Review Article - Journal of Physiology and Pharmacology
Transcription
Review Article - Journal of Physiology and Pharmacology
JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2003, 54, 3, 293317 www.jpp.krakow.pl Review Article 1 S.J. KONTUREK, 1J. PEPERA, 2K. ZABIELSKI, 3P.C. KONTUREK, 4T. PAWLIK, 1 A. SZLACHCIC, 3E.G. HAHN BRAIN-GUT AXIS IN PANCREATIC SECRETION AND APPETITE CONTROL 1 Department of Physiology, Jagiellonian University School of Medicine, Kraków, Poland The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences, Jablonna, Poland, 3First Department of Medicine, University Erlangen-Nuremberg, Erlangen, Germany 4Department of Gerontology, Jagiellonian University School of Medicine, Kraków, Poland 2 The stimulation of exocrine pancreatic secretion that has been attributed by Pavlov exclusively to various reflexes (nervism), was then found that it depend also on numerous enterohormones, especially cholecystokinin (CCK) and secretin, released by duodeno-jejunal mucosa and originally believed to act via an endocrine pathway. Recently, CCK and other enterohormones were found to stimulate the pancreas by excitation of sensory nerves and triggering vago-vagal and entero-pancreatic reflexes. Numerous neurotransmitters and neuropeptides released by enteric nervous system (ENS) of gut and pancreas have been also implicated in the regulation of exocrine pancreas. This article was designed to review the contribution of vagal nerves and entero-hormones, especially CCK and other enterohormones, involved in the control of appetitive behavior such as leptin and ghrelin and pancreatic polypeptide family (peptide YY and neuropeptide Y). Basal secretion shows periodic fluctuations with peals controlled by ENS and by motilin and Ach. Plasma ghrelin, that is considered as hunger hormone, increases under basal conditions, while plasma leptin falls to the lowest level. Postprandial pancreatic secretion, classically divided into cephalic, gastric and intestinal phases, involves predominantly CCK, which under physiological conditions acts almost entirely by activation of vago-vagal reflexes to stimulate the exocrine pancreas, being accompanied by the fall in plasma ghrelin and increase of plasma leptin, reflecting feeding behavior. We conclude that the major role in postprandial pancreatic secretion is played by vagus and gastrin in cephalic and gastric phases and by vagus in conjunction with CCK and secretin in intestinal phase. PP, PYY somatostatin, leptin and ghrelin that affect food intake appear to participate in the feedback control of postprandial pancreatic secretion via hypothalamic centers. K e y w o r d s : pancreas, cholecystokinin, secretin, gastrin releasing peptide, calcitonin gene related peptide, motilin, leptin, ghrelin, pancreatic polypeptide, peptide YY, neuropeptide Y. 294 INTRODUCTION Historical background Although Renier de Graf (1641-1679) had constructed first pancreatic and biliary fistula in the dog, it was not until Claude Bernard (1813-1878)(1, 2) emphasized the important role of pancreas in digestion of fat and attributed metabolic activities to liver, the discoveries which led him to formulate the concept of the stability of internal environment - milieu interieur - as a condition of health and well-being (Fig. 1). However, another 50 years passed before active physiology research on digestive system resumed starting at the turn of 19th and during early part of 20th century with discoveries by Ivan P. Pavlov of Russia of neural stimulation of digestive glands (3), attributing the mechanisms of this stimulation exclusively to neural reflexes (nervism). Soon, W.M. Bayliss and E.H. Starilng of England, discovered secretin in 1902 (4), a first hormonal substance, stimulating pancreatic secretion in response to duodenal acidification, while J.S. Edkins identified in 1905 (5) gastrin, another non-nervous stimulant, implicated in the stimulation of gastric acid secretion by food. These two later discoveries are millstones in development of modern endocrinology and for the first time the term hormone, originally invented by Hardy was applied to secretin. *C. BERNARD (1856) demonstrated that „pancreas plays an unique role in fat digestion”. * I.P. PAVLOV (1876) in his book: „The Work of the Digestive Gland” asserted that exocrine pancreatic secretion is exclusively regulated by neural reflex mechanism („nervism”). CNS *W.M. BAYLISS & E.H. STARLING (1902) discovered secretin, first hormone, released by H+ in gut to stimulate pancreas. *W. BOLDYREFF (1911) discovered motor and secretory cycles during interdigestive period and explained them by neuroreflexes. + - *J. MELLANBY (1925) postulated „dual hormonqal and nervous control; secretin controlling water and bicarbonate and vagus enzyme secretion. *A.A. HARPER & H.S. RAPER (1943) found „pancreozymine” the hormone that stimulates pancreatic enzyme secretion. *A.C. IVY & E. OLBERG (1928) isolated from intestines „cholecystokinin” the hormone that contracts the gallbladder and relaxes sphincter of Oddi. *E. JORPES & V. MUTT(1966) proved that pancreozymine and cholecystokin are the same substance and called it „cholecystokinin”. ENS *Numerous Investigators; discovered various gastro-enteric peptides (GEP) such as somatostatin, GRP, VIP, PP, PYY, NPY, galanin etc. released by neurons or endocrine-paracrine cells (APUD) in the gut and brain „Brain-Gut Axis”. Rene Dubois (1901): ”The past is not dead history, it is living material of which man buils its future” Fig. 1. Major discoveries in pancreatic physiology and brain-gut axis. 295 New discipline of gastrointestinal endocrinology flourished in the second part of 20th century when modern techniques of peptide biochemistry became available and successfully applied to identify major gut hormones such as gastrin, secretin, cholecystokinin (CCK), gastric inibitory peptide (GIP) and motilin, which have been isolated, chemically characterized and synthesized. In addition, numerous hormonal peptides have been recently discovered but still they lack full hormonal status such as pancreatic polypeptide (PP), peptide YY, neuropeptide Y (NPY), somatostatin, gastrin-releasing peptide (GRP), galanin, vasoactive intestinal peptide (VIP), pituitary adenylyl associated peptide (PACAP), substance P and other tachykinins, leptin and ghrelin (Fig. 2)(6). Recent findings that some of the gut hormones such as CCK, PP, PYY or NPY, somatostatin, originally believed to act on the target digestive organs via endocrine pathway, involve neural pathway to exert their biological action provide support for the concept of neuroendocrinology of the digestive system. Thus, the old Pavlovian nervism has been married with endocrinology and found to act together in full harmony to control the digestive activities. Enteric portion of autonomic nervous system (ENS) forms in the digestive system so called the brain of gut consisting of about 100 milion of neurons that cooperate with enterohormones, each of them contributing to the motor, secretory, absorptive, excretory, trophic and circulatory functions of this system. Most of the gut hormones originally identified in the central nervous system (CNS) were then also localized in the gut and vice versa, the brain neuropeptides and neutrotransmitters were later on identified in the gut forming so called the brain- Stimulation * Cholecystokinin (CCK) Secretin * Gastrin * Gastrin-Releasing Peptide (GRP) * Insulin * Vasoactive Intestinal peptide (VIP) * Cyclase-Activating peptide * Substance P and other tachykinines * Adenosine 5’-triphospate (ATP) Uridine triphosphate (UTP) * Histamine Panacreatic phospholipase A2 Inhibition * Pancreatic Polypeptide (PP) * Leptin * Ghrelin Peptyde YY * Neuropeptide Y * Calcitonin Gene-Related Peptide * Somatostatin Glucagon Glucagon-Like Peptide -1 (GLP-1) * Thyrotropin-Releasing Hormone * Enkephalin (Met- or Leu-) * Nitric Oxide (NO) * Dopamine *Present both in the brain and gut Acting through neural pathway Fig. 2. List of stimulatory and inhibitory hormones, neuropeptides and neurotransmitters involved in brain-gut axis, present in the gut and brain and acting through neural pathway 296 gut axis involved in the control of digestive activities and feeding behavior under normal and pathological conditions (Fig. 3). Structural basis of pancreatic secretion The pancreas is both an exocrine and endocrine organ. The exocrine pancreas combines two main functional elements; the acini comprizing about 85% of the total gland mass and the ductal and centroacinar cells (called also principal cells) that include only about 5% of gland cell mass. The acini of spheric or tubular shape consist of acinar cells that are specialized to synthesize and store digestive enzymes in secretory vesicles (zymogen granules) and release the enzymes in response to various secretagogues. On their basolateral membrane various receptors for secretagogues including CCK and neurotransmitters such as acetylcholine (Ach), gastrin releasing peptide (GRP) and vasoactive intestinal peptide (VIP) have been identified and found in in vitro studies on isolated cultured acinar cells to stimulate enzyme secretion (7). The centro-acinar and duct cells, that are largely responsible for the HCO3- and water secretion, become more columnar further down the ductal tree and are joined by other specialized cell types that are capable of secreting mucus and certain peptides such as trifoil peptides. Numerous unmyelinated nerve fibers are CNS Cerebral cortex, Limbic system Brain stem & Hypothalamus Vagal pathway Splanchnic pathway Enteric afferents & interneurons Sensors SECRETION MOTILITY BLOOD FLOW Afferent terminals Enteric Nervous System (ENS) Neurotrasmitters, Neuropeptides, Other chemical and Mechanical stimuli Fig. 3. Functional organization of enteric nervous system (ENS) with its sensory neurons, interneurons and effector neurons affecting the motility, secretion and blood flow to the gut and pancreas (on left) and the control of ENS activity by CNS affecting the effector organs in the gut and pancreas (on right). 