Identification and functional assay of an extracellular calcium-sensing receptor in Necturus gastric mucosa

Robert R. Cima1, Ivan Cheng1, Mary E. Klingensmith1, Naibedya Chattopadhyay2, Olga Kifor2, Steven C. Hebert3, Edward M. Brown2, and David I. Soybel1

1 Department of Surgery, West Roxbury Veterans Affairs Medical Center, and the 2 Renal and 3 Endocrine-Hypertension Divisions of the Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

In mammals and amphibians, increases in extracellular Ca2+ can activate bicarbonate secretion and other protective functions of gastric mucosa. We hypothesized that the recently cloned extracellular Ca2+-sensing receptor (CaR) is functioning in the gastric mucosa. In Necturus maculosus gastric mucosa, reverse transcription-polymerase chain reaction using primers based on previously cloned CaR sequences amplified a 326-bp DNA fragment that had 84% nucleotide sequence identity with the rat kidney CaR. Immunohistochemical localization of the CaR using specific anti-CaR antiserum revealed its presence on the basal aspect of gastric epithelial cells. In microelectrode studies of Necturus antral mucosa, exposure to elevated Ca2+ (4.8 mM) and the CaR agonists NPS-467 and neomycin sulfate resulted in significant hyperpolarizations of basal membrane electrical potentials and increases in apical-to-basal membrane resistance ratios. Circuit analysis revealed that these changes reflected specific decreases in basolateral membrane resistance. Inhibition of prostaglandin synthesis using indomethacin significantly attenuated these effects. We conclude that the CaR is present and functioning in Necturus gastric antrum.

surface epithelium; immunohistochemistry; circuit analysis; electrophysiology

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

EXTRACELLULAR CALCIUM LEVELS may regulate a variety of epithelial transport mechanisms and mucosal defense properties in the gastric mucosa. Previous studies in mammalian and amphibian models of gastric mucosa have shown that increases in extracellular Ca2+ can stimulate both H+ secretion from the acid-secreting oxyntic glands and HCO<SUP>−</SUP><SUB>3</SUB> secretion from the neighboring gastric surface epithelium (8, 9, 13, 25). In in vitro studies of gastric surface epithelium in Necturus maculosus, we observed marked increases in permeability properties and electromotive forces (EMFs) of the basolateral membranes of the gastric surface cells within 30 s of exposure to increases in Ca2+ in the serosal perfusate (20). Circuit analysis and ion substitution protocols indicated that these electrophysiological alterations were attributable to changes in basolateral conductance of K+ (20). Similar changes in basolateral conductance and potential were elicited by Sr2+, by La3+, and somewhat paradoxically, by high levels (5 mM) of Ba2+ in the serosal perfusate. These latter observations suggested that the effects of Ca2+ were elicited by its extracellular interactions and not by its passage into the cell interior.

The mechanisms permitting the gastric epithelium to detect and respond to changes in extracellular Ca2+ have remained uncharacterized. Recently, however, Brown et al. (3) have cloned and characterized an extracellular Ca2+-sensing receptor (CaR) from bovine parathyroid tissue. This ~120K cell surface receptor, which belongs to the superfamily of G protein-coupled receptors, is activated by changes in the extracellular concentrations of Ca2+, as well as Mg2+, Gd3+, and the polyvalent cation neomycin sulfate. With the use of Northern analysis, the tissue distribution of this receptor was also shown to include mammalian cerebral cortex, cerebellum, thyroid, renal cortex, and renal outer medulla. With the recent identification of this extracellular CaR and its wide tissue distribution, we hypothesized that the CaR may be present and functioning in the gastric mucosa. If so, then it may be responsible for initiating the cellular processes activated by changes in Ca2+ levels in the gastric mucosal subepithelial space.

For this study, we used the amphibian Necturus maculosus as our model of the gastric mucosa. Necturus was chosen because nearly all of the effects of extracellular Ca2+ on the transport and permeability properties of the gastric mucosa have been measured in amphibian as well as in mammalian preparations (6, 9, 20). Also, the cellular and epithelial electrophysiological properties of the Necturus gastric epithelium have been systematically characterized under a variety of experimental conditions including increases in nutrient Ca2+ (2, 10, 20, 22). In this study, we determined whether the CaR was present in the gastric mucosa of Necturus by subcloning and sequencing a polymerase chain reaction (PCR) product from the gastric mucosa using CaR-specific primers. In addition, immunohistochemical staining using specific anti-CaR antibodies was used to localize the CaR to the basal surfaces of the epithelium in the Necturus gastric mucosa. To determine functional activity of the CaR, we measured the intracellular electrophysiological changes induced in the surface cells in response to various CaR agonists. Intracellular microelectrode techniques were used to perform an equivalent intraepithelial circuit analysis of the antral surface cell in response to a CaR agonist, neomycin sulfate. Finally, interactions with other receptor-activated pathways were explored by evaluating effects of CaR activation after pretreatment with the cholinergic receptor antagonist atropine or the prostaglandin synthesis inhibitor indomethacin.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Preparation

