Down-regulation of pancreatic growth and gallbladder contractility by bile salts

Down-Regulation of Pancreatic Growth and Gallbladder Contractility by Bile Salts

Guillermo Gomez, MD, Courtney M. Townsend, Jr., MD, Roya Maani, MD, Pomila Singh, Pho, George G. Greeley, Jr., Pho, James C. Thompson, MD, Galveston, TW

Luminal sequestration of bile salts with cholestyramine and the oral administration of bile salts represent current forms of therapy for some diseases. We have recently reported that secretion of these salts exerts negative feedback control on the release of cholecystokinin (CCK) . The purpose of this study was to examine the effects of long-term alterations of huninal concentrations of bile salts on CCK target organs, the pancreas and gallbladder. The bile salt pool in adult guinea pigs was either enriched by feeding 0.5 percent sodium taurocholate or depleted by feeding 2 percent cholestyramine. Pancreatic growth, gallbladder contractility, the concentration of cholecystokinin receptors in the gallbladder muscle, and meal-stimulated plasma levels of cholecystokinin were significantly stimulated by feeding the bile salt sequestrant cholestyramine to guinea pigs. Administration of the bile salt taurocholate produced the opposite effects. Inhibition of CCK release by bile salts and up-regulation of CCK receptors by CCK may be the mechanisms responsible for the actions of bile salts on CCK target organs.

From the Department of Surgery, The University of Texas Medical Branch, Galveston, Texas.

Requests for reprints should be addressed to James C. Thompson, MD, Department of Surgery, The University of Texas Medical Branch, Galveston, Texas 77550. Supported in part by grants from the National Institutes of Health (5R37 DK 15241-17 PO1 DK 35608 and RCDA CA 00854), Bethesda, Maryland and from the American Cancer Soci- ety (PDT-220), Washington, DC.

Presented at the 29th Annual Meeting of the Society for Surgery of the Alimentary Tract, New Orleans, Louisiana, May 17-18, 1988.

A lterationsintheconcentrationofbilesaltsintheintes- tinal lumen represent a current form of therapy for

some diseases. Luminal sequestration of bile salts with cholestyramine resin is an effective method of lowering plasma cholesterol levels and thereby reducing the incidence of coronary heart disease in patients with hypercholesterolemia [I]. The oral administration of choles- tryamine is also used to relieve pruritus associated with hyperbileacidemia [2]. Bile acids, on the other hand, are administered orally to patients for medical dissolution treatment of cholesterol gallstones [3].

We have recently reported that bile salts in the intestinal lumen exert a physiologic negative feedback control on the release of cholecystokinin (CCK) [4]. The release of CCK stimulated by nutrients is recognized as a physiologic mechanism that stimulates pancreatic functions (growth and secretion) and gallbladder functions (contraction) [5]. The effects of long-term alterations in the luminal concentrations of bile salts on CCK target organs, the pancreas and gallbladder, are not known. To investigate this important question, pancreatic growth and the in vitro contraction of the gallbladder were examined in guinea pigs when the bile acid pool was either enriched by feeding sodium taurocholate or depleted by feeding cholestyramine. Cholestyramine is an anion-ex- change resin that adsorbs bile salts in the intestinal lumen [6]. It is neither digested nor absorbed. CCK receptors in the gallbladder muscle membrane and plasma levels were measured.

MATERIAL AND METHODS The following substances were purchased: cholestyra-

mine resin, taurocholic acid sodium salt (99 percent pure), soybean trypsin inhibitor, and bacitracin (all from Sigma Chemical, St. Louis, MO); CCK-8 sulfate (Ba- them, Torrance, CA); iodine- 125Bolton-Hunter-CCK- 8 sulfate (Amersham, Arlington, IL); chromatographi- tally purified collagenase (Worthington Biochemicals, Freehold, NJ); octadecylsilylsilica cartridges (Sep-Pak C- 18@; Waters Associates, Milford, MA); and Osmolite@ liquid nutrition (1 Cal/ml; protein 37 g/liter, fat 39 g/liter, and carbohydrate 145 g/liter; Ross Lab- oratories, Columbus, OH).

