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[ 17] P a n c r e a t i c S e c r e t i o n : In Vivo, P e r f u s e d G l a n d , and Isolated Duct Studies
By R. M. CASE and B. E. ARGENT Variations in Pancreatic Structure and Function The exocrine pancreas consists of two functional units, acini and ducts. Quantitatively, the acini dominate; duct cells have been calculated to occupy only 14% by volume of the gland in humans, 4% in guinea pig, and 2% in the rat. 1 The ductal tree begins with the centroacinar cells which line each acinus and which connect to the smallest elements of the true ductal system, the intercalated ducts. These open into intralobular ducts, which run within the pancreatic lobules, and which in turn join larger interlobular ducts.2,3 The final division of the ductal tree is usually the main pancreatic duct. However, in the rat, mouse, and hamster a variable number of interlobular ducts open into the bile duct forming a common bilepancreatic duct. In terms of experimental studies, the implications of this arrangement are clear. To collect pure pancreatic juice from a cannula inserted into the common duct, it is first necessary to hgate the duct beyond the limit of pancreatic tissue so as to prevent contamination with bile. In chronic animal experiments, cannulation of the bile duct at this point is obviously necessary in order to convey bile to the duodenum.4,5 Cats and dogs often have accessory ducts which open into the duodenum close to the main pancreatic duct. Rather than attempt to cannulate the accessory duct, it is usual to ligate it, and thereby direct all the secretion through the main duct, or ignore it. In most species the main pancreatic duct, or bile-pancreatic duct, empties into the second part of the duodenum. However, in the rabbit and guinea pig it enters the third part of the duodenum, a long way from the pylorus. It is generally agreed that acini secrete digestive enzymes and a variable (usually small) quantity of chloride-rich fluid in response to cholecystokinin-pancreozymin (CCK) and vagal stimulation (acting via acetylchoi S. Githens, J. Pediatr. Gastroenterol. Nutr. 7, 486 (1988). 2 R. M. Case and B. E. Argent, in "The Exocrine Pancreas: Biology, Pathobiology, and Diseases" (V. L. W. Go, J. D. Gardner, F. P. Brooks, E. Lebenthal, E. P. DiMagno, and G. A. Seheele, eds.), p. 213. Raven, New York, 1986. 3 R. M. Case and B. E. Argent, in "Handbook of Physiology: The Gastrointestinal System III" (S. G. Schultz, J. G. Forte, and B. B. Rauner, eds.), p. 383. Oxford University Press, New York, 1989. 4 F. J. Haberich, T. Bozkurt, and W. Reschke, Z. Gastroenterol. 18, 427 (1980). 5 S. Ormai, M. Sasv~-i, and E. Endr6czi, Scand, J. Gastroenterol. 21, 509 (1986).
METHODS IN ENZYMOLOGY, VOL. 192
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line), and that the ducts secret a bicarbonate-rich fluid in response to secretin. However, there are wide species variations in the volume of spontaneous secretion, the sensitivity to hormones and neurotransmitters, and the maximum bicarbonate concentration in pancreatic juice. 3,6 A knowledge o f these variations~ which are summarized in Table I, is essential for designing and interpreting both in vivo and in vitro experiments on the pancreas. In Vivo Studies
Today, in vivo studies are performed for three reasons. 1. To study physiological control mechanisms: Studies on the intact animal will always be necessary to probe the interaction between nerves, hormones, and paracrine agents in the regulation of pancreatic secretion.~,8 Most such studies are performed in conscious dogs or, to a lesser extent, cats and humans. Chronic animal experiments demand a permanent, satisfactory arrangement for collecting pancreatic juice. The simplest way to achieve this would be to create a fistula between the external body surface and duodenum. However, this is unsatisfactory because secreted pancreatic juice comes into contact with the duodenum. As a result, pancreatic proteases are activated, skin erosion occurs, and the flow of pancreatic juice becomes continuous? The solution to this problem is to install a wide cannula into the duodenal wall opposite the main pancreatic duct through which pancreatic juice can be collected temporarily, by cannulating the main duct, and returned to the duodenum. ~°,mlIn humans, either the duodenal contents are aspirated ~2 or the pancreatic duct is cannulated directly using endoscopic techniques. 13 6 R. M. Case, in "Experimental Pancreatitis" (G. Glazer and J. H. C. Ranson, eds.), p. 100. Balli6re Tindall, London, 1988. 7 Z. Itoh, R. Honda, and IC I-Iiwatashi, Am. Z Physiol. 238, G332 (1980). 8 M. Singer, in "The Exocrine Pancreas: Biology, Pathobiology, and Diseases" (V. L. W. Go, J. D. Gardner, F. P. Brooks, E. Lebenthal, E. P. DiMagno, and G. A. Scheele, eds.), p. 315. Raven, New York, 1986. 9 R. A. Gregory, "Secretory Mechanisms of the Gastro-Intestinal Tract." Arnold, London, 1962. l0 j. E. Thomas and J. O. Crider, Am. J. Physiol. 131, 349 (1940). i~ j. E. Thomas, "The External Secretion of the Pancreas." Thomas, Springfield, Illinois, 1950. 12E. P. DiMagno, in "The Exocrine Pancreas: Biology, Pathobiology, and Diseases" (V. L. W. Go, J. D. Gardner, F. P. Brooks, E. Lebenthal, E. P. DiMagno, and G. A. Scheele, eds.), p. 193. Raven, New York, 1986. 13S. Domschke, W. Domschke, W. Rosch, S. J. Konturek, E. Wunsch, and L. Demling, Gastroenterology 70, 533 (1976).
