Adenosine Mediates Sustained Adrenergic Desensitization in the Rat Heart via Activation of Protein Kinase C
Abstract—Adenosine attenuates the myocardial metabolic and contractile responses induced by β-adrenergic stimulation. Our study was conducted to investigate the longevity of this antiadrenergic action after adenosine exposure. Adenosine (33 μmol/L) was infused into isolated perfused rat hearts for 1, 5, 30, or 60 minutes, and the adrenergic responsiveness (AR) to isoproterenol (10−8 mol/L) was determined at the end of each infusion period and during a 45-minute adenosine washout period. Interstitial levels of adenosine, as determined from epicardial surface transudates, returned to preinfusion levels within 10 minutes of washout. The duration of adenosine infusion had no effect on the extent of attenuation of AR at the end of the infusion. Whereas AR returned to preadenosine levels with washout of shorter adenosine infusions (1 and 5 minutes), there was a slow and incomplete recovery of AR after the longer exposures (30 and 60 minutes) to adenosine. The magnitude of this persistent antiadrenergic effect (PAE) of adenosine at 15 minutes of washout was proportional to the epicardial concentration of adenosine during infusion of the nucleoside. Infusion of adenosine either with the nonselective adenosine receptor antagonist 8-p-sulfophenyl theophylline or with the selective A1-receptor antagonist 1,3-dipropyl, 8-cyclopentylxanthine, abolished the PAE during the washout period. In addition, the PAE could be demonstrated only with the selective A1-receptor agonist 2-chloro-N6-cyclopentyladenosine and not with the selective A3-receptor agonist 4-aminobenzyl-5′-N methylcarboxamido-adenosine. When the protein kinase C (PKC) inhibitor chelerythrine was coadministered with adenosine, the PAE of adenosine was not apparent during adenosine washout. A 30-minute infusion of phenylephrine, an α-adrenergic agonist that enhances PKC activity, produced a PAE that lasted for up to 30 minutes of washout. This effect was prevented by the coinfusion of chelerythrine. Thus, it is concluded that the PAE of adenosine is determined by the myocardial concentration of this nucleoside and is manifested when myocardial concentrations of adenosine returned to baseline levels. Moreover, a 5-minute duration of adenosine exposure is required for the expression of the PAE. This latter effect seems to be dependent on adenosine-induced PKC activation via A1-receptors.
Adenosine is an ubiquitous nucleoside that has important functions in the heart, many of which are homeostatic and protective in nature.1 2 A notable myocardial action of adenosine is the reduction of metabolic and contractile responses induced by β-adrenergic stimulation.3 4 5 6 7 8 The general perception is that this action of adenosine is short-lived,3 4 5 but there is evidence that adenosine may have effects that last beyond the time of receptor activation. In this regard, Newman et al9 showed that prolonged exposure to elevated levels of adenosine (90 to 120 minutes) in the isolated guinea pig heart elicited a persistent blunting of the inotropic responsiveness to isoproterenol after as much as 1 hour of washout with an adenosine-free buffer. Analogous to the data of Newman et al9 is evidence that adenosine A1- and A3-receptor activation mediates preconditioning,10 11 12 a phenomenon whereby a transient increase of myocardial adenosine concentrations during brief episodes of ischemia provides protection against the depressant effects of subsequent ischemia. Adenosine-induced preconditioning supports the notion that the myocardial effects of adenosine persist beyond the time of washout. Recent observations suggest that preconditioning is mediated via activation and translocation of protein kinase C (PKC) in rat and rabbit hearts13 14 and in isolated rabbit myocytes.15 We postulated that the adenosine-induced activation of PKC might mediate the persistent antiadrenergic actions of adenosine.
We hypothesized that the antiadrenergic effects of adenosine may be dependent not only on the extent of the increase but also on the duration of the increased myocardial adenosine concentrations. In addition, we examined whether the effect of adenosine, analogous to preconditioning, may persist beyond the time when adenosine levels return to baseline values, and that these actions may be mediated by A1- and/or A3-receptor(s)-mediated activation of PKC.
Materials and Methods
Animals used in this study were maintained and used 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, US Department of Health, Education, and Welfare, National Institutes of Health Publication No. 85-23, 1996, and the guidelines of the Institutional Animal Care and Use Committee of the University of Massachusetts Medical School, Worcester, Mass.
