Age-related changes in adenosine-mediated relaxation of coronary and aortic smooth muscle

We tested whether adenosine mediates nitric oxide (NO)-dependent and NO-independent dilation in coronary and aortic smooth muscle and whether age selectively impairs NO-dependent adenosine relaxation. Responses to adenosine and the relatively nonselective analog 5'-N-ethylcarboxamidoadenosine (NECA) were studied in coronary vessels and aortas from immature (1-2 mo), mature (3-4 mo), and moderately aged (12-18 mo) Wistar and Sprague-Dawley rats. Adenosine and NECA induced biphasic concentration-dependent coronary vasodilation, with data supporting high-sensitivity (pEC(50) = 5.2-5.8) and low-sensitivity (pEC(50) = 2.3-2.4) adenosine sites. Although sensitivity to adenosine and NECA was unaltered by age, response magnitude declined significantly. Treatment with 50 microM N(G)-nitro-L-arginine methyl ester (L-NAME) markedly inhibited the high-sensitivity site, although response magnitude still declined with age. Aortic sensitivity to adenosine declined with age (pEC(50) = 4.7 +/- 0.2, 3.5 +/- 0.2, and 2.9 +/- 0.1 in immature, mature, and moderately aged aortas, respectively), and the adenosine receptor transduction maximum also decreased (16.1 +/- 0.8, 12.9 +/- 0.7, and 9.6 +/- 0.7 mN/mm(2) in immature, mature, and moderately aged aortas, respectively). L-NAME decreased aortic sensitivity to adenosine in immature and mature tissues but was ineffective in the moderately aged aorta. Data collectively indicate that 1) adenosine mediates NO-dependent and NO-independent coronary and aortic relaxation, 2) maturation and aging reduce NO-independent and NO-dependent adenosine responses, and 3) the age-related decline in aortic response also involves a reduction in the adenosine receptor transduction maximum.     

Adenosine receptor-mediated coronary vascular protection in post-ischemic mouse heart

This study evaluated the ability of A1 and A3 adenosine receptor (AR) agonism, and A1, A2A, A2B and A3AR antagonism (revealing "intrinsic" responses), to modify post-ischemic coronary dysfunction in mouse heart. Vascular function was assessed before and after 20 min global ischemia and 30-45 min reperfusion in Langendorff perfused C57/Bl6 mouse hearts. Ischemic insult impaired coronary sensitivity to the endothelial-dependent dilators ADP (pEC50=6.8+/-0.1 vs. 7.6+/-0.1, non-ischemic) and acetylcholine (pEC50=6.1+/-0.1 vs. 7.3+/-0.1 in non-ischemic), and for the mixed endothelial-dependent/independent dilator 2-chloroadenosine (pEC50=7.5+/-0.1 vs. 8.4+/-0.1, non-ischemic). Endothelium-independent dilation in response to nitroprusside was unaltered (pEC50=7.0+/-0.1 vs. 7.1+/-0.1 in non-ischemic). Pre-treatment with a selective A1AR agonist (50 nM CHA) failed to modify coronary dysfunction, whereas A1AR antagonism (200 nM DPCPX) worsened the effects of I/R (2-chloroadenosine pEC50=6.9+/-0.1). Conversely, A3AR agonism (100 nM Cl-IB-MECA) did reduce effects of I/R (pEC50s=8.0+/-0.1 and 7.3+/-0.1 for 2-chloroadenosine and ADP, respectively), whereas antagonism (100 nM MRS1220) was without effect. While A2AAR agonism could not be assessed (due to pronounced vasodilatation), A2AAR antagonism (100 nM SCH58261) was found to exert no effect, and antagonism of A2BARs (50 nM MRS1754) was also ineffective. The protective actions of A3AR agonism were also manifest as improved reactive hyperemic responses. Interestingly, post-ischemic coronary dysfunction was also limited by: Na+-H+ exchange (NHE) inhibition with 10 or 50 microM BIIB-513 (2-chloroadenosine pEC50s=7.8+/-0.1, either dose), an effect not additive with A3AR agonism; Ca2+ antagonism with 0.3 microM verapamil (2-chloroadenosine pEC50=7.9+/-0.1); and Ca2+ desensitization with 5 mM BDM (2-chloroadenosine pEC50=7.8+/-0.1). In contrast, endothelin antagonism (200 nM PD142893) and anti-oxidant therapy (300 microM MPG+150 U/ml SOD+600 U/ml catalase) were ineffective. Our data collectively confirm that ischemia selectively impairs endothelial function and reactive hyperemia independently of blood cells. Vascular injury is intrinsically limited by endogenous (but not exogenous) activation of A1ARs, whereas exogenous A3AR activation further limits dysfunction (improving post-ischemic vasoregulation). Finally, findings suggest this form of post-ischemic coronary injury is unrelated to endothelin or oxidant stress, but may involve modulation of Ca2+ overload and/or related ionic perturbations.     

