Naunyn-Schmiedeberg's
Archivesof Pharmacology
Naunyn-Schmiedeberg's Arch. Pharmacol. 301, 145-147 (1977)
9 by Springer-Verlag 1977
Short Communication
The Role of Bile Acids in Phalloidin Poisoning* M. FRIMMER, E. PETZINGER, U. RUFEGER, and L. B. VEIL Institut ftir Pharmakologie und Toxikologie im Fachbereich Veterin/irmedizin und Tierzucht der Justus-Liebig-Universitfit Giessen, Frankfurter Strasse 107, D-6300 Giessen, Federal Republic of Germany
Summary. Glycocholate and other bile acids inhibit the response of isolated hepatocytes to phalloidin in a concentration dependent manner. It is suggested that the inhibition is due to a block of phalloidin uptake. This interaction might explain the high specificity of phalloidin for liver tissue.
Key words." Phalloidin poisoning - Isolated hepato-
Deoxycholic acid (sodium salt) and taurocholic acid (sodium salt) were purchased from Serva, Heidelberg; chenodeoxycholic acid (free acid) and the sodium salt of glycochenodeoxycholic acid, dehydrocholic acid, cholic acid and glycocholic acid were purchased from Sigma, St. Louis. The bile salts were dissolved in buffered tyrode in concentrations from 10 -s to 3 x 10 -4 M. Hepatocytes were incubated in presence of the bile salt 1 min prior to addition of phalloidin. After an incubation period of 20, 40 and 60 min with phalloidin the ratio of affected hepatocytes was counted.
cytes - Bile acids.
The high specificity of phalloidin for hepatocytes is an unexplained phenomenon. The interaction of phalloidin with microfilamentous structures on the inside of the membrane (Govindan et al., 1972, 1973; Agostini et al., 1975) can be regarded as a site of action, but not as a cause of selective toxicity on liver tissue. Many other types of cells contain microfilaments; but they are insensitive to phalloidin. Therefore the specificity of the cyclopeptide is caused rather by the mechanisms of recognition and uptake than by differences in the mode of interaction with intracellular components. Several findings suggest that phalloidin cannot be taken up by a simple diffusion 1. It is more probable that phalloidin uses the pathway of a physiological substrate. Systematical screening showed that bile acids inhibit the response of isolated liver cells to phalloidin in a dose dependent manner. MATERIAL AND METHODS All experiments were carried out on isolated hepatocytes from rat livers. The preparation of cells, the conditions of incubation and the quantitative measurment of the phalloidin response was described recently (Petzinger and Seeger, 1976; Frimmer et al., 1977). Send offprint requests to M. Frimmer at the above address * This paper was supported by the Deutsche Forschungsgemeinschaft 1 This problem was recently discussed (Frimmer et al., 1977)
RESULTS Glycocholic acid, added 1 min before phalloidin, markedly inhibited the development of protrusions due to phalloidin (Fig. 1). The inhibition depends as well on the concentration of glycocholic acid as on the concentration of phalloidin. Table i presents some data for the concentrations of several bile acids needed for 50 % inhibition of the phalloidin response, when the incubation period was limited to 20 rain. Obviously quantitative differences between the various bile acids are not remarkable. Apparently a ratio of 2 - 4 mole bile acids per mole phalloidin was able to block 50 ~ of the response to phalloidin when the incubation period was 20 min. After longer periods of incubation the inhibition by bile acids decreases gradually. E.g. 8 - 10 .5 M glycocholate reduced the response to phalloidin to 8 % of the controls, when the incubation period was 20 min; in contrast after an incubation of 40 and 60 min the rate of unaffected cells was 59 ~ and 93 % respectively. These findings suggest that bile acids inhibit the uptake of the toxin. The concentrations of bile acids used in the above experiments were not cytotoxic, as measured by exclusion of trypan blue in controls. Quantitative evaluation of the experiments presented in Figure I suggest that the interaction of glycocholic acid with phalloidin poisoned hepatocytes may
