Homocysteine (Hcy) is a sulfur-containing amino acid that is generated during the metabolism of methionine, an essential amino acid derived from dietary protein.1 During the methylation process, S-adenosylmethionine derived from methionine is converted to S-adenosylhomocysteine (SAH), which is hydrolyzed to Hcy and adenosine (Ado) by SAH hydrolase.2 It has been reported that this pathway provides a significant source of cytosolic Ado in the myocardium.3 Ado, an intracellular metabolite, is released into the extracellular fluid under physiologic and pathophysiologic conditions.4 Extracellular Ado induces several effects by stimulation of specific Ado receptors.5 By stimulation of these receptor, Ado exerts protective effects mainly in tissues, such as the heart, the brain, and the kidney, that are especially prone to ischemic injury.6 Recent progress has been made mainly regarding the role of Ado as cardioprotective agent, especially its implication in ischemic preconditioning, a process by which short periods of ischemia followed by reperfusion protect against otherwise lethal injury induced by sustained ischemia.7 Ado has also been reported to produce negative inotropic, chronotropic, and dromotropic cardiac effects.8,9 Ado produces acute inhibition of sinus node and atrioventricular nodal function. This profound but short-lived electrophysiologic effect makes Ado a suitable agent for treating supraventricular tachycardias that incorporate the sinus node or atrioventricular node as part of the arrhythmia circuit, or for unmasking atrial tachyarrhythmias or ventricular preexcitation. In addition to direct effects, a notable cardiac effect of Ado is the reduction of contractile responses induced by β-adrenergic stimulation.10,11 Ado antogonizes the effects of β-adrenergic stimulation, known as the indirect, anti-β-adrenergic action of Ado.12,13 Its antiadrenergic properties also make it an effective agent for use with some unique atrial and ventricular tachycardia.14 At the level of the vascular smooth muscle cell, Ado also inhibits the release of norepinephrine and reduces the postsynaptic vasoconstrictor response to α-adrenoceptor stimulation.15,16
It is important to remember that SAH hydrolase activity plays a critical role in the regulation of tissue Ado concentration. When concentrations of Hcy are high, the hydrolase reaction catalyzed by the SAH hydrolase reverses and SAH is formed from Ado and Hcy, which will result in a decrease in the Ado concentration.17 In this regard, hyperhomocysteinemia (hHcy) decreases plasma and tissue Ado concentration association with inhibition of SAH hydrolase.18 Subsequently, facilitated diffusion of extracellular Ado into cells through dipyridamole (DIP)-sensitive transporters is enhanced, thus limiting Ado receptor stimulation when most needed.19 Therefore, we hypothesized that elevations of plasma Hcy may lead to a change in reactivity to Ado in either heart or vessels, or both association with reduction of receptor stimulation in consequence of low plasma Ado levels. In a previous study, it has been clearly shown that prolonged exposure to elevated levels of Ado in the isolated guinea pig heart elicited a persistent blunting of the inotropic responsiveness to isoproterenol (ISO).20 Therefore, the reactivity of cardiovascular system to adrenoceptor agonists could be potentiated in hHcy on the basis of decreased Ado levels and this state could contribute to the detrimental cardiovascular effects of hHcy. To test this hypothesis, we investigated whether hHcy produces an alteration in reactivity to either adenosinergic or adrenergic agonists by using an in vitro model of rat right atrium and thoracic aorta. Also, cardiovascular responses to Ado have been evaluated in rats in vivo by measuring with a catheter that was inserted into their right carotid artery.
Induction of hHcy
Male Wistar rats (8 weeks of age) weighing 200 to 250 g were randomly allocated to experimental groups each consisting of 20 animals. The animals were kept in plastic cages at room temperature (23°C) under a day/night rhythm. The Animal Ethics Committee of Akdeniz University Medical Faculty registered the study. Rats were randomly divided into 2 groups. Twenty rats in the control group had free access to tap water. In the second group, hHcy was induced by administration of L-methionine (1 g/kg body weight/d) and succinylsulfathiazole (0.5 g/kg body weight/d) in the tap water for a period of 12 weeks. Succinylsulfathiazole was used to avoid bacterial proliferation and subsequent folate production.21,22 The dosage administered per animal was based on average daily fluid intake. Animals were housed separately, fed standard rat chow. The rats were killed at the end of the experiments with an overdose of urethane.
