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

Alexey E. Lyashkov, Tatiana M. Vinogradova, Ihor Zahanich, Yue Li, Antoine Younes, H. Bradley Nuss, Harold A. Spurgeon, Victor A. Maltsev, Edward Lakatta

Research output: Contribution to journalArticle

Abstract

Prior studies indicate that cholinergic receptor (ChR) activation is linked to beating rate reduction (BRR) in sinoatrial nodal cells (SANC) via 1) a Gi-coupled reduction in adenylyl cyclase (AC) activity, leading to a reduction of cAMP or protein kinase A (PKA) modulation of hyperpolarization- activated current (If) or L-type Ca2+ currents (I Ca,L), respectively; and 2) direct Gi-coupled activation of ACh-activated potassium current (IKACh). 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 μ10 nM, half maximal inhibition (IC50) was achieved at 100 nM, and 1,000 nM stopped spontaneous beating. Gi inhibition by pertussis toxin blocked all CCh effects on BRR. Using specific ion channel blockers, we established that If blockade did not affect BRR at any [CCh] and that IKACh 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 ∼50%. The dose response of BRR to CCh in the presence of IKACh 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 Gi activation and the extent of Gi coupling to Ca2+ cycling via PKA signaling or to IKACh: at low [CCh], IKACh 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 IKACh activation underlies a more pronounced BRR.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Heart and Circulatory Physiology
Volume297
Issue number3
DOIs
StatePublished - Sep 2009
Externally publishedYes

Fingerprint

Carbachol
Cholinergic Receptors
Cyclic AMP-Dependent Protein Kinases
Ion Channels
Adenylyl Cyclases
Inhibitory Concentration 50
Phosphorylation
Cholinergic Agonists
Pertussis Toxin
Sarcoplasmic Reticulum
Phosphoric Monoester Hydrolases
Cholinergic Agents
Potassium

Keywords

  • Ion channels
  • Protein kinase A phosphorylation
  • Signal transduction
  • Submembrane Ca release

ASJC Scopus subject areas

  • Physiology
  • Cardiology and Cardiovascular Medicine
  • Physiology (medical)

Cite this

Cholinergic receptor signaling modulates spontaneous firing of sinoatrial nodal cells via integrated effects on PKA-dependent Ca2+ cycling and IKACh. / Lyashkov, Alexey E.; Vinogradova, Tatiana M.; Zahanich, Ihor; Li, Yue; Younes, Antoine; Nuss, H. Bradley; Spurgeon, Harold A.; Maltsev, Victor A.; Lakatta, Edward.

In: American Journal of Physiology - Heart and Circulatory Physiology, Vol. 297, No. 3, 09.2009.

Research output: Contribution to journalArticle

Lyashkov, Alexey E. ; Vinogradova, Tatiana M. ; Zahanich, Ihor ; Li, Yue ; Younes, Antoine ; Nuss, H. Bradley ; Spurgeon, Harold A. ; Maltsev, Victor A. ; Lakatta, Edward. / Cholinergic receptor signaling modulates spontaneous firing of sinoatrial nodal cells via integrated effects on PKA-dependent Ca2+ cycling and IKACh. In: American Journal of Physiology - Heart and Circulatory Physiology. 2009 ; Vol. 297, No. 3.
@article{659ad005b1df4a968256d25ffb1e9303,
title = "Cholinergic receptor signaling modulates spontaneous firing of sinoatrial nodal cells via integrated effects on PKA-dependent Ca2+ cycling and IKACh",
abstract = "Prior studies indicate that cholinergic receptor (ChR) activation is linked to beating rate reduction (BRR) in sinoatrial nodal cells (SANC) via 1) a Gi-coupled reduction in adenylyl cyclase (AC) activity, leading to a reduction of cAMP or protein kinase A (PKA) modulation of hyperpolarization- activated current (If) or L-type Ca2+ currents (I Ca,L), respectively; and 2) direct Gi-coupled activation of ACh-activated potassium current (IKACh). 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 μ10 nM, half maximal inhibition (IC50) was achieved at 100 nM, and 1,000 nM stopped spontaneous beating. Gi inhibition by pertussis toxin blocked all CCh effects on BRR. Using specific ion channel blockers, we established that If blockade did not affect BRR at any [CCh] and that IKACh 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 ∼50{\%}. The dose response of BRR to CCh in the presence of IKACh 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 Gi activation and the extent of Gi coupling to Ca2+ cycling via PKA signaling or to IKACh: at low [CCh], IKACh 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 IKACh activation underlies a more pronounced BRR.",
keywords = "Ion channels, Protein kinase A phosphorylation, Signal transduction, Submembrane Ca release",
author = "Lyashkov, {Alexey E.} and Vinogradova, {Tatiana M.} and Ihor Zahanich and Yue Li and Antoine Younes and Nuss, {H. Bradley} and Spurgeon, {Harold A.} and Maltsev, {Victor A.} and Edward Lakatta",
year = "2009",
month = "9",
doi = "10.1152/ajpheart.01340.2008",
language = "English (US)",
volume = "297",
journal = "American Journal of Physiology",
issn = "0363-6135",
publisher = "American Physiological Society",
number = "3",