Six- to 8-month old male albino guinea pigs (Rich- Glo, El Campo, TX) were fed a blenderized guinea pig diet (Purina Mills, St. Louis, MO) containing either 4 percent (wt/wt) cholestyramine or 0.5 percent taurocholate. Control groups received the blenderized diet without the additives. The guinea pigs were fed ad libitum for a period of 2.5 or 5 weeks and were fasted overnight before pancreas harvest. The pancreas was weighed and frozen at -70°C until measurements of protein, DNA, and RNA contents were performed, as described previously

20 THE AMERICAN JOURNAL OF SURGERY VOLUME 157 JANUARY 1989

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lawel.--ofWMndlngd chdecyatm (CCK-8) to CCK mceptcra In tha @nea pla gattbladt!er. Total blndtng 9.2 percent ot lodhe-12% Both+tunter-CCK-8 e spectfk blndlng 61 percent of total binding.

[ 71. The in vitro gallbladder contraction in response to CCK-8 was investigated in the three groups after 5 weeks of treatment. We chose 5 weeks, although taurocholate inhibited pancreatic growth by 2.5 weeks, but cholestyramine did not cause pancreatic hyperplasia until 5 weeks. The gallbladders were removed immediately after the guinea pigs were sacrificed, opened along the longitudinal axis, and the mucosa was gently removed by blunt dissec- tion. Longitudinal muscle strips (approximately 5 by 20 mm, one per guinea pig) were prepared from the body of the gallbladder and were mounted in an organ bath that contained Krebs solution, gassed at 37OC with 95 percent oxygen and 5 percent carbon dioxide, as described previously [8]. After a l- to 2-hour equilibration period, each muscle strip was set to an initial tension of 1 g, and different doses (final concentration from lo-i4 to 10m7 M) of CCK-8 were added to the bath in random order. No cumulative dose-response curves were constructed. The isometric tensions of the strips were constantly regis- tered by force transducers (Gould Statham, model UC2) and recorded on a polygraph (Beckman Instruments, model R 611). The length and weight of each strip were measured at the end of the experiments, and the contractile responses were normalized to the cross-sectional area according to Herlihy and Murphy [9]. The density of the muscle tissue, as measured by specific gravity, was not different among the three experimental groups; a value of 1.05 g/ml was assumed for calculations [9]. Muscle strip contraction was expressed in grams per square centimeter and was a percentage of the response to maximal stimulation.

Some gallbladders were used for measurement of CCK receptors. After excision, the gallbladders were opened longitudinally, washed in ice-cold collection buffer (Tris 10 mM, sodium chloride 137 mM, potassium chloride 2 mM, magnesium chloride 2.5 mM, sucrose 0.25 mM containing 1 percent gelatin, 1 percent soybean trypsin inhibitor and 0.1 percent bacitracin; pH 7.4) and freed from the mucosa by gentle scraping. Crude gallbladder muscle membranes were prepared as described previously [IO]. The protein content of the membranes

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was measured by the method of Lowry et al [II]. Mem- brane aliquots containing 50 pg protein/100 ~1 incubation buffer (HEPES 25 mM, magnesium chloride 2.5 mM, potassium chloride 0.5 mM, sodium chloride 137 mM, sodium phosphate 0.7 mM, glucose 10 mM, 1 percent gelatin; pH 7.4) were incubated with increasing concentrations (10-i l to 1 0w9 M) of iodine- 125-Bolton-Hun- ter-CCK-8 sulfate in the presence (nonspecific binding) or absence (total binding) of a 1 ,OOO-fold excess of CCK- 8 sulfate. The specific activity of the radioiodinated ligand was reduced by the addition of nonlabeled CCK-8- sulfate to approximately 400 dpm/fmol, which allowed us to do a seven-point multisaturation binding analysis with less than 2 &i of the radiolabeled ligand per sample. The optimal incubation period for measuring total number of specific binding sites for CCK on guinea pig gallbladders was 30 minutes at 30°C, and the optimal pH for measuring the maximum concentration of CCK reccp- tors in vitro was 6.5 to 7.5, in contrast to an optimal pH of 5.5 reported for the bovine gallbladder [12]. In this system, we observed a dose-dependent inhibition and satura- tion of specific binding of CCK-8 to the CCK receptors at CCK-8 concentrations of less than 1 nM (Figure 1). The concentration of receptors and the equilibrium dissocia- tion constant (&) were determined from a Scatchard plot [13] of the specific binding data (Figure 2). The amount of CCK receptors was expressed as femtomoles of iodinated CCK bound per milligram of protein.

