549

J. Physiol. (1975), 245, pp. 549-565 With 8 text-ftgurew Printed in Great Britain

THE EFFECT OF SODIUM SALICYLATE ON BILE SECRETION IN THE DOG

BY SIGRID C. B. RUTISHAUSER AND THE LATE S. L. STONE From the Department of Physiology, University College, Cardiff CF1 1XL

(Received 3 May 1974) SUMMARY 1. The i.v. injection of sodium salicylate (100 mg/kg) in the dog caused a rapid and maintained choleresis of the order of 300-600% of control

levels. 2. The total amount of salicylate excreted in bile was only 1-2 % of that injected. 3. The secretion of bile salt into bile was not increased by salicylate. 4. The choleresis caused by salicylate was associated with a decrease in the concentrations of sodium and of bile salt in bile, and with an increase in the concentration of chloride; the biliary concentration of bicarbonate was either temporarily increased or unchanged. 5. The choleresis could not be inhibited by the intra-portal injection of

ouabain (0.1 mg/kg). 6. The secretion of bromsulphthalein into bile was not potentiated by the choleresis. 7. The choleretic efficiency of sodium taurocholate was not increased in the presence of salicylate. 8. The injection of acetazolamide (20 mg/kg), in the presence of a salicylate choleresis, caused an increase in the osmolality of bile and an increase in biliary sodium concentration, such that the composition of bile more nearly approached that of plasma. 9. The possible mechanisms underlying the choleretic effect of sodium salicylate are discussed. INTRODUCTION

A number of substances other than bile salts and secretin have been shown to increase the flow of hepatic bile in the dog. These include 2,4-dinitrophenol, and structurally related compounds (Pugh & Stone, 1968). Sodium salicylate has also been shown to act as a choleretic in a Address for correspondence: Dr S. C. B. Rutishauser, Department of Physiology,

Stopford Building, University of Manchester, Manchester M13 9PT.

550 S. C. B. RUTISHA USER AND THE LATE S. L. STONE variety of species including the dog (Okada, 1915). It is of interest that sodium salicylate resembles 2,4-dinitrophenol in its action as an uncoupler of oxidative phosphorylation (Miyahara & Karler, 1965). This suggests that both these substances may be acting at similar sites in the biliary system. The mechanism underlying their choleretic effect is unknown. As salicylate, unlike 2,4-dinitrophenol, may be determined easily in blood and in bile, a detailed study of its choleretic effect was undertaken in order to investigate the mechanism involved. A preliminary account of this work has been published (Rutishauser & Stone, 1973). METHODS

Surgery and design of experiments. Mongrel dogs of both sexes were anaesthetized with sodium pentobarbitone (30 mg/kg i.v.). The common bile duct was cannulated after ligation of the cystic duct, and drainage of the gall-bladder. The spleen was removed to ensure that the circulating blood volume remained constant during an experiment. In most experiments the ureters were cannulated for the collection of urine. In some experiments the animals were nephrectomized: these experiments are noted in the text. Drugs were administered i.v. through a cannula lying in the jugular vein. Infusions were given via a cannula lying in the femoral vein. Blood samples were obtained from the left carotid artery. Bile and urine samples were collected, in general, as 10 or 20 min samples in 10 ml. tubes, graduated in divisions of 0-1 ml. Blood pressure, e.c.g., heart rate, respiratory rate, and oxygen consumption were measured in some experiments. Oxygen consumption was measured using a rebreathing spirometer. Body temperature was recorded from a thermometer lying in the right axilla. Salicylic acid (100 mg/kg) was dissolved in its equivalent of 3M-NaOH, and some 0.1 m-(Na) phosphate buffer, pH 7 4, and administered as a single dose intravenously (total volume 10-20 ml.). Ouabain (0-1 mg/kg) was injected in small doses of 200-500 ,ag at a time, over a period of 1-2 hr, into the hepatic portal vein, via a cannula lying in the splenic vein. This was done to ensure that the liver would receive the first effect of the inhibitor, before it was distributed to the rest of the body. In the experiments with bromsulphthalein (BSP), a priming dose of BSP (5 mg/kg) was followed by a constant infusion of the dye (5 mg/min) designed to produce maximum excretion rates of BSP in the bile, and a rising concentration of BSP in the plasma. When the rate of excretion of BSP in bile was judged to have reached its plateau, a single dose of sodium salicylate was given intravenously. The solutions of BSP and of sodium taurocholate used for infusion were made up in 0.9% NaCl. Acetazolamide (20 mg/kg) was dissolved in 0-9 % NaCl solution, and injected i.v. as a single dose. Analytical methods. Salicylate in bile and in plasma was determined using the method of Routh & Dryer (1961). BSP in bile and in plasma was determined colorimetrically at 580 nm, after appropriate dilution and alkalinization. Sodium and potassium concentrations were determined by flame photometry; chloride was measured using a Buchler-Cotlove chloridometer; bicarbonate was determined in a Natelson micro-gasometer using the manometric method of Van Slyke; calcium and magnesium were determined in an atomic absorption spectrometer; osmolality was measured cryoscopically in an Advanced osmometer.

