Interplay between inhibition of adenosine uptake and phosphodiesterase type 3 on cardiac function by cilostazol, an agent to treat intermittent claudication

The authors have recently shown that cilostazol, a type 3 cyclic nucleotide phosphodiesterase (PDE3) inhibitor, has a much weaker positive inotropic effect than milrinone, a PDE3 inhibitor of similar potency. They have also shown that cilostazol inhibits adenosine uptake, whereas milrinone has no such effect. This study investigated the possible cardiac functional significance of cilostazol on adenosine uptake inhibition. In isolated rabbit hearts, 10 microM of cilostazol elevated adenosine concentration in interstitial dialysate (0.16 +/- 0.01 microM, or approximately 0.81 microM in the interstitial space when adjusted for recovery rate of microdialysis) and coronary effluent (0.69 +/- 0.03 microM ). The values are significantly higher than those for 10 microM of milrinone (0.11 +/- 0.1 microM in interstitial dialysate and 0.2 +/- 0.04 microM in coronary effluent). Although cilostazol increased contractility, heart rate, and coronary flow in isolated rabbit hearts, the effect on contractility and heart rate was significantly augmented in the presence of an adenosine A 1 receptor antagonist. Conversely, an adenosine A 1 receptor agonist or an adenosine uptake inhibitor attenuated the positive inotropic effect of milrinone. These results indicate that adenosine uptake inhibition by cilostazol increases interstitial and circulatory adenosine concentration, and antagonizes PDE3 inhibition-induced contractility and heart rate increases through an adenosine A 1 receptor-mediated mechanism.     

New mechanism of action for cilostazol: interplay between adenosine and cilostazol in inhibiting platelet activation

Cilostazol, a potent phosphodiesterase 3 inhibitor and anti-thrombotic agent, was recently shown to inhibit adenosine uptake into cardiac myocytes and vascular cells. In the present studies, cilostazol inhibited [ H]-adenosine uptake in both platelets and erythrocytes with a median inhibitory concentration (IC ) of 7 micro M. Next collagen-induced platelet aggregation was studied and it was found that adenosine (1 micro M ), having no effect by itself, shifted the IC of cilostazol from 2.66 micro M to 0.38 micro M (p < 0.01). This shifting was due to an enhanced accumulation of cAMP in platelets and was significantly larger than that by the combination of adenosine and milrinone, which has no effect on adenosine uptake. Similarly, cilostazol, by blocking adenosine uptake, enhanced the adenosine-mediated cAMP increase in Chinese hamster ovary cells that overexpress human A receptor. Furthermore, the inhibitory effect of cilostazol on platelet aggregation in whole blood was significantly reversed by ZM241385 (100 n ), an A adenosine receptor antagonist, and by adenosine deaminase (2 U/ml). These data suggest that the inhibitory effects of cilostazol on adenosine uptake and phosphodiesterase 3 together elevate intracellular cAMP, resulting in greater inhibition of agonist-induced platelet activation.     

Comparison of the effects of cilostazol and milrinone on intracellular cAMP levels and cellular function in platelets and cardiac cells.

Cilostazol is a potent cyclic nucleotide phosphodiesterase (PDE) type 3 (PDE3) inhibitor that was recently approved by the Food and Drug Administration (FDA) for the treatment of intermittent claudication. Its efficacy is presumed to be due to its vasodilatory and platelet activation inhibitory activities. Compared with those treated with placebo, patients treated with cilostazol showed a minimal increase in cardiac adverse events. Because of its PDE3 inhibitory activity, however, the possibility that cilostazol exerts positive cardiac inotropic effects is a safety concern. Therefore we compared the effects of cilostazol with those of milrinone, a selective PDE3 inhibitor, on intracellular cyclic adenosine monophosphate (cAMP) levels in platelets, cardiac ventricular myocytes, and coronary smooth muscle cells. We also compared the corresponding functional changes in these cells. Cilostazol and milrinone both caused a concentration-dependent increase in the cAMP level in rabbit and human platelets with similar potency. Furthermore, cilostazol and milrinone were equally effective in inhibiting human platelet aggregation with a median inhibitory concentration (IC50) of 0.9 and 2 microM, respectively. In rabbit ventricular myocytes, however, cilostazol elevated cAMP levels to a significantly lesser extent (p < 0.05 vs. milrinone). By using isolated rabbit hearts with a Langendorff preparation, we showed that milrinone is a very potent cardiotonic agent; it concentration-dependently increased left ventricular developed pressure (LVDP) and contractility. Cilostazol was less effective in increasing LVDP and contractility (p < 0.05 vs. milrinone), which is consistent with the cardiac cAMP levels. The cardiac effect of OPC-13015, a metabolite of cilostazol with about sevenfold higher PDE3 inhibition, was similar to cilostazol. Whereas milrinone concentration-dependently increased cAMP in rabbit coronary smooth muscle cells, cilostazol did not have such an effect. However, both compounds increased coronary flow equally in rabbit hearts. Our results show that although cilostazol and milrinone both inhibit PDE3, cilostazol preferentially acts on vascular elements (platelets and flow). This unique profile of cilostazol is consistent with its beneficial and safe clinical outcomes in patients with intermittent claudication.     