297 present in the pancreas and innervate the glandular cells as well as the vessels. These fibers may belong to cholinergic, adrenergic or peptidergic as well as sensory neurons and interneurons of the ENS, which transmit the signals from efferents fibers of external autonomic nerves to ENS or from the ENS in intestinal mucosa to the glandular cells of the pancreas through short enteropancreatic reflexes or through long vago-vagal reflexes with intergrative centers in the CNS. The nerve fibers in the pancreas are distributed through the interlobular connective tissue, where intrinsic ganglia, representing peripheral integrative centers are also found. The endocrine portion of the pancreas is located in the islets of Langerhans that contain A-, B-, D- and F-cells, releasing, respectively, glucagon, insulin, somatostatin, PP and many others. The islet hormones first perfuse the surrounding acinar cells by the insulino-acinar portal system and regulate digestive enzyme synthesis and transport, as well as the proliferation and growth of these cells. The disorders of this endocrine portion such as diabetes mellitus, resulting either from the lack of insulin production because of viral or autoimmune damage of B-cells (type I) or genetic defect that produces insensitive insulin receptors throughout the body (type II), greatly affect exocrine portion of the pancreas but this is beyond this review. The islet hormones reach general circulation with pancreatic blood flow and some of them (e.g. PP, NPY, CGRP, somatostatin) may also affect the pancreatic secretion via hypothalamic centers in CNS, were the receptors for these hormones have been identified (Fig. 4) Cellular regulation of HCO3 secretion Studies on pancreatic HCO3- secretion and volume flow in various species revealed many species-dependent variations in secretion of this component of secretion by ductal and centroacinar cells that are specifically equipped with receptors such as for secretin and VIP. They contain high activity of carbonic anhydrase, an enzyme which is important for their ability to secrete HCO3-. While human, canine and feline pancreas secretion is small under basal conditions, it respond with abundant amounts of HCO3- in response to secretin stimulation and little in response to CCK or vagal stimulation. In rats, which are so often used in physiological and pharmacological studies, the spontaneous pancreatic secretion is relatively higher, but secretin, CCK and vagal stimulation result in small increment of this secretion (8). Pigs, like humans, secrete little under basal conditions but respond with abundant secretion when secretin, CCK or vagus are stimulated ( 8). Maximum HCO3- secretion, which in humans or pigs may reach up to 150 mM/L, is only about 70 mM/L in rats. Whether this variation in HCO3water secretion reflects a variation in composition of primary fluid secretion or the rate of ductal cell HCO3/Cl exchange is unknown but surely the findings obtained from animals may not be relevant to those in humans an should be interpreted with caution. Secretin and VIP act on receptors to increase the 298 Fig. 4. Gastrointestinal hormones neurotransmitters inhibitng pancreatic secretion by acting on the hypothalamus and brain stem. concentration of cyclic adenosine monophosphate (cAMP) in ductal and centroacinar cells (8). The rise in cAMP increases HCO3- secretion by primary activation of a Cl channel on the luminal membrane resulting in the increase of chloride secretion into the lumen of acinus (9). The increased chloride transport in the lumen is coupled with Cl/HCO3- antiport resulting in an exchange of Cl for HCO3- at the lumanl membrane. This is the reason for the reciprocal relationship between pancreatic juice HCO3- and Cl concentrations that could be explained in part by the difference in water secretion in pancreatic juice. As the flow rate of secretion increases so does HCO3- concentrations, while Cl concentration declines. On baso-lateral membrane of the duct cell are a Na+/H + antiport, a Na+/K+-ATPase, a H+-ATPase and K+ channels. Following secretin or VIP stimulation, an apical Cl/HCO3- antiport is activated resulting in increased luminal formation of HCO3- that originates from the CO2, produced locally in the cell as metabolic product, as well as CO2 released in extracellular fluid by the action of H+ on plasma HCO3-. CO2 diffusing readily into the alkalinized ductal cells combines with water, the step catalyzed by high activity of carbonic anhydrase. The continued movement of H+ across the basolateral membrane leads to build up of HCO3- and the movement of HCO3- across the apical membrane in exchange for Cl. As HCO3- is secreted into the duct lumen, Na+ also moves across the epithelium to preserve electrical neutrality. Na+ moves mostly through the 299 intercellular pathway and water then rushes into the lumen along its osmotic gradient. With enhanced secretin or VIP stimulation of ductal and centroacinar cells there is continued increase in the HCO3- concentration in pancreatic juice as well as its pH combined with the fall in Cl concentration (Fig. 5). It is interest that CCK and vagal stimulation, though universally believed to promote predominantly protein enzyme secretion from acinar cells and in most species their effect on volume flow is negligible, however, in some species like pigs, rats, guinea pigs and rabbits, often used for pharmacological studies, both