Necturi (Nasco Biology Division, Ft. Atkinson, WI) were kept in filtered water at 4-8°C. Animals were anesthetized by immersion in oxygenated tank water containing 1% tricaine-methanesulfonate (Sigma Chemical, St. Louis, MO). For Northern analysis and reverse transcription (RT)-PCR, Necturus gastric antral and fundic mucosae were harvested by gross dissection and snap frozen in liquid nitrogen and stored at -70°C until further use. Before freezing, the mucosa was rapidly separated from the underlying muscularis by sharp dissection. For in vitro microelectrode studies, tissues were isolated and mounted as described previously (20-23). In brief, antral mucosae were isolated from underlying muscularis and mounted, mucosa side up, in a modified Ussing chamber. The mucosal perfusate (volume 0.5 ml) was continuously exchanged at a rate of 5-7 ml/min. The nutrient perfusate (volume 1.8 ml) was exchanged at a rate of 12-14 ml/min.

RNA Extraction and RT-PCR Analysis

To determine if transcripts for the CaR gene are present in the gastric mucosa of Necturus, gastric mucosa RNA was analyzed for expression of this gene product. Total cytoplasmic RNA was extracted from Necturus gastric mucosa following the method described previously (3, 5, 14, 19). Briefly, 1 g of tissue was homogenized in 8 ml of a solution containing 4 M guanidine isothiocyanate, 25 mM sodium citrate, and 1.12 g/ml beta -mercaptoethanol. The homogenate was layered onto 4 ml of a cushion of 5.7 mM CsCl and 25 mM sodium acetate and spun at 32,000 revolutions/min at 18°C for 18 h. The RNA pellet was resuspended into water and concentrated by precipitation with sodium acetate and ethanol. Total RNA (5 µg) was subjected to single-strand cDNA synthesis in a reaction volume of 20 µl using 25 pmol random hexamer primers, 50 mM tris(hydroxymethyl)aminomethane (Tris) · HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 0.5 mM deoxyribonucleotide triphosphates (dNTPs), and 200 U avian myeloblastoma virus reverse transcriptase (Invitrogen, San Diego, CA) at 42°C for 1 h. The resultant first-strand cDNA was used for the PCR reaction. PCR was performed in a total volume of 50 µl in a buffer solution containing 10 mM Tris · HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.25 mM dNTPs, and 10 U of cloned Pfu polymerase (Stratagene, La Jolla, CA), using 0.25 nM of sense and antisense primers with the sequences described below. The optimum temperature cycling protocol was determined to be 92°C for 1 min, 55°C for 1 min, and 72°C for 1.5 min for 30 cycles, using a programmable thermal cycler (Omnigene, Hybaid Instruments, Holbrook, NY). The products were then subcloned into the pCR-Script vector (Stratagene) following the supplier's recommendations. The first set of primers was based on a consensus sequence between rat kidney CaR and a partial clone of the Xenopus kidney CaR (M. Bei, unpublished observations): 5'-GGACCGAGCCCTTT-GGAATCGC-3', 5'-CGTAGGTGCTGGCATAGGCTGG-3'. RTPCR performed with this primer pair yielded a PCR product from Necturus gastric antrum that was 75% identical in its nucleotide sequence to that of RaKCaR. A second, nested set of primers was then designed based on the sequences of the presumptive gastric Necturus RT-PCR product and the Xenopus CaR (19): 5'-CTTGACCTCCTTTGTCCTGG-3', 5'-CGAAGGTGCTCAGAAAGACC-3'. PCR performed with the latter primers yielded the expected 326-bp products from both the gastric fundus and antrum of Necturus.

Nucleotide Sequencing of the Clone

Bidirectional sequencing was performed using the dideoxy chain termination method (3, 19) with an Applied Biosystems model 373A automated sequencer (Department of Genetics, Children's Hospital, Boston, MA). Further nucleotide and amino acid analyses were carried out using GeneWorks software (version 2.3.1, IntelliGenetics, Mountain View, CA).

Immunohistochemical Staining

Immunohistochemistry was performed using techniques modified from those described previously (11, 12). Frozen sections were prepared using a cryostat (International Equipment minotome, -20°C) and were postfixed in acetone for 10 min at -20°C.

