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Immunofluorescence demonstration of digoxin in human myocardial tissue and isolated rat cardiac myocytes with monoclonal antibodies
Forensic Elsevier
Science International, 47 (1990) 157Scientific Publishers Ireland Ltd.
157
163
IMMUNOFLUORESCENCE DEMONSTRATION OF DIGOXIN IN HUMAN MYOCARDIAL TISSUE AND ISOLATED RAT CARDIAC MYOCYTES WITH MONOCLONAL ANTIBODIES
P. S6TONYI., E. SOMOGYF, J. RAJSb, M. SUNDBERG” and B. ANDERSSON” aDepartment of Forensic Medicine of Semmelweis University of Medicine, H-1085 Budapest (Hungary) and bDepartment of Forensic Medicine, Karolinska Institutet, Box 60400, S-104 01 Stockholm (Sweden) (Received (Accepted
March 27th, 19901 April 12th, 19901
Summary The aim of the present investigation was to identify the morphological correlates of digoxin binding sites in human heart muscle tissue and isolated viable rat heart myocytes. Cardiac glycoside linked to myocardial cells was demonstrated by monoclonal digoxin specific antibody and by FITC-conjugated anti-mouse immunoglobin serum. This versatile immunofluorescence method can be used in diagnostic and experimental studies of cardiac glycoside binding. Key
words:
Digoxin;
Myocardium;
Immunofluorescence
method
Introduction Monoclonal glycoside specific antibodies are efficacious in the treatment of intoxication with glycosides. The use of purified digoxin specific monoclonal antibodies and Fab fragments are safe and effective means to reverse advanced, life-threatening digitalis intoxication and digoxin-induced arrhythmias [l-33]. Recently, on the basis of this principle, an immunofluorescence method using digoxin specific monoclonal antibody and FITC-conjugated anti-monoclonal antibody sera has been developed. The advent of morphological methods of studying the binding of cardiac glycoside will evidently be of importance in forensic medicine as well as in toxicology. The aim of the present study was to evaluate the binding of cardiac glycoside to the cell membrane receptor by means of immunohistochemistry. A comparative histochemical study was therefore carried out simultaneously on human myocardial tissue and on isolated rat cardiac myocytes in suspension. Address all correspondence and reprint requests to: Dr. P. Sotonyi, Department of Forensic Medicine, Medical Faculty, Semmelweis University, Ulloi Rd., 26 H-1085, Budapest, Hungary. Abbreviations: Fab; purified digoxin specific antibody fragment; FITC; fluorescein-isothiocyanate; PBS; phosphate-buffered saline: 0.15 mol/l NaCl, 0.01 mob1 phosphate, pH = 7.4.
Printed
and Published
in Ireland
158
Materials and Methods Digoxin was obtained from Richter Gedeon@ , Hungary. The anti-digoxin monoclonal antibody (clone No. Dl-221 was purchased from Sigma, St. Louis, MO, USA and FITC-conjugated antisera from goat from Biochemical Co., Hungary. Collagenase (type 111 was purchased from Worthington, NJ, USA. Other chemicals were obtained from local commercial sources. Frozen cryostat sections (15- 20pml from fixed heart tissues were used in the experiments. The fixation procedure was carried out with freshly prepared 1% paraformaldehyde solution in 72 mM Na-cacodylate buffer, pH 7.5, supplemented with 0.72% sucrose. The protocol used for preparation of samples to be analyzed by fluorescence microscopy is as follows: 1. Incubation of fixed cryostat prepared tissue sections with digoxin (1 mM or 0.01 mM1 in PBS buffer (150 mM NaCl and 10 mM phosphate, pH 7.41 for 30 min at 20°C. 2. Washing sections 3 x 2 min in PBS buffer supplemented with NaN, (0.1 g/l) at room temperature. 3. Incubation with the monoclonal digoxin specific antibody (1 mg/ml) in PBS buffer for 30 min at 20%. 