Adenosine In Rat Hearts Research Articles

Adenosine production and its role in protection against ischemic and reperfusion injury of the myocardium

Adenosine exerts cardioprotective effects on the ischemic myocardium. A flexibly mounted microdialysis apparatus was used to measure the concentration of interstitial adenosine and to assess the activity of ecto-5'-nucleotidase (a key enzyme responsible for adenosine production) in in vivo rat hearts. The level of adenosine during adenosine 5'-adenosine monophosphate (AMP) perfusion serves as an index of the activity of ecto-5'-nucleotidase in the tissue. Endogenous norepinephrine (NE) activates alpha 1-adrenoceptors to lead to the activation of protein kinase C (PKC), which, in turn, activates ecto-5'-nucleotidase via phosphorylation, thereby enhancing the production of interstitial adenosine. Histamine-induced release of NE activates PKC, which increases ecto-5'-nucleotidase activity and augments release of adenosine. Nicorandil, a hybrid of an ATP sensitive K+ (KATP) channel opener and nitrate, increases the level of interstitial adenosine via cGMP-mediated activation of ecto-5'-nucleotidase. Opening of cardiac KATP channels may cause hydroxyl radical (.OH) generation. Nitric oxide (NO) facilitates the production of interstitial adenosine, via activation of ecto-5'-nucleotidase. However, singlet oxygen (1O2) is a very powerful oxidant that causes inactivation of ecto-5'-nucleotidase to result in a decrease in the concentration of adenosine in rat heart. Adenosine plays an important role as a modulator of ischemic reperfusion injury. The production and action of adenosine are intimately linked to the release of NE.

Nicorandil increases adenosine 5'-monophosphate-primed interstitial adenosine via activation of ecto-5'-nucleotidase in rat hearts.

With the use of microdialysis techniques, we examined the effects of nicorandil, a hybrid of an ATP-sensitive K+ (K ATP) channel opener and a nitrate compound, on the production of interstitial adenosine in rat hearts in situ. The level of dialysate adenosine measured under a constant supply of adenosine 5'-monophosphate (AMP) reflected the activity of endogenous ecto-5'-nucleotidase. Nicorandil (0.3-3mM) increased the level of AMP (100 microM)-primed dialysate adenosine in a concentration-dependent manner, and this effect was completely abolished by the guanylate cyclase inhibitor, methylene blue (100 microM), but not by the K ATP channel blocker, glibenclamide (10 microM). Another K ATP channel opener, cromakalim (0.1-1mM), did not increase the production of AMP-primed dialysate adenosine. These results suggest that nicorandil increases the level of interstitial adenosine via cyclic guanosine monophosphate-mediated activation of ecto-5'-nucleotidase.

Reserpine Attenuates Interstitial Adenosine-Mediated Activation of Ecto-5′-nucleotidase in Rat Hearts in Vivo

We examined whether reserpine-induced norepinephrine (NE) depletion attenuated the products of adenosine in rat heart. A flexibly mounted microdialysis technique was used to measure the concentration of interstitial adenosine and to assess the activity of ecto-5′-nucleotidase in rat hearts in situ. The microdialysis probe was implanted in the left ventricular myocardium of anesthetized rats and perfused with Tyrode solution containing adenosine 5′-monophosphate (AMP) at rate of 1.0 μl/min. The baseline level of dialysate adenosine was 0.51 ± 0.09 μM. The introduction of AMP (100 μM) through the probe increased markedly the dialysate adenosine to 8.95 ± 0.86 μM, and this increase was inhibited by ecto-5′-nucleotidase inhibitor, α,β-methyleneadenosine 5′-diphosphate (AOPCP, 100 μM), to 0.66 ± 0.38 μM. Thus, the level of dialysate adenosine is a measure of the ecto-5′-nucleotidase activity in the tissue in situ. AMP concentration for the half-maximal effect of adenosine release (EC50) was 107.3 μM. The maximum attainable concentration of dialysate adenosine (Emax) by AMP was 21.1 μM. However, the EC50 and Emax values with reserpinized animals were 106.9 and 7.1 μM, respectively. Electrical stimulation of the left stellate ganglion increased significantly dialysate adenosine concentration, from the control level of 8.66 ± 0.96 μM to 12.38 ± 1.11 μM. After stimulation, dialysate adenosine returned to near the prestimulation level. When corresponding experiments were performed with reserpinized animals, the effect of electrical stimulation was abolished. Tyramine (endogenous catecholamine trigger) increased the adenosine concentration in a concentration-dependent manner. However, the elevation of adenosine concentration with reserpinized animals was not observed. These results suggest that reserpine attenuates NE-induced adenosine via stimulation of α1-adrenoceptor and protein kinase C mediated activation of ecto-5′-nucleotidase in rat heart.

