According to Sperber [1965], bile secretion in many species is mainly due to. South Wales, 2033, Australia.

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1 Quarterly Journal of Experimental Physiology (1974) 59, 93-2 REGULATION OF BILE FORMATION IN RABBITS AND GUINEA PIGS. By H. M. SHAW and T. J. HEATH. From the School of Physiology and Pharmacology, The University of New South Wales, Kensington, New South Wales, 2033, Australia. (Received for publication 25th June 1973) Despite interruption of the enterohepatic circulation of bile salts, bile flow in conscious rabbits and guinea pigs remained constant for several hours when saline was infused to compensate for loss of water and electrolytes in bile. Thus it appeared that biliary flow in these species is not as highly dependent on bile salt secretion as it is in dogs and man. The effects of other stimulants known to play an important role in regulating bile formation in other species were tested in rabbits and guinea pigs. Secretin, cholecystokinin-pancreozymin (CCK-PZ) and gastrin proved to be ineffective choleretics in rabbits although the doses of these exogenously-administered hormones were sufficient to siguificantly increase either pancreatic or gastric secretion. Trivial choleretic responses were recorded after administration of secretin and CCK-PZ to guinea pigs. Attempts were then made to elicit a choleresis by release of endogenous hormones. Again no changes in bile formation were recorded in rabbits although pancreatic secretion was stimulated. Bile flow in guinea pigs increased slightly, but the degree of duodenal acidification necessary to produce all of these responses to endogenous secretin release was probably unphysiological. Attempts to stimulate the release of endogenous CCK- PZ by infusing peptone were unsuccessful. It was concluded that if these herbivores do elaborate secretin and CCK-PZ, the factors responsible for their release may differ from those in other species. The present findings provide further evidence that the control of bile formation in rabbits and guinea pigs differs from that in other species, and it is possible that these differences could be related to the temporal pattern of their eating habits. According to Sperber [1965], bile secretion in many species is mainly due to active secretion of bile salts into the canaliculi and the subsequent osmotic flow of water. However, the primacy of bile salt secretion in the regulation of bile flow of herbivores such as rats and rabbits has been questioned by Klaassen [1972] and Erlinger, Dhumeaux, Berthelot and Dumont [1970]. Gastrointestinal hormones, particularly secretin, may also be important in the regulation of bile formation in dogs [Jones, Geist and Hall, 1971], cats [Scratcherd, 1965] and man [Konturek, Dabrowski, Adamozyk and Kulpa, 1969]. However, it would appear from the small amount of information existing on the subject, that herbivores, at least the non-ruminant type, may be relatively insensitive to the effects of gastrointestinal hormones [Scratcherd, 1965; Shaw and Heath, 1972]. It has been suggested that the failure of secretin to act as a choleretic agent in rabbits may be associated with the very high spontaneous rate of bile formation in this species [Scratcherd, 1965]. The present study was designed to provide more information on the regulation of bile formation in conscious rabbits and guinea pigs, two species which have the highest rates of bile formation known. VOL. LIX, NO

2 94 Shaw and Heath MATERIALS AND METHODS Thirty New Zealand white rabbits weighing125-2*8 kg were fasted for 24 hr, then anaesthetized with intravenous propanidid (Epontol,50 mg/mi., Bayer, Germany) at 20 mg/kg body weight. Twenty-seven varicoloured guinea pigs weighing g were anaesthetized with sodium pentobarbital (i.p. 40 mg/kg). In all animals, the abdomen was opened with a midline incision from the xiphoid process to the umbilicus. The cystic duct wasligated and the bile duct was cannulated at the hilus of the liver with a clear vinyl tube (0.86 mm i.d., 1-52 mm o.d.). In rabbits, the pancreatic duct was isolated close to its entrance into the duodenal wall, and cannulated with a polyethylene catheter (0.40 mm i.d., 0-80 mm o.d.). In each animal the pylorus was ligated and a clear vinyl tube fixed with a purse-string suture into the first part of the duodenum to permit infusion of saline or acid. In those animals which were to receive pentagastrin, a clear vinyl tube (2-0 mm i.d., 3 0 mm o.d.) was fixed into the most dependent portion of the stomach, and was held in place with a purse-string suture. All