4 Washing for 3 x 2 min in PBS buffer at room temperature. antisera, diluted 1:20, for 30 min at 5. Incubation with FITC-conjugated 2ooc. 6. Final washing for 3 x 2 min at room temperature. The sections were covered before evaluation by fluorescence microscopy. Cardiac myocytes were isolated from Sprague - Dawley rats (200 -300 g) according to the method described by Andersson et al. [4]. Briefly, the hearts were perfused with a modified Krebs-Henseleit buffer containing 11 mM Hepes (N,-hydroxyethyl-piperazine-iV’-2-ethanesulfonic acid), 10 mM glucose, amino acids and 50 PM CaCl,, pH 7.2, for about 5 min, followed by a 4 min perfusion with Ca2+-free Krebs buffer. The hearts were then perfused with a modified Krebs-buffer supplemented with collagenase (0.1%) for 15-20 min. A final incubation of pieces of the perfused hearts were carried out in collagenase containing Krebs-buffer for 3 x 10 min. The yield of myocytes was usually 5-8 x lO”/heart; 70-80% were viable by trypan blue exclusion, and 55- 75% showed rod-shaped configuration. Fixation of the isolated myocytes was carried out by drop-wise addition of 1.0 ml cell suspension (0.250.5 x lo6 myocytes) to 9.0 ml freshly prepared 1% paraformaldehyde solution in 72 mM Na-Cacodylate buffer, pH 7.5, containing 0.72% sucrose at 4%. After 10 min, the fixed cells were centrifuged at 125 g for 5 min at 20°C, resuspended and washed in PBS buffer for 2 x 3 min and centrifuged at 125 g. The cells were then resuspended and centrifuged on to slides by cytocentrifugation. Before cryostat sections were prepared, the fixed and centrifuged myocyte pellet was covered with Tissue Teok (Ames). Cryostat
159
sections (15-2Opml of fixed myocytes were incubated with Digoxin as described for the cryostat sections of heart tissue. After incubation with cardiac glycoside, the following steps were carried out. 1. Washing for 3 x 5 min in PBS at room temperature. 2. Incubation with monoclonal digoxin specific antibody (1 mg/l ml) in PBS for 20 min at 20 OC. 3. Washing for 3 x 5 min in PBS at room temperature. 4. Incubation with FITC-conjugated antisera (1:20 dilution1 for 30 min at 20 oc. 5. Washing for 3 x 5 min at room temperature, covering of the preparations and evaluation by fluorescence microcopy. The mounting medium consisted of glycerol-lepinol buffer (pH 8.61 and 0.1 mg paru-phenylendiamine/100 ml [5]. The specificity of the reaction was tested by incubating heart cells not treated with digoxin. No fluorescence was observed in such preparations. During the immunohistochemical procedures, sections were washed several times, sometimes with relatively high pH values, which resulted in the frequent problem that the whole section or parts thereof sank. Therefore, sections needed to be fixed, preferably on slides covered with ovalbumin or chromalum-gelatin. The mounting medium contained glycerol-lepinol buffer with added para-phenylendiamine [5] in order to diminish fading. For examination of the fluorescence, a Jenalumar fluorescence microscope (VEB Carl Zeiss Jena, GDR) was used with incident-light-narrow-band blue excitation (450<19490 nml narrowing the fluorescence spectrum by the filter KP 560, or with incident-light-narrow-band green excitation. For the investigation we used the objective Planchromat fl 50/0.09 and Planapochromat 25/ 0.66. Photomicrographs were taken with ORWO UK.17 colour and Fujicolor T.64 films. Sections, untreated with digoxin, were used as controls. Results Distinct fluorescence patterns were obtained in tissues and cells incubated for the demonstration of digoxin. The intensity of the reaction seemed to depend on the concentration of digoxin, both concerning intact heart tissue and isolated myocytes (Figs. 1- 41. The intensity of fluorescence in the intercalated disk was similar to that of the sarcolemmal membrane fluorescence. The distribution of fluorescent sites indicated that the intercalated disks strongly bound the glycoside, demonstrating the sensitivity of the method, and the fact that cardiac glycoside linked to the heart cell membrane is detectable in the range of concentrations occurring in therapeutic doses. No immunofluorescence reaction was seen either in myocardial tissue or in isolated cardiac myocytes not treated with digoxin.
