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Interaction of the organic anion 1-aniline 8-naphthalene sulfonate (ANS) with isolated rat hepatocytes
Comp. Biochem. Physiol. Vol. 86B, No. 1, pp. 7-10, 1987 Printed in Great Britain
0305-0491/87 $3.00 + 0.00 Pergamon Journals Ltd
INTERACTION OF THE ORGANIC ANION 1-ANILINE 8-NAPHTHALENE SULFONATE (ANS) WITH ISOLATED RAT HEPATOCYTES RUT M. AGOERO, GUILLERMO P I c r , EDGARDO GUIBERT a n d JUAN L. CORCHS* Instituto de Fisiologia Experimental, Consejo Nacional de Investigaciones Cientificas y Tecnicas, Universidad Nacional de Rosario, Rosario, Argentina (Received 27 January 1986)
Abstract--1. The interaction of ANS with rat hepatocytes in time was studied by fluorescence spectroscopy. 2. The intercept of the first linear portion of the time curve of interaction showed a positive value over all the ANS concentration range employed. 3. This value was maintained after cellular disruption by homogenization. 4. It was affected by ionic strength, pH, and divalent cation in the incubation medium, all conditions affecting the cellular surface. 5. These data suggest that this phenomenon might be a binding of the compound to the hepatocytes surface. 6. Due to the time constant and its disappearance after cellular disruption the other slower component of the curve seems to correspond to a process of translocation across the membrane.
INTRODUCTION Circulating organic a n i o n s b o u n d to serum a l b u m i n (Pfaff et al., 1974; Baker a n d Bradley, 1966) are excreted by the liver t h r o u g h m e c h a n i s m s involving binding, uptake, intracellular transport, conjugation a n d biliary excretion (Goresky, 1965). The first process is a n i m p o r t a n t one since it m i g h t represent a limiting step o n the sequence. Isolated rat hepatocytes have been s h o w n to possess specific sites for organic a n i o n s such as brom o s u l f o p h t h a l e i n (Tiribelli et al., 1978; Reichen et al., 1981; Lunazzi et al., 1982) a n d taurocholic acid (Schwarz et al., 1975). In the present p a p e r we analyze the interaction of a n organic a n i o n with isolated hepatocytes, which h a s also been employed as a n effective p r o b e in the study of a n i o n t r a n s p o r t in erythrocytes (Fortes a n d H o f f m a n , 1971) a n d Ehrlich ascites cells. A n early phase is described t h a t m i g h t be considered as binding since it is influenced by factors acting u p o n surface p h e n o m e n a , whereas the late phase of interaction could c o r r e s p o n d to a cellular u p t a k e o f the compound. MATERIALS AND METHODS Cell preparation IIM rats (NRE Lab., Carshalton, Surrey, Eng. 1964) weighing 200-300 g were used. Liver cells were obtained according to the procedure previously described (Guibert et al., 1983) with some modifications since in the second step *Correspondence to be addressed--Dr Juan L. Corchs, Chtedra de Fisiologia, Facultad de Ciencias Mrdicas, Santa Fe 3100a, 2000 Rosario, Argentina.