Endogenous peroxidase inhibition was carried out by incubating the sections in DAKO peroxidase blocking reagent (DAKO, Carpenteria, CA) for 10 min. Nonspecific immuno-cross-reactivity was blocked by DAKO protein block serum-free solution (DAKO) for 1 h. The sections were then incubated overnight at 4°C with primary (protein A purified) anti-CaR antiserum 4641 at a concentration of 10 µg/ml in blocking solution (DAKO). Characterization of the anti-bovine CaR antibodies has been detailed elsewhere (11, 12, 14). Control sections were prepared by incubation with protein A-purified preimmune serum and with anti-CaR antiserum preabsorbed with the synthetic CaR peptide (amino acids 215-237, 10 µg/ml) against which the antibody was raised. After the sections were washed three times with 0.5% bovine serum albumin in phosphate-buffered saline (PBS) for 20 min each, peroxidase-coupled goat anti-rabbit immunoglobulin G diluted 1:200 (Sigma Chemical) was added and incubated for 1 h at room temperature.

The slides were then washed in PBS three times for 20 min each, and the color reaction was developed using the DAKO AEC substrate system (DAKO) for ~5 min. The color reaction was stopped by washing three times in water. The peroxidase-stained specimens were examined by light microscopy, and photomicrographs were taken at a magnification of ×400 and ×630.

Microelectrode Techniques

Solutions. As previously described, the control solution used as the mucosal and nutrient perfusate during the microelectrode studies is amphibian Ringer solution (106.6 meq/l Na+, 4.0 meq/l K+, 1.8 mM Ca2+, 0.8 mM Mg2+, 101.9 mM Cl-, 13.9 mM HCO<SUP>−</SUP><SUB>3</SUB>, and 1.0 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 5% CO2--95% O2 gas) (2, 3, 10, 13). In the elevated Ca2+-Ringer solution, the Ca2+ concentration was increased by 3.0 mM to a total Ca2+ concentration of 4.8 mM. Solutions of the stereoisomers of the CaR agonist NPS-R-467 and NPS-S-467 (generously provided by Dr. Edward Nemeth, NPS Pharmaceuticals, Salt Lake City, UT) were prepared to a final concentration of 3.0 × 10-5 M in control Ringer solution ([Ca2+] = 1.8 mM) (24). The R-isomer of NPS-467 is known to be 100 times more potent as a CaR agonist than the S-isomer (24). Neomycin sulfate, another CaR agonist, was used both to measure a dose-response relationship at various nutrient concentrations of neomycin (0.25, 0.5, or 1.0 mM) in a Ringer salt-containing solution ([Ca2+] = 1.8 mM) and to perform an intraepithelial circuit analysis. In all subsequent experiments with neomycin, the concentration of neomycin in the nutrient perfusate was 1 mM. All solutions were titrated to pH 7.3. Unless specifically noted, all reagents used in these experiments were purchased from Sigma Chemical.

Tissue potential and resistance profile. The transepithelial potential (Vms, where m is mucosa and s is serosa) was measured using a high-impedance (>1012 Omega ) Duo-223 electrometer (World Precision Instruments, Saratoga, FL) providing digital readout. The nutrient solution was grounded, and the mucosal solution was connected to the electrometer by means of a 3.5% agar-Ringer bridge in contact with an Ag/AgCl pellet. Borosilicate glass capillaries were pulled on a two-stage puller (Sutter Instruments, Navato, CA) and back-filled with 3 M KCl. Electrodes with tip potentials and resistances of 15-60 MOmega were used for intracellular impalements. Impalements were obtained using remote-control micromanipulators (Narishige, Tokyo, Japan) on a vibration-free table (Micro G, Woburn, MA). As previously described, criteria for a satisfactory impalement included the following: 1) an abrupt change in voltage on entry into the cell, 2) a stable baseline for 60-90 s with a fluctuation of <2 mV while inside the cell, and 3) a return to the original baseline upon withdrawal of the electrode from the cell (17, 18, 21). The basolateral cell membrane potential (Vcs) was measured with reference to the nutrient solution. Apical membrane potential (Vmc) was determined from Vmc = Vcs - Vms. All measurements were corrected for junction potentials (17, 18, 21, 22).

