Quarterly Journal of Experimental Phy8iology (1971) 56, 197-209

FUNCTION OF THE GALL BLADDER IN SHEEP. By IVAN CAPLE and TREVOR HEATH. From the School of Physiology and Pharmacology, The University of New South Wales, Kensington, New South Wales, 2033, Australia.
(Received for publication 23rd March 1971) Bile collected from the gall bladder of sheep at surgery contained a higher concentration of solutes than hepatic bile collected at the same time. When 20 ml. bile was placed in the isolated gall bladder, fluid was absorbed at about 4 ml./hr in both sheep and dogs, and any absorption of lipids or bile salts was negligible. Pressure in the gall bladder of sheep fluctuated over a range of about 18 mm mercury: during fasting, pressure waves occurred at the rate of 3-6/hr and each lasted for five minutes; aft,er feeding they became more frequent, and sometimes lasted for 20 min. Injection of acid into the duodenum was followed by an increase in the pressure in the gall bladder, and this response, like most of the spontaneous waves, could be almost eliminated by atropine. The gall bladder did contract in response to cholecystokinin-pancreozymin, but this was not affected by atropine. It is concluded that the gall bladder in the sheep has appreciable absorptive and contractile activity. However, bile is produced at a more rapid and uniform rate in sheep than in animals with simple stomachs, and as a result in the sheep each unit volume of bile spends a shorter time in the gall bladder, and its organic constituents axe concentrated to a smaller extent.

The gall bladder in many animals including humans and dogs can absorb water and some ions and concentrate organic constituents of the bile; it can also contract to expel the concentrated bile into the common bile duct and the duodenum [Mann, 1924; Ivy, 1934]. However, the gall bladder in ruminant animals such as sheep has been believed to be virtually noncontractile under physiological conditions [Magee, 1965]. It has been assumed to have negligible absorptive ability [Dukes, 1955; Harrison, 1962; Harrison and Hill, 1962; Diamond, 1965, 1968], but this assumption has been based only on a comparison between the pigment concentration in gall bladder and hepatic bile [Schmidt and Ivy, 1937]. These assumptions have been tested in our experiments; the absorptive ability of the sheep gall bladder has been compared with that of the dog, and estimates made of the contractility of the gall bladder in response to various stimuli including acetylcholine chloride, cholecystokinin-pancreozymin (CCKPZ), feeding, and the presence in the duodenum of acid and abomasal contents.
MATERIALS AND METHODS Anim,als. Merino ewes and wethers that weighed between 38-45 kg, and mongrel dogs that weighed between 22-26 kg were used in the experiments. Sheep were kept indoors in metabolism cages and fed on lucerne chaff and oats. Anae.sthe&sia. All animals were starved for 12-24 hr, then anaesthesia was induced with sodium pentobarbitone. For acute experiments and surgical procedures of more than 15 min, an endotracheal tube was passed and anaesthesia continued with halothane and oxygen in a closed circuit.
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Surgical techniques. In all sheep an incision was made parallel to and just behind the last rib on the right side. The right dorsal lobe of the liver was retracted anteriorly and the intestines retracted posteriorly to gain access to the gall bladder and cystic duct. To study the absorptive ability of the sheep gall bladder, acute experiments were performed on eight sheep. In these sheep the cystic artery was separated from the cystic duct and the duct ligated. A PVC cannula was inserted into the fundus of the gall bladder and retained in position with a purse-string suture. The common bile duct was then cannulated proximal to the pancreatic duct, and hepatic bile collected under paraffin oil. The abdominal incision was closed with clamps and an electric blanket used to maintain body temperature. In similar experiments on four dogs, the gall bladder was isolated without interfering with its blood supply, and the common bile duct was cannulated [Ravdin, Johnston, Riegel and Wright, 1932]. To study the contractility of the sheep gall bladder, two different preparations were used. In two sheep the gall bladder was isolated from the biliary tract: the cystic duct was ligated without interfering with the cystic artery, then a PVC tube (O.D. 2-70 mm, I.D. 1L50 mm) was inserted into the gall bladder through a small hole in its neck, and was held in place with a purse-string suture. A tube was placed in the proximal duodenum and another in the jugular vein in each sheep, then the sheep was allowed to recover. Experimental methods. The anatomy of the blood vessels to the sheep gall bladder was studied to ensure that the gall bladder could be isolated without interfering with its blood supply. Four sheep were anaesthetized and exsanguinated, then neoprene latex of different colours was injected into the portal vein, caudal vena cava, hepatic artery and common bile duct. The abdominal organs were left in their normal positions and the sheep were stored at 40C to allow the latex to set before dissection. 1. Absorptive ability of the gall bladder. In these experiments, which are described in detail with their results, samples of bile were collected under paraffin oil and frozen shortly after collection. They were analysed for: (1) total long-chain fatty acids by the method of Heath and Hill [1969], (2) phospholipids by application of the method of Zilversmit and Davis [1950] to a lipid extract prepared according to Folch, Lees and Sloane-Stanely [1957], (3) total cholesterol by the method of Zlatkis, Zak and Boyle [1953] after the cholesterol had been extracted into n-hexane [Peric-Golia and Socic, 1968], (4) total cholates by the method of Irvin, Johnston and Kopala [1944], (5) chloride by potentiometric titration with silver nitrate using a silver electrode (Radiometer A/S, Copenhagen, Denmark), (6) sodium and potassium with a Baird-Atomic flame photometer, (7) bicarbonate by electrometric titration using a method similar to that described by Segal [1955] for plasma. 0 5 ml. freshly collected bile was added to 0 5 ml. of 0.1 N HCI and the mixture was swirled for 1 min and left standing for about 5 min. It was then titrated to the original pH of the bile with previously standardized NaOH of about 0-01 N. 2. Contractility ofstrips of sheep gall bladder in vitro. The gall bladders were removed from six sheep under pentobarbitone anaesthesia, and two longitudinal and two transverse strips, 0 4 cm wide and 3*5 cm long, were cut from each gall bladder and suspended in an organ bath that contained 15 ml. of continuously-oxygenated LockeRinger solution at 370C [Amer and Becvar, 1969]. In the experiments, performed after the strips had been in the bath for at least 3 hr, the tension on each strip was adjusted to 0-5 g: this was estimated with a Grass Force Displacement Transducer and Grass Model 7 Polygraph (Grass Instrument Co., Quincy, Mass.). Estimates were then made of the effects on this tension of acetycholine chloride (British Drug Houses Ltd.,

