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

0042-6989/82/121487-07903.00/0

I/ision Res. Vol. 22. pp. 1487 to 1493, 1982 Printed in Great Britain

Pergamon Press Ltd

TAURINE-CALCIUM INTERACTIONS IN FROG ROD OUTER SEGMENTS: TAURINE EFFECTS ON AN ATP-DEPENDENT CALCIUM TRANSLOCATION PROCESS H. PASANTES-MORALES Departamento Universidad National

de Neurociencias, Centro de Investigaciones en Fisiologia Celular, Aut6nona de Mtxico, Apartado Postal 70-600, 04510 Mkxico, D.F. Mtxico

Abstract-Frog rod outer segments (ROS) isolated in a Ca-free, EGTA-containing medium, showed a rapid ATP-dependent accumulation of 45Ca GTP , CTP, ITP, UTP, AMP, GMP and j?- y-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, for Ca accumulation was reduced in illuminated uptake was 66 PM with a V,,,,, of 12.5 nmoI/mg protein. %a ROS. The presence of 5-25 mM taurine, but not of GABA. glycine, histidine or proline, markedly enhanced Ca uptake by ROS.

INTRODUCTION

a sulfur amino acid that occurs universally in animals, is one of the most abundant components of the amino acid pools of most organs (Jacobsen and Smith, 1968). Excitable tissues, in particular, contain a high taurine concentration, l-10mM in the brain (Mandel and Pasantes-Morales, 1978), 3-38 mM in the heart and muscle (Kocsis et al., 1976) and l&lOOmM in the endocrine glands (Crabai et al., 1974). Taurine exists in the free amino acid form and is not found as a component of more complex molecules such as lipids or proteins. Elucidation of the role of taurine in animal tissues has been particularly difficult. Until now, except for its conjugation with cholic acid in the liver, it has not been possible to assign a physiological role to taurine. The retina is one of the tissues that contains a very high taurine concentration. Taurine levels in the retina of all species studied so far, range from 10 to 20 mM, and this one compound comprises more than 50% of the free amino acid pool (Pasantes-Morales et al., 1972). Studies on the intraretinal distribution of taurine have shown its prevalent concentrations at the photoreceptor layer (Orri et al., 1976). In the photoreceptor cells, taurine attains levels as high as 80 mM. Taurine is released from isolated retina upon light stimulation (Pasantes-Morales et al., 1973). Pharmacological and biochemical approaches suggest that this release occurs from the photoreceptors (Pasantes-Morales et al., 1981a,b; Salceda et al., 1977) thus relating taurine to the photoexcitation process. However, the steps or mechanisms of its possible action remain unknown. An involvement of taurine in the processes regulating calcium (Ca) levels in photoreceptors has been suggested by previous studies that show taurine effects on Ca fluxes in isolated retinal subcellular fractions (Pasantes-Morales et al., 1979). In frog rod outer segments (ROS), incubated in a

Taurine,

medium containing 1.5-2.5 mM external calcium, taurine reduces the accumulation of 45Ca, whereas at low external Ca concentration, below 100 PM taurine 4sCa translocation (Lbpez-Colornit and increases Pasantes-Morales, 1981). Studies on Ca fluxes in the ROS suggest the existence of two different Ca accumulation processes; one, which could correspond to a Ca/Ca-Na/Ca exchange, most likely occurring at the ROS external membrane (Schnetkamp et al., 1977), is similar to that described in isolated nerve endings, mitochondria and secretory tissues (Blaustein and Oborn, 1975; Crompton et ul., 1976; Rink, 1977); a second process, probably localized at the ROS disks, appears to be an active, ATP-dependent Ca translocation process (Schnetkamp et al., 1977). This latter mechanism has been observed only under certain conditions and is not reproducible in frog ROS (Szuts, 1980). We have observed an ATP-dependent 45Ca uptake by frog ROS isolated in a Ca-free EGTA containing medium. It is specifically stimulated by ATP and shows a strong Mg requirement. This Ca accumulation is activated by taurine (Pasantes-Morales and Ordbiiez, 1982). In the present work, further properties of this Ca translocation process and of the effect of taurine, are described.