Measurement of Hcy Levels
All blood samples were collected after an overnight fasting. Blood samples were stored at −80°C until the day of analysis. The levels of Hcy in plasma were all determined synchronously. Plasma Hcy level was studied with HP1100 (Hewlett Packard Avondale, PA) HPLC system using Chromsystem kits (Chromsystems Instruments & Chemicals GmbH, Martinsreid, Germany).
The right atria were separated from the ventricles and suspended vertically in a bath containing 20 mL physiologic salt solution (PSS) of the following composition (mM): sodium chloride 154, potassium chloride 5.6, calcium chloride 2.08, sodium carbonate 4.28, and glucose 5.5. The solution was maintained at 32°C and was bubbled with 95% O2 and 5% CO2, which produced a pH of 7.5. Resting tension was adjusted at 0.5 g and developed tension was recorded isometrically through a high sensitivity transducer and registered by a writing oscillograph (Harvard Universal Oscillograph, Cat No. 50-8622, Edenbridge, England, 2.5 mm/s rate). In another set of experiments, the full length of thoracic aorta was removed and cleaned of the connective tissue. Thoracic aorta was cut into 3 to 4 mm width rings. Then, the rings were carefully suspended by 2 stainless steel clips passed through the vessel lumen in 20 mL organ baths filled with PSS maintained at 37°C and gassed with 95% O2 and 5% CO2 to obtain a pH of 7.4. The rings were suspended under 1 g of tension, and the preparation was allowed to equilibrate for 60 minutes. Isometric tension was continuously measured with an isometric force transducer (FDT10-A, Commat Ltd, Ankara, Turkey), connected to a computer-based data acquisition system (TDA 97, Commat Ltd, Ankara, Turkey).
The experiments were performed on spontaneously beating right atria obtained from control and hHcy rats. After 60 minutes of equilibration, adenosine (Ado, 10−6 to 10−3 M) was added to the bathing fluid cumulatively. The chronotropic actions (beat/min) of atria were recorded for 30 seconds after reaching the maximum before adding a higher concentration. Atrial preparations were washed with PSS for every 15 minutes during the equilibration period. The effects of Ado on chronotropic action of atria were also investigated in the presence of dipyridamole (DIP, 10−6 M), an Ado uptake blocker, or 8-phenyltheophylline (8-PT, 10−5 M), an Ado receptor antagonist. On the other hand, the responses of right atrium to isoproterenol (ISO, 10−9 to 10−5 M), an β-adrenergic receptor agonist, were also investigated in 2 groups.
To investigate the effect of hHcy on Ado-induced relaxation responses in isolated rat thoracic aorta, concentration-response curves to Ado were constructed in control and hHcy rats. To evaluate Ado transporter activity, this protocol was repeated after the DIP incubation. Additionally, the effects of hHcy on α-adrenoceptor-mediated responses of rat thoracic aorta were evaluated by phenylephrine (Phe, 10−8–10−5 M).
In a separate set of experiments, heart rate (HR) and mean blood pressure (mBP) of rats anesthetized with i.p. injections of urethane (1.3 g/kg) were measured with a catheter that was inserted into their right carotid artery. Another cannula was inserted into femoral artery for the administration of Ado and DIP. All catheters were filled with heparinized saline. Body temperature was maintained at 37±1°C using a heating lamp. After stabilization for 15 to 20 minutes, arterial blood pressure was recorded with a pressure transducer connected to an amplifier (MAY GTA0303, Ankara, Turkey) and a recorder (BIOPAC system MP150, Ankara, Turkey). HR was calculated from the blood pressure tracing. Hemodynamic measurements were recorded using a converter and displayed on a personal computer. To measure the cardiovascular effect of Ado in vivo, after a 20-minute stabilization period, Ado (100 to 300 μg/kg) and DIP (125 to 250 mg/kg) were given as a rapid bolus into the femoral artery. Then, the HR and mBP were recorded.
