Biomedical subjects
M L Entman
Publications and source records attributed to M L Entman.
Glutathione disulfide formation and lipid peroxidation during cardiac ischemia and reflow in the dog in vivo.
The content of glutathione disulfide (GSSG) in tissue, coronary sinus blood plasma, and in cardiac lymph was measured in a well-characterized model of regional cardiac ischemia and reflow in dogs in vivo in order to assess the magnitude of the oxidant stress produced. No increase in GSSG content was observed during 60 min of occlusion of the circumflex or left anterior descending arteries, or during up to 70 min of reflow. The contents of 11-, 12-, and 15-hydroxyeicosatetraenoates (HETEs) in total lipids also were not increased following 60 min of regional ischemia and up to 60 min of reflow. In addition, global ischemia produced by aortic crossclamping and cardiopulmonary bypass did not increase HETE content. In contrast, infusion of tertiary butyl hydroperoxide (tBHP) into the left atrium produced readily measurable increases in GSSG content with or without prior induction of myocardial ischemia. Infusion of tBHP also increased tissue contents of the HETEs. These findings indicate that the canine myocardium subjected to ischemia-reflow conditions does not generate large amounts of reactive oxygen and does not form significant amounts lipid peroxidation products.
Nucleotide specificity of canine cardiac sarcoplasmic reticulum. Differential alteration of enzyme properties by detergent treatment.
We previously demonstrated that, in contrast to the hydrolysis of ATP, the hydrolysis of GTP by canine cardiac sarcoplasmic reticulum is not sensitive to calcium. Based on a variety of qualitative and quantitative considerations (cf. Tate, C. A., Bick, R. J., Chu, A., Van Winkle, W. B., and Entman, M. L. (1985) J. Biol. Chem. 260, 9618-9623), we suggested that the hydrolysis of ATP and GTP appears to be effected by the same enzyme. In the present paper, we examined the sensitivity of both enzymatic activities to low concentrations of detergent. With nonsolubilizing concentrations of the nonionic detergent, octaethylene glycol monododecyl ether, the hydrolysis of GTP was rendered partially calcium-sensitive resulting from a slightly increased total (Ca2+ + Mg2+)-GTPase activity and a markedly inhibited calcium-independent (Mg2+-dependent) GTPase activity. Calcium-dependent ATPase activity was increased with octaethylene glycol monododecyl ether, mimicking the effect of the ionophore, A23187. Calcium-dependent ATPase activity and detergent-induced calcium-dependent GTPase activity were similar in (a) calcium sensitivity, (b) sensitivity to mersalyl, and (c) pressure inactivation through dilution and centrifugation, all of which differed from the untreated calcium-independent GTPase activity. Calcium-dependent ATPase activity differed from calcium-dependent GTPase activity with (a) a higher nucleotide affinity, (b) a lower vanadate sensitivity, and (c) a calcium sensitivity for phosphoenzyme formation. Thus, the detergent-induced perturbation of the GTPase resulted in an enzyme with many characteristics qualitatively and quantitatively similar to the calcium ATPase.
Molecular basis of complement activation in ischemic myocardium: identification of specific molecules of mitochondrial origin that bind human C1q and fix complement.
Mitochondria may be a source of molecules that activate complement during ischemic injury to myocardium, providing therewith a stimulus for infiltration of polymorphonuclear leukocytes. To identify specific molecules that activate the classical complement pathway, detergent lysates of canine cardiac mitochondria were fractionated by polyacrylamide gel electrophoresis and transferred electrophoretically to nitrocellulose paper (NCP). The NCP replicas of the gels were incubated with isolated C1q and fresh sera as a source of complement, washed briefly, and overlaid with sensitized sheep erythrocytes (RBC) in agarose. A cluster of four to six molecules between 45 and 53 kDa as well as four others, 34, 30, 26, and 23 kDa, consumed complement thereby preventing complement-mediated lysis of sensitized sheep RBC in the agarose overlay. Additional molecules reactive with C1 were identified by their ability to bind isolated human C1q and to serve as assembly sites for later acting complement components. Sites of localization of complement were demonstrated by incubating NCP replicas of fractionated mitochondria with antisera specific for C1q, C3, C5, and C9, followed by peroxidase-conjugated anti-immunoglobulin and substrate. A total of 12 C1q binding molecules ranging in size from 67 kDa to 23 kDa, which can fix later acting complement components, were identified. At least two of these reacted with antisera prepared against canine cardiac lymph collected in the first 3-4 hours after a 45-minute coronary artery occlusion. These studies present direct evidence that specific molecules, released from subcellular fractions of myocardial cells rich in mitochondria, can activate the complement cascade.