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TABLE I SPECIES-DEPENDENT PATTERNS OF ELECTROLYTE SECRETION~b
Species
Stimulus
Volume
Dog (1,2), cat (3), human (4)
Spontaneous +Secretin +CCK +Vagus Spontaneous +Secretin +CCK +Vagus Spontaneous +Secretin +CCK +Vagus (carbachol) Spontaneous +Sccretin +CCK +Vagus Spontaneous +Secretin +CCK +Vagus
0(+) +++++ + + + ++ +++ ++ ++ +++ ++ +++ + +++++ ++ ++++ + +++++ +++ +++
Rat (5)
Rabbit (6)
Pig (7,8)
Guinea pig (9,10)
Hamster (11)
Spontaneous +Secretin +CCK +Vagus (carbachol)
+ ++++ + ++
Maximum [HCO3-] (mM) -145 60 .9 25 70 30 ? 60 130 110 120 .9 160 35 150 95 150 140 120 60 140 40 80
° This table gives an idea of the response to stimuli given alone: potentiation often occurs when stimuli are given together. The references are to key papers which illustrate most of the features. Except for Refs. 1, 2, 4, and 8 (see below), all data were obtained from studies on anesthetized animals: quantitative differences may occur in conscious animals, especially in the rat, in which secretion is increased fivefold in conscious animals (see Ref. 5). CCK, Cholecystokinin-pancreozymin. b Key to references: (1) W. M. Hart and J. E. Thomas, Gastroenterology4, 409 (1945); (2) H. T. Debas and M. I. Grossman, Digestion 9, 469 (1973); (3) R. M. Case, A. A. Harper, and T. Scratcher& J. Physiol. 201, 335 (1969); (4) S. Domschke, W. Domschke, W. Rosch, S. J. Konturek, E. Wunsch, and L. Demling, Gastroenterology 70, 533 (1976); (5) W. A. Sewelland J. A. Young, J. Physiol. 252, 379 (1975); (6) F. Seow and J. A. Young, Proc. Aast. Physiol. Pharmacol. Soc. 17, 199P (1986); and K. T. F. P. Seow, R. M. Case, and J. A. Young Pancreas, in press; (7) J. C. D. Hickson, J. Physiol. 206, 275, 299 (1970); (8) S. L. Jensen, J. F. Rehfeld, J. J. Hoist, O. V. Nielsen, J. Fahrenkrng, and O. B. Schaffalitsky de Muckadell, Acta Physiol. Scand. 111, 225 (1981); (9) J. S. Davison and V. Dickson, in "Secretion: Mechanisms and Control" (R. M. Case, J. M. Lingard, and J. A. Young, eds.), p. 225. Manchester University Press, Manchester, 1984; (10) P. J. Padfield, A. Garner, and R. M. Case, Pancreas 4, 204 (1989); (11) A. E. Aft, S. C. B. Rutishauser, and R. M. Case, Pancreas 5, 314 (1990).
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2. To assay the effects of newly discovered peptides, drugs, etc.: Although in vitro studies can provide much information, they cannot predict the influence of secondary phenomena such as changes in blood flow (e.g., Ref. 14). Usually anesthetized animals are used for this purpose. In this case the pancreatic duct can be cannulated permanently either from the duodenum or, more easily, through an incision in the pancreatic duct at the point where it passes obliquely through the duodenal wall. 3. To study pancreatic growth and adaption, and models of pancreatic disease: Most studies on growth and dietary adaptation 15and experimental pancreatitis ~6,~7 are carded out on rats, largely for reasons of economy, while studies on experimental cancer often use hamsters? s
Acinar Cell Atrophy As mentioned above, the majority of pancreatic bicarbonate secretion is derived from the ducts. Because duct cells comprise such a small proportion of the gland, studying their function is difficult. One way around this problem is to selectively destroy the acinar tissue. This can be achieved in two ways. 1. By duct ligation: In many species, including the mouse, rat, guinea pig, rabbit, and dog, ligation of the main duct causes atrophy of the acinar cells, but not the ductal cells or islets of Langerhans. Following duct ligation in the rat, for example, the acini essentially disappear within 72 hr and cuboidal duct cells proliferate so that by the fifth day ductlike structures form the bulk of the lobular structure. 19 2. By feeding a copper-deficient diet: As first noticed by M011er,2° rats fed a copper-free diet develop a noninflammatory acinar cell atrophy in the absence of changes in ductal tissue. Addition to the diet ofa copper-chelat-
14R. M. Case and T. Scratcherd, J. Physiol. (London) 226, 393 (1972). 15U. R. Frlsch, Clin. Gastroenterol. 13, 679 (1984). 16G. Adler, H. F. Kern, and G. A. Seheele, in "The Exocrine Pancreas: Biology, Pathobiology, and Diseases" (V. L. W. Go, J. D. Gardner, F. P. Brooks, E. Lebenthal, E. P. DiMagno, and G. A. Seheele, eds.), p. 407. Raven, New York, 1986. 17M. Steer, in "Experimental Panereatitis" (G. Glazer and J. H. C. Ranson, eds.), p. 207, Bailli~re Tindall, London, 1988. is D. S. Longnecker, in "The Exoerine Pancreas: Biology, Pathobiology, and Diseases" (V. L. W. Go, J. D. Gardner, F. P. Brooks, E. Lebenthal, E. P. DiMagno, and G. A. Scheele, eels.), p. 443, Raven, New York, 1986. 19A. W. Pound and N. I. Walker, Br. J. Exp. Pathol. 62, 547 (1981). 2oH. B. Mfdler, VirchowsArch. A:Pathol. Anat. 350, 353 (1970).
