567
J. Physiol. (1975), 245, pp. 567-582 With 7 text-figures Printed in Great Britain
ASPECTS OF BILE SECRETION IN THE RABBIT
BY SIGRID C. B. RUTISHAUSER* AND THE LATE S. L. STONE From the Department of Physiology, University College, Cardiff CF1 1XL
(Received 9 May 1974) SUMMARY
1. Bile secretion was studied in anaesthetized rabbits from whom hepatic bile was collected by cannulation of the common bile duct. 2. The flow and composition of bile formed by rabbits anaesthetized with urethane differed significantly from that formed by rabbits anaesthetized with pentobarbitone sodium. 3. The i.P. injection of a hypertonic solution of sucrose (3 M) decreased bile flow and produced changes in the ionic composition of bile and of plasma. 4. The infusion of sodium taurodeoxycholate (1.5-20 ,umole/min I.v.) gave higher rates of bile flow than did equimolar infusions of sodium taurocholate, and unlike taurocholate, increased the bicarbonate concentration of bile. 5. Acetazolamide (10-100 mg/kg) increased the concentration of bicarbonate both in bile and in plasma, and had little effect on bile flow. 6. The infusion of bromsulphthalein (5 mg/kg I.v.) decreased the excretion of bicarbonate into bile, and was associated with the formation of a hypotonic bile. 7. The implications of these results in relation to the mechanisms of bile secretion are discussed. INTRODUCTION
The rate of bile formation in the rabbit, and in the guinea-pig is about ten times greater than that in the dog, and in the cat (Pugh & Stone, 1969). The data of Pugh & Stone show that there is no obvious correlation between the ionic composition of bile and the rate at which bile is formed. Although the rate of secretion of bile salt into bile in the rabbit is greater than that in the cat and in the dog (Rutishauser, 1973), the disparity in the rates of formation of bile cannot be explained entirely in terms of differences in the rate of bile salt secretion. * Address for correspondence: Dr S. C. B. Rutishauser, Department of Physiology, Stopford Building, University of Manchester, Manchester M13 9PT.
568 S. C. B. RUTISHAUSER AND THE LATE S. L. STONE The major bile salt present in the bile of rabbits is deoxycholate, a dihydroxy bile salt (Gregg & Poley, 1966), whereas that in the cat and in the dog is cholate, a trihydroxy bile salt (Haslewood & Wootton, 1950; Wheeler & Ramos, 1960). Erlinger, Dhumeaux, Berthelot & Dumont (1970) found that glycodeoxycholate, a dihydroxy bile salt, had a choleretic efficiency of 70 ml./m-mole in the rabbit, a value which is very much higher than that of 6 ml./m-mole quoted by Wheeler, Ross & Bradley (1968) for taurocholate, a trihydroxy bile salt, in the dog. This raised the possibility that the dihydroxy bile salts were more efficient as choleretics than the trihydroxy bile salts. In view of this, it was of interest to compare the choleretic effect of a dihydroxy bile salt with that of a trihydroxy bile salt in the same species. Preliminary experiments indicated that sodium taurodeoxycholate was indeed a better choleretic than sodium taurocholate in the rabbit, and secondly that the mode of anaesthesia could affect both the composition of bile and its rate of formation in this species. The aim of the present work was to explore these effects in greater detail. A preliminary report of this work has already appeared (Rutishauser & Stone, 1974). METHODS
Experimental methods. Male and female rabbits weighing between 2 and 4 kg were fasted overnight, but were allowed free access to water. The animals were anaesthetized either with pentobarbitone sodium (30 mg/kg in 0-9 % NaCl injected into an ear vein) or with urethane (5 ml./kg of a 25 % solution i.P.). Occasionally this was supplemented with ether. Body temperature was maintained by surface warming. The animals were provided with a tracheal cannula, and the right jugular vein was cannulated for the injection of drugs. After ligation of the cystic duct, and drainage of the gallbladder, the common bile duct was cannulated. Bile samples were collected serially as 10 or 20 min samples in tubes graduated in divisions of 0.1 ml. Blood samples were obtained from a cannula inserted into the left carotid artery. Solutions were infused via a cannula lying in one of the femoral veins. At the end of each experiment the liver was removed and weighed. Sodium taurodeoxycholate (TDC) and sodium taurocholate (TC) (Maybridge Chemicals, Tintagel, Cornwall) were dissolved in 0 9 % NaCl and 0-1 M phosphate buffer pH 7 4, to which was added bovine albumin (4%) in order to reduce the haemolytic effects of the bile salts. The bile salts were infused at rates ranging from 1-5 to 20 ,mole/min. Bromsulphthalein (BSP) was dissolved in 0-9 % NaCl and 0-1 M phosphate buffer pH 7-4. This was infused at the rate of 5 mg/min after an initial priming dose of 10 mg/kg had been given. Acetazolamide was dissolved in water and given as a single intravenous injection (10-100 mg/kg). Analytical methods. The following determinations were performed on both bile and plasma: sodium and potassium concentrations were measured by flame photometry; chloride was determined by an electrometric titration method (Buchler-Cotlove chloridometer); bicarbonate was determined using the micromethod of Van Slyke in the Natelson gasometer; pH was determined using a Pye-Unicam micro-electrode;
BILE SECRETION IN THE RABBIT
569
osmolality was measured cryoscopically (Advanced Osmometer); BSP was determined by a spectrophotometric method, the samples being read at 580 nm after appropriate dilution and alkalinization with 01 M sodium hydroxide. The bile and blood samples for pH and bicarbonate determination were collected under paraffin. Total bile salts in bile were determined using the enzyme steroid dehydrogenase in a modification of the method described by Javitt & Emerman (1968). It was found to be necessary to incubate the enzyme-bile salt-reagent mixture at 370 C for 1 hr before determining the extinction of the solution at 340 nm. This was necessary because of the much slower reaction of TDC in this system as compared with TC. TABLE 1. The flow and composition of bile in two groups of rabbits shortly after the start of bile collection, one group having been anaesthetized with pentobarbitone i.v., and the other group anaesthetized with urethane i.P. The figures quoted give the mean + s.E. for flow and composition in each group
Bile flow
Pentobarbitone sodium 2-37±0'15
(#Il.Imin . g liver) Na K Cl (m-equivft.) HCO3 Bile salt Osmolality (m-osmole/kg) Bile salt secretion rate (n-mole/min. g liver) No. of animals
154-7 +07 3-47+0*19 83-0±+ 2-8
51-5+2*3 10 9+0 7 291-8 + 19 25-8 9
Urethane 1-47+0-24
158-3+ 14 4*09+0*19 82-0±+ 1-8
52*7+2-4
16-0+ 1-1 322-2 ± 3-1 23-6 9
RESULTS
Effects of mode of anaesthesia on the flow and composition of bile Urethane was originally used as the anaesthetic in these studies. It was found, however, that the blood pressure of these animals was undesirably low (60 mmHg), and that higher blood pressures could be maintained by the use of pentobarbitone sodium (90 mmHg). When the flow and composition of bile in these two groups was compared it was found that bile flow in the urethane group was significantly lower than that in group anaesthetized with pentobarbitone sodium (P < 0-025), and that significant differences were also apparent in the composition of bile. These differences are shown in Table 1. The concentration of bile salt in bile is significantly higher in the urethane group (P < 0-0125), and this is associated with a higher biliary osmolality (P < 0-005). In both groups of animals, bile was found to be isotonic with blood plasma. The data suggest that the biliary concentrations of sodium and potassium are also higher in the urethane group, but this could not be shown to be statistically significant (P < 0- 1). The concentrations of chloride and bicarbonate are similar in the two
570 S. C. B. RUTISHAUSER AND THE LATE S. L. STONE
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60 20 40 30 50 10 Time after start of bile collection (min) Fig. 1. Net changes in the concentration of bicarbonate in bile in the first 60 min after the start of bile collection, in rabbits anaesthetized with pentobarbitone (*-e) and in those anaesthetized with urethane (A-A). The concentration of bicarbonate in the first 20 min sample is set at zero, and the changes in concentration are referred to this. The points give the mean change ± s.E. of mean. 90 r Z.:
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10 20 30 Plasma HCO3 concn. (m-equiv/l.) Fig. 2. The relationship between the bicarbonate concentrations of bile and of plasma, during the first hour of bile collection in rabbits anaesthetized with pentobarbitone (I*-) and with urethane (A-A). The lines drawn connect the values obtained in the course of an individual experiment. The overall regression lines are: y = 2-24x+6 60 (r = 0-77) for anaesthesia with pentobarbitone; and y = 1-86x+ 21-76 (r = 0-73) for anaesthesia with urethane, where y is the concentration of bicarbonate in bile and is the concentration in plasma. x
571 BILE SECRETION IN THE RABBIT groups. It was observed, however, that there was a tendency for biliary bicarbonate concentration to decrease with time in the urethane group, and that this contrasted with a slight increase in biliary bicarbonate concentration over the same period of time in the animals anaesthetized with pentobarbitone. This is illustrated in Fig. 1. These changes in the concentration of bicarbonate in bile appeared to be related to similar directional changes in the concentration of bicarbonate in plasma (Fig. 2).