Intravenous adenosine protects the myocardium primarily by activation of a neurogenic pathway

Endogenous adenosine is a trigger for ischemic myocardial preconditioning (IPC). Although intravascular administration of adenosine has been used to further unravel the mechanism of protection by IPC, it is questionable whether adenosine and IPC employ the same signaling pathways to exert cardioprotection. We therefore investigated whether the active metabolic barrier of the endothelium prevents an increase in myocardial interstitial adenosine concentrations by intravenous adenosine, using microdialysis, and also the role of NO and activation of a neurogenic pathway in the cardioprotection by adenosine. In pentobarbital-anesthetized rats, area at risk and infarct size (IS) were determined 120 min after a 60-min coronary artery occlusion (CAO), using trypan blue and nitro-blue-tetrazolium staining, respectively. IPC with a single 15-min CAO and a 15-min adenosine infusion (ADO, 200 microg min(-1) i.v.) limited IS to the same extent (IS = 41 +/- 6% and IS = 40 +/- 4%, respectively) compared to control rats (IS = 63 +/- 3%, both P < 0.05). However, IPC increased myocardial interstitial adenosine levels seven-fold from 4.3 +/- 0.7 to 27.1 +/- 10.0 microM (P < 0.05), while ADO had no effect on interstitial adenosine (4.1 +/- 1.2 microM), or any of the other purines. The NO synthase inhibitor N(omega)-nitro-L-arginine (LNNA), which did not affect IS (IS = 62 +/- 3%), attenuated the protection by ADO (IS = 56 +/- 3%; P < 0.05 vs ADO, P = NS vs LNNA). The ganglion blocker hexamethonium, which had also no effect on IS (IS = 66 +/- 3%), blunted the protection by ADO (IS = 55 +/- 4%; P < 0.05 vs ADO and vs hexamethonium). These observations demonstrate that cardioprotection by ADO is dependent on NO, and is primarily mediated by activation of a neurogenic pathway.     

Cilostazol and dipyridamole synergistically inhibit human platelet aggregation

It has been previously shown that cilostazol (Pletal), a drug for relief of symptoms of intermittent claudication, potently inhibits cyclic nucleotide phosphodiesterase type 3 (PDE3) and moderately inhibits adenosine uptake. It elevates extracellular adenosine concentration, by inhibiting adenosine uptake, and combines with PDE3 inhibition to augment inhibition of platelet aggregation and vasodilation while attenuating positive chronotropic and inotropic effects on the heart. In the present study, we tested the hypothesis that cilostazol combined with a more potent adenosine uptake inhibitor, dipyridamole, synergistically inhibited platelet aggregation in human blood. In the presence of exogenous adenosine (1 microM), the combination of cilostazol and dipyridamole synergistically increased intra-platelet cAMP. Furthermore, cilostazol inhibited platelet aggregation in a washed platelet assay concentration-dependently with IC50s of 0.17 +/- 0.04 microM (P < 0.05 versus plus adenosine alone of 0.38 +/- 0.05 microM), 0.11 +/- 0.06 microM (P < 0.05), and 0.01 +/- 0.01 microM (P < 0.005) when combined with 1, 3, or 10 microM dipyridamole, respectively (n = 5). In whole blood, cilostazol (0.3 to 3 microM) and dipyridamole (1 or 3 microM) synergistically inhibited collagen- and ADP-induced platelet aggregation in vitro. Furthermore, the synergism was confirmed in an open-label, sequential study in healthy human subjects using ex vivo whole-blood collagen-induced platelet aggregation. Four hours after oral co-administration of cilostazol (100 mg) and dipyridamole (200 mg), platelet aggregation was inhibited by 45 +/- 17%, while no significant inhibition was observed from subjects treated with either drug alone. The combination may provide a potential treatment of arterial thrombotic disorders.     

Cilostazol (pletal): a dual inhibitor of cyclic nucleotide phosphodiesterase type 3 and adenosine uptake

