Signaling mechanisms of secretin receptor

Regulatory Peptides 137 (2006) 95 – 104 www.elsevier.com/locate/regpep

Review

Signaling mechanisms of secretin receptor Francis K.Y. Siu, Ian P.Y. Lam, Jessica Y.S. Chu, Billy K.C. Chow ⁎ Department of Zoology, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, PR China Received 10 January 2006; received in revised form 14 February 2006; accepted 27 February 2006 Available online 22 August 2006

Abstract Secretin, a 27-amino acid gastrointestinal peptide, was initially discovered based on its activities in stimulating pancreatic juice. In the past 20 years, secretin was demonstrated to exhibit pleiotropic functions in many different tissues and more importantly, its role as a neuropeptide was substantiated. To carry out its activities in the central nervous system and in peripheral organs, secretin interacts specifically with one known receptor. Secretin receptor, a member of guanine nucleotide-binding protein (G protein)-coupled receptor (GPCR) in the secretin/VIP/glucagon subfamily, possesses the characteristics of GPCR with seven conserved transmembrane domains, a relatively large amino-terminal extracellular domain and an intracellular carboxyl terminus. The structural features and signal transduction pathways of the secretin receptor in various tissues are reviewed in this article. © 2006 Elsevier B.V. All rights reserved. Keywords: Secretin receptor; Signal transduction; Pathway; Structure; cAMP; IP3

Contents 1. 2. 3.

Introduction . . . . . . . . . . . . Ligand binding . . . . . . . . . . Signal transduction pathways . . . 3.1. The central nervous system 3.2. Heart. . . . . . . . . . . . 3.3. Lung. . . . . . . . . . . . 3.4. Stomach . . . . . . . . . . 3.5. Liver. . . . . . . . . . . . 3.6. Intestine . . . . . . . . . . 3.7. Pancreas . . . . . . . . . . 3.8. Kidney. . . . . . . . . . . 3.9. Male reproductive system . 4. Receptor desensitization . . . . . 5. Summary . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . References . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

. . . . . . . . . .

95 96 96 96 98 98 98 98 99 99 100 100 101 101 101 101

1. Introduction

⁎ Corresponding author. Tel.: +852 2299 0850; fax: +852 2857 4672. E-mail address: [email protected] (B.K.C. Chow). 0167-0115/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2006.02.011

Secretin carries out gastrointestinal [1–4] and neuronal functions [5] via its specific interactions with a cell surface receptor, the secretin receptor. Secretin receptor together with receptors

96

F.K.Y. Siu et al. / Regulatory Peptides 137 (2006) 95–104

for other peptides in the same gene family including glucagon, glucagon-like peptides (GLP-1, GLP-2), glucose-dependent insulinotropic polypeptide (GIP), vasoactive intestinal peptide (VIP), pituitary adenylate cyclase-activating polypeptide (PACAP) and growth hormone-releasing hormone (GRF or GHRH) [6], is grouped in the B1 subclass in the G protein-coupled receptor (GPCR) superfamily. Rat secretin receptor cDNA was the first receptor cloned in the B1 subclass from rat/mouse hybrid NG108-15 cells by the expression cloning technique [7]. Later on, several identical human secretin receptor cDNAs were characterized from pancreatic adenocarcinoma cells and lung tissue [8–10]. The human secretin receptor contains 440 amino acids with a putative hydrophobic leader peptide (22 amino acids), a hydrophilic amino-terminal extracellular domain (122 amino acids), 7 transmembrane domains with 3 exo- and 3 endoloops (254 amino acids) and a carboxyl-terminal cytoplasmic tail (42 amino acids). It has been shown that the N-terminal extracellular domain and the first exoloop collectively constitute the ligand binding site [11–13]. The transmembrane domains that are highly conserved among members within the same family are involved in maintaining the structural conformation of the receptor in the lipid bilayer [14,15] while the third endoloop is responsible for G protein coupling and signal transduction [16]. Secretin receptor is highly expressed in both pancreatic acinar and ductal epithelial cells while in extremely low or undetectable levels within the islets and pancreatic vascular structures [17]. In liver, receptor transcripts are exclusively found in cholangiocytes [18]. In stomach, secretin receptor is present in the circular and longitudinal smooth muscle layers of the proximal nonglandular forestomach [19], on the fundic membranes [20], in the antral parts of the gastric mucosa [21] and on the vagal afferent fibers innervating the forestomach [22]. In the brain, expression of secretin receptor is high in the cerebellum, intermediate in the cortex, thalamus, striatum, hippocampus and hypothalamus while low in the midbrain, medulla, and pons [5,23,24]. Recent study by Nozaki S et al. demonstrated that secretin receptor is also expressed in the nucleus of the solitary tract (NTS), accumbens nucleus, lateral septal nucleus, olfactory bulb, amygdale, pineal gland, caudate/ putamen, pituitary; and also in cingulate, piriform, frontal, parietal, entorhinal, and orbital cortices [25]. In reproductive system, secretin receptor transcripts and receptor-like signals were observed throughout the epithelium of caput and cauda epididymis [26]. Additionally, these signals have also been located on the smooth musculature of the intestine as well as in the colon [27]. Secretin receptor transcripts and specific binding sites were also shown in heart [7], lung [28] and outer medulla of the kidney [29], clearly indicating a wide spectrum of biological functions played by secretin. The binding of secretin to its receptor triggers the activation of intracellular secondary messenger systems and hence various cellular processes [30]. Recently, it was shown that secretin could inhibit cell cycling and the binding of secretin with a dominant-negative receptor splice variant could lead to pancreatic carcinogenesis [31]. In summary, the understanding of the cellular mechanisms arising from secretin–receptor interaction

is crucial to future investigation of the physiology and pathophysiology of secretin. 2. Ligand binding By constructing chimeric secretin/VPAC1 receptors, the amino termini and the first exoloops of these receptors were found critical for ligand binding [11,12]. Within these domains, the first 10 amino acid residues as well as Lys173, Asp174, Arg166, His189 and Cys190 in the first extracellular loop were found important. In addition, Phe257, Leu258, Asn260 and Thr261 in the second extracellular domain also contributed to ligand interaction [13,32,33]. On the other hand, mutation of Cys11, Cys186, Cys193 or Cys263 to Serine residue in the rat secretin receptor resulted in a reduced affinity for secretin, suggesting that the disulphide linkages involving Cys11, Cys186, Cys193 and Cys263 played a role in maintaining the structural conformation of the receptor [34]. In addition, glycosylation of the high mannose-type carbohydrate side-chains of the receptor was found important for conformational integrity that led to ligand interactions; in the human secretin receptortransfected CHO cells, addition of tunicamycin and castanospermine, inhibitors for adding the core and high mannose-type sugars resulted in defective receptor functions. By mutation studies coupled to binding and confocal analyses, Asn72 was found to be the functional N-linked glycosylation site as mutation of Asn to Leu did not affect presentation of the mutant to the cell surface but still led to defective binding to iodinated secretin [35]. 3. Signal transduction pathways It is known that all the members in the B1 family of GPCR are capable of initiating intracellular accumulation of cAMP by coupling to adenylate cyclase via the Gs protein (Fig. 1) [6,36]. It was previously believed that the cAMP pathway was the sole signaling mechanism that gave rise to amylase release from pancreatic acinar cells and secretin had no effect on Ca2+ mobilization [37]. However, at high concentrations of secretin, cAMP accumulation was found maximum while amylase release was only half-maximum and there was a transient increase in intracellular Ca2+ [38]. These observations suggested the presence of an alternative signaling pathway and it was later demonstrated that secretin could also stimulate inositol triphosphate (IP3), intracellular calcium and diacylglycerol (DAG) pathways in pancreatic acinar cells (Fig. 1) [38–40]. The following is a detailed account of the signal transduction mechanisms and the functions of secretin in various tissues of our body. 3.1. The central nervous system The secretin receptor cAMP signaling pathway in neuronal cells was first demonstrated in rat/mouse neuroblastoma– glioma hybrid NG108-15 cells [41]. Similar results were obtained in subsequent studies by stimulating either the hybrid cells directly or receptor-transfected CHO or COS cells