Tissue electrical profile and circuit analysis. To measure transepithelial and cell membrane resistances, current pulses of 20 mA and 1-s duration were applied across the mucosa using a Pulsar 4i stimulator (Frederick Haer, Brunswick, ME). Transepithelial resistance (RT) was determined from the magnitude of the current-induced deflection of the transepithelial potential divided by the current density. The ratio of membrane resistances (Ra/Rb, where Ra is resistance of the apical membrane and Rb that of the basolateral membrane) was determined from the voltage divider ratio (Vmc/Vcs) as described previously (17, 18, 21). The same experimental protocol was performed for all calculations of tissue potential and resistance profiles (Vms, Vmc, Vcs, RT, and Ra/Rb). As previously described, the values of Ra, Rb, and the resistance of the paracellular pathway (Rs) were determined during a brief exposure of the tissue to a mucosal solution containing 10-4 M amiloride, a reversible blocker of apical Na+ conductances in this tissue (17, 21). The assumptions supporting this circuit analysis method have been tested previously and included direct comparison with measurements of the circuit resistances using cable analysis (20, 21).

Values for the EMF generated at the apical (Ea) and basolateral (Eb) cell membrane were calculated as previously described (16, 18, 20). Under control conditions, with both sides of the tissue bathed by identical Ringer solutions, the EMF across the paracellular, or shunt, pathway can be assumed to be 0 mV (18, 20). The apical and basolateral EMFs are then calculated from the following
<IT>E</IT><SUB>a</SUB> = <IT>V</IT><SUB>mc</SUB> − <IT>V</IT><SUB>ms</SUB>(<IT>R</IT><SUB>a</SUB>/<IT>R</IT><SUB>s</SUB>) (1)
<IT>E</IT><SUB>b</SUB> = <IT>V</IT><SUB>cs</SUB> + <IT>V</IT><SUB>ms</SUB>(<IT>R</IT><SUB>b</SUB> /<IT>R</IT><SUB>s</SUB>) (2)
Derivations of these expressions have been given previously (16, 18). These expressions are applicable when ion composition and pH of mucosal and nutrient perfusates are identical.

Experimental Protocols

In all experiments, tissues were mounted and allowed to equilibrate for 30 min, with both sides perfused with control Ringer solution. All tissues were evaluated for changes in potential, resistance, and EMF across individual cell membranes in response to changes in nutrient perfusate composition. In cases where a tissue was sequentially exposed to different agents, there was a recovery period of 30 min, during which both sides were perfused with control Ringer solution. Four series of microelectrode experiments were performed. The first series of experiments quantified the magnitude of electrophysiological changes induced in the surface cell after nutrient exposure to elevated Ca2+ or to the stereoisomers of the CaR agonist NPS-467. The second evaluated the dose-response relationship between exposure to the CaR agonist neomycin and electrical profile of the surface cell. In the third series of experiments, a circuit analysis was performed during exposure to neomycin (1 mM) in the nutrient perfusate. At the peak response to neomycin, amiloride (10-4 M) was added to the mucosal perfusate, and the circuit analysis was performed. During the last series of experiments, the tissue was incubated with atropine (10-4 M) or indomethacin (10-4 M) before nutrient neomycin exposure to determine whether the electrophysiological changes induced by CaR activation were altered by pretreatment with these agents.

Data Analysis and Statistical Methods

Data were summarized and analyzed using a standard software statistical package (Excel, Microsoft, Seattle, WA). Results are expressed as means ± SE. Comparisons of paired measurements performed in the same group of tissues were analyzed using an analysis of variance with significance set at P < 0.05.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Analysis of CaR Gene Expression

We could not identify specific CaR transcripts in Necturus gastric mucosa by Northern analysis, presumably because expression of the CaR was limited to a subset of cells (e.g., see Fig. 2) and the overall level of expression was low. Therefore, 5 µg of total RNA from the gastric antral and fundic mucosa of Necturus were subjected to RT-PCR analysis with the two sets of PCR primers described in MATERIALS AND METHODS and yielded similar 326-bp fragments corresponding to a region within the transmembrane domains of the CaR. Nucleic acid sequencing of PCR products from both gastric antrum and fundus revealed 84% identity in their nucleotide sequences with the corresponding region of rat kidney CaR (Fig. 1).


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Fig. 1.   Nucleotide sequence for Necturus 310-bp cDNA fragment (NecCaR) obtained from reverse transcription-polymerase chain reaction of antral mucosa using primers specific for Ca2+-sensing receptor (CaR). Comparison with rat kidney CaR gene (RaKCaR) revealed 84% homology (boxed areas).

Immunohistochemical Staining

Immunohistochemical staining of the gastric mucosa localized the CaR to the basal surface of the gastric epithelium and in the region of the myenteric plexus. Sections through the fundus revealed minimal staining in the region of the acid-secreting glands. However, staining of antral tissue demonstrated intense staining localized to the basal membrane of the surface cells (Fig. 2). Control sections that were stained with anti-CaR antiserum preabsorbed with the peptide against which it was raised showed no nonspecific binding (Fig. 2). These findings strongly suggest that the CaR is present and localizes predominantly to the basal surface of the gastric epithelium and, in particular, the antral surface cells.


