Role of Ca2+-activated K+ channels in alkalinization and β-glycerophosphate induced vascular smooth muscle cells calcification

Abstract

Objective To observe the role of intermediate conductance calcium - activated potassium channels (KCa3.1) in alkalinization and β - glycerophosphate induced vascular calcification. Methods Vascular smooth muscle cells (VSMCs) and aortic rings were obtained from rat thoracic aorta, and then randomly divided into control group (pH was provided into 7.4, 8.0), high phosphorus groups (pH was provided into 7.4, 7.7 and 8.0, VSMCs in three groups were treated with 10 mmol/L β-glycerophosphate; HCl and NaHCO3 were used to adjust the pH) and TRAM-34 group (20 nmol/L was added into pH8.0 high phosphorus dulbecco's modified eagle's medium). Calcium deposition and alkaline phosphatase (ALP) activity were measured by Alizarin red staining, calcium content and enzyme linked immunosorbent assay after cells were simulated for 12 days. Intracellular free Ca2 + was measured by ELISA. The expression of KCa3.1, runt - related transcription factor 2 (Runx2) were detected by RT-PCR and Western blotting 4 days after cells were stimulated. Calcium deposition was measured by von Kossa staining and calcium content after aortic rings were cultured for 12 days. The expressions of KCa3.1 and Runx2 were detected by immunohistochemistry after aortic rings were cultured for 4 days. Results Compared with control group, calcification in VSMCs and aortic rings were significantly increased in high phosphorus group (P <0.05) while decreased in TRAM-34 group (P <0.05). Compared with control group, the expressions of KCa3.1, Runx2 and the activity of ALP in high phosphorus groups were increased (P <0.05) while decreased in TRAM -34 group (P < 0.05). Besides, expressions of Runx2 and KCa3.1 were augmented as the pH was higher (P <0.05). The expression of Runx2 in aortic rings was the same situation. Besides, the Ca2+ influx was blocked by TRAM -34 (P <0.05). Conclusions Alkalinization contributes to β - glycerophosphate induced VSMCs calcification through increase of Ca2 + influx, up - regulation of KCa3.1 and promotion of osteogenic/chondrogenic differentiation. Key words: Myocytes, smooth muscle; Aorta, thoracic; Calcinosis; Osteogenic differentiation; Intermediate-conductance Ca2+-activated K+ channels