Am J Physiol. 1996 Jul;271(1 Pt 1):C103-11.  

Cytochrome P-450 metabolites mediate extracellular Ca(2+)-induced inhibition of
apical K+ channels in the TAL.

Wang WH, Lu M, Hebert SC.

Department of Pharmacology, New York Medical College, Valhalla 10595, USA.

We used the patch-clamp technique to study the effect of extracellular Ca2+
(Ca2+o) on the activity of the apical 70-pS K+ channel in the isolated
split-open thick ascending limb (TAL) of the rat kidney. Raising Ca2+o 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 microM
neomycin mimicked the effect of an increase in Ca2+o 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 microM 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 Ca2+o -induced inhibition of channel activity. Addition of
phorbol 12-myristate 13-acetate (2 microM) 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 Ca2+o on channel activity (370 +/- 40 s) and failed
to abolish the inhibitory effect of 5 mM Ca2+o on channel activity in
cell-attached patches, indicating that PKC was not mainly responsible for the
effect of Ca2+o on channel activity. In contrast, the effect of 5 mM Ca2+o on
the apical 70-pS K+ channel was completely abolished when TAL tubules were first
incubated in the 17-octadecynoic acid (5 microM)-containing solution, an agent
that specifically blocks cytochrome P-450 monooxygenase. In conclusion, these
data indicate that Ca2+o is an important regulator of the apical 70-pS K+
channel and that