the colagenase was recirculated. Cellular viability was tested by the tryptan blue exclusion test. All experiments were carried out on cell suspensions with viabilities of 85-95% and were done within 2 3 hr after cell isolation. Fluorescence measurements Fluorescence was measured by spectrofluorometry by means of a 93200 Spectral Fluorescence Accessory attached to a Beckman DU Spectrophotometer, with a GEF4T5BL mercury phosphor excitation lamp and a 7-60 Turner primary filter which provides a maximal transmittance at 360 nm. Light scattering effects were corrected by the use of an appropriate blank. Interaction studies Aliquots of ANS (final concn 3-60 #M) were added to 3 ml of cell suspension (0.30-0.40 x 106 cells/ml) in control or test medium, quickly mixed by cuvette inversion and the fluorescence intensity was recorded at 485 nm during 4 sec to 20 min. The cuvette was inverted previously to each determination in order to avoid cellular sedimentation. The experiments have been performed at least twice for each cell preparation. Fluorescence values were expressed as RFU (relative fluorescence units)x 106cell. These values were corrected for light scattering and fluorescence emitted by the ANS bound to proteins of supernatant. Both were constant during the time of the experiment. Media composition Control medium consisted of 150 mM NaCI; 5 mM KCI; 5 mM glucose; 10 mM HEPES (pH 7.5). High osmolality (400-600-900 mOsm/Kg H20 ) was obtained by addition of sucrose to the control medium. The pH (4 ~ 9) was adjusted with 10mM HCI or NaOH. Low ionic strength was obtained by substituting isosmotically NaCI by sucrose. The results were presented as difference between control and paired test media and expressed as mean values (__+SEM). Statistical analysis of the difference thus obtained was done according to standard methods (Snedecor, 1956).
RUT M. AGf3ERO et al.
8
I00
A - -
O t. O
== 03 Z
l
x -1
50 .la
O3 Z
IE b
15
I
0
I
320
I
45
ANS bound (UFRxlO6ceL)(r)
I
640
30
960
T i m e (sec)
Fig. 1. Kinetics of interaction between ANS (12#M) and intact (O) or disrupted (©) isolated rat hepatocytes (0.30-0.40 x 106cells/ml) suspended in control medium at room temperature. Each point is the mean 4-SEM of seven different experiments with intact cells and two with disrupted cells.
RESULTS
ANS associated to cells The fluorescence determined by the interaction of the ANS with the hepatocytes increased rapidly between 6 and 15 sec and then slowed until it became partly time independent at 20 min (Fig. 1). A linear regression was obtained with the first experimental points that fits the coefficient correlation (r = 0.95). A positive value of intercept in the experiment performed in the control medium was observed, which exhibited saturation mechanism in function of the ANS concentration in the medium (Fig. 2). The calculation of 1.0 x 10 -5 M of K d was performed by Scatchard analysis (Fig. 3).
Effect of cellular disruption N o significant modification of the intercept was
Fig. 3. Scatchard plot. Values of r/c were calculated from the data of Fig. 2. The dissociation constant was obtained from the slope of the line: kd: 1.0 x 10-5 M. r is expressed in RFU x 106cells. C is the total concentration of ANS (/*M) in the medium. Each point is the mean + SEM of five different experiments. observed with osmolalities up to 600 mOsm/kg H20. On the other hand at this osmolality value important reductions of the cellular diameter were observed (45%).
Effect of ionic strength Significant decrements of the intercept values were observed when reducing the ionic strength from 0.16 to 0.01 (Table 1).
Effect of pH The pH did not affect the viability of the suspension during the first 10min (Baur et al., 1975). The reduction of the pH enhanced the fluorescence which was more prominent at a lower pH and affected significantly all the components of the fluorescence curve in time. The increment of pH (pH 9) had the opposite effect (Fig. 4).
Effect of calcium The presence of Ca2C1 (0.3-1.6 mM) in the incubation medium determined an increment in fluorescence that affected the intercept value independent of the cation concentration (Table 2).
40
DISCUSSION
% 1¢
D
20
The organic anion ANS is virtually non-fluorescent in aqueous solution and becomes highly fluorescent upon combination with biological membranes and
t
_
tl.
+
0
I
20
I
40
I
60
£ANS1 ( p . M )
Fig. 2. Dependence of intercept value on ANS concentration in the medium. The data were obtained as referred to in the text, at different ANS concentrations in control medium at room temperature. Each point is the mean + SEM of five different experiments.
Table 1. Effect of ionic strength on the intercept value obtained as referred in the text. Each data is the mean + SEM (n) of the differences between control and paired test media. *P < 0.05 Ionic strength (M)
Intercept value (RFU x 106 cells)
0.16 (control) 0.11 0.08 0.01
9.6 -I- 1 (4)* I 1.3 +_ 3 (4)* 20.2 + 6 (4)*
Interaction of ANS with isolated hepatocytes
80-
60--
%
40
tl.
