Methylene Blue Is a Muscarinic Antagonist in Cardiac Myocytes

Abstract

SUMMARYWe 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 ϩ current (I K,ACh ) in rat ventricular and atrial myocytes, respectively, and on the binding of [3 H]quinuclidinyl benzylate in rat ventricular membranes. Superfusion, but not internal dialysis, with 30 M Mblue antagonized the inhibitory effect of acetylcholine (ACh, 1 M) on ␤-adrenergic stimulation of I Ca with isoprenaline (Iso, 10 nM or 1 M). 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 M 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-5Ј-O-(3-thio)-triphosphate or guanosine-5Ј-(␤,␥-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 [ 3 H]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 [ 3 H]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 Ca and 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.Mblue has been shown to antagonize the production of cGMP in various tissues and is generally accepted as a selective antagonist of the NO-sensitive isoform of guanylyl cyclase (1-5). As such, Mblue is often used to study the roles of NO and cGMP in the effects of neurotransmitters and hormones. For instance, Mblue antagonizes the effect of ACh on the relaxation of coronary vessels (6, 7).In the heart, Mblue was soon recognized as an inhibitor of the response to ACh (Ref. 8 and references therein). Because ACh, like NO donors, can increase cGMP levels in cardiac myocytes (for reviews, see Refs. 9 and 10), a likely mechanism for the cardiac effect of Mblue was the inhibition of guanylyl cyclase. Consistent with this hypothesis are the recent findings that Mblue (i) inhibits cGMP production induced by ACh in isolated guinea pig cardiomyocytes (11), (ii) prevents the inhibitory effect of ACh on the L-type Ca 2ϩ channel current (I Ca ) (12-15), (iii) prevents the inhibitory effect of ACh on the cAMP-stimulated Cl Ϫ current (16), and (iv) antagonizes the effects of NO donors on cardiac contractility (17, 18) and I Ca (12,14,19 Previous studies have questioned the mechanism of action of Mblue and suggested that Mblue is not a direct guanylyl cyclase inhibitor. Indeed, Mblue is a superoxide anion generator, and this anion, which is a potent NO scavenger, may

NSC23766, a Widely Used Inhibitor of Rac1 Activation, Additionally Acts as a Competitive Antagonist at Muscarinic Acetylcholine Receptors

“…8B), there is now an opportunity to rationalize the binding modes of the thus far uncharacterized ligands that bind to mAChRs. Thus, we calculated the docking results for NSC27366 and for methylene blue, an organic dye that has been shown to be an antagonist at mAChR (Abi-Gerges et al, 1997).…”

Pertussis Toxin-Sensitive G Protein but Not NO/cGMP Pathway Mediates the Negative Inotropic Effect of Carbachol in Adult Rat Cardiomyocytes

“…However, methylene blue and LY83583 exhibit several non-specific effects, including inhibition of nitric oxide synthase and generation of superoxide anions with accompanying auto-oxidation of NO [15]. In addition, methylene blue appears to act as a muscarinic receptor antagonist [16]. Thus, the literature in this area is contradictory.…”

Inhibitory Effects of Psychotropic Drugs on the Acetylcholine Receptor-Operated Potassium Current (I_K.ACh) in Guinea-Pig Atrial Myocytes

“…It was already reported that some psychotropic drugs with higher concentration ranges inhibited a cardiac type of GIRK channel in an over-expression system of Xenopus oocytes and CHO cells [16,17,28]. Moreover, it was reported that chlorpromazine inhibited the acetylcholine-induced I K.ACh with a threshold of ~30 nM in rat atrial cardiomyocyte [1]. In the present experiments, it was found that antipsychotics and antidepressants inhibited I K.ACh in native cardiac myocytes within or close to clinical plasma concentrations.…”

“…It was reported that a couple of antipsychotics and antidepressants inhibited GIRK channel current in an over-expression system [16,17]. Moreover, chlorpromazine inhibited I K.ACh in rat cardiac myocytes [1]. However, influences of most psychotropic drugs on native cardiac myocytes were not fully examined.…”