A complex of Ca(V)1.2/PKC is involved in muscarinic signaling in smooth muscle.
Here we present functional and biochemical evidence for a Ca(2+) channel (Ca(V)1.2)/protein kinase C (PKC) signaling complex being a key player in muscarinic regulation of urinary bladder smooth muscle. Muscarinic stimulation induced Ca(2+) signals and concomitant contractions in detrusor muscle from mice that were dependent on functional Ca(2+) channels. These signals were still present in muscles being depolarized by 85 mM extracellular K(+). Muscarinic-induced contractions were reduced by a PKC inhibitor [bisindolylmaleimide I (BIM-I)] and a phospholipase D (PLD) inhibitor (1-butanol). A phorbol ester (PDBu) enlarged muscarinic-induced Ca(2+) signals and contractions. The effects of BIM-I and PDBu were inhibited by isradipine and/or absent in muscles from Ca(V)1.2-deficient mice. Both carbachol and PDBu increased Ca(V)1.2 channel currents in isolated bladder myocytes. Blue native-PAGE electrophoresis revealed that Ca(V)1.2, PKC, and PLD are closely associated in muscles being previously stimulated by carbachol. Immunoprecipitation using anti-Ca(V)1.2 followed by Western blotting demonstrated that Ca(V)1.2 and PKC are coupled in stimulated muscles from wild-type mice. Autoradiography on immunoprecipitates showed that Ca(V)1.2 is a substrate for PKC-mediated phosphorylation. These findings suggest that a signaling complex consisting of Ca(V)1.2, PKC, and, probably, PLD controls muscarinic-mediated phasic contraction of urinary bladder smooth muscle.
Huster
Maria
M
Institut für Pharmakologie und Toxikologie, TU München, Biedersteiner Str. 29, 80802 Munich, Germany.
Frei
Eva
E
Hofmann
Franz
F
Wegener
Jörg W
JW
D000818:Animals; D020746:Calcium Channels, L-Type / Q000378:metabolism*; D008564:Membrane Potentials; D051379:Mice; D009119:Muscle Contraction; D009130:Muscle, Smooth / Q000378:metabolism* / Q000502:physiology; D010766:Phosphorylation; D011188:Potassium; D011493:Protein Kinase C / Q000378:metabolism*; D011976:Receptors, Muscarinic / Q000378:metabolism*; D015398:Signal Transduction; D013552:Swine; D001743:Urinary Bladder / Q000166:cytology
D020746:Calcium Channels, L-Type; C419385:L-type calcium channel alpha(1C); D011976:Receptors, Muscarinic; D011493:Protein Kinase C; D011188:Potassium