260
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ing agent, such as ~penicillamine 21 or triethylenetetramine, 22 accelerates the process. To produce atrophy, young, male rats (125- 125 g) are allowed free access to distilled water and are fed a copper-deficient diet (about 20 g daily for 6 - 1 0 weeks) containing no more than 3 mg kg--1 copper and between 10 and 30 mg kg-1 zinc. This diet can be purchased from SDS, Ltd. (Witham, Essex, England). Provided that the copper and zinc contents are as specified, it is unnecessary to add chelating agents. However, it is important to prevent coprophagy by housing the rats in wire-bottomed cages, and to ensure that cage fittings are not made from copper-containing alloys. 22
The secretory behavior of such atrophic glands in anesthetized rats is as would be predicted from Table I, i.e., the fluid secretory response to secretin is preserved while that to CCK is greatly attenuated. 2~,22Unfortunately attempts to apply this method to other laboratory species have so far failed (e.g., the hamster is not affected; U. R. F61sch, personal communication) so that it is unlikely to provide useful information about comparative aspects of regulation of duct cell function. However, it is a very convenient method for providing uncontaminated ductal tissue for biophysical studies (see below).
Ductal Perfusion The main pancreatic duct modifies the electrolyte composition of the primary secretions produced by the small ducts and acini. One way of studying the properties of the main pancreatic duct in vivo is to perfuse the duct lumen in anesthetized animals. In this method, first described in the cat) 3 the main duct is cannulated both at the tail of the gland and at the point where it traverses the duodenal wall. The duct is then perfused with an appropriate fluid (artificial "pancreatic juice") from tail to head using a motor-driven syringe. This technique is particularly suited to those species like the cat where there is no spontaneous secretion, so that perfusion fluids are not contaminated with endogenous secretions. However, with the inclusion of a volume marker in the perfusion fluid, it has also been used in other species such as the rabbit, u Localization of the duct at the tail of the gland is often difficult and requires microdissection. It is therefore tempting to thread a narrow flexible tube through the duct from the duodenal end and withdraw it from the tail. However, such treatment dramatically 21 U. R. F61sch and W. Crcutzfeldt, Gastroenterology 73, 1053 (1977). 22 p. A. Smith, J. P. Sunter, and R. M. Case, Digestion 23, 16 (1982). 23 R. M. Case, A. A. Harper, and T. Scratchcrd, J. Physiol. (London) 201, 335 (1969). u H. A. Reber, C. J. Wolf, and S. P. Lee, Surg. Forum 20, 382 (1969).
[17]
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changes the permeability of the ductal epithelium and should therefore be avoided. 25 Ductal perfusion was used first to demonstrate that significant passive fluxes of bicarbonate and chloride occur across the ductal epithelium and that such fluxes are responsible for the flow rate-dependent changes in anion composition of pancreatic juice (i.e., the decrease in bicarbonate and reciprocal increase in chloride which occur as secretory rate declines).2a As well as being used to characterize the physiological properties of the ductal epithelium, this technique has also been used to study the effect on ductal permeability of factors implicated in the pathogenesis of pancreatitis. 26 Isolated Gland Studies
The first in vitro preparation of the pancreas (essentially, pancreatic slices) was used in the 1930s to study pancreatic metabolism. Similar preparations have subsequently been used with huge success to study the cellular mechanisms and control of pancreatic enzyme secretion. More recently there has been a move toward using dissociated acini and single acinar cells for such studies. These in vitro techniques fall beyond the scope of this chapter and are described elsewhere in this volume. Pancreatic fluid secretion dearly cannot be studied in slices or dissociated acini because ductal integrity must be maintained in order to collect and measure the secretory product. To achieve this, isolated whole gland preparations are required. Such preparations have two major uses. 1. To study the function of the gland in the absence of interference from other organs which may complicate interpretation of the data: In this way it has been observed, for example, that the inhibitory effect of prostaglandin E on fluid secretion in the anesthetized cat is indirect (caused by reflex vasoconstriction), as the same substance stimulates fluid secretion in the perfused cat pancreas. 14 A number of peptides also have different effects on fluid secretion in vivo and in vitro, perhaps for the same reason. 2. To study the cellular mechanisms responsible for fluid secretion by observing the effects of ion substitution and transport inhibitors: Although direct studies of duct tissue are now possible (see below), isolated gland preparations have provided a great deal of useful information about pancreatic electrolyte secretion and its cellular control. 2,3 R. M. Case and T. Scratcherd,Biochim. Biophys. Acta 219, (1970). 26H. A. Reber, G. Adler, and IC R. Wedgwood, in "The Exocrine Pancreas: Biology, Pathobiology,and Diseases"(V. L. W. Go, J. D. Gardner,F. P. Brooks,E. Lebenthal,E. P. DiMagno, and G. A. Scheele,eds.), p. 255. Raven,New York, 1986.
262
GASTROINTESTINAL SYSTEM
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Although methods for isolating and perfusing the dog pancreas were described in the 1920s27,28and perfused rat preparations were first used to study insulin secretion in 1947,29 not until the 1960s were glands isolated from the rabbit and cat used to study pancreatic exocrine secretion. The techniques used in these two species are quite different. The Isolated Rabbit Pancreas The pancreas in rabbit lies between the descending and ascending limbs of the first intestinal loop. Taking advantage of the extreme thinness of this gland, Rothman and Brooks3° devised a method in which the gland was suspended in a bath of physiological salt solution. New Zealand White rabbits are fasted overnight and anesthetized with urethane. To isolate the pancreas, the attachments of other intestinal segments to the first intestinal loop are ligated and severed and its distal end cut away from the remaining intestine and from the mesentery medial to the portal vein. To prevent damage to the gland, a segment of rectum is left attached. The pancreatic duct which, as mentioned above, enters the third part of the duodenum is then cannulated with a fine polyethylene tube. Finally, the intestinal loop containing the pancreas is removed from the animal, attached by hooks to a Plexiglas frame, and the whole place in a heated chamber containing a physiological salt solution (Fig. 1). This preparation has been used in a number of laboratories for studies of pancreatic fluid secretion,a°-34 epithelial permeability,35 stimulussecretion coupling,36 and the "endocrine" secretion of pancreatic enzymes. 37 It is quick and easy to set up. However, it has one major disadvantage: its rate of spontaneous secretion is five- to sixfold greater than that of the gland in vivo and, consequently, it is almost insensitive to secretin stimulation.3°
27 B. P. Babkin and E. H. Starling, J. Physiol. (London) 61, 245 (1926). 2s B. Goldstein, Z. Gesamte Exp. Med. 61, 649 0928). 29 E. Anderson and J. A. Long, Endocrinology 40, 92 (1947). 3o S. S. Rothman and F. P. Brooks, Am. J. Physiol. 208, 1171 (1965). 31 K. A. Hub¢l, Am. J. Physiol. 212, 101 (1967). 32 A. S. Ridderstap, Pfluegers Arch. 311, 199 (1969). 33 C. H. Swanson and A. K. Solomon, J. Gen. Physiol. 62, 407 (1973). 34 C. R. Cafliscli, S. Solomon, and W. R. Galey, PfluegersArch. 380, 121 (1979). 35 j. W. C. M. Jansen, J. J. H. H. M. de Pont, and S. L. Bonting, Biochim. Biophys. Acta 551, 95 (1979). • s V. V. A. M. Schreurs, H. G. P. Swart.s, J. J. H. H. M. de Pont, and S. L. Bonting Biochim. Biophys. Acta 404, 257 (1975). 3~ L. D. Isenman and S. S. Rothman, Proc. Natl. Acad. Sci. U.S.A. 74, 4068 (1977).
