A mitochondrial nexus in major depressive disorder: Integration with the psycho-immune-neuroendocrine network.

Nervous system processes, including cognition and affective state, fundamentally rely on mitochondria. Impaired mitochondrial function is evident in major depressive disorder (MDD), reflecting cumulative detrimental influences of both extrinsic and intrinsic stressors, genetic predisposition, and mutation. Glucocorticoid 'stress' pathways converge on mitochondria; oxidative and nitrosative stresses in MDD are largely mitochondrial in origin; both initiate cascades promoting mitochondrial DNA (mtDNA) damage with disruptions to mitochondrial biogenesis and tryptophan catabolism. Mitochondrial dysfunction facilitates proinflammatory dysbiosis while directly triggering immuno-inflammatory activation via released mtDNA, mitochondrial lipids and mitochondria associated membranes (MAMs), further disrupting mitochondrial function and mitochondrial quality control, promoting the accumulation of abnormal mitochondria (confirmed in autopsy studies). Established and putative mechanisms highlight a mitochondrial nexus within the psycho-immune neuroendocrine (PINE) network implicated in MDD. Whether lowering neuronal resilience and thresholds for disease, or linking mechanistic nodes within the MDD pathogenic network, impaired mitochondrial function emerges as an important risk, a functional biomarker, providing a therapeutic target in MDD. Several treatment modalities have been demonstrated to reset mitochondrial function, which could benefit those with MDD.

Regulation of the ß-Adrenergic Receptor Signaling Pathway in Sustained Ligand-Activated Preconditioning.

Sustained ligand-activated preconditioning (SLP), induced with chronic opioid receptor (OR) agonism, enhances tolerance to ischemia/reperfusion injury in young and aged hearts. Underlying mechanisms remain ill-defined, although early data implicate phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) during the induction phase, and ß 2-adrenoceptor (ß 2-AR), Gs alpha subunit (Gα s), and protein kinase A (PKA) involvement in subsequent cardioprotection. Here, we tested for induction of a protective ß 2-AR/Gα s/PKA signaling axis with SLP to ascertain whether signaling changes were PI3K-dependent (by sustained cotreatment with wortmannin), and whether the downstream PKA target Rho kinase (ROCK) participates in subsequent cardioprotection (by acute treatment with fasudil). A protected phenotype was evident after 5 days of OR agonism (using morphine) in association with increased membrane versus reduced cytosolic levels of total and phosphorylated ß 2-ARs; increased membrane and cytosolic expression of 52 and 46 kDa Gα s isoforms, respectively; and increased phosphorylation of PKA and Akt. Nonetheless, functional sensitivities of ß 2-ARs and adenylyl cyclase were unchanged based on concentration-response analyses for formoterol, fenoterol, and 6-[3-(dimethylamino)propionyl]-forskolin. Protection with SLP was not modified by ROCK inhibition, and changes in ß 2-AR, Gα s, and PKA expression appeared insensitive to PI3K inhibition, although 5 days of wortmannin alone exerted unexpected effects on signaling (also increasing membrane ß 2-AR and PKA expression/phosphorylation and Gα s levels). In summary, sustained OR agonism upregulates cardiac membrane ß 2-AR expression and phosphorylation in association with increased Gα s subtype levels and PKA phosphorylation. While Akt phosphorylation was evident, PI3K activity appears nonessential to OR upregulation of the ß 2-AR signal axis. This opioidergic remodeling of ß 2-AR signaling may explain ß 2-AR, Gα s, and PKA dependence of SLP protection.

Health, pre-disease and critical transition to disease in the psycho-immune-neuroendocrine network: Are there distinct states in the progression from health to major depressive disorder?

The psycho-immune-neuroendocrine (PINE) network is a regulatory network of interrelated physiological pathways that have been implicated in major depressive disorder (MDD). A model of disease progression for MDD is presented where the stable, healthy state of the PINE network (PINE physiome) undergoes progressive pathophysiological changes to an unstable but reversible pre-disease state (PINE pre-diseasome) with chronic stress. The PINE network may then undergo critical transition to a stable, possibly irreversible disease state of MDD (PINE pathome). Critical transition to disease is heralded by early warning signs which are detectible by biomarkers specific to the PINE network and may be used as a screening test for MDD. Critical transition to MDD may be different for each individual, as it is reliant on diathesis, which comprises genetic predisposition, intrauterine and developmental factors. Finally, we propose the PINE pre-disease state may form a "universal pre-disease state" for several non-communicable diseases (NCDs), and critical transition of the PINE network may lead to one of several frequently associated disease states (influenced by diathesis), supporting the existence of a common Chronic Illness Risk Network (CIRN). This may provide insight into both the puzzle of multifinality and the growing clinical challenge of multimorbidity.