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Fig. 2.   Immunohistochemical peroxidase staining of full-thickness sections through Necturus gastric antrum. A: section has been stained with antibodies specific to amphibian CaR. Most intense staining is localized to basal surface of surface epithelium and in myenteric plexus. B: section is a nearby control section that has been stained with an anti-amphibian polyclonal immunoglobulin G solution rendered deficient in anti-CaR antibody by immunoabsorbent pretreatment. There is no nonspecific binding observed.

Electrophysiological Changes Induced by Elevated Ca2+ in the Nutrient Perfusate and NPS-467

Tissues were mounted as described in MATERIALS AND METHODS to evaluate the effects of the stereoisomers of the CaR agonist NPS-467 compared with increased Ca2+ in the nutrient perfusate on the electrophysiological properties of the surface cell. After the tissue had equilibrated, the tissue was exposed in random order to either elevated nutrient Ca2+-Ringer solution (total [Ca2+] = 4.8 mM) (n = 13) or to the R-isomer (n = 10) or S-isomer of NPS-467-Ringer solution (n = 7) at a concentration of 3.0 × 10-5 M (total [Ca2+] = 1.8 mM). Preliminary studies using NPS-467 in doses ranging from 3.0 × 10-7 to 3.0 × 10-5 M revealed that the most consistent electrophysiological response occurred at 3.0 × 10-5 M. Measurements of the potential difference and resistance profiles for the tissues exposed to elevated nutrient Ca2+ and to the stereoisomers of the Ca2+ receptor agonist NPS-467 are summarized in Table 1. Elevations of nutrient Ca2+ and exposure to the R-isomer of NPS-467 elicited significant (P < 0.05) and reversible hyperpolarizations of the apical and basal membrane potentials (Vmc and Vcs, respectively). There was no significant change in the transepithelial electrophysiological properties (Vms or RT) in response to either elevated Ca2+ or to NPS-467. Exposure to either Ca2+ or NPS-R-467 was accompanied by a significant increase (P < 0.05) in the membrane resistance ratio Ra/Rb. Serosal exposure to the S-isomer of NPS-467 did not significantly alter cellular or tissue electrical properties.

                              
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Table 1.   Potential and resistance profiles in Necturus antral mucosa during serosal exposure to agonists of the extracellular Ca2+-sensing receptor

Electrophysiological Effect of Neomycin in the Nutrient Perfusate

To further investigate the role of CaR activation and associated changes in surface cell electrophysiological properties, surface cell electrical parameters were measured during nutrient exposure to the CaR agonist neomycin. Tissue potential difference and resistance profiles were measured with control Ringer solution ([Ca2+] = 1.8 mM) and then during exposure to either 0.25 mM (n = 10), 0.5 mM (n = 10), or 1 mM (n = 13) neomycin ([Ca2+] = 1.8 mM) in the nutrient perfusate. A typical tracing of an intracellular recording from a surface cell during exposure to neomycin is shown in Fig. 3. The changes in Vcs and Ra/Rb in response to the different concentrations of neomycin in the nutrient perfusate are presented in Fig. 4. The data show that key electrophysiological properties (Vcs, Ra/Rb) were altered in a concentration-dependent fashion. As can be seen, at the lower neomycin (0.25 mM) concentration, there was no significant hyperpolarization in Vcs. However, as the concentration of neomycin increased (0.5 and 1.0 mM), the magnitude of the hyperpolarization became significant (P < 0.05). Similarly, the Ra/Rb increased in a dose-dependent fashion in response to higher concentrations of neomycin (P < 0.05). In this instance, even at the lower dose of neomycin there was a small but statistically significant increase in Ra/Rb (P < 0.05).


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Fig. 3.   Effects of nutrient 1 mM neomycin on potential and resistance profile in Necturus antral mucosa. VT, transepithelial potential; Vc, basolateral membrane potential; apical membrane potential (Vmc = Vc - VT) not shown. Nutrient solution is reference. Voltage deflections were generated by transepithelial current pulses of 20 µA at 10-s intervals. Recording begins with microelectrode already impaled. Ca2+ concentration is held constant at 1.8 mM.


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Fig. 4.   Summary of effects of progressive increases in nutrient neomycin levels on basolateral membrane potential (Vcs) in millivolts and on ratio of cell membrane resistances (Ra/Rb); n = 10 for 0.25 and 0.5 mM neomycin (NEO) groups and n = 14 for 1 mM neomycin group. * P < 0.05 compared with Ringer solution control using an analysis of variance.

