Abstract
Recently, we identified a novel signaling pathway involving Epac, Rap, and phospholipase C (PLC)epsilon that plays a critical role in maximal beta-adrenergic receptor (betaAR) stimulation of Ca2+-induced Ca2+ release (CICR) in cardiac myocytes. Here we demonstrate that PLCepsilon phosphatidylinositol 4,5-bisphosphate hydrolytic activity and PLCepsilon-stimulated Rap1 GEF activity are both required for PLCepsilon-mediated enhancement of sarcoplasmic reticulum Ca2+ release and that PLCepsilon significantly enhances Rap activation in response to betaAR stimulation in the heart. Downstream of PLCepsilon hydrolytic activity, pharmacological inhibition of PKC significantly inhibited both betaAR- and Epac-stimulated increases in CICR in PLCepsilon+/+ myocytes but had no effect in PLCepsilon-/- myocytes. betaAR and Epac activation caused membrane translocation of PKCepsilon in PLCepsilon+/+ but not PLCepsilon-/- myocytes and small interfering RNA-mediated PKCepsilon knockdown significantly inhibited both betaAR and Epac-mediated CICR enhancement. Further downstream, the Ca2+/calmodulin-dependent protein kinase II (CamKII) inhibitor, KN93, inhibited betaAR- and Epac-mediated CICR in PLCepsilon+/+ but not PLCepsilon-/- myocytes. Epac activation increased CamKII Thr286 phosphorylation and enhanced phosphorylation at CamKII phosphorylation sites on the ryanodine receptor (RyR2) (Ser2815) and phospholamban (Thr17) in a PKC-dependent manner. Perforated patch clamp experiments revealed that basal and betaAR-stimulated peak L-type current density are similar in PLCepsilon+/+ and PLCepsilon-/- myocytes suggesting that control of sarcoplasmic reticulum Ca2+ release, rather than Ca2+ influx through L-type Ca2+ channels, is the target of regulation of a novel signal transduction pathway involving sequential activation of Epac, PLCepsilon, PKCepsilon, and CamKII downstream of betaAR activation.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Benzylamines / pharmacology
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Calcium / metabolism*
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Calcium Channels, L-Type / genetics
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Calcium Channels, L-Type / metabolism
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Calcium Signaling / physiology*
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Calcium-Binding Proteins / genetics
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Calcium-Binding Proteins / metabolism
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Calcium-Calmodulin-Dependent Protein Kinase Type 2 / antagonists & inhibitors
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Calcium-Calmodulin-Dependent Protein Kinase Type 2 / genetics
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Calcium-Calmodulin-Dependent Protein Kinase Type 2 / metabolism*
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Cell Membrane / enzymology
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Cell Membrane / genetics
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Enzyme Activation / physiology
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Guanine Nucleotide Exchange Factors / genetics
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Guanine Nucleotide Exchange Factors / metabolism*
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Mice
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Mice, Knockout
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Myocardium / cytology
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Myocardium / enzymology*
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Myocytes, Cardiac / cytology
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Myocytes, Cardiac / enzymology
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Phosphatidylinositol 4,5-Diphosphate
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Phosphoinositide Phospholipase C / genetics
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Phosphoinositide Phospholipase C / metabolism*
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Phosphorylation / physiology
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Protein Kinase C-epsilon / genetics
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Protein Kinase C-epsilon / metabolism
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Protein Kinase Inhibitors / pharmacology
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Protein Transport / physiology
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Receptors, Adrenergic, beta / genetics
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Receptors, Adrenergic, beta / metabolism
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Ryanodine Receptor Calcium Release Channel / genetics
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Ryanodine Receptor Calcium Release Channel / metabolism
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Sarcoplasmic Reticulum / enzymology
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Sarcoplasmic Reticulum / genetics
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Sulfonamides / pharmacology
Substances
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Benzylamines
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Calcium Channels, L-Type
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Calcium-Binding Proteins
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Epac protein, mouse
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Guanine Nucleotide Exchange Factors
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Phosphatidylinositol 4,5-Diphosphate
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Protein Kinase Inhibitors
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Receptors, Adrenergic, beta
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Ryanodine Receptor Calcium Release Channel
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Sulfonamides
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phospholamban
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KN 93
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Prkce protein, mouse
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Protein Kinase C-epsilon
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Calcium-Calmodulin-Dependent Protein Kinase Type 2
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Phosphoinositide Phospholipase C
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phospholipase C epsilon
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Calcium