Sixteen-week-old male Sprague-Dawley rats, weighing 380 to 540 g, were anesthetized with an intraperitoneal injection of ketamine (75 mg/kg) and xylazine (15 mg/kg). The hearts were then excised and immediately rinsed and weighed in ice-cold physiological saline solution (PSS). The wet heart weights ranged from 1.2 to 1.9 g. The PSS contained the following (in mmol/L): NaCl 118, KCl 4.7, CaCl2 2.5, NaHCO3 25, KH2PO4 1.2, MgSO4 1.2, and glucose 10. The pH of the perfusate was maintained at 7.4 by gassing the PSS with 95% O2 and 5% CO2. The hearts were then perfused retrogradely with PSS maintained at 37°C. The flow rate of the perfusate was adjusted to 10 mL/min per g of wet heart weight and was kept constant throughout the duration of the experiment. The perfusion pressure was monitored from a side arm of the aortic perfusion cannula with a strain-gauge manometer (P23, Statham, Hato Rey, Puerto Rico). Then, the hearts were inverted and instrumented, as previously described.16 The hearts were held vertically by applying a mild vacuum generated by a sink aspirator to the surface of the apical tips of the hearts via a polyethylene tube (1 mm, i.d.). This manipulation had minimal effect on the perfusion pressure as measured before the inversion.
The hearts were paced at 300 bpm, with the voltage 10% above threshold, and a pulse duration of 3 to 5 milliseconds was supplied by a Grass SD 9B stimulator (Grass Instrument Co) via platinum wire electrodes attached to the left atrium and the apex of the heart. Developed left ventricular (LV) pressure was determined using a latex balloon–tipped cannula filled with water and attached to a Statham P23Db pressure transducer. An appropriately sized balloon (Hugo Sachs) was inserted via the left atrium into the LV cavity. The balloon volume was kept constant at a diastolic pressure of 10 mm Hg to allow isovolumic contractions. The maximum rate of LV pressure development (+dP/dt) was obtained from a differentiating circuit in the physiological recorder (model 13-4615-71, Gould Instrument Systems Inc) with a high-frequency cutoff set at 300 Hz. Coronary flow was determined volumetrically. Epicardial and coronary effluent levels of adenosine were determined by sampling transudates that appeared on the ventricular surface and the pulmonary effluent, respectively, as described previously.16 Effluents were obtained for 30 seconds and boiled for 5 minutes to denature adenosine deaminase that might be present. Epicardial transudates (5 μL) were collected by capillary action into glass disposable microsampling pipettes (Fisher Scientific). Samples were collected within 15 to 30 seconds from the entire surface of the LV and were left for the duration of the experiment on a chilled aluminum micropipette holder.
The different agents used in the study were delivered just proximal to the aortic cannula at a rate appropriate to achieve the desired drug concentration in the PSS perfusing the isolated heart preparation. This infusion rate never exceeded 1% of the PSS flow rate.
Adenosine Concentrations in the Coronary Effluent and Epicardial Transudate
After coronary effluents were boiled and centrifuged, the supernatant was air-dried. The residues were resuspended in deionized water to 33% of their original volume and filtered (Supor-450, 0.45 μm, Gelman Sciences). The reconstituted coronary effluent samples were analyzed isocratically for adenosine on a high-performance liquid chromatograph (HPLC, Waters Chromatography Division), using a 5 μm C-18 resolve column (Waters Chromatography Division) and a mobile phase of 10 mmol/L KH2PO4 and 5% methanol (pH 4, 1.0 mL/min) with ultraviolet absorption was measured at 254 nm by methods described previously.16 Chromatographic data were collected and analyzed using a Maxima 820 Chromatographic workstation (Waters Chromatography Division). The adenosine concentrations were expressed in picomoles per milliliter.
Adenosine in the epicardial fluid was derivatized by adding 5 μL of the transudate to polypropylene microvials containing 20 μL of 1.5 mmol/L chloroacetaldehyde and 15 μL of distilled water. The microvials were tightly capped, heated at 80°C for 40 minutes, and either analyzed immediately or stored at −80°C. The fluorescent adenosine derivative, ethenoadenosine, was subsequently separated by HPLC and detected with a McPherson spectrofluorometer (SF-749, McPherson) and a high-sensitivity attachment (Schoefel). The excitation and emission wavelengths used were 275 and >360 nm, respectively, and the fluorometer time constant was 5 seconds. The output was directed to an HPLC workstation for subsequent analysis.
