Diadenosine polyphosphates (APnAs) are members of a group of dinucleoside polyphosphates that are ubiquitous from bacteria to mammals (see Fig. 1). In recent years, the diadenosine polyphosphates have received increasing attention because they may act as extra- and intracellular messengers (1-6).
Intracellular APnAs are signal molecules that alert the cell under stress conditions. The amount of diadenosine polyphosphates increases after exposure to a wide variety of oxidants, and they have been suggested to act as "alarmones," alerting the cell to the onset of metabolic stress. Heat-shocked organisms are known to produce not only "heat-shock proteins" but also diadenosine tetraphosphate (AP4A) and related compounds (7).
Diadenosine polyphosphates can be stored in secretory granules of thrombocytes (8). They are present in the cytosol and can be transported to secretory granules, allowing their exocytotic release from synaptosomes. Extracellularly, they can act through P1- or P2-receptors. The dinucleotides are metabolized by soluble enzymes in the blood plasma as well as by membrane-bound ectoenzymes of endothelial cells, smooth muscle cells, and other cell types (9). The enzymatic cleavage of the dinucleotides plays a dual role for their biological function: (a) termination of the signal; and (b) generation of purinergically active products such as adenosine triphosphate (ATP), adenosine diphosphate (ADP), and finally adenosine. The effects of most diadenosine polyphosphates on force of contraction have been reported in animal and human cardiac preparations. Differences due to length of the phosphate chain have been noted. The differences were qualitative, quantitative, and between species. For instance, AP6A exerted a negative inotropic effect in human atria, whereas AP4A increased force of contraction in the same preparation. Moreover, AP3A was a less effective negative inotropic compound than AP6A. Finally, AP4A exerted a positive inotropic effect in human atrial preparations but a negative inotropic effect in guinea pig and canine atrial preparations (10,11). The diadenosine polyphosphates use at least two receptor types for mediation of their inotropic effects. The negative inotropic effects of AP4A and AP6A are mediated through A1-adenosine receptors (10,11). The positive inotropic effect of AP4A is mediated through a P2-purinoceptor and accompanied by an increase in inositol-1,4,5-triphosphate (IP3) (10,12).
Interestingly the shortest diadenosine phosphate congener, AP1A, has not been widely studied hitherto. As far as we know, the effect of AP1A on force of contraction in the heart has not been investigated. However, AP1A does have effects in the cardiovascular system because it can induce vasodilatation (13). Hence, we started to study the cardiac effects of AP1A in humans.
In this study we addressed the following questions:
• Does AP1A affect force of contraction in the human heart?
• Does AP1A interfere with the positive inotropic effect of β-adrenoceptor agonists like isoprenaline?
• Is the effect of AP1A mediated by degradation to adenosine?
• Which receptor mediates the inotropic effects of AP1A?
Contraction experiments were performed as described previously (14,15). In brief, trabeculae of right atria from patients with coronary heart disease undergoing cardiac surgery (diameter, <1 mm; length, 5-8 mm) were dissected in gassed bathing solution (composition, see later) at 4°C. The age of the patients ranged from 41 to 68 years (mean, 56 ± 12 years). All patients were in clinical class New York Heart Association II-III. Medical treatment consisted of nitrates and/or calcium antagonists. These studies were approved by the local ethical committee, and patients gave informed consent.
The bathing solution contained (in mM) NaCl, 119.8; KCl, 5.4; CaCl2, 1.8; MgCl2, 1.05; NaH2PO4, 0.42; NaHCO3, 22.6; Na2EDTA, 0.05; ascorbic acid, 0.28; and glucose, 5.05; continuously gassed with 95% O2 and 5% CO2 and maintained at 37°C and pH 7.4. Preparations were attached to a bipolar stimulating electrode and suspended individually in 10-ml glass tissue chambers for recording isometric contractions. Force of contraction was measured with inductive force transducers connected to a scriptor recorder (Föhr Medical Instruments, Seeheim/Ober-Beerbach, FRG). Single contractions were evaluated at high chart speed as before (15). Each muscle was stretched to the length of maximal force of contraction. Trabeculae carneae were electrically stimulated at 0.5 Hz with rectangular pulses of 5-ms duration (Grass stimulator SD9; Grass, Quincy, MA, U.S.A.), the voltage was ∼10-20% greater than threshold. All preparations equilibrated in bathing solution until complete stabilization (≥30 min). During this period, the bathing solution was changed every 15 min. Then adenosine deaminase (ADA, 0.2 U/ml) was added for additional 30 min to exclude the possibility that AP1A was degraded to adenosine. ADA converts adenosine to inactive inosine.