Cilostazol (Pletal), a quinolinone derivative, has been approved in the U.S. for the treatment of symptoms of intermittent claudication (IC) since 1999 and for related indications since 1988 in Japan and other Asian countries. The vasodilatory and antiplatelet actions of cilostazol are due mainly to the inhibition of phosphodiesterase 3 (PDE3) and subsequent elevation of intracellular cAMP levels. Recent preclinical studies have demonstrated that cilostazol also possesses the ability to inhibit adenosine uptake, a property that may distinguish it from other PDE3 inhibitors, such as milrinone. Elevation of interstitial and circulating adenosine levels by cilostazol has been found to potentiate the cAMP-elevating effect of PDE3 inhibition in platelets and smooth muscle, thereby augmenting antiplatelet and vasodilatory effects of the drug. In contrast, elevation of interstitial adenosine by cilostazol in the heart has been shown to reduce increases in cAMP caused by the PDE3-inhibitory action of cilostazol, thus attenuating the cardiotonic effects. Cilostazol has also been reported to inhibit smooth muscle cell proliferation in vitro and has been demonstrated in a clinical study to favorably alter plasma lipids: to decrease triglyceride and to increase HDL-cholesterol levels. One, or a combination of several of these effects may contribute to the clinical benefits and safety of this drug in IC and other disease conditions secondary to atherosclerosis. In eight double-blind randomized placebo-controlled trials, cilostazol significantly increased maximal walking distance, or absolute claudication distance on a treadmill. In addition, cilostazol improved quality of life indices as assessed by patient questionnaire. One large randomized, double-blinded, placebo-controlled, multicenter competitor trial demonstrated the superiority of cilostazol over pentoxifylline, the only other drug approved for IC. Cilostazol has been generally well-tolerated, with the most common adverse events being headache, diarrhea, abnormal stools and dizziness. Studies involving off-label use of cilostazol for prevention of coronary thrombosis/restenosis and stroke recurrence have also recently been reported.     

Protecting murine hearts from ischaemia-reperfusion using selective inhibitors of adenosine metabolism

1. By selectively modifying adenosine metabolism via adenosine deaminase or adenosine kinase inhibitors, it may be possible to enhance the receptor-mediated protective actions of adenosine in a site- and event-specific fashion. 2. We characterized cardioprotective actions of the adenosine deaminase inhibitor erythro-2-(2-hydroxy-3-non-yl)adenine (EHNA) and the adenosine kinase inhibitor iodotubercidin in C57/Bl6 mouse hearts subjected to 20 min global normothermic ischaemia and 40 min reperfusion. 3. Ventricular pressure development only recovered to 45 +/- 2% of baseline levels (67 +/- 5 mmHg) in untreated hearts, with sustained and pronounced diastolic contracture (25 +/- 2 mmHg). Treatment with 20 micromol/L EHNA increased recovery of ventricular pressure (107 +/- 9 mmHg), reduced postischaemic diastolic pressure (13 +/- 1 mmHg) and reduced loss of lactate dehydrogenase (LDH; an indicator of necrotic damage) by 50% (9 +/- 2 vs 19 +/- 2 IU/g). Adenosine kinase inhibition with 10 micromol/L iodotubercidin also improved pressure development (to 100 +/- 8 mmHg) and reduced LDH efflux (5 +/- 2 IU/g). 4. Protective actions were mimicked by adenosine and inhibited by adenosine receptor antagonism (50 micromol/L 8-rho-sulfophenyltheophylline) and mitochondrial K(ATP) channel inhibition (50 micromol/L 5-hydroxydecanoate). 5. Coinfusion of the inhibitors, 'trapping' formed adenosine, failed to exert protection and, in some instances, was detrimental. Although substantial benefit was gained by these agents in hearts from young animals, neither inhibitor was effective in 'aged' hearts (18 months). 6. Our data demonstrate that adenosine deaminase or kinase inhibition substantially limits injury during ischaemia-reperfusion. Protection involves adenosine receptor activation. However, cardioprotection via either enzyme inhibitor requires an alternative purine-salvage pathway to be functional and was reduced in aged hearts known to be increasingly susceptible to ischaemic damage.     

Protection from myocardial stunning by ischaemia and hypoxia with the adenosine A3 receptor agonist, IB-MECA

Guinea pig isolated working hearts were exposed to 30-min ischaemia by reducing coronary flow to 10%, followed by reperfusion. Aortic output fell to 4.5+/-4.5% of the pre-ischaemic value at reperfusion, recovering to 48.2+/-14.6% at 20-min post-reperfusion; the index of myocardial stunning. IB-MECA (N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide, 3 x 10(-7) M), infused from 10 min into ischaemia, did not affect recovery of aortic output 20 min after reperfusion (41.9+/-1.9%). IB-MECA infused at reperfusion, however, significantly protected against stunning, aortic output recovering to 79.6+/-3.9% at 20-min post-reperfusion. Hypoxic gassing (5% CO(2) in nitrogen, 30 min) of guinea pig isolated paced left atria and papillary muscles reduced the developed tension, recovering to 75% 5 min after re-oxygenation. This myocardial stunning was unaffected by IB-MECA (3 x 10(-7) M) added 10 min into hypoxia. IB-MECA added at reoxygenation significantly improved recovery, which was prevented by the adenosine A(3) receptor antagonist, 1-propyl-3-(3-iodo-4-aminobenzyl)-8-(4-oxyacetate)phenylxanthine (I-ABOPX, 1 x 10(-5) M). Thus, stimulation of adenosine A(3) receptors at reperfusion/reoxygenation in guinea pig cardiac preparations protects against myocardial stunning.     

