Adenosine Receptor Subtypes and Cardioprotection

Abstract

Brief ischemia prior to a sustained period of ischemia reduces myocardial infarct size, a phenomenon known as preconditioning. A cardiac ventricular myocyte model has been developed to investigate the role and signaling mechanism of adenosine receptor subtypes in cardiac preconditioning. A 5-min exposure of cardiac myocytes to simulated ischemia, termed preconditioning ischemia, prior to a subsequent 90-min period of ischemia protected them against injury incurred during the 90-min ischemia. Preconditioning ischemia preserved ATP content, reduced percentage of cells killed, and decreased release of creatine kinase into the medium. Activation of the adenosine A₁ receptor with CCPA or the A₃ receptor with IB-MECA can replace preconditioning ischemia and mimic the protective effect of preconditioning ischemia. Blockade of the A₁ receptor with its selective antagonist DPCPX or of the A₃ receptor with the A₃ selective antagonist MRS1191 during the preconditioning ischemia resulted in only a partial attenuation of the subsequent protection. Incubation with both DPCPX and MRS1191 or with the nonselective antagonist 8-SPT during the preconditioning ischemia completely abolished the protective effect of preconditioning ischemia. The K_ATP channel opener pinacidil caused a large activation of the K_ATP channel current and was able to precondition the myocyte. The K_ATP channel antagonist glibenclamide blocked the cardioprotective effect of preconditioning ischemia when it was included during myocyte exposure to the preconditioning ischemia, indicating that K_ATP channel is a requisite effector in mediating preconditioning. A receptor-mediated stimulation of phospholipase C or phospholipase D, with consequent activation of protein kinase C and K_ATP channel, appears to be the signaling mechanism linking adenosine A₁ and A₃ receptors to the induction of preconditioning. A model of how ischemic preconditioning is triggered and mediated is proposed. Evidence is accumulating to support its validity.

Keywords: adenosine; ischemia; myocardium; purinergic; receptor.

Fig. 1.

Induction of preconditioning of the cardiac myocytes. Preconditioning can be induced by a 5-min exposure of the cardiac myocytes to (A) simulated ischemia or (B) the adenosine A₁ agonist CCPA, the A₃ agonist Cl-IB-MECA or K_ATP channel opener pinacidil. In myocytes preconditioned by the brief ischemia, cells were incubated under normal O₂ for 10 min following the 5-min ischemia; cells were then exposed to 90 min of ischemia. In myocytes preconditioned by the receptor agonists or channel opener, the medium containing the receptor agonist or channel opener was replaced with fresh medium lacking the drug, and the myocytes were incubated for a further 10-min period before being exposed to 90 min of simulated ischemia.

Fig. 2.

Effects of prior exposure to preconditioning ischemia, CCPA or pinacidil on the myocyte ATP content, the percentage of cells killed, and the amount of CK released. Cardiac ventricular myocytes were prepared as described in Materials and Methods. Myocytes were preconditioned either by a 5-min preexposure to simulated ischemia, CCPA (30 nM), Cl-IB-MECA (10 nM), or pinacidil (10 μM) as described in Figure legend 1. Data were presented as (A) myocyte ATP content and as (B) percent cells killed or as amount of CK released. Data were mean ± SE of six to eight experiments. *Significantly different from nonpreconditioned cells (one-way ANOVA and Student-Newman-Keuls multiple comparison test, P < 0.001).

Fig. 3.

Pinacidil stimulates the K_ATP channel current in the cultured cardiac myocyte. Cardiac ventricular myocytes were cultured as described in Materials and Methods. After 24 h in culture, myocytes were patch-clamped in the whole-cell configuration. After a stable patch was achieved, the baseline K_ATP channel current was obtained. Myocytes were then superfused with the same extracellular solution containing 30 μM pinacidil. The same voltage steps were applied and an increase in the K_ATP channel current at the hyperpolarizing potential was recorded. The tracing is representative of three other myocytes. In response to pinacidil, the holding current at −40 mV increased significantly (data not shown).

Fig. 4.

Effects of 8-SPT and glibenclamide on the cardioprotective effect of ischemic preconditioning. Cardiac ventricular myocytes were prepared as described in Materials and Methods. Myocytes were preconditioned by a 5-min preexposure to ischemia in the presence or the absence of the nonselective adenosine receptor antagonist 8-SPT (100 μM) or the K_ATP channel antagonist glibenclamide (1 μM). Data were presented as (A) myocyte ATP content or (B) percentage of cells killed or amount of CK released and were the mean ± SE of seven experiments. *Significantly different from cells preconditioned in the absence of 8-SPT or glibenclamide (one-way ANOVA and Student-Newman-Keuls multiple comparison test, P < 0.001).

Fig. 5.

Effects of DPCPX on the cardioprotective effect of preconditioning ischemia. Cardiac ventricular myocytes were prepared as described in Materials and Methods. Myocytes were preconditioned in the presence or the absence of varying concentrations of DPCPX. Data were presented as percentage of cells killed or amount of CK released and were the mean ± SE of five experiments. *Significantly different from cells preconditioned in the absence of DPCPX (one-way ANOVA and Student-Newman-Keuls multiple comparison test, P < 0.001).

Fig. 6.

Signaling mechanisms underlying adenosine receptor-mediated preconditioning effect. The preconditioning (PC) effect can be divided into two phases. In the first phase, the preconditioning effect is initiated. Signaling mechanisms are activated and remain in an activated state during the sustained ischemia. During the sustained ischemia in which the second phase occurs, the actual cardioprotective effect of preconditioning is exerted. A series of signaling events take place during each phase as outlined in a hypothetical working model. During initiation phase, adenosine is released from the ischemic myocardium, which triggers the preconditioning by activating the adenosine receptor. The receptor is coupled to stimulation of diacylglyceride (DAG) via either phospholipase C or D. A sustained increase in the level of DAG stimulates protein kinase C (PKC), which in turn causes an activated K_ATP channel. Thus, inhibition of PKC blocks the preconditioning effect elicited by adenosine receptor activation. K_ATP channel antagonist blocks the preconditioning effect elicited by adenosine agonist or phorbol ester, establishing the sequential activation of adenosine receptor, PKC, and K_ATP channel as the sequence of signaling events during the initiation of preconditioning. During the second phase, activation of both adenosine receptor and K_ATP channel is required to exert the actual cardioprotective effect of preconditioning. Central to this hypothesis is the concept that the K_ATP channel is first primed in an activated state during the initiation phase, and becomes more sensitive to the protective effect of adenosine during the second phase. Ultimately, activation of the channel is responsible for protection against ischemia-induced injury.