Camp. Biochem. Physiol.
Vol. 102A,
No. 1, pp.
0300-9629/92$5.00+ 0.00 0 1992Pergamon Press plc
117-122,1992
Printed in Great Britain
TAURINE
FLUX
IN CHICKEN
ERYTHROCYTES*
DALE W. PORTER and WILLIAM G. MARTINt Division of Animal and Veterinary Sciences, West Virginia University, Morgantown, WV 26506-6108, U.S.A. Telephone: (304) 293-2631 (Received 12 August 1991)
Abstract-l. The intracellular taurine concentration in chick erythrocytes increased with age. 2. Erythrocyte taurine influx and efflux rates increased with age. 3. Erythrocyte taurine influx decreased when the extracellular sodium concentration was below normal physiological concentrations. 4. Under hypo-osmotic conditions, taurine efflux from erythrocytes increased. 5. The data suggest that chick erythrocyte taurine metabolism changes during early post-hatch development and that one taurine function may be as an osmoregulator.
MATERIALSAND
INTRODUCTION Taurine (TAU) is a p-amino acid found at high concentrations in various animal species, including avian species (Jacobsen and Smith, 1968). The chick embryo (Lowe and Roberts, 1955), chick (Martin et al., 1966) and hen (Mason et al., 1965) are able to synthesize TAU from inorganic sulfate (Martin et al., 1966; Miraglia and Martin, 1969; Sass and Martin, 1972), and the rate of TAU biosynthesis from inorganic sulfate is enhanced by L-methionine in the chick (Martin, 1972). TAU has not been extensively studied in the chicken erythrocyte (RBC), but the mammalian RBC is known to have a very low TAU concentration (McMenamy et al., 1960). The avian RBC differs physiologically and anatomically from the mammalian RBC on the basis that it has a shorter life span (28-35 days), is nucleated and contains mitochondria (Sturkie, 1986; Harris, 1971). Thus, the avian RBC may also differ from the mammalian RBC in terms of its TAU content. At hatch, the chick RBC population is composed of embryo-derived RBC which are gradually replaced by RBC which are produced after hatching. During the transition from embryonic to post-hatch conditions, alterations in some RBC intracellular regulatory molecules occur (Bartlett and Borgese, 1976). However, TAU has not been studied with regard to age-related changes in its intracellular concentration or function in the chick RBC. Therefore, experiments were conducted with chick RBC from hatch through 4 weeks of age to determine the effect of age and the extracellular sodium (Na+) concentration on the RBC intracellular TAU concentration and the rates of TAU influx and efflux.
*Published with approval of the director of the West Virginia Agricultural and Forestry Experiment Station as Scientific Article No. 2292. tTo whom correspondence should be addressed.
METHODS
Maintenance of animals
Broiler-type chicks (Gallus gallus) were reared from hatch the West Virginia University Poultry Farm until they were 4 weeks old. Chicks were fed a standard corn-soybean meal ration adequate in all nutrients ad lib., and provided free access to tap water. at
Whole blood collecrion and RBC isolation
Blood was collected from chicks at the following ages: hatch, 3, 7, 10, 14, 21 and 28 days. Blood collection was from individual chicks by cardiac puncture following chloroform anesthesia using a heparin (20mg/lOO ml solution) coated syringe. The RBC pellet was obtained from each blood sample after 10min centrifugation at 900 g. The plasma and buffy coat were aspirated and the RBC washed twice in a saline solution (0.9% NaCl). After washing, the RBC were resuspended in saline solution prior to counting (Coulter Counter Model Zm). Taurine concentration and frux studies
Plasma and RBC TAU content was determined by HPLC (Porter er al., 1988). The RBC intracellular water content was determined by wet cell - dry cell weight difference after correction for trapped extracellular water (Porter et al., 1991). TAU influx and efflux experiments were conducted simultaneously on RBC samples isolated from individual chicks. Initially, RBC samples (80 x lo6 cells per sample) were resuspended in 495~1 TAU flux buffer (1OmM N-2hydroxy ethylpiperazine-N’-2 ethane sulfonic acid, 145 mM NaCl, 5.5 mM glucose, 1 mM CaCl,, 5 mM KCl, 1 mM MgCI,, pH 7.4). The TAU flux buffer was supplemented with physiological concentrations of TAU (Porter et al., 1991): at hatch and 3 days (50pM), 7 and 10 days (75 PM), 14 to 28 days (100 PM). These RBC samples were incubated for 30 min at 41°C prior to the start of the TAU influx and efflux studies. The efflux studies were initiated by the addition of 0.10 PCi [2-‘H (N)]taurine in 5 ~1 TAU flux buffer into half of the RBC samples in order to load the cells with [‘Hltaurine. After 3.5 hr of [3H]taurine loading, the RBC were washed twice by alternate resuspension in 0.5 ml ice-cold TAU flux buffer followed by centrifugation at 14,OOOgfor 1 min. During [‘H]taurine loading for the efflux assay, the other half of the RBC samples to be used for the 117
DALE W. F’ORTERand WILLIAMG. MARTIN
118
influx studies were incubated in 500 ~1 TAU flux buffer and washed as described above. The cells for the influx assay were resuspended in 500 p 1 TAU flux buffer {containing 0.10 uCi 12-‘H (N)ltaurine) which had either normal (145 mM Naclj or low (j2.5 mM NaCl) Na+ concentrations, while the etTluxassay cells were resuspended in 1.2 ml of the normal Na+ or low Na+ TAU flux buffer. Samples (10 x lo6 RBC) were collected at 20 min intervals for 100 min and then centrifuged at 14,OOOgfor 1 min. To measure TAU influx, the RBC were washed twice by alternate resuspension in 0.5 ml ice-cold TAU flux buffer followed by centrifugation at 14,OOOg for 1 min. After completion of the assay, the cells were digested with 20 ~1 Solulene-350 (Packard Instrument Co.) for 2 hr at 56°C and the [)H]taurine activity determined. TAU efflux was determined as [‘H]taurine in the RBC supematant after centrifugation at 14,OOOgfor 1 min. The hyponatramic effect on chick RBC TAU efflux was investigated by incubating RBC isolated from a IO-day-old chick in 0.4, 0.6, 0.8 and normal physiological Na+ concentration (145mN NaCl). At 0.4 normal Na+ (58 mM) RBC lysis was observed as indicated by a reduction in cell counts. Therefore, a 50% decrease in the extracellular Na+ concentration (72.5 mM) was selected for comparative studies. Statistical
analysis
The RBC intracellular taurine concentration data were analysed by analysis of variance for a completely randomized design. All other data were analysed by analysis of
