INTRODUCTION

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ADENOSINE A_2a receptor (A_2aR)-mediated increases in myocyte shortening have been reported in myocytes in which the opposing adenosine A₁ receptor (A₁R)-inhibitory G protein (G_i) pathway was inhibited (17, 28) or remained intact (5). Recently, it was reported that the A_2aR-induced inotropic effects are mediated, in part, by cAMP-independent mechanisms (4, 5, 17). Furthermore, these inotropic effects are associated with a prolonged duration of myocyte shortening but with no change in the maximum rate of relaxation (5). In comparison, -adrenergic receptor-induced increases in myocyte shortening (mediated by cAMP) are characterized by a reduced duration of shortening and accelerated relaxation (5, 15). Hence, it has been suggested that these two inotropic responses are mediated by different pathways (5, 17).

Myocardial contractility can be enhanced by mechanisms that alter the intracellular Ca²⁺ concentration ([Ca²⁺]) (Ca²⁺-dependent pathway), modify the responsiveness of myofibrils to Ca²⁺ (Ca²⁺-independent pathway), or a combination of both (22). The relative contribution of these two stimulatory pathways is best determined by comparing the changes elicited in myocyte contractility and Ca²⁺ transients in response to an inotropic agent. -Adrenergic receptor activation results in an elevation in the amplitude of Ca²⁺ transients that surpasses the increase in myocyte contractility. This is indicative of a predominance of the Ca²⁺-dependent pathway. Alternatively, the force of contraction can be enhanced without changing Ca²⁺ transients (Ca²⁺-independent pathway), such as results from stretch-induced activation (e.g., Frank-Starling effects) (22). Stretch-induced elevations in myocyte contractility are characterized by a prolongation of contraction and delayed relaxation, which are caused by an enhanced responsiveness of myofilaments to Ca²⁺ (2, 22). The prolongation of contraction observed in combination with A_2aR-mediated increases in myocyte shortening (5) is suggestive of an increase in the affinity of the myofibrils to Ca²⁺ (2). Hence, it is possible that A_2aR stimulation enhances myocyte contractility via the Ca²⁺-independent pathway, as opposed to the Ca²⁺-dependent mechanisms characteristic of -adrenergic receptor activation (22). However, the effects of A_2aR stimulation on Ca²⁺ transients have not been investigated. Although A_2aR-mediated stimulation of L-type Ca²⁺ channels was reported to be responsible for an increase in ⁴⁵Ca influx in avian embryonic ventricular myocytes (17), influx is only one of the determinants of the amplitude and duration of Ca²⁺ transients (9). Furthermore, in mammalian cardiac muscle the release of Ca²⁺ from the sarcoplasmic reticulum (SR), rather than Ca²⁺ influx, is the largest contributor to the amplitude of Ca²⁺ transients (9).

The aim of this study was to investigate whether A_2aR-mediated increases in rat ventricular myocyte shortening are accompanied by alterations in myocyte Ca²⁺ transients. The effects of A_2aR stimulation on myocyte Ca²⁺ transients were determined in intact ventricular myocytes as well as in myocytes in which the opposing A₁R-G_i antiadrenergic pathway was inhibited to unmask, and thus facilitate, the quantitation of A_2aR-mediated responses. In addition, it was determined whether the effects of A_2aR stimulation on myocyte Ca²⁺ transients and myocyte shortening are proportional. The results of this study show that A_2aR activation in cardiomyocytes enhances shortening while only minimally increasing the Ca²⁺ transient magnitude. This observation suggests that the A_2aR-induced inotropic action is mediated primarily by a Ca²⁺-independent mechanism.

	METHODS

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The maintenance of the animals utilized in this study and the procedures employed were in accordance with recommendations in the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources, National Research Council (revised 1996) and the guidelines of the Institutional Animal Care and Use Committee of the University of Massachusetts Medical School, Worcester, MA.

