Prevention of oxidative damage to lens by caffeine.

PURPOSE: The primary objective of this study was to examine the possibility of inhibiting oxidative damage to the lens in vitro by caffeine. METHODS: Oxidative damage was inflicted by incubating mouse lenses in Tyrode medium containing 0.1 mM Fe(8)Br(8), an iron complex soluble in aqueous medium. Parallel incubations were conducted in the presence of caffeine (5 mM). RESULTS: Lenses incubated in the medium containing Fe(8)Br(8) undergo oxidative stress, as evidenced by the inhibition of Na(+)-K(+) ATPase-driven rubidium transport and the loss of tissue glutathione and ATP. These effects were prevented in presence of caffeine. That the effects are due to the oxyradicals produced was ascertained further by parallel studies with Tempol (5 mM), a well-known scavenger of reactive oxygen species (ROS) with its activity being more pronounced with hydroxyl radicals as compared to other ROS. CONCLUSIONS: Caffeine was found to be effective in preventing oxidative stress to the lens induced by iron under ambient conditions. The protective effect is attributable to its ability to scavenge ROS, particularly the hydroxyl radical.

[Il-2-regulated expression of Na+,K(+)-ATPase in activated human lymphocytes].

Expression of Na+,K(+)-ATPase alfal-subunit and of oubain-sensitive rubidium influxes has been investigated in human peripheral blood lymphocytes. Isolated lymphocytes were stimulated by phytogemagglutinin (PHA) or interleukin-2 (IL-2). It has been shown that during the early stage of the PHA-activation the alfal-subunit abundance in the membrane fractions of the human blood lymphocytes does not change, whereas at the late stages of Go-->G1-->S transition (16-48 h) the alfa1 protein content increases. A translation inhibitor cycloheximide was found to prevent the late increase in alfa1-subunit expression. An immunosuppressant cyclosporin A decreases both IL-2-dependent T-lymphocyte progression and alfa1-subunit abundance by 48 h of PHA-induced lymphocyte activation. In the lymphocytes pretreated by PHA in submitogenic concentration (0.8-1.0 microg/ml) exogenous IL-2 (100 U/ml) induces a proliferative response as well as alfal-protein accumulation. A decrease in alfa1-protein accumulation in the presence of specific inhibitors of separate signal transduction pathways enables us to conclude that protein kinases ERK1/2 (MAPK pathway) and JAK3 (JAK-STAT pathway) mediate the IL-2-dependent regulation of Na+,K(+)-ATPase expression during lymphocyte transition from resting stage to proliferation. A correlation between changes in ouabain-sensitive rubidium influxes and the alfal-subunit amount has been demonstrated. It is concluded that IL-2-dependent-progression of normal human lymphocytes from quiescence to proliferation is accompanied by the increase in Na+,K(+)-ATPase alfa1-subunits expression, and the enhanced transport activity of a sodium pump during the prereplicative stage is provided by the increased number of functional pump units in plasma membrane.

Sch-28080 depletes intracellular ATP selectively in mIMCD-3 cells.

Two H(+)-K(+)-ATPase isoforms are present in kidney: the gastric, highly sensitive to Sch-28080, and the colonic, partially sensitive to ouabain. Upregulation of Sch-28080-sensitive H(+)-K(+)-ATPase, or "gastric" H(+)-K(+)-ATPase, has been demonstrated in hypokalemic rat inner medullary collecting duct cells (IMCDs). Nevertheless, only colonic H(+)-K(+)-ATPase mRNA and protein abundance increase in this condition. This study was designed to determine whether Sch-28080 inhibits transporters other than the gastric H(+)-K(+)-ATPase. In the presence of bumetanide, Sch-28080 (200 microM) and ouabain (2 mM) inhibited (86)Rb(+) uptake (>90%). That (86)Rb(+) uptake was almost completely abolished by Sch-28080 indicates an effect of this agent on the Na(+)-K(+)-ATPase. ATPase assays in membranes, or lysed cells, demonstrated sensitivity to ouabain but not Sch-28080. Thus the inhibitory effect of Sch-28080 was dependent on cell integrity. (86)Rb(+)-uptake studies without bumetanide demonstrated that ouabain inhibited activity by only 50%. Addition of Sch-28080 (200 microM) blocked all residual activity. Intracellular ATP declined after Sch-28080 (200 microM) but recovered after removal of this agent. In conclusion, high concentrations of Sch-28080 inhibit K(+)-ATPase activity in mouse IMCD-3 (mIMCD-3) cells as a result of ATP depletion.

Muscarinic down-regulation of cAMP-stimulated potassium ion secretion by rabbit distal colon.

