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Inhibition of the intestinal absorption of taurocholic acid by a cardiac glycoside
425
PRELIMINARY NOTES
T. SUEMATSU for providing the adrenalectomized rats and to Miss T. MIYAJI for expert technical assistance. This investigation was supported b y a grant for scientific research from the Ministry of Education in Japan. FUMIO WADA Institute for Cancer Research, Osaka University Medical School, TANEAKI HIGASHI Fukushirna-ku, Osaka (Japan) TAMIKO UMEMOTO Y U K I Y A SAKAMOTO 1 C. F. STRITTMATTER AND E. G. BALL, Proc. Natl. dcad. Sci. U.S., 38 (1952) 19. C. F. STRITTMATTER AND E. G. BALL, J. Cellular Comp. Physiol., 43 (1954) 57. a M. KLINGXNBERG, Arch. Biochem. Biophys., 75 (1958) 376. 4 D. GARFINKEL, Arch. Biochem. Biophys., 71 (1957) I I I . 5 D. G-ARFINKEL, Arch. Biochem. Biophys., 77 (1958) 493. 6 p. 8TRITTMATTER AND S. F. VELICK, J. Biol. Chem., 221 (1956) 253. T. OMURA AIVD R. SA:rO, Biochim. Biophys. Acla, 71 (1963) 224. s T. OMORA AND R. SATO, J. Biol. Chem., 237 (1962) PC 1375. 9 R. SATO, Y. NISHIBAYASHI AND T. OMURA, Biochim. Biophys. Acta, 63 (I962) 55o. lo D. Y. COOPER, R. W. ESTABROOK AND O. ROSENTHAL, Federation Proc., 22 (1963) 5o2. 11 C. •ITOMA, H. S. POSNER, H. C. REITZ AND S. UDeNFRIEND, Arch. Biochem. Biophys., 61 (1956 ) 431 . 12 K. C_x.PAUL, H. THEORELL AND A. AKESSON, Acta Chem. Scand., 7 (1953) 1284. 13 R. GATO AND T. OMURA, personal c o m m u n i c a t i o n .
Received J a n u a r y 29th, 1964 Biochim. Biophys. Acla, 86 (1964) 422-425
PN 2 I 012
Inhibition of the intestinal absorption of taurocholic acid by a cardiac 91ycoside I t has been shown that the intestinal active transport of sugars:, amino acids, and pyrimidines 2 is inhibited b y the cardiac glycosides ouabain and thevitin. Evidence suggests that these compounds act at low concentrations (Io -7 M to Io -5 M) b y inhibiting cell membrane adenosine triphosphatase, which is implicated as an energyproducing component in the active transport system 3. PARKINSON AND 0LSON4 have reported inhibition of intestinal absorption of glucose and leucine b y glycocholic and taurocholic acid, and have proposed that cardiac glycosides and conjugated bile acids inhibit active transport b y a similar mechanism s. Since bile acids themselves are actively absorbed b y the intestine s, 7, it was of interest to determine the effect of a cardiac glycoside on their transport. Absorption of sodium taurocholate by sacs of everted guinea-pig intestine was studied using the procedures of LACK AND WEINER 6. Each sac (5-6 cm long) was filled with 2 ml of Krebs-Ringer bicarbonate buffer (pH 7.4) containing 2 mg of glucose per ml, and was incubated in IO ml of buffer for 9 ° rain under 95 % 0 ~ - 5 % CO s at 37 °. Initial taurocholate concentration on both sides of the intestinal wall was 0.2 raM. Absorptive activity was expressed in terms of the final ratio of the tauroeholate concentration of the serosal and mucosal fluids. As previously shown6, ~, bile acid transport is a function of the ileum rather than the more proximal portion of the small intestine (Fig. i). The uptake of taurocholate against a concentration gradient and the marked decrease in this uptake caused by Biochim. Biophys. Acta, 86 (1964) 425-426
426
PRELIMINARY NOTES
the m e t a b o l i c inhibitors 2,4-dinitrophenot a n d i o d o a c e t i c acid, indicate t h a t an a c t i v e t r a n s p o r t process is involved. I n h i b i t i o n of t a u r o c h o l a t e t r a n s p o r t b y o u a b a i n corresponds to the i n h i b i t o r y effects of c a r d i a c glycosides on t r a n s p o r t of other ciasses of substances. The dose response relationship of o u a b a i n on t a u r o c h o l a t e t r a n s p o r t was d e t e r m i n e d , a n d the c o n c e n t r a t i o n of o u a b a i n required to inhibit t r a n s p o r t b y 5o % was c a l c u l a t e d to be a p p r o x i m a t e l y i o -5 M. Since this c o n c e n t r a t i o n is cons i d e r a b l y less t h a n t h a t at which c a r d i a c glycosides uncouple o x i d a t i v e phosphor y l a t i o n (Io -a M), it a p p e a r s t h a t inhibition is n o t the result of genera1 c y t o t o x i c a c t i o n s. Results of p r e l i m i n a r y studies of the kinetics of inhibition indicate t h a t t h e y are of t h e c o m p e t i t i v e t y p e . CONTROLS
(12J
03 03 0'0
3
D
12
10-SM OUABAIN ;z u.
g o= o c~
(s]
f
Z
{O-4M
JODOACETIcIO-3M
ILEUM - -
~
JEJUNUM
Fig. I. Transport of taurocholic acid by sacs of everted guinea-pig intestine. Activity is expressed as the ratio of final tauroeholate concentration of the serosal and mucosa] fluid. Ratio of initial concentrations was i : 1. Inhibitors were added to the mucosal fluid only. The numbers in parentheses indicate the number of sacs studied. The I bars indicate the standard error of the means.
