Mediators of coronary reactive hyperaemia in isolated mouse heart

Abstract

1. Mechanisms regulating coronary tone under basal conditions and during reactive hyperaemia following transient ischaemia were assessed in isolated mouse hearts. 2. Blockade of NO-synthase (50 muM L-NAME), K(ATP) channels (5 muM glibenclamide), A(2A) adenosine receptors (A(2A)ARs; 100 nM SCH58261), prostanoid synthesis (100 muM indomethacin), and EDHF (100 nM apamin+100 nM charybdotoxin) all reduced basal flow approximately 40%. Effects of L-NAME, glibenclamide, and apamin+charybdotoxin were additive, whereas coadministration of SCH58261 and indomethacin with these inhibitors failed to further limit flow. 3. Substantial hyperaemia was observed after 5-40 s occlusions, with flow increasing to a peak of 48+/-1 ml min(-1) g(-1). Glibenclamide most effectively inhibited peak flows (up to 50%) while L-NAME was ineffective. 4. With longer occlusions (20-40 s), glibenclamide alone was increasingly ineffective, reducing peak flows by approximately 15% after 20 s occlusion, and not altering peak flow after 40 s occlusion. However, cotreatment with L-NAME+glibenclamide inhibited peak hyperaemia by 70 and 25% following 20 and 40 s occlusions, respectively. 5. In contrast to peak flow changes, sustained dilation and flow repayment over 60 s was almost entirely K(ATP) channel and NO dependent (each contributing equally) with all occlusion durations. 6. Antagonism of A(2A)ARs with SCH58261 reduced hyperaemia 20-30% whereas inhibition of prostanoid synthesis was ineffective. Effects of A(2A)AR antagonism were absent in hearts treated with L-NAME and glibenclamide, supporting NO and K(ATP)-channel-dependent effects of A(2A)ARs. 7. EDHF inhibition alone exerted minor effects on hyperaemia and only with longer occlusions. However, residual hyperaemia after 40 s occlusion in hearts treated with L-NAME+glibenclamide+SCH58261+indomethacin was abrogated by cotreatment with apamin+charybdotoxin. 8. Data support a primary role for K(ATP) channels and NO in mediating sustained dilation after coronary occlusion. While K(ATP) channels (and not NO) are also important in mediating initial peak flow adjustments after brief 5-10 s occlusions, their contribution declines with longer 20-40 s occlusions. Intrinsic activation of A(2A)ARs is important in triggering K(ATP) channel/NO-dependent hyperaemia. Synergistic effects of combined inhibitors implicate interplay between mediators, with compensatory changes occurring in K(ATP) channel, NO, and/or EDHF responses when one is individually blocked.