Adenosine A2a receptor (A2aR) stimulation enhances the shortening of ventricular myocytes. Whether the A2aR-mediated increase in myocyte contractility is associated with alterations in the amplitude of intracellular Ca2+ transients was investigated in isolated, contracting rat ventricular myocytes using the Ca2+-sensitive fluorescent dye fura 2-AM. In the presence of intact inhibitory G protein pathways, 10−4 M 2-p-(2-carboxyethyl)phenethyl-amino-5′-N-ethylcarboxamidoadenosine (CGS-21680), an A2aR agonist, insignificantly increased Ca2+transients by 8 ± 5%, whereas myocyte shortening increased by 54 ± 1%. In contrast, 2 × 10−7 M isoproterenol, a β-adrenergic receptor agonist, increased Ca2+ transients by 104 ± 15% and increased myocyte shortening by 61 ± 6%. When A2aR were stimulated in myocytes that had the antiadrenergic actions of adenosine (Ado) abolished by either treatment with pertussis toxin (PTx) or the presence of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), an adenosine A1-receptor antagonist, the maximum increases in Ca2+transients were similarly nominal (with PTx: 10−4 M CGS-21680, 14 ± 6% and 10−4 M Ado, 15 ± 4%; without PTx: 10−5 M Ado + 2 × 10−7 M DPCPX, 19 ± 1%). These results indicate that compared with β-adrenergic stimulation, which markedly increases myocyte Ca2+ transients and shortening, A2aR-mediated increases in myocyte shortening are accompanied by only modest increases in Ca2+ transients. These observations suggest that the A2aR-induced contractile effects are mediated predominantly by Ca2+-independent inotropic mechanisms.
- inotropic responses
- adenosine receptors
- intracellular calcium transients
adenosineA2a receptor (A2aR)-mediated increases in myocyte shortening have been reported in myocytes in which the opposing adenosine A1 receptor (A1R)-inhibitory G protein (Gi) pathway was inhibited (17,28) or remained intact (5). Recently, it was reported that the A2aR-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, β-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. β-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 A2aR-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 A2aR stimulation enhances myocyte contractility via the Ca2+-independent pathway, as opposed to the Ca2+-dependent mechanisms characteristic of β-adrenergic receptor activation (22). However, the effects of A2aR stimulation on Ca2+ transients have not been investigated. Although A2aR-mediated stimulation of L-type Ca2+ channels was reported to be responsible for an increase in45Ca 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 A2aR-mediated increases in rat ventricular myocyte shortening are accompanied by alterations in myocyte Ca2+ transients. The effects of A2aR stimulation on myocyte Ca2+ transients were determined in intact ventricular myocytes as well as in myocytes in which the opposing A1R-Giantiadrenergic pathway was inhibited to unmask, and thus facilitate, the quantitation of A2aR-mediated responses. In addition, it was determined whether the effects of A2aR stimulation on myocyte Ca2+ transients and myocyte shortening are proportional. The results of this study show that A2aR activation in cardiomyocytes enhances shortening while only minimally increasing the Ca2+ transient magnitude. This observation suggests that the A2aR-induced inotropic action is mediated primarily by a Ca2+-independent mechanism.
The maintenance of the animals utilized in this study and the procedures employed were in accordance with recommendations in theGuide 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.
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 ⋅ min−1 ⋅ heart−1for 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.
After 10 min of incubation, superfusion with PS-HEPES without fura 2-AM was commenced and the myocytes were washed for 15 min to remove unattached myocytes and incompletely deesterified fura 2-AM. Ventricular myocytes that were rod shaped, clearly striated, mechanically quiescent, and responsive to electrical stimulation with a vigorous and reversible contraction were selected for Ca2+ measurements. Fluorescent dye internalized within the cell was excited by incident light provided by a Delta Scan high-speed dual-wavelength scanning illuminator (xenon arc lamp, 75 W, Photon Technology International, Brunswick, NJ) capable of switching between excitation light of 340 and 380 nm at a speed of 650 ratios/s. Light emitted by the cells was detected at 510 nm by a photomultiplier tube (model 710) and recorded at 50 points/s using a computer-based data acquisition system (Felix, Fluorescence Analysis Software, Photon Technology International). Each myocyte selected for Ca2+ transient measurement was separated from the remainder of the field of view with the use of four shutters, thereby minimizing background fluorescence. Because the autofluorescence of the myocytes was negligible (∼1% of the individual 340- and 380-nm signals), there was no need to subtract this from the recorded signals. Calibration of the fluorescence signal was performed at the end of the experiment by repeated exposure of the myocyte to CaCl2 buffer containing detergent (1% Triton X-100) until no further increase in the 340-to-380 ratio was observed, thus obtaining the maximum cellular fluorescence ratio (Rmax). The buffer was then replaced with PS-HEPES supplemented with EGTA and detergent (1% Triton X-100) but no added Ca2+, to record the minimum cellular fluorescence ratio (Rmin). Values for intracellular [Ca2+] were calculated according to the formula [Ca2+] =K dβ[(R − Rmin)/(Rmax− R)] (11, 12), where the dissociation constant of fura 2 for Ca2+(K d) is 200 nM, β is the ratio of permeabilized myocyte 380-nm fluorescence in the presence of EGTA to the 380-nm fluorescence in the presence of the maximum concentration of CaCl2, and R is the ratio of myocyte fluorescence with excitation at 340 nm to that at 380 nm recorded at an emission wavelength of 510 nm (11).
