Requirement for hexose, unrelated to energy provision, in T-cell-mediated cytolysis at the lethal hit stage.

The requirement for D-glucose in T-cell-mediated cytolysis was studied using mouse spleen cells sensitized against alloantigens in vitro. Glucose was required for cytolysis: (a) cytolysis proceeded in a simple buffered salt solution containing Ca++ and Mg++ (low phosphate-buffered saline, LPBS) in the presence but not in the absence of added glucose; (b) 2-deoxy-D-glucose blocked cytolysis. The block by this agent was overcome by excess glucose added as late as 40 min after the inhibitor. This block was not due to inhibition of NADP reduction, since 2-deoxy-D-glucose failed to interfere with the rate of CO2 production by the pentose cycle which we found to be of significant activity in sensitized spleen cells; (c) dialyzed fetal bovine serum (DFBS) in LPBS supported cytolysis in the absence of added glucose. However, 2-deoxy-D-glucose was also inhibitory under these conditions, suggesting that carbohydrate was required here as well. Further results supported the conclusion that DFBS was not acting as a direct source of the required carbohydrate. The relationship between cytolysis, glucose requirement, and provision of energy was studied. As little as 0.1 mM D-glucose in LPBS supported cytolysis. At this glucose concentration, there was no measurable accumulation of lactate in sensitized spleen cells, but Krebs cycle activity was detectable. In 3 mM glucose or above, the range covered by standard tissue culture media, anaerobic glycolysis became a major source of energy in sensitized spleen cells. Consequently, it appears that in standard tissue culture medium, effector cells can generate sufficient energy for cytolysis either by aerobic or anaerobic metabolism. However, the addition of an energy source alone in the absence of glucose was insufficient to support cytolysis in LPBS. Pyruvate in LPBS did not support cytolysis but was shown to be a good substrate for aerobic metabolism in sensitized spleen cells. Glycogenic amino acids and glycerol also failed to support cytolysis. The stage of cytolysis at which glucose is required was investigated. Glucose was necessary for the calcium-dependent lethal hit phase, but not for the cytochalasin A-blockable recognition stage, nor for 51Cr release from injured target cells. Models for the lethal hit process are discussed, which are compatible with the observed requirement for certain hexoses unrelated to their capacity to serve as sources of energy.

Lack of dynein arms in immotile human spermatozoa.

Sermatozoa from two brothers who are not twins were found to be straight and immotile. Examinations of the sperm showed that oxygen consumption and lactic acid production were normal; viability tests showed that the percentage of dead sperm was not increased. The ultrastructural appearance of the sperm tail was normal except for a complete lack of dynein arms and some irregularities in the arrangement of the accessory fibers and the longitudinal columns of the fibrous sheath. The mitochondrial apparatus and the sperm head conform to the conventional model. According to the sliding-filament hypothesis first proposed by Afzelius (1959. J. Biophys. Biochem. Cytol. 5:269.), the arms are responsible for the bending movements of the tail. The simplest explanation for the simultaneous lack of arms and sperm motility appears to be that the two brothers have a genetic disorder involving production, assembly, or attachment of the dynein arms.

Interaction of fatty acid and glucose oxidation by cultured heart cells.

Chick embryo heart cells in tissue culture actively oxidize [1-(14)C]palmitate to (14)CO(2). Fatty acid oxidation by cell monolayers was linear with time and increasing protein concentration. The addition of carnitine to the assay medium resulted in a 30-70% increase in the rate of fatty acid oxidation. The specific activity of palmitic acid oxidation did not change significantly with time in culture and was also the same in rapidly proliferating and density-inhibited cell cultures. Addition of unlabeled glucose to the assay medium resulted in a 50% decrease in (14)CO(2) production from [1-(14)C]palmitate. Conversely, palmitate had a similar sparing effect on [(14)C]glucose oxidation to (14)CO(2). Lactate production accounted for most of the glucose depleted from the medium and was not inhibited by the presence of palmitate in the assay. Thus, the sparing action of the fatty acids on glucose oxidation appears to be at the mitochondrial level. The results indicate that although chick heart cells in culture are primarily anaerobic, they can oxidize fatty acid actively.

Evidence for a detrimental effect of bicarbonate therapy in hypoxic lactic acidosis.