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Poole, England), atropine sulphate (Sigma Chemical Co., St. Louis, Missouri) and CCK-PZ (Batch 26921, GIH Research Unit, Karolinska Institutet, Stockholm). The amount of CCK-PZ was expressed in clinical units (u.), one of which is equivalent to an Ivy Dog Unit of cholecystokinin [Ivy and Janecek, 1959], and is the amount of dried material which, when dissolved in normal saline solution and injected intravenously into a dog during 10-15 sec, causes within 1-5 min a rise in pressure of 10 cm bile. The concentrations in the organ bath were: CCK-PZ 0-025, 0 05, 0.1 u./ml., acetylcholine 0-67, 1-3 pg/ml., and atropine 0-13 mg/ml. 3. Pressure in the gall blder of conscious standing sheep. The cannula in the gall bladder was connected to a Statham P23AC pressure transducer and a Grass Polygraph, and the pressure recorded (in mm mercury) before and after feeding and after injections of CCK-PZ and of atropine to the jugular vein. The effects on gall bladder pressure of duodenal infusions of abomasal contents, obtained from sheep at slaughter and strained through a muslin cloth, and hydrochloric acid (pH 1.5) and acetic acid (pH 3) were also estimated.

RESULTS Anatomy of the blood supply to the sheep gall bladder. The sheep gall bladder is loosely attached to the right dorsal lobe of the liver and extends in a ventral direction beyond the margin of the liver, and slightly to the right of the midline of the body (Fig. 1). It receives arterial blood from the cystic artery, a branch of
PORTAL V

COMMON HEPATIC A.

COMMO BLE DUCTa GASTRODUODENAL A.

R. GASTRIC A

HCPATIC A. Ft PROPER

LBRANCH PORTAL VCYSTIC

L. PROPER HEPATIC A. R. 1
PORTAL V.

B
FIG. 1. Anatomy of the gall bladder and associated structures in sheep. These drawings were made from casts prepared by injecting latex of different colours into the portal vein, common hepatic artery, hepatic veins and common bile duct. (a) Arrangement of the bile ducts (black), the portal vein and its branches, and the common hepatic artery and its branches (hatched) near the hilus of the liver. The common hepatic artery divides to form the left proper hepatic artery, and the right proper hepatic artery, which supplies the cystic artery near the termination of the cystic duct. (b) Location and blood supply of gall bladder.

