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- Bonnevier, Johan, et al.
(författare)
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Actions downstream of cGMP/PKG can reverse PKC-mediated phosphorylation of CPI-17 and Ca2+ sensitization in smooth muscle.
- 2004
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 279:24, s. 28998-29003
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Tidskriftsartikel (refereegranskat)abstract
- Ca2+ sensitivity of smooth muscle contraction is modulated by several systems converging on myosin light chain phosphatase (MLCP). Rho-Rho kinase is considered to inhibit MLCP via phosphorylation, whereas protein kinase C (PKC) induced sensitization has been shown to be dependent on phosphorylation of the inhibitory protein CPI-17. We have explored the interaction of cGMP-dependent protein kinase (PKG) with Ca2+ sensitization pathways using permeabilized mouse smooth muscle. Three conditions giving ~50% of maximal active force were compared in small intestinal preparations: 1) Ca2+-activated unsensitized muscle (pCa 5.9 with Rho kinase inhibitor Y27632); 2) Rho-Rho kinase-sensitized muscle (pCa 6.1 with guanosine 5'-3-O-(thio)triphosphate); and 3) PKC-sensitized muscle (pCa 6.0 with Y27632 and PKC activator phorbol 12,13-dibutyrate). 8-Br-cGMP relaxed the sensitized muscles but had marginal effects on unsensitized preparations, showing that PKG reverses both PKC and Rho-mediated Ca2+ sensitization. CPI-17 was present in permeabilized intestinal tissue. In PKC-sensitized preparations, CPI-17 phosphorylation decreased in response to 8-Br-cGMP. The rate of PKC-mediated phosphorylation in the presence of the MLCP inhibitor microcystin-LR was not influenced by 8-Br-cGMP. PKC-induced Ca2+ sensitization also was reversed in vascular smooth muscle tissues (portal vein and femoral artery). We conclude that actions downstream of cGMP/PKG can reverse PKC-mediated phosphorylation of CPI-17 and Ca2+ sensitization in smooth muscle.
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| 3. |
- Shakirova, Yulia, et al.
(författare)
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Increased Rho activation and PKC-mediated smooth muscle contractility in the absence of caveolin-1.
- 2006
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Ingår i: American Journal of Physiology: Cell Physiology. - : American Physiological Society. - 1522-1563 .- 0363-6143. ; 291:6, s. 1326-1335
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Tidskriftsartikel (refereegranskat)abstract
- Abstract in UndeterminedCaveolae are omega-shaped membrane invaginations that are abundant in smooth muscle cells. Since many receptors and signaling proteins co-localize with caveolae, these have been proposed to integrate important signaling pathways. The aim of this study was to test whether RhoA/Rho-kinase and protein kinase C (PKC)-mediated Ca2+ sensitization depends on caveolae using caveolin (Cav)-1-deficient (KO) and wild-type (WT) mice. In WT smooth muscle, caveolae were detected and Cav-1, -2 and -3 proteins were expressed. Relative mRNA expression levels were ~15:1:1 for Cav-1, -2, and -3, respectively. Caveolae were absent in KO and reduced levels of Cav-2 and Cav-3 proteins were seen. In intact ileum longitudinal muscle, no differences in the responses to 5-HT or the muscarinic agonist carbachol were found, whereas contraction elicited by endothelin-1 was reduced. Rho activation by GTP{gamma}S was increased in KO compared with WT as shown using a pull-down assay. Following {alpha}-toxin permeabilization, no difference in Ca2+ sensitivity or in Ca2+ sensitization was detected. In KO femoral arteries, phorbol 12,13-dibutyrate (PDBu)-induced and PKC-mediated contraction was increased. This was associated with increased {alpha}1-adrenergic contraction. Following inhibition of PKC, {alpha}1-adrenergic contraction was normalized. PDBu-induced Ca2+ sensitization was not increased in permeabilized femoral arteries. In conclusion, Rho activation, but not Ca2+ sensitization, depends on caveolae in the ileum. Moreover, PKC driven arterial contraction is increased in the absence of caveolin-1. This depends on an intact plasma membrane and is not associated with altered Ca2+ sensitivity.Ca2+ sensitization; Rho-associated kinase; myosin phosphatase target protein; lipid rafts; CPI-17; G protein-coupled receptor
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