Dataset Information

Inhibition of cAMP-Dependent PKA Activates ?2-Adrenergic Receptor Stimulation of Cytosolic Phospholipase A2 via Raf-1/MEK/ERK and IP3-Dependent Ca2+ Signaling in Atrial Myocytes.

ABSTRACT: We previously reported in atrial myocytes that inhibition of cAMP-dependent protein kinase (PKA) by laminin (LMN)-integrin signaling activates ?2-adrenergic receptor (?2-AR) stimulation of cytosolic phospholipase A2 (cPLA2). The present study sought to determine the signaling mechanisms by which inhibition of PKA activates ?2-AR stimulation of cPLA2. We therefore determined the effects of zinterol (0.1 ?M; zint-?2-AR) to stimulate ICa,L in atrial myocytes in the absence (+PKA) and presence (-PKA) of the PKA inhibitor (1 ?M) KT5720 and compared these results with atrial myocytes attached to laminin (+LMN). Inhibition of Raf-1 (10 ?M GW5074), phospholipase C (PLC; 0.5 ?M edelfosine), PKC (4 ?M chelerythrine) or IP3 receptor (IP3R) signaling (2 ?M 2-APB) significantly inhibited zint-?2-AR stimulation of ICa,L in-PKA but not +PKA myocytes. Western blots showed that zint-?2-AR stimulation increased ERK1/2 phosphorylation in-PKA compared to +PKA myocytes. Adenoviral (Adv) expression of dominant negative (dn) -PKC?, dn-Raf-1 or an IP3 affinity trap, each inhibited zint-?2-AR stimulation of ICa,L in + LMN myocytes compared to control +LMN myocytes infected with Adv-?gal. In +LMN myocytes, zint-?2-AR stimulation of ICa,L was enhanced by adenoviral overexpression of wild-type cPLA2 and inhibited by double dn-cPLA2S505A/S515A mutant compared to control +LMN myocytes infected with Adv-?gal. In-PKA myocytes depletion of intracellular Ca2+ stores by 5 ?M thapsigargin failed to inhibit zint-?2-AR stimulation of ICa,L via cPLA2. However, disruption of caveolae formation by 10 mM methyl-?-cyclodextrin inhibited zint-?2-AR stimulation of ICa,L in-PKA myocytes significantly more than in +PKA myocytes. We conclude that inhibition of PKA removes inhibition of Raf-1 and thereby allows ?2-AR stimulation to act via PKC?/Raf-1/MEK/ERK1/2 and IP3-mediated Ca2+ signaling to stimulate cPLA2 signaling within caveolae. These findings may be relevant to the remodeling of ?-AR signaling in failing and/or aging heart, both of which exhibit decreases in adenylate cyclase activity.

SUBMITTER: Pabbidi MR

PROVIDER: S-EPMC5158063 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Inhibition of cAMP-Dependent PKA Activates β2-Adrenergic Receptor Stimulation of Cytosolic Phospholipase A2 via Raf-1/MEK/ERK and IP3-Dependent Ca2+ Signaling in Atrial Myocytes.

Pabbidi M R MR Ji X X Maxwell J T JT Mignery G A GA Samarel A M AM Lipsius S L SL

PloS one 20161215 12

We previously reported in atrial myocytes that inhibition of cAMP-dependent protein kinase (PKA) by laminin (LMN)-integrin signaling activates β2-adrenergic receptor (β2-AR) stimulation of cytosolic phospholipase A2 (cPLA2). The present study sought to determine the signaling mechanisms by which inhibition of PKA activates β2-AR stimulation of cPLA2. We therefore determined the effects of zinterol (0.1 μM; zint-β2-AR) to stimulate ICa,L in atrial myocytes in the absence (+PKA) and presence (-PKA ...[more]

