Effect of taurine on 45Ca transport in frog retinal rod outer segments

The addition of taurine produced different effects on ⁴⁵Ca²⁺ accumulation in frog retinal rod outer segments isolated in a Krebs-bicarbonate buffer depending on exogenous CaCl₂ concentration. At low external calcium (0·1 mm), 25 mm exogenous taurine produced an ATP-dependent increase in ⁴⁵Ca uptake of 60–80% over control values. At high external calcium (2·5 mm) exogenous taurine (2·5–25 mm) decreased ⁴⁵Ca uptake 25–50%; this effect was partially prevented by ATP. 10 mm GABA, glycine or β-alanine did not affect ⁴⁵Ca transport in similar conditions. Inhibition of Ca transport by taurine was maximal at pH 7·4, although it was observed at pH from 6·4 to 8·2. Both taurine effects were observed after 1 min of incubation and increased up to 10 min.     

Effects of the protein tyrosine kinase inhibitor genistein and taurine on retinal function in isolated superfused retina

Background Genistein has the potential to act as an intraocular antiangiogenic agent. Its therapeutical use, however, is limited by toxic side effects on the retina. This study was designed to evaluate the simultaneous use of taurine as a neuroprotective drug. Methods Bovine retinas were isolated and perfused with an oxygen-preincubated nutrient solution. The electroretinogram (ERG) was recorded as a transretinal electrical potential using Ag/AgCl electrodes. At stable ERG amplitudes, genistein at concentrations of 11, 37, and 150 μM was added to the nutrient solution for 45 min, in the absence or presence of taurine (3 mM). Thereafter, the retina was reperfused with the nutrient solution for another 100 min. The percentage of b-wave reduction during genistein and genistein/taurine application was calculated. Results The b-wave amplitude was reduced by a smaller amount during the application of genistein (11 and 37 μM) in the presence of taurine compared with genistein alone. For both, genistein/taurine and genistein alone the b-wave recovered completely during the wash-out of the drugs. However, during the application of the highest tested concentration of genistein (150 μM), taurine did not protect completely, leading to an irreversible b-wave reduction. Conclusions The adjuvant use of taurine reduces the genistein-induced retinal toxicity to a certain degree. However, the protective effect of taurine is limited and there is only a narrow therapeutic index for a combined intravitreal administration of genistein in coapplication with taurine to inhibit pathological ocular neovascularization. Dedicated to the late Prof. Dr. Werner Sickel. He passed away on December 18th, 2004     

Taurine-calcium interactions in frog rod outer segments: Taurine effects on an ATP-dependent calcium translocation process

Frog rod outer segments (ROS) isolated in a Ca-free, EGTA-containing medium, showed a rapid ATP-dependent accumulation of⁴⁵Ca. GTP, CTP, ITP, UTP, AMP, GMP and β- γ-methylene ATP did not substitute for ATP in energizing Ca uptake. This process required Mg, it was abolished in the presence of Ca ionophores A23187 and X537A and is not affected by external sodium. The Arrhenius activation energy was 7.9 kcal/mol and the pH optimum was approximately 7.2. The apparent K_m for Ca uptake was 66 μM with a V_max of 12.5 nmol/mg protein.⁴⁵Ca accumulation was reduced in illuminated ROS. The presence of 5–25 mM taurine, but not of GABA, glycine, histidine or proline, markedly enhanced Ca uptake by ROS.     