Effect of aging on the negative chronotropic and anti-beta-adrenergic actions of adenosine in the rat heart

The effect of aging on the antiadrenergic actions of adenosine was studied in vitro and in vivo by using adult (6-month-old) and old (24-month-old) male Fischer 344 rats. In anesthetized animals, adenosine (0.01-0.1 micromol/kg), given as a rapid bolus into the right atrium, exerted a negative chronotropic effect manifested by a dose-dependent transient prolongation of sinus cycle length (SCL). This effect was similar in both age groups (n = 6, each; i.e., the percentage maximal prolongation of SCL (%deltaSCL) ranged from 12 +/- 2% to 63 +/-14% in the adult and from 20 +/- 7% to 57 +/- 15% in the old rats. In the presence of isoproterenol (0.2 microg/kg/min), the negative chronotropic action of adenosine was potentiated in the adult rats much more than in the old rats [i.e., %deltaSCL ranged from 60 +/- 28% to 183 +/- 48% vs. 40 +/- 12% to 70 +/- 13%, respectively (p < 0.05, adult vs. old)]. In the isolated perfused hearts, isoproterenol (1 microM for 1 min) exerted similar chronotropic and inotropic effects in adult (n = 9) and old hearts [n = 6; i.e., heart rate, left ventricular pressure (LVP), and LVdp/dt increased by 56 +/- 3%, 17 +/- 1%, and 37 +/- 2%, and 57 +/- 2%, 17 +/- 1%, and 35 +/- 3%, respectively, in the absence of, and by 27 +/- 2%, 7 +/- 1%, and 19 +/- 2% and 41 +/- 3%, 12 +/- 1%, and 25 +/-2% in the presence of adenosine (5 microM for 1 min)]. Adenosine administration after isoproterenol caused only an insignificant increase in coronary blood flow. Finally, the adenosine attenuation of either isoproterenol- or forskolin-induced production of 3',5'-cyclic adenosine monophosphate (cAMP) was significantly less in atrial membranes isolated from old versus adult rats (n = 6, each). It was concluded that in the old Fischer 344 rat hearts, the antiadrenergic action of adenosine is attenuated as compared with its action in adult rat hearts.     

Effects of captopril on metabolic and hemodynamic alterations in global ischemia and reperfusion in the isolated working rat heart.

In the isolated working rat heart model, we studied metabolic and hemodynamic effects of 5- and 30-min global ischemia followed by reperfusion and assessed the potentially beneficial effect of captopril 80 micrograms/ml added throughout the experiment. Creatine kinase (CK) and catecholamines were measured in coronary effluent. De novo eicosanoids (prostaglandin E2) synthesis was assessed in endocardial explants. Hemodynamic alterations occurred after 30-min ischemia and were reflected most dramatically by a reduction in cardiac output (CO 72 +/- 10% of baseline values in captopril vs. 68 +/- 16% in controls) without significant differences as a result of treatment. Captopril shortened reperfusion ventricular fibrillation (VF) duration (6.9 +/- 1.2 vs. 13.6 +/- 8.7 min, p less than 0.05) but had no effect on VF incidence. No differences occurred in norepinephrine (NE) outflow, whereas total CK release was greater in controls. Five controls versus none of the treated hearts (p less than 0.05) released trace amounts of epinephrine during reperfusion. Increased de novo PGE2 synthesis was demonstrated after 5-min I (465 +/- 168 vs. 238 +/- 75 pg/100 mg tissue per hour, p less than 0.01). Captopril stimulated production of PGE2 in normoxic hearts (p less than 0.02), but the difference was no more apparent in ischemic hearts. We conclude that captopril produces some biochemical and electrophysiologic evidence of myocardial salvage, but these effects are not sufficient to induce hemodynamic improvement after global ischemia and reperfusion.     

Ischemic preconditioning protects post-ischemic renal function in anesthetized dogs： role of adenosine and adenine nucleotides

Aim: To investigate the effects of renal ischemic preconditioning (IPC) on both renal hemodynamics and the renal interstitial concentrations of adenosine and adenine nucleotides induced by ischemia-reperfusion injury. Methods: Renal hemodynamics responses to ischemia-reperfusion injury in mongrel dog models were determined with or without multiple brief renal ischemic preconditioning treatments, as well as the adenosine A1 receptor antagonist (KW-3902), respectively. The renal interstitial concentrations of adenosine and adenine nucleotides in response to ischemia-reperfusion injury, either following 1-3 cycles of IPC or not, were measured simultaneously using microdialysis sampling technology. Results: One 10-min IPC, adenosine A1 receptor antagonist (KW-3902) also shortened the recovery time of renal blood flow (RBF) and urine flow (UF), as well as mean blood pressure (BP). Advanced renal IPC attenuated the increment of adenosine and adenine nucleotides, as well as recovery time during the 60-min reperfusion which followed the 60-min renal ischemia. All of these recovery times were dependent on the cycles of 10-min IPC. The renal interstitial concentrations of adenosine and adenine nucleotides increased and decreased during renal ischemia and reperfusion, respectively. Conclusion: A significant relativity in dog models exists between the cycles of 10-min renal IPC and the recovery time of BP, UF, and RBF during the 60-min renal reperfusion following 60-min renal ischemia, respectively. Renal IPC can protect against ischemiareperfusion injury and the predominant effect of endogenous adenosine induced by prolonged renal ischemia; renal adenosine A1 receptor activation during the renal ischemia-reperfusion injury is detrimental to renal function.     