variance for a split-plot design. Trends in means were tested using orthogonal polynomial contrasts. RESULTS
Plasma and RBC taurine concentration
RBC intracellular TAU concentration ([TAU],) was calculated using the value of 0.37 f 0.055 ~1 Hz0/106 cells for the RBC intracellular water content (Porter et al., 1991). The [TAUli had a significant (P < 0.05) linear age response, increasing 2.5-fold from hatch to 21 days (Fig. 1). The plasma TAU concentration ([TAU],) also had a significant (P < 0.05) 3-fold increase during the first 14 days after hatch from 40 to 117 PM, then averaged 130 -
114 PM for the last 2 weeks (Fig. 1). The increase in [TAU], and [TAU], was approximately equal, as evidenced by a [TAU],: [TAU], ratio of 40-50 at all ages studied, except at day 3 when the ratio was 70. [TAU], was not reported at hatch due to excessive RBC lysis in the plasma samples. Taurine flux studies
A preliminary taurine efflux study was conducted on chick RBC at 10 days of age in which four concentrations of extracellular Na+ were tested (Fig. 2). This study indicated that when the extracellular Na+ concentrations were reduced to 87 mM or lower the TAU efflux was drastically increased vs the normal extracellular Na+ concentration (145 mM NaCl). Since RBC lysis was earlier detected at 58 mM NaCl, 72.5 mM was selected as sufficiently low to cause TAU efflux but not sufficiently hypo-osmotic to cause lysis. Data from the TAU influx studies was initially examined as a time course experiment. TAU uptake using RBC isolated from 21-day-old chicks demonstrated TAU uptake in normal Na+ was linear and higher than that used in low Na+, which was curvilinear (Fig. 3). This same pattern was observed when the RBC were obtained from chicks at the other ages used in this study. The RBC TAU influx and efflux rates in normal Na+ showed a significant (P < 0.05) linear age response (Fig. 4). The TAU influx rate was approximately 2-fold higher than the efflux rate from hatch to 21 days. By day 28, the TAU influx and efflux rates were nearly equivalent. In low Na+, the RBC TAU influx and efflux rates showed a significant (P < 0.05) linear age response (Fig. 4). In contrast to the observation in normal Na+, the TAU efflux rate was 5-fold higher than the influx rate in low Na+. The average TAU influx rate in normal Na+ was 0.025 f 0.004 pmol TAU/I06 cells/min, compared to 0.015 + 0.003 in low Na+. Thus, the TAU influx rate decreased significantly (P < 0.05) by 60% at the lower Na+ concentration. In contrast, the
6
p 8
-@- 145 mM NaCl -116mMNaCl -e87 mM NaCl
‘PO Y F ‘cc $ 9
loo-
: CL
30
’ 0
7
14
21
I
a
28’
Age (days) Fig. 1. Plasma (N = 17) and RBC intracellular taurine concentrations (N = 9-16). Values represent mean + SE.
Time (min)
Fig. 2. Chick RBC taurine efflux in four levels of extracellular Na+. Values are mean from one experiment.
Taurine flux in chicken erythrocytes
-+
145 mM NaCl
-
72.5 mM
NaCl
2-
Time (min)
Fig. 3. Chick RBC taurine uptake time course. Values are mean & SE (N = 3).
TAU efflux rates from RBC in low Na+ was 0.079 f 0.019 pmol TAU/106 cells/min, compared to 0.016 + 0.004 in normal Na+. This 5-fold higher TAU efflux rate in low Na+ significantly differed (P < 0.05) from the rate in normal Na+ and was consistent at each age. DISCUSSION
Our studies have demonstrated that chick RBC [TAUli increased 2.5fold from hatch to 21 days (Fig. 1). Previous studies with chicks have shown that heart and brain TAU content increases during embyonic development (Gonzales and Awapara, 1955) as does chick leukocyte [TAU], during post-hatch development (Porter et al., 1991). Similar changes have also been reported in rats (Macaione et al., 1975) and mice (Quilligan et al., 1984). -*Influx (145 mM NaCI) -o.-. - Efflux (145 mM NaCI) .-o-
$ 0.00;
Influx (72.5 mM NaCI) Efflux (72.5 mM NaCI)
I
I
I
7
14
21
I
28
Age (days)
Fig. 4. Chick RBC taurine influx and efflux rates in normal and low Na+. Values represent mean f SE (N = 3-4).
119
the chick RBC Three days after hatch [TAU],: [TAU], ratio ranged from 40 to 50. These data suggest the RBC can absorb and retain extracellular taurine, and maintain a relatively constant TAU gradient regardless of the age of the chick. The preliminary TAU efflux study indicated that a decrease in the extracellular Na+ concentration to 58 mM NaCl (148 mOsm) resulted in RBC lysis. This finding is in close agreement with the results of an earlier study using RBC from three different species of birds, which reported RBC lysis at 150 mOsm (Wood and Morgan, 1969). Consequently, subsequent studies with decreased extracellular Na+ concentrations were conducted at 72.5mM NaCl (177 mOsm) to avoid RBC lysis. It has been proposed that the mammalian cell TAU transport is linked with the co-transport of Na+ and chloride (Cl-) ions (Shain and Martin, 1990). In our low Nat buffer the average TAU influx rate was 60% lower than that measured in normal Nat, which supports the view that at least part of the chick RBC TAU influx is a Na+-dependent process. TAU influx and efflux rates in normal and low Nat increased from hatch to 14 days (Fig. 4). Studies on chick leukocytes (Porter et al., 1991) and B-cells (Porter and Martin, in press) have shown an increase in TAU uptake rates with age during the post-hatch period. Similarly, an age-related increase in the TAU release has been reported from the mouse hippocampus (Oja et al., 1990). The finding that chick RBC TAU influx and efflux rates increased with age may be related to the replacement of embryo-derived RBC after hatch suggesting that younger RBC may have fewer or different TAU transport proteins. TAU has been ascribed roles in different physiological processes (Wright et al., 1986) and so the increase in the [TAU], may reflect a greater requirement of TAU for these functions. One suggested physiological role for TAU is as a cellular osmoregulator. The osmolarity of the normal Na+ buffer (322mOsm) is approximately the same as that reported for birds (30&320mOsm) (Freeman, 1984), while the low Na + buffer osmolarity (177 mOsm) is 55% lower. The average TAU efflux rate in low Nat was 5-fold higher than that in normal Nat. The increased TAU efflux rate under hypo-osmotic conditions suggests TAU may function as an osmoregulator for the chick RBC. The chemical properties of TAU are consistent with its potential as an osmoregulator: small molecular weight, zwitterionic, and its existence as a free amino acid at a high intracellular concentration. Previous studies have also ascribed TAU as an osmoregulator in pigeon RBC (Shihabi et al., 1989), fish RBC (Fugelli, 1967; Fugelli and Zachariassen, 1976) and the mammalian brain (Wade et al., 1988). The increased TAU efflux rate under hypo-osmotic conditions may not be solely due to the decreased osmolarity of the buffer. In the preliminary taurine efflux study (Fig. 2), a 20% decrease in the osmolarity from 322 (145 mM NaCl) to 264 mOsm (116 mM NaCl) had essentially no effect on the taurine efflux rate while a 40% reduction in the osmolarity to 206 mOsm (87 mM) caused a marked increase in the efflux rate. Consequently, a metabolic function of taurine may be in membrane binding which
