The localization of the anion-sensitive ATPase activity in corneal endothelium.

The localization of the anion-sensitive ATPase (EC 3.6.1.3) of bovine corneal endothelium has been investigated. Homogenates were fractionated by differential and density gradient centrifugation, into fractions enriched in plasma membranes and mitochondria. (Na+ + K+)-ATPase (EC 3.6.1.3) and cytochrome oxidase (EC 1.9.3.1) were used as marker enzymes for these two cell components, and glucose-6-phosphatase (EC 3.1.3.5) was used to identify endoplasmic reticulum. 5'-Nucleotidase (EC 3.1.3.5) was also measured but was found not to be exclusively associated with any one cell component. The activity of the anion-sensitive ATPase (HCO3--ATPase) was measured in suspensions that were frozen and thawed before assay in order to expose latent enzyme activity. The fraction containing the greatest amount of (Na+ + K+)-ATPase (35%) contained only 6% of the cytochrome oxidase and HCO3--ATPase. Conversely, the mitochondrial fraction, containing 40% of the cytochrome oxidase, contained about 40% of the HCO3--ATPase, but only 7% of the (Na+ + K+)-ATPase. The recoveries and relative degree of purification of the cytochrome oxidase and HCO3--ATPase were also nearly identical in the other fractions examined. It was concluded that the anion-sensitive ATPase activity of the corneal endothelium is located solely in the mitochondria and not in the plasma membrane. Consequently, any role that the enzymes may have in the transport of bicarbonate across this tissue, which had been suggested in earlier studies, must be an indirect one.

Relative contributions of epithelial cells and fibers to rabbit lens ATP content and glycolysis.

The adenosine triphosphate (ATP) content was measured independently in separated capsule-epithelium and fibers from whole rabbit lenses, both fresh and after incubation under various combinations of glucose and oxygen deprivation. Lactate production was also measured during aerobic and anaerobic incubations of whole lenses and of monolayers of cultured epithelial cells. The fresh capsule-epithelium contained 3.3 nmoles ATP, whereas the decapsulated lens contained 410 nmoles ATP, a value that was indistinguishable from that of the whole, intact lens. In the presence of glucose, the fibers and epithelium each maintained their respective ATP content under aerobic and anaerobic conditions. In the absence of glucose, the ATP content in each fraction declined with time, but only in the epithelium was the rate of decline of ATP significantly faster in nitrogen than in oxygen. In whole lens, the rates of anaerobic and aerobic lactate production were similar, whereas in the cultured epithelial monolayers, the anaerobic rate was two-fold greater than in oxygen. From this it is concluded that approximately 50% of the ATP of the epithelial cells is derived from oxidative metabolism. A Pasteur response shown here for the first time with the cultured epithelium allows these cells to compensate for the loss of ATP production when mitochondrial oxidation is curtailed. The epithelium does not contribute to the ATP content of the lens fibers under aerobic or anaerobic conditions.

Does topical hydrogen peroxide penetrate the cornea?

PURPOSE: To determine under what conditions hydrogen peroxide (H2O2) can penetrate the cornea and cause increased concentrations of H2O2 in the aqueous humor. METHODS: Rabbit corneas were exposed in vitro and in vivo to H2O2 concentrations up to 60 mmol/l either in a 600 microliter volume or as consecutive drops. H2O2 was measured over time either in the endothelial superfusate or in tapped samples of aqueous humor, and in the fluid applied to the ocular surface. The stability of exogenous H2O2 added directly to aqueous humor was also determined. RESULTS: Exogenous H2O2 in aqueous humor decays with a half-life of 20 minutes, chiefly as a result of catalase activity. When applied to the entire ocular surface, 600 microliters of 60 mmol/l H2O2 caused no change in the concentration in aqueous humor, but when applied to the corneal surface alone, penetration occurred at 18 mmol/l and above. When applied as eight 40-microliters drops to the ocular surface the threshold for H2O2 penetration was above 36 mmol/l. CONCLUSIONS: H2O2 is rapidly eliminated on the ocular surface, chiefly by enzyme activity of the conjunctiva and cornea. Threshold for penetration of H2O2 into the aqueous humor depends on volume, concentration, duration, and ocular surface exposed. In a healthy eye, exogenously derived H2O2 is eliminated by enzyme activity of the aqueous humor and tissues surrounding the anterior chamber.

