Octavio Quesada

Taurine: An Osmolyte in Mammalian Tissues

One of the distinctive features of taurine is its presence at high levels in most animal tissues5,3. Although differences exist among cells and species, taurine is consistently found in mM concentrations. It is noteworthy that excitable tissues including brain, striatal muscle and heart contain large, inert, and intracellular taurine pools3,9. The most prominent example in this respect is the retina. In all species so far examined, retinal taurine levels exceed 20 mM and in some species its concentration is as high as 60 mM39. In addition to these high levels, taurine in excitable tissues has a very slow turnover rate. In rat organs, values for taurine turnover rate fit into three main groups: those with a relatively fast rate calculated in less than 1 day and include liver, kidney and pancreas; a second group, with a medium rate of about 2–3 days comprises lung, spleen, intestine, testes, bone marrow and the third group, with the slowest rate of more than 3 days and up to 7 days, is represented by brain, heart and muscle30;.

Treatment with Taurine, Diltiazem, and Vitamin E Retards the Progressive Visual Field Reduction in Retinitis Pigmentosa: A 3-Year Follow-Up Study

The purpose of this study to assess the effect of the formula taurine/diltiazem/vitamin E on the progression of visual field loss in retinitis pigmentosa. A double blind, placebo controlled study in 62 patients: visual field threshold values were obtained in a Humphrey Field Analyzer from center (30°) and periphery (30–60°), every 4 months during 3-year follow-up. Data were analyzed by univariate regression, with slopes obtained from the best fit lines. Based on slope values, three groups of patients were identified as those showing negative, positive, or zero slope: ≥-1 to ≤ + 1. In controls (32 patients), at central area, the distribution in negative, zero, or positive slope was, respectively, 16 (50%),11 (35%), and 5 (15%). In the treated group (30 patients) this distribution was 6 (20%) negative, 17 (53%) zero, and 7 (23%) positive slope. In periphery, 16 control patients were distributed as 11 (69%) negative, 4 (25%) zero, and 1 (6%) positive slope. In the treated group (17 patients), the distribution was opposite: 1 (6%) negative, 7 (41%) zero, and 9 (53%) positive slope. Nineteen patients receiving treatment up to 6 years showed similar distribution by slope values. Eight out of 9 patients switched from placebo (2 years) to treatment (2–3 years), showed improving changes in their slope values. A beneficial effect of the treatment decreasing the rate of visual field loss was observed, likely through a protective action from free radical reactions in affected photoreceptors.

Efflux of osmolyte amino acids during isovolumic regulation in hippocampal slices

The efflux of potassium (K+) and amino acids from hippocampal slices was measured after sudden exposure to 10% (270 mOsm), 25% (225 mOsm) or 50% (150 mOsm) hyposmotic solutions or after gradual decrease (−2.5 mOsm/min) in external osmolarity. In slices suddenly exposed to 50% hyposmotic solutions, swelling was followed by partial (74%) cell volume recovery, suggesting regulatory volume decrease (RVD). With gradual hyposmotic changes, no increase in cell water content was observed even when the solution at the end of the experiment was 50% hyposmotic, showing the occurrence of isovolumic regulation (IVR). The gradual decrease in osmolarity elicited the efflux of 3H‐taurine with a threshold at −5 mOsm and D‐[3H]aspartate (as marker for glutamate) and at −20 mOsm for [3H]GABA. The efflux rate of [3H]taurine was always notably higher than those of [3H]GABA and D‐[3H]aspartate, with a maximal increase over the isosmotic efflux of about 7‐fold for [3H]taurine and 3‐ and 2‐fold for [3H]GABA and D‐[3H]aspartate, respectively. The amino acid content in slices exposed to 50% hyposmotic solutions (abrupt change) during 20 min decreased by 50.6% and 62.6% (gradual change). Taurine and glutamate showed the largest decrease. An enhancement in 86Rb efflux and a corresponding decrease in K+ tissue content was seen in association with RVD but not with IVR. These results demonstrate the contribution of amino acids to IVR and indicate their involvement in this mechanism of cell volume control. J. Neurosci. Res. 61:701–711, 2000.