SODIUM SALICYLATE AND BILE SECRETION

551

Bile salts in bile were determined by one of two methods: either by the method of Irvin, Johnston & Kopala (1944), as modified by O'Maille, Richards & Short (1965), which measures the concentration of cholate, free and conjugated, in bile; or by the use of the enzyme steroid dehydrogenase, in the method detailed by Javitt & Emerman (1968), which measures all the bile salts present in bile bearing a 3-hydroxyl group.

RESULTS

General effects of salicylate The i.v. injection of sodium salicylate caused a prompt increase in bile flow. Flow increased to a maximum within 2-3 min after the injection of the drug, and was either maintained at this level for the remainder of the experiment (a further 2-3 hr) or declined gradually during this period. The rate of bile flow after the injection of salicylate was generally about 300-600 % of that in the control period. There was a temporary increase in systemic blood pressure (20-30 mmHg) immediately after the injection of the drug. A slight increase in heart rate was sometimes seen, but no obvious changes were noted in the pattern of the e.c.g. A temporary and quite marked diuresis also occurred in some experiments. The rate of oxygen consumption of the animal increased within a few minutes of the injection of salicylate, and remained elevated for the remainder of the experiment (Fig. 1). The increase in metabolic rate was accompanied by an increase in the production of heat, and the body temperature rose.

Excretion of salicylate in bile Only a small percentage of the administered dose of salicylate was recovered in the bile in the course of a 3 hr experiment. The mean percentage recovery was 1-56 + 0 33 (s.E. of mean; n = 6). Hydrolysis of the bile samples did not increase the percentage recovery. The mean concentration of salicylate in bile during a choleresis was 3 43 + 0'19 gtmole/ml. (S.E. of mean), and this was of the same order as that in the plasma. Ionic composition of bile The increase in bile flow caused by salicylate was accompanied by a marked decrease in the concentration of bile salt in bile (Fig. 2). If bile flow is multiplied by the bile salt concentration of bile, an estimate of the rate of bile salt secretion by the liver is obtained (biliary bile salt content). It can be seen from Fig. 2 that, apart from an initial 'wash-out' of bile salt from the dead space of the biliary system, the rate of bile salt secretion was not increased, but declined with time in a manner consistent with depletion of the bile salt pool. Both methods of bile salt determination gave the same results.