Effect of the pyridoindole SMe1EC2 and compounds affecting A(1) and A(2A) adenosine receptors in rat hippocampus under ischemia in vitro

The effect of the newly synthesized pyridoindole antioxidant SMe1EC2 (1 mumol/l) and drugs activating or inhibiting adenosine receptors was tested under ischemia. Synaptic transmission was recorded extracellularly before and under 6-min ischemia and 20-min reoxygenation in rat hippocampal slices in vitro. In untreated slices, ischemia elicited failure of synaptic transmission and excitability expressed by a population spike decay (t(0.5) = 1.7 +/- 0.1 min) and poor recovery of synaptic transmission at the end of reoxygenation, expressed as percentage of PoS amplitude of that at zero minute of ischemia (9.9 +/- 3.6%). The compound SMe1EC2 increased recovery of PoS amplitude in reoxygenation (31.2 +/- 7.0% of that at the beginning of ischemia) and decreased the number of irreversibly damaged slices in reoxygenation (64%) compared to untreated slices (80%). Co-administration of SMe1EC2 + SCH-58261 (1 mumol/l, A(2A) adenosine receptor antagonist) resulted in delayed synaptic transmission decay during 6-min ischemia (t(0.5) = 2.3 +/- 0.1 min), increased PoS amplitude recovery during reoxygenation (37.7 +/- 12.4% of that at zero minute of ischemia), and in a decreased number of slices with damaged synaptic transmission at the end of reoxygenation (54%), all data compared to untreated controls. Co-administration of pyridoindole with CGS 21680 (1 mumol/l, A(2A) adenosine receptor agonist) or with DPCPX (100 nmol/l, A(1) adenosine receptor antagonist) eliminated the described effect. Further studies are required to elucidate the putative influence of manipulation with adenosine receptors on the neuroprotective effect of SMe1EC2 under ischemia.     

Effects of the cardioselective KATP channel blocker HMR 1098 on cardiac function in isolated perfused working rat hearts and in anesthetized rats during ischemia and reperfusion

It has been argued that activation of KATP channels in the sarcolemmal membrane of heart muscle cells during ischemia provides an endogenous cardioprotective mechanism. In order to test whether the novel cardioselective KATP channel blocker HMR 1098 affects cardiac function during ischemia, experiments were performed in rat hearts during ischemia and reperfusion. Isolated perfused working rat hearts were subjected to 30 min of low-flow ischemia in which the coronary flow was reduced to 10% of its control value, followed by 30-min reperfusion. In the first set of experiments the hearts were electrically paced at 5 Hz throughout the entire protocol. At the end of the 30-min ischemic period the aortic flow had fallen to 44 +/- 2% (n=8) of its nonischemic value in vehicle-treated hearts, whereas in the presence of 0.3 micromol/l and 3 micromol/l HMR 1098 it had fallen to 29 +/- 7% (n=5, not significant) and 8 +/- 2% (n=12, P<0.05), respectively. Glibenclamide (3 micromol/l) reduced the aortic flow to 9.5 +/- 7% (n=4, P<0.05). In control hearts the QT interval in the electrocardiogram shortened from 63 +/- 6 ms to 36 +/- 4 ms (n=10, P<0.05) within 4-6 min of low-flow ischemia. This shortening was completely prevented by 3 micromol/l HMR 1098 (60 +/- 5 ms before ischemia, 67 +/- 6 ms during ischemia, n=9, not significant). When rat hearts were not paced, the heart rate fell spontaneously during ischemia, and HMR 1,098 (3 micromol/l) caused only a slight, statistically non-significant reduction in aortic flow during the ischemic period. In order to investigate whether HMR 1098 shows cardiodepressant effects in a more pathophysiological model, the left descending coronary artery was occluded for 30 min followed by reperfusion for 60 min in anesthetized rats. Treatment with HMR 1098 (10 mg/kg i.v.) had no statistically significant effects on mean arterial blood pressure and heart rate during the control, ischemia and reperfusion periods. At the end of the reperfusion period, aortic blood flow was slightly reduced by HMR 1098, without reaching statistical significance (two-way analysis of ANOVA, P=0.15). Myocardial infarct size as a percentage of area at risk was not affected by HMR 1098 (vehicle: 75 +/- 3%, HMR 1098: 72 +/- 2%, n=7 in each group). In conclusion, cardiodepressant effects of HMR 1098 were observed only in isolated perfused working rat hearts which were continuously paced during global low-flow ischemia. In the model of anesthetized rats subjected to regional ischemia, HMR 1098 had no significant effect on cardiac function or infarct size.     

