REVIEW

Secretin Historical Perspective and Current Status William Y. Chey, MD, DSc and Ta-Min Chang, PhD

Abstract: This review describes the history of secretin discovery, identification, purification, and structural determination; cloning of secretin and its receptor; synthetic secretin; and highly specific and sensitive radioimmunoassay to define the characteristic physiological role on postprandial pancreatic fluid and bicarbonate secretion, which requires robust potentiation by cholecystokinin. Secretin plays a key role in the negative and positive regulatory mechanisms of exocrine pancreatic secretion. Secretin-releasing peptides were discovered in duodenal acid perfusates of both rat and dog and in canine pancreatic juice. The release and action of secretin and secretin-releasing peptides are in part mediated via vagovagal reflex mechanism involving afferent sensory neurons in proximal intestine and efferent cholinergic neurons in the pancreas. Besides acetylcholine, many neurotransmitters or neuromodulators influence release and action of secretin. The action of secretin in the pancreas depends on insulin, which also suppresses local release of somatostatin and pancreatic polypeptide. Thus, release and action of secretin are mediated via neurohormonal interaction. Clinical conditions with hypersecretinemia and hyposecretinemia are discussed. Synthetic human secretin is used for studies of exocrine pancreatic secretion, secretin-enhanced magnetic resonance cholangiopancreatography combined with exocrine pancreatic function test and diagnosis of gastrinoma syndrome. Therapeutic use of secretin is considered for the relief of severe pain in chronic pancreatitis. Key Words: secretin history, physiology, pancreatic feedback mechanism, secretin-releasing peptides, neurohormonal regulation, pathophysiology (Pancreas 2014;43: 162Y182)

he direct experimental proof by Bayliss and Starling1 in 1902 that a substance in the small intestinal mucosal extract can drive secretion of pancreatic juice led to the discovery of secretin and established the hormonal mechanism for the stimulation of exocrine pancreatic secretion. Purification of secretin by Jorpes and Mutt2,3 from the extract of porcine intestinal mucosa and subsequent structural determination by Mutt et al4,5 in the 1960s confirmed secretin as a bioactive peptide capable of acting as a pancreatic secretagogue. Successful chemical synthesis of bioactive secretin by Bodanszky et al6 and Ondetti et al7 not only confirmed the finding of Jorpes and Mutt but also provided ample supply of pure secretin for studying its physiological and pharmacological actions. It also paved the way for the development of highly sensitive radioimmunoassay

T

From the Rochester Institute for Digestive Diseases and Sciences, Rochester, NY. Received for publication July 11, 2013; accepted September 6, 2013. Reprints: William Y. Chey, MD, DSc, 33 Harrington Dr, Fairport, NY (e-amidating enzyme in Escherichia coli, recombinant secretin synthesized in the bacteria lacked the C-terminal amidation at the valine residue but remained bioactive.77,78 Olson et al79 synthesized and purified recombinant human secretin with C-terminal Gly-Lys-Arg extention, which displayed at least 80% bioactivity of porcine

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secretin. Today, synthetic and recombinant secretins are available for research and clinical use from commercial sources.

The Conditions for the Proof of Secretin as a Hormone Efforts were made by many investigators to prove that secretin is indeed a circulating hormone. For a peptide to function as a hormone, as pointed by Grossman,80 (1) it must be released into the blood under physiological conditions to produce a defined physiological effect, and (2) the effect produced by its physiological release such as exocrine pancreatic secretion can be mimicked by intravenous infusion of exogenous hormone at a dose that attains the same plasma level of hormone immunoreactivity released endogenously. In the case of secretin, however, it is difficult to fulfill the second criterion. Under a typical physiological condition, such as postprandial state, the robust pancreatic secretion of fluid and bicarbonate results from the simultaneous actions on the pancreas of both endogenous secretin and CCK, which potentiate each other.37Y40 Thus, the pancreatic secretion achieved by exogenous secretin alone in a physiological dose is significantly less than that achieved by simultaneous action of both endogenous secretin and CCK. Accordingly, based on the effect of exogenous secretin, the hormone alone in a physiological dose range cannot mimic the pancreatic secretion in physiological condition, that is, after a meal or duodenal acid infusion. Thus, the task requires the direct proof that secretin is released from the intestine to the blood under a physiological condition, and only the elevated secretin level is indeed causatively involved in physiological stimulation of exocrine pancreatic secretion. The genuine role of endogenous secretin in physiological conditions can be determined only by a successful immunoneutralization with a high titer and specific antisecretin serum or by using a powerful secretin receptor-specific antagonist.

Proof by Radioimmunoassay and Immunoneutralization of Secretin It is necessary to have a highly sensitive secretin-specific radioimmunoassay method to determine elevation of secretin in the blood under a physiological condition such as after ingestion of a meal, increasing from fasting plasma concentration of 1 to 2 pM or lower to less than 5 pM in humans,81,82 which

FIGURE 1. Amino acid sequence of secretin from various species. Single-letter coded amino acid residues are used. The underlined residues indicate difference from porcine secretin.

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is also lower than other gut hormones. As summarized previously,81,82 a high-sensitivity secretin radioimmunoassay requires production of a high titer secretin-specific antibody and radioiodinated secretin of high specific activity and elimination of nonspecific interference in the plasma. Many studies in the early 1970s were able to show that secretin is released into circulation after duodenal acidification with HCl in the dose used by Bayliss and Starling1 or HCl solution in various high doses to demonstrate dose-response relationship but failed to detect elevation of secretin after ingestion of a meal. This fact led to the doubt that secretin might not be the hormonal stimulant of pancreatic secretion found in the crude intestinal extract of Bayliss and Starling.83 In 1978, 2 groups of investigators were able to detect initially the release of secretin after ingestion of a meal both in humans42,84 and in the dog.85 Moreover, postprandial release of secretin was abolished when gastric acid secretion was suppressed by administration of cimetidine, paralleled with a marked suppression of pancreatic secretion,42 indicating that gastric acid entering the duodenum after a meal is the major physiological stimulant of secretin release. Both laboratories also demonstrated that exogenous secretin given in a physiological dose to mimic postprandial secretin level was able to stimulate exocrine secretion.38,86,87 Although this observation is a prerequisite for secretin to function as a hormone, it cannot be regarded as a functional proof for secretin under physiological conditions. This argument is based on the fact that exocrine pancreatic secretion is under neurohormonal regulation and many regulatory peptides and neurotransmitters including both stimulators and inhibitors of pancreatic secretion are released after a meal.88,89 Therefore, the action of exogenous secretin alone to stimulate pancreatic secretion in the fasting state does not necessarily mimic the action of endogenous secretin in the presence of other elevated regulatory peptides released after a meal although the same plasma secretin levels are achieved by the exogenous secretin. Thus, we9 provided in 1979 the final proof that secretin is a hormone by observing in 9 dogs that when postprandial elevation of secretin in plasma was abolished by successful immunoneutralization with a high titer secretin-specific antiserum, pancreatic secretion of fluid and bicarbonate, both concentration and output, was markedly suppressed by more than 80% (Fig. 2). The remaining less than 20% of postprandial fluid and bicarbonate secretion is likely due to other neurohormonal regulatory mechanisms.88,89

Secretin, A Historical Review

prosecretin form suggested that there might be an alternative splicing of secretin messenger RNA (mRNA) in the pig, a notion supported by the detection of a shorter mRNA variant in porcine intestine.91 Secretin-Gly and secretin-Gly-Lys-Arg seemed to be the products of posttranslational processing of the C-terminally 41 amino acidYextended prosecretin before proteolytic cleavage and amidation into secretin. These forms were probably the products of posttranslational processing of preprosecretin after removal of signal and N-terminal peptides. On the other hand, the presence of the N-terminally extended secretin indicated that an alternative posttranslational processing pathway might exist in the pig in which C-terminal peptide was removed and amidation of the C-terminal valine had occurred before complete removal of the N-terminal peptide.94 All 4 forms of secretin precursors exhibited secretin bioactivity; particularly secretin-Gly95,98 and secretin-Gly-Lys-Arg79,96,98 had an activity nearly the same as or higher than that of secretin. Because of their high bioactivity, Solomon et al98 proposed that secretin-Gly and secretin-Gly-LysArg might be released under various physiological conditions and contributed to stimulation of exocrine pancreatic secretion. Alternatively, these 2 prosecretins might be preferentially expressed in secretin-expressing tissues other than upper small intestinal mucosa to exert their biological effects. This proposition needs to be tested by determining tissue distribution of these prosecretins

MOLECULAR BIOLOGY OF SECRETIN AND ITS PRECURSORS Complementary DNA of porcine and rat secretins were isolated, and their nucleotide sequences were determined by Kopin et al90 in 1990. From the nucleotide sequences, it was deduced that preprosecretin from both species consists of a signal peptide of different lengths, 11 amino acids of an N-terminal peptide, secretin, a Gly-Lys-Arg (GKR) amidation-cleavage sequence, and a C-terminal extension peptide of 72 amino acids. The rat secretin gene of 813 base pairs contains 4 exons separated by 3 introns with the entire secretin sequence encoded by a single exon.91,92 Human93 and mouse53 secretin genes have also been cloned and display similar coding structure. Four forms of secretin precursor peptides have been purified from porcine concentrate of thermostable intestinal peptide. These include a N-terminally 9 amino acidYextended secretin,94 a C-terminal glycineYextended secretin (secretin-Gly),95 secretinGly-Lys-Arg,96 and a secretin-Gly-Lys-Arg with 41 amino acids extension at the C-terminal region in which the first 32 amino acids of the secretin C-terminal peptide is substituted by a single Arg residue.97 The presence of the C-terminal 41 amino acidYextended * 2014 Lippincott Williams & Wilkins

FIGURE 2. Effect of normal rabbit serum (solid line) and antisecretin serum (dashed line) on pancreatic secretion of both bicarbonate concentration (A) and output (B) in response to a meat meal. Values represent means T SE of 9 experiments in 9 conscious dogs with chronic pancreatic fistulas. This figure was published in Gastroenterology, 77, Chey et al, Effect of rabbit antisecretin serum on postprandial pancreatic secretion in dog, Copyright Elsevier (1979).9 www.pancreasjournal.com

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relative to secretin and their release pattern under the physiological conditions of investigation.

