Numbers in parentheses indicate the number of myocytes studied
Numbers in parentheses indicate the number of myocytes studied. 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 inCPKA but not +PKA myocytes. Western blots showed that zint-2-AR stimulation increased ERK1/2 phosphorylation inCPKA 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. InCPKA 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 arousal of ICa,L inCPKA myocytes a lot more than in +PKA myocytes significantly. We conclude that inhibition of PKA gets rid of inhibition of Raf-1 and thus allows 2-AR arousal to do something via PKC/Raf-1/MEK/ERK1/2 and IP3-mediated Ca2+ signaling to stimulate cPLA2 signaling within caveolae. These results may be highly relevant to the redecorating of -AR signaling in declining and/or maturing center, both which display reduces in adenylate cyclase activity. Launch We previously reported that connection of atrial myocytes towards the extracellular matrix proteins laminin (LMN) works via 1 integrin receptors to diminish 1-adrenergic receptor (AR) and boost 2-AR arousal of L-type Ca2+ current (ICa,L) [1]. Cell connection to LMN reduces 1-AR signaling by inhibiting adenylate cyclase activity and diminishing cAMP amounts via integrin-dependent activation of focal adhesion kinase (FAK)/phosphatidyinositol-3 kinase (PI-3K)/proteins kinase B (Akt) signaling [2]. We also reported that atrial cell connection to LMN enhances 2-AR signaling by activating Gi/ERK/cytosolic phospholipase A2 (cPLA2)/arachidonic acidity (AA) arousal of ICa,L [3]. 2-AR activation of cPLA2 signaling would depend on concomitant LMN-mediated inhibition of adenylate cyclase/cAMP-dependent kinase (PKA) [3]. Quite simply, cell connection to LMN serves via inhibition of adenylate cyclase/PKA to both inhibit 1-AR signaling and enhance 2-AR signaling through activation of cPLA2. In embryonic chick ventricular myocytes [4] and rat ventricular myocytes [5] 2-AR arousal also activates cPLA2/AA signaling. Furthermore, these authors proposed that activation of 2-AR/cPLA2 signaling might compensate for despondent cAMP signaling [4]. Interestingly, in both these scholarly tests by Pavoine et al. (1999) and Ait-Mamar et al., (2005) cardiomyocytes had been cultured on LMN, helping our results that cell connection to LMN could be in charge of inhibition of PKA and activation of 2-AR/cPLA2 signaling. Nevertheless, the mechanism where PKA inhibition activates 2-AR/cPLA2 signaling isn’t clear. Our preliminary tests indicated that in atrial myocytes 2-AR activation of cPLA2 is normally Ca2+-reliant and mediated via ERK1/2 signaling [3]. That is consistent with research in embryonic chick ventricular myocytes (cultured on LMN) where 2-AR stimulation serves via ERK1/2 signaling to activate cPLA2 [6]. Furthermore, in a number of cell systems Raf-1 activates downstream PKA and ERK1/2 inhibits Raf-1 [7, 8]. As a result, inhibition of PKA should remove inhibition of Raf-1, enabling 2-AR arousal to do something via Raf-1/MEK/ERK1/2 signaling thereby. Moreover, proteins kinase C (PKC) activates Raf-1 [9, 10]. In.Particular binding was visualized by chemiluminescence (Immun-Star American C Package, Bio-Rad) utilizing a ChemiDoc XRS imager (Bio-Rad Lifestyle Science Analysis, Hercules, CA). (2-AR) arousal of cytosolic phospholipase A2 (cPLA2). Today’s study sought to look for the signaling systems where inhibition of PKA activates 2-AR arousal of cPLA2. We as a result determined the consequences of zinterol (0.1 M; zint-2-AR) to stimulate ICa,L in atrial myocytes in the lack (+PKA) and existence (-PKA) from the PKA inhibitor (1 M) KT5720 and compared these outcomes with atrial myocytes