Adenosine protects the heart from adrenergic overstimulation. This adenoprotection includes the direct anti-adrenergic action via adenosine A(1) receptors (A(1)R) on the adrenergic signaling pathway. An indirect A(1)R-induced attenuation of adrenergic responsiveness involves the translocation of PKC-epsilon to t-tubules and Z-line of cardiomyocytes. We investigated with sarcomere imaging, immunocytochemistry imaging, and coimmunoprecipitation (co-IP) whether A(1)R activation of PKC-epsilon induces the kinase translocation to receptor for activated C kinase 2 (RACK2) in isolated rat and mouse hearts and whether phospholipase C (PLC) is involved. Rat cardiomyocytes were treated with the A(1)R agonist chlorocyclopentyladenosine (CCPA) and exposed to primary PKC-epsilon and RACK2 antibodies with secondaries conjugated to Cy3 and Cy5 (indodicarbocyanine), respectively. Scanning confocal microscopy showed that CCPA caused PKC-epsilon to reversibly colocalize with RACK2 within 3 min. Additionally, rat and mouse hearts were perfused and stimulated with CCPA or phenylisopropyladenosine to activate A(1)R, or with phorbol 12-myristate 13-acetate to activate PKC. RACK2 was immunoprecipitated from heart extracts and resolved with SDS-PAGE. Western blotting showed that CCPA, phenylisopropyladenosine, and phorbol 12-myristate 13-acetate in the rat heart increased the PKC-epsilon co-IP with RACK2 by 186, 49, and >1,000%, respectively. The A(1)R antagonist 8-cyclopentyl-1,3-dipropylxanthine prevented the CCPA-induced co-IP with RACK2. In mouse hearts, CCPA increased the co-IP of PKC-epsilon with RACK2 by 61%. With rat cardiomyocytes, the beta-adrenergic agonist isoproterenol increased sarcomere shortening by 177%. CCPA reduced this response by 47%, an action inhibited by the PLC inhibitor U-73122 and 8-cyclopentyl-1,3-dipropylxanthine. In conclusion, A(1)R stimulation of the heart is associated with PLC-initiated PKC-epsilon translocation and association with RACK2.
Adenosine via an adenosine A1 receptor (A1R) is a negative feedback inhibitor of adrenergic stimulation in the heart, protecting it from toxic effects of overstimulation. Stimulation of the A 1R results in the activation of Gi protein, release of free G␥-subunits, and activation/translocation of PKC-ε to the receptor for activated C kinase 2 protein at the Z-line of the cardiomyocyte sarcomere. Using an anti-G␥ peptide, we investigated the role of these subunits in the A1R stimulation of phospholipase C (PLC), with the premise that the resulting diacylglycerol provides for the activation of PKC-ε. Inositol 1,4,5-triphosphate release was an index of PLC activity. Chlorocyclopentyl adenosine (CCPA), an A1R agonist, increased inositol 1,4,5-triphosphate production by 273% in mouse heart homogenates, an effect absent in A1R knockout hearts and inhibited by anti-G␥ peptide. In a second study, p38 MAPK and heat shock protein 27 (HSP27), found by others to be associated with the loss of myocardial contractile function, were postulated to play a role in the actions of A1R. Isoproterenol, a -adrenergic receptor agonist, increased the Ca 2ϩ transient and sarcomere shortening magnitudes by 36 and 49%, respectively. In the rat cardiomyocyte, CCPA significantly reduced these increases, an action blocked by the p38 MAPK inhibitor SB-203580. While CCPA significantly increased the phosphorylation of HSP27, this action was inhibited by isoproterenol. These data indicate that the activation of PKC-ε by A1R results from the activation of PLC via free G␥-subunits released upon A1R-induced dissociation of Gi␣␥. Attenuation of -adrenergic-induced contractile function by A1R may involve the activation of p38 MAPK, but not HSP27. G␥ subunits; antiadrenergic; rodent; contractility ADENOSINE IS RELEASED ENDOGENOUSLY in the heart that is stressed by hypoxia, ischemia, or adrenergic stimulation (13,15). In the interstitium, adenosine activates cell surface receptors that initiate signal cascades within the cardiomyocyte, ultimately reducing the cardiotoxic manifestations of the initial insult (16,43). For many years, study has focused on the antiadrenergic action of adenosine, or the ability of this nucleoside to protect the heart against excessive stimulation (34) by norepinephrine released during ischemia (36). In manifesting this action, adenosine A 1 receptors (A 1 R) attenuate -adrenergic catecholamine-elicited increases in G s protein cycling (17), adenylyl cyclase activity (33), cAMP formation (5), activation of protein kinase A (PKA) (6), protein phosphorylation (12), myocardial Ca 2ϩ transient magnitude (14), and ventricular contractility (6, 16).Adenosine also reduces the responsiveness of the myocardium to catecholamine stimulation by a mechanism independent of changes in adrenergic-stimulated cyclase activity. This adenoprotection by the A 1 R agonist chlorocyclopentyl adenosine (CCPA) has been demonstrated by us to involve the translocation of PKC-ε to receptor for activated C kinase 2 (RACK2) in rat and mouse myocard...