DALE W. PORTER and
120
terminates in release, a reaction which appears to accelerate under these conditions. Studies measuring TAU efflux from mammalian brain slices (Korpi and Oja, 1983) and neuroblastoma x glioma hybrid cells (Kiirzinger and Hamprecht, 1981) exposed to a hypo-osmotic environment have proposed that there is a reversal of the TAU uptake system. However, studies in astroglial cells (Shain et al., 1990) and Ehrlich ascites cells (Lambert and Hoffman, 1990) have indicated that TAU influx and efflux are independent processes. It has previously been established that there are at least three separate TAU transporters in a cell, and that their activity is dependent on cell type (Wright et al., 1986). Our data has demonstrated that the chick RBC TAU uptake system is Na+-dependent, but the type and number of transporters is not known. Further, the mechanism responsible for the efflux of TAU from the chick RBC cannot be characterized from these studies. The data presented on the age-related increases in [TAU], , TAU influx and efflux rates demonstrate that chick RBC TAU metabolism is changing during post-hatch development. In order to extend this finding, and to further investigate the RBC TAU flux and its physiological role in the chick, a figure depicting the RBC TAU flux was constructed (Fig. 5). In this figure, the RBC TAU influx in both normal and low Nat is depicted as pmol TAU/106 cells in relation to the RBC TAU content at each age studied. The increase in the quantity of TAU is shown within the chick RBC from hatch to 21 days, in the face of a declining influx of TAU from 3.2 to approximately 1.4%. In normal Na+, the amount of TAU entering the RBC is higher than the amount exiting the RBC from hatch to 21 days, which supports the observed increase in the RBC TAU content from 730 to 2100pmol TAU/lO”cells from
WILLIAM G. MARTIN hatch to day 28 (Fig. 5). A similar mechanism has been proposed in mice to explain the post-partum increase in heart TAU with age (Quilligan et al., 1984).
In view of the decreased chick RBC TAU influx rate in the low Na+ buffer, a significant proportion of the TAU transporters must be Na+-dependent. As seen in Fig. 5, a reduced but constant amount of TAU was transported at all ages in low Na+ buffer. This component of the TAU influx represents 33% of the total TAU influx measured in normal Na+ at hatch, approximately 50% at 7-14 days and 65% at days 21 and 28. For this reason only part of the TAU influx may be Na+-dependent and the RBC isolated from older chicks are able to maintain their TAU levels by other means. The increased TAU efflux under hypo-osmotic conditions suggests a role for TAU as an osmoregulator for the chick RBC. However, the effectiveness of TAU as an osmoregulator cannot be assessed solely on the increased efflux rate under hypo-osmotic conditions. In consideration of the availability and amount of TAU which can be released from the RBC (about 5% of the cell content at each age), RBC from young chicks can release about 50 pmol TAU/106cells/min as compared to older chicks releasing twice that amount. If TAU release is indeed involved in TAU metabolism and osmoregulation, then these chick cells are more capable of responding at the older ages. The results of these studies also suggest the source of TAU released in response to hypo-osmotic stress. Two pools of intracellular TAU exist, a large slowly exchangeable membrane-bound pool and a small rapidly exchangeable cytosolic free pool (Sturman et al., 1975). Shifts of free cytosolic TAU to membranebound have been demonstrated in response to a Mg2+ deficiency in rats (Robeson et al., 1980; Durlach et al., 1985). A similar shift in cellular TAU from
Taurine efflux rate Taurine influx rate
(pmoles/106 cells/min)
(pmoledl O6 celkdmin) as % of RBC Tau je%
of~z;uYS$j$$yB*
10 21 14
1.23 0.67 1.04
28
1.08
\ (pmoles/l naurinel O6 cell s) Age
-
_
lieu1
; j
\
Age H 3 7 IO 14 21 28
% RBC Tau 3.2 2.4 1.8 1.8 2.0 1.4 1.3
Taurine influx rate (pmoles/l06 cellslmin) as % of RBC Tau
1.0
14 21 28
6.2 6.5 4.0 4.9
Age H 3 7 IO 14 21 28
Fig. 5. Effect of Na+ on TAU flux as related to RBC TAU content.
% RBC Tau A:: 0.9 0.8 0.9 0.9 0.9
121
Taurine flux in chicken erythrocytes membrane-bound to free cytosolic was also reported in rat alveolar macrophages during oxidant stress (Banks er al., 1990). Since at least part of the chick RBC TAU transport is Nat-dependent, and TAU uptake is reduced in low Na+ buffer, the increase in TAU release probably results in a shift from the membrane-bound to the free cytosolic pool. This is further supported by a recent study using ozone exposed alveolar macrophages incubated in different levels of extracellular TAU. This study reported that in the absence of TAU Na+,K+-ATPase activity was low, but was significantly higher with 100pM TAU (Banks et al., 1991). Since the Na+,K+-ATPase activity was reduced by ozone exposure in the absence of TAU, the size of the inwardly directed Na+ concentration gradient should have decreased. Consequently, TAU uptake would have decreased. Thus, the source of the TAU responsible for the increase in the cytosolic pool in response to ozone was a shift of membrane-bound TAU to the cytosolic pool (Banks et al., 1991). In conclusion, our studies indicate that the chick RBC TAU flux increased during early post-hatch development concomitant with an increase in the [TAU], . Consequently, physiological processes requiring TAU in the chick RBC would be maintained during this developmental period. The results support the role of TAU as a possible osmoregulator, demonstrated by the increased TAU flux when stressed by hyponatremic conditions.
Reu. 48,424-S
11.