MATERIALS

AND METHODS

Retinas of dark adapted frogs were excised in a physiological medium containing 118 mM NaCl, 4.7mM KCl; 1.17mM MgSO,, 1.2mM KH2P04; 5.6 mM glucose and 2.5 mM NaHC03 (pH 7.4). ROS were isolated by mildly vortexing the retinas in tubes containing the physiological medium plus 1 mM EGTA, then pelleted by centrifugation at 900 g for 10 min. The pellet was washed once in an EGTA-free medium. In some experiments, ROS were purified on a ficoll gradient, according to the procedure of Szutz

1487

H. PASANTES-MORALES

148X

and Cone (1977). The ROS pellet was resuspended in 1 ml of lo”,, ficoll (w/v) and was layered over a gradient formed by 5 ml 2.5”,, ficoll and 5 ml lo”,;, ficoll. The gradient was centrifuged for 10min at 58.OOOg (SW 40 rotor, 4°C). and the floating ROS were collected at the interphase. pelleted by diluting with the medium and centrifuged at 900 g for 10 min: the pellet was washed twice with physioIogical solution. All these procedures were carried out under dim red light. “‘Ca accumulation was assayed in the physiological solution described. plus 0.2 LtCi of 45CaC12. 1 mM ATP. and the additions indicated at each experiment, in a final volume of 1 ml. The total free Ca concentration of the incubation mixture was controlled with a Ca EGTA buffer as a function of total Mg, pH and temperature according to Portzehl et ul. (1964). The pH of the incubation media was adjusted by gassing with a S’,, CO>-95”,, 0, mixture or with 100°Z~,OZ. The reaction was started by adding a ROS suspension containing 50m70j1g of protein. After IO min of incubation at 25 C, 0.3 ml aliquots of the suspension were withdrawn and centrifuged in a Beckman microfuge. Pellets were washed briefly with distilled water and solubilized with NCS (Tissue solubilizer, Amersham). Accumulated radioactivity was measured in the solubilized tissue after the addition of Tritosol (Fricke. 1975). In some experiments, the pellet was rapidly washed with a 2.5 mM unlabelled CaCl, and 1 mM EGTA containing medium. After this the total radioactivity decreased by treatment, 15-20”,,. The c~3ncentratioll of rhodopsin in ROS preparations was measured by difference spectra after solubilization in lol) Triton X-100 and bleaching. Cytochrome oxidase activity was measured by oximetry. according to Schnaikman et 01. (1967). The didansyl-cysteine assay was carried out following the method of Yoshikami et al. (1974) by exposing the ROS to the reagent (10 PM) for 1 min and then centrifuging the pellet prior the light microscope observation. Proteins were measured by the procedure of Lowry

(‘t LI/. ( 195 1).

Table

RESULTS Prepuration

ROS isolated by mild vortexing contain, as main contaminants, pigment epithelium cells, some erythrocytes and mitochondria from the inner segments. In order to establish the presence of Ca uptake in ROS, an estimate of the contribtltion of contaminants to this process is required. To this purpose; ROS were separated in a ficoll gradient and the rhodopsin content, the cytochrome oxidase activity and the “%a uptake were measured in purified ROS floating at the ficoll interphase and in the pellet containing the unbroken cells and mitochondria. Table 1 shows that rhodopsin was concentrated at the 1025”,, ficoll interphase whereas it was absent in the pellet. This contained 75% of the cytochrome oxidase originally present in the retinal homogenate. whereas no enzyme activity could be detected in the ficoll interphase. The distributiotl of “Ca acc~lmulation on the gradient, paralleled that of rhodopsin, the highest specific activity existing at the ficoll interphase. The contamination of the crude ROS preparation with erythrocytes was estimated by directly counting the number of red cells per number of ROS at the microscope and was found to be less than 0.3”/,. ROS in both, the crude and the purified preparation. were found to have their plasma membrane osmotically ruptured, as estimated by the didansyl cysteine assay. appears to be associated Since 45Ca accumulation with the ROS fraction. the crude preparation was routinely used.

The

time-course

of 45Ca

uptake

by

1. Distribution of rhodopsin content. cytochrome oxidase activity and ATP-dependent accumulation in a rod outer segment preparation fractioned on a ficoll gradient

Fraction I O-25”,, Ficoll interphase Pellet Crude preparation

Frog rod outer segments formed by 5 ml 25”” 58.GGU9. Cytochrome lation were measured the means I SEM of

frog

ROS

is

shown in Fig. 1. In the absence of ATP, approximately 0.3 nmol of Ca per mg protein were bound to ROS within 60 set and no significant additional binding occurred during the following 10 min. In the presence of 1 mM ATP, Ca uptake increased almost tenfold after I min of incubation and reached a maximum at approximately 3 min (Fig. 1). ATP-dependent 45Ca uptake by ROS was not observed when iono-