Influence of calcium on retinal ATPases.

The activity of Mg2+-ATPase and Na+-K+-ATPase was measured in media of calcium concentrations ranging from 10(-9)M to 10(-3)M, with retinal homogenates from rat and rabbit. In both species calcium stimulated the Mg2+-ATPase and inhibited Na+-K+-ATPase. In the rat, activity of Na+-K+-ATPase fell by 75% as calcium was increased from 10(-8)M to 10(-7)M and reached 90% inhibition only at 10(-3)M. By contrast, the activity in the rabbit fell gradually to a 90% inhibited state, over the range 10(-8)M to 10(-3)M. Calcium activated the Mg2+-ATPase by a maximum of 50% in each species, at a concentration of 10(-8)M in the rat and over a broader concentration range between 10(-5)M and 10(-4)M in the rabbit. It is postulated that maintenance of intracellular calcium at low levels by the Ca2+-activated Mg2+-ATPase or other cellular mechanisms is essential for the activity of the membrane-bound Na+-K+-ATPase of the retina.

Na+-K+ and HCO-3 ATPase activity in retina: dependence on calcium and sodium.

With the use of homogenates of whole rat retina, the activities of Na+-K+-- and HCO-3-stimulated ATPases were measured in media containing different concentrations of calcium and sodium ions. In comparison to the Na+-K+ ATPase activity observed in the presence of 2 mM calcium and 130 mM sodium, the omission of calcium from the medium led to a 16-fold increase in activity. Furthermore, the omission of calcium and the reduction of the concentration of sodium in the medium to 25 mM also resulted in an increase in activity of ninefold. This study also demonstrates, for the first time, the existence of a HCO-3 stimulated. ATPase activity in the retina. These results are discussed in relation to recent observations on the effects of calcium- and bicarbonate-free media on the electrical and metabolic activities of the rat retina.

Glutathione in the aqueous humor of human and other species.

Glutathione was measured in the aqueous humon of rabbit, dog, monkey, baboon, and man. The average concentrations were 17, 6, 6, 2, and 2 mumol/L of fluid, respectively. Except in the case of the monkey, where delay in aspiration of the aqueous apparently led to anomalous values, nearly all the glutathione was in the reduced state, the range being from 85% in the rabbit to over 99% in the human. Possible sources of the glutathione in the aqueous humor are discussed and also its significance for the physiology of the cornea.

Corneal endothelial glutathione after photodynamic change.

Rabbit corneal endothelial cells perfused with 5 X 10(-6)M rose bengal and exposed to incandescent light demonstrated no alteration of either total of or percent oxidized glutathione after 1 hr. Addition of 5400 U/ml catalase to the perfusing solution had no effect on total glutathione levels but caused a marked reduction in percent oxidized glutathione in corneas exposed to light as well as in those not exposed to light. Substitution of sucrose for glucose in the perfusing solution had no effect on total or percent oxidized glutathione. Perfusion of rabbit corneal endothelium with 0.5 mM chlorpromazine and exposure to ultraviolet (UV) light resulted in no change in total glutathione content. A marked reduction in percent oxidized glutathione occurred, however, in corneas perfused with 0.5 mM chlorpromazine both in the presence and absence of UV light. It is concluded that photodynamically induced swelling of corneas is not the result of a failure of the glutathione redox system.

The roles of bicarbonate and CO2 in transendothelial fluid movement and control of corneal thickness.