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the dorsal or right proper hepatic artery, which supplies blood to the right lateral and caudate lobes (Fig. 1). The cystic artery is loosely attached to the dorsal aspect of the cystic duct and supplies small branches to the duct. At the neck of the gall bladder it divides into three branches: one passes down the free margin of the gall bladder, and the other two pass ventrally along its right and left margins. These arteries divide into smaller branches which have tortuous paths around the wall of the gall bladder (Fig. 1). The veins from the gall bladder do not accompany the arteries; instead they extend around the circumference of the gall bladder from its free margin to its attachment to the liver, then enter the substance of the liver. Veins from the left side drain into the dorsal ramus of the ventral branch of the portal vein as described by Heath [1968]; and those on the right side, and a vein that extends dorsally along the free margin ofthe gall bladder, drain into the ventral ramus of the right branch of the portal vein (Fig. 1).
TABLE I. Composition of bile collected simultaneouwly from gall bladder and liver in ten anaesthetized sheep. Concentration (,umol/ml.) Hepatic bile Gall bladder bile P Total cholates 39 63 4 89.36.3 <0 001 Total fatty acids 16.82*3 23.14-4 NS Total cholesterol 0-780d14 <0 05 1D05014 Phospholipids 7-10-7 <0*05 8-71-1 Sodium 139-022-0 180.066-0 NS Potassium 3-20 5 4-50-8 <0*02 Chloride 98-22.9 74-0107 <0*05

Composition of hepatic and gall baldder bile in sheep (Table I). Bile was collected at surgery from 10 starved sheep in which the cystic duct had been ligated. The gall bladder was emptied with a syringe, and hepatic bile was collected from a cannula in the common bile duct. The concentration of total cholates in the bile from the gall bladder was more than twice that in the hepatic bile, and the concentrations of total cholesterol, phospholipids and potassium were significantly higher in bile from the gall bladder (P<0.050 001), but hepatic bile contained a significantly higher concentration of chloride (P<0.05). Changes in the volume and composition of hepatic bile in the gall bladder of sheep and dogs. In three acute experiments on each of four sheep, the isolated gall bladder was emptied through the cannula in its fundus and was washed out with 20 ml. saline, then refilled with 20 ml. freshly-collected hepatic bile. This was mixed each i hr with a syringe attached to the cannula, and left for 1, 2 or 3 hr. It was found that 434113 ml. was absorbed after 1 hr, and 7-4P14 and 9-8+1-9 ml. absorbed when the bile remained in the gall bladder for 2 hr and 3 hr. The total amount of sodium, chloride and bicarbonate decreased while the bile was in the gall bladder (P<0*02-0-005), but the amounts of potassium, total fatty acids and of cholates remained almost constant (Fig. 2). The concentration of sodium remained unaltered but that of potassium, total fatty

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acids and cholates increased and there were significant decreases in the concentrations of chloride and bicarbonate in the bile (P <0 05- 0 005; Fig. 3). In similar experiments on four dogs, 3 90 5 ml. and 8-00-4 ml. was absorbed after 1 and 2 hr, and 7-340-6 ml. was absorbed when the bile was in the gall bladder for 3 hr (Fig. 2). The amount of total cholates in the gall bladder did not change, but significant decreases occurred in the amounts of sodium, chloride and bicarbonate (P<005- 0001; Fig. 2). The concentration of sodium remained constant, but decreases occurred in the concentrations of chloride and bicarbonate (P<0.02; Fig. 3). The rate of absorption from the gall bladders of both sheep and dogs decreased as the time spent in the gall bladder increased (Fig. 2). In fact, when
SHEEP
a
w

DOG

1000
0

(U)

m 0

500

z E
0

1500
1000

en
1D z

500

a] w
Zn m
cz2 w
0
-

10

8 6 4 2 O0
TIME (hr)

TIME (hr)