OC 20
I 2
I 6
I
IO
pH
Fig. 4. Effect of pH on the intercept value obtained as referred to in the text. Each point is the mean _+SEM of four different experiments. certain proteins (Stryer, 1968; Radda and Vanderkooi, 1972). Taking advantage of this property, we have employed ANS as a conspicuous organic anion in order to study the kinetics and mechanisms of interaction with the isolated hepatocyte surface. In this experimental model all cells are exposed to the same concentration of solute and repeating sampling is possible, allowing the observation of the initial process of interaction between the anion and the structural components of the cellular membranes (Schwenk, 1980). Hence compared with other models classically employed in these studies (liver slices, perfused liver, whole animal) the isolated hepatocytes offer evident advantages (Harris and Cornell, 1983). When correlating the ANS-hepatocytes with time, two phases have been described by some authors. The first one ascribed to binding and the second, a slower one, to translocation (Cheng and Levy, 1978; Sugiyama et al., 1983). With reference to our results it is worthwhile to point out that ANS being a foreign anion and considering that the data have been corrected for unspecific fluorescence, a zero value for the intercept on the relative fluorescence axis could be expected. Notwithstanding, a positive value was obtained. We suggest that this positive intercept evaluates the binding of ANS to cellular surface since: (i) it persists after cellular disruption when the subsequent increase of fluorescence is not detected; (ii) the same behaviour was observed when interaction between ANS and purified plasma membranes was studied (unpublished data); (iii) the observed intercept is only slightly affected when the medium osmolality is raised up to 600 mOsm/Kg H20 (Kinsella et al., 1979). We have Table 2. Effect of the presence of calcium in the incubationmediumon the interceptvalueobtained as referred in the text. Each datum is the mean_+ SEM (n) of the differences between control and paired test media. *P < 0.05 Calciumconcentration in the medium Intercept value (mM) (RFU x 106cells) 0.3 15.2+_4 (5)* 0.6 17.0+ 6 (5)* 1.6 12.1 + 4(5)*
not estimated the intercept at lower temperature since in these conditions a change in the fluorescence quantum yield of the fixed anion could be expected (Brand and Witholt, 1967). Ionic strength, pH, and divalent cations, all variables that influence cellular surface phenomena, altered the intercept values. Considering the existence of fixed negative charges in the hepatocytes surface (Pfaff et al., 1980) it can be expected, as already observed, that lowering the ionic strength could increase the influence of these charges and consequently prevent the binding of negative molecules (Baskin, 1972). On the contrary as has been proved (Rubalcava et al., 1969; McLaughlin et al., 1971) divalent cations could partially neutralize the superficial charges and increase the interaction with ANS. The observed effects of pH are similar to those detected on the interaction between BSF and hepatocytes (Schwenk et al., 1976) or on ANS and liposomes (Gibrat and Grignon, 1982). The pK of the dye amino group has been shown to be around 1.5 (Flanagan and Hesketh, 1973). Then the influence of a variation in pH can be explained by a change in the protonation of superficial sites that would modify their electric charge and consequently the interaction with the anion (Schwenk et al., 1976). The binding process appears to have, at least, a specific component since with the Scatchard plot a saturation value is reached (Feidman, 1972). The slower component of the interaction-time plot are probably related to a translocation process since they are not detected after cellular disruption or when purified plasma membranes are used. If these slower interactions were the results of different phenomena superimposed the analysis in our experimental conditions would be rather complex and would exceed our present possibilities. Acknowledgements--This work was supported by Consejo Nacional de Investigaciones Cientificas y Trcnicas (CONICET) Republica Argentina. The authors wish to thank Miss Marta B. Bravo Luna for the revising of the English manuscript. REFERENCES
Baker K. J. and Bradley S. E. (1966) Binding of sulfobromophthalein (BSP) sodium by plasma albumin. Its role in hepatic BSP extraction. J. clin. Invest. 45, 281-286. Baskin K. J. (1972) Surface charge and calcium binding in sarcoplasmic reticulum membranes. Bioenergetics 3, 249-297. Baur H., Kasperek S. and Pfaff E. (1975) Criteria of viability of isolated liver cells. Hoppe Seyler's Z. physiol. Chem. 356, 827-838. Brand L. and Witholt B. (1967) Fluorescence measurement. In Methods in Enzymology Vol. XI, Enzyme Structure (Edited by Hirs C. H. W.) Chap 87, pp. 776-855. Academic Press, New York. Cheng S. and Levy D. (1978) The interaction of the anionic fluorescenceprobe, 1-anilinonaphthalene8-sulfonate with hepatocytes and hepatoma tissue culture cells. Biochim. biophys. Acta 511, 419-429. Feldman H. A. (1972) Mathematical theory of complex ligand-binding systems at equilibrium. Some methods for parameter fitting. Analyt. Biochem. 48, 317-338. Flanagan M. T. and Hesketh T. R. (1973) Electrostatic interactions in the binding of fluorescent probes to lipid membranes. Biochim. biophys. Acta 298, 535-545.