F.K.Y. Siu et al. / Regulatory Peptides 137 (2006) 95–104

97

Fig. 1. Schematic diagram of the secretin receptor cAMP (left) and PI (right) signal transduction pathways. cAMP pathway: Ligand and receptor binding triggers the GDP–GTP exchange of the Gs protein and the dissociation of the α-subunit which functions to activate AC. AC catalyzes the synthesis of cAMP from ATP, and cAMP elicits its cellular responses by the phosphorylation of a number of cellular proteins via cAMP-dependent protein kinase (PKA). PI pathway: Ligand and receptor binding is coupled to Gq protein. Dissociation of the α-subunit activates phospholipase Cβ, which cleaves PIP2 into DAG and IP3. DAG activates PKC which in turn phosphorylates cellular proteins. IP3 causes the release of Ca2+ from endoplasmic reticulum. The released Ca2+ leads to exocytosis and various other cellular responses. Abbreviations: cAMP: cyclic adenosine 5′-monophosphate; GDP: guanosine 5′-biphosphate; GTP: guanosine 5′-triphosphate; AC: adenylate cyclase; DAG: diacylglycerol; IP3: inositol-1,4,5-triphosphate; PIP2: phosphatidylinositol-4,5-bisphosphate; PLC-β: phospholipase C-β; PKC: protein kinase C; ER: endoplasmic reticulum.

[7,42,43]. In addition, the secretin receptor cAMP response was also studied in cultured mouse glioblast [44], mouse neuroblastoma C1300 [45], rat frontal cortex slice [46], mouse embryonic striatal neurons [47], mouse embryonic glial cells [47], rat neuroblastoma PC12 cells [48], rat hypothalamus [49], rat hippocampus [49] and rat cerebellum [5]. The secretin receptor that activated cAMP production in different brain regions established the diverse potential functions of secretin in the brain. It was shown that secretin could increase glutamate and GABA levels in the hippocampus [50]. The effects of secretin-stimulated cAMP formation via its receptor were also implicated in sympathetic nerve terminals by regulating neuronal tyrosine hydroxylase (TH) activities. For instance, secretin in the median eminence could elevate dopamine turnover

to alter prolactin and growth hormone plasma levels [51,52]. Besides, in the superior cervical ganglion (SCG), secretin could also act directly to potentiate TH activities, resulting in the activation of autonomic end organs innervated by SCG neurons such as the iris, submaxillary gland and pineal gland [53,54]. Other autonomic end organs like the right ventricle of the heart which is innervated by the middle and inferior cervical ganglia were also strongly correlated with secretin-induced TH activities [55]. Secretin could, mediated by cAMP, activate TH neurons in the NTS through a voltage-independent membrane channel, non-selective cationic conductance (NSCC) [56,57]. In the cerebellum, the depolarization-evoked release of secretin from the somatodendritic area of Purkinje cells serves as a retrograde messenger, interacting with receptors on the

98

F.K.Y. Siu et al. / Regulatory Peptides 137 (2006) 95–104

presynaptic basket cells, to increase the firing of GABAergic inhibitory signals. This increase in the probability of vesicular fusion of GABA vesicles is action potential-independent but is cAMP-dependent (Fig. 2) [5,58]. In addition, secretin could also activate glutamate release in the cerebellum in such a way that glutamate could act on AMPA receptors on basket cells to facilitate GABA release, and this action of secretin is similar to the direct activation of secretin receptors in the same terminal [59]. 3.2. Heart The effect of secretin on cAMP responsiveness in mammalian heart was demonstrated in rat heart disease models. In spontaneous hypertensive rat heart, responsiveness of cardiac secretin-stimulated adenylate cyclase was reduced by 50% to 80% in different reports [60–62]. In obese rat heart, secretinstimulated adenylate cyclase was significantly reduced by 60% to 90% [63,64]. Similar results were also obtained in hypertrophic rat heart. The reduced number of secretin receptors in the cardiac cells was suggested to be responsible for the reduced responsiveness of adenylate cyclase to secretin [65–67]. In human, cardiac output upon secretin stimulation was increased by an average of 20%; the stroke volume was also increased while the total systemic resistance decreased. However, systemic arterial pressure, heart rate and pulmonary capillary wedge pressure were unaltered [68]. In rat, secretin could affect heart rate and contractility [69,70]. In dog, secretin increased cardiac output and heart rate, while it decreased systemic arteriolar resistance and left ventricular end-diastolic pressure, with no significant change in stroke volume [71]. Although contradictory data were obtained in different species, secretin, however, has a potential in treating heart failure by increasing cardiac output and heart rate as well as decreasing systemic resistance [68]. 3.3. Lung Upon secretin stimulation, adenylate cyclase activation was demonstrated in the crude fractions prepared from rat and human lung tissues [28,72]. However, when using human lung cells Calu-1 and SW-900, the potencies of secretin to stimulate adenylate cyclase activity were several thousand times lower than those of VIP [73]. Despite its low potency, secretin could still significantly increase the bombesin/gastrin releasing peptide-like immunoreactivity (BLI) levels in small cell carcinoma of lung (SSCL) cells NCI-H345 and NCI-H209. More importantly, when secretin is i.v. injected into SSCL patients, drastic increase of BLI was obtained, probably due to the high expression level of secretin receptor in SSCL cells [74]. Furthermore, the secretinstimulated release of BLI leads to binding of the latter peptide to its cell surface receptor to activate cytosolic Ca2+ mobilization via phosphatidylinositol (PI) turnover [75]. A recent study has demonstrated the secretin receptor expression in specific lung regions. By using real-time quantitative PCR, in situ hybridization and immunohistochemistry, the receptor was found abundantly in tertiary bronchus, bronchial epithelial layer and some expression