Concentration-response curves of AP1A were obtained cumulatively, each step every 10 min. The IC50 values were determined by linear interpolation. In single-concentration experiments, the tested AP1A was applied for 10 min. The inotropic effect of AP1A was constant for another 20 min, the longest incubation time studied for this compound. In experiments with antagonists, preparations were preincubated with the selective adenosine-receptor antagonist 1,3-dipropyl-cyclopentyl-xanthine (DPCPX, 0.3 μM) for 30 min. DPCPX (0.3 μM) applied for 30 min did not affect contractility (16). Isoprenaline was given 10 min before adding AP1A.
AP1A was purchased from Sigma (München, Germany). 1,3-Dipropyl-cyclopentyl-xanthine (DPCPX; Research Biochemicals International, Natick, MA, U.S.A.), adenosine deaminase (Boehringer Mannheim, Germany), isoprenaline-HCl (Boehringer Ingelheim) were used as described. All other chemicals were of analytic or best commercial grade available. Deionized and bidistilled water was used throughout.
The experimental data given in text and figures are mean ± standard error of the mean (SEM). Force of contraction is given in percentage of the predrug values. These values are 2.4 ± 0.6 mN (concentration-response curve for the effects of AP1A, 1-100 μM, see also Fig. 3), 2.1 ± 0.7 mN (effects of 100 μM AP1A on force of contraction alone and in the presence of 0.3 μM DPCPX, see also Fig. 5) and 1.9 ± 0.5 mN (effects of 100 μM AP1A on force of contraction after β-adrenergic stimulation; see also Fig. 7). Hence basal values before stimulation were very comparable.
Statistical significance was estimated using one-way ANOVA (analysis of variance). Data of multiple groups (such as concentration-response curves) were compared using a one-way ANOVA for paired observations followed by the post hoc procedure of Dunnett. For pairwise comparisons of contractile responses with predrug values, a one-way ANOVA for repeated measurements was performed, followed by the post hoc test of Student-Newman-Keuls. A p value <0.05 was regarded as significant.
In isolated electrically driven human atrial trabeculae carneae, AP1A exerted a negative inotropic effect. This is clearly evident from a typical original recording (Fig. 2). For 100 μM AP1A, the time to 50% of the negative inotropic effect was 2.5 ± 0.5 min (n = 8). Although the decrease in force of contraction after the addition of AP1A was followed by a slight increase, force was not restored in the presence of AP1A. Contractile force was reported at 10 min after application of AP1A, and this level was maintained for ≥30 min (data not shown). A similar time course was observed after application of adenosine to atrial or ventricular human myocardium (17). Of note, all experiments were performed in the presence of adenosine deaminase. The concentration of adenosine deaminase used has been shown to block any negative inotropic effect of 100 μM adenosine under these experimental conditions (10,11). Hence the negative inotropic effect of AP1A cannot be mediated by degradation of AP1A to adenosine. In separate experiments, AP1A was cumulatively applied to isolated electrically driven human atrial preparations. AP1A exerted a concentration-dependent negative inotropic effect, starting at 3 μM. AP1A reduced force of contraction maximally to 14.4 ± 4.4% of control at 100 μM AP1A, the highest concentration tested (n = 5-8; Fig. 3), and the concentrations of that reduced force by 50 and 20% of baseline (IC50 and IC20) were 20.2 and 3.1 μM (n = 5-8). Conversely, after equilibrium had been reached (≥30 min after preloading each muscle to optimal length), force of contraction in untreated preparations declined in the mean only by 9.5 ± 2.1% of the initial value within 60 min. Hence the effect of AP1A is not overestimated in our experimental design. The negative inotropic effect of AP1A was not accompanied by a significant change in time parameters: In the presence of AP1A (100 μM), the following values were noted for time to peak tension, 83.8 ± 6.3 ms; time of relaxation, 182.5 ± 7.5 ms; and total contraction time, 266 ± 11.4 ms (predrug values: 85.0 ± 6.5, 198 ± 9.6, and 283 ± 11.1 ms; p > 0.05).
