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Am J Physiol Heart Circ Physiol 284: H204-H214, 2003. First published September 19, 2002; doi:10.1152/ajpheart.00068.2002
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Vol. 284, Issue 1, H204-H214, January 2003

Sole activation of three luminal adenosine receptor subtypes in different parts of coronary vasculature

Rafael Rubio1 and Guillermo Ceballos2

1 Departamento de Fisiologia, Faculdad de Medicina de la Universidad Autonoma de San Luis Potosi; and 2 Posgrado de la Escuela Superior de Medicina del Instituto Politecnico Nacional, San Luis Potosi ZP 78210, Mexico


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In isolated guinea pig hearts saline perfused at constant flow, adenosine A1, A2A, and A3 (Ax) agonists covalently bound to a large polymer (Pol; 2,000 kDa) were intracoronarily administered, and three effects were studied: dromotropic, vascular and inotropic. The rank order of potencies were the following: dromotropic (Pol-A2APol-A1>Pol-A3) and vascular and inotropic (Pol-A2A>= Pol-A1Pol-A3), where the rank order of potency for Pol-Ax depends on the part of the coronary vascular network involved; i.e., there is a vascular heterogeneity. The large size of Pol-Ax prevents extravascular diffusion and causes it to act solely in the endothelial luminal surface. This implies their cardiac effects are due to endothelial mediators. Inhibition of nitric oxide (NO) and prostaglandin (PG) synthesis with NG-nitro-L-arginine methyl ester and indomethacin, respectively, show that the three cardiac effects of Pol-A1 were mediated by NO and PG, whereas for Pol-A2A and Pol-A3 the mediator was mainly NO but not PG. These results suggest that if Pol-Ax activated the corresponding endothelial Ax-receptor subtype, a different mediator would be produced.

luminal endothelial receptors; endothelial control; endothelial mediators; endothelial heterogeneity; parenchymal function modulation; cardiac function control


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE CARDIAC EFFECTS OF INTRACORONARY administration of adenosine are mediated via activation of coronary luminal endothelial receptors (4, 5, 44), which results in the release of the bioactive messengers, including nitric oxide and prostaglandins (44). The relative importance of endothelium on adenosine cardiac effects becomes evident if one considers that intracoronary infusion of adenosine up to micromolar concentrations does not increase its interstitial levels. This is because of the impermeable metabolic barrier imposed by the endothelium (34, 51); however, maximal vasodilatory and negative dromotropic effects of adenosine are observed at even lower intravascular concentrations (7, 34). The pharmacokinetics of macromolecular adenosine analogs are similar to those of their low-molecular-weight counterparts during intracoronary infusion, suggesting an intravascular site of action (4, 5, 34, 48), and intracoronarily infused adenosine deaminase alters the vasodilator and dromotropic responses attributed to interstitial adenosine (42). However, the enzyme does not reach adequate interstitial concentrations to account for its pharmacological effects (51). These observations require one to consider whether the intracoronary adenosine-mediated cardiac effects have an endothelial component. Indeed, we have shown (3, 5, 44) that sole activation adenosine receptors via adenosine agonists covalently bound either to latex microspheres of 0.07 µm diameter or to an inert soluble polymer (Pol) (molecular mass >2,000 kDa) caused the same cardiovascular effects as free adenosine (4, 5, 44). Similarly, blockade of intravascular adenosine receptors via a receptor antagonist covalently bound to latex microspheres of 0.07 µm diameter blocked the cardiac effects of free adenosine. Yet, in both cases, these particles are confined to the vascular compartment because of their size. Furthermore, we demonstrated that activation of luminal receptors by intracoronary administration of adenosine caused negative dromotropic effects that are mediated by the release of nitric oxide and prostaglandins (44).

We asked whether the adenosine agonist subtypes A1, A2A, and A3 (Ax) covalently coupled to a inert soluble Pol (Pol-Ax; molecular mass >2,000 kDa) resulted in sole activation of intraluminal endothelial adenosine receptor subtypes in different parts of the coronary vascular network and whether these activations caused the same dromotropic, vascular, and inotropic effects as free adenosine. We also asked whether activation of each of these different receptor subtypes result in the release of the same bioactive messengers by the endothelium.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Isolated Saline-Perfused Hearts

Hearts from male guinea pigs (350-450 g) that had been anesthetized with Nembutal were isolated and the ascending aorta was cannulated and retrogradely perfused via a nonrecirculating perfusion system at constant flow, as previously described (4, 44). For the experiment, coronary flow was adjusted and maintained at 10 ml/min with a variable-speed peristaltic pump. The perfusion medium was Krebs-Henseleit solution of the following composition (in mM): 117.8 NaCl, 6 KCl, 1.75 CaCl2, 1.2 MgSO4, 1.2 NaH2PO4, 24.2 NaHCO3, 5 glucose, and 5 sodium pyruvate. The solution was equilibrated with 95% O2-5% CO2 (37°C at pH 7.4).