cannulas were brought out through the lower end of the abdominal incision and this was then closed in two layers. Animals were placed in restraining cages and allowed to recover from the immediate effects of the anaesthetic. During this recovery period, bile was returned to the animals through the duodenal cannula. During subsequent experiments, animals received an infusion of normal saline through the used for intravenous infusions of pure, natural porcine secretin, and cholecystokinin- duodenal cannula with a Palmer infusion pump. A Palmer infusion pump was also pancreozymin (CCK-PZ) (Gastrointestinal Hormone Research Unit, Stockholm, Sweden), pentagastrin (Imperial Chemical Industries, Cheshire, England), and for intraduodenal infusions of hydrochloric acid and peptone. Secretin dosage was measured in Clinical Units. According to the information supplied with the preparation of cholecystokinin-pancreozymin, each vial containing 75 Ivy Dog Units of CCK- PZ also contained 6 Clinical Units of secretin. Hormones were administered through a clear vinyl tube inserted into the marginal ear vein of rabbits and into the femoral vein of guinea pigs. In all experiments, samples of bile and pancreatic juice were collected under paraffin oil in plastic vials, and the volumes estimated from the net weight. The concentration of bicarbonate in bile and pancreatic juice and the total acidity in gastric juice were measured by the titrimetric methods described by Shaw and Heath [1972]. Protein concentration of bile and pancreatic juice was determined by the method of Lowry Rosebrough, Farr and Randall [1951] and the concentration of total bile salts was estimated by the method of Levin, Johnston and Boyle [1961]. To prevent pain during experiments, areas around all incisions were infiltrated several times during the course of the experiments with a local anaesthetic,lignocaine (Xylocaine, Astra Pharmaceuticals (Australia) Pty. Ltd., Evans Road, Salisbury Queensland), and solutions for infusion were warmed to 37 C just before use; no animal showed signs of distress during the 3-4 hr (or in one instance 7 hr) required for experiment, and all were killed promptly at the end of this time by intravenous barbiturate. Student's t tests coupled with analyses of variance were used to estimate whether significant differences existed between means obtained for the different parameters. The standard error of the mean (S.E.M.) for a single time interval or infusion rate, recorded at the left of Figs 1-5 was estimated from the error mean square (E.M.S.) and sample nnmber (N): S.E.M. = (E.M.S./N)t Control samples of bile and pancreatic juice from rabbits, for 3 RESULTS were collected from both rabbits and guinea pigs, 30-min periods after the animals had

3 Regulation of Bile Formation 95 recovered from the direct effects of anaesthesia, and were able to right themselves; this was generally 15 min after the end of the operation in rabbits and min in guinea pigs. In all animals each infusion of hormone or acid was given for 30 min. Saline was infused for the subsequent 2 30-min periods, then the rate of infusion of hormone was increased to the next level in the sequence. The results obtained from these animals were compared with those in control groups which were treated in an identical manner except that hormone or acid infusions were replaced by infusions of saline. Effects of interruption of the enterohepatic circulation of bile salts Saline was infused intraduodenally into 5 rabbits and 6 guinea pigs for 7 hr. In each animal, the rate of infusion was estimated to compensate for water and electrolytes lost in bile and pancreatic juice. There was no decline in bile flow rate in either species, although the output of bicarbonate and bile salts decreased significantly (P < 0 05-P < 0 01; Fig. 1). In rabbits, no changes occurred in the flow or bicarbonate content of pancreatic juice, although the output of protein declined significantly (P < 0*001; Fig. 1) )( ) II * 0 _J '3 0. im be0> CU. C! w 0 m _V 0 a _ 0Li WC) F,.c _, t) a <.C TIME FIG. 1. Effects of interruption of the enterohepatic circulation of bile salts on the flow, bicarbonate and bile salt output in the bile of rabbits (*) and guinea pigs (-), and the flow, bicarbonate and protein output in the pancreatic juice of rabbits (-). Vertical bars at the left represent the S.E.M. for a single time interval. In all figures 'kg' refers to bodyweight. (hr)