5'-Adenosine monophosphate and adenosine metabolism, and adenosine responses in mouse, rat and guinea pig heart.

We examined myocardial 5'-adenosine monophosphate (5'-AMP) catabolism, adenosine salvage and adenosine responses in perfused guinea pig, rat and mouse heart. MVO(2) increased from 71+/-8 microl O(2)/min per g in guinea pig to 138+/-17 and 221+/-15 microl O(2)/min per g in rat and mouse. VO(2)/beat was 0.42+/-0.03, 0.50+/-0.03 and 0.55+/-0.04 microl O(2)/g in guinea pig, rat and mouse, respectively. Resting and peak coronary flows were highest in mouse vs. rat and guinea pig, and peak ventricular pressures and Ca(2+) sensitivity declined as heart mass increased. Net myocardial 5'-AMP dephosphorylation increased significantly as mass declined (3.8+/-0.5, 9.0+/-1.4 and 11.0+/-1.6 nmol/min per g in guinea pig, rat and mouse, respectively). Despite increased 5'-AMP catabolism, coronary venous [adenosine] was similar in guinea pig, rat and mouse (45+/-8, 69+/-10 and 57+/-14 nM, respectively). Comparable venous [adenosine] was achieved by increased salvage vs. deamination: 64%, 41% and 39% of adenosine formed was rephosphorylated while 23%, 46%, and 50% was deaminated in mouse, rat and guinea pig, respectively. Moreover, only 35-45% of inosine and its catabolites derive from 5'-AMP (vs. IMP) dephosphorylation in all species. Although post-ischemic purine loss was low in mouse (due to these adaptations), functional tolerance to ischemia decreased with heart mass. Cardiovascular sensitivity to adenosine also differed between species, with A(1) receptor sensitivity being greatest in mouse while A(2) sensitivity was greatest in guinea pig. In summary: (i) cardiac 5'-AMP dephosphorylation, VO(2), contractility and Ca(2+) sensitivity all increase as heart mass falls; (ii) adaptations in adenosine salvage vs. deamination limit purine loss and yield similar adenosine levels across species; (iii) ischemic tolerance declines with heart mass; and (iv) cardiovascular sensitivity to adenosine varies, with increasing A(2) sensitivity relative to A(1) sensitivity in larger hearts.     

Reduced oxygen supply explains the negative force-frequency relation and the positive inotropic effect of adenosine in buffer-perfused hearts

In isolated rat hearts perfused with HEPES and red blood cell-enriched buffers, we examined changes in left ventricular pressure induced by increases in heart rate or infusion of adenosine to investigate whether the negative force-frequency relation and the positive inotropic effect of adenosine are related to an inadequate oxygen supply provided by crystalloid perfusates. Hearts perfused with HEPES buffer at a constant flow demonstrated a negative force-frequency relation, whereas hearts perfused with red blood cell-enriched buffer exhibited a positive force-frequency relation. In contrast, HEPES buffer-perfused hearts showed a concentration-dependent increase in left ventricular systolic pressure [EC50 = 7.0 +/- 1.2 nM, maximal effect (Emax) = 104 +/- 2 and 84 +/- 2 mmHg at 0.1 microM and baseline, respectively] in response to adenosine, whereas hearts perfused with red blood cell-enriched buffer showed no change in left ventricular pressure. The positive inotropic effect of adenosine correlated with the simultaneous reduction in heart rate (r = 0.67, P < 0.01; EC50 = 3.8 +/- 1.4 nM, baseline 228 +/- 21 beats/min to a minimum of 183 +/- 22 beats/min at 0.1 microM) and was abolished in isolated hearts paced to suppress the adenosine-induced bradycardia. In conclusion, these results indicate that the negative force-frequency relation and the positive inotropic effect of adenosine in the isolated rat heart are related to myocardial hypoxia, rather than functional peculiarities of the rat heart.     