To identify the pathways of ion permeation affected by neomycin exposure, an intraepithelial circuit analysis was performed under control conditions ([Ca2+] = 1.8 mM) and during exposure to 1 mM nutrient neomycin ([Ca2+] = 1.8 mM) (18). During these experiments, Ra, Rb, and Rs were measured. The apical and basal membrane EMFs were also measured. As previously described, the tissue was exposed to mucosal amiloride (10-4 M) Ringer solution either 30 min before or after the nutrient neomycin exposure (18). This amiloride exposure provided an independent measurement of Ra, Rb, and Rs. Data from 11 tissues are presented in Table 2. Exposure to nutrient neomycin caused a significant (P < 0.05) decrease in the basal membrane resistance (Rb) without affecting the apical membrane (Ra) or paracellular (Rs) resistance. There was also a marked hyperpolarization in Eb (P < 0.05). Ea was not significantly altered.

                              
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Table 2.   Equivalent circuit analysis of changes in potential and resistance profiles in Necturus antral mucosa during exposure to 1 mM nutrient neomycin

Effect of Atropine and Indomethacin on the Electrophysiological Response to Nutrient Perfusate Neomycin

The final series of studies evaluated possible interactions of CaR activation with other signaling pathways that have been identified in gastric surface epithelium. In these studies, cholinergic receptor antagonists and prostaglandin synthesis inhibitors were used before neomycin exposure. Membrane potential differences and resistances were measured in response to 1 mM neomycin after 10 min of exposure to either atropine (10-4 M) (n = 9) or indomethacin (10-4 M) in the nutrient perfusate (n = 9). As shown in Fig. 5, atropine had no effect on the hyperpolarization of Vcs or on the increase in Ra/Rb resulting from neomycin-induced CaR activation. However, pretreatment of tissues with the prostaglandin synthesis inhibitor indomethacin markedly attenuated the cellular electrophysiological response to CaR activation (P < 0.05).


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Fig. 5.   Summary of effects of preincubation of tissue with atropine (Atro) or indomethacin (Indo) on change in basolateral membrane potential (Vcs) and on membrane resistance ratio (Ra/Rb) induced by exposure to 1 mM nutrient neomycin (Neo); n = 9 for each experimental group. Atropine and indomethacin were both at a concentration of 10-4 M, [Ca2+] = 1.8 mM. * P < 0.05 compared with Ringer solution control using an analysis of variance.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The principal findings of this study were 1) detection of a putative CaR by RT-PCR amplification of a fragment resembling rat kidney CaR in the transmembrane domain of the extracellular CaR from the gastric mucosa of Necturus; 2) localization of CaR, using immunohistochemical staining, to the basolateral aspect of the gastric surface epithelium; and 3) characterization of the electrophysiological response induced in the surface cell due to CaR activation. These findings demonstrate that there is an extracellular CaR present and functionally active in the gastric mucosa of Necturus.

Molecular analysis of the gastric mucosa of Necturus revealed the presence of a CaR gene product closely homologous to that of the rat CaR (19). Similar to the work reported by Brown et al. (3) in bovine tissue, we were unable to detect any signal for the CaR from the stomach mucosa using RNA Northern analysis, presumably because of the lower overall level of expression of CaR transcript. However, we were able to identify a fragment of the CaR gene product amplified by RT-PCR. Subsequent immunohistochemical studies localized CaR to the basolateral surface of gastric surface cells and in the region of the myenteric plexus. In this preparation, there was very little staining in the region of the gastric glands. The localization of the CaR to the basal surface of the surface cells is consistent with our finding that Ca2+ and the receptor agonists neomycin and NPS-467 elicited their effects when present in the nutrient solution.

The 326-bp PCR product characterized in this study was taken from the highly conserved, transmembrane region of the CaR sequence and was 84% homologous to the corresponding rat sequence (19). This is a slightly higher level of homology than that observed between the chicken and mammalian sequences (7) and not so high as homologies observed between different mammalian species (3, 7, 19). Although the Xenopus gene product has not been fully characterized, the degree of homology between the corresponding transmembrane regions is 81% (unpublished observations). Similarly, the extracellular domain of CaR that is recognized by the anti-CaR 4641 antibody has been shown to be highly homologous in avian, amphibian, and mammalian species (3, 7, 19). The high degree of homology in the transmembrane region that was amenable to PCR amplification, taken together with the specificity of antibody staining for a highly homologous extracellular domain of the CaR molecule, indicates that the CaR gene product is highly homologous between Necturus and other species. Further characterization of the Necturus gene product and its regulatory sites will require development of a cDNA library.

