(Circulation. 1996;94:734-741.)
© 1996 American Heart Association, Inc.
Articles |
Modulation of the Hyperpolarization-Activated Current (If) by Adenosine in Rabbit Sinoatrial Myocytes
the Dipartimento di Fisiologia e Biochimica Generali, Universita di Milano, Milano, Italy.
Correspondence to Antonio Zaza, MD, Dipartimento di Fisiologia e Biochimica Generali, Via Celoria 26, 20133 Milano, Italy.
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Abstract |
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Background Modulation of sinoatrial pacemaking by adenosine (Ado) in the absence of concomitant adrenergic stimulation (direct modulation) has been attributed to activation of a K+ conductance. In the present study, we evaluated the direct effects of Ado on the pacemaking current If and tested their interaction with those of acetylcholine (ACh).
Methods and Results Rabbit sinoatrial myocytes were patch-clamped at 35°C in the presence of 1 mmol/L BaCl2 and 2 mmol/L MnCl2. Ado (1 µmol/L) reversibly reduced If by 33.1±5.7% of control (n=5; P<.05). Ado (1 µmol/L) reversibly shifted If midactivation potential by -6.63±1.18 mV (n=4; P<.05). Fully activated If conductance (0.262±0.037 versus 0.254±0.036 nS/pF; n=6, NS) and reversal potential (-17.35±0.99 versus -18.01±1.42 mV; n=6, NS) were not changed by 10 µmol/L Ado. The Ado receptor antagonist 8-PST (10 µmol/L) reversed the effect of 0.3 µmol/L Ado by 64.9±4.2% (n=6; P<.05). Ado maximally shifted the If activation curve by -5.85 mV, with a half-maximal concentration of 0.0796 µmol/L (n=93). The shifts in If activation induced by Ado (0.3 µmol/L) and ACh (1 µmol/L) separately were -4.89±0.05 and -8.84±0.51 mV, respectively; concomitant Ado and ACh superfusion shifted activation by -9.7±0.45 mV (NS versus ACh alone; n=9). Threshold Ado concentrations dose-dependently reduced the rate of spontaneous pacemaker activity (eg, -18.8±3.4% at Ado 0.03 µmol/L).
Conclusions Submicromolar Ado directly inhibits If and slows pacemaking in sinoatrial myocytes; the mode of If inhibition is similar to that previously described for ACh. Thus, Ado may exert local modulation of sinus rate through signaling pathways similar to those used by ACh.
Key Words: sinoatrial node • electrophysiology • adenosine • acetylcholine
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Introduction |
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In addition to autonomic neural activity, cardiac function is controlled by local modulating factors, among which Ado has a central role. Ado is a nucleoside, in part derived through ATP catabolism, whose production in cardiac muscle is increased when oxygen supply is inadequate.1 Ado is transported to the extracellular space, where, acting through specific membrane receptors, it exerts inhibitory effects on cardiac function, including negative chronotropy.2 3 4
In addition to antagonizing the action of catecholamines, Ado inhibits automaticity in sinoatrial myocytes under basal conditions.3 This "direct" chronotropic effect of Ado was entirely attributed to the activation of a K+ conductance.3 5 Indeed, If was reported to be insensitive to Ado under basal conditions.5 Because Ado inhibits adenylate-cyclase activity in cardiac myocytes,4 its lack of effect on If is in apparent contrast with the observation that this current is reduced by ACh under basal conditions through a decrease in intracellular cAMP levels.6
In the present study, we investigated the mode of Ado action on If in the sinus node and its relation to that of ACh. The results obtained indicate that Ado directly inhibits If in the SA node, with effects qualitatively comparable to those of ACh. Ado concentrations required for If inhibition are compatible with the hypothesis of negative feedback control of sinus rate exerted by the nucleoside during physiological metabolic stress.