Electrical Stimulation and Recording Procedures

Recording and stimulating electrodes consisted of small stainless steel wire vascular clamps (4, 5, 44) affixed to the epicardial tissue layer. Pacing was achieved through placement of a pair of stimulating electrodes on the right atrial appendage, and application electrical square pulses of 2-ms duration and twice the electrical threshold at a rate of 4 ± 0.3 Hz. An atrioventricular (AV) electrocardiogram was recorded placing two recording electrodes, one in the left atrium and the second on the apex of left ventricle. The two electrodes were connected to an oscilloscope synchronized with the atrial pacing stimulator. The interval between the application of the stimulus to the atrium and the rising phase of the atrial electrogram had a value of 19 ± 0.5 ms and remained constant under control conditions and throughout all of the different pharmacological manipulations used. Changes in the interval between atrial and ventricular electrocardiograms (AV delay) were used because the criterion of dromotropic effect indicates only changes in the delay that take place in the auricular-ventricular nodal area (2, 3, 7, 43). The AV delay (in ms) was continuously monitored and measured as the interval between the application of the stimulus to the atria and the rising phase of the ventricular electrical signal. Under control conditions, the AV delay had a mean value of 85 ± 3 ms.

Covalent Coupling of High-Molecular-Weight Pol Derived From Dextran to Ax Agonists

The adenosine agonists utilized were adenosine amine congener (ADAC), an A1 agonist (22, 23), 2-[p-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadenosine [CGS-21680 (CGS)], an A2A agonist (24, 38, 45, 48), and N-[2-(4-aminophenyl)ethyl]-adenosine (APNEA), an A3 agonist (9, 12). The procedure was basically the same for each of the agonists as described by Haga and Haga (17, 18) and modified by us (6, 44).

Synthesis of Pol

Briefly, dextran (2,000 kDa) was dissolved in distilled water, and, while the mixture was continuously stirred, NaIO4 was added and incubated for 90 min. This reaction formed a pair of reactive aldehyde endings per every glucose moiety. The multialdehyde Pol was precipitated by an excess of ice-cold methanol and then centrifuged. The pellet of Pol was dissolved in phosphate buffer (pH 7) and incubated either with 6-aminocaproic acid or lysine, thus forming a Schiff base linkage between the amino groups of aminocaproic acid or lysine and the aldehyde groups. Thereafter, to stabilize the Schiff base, NaCNBH3 was added and the reaction proceeded overnight at room temperature. The final solution was dialyzed against several changes of distilled water, precipitated with methanol, and dried. The final complexes Pol-aminocaproic (Pol-AC) or Pol-Lysine (Pol-Lys) were defined as Pol. The bound Pol-AC or Pol-Lys have two functions: to act as spacer molecules for a chain at least six carbons long, and as anchoring sites for the adenosine agonists. To the carboxyl-terminal groups of the Pol-AC complex were bound the amino groups of either ADAC or APNEA molecules. The amino groups of the Pol-Lys complex were bound to the carboxyl groups of CGS molecules.

Formation of Pol-ADAC or Pol-APNEA Complexes

Either ADAC (700 mg) or APNEA (500 mg) was dissolved in 10 ml of dioxane. Pol-AC was redissolved in 120 ml of a mixture of dioxane and 0.1 M sodium phosphate buffer (30/70) and divided into three equal aliquots. To one of the aliquots (control solution 1), no other addition was made; to a second aliquot, either 5 ml of ADAC or the APNEA dioxane solution was added under continuous stirring (control solution 2). To the third aliquot, 2 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide were added to activate the free carboxylate groups of Pol-AC (10), to react with the amino group of ADAC or APNEA molecules; thereafter, either 5 ml of the ADAC or the APNEA dioxane solution were added under continuous stirring, forming a Pol-AC-ADAC complex or a Pol-AC-APNEA complex, defined as Pol-ADAC and Pol-APNEA, respectively. The purpose of the solutions that did not receive the carbodiimide was to check for noncovalent binding of ADAC or APNEA to Pol-AC complex (control solution 2). The three different aliquots were left for 3 h at room temperature and were dialyzed against several changes of water-dioxane (70:30). The solutions of Pol-ADAC, Pol-APNEA, and control solution 2 were mixed with ice-cold acetone to precipitate the polymer complexes and then extracted several times with benzene to eliminate any free ADAC or APNEA.

Formation of Pol-Lys-CGS Complex

CGS (500 mg) was dissolved in 10 ml dioxane and mixed with a solution of Pol-Lys, and the procedures were identical to those described in the previous section. In this case, carbodiimide was added to activate the free carboxylate groups of CGS (10). The activated carboxylate groups reacted with the amino groups of Pol-Lys, forming a Pol-Lys-CGS complex (Pol-CGS).

Measurement of Dromotropic Effects

During sustained intracoronary infusion of the various pharmacological agents, AV delay increased to achieve a new steady-state value. This value was measured and AV delay control was subtracted. This difference was defined as the pharmacologically induced dromotropic response (in ms). Under control conditions, the AV delay had a mean value of 82 ± 3 ms.

Measurement of Coronary Vascular Effects

The coronary perfusion pressure was recorded continuously via a side arm of the perfusing cannula, and under control conditions it had a value of 67 ± 3 mmHg. Coronary vascular effects induced by the intracoronary sustained infusion of the various adenosine agonists were expressed as the decrease in coronary perfusion pressure (in mmHg).

Measurement of Inotropic Effects

Via the left atria, a fluid-filled latex balloon was introduced into the left ventricle. The fluid-filled balloon was connected to a pressure transducer. Diastolic pressure was adjusted to ~10 mmHg and the developed pressure amplitude was continuously monitored and taken as the contractile response. Under control conditions, the amplitude of the contraction trace was adjusted to 30 mm. Contraction amplitude changes induced by the intracoronary sustained infusion of the various adenosine agonists were determined as the decrease contraction amplitude (in mm).