Intraperitoneal injection of a hypertonic solution of sucrose The solution of urethane used for i.P. injection was very concentrated. Its osmolality was about ten times that of plasma. It was of interest therefore to examine the effect of the intraperitoneal injection of another hypertonic solution on the flow and composition of bile in a group of animals anaesthetized with pentobarbitone. Sucrose was chosen for this, and the volume and the tonicity of the solution was adjusted to resemble that of the urethane used as an anaesthetic. The results from a typical experiment are illustrated in Figs. 3 and 4. It can be seen from Fig. 3 that bile flow decreased rapidly after the intraperitoneal injection of sucrose, and that this was correlated with an increase in the osmolality of the blood plasma. It was again found that bile and plasma were always isotonic. The ionic changes occurring in bile and in plasma are shown in Fig. 4. The biliary concentrations of sodium, potassium and bicarbonate all increased despite the fact that the plasma concentrations of these ions declined. In some experiments an increase in bile salt concentration in bile also occurred. A considerable volume of fluid leaked out of the abdominal cavity after the injection of sucrose. The volume of this fluid was at least double that of the injected solution of sucrose. The composition of the intraperitoneal fluid before and after the injection of sucrose is also shown in Fig. 4. As equilibrium is achieved, the concentration of sodium and chloride in this fluid approached the plasma concentrations of these ions. A similar leakage of fluid from the abdominal cavity had been noticed previously in the animals anaesthetized with urethane.
Comparative effects of TDC and TC Within the range of infusion rates studied, TDC increased bile flow to a greater extend than did an equimolar infusion of TC (Fig. 5). The increase in the rate of bile salt secretion in bile was approximately equal to the rate at which each bile salt was infused. Thus TDC formed more bile per unit bile salt secreted than did TC. From Fig. 5 it appears that the choleretic efficiency of TDC is about 30 ml./m-mole, whereas that for TC is only 9 ml./m-mole. However, it is apparent that at higher rates of infusion the choleretic response to TDC falls off. The choleretic efficiency
572 S. C. B. RUTISHA USER AND THE LATE S. L. STONE of a bile salt may be defined as the increase in bile flow produced per unit increase in bile salt secretion rate. This was calculated for TDC at each individual infusion rate. Fig. 6 shows how the choleretic efficiency of TDC varies with the rate of infusion of bile salt. The choleretic efficiency of TDC decreased from a value of about 50 ml./m-mole at an infusion rate of 20 n-mole/min . g liver, to a value of 15 ml./m-mole at an infusion rate of 200 n-mole/min . g liver. On no occasion did the infusion of bile salt increase Sucrose I.P.
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Fig. 3a. The effect of an i.P. injection of 15 ml. of a hypertonic solution of sucrose (3 M) on bile flow, plasma osmolality ( --- -Q), and the osmolality of intraperitoneal fluid (+ ) in a rabbit anaesthetized with pentobarbitone. b, The relationship between plasma osmolality and bile flow from the results of the experiment shown in Fig. 3a.
the osmolality of bile or of plasma. Both TDC and TC increased the concentrations of sodium and bile salt in bile, and decreased the concentration of chloride (Table 2). However, the two bile salts differed markedly in their effects on the concentration of bicarbonate in bile. TDC consistently
BILE SECRETION IN THE RABBIT 573 increased the concentration of this ion in bile, whereas TC caused a net decrease in concentration. The quantitative data are given in Table 2. There was little net change in the osmolality of bile during the infusion of either TDC or TC. The ionic composition of plasma was not significantly altered during the infusion of either bile salt. In particular, it may be noted that the net change in the concentration of bicarbonate in plasma, in six experiments with TDC, was only + 0 7 + 0 3 m-equiv/l. (S.E. of mean). Bile
170 160_ 150 1 40
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2 1 2 Time (hr) F~ig. 4. The effects of an i.P. injection of 15 ml. of a hypertonic solution of sucrose (3 M) on the ionic composition of bile, of plasma, and of intraperitoneal fluid.. Na ( v-v), Cl (o - o), HCO, (I * ), bile salt ( x -- - x ), and K (V -V) concentrations. Sucrose was injected at the time marked by the arrows. ...
Effects of acetazolamide In view of the effect of TDC in increasing the concentration of bicarbonate in bile, it was of interest to determine the effect of an inhibitor of carbonic anhydrase on this response. The 'intravenous injection of acetazolamide alone significantly increased the bicarbonate concentration of both bile (P < 0 0025) and of plasma (P < 0-0125). The concentration of chloride in bile decreased, and the potassium concentrations of bile and of plasma increased. Acetazolamide had no significant effect on the rate of bile flow. The mean results from three experiments are shown in Table 3. The prior injection of acetazolamide (10-100 mg/kg) did not prevent the increase in the concentration of bicarbonate in bile caused by the infusion of TDC. In Fig. 7 it can be
574 S. C. B. RUTISHA USER AND THE LATE S. L. STONE 40
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Fig. 5. The relationship between the rate of infusion of bile salt and the increase in the rate of bile flow, for sodium taurocholate (A), and for sodium taurodeoxycholate (e), in rabbits anaesthetized with pentobarbitone. Lines of slope 30 ml./m-mole, and 9 ml./m-mole, have been drawn to indicate the choleretic efficiency of each bile salt. These lines are not regression lines.
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Fig. 6. The choleretic efficiency of sodium taurodeoxycholate at different rates of infusion of bile salt in rabbits anaesthetized with pentobarbitone.
'Choleretic efficiency' was calculated rate of secretion of bile salt in bile).
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BILE SECRETION IN THE RABBIT
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TABLE 2. Net changes in the composition of bile caused by the infusion of bile salt (TDC or TC 5 psmole/min i.v.). Figures listed give the mean change ± s.E. of mean Taurodeoxycholate Taurocholate Na +11-7 ±1-2 +11-8 ±1-2 K +0-12+0-05 +0-15±016 Cl -13-7 ± 1-7 (m-equiv/1.) -12-8 + 1-1 +8-2 ±1-9 HCO3 -2.9 + 1-8 Bile salt + 32-4 + 2-6 +16-0± 1-4 -3-3 + 1-5 + 0-2 + 1-3 Osmolality (m-osmole/kg) pH + 0-050 ± 0*015 TDC infused Acetazolamide 3-0
020 E
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Fig. 7. The effect of an infusion of sodium taurodeoxycholate (5 limole/min) on the concentration of bicarbonate in bile (LI---LI) and in plasma (U--), after the prior injection of a single dose of acetazolamide (300 mg i.v.) in a rabbit anaesthetized with urethane. The dotted line for the concentration of bicarbonate in bile ( .... ) indicates the level of bicarbonate concentration which would have been expected, after the injection of acetazolamide, had the bile salt not been infused.
576 S. C. B. RUTISHA USER AND THE LATE S. L. STONE seen that the effect of TDC was merely superimposed upon the effect of acetazolamide. Effects of BSP In the course of some experiments which were designed to examine the effects of TDC and TC on the secretion of BSP into bile, it was observed that BSP itself had some interesting effects on the composition of bile. The secretion of BSP into bile increased the biliary concentrations of sodium and of potassium. and decreased the concentrations of chloride and TABLE 3. The effects of acetazolamide (10 mg/kg body wt.) on the ionic composition of bile and of plasma, and on the rate of bile flow. The figures give the mean + S.E. for three experiments with the drug in animals anaesthetized with urethane Control
A. Bile Na K Cl
HCO3
158-7±2-8
\ j
(m-equiv/l.)
