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Departments of Surgery (G.W., W.W., X.Q., M.R.H., E.W.E., G.H.G.) and Human Biological Chemistry and Genetics (H.-Q.W.), The University of Texas Medical Branch, Galveston, Texas 77555; Department of Physiology (Y.A.), University of Toronto, Toronto, Ontario, Canada M5S 1A1; Shizuoka Cancer Center Hospital and Research Institute (T.M.), Shizuoka 420-0853, Japan; Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology (A.-M.O.), University of Bristol, Bristol, United Kingdom BS8 1TH
Address all correspondence and requests for reprints to: George H. Greeley, Jr., Ph.D., Department of Surgery, The University of Texas Medical Branch, 301 University Boulevard, Galveston, Texas 77555-0725. E-mail: ggreeley{at}utmb.edu.
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The purpose of the present study was to characterize apelin expression in the rat gastrointestinal (GI) tract, to localize apelin peptide in the rat and human stomachs by immunohistochemistry (IHC), to characterize the ontogeny of apelin expression and peptide in the rat stomach, and to assess the effect of apelin on gastric cell proliferation. Additionally, the influence of apelin on cholecystokinin (CCK) secretion, and the involvement of MAPK, protein kinase C (PKC), and intracellular Ca2+ signaling in apelin-induced CCK secretion in vitro were tested.
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Apelin expression in the adult rat GI tract and IHC localization of apelin peptide in the adult rat and human stomach
Apelin mRNA levels in the adult rat GI tract were characterized by Northern analysis. Ad libitum-fed adult male Sprague Dawley rats were killed, and the full thickness of the stomach fundus, duodenum, jejunum, ileum, and colon were extirpated, rinsed with cold saline, and extracted for total cellular RNA. Total cellular RNA samples were then processed for isolation of polyadenylated (A)+ [poly (A)+] RNA (5, 6). Apelin expression was also analyzed in extracts of the rat pancreas using rat pancreas poly (A)+ RNA that was prepared according to a previously described method (7). Apelin peptide in the adult rat and human stomachs was localized by means of IHC. Human stomach specimens were obtained from Brain and Tissue Banks for Developmental Disorders, University of Maryland (Baltimore, MD), and Birth Defects Research Laboratory, University of Washington (Seattle, WA).
Ontogeny of stomach apelin expression and peptide
Maximal apelin expression was found in the stomach fundus (i.e. oxyntic mucosa); therefore, the ontogeny of apelin expression was examined in the stomach. Fetal rat stomachs of both sexes at different stages of development [fetal d 16 (F16), F20, and F21] from timed-pregnant rats, stomachs of rat pups of both sexes at different ages [postnatal d 1 (P1), P3, P5, P7, P10, P16, and P24], and adult rat stomachs were harvested and extracted for RNA. In all dissections, care was taken not to include the pancreas. The entire fetal stomach was homogenized in a RNA extraction solution. For collection of pup and adult rat stomachs, the fundus was separated from the rumen and antrum, and the full-thickness fundus was homogenized in a RNA extraction solution. Total cellular RNA and then poly (A)+ RNA were prepared. Litters were born at approximately 22 d gestation and were kept with their mothers until 21 d postpartum. For fetal and some early postnatal samples the stomach specimens from three to four littermates were pooled to constitute one sample. For IHC localization of apelin peptide, stomach fundus specimens (P1, P6, P13, P20, P40, and P60) were fixed in buffered formalin and then processed for identification of apelin-containing cells.
Chemicals and peptides
All chemicals were obtained from Sigma Chemical Co. (St. Louis, MO) unless noted otherwise. Synthetic peptides were purchased from Bachem (Torrance, CA) and Phoenix (Belmont, CA) or synthesized by T. Mochizuki. The sequence of apelin-36 is LVQPRGPRSGPGPWQGGRRKFRRQRPRLSHKGPMPF. Pharmacological inhibitors of MAPK (PD98059) and PKC (GF109203X) were purchased from Calbiochem Novabiochem (San Diego, CA). Oligo dT cellulose was obtained from Stratagene (San Diego, CA). Media and calf and horse sera were purchased from Mediatech (Herndon, VA), Hyclone (Logan, UT), and Invitrogen (Carlsbad, CA), respectively.
RNA purification and Northern blotting analysis
All tissue samples were removed quickly after animals were killed and immediately homogenized in 4 M guanidinium isothiocyanate containing 25 mM sodium citrate (pH 7.0), 0.5% sodium lauroylsarcosine, and 0.1 M ß-mercaptoethanol. Extracts were frozen at -80 C until purification by ultracentrifugation over a cesium chloride cushion (2 ml, 5.7 M) (5, 6). Total cellular RNA samples were then extracted for poly (A)+ RNA using oligo dT chromography. Poly (A)+ RNA samples were separated on a 1% agarose gel (
10 µg/lane) in a 20 mM 3-[N-morpholino] propanesulfonic acid running buffer system (5, 6) and then transferred to a nylon membrane and subjected to Northern hybridization. Membranes were then stripped and rehybridized with a rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe as a reference mRNA species. 32P-labeled probes prepared from Strip-EZ RNA kits (catalog no. 1366, Ambion Inc., Austin, TX) were used for Northern hybridizations. The cDNA probe for rat apelin was supplied by Lee et al. (8). Expression levels of apelin were quantitated by phosphoimaging.
Real-time RT-PCR analysis of apelin expression
Real time RT-PCR assays were done as described (9). Real time RT-PCR assays were used to determine relative gene expression on an Applied Biosystems (Foster City, CA) 7000 sequence detection system. Applied Biosystems Assays-By-Design containing a 20x assay mix of primers and TaqMan MGB probes (6-FAM dye-labeled probe) were used for the target gene, rat apelin (accession no. AF179679), and a predeveloped rat 18S rRNA (VIC dye-labeled probe) TaqMan assay reagent (P/N 4319413E) was used for internal control. Primers were designed to span exon-exon junctions so as not to detect genomic DNA. Primers and probe sequence were searched against the Celera database. The probe and primer sequences were as follows: probe, TGGCACTCCACACACG; forward primer, GCTGCTCTGGCTCTCCTTGA; and reverse primer, CCATCTGGAGGCAGCATCA.
The efficiency of target gene amplification was validated using a reference amplification reaction. The absolute value of the slope of log input RNA amount vs. 
cyclin threshold (CT) was 0.06. One-step RT-PCR was performed with 40 ng total cellular RNA for both target gene and endogenous control. The TaqMan one-step RT-PCR master mix reagent kit (P/N 4309169) was used. The cycling parameters for one-step RT-PCR were as follows: RT at 48 C for 30 min, AmpliTaq activation at 95 C for 10 min, denaturation at 95 C for 15 sec, and annealing/extension at 60 C for 1 min for 40 cycles. Duplicate CT values were analyzed in Microsoft Excel using the comparative CT(CT) method as described by the manufacturer (Applied Biosystems).
IHC
Buffered formalin-fixed, paraffin-embedded tissue sections (5 µm) were deparaffinized and rehydrated by passage through xylene and graded ethanol solutions. After deparaffinization, slides were treated with 1% H2O2 in PBS for 15 min, followed by microwave antigen retrieval at 100 C for 10 min in Dako target retrieval solution (Dako Corp., Carpinteria, CA) in an H2800 microwave processor (Energy Beam Sciences, Inc., Agawam, MA). After sequential 15-min incubations with 0.1% avidin and 0.01% biotin (Vector Laboratories, Inc., Burlingame, CA) to block endogenous avidin and biotin, slides were then incubated in 0.05% casein (Sigma) in 0.05% Tween 20 (Dako) in PBS for 30 min to block nonspecific protein binding. Primary rabbit antisera for apelin-36 (Phoenix) were applied to sections at a 1:300 dilution for 60 min. This apelin antibody recognizes all carboxyl-terminal fragments. Rabbit serum Ready-to-Use (InnoGenex, San Ramon, CA) was applied as a negative control. Biotinylated F(ab')2 fragment of swine antirabbit Igs (Dako) served as the secondary antibody and was detected by streptavidin-horseradish peroxidase and colorized by diaminobenzidine (Dako). All antibody incubations and detection procedures were performed on a Dako Autostainer.