Canine neutrophil activation by cardiac lymph obtained during reperfusion of ischemic myocardium.
Cardiac lymph from a canine model of myocardial ischemia and reperfusion was examined for evidence of chemotactic activity. Lymph was continuously collected from awake animals before and during a 60-minute coronary artery occlusion and up to 6 hours after the initiation of reperfusion. It was assessed for the ability to activate the following proinflammatory functions in neutrophils isolated from the blood of healthy dogs: 1) morphological changes characteristic of chemotactic stimulation, which were assessed by phase contrast microscopy, 2) orientation of canine neutrophils in a gradient of cardiac lymph, which was assessed in Zigmond chambers, 3) the binding of monoclonal antibodies reactive with CD11b and CD18 adherence glycoproteins, which was assessed by flow cytometry, and 4) adherence of canine neutrophils to monolayers of canine jugular vein endothelium, which was assessed in vitro by a visual assay. Lymph samples collected after 1 hour of reperfusion in animals demonstrating ECG evidence of ischemia and histological evidence of infarction exhibited significant stimulatory activity for each of the functions tested. Shape change-inducing activity was evaluated at more frequent intervals than other functions and was found to peak at 1 hour after initiation of reperfusion and to disappear by 6 hours. In addition, the CD11b/CD18 levels on neutrophils isolated from cardiac lymph collected during reperfusion were significantly greater than neutrophils obtained before or during occlusion. Animals that failed to exhibit evidence of infarction also failed to exhibit increased stimulatory activity in lymph collected during reperfusion, and surface levels of CD11b/CD18 on neutrophils collected from reperfusion lymph were not elevated. This study provides direct evidence supporting the hypothesis that chemotactic activity is generated in ischemic and reperfused myocardium.
An animal model to examine the response to environmental stress as a factor in sudden cardiac death.
Sudden cardiac death is the leading mode of death in adults in the United States. While it appears intimately associated with coronary artery disease, the factors that render some subjects vulnerable to sudden cardiac death, while others with the same coronary disease do not die suddenly, are unknown. An approach is described which considers that sudden cardiac death represents a separate syndrome of coronary artery disease within certain vulnerable subjects. It is suggested that the response to psychosocial stress in vulnerable subjects results in deleterious cerebral cortical influences on the autonomic control of the heart which render it more vulnerable to ischemia-induced ventricular fibrillation. Studies in the pig, an animal known to be susceptible to sudden death resulting from an environmental stress, demonstrate that cerebral cortical influences on autonomic centers play a central role in vulnerability to ventricular fibrillation after coronary artery occlusion. In this model, the incidence of ventricular fibrillation can be reduced by psychologic adaptation of the animal to his environment, cryoblockade of frontal cortical brain stem pathways and by central administration of propranolol. The relative role of adrenergic and cholinergic innervation of the heart is an important consideration.
Phospholipid fatty acyl chain asymmetry in the membrane bilayer of isolated skeletal muscle sarcoplasmic reticulum.
