Abstract
We have investigated the mechanisms by which stimulation of cardiac muscarinic receptors result in paradoxical stimulatory effects on cardiac function, using cultured neonatal rat ventricular myocytes as a model system. Application of low concentrations of carbachol (CCh) (EC50 = 35 nM) produced an atropine-sensitive decrease in spontaneous contraction rate, while, in cells pretreated with pertussis toxin, higher concentrations of CCh (EC50 = 26 microM) elicited an atropine-sensitive increase in contraction rate. Oxotremorine, an m2 muscarinic acetylcholine receptor (mAChR) agonist, mimicked the negative but not the positive chronotropic response to CCh. Reverse transcription followed by polymerase chain reaction carried out on mRNA obtained from single cells indicated that ventricular myocytes express mRNA for the m1, m2, and, possibly, m4 mAChRs. The presence of m1 and m2 mAChR protein on the surface membranes of the cultured ventricular myocytes was confirmed by immunofluorescence. The CCh-induced positive chronotropic response was significantly inhibited by fluorescein-tagged antisense oligonucleotides directed against the m1, but not the m2 and m4, mAChR subtypes. The response was also inhibited by antisense oligonucleotides against Gqalpha protein. Finally, inhibition of CCh-induced phosphoinositide hydrolysis with 500 microM neomycin or 5 microM U73122 completely abolished the CCh-induced positive chronotropic response. These results are consistent with the stimulatory effects of mAChR activation on the rate of contractions in cultured ventricular myocytes being mediated through the m1 mAChR coupled through Gq to phospholipase C-induced phosphoinositide hydrolysis.
Highlights
Stimulation of postsynaptic muscarinic acetylcholine receptors1 on cardiac cells is able to produce dual effects on the heartbeat
It has been demonstrated that mRNA for muscarinic acetylcholine receptor (mAChR) subtypes that were missed by Northern blot studies were detected by use of reverse transcription followed by polymerase chain reaction (RT-PCR) in neuroblastoma cells as well as in guinea pig ventricular myocytes [7, 9]
1) Do cultured neonatal rat ventricular myocytes exhibit dual responses to mAChR stimulation in a manner similar to that observed in other cardiac cell preparations? 2) How many subtypes of mAChRs are expressed in cultured neonatal rat ventricular myocytes? 3) Which mAChR subtype mediates the stimulatory effects of muscarinic agonists on the rate of contraction? 4) What second messenger system is involved in generating this response?
Summary
Culture of Ventricular Myocytes—Cultured neonatal rat ventricular myocytes were prepared using modifications of a previously published procedure [13]. Cells were exposed to GPBS, containing either no primary antibody or affinity-purified rabbit polyclonal antibodies specific for the m1 and m2 mAChRs (diluted 1:200; 0.5 g/ml final concentration) for 48 h at 4 °C These antibodies have been characterized by immunoprecipitation and immunocytochemistry using the family of cloned mAChRs as well as receptors expressed in their native tissues [20]. At the end of the incubation period, excess primary antibody was removed by washing with GPBS and the cells were exposed to a fluorescein-labeled goat anti-rabbit secondary antibody, used at 1:500 dilution, for 45 min at room temperature. 200 –500 nM of fluorescein-tagged oligonucleotides and 8 g/ml Lipofectin (Life Technologies, Inc.) were each diluted in 100 l of serum-free Dulbecco’s modified Eagle’s medium prewarmed to 37 °C. 0.8 ml of serum-free medium was added to each tube containing the Lipofectin-oligonucleotide complexes and the resulting mixture overlaid onto the cultured cells. Intracellular organelles such as the nucleus were first identified by phase contrast microscopy and the images thereby attained compared with
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