DISTRIBUTION OF SECRETIN Before radioimmunoassay and immunocytochemistry became available, the distribution of secretin along gastrointestinal tract was assessed via bioassays in early studies. Bioassays of secretin were performed either by monitoring pancreatic fluid secretion in response to a stimulant (eg, HCl) applied into various segments of the small intestine or by measuring the level of secretin-like bioactivity in the mucosal extract isolated from various intestinal segments. Because intestinal stimulants are capable of eliciting the release of secretin and other pancreatic secretagogues such as CCK, vasoactive intestinal peptide and NT from the intestine and mucosal extracts also contain other hormones in addition to secretin; the results of these bioassays cannot represent the true distribution of the peptide hormone we now know as secretin. Radioimmunoassy of secretin in the extracts of the gastrointestinal tract indicated that secretin is found in the upper small intestine.99,100 Straus and Yallow100 reported that in several mammalian species, secretin-like immunoreactivity was most abundant in the mucosa of the distal duodenum and proximal jejunum. Others observed that terminal ileum contained high levels secretinlike immunoreactivity just second to the duodenum in rats101,102 and humans.103 Secretin-like immunoreactivity and bioactivity were also found in the antral mucosa of rats and dogs104,105 and in porcine and rat brains.46,106 Secretin-producing endocrine cells (S cells) are found in the upper small intestine mucosa by immunocytochemistry as pearshaped cells localized between the villous and crypt regions.107 In the dog duodenal mucosa, S cells are found to contain K-type granules with a wide halo distinct from secretin-negative endocrine cells containing S-type granules with a small halo. An electron micrograph of secretin cell108,109 is shown in Figure 3. Some intestinal S cells also produce serotonin in several mammalian species110; substance P (SP) and CCK in the mouse111,112; and CCK, GIP, GLP-I, peptide tyrosine tyrosinamide (PYY), and NT in the human intestinal mucosa.103 Secretin-immunoreactive cells were also found in the gastric mucosa104,105,113 and localized in Purkinje cells in the cerebellar cortex, central cerebellar nuclei, pyramidal cells in the motor cortex, and primary sensory neurons in humans and colchicine-treated rat brains.114 Secretin gene transcript was found by RT-PCR to distribute widely in the rat including the intestine, brain, heart, lung, kidney, testis, and epididymis115,116 and in humans including the stomach, duodenum, jejunum, ileum, ileocecum, colon, spleen, testis, ovary, lung, and various parts of the brain.93,115,117,118 In mice, secretin mRNA was detected by Northern blot analysis in the upper small intestine, ileum, and colon, with the highest transcript level seen in the ileum,90 whereas low level of secretin mRNA was detected in several regions of the brain by reverse transcription.90,91

ONTOGENY OF SECRETIN In the rat duodenum, secretin cells were identified as early as 17th embryonic day (E17),119 and secretin immunoreactivity and mRNA levels reached the highest levels at E20 immediately before birth and fall gradually after birth to the adult levels.91 Similarly secretin-like immunoreactivity was found at relatively high levels at birth in the guinea pig and rat intestines, decreasing in the postnatal period.120Y122 Secretin mRNA was also identified in beta cells of the developing rat pancreas, reaching maximal levels by E19 and falling to undetectable level by adulthood.123 Thus, the time course of increasing

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FIGURE 3. Electron micrograph of an S cell in human duodenal mucosa, showing secretin granules near blood vessel. From Kobeyashi et al108 with permission.

secretin gene expression in the developing pancreas occurs during a period of rapid growth and cytodifferentiation of its target organ and seems to conform to its trophic effects reported by various laboratories.124Y128 Secretin mRNA and immunoreactivity were also found widely distributed in several developing organs of the mouse embryo, including the intestine, several regions of the brain, heart, and kidney,129 suggesting that secretin might be involved in modulating the development of these organs.

SECRETIN RECEPTOR STRUCTURE, FUNCTIONAL CHARACTERISTICS, AND DISTRIBUTION The presence of a high affinity receptor for secretin was first demonstrated in isolated guinea pig pancreatic acini by Jensen et al69 in 1983. Binding of secretin to the receptor resulted in G proteinYcoupled activation of adenylate cyclase.69 The binding affinity of secretin to its receptor depends on the tracer being used. Early studies using 125I-secretin labeled at the N-terminal histidine residue at low efficiency would require rigorous purification of the tracer to separate from uniodinated secretin.81,82 Contamination of unlabeled secretin and the presence of a bulky iodine atom at the N-terminal in the tracer that might affect its binding affinity because this residue is critical for receptor binding (to be discussed later) would therefore result in a higher Ki values for secretin in a competitive binding study. Radioiodinated [Tyr10]secretin has been shown to have the same activity for receptor binding and cAMP production as native secretin.130 The Ki values of secretin measured with this tracer for the human secretin receptor on transfected cells were 1.4 T 0.2 nM, and half-maximal concentration (ED50) for cAMP production was 194 T 45 pM.131 The receptors for peptides of glucagon/VIP/secretin superfamily often cross-react with other peptides in the family with varying binding affinity; the binding studies occasionally resulted in the detection of multiple receptor subtypes not necessarily indicating different secretin receptor molecules. A rat secretin receptor cDNA was first cloned by Ishiihara et al132 from a mouse neuroblastoma/ rat glioma hybrid cell line, and identical to that was subsequently cloned by Ulrich et al133 from a cDNA library of rat pancreas. * 2014 Lippincott Williams & Wilkins

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The rat secretin receptor cDNA encodes a receptor protein of 427 amino acid residues with a molecular weight of 48,696 and contains 5 potential N-glycosylation sites, 7 cysteine residues, and 7 transmembrane regions characteristic of G proteinYcoupled receptors.132 Recombinant secretin receptor expressed in rat secretin receptor cDNA-transfected cells exhibited similar ligand binding affinity and activation of adenylate cyclase as the native pancreatic receptor132,133 and, when isolated after affinity labeling, had the same relative molecular weight (Mr) of 57,000 to 62,000,133 which were similar to the affinity cross-linked receptor isolated earlier from rat pancreatic acini134 and rat gastric glands.135 After treatment with endoglycosidase F, both the native and recombinant receptors displayed single homogenous band of Mr = 42,000, indicating an N-glycosylated receptor.133 Both human136Y138 and rabbit139 secretin receptors have also been cloned and shown to have structural organization similar to rat secretin receptor and the potency to mediate specific secretin binding and activation of adenylate cyclase. In addition, human secretin receptor was also shown to mediate secretin-stimulated elevation of intracellular Ca2+ concentration [Ca 2+]i via increased levels of inositol-1,4,5-triphosphate.137 Comparison of human, rat, and rabbit secretin receptor expressed in cDNAtransfected cells indicated that they have binding properties similar to porcine, rat, and rabbit secretins with minor difference owing to the sequence difference in the secretins.140 The secretin receptor is classified as a member of B family G proteinYcoupled receptor that includes receptors for secretin, VIP, glucagon, PACAP, GIP, GLP-1, parathyroid hormone, calcitonin, and corticotropin releasing factor.141

Secretin, A Historical Review

The putative structure of secretin receptor on the cell membrane includes an extracellular amino terminal end, with 3 extracellular loops and 3 intracellular loops alternatively interposing the 7 transmembrane segments and an intracellular C-terminal tail (Fig. 4).142 Early studies from the laboratories of P. Roberrecht and L. Miller have indicated that the N-terminal domain of the secretin receptor is essential for secretin-binding specificity and affinity by interacting with the C-terminal portion of secretin. On the other hand, the amino terminal of secretin interacts with the first and second transmembrane helices and the first extracellular loop of the receptor, whereas the extracellular disulfide bridges and N-glycosylation sites142 are also important for receptor binding activity. Before presenting to the cell surface membrane, the secretin receptor forms homodimer,143,144 and only one protomer of the homodimer binds secretin.145 The dimeric molecular complex seems to be important for achieving a high affinity ligand binding state and acquiring ligand-mediated negative cooperativity.143 In the past decade, Miller et al have conducted more extensive studies of how secretin and the secretin receptor interact on the cell surface, and the conclusions derived from their work are summarized briefly here. First, except for residues at positions 2, 16, 17, 21, 24, and 25 in secretin molecule, all other amino acid residues are required for high affinity receptor binding, among which the N-terminal His1 and Asp3 residues are critical for the activation of adenylate cyclase. Further fluorescencephotolabeling studies have revealed several likely contact points between secretin and the secretin receptor in the ligand-receptor complex and led to the conclusion that secretin binds to the secretin receptor through its C-terminal end to a disulfide-bonded

FIGURE 4. The structure of human secretin receptor. The putative N-linked glycosylation sites at residues 72, 100, 106, 128, and 291 are labeled with black boxes. The transmembrane domains of the receptor are labeled I to VII. Within the receptor, there is a putative hydrophobic leader peptide (22 amino acids), an N-terminal extracellular hydrophilic domain (122 amino acids), and a C-terminal hydrophilic cytoplasmic domain (42 amino acids). Filled circles represent amino acids that are conserved with rat secretin receptor, human, rat, mouse, and bovine PACAP type 1 receptor; the human and rat VIP1 receptor; and the human VIP2 receptor. From Pang et al142 with permission. * 2014 Lippincott Williams & Wilkins

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receptor N-terminal cleft and extension of the peptide toward the core helical bundle of the receptor. After secretin binding, the receptor N-terminal domain is then subjected to a conformational change resulting in the opening of the peptide binding cleft and some movement of the peptide toward the effector domain within the receptor core. Readers interested in this interaction in detail may refer to the relevant reviews.146,147 The interactions between the secretin receptor and Gs protein for activation of adenylate cyclase and Gq protein for mobilization of [Ca 2+]i have not yet been studied in detail. These interactions might involve the 3 intracellular loops and the C-terminal tail of the receptor. Recently, Garcia et al148 conducted a mutational study on the roles of intracellular loops of the secretin receptor. Thus, mutation of the conserved histidine residue (H157A and H157R) in the first intracellular loop resulted in reduced expression of the receptor on the cell surface with marked reduction in secretinstimulated cAMP production and no change in [Ca2+]i mobilization. Mutation of an arginine residue in the same intracellular loop (R157A) reduced [Ca2+]i response but had no effect on cAMP response. Mutation of Lys-Leu pair in the third intracellular loop (K302A/L303A) reduced cAMP response only, whereas the mutation (R318A/R312A) in the same intracellular loop reduced both responses. Combining the effective mutations resulted in an additive effect on the responses, whereas combining all effective mutations resulted in a mutant receptor that was capable of binding secretin normally but failed to elicit any second messenger response. These observations indicated that interaction of the secretin receptor with Gs and Gq proteins involves both overlapping and distinct points on the receptor. In addition, the formation of the receptor homodimer complex might also be involved in modulation of G protein coupling. The successful purification of recombinant human secretin receptor130 has provided a useful tool for future study of secretin-secretin receptor and the receptorYG protein molecular interactions via nuclear magnetic resonance or x-ray crystallography. To date, only a single secretin receptor is identified in the normal tissues of each species. Like secretin, the secretin receptor is widely distributed in mammalian species. For example, using more stringent Northern blot analysis, the human secretin receptor was found to express in descending order in the pancreas, kidney, small intestine, lung, and liver and at low levels in the brain, heart, and ovary.136,137 The human secretin receptor is even more widely distributed when assessed by quantitative RTPCR.118 Thus, in addition to the aforementioned tissues and organs, the receptor was detected with significant amount in the stomach, gallbladder, spleen, testis, and several regions of the brain. The rat secretin receptor was also found widely distributed in the brain examined by in situ hybridization.149 High affinity secretin receptor was detected only in acinar and ductal cells in the rat pancreas150 and together with secretin receptor mRNA in the human pancreas.151 In the rat liver, secretin receptor is detected in large cholangiocytes of the bile duct and is up-regulated after bile duct ligation152,153 without changing its molecular form.153 On the other hand, alternatively spliced variants of the secretin receptor with various extent of deletion were detected in several human cancers in addition to the native receptor.151,154Y158 Interestingly, a functionally inactive human secretin receptor spliced variants with exon 3 deletion (lacking residues 44Y79 of the N-terminal segment) was found to express on the cell surface and form a heterodimer with the native receptor and suppressed the receptor function.154,155 The pathophysiological role of this heterodimer formation is not clear at present although inhibition of the proliferation effect on tumor cells by the secretin receptor was proposed.155 Unlike the CCK receptor, no nonpeptide antagonist for the secretin receptor has been discovered. Early study has indicated

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that secretin(5Y27), which is capable of binding to the secretin receptor at very low affinity without activation of adenylate cyclase, is a very weak receptor antagonist for the secretin receptor. Recent studies through molecular modification of secretin(5Y27) have identified 2 derivatives, c[E16,K20]secretin(5Y27)159 and [I17,R 25]secretin(5Y27),160 as potential antagonists for the secretin receptor with improved binding affinity in the range of 10j8 M. These 2 antagonists may be useful for studying the in vivo function of secretin in small rodents such as the guinea pig, mouse, and rat.