Myocyte isolation. Ventricular myocytes were isolated from Sprague-Dawley rats (Charles River, Wilmington, MA, or Harlan, Indianapolis, IN) according to methods previously described (11) with several modifications. Briefly, male rats (175-275 g) were decapitated, and the hearts were rapidly excised and immediately perfused at a constant pressure (70 cmH₂O and nonrecirculated) for 5-10 min through the aorta with filtered (0.45-µm membrane filter) perfusion solution (PS) containing (in mM) 118.4 NaCl, 4.7 KCl, 25 NaHCO₃, 1.2 MgSO₄, 1.2 KH₂PO₄, 10 glucose, and 1.0 CaCl₂, pH 7.4, 37°C. After equilibration the hearts were constant-pressure perfused (70 cmH₂O and nonrecirculated) with nominally Ca²⁺-free PS until spontaneous contractions ceased (~30-60 s). Thereafter, the hearts were perfused with 48.4 µM Ca²⁺ PS containing (in mg/ml) 0.48 collagenase, 0.187 hyaluronidase, and 1.67 recrystallized BSA (fatty acid free) at a rate of 3-4 ml · min¹ · heart¹ for 4-10 min or until the hearts became pale and soft. Hearts were then removed, atria were dissected away, and ventricles were cut into small pieces that were placed in a flask with 5 ml of 50 µM Ca²⁺ PS containing (in mg/ml) 0.72 collagenase, 0.28 hyaluronidase, and 2.5 BSA (incubation solution). The flask was gently agitated at 40 oscillations/min in a reciprocating water bath for 7 min. The incubation solution was aspirated, and the 7-min oscillation procedure was repeated two to three times with the addition of fresh incubation solution. Thereafter, the flask was agitated at 120 oscillations/min for 12 min. Throughout the oscillation procedure the incubation solution was gassed with 95% O₂-5% CO₂ and maintained at 37°C. After the final shake, the contents of the flask were filtered through a 250-µm nylon mesh into a 50-ml polypropylene centrifuge tube, with the gradual addition of 20 ml of 100 µM Ca²⁺ PS containing 5 mg/ml BSA (wash solution). The myocytes were then allowed to settle for a 15-min period. Two-thirds of the supernatant was then aspirated and replaced with 20 ml of fresh wash solution, and a second 15-min period of settling was allowed. Thereafter, the myocytes were gradually exposed to increasing concentrations of Ca²⁺. This was achieved by combining nominally Ca²⁺-free PS with MEM to obtain the desired Ca²⁺ concentrations. Cardiomyocytes were washed and settled for 15 min in 200 µM Ca²⁺ PS-MEM followed by another wash and settling in 500 µM Ca²⁺ PS-MEM. During each of the settling steps Ca²⁺-intolerant myocytes remained suspended in the supernatant and were discarded. The Ca²⁺-tolerant myocytes remaining were resuspended and aliquoted into 30-mm culture dishes (Falcon) containing 500 µM Ca²⁺ PS-MEM (total vol 2-3 ml) and placed in an incubator (5% CO₂ in room air, 37°C) until use (between 0 and 4 h). Approximately 70-90% of the myocytes in the suspension were Ca²⁺-tolerant, rod-shaped myocytes.

Pertussis toxin treatment. To uncouple the A₁R from its effector, a sample of ventricular myocytes was treated with pertussis toxin (PTx, 300 ng/ml) for 3 h in the incubator before the measurement of Ca²⁺ transients. This dose and duration of PTx treatment were reported to be sufficient to uncouple the A₁R and, hence, abolish its antiadrenergic effects (13, 28).

Measurement of myocyte Ca²⁺ transients. Aliquots of ventricular myocytes were transferred to a 0.8-ml superfusion chamber and incubated for 10 min in PS-HEPES, which was PS modified by adding and changing the following (in mM): 1.0 glucose, 0.3 KCl, 0.25 CaCl₂, and 10.0 HEPES (pH 7.4) and 6.25 µM fura 2-AM. During this time period, the myocytes settled and adhered to the bottom of the chamber. The superfusion chamber was mounted on the stage of a Nikon inverted microscope (Diaphot 300), and the cells were viewed using a ×40 oil immersion fluorescence objective lens (Nikon Fluor 40). The temperature was maintained at 37°C using a heated microscope stage plate (Fryer A-50 Temperature Controller). Electrical field stimulation (model SD9, Grass Instruments, Quincy, MA) at 0.5 Hz was provided by platinum wire electrodes attached to the bottom of the chamber.

