Summary. The tissue distribution after repeated in- travenous administration of tritium-labelled digoxin,. /~-methyldigoxin and ouabain was examined in heart.
Naunyn-Schmiedeberg's
Archivesof
Naunyn-Schmiedeberg's Arch. Pharmacol. 307, 6 5 - 7 1 (1979)
Pharmacology
9 by Springer-Verlag 1979
Distribution of Cardiac Glycosides in Heart and Brain of Dogs and Their Affinity to the (Na § + K§ J. Kuhlmann 1., E. Erdmann 2, and N. Rietbrock 1.* 1 Institut ftir Klinische Pharmakologie der Freien Universit~it Berlin, D-1000 Berlin 2 Medizinische Klinik I der Universit/it Miinchen, Klinikum Grol3hadern, Postfach 701260, D-8000 Miinchen 70, Federal Republic of Germany
Summary. The tissue distribution after repeated intravenous administration of tritium-labelled digoxin, /~-methyldigoxin and ouabain was examined in heart and brain of 6 beagle dogs. In addition, the (Na + + K+)-ATPase activity was measured in various heart and brain areas, and its affinity to the cardiac glycosides was determined. The glycoside concentrations in the atria are lower than in the ventricles, and the left heart areas show higher concentrations than the right areas. Significant differences in the (Na + + K+) ATPase activity or its binding capacity in the various heart areas, which could be responsible for this characteristic distribution pattern, were not found. In agreement with its greater lipid-solubility, 3-methyldigoxin shows a higher accumulation in the brain than digoxin and ouabain. However, while 3-methyldigoxin is evenly distributed throughout all brain areas, concentration differences are found for digoxin and ouabain in the telencephalon, cerebellum and brain stem. This characteristic distribution of the more polar glycosides may be partly determined by the different structure of the capillaries in the central nervous system. In addition, the binding affinities for digoxin and ouabain also differ in the various crude brain preparations. In the diencephalon, pons, cerebellum and medulla the dissociation constants as a reciprocal measure of the binding affinity were lower for digoxin with 7.5 to 9.9 x 1 0 - 9 M than in the telencephalon, mesencephalon and spinal cord with dissociation constants of 1.1 to 1.45 x 10-8 M. Since, in these brain areas higher glycoside concentrations per g wet weight were also mea-
Send offprint requests to J. Kuhlmann at the above address.
Present addresses : *
Medizinische Universitgtsklinik Wgrzburg, Josef-SchneiderStr. 2, D-8700 Wtirzburg, Federal Republic of Germany ** Klinische Pharmakologie, Klinikum der Johann-Wolfgang-vonGoethe-Universitfit Frankfurt, Ludwig-Rehn-Str. 14, D-6000 Frankfurt/Main, Federal Republic of Germany
sured, the glycoside accumulation in the various brain areas could be dependent on the higher receptor affinity of these brain areas. On the other hand, the binding affinities for/3-methyldigoxin were the same inall brain areas, with a mean dissociation constant of 1.45 x 10-s M. Key words: Cardiac glycosides - Tissue distribution (Na + + K+)-ATPase - Heart - Brain.
Introduction
In a previous investigation glycoside-specific differences in distribution velocity and extent in the various brain areas have been discovered (Kuhlmann et al., 1979). Since it is not so much the absolute tissue concentration buth rather the component bound to specific receptors that is decisive in the action of drugs, it was the purpose of these investigations to determine to what extent the distribution of the various cardiac glycosides during steady state conditions correlate with the binding capacity of the glycoside receptors in the various areas of the heart and brain. The (Na § + K§ activated ATPase of the cell membrane is generally regarded as the specific cardiac-glycoside receptor (Portius and Repke, 1962; Repke and Portius, 1963; Schatzmann, 1963; Glynn, 1964; Greeff, 1968; Akera et al., 1970). According to studies by Erdmann and Schoner (1973), the individual cardiac glycosides accumulate with various affinities in this enzyme system. Since the extent of the ATPase activity and its glycoside sensitivity vary in the individual organs and tissues (Bonting et al., 1961; Dransfeld et al., 1966), the ATPase activity is determined in various areas of heart and brain, and its affinity to the cardiac glycosides is ascertained.