PHYSIOLOGY OF SECRETIN Release of Secretin It has been clearly established that endogenous secretin is released from the upper small intestinal mucosa in postprandial state to achieve significant rise in plasma secretin level in several mammalian species including humans, dogs, rats, guinea pigs, and pigs. The extensive studies were performed by many investigators in dogs, which share similar gastrointestinal physiological functions with humans. Secretin increases in plasma by duodenal infusion of HCl at as little as 0.05 N or 2 to 4 mmol/h. The pH threshold for secretin release in the upper small intestinal lumen is 4.5 in dogs.161 Because suppression of gastric acid secretion during postprandial period by an H2 receptor antagonist blocked the rise in plasma secretin levels41,42,85 as well as markedly suppressed pancreatic secretion,85 the major stimulant of secretin release after ingestion of a mixed meal is HCl. Secretin is also released by nonacid substances including bile and bile salts162Y164; digestive products of fat in man, dog, and rat165Y168; oligopeptide and mixed amino acid solution in rats169,170; certain antiulcer drugs including geranyl-geranyl acetone,171 MCI727,172 and plaunotol173; and certain herbal extracts such as those from Curcuma longa (1-phenyl-n-pentanol)174,175 and licorice root174,176 (Table 1).

Mechanism of Secretin Release and Feedback Regulation of Pancreatic Secretion It is now well established that the release of secretin, like CCK,177,178 is controlled through a negative feedback regulatory mechanism of exocrine pancreatic secretion which involves pancreatic proteases. Thus, diversion of pancreatic juice from the duodenum augments, whereas intraduodenal infusion of pancreatic juice or proteases suppresses the release of secretin as well as pancreatic secretion in rats,179Y184 humans,185 pigs,186 guinea pigs,187 and dogs188,189 in either fasting or postprandial state or intestinal phase. Interestingly in dogs167,188Y190 and guinea pigs,187 the feedback regulations of pancreatic secretion and the release of secretin were absent in fasting state. Similarly, in humans, the feedback regulation associated with CCK was not found in the fasting state.178 The negative feedback regulation associated with secretin is operative in the postprandial state189 as well as in the intestinal phase with duodenal infusion of emulsified oleic acid,188 or digestion products of fat in dogs,167 sodium oleate in guinea pigs,187 or proximal jejunal infusion of acidified mixed amino acid solution (Travasol) in humans.185 The absence of negative feedback regulation in fasting dogs was found by others.191 Moreover, in our 2 studies in dogs,188,189 the feedback regulation was not involved with CCK because diversion of pancreatic juice or duodenal trypsin administration failed to affect plasma level of CCK. The increases in both secretin release and pancreatic secretion of fluid and bicarbonate were markedly suppressed by duodenal or proximal jejunal administration of pancreatic juice or trypsin but not by infusion of bicarbonate in rats,146,192 dogs,188,189 and humans185 (Figs. 5 and 6). In humans, it was shown that the release of CCK was also * 2014 Lippincott Williams & Wilkins

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TABLE 1. Luminal Stimulants for Release of Secretin Acid in duodenum Nonacid stimulants Digestive products of fat Long chain fatty acids Sodium oleate Oligo peptide Mixed amino acids Bile salts

Antiulcer drugs Geranyl-geranyl acetone Plaunotol* MCI-727† Herbal extracts Curcuma longa (1-phenyl-n-pentanol) Licrorice root

The chemical names are as follows: *(2Z,6E)-2-[(3E)-4,8-Dimethyl-3, 7-nonadienyl]-6-methyl-2,6-octadien-1,8-diol and †[2-(4-methylpiperazin1-yl)-1-4-(2-phenylethyl)phenylethanone oxime.

significantly suppressed but not pancreatic polypeptide (PP) or NT (Fig. 5). Conversely, intraduodenal administration of a trypsin inhibitor such as Camostat, a synthetic soybean trypsin inhibitor, increases markedly the release of both secretin and CCK associated with significantly increased pancreatic secretion in rats (Fig. 7),184 emphasizing the important role of pancreatic proteases in the secretin-mediated feedback regulation of exocrine pancreatic secretion. It is of interest to note that there are species differences in the feedback regulation of exocrine pancreatic secretion associated with the release of secretin and CCK in the fasting and postprandial states or intestinal phase (Table 2). Of note, secretin is involved in the feedback regulation in all animal models so far studied.

Discovery and Significance of Secretin-Releasing Peptides Extensive studies in the rat revealed that the release of secretin is mediated by a secretin-releasing peptide (SRP) found in concentrated acid perfusates (CAP) of the upper small intestine in rats (Fig. 8).169 The SRP in CAP is a heat-stable and proteaselabile peptide of molecular mass between 1000 to 5000 Da.193 Thus, SRP plays an important role in the feedback regulation of exocrine pancreatic secretion of fluid and bicarbonate by stimulating the release of secretin. An SRP was also found in the concentrated perfusates from canine proximal small intestines during duodenal infusion of 0.02 N HCl.194 It is also highly significant to find that canine pancreatic juice also contains SRPs (p-SRPs), which stimulate the release of secretin in rats195 as well as in dogs.196 In both species, canine p-SRP increased the plasma level of secretin and pancreatic secretion of fluid and bicarbonate, which were completely blocked

Secretin, A Historical Review

by immunoneutralization of circulating secretin with our antisecretin serum.195,196 Two p-SRPs of 14 kDa were purified from canine pancreatic juice.197 The N-terminal sequence (31 residues) of p-SRP-1 was found identical to canine pancreatic phospholipase A2 (PLA2), and p-SRP-2 had 71% homology to PLA2. Both canine p-SRPs and porcine PLA2 stimulated secretin release from secretin-producing cells (STC-1 cells and rat S cellYenriched preparation) in vitro through activation of calcium influx and protein kinase C.198 Interestingly, HCl in the duodenum also releases PLA2-like immunoreactivity, which stimulates secretin release and pancreatic secretion of fluid and bicarbonate in rats.199 Indeed, when CAP of the duodenum was pretreated with a rabbit anti-PLA2 serum, the rise in both plasma secretin and pancreatic secretion was markedly suppressed (Fig. 9). It is apparent that one of the SRPs in both duodenal acid perfusates and canine pancreatic juice is PLA2. To summarize, the release of secretin by HCl in the duodenal lumen is mediated by trypsin-sensitive SRPs secreted from the duodenal mucosa and pancreatic juice. One of the 2 p-SRPs in canine pancreatic juice was found to be PLA2 in both duodenal fluid and pancreatic juice. Further studies are needed (1) to determine whether SRPs are released into the duodenal lumen not only by duodenal acidification but also by bile, digestive products of fat and protein, and herbal extracts and (2) to purify the peptides and determine their amino acid sequence. Radioimmunoassay of the peptides will have to be eventually developed to clarify their physiological significance.

BIOLOGICAL ACTIONS OF SECRETIN Secretin given intravenously exerts several known biological actions in the gastrointestinal tract (Table 3).

Physiological Actions Physiological actions of secretin is now defined as the biological actions of the endogenous secretin that are completely blocked or markedly suppressed by a specific and potent secretin receptor antagonist or when circulating free secretin is removed by a specific and high titer antisecretin serum given intravenously. Because no satisfactory secretin receptor antagonist has been used for in vivo studies, rabbit antisecretin sera have been used in experimental animal models including dogs, rats, and guinea pigs to determine if an expected biological effect was abolished or markedly suppressed under a given physiological condition. In humans, it is surmised that if a physiological dose of secretin, based on postprandial immunoreactive plasma secretin level,38,86 can exert the expected biological actions, it is considered

FIGURE 5. Plasma hormone concentrations in response to jejunal infusion in 10 healthy volunteers of either control solution (acidified mixed amino acid solution [Travasol] alternated with 0.1 N NaHCO3 solution) or test solution (acidified amino acid solution [open column] alternated with trypsin solution [hatched column]). Each column represents the net increase of mean T SE of the integrated plasma hormone levels in a 1-hour period (n = 10). Note that jejunal infusion of trypsin significantly inhibited both plasma secretin and CCK levels (P G 0.05) but not plasma PP or NT. A, Secretin; B, CCK; C, PP; and D; NT. *P G 0.05. This figure was published in Gastroenterology, 107, Jin et al, Roles of gut hormones in negative-feedback regulation of pancreatic exocrine secretion in humans, Copyright Elsevier (1994).185 * 2014 Lippincott Williams & Wilkins

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FIGURE 6. Summary of effect of jejunal infusion of trypsin on acidified mixed amino acid solutionYstimulated pancreatic bicarbonate and chymotrypsin secretion in 10 healthy volunteers. Each bar represents the net increase of mean T SE bicarbonate (A) or chymotrypsin (B) outputs during a 1-hour period. AA represents the control solution (acidified mixed amino acid solution [pH 2] alternated with 0.1 N NaHCO3 solution at 15-minute intervals]. Note that jejunal infusion of trypsin significantly inhibited both bicarbonate and chymotrypsin outputs. However, simultaneous intravenous infusion of secretin and CCK reversed this inhibitory effect. This figure was published in Gastroenterology, 107, Jin et al, Roles of gut hormones in negative-feedback regulation of pancreatic exocrine secretion in humans, Copyright Elsevier (1994).185

‘‘physiological.’’ However, until such a time when a reliable secretin receptor antagonist will be available, the real physiological role of secretin cannot be determined in humans.