Dose-Response Curves of Pol-ADAC, Pol-CGS, and Pol-APNEA Complexes and Corresponding Small-Size Agonist: ADAC, CGS, and APNEA

The dose-dependent dromotropic, vascular, and inotropic effects by each agonist were determined. These three responses were measured simultaneously. The three Pol agonists were dissolved in water as three stock solutions: 1, 10, and 100 mg/ml. Similarly, the three free agonists were dissolved in DMSO as concentrated solutions: 0.01, 0.1, and 1 µmol/ml. The final intracoronary agonist concentration was achieved by infusion of a stock solution at various rates (10-100 µl/min) for 5-min periods and the steady-state responses were then determined. Infusion was stopped, followed by a 10-min recovery period before the next drug administration. There were six hearts for each one of the Pol-adenosine agonists and six hearts for each of the free adenosine agonists. Values are expressed as means ± SE.

Control Studies on Effects of Pol-AC, Pol-Lys, Pol-ADAC, Pol-CGS, and Pol-APNEA Complexes

These experiments were designed to rule out any effect of the Pol-AC or Pol-Lys complexes alone (control solution 1) or noncovalent binding of the adenosine agonist to Pol-AC/Pol-Lys complexes (control solution 2). At time 0, a sustained intracoronary infusion of one of the control solutions (either 1 or 2) was started, all at a final concentration of 10 mg/ml. The three cardiac responses were continuously monitored during the period of infusion. There were five hearts for each of the control solutions.

To rule out the possibility that during the passage of the solution through the heart that Pol-ADAC, Pol-CGS, and Pol-APNEA would become hydrolyzed to the corresponding free adenosine agonist, 100 ml of venous effluent were collected during a 10-min infusion of a given Pol-adenosine agonist. In separate experiments, Pol-adenosine agonists were infused at the following final intracoronary concentrations (in mg/l): 100 Pol-ADAC, 100 Pol-CGS, and 500 Pol-APNEA. The venous effluents were extracted twice with 25 ml of benzene and the fractions were combined and evaporated to dryness. The residue, which could contain the corresponding free agonist, was resuspended in 100 ml of dioxane-Krebs-Henseleit (1/99). This solution was equilibrated with 95% O2-5% CO2 and brought to 37°C (control solution 3). The control solution 3 was perfused into a second heart preparation and the dromotropic, vascular, and inotropic effects were continuously recorded. There were five hearts for each control solution 3.

Blockade of Dextran Agonists Effects With NG-Nitro-L-Arginine Methyl Ester and Indomethacin

NG-nitro-L-arginine methyl ester (L-NAME) and indomethacin, respectively, are inhibitors of nitric oxide (41) and prostaglandin synthesis (35), respectively. To test whether intracoronary infusion of Pol-Ax produced their effects via release of nitric oxide and/or prostaglandins, the production of these substances was inhibited by either L-NAME or by indomethacin, respectively. A group of five hearts was challenged with a Pol-Ax to the final intracoronary concentration necessary to produce ~75% of the maximal effect. These concentrations were (in mg/l) 10 Pol-ADAC, 50 Pol-CGS, and 500 Pol-APNEA. These doses were given alone and during the infusion of a blocker.

A 5-min control infusion period of a Pol-Ax was performed, and the control responses were determined. This was followed by a 10-min washout period. Thereafter, an infusion of L-NAME (final concentration 0.5 mM) was sustained for 15 min, and at 10 min the Pol-Ax infusion was initiated for 5 min. The responses during L-NAME and Pol-Ax infusion were determined and compared with that induced by the Pol-Ax. At 15 min, L-NAME and Pol-Ax infusion was stopped and followed by a 20-min washout period. Thereafter, this cycle was repeated using indomethacin as the inhibitor (10 µm).

Pharmacological Activity Equivalence Between Concentration of Given Pol-Ax and Corresponding Ax

The pharmacological activity of a Pol-Ax (in mg/l) equivalent to that of the corresponding Ax (in mol/l) was determined. Dose-response curves for Pol-Ax and Ax were determined by using bioassay changes in smooth muscle tension of isolated guinea pig aortic rings denuded of endothelium. This preparation was chosen because the pharmacological agent added to the bathing solution has a direct access to the receptors of the smooth muscle cells.

The aortic rings were 3 mm wide, mounted between two stainless steel hooks, and stretched radially to their optimal length, as described by Herlihy et al. (21). The rings were precontracted with 30 mM K+, and the relaxing effects of different concentrations of Pol-Ax and Ax were determined. Two procedures were followed. In one case, in a set of rings, cumulative doses of a Pol-Ax were added to the bath and the response was determined at each concentration (n = 6). In a similar manner in a separate set of rings, the relaxing effects of the corresponding Ax were also determined (n = 6). In the second procedure, the administration of a dose of Pol-Ax and a dose of the corresponding Ax were alternated in the same ring. Each dose was administered for 5 min. The bathing solution was then removed and changed twice with Krebs-Henseleit. The washing period lasted 10 min, the other agent was administered, and the cycle was repeated. The two procedures gave the same results. The dose-response curves of a Pol-Ax agonist and the corresponding Ax agonist were plotted. From these curves, the concentration of Pol-Ax (in mg/l) to induce a given relaxing response was compared with the concentration of the corresponding Ax (mol/l) that cause the same relaxing response. These two concentrations were plotted against each other. Linear regression analysis shows the plots for the three Pol-Ax against Ax were straight lines with a zero intercept and r value ranging from 0.994 to 0.985. The slope of the straight line provided the pharmacological activity equivalence factor and show that 1 mg/l Pol-Ax agonist has an activity equal to a number of moles per liter of Ax agonist.