FIG. 2. Extent of absorption from the gall bladder. In four sheep and four dogs, 20 ml. bile was injected into the gall bladder after the cystic duct was ligated without interfering with the cystic artery. Estimates were made of the amount of fluid, sodium, potassium, chloride, bile salts (BS) and total fatty aids (TFA) that had been absorbed after 1, 2 and 3 hr. Bars at right represent SEM for a single time interval.
_

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20 ml. fresh hepatic bile was left in the gall bladder for 5 hr and mixed regularly, the mean volume absorbed was 8-2 ml. - only slightly more than that absorbed after 2 hr in the previous experiments. A preliminary account of this 5-hr experiment has already been presented [Caple and Heath, 1970].
SHEEP
DOG

z 0 H z

20

15
E.

10 5

0
LLU

-in

20 15
I5

0 z
cr

210

SO_t~ TFA
I
2 TIME (hr)

Caple and Heath

w
-J

0 3
1

2 TIME (hr)

FIG. 3. Effect of the gall bladder on the volume and composition of bile. In four sheep and four dogs, 20 ml. bile was injected into the gall bladder after the cystic duct was ligated without interfering with the cystic artery. After one, two or three hours, estimates were made of the volume of bile and of its concentration of sodium, potassium, chloride, bicarbonate, bile salts (BS) and total fatty acids (TFA). Bars at right represent SEM for a single time interval.

After each experiment was finished the gall bladder was distended with 60 ml. Evans Blue solution and inspected closely before and after it was removed from the sheep. No signs were found of either leaks or accessory bile ducts, or of any other ways by which bile may have escaped from the gall bladders.

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Contractility of the sheep gall bladder 1. Effect of CCK-PZ and acetylcholine chloride on sheep gall bladder muscle in vitro. Most strips of gall bladder showed little evidence of spontaneous activity, but contracted slowly when CCK-PZ was added to the organ bath (Fig. 4). The maximum tension, which was developed after about 15 min, was very variable as indicated by the standard errors of the means in Table II, and it did not appear to be related to the concentration of hormone in the bath (0-025-0*1 u./ml.; Table 2). However it was related to the orientation of the strips: those cut at right angles to the long axis of the gall bladder generally developed much more tension than longitudinal strips (P<0 001; Table 2).
TABLE II. Effect of CCK-PZ and of acetylcholine chloride on 8trip8 of sheep gall bladder.

Orientation of strip Number of strips Mass of strips (g) Increase in tension (g) over resting tension (0.5 g) after CCK-PZ 0-025 u./ml. CCK-PZ 0.05 u./ml. CCK-PZ 0.10 u./ml.

Transverse
8

Longitudinal
6

0-230-04

0-260-09

0-730 19 0 720 18
0 400*50

0-980*25

0-300*24

0*350 14

0-270-24

0 180*13 04180413 0-300*30 Four out of a total of 18 strips did show evidence of spontaneous rhythmic activity (see Fig. 4). These fluctuations in tension, which occurred each 20-30 sec and had an amplitude of up to 1 g, continued as the muscle contracted under the influence of CCK-PZ. The addition of acetylcholine chloride caused the strips to contract rapidly, and maximum tension was developed within 10 sec (Fig. 4). Atropine inhibited the response to acetylcholine, but not that to 13

ACh ACh

0.70 ,ug/m1.

,ug/ml.

CCK-PZ (Fig. 4). 2. Pressure in the gall bladder of conscious, standing sheep. Two sheep, in which a cannula had been placed into the gall bladder through the ligated cystic duct, were starved for periods between 12 and 48 hr before each experiment, then 20 ml. sterile normal saline was injected into the emptied gall bladder and pressure recorded for 1-3 hr (Fig. 5). During this period, each sheep appeared quiet and contented, and continued to ruminate. Fluctuations in pressure related to ruminal movements and rumination could be detected readily, but defaecating, urinating, drinking and moving in the cage did not cause major changes in the pressure recorded, and the responses to respiratory movements were negligible. However, rhythmic fluctuations in pressure in the gall bladder did occur, and if the sheep were starved for 12-15 hr about six large waves, with smaller fluctuations superimposed on them, occurred each hour. During each of the large fluctuations the pressure increased rapidly and reached a maximum of 16-17 mm mercury during the first minute, then decreased to the basal level after another 4 min (Fig. 5). When the sheep were starved for more than 24-30 hr, the frequency of the pressure waves decreased to about three/hr, and they were of smaller amplitude.

