Physiological Roles of Na+-Ca2+ Exchanger in Isolated Hepatocytes

초록

Na+-Ca2+ Exchanger (NCX) plays an important role in intracellular Ca2+ activity ([Ca2+]i) regulation and is expressed in variety of excitable and non-excitable cells. However, the role of NCX in the regulation of [Ca2+]i in hepatocytes is not understood clearly. The purpose of this study is to examine the activity and presence of NCX in hepatocytes. Isolated hepatocytes were grown on collagen-coated coverslips for 2 days. To identify the molecular characteristic of NCX in hepatocytes, SDS/PAGE of hepatocytes was performed under reducing conditions. The immunoblot results showed that the 33kDa band was detected and the expression of the NCX was increased in cells exposed to 0.5 mM H202. To examine the membrane current via NCX, the conventional whole cell patch clamp method was performed with ramp voltage pulses. The intersection point of l-V curves was determined between -50 and -40 mV. The inward current of NCX was blocked in hepatocytes treated with AS-oligos. The activity of NCX was investigated by measuring [Ca2+]i with Fura-2. When hepatocytes were perfused with 0 Na+/2.5 mM Ca2+ and 140 mM Na+/0 Ca2+ Tyrode solutions containing thapsigargin(l μM), caffeine (5 mM), and La3+ (250 μM), [Ca2+]i increased but not as much as shown in other cells. And the changes [Ca2+]i was similar in AS-oligos treated cells. However, the treatment of H2O2 enhanced the increase in [Ca2+]i significantly.