552 S. C. B. RUTISHA USER AND THE LATE S. L. STONE

The effects of sodium salicylate on the concentrations of the other major ions in bile is illustrated in Fig. 3. The fall in the concentration of bile salt was accompanied by a decrease in the concentration of sodium, and with a reciprocal rise in the concentration of chloride in bile. It was a characteristic feature of the response to sodium salicylate, that the concentration of sodium in bile often fell to very low levels (135-145 m-equiv/l.). In the experiment illustrated in Fig. 3 there was little change inthe concentrations of potassium, and of bicarbonate in bile. Neither of these two ions followed Salicylate

40

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80-

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1

1

2 3 Time (hr) Fig. 1. The effect of a single injection of sodium salicylate (100 mg/kg body wt.) on bile flow, total oxygen consumption and body temperature in the anaesthetized dog. Salicylate was injected i.v. at the time indicated by the arrow.

a consistent pattern in other experiments. A temporary increase in the concentration of bicarbonate in bile was seen in some experiments in the first hour after the injection of salicylate, but thereafter the concentration of bicarbonate fell below its pre-injection level, although the rate of bile flow at this point was still increased. The concentrations of calcium and magnesium in bile were also decreased after the injection of salicylate. The osmolality of bile either decreased or was unchanged. The total ionic concentration of bile was, however, considerably decreased. Thus the osmotic coefficient of bile (osmolality/total ionic concentration) was increased by the injection of salicylate. The osmotic coefficient gives a

553 SODIUM SALICYLATE AND BILE SECRETION measure of the proportion of the dissolved substances in a solution which are osmotically active. The results indicate that a greater proportion of the dissolved substances present in bile are osmotically active in the choleresis caused by salicylate than in the previous control period (Fig. 4). Salicylate 25 o0 bO 15

0c5 5.05 E

_

0 30 °Q E

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cO01017

i

25 .2 bO w: 15

F~ig. 2. The effect of a single injection of sodium salicylate (100 mgfkg body wt.) on the secretion of bile salt into bile. 'Bile salt secretion rate' was calculated as the product of the rate of bile flow and the bile salt concentration of each sample of bile. Salicylate wvas injected I.v. at the time indicated by the arrow.

Excretion of BSP into bile A typical experiment is illustrated in Fig. 5. The injection of salicylate was associated with a decrease in the concentration of BSP in bile, such that the total amount of BSP excreted per unit time was unchanged. The increase in the excretion of BSP immediately after the injection of salicylate is due to the 'wash-out' of BSP from the biliary dead space. The percentage changes in bile flow and BSP excretion rate in six experiments with salicylate are shown in Table 1.

554 S. C. B. RUTISHA USER AND THE LATE S. L. STONE It can be seen that although the rate of bile flow was approximately doubled in all but one of the experiments listed in Table 1, the rate of excretion of BSP in bile was not consistently increased by the salicylate choleresis. Salicylate 200 180 . 160 c 140 .° .' 120 w 100 _ 80.4--v8E 80i-A C U g 60 40 20 1

2 Time (hr)

3

Sodium

Chloride Bicarbonate Bile salt

4

Fig. 3. The effect of a single injection of sodium salicylate (100 mg/kg body wt.) on the ionic composition of bile. Salicylate was injected at the time indicated by the arrow. Sodium (0-*), chloride ( - - - A), bicarbonate (A-A), and bile salt ( x -x ) concentrations.

Effects of ouabain The injected dose of ouabain had considerable effects on the cardiovascular system: quite bizarre patterns were seen in the e.c.g., and the systemic blood pressure was increased by 20-S80 mmHg after the injection of the inhibitor. In spite of these very marked effects on the cardiovascular system, ouabain had little demonstrable effect on the choleresis caused by salicylate. One such experiment is illustrated in Fig. 6. The concentrations of sodium and of bile salt in bile showed little net change after ouabain, but the concentration of potassium in bile was consistently increased by 2-4 m-equiv/l. In only one experiment out of five did ouabain produce a decrease in bile flow coupled with an increase in the concentration of bile salt in bile. The effect of ouabain was also studied in dogs in the absence of salicylate. The biliary effects were again inconsistent. Apart from a consistent increase in the concentration of potassium in bile, no clear effects were seen on either the flow of bile, or on its composition.

