Swärd. Increased Rho activation and PKC-mediated smooth muscle contractility in the absence of caveolin-1. Am J Physiol Cell Physiol 291: C1326 -C1335, 2006; doi:10.1152/ajpcell.00046.2006.-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 2ϩ 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 ϳ15:1:1 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␥S was increased in KO compared with WT as shown using a pull-down assay. Following ␣-toxin permeabilization, no difference in Ca 2ϩ sensitivity or in Ca 2ϩ sensitization was detected. In KO femoral arteries, phorbol 12,13-dibutyrate (PDBu)-induced and PKC-mediated contraction was increased. This was associated with increased ␣1-adrenergic contraction. Following inhibition of PKC, ␣1-adrenergic contraction was normalized. PDBu-induced Ca 2ϩ sensitization was not increased in permeabilized femoral arteries. In conclusion, Rho activation, but not Ca 2ϩ 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 2ϩ sensitivity. Ca 2ϩ sensitization; Rho-associated kinase; myosin phosphatase target protein; lipid rafts; CPI-17; G protein-coupled receptor CAVEOLAE are 50 -100 nm flask-shaped membrane invaginations that are abundant in endothelial cells, adipocytes, and smooth muscle cells. Caveolae are characterized by high cholesterol and sphingolipid content, and a light buoyant density. They are stabilized by the caveolin proteins (9). Specific G protein-coupled and tyrosine kinase receptors, as well as downstream signaling intermediaries, have been shown to be caveolae associated (26,30). Such clustering has been envisioned to facilitate receptor signaling and has been proposed to play a role in receptor internalization. The scaffolding domain of caveolin-1 (Cav-1) may also function as a broad-spectrum kinase inhibitor (9).Compared with endothelial and adipocyte caveolae, smooth muscle caveolae have received relatively little attention. With the use of cholesterol depletion to disrupt caveolae in denuded caudal arteries from the rat, we demonstrated that serotonin (5-HT 2A ) as well as endothelin-1 (ET-1) receptor signaling was impaired by cholesterol depletion. Moreover, restoration of caveolae by cholest...

Caveolin-1 (Cav-1) is essential for the morphology of membrane caveolae and exerts a negative influence on a number of signaling systems, including nitric oxide (NO) production and activity of the MAP kinase cascade. In the vascular system, ablation of caveolin-1 may thus be expected to cause arterial dilatation and increased vessel wall mass (remodeling). This was tested in Cav-1 knockout (KO) mice by a detailed morphometric and functional analysis of mesenteric resistance arteries, shown to lack caveolae. Quantitative morphometry revealed increased media thickness and media-to-lumen ratio in KO. Pressure-induced myogenic tone and flow-induced dilatation were decreased in KO arteries, but both were increased toward wild-type (WT) levels following NO synthase (NOS) inhibition. Isometric force recordings following NOS inhibition showed rightward shifts of passive and active length-force relationships in KO, and the force response to alpha(1)-adrenergic stimulation was increased. In contrast, media thickness and force response of the aorta were unaltered in KO vs. WT, whereas lumen diameter was increased. Mean arterial blood pressure during isoflurane anesthesia was not different in KO vs. WT, but greater fluctuation in blood pressure over time was noted. Following NOS inhibition, fluctuations disappeared and pressure increased twice as much in KO (38 +/- 6%) compared with WT (17 +/- 3%). Tracer-dilution experiments showed increased plasma volume in KO. We conclude that NO affects blood pressure more in Cav-1 KO than in WT mice and that restructuring of resistance vessels and an increased responsiveness to adrenergic stimulation compensate for a decreased tone in Cav-1 KO mice.

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