After immunohistochemical localization of the CaR to the basolateral surface of Necturus gastric antral cells, our subsequent studies were directed toward demonstrating the electrophysiological responses of the surface epithelium to known CaR agonists. As can be seen from Table 1, increases in nutrient Ca2+ result in a marked transient hyperpolarization of both the apical and basolateral membrane potential without a significant change in the transepithelial potential or resistance. There was also a significant increase in Ra/Rb. Thus the current findings with 3.0 mM Ca2+ are consistent with our previous findings on the effect of elevated Ca2+ in the nutrient perfusate (20). Similar electrophysiological responses of surface cells to specific CaR agonists, such as the NPS-467 compound and neomycin, are strongly suggestive of a receptor-mediated process initiating the observed electrophysiological changes. Also noted in Table 1 are the effects of exposure to the R- and S-stereoisomers of the CaR agonist NPS-467. These data reveal that the R-isomer, which is known to be 100 times more potent than the S-isomer, elicits an electrophysiological response nearly identical to that produced by increased Ca2+ in the nutrient perfusate (24). The weaker S-isomer at the same concentration did not induce these electrophysiological changes. This stereospecific response to the NPS-R-467 compound is similar to the CaR activation induced by NPS-467 in bovine parathyroid cells (24). We view this as further evidence that the observed electrophysiological changes in the surface cell are activated through a receptor-mediated process.

To characterize further the electrophysiological response to CaR activation, a number of studies were conducted using the CaR agonist neomycin sulfate. Neomycin was chosen because it has been the best characterized of all of the CaR agonists other than Ca2+, because it is more potent than Ca2+, and because, as a large polyvalent cation, it does not readily cross cell membranes (3). Table 2 shows that, similar to the effects of increased Ca2+, neomycin (1 mM) in the nutrient perfusate caused a significant reversible hyperpolarization of the cell membrane potential and increased the membrane resistance ratio (Ra/Rb). Because exposure to 1 mM neomycin elicited electrophysiological responses similar to those induced by increased Ca2+ in the nutrient perfusate, an intraepithelial circuit analysis using nutrient neomycin was performed to measure the membrane EMFs and determine the pathways of ion permeation affected by neomycin. As presented in Table 2, exposure to neomycin in the nutrient perfusate elicited a significant and marked hyperpolarization restricted to the basolateral membrane EMF and a selective decrease in Rb. There was no significant change in the apical membrane EMF or resistance, nor was there any change in Rs, which is a direct measure of the paracellular pathway resistance. The direction and magnitude of these changes are nearly identical to our previously reported circuit analysis for exposure to 5 mM Ca2+ in the nutrient perfusate (20). These findings are highly suggestive that exposure to neomycin in the nutrient perfusate produces the observed electrophysiological effects by a mechanism similar to elevated nutrient Ca2+.

On the basis of the results of the circuit analysis, the effect of CaR activation can be distinguished from the effects of activation of cholinergic or prostaglandin pathways. Both cholinergic and prostaglandin exposure in the nutrient perfusate have been shown to induce significant cell membrane hyperpolarizations and decreases in basolateral resistance in Necturus surface cells (1, 10). However, the results of circuit analysis for these compounds have shown that the mechanisms responsible for these changes are quite different from that for Ca2+ or neomycin. In our previous circuit analysis for Ca2+ exposure in the nutrient perfusate, we showed that the effects of Ca2+ on membrane potentials and resistances can be attributed solely to an increase in K+ conductance across the basolateral membrane (20). This outwardly directed K+ conductance alone can account for both the hyperpolarization of the basolateral membrane potential and the selective decrease in Rb. In the case of exposure to carbachol (10-4 M) in the nutrient perfusate, a cholinergic receptor agonist, there was also a significant hyperpolarization of the basolateral cell membrane potential, as well as a decrease in basolateral membrane resistance (10). However, exposure to carbachol caused no apparent change in the basolateral membrane EMF. This was in contrast to exposure to Ca2+ and neomycin, both of which elicited significant hyperpolarizations in the basolateral membrane EMF. Using a circuit analysis such as that performed here, we provided evidence that cholinergic exposure results in activation of both a K+ and Cl- conductance across the basolateral membrane (10). With simultaneous activation of a K+ and Cl- conductance under baseline conditions, there would be a decrease in Rb because of the increased ion conductance. However, there would be no significant change in Eb under baseline conditions, because Eb is composed of two opposing forces: the hyperpolarizing K+ conductance and the depolarizing Cl- conductance. With regard to prostaglandin exposure, the circuit analysis performed by Ashley et al. (1) has shown that, similar to elevated Ca2+ and neomycin in the nutrient perfusates, there is hyperpolarization of both the apical and basolateral membrane potentials. However, unlike Ca2+ and neomycin, prostaglandin exposure causes a significant decrease in both Ra and Rb as well as an increase in Rs (1). Although the tissue potential and resistance profiles are qualitatively similar for CaR activation and for both cholinergic and prostaglandin stimulation, the circuit analysis indicates that there are clear differences in the mechanisms underlying these electrophysiological changes. Thus we conclude that the electrophysiological changes induced by elevated Ca2+ and the CaR agonist neomycin in the nutrient perfusate result from a similar intracellular mechanism, and both are initiated by activation of a basolateral membrane CaR.