146
Naunyn-Schmiedeberg's Arch. Pharmacol. 301 (1977)
% Inhibitionof phQttoidin response 100,90807060-
{ /
5040 30 20 10 0
lb
2b
3'0
4'0
5~
6'0 70 80 9'0 1~)0 ,u-M C g[ycocho[[c ,acid
Fig.]. Inhibition of the response of hepatocytes to 3 concentrations of phalloidin by glycocholic acid. Addition of various concentrations of glycocholic acid 1 min prior to phalloidin. (IB) 5 p.g phalloidin/ml (n = 5); (0) 10 gg phalloidin/mI (n = 5); (A) 20 pg phalloidin/ml (n = 4). Counting of affected cells 20 min after the addition of phalloidin. Controls ( = 100%) values in absence of glycocholate. After an incubation period of 20 min 5 pg phalloidin/ ml induced protrusions in 90 _+ 5% of total cells (10 gg/ml 92 + 5 %; 20 gg/ml 95 4- 3 %). These experimental values were corrected for 100%
Table 1. Concentration of bile acids needed for 50 % inhibition of the phalloidin response of isolated hepatocytes (5 pg phall./ml) Bite acids
~tM
n
Cholic acid Glycocholic acid Taurocholic acid Chenodeoxychotic acid Glycochenodeoxycholic acid Deoxychotic acid Dehydrocholic acid
26.0 _+ 5.7 23.5 4- 4.0 29.0, 35.0, 32.0 13.0, 11.0, 11.0 17.0, 15.0, 16.0 9.8 4- 1.6 13.0, 32.0, 36.0
4 8 3 3 3 4 3
generally a competitive one. Detailed mathematical studies are in progress and will be published in a separate paper by L. B. Veil. DISCUSSION Many nonphysiological reagents and drugs inhibit the response to phalloidin in liver cells when added simultaneously with the toxin (Frimmer and Kroker, 1975; Petzinger et al., 1975; Frimmer and Petzinger, 1975; Frimmer et al., 1975). None of those agents has any biological function in liver cells. In contrast bile acids represent physio-
logical substrates, highly typical for the normal function of the liver. The above findings demonstrate that physiological concentrations of bile acids, insufficient to induce unspecific cytolysis, inhibit the response of isolated cells to phalloidin. Probably this inhibition is due to a block of phalloidin uptake: Earlier studies with 3H-desmethyl-phalloin showed, that dehydrocholate inhibits the uptake of the toxin in the isolated perfused rat liver (Lutz et al., 1971). At present two explanations can be discussed: 1. The response to phalloidin might be inhibited in a nonspecific manner e.g. by detergent like interaction of bile acids with plasma membranes. 2. Phalloidin might pass the plasma membrane using the way of bile acids. Mathematical analysis of the diagrams of Figure i showed a nearly competitive character 2. The best approach convenient with the latter explanation is a competition of the uptake of phalloidin with the transport of bile acids. With respect to other findings one can suspect that the devebopment of protrusions occurs at a threshold concentration of phalloidin. In whole rat livers phalloidin poisoning becomes lethal when 20 gg toxin per gram liver are fixed (Wieland et al., 1972). In experiments on isolated hepatocytes the degree of inhibition of the phalloidin response by bile acids depends on the incubation period. The inhibition decreases after elongation of the incubation. This behavior is in agreement with the 2nd, but not with the first explanation. Further arguments for the 2nd working hypothesis base on earlier observations of O. Wieland (1965): In perfused livers phalloidin ceases the bile flow immediately after the addition of the toxin. Possibly the outward transport of bile acids might also involved in phatloidin poisoning. Furthermore many kinds of liver injury, probably followed by disturbances of bile acid transport, decrease the sensitivity of damaged livers to phalloidin (Floersheim, 1966, 1976). Recently an irreversible inhibition of the phalloidin response by DIDS 3 and H2-DIDS 4 was detected (Petzinger et al., unpublished). This protein reagent, binding preferentially to e-amino groups, blocks the anionic transport in red cells (Lepke et al., 1976). The anionic sulfobromophthalein (BSP) inhibits the uptake of taurocholate in isolated hepatocytes (Schwarz et al., 1975) and blocks binding of cholic acid to rat liver plasma membranes (Anwer et al., 2 Detailed studies will be published in a separate paper by L. B. Veil 3 DIDS = 4,4'-diisothiocyano stilbene-2,2'-disulfonic acid 4 H2-DIDS = 4,4'-diisothiocyano-],2-diphenyl ethane-2,2'-disulfonic acid