Control of vascular tone in notothenioid fishes is determined by phylogeny, not environmental temperature

We examined potential vasomotor control mechanisms in an Antarctic fish (Trematomus bernacchii; usual core temperature approximately -1 degrees C), comparing sensitivity to agonists by means of the cumulative dose response and potency with reference to depolarization by 50 mM KCl. In efferent branchial arteries, norepinephrine (NE) produced approximately 20% of the maximal KCl tension and ~40% in the presence of 10(-3)M sotalol, suggesting a modest contribution of alpha- and beta-adrenergic tonus [half-maximal response (pEC(50)) = 6.29 +/- 0.37 M]. Carbachol (CBC) and serotonin (5-HT) had different sensitivities (pEC(50) = 4.50 +/- 0.40 and 6.82 +/- 0.08 M, respectively) but similar potencies (21.6 +/- 11.1 and 31.1 +/- 5.3% of KCl). A related species from warmer waters around New Zealand, Paranotothenia angustata, had similar vascular reactivity for NE (pEC(50) = 5.48 +/- 0.31 M), CBC (pEC(50) = 4.94 +/- 0.22 M), and methysergide-sensitive vasoconstriction with 5-HT (pEC(50) = 6.22 +/- 0.40 M). Agonist potencies were 9, 65, and 45% that of KCl, respectively. Bovichtus variegatus, a member of the phylogenetic sister group to the notothenioids, also gave broadly similar responses. In contrast, Dissostichus mawsoni, a pelagic Antarctic notothenioid, showed a dominance of vasodilatation over vasoconstriction, with sensitive isoprenaline (pEC(50) = 6.66 +/- 0.05 M) but weak serotonergic (5.2 +/- 1.5% KCl) responses. The unusual dominance of serotonergic control appears to be primarily a consequence of evolutionary lineage rather than low environmental temperature, but the pattern may be modified according to functional demand.     

Intrinsic A(1) adenosine receptor activation during ischemia or reperfusion improves recovery in mouse hearts

We assessed the role of A(1) adenosine receptor (A(1)AR) activation by endogenous adenosine in the modulation of ischemic contracture and postischemic recovery in Langendorff-perfused mouse hearts subjected to 20 min of total ischemia and 30 min of reperfusion. In control hearts, the rate-pressure product (RPP) and first derivative of pressure development over time (+dP/dt) recovered to 57 +/- 3 and 58 +/- 3% of preischemia, respectively. Diastolic pressure remained elevated at 20 +/- 2 mmHg (compared with 3 +/- 1 mmHg preischemia). Interstitial adenosine, assessed by microdialysis, rose from approximately 0.3 to 1.9 microM during ischemia compared with approximately 15 microM in rat heart. Nonetheless, these levels will near maximally activate A(1)ARs on the basis of effects of exogenous adenosine and 2-chloroadenosine. Neither A(1)AR blockade with 200 nM 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) during the ischemic period alone nor A(1)AR activation with 50 nM N(6)-cyclopentyladenosine altered rapidity or extent of ischemic contracture. However, ischemic DPCPX treatment significantly depressed postischemic recovery of RPP and +dP/dt (44 +/- 3 and 40 +/- 4% of preischemia, respectively). DPCPX treatment during the reperfusion period alone also reduced recovery of RPP and +dP/dt (to 44 +/- 2 and 47 +/- 2% of preischemia, respectively). These data indicate that 1) interstitial adenosine is lower in mouse versus rat myocardium during ischemia, 2) A(1)AR activation by endogenous adenosine or exogenous agonists does not modify ischemic contracture in murine myocardium, 3) A(1)AR activation by endogenous adenosine during ischemia attenuates postischemic stunning, and 4) A(1)AR activation by endogenous adenosine during the reperfusion period also improves postischemic contractile recovery.     

