Reduction Of Intracellular ATP Research Articles

Impairment of integrin-mediated cell-matrix adhesion in oxidant-stressed PC12 cells

Oxidants are believed to play an important and complex role in neuronal injury and death in the aging process and various neurode generative diseases. We studied the effect of oxidative stress on integrin-mediated cell-extracellular matrix (ECM) interactions using the PC12 neuronal cell line. In assays in which attachment was measured between 30 and 90 min, addition of hydrogen peroxide (H 2O 2) to the attachment medium resulted in a dose-dependent inhibition of initial cell attachment to collagen. Addition of H 2O 2 also caused previously attached cells to detach from collagen. The inhibition by H 2O 2 was specific for integrin-mediated adhesion, since attachment to substrata coated with non-ECM molecules was much less affected. Exposure of cells to H 2O 2 resulted in a rapid and profound reduction of intracellular ATP, accompanied by only a slight increase in intracellular free Ca 2+ concentration ([Ca 2+] i). Treatment of cells with the microfilament-disrupting agent, cytochalasin B, like that with H 2O 2, inhibited cell adhesion to collagen. We propose that integrin-mediated cell adhesion, which requires interactions between cytoplasmic portions of integrin subunits and cytoskeletal microfilaments, is impaired by oxidative stress as a result of the depletion of intracellular ATP and that such depletion is an early event in the process of oxidant-induced neuronal injury.

Ca2+ and Mg-ATP activated potassium channels from rat pulmonary artery

We have observed a novel class of calcium-activated potassium channel which is activated by physiological levels of intracellular ATP. These KCa,ATP channels are found on smooth muscle cells isolated from the pulmonary artery. Since their activation by ATP is Mg2+ dependent and is poorly evoked by non- or slowly-hydrolyzed ATP analogues, we conclude that it involves phosphorylation. We suggest that in hypoxia a reduction of intracellular ATP may reduce KCa,ATP channel activity and thereby tend to depolarize the cells. This effect would increase Ca2+ entry through voltage-activated Ca2+ channels and contribute to vasoconstriction.

Energy depletion and autophagy. Cytochemical and biochemical studies in isolated rat hepatocytes

In this paper, data dealing with the sensitivity of autophagy towards partial ATP depletion in isolated rat hepatocytes are reviewed. Partial reduction of intracellular ATP causes: (1) a decrease of proteolytic flux; (2) a decrease in uptake of cytosolic components into the autophagic-lysosomal compartment; (3) either a decrease or no change in the ratio between volume densities of autophagosomes and lysosomes, depending on whether or not the cytosolic phosphate potential is affected; and (4) impairment of the lysosomal proton pump. It is concluded that the consecutive steps of autophagy all respond to relatively small changes of intracellular ATP concentration.

Role of cardiac ATP-regulated potassium channels in differential responses of endocardial and epicardial cells to ischemia.

Epicardial cells are more susceptible to the electrophysiological effects of ischemia than are endocardial cells. To explore the ionic basis for the differential electrophysiological responses to ischemia at the two sites, we used patch-clamp techniques to study the effects of ATP depletion on action potential duration and the ability of ATP-regulated K+ channels in single cells isolated from feline left ventricular endocardial and epicardial surfaces. During ATP depletion by treatment with 1 mM cyanide (CN-), shortening of action potential durations was significantly greater in epicardial cells than in endocardial cells. Thirty minutes after initiating exposure to 1 mM CN-, action potential duration at 90% repolarization was reduced to 0.70 +/- 0.12 of the control value for endocardial cells versus 0.39 +/- 0.18 for epicardial cells (p less than 0.01), and action potential duration at 20% repolarization was reduced to 0.72 +/- 0.13 for endocardial cells versus 0.12 +/- 0.09 for epicardial cells (p less than 0.01). In both endocardial and epicardial cells, the shortening of action potential by CN- treatment was partially reversed by 0.3 microM glibenclamide; the magnitude of reversal, however, was much greater in epicardial cells. After exposure to 1 mM CN-, the activity of ATP-regulated K+ channels in cell-attached membrane patches was significantly greater in epicardial cells than in endocardial cells. To study the dose-response relation between ATP concentration and open-state probability of the channels, intracellular surfaces of inside-out membrane patches containing ATP-regulated K+ channels were exposed to various concentrations of ATP (10-1,000 microM). The concentration of ATP that produced half-maximal inhibition of the channel was 23.6 +/- 21.9 microM in endocardial cells and 97.6 +/- 48.1 microM in epicardial cells (p less than 0.01). These data indicate that ATP-regulated K+ channels are activated by a smaller reduction in intracellular ATP in epicardial cells than in endocardial cells. The differential ATP sensitivity of ATP-regulated K+ channels in endocardial and epicardial cells may be responsible for the differential shortening in action potentials during ischemia at the two sites.