The isolated heart was equilibrated for 20 minutes before samples of the coronary effluent and epicardial transudate were collected. After equilibration, the contractile response to a 20-second infusion of isoproterenol (PSS concentration of 10−8 mol/L) was determined during 3 successive challenges (preinfusion control). A 10-minute recovery period was allowed between each infusion. A second set of effluent and interstitial fluid samples was collected 10 minutes after the last isoproterenol infusion. The experiment proceeded with 1 of the following infusion protocols (Figure 1⇓).
To assess the effect of duration of infusion and concentration of adenosine on the extent and persistence of the antiadrenergic effects of adenosine, the following experiments were designed. Adenosine was infused at a PSS concentration of 33 μmol/L for 1 and 5 minutes (set one), 30 minutes (set 2), and 60 minutes (set 3). The antiadrenergic action of adenosine was assessed by repeating the isoproterenol challenge at the end of the adenosine infusion and at 15, 30, and 45 minutes of adenosine washout. Because coronary effluent levels of adenosine cannot be presumed to reflect changes in the interstitial levels of this nucleotide,16 17 adenosine concentrations were determined in both the coronary effluent and epicardial transudate. Fluid samples were obtained every 5 minutes during the 60-minute infusion (set 3) and for ≈30 minutes after washout commenced to establish the time required for the adenosine concentrations to return to baseline levels.
The same protocol was repeated except that adenosine was infused at a lower dose (3.3 μmol/L) for 60 minutes (set 4). Also, to assess whether increases in endogenous adenosine produce antiadrenergic effects similar to exogenous adenosine, the same protocol was repeated with PSS containing the adenosine kinase inhibitor iodotubercidine (10 μmol/L) instead of adenosine (set 5). This intervention was expected to produce an increase in the interstitial levels of endogenous adenosine.18
On the basis of a demonstration that prolonged infusion of adenosine elicits a persistent antiadrenergic effect during the washout period, adenosine receptor antagonists were used to determine which adenosine receptor is involved in mediating this effect. Using the same protocol as before except that the infusion period was decreased to 30 minutes, the following receptor antagonists were coinfused with either 33 μmol/L adenosine or with the vehicle of adenosine: (1) the selective A1-receptor antagonist 1,3-dipropyl, 8-cyclopentylxanthine (DPCPX) at a dose of 1 μmol/L (sets 1 and 2), and (2) the nonselective adenosine receptor antagonist 8-p-sulfophenyl theophylline (8-SPT), at a dose of 5 μmol/L (sets 3 and 4). To further explore the pathways involved in mediating the persistent antiadrenergic action of adenosine, the effects of selective A1- and A3-receptor agonists on adrenergic responsiveness were assessed. The selective A1-receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA) was infused either for 1 minute (set 5) or for 30 minutes (set 6) at a dose of 10 μmol/L, and the same protocol as before was repeated during the 45-minute washout period. Then the selective A3-receptor agonist 4-aminobenzyl-5′-N methylcarboxamido-adenosine (AB-MECA), at a dose of 1 μmol/L, was either infused alone (set 7) or in combination with 10 μmol/L CCPA (set 8) for 30 minutes.
To explore whether activation of PKC is involved in these actions of adenosine, 2 μmol/L chelerythrine, a potent PKC inhibitor,19 was infused alone (set 1) or concurrently with the infusion of 33 μmol/L adenosine for 60 minutes (set 2). Chelerythrine was infused 10 minutes before the infusion of adenosine. To establish whether activation of PKC by other signal transduction pathways would also result in a persistent antiadrenergic effect, 5 μmol/L phenylephrine, an agonist which leads to enhancement of PKC activity,20 was infused for 30 minutes (set 3) and the same protocol was repeated with the coinfusion of 2 μmol/L chelerythrine (set 4). Isoproterenol challenges were repeated at the end of the infusion and at 5, 15, and 30 minutes of washout.
As a control for all the previous experiments and to verify the stability of the preparation, the adenosine vehicle, Milli-Q water, was infused for 60 minutes at the same flow rate as adenosine; isoproterenol challenges were repeated as before (set 1). Because the vehicle used to dissolve AB-MECA, CCPA, and DPCPX may have affected independently myocardial contractile function, the effect of a 60-minute infusion of dimethyl sulfoxide (0.01%) on the inotropic responsiveness to isoproterenol was evaluated in 5 hearts (set 2).