I n h i b i t i o n of t a u r o c h o l a t e t r a n s p o r t b y o n a b a i n , c o m p a r a b l e to inhibition of the active t r a n s p o r t of other water-soluble organic substances b y c a r d i a c glycosides, suggests t h a t t h e s a m e general t r a n s p o r t m e c h a n i s m is involved: The relationship b e t w e e n i n h i b i t i o n b y o u a b a i n a n d t a u r o c h o l a t e of glucose t r a n s p o r t in t h e j e j u n u m ~ a n d i n h i b i t i o n b y o u a b a i n of t a u r o c h o l a t e t r a n s p o r t in t h e ileum is being i n v e s t i g a t e &
Metabolic Diseases Research Laboratories, The Up2"oh~¢Compa~¢y, Katarnazoo, Mich. (U.S.A.)
1 T. T. a L. 4 T. s T. 6 L. 7 I. 8 T.
THOMAS M. PARKINSON
Z. CsXKY, H . G. HARTZOO AND C~. %V. F E R N A L D , A I ~ 4 , dr . Physiol., 2 0 0 ( t 9 6 1 ) 459Z. C S ~ K ¥ , Biochem. Pharmacol., 8 ( I 9 6 I ) 38. 17;. HOKXN AND M. R . HOKUM, Ann. Rev. Biochem., 32 ( I 9 6 3 ) 553. M. PAaKINSO?q AND J . A. OLSON, Life Scie~zces, 6 (I963) 393. M, P;~RKINSON AND J , A. OLSON, L / f e Scieuces, e (I964) lO 7. LACK AND I. IV[. WI~IN~R, Are. f . Physiol., 2 0 0 (1961) 3 1 3 . M. V~'EINIgR AND L. LACK, Are. J. Physiol., 2 0 2 ( I 9 6 2 ) I 5 5 . Z. CSXKY, Biochim. Biophys. Aclc~; 74 ( I 9 6 3 ) I 6 o .
R e c e i v e d March I 3 t h , I964
Biochim. Biophys. Aela, 86 (I964) 425--426
PRELIMINARY NOTES
T. SUEMATSU for providing the adrenalectomized rats and to Miss T. MIYAJI for expert technical assistance. This investigation was supported b y a grant for scientific research from the Ministry of Education in Japan. FUMIO WADA Institute for Cancer Research, Osaka University Medical School, TANEAKI HIGASHI Fukushirna-ku, Osaka (Japan) TAMIKO UMEMOTO Y U K I Y A SAKAMOTO 1 C. F. STRITTMATTER AND E. G. BALL, Proc. Natl. dcad. Sci. U.S., 38 (1952) 19. C. F. STRITTMATTER AND E. G. BALL, J. Cellular Comp. Physiol., 43 (1954) 57. a M. KLINGXNBERG, Arch. Biochem. Biophys., 75 (1958) 376. 4 D. GARFINKEL, Arch. Biochem. Biophys., 71 (1957) I I I . 5 D. G-ARFINKEL, Arch. Biochem. Biophys., 77 (1958) 493. 6 p. 8TRITTMATTER AND S. F. VELICK, J. Biol. Chem., 221 (1956) 253. T. OMURA AIVD R. SA:rO, Biochim. Biophys. Acla, 71 (1963) 224. s T. OMORA AND R. SATO, J. Biol. Chem., 237 (1962) PC 1375. 9 R. SATO, Y. NISHIBAYASHI AND T. OMURA, Biochim. Biophys. Acta, 63 (I962) 55o. lo D. Y. COOPER, R. W. ESTABROOK AND O. ROSENTHAL, Federation Proc., 22 (1963) 5o2. 11 C. •ITOMA, H. S. POSNER, H. C. REITZ AND S. UDeNFRIEND, Arch. Biochem. Biophys., 61 (1956 ) 431 . 12 K. C_x.PAUL, H. THEORELL AND A. AKESSON, Acta Chem. Scand., 7 (1953) 1284. 13 R. GATO AND T. OMURA, personal c o m m u n i c a t i o n .
Received J a n u a r y 29th, 1964 Biochim. Biophys. Acla, 86 (1964) 422-425
PN 2 I 012
Inhibition of the intestinal absorption of taurocholic acid by a cardiac 91ycoside I t has been shown that the intestinal active transport of sugars:, amino acids, and pyrimidines 2 is inhibited b y the cardiac glycosides ouabain and thevitin. Evidence suggests that these compounds act at low concentrations (Io -7 M to Io -5 M) b y inhibiting cell membrane adenosine triphosphatase, which is implicated as an energyproducing component in the active transport system 3. PARKINSON AND 0LSON4 have reported inhibition of intestinal absorption of glucose and leucine b y glycocholic and taurocholic acid, and have proposed that cardiac glycosides and conjugated bile acids inhibit active transport b y a similar mechanism s. Since bile acids themselves are actively absorbed b y the intestine s, 7, it was of interest to determine the effect of a cardiac glycoside on their transport. Absorption of sodium taurocholate by sacs of everted guinea-pig intestine was studied using the procedures of LACK AND WEINER 6. Each sac (5-6 cm long) was filled with 2 ml of Krebs-Ringer bicarbonate buffer (pH 7.4) containing 2 mg of glucose per ml, and was incubated in IO ml of buffer for 9 ° rain under 95 % 0 ~ - 5 % CO s at 37 °. Initial taurocholate concentration on both sides of the intestinal wall was 0.2 raM. Absorptive activity was expressed in terms of the final ratio of the tauroeholate concentration of the serosal and mucosal fluids. As previously shown6, ~, bile acid transport is a function of the ileum rather than the more proximal portion of the small intestine (Fig. i). The uptake of taurocholate against a concentration gradient and the marked decrease in this uptake caused by Biochim. Biophys. Acta, 86 (1964) 425-426
426
PRELIMINARY NOTES
the m e t a b o l i c inhibitors 2,4-dinitrophenot a n d i o d o a c e t i c acid, indicate t h a t an a c t i v e t r a n s p o r t process is involved. I n h i b i t i o n of t a u r o c h o l a t e t r a n s p o r t b y o u a b a i n corresponds to the i n h i b i t o r y effects of c a r d i a c glycosides on t r a n s p o r t of other ciasses of substances. The dose response relationship of o u a b a i n on t a u r o c h o l a t e t r a n s p o r t was d e t e r m i n e d , a n d the c o n c e n t r a t i o n of o u a b a i n required to inhibit t r a n s p o r t b y 5o % was c a l c u l a t e d to be a p p r o x i m a t e l y i o -5 M. Since this c o n c e n t r a t i o n is cons i d e r a b l y less t h a n t h a t at which c a r d i a c glycosides uncouple o x i d a t i v e phosphor y l a t i o n (Io -a M), it a p p e a r s t h a t inhibition is n o t the result of genera1 c y t o t o x i c a c t i o n s. Results of p r e l i m i n a r y studies of the kinetics of inhibition indicate t h a t t h e y are of t h e c o m p e t i t i v e t y p e . CONTROLS
(12J
03 03 0'0
3
D
12
10-SM OUABAIN ;z u.
g o= o c~
(s]
f
Z
{O-4M
JODOACETIcIO-3M
ILEUM - -
~
JEJUNUM
Fig. I. Transport of taurocholic acid by sacs of everted guinea-pig intestine. Activity is expressed as the ratio of final tauroeholate concentration of the serosal and mucosa] fluid. Ratio of initial concentrations was i : 1. Inhibitors were added to the mucosal fluid only. The numbers in parentheses indicate the number of sacs studied. The I bars indicate the standard error of the means.
I n h i b i t i o n of t a u r o c h o l a t e t r a n s p o r t b y o n a b a i n , c o m p a r a b l e to inhibition of the active t r a n s p o r t of other water-soluble organic substances b y c a r d i a c glycosides, suggests t h a t t h e s a m e general t r a n s p o r t m e c h a n i s m is involved: The relationship b e t w e e n i n h i b i t i o n b y o u a b a i n a n d t a u r o c h o l a t e of glucose t r a n s p o r t in t h e j e j u n u m ~ a n d i n h i b i t i o n b y o u a b a i n of t a u r o c h o l a t e t r a n s p o r t in t h e ileum is being i n v e s t i g a t e &
Metabolic Diseases Research Laboratories, The Up2"oh~¢Compa~¢y, Katarnazoo, Mich. (U.S.A.)
1 T. T. a L. 4 T. s T. 6 L. 7 I. 8 T.
THOMAS M. PARKINSON
Z. CsXKY, H . G. HARTZOO AND C~. %V. F E R N A L D , A I ~ 4 , dr . Physiol., 2 0 0 ( t 9 6 1 ) 459Z. C S ~ K ¥ , Biochem. Pharmacol., 8 ( I 9 6 I ) 38. 17;. HOKXN AND M. R . HOKUM, Ann. Rev. Biochem., 32 ( I 9 6 3 ) 553. M. PAaKINSO?q AND J . A. OLSON, Life Scie~zces, 6 (I963) 393. M, P;~RKINSON AND J , A. OLSON, L / f e Scieuces, e (I964) lO 7. LACK AND I. IV[. WI~IN~R, Are. f . Physiol., 2 0 0 (1961) 3 1 3 . M. V~'EINIgR AND L. LACK, Are. J. Physiol., 2 0 2 ( I 9 6 2 ) I 5 5 . Z. CSXKY, Biochim. Biophys. Aclc~; 74 ( I 9 6 3 ) I 6 o .
R e c e i v e d March I 3 t h , I964
Biochim. Biophys. Aela, 86 (I964) 425--426