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PANCREATIC SECRETION
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duodenum,
cannula"~7
stomach
pancreatic duct
spleen
rectum
FIG. 1. Schematic diagram of the isolated rabbit pancreas and attached duodenum mounted on a Plexiglas frame, which is then suspended in a chamber filled with a physiological salt solution. The stippled area represents pancreatic tissue. (Courtesy of Professor J. J. H. H. M. de Pont.)
Vascular Perfused Gland Preparations As an alternative to the rabbit gland preparation, Case et aL as developed a m o r e conventional preparation for studies o f electrolyte secretion in which the cat pancreas was surgically isolated a n d perfused through its arterial supply with physiological salt solutions. T h e pancreas receives its blood supply f r o m a n u m b e r o f sources: as an example the supply to the cat gland is illustrated in Fig. 2. Therefore considerable surgery is required in isolating the gland prior to perfusion. Although the principles involved in such surgery are similar in all species, the details will clearly differ according to m i n o r a n a t o m i c a l variations. In addition to the cat 3s details o f gland isolation a n d perfusion have been described for the following species: dog, 39-4t rat, 42 pig, 43 guinea pig, *~ a n d Syrian golden hamster. 45 Recent 38R. M. Case, A. A. Harper, and T. Scrateherd, J. Physiol. (London) 196, 133 (1968). 39G. L. Nardi, J. M. Grecp, D. A. Chambers, C. MeCrae, and D. B. Skinner, Ann. Surg. 158, 830 (1963). 40j. Hermon-Taylor, Gastroenterology55, 488 (1968). 4t D. Augier, J. P. Boucard, J. P. Pascal, A. Ribet, and N. Vaysse, J. Physiol. (London) 221, 55 (1972). 42T. Kanno, J. Physiol. (London) 226, 353 (1972). 43S. L. Jensen, J. Fahrenkrug, J. J. Hoist, C. Ktihl, O. V. Nielsen, and O. B. Schaffalitskyde Muckadell, Am. J. Physiol. 235, E381 (1978). 44T. Matsumoto and T. Kanno, Peptides 5, 285 (1984). 45R. H. Bell, S. Place, P. McCullough, M. B. Ray, and D. H. Rogers, Int. J. Pancreatol. 1, 71 (1986).
264
GASTROINTESTINAL SYSTEM
[ 1 7]
Pancrea duct car
Outflow
Fio. 2. Vascular supply of the cat pancreas. For clarity, the stomach is shown separated from the duodenum, displaced anteriorly and turned through 180 °. A, Aorta; CA, celiac axis; GDA, gastroduodenal artery; GSV, gastrosplenic vein; HA, hepatic artery; LA, lumbar artery; LGA, left gastric arte~; PV, portal vein; RGA, right gastric artery; SA, splenic artery; SMA, superior mesenteric arter~f, SMV, superior mesenteric vein. (Reproduced with permission from R. M. Case, A. A. Harper, and T. Scratcherd, Z Physiol. 196, 133, 1968.)
reviews of the techniques involved have been published for the cat~ and rap 7 and will not be repeated here. In addition to studies on the mechanisms and cellular control of pancreatic electrolyte secretion, 2,3 perfused glands can be used to study a 4~ T. Scratcherd, in "The Exocrine Pancreas: Biology, Pathobiology, and Diseases" (V. L W.
Go, J. D. Gardner, F. P. Brooks, E. Lebenthal, E. P. DiMagno, and G. A. Schccle, eds.), p. 245. Raven, New York, 1986. 47 T. Kanno in "In Vitro Methods for Studying Secretion" (A. M. Poisner and J. M. Trffar6, eds.) p. 45. Elsevier Biomedical, Amsterdam, 1987.