‘ZCa

Cytochrome oxidase p atoms of O,/min/mg protein

ATP-dependent Ca uptake nmol!mg

ND(h)

2.5 * 0.32 (6)

12.6 i 0.6 (6)

2.01 rt 0.16(4) 2.67 I: 0.19 (4)

0.9 Ifr O.lO(6) 4.0 * 0.01 (19)

ND(4) 2 I .o & 0.25 (4)

Rhodopsin protein

were isolated as described in the text and layered over a &oil gradient ficoll and 5 ml IO&, ficoll. The gradient was centrifuged for IO min at oxidase activity, rhodopsin content and ATP-dependent “‘Ca accumuin the pellet and in the fmCtiOn floating at the Ficoll interphase. Results are the numher of separate experiments indicated in parenthesis.

Taurine-calcium

interactions in frog rod outer segments

1489 T OC

t

,

I

1

I

I

3

5

IO

TIME

(min)

The time-course of 45Ca accumulation by frog rod outer segments Ca uptake activity was assayed in a medium containing 118 mM NaCl, 4.7 mM KCI, 1.2 mM KH,P04, 1.17 mM MgSO,, 1 mM ATP 2.5 mM NaHCO, and 20 PM 4sCaCl, in a final volume of 1 ml. The reaction was started by addition of the ROS suspension (5&7Opg) protein). ROS were incubated at 25°C with shaking; at the indicated times, 0.3 ml aliquots of the incubation mixture were withdrawn, centrifuged in a Microfuge and the radioactivity accumulated was measured in the solubilized pellets after the addition of Tritosol. Ca ionophores A23187 (2OnM) and X537A (24pM) were added to the medium dissolved in ethanol. (0) Control; (0) ATP omitted; (A) X537A, (a) A23187 added from the start (m--D) A23187 added at 10 min. Each point represent the mean & SEM of 4--19 separate experiments.

phores such as A23187 and X537A that abolish the Ca gradient, were present at the beginning of the incubation. The addition of A23187 after 15min of incubation caused a release of the accumulated 45Ca (Fig. 1). No ‘%a accumulation was observed when the ROS preparation was extensively sonicated or treated with 0.05% Triton X-100.

,301

31

32

33

34

35

ATP

GTP UTP CTP 3’5’ cyclic AMP 3’5’ cyclic .GMP .__ ~-y-methylene ATP

Concentration (mM) 0.5 1.0 2.0 3.0 1.0 2.0 1.0 2.0 1.0 2.0 1.0 1.0 1.0

37

1/Tx IO4 Fig. 2. Arrhenius plot of the ATP-dependent 45Ca uptake by frog rod outer segments. Isolated ROS were incubated described in Fig. 1 legend at various temperatures. Each point represent the mean of 4-19 experiments. The activation energy calculated from the plot was 7.9 kcal/mol.

Maximum stimulation by ATP was found at a 2 mM concentration of the nucleotide (Table 2). 4sCa uptake by ROS specificaliy required ATP, other nucleotides, such as GTP, CTP, ITP, UTP, added at various concentrations, did not support 45Ca uptake. Cyclic GMP and cyclic AMP were ineffective as well as /I- y-methylene ATP, the non-hydrolyzable analog of ATP (Table 2). The ATP-dependent Ca uptake was temperaturedependent, a marked reduction in the uptake measured at 25°C occurred at 4°C while an increase of the incubation temperature to 37°C increased 45Ca

Table 2. Effect of nucleotides on 45Ca uptake by frog rod outer segments Nucleotide

36

+ Nucleotide/basal 2.16 + 0.19(4) 4.57 + 0.37 (19) 5.23 k 0.61 (4) 5.16 + 0.62(3) 1.02 * 0.13 (4) 0.91 ? 0.12 (4) 1.19 + 0.19(4) 1.07 +0.16(4) 1.01 + 0.14 (4) 1.01 + 0.16(4) 0.93 & 0.10(4) 1.16 & 0.12(4) 1.29 f 0.16(6) 1.33 f 0.17(4)

+ Nucleotide/basal + taurine

9.47 + 0.03 (19)

I. 23 _C0.16(4) 1.16 t_:0.12(4)

1.36 f 0.12(4)

45Calcium uptake was assayed as described in Methods except that the nucleotide triphosphate was varied as indicated. Values of 45Ca a~umulation without nucleotide (basal) were 0.41 + 0.63 nmol/mg protein. Results are the means & SEM of the number of experiments indicated in parenthesis.