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Methods |
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Cell Isolation
Myocytes from the rabbit SA node region were isolated according to a procedure described previously.7 Briefly, female white rabbits (weight range, 0.8 to 1 kg) were anesthetized through exposure to cotton wool soaked in tribromoethanol solution (200 mg tribromoethanol dissolved in 10 mL of ether) and killed through cervical dislocation and exanguination. Hearts were quickly removed and placed in normal Tyrode's solution containing (in mmol/L) NaCl 140, KCl 5.4, CaCl2 1.8, MgCl2 1, D-glucose 5.5, and HEPES-NaOH 5, adjusted to pH 7.4. The ventricles were removed, and the SA node area was exposed. The region enclosed laterally by the crista terminalis and the interatrial septum and caudorostrally by the orifices of the inferior and superior venae cavae was isolated through the removal of surrounding atrial tissue and cut into a series of 1-mm-wide strips perpendicular to the crista terminalis border. These were rinsed several times with a nominally Ca2+-free solution containing (in mmol/L) NaCl 140, KCl 5.4, MgCl2 0.5, KH2PO4 1.2, D-glucose 5.5, taurine 50, and HEPES-NaOH 5, adjusted to pH 6.9, and transferred to an enzyme solution containing (in the same nominally Ca2+-free solution) 224 U/mL collagenase type I (tryptic activity, 0.36 U/mg), 1.9 U/mL elastase type I, 0.6 U/mL protease type XIV, 1 mg/mL BSA, and 200 µmol/L CaCl2. The tissue was triturated in the enzyme solution for 15 to 25 minutes at 37°C until the pieces became soft and filamentous. The tissue was next rinsed with a Ca2+-free, potassium glutamate-based salt solution containing (in mmol/L) KCl 20, glutamic acid 70, D-hydroxybutyric acid (Na salt) 10, KH2PO4 10, HEPES-KOH 10, KOH 80, taurine 10, and BSA 1 mg/mL, adjusted to pH 7.4, and then triturated for 15 minutes at 37°C in this solution. The cell suspension was filtered through a nylon mesh, and the calcium concentration was gradually raised to a final concentration of 1.3 mmol/L. The final storage solution contained (in mmol/L) NaCl 100, KCl 35, CaCl2 1.3, MgCl2 0.7, and BSA 1 mg/mL, pH 7.4. The cells were kept in this solution for
8 hours (4°C), until use.
Experimental Solutions and Data Acquisition
Isolated cells were allowed to settle to the bottom of a 30-mm polylysine-coated petri dish that contained a plastic ring to reduce total volume to 
1 mL. The dish was placed on the stage of an inverted microscope and continuously superfused at a rate of 2 mL/min with normal Tyrode's solution containing (in mmol/L) NaCl 140, KCl 5.4, CaCl2 1.8, MgCl2 1, D-glucose 5.5, and HEPES-NaOH 5, adjusted to pH 7.4. A thermostated pipette with multiple superfusion lines was positioned over the individual cell being studied to allow solution changes within 1 second. The temperature of the superfusing solution was monitored at the tip of the pipette with the use of a fast-response digital thermometer (BAT-12, Physitemp). Flow rate through the various lines was adjusted to maintain the temperature at 35±0.1°C. Currents were measured in the whole-cell configuration (Axopatch 200-A, Axon Instruments) through the use of pipettes with a tip resistance between 3 and 5 M
. Rs values were compensated to 85% to 90% of their initial value (5 to 15 M). During If measurements, 1 mmol/L BaCl2 and 2 mmol/L MnCl2 were added to the Tyrode's solution to minimize contamination by K+ and Ca2+ currents, respectively. The pipette solution contained (in mmol/L) NaCl 10, aspartic acid 130, KOH 146, MgCl2 2, ATP-Na salt 2, creatine phosphate 5, GTP 0.1, EGTA 5, CaCl2 2 (calculated free Ca2+=10-7 mol/L), and HEPES-KOH 10, pH 7.2. To avoid perturbations to the intracellular milieu, the spontaneous pacemaking rate was measured in the cell-attached configuration during superfusion with normal Tyrode's solution. To this end, the exterior of the membrane patch was held at 0 mV and the peak of the "action current," elicited by the spontaneous activity of the cell, was used as a marker of activation. The intervals between subsequent activations were then measured, with an accuracy of ±1 ms, through the use of custom-made software. In another group of cells, pacemaker activity was recorded in the whole-cell configuration to study Ado effects on membrane potential. In these experiments, the EGTA concentration of the pipette solution was reduced to 0.1 mmol/L (Ca2+ concentration adjusted to pCa=7) to minimize buffering of intracellular Ca2+ transients (cells were vigorously contracting long after achieving the whole-cell configuration).
