Tauroursodeoxycholic acid inhibits the cytosolic Ca++ increase in human neutrophils stimulated by formyl-methionyl-leucyl-phenylalanine

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September

3, 1990 28,

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Pages 1115-1121

1990

TAUROURSODEOXYCHOLIC

ACID

HUNAN NEUTROPEILS

INHIBITS

STIMULATED

THE CYTOSOLIC

CA++

INCREASE

IN

BY FORMYL-METBIONYL-LEUCYL-

PHENYLALANINE

U.Beuers, Departments Grosshadern,

M.Thiel*, of Medicine University

H.Bardenheuer*,

and G.Paumgartner

II and *Anesthesiology, of Munich, 8000 Munich

Klinikum 78, F.H.G.

Received August 6, 1990 Summary :

The effect of the cytoprotective bile acid tauroursodeoxycholic acid (TUDCA) on basal cytosolic free Ca++ (Ca++). and receptor-mediated (Ca++). increase was studied in human po&morphonuclear neutrophils uking the fluorescent dye guin2. Basal levels Of (Ca++) i were 96 f 6 nmol/l (mean f SEM, n=48). TUDCA and its cytotoxic epimer taurochenodeoxycholic acid (TCDCA) at 500 pmol/l increased (Ca++). by 31 f 12 and 27 & 7 nmol/l, respectively (n=6, ~~0.05). Stimulation of neutrophils with the chemotactic tripeptide N-foy$l-methionyl-leucyl-phenylalanine (FMLP;10-7 mol/l) induced a (Ca ). increase of 200 + 32 nmol/l which was inhibited after preincuba 1.ion with TUDCA (500 @mol/l) or TUDCA+TCDCA (500 pmol/l, each) by 60.1 % and 59.5 %, respectively, but not with TCDCA (500 &mol/l) alone. The inhibitory effect of TUDCA on FMLP-induced (Ca )i increase was strongly concentrationdependent and was nearly complete at 1000 I.tmol/l. Since (Ca++). is discussed as a mediator of cellular injury we hypothesize k hat TUDCA may exert its protective effects at least partly via inhibition of (Ca++) i-mediated cytotoxic processes. 01990 Academic ecess, Inc. Ursodeoxycholic acid (UDCA), a hydrophilic dihydroxy bile acid, induces marked improvement of liver function indices in patients with cholestatic liver disease and may be an effective drug for the treatment of cholestatic liver disease (reviewed in 1). UDCA is known to have a protective effect against cell damage induced by other bile acids in vitro. Some evidence suggested that the physiological glycine and taurine conjugates of UDCA represent the 'I Protective" form of UDCA (2). The mechanism of its potentially cytoprotective action, however, is unclear. Cytosolic free calcium (Ca++)i plays a key role in the control of many different cell functions (3) and may be involved in cellular A possible role of (Ca++)i in the mediation of bile injury (4,5). acid induced cytotoxicity has been suggested in recent studies

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0006-291x/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