Parallel modification of adenosine extracellular metabolism and modulatory action in the hippocampus of aged rats

The neuromodulator adenosine can be released as such, mainly activating inhibitory A1 receptors, or formed from released ATP, preferentially activating facilitatory A2A receptors. We tested if changes in extracellular adenosine metabolism paralleled changes in A1/A2A receptor neuromodulation in the aged rat hippocampus. The evoked release and extracellular catabolism of ATP were 49-55% lower in aged rats, but ecto-5'-nucleotidase activity, which forms adenosine, was 5-fold higher whereas adenosine uptake was decreased by 50% in aged rats. The evoked extracellular adenosine accumulation was 30% greater in aged rats and there was a greater contribution of the ecto-nucleotidase pathway and a lower contribution of adenosine transporters for extracellular adenosine formation in nerve terminals. Interestingly, a supramaximal concentration of an A1 receptor agonist, N6-cyclopentyladenosine (250 nM) was less efficient in inhibiting (17% in old versus 34% in young) and A2A receptor activation with 30 nM CGS21680 was more efficient in facilitating (63% in old versus no effect in young) acetylcholine release from hippocampal slices of aged compared with young rats. The parallel changes in the metabolic sources of extracellular adenosine and A1/A2A receptor neuromodulation in aged rats further strengthens the idea that different metabolic sources of extracellular adenosine are designed to preferentially activate different adenosine receptor subtypes.     

Targeted deletion of A2A adenosine receptors attenuates the protective effects of myocardial postconditioning

Endogenous adenosine is an important ligand trigger for the cardioprotective effects of postconditioning (POC), yet it is unclear which adenosine receptor subtype is primarily responsible. To evaluate the role of A(2A) adenosine receptors in POC-induced protection, global ischemia-reperfusion was performed with and without POC in isolated wild-type (WT) and A(2A) adenosine receptor knockout (A(2A)KO) mouse hearts. Injury was measured in terms of postischemic functional recovery and release of cardiac troponin I (cTnI). Activation of protective signaling with POC was assessed by Akt and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation. In WT hearts, POC improved recovery of postischemic developed pressure in early (81.6 +/- 6.4% of preischemic baseline vs. 37.5 +/- 5.6% for non-POC WT at 1 min) and late (62.2 +/- 4.2% of baseline vs. 45.5 +/- 5.3% for non-POC WT at 30 min) reperfusion, reduced cTnI release by 37%, and doubled the phosphorylation of both Akt and ERK1/2. These beneficial effects of POC were blocked by treatment with the selective A(2A) adenosine receptor antagonist ZM-241385 during reperfusion. Postischemic functional recovery, cTnI release, and phosphorylation of Akt and ERK1/2 were not different between non-POC WT and A(2A)KO hearts. In A(2A)KO hearts, POC did not improve functional recovery, reduce cTnI release, nor increase phosphorylation of Akt or ERK1/2. Thus the protective effects of POC are attenuated by both selective A(2A) receptor antagonism and targeted deletion of the gene encoding A(2A) adenosine receptors. These observations support the conclusion that endogenous activation of A(2A) adenosine receptors is an essential trigger leading to the protective effects of POC in isolated murine hearts.     

Receptor and non-receptor-dependent mechanisms of cardioprotection with adenosine

The relative roles of mitochondrial (mito) ATP-sensitive K(+) (mitoK(ATP)) channels, protein kinase C (PKC), and adenosine kinase (AK) in adenosine-mediated protection were assessed in Langendorff-perfused mouse hearts subjected to 20-min ischemia and 45-min reperfusion. Control hearts recovered 72 +/- 3 mmHg of ventricular pressure (50% preischemia) and released 23 +/- 2 IU/g lactate dehydrogenase (LDH). Adenosine (50 microM) during ischemia-reperfusion improved recovery (149 +/- 8 mmHg) and reduced LDH efflux (5 +/- 1 IU/g). Treatment during ischemia alone was less effective. Treatment with 50 microM diazoxide (mitoK(ATP) opener) during ischemia and reperfusion enhanced recovery and was equally effective during ischemia alone. A(3) agonism [100 nM 2-chloro-N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide], A(1) agonism (N(6)-cyclohexyladenosine), and AK inhibition (10 microM iodotubercidin) all reduced necrosis to the same extent as adenosine, but less effectively reduced contractile dysfunction. These responses were abolished by 100 microM 5-hydroxydecanoate (5-HD, mitoK(ATP) channel blocker) or 3 microM chelerythrine (PKC inhibitor). However, the protective effects of adenosine during ischemia-reperfusion were resistant to 5-HD and chelerythrine and only abolished when inhibitors were coinfused with iodotubercidin. Data indicate adenosine-mediated protection via A(1)/A(3) adenosine receptors is mitoK(ATP) channel and PKC dependent, with evidence for a downstream location of PKC. Adenosine provides additional and substantial protection via phosphorylation to 5'-AMP, primarily during reperfusion.     

