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.

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.

[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.

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.

Mice lacking the vascular endothelial growth factor-B gene (Vegfb) have smaller hearts, dysfunctional coronary vasculature, and impaired recovery from cardiac ischemia.

Vascular endothelial growth factor-B (VEGF-B) is closely related to VEGF-A, an effector of blood vessel growth during development and disease and a strong candidate for angiogenic therapies. To further study the in vivo function of VEGF-B, we have generated Vegfb knockout mice (Vegfb(-/-)). Unlike Vegfa knockout mice, which die during embryogenesis, Vegfb(-/-) mice are healthy and fertile. Despite appearing overtly normal, Vegfb(-/-) hearts are reduced in size and display vascular dysfunction after coronary occlusion and impaired recovery from experimentally induced myocardial ischemia. These findings reveal a role for VEGF-B in the development or function of coronary vasculature and suggest potential clinical use in therapeutic angiogenesis.

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.

Differences in nucleotide compartmentation and energy state in isolated and in situ rat heart: assessment by 31P-NMR spectroscopy.

Free cytosolic concentrations of ATP, PCr, ADP and 5'-AMP, and the cytosolic [ATP]/[ADP].[Pi] ratio, were determined in isolated and in situ rat hearts using 31P-NMR spectroscopy. Total tissue metabolite concentrations were determined by HPLC analysis of freeze-clamped, perchloric acid-extracted tissue. In in situ myocardium the PCr/ATP ratio was 2.7 +/- 0.2 determined from 31P-NMR data (using either PCr/beta-NTP or PCr/gamma-NTP), and 1.9 +/- 0.1 (P < 0.01) determined from total tissue concentrations. 31P-NMR-determined and total tissue [PCr] were in excellent agreement (49.6 +/- 8.4 and 49.5 +/- 1.0 mumol.g-1 dry wt, respectively), whereas 31P-NMR-determined [ATP] (18.6 +/- 3.2 mumol.g-1 dry wt) was only 71% of the total tissue concentration (26.1 +/- 1.7 mumol.g-1 dry wt, P < 0.01). Isolation and Langendorff perfusion of rat hearts with glucose as substrate reduced total tissue [ATP] and [PCr] and the 31P-NMR-determined PCr/ATP ratio fell to 1.5 +/- 0.1. This value agreed well with the total tissue ratio of 1.4 +/- 0.1, and there was excellent agreement between 31P-NMR-determined and total tissue [PCr] and [ATP] values in the perfused heart. Addition of pyruvate to perfusate increased the 31P-NMR-determined PCr/ATP ratio to 1.7 +/- 0.1 due to elevated [PCr], and there remained excellent agreement between NMR-determined and total tissue [PCr] and [ATP] values. Free cytosolic [ADP] (from the creatine kinase equilibrium) was 5% of total tissue ADP, and free cytosolic [5'-AMP] (from the adenylate kinase equilibrium) ranged from 0.2-0.3% of total tissue 5'-AMP. Bioenergetic state, indexed by [ATP]/[ADP].[Pi], was much lower in isolated perfused hearts (30 mM-1) vs. in situ myocardium (approximately 150 mM-1). In summary, we observe a substantial disproportionality between total tissue PCr/ATP and 31P-NMR-determined PCr/ATP in highly energised in situ myocardium but not in isolated perfused hearts. This appears due to an NMR invisible ATP compartment approximating 29% of total tissue ATP in situ. Additionally, more than 95% of ADP and more than 99% of 5'-AMP exist in bound forms in perfused and in situ myocardium. The physiological significance of these observations is unclear. However, substantial differences between 31P-NMR visible and total tissue [ATP] introduces significant errors in conventional estimation of free cytosolic [ADP], [5'-AMP] and [ATP]/[ADP].[Pi] from in vivo 31P-NMR data.

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.

Extracellular adenosine levels and cellular energy metabolism in ischemically preconditioned rat heart.