SODIUM SALICYLATE AND BILE SECRETION

555

Choleretic efficiency of sodium taurocholate The choleretic efficiency of sodium taurocholate was determined in one of two ways: in single injection studies, the total volume of bile produced in response to sodium taurocholate, in excess of the control secretion levels, was divided by the total amount of the injected dose of bile salt which was recovered in the bile; in infusion studies, the rate of bile flow was plotted against the rate of bile salt secretion in bile, the slope of the line giving a measure of the choleretic efficiency of the bile salt. Salicylate

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3 2 Time (hr) Fig. 4. The effect of a single injection of sodium salicylate (100 mg/kg body wt.) on the osmolality, total ionic concentration, and osmotic coefficient of bile. Salicylate was injected i.v. at the arrow. 'Total ionic concentration' was calculated as twice the sum of the concentrations of sodium, potassium, calcium and magnesium in bile. 'Osmotic coefficient' was calculated by dividing the bile osmolality by the 'total ionic concentration'.

1

556 S. C. B. RUTISHA USER AND THE LATE S. L. STONE

Both methods of determination showed that the choleretic efficiency of sodium taurocholate was the same in the presence and absence of a salicylate choleresis. In the single injection studies (sodium taurocholate 50 mg/kg), the choleretic efficiency of the bile salt was 9-9 + 0'7 ml./m-mole in the presence of a salicylate choleresis, and 10-9 + 0 4 ml./m-mole in the Salicylate

BSP priming dose and constant infusion 1.08 Z 068 E 0.4

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2 Time (hr)

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Fig. 5. The effect of sodium salicylate on the excretion of BSP into bile. At the first arrow a priming dose of BSP was given (5 mg/kg) followed by a constant infusion of BSP (5 mg/min). Sodium salicylate (100 mg/kg) was injected I.v. at the second arrow. Bile BSP content was calculated as the product of the flow and the BSP concentration of each sample of bile.

absence of the drug (S.D. of mean). Similarly in the infusion studies at much lower rates of bile salt infusion (15-30 n-mole/min. g liver), the choleretic efficiency of taurocholate was effectively the same before and after the injection of salicylate (Fig. 7). The secretion of bile salt into bile

557 SODIUM SALICYLATE AND BILE SECRETION was not apparently affected by salicylate because the increase in the rate of bile salt secretion into bile following the infusion of bile salt, was the same with and without salicylate. TABLE 1. Effect of sodium salicylate on bile flow, and on BSP excretion into bile in six individual experiments. The changes in bile flow and BSP excretion have been expressed as percentages of the control rates

Change in BSP excretion

Dose

Increase in bile flow

(mg/kg)

(%)

(%/0)

50

+86X8 + 76X7 + 37-6 +118-1 +81-2 +139-2

+13X1 +1-5 - 203* -5-5

100 100 100 100 *

+ 21P5

+30

Indicates that bile flow only increased temporarily.

The effects of acetazolamide The injection of acetazolamide during the choleresis caused by salicylate had several consistent effects: it produced a maintained increase in bile flow; it increased the concentration of sodium in bile, such that this now approached the sodium concentration of plasma; it increased the osmolality of bile, such that in the experiments where bile had become hypotonic to plasma after the injection of salicylate, the formation of bile more nearly isotonic with plasma was restored; it increased the concentration of chloride in bile, and this was inversely related to a decrease in the concentration of bicarbonate in bile. These changes are illustrated in Fig. 8 which depicts the results of a typical experiment. In these experiments all the dogs were previously nephrectomized. It was observed that where the injection of acetazolamide did not produce very marked changes in the osmolality of bile (+ 9 m-osmole/kg), and in its sodium concentration ( +4 m-equiv/l.), the effect on the concentration of bicarbonate in bile was very much more marked (-34 m-equiv/l.) than in the experiment shown. The amounts of bicarbonate excreted in bile before and after the injection of acetazolamide are shown in Table 2. Other experiments Bilateral cervical vagotomy, and atropinization (atropine sulphate 1-0-1.5 mg iv.) did not prevent the choleretic effect of salicylate.