Brain Amino Acids During Hyponatremia In Vivo: Clinical Observations and Experimental Studies

Hyponatremia is a highly morbid condition, present in a wide range of human pathologies, that exposes patients to encephalopathic complication and the risk of permanent brain damage and death. Treating hyponatremia has proved to be difficult and still awaits safe management, avoiding the morbid sequelae of demyelinizing and necrotic lesions associated with the use of hypertonic solutions. During acute and chronic hyponatremia in vivo, the brain extrudes the excessive water by decreasing its content of electrolytes and organic osmolytes. At the cellular level, a similar response occurs upon cell swelling. Among the organic osmolytes involved in both responses, free amino acids play a prominent role because of the large intracellular pools often found in nerve cells. An overview of the changes in brain amino acid content during hyponatremia in vivo is presented and the contribution of these changes to the adaptive cell responses involved in volume regulation discussed. Additionally, new data are provided concerning changes in amino acid levels in different regions of the central nervous system after chronic hyponatremia. Results favor the role of taurine, glutamine, glutamate, and aspartate as the main amino acid osmolytes involved in the brain adaptive response to hyponatremia in vivo. Deeper knowledge of the adaptive overall and cellular brain mechanisms activated during hyponatremia would lead to the design of safer therapies for the hyponatremic patient.

Isovolumetric regulation mechanisms in cultured cerebellar granule neurons

Cultured cerebellar granule neurons exposed to gradual reductions in osmolarity (− 1.8 mOsm/min) maintained constant volume up to − 50% external osmolarity (πo), showing the occurrence of isovolumetric regulation (IVR). Amino acids, Cl−, and K+ contributed at different phases of IVR, with early efflux threshold for [3H]taurine, d‐[3H]aspartate (as marker for glutamate) of πo− 2% and − 19%, respectively, and more delayed thresholds of − 30% for [3H]glycine and − 25% and − 29%, respectively, for Cl− (125I) and K+ (86Rb). Taurine seems preferentially involved in IVR, showing the lowest threshold, the highest efflux rate (five‐fold over other amino acids) and the largest cell content decrease. Taurine and Cl− efflux were abolished by niflumic acid and 86Rb by 15 mm Ba2+. Niflumic acid essentially prevented IVR in all ranges of πo. Cl−‐free medium impaired IVR when πo decreased to − 24% and Ba2+ blocked it only at a late phase of − 30% πo. These results indicate that in cerebellar granule neurons: (i) IVR is an active process of volume regulation accomplished by efflux of intracellular osmolytes; (ii) the volume regulation operating at small changes of πo is fully accounted for by mechanisms sensitive to niflumic acid, with contributions of both Cl− and amino acids, particularly taurine; (iii) Cl− contribution to IVR is delayed with respect to other niflumic acid‐sensitive osmolyte fluxes (osmolarity threshold of − 25% πo); and (iv), K+ fluxes do not contribute to IVR until a late phase (< − 30% πo).

Amino Acids as Osmolytes in the Retina

Amino acids play a role as osmolytes during the regulatory volume decrease subsequent to hyposmotic swelling, but less is known about its role when swelling occurs in isosmotic conditions. In this work we examined the efflux of labelled GABA, taurine and glutamate (traced as D-aspartate) from the chick retina, after isosmotic swelling evoked by KCl-containing solutions, and compared its features to those in hyposmotic swelling. In both conditions, GABA and taurine efflux were more sensitive to swelling than glutamate, as assessed by the activation threshold and the amount released. The amino acid efflux in hyposmotic media was decreased by DIDS, tamoxifen and NPPB, agents acting as Cl channels blockers, which also inhibit the osmosensitive Cl efflux. The component associated with swelling in the KCl-stimulated efflux was assessed by the reduction observed when Cl is replaced by an impermeant anion, or by the influence of hyperosmotic media. GABA and taurine efflux exhibited a large swelling-dependent component, which was lower for D-aspartate. This component was markedly decreased by NPPB, but this was due to an effect of the blocker preventing swelling. These results suggest that the influx of Cl, acting as K counterion, which is responsible for cell swelling, occurs through a pathway sensitive to NPPB, similarly to that activated by hyposmolarity. This finding may be of interest in studies aiming at preventing the cell edema which occurs in a number of pathologies.

Higher susceptibility of taurine-deficient rats to seizures induced by 4-aminopyridine

The susceptibility of rats made deficient of taurine by treatment with guanidinoethane sulfonate (GES), to seizures induced by 4-aminopyridine was examined. Guanidinoethane sulfonate, at a concentration of 1% was administered to pregnant rats, in the drinking water 2-3 days prior to delivery and the treatment was continued during nursing. Pups were weaned to the same treatment until 6 weeks of age. This treatment decreased levels of taurine in the cerebral cortex by 70%. 4-Aminopyridine was injected intraperitoneally at doses ranging from 4-7 mg/kg. Taurine-deficient rats showed a greater susceptibility to seizures, as demonstrated by a lowered latency for clonic seizures, an increased incidence of tonic seizures and a higher postseizure mortality. These results suggest an involvement of endogenous taurine in nervous excitability.

