Effects of ryanodine on skeletal muscle lactate and pyruvate in malignant hyperthermia-susceptible and normal swine as assessed by microdialysis.

BACKGROUND: The caffeine/halothane contracture test in North America and the in vitro contracture test in Europe are currently the only validated bioassays for diagnosing malignant hyperthermia susceptibility and phenotyping families. Both tests are invasive requiring surgical muscle biopsy. Here, we report first use of the selective ryanodine receptor type I agonist ryanodine in a percutaneous microdialysis protocol designed to test whether microdialysis-induced local metabolic responses of skeletal muscle due to ryanodine receptor activation can differentiate between malignant hyperthermia-sensitive and normal pigs. METHODS: Six microdialysis catheters were implanted percutaneously into the adductor muscles of the right and left thighs of malignant hyperthermia-susceptible (n = 9) and normal (n = 8) anaesthetized (ketamine/propofol) and mechanically ventilated swine. Systemic blood gases, haemodynamic parameters and creatine kinase levels were measured before, during and after microdialysis perfusion of ryanodine. After a post-implantation equilibration period of 30 min, one catheter perfused (2 micro min-1) with 0.9% NaCl (control) and was compared with the remaining five catheters perfused with increasing concentrations of ryanodine (0.2-100 micromol). Lactate and pyruvate levels were measured enzymatically. RESULTS: Continuous perfusion with ryanodine revealed dose-dependent sigmoidal increases in the dialysate lactate and lactate-pyruvate ratio parameters; these effects were greatly augmented in malignant hyperthermia-susceptible pigs compared to normal pigs (two- to threefold): estimated EC50 greatly decreased (>19-fold) while the maximum effect increased (>twofold) in the malignant hyperthermia-susceptible group. CONCLUSION: The in vivo percutaneous microdialysis protocol for skeletal muscle, using ryanodine as the ryanodine receptor type I agonist and dialysed lactate-pyruvate parameters as metabolic index, can reproducibly differentiate between malignant hyperthermia-susceptible and normal swine.

Role of complement activation in hypersensitivity reactions to doxil and hynic PEG liposomes: experimental and clinical studies.

Pegylated liposomal doxorubicin (Doxil) and 99mTc-HYNIC PEG liposomes (HPL) were reported earlier to cause hypersensitivity reactions (HSRs) in a substantial percentage of patients treated i.v. with these formulations. Here we report that (1) Doxil, HPL, pegylated phosphatidylethanolamine (PEG-PE)-containing empty liposomes matched with Doxil and HPL in size and lipid composition, and phosphatidylglycerol (PG)-containing negatively charged vesicles were potent C activators in human serum in vitro, whereas small neutral liposomes caused no C activation. (2) Doxil and other size-matched PEG-PE and/or PG-containing liposomes also caused massive cardiopulmonary distress with anaphylactoid shock in pigs via C activation, whereas equivalent neutral liposomes caused no hemodynamic changes. (3) A clinical study showed more frequent and greater C activation in patients displaying HSR than in non-reactive patients. These data suggest that liposome-induced HSRs in susceptible individuals may be due to C activation, which, in turn, is due to the presence of negatively charged PEG-PE in these vesicles.

Pyruvate improves cerebral metabolism during hemorrhagic shock.

Pyruvate (PYR) improves cellular and organ function hypoxia and ischemia by stabilizing the reduced nicotinamide adenine dinucleotide redox state and cytosolic ATP phosphorylation potential. In this in vivo study, we evaluated the effects of intravenous pyruvate on neocortical function, indexes of the cytosolic redox state, cellular energy state, and ischemia during a prolonged (4 h) controlled arterial hemorrhage (40 mmHg) in swine. Thirty minutes after the onset of hemorrhagic shock, sodium PYR (n = 8) was infused (0.5 g x kg(-1) x h(-1)) to attain arterial levels of 5 mM. The volume and osmotic effects were matched with 10% NaCl [hypertonic saline (HTS)] (n = 8) or 0.9% NaCl [normal saline (NS)] (n = 8). During the hemorrhage protocol, the time to peak hemorrhage volume was significantly delayed in the PYR group compared with the HTS and NS groups (94 +/- 5 vs. 73 +/- 6 and 72 +/- 4 min, P < 0.05). In addition to the early onset of the decompensatory phase of hemorrhagic shock, the complete return of the hemorrhage volume during decompensatory shock resulted in the death of five and four animals, respectively, in the HTS and NS groups. In contrast, in the PYR group, reinfusion of the hemorrhage volume was slower and all animals survived the 4-h hemorrhage protocol. During hemorrhage, the PYR group also exhibited improved cerebral cortical metabolic and function status. PYR slowed and reduced the rise in neocortical microdialysis levels of adenosine, inosine, and hypoxanthine and delayed the loss of cerebral cortical biopsy ATP and phosphocreatine content. This improvement in energetic status was evident in the improved preservation of the electrocorticogram in the PYR group. PYR also prevented the eightfold increase in the excitotoxic amino acid glutamate observed in the HTS group. The findings show that PYR administered after the onset of hemorrhagic shock markedly improves cerebral metabolic and functional status for at least 4 h.