Data were recorded with a PCM videorecording system during the experiment and then digitally analyzed off-line by replaying the data into a 12-bit A/D board.
Collagenase was obtained from Worthington Biochemical; elastase, protease, Ado, and ACh were obtained from Sigma; and 8-PST was obtained from RBI.
Experimental Protocols
Steady-state If activation curves were constructed by measuring tail currents at 15 mV after activating steps of an appropriate duration at various potentials (see Fig 2
). Fully activated I-V relations (see Fig 3) were obtained by measuring tail currents at various potentials after activation of If by a conditioning pulse at a very negative potential (eg,-125 mV). In both protocols, contamination by currents other than If was removed by subtracting tails recorded after a conditioning pulse to potentials where If activation was negligible (eg,-15 mV).7
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The effect on If of agonists modulating its gating properties is best estimated in terms of the shift in V dependence of activation induced by the agonist. However, the presence of even reduced current rundown biases the appraisal of changes in If activation by protocols requiring several minutes, such as the one for construction of the full activation curve mentioned above. Thus, after demonstration through complete activation protocols that Ado modulates If via a parallel shift of the activation curve (see Fig 2
), quantitative evaluation of the effect of this agonist on the current was obtained with a faster method.8 From -35 mV, steps of constant amplitude were applied in the control solution at a rate of 1/6 Hz to a voltage E near the midactivation point. During superfusion of Ado, the If inhibition was compensated for by adjusting the holding potential while keeping the step magnitude constant, and the sm was measured. Under these conditions, the compensated current in Ado equals the control current, ie:
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(E) is the steady-state activation variable at voltage E, and s is the actual shift induced by Ado. This relation can be linearized by developing to the first term of Taylor series its right end term, which after rearranging yields:
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=0.5, Ef=-17.35 mV (measured If reversal potential in this set of experiments; see "Results"), and y'=dy/dE=-0.0988 mV-1 (mean value of Boltzmann slopes measured from mean activation curves in control and after washout; see Fig 2). This procedure allowed quick evaluation of shifts with a resolution of tenths of a millivolt due to the high steepness of the If activation curve. The difference between measured (sm) and actual shift (s) did not exceed 9% of sm in the worst case. Unless otherwise specified, this method was used throughout the study for quick assessment of agonist-induced shift of activation. Only measurements in which the agonist effect was completely reversible were used. Cm was estimated from the capacity compensation current required after compensation of Rs to eliminate capacity transients during small hyperpolarizing V-clamp pulses.
Statistical Analysis
One-way ANOVA for repeated measurements was used for comparison of multiple mean values. Post hoc comparison between individual mean values was performed with Tukey's MSD test. Curve parameter estimation was performed by a nonlinear, least-squares fitting routine (developed by D. Goldsby, Emory University, Atlanta, Ga). The 95% CIs of parameter estimates were used to test for the difference between individual curves within each cell. A probability level of <.05 was used to define significance throughout the study. In the text and figures, values are presented as mean±SEM.
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Results |
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Effect of Ado on If
Initially, experiments were performed to test whether Ado had a direct effect on If. Contrary to previous reports,5 Ado alone inhibited If activated by hyperpolarizing pulses in the midactivation potential range. Ado (1 µmol/L) reduced If, measured as the time-dependent current activated 1.5 seconds after the beginning of the hyperpolarizing step, by 33.1±5.7% (n=5; P<.05). As shown in Fig 1,
both the amplitude and the rate of activation of If were reversibly reduced by the nucleoside.
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Mode of Ado Inhibition of If
Ado inhibition of If might result from a negative shift in the voltage dependence of activation, a reduction of fully activated conductance, or both of these actions simultaneously. To discriminate between these possibilities, we studied separately the effects of Ado on the If activation curve and on the If fully activated I-V relation.
