Calcilytic NPS2143 promotes proliferation and inhibits apoptosis of spontaneously hypertensive rat vascular smooth muscle cells via activation of the renin-angiotensin system.

Abstract

Vascular smooth muscle cell (VSMC) proliferation and apoptosis and the renin-angiotensin system (RAS) play critical roles in the development of essential hypertension. The activation of calcium-sensing receptor (CaSR), functionally expressed in VSMCs, inhibits cyclic adenosine monophosphate (cAMP) formation by elevating intracellular calcium ([Ca2+]i) and then suppressing renin release. The present study aimed to investigate the effects of NPS2143-mediated inhibition of CaSR on VSMC proliferation and apoptosis in spontaneously hypertensive rat (SHR) VSMCs and to assess whether these effects were mediated by alterations to RAS signaling. Primary VSMCs were isolated from the aortas of SHRs and Wistar-Kyoto rats. SHR VSMCs were treated with CaSR antagonist NPS2143 and cell proliferation and CaSR and RAS-related protein expression levels were measured to assess the effect. The results indicated that NPS2143 treatment promoted SHR VSMC proliferation, lower CaSR expression levels and higher RAS-related proteins levels when compared with control treatment. Additional measurement of the expression levels of proteins related to proliferation, remodeling, apoptosis and RAS related proteins, as well as cell viability, cell cycle, cell apoptosis ratio, [Ca2+]i, and the concentration of cAMP was performed after treatment with NPS2143, PLC inhibitor U73122, IP3 receptor antagonist 2-aminoethoxydiphenylborane (APB), adenylyl cyclase-V inhibitor MDL12330A, angiotensin converting enzyme inhibitor captopril, angiotensin I receptor (AT1R) inhibitor losartan, NPS2143 + U73122, NPS2143 + 2-APB, NPS2143 + MDL12330A, NPS2143 + captopril and NPS2143 + losartan. The results suggested that NPS2143 promoted cell proliferation, inhibited cell apoptosis, decreased [Ca2+]i and increased the expression of RAS compared with control treatments. NPS2143 + U73122 and NPS2143 + 2-APB enhanced the effects of NPS2143, while NPS2143 + MDL12330A, NPS2143 + captopril, NPS2143 + losartan attenuated the effected of NPS2143 in SHR VSMCs. Furthermore, the knockdown of AT1R by AT1R-short hairpin RNA also attenuated the effects of NPS2143 compared with NPS2143 alone. Collectively, these data indicated that NPS2143 promoted proliferation and inhibited apoptosis of VSMCs in SHRs, the effect of which was achieved by activation of RAS signaling.