4-2+0-4 90-2 + 1-2
40-8+220
Bilesalt 11-4+1-1 Bile flow (fl./min.g liver) 1-028 + 0-178 B. Plasma K 2-9+0-4 1 Cl 3 (m-equiv/l.) 97.7 + 1-2 HCO3I 12-6 +12
After acetazolamide
160-6+1 9 5-3+0-6 84-8 + 2-5 51-7+0 9 8-1+0-5 1-136 + 0-078
3-5+0 5 95-7 + 2-0 18-3 + 1-3
TABLE 4. Net changes in the composition of bile and of plasma, caused by the infusion of bromsulphthalein (5 mg/min). The figures give the mean change in composition + s.E. of the mean in six experiments. * indicates results from only three experiments Bile Plasma Na +36-0±1-5 K* | +1-76±0-28 Cl -10-7 2-3 (m-equiv/l.) HCO3 -26-3+1-2 -2-3+0 -4 BSP +53-9 3-0 Osmolality (m-osmole/kg) -17-5 ± 1-2 -2-3 + 0-9 pH* -0-18±0-05 +0-07±0-03
of bicarbonate. In every case the osmolality of bile also decreased such that bile became hypotonic with respect to plasma. The quantitative data for the changes observed in the composition of bile and of plasma are shown in Table 4. The concentrations of sodium, potassium and chloride in plasma were not affected by the infusion of BSP. The decrease in the biliary concentration of bicarbonate is quite considerable. Although the concentration of bicarbonate in the plasma also decreased, the decrease in biliary bicarbonate is much greater, such that
577 BILE SECRETION IN THE RABBIT the bile: plasma ratio for this ion decreased from 2-74 + 023 to 1'58 + 0d14 (s.E. of mean) after the infusion of BSP. In fact the total bicarbonate excreted in bile per unit time decreased in each of the six experiments with BSP, despite the fact that the flow of bile was increased in three of these. The effect of BSP on the flow of bile was somewhat variable, in that flow was increased in three experiments, decreased in one, and was unchanged in the remaining two. The maximum concentration of BSP in bile was 23-1 + 3.5 mg/ml. (S.D. of an observation) in six experiments. The maximum rate of excretion of BSP into bile, obtained in the presence of a rising concentration of BSP in the plasma was 59-9 + 12-6 n-mole/min . g liver (S.D. of an observation). This is roughly equivalent to 1-7 + 04 ptmole/ min. kg body wt. DISCUSSION
Bile secretion has been studied in a variety of species including the rabbit, guinea-pig, rat, cat and dog, and it is clear from a number of studies, that significant differences exist between these species, in terms of the rate at which bile is formed, the composition of bile, and the responsiveness of the biliary system to different agents (Pugh & Stone, 1969; Scratcherd, 1965; Bizard, 1965; Stone, 1965; Klaassen, 1972). The picture is further complicated by the fact that a variety of anaesthetics have been used. For example, urethane is commonly used as an anaesthetic in rabbits, and guinea-pigs, whereas pentobarbitone sodium is generally the anaesthetic of choice for the dog. Mode of anaesthesia It is clear from the results presented in this paper, that the mode of anaesthesia has a profound effect on the flow and composition of bile in at least one species, namely the rabbit. This may be interpreted in at least one of two ways: either pentobarbitone stimulates the secretion of bile, or urethane depresses it. In order to test the first possibility it would be necessary to compare the rate of flow of bile in anaesthetized and unanaesthetized animals. This was not possible in the present work. However, the results presented strongly suggest that the intraperitoneal injection of urethane decreases bile flow, and that this is because the solution injected is considerably hypertonic with respect to the blood plasma. The results obtained with urethane are of interest in clarifying some points in literature. Scratcherd (1965) observed that bile flow could be maintained in rabbits, provided a suitable solution of electrolytes was infused. As he too used urethane as anaesthetic, it may be that the electrolyte solution successfully replaced the fluid which was lost from the abdominal cavity, and thus bile flow was maintained. Pugh & Stone (1969) quoted values for the flow and composition of bile
578 S. C. B. RUTISHAUSER AND THE LATE S. L. STONE in rabbits anaesthetized with urethane. Although the value given for the rate of flow of bile (1 67 #t1./min.g liver) is consistent with the present data, their values for biliary bicarbonate concentration are somewhat lower (32.7 m-equiv/l.) than those quoted here. This may be partly explained by the observation, that biliary bicarbonate concentration tended to decline with time in the animals anaesthetized with urethane. The extent of the decline will depend on the length of time elapsing between the injection of the anaesthetic and the collection of bile for analysis, and the amount of bicarbonate lost in that time in the i.P. fluid. It may be that this time was somewhat longer in the experiments of Pugh & Stone. Plasma osmolality and bile flow The effects of the presence of a hypertonic solution in the duodenum on the rate of bile flow have been described (Knightly, Vanamee & Lawrence, 1960), and it has been demonstrated that the i.v. injection of hypertonic solutions decreases bile flow in nephrectomized guinea-pigs (Chenderovitch, Phocas & Rautureau, 1963). Clearly, the presence of a hypertonic solution in the abdominal cavity will cause the withdrawal of fluid and diffusible electrolyte from the plasma, and that this, together with the absorption of the injected solute into the plasma, will increase plasma osmolality. In the present experiments the increase in plasma osmolality was linearly related to the decrease in bile flow, such that an increase in osmolality of 37 m-osmole/kg decreased bile flow by 1 OIjc./min.g liver. It is surprising that so small an increase in plasma osmolality should have such a marked effect on bile flow. This suggests that the underlying mechanism for the formation of the fluid fraction of bile is particularly sensitive to changes in plasma osmolality. Sperber (1959) originally suggested that the driving force for the formation of bile was the creation of an osmotic gradient between bile and plasma, by the active secretion of non-diffusible bile salt anions into the biliary canaliculus. Clearly, an increase in plasma osmolality will affect the osmotic gradient between bile and plasma, but whether this is an adequate explanation of the effect remains to be seen.
Choleretic efficiency of bile salts Several bile salts have been shown to differ in their choleretic efficiency. O'Maille, Richards & Short (1965) found that cholate was more efficient than taurocholate in increasing bile flow, and Sperber (1959) and others have presented data which show that dehydrocholate is exceptionally good as a choleretic. It has been assumed that the underlying reason for such differences in choleretic efficiency has been the different physico-chemical properties of the bile salts. The greater choleretic efficiency of cholate and
.579 BILE SECRETION IN THE RABBIT dehydrocholate is assumed to be due to the lesser ability of these bile salts to form micelles. More osmotically active particles are thus thought to be available per mole of bile salt secreted, than for taurocholate which forms larger aggregates. The tendency for bile salts to form micelles depends on their structure and their polarity, and it has been shown in simple physicochemical systems that deoxycholate, conjugated with either glycine or taurine, forms much larger aggregates than taurocholate (Small, 1971). It would therefore be expected that these bile salts would prove to be less efficient as choleretics than taurocholate. It is surprising, therefore, that both taurodeoxycholate, and glycodeoxycholate (Erlinger et al. 1970) should in fact be more efficient than taurocholate in the rabbit. Unless these bile salts exhibit some rather unusual physico-chemical properties in rabbit bile, it would seem very unlikely that their choleretic efficiency can be explained in terms of reduced micelle formation. It is therefore of interest, that taurodeoxycholate, unlike taurocholate, consistently increased the bicarbonate concentration of bile. This suggests that taurodeoxycholate may stimulate a bicarbonate pump in the biliary system, and that this is responsible for the increased output of water and electrolyte in bile. It may also be noted that the choleretic efficiency of taurodeoxycholate decreased as the rate of infusion of bile salt increased. Such a change in choleretic efficiency might be expected if the increase in bile flow was the summed effect of taurodeoxycholate on two independent mechanisms of bile secretion. The total bile flow, at any given infusion rate, would then depend on the individual contributions made by each mechanism at that infusion rate. It is conceivable that the formation of the electrolyte component is maximally stimulated at low rates of bile salt infusion. As the rate of infusion of bile salt is increased, that fraction of bile which is directly dependent on the active secretion of bile salt into the canaliculus will form an increasingly greater fraction of the total flow. Hence at low rates of bile salt infusion the calculated choleretic efficiency of taurodeoxycholate would be high, but this would decrease as the rate of infusion of bile salt was increased. Alternatively, it may be argued, that at high rates of infusion of bile salt, the permeability of the biliary system is affected, and thus the choleretic efficiency of the bile salt declines. But the fact remains that taurodeoxycholate is a more efficient choleretic than taurocholate in the rabbit, and this implies that the two bile salts exert very different effects on the biliary system, regardless of the mechanism involved. Erlinger et al. (1970) postulated that 60 % of spontaneous bile flow in the rabbit is dependent on the activity of a sodium pump believed to be located at the level of the canaliculus. It could be suggested therefore that taurodeoxycholate is able to stimulate this pump, and that the increase in
580 S. 0. B. RUTISHA USER AND THE LATE S. L. STONE biliary bicarbonate concentration is either a result of the effect of a change in flow rate on some ductular exchange mechanisms, or is secondary to a local increase in the Pco2. The second possibility is unlikely in that the pH of bile would be expected to decrease or remain the same. In fact the infusion of taurodeoxycholate was usually associated with a slight increase in the pH of bile. It would be of interest to examine the effect of the inhibitors used by Erlinger and his group, on the biliary response to taurodeoxycholate. If taurodeoxycholate is stimulating a sodium pump, then the choleretic efficiency of the bile salt should be reduced by substances such as ouabain, amiloride, and ethacrynic acid, all of which inhibit the Na/K ATPase involved in sodium transport. It is of interest, however, that Erlinger et al. (1970) found that the choleretic efficiency of glycodeoxycholate was unchanged by the presence of these inhibitors.