Mechanical measurements of changes in myocyte length. The mechanical function of myocytes was assessed as previously described (5). In brief, changes in individual myocyte length were assessed by placing 50-100 cells in a 0.8-ml superfusion chamber (similar to that used for myocyte Ca²⁺ determinations) that contained platinum wire electrodes for field stimulation of myocytes at 0.5 Hz. The chamber was continuously suffused with fresh solution containing (in mM) 136.4 NaCl, 4.7 KCl, 1.0 CaCl₂, 10 HEPES, 1.0 NaHCO₃, 1.2 MgSO₄, 1.2 KH₂PO₄, 10 glucose, 0.6 ascorbate, and 1.0 pyruvate. The chamber was mounted on the stage of an inverted microscope and the image of a single myocyte projected at ×300 onto a line-scan camera (Fairchild, model 1600R) containing a linear array of photodiodes (1 × 3,456) operating at 200 Hz. The signals from the line-scan camera were displayed on an oscilloscope (model V-660, Hitachi), thus permitting optimal positioning of the camera in alignment with the longitudinal axis of the cell. On transillumination of the myocyte, both ends of the cell were easily discerned. Measurement of myocyte length and change in length with respect to time for a single contraction was achieved by determining the pixels in which the appropriate transitions between light and dark occurred. A stage micrometer scaled in 10-µm divisions was used to calibrate the line-scan camera. The signals from the camera were directed to a Hewlett-Packard computer (model Vectra RS/20C) that utilized custom computer programming (MCS Computer Consulting, Keene, NH) to determine the maximum change in myocyte length (shortening).

Protocols for ventricular myocyte Ca²⁺ and myocyte length responses. All responses to agonists were elicited after baseline myocyte Ca²⁺ transients or myocyte shortening had stabilized in the presence of the appropriate vehicles for each of the agonists. To determine whether A_2aR agonists elicit alterations in myocyte Ca²⁺ transients, responses to 2-p-(2-carboxyethyl)phenethyl-amino-5'-N-ethylcarboxamidoadenosine (CGS-21680) or adenosine (Ado) were assessed with concentrations ranging from 10⁷ to 10⁴ M. Alterations in myocyte shortening in response to CGS-21680 at concentrations increasing from 10⁷ to 10⁴ M were also determined. To maximize these responses, the opposing effects mediated by A₁R were abolished either by treatment of the myocytes with PTx or by administration of 2 × 10⁷ M 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), an A₁R antagonist. For comparison, the effects of 10⁸ to 2 × 10⁷ M isoproterenol (Iso), a -adrenergic receptor agonist, on myocyte Ca²⁺ transients and myocyte shortening were ascertained.

Materials. Buffer salts and acids were supplied by Fisher Scientific (Medford, MA). Iso, HEPES, EGTA, DMSO, BSA, and Triton X-100 were purchased from Sigma Chemical (St. Louis, MO). Fura 2-AM was obtained from Calbiochem (La Jolla, CA) and MEM from GIBCO Laboratories (Grand Island, NY). Crude collagenase and hyaluronidase were purchased from Worthington Biochemical (Freehold, NJ). Adenosine was obtained from Boehringer-Mannheim (Indianapolis, IN), and CGS-21680, 8-(3-chlorostyryl)caffeine (CSC), and DPCPX from Research Biochemicals (Natick, MA).

Statistics. The amplitude of the Ca²⁺ transient was calculated as the difference between maximum [Ca²⁺] attained during systole and minimum [Ca²⁺] recorded during diastole. Absolute changes as well as percent changes in the amplitudes of the Ca²⁺ transients or myocyte length in response to increasing concentrations of the various agonists were calculated and compared with a value of zero (no change, as occurred with addition of appropriate vehicle). Results are expressed as means ± SE. Statistical significance was assessed by using ANOVA, Tukey-Kramer, and unpaired Student's t-test. A P value <0.05 was selected to indicate a significant difference.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Effects of CGS-21680, Ado, and Iso on [Ca²⁺], amplitude of Ca²⁺ transient, and changes in myocyte length. The alterations elicited in the maximum [Ca²⁺] attained during systole and the minimum [Ca²⁺] present during diastole by each of the agonists CGS-21680, Ado, and Iso are detailed in Table 1. CGS-21680 and Ado had no effect on diastolic [Ca²⁺], which was augmented in the presence of Iso. Both CGS-21680 and Ado modestly increased systolic [Ca²⁺], although only the effect of Ado was significant. In contrast, systolic [Ca²⁺] was markedly increased in response to Iso.