The sustained effects of the cholinergic agonist carbachol (CCh) on electrolyte transport across the isolated, short-circuited rabbit distal colon were examined in the absence and presence of secretagogue (di-butyryl cyclic-adenosine monophosphate, dB-cAMP). Steady-state, basal absorption of 22Na+, 42K+ (86Rb+), and 36Cl- were not significantly altered by addition of the CCh (10(-4) mmol/l) to the serosal reservoir. Stimulation with dB-cAMP (1.0 mmol/l, serosal) promoted K+ (or Rb+) and Cl- secretion across the colon, without significantly affecting the unidirectional or net fluxes of Na+. Serosal (but not mucosal) addition of CCh to dB-cAMP-stimulated tissues reduced the serosal to mucosal flux of Rb+ (J(Rb)SM) in a concentration-dependent manner with a half-maximum concentration approximately equal 5 micromol/l. Pretreatment with CCh (100 micromol/l, serosal) inhibited dB-cAMP-induced K+ secretion, but had no significant effect on the steady-state unidirectional fluxes of Na+ or Cl-. Serosal histamine (20 micromol/l) also inhibited J(Rb)SM in dB-cAMP-stimulated tissues. Serosal epinephrine (10 micromol/l) promoted a decrease in short-circuit current (Isc) and transepithelial potential (VT) that was mirrored by increases in J(Rb)SM. Both Isc, and VT became more positive and J(Rb)SM was reduced when CCh was added to the epinephrine-stimulated tissues. Serosal muscarine (50 micromol/l) mimicked the CCh-induced inhibition of J(Rb)SM, but serosal nicotine (50 micromol/l) had no effect. In atropine-treated tissues (1 micromol/l, serosal), CCh failed to block dB-cAMP-stimulated increases in J(Rb)SM. The inhibitory action of CCh was observed in tissues that had been pretreated with 50 micromol/l serosal hexamethonium (a ganglionic transmission blocker) or 2 micromol/l serosal tetrodotoxin (a voltage-gated Na+ channel blocker), indicating that the inhibitory action of this cholinergic agonist does not depend on remnant enteric neural pathways. Rubidium ion transport across confluent monolayers of T84 cells was similarly affected by dB-cAMP and CCh, supporting the notion that enteric neural pathways are not required. Serosal charybdotoxin (20 nmol/l) mitigated the inhibitory action of CCh on J(Rb)SM in dB-cAMP-stimulated tissues, suggesting a role for basolateral, Ca2+-dependent K+ channels in the actions of CCh. It is concluded that basolateral muscarinic receptors (and possibly other Ca2+-dependent receptor pathways) of secretory colonocytes mediate the down-regulation of potassium ion secretion by rabbit distal colon, possibly by increasing basolateral membrane K+ conductance.

Inhibition of Ca(2+)-dependent Cl- secretion in T84 cells: membrane target(s) of inhibition is agonist specific.

Previous studies have indicated that Ca(2+)-dependent Cl- secretion across monolayers of T84 epithelial cells is subject to a variety of negative influences that serve to limit the overall extent of secretion. However, the downstream membrane target(s) of these inhibitory influences had not been elucidated. In this study, nuclide efflux techniques were used to determine whether inhibition of Ca(2+)-dependent Cl- secretion induced by carbachol, inositol 3,4,5,6-tetrakisphosphate, epidermal growth factor, or insulin reflected actions at an apical Cl- conductance, a basolateral K+ conductance, or both. Pretreatment of T84 cell monolayers with carbachol or a cell-permeant analog of inositol 3,4,5,6-tetrakisphosphate reduced the ability of subsequently added thapsigargin to stimulate apical 125I-, but not basolateral 86Rb+, efflux. These data suggested an effect on an apical Cl- channel. Conversely, epidermal growth factor reduced Ca(2+)-stimulated 86Rb+ but not 125I- efflux, suggesting an effect of the growth factor on a K+ channel. Finally, insulin inhibited 125I- and 86Rb+ effluxes. Thus effects of agents that inhibit transepithelial Cl- secretion are also manifest at the level of transmembrane transport pathways. However, the precise nature of the membrane conductances targeted are agonist specific.

Phorbol esters stimulate and inhibit Cl- secretion by different mechanisms in a colonic cell line.

Phorbol 12-myristate 13-acetate (PMA) was found to increase both the short-circuit current (Isc) and the efflux of 125I- or 36Cl- in the colonic epithelial cell line HT-29.cl19A. Neither the PMA-provoked rise in Isc nor the stimulation of 125I- efflux was affected by the cyclooxygenase inhibitor indomethacin. The PMA-induced increase in Cl- efflux was not accompanied by a rise in adenosine 3',5'-cyclic monophosphate (cAMP) levels. A prolonged incubation with PMA (3 h), however, inhibited the PMA- and the cAMP-stimulated Isc by greater than 90%, whereas the cAMP-provoked 125I- and 36Cl- efflux was not inhibited. The long-term PMA treatment was found to inhibit the basal and cAMP-provoked 86Rb+ efflux by 65 +/- 9 and 86 +/- 7%, respectively. A 3-h incubation with PMA also strongly inhibited the Ca2+ ionophore A23187-induced increase in 86Rb+ efflux, whereas the A23187-stimulated 125I- efflux was only marginally inhibited. These data suggest that phorbol esters, presumably by activation of protein kinase C, can provoke Cl- secretion in HT-29.cl19A colonocytes independently of a prostaglandin- or cAMP-mediated pathway. Prolonged exposure to PMA, however, causes an inhibition of net electrogenic Cl- secretion by downregulation of the activity of K+ transporters.