(A1R) is antiadrenergic by reducing the adrenergic 1 receptor (1R)-elicited increase in contractility. In this study we compared the A2AR-, A1R-, and 1R-elicited actions on isolated rat ventricular myocytes in terms of Ca transient and contractile responses involving PKA and PKC. Stimulation of A2AR with 2 M (ϳEC50) CGS-21680 (CGS) produced a 17-28% increase in the Ca transient ratio (CTR) and maximum velocities (Vmax) of transient ratio increase (ϩMVT) and recovery (ϪMVT) but no change in the time-to-50% recovery (TTR). CGS increased myocyte sarcomere shortening (MSS) and the maximum velocities of shortening (ϩMVS) and relaxation (ϪMVS) by 31-34% with no change in time-to-50% relengthening (TTL). 1R stimulation using 2 nM (ϳEC50) isoproterenol (Iso) increased CTR, ϩMVT, and ϪMVT by 67-162% and decreased TTR by 43%. Iso increased MSS, ϩMVS, and ϪMVS by 153-174% and decreased TTL by 31%. The A2AR and 1R Ca transient and contractile responses were not additive. The PKA inhibitor Rp-adenosine 3Ј,5Ј-cyclic monophosphorothioate triethylamonium salt prevented both the CGS-and Iso-elicited contractile responses. The PKC inhibitors chelerythrine and KIE1-1 peptide (PKCε specific) prevented the antiadrenergic action of A 1R but did not influence A2AR-mediated increases in contractile variables. The findings suggest that cardiac A 2AR utilize cAMP/PKA like 1R, but the Ca transient and contractile responses are less in magnitude and not equally affected. Although PKC is important in the A1R antiadrenergic action, it does not seem to play a role in A2AR-elicited Ca transient and contractile events. adenosine A1 receptor; contractility; protein kinase A; protein kinase C; sarcomere shortening ADENOSINE VIA ADENOSINERGIC receptors plays an important role in heart function. This naturally occurring nucleoside in the myocardium (8, 16) is capable of interacting with both the adenosine A 2A receptor (A 2A R) and adenosine A 1 receptor (A 1 R) to modulate contractile function. The A 2A R have been reported to increase contractile performance in rat (12) and neonatal avian (29) ventricular myocytes as well as increase the contractility of perfused rat (32) and mouse (42) hearts. A 2A R have been postulated to activate adenylyl cyclase (29) or initiate mechanisms that are Ca dependent and -independent (11, 44) and cAMP independent (12, 30). In those studies reporting an effect of cAMP, PKA may be activated in a manner similar to that resulting from adrenergic  1 receptor ( 1 R) simulation (5, 6). Although previous studies have focused on the involvement of cAMP, little regard has been shown concerning the involvement of PKA and PKC in the A 2A R-mediated Ca and contractile effects in the myocardium. In a previous report, only an increase in Ca transient and myocyte shortening were reported with A 2A R stimulation of rat ventricular myocytes (44). Thus in this study the Ca transient magnitude and maximum velocities of both the Ca transient increase and recovery as well as the time-to-50% recovery of the Ca transient were ex...
Adenosine protects the myocardium of the heart by exerting an antiadrenergic action via the adenosine A1 receptor (A1R). Because beta 1-adrenergic receptor (beta 1R) stimulation elicits myocardial protein phosphorylation, the present study investigated whether protein kinase A (PKA) catalyzed rat heart ventricular membrane phosphorylation affects the beta 1R adrenergic and A1R adenosinergic actions on adenylyl cyclase activity. Membranes were either phosphorylated with PKA in the absence/presence of a protein kinase inhibitor (PKI) or dephosphorylated with alkaline phosphatase (AP) and assayed for adenylyl cyclase activity (AC) in the presence of the beta 1R agonist isoproterenol (ISO) and/or the A1R agonist 2-chloro-N6-cyclopentyladenosine (CCPA). 32P incorporation into the protein substrates of 140-120, 43, and 29 kDa with PKA increased both the ISO-elicited activation of AC by 51-54% and the A1R-mediated reduction of the ISO-induced increase in AC by 29-50%, thereby yielding a total antiadrenergic effect of approximately 78%. These effects of PKA were prevented by PKI. AP reduced the ISO-induced increase in AC and eliminated the antiadrenergic effect of CCPA. Immunoprecipitation of the solubilized membrane adenylyl cyclase with the use of a polyclonal adenylyl cyclase VI antibody indicated that the enzyme is phosphorylated by PKA. These results indicate that the cardioprotective effect of adenosine afforded by its antiadrenergic action is facilitated by cardiac membrane phosphorylation.
Adenosine A1 receptor (A1R)-induced translocation of PKCε to transverse (t) tubular membranes in isolated rat cardiomyocytes is associated with a reduction in β1-adrenergic-stimulated contractile function. The PKCε-mediated activation of protein kinase D (PKD) by endothelin-1 is inhibited by β1-adrenergic stimulated protein kinase A (PKA) suggesting a similar mechanism of A1R signal transduction modulation by adrenergic agonists may exist in the heart. We have investigated the influence of β1-adrenergic stimulation on PKCε translocation elicited by A1R. Immunofluorescence imaging and Western blotting with PKCε and β-COP antibodies were used to quantify the co-localization of PKCε and t-tubular structures in isolated rat cardiomyocytes. The A1R agonist CCPA increased the co-localization of PKCε and t-tubules as detected by imaging. The β1-adrenergic receptor agonist isoproterenol (ISO) inhibited this effect of CCPA. Forskolin, a potent activator of PKA, mimicked, and H89, a pharmacological PKA inhibitor, and PKI, a membrane-permeable PKA peptide PKA inhibitor, attenuated the negative effect of ISO on the A1R-mediated PKCε translocation. Western blotting with isolated intact hearts revealed an increase in PKCε/β-COP co-localization induced by A1R. This increase was attenuated by the A1R antagonist DPCPX and ISO. The ISO-induced attenuation was reversed by H89. It is concluded that adrenergic stimulation inhibits A1R-induced PKCε translocation to the PKCε anchor site RACK2 constituent of a coatomer containing β-COP and associated with the t-tubular structures of the heart. In that this translocation has been previously associated with the antiadrenergic property of A1R, it is apparent that the interactive effects of adenosine and β1-adrenergic agonists on function are complex in the heart.
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