The negative inotropic effects of AP1A were reversible on washout within 5 min (data not shown). DPCPX, a typical A1-adenosine receptor antagonist (0.3 μM) abolished the negative inotropic effects. An original recording illustrating the effect of AP1A (100 μM) and DPCPX (0.3 μM) + AP1A (100 μM) on force of contraction in an isolated electrically driven human atrial trabecula carnea is shown in Fig. 4. The experiments are summarized in Fig. 5.
Next we asked whether AP1A functionally affects the positive inotropic effect of β-adrenergic stimulation. The β-adrenoceptor agonist isoprenaline increased force in isolated atrial preparations, and this positive inotropic effect was attenuated as exemplified in Fig. 6. To summarize data, isoprenaline (10 nM) increased force of contraction to 446 ± 65% of predrug value (n = 8). AP1A reduced isoprenaline-stimulated force of contraction significantly to 308 ± 66% of predrug value (n = 7; Fig. 7). The time for 100 μM AP1A to reduce force of contraction to 50% of value before addition of AP1A to the organ bath (isoprenaline effect) was 2.3 ± 0.2 min (n = 5) and is similar to values without isoprenaline. These negative inotropic effects of AP1A in the presence of isoprenaline were studied in the presence of ADA and thus cannot be mediated by adenosine formed by degradation of AP1A. Isoprenaline (10 nM) shortened time to peak tension and time of relaxation to 79.6 ± 4.6% and 81.0 ± 2.3% of predrug value (n = 8; p < 0.05). Additionally applied AP1A reversed these effects of isoprenaline on time parameters, prolonging time to peak tension and time of relaxation to 99.5 ± 15.7% and 92.4 ± 5.1% of predrug value (n = 7; p < 0.05). DPCPX (0.3 μM) abolished the negative inotropic effect of AP1A in the presence of isoprenaline.
To investigate whether the negative inotropic effect of AP1A on isoprenaline-enhanced force is antiadrenergic per se or nonspecific, additional experiments were performed in the presence of Ca2+; 16.2 mM Ca2+ increased force of contraction to 399 ± 69% of predrug value (n = 7). The addition of AP1A (100 μM) did not change this inotropic effect (data not shown).
We have reached the following conclusions. AP1A (alone) can exert a negative inotropic (direct) effect in human cardiac preparations. This effect is not mediated by a degradation of AP1A to adenosine. Moreover, AP1A has an anti-β-adrenergic (indirect) effect. Both the direct and indirect negative inotropic effects are mediated by DPCPX-sensitive receptors. Hence there is compelling evidence that the inotropic effect of AP1A is mediated through A1-adenosine receptors in the human atrium.
How does our work compare with previous investigations? It is well known that adenosine exerts both direct and indirect negative inotropic effects through A1-receptors in the human atrium. In the human ventricle, adenosine alone does not affect force of contraction; however, it attenuated the positive inotropic effect of β-adrenoceptor agonists or other cyclic adenosine monophosphate (cAMP)-increasing agents (17,18). Whether the effect of adenosine on isoprenaline-enhanced force is a nonspecific effect or antiadrenergic per se has already been studied. Böhm et al. (19) showed in isolated electrically driven papillary muscles of guinea pigs that adenosine and the adenosine-receptor agonist (−)-N6-phenylisopropyl-adenosine (PIA) did not affect the increase in force of contraction produced by elevating extracellular Ca2+ concentration. Corresponding results were found by Rockoff and Dobson (20) in isolated atria. As AP1A acts through A1 receptors, a similar pharmacology was expected.
