Control of heat production by the Ca2+-ATPase of rabbit and trout sarcoplasmic reticulum

Leopoldo De Meis

Instituto de Ciências Biomédicas, Departamento de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Cidade Universitária, Rio de Janeiro 21941-590, Brazil

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

The sarcoplasmic reticulum Ca2+-ATPase of rabbit skeletal muscle can convert the energy derived from a Ca2+ gradient into heat (L. de Meis, M. L. Bianconi, and V. A. Suzano. FEBS Lett. 406: 201-204, 1997). In this report, it is shown that this conversion varies depending on the temperature and on whether rabbit (endotherm) or trout (poikilotherm) sarcoplasmic reticulum vesicles are used. The gradient doubled the yield of heat produced during ATP hydrolysis and the calorimetric enthalpy of ATP hydrolysis (Delta Hcal) value found with both rabbit and trout varied between -10 and -12 kcal/mol in leaky vesicles (no gradient) and between -20 and -22 kcal/mol with intact vesicles (gradient). For the rabbit, the difference of Delta Hcal measured with and without gradient was detected in the range of 30-35°C and disappeared when the temperature was decreased below 30°C. For the trout, the difference was detected between 20 and 25°C and disappeared below 20°C. The effect of the gradient on the Delta Hcal for ATP hydrolysis was modified by DMSO, trifluoperazine, and heparin sodium.

calorimetric enthalpy of adenosine 5'-triphosphate hydrolysis

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

IN ANIMALS LACKING brown adipose tissue, the principal source of heat during nonshivering thermogenesis is derived from the hydrolysis of ATP by the sarcoplasmic reticulum Ca2+-ATPase of skeletal muscles (3, 32, 36). The evidence supporting this proposal is as follows. 1) Calorimetric measurement of rat soleus muscle (7) indicated that 25-45% of the heat produced in resting muscle is related to Ca2+ recirculation between sarcoplasm and sarcoplasmic reticulum. 2) In cold-acclimated ducklings, 70% of the total heat production is derived from muscle (24, 25). In these birds, the development of muscular nonshivering thermogenesis was associated with a 30-50% increase of both sarcoplasmic reticulum Ca2+-ATPase and ryanodine-sensitive Ca2+ release channel. 3) In billfish (marlin, swordfish), ocular muscles are transformed into specialized heater tissues (2, 4-6, 38). During the daily fluctuations in temperature, the swordfish reduces the temperature changes experienced by the brain and retina by warming these tissues with the heater organ. The heater tissues are composed of modified muscle cells in which the contractile filaments are virtually absent and the cell volume is packed with mitochondria and a highly developed sarcoplasmic reticulum. Activation of thermogenesis seems to be associated with the ATP-dependent cycling of Ca2+ at the sarcoplasmic reticulum.