Phosphofructokinase of calf trabecular meshwork

The activity of phosphofructokinase (PFK), a key regulatory enzyme of glycolysis, has been measured in the 27,000 X g supernatant of homogenates prepared from excised calf trabecular meshwork. The enzyme required NH4+, both at pH 8.5 and pH 7.2. This requirement was not relieved by K+ or AMP. At pH 7.2 and ATP levels of 0.1 to 2.5 mM, PFK was completely inactive in the absence of added AMP or NH4+. PFK was only weakly activated by 0.5 mM AMP or by 5 mM NH4+, but in the presence of both AMP and NH4+, PFK was highly active up to 1 mM ATP. At pH 8.5 and ATP levels of 0.1-12.5 mM, PFK was weakly active in the absence of added NH4+, with or without AMP. With the addition of 5 mM NH4+, PFK was highly active up to 2.5 mM ATP, while AMP was largely without effect. Concentrations of NH4+ as low as 0.03 mM stimulated PFK activity to 20% of maximal, yet the maximum was not reached until NH4+ levels were 10-30 mM. The activation of PFK by AMP and its inhibition by ATP is profoundly modified by pH. In contrast, the requirement for NH4+ is unaffected. This requirement suggests a regulatory role for ammonium ion in controlling the rate of glycolysis in trabecular meshwork. The concentration of ammonium in calf aqueous humor was found to be 0.18 mM, which is in the right range to have an effect.     

Chloride dependent intracellular pH effects of external ATP in cultured human non-pigmented ciliary body epithelium

PURPOSE: To examine the effects of extracellular adenosine 5-triphosphate (ATP) on intracellular pH ([pH](i)) in cultured human non-pigmented ciliary body epithelium (HNPE). METHODS: Intracellular pH was measured using spectrofluorescence video microscopy in isolated HNPE cells loaded with the cell-permeable acetoxymethyl ester form of the fluorescent probe BCECF. RESULTS: In 5%CO(2)/HCO(3)(-) buffered Ringer's the resting [pH](i) was 7.25 +/- 0.006 (mean +/- SEM). Application of 10 microM ATP significantly decreased [pH](i) to 7.00 +/- 0.007 (P < 10(-5), n = 14). In the presence of 1 mM suramin, a P(2) receptor inhibitor, this process was significantly blocked. This [pH](i) effect required the presence of Cl(-) and was significantly inhibited by 0.1 mM diisothiocyanatostilbene-2-2'-disulfonic acid or acetazolamide (500 microM), indicating the involvement of a Cl(-)/HCO(3)( +) exchange mechanism. This response exhibited little dependence on external Na(+) and remained unaffected by the addition of the Na(+)/H( +) exchanger inhibitor amiloride (1 mM). Clamping intracellular calcium levels by incubation in the cell permeable calcium chelator, the acetoxymethyl ester form of BAPTA (100 microM) in low extracellular calcium solution (pCa9) did not affect the ATP-induced [pH](i) signal. In addition, the vacuolar H(+)-ATPase (V-ATPase) inhibitor, bafilomycin A(1) (1 microM), failed to alter the [pH](i) transient. CONCLUSION: We have demonstrated that extracellular ATP leads to a sustained increase in [H(+)](i) in HNPE cells via a purinergic receptor activated pathway which is independent of the intracellular calcium signaling system. This study demonstrates that the ATP induced [pH]( i) transient is mediated through an upregulation in Cl(-)/HCO( 3)(-) exchange across the plasmamembrane in HNPE cells.     

Phosphorylated metabolites and effects of amphotericin B and ouabain on phosphorylation state in amphibian cornea

The phosphorylated metabolite content of endothelial denuded frog corneas and toad corneal epithelial cells was measured. The extract of frog corneas contained adenine, uridine and guanine nucleotides, in addition to various glycolytic intermediates, orthophosphate (Pi) and creatine phosphate. The relationship between the phosphorylation state: PS = [ATP]/[ADP] X [Pi] and the rate of active ion transport was studied in the toad cornea. This was considered by measuring the effects of 10(-5) M-amphotericin B and 10(-2) M-ouabain on the adenine nucleotide and Pi content of toad epithelial cells since these drugs selectively stimulate and inhibit active ion transport and metabolic rates, respectively. In order to calculate PS values, we derived and validated a relationship for converting content in terms of nmol/mg protein into concentration (mM). Incubation with amphotericin B in six different cell suspensions resulted in decreases of the ATP and ADP concentrations from 2.3 to 1.2 mM and 0.7 and 0.4 mM, respectively. The Pi concentration increased more than the ATP decrease from 6.2 to 12.2 mM. The average of these individual changes in each of the suspensions resulted in a significant decrease of the calculated PS from 599 +/- 226 to 342 +/- 127 M-1. However, ouabain increased the ATP concentration from 2.3 to 2.6 mM and decreased the ADP and Pi concentrations from 0.7 to 0.3 mM and 6.2 to 5.4 mM, respectively. The average of these individual changes in each of the suspensions resulted in a significant increase of the PS from 599 +/- 226 to 2975 +/- 1639 M-1. These results support the current notion that the PS is the feedback signal synchronizing metabolic rates with the energetic requirements of active Na and K transport.     