Korpi E. R. and Oja S. S. (1983) Characteristics of tamine release from cerebral cortex slices induced by sodiumdeficient media. Brain Res. 289, 197-204. Kiirzinger K. and Hamprecht B. (1981) Na+-dependent uptake and release of taurine by neuroblastoma x ghoma hybrid cells. J. Neurochem. 37, 956-967. Lambert I. H. and Hoffman E. K. (1990) Tauriner Functional neurochemistry,
Physjoiogy and Cardiology
(Edited by Pasantes-Morales H., Martin D. L., Shain W. and Martin de1 Rio R.), pp. 267-276. Wiley-Liss, New York. Lowe I. P. and Roberts E. (1955) Incorporation of radioactive sulfate into taurine and other substances in the chick embryo. J. biol. Chem. 212, 477-483. Macaione S., Tucci G. and DiGiorgio R. M. (1975) Taurine distribution in rat tissues during development. Ital. J. Biochem. 24, 162-173.
Martin W. G. (1972) Sulfate metabolism and taurine synthesis in the chick. Poultry Sci. 51, 608612. Martin W. G., Miragha R. J., Spaeth D. G. and Patrick H. (1966) Synthesis of taurine from sulfate by the chick. Proe. Sac. exp. Biol. Med. 122, 841844. Mason V. C., Hansen J. G. and Weidner K. (1965) Studies on the quantitative incorporation of sulfate sulfur into methionine, cystine and taurine in the hen. Acta. Agri. Stand. 15, 3-15. McMenamy R. H., Lund C. C., Neville G. J. and Waliach D. F. H. (1960) Studies of unbound amino acid distributions in plasma, erythr~ytes, leukocytes and urine of normal human subjects. J. c&n. Imesr. 39, 1675-1687.
Miraglia R. J. and Martin W. G. (1969) The synthesis of taurine from sulfate: II. Chick liver PAPS-transferase.
REFERENCES
Proc. Sot. exp. Biol. Med. 132, 640-644.
Banks M. A., Porter D. W., Martin W. G. and Castranova V. 11990)Effects of in uitro ozone exnosure on neroxidativd damage, membrane leakage and taurine c&tent of rat alveolar macrophages. J. Toxicol. appt. P/tarmac. 105, 5565.
Banks M. A., Porter D. W., Martin W. G. and Castranova V. (1991) Ozone-induced lipid peroxidation and membrane leakage in isolated rat alveolar macrophages: protective effects of taurine. J. Nufr. &&em. 2,308-313. Bartlett G. R. and Borgese T. A. (1976) Phosphate compounds in red cells of the chicken and duck embryo and hatchhng. Camp. Biochem. Physiol. JSA, 2077210. Durlach J., Rapin J. R., LePoncin-LaFitte M., Rayssiguier Y., Bata M. and Guiet-Bara A. (1985) Magnesium Defyciency. Physiopathology and Treutment Implications, &I European Congress on Magnesia (Edited bv Halnern
M. J and Duaach J.), pp.4653. Larger, P&s. * Freeman B. M. (1984) Physiology and Biochemistry of the Domestic Fowl (Edited by Freeman
Jacobsen J. G. and Smith L. H. Jr. (1968) Biochemistry and physiology of taurine and taurine derivatives. Physiof.
B. M.). Academic
Press, New York. Fugelli K. (1967) Regulation of cell volume in flounder (Pleuronectes flesus) erythrocytes accompanying a decrease in plasma osmoiarity. Comp. Biochem. Physiol. 22, 253-260.
Fugelli K. and Zachariassen K. E. (1976) The distribution of taurine, gamma-aminobutyric acid and inorganic ions between plasma and erythrocytes in flounder (Platichthys Jesus) at different plasma osmolarities. Camp. Biochem. Physiol. %A, 173-177. Gonzales F. and Awapara J. (1955) The taurine concentration of developing chick embryo. Exp. Cell. Res. 9, 353-35s.
Harris J. R. (1971) Physiology and Biochemistry of the Domesfic Fawl (Edited by Bell D. J. and Freeman B. M.), pp. 853-862. Academic Press, New York.
Oja S. S., Holopainen I. and Kontro P. (1990) Taurine: Functional ~~roehemistry,
Physiology and Cardiology
(Edited by Pa~ntes-Morales H., Martin D. L., Shain W. and Martin de1 Rio, R.), pp. 277-288. Wiley-Liss, New York. Porter D. W., Banks M. A., Castranova V. and Martin W. G. (1988) Reversed-phase high performance liquid chromatography technique for taurine quantitation. J. Chrom. 454, 311-316.
Porter D. W. and Martin W. G. (in press) Taurine uptake into chick B-cells. Proc. Sot. exp. Biol. Med. Porter D. W., Walker S. A., Martin W. G., Lee P. and Kaczmarczyk W. (1991) Taurine uptake in chicken leukocytes and erythrocytes. Comp. Biochem. Physiol. 98A, 305-309.
Quilligan C. J., Hilton F. K. and Hilton M. A. (1984) Taurine in hearts and bodies of embyonic through early post partum CFI mice. Proc. Sot. exp. Biol. Med. 177, 143-150.
Robeson B. L., Martin W. G. and Friedman M. H. (1980) A biochemical and ultrastructural study of skeletal muscle from rats fed a magnesium-deficient diet. J. Nutr. 110, 2078-2084.
Sass N. L. and Martin W. G. (1972)The synthesis of taurine from sulfate: III. Further evidence for the enzymatic pathway in chick liver. Proc. Sot. exp. Biol. Med. 139, 755-761. Shain W., Madehan V., Waniewski R. A. and Martin D. L. (1990) Tavrine: Functional Neurochemistry, Physiology and Cardiology (Edited by Pasantes-Morales H., Martin D. L., Shain W. and Martin del Rio R.), pp. 299-306. Wiley-Liss, New York. Shain W. and Martin D. L. (1990) Taurine: Functionai Neurochemistry, Physiology, and Cardioiogy (Edited by Pasantes-Morales H., Martin D. L., Shain W. and Martin de1 Rio R.), pp. 243-252. Wiley-Liss, New York.
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DALE W. PORTER and WILLIAMG. MARTIN
Shihabi Z. K., Goodman H. O., Holmes R. P. and O’Connor M. L. (1989) The taurine content of avian erythrocytes and its role in osmoregulation. Comp. Biothem. Physiol. 92A, 545-549.