0028-1298/79/0307/0065/$01.40
66
Materials and Methods A. Determination o f Glycoside Concentration in Plasma and Tissue Substances. 12-c~-H3-digoxin, specific activity 750-800 ~tCi/mg; 12a-H3-/3-methyldigoxin, specific activity 738-768gCi/mg; H 3ouabain, nonspecifically labelled, specific activity 750- 800 gCi/mg. The compounds migrated as a single spot and their radiochemical purity averaged 9 6 - 98 ~ as determined by thin-layer systems I, II and 1II (see below). The following were available as unlabelled reference substance in purified, crystalline form: digoxin, /3methyldigoxin, ouabain, digoxigenin-bis-digitoxoside, digoxigeninmono-digitoxoside, digoxigenin, 3-c~-epi-digoxigenin. Ouabain was obtained from NEN, Boston. All other substances were kindly provided by the companies Beiersdorf, Hamburg, and Boehringer, Mannheim. Experimental Course and Analytical Methods. Experimental studies were performed on 1 - 2 year old beagle dogs with an average body weight of 15kg. Two dogs were treated with digoxin, 2 with /3methyldigoxin and 2 with ouabain. Tritium-labelled glycoside was intravenously administered once a day over a period of 10 days. Blood samples were always taken 24h after the daily glycoside application. The dogs were killed 24 h after the 10th dose by an overdose ofpentobarbital and full-thickness tissue samples, weighing between 0.5 -2.0 g were taken from various heart and brain areas in order to determine the total radioactivity. The detailed preparation and extraction methods have been described comprehensively in an earlier publication (Kuhlmann et at., 1979). The separation of the total radioactivity of the individual tissue samples into a chloroform-soluble and a chloroform-insoluble fraction was achieved by 3 extractions with chloroform. Before and after the triple extraction, the total radioactivity in the aqueous phase was determined. For further qualitative analysis the chloroform-phases were combined and evaporated. The residues were taken up in 50-100 ~tl of a chloroform/methanol (1 : 1) mixture and applied to precoated silica gel plates (Merck, Darmstadt) for thin layer chromatographic analysis. Total radioactivity after H3-ouabain administration was directly applied without previous chloroform-extraction. System I: acetone/chloroform 1 : i, 4-fold development System II: pyridine/chloroform 1:6, 2-fold development System III: chloroform/methanol/water 65:30:5, 2-fold development. The systems I and II permit a clear separation of digoxin, methyldigoxin and their known metabolites. System III was used for TLC analysis of the total radioactivity after 3H-ouabain administration. The radioactive zones on the plates were localized with the thin-layer scanner II (Berthold-Friesecke) and compared with the position of the unlabelled reference substances. The reference substances were visualized by UV-fiuorescence at 366 nm after spraying the plates with Kaiser's (1955) reagent (10 ml of 0.3 % chloramine-T and 40 ml of 25 % trichloracetic acid) and activation at 110~ C for 10 min. The quantitative evaluation of the thin-layer chromatograms were done by automatic integration of the activity peaks (Integrator: Berthold LB 2437). Especially for lower activity chromatograms, the results were controlled by scraping out the individual tracks at 0.5 cm intervals. The silica gel was dissolved in 1 ml of distilled water by 24 h incubation and the radioactivity determined in the liquid scintillation counter by the addition of 15 ml of Instagel ~.
B. Determination of the (Na + + K+ )-ATPase Activity in the Heart and Brain of Dogs and Its Affinity to Digoxin, ~-Methyldigoxin and Ouabain Substances. 12-c~-H3-digoxin, specific activity 10.7Ci/mMol; 12-c~H3-methyldigoxin, specific activity 0.8 Ci/mMol; H3-ouabain, nonspecifically labelled, specific activity 30 Ci/mMol.
Naunyn-Schmiedeberg's Arch. Pharmacol. 307 (1979) The experiments were done on beagle dogs of the same strain, as had been used for the preliminary distribution studies. After the untreated animals had been killed, the heart and brain were immediately removed and prepared in the same manner as described in an earlier publication (Kuhlmann et at., 1979).
Cell Membrane Concentration. When a "pure" membrane preparation is isolated, only about 100 mg of membrane protein is gained from 100 g wet weight. This small and inconstant yield does not permit a quantitative statement correlating to the wet weight. Since, however, besides the qualitative differences, quantitative differences were also to be examined, a highly purified membrane preparation of these organs was not prepared. One gram wet weight of both atria and ventricles as well as various brain areas were homogenized for 30 s with an Ultra-Turrax in 30 ml of 0.01 M imidazole/HCl-buffer pH 6.9 and 3 mM EDTA. The homogenate were centrifuged for 10 rain at 2000 x g, the sediment discarded and the supernatant centrifuged again for 30 min at 80.000 • g. The sediment, thus obtained, was again homogenized in 30 ml of 0.01 M imidazole/HCl-buffer pH 6.9 and 3 M EDTA, and this homogenate of the various tissue samples was used for all examinations. Analytical Methods. The (Na + + K+)-ATPase activity in the heart and brain homogenates was measured in the optical coupled test (Schoner et al., 1967). An enzyme unit (U) is defined as the conversion of 1 gMol of ATP/min at 37~ C. Protein was determined according to Lowry et al. (1951). The digoxin, /3-methyldigoxin and ouabain binding was determined according to the method described by Erdmann and Schoner (1973, 1974). Each experiment was performed with at least 2 membrane preparations, and each measurement is the mean value of 2 - 3 analyses.
