RhoA localization with caveolin-1 regulates vascular contractions to serotonin

Abstract

Vascular smooth muscle contraction occurs following an initial response to an increase in intracellular calcium concentration and a sustained response following increases in the sensitivity of contractile proteins to calcium (calcium sensitization). This latter process is regulated by the rhoA/rho kinase pathway and activated by serotonin. In multiple cell types, signaling molecules compartmentalize within caveolae to regulate their activation. We hypothesized that serotonin differentially compartmentalizes rhoA within caveolar versus noncaveolar lipid rafts to regulate sustained vascular contractions. To test this hypothesis, we measured aortic contractions in response to serotonin in wild-type (WT) and cav-1-deficient mice (cav-1 KO). RhoA-dependent contractions in response to serotonin were markedly augmented in arteries from cav-1 KO mice despite a modest reduction in rhoA expression compared with WT. We found that under basal conditions, rhoA in WT arteries was primarily localized within high-density sucrose gradient fractions but temporally shifted to low-density fractions in response to serotonin. In contrast, rhoA in cav-1 KO arteries was primarily in low-density fractions and shifted to high-density fractions in a similar timeframe as that seen in WT mice. We conclude that localization of rhoA to caveolar versus noncaveolar lipid rafts differentially regulates its activation and contractions to rhoA-dependent agonists with greater activation associated with its localization to noncaveolar rafts. Disruption of rhoA localization within caveolae may contribute to increased activation and enhanced vascular contractions in cardiovascular disease.

Fig. 1.

Inhibition of rhoA with cell-permeable exoenzyme C3 transferase, CT04 (A), or rho kinase with H1152 (1 μM, B), reduced contractions to serotonin in both wild-type (WT) and cav-1 knockout mice (cav-1 KO). Contractions to serotonin did not differ between WT and cav-1 KO following H1152. Contractions to KCl were inhibited by H1152 (C) but not CT04 (not shown). Values are expressed as means ± SE. *P < 0.05 vs. WT. †P < 0.05 vs. respective control in the absence of inhibitor; n = 3–12.

Fig. 2.

Disruption of lipid rafts with methyl-β-cyclodextrin (MβCD, 10 mM) reduced contractions to serotonin (A) but not to KCl (B) in arteries from both wild-type (WT) and cav-1 knockout mice (cav-1 KO). Values are expressed as means ± SE. *P < 0.05 vs. WT. †P < 0.05 vs. respective control in the absence of MβCD; n = 3–6.

Fig. 3.

Representative (A) and mean (B) levels of protein expression of rhoA, rho kinase (ROCK1 and ROCK2), caveolin-1 (Cav-1), and β-actin in whole cell lysate of aorta from wild-type (WT) and cav-1 KO mice. Values are expressed as means ± SE of 4 aorta. *P < 0.05 vs. WT. C: representative immunoblot (IB) from immunoprecipitations (IP) using cav-1, rhoA, or IgG in aorta from WT mice. Lane 1 shows positive control using whole cell lysate from aorta (WC); lane 2 shows IP with cav-1; lane 3 shows IP with rhoA; and lane 4: shows IP with IgG. (n = 2 for each IP).

Fig. 4.

Under basal conditions, neither rhoA nor ROCK1 nor ROCK2 localized with caveolin-1 (cav-1) in low-density fractions in the aorta from wild-type mice (A, representative blots, B, levels as percent of total; values are expressed as means ± SE; n = 3–6). Conversely, in aorta from cav-1 KO mice, levels of rhoA and ROCK2 were higher in low-density fractions compared with wild-type mice (C, representative blots, D, levels as percent of total, mean ± SE; n = 3 or 4 pooled samples).

Fig. 5.

Levels of RhoA (A), cav-1 (B), and ROCK2 (C), measured in low-density fractions (3–5) versus high-density fractions (8–10) from sucrose density centrifugation of aorta from wild-type mice under basal conditions, and early (30–60 s) and late (5 min) after the addition of serotonin (1 μM). Values are expressed as means ± SE; n = 3–6 pooled samples. *P < 0.05 vs. basal.

Fig. 6.

Levels of RhoA (A), ROCK2 (B), and β-actin (C) measured in low-density fractions (3–5) versus high-density fractions (8–10) from sucrose density centrifugation of aorta from caveolin-1 KO mice. Samples were flash frozen under basal conditions, and early (30–60 s) and late (5 min) after the addition of serotonin (1 μM). Values are expressed as means ± SE; n = 3–5 pooled samples. *P ≤ 0.05 vs. basal. †P < 0.07 vs. basal.

Fig. 7.

Levels of RhoA (A and C) and ROCK2 (B and D) measured in wild-type and cav-1 KO low-density fractions (3–5) versus high-density fractions (8–10) of aorta under basal conditions (A and B) and late (5 min, C and D) after addition of serotonin (1 μM). Values are expressed as means ± SE. *P < 0.05 vs. wild-type; n = 3–6 pooled samples.