Taurine uptake in apical membrane vesicles from the bovine retinal pigment epithelium

Characteristics of taurine uptake were investigated in apical membrane vesicles isolated from bovine retinal pigment epithelium. Uptake of taurine into these vesicles was stimulated markedly by the presence of an inwardly directed NaCl gradient across the membrane. The time course of the NaCl gradient-dependent uptake showed an overshoot, indicating a transient accumulation of taurine in the vesicles against a concentration gradient. Monovalent cations other than Na+ did not support taurine uptake. There was also an anion requirement for the uptake system, and Cl- was the most potent among several monovalent anions tested. The NaCl-dependent taurine uptake was stimulated by inside-negative, K+, and H+ diffusion potentials, demonstrating the electrogenic nature of the system. Stoichiometric analysis revealed that two Na+ and one Cl- ions were involved in the translocation of one taurine molecule. The system had an apparent dissociation constant (Kt) of 96 +/- 17 microM and a maximal velocity of 267 +/- 24 pmol/mg of protein over 15 sec. Uptake of radiolabeled taurine was inhibited by excess amounts of unlabeled taurine, hypotaurine, beta-alanine, and gamma-aminobutyric acid. The relative potencies of the amino acids to inhibit taurine uptake were compared in three tissues, the bovine retinal pigment epithelium, the rat kidney, and the human placenta. In the apical membrane vesicles of the pigment epithelium, gamma-aminobutyric acid was many times more potent than taurine itself in inhibiting radiolabeled taurine uptake. However, in the brush border membrane vesicles of the kidney and the placenta, taurine was many times more potent than gamma-aminobutyric acid.(ABSTRACT TRUNCATED AT 250 WORDS)     

Further investigation of the role of calcium in human lens protein aggregation.

High-molecular-weight (HMW) protein from human cataractous lenses, isolated by differential centrifugation, was deaggregated in 7M urea and then reaggregated in either the presence or absence of 10 mM CaCl2. Over 90% of the material reaggregated in the presence of calcium appears to have a size greater than 50 X 10(6) daltons. By contrast, only 20% to 25% of the material reaggregated in the absence of calcium has molecular weight greater than 50 X 10(6) daltons. Disulfide formation during reaggregation is unlikely in the latter experiment, since the addition of 50 mM mercaptoethanol caused no change in results. About 60% to 70% of the low-molecular-weight (LMW) protein fraction deaggregated in 7M urea buffer can be converted to HMW species in the presence of 10 mM CaCl2, when the deaggregating agent is removed. However, only 5% to 10% of this protein is converted to HMW species if the deaggregation step is eliminated. Experiments with 45 Ca indicate that whereas calcium is necessary for the formation of the HMW aggregates, only one calcium per approximately 5 X 10(5) daltons remains bound in the reaggregated material. The data suggest that although calcium may be required to induce aggregation to HMW species, it is not required to stabilize such macromolecules. SDS-polyacrylamide gel electrophoresis of the HMW species formed upon reaggregation of the dissociated HMW species with calcium indicates the presence of all the major polypeptide subunits of the original HMW species present in the lens; however, reaggregation in the absence of calcium yields HMW species lacking in the 9600 dalton component.     