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variety of functions, including control of enzyme secretion,4s,49the "endocrine" secretion of pancreatic enzymes,5°,5~ amino acid transport into the gland, 52 determination of the neurotransmitters involved in regulation,4s,53 assaying secretin,54 tissue metabolism,55 hemodynamics and vasomotor phenomena,4~,56,57 production of circulatory shock factors, 5s and experimental pancreatitis. 59,6° The type of solution used as a perfusion fluid is determined to some extent by the needs of the experiment, by convenience, and by cost. For most studies a simple bicarbonate-buffered physiological salt solution is adequate: in the case of ion substitution studies it is essential. With such solutions it is necessary to add a small amount of albumin (0.1%) to prevent binding of stimulatory peptides to the glassware of the perfusion circuit. The low viscosity of these simple salt solutions allows rapid perfusion at low perfusion pressures (
GASTROINTESTINAL
SYSTEM
[ 17]
[ 17] P a n c r e a t i c S e c r e t i o n : In Vivo, P e r f u s e d G l a n d , and Isolated Duct Studies
By R. M. CASE and B. E. ARGENT Variations in Pancreatic Structure and Function The exocrine pancreas consists of two functional units, acini and ducts. Quantitatively, the acini dominate; duct cells have been calculated to occupy only 14% by volume of the gland in humans, 4% in guinea pig, and 2% in the rat. 1 The ductal tree begins with the centroacinar cells which line each acinus and which connect to the smallest elements of the true ductal system, the intercalated ducts. These open into intralobular ducts, which run within the pancreatic lobules, and which in turn join larger interlobular ducts.2,3 The final division of the ductal tree is usually the main pancreatic duct. However, in the rat, mouse, and hamster a variable number of interlobular ducts open into the bile duct forming a common bilepancreatic duct. In terms of experimental studies, the implications of this arrangement are clear. To collect pure pancreatic juice from a cannula inserted into the common duct, it is first necessary to hgate the duct beyond the limit of pancreatic tissue so as to prevent contamination with bile. In chronic animal experiments, cannulation of the bile duct at this point is obviously necessary in order to convey bile to the duodenum.4,5 Cats and dogs often have accessory ducts which open into the duodenum close to the main pancreatic duct. Rather than attempt to cannulate the accessory duct, it is usual to ligate it, and thereby direct all the secretion through the main duct, or ignore it. In most species the main pancreatic duct, or bile-pancreatic duct, empties into the second part of the duodenum. However, in the rabbit and guinea pig it enters the third part of the duodenum, a long way from the pylorus. It is generally agreed that acini secrete digestive enzymes and a variable (usually small) quantity of chloride-rich fluid in response to cholecystokinin-pancreozymin (CCK) and vagal stimulation (acting via acetylchoi S. Githens, J. Pediatr. Gastroenterol. Nutr. 7, 486 (1988). 2 R. M. Case and B. E. Argent, in "The Exocrine Pancreas: Biology, Pathobiology, and Diseases" (V. L. W. Go, J. D. Gardner, F. P. Brooks, E. Lebenthal, E. P. DiMagno, and G. A. Seheele, eds.), p. 213. Raven, New York, 1986. 3 R. M. Case and B. E. Argent, in "Handbook of Physiology: The Gastrointestinal System III" (S. G. Schultz, J. G. Forte, and B. B. Rauner, eds.), p. 383. Oxford University Press, New York, 1989. 4 F. J. Haberich, T. Bozkurt, and W. Reschke, Z. Gastroenterol. 18, 427 (1980). 5 S. Ormai, M. Sasv~-i, and E. Endr6czi, Scand, J. Gastroenterol. 21, 509 (1986).
METHODS IN ENZYMOLOGY, VOL. 192
Copyright© 1990by AcademicPress,Inc. All rightsof reproductionin any formreserved.
[17]
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line), and that the ducts secret a bicarbonate-rich fluid in response to secretin. However, there are wide species variations in the volume of spontaneous secretion, the sensitivity to hormones and neurotransmitters, and the maximum bicarbonate concentration in pancreatic juice. 3,6 A knowledge o f these variations~ which are summarized in Table I, is essential for designing and interpreting both in vivo and in vitro experiments on the pancreas. In Vivo Studies
Today, in vivo studies are performed for three reasons. 1. To study physiological control mechanisms: Studies on the intact animal will always be necessary to probe the interaction between nerves, hormones, and paracrine agents in the regulation of pancreatic secretion.~,8 Most such studies are performed in conscious dogs or, to a lesser extent, cats and humans. Chronic animal experiments demand a permanent, satisfactory arrangement for collecting pancreatic juice. The simplest way to achieve this would be to create a fistula between the external body surface and duodenum. However, this is unsatisfactory because secreted pancreatic juice comes into contact with the duodenum. As a result, pancreatic proteases are activated, skin erosion occurs, and the flow of pancreatic juice becomes continuous? The solution to this problem is to install a wide cannula into the duodenal wall opposite the main pancreatic duct through which pancreatic juice can be collected temporarily, by cannulating the main duct, and returned to the duodenum. ~°,mlIn humans, either the duodenal contents are aspirated ~2 or the pancreatic duct is cannulated directly using endoscopic techniques. 13 6 R. M. Case, in "Experimental Pancreatitis" (G. Glazer and J. H. C. Ranson, eds.), p. 100. Balli6re Tindall, London, 1988. 7 Z. Itoh, R. Honda, and IC I-Iiwatashi, Am. Z Physiol. 238, G332 (1980). 8 M. Singer, in "The Exocrine Pancreas: Biology, Pathobiology, and Diseases" (V. L. W. Go, J. D. Gardner, F. P. Brooks, E. Lebenthal, E. P. DiMagno, and G. A. Scheele, eds.), p. 315. Raven, New York, 1986. 9 R. A. Gregory, "Secretory Mechanisms of the Gastro-Intestinal Tract." Arnold, London, 1962. l0 j. E. Thomas and J. O. Crider, Am. J. Physiol. 131, 349 (1940). i~ j. E. Thomas, "The External Secretion of the Pancreas." Thomas, Springfield, Illinois, 1950. 12E. P. DiMagno, in "The Exocrine Pancreas: Biology, Pathobiology, and Diseases" (V. L. W. Go, J. D. Gardner, F. P. Brooks, E. Lebenthal, E. P. DiMagno, and G. A. Scheele, eds.), p. 193. Raven, New York, 1986. 13S. Domschke, W. Domschke, W. Rosch, S. J. Konturek, E. Wunsch, and L. Demling, Gastroenterology 70, 533 (1976).