We previously showed [Herbette, L. G., Blasie, J. K., DeFoor, P., Fleischer, S., Bick, R. J., Van Winkle, W. B., Tate, C. A., & Entman, M. L. (1984) Arch. Biochem. Biophys. 234, 235-242; Herbette, L. G., DeFoor, P., Fleischer, S., Pascolini, D., Scarpa, A., & Blasie, J. K. (1985) Biochim. Biophys. Acta 817, 103-122] that the phospholipid head-group distribution in the membrane bilayer of isolated sarcoplasmic reticulum is asymmetric. From these studies, both the total number of phospholipid head groups and the total lipid, as well as the head-group species for these lipids, were found to be different for each monolayer of the membrane bilayer. In this paper, we demonstrate for the first time that there is significant asymmetry in the distribution of unsaturated fatty acids between the two monolayers; i.e., the outer monolayer of the sarcoplasmic reticulum contained more unsaturated and polyunsaturated chains when compared to the inner monolayer. X-ray diffraction measurements demonstrated that the time-averaged fatty acyl chain extension for the outer monolayer was approximately 20% less than for the inner monolayer. This is consistent with the concept that the greater degree of unsaturation in the outer monolayer may provide for a decreased average fatty acyl chain extension for that layer. This architecture for the bilayer may be related to both the "resting" state mass distribution of the calcium pump protein within the membrane bilayer and possible "conformational" states of the calcium pump protein during calcium transport by the sarcoplasmic reticulum.
G-protein distribution in canine cardiac sarcoplasmic reticulum and sarcolemma: comparison to rabbit skeletal muscle membranes and to brain and erythrocyte G-proteins.
Herein we describe the distribution of G-proteins in canine cardiac sarcolemma (SL) and sarcoplasmic reticulum (SR) and in rabbit skeletal muscle SL, T-tubules, and junctional and longitudinal SR in comparison to G-proteins of human erythrocyte and bovine brain. G-proteins were unequivocally present in cardiac SL and SR and in skeletal T-tubules. Both cardiac fractions had two substrates specifically ADP-ribosylated by cholera toxin migrating on a sodium dodecyl sulfate-polyacrylamide gel at about 42 and 45 kDa. In skeletal muscle membranes, cholera toxi-labeled substrates migrated at about 42 and 62 kDa. Three substrates for pertussis toxin were resolved by sodium dodecyl sulfate/urea-polyacrylamide gel electrophoresis in cardiac SL at about 38, 40, and 43 kDa. Only the two higher molecular weight substrates were detected in cardiac SR and in any of several skeletal muscle membrane fractions. Comparison of G-proteins in muscle membrane fractions with G-proteins isolated from bovine brain and human erythrocyte as well as their reaction with antisera to either a common sequence of alpha subunits of G-proteins (G alpha common antibody) or to a unique sequence of the alpha subunit of Go (G alpha o antibody) indicated that the two lower molecular weight bands in cardiac SL are Go or Go-like, and therefore the upper band is probably Gi. These data demonstrate that pertussis toxin substrates are more heterogeneous than previously described and have implications for studies attempting to attribute physiological functions to G-protein isolates.
First-pass hepatic extraction and metabolic effects of insulin and insulin analogues.
First-pass hepatic extraction of insulin and hepatic and peripheral contributions to hypoglycemia were compared in conscious dogs during portal infusion of insulin A1, B29 diacetyl insulin, or A1-B29 dodecoyl insulin at 7 and 14 pmol X kg-1 X min-1. The liver removed 43 +/- 2% of insulin, 12 +/- 1% of dodecoyl, and 8 +/- 1% of diacetyl insulin, in a single transhepatic circulation. The hypoglycemia induced by insulin and diacetyl insulin and the ensuing glucagon response were greater than that produced by the dodecoyl analogue. Diacetyl insulin primarily increased glucose utilization, dodecoyl insulin solely inhibited hepatic production, and insulin affected both. The lack of hepatic effect of diacetyl insulin during hypoglycemia can be ascribed to greater counterregulation, because under euglycemic clamp conditions, this analogue caused suppression of glucose production. The different patterns of hypoglycemia exhibited can be explained by the combined effects of altered distribution between the liver and peripheral tissues caused by differences in hepatic extraction, the effect of this phenomenon on the counterregulatory response, and the intrinsic biological potency of the analogues.
Protection by verapamil of mitochondrial glutathione equilibrium and phospholipid changes during reperfusion of ischemic canine myocardium.