Role of Circulating Endogenous Secretin on Exocrine Pancreatic Secretion In 1977 to 1978, we9 observed that when the circulating free endogenous secretin after ingestion of a meat meal was bound completely with a rabbit antisecretin antiserum in 9 conscious dogs, pancreatic secretion of both fluid and bicarbonate was profoundly suppressed (Fig. 2) and protein output was significantly suppressed (Fig. 10). The postprandial rise in bicarbonate

concentration was virtually abolished (Fig. 2), but protein concentration was not (Fig. 10), indicating convincingly that the specific role of endogenous secretin is to stimulate postprandial pancreatic secretion of fluid and bicarbonate in the dog.9 Similar observations on the role of endogenous secretin were made with the antisecretin serum during diversion of pancreatic juice in guinea pigs187 and intraduodenal infusion of 0.05 N HCl in both guinea pigs187 and rats200 and postprandial state in rats (Chey, Chang, Lee, unpublished data) in our laboratory. Thus, secretin is indeed the major gut hormone that drives secretion of fluid and bicarbonate. Similar studies in humans await the availability of a specific and potent secretin receptor antagonist. It is clear, however, that the pancreatic secretion of fluid and bicarbonate in response to a meat meal or in intestinal phase results from the interaction between secretin and CCK, which

TABLE 2. Various Feed Back Regulation of Pancreatic Secretion and Hormone Release in Several Mammalian Species Feedback Regulation of Pancreatic Secretion Species Rats Dogs Guinea pigs Pigs FIGURE 7. Effect of synthetic trypsin inhibitor, camostat, on pancreatic secretion of bicarbonate and chymotrypsin in rats. The data were obtained from 6 experimental groups, including fasted, sham-operated, saline-infused, and camostat (25 mg, 50 mg, and 100 mg)Yinfused groups. *Significant differences from values of the fasting group with P G 0.01. Each bar represents mean T SE of 10 rats. This figure was published in Gastroenterology, 102, Watanabe et al, Mediation of trypsin inhibitor-induced pancreatic hypersecretion by secretin and cholecystokinin in rats, Copyright Elsevier (1992).184

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Humans

Hormone Release

State

Bicarbonate

Protein*

Secretin

CCK

1 2 1 2 1 2 1 2 1 2

Yes Yes No Yes No Yes No No NT Yes

Yes Yes No Yes No Yes No No No Yes

Yes Yes No Yes No Yes Yes No NT Yes

Yes Yes No No No Yes No No No† Yes

1 indicates fasting state; 2, postprandial state or intestinal phase. *Includes enzymes. † One patient with complete obstruction of pancreatobilliary duct with cancer at papillar of Vater. NT, not tested.

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TABLE 3. Biological Actions of Secretin in the Gastrointestinal Tract Stimulation

FIGURE 8. Pancreatic fluid volume (A), bicarbonate output (B), and plasma secretin concentration (C) in response to intraduodenal infusion of CAP (concentrates of acid perfusates) or CSP (concentrates of saline perfusate) in 7 recipient rats. Each bar represents a mean T SE. * and ** indicate significant difference from the percentage change of CSP group with P G 0.05 and P G 0.0 1, respectively. From Li et al193 with permission.

are simultaneously released into the circulation, for the increase in pancreatic secretion of both fluid and bicarbonate is virtually abolished by either antisecretin antibodies (Fig. 2) or CCK receptor antagonist, proglumide in dogs201 (Fig. 11). In addition, dietary fat that elicited pancreatic fluid secretion was also markedly suppressed by proglumide in rats.168 Thus, CCK is the other equally important and indispensable hormone for the secretion of fluid and bicarbonate. There is almost no physiological or biological condition in which either secretin or CCK alone circulates in the blood. The 2 hormones are always released together from proximal small intestinal mucosa, even in response to duodenal acidification.36 Thus, pancreatic secretion of both fluid and bicarbonate is stimulated by simultaneous joint actions of secretin and CCK, which are inseparable in given physiological or biological conditions, namely, postprandial state, duodenal acidification, duodenal infusion of sodium oleate, oleic acid, and digestion products of fat. It has been well recognized that pancreatic secretion of fluid and bicarbonate by intravenous administration of secretin in physiological doses is markedly potentiated by CCK-8 in humans,38 dogs,37 guinea pigs,202 and rats.39,203 Therefore, in driving pancreatic secretion of fluid that transports

Inhibition

Pancreatic secretion: fluid and bicarbonate* Billiary secretion: fluid and bicarbonate Gastric secretion: mucus Gastric secretion: pepsinogen* Brunner’s gland secretion Endocrine pancreas: somatostatin Endocrine pancreas: PP Gastric release: somatostatin Gastric release: prostaglandins

Gastric release: gastrin* Gastric acid secretion* Gastrointestinal motility: Lower esophageal sphincter Stomach* Small intestine Colon

*Experimental proof of physiological action.

enzymes and bicarbonate to the duodenum after a meal, the 2 hormones are essential partners. One must recognize that the function of CCK on exocrine pancreas is as equally important for the bicarbonate-rich fluid secretion as its stimulatory action on enzyme secretion. Less than 20% of the secretion of fluid and bicarbonate is driven by nonsecretin stimulants, which may include acetylcholine, CCK, gastrin-releasing peptide (GRP), and PACAP, which are potential candidates. In recent years, both secretin-deficient204,205 and secretin receptorYdeficient mouse models became available for investigations on the actions of secretin on exocrine pancreas,205 biliary ducts,206 central nervous system,204,207 and the kidney.208 Sans et al205 found that in secretin receptorYdeficient mice, basal secretory volume was significantly lower than in the control mice, suggesting that endogenous secretin exerts a significant stimulatory action on basal pancreatic secretion of fluid in mice. Secretin must have been released in a small quantity by mild duodenal acidification in fasting state. More significantly, in the same mouse model, pancreatic fluid secretion stimulated by secretin even in a large dose, 2 CU/kg, given intravenously was markedly less than that in the control mice. It is highly desirable to confirm their novel finding in other species that are widely used for in vivo experiments. Nonetheless, their exciting observation opens a new era for investigations of exocrine pancreas.

Gastric Acid Secretion: Secretin is a Physiological Enterogastrone

FIGURE 9. Plasma secretin concentration and pancreatic secretion in response to upper small intestinal acid perfusate preincubated with anti-PLA2 serum in recipient rats. The proximal jejunum was perfused with 0.02 N HCl at 0.3 mL/min for 1.5 hours, whereas bile was diverted in donor rats. The acid perfusates were concentrated 10-fold and incubated with anti-PLA2 serum or normal rabbit serum [NRS] (1:10) at 37-C for 30 minutes. The materials were filtered through a PM-10 Amicon membrane to remove anti-PLA2 antibodies and reinfused into recipient rats. A, Volume output; B, Bicarbonate output; C, Plasma secretin. *P G 0.05 and **P G 0.01 versus basal pancreatic secretion or basal plasma concentration of secretin. From Li et al.199 * 2014 Lippincott Williams & Wilkins

The concept of enterogastrone was proposed by Kosaka and Lim209 in 1930 when they found inhibition of gastric acid secretion by intraduodenal infusion of fat emulsion in dogs. Indeed, when their upper intestinal mucosal extracts were given intravenously, the acid secretion was markedly reduced. Thus, they coined this humoral substance as ‘‘enterogastrone.’’ Since Greenlee et al210 in 1957 reported the inhibitory effect of a partially pure secretin on gastric acid secretion in dogs, many workers confirmed their observations in dogs and humans.211,212 Most workers, however, used secretin in a pharmacological dose range. Only after pure secretin or synthetic secretin became available and the physiological dose ranges were established were they able to determine whether secretin is indeed an enterogastrone. In conscious dogs with a vagally innervated gastric pouch,11,213 the pouch acid secretion stimulated by intravenous human synthetic gastrin or pentagastrin was significantly inhibited by intravenous porcine synthetic secretin in doses of 0.03, 0.06, and 0.125 CU/kg per hour. Moreover, when circulating free www.pancreasjournal.com

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FIGURE 10. Effect of normal rabbit serum (solid line) and antisecretin serum (dotted line) on pancreatic protein output (A) and concentration (B) during postprandial period. Each value represents a mean T SE value of 9 experiments in 9 dogs with chronic pancreatic fistulas. This figure was published in Gastroenterology, 77, Chey et al, Effect of rabbit antisecretin serum on postprandial pancreatic secretion in dog, Copyright Elsevier (1979).9

secretin was immunoneutralized with a rabbit antisecretin serum,11 both acid output and gastrin release after a meal were significantly augmented. Thus, both gastrin release and acid secretion are in part regulated by circulating endogenous secretin in postprandial state. Similar observations were made in conscious

dogs with intact stomach (Fig. 12)12 in which endogenous secretin was shown to inhibit not only gastric acid secretion but also gastric emptying. Similarly, immunoneutralization of endogenous secretin released by intraduodenal infusion of HCl in rats resulted in the augmentation of gastric acid secretion.214 In humans, gastric acid secretion is inhibited by intravenous secretin in physiological doses.215 In the studies with rats216Y218 and isolated perfused rat stomach preparations,219 the inhibitory mechanism of secretin seems to be mediated by the release of somatostatin and prostaglandins.217Y219 It seems that so far as we know, the 2 gut hormones, namely secretin and CCK,220,221 are qualified as enterogastrone. Because the 2 major gut hormones are released simultaneously in physiological conditions, one must pursue to determine whether a combined effect of secretin and CCK in physiological doses would produce more potent enterogastrone effect. Another candidate enterogastrone is peptide YY, which deserves further studies.

Gastrointestinal Motility

FIGURE 11. Effect of proglumide on pancreatic bicarbonate secretion in response to intraduodenal [ID] administration of digested Lipomul in 6 dogs with chronic pancreatic fistulas. Each value represents a mean T SE. From Jo et al201 with permission.

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Secretin given intravenously inhibits contractile activity of the lower esophageal sphincter in humans222 and cats,223 stomach in dogs,224 small intestine in humans225 and dogs,226 and colon in humans.227 It also inhibits gastric emptying of liquid in humans.228 The doses of secretin used in these studies, however, were in pharmacological doses. With the use of intravenous administration in a physiological dose range, exogenous secretin was found to delay gastric emptying of liquid meal in conscious dogs.12 Moreover, when circulating endogenous secretin was immunoneutralized with a high titer rabbit antisecretin serum, gastric emptying time was shortened.12 In humans, intravenous secretin in physiological doses was shown to inhibit gastric emptying of liquid.229 These data * 2014 Lippincott Williams & Wilkins

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FIGURE 12. Effects of exogenous secretin and antisecretin serum on gastric acid secretion (A) and plasma gastrin concentration (B) in response to a mixed amino acid meal (Travasol) in 7 dogs with chronic gastric cannulas. *P G 0.05, ** P G 0.01 compared with saline infusion (A). From Jin et al.12

indicate that secretin is a physiological inhibitor of gastric motility in dogs, humans, and rats. Possible physiological roles of secretin on the motility of the small and large intestines await future investigations.

Biliary Secretion Among several gastrointestinal peptides or hormones including secretin, gastrin, and bombesin/GRP, secretin plays a key role in the biliary secretion of water and bicarbonate, and its receptor is expressed in the rodent and human liver by larger bile ducts.230 Since the last review of this subject in 1989,81 there has been little progress made in vivo study other than those of rodents. Although increased bile secretion by duodenal acidification was first noticed by Rutherford in 1880, the ‘‘crude secretin’’ in canine duodenojejunal mucosal extracts of Bayliss and Starling in 1902 was shown to stimulate bile flow in dogs. Their observation was confirmed by numerous investigators. With the use of relatively pure secretin preparations, secretin was confirmed to stimulate bile secretion of water and bicarbonate and chloride from bile ducts and ductules in animals,231Y234 isolated perfused livers of pigs235 and dogs,236 and in humans,237Y239 but secretin does not influence biliary secretion of bile acids.236 There is also evidence that bile salt-independent fraction of canalicular bile secretion is increased during secretininduced choleresis.240 These studies, however, were performed, using mostly impure secretin preparations in pharmacological doses. They must be confirmed by the studies using synthetic secretin in physiological dose range,10,36,37,86,87,213,241 and the role of circulating endogenous secretin must be confirmed using either antisecretin antibodies for immunoneutralization approach9,11,12 or a secretin receptor antagonist if available. It is surprising to find that in rats, intravenous secretin at 10 CU/kg per hour produced minimal choleretic effect,242 although secretin receptors are found in bile ductular cells. The plasma level of secretin achieved by this superpharmacological dose does not exist in physiological conditions of mammals or rodents.10,37,86,87,213,241 Alternatively, secretin cells have recently been found in the bile duct epithelium.243 It remains to be * 2014 Lippincott Williams & Wilkins

determined whether a high local secretin level can be achieved by the release from ductal S cells. A recent study with secretin receptor-deficient mice suggested that secretin might be involved in the modulation of proliferation of large cholangiocytes, suggesting that secretin/secretin receptor pathway may be of pathophysiological significance in hepatic ductopenia pathologies206 such as primary sclerosing cholangitis or primary biliary cirrhosis.

