Isotopic differences in the lithium transport rate in human erythrocytes during simultaneous incubations with the stable isotopes 6Li and 7Li.

The membrane transport of the two stable lithium isotopes, 6Li and 7Li, by erythrocytes has been studied using a dual channel atomic absorption spectroscopic technique. 6Li appears to be taken up preferentially to 7Li, in the ratio of 10 to 40%, depending on the concentration of total lithium and on the lithium isotopic ratio in the external medium.     

Cytotoxic ether phospholipids. Different affinities to lysophosphocholine acyltransferases in sensitive and resistant cells

Alkyllysophospholipids (ALP) which are 1-O-alkyl analogs of the cell membrane component 1-acyl-sn-glycero-3-phosphocholine (1-acyl-GPC) represent a family of new antitumor drugs. Susceptibility of cells to ALP is correlated to a selective inhibition of fatty acid incorporation into 1,2-diacyl-sn-glycero-3-phosphocholine in intact cells. This report examines oleoyl-CoA-1-acyl-GPC acyl-transferase activities in cell-free systems of ALP-sensitive methylcholanthrene-induced fibrosarcoma cells (MethA cells) and ALP-resistant bone marrow-derived murine macrophages (BMM phi). The specific activities for the oleoyl-CoA-1-acyl-GPC acyltransferases were 1.05 +/- 0.06 nmol X mg-1 X min-1 and 2.98 +/- 0.27 nmol X mg-1 X min-1, respectively. The kinetic parameters for 1-palmitoyl-GPC were Km = 16.6 microM, Vmax = 4.3 nmol X mg-1 X min-1 (BMM phi) and Km = 7.6 microM, Vmax = 2.0 nmol X mg-1 X min-1 (MethA cells). In the presence of 1-O-octadecyl-2-O-methyl racemic glycero-3-phosphocholine (ET-18-OCH3), one of the most potent cytotoxic ALP, the acyltransferase was dose dependently inhibited in MethA cells with a 50% inhibition concentration at 40 micrograms/ml. The BMM phi-acyltransferase was not affected up to 80 micrograms of ET-18-OCH3/ml. The kinetic parameters (Km' = 15.4 microM, Vmax' = 2.2 nmol X mg-1 X min-1) suggest that ET-18-OCH3 is a competitive inhibitor in MethA cells. Inhibitor constants for ET-18-OCH3, calculated from Dixon plots, were found to be 423 microM (BMM phi) and 13 microM (MethA cells) indicating a 33-fold larger affinity of ET-18-OCH3 to the MethA cells than to the BMM phi acyltransferase. From these data we assume that the inhibition of oleic acid incorporation into cellular phosphocholine during the antineoplastic action of ALP may be due to different affinities of the inhibitor to the 1-acyl-GPC acyltransferases in different cell types.     

Calcium modulation of inositol 1,4,5-trisphosphate-induced calcium release from neuroblastoma x glioma hybrid (NG108-15) microsomes

Subcellular fractions of neuroblastoma x glioma (NG108-15) hybrid cells were used to study the mechanism of inositol 1,4,5-trisphosphate-induced calcium release. A microsomal fraction, enriched in endoplasmic reticulum and plasma membranes and almost devoid of mitochondria, was the most active in inositol trisphosphate- or GTP-dependent release of calcium. Neither GTP nor inositol 1,4,5-trisphosphate affected the calcium efflux mediated by the other reagent, suggesting that inositol trisphosphate and GTP act on different calcium-sequestrating vesicles. The stimulation of calcium release by GTP was relatively slow (t1/2 = 90 s), dependent on polyethyleneglycol, and greater at 2 X 10(-5) M calcium (5 nmol X min-1 X mg-1) than at 10(-6) M calcium (0.8 nmol X min-1 X mg-1). The inositol trisphosphate-induced calcium efflux was not mimicked by inositol monophosphate; it was fast (t1/2 less than 10 s) and unaffected by 3% polyethyleneglycol. The amount of calcium released by inositol trisphosphate was greatest at 10(-6) M external calcium (1 nmol X min-1 X mg-1) and it was undetectable at 2 X 10(-5) M calcium. A feedback inhibition of the inositol trisphosphate-induced calcium release by cytoplasmic calcium provides a safety mechanism preventing deleterious effects of abnormally high calcium levels.     

