Role of neuropeptide-sensitive L-type Ca2+ channels in histamine release in gastric enterochromaffin-like cells

Ningxin Zeng, Christoph Athmann, Tao Kang, John H. Walsh, and George Sachs

Wadsworth Veterans Affairs Hospital, Los Angeles, California 90073


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Peptides release histamine from enterochromaffin-like (ECL) cells because of elevation of intracellular Ca2+ concentration ([Ca2+]i) by either receptor-operated or voltage-dependent Ca2+ channels (VDCC). To determine whether VDCCs contribute to histamine release stimulated by gastrin or pituitary adenylate cyclase-activating polypeptide (PACAP), the presence of VDCCs and their possible modulation by peptides was investigated in a 48-h cultured rat gastric cell population containing 85% ECL cells. Video imaging of fura 2-loaded cells was used to measure [Ca2+]i, and histamine was assayed by RIA. Cells were depolarized by increasing extracellular K+ concentrations or by 20 mM tetraethylammonium (TEA+). Cell depolarization increased transient and steady-state [Ca2+]i and resulted in histamine release, dependent on extracellular Ca2+. These K+- or TEA+-dependent effects on histamine release from ECL cells were coupled to activation of parietal cells in intact rabbit gastric glands, and L-type channel blockade by 2 µM nifedipine inhibited 50% of [Ca2+]i elevation and histamine release. N-type channel blockade by 1 µM omega -conotoxin GVIA inhibited 25% of [Ca2+]i elevation and 14% of histamine release. Inhibition was additive. The effects of 20 mM TEA+ were fully inhibited by 2 µM nifedipine. Both classes of Ca2+ channels were found in ECL cells, but not in parietal cells, by RT-PCR. Nifedipine reduced PACAP-induced (but not gastrin-stimulated) Ca2+ entry and histamine release by 40%. Somatostatin, peptide YY (PYY), and galanin dose dependently inhibited L-type Ca2+ channels via a pertussis toxin-sensitive pathway. L-type VDCCs play a role in PACAP but not gastrin stimulation of histamine release from ECL cells, and the channel opening is inhibited by somatostatin, PYY, and galanin by interaction with a Gi or Go protein.

acid secretion; gastric glands; calcium channels


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

CALCIUM CHANNELS in neuroendocrine cells are important because Ca2+ entry through these channels controls a wide variety of their physiological functions, such as transmitter release, firing patterns, biochemical processes, bioenergetics, and gene expression (28). The three types of known Ca2+ entry pathways are voltage-dependent Ca2+ channels (VDCC), receptor-operated Ca2+ channels (ROCC), and reversal of Na+/Ca2+ exchange (21, 28, 38, 42). VDCCs are expressed in excitable neurons, muscle cells, and all neuroendocrine cells so far studied, such as hypothalamic, pituitary, glomus corticum, thyroid C, neuroendocrine pulmonary, chromaffin, enterochromaffin, CCK-secreting, and pancreatic alpha - and beta -cells (28).

Distinct types of VDCCs have been defined according to various pharmacological and electrophysiological criteria: L-type channels are dihydropyridine sensitive, whereas N-type channels are omega -conotoxin GVIA (omega -CTx-GVIA) sensitive. P- and Q-type channels are distinguished on the basis of differential sensitivities to omega -agatoxin IVA, and the R-type component of the high-voltage-activated current is resistant to dihydropyridines and toxins (28, 54). Cloning has shown an even greater diversity among VDCCs. At least six different genes have been identified, and even greater functional diversity might be produced by alternative splicing of the different gene products (28, 54). These channels differ considerably in their responsiveness to neuromodulators, their distribution among various cell types, and their localization in different regions within individual cells (9, 14, 35). There is now general agreement that, even in a single cell, different subtypes of VDCCs can be coexpressed (9, 14, 35).

The enterochromaffin-like (ECL) cell is a histamine-containing neuroendocrine cell in fundic gastric mucosa that plays a central role in the peripheral regulation of mammalian gastric acid secretion (19, 20, 52). Studies using a preparation of isolated, highly enriched ECL cells have shown that these cells display some of the characteristic features of neuroendocrine cells, but their stimulus-secretion coupling is often like that of nonexcitable cells such as the mast cell (3, 34, 47). Activation of histamine secretion requires specific signaling dependent on a distinct receptor distribution on ECL cells (32, 47, 48, 52, 61). ECL cells respond to the antral hormone gastrin with histamine secretion, activation of the histamine-synthesizing enzyme histidine decarboxylase (HDC), and cellular proliferation (49). Other stimulatory receptors such as pituitary adenylate cyclase-activating polypeptide (PACAP), adrenergic, and cytokine receptors have been found, as well as inhibitory receptors such as somatostatin, galanin, and peptide YY (PYY) receptors (59-62). Although several studies clearly indicate that an intracellular Ca2+ concentration ([Ca2+]i)-dependent pathway is required for histamine release, the specific cellular mechanisms that regulate histamine release have yet to be delineated (47, 61).