Statistics

Values are expressed as means ± SE. In these experiments, each heart and each group served as its own control, and responses under control conditions and during specific manipulations were compared in the same heart. For these reasons, statistical significance was determined with a paired t-test with a Bonferroni correction factor for multiple comparisons. A statistically significant difference was defined for values of P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Negative Dromotropic, Vasodilatory, and Negative Inotropic Effects of Three Pol-Ax

The dromotropic (Fig. 1A), vascular (Fig. 1B), and inotropic (Fig. 1C) cardiac effects of the different Pol-Ax (in mg/l) are shown in Fig. 1. These results show that Pol-ADAC, Pol-CGS, and Pol-APNEA can induce a dromotropic, vascular, and inotropic effect, the exception being Pol-CGS, which lacks a dromotropic action.


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  		Fig. 1.   Dose-response curves for the three polymer (Pol)-A1, A2A, and A3 (Pol-Ax) agonists on three different cardiac functions: auricular-ventricular delay (A), vascular dilatation (B), and inotropic effect (C). Pol + 2-[p-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadenosine (CGS-21680) (Pol-CGS) does not exert a negative dromotropic effect, but it causes vascular and inotropic effects. ADAC, adenosine amine congener; APNEA, N-[2-(4-aminophenyl)ethyl]-adenosine.

The results in Fig. 1, A-C, suggest that Pol-ADAC would required a lesser concentration (in mg/l) to produce the same effects as Pol-CGS and Pol-APNEA. However, it is not possible to compare their pharmacological activities when their concentrations are expressed in milligrams per liter because each Pol-Ax is pharmacologically a complex molecule not with a single active site but with multiple sites. The estimated molecular weight for each Pol-Ax differs because the number of bound Ax moieties may vary, and each Ax has a different molecular weight. These are the reasons for which we decided to adapt a system to be able to convert the activity (in mg/l) of a given Pol-Ax into moles per liter and for that we used as standard the activity of the corresponding Ax (in mol/l), i.e., to calculate mass per volume in milligrams per liter of a given Pol-Ax with the same activity as a Ax value (in mol/l), the pharmacological activity equivalence. It is evident that this standardization procedure assumes that the Pol-Ax activates Ax receptors.

Pharmacological Activity Equivalences of Pol-Ax in Isolated Guinea Pig Aortic Rings

The pharmacological activity equivalence for Pol-ADAC was 1 mg/l = 5.2 × 10-8 mol/l of free ADAC, for Pol-CGS was 1 mg/l = 0.5 × 10-8 mol/l of free CGS, and for Pol-APNEA was 1 mg/l = 2 × 10-8 mol/l of free APNEA. These three numerical factors were used to change the y-axes from milligrams per liter to moles per liter (see Fig. 1, A-C).

Cardiac Effects of Pol-ADAC, Pol-CGS, and Pol-APNEA and Same Effects of Corresponding Ax

Negative dromotropic effects. The dromotropic effects of the three Ax and the three Pol-Ax are shown in Fig. 2. Figure 2A shows the effects of the three free Ax and Fig. 2B illustrates the effects of Pol-Ax. The most potent of the free agonists was CGS, an A2A receptor agonist. The order of potencies was CGSADAC> APNEA (A2AA1>A3). In contrast, CGS bound to Pol was inactive even at a high concentration (10-5 M), and the effects were not statistically different from zero. However, Pol-CGS was active because at the same time it caused vasodilation and an inotropic effect. The order of potency of the Pol-Ax was Pol-CGS Pol-ADAC>Pol-APNEA (Pol-A2APol-A1>Pol-A3).


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  		Fig. 2.   Dose-dependent negative dromotropic effects of the three small diffusible Ax agonists (A) and the three Pol-Ax agonists (B). CGS is the most active (A), whereas Pol-CGS is not (B).

Vasodilatory effects. The dose-dependent vascular effects of the three Ax and the three Pol-Ax agonists are shown in Fig. 3, A and B, respectively. The curve for CGS was slightly to the left, as was that for ADAC, and the order of potencies was CGS>= ADACAPNEA (A2A>= A1A3). A similar ranking was maintained for Pol-Ax agonists except that Pol-CGS was more potent than Pol-ADAC and much more than Pol-APNEA. The order of potencies was Pol-CGS>Pol-ADAC>Pol-APNEA (Pol-A2A>Pol-A1>Pol-A3).


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  		Fig. 3.   Dose-dependent vascular dilation effects of the three small diffusible Ax agonists (A) and the three Pol-Ax agonists (B).

Negative inotropic effects. The dose-dependent inotropic effects of the three Ax agonists and the three Pol-Ax agonists are shown in Fig. 4, A and B, respectively. The order of potencies for the Ax agonists was CGS>ADACAPNEA (A2A>A1A3). The order of potency for the Pol-Ax agonists was Pol-CGS = Pol-ADACPol-APNEA (Pol-A2A = Pol-A1Pol-A3).


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  		Fig. 4.   Dose-dependent negative inotropic effects of the three small diffusible Ax agonists (A) and the three Pol-Ax agonists (B).

