Bile-salt-dependentand independent choleresis induced by Bsucdome in the rat
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K.Ka-FANI,Y. h f o ~ r ~R. r ~MIURA, , AND S. KANAI Firsr 6,uborufory of C l i ~ ~ iPhy.tiology, c~l T o k y o Metropolifan Insrhtlstc of Gerontology, 35-2, ,%'eakn~c.ho, btuhashi-ku, Tokyo, Buptan-I 73
Received July 2 1, I976
KITANI,K., MORIIA,Y.,~ ~ I U R A W.. , and KAWAI,S. 1977. Bile-salt-dependent and independent choleresis induced by Isucolome in the rat. Can. 9. Physiol. Pharnsacol. 55, 1155-1 161. Choleresis induced by bucolsrne ( RC ) ( I -cyclohexyl-5-rz-b~ity1-2~4,6-1rioxoperhycBropyrimidine) was studied in male Wistar rats. [' 'CIErythritol and rnannitol clearance studies indicated this choleresis to bc of canalicular origin. In 1-h continuo~nsbile coIlection studies. immediately after the interruption of enterohepatic circulation (acute interniption), both bile flaw and bile salt excretion rates were significantly increased in rats administered BC. However, the bile salt excretioia rate fell rather rapidly in IBC-administered rats, while the bile flow rate was fairly constant during this 8-h period. Thus, unlike thc situation in control rats, bile flow rate was not significantly correlated with the bile salt excretion rate in BC-adn-iinistered rats. In rats that had an external bile fistirla open for 16-20 h (chronic interruption of entes-ohepatic circulatiors) the bile flow rate was also significantly increased by BC administration, while the bile salt excretion rate was not changed after BC administration. It is suggested that BC induced bile-salt-independent choleresis in both experimental rat groups (acute and chronic interruption of enterohepatic circulation). Bn addition, BC appeared to increase the bile-salt-dependent fraction of bile in rats with acute interruption of enterohepatic circulation, possibly by mobilizing the bile salt pooled in the intestinal content and (or) intestinal wall. KITANI, K., MOWITA,Y.,MIURA,W . et KANAI,S. 1977. Bile-salt-dependent and independent choleresis induced by \~ueolomein the rat. Can. 5 . Physiol. Pharmacol. 55, 1155-1 161. La cholCr2se induite par le bucolome (BC) ( I-eyc~ohexyl-5-r~-biityl-2~4,6-trioxnperhydropyrirnidine) est Ctudiie chez des rats mgles Wistar. Ides Ctiides d96puration ail ["'@Itrythritol et au mannitol indiquent qaie cette cholCr6se est d'origine canaliculaire. En 1 h d'ktiades portant sur la bile collectie d e fagon continue, ~rninCdiatementaprits l'interruptisn de la circulation enttrohipatique (interruption aiguE). Be dCbit biliaire et 11excr6tion Be sels biliaires sant significativement augrnelst6s chez le rat ayant regu du BC. Cepeasdant, le taux d'excrktion des sels biliaires tonabe rapidement chez les rats ayant recu du BC alors que le dCbit biliaire est constant d~arantcettc pkriode de I h. Bone, contrairerntnt ?i ce qui se passe chez les rats t h o i n s , 1e dtbit biliaire n'est pas en corrtlatian significative avec le dCbit d'excrition des sels biliaires chez les rats ayant requ dm BC. Chez les rats ayant une fistule biliaire externe ouverte pendant 16-20 h (interruption chronique de la circulation ent6rohepatiqale), le &bit biliaire est aussi significativernent augment6 par l'administration de BC, alors qtae le dtbit d9excrCtion des sels biliaires demeure inchangi aprks administration de BC. II est sug&rC que le BC provoqile une cholCsise indtpendante des sels biliaires dans l a deux groupes expCrimentaux (interruption aiguE et cbronique de la circulation entirohkpatique). De plus. le BC sertable augmenter la fraction biliaire dtpendante des sels biliaires chez les rats ayant subi une interruption aigui;: de la circulation ent6rohCpatiqtae, possiblement pas rmobilisation des sels biliaires en rtserve dans Te contenu intestinal oan la paroi intestinale. [Traduit par le journal]
Introduction It has been established that bile flcaw can be classified according- to the site of its secretion, ABBREVIATIONS: BC, bucolome ( I-cyclohexyl-5-nbut~71-2,4,6-tri~~~perhydropyrimidine; ~ / ratio, p bileto-plasma ratio.
namely canalicular bile and distal (ductal or ductular) bile (Forker 1967; Wheeler et ak. 1968). Canalicular bile can be classified further according to its dependence on bile salt excretion into the bile (Bedhelot ef nl. 1970; Boyer and Klatskin 1970; ErHinger et al. 1970).
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Bile-salt-dependent bile is presumed to be secreted by the osmotic force of bile salts actively transported through the canalicular membrane (09Miiile et at. 1969), while another fraction of canaIicuIar bile (bile-saltindependent bile) is considered to be unrelated to bile salt excretion (Erlinger el nb. 1948; Boyer and Klatskin 1970). Thus, the choleresis produced by many drugs has been described according to the site of choleresis (Forker 1967; Wheeler et a&. 1968). and on its dependcnce on bile salt excretion (Berthelot et al. 1940; Erlinger et al. 1970; Erlinger and Bearno~at1973; Barnhart and Csmbes 1974). BC was originally developed as a monsteroid antiinflammatory agent (Senda et al. 1967), but it is also known to induce choleresis in several species of animals including man (Kondo et a&. 1974; Tsumoka et aH. 1945; Kitani et RI. I976a, 1976b). Recently the authors havc demonstrated that BC can induce canalicular choleresis in the dog withorit increasing the bile salt excretion rate (Kitani sf a [ . 1976b). On the other hand, we also previously found that bile salt excretion was increased in rats injected with BC (Kitani et ai. 1976a). Thus, the question arises whether BCinduced cho!eresis is caused by accelerated bile salt excretion (bile-salt-dependent choleresis) or is independent of bile salt excretion (bilesalt-independent choleresis) . The apparent discrepancy in the observed bile salt dependence of BC-induced choleresis between dog and rat in the previous studies could be due to species differences in response to this drug. 'To study ehis problem further, we performed experiments on rats under different conditions with regard to the bile salt pool in the body. Mate~aL5aand Methods Male Wistar rats weighing 180-250 g were used in the study. The rats were kept on commercial rat pellets and tap water until use. Anesthesia was performed by intraperitoneal injection of sodium pentobarbital 44.5 mg/lOO g ) plus additional small doses as needed. Rectal temperature was mntinuousIy monitored and kept between 37 and 38°C by a heating lamp throughoaat the experiments. In all the experiments, BC was administered by intraperitoneal injection of a saline solution of the sodium salt of BC1 (20 nag/lOO g body weight). 'Kindly provided by Takeda Chemical Industries for experimental use.