M. Frimmer et al. : Role of Bile Acids in Phalloidin Poisoning
1977). BSP is also a potent inhibitor of phalloidin response (Frimmer and Petzinger, unpublished). Detailed studies concerning the influence of phalloidin poisoning on the uptake of bile acids by liver cells are in progress. REFERENCES Agostini, B., Govindan, V. M., Hofmann, W , Wieland, Th. : Phalloidin-induced proliferation of actin filaments within rat hepatocytes. Z. Naturforsch. 30c, 793-795 (1975) Anwer, S. M., Kroker, R., Hegner, D., Pelter, A. : Cholic acid binding to isolated rat liver plasma membranes. Hoppe-Seyler's Z. Physiol. Chem. 358, 543-553 (1977) Frimmer, M., Kroker, R.: Phalloidin-Antagonisten, 1. Mitt. Arzneim.-Forsch. (Drug Res.) 25, 394-396 (1975) Frimmer, M., Petzinger, E. : PhalloidinhAntagonisten, 3. Mitt. Arzneim.-Forsch. (Drug Res.) 25, ~423-1426 (1975) Frimmer, M., Petzinger, E., Homann, J. : Phalloidin-Antagonisten, 4. Mitt. Arzneim.-Forsch. (Drug Res.) 25, 1881-1884 (t975) Frimmer, M., Petzinger, E., Rufeger, U., Veil, L. B.: Trypsin protection of hepatocytes against phalloidin. Naunyn-Schmiedeberg's Arch. Pharmacol. 300, 163-171 (1977) Floersheim, G. L.: Schutzwirkung hepatotoxischer Stoffe gegen letale Dosen eines Toxins aus Amanita phalloides (Phalloidin). Biochem. Pharmacol. 15, 1589-1593 (1966) Floersheim, G. L. : Antagonistic effects against single lethal doses of amanita phalloides. Naunyn-Schmiedeberg's Arch. Pharmacol. 293, 171 - 174 (1976) Govindan, V. M., Faulstich, H., Wieland, Th., Agostini, B., Hasselbach, W. : In-vitro effect of phalloidin an a plasma membrane
147 preparation from rat liver. Naturwissenschaften 59, 521- 522 (1972) Govindan, V. M., Rohr, G., Wieland, Th., Agostini, B.: Binding of phallotoxin to protein filaments of plasma membrane of liver cell. Hoppe-Seyler's Z. Physiol. Chem. 354, 1159-1161 (1973) Lepke, S., Fasold, H., Pring, M., Passow, H. : A study of the relationship between inhibition of anion exchange and binding to the red blood cell membrane of 4,4'-diisothiocyano stilbene-2,2'disulfonic acid (DIDS) and its dihydro derivative (H2-DIDS). J. Membr. Biol. 29, 147-177 (1976) Lutz, F., Hermann, S., Frimmer, M. : The influence of sodium dehydrocholate on the uptake of 3H-desmethylphalloin by the perfused rat liver. Naunyn-Schmiedeberg's Arch. PharmacoI. 270, 310--312 (1971) Petzinger, E., Homann, J., Frimmer, M. : Phalloidin-Antagonisten. 2. Mitt. Arzneim.-Forsch. (Drug Res.) 25, 571- 576 (1975) Petzinger, E., Seeger, R. : Scanning electron microscopic studies on the cytolytic effect of phallolysin on isolated rat hepatocytes and AS-30 D hepatoma cells. Naunyn-Schmiedeberg's Arch. Pharmacol. 295, 211-213 (1976) Schwarz, L. R., Buss, R., Schwenk, M., Pfaff, E., Greim, H. : Uptake of taurocholic acid into isolated rat-liver cells. Eur. J. Biochem. 55, 617-623 (1975) Wieland, O. : Changes in liver metabolism induced by the poisons of amanita phalloides. Clin. Chem. 2, 323-338 (1965) Wieland, Th., Faulstich, H., Jahn, W., Govindan, M. V., Puchinger, H., Kopitar, Z., Schmaus, H., Schmitz, A. : Zur Wirkungsweise des Antamanids. Hoppe-Seyler's Z. Physiol. Chem. 353, 13371345 (1972)
Received September 19~Accepted October 3, 1977
Archivesof Pharmacology
Naunyn-Schmiedeberg's Arch. Pharmacol. 301, 145-147 (1977)
9 by Springer-Verlag 1977
Short Communication
The Role of Bile Acids in Phalloidin Poisoning* M. FRIMMER, E. PETZINGER, U. RUFEGER, and L. B. VEIL Institut ftir Pharmakologie und Toxikologie im Fachbereich Veterin/irmedizin und Tierzucht der Justus-Liebig-Universitfit Giessen, Frankfurter Strasse 107, D-6300 Giessen, Federal Republic of Germany
Summary. Glycocholate and other bile acids inhibit the response of isolated hepatocytes to phalloidin in a concentration dependent manner. It is suggested that the inhibition is due to a block of phalloidin uptake. This interaction might explain the high specificity of phalloidin for liver tissue.