258
GASTROINTESTINAL SYSTEM
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TABLE I SPECIES-DEPENDENT PATTERNS OF ELECTROLYTE SECRETION~b
Species
Stimulus
Volume
Dog (1,2), cat (3), human (4)
Spontaneous +Secretin +CCK +Vagus Spontaneous +Secretin +CCK +Vagus Spontaneous +Secretin +CCK +Vagus (carbachol) Spontaneous +Sccretin +CCK +Vagus Spontaneous +Secretin +CCK +Vagus
0(+) +++++ + + + ++ +++ ++ ++ +++ ++ +++ + +++++ ++ ++++ + +++++ +++ +++
Rat (5)
Rabbit (6)
Pig (7,8)
Guinea pig (9,10)
Hamster (11)
Spontaneous +Secretin +CCK +Vagus (carbachol)
+ ++++ + ++
Maximum [HCO3-] (mM) -145 60 .9 25 70 30 ? 60 130 110 120 .9 160 35 150 95 150 140 120 60 140 40 80
° This table gives an idea of the response to stimuli given alone: potentiation often occurs when stimuli are given together. The references are to key papers which illustrate most of the features. Except for Refs. 1, 2, 4, and 8 (see below), all data were obtained from studies on anesthetized animals: quantitative differences may occur in conscious animals, especially in the rat, in which secretion is increased fivefold in conscious animals (see Ref. 5). CCK, Cholecystokinin-pancreozymin. b Key to references: (1) W. M. Hart and J. E. Thomas, Gastroenterology4, 409 (1945); (2) H. T. Debas and M. I. Grossman, Digestion 9, 469 (1973); (3) R. M. Case, A. A. Harper, and T. Scratcher& J. Physiol. 201, 335 (1969); (4) S. Domschke, W. Domschke, W. Rosch, S. J. Konturek, E. Wunsch, and L. Demling, Gastroenterology 70, 533 (1976); (5) W. A. Sewelland J. A. Young, J. Physiol. 252, 379 (1975); (6) F. Seow and J. A. Young, Proc. Aast. Physiol. Pharmacol. Soc. 17, 199P (1986); and K. T. F. P. Seow, R. M. Case, and J. A. Young Pancreas, in press; (7) J. C. D. Hickson, J. Physiol. 206, 275, 299 (1970); (8) S. L. Jensen, J. F. Rehfeld, J. J. Hoist, O. V. Nielsen, J. Fahrenkrng, and O. B. Schaffalitsky de Muckadell, Acta Physiol. Scand. 111, 225 (1981); (9) J. S. Davison and V. Dickson, in "Secretion: Mechanisms and Control" (R. M. Case, J. M. Lingard, and J. A. Young, eds.), p. 225. Manchester University Press, Manchester, 1984; (10) P. J. Padfield, A. Garner, and R. M. Case, Pancreas 4, 204 (1989); (11) A. E. Aft, S. C. B. Rutishauser, and R. M. Case, Pancreas 5, 314 (1990).
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2. To assay the effects of newly discovered peptides, drugs, etc.: Although in vitro studies can provide much information, they cannot predict the influence of secondary phenomena such as changes in blood flow (e.g., Ref. 14). Usually anesthetized animals are used for this purpose. In this case the pancreatic duct can be cannulated permanently either from the duodenum or, more easily, through an incision in the pancreatic duct at the point where it passes obliquely through the duodenal wall. 3. To study pancreatic growth and adaption, and models of pancreatic disease: Most studies on growth and dietary adaptation 15and experimental pancreatitis ~6,~7 are carded out on rats, largely for reasons of economy, while studies on experimental cancer often use hamsters? s
Acinar Cell Atrophy As mentioned above, the majority of pancreatic bicarbonate secretion is derived from the ducts. Because duct cells comprise such a small proportion of the gland, studying their function is difficult. One way around this problem is to selectively destroy the acinar tissue. This can be achieved in two ways. 1. By duct ligation: In many species, including the mouse, rat, guinea pig, rabbit, and dog, ligation of the main duct causes atrophy of the acinar cells, but not the ductal cells or islets of Langerhans. Following duct ligation in the rat, for example, the acini essentially disappear within 72 hr and cuboidal duct cells proliferate so that by the fifth day ductlike structures form the bulk of the lobular structure. 19 2. By feeding a copper-deficient diet: As first noticed by M011er,2° rats fed a copper-free diet develop a noninflammatory acinar cell atrophy in the absence of changes in ductal tissue. Addition to the diet ofa copper-chelat-
14R. M. Case and T. Scratcherd, J. Physiol. (London) 226, 393 (1972). 15U. R. Frlsch, Clin. Gastroenterol. 13, 679 (1984). 16G. Adler, H. F. Kern, and G. A. Seheele, in "The Exocrine Pancreas: Biology, Pathobiology, and Diseases" (V. L. W. Go, J. D. Gardner, F. P. Brooks, E. Lebenthal, E. P. DiMagno, and G. A. Seheele, eds.), p. 407. Raven, New York, 1986. 17M. Steer, in "Experimental Panereatitis" (G. Glazer and J. H. C. Ranson, eds.), p. 207, Bailli~re Tindall, London, 1988. is D. S. Longnecker, in "The Exoerine Pancreas: Biology, Pathobiology, and Diseases" (V. L. W. Go, J. D. Gardner, F. P. Brooks, E. Lebenthal, E. P. DiMagno, and G. A. Scheele, eels.), p. 443, Raven, New York, 1986. 19A. W. Pound and N. I. Walker, Br. J. Exp. Pathol. 62, 547 (1981). 2oH. B. Mfdler, VirchowsArch. A:Pathol. Anat. 350, 353 (1970).