The following drugs were obtained from the Sigma Chemical Company (St Louis, MO): Adenosine, DL-homocysteine, dipyridamole, 8-phenyltheophylline, phenylephrine, sodium chloride, potassium chloride, calcium chloride, sodium carbonate, and glucose. All drugs were freshly prepared daily during the experiments and dissolved in distilled water before use with the exception of dipyridamole (dissolved in ethanol) and 8-PT (dissolved in dimethyl sulfoxide).
Analysis of Results
All values are expressed as mean±standard error of the mean (SEM). Responses to Ado are expressed as percentages of the reversal of the tension developed in response to Phe. The logarithm of concentration of agonists that elicited a 50% of maximal response (Emax) was designated as the EC50. These values were determined by regression analysis of the linear portions of the log concentration-response curves. Sensitivity was expressed as pD2 (−log EC50). The statistical significance was calculated by analysis of variance or Student t test where appropriate. P<0.05 considered significant.
Body weights and levels of plasma Hcy in control and methionine-administered rats are shown in Table 1. Body weights were found to be significantly lower in the hHcy group compared with the control. On the other hand, plasma Hcy levels of hHcy rats were found to be significantly higher than in control rats (P<0.05). Oral methionine diet induced a marked hHcy with an approximately 6-fold increment in plasma Hcy levels as compared with control rats (P<0.05).
Change in the Reactivity of Atrium and Thoracic Aorta to Ado in hHcy
The beating rates were not significantly different between the groups (300±21 and 338±19 beats/min for the control and hHcy atria, respectively). Ado produced concentration-dependent decreases in chronotropic action both in control and in hHcy groups. However, the inhibitory effect of Ado on chronotropic action was found to be significantly greater in hHcy rats than in controls (Fig. 1A). pD2 values for Ado were 4.09±0.28 and 4.79±0.18 for control and hHcy groups, respectively. Emax values for Ado were 91.8±3.9% inhibition for control and 96.9±2.1% inhibition for hHcy groups. DIP significantly potentiated the negative chronotropic response to Ado in both groups, but it was more pronounced in hHcy rats (Fig. 1B, P<0.05). Additionally, incubation of atrial preparations with 8-PT caused a significant inhibition on negative chronotropic effect of Ado in both groups (Fig. 1C, P<0.05).
Although Ado caused a concentration-dependent relaxation in endothelium-intact rat aorta of 2 groups, Ado-induced relaxation responses were significantly more pronounced in rats treated with oral methionine for 12 weeks (Fig. 2A, P<0.05). The maximum relaxation induced by Ado was 40.8±2.4 and 53.8±3.7% for control and hHcy rats, respectively. The concentration-response curve for Ado was shifted to the left in aorta rings obtained from either control or hHcy rats after the incubation of DIP (Fig. 2B). However, Ado-induced relaxation response in the presence of DIP was still more pronounced in hHcy rats than in controls Emax values for Ado in the presence of DIP were 55.7±4.8 and 73.9±4.3% for control and hHcy groups, respectively.
In vivo administration of either Ado or DIP caused a significant decrease in mBP and HR of control and hHcy rats (Table 2). However, either Ado-evoked or DIP-evoked reduction in mBP and HR was significantly pronounced in hHcy rats when compared with control animals (P<0.05).
Change in the Reactivity of Atrium and Thoracic Aorta to Adrenoceptor Agonists in hHcy
ISO produced concentration-dependent increases in chronotropic action both in control and in hHcy groups. However, the effect of ISO on chronotropic action was found to be significantly different in atria obtained from hHcy rats compared with controls (Fig. 3, P<0.05). pD2 values for ISO were 7.72±0.26 and 7.95±0.24 for control and hHcy groups, respectively. Emax values for ISO were 638±22 and 742±40 beats/min for control and hHcy atria, respectively. Otherwise, Phe-induced contraction responses were not significantly changed in the aorta rings isolated from the hHcy rats (Fig. 4, P>0.05). The concentration-response curve and maximum contraction response for Phe were similar in aorta rings collected from hHcy rats when compared with controls.
