Regulation of NO-dependent acetylcholine relaxation by K channels and the Na-K ATPase pump in porcine internal mammary artery

This study was designed to determine whether K⁺ channels play a role in nitric oxide (NO)-dependent acetylcholine relaxation in porcine internal mammary artery (IMA). IMA segments were isolated and mounted in organ baths to record isometric tension. Acetylcholine-elicited vasodilation was abolished by muscarinic receptor blockade with atropine (10^-6 M). Incubation with indomethacin (3 × 10⁻⁶ M), superoxide dismutase (150 U/ml) and bosentan (10⁻⁵ M) did not modify the acetylcholine response ruling out the participation of cyclooxygenase-derivates, reactive oxygen species or endothelin. The relaxation response to acetylcholine was strongly diminished by NO synthase- or soluble guanylyl cyclase-inhibition using l-NOArg (10⁻⁴ M) or ODQ (3 × 10⁻⁶ M), respectively. The vasodilation induced by acetylcholine and a NO donor (NaNO₂) was reduced when rings were contracted with an enriched K⁺ solution (30 mM), by voltage-dependent K⁺ (K_v) channel blockade with 4-amynopiridine (4-AP; 10⁻⁴ M), by Ca²⁺-activated K⁺ (K_Ca) channel blockade with tetraethylammonium (TEA; 10⁻³ M), and by apamin (5 × 10⁻⁷ M) plus charybdotoxin (ChTx; 10⁻⁷ M) but not when these were added alone. In contrast, large conductance K_Ca (BK_Ca)_, ATP-sensitive K⁺ (K_ATP) and inwardly rectifying K⁺ (K_ir) channel blockade with iberiotoxin (IbTx; 10⁻⁷ M), glibenclamide (10⁻⁶ M) and BaCl₂ (3 × 10⁻⁵ M), respectively, did not alter the concentration-response curves to acetylcholine and NaNO₂. Na⁺−K⁺ ATPase pump inhibition with ouabain (10⁻⁵ M) practically abolished acetylcholine and NaNO₂ relaxations. Our findings suggest that acetylcholine-induced relaxation is largely mediated through the NO-cGMP pathway, involving apamin plus ChTx-sensitive K⁺ and K_v channels, and Na⁺−K⁺-ATPase pump activation.