Role of Muscarinic M1, M2, and M3 Receptors in the Regulation of Electrical Activity of Myocardial Tissue of Caval Veins during the Early Postnatal Ontogeny.

We studied the influence of blockers of muscarinic M1, M2, and M3 receptors on the effect of acetylcholine in the myocardial tissue of caval veins in rats at the early stage of ontogeny. The experiments were performed on isolated preparations of the right superior vena cava working under their own rhythm. Action potentials were recorded using the standard microelectrode technique. Acetylcholine (1 µM) suppressed automatic activity in the superior vena cava myocardium. Preliminary perfusion of the preparation with non-selective blocker atropine (1 µM) completely abolished the effect of acetylcholine, treatment with M2 receptor blocker AQ-RA 741 (1 µM) led to partial suppression of the effect of acetylcholine. Blockers of M1 and M3 receptors pirenzepine (1 µM) and 4DAMP (0.1 µM) did not suppress the effect of acetylcholine. Thus, the effect of acetylcholine is predominantly realized via M2 receptors, but M3 receptors can also partially mediate its effect in the superior vena cava myocardium in rats at the early stages of ontogeny.

Decrease in the Sensitivity of Myocardium to M3 Muscarinic Receptor Stimulation during Postnatal Ontogenisis.

Type 3 muscarinic receptors (M3 receptors) participate in the mediation of cholinergic effects in mammalian myocardium, along with M2 receptors. However, myocardium of adult mammals demonstrates only modest electrophysiological effects in response to selective stimulation of M3 receptors which are hardly comparable to the effects produced by M2 stimulation. In the present study, the effects of selective M3 stimulation induced by application of the muscarinic agonist pilocarpine (10 µM) in the presence of the selective M2 blocker methoctramine (100 nM) on the action potential (AP) waveform were investigated in isolated atrial and ventricular preparations from newborn and 3-week-old rats and compared to those in preparations from adult rats. In the atrial myocardium, stimulation of M3 receptors produced a comparable reduction of AP duration in newborn and adult rats, while in 3-week-old rats the effect was negligible. In ventricular myocardial preparations from newborn rats, the effect of M3 stimulation was more than 3 times stronger compared to that from adult rats, while preparations from 3-week old rats demonstrated no definite effect, similarly to atrial preparations. In all studied types of cardiac preparations, the effects of M3 stimulation were eliminated by the selective M3 antagonist 4-DAMP (10 nM). The results of RT-PCR show that the amount of product of the M3 receptor gene decreases with the maturation of animals both in atrial and ventricular myocardium. We concluded that the contribution of M3 receptors to the mediation of cardiac cholinergic responses decreases during postnatal ontogenesis. These age-related changes may be associated with downregulation of M3 receptor gene expression.

Different Myocardial Sensitivity in Newborn and Mature Rats to Selective Stimulation of M3 Cholinoreceptors.

The effects of selective activation of subtype 3 muscarinic (M3) receptors on electrical activity of isolated preparations of the atrial and ventricular myocardium of the newborn and 4-month-old rats were examined. Application of muscarinic receptor agonist pilocarpine (10(-5) M) in preparations with M2 cholinoreceptors blocked by selective antagonist methoctramine (10(-7) M) decreased the duration of action potentials in the atrial and ventricular myocardium. Selective blocker of M3 cholinoreceptors 4-DAMP (10(-8) M) prevented this effect. While stimulation of ventricular M3 cholinoreceptors with pilocarpine was significantly stronger in newborn pups than in mature rats, similar stimulation of atrial receptors revealed no significant difference in both groups.

Effect of selective stimulation of muscarinic M3 cholinoceptors on electrical and contractile activity of rat ventricular myocardium.

We studied the effect of selective activation of muscarinic M3receptors on electrical activity in the isolated preparation of rat ventricular myocardium as well as contractile activity of the left ventricle of Langendorff-perfused isolated heart. Application of muscarinic agonist pilocarpine (10(-5)M) against the background of selective blockade of subtype 2 muscarinic receptors with methoctramine (10(-7)M) markedly shortened the duration of action potentials in the isolated ventricular myocardium and reduced the amplitude and maximum rates of left-ventricular pressure rise and decay in the isolated heart paced at a fixed rate. All these effects were significantly suppressed by selective M3receptor blocker 4-DAMP (10(-8)M), which attested to the involvement of M3muscarinic receptors.