4 96 Shaw and Heath Efects of exogenous and endogenous secretin Secretin was infused intravenously into 5 rabbits and 4 guinea pigs at 0 3, 3 0, 30 and 300 m-u. min-' kg-'. In rabbits, secretin had no demonstrable effect on bile formation. However, the flow and bicarbonate output of pancreatic juice increased significantly over control values at each dose of secretin used (P < 0-05-P < 0 001). There appeared to be a linear relationship between the responses and the log dose of secretin and there was no indication that a plateau was being reached (Fig. 2). In guinea pigs, significant increases over control values occurred in the flow and bicarbonate output in bile at eachdoseofsecretinused (P < 0 05-P < ) A plateau was reached at about 30 m-u. min-' kg-' when increases amounting to 30-59% of control values were recorded (Fig. 2). >300 o E 200 I - 1.0K w X Is 050 I :, 025. m XA Xs FIG I _ 03 3* SECRETIN INFUSION RATE (m-u./min.kg) Effects of secretin infusion on the flow, bicarbonate and bile salt output in the bile of rabbits (-) and guinea pigs (-), and the flow, bicarbonate and protein output in the pancreatic juice of rabbits (*). Vertical bars at the left represent the S.E.M. for a single infusion rate, and each point at the extreme left represents the value before commencement of secretin infusion. Attempts were made to stimulate the release of endogenous secretin by infusing hydrochloric acid into the duodenum of 5 rabbits and 5 guinea pigs at 0*3, 3*0 and 30,uEq. min-' kg-'. In rabbits, infusion of acid had no demonstrable effect on bile formation, and only at the highest rate of infusion were

5 Regulation of Bile Formation 97 significant increases recorded in the flow and bicarbonate output of pancreatic juice (P < 0 001; Fig. 3). Similarly in guinea pigs, significant increases (P < 0 01) amounting to 17-29% of control values in the flow and bicarbonate output of bile were recorded, but not until acid was infused at the highest rate (Fig. 3). v 300 o 200 I- * Li- 30 :0 20 I DX 1 5 I, 8 a5 5. Q0.c157. z ~ '~~~~AI 30 INUINRT a. 20 FIG. 3. ACID INFUSION RATE (uequiv/min.kg) Effects of acid infusion on the flow, bicarbonate and bile salt output in the bile of rabbits (0) and guinea pigs (A), and the flow, bicarbonate and protein output in the pancreatic juice of rabbits (U). Vertical bars at the left represent the S.E.M. for a single infusion rate, and each point at the extreme left represents the value before commencement of acid infusion. Effects of CCK-PZ CCK-PZ was infused intravenously into 7 rabbits and 4 guinea pigs at 0-036, 0-36, 0 9, 1'8 and 3'6 u. min-' kg-'. In rabbits, CCK-PZ had no demonstrable effect on bile formation, and although the protein output in pancreatic juice doubled with 0-36 u. min-' kg-1, higher rates of infusion had no additional effect (Fig. 4). The flow and bicarbonate output of pancreatic juice increased significantly over control values at each dose of CCK-PZ used (P < P < 0*01), but there was no evidence of a plateau being reached for these responses. In guinea pigs, significant increases over control values occurred in the flow and bicarbonate output of bile at each dose of CCK-PZ used (P < P < 0.001). When the highest dose rate was used, increases amounting to.,/

6 98 Shaw and Heath 61-1% of control values were recorded, but there was no indication that a plateau was being reached (Fig. 4). In both rabbits and guinea pigs, attempts were made to stimulate the release of endogenous CCK-PZ by infusing 5 % peptone intraduodenally at 0 12 ml./min for 30 min. However, no changes in the protein output of pancreatic juice in rabbits or bile flow in guinea pigs could be demonstrated. 2' 300 m g 200 ZS 0 w Xk 30 1; -U- a twiii I *_ XE ** FIG CCK-PZ 0-36 INFUSION 36 RATE (u/minkg) Effects of CCK-PZ infusion on the flow, bicarbonate and bile salt output in the bile of rabbits (-) and guinea pigs (-), and the flow, bicarbonate and protein output in the pancreatic juice of rabbits (*). Vertical bars at the left represent the S.E.M. for a single infusion rate, and each point at the extreme left represents the value before commencement of CCK-PZ infusion. Effects of pentagastrin Pentagastrin was infused intravenously into 4 rabbits and 4 guinea pigs at 0-45, 09, 4.5 and 9-0,tg min-' kg-'. Four rabbits and 4 guinea pigs provided with gastric fistulas acted as controls. Pentagastrin had no demonstrable effect on the formation of either bile or pancreatic juice in rabbits, or of bile in guinea pigs despite the fact that increases over control values occurred in gastric acid output at each dose of pentagastrin used (P < 0-001; Fig. 5). In both species, a linear relationship appeared to exist between log dose of pentagastrin and gastric acid output up to 4-5,ug min-' kg-' when a plateau was reached.

