Cholinergic receptor signaling modulates spontaneous firing of sinoatrial nodal cells via integrated effects on PKA-dependent Ca(2+) cycling and I(KACh)

Am J Physiol Heart Circ Physiol. 2009 Sep;297(3):H949-59. doi: 10.1152/ajpheart.01340.2008. Epub 2009 Jun 19.

Abstract

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 Ca(2+) 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 Ca(2+) cycling by the sarcoplasmic reticulum within SANC (referred to as a Ca(2+) clock) generates rhythmic, spontaneous local Ca(2+) releases (LCR) that are AC-PKA dependent. LCRs activate Na(+)-Ca(2+) 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 approximately 10 nM, half maximal inhibition (IC(50)) 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 IC(50), CCh reduced cAMP and reduced PKA-dependent phospholamban (PLB) phosphorylation by approximately 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 IC(50), 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 IC(50) CCh on SANC LCRs and BRR. Numerical model simulations demonstrated that Ca(2+) 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 Ca(2+) 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 Ca(2+) signaling; as [CCh] increases beyond 30 nM, a tight coupling between suppression of PKA-dependent Ca(2+) signaling and I(KACh) activation underlies a more pronounced BRR.

Publication types

  • Research Support, N.I.H., Intramural

MeSH terms

  • Action Potentials / drug effects
  • Action Potentials / physiology
  • Animals
  • Atropine / pharmacology
  • Bee Venoms / pharmacology
  • Calcium / metabolism
  • Calcium Channels, L-Type / physiology*
  • Calcium Signaling / drug effects
  • Calcium Signaling / physiology*
  • Calcium-Binding Proteins / metabolism
  • Cells, Cultured
  • Cesium / pharmacology
  • Chlorides / pharmacology
  • Cholinergic Agonists / pharmacology
  • Cyclic AMP / metabolism
  • Cyclic AMP-Dependent Protein Kinases / metabolism*
  • Cyclic GMP / metabolism
  • Parasympatholytics / pharmacology
  • Patch-Clamp Techniques
  • Pertussis Toxin / pharmacology
  • Phosphorylation / drug effects
  • Phosphorylation / physiology
  • Potassium Channel Blockers / pharmacology
  • Rabbits
  • Receptors, Cholinergic / physiology*
  • Sinoatrial Node / cytology
  • Sinoatrial Node / physiology*
  • Stochastic Processes

Substances

  • Bee Venoms
  • Calcium Channels, L-Type
  • Calcium-Binding Proteins
  • Chlorides
  • Cholinergic Agonists
  • Parasympatholytics
  • Potassium Channel Blockers
  • Receptors, Cholinergic
  • phospholamban
  • Cesium
  • tertiapin
  • Atropine
  • Cyclic AMP
  • Pertussis Toxin
  • Cyclic AMP-Dependent Protein Kinases
  • cesium chloride
  • Cyclic GMP
  • Calcium