558 S. C. B. RUTISHA USER AND THE LATE S. L. STONE Salicylate

^ 06

Ouabain00

EIF1

05

02~~~~~~~~~~~~04 iE 0 4 =2 g

2

150_

Sodium

I? 100_ Chloride

*' CucS .0

U

,Bile salt 1

2 3 Time (hr) Fig. 6. The effect of ouabain (total dose 1 mg/lO kg body wt.) on the choleresis caused by sodium salicylate (100 mg/kg body wt.). Salicylate was injected I.v. at the first arrow. Ouabain was injected intra-portally in small doses of 0-2-0-5 mg at each of the points marked by the succeeding arrows. The length of the arrow and the scale at the top right-hand side of the diagram gives the amount in mg injected each time. Sodium (0-a), chloride (A-A), and bile salt (x - x ) concentrations in bile.

SODIUM SALICYLATE AND BILE SECRETION

559

0-5

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0-24_

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0-3

bo

A

0.1 I

,

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l

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10 20 30 40 50 Bile salt secretion rate (n-molejmin.g liver) Fig. 7. The relationship between bile flow and bile salt secretion rate during the constant infusion of sodium taurocholate at two known rates in the presence (0-0) and in the absence (A-A) of a salicylate choleresis. The choleretic efficiency of sodium taurocholate was calculated from the slopes of the lines and was 10-3 ml.fm-mole in the absence of salicylate, and 9.8 ml./m-mole in the presence of salicylate (mean slope of the two lines). The arrows indicate increase and decrease of infusion rate. The control bile flows before and after the infusion of sodium taurocholate are not identical in the presence of salicylate because of the tendency for bile flow to decrease with time (see Figs. 1 and 2). TABLE 2. The effect of acetazolamide (20 mg/kg) on the excretion of bicarbonate in bile, and on bile flow in three nephrectomized dogs

Bicarbonate excretion

(/sequiv/min)

Bile flow ul./min.g liver

A-

A B C

Control 5.3

10*0 18.1

After acetazolamide 10-4 8-4

19*2

Control 0-302 0-254 0-464

After acetazolamide 0-616

0*381 0-580

DISCUSSION

At least three mechanisms are believed to be involved in the formation of the fluid fraction of bile. First, the active transport of bile salt into the biliary canaliculus is thought to create an osmotic force which causes the passage of water and electrolytes into bile (Sperber, 1959). Secondly, it has been shown that the hormone, secretin, is able to stimulate the

560 S. C. B. RUTISHA USER AND THE LATE S. L. STONE Salicylate Acetazolamide

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if

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~~~~I\_M_'Bicarbonate A

~Bile salt I~ X_.ZX 1 2 3 Time (hr)

Fig. 8. The effects of acetazolamide (20 mg/kg i.v.) on the flow and composition of bile after the prior administration of sodium salicylate (100 mg/kg i.v.). Salicylate was given at the first arrow; acetazolamide was given at the second arrow. Sodium (e*-), chloride (A A ), bicarbonate (f-f*), and bile salt ( x x ) concentrations in bile, and bile osmolality (-+). Open symbols represent values for plasma sodium (0-0) and plasma osmolality ---

-

(

>-

>).

561 SODIUM SALICYLATE AND BILE SECRETION production of a bicarbonate rich bile (Wheeler & Ramos, 1960). It is believed that this hormone may act on the ductular part of the biliary system (Wheeler, 1965; O'Maille, Richards & Short, 1966). Thirdly, to account for the exceptionally high rates of bile formation found in animals such as rabbits, guinea-pigs and rats, a third mechanism must be postulated. In the rabbit, it has been shown that inhibitors of the Na/K dependent ATPase, such as ouabain, ethacrynic acid, and amiloride, are able to depress the rate of bile formation without any change in the rate of bile salt secretion (Erlinger, Dhumeaux, Berthelot & Dumont, 1970). It is therefore suggested, that a sodium pump, sited at the canaliculus, may also contribute to the formation of the fluid fraction of bile in this species. The choleresis produced by salicylate has several characteristic features: it is rapid in onset, may be maintained for several hours, and is associated with a marked decrease in the concentrations of sodium and bile salt in bile, and with an increase in the concentration of chloride. The precise mechanism involved in its choleretic effect remains obscure, but the results of the present work have ruled out several possibilities: Firstly, analysis of the bile showed that the secretion of bile salt was not increased by salicylate. It is unlikely that the choleresis is produced by a bile salt anion which cannot be detected by either of the methods of bile salt analysis used, partly because both methods of analysis gave similar qualitative results, but also because no significant discrepancy was found between the sum of the anions, and the sum of the cations in bile. The choleresis therefore, is not due to an increased rate of bile salt secretion in bile. The ionic data argues strongly against the possibility that salicylate is releasing secretin, and thereby stimulating bile flow. The choleresis caused by secretin is generally associated with a maintained increase in the bicarbonate concentration of bile; in addition the sodium concentration shows little change, and there is a slight increase in the osmolality of the bile (Wheeler & Ramos, 1960; Preisig, Cooper & Wheeler, 1962). These effects are different from those obtained with salicylate. In this case, although there was a temporary increase in the bicarbonate concentration of bile in some experiments, the final concentration of this ion in bile after salicylate was less than in the control period, even though bile flow at this point was still elevated. Furthermore, salicylate consistently decreased the concentration of sodium in bile. No clear evidence was obtained for the involvement of a sodium pump in the choleretic effect of salicylate. The fact that ouabain did not inhibit the choleresis does not however rule out the possibility that salicylate may be stimulating bile flow through increasing the activity of a sodium pump. It is conceivable that the ouabain was unable to reach its appropriate site 24

P HY 245

562 S. C. B. RUTISHAUSER AND THE LATE S. L. STONE of action, or that the sodium/potassium ATPase in dog liver is not sensitive to this inhibitor. Thus it would seem that the salicylate choleresis cannot be readily explained in terms of stimulation of one of the known mechanisms of bile secretion. It may also be noted that the increase in bile flow was not secondary to the increase in body temperature. Bile flow increased within a few minutes of the injection of the drug, whereas the body temperature rose over 2-3 hr. Stone (1965) has previously shown that raising the body temperature to the level found in these experiments, by extracorporeal warming of the blood, has very little effect on bile flow in the dog. The choleresis also cannot be accounted for in terms of an osmotic choleresis caused by the salicylate anion itself, because the total amount of salicylate excreted in bile is too small. As salicylate is still able to cause a choleresis after vagotomy and atropinization, it is inferred that cholinergic mechanisms are not of primary importance in the response to the drug. It was of interest to try and determine the site at which salicylate was adding fluid to bile. O'Maille et al. (1966), showed that the addition of fluid to bile at the level of the canaliculus increased the maximum rate of secretion of BSP into bile, whereas the addition of water downstream in the ductular region of the biliary system, as for example in a secretin choleresis, had no effect on the rate of secretion of BSP. In the present work salicylate potentiated the excretion of BSP in only two out of five experiments and then only to a small extent (23 % increase in BSP excretion, with an 87 % increase in bile flow). These experiments may be compared with one in which a choleresis was induced with bile salt. In this case a 200 % increase in bile flow produced a 150 % increase in the excretion of bromsulphthalein. The results suggest therefore that salicylate may be adding fluid to bile at a site downstream from the canaliculus. However, recent work indicates that the potentiation of BSP secretion in bile may not, in fact, be a good criterion for determining the site at which fluid is added to bile. Forker & Gibson (1972), have shown that choleresis per se is not an important determinant of BSP secretion in the rat. It would be of interest, therefore, to determine the effect of sodium salicylate on the clearance of labelled erythritol into bile, as this has been used as a measure of canalicular flow rate. If salicylate does add fluid and electrolyte at the ductular level of the biliary system, at least three possible mechanisms could be involved: Firstly, salicylate could be stimulating a ductular secretary mechanism. At present, the only one known is the elaboration of a bicarbonate-rich fluid under the action of secretin. The evidence against such a mechanism has already been presented. Secondly, salicylate may be affecting the formation of micelles in bile. It is conceivable that the salicylate anion could