Measurement of the Radioactivity. All radioactivity measurements were carried out in a Tri-Carb (Packard Model 3380) or in a Marck II (Nuclear Chicago) scintillation counter. The counting yield and the quench correction were determined according to the channel ratio method with the application of an external standard. The counting times were chosen in such a manner that the deviation for multiple determinations was always less than 5 %. The scintillation fluid was either Instagel | or a dioxane scintillation fluid with the following composition: 10% naphthalene, 0.98% diphenyloxazole, 0.02% 2,2-p-phenylene-bis (5-phenyloxazole) in dioxane.
Results A. Glycoside Concentrations in Plasma, Heart and Brain G l y c o s i d e c o n c e n t r a t i o n s in p l a s m a after daily int r a v e n o u s a d m i n i s t r a t i o n s o f 0.0125 m g / k g t r i t i u m labelled digoxin,/~-methyldigoxin or ouabain increase continously and reach a plateau after 6- 8 days (Table 1). T h e m e a n s t e a d y state p l a s m a c o n c e n t r a t i o n s ( 6 t h 10th d a y a f t e r i n i t i a t i o n o f g l y c o s i d e a d m i n i s t r a t i o n ) a r e : / % m e t h y l d i g o x i n 1.87 + 0.2 n g / m l , o u a b a i n 1.47 + 0.2 n g / m l a n d d i g o x i n 1.0 + 0.1 n g / m l . I n t h e m y o c a r d i u m the h i g h e s t c o n c e n t r a t i o n s c o u l d be m e a s u r e d for o u a b a i n a f t e r c o n s t a n t daily i.v. doses for 10 days. T h e c o n c e n t r a t i o n s r e a c h e d b y / 3 - m e t h y l d i g o x i n a n d d i g o x i n w e r e n o t q u i t e as h i g h ( T a b l e 2). I n the v a r i o u s h e a r t s e c t i o n s t h e r e exist c o n c e n t r a t i o n d i f f e r e n c e s for all t h r e e glycosides. T h e c o n c e n t r a t i o n s in the a t r i a are 3 0 - 50 % l o w e r t h a n in
J. Kuhlmann et al. : Cardiac Glycosides: Distribution and Affinity to (Na § + K+)-ATPase
67
Table 1. Time course of total radioactivity in plasma of beagle dogs. Measurements of concentrations 24 h after each daily intravenous administration of 0.0125 mg/kg tritium-labelled digoxin (n = 2),/~-methyldigoxin (n = 2) and onabain (n = 2) Glycoside
Digoxin Digoxin /~-Methyldigoxin /3-Methyldigoxin Ouabain Ouabain
Plasma concentration (rig/m1) 1
2
3
4
5
6
7
8
9
10 application
0.40 0.35 0.79 1.15 0.88 0.69
0.70 0.62 1.33 1.00 1.13 0.76
0.60 1.04 1.35 1.05 1.47 1.05
0.68 0.90 1.44 1.25 1.42 1.15
0.75 1.15 1.43 1.30 1.58 1.13
0.85 0.85 1.73 1.85 1.65 1.20
0.82 0.94 2.10 1.80 1.70 1.28
1.02 1.07 1.80 2.25 1.82 1.36
1.08 1.00 1.90 1.55 1.53 1.37
1.14 1.18 1.85 1.90 1.50 1.27
Table 2. Glycoside concentration (ng/g wet weight) in various heart and brain areas of beagle dogs 24 h after the last daily i.v. dose of 0.0125 mg/kg tritium-labelled digoxin,/~-methyldigoxinand ouabain over a period of 10 days Tissue
Glycoside concentration (ng/g) Digoxin
/~-Methyldigoxin
Ouabain
Heart r. atria 1. atria r. ventricle 1. ventricle
25.7 27.5 42.5 43.1
28.3 36.7 45.2 54.4
30.0 36.9 63.9 73.6
29.1 33.0 49.6 59.7
31.5 44.6 78.3 83.7
47.1 54.3 95.3 98.2
Telencephalon 1. frontalis 1. parietalis 1. occipitalis hippocampus
7.9 9.5 7.9 21.0
7.7 9.6 9.4 12.4
102.4 104.7 129.5 90.3
93.8 118.4 110.8 101.4
2.7 2.8 3.0 4.4
4.2 4.0 4.3 6.1
Diencephalon hypothalamus thalamus
32.7 18.6
23.8 13.8
90.6 107.1
90.5 111.2
5.4 3.0
8.7 6.0
Mesencephalon
20.9
19.3
112.2
94.6
3.6
4.9
Rhombencephalon pons cerebellum vermis cerebelli medulla
17.4 22.4 14.6 36.7
14.7 22.1 15.4 20.7
82.0 97.4 108.7 95.2
93.6 98.9 105.3 82.7
4.5 4.5 3.9 6.7
5.4 5.3 5.0 8.5
Spinal cord
17.4
15.2
50.5
45.7
5.1
7.0
the ventricles a n d 1 0 - 2 0 ~ higher in the left sections t h a n in the right sections of the heart. The digoxin c o n c e n t r a t i o n s in all b r a i n areas are distinctly lower t h a n in the m y o c a r d i u m . The digoxin c o n c e n t r a t i o n in the t e l e n c e p h a l o n reached only one sixth of the digoxin c o n c e n t r a t i o n in the left ventricle. I n the cerebellum a n d b r a i n stem 2 - 3 times higher c o n c e n t r a t i o n s could be measured. C o m p a r e d to digoxin the o u a b a i n c o n c e n t r a t i o n s in the various b r a i n areas are distinctly lower a n d reached only one f o u r t h to one fifth of the digoxin concentrations. The highest c o n c e n t r a t i o n s in all b r a i n areas could be m e a s u r e d after /%methyldigoxin a d m i n i s t r a t i o n . I n the telenc e p h a l o n the /?-methyldigoxin c o n c e n t r a t i o n s are 13 times a n d in the m e d u l l a 3 times higher t h a n after
digoxin. C o n c e n t r a t i o n differences between telencephalon, cerebellum a n d b r a i n stem could only be f o u n d for digoxin a n d o u a b a i n while the more lipophilic ]~-methyldigoxin is distributed evenly over all b r a i n areas. The total radioactivity after digoxin a n d methyldigoxin a d m i n i s t r a t i o n in heart a n d b r a i n falls completely within the c h l o r o f o r m - s o l u b l e fraction, which could be identified by thin layer c h r o m a t o g r a p h y . The total with e t h a n o l extracted radioactivity after H 3 - o u a b a i n was directly applied to silica gel plates w i t h o u t previous s e p a r a t i o n in a c h l o r o f o r m - s o l u b l e a n d -insoluble fraction a n d identified by thin layer c h r o m a t o g r a p h y . I n all heart a n d b r a i n areas only u n c h a n g e d glycosides are detected.
68
Naunyn-Schmiedeberg's Arch. Pharmacol. 307 (1979)
Table 3. (Na + + K+)-ATPase activity in different heart and brain areas of beagle dogs as well as dissociation constants and binding capacity of the cell membranes for digoxin,/~-methyldigoxinand ouabain (mean of 2 experiments) Tissue
(Na + + K+)-ATPase activity (U/mg protein)
Dissociation constant KD (Mol)
Binding capacity (picomol/mg protein)
Digoxin
B - M e t h y l - Ouabain digoxin
Digoxin
/~-Methyl- Ouabain digoxin
Heart r. atria I. atria r. ventricle I. ventricle
0.047 0,036 0.035 0.022
3.0 xl0 9 t.J • 10 - 9 2.1 • 10-9 6.3 x 10-9
1.06x10-9 1.9 • 10-8 3.7 • 10-9 5.5 x 10-9
3.0 xl0 -8 n.u. 1.6 x 10-8 1.5 x 10-s
1.7 1.8 1.5 1.3
1.7 1.5 2.0 1.1
n.u. 1.7 1.6 n.u.
Telencephalon 1. frontalis 1. parietalis 1. occipitalis hippocampus
0,640 0.389 0.600 0.572
1.45 • 10-8 1.10 x 10-s 1.39 x 10-8 1.28 x 10 8
1.34x 10-8 1.19 • 10-8 1.65 x 10 -8 1.47 x 10-8
1.14x 10-8 9.60x 10-9 1.04 x 10-8 1.21 x 10-8
54.1 30.1 41.7 50.2
49.9 38.8 42.4 42.0
42.7 33.7 51.9 48.5
Diencephalon hypothalamus thalamus
0.278 0,494
7,49 x 10 9 8.70 x 10-9
1,18 x 10-8 1.10 x 10-8
8.23 x 10-9 8.80x 10-9
16.6 40.5
19.2 44.6
22.4 35.2
Mesencephalon
0.728
1.20 x 10-8
1.49 x 10 8
1.19 x 10-8
52.2
50.4
55.4
Rhombencephalon pons cerebellum vermis cerebelli medulla
0.204 0.509 0.700 0.526
8.82 x 10-9 8.04 x 10-9 1.00 x 10-8 9.90 x 10 - 9
1.89 • 10-8 1.03 x 10-8 1.86 x 10-8 1.95 x 10-8
8.99 x 10 9 7.78 • 10 - 9 9.20 x 10-9 1.28 x 10-8
33.6 47.5 50.9 36.9
43.0 53.9 52.2 44.8
63.1 45.7 39.0 31.8
Spinal cord
0.192
1.22 x I0 -8
1.21 x 10 8
1.65 x 10-8
22.2
23.6
25.0
B. Activity of the (Na + + K+)-ATPase in Heart and Brain and Its Affinity for Digoxin, ~-Methyldigoxin and Ouabain The atria a n d ventricles show (Na + + K + ) - A T P a s e activities of 0.022 to 0.047 U / m g of p r o t e i n with the p r e p a r a t i o n used i n these experiments (Table 3). The differences between the individual sections of the heart are n o t significant. The cell m e m b r a n e s o f the various b r a i n areas have, o n the average, a 15 times higher ATPase-activities per m g o f protein. The experiments show in detail that there are some b r a i n areas with a rather high ( N a + + K + ) - A T P a s e activity of more t h a n 0.6 U / m g of protein, such as the frontal a n d occipital lobes of the t e l e n c e p h a l o n as well as the m e s e n c e p h a l o n a n d the vermis cerebelli. The other b r a i n areas show a lower A T P a s e activity o f 0 . 2 - 0 . 5 7 U / m g of protein. C o r r e s p o n d i n g to the higher A T P a s e activity in the b r a i n as c o m p a r e d to t h a t in the heart, the b r a i n has a greater b i n d i n g capacity for cardiac glycosides, i.e. the receptor density of b r a i n cell m e m b r a n e s is 10 to 38 times higher t h a n that of heart cell m e m b r a n e s . The b r a i n areas with the highest A T P a s e activity also show the highest b i n d i n g capacity. The m a x i m a l a m o u n t of the total ATPases, which could be inhibited by cardiac glycosides a n d expresses the percentage of the ( N a + + K +)-ATPase o f the total A T P a s e , reached 8 0 - 90 ~ in
the b r a i n a n d is 2 to 3 times higher t h a n in the m y o c a r d i u m . Preliminary experiments h a d shown that digoxin,/~-methyldigoxin a n d o u a b a i n are specifically b o u n d by the m e m b r a n e p r e p a r a t i o n s of the various heart a n d b r a i n areas. A l t h o u g h the receptor density of the m e m b r a n e s varies a n d thus also the total a m o u n t of b o u n d glycoside, this should n o t be regarded as a f u n d a m e n t a l difference. T h e nonspecific b i n d i n g , which is defined as the m e m b r a n e - b o u n d radioactivity in the presence of 1 0 - S M of u n l a b e l l e d cardiac glycoside or after h e a t i n g o f the m e m b r a n e p r o t e i n for 1 0 m i n to 80~ a m o u n t s to 1 - 3 ~ o of the total m e m b r a n e - b o u n d glycoside for digoxin, /~-methyldigoxin a n d o u a b a i n . The cardiac glycoside receptor b i n d i n g reaches a n e q u i l i b r i u m after 60 m i n a n d rem a i n s c o n s t a n t for a considerable testing period ( < 240 rain). U n d e r the chosen conditions, 2 receptors each with different affinities for digoxin a n d / ~ - m e t h yldigoxin can be f o u n d in dog hearts for the c o n c e n t r a t i o n - d e p e n d e n t glycoside b i n d i n g u n d e r equil i b r i u m c o n d i t i o n s (Fig. 1). The average dissociation c o n s t a n t s a m o u n t to 2.2 x 1 0 - 6 M a n d 3 x 1 0 - 9 for digoxin a n d 2 x 10 - v M a n d 1.1 x 1 0 - 9 M for methyldigoxin. Even if one does n o t w a n t to regard this second type of receptor with lower affinity a n d higher b i n d i n g capacity as a n artefact, it m u s t nevertheless be p o i n t e d out that this 2 n d receptor type in dog hearts c a n be
J. Kuhlmann et al. : Cardiac Glycosides: Distribution and Affinity to (Na++ K+)-ATPase
69
1,0KD2%2,2x10-6M 1,5
c~
E
-5 E
X
0-7M
v
0,5.