Targeted deletion of A(3) adenosine receptors improves tolerance to ischemia-reperfusion injury in mouse myocardium

A(3) adenosine receptors (A(3)ARs) have been implicated in regulating mast cell function and in cardioprotection during ischemia-reperfusion injury. The physiological role of A(3)ARs is unclear due to the lack of widely available selective antagonists. Therefore, we examined mice with targeted gene deletion of the A(3)AR together with pharmacological studies to determine the role of A(3)ARs in myocardial ischemia-reperfusion injury. We evaluated the functional response to 15-min global ischemia and 30-min reperfusion in isovolumic Langendorff hearts from A(3)AR(-/-) and wild-type (A(3)AR(+/+)) mice. Loss of contractile function during ischemia was unchanged, but recovery of developed pressure in hearts after reperfusion was improved in A(3)AR(-/-) compared with wild-type hearts (80 +/- 3 vs. 51 +/- 3% at 30 min). Tissue viability assessed by efflux of lactate dehydrogenase was also improved in A(3)AR(-/-) hearts (4.5 +/- 1 vs. 7.5 +/- 1 U/g). The adenosine receptor antagonist BW-A1433 (50 microM) decreased functional recovery following ischemia in A(3)AR(-/-) but not in wild-type hearts. We also examined myocardial infarct size using an intact model with 30-min left anterior descending coronary artery occlusion and 24-h reperfusion. Infarct size was reduced by over 60% in A(3)AR(-/-) hearts. In summary, targeted deletion of the A(3)AR improved functional recovery and tissue viability during reperfusion following ischemia. These data suggest that activation of A(3)ARs contributes to myocardial injury in this setting in the rodent. Since A(3)ARs are thought to be present on resident mast cells in the rodent myocardium, we speculate that A(3)ARs may have proinflammatory actions that mediate the deleterious effects of A(3)AR activation during ischemia-reperfusion injury.     

A1 adenosine receptor overexpression attenuates ischemia-reperfusion-induced apoptosis and caspase 3 activity

We tested the hypothesis that myocardial ischemia-reperfusion (I/R)-induced apoptosis is attenuated in transgenic mice overexpressing cardiac A(1) adenosine receptors. Isolated hearts from transgenic (TG, n = 19) and wild-type (WT, n = 22) mice underwent 30 min of ischemia and 2 h of reperfusion, with evaluation of apoptosis, caspase 3 activity, function, and necrosis. I/R-induced apoptosis was attenuated in TG hearts. TG hearts had less I/R-induced apoptotic nuclei (0.88 +/- 0.10% vs. 4.22 +/- 0.24% terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells in WT, P < 0.05), less DNA fragmentation (3.30 +/- 0.38-fold vs. 4.90 +/- 0.39-fold over control in WT, P < 0.05), and less I/R-induced caspase 3 activity (145 +/- 25% over nonischemic control vs. 234 +/- 31% in WT, P < 0.05). TG hearts also had improved recovery of function and less necrosis than WT hearts. In TG hearts pretreated with LY-294002 (3 microM) to evaluate the role of phosphosinositol-3-kinase in acute signaling, there was no change in the functional protection or apoptotic response to I/R. These data suggest that cardioprotection with transgenic overexpression of A(1) adenosine receptors involves attenuation of I/R-induced apoptosis that does not involve acute signaling through phosphoinositol-3-kinase.     

Age-dependent decrease in adenosine A1 receptor binding sites in the rat brain. Effect of cis unsaturated free fatty acids.