OBJECTIVE: Microdialysis and 31P-NMR spectroscopy were used to test opposing hypotheses that ischemic preconditioning inhibits adenine nucleotide degradation and purine efflux, or that preconditioning activates cardiovascular adenosine formation to provide enhanced cardioprotection. METHODS: 31P-NMR spectra and matching interstitial fluid (ISF) or venous effluent samples were obtained from Langendorff perfused rat hearts. Control hearts (n = 9) underwent 30 min of global normothermic ischemia and 30 min reperfusion. Preconditioned hearts (n = 6) were subjected to a 5 min ischemic episode and 10 min reflow prior to 30 min ischemia and 30 min reperfusion. Effects of repetitive ischemia-reperfusion (3 x 5 min ischemic episodes) on adenosine levels and energy metabolism were also assessed (n = 8). RESULTS: Preconditioning improved post-ischemic recovery of heart rate x left ventricular developed pressure (71 +/- 5 vs 43 +/- 8%, P < 0.05) and end-diastolic pressure (14 +/- 3 vs 29 +/- 4 mmHg, P < 0.05) compared with control hearts, respectively. Preconditioning did not alter intracellular ATP, phosphocreatine (PCr), inorganic phosphate (Pi), H+ or free Mg2+ during global ischemia, but improved recoveries of PCr, Pi, and delta GATP on reperfusion. ISF adenosine increased more than 20-fold during 30 min ischemia. The 5 min preconditioning episode increased ISF adenosine 3-fold, and reduced ISF adenosine and inosine during subsequent prolonged ischemia by up to 75%. Venous purine levels during reperfusion were also reduced by preconditioning. Accumulation of adenosine in ISF and venous effluent during repetitive ischemia was progressively reduced despite comparable changes in substrate for adenosine formation via 5'-nucleotidase, (5'-AMP), and in allosteric modulators of this enzyme (Mg2+, H+, Pi, ADP, ATP). CONCLUSIONS: (i) Ischemic preconditioning reduces interstitial and vascular adenosine levels during ischemia-reperfusion, (ii) reduced ISF adenosine during ischemia is not due to reduced ischemic depletion of adenine nucleotides in preconditioned rat hearts, (iii) preconditioning may inhibit adenosine formation via 5'-nucleotidase in ischemic rat hearts, and (iv) improved functional recovery with preconditioning is unrelated to metabolic/bioenergetic changes during the ischemic insult, but may be related to improved post-ischemic recovery of [Pi] and delta GATP in this model.

Interstitial adenosine and cellular metabolism during beta-adrenergic stimulation of the in situ rabbit heart.

OBJECTIVE: Adenosine, derived from hydrolysis of 5'-AMP, may be involved in coupling coronary blood flow to cardiac function and metabolism. The purpose of this study was to measure interstitial fluid (ISF) adenosine and 5'-AMP levels, and cytosolic 5'-AMP in in situ rabbit heart during beta-adrenergic stimulation. METHODS: Isoproterenol was infused into open chest rabbits (n = 7) at 1 and 4 micrograms.kg-1.min-1. Left ventricular ISF adenosine and 5'-AMP levels were measured using microdialysis, and energy metabolism simultaneously monitored using 31P-NMR spectroscopy. RESULTS: Graded beta-stimulation increased heart rate (by 50% and 70%), arterial pulse-pressure (by 55% and 45%), and the rate-pressure product (by 45% and 70%). Dialysate [adenosine] increased 300-400% from a control value of 0.44 +/- 0.13 microM. Dialysate [5'-AMP] increased 200% from a control value of 0.94 +/- 0.22 microM. Cytosolic [ATP], pH and free [Mg2+] remained stable, whereas [PCr] declined by 10-20%. Free cytosolic [5'-AMP] increased 300-400% from a control value of 0.40 microM. Competitive inhibition of ecto-5'-nucleotidase with alpha,beta-methylene-ADP (0.6 mg.kg-1.min-1) significantly reduced ISF adenosine (and enhanced ISF 5'-AMP) during beta-stimulation, but not under basal conditions. CONCLUSIONS: Beta-adrenergic stimulation increases ISF adenosine levels and depresses bioenergetic state in in situ rabbit heart without altering [ATP], pH or [Mg2+], indicating an absence of "demand" ischemia. ISF adenosine originates primarily from cytosolic 5'-AMP under basal conditions whereas increased adenosine during beta-stimulation appears to occur via hydrolysis of both ISF and cytosolic 5'-AMP. ISF adenosine levels achieved during stimulation are appropriate for stimulation of cardiovascular A1 and A2 receptors.

