Small chemical compounds Y16 and Rhosin can inhibit calcium sensitization pathway in vascular smooth muscle cells of spontaneously hypertensive rats

Abstract

The small-molecule compounds Y16 and Rhosin can inhibit the activation of leukemia-associated Rho guanine nucleotide exchange factor (LARG) and small G-protein RhoA, respectively, in breast cancer cells and inhibit their growth and migration. However, it remains unclear whether they have inhibitory effects on the vascular smooth muscle cells (VSMCs) of spontaneously hypertensive rats (SHRs). Primary cultured VSMCs from SHRs were treated with different concentrations of Y16 or Y16 plus Rhosin for 24h, followed by 10-min stimulation with 10-7M angiotensin II (Ang II). The cells were then harvested, and the total protein was extracted. The co-immunoprecipitation method, Western blot analysis, and MTT assay were performed to determine the LARG-RhoA interaction, the protein levels of RhoA and MYPT1, and cell viability, respectively. Y16 dose-dependently inhibited the LARG-RhoA complex formation induced by Ang II. With 50μM of Y16, the effect of inhibition was statistically significant. Y16 also reduced the formation of phospho-MYPT1 stimulated by Ang II. With 5μM of Y16, the inhibitory effect was statistically significant. When 25μM of Y16 and 25μM of Rhosin were combined, the inhibitory effect on LARG-RhoA interaction was statistically significant. When Y16 and Rhosin were combined, a significantly reduced concentration could effectively inhibit MYPT1 phosphorylation (2.5μM compared with 5μM for Y16 alone). Treating SHR VSMCs with Y16 can suppress the activation of LARG, prevent LARG binding to RhoA, and decrease the phosphorylation of MYPT1, thus weakening the activation of the calcium (Ca2+) sensitization pathway in SHR VSMCs.