Bicarbonate in bile The enzyme, carbonic anhydrase is usually involved in most systems which are responsible for the formation and transport of bicarbonate in the body. As the concentration of bicarbonate in rabbit bile is about twice that in the plasma, it is to be expected that a bicarbonate pump of some description must be present in the biliary system. The results presented here have shown that the carbonic anhydrase inhibitor, acetazolamide, does not depress the excretion of bicarbonate in bile, and that the increase in bicarbonate concentration produced by taurodeoxycholate also cannot be prevented by the prior injection of this inhibitor. Maren (1967, and personal communication) found that the carbonic anhydrase which may be extracted from rabbit liver is refractory to acetazolamide and other sulfonamides. The in vivo results presented here correlate well with Maren's in vitro data. It would seem that the mechanism involved in the secretion of bicarbonate in the biliary system of the rabbit possesses some unusual features. In this context it is of interest that the hormone secretin, which is able to stimulate the production of a bicarbonate rich fluid in the biliary systems of the dog, cat and guinea-pig (Wheeler & Ramos, 1960; Scratcherd, 1965; Forker, 1967) is completely without effect on the biliary system of the rabbit (Scratcherd, 1965). Indeed it would seem that the role of this hormone has been adopted by the dihydroxy bile salt, deoxycholate, and it is tempting to speculate that the dihydroxy bile salts may in fact act as the 'secretin' of the rabbit in relation to the biliary system.
Bromsulphthalein Finally, the effect of the biliary excretion of another anion, bromsulphthalein (BSP), on the composition of bile may be compared with the
581 BILE SECRETION IN THE RABBIT effects of the bile salt anions. BSP is frequently used as a model of organic anion transport in the liver. The maximum rate at which it is transported into bile may be affected by the excretion of bile salt. It is believed that this is due to the decrease in the biliary concentration of BSP which occurs as bile flow is increased (O'Maille, Richards & Short, 1966). BSP, unlike the bile salts, is a disodium salt, and thus the marked increase in the concentration of sodium in bile, during the excretion of BSP in bile, is not unexpected. It is also expected that the secretion of an anion into bile will decrease the concentration of the other diffusible anions present. However, the degree to which BSP decreased the concentration of bicarbonate in bile is surprising, particularly when this is compared with the effect of taurocholate on the biliary concentration of bicarbonate. An increase in the taurocholate concentration of bile of 50 m-equiv/l. only decreased the concentration of bicarbonate in bile by about 5 m-equiv/l., whereas an increase of 56 m-equiv/l. in the concentration of BSP in bile decreased biliary bicarbonate concentration by about 25 m-equiv/l. Taurocholate and BSP also have different effects on the osmolality of bile. The infusion of bile salt never caused any significant changes in the osmolality of bile or of plasma, whereas the infusion of BSP consistently decreased the osmolality of bile such that bile became hypotonic with respect to plasma. The low osmolality of the bile samples is unlikely to be due to aggregation of the BSP molecules at the low temperatures obtained in the freezing point osmometer because the osmolality of a pure solution of BSP was determined both in a freezing point osmometer, and in a vapour pressure osmometer, and the values obtained were practically identical. The simplest explanation of the effects of BSP on the composition of bile would be that at some point in the biliary system acid is being added to bile, and bicarbonate is thus being removed. Isotonicity is possibly not maintained, because the permeability of the biliary system at that point, and the rate of flow of bile is such, that proper equilibration of the bile with plasma is not achieved. Clearly, the three anions studied, taurodeoxycholate, taurocholate, and bromsulphthalein, differ profoundly in the effects that they have on the flow and composition of bile. This raises some questions in terms of the kinetics of the underlying transport mechanisms. For if the anions differ in this way, then the factors limiting their secretion into bile may not be simply the availability of carrier sites at the biliary membrane, but may be the prevailing conditions within the canaliculus itself. Thanks are due to Mr B. M. Shears for his very capable and willing assistance, to Mr T. J. Surman for his help in the initial stages of the work, and to Dr J. S. Thomas for very kindly reading the manuscript. S.C.B.R. was in receipt of an M.R.C. Scholarship during part of this work.
582 S. C. B. RUTISHA USER AND THE LATE S. L. STONE REFERENCES BIZARD, G. (1965). Enzyme inhibitors and biliary secretion. In The Biliary System, ed. TAYLOR, W., pp. 315-324. Oxford: Blackwell. CHENDEROVITCH, J., PHOCAS, E. & RAUTUIREAu, M. (1963). Effects of hypertonic solutions on bile formation. Am. J. Physiol. 205, 863-867. ERLINGER, S., DHIUMEAuX, D., BERTHELOT, P. & DUMONT, M. (1970). Effect of inhibitors of sodium transport on bile formation in the rabbit. Am. J. Physiol. 219, 416-422. FORKER, E. L. (1967). Two sites of bile formation as determined by mannitol and erythritol clearance in the guinea-pig. J. clin. Invest. 46, 1189-1195. GREGG, J. A. & PoLEY, J. R. (1966). Excretion of bile acids in normal rabbits. Am. J. Physiol. 211, 1147-1151. HASLEWOOD, G. A. D. & WOOTTON, V. (1950). Comparative studies of bile salts. Biochem. J. 47, 584-597. JAVITT, N. B. & EMERMAN, S. (1968). Effect of sodium taurolithocholate on bile flow and bile acid excretion. J. clin. Invest. 47, 1002-1014. KLAASSEN, C. D. (1972). Species differences in the choleretic response to bile salts.
J. Physiol. 224, 259-269. KNIGHTLY, J. J., VANAMEE, P. & LAWRENCE, W. (1960). The effect of intraduodenal hypertonic glucose on biliary and pancreatic secretion. Surge. Forum 11, 371-373. MAREN, T. H. (1967). Carbonic anhydrase: chemistry, physiology and inhibition. Physiol. Rev. 47, 595-781. O'MAILLE, E. R. L., RICHARDS, T. G. & SHORT, A. H. (1965). Acute taurine depletion and maximal rates of hepatic conjugation and secretion of cholic acid in the dog. J. Physiol. 180, 67-79. O'MAILLE, E. R. L., RICHARDS, T. G. & SHORT, A. H. (1966). Factors determining the maximal rate of organic anion secretion by the liver and further evidence on the hepatic site of action of the hormone secretin. J. Physiol. 186, 424-438. PUGH, P. M. & STONE, S. L. (1969). The ionic composition of bile. J. Physiol. 201, 50-51 P. RUTISHAUSER, S. C. B. (1973). Studies in the physiology of the liver. Ph.D. Thesis, University of Wales. RUTISHAUSER, S. C. B. & STONE, S. L. (1974). Aspects of bile secretion in the rabbit. J. Physiol. 238, 46-47P. SCRATCHERD, T. (1965). Electrolyte composition and control of biliary secretion in the cat and rabbit. In The Biliary System, ed. TAYLOR, W., pp. 515-528. Oxford: Blackwell. SMALL, D. M. (1971). The physical chemistry of cholanic acids. In The Bile Acids, vol. 1, ed. NAn, P. P. & KRITCHEVSKY, D., pp. 249-355. New York and London: Plenum Press. SPERBER, I. (1959). Secretion of organic anions in the formation of urine and bile. Pharmac. Rev. 11, 109-134. STONE, S. L. (1965). Energy requirements for bile secretion. In The Biliary System, ed. TAYLOR, W., pp. 277-292. Oxford: Blackwell. WHEELER, H. 0. & RAMOS, 0. L. (1960). Determinants of the flow and composition of bile in the unanaesthetized dog during constant infusion of sodium taurocholate. J. clin. Invest. 39, 161-170. WHEELER, H. O., Ross, E. D. & BRADLEY, S. E. (1968). Canalicular bile production in dogs. Am. J. Physiol. 214, 866-874.
J. Physiol. (1975), 245, pp. 567-582 With 7 text-figures Printed in Great Britain
ASPECTS OF BILE SECRETION IN THE RABBIT
BY SIGRID C. B. RUTISHAUSER* AND THE LATE S. L. STONE From the Department of Physiology, University College, Cardiff CF1 1XL
(Received 9 May 1974) SUMMARY
1. Bile secretion was studied in anaesthetized rabbits from whom hepatic bile was collected by cannulation of the common bile duct. 2. The flow and composition of bile formed by rabbits anaesthetized with urethane differed significantly from that formed by rabbits anaesthetized with pentobarbitone sodium. 3. The i.P. injection of a hypertonic solution of sucrose (3 M) decreased bile flow and produced changes in the ionic composition of bile and of plasma. 4. The infusion of sodium taurodeoxycholate (1.5-20 ,umole/min I.v.) gave higher rates of bile flow than did equimolar infusions of sodium taurocholate, and unlike taurocholate, increased the bicarbonate concentration of bile. 5. Acetazolamide (10-100 mg/kg) increased the concentration of bicarbonate both in bile and in plasma, and had little effect on bile flow. 6. The infusion of bromsulphthalein (5 mg/kg I.v.) decreased the excretion of bicarbonate into bile, and was associated with the formation of a hypotonic bile. 7. The implications of these results in relation to the mechanisms of bile secretion are discussed. INTRODUCTION
The rate of bile formation in the rabbit, and in the guinea-pig is about ten times greater than that in the dog, and in the cat (Pugh & Stone, 1969). The data of Pugh & Stone show that there is no obvious correlation between the ionic composition of bile and the rate at which bile is formed. Although the rate of secretion of bile salt into bile in the rabbit is greater than that in the cat and in the dog (Rutishauser, 1973), the disparity in the rates of formation of bile cannot be explained entirely in terms of differences in the rate of bile salt secretion. * Address for correspondence: Dr S. C. B. Rutishauser, Department of Physiology, Stopford Building, University of Manchester, Manchester M13 9PT.
568 S. C. B. RUTISHAUSER AND THE LATE S. L. STONE The major bile salt present in the bile of rabbits is deoxycholate, a dihydroxy bile salt (Gregg & Poley, 1966), whereas that in the cat and in the dog is cholate, a trihydroxy bile salt (Haslewood & Wootton, 1950; Wheeler & Ramos, 1960). Erlinger, Dhumeaux, Berthelot & Dumont (1970) found that glycodeoxycholate, a dihydroxy bile salt, had a choleretic efficiency of 70 ml./m-mole in the rabbit, a value which is very much higher than that of 6 ml./m-mole quoted by Wheeler, Ross & Bradley (1968) for taurocholate, a trihydroxy bile salt, in the dog. This raised the possibility that the dihydroxy bile salts were more efficient as choleretics than the trihydroxy bile salts. In view of this, it was of interest to compare the choleretic effect of a dihydroxy bile salt with that of a trihydroxy bile salt in the same species. Preliminary experiments indicated that sodium taurodeoxycholate was indeed a better choleretic than sodium taurocholate in the rabbit, and secondly that the mode of anaesthesia could affect both the composition of bile and its rate of formation in this species. The aim of the present work was to explore these effects in greater detail. A preliminary report of this work has already appeared (Rutishauser & Stone, 1974). METHODS
Experimental methods. Male and female rabbits weighing between 2 and 4 kg were fasted overnight, but were allowed free access to water. The animals were anaesthetized either with pentobarbitone sodium (30 mg/kg in 0-9 % NaCl injected into an ear vein) or with urethane (5 ml./kg of a 25 % solution i.P.). Occasionally this was supplemented with ether. Body temperature was maintained by surface warming. The animals were provided with a tracheal cannula, and the right jugular vein was cannulated for the injection of drugs. After ligation of the cystic duct, and drainage of the gallbladder, the common bile duct was cannulated. Bile samples were collected serially as 10 or 20 min samples in tubes graduated in divisions of 0.1 ml. Blood samples were obtained from a cannula inserted into the left carotid artery. Solutions were infused via a cannula lying in one of the femoral veins. At the end of each experiment the liver was removed and weighed. Sodium taurodeoxycholate (TDC) and sodium taurocholate (TC) (Maybridge Chemicals, Tintagel, Cornwall) were dissolved in 0 9 % NaCl and 0-1 M phosphate buffer pH 7 4, to which was added bovine albumin (4%) in order to reduce the haemolytic effects of the bile salts. The bile salts were infused at rates ranging from 1-5 to 20 ,mole/min. Bromsulphthalein (BSP) was dissolved in 0-9 % NaCl and 0-1 M phosphate buffer pH 7-4. This was infused at the rate of 5 mg/min after an initial priming dose of 10 mg/kg had been given. Acetazolamide was dissolved in water and given as a single intravenous injection (10-100 mg/kg). Analytical methods. The following determinations were performed on both bile and plasma: sodium and potassium concentrations were measured by flame photometry; chloride was determined by an electrometric titration method (Buchler-Cotlove chloridometer); bicarbonate was determined using the micromethod of Van Slyke in the Natelson gasometer; pH was determined using a Pye-Unicam micro-electrode;
BILE SECRETION IN THE RABBIT
569
osmolality was measured cryoscopically (Advanced Osmometer); BSP was determined by a spectrophotometric method, the samples being read at 580 nm after appropriate dilution and alkalinization with 01 M sodium hydroxide. The bile and blood samples for pH and bicarbonate determination were collected under paraffin. Total bile salts in bile were determined using the enzyme steroid dehydrogenase in a modification of the method described by Javitt & Emerman (1968). It was found to be necessary to incubate the enzyme-bile salt-reagent mixture at 370 C for 1 hr before determining the extinction of the solution at 340 nm. This was necessary because of the much slower reaction of TDC in this system as compared with TC. TABLE 1. The flow and composition of bile in two groups of rabbits shortly after the start of bile collection, one group having been anaesthetized with pentobarbitone i.v., and the other group anaesthetized with urethane i.P. The figures quoted give the mean + s.E. for flow and composition in each group
Bile flow
Pentobarbitone sodium 2-37±0'15
(#Il.Imin . g liver) Na K Cl (m-equivft.) HCO3 Bile salt Osmolality (m-osmole/kg) Bile salt secretion rate (n-mole/min. g liver) No. of animals
154-7 +07 3-47+0*19 83-0±+ 2-8
51-5+2*3 10 9+0 7 291-8 + 19 25-8 9
Urethane 1-47+0-24
158-3+ 14 4*09+0*19 82-0±+ 1-8
52*7+2-4
16-0+ 1-1 322-2 ± 3-1 23-6 9
RESULTS
Effects of mode of anaesthesia on the flow and composition of bile Urethane was originally used as the anaesthetic in these studies. It was found, however, that the blood pressure of these animals was undesirably low (60 mmHg), and that higher blood pressures could be maintained by the use of pentobarbitone sodium (90 mmHg). When the flow and composition of bile in these two groups was compared it was found that bile flow in the urethane group was significantly lower than that in group anaesthetized with pentobarbitone sodium (P < 0-025), and that significant differences were also apparent in the composition of bile. These differences are shown in Table 1. The concentration of bile salt in bile is significantly higher in the urethane group (P < 0-0125), and this is associated with a higher biliary osmolality (P < 0-005). In both groups of animals, bile was found to be isotonic with blood plasma. The data suggest that the biliary concentrations of sodium and potassium are also higher in the urethane group, but this could not be shown to be statistically significant (P < 0- 1). The concentrations of chloride and bicarbonate are similar in the two
570 S. C. B. RUTISHAUSER AND THE LATE S. L. STONE
+4.0 r
030.0 bo = bO C
._
Zo U .-
Xuw
c
Z cU
-4 0 -6-0 .H-U
l
I
I
I
I
I
I
I
I
I
I
1
1
60 20 40 30 50 10 Time after start of bile collection (min) Fig. 1. Net changes in the concentration of bicarbonate in bile in the first 60 min after the start of bile collection, in rabbits anaesthetized with pentobarbitone (*-e) and in those anaesthetized with urethane (A-A). The concentration of bicarbonate in the first 20 min sample is set at zero, and the changes in concentration are referred to this. The points give the mean change ± s.E. of mean. 90 r Z.:
60
1.