Electrophysiological studies have shown that the ECL cell has a resting potential of -58 mV, and pharmacological studies demonstrate that L-type Ca2+ channels are the main VDCCs presented in rat ECL cells (3, 34). L-type Ca2+ channels are believed to contribute to exocytosis in many neuroendocrine cells, such as insulin secretion from pancreatic beta -cells (34), catecholamines from chromaffin cells (14, 30, 33, 35, 50), 5-HT from enterochromaffin cells (59), and many other cell types (9-11, 27, 39). However, whether L-type Ca2+ channels contribute to activation of secretion of histamine from rat ECL cells is not known. In the present study, this issue was addressed by RT-PCR to analyze the expression of L-type Ca2+ channels and by video imaging to monitor single cell [Ca2+ ]i responses. Contributions of the distinct Ca2+ channel subtypes to the release of histamine measured by RIA were evaluated by using L- and N-type antagonists after cell depolarization by high K+ or by tetraethylammonium (TEA+). In addition, we also investigated whether L-type Ca2+ channels are functionally altered by gastrin and neuropeptides such as PACAP, PYY, somatostatin, and galanin and whether histamine release from ECL cells induced by depolarization affects parietal cells in intact gastric gland preparations.

We found that high-K+ solution induced dose-related histamine release from rat ECL cells and H2 histamine-receptor antagonist-sensitive Ca2+ signals in rabbit glands. L-type VDCCs were found to be the main contributors, accounting for 50% of K+ depolarization and 95% of TEA+-induced histamine release. L-type Ca2+ channels also contribute 40% of the PACAP-induced Ca2+ entry signal, as well as histamine release, but are probably not involved in gastrin-induced Ca2+ entry and histamine release (<10%). In ECL cells, L-type Ca2+ channels are completely inhibited by somatostatin, galanin, and PYY.


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Isolation and purification of ECL cells. The rat gastric ECL cells were isolated by a combination of elutriation and density gradient centrifugation as described previously (47, 61). Five rat stomachs provided 1 × 106 small cells in the low-density layer. Cell viability was >95% as determined by trypan blue exclusion. Cell purification was quantified by immunostaining with anti-HDC antibody and antihistamine antibody and by using the fluorescent dye acridine orange, which accumulates with a red shift in fluorescence in the acidic, histamine-containing vacuoles. A majority (65-75%) of cells in the freshly isolated cell population were ECL cells. Freshly isolated ECL cells were rinsed by gentle centrifugation in growth medium containing DMEM/F-12, supplemented with 2 mg/ml BSA, 2.5% FCS, 100 µM hydrocortisone, 1% penicillin, streptomycin, and 5 mg/ml insulin, 5 mg/ml transferrin, and 5 µg/l sodium selenite. After 48 h in culture, the ECL cell population was enriched to >85%. All Ca2+ imaging experiments and histamine release studies were carried out on these cultured cells after 2-4 days in culture at 37°C.

RT-PCR analysis of dihydropyridine- and omega -CTx-GVIA-sensitive Ca2+ channels. Molecular cloning and heterologous expression systems have shown that the different alpha 1-subunits give rise to Ca2+ channels that reflect the electrophysiological and pharmacological classification. The alpha 1C- and alpha 1D-subunits produce L-type Ca2+ currents that are sensitive to dihydropyridines, and the alpha 1B-subunit produces an N-type Ca2+ channel that is sensitive to omega -CTx-GVIA. The alpha 1A-subunit expresses a omega -agatoxin-IVA-sensitive current resembling both P- and Q-type Ca2+ channels, possibly differentiated by alternative splicing of the alpha 1A-subunit transcript or by association with different auxiliary subunits. The alpha 1E-subunit encodes for a Ca2+ channel characterized by voltage activation properties at relatively negative potentials, fast kinetics, and insensitivity to known toxins or drugs. Whether the alpha 1E-subunit represents the R-type or other types of Ca2+ currents remains to be determined.

RT-PCR was used to identify the presence of VDCCs in ECL cells and parietal cells. The same amount of total RNA (5 µg) isolated from enriched rat ECL cells or from parietal cells was used to make cDNA by RT with oligo(dT)15 primers. The sample without RT was used as a control. PCR was performed in low-salt Taq+ DNA polymerase buffer and 5 units Taq+ DNA polymerase (Stratagene) in the presence of oligonucleotide primers under the following conditions: initial step (one cycle), 94°C for 2 min, 57°C for 1 min, and 72°C for 2 min; repeating steps (35 cycles), 94°C for 1 min, 57°C for 1 min, and 72°C for 2 min; and extension step (one cycle), 94°C for 1 min, 57°C for 1 min, and 72°C for 5 min. The primers were designed based on the cloned rat Da1 sequence, and the primer sense and antisense sequences are summarized in Table 1. The RT-PCR products were amplified and cloned into pCRII vector (Invitrogen, San Diego, CA). The cloned PCR products were sequenced on both strands by the chain termination method using T7 and SP6 primers to confirm that the fragments were the portions of the Ca2+ channels.

