Disulfide stimulation of fluid transport and effect on ATP level in rabbit corneal endothelium

doi:10.1016/0014-4835(74)90066-9

Experimental Eye Research

Volume 19, Issue 1, July 1974, Pages 1-10

https://doi.org/10.1016/0014-4835(74)90066-9 Get rights and content

Abstract

Transendothelial fluid transport in rabbit corneal preparations is known to be improved when perfusates are supplemented with reduced glutathione. Evidence is now presented showing that oxidized glutathione which arises by autoxidation of reduced glutathione under the conditions of perfusion is responsible for this effect. An optimal level of about 10⁻⁵m-oxidized glutathione is necessary to support fluid transport while only 10⁻⁷m oxidized glutathione will sustain endogenous endothelial adenosine triphosphate levels. Of the structurally related disulfides tested, cystine and to a lesser degree homocystine, can replace oxidized glutathione.

Levels of adenosine triphosphate and activities of (Na⁺+K⁺)-activated and Mg²⁺-activated adenosine triphosphatase were measured in individual endothelia after maximal thinning of preswollen corneal preparations had been achieved and the criteria for pump failure had been met. Data for these physiological and biochemical parameters after perfusion with basal salts, glucose or adenosine in the absence or presence of glutathione have been obtained. Adenosine triphosphate levels in the presence of basal salt perfusates are comparably elevated by supplementation with either glutathione or glucose and remain unchanged when both substrates are simultaneously available. Similarly, the elevated ATP levels that accompany adenosine supplementation are not affected by the additional presence of glutathione or glucose. Adenosine triphosphatase activities are somewhat depressed in the absence of all metabolites and are not improved by glutathione alone. These activities remain essentially normal when glucose or adenosine is present.

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Cited by (38)

