M₃ Muscarinic Receptors Mediate Positive Inotropic Responses in Mouse Atria: A Study with Muscarinic Receptor Knockout Mice

Abstract

The potential functional roles of M 3 muscarinic receptors in mouse atria were examined by pharmacological and molecular biological techniques, using wild-type mice, muscarinic M 2 or M 3 receptor single knockout (M 2 KO, M 3 KO), and M 2 and M 3 muscarinic receptor double knockout mice (M 2 /M 3 KO). Realtime quantitative reverse transcriptase-polymerase chain reaction analysis showed that the M 2 receptor mRNA was expressed predominantly in mouse atria but that the M 1 , M 3 , M 4 , and M 5 receptor subtypes were also expressed at low levels. Carbachol (10 nM-30 M) decreased the spontaneous beating frequency of right atria isolated from wild-type mice. Studies with subtype-preferring antagonists and atria from M 2 KO mice confirmed that this activity is mediated by the M 2 receptor subtype. In left atria from wild-type mice, carbachol decreased the amplitude of electrical field stimulation-evoked contractions (negative inotropic action), but this inhibition was transient and was followed by a gradual increase in contraction amplitude (positive inotropic response). In atria from M 3 KO mice, the transient negative inotropic action of carbachol changed to a sustained negative inotropic action. In contrast, in atria from M 2 KO mice, carbachol showed only positive inotropic activity. In atria from M 2 /M 3 double KO mice, carbachol was devoid of any inotropic activity. These observations, complemented by functional studies with subtype-preferring antagonists, convincingly demonstrate that atrial M 3 muscarinic receptors mediate positive inotropic effects in mouse atria. Physiologically, this activity may serve to dampen the inhibitory effects of M 2 receptor activation on atrial contractility.Muscarinic receptor stimulation by acetylcholine plays an important role in parasympathetic control of cardiac functions such as heart rate (chronotropic action), conduction velocity (dromotropic action), and contractility (inotropic action). Muscarinic receptors are prototypic members of the superfamily of G protein-coupled receptors, and molecular cloning studies have demonstrated the existence of five distinct mammalian muscarinic receptor subtypes (M 1 -M 5 ) (Caulfield and Birdsall, 1998). Based on their differential G protein-coupling properties, the five receptors can be subdivided into two major functional classes. The M 1 , M 3 , and M 5 receptors preferentially couple to G q/11 proteins, whereas the M 2 and M 4 receptors are selectively linked to G i/o proteins (Caulfield and Birdsall, 1998;Lanzafame et al., 2003). It is well documented that the heart predominantly expresses the M 2 receptor subtype (Brodde and Michel, 1999;Dhein et al., 2001). After activation of cardiac M 2 receptors, the activated ␣ subunit of G i proteins inhibits adenylate cyclase activity, resulting in a decrease of cytoplasmic cAMP, whereas the ␤␥ subunit of the G i proteins directly activates the inwardly rectifying muscarinic K ϩ channel (Yamada et al., 1998;Dhein et al., 2001). However, the M 2 receptor is not the only muscarinic ...

Identification and Role of Muscarinic Receptor Subtypes Expressed in Rat Adrenal Medullary Cells

“…Thus, results obtained from knockout mice may not always be the case in rats. Second, genetic ablation of a certain receptor subtype may induce compensational changes in expression of other subtypes (21) and/or uncover involvement of other subtypes whose physiological significance is not apparent (22). The second aim was to clarify whether muscarinic receptors mediate neuronal transmission from the splanchnic nerve.…”

Involvement of Cyclooxygenase-2 in Carbachol-Induced Positive Inotropic Response in Mouse Isolated Left Atrium

“…Muscarinic M 3 -receptor blockade by 4-DAMP almost completely abolished the positive response to CCh with slight inhibition of the negative response in the present study. It was recently demonstrated in the M 3 -receptor knockout mice that M 3 receptors mediate positive inotropic responses in mouse atria (Kitazawa et al, 2009). The negative response persisted for a long duration in the 4-DAMP-treated and the endotheliumdeprived mouse atria similar to the response observed in rat and guinea pig.…”

“…The transient negative phase is the standard M 2 receptor-mediated pertussis toxin (PTX)-sensitive response. Although it was reported that the late positive response is mediated by M 3 receptor (Kitazawa et al, 2009) and production of prostaglandins (PGs) (Tanaka et al, 2001), precise mechanisms of this positive inotropic response to muscarinic stimulation in mouse left atrial tissue remained to be clarified. Therefore, the purpose of the present study is to confirm the biphasic response induced by a muscarinic agonist, carbachol (CCh), in mouse left atria and to examine the mechanisms underling the biphasic response, especially relating to PG production.…”

A new potassium ion current induced by stimulation of M2-cholinoreceptors in fish atrial myocytes

“…However, the presence of physiologically relevant M 3 cholinoreceptors was confirmed during the last decade for mammalian myocardium (Hellgren et al, 2000;Kitazawa et al, 2009), and therefore cannot be neglected as a possible signaling pathway in fish cardiac myocytes. To this end, we have investigated effects of the selective M 3 blocker 4-DAMP (10 −9 mol l )-induced current.…”