Calfaliculur Bile Flow E~tirnutiePn Canalicular bile flow was estimated by the technique reported by Forker ( 1967) using radioactive tracers of erythritol and mannnitol. ["6:]Erytlaritol ( 5 pCi ( 1 Ci = 47 GBq)) QNEN. U.S.A.) or [l'C]mannitol (5 pCi) ( N E N , U.S.A.) in saline solution wa\ given intravenoaisly to anesthetized rats through a femoral vein catheter (PE-10 tubing). After a period of 2 h, in which the tracer is assumed to have been evenly distributed in the animals' body water, the sornmon bile duct was cann~alatedQPE-I0 tubing) through an abdominaI incision, and bile collection was kcgila~.BC' administration was performed either 40 min or 2 h before the start of bile collection. In control groups, an amount of saline equal to the R C solution was injected intraperitoncally. In all experiments, four to six cumulative biic samples were collected every 10 rrain. Arterial blood (50-60 p l ) was taken through a femoral artery catheter at the naidpoint of every bile collection period. The B/P efythritol o r anaalnikol concentration ratio was calculated for each period from the specific activities of the pIasma and bile. The biliary clearances of ['T]erythritol and [14C'jmannitol were calculated as the product of bile Wow times the H/P ratio s f these slabstances.
Acute Pnterruprion o j Enterohepcatic CircuEntion Jn separate experiments, cunaulative bile samples were collected every 10 nain for an hour in control and BC-treated rats inamediately after cannulation of the bile da~ct.They were infused with physiologic saline during bile collection. l n thc experimental group, RC was administered 40 min before the bile collection. The bile salt excretion rate was obtained from the bile flow and bile salt concentration. The changes of bile flow were compared between the two g o u p s in reIation to the bilis. salt excretion changcs. Cllroraic It~r~rrldption o f Ent~roh~parb'c Circ~alatiotz In another group of rats, an external common bile duct fistula was constructed under pentobarbital anesthesia. This fistula was kept open for 16-20 h. During this period, rats were kept awake and allowed foczd and drinking water. The rats were again anesthetized. and the bile was collected every 461 min. After a baseline period of I h, BC was adrninistcred and bile coIlection was continued for another 24 h. During ehis bile collection period, an appropriate amount of physiologic saline was infalsed throaagh a venous catheter to compensate for the loss of body water. Bile flow and bile salt excretion rates were compared before and after BC adnainistration. /1 truly tica 1 Prc?c.edures
Bile samples were weighed and bile flow rate was ot~tainedassuming the specific gravity of bile to be 1.0. Twenty nlicrolitres of bile or plasma was mixed with 10 ml sf scintillator (Instagel, Packard: Downers Grove, I L ) . The radioactivity was counted by a liquid sciniilllation counter (ABoka, Tokyo). An automatic external standard was used for quench correction. The bile salt concentration in bile was determined enzymatically by using 3a-hydroxysteroid dehydrogenase
KITANH ET Ak.
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TABLE I . Camalicular clearances of ['CCqthritol and [L4G]mannitol in control and treated rats
Bile WOW rate, y l i n ~ i nper 100 g
Erythritol study Control rats (24)" BC ratst (21)
14.13f H . 5 5 $
Mannitol study Comtrol rats (19) BC rats4i (20)
13.31 rt_l.Q5$
8.8690.75
8.3058.63
B/P ratio
Camalicular clearance, prlitnin per 100 g
1.0050.08 I .O% +0.0!2s
8.94k1.20 14.04k 1.41%
0.83 k0.061) 0.84kO.10s
11.13+1.33$
*Number in parenthesis is the numbcr of' bile samples obtained from five to t B C was ntirninistered 40 nlin before the start of bile collection.
6.95 k0.87 six rats.
%Significantlydifferent from respective control value ( p i 0.05). $Not significantly ditTcrcrlt from respective ccmtrol v;ilue ( p > 0.05). (ISignifie~ntly tlifi'erent from erythritol B / P ratio in ccr~trolrats ( p < 0.05). THC was administered L20 rnin before the start of bile collection. W C I I EAll : values arc nlcan SJ.9.
+
(EG 1.1.I.SBbB (Worthington Riochern., W9, U.S.A.). according PO the method of Hurlock and TaIalay (1957) with the modification of Paumgarfner t.? a / .
(1971). Steatisticui A I K N ~ . Y . G ~ S
The remits were atiiaByzcd by Student's r test. The difference between two groups was considered to be significant at a P !eve1 of less than 0.05.
Results
Cnnabicubar Clearance Study The results of the canalicular clearance study are sumlnarized in Table 1. The B/B ratio in the control erythritol study ( I .00 + 0.08) was slightly but significantly higher than the mannitol B/P ratio (0.83 a 0.06). In BC-atfministered rats the bile Aow increased about 5060% in the erythritol study (48-106) rnin after BC administration) as well as in the mannits1 study 120-1 80 man after BC administration), while the B/P ratios did not diEer significantly from their respective control values. Consep quently, the ca~malicular clearances of both erythritol and marnnitol were significantly increased by BC administration almost in proportion to the increase of bile flow. Acute Interruption of Enterohepatic Circula-
tion Figure 1 shows the changes of bile flow, bile salt concentratiotm, and excretion rates in rats with continuous bile sampling for 60 min immediately after the common bile duct cawnulation. In the control rats, the bile flow rate remained relatively constant throughout the
hour (Fig. BA). The rats given BC 40 rnin before bile collection showed a considerable increase of bile flow, which was relatively constant and continued to be high throughout the observation period (first sample, 1 2 -62 k 0.96 pI/min per 100 g; sixth sample, J 1.28 k 0.86 pl/min per 100 g; Fig. 1B). The constantly high bile flow rate was observed not only on the average in this group, but even in individual rats during the observation period. Moreover, it usually continued for another hour and then began to decline very gradually. The initial bile salt excretion rate in Be-treated rats was 60% higher than the control value (first sample, 0.24 2 0.08 prnol/rnin per 100 g in control rats and 0.39 8.05 pmo%/rnin per 100 g in BC-administered rats), but decreased more rapidly than that in control rats during the experimental period. However, at the end of the experiment (60 min after the start of bile collection, and 100 man after BC administration) the bile salt excretion rate was still significantly higher than the control value at the corresponding time period (sixth sample; 0.15 2 0.05 pmsl/min per 100 g in control rats and 0.20 -t- 0.85 prnol/rnin per 400 g in BC-administered rats, P < 0.05). In Fig. 2, the bile flow rate is replotted against the bile salt excretion rate in each bile sample obtained from control and BC-adrninistered rat groups. In the bile salnples obtained from the control rats (Fig. 2, closed circles), there is a significant linear relation expressed in the following quation: Y = 11.2X 5.37 (n 41, r 8.861, where Y is a bile flow rate
-
+
;
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CAN. J. PHYSIOL. PHARMACOL. VOL. 5 5 , 1993
FIG.1. Sequential changes in bile flow and bile salt excretion rate in control ( A ) and BCadministered ( B ) rats with acute interruption of enterohepatic circulation. Each value represents the ancan obtained from seven rat experiments. Vertical bar indicates B SD.
FIG.2. The relation between bile flow and bile salt excretion rates obtained from bile samples in control and BC-administered rats with acute interruption of enterohepatic circulation (data for Fig. 1 ) .
(pl/min per It 80 g ) and X is a bile salt excretion rate (pmo%/minper 100 g ) . There was a significant relationship between bile flow rate and bile salt excretion rate in control rats, but in rats given BC no such reIationship was present (Fig. 2, open circles, n = 42, r = 0.84). Chronic Interruption of Enterohepatic Circulation
The bile flow rate and the bile salt excretion rate in rats with an external bile fistula kept open for 16-20 h before BC administration
are summarized in Fig. 3. The average bile flow before BC administration was 4.41 2 1.08 pl/min per 100 g, which was significantly increased 30 min after BC administration to 7.29 & 1.41 pl/min per I00 g ( n = 8, P < 0.01). The bile salt excretion rate before BC adrninistraticzn was 23.1 -t- 4.7 nrnoI/nnin per 100 g, which was about 10 times less than the value in control rats with acute interruption of enterohepatic circulation. After BC administration, there was no significant change observed in the bile salt excretion rate (21.0 t- 5.11 5
BC (20 mg /I00 g body weight, ip]
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I
Time after BueoIorne injection ( min )
FIG.3. Bile flow and bile salt excretion rates before and after B C adminaistratisna in rats with a bile fistula kept open for 16-20 h (chronic interruption of enterohepatic circulation ). Each value represents the mean s f eight rat experiments. Vertical bar indicates 1 SD.
nmol/min per 100 g, P > 0.05) in spite of the significant increase in the bile flow rate.
the B/P ratios of erytkritol and rnannitol were unchanged by BC-induced clnoleresis. Thus, the biliary clearances of both erythritol and mannit01 were significantly increased by BC Discussion administration. Assuming that the principle of The theoretical basis for the use of erythritol the inert substance clearance method is also or mannitol for canalicular bile flow estimation valid in the rat, the present data could be interhas been established in the guinea pig by Forker preted as showing that the BC-induced (1967) and in the dog by Wheeler et al. ckoleresis is due to an increase of canalicular (1968). This method is based on the assump- bile. This is in agreement with our observations tion that these substances are impermeable to in the dog (Kitani et al. 1976b) and in the the bile duct (or ductules). In these animals, guinea pig (Tsmruoka et a%. 8975), in which secretin induces a significant cholcresis without the increase of bile flow was mostly due to an increasing clearances of erythritol or mannitol, increase in canalicular biIe production. which has been accepted as an experimental As can be seen from Fig. 8, the choleresis proof for this assumption. In the rat, there is induced by BC in rats with acute interruption no direct evidence for the impermeability of of enterohepatic circulation is accompanied by these substances in the ductules (or ducts), an increase in bile salt excretion, and thus apbecause secretin does not cause a significant pears to be bile salt dependent. However, choIeresis in this species (Forker et a%.8967). while the biIe flow continued to be high, the However, based on the assumption of im- bile salt concentration and excretion fell rather permeability, several studies (Forker et a%. rapidly in BC-administered rats. This could be 1967; Berthelot et a%. 1970; Boyer and interpreted as BC-stimulated bile-salt-dependKlatskin 1970; Klaassen 1971) have reported ent bile flow decreasing, while BC-stimulated the biliary clearances of these substances in bile-salt-independent bile flow is increasing the rat as an expression of canalicular bile with time. The absence of the significant corproduction. Mannitol clearance is reported to relation between the bile flow rate and bile salt be slightly lower than erythritol clearance in excretion rate in BC-administered rats (Fig. 2, many species of animals (Forker et a%. 1967; open circles) may be due to this unsteady Forker 1968), and this has been explained by choleretic condition. the difference in molecular size (Forker 8968). Since the total bile flow rate was relatively In the present study, the control value of the constant in BC-administered rats during the obB/P ratio was also slightly but significantly servation period, it is possible that some different for these two substances. However, mechanism is working to maintain the increased
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1160
CAN. B.
PHYSIOL. PNARMACOL.
bile flow rate constant for a considerable time period, while the contribution of two (bils-saltdependent and independent) bile fractions is changing with time. 111 rats with a bile fistula open for 16-20 h (chronic interruption of enterohepatic circulation). the bile Wow was also significantly increased after BC adnministratioa, but the bile salt excretion was not increased (Fig. 3). Thus, the choleresis observed in this experiment would seem to be totally bile salt independent, This agrees with the authors9 previous study in the dog (Kitani et wH. 1976b) in which a canalicular choleresis of the bile-salt-independent type was demonstrated for BC. Therefore, BG appears to have a choleretic effect of the bile-salt-independent type in both rats and dogs. However, the bile salt excretion and probably the bile-salt-dependent bile flow were increased by BC in rats with acute interruption of enterohepatic circulation (Fig. 1B) . This difference (Kitani et a&. 19'76a9 1976b)l is probably due to different experimental conditions rather than interspecies variation. It laas been established that a chronic interruption of enterohepatic circulation of bile salt for 1620 h is long enough to eliminate the bile salt pool in the rat (Thompson and Vars 1953, 1954; Eriksson 1957a, 1957b) and that the bile salt in the bile in such an animal is mostly newly synthetized bile salt from the liver. In the present study, the bile salt excretion rate in rats with chronic interruption of enterohepatic circulation was 10 times less than that observed in the control rats with acute interruption where there is a minimal loss of the bile salt pool. Thus, the increase in the bile salt excretion in the bile iaa BC-administered rats with acute interruption of enterohepatic circulation might be most likely due to tlne mobilization of a bile salt pool in thc intestinal content and in the intestinal wall of these animals. The rapid decline of bile salt excretion after an initial rise in BC-administered rats may be due to the rapid washout of the pool. On the other hand, in rats with chronic interruption of enterohepatic circulation, bile salt excretion may not be increased by BC because this bile salt pool is already depleted and thus cannot be mobilized. The mechanism for this hypothesized mobilization of a bile salt pool by BC was not elucidated in the present study.
VOL. 5 5 , 1977
Hcrwever, an increase in hepatic blood flow was suggested to increase the bile-salt-dependent bile (Preisig 1972) by augmenting the hepatic load of circulating bile salts. In this regard, we recently demonstrated a 50% increase in the small intestinal fraction of cardiac output in rats by BC, as determined by the radioisotopelabeled microsphere method (Miura and Kitani 1976). This might accelerate the intestinal absorption of bile salt pool and (or) the transfer crf bile salt pool in the intestinal wall to the liver, thus contributing to the increase in bile salt excretion. This study suggests that BC induces both a bile-salt-dependent and independent choleresis. This contrasts with other types of choleresis reported in the past, which were classified either as bile-salt-dependent or independent. For example, phenobarbital (Berthelot et ak. 1 976; Klaassen 1971 ), SC 2644 (Gibson and Farker 1974), or theophylline (Erlinger and Dumont 1973; Barnhart and Coslbes 1974) have been shown to induce canalicular choleresis of tlae bile-salt-independent type, while bile salt administration is known to produce bile-salt-dependent choleresis ( O'MiiilIe et ak. 1 969; Bcrthelot et ak. 1970; Erlinger and Dumont 1973). Recently, however, Gardiner et a&. (1975 ) reported that secretin increased bile salt excretion in the bile in monkeys in which the bile salt pool was maintained during the experimental period. Secretin was generally believed to increase only noncana'ticular (distal bile duct) bile Wow independent of bile salt excretion. Thus, BG and secretin may be classified as a bile-salt-dependent and independent choleretic, although there is a diEerence between these two in the site of bile-saltindependent choleresis (canalicular and distal bile duct). The biliary excretion of a drug s r its metabolites can cause an osmotic driving force increasing bile flow independent of bile salt excretion (Klaassen and Fitzgerald 1974). In this regard, one should consider the biliarqr excretion of BC or its metabolites, as a possible cause of BG-induced choleresis. However, the biliary excretion of BC is reported to be very small, i.e., less than 2% of the dose in the rabbit in the first 8 h (Mima e&al, 1965 ) . Only 6% was recovered in the feces, while 72% was recovered in the urine sf rats in 96 kt, when
KITANI EF AL.
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studied by using [2-14C]BC (Yashiki 1971). This problem is currently under reinvestigation in our laboratory in relation to the choleretic activity of BC.
1161
1976a. Increased bile salt excretion by bucolome. Kanzcd (Acta Hepatol. Jpn.), 87, 224. -- 1976b. The effect of bucolome on the candicular bile formation and sulfobron~ophthaleintransport r n a x i n ~ ~ ~inr ntbe d o g Biocbem. Pharmacol. 25, 3 377-1381, Acknow!edgments KLAASSEN, C. D. 1971. Studies on the increased biliary flow produced by phenobarbital in rats. J. PharThe authors are deeply indebted to Dr. macol. Exp. Ther. 176, 743-751. C. D. Klaassen and Mr. J. Ek for their careful KI AASSFN. C. D., and $;ITZGERALD, T. J. 1974. Metabreview of the manuscript* olistn 2nd biliary excretion of ethacrynic acid. J* Pharmacel. Exp. There 1917548-556. E. 1974. B A R N H ~ R T , J . L.. and C o ~ u ~ B. s , 1974. Effect of KONDO,T., MIYOSIIH,K., and HW~QHATA, Cholesetic eflcct of bucolome. Kanzo (Acts Hepatole theophylline on hepatic excretory function. Am* J * Jpm.) 15. 670-675. Physiol. 227. 194-199. ~ ~ I M H., A , ~ ~ A T S U Z A KT., I , TERADA, K.? and KONDQ.T. ~ E X T I ~ E I C ) I ' , ERLINGER, S . , DKUMEAUX, D., and 1965. Gastrointestinal absorption and excretion of PRFALJX, As M. 1970. Mechanism of phenobarbital5 -n-butyl- 1-cyclohexyI-2,4,6-trioxoperhydropyrinninduced hyperchoieresis in the rat. Am. J e Physiol. dine. Annai. Rep. Takeda Res. Lab. 24. 9-24. 21 9, 809-8 12. BoYI-R, J * I>., and K r ATSKIN, G. 1970. Canalicular bile hfnwxA, R., and KITANI, K . 1976. Study on the splanchflow and bile secretory pressure. Evidence for 21 nic circl~lationin the rat using microspheres labeled non-bile salt dependent fraction in the isolated rat with radioisotopes. The effects of pentobarbital and liver. Gahtrcxmterology, 59. 853-859* bucolome. Jpn. 9.Nucl. Med. 13, 707. ERIKSSON, S. 1957a. Biliary excretion of bile acids and O ' ~ ~ . ~ H IE. 437-439. and guinea pigs- Jpn- J . Nucl. Med. 12, 517. GIBSON, G. F... and FORKER, E. L. 1974. Canzilic~ilar bile flow and bromosulfophthalein transport maxi- W H E E B ER, M . O., ROSS, E. D., and BRADLEY, S. E. 196g0 Canalicular bile production in dogs. Am, Je n-tw-ri: The effect of a bile salt-independent choleretic, Physiol. 214, 866-874. SC-2644. Gastroenterology, 46, 1046-1053. H e r ~ a o c K ,H.. and TALAI AY, B. 1957. Principles of the YASIHKH, T. 1971. Metabolic fate and the pharmacokinetics of 5-PI-butyl-1-cycI~~hexyl-2,4,6-trioxo-perhyenzymatic ineasurement of steroids. 9. Biol. C h e n 227, 37-52. dropyrimidine (BCP) in m a n J. Takeda Res. Lab. KITANI, K . , I'V~IURA~ R., KANAI.S., and MORITA? Yo 30,8011-840.