Isoproterenol was purchased from Sigma Chemical Co and dissolved in 0.1% sodium metabisulfite. Other agents were obtained from Research Biochemical International. Chloroacetaldehyde was purchased from Aldrich Chemical Co and purified by distillation. Chelerythrine, iodotubercidine, and 8-SPT were dissolved in purified water (Milli-Q system, Waters Co) whereas AB-MECA, CCPA, and DPCPX, were dissolved in dimethyl sulfoxide (Sigma Chemical Co) to make a 10−2 mol/L stock solution. The final concentration of dimethyl-sulfoxide in the perfusion solution was 0.01%.
Data are expressed as mean±SEM. During the isoproterenol challenges, the antiadrenergic effect of adenosine was expressed as the percentage change in +dP/dtmax relative to preinfusion control (average of 3) and was calculated as follows: (+dP/dtmax/+dP/dtmax during the preinfusion control period)×100.
ANOVA for repeated measurements was used to determine statistical significance within each experimental group, and factorial ANOVA was used for comparisons between the different interventions. Bonferroni’s correction was used for multiple comparisons. A Student t test was applied to paired comparisons. A probability of P<0.05 was used to indicate statistical significance.
Epicardial and Effluent Adenosine Concentrations
Preinfusion baseline concentrations of adenosine in the coronary effluent and interstitial fluid were within the same range in each group, increasing significantly within 1 to 5 minutes of infusion of adenosine, remaining elevated throughout the infusion period, and returning to baseline levels within 10 minutes of washout in all experiments (Figure 2⇓). Adenosine concentrations in both fluid compartments during 33 μmol/L adenosine infusion were ≈10-fold higher than with the 3.3 μmol/L adenosine.
At 60 minutes of iodotubercidine infusion, the adenosine concentration in the coronary effluent increased significantly from the baseline level of 46±8 to 80±18 pmol/mL, and in the epicardial transudate the concentration increased from 127±10 to 353±88 pmol/mL. Both levels returned to control values within 5 minutes of cessation of iodotubercidine administration. Adenosine concentrations attained in the epicardial transudate and coronary effluent with iodotubercidine were 3-fold and 8-fold lower, respectively, than the levels obtained during 3.3 μmol/L of adenosine infusion.
Antiadrenergic Actions of Adenosine
Effects of Duration of Infusion and Concentration of Adenosine
Overall, the duration of adenosine infusion had no effect on the extent by which adrenergic responsiveness to isoproterenol was attenuated, but had a significant impact on the rate at which adrenergic responsiveness returned to baseline levels following adenosine washout (Figure 3⇓). A 1-minute infusion of adenosine suppressed the response to isoproterenol by 50%. By 15 minutes of washout, the response to isoproterenol was of similar magnitude to the preadenosine infusion response (Table 1⇓). With 5 minutes of the same dose of adenosine, a similar effect on the extent of adrenergic suppression to the 1-minute infusion occurred. However, in contrast to the latter, the response to isoproterenol was still significantly suppressed at 15 minutes and only returned to preadenosine infusion levels at 30 minutes of washout. Both 1- and 5-minute infusions of 33 μmol/L of adenosine significantly decreased the coronary perfusion pressure by 13±1%. This was associated with a significant decrease (12±1%) in both developed left ventricular pressure and +dP/dt.
The 30 minutes of 33 μmol/L adenosine did not affect the coronary perfusion pressure or +dP/dt but significantly suppressed the contractile response to isoproterenol by 52%. In contrast to the shorter infusions, there was a slow and incomplete recovery of the contractile response to isoproterenol. At 45 minutes of washout, there was still a significant depression (−16%) of adrenergic responsiveness. Compared with the 30-minute infusion, 60 minutes of adenosine did not further change either the extent of attenuation of adrenergic responsiveness or the rate and completeness of recovery of the contractile responsiveness to isoproterenol during the washout period. This persistence of contractile suppression after prolonged adenosine infusions occurred at the time when adenosine concentrations in the epicardial fluid had returned to baseline levels.
Developed LV pressure and +dP/dt did not change during vehicle infusion and up to 30 minutes of its washout (Table 1⇑). However, at 45 minutes of washout, the coronary perfusion pressure significantly increased by a mean of 6 mm Hg. This change in perfusion pressure was accompanied by a small, but significant, increase in +dP/dt.