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5 TIME (min)

10

15

FIa. 4. Development of tension above resting tension (0.5 g) by circular strips take from sheep gall bladders and suspended in continuously-oxygenated Locke-Ringer in an organ bath. Top panel: Effect of CCK-PZ, present in the bath in a concentration of 0-05u./ml. Middle panel: Effect of CCK-PZ during spontaneous activity. Bottom panel: The effect of acetylcholine (0-7 &g/ml.) was estimated, then the bath was washed out ('W'), and estimates made of the effects of acetylcholine (0-7 pg/ml.) and of CCK-PZ (0-05 u./ml.) in the presence of atropine (0.13 mg/ml.).

EFFECT OF FEEDING ON PRESSURE IN THE SHEEP GALL BLADDER

30

40

TIME AFTER FEEDING (min)

50

60

FIG. 5.

Effect of feeding on pressure in the sheep gall bladder. Recordings were made from sheep with an isolated gall bladder, in which the cannula was inserted through the cystic duct without interfering with the cystic artery, and from sheep in which the biliary tract was intact and the cannula tied into the gall bladder through a small incision near its neck.

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ACID

TO

DUODENUM

E
w

t0
lm

ABOMASAL CONTENTS TO DUODENUM

1 20 0

w w
I CCK-PZ TO JUGULAR VEIN

z
ATROPINE TO JUGULAR VEIN
D 20 (0 (0

w
0.

+
CCK-PZ AFTER ATROPINE TO JUGULAR VEIN
20

10

15

20

TIME (min)
FIG. 6. Effect of various stimuli on pressure in the sheep gall bladder. The biliary tract was intact, and the recording cannula tied into the gall bladder through a small incision near its neck. Top panel: 20 ml. HCI (pH 1-5) was injected through a duodenal cannula after the sheep
had been starved for 15-18 hr. Second panel: 50 ml. abomasal contents (pH 3.1) was injected through a duodenal cannula after the sheep had been starved for 15-18 hr. Third panel: 75 u. CCK-PZ was injected intravenously. Pressure changes similar to those marked 'R' correspond with ruminal movements. Fourth panel: 2 mg atropine sulphate was injected intravenously to a sheep 4 hr after feeding. Bottom panel: 75 u. CCK-PZ injected intravenously to a sheep 30 min after the intravenous injection of 2 mg atropine sulphate.

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When the sheep were fed, they began to eat almost immediately, and the fluctuations in pressure increased in frequency and amplitude. After 20-40 min the rhythm was interrupted by occasional periods in which the pressure increased rapidly to 15-18 mm mercury but decreased slowly, and did not reach the basal level for about 20 min (Fig. 5). As the mean pressure decreased towards the basal level, additional fluctuations in pressure, over a range of 5-8 mm mercury and occurring each 15-30 sec, became more prominent (Fig. 5). Spontaneous fluctuations in pressure also occurred occasionally in two sheep in which the biliary tract was intact and in which a cannula had been tied into the lumen of the gall bladder through a small incision near the origin of the cystic duct. In these sheep, as in the sheep with the isolated gall bladders, the pressure waves increased in frequency and amplitude after feeding, and sometimes the pressure remained elevated for about 20 min (Fig. 5). When 20 ml. of acetic acid (pH 3) or hydrochloric acid (pH 1.5) was infused into the duodenum in sheep with intact biliary tracts, and in those with isolated gall bladders, the pressure in the gall bladder increased between the second and fifth minute to a maximum of about 17 mm mercury, then decreased slowly to reach the basal level by 30 min (Fig. 6). The pressure in the gall bladder did not change when 20 ml. saline was infused into the duodenum, but when 50 ml. strained abomasal contents (pH 3) was infused into the duodenum after the sheep had been starved for 15-18 hr, the pressure fluctuated in a manner similar to that after feeding. If CCK-PZ was injected intravenously in doses between 15 and 75 u. the pressure increased rapidly (Fig. 6). It reached a maximum within the first minute, but decreased slowly and with regular fluctuations, and reached the basal level by 40-60 min. The responses to feeding and to the duodenal infusions were all inhibited for 50-60 min after the intravenous injection of 2 mg of atropine sulphate, but the response to CCK-PZ was not affected (Fig. 6). When the atropine was given the pupils of the sheep became widely dilated and the pupillary light reflex could not be elicited, but the sheep continued to eat and did not appear to be distressed. DISCUSSION Analysis of samples of bile collected from man and animals at surgery or slaughter has shown that the concentrations of some constituents of hepatic bile, especially bile salts, lipids and bilirubin, are less than those of gall bladder bile [Schmidt and Ivy, 1937; Small and Rapo, 1970]. Most of this difference has been attributed to absorption of some constituents of the hepatic bile by the wall of the gall bladder. However, part of the difference could be due to variations in the composition of the bile secreted by the liver; bile that entered the gall bladder initially may have had a different composition from that collected from the hepatic duct for analysis. Bile produced during the period shortly after a meal when bile salts are being absorbed from the intestine will enter the gall bladder if the tone in the sphincter of Oddi is high [see Magee, 1965]. Because of the role of reabsorbed bile salts in stimulating bile secretion [Sperber, 1959; Preisig, Cooper and Wheeler, 1962; Heath, Caple and Redding, 1970],