Activation curves were constructed with the complete protocol (see Fig 2 and "Methods") before, during, and after exposure to Ado. Activation parameters, estimated through Boltzmann fits of normalized data points from individual cells, showed that 1 µmol/L Ado shifted the mid-activation potential (E50) by -6.63±1.18 mV (from -69.64±2.04 to -76.27±3.18 mV; n=4, P<.05) without changing the slope factor (from 0.101±0.01 to 0.110±0.008 mV-1; NS); 97.4±1.6% of the change in mid-activation potential reversed on Ado wash-out.
The effect of Ado on the conductance of fully activated If is shown in Fig 3. Neither the steepness of the I-V relationship nor the reversal potential was affected by an Ado concentration as high as 10 µmol/L. The channel conductance, normalized to cell capacitance (Cm: mean=38.9±8.7 pF, n=6), was 0.262±0.037 nS/pF in control, 0.256±0.041 nS/pF during exposure to 10 µmol/L Ado, and 0.254±0.036 nS/pF after washout (n=6; NS). The If reversal potential, estimated from I-V relations, was -17.35±0.99 mV in control, -18.01±1.42 mV during 10 µmol/L Ado, and -17.98±1.47 mV after washout (n=6; NS).
The mechanism of Ado modulation of If was also investigated by a different protocol, which could not provide an exact estimation of the shift induced by Ado on If kinetics but was sufficiently fast to avoid possible interference of current rundown9 (Fig 4A). From a holding potential of -35 mV, the potential was stepped for 4 seconds to near the current midactivation value and then further hyperpolarized to produce full If activation. Ado (10 µmol/L) changed the proportion of total current activated in each of the two steps reciprocally (Fig 4C) without affecting the fully activated current at -135 mV (Fig 4B). This observation, reproduced in all the cells tested with this protocol (n=11), is again consistent with an action of Ado on the voltage dependence of If activation with a lack of action on the fully activated conductance.
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Dose Dependence of Ado Effects on If
Dose dependence of Ado effects (Fig 5A) was tested by exposing cells to Ado concentrations of 0.01 µmol/L (n=5), 0.03 µmol/L (n=13), 0.1 µmol/L (n=14), 0.3 µmol/L (n=13), 1 µmol/L (n=11), 3 µmol/L (n=8), and 10 µmol/L (n=29). Ado effect was expressed as the shift of activation curve estimated as detailed in "Methods." Average shift values were best fitted by the use of a Hill function (Fig 5B), which yielded a half-effective Ado concentration of 0.0796±0.0121 µmol/L, a maximal shift of -5.85±0.26 mV, and a Hill coefficient (n) of 1.42.
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The effect of increasing Ado concentrations on If can be better appreciated from Fig 5C, where the If steady-state I-V relations at various Ado concentrations ([Ado]) for a cell with a membrane capacitance of 38.9 pF (average measured value) are shown. The curves were estimated from the following relation:
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is the steady-state activation variable, gf is fully activated If conductance, and Ef is If reversal potential. y(E,[Ado]) was computed from the activation curve parameters, with E50 shifted, at each Ado concentration, according to the dose-response curve (Fig 5B). gf and Ef were obtained from the mean values measured from fully activated I-V relations (Fig 3).
Dependence of Ado Effect on Receptor Stimulation
8-PST, a polar theophylline derivative, was used to identify the type of receptor mediating Ado modulation of If (Fig 6). We chose a charged Ado receptor antagonist to prevent crossing of the cell membrane and inhibition of intracellular phosphodiesterases, an action common to most xanthines, to restrict the action of 8-PST to membrane receptor antagonism.
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In six cells (an example is shown in Fig 6A), 0.3 µmol/L Ado induced a negative shift of activation of -5.07±0.59 mV; 10 µmol/L 8-PST reduced Ado-induced shift to -1.80±0.31 mV (P<.05), thus antagonizing 64.92±4.19% of the nucleoside effect. 8-PST alone did not modify If, and its antagonistic effect was readily reversible on washout (Fig 6B).
