P450 metabolites mediate extracellular ca2+-induced inhibition of
apical k+ channels in the thick ascending limb of the rat kidney.
Wang, Wen-Hui, Ming Lu, and Steven C. Hebert.
Department of Pharmacology, New York Medical College, Valhalla, NY
10595 and Department of Medicine, Renal Division, Brigham &
Women's Hospital, Harvard Medical School, Boston, MA 02115
APStracts 3:0018C, 1996.
We used the patch-clamp technique to study the effect of extracellular
Ca2+ (Cao2+) on the activity of the apical 70 pS K+ channel in the
isolated split-open thick ascending limb (TAL) of the rat kidney.
Raising Cao2+ from 1.1 mM 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 seconds. In addition, 50 [mu]M neomycin
mimicked the effect of an increase in Cao2+ 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 P450-dependent metabolites of arachidonic acid in mediating
the Cao2+-induced inhibition of channel activity. Addition of phorbol
12-myristate 13-acetate (PMA, 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 Cao2+ on channel activity (370+/-40 seconds) and failed
to abolish the inhibitory effect of 5 mM Cao2+ on channel activity in
cell-attached patches, indicating that PKC was not mainly responsible
for the effect of Cao2+ on channel activity. In contrast, the effect
of 5 mM Cao2+ on the apical 70 pS K+ channel was completely abolished
when TAL tubules were first incubated in the 17-octadecynoic acid (5
[mu]M) containing solution, an agent which specifically blocks
cytochrome P450-monooxygenase. In conclusion, these data indicate
that Cao2+ is an important regulator of the apical 70 pS K+ channel
and that a cytochrome P450-dependent metabolite of arachidonic acid
is involved in mediating this inhibitory effect.
Received 26 September 1995; accepted in final form 2 January
1996.
APS Manuscript Number C587-5.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 25 January 96