Effects of intermediate conductance calcium-activated potassium channel blocker TARAM-34 on β-glycerophosphate induced vascular smooth muscle cells calcification

Abstract

To observe the role of TRAM-34 (1-((2-chlorophenyl)diphenylmethyl)-1H-pyrazole), the blocker of intermediate conductance calcium-activated potassium channel (KCa3.1), on β-glycerophosphate induced vascular calcification in vitro. Vascular smooth muscle cells(VSMCs) were obtained from rat thoracic aorta, and VSMCs after the fourth passage and aortic rings were divided into control group (cultured in DMEM with 10% fetal bovine serum), high phosphorus group (cultured in DMEM with 10% fetal bovine serum and 10% β-glycerophosphate) and TRAM-34 group(20 nmol/L TRAM-34 was added into high phosphorus DMEM). Calcium deposition of VSMCs and aortic rings were measured by o-cresolphthalein complexone method.Calcium influx of VSMCs was measured by immunofluorescence probe Fluo-3 AM.The expression of runt-related transcription factor 2(Runx2)was detected by RT-PCR and Western blot for cells and immunohistochemistry for aortic rings.ALP activity was measured by alkaline phosphatase activity detection kit. (1) Compared with control group, calcification was significantly increased in high phosphorus group ((121.67±6.17) mg/g vs. (84.38±8.17) mg/g, P<0.05) and this effect could be attenuated by TRAM-34 ((93.31±11.36) mg/g, P<0.05 vs. high phosphorus group) after 12 days culture. Similar results were found in aortic rings cultured for 12 days-high phosphorus group: (7.17±0.57) mg/g vs. (1.18±0.13) mg/g (P<0.05) and TRAM-34: (4.71±0.42) mg/g, P<0.05 vs. high phosphorus group.(2) Compared with control group, the calcium influx was higher in high phosphorus group (349.22±40.47 vs. 151.67±16.94, P<0.05) and reduced in TRAM-34 group (194.67±22.21, P<0.05 vs. high phosphorus group) in VSMCs simulated for 4 days. (3) Both mRNA and protein expressions of Runx2 in high phosphorus groups were higher than in control group (0.630±0.033 vs.0.340±0.058 and 0.865±0.031 vs.0.414±0.011, both P<0.05) and lower in TRAM-34 group (0.399±0.023 and 0.575±0.014, both P<0.05 vs. high phosphorus group) in VSMCs simulated for 4 days.Besides, compared with high phosphorus group, the expression of Runx2 was decreased in control group(0.113±0.010 vs.0.067±0.008, P<0.05) and TRAM-34 group (0.069±0.006, P<0.05) after aortic rings were cultured for 4 days. (4) Compared with control group, the activity of ALP was significantly increased in high phosphorus group (96.56±9.84 vs.46.92±4.60, P<0.05) and decreased in TRAM-34 group(70.20±8.41, P<0.05 vs. high phosphorus group) in VSMCs simulated for 12 days. KCa3.1 blocker TRAM-34 can inhibit β-glycerophosphate induced VSMCs and aortic ring calcification through inhibiting calcium influx, downregulating Runx2 expression and attenuating osteogenic differentiation.