Intramitochondrial pyruvate attenuates hydrogen peroxide-induced apoptosis in bovine pulmonary artery endothelium.

In the hydrogen peroxide (H2O2) apoptosis model of the murine thymocyte, redox reactant and antioxidant pyruvate prevents programmed cell death. We tested the hypothesis that such protection was mediated, at least in part, via pyruvate handling by mitochondrial metabolism. Cultured bovine pulmonary artery endothelial cells were incubated for 30 min with 0.5 mM H2O2 in the absence and presence of 0.5 mM alpha-cyano-3-hydroxycinnamate, as a selective inhibitor of the mitochondrial pyruvate transporter. In controls H2O2 decreased cell viability by 30% within 24 h; this was associated with apoptosis-like bodies, nuclear condensation, and biochemical DNA damage consistent with programmed cell death. Pyruvate (0.1-20 mM) enhanced cell viability in a dose-dependent manner, with > or = 85% viable cells at > or = 3 mM and no DNA laddering, no positive nick-end labeling (TUNEL), and no detectable Annexin V or propidium iodide staining. In contrast, using > or = 5 mM L-lactate as a cytosolic reductant or acetate as a redox-neutral substrate, cell death increased to approximately 40%, which was associated with intense DNA laddering, positive TUNEL and Hoechst 33258 assays. Alpha-cyano-3-hydroxycinnamate alone did not significantly decrease endothelial viability but reduced viability from 85+/-3 to 71+/-4% (p = 0.023) in presence of 3 mM pyruvate plus H2O2; pathological cell morphology and DNA laddering under the same conditions suggested loss of pyruvate protection against apoptosis. Since alpha-cyano-3-hydroxycinnamate re-distributed medium pyruvate and L-lactate consistent with selective blockade of pyruvate uptake into the mitochondria, the findings support the hypothesis that pyruvate protection against H2O2 apoptosis is mediated in part via the mitochondrial matrix compartment. Possible mediators include anti-apoptotic bcl-2 and/or products of mitochondrial pyruvate metabolism such as citrate that affect metabolic regulation and anti-oxidant status in the cytoplasm.

Antioxidant pyruvate inhibits cardiac formation of reactive oxygen species through changes in redox state.

Myocardial ischemia-reperfusion is associated with bursts of reactive oxygen species (ROS) such as superoxide radicals (O(2)(-).). Membrane-associated NADH oxidase (NADHox) activity is a hypothetical source of O(2)(-)., implying the NADH concentration-to-NAD(+) concentration ratio ([NADH]/[NAD(+)]) as a determinant of ROS. To test this hypothesis, cardiac NADHox and ROS formation were measured as influenced by pyruvate or L-lactate. Pre- and postischemic Langendorff guinea pig hearts were perfused at different pyruvate/L-lactate concentrations to alter cytosolic [NADH]/[NAD(+)]. NADHox and ROS were measured with the use of lucigenin chemiluminescence and electron spin resonance, respectively. In myocardial homogenates, pyruvate (0.05, 0.5 mM) and the NADHox blocker hydralazine markedly inhibited NADHox (16 +/- 2%, 58 +/- 9%). In postischemic hearts, pyruvate (0.1-5.0 mM) dose dependently inhibited ROS up to 80%. However, L-lactate (1.0-15.0 mM) stimulated both basal and postischemic ROS severalfold. Furthermore, L-lactate-induced basal ROS was dose dependently inhibited by pyruvate (0.1-5.0 mM) and not the xanthine oxidase inhibitor oxypurinol. Pyruvate did not inhibit ROS from xanthine oxidase. The data suggest a substantial influence of cytosolic NADH on cardiac O(2)(-). formation that can be inhibited by submillimolar pyruvate. Thus cytotoxicities due to cardiac ischemia-reperfusion ROS may be alleviated by redox reactants such as pyruvate.

