Does the activity of ecto-5'-nucleotidase depend on the membrane potential?

缺血时，腺苷是由5′-核苷酸酶对5′-单磷酸腺苷（5′-AMP）进行酶解磷酸化而产生的。我们研究了钾离子对腺苷生产的影响，采用灵活安装微透析技术。将微透析探针植入麻醉开胸大鼠左心室心肌，然后灌注含有5′-AMP的Tyrode溶液。我们澄清了在持续供应5'-AMP期间测量的透析液腺苷水平反映了内源性外泌-5'-核苷酸酶的活性。当Tyrode溶液中的KCI浓度从5.4 mM（对照）逐步增加到140 mM时，透析液腺苷水平显著增加，且呈浓度依赖性，后一效应在1 mM CsCI （aK^+通道抑制剂）存在下被消除。通过添加270 mM的蔗糖来增加Tyrode溶液的渗透浓度，不影响日增。在离体豚鼠心室乳头肌中，向内整流K^+电流阻滞剂（I_<K1>），即CsCI和BaCI_2 （20mM）可显著去极化静息膜电位。为了排除高浓度KCI增强腺苷生成的可能性是由去甲肾上腺素的释放引起的，继发于交感神经末梢的去极化，我们在30mum α ^1受体阻滞剂吡唑嗪的存在下重复了相同的实验。即使在这种情况下，引入含有140 mM KC1的溶液也显著提高了腺苷的产生，从而排除了由去甲肾上腺素诱导的、alpha_1受体介导的蛋白激酶C活性激活介导的腺苷产生的可能性。这些结果表明，细胞外K^+浓度的增加使心肌细胞静息膜电位去极化，并增加体外5′-核苷酸酶的活性，最终增加原位大鼠心脏中腺苷的产生。少

Under ischemia, adenosine is produced by enzymatic dephosphorylation of adenosine 5'-monophosphate (5'-AMP) by 5'-nucleotidase. We studied the effect of potassium ions on the production of adenosine, using flexibly mounted microdialysi techniques. The microdialysisi probe was implanted into the left ventricular myocardium of anesthetized open-chested rats, followed by a perfusion with Tyrode solution containing 5'-AMP.We clarified that the level of dialysate adenosine measured during continued supply of 5'-AMP reflects the activity of endogenous ecto-5'-nucleotidase. When the concentration of KCI in the Tyrode solution was increased stepwise from 5.4 mM (control) to up to 140 mM,the level of dialysate adenosine significantly increased, in a concentration-dependent manner and the latter effect was abolished in the presence of 1 mM CsCI,aK^+ channel inhibitor. Equivalent increases in osmotic concentration of the Tyrode solution, made by adding sucrose (270 mM) , did not affect the dialys … More ate adenosine concentration. In isolated guinea pig ventricular papillary muscles, the blockers of inward rectifier K^+ current (I_<K1>), i.e., CsCI and BaCI_2 (20mM) depolarized the resting membrane potential significantly. To rule out the possibility that the enhanced production of adenosine by high concentration of KCI is caused by the release of noradrenaline, secondary to the depolarization of sympathetic nerve terminals, we repeated the same experiments in the presence of 30 muM prazocin, an alpha^1-blocker. Even under this condition, introduction of 140 mM KC1 containing solution significantly enhanced the adenosine production, thereby dismissing the possibility of adenosine production mediated by noradrenaline-induced, alpha_1-receptor-mediated activation of protein kinase C activity.These results suggest that increases in extracellular K^+ concentration depolarizes the resting membrane potential of cardiomyocytes and increase the activity of ecto-5'-nucleotidase with eventual increases of adenosine production in in situ rat's hearts. Less