Light D B, Capes T L, Gronau R T, Adler M R
Department of Biology, Ripon College, Ripon, Wisconsin 54971, USA.
Am J Physiol. 1999 Sep;277(3):C480-91. doi: 10.1152/ajpcell.1999.277.3.C480.
This study examined whether extracellular ATP stimulates regulatory volume decrease (RVD) in Necturus maculosus (mudpuppy) red blood cells (RBCs). The hemolytic index (a measure of osmotic fragility) decreased with extracellular ATP (50 microM). In contrast, the ATP scavenger hexokinase (2.5 U/ml, 1 mM glucose) increased osmotic fragility. In addition, the ATP-dependent K+ channel antagonist glibenclamide (100 microM) increased the hemolytic index, and this inhibition was reversed with ATP (50 microM). We also measured cell volume recovery in response to hypotonic shock electronically with a Coulter counter. Extracellular ATP (50 microM) enhanced cell volume decrease in a hypotonic (0.5x) Ringer solution. In contrast, hexokinase (2.5 U/ml) and apyrase (an ATP diphosphohydrolase, 2.5 U/ml) inhibited cell volume recovery. The inhibitory effect of hexokinase was reversed with the Ca2+ ionophore A-23187 (1 microM); it also was reversed with the cationophore gramicidin (5 microM in a choline-Ringer solution), indicating that ATP was linked to K+ efflux. In addition, glibenclamide (100 microM) and gadolinium (10 microM) inhibited cell volume decrease, and the effect of these agents was reversed with ATP (50 microM) and A-23187 (1 microM). Using the whole cell patch-clamp technique, we found that ATP (50 microM) stimulated a whole cell current under isosmotic conditions. In addition, apyrase (2.5 U/ml), glibenclamide (100 microM), and gadolinium (10 microM) inhibited whole cell currents that were activated during hypotonic swelling. The inhibitory effect of apyrase was reversed with the nonhydrolyzable analog adenosine 5'-O-(3-thiotriphosphate) (50 microM), and the effect of glibenclamide or gadolinium was reversed with ATP (50 microM). Finally, anionic whole cell currents were activated with hypotonic swelling when ATP was the only significant charge carrier, suggesting that increases in cell volume led to ATP efflux through a conductive pathway. Taken together, these results indicate that extracellular ATP stimulated cell volume decrease via a Ca2+-dependent step that led to K+ efflux.
本研究检测了细胞外ATP是否能刺激斑泥螈红细胞的调节性容积减小（RVD）。溶血指数（一种渗透脆性的度量指标）随细胞外ATP（50微摩尔）而降低。相反，ATP清除剂己糖激酶（2.5单位/毫升，1毫摩尔葡萄糖）增加了渗透脆性。此外，ATP依赖性钾通道拮抗剂格列本脲（100微摩尔）增加了溶血指数，而这种抑制作用可被ATP（50微摩尔）逆转。我们还用库尔特计数器以电子方式测量了低渗休克后细胞容积的恢复情况。细胞外ATP（50微摩尔）增强了在低渗（0.5倍）林格氏液中细胞容积的减小。相反，己糖激酶（2.5单位/毫升）和腺苷三磷酸双磷酸酶（一种ATP二磷酸水解酶，2.5单位/毫升）抑制了细胞容积的恢复。己糖激酶的抑制作用可被钙离子载体A - 23187（1微摩尔）逆转；在胆碱 - 林格氏液中，阳离子载体短杆菌肽（5微摩尔）也能逆转这种作用，这表明ATP与钾离子外流有关。此外，格列本脲（100微摩尔）和钆（10微摩尔）抑制了细胞容积的减小，而ATP（50微摩尔）和A - 23187（1微摩尔）可逆转这些药物的作用。使用全细胞膜片钳技术，我们发现ATP（50微摩尔）在等渗条件下刺激了全细胞电流。此外，腺苷三磷酸双磷酸酶（2.5单位/毫升）、格列本脲（100微摩尔）和钆（10微摩尔）抑制了低渗肿胀期间激活的全细胞电流。腺苷三磷酸双磷酸酶的抑制作用可被不可水解的类似物腺苷5'-O-（3-硫代三磷酸）（50微摩尔）逆转，而格列本脲或钆的作用可被ATP（50微摩尔）逆转。最后，当ATP是唯一重要的电荷载体时，低渗肿胀激活了阴离子全细胞电流，这表明细胞容积的增加导致ATP通过一条传导途径外流。综上所述，这些结果表明细胞外ATP通过一个导致钾离子外流的钙离子依赖性步骤刺激了细胞容积减小。