Further characterization of an adenosine transport system in the mitochondrial fraction of rat testis

Previous work from our laboratory has demonstrated the presence of high-affinity binding sites for [3H]nitrobenzylthioinosine ([3H]NBTI), a marker of adenosine uptake systems, in the mitochondrial fraction of rat testis. Here, we characterize this system functionally through [3H]adenosine uptake assays. This system (K(m)=2+/-1.3 microM; V(max)=86.2+/-15.5 pmol/mg protein/min) was found to be saturable, non sodium-dependent and sensitive to temperature, pH and osmolarity. [3H]Adenosine incorporation was potently inhibited by hydroxynitrobenzylthioguanosine (HNBTG, IC(50)=3 nM) although NBTI inhibited this uptake weakly (IC(50)=72. 7+/-37.1 microM). Dilazep>dipyridamole>/=hexobendine inhibited [3H]adenosine incorporation at low micromolar concentrations. The nucleosides inosine and uridine were weak inhibitors of this system. The adenosine receptor ligands N(6)-phenylisopropyladenosine (PIA) and 2-chloroadenosine inhibited the uptake only at micromolar concentrations. Neither 5'-(N-ethylcarboxamido)-adenosine (NECA) nor theophylline inhibited adenosine uptake by more than 60% but the mitochodrial benzodiazepine receptor ligands 4'-chloro-diazepam (Ro 5-4864) and 1-(2-chlorophenyl)-N-methyl-N-(1-methyl-propyl) isoquinoline carboxamide (PK 11195) were able to inhibit it. The lack of inhibition by the blockers of the mitochondrial adenine-nucleotide carrier, atractyloside and alpha, beta-methylene-ATP, indicates that [3H]adenosine uptake occurs via a transporter other than this carrier. All these results support the existence of an equilibrative adenosine transport system, which might mediate the passage of adenosine formed in the mitochondria to the cytoplasm.     

Functional characterization of coronary vascular adenosine receptors in the mouse

Coronary responses to adenosine agonists were assessed in perfused mouse and rat hearts. The roles of nitric oxide (NO) and ATP-dependent K(+) channels (K(ATP)) were studied in the mouse. Resting coronary resistance was lower in mouse vs rat, as was minimal resistance (2.2+/-0.1 vs 3.8+/-0.2 mmHg ml(-1) min(-1) g(-1)). Peak hyperaemic flow after 20 - 60 s occlusion was greater in mouse. Adenosine agonists induced coronary dilation in mouse, with pEC(50)s of 9.4+/-0.1 for 2-[p-(2-carboxyethyl)phenethylamino]-5'-N-ethyl carboxamidoadenosine (CGS21680, A(2A)-selective agonist), 9.3+/-0.1 for 5'-N-ethylcarboxamidoadenosine (NECA, A(1)/A(2) agonist), 8.4+/-0.1 for 2-chloroadenosine (A(1)/A(2) agonist), 7.7+/-0.1 for N(6)-(R)-(phenylisopropyl)adenosine (R-PIA, A(1)/A(2B) selective), and 6.8+/-0.2 for adenosine. The potency order (CGS21680=NECA>2-chloroadenosine>R-PIA>adenosine) supports A(2A) adenosine receptor-mediated dilation in mouse coronary vessels. 0.2 - 2 microM of the A(2B)-selective antagonist alloxazine failed to alter CGS21680 or 2-chloroadenosine responses. pEC(50)s in rat were 6.7+/-0.2 for CGS21680, 7.3+/-0.1 for NECA, 7.6+/-0.1 for 2-chloroadenosine, 7.2+/-0.1 for R-PIA, and 6.2+/-0.1 for adenosine (2-chloroadenosine>NECA=R-PIA>CGS21680> adenosine), supporting an A(2B) adenosine receptor response. NO-synthase antagonism with 50 microM N(G)-nitro L-arginine (L-NOARG) increased resistance by approximately 25%, and inhibited responses to CGS21680 (pEC(50)=9.0+/-0.1), 2-chloroadenosine (pEC(50)=7.3+/-0.2) and endothelial-dependent ADP, but not sodium nitroprusside (SNP). K(ATP) channel blockade with 5 microM glibenclamide increased resistance by approximately 80% and inhibited responses to CGS21680 in control (pEC(50)=8.3+/-0.1) and L-NOARG-treated hearts (pEC(50)=7.3+/-0.1), and to 2-chloroadenosine in control (pEC(50)=6.7+/-0.1) and L-NOARG-treated hearts (pEC(50)=5.9+/-0.2). In summary, mouse coronary vessels are more sensitive to adenosine than rat vessels. A(2A) adenosine receptors mediate dilation in mouse coronary vessels vs A(2B) receptors in rat. Responses in the mouse involve a sensitive NO-dependent response and K(ATP)-dependent dilation.     