CCK and vagal nerves are potent stimulants of fluid secretion. Furthermore, in pigs and guinea pigs large number of VIP-ergic neurons are present in the pancreas and during vagal stimulation, these fibers release large amounts of VIP, which evokes HCO3- secretion in manner analogous to secretin (8,10-13 ). The primary goal of pancreatic HCO3- secretion in response to secretin in man or dogs is the neutralization of gastric acid entering the duodenum. In this process, HCO3- originating from the pancreatic and biliary duct cells and duodenal mucosa provide optimal intraduodenal pH for protein, lipid and carbohydrate digestion by pancreatic and intestinal brush border enzymes and for the absorption of digestion products. Duodenal pH in the major regulator of secretin release and the threshold value for secretin release and stimulation of pancreatic HCO3- is 4.5 (12-14). Below this threshold the amount of secreted HCO3- is related to the increment in plasma secretin and this increment depends upon the total B A C R VIP Fig. 5. Mechanisms of secretion of HCO3 by centro-acinar and ductal cells (A), The relation between HCO3 and Cl secretion as related to the pancreatic volume flow (B) and the acinar fluid and plasma levels of major electrolytes (C). 300 amounts of titratable acid delivered to the duodenum (14, 15). Chey and his coworkers (16) obtained an evidence that the release of secretin occurring postprandially plays a crucial role in the secretion of HCO3- and fluid because immunoneutralization of secretin by specific antibody actually blocked the increase in the secretion of fluid and HCO3-. Li at al (17,18) found that concentrated acid perfusate in duodenum results in the rise of plasma secretin that is mediated by secretin-releasing peptides (S-RP) present in the proximal intestinal lumen or in the pancreatic juice (Fig. 6). Partially purified S-RP infused into the duodenum of anesthetized rats increased pancreatic fluid and HCO3- secretion and raised plasma secretin level. These secretory effects were blocked by vagotomy, tetradotoxin and capsaicin, suggesting neutral control of S-RP release by intestinal mucosa (17-20). This peptide has some homology to pancreatic phospholipase, therefore, it is not excluded that phospholipase A2 is capable of specific receptor-mediated action on secretin-releasing cells and this may represent an alternative function of the enzyme on intestinal endocrine cells (21-23).` Fig. 6. Schematic presentation of the mechanism of release of secretin by acid in duodenum and mediation of secretin-releasing peptide (S-RP) in this release (on left) and pancreatic bicarbonate outputs as related to plasma levels of secretin alone or to combined with acetylcholine or CCK or to acetylcholine or CCK alone (on right) [modified from Li et al (17, 18)]. 301 Cellular regulation of enzyme secretion Pancreatic enzymes are synthesized, packaged, stored and released from acinar cells by the process of exocytosis which involves multiple subcellular organelles. Most of the packaging of newly synthesized enzymatic protein occurs in the Golgy stack. A G protein facilitates the transfer of enzymatic protein from endoplasmic vesicles into the cisternae. Secretory granules exist in the cytoplasm or migrate to apical cell membrane to be released into the lumen of acinus (Fig. 7). Hormones such as CCK and secretin and neuro-transmitters such as Ach or GRP induced the fusion of zymogen granules with the apical plasma membrane of acinar cells resulting in the release by exocytosis of digestive enzymes into the pancreatic duct system. The process of binding of secretagogues to membrane receptors, ultimately lead to exocytosis includes the receptor-mediated generation of intracellular messengers and exocytosis (24-27). Receptors for pancreatic secretagogues are believed to belong to the receptor family characterized structurally by seven hydrophobic transmembrane domains and functionally by Fig. 7. Stimuli for acinar cells bind to membrane receptors and initiate intracellular events that increase enzyme secretion. Schematic presentation of stimulus-secretioin coupling of pancreatic acinar cell protein secretion. 302 their interaction with G-proteins. The CCKA-, muscarinic M3- and GRP-receptors interact with heterotrimetric G-protein complex leading to the stimulation of phosphoinositide-specific phospholipase Cβ (PLCβ) activity (Fig. 7). Phospholipase C activity leads to hydrolysis of phosphatidylinositol-4,5biphosphate (PI-P2) and the formation of inositol 1,4,5 triphosphate (IP3) and 1,2diacylglycerol (DAG). This occurs within seconds in response to high secretagogue concentrations. IP3 then binds intracellular receptors forming channels activating the release of Ca2+ from intracellular stores. The DAG produced by phosholipase C (PLC) activates protein kinase C (PKC). The major intracellular messengers involved in the regulation of pancreatic secretion are IP3, Ca2+, DAG and cAMP (8). The first three are predominant in the acinar cells and increase after the activation of phosphoinositide specific phosphalipase C by CCK+ or Ach while cAMP in the predominant messenger in centro-acinar and