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This Article Full Text Full Text (PDF) Supplemental Material All Versions of this Article: 297/3/H949    most recent 01340.2008v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Lyashkov, A. E. Articles by Lakatta, E. G. PubMed PubMed Citation Articles by Lyashkov, A. E. Articles by Lakatta, E. G.

Cholinergic receptor signaling modulates spontaneous firing of sinoatrial nodal cells via integrated effects on PKA-dependent Ca²⁺ cycling and I_KACh

Laboratory of Cardiovascular Science, Gerontology Research Center, National Institute on Aging, National Institutes of Health, Baltimore, Maryland

Submitted 30 December 2008 ; accepted in final form 15 June 2009

Prior studies indicate that cholinergic receptor (ChR) activation is linked to beating rate reduction (BRR) in sinoatrial nodal cells (SANC) via 1) a G_i-coupled reduction in adenylyl cyclase (AC) activity, leading to a reduction of cAMP or protein kinase A (PKA) modulation of hyperpolarization-activated current (I_f) or L-type Ca²⁺ currents (I_Ca,L), respectively; and 2) direct G_i-coupled activation of ACh-activated potassium current (I_KACh). More recent studies, however, have indicated that Ca²⁺ cycling by the sarcoplasmic reticulum within SANC (referred to as a Ca²⁺ clock) generates rhythmic, spontaneous local Ca²⁺ releases (LCR) that are AC-PKA dependent. LCRs activate Na⁺-Ca²⁺ exchange (NCX) current, which ignites the surface membrane ion channels to effect an AP. The purpose of the present study was to determine how ChR signaling initiated by a cholinergic agonist, carbachol (CCh), affects AC, cAMP, and PKA or sarcolemmal ion channels and LCRs and how these effects become integrated to generate the net response to a given intensity of ChR stimulation in single, isolated rabbit SANC. The threshold CCh concentration ([CCh]) for BRR was 10 nM, half maximal inhibition (IC₅₀) was achieved at 100 nM, and 1,000 nM stopped spontaneous beating. G_i inhibition by pertussis toxin blocked all CCh effects on BRR. Using specific ion channel blockers, we established that I_f blockade did not affect BRR at any [CCh] and that I_KACh activation, evidenced by hyperpolarization, first became apparent at [CCh] > 30 nM. At IC₅₀, CCh reduced cAMP and reduced PKA-dependent phospholamban (PLB) phosphorylation by 50%. The dose response of BRR to CCh in the presence of I_KACh blockade by a specific inhibitor, tertiapin Q, mirrored that of CCh to reduced PLB phosphorylation. At IC₅₀, CCh caused a time-dependent reduction in the number and size of LCRs and a time dependent increase in LCR period that paralleled coincident BRR. The phosphatase inhibitor calyculin A reversed the effect of IC₅₀ CCh on SANC LCRs and BRR. Numerical model simulations demonstrated that Ca²⁺ cycling is integrated into the cholinergic modulation of BRR via LCR-induced activation of NCX current, providing theoretical support for the experimental findings. Thus ChR stimulation-induced BRR is entirely dependent on G_i activation and the extent of G_i coupling to Ca²⁺ cycling via PKA signaling or to I_KACh: at low [CCh], I_KACh activation is not evident and BRR is attributable to a suppression of cAMP-mediated, PKA-dependent Ca²⁺ signaling; as [CCh] increases beyond 30 nM, a tight coupling between suppression of PKA-dependent Ca²⁺ signaling and I_KACh activation underlies a more pronounced BRR.

submembrane Ca²⁺ release; ion channels; protein kinase A phosphorylation; signal transduction