The Ca2+-ATPase of the sarcoplasmic reticulum of skeletal muscle is able to interconvert different forms of energy. During Ca2+ transport, the chemical energy derived from ATP hydrolysis is converted into osmotic energy. After Ca2+ has accumulated inside the reticulum, a Ca2+ gradient is formed across the membrane, and this promotes the reversal of the catalytic cycle of the enzyme, during which osmotic energy is converted back into chemical energy (1, 9, 13, 21, 30, 33, 34). During cycle reversal, Ca2+ leaves the reticulum through the Ca2+-ATPase, and this is coupled with the synthesis of ATP from ADP and Pi. The Ca2+ release is referred to as active Ca2+ efflux. Not all the Ca2+ that leaves the vesicles is active; a part of it leaks through the Ca2+-ATPase without promoting synthesis of ATP, and this is referred to as uncoupled Ca2+ efflux (12, 17, 19, 20, 22, 27-29, 31). In a previous report (14), it was shown that heat is produced when Ca2+ leaks from the vesicles through the uncoupled efflux. In this process, the Ca2+-ATPase converts osmotic energy into heat. These experiments were performed at 35°C using vesicles derived from rabbit skeletal muscle. In this report, the effect of temperature and drugs on the heat released during ATP hydrolysis was studied using the Ca2+-ATPase of two animals that in physiological conditions have different body temperatures, the rabbit, an endothermic animal, and the trout, a poikilothermic animal.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Preparation of intact and permeated vesicles. Vesicles derived from the longitudinal sarcoplasmic reticulum of rabbit hindleg white skeletal muscle and from rainbow trout (Salmo gairdnerii) dorsolateral muscles were prepared as previously described (8, 26). Electrophoretic analysis revealed that the two preparations have practically no junctional proteins (8, 31). The efflux of Ca2+ measured in the rabbit and trout vesicles was not altered by ryanodine, indicating that they did not contain significant amounts of ryanodine-sensitive Ca2+ channels. The two vesicle preparations also did not exhibit the phenomenon of Ca2+-induced Ca2+ release found in the heavy fraction of sarcoplasmic reticulum. The fish used were adapted to the temperature range of 10-18°C. Rabbit permeated vesicles were prepared by incubating the vesicles at pH 9.0 in the presence of 2 mM EGTA at room temperature for 20 min. After that, the pH was readjusted to 7.0; the ATPase activity of the vesicles was maintained, but the permeability of the membrane was increased and the vesicles were no longer able to accumulate Ca2+ (23). To ensure that the vesicles did not retain any Ca2+ in the lumen, the divalent cation ionophore A-23187 (5-10 µM) was included in the assay medium in addition to the treatment with EGTA at pH 9.0.

ATPase activity, Ca2+ uptake, ATP synthesis, and heat of reaction. The methods for measuring the ATPase activity using [gamma -32P]ATP, calcium uptake using 45Ca, and ATP synthesis from ADP and 32Pi are described elsewhere (10). Heats of reaction were measured using an OMEGA isothermal titration calorimeter from Microcal (Northampton, MA) (14, 41). The calorimeter cell was filled with a reaction medium containing ATP, and the reference cell was filled with Milli-Q water. After equilibration at the desired temperature, the reaction was started by injecting the vesicles into the reaction cell, and the heat change due to the ATP hydrolysis was recorded. The calorimetric enthalpy of ATP hydrolysis (Delta Hcal) was calculated by dividing the amount of heat released by the amount of ATP hydrolyzed. The units used were moles for ATP hydrolyzed and kilocalories for the heat released. A negative value indicates that the reaction was exothermic.

Figures 1-7 are resentative experiments, and the values of Tables 1-3 are means ± SE of the principal finidings described in this study.

    RESULTS
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Introduction
Methods
Results
Discussion
References

Ca2+ transport by rabbit and trout vesicles. The activity of the Ca2+-ATPases increased with the temperature, and after 40-min incubation at 25°C, the amounts of Ca2+ retained by the rabbit and trout vesicles were practically the same (Figs. 1 and 2 and Table 1). The trout Ca2+-ATPase is unstable at temperatures higher than 25°C and is inactivated after a few minutes of incubation at 35°C (8). The rabbit ATPase, however, was stable for >1 h at 35°C. The physiological body temperature of the trout varies between 20 and 25°C, whereas that for the rabbit is 37°C. Thus, despite the fact that the two enzymes can pump similar amounts of Ca2+ at 25°C, at the physiological body temperature the rabbit sarcoplasmic reticulum was able to pump more Ca2+ and at a faster rate than the reticulum of the trout (Table 1 and Figs. 1A and 2A). After formation of the gradient, both the rabbit and the trout Ca2+-ATPases were able to synthesize a small amount of ATP, and in all conditions tested, the rate of synthesis was 45 to 25 times smaller than the rate of ATP hydrolysis (Table 1).


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Fig. 1.   Rabbit: heat released during ATP hydrolysis and Ca2+ uptake at 35°C (bullet ) and 25°C (open circle ). Assay medium composition was 50 mM MOPS-Tris buffer, pH 7.0, 0.1 mM CaCl2, 1 mM ATP, 4 mM MgCl2, and 10 mM Pi. Medium was divided into 3 samples. One was used for heat measurements (C). To the other 2 samples, trace amounts of either 45Ca or [gamma -32P]ATP were added for measurement of Ca2+ uptake (A) and ATPase activity (B). Three reactions were started simultaneously by addition of either 10 µg/ml (35°C; bullet ) or 40 µg/ml (25°C; open circle ) rabbit intact sarcoplasmic reticulum vesicles. In D, data of B and C were replotted.