Lactic acidosis, a clinical syndrome caused by the accumulation of lactic acid, is characterized by lactate concentration in blood greater than 5 mM. Therapy usually consists of intravenous sodium bicarbonate (NaHCO3), but resultant mortality is greater than 60 percent. The metabolic and systemic effects of NaHCO3 therapy of hypoxic lactic acidosis in dogs were studied and compared to the effects of sodium chloride or no therapy. Sodium bicarbonate elevated blood lactate concentrations to a greater extent than did either sodium chloride or no treatment. Despite the infusion of NaHCO3, both arterial pH and bicarbonate concentration decreased by a similar amount in all three groups of dogs. Additional detrimental effects of NaHCO3 were observed on the cardiovascular system, including decreases in cardiac output and blood pressure that were not observed with either sodium chloride or no treatment. Thus there is evidence for a harmful effect of NaHCO3 in the treatment of hypoxic lactic acidosis.

Intracellular edema and dehydration: effects on energy metabolism in alveolar macrophages.

The effects of intracellular edema and dehydration on energy metabolism in alveolar macrophages were studied. Intracellular edema increased lactate production and reversibly decreased oxygen consumption. Dehydration caused no significant change in lactate production but irreversibly decreased oxygen consumption. These phenomena may be applicable to a wide variety of clinical problems including lung and brain edema.

Ribosome-catalyzed polyester formation.

Deamination of phenylalanyl-transfer RNA with nitrous acid yields the alpha-hydroxyacyl analog, phenyllactyl-transfer RNA. When this is incubated in a protein-synthesizing system directed by polyuridylic acid, it yields an acid-precipitable, alkali-labile polyester of phenyllactic acid.

Leukocyte oxidase: defective activity in chronic granulomatous disease.

The intact leukocytes of two children with chronic granulomatous disease fail to reduce nitroblue tetrazolium during phagocytosis. This is due to defective operation of an oxidase of reduced nicotinamide adenine dinucleotide that is insensitive to cyanide and that indirectly stimulates the oxidation of glucose-6-phosphate in leukocytes. Such leukocytes undergo no increase in oxygen consumption or in activity of the hexose monophosphate shunt during phagocytosis, although lactate production is normal. The addition of nitroblue tetrazolium to a leukocyte suspension appears to provide a sensitive diagnostic screening test for this disease.

Cytochalasin B: inhibition of D-2-deoxyglucose transport into leukocytes and fibroblasts.

Cytochalasin B inhibits transport of (D)-2-deoxyglucose and of glucosamine into kukocytes, but does not impair uptake of kucine by these cells. This inhibitory action is rapid and reversible, and results in suppression of glycolysis. Cytochalasin B also blocks transport of (D)-2-deoxyglucose, but not of leucine, into mouse L strain fibroblasts.

A physiological approach to the assessment of disease activity in rheumatoid arthritis.

A method is described for assessing the in vivo oxygen consumption and lactate production rates of human knee joints. It is based on the rate of fall of P(o2) and the rate of rise in lactate concentration in an intra-articular saline pool after interruption of the circulation to the joint with an arterial tourniquet. Studies in 5 control patients with degenerative joint disease and 29 patients with rheumatoid arthritis showed a 2- to 3-fold higher mean oxygen uptake rate and a 10- to 12-fold higher mean lactate appearance rate in the saline in the rheumatoid joints with severe disease compared to the control joints. These metabolic variables correlated with tissue metabolic demand as estimated in synovial biopsies. (133)Xe washout from the intra-articular space, which reflects joint circulatory flow, showed a 3-fold greater mean washout rate from the rheumatoid joints (48 studies) than control joints (7 studies) with extensive overlap between the two groups. (133)Xe washout rate correlated with knee joint inflammation estimated both clinically and histologically. After synovectomy in four patients, the operated knee showed a greater fall in lactate production than the opposite knee in three of these patients. Neither knee joint oxygen uptake nor (133)Xe washout rate changed significantly. Intra-articular corticosteroid injection (eight patients) resulted in decreased lactate production and a decreased (133)Xe washout rate in the injected knee and variable results in the untreated knee. Oxygen uptake again was unchanged after therapy.

Studies of the metabolic activity of leukocytes from patients with a genetic abnormality of phagocytic function.

Polymorphonuclear leukocytes from patients with chronic granulomatous disease respond to the phagocytosis of latex particles with normal increments in glucose consumption, lactate production, Krebs' cycle activity, and lipid turnover. The leukocytes of these patients fail to show normal increments in respiration, direct oxidation of glucose, and hydrogen peroxide formation during particle uptake. It appears that the stimulation of respiration with the formation of hydrogen peroxide and stimulation of the direct oxidative pathway of glucose metabolism are closely linked to degranulation and intracellular killing of bacteria by polymorphonuclear leukocytes.