10
RUT M. AGOERO et al.
Fortes G. and Hoffman J. (1971) Interaction of the fluorescent anion l-anilino 8-naphthalene sulfonate with membrane charges in human red cell ghosts. J. Membr. Biol. 5, 154-168. Gibrat R. and Grignon C. (1982) Effect of pH on the surface charge density of plant membranes. Biochim. biophys. Acta 692, 462-468. Goresky C. (1965) The hepatic uptake and excretion of sulfobromophthalein and bilirubin. Can. Med. Ass. J. 92, 851-857. Guibert E. E., Morisoli L. S., Monti J. A. and Rodriguez Garay E. (1983) Effect of spironolactone on pnitropbenol glucuronidation in isolated rat hepatocytes. Experientia 39, 527-528. Harris R. A. and Cornell W. (1983) Isolation, Characterization and Use of Hepatocytes. Elsevier, Amsterdam, pp. 205-277. Kinsella J. L., Peter D. H., Pessah I. and Ross Ch. R. (1979) Transport of organic ions in renal cortical luminal and antiluminal membrane vesicles. J. Pharmac. exp. Ther. 209, 443-450. Lunazzi G., Tiribelli C., Gazzin B. and Sottocasa G. (1982) Further studies on bilitranslocase, a plasma membrane protein involved in hepatic organic anion uptake. Biochim. biophys. Acta 685, 117-122. McLaughlin S. G. A., Szabo G. and Eisenman G. (1971) Divalent ions and the surface potential of charged phospholipid membranes. J. gen. Physiol. 58, 667687. Pfaff E., Schuler B., Krell H. and H6ke H. (1980) Viability control and special properties of isolated rat hepatocytes. Archs Toxicol. 44, 3-21. Pfaff E., Schwenk M., Burr R. and Returner H. (1974) Molecular aspects of the interaction of bromosulfophthalein with high-affinity binding sites of bovine serum albumin. Molec. Pharmac. l l , 144-152.