Influence of CA2+ on K+ efflux during regulatory volume decrease in cultured astrocytes

The calcium (Ca2+) dependence of potassium (K+) efflux activated by hyposmolarity in cultured cerebellar astrocytes was investigated, measuring in parallel experiments 86Rb release and changes in cytosolic Ca2+ ([Ca2+]i). Hyposmotic (50%) medium increased [Ca2+]i from 117 to 386 nM, with contributions of extracellular Ca2+ and Ca2+ from the endoplasmic reticulum. Hyposmotic medium increased 86Rb efflux rate from 0.015 min−1 to a maximal of 0.049 min−1 and a net release of 30%. This osmosensitive efflux was inhibited by Ba2+ (0.028 min−1), quinidine (0.024 min−1), and charybdotoxin (0.040 min−1), but was unaffected by TEA, 4‐AP, or apamin. Removal of external Ca2+ from the hyposmotic medium increased 86Rb efflux to a maximal rate constant of 0.056 min−1 and a net release of 38% and caused a delay of inactivation. These changes were due to the overlaping of an efflux activated by Ca2+ removal in isosmotic medium. This isosmotic 86Rb efflux was unaffected by TEA or 4‐AP, reduced by verapamil, and abolished by Ba2+, nitrendipine, and Mg2+. With the swelling‐induced [Ca2+]i rise suppressed by ethyleneglycoltetraacetic acid‐acetoxy‐methyl ester (EGTA‐AM), hyposmotic 86Rb was 30% reduced. The Ca2+ entry blockers Cd2+, Ni2+, La3+, and Gd3+ did not affect 86Rb efflux. A 40% decrease observed with verapamil and nitrendipine was found unrelated to Ca2+, because these agents did not affect the [Ca2+]i rise and the inhibition persisted in the absence of external Ca2+. The phospholipase C blocker U‐73122 did not affect [Ca2+]i nor 86Rb efflux. Blockers of Ca2+/calmodulin W7 and KN‐93 decreased 86Rb efflux to the same extent as EGTA‐AM. Ionomycin markedly potentiated 86Rb release in hyposmotic conditions only when [Ca2+]i was raised to about 1 μM, suggesting the implication of maxi‐K+ channels at this [Ca2+]i threshold, which nonetheless, was not attained during hyposmotic swelling. It is concluded that 86Rb efflux in cerebellar astrocytes is largely (70%) Ca2+‐independent and the Ca2+‐dependent fraction is sustained essentially by Ca2+ released from the endoplasmic reticulum and mediated by a mechanism involving Ca2+/calmodulin. J. Neurosci. Res. 57:350–358, 1999.

Light‐Stimulated Release of Taurine from Retinas of Kainic Acid‐Treated Chicks

Abstract: The light‐stimulated release of [3H]taurine from chick retina was studied in chicks intraocularly injected with kainic acid (60 nmol). This treatment produced a loss of more than 80% of the inner nuclear and the inner synaptic layers, sparing the outer retinal layers. Concomitantly, the treatment produced a marked decrease of endogenous GABA and glycine but not of taurine. The activity of glutamate decarboxylase was also markedly decreased in the kainic acid‐treated retinas. The release of [3H]taurine, either spontaneous or stimulated by light, was unaffected by the treatment. These results suggest that the light‐stimulated efflux of taurine occurs from the retinal layers which are not affected by the kainic acid treatment.

CA2+ CHANGES AND 86RB EFFLUX ACTIVATED BY HYPOSMOLARITY IN CEREBELLAR GRANULE NEURONS

Hyposmotic swelling increased 86Rb release in cultured cerebellar granule neurons (1 day in vitro [DIV]) with a magnitude related to the change in osmolarity. 86Rb release was partially blocked by quinidine, Ba2+, and Cs+ but not by TEA, 4‐AP, or Gd3+. 86Rb efflux decreased in Cl−‐depleted cells or cells treated with DDF or DIDS, suggesting an interconnection between Cl− and K+ fluxes. Swelling induced a substantial increase in [Ca2+]i to which both external and internal sources contribute. However, 86Rb efflux was independent of [Ca2+]o, unaffected by depleting the endoplasmic reticulum (ER) by ionomycin or thapsigargin and insensitive to charybdotoxin, iberiotoxin, and apamin. Swelling‐activated 86Rb efflux in differentiated granule neurons after 8 DIV, which express Ca2+‐sensitive K+ channels, was not different from that in 1 DIV neurons, nor in time course, net release, Ca2+‐dependence, or pharmacological sensitivity. We conclude that the swelling‐activated K+ efflux in cerebellar granule neurons is not mediated by Ca2+‐sensitive large conductance K+ channels (BK) as in many cell types but resembles that in lymphocytes where it is possibly carried by voltage‐gated K+ channels. J. Neurosci. Res. 53:626–635, 1998.