PURPOSE: To determine whether maintenance of corneal hydration is dependent on bicarbonate ions and whether these ions can be derived from metabolic or exogenous CO2, and to investigate the relationship of transendothelial fluid movement to control of hydration. METHODS: The thickness of intact or deepithelialized rabbit corneas was measured while superfused on the endothelial surface with either 33 mM HGO3-/5% CO2 buffered media or 10 mM HPO4- buffered media in the presence and absence of inhibitors of ion transport and respiration. The corneal surface was covered with either silicone oil ("normal" corneas) or with the same media used for superfusion ("swollen" corneas). ATP and Na+,K(+)-ATPase activity were measured in endothelia scraped from the tissues after superfusion. RESULTS: Intact and deepithelialized corneas covered with oil swelled at a negligible rate (4 to 8 microns/hour) in 33 mM HCO3- medium but at 45 to 60 microns/hour in HPO4- medium. Antimycin A altered neither of these swelling rates, but ethoxzolamide (0.1 mM) caused swelling in HCO3-/CO2 (approximately 12 microns/hour above controls) with no change of rate in HPO4-. Ouabain (0.1 mM) increased swelling to 45 to 50 microns/hour in HCO3-/CO2 but had no effect in HPO4-. Saturating the oil on deepithelialized corneas with 5% CO2, or putting HCO3-/CO2 medium on the epithelial surface of intact corneas, did not alter the swelling rates seen with HPO4- superfusion. The equilibrium thickness of deepithelialized corneas swollen with HCO3-/CO2 on both surfaces was 35 microns less than that of corneas swollen in HPO4-. The difference was abolished by ouabain, which caused corneas in HCO3-/CO2 to swell an additional 30 microns but did not alter the equilibrium thickness of corneas swollen in HPO4-. Ethoxzolamide and DIDS (0.2 mM) increased the thickness in HCO3-/CO2 but not in HPO4-. Na+,K(+)-ATPase activities of endothelia were similar after HCO3-/CO2 and HPO4- superfusions, but the concentration of ATP in the HPO4(-)-superfused tissues was increased 35%. CONCLUSIONS: Normal corneal thickness can be maintained in vitro only in media that contain HCO3- at concentrations of more than 20 mM. Neither metabolic CO2 nor CO2 present in air-equilibrated, nominally HCO3(-)-free media can supply this requirement for HCO3-, even though these sources support the presumably related processes of transendothelial fluid movement and intracellular pH regulation.

Adenosine promotes regulation of corneal hydration through cyclic adenosine monophosphate.

PURPOSE: To investigate the cellular mechanisms whereby adenosine increases net transendothelial fluid transport by the endothelial cells of the cornea. METHODS: Rabbit corneas were isolated and the endothelial surface was superfused while thickness was measured with the specular microscope. Cyclic adenosine monophosphate (cAMP) was measured in endothelia from fresh and incubated corneas, and adenylyl cyclase and phosphodiesterase activities were measured in homogenates or the particulate fraction of endothelia from bovine or rabbit. Adenosine, adenosine-receptor agonists, dibutyryl cAMP, forskolin, and phosphodiesterase inhibitors were used to modulate physiological and biochemical parameters. RESULTS: Adenosine, N-ethyl(carboxamido)adenosine, dibutyryl cAMP, forskolin, and phosphodiesterase inhibitors all promoted deturgescence of swollen corneas and maintained fresh corneas at lower steady state thicknesses than in controls. These effects were abolished in the presence of ouabain or 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid or after complete removal of HCO3- from the media. Intracellular cAMP was significantly increased by forskolin and phosphodiesterase inhibitors and, to a lesser extent, by agonists. Increases in cAMP concentration declined rapidly with time. Cyclase activity in the bovine tissue was enhanced by agonists and by G-protein activators. Dose-response curves of corneal swelling indicated a greater sensitivity to N-ethyl(carboxamido)adenosine than to the A2 alpha specific agonist CGS 21680. CONCLUSIONS: Adenosine increases net endothelial fluid transport through an increase in cAMP. The effects are mediated by stimulation of adenylyl cyclase through a G-protein coupled to an adenosine receptor, which is most probably of the A2 beta subtype. Results suggest that the regulation of corneal hydration by adenosine is more probably through stimulation of active transport than through a change in permeability, involving either transmembrane fluxes of Na+ or HCO3- or another step tightly coupled to these primary events in fluid movement.

Relationship between fluid transport and in situ inhibition of Na(+)-K+ adenosine triphosphatase in corneal endothelium.