Purification and characterization of S-adenosylhomocysteine hydrolase from mouse mastocytoma P-815 cells. Evidence for cell cycle-specific fluctuation of the enzyme activity

S-Adenosylhomocysteine hydrolase [EC 3.3.1.1] was purified to electrophoretic homogeneity from mastocytoma P-815 cells. The purified enzyme had a molecular weight of 190,000, as estimated by Sephadex G-200 chromatography, and a monomer molecular weight of 45,000, as determined by polyacrylamide gel electrophoresis in the presence of SDS. The Km value for adenosine was 0.29 microM and the Vmax value 4.5 mumol S-adenosylhomocysteine X min-1 X mg-1 in the synthetic reaction, while the Km value for S-adenosylhomocysteine was 0.77 microM and the Vmax 0.48 mumol adenosine X min-1 X mg-1 in the hydrolytic reaction. The purified enzyme also had one binding site for adenosine (KD = 2.61 X 10(-7) M) and one for cAMP (KD = 1.6 X 10(-7) M). Using rabbit antiserum raised against the purified enzyme, it was shown that the enzyme activity and enzyme synthesis fluctuated during the cell cycle of mastocytoma cells, reaching the maximum levels as the cells changed from the G1/S phase to the G2 phase.     

Glucose turnover and hormonal changes during insulin-induced hypoglycemia in trained humans

Eight athletes (T), studied the third morning after the last exercise session, and seven sedentary males (C) (maximal O2 consumption 65 +/- 4 vs. 49 +/- 4 (SE) ml X kg-1 X min-1, for T and C men, respectively) had insulin infused until plasma glucose, at an insulin level of 1,600 pmol X l-1, was 1.9 mmol X l-1. Glucose turnover was determined by primed constant rate infusion of 3-[3H]glucose. Basal C-peptide (0.46 +/- 0.04 vs. 0.73 +/- 0.06 pmol X ml-1) and glucagon (4 +/- 0.4 vs. 10 +/- 2 pmol X l-1) were lower (P less than 0.05) and epinephrine higher (0.30 +/- 0.06 vs. 0.09 +/- 0.03 nmol X l-1) in T than in C subjects. During and after insulin infusion production, disappearance and clearance of glucose changed identically in T and C subjects. However, in spite of identical plasma glucose concentrations, epinephrine (7.88 +/- 0.99 vs. 3.97 +/- 0.40 nmol X l-1), growth hormone (97 +/- 17 vs. 64 +/- 6 mU X l-1), and pancreatic polypeptide (361 +/- 84 vs. 180 +/- 29 pmol X l-1) reached higher levels (P less than 0.05) and glucagon (28 +/- 3 vs. 47 +/- 10 pmol X l-1) lower levels in T than in C subjects. Blood pressures changed earlier in athletes during insulin infusion, and early recovery of heart rate, free fatty acid, and glycerol was faster. Responses of norepinephrine, cortisol, C-peptide, and lactate were similar in the two groups. Training radically changes hormonal responses but not glucose kinetics in insulin hypoglycemia.     

Production of superoxide and activity of superoxide dismutase in rabbit epididymal spermatozoa

Mature rabbit spermatozoa from the cauda epididymidis suspended in potassium Tris phosphate buffer at 24 degrees C produced O2.-, as measured by reduction of acetylated ferricytochrome c, with an intrinsic rate of 0.20 nmol/min per 10(8) cells. This rate increased to 1.80 nmol/min per 10(8) cells in the presence of 10 mM cyanide. These spermatozoa contain 2.8 units per 10(8) cells of superoxide dismutase activity, 95% of which is sensitive, and 5% of which is insensitive, to cyanide inhibition. These activities correspond to the cytosolic Cu-Zn form and the mitochondrial Mn form of the dismutase, respectively. Only the cyanide-sensitive form is released from the sperm on hypo-osmotic treatment or sonication. Hypo-osmotically treated rabbit epididymal spermatozoa produced O2.- with an intrinsic rate of 0.24 nmol/min per 10(8) cells, which increased to 0.58 nmol/min per 10(8) cells in the presence of 10 mM cyanide. Both intact and hypo-osmotically treated cells react with O2.- in a second order reaction as inferred from the hyperbolic dependence on cell concentration of O2.- production rate in both the absence and presence of cyanide. The second order rate constant for this reaction with intact cells, kS, was calculated to be 22.9 X 10(-8) (cells/ml)-1 min-1 in its absence. For hypo-osmotically treated cells, the values of kS were 10.8 X 10(-8) (cells/ml)-1 min-1 and 8.2 X 10(-8) (cells/ml) -1 min-1, respectively. Since hypo-osmotically treated cells have lost much of their plasma membrane, the lower value of kS for the treated cells implies that this membrane is one site of reaction of O2.- with the cells. The increase in kS in the presence of cyanide, which inhibits superoxide dismutase and so increases O2.- production, suggests that the cells become more reactive with O2.- as its production rate increase, as would be expected for the occurrence of radical chain oxidation. This in turn suggests that superoxide dismutase plays a major role in protecting rabbit sperm against damage from lipid peroxidation.     

