Role of nitric oxide and K(ATP) channels in pharmacological preconditioning of the heart

The role of NO and mitochondrial K_ATP channels in cardioprotection was investigated by using Diazoxide, a specific opener of mitochondrial_ATP channels. Diazoxide (1 mg/Kg) was administered either 30 min or 24 h before lethal ischemia in rabbits. Blockers of mitochondrial K_ATP channel [5-hydroxydecanoate (5-HD, 5 mg/kg, iv)] or NO synthase [N(G)-nitro-L-arginine methyl ester (L-NAME, )] were given 10 min before ischemia-reperfusion and infarct size was measured by triphenyltetrazolium chloride (TTC) staining. Diazoxide decreased infarct size from 27.8 ± 4.2% and 30.4 ± 4.2% in the vehicle groups to 12.9 ± 1.2% and 19.6 ± 2.4% during early and delayed preconditioning, respectively (P < 0.05). Infarct size increased to 31.3 ± 1.1% and 27.9 ± 1.0% (early preconditioning); 29.9 ± 2.3% and 35.1 ± 1.8% (delayed preconditioning) with 5-HD and L-NAME, respectively (P < 0.05). To indentify the key NOS isoform, isolated perfused mouse hearts were subjected to 20 min of global ischemia and 30 min of reperfusion. Adult male mice (ICR and iNOS knockout) were pretreated with diazoxide (1 mg/Kg, i.p.) or volume-matched vehicle. Twenty-four hrs later, the hearts were perfused in Langendorff mode and subjected to 20 min global ischemia and 30 min reperfusion. S-methylisothiourea (SMT; 3 mg/Kg, i.p.) was given 30 min before ischemia to block iNOS. Infarct size was measured by TTC staining and expression of iNOS and endothelial NOS (eNOS) were measured by Western blots. Infarct size was reduced from 32.9 ± 4.3% in vehicle group to 12.7 ± 6.1% with diazoxide (P < 0.05, n = 5–7). The protective effect of diazoxide was blocked in SMT-treated as well as iNOS gene knockout mice with a resultant infarct size 29.0 ± 5.7% and 26.3 ± 9.8% respectively which was not significantly different from control. Western blot analysis showed increase in iNOS protein in both diazoxide and diazoxide + SMT groups (Arbitrary units; 0.85 ± 0.20 and 0.75 ± 0.13) as compared to the vehicle 0.49 ± 0.11 (mean ± SE, P < 0.05) while eNOS expression remained.; In the second part, the cardioprotective effect of Sildenafil citrate (Viagra) was studied. Rabbits were treated with sildenafil citrate (0.7 mg/Kg, i.v.) either 30 min or 24 h before ischemia/reperfusion. The infarct size decreased from 33.8 ± 1.7 in control rabbits to 10.8 ± 0.9 during the early phase and 19.9 ± 2.0 during the delayed phase. 5-HD (5mg/Kg, i.v.) abolished protection with an increase in infarct size to 35.6 ± 0.4% and 36.8 ± 1.6% during the early and delayed phase, respectively (P < 0.05). Furthermore, oral administration of sildenafil resulted in similar cardioprotective effects. Selective inhibitor of guanylate cyclase, quinoxalin derivative 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 5mg/kg, i.p.) given 60 minutes prior to ischemia abolished the cardioprotective effect of Sildenafil, suggesting an important role of cGMP in sildenafil-induced preconditioning.