Role of diacylglycerol in adrenergic-stimulated 86Rb uptake by proximal tubules.

We used rat proximal tubule fragments purified by Percoll centrifugation to examine the role of diacylglycerol (DAG) in noradrenergic-stimulated Na+ reabsorption. Tubular DAG concentration and ouabain-inhibitable 86Rb uptake increased within 30 s after adding norepinephrine (NE) and remained elevated for at least 5 min. NE (1 microM) increased DAG content 17% and ouabain-inhibitable 86Rb uptake 23%. Cirazoline-stimulated 86Rb uptake was not inhibited by BaCl, quinidine, or bumetanide (1-10 microM) or by the omission of HCO3- or Cl- from the medium, but it was completely inhibited by ouabain and furosemide. Oleoyl-acetyl glycerol, L-alpha-1,2-dioctanoylglycerol, and L-alpha-1,2-dioleoylglycerol (DOG) increased total 86Rb uptake 8-11%. 12-O-tetradecanoylphorbol-13-acetate (TPA) (5 nM) increased uptake by only 4%. Staurosporine at 5 nM inhibited DOG stimulation completely, whereas 50 nM staurosporine was required to inhibit NE stimulation completely. Sphingosine inhibited DOG stimulation by 66% but did not inhibit NE stimulation. Amiloride (1 mM) completely blocked DOG stimulation. Monensin increased 86Rb uptake 31% and completely blocked the DOG effect but reduced the NE effect by only 26% (P = 0.08). In tubules from salt-loaded rats, NE did not increase DAG concentration, but NE-stimulated 86Rb uptake was reduced by only 23% (P = 0.15). Thus DAG released by NE may stimulate Na+ entry through Na(+)-H+ exchange. NE predominantly stimulates Na(+)-K(+)-adenosinetriphosphatase (ATPase) by activating a protein kinase that is insensitive to DAG and TPA and is inhibited by staurosporine but not by sphingosine. NE may also stimulate K+ efflux through a BaCl-insensitive K+ channel that is inhibited by millimolar furosemide.(ABSTRACT TRUNCATED AT 250 WORDS)

Presence of a charybdotoxin sensitive Ca2+-activated K+ channel in rat glioma C6 cells.

A study was made of the 86Rb+ influx and efflux through Ca2+-activated K+ channels of intact rat glioma C6 cells after addition of a Ca2+ ionophore to the incubation medium. Half-maximal activation of the channels was obtained at a cytoplasmic Ca2+ concentration of approximately 400 nM. The 86Rb+ ion flux through the Ca2+-activated K+ channels was insensitive to apamin, but was inhibited by low concentrations of charybdotoxin (IC50 = 1.6 nM). This is the first evidence for the presence of charybdotoxin-sensitive Ca2+-activated K+ channels in glial cells.

The effect of digoxin on 86rubidium uptake by erythrocytes from mothers and babies.

1 The uptake of 86rubidium by erythrocytes from mothers and babies has been used as a model system to investigate possible differences in sensitivity to digoxin in the very young. 2 While total 86rubidium uptake was not significantly different between mothers and babies, the digoxin-sensitive proportion was higher in neonatal erythrocytes. Neonatal cells were less sensitive to digoxin, demonstrated by the requirement for a larger amount of digoxin to inhibit 86rubidium uptake and this was accompanied by an increase in numbers of specific erythrocyte binding sites for digoxin. 3 These results provide further evidence in support of the hypothesis of decreased sensitivity to digoxin in the very young.

Alloxan cytotoxicity in vitro. Inhibition of rubidium ion pumping in pancreatic beta-cells.

Exposing micro-dissected pancreatic islets of non-inbred ob/ob mice to 2-5 mM-alloxan for 10 min decreased the ability of the islets to accumulate Rb+. Rb+ accumulation in pieces of exocrine pancreas was unaffected by alloxan. When islets were treated with alloxan in the presence of 2-20 mM-D-glucose, the Rb+-accumulating ability was protected in a dose-dependent manner. The protective action of D-glucose was reproduced with 3-O-methyl-D-glucose but not with L-glucose or D-mannoheptulose; mannoheptulose prevented D-glucose from exerting its protective action. The inhibition of Rb+ accumulation was due to a decreased inward pumping, since alloxan did not affect Rb+ efflux from pre-loaded islets. The inhibitory effect of alloxan had a latency of about 1 min, as revealed by experiments with dispersed islet cells in suspension. Alloxan-treated islets showed only a marginal decrease in ATP and no change in glucose 6-phosphate concentration. Although alloxan slightly decreased the hydrolysis of ATP in a subcellular fraction enriched in plasma membranes, this effect could not be attributed to a ouabain-sensitive adenosine triphosphatase. The plasma membranes exhibited a K+-activated hydrolysis of p-nitrophenyl phosphate; this enzyme activity too was insensitive to alloxan. Glucose may protect the univalent-cation pump by preventing permeation of alloxan via a path coupled to the hexose-transport system. Inhibition of the pump may be fundamental to the induction of alloxan-diabetes.