View larger version (35K): [in this window] [in a new window]   Fig. 1.   A: representative traces depicting comparison of CGS-21680 (CGS)-, adenosine (Ado)-, Ado + 8-cyclopentyl-1,3-dipropylxanthine (DPCPX)-, and isoproterenol (Iso)-elicited increases in amplitude of Ca2+ transients. Traces are myocyte Ca2+ transients of representative non-pertussis toxin (PTx)-treated myocytes exposed to vehicle (Veh) followed by 2 × 107 M Iso; Veh followed by 106 M CGS; and Veh followed by 105 M Ado and 105 M Ado + 2 × 107 M DPCPX. Representative Ca2+ transients (B) and tracings of changes in myocyte length (C) in presence of Veh, CGS, or Iso are depicted on an expanded time scale. [Ca2+], Ca2+ concentration.

Effects of PTx treatment on antiadrenergic action of Ado. The antiadrenergic receptor effects of Ado, which are mediated by an A₁R-G_i mechanism, were abolished by PTx treatment of ventricular myocytes. The -adrenergic receptor agonist Iso, at 2 × 10⁷ M, elicited increases in the amplitude of ventricular myocyte Ca²⁺ transients that were reduced by the addition of 10⁵ M Ado (Fig. 2A). In ventricular myocytes treated with PTx, similar elevations in the amplitude of Ca²⁺ transients were produced by 2 × 10⁷ M Iso, but the administration of 10⁵ M Ado did not reduce the amplitude of the Ca²⁺ transients (Fig. 2B).

View larger version (21K): [in this window] [in a new window]   Fig. 2.   . Comparison of effects of 105 M Ado on 2 × 107 M Iso-elicited increases in amplitude of Ca2+ transients in non-PTx-treated (A) and PTx-treated (B) ventricular myocytes. Values are means ± SE for 7 (A) and 5 (B) ventricular myocytes. * Significant difference from vehicle;  significant difference from Iso alone.

Effects of CGS-21680 and Iso on amplitude of Ca²⁺ transients. In the presence of an intact A₁R-G_i inhibitory mechanism, the A_2aR agonist CGS-21680 alone, at concentrations ranging from 10⁷ to 10⁴ M, did not significantly increase the amplitude of Ca²⁺ transients (Fig. 3A). In comparison, Iso produced profound increases in the amplitude of Ca²⁺ transients that ranged from 52 ± 14% at 10⁸ M to 104 ± 15% at 2 × 10⁷ M. These increases were considerable in contrast to the minimal responses (ranging from 7 ± 3% at 10⁷ M to 8 ± 5% at 10⁴ M) observed in non-PTx-treated myocytes in the presence of CGS-21680.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Comparison of effects of A2 receptor (A2aR) agonist CGS with effects of -adrenergic receptor agonist Iso on change in amplitude of Ca2+ transients (A) and on changes in myocyte length (B) in ventricular myocytes not treated with PTx. Values are means ± SE for 4-10 ventricular myocytes. * Significant difference from zero agent;  significant difference from corresponding CGS value.

Effects of CGS-21680 and Iso on myocyte shortening. Both the A_2aR agonist CGS-21680 and the -adrenergic receptor agonist Iso significantly enhanced ventricular myocyte shortening (Fig. 3B). These responses were observed in the absence of any inhibition of opposing A₁R-mediated effects. The increases in response to CGS-21680 (from 22 ± 1% at 10⁷ M to 54 ± 1% at 10⁵ M) were similar in magnitude to those elicited by Iso (from 32 ± 7% at 10⁸ M to 61 ± 6% at 2 × 10⁷ M).