PURPOSE: To examine the relationship between the activity of the sodium pump of the corneal endothelium and corneal thickness. It was postulated that because inhibition pressure of the stroma decreases as thickness increases, a partially inhibited sodium pump would result in a new steady-state thickness of the cornea when reduced rates of fluid influx and efflux were equal. Measurements of physiologic behavior and biochemical activity were to be made in the same tissue and thus establish the relationship directly. METHODS: Rabbit corneas were superfused with a bicarbonate Ringer solution containing different concentrations of ouabain. Exposure to ouabain was either continuous for 4 hours or for an initial 10 minutes followed by ouabain-free superfusion. Thickness was measured, and, after superfusion, endothelium was removed from the corneas, sonicated, and assayed for Na(+)-K+ adenosine triphosphatase (ATPase) activity without further addition of ouabain to the assay medium. Thickness was also measured during superfusion with suboptimal concentrations of Na+ or HCO3- and with brefeldin A, an inhibitor of protein trafficking. RESULTS: Continuous exposure to ouabain caused corneas to swell, but no new steady-state thickness was reached. At low concentrations, swelling rates increased with time, as did the extent of inhibition of the Na(+)-K+ ATPase. With only a 10-minute exposure to ouabain, swelling rates with 10(-4) M to 10(-5) M decreased with the duration of ouabain-free superfusion. Similar swelling curves were obtained by reductions in Na+ or HCO3- concentrations in the superfusion medium, indicating that partial inhibition of the endothelial fluid transport processes, whether via the Na(+)-K+ ATPase or by suboptimal ionic conditions, led toward a new equilibrium thickness of the cornea. However, when superfusion was continued for more than 4 hours, the corneas exposed for 10 minutes to 3 x 10(-5) M or lower-concentration ouabain showed increasing Na(+)-K+ ATPase activity and began to thin, indicating a recovery of fluid transport capability. This recovery was blocked by addition of brefeldin A during the ouabain-free superfusion. CONCLUSIONS: Inhibition of Na(+)-K+ ATPase by low concentrations of ouabain increases with time. Temporary exposure to ouabain causes swelling at rates that decline with time as ouabain dissociates from enzyme sites. This dissociation, together with the turnover of Na(+)-K+ ATPase in the plasma membrane, can lead to recovery of normal thickness in ouabain-exposed corneas. Twenty percent of Na(+)-K+ ATPase in the endothelium is estimated to be intracellular, and about 20% of the activity can be inhibited without inducing swelling.

Intracellular pH and glutathione levels in rabbit corneal endothelium following storage in moist chamber and MK medium.

Rabbit corneas were stored for up to 14 days at 4 C either as the whole eye in a moist chamber or as the isolated cornea in MK medium with HEPES buffer. The intracellular pH, the glutathione content, and its oxidation state were determined in the endothelial cells of fresh and stored tissue. The endothelial pH was found to be unchanged following storage of up to 7 days by either method, but after 14 days the pH rose slightly but statistically significantly in corneas stored by both techniques. The intracellular pH was similar in endothelia of those corneas stored in MK medium and of those stored as the whole eye in a moist chamber, for all time periods studied. The intracellular total and percent oxidized glutathione of the endothelium were increased by 50 and 180%, respectively, following 7 days of moist chamber storage. Over this time period there was a 50-fold increase in total glutathione content of the aqueous humor in the stored eyes. In contrast, corneas stored in MK medium for 7 days maintained intracellular total glutathione at levels similar to those of fresh corneas. A gradual but constant decrease in percent oxidized glutathione was observed with increasing length of storage. In terms of pH and glutathione content, the MK medium provided a much more stable environment for the stored cornea than did the aqueous humor in the stored eye.

Regulation of corneal endothelial barrier function by adenosine, cyclic AMP, and protein kinases.