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 A2aR 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 10−7 to 10−4 M. Alterations in myocyte shortening in response to CGS-21680 at concentrations increasing from 10−7 to 10−4 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 × 10−7 M 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), an A1R antagonist. For comparison, the effects of 10−8 to 2 × 10−7 M isoproterenol (Iso), a β-adrenergic receptor agonist, on myocyte Ca2+ transients and myocyte shortening were ascertained.
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).
Stock solutions of 10 mM CGS-21680, 1 mM CSC, and 1 mM DPCPX were prepared in DMSO. Iso and Ado were prepared at 1 mM in 0.1% sodium metabisulfite (Sigma Chemical) and distilled, deionized water, respectively. Stock solutions were serially diluted in the appropriate buffer to the desired concentrations.
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’st-test. AP value <0.05 was selected to indicate a significant difference.
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.
Figure1 Adepicts examples of myocyte Ca2+transients in non-PTx-treated ventricular myocytes in the presence of Iso, CGS-21680, Ado, or Ado plus DPCPX, and their appropriate vehicles. Figure 1 B typifies Ca2+ transients recorded in the presence of CGS-21680 in comparison to those recorded in the presence of Iso. The effects of CGS-21680 on changes in ventricular myocyte length are depicted in comparison to the inotropic effects characteristic of the β-adrenergic receptor agonist Iso, using a representative trace obtained in the presence of each agonist (Fig.1 C).
Effects of PTx treatment on antiadrenergic action of Ado.
The antiadrenergic receptor effects of Ado, which are mediated by an A1R-Gimechanism, were abolished by PTx treatment of ventricular myocytes. The β-adrenergic receptor agonist Iso, at 2 × 10−7 M, elicited increases in the amplitude of ventricular myocyte Ca2+ transients that were reduced by the addition of 10−5 M Ado (Fig.2 A). In ventricular myocytes treated with PTx, similar elevations in the amplitude of Ca2+ transients were produced by 2 × 10−7 M Iso, but the administration of 10−5 M Ado did not reduce the amplitude of the Ca2+transients (Fig. 2 B).
Effects of CGS-21680 and Iso on amplitude of Ca2+ transients.
In the presence of an intact A1R-Giinhibitory mechanism, the A2aR agonist CGS-21680 alone, at concentrations ranging from 10−7 to 10−4 M, did not significantly increase the amplitude of Ca2+ transients (Fig.3 A). In comparison, Iso produced profound increases in the amplitude of Ca2+ transients that ranged from 52 ± 14% at 10−8 M to 104 ± 15% at 2 × 10−7 M. These increases were considerable in contrast to the minimal responses (ranging from 7 ± 3% at 10−7 M to 8 ± 5% at 10−4 M) observed in non-PTx-treated myocytes in the presence of CGS-21680.
Effects of CGS-21680 and Iso on myocyte shortening.
Both the A2aR agonist CGS-21680 and the β-adrenergic receptor agonist Iso significantly enhanced ventricular myocyte shortening (Fig.3 B). 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 10−7 M to 54 ± 1% at 10−5 M) were similar in magnitude to those elicited by Iso (from 32 ± 7% at 10−8 M to 61 ± 6% at 2 × 10−7 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 10−5 M and 10−4 M increased the amplitude of Ca2+ transients (Fig.4 A). The percent increases induced were 15 ± 7% at 10−5 M and 14 ± 6% at 10−4 M. The addition of the A2aR-specific antagonist CSC prevented the increases in the amplitude of Ca2+ transients produced by 10−4 M CGS-21680, indicating that these modest elevations were mediated by A2aR.