The 60 minutes of 3.3 μmol/L adenosine did not alter the coronary perfusion pressure or +dP/dt and was associated with less marked contractile depression in response to isoproterenol than the higher dose of adenosine (Table 2⇓). But, as with the higher adenosine concentration, there was a slow and incomplete recovery during the washout period. Infusion of 10 μmol/L iodotubercidine did not change the coronary perfusion pressure or +dP/dt, but resulted in a significant (21%) attenuation of the contractile response to isoproterenol (Table 2⇓). There was a continued significant depression of the contractile response to isoproterenol at 15 minutes of washout at a time when the adenosine concentration in the epicardial transudate had returned to baseline levels.
When data from the high- and low-dose adenosine and iodotubercidine infusions were combined, it was apparent that the magnitude of adrenergic desensitization at 15 minutes of washout was proportional to the prewashout concentration of adenosine in the epicardial transudate (Figure 4⇓).
Effect of A1- and Nonselective Adenosine-Receptor Antagonists
The simultaneous infusion of the A1-receptor antagonist, DPCPX (1 μmol/L), with 33 μmol/L adenosine completely prevented the adenosine-induced attenuation of the contractile response to isoproterenol at 60 minutes of infusion compared with DPCPX alone (Table 3⇓, Figure 5A⇓). This selective A1-antagonist alone was associated with a significant increase in the coronary perfusion pressure (+17%) and developed left ventricular pressure (+30%) at the end of the infusion (Table 3⇓).
In a way similar to DPCPX, the nonselective adenosine receptor antagonist 8-SPT also completely prevented the adenosine-induced attenuation of the isoproterenol-elicited contractile responses (Table 3⇑, Figure 5B⇑). This antagonist, when infused alone, caused an even greater increase in developed left ventricular pressures (+73%) and baseline +dP/dt (+144%) than DPCPX alone (Table 3⇑). These values returned to preinfusion control levels during the washout period.
Effects of Adenosine A1- and A3-Receptor Agonists
A 1-minute infusion of the selective A1-receptor agonist CCPA at 10 μmol/L suppressed the response to isoproterenol by 42% (Table 4⇓). As with 1 minute of adenosine, the contractile response to isoproterenol was similar to the preinfusion control levels at 30 minutes of washout and were only moderately reduced at 15 minutes washout (Figure 6⇓). The 30 minutes of CCPA suppressed the response to isoproterenol to a similar extent as the 1-minute infusion. However, as with prolonged adenosine infusions, the contractile responses to isoproterenol remained significantly depressed at 30 minutes of washout.
The 30-minute infusion of the A3-receptor agonist, AB-MECA, had no effect on the contractile responses to isoproterenol at the end of the infusion (Table 4⇑) and during the washout period (data not shown). Furthermore, the addition of AB-MECA to CCPA did not alter the extent of adrenergic desensitization during the infusion and washout period (Table 4⇑, Figure 6⇑).
Effects of PKC Activation and Inhibition
Compared with adenosine alone, the simultaneous infusion of 33 μmol/L of adenosine and 2 μmol/L of chelerythrine did not affect the extent of adrenergic desensitization at the end of the infusion, but this combination prevented the adenosine-induced sustained adrenergic desensitization during the washout period (Table 5⇓, Figure 7⇓). Chelerythrine alone caused a significant increase in baseline developed left ventricular pressure (+66%) and +dP/dt (+90%). However, during the washout period, there was a progressive, although modest, decrease in the contractile response to isoproterenol.
To activate PKC independently, phenylephrine was used to assess whether it produced a persistent antiadrenergic effect. Phenylephrine alone resulted in an equally significant increase baseline-developed left ventricular pressure (+81%) and +dP/dt (+89%), and, similar to chelerythrine alone, there was a progressive decrease in the contractile responses to isoproterenol at the end of the infusion and during the washout period. These effects on adrenergic responsiveness were largely abolished when 2 μmol/L of chelerythrine was coinfused with phenylephrine (Figure 8⇓).
Effects of Vehicle Infusion
In 5 animals, infusion of 0.01% dimethylsulfoxide, the solvent used to dissolve DPCPX and iodotubercidine, altered neither the baseline hemodynamics nor the response to isoproterenol (data not shown).
This study demonstrates that the antiadrenergic actions of adenosine are more complex than previously appreciated. The major finding is that there seems to be 2 distinct components. First, in the presence of adenosine, there is a PKC-independent effect that is reversible on normalization of adenosine concentrations in the interstitial fluid. Second, there is a persistence of adrenergic desensitization that is evident even though interstitial adenosine levels have returned to preinfusion levels and the β-adrenergic desensitization seems to be mediated by PKC activation. This latter response seems to be mediated principally via adenosine A1-receptor activation.