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Fig. 2.   Trout: Ca2+ uptake (A) and heat released (C) during ATP hydrolysis at 25°C (bullet ) and 17°C (open circle ). Assay medium composition and other experimental conditions were as described in Fig. 1 using either 20 µg/ml (25°C; bullet ) or 40 µg/ml (17°C; open circle ) trout intact sarcoplasmic reticulum vesicles. In D, calorimetric enthalpy of ATP hydrolysis (Delta Hcal) was calculated by dividing amount of heat released measured in C by amount of ATP hydrolyzed in B.

                              
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Table 1.   Ca2+ uptake, ATPase activity, and ATP synthesis by rabbit and trout sarcoplasmic reticulum vesicles at different temperatures

Heat production in the presence and absence of a Ca2+ gradient. Both in the presence and in the absence of a Ca2+ gradient, the amount of heat released was proportional to the amount of ATP hydrolyzed (Figs. 1-3). This could be visualized by either plotting the heat released as a function of the amount of ATP hydrolyzed (Figs. 1D and 3) or calculating the Delta Hcal at each incubation interval (Fig. 2D). In an earlier report (14), it was shown that the heat released for each ATP molecule hydrolyzed by rabbit intact vesicles at 35°C was larger than that measured with leaky vesicles. We now show that the difference between intact and leaky vesicles varied depending on the temperature of the assay and the source of the vesicles used (Fig. 4 and Table 2). For the rabbit, the value of Delta Hcal measured at 35°C with intact vesicles was double that measured with leaky vesicles. This difference was no longer detected when the temperature was decreased to 25°C, as if in the rabbit the mechanism that converts osmotic energy into heat production were turned off when the temperature was decreased to a level far from the physiological body temperature. For the trout vesicles (poikilotherm), formation of a transmembrane Ca2+ gradient at 25°C leads to a change of the Delta Hcal for ATP hydrolysis to a value similar to that measured with the rabbit vesicles at 35°C. The difference of Delta Hcal values measured with trout vesicles in the presence and absence of a Ca2+ gradient was also abolished when the temperature of the medium was decreased, in this case to a value below 17°C. The Delta Hcal measured with leaky vesicles did not vary with the temperature or with the source of the vesicles used (Figs. 3 and 4 and Table 2).


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Fig. 3.   Heat released during ATP hydrolysis by rabbit and trout leaky vesicles (no gradient) at different temperatures. Ionophore A-23187 (5 µM) was included in assay medium. Other additions to assay medium and experimental conditions were as described in Figs. 1 and 2. open circle  and bullet , Rabbit leaky vesicles at 35 and 25°C, respectively; square  and black-square, trout vesicles at 25 and 17°C, respectively.


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Fig. 4.   Effects of gradient and temperature on Delta Hcal of ATP hydrolysis measured with trout (black-square, square ) and rabbit (open circle , bullet ) vesicles. Assay medium composition and other experimental conditions were as described in Figs. 1-3 for intact (solid symbols) and leaky vesicles (open symbols).

                              
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Table 2.   Delta Hcal of ATP hydrolysis: effects of gradient, temperature, and DMSO in rabbit and trout vesicles

Controls. Pi was included in the assay medium in most of the experiments described in this report. During transport, this anion diffuses through the membrane and forms calcium phosphate crystals inside the vesicles (15). The difference in heat release measured with intact and leaky vesicles was not related to the formation of calcium precipitates inside the vesicles because 1) the Delta Hcal values found without added Pi or in the presence of 10 mM Pi were the same (data not shown and Ref. 14) and 2) the amount of calcium phosphate precipitate formed inside the vesicles is proportional to the amount of Ca2+ accumulated by the vesicles (9, 15, 30). At 25°C, the amounts of Ca2+ accumulated by the vesicles of trout and rabbit were practically the same (Table 2), but only in the trout did the accumulation of Ca2+ lead to an increase of heat production during ATP hydrolysis.