The effect of endogenous bile salts on plasma CCK levels was investigated during fasting and stimulation. Fasting levels of CCK were measured in the guinea pigs that received the control or the cholestyramine diet for 5 weeks. For measurement of stimulated levels of CCK, a balanced liquid meal (Osmolite) was given intraduode- nally at 2 ml/hour for 30 minutes, either alone or in combination with cholestyramine (40 mg/ml), to fasted, conscious guinea pigs. The ability for sodium taurocholate (38 mg/ml) to inhibit meal-stimulated levels of plasma CCK was also tested. We have previously determined that the mean concentration of bile salts in the gallbladder of fasted guinea pigs is 19 mg/ml [unpublished data]. Since the mean gallbladder volume of fasted guinea pigs is 2 ml (range 1.5 to 2.5 ml), taurocholate (38 mg)

THE AMERICAN JOURNAL OF SURGERY VOLUME 157 JANUARY 1989 21

TABLE I Effects of Feedlng Sodium Taurocholate or Cholestyramine on Pancreatic Growth In Guinea Pigs’

Control 2.5 Weeks

Cholestyramine Taurocholate Control 5 Weeks

Cholestyramine

Pancreas weight (g) 1.74 i 0.09 I.84 f 0.11 1.27 f 0.06+ 1.84 f 0.12 2.49 f 0.17

Total protein (mg) 363 f 25 416 f 33 252 f 14+ 294 & 14 407 f 20’

Total DNA (mg) 6.17 i 0.5 7.28 f 0.7 5.22 f 0.2 6.82 f 0.4 9.45 f 0.4’ Total RNA (mg) 23.5 f 1.2 23.1 f 1.2 15 f 0.8’ 23.9 f 1.7 30.3 f 2.4’

e Values indicate the mean f SE (eight rodents in each group). t Significant difference from control value. 1

given over 30 minutes corresponds to the average total content of bile salts in the gallbladder during fasting.

Intraduodenal infusions were accomplished by way of duodenal fistulas that were prepared 3 to 5 hours before the experiments. Under ether anesthesia, an upper mid- line laparotomy was performed, and a 16-gauge polyeth- ylene catheter was inserted into the second portion of the duodenum; the catheter was exteriorized through the left posterior abdominal wall and the abdomen was closed by continuous suture with 3-O silk in two layers.

Levels of plasma CCK were measured using an in vitro bioassay described by Liddle et al [14]. This bioassay measures plasma CCK bioactivity based on the release of amylase stimulated by plasma extracts from isolated rat pancreatic acini. In brief, mixed arteriovenous blood was collected into iced heparinized tubes; CCK obtained from 3 to 6 ml of plasma was extracted by adsorption onto activated octadecylsilylsilica cartridges and eluted with 1 ml of 100 percent ethanol-to-l percent trifluoroacetic acid (volume 4:l) into incubation vials; after the eluants were dried under a stream of nitrogen at 45 to 50°C a l-ml acini suspension was added into each vial and incubated for 30 minutes at 37’C. Amylase release stimulated by the plasma extracts was compared with that stimulated by known amounts (dose-response curve) of CCK-8. Amylase released into the medium was measured with the Phadebas Amylase Test@ (Pharmacia Diagnostics, Pharmacia, Piscataway, NJ) and calculated as a percentage of total amylase content. Basal (nonsti- mulated) amylase release in this preparation was 3 f 0.1 percent. Isolated pancreatic acini were prepared from male Sprague-Dawley rats (280 to 320 g) by enzymatic digestion of pancreas with collagenase as described [ 141. Levels of plasma CCK are expressed as CCK-8 equiva- lents (picamoles per liter).

Data were analyzed by analysis of variance, with the Student’s t test used post hoc in order to determine which differences were significant. p <0.05 was considered significant.

RESULTS Body weights were not significantly different after

feeding taurocholate or cholestyramine for 2.5 weeks or feeding cholestyramine for 5 weeks when compared with the control group (data not shown). However, taurocholate and cholestyramine had opposite effects on growth of the pancreas. As shown in Table I, taurocholate inhibited

pancreatic growth by 2.5 weeks of treatment, whereas cholestyramine stimulated both hypertrophy (weight) and hyperplasia (DNA content) of the pancreas by week 5. The tendency for increased pancreatic size after 2.5 weeks of cholestyramine treatment was not statistically significant.