Pretreatment of the ischemic myocardium with verapamil protects against mitochondrial respiratory depression observed during ischemic arrest as well as during reperfusion. Since ischemic mitochondrial function appears not to be altered further by reperfusion, the purpose of this study is to identify a biochemical event affecting mitochondria that is specifically associated with reperfusion injury. It has been proposed that increased cellular Ca2+ influx and oxygen toxicity may result from reintroduction of coronary flow. Increased cytosolic Ca2+ is transmitted to the mitochondria with subsequent activation of Ca2+-dependent events, including phospholipase A2. Net production of lysophospholipids (and loss of total diacylphospholipids from the mitochondria) will proceed when reacylation mechanisms are inhibited. Since acyl-CoA:lysophospholipid acyltransferase is a sulfhydryl-sensitive enzyme and since increased activity of glutathione peroxidase shifts the levels of the mitochondrial sulfhydryl buffer, glutathione, towards oxidation, levels of glutathione and its oxidation state were measured during reperfusion in the absence or presence of verapamil pretreatment. Ischemia lowers total glutathione and reduces the redox ratio (reduced glutathione: oxidized glutathione) by 85%. Reperfusion partially returns the redox ratio to control by causing oxidized glutathione to disappear from the matrix. Verapamil maintains both the concentration and the redox potential of glutathione at control levels. Concomitant with alterations in reduced glutathione:oxidized glutathione is a decrease in ischemic mitochondrial phospholipid content. During reperfusion, phosphatidylethanolamine and its major constituent fatty acids (C 18:0 and C 20:4) are specifically lost from the mitochondrial membrane. Accompanying the significant loss of arachidonic acid during reperfusion is the decreased content of 11-OH, 12-OH, and 15-OH arachidonate. These lipid peroxidation products are not increased in ischemia. It is proposed that oxidation of matrix glutathione to glutathione disulfide during ischemia results in formation of glutathione-protein mixed disulfides and inhibition of sulfhydryl-sensitive proteins, including acyl-CoA lysophosphatide acyltransferase. Thus, metabolic events occurring within the ischemic period set the stage for prolonged dysfunction during reperfusion.
Electron spin resonance studies on the spin label of rat cardiac mitochondria treated with adriamycin.
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No evidence for reactive oxygen damage in ischemia-reflow injury.
The present studies show that ischemia and reperfusion cause severe injury without evidence of generation of large amounts of reactive oxygen. Calculations show that it is necessary that amounts of reactive oxygen several orders of magnitude greater than that measured during ischemia-reflow injury be generated intracellularly by redox-cycling compounds such as diquat or by direct oxidants such as t-butyl hydroperoxide before endogenous cellular antioxidant systems in ischemia-reperfused rat liver or canine heart are overwhelmed. Thus, our data are incompatible with the hypothesis that oxygen radicals cause ischemia-reflow injury by lipid peroxidation or tissue thiol oxidation as postulated in Figure 1. How might one reconcile these results with the important experiments documenting protection against ischemia-reflow injury by treatment with antioxidants, antioxidant enzymes, allopurinol, cyclooxygenase/lipoxygenase inhibitors, cobra venom factor, and antisera against leukocytes (Table 1)? It seems most likely that the small amounts of reactive oxygen that can be generated in a reduced, acidic environment, such as occurs in tissue ischemia, could only be playing a catalytic function and not producing a massive oxidative attack on tissue membranes. Thus, future studies might best be directed at the catalytic function of superoxide/hydrogen peroxide in modulating the availability of substances such as lipoxygenase and cyclooxygenase products and of endothelium-derived relaxing factor during ischemia-reflow injury.
Factors that determine the occurrence of arrhythmias during acute myocardial ischemia.