In summary, for the three cardiac responses studied, the corresponding rank order of potencies of the free Ax agonists were different and depended on the function measured. The same results were observed for the Pol-A1, Pol-A2A, and Pol-A3 agonists, i.e., the order of potencies were function dependent. The three different cardiac responses reflect functions that occur in distinct parts of the heart/coronary vascular network. The order of potencies for the Ax agonist and the Pol-Ax agonists for the three cardiac responses studied are summarized in Table 1.

                              
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  		Table 1.   Order of potencies of small adenosine agonists and Pol agonists and endothelial mediators for Pol agonists in different parts of coronary vascular tree

Control Studies

Infusion of the three control solutions did not have any effect (not shown). These results indicate that Pol (control solution 1) was not active and that there was no noncovalent absorption of ADAC or CGS or APNEA to Pol (control solution 2). In addition, Pol-ADAC, Pol-CGS, and Pol-APNEA were not hydrolyzed to the corresponding free Ax agonist during their passage through the heart because the venous effluents of hearts perfused with supramaximal concentration of any of the Pol-Ax agonists showed no benzene-extracted free Ax agonists. The benzene fractions (which could contain the corresponding free Ax agonist) that were resuspended in Krebs-Henseleit solution and perfused into an assay heart showed no activity (control solution 3).

Partial Blockade of Pol-ADAC, Pol-CGS, and Pol-APNEA Dromotropic, Vascular, and Inotropic Effects With L-NAME and Indomethacin

These results are shown in Fig. 5, A-C, respectively. Figure 5A shows the level of blockade induced by L-NAME and indomethacin on the negative dromotropic, vascular, and inotropic effects of Pol-ADAC. L-NAME and indomethacin caused ~50% depression of the dromotropic effects of Pol-ADAC and there was no difference between the level of blockade produced by these two agents. In a previous study (43), we demonstrated that the dromotropic effects of adenosine and Pol-ADAC were blocked by L-NAME and indomethacin and that their blocking effects were additive. The dilatory and inotropic effects of Pol-ADAC in Fig. 5A show that L-NAME caused a significantly greater level of blockade than that caused by indomethacin, suggesting a more important role of nitric oxide than prostaglandins. Nevertheless, L-NAME and indomethacin caused important levels of blockade of three different cardiac effects of Pol-ADAC.


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  		Fig. 5.   Levels of blockade of dromotropic, dilatory, and inotropic effects of the three Pol-Ax agonists either by NG-nitro-L-arginine methyl ester (L-NAME) or indomethacin (Indom). Inhibition of the effects of Pol-ADAC (A), Pol-CGS (B), and Pol-APNEA (C) is shown. Indomethacin was ineffective in the cases of Pol-CGS and Pol-APNEA. * Different from 0; # not different from 0.

Figure 5B shows the levels of blockade by L-NAME and indomethacin of the vascular and negative inotropic effects of Pol-CGS. Pol-CGS did not cause a negative dromotropic effect. L-NAME caused an 80% blockade of both the vascular and inotropic effects of Pol-CGS. However, indomethacin caused only an 18% blockade of the vascular effects and had no effect in the inotropic response. These results indicate that the Pol-CGS vasodilatory and inotropic effects are mainly mediated by nitric oxide.

Figure 5C shows the levels of blockade by L-NAME and indomethacin of the negative dromotropic, vascular, and inotropic effects of Pol-APNEA. In the three cases, L-NAME caused a significant level of blockade. In contrast, none of the effects of Pol-APNEA were diminished by indomethacin.

Table 1 summarizes these results. The cardiac dromotropic, vascular, and inotropic effects of Pol-A1 and Pol-A2A have no AV effects, and Pol-A3 is mostly mediated by nitric oxide. Prostaglandins are also important mediators of the dromotropic, vascular, and inotropic effects of Pol-A1 but do not participate in the effects of Pol-A2A and Pol-A3.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our results show that functions of the three distinct groups of cardiac cells, AV nodal cells, vascular smooth muscle cells of coronary resistance vessels, and contractile ventricular myocytes, are modulated as a result of intravascular administration of three types of freely diffusible Ax receptor agonists and the three intracoronarily confined Pol-Ax receptor agonist subtypes. The exception was that Pol-CGS did not have a nodal effect. The same effects produced by all these adenosine receptor agonists are produced by intracoronary administration of the purine nucleoside adenosine (4, 5).

The dromotropic, vascular, and inotropic effects reflect functions taking place in distinct parts of the heart. The AV node is a small region (0.1 mm thick and 1 mm long) made of specialized cardiac cells that are solely responsible for the changes in the AV delay interval (2, 3, 7, 43). The changes in coronary vascular resistance reflects the changes in tone of the vascular smooth muscle of small coronary arterial vessels, mainly arterioles, and the changes in left ventricular contraction reflect changes in contractility of the ventricular contractile myocytes (27). Each of these distinct cardiac cell groups is associated with its own endothelial coronary capillary network. These three different endothelial networks are responsive to the luminally confined polymer Pol-A1, Pol-A2A, and Pol-A3 agonists, and each network shows a characteristic rank order of potency (Table 1). This observation suggests that the endothelium in these distinct vascular regions has three different adenosine receptor subtypes. The exception was the AV nodal coronary endothelium, which does not appear to respond to the large-size Pol-A2A. The three Pol-Ax agonists exerted their effects importantly through mediation by nitric oxide. In the case of Pol-A1, prostaglandins were also important mediators of its dromotropic, vascular, and inotropic effects. For Pol-A2A and Pol-A3, another mediator of unknown nature may exist.