7 Regulation of Bile Formation 99 FIG. 5. F1-S3.v G GASTRIN INFUSION RATE (4tnin.kg) Effects of pentagastrin infusion on the acid output in the gastric juice of rabbits (U) and guinea pigs (-). The vertical bar at the left represents the S.E.M. for a single infusion rate, and each point at the extreme left represents the value before commencement of pentagastrin infusion. DIscusSION It was essential to ensure that the doses of secretin and CCK-PZ were sufficient to stimulate pancreatic secretion. In rats at least, secretion is depressed during anaesthesia [Colwell, 1951; Love, 1957; Lin and Alphin, 1962], and so the present work was done on conscious animals. The short time after operation was not ideal, but our animals appeared comfortable and we have not been able consistently to keep such operated animals healthy for more than 24 hr after operation. In the present experiments on rabbits and guinea pigs, bile flow rate remained constant for at least 7 hr after interruption of the enterohepatic circulation of bile salts. Weak choleretic responses were obtained from guinea pigs, but none of the substances tested produced a choleresis in the rabbit, despite the fact that the doses chosen were associated with significant increases in the formation of either pancreatic juice or gastric acid. Bile salts may play a major role in the control of bile formation in dogs, cats and man [Sperber, 1965], and interruption of the enterohepatic circulation of bile salts in these species results in an immediate reduction in bile flow [Wheeler and Ramos, 1960; Scratcherd, 1965; Thureborn, 1962]. In herbivores such as rats and rabbits, however, diverting bile from the duodenum has very little effect on flow rate [Klaassen, 1971; Shaw and Heath, 1972; Scratcherd, 1965]. In the present experiments, it was found that biliary flow is not highly dependent on bile salt secretion in these species and it is possible that, as postulated by Wheeler [1967], active transport of other solutes, particularly inorganic ions, could be responsible for the high flow rate in these species even after interruption of the enterohepatic circulation of bile salts. Many studies have emphasized that factors other than the enterohepatic circulation of bile salts may play a role in regulating bile formation. Secretin may be an important stimulus to the secretion of water and electrolytes into the bile of dogs [Jones, Geist and Hall, 1971], cats [Scratcherd, 1965] and man [Konturek, Dabrowski, Adamczyk and Kulpa, 1969], but it appears to be an ineffective choleretic agent in rabbits [see Scratcherd, 1965; Kirchmayer, Tarnawski, Drozdz and Cichecka, 1972]. Similarly, we could not produce a choleresis in rabbits either by the intravenous infusion of porcine secretin, or by the intraduodenal infusion of acid. Unless one postulates that there are different receptors in the pancreas and liver for porcine secretin, the absence of

8 0 Shaw and Heath a choleretic response in our experiments could not be attributed to the use of this secretin preparation, since the flow and bicarbonate output of pancreatic juice increased significantly in the presence of both exogenously-administered and endogenously-released secretin. It must be stressed, however, that it is not clear whether the presence of acid in the duodenum is an important physiological factor concerned in the release of this hormone in these species. This is because a much higher degree of duodenal acidification was necessary to stimulate pancreatic secretion in rabbits than in dogs [Brooks and Grossman, 1970]. Thus to assign a physiological role to acid in the release of secretin from the duodenum of rabbits, one must demonstrate that amounts of acid sufficient to release secretin normally occur within the duodenum. No information appears to exist about this in rabbits. In guinea pigs, not only was the choleresis after infusion of porcine secretin of a trivial nature, but the responses to infusions of acid in attempts to stimulate release of secretin