Divergent inotropic effects of diadenosine polyphosphates have been noted. In guinea pig left atria, diadenosine polyphosphates exerted negative inotropic effects through P1-purinoceptors and positive inotropic effects mediated through a suramin-sensitive P2-purinoceptor (21). The rank order of potency for negative inotropic responses was AP2A ≥ AP4A = AP3A = AP5A. However, AP1A was not studied. The P1-purinoceptor antagonist 8-para-sulphophenyltheophylline (8-pSPT; 20 μM) caused a rightward shift in the concentration-response curves for AP2A, but converted the negative inotropic responses to AP3A, AP4A, and AP5A into positive inotropic effects. The nonselective P2-purinoceptor antagonist suramin (300 μM) abolished the positive inotropic responses evoked by these dinucleotides (21). Likewise, Rubino et al. (22) described direct negative inotropic effects of AP2A, AP3A, AP4A, AP5A, and AP6A in isolated guinea pig atria. These effects were antagonized by 1,3-dipropyl-cyclopentyl-xanthine (DPCPX, 1 μM), an A1-receptor antagonist, and thus suggest an activation of A1-adenosine receptors. AP6A exerts, like adenosine, a direct and an indirect negative inotropic effect in the atrium and an indirect negative inotropic effect in the ventricle [human heart (10), guinea pig atrium (13). Table 1 gives a synopsis of inotropic effects produced by APnAs.
It is important to study human tissue for the characterization of the inotropic effect of AP1A. We have noted remarkable species differences in the past. Whereas AP4A alone has a negative inotropic effect in guinea pig or canine atrium through A1-receptors, it exerts a positive inotropic effect in human atria through P2-purinoceptors (10,11). For lack of tissue, we have not yet been able to study the inotropic effect of AP1A in preparations from human ventricles, and thus the ventricular effect of AP1A remains to be elucidated. Moreover, the physiologic importance of the negative inotropic effect of AP1A is at present speculative. The IC20 of AP1A (3.1 μM) is in the range of the IC20 values of other APnAs in cardiac preparations (see Table 1). The tissue concentration of AP1A in the heart of any species is at present unknown. However, Luo et al. (23) reported the identification of substantial amounts of AP2A in the human heart. These concentrations were sufficient to affect A1-or P2-receptors. Comparable work for AP1A is currently lacking. In any case, a regulatory function for endogenous AP1A is a reasonable assumption from precedents. It is still unclear where AP1A is physiologically generated. Although biochemical synthesis and metabolism of several diadenosine polyphosphates are in part known [for review, see (24)], corresponding information concerning AP1A is currently lacking.
It is not evident how and to what extent AP1A can be liberated from the different sources and the final concentrations that could be achieved in vivo in cardiac tissue. It is conceivable that under certain pathophysiologic circumstances, the smallest member of the "alarmone family" diadenosine polyphosphates, AP1A, will be formed and released (to a greater extent than normal). Further work is required to investigate the physiologic or potential pathophysiologic role of the APnAs in cardiac function. For example, increased concentrations of other diadenosine polyphosphates, like AP4A and AP5A, were found in the coronary venous blood during ischemia and reperfusion (25,26). One can speculate that AP1A levels may also increase during ischemia. Moreover, it is conceivable that AP1A, like adenosine, might protect the heart against detrimental adrenergic stimuli on force of contraction. Finally, the positive chronotropic effects of β-adrenergic stimulation, which can deteriorate into arrhythmias, might be physiologically attenuated by AP1A. It is not known whether AP1A, like adenosine, exerts effect on the tone of human vessels for instance in the coronary circulation.
In summary, we have shown that the shortest APnA, AP1A, exerts pharmacologic effects in a way reminiscent of adenosine. The (patho)physiologic effects of AP1A in the human heart remain to be elucidated.
Acknowledgment: The technical assistance of Susanne Heck and Okja Bossems is greatly appreciated. We gratefully acknowledge the expert assistance of Dr. Dieter Hafner in statistic analysis.
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