Sole Activation of Three subtypes of Intravascular Endothelial Adenosine Receptors by Pol-A1, Pol-A2A, and Pol-A3 Agonists

We (4, 5, 44) established that the vascular endothelium is possibly the sole component of the cardiac effects of intravascular adenosine. This conclusion is supported by our previous observation (23), where the adenosine receptor blocker xanthine amino congener (XAC) covalently bound to 0.07-µm-diameter latex microspheres when infused intracoronarily blocked fully the dromotropic, inotropic, and vascular effects of supramaximal intracoronary adenosine (5). Our present results also support this concept because Pol-ADAC, Pol-CGS, and Pol-APNEA remain intravascularly confined. To ensure intravascular confinement of the adenosine agonists ADAC, CGS, and APNEA, they were coupled to multiple sites of a polymer with an estimated molecular mass of >4,000 kDa and a diameter of >= 0.1 µm.

Pore theory modeling studies that use macromolecular dextran or microspheres in the dog myocardium and dog leg have defined an upper limit of the capillary large-pore diameter to be 0.024-0.032 µm for the myocardium (50) and 0.04 µm for the leg (14). Our Pol-Ax complexes, with a mean diameter >0.1 µm, exceed the upper limit of the capillary large-pore diameter. Moreover, in our previous studies (4, 5), ADAC and XAC (23) were covalently bound to 0.07-µm-diameter latex microspheres and shown to be biologically active. After 15 min of microsphere infusion, electron microscopic studies revealed that the microspheres were uniformly localized exclusively around the capillary endothelial luminal surface and were not found within the myocardial interstitium. Therefore, theoretical model studies (14, 50) and our experimental evidence support our conclusion that the Pol-Ax complexes and microsphere conjugates are restricted to the intravascular space because of their size and thereby can only act via the intravascular endothelium.

Our present and previous (4, 5, 44) control experiments attest to the fact that Pol-Ax agonist complexes are responsible for the activation of intravascular endothelial adenosine receptors. Venter (55) proposed three possible mechanisms by which immobilized drug preparations may yield pharmacological activity: 1) action/effect of noncovalently bound drug (adsorbed drug); 2) action/effect of locally released drug (either from adsorbed or hydrolyzed drug); and 3) action/effect of the immobilized drug itself. In addition, one should also consider the possible activity of the matrix used for binding. We found no effect of the binding matrix (Pol = Pol-AC or Pol-Lys; control solution 1). In an attempt to rule out the first two mechanisms proposed by Venter (55), several control studies were performed. For mechanism 1, which tested for ADAC, CGS, or APNEA adsorption to Pol, we examined the effects of a control Pol preparation (control solution 2), where the coupling agent was omitted from the reaction mixture (Pol plus the adenosine agonist) and found no biological activity. Therefore, these experiments ruled out the possibility of adsorbed adenosine agonists in our preparations. To rule out mechanism 2 proposed by Venter (55), we performed a bioassay analysis using both donor and recipient guinea pig hearts. It could be proposed that if the intracoronarily infused Pol-Ax agonist complexes were locally hydrolyzed (in situ), "free" ADAC or CGS or APNEA would be released into the venous effluent, from which they could be extracted with benzene. The benzene-extracted fractions (control solution 3) had none of the cardiac effect of the Pol agonists in an assay heart. These results are similar to those of previous experiments with ADAC and XAC bound to latex microspheres, which showed that the venous effluent, once filtered of microspheres and assayed in a recipient heart, had no biological effect (4, 5). The stability of our Pol-ADAC, Pol-CGS, and Pol-APNEA complexes is due to the formation of an amide bond between carboxylic acid groups and amine moieties (10). This bond is stable during intravascular infusion because in situ there was no hydrolysis of our compounds. From the above discussion, it follows that the biological activities of the Pol-ADAC, Pol-CGS, and Pol-APNEA are due to the immobilized ligands that activate three types of intravascular endothelial adenosine receptors.

Ambiguous Significance of Rank Order of Potency of Intravascularly Administered Freely Diffusible ADAC, CGS, and APNEA

Table 1 illustrates the rank order of potencies of the Ax agonists and the Pol-Ax agonists for the three cardiac functions studied. For a given function, the rank order of potency of the three Ax agonists cannot be compared with that of the Pol-Ax agonists for various reasons.

First, the Ax moieties bound to Pol-Ax may as a result of the covalently attachment to Pol have altered their pharmacological selectivity. Although it is likely that Pol-ADAC, Pol-CGS, and Pol-APNEA agonists may retain levels of pharmacological selectivity to preferentially activate their respective Ax receptors, this has to be established before a comparison of pharmacological of rank order of potency between Ax agonists and Pol-Ax agonists is accepted. So far, our results indicate only that Pol-ADAC, Pol-CGS, and Pol-APNEA behave as though they were three distinct adenosine receptor subtype agonists.

