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.

Frei E, Huster M, Smital P, Schlossmann J, Hofmann F, Wegener JW. Calcium-dependent and calcium-independent inhibition of contraction by cGMP/cGKI in intestinal smooth muscle. Am J Physiol Gastrointest Liver Physiol 297: G834 -G839, 2009. First published July 23, 2009 doi:10.1152/ajpgi.00095.2009.-cGMP-dependent protein kinase I (cGKI) induces relaxation of smooth muscle via several pathways that include inhibition of intracellular Ca 2ϩ signaling and/or involve activation of myosin phosphatase. In the present study, we investigated these mechanisms comparatively in colon and jejunum longitudinal smooth muscle from mice. In simultaneous recordings from colon muscle, 8-bromo-cGMP (8-Br-cGMP) reduced both carbachol-induced tension and carbachol-induced increase in intracellular Ca 2ϩ concentration ([Ca 2ϩ ]i). These effects of 8-Br-cGMP were absent in colon from mice carrying a mutated inositol-1,4,5 trisphosphate receptor I-associated G kinase substrate (IRAG) gene or lacking cGKI. However, in jejunum, 8-Br-cGMP reduced carbachol-induced tension but did not change corresponding [Ca 2ϩ ]i signals. This setting was also observed in jejunum from mice carrying a mutated IRAG gene, whereas no response to 8-Br-cGMP was observed in jejunum from mice lacking cGKI. After inhibition of phosphatase activity by calyculin A, 8-Br-cGMP did not relax jejunum but still relaxed colon muscle. In Western blot analysis, 8-BrcGMP reduced the signal for phosphorylated MYPT-1 in carbacholstimulated jejunum but not in colon. These results suggest that cGMP/cGKI signaling differentially inhibits contraction in the muscles investigated: in jejunum, inhibition is performed without changing [Ca 2ϩ ]i and is dependent on phosphatase activity, whereas in colon, inhibition is mediated by inhibition of [Ca 2ϩ ]i signals.inositol-1,4,5 trisphosphate receptor I-associated G kinase substrate; myosin phosphatase targeting subunit 1; guanosine 3Ј,5Ј-cyclic monophosphate-dependent protein kinase I; colon; jejunum NONADRENERGIC, NONCHOLINERGIC NEURONS of the gut use nitric oxide (NO) as a neurotransmitter to regulate smooth muscle contractility via cGMP/cGMP-dependent protein kinase I (cGKI) signaling (3,13,15,25). Since NO is gaseous, it diffuses directly from the neurons to its receptor, the soluble guanylyl cyclase (sGC) located in smooth muscle cells. Stimulation of sGC increases intracellular levels of cGMP and induces relaxation via activation of cGKI (10). Smooth muscle contraction is controlled by Ca 2ϩ -dependent and Ca 2ϩ -independent signaling pathways (6,15,22,29). It is therefore likely that relaxation by NO/cGMP/cGKI interferes with both Ca 2ϩ -dependent and -independent signaling pathways. Indeed, several mechanisms have been reported by which cGKI mediates relaxation in smooth muscle. Ca 2ϩ -dependent mechanisms involve cGMP/cGKI inhibition of hormone-induced Ca 2ϩ release from intracellular stores that is not observed after deletion of the cGKI gene in mice (18). Further studies have shown that the latter mechanism involves a...

cGMP dependent protein kinase I (cGKI) in intestinal smooth muscle may induce relaxation via several mechanisms that include inhibition of intracellular Ca2+ release by phosphorylation of the inositol‐1,4,5‐phosphat receptor associated G‐kinase substrate (IRAG) and increasing myosin phosphatase activity by phosphorylation of the myosin phosphatase targeting subunit 1 (MYPT1). In the present study, we investigated whether one or both mechanisms are involved in cGMP‐mediated inhibition of carbachol‐stimulated contraction of colon and jejunum. In simultaneous recordings of tension and intracellular Ca2+ signals, 8‐Br‐cGMP reduced both carbachol‐induced tension and intracellular Ca2+ signals in colon muscle. In contrast, in jejunum muscle, 8‐Br‐cGMP reduced tension but did not change intracellular Ca2+ signals. In colon muscles from mice exhibiting a mutated IRAG (IRAG 12), 8‐Br‐cGMP did not reduce carbachol‐induced tension and Ca2+ signals. Inhibition of phosphatase activity by calyculin A abolished relaxation of carbachol‐induced contraction by 8‐Br‐cGMP in jejunum but not in colon muscle. Western blot analysis showed a larger signal for phosphorylated MYPT1 in carbachol‐stimulated jejunum than in colon muscle. These result suggest that cGMP/cGKI induces relaxation mostly by inhibition of intracellular Ca2+ release in colon and by activation of myosin phosphatase in jejunum.

Muscarinic agonists like carbachol induce contraction of urinary bladder. This contraction is blocked by L‐type Ca2+ channel antagonists and is abolished in detrusor muscle from Cav1.2 deficient mice, demonstrating that this type of contraction depends on functional L‐type Ca2+ channels. However, it is unclear how muscarinic stimulation couples to the L‐type Ca2+ channel. In this study, we present several lines of evidence that protein kinase C is involved in this signalling pathway: (i) carbachol‐induced contraction was attenuated by the protein kinase C inhibitor Gö6850; (ii) contraction and (iii) intracellular Ca2+ signals induced by carbachol were increased after activation of protein kinase C with phorbol ester; (iv) these effects of phorbol ester were inhibited by Gö6850; (v) co‐immunoprecipitation using a Cav1.2 antibody and (vi) analysis of membrane preparations using blue native gelelectrophoresis revealed an association of protein kinase C to the Cav1.2 channel if the muscles had been stimulated by carbachol; (vi) phosphorylation of the Cav1.2 channel was increased in carbachol‐stimulated preparations and attenuated by Gö6850. These results suggest that association of protein kinase C to the Cav1.2 Ca2+ channel participates, at least partially, in the signalling pathway from the muscarinic receptor to the L‐type Ca2+ channel during micturition.