Methylene Blue Is a Muscarinic Antagonist in Cardiac Myocytes
- Najah Abi-Gerges,
- Thomas Eschenhagen1,
- Leif Hove-Madsen2,
- Rodolphe Fischmeister and
- Pierre-François Mery
- Laboratoire de Cardiologie Cellulaire et Moléculaire, Institut National de la Santé et de la Recherche Médicale U-446,Université de Paris-Sud, Faculté de Pharmacie, F-92296 Châtenay-Malabry, France
Abstract
We studied the mechanism of action of methylene blue (Mblue), a putative guanylyl cyclase inhibitor, on the L-type calcium current (ICa) and the muscarinic activated K+ current (IK,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 ICa with isoprenaline (Iso, 10 nm or 1 μm). However, Mblue had no effect on the basal ICa or on the stimulation of ICa by Iso in the absence of ACh. The activation of IK,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 IK,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 IK,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 (Ki) values of 187 nm for Mblue and 366 nm for CPZ. In conclusion, Mblue exerts antimuscarinic effects on ICaand IK,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.
Footnotes
-
Send reprint requests to: Dr. Pierre-François Mery, INSERM U-446, Faculté de Pharmacie, F-92296 Châtenay-Malabry, France. E-mail: u446{at}vjf.inserm.fr
-
↵1 Current affiliation: Pharmakologisches Institut, Universitäts-Krankenhaus Eppendorf, D-2000 Hamburg 20–38, Germany.
-
↵2 Current affiliation: Department of Biologia Celular, Fisiologia Animal, Fac. Ciencias, Universidad Autonoma de Barcelona, E-08193 Cerdanyola, Spain.
-
This work was supported by a grant from the Association Française contre les Myopathies. L.H.-M. was a Fellowship recipient of the Carlsberg Foundation. T.E. was supported by a grant from the Deutsche Forschungsgemeinschaft.
-
↵3 86/609/CEE, CE off J L358, December 18, 1986.
-
↵4 J Off République Française, October 20, 1987, pp. 12245–12248.
-
↵5 No. 04226, April 12, 1991.
- Abbreviations:
- NO
- nitric oxide
- ACh
- acetylcholine
- EGTA
- ethylene glycol bis(α-aminoethyl ether)-N,N,N′,N′-tetraacetic acid
- HEPES
- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- CCh
- carbachol
- Iso
- isoprenaline
- Mblue
- methylene blue
- CPZ
- chlorpromazine
- ICa
- L-type calcium current
- IK,ACh
- muscarinic activated potassium current
- SOD
- superoxide dismutase
- NOS
- nitric oxide synthase
- GTPγS
- guanosine-5′-O-3-triphosphate
- Gpp(NH)p
- guanosine-5′-(β,γ-imido)triphosphate
- DTT
- dl-dithiotreitol
- QNB
- quinuclidinyl benzylate
- AF-DX 116
- 11-[((2-diethylamino)methyl-1-piperidinyl)acetyl]5,11-dihydro-6H-pyrido-2,3-b)(1,4)-benzodiazepine-6-one
-
- Received January 23, 1997.
- Accepted June 2, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
