|Budget Amount *help
¥3,200,000 (Direct Cost : ¥3,200,000)
Fiscal Year 1999 : ¥1,500,000 (Direct Cost : ¥1,500,000)
Fiscal Year 1998 : ¥1,700,000 (Direct Cost : ¥1,700,000)
Vascular endothelium is known to secrete various biologically active mediators in response to mechanical stress. However, the pathways for secreting these agents are still not clarified. On the other hand, volume-regulated chloride channel (VRAC) is activated by mechanical stress, especially hypotonic stress, in vascular endothelium. The aim of this study was to investigate the possible relationship between the activation of VRAC and the secretion of biological mediators. Hypotonic stress (HTS) elicited CaィイD12+ィエD1 oscillations in bovine aortic endothelial cells, which was suppressed by phospholipase C inhibitors and PィイD22ィエD2 antagonist, thereby suggesting the involvement of ATP release in HTS-induced CaィイD12+ィエD1 oscillations. The amount of ATP release was 91amol/cell/10min. On the other hand, HTS induced the activation of VRAC current, which was inhibited by high concentrations of glibenclamide, fluoxetine and verapamil. These agents also abolished HTS-induced CaィイD12+ィエD1 oscillations and ATP release. Furthermore, high KィイD1+ィエD1 depolarization also inhibited VRAC and CaィイD12+ィエD1 oscillations/ATP release. These suggest that ATP release largely depends on the activation of VRAC. However, previous reports have indicated that VRAC is inhibited by extracellular ATP, therefore ATP has been considered as an inhibitor of VRAC. We also observed that extracellular ATP blocked VRAC current, but its blocking action was less prominent at highly depolarized membrane potentials. By the mathematical analysis of these data, we succeeded to show that ATP has dual actions on VRAC, I. E., ATP partially blocks but partially permeates VRAC. Therefore, present study strongly suggests that HTS-induced ATP release is probably through VRAC.