1 Department of Surgery, 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
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 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.
Preparation
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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/mlNucleotide 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,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
, 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 ) 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 M
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 104 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).
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(1) |
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(2) |
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 (10Data 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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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).
|
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.
|
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
|
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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|
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 (104 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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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
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DISCUSSION |
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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
(104 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.
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ACKNOWLEDGEMENTS |
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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).
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FOOTNOTES |
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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.
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