in bronchial smooth muscle [76]. In primary cultures of human tertiary bronchial epithelial cells, secretin was demonstrated to potently stimulate channel-mediated Cl− efflux in a concentration-dependent manner. Secretin was also shown to cause a concentration-dependent relaxation of human tertiary bronchial smooth muscle [76]. 3.4. Stomach It was demonstrated that upon secretin stimulation, mucus secretion from cultured rat gastric epithelium was strongly induced [77] with a concomitant increase in intracellular cAMP, but not in intracellular Ca2+ [78]. Similar stimulatory effects of secretin on cAMP production were observed in isolated gastric glands from rat fundic and antral mucosal regions [79]. In dispersed chief cells from guinea pig and rat, a close correlation between secretin dose-dependent increase of intracellular cAMP and pepsinogen secretion was identified [80,81]. To confirm the involvement of cAMP pathway in gastric epithelial mucus secretion, H-89 and chelerythrine were used to inhibit PKA and PKC, respectively [78]. Among these drugs, only H-89 was able to inhibit the secretin-induced mucus secretion [78], suggesting that cAMP–PKA but not DAG–PKC pathway was involved. 3.5. Liver Secretin regulates ductal bile secretion [82] by interacting with cholangiocytes rather than hepatocytes to stimulate a bicarbonaterich bile flow [83]. This was further confirmed by studies which revealed that secretin receptors were expressed only on the basolateral membrane of rat cholangiocytes [18,84,85]. According to the size, there are three subpopulations (small, medium and large) of cholangiocytes [83]. It was demonstrated that only medium and large cholangiocytes expressed secretin receptor with increased cAMP levels in response to secretin stimulation [86,87]. By fluorescent unquenching assay, it was shown that secretin induced exocytosis and a dose-dependent increase in cAMP levels in isolated rat cholangiocytes with an EC50 of 6 nM [88]. On the other hand, exposure of cholangiocytes to secretin had no effect on intracellular Ca2+ and cGMP [88]. Similarly, it was also demonstrated that secretin-induced bile secretion in cholangiocytes did not involve the PI signal transduction pathway [89]. Since the identification of secretin–cAMP signaling pathway in cholangiocytes, it has been used as one of the parameters to measure the functionality of isolated or cultured cholangiocytes from normal or bile duct ligation rat [90–92]. The elevated cytosolic cAMP opens the cAMP-dependent Cl− channels, creating a Cl− gradient which activates the Cl−/HCO3− exchanger at the apical membrane and Na+/HCO3− symport at the basolateral membrane for high-bicarbonate choleresis and intracellular pH maintenance, respectively. Meanwhile, increased PKA activity activates the cystic fibrosis transmembrane regulator (CFTR), which can also insert the Cl−/HCO3− exchanger into the apical membrane [83]. In terms of water movement in cholangiocytes, secretin activates aquaporin 1 (AQP1) insertion into the apical membrane to facilitate water movement across the apical membrane [93,94].

F.K.Y. Siu et al. / Regulatory Peptides 137 (2006) 95–104

99

Fig. 2. A working model for the neuroactive function of secretin in the cerebellum. Secretin (in triangle) is synthesized and released upon depolarization from the somatodendritic region of Purkinje cells. The released secretin binds to secretin receptors at the presynaptic basket cell membrane, activates adenylate cyclase and increases cAMP level. The increased cAMP facilitates GABA (in circle) release. The binding of GABA to GABAA receptor on the Purkinje cell membrane opens the Cl− channel, which allows the entering of Cl− resulting in inhibitory postsynaptic current. SR: secretin receptor; AC: adenylate cyclase; Gs: stimulatory G protein; GAD: glutamic acid decarboxylase [58].

3.6. Intestine

3.7. Pancreas

As a gastrointestinal peptide, secretin is capable of inducing chloride ion and bicarbonate secretion in the small intestine in rodents [95–98]. These effects are mediated through adenylate cyclase and are less potent than VIP [99]. In some of these studies, secretin was also shown to inhibit the reabsorption of bicarbonate, water, glucose and electrolytes [95–98,100]. In addition, secretin inhibits small intestine and colon contraction activity in dog [101,102] and human [103]. Secretin also stimulates secretion from the Brunner's gland in dog, cat and rat [2,104,105] and increases the weight, DNA, and protein content of the rat small intestine [106].

The effects of secretin in the pancreas are well established. It has long been recognized that the principal function of secretin is to stimulate the secretion of bicarbonate, water, and electrolytes from pancreatic ductal epithelial cells in response to gastric acid and fatty acids in the duodenum [1,107]. Upon stimulation, the receptor, which is located at the basolateral membrane of the ductal cells, mediates an increase in cytosolic cAMP and PKA activity [108]. The increased intracellular cAMP activates CFTR to recirculate Cl− back into the glandular lumen and hence depolarizes both luminal and basolateral membranes [109]. Depolarization of the basolateral membrane

100

F.K.Y. Siu et al. / Regulatory Peptides 137 (2006) 95–104

Fig. 3. A model of secretin-stimulated pancreatic bicarbonate secretion. Bicarbonate is uptaken by Na+–HCO−3 cotransporter. Accumulated bicarbonate is secreted into the ductal lumen through Cl−/HCO−3 exchanger at the apical membrane. The rate of this exchanger depends on the availability of the luminal Cl−, which is in turn controlled by CFTR [108].

increases the driving force of an electrogenic Na+–HCO3− cotransporter on the basolateral membrane leading to the entry of HCO3− which is then secreted at the apical membrane via the Cl−/HCO3− exchanger (Fig. 3) [109–111]. Besides, secretin also increases permeability of tight junctions in CAPAN-1 pancreatic ductal carcinoma through a cAMP-dependent pathway [112]. Similarly, in dispersed pancreatic rat and guinea pig acinar cells, cAMP signaling pathways of secretin receptor were implicated [38,39,113–118]. Nevertheless, both rat primary acinar cells and acinar cell-line AR4-2J were coupled to the cAMP pathway at low secretin concentrations (10− 10 M) whereas these cells were coupled to the PI pathway at high secretin concentrations (10− 8 M) [38,39,119]. Activation of the cAMP pathway leads to a modest increase in amylase secretion, while activation of the PI pathway potentiates CCK-stimulated amylase secretion [119,120]. In addition, secretin can also mediate a moderate dose-dependent increase in basal insulin secretion in mouse [121], a suppression of glucagon release in dog [122], and an increase of the potassium outward voltagedependent K+ current [123].

Ca2+ as well as PI hydrolysis were activated by secretin [10]. When rat kidney membrane fractions were incubated with secretin, adenylate cyclase activity was dose-dependently increased and a high density secretin binding site was located in the outer medulla of the kidney as revealed by autoradiography [29]. The secretin signaling pathway directs various functions of the kidney. For example, secretin was suggested to have both diuretic and anti-diuretic activities in the kidney depending on the route of secretin administration. Microinjection of secretin into the tubular lumen increased nephron filtration and proximal reabsorption, while microperfusion of secretin into peritubular capillaries had opposite effects [124]. In patients with cystic fibrosis or on hemodialysis, plasma concentrations of secretin were significantly increased [125,126]. Besides, secretin stimulated the proliferation of cultured kidney cells from patients with autosomal dominant polycystic kidney disease (ADPKD) through the cAMP-dependent pathway [127], indicating that secretin may play a role in some of these diseases.

3.8. Kidney

Information about secretin receptor signaling in male reproductive system was limited. In epididymis, secretin was proposed to act on the apical and basolateral sides to stimulate chloride and bicarbonate secretion. Secretin was found to be

By transfecting human secretin receptor into human embryonic kidney (HEK) 293 cells, both intracellular cAMP and

3.9. Male reproductive system

F.K.Y. Siu et al. / Regulatory Peptides 137 (2006) 95–104

localized in the principal cells of the initial segment and caput epididymis, whereas secretin receptor was present in the principal cells of the proximal as well as in the distal part of the epididymis. This pattern of distribution is consistent with the idea that secretin is secreted by the proximal epididymis and acts on secretin receptor in proximal and distal epididymis which mediates the secretory responses through cAMP [26,128]. In addition, secretin was reported to stimulate progesterone and testosterone production in Leydig cells [129]. 4. Receptor desensitization Desensitization of receptor to ligand stimulation is a phenomenon to protect cells from overstimulation which may result in cell damage. It could occur at many levels during the signaling cascade and one of those sites is at the level of receptor itself by rapid phosphorylation or internalization [130]. The mechanism of desensitization of GPCR was best established for β2-adrenergic receptor (β2AR). Agonist binding induces conformational change in GPCR which is necessary for the phosphorylation of serine and threonine residues on the receptor carboxyl-terminal (C-terminal) tail by G protein-coupled receptor kinases (GRKs). Phosphorylation by GRKs also allows the arresting proteins (e.g β-arrestins) to uncouple the G protein from the GPCRs. GPCRs then undergo GRKs–β-arrestins initiated and mediated receptor sequestration which leads to the removal of GPCRs from the cell surface to endosomes followed by recycling back to cell surface. Also, β-arrestins were demonstrated to target GPCRs through clathrin coated vesicles for endocytosis [131]. The desensitization mechanism of secretin receptor was studied by transfecting wild-type receptor and C-terminal truncated receptor into CHO cells. The receptors bearing cells were exposed to secretin prior to washing and restimulation. cAMP response was absent in cells bearing wildtype receptor while 25% to 30% of response was retained in Cterminal truncated receptor bearing cells [132]. The stimulated receptor was located at the level of plasmalemma when the receptor was labeled with fluorescent secretin [132]. Hence, it was suggested that phosphorylation-independent receptor internalization was the major desensitization mechanism of secretin receptor [132]. However, another group has found that phosphorylation by PKA and PKC has little effect on receptor desensitization whereas GRKs potently enhanced the secretin receptor desensitization in HEK293 cells. Pre-treatment by PKA and PKC inhibitors failed to alter the secretin-stimulated cAMP accumulation. But, GRK 2 caused a 40% reduction in maximal cAMP response while GRK 5 caused a shift in EC50 to the right [133]. Furthermore, using wild-type and C-terminal truncated receptors as models, the expression of dominantnegative β-arrestin and clathrin coated vesicle-mediated internalization inhibitor did not reduce secretin receptor internalization. This suggested that secretin receptor underwent an endocytosis process in which GRKs and β-arrestins were not involved [133,134]. The desensitization of the human secretin receptor was also investigated using Cytosensor microphysiometry, which indirectly measures cellular metabolic rates. CHO cells bearing human secretin receptor were continuously ex-

101

posed to secretin and the response of the cells increased rapidly at first and then leveled off, suggesting the occurrence of desensitization which acts to limit the maximal response [135]. However, when the cells were challenged by repeated doses of secretin at three different concentrations (0.03 nM, 3 nM and 300 nM), the peak responses of the cells did not change significantly from the first to the third dose, suggesting that the secretin receptor did not exhibit robust homologous desensitization. It appears that the desensitization event could be reversed by washing off the ligand since re-stimulation with successive doses of secretin could still elicit responses of similar magnitude and kinetics. It is likely that desensitization of the secretin receptor may be followed by a rapid resensitization process [135]. Thus, the desensitization mechanism of secretin receptor remains controversial. 5. Summary It becomes more and more evident that secretin exhibits diverse physiological functions in multiple tissues including the central nervous system [30]. There were also supporting arguments for the involvement of secretin and its receptor in cardiovascular emergencies [136] and pancreatic cancer [31]. Its neuronal activities suggest its potential importance in the central nervous system [5]. Future investigation and hence understanding the signaling mechanisms of its receptor will definitely provide crucial information on the potential use of secretin and its agonists and antagonists as a central or peripheral agent to modulate functions carried out by this pleiotropic hormone. Acknowledgements This work was supported by the HK government RGC HKU 7384/04M and RGC HKU 7501/05M to Billy K.C. Chow. References [1] Meyer JH, Way LW, Grossman MI. Pancreatic response to acidification of various lengths of proximal intestine in the dog. Am J Physiol 1970;219:971–7. [2] Kirkegaard P, Skov OP, Seier PS, Holst JJ, Schaffalitzky de Muckadell OB, Christiansen J. Effect of secretin and glucagon on Brunner's gland secretion in the rat. Gut 1984;25:264–8. [3] Watanabe S, Chey WY, Lee KY, Chang TM. Secretin is released by digestive products of fat in dogs. Gastroenterology 1986;90:1008–17. [4] You CH, Chey WY. Secretin is an enterogastrone in humans. Dig Dis Sci 1987;32:466–71. [5] Yung WH, Leung PS, Ng SSM, Zhang J, Chan SCY, Chow BKC. Secretin facilitates GABA transmission in the cerebellum. J Neurosci 2001;21:7063–8. [6] Harmar AJ. Family-B G-protein-coupled receptors. Genome Biol 2001;2 [REVIEWS 3013.1-3013.10]. [7] Ishihara T, Nakamura S, Kaziro Y, Takahashi T, Takahashi K, Nagata S. Molecular cloning and expression of a cDNA encoding the secretin receptor. EMBO J 1991;10:1635–41. [8] Chow BKC. Molecular cloning and functional characterization of a human secretin receptor. Biochem Biophys Res Commun 1995;212:204–11. [9] Jiang S, Ulrich C. Molecular cloning and functional expression of a human pancreatic secretin receptor. Biochem Biophys Res Commun 1995;207:883–90.