Sturkie P. D. (1986) Aoiun Physiology (Edited by Sturkie P. D.), pp. 102-121. Springer, New York. Sturman J. A., Hepner G. W., Hufman A. L. and Thomas P. J. (1975) Metabolism of 3?S-taurine in man. J. Nutr. 105, 120661214.
Wade J. V., Olson J. P., Samson F. E., Nelson S. R. and Pazdemik T. L. (1988) A possible role for taurine in osmoregulation within the brain. .I. Neurochem. 51, 740-745.
Wood R. E. and Morgan H. E. (1969) Regulation of sugar transport in avian erythrocytes. J. biol. Chem. 244, 1451-1460. Wright C. E., Tallan H. H. and Lin Y. Y. (1986) Taurine: biological update. A. Rev. Biochem. 207, 421-453.
Vol. 102A,
No. 1, pp.
0300-9629/92$5.00+ 0.00 0 1992Pergamon Press plc
117-122,1992
Printed in Great Britain
TAURINE
FLUX
IN CHICKEN
ERYTHROCYTES*
DALE W. PORTER and WILLIAM G. MARTINt Division of Animal and Veterinary Sciences, West Virginia University, Morgantown, WV 26506-6108, U.S.A. Telephone: (304) 293-2631 (Received 12 August 1991)
Abstract-l. The intracellular taurine concentration in chick erythrocytes increased with age. 2. Erythrocyte taurine influx and efflux rates increased with age. 3. Erythrocyte taurine influx decreased when the extracellular sodium concentration was below normal physiological concentrations. 4. Under hypo-osmotic conditions, taurine efflux from erythrocytes increased. 5. The data suggest that chick erythrocyte taurine metabolism changes during early post-hatch development and that one taurine function may be as an osmoregulator.
MATERIALSAND
INTRODUCTION Taurine (TAU) is a p-amino acid found at high concentrations in various animal species, including avian species (Jacobsen and Smith, 1968). The chick embryo (Lowe and Roberts, 1955), chick (Martin et al., 1966) and hen (Mason et al., 1965) are able to synthesize TAU from inorganic sulfate (Martin et al., 1966; Miraglia and Martin, 1969; Sass and Martin, 1972), and the rate of TAU biosynthesis from inorganic sulfate is enhanced by L-methionine in the chick (Martin, 1972). TAU has not been extensively studied in the chicken erythrocyte (RBC), but the mammalian RBC is known to have a very low TAU concentration (McMenamy et al., 1960). The avian RBC differs physiologically and anatomically from the mammalian RBC on the basis that it has a shorter life span (28-35 days), is nucleated and contains mitochondria (Sturkie, 1986; Harris, 1971). Thus, the avian RBC may also differ from the mammalian RBC in terms of its TAU content. At hatch, the chick RBC population is composed of embryo-derived RBC which are gradually replaced by RBC which are produced after hatching. During the transition from embryonic to post-hatch conditions, alterations in some RBC intracellular regulatory molecules occur (Bartlett and Borgese, 1976). However, TAU has not been studied with regard to age-related changes in its intracellular concentration or function in the chick RBC. Therefore, experiments were conducted with chick RBC from hatch through 4 weeks of age to determine the effect of age and the extracellular sodium (Na+) concentration on the RBC intracellular TAU concentration and the rates of TAU influx and efflux.
*Published with approval of the director of the West Virginia Agricultural and Forestry Experiment Station as Scientific Article No. 2292. tTo whom correspondence should be addressed.
METHODS
Maintenance of animals
Broiler-type chicks (Gallus gallus) were reared from hatch the West Virginia University Poultry Farm until they were 4 weeks old. Chicks were fed a standard corn-soybean meal ration adequate in all nutrients ad lib., and provided free access to tap water. at
Whole blood collecrion and RBC isolation
Blood was collected from chicks at the following ages: hatch, 3, 7, 10, 14, 21 and 28 days. Blood collection was from individual chicks by cardiac puncture following chloroform anesthesia using a heparin (20mg/lOO ml solution) coated syringe. The RBC pellet was obtained from each blood sample after 10min centrifugation at 900 g. The plasma and buffy coat were aspirated and the RBC washed twice in a saline solution (0.9% NaCl). After washing, the RBC were resuspended in saline solution prior to counting (Coulter Counter Model Zm). Taurine concentration and frux studies
Plasma and RBC TAU content was determined by HPLC (Porter er al., 1988). The RBC intracellular water content was determined by wet cell - dry cell weight difference after correction for trapped extracellular water (Porter et al., 1991). TAU influx and efflux experiments were conducted simultaneously on RBC samples isolated from individual chicks. Initially, RBC samples (80 x lo6 cells per sample) were resuspended in 495~1 TAU flux buffer (1OmM N-2hydroxy ethylpiperazine-N’-2 ethane sulfonic acid, 145 mM NaCl, 5.5 mM glucose, 1 mM CaCl,, 5 mM KCl, 1 mM MgCI,, pH 7.4). The TAU flux buffer was supplemented with physiological concentrations of TAU (Porter et al., 1991): at hatch and 3 days (50pM), 7 and 10 days (75 PM), 14 to 28 days (100 PM). These RBC samples were incubated for 30 min at 41°C prior to the start of the TAU influx and efflux studies. The efflux studies were initiated by the addition of 0.10 PCi [2-‘H (N)]taurine in 5 ~1 TAU flux buffer into half of the RBC samples in order to load the cells with [‘Hltaurine. After 3.5 hr of [3H]taurine loading, the RBC were washed twice by alternate resuspension in 0.5 ml ice-cold TAU flux buffer followed by centrifugation at 14,OOOgfor 1 min. During [‘H]taurine loading for the efflux assay, the other half of the RBC samples to be used for the 117