Glutathathione synthetase of bovine lens: anomalies of the enzyme-catalyzed formation of ophthalmic acid

The activity of glutathione synthetase from bovine lens was examined as a functions of the concentration of L-gamma-glutamyl-L-alpha-aminobutyrate, the dipeptide substrate required in the formation of ophthalmic acid. Several significant anomalies of the glutathione synthetase-catalyzed formation of ophthalmic acid were found. Curvilinearity of double reciprocal plots occurred with this substrate; this curvilinearity shows substrate activation of the reaction which is likely a result of negative cooperativity. Both ATP4- and, to a lesser extent Mg2+ inhibited the reaction, whereas MgATP2- is the substrate; maximum activity occurred with 2 mM Mg2+ in excess of the concentration of added ATP. This investigation shows that it is necessary to establish a defined set of conditions for reporting enzyme activity and that the usual practice of using very large concentrations of Mg2+ relative to ATP, and 5- to 20-fold excess of the dipeptide will give less than optimum activity. The unit of enzyme activity is suggested to be that activity in ml using 2 mM ATP, 4 mM Mg2+, 30 mM glycine and 15 mM L-gamma-glutamyl-alpha-aminobutyrate, which results in the formation of 1 nmole/minute of ADP or P(i). In this study, 5'-AMP was for the first time, shown to be an inhibitor of the reaction with a K(i) of 0.9 mM.     

Reductions in taurine secondary to photoreceptor loss in Irish setters with rod-cone dysplasia

These studies show that onset of photoreceptor cell degeneration preceded the loss of taurine in retinas of Irish setters with rod-cone dysplasia. The numbers of photoreceptor cell nuclei were within the normal adult range in affected setters at 10 through 26 days of age but declined rapidly between 26 and 45 days and more gradually thereafter; their numbers became reduced to 50% of normal at 45 days and then to 12-20% and 3-10% of normal at 192 and 346 days, respectively. Taurine concentrations increased within the photoreceptor cell layer during normal development to peak values (50 mM) at a time (45 days of age) corresponding to the development of adult photoreceptor function. In the affected setters, taurine levels increased as in the normal until 26 days of age and then remained at that value until 40-50% of the photoreceptor cells had degenerated. Thereafter, taurine levels declined gradually throughout the period of photoreceptor cell degeneration and were reduced to 30-40% of the normal adult level at the time (346 days) when the thickness of the outer nuclear layer was reduced to less than one complete row of nuclei. These observations agree with findings in retinal degeneration (rd) mice and RCS rats and indicate that in all three of these animal models of hereditary retinal degenerations, reductions in retinal taurine levels occur secondary to the loss of photoreceptor cells.     

Taurine binding by rat retinal membranes

Retinal membrane preparations contain endogenous taurine which is difficult to remove. By repeatedly washing the membranes in buffer the taurine content was reduced from 11.7 +/- 2.5 to 2.5 +/- 0.5 nmol/mg protein. However, complete elimination of the endogenous taurine from the membrane preparation was not achieved. Binding of [3H]-taurine to rat retinal membrane preparations revealed both high and low affinity binding sites. A Hill coefficient of 0.71 suggested that cooperativity may be involved in the taurine binding process. Taurine binding was sodium dependent with maximum binding achieved at 118 mM. At suboptimal concentrations of sodium ions (30 mM) only one binding site was observed which appears to be the high affinity binding site. Analogues of taurine were tested for their effectiveness in displacing taurine from its binding site. Hypotaurine, beta-alanine, gamma-aminobutyric acid, and guanidinoethanesulfonic acid were the most potent displacers.     