102

F.K.Y. Siu et al. / Regulatory Peptides 137 (2006) 95–104

[10] Patel DR, Kong Y, Sreedharan SP. Molecular cloning and expression of a human secretin receptor. Mol Pharmacol 1995;47:467–73. [11] Holtmann MH, Hadac EM, Miller LJ. Critical contributions of aminoterminal extracellular domains in agonist binding and activation of secretin and vasoactive intestinal polypeptide receptors. Studies of chimeric receptors. J Biol Chem 1995;270:14394–8. [12] Vilardaga JP, De Neef P, Di Paolo E, Bollen A, Waelbroeck M, Robberecht P. Properties of chimeric secretin and VIP receptor proteins indicate the importance of the N-terminal domain for ligand discrimination. Biochem Biophys Res Commun 1995;211:885–91. [13] Vilardaga JP, Di Paolo E, De Neef P, Waelbroeck M, Bollen A, Robberecht P. Lysine 173 residue within the first exoloop of rat secretin receptor is involved in carboxylate moiety recognition of Asp 3 in secretin. Biochem Biophys Res Commun 1996;218:842–6. [14] Donnelly D. The arrangement of the transmembrane helices in the secretin receptor family of G-protein-coupled receptors. FEBS Lett 1997;409:431–6. [15] Frimurer TM, Bywater RP. Structure of the integral membrane domain of the GLP1 receptor. Proteins 1999;35:375–86. [16] Chan KYY, Pang RTK, Chow BKC. Functional segregation of the highly conserved basic motifs within the third endoloop of the human secretin receptor. Endocrinology 2001;142:3926–34. [17] Ulrich CD, Wood P, Hadac EM, Kopras E, Whitcomb DC, Miller LJ. Cellular distribution of secretin receptor expression in rat pancreas. Am J Physiol 1998;275:G1437–44. [18] Alpini G, Ulrich CD, Phillips JO, Pham LD, Miller LJ, LaRusso NF. Upregulation of secretin receptor gene expression in rat cholangiocytes after bile duct ligation. Am J Physiol 1994;266:G922–8. [19] Steiner TS, Mangel AW, McVey DC, Vigna SR. Secretin receptors mediating rat forestomach relaxation. Am J Physiol 1993;264:G863–7. [20] Gespach C, Bataille D, Vauclin N, Rosselin G, Moroder L, Wunsch E. Secretin binding sites coupled with adenylate cyclase in rat fundic membranes. Peptides 1981;2(Suppl 2):247–51. [21] Bawab W, Gespach C, Marie JC, Chastre E, Rosselin G. Pharmacology and molecular identification of secretin receptors in rat gastric glands. Life Sci 1988;42:791–8. [22] Li P, Chang TM, Chey WY. Secretin inhibits gastric acid secretion via a vagal afferent pathway in rats. Am J Physiol 1998;275:G22–8. [23] Fremeau Jr RT, Jensen RT, Charlton CG, Miller RL, O'Donohue TL, Moody TW. Secretin: specific binding to rat brain membranes. J Neurosci 1983;3:1620–5. [24] Koves K, Kausz M, Horvath K. What may be the anatomical basis that secretin can improve the mental functions in autism? Regul Pept 2002;109:167–72. [25] Nozaki S, Nakata R, Mizuma H, Nishimura N, Watanabe Y, Kohashi R, Watanabe Y. In vitro autoradiographic localization of (125)i-secretin receptor binding sites in rat brain. Biochem Biophys Res Commun 2002;292:133–7. [26] Chow BK, Cheung KH, Tsang EM, Leung MC, Lee SM, Wong PY. Secretin controls anion secretion in the rat epididymis in an autocrine/ paracrine fashion. Biol Reprod 2004;70:1594–9. [27] Andersson A, Sundler F, Ekblad E. Expression and motor effects of secretin in small and large intestine of the rat. Peptides 2000;21:1687–94. [28] Christophe J, Chatelain P, Taton G, Delhaye M, Waelbroeck M, Robberecht P. Comparison of VIP-secretin receptors in rat and human lung. Peptides 1981;2(Suppl 2):253–8. [29] Charlton CG, Quirion R, Handelmann GE, Miller RL, Jensen RT, Finkel MS, O'Donohue TL. Secretin receptors in the rat kidney: adenylate cyclase activation and renal effects. Peptides 1986;7:865–71. [30] Chey WY, Chang TM. Secretin, 100 years later. J Gastroenterol 2003;38:1025–35. [31] Ding WQ, Cheng ZJ, McElhiney J, Kuntz SM, Miller LJ. Silencing of secretin receptor function by dimerization with a misspliced variant secretin receptor in ductal pancreatic adenocarcinoma. Cancer Res 2002;62:5223–9. [32] Holtmann MH, Ganguli S, Hadac EM, Dolu V, Miller LJ. Multiple extracellular loop domains contribute critical determinants for agonist binding and activation of the secretin receptor. J Biol Chem 1996;271:14944–9.

[33] Di Paolo E, De Neef P, Moguilevsky N, Petry H, Bollen A, Waelbroeck M, Robberecht P. Contribution of the second transmembrane helix of the secretin receptor to the positioning of secretin. FEBS Lett 1998;424:207–10. [34] Vilardaga JP, Di Paolo E, Bialek C, De Neef P, Waelbroeck M, Bollen A, Robberecht P. Mutational analysis of extracellular cysteine residues of rat secretin receptor shows that disulfide bridges are essential for receptor function. Eur J Biochem 1997;246:173–80. [35] Pang RTK, Ng SSM, Cheng CHK, Holtmann MH, Miller LJ, Chow BKC. Role of N-linked glycosylation on the function and expression of the human secretin receptor. Endocrinology 1999;140:5102–11. [36] Gether U. Uncovering molecular mechanisms involved in activation of G protein-coupled receptors. Endocr Rev 2000;21:90–113. [37] Williams JA. Regulatory mechanisms in pancreas and salivary acini. Annu Rev Physiol 1984;46:361–75. [38] Trimble ER, Bruzzone R, Biden TJ, Farese RV. Secretin induces rapid increases in inositol trisphosphate, cytosolic Ca2+ and diacylglycerol as well as cyclic AMP in rat pancreatic acini. Biochem J 1986;239:257–61. [39] Trimble ER, Bruzzone R, Biden TJ, Meehan CJ, Andreu D, Merrifield RB. Secretin stimulates cyclic AMP and inositol trisphosphate production in rat pancreatic acinar tissue by two fully independent mechanisms. Proc Natl Acad Sci U S A 1987;84:3146–50. [40] Neves SR, Ram PT, Iyengar R. G protein pathways. Science 2002;296:1636–9. [41] Propst F, Moroder L, Wunsch E, Hamprecht B. The influence of secretin, glucagon and other peptides, of amino acids, prostaglandin endoperoxide analogues and diazepam on the level of adenosine 3′,5′cyclic monophosphate in neuroblastoma × glioma hybrid cells. J Neurochem 1979;32:1495–500. [42] Gossen D, Tastenoy M, Robberecht P, Christophe J. Secretin receptors in the neuroglioma hybrid cell line NG108-15. Characterization and regulation of their expression. Eur J Biochem 1990;193:149–54. [43] Vilardaga JP, Ciccarelli E, Dubeaux C, De Neef P, Bollen A, Robberecht P. Properties and regulation of the coupling to adenylate cyclase of secretin receptors stably transfected in Chinese hamster ovary cells. Mol Pharmacol 1994;45:1022–8. [44] van Calker D, Muller M, Hamprecht B. Regulation by secretin, vasoactive intestinal peptide, and somatostatin of cyclic AMP accumulation in cultured brain cells. Proc Natl Acad Sci U S A 1980;77:6907–11. [45] Roth BL, Beinfeld MC, Howlett AC. Secretin receptors on neuroblastoma cell membranes: characterization of 125I-labeled secretin binding and association with adenylate cyclase. J Neurochem 1984;42:1145–52. [46] Fremeau Jr RT, Korman LY, Moody TW. Secretin stimulates cyclic AMP formation in the rat brain. J Neurochem 1986;46:1947–55. [47] Chneiweiss H, Glowinski J, Premont J. Do secretin and vasoactive intestinal peptide have independent receptors on striatal neurons and glial cells in primary cultures? J Neurochem 1986;47:608–13. [48] Martin PT, Chung BT, Koshland Jr DE. Regulation of neurosecretory habituation by cAMP. Role of adaptation of cAMP signals. Eur J Biochem 1993;217:259–65. [49] Karelson E, Laasik J, Sillard R. Regulation of adenylate cyclase by galanin, neuropeptide Y, secretin and vasoactive intestinal polypeptide in rat frontal cortex, hippocampus and hypothalamus. Neuropeptides 1995;28:21–8. [50] Kuntz A, Clement HW, Lehnert W, van Calker D, Hennighausen K, Gerlach M, Schulz E. Effects of secretin on extracellular amino acid concentrations in rat hippocampus. J Neural Transm 2004;111:931–9. [51] Fuxe K, Andersson K, Hokfelt T, Mutt V, Ferland L, Agnati LF, Ganten D, Said S, Eneroth P, Gustafsson JA. Localization and possible function of peptidergic neurons and their interactions with central catecholamine neurons, and the central actions of gut hormones. Fed Proc 1979;38:2333–40. [52] Babu GN, Vijayan E. Plasma gonadotropin, prolactin levels and hypothalamic tyrosine hydroxylase activity following intraventricular bombesin and secretin in ovariectomized conscious rats. Brain Res Bull 1983;11:25–9. [53] Ip NY, Ho CK, Zigmond RE. Secretin and vasoactive intestinal peptide acutely increase tyrosine 3-monooxygenase in the rat superior cervical ganglion. Proc Natl Acad Sci U S A 1982;79:7566–9.

F.K.Y. Siu et al. / Regulatory Peptides 137 (2006) 95–104 [54] Schwarzschild MA, Zigmond RE. Secretin and vasoactive intestinal peptide activate tyrosine hydroxylase in sympathetic nerve endings. J Neurosci 1989;9:160–6. [55] Schwarzschild MA, Zigmond RE. Effects of peptides of the secretinglucagon family and cyclic nucleotides on tyrosine hydroxylase activity in sympathetic nerve endings. J Neurochem 1991;56:400–6. [56] Yang B, Goulet M, Boismenu R, Ferguson AV. Secretin depolarizes nucleus tractus solitarius neurons through activation of a nonselective cationic conductance. Am J Physiol Regul Integr Comp Physiol 2004;286: R927–34. [57] Yang H, Wang L, Wu SV, Tay J, Goulet M, Boismenu R, Czimmer J, Wang Y, Wu S, Ao Y, Tache Y. Peripheral secretin-induced Fos expression in the rat brain is largely vagal dependent. Neuroscience 2004;128:131–41. [58] Ng SSM, Yung WH, Chow BKC. Secretin as a neuropeptide. Mol Neurobiol 2002;26:97–107. [59] Lee SM, Chen L, Chow BK, Yung WH. Endogenous release and multiple actions of secretin in the rat cerebellum. Neuroscience 2005;134:377–86. [60] Chatelain P, Robberecht P, De Neef P, Claeys M, Christophe J. Low responsiveness of cardiac adenylate cyclase activity to peptide hormones in spontaneously hypertensive rats. FEBS Lett 1979;107:86–90. [61] Chatelain P, Robberecht P, De Neef P, Camus JC, Heuse D, Christophe J. Secretin and VIP-stimulated adenylate cyclase from rat heart. II. Impairment in spontaneous hypertension. Pflugers Arch 1980;389:29–35. [62] Chatelain P, Robberecht P, Camus JC, De Neef P, Waelbroeck M, Roba J, Christophe J. The adenylate cyclase activity in heart membranes from normotensive and spontaneously hypertensive rats, after chemical sympathectomy, suggests the presence of presynaptic secretin receptors. Eur J Pharmacol 1983;93:271–6. [63] Chatelain P, Robberecht P, De Neef P, Camus JC, Poloczek P, Christophe J. Impairment of hormone-stimulated cardiac adenylate cyclase activity in the genetically obese (fa/fa) Zucker rat. Pflugers Arch 1981;390:10–6. [64] Robberecht P, De Neef P, Camus JC, Waelbroeck M, Fontaine J, Christophe J. The cardiac inotropic response to secretin is lower in genetically obese (fa/fa) than in lean (fa/?) Zucker rats. Pflugers Arch 1983;398:217–20. [65] Robberecht P, Pochet R, Chatelain P, Verloes A, Camus JC, De Neef P, Christophe J. Decreased secretin and glucagon responsiveness of adenylate cyclase in cardiac membranes from hypothyroid rats. FEBS Lett 1981;132:33–6. [66] Chatelain P, Robberecht P, De Neef P, Camus JC, Christophe J. Early decrease in secretin-, glucagon-, and isoproterenol-stimulated cardiac adenylate cyclase activity in rats treated with isoproterenol. Biochem Pharmacol 1982;31:347–52. [67] De Fontaine S, Robberecht P, De Neef P, Christophe J. Alteration of secretin-stimulated cardiac adenylate cyclase activity in rats with portacaval shunt. Peptides 1986;7:645–9. [68] Gunnes P, Waldum HL, Rasmussen K, Ostensen H, Burhol PG. Cardiovascular effects of secretin infusion in man. Scand J Clin Lab Invest 1983;43:637–42. [69] Robberecht P, De Neef P, Waelbroeck M, Camus JC, Fontaine J, Christophe J. Secretin-induced changes in rate, contractility and adenylate cyclase activity in rat heart atria. Pflugers Arch 1984;401:1–5. [70] Christophe J, Waelbroeck M, Chatelain P, Robberecht P. Heart receptors for VIP, PHI and secretin are able to activate adenylate cyclase and to mediate inotropic and chronotropic effects. Species variations and physiopathology. Peptides 1984;5:341–53. [71] Gunnes P, Smiseth OA, Lygren I, Jorde R. Effects of secretin infusion on myocardial performance and metabolism in the dog. J Cardiovasc Pharmacol 1985;7:1183–7. [72] Taton G, Delhaye M, Camus JC, De Neef P, Chatelain P, Robberecht P, Christophe J. Characterization of the VIP- and secretin-stimulated adenylate cyclase system from human lung. Pflugers Arch 1981;391:178–82. [73] Laburthe M, Boissard C, Chevalier G, Zweibaum A, Rosselin G. Peptide receptors in human lung tumor cells in culture: vasoactive intestinal peptide (VIP) and secretin interaction with the Calu-1 and SW-900 cell lines. Regul Pept 1981;2:219–30. [74] Korman LY, Carney DN, Citron ML, Moody TW. Secretin/vasoactive intestinal peptide-stimulated secretion of bombesin/gastrin releasing