Ado Versus ACh Modulation of If
This set of experiments aimed at testing the extent to which Ado further inhibited If after the current had been reduced by saturating concentrations of the autonomic neuromediator ACh. To this end, we evaluated the If activation shift induced by 0.3 µmol/L Ado either alone or in the presence of a saturating concentration of ACh (1 to 2 µmol/L). As illustrated by the example in Fig 7A and 7B, If was activated by hyperpolarizing steps to the steep portion of the activation curve; ACh and Ado were first applied separately and then in combination. The average activation shifts produced by these interventions in nine cells were (see Fig 7C) -8.84±0.51 mV (ACh alone), -4.89±0.05 mV (Ado alone), and -9.70±0.45 mV (Ado plus ACh; NS versus ACh alone). Thus, exposure to saturating ACh concentrations almost completely occluded the effect of Ado on If.
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Ado Modulation of Pacemaking Rate
To test whether If inhibition at low Ado concentrations was associated with changes in pacemaking rate, we measured the BR in the presence of three Ado concentrations (0.01, 0.03, and 0.1 µmol/L) corresponding to the lower end of the If dose-response relation. Ado-induced changes are reported in terms of BR rather than CL because the rate of diastolic depolarization is linearly related to BR (A. Zaza, unpublished observation). To prevent intracellular dialysis by pipette solution, cells were studied in the cell-attached configuration (see "Methods"). Washout was checked after each Ado concentration, and only fully reversible effects were considered. Results (Fig 8A and 8B) are reported for a total of 24 measurements from 10 cells. Average BR in control was 167.9±3.1 bpm. Ado (0.01 µmol/L) slightly but consistently slowed pacemaking (BR, -3.3±0.7% versus control; n=8, P<.05). A substantial decrease in BR was observed at 0.03 µmol/L Ado (-18.8±3.4% versus control; n=9, P<.05), whereas increasing Ado to 0.1 µmol/L resulted in a smaller further decrement (-20.1±3.4% versus control; n=7, P<.05).
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The effects of Ado on transmembrane action potentials were studied in another set of experiments performed in the whole-cell configuration on nine cells. From a control mean value of 200.3±8.5 bpm, Ado reduced BR by 7.5±3.1% at 0.01 µmol/L (n=9; P=.05), by 13.1±3.7% at 0.03 µmol/L (n=9; P<.05), and by 19.7±4.9% at 0.1 µmol/L (n=8; P<.05). Except for the higher control BR, these values were similar to those obtained in the cell-attached configuration. A decrease in the steepness of the whole diastolic depolarization would be expected if Ado effects on BR were primarily due to If inhibition. Such expectation was fulfilled in the majority of cells; however, in three cases, Ado mainly (although not exclusively) depressed the late portion of diastolic depolarization. Examples of these different response patterns are illustrated in Fig 8C. Changes in membrane potential, underlying Ado chronotropic effect, were analyzed by plotting Ado-induced changes in BR against the corresponding changes in the steepness of the early half of diastolic depolarization (SL50) (Fig 8D). Linear regression analysis showed that these parameters were rather strongly correlated (r=.89; P<.05), thus suggesting that a decrease in the steepness of the diastolic depolarization, including its early portion, was the prevailing mechanism of Ado-induced bradycardia.
A slight prolongation of action potential duration, probably reflecting rate-dependency of repolarization, occurred only when BR was strongly depressed by Ado. The maximum diastolic potential was -61.1±1.1 mV in control and remained unchanged at all Ado concentrations (eg, -62.1±1.3 mV at 0.1 µmol/L Ado; NS).
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Discussion |
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Ado Directly Modulates If and Pacemaking Rate
The results of the present study indicate that submicromolar concentrations of Ado decrease If and pacemaking rate in isolated rabbit SA myocytes directly, ie, independent of coexisting adrenergic stimulation. The implications of this finding are relevant to the understanding of intracellular transduction mechanisms and of the role of purinergic modulation of heart rate.