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which demonstrated that different toxic bile acids induced elevation Of (Ca++)i in human erythrocytes and rat hepatocytes concomitant with signs of cell damage (6,7). A possible role of (ca++)i in the cytoprotective action of UDCA has to be considered but has not yet been investigated. Therefore, we studied the action of the taurine conjugate of UDCA (TUDCA) on basal as well as receptor agonist-stimulated (Ca++)i levels in comparison to that of its physiological epimer taurochenodeoxycholic acid (TCDCA). Effects of bile acids on (Ca++)i (6,8) as well as cytoprotection of TUDCA (1,2) have been observed in different types of cells. We have chosen neutrophils for our investigations since they are not only one of the most easily accessible human cell types, but provide a representative pharmacological model for the study of complex cellular signal transduction processes. Methods Reasents. Quin2-tetra(acetoxymethyl)ester (quina-AM), quin2, Nformyl-methionyl-leucyl-phenylalanine (FMLP), Triton X-100, and ethylene glycol-bis(R-aminoethylether)-tetraacetic acid (EGTA) were obtained from Sigma Biochemical Co. (St.Louis,MO). Sodium salts of tauroursodeoxycholate (TUDCA) and taurochenodeoxycholate (TCDCA) were obtained from Calbiochem-Behring (San Diego, CA). Fitoll-hypaque 400 (density 1.077) was from Biochrom (Berlin, F.R.G.). All other chemicals were of reagent grade and from commercial sources. Isolation of oolvmorohonuclear neutroohils was performed in heparinized blood (50 IU/ml) of healthy human volunteers by the method of A.Boyum (9). Ficoll-hypaque gradient centrifugation (1200 g/min for 20 min) was followed by dextran (6% w/v) sedimentation (90 min at room temperature) and hypotonic lysis of erythrocytes. Cellular suspensions were made in Hank's balanced salt solution (HBSS) containing 1 mmol/l Ca++. Determination of (Ca++l.. After isolation, polymorphonuclear neutrophils (5x106 cells/m 1 ) were incubated for 60 min at 37°C in HBSS containing QuinZ/AM at a concentration of 60 pmol/l. Cells were then extensively washed and diluted to 2.5~10~ cells/ml. Intracellular content of quin2 free acid was 0.85 t 0.15 nmol/106 cells (n=lO). The fluorescence of control and quin2-loaded cells was measured using a Zeiss spectrofluorometer (PMQ3): Excitation wavelength was 339 nm and emission wavelength was 500 nm (10). Cuvettes containing 1 ml of cell suspension were kept at 37°C in a thermostated cuvette holder and stirred. After fluorescence had stabilized for at least 5 min, bile acids (dissolved in HBSS) or HBSS were added to the cell suspension and fluorescence was continuously recorded. FMLP (in HBSS) was added when indicated. (Ca++)i was calculated using the formula (Ca++) i = KD X (F - Fmin)/(Fmax

where

KD is

115 nm (lo),

and F is 1116

the

- F) 1

fluorescence

(10)

measured

in

ar-

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the experiment. F,, is the fluorescence units duri+ng concentrations after fysis of cells by addimeasured at high Ca tion of Triton X-100 at the end of each experiment. F . fluorescence at low Ca++ concentrations measured after amd&20ntZ EGTA to the Triton X-100 containing cell lysates.

bitrary

Results

Basal (Ca++)i levels in polymorphonuclear neutrophils of healthy subjects were 96 + 6 nmol/l (n=48). Addition of the taurine conjugates of UDCA or CDCA (500 fimol/l) led to comparable increments Of (ca++) i of 31 + 12 and 27 k 7 nmol/l after 3 minutes for either bile acid (~~0.05; figure 1). Addition of both TCDCA and TUDCA in equimolar concentrations (500 pmol/l, each) led to an increase of (Ca++)i comparable to that seen after TUDCA alone (figure 1). When 62.5 to 1000 pmol/l of TUDCA was added the increase of (Ca++)i was correlated with the concentration of the bile acid according to a 2nd order polynomial function (r=0.9817; figure 2a).

[Ca' .+I (nmolll)

2oc

I-

1X

)-

1oc

k

5c

I-

, -!

0

5

10

15

20

25

(mid

Ca++ levels (Ca++). in human polymorphoFigure 1 . Intracellular nuclear neutrophils during incubation with'taurochenodeoxycholic tauroursodeoxycholic acid (TUDCA: 500 acid (TCDCA; 500 fimol/l), of both (500 pmol/l, each), and no bile wol/l) , a combination acids (control) before and after addition of the potent chemotacN-formyl-methionyl-leucyl-phenylalanine tic stimu_$ant. tripeptide Cells were isolated from blood of 6 different mol/l). (FMLP: 10 healthy subjects. .Fluorescence signals of the 4 assays of one cell suspension using the dye guin2 were measured simultaneously in a Results are given as mean f SEM of 6 experispectrofluorometer. ments. 1117

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a

I

0

500

250

0

TUDCA

750

1OC

(urnoIl

of the TUDCA-induced (Ca++). increase with Figure 2. a. Correlation the concentration of TUDCA used. The (Ca++). values'are the diffemeasured before addition if TUDCA and 3 minutes rence of (ca++). Resu 1 ts are given as mean t SEM for different concenthereafter. trations between 0 and 1000 )imol/l TUDCA. The number of experiments is given in the figure. The increases of (Ca++)i and the concentrations of TUDCA can be described by a 2nd order polynomial

function (r=0.9817). b. Correlation of the amount of (Ca++).