mounted on 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 inCPKA however, not +PKA myocytes. Traditional western blots demonstrated that zint-2-AR arousal elevated ERK1/2 phosphorylation inCPKA in comparison to +PKA myocytes. Adenoviral (Adv) appearance of dominant detrimental (dn) -PKC, dn-Raf-1 or an IP3 affinity snare, each inhibited zint-2-AR arousal of ICa,L in + LMN myocytes in comparison to control +LMN myocytes contaminated with Adv-gal. In +LMN myocytes, zint-2-AR arousal of ICa,L was improved by adenoviral overexpression of wild-type cPLA2 and Doxercalciferol inhibited by dual dn-cPLA2S505A/S515A mutant in comparison to control +LMN myocytes contaminated with Adv-gal. InCPKA myocytes depletion of intracellular Ca2+ shops by 5 M thapsigargin didn’t inhibit zint-2-AR arousal of ICa,L via cPLA2. Nevertheless, disruption of caveolae development by 10 mM methyl–cyclodextrin inhibited zint-2-AR arousal of ICa,L inCPKA myocytes more than in +PKA myocytes. We conclude that inhibition of PKA gets rid of inhibition of Raf-1 and thus allows 2-AR arousal to do something via PKC/Raf-1/MEK/ERK1/2 and IP3-mediated Ca2+ signaling to stimulate cPLA2 signaling within caveolae. These results could be highly relevant to the redecorating of -AR signaling in declining and/or aging center, both which display reduces in adenylate cyclase activity. Launch We previously reported that connection of atrial myocytes towards Doxercalciferol the extracellular matrix proteins laminin (LMN) works via 1 integrin receptors to diminish 1-adrenergic receptor (AR) and boost 2-AR arousal of L-type Ca2+ current (ICa,L) [1]. Cell connection to LMN reduces 1-AR signaling by inhibiting adenylate cyclase activity and diminishing cAMP amounts via integrin-dependent activation of focal adhesion kinase (FAK)/phosphatidyinositol-3 kinase (PI-3K)/proteins kinase B (Akt) signaling [2]. We also reported that atrial cell connection to LMN enhances 2-AR signaling by activating Gi/ERK/cytosolic phospholipase A2 (cPLA2)/arachidonic acidity (AA) arousal of ICa,L [3]. 2-AR activation of cPLA2 signaling would depend on concomitant LMN-mediated inhibition of adenylate cyclase/cAMP-dependent kinase (PKA) [3]. Quite simply, cell connection to LMN serves via inhibition of adenylate cyclase/PKA to both inhibit 1-AR signaling and enhance 2-AR signaling through activation of cPLA2. In embryonic chick ventricular myocytes [4] and rat ventricular myocytes [5] 2-AR arousal also activates cPLA2/AA signaling. Furthermore, these authors proposed that activation of 2-AR/cPLA2 signaling may compensate for stressed out cAMP signaling [4]. Interestingly, in both of these studies by Pavoine et al. (1999) and Ait-Mamar et al., (2005) cardiomyocytes were cultured on LMN, supporting our findings that cell attachment to LMN may be responsible for inhibition of PKA and activation of 2-AR/cPLA2 signaling. However, the mechanism by which PKA inhibition activates 2-AR/cPLA2 signaling is not clear. Our initial experiments indicated that in atrial myocytes 2-AR activation of cPLA2 is usually Ca2+-dependent and mediated via ERK1/2 signaling [3]. This is consistent with studies in embryonic chick ventricular myocytes (cultured on LMN) in which 2-AR stimulation functions via ERK1/2 signaling to activate cPLA2 [6]. Moreover, in a variety of cell systems Raf-1 activates downstream ERK1/2 and PKA.* = P 0.05. In another approach we infected atrial myocyte with an Adv expressing a dominant-negative mutant of Ca2+-dependent PKC (dn-PKC). activation elicited a typically enhanced increase in ICa, L and 2-APB significantly inhibited zint-2-AR activation of ICa,L. Figures in parentheses show the number of myocytes analyzed. Doxercalciferol * = P 0.01.