OTHER BIOLOGICAL ACTIONS OF SECRETIN Gastric Pepsin Secretion Secretin receptors were found in chief cells of guinea pigs244 and in canine chief cells in primary monolayer culture.245 In humans, pure natural secretin in a dose as little as 0.05 CU/kg per hour significantly increases both serum group I pepsinogen concentration and secretion of pepsin in unstimulated stomachs of young healthy volunteers.246,247 These studies strongly suggest that secretin, either exogenous or endogenous, is a physiological pepsinogogue. Because secretin and its mRNA are known to be present in gastric mucosa,113 a possible paracrine role of secretin on pepsinogen secretion remains to be elucidated. Thus, more convincing experiment to prove secretin’s role on pepsin secretion requires the use of an antisecretin serum for immunoneutralization, a potent secretin receptor antagonist, or tissue-specific secretin or secretin receptor knockout animal models in an appropriate experimental setting.

Gastric Mucus and Bicarbonate Secretion Gastric mucusYbicarbonate barrier is an important first line of defense against mucosal damage by acid and pepsin.248,249 Exogenous secretin increases sugars of mucus glycoproteins in the gastric juice of humans,250 dogs,251,252 and cats.253Y258 Secretin at 10 nM also stimulated mucin secretion from cultured rat gastric epithelial cells via a cAMP-mediated mechanism.259 In addition, secretin was found to stimulate gastric lipase secretion from dog gastric gland pit cells in parallel to mucus secretion.260 It is interesting to note that an intravenous secretin www.pancreasjournal.com

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at 0.3 CU/kg increased gastric bicarbonate secretion in the Heidenhain pouch of dogs.261 It is not certain, however, whether exogenous secretin in a physiological dose or endogenous secretin in a physiological setting stimulates the mucus and bicarbonate secretion.

Secretion of Brunner’s Glands Secretin in pharmacological doses was shown to stimulate Brunner’s glands’ secretion of fluid and bicarbonate in dogs.262Y264 In anesthetized rats with proximal duodenal pouch,265 pure natural porcine secretin given intravenously at as little as 15 ng (0.06 CU/kg/h) increased significantly both bicarbonate and protein output from the pouch and depleted the glands’ periodic acidSchiff stain-positive mucin. In rats, secretin at 50 pmol/kg per hour stimulated volume and epidermal growth factor secretion from Brunner’s gland that was inhibited by somatostatin.266 In anesthetized rabbits, duodenal acidification and exogenous secretin also stimulated Brunner’s gland secretion.267 It was suggested265 that secretin is a physiological stimulant of Brunner’s gland secretion. If their observation is confirmed in other species including dogs and humans, secretin is likely to play a significant role in the cytoprotection of the proximal duodenal mucosa as proposed recently.268

Is Secretin a Trophic Hormone? Secretin in superpharmacological doses was reported to produce both pancreatic hyperplasia and hypertrophy in rats.125,126 A recent study205 using both secretin- and secretin receptorYdeficient mice, however, showed no modification of the pancreatic weight and growth parameters including total protein and DNA and the protein/DNA ratio compared with those of control mice. Thus, in mice, secretin is not a growth hormone for exocrine pancreas. This is a novel finding, which needs to be confirmed in other animal species.

NEUROHORMONAL REGULATION ON THE RELEASE AND ACTION OF SECRETIN In rats,88,269 pancreatic secretion of fluid and bicarbonate in response to intravenous secretin in physiological doses, but not in pharmacological dose, is suppressed significantly by perivagal capsaicin application as well as mucosal application of proximal

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small intestine (Fig. 13) or subdiaphragmatic vagotomy.269 Similar observations were made with CCK on pancreatic enzyme secretion.270,271 These observations indicate that the action of secretin in physiological doses on exocrine pancreas is mainly mediated via vagal capsaicin-sensitive afferent pathway in the rat. Indeed, Li et al272 reported that secretin in physiological doses activates vagal primary afferent neurons in the rat, based on their electrophysiological and immunohistochemical studies, which showed increased c-Fos protein expression in nodose neurons. Moreover, secretin receptors are found on vagal afferent neurons and area postrema outside the blood-brain barrier.273 It is yet to be determined whether the action of secretin is mediated via vagal afferent pathway in other species. Several neurotransmitters and/or neuromodulators are known to influence the release and action of secretin. They are found in both vagal and adrenergic neurons in the proximal small intestine and the pancreas. They either stimulate or inhibit release and action of secretin (Table 4). These include, in addition to cholinergic and adrenergic transmitters, GRP and PACAP, enkephalins, somatostatin, galanin, serotonin, and nitric oxide, which are probably major parts of neurotransmitters/neuromodulators for the release and/or action of secretin and SRP. For example, in both a secretin cell-enriched preparation isolated from the rat upper small intestinal mucosa and the secretin-producing murine neuroendocrine cell line, STC-1, the release of secretin was stimulated by bombesin/GRP and PACAP-27 that was suppressed by galanin.274

Stimulants for Release and Action of Secretin Acetylcholine The release of secretin in response to either duodenal acidification or ingestion of a meal is not influenced by atropine in humans, dogs and rats, indicating that the release is not affected by cholinergic tone (Table 4). The pancreatic secretion of fluid and bicarbonate stimulated by intravenous secretin in a physiological dose or endogenous secretin is profoundly inhibited by atropine, as much as 80% in humans (Fig. 14),87 dogs,275Y277 and rats.269,278 In guinea pigs, sodium oleate-stimulated pancreatic secretion of bicarbonate and protein was completely blocked by atropine.187 These observations indicate that the action of secretin on pancreatic bicarbonate and fluid secretion from ductal cells

FIGURE 13. Effect of perivagal capsaicin (PVC) and proximal intestinal mucosal capsaicin (IMC) application on pancreatic secretion stimulated by intravenous secretin in rats. The doses of secretin were 0 (open bars), 5 (striped bars), and 10 pmol/kg per hour (solid bars). Both methods of capsaicin applications markedly suppressed the fluid volume (A) and bicarbonate output (B) stimulated by secretin at a physiological dose (5 pmol/kg/h) but not at a pharmacological dose (10 pmol/kg/h). Each value represents mean T SE of 7 rats in each group. *P G 0.05 compared with treatment without secretin. From Li et al.269

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TABLE 4. Neurotransmitter and Neuropeptide Modulators of Secretin Release and Pancreatic Secretion Stimulation or Inhibition of Pancreatic Secretion

Release of Secretin Fluid Bicarbonate Stimulators Acetyl choline VIP CCK-8 GRP/bombesin PACAP Serotonin Nitric oxide Inhibitors A-Adrenergic agonists Somatostatin Met-enkephalin SP Galanin Neuropeptide Y

Protein/ Enzyme

No No No Yes Yes Yes No

Yes Yes Yes Yes Yes Yes Yes

Yes Yes Yes Yes Yes Yes Yes

Yes Yes Yes Yes Yes Yes Yes

No

Yes

Yes

NT

Yes Yes NT Yes* NT

Yes Yes Yes Yes Yes

Yes Yes Yes Yes Yes

Yes Yes Yes Yes Yes

*Observed in STC-1 cells, in vitro. NT, not tested.

requires cholinergic tone. In anesthetized rats, however, the release and actiovn of SRP on secretin release was not sensitive to atropine or hexamethonium but highly dependent on vagal afferent sensory neurons.269 The pancreatic fluid and bicarbonate secretion stimulated by intravenous secretin in a physiological dose, 5 pmol/kg per hour, was also inhibited by perivagal treatment with capsaicin, whereas that stimulated by a pharmacological dose of secretin was not vagal dependent.269 The importance of vagal afferent pathway was not considered at the time ‘‘secretin’’ was discovered by Bayliss and Stahling in 1902.1 They used HCl in high concentration (0.4%) for the release of secretin and probably used crude secretin preparation in supraphysiological doses to evoke pancreatic secretion, which surpassed the influence of vagal afferent sensory neurons. In isolated guinea pig ductules, a low physiological concentration of secretin (1 pM) could not stimulate fluid secretion but was potentiated by 4 HM acetylcholine, which by itself was also unable to stimulate fluid secretion.279 In addition, in conscious and anesthetized rats, inhibition of pentagastrin-stimulated gastric acid secretion by both exogenous and endogenous secretin was abolished by perivagal capsaicin treatment but not by periceliac ganglionic capsaicin treatment, indicating that vagal afferent but not splanchnic afferent pathway mediates the action of secretin.280 In addition, inhibition of gastric motility at a physiological dose was also dependent on the vagal afferent pathway,281 and vagal input was found to modulate secretin binding site in rat forestomach.282 These observations indicate that the release and action of secretin is mediated via vagal afferent pathway in the rat. It is possible that secretin triggers vagal ascending sensory neurons, which signal vagal efferent cholinergic pathway to release acetylcholine, GRP, and/or CCK-8,283 which are stimulants of pancreatic ductal secretion of fluid and bicarbonate. Secretin alone at physiological concentration in plasma, such as 5 to 10 pM delivered to the pancreatic ductal cells may be a weak stimulant to drive adequate amount of bicarbonate rich isotonic fluid into the duct lumen. The * 2014 Lippincott Williams & Wilkins

local action of secretin is potentiated by CCK and acetylcholine in humans, dogs, guinea pigs, and rats.

Gastrin-Releasing Peptide In isolated perfused pig pancreas, GRP infused intraarterially increased significantly the secretion of fluid, bicarbonate, and protein.284 Porcine pancreas was shown to contain GRP in intrapancreatic nerves.285 In totally isolated perfused rat pancreas, electrical field stimulation produced a significant increase in portal venous immunoreactive GRP level.286 Gastrin-releasing peptide given intra-arterially exerted additive increase on secretinstimulated fluid and bicarbonate secretion and potentiated amylase secretion.286 In anesthetized pigs, intravenous infusion of GRP increased both plasma secretin concentration and pancreatobiliary secretion of fluid and bicarbonate287 but did not affect the release of secretin by duodenal acidification.288 In anesthetized rats, GRP given intravenously also increased plasma secretin level to stimulate pancreatic secretion of fluid and bicarbonate.289 When the circulating secretin was immunoneutralized with a rabbit antisecretin serum, the pancreatic secretion was markedly suppressed. Both GRP-stimulated release of secretin and pancreatic secretion of fluid and bicarbonate were significantly suppressed by subdiaphragmatic vagotomy, perivagal capsaicin application, and atropine, indicating that GRP-induced secretin release and pancreatic secretion are mediated via vagal innervation involving both afferent and efferent pathways. However, it is unclear under which physiological condition endogenous GRP is released and stimulates secretin release.