mol/l)

with

1000 pmol/l.

the

concentration (Ca++). values

increase

due to FMLP (10s7

0 and of TUDCi in a range between are the difference of (Ca++). measured

&lls were FMLP and 2 minutes thereafter. before addition 08 preincubated for 15 min with TUDCA (s.figure 1). Results are.given is+$iven in the figure. as mean f SEM. The number of experiments The relation between the increases of (ca Ii and the concentrations of TUDCA can be described by a 2nd order polynomial function

(r=0.9962).

After addition of the chemotactic tripeptide N-formyl-methionyl(FMLP; 10B7 mol/l), a receptor-mediated leucyl-phenylalanine (Ca++)i increase of 200 f 32 nmol/l (n=6) was seen in neutrophils not pretreated by bile acids (figure 1). Preincubation with TCDCA (500 pmol/l) for 15 min did not alter the (Ca++)i increase caused by stimulation with FMLP. Preincubation with TUDCA (500 pmol/l) or with a combination of TUDCA and TCDCA (500 pmol/l, each), however, significantly inhibited the increase of (Ca++)i following FMLP (figure 1). This stimulation by 60.1 % and 59.5 %, respectively inhibitory effect of TUDCA on the increase of (Ca++)i after FMLP was dose-dependent (r=0.9962; figure 2b). Discussion Our data increases

show that the basal cytosolic

putatively cytoprotective free Ca+' levels in 1118

human

bile acid cells to

TUDCA a si-

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degree as its CytOtOXiC epimer TCDCA. TCDCA, however, inhibits the receptor-mediated human neutrophils in a concentration-dependent

milar

RESEARCH COMMUNICATIONS

TUDCA in (Ca++)i manner.

contrast increase

to in

Basal (Ca++)i levels in our study are in accordance with those reported in the literature (3). Experimental pitfalls commonly observed when fluorescent dyes are used (10) were excluded : i) addition of Mn++ to the medium did not alter the results indicating absence o:f significant dye leakage: such leakage, in general, is not seen in neutrophils as opposed to mononuclear cells or hepatocytes (10); ii) bile acids or FMLP did not cause alterations of fluorescence in the concentration range used; iii) quenching effects like those of hemoglobin in erythrocytes are not known to disturb fluorescence measurement in neutrophils (10) and have not been observed in bile acid-containing media. The degree of hydrophobicity of a bile acid has previously been postulated to be an important factor of bile acid-induced (Ca++)i increase (7,8). The comparable changes in (Ca++)i caused by TUDCA and TCDCA, which differ in their degree of hydrophobicity (11) contradict this contention, but are in line with comparable Ca++ binding capacities of TUDCA and TCDCA in vitro (12) - Moreover, they support the hypothesis that the bile acid-induced (Ca++)i increase per se does not initiate cytotoxicity (13). The mechanisms of Ca++ mobilization in neutrophils caused by the chemoattractant tripeptide FMLP have been well studied. They have been suggested to be similar to those of other Ca++ mobilizing angiotensin II or the A-adrenergic agent agents like vasopressin, (14): (i) specific binding of the phenylephrine in hepatocytes agonist - FMLP to the tlchemoattractant receptor" of neutrophils, (ii) intramembraneous signal transduction via a receptor - G protein - phospholipase C system, (iii) stimulation of phosphoinositide turnover, (iv) increase of (Ca++)i due to a release of Ca++ from intracellular storage sides in combination with the influx of Preincubation of cells with TCDCA at a extracellular Ca++ (14). concentration of 500 pmol/l did not alter the FMLP-induced (Ca++)i increase. TCDCA at the concentration chosen has previously been shown to be cytotoxic in a Ca++-dependent manner (7). TUDCA inhibited the FMLP-induced (Ca+")i increase in a wide range of concentrations (62.5 to 1000 pmol/l) in which no major damaging effects on human hepatocytes have been noted (16). Incubation of neutroconcentrations led to with TUDCA and TCDCA in equimolar phils (Ca++)i levels changes of basal and receptor agonist-mediated 1119

