Cytochrome P-450 metabolites mediate extracellular Ca (2+) -induced inhibition of apical K+ channels in the TAL
Wang, W.H.; Lu, M.; Hebert, S.C.
American Journal of Physiology 271(1 Pt 1): C103-C111
1996
ISSN/ISBN: 0002-9513 PMID: 8760035 Document Number: 462383
We used the patch-clamp technique to study the effect of extracellular Ca-2+ (Ca-o-2+) on the activity of the apical 70-pS K+ channel in the isolated split-open thick ascending limb (TAL) of the rat kidney. Raising Ca-o-2+ from 1.1 to 5 mM reversibly reduced the activity of the 70-pS K+ channel in cell-attached patches to 16 +- 2% of the control value within 300 s. In addition, 50 mu-M neomycin mimicked the effect of an increase in Ca-o-2+ on channel activity in cell-attached patches and completely inhibited channel activity. The effect of neomycin on the channel activity in cell-attached patches is an indirect effect, since addition of 50 mu-M neomycin on the 70-pS K+ channel in inside-out patches reduced only the apparent amplitude of the channel current without changing channel open probability. We examined further the role of protein kinase C (PKC) and the cytochrome P-450-dependent metabolites of arachidonic acid in mediating the Ca-o-2+-induced inhibition of channel activity Addition of phorbol 12-myristate 13-acetate (2 mu-M) reversibly blocked channel activity in cell-attached patches to 4 +- 1% of the control value, whereas 75 nM calphostin C increased the channel activity by 115 +- 10%. Moreover, addition of 1 nM exogenous PKC reversibly and completely inhibited the 70-pS K+ channel. However, inhibition of PKC with calphostin C (75 nM) only slightly prolonged the time course of the effect of Ca-o-2+ on channel activity (370 +- 40 s) and failed to abolish the inhibitory effect of 5 mM Ca-o-2+ on channel activity in cell-attached patches, indicating that PKC was not mainly responsible for the effect of Ca-o-2+ on channel activity. In contrast, the effect of 5 mM Ca-o-2+ on the apical 70-pS K+ channel was completely abolished when TAL tubules were first incubated in the 17-octadecenoic acid (5 mu-M)-containing solution, an agent that specifically blocks cytochrome P-450 monooxygenase. In conclusion, these data indicate that Ca-o-2+ is an important regulator of the apical 70-PS K+ channel and that a cytochrome P-450-dependent metabolite of arachidonic acid is involved in mediating this inhibitory effect.