                              
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Table 1.   Primer sequences

Measurement of [Ca2+]i in isolated cells using video microscopy. After 48-h culture, cells were loaded with 2 µM fura 2-AM for 30 min at 37°C, and then the coverslip was washed with growth medium and placed in a heated chamber (Medical Systems, Greenvale, NY). The temperature was maintained constant at 37°C. The coverslip was perfused with Ringer buffer with different concentrations of KCl or TEA+ at a rate of 5 ml/min, and the effluent was constantly removed with a peristaltic pump. Fura 2 fluorescence was measured by using a Nikon Fluo ×40 objective with a Zeiss Axiovert 100TV microscope (Zeiss, Thornwood, NY) connected to a PC-XT computer programmed to rapidly alternate between the excitation wavelengths of 340 and 380 nm. The emission wavelength (505 nm) was constant. Image pairs were captured under the control of Image-1/FL software (Universal Imaging, West Chester, PA) and expressed as the ratio of fluorescence level in the chosen field. Maximal and minimal fluorescence was achieved by adding 10 µM ionomycin followed by 20 mM EGTA. [Ca2+]i was calculated using previously described formulas with slight modification (47, 61). All data presented in the Figs. 1-10 are representative of at least four experiments.

Confocal microscopy of rabbit gastric glands. The preparation of rabbit fundic gastric glands and measurement of [Ca2+]i by confocal microscopy are the same as described elsewhere (N. Zeng, unpublished observations). Briefly, under anesthesia, high-pressure aortic perfusion with mammalian Ringer was used to loosen the epithelial layer from the submucosa. The stomach was removed, and the epithelium was stripped off, minced with scissors, and digested with collagenase. The glands are washed by allowing them to settle on the coverslips and then kept at room temperature until use. This is a preparation that responds to histamine, gastrin, and carbachol in terms of stimulation of acid secretion and changes of intracellular messengers in the parietal cells. Furthermore, as in humans, gastrin stimulation but not carbachol stimulation of acid secretion is ablated by H2-receptor antagonists. Hence, rabbit glands were chosen as an integrated model in which to investigate the action of high-K+ depolarization on ECL cells in situ and consequent effects on parietal cells. It was expected that the ECL cell of rabbit has a generally similar response to that of rat, and this expectation is borne out by our experiments.

The glands were loaded with fluo 4-AM at a concentration of 5 µM for 30 min on a coverslip coated with Cell-TAK before they were transferred to a superfusion chamber at 37°C. The dye fluorescence was measured at 525 nm, with excitation at 488 nm. The high-K+ solutions (40 mM) were added during the perfusion with Ringer buffer as noted. The relative changes in [Ca2+]i were monitored on a selected region during the experiment, visualizing individual gastric glands at ×63 magnification, scanning 512 × 512 pixels every other second. The region of interest was highlighted on the image, with numbers indicating the position of each image on the scan. Changes in Ca2+ in ECL cells and parietal cells were measured. The images are representative of at least four experiments.

Histamine release. Histamine release was determined following 48-h culture by incubating ECL cells on Cell-TAK-precoated coverslips in six-well plates. Growth medium was replaced 3 h before the experiments. The cultured ECL cells were rinsed with Ringer buffer and incubated in test medium for 60 min at 37°C. Approximately 20,000 cells per well were seeded for the experiment. The test medium contained, as necessary, different concentrations of K+ or TEA+ in Ringer medium. Na+ was replaced by K+ or TEA+, retaining the same osmolarity in different test solutions. Histamine concentrations were determined as previously described by using a commercially available kit (AMAC, Westbrook, MA). The data are presented as percent over basal (control).

Statistical analysis. Results are means ± SE. Where appropriate, statistical analysis of the data was performed by Mann-Whitney U test if the Kruskali-Willis indicated a significant difference between multiple groups. For multiple comparisons with the same control group, the limit of significance was divided by the number of comparisons according to Bonferroni. Differences between paired groups were determined using a paired t-test. Median effective concentration and IC50 were calculated by using linear regression analysis. Values were considered statistically significant when P < 0.05.

Materials. Rabbit anti-HDC antibody and monoclonal antibody against somatostatin (S6) were obtained from the Antibody Core of the CURE Digestive Diseases Research Center. All other reagents were analytical grade and were purchased from the indicated sources. Pronase E was from Boehringer Mannheim. Taq+ DNA polymerase and pCR-Script Amp SK(+) plasmid were from Stratagene (La Jolla, CA). PCR primers for beta -actin were from Clontech (Palo Alto, CA). Model 391 PCR-MATE ABI was from Perkin-Elmer (Foster City, CA). Gastrin-17 and galanin were from Peninsula. PACAP-27 was from American Peptide. BSA, acridine orange, DMEM/F-12, insulin-transferrin-sodium selenite media supplement, hydrocortisone, trypan blue, gastrin, somatostatin, goat anti-rabbit fluorescein-conjugated IgG, ionomycin, TEA, and EGTA were all from Sigma; Cell-TAK was from Collaborative Research; Nycodenz was from Accurate Chemical (Westbury, NY); and fura 2 was from Molecular Probes (Eugene, OR).