Figs. l-2. fluorescence. intercalated
Localization of digoxin (0.01 mM and 1 mM) in The images provide evidence for binding to the disks. (x 160, x 160).
heart tissues sarcolemmal
with immunomembrane and
161
Figs. 3-4. Localization of digoxin (0.01 mM and 1 mM) in isolated cardiac immunofluorescence. The fluorescent compound is located to the sarcolemmal the intercalated disks (as in Figs. 1- 2). ( x 160, x 160).
myocytes with membrane and
162
Discussion Digitalis glycosides and related compounds are widely used in the treatment of various cardiac disorders. A wide variety of irreversible ultrastructural changes of the myocardium have been reported in different experimental conditions following acute cardiac glycoside intoxication [6.7]. In forensic pathology, there is a great need for a method allowing an accurate morphological localization of cardiac glycoside in heart muscle cells [8,9]. The pathogenesis of cell damage and cell death, as well as the functionalmorphological expression after acute cardiac glycoside intoxication, has always been of great interest to the forensic pathologist. Undoubtedly, the use of monoclonal antibodies has exerted great effects on the recent developments of immunohistochemistry, and greater specificity is usually obtained with immunohistochemical methods than with histochemical procedures. However, the specificity in immunohistochemistry has its limits. With these methodologic limits in mind, an immune histochemical approach can be a reliable means of studying the presence of certain molecules and their localization in tissues and cells [lO,ll]. The monoclonal digoxin specific antibody and FITC-conjugated antisera were found to be useful for the morphological demonstration of the localization of digoxin in cardiac tissue. The specificity of the immunofluorescence reaction was proved by the negative result with heart muscle unexposed to digoxin. The intensity of reaction appeared to be dependent of the concentration of cardiac glycoside. This issue could be further studied by photometric determination of the intensity of fluorescence. Besides, with FITC, the antisera could be marked by other fluorochromes, like Texas-Red, Lizeminrodemin B 200 sulphonylchloride (RB 2001 and 1-dimethyl-amino-naphtyl-5 sulphonyl-chloride (DANS). It is easy to envisage that with electronmicroscopic adaption of immunofluorescence technology, it would be possible to study the exact ultrastructural localization of the cardiac glycoside by application of peroxidase-anti-peroxidase (PAP) complex, alkaline phosphatase, or ferritin labelling techniques. Furthermore, labelling of sera with radioactive isotopes could provide the possibility of detecting cardiac glycoside in cells and tissues by use of light and electronmicroscopic autoradiography. The sensitivity of the present method allows the detection of cardiac glycoside bound to the heart muscle cell membrane in the range of the concentrations used in therapeutic situations. On the basis of our own experience, it is possible to study the kinetics of digoxin binding to the cells, especially to cell membrane. In recent papers [12,13], we have described a method by which Romhbnyi’s topooptical aldehyde-bisulphite-toluidine blue (ABT) reaction has been adapted to the demonstration of membrane-bound glycoside oligosaccharides and their molecular orientation [14,15]. Digoxin pretreatment induced an accelerated ABT-reaction with negative dichroism, metachromasia and intensive basophilia. ABT reaction and labelled lectin (concanavaline A-FITC)
163
binding proved to be a suitable method for the microscopical topical localization of cardiac glycoside [16]. The development cytochemical method opens new perspectives for the demonstrating the localization of glycoside.
and submicrosof an immunopossibilities of
Acknowledgments This work was supported Karolinska Institutet.
by grants
from
Semmelweis
University
and
References 1
P.L. Lechat, M. Mudgett-Hunter, M.N. Margolies, E. Haber and T. Smith, Reversal of lethal digoxin toxicity in guinea pigs using monoclonal antibodies and Fab fragments. J. Pharmacol Exp. Z’her., 29 (1984) 210 - 213.