Tyramine produces interstitial adenosine-mediated activation of ecto-5′-nucleotidase in rat heart in vivo

We examined the effect of tyramine on the production of adenosine in rat heart. A flexibly mounted microdialysis setup was used to measure the concentration of interstitial adenosine and to assess the activity of ecto-5′-nucleotidase in in vivo rat hearts. The microdialysis probe was implanted in the left ventricular myocardium of anesthetized rats and perfused with Tyrode solution containing adenosine 5′-monophosphate (AMP) at a rate of 1.0 μl/min. The concentration of adenosine in the effluent (dialysate) was measured by high-performance liquid chromatography (HPLC). Dialysate adenosine obtained during perfusion with the AMP-containing solution through the probe originated from the hydrolysis of AMP by endogenous ecto-5′-nucleotidase, and the level of adenosine reflected the activity of ecto-5′-nucleotidase in the tissue. Tyramine (0–4 mM) increased the adenosine concentration measured during the perfusion of AMP (100 μM) in a concentration-dependent manner. α,β-Methyleneadenosine 5′-diphosphate (α,β-meADP, 100 μM), an inhibitor of ecto-5′-nucleotidase, abolished the AMP-induced increase in dialysate adenosine. Tyramine (1 mM) increased the adenosine concentration measured in the presence of 100 μM AMP (i.e., the activity of ecto-5′-nucleotidase) by 65.8±19.9% ( n=6, P<0.05), an increase which was inhibited by an antagonist of the α 1-adrenoceptor (prazosin, 50 μM) or of protein kinase C (chelerythrine, 10 μM). These data provide the first evidence that α 1-adrenoceptor stimulation and the subsequent activation of protein kinase C can increase adenosine concentrations in the interstitial space of ventricular muscle in vivo, through activation of endogenous ecto-5′-nucleotidase. To examine the effect of tyramine on the production of adenosine by ischemia–reperfusion of the rat myocardium, the heart was subjected to myocardial ischemia for 15 min by occlusion of the left anterior descending coronary artery. When the heart was reperfused, elevation of the level of adenosine in the ischemic zone was observed, but this change was not significant. However, when corresponding experiments were performed with a subsequent systemic administration of tyramine (1 mM), a marked elevation in the level of adenosine was observed. The results suggest that tyramine elevates adenosine via stimulation of α 1-adrenoceptors and protein kinase C-mediated activation of ecto-5′-nucleotidase in rat heart.

NO and cGMP facilitate adenosine production in rat hearts via activation of ecto-5'-nucleotidase.