Liposome-induced pulmonary hypertension: properties and mechanism of a complement-mediated pseudoallergic reaction.

Intravenous injection of liposomes can cause significant pulmonary hypertension in pigs, a vasoconstrictive response that provides a sensitive model for the cardiopulmonary distress in humans caused by some liposomal drugs. The reaction was recently shown to be a manifestation of "complement activation-related pseudoallergy" (CARPA; Szebeni J, Fontana JL, Wassef NM, Mongan PD, Morse DS, Dobbins DE, Stahl GL, Bünger R, and Alving CR. Circulation 99: 2302-2309, 1999). In the present study we demonstrate that the composition, size, and administration method of liposomes have significant influence on pulmonary vasoactivity, which varied between instantaneously lethal (following bolus injection of 5 mg lipid) to nondetectable (despite infusion of a 2,000-fold higher dose). Experimental conditions augmenting the pulmonary hypertensive response included the presence of dimyristoyl phosphatidylglycerol, 71 mol% cholesterol, distearoyl phosphatidylcholine, and hemoglobin in liposomes, increased vesicle size and polydispersity, and bolus injection vs. slow infusion. The vasoactivity of large multilamellar liposomes was reproduced with human C3a, C5a, and xenoreactive immunoglobulins, and it correlated with the complement activating and natural antibody binding potential of vesicles. Unilamellar, monodisperse liposomes with 0.19 +/- 0.10 microm mean diameter had no significant vasoactivity. These data indicate that liposome-induced pulmonary hypertension in pigs is multifactorial, it is due to natural antibody-triggered classic pathway complement activation and it can be prevented by appropriate tailoring of the structure and administration method of vesicles.

Myocardial oxygen consumption modulates adenosine formation by canine right ventricle in absence of hypoxia.

Myocardial adenosine formation varies with myocardial oxygen consumption (MVO(2)), but whether concurrent hypoxia is required for adenosine formation is uncertain. Changes in right coronary (RC) perfusion pressure (RCP) produce directionally similar alterations in right ventricular (RV) MVO(2)and in RC venous P O(2)(P(v)O(2)), an index of myocardial P O(2). RCP was varied in 10 anesthetized, open chest dogs to determine if, under these conditions, RV formation of adenosine would increase with MVO(2)in absence of myocardial hypoxia. Dialysis probes were implanted in the mid myocardium of RV free wall for collecting dialysate samples for HPLC analyses to estimate interstitial adenosine and other purines. Coronary venous blood was sampled from a superficial vein draining the RC artery (RCA) perfusion territory. At 115+/-3 mmHg baseline RCP, RC blood flow (RCBF)=0.51+/-0.04 ml/min/g, MVO(2)=4.6+/-0.5 ml/min/100 g, P(v)O(2)=34+/-1.5 mmHg, and dialysate adenosine=0. 27+/-0.03microM. When RCP was lowered to 61+/-1 mmHg by adjusting an occluder on the proximal RCA, RCBF decreased to 0.36+/-0.03 ml/min/g, MVO(2)fell to 3.7+/-0.4 ml/min/100 g, lactate uptake remained positive, P(v)O(2)fell to 30+/-1.7 mmHg, and dialysate adenosine decreased to 0.20+/-0.03microM. Reactive hyperemia of 1.25+/-0.13 ml/min/g was observed when the RCA constriction was released, although dialysate adenosine had fallen. When RCP was elevated to 164+/-2 mmHg by inflating a balloon catheter in the descending aorta, RCBF increased to 0.70+/-0.06 ml/min/g, MVO(2)increased to 5.8+/-1. 0 ml/min/100 g, P(v)O(2)rose to 39+/-2.3 mmHg, and dialysate adenosine increased to 0.33+/-0.04microM. These data indicate that (1) RV oxygen demand varies with RCP; (2) if RV ischemia is absent, myocardial adenosine formation is modulated by MVO(2), with no requirement for hypoxia; (3) pressure-flow autoregulation is relatively ineffective in the RC circulation, where adenosine does not mediate and may even blunt autoregulation.

Intravenous pyruvate prolongs survival during hemorrhagic shock in swine.