Effects of CGS-21680 and Ado on amplitude of Ca²⁺ transients in PTx-treated myocytes. The absence of significant increases in Ca²⁺ transients in response to CGS-21680 may have resulted from the influence of simultaneous A₁R activation, especially at the higher doses of CGS-21680. Hence, the effects of CGS-21680 were assessed in ventricular myocytes that had been treated with PTx. In PTx-treated ventricular myocytes, CGS-21680 at concentrations of 10⁵ M and 10⁴ M increased the amplitude of Ca²⁺ transients (Fig. 4A). The percent increases induced were 15 ± 7% at 10⁵ M and 14 ± 6% at 10⁴ M. The addition of the A_2aR-specific antagonist CSC prevented the increases in the amplitude of Ca²⁺ transients produced by 10⁴ M CGS-21680, indicating that these modest elevations were mediated by A_2aR.

View larger version (31K): [in this window] [in a new window]   Fig. 4.   Effects of A2aR agonists CGS (A) and Ado (B) on amplitude of Ca2+ transients in PTx-treated ventricular myocytes in absence () or presence () of 106 M 8-(3-chlorostyryl)caffeine (CSC), an A2aR specific antagonist. Values are means ± SE for 4-9 myocytes. * Significant difference from no change (zero);  significant difference from 104 M CGS or 104 M Ado value without CSC.

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Effects of DPCPX on Ado-mediated changes in Ca²⁺ transients and myocyte length. In non-PTx-treated ventricular myocytes, Ado at 10⁶ M produced no effect (4 ± 4%), at 10⁵ M marginally increased (5 ± 2%), and at 10⁴ M markedly diminished (12 ± 2%) the amplitude of Ca²⁺ transients (Fig. 5A). With myocytes treated with the A₁R antagonist DPCPX, 10⁵ M Ado augmented the amplitude of Ca²⁺ transients by 19 ± 1%. The depression of the Ca²⁺ transient by Ado at 10⁴ M was reversed by DPCPX. The degree of shortening was increased by 9 ± 1% in response to 10⁴ M Ado (Fig. 5B). However, in the presence of A₁R antagonism with DPCPX, the increase in myocyte shortening was enhanced 51 ± 2%.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Effect of 104 M Ado in absence or presence of 2 × 107 M DPCPX, an A1R antagonist, on amplitude of Ca2+ transients (A) or on change in myocyte length (B) in non-PTx-treated ventricular myocytes. Values are means ± SE for 4-14 myocytes. * Significant difference from no response (zero);  significant difference from Ado alone.

Comparison of percent changes in Ca²⁺ transients or myocyte lengths elicited by A_2aR or -adrenergic receptor agonists. Although increases in the amplitude of Ca²⁺ transients were produced by CGS-21680 and Ado in PTx-treated ventricular myocytes and by Ado plus DPCPX in non-PTx-treated myocytes, the maximum percent changes recorded (14 ± 6, 15 ± 4, and 19 ± 1%, respectively) were modest in comparison to the maximum percent changes in myocyte length (54 ± 1% with CGS-21680 and 51 ± 2% with Ado + DPCPX in non-PTx-treated myocytes; Fig. 6). Thus it is apparent that A_2aR activation results in profound increases in myocyte shortening that are accompanied by only marginal changes in the amplitude of Ca²⁺ transients. By comparison, in response to Iso, the maximum percent increase in the amplitude of Ca²⁺ transients was greater than the maximum percent increase in myocyte shortening (104 ± 15% vs. 61 ± 6%).

View larger version (24K): [in this window] [in a new window]   Fig. 6.   Comparison of maximum percent changes in amplitude of Ca2+ transients (left) or myocyte length (right) in response to 104 M CGS, 104 M Ado, 105 M Ado + 2 × 107 M DPCPX, or 2 × 107 M Iso in either absence () or presence (+) of PTx as indicated. * Significant difference from no response (zero).

	DISCUSSION

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The major finding of the present study is that A_2aR-induced increases in myocyte shortening are mediated by Ca²⁺-independent inotropic pathways. In isolated, contracting rat ventricular myocytes, A_2aR activation produced increases in myocyte shortening that were accompanied by only modest elevations in ventricular myocyte Ca²⁺ transients. In comparison, increases in ventricular myocyte Ca²⁺ transients that surpassed the elevations in myocyte shortening, indicative of a Ca²⁺-dependent inotropic response, were elicited by -adrenergic receptor activation.