H. PASANTES-MORALES

1490 Table

3. Effects of cations on the ATP-induced uptake by frog rod outer segments

4’Ca uptake nmol;mg protein

100 65 63 19 22 101 29 100

chloride

118 mM NaCI, no Mg 118 mM NaCI, Mg 0.5 mM 118mM NaCI, Mg l.OmM 118 mM NaCI, No Mg, 2 mM Ca 118 mM NaCl, no phosphate The

Table 4. The effect of illumination on the ATP-dependent 45Ca accumulation by frog rod outer segments

“i, of 45Ca accumulated

Medium 118 mM NaCl 118 mM KCI 118 mM Choline

“‘Ca

+ + * * + k *

4.5 7.1 2.3 1.9 6.0 2.1 7.1

medium contained in addition bicarbonate buffer, 2.5 mM, I mM ATP; 1.47 mM MgSO,, and 20pM 45CaCI,, pH 7.6 incubation was carried out at 25°C for 10 min in the dark. Results are the means of 3-19 separate experiments + SEM.

Control Dark Light

3.52 i 0.27 (6) 1.55 + 0.12(8)

Taurine

(25 mM)

7.61 + 0.41 (6) 2.65 _I 0.24 (8)

Frog rod outer segments. isolated in the dark. were incubated at 37°C. during IO min. in the medium described in Methods, in darkness or under continuous illumination. Light intensity was so as to give maximal bleaching. Results correspond to the ATP-dependent 45Ca accumulation; “‘Ca accumulation in tubes incubated without ATP was subtracted from values obtained in the presence of ATP. Results are the means & SEM of the number of experiments indicated in parenthesis.

(Fig. 2). The activation energy calculated from the Arrhenius plot was 7.9 kcal/mol. The effect of different cations on 45Ca uptake is shown in Table 3. Ca accumulation showed a strict dependence on Mg ions. Incubation in a Mg-free medium reduced 45Ca uptake to less than 2O”/, of the controls incubated with Mg. Ca could not replace Mg. In other Ca transport systems (Blaustein and Oborn, 1975; Crompton et al., 1976) monovalent cations depress or stimulate Ca fluxes. Sodium. in particular, has been observed to compete with Ca, and therefore decrease 45Ca accumulation. In the ROS preparation, omission of sodium decreased the ATP-dependent 4”Ca uptake only by about 35”“. Similar results were obtained when choline chloride or potassium chloride replaced the sodium chloride (Table 3). The ATP-dependent Ca uptake was measured as a function of the free Ca concentration in the medium (Fig. 3). Ca uptake reached a maximum at an approximately 20 LAM external concentration. A maximal velocity, (L’,,,) of 12.5 nmol of Cajmg of protein and an apparent K, for free Ca of 66 PM were calculated from a double reciprocal plot (Fig. 3). accumulation

-

Log

[Cal

. 2-

%

I 0.1

I 0.6

CL2 x

O4

Fig. 3. Effect of free Ca concentration on Ca uptake by ROS. Experimental conditions as described in Fig. I legend. Free [Ca] was controlled with a Ca EGTA buffer as a function of total Mg, pH and temperature. The kinetic features of the ATP-dependent Ca uptake were calculated from a double reciprocal plot (B) of the results in A. The apparent K, of the Ca transport system was 66 PM with a V,,, of 12.5 nmol of Ca/mg of protein. Solid line, control. Dotted line, 25 mM taurine.

Table 4 shows the effect of illumination on the ROS ATP-dependent Ca uptake. The experiments were carried out both at 25 and at 37 C, since Ostwald Heller (1972) have reported that the effects of light on a Ca-Mg ATPase activity of a frog ROS preparation are only observed when the preparation is assayed at 37’ C but not at 15‘ or at 24 ‘C. In a ROS preparation incubated at 37’ C. illumination significantly reduced the ATP-dependent 4”Ca accumulation.