-
d C
0
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0 U I ._
I
I
I
I
I
I
l
I
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10 20 30 Plasma HCO3 concn. (m-equiv/l.) Fig. 2. The relationship between the bicarbonate concentrations of bile and of plasma, during the first hour of bile collection in rabbits anaesthetized with pentobarbitone (I*-) and with urethane (A-A). The lines drawn connect the values obtained in the course of an individual experiment. The overall regression lines are: y = 2-24x+6 60 (r = 0-77) for anaesthesia with pentobarbitone; and y = 1-86x+ 21-76 (r = 0-73) for anaesthesia with urethane, where y is the concentration of bicarbonate in bile and is the concentration in plasma. x
571 BILE SECRETION IN THE RABBIT groups. It was observed, however, that there was a tendency for biliary bicarbonate concentration to decrease with time in the urethane group, and that this contrasted with a slight increase in biliary bicarbonate concentration over the same period of time in the animals anaesthetized with pentobarbitone. This is illustrated in Fig. 1. These changes in the concentration of bicarbonate in bile appeared to be related to similar directional changes in the concentration of bicarbonate in plasma (Fig. 2).
Intraperitoneal injection of a hypertonic solution of sucrose The solution of urethane used for i.P. injection was very concentrated. Its osmolality was about ten times that of plasma. It was of interest therefore to examine the effect of the intraperitoneal injection of another hypertonic solution on the flow and composition of bile in a group of animals anaesthetized with pentobarbitone. Sucrose was chosen for this, and the volume and the tonicity of the solution was adjusted to resemble that of the urethane used as an anaesthetic. The results from a typical experiment are illustrated in Figs. 3 and 4. It can be seen from Fig. 3 that bile flow decreased rapidly after the intraperitoneal injection of sucrose, and that this was correlated with an increase in the osmolality of the blood plasma. It was again found that bile and plasma were always isotonic. The ionic changes occurring in bile and in plasma are shown in Fig. 4. The biliary concentrations of sodium, potassium and bicarbonate all increased despite the fact that the plasma concentrations of these ions declined. In some experiments an increase in bile salt concentration in bile also occurred. A considerable volume of fluid leaked out of the abdominal cavity after the injection of sucrose. The volume of this fluid was at least double that of the injected solution of sucrose. The composition of the intraperitoneal fluid before and after the injection of sucrose is also shown in Fig. 4. As equilibrium is achieved, the concentration of sodium and chloride in this fluid approached the plasma concentrations of these ions. A similar leakage of fluid from the abdominal cavity had been noticed previously in the animals anaesthetized with urethane.
Comparative effects of TDC and TC Within the range of infusion rates studied, TDC increased bile flow to a greater extend than did an equimolar infusion of TC (Fig. 5). The increase in the rate of bile salt secretion in bile was approximately equal to the rate at which each bile salt was infused. Thus TDC formed more bile per unit bile salt secreted than did TC. From Fig. 5 it appears that the choleretic efficiency of TDC is about 30 ml./m-mole, whereas that for TC is only 9 ml./m-mole. However, it is apparent that at higher rates of infusion the choleretic response to TDC falls off. The choleretic efficiency
572 S. C. B. RUTISHA USER AND THE LATE S. L. STONE of a bile salt may be defined as the increase in bile flow produced per unit increase in bile salt secretion rate. This was calculated for TDC at each individual infusion rate. Fig. 6 shows how the choleretic efficiency of TDC varies with the rate of infusion of bile salt. The choleretic efficiency of TDC decreased from a value of about 50 ml./m-mole at an infusion rate of 20 n-mole/min . g liver, to a value of 15 ml./m-mole at an infusion rate of 200 n-mole/min . g liver. On no occasion did the infusion of bile salt increase Sucrose I.P.
A
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Fig. 3a. The effect of an i.P. injection of 15 ml. of a hypertonic solution of sucrose (3 M) on bile flow, plasma osmolality ( --- -Q), and the osmolality of intraperitoneal fluid (+ ) in a rabbit anaesthetized with pentobarbitone. b, The relationship between plasma osmolality and bile flow from the results of the experiment shown in Fig. 3a.
the osmolality of bile or of plasma. Both TDC and TC increased the concentrations of sodium and bile salt in bile, and decreased the concentration of chloride (Table 2). However, the two bile salts differed markedly in their effects on the concentration of bicarbonate in bile. TDC consistently
BILE SECRETION IN THE RABBIT 573 increased the concentration of this ion in bile, whereas TC caused a net decrease in concentration. The quantitative data are given in Table 2. There was little net change in the osmolality of bile during the infusion of either TDC or TC. The ionic composition of plasma was not significantly altered during the infusion of either bile salt. In particular, it may be noted that the net change in the concentration of bicarbonate in plasma, in six experiments with TDC, was only + 0 7 + 0 3 m-equiv/l. (S.E. of mean). Bile
170 160_ 150 1 40
Plasma
Intraperitoneal fluid
t
-
110 0 0
90 :
j
1
2
1
2 1 2 Time (hr) F~ig. 4. The effects of an i.P. injection of 15 ml. of a hypertonic solution of sucrose (3 M) on the ionic composition of bile, of plasma, and of intraperitoneal fluid.. Na ( v-v), Cl (o - o), HCO, (I * ), bile salt ( x -- - x ), and K (V -V) concentrations. Sucrose was injected at the time marked by the arrows. ...
Effects of acetazolamide In view of the effect of TDC in increasing the concentration of bicarbonate in bile, it was of interest to determine the effect of an inhibitor of carbonic anhydrase on this response. The 'intravenous injection of acetazolamide alone significantly increased the bicarbonate concentration of both bile (P < 0 0025) and of plasma (P < 0-0125). The concentration of chloride in bile decreased, and the potassium concentrations of bile and of plasma increased. Acetazolamide had no significant effect on the rate of bile flow. The mean results from three experiments are shown in Table 3. The prior injection of acetazolamide (10-100 mg/kg) did not prevent the increase in the concentration of bicarbonate in bile caused by the infusion of TDC. In Fig. 7 it can be
574 S. C. B. RUTISHA USER AND THE LATE S. L. STONE 40
,Z, 0
0
bo 30
A 0
-0 C 0 0
.
0
100 200 300 Bile salt infusion rate (n-molelmin .g liver)
400
Fig. 5. The relationship between the rate of infusion of bile salt and the increase in the rate of bile flow, for sodium taurocholate (A), and for sodium taurodeoxycholate (e), in rabbits anaesthetized with pentobarbitone. Lines of slope 30 ml./m-mole, and 9 ml./m-mole, have been drawn to indicate the choleretic efficiency of each bile salt. These lines are not regression lines.
