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Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents
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Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

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Inhibition of cAMP-Dependent PKA Activates β2-Adrenergic Receptor Stimulation of Cytosolic Phospholipase A2 via

M R Pabbidi1, X Ji2, J T Maxwell2

  • 1Department of Pharmacology, University of Mississippi Medical Center, Jackson, MS, United States of America.

Plos One
|December 16, 2016
PubMed

Insights

Inhibition of protein kinase A (PKA) in atrial myocytes removes a block on Raf-1, enabling beta-2 adrenergic receptor (β2-AR) signaling via PKCα/Raf-1/MEK/ERK1/2 and IP3 to activate cPLA2 within caveolae.

Area of Science:

  • Cardiovascular Physiology
  • Cell Signaling
  • Molecular Pharmacology

Background:

  • Laminin-integrin signaling inhibits cAMP-dependent protein kinase (PKA), which normally activates cytosolic phospholipase A2 (cPLA2) downstream of beta-2 adrenergic receptor (β2-AR) stimulation in atrial myocytes.
  • Understanding the precise signaling cascade initiated by PKA inhibition is crucial for elucidating β2-AR pathway regulation.

Purpose of the Study:

  • To delineate the signaling mechanisms through which PKA inhibition potentiates β2-AR-mediated cPLA2 activation in atrial myocytes.
  • To investigate the roles of specific signaling molecules (Raf-1, PLC, PKC, IP3R, ERK1/2) and cellular structures (caveolae) in this pathway.

Main Methods:

  • Utilized atrial myocytes treated with a PKA inhibitor (KT5720) or laminin (LMN) to mimic PKA inhibition.
  • Employed pharmacological inhibitors for Raf-1, PLC, PKC, and IP3 receptors, alongside Western blotting for ERK1/2 phosphorylation.
  • Investigated signaling pathways using adenoviral vectors expressing dominant-negative constructs and wild-type/mutant cPLA2.
  • Assessed the role of intracellular calcium stores and caveolae disruption using thapsigargin and methyl-β-cyclodextrin, respectively.

Main Results:

  • β2-AR stimulation of ICa,L was significantly inhibited by blocking Raf-1, PLC, PKC, or IP3R signaling in PKA-inhibited myocytes, but not in PKA-active myocytes.
  • β2-AR stimulation increased ERK1/2 phosphorylation in PKA-inhibited myocytes.
  • Overexpression of wild-type cPLA2 enhanced β2-AR-stimulated ICa,L, while a double mutant (cPLA2S505A/S515A) inhibited it.
  • Disruption of caveolae significantly inhibited β2-AR-stimulated ICa,L in PKA-inhibited myocytes more than in PKA-active myocytes.

Conclusions:

  • PKA inhibition removes a negative regulation on Raf-1, allowing β2-AR stimulation to proceed via a PKCα/Raf-1/MEK/ERK1/2 and IP3-mediated pathway to activate cPLA2 within caveolae.
  • These findings offer insights into the altered β-AR signaling observed in aging or failing hearts, characterized by reduced adenylate cyclase activity.

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