Radda G. K. and Vanderkooi J. (1972) Can fluorescent probes tell us anything about membranes? Biochim. biophys. Acta 265, 509-549. Reichen J., Blitzer B. and Berk P. (1981) Binding of unconjugated and conjugated sulfobromophthalein to rat liver plasma membrane fractions in vitro. Biochim. biophys. Acta 640, 298-312. Rubalcava B., Martinez de Mufioz D. and Gitler C. (1969) Interaction of fluorescent probe with membranes. I. Effect of ions on erythrocytes membranes. Biochemistry 8, 2742-2747. Schwarz L. R., Burr R., Schwenk M., Pfaff E. and Greim H. (1975) Uptake of taurocholic acid into isolated ratliver cells. Eur. J. Biochem. 55, 617-623. Scbwenk M., Burr R. and Pfaff E. (1976) Uptake of sulfobromophthalein by isolated liver cells. Eur. Bioehem. 64, 189-197. Sehwenk M. (1980) Transport systems of isolated hepatocytes. Archs. Toxieol. 44, 113-126. Snedecor G. N. (1956) Statistical Methods. The Iowa State University Press, USA. Stryer L. (1968) Fluorescence spectroscopy of proteins. Science 162, 526-532. Sugiyama Y., Kimura S., Hueilin J., Izukura M., Awazu S. and Hanan O. (1983) Effects of organic anions on the uptake of 1-anilino 8-naphthalene sulfonate by isolated liver cells. J. Am. pharm. Assn. 72, 871-875. Supplement IV of the International Survey of the Supply Quality and use of laboratory animals. November, 1964, on the advice of the International Committee on Laboratory Animals (N.R.E., Lab., Carshalton, Surrey, England). Tiribelli C., Lunazzi G., Luciani M. et al. (1978) Isolation of a sulfobromophthalein binding protein from hepatocytes plasma membrane. Biochim. biophys. Acta 532, 105-112.
0305-0491/87 $3.00 + 0.00 Pergamon Journals Ltd
INTERACTION OF THE ORGANIC ANION 1-ANILINE 8-NAPHTHALENE SULFONATE (ANS) WITH ISOLATED RAT HEPATOCYTES RUT M. AGOERO, GUILLERMO P I c r , EDGARDO GUIBERT a n d JUAN L. CORCHS* Instituto de Fisiologia Experimental, Consejo Nacional de Investigaciones Cientificas y Tecnicas, Universidad Nacional de Rosario, Rosario, Argentina (Received 27 January 1986)
Abstract--1. The interaction of ANS with rat hepatocytes in time was studied by fluorescence spectroscopy. 2. The intercept of the first linear portion of the time curve of interaction showed a positive value over all the ANS concentration range employed. 3. This value was maintained after cellular disruption by homogenization. 4. It was affected by ionic strength, pH, and divalent cation in the incubation medium, all conditions affecting the cellular surface. 5. These data suggest that this phenomenon might be a binding of the compound to the hepatocytes surface. 6. Due to the time constant and its disappearance after cellular disruption the other slower component of the curve seems to correspond to a process of translocation across the membrane.
INTRODUCTION Circulating organic a n i o n s b o u n d to serum a l b u m i n (Pfaff et al., 1974; Baker a n d Bradley, 1966) are excreted by the liver t h r o u g h m e c h a n i s m s involving binding, uptake, intracellular transport, conjugation a n d biliary excretion (Goresky, 1965). The first process is a n i m p o r t a n t one since it m i g h t represent a limiting step o n the sequence. Isolated rat hepatocytes have been s h o w n to possess specific sites for organic a n i o n s such as brom o s u l f o p h t h a l e i n (Tiribelli et al., 1978; Reichen et al., 1981; Lunazzi et al., 1982) a n d taurocholic acid (Schwarz et al., 1975). In the present p a p e r we analyze the interaction of a n organic a n i o n with isolated hepatocytes, which h a s also been employed as a n effective p r o b e in the study of a n i o n t r a n s p o r t in erythrocytes (Fortes a n d H o f f m a n , 1971) a n d Ehrlich ascites cells. A n early phase is described t h a t m i g h t be considered as binding since it is influenced by factors acting u p o n surface p h e n o m e n a , whereas the late phase of interaction could c o r r e s p o n d to a cellular u p t a k e o f the compound. MATERIALS AND METHODS Cell preparation IIM rats (NRE Lab., Carshalton, Surrey, Eng. 1964) weighing 200-300 g were used. Liver cells were obtained according to the procedure previously described (Guibert et al., 1983) with some modifications since in the second step *Correspondence to be addressed--Dr Juan L. Corchs, Chtedra de Fisiologia, Facultad de Ciencias Mrdicas, Santa Fe 3100a, 2000 Rosario, Argentina.
