Increased Rho activation and PKC-mediated smooth muscle contractility in the absence of caveolin-1.
Caveolae 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 Ca(2+) 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 approximately 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 GTPgammaS was increased in KO compared with WT as shown using a pull-down assay. Following alpha-toxin permeabilization, no difference in Ca(2+) sensitivity or in Ca(2+) 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 Ca(2+) sensitization was not increased in permeabilized femoral arteries. In conclusion, Rho activation, but not Ca(2+) 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 Ca(2+) sensitivity.
Shakirova
Yulia
Y
Department of Experimental Medical Science, Lund University, Lund, Sweden.
Bonnevier
Johan
J
Albinsson
Sebastian
S
Adner
Mikael
M
Rippe
Bengt
B
Broman
Jonas
J
Arner
Anders
A
Swärd
Karl
K
D000316:Adrenergic alpha-Agonists / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D021941:Caveolae / Q000378:metabolism; D051242:Caveolin 1 / Q000235:genetics / Q000378:metabolism*; D051243:Caveolin 2 / Q000235:genetics / Q000378:metabolism; D051244:Caveolin 3 / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000378:metabolism; D005263:Femoral Artery / Q000166:cytology / Q000378:metabolism; D006801:Humans; D007082:Ileum / Q000166:cytology / Q000378:metabolism; D007093:Imidazoles / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009119:Muscle Contraction / Q000502:physiology*; D009130:Muscle, Smooth / Q000166:cytology / Q000502:physiology*; D032389:Myocytes, Smooth Muscle / Q000378:metabolism / Q000648:ultrastructure; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D010738:Type C Phospholipases / Q000378:metabolism; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
D000316:Adrenergic alpha-Agonists; D051242:Caveolin 1; D051243:Caveolin 2; D051244:Caveolin 3; D004791:Enzyme Inhibitors; D007093:Imidazoles; D047908:Intracellular Signaling Peptides and Proteins; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D011493:Protein Kinase C; D010738:Type C Phospholipases; D020742:rhoA GTP-Binding Protein; C014282:cirazoline; D002118:Calcium