Pyruvate improves cellular and organ function during hypoxia and ischemia and stabilizes the NADH redox state and cytosolic ATP phosphorylation potential. In this in vivo study, we evaluated the effects of intravenous pyruvate on cardiovascular and neocortical function, indexes of the cytosolic redox state (lactate/pyruvate ratio, L/P) and cellular energy state (adenosine and degradative products hypoxanthine and inosine, ADO + HX + Ino) during controlled arterial hemorrhage (40 mmHg) in sedated swine (45 kg). Na+ pyruvate was infused 1 h before (1 g. kg(-1). h(-1)) and 2 h during (0.5 g. kg(-1). h(-1)) hemorrhage to attain arterial pyruvate levels of 6 mM. Volume (0.9% NaCl) and osmotic (10% NaCl) effects were matched in controls. Time to peak hemorrhage (57 min) and peak hemorrhage volume (43 ml/kg) were similar in all groups. The volume and osmotic groups experienced spontaneous cardiovascular decompensation between 60 and 90 min, with an average time until death of 82.7 +/- 5.5 and 74.8 +/- 8.2 min. In contrast, survival in the pyruvate group was 151.2 +/- 10.0 min (P < 0.001). During hemorrhage, the pyruvate group had better cardiovascular and cerebrovascular function with significantly higher systemic and cerebral oxygen consumption and less attenuation of the amplitude and frequency of the electrocorticogram. In addition, pyruvate prevented metabolic acidosis and stabilized the L/P. Pyruvate slowed the rise in neocortical microdialysis levels of ADO + HX + Ino, and prevented the net efflux of ADO + HX + Ino into the sagittal sinus. The findings reveal considerable metabolic and functional enhancement by pyruvate during severe hemorrhagic shock with a 75-min delay in spontaneous cardiovascular decompensation and death.

Hemodynamic changes induced by liposomes and liposome-encapsulated hemoglobin in pigs: a model for pseudoallergic cardiopulmonary reactions to liposomes. Role of complement and inhibition by soluble CR1 and anti-C5a antibody.

BACKGROUND: Intravenous administration of some liposomal drugs can trigger immediate hypersensitivity reactions that include symptoms of cardiopulmonary distress. The mechanism underlying the cardiovascular changes has not been clarified. METHODS AND RESULTS: Anesthetized pigs (n=18) were injected intravenously with 5-mg boluses of large multilamellar liposomes, and the ensuing hemodynamic, hematologic, and laboratory changes were recorded. The significant (P<0.01) alterations included 79+/-9% (mean+/-SEM) rise in pulmonary arterial pressure, 30+/-7% decline in cardiac output, 11+/-2% increase in heart rate, 236+/-54% increase in pulmonary vascular resistance, 71+/-27% increase in systemic vascular resistance, and up to a 100-fold increase in plasma thromboxane B2. These changes peaked between 1 and 5 minutes after injection, subsided within 10 to 20 minutes, were lipid dose-dependent (ED50=4. 5+/-1.4 mg), and were quantitatively reproducible in the same animal several times over 7 hours. The liposome-induced rises of pulmonary arterial pressure showed close quantitative and temporal correlation with elevations of plasma thromboxane B2 and were inhibited by an anti-C5a monoclonal antibody (GS1), by sCR1, or by indomethacin. Liposomes caused C5a production in pig serum in vitro through classic pathway activation and bound IgG and IgM natural antibodies. Zymosan- and hemoglobin-containing liposomes and empty liposomes caused essentially identical pulmonary changes. CONCLUSIONS: The intense, nontachyphylactic, highly reproducible, complement-mediated pulmonary hypertensive effect of minute amounts of intravenous liposomes in pigs represents a unique, unexplored phenomenon in circulation physiology. The model provides highly sensitive detection and study of cardiopulmonary side effects of liposomal drugs and many other pharmaceutical products due to "complement activation-related pseudoallergy" (CARPA).

Pyruvate prevents hydrogen peroxide-induced apoptosis.

Studies were carried out to investigate the protective effects of pyruvate, a key glycolytic intermediate and alpha-keto-monocarboxylate, against oxidative stress-induced apoptosis. Oxidative stress was induced by treating mouse thymocytes with 25 microM hydrogen peroxide for 15 min at 37 degrees C under 5% CO2 in air. Pre- and post-treatment of cells with 10 mM pyruvate inhibited morphological changes, internucleosomal DNA fragmentation, and translocation of phosphatidylserine to the plasma membrane surface, which are characteristic features of apoptosis. L-lactate (10 mM) and acetate (10 mM) were ineffective in inhibiting apoptosis and appeared to be toxic to the cells under similar conditions. The results suggest that pyruvate has therapeutic potential for use in the treatment of oxidative stress-induced disorders associated with increased apoptosis.