1,0
.E x
._c
~
0,5 -
.o~ "t3
1~1,1x10-9M
E J
0,02
i
I
I
l
i
i
0,04
0,06
0,08
0,002
0,004
0,006
digoxin bound digoxin free
1
0,008
I
0,01
~- rnethyldigoxin bound /3- methyldigoxin free
Fig. 1. Digoxin and fl-methyldigoxin binding to beagle dog heart muscle in relation to the glycoside concentration. Cell membranes (0.96 mg) were incubated. The data are plotted according to Scatchard (1949)
10-
i 88 g
•r
%
tween digoxin, fi-methyldigoxin and ouabain with regard to their binding affinity to the heart cell membranes (Table 3). The binding affinities of fi-methyldigoxin are the same for all areas of the brain and show a mean dissociation constant of 1.45 • 10-8 M. The various brain areas show different binding affinities for digoxin and ouabain. The dissociation constants are clearly lower for both glycosides in the diencephalon and rhombencephalon than in the telencephalon, mesencephalon and spinal cord. Since higher glycoside concentrations per g of wet weight are also measured in these brain areas, the glycoside concentration in the various brain areas could depend on the receptor affinity of these areas.
X =/3-methyldigoxin 9 = digoxin
X
KD'~10-8M
6-
~3
4-
i
I
i
0,1
0,3 glycoside
glycoside
i
I
0,5
bound
Discussion
free
Fig. 2. Digoxin and fi-methyldigoxin binding to brain cell membranes (thalamus) of beagle dogs in relation to the glycoside concentration. Cell membranes (0.4 mg protein) were incubated. The data are plotted according to Scatchard (1949)
contingent on the relatively crude membrane preparation and the composition of the incubation medium (Erdmann et al., 1976). For ouabain, only one type of receptor is found in the heart. On the cell membranes in all brain areas, only one type of receptor is detected for digoxim, fi-methyldigoxin and ouabain. If the data obtained are plotted according to Scatchard (1949), the result is a straight line (Fig. 2). There are no significant differences be-
The distribution of cardiac glycosides shows distinct concentration differences in various areas of the heart. These differences characteristic for the heart can already be observed in the early distribution phase, i.e., 60 rain after a single glycoside administration (Kuhlmann et al., 1975). While the higher blood supply of the ventricles seems to be responsible for the higher accumulation of glycoside compared to the atria before the distribution equilibrium is reached, the distribution in the steady state is independent of the circulation under physiological conditions. No noteworthy differences could be found in the (Na § + K§ activity or its binding capacity between the various heart areas. On the other hand, it cannot be ruled out that the number of non-specific
70
binding sites differ between the atrium and ventricle and decisively influence the glycoside distribution in steady state. In the central nervous system, considerable differences have been discovered in the distribution pattern for the individual glycosides. In all areas of the dog brain, the highest concentrations were measured for fimethyldigoxin, followed by digoxin and ouabain in that order. Similar concentration differences between more polar and more lipophilic glycosides were also found by Flasch and Heinz (1976) after repeated administration in the cat and by Haasis and Larbig (1976) in man. So far there have been no studies on the selective uptake in various areas of the brain, which could explain centrally determined glycoside effects. Dutta et al. (1977) have determined radioactivity directly after l-h infusions of tritium-labelled ouabain and digitoxin in dogs and did not find any concentration differences between cerebellum, hypothalamus, mesencephalon, pons and medulla. The absence of differences early after a single administration seems reasonable because of the slow glycoside penetration into the brain. In addition, the glycoside levels showed considerable variations between individual animals. In our experiments distinct concentration differences between telencephalon and cerebellum as well as between the various sections of the brain stem could be found for digoxin and ouabin 24 h after single and repeated application (Kuhlmann, 1978). In contrast, the more lipophilic glycosides fl-methyldigoxin