Phospholipase A2 is involved in mediating the effect of extracellular Ca2+ on apical K+ channels in rat TAL
Wang, W.; Lu, M.; Balazy, M.; Hebert, S.C.
American Journal of Physiology 273(3 Pt 2): F421-F429
1997
ISSN/ISBN: 0002-9513 PMID: 9321915 Document Number: 480396
Raising extracellular Ca-2+ (Ca-o-2+) 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 mu-M gadolinium (Gd-3+), 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 Ca-o-2+ from 1.1 to 5 mM on channel activity. Effects of the high Ca-o-2+ and Gd-2+ were abolished by blockade of phospholipase A-2 (PLA-2) but not by inhibition of phospholipase C (PLC). Raising Ca-o-2+ also increased 20-hydroxyeicosatetraenoic acid production significantly. To investigate the effect of stimulation of the CaR on intracellular Ca-2+ (Ca-i-2+), we used the acetoxymethyl ester of fura 2 to monitor the Ca-i-2+. Raising Ca-o-2+ from 1.1 to 5 mM increased the Ca-i-2+ significantly from 50 to 150 nM. However, addition of thapsigargin failed to abolish the effect of 5 mM Ca-o-2+ on Ca-i-2+. Also, application of Gd-3+ only slightly increased the Ca-i-2+, suggesting that elevation of the Ca-i-2+ by high Ca-o-2+ was the result of an influx of Ca-2+ rather than enhanced Ca-2+ release from Ca-2+ stores. That the increase in Ca-2+ influx is not mainly responsible for the effect of stimulating the CaR on channel activity is further supported by experiments in which 500 mu-M Gd-3+ inhibited the K+ channel in cell-attached patches in a Ca-2+-free bath. Furthermore, addition of 500 mu-M Gd-3+ or 5 mM Ca-2+ decreased intracellular Na+ measured with fluorescent sodium indicator, suggesting inhibition of Na+ transport. We conclude that PLA-2 is involved in the stimulation of the CaR-induced inhibition of apical K+ channels in the TAL.