duct cells originating from ATP due to the action of adenylyl cyclase (AC) and activated by secretin. The central role in stimulus-secretion coupling in acinar cells is played by Ca2+ that originates both from the intracellular stores followed by a small sustained increase mediated by entrance of Ca2+ through the plasma membrane channels. Physiological concentrations of secretagogues induce only transient and repetitive increase in Ca2+ named Ca2+ oscillations or spikes. The increase in Ca2+ spreads around the acini with both Ca2+ and IP3 traversing through gap-junctions from one acinar cell to another (24). Ca2+ activates then kinases and phosphatases and this activation involves calmodulin (CAM) as a Ca2+ receptor and a catalytic kinase or phosphatase domain. Ca2+ can also activate protein phosphatase 2B (PP) which is identical to calcineurin. Secretin and VIP binds with the membrane receptors to increase intracellular cAMP which in turn stimulates protein kinase A (Fig. 8). Fig. 8. Functional unit of pancreas consisting of the acinus and draining pancreatic duct. The site of action of hormones and neuropeptides mediating the secretory response of acinar and ductal cells. 303 The pancreas produces several important digestive enzymes that are responsible for about half of overall digestion of nutrients in the gastrointestinal tract. Alphaamylase digests starch, the most prevalent carbohydrate in the human diet. Lipase cleaves the ester linkages of dietary fats, resulting in the conversion of triglycerides to diglycerides and then to 2-monoglycerides and fatty acids that are absorbed from the intestines. Trypsin, secreted primarily as trypsinogen, a proenzyme, is converted to its active form, trypsin, that in turn is responsible for conversion of other proenzymes to active enzymes. Carboxypeptidase is also secreted as a proenzyme, procarboxypeptidase, which is converted to active enzyme by trypsin to cleave Cterminal linkages of proteins into amino acids that can be absorbed from the gut. Pancreatic juice also contains a low concentration of trypsin inhibitor, a polypeptide that at pH 3 to 7 combines with trypsin and inactivates this enzyme in a 1:1 ratio. It also partially inhibits chymotrypsin. The presence of trypsin inhibitor in the pancreas is thought to protect the pancreas against autodigestion by small amounts of active trypsin within the organ. Several studies demonstrated that the relative synthesis of specific digestive enzymes change as a function of dietary intake. The mechanism of this adaptation is not understood but for example the amylase gene expression was reported to be regulated by both insulin and gene (28). Interdigestive pancreaticl secretion Pancreatic enzyme secretion occurs continuously in the interdigestive (fasting) state when upper g.i. tract is cleared of food as well as after meal. The interdigestive secretion in humans and in other species (carnivore) is cyclic and follows the pattern of the migrating myoelectric complex (MMC) (Fig. 9). The patterns recur every 90-100 min with burst of motor activity and pancreatic enzyme secretion temporally associated with phase II and III of MMC in the duodenum. The underlying mechanism of this cycling is not clear but since atropine blocked the responses it is believed that cholinergic activation is involved and accompanied by the rise in plasma motilin and pancreatic polypeptide (PP). Motilin by itself is capable of activating premature MMC cycle and this does not depend upon cholinergic blockade so it may be that motilin release is not dependent upon the cholinergic activity but its action on the musculature of the gut requires this enhanced motor activity (29). The role of fluctuations of pancreatic secretion is not known but it may be related to the housekeeping function of MMC. Fluctuations in basal pancreatic secretion are maintained in early phase of acute pancreatitis in humans (30). Basal pancreatic secretion is relatively small and the enzymatic component does not exceed 1015% of maximal response of the organ to exogenous secretagogue. Posprandial pancreaticl secretion Posprandially, the pancreatic secretion starts almost immediately after the meal (Figs. 9 and 10) and it is accompanied by the significant elevation of plasma 304 Fig. 9. Interdigestive and postprandial pancreatic trypsin secretion and related to phases of MMC (34). The fluctuations in pancreatic enzyme secretion under basal conditions probably depend upon the alterations in the activity of ENS and in the release of motilin (11). Pancreatic secretion Fig. 10. Pancreatic protein and bicarbonate secretion before and after meat meal in dogs as compared to maximal CCK-induced protein secretion and maximal secretin-induced bicarbonate secretion in these animals (unpublished data). 