| Objective:Hypertension is one of the most common cardiovascular diseases, and it is shown that hypertension is an independent risk factor for coronary artery diseases. Vascular smooth muscle cells (VSMCs) have played a key role in the course of coronary artery diseases caused by hypertension. The study of potassium channels not only can understand the physiological function of VSMCs, but also can demonstrate the role of VSMCs in the process of disease. So far, it is still not entirely clear about the change of ion channels on the coronary artery smooth muscle cells (CASMCs) during hypertension. In light of that, it is necessary to investigate the electrophysiological characteristics of CASMCs during hypertension.Methods:(1) In this study, we choose spontaneously hypertensive rats (SHR) as animal model of hypertension. CASMCs were isolated by "three-step" enzyme digestion, and were identified by monoclonal anti-a smooth muscle actin.(2) The contraction of VSMCs depends on the rise of cytosolic calcium owing to calcium influx, and the diastolic depends on the efflux of potassium. To date, four distinct types of potassium channel have been identified in VSMCs:voltage dependent potassium channel (Kv), calcium activated potassium channel (Kca), inward rectifier potassium channel (Kir) and ATP-sensitive potassium channel (KATP).This study was to focus on Kv channel in CASMCs of SHR by means of electrophysiology and molecular biology, and to illustrate the change of Kv channel current and the alternation of RNA and protein expression during hypertension.(3) Whole cell current recordings were performed at room temperature. In this way. we got the overall K+current, Kv channel current, tail current and resting potential in CASMCs of WKY and SHR. and drew I-V curve, steady-state activation curve and steady-state inactivation curve. (4) Extracted the RNA from WKY and SHR coronaries and performed reverse transcription, then compared Kv1.2 and Kv1.5 mRNA with real time fluorescence quantitative PCR (FQ-PCR).(5) Extracted the protein from WKY and SHR coronaries. compared the Kv1.2 and Kvl.5 proteins express in the way of Western Blot.Results:(1) The mean weight of WKY (n=79) was 317±24.9 g, average tail artery systolic pressure was 118.44±3.75 mmHg. The mean weight of SHR (n=66) was 281±21.4 g, average tail artery systolic pressure was 192.52±11.06 mmHg. The level of average tail artery systolic pressure between WKY and SHR had a remarkable significance (P< 0.05).(2) The average cell capacitance of CASMCs from WKY was 17.48±2.59 pF (n=30), and the average cell capacitance of the same cells from SHR was 15.44±2.68 pF (n=25). No significant difference of the average cell capacitance was found between these two strains (P> 0.05).(3) The average resting membrane potential of WKY was-47.7±3.52 mV (n=11), and SHR was-34.5±1.69 mV (n=9). The value of membrane potential was more negative in WKY (P< 0.05).(4) At the test potential of+60 mV,+50 mV,+40 mV,+30 mV and+20 mV, the overall K+current average current densities in WKY (n=7) were 20.21±1.38 pA/pF, 15.82d=0.91 pA/pF,12.95±0.94 pA/pF,10.15±1.35 pA/pF and 7.47±0.54 pA/pF, and in SHR (n=6) were 30.36±2.29 pA/pF,26.55±2.28 pA/pF,23.05±2.30 pA/pF,18.19±1.98 pA/pF and 13.30±1.03 pA/pF. The average current densities were smaller in WKY compared to those in SHR(P< 0.05).(5) After added IBTX, a special inhibitor of large conductance calcium activated potassium channel (BKCa), at the test potential of+60 mV,+50 mV.+40 mV and+30 mV, the Kv channel average current densities in WKY were 19.21±1.53 pA/pF,15.62±1.40 pA/pF,12.84±1.33 pA/pF and 10.09±1.06 pA/pF, and in SHR were 28.56±1.93 pA/pF. 24.70±1.31 pA/pF,19.90±1.16 pA/pF and 15.79±1.09 pA/pF. The Kv channel average current densities were significant smaller in WKY (P< 0.05).(6) The percentage of current densities sensitive to IBTX in the overall K+current average current densities were 4.94%,1.25%,0.87% and 0.55% in WKY versus 5.94%. 6.97%,13.67% and 13.18% in SHR (P> 0.05) at the test potential of+60 mV,+50 mV. +40 mV and+30 mV. And therefore, the BKca average current density was not different between these two groups (P>0.05).(7) At the test potential of+60 mV,+50 mV.+40 mV,+30 mV and+20 mV, the tail current of Kv channel average current densities in WKY (n=5) were 17.54±0.76 pA/pF, 11.89±1.25 pA/pF,9.85±0.55 pA/pF,7.54±0.42 pA/pF and 5.20±0.95 pA/pF,and in SHR (n=5) were 26.95±2.46 pA/pF,24.40±1.37 pA/pF,19.73±1.56 pA/pF,16.59±0.55 pA/pF and 9.08±0.54 pA/pF. The tail current average current densities were larger in SHR than in WKY (P< 0.05).(8) The half maximal activation voltage in WKY was 33.93±1.87 mV, and in SHR was 20.68±1.02 mV. The voltage dependence of activation was smaller in SHR (P<0.05). The value for slope factor of steady-state activation curve in WKY was 15.19±1.81 mV, and in SHR was 15.74±0.93 mV. The difference was no statistics significance between WKY and SHR (P> 0.05).(9) The half maximal inactivation voltage in WKY (n=7) rats was-23.8±1.1 mV, and in SHR (n=5) was-20.08±1.28 mV. The value for slope factor of steady-state inactivation curve in WKY rats was 14.72±1.01 mV, and in SHR was 16.71±1.20 mV. No significant differences were found in either half maximal inactivation voltage or slope factor between WKY and SHR (P> 0.05).(10) As compared to WKY, the results of the FQ-PCR showed a dramatic increase in Kv1.2 and Kv1.5 mRNA in SHR (P<0.05).(11) The Western Blot showed the grey scale of Kv1.2 and Kv1.5 protein in SHR was increased than in WKY, 0.61±0.13 versus 0.84±0.10 and 0.47±0.10 versus 0.96±0.07, respectively. The proteins expression of Kv1.2 and Kv1.5 were significantly lower in CASMCs from WKY than SHR (P<0.05).Conclusions:The value of membrane potential was more positive in SHR which suggested that the coronary artery tension is significantly increased. The overall K+current and Kv channel average current densities in CASMCs of SHR were larger than in WKY which suggested a higher Kv channel activity during hypertension. The higher express of Kv1.2 and Kv1.5 mRNA and proteins in SHR can account for this change. The increased Kv channel current in SHR can enhance membrane potential of CASMCs, reduce the influx of calcium, diminish vascular tension and improve myocardial blood supply. Therefore, we consider that Kv channel may provide a compensatory mechanism to buffer vasoconstriction during hypertension. |
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