Effect of organic contamination upon microbial distributions and heterotrophic uptake in a Cape Cod, Mass., aquifer.

Bacterial abundance, distribution, and heterotrophic uptake in a freshwater aquifer contaminated by treated sewage were determined from analyses of groundwater and sediment-core samples. The number of free-living (unattached) bacteria in contaminated groundwater declined steadily with increasing distance from the source of sewage infiltration, from 1.94 (+/- 0.20) X 10(6) ml-1 at 0.21 km to 0.25 (+/- 0.02) X 10(6) ml-1 at 0.97 km. Bacterial abundance in groundwater sampled at 0.31 km correlated strongly with specific conductance and increased sharply from 4.0 (+/- 0.3) X 10(4) ml-1 at a depth of 6 m to 1.58 (+/- 0.12) X 10(6) ml-1 at 14 m, then declined at 20 and 31 m to 1.29 (+/- 0.12) X 10(6) and 0.96 (+/- 0.12) X 10(6) ml-1, respectively. A majority of the bacteria in contaminated and uncontaminated zones of the aquifer were bound to the surfaces of particulates, less than 60 micron in diameter. The glucose uptake rate, assayed at in situ and 5 microM concentrations, declined steadily in contaminated groundwater sampled along a transect. A preparative wet-sieving technique for use in processing core samples for bacterial enumeration is described and evaluated.     

Hepatic antioxidant-sensitive respiration. Effect of ethanol, iron and mitochondrial uncoupling.

The addition of the antioxidants (+)-cyanidanol-3, butylated hydroxyanisole and ascorbate to the perfused rat liver resulted in a decrease in the rate of oxygen consumption. This basal antioxidant-sensitive respiration of 110-130nmol X min-1 X (g of liver)-1 represents 5-7% of total respiration. Increased antioxidant-sensitive respiratory rates are found after the infusion of increasing concentrations of ethanol (1.8-72.2mM) or iron (35.5-248.5 microM). This respiratory component exhibits a dependence on ethanol or iron concentration, with maximal rates of 200-255 and 330nmol X min-1 X (g of liver)-1 respectively. After the addition of 100 microM-2,4-dinitriphenol, an antioxidant-sensitive respiratory component of 230nmol X min-1 X (g of liver)-1 is found, which is not observed at lower concentrations of the uncoupler (5-50 microM). The lack of effect of the antioxidants used on mitochondrial respiration [the preceding paper, Videla, Villena, Donoso, Giulivi & Boveris (1984) Biochem. J. 223, 879-883] and on the glycolytic rate of the perfused liver suggests that the basal and chemically induced antioxidant-sensitive respiration observed are related to oxygen required for one-electron transfer reactions associated with the generation of active species of oxygen and lipid peroxidation in the liver cell.     

The role of glutathione and glutathione S-transferases in fatty acid ozonide detoxification

