Raising extracellular Ca2+ (Ca2+o) stimulating the Ca(2+)-sensing receptor (CaR) decreased the activity of the apical 70-pS K+ channel via a cytochrome P-450-dependent mechanism in the thick ascending limb (TAL) of the rat kidney [W. H. Wang, M. Lu, and S. C. Hebert. Am. J. Physiol. 270 (Cell Physiol. 39): C103-C111, 1996]. We have now used the patch-clamp technique and fluorescent dyes to investigate the signaling mechanism by which this effect is produced. Addition of 500 microM gadolinium (Gd3+), an agent which has been shown to activate the CaR (E. M. Brown, G. Gamba, D. Riccardi, M. Lombardi, R. Butters, O. Kifor, A. Sun, M. A. Hediger, J. Lytton, and S. C. Hebert. Nature 366: 575-580, 1993), mimics the inhibitory effect of raising Ca2+o from 1.1 to 5 mM on channel activity. Effects of the high Ca2+o and Gd3+ were abolished by blockade of phospholipase A2 (PLA2) but not by inhibition of phospholipase C (PLC). Raising Ca2+o also increased 20-hydroxyeicosatetraenoic acid production significantly. To investigate the effect of stimulation of the CaR on intracellular Ca2+ (Ca2+i), we used the acetoxymethyl ester of fura 2 to monitor the Ca2+i. Raising Ca2+o from 1.1 to 5 mM increased the Ca2+i significantly from 50 to 150 nM. However, addition of thapsigargin failed to abolish the effect of 5 mM Ca2+o on Ca2+i. Also, application of Gd3+ only slightly increased the Ca2+i, suggesting that elevation of the Ca2+i by high Ca2+o was the result of an influx of Ca2+ rather than enhanced Ca2+ release from Ca2+ stores. That the increase in Ca2+ influx is not mainly responsible for the effect of stimulating the CaR on channel activity is further supported by experiments in which 500 microM Gd3+ inhibited the K+ channel in cell-attached patches in a Ca(2+)-free bath. Furthermore, addition of 500 microM Gd3+ or 5 mM Ca2+o decreased intracellular Na+ measured with fluorescent sodium indicator, suggesting inhibition of Na+ transport. We conclude that PLA2 is involved in the stimulation of the CaR-induced inhibition of apical K+ channels in the TAL.
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