SODIUM SALICYLATE AND BILE SECRETION

563 in some way either disrupt the bile salt micelles present in bile, or prevent their formation. This would increase the number of osmotically active particles in bile, and therefore provide a gradient for the addition of more fluid and electrolyte to bile. The results have shown that there is an increase in the proportion of osmotically active particles in bile after the injection of salicylate. However as dilution of bile will also cause disruption of micelles, it could be that the increase in the osmotic coefficient was secondary to the increase in bile flow and not the primary cause of it. If salicylate were affecting the formation of micelles it would be expected that the choleretic efficiency of the bile salts secreted in bile would be altered. No evidence could be obtained for this, even at fairly low rates of bile secretion when such an effect might be expected to be most evident. This leaves the final possibility that salicylate may be inhibiting a reabsorptive mechanism. Other workers have suggested the existence of reabsorptive mechanisms in the biliary system of the dog (Wheeler, Ross & Bradley, 1968). Their results indicate that the rate of reabsorption is about 0-1 ml./min in a 20 kg dog which is equivalent to a bile flow of 0*167 #I/min . g liver. This is considerably less than the increase in bile flow obtained with sodium salicylate which was of the order of 0 5 ,uI/min . g liver. Salicylate has been shown to be effective as a choleretic in the rat also. Bullock, Delaney, Sawyer & Slater (1970) showed that the parenteral administration of sodium salicylate in the rat caused the proliferation of multivesicular bodies, and the Golgi apparatus in the vicinity of the canaliculi. They suggested that this might be related to the choleretic effect of the drug. These results are of interest, in view of the fact that the present results have indicated that salicylate may not be acting at the canaliculus, and that the rate of bile flow reached a maximum within 2-3 min of the injection of the drug - a length of time too short for significant structural changes to have occurred. The mechanism whereby salicylate increases bile flow is unclear: if it increases the activity of a canalicular secretary mechanism, then the lack of effect of the choleresis on the excretion of BSP in bile is of interest; if it stimulates the secretion of fluid in the ductular part of the biliary system, then a new secretary mechanism must be postulated; finally if it inhibits reabsorption of fluid in the ducts, then the extent of normal reabsorption in the dog is far in excess of anything yet quoted in the literature. Finally, the action of acetazolamide in the presence of a salicylate choleresis is of interest. The effects of acetazolamide on the flow and composition of bile are similar to those reported by Wheeler & Ramos (1960) and Stone (1965) in that bile flow increased, and that an increase in the concentration of chloride in bile was coupled with a decrease in the concentration of bicarbonate. No mention has, however, been made 24-2

564 S. C. B. RUTISHAUSER AND THE LATE S. L. STONE previously of an effect of acetazolamide on either the sodium concentration of bile, or on biliary osmolality. The present results suggest that sodium may be actively reabsorbed from bile, and that acetozolamide inhibits the active transport mechanism involved. The changes in the osmolality of bile are explicable if it is postulated that the movement of water is somewhat restricted at that part of the biliary system at which the active reabsorption of sodium is taking place. At high rates of bile flow this could cause the bile to become hypotonic while the reabsorption of sodium was occurring, but in the presence of acetazolamide, with the reabsorptive mechanism inhibited, biliary osmolality would be expected to increase to a value closer to that of the blood plasma. The fact that an effect of acetazolamide on the concentration of sodium in bile has not been noted before, maybe because of the different conditions under which the experiments were performed. For example, where the concentration of bile salt in bile is high, as in the experiments of Wheeler & Ramos, and of Stone, the sodium present in bile may be considered to exist in two forms, namely that which is freely diffusible, and that which is associated with the bile salt micelles and which is therefore restricted in its diffusion. Under these conditions the reabsorption of sodium, accompanied by some water, would lead to a decrease in the concentration of diffusible sodium in bile and to an increase in the concentration of sodium 'trapped' by the bile salt micelles. No net change in the concentration of sodium in bile might therefore be apparent. However, in the experiments with sodium salicylate, in which the concentration of bile salt in bile is low, the proportion of the sodium in bile which is 'trapped' by the bile salt micelles is very much less. Therefore changes in the concentration of diffusible sodium in bile would be expected to be made clear. It is of interest that acetazolamide did not decrease the excretion of bicarbonate in bile. The decrease in bilary bicarbonate concentration would seem to be due to the dilution of a bicarbonate-containing fluid with a solution of sodium chloride. Sullivan & Berndt (1973a, b) have studied the reabsorptive properties of the isolated rabbit gall-bladder, and have found that acetazolamide and 2,4-DNP depress volume transport in this organ. In the present work, salicylate, which resembles 2,4-DNP in its action on the biliary system, and acetazolamide have been shown to act as choleretics. By analogy with the work of Sullivan & Berndt it may be suggested that both salicylate and acetazolamide increase bile flow in the dog by inhibiting reabsorptive mechanisms in the ductular part of the biliary system. Thanks are due to Mr T. J. Surman and Mr B. M. Shears for their most able technical assistance, to Dr J. S. Thomas for his help in preparing the manuscript, and to the Medical Research Council for their award of a scholarship to S.C.B.R.