PURPOSE: To determine which processes or factors that regulate corneal hydration are responsible for the hydration-modulating effects of adenosine. Influx of fluid to the stroma and efflux to the aqueous humor are governed, respectively, by the imbibition pressure of the stromal matrix and the transendothelial ionic gradients determined by the permeability and active transport characteristics of this monolayer. The focus of this study was to assess the effects of adenosine on these endothelial parameters. METHODS: Isolated corneas freshly dissected from rabbit eyes were used throughout. Active ion transport was assessed by measurement of 86Rb+ uptake by the endothelial cells of intact corneas incubated for 30 minutes in 25 mM HCO3(-)-Ringer with agents promoting corneal deturgescence or corneal swelling. Intracellular and extracellular fluid in the scraped endothelial cell mass was estimated from simultaneous counts of 3H-mannitol and 14C-urea, allowing calculation of tissue-to-medium (T-M) ratios of 86Rb+ in cell water. Permeability of the endothelium was determined by measuring the efflux into the superfusate of 5-carboxyfluorescein (CF) applied to the stroma of deepithelialized corneas superfused at the endothelial surface with the same media described for 86Rb+ uptake. Thickness of these corneas and of others fixed for scanning electron microscopy was monitored with a specular microscope. RESULTS: In the control medium, 25 mM HCO3(-)-Ringer, 86Rb+ was accumulated to yield a T-M ratio of 6.21. Neither adenosine nor other agents that increase cyclic adenosine monophosphate (cAMP)--that is, forskolin and dibutyryl cAMP--changed this value to a significant extent. Bumetanide had no effect, but ouabain caused a decrease in T-M to 1.30, a 79% inhibition. Elimination of Na+ or HCO3- also caused marked decreases in uptake. Permeability to CF in control medium was 3.40 x 10(-4) cm/min. A decrease of more than 20% (P < 0.05) was seen in the presence of adenosine and cAMP promoters and also with the protein kinase inhibitor H-8, whereas phorbol myristate acetate caused an increase to 4.50 x 10(-4) cm/min (P < 0.01). Ouabain caused no change, but blocked the effects of adenosine. Reducing the Ca2+ concentration of the superfusing medium caused time-dependent increases in permeability to 4.57 at 15 to 45 minutes and 12.5 at 80 to 110 minutes. At the earlier time, this increase in permeability could be prevented by the addition of adenosine or H-8. Elimination of Na+ or HCO3- ions from the medium caused a small decrease in permeability and, like ouabain, blocked the effect of adenosine. Changes in thickness of corneas were consistent, in most cases, with the observed alterations in 86Rb+ uptake or permeability to CF. Scanning electron microscopy showed contraction and rounding of endothelial cells in low Ca2+ medium, with stretching of intercellular borders, features that were largely eliminated when adenosine was also present. CONCLUSIONS: Adenosine, through increasing cAMP, decreases permeability of the corneal endothelium. This effect, rather than a change in the active transport (fluid pump) mechanism, is responsible for the promotion of deturgescence and maintenance of lower steady state thickness of corneas exposed to adenosine. The mechanism may involve the phosphorylation state of cytoskeletal proteins and seems to be dependent on an undisturbed environment of monovalent ions.

Toxic effects of hydrogen peroxide on corneal endothelium.

We have measured the effects of hydrogen peroxide on the ability of the isolated rabbit cornea to maintain normal hydration during perfusion, on the structure of the endothelial cells, and on the redox state of glutathione and the activity of the hexose monophosphate shunt in these cells. Swelling of isolated corneas was immediate and severe upon exposure to 50 microM H2O2 in the absence of glucose, or to 200 microM H2O2 in the presence of glucose. The presence of glucose with 50 microM H2O2 delayed the onset of swelling for two hours and markedly decreased its severity. The concentration of glutathione in the endothelium, and its redox state, were unaltered by 50 microM H2O2 in the presence of glucose, but in its absence glutathione loss was significant and the fraction in the oxidized state was greatly increased. H2O2 was removed from the medium by reaction with the cornea at a concentration-dependent rate. It was calculated that the reaction rate at 50 microM H2O2 was about twice that which could be accounted for by the increased yield of NADPH generated by the stimulation of the hexose monophosphate shunt. H2O2 may also react with endothelial cell membranes, which could account for the marked changes in cell structure seen in the scanning electron microscope.

The release of a neutrophil chemotactic factor from UV-B irradiated rabbit corneas in vitro.