The ozonide derived from methyl linoleate was shown to cause a dose dependent inhibition of the phagocytosis of rat alveolar macrophages exposed in vitro to concentrations varying from 10(-5) to 10(-4) M. Vitamin C was demonstrated to detoxify the ozonide. In analogy to their behaviour on exposure to ozone, vitamin E supplemented cells demonstrated a decreased and glutathione depleted cells an increased sensitivity towards the compound. The characteristics of antioxidant protection of cells against the ozonide were thus comparable to those for protection against ozone. Preincubation with glutathione also detoxified the ozonide model compound. Survival of rat alveolar macrophages exposed to a toxic concentration of the ozonide (86 microM final concentration), measured by phagocytosis of the cells, increased significantly (P less than 0.01) from 23 to 54% after a 2.5-h preincubation of the ozonide with glutathione (5 mM final concentration). The detoxification of methyl linoleate ozonide by glutathione could be catalyzed by the rat liver glutathione S-transferases. After a 2.5-h preincubation of the ozonide (86 microM final concentration) with glutathione and glutathione S-transferases (final concentrations, respectively, 5 mM and 0.01 mg/ml), its toxicity was completely abolished, as demonstrated by the 98% survival (P less than 0.001) of subsequently exposed cells. A Km(app) (at 1 mM glutathione) for the ozonide of 0.80 mM and a Vmax(app) (at pH 6.5) of 94 nmol glutathione converted X min-1 X mg protein-1 or (at pH 7.4) of 34 nmol glutathione converted X min-1 X mg protein-1, were found. This glutathione S-transferase catalyzed detoxification of the potential intermediates in ozone induced cell damage, offers a new viewpoint on the role of glutathione in the protection of cells against ozone.     

Degradation of the 34 amino acid gastrin by rat tissue homogenates

Extracts of rat kidney contain an enzyme (gastrinase) that is highly specific for degradation of the 34 amino acid gastrin (G34). The Michaelis constant (Km) for kidney is 0.36 +/- 0.04 microM and the Vmax is 9.5 +/- 2.4 nmol X g-1 X min-1. Extracts of liver and brain also have gastrin degrading activity but the enzymes responsible appear to be different from the kidney gastrinase. Km for the liver enzyme is 0.08 +/- 0.02 microM but its Vmax (0.10 +/- 0.02 nmol X g-1 X min-1) is only 1% of the kidney gastrinase; Km for the brain enzyme is 0.10 +/- 0.03 microM but its Vmax (0.023 +/- 0.007 nmol X g-1 X min-1) is even lower than for the liver enzyme. The liver and brain enzymes appear to be less specific than the kidney enzyme with respect to competitive inhibition by insulin and glucagon. Cholecystokinin octapeptide is less inhibitory than the other peptides even though it shares a common C-terminal pentapeptide with G34. These findings are consistent with in vivo studies which have demonstrated that the dog kidney is an important site for extraction and degradation of endogenous dog gastrin but there is little or no hepatic removal of G34.     

Differential Uptake of Lithium Isotopes by Rat Cerebral Cortex and Its Effect on Inositol Phosphate Metabolism

Twenty hours following the subcutaneous administration of 5 mEq/kg doses of 6LiCl and 7LiCl to two groups of rats, the cerebral cortex molar ratio of 6Li+/7Li+ is 1.5. The effects of the lithium isotopes on cortex myo-inositol and myo-inositol-l-phosphate levels are the same as we have reported earlier: a Li+ concentration-dependent lowering of myo-inositol and increase in myo-inositol-1-phosphate. Thus 6LiCl, when administered at the same dose as 7LiCl, produces the larger effect on inositol metabolism. When the 6LiCl and 7LiCl doses were adjusted to 5 mEq/kg and 7 mEq/kg, respectively, the cortical lithium myo-inositol and myo-inositol-1-phosphate levels of each group of animals became approximately equal, suggesting that the isotope effect occurs at the level of tissue uptake, but not on inositol phosphate metabolism. The inhibition of myo-inositol-1-phosphatase by the two lithium isotopes in vitro showed no differential effect. The isotope effect on cerebral cortex uptake of lithium is in the same direction as that reported by others for erythrocytes and for the CSF/plasma ratio, but of larger magnitude.     

Differences in the effect of arachidonic acid on polymorphonuclear and mononuclear leukocyte function