Enzymatic degradation of uric acid by uricase-loaded human erythrocytes.

Erythrocytes containing pig liver uricase have been prepared by hypotonic hemolysis in the presence of the enzyme. Uricase is shown to be active within the erythrocytes and to degrade uric acid as rapidly as it enters the cells when high intracellular enzyme concentrations are employed. The kinetics and characteristics of uric acid entry are shown to be the same for hemolysed and normal erythrocytes. At physiological concentrations of uric acid, loaded erythrocytes can degrade a maximum of about 21 mumol uric acid/liter erythrocytes per min. The possible application of enzyme-loaded erythrocytes to medicine is discussed.

Altered membrane sodium transport in Bartter's syndrome.

To explore the possibility that Bartter's syndrome is the manifestation of an inherited abnormality of sodium transport, we have measured various parameters of sodium transport in erythrocytes from patients with Bartter's syndrome, their siblings, and their parents. Sodium transport in six of the eight patients with Bartter's syndrome differed significantly from that in the other two patients. On the basis of this difference, the patients were divided into two groups (type I and type II). In the six type I patients, fractional sodium outflux (0.38+/-0.05/hr [SD]) was significantly less than normal (0.50+/-0.07) and erythrocyte sodium concentration (9.48+/-0.84 mmoles/liter cells per hr) was significantly greater than normal (5.24+/-0.66). In the two type II patients, none of the measured parameters of sodium transport differed significantly from normal. Erythrocyte sodium transport in the relatives of three type I patients was altered in a way similar to that in the type I patients and was significantly different from that in the relatives of a type II patient. These findings indicate the presence of inherited alterations of erythrocyte sodium transport in certain patients with Bartter's syndrome.

Morphology and metabolism of an aortic intima-media preparation in which an intact endothelium is preserved.

An in vitro preparation of rabbit aortic "intima-media" previously shown to exhibit stable rates of respiration and glucose metabolism and the high rate of aerobic glycolysis considered characteristic of the metabolism of this tissue was subjected to electron microscopic examination. In samples examined immediately after the aortae were dissected free of adipose tissue and adventitia, under conditions similar to those now in common use, marked and widespread alterations in endothelial cell structure were present, including loss of cell integrity. The vascular smooth muscle cells retained a normal electron microscopic (EM) appearance. During subsequent incubation with 5 mM glucose in Krebs-Ringer bicarbonate (KRB), pH 7.4, under the conditions usually employed in studies of this preparation, large zones of the luminal surface were rapidly denuded of endothelium, and the remaining endothelial cells exhibited a wide range of ultrastructural alterations. The smooth muscle cells, however, continued to maintain a normal EM appearance. A method was developed to prepare segments of rabbit aortic intima-media which retained an intact layer of endothelium resembling that observed in tissue fixed in situ. During a 1-h incubation with 5 mM glucose in KRB, pH 7.4, gas phase 5% CO2/95% O2, containing 6% bovine serum albumin, the intact aortic intima-media preparation retains an essentially unmodified EM appearance and exhibits linear rates of respiration. Under these conditions the intact aortic intima-media preparation exhibits significantly higher rates of O2 uptake and glucose uptake than those observed in our previous preparation or in other reported aortic intima-media preparations. The intact aortic intima-media does not exhibit the high rate of aerobic glycolysis during in vitro incubation that has been considered characteristic of the metabolism of rabbit, rat, and swine aortic intima-media. In addition, the magnitude of the Pasteur effect was far greater than that observed in other aortic intima-media preparations. The data suggest that component cells of the aortic intima-media may derive a major fraction of their energy requirements from respiration; they raise further questions concerning the significance of the high rate of aerobic glycolysis observed when aortic intima-media preparations are incubated in vitro, and they suggest that documentation of the EM appearance of the endothelium in such preparations is desirable.

Impaired carbohydrate metabolism of polymorphonuclear leukocytes in glycogen storage disease Ib.

