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. 2016 Dec 15;11(12):e0168505.
doi: 10.1371/journal.pone.0168505. eCollection 2016.

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

M R Pabbidi  1 , X Ji  2 , J T Maxwell  2 , G A Mignery  2 , A M Samarel  3 , S L Lipsius  2
Affiliations

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

M R Pabbidi et al. PLoS One. .

Abstract

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) of the PKA inhibitor (1 μM) KT5720 and compared these results with atrial myocytes attached to laminin (+LMN). Inhibition of Raf-1 (10 μM GW5074), phospholipase C (PLC; 0.5 μM edelfosine), PKC (4 μM chelerythrine) or IP3 receptor (IP3R) signaling (2 μM 2-APB) significantly inhibited zint-β2-AR stimulation of ICa,L in-PKA but not +PKA myocytes. Western blots showed that zint-β2-AR stimulation increased ERK1/2 phosphorylation in-PKA compared to +PKA myocytes. Adenoviral (Adv) expression of dominant negative (dn) -PKCα, dn-Raf-1 or an IP3 affinity trap, each inhibited zint-β2-AR stimulation of ICa,L in + LMN myocytes compared to control +LMN myocytes infected with Adv-βgal. In +LMN myocytes, zint-β2-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-βgal. In-PKA myocytes depletion of intracellular Ca2+ stores by 5 μM thapsigargin failed to inhibit zint-β2-AR stimulation of ICa,L via cPLA2. However, disruption of caveolae formation by 10 mM methyl-β-cyclodextrin inhibited zint-β2-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 β2-AR stimulation to act via PKCα/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 β-AR signaling in failing and/or aging heart, both of which exhibit decreases in adenylate cyclase activity.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1
Effects of Raf-1 inhibition (10 μM GW5074) on zint-β2-AR stimulation of ICa,L in +PKA (A), -PKA (B) atrial myocytes. A; In +PKA myocytes, GW5074 (30 min) had no significant effect on zint-β2-AR stimulation of ICa,L. B; In -PKA myocytes, zint-β2-AR stimulation of ICa,L was enhanced compared to control responses (A) and GW5074 significantly inhibited zint-β2-AR stimulation of ICa,L. Numbers in parentheses indicate the number of myocytes studied. * = P<0.05.
Fig 2
Fig 2. Effects of dn-Raf-1-Adv on zint-β2-AR stimulation of ICa,L in +LMN myocytes.
In control +LMN myocytes (βgal) zint-β2-AR stimulation elicited a typically enhanced increase in ICa,L. In +LMN myocytes infected with dn-Raf-1-Adv zint-β2-AR stimulation of ICa,L was significantly inhibited compared with controls. Numbers in parentheses indicate that number of cells studied. * = P<0.05.
Fig 3
Fig 3. Effect of PKA inhibition on β2-AR-medited phosphorylation of ERK1/2.
Western blots of atrial cell homogenates show that in control -PKA myocytes zinterol modestly increased ERK1/2 phosphorylation (solid bars). Inhibition of PKA by 1 μM KT5720 (KT) increased basal ERK/1/2 phosphorylation. In the presence of KT5720, zinterol prominently stimulates ERK1/2 phosphorylation. Zinterol only modestly stimulated p38MAPK phosphorylation (open bars), and without significant effect by KT5720. The last lane on the Western blot is a positive control showing ERK1/2 and p38MAPK phosphorylation in A7r5 cells stimulated by 1 μM angiotensin II (5 min). Similar results were obtained in a total of 3 experiments. * = P<0.05.
Fig 4
Fig 4
Effects of 0.5 μM edelfosine on zint-β2-AR stimulation of ICa,L in +PKA (A), -PKA(B) and atrial myocytes. A; in control +PKA myocytes edelfosine had no significant effects on zint-β2-AR stimulation of ICa,L. B; in -PKA myocytes zint-β2-AR stimulation elicited a typically enhanced increase in ICa,L compared to controls (A) and edelfosine significantly inhibited zint-β2-AR stimulation of ICa,L. Numbers in parentheses indicate the number of myocytes studied. * = P<0.05.
Fig 5
Fig 5