305 concentrations of stimulatory hormones such as gastrin, CCK, secretin and slightly GRP (Fig. 11). Concurrently, there is an increase in plasma levels of pancreatic inhibitory hormones such as PP, leptin and decrease in plasma ghrelin. The postprandial stimulation of pancreatic secretion includes three overlapping phases; cephalic, gastric and intestinal contributing, respectively, to about 20%, 10% and 70% of total postprandial response (19). In the past the postprandial phases have been entirely attributed to vagal stimulation with the efferent nerves releasing Ach in the pancreas and to cholinergically released gastrin that stimulate the acinar cells together with cholecystokinin (CCK) and secretin released from duodeno-jejunal mucosa by products of protein and fat digestion and gastric acid entering the duodeum (14, 15). Cephalic phase, that can be induced in dogs by sham-feeding and in humans by modified sham-feeding, is characterized by a prompt rise in pancreatic enzyme protein secretion and small increase in HCO3- secretion (Fig. 12). It is accompanied by the elevation of plasma concentrations of stimulatory hormones such as gastrin and CCK and slight rise in plasma secretin. Among the inhibitory Stimulating hormones A Inhibiting hormones B Ghrelin Fig. 11. Major gut stimulatory hormones (CCK, secretin, gastrin and GRP) and inhibitory hormones (PP, PYY, ghrelin and leptin) involved in the control of pancreatic secretion (unpublished data). Inhibitory hormones Stimulatory hormones Fig. 12. Cephalic phase of gastric and pancreatic secretion in dogs with esophageal, gastric and pancreatic fistulas (on left) and plasma levels of pancreatic stimulatory (gastrin, CCK and secretin) and inhibitory hormones (PP, leptin and ghrelin) (on right) (unpublished data). Pancreatic secretion Gastric secretion 306 307 hormones, both PP and leptin tend to increase, while ghrelin after short rise declines (21). Similar changes in pancreatic secretion can be induced by insulin hypoglycemia or glycocytopenia induced by 2-deoxy-D-glucose (2-DG) (Fig. 13). Since the rise in pancreatic secretion can be significantly reduced using either atropine to clock muscarinic receptors or S-0509, a specific CCKB-receptors, it is reasobnaly to conclude that the major stimulants of the cephalic phase induced by sham-feeding, insulin or 2-DG include vagal-cholinergic stimulation and vagally released gastrin and possibly GRP, acting directly on the acinar cells to stimulate enzyme secretion. The major part of the postprandial pancreatic secretion is usually attributed to intestinal phase during which the major role has been attributed to CCK and secretin, released from the endocrine I and S cells by protein and fat digestion products and by gastric acid entering the duodenum. The question remains whether CCK, the most importent stimulant of pancreatic enzyme secretion, acts through the endocrine pathway following the release into the circulation and stimulation of acinar cells via CCK2-receptors or through the neurocrine pathway Fig. 13. Cephalic phase of gastric and pancreatic secretion in dogs with esophageal, gastric and pancreatic fistulas in response to sham-feeding, insulin or 2-DG alone and after administration of atropine (25 µg/kg sc), L-364,718 (CCK1-receptor blocker) or S-0509 (CCK2-receptor blocker) (unpublished data). Schematic presentation of the mechanisms responsible for gastric and pancreatic secretion in response to cephalic stimulation. 308 via neural reflexes (Fig. 14). Classic concept of postprandial pancreatic secretion that CCK acts primarily via endocrine pathway was changed with the discovery that efferent vagal nerves to the pancreas release not only Ach but also other neurotransmitters such as GRP and VIP (31). Moreover, CCK together with secretin were found to play a major role in the postprandial pancreatic secretion (32). Studies on rats with the diversion of pancreatic juice from the intestine showed an increased pancreatic secretion (33, 34), the release of CCK appears to be controlled by at least two CCK-releasing factors, one monitor peptide secreted by acinar cells and another CCK-releasing peptide produced by enterocytes. These releasing peptides are inactive in the interdigestive period because of their digestion by free luminal trypsin. Both peptides appear to remain under neuronal (cholinergic) and neurocrine (somatostatin) control. After meal, when trypsin is bound by food products, the releasing peptides are secreted in large amounts causing excessive release of CCK that may act via stimulation of afferent nerves and via endocrine pathway on acinar cells. Actually, at present, not only the pancreatic protein secretion but also other effects of CCK such as relaxation of proximal stomach combined with increased antral and pyloric contraction, increased duodenal motility, gall bladder contraction and sphincter Oddi Fig. 14. The question remains whether CCK released from the I-cells is acting through the endocrine or neurocrine pathway. 