We examined whether nitric oxide (NO), a possible cardioprotective substance, can increase the production of interstitial adenosine in the ventricular myocardium. A flexibly mounted microdialysis technique was used to measure the concentration of interstitial adenosine and to assess the activity of ecto-5'-nucleotidase in in vivo rat hearts. The microdialysis probe was implanted in the left ventricular myocardium of anesthetized rats and perfused with Tyrode solution containing adenosine 5'-monophosphate (AMP) at a rate of 1.0 microl min-1. The concentration of adenosine in the effluent (dialysate) was measured by high-performance liquid chromatography. Dialysate adenosine obtained during perfusion with the AMP-containing solution through the probe originated from the hydrolysis of AMP by endogenous ecto-5'-nucleotidase, and the level of adenosine reflected the activity of ecto-5'-nucleotidase in the tissue. S-Nitroso-N-acetylpenicillamine (SNAP, 0.3-3 mM), an NO donor, increased the dialysate adenosine measured in the presence of AMP (100 microM) in a concentration-dependent manner. However, in the presence of an NO-oxidizing agent, 2-(4-carboxyphenyl-4,4,5, 5-tetramethylimidazoline)-1-oxyl 3-oxide (carboxy-PTIO, 1 mM), the effect of SNAP was abolished. Another NO donor, (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (FK409, 1 mM) also increased adenosine production. 8-Bromo-cGMP (0.1-3 mM), a membrane-permeable cGMP analogue and a potent activator of cGMP-dependent protein kinase, increased the level of AMP-primed dialysate adenosine in a concentration-dependent manner. These results suggest that NO facilitates the production of interstitial adenosine in rat hearts in situ, via cGMP-mediated activation of ecto-5'-nucleotidase.

Effects of lysophosphatidylcholine on the production of interstitial adenosine via protein kinase C-mediated activation of ecto-5'-nucleotidase.

1. Adenosine plays a crucial role in the evolution of ischemic preconditioning. With the use of microdialysis techniques in in situ rat hearts, we assessed the activity of ecto-5'-nucleotidase (a key enzyme responsible for adenosine production), and examined the effects of lysophosphatidylcholine (LPC) on the production of interstitial adenosine. 2. The microdialysis probe was implanted in the left ventricular myocardium of anesthetized rat hearts and perfused with Tyrode solution containing adenosine 5'-monophosphate (AMP, 100 microM). With this system, the dialysate adenosine originates from the dephosphorylation of AMP, catalyzed by endogenous ecto-5'-nucleotidase. The level of dialysate adenosine is a measure of the ecto-5'-nucleotidase activity in vivo. 3. LPC at concentrations of 25 and 50 microM significantly increased the level of dialysate adenosine to 122.7+/-4.3% (n=4, P<0.05) and 158.6+/-7.2% (n=5, P<0.05) of the control, respectively. Chelerythrine (200 microM), a protein kinase C (PKC) inhibitor, completely abolished the increase of dialysate adenosine afforded by LPC (50 microM) (n=5). 4. These data provide the first evidence that LPC does increase the concentration of interstitial adenosine in rat hearts in situ, through the PKC-mediated activation of endogenous ecto-5'-nucleotidase.

Effect of antiarrhythmics on the release of adenosine in rat hearts with coronary occlusion and reperfusion

In isolated perfused rat hearts the left coronary artery was occluded for 5 min, with subsequent reperfusion for 20 min. During the reperfusion severe tachyarrhythmias were observed, with ventricular fibrillation occurring in all hearts. Simultaneously, large amounts of adenosine and its degradation products inosine, hypoxanthine, xanthine and uric acid were released into the coronary perfusate. The antiarrhythmics quinidine, lidocaine and gallopamil significantly decreased the release. The effect of quinidine and lidocaine was linked with the antifibrillatory action of these drugs. Also the interruption of fibrillation immediately after its appearance by potassium chloride decreased the release of adenosine and its metabolites in a highly significant way. The effect of gallopamil on the release was independent of an antifibrillatory action. The findings indicate that different kinds of antiarrhythmic drugs can affect the release of nucleosides and oxypurines in hearts with ischaemia and reperfusion.

