title: Cytochrome P-450 metabolites mediate extracellular Ca(2+)-induced inhibition of apical K+ channels in the TAL.
abstract: 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 a cytochrome P-450-dependent metabolite of arachidonic acid is involved in mediating this inhibitory effect.
authors: Department of Pharmacology, New York Medical College, Valhalla 10595, USA.
authors: W H
authors: WH
authors: Wang
authors: M
authors: M
authors: Lu
authors: S C
authors: SC
authors: Hebert
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000494:pharmacology / Q000502:physiology*; D002462:Cell Membrane / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D005110:Extracellular Space / Q000378:metabolism*; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D066298:In Vitro Techniques; D008138:Loop of Henle / Q000378:metabolism*; D009355:Neomycin / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D026902:Potassium Channel Blockers; D015221:Potassium Channels / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D005231:Fatty Acids, Unsaturated; D026902:Potassium Channel Blockers; D015221:Potassium Channels; C047008:17-octadecynoic acid; D000255:Adenosine Triphosphate; D003577:Cytochrome P-450 Enzyme System; D011493:Protein Kinase C; D009355:Neomycin; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium