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Published ahead of print on June 10, 2004, doi:10.1165/rcmb.2004-0035OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 31, pp. 302-308, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2004-0035OC

Expression and Functions of the Duodenal Peptide Secretin and its Receptor in Human Lung

Richard J. Davis, Keith J. Page, Gabriela J. Dos Santos Cruz, Dan W. Harmer, Peter W. Munday*, Sandra J. Williams, Joanna Picot, Tom J. Evans{dagger}, Robert L. Sheldrick, Robert A. Coleman and Kenneth L. Clark

Pharmagene Laboratories, Royston, Hertfordshire; and Department of Infectious Disease, Faculty of Medicine, Imperial College, Hammersmith Hospital, London, United Kingdom

Address correspondence to: Richard Davis, Pharmagene Laboratories, 2 Orchard Road, Royston, Hertfordshire SG8 5HD, UK. E-mail: richard.davis{at}pharmagene.com


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The physiological role of the duodenal peptide secretin is as a potent stimulant of electrolyte and water movement in pancreatic and biliary epithelium, via activation of G protein–coupled secretin receptors (hSCTR). However, the distribution and potential function of hSCTR in human lung has not previously been addressed. Using real-time quantitative reverse transcriptase–polymerase chain reaction profiling, in situ hybridization, and immunohistochemistry, we demonstrated that the hSCTR is abundantly expressed within the distal regions of human lung (tertiary bronchus and parenchyma), with negligible expression detected in more proximal regions (trachea, primary, and secondary bronchus). Expression was observed predominantly on the basolateral membrane of the bronchial epithelial layer, with some expression also observed in bronchial smooth muscle. 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 concentration-dependent relaxation of human tertiary bronchial smooth muscle. In summary, these data demonstrate that secretin receptors are present in human lung, and that activation of these receptors with human secretin potently stimulates concentration-dependent Cl– efflux from bronchial epithelial cells and bronchorelaxation.

Abbreviations: airway surface liquid, ASL • cystic fibrosis, CF • CF transmembrane regulator, CFTR • chloride ion, Cl– • Dulbecco's minimum essential medium, DMEM • digoxygenin, DIG • human secretin receptor, hSCTR • industrial methylated spirits, IMS • pituitary adenylate cyclase–activating polypeptide, PACAP • real-time, quantitative reverse transcriptase-polymerase chain reaction, rt-PCR • vasoactive intestinal peptide, VIP


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Secretin is a 27–amino acid peptide hormone that is secreted from duodenal S endocrine cells in response to acidic contents leaving the stomach, and potently stimulates electrolyte and water secretion from pancreatic and biliary epithelial cells. These effects result in neutralization of acidic chyme in the intestine, and regulation of bile volume and composition. Secretin's actions are mediated via interaction with the human secretin receptor (hSCTR), a cell surface, GS-coupled receptor, for which it exhibits nanomolar affinity. The hSCTR is a member of the class II G protein–coupled receptor family, which includes vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase–activating polypeptide (PACAP). Receptors of this family are structurally related, show sequence homology, and are all physiologically activated by peptide ligands (1).

Semiquantitative analysis of mRNA distribution has shown secretin itself to be widely distributed (2, 3) and the hSCTR to be highly expressed in the pancreas, kidney, and small intestine, with limited expression in the lung and liver, and trace levels in the brain, heart, and ovary (4, 5). The potential role(s) of secretin and its receptor in a number of these tissues is currently under investigation. Clinically, secretin is used diagnostically to assess pancreatic exocrine function, although its therapeutic potential has also been evaluated for autism (6).

Physiologically, secretin receptor activation in cholangiocytes prototypically stimulates channel-mediated chloride ion (Cl–) efflux and bicarbonate (HCO3–) secretion via cAMP activation of the cystic fibrosis transmembrane regulator (CFTR) Cl– channel and electroneutral Cl–/HCO3– exchangers (7). Secretin can also induce the microtubule-mediated translocation and exocytic insertion of vesicles containing functional aquaporin water channel proteins into the apical plasma membrane of cholangiocytes, resulting in concentration-dependent increases in osmotic movement into the bile duct lumen (8). The importance of this secretin function is illustrated after bile duct ligation in rats, which results in increased SCTR mRNA and protein expression (9) that correlates with increased function, leading to the state of hypercholeresis (10).

The aim of the present study was to quantify the level of hSCTR mRNA and examine its expression as a protein product throughout the human body in vitro, focusing in particular on the respiratory tract, as few studies have examined its expression or function in this system. To date, a minor VIP-preferring receptor population that can mediate secretin-stimulated cAMP accumulation in human lung membranes has been suggested (11). Unlike secretin, VIP is known to mediate a number of functional effects within the lung, including ionic movement (12) and bronchorelaxation (13). In this present study, potential functions of hSCTR stimulation in respiratory tissues were examined.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
All human tissue samples were removed with the informed consent of the donor or donor's next of kin, and with the approval of the appropriate local ethics committee. All tissues were accompanied by a brief clinical history of the donor, and were histopathologically assessed by an independent pathologist.

Polymerase Chain Reaction
Real-time, quantitative reverse transcriptase–polymerase chain reaction (rt-PCR) analysis of secretin or hSCTR mRNA in human tissue samples was performed as previously described (14). Total RNA samples were annealed to optimized concentrations of specific primer probe sets that exhibit no significant homology to any other mRNA as determined by Blast searches, and reverse transcribed using MuLV reverse transcriptase. Primer probe sets for hSCTR were: forward (5'-GACCAGCATCATCTGAGAGGCT-3') and reverse primers (5'-CCTTCGCAGGACCTCTCTTG-3'), and probe (5'-TCTCTGTCCGTGGGTGACCCTGCT-3'). For secretin, these were: forward (5'-CCCTGGGCCTATGGTTTCA-3') and reverse primers (5'-CATCTGGGCCGCAAGG-3'), and probe (5-TCCTTCTGCAGCAGCGCCAGCT-3'). Quantitative sequence detection was performed on the resulting cDNA using an ABI 7700 (Applied Biosystems, Warrington, UK).

In Situ Hybridization
An hSCTR cDNA template was generated using two rounds of rt-PCR and two sets of primers. The first set of primers (forward 5'-GCTGCTGGCCCTCTTCTGTG-3' and reverse 5'-CTTGCAGCCCGCCCTGT-3') amplified a 428-b amplicon that was subsequently purified by phenol:ethanol precipitation and used as template DNA for a second round of PCR. rt-PCR was performed using the Perkin Elmer (Warrington, UK) rTth (Thermus thermophilus) DNA polymerase kit and 1 ng total RNA (extracted using Trizol). PCR conditions were as follows; 60°C for 30 min, 94°C for 60 s followed by 40 cycles of 94°C for 30 s and 70°C for 30 s, followed by 60°C for 7 min and then a 4°C soak. The primers used in the second round of PCR (forward 5'-CAGAAATGGTTCCTTGTTCCGAAACTGCACACAGGATGGC-3' and reverse 5'-TGATGAAGTTGGACAGG GC ACGAAGGATGAAGGACACG-3') were modified at the 5'- to incorporate the minimum sequence of SP6 RNA polymerase required for efficient transcription of RNA (5'-ATTTAGGTGACACTATAGAGG-3'). These primers amplified a 290-base amplicon specific to hSCTR mRNA, excluding the SP6 transcription initiation sequences. The master mix and conditions for the second round of PCR were identical as before, with the exclusion of the initial reverse transcriptase step (60°C, 30 min).

Riboprobes were labeled using a digoxygenin (DIG)-labeled uridine 5'-triphosphate (UTP) using SP6 RNA polymerase mediated in vitro transcription. The transcription reaction took place over 2 h at 37°C, with additional SP6 RNA polymerase and RNAse inhibitor being added at 1 h (Roche, Welwyn Garden City, UK). The transcribed RNAs were treated with DNase and precipitated in ethanol/LiCl for 60 min at –80°C.

Localization of hSCTR mRNA was examined in 6-µm formalin-fixed/paraffin-embedded sections of tertiary bronchus using empirically optimized prehybridization steps for de-fatting, rehydration, and proteolysis before incubation in a standard DIG-riboprobe hybridization buffer (50% formamide, 5x Denhardt reagent, 10% Dextran sulfate, herring sperm DNA and 5x saline sodium citrate) overnight at 55°C containing 0.5 ng/µl sense or antisense riboprobe. Posthybridization stringency washing comprised serial washes in decreasing concentration of saline sodium citrate at 55°C (2x, 1x, 0.5x) and exposure to RNAse A to digest incomplete hybrids and excess riboprobes. Specific RNA–riboprobe hybrids were detected immunocytochemically using monoclonal anti–DIG-alkaline phosphatase (-AP) and -AP–mediated chromagenesis.

Antibody Specificity
A rabbit–anti-human polyclonal antibody (Ab-hSCTR) was generated to a 16–amino acid peptide sequence (ELSREQTGDLGTEQPV) corresponding to residues 48–63 of the hSCTR. Specificity of Ab-hSCTR was evaluated by immunocytochemistry of HEK293 cells transiently transfected with human hSCTR cDNA.

Immunohistochemistry
Paraffin wax–embedded tertiary bronchial lung sections, 6 µm thick, were cut using a microtome (RM215RTS; Leica, Milton Keynes, UK), and allowed to air dry to slides. Sections were dewaxed and rehydrated (2 x 5 min 100% xylene wash, 2 x 5 min 98–100% industrial methylated spirits [IMS] wash, 2 x 5 min 90% IMS wash), followed by 2 x 5 min phosphate-buffered saline wash. Antigen retrieval was by 0.1% chymotrypsin digestion (37°C, pH 7.8 in 0.1% CaCl2). Endogenous peroxidases, nonspecific protein binding sites, and endogenous biotin were all blocked in order (100% methanol, 0.3% H2O2, 20 min, RT; 1% normal horse serum, 15 min, RT; biotin block [Vector avidin/biotin blocking kit; Vector, Peterborough, UK], 5 min, RT; respectively). Ab-hSCTR (7 µg/ml), Ab-hSCTR plus 100 x blocking peptide, or purified polyclonal rabbit IgG (7 µg/ml) was applied (1 h, RT), followed by a universal biotinylated secondary antibody followed a tertiary horseradish peroxidase antibody (Vectastain Universal Elite Kit, Vector; 30 min at RT). Sections were incubated in diaminobenzidine (4 min, RT), and then counterstained in Mayers hematoxylin solution (1 min, RT). Slides were dehydrated (2 x 90% IMS wash, 5 min, RT, 2 x 98–100% IMS, 5 min RT; 2 x 100% xylene wash, 5 min, RT), then mounted and coverslipped with DePeX. For immunnofluorescence, an anti-rabbit Alexa-546–conjugated secondary antibody was used (1:200 in phosphate-buffered saline; Molecular Probes, Leiden, The Netherlands). Conventional fluorescence microscopy was performed using a Zeiss Axioplan 2, and images captured using Snapper software (Datacell, Austin, TX), under bright field settings. Sections were viewed at x1000 magnification using a laser scanning confocal microscope (Leica TSC-SP-MP, Leica). All measurements were taken at the same fluorescence settings to allow comparison of antibody fluorescence labeling between conditions.

Tissue Pharmacology
Human bronchial rings (1–3 mm internal diameter, 3–4 mm long) were prepared from sections of tertiary bronchus (defined as the third branching bronchus from the trachea). The preparations were mounted in 10 ml organ baths (Linton, Norfolk, UK) under a resting tension of 10 mN (1 g), bathed with oxygenated (5% CO2/95% O2) Krebs solution of composition (mM): NaCl (118.4), KCl (4.69), MgSO4.7H2O (1.18), KH2PO4 (1.18), D-glucose (11.1), NaHCO3 (25.0), and CaCl2.6H2O (2.5), at 37°C. Responses were recorded isometrically using transducers (Pioden, Canterbury, UK) coupled to an Apple Macintosh computer via a MacLab interface (Powerlab, Oxford, UK). After a 60-min equilibration period, tissues were precontracted via addition of cumulatively increasing concentrations of the muscarinic agonist, carbachol (0.01–100 µM), followed by the ß-adrenoceptor agonist isoprenaline (1 µM) to induce bronchodilatation. Preparations were washed, and in some tissues a single EC60/70 concentration of carbachol (1 µM) was added to contract the bronchus, before cumulative challenge with human secretin (0.01–10 µM). Once the final response to secretin had reached a plateau, all preparations were challenged with isoprenaline (1 µM) to obtain a maximum relaxation.

Bronchial Epithelial Cells
Human tertiary bronchus was incubated overnight at 4°C in sterile Hanks' buffered saline solution containing 0.1% protease, then rinsed with Dulbecco's minimum essential medium (DMEM) supplemented with fetal bovine serum (10%) to neutralize the protease, before epithelial cells lining the bronchial lumen were removed by gentle scraping. The recovered cells were twice washed in Hanks' buffered saline solution (supplemented with 100 U/ml penicillin G, 100 µg/ml streptomycin, and 100 µg/ml Fungizone) and centrifuged (120 x g, 8 min, 4°C). The subsequent cell pellet was resuspended in complete serum-free Keratinocyte medium (Sigma, Dorset, UK) supplemented with 50 U/ml penicillin G, 50 µg/ml streptomycin, and 50 µg/ml Fungizone, and plated into rat tail type 1 collagen–coated, 96-well black-walled, flat-bottomed plates (Fahrenheit, Milton Keynes, UK) at a density of 3 x 104 cells/well. Cell yield and viability assessment were performed using a hemocytometer. Cells were cultured to confluence (3–5 d) before use.

Cl– Efflux
Secretin stimulated Cl– efflux was determined using the Cl– chelating fluorescent dye, MQAE (Molecular Probes) and was essentially performed as previously described (15). In brief, culture media were removed, and confluent human tertiary bronchial epithelial cells were washed twice in DMEM before the addition of 95 µl DMEM and 5 µl of 80 mM MQAE (final concentration 4 mM) for 5 h at 37°C. Cells were washed (3 x 100 µl/well) in an extracellular buffer containing (in mM) NaCl (130), Hepes (10), MgSO4.7H2O (1), K2HPO4.3H2O (2.4), KH2PO4 (0.6), CaSO4.2H2O (1), and D-Glucose (1), pH 7.4 at 37°C, then incubated for 15 min at 37°C. If required, the nonselective Cl– channel blocker, 5-nitro-2-(3-phenylpropyl-amino)benzoic acid (NPPB, 100 µM), the secretin receptor antagonist, secretin (7–27) (10 µM), or the VIP receptor antagonist [4-Cl-D-Phe-6, Leu17]-VIP (10 µM) was also preincubated at this time. An unstimulated basal fluorescence reading was taken at excitation and emission wavelengths of 360 and 460 nm, respectively (Cytofluor multiwell plate reader, series 4,000; Applied Biosystems). Buffer was removed and 100 µl/well of secretin (1–1,000 nM) prepared in a Cl–-free buffer (containing components as previously described but with 130 mM NaNO3 substituted for NaCl). For studies examining the effects of antagonists, cells were stimulated with an approximate EC80 concentration of secretin (100 nM). The plate was read immediately and every 1 min for 16 min, by which time Cl– efflux had reached an equilibrated maximum. Cells were subsequently lysed (10 µl 10% sodium dodecyl sulfate, 2 min, room temperature) and re-read to determine total MQAE fluorescence. All 37°C incubations were performed in a 5%CO2/95% air environment.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
A highly sensitive and quantitative rt-PCR technique was used to measure mRNA expression of secretin and the hSCTR. Secretin mRNA was found to be widely distributed throughout the peripheral and central nervous systems, with highest expression observed within the duodenum, jejunum, choroid plexus, and testis (Figure 1A). hSCTR mRNA was found to be more discretely expressed within the body (Figure 1B). High expression was observed within the duodenum, jejunum, and pancreas, consistent with secretin's well established physiologic function. Of note, however, was the novel observation of high, regional expression of hSCTR mRNA within the tertiary bronchus, lung parenchyma, and pulmonary artery. In a larger number of donors, the latter data were substantiated, with negligible levels of hSCTR mRNA expression observed in the upper conducting airways and higher expression levels in the tertiary bronchus and lung parenchyma (Table 1).




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  		Figure 1. Quantitative mRNA expression profile of (A) secretin and (B) hSCTR in 72 macroscopically normal human tissues from all major regions of the body arranged as follows: reproductive (female), (1) ovary, (2) fallopian tube, (3) uterus myometrium, (4) cervix; reproductive (male), (5) prostate, (6) vas deferens, (7) testis; respiratory, (8) trachea, (9) lung parenchyma, (10) primary bronchus, (11) tertiary bronchus; endocrine, (12) pineal gland, (13) pituitary gland, (14) adrenal gland, (15) thyroid gland; hemochorial, (16) umbilical cord, (17) placenta; hemolymphoid, (18) spleen, (19) mononuclear blood, (20) tonsil; integumental, (21) skin, (22) breast; musculoskeletal, (23) skeletal muscle; apidose, (24) adipose; alimentary, (25) esophagus, (26) stomach fundus, (27) body, (28) antrum, (29) pyloric-canal, (30), duodenum, (31) jejunum, (32) ileum, (33) cecum, (34) colon, (35) rectum, (36) gallbladder, (37) pancreas, (38) liver; cardiovascular, (39) heart left atria, (40) left ventricle; (41) pulmonary artery, (42) mesenteric artery, (43) choroids plexus, (44) coronary artery, (45) renal artery, (46) cerebral artery; nervous, (47) cerebellum, (48) hippocampus, (49) medulla-oblongata, (50) locus-coeruleus (51) amygdala, (52) caudate, (53) hypothalamus (anterior), (54) hypothalamus (posterior), (55) cingulate (anterior), (56) cingulate (posterior), (57) frontal lateral cortex, (58) frontal medial cortex, (59) occipital cortex, (60) parietal cortex, (61) temporal cortex, (62) nucleus-accumbens, (63) substantia-nigra, (64) dorsal-raphe nucleus, (65) spinal cord, (66) dorsal root ganglia; urinary, (67) kidney cortex, (68) medulla, (69) pelvis, (70) ureter, (71) bladder, (72) trigone. Data for each tissue are represented as the geometric mean copy number of hSCTR per 100 ng cDNA from total RNA derived from three donors.

 

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  		TABLE 1. Expression of hSCTR mRNA in human respiratory tract

 
In situ hybridization was used to demonstrate the localization of hSCTR mRNA in human tertiary bronchus. Duodenal sections from a single donor were used as positive control tissues to demonstrate the specificity of antisense hSCTR riboprobes, with hybridization to the antisense probe observed in the ductal epithelial layer of the duodenum, but not in the smooth muscle, in keeping with the recognized physiological role of secretin (data not shown). In tertiary bronchus sections from six lung donors, specific hybridization to the hSCTR antisense riboprobe was primarily observed in the epithelial cell layer of the tertiary bronchus and mucosal inflammatory cells, with low levels in bronchial smooth muscle (Figure 2A). Of note, the expression of hSCTR mRNA was not uniform in all epithelial cells lining the bronchial lumen.



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  		Figure 2. (A) Localization of hSCTR mRNA as determined by in situ hybridization in representative sections of human tertiary bronchi (left panel). Similar patterns of hybridization were observed in all lung donors, where hSCTR mRNA was localized predominantly in the epithelial cell layer and smooth muscle. Letters denote lung regions, bronchial lumen (L), epithelial cell layer (E), and smooth muscle (SM). Negligible hybridization was observed in the presence of hSCTR sense probes (right panel). (B) hSCTR protein localization in representative tertiary bronchial sections (x200 magnification; left panel). Immunoreactivity to Ab-hSCTR was observed primarily in the epithelial cell layer, with lower levels seen in the bronchial smooth muscle. Negligible expression was observed in the presence of 10x blocking peptide (right panel). (C) Confocal micrograph of tertiary bronchus demonstrates hSCTR immunoreactivity primarily on the basolateral membranes of the epithelial cell layer (x1000 magnification). Letters denote lung regions: bronchial lumen, L; epithelial cell, E; basement membrane, B; and smooth muscle, SM. Right panel denotes lack of immunoreactivity in the presence of 10x blocking peptide.

 
To evaluate the protein expression of the hSCTR in human lung, a rabbit anti-human polyclonal antibody (Ab-hSCTR) was raised. The specificity of the antibody was demonstrated in cells transiently transfected with hSCTR cDNA. Intense immunoreactivity to Ab-hSCTR was observed at the plasma membrane of hSCTR transfected HEK293 cells but not in null-transfected cells (data not shown).

Immunohistochemical analysis of three tertiary bronchial lung sections demonstrated specific hSCTR immunoreactivity on epithelial cells lining the airways (Figure 2B), in good agreement with that observed from hSCTR mRNA in situ hybridization. Negligible expression was observed in the presence of blocking peptide (Figure 2B, right panel). Specific Ab-hSCTR staining appeared to be primarily localized to the epithelial plasma membrane, with lower, but significant levels of staining within the bronchial smooth muscle. These data were further confirmed by confocal microscopy; the most intense epithelial cell immunoreactivity was observed primarily on the basolateral epithelial cell membrane (Figure 2C). A greater donor variability and lower intensity of immunoreactivity to Ab-hSCTR was observed in the primary and secondary bronchus (n = 3 donors per region, data not shown).

Addition of human secretin to tertiary bronchial epithelial cells caused a concentration-dependent increase in MQAE fluorescence, indicative of Cl– efflux (Figure 3A). Secretin was demonstrated to exhibit nanomolar potency in epithelial cells derived from six donors (Log EC50 [M] –7.6 ± 0.2; 22.9 nM; Figure 3B). Maximal secretin-stimulated Cl– efflux was of a similar magnitude to that observed in response to prostaglandin E2 (PGE2, 10 µM) (81.3 ± 8.9%, 79.3 ± 14.1%, respectively). Incubation of the secretin antagonist, secretin (7–27; 10 µM), 30 min before stimulation of cells with 100 nM secretin resulted in a 62% mean reduction in the maximal response observed (31.7 ± 14.0%; n = 7). Incubation of cells with the VIP receptor antagonist [4-Cl-D-Phe6, Leu17]-VIP (10 µM) had no effect on maximal secretin-stimulated increases in MQAE fluorescence (76.1 ± 8.6%; n = 3). Agonist-stimulated and basal Cl– efflux were both found to be abolished by the addition of the nonselective Cl– channel blocker 5-nitro-2-(3-phenylpropyl-amino)benzoic acid (NPPB, 100 µM; n = 3; Figure 3C).





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  		Figure 3. (A) Secretin concentration-dependently stimulates an increase in MQAE fluorescence in human tertiary bronchial epithelial cells, indicative of agonist stimulated Cl– efflux. Representative data from a single experiment of six independent experiments are shown. Concentrations were secretin 1,000 nM (closed circles), 300 nM (open diamonds), 100 nM (inverted triangles), 30 nM (triangles), 10 nM (closed squares), 3 nM (open circles), 1 nM (closed diamonds); PGE2 100 nM (open squares). (B) Concentration-response curve to secretin (closed circles). Responses at each concentration were normalized to those observed to PGE2 (open circle, 10 µM) and are the mean ± SEM from three to six independent experiments on cells derived from six donors. (C) Secretin-mediated increases in MQAE fluorescence are inhibited by the secretin antagonist, secretin (7–27) (10 µM), but not the VIP receptor antagonist [4-Cl-D-Phe6, Leu17]-VIP (10 µM). Secretin responses were abolished in the presence of the nonselective Cl– channel blocker NPPB (100 µM, n = 3). Responses at each concentration were normalised to the maximal responses observed in each experiment and are the mean ± SEM from three to eight independent experiments on cells derived from eight donors.

 
The effect of secretin was examined in human tertiary bronchial rings under basal tone and in preparations precontracted with the muscarinic agonist, carbachol. Under both basal and precontracted conditions, secretin was equi-effective in concentration-dependently causing relaxation of bronchial smooth muscle, with Log EC50 values (M) of –6.7 ± 0.1 (208 nM) and –7.0 ± 0.1 (106 nM), respectively (Figure 4). The magnitude of the relaxatory responses were equal to 45 ± 13% and 46 ± 9%, respectively, of the relaxation elicited by the ß-adrenoceptor agonist isoprenaline.



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  		Figure 4. Secretin-induced relaxation of basal tone (squares) and carbachol (1 µM)-induced tone (circles) in human tertiary bronchial rings. Data are shown as the mean ± SEM of seven preparations from four donors, under each experimental condition, and are expressed as a percentage of the relaxation to isoprenaline (1 µM).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Secretin is a 27–amino acid peptide hormone that plays a key role in gastrointestinal, pancreatic, and biliary physiology via specific activation of hSCTR, a cell surface, GPCR for which secretin exhibits nanomolar affinity. The hSCTR is a member of a family of highly homologous GPCRs that includes the VIP receptor and PACAP, for which secretin can bind to and activate, albeit with low affinity (16, 25). Primarily, hSCTR stimulation leads to elevations in cAMP, although independent activation of alternative Ca2+ signaling pathways in the pancreatic epithelium and recombinant systems are described (17, 18). Physiologically, agonist binding to the hSCTR results in potent stimulation of electrolyte (Cl–, HCO3–, K+, and Na+) and water movement, and enzymatic secretion in the pancreatic and bile duct epithelium (7).

Although much is known about the actions of secretin in these epithelia, there is less information about the action of this hormone elsewhere in the human body. The aim of the present study therefore was to examine the distribution and function of secretin and the hSCTR within other tissues of the human body, focusing in particular on the respiratory tract. Using the highly sensitive and quantitative technique of rt-PCR, our studies examining the distribution of secretin mRNA in human tissues are in good agreement with previously published results from Northern blot analysis of human and rat prosecretin mRNA tissue expression, demonstrating widespread peripheral and CNS expression (2, 3). Particularly high expression was observed in the choroid plexus, where via a saturable transport system, secretin could reportedly cross into the brain (19). Negligible secretin mRNA expression was observed in the trachea, bladder, ureter, skin, and adipose tissues.

In contrast, previous semiquantitative analysis of the distribution of hSCTR mRNA suggests a more discrete expression profile. Abundant expression has been observed in the pancreas, kidney, and small intestine, with lower levels observed in the lung and liver, and trace levels in the brain, heart, and ovary (4, 5). Despite significant conservation of homology of secretin receptors across mammalian species, in some respects the distribution of SCTR mRNA in the rat is somewhat different from that in humans, such as abundant expression within the rat heart (20).

In the present study using quantitative rt-PCR, hSCTR mRNA was found to be expressed in tissues as previously described. However, to our surprise, we also observed abundant expression of hSCTR mRNA in human tertiary bronchus and lung parenchyma. In contrast, only negligible hSCTR mRNA expression was detected in the upper respiratory tract, trachea, primary and secondary bronchus, and nasal cavity (as exemplified by nasal polyps). Visualization of the distribution of hSCTR mRNA expression by in situ hybridization demonstrated localization primarily to the epithelial cell layer lining the lumen of the tertiary bronchus, and to a lesser, but still significant degree, the smooth muscle. hSCTR mRNA was also discretely expressed at moderate levels in a number of cortical brain regions, including the frontal cortex and the posterior but not anterior hypothalamus. In contrast, in the rat hypothalamus but not frontal cortex, functional secretin receptors are known to present (21). The functional relevance of this expression, and potential therapeutic benefit of secretin in the treatment of autism is currently under clinical evaluation (6).

We went on to determine whether expression of hSCTR mRNA reflected expression of receptor protein using an immunohistochemical approach. Recently, a variant spliceoform of hSCTR missing the third exon (amino acid residues 70–77) has been identified from ductal pancreatic adenocarcinomas (22). This has been shown to have no capability of binding secretin or signaling, but acts as a dominant-negative inhibitor of wild-type secretin function (23). In the present study, therefore, an hSCTR antibody was designed to residues containing part of this missing exon to ensure that observed immunoreactivity was only to that of a potentially functional hSCTR protein. In lung tertiary bronchial sections, hSCTR immunoreactivity confirmed the data obtained by in situ hybridization, showing that receptor protein was abundantly observed in the epithelial cell layer. Higher magnification confocal micrographs demonstrated the epithelial cell distribution of the hSCTR protein to be primarily on the basolateral epithelial membrane. Low levels of immunoreactivity were also observed in the bronchial smooth muscle. In the presence of a blocking peptide, all immunoreactive staining was abolished. Specificity of the antibody for the hSCTR was demonstrated using immunocytochemistry and immunoblotting of HEK293 cells transfected with human hSCTR cDNA. The present data suggest that hSCTR mRNA and protein expression are colocalized, primarily to the basolateral membrane of bronchial epithelial cells and bronchial smooth muscle.

A primary role of the lung epithelium is to determine the depth of the airway surface liquid (ASL) by actively regulating its ionic composition. Loss of function and regulation by the CFTR results in enhanced absorption of Na+ ions and a decrease in Cl– and HCO3– secretion by the bronchial epithelium. This in turn leads to a reduction in the depth of the ASL and impaired mucociliary clearance (24), as observed in cystic fibrosis (CF).

Given our identification of hSCTR expression in the bronchial epithelium, we examined whether secretin possessed a similar secretory function as observed in cholangiocytes. Indeed, secretin was shown to cause concentration-dependent increases in the fluorescence of the Cl– sensitive dye MQAE, indicative of Cl– efflux, from human cultured tertiary bronchial epithelial cells. The potency of secretin was unexpectedly ~10 fold less than may have been expected from previously determined secretin-mediated responses in recombinant cell lines (16) and the pancreatic epithelium (7). However, while secretin's affinity for its own receptor in human lung membranes has not been previously reported, secretin has been shown to exhibit over 1,000 fold lower affinity for the PACAP receptor than PACAP and ~ 200 fold lower affinity for VIP receptors than VIP (25), stimulating VIP receptor mediated cAMP accumulation only at micromolar concentrations (11). Taken together, these data would suggest that the observed response appears to be primarily hSCTR mediated, but the efficiency of receptor coupling may be sub-optimal, especially in comparison to cell based systems in which these receptors are overexpressed. Furthermore, incubation of cells with a secretin receptor antagonist secretin (7–27) reduced secretin-stimulated Cl– efflux, a response that was unaffected by the VIP receptor antagonist [4-Cl-D-Phe-6, Leu17]-VIP (26). These data strongly suggest that secretin directly stimulates its own receptor rather than acting via VIP receptors.

Secretin-stimulated Cl– efflux was channel mediated, as demonstrated by the abolition of responses in the presence of the nonselective Cl– channel blocker, NPPB. Secretin-stimulated epithelial Cl– efflux in non-lung tissue has been previously described. Cl– efflux is reportedly mediated via CFTR Cl– channels in gallbladder epithelial cells (27) and low conductance (13pS), cAMP-sensitive Cl– selective channels on bile duct epithelial cells (28). Non-CFTR mechanisms such as NPPB-sensitive large conductance Cl– channels on human immortalized intestinal epithelial cells and rat bile duct epithelial cells mediated via cAMP and Ca2+-sensitive pathways have also been suggested (29, 30). Other uncharacterized Cl– channels stimulated by secretin in the rabbit pancreatic acini are also described (31).

The most common genetic mutation causing CF results in defective trafficking and lack of insertion of {Delta}508 CFTR containing vesicles into the plasma membrane (32), which may account in part for the reduced Cl– efflux and lack of cellular ionic regulation by the CFTR (33). As such, therapeutic agents like 4-phenylbutyrate that direct CFTR transport to the plasma membrane have been clinically evaluated in CF (34). Interestingly, in rat cholangiocytes, basolateral secretin receptor stimulation can induce the microtubule-mediated translocation and exocytic insertion of intracellular vesicles co-containing functional CFTR, Cl–/HCO3– exchanger, and aquaporin 1 water channel protein into the apical plasma membrane. This results in a 60% concentration-dependent increase in osmotic water-selective movement into the bile duct lumen (8), facilitated by an increase in CFTR-mediated Cl–-driven HCO3– secretion (35). This does not appear to be a unique function of secretin, as VIP-stimulated apical Cl– efflux can be quantifiably accompanied by acute CFTR trafficking to the apical epithelial membrane in primary (36), but not in immortalized cell lines (37). Whether secretin stimulates CFTR-dependent trafficking to the plasma membrane of human airway cells, and induces ion and water transport to influence the depth of the ASL layer is currently under investigation.

The expression of the hSCTR mRNA and protein in human bronchial smooth muscle prompted us to examine potential bronchodilator functions of secretin. Secretin was found to be equi-effective in stimulating relaxation of either carbachol precontraction or endogenous tone in human bronchial rings, producing a maximal relaxation of approximately half of that observed to the ß-adrenoceptor agonist, isoprenaline. VIP activation of the VIP receptor is also known to relax human bronchus in vitro (13), with a similar nanomolar potency and efficacy to that observed with secretin in the present study. Based on the observed potency, this secretin-stimulated bronchorelaxation is likely to be mediated via the hSCTR, although a contributory role of secretin-activated VIP receptors cannot be excluded. In conclusion, we have, for the first time, identified the distribution and characterized the function of the secretin receptor in human lung. The potential therapeutic significance of secretin in the treatment of human respiratory disease is now under clinical investigation.


   Acknowledgments
 
The authors thank Mark Eagle, Colin Murdoch, and Joanne Mitchell for their excellent technical contributions.


   Footnotes
 
Conflict of Interest Statement: R.J.D., K.J.P, G.J.D.S.C., D.W.H., S.J.W., R.L.S., and K.L.C. are full-time salaried employees of Pharmagene. R.J.D. and K.J.P. have also published patents (which are currently not granted) relating to the use of secretin for respiratory disease submitted on the basis of data included in this paper. T.J.E. was principal investigator of a grant of £65,950 from Pharmagene plc to Imperial College London in 2002–2003, to carry out work presented in this paper. R.A.C. was a joint founder of Pharmagene and remains a full-time employee; his full financial interests are reported in the company's Annual Report (a copy will be provided on request). P.W.M. and J.P. have no declared conflicts of interest.

* Present affiliation: Inpharmatica, London, UK Back

{dagger} Present affiliation: Division of Immunology, Infection and Inflammation, University of Glasgow, Western Infirmary, Glasgow, UK Back

Received in original form February 2, 2004

Received in final form June 9, 2004


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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