The difference of Delta Hcal for ATP hydrolysis measured with leaky and intact vesicles does not seem to be related to Ca2+ efflux through the ryanodine and/or the Ca2+ channel regulated by inositol 3-phosphate for two reasons. 1) The rabbit and trout vesicle preparations used were derived primarily from the longitudinal sarcoplasmic reticulum. As previously shown (8, 31), electrophoretic analysis of the two preparations revealed that the dominant protein found in both rabbit and trout vesicles was the Ca2+-ATPase, with no ryanodine-sensitive Ca2+ channels. 2) The Ca2+ transport of the vesicles was not sensitive to inositol 3-phosphate, ryanodine, or caffeine, nor did it exhibit the phenomenon of activation of Ca2+ efflux by external Ca2+, i.e., Ca2+-induced Ca2+ release (data not shown).

Effect of DMSO. In early reports (11, 16, 18), it was shown that DMSO promotes the coupling of enzyme units that are leaky and abolished the difference of Delta Hcal for ATP hydrolysis measured with intact and leaky vesicles (14). These measurements were done only with rabbit vesicles at 35°C. We now show that DMSO only modified the Delta Hcal of ATP hydrolysis in the temperature range in which the gradient enhanced the yield of heat released during ATP hydrolysis (Table 2). Thus it had no effect 1) on the Delta Hcal measured with rabbit intact vesicles (gradient) below 25°C, 2) on trout vesicles at temperatures below 20°C, and 3) at all temperatures when the vesicles were leaky (data not shown).

Effect of uncoupling drugs. Heparin and trifluoperazine inhibit the Ca2+ uptake and the synthesis of ATP measured with rabbit vesicles at 35°C (Figs. 5 and 6). The two drugs modifiy the mechanism of Ca2+ transport in different ways (12, 19, 37). Heparin promotes a small increase of the Ca2+ efflux rate (19) and inhibits the rate of ATP hydrolysis (Fig. 5), whereas trifluoperazine promotes a large increase of Ca2+ efflux (12, 22) and enhances the rate of ATP hydrolysis (Fig. 6C). With heparin, the inhibition of Ca2+ uptake is mostly due to a decrease of the ATPase activity, whereas with trifluoperazine it is promoted by the large increase of the Ca2+ efflux (12, 19). In a recent report, it was shown that at 35°C heparin increases the yield of heat produced during ATP hydrolysis by rabbit intact vesicles (14). The effects of heparin and trifluoperazine on Ca2+ uptake and ATP hydrolysis at 25°C with both rabbit and trout vesicles were the same as those previously measured with rabbit at 35°C (Figs. 5 and 6). The effect on the Delta Hcal for ATP hydrolysis, however, was found to vary depending on the temperature and the vesicle preparation used (Fig. 7A and Table 3). For the rabbit and in presence of a gradient, the Delta Hcal for ATP hydrolysis became more negative after the addition of 2-4 µg/ml heparin, but this was only detected at 35°C. At 30°C and at 25°C, 2-4 µg/ml heparin did not modify the Delta Hcal measured with and without a gradient, despite the fact that it decreased both the amount of Ca2+ uptake and the rate of ATP hydrolysis (Table 3). For the trout vesicles at 25°C, the effect of heparin was opposite to that found with rabbit at 35°C, the Delta Hcal value measured with intact vesicles becoming less negative with increasing heparin concentrations, reaching the same value as that found with leaky vesicles (Fig. 7A). Both the rabbit and trout vesicles were no longer able to accumulate Ca2+ when the heparin concentration was raised from 4 to 10 µg/ml. In these concentrations, the vesicles were still able to hydrolyze ATP, and the Delta Hcal for ATP hydrolysis was the same as that measured with leaky vesicles in the range of 15-35°C (Table 3). The effect of heparin in rabbit skeletal muscle is antagonized by K+ (19, 37). K+ had no effect on the heat produced during ATP hydrolysis measured with either leaky vesicles (data not shown) or with intact vesicles (Table 3), but it did antagonize the effects of heparin on the Delta Hcal for ATP hydrolysis and transport of Ca2+ in both rabbit at 35°C and trout vesicles at 25°C (Table 3).