Dissociation of adenosine levels from bioenergetic state in experimental brain trauma: potential role in secondary injury.

Intracellular bioenergetic state and extracellular adenosine levels were monitored in rat brain prior to and following traumatic brain injury (TBI) using phosphorus magnetic resonance spectroscopy and microdialysis, respectively. Fluid percussion-induced TBI (2.6 +/- 0.2 atm) resulted in significant reductions in free cytosolic [Mg2+], cytosolic [ATP]/[ADP] [P(i)], and delta GATP and elevations in cytosolic [ADP] and [5'-AMP]. Intracellular ATP concentration and pH did not change significantly after trauma. Mitochondrial capacity for oxidative phosphorylation (indexed by V/Vmax) increased significantly from approximately 0.45 prior to injury to approximately 0.58 following TBI. All metabolic changes were maximal at 2-3 h post-TBI. Conversely, extracellular adenosine concentrations increased transiently following TBI, with levels peaking at 10 min posttrauma, then declining rapidly to preinjury values by 50 min. Thus, despite pronounced long-term depression in bioenergetic status and a marked rise in [5'-AMP], formation and release of adenosine were elevated only transiently within the first hour following TBI. Since steady-state adenosine levels were essentially unchanged beyond 1 h posttrauma, mooted neuroprotective actions of endogenous adenosine would be minimized. Intracerebroventricular injections of 2-chloroadenosine (0.5 and 2.5 nmol) immediately prior to TBI dose-dependently attenuated metabolic disturbances and improved posttraumatic neurologic outcome (p < 0.05). The observations indicate that (a) TBI results in dissociation of adenosine release from intracellular bioenergetic state, a phenomenon possibly contributing to secondary injury following TBI; and (b) supplementing brain with an adenosine agonist attenuates irreversible injury.

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.

Bioenergetic analysis of oxidative metabolism following traumatic brain injury in rats.

Studies of fluid percussion-induced traumatic brain injury have shown that moderate trauma results in ionic imbalances, with resultant increases in energy demand to restore these ion gradients. Because there are also increased rates of glucose metabolism during periods of focal decline in blood flow, it has been suggested that the mitochondria may be incapable of sufficient oxidative metabolism to cope with this increased energy demand after injury and that ATP derived from substrate level phosphorylation must meet this demand. In the present study, we used phosphorus magnetic resonance spectroscopy to determine the mitochondrial capacity for oxidative phosphorylation after moderate brain trauma. Before injury, mean oxidative capacity was 54% +/- 1%. After injury, mean capacity increased significantly (p < 0.001) to a maximum of 61% +/- 1%, indicating that mitochondrial oxidative metabolism was enhanced after trauma. Increased oxidative capacity was accompanied by increases in ADP, AMP, and inorganic phosphate concentrations and was correlated to decreases in cytosolic phosphorylation ratio. We conclude that moderate brain trauma increases mitochondrial rate of ATP synthesis over the first 4 h posttrauma, and that during this time of increased ATP turnover, positive feedback regulation of glycolysis by increased concentrations of ADP, AMP, and inorganic phosphate contributes to maintenance of metabolic steady state.

Intrathecal dynorphin-A infusion in rat spinal cord causes energy depletion, edema and neurologic dysfunction.

The opioid dynorphin-A (dynA) is thought to contribute to the secondary injury process following spinal cord trauma although little is known about the biochemical mechanisms involved. In the present study, we have used a combination of magnetic resonance imaging (MRI) and spectroscopy (MRS) and hindlimb motor function tests to examine the effects of intrathecal dynA infusion on rat spinal cord. Infusion of 100 nmol of dynA (1-17) caused pronounced edema development as determined by MRI at 24 h after infusion. Infusion of 100 nmol of the dynA (2-17) fragment, which does not have any activity at opiate receptors, also produced profound edema whereas 100 nmol of the low potency kappa opiate receptor ligand dynA (1-8) or artificial CSF (ACSF) did not produce any edema. Both dynA (1-17) and dynA (2-17) produced significant hindlimb motor deficits at 24 h when compared to dynA (1-8) and ACSF (P < 0.05), but the deficits in the dynA (1-17) group were significantly worse than in the dynA (2-17) treated animals (P < 0.05). Similarly, mortality in the dynA (1-17) treated animals was significantly higher than in the other groups (P = 0.002). Phosphorus MRS demonstrated that the dynA (1-17) and dynA (2-17) treated animals also had a pronounced decline in high energy phosphates in the spinal cord 24 h after infusion. We conclude that dynA contributes to spinal cord cell death by causing metabolic failure and edema development.