Ado at concentrations ranging from 10−6 to 10−4 M enhanced the amplitude of Ca2+ transients in PTx-treated ventricular myocytes (Fig.4 B). The percent changes observed (from 8 ± 2% at 10−6 M to 15 ± 4% at 10−4 M) were similar in magnitude to those induced by CGS-21680. Furthermore, the addition of CSC prevented the increase observed in response to 10−4 M Ado, confirming that these modest changes in the amplitude of Ca2+ transients were mediated by A2aR.
Effects of DPCPX on Ado-mediated changes in Ca2+ transients and myocyte length.
In non-PTx-treated ventricular myocytes, Ado at 10−6 M produced no effect (4 ± 4%), at 10−5 M marginally increased (5 ± 2%), and at 10−4 M markedly diminished (−12 ± 2%) the amplitude of Ca2+ transients (Fig.5 A). With myocytes treated with the A1R antagonist DPCPX, 10−5 M Ado augmented the amplitude of Ca2+transients by 19 ± 1%. The depression of the Ca2+ transient by Ado at 10−4 M was reversed by DPCPX. The degree of shortening was increased by 9 ± 1% in response to 10−4 M Ado (Fig.5 B). However, in the presence of A1R antagonism with DPCPX, the increase in myocyte shortening was enhanced 51 ± 2%.
Comparison of percent changes in Ca2+ transients or myocyte lengths elicited by A2aR or β-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 A2aR 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%).
The major finding of the present study is that A2aR-induced increases in myocyte shortening are mediated by Ca2+-independent inotropic pathways. In isolated, contracting rat ventricular myocytes, A2aR 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 β-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 β-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, A2aR have been shown to mediate increases in ventricular myocyte shortening (5, 17, 28). Recently, it was suggested that the A2aR-induced positive inotropic effects are mediated, in part, by a cAMP-independent mechanism that may differ from that of β-adrenergic receptor inotropic responses (5,17).
Differences between the characteristics of β-adrenergic- and A2a-adenosinergic-induced contractile responses suggest that these inotropic effects are mediated by different mechanisms (5). β-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, β-adrenergic receptor-mediated inotropic responses are characterized by a shortened duration of contraction and an increased rate of relaxation (5, 15). In comparison, A2aR stimulation has no effect on the maximum rate of relaxation and the duration of shortening is prolonged (5). Actually, the characteristics of A2aR-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 A2aR 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 A2aR activation are not accompanied by changes in Ca2+transients. Although A2aR-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 A2aR agonists reported previously may have resulted from the simultaneous activation of opposing adenosine A1R (27). Adenosine A1R mediate inhibitory effects on β-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 β-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 10−4 M. To unmask possible A2aR-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 A2aR agonist CGS-21680 or Ado to myocytes that had been incubated in PTx. PTx uncouples the A1R from its effector (Gi) (13), thereby inhibiting A1R-mediated effects. To ensure that inhibition of the A1R-Gipathway was achieved by the incubation of ventricular myocytes in PTx, the effects of A1R activation on β-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-Gipathway 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 A2aR-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 A2aR 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 β-adrenergic receptor activation, which confirms previous reports (9,22). Furthermore, the β-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 β-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 β-adrenergic receptor-mediated inotropic responses, A2aR 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 A2aR activation (5) further supports the notion that A2aR mediate their effects via Ca2+- independent inotropic pathways.
Ca2+-independent inotropic effects, such as those elicited by α1-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 A2aR-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 A2aR 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 A2aR inotropic responses warrant further investigation.
In summary, this study demonstrates that with similar contractile responses in cardiomyocytes to A2aR and β-adrenoceptor agonists the increases in Ca2+ transients are minimal with A2a-adenosinergic compared with β-adrenergic stimulation. These data suggest that A2aR effects are mediated by a Ca2+-independent inotropic pathway, as opposed to the Ca2+-dependent pathway of β-adrenergic inotropic responses.
The authors thank Lynne G. Shea for excellent technical assistance.
Address for reprint requests and other correspondence: J. G. Dobson, Jr., Dept. of Physiology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655-0127.
This study was supported by National Institutes of Health Grants HL-22828 and AG-11491. The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of the awarding agencies.
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- Copyright © 1999 the American Physiological Society