Persistence of the Antiadrenergic Action of Adenosine
In accordance with earlier observations,21 this study showed that in the presence of adenosine the antiadrenergic effect is dose-dependent and, to a far lesser extent, dependent on the duration of exposure. Furthermore, the depression of β-adrenergic responsiveness induced by prolonged (30 or 60 minutes), as opposed to short-term (1-minute), exposure to adenosine persisted beyond the time when adenosine concentrations in the effluent and epicardial transudate returned to baseline levels. This suggests that the depressed contractile responsiveness observed during the washout period after the infusion of adenosine or iodotubercidine is unlikely to be caused by a delayed clearance of adenosine. Although we acknowledged that the concentration of adenosine in the epicardial transudate may be different from the actual interstitial values, this underestimation is thought to be minimal.22 Persistence was evident in response to increases in both exogenous and endogenous adenosine. However, increases in endogenous adenosine levels attained with the adenosine kinase inhibitor, iodotubercidine, were much lower than those achieved during infusion of adenosine.
The extent and duration of the adrenergic desensitization after adenosine washout seemed to be related to the prewashout concentration of adenosine in the transudate, as evidenced by the greater and more prolonged depression after high-dose, versus low-dose adenosine and iodotubercidine infusions. Furthermore, the recovery of the contractile response to isoproterenol at 15 minutes of washout was complete after the 1-minute adenosine infusion, moderately depressed after the 5-minute adenosine infusion, and more severely depressed after the longer infusions (30 and 60 minutes). The data suggest that the persistence of adrenergic desensitization seems to be determined by the prewashout myocardial concentration of adenosine and dependent on the duration of adenosine exposure.
Newman et al9 came to a similar conclusion about the time-dependent nature of the antiadrenergic actions of adenosine. However, in contrast to the present work, they concluded that the desensitizing effect on inotropic responsiveness required a minimum of 90 minutes of 10 μmol/L adenosine in the guinea pig heart. The explanation for the discrepancy in the time required for the desensititizing effect to be expressed between the observations of Newman et al9 and the data presented in this study is not readily apparent. A possible explanation is that unlike in this study in which a single dose of 10−8 mol/L isoproterenol was used to characterize adrenergic responsiveness, Newman et al9 obtained a dose-response curve using 24, 47, and 94 pmol/L isoproterenol at 15 minutes of adenosine washout. Possibly, the last isoproterenol challenge was completed long after 15 minutes of washout. This may have allowed more time for contractile responsiveness to recover during the time when data were acquired in a dose-dependent fashion. Thus, Newman et al9 may have been unable to detect a subtle depression of the contractile response at 15 minutes of washout as opposed to a protocol in which a single dose of isoproterenol was used. In the present study, a minimum duration threshold of 5 to 30 minutes was needed to exhibit persistence of adrenergic desensitization. This is comparable with the time required for adenosine to induce preconditioning.23 24 25 26
Notwithstanding some differences, the data of this study, together with the findings of Newman et al,9 suggest that prolonged exposure to adenosine induces a sustained depression of isoproterenol-elicited myocardial contractile activity. Hence, adenosine exerts a myocardial antiadrenergic effect via the induction of second messengers that persist after removal of adenosine from its membrane receptor(s).
To elucidate the pathway(s) involved in the expression of the aforementioned effect, we wished to establish which of the adenosine receptors known to exist on myocyte cell membranes are responsible for the transduction of this phenomenon. Four subtypes of adenosine receptors have been identified.27 These receptors are classified on the basis of their inhibitory or stimulatory effects via G proteins on adenylyl cyclase. The A1- and A3-subtypes are coupled to the inhibitory pertussis toxin-sensitive G protein, while the A2a- and A2b-receptors are coupled, in a high- and low-affinity manner, respectively, to the stimulatory G protein. Data from the studies in which adenosine was coadministered with a selective A1-receptor and a nonselective adenosine receptor antagonist, as well as from studies in which selective A1- and A3-receptor agonists were infused for the same period of time, suggest that the sustained antiadrenergic action of adenosine is principally transduced via the adenosine A1-receptor. Nonselective adenosine receptor blockade with 8-SPT and selective A1-receptor blockade with DPCPX completely abolished the persistent adrenergic desensitization during the washout period. In addition, persistent adrenergic desensitization could be demonstrated only with the selective A1-receptor agonist CCPA and not with the infusion of the selective A3-receptor agonist AB-MECA. Furthermore, the antiadrenergic effects of CCPA were not enhanced with the coinfusion of AB-MECA.