(TIF) pone.0168505.s003.tif (154K) GUID:?0331C13A-6F10-467C-ADA9-7A363DDDD249 Data Availability StatementAll relevant data are within the paper and its Supporting Information files. 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) activation of cytosolic phospholipase A2 (cPLA2). The present study sought to determine the signaling mechanisms by which inhibition of PKA activates 2-AR activation 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 inCPKA but not +PKA myocytes. Western blots showed that zint-2-AR activation increased ERK1/2 phosphorylation inCPKA compared to +PKA myocytes. Adenoviral (Adv) expression of dominant unfavorable (dn) -PKC, dn-Raf-1 or an IP3 affinity trap, each inhibited zint-2-AR activation of ICa,L in + LMN myocytes compared to control +LMN myocytes infected with Doxercalciferol Adv-gal. In +LMN myocytes, zint-2-AR activation 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. InCPKA myocytes depletion Doxercalciferol of intracellular Ca2+ stores by 5 M thapsigargin failed to inhibit zint-2-AR activation of ICa,L via cPLA2. However, disruption of caveolae formation by 10 mM methyl–cyclodextrin inhibited zint-2-AR activation of ICa,L inCPKA myocytes significantly more than in +PKA myocytes. We conclude that inhibition of PKA removes inhibition of Raf-1 and thereby allows 2-AR activation 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. Introduction We previously reported that attachment of atrial myocytes to the extracellular matrix protein laminin (LMN) acts via 1 integrin receptors to decrease 1-adrenergic receptor (AR) and increase 2-AR activation of L-type Ca2+ current (ICa,L) [1]. Cell attachment to LMN decreases 1-AR signaling by inhibiting adenylate cyclase activity and diminishing cAMP levels via integrin-dependent activation of focal adhesion kinase (FAK)/phosphatidyinositol-3 kinase (PI-3K)/protein kinase B (Akt) signaling [2]. We also reported that atrial cell attachment to LMN enhances 2-AR signaling by activating Gi/ERK/cytosolic phospholipase A2 (cPLA2)/arachidonic acid (AA) activation of ICa,L [3]. 2-AR activation of cPLA2 signaling is dependent on concomitant LMN-mediated inhibition of adenylate cyclase/cAMP-dependent kinase (PKA) [3]. In other words, cell attachment to LMN functions via inhibition of adenylate cyclase/PKA to both inhibit 1-AR signaling and enhance 2-AR signaling through activation of cPLA2. In embryonic chick ventricular myocytes [4] and rat ventricular myocytes [5] 2-AR activation also activates cPLA2/AA signaling. Moreover, these authors proposed that activation of 2-AR/cPLA2 signaling may compensate for stressed out cAMP signaling [4]. Interestingly, in both of these studies by Pavoine et al. (1999) and Ait-Mamar et al., (2005) cardiomyocytes were cultured on LMN, supporting our findings that cell attachment to LMN may be responsible for inhibition of PKA and activation of 2-AR/cPLA2 signaling. However, the mechanism by which PKA inhibition activates 2-AR/cPLA2 signaling is not clear. Our initial experiments indicated that in atrial myocytes 2-AR activation of cPLA2 is usually Ca2+-dependent and mediated via ERK1/2 signaling [3]. This is consistent with studies in embryonic chick ventricular myocytes (cultured on LMN) in which 2-AR stimulation functions via ERK1/2 signaling to activate cPLA2 [6]. Moreover, in a variety of cell systems Raf-1 activates downstream ERK1/2 and PKA inhibits Raf-1 [7, 8]. Therefore, inhibition of PKA should remove inhibition of Raf-1, thereby allowing 2-AR activation to act via Raf-1/MEK/ERK1/2 signaling. Moreover, protein kinase C (PKC) activates Raf-1 [9, 10]. In other words, PKA inhibits and PKC activates Raf-1/MEK/ERK1/2 signaling. Based on these considerations we sought to determine whether inhibition of PKA facilitates 2-AR stimulation to act via PKC/Raf-1/MEK/ERK1/2 to activate cPLA2. These findings may be relevant to the remodeling of 2-AR signaling in the failing and/or aging heart, both of which.InCPKA myocytes, GW5074 had no effects on basal ICa,L amplitudes (open bars) but in contrast to +PKA myocytes, GW5074 significantly inhibited zint-2-AR stimulation of ICa,L (B; 405%, N = 3). 