the colagenase was recirculated. Cellular viability was tested by the tryptan blue exclusion test. All experiments were carried out on cell suspensions with viabilities of 85-95% and were done within 2 3 hr after cell isolation. Fluorescence measurements Fluorescence was measured by spectrofluorometry by means of a 93200 Spectral Fluorescence Accessory attached to a Beckman DU Spectrophotometer, with a GEF4T5BL mercury phosphor excitation lamp and a 7-60 Turner primary filter which provides a maximal transmittance at 360 nm. Light scattering effects were corrected by the use of an appropriate blank. Interaction studies Aliquots of ANS (final concn 3-60 #M) were added to 3 ml of cell suspension (0.30-0.40 x 106 cells/ml) in control or test medium, quickly mixed by cuvette inversion and the fluorescence intensity was recorded at 485 nm during 4 sec to 20 min. The cuvette was inverted previously to each determination in order to avoid cellular sedimentation. The experiments have been performed at least twice for each cell preparation. Fluorescence values were expressed as RFU (relative fluorescence units)x 106cell. These values were corrected for light scattering and fluorescence emitted by the ANS bound to proteins of supernatant. Both were constant during the time of the experiment. Media composition Control medium consisted of 150 mM NaCI; 5 mM KCI; 5 mM glucose; 10 mM HEPES (pH 7.5). High osmolality (400-600-900 mOsm/Kg H20 ) was obtained by addition of sucrose to the control medium. The pH (4 ~ 9) was adjusted with 10mM HCI or NaOH. Low ionic strength was obtained by substituting isosmotically NaCI by sucrose. The results were presented as difference between control and paired test media and expressed as mean values (__+SEM). Statistical analysis of the difference thus obtained was done according to standard methods (Snedecor, 1956).
RUT M. AGf3ERO et al.
8
I00
A - -
O t. O
== 03 Z
l
x -1
50 .la
O3 Z
IE b
15
I
0
I
320
I
45
ANS bound (UFRxlO6ceL)(r)
I
640
30
960
T i m e (sec)
Fig. 1. Kinetics of interaction between ANS (12#M) and intact (O) or disrupted (©) isolated rat hepatocytes (0.30-0.40 x 106cells/ml) suspended in control medium at room temperature. Each point is the mean 4-SEM of seven different experiments with intact cells and two with disrupted cells.
RESULTS
ANS associated to cells The fluorescence determined by the interaction of the ANS with the hepatocytes increased rapidly between 6 and 15 sec and then slowed until it became partly time independent at 20 min (Fig. 1). A linear regression was obtained with the first experimental points that fits the coefficient correlation (r = 0.95). A positive value of intercept in the experiment performed in the control medium was observed, which exhibited saturation mechanism in function of the ANS concentration in the medium (Fig. 2). The calculation of 1.0 x 10 -5 M of K d was performed by Scatchard analysis (Fig. 3).
Effect of cellular disruption N o significant modification of the intercept was
Fig. 3. Scatchard plot. Values of r/c were calculated from the data of Fig. 2. The dissociation constant was obtained from the slope of the line: kd: 1.0 x 10-5 M. r is expressed in RFU x 106cells. C is the total concentration of ANS (/*M) in the medium. Each point is the mean + SEM of five different experiments. observed with osmolalities up to 600 mOsm/kg H20. On the other hand at this osmolality value important reductions of the cellular diameter were observed (45%).
Effect of ionic strength Significant decrements of the intercept values were observed when reducing the ionic strength from 0.16 to 0.01 (Table 1).
Effect of pH The pH did not affect the viability of the suspension during the first 10min (Baur et al., 1975). The reduction of the pH enhanced the fluorescence which was more prominent at a lower pH and affected significantly all the components of the fluorescence curve in time. The increment of pH (pH 9) had the opposite effect (Fig. 4).
Effect of calcium The presence of Ca2C1 (0.3-1.6 mM) in the incubation medium determined an increment in fluorescence that affected the intercept value independent of the cation concentration (Table 2).