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Selective expression of dihydropyridine- and omega -CTx-GVIA-sensitive Ca2+ channels on ECL cells. For analysis of dihydropyridine- and omega -CTx-GVIA-sensitive Ca2+ channel expression by RT-PCR, 5 µg total RNA extracted from purified rat ECL or parietal cells was used to make cDNAs as described previously using RT. The same amount of cDNA from purified ECL cells and parietal cells was used for PCR and amplification of HDC; H+-K+-ATPase and beta -actin were used as internal controls.

By using dihydropyridine-sensitive Ca2+ channel primers, as shown in Fig. 1, a band of 437-bp DNA fragment was obtained from ECL cDNA but not from the purified parietal cell cDNA.


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Fig. 1.   Selective expression of dihydropyridine- and omega -conotoxin-GVIA (omega -CTx-GVIA)-sensitive Ca2+ channels on enterochromaffin-like (ECL) cells as determined by RT-PCR. RNA isolated from parietal cell and ECL cell preparations (top) was probed with selective primers for these channels. Bands shown by ethidium bromide staining (bottom) were confirmed by dideoxynucleotide sequencing.

Amplification of the same cDNA samples using primers for omega -CTx-GVIA-sensitive Ca2+ channels provided a 671-bp product from ECL cDNA but not from parietal cell cDNA. These products were identical in size to that calculated from the cDNA sequences of rat dihydropyridine- and omega -CTx-GVIA-sensitive Ca2+ channels, respectively (28, 45, 54). No products were obtained from negative controls, which contained all components except RT, ruling out the possibility of genomic DNA contamination (data not shown). The products of PCR were then sequenced and were 100% identical to dihydropyridine- and omega -CTx-GVIA-sensitive Ca2+ channels. These data demonstrated that the alpha 1-subunits of dihydropyridine-sensitive and of omega -CTx-GVIA-sensitive Ca2+ channels are expressed on rat ECL cells but not on parietal cells.

K+ depolarization induced elevation of [Ca2+]i. Elevation of extracellular K+ concentration depolarizes cells because of changes in the K+ equilibrium potential. To determine whether VDCCs are involved in the Ca2+ entry in ECL cells, fura 2-loaded ECL cells were perfused with HEPES buffer containing from 5 (basal) to 60 mM K+ that substituted for Na+. In the presence of Ringer buffer containing 5 mM extracellular K+, the resting [Ca2+]i in ECL cells was found to be from 70 to 146 nM and remained unchanged for up to 3-5 h at 37°C as described previously (61). Brief exposure (1 min) to elevated extracellular K+ from 5 (basal) to 10, 20, 40, and 60 mM resulted in a rapid (peak in 5-10 s) transient increase in [Ca2+]i that fell rapidly, as shown in Fig. 2A. With prolonged exposure to an increase in K+, for example 40 mM, a typical biphasic [Ca2+]i increase was found in >85% of the cells observed as an initial transient followed by a plateau phase (Fig. 2B), reminiscent of the gastrin or PACAP response of the ECL cells. Some cells even had a sustained [Ca2+]i increase when the K+ concentration was <20 mM (data not shown).


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Fig. 2.   K+ depolarization-induced elevation of intracellular Ca2+ concentration ([Ca2+]i). A: brief exposure (1 min) to an increase in extracellular K+ from 5 (basal) to 10-60 mM resulted in a fast (peak in 5-10 s) increase in [Ca2+]i and a rapid fall. B: with prolonged exposure to high-K+ solution, a typical biphasic [Ca2+]i increase was found, namely an initial transient followed by a plateau phase. C: increase in [Ca2+]i induced by K+ was dependent on extracellular Ca2+ because addition of an excess of EGTA (3 mM) during sustained plateau phase resulted in a rapid decline of [Ca2+]i toward basal, prestimulated levels.

The increase of [Ca2+]i induced by K+ was dependent on extracellular Ca2+ because addition of an excess of EGTA (3 mM) during the plateau phase resulted in a rapid decline of [Ca2+]i toward basal levels (Fig. 2C). Both initial and sustained Ca2+ signals were absent during brief or prolonged K+ stimulation when Ca2+-free solutions were used containing 100 µM EGTA (data not shown).

TEA+, which blocks outward K+ currents and depolarizes the membrane potential, evoked Ca2+ oscillations, which were dependent on extracellular Ca2+ at concentrations up to 30 mM. At 40 mM, the effect mimicked that of K+ depolarization, inducing a transient and steady-state response (Fig. 3). Hence, membrane depolarization activates Ca2+-signaling pathways in ECL cells, whether typically biphasic or oscillatory.


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Fig. 3.   Tetraethylammonium (TEA+)-induced Ca2+ signals. TEA+ (10-30 mM) induced dose-dependent Ca2+ oscillations requiring external Ca2+. At 40 mM, TEA+ induced biphasic Ca2+ signals rather than Ca2+ oscillations.

Flufenamic acid has been shown to stimulate outward K+ currents and to hyperpolarize the resting membrane potential of a variety of cell types as a result of activation of K+ channels (51). The effects of flufenamic acid (200 µM) on [Ca2+]i are shown in Fig. 4. Perfusion with flufenamic acid significantly reduced basal [Ca2+]i and K+ depolarization-induced increase of [Ca2+]i.