Ado and other adenine compounds reduce coronary vascular resistance (1) via A₂R (18) and mediate, via A₁R, negative inotropic effects in the heart and ventricular myocytes both in the presence (3, 5, 6) and in the absence (14, 24, 25) of -adrenergic receptor activation. In addition, Ado at high concentrations may have positive inotropic effects in ventricular preparations (4). Subsequent to the development of specific Ado receptor agonists, A_2aR have been shown to mediate increases in ventricular myocyte shortening (5, 17, 28). Recently, it was suggested that the A_2aR-induced positive inotropic effects are mediated, in part, by a cAMP-independent mechanism that may differ from that of -adrenergic receptor inotropic responses (5, 17).

Differences between the characteristics of -adrenergic- and A_2a-adenosinergic-induced contractile responses suggest that these inotropic effects are mediated by different mechanisms (5). -Adrenergic receptor stimulation results in an increase in myocardial cAMP concentration, one of the consequences of which is an enhanced Ca²⁺-induced Ca²⁺ release from the SR (19). As a result, [Ca²⁺] is markedly elevated, as reflected by an increase in Ca²⁺ transients that is proportionately greater than the increase in the force of contraction (10, 21, 22). In addition, cAMP, by reducing the affinity of troponin C for Ca²⁺ (23), mediates a decrease in the Ca²⁺ sensitivity of the myofibrils, which works in concert with the cAMP-induced acceleration of Ca²⁺ sequestration by the SR to hasten myocardial relaxation (19). Hence, -adrenergic receptor-mediated inotropic responses are characterized by a shortened duration of contraction and an increased rate of relaxation (5, 15). In comparison, A_2aR stimulation has no effect on the maximum rate of relaxation and the duration of shortening is prolonged (5). Actually, the characteristics of A_2aR-mediated inotropic responses resemble those of the enhanced contractility elicited by the stretching of ventricular muscle strips (2, 22). Stretch-induced increases in myocyte shortening are mediated by Ca²⁺-independent pathways of activation, as evidenced by an enhancement in the force of contraction with no significant change in Ca²⁺ transients (2, 22).

The present study shows that the administration of the A_2aR agonist CGS-21680 did not alter Ca²⁺ transients in ventricular myocytes. Eckert et al. (7) reported similarly that CGS-21680 had no effect on Ca²⁺ transients. However, at equivalent doses in the present study CGS-21680 enhanced myocyte shortening, thus confirming previous reports (5, 17). These data suggest that increases in myocyte shortening as a result of A_2aR activation are not accompanied by changes in Ca²⁺ transients. Although A_2aR-mediated augmentations in ⁴⁵Ca uptake have been demonstrated in avian embryonic ventricular myocytes (17), in mammalian heart muscle L-type Ca²⁺ channel activity is considered as only a minor determinant of the Ca²⁺ transient (9).

The absence of quantifiable inotropic responses to A_2aR agonists reported previously may have resulted from the simultaneous activation of opposing adenosine A₁R (27). Adenosine A₁R mediate inhibitory effects on -adrenergic receptor-induced increases in myocyte shortening (3, 5) and myocyte Ca²⁺ transients (11). In addition, at high concentrations (0.01-1 mM) A₁R agonists, in the absence of -adrenergic responses, were reported to decrease myocyte shortening and the amplitude of Ca²⁺ transients (14). In fact, the present results confirm a reduction in the amplitude of Ca²⁺ transients in response to Ado at a concentration of 10⁴ M. To unmask possible A_2aR-mediated responses, the opposing A₁R-mediated effects were inhibited (17, 28). Two different approaches were employed to prevent A₁R activation: 1) administration of Ado in the presence of an A₁R antagonist (DPCPX), and 2) administration of either the A_2aR agonist CGS-21680 or Ado to myocytes that had been incubated in PTx. PTx uncouples the A₁R from its effector (G_i) (13), thereby inhibiting A₁R-mediated effects. To ensure that inhibition of the A₁R-G_i pathway was achieved by the incubation of ventricular myocytes in PTx, the effects of A₁R activation on -adrenergic receptor-induced responses were compared in non-PTx-treated versus PTx-treated myocytes. In non-PTx-treated myocytes, Ado reduced the increase in Ca²⁺ transients elicited by Iso, hence confirming the antiadrenergic effects mediated by A₁R (5, 11, 14). In PTx-treated myocytes, the amplitudes of the Ca²⁺ transients in the presence of Iso plus Ado were not different from those in the presence of Iso alone, thus demonstrating that in PTx-treated myocytes the antiadrenergic effects of Ado were inhibited. These results confirm that the A₁R-G_i pathway was inhibited in the present study with incubation of myocytes in PTx.