The beta-blocker carteolol inhibits contractions induced by KCl in pig ciliary arteries: an effect modulated by extracellular Ca++

BACKGROUND: To assess if the vasorelaxing properties of the ocular hypotensive beta-blocker carteolol could share some pharmacological features of calcium-channel antagonists in isolated porcine ciliary arteries. METHODS: In a myograph system (isometric force measurement), quiescent vessels were contracted with increasing concentrations of potassium chloride (KCl: 4.7 - 30 mM) in the presence of different concentrations of extracellular calcium (Ca (++): 0 - 10 mM) and/or carteolol (0 - 1 mM). Vessels precontracted with 20 mM KCl were exposed (cumulative manner) to carteolol (0.03 - 3 mM) in the presence of different concentrations of Ca (++) (0.25, 2.5, 10 mM). Contractions and relaxations are expressed in percent of 100 mM and 20 mM KCl-induced contractions, respectively (mean +/- SEM). RESULTS: Contractions induced by 30 mM KCl (99.0 +/- 4.8 %) were abolished in the absence of Ca (++) and markedly inhibited in the presence of carteolol (1 mM: 40.6 +/- 4.0 %). The inhibitory effect of carteolol on KCl-induced contractions was potentiated (0.25 mM: 6 +/- 2 %) by decreasing and reversed (10 mM: 94 +/- 5 %) by increasing Ca (++) concentrations. Vessels precontracted with KCl were relaxed by carteolol (maximum: 82 +/- 9 %), an effect potentiated (102 +/- 10 %) by lowering or inhibited (27 +/- 11 %) by increasing Ca (++) concentrations. DISCUSSION: The present in vitro pharmacological study, for which in principle no clinical interpretation should be made, suggests that carteolol shares, at rather high concentrations, some characteristics that apparently could mimic certain effects of calcium-channel blockers.     

A comparison of the effects of olopatadine and ketotifen on model membranes

Olopatadine is a human conjunctival mast cell stabilizer with antihistaminic activity. Ketotifen is an older molecule that possesses antihistaminic activity and is reported to have additional pharmacological properties. The interactions of these two compounds with model membranes (i.e., monolayers of 1-stearoyl-2-oleoyl-sn-glycerophosphocholine at the argon-buffer interface), and natural (i.e., erythrocyte) membranes were compared in an effort to understand the differences in their biological activities. Drug-lipid interaction with monolayers was determined by monitoring the surface pressure as a function of the drug concentration in the aqueous phase supporting the monolayer. Drug interaction with erythrocyte membranes was determined by monitoring changes in the permeability of the membranes to hemoglobin and 6-carboxyfluorescein as a function of drug concentration in the medium. Olopatadine and ketotifen are both intrinsically surface active and both interact with phospholipid monolayers. However, in both the presence and absence of lipid monolayers, the changes in surface pressure induced by olopatadine are lower than those caused by ketotifen. The effects of these two drugs on cell membranes were dramatically different. Exposure of bovine erythrocytes to increasing concentrations of ketotifen (1-10 mM) resulted in complete hemolysis of the cells, whereas olopatadine (1-10 mM) caused only minimal hemolysis (< 8%). Consistent results were obtained in experiments measuring the leakage of 6-carboxyfluorescein from erythrocyte ghosts as a more sensitive marker of membrane perturbation. Olopatadine treatment (0.1-10 mM) minimally perturbed the cell membrane while ketotifen (1-10 mM) caused a concentration dependent release of the fluorescent marker. These data demonstrate fundamental differences between the two drugs in their effects on cell membranes. Moreover, the differences are consistent with the surface activities of the two compounds measured in monolayers and with reported differences in their pharmacological activities. These findings offer an explanation for the biphasic non-specific cytotoxic effect of ketotifen on histamine release from mast cells and may account for the nonlytic mast cell stabilizing activity of olopatadine.     

