Kim C S, Coyne M D, Gwathmey J K
Institute for Cardiovascular Disease and Muscle Research, Cambridge MA 02138, USA.
Am J Physiol Regul Integr Comp Physiol. 2000 Jun;278(6):R1524-34. doi: 10.1152/ajpregu.2000.278.6.R1524.
Voltage-dependent calcium channels (VDCC) in ventricular myocytes from rainbow trout (Oncorhynchus mykiss) were investigated in vitro using the perforated patch-clamp technique, which maintains the integrity of the intracellular milieu. First, we characterized the current using barium as the charge carrier and established the doses of various pharmacological agents to use these agents in additional studies. Second, we examined the current at several physiological temperatures to determine temperature dependency. The calcium currents at 10 degrees C (acclimation temperature) were identified as L-type calcium currents based on their kinetic behavior and response to various calcium channel agonists and antagonists. Myocytes were chilled (4 degrees C) and warmed (18 and 22 degrees C), and the response of VDCC to varying temperatures was observed. There was no significant dependency of the current amplitude and kinetics on temperature. Amplitude decreased 25-36% at 4 degrees C (Q(10) approximately 1.89) and increased 18% at 18 degrees C (Q(10) approximately 1.23) in control, Bay K8644 (Bay K)-, and forskolin-enhanced currents. The inactivation rates (tau(i)) did not demonstrate a temperature sensitivity for the VDCC (Q(10) 1.23-1. 92); Bay K treatment, however, increased temperature sensitivity of tau(i) between 10 and 18 degrees C (Q(10) 3.98). The low Q(10) values for VDCC are consistent with a minimal temperature sensitivity of trout myocytes between 4 and 22 degrees C. This low-temperature dependency may provide an important role for sarcolemmal calcium channels in adaptation to varying environmental temperatures in trout.
利用穿孔膜片钳技术在体外研究了虹鳟(Oncorhynchus mykiss)心室肌细胞中的电压依赖性钙通道(VDCC),该技术可维持细胞内环境的完整性。首先,我们以钡作为载流子对电流进行了表征,并确定了各种药理剂的剂量,以便在后续研究中使用这些药剂。其次,我们在几个生理温度下检测了电流,以确定温度依赖性。基于其动力学行为以及对各种钙通道激动剂和拮抗剂的反应,10℃(驯化温度)下的钙电流被确定为L型钙电流。将肌细胞冷却至4℃并加热至18℃和22℃,观察VDCC对不同温度的反应。电流幅度和动力学对温度没有显著依赖性。在对照、Bay K8644(Bay K)和福斯高林增强的电流中,4℃时幅度降低25 - 36%(Q10约为1.89),18℃时幅度增加18%(Q10约为1.23)。失活速率(tau(i))对VDCC未表现出温度敏感性(Q10为1.23 - 1.92);然而,Bay K处理增加了10℃至18℃之间tau(i)的温度敏感性(Q10为3.98)。VDCC的低Q10值与虹鳟肌细胞在4℃至22℃之间的最小温度敏感性一致。这种低温依赖性可能在虹鳟肌膜钙通道适应不同环境温度方面发挥重要作用。