309 relaxation are mediated by vago-vagal reflexes activated by stimulation of by CCK of the CCKA-receptors at sensory nerve terminals in the digestive system. The major change in the mechanism of pancreatic secretion concerns the action of CCK on exocrine pancreas through vago-vagal reflexes (32-36) (Fig. 15). The evidence for such neuronal rather than endocrine pathway of the action of CCK on the pancreas stems from earlier studies in animals such as dogs in which atropine was found to inhibit the pancreatic secretion induced by endogenous stimulants of CCK such as leucine or tryptophan, but also by lower, more physiological doses of CCK (34, 37). Our studies on humans performed in collaboration with Gabryelewicz showed that pancreatic enzyme secretion induced by CCK + secretin was inhibited not only by loxiglumide but also by atropine indicating that neuronal pathway in men does play a role in the action of postprandially released CCK on pancreatic enzyme secretion (38). CCK release & action on pancreas CCK release in peptides Fig. 15. The release of CCK from duodenal I-cells by products of protein (amino acids) and fat digestion (fatty acids) (upper panel) and by monitor peptide originating from the pancreas and by CCK-releasing peptide produced by the enterocytes (lower panel). The physiological effects of CCK on the pancreas (stimulation of enzyme secretion), the stomach (relaxation of proximal stomach and inhibition of acid secretion), gall-bladder (contraction) and sphincter of Oddi (relaxation) (not shown) are mediated by long vago-vagal and short entero-pancreatic reflexes. 310 Fig. 16. Schematic presentation of pancreatic enzyme secretion in response to CCK administered intraduodenally that is mediated via neurocrine route acting through long vago-vagal and short entero-pancreatic reflexes. Exogenous CCK in large dose may stimulate pancreatic secretion by direct action on acinar cell receptors. Owyang (32) using anesthetized rats demonstrated that atropine and hexamethonium almost completely abolished the pancreatic protein response to low but not high supraphysiological doses of CCK. Similar effects were obtained using local capsaicin deactivation of afferent nerves or transection of afferent nerves. Thus, there is strong evidence for neuronal rather than endocrine action of CCK on pancreatic secretion but further studies are needed to clarify the relative contribution of neuronal and endocrine pathway of the action of CCK on exocrine pancreas. Zabielski et al (35 ) were first to find that intraintestinally applied CCK-8 resulted in the stimulation of pancreatic secretion, the effect that was abolished following blockade of muscarinic and CCK1-receptors, suggesting that these receptors are involved in the mediation of the stimulatory effect of luminal CCK on exocrine pancreatic secretion. These results could be summarized that the major stimulant in the postprandial pancreatic secretion originates from the action of CCK, that at large pharmacological doses applied exogenously may stimulate the exocrine pancreas by direct activatiuon of the acinar cells. However, under physiological conditions CCK acts primarily via long vago-vagal and short intestino-pancreatic reflexes to release at the terminal of postsynaptic nerves such neuropeptides as GRP and VIP and neurotransmitters such as Ach (Fig. 15). 311 Fig. 17. Schematic presentation of the complex neuro-hormonal mechanisms involved in the pancreatic stimulation (on left) and inhibition (on right). Nitric oxide is one of the factors released by neurons and CGRP to increase pancreatic blood flow and secretion (left panel); Neurohormonal factors affecting food intake acting through endocrine and neuronal pathways on hypothalamus to control the release and action of NPY and AgRP responsible for the appetitive behavior. Leptin and ghrelin acting in opposite direction (leptin-ghrelin tango) on satiety and appetite (right panel). Recently the role of nitric oxide (NO) and CGRP has been suggested to play a role in the control of pancreatic secretion and pancreatitis (39-43). NO is produced in epithelium, endothelium and macrophages of the GI tract and the pancreas from L-arginine by constitutive NO synthase (cNOS). GI tract is very active in NO release, therefore attempts have been made to define the possible role of NO in exocrine pancreatic secretion (Fig. 16). Using cNOS inhibitor, Lnitro-arginine derivative L-NNA, we found that such blockade of NOS in vivo dogs resulted in the dose-dependent reduction in CCK + secretin stimulated pancreatic secretion and this was accompanied by the reduction in insulin and PP secretion, the effects that were reversed by addition of L-arginine. (42) These results indicate that NO alters the exocrine pancreatic secretion and this alteration is mediated by changes in pancreatic blood flow. 312 It should be mentioned that L-nitro-arginine (L-NMMA) was also highly effective in the inhibition of pancreatic enzyme secretion in humans, the effect that was also reversed by the addition of L-arginine to L-NMMA (43). In rats, we have an evidence that endogenous NO may be released from afferent nerves by stimulation with small dose of capsaicin and that such stimulation attenuates the development of pancreatitis induced by caerulein (44,45). This is supported by the fact that small dose of capsaicin that stimulates the release of CGRP and NO or exogenously applied calcitonin-gene related peptide (CGRP) prevents the damage of pancreatic acini provoked by caerulein overstimulation (45). With recent discovery of ghrelin (46,47) and leptin (48), that are involved in appetitive behavior, ghrelin as hunger hormone and leptin as satiety