DALE W. F’ORTERand WILLIAMG. MARTIN
118
influx studies were incubated in 500 ~1 TAU flux buffer and washed as described above. The cells for the influx assay were resuspended in 500 p 1 TAU flux buffer {containing 0.10 uCi 12-‘H (N)ltaurine) which had either normal (145 mM Naclj or low (j2.5 mM NaCl) Na+ concentrations, while the etTluxassay cells were resuspended in 1.2 ml of the normal Na+ or low Na+ TAU flux buffer. Samples (10 x lo6 RBC) were collected at 20 min intervals for 100 min and then centrifuged at 14,OOOgfor 1 min. To measure TAU influx, the RBC were washed twice by alternate resuspension in 0.5 ml ice-cold TAU flux buffer followed by centrifugation at 14,OOOg for 1 min. After completion of the assay, the cells were digested with 20 ~1 Solulene-350 (Packard Instrument Co.) for 2 hr at 56°C and the [)H]taurine activity determined. TAU efflux was determined as [‘H]taurine in the RBC supematant after centrifugation at 14,OOOgfor 1 min. The hyponatramic effect on chick RBC TAU efflux was investigated by incubating RBC isolated from a IO-day-old chick in 0.4, 0.6, 0.8 and normal physiological Na+ concentration (145mN NaCl). At 0.4 normal Na+ (58 mM) RBC lysis was observed as indicated by a reduction in cell counts. Therefore, a 50% decrease in the extracellular Na+ concentration (72.5 mM) was selected for comparative studies. Statistical
analysis
The RBC intracellular taurine concentration data were analysed by analysis of variance for a completely randomized design. All other data were analysed by analysis of
variance for a split-plot design. Trends in means were tested using orthogonal polynomial contrasts. RESULTS
Plasma and RBC taurine concentration
RBC intracellular TAU concentration ([TAU],) was calculated using the value of 0.37 f 0.055 ~1 Hz0/106 cells for the RBC intracellular water content (Porter et al., 1991). The [TAUli had a significant (P < 0.05) linear age response, increasing 2.5-fold from hatch to 21 days (Fig. 1). The plasma TAU concentration ([TAU],) also had a significant (P < 0.05) 3-fold increase during the first 14 days after hatch from 40 to 117 PM, then averaged 130 -
114 PM for the last 2 weeks (Fig. 1). The increase in [TAU], and [TAU], was approximately equal, as evidenced by a [TAU],: [TAU], ratio of 40-50 at all ages studied, except at day 3 when the ratio was 70. [TAU], was not reported at hatch due to excessive RBC lysis in the plasma samples. Taurine flux studies
A preliminary taurine efflux study was conducted on chick RBC at 10 days of age in which four concentrations of extracellular Na+ were tested (Fig. 2). This study indicated that when the extracellular Na+ concentrations were reduced to 87 mM or lower the TAU efflux was drastically increased vs the normal extracellular Na+ concentration (145 mM NaCl). Since RBC lysis was earlier detected at 58 mM NaCl, 72.5 mM was selected as sufficiently low to cause TAU efflux but not sufficiently hypo-osmotic to cause lysis. Data from the TAU influx studies was initially examined as a time course experiment. TAU uptake using RBC isolated from 21-day-old chicks demonstrated TAU uptake in normal Na+ was linear and higher than that used in low Na+, which was curvilinear (Fig. 3). This same pattern was observed when the RBC were obtained from chicks at the other ages used in this study. The RBC TAU influx and efflux rates in normal Na+ showed a significant (P < 0.05) linear age response (Fig. 4). The TAU influx rate was approximately 2-fold higher than the efflux rate from hatch to 21 days. By day 28, the TAU influx and efflux rates were nearly equivalent. In low Na+, the RBC TAU influx and efflux rates showed a significant (P < 0.05) linear age response (Fig. 4). In contrast to the observation in normal Na+, the TAU efflux rate was 5-fold higher than the influx rate in low Na+. The average TAU influx rate in normal Na+ was 0.025 f 0.004 pmol TAU/I06 cells/min, compared to 0.015 + 0.003 in low Na+. Thus, the TAU influx rate decreased significantly (P < 0.05) by 60% at the lower Na+ concentration. In contrast, the
6
p 8
-@- 145 mM NaCl -116mMNaCl -e87 mM NaCl
‘PO Y F ‘cc $ 9
loo-
: CL
30
’ 0
7
14
21
I
a
28’
Age (days) Fig. 1. Plasma (N = 17) and RBC intracellular taurine concentrations (N = 9-16). Values represent mean + SE.
Time (min)
Fig. 2. Chick RBC taurine efflux in four levels of extracellular Na+. Values are mean from one experiment.
Taurine flux in chicken erythrocytes
-+
145 mM NaCl
-
72.5 mM
NaCl
2-
Time (min)
Fig. 3. Chick RBC taurine uptake time course. Values are mean & SE (N = 3).
TAU efflux rates from RBC in low Na+ was 0.079 f 0.019 pmol TAU/106 cells/min, compared to 0.016 + 0.004 in normal Na+. This 5-fold higher TAU efflux rate in low Na+ significantly differed (P < 0.05) from the rate in normal Na+ and was consistent at each age. DISCUSSION
Our studies have demonstrated that chick RBC [TAUli increased 2.5fold from hatch to 21 days (Fig. 1). Previous studies with chicks have shown that heart and brain TAU content increases during embyonic development (Gonzales and Awapara, 1955) as does chick leukocyte [TAU], during post-hatch development (Porter et al., 1991). Similar changes have also been reported in rats (Macaione et al., 1975) and mice (Quilligan et al., 1984). -*Influx (145 mM NaCI) -o.-. - Efflux (145 mM NaCI) .-o-
$ 0.00;
Influx (72.5 mM NaCI) Efflux (72.5 mM NaCI)
I
I
I
7
14
21
I
28
Age (days)
Fig. 4. Chick RBC taurine influx and efflux rates in normal and low Na+. Values represent mean f SE (N = 3-4).