Unsaturated free fatty acids and adenosine operate two neuromodulatory systems with opposite effects on neuronal function. Here, we tested if fatty acids controlled inhibitory adenosine A1 receptors. Arachidonate (AA, 10 microM) decreased the Bmax of an A1 receptor agonist, (R)-[3H]phenylisopropyladenosine (PIA; from 812 to 267 fmol x mg(-1) protein), and antagonist, [3H]1,3-dipropyl-8-cyclopentylxanthine (DPCPX; from 994 to 311 fmol x mg(-1) protein) and decreased the Kd of [3H]PIA (from 1.20 to 0.57 nM) binding to brain membranes of young adult rats (2 months old), these effects being mimicked by other cis but not trans unsaturated or saturated fatty acids. AA (10 microM) increased the potency of the A1 receptor agonist, 2-chloroadenosine to inhibit hippocampal synaptic transmission in young adult rats (EC50 decreased from 337 to 237 nM), which may constitute a safety feedback mechanism to control AA-induced neurotoxicity. Upon aging, there were increased free fatty acid levels and a concomitant decreased density of A1 receptors. This was more marked in hippocampal nerve terminals of aged rats (24 months old) and may be the determinant factor contributing to the lower potency of 2-choloroadenosine in aged rats (EC50 = 955 nM), in spite of the decreased Kd of PIA binding upon aging. The effects of AA on A1 receptor binding were attenuated upon aging, AA being devoid of effects in aged rats. Accordingly, AA (10 microM) failed to modify the potency of 2-choloroadenosine in aged rats (EC50 = 997 nM). However, albumin, which quenches free fatty acids, increased A1 receptor density by 65% and 2-chloroadenosine potency (EC50 = 703 nM) in aged rats, suggesting that the increased fatty acids levels in aged rats may contribute to the decreased potency of A1 receptor agonists in aged rats. Also, the observed saturation of the control by AA of A1 receptors may contribute to the decreased adaptability of neuromodulation to different firing conditions in aged rats.     

Cardiac dysfunction in aging conscious rats: altered cardiac cytoskeletal proteins as a potential mechanism

The objective of this study was to test the hypothesis that the mechanism mediating left ventricular (LV) dysfunction in the aging rat heart involves, in part, changes in cardiac cytoskeletal components. Our results show that there were no significant differences in heart rate, LV pressure, or LV diameter between conscious, instrumented young [5.9 +/- 0.3 mo (n = 9)] and old rats [30.6 +/- 0.1 mo (n = 10)]. However, the first derivative of LV pressure (LV dP/dt) was reduced (8,309 +/- 790 vs. 11,106 +/- 555 mmHg/s, P < 0.05) and isovolumic relaxation time (tau) was increased (8.7 +/- 0.7 vs. 6.3 +/- 0.6 ms, P < 0.05) in old vs. young rats, respectively. The differences in baseline LV function in young and old rats, which were modest, were accentuated after beta-adrenergic receptor stimulation with dobutamine (20 mug/kg), which increased LV dP/dt by 170 +/- 9% in young rats, significantly more (P < 0.05) than observed in old rats (115 +/- 5%). Volume loading in anesthetized rats demonstrated significantly impaired LV compliance in old rats, as measured by the LV end-diastolic pressure and dimension relationship. In old rat hearts, there was a significant (P < 0.05) increase in the percentage of LV collagen (2.4 +/- 0.2 vs. 1.3 +/- 0.2%), alpha-tubulin (92%), and beta-tubulin (2.3-fold), whereas intact desmin decreased by 51%. Thus the cardiomyopathy of aging in old, conscious rats may be due not only to increases in collagen but also to alterations in cytoskeletal proteins.     

Cardioprotection by K(ATP) channels in wild-type hearts and hearts overexpressing A(1)-adenosine receptors

We studied the role of mitochondrial ATP-sensitive K(+) (K(ATP)) channels in modifying functional responses to 20 min global ischemia and 30 min reperfusion in wild-type mouse hearts and in hearts with approximately 250-fold overexpression of functionally coupled A(1)-adenosine receptors (A(1)ARs). In wild-type hearts, time to onset of contracture (TOC) was 303 +/- 24 s, with a peak contracture of 89 +/- 5 mmHg. Diastolic pressure remained elevated at 52 +/- 6 mmHg after reperfusion, and developed pressure recovered to 40 +/- 6% of preischemia. A(1)AR overexpression markedly prolonged TOC to 517 +/- 84 s, reduced contracture to 64 +/- 6 mmHg, and improved recovery of diastolic (to 9 +/- 4 mmHg) and developed pressure (to 82 +/- 8%). 5-Hydroxydecanoate (5-HD; 100 microM), a mitochondrial K(ATP) blocker, did not alter ischemic contracture in wild-type hearts, but increased diastolic pressure to 69 +/- 8 mmHg and reduced developed pressure to 10 +/- 5% during reperfusion. In transgenic hearts, 5-HD reduced TOC to 348 +/- 18 s, increased postischemic contracture to 53 +/- 4 mmHg, and reduced recovery of developed pressure to 22 +/- 4%. In summary, these data are the first to demonstrate that endogenous activation of K(ATP) channels improves tolerance to ischemia-reperfusion in murine myocardium. This functional protection occurs without modification of ischemic contracture. The data also support a role for mitochondrial K(ATP) channel activation in the pronounced cardioprotection afforded by overexpression of myocardial A(1)ARs.     