Adenosine attenuates in vivo myocardial stunning with minimal effects on cardiac energetics.

Adenosine has been shown to modulate myocardial intermediary metabolism. The purpose of this study was to determine whether adenosine-mediated attenuation of in vivo myocardial stunning is associated with improved myocardial phosphorylation potential. Adult, open chest pigs were subjected to 10 minutes of regional myocardial ischemia and 90 minutes reperfusion. Regional ventricular function was assessed by measuring systolic wall thickening. Myocardial phosphorylation potential was estimated from the tissue (CrP/CrxPi) ratio determined in rapid-frozen tissue biopsy samples from normal and stunned myocardium. Control pigs were compared to animals treated prior to ischemia with intracoronary adenosine (50 micrograms/kg/min). Postischemic regional systolic wall thickening in adenosine treated pigs was significantly improved (40 +/- 3% of preischemic values) compared to control untreated pigs (26 +/- 3%). Myocardial stunning was associated with decreased ATP levels, but neither the total creatine pool (CrP + Cr) nor the (CrP/CrxPi) ratio was reduced. Adenosine pretreatment was associated with decreased Pi and Cr contents resulting in improved postischemic (CrP/CrxPi) ratio in the stunned bed compared to controls, but this effect occurred only after postischemic function had attained maximal improvement. These results suggest that adenosine attenuation of in vivo myocardial stunning is independent of elevated myocardial phosphorylation potential.

Ecto-5'-nucleotidase is not required for ischemic preconditioning in rabbit myocardium in situ.

This study tested the hypothesis that cardiac ecto-5'-nucleotidase (ecto-5'-NT) activity during ischemic preconditioning (PC) contributes to augmented tolerance against ischemia, thereby reducing infarct size in the rabbit heart in situ. The effects of alpha,beta-methylene-adenosine diphosphate (AOPCP), a selective inhibitor of ecto-5'-NT, on cardiovascular responses to AMP were measured to establish in vivo activities of the enzyme and its inhibitor. Left atrial infusion of AOPCP (0.75 mg . kg-1 . min-1) raised AOPCP plasma levels to 138 microM; under these conditions negative chronotropic and inotropic effects of AMP were blocked, demonstrating essentially full inhibition of ecto-5'-NT in the heart in situ. This AOPCP-blocked heart in situ model was used to examine the proposed contribution of ecto-5'-NT in ischemic PC. Myocardial infarction caused by 30-min ischemia was followed by 3-h reperfusion. Infarct size (IS) was measured and expressed as a percentage of the size of the area at risk (%IS/AR). In untreated controls, %IS/AR was 38.1 +/- 3.8%; PC (5-min ischemia, 5-min reperfusion) markedly reduced %IS/AR to 10.0 +/- 2.0%. Essentially identical IS reductions by PC were observed in AOPCP-blocked animals (%IS/AR = 13.8 +/- 2.2 and 13.3 +/- 1.8% in rabbits receiving AOPCP at 0.75 and 1.50 mg . kg-1 . min-1, respectively); here plasma AOPCP levels were established before and during PC but not during the subsequent prolonged ischemia. As expected, AOPCP also did not affect %IS/AR in non-PC controls (%IS/AR = 35.5 +/- 3.7%). In contrast but as predicted, adenosine-receptor blockade by 8-phenyltheophylline (10 mg/kg iv) substantially attenuated IS reduction by PC in both AOPCP-blocked and control hearts (%IS/AR = 25.2 +/- 4.3 and 21.8 +/- 2.2%, respectively; P < 0.05 vs. PC alone). The results demonstrate that cardiac ecto-5'-NT is not required for ischemic PC against infarction in the rabbit.

Pyruvate augments calcium transients and cell shortening in rat ventricular myocytes.