Ado effects were antagonized by 8-PST, a receptor antagonist with constants of inhibition (Ki) of 2.63 and 15.3 µmol/L for A1 and A2 receptor subtypes, respectively. The inhibition of the effect of Ado by 8-PST expected on the basis of such Ki can be computed as follows10 :
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The effects exerted by Ado through the A1 receptor subtype on ionic currents of atrial and ventricular myocytes closely resemble those of ACh.4 Indeed, the evidence available so far suggests that both Ado A1 and muscarinic M2 receptors exert their cardiac electrophysiological effects through G protein-mediated inhibition of the adenylate cyclase system.4 6 ACh has prominent direct inhibitory effects on If in rabbit SA myocytes6 8 9 ; thus, the absence of similar Ado effects, as previously reported,5 is difficult to reconcile with its specific inhibitory action on adenylate cyclase. In addition to demonstrating that Ado does inhibit If directly, the present study shows that its mode of action is similar to that previously reported for ACh and that saturating ACh concentrations occlude the inhibitory action of Ado. This supports the hypothesis that ACh and Ado receptors may share a common transduction pathway in the SA node, too.
The reason for the discrepancy between this and previous evidence3 5 is not obvious. Because If is activated by intracellular cAMP,11 the substrate for the direct inhibitory effects of Ado would be removed if basal cAMP levels were insufficient to support tonic If stimulation in the isolated cell. Thus, the lack of evidence for a direct Ado action on If might depend on different metabolic conditions of the myocytes studied, resulting in different cellular cAMP levels. At variance with previous work, 2 mmol/L MnCl2 was used to minimize contamination by Ca2+ currents. MnCl2 may induce small positive shifts in the If activation curve and slightly blunt the effects of ACh.12 Since MnCl2 concentration was constant through control and test solutions, the effects observed cannot be influenced by the ion. Based on the interaction with ACh, the presence of MnCl2 might have led to a slight underestimation of the Ado effects on If. It should also be considered that direct inhibition of If by Ado has been recently reported in myocytes isolated from the rabbit atrioventricular node.13
Relevance of If and IKAdo in Ado Modulation of Sinus Rate
A consequence of the evidence presented here is that the Ado-induced modulation of If may have a role in the direct negative chronotropic effect of this agent, previously entirely attributed to activation of a K+ current (IKAdo).3 5
The observation that the chronotropic effect of Ado was correlated with a decrease in the steepness of the initial phase of diastolic depolarization (Fig 8D) and the absence of changes in the maximum diastolic potential are both consistent with a role of If inhibition. In a few cells, Ado primarily (even if not exclusively) affected the late portion of diastolic depolarization (Fig 8C). A closer examination of Fig 8D shows that cells may deviate from the "average" response, as defined by the regression line, in both directions. That is, the same change in SL50, especially if small, may be associated to either smaller- or large-than-average changes in BR. If these deviations were caused by Ado, we should conclude that Ado had variable effects on late diastolic currents in different cells. Alternatively, cells might respond differently to a small, Ado-induced decrease in the rate at which the threshold potential is approached. Even subtle interindividual differences in the kinetics of ICaL or in the rectification of K+ currents might account for such a variability. The observation that take-off potential has a strong spontaneous beat-to-beat variability in isolated SA myocytes14 suggests that the late portion of diastolic depolarization may indeed be very unstable. In the intact sinus node, such a variability should be largely blunted by electrotonic interaction among cells. An effect of Ado on ICaL, the main late diastolic current, is also unlikely based on previous work showing that this current is only slightly inhibited by high Ado concentrations (10 µmol/L) in guinea pig atrial muscle15 and is unaffected in the rabbit sinus node.5
Ado inhibitory effect on If and on pacemaking rate occurred at similar threshold concentrations (0.01 µmol/L), and these concentrations were much smaller than those reported to activate IKAdo in the same cell type (10 to 50 µmol/L).5 On the other hand, as previously suggested,16 If inhibition may actually be an essential complement to the activation of IKAdo in reducing the automatic rate of SA myocytes at higher Ado concentrations. Fig 9 shows the results of a numerical simulation performed with software (OXSOFT HEART) based on the model of SA activity.17 18 This simulation shows how IKAdo activation and If inhibition, applied either concomitantly (Fig 9A) or separately (Fig 9B and 9C), would affect SA pacemaking rate. According to the model, the effect of IKAdo activation alone (Fig 9C) is very small; furthermore, the decrease in rate obtained by concomitant IKAdo activation and If inhibition (Fig 9A) is substantially larger than the sum of those resulting from either action separately. This is due to the fact that IKAdo activation causes membrane hyperpolarization; thus, the primary inhibitory action of this current may be largely offset by an increased activation of If, resulting from the more negative diastolic potential. If, on the other hand, the If activation curve is concomitantly shifted to more negative potentials, as experimentally verified in this work, the resulting depressant effect is markedly enhanced. For the same reason, for example, experiments that used If blockade (eg, by Cs+) as a method of testing the role of this current in pacemaking modulation3 19 should be interpreted with caution. Indeed, the effect of activation of K+ currents (IKACh or IKAdo) on membrane potential may be artifactually enhanced when the influence of If is strongly depressed by the addition of blockers.
