The taurine content of avian erythrocytes and its role in osmoregulation

Camp. Biochem. Physiol. Vol. 92A, No. 4, pp. M-549, Printed I” Great Britain

THE

1989 0

0300-9629/89 $3.00 + 0.00 1989 Pergamon Press plc

TAURINE CONTENT OF AVIAN ERYTHROCYTES AND ITS ROLE IN OSMOREGULATION Z. K. SHIHABI,* H. 0.

Departments of *Pathology, Wake Forest University,

GooDmw,t

R. P. HOLMES~ and M. L. O’CONNOR*

tpediatrics and $Urology, Bowman Gray School of Medicine, Winston-Salem, NC 27103, USA. Telephone: (919) 748-4311

(Received 26 September

arest

1988)

The taurine content of erythrocytes from 15 avian species contained levels of taurine in the range of 20 70 mmol/kg of hemoglobin, about IOO-fold that of mammalian red blood cells. 2. This high taurine content did not appear to be related to the nucleation of these cells as nucleated amphibian erythrocytes and human reticulocytes contained low levels. 3. The erythrocytes lacked cysteine sulfinic acid decarboxylase, a key enzyme in the synthesis of taurine from cysteine, indicating a probable lack of synthetic capabilities. 4. The cells were able to accumulate labeled taurine against a concentration gradient. This uptake was inhibited by /?-alanine and was Nat-dependent. 5. When incubated in hypotonic medium, the cell volume of pigeon erythrocytes rapidly increased and was followed by a much slower return to normal size. The cell volume reduction was accompanied by a slow efflux of taurine into the medium. 6. These data suggest that taurine plays a role in cell volume maintenance and osmotic regulation in avian erythrocytes. Abstract-l,

INTRODUCTION

MATERIALS

AND METHODS

Red blood cell isolation

Taurine

is an amino acid abundant in many animal cells. Because it is not incorporated in proteins or utilized for energy. its biological role is not clear. White blood cells and cardiac cells contain large (millimolar) concentrations of taurine while other cells, such as fibroblasts and mammalian erythrocytes, contain small (micromolar) concentrations of this amino acid (Jacobsen and Smith, 1968). In contrast to mammalian erythrocytes, Fugelli and Zachariassen (1976) found that erythrocytes from the European flounder (Plrrtichthps Flesus) contain high intracellular levels of taurine. This observation was extended by Fincham et al. (1987), who detected high levels in erythrocytes from the starry flounder (Plutichthys stellutus) and the eel (Anguilla japonica). This is in contrast to low levels observed in mammalian erythrocytes (Jacobsen and Smith, 1968). Its small size, its solubility, its non-utilization, its zwitterionic properties, and the presence of a transporter for its rapid uptake in cell membranes render taurine a potentially effective cell osmoregulator. In muscles (Schoffeniels and Gilles, 1972), and flounder erythrocytcs (Fugelli, 1967; Fugelli and Zachariassen, 1976) taurine acts as an osmoregulator. In these cells, the level of this amino acid changes relative to the salinity of the medium. In Ehrlich ascites tumor cells (Hoffman and Hendil, 1976) and heart muscle cells (Thurston PI ul., 1981) taurine has also been implicated in cellular osmoregulation. Taurine-depleted kittens have lower brain cell water and high mortality induced by chronic hypernatremia which suggests that taurine is an osmoprotective agent (Trachtman t>r ul., 1988). In this study we have measured the taurine content of erythrocytes isolated from several avian species, determined its likely origin, and examined its role in osmoregulation in pigeon erythrocytes.

Blood was collected in tubes containing either citrate or EDTA as an anticoagulant. After centrifugation at 400g for 10 min, the plasma and buffy cozt were discarded. The red blood cells were washed three times with phosphate-buffered saline (PBS) consisting of 0.145 M NaCI, 6 mM Na phosphate, 6mM glucose, pH 7.4 (320 mosMol). Cell counts were measured with a model S Plus IV Coulter Counter (Coulter Electronics, Hialeah, Fl). Cell volume

As an index of cell volume, hematocrits were determined by centrifugation of cell suspensions in hematocrit capillary tubes at 8000 g for 5 min. Eflux from cells Erythrocytes (3 x lO’/ml) were incubated in the phosphate-buffered saline of desired osmolarity at room temperature (22°C) for various periods of time. After centrifugation for 1min at lO,OOOg, the supernatant was analysed for taurine and other amino acids.

Taurine uptake Washed erythrocytes were resuspended at 5% hematocrit in PBS containing 50 PM 14C-taurine (0.5 PCi) in a final volume of 3ml in a 25ml flask. The flask was shaken at 100 rpm at 22°C. To measure uptake 0.1 ml aliquots were withdrawn and added to 1.2 ml of ice-cold PBS. Cells were pelleted by centrifugation at 6000g in an Eppendorf microfuge for 30 set and washed twice with ice-cold PBS. Control experiments in which 14C-taurine was added to a chilled erythrocyte suspension verified that this washing procedure was satisfactory. The pellet was resuspended in 0.5 ml of H,O and 0.5 ml of 15% perchloric acid was added. After 30 min on ice, tubes were centrifuged and the radioactivity was measured in 0.5 ml of the supernatant with the addition of 3 ml of Beckman Ready-Safe scintillation cocktail. Analytical

methods

Taurine was assayed Instruments, Tarrytown, 545

by an NY)

autoanalyser (Technicon as previously described

546

Z. K.

et

SHIHABI

al

Table

(Goodman and Shihabi, 1987). In this instrument, taurine is dialysed through a membrane to remove proteins and peptides. All the amino acids except taurine are subsequently trapped on a mixed cation and anion exchange column. Taurine in the eluate reacts with ophthaldialdehyde (OPA). The instrument analyses twenty samples per hour. Selected amino acids were measured with automated instruments (TSM. Technicon) and the Perkin-Elmer amino

Amino acid

RESULTS

Taurine content

qf aCan erythrocytes

We measured the taurine content of erythrocytes from a wide variety of avian species. The taurine levels in these cells in general were 100 times higher than in the mammalian cells tested (Table 1). Similar results were obtained utilizing either an automated amino acid analyser or a HPLC technique to assay taurine. The values obtained for human and rat cells were similar to those reported by others (Jacobsen and Smith, 1968). Taurine concentrations in pigeon erythrocytes were approximately 17 mmol/l. This value is similar to the 16 mmol/l reported in Ehrlich ascites tumor cells (Hoffman and Hendil, 1976) and 14 mmol/l in HeLa cells (Jacobsen and Smith, 1968). The elevated taurine level in avian cells was not related to the nucleation of these cells or to the Table

I. Taurine

of

mmol,‘kg Hb

TaUri”e Aspartlc acid Glycine Glutamic aud Anserine Glutamine Carnosine Alanine Serine Threonine /I-alanine Other amino acids

acid analyser (Norwalk, CT).

Small dialysable compounds reactive with OPA were measured on the same taurine autoanalyser with the removal of the ion exchange column to allow all the dialysable amino acids to react with the OPA. This procedure detects essentially all the free amino acids (AA). However, because of their different rates of reaction with the OPA and dialysis through the membrane, it is not an exact measure of total free amino acids. Cysteinesulfinate decarboxylase was assayed by incubating the red blood cells or tissue homogenates with 10 mM cysteic acid or cysteine sulfinic acid as substrate in 0.1 M phosphate buffer (pH 6.8) with 0.1 mM pyridoxal phosphate and 1 mM dithiothreitol. The liberated taurine or hypotaurine was measured after derivatization with OPA. Hemoglobin was measured spectrophotometrically by the absorbance of cyanmethemoglobin at 540 nm.

2. Free amino acid content pigeon erythrocytes

59.0 12.7 8.X 6.0 5.9 4.4 2.0 1.7 1.5 1.2 0 5.0

presence of DNA since frog erythrocytes, which are nucleated, and the human blood samples with elevated reticulocytes, did not contain high taurine levels (Table 1). The taurine content of avian plasma was ten times that of mammalian and frog plasma (Table 1). The level of taurine in erythrocytes was correlated with the plasma value with r = 0.581 (P < 0.02). We also measured other free amino acids in pigeon erythrocytes using an automated analyser. Taurine comprised more than half of the free amino acids in these cells (Table 2). Aspartic acid, glycine, glutamic acid, anserine and glutamine were other free amino acids present in relatively significant concentrations. Source of erythrocyte

taurine

The taurine in avian erythrocytes could be derived from synthesis within the cell or uptake from serum. The ability of pigeon erythrocytes to synthesize taurine was assessed by the presence of cysteine sulfinic acid decarboxylase activity, a key enzyme in taurine biosynthesis in most tissues (Weinstein and Griffith, 1987). We were unable to detect any activity in these cells as assayed by the conversion of either cysteine sulfinic acid or cysteic acid to hypotaurine or taurine utilizing a HPLC assay. We were able to demonstrate the presence of this enzyme in cultured renal tubular cells and liver homogenates. These cells were shown, however, to accumulate taurine against a concentration gradient (Fig. I).

concentration

in plasma

and erythrocytes

Plasma taurine (PM)

Erythrocyte (mm&kg Hb k SD)

Species

”

HU”Xl” Rabbit Rat Pigeon, white carneau (Columha liuia) Chicken. white leghorn (CaNus gal/us) Wild turkey (MeLagris ga/lopar,o) Domestic turkey (Melragris gallopam) Barred rock (CaNus gal/us) Golden pheasant (Chrysolophu.~ pictus) Mallard duck (Anus platyrhynchos) Chukar partridge (Alecroris chukar) Bob-white quail (Cdinus virginianus) Coturnix quail (Coturnix coturnix) Mambo” stork (Leptoptilus crumenferus) Kori bustard (Choriofis kori) Flamingo (Phoenicopterus ruher) Korhaan (A.frori.7 am) Macaw (Ara ararauna) Frog (Runcr pip&u) Human (24% reticulocytes) Human (20% reticulocytes)

7 3 4

45 36

8

200

62 i X.0

3 I I I I I I I I I I I I I

275 285 265 321

46 37 38 20

44x 749 571 1345 625

20 74 43 72 52

3

I I

0.2 * 0.1 1.3 0.3

32

500

49

625

57

150

53

125

37

24

0.6

0.4 0.3

Osmoregulation and taurine in avian erythrocytes

i

*

547

0

6

3

0 0

t0

20

30

40

50

60

70

Incubation Time tMin)

Incubation

Fig, I. Uptake of ‘%-taurine (50pM) by pigeon erythrocytes. Incubation media contained either Nat salts (a), Na’ salts + 500 PM [I-alanine (0) or K+ salts (+).

Whilst the cells contained in excess of 17 mM taurine, they rapidly took up taurine when it was only 50 FM in the medium. Little or no efhux occurred under these conditions, indicating it was not an exchange process. Negligible uptake occurred at 4°C. A double reciprocal plot revealed that the uptake process had a K, of 62.5 /IM and a V,,, of 0.9 1 nmol/min/109 cells (Fig. 2). Uptake was inhibited by /I-analine, suggesting that the process is similar to that described for mammalian cells and fish erythrocytes (Tallan et al., 1983; Fincham et al., 1987). Uptake required Na+, as illustrated by the low uptake in K+-containing media (Fig. 1). There was a small amount (8% of the total) of Na+-independent uptake consistent with that observed in fish erythrocytes (Fincham et al., 1987) and in lymphoblastoid cells (Tallan et al., 1983).

Kregeuow (I 97 1) observed that duck erythrocytes respond to hypotonic solutions in the first few minutes by swelling followed by a slow decrease to normal size. Figure 3 illustrates that pigeon erythrocytes also respond to hypotonic solutions with a rapid increase in cell volume during the first few minutes as measured by an increase in the hematocrit.

OS0 I 0.40

-

+

0.30 -

//I

+

Time

(Hours)

Fig. 3. Changes in erythrocyte volume as a function of time and osmolarity. Cells were incubated with 157 mM NaCl ( + ), 103 mM (A) or 87 mM (A). After 5 hr incubation, the salt concentration of the media was adjusted to isotonicity with a concentrated saline solution.

This was then followed by a slow decrease in cell volume. There was a slow decrease in cell volume in erythrocytes maintained under iso-osmotic conditions over the course of the incubation in this buffered salt solution. On restoration of iso-osmotic conditions, hypotonically shocked cells regained a cell volume similar to that of controls, indicating that cell volume regulatory mechanisms had not deteriorated during the course of the experiment. The rapid swelling in hypotonic media was accompanied by a release of taurine into the medium. During the cell shrinkage that followed the initial cell swelling, taurine continued to be released into the medium (Fig. 4). This efRux was abolished by a restoration of isotonicity (Fig. 4). Cells in the taurine-free isotonic saline also released taurine into the medium but at a much slower rate than did the hypotonically shocked cells. Other amino acids were released into the medium following the hypotonic shock (Table 3). However, the ratio of taurine relative to the other amino acids (as OPA reactive material) in the hypotonic incubation medium was higher than that in the isoosmotic medium (Fig. 4). Some amino acids, such as isoleucine or leucine, did not efflux from the cell with incubation in the hypotonic medium whereas glycine was released, indicating some selectivity in this process (Table 3).

+

)r, =: 0.20 _

I&l. -0.20

0.00

0.40

0.20

Table 3. Amino Amino acid

0.60

0.60

1 ,ISl

Fig. 2. Double reciprocal plot of the dependence of the rate of taurine uptake on the concentration of taurine. As Fig. 1demonstrates that uptake is linear for at least 40 min, the initial rate of uptake was estimated from the uptake in 30 min. The concentration of taurine is expressed in pM and the uptake rate in pmol/min/lO’ cells.

Taurine GlyCi!X Isoleucine Lelkne Aspartic Glutamic Serine Alanine

acid efflux from pigeon erythrocytes Efflux in 5 min (nmol)

Efflux in 120 min (nmol)

25.0 10.8 12.8 10.6 5.5 2.1 4.4 0.3

75.0 21.4

-~

Il.8 12.1 1.6 5.6 7.4 0.6

Etythrocytes (3 x 10’) were incubated in 1ml of hypotonic buffer containing 87 mM Na+ The concentration of amino acids in the medium was measured after centrifugation of the cells.

Z. K. SHIHABI

548

+

0

.

1

A

3

2

Incubation

4

5

( Hours)

Time

(4 _.-_ 0.5.3

-

0.20

0

1

2

Incubation

3

Time

4

5

( Hours)

(b) Fig. 4. Efflux of taurine and amino acids from erythrocytes as a function of time and osmolarity. (a) Illustrates changes in the taurine content of the medium under isotonic ( + ) (157mM Nat) or hypotonic (A) (87mM Na+), and (b) illustrates changes in the ratio of taurine to the total amino acid content of the medium. In one set of incubations isotonicity was restored after a 2 hr incubation (A).

DISCUSSION

Studies with taurine-deficient animals have suggested that taurine has important roles in retinal and cardiac tissues @chaffer et al., 1984; PasantesMorales and Cruz, 1984) although its precise biological function remains uncertain. Evidence has been presented that it acts as a neurotransmitter, stabilizes membranes, and could be an important antioxidant, osmoregulator, and detoxifier (Jacobsen and Smith, 1968). The inter-relationships between these different functional roles, if any, are not clear. Our studies have revealed that the erythrocytes of avian species contain a high taurine content. The high taurine content did not appear to be related to their nucleation, as nucleated amphibian erythrocytes and human preparations enriched with reticulocytes contained low levels. Avian erythrocytes are thus similar to fish erythrocytes in having a high taurine content. Their transport systems also have similar kinetic properties (Fincham et al., 1987). Regulatory volume control has been observed previously in avians. When placed in hypotonic media, duck erythrocytes initially swell as a result of osmotic forces but subsequently shrink due to regulatory volume mechanisms (Kregenow, 1977). This shrink-

et ul.

age is primarily due to a net loss of KC1 and an associated loss of water. In most cells studied, efflux of small, zwitterionic amino acids such as taurine and glycine, also contributes to this regulated volume decrease. We investigated the role of taurine in osmoregulation in pigeon erythrocytes and found that changes in taurine content occurred in concert with osmotic changes. Similar responses were observed in flounder red blood cells which also have a high taurine content (Fugelli, 1967; Fugelli and Zachariassen, 1976). When fish are removed to a low salt water medium the plasma osmolarity decreases. The adaptation of the erythrocytes to the new environment results in a decrease in taurine content and in the concentration of some other amino acids. When Ehrlich ascites mouse tumor cells are subjected to hypotonic solutions they also swell and then reduce their volume towards that seen in isotonic medium. Taurine has been shown to be involved in this regulation (Hoffman and Hendil, 1976). Taurine is one of the main amino acids to decrease rapidly in a rat lens incubated in a medium containing galactose (Kinoshita et al., 1969). Under these conditions galactose is metabolized to galactitol, a nonmetabolizable substance which increases osmotic pressure. This change in taurine concentration can be inhibited by compensating for the osmolarity change in the medium. Trachtman et al. (1988) also demonstrated that taurine plays a major role in osmotic pressure regulation in the brain of the cat. Chesney (1988) pointed to the importance of taurine in osmoregulation in general. The high millimolar concentration of taurine contributes to the total osmolarity of avian red blood cells. Under hypotonic conditions taurine effluxes from cells at a higher rate than other amino acids, thus sparing more important amino acids. This may be a major role of taurine in cells such as leukocytes and cardiac cells which contain high taurine levels. However, this does not exclude other important roles which taurine might play, such as neuromodulation and detoxification, in different cells. This multifunctional role would be analogous to that of albumin which has a primary role in regulating plasma osmotic pressure and a secondary role of binding fatty acids, drugs, hormones, bilirubin etc. In agreement with the observations of Kregenow (1971, 1981) we have noted, using an ion-selective electrode, that K+ ions exit from the pigeon erythrocytes in response to a hypotonic incubation medium. Taurine contributes only a minor fraction to the osmolarity of the cell compared to K+ (17 vs 120 mmol/l). It is difficult to speculate on the importance of taurine efflux relative to K+ without further studies including the effects of different transport inhibitors on taurine efflux. The selective taurine fluxes relative to other amino acids, the high taurine concentration within the cell, and a high affinity, specific transport mechanism suggest that taurine participates with K+ in regulating cell size and osmotic pressure. It is also worth noting that taurine efflux from the avian erythrocyte is a slow process compared to that seen in similar experiments we performed on human platelets, possibly reflecting differences in cell volume/cell surface ratios. Our data demonstrate that a primary role of

Osmoregulation and tautpine in avian erythrocytes

549

taurine in pigeon erythrocytes may be in the regulation of osmotic pressure and cell volume. This

between plasma and erythrocytes in flounder (Platichthys flesus) at different plasma osmolalities. Comp. Biochem.

reinforces the concept that taurine plays an important role in size and osmotic regulation in a wide range of cells. This study also raises several important questions: why do some cells but not others opt to use taurine for osmotic regulation? What are the factors which regulate taurine efFiux and uptake under iso-osmotic conditions? What happens if such cells are deprived of taurine? Can taurine be replaced by other compounds? Since avian erythrocytes are readily isolated, undergo regulatory volume control, and transport taurine, this model should be useful in further investigations of the role of taurine in cells and in osmoregulation.

P&oi. 55A, 173-177. Goodman H. 0. and Shihabi Z. S. (1987) Automated analysis for taurine in biological fluids and tissues. Clin.

A~kno~~,fedgement.~--Thy authors thank Dr C. Parkhurst, Department of Poultry Science, NC State University, Raleigh. North Carolina; Dr M. Loomis of NC State Zoo, Asheboro, North Carolina; Dr W. M. Colwell, Holly Farms, North Wilkesboro, North Carolina; and Dr. Jon Lewis and his group, Department of Pathology, Wake Forest University, Winston-Salem, North Carolina for their help in providing blood samples from several species, We thank Dr R. W. Prichard for his encouragement of this work.

Chem. 33, 835-837.

Hoffman E. K. and Hendil K. B. (1976) The role of amino acids and taurine in isosmotic intracellular regulation in Ehrlich ascites mouse tumour cells. J. conzp. Physioi. 108, 279-286.

Jacobsen J. G. and Smith Jr. L. H. (1968) Biochemistry and physiology of taurine and taurine derivatives, Physiol. Rev. 48, 424-5 1I. Kinoshita J. H., Barber G. W., Merola L. 0. and Tung B. (1969) Changes in levels of free amino acids and mvoinositol in gsactose-exposed lens. Invest. Ophthalmology 8, 625-632.

Kregenow F. W. (1971) The response of duck erythrocytes to non-~emolyti~ hypotoni~ media. Evidence for a volume-controlling mechanism. J. gen. Physiof. 58, 372-395.

Kregenow F. M. (1981) Osmoregulatory salt transporting mechanisms: Control of celi volume in anisotonic media. Ann. Rev. Physiol. 43, 493-505.

Kulakowski E. C., Maturo J. and Schaffer S. W. (1984) The low affinity taurine-binding protein may be related to the insulin receptor. Frog. Clin. Biof. Res. 179, 1277136. Pasantes-Morales H. and Cruz C. (1984) j , Taurine: a ohvs. iological stabilizer of photor~eptor membranes, Prog. Cfin. Biof. Res. 179, 371-381. Schaffer S. W., Seyed-Mozaffari K., Kramer J. and Tan B. H. (1984) EtTect of taurine depletion and treatment on cardiac contractility and metabolism. Prog. Clint. Biol. 1

REFERENCES

Bucuvalas J. C., Goodrich A. L. and Suchy F. J. (1988) Enhanced uptake of taurine by basolateral plasma membrane vesicles isolated from develoning . - rat liver. Ped. Res. 23, 172-175. Chesney R. W. (1988) Taurine: is it required for infant nutrition? J. Nutr. 118, 610. Chesney R. W., Gusowski N. and Dabbagh S. (1985) Rena1 cortex taurine content regmates renal adaptive response to altered dietary intake of sulfur amino acids. J. cfin. Invest. 76, 22 13-222 1. Fincham D. A., Wolowyk M. W. and Young J. D. (1987) Volume-sensitive taurine transport in fish erythrocytes. J. Membr. L&f, 96, 45-56. Fugelli K. (1967) Regulation of cell volume in Rounder (Pleuronectes flesus) erythrocytes accompanying a decrease in plasma osmolarity. Camp. Biochem. Physiol. 22, 253-260.

Fugelli K. and Zachariassen K. E. (1976) The distribution of taurine, Emma-aminobutyri~ acid and inorganic ions

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Schoffenieis E. and Gilles R. (1972) Ionoregulation and osmoregulation in mollusca. In Chemical Zoology (Edited by Florkin M. and Scheer B. T.), Vol. 7, pp. 3933420. Academic Press, New York. Tallan H. H., Jacobson E., Wright C. E., Schneidman K. and Gaul1 G. E. (1983) Taurinc uptake by cultured human lymphoblastoid cells. Lije Sci. 33, 1853-1860. Thurston J. H., Hauhart R. E. and Naccarato E. F. (1981) Taurine: possible role in osmotic regulation of mamalian heart. Science 214, 1373-1374. Trachtman Ii., Barbour R., Sturman J. A. and Finberg L. (1988) Taurine and osmoregulation; Taurine is a cerebral osmoprotective molecule in chronic hypernatremic dehydration Ped. Res. 23, 35-39. Weinstein C. L. and Griffith 0. W. (1987) Cysteinesuifinate decarboxylase. Meth. Enzymof. 143, 4044 t 0.