The effect of feeding cholestyramine or taurocholate for 5 weeks on the in vitro contractile response of gallbladders to CCK-8 is shown in Figures 3 and 4. The threshold for contraction (Figure 3) was 100 times lower with cholestyramine and 10 times higher with taurocholate compared with the control threshold. The contractile response of gallbladder strips, however, was not significantly different in the three groups at the highest doses of CCK-8 tested (greater than 10e9 M) (Figure 3). Nor- malization of the data to 100 percent of the contraction in response to the maximum dose of CCK-8 used (lo-’ M) (Figure 4) demonstrated that the right and left shifts of the dose-response curves induced by taurocholate and cholestyramine, respectively, were statistically significant over a physiologic range of CCK-8 concentrations ( lo-t3 to 1O-9 M).

Only one type of CCK receptors (high-affinity specific CCK-binding sites) was found in the gallbladder muscle membranes from the three groups. A representative Scatchard plot of the binding data from the control groups is shown in Figure 2. In contrast to the effect on the CCK-8 threshold for contraction, taurocholate reduced and cholestyramine increased the concentration of CCK receptors in the gallbladders significantly (Figure 5). The binding affinity of the CCK receptors (& = 0.1 to 0.3 mM) was not significantly affected by the treatments. Figure 6 shows the suppressive effect of endogenous and exogenous bile salts on meal-stimulated levels of plasma CCK. Depletion of bile salts with cholestyramine did not influence the levels of plasma CCK during fasting.

COMMENTS Administration of the bile salt sequestrant, cholesty-

ramine, stimulated the growth of the pancreas and increased the in vitro contractile response of the gallbladder to CCK-8. Cholestyramine binds anions nonspecifically; therefore, we tested the cause-and-effect relationship with bile salts by giving pure bile salt to the guinea pigs. The result was that taurocholate inhibited pancreatic growth and gallbladder contractility. Since cholestyra-


BILE, PANCREATIC GROWTH, AND GALLBLADDER CONTRACTILITY

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mine acted on the pool of endogenous bile salts, we con- cluded that the amount of luminal bile salts is a physiologic determinant of the maintenance of normal pancreatic growth and gallbladder contractility.

We have recently reported that the secretion of bile salts exerts a physiologic negative-feedback control on the release of CCK in dogs and human subjects [4]. The inhibition of the release of CCK by endogenous bile salts is paralleled by the in vivo inhibition of pancreatic protein secretion and gallbladder contraction. The present study shows that this feedback control mechanism for the release of CCK is also physiologically operative in the guinea pig. Administration of bile salt in a dose equivalent to the total content of bile salts in the gallbladder prevented the release of CCK stimulated by a meal. Conversely, the neutralization of endogenous bile salts with cholestyramine caused a significant 2.4-fold increase in meal-stimulated plasma levels of CCK compared with control levels. Interestingly, this magnitude of increase in CCK release, as measured with a bioassay system, is absolutely compa- rable to the increases in CCK immunoreactivity we have reported previously in related experiments in dogs and

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human subjects [4,15]. The amount of CCK released by nutrients is, in part, a function of the length of the intestinal mucosa exposed to the stimulants [16]. Bile salt- induced alterations in the rate of fat digestion and absorp- tion, however, could not explain most of the inhibitory effect of bile salts on CCK release. In the experiment with intraduodenal infusion of the liquid meal, we observed that at the time of sacrifice, the small intestines of guinea pigs in the three groups were filled with meal to the same extent: the entire duodenum and most of the jejunum but not the ileum. Furthermore, taurocholate can abolish the release of CCK when the duodenum is full of food (Fig- ure 6). Ohta et al [Z7] have reported recently that luminal taurocholate also inhibits the release of CCK that is stimulated by diversion of bile and pancreatic juice from the duodenum in the rat. Intraluminal trypsin inhibits the release of CCK in man and rat [18,19]. Since bile salts can protect trypsin from autodigestion, a reduction in the concentration of these salts in the intestinal lumen might increase CCK release by disinhibition of protease activity


[20]. Our studies [4] and the study of Ohta et al [17] have demonstrated, however, that the inhibitory effect of bile salts on CCK release is independent of the presence of pancreatic protease. The mechanism by which bile salt inhibits release of CCK is still unknown.