Incubation of human polymorphonuclear leukocytes with arachidonic acid resulted in a stimulation of the oxidative metabolism of the cells. Upon stimulation with 80 microM arachidonic acid, neutrophils (5 X 10(6) cells/ml) produced superoxide (53 +/- 8 nmol/5 X 10(6) cells per 15 min), generated chemiluminescence (1211 100 +/- 157 000 cpm) and consumed oxygen (20 +/- 1 nmol/10(6) cells per 5 min). The stimulation of the cell metabolism could be reduced 40-60% by prior incubation of the cells with 10 microM indomethacin. Incubating polymorphonuclear leukocytes with arachidonic acid also resulted in a diminished chemotaxis towards an attractant, a decreased uptake of opsonized staphylococci and aggregation of the cells. This may be due to inhibitory products of arachidonic acid metabolism and toxic oxygen species produced during stimulated oxidative metabolism. The effects of arachidonic acid are specific for neutrophils, as mononuclear phagocytes only produced 17 +/- 8 nmol superoxide/5 X 10(6) cells per 15 min and generated 27 000 +/- 15 000 cpm chemiluminescence when stimulated with 80 microM arachidonic acid. When monocytes and neutrophils were stimulated with particles such as opsonized staphylococci, the amount of superoxide produced, oxygen consumed and chemiluminescence generated were similar. The phagocytic activity of the monocytes was also not affected by prior incubation with arachidonic acid. We conclude that in contrast to monocytes, neutrophil metabolism can be stimulated with arachidonic acid and this stimulation resulted in a decreased phagocytic activity of these cells.     

The effect of Li+ on Na-Ca exchange in cardiac sarcolemmal vesicles

The effects of Li+ on Na-Ca exchange in bovine cardiac sarcolemmal vesicles were examined. The initial rate of Na(+)-dependent Ca2+ uptake and efflux was inhibited by Li+ in a dose dependent manner. The initial rate of Na(+)-dependent Ca2+ uptake was inhibited 49.8 +/- 2.9% (S.E.) (n = 6) in the presence of Li+ compared to activity in external K+ or choline+. Kinetic analysis indicated that Li+ increased the Km for Ca2+ (96.3 microM) compared to K+ and choline+ (25.5 and 22.9 microM respectively) while Vmax (1.4, 1.2 and 1.1 nmol Ca2+/mg protein/sec respectively) remained unchanged. Li+ did not alter the experimentally derived stoichiometry of the exchange reaction of 3 Na+ for 1 Ca2+.     

Vasoconstrictor effects in vivo and plasma disappearance rate of neuropeptide Y in man

Vascular effects of neuropeptide Y (NPY) and noradrenaline (NA) were studied in six human volunteers. Systemic infusion of human NPY for 40 min (5 pmol X kg-1 X min-1) increased arterial plasma NPY-like immunoreactivity (NPY-LI) from 12 +/- 2 to 356 +/- 30 pM. This concentration caused no systemic cardiovascular effects. The disappearance curve for NPY-LI was biphasic; the slopes of the two phases corresponding to half lives of 4.1 +/- 0.4 and 20 +/- 2 min respectively. Close i.a. infusion of human NPY in the forearm caused a slowly developing and dose dependent decrease in forearm blood flow (FBF) and increase in venous tone with maximal values of 44 +/- 6 and 235 +/- 81% of control respectively at 5 nmol X min-1. The corresponding values for NA (5 nmol X min-1) were 21 +/- 9 and 489 +/- 78% of control. A threshold concentration for a decrease in FBF was obtained at a plasma NPY-LI of 3.7 +/- 0.6 nM. The decrease in FBF caused by NPY was maintained for a much longer period compared to that of NA.     

Pulmonary fibrin microembolism with Echis carinatus venom in dogs: effects of a synthetic thrombin inhibitor

We produced pulmonary fibrin microembolism using an infusion of a prothrombin activator (Echis carinatus venom, 30 min, 0.5 NIH thrombin equivalent units/kg) in open-chest mongrel dogs. To determine the nonclotting effects of this venom on edemagenesis we infused an irreversible thrombin inhibitor, D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (PPACK, 57 nmol X kg-1 X min-1 for 120 min), alone (n = 5) or with venom (Echis + PPACK, n = 5). The control group (n = 5) was given 1 ml of 0.9% NaCl. A decline in left atrial pressure (means +/- SE, 5.3 +/- 0.4 to 4.0 +/- 0.5 mmHg, P less than 0.05) and cardiac index (149 +/- 10 to 82 +/- 13 ml X min-1 X kg-1, P less than 0.01) in association with a marked increase in pulmonary arterial pressure (14.5 +/- 0.6 to 26.6 +/- 2.5 mmHg, P less than 0.001) and pulmonary vascular resistance (64 +/- 5 to 304 +/- 42 mmHg X ml-1 X min-1 X kg-1, P less than 0.001) was observed after 20 min of venom infusion. During this interval, pulmonary artery wedge pressure increased (4 +/- 1 to 12 +/- 4 mmHg, P less than 0.01) in four of eight animals. Fibrinogen declined below measurable levels and fibrin microemboli were seen in many pulmonary arterioles. These changes were not observed in the Echis + PPACK, PPACK, or control groups. Leukopenia and thrombocytopenia were observed in the Echis and Echis + PPACK groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Leukotriene C4 formation catalyzed by three distinct forms of human cytosolic glutathione transferase