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identical to those after incubation with TUDCA alone. This finding is in line with the observation that TUDCA may reverse the potentoxic effects of other bile acids in the pertially Ca++ -mediated fused liver (2). of cells with hydrophobic It may be speculated that incubation bile acids leads to structural destabilization of cell membranes enhanced by receptor-mediated elevation of which is further increase activate Ca++-dependent (Ca++) i. This (Ca++)i may phospholipases (16) and proteases (17) resulting in the breakdown (Ca++)i inof cell membranes. Inhibition of receptor-mediated crease has been suggested to represent a crucial factor of cytoprotection in the model of norepinephrine-induced renal failure. The Ca+' entry blockers verapamil and nifedipine have been shown functional and morphological protection to exert substantial against norepinephrine-induced ischemic acute renal failure (18). In search of the molecular mechanism of cytoprotection of TUDCA in potential interactions with membile acid-mediated cytotoxicity, brane receptor binding sites, membrane channels, G binding proteins or intracellular structures have been discussed (1). Almost all of these factors are clearly involved in the FMLP-stimulated rise of (ca++)i in human neutrophils. In conclusion, our data show that the putatively cytoprotective bile acid TUDCA increases basal (Ca++)i levels in human neutrophils to a similar degree as its cytotoxic epimer TCDCA, but that, in contrast to TCDCA, it inhibits the receptor-mediated (Ca++)i increase. We hypothesize that TUDCA exerts its protective effects at least partly via inhibition of (Ca++)i- mediated cytotoxic processes. Acknowledament: Claudia Made1

The is gratefully

skillful technical acknowledged.

assistance

of

Miss

References 1.

2.

3. 4. 5.

Hofmann, A.F. (1990) In Strategies for the treatment of hepatobiliary diseases (G. Paumgartner, A. Stiehl, L. Barbara, E. Roda, Ed.) pp 13-34. Kluwer Academic Publishers, Lancaster, UK. Kitani, K. (1990) In Strategies for the management of hepatobiliary diseases (G. Paumgartner, A. Stiehl, L. Barbara, E. Roda, Eds.), pp 43-56. Kluwer Academic Publishers, Lancaster, UK. Carafoli, E. (1987) Ann. Rev. Biochem. 56, 395-433. Cheung, J.Y., Bonventre, J.V., Malis, C.D., Leaf, A. (1986) N. Engl. J. Med. 314, 1670-1676. Thomas, C.E., Reed, D.J. (1989) Hepatology 10, 375-384. 1120

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Delberg, D-G., Dubinsky, W.P., Sackman, J.W., Wang, L.B., Adcock, E.W., Lester R. (1987) Hepatology 7, 245-252. Anwer, M.S., Engelking, L.R., Nolan, K., Sullivan, D., Zimniak, 'P., Lester, R. (1988) Hepatology 8, 887-891, Combettes, L., Berthon, B., Doucet, E., Erlinger, S., Claret, M. (1989) J. Biol. Chem. 264, 157-167. Boyum, A. (1968) Scand.J. Clin. Lab. Invest. Suppl. 97, 7789. Tsien, R., Pozzan, T. (1989) Meth. Enzymol. 172, 230-262. Heuman, D. (1989) J. Lipid. Res. 30, 719-730. Gleeson, D., Murphy, G.M., Dowling, R.H. (1990) J. Lipid. Res. 31, 781-791. Farrell, G.C., Duddy, S.K., Kass, G.E.N., Llopis, J., Orrenius, S. (1990) J. Clin. Invest. 85, 1255-1259. Williamson, J.R., Monck, J.R. (1989) Annu. Rev. Physiol. 51, 107-124. Miyazaki, K., Nakayama, F., Koga, A. (1984) Dig. Dis. Sci. 29, 1123-1130. Chien, K-R., Abrams, J., Serroni, A., Martin, J.T., Farber, J.L. (1978) J. Biol. Chem. 253,4809-4817. Nicotera, P., Hartzell, P., Davis, G., et al. (1986) FEBS Lett. 209, 139-144. Gordon, J-A., Burke, T.J., Arnold, P.E., Bulger, R.E., Dobyan, D.C., Schrier, R.W. (1984) J. Clin. Invest. 74, 18301841.

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