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.