Effects of muscarinic receptor stimulation on Ca(2+) transient, cAMP production and pacemaker frequency of rabbit sinoatrial node cells

We investigated the contribution of the intracellular calcium (Ca(i)(2+)) transient to acetylcholine (ACh)-mediated reduction of pacemaker frequency and cAMP content in rabbit sinoatrial nodal (SAN) cells. Action potentials (whole cell perforated patch clamp) and Ca(i)(2+) transients (Indo-1 fluorescence) were recorded from single isolated rabbit SAN cells, whereas intracellular cAMP content was measured in SAN cell suspensions using a cAMP assay (LANCE(®)). Our data show that the Ca(i)(2+) transient, like the hyperpolarization-activated “funny current” (I (f)) and the ACh-sensitive potassium current (I (K,ACh)), is an important determinant of ACh-mediated pacemaker slowing. When I (f) and I (K,ACh) were both inhibited, by cesium (2 mM) and tertiapin (100 nM), respectively, 1 μM ACh was still able to reduce pacemaker frequency by 72%. In these I (f) and I (K,ACh)-inhibited SAN cells, good correlations were found between the ACh-mediated change in interbeat interval and the ACh-mediated change in Ca(i)(2+) transient decay (r (2) = 0.98) and slow diastolic Ca(i)(2+) rise (r (2) = 0.73). Inhibition of the Ca(i)(2+) transient by ryanodine (3 μM) or BAPTA-AM (5 μM) facilitated ACh-mediated pacemaker slowing. Furthermore, ACh depressed the Ca(i)(2+) transient and reduced the sarcoplasmic reticulum (SR) Ca(2+) content, all in a concentration-dependent fashion. At 1 μM ACh, the spontaneous activity and Ca(i)(2+) transient were abolished, but completely recovered when cAMP production was stimulated by forskolin (10 μM) and I (K,ACh) was inhibited by tertiapin (100 nM). Also, inhibition of the Ca(i)(2+) transient by ryanodine (3 μM) or BAPTA-AM (25 μM) exaggerated the ACh-mediated inhibition of cAMP content, indicating that Ca(i)(2+) affects cAMP production in SAN cells. In conclusion, muscarinic receptor stimulation inhibits the Ca(i)(2+) transient via a cAMP-dependent signaling pathway. Inhibition of the Ca(i)(2+) transient contributes to pacemaker slowing and inhibits Ca(i)(2+)-stimulated cAMP production. Thus, we provide functional evidence for the contribution of the Ca(i)(2+) transient to ACh-induced inhibition of pacemaker activity and cAMP content in rabbit SAN cells.