From feedback loop transitions to biomarkers in the psycho-immune-neuroendocrine network: Detecting the critical transition from health to major depression.

BACKGROUND: Biological pathways underlying major depressive disorder (MDD) can be viewed as systems biology networks. The psycho-immune-neuroendocrine (PINE) network comprises central nervous, immune, endocrine and autonomic systems, integrating biological mechanisms of MDD. Such networks exhibit recurrent motifs with specific functions, including positive and negative feedback loops, and are subject to critical transitions, influenced by feedback loop transitions (FLTs). AIMS: We aim to identify critical feedback loops and their FLTs, as well sentinel network nodes (SNNs), key network nodes that drive FLTs, within the PINE network. Examples of biomarkers are provided which may reflect early warning signs of impending critical transition to MDD. RESULTS: Disruption of homeostatic feedback loops reflects the physiological transition to MDD. Putative FLTs are identified within hypothalamic-pituitary-adrenal (HPA) and sympathetic-parasympathetic axes, the kynurenine pathway, gut function and dysbiosis. CONCLUSIONS: Progression from health to disease is driven by FLTs in the PINE network, which is likely to undergo changes characteristic of system instability. Biomarkers of system instability may effectively predict the critical transition to MDD.

Chronically elevated bilirubin protects from cardiac reperfusion injury in the male Gunn rat.

AIMS: Bilirubin is associated with reduced risk of cardiovascular disease, as evidenced in conditions of mild hyperbilirubinaemia (Gilbert's Syndrome). Little is known regarding myocardial stress resistance in hyperbilirubinaemic conditions or whether life-long exposure modifies cardiac function, which might contribute to protection from cardiovascular disease. METHODS: Hyperbilirubinaemic rats and littermate controls underwent echocardiography at 3, 6 and 12 months of age, with hearts subsequently assessed for resistance to 30 min of ischaemia. Heart tissue was then collected for assessment of bilirubin content. RESULTS: No difference in baseline cardiac function was evident until 6 months onwards, where Gunn rats demonstrated aortic dilatation and reduced peak ejection velocities. Additionally, duration of ventricular ejection increased progressively, indicating a negative inotropic effect of bilirubin in vivo. Ex vivo analysis of baseline function revealed reduced left ventricular pressure development (LVDP) and contractility in hyperbilirubinaemic rats. Furthermore, stress resistance was improved in Gunn hearts: post-ischaemic recoveries of LVDP (76 ± 22% vs. 29 ± 17% Control, P < 0.01) and coronary flow (96 ± 9% vs. 86 ± 16% Control, P < 0.01) were improved in Gunn hearts, accompanied by reduced infarct area (21 ± 5% vs. 47 ± 15% Control, P < 0.01), and ventricular malondialdehyde and protein carbonyl content. Expression of myocardial nitric oxide-regulating genes including Nos1 and Noa1 were not significantly different. CONCLUSIONS: These data reveal life-long hyperbilirubinaemia induces age-dependent hypocontractility in male Gunn rats, and improved stress resistance. In addition, bilirubin exerts sex-independent effects on vascular structure, myocardial function and ischaemic tolerance, the latter likely mediated via bilirubin's antioxidant properties.

Coupling of myocardial stress resistance and signalling to voluntary activity and inactivity.

AIMS: We examined coupling of myocardial ischaemic tolerance to physical activity and inactivity, and whether this involves modulation of survival (AKT, AMPK, ERK1/2, HSP27, EGFR) and injury (GSK3ß) proteins implicated in ischaemic preconditioning and calorie restriction. METHODS: Proteomic modifications were assessed in ventricular myocardium, and tolerance to 25-min ischaemia in ex vivo perfused hearts from C57Bl/6 mice subjected to 14-day voluntary activity in running-naïve animals (Active); 7 days of subsequent inactivity (Inactive); brief (day 3) restoration of running (Re-Active); or time-matched inactivity. RESULTS: Active mice increased running speed and distance by 75-150% over 14 days (to ~40 m min(-1) and 10 km day(-1) ), with Active hearts resistant to post-ischaemic dysfunction (40-50% improvements in ventricular pressure development, diastolic pressure and dP/dt). Cardioprotection was accompanied by ~twofold elevations in AKT, AMPK, HSP27 and GSK3ß phosphorylation and EGFR expression. Ischaemic tolerance was reversed in Inactive hearts, paralleling reduced EGFR expression and GSK3ß and ERK1/2 phosphorylation (AKT, AMPK, HSP27 phosphorylation unaltered). Running characteristics, ischaemic tolerance, EGFR expression and GSK3ß phosphorylation returned to Active levels within 1-3 days of restored activity (without changes in AKT, AMPK or HSP27 phosphorylation). Transcriptional responses included activity-dependent Anp induction vs. Hmox1 and Sirt3 suppression, and inactivity-dependent Adora2b induction. CONCLUSIONS: Data confirm the sensitive coupling of ischaemic tolerance to activity: voluntary running induces cardioprotection that dissipates within 1 week of inactivity yet recovers rapidly upon subsequent activity. While exercise in naïve animals induces a molecular profile characteristic of preconditioning/calorie restriction, only GSK3ß and EGFR modulation consistently parallel activity- and inactivity-dependent ischaemic tolerance.