Cholecystokinin is a known stimulant of pancreatic growth [5]. Evidence that CCK exerts this trophic effect at a physiologic level has been obtained in rodents by two approaches: administration of protease inhibitors, which increases the circulating levels of endogenous CCK, and counteraction of endogenous CCK with CCK-receptor antagonists [21,22]. In our study, three different pancreas sizes (large, normal, and small) coexisted with three different patterns of CCK release (increased, normal, and decreased) in a direct relationship. The common denominator was the amount of bile salt in the intestinal lumen. Therefore, inhibition of CCK release by bile salts is the main mechanism that may explain the suppressive action of luminal bile salts on the growth of the pancreas. Reciprocally, this study adds new evidence that CCK may play a physiologic role in regulating pancreatic growth, at least in rodents. Our results agree with previous observations from other investigators. Brand and Morgan [23] reported stimulation of pancreatic growth in rats fed cholestyramine over a long period of time. However, these investigators did not examine directly the effect of bile salt and they did not measure plasma levels of CCK. Baba et al [24] reported stimulation of pancreatic growth in rats during experimental obstructive jaundice. The relationship with changes in CCK release was not established in that study since plasma levels of CCK in the jaundiced rats did not differ from control levels. Unfortunately, these investigators measured CCK during fasting and not after luminal stimulation. We have found that nutrient-stimulated, but not fasting levels, of CCK are increased by bile diversion or cholestyramine administration, as have others [4,15].

The concentration of CCK receptors in the gallbladder muscle was directly related to the pattern of CCK release. Both the receptor population and meal-stimulated plasma levels of CCK were increased nearly twofold by cholestyramine and decreased sixfold by taurocholate (Figures 5 and 6). These results strongly support the concept that endogenous CCK up-regulates its receptor, at least, in the gallbladder.

The sensitivity of the gallbladder muscle cells to CCK was increased by feeding cholestyramine and decreased by feeding taurocholate, as shown by the changes in the threshold for contraction and the displacement of dose- response curves to CCK-8 within a physiologic dose range. The mechanism responsible for the different sensi- tivities of the gallbladder to CCK, most likely, was a bile salt-induced reduction in the total concentration of CCK receptors. Biologic responses are generally proportional to the number of occupied receptors [25]. Then, a reduction in the number of receptors increases the amount of hormone required to till the number of receptors necessary to elicit an equal biologic response. As the hormone concentration increases, however, a sufficient occupancy of receptors may result in responses whose magnitudes

are less dependent on the number of receptors. Accord- ingly, the in vitro contractile responses of the gallbladders to the highest doses of CCK-8 in the three groups of guinea pigs were not different. This observation also indi- cates that there was no impairment of the intrinsic mechanisms of contraction within the muscle cells. Under in vivo conditions, however, the amount of CCK required to compensate for a reduction in the number of CCK receptors may not be provided by the endogenous release. Thus, an overall impairment of contraction of the gallbladder could be observed after long-term administration of bile salts.

Forgacs et al [26] and Sylwestrowicz et al [27] have reported gallbladder emptying reductions in normal vol- unteers and in patients with gallstones during oral therapy with bile acids. Our study provides insights into possi- ble mechanisms that might explain this important clinical observation. Bile salt-induced inhibition of gallbladder contraction may account for part of the relatively low success rate of chemical dissolution treatment of gallstones with oral bile acids. Therefore, we advise the devel- opment of therapeutic regimens that can prevent the po- tential deficit of CCK produced by the administration of bile salts. Also, bile salts may have a therapeutic role in the relief of symptoms seen in diseases of CCK target organs, for example, the treatment of pain in chronic pancreatitis.

REFERENCES 1. Lipid Research Clinics Program. The lipid research clinics coronary primary prevention trial results. I. Reduction in incidence of coronary heart disease. II. The relationship of reduction in incidence of coronary heart disease to cholesterol lowering. JAMA 1984; 251: 351-74. 2. Carey JB Jr, Williams G. Relief of the pruritus of jaundice with a bile-acid sequestering resin. JAMA 1961; 176; 114-7. 3. Fromm H. Gallstone dissolution therapy. Current status and future prospects. Gastroenterology 1986; 91: 1560-7. 4. Gomez G, Upp JR Jr, Lluis F, et al. Regulation of the release of cholecystokinin by bile salts in dogs and humans. Gastroenterology 1988; 94: 1036-46. 5. Marx M, Gomez G, Lonovics J, Thompson JC. Cholecystokinin. In: Thompson JC, Greeley GH Jr, Rayford PL, Townsend CM Jr, eds. Gastrointestinal endocrinology. New York: McGraw-Hill, 1987: 213-22. 6. Grundy SM. Treatment of hypercholesterolemia by interference with bile acid metabolism. Arch Intern Med 1972; 130: 638-48. 7. Upp JR Jr, Poston GJ, MacLellan DG, Townsend CM Jr, Barranco SC, Thompson JC. Mechanisms of the trophic actions of bombesin on the pancreas. Pancreas 1988; 3: 193-8. 8. Guice KS, Lluis F, Thompson JC. Bioassay of gut peptides. In: Thompson JC, Grceley GH Jr, Rayford PL, Townsend CM Jr, eds. Gastrointestinal endocrinology. New York: McGraw-Hill, 1987: 26-44. 9. Herlihy JT, Murphy RA. Length-tension relationship of smooth muscle of the hog carotid artery. Circ Res 1973; 33: 275-83. 10. Singh P, Rae-Venter B, Townsend CM Jr, Khalil T, Thompson JC. Gastrin receptors in normal and malignant gastrointestinal mucosa: age-associated changes. Am J Physiol 1985; 249: 76 l-9. 11. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193: 265-75. 12. Steigerwalt RW, Goldline ID, Williams JA. Characterization of choleeystokinin receptors on bovine gallbladder membranes. Am