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Fig. 5.   Inhibition of Ca2+ uptake (A), ATP synthesis (B), and ATP hydrolysis (C) by heparin sodium. Assay medium composition and other experimental conditions were as described in Figs. 1 and 2 using intact rabbit vesicles at either 35°C (open circle ) or 25°C (triangle ) and trout intact vesicles at 25°C (bullet ). On figure, 100% refers to activity measured in absence of heparin sodium.


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Fig. 6.   Inhibition of Ca2+ uptake (A), ATP synthesis (B), and ATP hydrolysis (C) by trifluoperazine. Assay medium composition and other experimental conditions were as described in Figs. 1 and 2 using intact rabbit vesicles at either 35°C (open circle ) or 25°C (triangle ) and trout intact vesicles at 25°C (bullet ). On figure, 100% refers to activity measured in absence of trifluoperazine.


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Fig. 7.   Effects of heparin sodium (A) and trifluoperazine (B) on Delta Hcal of ATP hydrolysis. Assay medium composition and other experimental conditions were as described in Figs. 1 and 2 using intact rabbit vesicles at either 35°C (open circle ) or 20°C (triangle ) and trout intact vesicles at 25°C (bullet ).

                              
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Table 3.   Effect of heparin at different temperatures in rabbit and trout vesicles

The effect of trifluoperazine on the Delta Hcal for ATP hydrolysis has not been measured previously. With the rabbit at 35°C and the trout at 25°C, it was found that the Delta Hcal for ATP hydrolysis measured in the presence of a gradient became less negative with trifluoperazine, reaching the same value as that measured with leaky vesicles (Fig. 7B). At 25°C, trifluoperazine inhibited the Ca2+ uptake measured with rabbit vesicles (Fig. 6) but had no effect on the Delta Hcal for ATP hydrolysis (Fig. 7B).

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

The data reported show that the amount of heat produced during ATP hydrolysis by the Ca2+-ATPase increases when a gradient is formed across the sarcoplasmic reticulum membrane. The gradient-dependent heat production, however, seems to be arrested when the temperature of the medium is decreased more than 5°C below the physiological body temperature, i.e., below 30°C for the rabbit and below 20°C for the trout. The enhancement of heat production associated with the gradient could therefore play a physiological role in the maintenance of the body temperature but would not be a good emergency system to raise the body temperature after rapid cooling of the animal to an extreme point that leads to a large variation of the body temperature. More experiments with different animal species are needed to establish whether or not the temperature limits observed in Table 2 and Fig. 4 are specific characteristics of rabbit and trout or represent a common feature of endotherms and poikilotherms.