Transcapillary adenosine transport in isolated guinea pig and rat hearts

Transcapillary adenosine transport was studied in isolated guinea pig and rat hearts perfused with a colloid-free solution. High-performance liquid chromatography techniques were used to measure adenosine concentration of venous and interstitial (epicardial surface) fluid during steady-state perfusion with various concentrations of adenosine. A mathematical model was used to analyze these data to obtain estimates of the following parameters of transcapillary adenosine transport: PSg, permeability-surface area product for adenosine movement through interendothelial cell channels; PSecl, permeability-surface area product for adenosine movement through the luminal plasma membrane of endothelial cells; and Gec, clearance rate constant for endothelial cell metabolism and/or sequestration of adenosine. In both guinea pig and rat hearts, PSg was estimated to be less than or equal to 3 ml.min-1.g-1. Estimates of PSecl and Gec of guinea pig hearts (7.2 +/- 0.4 and 230 +/- 157 ml.min-1.g-1) were significantly less than those of rat hearts (66 +/- 11 and 2,490 +/- 1,360 ml.min-1.g-1). That PSecl is greater than PSg in both species indicates that endothelial cells represent an important pathway for transcapillary adenosine transport. That Gec is much greater than PSecl in both species implies that endothelial cells act as a sink for adenosine from surrounding areas. Our results indicate that endothelium is a stronger sink for adenosine in rat hearts than in guinea pig hearts. Inosine infusion (10(-4)M) had little effect on the estimated PSecl and Gec in guinea pig hearts but reduced these parameters several-fold in rat hearts, suggesting that different transport mechanisms for adenosine exist in endothelia of guinea pig and rat hearts.

Evidence for extracellular deamination of adenosine in the rat heart

1. 1. In rat heart perfused with adenosine (10 −6M), dilazep (10 −4M) inhibited incorporation of adenosine into nucleotides (an index of nucleoside transport and phosphorylation) to a greater extent (70%) than metabolism to inosine and uric acid (40%) and actually increased the recovery of inosine to 30% of the adenosine infused. 2. 2. Extrapolating for complete inhibition of transport suggested that 60% of adenosine metabolism was intracellular and 40% extracellular. 3. 3. Static incubations of atria also gave an estimate for extracellular metabolism of 40%. 4. 4. Adenosine deaminase was localised by immunocytochemistry to the extracellular surface of endothelial cells of small coronary arteries. 5. 5. Extracellular deamination may explain the lack of effect of nucleoside transport inhibitors on responses to adenosine in rat heart.

Do different metabolisation rates of adenosine in rat hearts and isolated myocytes reflect a barrier function of endothelium for high intravasal adenosine concentrations?

W.-J. Cai, S. Koltai, E. Kocsis, D. Scholz, W. Schaper and J. Schaper. Connexin37, not Cx40 and Cx43, is Induced in Vascular Smooth Muscle Cells During Coronary Arteriogenesis. Journal of Molecular and Cellular Cardiology (2001) 33, 957–967. The hypothesis that an altered expression of gap junction (GJ) proteins, connexin37 (Cx37), Cx40 and Cx43 will contribute to adaptive arteriogenesis was tested in growing coronary collateral vessels (CV) of the dog heart by immunoconfocal microscopy and transmission electron microscopy (TEM). We found that: (1) in the normal coronary system Cx37 and Cx40 were only expressed in endothelial cells (EC) from artery to capillary; (2) during collateral growth Cx37 was significantly induced in smooth muscle cells (SMC) from small-large arteries to precapillary arterioles (Ø=15 μ m), while Cx40 was still only present in EC; (3) both homogeneous and heterogeneous distribution of Cx37 was observed in normal vessels (NV) and growing vessels (GV); (4) in mature vessels (MV), Cx37 was downregulated, similar to NV; (5) dual immunostaining revealed an inverse correlation between expression of Cx37 and desmin in GV occurring prior to downregulation of α -smooth actin and calponin; (6) Cx43 was undetectable in any vascular cells, both in NV and GV; (7) GJ were not found in SMC by TEM. Our data for the first time show the profile of connexin expression in the coronary system and provide evidence for existence of GJ proteins in capillaries. It is a novel finding that an altered expression of Cx37 is characteristic of adaptive arteriogenesis in the dog heart and may be used as a marker of vascular growth. Induced Cx37 may be an early signal indicating that SMC are responding to haemodynamic changes, i.e. increased shear stress.

Phosphorylation of adenosine in rat heart—inhibition by dipyridamole at the enzymatic and tissue level

Phosphorylation of adenosine in rat heart—inhibition by dipyridamole at the enzymatic and tissue level