ADDITIVE INHIBITION OF AFFERENT AND EFFERENT IMMUNOLOGICAL RESPONSES OF HUMAN PERIPHERAL BLOOD MONONUCLEAR CELLS BY VERAPAMIL AND CYCLOSPORINE

We have studied the effects of verapamil (0-50 microM) on the in vitro immunological function of human peripheral blood mononuclear cells in the presence or absence of cyclosporine (0-600 ng/ml). The proliferative response to phytohemagglutinin, OKT3, and alloantigens, the generation of cytotoxic T lymphocytes following allogeneic stimulation, and mitogen-induced reduction of intracellular ATP were inhibited in a concentration-dependent fashion by verapamil alone and by cyclosporine alone. When the two drugs were added to the same culture, additive inhibition was observed. A verapamil concentration of 5 microM usually reduced by at least 50% the amount of cyclosporine necessary to cause the same level of inhibition seen when no verapamil was present. The additive inhibition of the two drugs was likely not due to additive inhibition of IL-2 responsiveness, since neither drug alone inhibited the response of an IL-2-dependent T cell clone (CTLL-2) to recombinant IL-2 except at the highest concentrations tested, where a mild additive effect was noted. Nor was the additive inhibition related to an additive effect on total IL-2 receptor expression since an additive inhibitory effect on PHA-induced IL-2 receptor expression was only seen with 50 microM verapamil, while additive functional effects on mitogen- and antigen-induced proliferation and alloantigen-induced CTL generation were seen with 5 microM verapamil doses. Verapamil or cyclosporine alone inhibited IL-2 production of PHA- and phorbol ester-stimulated peripheral blood mononuclear cells--however, no additive effect was seen when the two drugs were both added to culture, probably because of the very potent inhibition by cyclosporine alone. Natural killer cell activity of human peripheral blood mononuclear cells against K562 target cells was significantly inhibited by verapamil in a concentration-dependent fashion, while cyclosporine had a more modest concentration-dependent effect. The combination of both drugs demonstrated additive inhibition. Effector function of cytotoxic T lymphocytes was modestly inhibited by either verapamil or cyclosporine alone. A combination of the highest concentrations of verapamil and cyclosporine caused an additive inhibitory effect. In summary, these data demonstrate that verapamil and cyclosporine have concentration-dependent inhibitory activities on both the afferent and efferent limbs of immunity that were additive when verapamil was used in a concentration of at least 5 microM. The additive effects are probably not related to effects on IL-2 circuitry.

(Na+,K+)-cotransport in the Madin-Darby canine kidney cell line. Kinetic characterization of the interaction between Na+ and K+.

Confluent monolayer cultures of the differentiated kidney epithelial cell line, Madin-Darby canine kidney cells (MDCK), have been used to study ion transport mechanisms involved in transepithelial transport. We have investigated the previously reported K+-stimulation of 22Na+ uptake by confluent monolayers of Na+ depleted cells (Rindler, M. J., Taub, M., and Saier, M. H., Jr. (1979) J. Biol. Chem. 254, 11431-11439). This component of Na+ uptake was insensitive to ouabain and amiloride, but was strongly inhibited by furosemide or bumetanide. Ouabain-insensitive 86Rb+ uptake was also inhibitable by furosemide or bumetanide and stimulated by extracellular Na+. The synergistic effect of Na+ and 86Rb+ uptake and K+ on 22Na+ uptake was reflected by an increase in the apparent Vmax and a decrease in the apparent Km as the concentration of the other cation was increased. The extrapolated Km for either 86Rb+ or 22Na+ uptake in the absence of the other cation was 30 mM while the Km in the presence of a saturating concentration of the other cation was 9 mM. The absolute Vmax values for 22Na+ and 86Rb+ uptake suggest a cotransport system with a stoichiometry of 2Na+:3K+. However, because of the experimental design, the actual ratio may be closer to 1:1. Competition with, and stimulation by, a variety of unlabeled cations indicated that Na+ could be partially replaced by Li+, while K+ could be fully replaced by Rb+ and partially replaced by NH4+ and CS+. Uptake by this system was dependent upon cellular ATP. Reduction of intracellular ATP to 3% of normal abolished both K+-stimulated 22Na+ uptake and Na+-stimulated 86Rb+ uptake.