Compartmental analysis of taurine transport to the outer retina in the bovine eye

PURPOSE: To assess the relative resistance presented individually by Bruch's membrane-choroid (BC) and the retinal pigment epithelium (RPE) to movement of taurine between the choroidal circulation and the outer retina. To quantify the effect of light-evoked changes in subretinal potassium concentration on the transepithelial transport of taurine across bovine RPE. METHODS: Transport studies were performed in Ussing chambers with intact and RPE-denuded specimens of BC. RPE viability was monitored by recording transepithelial potential (TEP) and transepithelial resistance (TER). Taurine transport with substrate concentrations in the micro- and millimolar range, reflecting physiological taurine concentrations in plasma, retina, and subretinal space was quantified by high-performance liquid chromatography (HPLC) and radiotracer techniques. Taurine transport was also assessed after apical potassium concentration was lowered from 6.0 to 2.2 mM to mimic the effects of light. RESULTS: Transport of taurine across RPE-BC at a 10-mM substrate concentration increased from 32.92 before to 111.72 nanomoles/4 mm per hour after removal of the RPE. Similarly, at 50 microM taurine, transport rates increased from 0.158 to 0.439 nanomoles/4 mm per hour after removal of the RPE. At both high (10 mM) and low (50 microM) substrate concentrations, lowering of apical potassium was associated with decreased transport of taurine across the RPE. For taurine concentrations greater than 42 microM, the rate-limiting compartment for transport of taurine to the outer retina was the RPE monolayer. Similar rates were observed across each compartment for concentrations <42 microM. CONCLUSIONS: The magnitude and directionality of taurine transport across the RPE is determined solely by the driving taurine concentration gradient and is modulated by subretinal levels of potassium. Such modulation may provide a mechanism for conserving retinal taurine. Processes that increase the resistance to diffusion across Bruch's membrane such as human ageing and increased thickening and deposition of debris associated with age-related macular degeneration (AMD) are likely to affect transport across the RPE, culminating in a secondary retinal taurine deficiency.     

Horizontal cell glutamate receptor modulation by NO: mechanisms and functional implications for the first visual synapse.

Neurons of the horizontal cell retinal neural network are subject to modulation by the neurotransmitter nitric oxide (NO). We have examined the effects of NO on glutamate receptor function in isolated horizontal cells from the perch (Perca fluviatilis) using the concentration ramp technique to simultaneously record receptor current and agonist concentration. Dose-response curves for glutamate (0-1 mM) and kainate (0-200 microM) were measured in the presence and absence of 1-2 mM sodium nitroprusside (SNP), 1 mM 8-Br-cGMP, 100 microM cyclothiazide or 200 microM dopamine as modulators. SNP increased the EC50 (i.e. decreased affinity) for glutamate and increased Imax (i.e. increased efficacy), whereas 8-Br-cGMP increased EC50, but not Imax. In the presence of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor desensitization blocker cyclothiazide, the SNP-induced increase in EC50 persisted, but the increase in Imax was blocked. The increase in EC50, but not the increase in Imax was also observed when the non-desensitizing agonist kainate (100-200 microM) was applied in the presence of SNP. When 2 mM SNP and 200 microM dopamine were applied together, they increased Imax (740 vs. 2455 pA) and EC50 (422 vs. 682 microM). Our findings indicate that NO modulates horizontal cell glutamate responses by reducing the affinity of receptors for glutamate while simultaneously increasing the maximal current. The shift in affinity is cGMP-mediated and independent of desensitization. The action of NO on horizontal cell glutamate receptors is distinct from, but synergistic with. that of dopamine. Glutamate receptor modulation by NO qualitatively predicts the action of NO on horizontal cell light responses in situ and may alter transmission at visual synapses according to adaptational conditions.     