Pituitary Adenylate Cyclase-Activating Polypeptide This neuropeptide exists in 2 variants, PACAP-27 and PACAP-38. Pituitary adenylate cyclase-activating polypeptide is a potent stimulant of exocrine pancreatic secretion in rats, rabbits, pigs, and sheeps,290Y293 but in conscious dogs, both PACAP-27 and PACAP-38 are reported to be a weak stimulant of pancreaic fluid and bicarbonate secretion.294 This observation needs confirmation. In anesthetized rats, PACAP-27 given intravenously produced significant increases in both plasma levels of secretin and

FIGURE 14. Effect of atropine on pancreatic bicarbonate secretion (mean T SE) in response to intravenous administration of secretin in 5 healthy subjects. Secretin was given in 3 graded doses as indicated. Differences between values of the 2 experiments at each corresponding time period were statistically significant as indicated. From You et al.87 www.pancreasjournal.com

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CCK, which paralleled with significant increases in pancreatic secretion of fluid, bicarbonate, and protein. Immunoneutralization of secretin profoundly suppressed the secretion of fluid, bicarbonate, and protein, which were completely abolished by a combination of antisecretin serum and loxiglumide, suggesting that the effect of PACAP on pancreatic secretion is mediated by the releases of both secretin and CCK.295 Because PACAP is a potent stimulant of insulin release,296 pancreatic secretion stimulated by PACAP might also involve the release of insulin, which plays an important modulating role on actions of secretin and CCK (as will be discussed later). Because PACAP is expressed in myenteric plexus of the guinea pig duodenum,297 it would be interesting to determine if activation of these neurons will elicit secretin and CCK release. In isolated, perfused rat stomach, inhibition of pentagastrin-stimulated gastric acid secretion by PACAP-27 was reduced by 33% by an antisecretin serum, suggesting that the effect of PACAP was in part mediated by a release of the endogenous secretin.298

Serotonin (5-HT) In anesthetized rats, 5-HT2 receptor antagonist, ketanserin, and 5-HT3 receptor antagonist, ondansetron, dose-dependently inhibit the release of secretin and pancreatic secretion of fluid and bicarbonate elicited by duodenal acidification.299 The finding implies that serotonin participates both in the release mechanism of secretin as a paracrine messenger or neurotransmitter to secretin cells via 5-HT2 and 5-HT3 receptors and in the pancreatic secretion stimulated by endogenous secretin during duodenal acidification as well as administration of exogenous secretin.299

Nitric Oxide In conscious rats, the pancreatic secretion of fluid, bicarbonate, and protein stimulated by exogenous secretin, duodenal acidification, or a liquid meal (ENSURE) was dose-dependently inhibited by a nitric oxide (NO) synthase inhibitor, N-nitro-Larginine, and the inhibition was reversed by the substrate of the enzyme arginine. The release of secretin in these experiments, however, was not affected by N-nitro-L-arginine. The study implies that NO mediates the action of secretin on exocrine pancreas but the release of secretin is not.300 These observations made in the rat are consistent with the concept that the pancreatic secretion stimulated by secretin in physiological doses or endogenous secretin under physiological conditions is mediated through vagal afferent neural pathways

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that may involve mediation of the 2 known neuropeptides (GRP and PACAP) and other transmitters such as serotonin and NO. Again, whether pancreatic ductular cells are the primary targets for circulating endogenous secretin in physiological condition is not convincingly settled. In dogs, guinea pigs, rats, and humans, however, the action of secretin in physiological doses or endogenous secretin in physiological conditions is mediated via vagal efferent postganglionic cholinergic pathway in the pancreas, and thus, the secretin action is atropine sensitive. The mechanism associated with the role of capsaicin-sensitive vagal afferent pathway on the release and action of secretin is unknown. Nonetheless, secretin stimulates pancreatic fluid and bicarbonate secretion via vagovagal reflex, and the local action of secretin is potentiated by CCK and acetylcholine in the pancreas.

Insulin (Pancreatic Endocrine-Exocrine Axis) The exocrine pancreas is in close contact with pancreatic islets through the portal system.301 It was proposed that islet hormones reach the acinar cells at relatively high concentration to affect acinar function.302 Exogenous insulin was shown to potentiate CCK- and acetylcholine-stimulated amylase secretion in rats.303Y305 We found that endogenous insulin plays a pivotal role on exocrine pancreatic secretion of fluid and bicarbonate stimulated by secretin and CCK in rats40,306 and in totally isolated vascularly perfused canine pancreas preparations.307 In conscious rats, immunoneutralization of endogenous insulin with a high titer, specific rabbit anti-insulin serum given intravenously abolished the pancreatic secretion of fluid, bicarbonate (both concentration and output) (Fig. 15), and amylase stimulated by ingestion of a meal.40 Moreover, even basal secretion was significantly suppressed in the same experiment. Similarly, the pancreatic secretion stimulated by intravenous administration of secretin and CCK was completely blocked by the anti-insulin serum. This highly significant finding was confirmed in isolated, perfused pancreas preparations of both rats306 and dogs,307 in which pancreatic secretion of fluid, bicarbonate, and protein was stimulated by intra-arterial infusion of secretin and CCK in physiological doses (Fig. 16). Thus, endogenous insulin plays a pivotal role on pancreatic secretion of bicarbonaterich fluid stimulated by a meal or exogenous secretin and CCK as well as basal pancreatic secretion. Furthermore, after the antiinsulin serum administration, there was significant increases in concentrations of both immunoreactive somatostatin and PP in portal venous effluent in canine pancreas preparation307 and a similar increase in somatostatin level in the rat pancreas preparation,

FIGURE 15. Effect of anti-insulin serum and normal rabbit serum [NRS] on meal-stimulated pancreatic bicarbonate secretion in rats. Each data point indicate a mean T SE of bicarbonate output (A) and concentration (B). * and ** indicate significant difference from basal secretion at each time point with P G 0.05 and P G 0.01, respectively. From Lee et al.40

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afferent sensory neurons that signal vagal efferent, postganglionic cholinergic neurons in the pancreas, and the other is its direct action on pancreatic duct cells, which is probably potentiated by acetylcholine, CCK-8, and CCK-58. The 2 secretory mechanisms require paracrine or local action of insulin (Fig. 17).

Inhibitors of Secretin Release and Action Somatostatin and Met-enkephalin Somatostatin inhibits the release and action of secretin on exocrine pancreas in rats,310 dogs,311,312 and humans313 (Table 4). Met-enkephalin314 also inhibits acid-induced release of secretin and exocrine pancreatic secretion in rats314 and dogs.315 In humans, it inhibits pancreatic secretion elicited by sham feeding or exogenous secretin.316 The inhibition by Met-enkephalin of SRP release seems to be mediated by somatostatin.314 Thus, the inhibitory action by Met-enkephalin on the release of secretin may be mediated by local release of somatostatin, whereas the inhibition of exocrine pancreatic secretion may result from the inhibition of local release of acetylcholine.317Y319

Pancreatic Polypeptide Pancreatic polypeptide, a hormone first isolated from chicken pancreas,320 significantly inhibits postprandial and intravenous secretin and CCK-stimulated pancreatic secretion of both bicarbonate and protein in dogs. The postprandial pancreatic secretion was significantly augmented when circulating PP was immunoneutralized with a specific and high titer rabbit anti-PP serum. The inhibitory effect of PP on the pancreatic secretion stimulated by intravenous secretin and CCK was also reversed by the antiserum.321 FIGURE 16. Effect of normal rabbit serum, anti-insulin serum, or combination of anti-insulin, antisomatostatin, and anti-PP sera on pancreatic secretion of fluid volume, bicarbonate, and protein in response to intra-arterial infusion of secretin (S) + CCK-8 in isolated and perfused dog pancreas. The dose of S and CCK-8 were 2.5 ng/min each for 10 minutes administered under 3 conditions: background infusion of normal rabbit serum [NRS] (n = 6), anti-insulin serum [Anti-Ins] (n = 6), and Anti-Ins + anti-SS + anti-PP sera (n = 3). Anti-insulin significantly suppressed S + CCK-8Ystimulated pancreatic secretion of all 3 variables (*P G 0.05). When Anti-Ins was administered with anti-SS and anti-PP, the suppressed secretions of fluid and bicarbonate were completely reversed, but protein output was partially reversed (**P G 0.05). From Lee et al307 with permission.

which was the only peptide assayed.306 The simultaneous intraarterial administration of antisera of insulin, somatostatin, and PP307 reversed the marked suppression of pancreatic secretion of bicarbonate and protein stimulated by secretin and CCK (Fig. 16). The findings imply that the action of secretin and CCK on exocrine pancreatic secretion requires insulin as a local or paracrine hormone for both ductal cells as well as acinar cells. In addition, insulin seems a local regulator of somatostatin and PP release, and thus, insulin as a paracrine or local hormone may regulate local release of somatostatin and PP to regulate exocrine pancreatic secretion. This notion is supported by the presence of endocrine cells expressing insulin, somatostatin, and PP in pancreatic ducts.308,309 Based on the findings from isolated perfused pancreata of both rats and dogs, secretin and CCK in physiological doses seem to be capable of stimulating pancreatic secretion of bicarbonate-rich fluid and protein/enzymes without the influence of vagal afferent neurons and vagal efferent preganglionic cholinergic neurons. The study strongly suggests that the action of secretin on the pancreatic duct is mediated via 2 pathways, namely, the main pathway via vagal * 2014 Lippincott Williams & Wilkins

Peptide Tyrosine Tyrosinamide The plasma levels of PYY, which was first isolated from porcine intestine as a candidate hormone,322 increase postprandially. When circulating PYY was immunoneutralized with a specific rabbit anti-PYY serum, the postprandial pancreatic secretion of bicarbonate and protein was significantly augmented in conscious rats. The pancreatic secretion stimulated by exogenous secretin and CCK in physiological doses was also significantly inhibited by a physiological dose of PYY.323

Substance P In conscious dogs, SP given intravenously caused dosedependent inhibition of secretin-stimulated pancreatic secretion of fluid and bicarbonate and suppressed protein secretion stimulated by caerulein, feeding, and duodenal acidification. The inhibitory effect of SP occurred at a dose range that did not cause a significant effect on blood pressure.324 A similar inhibitory effect was found in isolated pancreatic duct preparation of guinea pig,325 rats, and mice.326

Galanin The inhibitory effect of galanin on pancreatic enzyme secretion is well established in rats.327 In dogs, galanin was shown to inhibit bicarbonate secretion stimulates by intravenous secretin, CCK, and a mixed meal.328 It also inhibited pancreatic protein secretion induced by the 3 stimulants.