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K.Ka-FANI,Y. h f o ~ r ~R. r ~MIURA, , AND S. KANAI Firsr 6,uborufory of C l i ~ ~ iPhy.tiology, c~l T o k y o Metropolifan Insrhtlstc of Gerontology, 35-2, ,%'eakn~c.ho, btuhashi-ku, Tokyo, Buptan-I 73
Received July 2 1, I976
KITANI,K., MORIIA,Y.,~ ~ I U R A W.. , and KAWAI,S. 1977. Bile-salt-dependent and independent choleresis induced by Isucolome in the rat. Can. 9. Physiol. Pharnsacol. 55, 1155-1 161. Choleresis induced by bucolsrne ( RC ) ( I -cyclohexyl-5-rz-b~ity1-2~4,6-1rioxoperhycBropyrimidine) was studied in male Wistar rats. [' 'CIErythritol and rnannitol clearance studies indicated this choleresis to bc of canalicular origin. In 1-h continuo~nsbile coIlection studies. immediately after the interruption of enterohepatic circulation (acute interniption), both bile flaw and bile salt excretion rates were significantly increased in rats administered BC. However, the bile salt excretioia rate fell rather rapidly in IBC-administered rats, while the bile flow rate was fairly constant during this 8-h period. Thus, unlike thc situation in control rats, bile flow rate was not significantly correlated with the bile salt excretion rate in BC-adn-iinistered rats. In rats that had an external bile fistirla open for 16-20 h (chronic interruption of entes-ohepatic circulatiors) the bile flow rate was also significantly increased by BC administration, while the bile salt excretion rate was not changed after BC administration. It is suggested that BC induced bile-salt-independent choleresis in both experimental rat groups (acute and chronic interruption of enterohepatic circulation). Bn addition, BC appeared to increase the bile-salt-dependent fraction of bile in rats with acute interruption of enterohepatic circulation, possibly by mobilizing the bile salt pooled in the intestinal content and (or) intestinal wall. KITANI, K., MOWITA,Y.,MIURA,W . et KANAI,S. 1977. Bile-salt-dependent and independent choleresis induced by \~ueolomein the rat. Can. 5 . Physiol. Pharmacol. 55, 1155-1 161. La cholCr2se induite par le bucolome (BC) ( I-eyc~ohexyl-5-r~-biityl-2~4,6-trioxnperhydropyrirnidine) est Ctudiie chez des rats mgles Wistar. Ides Ctiides d96puration ail ["'@Itrythritol et au mannitol indiquent qaie cette cholCr6se est d'origine canaliculaire. En 1 h d'ktiades portant sur la bile collectie d e fagon continue, ~rninCdiatementaprits l'interruptisn de la circulation enttrohipatique (interruption aiguE). Be dCbit biliaire et 11excr6tion Be sels biliaires sant significativement augrnelst6s chez le rat ayant regu du BC. Cepeasdant, le taux d'excrktion des sels biliaires tonabe rapidement chez les rats ayant recu du BC alors que le dCbit biliaire est constant d~arantcettc pkriode de I h. Bone, contrairerntnt ?i ce qui se passe chez les rats t h o i n s , 1e dtbit biliaire n'est pas en corrtlatian significative avec le dCbit d'excrition des sels biliaires chez les rats ayant requ dm BC. Chez les rats ayant une fistule biliaire externe ouverte pendant 16-20 h (interruption chronique de la circulation ent6rohepatiqale), le &bit biliaire est aussi significativernent augment6 par l'administration de BC, alors qtae le dtbit d9excrCtion des sels biliaires demeure inchangi aprks administration de BC. II est sug&rC que le BC provoqile une cholCsise indtpendante des sels biliaires dans l a deux groupes expCrimentaux (interruption aiguE et cbronique de la circulation entirohkpatique). De plus. le BC sertable augmenter la fraction biliaire dtpendante des sels biliaires chez les rats ayant subi une interruption aigui;: de la circulation ent6rohCpatiqtae, possiblement pas rmobilisation des sels biliaires en rtserve dans Te contenu intestinal oan la paroi intestinale. [Traduit par le journal]
Introduction It has been established that bile flcaw can be classified according- to the site of its secretion, ABBREVIATIONS: BC, bucolome ( I-cyclohexyl-5-nbut~71-2,4,6-tri~~~perhydropyrimidine; ~ / ratio, p bileto-plasma ratio.
namely canalicular bile and distal (ductal or ductular) bile (Forker 1967; Wheeler et ak. 1968). Canalicular bile can be classified further according to its dependence on bile salt excretion into the bile (Bedhelot ef nl. 1970; Boyer and Klatskin 1970; ErHinger et al. 1970).
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CAN. b. P W S I O L . PHARMACOL. V 8 L . 5 5 , I977
Bile-salt-dependent bile is presumed to be secreted by the osmotic force of bile salts actively transported through the canalicular membrane (09Miiile et at. 1969), while another fraction of canaIicuIar bile (bile-saltindependent bile) is considered to be unrelated to bile salt excretion (Erlinger el nb. 1948; Boyer and Klatskin 1970). Thus, the choleresis produced by many drugs has been described according to the site of choleresis (Forker 1967; Wheeler et a&. 1968). and on its dependcnce on bile salt excretion (Berthelot et al. 1940; Erlinger et al. 1970; Erlinger and Bearno~at1973; Barnhart and Csmbes 1974). BC was originally developed as a monsteroid antiinflammatory agent (Senda et al. 1967), but it is also known to induce choleresis in several species of animals including man (Kondo et a&. 1974; Tsumoka et aH. 1945; Kitani et RI. I976a, 1976b). Recently the authors havc demonstrated that BC can induce canalicular choleresis in the dog withorit increasing the bile salt excretion rate (Kitani sf a [ . 1976b). On the other hand, we also previously found that bile salt excretion was increased in rats injected with BC (Kitani et ai. 1976a). Thus, the question arises whether BCinduced cho!eresis is caused by accelerated bile salt excretion (bile-salt-dependent choleresis) or is independent of bile salt excretion (bilesalt-independent choleresis) . The apparent discrepancy in the observed bile salt dependence of BC-induced choleresis between dog and rat in the previous studies could be due to species differences in response to this drug. 'To study ehis problem further, we performed experiments on rats under different conditions with regard to the bile salt pool in the body. Mate~aL5aand Methods Male Wistar rats weighing 180-250 g were used in the study. The rats were kept on commercial rat pellets and tap water until use. Anesthesia was performed by intraperitoneal injection of sodium pentobarbital 44.5 mg/lOO g ) plus additional small doses as needed. Rectal temperature was mntinuousIy monitored and kept between 37 and 38°C by a heating lamp throughoaat the experiments. In all the experiments, BC was administered by intraperitoneal injection of a saline solution of the sodium salt of BC1 (20 nag/lOO g body weight). 'Kindly provided by Takeda Chemical Industries for experimental use.
Calfaliculur Bile Flow E~tirnutiePn Canalicular bile flow was estimated by the technique reported by Forker ( 1967) using radioactive tracers of erythritol and mannnitol. ["6:]Erytlaritol ( 5 pCi ( 1 Ci = 47 GBq)) QNEN. U.S.A.) or [l'C]mannitol (5 pCi) ( N E N , U.S.A.) in saline solution wa\ given intravenoaisly to anesthetized rats through a femoral vein catheter (PE-10 tubing). After a period of 2 h, in which the tracer is assumed to have been evenly distributed in the animals' body water, the sornmon bile duct was cann~alatedQPE-I0 tubing) through an abdominaI incision, and bile collection was kcgila~.BC' administration was performed either 40 min or 2 h before the start of bile collection. In control groups, an amount of saline equal to the R C solution was injected intraperitoncally. In all experiments, four to six cumulative biic samples were collected every 10 rrain. Arterial blood (50-60 p l ) was taken through a femoral artery catheter at the naidpoint of every bile collection period. The B/P efythritol o r anaalnikol concentration ratio was calculated for each period from the specific activities of the pIasma and bile. The biliary clearances of ['T]erythritol and [14C'jmannitol were calculated as the product of bile Wow times the H/P ratio s f these slabstances.
Acute Pnterruprion o j Enterohepcatic CircuEntion Jn separate experiments, cunaulative bile samples were collected every 10 nain for an hour in control and BC-treated rats inamediately after cannulation of the bile da~ct.They were infused with physiologic saline during bile collection. l n thc experimental group, RC was administered 40 min before the bile collection. The bile salt excretion rate was obtained from the bile flow and bile salt concentration. The changes of bile flow were compared between the two g o u p s in reIation to the bilis. salt excretion changcs. Cllroraic It~r~rrldption o f Ent~roh~parb'c Circ~alatiotz In another group of rats, an external common bile duct fistula was constructed under pentobarbital anesthesia. This fistula was kept open for 16-20 h. During this period, rats were kept awake and allowed foczd and drinking water. The rats were again anesthetized. and the bile was collected every 461 min. After a baseline period of I h, BC was adrninistcred and bile coIlection was continued for another 24 h. During ehis bile collection period, an appropriate amount of physiologic saline was infalsed throaagh a venous catheter to compensate for the loss of body water. Bile flow and bile salt excretion rates were compared before and after BC adnainistration. /1 truly tica 1 Prc?c.edures
Bile samples were weighed and bile flow rate was ot~tainedassuming the specific gravity of bile to be 1.0. Twenty nlicrolitres of bile or plasma was mixed with 10 ml sf scintillator (Instagel, Packard: Downers Grove, I L ) . The radioactivity was counted by a liquid sciniilllation counter (ABoka, Tokyo). An automatic external standard was used for quench correction. The bile salt concentration in bile was determined enzymatically by using 3a-hydroxysteroid dehydrogenase
KITANH ET Ak.
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TABLE I . Camalicular clearances of ['CCqthritol and [L4G]mannitol in control and treated rats
Bile WOW rate, y l i n ~ i nper 100 g
Erythritol study Control rats (24)" BC ratst (21)
14.13f H . 5 5 $
Mannitol study Comtrol rats (19) BC rats4i (20)
13.31 rt_l.Q5$
8.8690.75
8.3058.63
B/P ratio
Camalicular clearance, prlitnin per 100 g
1.0050.08 I .O% +0.0!2s
8.94k1.20 14.04k 1.41%
0.83 k0.061) 0.84kO.10s
11.13+1.33$
*Number in parenthesis is the numbcr of' bile samples obtained from five to t B C was ntirninistered 40 nlin before the start of bile collection.
6.95 k0.87 six rats.
%Significantlydifferent from respective control value ( p i 0.05). $Not significantly ditTcrcrlt from respective ccmtrol v;ilue ( p > 0.05). (ISignifie~ntly tlifi'erent from erythritol B / P ratio in ccr~trolrats ( p < 0.05). THC was administered L20 rnin before the start of bile collection. W C I I EAll : values arc nlcan SJ.9.
+
(EG 1.1.I.SBbB (Worthington Riochern., W9, U.S.A.). according PO the method of Hurlock and TaIalay (1957) with the modification of Paumgarfner t.? a / .
(1971). Steatisticui A I K N ~ . Y . G ~ S
The remits were atiiaByzcd by Student's r test. The difference between two groups was considered to be significant at a P !eve1 of less than 0.05.
Results
Cnnabicubar Clearance Study The results of the canalicular clearance study are sumlnarized in Table 1. The B/B ratio in the control erythritol study ( I .00 + 0.08) was slightly but significantly higher than the mannitol B/P ratio (0.83 a 0.06). In BC-atfministered rats the bile Aow increased about 5060% in the erythritol study (48-106) rnin after BC administration) as well as in the mannits1 study 120-1 80 man after BC administration), while the B/P ratios did not diEer significantly from their respective control values. Consep quently, the ca~malicular clearances of both erythritol and marnnitol were significantly increased by BC administration almost in proportion to the increase of bile flow. Acute Interruption of Enterohepatic Circula-
tion Figure 1 shows the changes of bile flow, bile salt concentratiotm, and excretion rates in rats with continuous bile sampling for 60 min immediately after the common bile duct cawnulation. In the control rats, the bile flow rate remained relatively constant throughout the
hour (Fig. BA). The rats given BC 40 rnin before bile collection showed a considerable increase of bile flow, which was relatively constant and continued to be high throughout the observation period (first sample, 1 2 -62 k 0.96 pI/min per 100 g; sixth sample, J 1.28 k 0.86 pl/min per 100 g; Fig. 1B). The constantly high bile flow rate was observed not only on the average in this group, but even in individual rats during the observation period. Moreover, it usually continued for another hour and then began to decline very gradually. The initial bile salt excretion rate in Be-treated rats was 60% higher than the control value (first sample, 0.24 2 0.08 prnol/rnin per 100 g in control rats and 0.39 8.05 pmo%/rnin per 100 g in BC-administered rats), but decreased more rapidly than that in control rats during the experimental period. However, at the end of the experiment (60 min after the start of bile collection, and 100 man after BC administration) the bile salt excretion rate was still significantly higher than the control value at the corresponding time period (sixth sample; 0.15 2 0.05 pmsl/min per 100 g in control rats and 0.20 -t- 0.85 prnol/rnin per 400 g in BC-administered rats, P < 0.05). In Fig. 2, the bile flow rate is replotted against the bile salt excretion rate in each bile sample obtained from control and BC-adrninistered rat groups. In the bile salnples obtained from the control rats (Fig. 2, closed circles), there is a significant linear relation expressed in the following quation: Y = 11.2X 5.37 (n 41, r 8.861, where Y is a bile flow rate
-
+
;
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CAN. J. PHYSIOL. PHARMACOL. VOL. 5 5 , 1993
FIG.1. Sequential changes in bile flow and bile salt excretion rate in control ( A ) and BCadministered ( B ) rats with acute interruption of enterohepatic circulation. Each value represents the ancan obtained from seven rat experiments. Vertical bar indicates B SD.