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A current named IKDD has been recently proposed to support pacemaking in Purkinje myocytes.20 However, the relevance of such a mechanism to SA pacemaking and to its modulation by receptor agonists remains unknown.
Physiological and Pathophysiological Relevance of Ado Effect on If
The dose dependence of the Ado effect tested in the present study shows that If and pacemaking modulation occur at concentrations of the nucleoside between 0.01 and 0.1 µmol/L, comparable to those measured in cardiac interstitial fluid under basal conditions.21 22 This implies that Ado should exert a tonic restraint on sinus rate at rest. On the other hand, when tested in vivo23 24 or in isolated and perfused hearts,25 Ado receptor antagonists did not induce significant changes in basal heart rate. Such a discrepancy is subject to several interpretations. Direct Ado effects on sinus node may be concealed in vivo through activation of autonomic reflexes. On the other hand, due to different work loads and metabolic conditions, the levels of interstitial Ado in the isolated heart may not reflect the physiological ones. Therefore, the interpretation of sinus rate changes resulting from Ado receptor blockade in these experimental conditions may not be straightforward. Alternatively, interstitial Ado concentrations, measured from the whole heart, might not be representative of those present in the SA node. Indeed, with the same workload, the metabolic demand and, thus, Ado production might be smaller in myocytes with weaker contractile activity, such as SA myocytes.
Since, particularly during ischemia, but even in the normal heart, Ado production is a steep function of heart rate,22 inhibition of If by this nucleoside may be viewed as a component of the local feedback system preventing mismatch between cardiac oxygen supply and consumption during neurally mediated tachycardia.26
In the heart in situ, at least some adrenergic stimulation is tonically present; thus, the physiological relevance of a direct inhibitory effect of Ado, compared with that resulting from antagonism of adrenergic activation,5 may be questioned. However, in addition to its postsynaptic effects, Ado presynaptically inhibits neuromediator release from adrenergic synapses.1 Thus, the existence of a component of Ado modulatory effect independent of concomitant adrenergic stimulation may be functional to the physiological actions of the nucleoside. Furthermore, Ado concentrations reported to inhibit the effects of adrenergic activation of If5 are higher than those found to directly inhibit If in this study. Thus, direct and antiadrenergic effects of Ado may have different threshold concentrations.
Clinical Implications
According to the evidence presented, Ado modulation of sinus pacemaking may occur at very low concentrations, with a mechanism different from the one causing inhibition of AV nodal conduction (activation of IKAdo). Thus, negative dromotropic effects on AV node, instrumental to the Ado antiarrhythmic effect, may have a higher dose threshold than depression of sinus function. Indeed, intense bradycardia occasionally occurs after Ado administration.27 In normal subjects, chemoreceptor-induced reflex sympathetic activation usually overrides most of the direct cardiovascular effects of Ado.28 Thus, the prevailing negative chronotropic effects of Ado become manifest in subjects with weak reflex responses, such as recipients of cardiac transplants,29 patients affected by autonomic failure,28 and those under general anesthesia.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by grants MURST, CNR CT92.02582, and Telethon 396. We are grateful to Prof Arnaldo Ferroni for his thoughtful advice and criticism and to Gaspare Mostacciuolo for expert technical assistance.
Received December 11, 1995; revision received February 13, 1996; accepted February 19, 1996.
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