Slides were counterstained with Mayer’s modified hematoxylin (Poly Scientific, Bay Shore, NY) before mounting and viewed under a Nikon Eclipse E600 microscope. Images were captured with a Nikon DXM1200 digital camera and ACT-1 (version 2.00) program.
Cell culture
A murine intestinal enteroendocrine cell line (STC-1) (10) that expresses and secretes CCK was grown in DMEM containing 2.5% fetal bovine serum, 15% horse serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were maintained at 37 C in 5% CO2, and media were changed every 3–4 d. Cells were trypsinized, plated in six-well culture plates, and allowed to reach 60–80% confluence. On the day of the experiment, cells were washed twice with media (without serum) and then incubated with test agents in media. A human gastric epithelial cell line (SIIA) was used to study the influence of apelin on cell proliferation. SIIA cells were cultured in HAM’s F-10 media with 5% fetal bovine serum. To examine the influence of apelin on cell proliferation, SIIA cells were cultured with synthetic apelin-36 or apelin-13 (10-8 M). Cells were plated in six-well plates initially at a density of 15 x 103 cells per well. Apelin was added daily starting 24 h after plating. Cells were counted daily.
RIAs
Media levels of CCK were measured using a standard RIA protocol (11) and an antiserum that detects CCK (antibody 5135, gift of J. Walsh, UCLA, Los Angeles, CA). The intra- and interassay coefficients of variance are 5–7 and 10–12%, respectively.
Calcium imaging
Real-time recording of intracellular Ca2+ concentration ([Ca2+]i) was done in single STC-1 cells using a method reported previously (12, 13). STC-1 cells were plated on glass coverslips (25 mm) at a density of approximately 1.5–3 x 105 cells per coverslip, cultured for 48 h, washed with KRH (125 mM NaCl, 5 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 2 mM CaCl2, 6 mM glucose, and 25 mM HEPES, pH 7.4) and loaded with 2 µM fura-2 AM (Molecular Probes, Eugene, OR) for 50 min at 25 C. Loaded cells were washed three times with KRH and incubated in KRH plus 0.1% BSA for 60 min at 25 C in the dark. Cells were then exposed to apelin or bombesin and imaged using a Nikon Diaphot inverted microscope (Garden City, NJ). The microscope is coupled to a dual monochromator system via a fiber optic cable (Photon Technology International, South Brunswick, NJ). Fluorescence was detected using an intensified charged coupled device camera (Dage-MTI Inc., Michigan City, IN), and images were processed using ImageMaster software (Photon Technology).
Statistics
Results are shown as means ± SE. Data were analyzed by a one-way or two-way ANOVA followed by the Newman-Keuls test where pertinent. Differences with a value of P < 0.05 were considered significant.
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). Apelin mRNA levels were significantly (P < 0.05) higher in the ileum when compared with apelin mRNA levels in the duodenum and colon. Apelin is not expressed in the rat pancreas (data not shown). IHC examination of the adult rat stomach showed abundant apelin peptide in the cytoplasm of epithelial cells in the oxyntic mucosa (Fig. 2). A high-power magnification indicates that apelin peptide is stored in vesicle-like structures and that apelin staining is localized adjacent to the lumen of the gastric glands (Fig. 3). IHC examination of the adult human stomach showed apelin staining in the gastric epithelium (Fig. 2). IHC examination of the rat duodenum failed to identify apelin-containing cells (data not shown).
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). Apelin mRNA levels in fetal and postnatal stomach samples did not differ significantly. IHC did not detect apelin peptide in stomachs harvested from pups at 2, 7, and 13 d of age (data not shown). Apelin-containing cells were observed initially at 20 d of age in the rat stomach (Fig. 5), and apelin staining intensity (i.e. number of apelin-containing cells) in the stomach increased progressively until adulthood. Apelin peptide was not detectable by IHC in the human fetal stomach (data not shown).
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). The proliferative action of apelin-36, at 10-8 M, was also tested. The stimulatory action of apelin-36 (data not shown) was significantly less (105–108% of controls) when compared with apelin-13 (120–145% of controls).
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). Apelin at 10-7 M and 10-9 M caused 2-fold and 1-fold elevations in media CCK levels, respectively. Apelin at 10-9 M did not increase CCK levels when compared with control treatment. Apelin-19 and -12 also stimulated CCK secretion equipotently with apelin-13 (data not shown).
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). In contrast, exposure of STC-1 cells to bombesin produced a calcium response characterized by an initial rapid elevation in [Ca2+]i followed by a second sustained elevation in [Ca2+]i that decreased to resting levels over 10–20 min. Neither a brief exposure of STC-1 cells to apelin before bombesin treatment nor exposure of cells to apelin with bombesin simultaneously altered the bombesin-induced elevation in [Ca2+]i (data not shown).
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30% homology). Apelin has no sequence homology with angiotensin II, and angiotensin II does not have acidification-promoting activity in cells expressing the APJ receptor, indicating that the APJ receptor is not related functionally to the angiotensin II receptor (2). Like many other regulatory peptides, pharmacological studies indicate that apelin has multiple biological activities. Reported actions for apelin include inhibition of proinflammatory cytokine production by mouse spleen cells (4), chemotactic activity on CHO-A10 cells (15), and lowering of blood pressure (8) and stimulation of drinking behavior in rats (16), and apelin is thought to function as a coreceptor with CD4 in the process of HIV infection (1, 8, 15, 17). The effects of apelin on the GI tract have not been investigated previously, although there is an abstract describing its CCK-releasing activity (18).
Apelin is produced in several tissues of the body, including the heart, brain, lung, pregnant and lactating breast, and GI tract (4, 19). In the present study, Northern analysis demonstrated the highest apelin expression in the stomach fundus with lower expression levels in the intestine. IHC shows abundant apelin peptide in the glandular epithelium of the rat and human stomachs. Although apelin peptide was not detected in the rat duodenum by IHC (data not shown), production of marginal levels of apelin peptide in the intestine may exceed the lower limit of sensitivity for IHC. The abundant pattern of apelin peptide staining in the stomach mucosa suggests that apelin is produced in multiple gastric mucosa cell types. Although we do not characterize the cell types in the gastric epithelium that produce apelin, preliminary findings show that chromogranin A, a marker of enteroendocrine cells (20) is colocalized with apelin in a portion of the gastric epithelial cells, indicating that some of the gastric apelin cells are endocrine in nature.
Northern analysis and real-time RT-PCR showed that stomach apelin expression was activated early during development because stomach apelin mRNA levels in the rat fetus were elevated at d 16 of gestation and remained elevated during the perinatal period. Despite the high expression levels of apelin in the fetal and postnatal stomachs, apelin peptide was detectable by IHC only at weaning and increased progressively with age. The finding that gastric stores of apelin peptide increased at the time of weaning suggests that apelin peptide synthesis is activated by the switch from milk to solid food. Interestingly, short-term primary cultures of dispersed stomach cells harvested from rat pups 5–17 d of age showed apelin peptide production and secretion within 2–3 d of culture (data not shown), suggesting that translation of stomach apelin is inhibited during the perinatal period. The inhibitory mechanism may involve apelin itself because nursing pups consume large amounts of apelin (300–600 ng/ml) (Greeley, Jr., G. H., unreported data) contained in milk ingested from their lactating mothers. The observations that rat pups consume large amounts of apelin and that there is a developmental increase in apelin peptide in the stomach after weaning suggest that apelin has an important developmental role in the GI tract. Because the present study shows that apelin stimulates stomach cell proliferation in vitro, one obvious possibility is a tropic role in regulation of GI epithelial proliferation. Interestingly, our findings show that both apelin-36 and apelin-13 stimulate cell growth; however, apelin-36, the parent peptide of apelin-13, is less potent.
Interestingly, the developmental appearance of stomach apelin peptide differs when compared with the developmental patterns of stomach ghrelin and gastrin and an ileo-colonic hormone, peptide YY. In the rat, gastrin, ghrelin, and peptide YY peptides appear in late gestation initially and increase to maximal levels at approximately 18–21 d of age (5, 6, 21). Of further interest is the finding that apelin expression in the lung shows a developmental pattern opposite to that observed for the stomach (data not shown). In contrast to the relatively high expression levels of apelin in the fetal and postnatal stomachs, apelin expression is low and increases progressively with age after birth in the lung. Together, the findings that apelin expression is maximally activated in the fetal stomach and pregnant breast, whereas apelin expression in the lung is increased postnatally, indicates that transcriptional regulation of apelin is complex and tissue specific.
The present findings show that apelin-13 stimulates CCK release from a murine small-intestinal cell line (STC-1) that expresses and secretes CCK. The finding that apelin stimulates CCK secretion from STC-1 cells indicates that apelin can act directly on CCK cells to stimulate CCK secretion. Although the apelin receptor, the APJ receptor, has not been localized to CCK cells, the present findings implicate that the APJ receptor is found on CCK cells. Pharmacological blockade of the MAPK pathway but not the PKC pathway inhibited apelin-13-induced CCK secretion from STC-1 cells, indicating that the MAPK pathway is involved in apelin-induced CCK secretion. It should be pointed out that the PKC inhibitor GF-109023X is known to inhibit both conventional and novel PKC isoforms (22), leaving the possibility that atypical PKC isoforms might participate in the apelin-induced CCK secretion from STC-1 cells. The present findings also indicate that apelin-induced CCK secretion is not dependent upon a concurrent elevation in [Ca2+]i because intracellular calcium levels of STC-1 cells do not change when challenged by apelin exposure. This finding agrees with an earlier report showing lack of involvement of [Ca2+]i in apelin activation of the APJ receptor (23). Apelin-13 was used in the CCK secretion studies because apelin-36 failed to activate CCK secretion in vitro from STC-1 cells. Although apelin processing has not been described, apelin-36 presumably represents a parent form of apelin-13. Earlier reports indicate that the smaller apelin variants such as apelin-17 and apelin-13 show a greater biological activity when compared with apelin-36 in the extracellular acidification assay (2, 15). We have also found that apelin-19 and apelin-12 can stimulate CCK secretion potently from STC-1 cells (Greeley, Jr., G. H., unreported data).
The findings that apelin stimulates CCK secretion and the measurement of apelin peptide in a luminal perfusate of the rat intestine (data not shown) suggests that apelin is a potential luminal factor, i.e. a lumone that stimulates CCK secretion. Previously documented luminal CCK-releasing factors include monitor peptide, luminal CCK-releasing factor, and diazepam-binding inhibitor (24, 25, 26). The present IHC data indicate that apelin is not produced by the intestine; however, apelin may travel by a luminal route from the stomach to the intestine to stimulate CCK release. Other gastric peptides, including proxenopsin and leptin, have been shown to be secreted into the gastric lumen where they are either processed by pepsin or travel to the duodenal lumen to stimulate CCK secretion (27, 28, 29, 30).
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Footnotes |
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Abbreviations: [Ca2+]i, Intracellular calcium concentration; CCK, cholecystokinin; CT, cyclin threshold; F16, fetal d 16; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GI, gastrointestinal; IHC, immunohistochemistry; P1, postnatal d 1; PKC, protein kinase C; poly (A)+, polyadenylated (A)+.
Received August 26, 2003.
Accepted for publication November 18, 2003.
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, P < 0.05 vs. duodenum, jejunum, and colon.