FIG.2. The relation between bile flow and bile salt excretion rates obtained from bile samples in control and BC-administered rats with acute interruption of enterohepatic circulation (data for Fig. 1 ) .
(pl/min per It 80 g ) and X is a bile salt excretion rate (pmo%/minper 100 g ) . There was a significant relationship between bile flow rate and bile salt excretion rate in control rats, but in rats given BC no such reIationship was present (Fig. 2, open circles, n = 42, r = 0.84). Chronic Interruption of Enterohepatic Circulation
The bile flow rate and the bile salt excretion rate in rats with an external bile fistula kept open for 16-20 h before BC administration
are summarized in Fig. 3. The average bile flow before BC administration was 4.41 2 1.08 pl/min per 100 g, which was significantly increased 30 min after BC administration to 7.29 & 1.41 pl/min per I00 g ( n = 8, P < 0.01). The bile salt excretion rate before BC adrninistraticzn was 23.1 -t- 4.7 nrnoI/nnin per 100 g, which was about 10 times less than the value in control rats with acute interruption of enterohepatic circulation. After BC administration, there was no significant change observed in the bile salt excretion rate (21.0 t- 5.11 5
BC (20 mg /I00 g body weight, ip]
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I
Time after BueoIorne injection ( min )
FIG.3. Bile flow and bile salt excretion rates before and after B C adminaistratisna in rats with a bile fistula kept open for 16-20 h (chronic interruption of enterohepatic circulation ). Each value represents the mean s f eight rat experiments. Vertical bar indicates 1 SD.
nmol/min per 100 g, P > 0.05) in spite of the significant increase in the bile flow rate.
the B/P ratios of erytkritol and rnannitol were unchanged by BC-induced clnoleresis. Thus, the biliary clearances of both erythritol and mannit01 were significantly increased by BC Discussion administration. Assuming that the principle of The theoretical basis for the use of erythritol the inert substance clearance method is also or mannitol for canalicular bile flow estimation valid in the rat, the present data could be interhas been established in the guinea pig by Forker preted as showing that the BC-induced (1967) and in the dog by Wheeler et al. ckoleresis is due to an increase of canalicular (1968). This method is based on the assump- bile. This is in agreement with our observations tion that these substances are impermeable to in the dog (Kitani et al. 1976b) and in the the bile duct (or ductules). In these animals, guinea pig (Tsmruoka et a%. 8975), in which secretin induces a significant cholcresis without the increase of bile flow was mostly due to an increasing clearances of erythritol or mannitol, increase in canalicular biIe production. which has been accepted as an experimental As can be seen from Fig. 8, the choleresis proof for this assumption. In the rat, there is induced by BC in rats with acute interruption no direct evidence for the impermeability of of enterohepatic circulation is accompanied by these substances in the ductules (or ducts), an increase in bile salt excretion, and thus apbecause secretin does not cause a significant pears to be bile salt dependent. However, choIeresis in this species (Forker et a%.8967). while the biIe flow continued to be high, the However, based on the assumption of im- bile salt concentration and excretion fell rather permeability, several studies (Forker et a%. rapidly in BC-administered rats. This could be 1967; Berthelot et a%. 1970; Boyer and interpreted as BC-stimulated bile-salt-dependKlatskin 1970; Klaassen 1971) have reported ent bile flow decreasing, while BC-stimulated the biliary clearances of these substances in bile-salt-independent bile flow is increasing the rat as an expression of canalicular bile with time. The absence of the significant corproduction. Mannitol clearance is reported to relation between the bile flow rate and bile salt be slightly lower than erythritol clearance in excretion rate in BC-administered rats (Fig. 2, many species of animals (Forker et a%. 1967; open circles) may be due to this unsteady Forker 1968), and this has been explained by choleretic condition. the difference in molecular size (Forker 8968). Since the total bile flow rate was relatively In the present study, the control value of the constant in BC-administered rats during the obB/P ratio was also slightly but significantly servation period, it is possible that some different for these two substances. However, mechanism is working to maintain the increased
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1160
CAN. B.
PHYSIOL. PNARMACOL.
bile flow rate constant for a considerable time period, while the contribution of two (bils-saltdependent and independent) bile fractions is changing with time. 111 rats with a bile fistula open for 16-20 h (chronic interruption of enterohepatic circulation). the bile Wow was also significantly increased after BC adnministratioa, but the bile salt excretion was not increased (Fig. 3). Thus, the choleresis observed in this experiment would seem to be totally bile salt independent, This agrees with the authors9 previous study in the dog (Kitani et wH. 1976b) in which a canalicular choleresis of the bile-salt-independent type was demonstrated for BC. Therefore, BG appears to have a choleretic effect of the bile-salt-independent type in both rats and dogs. However, the bile salt excretion and probably the bile-salt-dependent bile flow were increased by BC in rats with acute interruption of enterohepatic circulation (Fig. 1B) . This difference (Kitani et a&. 19'76a9 1976b)l is probably due to different experimental conditions rather than interspecies variation. It laas been established that a chronic interruption of enterohepatic circulation of bile salt for 1620 h is long enough to eliminate the bile salt pool in the rat (Thompson and Vars 1953, 1954; Eriksson 1957a, 1957b) and that the bile salt in the bile in such an animal is mostly newly synthetized bile salt from the liver. In the present study, the bile salt excretion rate in rats with chronic interruption of enterohepatic circulation was 10 times less than that observed in the control rats with acute interruption where there is a minimal loss of the bile salt pool. Thus, the increase in the bile salt excretion in the bile iaa BC-administered rats with acute interruption of enterohepatic circulation might be most likely due to tlne mobilization of a bile salt pool in thc intestinal content and in the intestinal wall of these animals. The rapid decline of bile salt excretion after an initial rise in BC-administered rats may be due to the rapid washout of the pool. On the other hand, in rats with chronic interruption of enterohepatic circulation, bile salt excretion may not be increased by BC because this bile salt pool is already depleted and thus cannot be mobilized. The mechanism for this hypothesized mobilization of a bile salt pool by BC was not elucidated in the present study.