119
the chick RBC Three days after hatch [TAU],: [TAU], ratio ranged from 40 to 50. These data suggest the RBC can absorb and retain extracellular taurine, and maintain a relatively constant TAU gradient regardless of the age of the chick. The preliminary TAU efflux study indicated that a decrease in the extracellular Na+ concentration to 58 mM NaCl (148 mOsm) resulted in RBC lysis. This finding is in close agreement with the results of an earlier study using RBC from three different species of birds, which reported RBC lysis at 150 mOsm (Wood and Morgan, 1969). Consequently, subsequent studies with decreased extracellular Na+ concentrations were conducted at 72.5mM NaCl (177 mOsm) to avoid RBC lysis. It has been proposed that the mammalian cell TAU transport is linked with the co-transport of Na+ and chloride (Cl-) ions (Shain and Martin, 1990). In our low Nat buffer the average TAU influx rate was 60% lower than that measured in normal Nat, which supports the view that at least part of the chick RBC TAU influx is a Na+-dependent process. TAU influx and efflux rates in normal and low Nat increased from hatch to 14 days (Fig. 4). Studies on chick leukocytes (Porter et al., 1991) and B-cells (Porter and Martin, in press) have shown an increase in TAU uptake rates with age during the post-hatch period. Similarly, an age-related increase in the TAU release has been reported from the mouse hippocampus (Oja et al., 1990). The finding that chick RBC TAU influx and efflux rates increased with age may be related to the replacement of embryo-derived RBC after hatch suggesting that younger RBC may have fewer or different TAU transport proteins. TAU has been ascribed roles in different physiological processes (Wright et al., 1986) and so the increase in the [TAU], may reflect a greater requirement of TAU for these functions. One suggested physiological role for TAU is as a cellular osmoregulator. The osmolarity of the normal Na+ buffer (322mOsm) is approximately the same as that reported for birds (30&320mOsm) (Freeman, 1984), while the low Na + buffer osmolarity (177 mOsm) is 55% lower. The average TAU efflux rate in low Nat was 5-fold higher than that in normal Nat. The increased TAU efflux rate under hypo-osmotic conditions suggests TAU may function as an osmoregulator for the chick RBC. The chemical properties of TAU are consistent with its potential as an osmoregulator: small molecular weight, zwitterionic, and its existence as a free amino acid at a high intracellular concentration. Previous studies have also ascribed TAU as an osmoregulator in pigeon RBC (Shihabi et al., 1989), fish RBC (Fugelli, 1967; Fugelli and Zachariassen, 1976) and the mammalian brain (Wade et al., 1988). The increased TAU efflux rate under hypo-osmotic conditions may not be solely due to the decreased osmolarity of the buffer. In the preliminary taurine efflux study (Fig. 2), a 20% decrease in the osmolarity from 322 (145 mM NaCl) to 264 mOsm (116 mM NaCl) had essentially no effect on the taurine efflux rate while a 40% reduction in the osmolarity to 206 mOsm (87 mM) caused a marked increase in the efflux rate. Consequently, a metabolic function of taurine may be in membrane binding which
DALE W. PORTER and
120
terminates in release, a reaction which appears to accelerate under these conditions. Studies measuring TAU efflux from mammalian brain slices (Korpi and Oja, 1983) and neuroblastoma x glioma hybrid cells (Kiirzinger and Hamprecht, 1981) exposed to a hypo-osmotic environment have proposed that there is a reversal of the TAU uptake system. However, studies in astroglial cells (Shain et al., 1990) and Ehrlich ascites cells (Lambert and Hoffman, 1990) have indicated that TAU influx and efflux are independent processes. It has previously been established that there are at least three separate TAU transporters in a cell, and that their activity is dependent on cell type (Wright et al., 1986). Our data has demonstrated that the chick RBC TAU uptake system is Na+-dependent, but the type and number of transporters is not known. Further, the mechanism responsible for the efflux of TAU from the chick RBC cannot be characterized from these studies. The data presented on the age-related increases in [TAU], , TAU influx and efflux rates demonstrate that chick RBC TAU metabolism is changing during post-hatch development. In order to extend this finding, and to further investigate the RBC TAU flux and its physiological role in the chick, a figure depicting the RBC TAU flux was constructed (Fig. 5). In this figure, the RBC TAU influx in both normal and low Nat is depicted as pmol TAU/106 cells in relation to the RBC TAU content at each age studied. The increase in the quantity of TAU is shown within the chick RBC from hatch to 21 days, in the face of a declining influx of TAU from 3.2 to approximately 1.4%. In normal Na+, the amount of TAU entering the RBC is higher than the amount exiting the RBC from hatch to 21 days, which supports the observed increase in the RBC TAU content from 730 to 2100pmol TAU/lO”cells from
WILLIAM G. MARTIN hatch to day 28 (Fig. 5). A similar mechanism has been proposed in mice to explain the post-partum increase in heart TAU with age (Quilligan et al., 1984).
In view of the decreased chick RBC TAU influx rate in the low Na+ buffer, a significant proportion of the TAU transporters must be Na+-dependent. As seen in Fig. 5, a reduced but constant amount of TAU was transported at all ages in low Na+ buffer. This component of the TAU influx represents 33% of the total TAU influx measured in normal Na+ at hatch, approximately 50% at 7-14 days and 65% at days 21 and 28. For this reason only part of the TAU influx may be Na+-dependent and the RBC isolated from older chicks are able to maintain their TAU levels by other means. The increased TAU efflux under hypo-osmotic conditions suggests a role for TAU as an osmoregulator for the chick RBC. However, the effectiveness of TAU as an osmoregulator cannot be assessed solely on the increased efflux rate under hypo-osmotic conditions. In consideration of the availability and amount of TAU which can be released from the RBC (about 5% of the cell content at each age), RBC from young chicks can release about 50 pmol TAU/106cells/min as compared to older chicks releasing twice that amount. If TAU release is indeed involved in TAU metabolism and osmoregulation, then these chick cells are more capable of responding at the older ages. The results of these studies also suggest the source of TAU released in response to hypo-osmotic stress. Two pools of intracellular TAU exist, a large slowly exchangeable membrane-bound pool and a small rapidly exchangeable cytosolic free pool (Sturman et al., 1975). Shifts of free cytosolic TAU to membranebound have been demonstrated in response to a Mg2+ deficiency in rats (Robeson et al., 1980; Durlach et al., 1985). A similar shift in cellular TAU from
Taurine efflux rate Taurine influx rate
(pmoles/106 cells/min)
(pmoledl O6 celkdmin) as % of RBC Tau je%
of~z;uYS$j$$yB*
10 21 14
1.23 0.67 1.04
28
1.08
\ (pmoles/l naurinel O6 cell s) Age
-
_
lieu1
; j
\
Age H 3 7 IO 14 21 28
% RBC Tau 3.2 2.4 1.8 1.8 2.0 1.4 1.3
Taurine influx rate (pmoles/l06 cellslmin) as % of RBC Tau
1.0
14 21 28
6.2 6.5 4.0 4.9
Age H 3 7 IO 14 21 28
Fig. 5. Effect of Na+ on TAU flux as related to RBC TAU content.
% RBC Tau A:: 0.9 0.8 0.9 0.9 0.9
121
Taurine flux in chicken erythrocytes membrane-bound to free cytosolic was also reported in rat alveolar macrophages during oxidant stress (Banks er al., 1990). Since at least part of the chick RBC TAU transport is Nat-dependent, and TAU uptake is reduced in low Na+ buffer, the increase in TAU release probably results in a shift from the membrane-bound to the free cytosolic pool. This is further supported by a recent study using ozone exposed alveolar macrophages incubated in different levels of extracellular TAU. This study reported that in the absence of TAU Na+,K+-ATPase activity was low, but was significantly higher with 100pM TAU (Banks et al., 1991). Since the Na+,K+-ATPase activity was reduced by ozone exposure in the absence of TAU, the size of the inwardly directed Na+ concentration gradient should have decreased. Consequently, TAU uptake would have decreased. Thus, the source of the TAU responsible for the increase in the cytosolic pool in response to ozone was a shift of membrane-bound TAU to the cytosolic pool (Banks et al., 1991). In conclusion, our studies indicate that the chick RBC TAU flux increased during early post-hatch development concomitant with an increase in the [TAU], . Consequently, physiological processes requiring TAU in the chick RBC would be maintained during this developmental period. The results support the role of TAU as a possible osmoregulator, demonstrated by the increased TAU flux when stressed by hyponatremic conditions.