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this bile would be likely to have a higher concentration of organic constituents including bile salts than bile produced during the period of fasting likely to be associated with surgery or slaughter. In the present experiments the rate of absorption of water and ions from the isolated gall bladder was found to be similar in sheep and dogs, and the maximum rate of absorption of fluid was similar to that reported by Ravdin, Johnston, Riegel and Wright [1932] in the isolated gall bladder of conscious dogs. Although the absorptive capacity of the gall bladder is important, the extent to which the organic constituents of the bile will be concentrated will also depend on the rate of secretion of bile and on the frequency with which the gall bladder empties. In sheep, bile is formed at a greater rate than in dogs [see Wheeler and Ramos, 1960; Nahrwold and Grossman, 1967; Heath, Caple and Redding, 1970], but in dogs it is usually stored in the gall bladder for a considerable time until released in response to a meal [see Magee, 1965]. In sheep, mixed bile and pancreatic juice enter the duodenum in a series of spurts, and the rate of entry increases as the sheep are fed more frequently [Harrison and Hill, 1962]. The pattern of contractions of the sheep gall bladder, which is similar to the pattern described in other animals [Ivy, 1934], is accentuated after feeding. Under most conditions sheep do eat more or less continuously, and thus bile may be expected to enter the duodenum at frequent intervals throughout the day. As a result, each unit volume of bile is likely to spend a much shorter time in the gall bladder in sheep than in animals such as dogs, and the organic constituents will be concentrated to a smaller extent. However, the sheep gall bladder does have significant absorptive ability, and this must be considered when bile is collected from gall bladder fistulae in studies of normal or abnormal liver function [Harrison, 1962; Leaver, 1968]. Some of the contractions of the sheep gall bladder are eliminated by atropine, and are probably due to vagal reflexes associated with the ingestion of food or with the presence of chyme in the gut. The motility of the abomasum increases after feeding and this results in an increased rate of passage of chyme into the duodenum [Hill, 1968; Ruckebusch, 1970]. The presence of chyme in the duodenum could also stimulate the release of hormones, possibly CCK-PZ, capable of stimulating the musculature of the gall bladder. Although there is no direct evidence for the existence of CCK-PZ in sheep, a substance capable of causing contraction of the dog gall bladder has been isolated from the sheep intestine [Ivy, Kloster, Lueth and Drewyer, 1929; Kloster, Ivy and Lueth, 1929] and CCK-PZ will cause contraction of the gall bladder in sheep. Thus it is likely that in the sheep as in other animals, both nervous and hormonal influences regulate the contractions of the gall bladder, and influence the concentration of organic matter in the bile.
ACKNOWLEDGMENT We record our sincere thanks to the Australian Wool Board and the George Aitkens Pastoral Research Trust for financial assistance, and to Mr. John Reynolds and Miss Jenny Daynes for excellent technical assistance. I. Caple was on study leave from the Victorian Department of Agriculture.