Key words." Phalloidin poisoning - Isolated hepato-
Deoxycholic acid (sodium salt) and taurocholic acid (sodium salt) were purchased from Serva, Heidelberg; chenodeoxycholic acid (free acid) and the sodium salt of glycochenodeoxycholic acid, dehydrocholic acid, cholic acid and glycocholic acid were purchased from Sigma, St. Louis. The bile salts were dissolved in buffered tyrode in concentrations from 10 -s to 3 x 10 -4 M. Hepatocytes were incubated in presence of the bile salt 1 min prior to addition of phalloidin. After an incubation period of 20, 40 and 60 min with phalloidin the ratio of affected hepatocytes was counted.
cytes - Bile acids.
The high specificity of phalloidin for hepatocytes is an unexplained phenomenon. The interaction of phalloidin with microfilamentous structures on the inside of the membrane (Govindan et al., 1972, 1973; Agostini et al., 1975) can be regarded as a site of action, but not as a cause of selective toxicity on liver tissue. Many other types of cells contain microfilaments; but they are insensitive to phalloidin. Therefore the specificity of the cyclopeptide is caused rather by the mechanisms of recognition and uptake than by differences in the mode of interaction with intracellular components. Several findings suggest that phalloidin cannot be taken up by a simple diffusion 1. It is more probable that phalloidin uses the pathway of a physiological substrate. Systematical screening showed that bile acids inhibit the response of isolated liver cells to phalloidin in a dose dependent manner. MATERIAL AND METHODS All experiments were carried out on isolated hepatocytes from rat livers. The preparation of cells, the conditions of incubation and the quantitative measurment of the phalloidin response was described recently (Petzinger and Seeger, 1976; Frimmer et al., 1977). Send offprint requests to M. Frimmer at the above address * This paper was supported by the Deutsche Forschungsgemeinschaft 1 This problem was recently discussed (Frimmer et al., 1977)
RESULTS Glycocholic acid, added 1 min before phalloidin, markedly inhibited the development of protrusions due to phalloidin (Fig. 1). The inhibition depends as well on the concentration of glycocholic acid as on the concentration of phalloidin. Table i presents some data for the concentrations of several bile acids needed for 50 % inhibition of the phalloidin response, when the incubation period was limited to 20 rain. Obviously quantitative differences between the various bile acids are not remarkable. Apparently a ratio of 2 - 4 mole bile acids per mole phalloidin was able to block 50 ~ of the response to phalloidin when the incubation period was 20 min. After longer periods of incubation the inhibition by bile acids decreases gradually. E.g. 8 - 10 .5 M glycocholate reduced the response to phalloidin to 8 % of the controls, when the incubation period was 20 min; in contrast after an incubation of 40 and 60 min the rate of unaffected cells was 59 ~ and 93 % respectively. These findings suggest that bile acids inhibit the uptake of the toxin. The concentrations of bile acids used in the above experiments were not cytotoxic, as measured by exclusion of trypan blue in controls. Quantitative evaluation of the experiments presented in Figure I suggest that the interaction of glycocholic acid with phalloidin poisoned hepatocytes may
146
Naunyn-Schmiedeberg's Arch. Pharmacol. 301 (1977)
% Inhibitionof phQttoidin response 100,90807060-
{ /
5040 30 20 10 0
lb
2b
3'0
4'0
5~
6'0 70 80 9'0 1~)0 ,u-M C g[ycocho[[c ,acid
