Gastrointestinal Functions, edited by Edgard E. Delvin and Michael J. Lentze. Nestle Nutrition Workshop Series, Pediatric Program, Vol. 46, Nestec Ltd., Vevey/Lippincott Williams & Wilkins, Philadelphia © 2001. Exocrine Pancreatic Function Jean Morisset Department of Medicine, University of Sherbrooke, Sherbrooke, Quebec, Canada The control of human pancreatic enzyme secretion is still a matter of open debate, as indicated by a recent statement by Adler: "Human pancreatic secretion is regulated through a complicated coordination of neural, hormonal and possibly paracrine effects. Cholinergic input is essential for full action of any other agonist like cholecystokinin (CCK) and secretin" (1). Indeed, confusion exists over the type of CCK receptors present on human pan- creatic cells. Thus, it was suggested by in vivo studies in the early 1990s that exocrine pancreatic enzyme secretion was mediated by occupation of the CCKA receptor subtypes (2), but more recently Tang et al. showed that the human pancreas appeared predominantly to expresses the CCKB subtype (3), an observation later confirmed by Weinberg et al. (4). Compounding this problem, it was then reported that infusion of postprandial concentrations of human gastrin, the natural ligand of the CCKB receptor, failed to stimulate human pancreatic secretion (5). As indicated recently by Miller (6) in an editorial, "... the more prominent existence of type B than type A CCK receptors within the human pancreas raises a number of important questions such as: if the receptor resides on the surface of the pancreatic acinar cells, why would it not be coupled to the secretory machinery of the cell? What functional role does it play?" These questions are still unanswered and progress has been hampered by the difficulties in obtaining sufficient quantities of healthy human pancreatic tissue that has not been damaged by autolysis. In the meanwhile, pancreatic exocrine functions have been investigated in animal models, mostly in the rat, mouse, and guinea pig, all of which are rodents. More recently, the pig has been chosen in an effort to establish its suitability as a human model for the study of pancreatic physiology. In this chapter, is summarized current knowledge on pancreatic development, pancreatic enzyme synthesis, and secretion, and the implications of CCK and its receptors for the pancreatic response to duodenal hormone stimulation. 165 766 EXOCR1NE PANCREATIC FUNCTION PANCREATIC FUNCTIONS The exocrine pancreas supplies digestive enzymes for food digestion in the gut and ensures that the milieu of the intestine is sufficiently alkaline for maximal enzyme activity to hydrolyze the various substrates. The water needed to carry the digestive enzymes through the pancreatic duct system and the bicarbonate necessary to buffer the acidic stomach chyme are produced in and released from the pancreatic duct cells under the control of the parasympathetic nervous system and secretin. The pancreatic acinar cells, on the other hand, perform two major functions—the synthesis and secretion of the digestive enzymes. In rodents at least, the secretion of these enzymes into the intestine is controlled by the parasympathetic nervous system through acetylcholine and the gastrointestinal hormone CCK (7). The acinar cells, therefore are equipped with muscarinic (8) and CCK (9) receptors, among other receptor types. DEVELOPMENT OF PANCREATIC COMPONENTS AND FUNCTIONS At birth, the rat pancreatic gland is well developed and ready to assume its endo- crine and exocrine functions. However, early in life, the pancreas remains in a state of active development to ensure that the strong demand for digestive enzymes to deal with the increased nutrient load necessary for proper body and organ development is 50 10 15 20 25 AGE (days) FIG. 1. 3H-thymidine incorporation into pancreatic DNA with age. Pieces of pancreatic tissue excised from newborn rats up to 23 days after birth were incubated in vitro and 3H-thymidine incorporation into DNA was measured as described in reference 47. Results are the means ± SE of six animals per point. EXOCR1NE PANCREATIC FUNCTION 167 150 100 50 0.2 0.4 0.6 0.8 10 1.2 1.4 TOTAL DNA (mg) FIG. 2. Correlation between total pancreatic DNA content and pancreatic weight developments in rats. Newborn and neonatal rat pancreata obtained up to 23 days after birth were weighed and their total DNA extracted as described by Morisset et at. (47). These data come from the same animals used in Fig. 1. met. As shown in Fig. 1, total thymidine incorporation, a marker of cell division, is relatively important at birth but decreases to a minimal level by day 5. From that point, an almost linear increase in DNA synthesis can be observed up to 25 days after birth. Interestingly, this active DNA synthesizing activity results in a linear increase in total DNA content when plotted against pancreatic weight, as shown in Fig. 2. From birth up to 1 year of age, development of pancreatic DNA and RNA total contents are