A partial agonist of the A(1)-adenosine receptor selectively slows AV conduction in guinea pig hearts

The use of full agonists of the A(1)-adenosine receptor (A(1)-ADOR) as antiarrhythmic agents is limited by their actions to cause high-grade atrioventricular (AV) block, profound bradycardia, atrial fibrillation, and vasodilation. It may be possible to avoid these undesired actions by use of partial agonists. We determined the effects of CVT-2759, a potential partial agonist of A(1)-ADORs, on guinea pig hearts. CVT-2759 (0.1-100 microM) increased the S-H interval of the isolated heart from 45 +/- 1 to 60 +/- 3 ms (P < 0. 01) with a half-maximal effect at 3.1 microM. CVT-2759 did not cause second-degree AV block. CVT-2759 significantly attenuated the actions of the full agonists N(6)-cyclopentyladenosine and adenosine. CVT-2759 caused a moderate slowing of atrial rate by 10 microM. In contrast, CVT-2759 was a full agonist to decrease cAMP content of rat adipocytes and Fischer rat thyroid line 5 cells. Results of radioligand binding assays indicated that CVT-2759 stabilized a high-affinity, G protein-coupled state of the A(1)-ADOR in membranes prepared from rat adipocytes but not in membranes prepared from the guinea pig brain. The results suggest that a weak A(1)-ADOR agonist, such as CVT-2759, may be useful to slow AV nodal conduction and thereby ventricular rate without causing AV block, bradycardia, atrial arrhythmias, or vasodilation.     

Loss of ischemic preconditioning's cardioprotection in aged mouse hearts is associated with reduced gap junctional and mitochondrial levels of connexin 43

Connexin 43 (Cx43) is localized at left ventricular (LV) gap junctions and in cardiomyocyte mitochondria. A genetically induced reduction of Cx43 as well as blockade of mitochondrial Cx43 import abolishes the infarct size (IS) reduction by ischemic preconditioning (IP). With progressing age, Cx43 content in ventricular and atrial tissue homogenates is reduced. We now investigated whether or not 1) the mitochondrial Cx43 content is reduced in aged mice hearts and 2) IS reduction by IP is lost in aged mice hearts in vivo. Confirming previous results, sarcolemmal Cx43 content was reduced in aged (>13 mo) compared with young (<3 mo) C57Bl/6 mice hearts, whereas the expression levels of protein kinase C epsilon and endothelial nitric oxide synthase remained unchanged. Also in mitochondria isolated from aged mice LV myocardium, Western blot analysis indicated a 40% decrease in Cx43 content compared with mitochondria isolated from young mice hearts. In young mice hearts, IP by one cycle of 10 min ischemia and 10 min reperfusion reduced IS (% of area at risk) following 30 min regional ischemia and 120 min reperfusion from 67.7 +/- 3.3 (n = 17) to 34.2 +/- 6.6 (n = 5, P < 0.05). In contrast, IP's cardioprotection was lost in aged mice hearts, since IS in nonpreconditioned (57.5 +/- 4.0, n = 10) and preconditioned hearts (65.4 +/- 6.3, n = 8, P = not significant) was not different. In conclusion, mitochondrial Cx43 content is decreased in aged mouse hearts. The reduced levels of Cx43 may contribute to the age-related loss of cardioprotection by IP.     