[Protein kinases role in adaptive phenomenon of heart ischemic postconditioning development].

Authors submitted an analysis of papers given up an involvement of protein kinases in heart ischemic postconditioning. This analysis of literature source allowed to authors affirms that signaling system of postconditioning can involve kinases: PKC, PI3K, Akt, MEKl/2, ERK1/2, MTOR, p70s6K, GSK3b, PKG and also eNOS, NO, GC, motoKATP channel, ROS, MPT pore. At the same time it is unclear a real contributions of kinases mTOR, p70s6, AMPK and GSK3b in the mechanism of infarct limiting impact of postconditioning. It is required a further study of the chain of signaling events following JAK2 and p38 kinase activation. The knowledge of Ras and Raf-1 role in postconditioning has hypothetical character. The tyrosine kinase significance in postcondi-tioning is unclear, particular Src kinase, which plays an important role in the regulation of cardiac tolerance to an impact of ischemia and reperfusion.

[Perspective of creation of drugs on basis of opioids increasing cardiac tolerance to pathogenic impact of ischemia reperfusion].

It was established that delta- and kappa1-opioid receptor (OR) stimulation both in vivo and in vitro promotes a decrease of infarct size/area at risk (IS/AAR) ratio during ischemia and reperfusion of heart. mu-OR activation increases a tolerance of isolated perfused heart to impact of ischemia and reperfusion but has no effect on IS/AAR index in vivo. The ORL1-receptor agonist nociceptin does not exert IS/AAR ratio in vivo. Delta- and kappa1-OR stimulation prevents cardiomyocyte apoptosis during ischemia and reperfusion of heart. The delta- and kappa1-OR agonists mimic infarct-reducing effect of postconditioning. The OR inhibition does not impact IS/AAR index both in vivo and in vitro. The delta1-, delta2- and kappa1-OR agonists are the most perspective group of opioids for creation of drugs increasing cardiac tolerance to pathogenic impact of ischemia and reperfusion.

[Role of receptor transactivation in the cardioprotective effects of preconditioning and postconditioning].

Analysis of published data indicates that the activity of receptors for adenosine, opioids, bradykinin, calcitonin-gene related peptides (CGRP) and epidermal growth factor (EGF) play important role in triggering the cardioprotective effects of ischemic preconditioning. Cannabinoids mimic the infarct-sparing effects of preconditioning. Endogenous adenosine, opioids, bradykinin and CGRP have also been implicated in infarct-reduction with ischemic postconditioning. Again, cannabinoids also mimic the protective effect of postconditioning. Recent works support heterodimerization of G-protein coupled receptors (GPCRs), and GPCR transactivation of EGF receptors. It was found that cross-talk between delta(j)-opioid receptors and adenosine A(1)-receptors is essential to cardiac protection. Furthermore, evidence implicates EGF receptor transactivation in cardioprotective effect of multiple GPCrs including adenosine, acetylcholine, bradykinin, and opioid receptors. Such findings support a convergent pathway in which multiple GPCRs may interact (or function independently) to transactivate EGF receptor-dependent kinase signaling and cytoprotection.

Functional properties and responses to ischaemia-reperfusion in Langendorff perfused mouse heart.