Pyruvate has been shown to be a metabolic inotrope in the myocardium. In millimolar concentrations, it has been shown to increase both myocardial phosphorylation potential and the cytosolic [NAD+]-to-[NADH] ratio. To determine if changes in these parameters can alter intracellular Ca2+ concentration ([Ca2+]i) and hence contractile function, Ca2+ transients and cell shortening (CS) were measured in isolated rat ventricular myocytes superfused with a physiological N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer (11 mmol/l glucose) with and without additional pyruvate, L-lactate, acetate, or isoproterenol. The addition of 5 mmol/l pyruvate resulted in a 33% increase in CS and a 39% increase in systolic [Ca2+]i. These pyruvate effects were 70% of those observed with 100 nmol/l isoproterenol. The mitochondrial monocarboxylate transport inhibitor alpha-cyano-4-hydroxycinnamate (250 mumol/l) strongly inhibited pyruvate inotropy, suggesting a substantial obligatory coupling between pyruvate inotropism and its oxidation by the mitochondria. A possible role of the cytosolic [NAD+]-to-[NADH] ratio was assessed by comparing the effects of 20 mmol/l L-lactate to those of equimolar pyruvate. In contrast to 20 mmol/l pyruvate, excess L-lactate failed to appreciably increase CS or systolic [Ca2+]i. The findings imply that, at levels substantially above 5 mmol/l, a portion of pyruvate inotropism might be due to extreme cytosolic [NAD+]-to-[NADH] ratios. This study is the first evidence that augmented [Ca2+]i transients are most likely the mechanism of cardiac pyruvate inotropism.

Profound normovolemic hemodilution: hemostatic effects in patients and in a porcine model.

Previous systematic investigations of the hemostatic effects of normovolemic hemodilution (NHD) have not explored the influence of hematocrits less than 20% in humans or animals. However, clinical interest in maximizing the perioperative conservation of erythrocytes may involve profound NHD beyond traditionally accepted empiric end points. We report here on coagulation data in eight healthy adolescent patients undergoing profound NHD in concert with surgical correction of idiopathic scoliosis, and in 29 swine undergoing experimental stepwise NHD until death. Blood was replaced with 5% albumin in 0.9% saline in our patients, and with 5% albumin in lactated Ringer's solution in our pigs. A 75% blood volume exchange in our patients yielded a platelet count (PLT) of 158 +/- 26 x 10(3)/microL, fibrinogen concentration (FIB), 50 +/- 7 mg/dL, prothrombin time (PT), 25.4 +/- 2.6 s, activated partial thromboplastin time (aPTT), 87 +/- 15s, and a nadir hemoglobin of 2.8 +/- 0.2 g/dL; however, global oxygen delivery as assessed by body oxygen consumption remained adequate. Coagulation during the experimental porcine hemodilution was assessed by measuring PLT, FIB, PT, and aPTT, as well as by measurement of coagulation factor activities. In neither species did clinically significant thrombocytopenia (PLT < 100 x 10(3)/ microL) become manifest prior to clinical or other laboratory evidence of coagulopathy. Rather, a combined deficiency of coagulation factors explains the coagulopathy developing during NHD in both patients and swine. Abnormal hemostasis develops prior to compromise of global tissue oxygenation, assessed by mixed venous oxygen saturation and total body oxygen consumption, during NHD in healthy patients anesthetized as described. Therefore, NHD may be more limited by preservation of normal coagulation than of global oxygen delivery and consumption.

Magnesium activated adenosine formation in intact perfused heart: predominance of ecto 5'-nucleotidase during hypermagnesemia.