Osmoregulatory alterations in taurine uptake by cultured human and bovine lens epithelial cells

PURPOSE: Comparative assessment of cultured human lens epithelial cells (HLECs) and bovine lens epithelial cells (BLECs) established the nature of the relationship between taurine-concentrating capability and intracellular polyol accumulation or extracellular hypertonicity. METHODS: The kinetic characteristics of active taurine accumulation based on the measurement of in vitro [3H]-taurine uptake were resolved by side-to-side review of cultured HLECs and BLECs pre-exposed to either galactose-supplemented medium or extracellular hypertonicity. Competitive RT-PCR was used to appraise variation in taurine transporter (TauT) mRNA abundance from cells maintained in hyperosmotic medium over a 72-hour exposure period. RESULTS: The capacity to accumulate [3H]-taurine was significantly lowered after prolonged (20-hour) incubation of cultured BLECs in 40 mM galactose in contrast to HLECs, the latter cells' velocity curve being indistinguishable from control cells in physiological medium. Inhibition of the intracellular taurine transport site appeared to be noncompetitive, in that there was a marked reduction in the V(max) without significant alteration in the K(m) to a high-affinity transport site. Galactitol content in BLECs exceeded five times that found in HLECs. The coadministration of the aldose reductase inhibitor, sorbinil, with 40 mM galactose completely prevented the inhibitory effect of galactose on [3H]-taurine uptake. Acute exposure (3 hours) of HLECs and BLECs to a range of 10 to 40 mM galactitol or 10 to 40 mM galactose plus sorbinil-supplemented medium suggested by Dixon plot that neither galactitol nor galactose interacted with the extracellular taurine transport site. In contrast, [3H]-taurine accumulation was markedly elevated in both HLECs and BLECs after prolonged exposure to galactose-free medium made hyperosmotic by supplementation with sodium chloride. The enhanced taurine uptake capacity involved increase in peak velocity (V(max)) without significant change in Michaelis-Menten constant (K(m)). Cultured HLECs and BLECs responded to hypertonicity with an inducible but transitory upregulation of TauT mRNA. CONCLUSIONS: These results demonstrate that lens epithelial cells express a high-affinity TauT protein capable of active uptake, but predisposed to inhibition by intracellular galactitol when the sugar alcohol is present in sufficiently high concentration to interfere with cell metabolism. Furthermore, lens epithelial cells respond to hypertonic stress by raising taurine transport activity. The increase in taurine uptake is due to an increase in the number of high-affinity TauTs expressed as a result of an increase in the manifestation of taurine mRNA stemming from exposure to hypertonic medium.     

Ascorbic acid. Cytotoxic effect on cultivated bovine lens epithelium cells]

Ascorbic acid is one of the main components (1.16 mM/l) of the aqueous humor. The molarity of this molecule is 25 times higher than in the plasma of the cow, man or horse. Now the question arises as to which function ascorbic acid has in this extremely high concentration referring to the proliferation of the lens epithelial cells. Thus, the effect of ascorbic acid was investigated upon bovine lens epithelial cells (BLEC) in the range of 0-3 mM/l. These cells were cultivated under various culture conditions (serum-free, serum-containing, aqueous-humor-containing medium) and also incubated with such mitogens as retinal extract (RE), crude endothelial cell growth factor (cECGF), basic fibroblast growth factor (bFGF) or with calcium. In each culture condition 1 mM/l ascorbic acid caused remarkable inhibition of the proliferation of BLEC. Higher concentrations (> 1.5 mM/l) revealed cytotoxic effects. These effects were independent of small variations in the pH value caused by ascorbic acid. In addition, the effect of 2 mM/l ascorbic acid in combination with catalase in a concentration of 500 Um/ml and 1000 Um/ml, respectively, was investigated. It could be shown that catalase is capable of preventing the cytotoxic effect of ascorbic acid. These results show the inhibitory effect of ascorbic acid in its physiological concentration in the proliferation of BLEC.     