Second, in the case of the free agonists, the sequence for dromotropic effects, A2AA1>A3, if taken at face value, would indicate that the dromotropic effect of adenosine is mediated via an A2A adenosine receptor. This inference contrast with that reached by other investigators (7, 8) who used a similar preparation as ours and concluded that adenosine produces its dromotropic effect via activation of the A1 receptor (7, 8). It is not difficult to explain this seeming controversy. Clemo and Belardinelli (7, 8) used adenosine agonists different from those now available and from the obtained rank order of potency they concluded that the adenosine dromotropic effect was via activation of an A1-type receptor (7). In addition, these investigators (8), by using a series of alkylxanthines with different affinities for A1 and A2 receptors (those available at that time), concluded that intravascularly administered adenosine exerted a direct action on the AV node via an A1-adenosine receptor (38). Since the 1980s, when these studies were published, many more adenosine receptors and their molecular biology have been described and the chemistry of agonists and antagonists has further evolved (46, 49).

Third, the most important source for controversy on adenosine receptor subtype mediation in whole heart studies is that Ax agonists or antagonists are highly lipid soluble and diffusible and are poorly metabolized by cells that will have an extracellular and intracellular large volume of tissue distribution. Consequently, the same agonist can act at intravascular and at extravascular sites of diverse cell types. Intracoronary administration of a diffusible agonists such as CGS is going to have effect in different cell types located intra- and extravascularly and the integrated effect on the function of the cell under study (AV nodal cells) results from the direct action of the drug in that cell plus the effects of paracrine signals arising in neighboring cells of different types. Simply because there is a rank order of potency and not an all-or-none action by each agonist subtype indicates the available agonists are not absolutely specific and because of the multiple sites of actions and diversity of neighboring cell types and paracrine signals involved, the coronary administration of a freely diffusible adenosine agonist/antagonist does not permit to define precisely the dominant receptor subtype of the cell whose function is affected. At most, one can conclude that the rank order of potency observed, the result of the sum of multiple effects, would be similar to that obtained if indeed the cells of interest had a dominant receptor subtype defined by the ranking order. Our results with the freely diffusible Ax agonists would seem that the A2A receptor subtype is dominant but not the A1 receptor subtype.

Both the direct effect on AV nodal cells (33) and the endothelial-mediated effect of adenosine on the AV nodal transmission are mediated by the preferred activation of A1 adenosine receptor (5). The work of Martynyuk et al. (33) in isolated rabbit AV nodal cells indicates that the direct dromotropic effects of adenosine are via A1 receptor activation because 8-cyclopentyl-1-3-dypropylxanthine, an A1 adenosine antagonist, reversed the hyperpolarizing current induced by the A1 receptor agonist (-)-N6-(2-phenylisopropyl)-adenosine and adenosine. Our previous work (5) indicated that the endothelial-mediated effect of adenosine on the AV nodal transmission is also mediated via the A1 adenosine receptor (5). In these studies, intracoronary adenosine acts solely in endothelial luminal receptors (4, 5, 44). However, its dromotropic effects were blocked by the A1 antagonist XAC and by XAC covalently bound to microbeads. Our present results, which used stable, nondiffusible Ax agonists that act only on the intravascular lumen of the coronary endothelium, suggest that in the AV nodal region activation of luminal endothelial A1 receptors results in a negative dromotropic effect, as indicated by the rank order of potency of Pol-A1>Pol-A3Pol-A2A. However, it remains to be established that Pol-ADAC, Pol-CGS, and Pol-APNEA agonists still retain ranking order levels of pharmacological selectivity to activate Ax receptor subtypes, respectively.

Rank Order of Potencies of Pol-Ax Agonists Are Different in Three Distinct Functional Areas of Heart: Heterogeneity of Coronary Vasculature

Although the pharmacological selectivity of Ax agonists may have been modified as a result of their chemical alteration once covalently attached to Pol, it is likely that the resulting agonists Pol-A1, Pol-A2A, and Pol-A3 may still retain a higher affinity for the respective Ax receptors. That is, the rank order of potencies of the Pol-Ax agonist may differ only quantitatively but not qualitatively from those of the Ax agonists. This is because the Ax agonists have large differences in their affinities for the Ax receptors. At any rate, our results indicate that Pol-A1, Pol-A2A, and Pol-A3 behave as distinct agonists that act selectively in specific receptors, which at this point we cannot ascertain the subtypes.

It is well established that endothelial cells have adenosine receptors (22, 30, 45) and possibly the three subtypes. The Pol-Ax agonists act on the luminal surface of the coronary endothelium neighboring the parenchymal cells responsible for the function studied. Comparison of the rank order of potencies of Pol-ADAC, Pol-CGS, and Pol-APNEA agonists on the functional areas (AV node, contractile ventricular muscle, and coronary resistance vessels; Table 1) shows that the ranking orders are different. This implies that the dominant adenosine receptor on the endothelial lumen in each functional area is different, i.e., the luminal surface of the coronary vascular network is biochemically heterogeneous. However, the three coronary networks studied responded to the action of the three Pol-Ax agonists; the exception was the AV nodal vascular area, which seem not to possess adenosine receptors activated by Pol-A2A, at least judging by the lack of dromotropic effect. However, alteration of other functions is possible.

The coronary vascular network is biochemically heterogenous, as indicated by extensive evidence. Intramural coronary arterioles are responsive to adenosine and beta -catecolamines; in contrast, large coronary distributing arteries are not (52, 53). Studies on intracoronary perfusion of hydrolyzing enzymes or lectins with specifities toward different luminal endothelial glycosidic groups or perfusion of antibodies against intravascular endothelial cell membrane proteins show that these agents differentially modulated the effect of coronary flow on different functional regions of the coronary vasculature. This indicates that there is chemical and functional heterogeneity of the coronary vascular lumen (43). Furthermore, extensive evidence in the mechanism of hemostasis supports the concept that disorders in coagulation may be associated with distinct vascular beds as a result of heterogeneous regulation of local endothelial cell activity (11).