[75] [76]

[77] [78]

[79]

[80]

[81]

[82]

[83] [84]

[85]

[86]

[87]

[88]

[89]

[90]

[91]

[92]

[93]

[94]

103

peptide from human small cell carcinoma of the lung. Cancer Res 1986;46:1214–8. Moody TW, Korman LY. The release of bombesin-like peptides from small cell lung cancer cells. Ann N Y Acad Sci 1988;547:351–9. Davis RJ, Page KJ, Dos Santos Cruz GJ, Harmer DW, Munday PW, Williams SJ, Picot J, Evans TJ, Sheldrick RL, Coleman RA, Clark KL. Expression and functions of the duodenal peptide secretin and its receptor in human lung. Am J Respir Cell Mol Biol 2004;31:302–8. Tani S, Okuda M, Morishige R, Tanaka T. Gastric mucin secretion from cultured rat epithelial cells. Biol Pharm Bull 1997;20:482–5. Tani S, Suzuki T, Kano S, Tanaka T, Sunaga K, Morishige R, Tsuda T. Mechanisms of gastric mucus secretion from cultured rat gastric epithelial cells induced by carbachol, cholecystokinin octapeptide, secretin, and prostaglandin E2. Biol Pharm Bull 2002;25:14–8. Gespach C, Bataille D, Dupont C, Rosselin G, Wunsch E, Jaeger E. Evidence for a cyclic AMP system highly sensitive to secretin in gastric glands isolated from the rat fundus and antrum. Biochim Biophys Acta 1980;630:433–41. Sutliff VE, Raufman JP, Jensen RT, Gardner JD. Actions of vasoactive intestinal peptide and secretin on chief cells prepared from guinea pig stomach. Am J Physiol 1986;251:G96–G102. Tanaka T, Tani S. Inhibitory action of somatostatin on cAMP dependent pepsinogen secretion from rat gastric chief cells: involvement of pertussis toxin-sensitive G-protein. Biol Pharm Bull 1994;17:415–8. Alpini G, Lenzi R, Sarkozi L, Tavoloni N. Biliary physiology in rats with bile ductular cell hyperplasia. Evidence for a secretory function of proliferated bile ductules. J Clin Invest 1988;81:569–78. Fitz JG. Regulation of cholangiocyte secretion. Semin Liver Dis 2002;22:241–9. Farouk M, Vigna SR, McVey DC, Meyers WC. Localization and characterization of secretin binding sites expressed by rat bile duct epithelium. Gastroenterology 1992;102:963–8. Farouk M, Vigna SR, Haebig JE, Gettys TW, McVey DC, Chari R, Pruthi RS, Meyers WC. Secretin receptors in a new preparation of plasma membranes from intrahepatic biliary epithelium. J Surg Res 1993;54:1–6. Alpini G, Roberts S, Kuntz SM, Ueno Y, Gubba S, Podila PV, LeSage G, LaRusso NF. Morphological, molecular, and functional heterogeneity of cholangiocytes from normal rat liver. Gastroenterology 1996;110:1636–43. Alpini G, Glaser S, Robertson W, Rodgers RE, Phinizy JL, Lasater J, LeSage GD. Large but not small intrahepatic bile ducts are involved in secretin-regulated ductal bile secretion. Am J Physiol 1997;272: G1064–74. Kato A, Gores GJ, LaRusso NF. Secretin stimulates exocytosis in isolated bile duct epithelial cells by a cyclic AMP-mediated mechanism. J Biol Chem 1992;267:15523–9. Lenzen R, Alpini G, Tavoloni N. Secretin stimulates bile ductular secretory activity through the cAMP system. Am J Physiol 1992;263: G527–32. LeSage G, Glaser SS, Gubba S, Robertson WE, Phinizy JL, Lasater J, Rodgers RE, Alpini G. Regrowth of the rat biliary tree after 70% partial hepatectomy is coupled to increased secretin-induced ductal secretion. Gastroenterology 1996;111:1633–44. Alpini G, Ulrich C, Roberts S, Phillips JO, Ueno Y, Podila PV, Colegio O, LeSage GD, Miller LJ, LaRusso NF. Molecular and functional heterogeneity of cholangiocytes from rat liver after bile duct ligation. Am J Physiol 1997;272:G289–97. Alpini G, Phinizy JL, Glaser S, Francis H, Benedetti A, Marucci L, LeSage G. Development and characterization of secretin-stimulated secretion of cultured rat cholangiocytes. Am J Physiol Gastrointest Liver Physiol 2003;284:G1066–73. Marinelli RA, Pham L, Agre P, LaRusso NF. Secretin promotes osmotic water transport in rat cholangiocytes by increasing aquaporin-1 water channels in plasma membrane. Evidence for a secretin-induced vesicular translocation of aquaporin-1. J Biol Chem 1997;272:12984–8. Marinelli RA, Tietz PS, Pham LD, Rueckert L, Agre P, LaRusso NF. Secretin induces the apical insertion of aquaporin-1 water channels in rat cholangiocytes. Am J Physiol 1999;276:G280–6.