2
T.W. Smith, E.M. Auzman, P. Friedman, C.M. Blatt, J.D. Marsh, Digitalis glycoside mechanism and manifestation of toxicity. Basic mechanism of cardiac glycoside action. Prog. Cardiovasc. Dis., 26 (19841 495- 540. E. Haber, Antibodies and digatalis: The modern revolution in the use of an ancient drug. J. Am. Cell Cardiol, 5 (1985) 111-117. B.S. Andersson, M. Sundberg and J. Rajs, Tert-Butyl hydroperoxide-induced toxicity in isolated adult cardiac myocytes, manuscript in preparation. H. Kupper and H. Storz, Double staining technique using combination of indirect and direct Acta histochem., 78 (1986) 185- 188. immunfluorescence with monoclonal antibodies. E. Somogyi and P. Sotonyi, Electron microscopic studies of the myocardium in experimental acute heart glycoside intoxication. Festschrift fur Horst Leitoff. Kriminalistik Verlag Hei-
3 4 5 6
7
8 9 10 11 12 13 14
15
16
delberg (19851. P. Sotonyi and E. Somogyi, Electron microscopic autoradiographic study of heart muscle calcium in experimental cardiac glycoside poisoning. Acta Morph. Acad. Sci Hung., 33 (1985) 203-209. P. Sbtonyi and E. Somogyi, Compartive topooptical investigation of cardiac glycoside localization. Z Rechtsmed., 90 (19831 87 - 94. P. Sotonyi and S.N. JuhLsz, Effect of cardiac glycoside on the coronaries: physiologic and morphologic studies in the dog heart. Acta PhysioL Acad. Sci. Hung., 62 (1984) 153- 160. B.B. Mishell and S.M. Shiigi, Selected Methods in Cellular Immunology, W.H. Freeman and Company. New York (19801. A.M. Campbell, Monoclonal Antibody Technology, Elsevier Science publishers, Amsterdam, The Netherlands, 1984. P. Sbtonyi and E. Somogyi, Cytochemical demonstration of the molecular forms of cardiac glycoside in the heart muscle. Acta histochem., 72 (19831 117-122. P. Sbtonyi, M. Oberna and E. Keller, Topooptical investigation of cardiac glycoside in heart muscle. Acta Med. Leg., 35 (19851 199-202. Gy. Romhbnyi, Gy. Delk and J. Fischer, Aldehyde-Bisulfite-Toluidineblue (ABT) staining as topooptical reaction for demonstration of linear order of vicinal-OH groups in biological structures. Histochemistry, 43 (1975) 333-348. J. Fischer and L. Emody, Molecular order of carbohydrate components in cell walls of bacteria, fungi and algae according to the topooptical reaction of the vicinal OH groups. Acta Morphol. Sci Hung., 23 (1976) 97- 108. P. Sotonyi, E. Somogyi and A. Nemeth, Morphological investigation of cardiac glycosides. In L. Neoral und A. Loyka teds.), Sammelbuch der Symposium der Gerichtsmedizin. Avicenum, Olomucz, 1987.
Science International, 47 (1990) 157Scientific Publishers Ireland Ltd.
157
163
IMMUNOFLUORESCENCE DEMONSTRATION OF DIGOXIN IN HUMAN MYOCARDIAL TISSUE AND ISOLATED RAT CARDIAC MYOCYTES WITH MONOCLONAL ANTIBODIES
P. S6TONYI., E. SOMOGYF, J. RAJSb, M. SUNDBERG” and B. ANDERSSON” aDepartment of Forensic Medicine of Semmelweis University of Medicine, H-1085 Budapest (Hungary) and bDepartment of Forensic Medicine, Karolinska Institutet, Box 60400, S-104 01 Stockholm (Sweden) (Received (Accepted
March 27th, 19901 April 12th, 19901
Summary The aim of the present investigation was to identify the morphological correlates of digoxin binding sites in human heart muscle tissue and isolated viable rat heart myocytes. Cardiac glycoside linked to myocardial cells was demonstrated by monoclonal digoxin specific antibody and by FITC-conjugated anti-mouse immunoglobin serum. This versatile immunofluorescence method can be used in diagnostic and experimental studies of cardiac glycoside binding. Key
words:
Digoxin;
Myocardium;
Immunofluorescence
method
Introduction Monoclonal glycoside specific antibodies are efficacious in the treatment of intoxication with glycosides. The use of purified digoxin specific monoclonal antibodies and Fab fragments are safe and effective means to reverse advanced, life-threatening digitalis intoxication and digoxin-induced arrhythmias [l-33]. Recently, on the basis of this principle, an immunofluorescence method using digoxin specific monoclonal antibody and FITC-conjugated anti-monoclonal antibody sera has been developed. The advent of morphological methods of studying the binding of cardiac glycoside will evidently be of importance in forensic medicine as well as in toxicology. The aim of the present study was to evaluate the binding of cardiac glycoside to the cell membrane receptor by means of immunohistochemistry. A comparative histochemical study was therefore carried out simultaneously on human myocardial tissue and on isolated rat cardiac myocytes in suspension. Address all correspondence and reprint requests to: Dr. P. Sotonyi, Department of Forensic Medicine, Medical Faculty, Semmelweis University, Ulloi Rd., 26 H-1085, Budapest, Hungary. Abbreviations: Fab; purified digoxin specific antibody fragment; FITC; fluorescein-isothiocyanate; PBS; phosphate-buffered saline: 0.15 mol/l NaCl, 0.01 mob1 phosphate, pH = 7.4.
