G protein-mediated signaling plays a decisive role in blood pressure regulation and the phenotype of vascular smooth muscle cells (VSMCs); however, the relevance of proteins that restrict G protein activity is not well characterized in this context. Here, we investigated the influence of regulator of G protein signaling 5 (RGS5), an inhibitor of Gα and Gα activity, on blood pressure and the VSMC phenotype during experimental hypertension. In mice, loss of RGS5 did not affect baseline blood pressure, but prevented hypertension-induced structural remodeling. RGS5-deficient arterial VSMCs did not acquire a synthetic phenotype as evidenced by their inability to decrease the abundance of contractile markers-α-smooth muscle actin and smooth muscle-myosin heavy chain-or to proliferate under these conditions. Mechanistically, hypertensive pressure levels or biomechanical stretch are sufficient to increase the expression of RGS5. Loss of RGS5 severely impairs the activation of RhoA and stress fiber formation. In stretch-exposed VSMCs, RhoA activity was amplified upon inhibition of PKC, which mimics the downstream effects evoked by RGS5-mediated inhibition of Gα signaling. Collectively, our findings underline that RhoA activation may depend on the restriction of G protein activity and identify RGS5 as a mechanosensitive regulatory protein that is required to promote the synthetic VSMC phenotype as a prerequisite for structural renovation of the arterial wall during hypertension.-Arnold, C., Demirel, E., Feldner, A., Genové, G., Zhang, H., Sticht, C., Wieland, T., Hecker, M., Heximer, S., Korff, T. Hypertension-evoked RhoA activity in vascular smooth muscle cells requires RGS5.
The regulator of G-protein signaling 5 (RGS5) acts as an inhibitor of Gαq/11 and Gαi/o activity in vascular smooth muscle cells (VSMCs), which regulate arterial tone and blood pressure. While RGS5 has been described as a crucial determinant regulating the VSMC responses during various vascular remodeling processes, its regulatory features in resting VSMCs and its impact on their phenotype are still under debate and were subject of this study. While Rgs5 shows a variable expression in mouse arteries, neither global nor SMC-specific genetic ablation of Rgs5 affected the baseline blood pressure yet elevated the phosphorylation level of the MAP kinase ERK1/2. Comparable results were obtained with 3D cultured resting VSMCs. In contrast, overexpression of RGS5 in 2D-cultured proliferating VSMCs promoted their resting state as evidenced by microarray-based expression profiling and attenuated the activity of Akt- and MAP kinase-related signaling cascades. Moreover, RGS5 overexpression attenuated ERK1/2 phosphorylation, VSMC proliferation, and migration, which was mimicked by selectively inhibiting Gαi/o but not Gαq/11 activity. Collectively, the heterogeneous expression of Rgs5 suggests arterial blood vessel type-specific functions in mouse VSMCs. This comprises inhibition of acute agonist-induced Gαq/11/calcium release as well as the support of a resting VSMC phenotype with low ERK1/2 activity by suppressing the activity of Gαi/o.
We investigated the effects of calcium ions (Ca2+) on the adenylyl cyclase activity in purified turkey erythrocyte membranes. Results showed the following: (i) Ca2+ inhibits cAMP accumulation stimulated by isoproterenol (1 µmol/l), NaF + AlCl3 (10 mmol/l + 20 µmol/l) or forskolin (10 µmol/l) in EGTA-washed turkey erythrocyte membranes. IC50 of free [Ca2+] is approximately 0.1 mmol/l in the presence of Mg2+ (2.5 mmol/l) and isobutylmethylxanthine (1 mmol/l). (ii) The potency of Ca2+ to inhibit cAMP accumulation is independent of the type of stimulus used to activate the adenylyl cyclase. We also evaluated the calcium sensitivity of the basal cAMP accumulation in the presence of GTP (10 µmol/l) and Mg2+ (2.5 mmol/l) which was also inhibited by Ca2+ with the same potency. (iii) The inhibition pattern of cAMP accumulation is not affected by the presence of added calmodulin (100 nmol/l). (iv) Ca2+ is ineffective on the binding of isoproterenol to the β-adrenoceptors. (v) Increasing the concentration of Ca2+ does not induce an observable activation of cyclic nucleotide phosphodiesterase in the present experimental conditions. Thus, we concluded that the inhibition of cAMP accumulation is due to an inhibition of the adenylyl cyclase rather than the activation of phosphodiesterase(s). The presence of a yet unidentified isoform of adenylyl cyclase that can be directly inhibited by Ca2+ or a Gi protein that can be activated by Ca2+ seems to explain these results. In either case, these results provide an additional mode of cross-talk that can take place between the Ca2+- and cAMP-signaling systems.
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