title: Effects of muscarinic receptor stimulation on Ca2+ transient, cAMP production and pacemaker frequency of rabbit sinoatrial node cells.
abstract: 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((R))). 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 micro M 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 microM) or BAPTA-AM (5 microM) 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 microM ACh, the spontaneous activity and Ca (i) (2+) transient were abolished, but completely recovered when cAMP production was stimulated by forskolin (10 microM) and I (K,ACh) was inhibited by tertiapin (100 nM). Also, inhibition of the Ca (i) (2+) transient by ryanodine (3 microM) or BAPTA-AM (25 microM) 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.
authors.affiliation: Heart Failure Research Center, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. m.m.vanborren@amc.uva.nl
authors.forename: Marcel M G J
authors.initials: MM
authors.lastname: van Borren
authors.forename: Arie O
authors.initials: AO
authors.lastname: Verkerk
authors.forename: Ronald
authors.initials: R
authors.lastname: Wilders
authors.forename: Najat
authors.initials: N
authors.lastname: Hajji
authors.forename: Jan G
authors.initials: JG
authors.lastname: Zegers
authors.forename: Jan
authors.initials: J
authors.lastname: Bourier
authors.forename: Hanno L
authors.initials: HL
authors.lastname: Tan
authors.forename: Etienne E
authors.initials: EE
authors.lastname: Verheijck
authors.forename: Stephan L M
authors.initials: SL
authors.lastname: Peters
authors.forename: Astrid E
authors.initials: AE
authors.lastname: Alewijnse
authors.forename: Jan-Hindrik
authors.initials: JH
authors.lastname: Ravesloot
mesh_terms: D000109:Acetylcholine / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism*; D004533:Egtazic Acid / Q000031:analogs & derivatives; D018721:Muscarinic Agonists; D018408:Patch-Clamp Techniques; D011817:Rabbits; D011976:Receptors, Muscarinic / Q000378:metabolism*; D012433:Ryanodine; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D012849:Sinoatrial Node / Q000166:cytology / Q000378:metabolism*
chemical_list: D018721:Muscarinic Agonists; D011976:Receptors, Muscarinic; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D012433:Ryanodine; D004533:Egtazic Acid; D000242:Cyclic AMP; D000109:Acetylcholine; D002118:Calcium