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Fig. 4.   Effect of flufenamic acid. Perfusion with flufenamic acid significantly reduced basal [Ca2+]i (top) and K+ depolarization induced increase of [Ca2+ ]i (bottom). Top: typical set of basal Ca2+ recordings from different ECL cells. Bottom: effect on peak Ca2+ induced by K+ depolarization.

High-K+ solution- and TEA+-induced histamine release requires external Ca2+. To determine the functional effect of high-K+ and TEA+ depolarization on isolated gastric ECL cells, the release of histamine from ECL cells was examined after exposure to different concentrations of KCl and TEA+.

Under basal conditions, cultured ECL cells release 40 pmol histamine per well. In the presence of 1.8 mM Ca2+ in the incubation medium, K+ depolarization elevated histamine release from cultured ECL cells in a dose-related manner, with maximal release being 500% of basal at 40 mM (Fig. 5A). Addition of TEA+ to the incubation medium induced a dose-dependent increase in histamine release, with a maximal threefold increase at 30 mM (Fig. 5B). Increasing TEA+ to 40 mM did not increase the histamine release further. The minimum effective dose of TEA+ was 10 mM, and the EC50 was 20 mM. However, in Ca2+-deficient medium, high-K+ depolarization- and TEA+-induced histamine release were completely abolished (Fig. 5C). These results suggested that K+ depolarization- and TEA+-induced histamine release from ECL cells depended on Ca2+ entry from the extracellular space.


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Fig. 5.   High K+- and TEA+-induced histamine release requires external Ca2+. A: in presence of 1.8 mM Ca2+ in incubation medium, K+ depolarization induced histamine release from cultured ECL cells in a dose-related manner, with maximal release (fivefold above basal) at 40 mM. B: addition of TEA at incubation medium from 5 to 30 mM induced a dose-dependent increase in histamine release, with maximal release at 30 mM (threefold of basal). C: in Ca2+-deficient medium, high-K+ depolarization- and TEA+-induced histamine release was completely abolished.

Effects of high-K+ depolarization on rabbit gastric glands. As shown in Fig. 6, ECL cells load with the fluo 4 dye to a greater extent than parietal cells, presumably because of accumulation of the dye in the secretory vacuoles. In the confocal microscope, only single-plane sections are shown here, visualizing only a single ECL cell but several parietal cells, reflecting the relative numbers of these two cell types. The scan of a defined region of the images in the whole sequence shows the Ca2+ signal in a specific cell type.


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Fig. 6.   Effect of high-K+ depolarization on ECL and parietal cells in rabbit gastric glands. A: an increase of [Ca2+]i in the ECL cells (defined either by high fluorescence or subsequent accumulation of acridine orange in their vacuoles) due to depolarization by addition of 40 mM KCl was followed by a Ca2+ transient in parietal cells, as shown in scan at bottom. Circled numbers correspond to images recorded at that particular time, shown at top. Green line shows Ca2+ signal in highlighted ECL cell, and orange and red lines show Ca2+ signal in 2 highlighted parietal cells. It can be seen that several other parietal cells also increased their [Ca2+]i. B: transients in parietal cells but not in ECL cells were blocked by H2-receptor antagonist ranitidine.

As shown in Fig. 6A, there was a biphasic increase of [Ca2+]i in the ECL cells (identified either by high-fluo 4 fluorescence or subsequent accumulation of acridine orange in their vacuoles) due to the addition of 40 mM KCl to the perfusate (green line). This is visualized in the displayed images selected from the recorded sequence and numbered on the recording of the signal. The increase of [Ca2+]i in the ECL cells was followed, after a lag phase, by a Ca2+ transient in parietal cells, as shown in the Ca2+ scans of selected cells illustrated (red and orange lines). The data presented are typical of several experiments in which both cell types were stimulated, but the parietal cell signal always followed the ECL cell signal with a significant delay (~100 s).

The transient in the parietal cells but not in the ECL cells was blocked by the H2-receptor antagonist ranitidine (Fig. 6B). Histamine itself produced a transient signal in parietal cells but not in ECL cells, as has been shown previously. Thus high-K+ depolarization in this more integrated model stimulates ECL cell Ca2+ signaling and then, via histamine release and activation of the H2 receptor, parietal cell Ca2+ signals. Because the presence of H2-receptor antagonists ablated the effect of depolarization by high K+ on parietal cells but not on ECL cells, there can be no functional VDCCs on parietal cells, confirming the RT-PCR results.

Contribution of L-type Ca2+ channels to high K+- and TEA+-induced Ca2+ entry. K+ depolarization induces Ca2+ entry via VDCCs in many types of neuroendocrine cells. VDCCs have been classified into at least six types (L, N, P, Q, T, and O) on the basis of electrophysiological and pharmacological criteria. Multiple brief applications of 40 mM K+ (1 min) produce [Ca2+]i transients of identical amplitude, as described. Therefore, we pulsed KCl to examine the pharmacology of subtypes of VDCCs in ECL cells.

L-type channel blockade by 2 µM nifedipine inhibited 50% of [Ca2+]i elevation (Fig. 7A) and histamine release (Fig. 7B). N-type channel blockade by 1 µM omega -CTx-GVIA inhibited 25% of [Ca2+]i elevation and 14% of histamine release, which was not statistically significant (Fig. 7, A and B). The combination was apparently additive, resulting in 70% inhibition. The elevation of [Ca2+]i due to TEA+ blockade and the histamine release were inhibited by 2 µM nifedipine and Ca2+ removal (Fig. 7, C and D). BAY K 8644, a compound that activates L-type Ca2+ channels, at 1 µM alone also induced a biphasic Ca2+ increase in 48% of the ECL cell population as well as a significant increase in basal and high K+- or TEA+-induced histamine release (Fig. 7, E and F).


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Fig. 7.   Contribution of L-type Ca2+ channels to high-K+- and TEA+-induced Ca2+ signals. L-type channel blockade by 2 µM nifedipine inhibited 50% of [Ca2+]i elevation due to a pulse of 40 mM KCl (A) and basal histamine release (B). N-type channel blockade by 1 µM omega -CTx-GVIA inhibited only 25% of [Ca2+]i elevation (A) and 14% of histamine release (B). Combination was additive, resulting in 70% inhibition. TEA+ (20 mM) also elevated [Ca2+]i because of K+ channel blockade, but this effect as well as histamine release were almost fully inhibited by 2 µM nifedipine (C and D). BAY K 8644 induced a biphasic Ca2+ signal and histamine release, which were totally blocked by nifedipine (E and F). BAY K 8644 also significantly increased high-K+- and TEA+-induced histamine release (E and F). * P < 0.05.

Modulation of L-type Ca2+ channels by peptide ligands. Gastrin and PACAP showed the same efficacy in stimulation of histamine release, namely a fourfold increase over basal from isolated rat ECL cells (49, 59). As shown in Fig. 8, both gastrin (10-9 M) and PACAP (10-9 M) stimulated a biphasic Ca2+ signal. Nifedipine gave <10% inhibition on gastrin-induced Ca2+ entry and histamine release (Fig. 8A). However, nifedipine partially inhibited the PACAP-induced Ca2+ transient and 40% of the PACAP-induced histamine release (Fig. 8B). Part of the action of PACAP is via modulation of other Ca2+ pathways.


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Fig. 8.   Modulation of L-type Ca2+ channels by neuropeptides. Gastrin (A) and pituitary adenlyate cyclase-activating peptide (PACAP; B) stimulated a biphasic Ca2+ signal, but nifedipine had <10% inhibition on gastrin-induced Ca2+ entry and histamine release (A). However, nifedipine partially inhibited PACAP-induced Ca2+ transient and 40% of histamine release (B). *P < 0.05.

The effect of inhibitory peptides on L-type Ca2+ channels was also investigated in this isolated cell model. As shown in Fig. 9, somatostatin, PYY, and galanin completely inhibited TEA+-induced Ca2+ oscillations as well as histamine release, with maximal inhibition of 90%. The BAY K 8644-induced histamine release was also totally inhibited by these neuropeptides (Fig. 9B). Somatostatin, PYY, and galanin also inhibited high-K+-induced [Ca2+]i increases and histamine release, with an EC50 of 10-9, 10-9, and 10-8 M, respectively (data not shown). These are universal inhibitors of ECL cell function.


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Fig. 9.   Inhibition of L-type Ca2+ channels by neuropeptides. Somatostatin (SS), peptide YY (PYY), and galanin (Gal) completely inhibited 20 mM TEA+-induced Ca2+ oscillations (A) as well as histamine release (B). BAY K 8644-induced histamine release was also totally inhibited by these neuropeptides (B). *P < 0.05.

Modulation of G proteins in VDCCs. The inhibitory effects of somatostatin, galanin, and PYY on gastrin-induced Ca2+ entry and histamine release are sensitive to pertussis toxin, indicating that these neuropeptide receptors are coupled to Gi or Go proteins (12, 23, 25, 26, 37). Pretreatment of the cultured ECL cells with pertussis toxin (200 ng/ml) for 16 h abolished the inhibitory action of somatostatin and PYY on both Ca2+ signaling and histamine release and partially reduced the inhibition by galanin, as shown in Fig. 10.


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Fig. 10.   Role of G proteins in modulation of L-type activity by neuropeptides. ECL cells pretreated with pertussis toxin (200 ng/ml) for 16 h abolished inhibitory action of somatostatin (SS) and peptide YY (PYY) on BAY K 8644-induced Ca2+ entry (A) and TEA+-induced histamine release (B) as well as partially reduced inhibition by galanin (Gal; B). P < 0.05.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Some neuroendocrine cells share characteristics with neuronal cells, such as their secretory mechanisms, membrane trafficking proteins, and electrical excitability (11, 28, 52, 58). Some neuroendocrine cells can fire action potentials in response to membrane depolarization (11, 28, 52, 58). The depolarization-induced action potentials cause corresponding rises in [Ca2+]i and thereby Ca2+-dependent hormone or transmitter release (11). The Ca2+ influx due to membrane depolarization occurs mainly by voltage-dependent Ca2+ channels in neurons as well as in most types of neuroendocrine cells.