were also trivial. In addition, significant increases in bile formation only occurred when very large amounts of titratable acid were introduced into the duodenum. Scratcherd [1965] and Forker, Hicklin and Sornson [1967] recorded larger choleretic responses to secretin infusion in guinea pigs. The reason for this difference in response to secretin is obscure, but may be related to differences in the purity of the secretin preparations used in these studies. Although the cholecystokinetic effects of CCK-PZ are well documented for most species, there are only isolated reports regarding the actions of this hormone on the liver. One difficulty in assessing these actions is that preparations free from contamination with secretin are not readily available. Jones and Grossman [1970], using CCK-PZ from the Gastrointestinal Hormone Research Unit, reported this preparation to be an effective choleretic agent in dogs. Although we could not elicit a choleresis in rabbits with the same preparation of CCK-PZ in doses which were sufficient to double the output of protein in pancreatic juice, Kirchmayer, Tarnawski, Drozdz and Cichecka [1972] and Murillo and Lopez [1971] did find that other preparations of CCK-PZ had choleretic effects in anaesthetized rabbits. It is possible that the rabbit, in which large quantities of dilute bile constantly trickle into the duodenum, does not elaborate CCK-PZ. In this connection, it should be noted that Magee [1965] could not demonstrate a gallbladder contraction after infusing acids, fats or amino acids into the duodenum of spinal rabbits. Similarly, we could not detect changes in the formation of bile or pancreatic juice in rabbits after infusing 5% peptone, which has been employed successfully to stimulate release of endogenous CCK-PZ in dogs and cats [Berry and Flower, 1971]. Since increases in bile salt output were recorded after infusion of CCK-PZ by Kirchmayer, Tarnawski, Drozdz and Cichecka [1972] and Murillo and Lopez [1971], it is conceivable that the choleresis they obtained was due, at least in part, to impurities such as bile salts contained in their hormone preparations. A choleresis was recorded with the highest doses of CCK-PZ in guinea pigs, but it is not clear whether the secretin contaminating the CCK-PZ preparation contributed to this choleresis. Again there are doubts regarding the physio-

9 Regulation of Bile Formation logical significance of this choleresis. In trial experiments, we failed to elicit a gallbladder contraction after infusing 5% peptone intraduodenally and thus it is possible that the guinea pig, like the rabbit, may not produce CCK-PZ. Gastrin and gastrin analogues have been shown to be weak but effective choleretic agents in dogs [Jones and Grossman, 1970] and cats [Konturek, Kieta-Fyda and Moczurad, 1967]. In those species in which gastrin has choleretic effects, doses generally considered to be maximal for gastric acid secretion have to be employed. However, even with supramaximal doses, no choleresis occurs in man [Konturek, Dabrowski, Adamczyk and Kulpa, 1969]. No ready explanation exists for the difference at this time. In the present experiments, no choleresis was recorded either in rabbits or guinea pigs after infusing pentagastrin in doses necessary to stimulate maximum gastric acid secretion. This is perhaps not surprising in view of the insensitivity of the stomachs of herbivores to infusion of gastrin [Shaw and Heath, 1972; Capoferro and Torgersen, 1971]. The present findings provide further evidence that factors influencing bile formation in rabbits and guinea pigs differ from those in other species. This data can be most easily explained on the assumption that since carnivores eat intermittently, delivery of bile to the duodenum is intermittent and peaks in bile output are induced by the release of gastrointestinal hormones. In herbivores, however, food probably enters the intestine more or less continuously, bile is formed at a constant high rate and these hormones are less likely to play an essential role in bile formation. 