Estrogen improves biochemical and neurologic outcome following traumatic brain injury in male rats, but not in females.

Phosphorus magnetic resonance spectroscopy was used in conjunction with neurologic motor function tests to assess the effects of estrogen on biochemical and neurologic outcome following traumatic brain injury in male and female rats. Male (n = 18) and female (n = 18) rats were randomly assigned into three groups, and 4 h prior to injury received either 17 beta-estradiol (144 micrograms/kg intraperitoneally), equal volume vehicle (30% ethanol in saline), or no treatment. Traumatic brain injury was induced at 2.8 atm using a fluid percussion injury device, and animals monitored for 4 h using phosphorus magnetic resonance spectroscopy to determine brain intracellular pH, free magnesium concentration and cytosolic phosphorylation potential. Males treated with estrogen demonstrated a significant improvement in free magnesium concentration, and slightly improved values of cytosolic phosphorylation potential after trauma when compared to controls. There was also a significant improvement in post-traumatic motor function at 1 week after trauma. In contrast, estrogen treatment in females lowered cytosolic phosphorylation potential after trauma, but did not affect free magnesium concentration after trauma. Mortality in all female groups was significantly worse than in males. We conclude that estrogen is protective in males, but exacerbates brain injury in females through effects mediated by estrogen receptor binding.

[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.

[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.

Interstitial adenosine and function in rat heart in vivo: effects of adrenaline and 8-cyclopentyl-1,3-dimethylxanthine.

1. Left ventricular interstitial adenosine and cardiac function were studied in open chest rats during adrenaline stimulation and P1-purinoceptor antagonism with 8-cyclopentyl-1,3-dimethylxanthine (8-CPT). 2. Cardiac microdialysate adenosine concentration was 0.10 +/- 0.01 mumol/L (n = 24) under basal conditions, giving an estimated interstitial adenosine concentration of 0.27 mumol/L. Stimulation with 3.2 and 8.0 micrograms/kg per min adrenaline increased the rate-pressure product (heart rate x systolic blood pressure) by 72 and 157%, respectively, and increased dialysate adenosine to 0.26 +/- 0.04 and 0.65 +/- 0.11 mumol/L (n = 12), respectively (interstitial concentrations of approximately 0.70 and 1.76 mumol/L). 3. Treatment with 60 micrograms/kg per min 8-CPT did not alter basal adenosine concentrations, but potentiated elevations in dialysate adenosine during infusion of 3.2 and 8.0 micrograms/kg per min adrenaline to 0.54 +/- 0.10 and 1.30 +/- 0.22 mumol/L, respectively (n = 12). Basal function and the response to 8.0 micrograms/kg per min adrenaline were unaltered by 8-CPT, whereas elevations in heart rate and rate-pressure product during stimulation with 3.2 micrograms/kg per min adrenaline were enhanced by 8-CPT (by up to 30%). 4. Studies in isolated hearts confirmed the inhibitory potency of 8-CPT at A1 vs A2 P1-purinoceptors (e.g. pK(B) of 7.7 +/- 0.2 and 6.4 +/- 0.1 for 5'-N-ethyl carboxamidoadenosine-mediated bradycardia and vasodilatation, respectively; n = 6). Studies in intact animals verified effective A1 blockade by 60 micrograms/kg per min 8-CPT, but also revealed some inhibition of A2-mediated responses. 5. In conclusion, the data show that cardiac interstitial adenosine levels exist within a physiologically active range in vivo and increase dose-dependently during graded adrenaline stimulation. Adenosine receptor antagonism enhances elevations in interstitial adenosine and modifies functional responses to moderate, but not high, doses of adrenaline. Whether 8-CPT-dependent elevations in interstitial adenosine are due to A1 inhibition vs inhibition of A2-mediated vasodilatation requires further investigation.