Inhibition of growth of Escherichia coli by lactose and other galactosides

A study has been made of the inhibition of growth caused by the addition of lactose or other galactosides to lac constitutive Escherichia coli growing in glycerol minimal medium. The effect was greater at pH 5.9 and pH 7.9 than at pH 7.0. Inhibition of growth by lactose was observed also in the case of a β-galactosidase negative mutant. However, a lac Y mutant, which has a defect in the entry of protons normally coupled with galactoside transport, showed only slight inhibition of growth on the addition of galactosides. In the case of the parental strain the addition of lactose resulted in a sharp fall in ΔpH across the cell membrane and a reduction in intracellular ATP, and the recovery was slow. Under the same conditions the lac Y mutant showed a smaller and only transient effect. It is postulated that the sudden entry of protons associated with lactose uptake lowers the protonmotive force, reducing the ATP levels and inhibiting growth of the cells. This hypothesis would account also for the selection of lac Y mutants found when E. coli is grown in the presence of isopropyl-β- d-thiogalactoside.

Biochemical factors influencing erythrocyte deformability and capillary entrance phenomena.

Evaluation of the effects of biochemical modification of erythrocytes by low pressure filtration through Nuclepore filters of 2.8 and 3.0 micrometer pore diameter and glass micropipettes of 2.7 and 3.0 micrometer internal diameter and channel length greater than 100 micrometer indicates the relative sensitivity of the micropipette in detection of reduction of cellular deformability. In dynamic studies of erythrocyte entrance behaviour on micropipettes, the significance of induced relative sphericity on cellular deformability was evident. Reduction of intracellular ATP, cellular pH, sphere induction by bifunctional membrane protein cross-linking reagents and lead caused diminution in cellular deformability, attributable to the more spherical shape. Similarly echinocyte formation by effects of aged plasma, oleate and senscence, and stomatocyte induction by chlorpromazine caused marked decrease in capillary entry of cells having flow velocities of approximately 100 micrometer/s in contrast inhibitors of the glucose carrier, of calmodulin and manipulated decrease in membrane lipid of bilayer fluidity did not cause detectable change in cellular deformability. These data emphasize the relative importance of erythrocytic shape as a determinant of cellular deformability and suggest that necessity for adequate control of shape change is necessary when examining effects of biochemical manipulations on membrane elasticity and cellular deformability. The preliminary date from capillary entrance experiment indicate the importance of dynamic experiments in in vitro studies of microcirculation.

Decreased insulin binding and degradation associated with depressed intracellular ATP content.

The effect of metabolic inhibitors, 2,4-dinitrophenol (DNP) and NaF, on insulin binding and degradation has been studied in cultured Buffalo rat liver (BRL) cells. In control studies, 1.8 fmol of 125I-insulin binds to 1.2 x 10(6) cells, possessing approximately 40,000 receptor sites per cell with binding affinity of 5.52 x 10(-8) M. When the cells were preincubated with increasing concentrations of either DNP or NaF, a dose- and time-dependent decrease in both insulin binding and degradation was observed. The total amount of 125I-insulin bound to BRL cells preincubated with metabolic inhibitors was reduced to 1.2 fmol per 1.2 x 10(6) cells. The point of 1/2 B max was achieved in the presence of 50 ng/ml of native insulin, 1.7 times that of the control level. The number of receptor sites was unaffected by either DNP or NaF, but an average affinity profile revealed a decrease in the affinity of the ATP-depleted cells for insulin (KD: 7.31 x 10(-8) M and 7.06 x 10(-8) M in DNP- and NaF-treated cells, respectively). The decrease in insulin binding and degradation following the exposure of the BRL cells to the metabolic inhibitors was associated with a 20% reduction in intracellular ATP and adenylate energy charge. DNP and NaF did not affect the equilibrium constant for the myokinase catalyzed reaction and the intracellular concentration of hypoxanthine was stable, confirming the integrity of the cells during the experiments. It is suggested that ATP levels must remain intact to maintain normal insulin receptor affinity. Furthermore, the rate of insulin degradation by ATP-depleted cells is slower than that of intact cells. It is conceivable that the depression of insulin degradation by partially ATP-depleted cells results from either diminished binding or decreased endocytosis and lysosomal activity, all of which appear to be energy dependent.