Fig.]. Inhibition of the response of hepatocytes to 3 concentrations of phalloidin by glycocholic acid. Addition of various concentrations of glycocholic acid 1 min prior to phalloidin. (IB) 5 p.g phalloidin/ml (n = 5); (0) 10 gg phalloidin/mI (n = 5); (A) 20 pg phalloidin/ml (n = 4). Counting of affected cells 20 min after the addition of phalloidin. Controls ( = 100%) values in absence of glycocholate. After an incubation period of 20 min 5 pg phalloidin/ ml induced protrusions in 90 _+ 5% of total cells (10 gg/ml 92 + 5 %; 20 gg/ml 95 4- 3 %). These experimental values were corrected for 100%
Table 1. Concentration of bile acids needed for 50 % inhibition of the phalloidin response of isolated hepatocytes (5 pg phall./ml) Bite acids
~tM
n
Cholic acid Glycocholic acid Taurocholic acid Chenodeoxychotic acid Glycochenodeoxycholic acid Deoxychotic acid Dehydrocholic acid
26.0 _+ 5.7 23.5 4- 4.0 29.0, 35.0, 32.0 13.0, 11.0, 11.0 17.0, 15.0, 16.0 9.8 4- 1.6 13.0, 32.0, 36.0
4 8 3 3 3 4 3
generally a competitive one. Detailed mathematical studies are in progress and will be published in a separate paper by L. B. Veil. DISCUSSION Many nonphysiological reagents and drugs inhibit the response to phalloidin in liver cells when added simultaneously with the toxin (Frimmer and Kroker, 1975; Petzinger et al., 1975; Frimmer and Petzinger, 1975; Frimmer et al., 1975). None of those agents has any biological function in liver cells. In contrast bile acids represent physio-
logical substrates, highly typical for the normal function of the liver. The above findings demonstrate that physiological concentrations of bile acids, insufficient to induce unspecific cytolysis, inhibit the response of isolated cells to phalloidin. Probably this inhibition is due to a block of phalloidin uptake: Earlier studies with 3H-desmethyl-phalloin showed, that dehydrocholate inhibits the uptake of the toxin in the isolated perfused rat liver (Lutz et al., 1971). At present two explanations can be discussed: 1. The response to phalloidin might be inhibited in a nonspecific manner e.g. by detergent like interaction of bile acids with plasma membranes. 2. Phalloidin might pass the plasma membrane using the way of bile acids. Mathematical analysis of the diagrams of Figure i showed a nearly competitive character 2. The best approach convenient with the latter explanation is a competition of the uptake of phalloidin with the transport of bile acids. With respect to other findings one can suspect that the devebopment of protrusions occurs at a threshold concentration of phalloidin. In whole rat livers phalloidin poisoning becomes lethal when 20 gg toxin per gram liver are fixed (Wieland et al., 1972). In experiments on isolated hepatocytes the degree of inhibition of the phalloidin response by bile acids depends on the incubation period. The inhibition decreases after elongation of the incubation. This behavior is in agreement with the 2nd, but not with the first explanation. Further arguments for the 2nd working hypothesis base on earlier observations of O. Wieland (1965): In perfused livers phalloidin ceases the bile flow immediately after the addition of the toxin. Possibly the outward transport of bile acids might also involved in phatloidin poisoning. Furthermore many kinds of liver injury, probably followed by disturbances of bile acid transport, decrease the sensitivity of damaged livers to phalloidin (Floersheim, 1966, 1976). Recently an irreversible inhibition of the phalloidin response by DIDS 3 and H2-DIDS 4 was detected (Petzinger et al., unpublished). This protein reagent, binding preferentially to e-amino groups, blocks the anionic transport in red cells (Lepke et al., 1976). The anionic sulfobromophthalein (BSP) inhibits the uptake of taurocholate in isolated hepatocytes (Schwarz et al., 1975) and blocks binding of cholic acid to rat liver plasma membranes (Anwer et al., 2 Detailed studies will be published in a separate paper by L. B. Veil 3 DIDS = 4,4'-diisothiocyano stilbene-2,2'-disulfonic acid 4 H2-DIDS = 4,4'-diisothiocyano-],2-diphenyl ethane-2,2'-disulfonic acid