104

F.K.Y. Siu et al. / Regulatory Peptides 137 (2006) 95–104

[95] Barbezat GO, Grossman MI. Intestinal secretion: stimulation by peptides. Science 1971;174:422–4. [96] Hubel KA. Effects of secretin and glucagon on intestinal transport of ions and water in the rat. Proc Soc Exp Biol Med 1972;139:656–8. [97] Pansu D, Bosshard A, Dechelette MA, Vagne M. Effect of pentagastrin, secretin and cholecystokinin on intestinal water and sodium absorption in the rat. Digestion 1980;20:201–6. [98] Isenberg JI, Wallin B, Johansson C, Smedfors B, Mutt V, Tatemoto K, Emas S. Secretin, VIP, and PHI stimulate rat proximal duodenal surface epithelial bicarbonate secretion in vivo. Regul Pept 1984;8:315–20. [99] Reimer R, Odes HS, Beil W, Schwenk M, Muallem R, Sewing KF. Bicarbonate secretion in the guinea pig duodenum: functional characterization of peptide hormone receptors in duodenal enterocytes. Pharmacology 1996;52:339–46. [100] Hirose S, Shimazaki K, Hattori N. Effect of secretin and caerulein on the absorption of water, electrolytes and glucose from the jejunum of dogs. Digestion 1986;35:205–10. [101] Hermon-Taylor J, Code CF. Effect of secretin on small bowel myoelectric activity of conscious healthy dogs. Am J Dig Dis 1970;15:545–50. [102] Ramirez M, Farrar JT. The effect of secretin and cholecystokininpancreozymin on the intraluminal pressure of the jejunum in the unanesthetized dog. Am J Dig Dis 1970;15:539–44. [103] Dinoso Jr VP, Meshkinpour H, Lorber SH, Gutierrez JG, Chey WY. Motor responses of the sigmoid colon and rectum to exogenous cholecystokinin and secretin. Gastroenterology 1973;65:438–44. [104] Love JW, Walder AI, Bingham C. Effect of pure natural and synthetic secretion on Brunner's gland secretion in dogs. Nature 1968;219:731–2. [105] Stening GF, Grossman MI. Hormonal control of Brunner's glands. Gastroenterology 1969;56:1047–52. [106] Hoang HD, Wood JG, Bussjaeger LJ, Solomon TE. Interaction of neurotensin with caerulein or secretin on digestive tract growth in rats. Regul Pept 1988;22:275–84. [107] Watanabe SI, Chey WY, Lee KY, Chang TM. Release of secretin by licorice extract in dogs. Pancreas 1986;1:449–54. [108] Raeder MG. The origin of and subcellular mechanisms causing pancreatic bicarbonate secretion. Gastroenterology 1992;103:1674–84. [109] Gray MA, Greenwell JR, Argent BE. Secretin-regulated chloride channel on the apical plasma membrane of pancreatic duct cells. J Membr Biol 1988;105:131–42. [110] Ishiguro H, Steward MC, Lindsay AR, Case RM. Accumulation of intracellular HCO−3 by Na(+)–HCO−3 cotransport in interlobular ducts from guinea-pig pancreas. J Physiol 1996;495(Pt 1):169–78. [111] Shumaker H, Amlal H, Frizzell R, Ulrich CD, Soleimani M. CFTR drives Na+–nHCO−3 cotransport in pancreatic duct cells: a basis for defective HCO−3 secretion in CF. Am J Physiol 1999;276:C16–25. [112] Rotoli BM, Bussolati O, Dall'Asta V, Orlandini G, Gatti R, Gazzola GC. Secretin increases the paracellular permeability of CAPAN-1 pancreatic duct cells. Cell Physiol Biochem 2000;10:13–25. [113] Robberecht P, Waelbroeck M, Noyer M, Chatelain P, De Neef P, Konig W, Christophe J. Characterization of secretin and vasoactive intestinal peptide receptors in rat pancreatic plasma membranes using the native peptides, secretin-(7-27) and five secretin analogues. Digestion 1982;23:201–10. [114] Jensen RT, Charlton CG, Adachi H, Jones SW, O'Donohue TL, Gardner JD. Use of 125I-secretin to identify and characterize high-affinity secretin receptors on pancreatic acini. Am J Physiol 1983;245:G186–95. [115] Bissonnette BM, Collen MJ, Adachi H, Jensen RT, Gardner JD. Receptors for vasoactive intestinal peptide and secretin on rat pancreatic acini. Am J Physiol 1984;246:G710–7. [116] Gillet L, Robberecht P, Waelbroeck M, Camus JC, De Neef P, Konig W, Christophe J. Interaction of Gila monster venom with secretin receptors in rat pancreatic membranes. Peptides 1984;5:407–9.

[117] Robberecht P, Waelbroeck M, Camus JC, De Neef P, Christophe J. Importance of disulfide bonds in receptors for vasoactive intestinal peptide and secretin in rat pancreatic plasma membranes. Biochim Biophys Acta 1984;773:271–8. [118] Zhou ZC, Gardner JD, Jensen RT. Receptors for vasoactive intestinal peptide and secretin on guinea pig pancreatic acini. Peptides 1987;8:633–7. [119] Bold RJ, Ishizuka J, Townsend Jr CM, Thompson JC. Secretin potentiates cholecystokinin-stimulated amylase release by AR4-2J cells via a stimulation of phospholipase C. J Cell Physiol 1995;165:172–6. [120] Gardner JD, Jackson MJ. Regulation of amylase release from dispersed pancreatic acinar cells. J Physiol 1977;270:439–54. [121] Ahren B, Lundquist I. Effects of vasoactive intestinal polypeptide (VIP), secretin and gastrin on insulin secretion in the mouse. Diabetologia 1981;20:54–9. [122] Hisatomi A, Unger RH. Secretin inhibits glucagon in the isolated perfused dog pancreas. Diabetes 1983;32:970–3. [123] Kim SJ, Kim JK, Pavenstadt H, Greger R, Hug MJ, Bleich M. Regulation of slowly activating potassium current (I(Ks)) by secretin in rat pancreatic acinar cells. J Physiol 2001;535:349–58. [124] Romano G, Giagu P, Favret G, Bartoli E. Dual effect of secretin on nephron filtration and proximal reabsorption depending on the route of administration. Peptides 2000;21:723–8. [125] Grekas DM, Raptis S, Tourkantonis AA. Plasma secretin, pancreozymin, and somatostatin-like hormone in chronic renal failure patients. Uremia Invest 1984;8:117–20. [126] Windstetter D, Schaefer F, Scharer K, Reiter K, Eife R, Harms HK, Bertele-Harms R, Fiedler F, Tsui LC, Reitmeir P, Horster M, Hadorn HB. Renal function and renotropic effects of secretin in cystic fibrosis. Eur J Med Res 1997;2:431–6. [127] Yamaguchi T, Pelling JC, Ramaswamy NT, Eppler JW, Wallace DP, Nagao S, Rome LA, Sullivan LP, Grantham JJ. cAMP stimulates the in vitro proliferation of renal cyst epithelial cells by activating the extracellular signal-regulated kinase pathway. Kidney Int 2000;57:1460–71. [128] Wong PY, Huang SJ. Secretory agonists stimulate a rise in intracellular cyclic AMP but not Ca2+ and inositol phosphates in cultured rat epididymal epithelium. Exp Physiol 1990;75:321–37. [129] Kasson BG, Lim P, Hsueh AJ. Vasoactive intestinal peptide stimulates androgen biosynthesis by cultured neonatal testicular cells. Mol Cell Endocrinol 1986;48:21–9. [130] Ulrich CD, Holtmann M, Miller LJ. Secretin and vasoactive intestinal peptide receptors: members of a unique family of G protein-coupled receptors. Gastroenterology 1998;114:382–97. [131] Ferguson SS, Zhang J, Barak LS, Caron MG. Molecular mechanisms of G protein-coupled receptor desensitization and resensitization. Life Sci 1998;62:1561–5. [132] Holtmann MH, Roettger BF, Pinon DI, Miller LJ. Role of receptor phosphorylation in desensitization and internalization of the secretin receptor. J Biol Chem 1996;271:23566–71. [133] Shetzline MA, Premont RT, Walker JK, Vigna SR, Caron MG. A role for receptor kinases in the regulation of class II G protein-coupled receptors. Phosphorylation and desensitization of the secretin receptor. J Biol Chem 1998;273:6756–62. [134] Walker JK, Premont RT, Barak LS, Caron MG, Shetzline MA. Properties of secretin receptor internalization differ from those of the beta(2)-adrenergic receptor. J Biol Chem 1999;274:31515–23. [135] Ng SS, Pang RT, Chow BK, Cheng CH. Real-time evaluation of human secretin receptor activity using cytosensor microphysiometry. J Cell Biochem 1999;72:517–27. [136] Redfern JS, O'Dorisio TM. Therapeutic uses of gastrointestinal peptides. Endocrinol Metab Clin North Am 1993;22:845–73.