Magnesium ion is an allosteric effector of 5'-nucleotidase and thus activates adenosine production from AMP. Two distinct 5'-nucleotidase systems, the membrane-bound ecto and the soluble cytosolic isoforms, exist in mammalian myocardium. The aim of this study was to delineate the contributions of the ecto vs. cytosolic isoforms to Mg2+-stimulated cardiac purine nucleoside formation and release. Isolated guinea pig hearts were retrogradely perfused at their physiological aortic pressure with Krebs-Henseleit bicarbonate buffer fortified with 10 mM glucose. AMP and the adenylate degradatives adenosine and inosine were measured in coronary venous effluent and in epicardial transudate, which was sampled to estimate concentrations of adenylate degradatives in the interstitium. When perfusate Mg2+ was increased from 0.6 to 6 mM, coronary vascular resistance and spontaneous heart rate fell, and steady-state coronary venous release of adenosine + inosine rose severalfold. Cytosolic free magnesium, as estimated by 31P-NMR after 15 min of perfusion with 6 mM Mg2+ or from chemically measured indicator metabolites after 30 min, rose 60 and 144% respectively (P < 0.05). Excess Mg2+ stimulated purine nucleoside release nearly threefold in coronary venous effluent and four- to sevenfold in epicardial transudate. 50 microM, alpha,beta-methylene adenosine 5'-diphosphate (AOPCP), a selective inhibitor of ecto 5'-nucleotidase, elevated interstitial AMP concentration tenfold, did not attenuate basal nucleoside release, but completely inhibited Mg2+-stimulated coronary venous purine nucleoside release and blunted Mg2+-stimulated interstitial purine nucleoside formation by 69%. During perfusion with exogenous 1 microM [8-14C]AMP, excess perfusate MgCl2 increased [14C]adenosine release by 63% in coronary effluent and 133% in epicardial transudate. AOPCP decreased baseline [14C]adenosine release in coronary effluent and epicardial transudate by 85-90%, caused equilibration of arterial and epicardial AMP, and attenuated MgCl2 activation of p[14C]adenosine formation by approx. 75%, in both the vascular and interstitial compartments. Intramyocytic concentrations of allosteric regulators of the cytosolic 5'-nucleotidases were evaluated in stop-frozen myocardium. Excess magnesium did not appreciably alter intracellular pH and ATP concentration, but lowered free cytosolic ADP and AMP concentrations by 50 and 70%, respectively. A simplified model of compartmentalized adenosine metabolism is proposed in which magnesium ion-activated cardiac purine release originates predominantly from the ecto 5'-nucleotidase; magnesium ion stimulation of metabolic flux through the cytosolic isoforms was constrained by concomitant reductions in intracellular AMP substrate and allosteric activator ADP. Magnesium ion-enhanced adenosine formation by 5'-nucleotidase could contribute to the known cardioprotective effects of this clinically used cation.

Myocardial stunning: a therapeutic conundrum.

Dobutamine and pyruvate are two inotropic agents with different mechanisms of action. Although both agents alter postischemic myocardial dysfunction, their potential metabolic effects in the setting of in vivo myocardial stunning have not been addressed. In this study, the effects of dobutamine and pyruvate on systolic wall thickening, myocardial phosphorylation potential index, interstitial fluid adenosine level, and myocardial oxygen consumption in in vivo stunned porcine myocardium were assessed. Stunning was induced with a 10-minute occlusion of the left anterior descending coronary artery. After 30 minutes of reperfusion, pigs were treated with either intravenous dobutamine (10 micrograms/kg per minute) or intracoronary pyruvate (1 ml/min, 150 mmol/L solution, pH 7.4). Infusion of both agents resulted in a marked improvement in regional systolic wall thickening. The dobutamine effect, however, produced a marked increase in myocardial oxygen consumption and was associated with an increase in interstitial adenosine caused by myocardial de-energization, because the myocardial phosphorylation potential index ratio decreased from 0.17 +/- 0.02 to 0.09 +/- 0.02 (p < 0.05). In contrast, pyruvate enhanced myocardial energy status, because the myocardial phosphorylation potential index ratio increased from 0.20 +/- 0.03 to 0.55 +/- 0.08 (p < 0.01). These experimental findings suggest that under certain circumstances the use of beta-receptor agonists to treat myocardial stunning may be suboptimal, if not undesirable. Further investigation is warranted to determine the optimum therapy for the stunned heart.

Oxygen consumption and cardiovascular function in children during profound intraoperative normovolemic hemodilution.

The clinically acceptable limit of acute normovolemic, normothermic hemodilution, a standard procedure in scoliosis surgery, is not yet well defined. Eight ASA class I patients undergoing idiopathic scoliosis correction were administered a standard anesthetic with 100% oxygen and controlled ventilation. Hemodilution was accomplished by exchanging whole blood for 5% albumin in 0.9% saline. Blood gases, acid-base status, and circulatory variables were recorded prior to and after hemodilution, and every 30 min throughout surgery. The impact of hemodilution was judged by mixed venous oxygen saturation which was maintained at > or = 60%, while intravascular volume was maintained with the 5% albumin solution. Reinfusion of the autologous blood was completed by the end of surgery. In the eight controlled cases in which normovolemic hemodilution was studied, hemoglobin levels decreased from 10.0 +/- 1.6 g/dL to 3.0 +/- 0.8 g/dL. Mixed venous oxygen saturation decreased from 90.8% +/- 5.4% to 72.3% +/- 7.8%. Oxygen extraction ratio increased from 17.3% +/- 6.2% to 44.4% +/- 5.9%. Oxygen delivery decreased from 532.1 +/- 138.1 mL.min-1.m-2 to 260.2 +/- 57.1 mL.min-1.m-2, while global oxygen consumption did not decrease and plasma lactate did not appreciably increase. Central venous pressure increased and peripheral resistance decreased during hemodilution. Cardiac index increased, heart rate remained essentially constant, and left ventricular stroke work index did not decrease significantly. No patients suffered clinically adverse outcomes. Global oxygen transport and myocardial work can be maintained at extreme normovolemic anemia. Our evidence suggests that stages of normovolemic hemodilution more severe than previously reported may be clinically acceptable for young, healthy patients during normocarbic anesthesia.