Neural Regulation on the Release and Action of SRP The CAP prepared from donor rats treated with either tetrodotoxin (TTX), bilateral subdiaphragmatic vagotomy (BV) or perivagal or intestinal mucosal application of capsaicin (PVC or IMC) was unable to stimulate both the release of secretin and its www.pancreasjournal.com

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FIGURE 17. Proposed neurohormonal action of secretin. Secretin released from S cells in the duodenal mucosa enters the blood vessel as a hormone to reach the pancreatic duct. The released secretin also acts on local vagal afferent sensory fibers to send ascending signals through nodose ganglia to reach dorsal motor nucleus of vagus [DMV] in the brain stem, which in turn sends descending signals via vagal efferents to reach the pancreas to activate intrapancreatic postganglionic cholinergic neurons to release acetylcholine [Ach] and CCK-8 to potentiate the effect of hormonal secretin. Hormonal CCK (CCK-58) may also potentiate with the effect of secretin. In addition, locally released insulin provides a pivotal permissive role for hormonal actions of both secretin and CCK-58.

action on pancreatic secretion of fluid and bicarbonate in recipient rats (Fig. 13). Likewise, the CAP prepared from intact donor rats was unable to stimulate the release and action of secretin on the pancreatic secretion in the recipient rats pretreated with TTX, BV, or PVC/IMC. The findings indicate that the release and action of SRP are significantly inhibited under these experimental conditions with TTX, BV, or PVC/IMC.269 The capsaicin treatment caused approximately 75% reduction in the plasma secretin concentration and approximately 85% reduction in the bicarbonate output. It was also found that CAP from propranolol-treated donor rats produced approximately 50% decrease in the plasma secretin concentration and pancreatic bicarbonate secretion achieved by CAP of control rats, whereas treatment with phentolamine showed no effect. However, like atropine, neither of the 2 adrenergic blockers affected SRP-stimulated release of secretin or pancreatic bicarbonate secretion. Thus, in the rat, the release and action of SRP are under neural control, particularly by the capsaicin-sensitive vagal sensory neurons in the proximal small intestinal mucosal layer, and exocrine pancreatic secretion of fluid and bicarbonate is regulated by an interaction between neural and hormonal mechanisms.88,89,329Y331 In summary, the release and action of SRP are regulated by neural mechanisms. The vagal afferent pathway and sympathetic pathway via A-adrenergic receptors play an important role in the release of SRP. The release of secretin by SRP also depends heavily on the vagal afferent pathway. Thus, the vagal afferent pathway seems to play an important permissive role in the release of SRP as well as secretin release in rats. Hence, both release and action of SRP are also dependent on vagal-vagal reflex mechanisms similar to the neural regulatory pathway for the action of secretin as depicted in Figure 17, with the exception that a separate vagal efferent noncholinergic pathway may act on SRP-containing cells and S cells in the proximal intestinal mucosa. In addition, the local neuromodulators might

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be GRP, PACAP, VIP, Met-enkephalin, galanin, and 5-HT released from vagal efferent terminals or local neurons receiving vagal efferent signal. The future investigations will unravel their physiological relevance.

PATHOPHYSIOLOGY OF SECRETIN Hypersecretinemia Pancreatic Hypersecretion of Fluid and Bicarbonate in a Patient With Verner-Morrison (Pancreatic Cholera) Syndrome A marked hypersecretinemia with plasma secretin concentration of greater than 5000 pg/mL was found in a 27-year-old white male with a non-beta islet cell carcinoma of the pancreas and extensive metastases including the liver.332 His main symptoms were massive watery diarrhea ranging up to 7 to 14 L/d, hypokalemia, dehydration, and 25 lbs of weight loss in 6 months. Repeated stool water analysis revealed large loss of bicarbonate with a pH of 8.79; sodium 45, potassium 104, chloride 44, HCO3 104 mEq/L, and osmolality of 270 mOsm. Aspiration of duodenal fluid in fasting state revealed hourly volume and bicarbonate output of 557 mL (approximately Q13,000 mL per 24 hours) and 54.9 mEq (approximately Q1300 mEq per 24 hours), respectively, which exceeded far above the secretin-stimulated pancreatic secretion in healthy subjects and patients with duodenal ulcer (G300 mL/h). In response to intravenous bolus injection of GIH secretin at 1 CU/kg, the increases in both volume and bicarbonate output were no greater than 20% above the basal values, indicating that his pancreas was at a near-maximum secretory state before a pharmacological dose of secretin was administered intravenously to determine the post-secretin pancreatic secretion of fluid and bicarbonate. This pancreatic exocrine hypersecretory pattern was similar to that of gastric acid * 2014 Lippincott Williams & Wilkins

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hypersecretion in patients with gastrinoma (Zollinger-Ellison) syndrome333 in which basal acid hypersecretion is at near-maximum secretory state, so that the maximum acid output produced by either intravenous human synthetic gastrin or percutaneous injection of betazole hydrochloride (Hsitalog) is usually insignificantly higher than basal acid output.334 Continuous nasogastric and duodenal aspiration had resulted in marked decreases in daily stool volume to less than 1000 mL, and massive diarrhea stopped. The immunofluorescence study with specific, high titer rabbit antisecretin serum81,82 revealed numerous fluorescing cells in a frozen hepatic metastatic tumor tissue.331 The tumor tissue contained 10-ng/g immunoreactive secretin. His plasma VIP was marginally elevated, and blood serotonin concentration was 2.3 Hg/mL before and 1.8 Hg/mL after 10 Hg of intravenous epinephrine. Neither VIP nor serotonin is a potent stimulant of pancreatic exocrine secretion of fluid and bicarbonate in humans. Thus, the patient had a marked hypersecretion of exocrine pancreas consisting mainly of fluid and bicarbonate driven by a very high secretin level in plasma, resulting in massive watery diarrhea and dehydration. This condition is indeed consistent with a true ‘‘pancreatic cholera syndrome.’’335 In diagnostic workup for watery diarrhea syndrome or Verner-Morrison syndrome334Y336 therefore, the measurement of plasma secretin level is as important as other diarrheal hormones such as VIP and serotonin. In patients with Zollinger-Ellison syndrome, fasting plasma level of secretin (usually 920 pg/mL) is significantly higher than that of healthy subjects or most of patients with duodenal ulcer (G10 pg/mL),337,338 provided that H2 receptor blocker or proton pump inhibitor therapy is temporally discontinued for several days. Some patients with duodenal ulcer with gastric acid hypersecretion were also found with elevated secretin levels, which are usually less than 20 pg/mL. The secretin levels are elevated in patients with advanced renal failure, probably attributable to impaired clearance of secretin by the kidney.337,339 Plasma secretin levels were also elevated in anephric rats.340 However, none of these patients or rats exhibited hypersecretion of pancreatic juice.

Hyposecretinemia There are 2 clinical conditions in which hyposecretinemia is found. Patients with untreated celiac disease341Y344 fail to exhibit increase in plasma level of secretin in response to duodenal acidification with HCl solution and ingestion of a mixed meal.344 It was reported that mucosal inflammation and atrophy of the upper small intestine result in significant loss of S-cell population in adult celiacs.341 Thus, the malabsorption in this clinical condition involves both intestinal malabsorption and potential maldigestion associated with some degree of exocrine pancreatic insufficiency because the major driving force of pancreatic secretion of fluid, which transports pancreatic enzymes to the upper small intestine, is circulating endogenous secretin.9 As the mucosal abnormality improves with gluten-free diet, the patients display normal postprandial release of secretin342 and have significant rise in plasma secretin concentration in response to duodenal acidification, which paralleled a significant increase in bicarbonate output.343 No significant postprandial increase in plasma secretin level was observed in patients with achlorhydria.342 When their duodenum was acidified with diluted HCl solution, plasma secretin level significantly increased. Because digestion products of fat in the upper small intestine stimulate the release of secretin in humans,165 dogs,167 and rats,168 a mild increase in circulating secretin may still exert pancreatic secretion of fluid and bicarbonate in association with circulating CCK released by digestion products of fat and protein in diet, which potentiates the action * 2014 Lippincott Williams & Wilkins

Secretin, A Historical Review

of secretin.37Y40 A minute postprandial increase in plasma secretin level by digestive products in achlorhydric patients may be difficult to detect with the available radioimmunoassay method of secretin. Yet, such a small rise in the secretin level in potentiation with circulating CCK may produce a sufficient quantity of pancreatic juice for digestion. Further studies are needed to clarify the hormonal mechanism of exocrine pancreatic secretion in these patients.

Clinical Use of Secretin Secretin or Secretin-CCK Test Secretin has been used for clinical investigation and diagnosis of pancreatic diseases using a double-lumen tube to collect separately duodenal juice and gastric juice since 1930s344Y349 when Lagero¨f (Supplemental Figure 4 http://links.lww.com/MPA/A254) introduced secretin test in 1930s.350 The test is still the most reliable for clinical diagnosis of chronic pancreatitis. Human synthetic secretin has been available for clinical use since early 2000s. Because the test is laborious and time consuming and requires fluoroscopy and technical expertise, besides patient’s discomfort, 2 relatively new tests using secretin were introduced, including ‘‘intraductal secretin test’’351 and ‘‘endoscopic secretin test.’’352 It seems that the result of exocrine pancreatic secretion of bicarbonate with endoscopic secretin test parallel that with the conventional secretin test using a double-lumen tube (Dreiling tube)347 (Late Dr David Dreiling at Mount Sinai Hospital New York probably performed the largest number of secretin test in the world in the 1950s. Using his designed double-lumen tube, he often performed secretin test on 2 patients simultaneously in his G.I. Laboratory. I had a pleasure and honor to observe his performance and discuss with him the test results and physiology of pancreatic secretion in 1956 to 1957.)

Secretin Provocation Test for Zollinger-Ellison (Gastrinoma) Syndrome Secretin inhibits gastric acid secretion in humans11,212,215 and inhibits gastrin release in humans353 and dogs.11 In 1973,

FIGURE 18. Effect of intravenous secretin (2 CU/kg) on plasma gastrin concentration and gastric acid secretion in a patient with gastric acid hypersecretion, duodenal ulcer disease, and nonbeta islet cell cancer of the pancreas (Zollinger-Ellison syndrome). After secretin injection, both plasma gastrin concentration and the acid secretion markedly increased. From Chey et al356 with permission. www.pancreasjournal.com

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Isenberg et al354 found significant paradoxical increases in both gastric acid secretion and serum gastrin concentration in a patient with Zollinger-Ellison syndrome. This novel finding was confirmed in many patients with the syndrome.355 The secretinstimulated gastrin increase occurs exclusively in this syndrome. Thus, the test has been recognized as the most sensitive and specific diagnostic method. The dose of pure porcine secretin used for the test was 1 to 2 CU/kg given intravenously. Synthetic human secretin (ChiRhoStim) is now used at a dose of 0.4 Hg/kg given intravenously over 1 minute. The test result is positive when serum gastrin level increases from the basal level to a level of more than 110 pg/mL (Fig. 18). The test is not necessary if fasting serum gastrin level is greater than 1000 pg/mL with proven gastric acid hypersecretion in the fasting state.355,356 However, the gastric acid hypersecretion still remains as the cardinal pathognomonic marker for this syndrome because in some patients, the secretin provocation test result may not be positive with normal serum or plasma gastrin concentrations.357Y360

Secretin-Enhanced MRCP The test has been developed to examine both morphological abnormalities of pancreatobiliary duct system and exocrine pancreatic function in patients with chronic pancreatic diseases, particularly chronic pancreatitis.361Y364 The pancreatic secretory volume was measured from the fluid accumulated in the duodenum for 10 minutes of postsecretin stimulation period (Fig. 19). The measurement of pancreatic juice volume should be nearly accurate without significant loss of the fluid to the jejunum because it has been known that secretin given intravenously inhibits the motility of the duodenum and jejunum365Y367 and minimizes the rapid escape of pancreatic juice from the duodenum. Although secretin-enhanced MRCP cannot match the subtle ductal abnormalities found by endoscopic retrograde cholangiopancreatography (ERCP), ductal distention by T2 bright fluid after secretin stimulation provides more complete visualization of the main pancreatic duct, the duct of Santorini, and side branches, compared with conventional MRCP without secretin stimulation. It seems that secretin-enhanced MRCP has an advantage over ERCP because of inability of ERCP to delineate the duct proximal to a very severe stenosis,363 not to mention the risk of post-ERCP pancreatitis. Sanyal et al363 reported recently that among the 134 patients they investigated, there was a significant association between the clinical groups, including normal, equivocal, early pancreatitis, and established pancreatitis and volume measurement (P = 0.0003). Mean volumes were progressively smaller with increasing severity of pancreatitis (mean volume of 57.3 mL in normal vs

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22.1 mL in established pancreatitis). Thus, significant volume differences were found between the normal and the established pancreatitis groups as well as the equivocal and the established pancreatitis groups. Marginally significant differences were also found between the normal and the early pancreatitis groups as well as the early and the established pancreatitis groups. Thus, a highly significant correlation was found between the clinical groups and the volume measurement on secretin-enhanced MRCP (r = j0.324, P G 0.0001). They further found in 65 patients that the maximum bicarbonate concentrations obtained on endoscopic secretin test showed a statistically significant correlation with volume measurements on secretin-enhanced MRCP (r = 0.270, P = 0.0017). If their observations were confirmed in a larger number of a comparable group of patients with chronic pancreatitis, secretin-enhanced MRCP could be a useful test for the diagnosis of early or mild chronic pancreatitis.