1 REFERENCES BERRY, H. and FLOWER, R. J. (1971). The assay of endogenous cholecystokinin and factors influencing its release in the dog and cat. Gastroenterology, 60, BROOKS, A. M. and GROSSMAN, M. I. (1970). Post prandial ph and neutralizing capacity of the proximal duodenum in dogs. Gastroenterology, 59, CAPOFERRO, R. and TORGERSEN, 0. (1971). A gastric secretion test in the intact guinea-pig. Scandinavian Journal of Gastroenterology, 6, COLWEL, A. R. (1951). Collection of pancreatic juice from rats and consequences of its continued loss. American Journal of Physiology, 164, ERLINGER, S., DHUMEAUX, D., BERTHELOT, P. and DUMONT, D. (1970). Effect of inhibitors of sodium transport on bile formation in the rabbit. American Journal of Physiology, 219, FORKER, E. L., HICKLIN, T. and SORNSON, H. (1967). The clearance of mannitol and erythritol in rat bile. Proceedings of the Society for Experimental Biology and Medicine, 126, JONES, R. S. and GROSSMAN, M. I. (1970). Choleretic effects of cholecystokinin, gastrin II, and caerulein in the dog. American Journal of Physiology, 219, JONES, R. S., GEIST, R. E. and HALL, A. D. (1971). The choleretic effects of glucagon and secretin in the dog. Gastroenterology, 60, KIRCHMAYER, S., TARNAWSKI, A., DROZDZ, H. and CICHECKA, K. (1972). Effect of cholecystokinin-pancreozymin and secretin on the volume, composition, and enzymatic activity of hepatic bile in rabbits. Gut, 13, KLAAsSEN, C. D. (1971). Does bile acid secretion determine canalicular bile production in rats? American Journal of Physiology, 220, KLAASSEN, C. D. (1972). Species differences in the choleretic response to bile salts. Journal of Physiology, 224, KONTUREK, S. J., KIETA-FYDA, A. and MOCZURAD, K. (1967). The influence of gastrin analogues and 2-deoxy-D-glucose on bile secretion. American Journal of Digestive Diseases, 12,

10 2 Shaw and Heath KONTUREK, S. J., DABROWSKI, A., ADAMCZYK, B. and KULPA, J. (1969). The effect of secretin, gastrin-pentapeptide, and histamine on gastric acid and hepatic bile secretion in man. American Journal of Digestive Diseases, 14, LEvIN, S. J., JOHNSTON, C. G. and BOYLE, A. J. (1961). Spectrophotometric determination of several bile acids as conjugates. Analytical Chemistry, 33, LIN, T. M. and ALPHIN, R. S. (1962). Comparative bioassay of secretin and pancreozymin in rats and dogs. American Journal of Physiology, 203, LovE, J. W. (1957). A method for the assay of secretin, using rats. Quarterly Journal of Experimental Physiology, 42, LowRY, 0. H., ROSEBROUGH, N. J., FARR, A. L. and RANDALL, R. J. (1951). Protein measurement with the folin phenol reagent. Journal of Biological Chemistry, 193, MAGEE, D. F. (1965). Physiology of gallbladder emptying. In: The Biliary System. Ed. W. Taylor. Blackwell, Oxford. Pp MURILLO, A. and LOPEZ, M. A. (1971). Contribucion al estudio de la regulaci6n hormonal de la secreci6n pancreatica en el conejo. Revista Espanola de Fisiologia, 27, SCRATCHERD, T. (1965). Electrolyte composition and control of biliary secretion in the cat and rabbit. In: The Biliary System. Ed. W. Taylor. Blackwell, Oxford. Pp SHAW, H. M. and HEATH, T. (1972). The significance of hormones, bile salts, and feeding in the regulation of bile and other digestive secretions in the rat. Australian Journal of Biological Sciences, 25, SPERBER, I. (1965). Biliary secretion of organic anions and its influence on bile flow. In: The Biliary System. Ed. W. Taylor. Blackwell, Oxford. Pp THUREBORN, E. (1962). Human hepatic bile. Composition changes due to altered enterohepatic circulation. Acta Chirurgica Scandinavica, Supplementum, 303, WHEELER, H. 0. (1967). Water and electrolytes in bile. In: Handbook of Physiology. Ed. by C. F. Code. Washington American Physiological Society, Pp WHEELER, H. 0. and RAmos, 0. L. (1960). Determinants of the flow and composition of bile in the unanesthetized dog during constant infusion of sodium taurocholate. Journal of Clinical Investigation, 39,