}

TY - JOUR

T1 - Cholinergic receptor signaling modulates spontaneous firing of sinoatrial nodal cells via integrated effects on PKA-dependent Ca2+ cycling and IKACh

AU - Lyashkov, Alexey E.

AU - Vinogradova, Tatiana M.

AU - Zahanich, Ihor

AU - Li, Yue

AU - Younes, Antoine

AU - Nuss, H. Bradley

AU - Spurgeon, Harold A.

AU - Maltsev, Victor A.

AU - Lakatta, Edward

PY - 2009/9

Y1 - 2009/9

N2 - Prior studies indicate that cholinergic receptor (ChR) activation is linked to beating rate reduction (BRR) in sinoatrial nodal cells (SANC) via 1) a Gi-coupled reduction in adenylyl cyclase (AC) activity, leading to a reduction of cAMP or protein kinase A (PKA) modulation of hyperpolarization- activated current (If) or L-type Ca2+ currents (I Ca,L), respectively; and 2) direct Gi-coupled activation of ACh-activated potassium current (IKACh). 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 μ10 nM, half maximal inhibition (IC50) was achieved at 100 nM, and 1,000 nM stopped spontaneous beating. Gi inhibition by pertussis toxin blocked all CCh effects on BRR. Using specific ion channel blockers, we established that If blockade did not affect BRR at any [CCh] and that IKACh 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 ∼50%. The dose response of BRR to CCh in the presence of IKACh 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 Gi activation and the extent of Gi coupling to Ca2+ cycling via PKA signaling or to IKACh: at low [CCh], IKACh 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 IKACh activation underlies a more pronounced BRR.

AB - Prior studies indicate that cholinergic receptor (ChR) activation is linked to beating rate reduction (BRR) in sinoatrial nodal cells (SANC) via 1) a Gi-coupled reduction in adenylyl cyclase (AC) activity, leading to a reduction of cAMP or protein kinase A (PKA) modulation of hyperpolarization- activated current (If) or L-type Ca2+ currents (I Ca,L), respectively; and 2) direct Gi-coupled activation of ACh-activated potassium current (IKACh). 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 μ10 nM, half maximal inhibition (IC50) was achieved at 100 nM, and 1,000 nM stopped spontaneous beating. Gi inhibition by pertussis toxin blocked all CCh effects on BRR. Using specific ion channel blockers, we established that If blockade did not affect BRR at any [CCh] and that IKACh 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 ∼50%. The dose response of BRR to CCh in the presence of IKACh 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 Gi activation and the extent of Gi coupling to Ca2+ cycling via PKA signaling or to IKACh: at low [CCh], IKACh 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 IKACh activation underlies a more pronounced BRR.

KW - Ion channels

KW - Protein kinase A phosphorylation

KW - Signal transduction

KW - Submembrane Ca release

UR - http://www.scopus.com/inward/record.url?scp=69249150907&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=69249150907&partnerID=8YFLogxK

U2 - 10.1152/ajpheart.01340.2008

DO - 10.1152/ajpheart.01340.2008

M3 - Article

VL - 297

JO - American Journal of Physiology

JF - American Journal of Physiology

SN - 0363-6135

IS - 3

ER -