This experimental research was conducted to investigate the effects of hHcy on cardiovascular reactivity to adenosinergic and adrenoceptor agonists in rats. There are sound reasons to believe that the animals treated with oral methionine for 12 weeks in the present study were fully hHcy.
Increasing epidemiologic and experimental studies indicate that even a mild elevation of plasma Hcy concentration is an independent risk factor for cardiovascular diseases.23 The detrimental effects of increased plasma Hcy levels in hHcy patients may be due to several mechanisms. Recently, Riksen and coworkers17 have drawn attention to a new hypothesis, focusing on the influence of Hcy on the metabolism of the endogenous nucleoside Ado. The authors have suggested that hHcy decreases plasma and tissue Ado concentrations, and a decrease in Ado level may be an important mechanism mediating the pathogenic effects of hHcy.18 Till date, however, it remains to be established whether there is a change in reactivity of the cardiovascular system to Ado in hHcy. In the present study, we show for the first time that hHcy causes an alteration in reactivity of cardiovascular system to Ado. In our study, Ado caused a significant decrease in chronotropic action rate in isolated both control and hHcy rat atrium. Also, it produced a significant reduction in mBP and HR in vivo. Importantly, the negative chronotropic effect of Ado was found to be significantly greater in atria obtained from hHcy rats than those from control group. In addition, our study showed that hHcy rat thoracic aorta was significantly more sensitive to the vasorelaxant effect of Ado. In agreement with these results, Ado also produced more pronounced effect on mBP and HR in vivo. The finding of a similar increment in sensitivity to negative chronotropic and vasodilatory effect of Ado may suggest that alteration in sensitivity to effect of Ado in rat atria and thoracic aorta is an important finding in the course of the hHcy process. As the A1 and A2 Ado receptors mediate the negative chronotropic and the vasodilatory effect of Ado, respectively24, we suggest that both A1 and A2-receptor-mediated cardiovascular responses were concomitantly augmented by the high-methionine diet. Possible changes in the process of nucleoside uptake, breakdown, or the receptor and/or postreceptor mechanisms in experimental hHcy might explain the observed hyperreactivity to Ado in rat atria and thoracic aorta. In the present study, the Ado uptake inhibitor, DIP, caused a significant increment in the inhibitory effects of Ado in both control and hHcy rats. Additionally, DIP in vivo caused a significant reduction in mBP and HR. Importantly, this effect is also more pronounced in hHcy rats. The enhanced potentiating effect of DIP on the negative chronotropic action of Ado that was more pronounced in the presence of hHcy, supports the hypothesis that hHcy is associated with accelerated cellular Ado uptake. Although the further increase in inhibitory effect of Ado in hHcy rats does not exclude a possible concurrent increase in Ado uptake as an explanation for the observed effects, it also indicates a possible concurrent change in the receptor and/or postreceptor mechanisms of Ado in hHcy. As the Ado-evoked relaxant and negative chronotropic responses were still more pronounced in hHcy rats in the presence of DIP, we suggested that hHcy may lead to increased reactivity to Ado on the cardiovascular system via receptor up-regulation and/or increased signaling, in addition to accelerated cellular Ado uptake. In contrast to our findings, Riksen et al19 have found that the Ado-induced vasodilatation is impaired but is restored by DIP. This discrepancy could be because of the differences in either the methods used or the course of the hHcy process. Also, the experimental methionine-induced hHcy in the rat model may differ from the hHcy in man. However, the aim of our study was to investigate the possible mechanisms responsible for Hcy-induced changes in reactivity to Ado. Therefore, the use of experimental hHcy model is sometimes required in vitro to reproduce the in vivo situation. Most importantly, the experiments in vitro could not completely mimic the conditions in vivo, but we can exclude the impact of many factors in vivo and are widely used to evaluate the effects of nucleosides per se on the mechanisms underlying them.