60 r 0
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100
200
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300
Bile salt infusion rate (n-mole/min.g liver)
Fig. 6. The choleretic efficiency of sodium taurodeoxycholate at different rates of infusion of bile salt in rabbits anaesthetized with pentobarbitone.
'Choleretic efficiency' was calculated rate of secretion of bile salt in bile).
as
(increase in bile
flow)/(increase in
BILE SECRETION IN THE RABBIT
575
TABLE 2. Net changes in the composition of bile caused by the infusion of bile salt (TDC or TC 5 psmole/min i.v.). Figures listed give the mean change ± s.E. of mean Taurodeoxycholate Taurocholate Na +11-7 ±1-2 +11-8 ±1-2 K +0-12+0-05 +0-15±016 Cl -13-7 ± 1-7 (m-equiv/1.) -12-8 + 1-1 +8-2 ±1-9 HCO3 -2.9 + 1-8 Bile salt + 32-4 + 2-6 +16-0± 1-4 -3-3 + 1-5 + 0-2 + 1-3 Osmolality (m-osmole/kg) pH + 0-050 ± 0*015 TDC infused Acetazolamide 3-0
020 E
110
-
60
so E 0 U
303
0
020 ._
0
.0
(U
L.10
1
2. Time (hr)
3
4
Fig. 7. The effect of an infusion of sodium taurodeoxycholate (5 limole/min) on the concentration of bicarbonate in bile (LI---LI) and in plasma (U--), after the prior injection of a single dose of acetazolamide (300 mg i.v.) in a rabbit anaesthetized with urethane. The dotted line for the concentration of bicarbonate in bile ( .... ) indicates the level of bicarbonate concentration which would have been expected, after the injection of acetazolamide, had the bile salt not been infused.
576 S. C. B. RUTISHA USER AND THE LATE S. L. STONE seen that the effect of TDC was merely superimposed upon the effect of acetazolamide. Effects of BSP In the course of some experiments which were designed to examine the effects of TDC and TC on the secretion of BSP into bile, it was observed that BSP itself had some interesting effects on the composition of bile. The secretion of BSP into bile increased the biliary concentrations of sodium and of potassium. and decreased the concentrations of chloride and TABLE 3. The effects of acetazolamide (10 mg/kg body wt.) on the ionic composition of bile and of plasma, and on the rate of bile flow. The figures give the mean + S.E. for three experiments with the drug in animals anaesthetized with urethane Control
A. Bile Na K Cl
HCO3
158-7±2-8
\ j
(m-equiv/l.)
4-2+0-4 90-2 + 1-2
40-8+220
Bilesalt 11-4+1-1 Bile flow (fl./min.g liver) 1-028 + 0-178 B. Plasma K 2-9+0-4 1 Cl 3 (m-equiv/l.) 97.7 + 1-2 HCO3I 12-6 +12
After acetazolamide
160-6+1 9 5-3+0-6 84-8 + 2-5 51-7+0 9 8-1+0-5 1-136 + 0-078
3-5+0 5 95-7 + 2-0 18-3 + 1-3
TABLE 4. Net changes in the composition of bile and of plasma, caused by the infusion of bromsulphthalein (5 mg/min). The figures give the mean change in composition + s.E. of the mean in six experiments. * indicates results from only three experiments Bile Plasma Na +36-0±1-5 K* | +1-76±0-28 Cl -10-7 2-3 (m-equiv/l.) HCO3 -26-3+1-2 -2-3+0 -4 BSP +53-9 3-0 Osmolality (m-osmole/kg) -17-5 ± 1-2 -2-3 + 0-9 pH* -0-18±0-05 +0-07±0-03
of bicarbonate. In every case the osmolality of bile also decreased such that bile became hypotonic with respect to plasma. The quantitative data for the changes observed in the composition of bile and of plasma are shown in Table 4. The concentrations of sodium, potassium and chloride in plasma were not affected by the infusion of BSP. The decrease in the biliary concentration of bicarbonate is quite considerable. Although the concentration of bicarbonate in the plasma also decreased, the decrease in biliary bicarbonate is much greater, such that
577 BILE SECRETION IN THE RABBIT the bile: plasma ratio for this ion decreased from 2-74 + 023 to 1'58 + 0d14 (s.E. of mean) after the infusion of BSP. In fact the total bicarbonate excreted in bile per unit time decreased in each of the six experiments with BSP, despite the fact that the flow of bile was increased in three of these. The effect of BSP on the flow of bile was somewhat variable, in that flow was increased in three experiments, decreased in one, and was unchanged in the remaining two. The maximum concentration of BSP in bile was 23-1 + 3.5 mg/ml. (S.D. of an observation) in six experiments. The maximum rate of excretion of BSP into bile, obtained in the presence of a rising concentration of BSP in the plasma was 59-9 + 12-6 n-mole/min . g liver (S.D. of an observation). This is roughly equivalent to 1-7 + 04 ptmole/ min. kg body wt. DISCUSSION
Bile secretion has been studied in a variety of species including the rabbit, guinea-pig, rat, cat and dog, and it is clear from a number of studies, that significant differences exist between these species, in terms of the rate at which bile is formed, the composition of bile, and the responsiveness of the biliary system to different agents (Pugh & Stone, 1969; Scratcherd, 1965; Bizard, 1965; Stone, 1965; Klaassen, 1972). The picture is further complicated by the fact that a variety of anaesthetics have been used. For example, urethane is commonly used as an anaesthetic in rabbits, and guinea-pigs, whereas pentobarbitone sodium is generally the anaesthetic of choice for the dog. Mode of anaesthesia It is clear from the results presented in this paper, that the mode of anaesthesia has a profound effect on the flow and composition of bile in at least one species, namely the rabbit. This may be interpreted in at least one of two ways: either pentobarbitone stimulates the secretion of bile, or urethane depresses it. In order to test the first possibility it would be necessary to compare the rate of flow of bile in anaesthetized and unanaesthetized animals. This was not possible in the present work. However, the results presented strongly suggest that the intraperitoneal injection of urethane decreases bile flow, and that this is because the solution injected is considerably hypertonic with respect to the blood plasma. The results obtained with urethane are of interest in clarifying some points in literature. Scratcherd (1965) observed that bile flow could be maintained in rabbits, provided a suitable solution of electrolytes was infused. As he too used urethane as anaesthetic, it may be that the electrolyte solution successfully replaced the fluid which was lost from the abdominal cavity, and thus bile flow was maintained. Pugh & Stone (1969) quoted values for the flow and composition of bile
578 S. C. B. RUTISHAUSER AND THE LATE S. L. STONE in rabbits anaesthetized with urethane. Although the value given for the rate of flow of bile (1 67 #t1./min.g liver) is consistent with the present data, their values for biliary bicarbonate concentration are somewhat lower (32.7 m-equiv/l.) than those quoted here. This may be partly explained by the observation, that biliary bicarbonate concentration tended to decline with time in the animals anaesthetized with urethane. The extent of the decline will depend on the length of time elapsing between the injection of the anaesthetic and the collection of bile for analysis, and the amount of bicarbonate lost in that time in the i.P. fluid. It may be that this time was somewhat longer in the experiments of Pugh & Stone. Plasma osmolality and bile flow The effects of the presence of a hypertonic solution in the duodenum on the rate of bile flow have been described (Knightly, Vanamee & Lawrence, 1960), and it has been demonstrated that the i.v. injection of hypertonic solutions decreases bile flow in nephrectomized guinea-pigs (Chenderovitch, Phocas & Rautureau, 1963). Clearly, the presence of a hypertonic solution in the abdominal cavity will cause the withdrawal of fluid and diffusible electrolyte from the plasma, and that this, together with the absorption of the injected solute into the plasma, will increase plasma osmolality. In the present experiments the increase in plasma osmolality was linearly related to the decrease in bile flow, such that an increase in osmolality of 37 m-osmole/kg decreased bile flow by 1 OIjc./min.g liver. It is surprising that so small an increase in plasma osmolality should have such a marked effect on bile flow. This suggests that the underlying mechanism for the formation of the fluid fraction of bile is particularly sensitive to changes in plasma osmolality. Sperber (1959) originally suggested that the driving force for the formation of bile was the creation of an osmotic gradient between bile and plasma, by the active secretion of non-diffusible bile salt anions into the biliary canaliculus. Clearly, an increase in plasma osmolality will affect the osmotic gradient between bile and plasma, but whether this is an adequate explanation of the effect remains to be seen.