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 inCPKA but not +PKA myocytes. Western blots showed that zint-2-AR stimulation increased ERK1/2 phosphorylation inCPKA compared to +PKA myocytes. Adenoviral (Adv) expression of dominant unfavorable (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. InCPKA 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 inCPKA 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. Introduction We previously reported that attachment of atrial myocytes to the extracellular matrix protein laminin (LMN) acts via 1 integrin receptors to decrease 1-adrenergic receptor (AR) and increase 2-AR stimulation of L-type Ca2+ current (ICa,L) [1]. Cell attachment to LMN decreases 1-AR signaling by inhibiting adenylate cyclase activity and diminishing cAMP levels via integrin-dependent activation of focal adhesion kinase (FAK)/phosphatidyinositol-3 kinase (PI-3K)/protein kinase B (Akt) signaling [2]. We also reported that atrial cell attachment to LMN enhances 2-AR signaling by activating Gi/ERK/cytosolic phospholipase A2 (cPLA2)/arachidonic acid (AA) stimulation of ICa,L [3]. 2-AR activation of cPLA2 signaling is dependent on concomitant LMN-mediated inhibition of adenylate cyclase/cAMP-dependent kinase (PKA) [3]. In other words, cell attachment to LMN acts via inhibition of adenylate cyclase/PKA to both inhibit 1-AR signaling and enhance 2-AR signaling through activation of cPLA2. In embryonic chick ventricular myocytes [4] and rat ventricular myocytes [5] 2-AR stimulation also activates cPLA2/AA signaling. Moreover, these authors proposed that activation of 2-AR/cPLA2 signaling may compensate for depressed cAMP signaling [4]. Interestingly, in both of these studies by Pavoine et al. (1999) and Ait-Mamar et al., (2005) cardiomyocytes were cultured on LMN, supporting our findings that cell attachment to LMN may be responsible for inhibition of PKA and activation of 2-AR/cPLA2 signaling. However, the mechanism by which PKA inhibition activates 2-AR/cPLA2 signaling is not clear. Our initial experiments indicated that in atrial myocytes 2-AR activation of cPLA2 is usually Ca2+-dependent and mediated via ERK1/2 signaling [3]. This is consistent with studies in embryonic chick ventricular myocytes (cultured on LMN) in which 2-AR stimulation acts via ERK1/2 signaling to activate cPLA2 [6]. Moreover, in a variety of cell systems Raf-1 activates downstream ERK1/2 and PKA inhibits Raf-1 [7, 8]. Therefore, inhibition of PKA should remove inhibition of Raf-1, thereby allowing 2-AR stimulation to act via Raf-1/MEK/ERK1/2 signaling. Moreover, protein kinase C (PKC) activates Raf-1 [9, 10]. In other words, PKA inhibits and PKC activates Raf-1/MEK/ERK1/2 signaling. Based on these considerations we sought to determine whether inhibition of PKA facilitates 2-AR stimulation to act via PKC/Raf-1/MEK/ERK1/2 to activate cPLA2. These findings may be relevant to the remodeling of 2-AR signaling in the failing and/or aging heart, both of which exhibit decreases in adenylate cyclase activity. Materials and Methods Ethics Statement The animal and experimental protocols used in this study were approved by the Institutional Animal Care and Use Committee (IACUC) of Loyola University Medical Center, Maywood, IL. IACUC prescribed the rules for the animal care and supervised their enforcement. Animals were obtained from a licensed vendor.Multiple comparisons were performed by ANOVA followed by a StudentCNewmanCKeuls test with significance at P 0.05. Results Role of Raf-1 in +PKA and CPKA atrial myocytes As shown in Fig 1we determined the effects of 10 M GW5074, a potent Raf-1 inhibitor [8] on zint-2-AR stimulation of ICa,L in +PKA andCPKA atrial myocytes. 