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

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