Calcium Sensing Receptor Regulating Smooth Muscle Cells Proliferation Through Initiating Cystathionine-Gamma-Lyase/Hydrogen Sulfide Pathway in Diabetic Rat

Abstract

Aims: Hydrogen sulfide (H₂S) inhibits the proliferation of vascular smooth muscle cells (VSMCs). However, how cystathionine-gamma-lyase (CSE), a major enzyme that produces H₂S, is regulated remains unknown. Whether calcium-sensing receptor (CaSR) inhibits the proliferation of VSMCs by regulating the endogenous CSE/H₂S pathway in diabetic rat has not been previously investigated. Methods and Results: The morphological and ultrastructure alterations were tested by transmission electron microscopy, changes in the H₂S concentration and the relaxation of the mesenteric secondary artery loop of diabetic rats were determined by Multiskan spectrum microplate spectrophotometer and isometric force transducer. Additionally, the expression levels of CaSR, CSE and Cyclin D1 in the mesenteric arteries of rats were examined by western blotting. The intracellular calcium concentration, the expression of p-CaMK II (phospho-calmodulin kinases II), CSE activity, the concentration of endogenous H₂S and the proliferation of cultured VSMCs from rat thoracic aortas were measured by using confocal microscope, western blotting, microplate spectrophotometer, MTT and BrdU, respectively. The VSMC layer thickened, the H₂S concentration dropped, the relaxation of the mesenteric secondary artery rings weakened, and the expression of CaSR and CSE decreased whereas the expression of Cyclin D1 increased in diabetic rats compared with the control group. The [Ca²⁺]_i of VSMCs increased upon treatment with CaSR agonists (10 µM Calindol and 2.5 mM CaCl₂), while it decreased upon administration of calhex231, U73122 and 2-APB. The expression of p-CaMK II and CSE increased upon treatment with CaSR agonists in VSMCs. CSE activity and the endogenous H₂S concentration decreased in response to high glucose, while it increased with treatment of CaSR agonists. The proliferation rate increased in response to high glucose, and CaSR agonists or NaHS significantly reversed the proliferation of VSMCs caused by high glucose. Conclusions: Our results demonstrated that CaSR regulated the endogenous CSE/H₂S pathway to inhibit the proliferation of VSMCs in both diabetic and high glucose models.