Amp 579 reduces contracture and limits infarction in rabbit heart by activating adenosine A2 receptors

To determine the mechanism by which AMP 579, an adenosine A1/A2 agonist, administered at reperfusion protects ischemic myocardium, buffer-perfused rabbit hearts were subjected to 30 min of global ischemia and 2 h of reperfusion. AMP 579 (500 nM) was included in the reperfusate for the first 70 min. Average left ventricular diastolic pressure during reperfusion in hearts receiving AMP 579 was lower than that in control hearts (17.9 +/- 2.4 vs. 39.0 +/- 6.5 mm Hg, p < 0.05), indicating attenuation of contracture. Left ventricular developed pressure and coronary flow during reperfusion were also significantly improved with AMP 579 treatment. AMP 579's anti-contracture effect was blocked by the adenosine A2-receptor antagonist 8-(3-chlorostyryl)caffeine (CSC), but not by the A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). CSC, but not DPCPX, also blocked AMP 579's ability to preserve developed pressure and coronary flow in these hearts. AMP 579 significantly reduced infarction in isolated hearts subjected to regional ischemia. The anti-infarct effect again was abolished by CSC but not by DPCPX. Finally, we tested whether 5'-(N-ethylcarboxamido)adenosine (NECA), another A1/A2 agonist, also administered for the initial 70 min of reperfusion, could duplicate the anti-infarct effect of AMP 579. One-hundred-nanomolar NECA duplicated the protection, but neither 50 nM CGS21680, a selective A2 agonist, nor 100 microM adenosine was protective. Therefore, AMP 579 given at reperfusion reduces contracture and infarction. Anti-contracture and anti-infarct effects require the adenosine A2, but not the A1, receptor suggesting that prevention of contracture and tissue salvage are mechanistically related. Not all A2 agonists were able to duplicate the anti-infarct effect, suggesting something unique about AMP579.     

Chronotropic and vasodilatory responses to adenosine and isoproterenol in mouse heart: effects of adenosine A1 receptor overexpression

1. Chronotropic and vasodilatory effects of adenosine receptor activation with 2-chloroadenosine (2-ClAdo) and beta-adrenoceptor activation with isoproterenol were studied in wild-type murine hearts and transgenic hearts overexpressing the A1 adenosine receptor. 2. Treatment of wild-type hearts with 2-ClAdo induced bradycardia (pEC50 6.4+/-0.2) and vasodilatation (pEC50 7.9+/-0.1; minimal resistance 2.2+/-0.2 mmHg/mL per min per g). The A1 receptor-mediated bradycardia was 20-fold more sensitive in transgenic hearts (pEC50 7.7+/-0.2), whereas coronary vasoactivity of 2-ClAdo was unaltered (pEC50 7.6+/-0.1). 3. beta-Adrenoceptor stimulation with isoproterenol increased heart rate (pEC50 8.5+/-0.2; maximal rate 594+/-23 b.p.m.) and produced vasodilation (pEC50 8.7+/-0.1; minimal resistance 1.7 +/-0.2 mmHg/ml, per min per g) in wild-type hearts. Treatment with 10 IU/mL adenosine deaminase increased the magnitude of the tachycardia (maximal rate 653+/-27 b.p.m.) without altering potency (pEC50 8.5+/-0.1). Antagonism of A1 receptors with 10nmol/L 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) produced a comparable increase in the magnitude of the chronotropic response (maximal rate 695+/-26b.p.m.) without altering potency (pEC50 8.3+/-0.1). 4. Isoproterenol-mediated vasodilatation was unaltered by transgenic A1 receptor overexpression. Overexpression of A1 receptors significantly reduced the maximal heart rate during beta-adrenoceptor stimulation by 35% (to 381 +/-28 b.p.m.) without altering potency (pEC50 8.4+/-0.2). At 10nmol/L, DPCPX increased the magnitude of the chronotropic response to isoproterenol in transgenic hearts (maximal heart rate 484+/-36 b.p.m.) without altering potency (pECs50 8.3+/-0.2). 5. The data show that transgenic A1 receptor overexpression selectively sensitizes the cardiovascular A1 receptor response and that A1 receptor activation by endogenous adenosine depresses the magnitude, but not potency, of the beta-adrenoceptor-mediated chronotropic response in mouse heart. The A1 receptor-mediated depression of beta-adrenoceptor responsiveness is non-competitive (reduced response magnitude with no change in sensitivity). This indicates that A1 receptor activation non-competitively inhibits effector mechanisms activated by beta-adrenoceptors (e.g. adenylate cyclase) and/or A1 receptors activate unrelated but opposing mechanisms. This inhibitory response may have physiological importance during periods of sympathetic stimulation of cardiac work.     