hormone (47), the concept of negative interaction between these peptides on food intake so called ghrelin-leptin tango has been proposed (49) to explain their contribution in the release of hypothalamic neuropeptide Y (NPY) and agouti-related protein (AgRP), controlling food intake (Fig. 17). Using the technique of feeding with liquid meal and measuring the amounts of food ingested by collection of the meal dropping from the large gastric fistula, we showed that under basal conditions, ghrelin prevails and its plasma level in fasted animals reaches the peak, while plasma leptin is strongly attenuated, indicating that ghrelin diminishes the release of leptin from the stomach and other sources such as adipocytes. Following food intake, there is an immediate Fig. 18. Changes in food intake and plasma ghrelin after feeding and administration of leptin without and with specific antibody against leptin in conscious rats with gastric fistula to collect and measure the amounts of ingested liquid food (unpublished data). 313 Fig. 19. Changes in food intake and plasma leptin after feeding and administration of ghrelin without and with specific antibody against ghrelin in conscious rats with gastric fistula to collect and measure the amount of ingested liquid food (unpublished data). fall in plasma ghrelin, whereas plasma leptin increases during and after feeding indicating that circulating leptin suppresses ghrelin release and plays a role of the satiety signal (Figs 18 and 19). Immunonetralization of either peptide leads to the changes in eating behavior and similar effects can be obtained with reduction of NPY activity using specific antagonist of NPY. These changes result also in alterations in exocrine pancreatic secretion, but both ghrelin and leptin appear to inhibit exocrine pancreatic secretion (50-54). Both ghrelin and leptin appear to act through normal endocrine pathway to affect the release of NPY as well as via activation of peripheral, namely gastric receptors and signaling to the hypothalamic feeding centers via the neural pathway. This double signaling routes, one endocrine and another neuronal, emphasizes the importance of the feeding behavior in the homeostasis. Furthermore, both peptides contribute not only to the control of food intake but also to the adjustment of gastric and pancreatic secretion to the anticipation and ingestion of food to provide the optimal conditions for the digestion and absorption of ingested food (54-55). In addition to the control of food intake both ghrelin and leptin were shown to affect gastric mucosal integrity by enhancing the mucosal blood flow and protection against topical irritants (57). Both hormonal peptides were shown to reduce pancreatic damage in the course of acute pancreatitis (50-52). 314 CONCLUSIONS In conclusion, the mechanism of pancreatic secretion under basal conditions and following physiological stimulus such as meal is very complex. The major role appears to be played by vagal nerves and CCK but other neuromediators including Ach, GRP, CGRP, NO are also involved as modulators of this secretion, partly by affecting pancreatic blood flow. Under physiological conditions cholinergic afferent pathways rather than pancreatic acinar cells represent the primary targets on which gut peptides such as CCK affects the postprandial pancreatic secretion. This supports an old Pavlovian concept that the neural system is the major regulator of pancreatic enzyme secretion. Pancreatic bicarbonate secretion appears to be dependent upon the pH of duodenal content and duodenal acid loads and the mediator of this secretion is mainly secretin acting by endocrine way on centro-acinar or duct cells of the pancreas. A feedback system involving the release from the pancreas and the gut of peptides stimulating the release of CCK from I cells and secretin from S cells has been postulated to control of pancreatic enzyme and bicarbonate secretion but its physiological role in the overall regulation of exocrine pancreas, at least in humans, has not been fully elucidated. Reflex pancreatic stimulation involving CGRP and NO and induced by capsaicin in the duodenum indicates that enteropancreatic reflexes are also operating to control exocrine pancreatic secretion. Feeding behavior is accompanied by the release from gastric mucosa of two opposing peptides, ghrelin activating the hunger mechanisms and leptin responsible for the satiety mechanisms. 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Inhibition of pancreatic protein secretion by ghrelin in the rat. J Physiol (London) 2001; 15: 231-236. 54. Konturek PC, Konturek SJ, Brzozowski T, Jaworek J, Hahn EG. Role of leptin in the stomsch and the pancreas. J Physiol(Paris) 2001; 95: 345-354. 55. Garlicki J, Konturek PC, Majka J, Kwiecien N, Konturek SJ. Cholecystokinin receptors and vagal nerves in control of food intake in rats. Am J Physiol. 1990; 258: E40-45. R e c e i v e d : July 8, 2003 A c c e p t e d : July 31, 2003 Authors address: Prof. Dr S.J. Konturek, Department of Physiology Univ. Med. College, ul. Grzegorzecka 16, 31-531 Krakow, Poland. Tel: +48-12-4211006, fax: 48-12-4211578. E-mail; [email protected]