Despite minimal model characterisation Langendorff perfused murine hearts are increasingly employed in cardiovascular research, and particularly in studies of myocardial ischaemia and reperfusion. Reported contractility remains poor and ischaemic recoveries variable. We characterised function in C57/BL6 mouse hearts using a ventricular balloon or apicobasal displacement and assessed responses to 10-30 min global ischaemia. We examined the functional effects of pacing, ventricular balloon design, perfusate filtration, [Ca(2+)] and temperature. Contractility was high in isovolumically functioning mouse hearts (measured as the change in pressure with time (+dP/dt), 6000-7000 mmHg s(-1)) and was optimal at a heart rate of approximately 420 beats min(-1), with the vasculature sub-maximally dilated, and the cellular energy state high. Post-ischaemic recovery (after 40 min reperfusion) was related to the ischaemic duration: developed pressure recovered by 82 +/- 5 %, 73 +/- 4 %, 68 +/- 3 %, 57 +/- 2 % and 41 +/- 5 % after 10, 15, 20, 25 and 30 min ischaemia, respectively. Ventricular compliance and elastance were both reduced post-ischaemia. Post-ischaemic recoveries were lower in the apicobasal model (80 +/- 4 %, 58 +/- 7 %, 40 +/- 3 %, 32 +/- 7 % and 25 +/- 5 %) despite greater reflow and lower metabolic rate (pre-ischaemic myocardial O(2) consumption (V(O2,myo)) 127 +/- 15 vs. 198 +/- 17 microl O(2) min(-1) g(-1)), contracture, enzyme and purine efflux. Electrical pacing slowed recovery in both models, small ventricular balloons (unpressurised volumes < 50-60 microl) artificially depressed ventricular function and recovery from ischaemia, and failure to filter the perfusion fluid to < 0.45 microm depressed pre- and post-ischaemic function. With attention to these various experimental factors, the buffer perfused isovolumically contracting mouse heart is shown to be stable and highly energized, and to possess a high level of contractility. The isovolumic model is more reliable in assessing ischaemic responses than the commonly employed apicobasal model.

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.

Cardioprotection with adenosine metabolism inhibitors in ischemic-reperfused mouse heart.

OBJECTIVES: To characterize the 'anti-ischemic' effects of adenosine metabolism inhibition in ischemic-reperfused myocardium. METHODS: Perfused C57/B16 mouse hearts were subjected to 20 min ischemia 40 min reperfusion in the absence or presence of adenosine deaminase inhibition (50 microM erythro-2-(2-hydroxy-3-nonyl)adenine; EHNA) adenosine kinase inhibition (10 microM iodotubercidin; IODO), or 10 microM adenosine. Hearts overexpressing A(1) adenosine receptors (A(1)ARs) were also studied. RESULTS: EHNA treatment reduced ischemic contracture and post-ischemic diastolic pressure (14+/-2 vs. 20+/-1 mmHg), increased recovery of developed pressure (66+/-3 vs. 53+/-2%) and reduced LDH efflux (8.9+/-1.6 vs. 18.0+/-1.7 I.U./g). IODO also improved functional recovery (to 60+/-2%) and reduced LDH efflux (5.3+/-1.7 I.U./g), as did treatment with 10 microM adenosine. Protection with EHNA was reversed by co-infusion of IODO or 50 microM 8-rho-sulfophenyltheophylline (adenosine receptor antagonist), but unaltered by 20 microM inosine+10 microm hypoxanthine. Similarly, effects of iodotubercidin were inhibited by EHNA and 8-rho-sulfophenyltheophylline. A(1)AR overexpression exerted similar effects to EHNA and EHNA or IODO alone enhanced recovery while EHNA+IODO reduced recovery in transgenic hearts. Functional recoveries and xanthine oxidase reactant levels were unrelated in the groups studied. CONCLUSIONS: Adenosine deaminase or kinase inhibition protects from ischemia-reperfusion. Cardioprotection via these enzyme inhibitors requires a functioning purine salvage pathway and involves enhanced adenosine receptor activation. Reduced formation of inosine is unimportant in EHNA-mediated protection.

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.

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.

Acute but not chronic caffeine impairs functional responses to ischaemia-reperfusion in rat isolated perfused heart.

1. The effect of acute (50 micromol/L) and chronic (0.06% in drinking water for 14 days) caffeine on the response to ischaemia-reperfusion was studied in Wistar rat isolated perfused hearts. 2. Neither acute nor chronic caffeine modified normoxic heart rate or left ventricular pressures. However, acute caffeine decreased coronary flow by up to 20%, while chronic caffeine consumption increased coronary flow by approximately 15% and abolished the vasoconstrictor effect of acute caffeine (P<0.05). 3. After 15 min global ischaemia, chronic caffeine treatment did not alter the recovery of left ventricular diastolic pressure (LVDP), end-diastolic pressure (EDP) or heart rate during reperfusion, but did enhance coronary flow rate (P<0.05). Acute caffeine inhibited the recovery of LVDP and elevated postischaemic EDP in both caffeine-naive and chronic caffeine-treated groups. Acute caffeine also significantly inhibited coronary reflow in naive but not chronic caffeine-treated groups and produced a transient tachycardia during reperfusion in hearts from chronic caffeine-treated rats. 4. The incidence of arrhythmias was unaltered by chronic caffeine treatment, but was increased by acute caffeine in both naive and chronic caffeine hearts. 5. In conclusion, chronic caffeine intake alone has no detrimental effects on recovery from ischaemia; however, acute caffeine worsens postischaemic contractile function in hearts from naive and chronic caffeine-treated rats.