Attenuation of trace element-mediated injury during ischemia and reperfusion by an N-terminus analogue of human albumin (H4DUS60131)

The N-terminus region of human albumin binds strongly to trace metals (Co, Cu, Ni). Ischemia, acidosis and reperfusion can cause a marked increase in plasma free Cu and its normal regulation by plasma proteins may be overwhelmed and predispose to oxidative injury by Cu-catalyzed oxyradical production. H4DUS60131 is an analogue of the N-terminus of human albumin, it binds copper tightly and in vitro, is a potent inhibitor of Cu-catalyzed radical formation. We have tested the ability of H4DUS60131 to reduce injury during ischemia and reperfusion in isolated blood-perfused rat hearts (n = 6/group) subjected to 20-min aerobic perfusion, followed by a 2-min infusion of saline or saline plus H4DUS60131. Following infusion, hearts were subjected to 30-min global ischemia plus 40-min reperfusion. The 2-min infusion was repeated in both groups at the start of reperfusion. In the vehicle controls, left ventricular developed pressure recovered to only 15.3 +/- 3.2%, whereas the H4DUS60131 group recovered to 50.5 +/- 9.3% (p < 0.005). The H4DUS60131 group normalised their left ventricular end diastolic pressure more quickly and completely than the controls (44.1 +/- 11.5 vs. 91.5 +/- 5.5 mm Hg). In conclusion, H4DUS60131 greatly improves the recovery of the rat heart from ischemia and reperfusion and may represent a novel approach to the limitation of myocardial injury.     

Increased sensitivity of neonate atrial myocytes to adenosine A1 receptor stimulation in regulation of the L-type Ca2+ current

Adenosine has cardioprotective effects against ischemia, and newborn hearts show high resistance to ischemia. The effects of purinoceptor stimulation by adenosine and ATP on the L-type Ca2+ current (ICa) were examined in atrial cells from neonate and adult rabbits. ICa was measured by the membrane-perforated patch method. Adenosine inhibited the isoproterenol-stimulated ICa more potently in neonate cells than in adult cells. The high sensitivity of neonate myocytes to adenosine was accompanied not only by an increased maximum response but also by a lower IC50 concentration. ATP also inhibited isoproterenol-stimulated ICa. The effect of ATP on neonate cells was stronger than that on adult cells at high concentrations (greater than or = 100 microM). The effect of adenosine was antagonized by an A1 adenosine receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX). DPCPX or an ecto-5'-nucleosidase inhibitor (alpha,beta-methylene-ADP) blocked most (approximately 60%) of the effect of ATP (30 microM), and co-addition of DPCPX and suramin (P2 receptor blocker) abolished the effect of ATP. Suramin alone did not reduce the effect of ATP significantly in neonate cells. Both the effects of adenosine and ATP were eliminated by pre-treatment with pertussis toxin or by superfusion with forskolin plus 3-isobutyl-1-methylxanthine (IBMX). Inhibitors of the nitric oxide-cyclic GMP pathway did not affect the adenosine inhibition of ICa. In summary, neonatal myocardial cells are highly sensitive to adenosine A1 receptor stimulation. ATP stimulates both the adenosine A1 and P2 receptors. Adenosine A1 receptor stimulation, as a result of hydrolysis of ATP, predominantly mediates the effect of ATP, and the role of P2 receptors in the ATP inhibition of ICa is relatively small in neonate cells. The high sensitivity to adenosine may contribute to the ischemic tolerance of newborn hearts.     

Effects of levosimendan and milrinone on oxygen consumption in isolated guinea-pig heart

Levosimendan is a novel calcium sensitizer that increases contraction force without change in intracellular calcium ([Ca2+]i); milrinone is a phosphodiesterase inhibitor that exerts a positive inotropic effect by increasing [Ca2+]i. The effects of levosimendan and milrinone on oxygen consumption in the isolated guinea-pig heart were studied. Isolated guinea-pig hearts were paced (280 beats/min) and perfused according to the Langendorff technique. Levosimendan (0.01-1 microM) or milrinone (0.1-10 microM) were added cumulatively and changes from baseline for diastolic and systolic pressure (LVEDP and LVSP), contractility and relaxation (+dP/dt and -dP/dt), and coronary flow and oxygen consumption (CF and VO2) were calculated. Levosimendan was found to be 10 to 30 times more potent than milrinone as an inotropic agent. The effect on VO2 was markedly lower in levosimendan-perfused hearts than in milrinone-perfused hearts (P = 0.031 between the concentration-dependent effects of the two drugs). The maximum increase in VO2 was 10 +/- 4% in the levosimendan group and 38 +/- 15% in the milrinone group. The economy of the contraction was more advantageous in levosimendan-perfused hearts (P 相似文献