VOL. 5 5 , 1977
Hcrwever, an increase in hepatic blood flow was suggested to increase the bile-salt-dependent bile (Preisig 1972) by augmenting the hepatic load of circulating bile salts. In this regard, we recently demonstrated a 50% increase in the small intestinal fraction of cardiac output in rats by BC, as determined by the radioisotopelabeled microsphere method (Miura and Kitani 1976). This might accelerate the intestinal absorption of bile salt pool and (or) the transfer crf bile salt pool in the intestinal wall to the liver, thus contributing to the increase in bile salt excretion. This study suggests that BC induces both a bile-salt-dependent and independent choleresis. This contrasts with other types of choleresis reported in the past, which were classified either as bile-salt-dependent or independent. For example, phenobarbital (Berthelot et ak. 1 976; Klaassen 1971 ), SC 2644 (Gibson and Farker 1974), or theophylline (Erlinger and Dumont 1973; Barnhart and Coslbes 1974) have been shown to induce canalicular choleresis of tlae bile-salt-independent type, while bile salt administration is known to produce bile-salt-dependent choleresis ( O'MiiilIe et ak. 1 969; Bcrthelot et ak. 1970; Erlinger and Dumont 1973). Recently, however, Gardiner et a&. (1975 ) reported that secretin increased bile salt excretion in the bile in monkeys in which the bile salt pool was maintained during the experimental period. Secretin was generally believed to increase only noncana'ticular (distal bile duct) bile Wow independent of bile salt excretion. Thus, BG and secretin may be classified as a bile-salt-dependent and independent choleretic, although there is a diEerence between these two in the site of bile-saltindependent choleresis (canalicular and distal bile duct). The biliary excretion of a drug s r its metabolites can cause an osmotic driving force increasing bile flow independent of bile salt excretion (Klaassen and Fitzgerald 1974). In this regard, one should consider the biliarqr excretion of BC or its metabolites, as a possible cause of BG-induced choleresis. However, the biliary excretion of BC is reported to be very small, i.e., less than 2% of the dose in the rabbit in the first 8 h (Mima e&al, 1965 ) . Only 6% was recovered in the feces, while 72% was recovered in the urine sf rats in 96 kt, when
KITANI EF AL.
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studied by using [2-14C]BC (Yashiki 1971). This problem is currently under reinvestigation in our laboratory in relation to the choleretic activity of BC.
1161
1976a. Increased bile salt excretion by bucolome. Kanzcd (Acta Hepatol. Jpn.), 87, 224. -- 1976b. The effect of bucolome on the candicular bile formation and sulfobron~ophthaleintransport r n a x i n ~ ~ ~inr ntbe d o g Biocbem. Pharmacol. 25, 3 377-1381, Acknow!edgments KLAASSEN, C. D. 1971. Studies on the increased biliary flow produced by phenobarbital in rats. J. PharThe authors are deeply indebted to Dr. macol. Exp. Ther. 176, 743-751. C. D. Klaassen and Mr. J. Ek for their careful KI AASSFN. C. D., and $;ITZGERALD, T. J. 1974. Metabreview of the manuscript* olistn 2nd biliary excretion of ethacrynic acid. J* Pharmacel. Exp. There 1917548-556. E. 1974. B A R N H ~ R T , J . L.. and C o ~ u ~ B. s , 1974. Effect of KONDO,T., MIYOSIIH,K., and HW~QHATA, Cholesetic eflcct of bucolome. Kanzo (Acts Hepatole theophylline on hepatic excretory function. Am* J * Jpm.) 15. 670-675. Physiol. 227. 194-199. ~ ~ I M H., A , ~ ~ A T S U Z A KT., I , TERADA, K.? and KONDQ.T. ~ E X T I ~ E I C ) I ' , ERLINGER, S . , DKUMEAUX, D., and 1965. Gastrointestinal absorption and excretion of PRFALJX, As M. 1970. Mechanism of phenobarbital5 -n-butyl- 1-cyclohexyI-2,4,6-trioxoperhydropyrinninduced hyperchoieresis in the rat. Am. J e Physiol. dine. Annai. Rep. Takeda Res. Lab. 24. 9-24. 21 9, 809-8 12. BoYI-R, J * I>., and K r ATSKIN, G. 1970. Canalicular bile hfnwxA, R., and KITANI, K . 1976. Study on the splanchflow and bile secretory pressure. Evidence for 21 nic circl~lationin the rat using microspheres labeled non-bile salt dependent fraction in the isolated rat with radioisotopes. The effects of pentobarbital and liver. Gahtrcxmterology, 59. 853-859* bucolome. Jpn. 9.Nucl. Med. 13, 707. ERIKSSON, S. 1957a. Biliary excretion of bile acids and O ' ~ ~ . ~ H IE. 437-439. and guinea pigs- Jpn- J . Nucl. Med. 12, 517. GIBSON, G. F... and FORKER, E. L. 1974. Canzilic~ilar bile flow and bromosulfophthalein transport maxi- W H E E B ER, M . O., ROSS, E. D., and BRADLEY, S. E. 196g0 Canalicular bile production in dogs. Am, Je n-tw-ri: The effect of a bile salt-independent choleretic, Physiol. 214, 866-874. SC-2644. Gastroenterology, 46, 1046-1053. H e r ~ a o c K ,H.. and TALAI AY, B. 1957. Principles of the YASIHKH, T. 1971. Metabolic fate and the pharmacokinetics of 5-PI-butyl-1-cycI~~hexyl-2,4,6-trioxo-perhyenzymatic ineasurement of steroids. 9. Biol. C h e n 227, 37-52. dropyrimidine (BCP) in m a n J. Takeda Res. Lab. KITANI, K . , I'V~IURA~ R., KANAI.S., and MORITA? Yo 30,8011-840.