and digitoxin are distributed evenly over all brain areas (Kuhlmann, 1978). The characteristic distribution of more polar glycosides may be partially determined by the different structure of the capillaries in the central nervous system. According to studies by Wilson and Brodie (1961) and Oldendorf (1974), no real bloodbrain barrier exists for some areas of the brain, including the neurohypophysis, tuber cinereum, hypothalamus, pineal body and area postrema. In addition, the higher receptor affinity for digoxin and ouabain in these brain areas might also influence glycoside distribution. Since the central nervous system shows an activity of cardiac glycoside-suppressible (Na § + K+) ATPase which is more than 10 times higher than that in the myocardium, it can be conjectured that the glycoside fraction of all glycosides specifically bound in the brain is higher than, for example, in the myocardium. Thus differences in the specifically bound glycoside fraction between the various brain areas could also influence global distribution and become evident during determination of the glycoside concentration in the tissue homogenate. Examinations by Williams et al. (1976) in the course of distribution studies with digoxin in various brain tumors also draw attention to the
Naunyn-Schmiedeberg's Arch. Pharmacol. 307 (I979)
significance of the blood-brain barrier and (Na + + K +)-ATPase for the glycoside concentration. In accordance with a significantly higher ATPase-activity in meningiomas than in malignant blastomas (Laws and O'Connor, 1970; •gren et al., 1971), higher digoxin concentrations were measured in the meningiomas with 21.8 _+ 7.3 ng/g wet weight than in the blastomas with 5.7 +_ 5.2 ng/g. The considerable discrepancy between the brain concentrations of fl-methyldigoxin and digoxin gives rise to the question for the extent to which there is a correlation between the degree of organ concentration and the central nervous effect. In a comparative study by Rietbrock et al. (1978) in 196 patients, who were treated with 0.2 mg of fi-methyldigoxin (Lanitop | or 0.3 mg of fi-acetyldigoxin (Novodigal | per os, there was no difference in the number of patients with undesirable extracardial glycoside effects. If one proceeds on the assumption that (Na + + K+)-ATPase is also the receptor for the glycoside effect in the brain, then a glycoside-specific "intrinsic activity" according to Ariens et al. (1964) could explain the missing relation between the degree of glycoside concentration in the brain and the neurotoxicity. Determinations of the binding capacity and binding affinity of the (Na + + K+)-ATPase in the various brain areas of the dog showed no significant differences between digoxin, fl-methyldigoxin and ouabain. Since only a part of the glycoside is specifically bound in the brain, as in the heart, the considerable differences in the lipid solubility of fi-methyldigoxin and digoxin could lead to a different degree of nonspecific binding. Since we do not know the "turn over" rate of the specific glycoside-receptor binding, an exchange with the non-specific binding can not be excluded.
Reference Agren, G., Ponten, J., Ronquist, G., Westermark, B. : Demonstration of an ATPase at the cell surface of intact normal and neoplastic human cells in culture. J. Cell. Physiol. 78, 1 7 1 - 1 7 6 (1971) Akera, T., Larsen, F. S., Brody, T. M.: Correlation of cardiac sodium- and potassium-activated adenosine triphosphatase activity with ouabain induced inotropic stimulation. J. Pharmacol. Exp. Ther. 173, 145-151 (1970) Ariens, E. J., Simonis, A. M., Rossum, J. M. van: Drug-receptor interaction : Interaction of one or more drugs with one receptor system. In: Molecular pharmacology. The mode of action of biologically active compounds. Vol. I. pp. 1 1 9 - 269 (Ariens, E. J., ed.). New York ancl London: Academic Press 1964 Bonting, S. L., Simon, K. A., Hawkins, N. M. : Studies on sodiumpotassium-activated adenosine triphosphatase. I. Quantitative distribution in several tissues of the cat. Arch. Biochem. Biophys. 95, 4 1 6 - 4 2 3 (1961)