Effects of 4 μM chelerythrine (cheler) on zint-β2-AR stimulation of ICa,L in +PKA(A) and -PKA (B) myocytes and effects of dn-PKCα-Adv (C) on zint-β2-AR stimulation of ICa,L in +LMN myocytes. A; in control +PKA myocytes, chelerythrine had no significant effects on zint-β2-AR stimulation of ICa,L. B; in—PKA myocytes, zint-β2-AR stimulation elicited a typically enhanced in increase in ICa,L compared to controls (A) and chelerythrine significantly inhibited zint-β2-AR stimulation of ICa,L. C; in control +LMN myocytes (βgal) zint-β2-AR stimulation of ICa,L was typically enhanced compared to controls (A). In +LMN myocytes infected with dn-PKCα-Adv, zint-β2-AR stimulation of ICa,L was significantly inhibited. Numbers in parentheses indicate that number of cells studied. * = P<0.05.
Fig 6
Fig 6. Effects of dn-cPLA2S515A/S505A and wt-cPLA2 on zint-β2-AR stimulation of ICa,L in +LMN myocytes.
Compared to control +LMN myocytes (βgal), zint-β2-AR stimulation of ICa,L was significantly inhibited and enhanced in myocytes overexpressing dn-cPLA2S515A/S505A and expressing wt-cPLA2, respectively. Numbers in parentheses indicate that number of cells studied. * = P<0.05.
Fig 7
Fig 7
Effects of 2 μM 2-APB on zint-β2-AR stimulation of ICa,L in +PKA (A), -PKA atrial myocytes. A; in control +PKA myocytes, 2-APB had no significant effects on zint-β2-AR stimulation of ICa,L. B; in -PKA myocytes, zint-β2-AR stimulation elicited a typically enhanced increase in ICa,L compared to controls (A) and 2-APB significantly inhibited zint-β2-AR stimulation of ICa,L. Numbers in parentheses indicate the number of myocytes studied. * = P<0.05.
Fig 8
Fig 8. Effect of adenovirus IP3 affinity trap on zint-β2-AR stimulation of ICa,L in +LMN myocytes.
Compared with control +LMN myocytes (βgal), zint-β2-AR stimulation of ICa,L was significantly inhibited in +LMN myocytes infected with adenovirus expressing the IP3 affinity trap. Numbers in parentheses indicate the number of myocytes studied. * = P<0.05.
Fig 9
Fig 9
Effects of 5 μM thapsigargin (10 min; Thaps) on zint-β2-AR stimulation of ICa,L in +PKA(A) and -PKA (B) myocytes. A; in +PKA myocytes, compared to control responses, thapsigargin slightly enhanced zint-β2-AR stimulation of ICa,L. B; in -PKA myocytes, zint-β2-AR stimulation elicited a typically enhanced increase in ICa,L compared to control (A) that was not prevented by treatment with thapsigargin. The addition of 10 μM AACOCF3 (+ thaps) significantly inhibited zint-β2-AR stimulation of ICa,L indicating that thapsigargin did not prevent zint-β2-AR stimulation of cPLA2. Numbers in parentheses indicate the number of myocytes studied. * = P<0.05.
Fig 10
Fig 10
Effect of 2 mM methyl-β-cyclodextrin (MCD; 30 min) on zint-β2-AR stimulation of ICa,L in +PKA (A) and -PKA (B) myocytes. A; in +PKA myocytes, MCD significantly decreased zint-β2-AR stimulation of ICa,L compared to control responses (-24%). B; in—PKA myocytes, zint-β2-AR stimulation elicited a typically enhanced increase in ICa,L compared to +PKA myocytes (A) and the effect of MCD to decrease zint-β2-AR stimulation of ICa,L was enhanced (-77%) compared to +PKA myocytes (A). Numbers in parentheses indicate the number of myocytes studied. * = P<0.05.
Fig 11
Fig 11
Schematic summary showing the proposed signaling mechanisms underlying zint-β2-AR stimulation of ICa,L in +PKA (A), -PKA (B) atrial myocytes. A; in +PKA myocytes, zint-β2-AR stimulation acts via Gs to activate adenylate cyclase (AC)/cAMP-dependent kinase (PKA) which in turn stimulates ICa,L. Both basal and stimulated PKA activity inhibits Raf-1 signaling, thereby preventing zint-β2-AR stimulation of ICa,L via cPLA2. B; in cells not attached to LMN, inhibition of PKA by KT5720 removes inhibition of Raf-1. C; cell attachment to LMN acts via β1 integrins and FAK/PI-(3)K/Akt signaling to inhibit adenylate cyclase (AC)/cAMP-dependent kinase (PKA) activity, thereby removing inhibition of Raf-1. β2-AR stimulation acts via Gi to stimulate PLC leading to activation of PKCα and IP3R-mediated Ca2+ signaling within caveolae. With PKA inhibited, PKCα stimulates Raf-1/MEK/ERK1/2 signaling. Together, ERK1/2 and IP3-mediated Ca2+ signaling activate cPLA/AA, resulting in stimulation of ICa,L.
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