Transcellular Mediators: Nitric Oxide and Prostaglandins

We previously established that in isolated perfused guinea pig hearts, intracoronary adenosine activates solely endothelial intravascular receptors (4, 5, 44), and we demonstrated the existence of indirect negative dromotropic, vascular, and negative inotropic effects of adenosine. The negative dromotropic effect resulted from an A1 adenosine luminal endothelial receptor that was the consequence of release of nitric oxide and prostaglandins, both possibly of endothelial origin (44). It seems reasonable to see whether nitric oxide and prostaglandins also mediate the cardiac effects of adenosine Ax agonists when coupled to Pol.

Nitric Oxide

Dromotropic effect. Nitric oxide plays an important role in the cholinergic and purinergic modulation of auricular-ventricular nodal cells in the adult rabbit (19) and guinea pig heart (6). Nitric oxide inhibits the L-type calcium channel current in auricular-ventricular nodal cells, which may result in prolongation of auricular-ventricular interval (19). Indeed, endothelium of the auricular-ventricular nodal region has a strong constitutive nitric oxide synthase activity (19). We established that adenosine (44) and acetylcholine (6), through activation of intravascular endothelial receptors, exert their negative dromotropic effect partially via the release of nitric oxide. The effects of adenosine and acetylcholine were reduced or magnified either by reducing or increasing nitric oxide accumulation, respectively (13, 32, 41, 44, 47). Our present results show that three intraluminal endothelial adenosine receptor subtypes in the coronary vasculature neighboring the auricular-ventricular myocytes show a rank order of potency Pol-A2APol-A1>Pol-A3 and that 55% of the effects of Pol-A1 and Pol-A3 were mediated by nitric oxide. Pol-A2A was without a dromotropic effect.

Vasodilator effect. Many studies (1, 13, 26, 36, 37, 54) have shown that nitric oxide derived from coronary endothelium causes dilation of the coronary vasculature. Through this mechanism, adenosine induces coronary dilation, activating an A2-type receptor (1, 4, 5, 20, 36, 37, 53, 54). Our present results show that the three intraluminal endothelial adenosine receptors subtypes in the coronary resistance vessels show a rank order of potency of Pol-A2A>Pol-A1>Pol-A3 and that 60-80% of their effect is mediated by nitric oxide.

Inotropic effect. Endogenous and exogenous nitric oxide causes either a negative (56) or a positive inotropic effect (28, 40, 56). Vila-Petroff et al. (56) demonstrated in isolated left ventricular rat myocytes that nitric oxide donors exert a concentration-dependent biphasic inotropic effect. The positive inotropic phase was observed at low concentrations, whereas higher concentrations caused the negative inotropic phase. Our present results show that the three intraluminal endothelial adenosine receptors subtypes in the coronary vasculature neighboring the contractile myocytes show a rank order of potency of Pol-A2A = Pol-A1Pol-A3 and that these effects are mediated by nitric oxide by 60-80%. These effects were negative monophasic and not biphasic. The reason for this apparent discrepancy with the reports using exogenous nitric oxide donors in isolated myocytes (28, 56) is unknown.

Prostaglandins

Our results show that inhibition of cyclooxygenase by indomethacin, a prostaglandin-forming enzyme, depresses the three functional effects of Pol-A1. Indomethacin has a small blockade of the dilatory effect of Pol-A2A and was without effect on its inotropic response and in the three functional effects of Pol-A3, i.e., only in the case of Pol-A1 are prostaglandins important paracrine participants.

The three functional actions of intravascular Pol-A1 result from the sum of the individual negative effects of two mediators: nitric oxide and a prostaglandin. The sum of individual effects L-NAME and the effects of indomethacin added to a value that was not significantly different from 100%. This finding is compatible with the fact that indomethacin and L-NAME both individually depress the negative dromotropic effect of adenosine and that their effects are additive (44).

In summary, our results indicate that different sections of the coronary vascular network/functional areas possess three subtypes of luminal coronary endothelial receptors activated by Pol-A1, Pol-A2A, and Pol-A3 (agonists acting intravascularly). Because the rank order of potencies of Pol-A1, Pol-A2A, and Pol-A3 is different for each coronary endothelial network, it indicates that the coronary endothelium throughout the vascular tree expresses a different dominant adenosine receptor subtype, i.e., the coronary vascular network is biochemically heterogenous. A large fraction of the effects of Pol-A1, Pol-A2A, and Pol-A3 is mediated by nitric oxide and only in the case of Pol-A1 are prostaglandins also mediators.


    ACKNOWLEDGEMENTS

We acknowledge the technical assistance of Luvenia Wigginton.


    FOOTNOTES

This work was supported by Consejo Nacional de Ciencia y Tecnologia Grants G34998-N, 25963-N, and 0433P-N.

Address for reprint requests and other correspondence: R. Rubio, Depto. de Fisiologia, Faculdad de Medicina, Universidad Autonoma de San Luis Potosi, Av. V. Carranza 2405, San Luis Potosi, ZP 78210, Mexico (E-mail: rrubio{at}deimos.tc.uaslp.mx).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published September 19, 2002;10.1152/ajpheart.00068.2002

Received 25 January 2002; accepted in final form 12 September 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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