Mechanisms were considered by which adenosine may induce rapid covalent modification of β-adrenergic receptors responsible for short-term desensitization and/or affect isoproterenol-induced activation of adenylate cyclase. Newman et al9 demonstrated that prolonged adenosine exposure was associated with reduced activation of adenylyl cyclase by isoproterenol in membrane fractions but not with a reduction in the density or in the affinity of the β1-adrenergic receptors.9 However, the adenosine agonist phenylisopropyladenosine has demonstrated also the ability to reduce high-affinity isoproterenol binding to the β-adrenergic receptor of rat myocardial membranes.28 Because PKC activation has been demonstrated to desensitize β-receptor–mediated stimulation of adenylyl cyclase and to promote phosphorylation of the β-adrenergic receptor,29 30 and because adenosine seems to activate PKC,31 it was hypothesized that PKC may be involved in the mediation of this persistent adrenergic desensitization. The demonstration that the PKC inhibitor, chelerythrine, completely abolished the persistence of the adrenergic desensitization after adenosine washout strongly suggests that PKC is involved in this phenomenon. However, the addition of this inhibitor did not alter the extent of contractile depression at the end of the infusion of adenosine.
Further support for the role of PKC activation in mediating the phenomenon of sustained β-adrenergic desensitization is found in data which show that a 30-minute infusion of the α-adrenergic agonist, phenylephrine, produces a persistent antiadrenergic effect that lasts up to 30 minutes of washout. This effect was prevented by the coinfusion of chelerythrine. The noticeable similarity between the persistent antiadrenergic actions of adenosine and phenylephrine may be explained by the latter agonist, similar to adenosine, which has been shown to mimic ischemic preconditioning via activation of the pertussis toxin–sensitive G protein, which leads to the subsequent enhancement of PKC activity.20
Physiological Significance of the Longer-Term Effects of Adenosine
At first glance, the demonstration of the delayed effects of adenosine after the infusion of supraphysiological (33 μmol/L) concentrations of this purine may have less relevance in the context of the intact heart where fluctuations in adenosine levels under normal and pathological conditions are unlikely to be within a similar range. However, the persistence of adrenergic desensitization was also observed after the infusion of a lower dose (3.3 μmol/L) of adenosine. This dose achieved concentrations in the epicardial transudate that were comparable with those measured in the rat heart during hypoxia.32 Moreover, data from the iodotubercidine experiment illustrated that the persistent antiadrenergic effects can also be detected after only modest increases in endogenous adenosine concentrations.
The striking analogy of this phenomenon to adenosine-induced cardiac preconditioning lends further credence to its physiological importance. These 2 effects of adenosine are similar in that both are dose-dependent, involve adenosine A1-receptor–mediated PKC activation, and are manifest at a time when adenosine concentrations are within a physiological range. However, the persistent antiadrenergic effects of adenosine may only be apparent in rats and guinea pigs9 and not in larger animals, because adenosine does not seem to increase protein kinase C activity in dogs.33
This study showed that the antiadrenergic actions of adenosine involve both short-term and sustained cellular responses. The short-term effect is expressed in the presence of adenosine and is not mediated by PKC activation. The sustained antiadrenergic effect is determined by the myocardial concentration of adenosine, is manifested when adenosine concentration had returned to baseline levels, and a minimum duration of adenosine exposure of ≈5 minutes is required for the expression of this effect. This latter effect is dependent on adenosine-induced PKC activation, via A1-receptor–mediated mechanisms. Therefore, adenosine induces a sustained antiadrenergic effect that may extend its cardioprotective actions beyond the time when adenosine concentrations have returned to normal levels.
Dr Perlini was supported, in part, by the Salvatore Campus Award from the Italian Society of Hypertension. The authors thank Robert J. McGinn, Julius Yang, and Filip Bartl for their excellent technical assistance.
Presented in part at the 69th Scientific Sessions of the American Medical Association, New Orleans, La, November 10–13, 1996, and published in abstract form (Circulation. 1996;94[suppl I]:I-183).
- Received June 23, 1998.
- Accepted July 8, 1998.
- © 1998 American Heart Association, Inc.
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