Regulation of ATP-sensitive K(+) channels by protein kinase C in murine colonic myocytes.
We investigated the regulation of ATP-sensitive K(+) (K(ATP)) currents in murine colonic myocytes with patch-clamp techniques. Pinacidil (10(-5) M) activated inward currents in the presence of high external K(+) (90 mM) at a holding potential of -80 mV in dialyzed cells. Glibenclamide (10(-5) M) suppressed pinacidil-activated current. Phorbol 12,13-dibutyrate (PDBu; 2 x 10(-7) M) inhibited pinacidil-activated current. 4-alpha-Phorbol ester (5 x 10(-7) M), an inactive form of PDBu, had no effect on pinacidil-activated current. In cell-attached patches, the open probability of K(ATP) channels was increased by pinacidil, and PDBu suppressed openings of K(ATP) channels. When cells were pretreated with chelerythrine (10(-6) M) or calphostin C (10(-7) M), inhibition of the pinacidil-activated whole cell currents by PDBu was significantly reduced. In cells studied with the perforated patch technique, PDBu also inhibited pinacidil-activated current, and this inhibition was reduced by chelerythrine (10(-6) M). Acetylcholine (ACh; 10(-5) M) inhibited pinacidil-activated currents, and preincubation of cells with calphostin C (10(-7) M) decreased the effect of ACh. Cells dialyzed with protein kinase C epsilon-isoform (PKCepsilon) antibody had normal responses to pinacidil, but the effects of PDBu and ACh on K(ATP) were blocked in these cells. Immunofluorescence and Western blots showed expression of PKCepsilon in intact muscles and isolated smooth muscle cells of the murine proximal colon. These data suggest that PKC regulates K(ATP) in colonic muscle cells and that the effects of ACh on K(ATP) are largely mediated by PKC. PKCepsilon appears to be the major isozyme that regulates K(ATP) in murine colonic myocytes.
Jun
J Y
JY
Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada 89557, USA.
Kong
I D
ID
Koh
S D
SD
Wang
X Y
XY
Perrino
B A
BA
Ward
S M
SM
Sanders
K M
KM
D000109:Acetylcholine / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000470:Alkaloids; D000818:Animals; D053119:Benzophenanthridines; D003106:Colon / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005905:Glyburide / Q000494:pharmacology; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D051379:Mice; D008807:Mice, Inbred BALB C; D009130:Muscle, Smooth / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D009281:Naphthalenes / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D010617:Phenanthridines / Q000494:pharmacology; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D020110:Pinacidil / Q000494:pharmacology; D015221:Potassium Channels / Q000187:drug effects / Q000502:physiology*; D011493:Protein Kinase C / Q000378:metabolism*; D051744:Protein Kinase C-epsilon
D000470:Alkaloids; D053119:Benzophenanthridines; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D009281:Naphthalenes; D010617:Phenanthridines; D015221:Potassium Channels; D015240:Phorbol 12,13-Dibutyrate; D020110:Pinacidil; D000255:Adenosine Triphosphate; C016299:chelerythrine; C497256:Prkce protein, mouse; D011493:Protein Kinase C; D051744:Protein Kinase C-epsilon; C058819:calphostin C; D000109:Acetylcholine; D005905:Glyburide