PMID: 27977772

Similar Datasets

Project description:Mutations in polycystins are a cause of polycystic liver disease. In polycystin-2 (PC2)-defective mice, cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)-dependent activation of the Rat Sarcoma (Ras)/rapidly accelerated fibrosarcoma (Raf)/mitogen signal-regulated kinase-extracellular signal-regulated kinase (ERK) 1/2 pathway stimulates the growth of liver cysts. To test the hypothesis that sorafenib, a Raf inhibitor used for the treatment of liver and kidney cancers, inhibits liver cyst growth in PC2-defective mice, we treated PC2 (i.e., Pkd2(flox/-) :pCxCreER(TM) [Pkd2cKO]) mice with sorafenib-tosylate for 8 weeks (20-60 mg/kg/day). Sorafenib caused an unexpected increase in liver cyst area, cell proliferation (Ki67), and expression of phosphorylated ERK (pERK) compared with Pkd2cKO mice treated with vehicle. When given to epithelial cells isolated from liver cysts of Pkd2cKO mice (Pkd2cKO-cells), sorafenib progressively stimulated pERK1/2 and cell proliferation [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and bromodeoxyuridine assay (MTS)] at doses between 0.001 and 1 ?M; however, both pERK1/2 and cell proliferation significantly decreased at the dose of 10 ?M. Raf kinase activity assay showed that whereas B-Raf is inhibited by sorafenib in both wild-type (WT) and Pkd2cKO cells, Raf-1 is inhibited in WT cells but is significantly stimulated in Pkd2cKO cells. In Pkd2cKO cells pretreated with the PKA inhibitor 14-22 amide, myristolated (1 ?M) and in mice treated with octreotide in combination with sorafenib, the paradoxical activation of Raf/ERK1/2 was abolished, and cyst growth was inhibited.In PC2-defective cells, sorafenib inhibits B-Raf but paradoxically activates Raf-1, resulting in increased ERK1/2 phosphorylation, cell proliferation, and cyst growth in vivo. These effects are consistent with the ability of Raf inhibitors to transactivate Raf-1 when a PKA-activated Ras promotes Raf-1/B-Raf heterodimerization, and are inhibited by interfering with cAMP/PKA signaling both in vitro and in vivo, as shown by the reduction of liver cysts in mice treated with combined octreotide and sorafenib.

Project description:AimsCardiomyocyte β2-adrenergic receptor (β2AR) cyclic adenosine monophosphate (cAMP) signalling is regulated by the receptors' subcellular location within transverse tubules (T-tubules), via interaction with structural and regulatory proteins, which form a signalosome. In chronic heart failure (HF), β2ARs redistribute from T-tubules to the cell surface, which disrupts functional signalosomes and leads to diffuse cAMP signalling. However, the functional consequences of structural changes upon β2AR-cAMP signalling during progression from hypertrophy to advanced HF are unknown.Methods and resultsRat left ventricular myocytes were isolated at 4-, 8-, and 16-week post-myocardial infarction (MI), β2ARs were stimulated either via whole-cell perfusion or locally through the nanopipette of the scanning ion conductance microscope. cAMP release was measured via a Förster Resonance Energy Transfer-based sensor Epac2-camps. Confocal imaging of di-8-ANNEPS-stained cells and immunoblotting were used to determine structural alterations. At 4-week post-MI, T-tubule regularity, density and junctophilin-2 (JPH2) expression were significantly decreased. The amplitude of local β2AR-mediated cAMP in T-tubules was reduced and cAMP diffused throughout the cytosol instead of being locally confined. This was accompanied by partial caveolin-3 (Cav-3) dissociation from the membrane. At 8-week post-MI, the β2AR-mediated cAMP response was observed at the T-tubules and the sarcolemma (crest). Finally, at 16-week post-MI, the whole cell β2AR-mediated cAMP signal was depressed due to adenylate cyclase dysfunction, while overall Cav-3 levels were significantly increased and a substantial portion of Cav-3 dissociated into the cytosol. Overexpression of JPH2 in failing cells in vitro or AAV9.SERCA2a gene therapy in vivo did not improve β2AR-mediated signal compartmentation or reduce cAMP diffusion.ConclusionAlthough changes in T-tubule structure and β2AR-mediated cAMP signalling are significant even at 4-week post-MI, progression to the HF phenotype is not linear. At 8-week post-MI the loss of β2AR-mediated cAMP is temporarily reversed. Complete disorganization of β2AR-mediated cAMP signalling due to changes in functional receptor localization and cellular structure occurs at 16-week post-MI.

Dataset Information

Inhibition of cAMP-Dependent PKA Activates ?2-Adrenergic Receptor Stimulation of Cytosolic Phospholipase A2 via Raf-1/MEK/ERK and IP3-Dependent Ca2+ Signaling in Atrial Myocytes.

Publications

Inhibition of cAMP-Dependent PKA Activates β2-Adrenergic Receptor Stimulation of Cytosolic Phospholipase A2 via Raf-1/MEK/ERK and IP3-Dependent Ca2+ Signaling in Atrial Myocytes.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets