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DRUG DEVELOPMENT RESEARCH 59:386–394 (2003)

DDR Intestinal Epithelial Secretory Function: Role of Proteinase-Activated Receptors Michelle C. Buresi and Wallace K. MacNaughtonn Mucosal Inflammation Research Group, University of Calgary, Calgary, Canada

Strategy, Management and Health Policy

Preclinical Development Clinical Development Venture Capital Preclinical Toxicology, Formulation Phases I-III Postmarketing Enabling Research Drug Delivery, Regulatory, Quality, Phase IV Technology Pharmacokinetics Manufacturing

ABSTRACT The ability of to secrete electrolytes and water into the intestinal lumen represents a critical feature of mucosal defense. During disease, this function may be altered and may initiate or exacerbate pathological conditions. Although many of the intracellular mechanisms linking stimulation to have been elucidated, novel pathways continue to be revealed. These pathways provide potential for therapeutic manipulation of cellular function. In addition, the importance of the microenvironment surrounding enterocytes is increasingly being acknowledged, and the interactions between epithelial cells and their milieu are proving to be essential to the regulation of secretory function, both in health and disease. In this way, epithelial ion transport functions can be modulated by mediators released from neighboring nerves, inflammatory cells, and pathogens, or by endocrine factors. Much interest has recently been elicited by the discovery that proteinases can regulate cellular functions through the activation of proteinase-activated receptors (PARs). Because of the abundance of within the , particularly in the setting of development, inflammation, and healing, it is likely that PARs have an important role to play in these processes. PARs have been localized to a variety of types in the gastrointestinal tract, and have been shown to influence epithelial secretory function on several levels. In this review, we discuss the mechanisms by which proteases and PARs regulate intestinal secretory function, and the manner in which these modulations might contribute to inflammatory processes. Drug Dev. Res. 59:386–394, 2003. c 2003 Wiley-Liss, Inc.

Key words: epithelium; secretion; chloride; MAP kinase

THE ROLE OF THE INTESTINAL EPITHELIUM IN conditions. Excessive secretion due to a hyperrespon- HOST DEFENSE siveness to secretagogues, as exemplified by the The gastrointestinal epithelium represents the response to cholera toxin, can result in leading first line of defense against ingested , antigens, to excess water and electrolyte loss. Conversely, the drugs, and toxins. An important aspect of intestinal inability to secrete chloride and water due to a barrier function is the ability of a subpopulation of hyporesponsiveness to secretagogues, exemplified by enterocytes to secrete electrolytes such as chloride in a basolateral to apical direction, promoting the move- Grant sponsor: Canadian Institutes of Health Research ment of water into the lumen. The movement of (CIHR); Grant number: MOP 53311. chloride and water into the lumen of the intestinal n crypts prevents their colonization by bacteria and slows Correspondence to: Wallace MacNaughton, PhD, Muco- sal Inflammation Research Group, University of Calgary, the translocation of antigens into the lamina propria 3330 Hospital Dr. NW, Calgary, AB, Canada T2N 1N4. [Cirino et al., 1996; Asfaha et al., 2001]. E-mail: [email protected] Responsiveness of the epithelium to secretago- Published online in Wiley InterScience (www.interscience.wiley. gues can be significantly altered during pathological com) DOI: 10.1002/ddr.10308

c 2003 Wiley-Liss, Inc. PARs AND INTESTINAL ION TRANSPORT 387 models of intestinal inflammation [MacNaughton et al., [Keast et al., 1986; Cooke, 1992; MacNaughton et al., 1998; Asfaha et al., 2001; Freeman and MacNaughton, 1996, 1997; Green et al., 2000]. Thus, substances that 2000] and as observed in human inflammatory bowel affect enteric neural function also have the potential to disease [Archampong et al., 1972; Hubel and Renquist, alter epithelial secretion of chloride and water. 1990; Sandle et al., 1990], may allow the translocation of bacteria, antigens, or toxins across the epithelium PROTEINASES AND EPITHELIAL FUNCTION: and into the lamina propria where they may then MICROENVIRONMENT AND NEUROIMMUNE trigger, exacerbate, or prolong inflammation. Further- CONTROL SYSTEMS more, defective chloride secretion in the gut, such as How can the secretory function of enterocytes be that which occurs in congenital deficiencies such as regulated by serine proteinases and PARs? Serine cystic fibrosis, leads to intestinal obstruction and proteinases often have activities related to inflamma- malabsorption [O’Loughlin et al., 1991; Grubb and tion, tissue remodeling, and healing [reviewed in Gabriel, 1997]. Macfarlane et al., 2001]. The PARs in different tissues might thus respond selectively to site-targeted protei- REGULATION OF CHLORIDE SECRETION nases, either in the setting of tissue injury or in the Many of the intracellular mechanisms regulating course of development [Werb, 1997]. The gastrointest- chloride secretion by intestinal epithelial cells have inal (GI) tract is exposed to a particularly large array of been characterized. While the details are beyond the proteinases, whether in physiological or pathological scope of this review, of relevance are the second settings, due to its proximity to digestive , messenger systems that regulate apical chloride secre- proteinases from pathogens and proteinases derived tion. Typically, chloride secretion in the gut is mediated from inflammatory cells or the microvasculature. by intracellular signaling pathways involving either an Proteinases such as and mast cell increase in intracellular calcium levels through the are abundant in the gastrointestinal mucosa and action of C and inositol triphosphate, or submucosa during inflammatory conditions where an increase in cyclic AMP (cAMP) through the action hemorrhage, vascular disruption, and immune cell of adenylyl cyclase. These signaling systems can be infiltration are evident [reviewed in Vergnolle, 2000]. modified by mediators originating from nerves, im- Luminal may also act on enterocytes via PAR2 mune cells, myofibroblasts, or other neighboring cells, localized to the apical surface [Kong et al., 1997] or and responsiveness to these mediators may be altered may access the basolateral surface of the epithelium, as during disease states [reviewed in Barrett and Keely, well as other cell types, as a result of increased 2000]. For example, various reactive oxygen species, epithelial permeability. Other proteinases, such as platelet activating factor, lipoxygenase metabolites of , A, and G have also been arachidonic , and certain cytokines including IL-1, shown to cleave and activate PARs, albeit less potently all induce chloride secretion. Interestingly, the secre- [Suidan et al., 1994, 1996; Molino et al., 1995; Vouret- tion elicited by each of these factors is sensitive to Craviari et al., 1995]. In addition, it has been shown inhibition by cyclooxygenase inhibitors such as non- that allergenic house dust mite proteinases are capable steroidal anti-inflammatory drugs (NSAIDs). This of cleaving and activating PARs to affect epithelial observation suggests that arachidonic acid metabolites, function in the respiratory tract [Sun et al., 2001; such as the prostaglandins, may act as final common Asokananthan et al., 2002b]. Some bacterial protei- mediators of many stimulants of intestinal ion transport nases have also been demonstrated to activate PARs [Hinterleitner and Powell, 1991; Montrose et al., 1999]. situated on oral epithelial cells, and to affect secretion This has implications for the mediation of epithelial of inflammatory mediators [Lourbakos et al., 2001]. secretory function by PARs as discussed below. Regulation of epithelial function by PAR1 may Regulation of epithelial chloride secretion is also furthermore be influenced by proteinases, such as under tight neural control, especially through the neutrophil , , and , that action of submucosal secretomotor neurons of the are able to cleave PAR1 downstream of the thrombin . Specifically, release of vasoac- cleavage site, resulting in the deactivation of the tive intestinal polypeptide or stimulates receptor, and rendering it unresponsive to further chloride secretion through activation of cAMP- and activation by thrombin. Caþþ-dependent pathways respectively. In addition, Thus, a variety of proteinases may be in a position the rate of chloride secretion can be influenced by a to act on PARs and to influence secretory variety of neurally derived mediators including opiates, function on several levels, either by direct interaction gamma-aminobutyric acid (GABA), calcitonin gene with the epithelium, or by altering the release of related (CGRP), substance P, and serotonin secretagogues from other cell types. Although PAR 388 BURESI AND MACNAUGHTON activation by a is an irreversible event, and stimulus-secretion coupling pathway, wherein EGF resensitization is dependent on mobilization of intra- receptor transactivation and Src activation precede a cellular stores and on synthesis of new receptors, the MEK-ERK1/2 dependent activation of cytosolic PLA2 turnover of PARs in native tissue is quite rapid and and COX-1/COX-2 dependent chloride secretion supports a role for these receptors in the regulation of [Buresi et al., 2002] (Fig. 1). Although it is a novel ion transport [Bohm et al., 1996; Cocks et al., 1999a,b; finding that a mitogenic signaling pathway is involved Mall et al., 2002]. in stimulus-secretion coupling in the intestine, PARs have been linked to mitogen-activated (MAP) MUCOSAL DISTRIBUTION OF PARS AND kinase pathways in other systems. Thrombin is mito- CONTROL OF CHLORIDE SECRETION genic in many cells and furthermore induces inter- leukin-8 production in endothelial cells via activation of PAR1 PAR1 and subsequent activation of MAP kinases PAR1 has been localized to various epithelia and [L’Allemain et al., 1991; Molloy et al., 1996; Takata its activation has been demonstrated to induce the et al., 2001]. secretion of growth factors and cytokines in airway and The signaling mechanisms by which GPCRs such corneal epithelia [Asokananthan et al., 2002a; Shimizu as the PARs activate the MAP kinase pathway is et al., 2000; Lang et al., 2003]. Furthermore, a role complex and not fully understood, but much evidence for PAR1 in electrolyte transport has been suggested, has accumulated for the transactivation of a receptor and may occur via the release of arachidonic acid tyrosine (tyr) kinase, and in particular, the epidermal metabolites, which are known to stimulate secretion. As growth factor (EGF) receptor. The mechanism by mentioned above, arachidonic acid metabolites and which GPCR signaling leads to the transactivation of the cyclooxygenase (COX) pathways are essential to the EGF receptor has been hypothesized to occur in a the mechanism of action of many secretagogues. The number of ways and is reviewed elsewhere [Gschwind induction by PARs of a COX-dependent pathway leading to the release of prostaglandins has been reported in several systems including platelets, Chinese hamster ovary cells, murine tracheal smooth muscle, and human urothelial carcinoma cells [Winitz et al., 1994; Seiler et al., 1995; Lan et al., 2001; McHowat et al., 2001]. In addition, we have recently shown that an intestinal myofibroblast cell line is capable of responding to thrombin with increased expression of COX-2 and synthesis of PGE2. This effect of thrombin had both PAR1-dependent and independent compo- nents [Seymour et al., 2003]. The ability of mediators such as PGE2 that are released from neighboring cells to modulate epithelial secretory function has been well described [Berschneider and Powell, 1992; Powell et al., 1999], and now seems to be a component of the response to mucosal activation of PAR1 also. In the GI tract, PAR1 has been localized to smooth muscle cells, myenteric neurons [Kawabata et al., 1999; Gao et al., 2002], and endothelial cells of the lamina propria and submucosa [Wang et al., 2002]. Fig. 1. Proposed model of PAR1 induced chloride secretion in intestinal epithelial cells. PAR1 activation by thrombin (or by PAR1- Recently, PAR1 was also found to be expressed in a þþ non-tumorogenic, chloride-secreting intestinal epithe- activating ) leads to increased intracellular Ca , likely through a PLC-IP3/DAG mechanism. This leads to a Src-associated lial cell line, SCBN [Buresi et al., 2001]. Furthermore, EGF receptor transactivation, which triggers a Raf-dependent activa- we have shown that exposure of the basolateral (but not tion of the ERK1/2 MAP kinase pathway. ERK1/2 mediates PLA2 apical) SCBN cell membrane to thrombin or PAR1- activation and subsequent liberation of arachidonic acid from the activating peptides results in calcium-dependent chlor- plasma membrane. Arachidonic acid by cyclo-oxygenase ide secretion. Cross-desensitization between thrombin (COX) results in a prostanoid (PG)-dependent increase in apically directed chloride secretion. The chloride channel involved is not and PAR1-activating peptides strongly suggests that known, but is unlikely to be the cystic fibrosis transmembrane the effect is mediated by PAR1 [Buresi et al., 2001]. conductance regulator (CFTR). With data from [Buresi et al., 2001, Interestingly, PAR1 activation results in a novel 2002; Dery et al., 1998]. PARs AND INTESTINAL ION TRANSPORT 389 et al., 2001]. The events occurring subsequent to physiological functions including ion transport pro- EGF receptor activation, and that lead to MAP kinase cesses. Basolateral PAR2 activation on pancreatic duct signaling, are fairly well characterized [van Biesen epithelial cells leads to calcium dependent activation of et al., 1996; Luttrell, 2002]. Briefly, EGF binding to its ion channels [Nguyen et al., 1999] and activation of the receptor leads to receptor dimerization and autophos- receptor in the mouse distal colon stimulates Cl and phorylation, followed by recruitment of adaptor Kþ secretion while inhibiting baseline Naþ absorption and non-receptor tyrosine kinases such as [Cuffe et al., 2002]. The mechanisms by which PAR2 src. A phosphorylation cascade ensues, involving Raf-1 regulates epithelial ion secretion are once again likely kinase (MAPKKK), MEK-1/2 (MAPKK), and extra- to occur at several levels. For instance, tumor cellular signal response kinases (ERK) -1/2. ERK1/2 is factor-a (TNF-a) induces PAR2 expression and may a MAP kinase that, once activated, can phosphorylate thus contribute to the altered secretory responsiveness transcription factors such as Elk-1, or other enzymes observed in inflammatory processes such as chronic rsk such as p90 S6 kinase or phospholipase A2 (PLA2). inflammatory bowel disease [Nystedt et al., 1996; Mall The end result is activation of transcription factors and et al., 2002]. It has also been suggested that PAR2 may consequent up-regulation of immediate early genes have an important role in intestinal neuroimmune that regulate growth [Dery et al., 1998]. modulation. Tryptase derived from mast cells can There is considerable evidence linking MAP activate PAR2 and induce long-term hyperexcitability kinase signaling with COX activity. COX expression of guinea pig submucosal neurons [Reed et al., 2003], can be upregulated via MAP kinase pathways [Tessner raising the possibility that the action of this proteinase et al., 2003], and as mentioned, COX activity can be at PAR2 could thus alter secretory function. Indeed, modulated through serine-phosphorylation of cPLA2. cholinergic and noncholinergic submucosal neurons in Several studies have shown a link between MAP kinase porcine have been shown to express PAR2 and cascades, activation of PLA2, and the biosynthesis of agonists of the receptor stimulate active anion secretion PGE2 in a variety of systems [Laporte et al., 1999; by a neurogenic mechanism. This effect can further- Molina-Holgado et al., 2000; Newton et al., 2000; Yang more be modulated by prostanoids and opioids [Green et al., 2000]. The activation of PLA2 by ERK thus et al., 2000]. In addition, PAR activation in cultured provides a mechanism by which induction of MAP neurons results in release of substance P and CGRP, kinases also regulates secretory function. Although our both of which modulate intestinal ion transport study is the first to do so in the intestine, MAP kinase [Perdue et al., 1987; Cooke, 1992; Cox and Tough, signaling pathways have been linked to the regu- 1994; Kawabata et al., 2001; Coelho et al., 2002]. PAR2 lation of ion secretion in other systems. It has been activation may also occur on fibroblasts and inflamma- demonstrated that potassium and calcium ion channels tory cells, inducing the release of factors that regulate can be regulated by epidermal growth factor [Peppe- epithelial secretory function. For instance, PAR2 lenbosch et al., 1991; Peralta, 1995; Bowlby et al., 1997; activation of primary gastrointestinal mucosal myofi- Tsai et al., 1997; Wischmeyer et al., 1998] and a Src-like broblasts stimulates PGE2 synthesis, which would then kinase was able to activate a chloride conductance in be able to stimulate chloride secretion from nearby lymphocytes [Lepple-Wienhues et al., 2001]. epithelial cells [Seymour et al., 2001]. While activation of epithelial PARs results in the In addition to its effects on cells that regulate stimulation of chloride secretion, activation of PARs on epithelial secretory function, PAR2 may also be other cells in the intestinal mucosal lamina propria may expressed and activated on the epithelium itself to inhibit secretion. Activation of PAR1 in isolated segments directly stimulate chloride secretion [Kong et al., 1997; of mouse colon resulted in a decrease in neurally Mall et al., 2002] (Buresi, unpublished observations). dependent secretion stimulated by electrical field Indeed, PAR2 is expressed in human colonic epithe- stimulation [Buresi et al., 2003]. While the nature of lium and mediates intestinal electrolyte secretion this response remains to be more fully elucidated, it induced by the release of mast cell tryptase [Mall may represent a control mechanism for dampening the et al., 2002]. The signaling mechanisms involved in the secretion elicited by activation of epithelial PAR1. regulation of chloride secretion by epithelial PAR2 have not been fully elucidated. However, all of the PARs PAR2 cloned to date appear able to couple to both Gq and Gi The role of PAR2 in secretory function has been [Hollenberg, 1999], and thus receptor agonism typi- more extensively studied, and has been demonstra- cally results in Ca2þ mobilization and activation of ted in various epithelia including those of the lung and protein kinase C (PKC). Concurrently, coupling to kidney [Bertog et al., 1999]. PAR2 is widely expressed Gi proteins leads to inhibition of adenylyl cyclase in the gastrointestinal tract and contributes to various and hence suppression of cAMP [Hung et al., 1992; 390 BURESI AND MACNAUGHTON Baffy et al., 1994; Benka et al., 1995]. Hence, as for inflammatory conditions of the gut. Due to the vascular several of the examples discussed above, PAR2 disruption, hemorrhage, and increased vascular perme- mediated effects often demonstrate a calcium depen- ability that are characteristic of inflammation, thrombin dency. Furthermore, luminal trypsin is able to activate would be in a position to interact with many cell types PAR2 at the apical surface of enterocytes to stimulate in the lamina propria and submucosa. Thrombin has secretion of eicosanoids. These, in turn, may regulate long been known to be involved in inflammation multiple cell types through paracrine and autocrine [Cirino et al., 1996; Dery et al., 1998] and has been processes [Kong et al., 1997]. implicated in various aspects of the pathogenesis of An anti-secretory role for PAR2 has also been inflammatory bowel disease [Wakefield et al., 1989; reported. Activation of PAR2 inhibited amiloride- Chamouard et al., 1995; Thompson et al., 1995; sensitive ion transport in monolayers of bronchial Stadnicki et al., 1997; Carty et al., 2000; Swiatkowski epithelial cells [Danahay et al., 2001]. Furthermore, in et al., 2000; van Bodegraven et al., 2001]. Given that the human colonic carcinoma cell line, T84, trypsin thrombin plays numerous roles in the inflammatory causes an inhibition of forskolin stimulated chloride process in the intestine, it is not unreasonable to secretion [Danahay et al., 2000]. The effects of trypsin assume that it would also be in a position to affect appear to be mediated at the basolateral membrane epithelial secretory function in the inflamed gut. where trypsin causes an initial activation followed by an Trypsin is an abundant digestive in the inactivation of the Kþ conductance. These effects are upper GI tract and so could readily interact with the mirrored by a transient increase and subsequent luminal surface of enterocytes even under physiological decrease in Isc. conditions [Kong et al., 1997], and in circumstances where the epithelial barrier is disrupted and perme- Other PARs ability is increased, trypsin would be allowed to access PAR3 has been detected in the and small the intestinal mucosa. Furthermore, trypsin levels are intestine [Ishihara et al., 1997] and PAR4 is expressed increased during inflammation, and thus might con- in high levels in the and to a more tribute to altered secretory function under such moderate extent in the colon [Xu et al., 1998], conditions [reviewed in Vergnolle, 2000]. In addition, including on the epithelium [Ferazzini et al., 2003]. the regulation of ion secretion by this proteinase has A potential role for these PARs in the regulation of been suggested to elicit either pro- or anti-inflamma- intestinal ion transport has yet to be examined, tory effects in epithelia of the pancreatic ducts, airways, however. and urinary tract [Bertog et al., 1999; Cocks et al., Pharmacological evidence suggests that even more 1999a; Nguyen et al., 1999; Danahay et al., 2001]. It is PARs exist, but have yet to be cloned [Vergnolle et al., unlikely that trypsin is the endogenous activator at all 1998]. The structure-activity relationship (SAR) data of these sites however, and in many tissues, the obtained with a spectrum of PAR-APs continue to identities of proteases that stimulate PAR2 in vivo provide evidence for the existence of novel PARs in require further clarification [Bertog et al., 1999]. target tissues such as the endothelium and the Tryptase is released during mast cell degranula- gastrointestinal tract [Tay-Uyboco et al., 1995; Verg- tion. These cells, of which there are many in the nolle et al., 1998; Roy et al., 1998]. Indeed, the mucosa of the gut, may furthermore secrete mediators, presence of a ‘‘PAR2-like’’ receptor, that is pharmaco- such as TNF-a, that have been shown to upregulate logically distinct from PAR1 or PAR2, and that regulates PAR2 expression, and that are critically involved in intestinal transport via a cyclo-oxygenase-dependent inflammatory processes [Nystedt et al., 1996; Bischoff mechanism, has been demonstrated in rat jejunum et al., 1999]. Indeed, an important characteristic of [Vergnolle et al., 1998], but has yet to be fully inflammatory bowel disease is mast cell infiltration, and characterized. mast cell regulation of intestinal ion transport is altered in patients with IBD [Levo and Livni, 1978; Crowe and THE ROLE OF PROTEINASES AND SECRETORY Perdue, 1992]. FUNCTION IN INTESTINAL INFLAMMATION Finally, numerous other proteases that act on As we have discussed, proteases have the PARs, including and cathepsin G, potential to influence secretory function through are also characteristic of inflammatory cell activity, and various mechanisms. These pathways may be substan- have been implicated in epithelial secretory function in tially altered during inflammation and may thus impact the airway [Lundgren et al., 1994; Nadel et al., 1999]. significantly on epithelial barrier function in disease. More work needs to be done in order to determine the Thrombin has been found in abundance in the lumen role of these proteases in intestinal epithelial function and lamina propria of the gastrointestinal tract during in inflammatory diseases of the intestine. PARs AND INTESTINAL ION TRANSPORT 391

RELEVANCE AND FUTURE DIRECTIONS Asokananthan N, Graham PT, Stewart DJ, Bakker AJ, Eidne KA, Thompson PJ, Stewart GA. 2002b. House dust mite allergens In addition to their degradative functions in induce proinflammatory cytokines from respiratory epithelial , , and tissue remodeling, serine cells: the cysteine protease allergen, Der p 1, activates protease- proteinases also act as signaling molecules via activation activated receptor PAR2 and inactivates PAR1. J Immunol. of PARs, and may thus affect a wide array of 169:4572–4578. physiological functions. In this way, proteinases may Baffy G, Yang L, Raj S, Manning DR, Williamson JR. 1994. G act on enterocytes or on neighboring cells to alter protein coupling to the thrombin receptor in Chinese hamster secretory function and modulate inflammatory patho- lung fibroblasts. J Biol.Chem. 269:8483–8487. genesis. In the mouse colon, activation of PAR may Barrett KE, Keely SJ. 2000. Chloride secretion by the intestinal 1 epithelium: molecular basis and regulatory aspects. Ann Rev initially evoke chloride secretion but subsequently Physiol 62:535–572. suppress responses to other secretagogues and thus Benka ML, Lee M, Wang GR, Buckman S, Burlacu A, Cole L, contribute to the hyporesponsiveness seen in IBD. DePina A, Dias P, Granger A, Grant B. 1995. The thrombin Elucidation of the mechanisms underlying the control receptor in human platelets is coupled to a GTP binding protein of secretory function and responsiveness by PARs of the G alpha q family. FEBS Lett 363:49–52. will lead to a better understanding of ion transport Berschneider HM, Powell DW. 1992. Fibroblasts modulate processes in mucosal tissues in general. Furthermore, intestinal secretory responses to inflammatory mediators. J Clin Invest 89:484–489. the use of inhibitors of PAR1, or of signaling proteins, could lead to decreased secretory dysfunction during Bertog M, Letz B, Kong W, Steinhoff M, Higgins MA, Bielfeld- inflammatory conditions of the gut. In addition, Ackermann A. Fromter E, Bunnett NW, Korbmacher C. 1999. Basolateral proteinase-activated receptor PAR2 induces chloride elucidation of the intracellular stimulus-secretion secretion in M-1 mouse renal cortical collecting duct cells. coupling mechanisms that are induced by PAR J Physiol 521:3–17. activation is of importance. Given that PAR cleavage Bischoff SC, Lorentz A, Schwengberg S, Weier G, Raab R, Manns typically leads to increased intracellular calcium, it MP. 1999. Mast cells are an important cellular source of tumour appears unlikely that the route of apical chloride necrosis factor alpha in human intestinal tissue. Gut 44:643–652. secretion is through the cAMP-dependent CFTR. 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