To elucidate the determinants of arrhythmias during acute myocardial ischemia, a 25-minute coronary artery occlusion was performed in 101 open-chest dogs. Heart rate correlated positively with the number of premature ventricular complexes (PVCs) and the occurrence of ventricular tachycardia (VT) and fibrillation (VF). The size of the occluded coronary bed was positively correlated with the occurrence of VF, but exhibited only a weak association with PVCs and VT. Coronary collateral flow exhibited a strong negative correlation with all forms of arrhythmias. Importantly, a sensitive coupling was present, whereby small differences in flow were associated with large differences in rhythm disorders. VT occurred in all ranges of occluded bed size examined, but within any given range, its incidence was inversely related to collateral flow. Thus, VT is relatively independent of occluded bed size, and is determined primarily by the degree of myocardial hypoperfusion. In contrast, even in the presence of low flows, VF did not develop with occluded beds less than 28% of left ventricular mass; above this critical occluded bed size, the incidence of VF was inversely related to collateral perfusion. VF is therefore determined by the association of a large occluded bed with a poor collateral function. This study identifies and systematically analyzes three major determinants of arrhythmias during acute myocardial ischemia: the heart rate, the amount of ischemic myocardium, and the severity of myocardial flow reduction. The finding that these factors account for most of the variability in the occurrence of malignant tachyarrhythmias has important methodological and clinical implications.
Roussel award for cardiology. The mechanism of nucleotide induced calcium translocation across sarcoplasmic reticulum membranes: evidence for a non-translocated intermediate pool of calcium.
Our previous data suggested that, in cardiac muscle sarcoplasmic reticulum fragments, GTP hydrolysis occurs by an alternative enzyme cycle of the Ca2+ ATPase which is insensitive to (Ca2+) and does not involve an acyl phosphate intermediate. Despite this, GTP induces the incorporation of calcium into a membrane pool that is not translocated to the vesicular lumen. The present study suggests that this GTP-induced intermediate calcium pool is identical to a modulable component of the calcium translocation process in that: it has an identical pH sensitivity; the initial incorporation of calcium in response to GTP eliminates the initial rapid burst and lag component of the typical ATP-induced calcium uptake curve when ATP is added during GTP-induced calcium accumulation. Instead, the addition of ATP during GTP-induced calcium accumulation results in the prompt onset of the linear phase of calcium translocation; GTP-induced calcium accumulation directly affects the pH sensitivity of subsequent ATP-induced calcium accumulation. We suggest that the intermediate calcium pool is in series with calcium translocation and is the site of the pH sensitivity observed in calcium flux.
Association of clathrin with microsomes isolated from canine myocardium.
We and others have observed specialized regions of sarcoplasmic reticulum membranes that resemble coated vesicles, in the I-band region of myocardial cells. These structures have been named "corbular" sarcoplasmic reticulum, and are distinct in appearance from Golgi-associated coated vesicles, in that they are larger and contain a flocculent material that has been identified as calsequestrin. Whereas it has been suggested that these structures have a role in cardiac calcium metabolism, their function(s) and the molecular identity of the characteristic "bristle" coat remain unknown. Microsomes enriched in sarcoplasmic reticulum were prepared from canine ventricular muscle by Polytron homogenization in pH 6.5 buffer, followed by differential centrifugation. Protein was released by incubation in 50 mM Tris/HCl, pH 8, followed by centrifugation. We found these extracts to be enriched in a protein that was identical to brain clathrin in mobility on a Sepharose 4B gel filtration column, final position of the native protein following nondenaturing electrophoresis, relative mobility in denaturing (sodium dodecyl sulfate) electrophoresis on 6% and 7.5% gels, and antigenicity to anti-clathrin IgG. These findings confirmed the presence of clathrin triskelions in the cardiac microsome extract. On this basis, we suggest that clathrin may be a component of the electron dense "coat" of corbular sarcoplasmic reticulum.
Myocardial ischemia: platelet and thromboxane concentrations in cardiac lymph and the effects of ibuprofen and prostacyclin.