The ability of three distinct types of human cytosolic glutathione transferase to catalyze the formation of leukotriene C4 from glutathione and leukotriene A4 has been demonstrated. The near-neutral transferase (mu) was the most efficient enzyme with Vmax= 180 nmol X min-1 X mg-1 and Km= 160 microM. The Vmax and Km values for the basic (alpha-epsilon) and the acidic (pi) transferases were 66 and 24 nmol X min-1 X mg-1 and 130 and 190 microM, respectively. The synthetic methyl ester derivative of leukotriene A4 was somewhat more active as a substrate for all the three forms of the enzyme.     

The effects of lithium isotopes on the myo-inositol 1-phosphatase reaction in rat brain, liver, and testes.

Enzyme inhibition studies were performed with several lithium isotopes in order to more precisely define how lithium inhibits the enzyme myo-inositol 1-phosphatase. This lithium-induced inhibition is thought to be central to the therapeutic effects of lithium in the treatment of manic-depressive disorder. Naturally occurring lithium (NLi) exists as a combination of isotopes: 6Li and 7Li. Lethality studies were performed comparing 6LiCl, 7LiCl, and NLiCl, did not demonstrate a differential effect as previous studies had suggested. Enzyme inhibition studies were performed with these individual lithium isotopes, and compared to the effects of the naturally occurring combination (NLi) on the inhibition of myo-inositol 1-phosphatase using a partially purified enzyme preparation from rat brain, liver and testes. Identical inhibition was observed with all lithium isotopes and their combinations. In addition, both D- and L-myo-inositol 1-phosphates were used as enzyme substrates and found to be equivalent. These experiments, along with previous work demonstrating lithium acting as an uncompetitive inhibitor in the reaction, and the lack of lithium binding sites on the enzyme, suggests the hypothesis that lithium is possibly inhibiting this reaction by interfering with the formation of a transition cyclic intermediate, myo-inositol 1,3-cyclic phosphate, which may be formed from either the D- or L-substrates. This proposal is in contrast to previous suggestions regarding the inhibitory mechanism of action of lithium on the myo-inositol 1-phosphatase reaction.     

Blood flow to contracting human muscles: influence of increased sympathetic activity

The purpose of this study was to examine the effects of the increased sympathetic activity elicited by the upright posture on blood flow to exercising human forearm muscles. Six subjects performed light and heavy rhythmic forearm exercise. Trials were conducted with the subjects supine and standing. Forearm blood flow (FBF, plethysmography) and skin blood flow (laser Doppler) were measured during brief pauses in the contractions. Arterial blood pressure and heart rate were also measured. During the first 6 min of light exercise, blood flow was similar in the supine and standing positions (approximately 15 ml.min-1.100 ml-1); from minutes 7 to 20 FBF was approximately 3-7 ml.min-1.100 ml-1 less in the standing position (P less than 0.05). When 5 min of heavy exercise immediately followed the light exercise, FBF was approximately 30-35 ml.min-1.100 ml-1 in the supine position. These values were approximately 8-12 ml.min-1.100 ml-1 greater than those observed in the upright position (P less than 0.05). When light exercise did not precede 8 min of heavy exercise, the blood flow at the end of minute 1 was similar in the supine and standing positions but was approximately 6-9 ml.min-1.100 ml-1 lower in the standing position during minutes 2-8. Heart rate was always approximately 10-20 beats higher in the upright position (P less than 0.05). Forearm skin blood flow and mean arterial pressure were similar in the two positions, indicating that the changes in FBF resulted from differences in the caliber of the resistance vessels in the forearm muscles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of a pentosan polysulphate upon thrombin and factor Xa inactivation by antithrombin III.