This study measures hexose monophosphate (HMP) shunt activity, glycolytic rate, and glucose transport in PMN and lymphocytes of patients with glycogen storage disease (GSD) type Ib as compared with controls and with GSD Ia patients. HMP shunt activity and glycolysis were significantly lower in intact PMN cells of GSD Ib patients as compared with GSD Ia patients and with controls. These activities were above normal levels in disrupted GSD Ib PMN. HMP shunt activity and glycolytic rates in lymphocytes were similar in all three groups studied. The rate of 2-deoxyglucose transport into GSD Ib PMN was 30% of that into cells of normal controls. In GSD Ib lymphocytes or in GSD Ia PMN and lymphocytes transport was normal. The striking limitation of glucose transport across the cell membrane of the PMN of GSD Ib patients may account for the impairment of leukocyte function that is characteristic of GSD Ib, but not found in GSD Ia patients.

Glycogenesis and glyconeogenesis in human platelets. Incorporation of glucose, pyruvate, and citrate into platelet glycogen; glycogen synthetase and fructose-1,6-diphosphatase activity.

Washed human platelets are capable of depositing 1-4 as well as probable 1-6 glucosyl linkages onto preexistent glycogen primer. They are also capable of degrading (glycogenolysis) newly synthesized 1-4 as well as probable 1-6 glucosyl linkages. A higher rate of glycogen synthesis was found in platelet suspensions containing lower concentrations of platelets. This was shown to result from decreased glycogen degradation and consequent increased residual glycogen primer in low platelet suspensions. The increased glycogen content of low platelet suspensions was not a result of platelet washing, removal of platelets from plasma, or release of platelet metabolites into the media. The enzyme glycogen synthetase was found to be present at a rate of 5.2 mumoles of uridine diphosphate (UDP) glucose incorporated into glycogen per gram platelets per hour at 37 degrees C. The K(m) for UDP glucose was 6.6 mmoles/liter. At optimum concentration of glucose 6-phosphate, the K(m) was reduced 4.6 fold and V(max) was increased 4.3-fold. Human platelets contain the glyconeogenic pathway. They incorporate pyruvate-(14)C and citrate-(14)C into platelet glycogen and contain an apparent fructose-1,6-diphosphatase. The apparent fructose-1,6-diphosphatase was activated by adenosine monophosphate (AMP) and adenosine diphosphate (ADP), inhibited by adenosine triphosphate (ATP), and shown to be rate limiting for glyconeogenesis at physiologic concentration of adenine nucleotide.

Anaerobic performance and metabolism of the hyperthyroid heart.

Anaerobically periused hearts from rats with experimentally induced hyperthyroidism exhibited accelerated deterioration of pacemaker activity and ventricular performance. The diminished anaerobic performance of hyperthyroid hearts was associated with decreased adenosine triphosphate (ATP) levels and a reduced rate of anaerobic glycolysis as reflected in decreased lactic acid production during 30 min of anoxic perfusion.Studies on whole heart homogenates demonstrated inhibition at the phosphofructokinase (PFK) step of the glycolytic pathway. Such inhibition was not demonstrated in the hyperthyroid heart cytosol. It is postulated that an inhibitor of PFK which resides dominantly in the particulate fraction is probably responsible for the diminished anaerobic glycolysis and performance of the hyperthyroid heart.

Simultaneous measurements of lactate turnover rate and umbilical lactate uptake in the fetal lamb.

Lactic acid represents a major exogenous nutrient for the developing fetal lamb in utero. Our study was undertaken (a) to quantitate the net consumption of lactate by the fetus, (b) to quantitate the net lactate production and metabolism by the placenta, and (c) to compare the net fetal lactate consumption with fetal lactate use, measured simultaneously with radioactive tracers. 14 pregnant sheep were prepared with catheters in the maternal femoral artery and uterine vein and in the fetal aorta and umbilical vein. By simultaneous application of the Fick principle to the uterine and umbilical circulations, placental glucose consumption and placental lactate production were rapid, averaging 39.8 +/- 5.1 and 11.8 +/- 0.7 mg.min-1. Net lactate umbilical uptake averaged 1.95 +/- 0.16 mg-1.kg.min-1. During infusion of L-[14C(U)]lactate, fetal lactate turnover was much more rapid, averaging 6.5 +/- 0.8 mg.kg-1.min-1, and lactate utilization within the anatomic fetus was 5.9 +/- 0.7 mg.kg-1.min-1. During infusion of tracer glucose, endogenous fetal lactate production from glucose and nonglucose substrates averaged 3.0 and 1.5 mg.kg-1.min-1, respectively. The present studies have quantitated under well oxygenated, steady-state conditions, the rapid placental metabolism and production of lactate, the net fetal consumption of lactate, and the rapid endogenous fetal lactate production from glucose and nonglucose substrates.