Effects of intracellular calcium on lens membrane permeability

The present investigation was designed to assess whether lens membrane permeability is affected by changes in levels of intracellular calcium. Lanthanum, an inhibitor of Ca-ATPase, affected an increase in the concentration of intracellular calcium (Cai) measured in cortical fiber cells. Preculture of lenses in lanthanum (1.0mM) caused an accumulation of 36Cl during subsequent culture at a rate three-fold higher than control lenses. Changes in calcium levels, however, were not responsible for the observed flux changes because a 40mV depolarization was observed to occur prior to a significant increase in calcium levels. The non-specific effects of lanthanum and other potential inhibitors of calcium transport were avoided by preculturing lenses in an ion-HEPES medium containing 20mM calcium chloride. In lenses with a six-fold increase in calcium levels there resulted only a 10% increase in 36Cl uptake over a 3 hr period. 86Rb efflux was also measured and the rate constant was unchanged compared to control lenses. Calcium accumulation did lead to a small (8mV) depolarization which may account for the small increase in chloride accumulation. By light microscopy, morphology of cortical lens fibers and the epithelium appeared unchanged in the calcium-loaded lens. The results provide little evidence that an increase in Cai leads to acute changes in lens membrane permeability.     

Energy metabolism in human retinal Müller cells

PURPOSE: To measure selected parameters of energy metabolism and adenosine triphosphate (ATP) production in passaged monolayer cultures of human retinal glial (Müller) cells to assess the effects of varying substrate and oxygen availability on the biochemistry and histologic integrity of these cells. METHODS: Confluent Müller cell cultures were incubated for up to 4 hours at 37 degrees C in a modified minimal essential medium (no serum) under aerobic or mitochondrial-inhibited conditions in the presence and absence of 5 mM glucose or in the presence of lactate, pyruvate, glutamate, or glutamine. Cellular ATP levels, lactic acid production, and (14)CO(2) production from labeled glucose or glutamate were measured along with an examination of cellular morphology. Immunohistochemistry with antibodies to glial cell-specific proteins was also performed. Cells were positive for vimentin, but negative for glial fibrillary acidic protein and glutamine synthetase. RESULTS: Human Müller cells maintained ATP content aerobically at the same level for 4 hours in the presence and absence of glucose. ATP content was also maintained anaerobically at a value equal to that found aerobically, but only in the presence of glucose. ATP content in human Müller cells declined to a very low level when glycolysis was blocked by iodoacetate, and inclusion of lactate, pyruvate, glutamate, or glutamine did not restore the level of ATP. Aerobically, lactic acid production accounted for 99% of the total glucose used, whereas the oxidation of glucose by the mitochondria accounted for only 1%. When mitochondria were inhibited with antimycin A, there was only a modest (1.3-fold) increase in the rate of lactic acid production. No significant differences were found in the histologic appearance of the cells after mitochondrial blockade, but there was massive death of cells after inhibition of glycolysis with iodoacetate. CONCLUSIONS: These results suggest that, in the presence of glucose and oxygen, cultured Müller cells obtain their ATP principally from glycolysis and have a low rate of oxygen consumption. This metabolic pattern may spare oxygen for retinal neurons, particularly in the inner nuclear and ganglion cell layers under normal physiological conditions. Furthermore, retinal Müller cells in culture are resistant to anoxia or absence of glucose, which provides a basis for understanding why Müller cells are less susceptible than neurons to ischemia or hypoglycemia.     

Calcium transport in the lens

Evidence based on the following three observations suggests the existence of a calcium transport system in the mammalian lens: calcium levels in the lens are lower than that measured in the aqueous humor; calcium efflux is temperature-dependent and is reduced by inhibitors of Ca++ transport; and there exists a calcium-acivated, magnesium-dependent ATPase. In rat, bovine, dog, and rabbit lenses, the concentration of total calcium was found to be approximately 0.2 mM, at least an order of magnitude lower than that found in the aqueous humor. To determine the nature of the mechanism responsible for maintaining these low levels, calcium fluxes were measured. During the initial rapid phase of 45Ca efflux, the rate at 4 degrees C was reduced by 85% compared with that found at 37 degrees C. Efflux was not altered in the absence of external Na+. Calcium efflux was reduced, however, by lanthanum and propranolol, inhibitors of Ca/Mg ATPase. The presence of Ca/Mg ATPase was also demonstrated in the rat, bovine, and rabbit lens and was likewise inhibited by both lanthum and propranolol.