Effects of oxidized-low density lipoprotein on expression of urotensin II receptor GPR14 in rat aortic smooth muscle cells

Abstract

To investigate the effects of oxidized-low density lipoprotein (ox-LDL) and urotensin II (U II) on the proliferation of rat aortic smooth muscle cells (VSMCs) and the effect of ox-LDL on the mRNA expression of U II receptor GPR14 in. Rat VSMCs were incubated with UII and ox-LDL at different concentrations. The viability of rat VSMCs was detected by MTT assay. Rat VSMCs were incubated with ox-LDL at the concentrations of 0, 0.1, 1, 10, and 50 microg/ml. Twelve hours later competitive RT-PCR was used to detect the effects of concentration of ox-LDP on the mRNA expression of GPR14 in the VSMCs. Another VSMCs were incubated with ox-LDL at the final concentration of 50 microg/ml, and 0, 2, 6, 12, and 24 hours later competitive RT-PCR was used to detect the mRNA expression of GPR14 in the VSMCs. (125)I-labelled U II was added into the culture fluid of VSMCs, and radioligand binding assay was used to calculate the maximum binding (B(max)). UII of the concentration of 50 nmol/L significantly increased the proliferation of VSMCs. When the concentration of ox-LDL was 10 microg/ml the proliferation of VSMCs (P < 0.01) was 0.678 +/- 0.061, increased by 121% compared with the control group (0.325 +/- 0.052, P < 0.01). 0.01 microg/ml of ox-LDL and 10 nmol/L of UII showed a synergistic effect on the proliferation of VSMCs with the MTT value increased to 162% +/- 29% that of the control group (P < 0.01). When the concentration of ox-LDL increase to 0.1 microg/ml the synergistic effect was attenuated with the MTT value of 153% +/- 22% that of the control group (P < 0.05) and when the concentration of ox-LDL increased to 1 microg/ml the synergistic effect diminished, with a MTT value of 123% +/- 13% that of the control group (P > 0.05). When the concentration of ox-LDL was 50 microg/ml the proliferation of VSMCs the MTT value decreased to 59% that of the of control group, however, ox-LDL of the concentration of 50 microg/ml combined with UII of the concentration of 10 nmol/L the MTT value increased to 68% that of the control group, showing that UII of that concentration significantly inhibited the cytotoxic effect of ox-LDL. Incubation of VSMCs with the ox-LDL at the concentration of 0.1 microg/ml the GPR14 mRNA expression of the VSMCs was 125.1% that of the control group (P < 0.01) as assessed by competitive RT-PCR, however, when the concentration of ox-LDL increased over 1 microg/ml the GPR14 mRNA expression does-dependently decreased as much as 72.6% that of the control group (all P < 0.01), with the maximal effect when the concentration of ox-LDL reached 50 microg/ml. ox-LDL at the concentration of 50 microg/ml time-dependently down-regulated the GPR14 mRNA expression with the maximal effect 12 hours after addition of ox-LDL and this effect was sustained for up to 24 hours. After incubation for 12 hours the binding of (125)I-UII to VSMCs was 117.5% that of the control group (P < 0.01) with the ox-LDL at the concentration of 0.1 microg/ml, and was 68.8% that of the control group (P < 0.01) with the ox-LDL at the concentration of 50 microg/ml. ox-LDL and with U-II. GPR14 at certain concentrations show synergetic effects on the proliferation of VSMCs. The GPR14 mRNA expression in VSMCs can be upregulated by low concentration of ox-LDL, while downregulated by high concentration of ox-LDL.