40
DISCUSSION
% 1¢
D
20
The organic anion ANS is virtually non-fluorescent in aqueous solution and becomes highly fluorescent upon combination with biological membranes and
t
_
tl.
+
0
I
20
I
40
I
60
£ANS1 ( p . M )
Fig. 2. Dependence of intercept value on ANS concentration in the medium. The data were obtained as referred to in the text, at different ANS concentrations in control medium at room temperature. Each point is the mean + SEM of five different experiments.
Table 1. Effect of ionic strength on the intercept value obtained as referred in the text. Each data is the mean + SEM (n) of the differences between control and paired test media. *P < 0.05 Ionic strength (M)
Intercept value (RFU x 106 cells)
0.16 (control) 0.11 0.08 0.01
9.6 -I- 1 (4)* I 1.3 +_ 3 (4)* 20.2 + 6 (4)*
Interaction of ANS with isolated hepatocytes
80-
60--
%
40
tl.
OC 20
I 2
I 6
I
IO
pH
Fig. 4. Effect of pH on the intercept value obtained as referred to in the text. Each point is the mean _+SEM of four different experiments. certain proteins (Stryer, 1968; Radda and Vanderkooi, 1972). Taking advantage of this property, we have employed ANS as a conspicuous organic anion in order to study the kinetics and mechanisms of interaction with the isolated hepatocyte surface. In this experimental model all cells are exposed to the same concentration of solute and repeating sampling is possible, allowing the observation of the initial process of interaction between the anion and the structural components of the cellular membranes (Schwenk, 1980). Hence compared with other models classically employed in these studies (liver slices, perfused liver, whole animal) the isolated hepatocytes offer evident advantages (Harris and Cornell, 1983). When correlating the ANS-hepatocytes with time, two phases have been described by some authors. The first one ascribed to binding and the second, a slower one, to translocation (Cheng and Levy, 1978; Sugiyama et al., 1983). With reference to our results it is worthwhile to point out that ANS being a foreign anion and considering that the data have been corrected for unspecific fluorescence, a zero value for the intercept on the relative fluorescence axis could be expected. Notwithstanding, a positive value was obtained. We suggest that this positive intercept evaluates the binding of ANS to cellular surface since: (i) it persists after cellular disruption when the subsequent increase of fluorescence is not detected; (ii) the same behaviour was observed when interaction between ANS and purified plasma membranes was studied (unpublished data); (iii) the observed intercept is only slightly affected when the medium osmolality is raised up to 600 mOsm/Kg H20 (Kinsella et al., 1979). We have Table 2. Effect of the presence of calcium in the incubationmediumon the interceptvalueobtained as referred in the text. Each datum is the mean_+ SEM (n) of the differences between control and paired test media. *P < 0.05 Calciumconcentration in the medium Intercept value (mM) (RFU x 106cells) 0.3 15.2+_4 (5)* 0.6 17.0+ 6 (5)* 1.6 12.1 + 4(5)*
not estimated the intercept at lower temperature since in these conditions a change in the fluorescence quantum yield of the fixed anion could be expected (Brand and Witholt, 1967). Ionic strength, pH, and divalent cations, all variables that influence cellular surface phenomena, altered the intercept values. Considering the existence of fixed negative charges in the hepatocytes surface (Pfaff et al., 1980) it can be expected, as already observed, that lowering the ionic strength could increase the influence of these charges and consequently prevent the binding of negative molecules (Baskin, 1972). On the contrary as has been proved (Rubalcava et al., 1969; McLaughlin et al., 1971) divalent cations could partially neutralize the superficial charges and increase the interaction with ANS. The observed effects of pH are similar to those detected on the interaction between BSF and hepatocytes (Schwenk et al., 1976) or on ANS and liposomes (Gibrat and Grignon, 1982). The pK of the dye amino group has been shown to be around 1.5 (Flanagan and Hesketh, 1973). Then the influence of a variation in pH can be explained by a change in the protonation of superficial sites that would modify their electric charge and consequently the interaction with the anion (Schwenk et al., 1976). The binding process appears to have, at least, a specific component since with the Scatchard plot a saturation value is reached (Feidman, 1972). The slower component of the interaction-time plot are probably related to a translocation process since they are not detected after cellular disruption or when purified plasma membranes are used. If these slower interactions were the results of different phenomena superimposed the analysis in our experimental conditions would be rather complex and would exceed our present possibilities. Acknowledgements--This work was supported by Consejo Nacional de Investigaciones Cientificas y Trcnicas (CONICET) Republica Argentina. The authors wish to thank Miss Marta B. Bravo Luna for the revising of the English manuscript. REFERENCES
Baker K. J. and Bradley S. E. (1966) Binding of sulfobromophthalein (BSP) sodium by plasma albumin. Its role in hepatic BSP extraction. J. clin. Invest. 45, 281-286. Baskin K. J. (1972) Surface charge and calcium binding in sarcoplasmic reticulum membranes. Bioenergetics 3, 249-297. Baur H., Kasperek S. and Pfaff E. (1975) Criteria of viability of isolated liver cells. Hoppe Seyler's Z. physiol. Chem. 356, 827-838. Brand L. and Witholt B. (1967) Fluorescence measurement. In Methods in Enzymology Vol. XI, Enzyme Structure (Edited by Hirs C. H. W.) Chap 87, pp. 776-855. Academic Press, New York. Cheng S. and Levy D. (1978) The interaction of the anionic fluorescenceprobe, 1-anilinonaphthalene8-sulfonate with hepatocytes and hepatoma tissue culture cells. Biochim. biophys. Acta 511, 419-429. Feldman H. A. (1972) Mathematical theory of complex ligand-binding systems at equilibrium. Some methods for parameter fitting. Analyt. Biochem. 48, 317-338. Flanagan M. T. and Hesketh T. R. (1973) Electrostatic interactions in the binding of fluorescent probes to lipid membranes. Biochim. biophys. Acta 298, 535-545.
10
RUT M. AGOERO et al.
Fortes G. and Hoffman J. (1971) Interaction of the fluorescent anion l-anilino 8-naphthalene sulfonate with membrane charges in human red cell ghosts. J. Membr. Biol. 5, 154-168. Gibrat R. and Grignon C. (1982) Effect of pH on the surface charge density of plant membranes. Biochim. biophys. Acta 692, 462-468. Goresky C. (1965) The hepatic uptake and excretion of sulfobromophthalein and bilirubin. Can. Med. Ass. J. 92, 851-857. Guibert E. E., Morisoli L. S., Monti J. A. and Rodriguez Garay E. (1983) Effect of spironolactone on pnitropbenol glucuronidation in isolated rat hepatocytes. Experientia 39, 527-528. Harris R. A. and Cornell W. (1983) Isolation, Characterization and Use of Hepatocytes. Elsevier, Amsterdam, pp. 205-277. Kinsella J. L., Peter D. H., Pessah I. and Ross Ch. R. (1979) Transport of organic ions in renal cortical luminal and antiluminal membrane vesicles. J. Pharmac. exp. Ther. 209, 443-450. Lunazzi G., Tiribelli C., Gazzin B. and Sottocasa G. (1982) Further studies on bilitranslocase, a plasma membrane protein involved in hepatic organic anion uptake. Biochim. biophys. Acta 685, 117-122. McLaughlin S. G. A., Szabo G. and Eisenman G. (1971) Divalent ions and the surface potential of charged phospholipid membranes. J. gen. Physiol. 58, 667687. Pfaff E., Schuler B., Krell H. and H6ke H. (1980) Viability control and special properties of isolated rat hepatocytes. Archs Toxicol. 44, 3-21. Pfaff E., Schwenk M., Burr R. and Returner H. (1974) Molecular aspects of the interaction of bromosulfophthalein with high-affinity binding sites of bovine serum albumin. Molec. Pharmac. l l , 144-152.
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