Rabbit corneas were isolated, mounted on plastic rings to form a cup and the endothelium was covered with RPMI tissue culture medium. The preparation was then irradiated with 1 J. cm-2 of 300 nm light over 1 hour and then incubated for a further two hours in the dark. The supernatant fluid was assayed for chemotactic activity toward rabbit neutrophils in an in vitro Boyden chamber assay. The results indicated that medium from irradiated corneas had a chemotactic activity that was 42% of that produced by the standard chemoattractant f-met-leu-phe, (10(-9) M) while medium from unexposed corneas and exposed medium alone had less than 3% activity. An in vivo assay using sub-epidermal injection into the back of a rabbit gave qualitatively similar results, only f-met-leu-phe and the medium from irradiated corneas causing neutrophil infiltration of the tissue. A checkerboard analysis confirmed that the activity was chemotactic rather than chemokinetic. Release of a chemotactic factor following UV-B irradiation provides a mechanism for the recruitment of neutrophils, at specific localized areas of the endothelium, that is seen after discrete in vivo irradiation. The results also confirm the importance of corneal inflammatory mediators in the development of tissue damage subsequent to exposure to toxic agents.

Regional distribution of calcium in alloxan diabetic rabbit lens.

A diabetic rabbit model was developed for investigation of cataractogenesis and other changes in the anterior segment. Rabbits were fasted, injected with 0.7 mg/kg alloxan, fed 1% glucose solution for 24 hrs and returned to a normal diet. Animals showing and maintaining blood glucose of greater than 300 mg% within two days were used in this study. Concomitant with increase in blood glucose was a rise in aqueous humor glucose and osmolality, together with a decrease in ascorbate concentration. Vacuoles and small discrete opacities developed, and in some cases, at longer time periods complete opacity of anterior or posterior aspects was found. Total calcium content of the whole lens increased up to 2-fold, especially after 60 days, and was correlated with a decrease in lens transmittance of a He/Ne laser beam and also with high osmolality of the aqueous humor. Free calcium was six-fold higher in opaque areas than clear areas, and was 100-fold higher in vacuoles. It is suggested that, in addition to the recognized role in sugar cataractogenesis of osmotic stress due to sorbitol accumulation in the lens, changes of intracellular calcium in localized areas of the lens and stresses imposed by changes in aqueous humor osmolality may also be important.

The effects of UV-B irradiation on the corneal endothelium.

Rabbit eyes, in vivo and in vitro, were exposed to UV-B irradiation at 300 nm, from a mercury arc lamp with an 11 nm bandpass filter. Radiant exposure ranged from 0.1 J/cm2 to 0.5 J/cm2. In vivo, swelling of the cornea resulted over a 12 to 40 hr period, the extent and duration being directly related to exposure. Recovery of normal thickness was complete within four days. Corneas removed at 18 hr after exposure recovered normal thickness during a five hour perfusion period, except for those most heavily exposed. When removed at 42 hr post exposure all corneas thinned to almost normal thickness. SEM showed the endothelial cells of exposed eyes to have either exaggerated villi on the surface and a disorganized mosaic or, after higher exposures, to be devoid of villi and have loose, flap like cell borders and large "blebs." After exposure of isolated corneas mounted for perfusion, swelling again ensued and similar changes were observed in the appearance of the cells, except that "blebs" were not found. No significant changes were observed in the metabolic components ATP, ascorbate and glutathione, nor was there any indication of lipid peroxidation. At higher in vivo exposures, the aqueous humor did show a decrease in ascorbate concentration and an increase in protein content, which probably result from a breakdown of the blood-aqueous barrier. UV-B irradiation may cause or promote changes in the endothelium associated with aging, but the one time radiant exposures of the magnitude used in this study, appear to have no severe or permanently toxic effects.

Interactions of ascorbate and H2O2: implications for in vitro studies of lens and cornea.