Printed
and Published
in Ireland
158
Materials and Methods Digoxin was obtained from Richter Gedeon@ , Hungary. The anti-digoxin monoclonal antibody (clone No. Dl-221 was purchased from Sigma, St. Louis, MO, USA and FITC-conjugated antisera from goat from Biochemical Co., Hungary. Collagenase (type 111 was purchased from Worthington, NJ, USA. Other chemicals were obtained from local commercial sources. Frozen cryostat sections (15- 20pml from fixed heart tissues were used in the experiments. The fixation procedure was carried out with freshly prepared 1% paraformaldehyde solution in 72 mM Na-cacodylate buffer, pH 7.5, supplemented with 0.72% sucrose. The protocol used for preparation of samples to be analyzed by fluorescence microscopy is as follows: 1. Incubation of fixed cryostat prepared tissue sections with digoxin (1 mM or 0.01 mM1 in PBS buffer (150 mM NaCl and 10 mM phosphate, pH 7.41 for 30 min at 20°C. 2. Washing sections 3 x 2 min in PBS buffer supplemented with NaN, (0.1 g/l) at room temperature. 3. Incubation with the monoclonal digoxin specific antibody (1 mg/ml) in PBS buffer for 30 min at 20%. 4 Washing for 3 x 2 min in PBS buffer at room temperature. antisera, diluted 1:20, for 30 min at 5. Incubation with FITC-conjugated 2ooc. 6. Final washing for 3 x 2 min at room temperature. The sections were covered before evaluation by fluorescence microscopy. Cardiac myocytes were isolated from Sprague - Dawley rats (200 -300 g) according to the method described by Andersson et al. [4]. Briefly, the hearts were perfused with a modified Krebs-Henseleit buffer containing 11 mM Hepes (N,-hydroxyethyl-piperazine-iV’-2-ethanesulfonic acid), 10 mM glucose, amino acids and 50 PM CaCl,, pH 7.2, for about 5 min, followed by a 4 min perfusion with Ca2+-free Krebs buffer. The hearts were then perfused with a modified Krebs-buffer supplemented with collagenase (0.1%) for 15-20 min. A final incubation of pieces of the perfused hearts were carried out in collagenase containing Krebs-buffer for 3 x 10 min. The yield of myocytes was usually 5-8 x lO”/heart; 70-80% were viable by trypan blue exclusion, and 55- 75% showed rod-shaped configuration. Fixation of the isolated myocytes was carried out by drop-wise addition of 1.0 ml cell suspension (0.250.5 x lo6 myocytes) to 9.0 ml freshly prepared 1% paraformaldehyde solution in 72 mM Na-Cacodylate buffer, pH 7.5, containing 0.72% sucrose at 4%. After 10 min, the fixed cells were centrifuged at 125 g for 5 min at 20°C, resuspended and washed in PBS buffer for 2 x 3 min and centrifuged at 125 g. The cells were then resuspended and centrifuged on to slides by cytocentrifugation. Before cryostat sections were prepared, the fixed and centrifuged myocyte pellet was covered with Tissue Teok (Ames). Cryostat
159
sections (15-2Opml of fixed myocytes were incubated with Digoxin as described for the cryostat sections of heart tissue. After incubation with cardiac glycoside, the following steps were carried out. 1. Washing for 3 x 5 min in PBS at room temperature. 2. Incubation with monoclonal digoxin specific antibody (1 mg/l ml) in PBS for 20 min at 20 OC. 3. Washing for 3 x 5 min in PBS at room temperature. 4. Incubation with FITC-conjugated antisera (1:20 dilution1 for 30 min at 20 oc. 5. Washing for 3 x 5 min at room temperature, covering of the preparations and evaluation by fluorescence microcopy. The mounting medium consisted of glycerol-lepinol buffer (pH 8.61 and 0.1 mg paru-phenylendiamine/100 ml [5]. The specificity of the reaction was tested by incubating heart cells not treated with digoxin. No fluorescence was observed in such preparations. During the immunohistochemical procedures, sections were washed several times, sometimes with relatively high pH values, which resulted in the frequent problem that the whole section or parts thereof sank. Therefore, sections needed to be fixed, preferably on slides covered with ovalbumin or chromalum-gelatin. The mounting medium contained glycerol-lepinol buffer with added para-phenylendiamine [5] in order to diminish fading. For examination of the fluorescence, a Jenalumar fluorescence microscope (VEB Carl Zeiss Jena, GDR) was used with incident-light-narrow-band blue excitation (450<19490 nml narrowing the fluorescence spectrum by the filter KP 560, or with incident-light-narrow-band green excitation. For the investigation we used the objective Planchromat fl 50/0.09 and Planapochromat 25/ 0.66. Photomicrographs were taken with ORWO UK.17 colour and Fujicolor T.64 films. Sections, untreated with digoxin, were used as controls. Results