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ing agent, such as ~penicillamine 21 or triethylenetetramine, 22 accelerates the process. To produce atrophy, young, male rats (125- 125 g) are allowed free access to distilled water and are fed a copper-deficient diet (about 20 g daily for 6 - 1 0 weeks) containing no more than 3 mg kg--1 copper and between 10 and 30 mg kg-1 zinc. This diet can be purchased from SDS, Ltd. (Witham, Essex, England). Provided that the copper and zinc contents are as specified, it is unnecessary to add chelating agents. However, it is important to prevent coprophagy by housing the rats in wire-bottomed cages, and to ensure that cage fittings are not made from copper-containing alloys. 22
The secretory behavior of such atrophic glands in anesthetized rats is as would be predicted from Table I, i.e., the fluid secretory response to secretin is preserved while that to CCK is greatly attenuated. 2~,22Unfortunately attempts to apply this method to other laboratory species have so far failed (e.g., the hamster is not affected; U. R. F61sch, personal communication) so that it is unlikely to provide useful information about comparative aspects of regulation of duct cell function. However, it is a very convenient method for providing uncontaminated ductal tissue for biophysical studies (see below).
Ductal Perfusion The main pancreatic duct modifies the electrolyte composition of the primary secretions produced by the small ducts and acini. One way of studying the properties of the main pancreatic duct in vivo is to perfuse the duct lumen in anesthetized animals. In this method, first described in the cat) 3 the main duct is cannulated both at the tail of the gland and at the point where it traverses the duodenal wall. The duct is then perfused with an appropriate fluid (artificial "pancreatic juice") from tail to head using a motor-driven syringe. This technique is particularly suited to those species like the cat where there is no spontaneous secretion, so that perfusion fluids are not contaminated with endogenous secretions. However, with the inclusion of a volume marker in the perfusion fluid, it has also been used in other species such as the rabbit, u Localization of the duct at the tail of the gland is often difficult and requires microdissection. It is therefore tempting to thread a narrow flexible tube through the duct from the duodenal end and withdraw it from the tail. However, such treatment dramatically 21 U. R. F61sch and W. Crcutzfeldt, Gastroenterology 73, 1053 (1977). 22 p. A. Smith, J. P. Sunter, and R. M. Case, Digestion 23, 16 (1982). 23 R. M. Case, A. A. Harper, and T. Scratchcrd, J. Physiol. (London) 201, 335 (1969). u H. A. Reber, C. J. Wolf, and S. P. Lee, Surg. Forum 20, 382 (1969).
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changes the permeability of the ductal epithelium and should therefore be avoided. 25 Ductal perfusion was used first to demonstrate that significant passive fluxes of bicarbonate and chloride occur across the ductal epithelium and that such fluxes are responsible for the flow rate-dependent changes in anion composition of pancreatic juice (i.e., the decrease in bicarbonate and reciprocal increase in chloride which occur as secretory rate declines).2a As well as being used to characterize the physiological properties of the ductal epithelium, this technique has also been used to study the effect on ductal permeability of factors implicated in the pathogenesis of pancreatitis. 26 Isolated Gland Studies
The first in vitro preparation of the pancreas (essentially, pancreatic slices) was used in the 1930s to study pancreatic metabolism. Similar preparations have subsequently been used with huge success to study the cellular mechanisms and control of pancreatic enzyme secretion. More recently there has been a move toward using dissociated acini and single acinar cells for such studies. These in vitro techniques fall beyond the scope of this chapter and are described elsewhere in this volume. Pancreatic fluid secretion dearly cannot be studied in slices or dissociated acini because ductal integrity must be maintained in order to collect and measure the secretory product. To achieve this, isolated whole gland preparations are required. Such preparations have two major uses. 1. To study the function of the gland in the absence of interference from other organs which may complicate interpretation of the data: In this way it has been observed, for example, that the inhibitory effect of prostaglandin E on fluid secretion in the anesthetized cat is indirect (caused by reflex vasoconstriction), as the same substance stimulates fluid secretion in the perfused cat pancreas. 14 A number of peptides also have different effects on fluid secretion in vivo and in vitro, perhaps for the same reason. 2. To study the cellular mechanisms responsible for fluid secretion by observing the effects of ion substitution and transport inhibitors: Although direct studies of duct tissue are now possible (see below), isolated gland preparations have provided a great deal of useful information about pancreatic electrolyte secretion and its cellular control. 2,3 R. M. Case and T. Scratcherd,Biochim. Biophys. Acta 219, (1970). 26H. A. Reber, G. Adler, and IC R. Wedgwood, in "The Exocrine Pancreas: Biology, Pathobiology,and Diseases"(V. L. W. Go, J. D. Gardner,F. P. Brooks,E. Lebenthal,E. P. DiMagno, and G. A. Scheele,eds.), p. 255. Raven,New York, 1986.