The kinetics of inhibition of human and bovine alpha-thrombin and human factor Xa by antithrombin III were examined under pseudo-first-order conditions as a function of the concentration of pentosan polysulphate [a fully sulphated (beta 1-4)-linked D-xylopyranose with a single laterally positioned 4-O-methyl-alpha-D-glucuronic acid]. Double-reciprocal plots of the observed first-order rate constant against concentration of pentosan polysulphate gave straight lines, intercepts on the axes giving values for maximum increase in second-order rate constant (by calculation) and apparent dissociation constant. These values were: for human alpha-thrombin 1.52 X 10(7) M-1 . min-1 and 3.6 microM respectively, for bovine alpha-thrombin 6.56 X 10(6) M-1 . min-1 and 0.16 microM and for factor Xa 6.86 X 106 M-1 . min-1 and 20 microM. In the presence of pentosan polysulphate the dissociation constant for the initial complex of antithrombin III and thrombin was shown to be reduced from approx. 2 X 10(-3) M to 61 X 10(-6) M without apparent change in the limiting rate constant of 750 min-1. An oligosaccharide (primarily 8-10 saccharide units) prepared from heparin and with high affinity for antithrombin III but low potency in the thrombin-antithrombin III interaction did not diminish the rate of interaction catalysed by pentosan polysulphate. The catalysis was shown to be due to a weak electrostatic interaction, since it was completely reversed by concentrations of NaCl greater than 0.3 M. It is concluded that the mechanism is independent of the heparin high-affinity binding site on antithrombin III and is probably due to binding of the high-charge-density polysaccharide to the proteinase. It is calculated that the acceleration in rate achieved, although lower than that of heparin, approaches that required to be of physiological significance and may be of importance in the anticoagulation role of antithrombin III at sites of high charge density which may occur in vivo.     

Effects of capillary red cell density on gas conductance of frog skin.

We tested experimentally the hypothesis that decreasing capillary red blood cell (RBC) density (dRBC) reduces the tissue diffusing capacity of frog skin to CO (DtiCO) and O2 (DtiO2). The effects of dRBC on CO2 transport were also assessed. C18O, O2, and CO2 transport between the skin and a cutaneous sample chamber on the belly of anesthetized (halothane) frogs (Rana pipiens) was measured by mass spectrometry, and the cutaneous conductances to C18O (GCO), O2 (GO2), and CO2 (GCO2) were calculated. The dRBC of the planar cutaneous capillary bed was measured by intravital fluorescent video microscopy. DtiCO and DtiO2 were calculated from a modification of the Roughton-Foster equation: 1/G = 1/Dti + 1/(theta RBC.dRBC), where theta RBC values were estimated from literature values. In one group of animals (n = 6), measurements were made before hemodilution (dRBC = 630 +/- 56 cells/mm2), after one hemodilution (dRBC = 349 +/- 50 cells/mm2), and after a second hemodilution (dRBC = 150 +/- 31 cells/mm2). In controls, time had no effect on GCO, GO2, or GCO2 (P greater than 0.42). Before hemodilution, GCO, GO2, and GCO2 were 0.069 +/- 0.010, 0.088 +/- 0.0012, and 1.23 +/- 0.010 nmol.min-1.Torr-1.cm-2, respectively, and lowering dRBC by hemodilution decreased all these parameters (P less than 0.025). The mean slopes of GCO, GO2, and GCO2 vs. dRBC were 6.0 +/- 1.3 x 10(-7), 7.2 +/- 2.3 x 10(-7), and 7.8 +/- 3.0 x 10(-6) nmol.min-1.Torr-1.RBC-1, respectively. Lowering dRBC also decreased DtiCO and DtiO2 (P less than 0.034). DtiCO and DtiO2 were 0.080 +/- 0.012 and 0.096 +/- 0.013 nmol.min-1.Torr-1.cm-2, respectively, before hemodilution. The mean slopes of DtiCO and DtiO2 vs. dRBC were 4.9 +/- 2.1 x 10(-7) and 6.5 +/- 2.8 x 10(-7) nmol.min-1.Torr-1.RBC-1, respectively. Hemodilution had no effect on perfused capillary density (P = 0.38). These results indicate that tissue diffusive conductance is proportional to dRBC. Regulation of dRBC may be an important mechanism modulating diffusive gas transport in tissue.