The effect of pressure or flow stress on right ventricular protein synthesis in the face of constant and restricted coronary perfusion.

Cardiac stress produced by hypertension or excess volume loading results in different types of hypertrophy. Elevated left ventricular pressure rapidly results in increased myocardial protein synthesis in vivo and in vitro, but such rapid alterations are not consistently seen in volume loading. The difference in response is difficult to clarify since it is not possible to effect alterations in left ventricular pressure or perfusion without profoundly affecting coronary perfusion. The present study describes cardiac protein synthesis in the right ventricle of the young guinea pig heart in vitro by utilizing a perfusion model in which the right ventricle could be stressed by elevations of pressure or volume loading in the presence of constant and restricted coronary perfusion. With coronary flow maintained at 4 ml/min per heart equivalent to 25 ml/min/g dry wt, an increase in right ventricular pressure from normal levels of 3 mm Hg to 11 mm Hg resulted in a 60 percent increase of myocardial incorporation of (14C)lysine into protein. However, with further increases of right ventricular pressure to 22 mm Hg, protein synthesis dropped back to normal levels. The falloff in protein synthesis was not due to decreased contractility, alterations in intracellular lysine pool specific activity, or alterations in distribution of coronary flow. a 60 percent increase in coronary perfusion was again associated with a similar response of protein synthesis to progressive elevations of pressure despite a rise in the ATP levels and a fall in lactate production. Thus, a deficiency of O2 did not entirely explain the decline of protein synthesis with maximal pressures. At all levels of coronary perfusion, volume loading for 3 h did not result in increased protein incorporation of (14C)lysine. The studies support a relationship between ventricular pressure and protein synthesis unrelated to coronary flow per se. A pressure receptor triggering protein synthesis within the ventricular wall is postulated. Such a relationship is not apparent in short-term volume loading in vitro.

Energy metabolism in human erythrocytes. II. Effects of glucose depletion.

Normal red cells were incubated in the absence of glucose to develop a system in which total adenosine triphosphate (ATP) turnover could be assessed. After 1 hr, the triose pool had been completely consumed. Thereafter, the metabolism of 2,3-diphosphoglycerate (DPG) to pyruvate and lactate was the sole significant source of ATP synthesis.10(-3)M CuCl(2), which did not enter the cells, diminished ATP utilization by more than 50%. This could be only partially attributed to the inhibition by copper of residual acylation and cation pumping, which were already reduced by glucose depletion. Other membrane enzymes, which presumably function in the maintenance of membrane integrity, must, therefore, use a significant portion of erythrocyte ATP. The behavior of glucose-depleted red cells with respect to cation transport was complex. The addition of ouabain did not decrease ATP utilization in these red cells. Ouabain inhibitable potassium influx was nearly normal after triose depletion, but total potassium influx was decreased. In contrast, the ouabain inhibitable sodium efflux was markedly reduced after triose depletion, although the concentration of ATP was 70% of normal. The dissociation of monovalent cation pumping suggests that the energy for active sodium transport is derived from a specific source (such as the ATP produced by the phosphoglycerate kinase reaction) distinct from that for potassium transport.

Heterogeneity of human platelets. I. Metabolic and kinetic evidence suggestive of young and old platelets.

Human platelets have been separated into two extreme density populations by centrifugation in specific density media. A large-heavy platelet population with specific gravity > 1.055 and a light-small population with specific gravity < 1.046 were obtained, each representing approximately 15-20% of the total population volume. The average volume per platelet of the separated large-heavy and light-small platelet populations was 12 and 5 mu(3) respectively. When data are expressed per milliliter platelets or per gram wet weight, the large-heavy platelet population had a 2-fold greater glycogen content, 1.3-fold greater orthophosphate content, 1.3-fold greater total adenine nucleotide content, 4.2-fold greater rate of glycogenolysis, 2.6-fold greater rate of glycolysis, 2.9-fold greater rate of protein synthesis, and 5.7-fold greater rate of glycogen synthesis. Significant differences were not obtained with respect to total lipid content or total lipid synthesis. The large-heavy platelet had a 2.5-fold greater resistance to osmotic shock as measured by adenosine triphosphate (ATP) or adenosine diphosphate (ADP) release. These data, as well as diisopropyl fluorophosphate (DFP(32)) survival curves in rabbits, indicate that large-heavy platelets have a greater metabolic potential and suggest that they may be the young platelets which progress with age to light-small platelets with a diminished metabolic potential.