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 inCPKA but not +PKA myocytes. Western blots showed that zint-2-AR stimulation increased ERK1/2 phosphorylation inCPKA 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. InCPKA 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 inCPKA 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. Introduction We previously reported that attachment of atrial myocytes to the extracellular matrix protein laminin (LMN) acts via 1 integrin receptors to decrease 1-adrenergic receptor (AR) and increase 2-AR stimulation of L-type Ca2+ current (ICa,L) [1]. Cell attachment to LMN decreases 1-AR signaling by inhibiting adenylate cyclase activity and diminishing cAMP levels via integrin-dependent activation of focal adhesion kinase (FAK)/phosphatidyinositol-3 kinase (PI-3K)/protein kinase B (Akt) signaling [2]. We also reported that atrial cell attachment to LMN enhances 2-AR signaling by activating Gi/ERK/cytosolic phospholipase A2 (cPLA2)/arachidonic acid (AA) stimulation of ICa,L [3]. 2-AR activation of cPLA2 signaling is dependent on concomitant LMN-mediated inhibition of adenylate cyclase/cAMP-dependent kinase (PKA) [3]. In other words, cell attachment to LMN acts via inhibition of adenylate cyclase/PKA to both inhibit 1-AR signaling and enhance 2-AR signaling through activation of cPLA2. In embryonic chick ventricular myocytes [4] and rat ventricular myocytes [5] 2-AR stimulation also activates cPLA2/AA signaling. Moreover, these authors proposed that activation of 2-AR/cPLA2 signaling may compensate for depressed cAMP signaling [4]. Interestingly, in both of these studies by Pavoine et al. (1999) and Ait-Mamar et al., (2005) cardiomyocytes were cultured on LMN, supporting our findings that cell attachment to LMN may be responsible for inhibition of PKA and activation of 2-AR/cPLA2 signaling. However, the mechanism by which PKA inhibition activates 2-AR/cPLA2 signaling is not clear. Our initial experiments indicated that in atrial myocytes 2-AR activation of cPLA2 is Ca2+-dependent and mediated via ERK1/2 signaling [3]. This is consistent with studies in embryonic chick ventricular myocytes (cultured on LMN) in which 2-AR stimulation acts via ERK1/2 signaling to activate cPLA2 [6]. Moreover, in a variety of cell systems Raf-1 activates downstream ERK1/2 and PKA inhibits Raf-1 [7, 8]. Therefore, inhibition of PKA should remove inhibition of Raf-1, thereby allowing 2-AR stimulation to act via Raf-1/MEK/ERK1/2 signaling. Moreover, protein PSEN2 kinase C (PKC) activates Raf-1 [9, 10]. In other words, PKA inhibits and PKC activates Raf-1/MEK/ERK1/2 signaling. Based on these considerations we sought to determine whether inhibition of PKA facilitates 2-AR stimulation to act via PKC/Raf-1/MEK/ERK1/2 to activate cPLA2. These findings may be relevant to the remodeling of 2-AR signaling in the failing and/or aging heart, both of which exhibit decreases in adenylate cyclase activity. Materials and Methods Ethics Statement The animal and experimental protocols used in this study were approved by the Institutional Animal Care and Use Committee (IACUC) of Loyola University Medical.