Distinct fluorescence patterns were obtained in tissues and cells incubated for the demonstration of digoxin. The intensity of the reaction seemed to depend on the concentration of digoxin, both concerning intact heart tissue and isolated myocytes (Figs. 1- 41. The intensity of fluorescence in the intercalated disk was similar to that of the sarcolemmal membrane fluorescence. The distribution of fluorescent sites indicated that the intercalated disks strongly bound the glycoside, demonstrating the sensitivity of the method, and the fact that cardiac glycoside linked to the heart cell membrane is detectable in the range of concentrations occurring in therapeutic doses. No immunofluorescence reaction was seen either in myocardial tissue or in isolated cardiac myocytes not treated with digoxin.
Figs. l-2. fluorescence. intercalated
Localization of digoxin (0.01 mM and 1 mM) in The images provide evidence for binding to the disks. (x 160, x 160).
heart tissues sarcolemmal
with immunomembrane and
161
Figs. 3-4. Localization of digoxin (0.01 mM and 1 mM) in isolated cardiac immunofluorescence. The fluorescent compound is located to the sarcolemmal the intercalated disks (as in Figs. 1- 2). ( x 160, x 160).
myocytes with membrane and
162
Discussion Digitalis glycosides and related compounds are widely used in the treatment of various cardiac disorders. A wide variety of irreversible ultrastructural changes of the myocardium have been reported in different experimental conditions following acute cardiac glycoside intoxication [6.7]. In forensic pathology, there is a great need for a method allowing an accurate morphological localization of cardiac glycoside in heart muscle cells [8,9]. The pathogenesis of cell damage and cell death, as well as the functionalmorphological expression after acute cardiac glycoside intoxication, has always been of great interest to the forensic pathologist. Undoubtedly, the use of monoclonal antibodies has exerted great effects on the recent developments of immunohistochemistry, and greater specificity is usually obtained with immunohistochemical methods than with histochemical procedures. However, the specificity in immunohistochemistry has its limits. With these methodologic limits in mind, an immune histochemical approach can be a reliable means of studying the presence of certain molecules and their localization in tissues and cells [lO,ll]. The monoclonal digoxin specific antibody and FITC-conjugated antisera were found to be useful for the morphological demonstration of the localization of digoxin in cardiac tissue. The specificity of the immunofluorescence reaction was proved by the negative result with heart muscle unexposed to digoxin. The intensity of reaction appeared to be dependent of the concentration of cardiac glycoside. This issue could be further studied by photometric determination of the intensity of fluorescence. Besides, with FITC, the antisera could be marked by other fluorochromes, like Texas-Red, Lizeminrodemin B 200 sulphonylchloride (RB 2001 and 1-dimethyl-amino-naphtyl-5 sulphonyl-chloride (DANS). It is easy to envisage that with electronmicroscopic adaption of immunofluorescence technology, it would be possible to study the exact ultrastructural localization of the cardiac glycoside by application of peroxidase-anti-peroxidase (PAP) complex, alkaline phosphatase, or ferritin labelling techniques. Furthermore, labelling of sera with radioactive isotopes could provide the possibility of detecting cardiac glycoside in cells and tissues by use of light and electronmicroscopic autoradiography. The sensitivity of the present method allows the detection of cardiac glycoside bound to the heart muscle cell membrane in the range of the concentrations used in therapeutic situations. On the basis of our own experience, it is possible to study the kinetics of digoxin binding to the cells, especially to cell membrane. In recent papers [12,13], we have described a method by which Romhbnyi’s topooptical aldehyde-bisulphite-toluidine blue (ABT) reaction has been adapted to the demonstration of membrane-bound glycoside oligosaccharides and their molecular orientation [14,15]. Digoxin pretreatment induced an accelerated ABT-reaction with negative dichroism, metachromasia and intensive basophilia. ABT reaction and labelled lectin (concanavaline A-FITC)
163
binding proved to be a suitable method for the microscopical topical localization of cardiac glycoside [16]. The development cytochemical method opens new perspectives for the demonstrating the localization of glycoside.