J Physiol 1984; 247: 709-14. 13. Scatchard G. The attractions of proteins for small molecules and ions. Ann NY Acad Sci 1949; 51: 660-72. 14. Liddle RA, Goldfine ID, Williams JA. Bioassay of plasma cholecystolcinin in rats: effects of food, trypsin inhibitor, and alto- hol. Gastroenterology 1984; 87: 542-9. 15. Gomez G, Lluis F, Guo Y-S, Greeley GH Jr, Townsend CM Jr, Thompson JC. Bile inhibits release of cholecystokinin and neuro- tensin. Surgery 1986; 100: 363-8. 16. Meyer JH. Release of secretin and cholecystokinin. In: Thomp son JC, ed. Gastrointestinal hormones. Austin: University of Texas Press, 1975: 475-489. 17. Ohta H, Guan T, Tawil T, Liddle R, Green G. Inhibition of rat pancreatic secretion and CCK release by bile acids (abstr). Gastro- enterology 1988; 94: 331. 18. Owyang C, Louie DS, Tatum D. Feedback regulation of pancreatic enzyme secretion. Supression of cholecystokinin release by trypsin. J Clin Invest 1986; 77: 2042-7: 19. Shiratori K, Chen YF, Chey WY, Lee KY, Chang T-M. Mechanism of increased exocrine pancreatic secretion in pancreatic juice-diverted rats. Gastroenterology 1986; 91: 117 l-8. 20. Green GM, Nasset ES. Importance of bile in regulation of intraluminal proteolytic enzyme activities in rat. Gastroenterology 1980; 79: 695-702. 21. Niederau C, Liddle RA, Williams JA, Grendell JH. Pancreatic growth: interaction of exogenous cholecystokinin, a protease inhibitor, and a cholecystokinin receptor antagonist in mice. Gut 1987; 28: 63-9. 22. Wisner JR Jr, McLaughlin RE, Rich KA, Ozawa S, Renner IG. Effects of L-364,718, a new cholecystokinin receptor antagonist, on camostate-induced growth of the rat pancreas. Gastroenter- ology 1988; 94: 109-13. 23. Brand SJ, Morgan RGH. Stimulation of pancreatic secretion and growth in the rat after feeding cholestyramine. Gastroenterolo- gy 1982; 83: 851-9. 24. Baba N, Suzuki T, Tobe T, et al. Influence of obstructive jaundice. on pancreatic growth and on basal plasma levels of cholecystokinin and gastrin in rats. Dig Dis Sci 1986; 31: 1233-41. 25. Kahn CR. Role of insulin receptors in insulin-resistant states. Metabolism 1980; 29: 455-66. 26. Forgacs IC, Maisey MN, Murphy GM, Dowling RH. Influ- ence of gallstones and ursodeoxycholic acid therapy on gallbladder emptying. Gastroenterology 1984; 87: 299-307. 27. Sylwestrowicz T, Logan K, Kloiber R, Shaffer E. Effect of bile acid therapy and gallstone dissolution on gallbladder motility (abstr). Gastroenterology 1987; 92: 1784.

