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.

Opioids and myocardial reperfusion injury.

It is well-established that reperfusion is the major method of salvaging ischemic myocardium following prolonged coronary artery occlusion, although the idea of reperfusion injury remains controversial. Moreover, more recent evidence strongly suggests that reperfusion per se is thought to result in further damage to the myocardium and blood vessel endothelium by various biochemical and physical factors including a burst of oxygen-derived free radicals (ROS), cellular or mitochondrial calcium overload and shear stress, to name a few. This has been termed lethal reperfusion injury. It has become increasingly evident that strategies in which interventions are administered during the early stages of reperfusion produce a reduction in reperfusion-mediated damage primarily by reducing massive calcium overload or by altering the intracellular milieu (pH, osmotic stress, etc.) and ROS release upon reperfusion. Furthermore, it is apparent that activation of blood borne elements such as neutrophils and macrophages and factors released by these cells such as cytokines may be responsible for a continuing expansion of infarction in the hours or even days following timely reflow and that inhibiting these factors may attenuate reperfusion injury. The present review will focus on the effect of endogenous and exogenous opioids on ischemic and reperfusion injury since these compounds are routinely used in the surgical arena and may have unappreciated cardioprotective effects in this subset of patients. Particular emphasis will be on the role of opioids in reperfusion injury and their relationship to the newly discovered phenomenon of postconditioning.

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.

Effects of energy intake during late pregnancy and of genotype on immunoglobulin transfer to calves in suckler herds.

Blue-Grey (Shorthorn x Galloway) and Hereford x Friesian cows were given levels of feeding, during the last 12 weeks of pregnancy, varying between 75.2 per cent and 171.5 per cent of the maintenance requirements in one experiment and between 65 per cent and 125 per cent of the maintenance requirements in a second experiment. The concentrations of the immunoglobulin classes IgG1, IgG2 and IgM, in their colostrum and in their calves' serum were measured. The IG1 and IgM, but not the IgG2, concentrations were significantly higher in the Blue-Grey than in the Hereford x Friesian colostrum and serum. The relative concentrations of the three classes in the serum were almost identical to those in the colostrum, suggesting that absorption by the calves was non-selective. The amounts of immunoglobulin transferred to the calves were not affected by the cows' levels of feeding in the first year, but there was a small increase in transfer with increasing levels of feeding during the second year. Cows that transferred large amounts to their calves in the first year tended to do so in the second year. The milk intake and growth of the calves during the first 42 days were significantly correlated with their immunoglobulin concentrations.

Cardioprotection following adenosine kinase inhibition in rat hearts.

Adenosine kinase phosphorylates adenosine to AMP, the primary pathway for adenosine metabolism under basal conditions. Inhibition of adenosine kinase results in a site-specific increase in interstitial adenosine. Using a rat model of myocardial infarction, we examined the protective effects of adenosine kinase inhibition. Male Sprague-Dawley rats underwent 30 min regional occlusion followed by 90 min reperfusion. Infarct size, expressed as a percent of the area-at-risk, IS/AAR(%), was 58.0 +/- 2.1 % in untreated rats. Pretreatment with the adenosine kinase inhibitor, 5-iodotubercidin (1 mg/kg), limited infarct development to 37.5+/-3.7% (P < 0.001). The A(1) adenosine receptor (A(1)AR) antagonist, DPCPX (100 microg/kg), abolished the infarct-sparing effect of 5-iodotubercidin (IS, 62.8 +/- 1.3%). Similarly, the A(3) adenosine receptor (A(3)AR) antagonist, MRS-1523 (2 mg/kg), and the delta-opioid receptor (DOR) antagonist, BNTX, (1 mg/kg) abolished the reduction of IS produced by iodotubercidin. Pretreatment with the ROS scavenger, 2-MPG (20 mg/kg), or the PKC-delta antagonist, rottlerin (0.3 mg/kg) also abolished iodotubercidin-mediated cardioprotection. Furthermore, pretreatment with 5-HD, a mitochondrial K(ATP) (mitoK(ATP)) channel inhibitor, but not the sarcolemmal K(ATP) channel blocker, HMR-1098, abrogated the beneficial effects of adenosine kinase inhibition (IS, 59.5 +/- 3.8%). These data suggest that inhibition of adenosine kinase is effective in reducing infarct development via A(1)AR, A(3)AR and DOR activation. Data also suggest that this protection is mediated via ROS, PKC-delta and mitoK(ATP) channels.

Sarcolemmal and mitochondrial K(ATP) channels and myocardial ischemic preconditioning.

Ischemic preconditioning (IPC) is the phenomenon whereby brief periods of ischemia have been shown to protect the myocardium against a sustained ischemic insult. The result of IPC may be manifest as a marked reduction in infarct size, myocardial stunning, or incidence of arrhythmias. While many substances and pathways have been proposed to play a role in the signal transduction mediating the cardioprotective effect of IPC, overwhelming evidence indicates an intimate involvement of the ATP-sensitive potassium channel (K(ATP) channel) in this process. Initial hypotheses suggested that the surface or sarcolemmal K(ATP) (sarcK(ATP)) channel mediated the cardioprotective effects of IPC. However, much research has subsequently supported a major role for the mitochondrial K(ATP) channel (mitoK(ATP)) as the one involved in IPC-mediated cardioprotection. This review presents evidence to support a role for the sarcK(ATP) or the mitoK(ATP) channel as either triggers and/or downstream mediators in the phenomenon of IPC.

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.