Choleretic efficiency of bile salts Several bile salts have been shown to differ in their choleretic efficiency. O'Maille, Richards & Short (1965) found that cholate was more efficient than taurocholate in increasing bile flow, and Sperber (1959) and others have presented data which show that dehydrocholate is exceptionally good as a choleretic. It has been assumed that the underlying reason for such differences in choleretic efficiency has been the different physico-chemical properties of the bile salts. The greater choleretic efficiency of cholate and
.579 BILE SECRETION IN THE RABBIT dehydrocholate is assumed to be due to the lesser ability of these bile salts to form micelles. More osmotically active particles are thus thought to be available per mole of bile salt secreted, than for taurocholate which forms larger aggregates. The tendency for bile salts to form micelles depends on their structure and their polarity, and it has been shown in simple physicochemical systems that deoxycholate, conjugated with either glycine or taurine, forms much larger aggregates than taurocholate (Small, 1971). It would therefore be expected that these bile salts would prove to be less efficient as choleretics than taurocholate. It is surprising, therefore, that both taurodeoxycholate, and glycodeoxycholate (Erlinger et al. 1970) should in fact be more efficient than taurocholate in the rabbit. Unless these bile salts exhibit some rather unusual physico-chemical properties in rabbit bile, it would seem very unlikely that their choleretic efficiency can be explained in terms of reduced micelle formation. It is therefore of interest, that taurodeoxycholate, unlike taurocholate, consistently increased the bicarbonate concentration of bile. This suggests that taurodeoxycholate may stimulate a bicarbonate pump in the biliary system, and that this is responsible for the increased output of water and electrolyte in bile. It may also be noted that the choleretic efficiency of taurodeoxycholate decreased as the rate of infusion of bile salt increased. Such a change in choleretic efficiency might be expected if the increase in bile flow was the summed effect of taurodeoxycholate on two independent mechanisms of bile secretion. The total bile flow, at any given infusion rate, would then depend on the individual contributions made by each mechanism at that infusion rate. It is conceivable that the formation of the electrolyte component is maximally stimulated at low rates of bile salt infusion. As the rate of infusion of bile salt is increased, that fraction of bile which is directly dependent on the active secretion of bile salt into the canaliculus will form an increasingly greater fraction of the total flow. Hence at low rates of bile salt infusion the calculated choleretic efficiency of taurodeoxycholate would be high, but this would decrease as the rate of infusion of bile salt was increased. Alternatively, it may be argued, that at high rates of infusion of bile salt, the permeability of the biliary system is affected, and thus the choleretic efficiency of the bile salt declines. But the fact remains that taurodeoxycholate is a more efficient choleretic than taurocholate in the rabbit, and this implies that the two bile salts exert very different effects on the biliary system, regardless of the mechanism involved. Erlinger et al. (1970) postulated that 60 % of spontaneous bile flow in the rabbit is dependent on the activity of a sodium pump believed to be located at the level of the canaliculus. It could be suggested therefore that taurodeoxycholate is able to stimulate this pump, and that the increase in
580 S. 0. B. RUTISHA USER AND THE LATE S. L. STONE biliary bicarbonate concentration is either a result of the effect of a change in flow rate on some ductular exchange mechanisms, or is secondary to a local increase in the Pco2. The second possibility is unlikely in that the pH of bile would be expected to decrease or remain the same. In fact the infusion of taurodeoxycholate was usually associated with a slight increase in the pH of bile. It would be of interest to examine the effect of the inhibitors used by Erlinger and his group, on the biliary response to taurodeoxycholate. If taurodeoxycholate is stimulating a sodium pump, then the choleretic efficiency of the bile salt should be reduced by substances such as ouabain, amiloride, and ethacrynic acid, all of which inhibit the Na/K ATPase involved in sodium transport. It is of interest, however, that Erlinger et al. (1970) found that the choleretic efficiency of glycodeoxycholate was unchanged by the presence of these inhibitors.
Bicarbonate in bile The enzyme, carbonic anhydrase is usually involved in most systems which are responsible for the formation and transport of bicarbonate in the body. As the concentration of bicarbonate in rabbit bile is about twice that in the plasma, it is to be expected that a bicarbonate pump of some description must be present in the biliary system. The results presented here have shown that the carbonic anhydrase inhibitor, acetazolamide, does not depress the excretion of bicarbonate in bile, and that the increase in bicarbonate concentration produced by taurodeoxycholate also cannot be prevented by the prior injection of this inhibitor. Maren (1967, and personal communication) found that the carbonic anhydrase which may be extracted from rabbit liver is refractory to acetazolamide and other sulfonamides. The in vivo results presented here correlate well with Maren's in vitro data. It would seem that the mechanism involved in the secretion of bicarbonate in the biliary system of the rabbit possesses some unusual features. In this context it is of interest that the hormone secretin, which is able to stimulate the production of a bicarbonate rich fluid in the biliary systems of the dog, cat and guinea-pig (Wheeler & Ramos, 1960; Scratcherd, 1965; Forker, 1967) is completely without effect on the biliary system of the rabbit (Scratcherd, 1965). Indeed it would seem that the role of this hormone has been adopted by the dihydroxy bile salt, deoxycholate, and it is tempting to speculate that the dihydroxy bile salts may in fact act as the 'secretin' of the rabbit in relation to the biliary system.
Bromsulphthalein Finally, the effect of the biliary excretion of another anion, bromsulphthalein (BSP), on the composition of bile may be compared with the
581 BILE SECRETION IN THE RABBIT effects of the bile salt anions. BSP is frequently used as a model of organic anion transport in the liver. The maximum rate at which it is transported into bile may be affected by the excretion of bile salt. It is believed that this is due to the decrease in the biliary concentration of BSP which occurs as bile flow is increased (O'Maille, Richards & Short, 1966). BSP, unlike the bile salts, is a disodium salt, and thus the marked increase in the concentration of sodium in bile, during the excretion of BSP in bile, is not unexpected. It is also expected that the secretion of an anion into bile will decrease the concentration of the other diffusible anions present. However, the degree to which BSP decreased the concentration of bicarbonate in bile is surprising, particularly when this is compared with the effect of taurocholate on the biliary concentration of bicarbonate. An increase in the taurocholate concentration of bile of 50 m-equiv/l. only decreased the concentration of bicarbonate in bile by about 5 m-equiv/l., whereas an increase of 56 m-equiv/l. in the concentration of BSP in bile decreased biliary bicarbonate concentration by about 25 m-equiv/l. Taurocholate and BSP also have different effects on the osmolality of bile. The infusion of bile salt never caused any significant changes in the osmolality of bile or of plasma, whereas the infusion of BSP consistently decreased the osmolality of bile such that bile became hypotonic with respect to plasma. The low osmolality of the bile samples is unlikely to be due to aggregation of the BSP molecules at the low temperatures obtained in the freezing point osmometer because the osmolality of a pure solution of BSP was determined both in a freezing point osmometer, and in a vapour pressure osmometer, and the values obtained were practically identical. The simplest explanation of the effects of BSP on the composition of bile would be that at some point in the biliary system acid is being added to bile, and bicarbonate is thus being removed. Isotonicity is possibly not maintained, because the permeability of the biliary system at that point, and the rate of flow of bile is such, that proper equilibration of the bile with plasma is not achieved. Clearly, the three anions studied, taurodeoxycholate, taurocholate, and bromsulphthalein, differ profoundly in the effects that they have on the flow and composition of bile. This raises some questions in terms of the kinetics of the underlying transport mechanisms. For if the anions differ in this way, then the factors limiting their secretion into bile may not be simply the availability of carrier sites at the biliary membrane, but may be the prevailing conditions within the canaliculus itself. Thanks are due to Mr B. M. Shears for his very capable and willing assistance, to Mr T. J. Surman for his help in the initial stages of the work, and to Dr J. S. Thomas for very kindly reading the manuscript. S.C.B.R. was in receipt of an M.R.C. Scholarship during part of this work.
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