Reu. 48,424-S
11.
Korpi E. R. and Oja S. S. (1983) Characteristics of tamine release from cerebral cortex slices induced by sodiumdeficient media. Brain Res. 289, 197-204. Kiirzinger K. and Hamprecht B. (1981) Na+-dependent uptake and release of taurine by neuroblastoma x ghoma hybrid cells. J. Neurochem. 37, 956-967. Lambert I. H. and Hoffman E. K. (1990) Tauriner Functional neurochemistry,
Physjoiogy and Cardiology
(Edited by Pasantes-Morales H., Martin D. L., Shain W. and Martin de1 Rio R.), pp. 267-276. Wiley-Liss, New York. Lowe I. P. and Roberts E. (1955) Incorporation of radioactive sulfate into taurine and other substances in the chick embryo. J. biol. Chem. 212, 477-483. Macaione S., Tucci G. and DiGiorgio R. M. (1975) Taurine distribution in rat tissues during development. Ital. J. Biochem. 24, 162-173.
Martin W. G. (1972) Sulfate metabolism and taurine synthesis in the chick. Poultry Sci. 51, 608612. Martin W. G., Miragha R. J., Spaeth D. G. and Patrick H. (1966) Synthesis of taurine from sulfate by the chick. Proe. Sac. exp. Biol. Med. 122, 841844. Mason V. C., Hansen J. G. and Weidner K. (1965) Studies on the quantitative incorporation of sulfate sulfur into methionine, cystine and taurine in the hen. Acta. Agri. Stand. 15, 3-15. McMenamy R. H., Lund C. C., Neville G. J. and Waliach D. F. H. (1960) Studies of unbound amino acid distributions in plasma, erythr~ytes, leukocytes and urine of normal human subjects. J. c&n. Imesr. 39, 1675-1687.
Miraglia R. J. and Martin W. G. (1969) The synthesis of taurine from sulfate: II. Chick liver PAPS-transferase.
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Banks M. A., Porter D. W., Martin W. G. and Castranova V. (1991) Ozone-induced lipid peroxidation and membrane leakage in isolated rat alveolar macrophages: protective effects of taurine. J. Nufr. &&em. 2,308-313. Bartlett G. R. and Borgese T. A. (1976) Phosphate compounds in red cells of the chicken and duck embryo and hatchhng. Camp. Biochem. Physiol. JSA, 2077210. Durlach J., Rapin J. R., LePoncin-LaFitte M., Rayssiguier Y., Bata M. and Guiet-Bara A. (1985) Magnesium Defyciency. Physiopathology and Treutment Implications, &I European Congress on Magnesia (Edited bv Halnern
M. J and Duaach J.), pp.4653. Larger, P&s. * Freeman B. M. (1984) Physiology and Biochemistry of the Domestic Fowl (Edited by Freeman
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Fugelli K. and Zachariassen K. E. (1976) The distribution of taurine, gamma-aminobutyric acid and inorganic ions between plasma and erythrocytes in flounder (Platichthys Jesus) at different plasma osmolarities. Camp. Biochem. Physiol. %A, 173-177. Gonzales F. and Awapara J. (1955) The taurine concentration of developing chick embryo. Exp. Cell. Res. 9, 353-35s.
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(Edited by Pa~ntes-Morales H., Martin D. L., Shain W. and Martin de1 Rio, R.), pp. 277-288. Wiley-Liss, New York. Porter D. W., Banks M. A., Castranova V. and Martin W. G. (1988) Reversed-phase high performance liquid chromatography technique for taurine quantitation. J. Chrom. 454, 311-316.
Porter D. W. and Martin W. G. (in press) Taurine uptake into chick B-cells. Proc. Sot. exp. Biol. Med. Porter D. W., Walker S. A., Martin W. G., Lee P. and Kaczmarczyk W. (1991) Taurine uptake in chicken leukocytes and erythrocytes. Comp. Biochem. Physiol. 98A, 305-309.
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Robeson B. L., Martin W. G. and Friedman M. H. (1980) A biochemical and ultrastructural study of skeletal muscle from rats fed a magnesium-deficient diet. J. Nutr. 110, 2078-2084.
Sass N. L. and Martin W. G. (1972)The synthesis of taurine from sulfate: III. Further evidence for the enzymatic pathway in chick liver. Proc. Sot. exp. Biol. Med. 139, 755-761. Shain W., Madehan V., Waniewski R. A. and Martin D. L. (1990) Tavrine: Functional Neurochemistry, Physiology and Cardiology (Edited by Pasantes-Morales H., Martin D. L., Shain W. and Martin del Rio R.), pp. 299-306. Wiley-Liss, New York. Shain W. and Martin D. L. (1990) Taurine: Functionai Neurochemistry, Physiology, and Cardioiogy (Edited by Pasantes-Morales H., Martin D. L., Shain W. and Martin de1 Rio R.), pp. 243-252. Wiley-Liss, New York.
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DALE W. PORTER and WILLIAMG. MARTIN
Shihabi Z. K., Goodman H. O., Holmes R. P. and O’Connor M. L. (1989) The taurine content of avian erythrocytes and its role in osmoregulation. Comp. Biothem. Physiol. 92A, 545-549.
Sturkie P. D. (1986) Aoiun Physiology (Edited by Sturkie P. D.), pp. 102-121. Springer, New York. Sturman J. A., Hepner G. W., Hufman A. L. and Thomas P. J. (1975) Metabolism of 3?S-taurine in man. J. Nutr. 105, 120661214.
Wade J. V., Olson J. P., Samson F. E., Nelson S. R. and Pazdemik T. L. (1988) A possible role for taurine in osmoregulation within the brain. .I. Neurochem. 51, 740-745.
Wood R. E. and Morgan H. E. (1969) Regulation of sugar transport in avian erythrocytes. J. biol. Chem. 244, 1451-1460. Wright C. E., Tallan H. H. and Lin Y. Y. (1986) Taurine: biological update. A. Rev. Biochem. 207, 421-453.