When ATP is hydrolyzed by the Ca2+-ATPase, part of the chemical energy released is dissipated into the surrounding medium as heat. The other part is used to pump Ca2+ across the vesicle membrane, and in this process, chemical energy is converted into osmotic energy. When Ca2+ flows back from the vesicle lumen to the medium driven by the Ca2+ gradient, the osmotic energy can be used to synthesize ATP, or it can be converted into other forms of energy that cannot be detected with the methods used in this report. An example is molecular work in which a protein cycles between two or more different conformations (35, 39, 40). The data described in this and a previous report (14) suggest that the energy derived from the gradient can also be converted into heat. However, it seems that osmotic energy cannot be transformed spontaneously into heat and that a device is needed for this conversion. For the sarcoplasmic reticulum, the device is probably the Ca2+-ATPase itself, which, in addition to interconverting chemical into osmotic energy, could also convert osmotic energy into heat. The following data support this proposal. 1) In leaky vesicles, Ca2+ is translocated across the membrane during ATP hydrolysis and afterward flows back to the medium through the permeabilized membrane. If the simple diffusion of Ca2+ through any kind of pore in the membrane would lead to heat production, then the same Delta Hcal value should have been found with intact and permeabilized vesicles (Table 2). 2) Trifluoperazine greatly enhances the Ca2+ efflux from both rabbit and trout vesicles (12; Fig. 6). If the simple diffusion of Ca2+ through the membrane were to produce heat, then in steady-state conditions the Delta Hcal for ATP hydrolysis should become more negative and not less negative as observed in Fig. 7B with 40 and 70 µM trifluoperazine. 3) In previous reports (12, 19, 37), it was shown that heparin and trifluoperazine uncoupled the reversal of the Ca2+ pump in different manners. At the concentration range used in Figs. 5 and 7, heparin promoted the same increase of Ca2+ efflux as that induced by ADP during reversal of the pump. Trifluoperazine, on the other hand, promotes an increase of the efflux orders of magnitude larger than that measured with either heparin or ADP. At present, we do not know why heparin modifies the Delta Hcal for ATP hydrolysis in rabbit and trout differently (Fig. 7 and Table 3). Perhaps this is related to differences in the protein structure of the two Ca2+-ATPase isoforms. In spite of this difference, however, if the simple diffusion of Ca2+ through the membrane were to produce heat, then it would be expected that heparin should have the same effect on the Delta Hcal value measured with rabbit and trout vesicles.

A possible interpretation of the data presented in this and previous reports (12, 14, 16-22) is that during transport in intact vesicles, the different Ca2+-ATPase units could operate in four different modes as follows. 1) Most of the enzyme units would work in the forward mode totally coupled, cleaving ATP and accumulating Ca2+ inside the vesicles. In this mode, one part of the energy derived from the hydrolysis of ATP would be dissipated as heat (10-12 kcal/mol), and the other part would be converted into osmotic energy. 2) A small part of the enzyme units would work in the reverse direction and synthesize ATP in a process coupled with a slow Ca2+ efflux. In this condition, there would be no heat production. 3) A few enzyme units would be partially uncoupled, working in the reverse direction but without synthesizing ATP, and the slow Ca2+ release would produce heat. In this form, the ATPase would convert osmotic energy into heat, and this would account for the difference of heat production measured during the hydrolysis of ATP by intact and leaky vesicles in Table 2. This enzyme form is thermosensitive and becomes incapable of converting osmotic energy into heat below 30 or 20°C depending on the animal species studied. As shown previously (16, 18, 20), DMSO couples these leaky enzyme units, arresting the heat production and increasing both the amount of Ca2+ retained by the vesicles and the amount of ATP synthesized. In the rabbit, heparin increases the fraction of enzyme units that are partially uncoupled and produces heat. 4) The enzyme would be totally uncoupled as observed with trifluoperazine (12). Now Ca2+ leaves the vesicles at a fast rate in a process that is not coupled to either ATP synthesis or heat production (Figs. 6 and 7B).

Finally, the effects of DMSO, heparin, and trifluoperazine on the Delta Hcal of ATP hydrolysis raise the possibility that in living animals drugs may change the yield of heat derived from the transport of Ca2+ and the hydrolysis of ATP by the sarcoplasmic reticulum, thus characterizing a new mechanism of thermoregulation in which the drugs act directly at the source of heat production, changing the amount of heat generated from each molecule of ATP cleaved during the transport of Ca2+ and not indirectly through hormones, receptors, and nervous system that enhance the metabolic activity of the cell.

    ACKNOWLEDGEMENTS

I thank Valdecir A. Suzano for technical assistance.

    FOOTNOTES

This work was supported by grants from PRONEX-Financiadora de Estudos e Projetos, Conselho Nacional de Desenvolvimento Científico e Tecnológico, and Fundaçao de Amparo à Pesquisa do Estado do Rio de Janeiro.

Address for reprint requests: L. de Meis, Instituto de Ciências Biomédicas, Departamento de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Cidade Universitária, RJ 21941-590, Brazil.

Received 13 November 1997; accepted in final form 23 February 1998.

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Abstract
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Methods
Results
Discussion
References

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Am J Physiol Cell Physiol 274(6):C1738-C1744
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