Abstract

We studied the mechanism of action of methylene blue (Mblue), a putative guanylyl cyclase inhibitor, on the L-type calcium current (I_Ca) and the muscarinic activated K⁺ current (I_K,ACh) in rat ventricular and atrial myocytes, respectively, and on the binding of [³H]quinuclidinyl benzylate in rat ventricular membranes. Superfusion, but not internal dialysis, with 30 μm Mblue antagonized the inhibitory effect of acetylcholine (ACh, 1 μm) on β-adrenergic stimulation of I_Ca with isoprenaline (Iso, 10 nm or 1 μm). However, Mblue had no effect on the basal I_Ca or on the stimulation of I_Ca by Iso in the absence of ACh. The activation of I_K,ACh by 3 μm ACh was also antagonized by Mblue in a dose-dependent manner. In contrast, Mblue had no effect on the activation of I_K,ACh by either guanosine-5′-O-(3-thio)triphosphate or guanosine-5′-(β,γ-imido)triphosphate. Chlorpromazine (CPZ), a piperazine derivative like Mblue, also inhibited the muscarinic activation of I_K,ACh in a dose-dependent manner. The specific binding of [³H]QNB, a muscarinic ligand, to rat ventricular membranes was displaced in a dose-dependent manner by Mblue and CPZ. The piperazine derivatives behaved like competitive antagonists of [³H]QNB binding, exhibiting equilibrium dissociation constant (K_i ) values of 187 nm for Mblue and 366 nm for CPZ. In conclusion, Mblue exerts antimuscarinic effects on I_Caand I_K,ACh in rat cardiac myocytes that are best explained by the binding of Mblue to the M2 subtype of muscarinic receptors. This property probably contributes to the antimuscarinic effect of the putative guanylyl cyclase inhibitor reported in previous studies.