Regional differences in glutathione accumulation pathways in the rat cornea: Mapping of amino acid transporters involved in glutathione synthesis
2017, Experimental Eye Research
Citation Excerpt :
While ascorbic acid is found at higher levels in the epithelium compared to GSH (Brubaker et al., 2000; Riley and Yates, 1977), GSH is essential for reducing oxidised ascorbic acid to ascorbic acid to maintain ascorbic acid levels (Wu et al., 1998). GSH and oxidised glutathione (GSSG) are also essential for protecting cellular integrity and pump function in the corneal endothelium and thus regulating stromal deturgescence (Anderson et al., 1974; Araie et al., 1988; Nakamura et al., 1994; Riley, 1984). At low or physiological concentrations of H2O2 in the aqueous humour, it has been shown that GSH is preferentially used to protect and maintain endothelial integrity (Umapathy et al., 2013), further highlighting the importance of GSH in the cornea.
In this study we have sought to complete the identification and localisation of uptake pathways involved in accumulating precursor amino acids involved in GSH synthesis in the rat cornea. To do this, we performed reverse transcription PCR (RT-PCR) to identify the Excitatory Amino Acid Transporters (EAAT 1–5) responsible for glutamate uptake, and glycine transporters (GLYT 1–2) at the transcript level. Western blotting was used to verify protein expression, while immunolabelling of sagittal sections was used to localise transporters to the different layers of the cornea. Immunolabelling of en face sections was used to examine the subcellular distribution of proteins in the corneal endothelium. Our findings revealed EAAT 1–5 and GLYT 1–2 to be expressed at the transcript and protein level in the rat cornea. Immunohistochemistry revealed all amino acid transporters to be localised to the epithelium. In the majority of cases, labelling was restricted to the epithelium, and labelling absent from the stroma or endothelium. However, EAAT 4 and GLYT 2 labelling was detected in the stroma with EAAT 4 labelling also present in the endothelium. Overall, the identification of amino acid transporters strongly supports the existence of an intracellular GSH synthesis pathway in the rat corneal epithelium. This suggests that regional differences in GSH accumulation pathways exist, with direct uptake of GSH and intracellular synthesis of GSH restricted to the endothelial and epithelial cell layers, respectively. This information is important in the design of targeted strategies to enhance GSH levels in specific layers of the cornea to prevent against oxidative damage, corneal swelling and loss of corneal transparency.
Influences of glutathione and sulfhydryl containing compounds on aqueous humor outflow function
1990, Experimental Eye Research
Several sulfhydryl reactive compounds have previously been shown to influence aqueous humor outflow facility. The purpose of the current study was to investigate the effect of glutathione depletion on certain of these sulfhydryl actions. Enucleated calf, monkey, and human eyes were perfused via the anterior chamber by the constant pressure (15 mmHg) technique. In calf eyes, perfusion of 10 mm cysteamine produced a small (−23%, P = 0·03) decrease in outflow facility that was also observed after hyaluronidase pretreatment. In contrast, following pretreatment with 1 mm BCNU [1,3 bis(2 chlorethyl)-1-nitrosourea], an inhibitor of glutathione reductase, and 10 mm diamide, a glutathione oxidant, which did not by themselves significantly affect outflow facility, perfusion of cysteamine resulted in an opposite effect—a remarkably large (+90%, P < 0·001) increase in outflow facility. Other reduced and oxidized sulfhydryl-containing compounds such as cysteine, β-mercaptoethanol, and glutathione, itself, as well as the non-sulfhydryl reducing agent, ascorbic acid, were substituted for cysteamine in this protocol and found to produce similar effects of varying magnitudes. In general, the reduced sulfhydryl containing compounds and ascorbic acid were the most effective. Pretreatment with BCNU alone without diamide did not produce this effect.
Treatment with BCNU and diamide resulted in a greater than 75% decrease in reduced glutathione levels and a concomitant tenfold increase in glutathione mixed disulfide levels (0·229 vs. 0·030 μmol g⁻¹ wet weight) in the calf trabecular meshwork. The subsequent perfusion with cysteamine reversed this mixed disulfide formation.
In one pair of enucleated human eyes treated with BCNU, diamide and then cysteamine, loss of cell to cell attachments and breaks in the inner wall of Schlemm's canal were observed, a morphological effect similar to that of certain sulfhydryl binding compounds.
Data from combination studies of BCNU and diamide with the sulfhydryl binding agents, ethacrynic acid and PCMBS (p-chloromercuribenzene sulfonate), are presented which suggest that there are multiple sulfhydryl-sensitive sites in the outflow pathway that may be involved in the regulation of outflow resistance.
A note of caution to those who add reduced glutathione (GSH) to incubation media
1986, Experimental Eye Research
A role for glutathione and glutathione reductase in control of corneal hydration
1984, Experimental Eye Research
We have assessed the importance of the glutathione redox system of the corneal endothelial cells in the control of stromal hydration. The ability of freshly isolated corneas to maintain normal hydration during perfusion, while the activity of glutathione reductase was inhibited with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), was tested under a number of conditions. BCNU at 125 μm led to mild swelling and at 500 μm to more severe swelling. Swelling was also severe when 50 μm H₂O₂ was added together with 125 μm BCNU. The concentration of total glutathione (GSH + GSSG) in the endothelia of these corneas was decreased and the fraction found in the oxidized form was higher than in controls without BCNU. Glutathione reductase activity was inhibited in the perfused endothelia by 90%. With the addition of 0·5 mm GSSG during perfusion, swelling due to 125 μm BCNU was at a slightly lower rate and reductase activity was inhibited only 79%. Complete proteetion was afforded against 125 μm BCNU induced swelling by 1·0 mm GSH, corneas maintaining normal thickness for over 4 hr. The endothelial surface of the perfused corneas was shown by seanning electron microscopy to be little disturbed by the low concentration of BCNU, but at high concentration, or with H₂O₂ added, the cells were collapsed and had a heavily pitted appearance. We conclude that when the cornea is under oxidative stress (e.g. in the presence of H₂O₂) a rapid turnover of endothelial GSH via glutathione reductase and the hexose monophosphate shunt is required. Under conditions of less stress, the reduced need for GSH to counteract oxidative threats can be supplied either exogenously or by the partially inhibited reductase.
Pyridine nucleotides of rabbit cornea with histotoxic anoxia: Chemical analysis, non-invasive fluorometry and physiological correlates
1983, Experimental Eye Research
The pyridine nucleotides from both the epithelium and the endothelium of rabbit cornea were measured by the cycling assay. Sodium azide (10 mm) applied for 1 hr to induce histotoxic anoxia decreased the endothelial $NAD^{+} NADH$ ratio from 4·62 to 1·49 and decreased the epithelial $NAD^{+} NADH$ ratio from 2·56 to 1·08. The larger $NAD^{+} NADH$ ratio for the endothelium as compared to the epithelium corresponds to a more oxidized state. The corresponding ratios for $NADP^{+} NADPH$ were 1·2 for the endothelium and 0·70 for the epithelium. Sodium azide had no effect on the $NADP^{+} NADPH$ ratio for the endothelium, but decreased the epithelial ratio to 0·62. Pyridine nucleotide fluorescence was measured with a difference corneal fluorometer on the perfused whole cornea preparation and the perfused everted corneal preparation. Sodium azide (10 mm) for 30 min resulted in a 19·4 ± 0·7% increase in the pyridine nucleotide fluorescence from the whole corneal preparation and a 4·5 ± 0·6% increase from the everted endothelial preparation. Corneal anoxia induced by stopping the perfusion on the endothelial side resulted in a 18·7 ± 0·6% increase in pyridine nucleotide fluorescence for the whole corneal preparation. Sodium azide (10 mm) resulted in a 35% decrease in the transendothelial potential difference and a 76% decrease in the rate of transendothelial fluid transport. A comparison is made between invasive chemical analysis and real time, non-invasive fluorometry to measure histotoxic corneal anoxia.
The roles of glutathione reductase and γ-glutamyl transpeptidase in corneal transendothelial fluid transport mediated by oxidized glutathione and glucose
1982, Experimental Eye Research
Complete inhibition of corneal endothelial glutathione reductase by 1, 3-bis-(2-chloroethyl)-1-nitrosourea has been shown to be without effect on the endothelial fluid pump maintained by perfusion with either 0·10 mm-oxidized glutathione or 5·0 mm-glucose. Some dependence of pump function on γ-glutamyl transpeptidase is indicated by the 33% reduction in the amount of stromal deturgescence that was achieved by co-perfusion of GSSG with the transpeptidase inhibitor, 6-diazo-5-oxo-l-norleucine. The apparent 22% improvement in pump function found by coperfusion of glucose with 6-diazo-5-oxo-l-norleucine is likely the result of a reduced leak effected by reaction of the inhibitor with functional groups on the membrane.
Endothelial levels of total glutathione after 3-hr perfusion periods with either glucose or oxidized glutathione were not statistically different, but a shift of the redox state of glutathione occurred in oxidized glutathione-perfused endothelia that resulted in a 4-fold increase in the cellular content of oxidized glutathione relative to that maintained by glucose. This and others' results are interpreted to indicate that glutathione is taken up by the endothelium and that oxidized glutathione is the privileged redox state for this process.