Even though the opposing effects of A₁R were inhibited, the administration of CGS-21680 to PTx-treated myocytes produced only modest increases in the amplitude of Ca²⁺ transients. The Ca²⁺ transient responses elicited by Ado in PTx-treated myocytes were similarly nominal. In comparison, CGS-21680 produced marked increases in myocyte shortening. Furthermore, the administration of Ado in the presence of A₁R inhibition with DPCPX enhanced myocyte Ca²⁺ transients to a minimal extent in comparison to the increases elicited in myocyte shortening. Thus A_2aR-mediated increases in myocyte shortening are accompanied by only modest increases in the amplitude of Ca²⁺ transients.

It could be argued that the modest increases in Ca²⁺ transients in response to A_2aR agonists, as presently reported, indicate an insensitivity of the technique to detect changes in Ca²⁺ transients. However, the technique demonstrated considerable increases (104%) in the amplitude of Ca²⁺ transients in response to -adrenergic receptor activation, which confirms previous reports (9, 22). Furthermore, the -adrenergic receptor-mediated elevations in the amplitude of Ca²⁺ transients were proportionately greater in magnitude than the increases in myocyte shortening (61%) at similar concentrations of agonist. The results confirm that -adrenergic receptor-induced inotropic responses are mediated by Ca²⁺-dependent pathways (9, 22). In other words, the increase in contractility is reliant on elevations in [Ca²⁺] and an associated decrease in myofibrillar Ca²⁺ sensitivity (22).

In comparison to -adrenergic receptor-mediated inotropic responses, A_2aR activation elicits increases in myocyte shortening that surpass the increments in Ca²⁺ transients. Similar responses are produced by endothelin and phenylephrine (2) and by stretch-induced activation (22). An increase in contractility that is accompanied by either no change or only modest elevations in Ca²⁺ transients is indicative of a Ca²⁺-independent inotropic pathway (22). Such inotropic responses rely on an increase in myofibrillar Ca²⁺ sensitivity (2). An enhanced myofibrillar Ca²⁺ sensitivity is identified by an enhanced duration of contraction and a delayed relaxation such as occur in response to endothelin, phenylephrine (2), and stretch-induced activation (22). The demonstration of an increase in the duration of shortening in response to A_2aR activation (5) further supports the notion that A_2aR mediate their effects via Ca²⁺- independent inotropic pathways.

Ca²⁺-independent inotropic effects, such as those elicited by ₁-adrenoreceptors, endothelin, and angiotensin II, are thought to be mediated via the activation of phospholipase C with subsequent acceleration of the hydrolysis of phosphoinositide (PI) and the resultant production of inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol (9). Diacylglycerol, in turn, activates protein kinase C which, by reducing intracellular H⁺ concentrations, enhances the responsiveness of myofibrils to Ca²⁺ (8). In support of the involvement of these pathways in A_2aR-mediated inotropic effects, adenine compounds, including Ado, via P₂-purinoceptors are reported to induce positive inotropic effects and stimulate IP₃ synthesis (16), the consequences of which include facilitated release of Ca²⁺ from the SR (20). However, other studies report no changes in IP₃ accumulation after A_2aR activation (7, 17). It has also been proposed that cGMP plays a role as a regulator of the responsiveness of myofibrils to Ca²⁺ (26). Therefore, the molecular mechanisms mediating Ca²⁺-independent A_2aR inotropic responses warrant further investigation.

In summary, this study demonstrates that with similar contractile responses in cardiomyocytes to A_2aR and -adrenoceptor agonists the increases in Ca²⁺ transients are minimal with A_2a-adenosinergic compared with -adrenergic stimulation. These data suggest that A_2aR effects are mediated by a Ca²⁺-independent inotropic pathway, as opposed to the Ca²⁺-dependent pathway of -adrenergic inotropic responses.