J. Kuhlmann et al. : Cardiac Glycosides: Distribution and Affinity to (Na + + K+)-ATPase Dransfeld, H., Greeff, K., Cautius, V.: Die verschiedene Empfindlichkeit der Na § + K+-aktivierten ATPase des Herzund Skelettmuskels gegen k-Strophanthin. Naunyn-Schmiedeberg's Arch. Pharmak. exp. Path. 254, 225-234 (1966) Dutta, S., Marks, B. H., Schoener, E. 1}.: Accumulation of radioactive cardiac glycosides by various brain regions in relation to the dysrhythmogenic effect. Br. J. Pharmacol. 59,'101 - 106 (1977) Erdmann, E., Schoner, W. : Ouabain-receptor interactions in (Na § + K +)-ATPase preparations from different tissues and species. Determination of kinetic constants and dissociation constants. Biochim. Biophys. Acta 30"7, 386-398 (1973) Erdmann, E., Schoner, W.: Eigenschaften des Rezeptors fiir Herzglykoside. Klin. Wschr. 52, 705-718 (1974) Erdmann, E., Philipp, G., Tanner, G. : Ouabain-receptor interactions in (Na § + K§ preparations. A contribution to the problem of nonlinear Scatchard plots. Biochim. Biophys. Acta 455, 287-296 (1976) Ftasch, H., Heinz, N.: Konzentrationen yon Herzglykosiden im Myokard und im Gehirn. Arzneim.-Forsch. 26, 1213-1216 (1976) Glynn, J. M. : The action of cardiac glycosides on ion movements. Pharmacol Rev. 16, 381-407 (1964) Greeff, K. : Zmn Wirkungsmechanismus der Digitalisglykoside. In: Probleme der klinischen Pr~fung herzwirksamer Glykoside. (Greeff, K., Bahrmann, H., Benthe, H. F., Haan D., Kreuzer, H., Hrsg.), pp. 12-24. Darmstadt: D. Steinkopff 1968 Haasis, R., Larbig, D. : Radioimmunologische Bestimmungen der Glykosidkonzentrationen im menschlichen Gehirngewebe. Verh. Dtsch. Ges. Kreisl.-Forsch. 42, 275-277 (1976) Kaiser, F. : Die papierchromatographische Trennung von Herzgiftglykosiden. Chem. Ber. 88, 556-563 (1955) Kuhlmann, J. : Verteilung von Herzglykosiden im Organismus, klinisch-pharmakologische Grundlagen der Therapie. Habilitation work, pp. 78-117, Berlin 1978 Kuhlmann, J., K6tter, V., yon Leitner, E., Arbeiter, G., Schr6der, R., Rietbrock, N. : Concentration of digoxin, methyldigoxin, digitoxin and ouabain in the myocardium of the dog following coronary occlusion. Naunyn-Schmiedeberg's Arch. Pharmacol. 287, 399-411 (1975) Kuhlmann, J., Rietbrock, N., Schnieders, B. : Tissue distribution and elimination of digoxin and methyldigoxin after single and multiple doses in dogs. J. Cardiovasc. Pharmacol. (in press, 1979)
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Laws, E. R., O'Connor, J. S.: ATPase in human brain tumors. J. Neurosurg. 33, 167-171 (1970) Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J. : Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265-275 (1951) Oldendorf, W. H. : Drug penetration of the blood-brain barrier. In: Narcotics and the hypothalamus (Zimmermann, E., George, R., eds.), pp. 213-223. New York: Raven Press 1974 Portius, H. J., Repke, K. : Die Wirkung yon Herzglykosiden auf verschiedene ATPasen des Herzmuskels in AbMngigkeit vom Ionenmilieu. Naunyn-Schmiedeberg's Arch. exp. Path. Pharmak. 243, 335-336 (1962) Repke, K., Portius, H. J. : lJber die Identit/it der IonenpumpenATPase in der Zellmembran des Herzmuskels mit einem Digitalis-Rezeptorenzym. Experientia 19, 452-458 (1963) Rietbrock, N., Oeff, F., Maertin, K., Kuhlmann, J. : Glykosidkonzentrationen im Plasma und Intoxikationsh~iufigkeit nach flMethyldigoxin und fl-Acetyldigoxin unter standardisierten Bedingungen. Eine prospektive Studie. Herz/Kreisl. 10, 267-273 (1978) Scatchard, G. : The attractions of proteins for small molecules and ions. Ann. N.Y. Acad. Sci. 51, 660-671 (1949) Schatzmann, H. J.: Die Bedeutung yon Na + und K + fiir die Herzglykosidwirkung auf die Membran-ATPase des Herzmuskels. Helv. Physiol. Pharmacol. 21, 6 0 - 6 3 (1963) Schoner, W., Ilberg, C. von, Kramer, R., Seubert, W.: On the mechanism of N a +- and K +-stimulated hydrolysis of adenosine triphosphate. 1. Purification and properties of a N +- and K +activated ATPase from ox brain. Eur. J. Biochem. 1, 3 3 4 - 343 (1967) Williams, R., Flanigen, St., Bissett, J., Doherty, J.: Differential uptake of tritiated digoxin in benign and malignant central nervous system neoplasma. Am. J. Med. Sci. 272, 132--137 (1976) Wilson, C. W. M., Brodie, B. B. : The absence of blood-brain barrier from certain areas of the central nervous system. J. Pharmacol. Exp. Ther. 133, 332-334 (1961)
Received September 21, 1978/Accepted February 7, 1979