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Experimental Eye Research

Abstract

References (25)

Fluid transport, ATP level and ATPase activities in isolated rabbit corneal endothelium

Biochim. Biophys. Acta

A glutathione reductase from Escherichia coli

J. Biol. Chem.

The metabolism of ribonucleoside by the human erythrocyte

Biochim. Biophys. Acta

Ribonucleotide reductase and cell proliferation

J. Biol. Chem.

Potential difference and fluid transport across rabbit corneal endothelium

Biochim. Biophys. Acta

Active and passive properties of the rabbit corneal endothelium

Exp. Eye Res.

Evidence for a bicarbonate-dependent sodium pump in corneal endothelium

Exp. Eye Res.

Glutathione in the regulation of the hexose monophosphate pathway

J. Biol. Chem.

The oxidation of C¹⁴-labeled glucose and lactate by the rabbit cornea

Arch. Biochem. Biophys.

Glutathione-homocystine transhydrogenase

J. Biol. Chem.

The role of the epithelium in control of corneal hydration

Exp. Eye Res.

Levels of (Na⁺+K⁺)-activated and Mg²⁺-activated ATPase activity in bovine and feline corneal endothelium and epithelium

Biochim. Biophys. Acta

Cited by (38)

Regional differences in glutathione accumulation pathways in the rat cornea: Mapping of amino acid transporters involved in glutathione synthesis

Influences of glutathione and sulfhydryl containing compounds on aqueous humor outflow function

A note of caution to those who add reduced glutathione (GSH) to incubation media

A role for glutathione and glutathione reductase in control of corneal hydration

Pyridine nucleotides of rabbit cornea with histotoxic anoxia: Chemical analysis, non-invasive fluorometry and physiological correlates

The roles of glutathione reductase and γ-glutamyl transpeptidase in corneal transendothelial fluid transport mediated by oxidized glutathione and glucose