Although direct stimulation by either cholinergic receptor agonists or prostaglandins results in different electrophysiological responses in the surface cells, it does not rule out the possibility that either of these major intraepithelial signaling pathways may influence the efficacy or ability of the CaR to cause the observed electrophysiological response. As shown in Fig. 5, atropine, which is a nonselective muscarinic receptor antagonist, does not alter the electrophysiological response induced by neomycin exposure. However, indomethacin, which inhibits prostaglandin synthesis, markedly attenuates the response. These data suggest that the signaling pathway that links the activation of the CaR to the activation of the K+ conductance is dependent on prostaglandin synthesis. Further experiments are required to better define the relationship between activation of the CaR and activation of the basolateral membrane K+ conductance.

In summary, we have reported evidence for the presence of a functionally active extracellular CaR in the basal membrane of the gastric mucosa of Necturus. We have identified the presence of the CaR in the gastric mucosa by 1) using RT-PCR to amplify and then sequence a fragment of the CaR gene from the gastric mucosa, and 2) localizing the CaR to the basal membrane of the gastric antral surface cells by immunohistochemical staining. Intracellular microelectrode techniques were used to measure an electrophysiological response of the surface cell to activation of the CaR. The intracellular hyperpolarization and selective increase in basal membrane conductance in response to CaR activation by increased Ca2+ and known CaR agonists such as the NPS-467 compound and neomycin in the nutrient perfusate provide evidence that this is a receptor-mediated process. The stereospecific response to the R-isomer of the NPS-467 CaR agonist as opposed to the S-isomer is further suggestive of a receptor-mediated process.

Although we have presented evidence for the existence of a functionally active extracellular CaR in the basal surface of Necturus gastric mucosa, we have not demonstrated a physiological role for the CaR in the gastric mucosa. In this regard, it should be noted that, across several species, CaR sequences have now been identified not only in tissues that regulate Ca2+ homeostasis, such as parathyroid, thyroid C cells, and kidney, but also in tissues of the central and peripheral autonomic nervous systems (4), as well as endocrine cells such as the gastrin-secreting G cells of gastric antral mucosa (15). Thus CaR is not localized only in tissues that contribute directly to absorption or excretion of Ca2+.

In such tissues, one possible function of the CaR may be to communicate the level of Ca2+ in the extracellular microenvironment, which may in turn alter intracellular processes. Changes in the microenvironment that may occur during periods of acid or bicarbonate secretion may change the level of ionized Ca2+, and thus the CaR may serve as a mechanism to regulate these processes. Alternatively, the Ca2+ concentration in this microenvironment may increase during injury to the epithelium, and thus the CaR may serve to sense these changes in Ca2+ and then activate cellular defense mechanisms. The suggestion that inhibitors of prostaglandin synthesis may interfere with one of the signaling mechanisms affected by activation of the CaR is interesting considering the role of prostaglandins in maintaining the integrity of the gastric mucosa. With the evidence that we have presented in regard to the presence of the CaR in the gastric surface epithelium, a role for Ca2+ as a true first messenger may be postulated. Further studies are needed to elucidate the exact role of the CaR in the gastric mucosa and to determine what role this receptor may play, if any, in maintaining the integrity of the gastric mucosa.

    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-09288-02 (to R. R. Cima), DK-09033 (to M. E. Klingensmith), and DK-44571-02 (to D. I. Soybel); Howard Hughes Medical Institute Research Training Fellowship for Medical Students (to I. Cheng); and I-S R- & S-467 individual and NPS Pharmaceutical Grants DK-41415 and DK-48330 (to E. M. Brown).

    FOOTNOTES

Portions of this work have been presented in abstract form at the Fundamental Forum, American College of Surgeons Meeting (San Francisco, CA, October 1996) and Digestive Diseases Week (Washington, DC, May 1997).

Address for reprint requests: D. I. Soybel, Dept. of Surgery/112, West Roxbury Veterans Administration Medical Center, 1400 VFW Parkway, West Roxbury, MA 02132.

Received 13 May 1997; accepted in final form 18 August 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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AJP Gastroint Liver Physiol 273(5):G1051-G1060
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