Blood platelets have been implicated in several mechanisms leading to and/or modifying myocardial ischemia. Cardiac lymph examination allows insight into the extracellular fluid that is in equilibrium with the capillary blood. In order to obtain an index of platelet activation during coronary artery events in the awake chronic animal, we wished to ascertain whether evaluation of cardiac lymph would detect changes in platelet activation resulting from a vascular occlusion. The study used conscious dogs in which cardiac lymph vessels had been previously cannulated by open-chest surgical protocol. The concentrations of immunoreactive thromboxane B2 and platelet counts were assessed in the cardiac lymph during the control period, the 10-60 minute occlusions, and the reperfusion periods. The same protocols were effected on another series of dogs after infusion of ibuprofen or prostacyclin. Initially, immunoreactive thromboxane B2 concentrations in the systemic blood and cardiac lymph were identical. A three-fold increase in immunoreactive thromboxane B2 concentrations occurred in untreated animals and was accompanied by a fall in platelet count in the lymph. The infusion of ibuprofen or prostacyclin, which inhibit platelet aggregation by different mechanisms, prevented both the decrease in platelets and the increase in immunoreactive thromboxane B2. In this study, intravascular events resulting from coronary occlusion invoke a rapid rise of immunoreactive thromboxane B2 in the extravascular fluid. A decrease in platelet escape into the extravascular compartment is interpreted as a result of intravascular aggregation promoting decreased platelet numbers. Thus, examination of continuously flowing cardiac lymph allows rapid detection of intravascular activation of platelets in the awake animal in the absence of surgical trauma.
Effect of dexamethasone on hepatic glucose and insulin metabolism after oral glucose in conscious dogs.
To examine whether hyperinsulinemia associated with glucocorticoid treatment results solely from hypersecretion of insulin or also involves altered fractional hepatic extraction, oral glucose (1 g/kg body wt) was administered to dogs with or without dexamethasone treatment (2 mg/d for 2 d). Dexamethasone significantly increased basal glucose and insulin concentrations in the portal vein, hepatic vein, and femoral artery, reduced basal fractional hepatic extraction of insulin from 43 +/- 4% to 22 +/- 4%, and, after oral glucose, increased retention by the liver of net glucose released into the portal system from 27 +/- 4% to 53 +/- 13%. Intraportal insulin infusion (1 and 2 mU/kg per min) after 7 d of dexamethasone treatment (2 mg/d) caused less suppression of endogenous glucose production, and less exogenous glucose was required to maintain an euglycemic clamp than in control animals. Dexamethasone treatment is associated with: decreased basal fractional hepatic insulin extraction contributing to hyperinsulinemia; and less suppression of endogenous glucose production and increase in peripheral uptake in response to insulin, but no reduction in net hepatic glucose uptake after oral glucose.
Evidence for increased membrane permeability of plasmalemmal vesicles from livers of phenobarbital-induced CCl4-intoxicated rats.
We have observed a marked increase in Ca2+ permeability of plasma membranes isolated from rats treated in vivo with CCl4 (2 ml/kg), after phenobarbital induction and overnight fast. Regulation of intracellular free Ca2+ is vital to cell viability and function, and the increased plasma membrane permeability, if representative of a change occurring in vivo, may be a critical biochemical determinant of CCl4-induced hepatic necrosis. Permeability to small cations of liver plasma membrane vesicles of control and CCl4-dosed rats was tested by two independent methods: 1) Ca2+ efflux after passive loading in 1 mM Ca2+, and 2) 86Rb+ uptake driven by valinomycin-induced K+ diffusion potential after 100 mM KCl-equilibrated vesicles were stripped of external K+ by cation exchange. Both indicated markedly increased permeability in plasma membranes after CCl4 in vivo. First order rate constants of biphasic Ca2+ efflux were 0.272 and 0.0516 min-1 for controls and 1.78 and 0.171 min-1 for vesicles from CCl4-treated animals. 86Rb+ uptake by CCl4 vesicles was 47% of control. Total calcium contents of plasma membranes (prepared in the absence of EGTA) by atomic absorption were 17.4 +/- 2.0 (control) and 10.9 +/- 1.2 (CCl4) nmol/mg of protein (means +/- SE, p less than 0.025). In correlation with altered biochemical function, we found 4-fold increases in the content of 11-, 12-, and 15-hydroxyeicosatetraenoic acids in plasma membranes of CCl4-treated rats. Although these specific oxidized fatty acids are unlikely to be ionophores, the ionophoretic properties of certain other oxygenated polyunsaturated fatty acids suggest a mechanism whereby accumulation of lipid oxidation products may be responsible for the altered membrane permeability we have observed after CCl4, and perhaps ultimately for cell death in CCl4-induced hepatic necrosis.