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.

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.

Transgenic overexpression of cardiac A(1) adenosine receptors mimics ischemic preconditioning.

The role of A(1) adenosine receptors (A(1)AR) in ischemic preconditioning was investigated in isolated crystalloid-perfused wild-type and transgenic mouse hearts with increased A(1)AR. The effect of preconditioning on postischemic myocardial function, lactate dehydrogenase (LDH) release, and infarct size was examined. Functional recovery was greater in transgenic versus wild-type hearts (44.8 +/- 3.4% baseline vs. 25.6 +/- 1.7%). Preconditioning improved functional recovery in wild-type hearts from 25.6 +/- 1.7% to 37.4 +/- 2.2% but did not change recovery in transgenic hearts (44.8 +/- 3.4% vs. 44.5 +/- 3.9%). In isovolumically contracting hearts, pretreatment with selective A(1) receptor antagonist 1, 3-dipropyl-8-cyclopentylxanthine attenuated the improved functional recovery in both wild-type preconditioned (74.2 +/- 7.3% baseline rate of pressure development over time untreated vs. 29.7 +/- 7.3% treated) and transgenic hearts (84.1 +/- 12.8% untreated vs. 42.1 +/- 6.8% treated). Preconditioning wild-type hearts reduced LDH release (from 7,012 +/- 1,451 to 1,691 +/- 1,256 U. l(-1). g(-1). min(-1)) and infarct size (from 62.6 +/- 5.1% to 32.3 +/- 11.5%). Preconditioning did not affect LDH release or infarct size in hearts overexpressing A(1)AR. Compared with wild-type hearts, A(1)AR overexpression markedly reduced LDH release (from 7,012 +/- 1,451 to 917 +/- 1,123 U. l(-1). g(-1). min(-1)) and infarct size (from 62.6 +/- 5.1% to 6.5 +/- 2.1%). These data demonstrate that murine preconditioning involves endogenous activation of A(1)AR. The beneficial effects of preconditioning and A(1)AR overexpression are not additive. Taken with the observation that A(1)AR blockade equally eliminates the functional protection resulting from both preconditioning and transgenic A(1)AR overexpression, we conclude that the two interventions affect cardioprotection via common mechanisms or pathways.

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.

Age-related changes in A(1)-adenosine receptor-mediated bradycardia.

The impact of age on functional sensitivity to A(1)-adenosine receptor activation was studied in Langendorff-perfused hearts from young (1-2 mo) and old (12-18 mo) male Wistar rats. Adenosine mediated bradycardia in young and old hearts, with sensitivity enhanced approximately 10-fold in old [negative logarithm of EC(50) (pEC(50)) = 4.56 +/- 0.11] versus young hearts (pEC(50) = 3.70 +/- 0. 09). Alternatively, the nonmetabolized A(1) agonists N(6)-cyclohexyladenosine and (R)-N(6)-phenylisopropyladenosine were equipotent in young (pEC(50) = 7.43 +/- 0.12 and 6.61 +/- 0.19, respectively) and old hearts (pEC(50) = 7.07 +/- 0.10 and 6.80 +/- 0. 11, respectively), suggesting a role for uptake and/or catabolism in age-related changes in adenosine sensitivity. In support of this suggestion, [(3)H]-adenosine uptake was approximately twofold greater in young than in old hearts (from 3-100 microM adenosine). However, although inhibition of adenosine deaminase and adenosine transport with 10 microM erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride and 10 microM S-(4-nitrobenzyl)-6-thioinosine increased adenosine sensitivity three- to fourfold, it failed to abolish the sensitivity difference in old (pEC(50) = 4.95 +/- 0.08) versus young (pEC(50) = 4.29 +/- 0.13) hearts. Data indicate that 1) age increases functional A(1) receptor sensitivity to adenosine without altering the sensitivity of the A(1) receptor itself, and 2) age impairs adenosine transport and/or catabolism, but this does not explain differing functional sensitivity to adenosine. This increased functional sensitivity to adenosine may have physiological significance in the older heart.