M. Frimmer et al. : Role of Bile Acids in Phalloidin Poisoning
1977). BSP is also a potent inhibitor of phalloidin response (Frimmer and Petzinger, unpublished). Detailed studies concerning the influence of phalloidin poisoning on the uptake of bile acids by liver cells are in progress. REFERENCES Agostini, B., Govindan, V. M., Hofmann, W , Wieland, Th. : Phalloidin-induced proliferation of actin filaments within rat hepatocytes. Z. Naturforsch. 30c, 793-795 (1975) Anwer, S. M., Kroker, R., Hegner, D., Pelter, A. : Cholic acid binding to isolated rat liver plasma membranes. Hoppe-Seyler's Z. Physiol. Chem. 358, 543-553 (1977) Frimmer, M., Kroker, R.: Phalloidin-Antagonisten, 1. Mitt. Arzneim.-Forsch. (Drug Res.) 25, 394-396 (1975) Frimmer, M., Petzinger, E. : PhalloidinhAntagonisten, 3. Mitt. Arzneim.-Forsch. (Drug Res.) 25, ~423-1426 (1975) Frimmer, M., Petzinger, E., Homann, J. : Phalloidin-Antagonisten, 4. Mitt. Arzneim.-Forsch. (Drug Res.) 25, 1881-1884 (t975) Frimmer, M., Petzinger, E., Rufeger, U., Veil, L. B.: Trypsin protection of hepatocytes against phalloidin. Naunyn-Schmiedeberg's Arch. Pharmacol. 300, 163-171 (1977) Floersheim, G. L.: Schutzwirkung hepatotoxischer Stoffe gegen letale Dosen eines Toxins aus Amanita phalloides (Phalloidin). Biochem. Pharmacol. 15, 1589-1593 (1966) Floersheim, G. L. : Antagonistic effects against single lethal doses of amanita phalloides. Naunyn-Schmiedeberg's Arch. Pharmacol. 293, 171 - 174 (1976) Govindan, V. M., Faulstich, H., Wieland, Th., Agostini, B., Hasselbach, W. : In-vitro effect of phalloidin an a plasma membrane
147 preparation from rat liver. Naturwissenschaften 59, 521- 522 (1972) Govindan, V. M., Rohr, G., Wieland, Th., Agostini, B.: Binding of phallotoxin to protein filaments of plasma membrane of liver cell. Hoppe-Seyler's Z. Physiol. Chem. 354, 1159-1161 (1973) Lepke, S., Fasold, H., Pring, M., Passow, H. : A study of the relationship between inhibition of anion exchange and binding to the red blood cell membrane of 4,4'-diisothiocyano stilbene-2,2'disulfonic acid (DIDS) and its dihydro derivative (H2-DIDS). J. Membr. Biol. 29, 147-177 (1976) Lutz, F., Hermann, S., Frimmer, M. : The influence of sodium dehydrocholate on the uptake of 3H-desmethylphalloin by the perfused rat liver. Naunyn-Schmiedeberg's Arch. PharmacoI. 270, 310--312 (1971) Petzinger, E., Homann, J., Frimmer, M. : Phalloidin-Antagonisten. 2. Mitt. Arzneim.-Forsch. (Drug Res.) 25, 571- 576 (1975) Petzinger, E., Seeger, R. : Scanning electron microscopic studies on the cytolytic effect of phallolysin on isolated rat hepatocytes and AS-30 D hepatoma cells. Naunyn-Schmiedeberg's Arch. Pharmacol. 295, 211-213 (1976) Schwarz, L. R., Buss, R., Schwenk, M., Pfaff, E., Greim, H. : Uptake of taurocholic acid into isolated rat-liver cells. Eur. J. Biochem. 55, 617-623 (1975) Wieland, O. : Changes in liver metabolism induced by the poisons of amanita phalloides. Clin. Chem. 2, 323-338 (1965) Wieland, Th., Faulstich, H., Jahn, W., Govindan, M. V., Puchinger, H., Kopitar, Z., Schmaus, H., Schmitz, A. : Zur Wirkungsweise des Antamanids. Hoppe-Seyler's Z. Physiol. Chem. 353, 13371345 (1972)
Received September 19~Accepted October 3, 1977