and submicrosof an immunopossibilities of
Acknowledgments This work was supported Karolinska Institutet.
by grants
from
Semmelweis
University
and
References 1
P.L. Lechat, M. Mudgett-Hunter, M.N. Margolies, E. Haber and T. Smith, Reversal of lethal digoxin toxicity in guinea pigs using monoclonal antibodies and Fab fragments. J. Pharmacol Exp. Z’her., 29 (1984) 210 - 213.
2
T.W. Smith, E.M. Auzman, P. Friedman, C.M. Blatt, J.D. Marsh, Digitalis glycoside mechanism and manifestation of toxicity. Basic mechanism of cardiac glycoside action. Prog. Cardiovasc. Dis., 26 (19841 495- 540. E. Haber, Antibodies and digatalis: The modern revolution in the use of an ancient drug. J. Am. Cell Cardiol, 5 (1985) 111-117. B.S. Andersson, M. Sundberg and J. Rajs, Tert-Butyl hydroperoxide-induced toxicity in isolated adult cardiac myocytes, manuscript in preparation. H. Kupper and H. Storz, Double staining technique using combination of indirect and direct Acta histochem., 78 (1986) 185- 188. immunfluorescence with monoclonal antibodies. E. Somogyi and P. Sotonyi, Electron microscopic studies of the myocardium in experimental acute heart glycoside intoxication. Festschrift fur Horst Leitoff. Kriminalistik Verlag Hei-
3 4 5 6
7
8 9 10 11 12 13 14
15
16
delberg (19851. P. Sotonyi and E. Somogyi, Electron microscopic autoradiographic study of heart muscle calcium in experimental cardiac glycoside poisoning. Acta Morph. Acad. Sci Hung., 33 (1985) 203-209. P. Sbtonyi and E. Somogyi, Compartive topooptical investigation of cardiac glycoside localization. Z Rechtsmed., 90 (19831 87 - 94. P. Sotonyi and S.N. JuhLsz, Effect of cardiac glycoside on the coronaries: physiologic and morphologic studies in the dog heart. Acta PhysioL Acad. Sci. Hung., 62 (1984) 153- 160. B.B. Mishell and S.M. Shiigi, Selected Methods in Cellular Immunology, W.H. Freeman and Company. New York (19801. A.M. Campbell, Monoclonal Antibody Technology, Elsevier Science publishers, Amsterdam, The Netherlands, 1984. P. Sbtonyi and E. Somogyi, Cytochemical demonstration of the molecular forms of cardiac glycoside in the heart muscle. Acta histochem., 72 (19831 117-122. P. Sbtonyi, M. Oberna and E. Keller, Topooptical investigation of cardiac glycoside in heart muscle. Acta Med. Leg., 35 (19851 199-202. Gy. Romhbnyi, Gy. Delk and J. Fischer, Aldehyde-Bisulfite-Toluidineblue (ABT) staining as topooptical reaction for demonstration of linear order of vicinal-OH groups in biological structures. Histochemistry, 43 (1975) 333-348. J. Fischer and L. Emody, Molecular order of carbohydrate components in cell walls of bacteria, fungi and algae according to the topooptical reaction of the vicinal OH groups. Acta Morphol. Sci Hung., 23 (1976) 97- 108. P. Sotonyi, E. Somogyi and A. Nemeth, Morphological investigation of cardiac glycosides. In L. Neoral und A. Loyka teds.), Sammelbuch der Symposium der Gerichtsmedizin. Avicenum, Olomucz, 1987.