No confirmation for a causal role of volume-regulated chloride channels in ischemic preconditioning in rabbits

Volume-regulated chloride channels have recently been proposed to be end-effecters in ischemic preconditioning. The present study attempted to confirm this hypothesis by looking both at cardioprotection and channel activity. In isolated rabbit cardiomyocytes, hypo-osmotic stress (167 mosm/I) induced a current with a magnitude of 2-5 pA/pF at 60 mV. That current could be blocked by the selective chloride channel blockers 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) or indanyloxyacetic acid 94 (IAA-94). but only at 100 muM and 1 mM respectively. Lower concentrations were not effective. Because the channel-blocking concentrations were toxic in isolated perfused rabbit hearts, as evidenced by cessation of cardiac contraction and massive infarction, neither agent could be tested against preconditioning's anti-infarct effect. NPPB and IAA-94 at 1 muM and 10 muM, respectively (the doses used in a previous report), did not affect coronary flow, heart rate and developed pressure, and also did not prevent the infarct size reduction of ischemic preconditioning with 5 min global ischemia/10 min reperfusion preceding 30 min of regional ischemia and 120 min of reperfusion [11.4(+/- 3.6) and (11.1(+/- 3.7)% infarction of risk area, respectively]. The volume-regulated chloride and organic osmolyte channel blocker 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) at 100 muM blocked the hypoosmotically induced current in myocytes, but again could not be used, since it induced total cessation of cardiac contraction and reduced infarct size in non-preconditioned hearts. Our data do not confirm a prior study on a causal role for volume-regulated chloride channels in the protection of ischemic preconditioning. This hypothesis remains to be adequately tested. (C) 2000 Academic Press.