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REFERENCES BULLOCK, G. R., DELANEY, V. B., SAWYER, B. C. & SLATER, T. F. (1970). Biochemical and structural changes in rat liver resulting from the parenteral administration of a large dose of sodium salicylate. Biochem. Pharmac. 19, 245-253. ERLINGER, S., DHUI)EAUx, 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. & GIBSON, G. (1972). The Liver. Quantitative aspects of structure and function. 1st International Gstaad Symposium, pp. 326-336. Basel: Karger. IRVIN, J. L., JOHNSTON, C. G. & KOPALA, J. (1944). A photometric method for the determination of cholates in bile and blood. J. biol. Chem. 153, 439-457. JAVITT, N. B. & EMERMAN, S. (1968). Effect of sodium taurolithocholate on bile flow and bile acid excretion. J. clin. Invest. 47, 1002-1014. MIYAHARA, J. T. & KARLER, R. (1965). Effect of salicylate on oxidative phosphorylation and respiration of mitochondrial fragments. Biochem. J. 97, 194-198. OKADA, S. (1915). On the secretion of bile. J. Physiol. 49, 457-482. 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'MAIETa, 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. PREISIG, R., CooPER, H. L. & WHEELER, H. 0. (1962). The relationship between taurocholate secretion rate and bile production in the unanaesthetized dog during cholinergic blockade and during secretin administration. J. clin. Invest. 41, 1152-1162. PUGH, P. M. & STONE, S. L. (1968). The effect of 2,4-dinitrophenol and related compounds on bile secretion. J. Physiol. 198, 39-49. ROUTH, J. I. & DRYER, R. L. (1961). Salicylate. In Standard Methods of Clinictl Chemistry, vol. 3, ed. SELIGSON, D., pp. 194-199. New York and London: Academic Press. RUTISHAUSER, S. C. B. & STONE, S. L. (1973). The effect of sodium salicylate on bile secretion in the dog. J. Physiol. 230, 53-54P. 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. SULLIVAN, B. & BERNDT, W. 0. (1973a). Transport by isolated rabbit gallbladders in phosphate-buffered solutions. Am. J. Physiol. 225, 838-844. SULLIVAN, B. & BERNDT, W. 0. (1973b). Transport by isolated rabbit gallbladders in bicarbonate-buffered solutions. Am. J. Physiol. 225, 845-848. WHEELER, H. 0. (1965). Inorganic ions in bile. In The Biliary System, ed. TAYLOR, W., pp. 481-492. Oxford: Blackwell. WHEELER, H. 0. & RAMOS, 0. L. (1960). Determinants of the flow and composition of bile in the unanaesthetized dog during constant infusions 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.

The effect of sodium salicylate on bile secretion in the dog.

1. The I.V. injection of sodium salicylate (100 mg/kg) in the dog caused a rapid and maintained choleresis of the order of 300-600 percent of control ...
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