Addition of taurine to the incubation medium at 5-25 mM, concentrations produced a marked increase in the ATP-dependent Ca accumulation. The taurine effect increased with increasing time. and was maximal after 15 min of incubation. The stimulatory effect of taurine was concentration-dependent. since an in-

Taurine-calcium

interactions in frog rod outer segments

Table 5. The effect of taurine and other amino acids on the ATP-dependent 45Ca accumulation by rod outer segments “Ca uptake nmol/mg protein

Amino acids (10 mM) Control Glycine /I-alanine Histidine Proline Glutamic acid Cysteine sulfinic acid Guanidinoethyl sulfonate Taurine (25 mM) Taurine (10 mM) Taurine (5 mM)

3.9 f 3.7 + 4.8 + 3.6 + 3.8 i 6.3 f 6.0 + 3.7 + 8.2 k 6.7 * 5.7 +

0.16(19) 0.35 (4) 0.67 (6) 0.41 (4) 0.42 (4) 0.54 (4) 0.62 (4) 0.29 (4) 0.52 (19) 0.66 (4) 0.54 (4)

ROS were incubated in the presence of the amino acids at the concentrations indicated, during IOmin, at 25°C. The incubation medium contained NaCI, 118mM; KCI, 4.7mM; MgS04, 1.17mM; NaHCO,, 2.5mM; ATP, 1mM and 20pM 45CaCI,. Results are the means f SEM of the number of experiments indicated

in parenthesis.

crease of 41% over control values without taurine was observed in the presence of a 5 mM amino acid concentration, At 10 mM and 25 mM, taurine increased 45Ca uptake by 21% and 110x, respectively (Table 5). In order to test the specificity of the taurine effect, other amino acids were tested for their ability to promote 45Ca accumulation. At a 10 mM, concentration, glycine, GABA, histidine and proline, did not affect Ca accumulation; guanidino-ethyl sulfonate, the sulfur taurine analog, did not increase 45Ca uptake, whereas p-alanine, the carboxyl analog, produced a slight increase. The acidic amino acids, glutamic acid, and cysteine sulfinic acid also had a stimulatory effect (Table 5). As we have previously described (LopezColomt and Pasantes-Morales, 1981), taurine reduces 45Ca accumulation, where ROS are incubated in a medium containing 2.5 mM CaC12 whereas the opposite is true when the Ca concentration in the medium is lower than SoOpM. The effect of taurine on the kinetic constants of the ATP-dependent 45Ca accumulation was studied. Taurine was found to affect the V,,, without modifying K, values (Fig. 3). The taurine effect on illuminated ROS was also examined. The taurine activating effect was higher in dark incubated ROS in this case, taurine increased 45Ca accumulation by about 130% whereas in the illuminated RQS, the taurine produced increase was less than 900/;,(Table 4). DISCUSSION Intracellular and extracellular Ca activity have been implicated in the regulation of multiple functions in ROS. In particular, Ca has been proposed as the messenger initiating the changes in permeability of the cell membrane which lead to the hyperpolarization of the photoreceptor; according to the Ca hy-

1491

pothesis of phototransduction, Ca ions, sequestered in the ROS disks are released into the outer segment cytoplasm when light bleaches rhodopsin, and cause the sodium dark current to drop, thus producing ROS hyperpolarization (Hagins and Yoshikami, 1974). The Ca content of the ROS has been measured and found to be about 5 mM inside the disks, whereas the Ca concentration in the dark adapted outer segment cytoplasm is about 1 PM (Szutz and Cone, 1977). The mechanisms by which ROS maintain this asymmetric distribution of Ca are still unknown. Schnetkamp et al. (1977) have shown that cattle rods with a leaky plasma membrane, contain a specific calcium translocation system sensitive to sodium but not to magnesium or potassium. In frog ROS we find a mechanism of 45Ca accumulation very similar to the transfocation system described by Schnetkamp et al. (1977). Ca accumulated by this mechanism is not affected by ATP, ruthenium red, oligomycin, ethylmaleimide or DCCD and it is inhibited when sodium gradients are reversed. This process of 45Ca accumulation shows marked differences from the ATP-dependent Ca translocation process whose properties have been previously described (Pasantes-Morales and Ordoiiez, 1981) and extended in the present study. The main differences concern: (1) the activating affect of ATP, (2) its pharmacological sensitivity, (3) its requirement for magnesium, (4) its insensitivity to monovalent cations and (5f the optimum pH values. Altogether, these differences clearly indicate that two mechanisms are operating for ?Za accumulation in frog ROS. The ATP-dependent “Ca uptake herein described exhibits the properties of an active system and rep resents most likely a Ca translocation process across membranes. The effect of Ca ionophores as well as that of disrupting agents, suggests that Ca is being sequestered into vesicular structures. Since we are dealing with a leaky preparation, these structures probably involve the ROS disks. The specific requirement for ATP and the inability of the non hydrolyzable-ATP analog to substitute for ATP, raises the possibility of an ATPase involvement in the Ca accumulation. ATP is known to alter Ca binding to membrane preparations, but in our system it seems unlikely that the observed increase in 45Ca is only a manifestation of calcium binding as Ca ATP to superficial sites of ROS membranes. The specificity for ATP and the requirement of Mg would be contrary to this interpretation. Since Ca and Mg bind to ATP with nearly equal affinity (O’Sullivan and Perrin, 19&l), lowering the Mg concentration should have increased Ca ATP binding and therefore, in the absence of Mg more and not less Ca should have been bound. The inhibitory effect of compounds known to block active Ca transport in other systems, such as DCCD, oligomycin, ethylmaleimide and mersalyl (PasantesMorales and Ordoiiez, 1982) strongly support the suggestion that an active calcium translocation, probably dependent on the hydrolysis of ATP is occurring