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Although methods for isolating and perfusing the dog pancreas were described in the 1920s27,28and perfused rat preparations were first used to study insulin secretion in 1947,29 not until the 1960s were glands isolated from the rabbit and cat used to study pancreatic exocrine secretion. The techniques used in these two species are quite different. The Isolated Rabbit Pancreas The pancreas in rabbit lies between the descending and ascending limbs of the first intestinal loop. Taking advantage of the extreme thinness of this gland, Rothman and Brooks3° devised a method in which the gland was suspended in a bath of physiological salt solution. New Zealand White rabbits are fasted overnight and anesthetized with urethane. To isolate the pancreas, the attachments of other intestinal segments to the first intestinal loop are ligated and severed and its distal end cut away from the remaining intestine and from the mesentery medial to the portal vein. To prevent damage to the gland, a segment of rectum is left attached. The pancreatic duct which, as mentioned above, enters the third part of the duodenum is then cannulated with a fine polyethylene tube. Finally, the intestinal loop containing the pancreas is removed from the animal, attached by hooks to a Plexiglas frame, and the whole place in a heated chamber containing a physiological salt solution (Fig. 1). This preparation has been used in a number of laboratories for studies of pancreatic fluid secretion,a°-34 epithelial permeability,35 stimulussecretion coupling,36 and the "endocrine" secretion of pancreatic enzymes. 37 It is quick and easy to set up. However, it has one major disadvantage: its rate of spontaneous secretion is five- to sixfold greater than that of the gland in vivo and, consequently, it is almost insensitive to secretin stimulation.3°
27 B. P. Babkin and E. H. Starling, J. Physiol. (London) 61, 245 (1926). 2s B. Goldstein, Z. Gesamte Exp. Med. 61, 649 0928). 29 E. Anderson and J. A. Long, Endocrinology 40, 92 (1947). 3o S. S. Rothman and F. P. Brooks, Am. J. Physiol. 208, 1171 (1965). 31 K. A. Hub¢l, Am. J. Physiol. 212, 101 (1967). 32 A. S. Ridderstap, Pfluegers Arch. 311, 199 (1969). 33 C. H. Swanson and A. K. Solomon, J. Gen. Physiol. 62, 407 (1973). 34 C. R. Cafliscli, S. Solomon, and W. R. Galey, PfluegersArch. 380, 121 (1979). 35 j. W. C. M. Jansen, J. J. H. H. M. de Pont, and S. L. Bonting, Biochim. Biophys. Acta 551, 95 (1979). • s V. V. A. M. Schreurs, H. G. P. Swart.s, J. J. H. H. M. de Pont, and S. L. Bonting Biochim. Biophys. Acta 404, 257 (1975). 3~ L. D. Isenman and S. S. Rothman, Proc. Natl. Acad. Sci. U.S.A. 74, 4068 (1977).
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duodenum,
cannula"~7
stomach
pancreatic duct
spleen
rectum
FIG. 1. Schematic diagram of the isolated rabbit pancreas and attached duodenum mounted on a Plexiglas frame, which is then suspended in a chamber filled with a physiological salt solution. The stippled area represents pancreatic tissue. (Courtesy of Professor J. J. H. H. M. de Pont.)
Vascular Perfused Gland Preparations As an alternative to the rabbit gland preparation, Case et aL as developed a m o r e conventional preparation for studies o f electrolyte secretion in which the cat pancreas was surgically isolated a n d perfused through its arterial supply with physiological salt solutions. T h e pancreas receives its blood supply f r o m a n u m b e r o f sources: as an example the supply to the cat gland is illustrated in Fig. 2. Therefore considerable surgery is required in isolating the gland prior to perfusion. Although the principles involved in such surgery are similar in all species, the details will clearly differ according to m i n o r a n a t o m i c a l variations. In addition to the cat 3s details o f gland isolation a n d perfusion have been described for the following species: dog, 39-4t rat, 42 pig, 43 guinea pig, *~ a n d Syrian golden hamster. 45 Recent 38R. M. Case, A. A. Harper, and T. Scrateherd, J. Physiol. (London) 196, 133 (1968). 39G. L. Nardi, J. M. Grecp, D. A. Chambers, C. MeCrae, and D. B. Skinner, Ann. Surg. 158, 830 (1963). 40j. Hermon-Taylor, Gastroenterology55, 488 (1968). 4t D. Augier, J. P. Boucard, J. P. Pascal, A. Ribet, and N. Vaysse, J. Physiol. (London) 221, 55 (1972). 42T. Kanno, J. Physiol. (London) 226, 353 (1972). 43S. L. Jensen, J. Fahrenkrug, J. J. Hoist, C. Ktihl, O. V. Nielsen, and O. B. Schaffalitskyde Muckadell, Am. J. Physiol. 235, E381 (1978). 44T. Matsumoto and T. Kanno, Peptides 5, 285 (1984). 45R. H. Bell, S. Place, P. McCullough, M. B. Ray, and D. H. Rogers, Int. J. Pancreatol. 1, 71 (1986).
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Pancrea duct car
Outflow
Fio. 2. Vascular supply of the cat pancreas. For clarity, the stomach is shown separated from the duodenum, displaced anteriorly and turned through 180 °. A, Aorta; CA, celiac axis; GDA, gastroduodenal artery; GSV, gastrosplenic vein; HA, hepatic artery; LA, lumbar artery; LGA, left gastric arte~; PV, portal vein; RGA, right gastric artery; SA, splenic artery; SMA, superior mesenteric arter~f, SMV, superior mesenteric vein. (Reproduced with permission from R. M. Case, A. A. Harper, and T. Scratcherd, Z Physiol. 196, 133, 1968.)
reviews of the techniques involved have been published for the cat~ and rap 7 and will not be repeated here. In addition to studies on the mechanisms and cellular control of pancreatic electrolyte secretion, 2,3 perfused glands can be used to study a 4~ T. Scratcherd, in "The Exocrine Pancreas: Biology, Pathobiology, and Diseases" (V. L W.
Go, J. D. Gardner, F. P. Brooks, E. Lebenthal, E. P. DiMagno, and G. A. Schccle, eds.), p. 245. Raven, New York, 1986. 47 T. Kanno in "In Vitro Methods for Studying Secretion" (A. M. Poisner and J. M. Trffar6, eds.) p. 45. Elsevier Biomedical, Amsterdam, 1987.
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variety of functions, including control of enzyme secretion,4s,49the "endocrine" secretion of pancreatic enzymes,5°,5~ amino acid transport into the gland, 52 determination of the neurotransmitters involved in regulation,4s,53 assaying secretin,54 tissue metabolism,55 hemodynamics and vasomotor phenomena,4~,56,57 production of circulatory shock factors, 5s and experimental pancreatitis. 59,6° The type of solution used as a perfusion fluid is determined to some extent by the needs of the experiment, by convenience, and by cost. For most studies a simple bicarbonate-buffered physiological salt solution is adequate: in the case of ion substitution studies it is essential. With such solutions it is necessary to add a small amount of albumin (0.1%) to prevent binding of stimulatory peptides to the glassware of the perfusion circuit. The low viscosity of these simple salt solutions allows rapid perfusion at low perfusion pressures (