Several mechanisms may be involved in the increased reactivity of cardiovascular system to Ado in hHcy. It was reported that the Ado receptor antagonist, caffeine, induces a compensatory sensitization of the A1 receptor-adenylate cyclase system.25 Besides, exposure of the myocardium to theophylline increases the number of Ado receptors.26 These results supported the findings of Varani et al,27 who suggested that blockade by caffeine of Ado receptors can lead to up-regulation of Ado A2A receptors, which is accompanied by sensitization to the actions of the Ado receptor agonist 2-hexynyl-5′-N-ethylcarboxamidoadenosine. In light of these explanations, it could be suggested that hHcy may increase Ado receptor-mediated responses by alterations at the level of Ado receptor (up-regulation of Ado receptors) and/or by compensatory sensitization of Ado receptor-G protein-adenylate cyclase system. However, our findings have clearly shown that hHcy had a significant effect on both cardiac A1 and vascular A2 Ado receptors. Recent evidence suggests that an increase in the number of G protein or the efficiency of their coupling with receptors could lead to an increased maximum response.28 In general, Ado A1 receptor subtype is believed to be coupled to Gi and Go proteins.29 The transduction system that has been associated with the activation of A1 receptors is inhibition of adenylate cyclase.30 In contrast to Ado A1 receptors, vasodilatory A2 receptors couple the Gs-protein,31 and the transduction system involves the stimulation of adenylyl cyclase.32 In view of these facts, it could be suggested that the increased sensitivity to both Ado A1 and A2 receptor stimulation in hHcy may be due to possible changes in Ado receptor-G protein system, but probably not in adenylate cyclase activity.
Besides that, we have also shown that responses of cardiovascular system to adrenergic receptor agonists were potentiated by hHcy. In the present study, the positive chronotropic effects of ISO were found to be significantly greater in hHcy rats than in control group. The adrenergic and Ado receptors belong to the superfamily of G protein-coupled receptors that transduce extracellular stimuli to activate intracellular signaling pathway.33 It has been accepted that the Ado A1 receptors seem to act on many effectors or second messenger systems.24 The activation of β-adrenergic receptor stimulates cyclic AMP accumulation, whereas agonist activation of Ado A1 receptor inhibits cyclic AMP accumulation to provide antagonistic control on receptor-mediated adenylate cyclase activity at the cellular level. An inadequate stimulation of A1 receptors in hHcy may lead to disruption of the balance between Ado A1 and β-adrenergic receptor signaling and cyclic AMP accumulation in atrium, resulting in increased reactivity to β-adrenergic receptor stimulation. As mentioned above, inadequate Ado receptor stimulation in consequence of enhanced cellular Ado uptake might lead to changes in Ado receptor-G protein activity in these tissues and probably accounts for the observed increased atrial reactivity to ISO in hHcy rats. In line with these explanations, we suggest that the accelerated cellular Ado uptake in the course of the hHcy process is likely to be involved in the potentiated response to ISO. Interestingly, hHcy rat thoracic aorta was not significantly more sensitive to the effect of Phe, indicating the unvariable reactivity of aorta to α-adrenergic receptor agonists. As the α-adrenergic receptor is thought to be coupled to G proteins,34,35 a possible change in G proteins is largely excluded in hHcy conditions. The reason that there is no increase in the reactivity of cardiovascular system to α-receptor stimulation might indicate a possible change in Ado receptors than those in G proteins in hHcy rats. This possibility is further supported by the fact that the potentially different signal-transduction pathways mediate the direct negative chronotropic and indirect antiadrenergic actions of Ado.36 More extensive studies investigating the molecular mechanisms of cardiovascular hyperreactivity to Ado and adrenoceptor agonists in hHcy are required to elucidate the exact mechanism responsible for this effect and further studies are need to clarify the issue.
In conclusion, both atria and thoracic aorta isolated from 12-week high-methionine diet-induced hHcy rats were found to be significantly more sensitive to the negative chronotropic and vasorelaxant effects of Ado, possibly because of accelerated cellular Ado uptake and/or a change in Ado receptor-G protein system. Moreover, the effects of β-adrenergic agonists on cardiovascular system are significantly potentiated by hHcy, which might contribute to the harmful cardiac effects of hHcy.
The authors thank the support provided by the Novartis, Turkey, and Akdeniz University Research Foundation.
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