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REFERENCES AMER, M. S. and BECVAR, W. E. (1969). 'A sensitive in-vitro method for the assay of cholecystokinin.' Journal of Endocrinology 43, 637. CAPLE, I. W. and HEATH, T. J. (1970). 'Effect of the gall bladder on the volume and composition of bile in sheep.' Proceedings of the Australian Physiological and Pharmacological Society 1, 33. DIAMOND, J. M. (1965). 'The concentrating activity of the gall bladder.' In The Biliary System, pp. 495-514. Oxford: Blackwell Scientific Publications. DIAMOND, J. M. (1968). 'Transport mechanisms in the gall bladder.' In Handbook of Physiology, pp. 2451-2482. Washington: American Physiological Society. DUKES, H. H. (1955). The Physiology of Domestic Animals. London: Bailliere, Tindall and Cox. FOLCH, J., LEES, M. and SLOANE-STANLEY, G. H. (1957). 'A simple method for the isolation and purification of total lipides from animal tissues.' Journal of Biological Chemistry 226, 497. HAPRISON, F. A. (1962). 'Bile secretion in the sheep.' Journal of Physiology 162, 212. HARRISON, F. A. and HILL, K. J. (1962). 'Digestive secretions and the flow of digesta along the duodenum of the sheep.' Journal of Physiology 162, 225. HEATH, T. (1968). 'Origin and distribution of portal blood in the sheep.' American Journal of Anatomy 122, 95. HEATH, T. J. and HILL, L. N. (1969). 'Dietary and endogenous long-chain fatty acids in the intestine of sheep, with an appendix on their estimation in feeds, bile and faeces.' Australian Journal of Biological Sciences 22, 1015. HEATH, T. J., CAPLE, I. W. and REDDING, P. M. (1970). 'Effect of the enterohepatic circulation of bile salts on the flow of bile and its content of bile salts and lipids in sheep.' Quarterly Journal of Experimental Physiology 55, 93. HILL, K. J. (1968). 'Abomasal function.' In Handbook of Physiology, pp. 2747-2760. Washington: American Physiological Society. IRVIN, J. L., JOHNSTON, C. G. and KOPALA, J. (1944). 'A photometric method for the determination of cholates in bile and blood.' Journal of Biological Chemistry 153, 439. Ivy, A. C. (1934). 'The physiology of the gall bladder.' Physiological Reviews 14, 1. Ivy, A. C., KLOSTER, G., LUETH, H. C. and DREWYER, G. E. (1929). 'On the preparation of "cholecystokinin".' American Journal of Physiology 91, 336. Ivy, A. C. and JANECEK, H. M. (1959). 'Assay of Jorpes-Mutt secretin and cholecystokinin.' Acta Physiologica Scandinavica 45, 220 KLOSTER, G., Ivy, A. C. and LUETH, H. C. (1929). 'The preparation of solutions containing "cholecystokinin".' American Journal of Physiology 90, 415. LEAVER, D. D. (1968). 'Sporidesmin poisoning in sheep.' Research in Veterinary Science 9, 255. MAGEE, D. F. (1965). 'Physiology of gall bladder emptying.' In The Biliary System, ed. W. Taylor, pp. 233-245. Oxford: Blackwell Scientific Publications. MANN, F. C. (1924). 'The functions of the gall bladder.' Physiological Reviews 4, 251. NAHRWOLD, D. L. and GROSSMAN, M. I. (1967). 'Secretion of bile in response to food with and without bile in the intestine.' Gastroenterology 53, 11. PERIC-GOLIA, L. and Socic, H. (1968). 'Biliary bile acids and cholesterol in developing sheep.' American Journal of Physiology 215, 1284. PREISIG, R., COOPER, H. L. and WHEELER, H. 0. (1962). 'The relationship between taurocholate secretion rate and bile production in the unanesthetised dog during choline rgic blockade and during secretin administration.' Journal of Clinical Investigation 41, 1152. RAVDIN, I. S., JOHNSTON, C. G., RIEGEL, C. and WRIGHT, S. L. Jr (1932). 'Studies of gall bladder function. VII. The anion-cation content of hepatic and gall bladder bile.' American Journal of Physiology 100, 317. RUCKEBUSCH, Y. (1970). 'The electrical activity of the digestive tract of the sheep as an indication of the mechanical events in various regions.' Journal of Physiology 210, 857. SCHMIDT, C. R. and Ivy, A. C. (1937). 'The general function of the gall bladder.' Journal of Cellular and Comparative Physiology 10, 365. SEGAL, M. A. (1955). 'A rapid electrotitrimetric method for determining CO2 combining power in plasma or serum.' American Journal of Clinical Pathology 25, 1212.

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