1492

H. PASANTES-MORALES

in ROS. The presence of Mg ATPase activities has been reported in vertebrate retinas as well as in ROS obtained from bovine retinas, and from toad retinas (Ostwald and Heller, 1972; Thacher, 1978, Winkler and Riley, 1977). An oligomycin and DCCD-sensitive Mg ATPase, most likely present at the photoreceptor disk membrane has been recently reported by Uhl et al. (1979). This ATP-hydrolyzing activity has been related to structural changes occurring within the disk membrane upon illumination. The possibility that some of these ATPase activities are related to Ca translocation seems likely; evidently, the direct demonstration of a coupling between ATP hydrolysis and Ca accumulation in the same preparation, showing identical kinetic constants and similar pharmacological sensitivity, is necessary in order to support this suggestion. The effect of taurine on increasing ATP-promoted 45Ca accumulation at concentrations which are within the range of its physiological levels inside the ROS, provides further support for the involvement of this amino acid in mechanisms regulating photoreceptor Ca levels. The effect of taurine is not shared by amino acids like GABA, glycine or b-alanine, which, like taurine, depress neuronal activity. This suggests that the effect observed in ROS is different from that occurring at the postsynaptic receptors. Acidic amino acids such as glutamic acid and cysteine sulfinic acid, also show some stimulatory effect; however, since these compounds are not present in the ROS, their effect cannot be related to a physiological action. It is certainly difficult at this point, to define the mechanism whereby taurine is affecting Ca fluxes, as well as its relationship with the physiological role of the amino acid in the photoreceptors. Taurine has also been implicated in the mechanisms responsible for maintaining photoreceptor structure. This has been suggested by the studies of Hayes et al. (1975) in cats deprived of dietary taurine. This species cannot synthesize taurine and therefore depends on the dietary supply for maintaining normal tissue levels. Depletion of taurine in retinas of taurinedeprived cats is accompanied by a severe disturbance in the structure of the outer segment of the photoreceptor which finally leads to cell death and blindness. Taurine, but no other amino acid or precursor, prevents or arrests the degeneration produced by this deficiency. Taurine also protects the structure of isolated frog ROS exposed to continuous illumination (PasantesMorales et al., 1981a,b) in an in dro system. The mechanism of this action of taurine remains unknown. The findings of the present study might relate taurine effects on Ca uptake to its protective action in ROS structure. The light regulated sodium channels responsible for the polarization of the photoreceptor are highly sensitive to cytoplasmic Ca levels (Wormington and Cone, 1978) which have to be kept low in order to maintain the photoreceptor resting properties. Ca sequestering in the intradiskal space has been

considered cytoplasmic

as a possible mechanism calcium

concentration,

for regulating and

therefore,

the a

would simulate continuous illumination and might lead to an alteration of the ROS structure, similar to that observed in continuously illuminated retinas. Taurine, contributing to maintain Ca inside the disks, might thereby be protecting the ROS structure. Interestingly, the degenerative pattern of taurine-deficient photoreceptors shows strong similarities with the structural alterations produced by light. failure

of this mechanism,

Acknowledgenlents-This work was supported in part by grant 2,Rdl EY 02540-03 from the National Eye Institute and bv grant ICAINAL-800792 from CONACvT. 1 am please2 ts acknowledge the excellent technical asiistance of MS A. Ord&ez. I would also like to thank Dr C. D. B. Bridges for a gift of N-dansylcysteine.

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