Therapeutic Use of Secretin Autistic Spectrum Disorders The efficacy of intravenous synthetic human secretin for the treatment of autistic spectrum disorders (ASDs) had been investigated extensively in multiple randomized controlled trials since 1998.368 The evidence from 7 randomized controlled trials supports a lack of effectiveness of secretin for the treatment of ASD symptoms including language and communication impairment, symptom severity, as well as cognitive and social skill deficits. No significant improvements in the measures of language, cognition, or autistic symptoms were observed.369 Thus, secretin is not recommended for the treatment of ASD in the year 2013. Nevertheless, secretin may be of use in a subgroup of ASD, as secretin was found effective in some patients with treatment-resistant schizophrenia.370,371 Further clinical studies are needed.

Potential Use of Secretin for Relief of Refractory Pain in Patients With Chronic Pancreatitis There have been 2 studies evaluating the efficacy of secretin for the relief of type B pain,372 severe unremitting pain, in patients with chronic pancreatitis.373,374 The rationale of using secretin for pain is that increasing bicarbonate-rich fluid secretion from pancreatic ductular cells stimulated by secretin in a therapeutic dose may reduce intraductal viscosity, thereby may reduce ductal pressure by flushing out viscous fluid from the ductal system. Thus, ductal hypertension may decrease to improve the pain symptoms. In a placebo-controlled study, 20 patients with chronic recurrent pancreatitis received daily subcutaneous injections of controlled depot porcine secretin at a dose of 800 CU or 160 Hg

FIGURE 19. Secretin-enhanced MRCP with quantification of exocrine function. Postsecretin coronal HASTE image shows duodenum distended with T2 bright fluid accumulation (arrows) (A) in a 43-year-old patient with normal pancreatic function and a collapsed duodenum (arrows) (B) in presecretin injection period of the same patient. From Sanyal et al363 with permission and courtesy of Drs Joseph C. Vaniero and Tyler Steven who provided the pictures. Reprinted with permission form the American Journal of Roentgenology.

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for 7 days. Pain levels were significantly reduced after secretin treatment compared with placebo, as pancreatic juice viscosity, lactoferrin, and trypsin decreased.372 Recently, a phase II trial of human synthetic secretin (ChiRhoStim) for refractory type B pain was performed in 12 patients with chronic pancreatitis, and 11 patients completed the study.372 The patients received 3 intravenous bolus injections of secretin at 3 different doses (0.05Y0.8 Hg/kg), 2 hours apart for 3 days. During 90 days of monitoring, the study did not find a statistically significant difference in pain scores or opioid use, but there was a strong trend toward improvement in both outcome measures. It was of interest to note that women seemed to respond more than male. It is desirable to expand the study in a larger number of patients with chronic pancreatitis with agonizing pain. One additional benefit of secretin may be that secretin may relax pancreatic sphincter tone because secretin does suppress duodenal contractile activity.365,366

SUMMARY AND CONCLUSIONS Since the discovery of a hypothetic hormone, secretin in 1902, it took 60 years to have it purified and sequenced, and another 16 years before its decisive hormonal status was established in the dog, pig, guinea pig, and rat. With the advent of radioimmunoassay methodology of secretin and subsequent cloning of secretin gene, secretin is found to distribute widely in many tissues and organs, with highest concentration found in the upper small intestinal mucosa. The secretin receptor has also been cloned and extensively studied for its interaction with secretin and the development of potent receptor antagonist. Both secretin and secretin receptor knockout mouse models have also been developed and should be useful for future study of the physiological roles of secretin not only in the gastrointestinal tract but also in other tissues/organs.

Physiological Significance of Circulating Endogenous Secretin When circulating free secretin was eliminated with a highly specific and high titer antisecretin serum, the postprandial pancreatic secretion of bicarbonate-rich fluid was markedly suppressed, whereas gastric acid secretion and emptying and the release of gastrin were augmented. Thus, the genuine physiological action of circulating secretin is, indeed, its stimulation of exocrine pancreatic secretion of bicarbonate-rich fluid that transports pancreatic enzymes to the duodenum and modulation of gastric motility and acid secretion. Works remain to be done to determine its potential physiological actions on biliary and intestinal secretions and the motility of remaining gastrointestinal tract.

Exocrine Pancreas Needs Both Secretin and CCK for Secretion of Fluid and Bicarbonate The meal-stimulated pancreatic secretion of fluid and bicarbonate is mainly the net result of potentiation between secretin and CCK. Therefore, when 1 of the 2 hormones is either eliminated from circulation (secretin) or action blocked by a receptor antagonist (CCK), postprandial pancreatic secretion of bicarbonate-rich fluid is virtually abolished, indicating that both secretin and CCK are essential for secretion of fluid and bicarbonate in physiological conditions.

Role of Secretin in Feedback Regulation of Pancreatic Secretion and Discovery of SRP Negative feedback mechanism by pancreatic trypsin/ chymotrypsin in the duodenum of exocrine pancreatic * 2014 Lippincott Williams & Wilkins

Secretin, A Historical Review

secretion including both bicarbonate and protein/enzymes involves not only CCK but also secretin. Secretin is released by SRPs that are found in both acid perfusate of the proximal small intestine in rats and dogs and canine pancreatic juice (positive feedback). One of them is PLA2.

Neurohormonal Regulation of the Release and Action of SRP and Secretin The release and action of SRP are mediated through vagal afferent sensory neurons and in part adrenergic neurons via A-adrenergic receptor. Future research will determine if they exist in the duodenal lumen in other species and are operative in postprandial state or physiological conditions other than duodenal acidification. It is highly likely that they are involved in the negative and positive feedback mechanisms of exocrine pancreatic secretion. The release and action of secretin are also regulated by neural and hormonal mechanisms. Secretin in physiological dose activates vagal afferent neurons to express c-Fos protein and secretin receptors. When vagal afferent nerve is inactivated by capsaicin, both release and action of secretin are markedly suppressed, and action of secretin on pancreatic fluid and bicarbonate secretion is inhibited by atropine, indicating dependence on cholinergic tone. Thus, the release and action of secretin are heavily dependent on vagal afferent capsaicin-sensitive neurons and efferent cholinergic neurons in the pancreas. Many neuropeptides, which are found in the neurons in the small intestinal mucosa and pancreas, either stimulate or inhibit the release and/or action of secretin (Table 4). It is likely that these neuropeptides are released from the nerve endings of vagal afferent and efferent pathways to affect the release and action of secretin, in particular, PACAP and GRP for the stimulation as well as enkephalins, SP, and galanin for the inhibition of secretin release. Exocrine pancreatic secretion stimulated by a meal as well as exogenous secretin and CCK is completely abolished by immunoneutralization of insulin, indicating that insulin plays a pivotal permissive role on the action of secretin/CCK on the exocrine pancreas. The mechanism of insulin action involves, at least in part, its tonic suppression on local release of somatostatin and PP. Other neuropeptide found in the nerve network of the pancreas such as galanin, pancreastatin, SP, PACAP, GRP, VIP, CGRP, and neuropeptide Y may also be involved in the mechanism.

Pathophysiology and Clinical Use of Secretin Hypersecretinemia and hyposecretinemia are found in various clinical conditions. A marked hypersecretinemia may be found in some patients with Verner-Morrison syndrome associated with massive pancreatic hypersecretion of water and bicarbonate. Radioimmunoassay of secretin in plasma is necessary for the diagnosis of non-beta islet cell tumor of the pancreas secreting excessive secretin without elevated plasma VIP or serotonin. Secretin in pharmacological doses is being investigated for a possible relief of chronic pancreatitisYassociated severe pain. The development of secretin-enhanced MRCP combined with pancreatic function test may become a useful diagnostic modality for chronic pancreatitis.

Idea for Future Secretin Research 1. Development of highly specific and highly sensitive radioimmunoassay of secretin, secretin-Gly95,98 and secretin Gly-Lys-Arg79,96,98 capable of measuring secretin in the concentration of fentamol range. One must rule out the possibility of more than one secretin in circulation. www.pancreasjournal.com

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2. Development of specific and potent secretin receptor antagonists and tissue/organ-specific secretin or secretin receptor knockout mouse or rat models for many physiological and pathophysiological investigations. 3. Investigations on the combined effect of secretin and CCK in physiological doses on gastric secretory (including HCl, pepsinogen, mucus, and bicarbonate) and motor functions. 4. Physiological roles of secretin and the combination of secretin and CCK on the motility of gastrointestinal tract. 5. Physiological roles of secretin and CCK on biliary duct secretion. 6. Further investigations on SRP include purification, structural determination, development of synthetic SRPs and development of radioimmunoassay to measure immunoreactive SRPs in the duodenal secretion and pancreatic juice, and identification of SRP cells in the intestinal mucosa and pancreas including the pancreatic duct. 7. Investigations on neurohormonal interaction between SRP or secretin and vagovagal reflex pathways, in particular in the duodenum for the identification of the neurotransmitters or neuropeptides involved.

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8. Clinical investigations to improve the diagnosis of chronic pancreatic diseases, using human synthetic secretin; (1) exocrine pancreatic function test with a combination of secretin and CCK in physiological doses and (2) further assessment of secretin-enhanced MRCP combined with pancreatic function test. 9. Search for watery diarrhea syndrome without VIP or serotonin elevation in plasma. Some may have ‘‘secretinoma’’ of the pancreas. ACKNOWLEDGMENT The authors express their heartfelt appreciation for the enthusiastic support of Alvin L. Ureles, MD, professor emeritus of medicine, University of Rochester School of Medicine and Dentistry, and former physician-in-chief at the Genesee Hospital, Rochester, NY. REFERENCE References are available online at: http://links.lww.com/MPA/A287.

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Secretin: historical perspective and current status.

This review describes the history of secretin discovery, identification, purification, and structural determination; cloning of secretin and its recep...
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