The interaction of ascorbate, hydrogen peroxide and oxygen has been examined in order to understand the equilibrium between these compounds that exists in the aqueous humor of the eye and their influence on function of the corneal endothelium. Ascorbate was found to promote corneal swelling when isolated corneas were perfused with a medium lacking glucose. This was found to be due to the rapid oxidation of ascorbate in the medium, yielding H2O2 which is toxic to the endothelial cells. In the absence of oxygen, or if EDTA was added to the medium, no H2O2 was produced from ascorbate, but ascorbate reacted with any pre-existing H2O2. Oxidation of ascorbate in the aqueous humor is limited by the presence of glutathione (and, possibly, other compounds) and no significant increase in H2O2 concentration occurs on standing in air. Nevertheless, the concentration of H2O2 in the aqueous is directly dependent on the concentration of ascorbate secreted in the aqueous humor. Therefore, there must be a dynamic equilibrium in this fluid between ascorbate, H2O2 and oxygen, and it may be modulated by glutathione. Each of these substances is important in redox reactions, including free-radical production or scavenging. Consequently, when studying the effects on corneal or lenticular function of other agents which cause or relieve oxidant stress, it is critical that the modifying effects of ascorbate and H2O2, as they occur in vivo, be considered. A perfusion system is described which permits an approximation in vitro of stable concentrations of ascorbate, H2O2, GSH and O2 similar to those found in the aqueous humor.

Hydrogen peroxide in the rabbit anterior chamber: effects on glutathione, and catalase effects on peroxide kinetics.

Intracameral hydrogen peroxide (H2O2) is cleared at a faster rate in young (t1/2, 93 seconds) than in adult (t1/2, 109 seconds) rabbits. Extrapolated zero time concentrations of H2O2 were 3.3 mM in adults and 3.2 mM in young. The more rapid disappearance of H2O2 correlated with greater catalase levels in iris (35%) and corneal endothelium (50%) in young as compared to adult animals. Catalase levels have been found to be reduced in ocular tissues with 3-amino-1H-1,2,4-triazole (3AT) in a dose-related manner up to 6 ml/kg of an intravenous 3M solution. Iris and ciliary processes showed a linear reduction with dose, while corneal endothelium, liver and lung reached near maximal decreases in catalase activity at 2, 4, and 6 ml/kg, respectively. 3AT caused a significant dose-dependent extension of the rate of clearance of H2O2 from the anterior chamber, that was directly related to catalase loss. The t1/2 for H2O2 disappearance in adult animals increased from 109 seconds with no 3AT, to 147 seconds after 2 ml/kg 3M 3AT, to 161 seconds after 4 ml/kg 3M 3AT and 184 seconds after 6 ml/kg 3M 3AT. Corneal endothelial oxidized glutathione levels were transiently increased after intracameral hydrogen peroxide. Considering the sum total of all tissues of the anterior segment, specific incremental decreases of catalase generated by intravenous 3AT caused the t1/2 of H2O2 clearance from the anterior chamber to become longer, while the reducing power of anterior segment tissues excluding lens epithelium is related clearly to the systemic dose of 3AT.(ABSTRACT TRUNCATED AT 250 WORDS)

Marihuana-derived material: biochemical studies of the ocular responses.

Some biochemical factors of the iris-ciliary body of the rabbit have been examined for effects induced by water-soluble marihuana-derived material (MDM). Adenylate cyclase activity and sensitivity to beta-adrenergic agonists were unchanged, as measured 4 hours after MDM administration in vivo. Magnesium-dependent and anion-sensitive, but not sodium-potassium, ATPase activities were inhibited 6 hours after MDM administration in vivo, although they were unaffected by in vitro incubation. Topical administration of a potent substance P antagonist had no effect on the time course or magnitude of intravenous MDM-induced ocular effects in rabbit. Intravenously administered sugars antagonized the effects of MDM on intraocular pressure. A variety of drugs which display a range of biochemical effects varying from beta-adrenergic receptor agonism, to alteration of glycoprotein residues were employed. None of the agents employed, ranging from cAMP modifiers to protein synthesis blockers, had any effect on the MDM-induced response. It is apparent that the mechanism underlying the ocular hypotensive effect of MDM does not reside in mediation through adenylate cyclase, ATPase or substance P, but rather through a mechanism mediated by terminal sugar moieties on the molecule. The data suggest that modification of the surface membrane glycoprotein residues on the ciliary epithelium can induce marked alterations in aqueous humor flow rate.