DISCUSSION R. Scott Jones (Charlottesville, VA): Dr. Gomez, the

findings you have described are clearly important and have physiologic significance. I would like to ask a couple of questions and make a few comments about the study which actually don’t detract from the conclusions. In terms of the effects of ingestion of bile acids, in addition to the results you have measured, there are a couple of things that may be worth considering. One is the possibili- ty that certain amounts of bile acids may produce diarrhea when ingested. Did your guinea pigs have diarrhea? I would also have anticipated that if you made guinea pig chow containing 0.5 percent sodium taurocholate that it would alter palatability and, therefore, food intake. In terms of the pancreas weight changes and changes in the composition of the pancreas, I am curious to know wheth- er that occurred in exocrine or endocrine pancreas or both. I would think, from your hypothesis and from your data, that these changes were confined to the exocrine

pancreas; were there trophic alterations in the islets? You inferred in your conclusion that the results were due to alterations in bile salt pool size. Did you actually quantify control and posttaurocholate and postcholestyramine bile salt pool sizes in any of your animals to determine what quantitative changes you were producing? If you did, in fact, alter bile salt pool size, it is probable that you would have altered resting gallbladder or fasting gallbladder volume if you increased the bile salt pool size. I wonder if you have any information about what the effect of resting gallbladder volume might have on responsiveness to CCK, irrespective of the bile salt issue. Overall, I think you and your colleagues should be proud of this work.

Travis Solomon (Kansas City, MO): I think this study adds to our knowledge about one area of regulation of CCK release: negative feedback regulation of CCK by the products of its target organs, the gallbladder and bile. As you have indicated, there are clear implications for both normal physiologic and pathophysiologic regulation of CCK targets and of CCK secretion itself. It was not clear to me that the changes you measured in plasma CCK in response to a meal were after long-term treatment; that is, were the effects of food, cholestyramine, and sodium taurocholate studied in the short-term or after long-term feeding to demonstrate that the changes in CCK secretion were maintained over the long-term? Do you have information indicating that blocking CCK action would have the same effect that sodium taurocholate did on the measurements you made? For example, if you administer a CCK-receptor blocker, does it repro- duce some of the effects you measured? The hypothesis regarding up-regulation of CCK receptors by CCK is extremely interesting. Could you give us information about the effects of long-term administration of CCK on CCK receptors in this model or other models to help us understand this phenomenon?

Joel J. Roslyn (Los Angeles, CA): Dr. Gomez, in regard to the effects of cholestyramine and cholic acid on the enterohepatic circulation, presumably both agents may affect biliary lipid composition as well as individual bile acid concentration. Were these measured? You elected to use taurocholic acid. Is this a primary bile acid in the guinea pig? Did you look at any of the other bile acids that perhaps are endogenous bile acids in this model?

Guillermo Gomez (closing): Dr. Jones, diarrhea was not observed with either treatment. Indeed, the differences in body weight were not significant. We did not quantitate the pool of bile salts, but other studies have shown that feeding cholestyramine does reduce the size of the bile salts pool. The gallbladders were immediately removed for the in vitro studies of gallbladder contraction or the measurement of CCK receptors; therefore, we did not measure the fasting gallbladder volume. However, on macroscopic examination, the gallbladders from the guinea pigs fed cholestyramine were smaller than those in the control and taurocholate groups.

BILE,PANCREATlCGROWTH,ANDCALLBLADDERCONTRACTILITY


GOMEZETAL

Dr. Solomon, we also measured plasma CCK levels during long-term feeding of cholestyramine or taurocholate in some guinea pigs. Although these levels were more increased during bile salt sequestration, the variability between animals was quite considerable. Therefore, to demonstrate the differences in CCK release among the three treatments, it was necessary to standardize the stimulation; we gave a liquid meal after fasting. The effect of CCK antagonists was not tested in our study. Wisner and coworkers [22] have recently reported inhibition of pancreatic growth in rats using a specific CCK- receptor antagonist, which provides further evidence that endogenous CCK participates in pancreatic growth. We

did not test the effect of exogenous CCK on the CCK- receptor population of the gallbladder. The usual regimens for administration of CCK may not be physiologic. The normal pattern of CCK release in guinea pigs is not known. Although speculative, pulsatile and supraphysio- logic levels of exogenous CCK could also result in down- regulation of CCK receptors. Our results suggest that the level of endogenous CCK,up-regulates CCK receptors in the gallbladder.

Dr. Roslyn, we have not examined the effects of biliary lipids. We chose taurocholic acid based on our previous experience with studies in dogs and human subjects.


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Down-regulation of pancreatic growth and gallbladder contractility by bile salts

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