Intermittent v continuous ischemia decelerates adenylate breakdown and prevents norepinephrine release in reperfused rabbit heart.

Myocardium tolerates intermittent ischemia followed by short reperfusions better than continuous ischemia of the same duration. We attempted to delineate the differential mechanism(s) involved in intermittent v continuous ischemia. Isolated, paced rabbit hearts were perfused at 22 ml/min. Coronary flow was stopped intermittently 12 x for 2 or 4 min, with 3-min reperfusions (total reperfusion period: 36 min). In two other groups, flow was stopped continuously for 24 or 36 min followed by a flat 36-min reperfusion. Following the first intermittent 2-min ischemia, adenosine efflux increased ninefold; in all subsequent ischemia/reperfusion cycles, adenosine and total purine releases were substantially less despite identical heart rates, coronary flows and ischemic periods. The rate-pressure product prior to the intermittent ischemias exhibited exponential correlations with total purine efflux during the 3 min of reperfusion. When intermittent ischemia was extended to 4 min, the initial attenuation of ATP breakdown during the prior 2-min occlusions was overcome, but during subsequent 4-min ischemia/reperfusion cycles, ATP breakdown was again attenuated relative to the first 4-min ischemia. After the prolonged continuous ischemias, purine efflux was up to 6 x higher than with intermittent ischemias of the same total time of zero flow. Lactate release and hence cellular H+ export after intermittent ischemias remained consistently elevated, but following the continuous ischemia of 36 min, release of lactate, and thus H+, was subsequentially decreased. Glycogen mobilization occurred regardless of the ischemia's nature, but it was markedly enhanced during continuous ischemias, where no fall in proglycogen levels occurred. Similarly, myocardial norepinephrine release increased substantially only during the prolonged continuous ischemias. Thus short intermittent ischemia attenuates cardiac adenylate degradation and glycogen mobilization; such ischemic insult also provides for better lactate and H+ washouts immediately upon reperfusion. Another beneficial effect of intermittent ischemia was the near-complete absence of free interstitial norepinephrine, which exacerbates myocardial ischemic insults. In addition, the exponential correlations between preischemic rate-pressure product and postischemic purine release suggest that preischemic energy demand may determine ATP breakdown in ischemic rabbit myocardium.

Protection by pyruvate against inhibition of Na+, K(+)-ATPase by a free radical generating system containing t-butylhydroperoxide.

Global tissue damage due to oxygen-derived free radicals has been implicated in several pathological processes including exposure to ionizing radiation, and postischemic reperfusion of the heart or kidney. Recently pyruvate, a hydroperoxide scavenger, has been shown to protect against functional damage during postischemic reperfusion of the heart and in acute renal failure. In the present study, pyruvate was found to protect against inactivation of partially purified guinea pig renal and rat cardiac Na+,K(+)-ATPase which occurred when microsomal membranes were assayed for 1 hr at 37 degrees C (pH 7.5) in the presence of a free radical generating system (FRGS) containing 0.3 mM t-butylhydroperoxide and horseradish peroxidase. The presence of the FRG system inhibited the guinea pig renal Na+,K(+)-ATPase activity by 48.2 +/- 4.8% (N = 10, P < .05) and the presence of 0.2 to 20 mM pyruvate partially protected the Na+,K(+)-ATPase. At 5 mM pyruvate Na+,K(+)-ATPase was inhibited by only 18.8 +/- 2.5% (N = 10, P < .05) but increasing the pyruvate concentration gave no further protection. Equimolar concentrations of glucose, mannitol or lactate were without effect. The protection appeared to require an alpha-keto acid since alpha- but not beta-ketoglutarate was also effective and the mechanism is most probably the scavenging of t-BHO2. The results of the present study therefore support the hypothesis that, if free radical damage to native Na+,K(+)-ATPase does contribute to global tissue injury in certain pathological processes, pyruvate, in addition to being a powerful metabolic effector of recovery, may also protect against oxidative damage.