The effects of extracellular adenosine triphosphate (ATP) on the basal L-type Ca2+ current (ICa) were investigated in ferret isolated right ventricular myocytes using the gigaohm seal voltage clamp in the whole-cell and cell-attached configurations. 2. Micromolar levels of extracellular ATP reversibly inhibited ICa in a concentration-dependent manner, without any significant changes in the voltage dependence of either the peak ICa I-V relationship or steady-state activation curve. 3. In contrast, micromolar levels of extracellular ATP did significantly alter the inactivation characteristics of ICa. Ten micromolar ATP: (i) increased the degree of steady-state inactivation of ICa; (ii) altered the time constants of ICa inactivation at 0 mV; and (iii) decreased the time constant of ICa recovery from inactivation at -70 mV. 4. The inhibitory effect of ATP on ICa was not blocked by atropine, a muscarinic cholinergic receptor antagonist, or CPDPX (8-cyclopentyl-3,4-dipropylxanthine), an A1 adenosine receptor antagonist. In contrast, the inhibitory effect of 10 microM ATP could be nearly completely antagonized by 100 microM suramin, a purinergic P2 receptor antagonist. 5. The potency order of ATP analogues in inhibiting ICa was 2-methyl-thio-ATP > ATP > alpha,beta-methylene-ATP, indicating involvement of a P2Y-type ATP receptor. 6. Pretreatment of cells with pertussis toxin (PTX) did not prevent the ATP-induced decrease in ICa. However, (i) ATP produced an irreversible decrease of ICa in the presence of intracellular GTP gamma S, and (ii) the inhibitory effect was significantly attenuated in the presence of intracellular GDP beta S, indicating the involvement of a PTX-insensitive G protein in the P2Y receptor-coupling process. 7. Neither (i) replacing extracellular Ca2+ with 1 mM Ba2+, nor (ii) intracellular perfusion of 10 mM BAPTA for at least 30 min attenuated the inhibitory effect of ATP on the current through Ca2+ channels, suggesting that the inhibitory effect was not obligatorily dependent upon influx of Ca2+ or changes in [Ca2+]i. 8. Ensemble-average current behaviour constructed from cell-attached patch recordings of single L-type Ca2+ channels (110 mM BaCl2) demonstrated that when 10 microM ATP was added to the superfusate on the outside of the patch electrode the inhibition of ICa was still observed, providing evidence for the involvement of intracellular diffusible second messenger(s).(ABSTRACT TRUNCATED AT 400 WORDS)
摘要
运用全细胞和细胞贴附式配置的千兆欧封接电压钳技术，在雪貂分离的右心室肌细胞中研究了细胞外三磷酸腺苷（ATP）对基础L型钙电流（ICa）的影响。2. 微摩尔浓度的细胞外ATP以浓度依赖的方式可逆性抑制ICa，而ICa峰值电流-电压关系或稳态激活曲线的电压依赖性未发生任何显著变化。3. 相比之下，微摩尔浓度的细胞外ATP确实显著改变了ICa的失活特性。10微摩尔ATP：（i）增加了ICa的稳态失活程度；（ii）改变了0 mV时ICa失活的时间常数；（iii）降低了-70 mV时ICa从失活状态恢复的时间常数。4. ATP对ICa的抑制作用未被毒蕈碱胆碱能受体拮抗剂阿托品或A1腺苷受体拮抗剂CPDPX（8-环戊基-3,4-二丙基黄嘌呤）阻断。相反，100微摩尔嘌呤能P2受体拮抗剂苏拉明几乎可以完全拮抗10微摩尔ATP的抑制作用。5. ATP类似物抑制ICa的效力顺序为2-甲基硫代-ATP > ATP > α,β-亚甲基-ATP，表明涉及P2Y型ATP受体。6. 用百日咳毒素（PTX）预处理细胞并不能防止ATP诱导的ICa降低。然而，（i）在细胞内存在GTPγS的情况下，ATP使ICa产生不可逆的降低，并且（ii）在细胞内存在GDPβS的情况下，抑制作用显著减弱，表明在P2Y受体偶联过程中涉及一种对PTX不敏感的G蛋白。7. （i）用1 mM Ba2+替代细胞外Ca2+，以及（ii）细胞内灌注10 mM BAPTA至少30分钟，均未减弱ATP对通过钙通道电流的抑制作用，这表明抑制作用不一定依赖于Ca2+内流或细胞内Ca2+浓度（[Ca2+]i）的变化。8. 从单个L型钙通道（110 mM BaCl2）的细胞贴附式膜片钳记录构建的总体平均电流行为表明，当向膜片电极外部的灌流液中添加10微摩尔ATP时，仍可观察到对ICa的抑制作用，这为细胞内可扩散第二信使的参与提供了证据。（摘要截断于400字）