A rise in [Ca2+]i is an obligatory step in stimulus-secretion coupling in ECL cells due to gastrin or PACAP (47, 52, 61). The ECL cells produce and store histamine (19, 20). This biogenic amine is stored in vesicles with a total content of 2.8-4.3 pg/cell of histamine, which is a relatively low amount compared with mast cells (12-20 pg/cell). The cytoplasm of ECL cells shows numerous hollow vesicles, which contain histamine, and a characteristic electron-dense core inside these electron-lucent vesicles, which is thought to represent the storage of a peptide hormone (19, 20). The exocytotic process of histamine release involves members of membrane-trafficking protein families such as vesicle-associated membrane protein, synaptosome-associated protein, or syntaxins (24). In isolated rat ECL cells, the removal of extracellular Ca2+ abolishes the secretion of histamine in response to gastrin, showing that influx of Ca2+ into the ECL cell is required for histamine release (47, 61). Patch-clamp analysis has shown the presence of voltage-gated Ca2+ currents in rat gastric ECL cells, inhibited by verapamil and augmented by the L-type Ca2+ channel activator BAY K 8644 (3), but their functional significance was not analyzed.

Excitation-secretion coupling in many endocrine cells is dependent on changes in membrane voltage, which is controlled by ion channels (57, 58). Modulation of ion channels causes changes in resting membrane potential that lead to alteration in Ca2+ influx and hormone secretion. In most endocrine cells, the resting membrane voltage is regulated by K+ channels (57, 58). ECL cells have a resting potential of -58 mV. Electrophysiological and pharmacological studies have provided evidence for depolarization-activated K+ channels and Ca2+ channels in gastric ECL cells (3). It has been established that K+ channels are important modulators of hormone secretion in many types of endocrine cells (3, 34). Blocking K+ channels induces catecholamine release from adrenal medullary cells, corticoid secretion from adrenocortical cells, insulin secretion from pancreatic beta -cells, and prolactin secretion from pituitary cells (4, 17, 57).

In this study, we confirmed that L- and N-type Ca2+ channels are selectively expressed in the gastric ECL cells by measurements of Ca2+ signals and RT-PCR. In addition, we found that Ca2+ influx through L-type VDCCs induced increases in [Ca2+]i and exocytosis in gastric ECL cells.

First, both high-K+ and TEA+ depolarization-induced histamine release depended on external Ca2+ and were partially or completely inhibited by nifedipine, respectively, at the concentrations that target L-type channels. Second, BAY K 8644 significantly enhanced the depolarization-induced histamine release. BAY K 8644 alone also induced a biphasic Ca2+ signal and histamine release from ECL cells, which was totally inhibited by nifedipine. Therefore, an L-type Ca2+ channel, as in other neuroendocrine cells, is present in ECL cells and may play a role in histamine release. This depolarization-induced Ca2+ elevation in isolated ECL cells is also found in intact rabbit gastric glands, and the subsequent [Ca2+]i response of the parietal cell in this integrated preparation shows that there is depolarization-dependent signaling between these two cell types, implying a physiological significance to the VDCCs found only in the ECL cell.

In the present study, we have also evaluated the effect of blocking K+ channels on histamine release from isolated gastric ECL cells by using the K+ channel blocker TEA+. We found that TEA+ (10-40 mM) evoked histamine release from cultured ECL cells. We have also shown that TEA+-induced histamine release is absolutely dependent on the presence of external Ca2+ and that this histamine release is completely blocked by nifedipine and profoundly enhanced by the L-type Ca2+ channel agonist BAY K 8644.

Gastrin is the major endocrine ligand that serves to stimulate histamine secretion. This effect is mediated by the binding of gastrin to CCK-B receptors located on the surface of the ECL cell, and secretion can be detected within 5 min (52). The effect of binding the ligand is displayed as a typical G7 receptor-mediated change in [Ca2+]i, namely a transient followed by a steady-state elevation. The elevation of Ca2+ during cellular activation by Ca2+-mobilizing hormones may originate from both release from intracellular Ca2+ stores and entry via membrane Ca2+ channels (61). Patch-clamp experiments have shown that both L- and N-type VDCCs could be activated during exocytosis because of electrical changes after fusion of the histamine-containing vacuole with the plasma membrane of ECL cell (3). The histamine-containing vacuole has a membrane V-type ATPase (VMAT2) that is an electrogenic proton pump. Acidification by this pump depends on the presence of a Cl- conductance that allows electrogenic proton pumping. Stimulation of histamine release by gastrin resulted in activation of a Cl- current, presumably because of fusion of the vacuole membrane with the plasma membrane (34). The depolarization because of this channel could activate VDCCs. However, our data show that the L-type channel is not important in the gastrin-induced histamine release in vitro because nifedipine had no effects on gastrin-induced Ca2+ entry and histamine release. Different results were obtained for stimulation of ECL cells by PACAP.

We previously found that PAC1 receptors are expressed on gastric ECL cells by using RT-PCR and by pharmacological studies measuring Ca2+ signals and histamine release in isolated ECL cells and gastric glands (59). Influx of Ca2+ through different VDCCs has been reported to mediate PACAP-induced catecholamine release from bovine chromaffin cells or insulin release from pancreatic islet beta -cells (6, 15, 18, 40, 59). Here, activation of L-type VDCCs also contributed to PACAP-induced Ca2+ entry and histamine release because nifedipine significantly reduced histamine release as well as Ca2+ entry induced by PACAP. It is of interest that PACAP, in contrast to gastrin, is coupled to both adenylylcyclase and Ca2+ elevation. Elsewhere, we have shown that PACAP addition to rabbit gastric glands elevates ECL cell Ca2+ and also parietal cell Ca2+, the latter effect being completely blocked by H2-receptor antagonists. Furthermore, PACAP injection can result in gastric acid stimulation in intact rats, provided the response of the fundic D cell is blunted by the presence of neutralizing somatostatin antibody (60a). It appears that VDCCs may play a role in PACAP activation of acid secretion.

There are many neuropeptides involved in the inhibition of ECL cell function (48, 60, 62). Somatostatin is known to be a major peptide inhibitor of gastric acid secretion with distinct cellular targets, such as the G cell of the antrum and the ECL cell of the fundus. In the case of the ECL cell, somatostatin, by binding at a somatostatin 2 subtype receptor, inhibits both histamine release and Ca2+ signaling. PYY is a peptide that also inhibits gastric acid secretion by acting at a variety of locations. Pharmacological evidence in the rat suggests that its site of action on the ECL cell is at a Y1 receptor of PYY. Interestingly, this peptide blocks the plateau phase of Ca2+ signaling but is much less active against the transient elevation of [Ca2+]i (23). The PYY effect may account for much of the inhibitory effect of nutrient presentation to the intestine, classified as an "enterogastrone" effect. Recently, we found that galanin has an equipotent effect to somatostatin in inhibition of ECL function (60).

In the beta -cell of the pancreas, somatostatin and galanin inhibit insulin release by blockade of the L-type Ca2+ channels (12, 22, 23, 25, 26, 29, 39, 41, 43, 44, 53, 56, 60). PYY is also effective in inhibition of L-type Ca2+ channels in PC-12 cells and other neuroendocrine cell lines (55, 62). In our study, we found that these peptides inhibit high-K+ and TEA+ depolarization-induced Ca2+ signals and histamine release as well as BAY K 8644-induced Ca2+ signals and histamine release, indicating that, in this cell type, these inhibitory peptides are able to inhibit L-type Ca2+ channels. Their effects on TEA+- or BAY K 8644-induced Ca2+ signals and histamine release were abolished by pretreatment with pertussis toxin, similar to their effects on gastrin stimulation of the ECL cell. Hence, inhibition of either VDCCs or ROCCs of these inhibitory peptides is mediated by Gi trimeric proteins (12, 22, 23, 26, 29, 39, 41, 53).

The importance of N-type Ca2+ channels in the stimulation of neuronal transmitter release has been well demonstrated, and numerous neurotransmitters have been found to inhibit transmitter release through inhibition of this channel subtype (9, 14, 30, 33). Our data indicate that N-type Ca2+ channels are expressed on ECL cells, but, functionally, they seem less important in stimulus secretion coupling than the L-type Ca2+ channels, similar to data obtained for catecholamine release from chromaffin cells (14, 30, 35).

In vivo studies have implied that VDCCs may be involved in the regulation of gastric acid secretion because nifedipine, an L-type Ca2+ channel antagonist, could significantly reduce gastric acid secretion in humans and rats (1, 2, 5, 8, 13, 16, 31, 36). This investigation therefore suggests that L-type Ca2+ channels, expressed on gastric ECL cells, are functionally coupled to histamine release induced by a neuropeptide, PACAP. Because this peptide elevates both cAMP and [Ca2+]i and gastrin is not coupled to adenylylcyclase, perhaps the cAMP component of PACAP signaling is coupled to activation of L-type Ca2+ channels (6, 7, 15, 18, 40, 46). On the other hand, other Ca2+ pathways are involved in ECL function, such as the stores and also the receptor-operated channels activated by gastrin binding to the CCK-B receptor. PAC 1 receptors are also coupled to this system, explaining why inhibitory peptides such as somatostatin are more effective inhibitors than L-type channel blockade.

This investigation shows that L-type Ca2+ channels are indeed expressed on gastric ECL cells and are coupled to histamine release activated by a neuropeptide, PACAP. The finding that activation of these L-type Ca2+ channels is blocked by somatostatin, galanin, and PYY is pertussis toxin sensitive is in agreement with our previous investigations into mechanisms of inhibition of ECL cell function by these neuropeptides. This study, in combination with our previous work, provides additional evidence that regulation of gastric acid secretion by neuropeptides from enteric neurons is exerted via regulation of ECL cell function, in part by activation of VDCCs.


    ACKNOWLEDGEMENTS

This work was supported by United States Veterans Affairs Senior Medical Investigation and National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-46917, DK-53462, DK-41301, and DK-17294.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: G. Sachs, Wadsworth VA Hospital, Los Angeles, CA 90073 (E-mail: gsachs{at}ucla.edu).

Received 3 June 1999; accepted in final form 18 August 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Gastroint Liver Physiol 277(6):G1268-G1280
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