parallel, whereas total protein content remains relatively low until weaning at 21 days, and increases tremendously thereafter, as shown in Fig. 3. The content of amylase and of chymotrypsinogen develops almost in parallel, although the pancreas is richer in amylase than in chymotrypsinogen (Fig. 4). This may result from the fact that amylase is the only enzyme responsible for starch and glycogen digestion, whereas protein digestion can be achieved by multiple proteases, including trypsinogen, procarboxypeptidases A and B, and elastases. Once the pancreatic gland has reached its full development, turnover rates of its different cell populations are comparable. Indeed, acinar cells show a labeling index of 6%, ductal cells 6%, endothelial cells 4%, interstitial cells 4% to 8%, and endocrine cells 2.5% to 4% (10). 168 EXOCRINE PANCREATIC FUNCTION 300g 30 H 6.0 25 150 3 SO 20 120-r I 4JO 15 * 100 1 ao I A 2JO DN 60 3 L ttf 40 m TOTA 0.6 •6 * 20 | 0.2 2 T 13 17 21 25 27 30 45 60 3 6 9 12 days months AGE FIG. 3. Development of rat total pancreatic DNA, RNA and protein contents up to a year of age. Rats of different ages were killed and their pancreases used to evaluate DNA, RNA, and protein contents, as described by Morisset and Jolicoeur (48). The acquisition of a secretory capacity in response to different stimuli occurs after birth. A secretory response to the muscarinic neurotransmitter acetylcholine appears after birth and reaches a maximum just before weaning in the rat (11). A good correlation has been established in the rat between acetylcholine-induced amy- lase output from the exocrine pancreas and the concentration of muscarinic receptors on the acinar cells (12). Premature weaning does not seem to modify the capacity of the pancreas to secrete enzymes under conditions of basal and acetylcholine stimulation or to increase its amylase and chymotrypsinogen contents (13). The secretory response to CCK is also absent in rat fetal pancreas and develops after birth (14). This lack of responsiveness to CCK in the early stage of life may result from a low binding capacity of the high-affinity component of the CCK receptor (15). In the human exists a refractoriness to secretagogs in the pancreas of young infants for which no explanation is found (16). In adult rats, the secretory capacity of the exocrine pancreas can be either increased or severely diminished. Indeed, an increase in pancreatic weight produced by re- peated injections of CCK is accompanied by proportional increases in functional capacity, as reflected by the increased maximal protein output in response to CCK (17). On the other hand, the rat secretory response to the acetylcholine analog carba- mylcholine was severely impaired during the induction of acute pancreatitis by high doses of cerulein, a CCK analog (18). This pathology resulted in major decreases EXOCRINE PANCREATIC FUNCTION 169 9 13 17 21 25 27 30 45 60 3 6 9 12 days months AGE FIG. 4. Development of rat total pancreatic amylase and chymotrypsin contents up to 1 year of age. Enzyme assays were performed as described by Morisset and Jolicoeur (48). in pancreatic amylase concentrations after 2 days of treatment, loss of acetylcholine potency and efficacy in stimulating amylase release, and an important reduction in acetylcholine muscarinic receptor concentration, although with no effect on their affinity for the agonist. PANCREATIC ENZYME SYNTHESIS Pancreatic enzyme synthesis—the major and most important function of the pan- creatic acinar cells—concerns the replenishment of the different pancreatic digestive enzymes after their release into the duodenum. It is logical to assume that changes in the relative amounts of enzymes packaged in the zymogen granules of the acinar cells result from altered rates of specific synthesis. The capacity of the pancreatic gland to synthesize enzymes can be affected by various factors, including feeding, starvation, diet composition, and the administration of gastrointestinal hormones and cholinergic agents. The synthesizing response of the pancreas to feeding has been studied in different animal models but little experimental evidence is seen for major variation in enzyme synthesis rate after meals. When fed rats are compared with 24-hour fasted rats, little (19) or no (20) change is seen in incorporation of labeled phenylalanine into protein as measured in vitro. In rats trained to eat for 1 hour every 12 hours for 3 170 EXOCRINE PANCREA TIC FUNCTION days, and then fasted for 24 hours, refeeding for 15 minutes resulted in a small decrease in amino acid incorporation into protein in vivo 45 minutes after the meal, followed by a small increase 90 to 105 minutes after the meal (21). Other studies indicated that refeeding after prolonged fasting (48 hours) increased amino acid incorporation into pancreatic protein in rats (20) and depleted pancreatic stores of amylase (22). Prolonged periods of fasting have dramatic effects on the exocrine pancreas, in- cluding major loss of protein and amylase (19,23).