA is involved in inhibiting the apical K conductance of the TAL in rats on a K-deficient diet. The second hypothesis of Ca2+ sensing receptor is responsible for decreasing the apical K conductance in the TAL from animals on a K-deficient diet. This hypothesis is based on the observation that extracellular Ca2+ concentrations required for inducing a 50% inhibition of the apical 70 pS K channel were significantly lower in the tubules from rats on a K-deficient diet than those on a high K diet. The third hypothesis of the proposal is that a decrease in the activity of nitric oxide synthase (NOS) and heme oxygenase (HO) is responsible for reducing the activity of apical K channels in the TAL from rats on a K- deficient diet. Preliminary data shows that the expression of iNOS and HO-2 decreases in the renal medulla from animals on a K-deficient diet. Moreover, previous studies have shown that NO and CO stimulate the apical 70pS K channels in the mTAL. To achieve our goals, the patch clamp technique, ion-sensitive dye, biochemical approaches and microperfusion techniques will be used to assess effects of AA metabolites, CO and NO on channel activity as well as Na transport in the mTAL.
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