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 microliter/min. The baseline level of dialysate adenosine was 0.51 +/- 0.09 microM. The introduction of AMP (100 microM) through the probe increased markedly the dialysate adenosine to 8.95 +/- 0.86 microM, and this increase was inhibited by ecto-5'-nucleotidase inhibitor, alpha, beta-methyleneadenosine 5'-diphosphate (AOPCP, 100 microM), to 0.66 +/- 0.38 microM. 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 (EC(50)) was 107.3 microM. The maximum attainable concentration of dialysate adenosine (E(max)) by AMP was 21.1 microM. However, the EC(50) and E(max) values with reserpinized animals were 106.9 and 7.1 microM, respectively. Electrical stimulation of the left stellate ganglion increased significantly dialysate adenosine concentration, from the control level of 8.66 +/- 0.96 microM to 12.38 +/- 1.11 microM. 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 alpha(1)-adrenoceptor and protein kinase C mediated activation of ecto-5'-nucleotidase in rat heart.     

Electrophysiological evidence for the coexistence of alpha1 and alpha6 subunits in a single functional GABA(A) receptor

The subunit combinations alpha1beta2gamma2, alpha6beta2gamma2, and alpha1alpha6beta2gamma2 of the GABA(A) receptor were functionally expressed in Xenopus oocytes. The properties of the resulting ion currents were characterized by using electrophysiological techniques. The concentration-response curve of the channel agonist GABA for alpha1alpha6beta2gamma2 showed a single apparent component characterized by an EC(50) of 107 +/- 26 microM (n = 4). It was different from the one for alpha1beta2gamma2, which had an EC(50) of 41 +/- 9 microM (n = 4), that for alpha6beta2gamma2, with an EC(50) of 6.7 +/- 1.9 microM (n = 5), and those for alpha1beta2 and alpha1alpha6beta2. There was no appreciable functional expression of alpha6beta2. Allosteric responses of alpha1alpha6beta2gamma2 to diazepam were intermediate to those of alpha1beta2gamma2 and alpha6beta2gamma2, and allosteric responses to flumazenil were comparable to the ones for alpha1beta2gamma2. The inhibition by furosemide of the currents elicited by GABA in alpha1alpha6beta2gamma2 [IC(50) = 298 +/- 116 microM (n = 7), assuming only one component] was not identical with inhibition of alpha6beta2gamma2 (IC(50) = 38 +/- 2 microM, n = 4), alpha1beta2gamma2 (IC(50) = 5,610 +/- 910 microM, n = 5), or a mixture of these components (assuming two components). These findings indicate unambiguously the formation of functional GABA(A) receptors containing two different alpha subunits, alpha1 and alpha6, with properties different from those of alpha1beta2gamma2 and alpha6beta2gamma2. Furthermore, we provide evidence for the facts that in the Xenopus oocyte (a) the formation of the different receptor types depends on the relative abundance of cRNAs coding for the different receptor subunits and (b) that functional dual subunit combinations alphabeta do not form in the presence of cRNA coding for the gamma subunit.     

Age-related changes in conducted vasodilation: effects of exercise training and role in functional hyperemia

Conducted vasodilation may coordinate blood flow in microvascular networks during skeletal muscle contraction. We tested the hypotheses that 1) exercise training enhances conducted vasodilation and 2) age-related changes in the capacity for conduction affect muscle perfusion during contractions. To address hypothesis 1, young (4-5 mo), adult (12-14 mo), and old (19-21 mo) C57BL6 male mice were sedentary or given access to running wheels for 8 wk. Voluntary running distances were significantly different (in km/day): young = 5.8 +/- 0.1, adult = 3.9 +/- 0.1, old = 2.2 +/- 0.1 (P < 0.05). In gluteus maximus muscles, conducted vasodilation was greater in adult than in young or old mice (P < 0.05) and greater in young sedentary than in old sedentary mice but was not affected by exercise training. Citrate synthase activity was greater with exercise training at all ages (P < 0.05). mRNA for endothelial nitric oxide synthase did not differ among ages, but endothelial nitric oxide synthase protein expression was greater in adult and old mice with exercise training (P < 0.05). Connexin 37, connexin 40, and connexin 43 mRNA were not affected by exercise training and did not differ by age. To address hypothesis 2, perfusion of the gluteus maximus muscle during light to severe workloads was assessed by Doppler microprobe at 3-26 mo of age. Maximum perfusion decreased linearly across the lifespan. Perfusion at the highest workload, absolute and relative to maximum, decreased across the lifespan, with a steeper decline beyond approximately 20 mo of age. In this model, 1) exercise training does not alter conducted vasodilation and 2) muscle perfusion is maintained up to near maximum workloads despite age-related changes in conducted vasodilation.