Myocardial adenosine A(1)-receptor-mediated adenoprotection involves phospholipase C, PKC-epsilon, and p38 MAPK, but not HSP27

Abstract

Adenosine via an adenosine A(1) receptor (A(1)R) is a negative feedback inhibitor of adrenergic stimulation in the heart, protecting it from toxic effects of overstimulation. Stimulation of the A(1)R results in the activation of G(i) protein, release of free Gbetagamma-subunits, and activation/translocation of PKC-epsilon to the receptor for activated C kinase 2 protein at the Z-line of the cardiomyocyte sarcomere. Using an anti-Gbetagamma peptide, we investigated the role of these subunits in the A(1)R stimulation of phospholipase C (PLC), with the premise that the resulting diacylglycerol provides for the activation of PKC-epsilon. Inositol 1,4,5-triphosphate release was an index of PLC activity. Chlorocyclopentyl adenosine (CCPA), an A(1)R agonist, increased inositol 1,4,5-triphosphate production by 273% in mouse heart homogenates, an effect absent in A(1)R knockout hearts and inhibited by anti-Gbetagamma 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 A(1)R. Isoproterenol, a beta-adrenergic receptor agonist, increased the Ca(2+) 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-epsilon by A(1)R results from the activation of PLC via free Gbetagamma-subunits released upon A(1)R-induced dissociation of G(i)alphabetagamma. Attenuation of beta-adrenergic-induced contractile function by A(1)R may involve the activation of p38 MAPK, but not HSP27.

Fig. 1.

Effect of chlorocyclopentyl adenosine (CCPA) and anti-Gβγ peptide on inositol 1,4,5-triphosphate (IP₃) produced in homogenates of hearts obtained from wild-type and adenosine A₁ receptor (A₁R) knockout (KO) mice. CCPA and peptide were administered at 1 and 50 μM, respectively. The A₁R antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) was administered at 100 nM. Values are means ± SE for 3–4 hearts. *Mean is significantly different from the agent-free value (P < 0.05). †Means are significantly different from the CCPA-alone value (P < 0.05).

Fig. 2.

Effect of p38 MAPK inhibition with SB-203580 (SB) on changes in rat ventricular cardiomyocyte Ca²⁺ transient ratio magnitudes elicited by isoproterenol (ISO) and CCPA. β-Adrenoceptors were stimulated with 1 nM ISO for 3 min in the absence or presence of the A₁R agonist CCPA at 1.0 μM. Experiments were conducted in the absence (A) or presence (B) of the p38 MAPK inhibitor SB (1 μM). Representative calcium transients are depicted above the corresponding bars. Horizontal bar (s) indicates 1 s. Values are means ± SE for 7 (without SB) or 11 (with SB) cardiomyocytes. *Means are significantly different from the appropriate ISO-free control (P < 0.05). †Mean is significantly different from the ISO value in the absence of CCPA (P < 0.05).

Fig. 3.

Effect of SB-203580 on changes in rat cardiomyocyte sarcomere shortening elicited by ISO and CCPA. β-Adrenoceptors were stimulated with 1 nM ISO for 3 min in the absence or presence of the A₁R agonist CCPA at 1.0 μM. Experiments were conducted in the absence (A) or presence (B) of the p38 MAPK inhibitor SB (1 μM). The mean resting sarcomere length was 1.76 ± .010 (SE) μm for 19 cells. Values are means ± SE for 7 (without SB) or 11 (with SB) cardiomyocytes. *Means are significantly different from the appropriate ISO-free control (P < 0.05). †Mean is significantly different from the ISO value in the absence of CCPA (P < 0.05).

Fig. 4.

Effect of SB-203580 on changes in rat cardiomyocyte sarcomere maximum shortening and relaxation velocities elicited by ISO and CCPA. One nanomole of ISO was administered for 3 min in the absence or presence of the A₁R agonist CCPA at 1.0 μM. Experiments were conducted in the absence (A) or presence (B) of the p38 MAPK inhibitor SB (1 μM). Positive values indicate the velocity of shortening. Negative values indicate the velocity of relaxation. Values are means ± SE for 7 (without SB) or 11 (with SB) cardiomyocytes. *Means are significantly different from the appropriate ISO-free control (P < 0.05). †Means are significantly different from the ISO value in the absence of CCPA (P < 0.05).

Fig. 5.

Effect of CCPA and SB-203580 on the phosphorylation of heat shock protein 27 (HSP27) in the isolated heart obtained from wild-type and A₁R KO mice. CCPA (1.0 μM) was administered for 5 min in the presence or absence of SB (0.265 μM). Representative film densities are indicated above the corresponding bars for Western blots utilizing general (total HSP27) and phospho-specific antibodies (phospho-HSP27). Density ratios are depicted for each respective bar graph. Values are means ± SE of 4–10 hearts and are the ratio of phosphorylated HSP27 to total HSP27. *Mean is significantly different from the agent-free value (P < 0.05). †Means are significantly different from the wild-type CCPA-alone value (P < 0.05).

Fig. 6.

Effect of exposure time on CCPA-induced phosphorylation of HSP27 in the isolated, perfused mouse heart. Hearts were perfused in the absence or presence of CCPA (1.0 μM) for 5 or 45 min before freeze-clamping and analysis. See legend of Fig. 5 for details. Values are means ± SE of 4–9 hearts. *Means are significantly different from the appropriate agent-free value (P < 0.05). †Mean is significantly different from the corresponding 5-min value (P < 0.05).

Fig. 7.

Effect of ISO and CCPA on the phosphorylation of HSP27 in the isolated mouse heart. Hearts were perfused in the absence or presence of ISO (1 min) at 10 or 100 nM. When CCPA was administered, this A₁R agonist was infused for 5 min alone or 4 min before and during the ISO administration. Control and CCPA-alone values are repeated from Fig. 2 for comparison. See legend of Fig. 5 for details. Values are means ± SE of 3–9 hearts. *Mean is significantly different from the agent-free value (P < 0.05). †Mean is significantly different from the CCPA-alone value (P < 0.05).