METHYLENE-BLUE IS A MUSCARINIC ANTAGONIST IN CARDIAC MYOCYTES

We studied the mechanism of action of methylene blue (Mblue), a putati ve guanylyl cyclase inhibitor, on the L-type calcium current (I-Ca) an d the muscarinic activated K+ current (I-K,I-ACh) in rat ventricular a nd atrial myocytes, respectively, and on the binding of [H-3]quinuclid inyl benzylate in rat ventricular membranes. Superfusion, but not inte rnal dialysis, with 30 mu M Mblue antagonized the inhibitory effect of acetylcholine (ACh, 1 mu M) on beta-adrenergic stimulation of I-Ca wi th isoprenaline (Iso, 10 nM or 1 mu M). However, Mblue had no effect o n the basal I-Ca or on the stimulation of I-Ca by Iso in the absence o f ACh. The activation of I-K,I-ACh by 3 mu M ACh was also antagonized by Mblue in a dose-dependent manner. In contrast, Mblue had no effect on the activation of I-K,I-ACh by either guanosine-5'-O-(3-thio)-triph osphate or guanosine-5'-(beta,gamma-imido)triphosphate. Chlorpromazine (CPZ), a piperazine derivative like Mblue, also inhibited the muscari nic activation of I-K,I-ACh in a dose-dependent manner. The specific b inding of [H-3]QNB, a muscarinic ligand, to rat ventricular membranes was displaced in a dose-dependent manner by Mblue and CPZ. The piperaz ine derivatives behaved like competitive antagonists of [H-3]QNB bindi ng, exhibiting equilibrium dissociation constant (K-i) values of 187 n M far Mblue and 366 nM for CPZ. In conclusion, Mblue exerts antimuscar inic effects on I-Ca and I-K,I-ACh in rat cardiac myocytes that are be st explained by the binding of Mblue to the M2 subtype of muscarinic r eceptors. This property probably contributes to the antimuscarinic eff ect of the putative guanylyl cyclase inhibitor reported in previous st udies.