Voltage-gated Ca2+ currents in rat gastric enterochromaffin-like cells

J. Bufler1, G. C. Choi2, C. Franke1, W. Schepp2, and C. Prinz2

1 Department of Neurology and 2 Second Department of Medicine, Technical University of Munich, D-81675 Munich, Germany

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Enterochromaffin-like (ECL) cells are histamine-containing endocrine cells in the gastric mucosa that maintain a negative membrane potential of about -50 mV, largely due to voltage-gated K+ currents [D. F. Loo, G. Sachs, and C. Prinz. Am. J. Physiol. 270 (Gastrointest Liver Physiol. 33): G739-G745, 1996]. The current study investigated the presence of voltage-gated Ca2+ channels in single ECL cells. ECL cells were isolated from rat fundic mucosa by elutriation, density gradient centrifugation, and primary culture to a purity >90%. Voltage-gated Ca2+ currents were measured in single ECL cells using the whole cell configuration of the patch-clamp technique. Depolarization-activated currents were recorded in the presence of Na+ or K+ blocking solutions and addition of 20 mM extracellular Ca2+. ECL cells showed inward currents in response to voltage steps that were activated at a test potential of around -20 mV with maximal inward currents observed at +20 mV and 20 mM extracellular Ca2+. The inactivation rate of the current decreased with increasingly negative holding potentials and was totally abolished at a holding potential of -30 mV. Addition of extracellular 20 mM Ba2+ instead of 20 mM Ca2+ increased the depolarization-induced current and decreased the inactivation rate. The inward current was fully inhibited by the specific L-type Ca2+ channel inhibitor verapamil (0.2 mM) and was augmented by the L-type Ca2+ channel activator BAY K 8644 (0.07 mM). We conclude that depolarization activates high-voltage-activated Ca2+ channels in ECL cells. Activation characteristics, Ba2+ effects, and pharmacological results imply the presence of L-type Ca2+ channels, whereas inactivation kinetics suggest the presence of additional N-type channels in rat gastric ECL cells.

high-voltage-activated calcium channels; histamine; calcium; patch clamp

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

ENTEROCHROMAFFIN-LIKE (ECL) cells are histamine-producing cells in the gastric mucosa that control the peripheral regulation of acid secretion. This cell type is responsible for histamine secretion during food-stimulated acid secretion (11, 16). Histamine in ECL cells is stored in electron-empty cytoplasmatic vesicles. Thereby, ECL cells display a close similarity to chromaffin cells of the adrenal gland or neuroendocrine pancreatic cells (5, 11, 16). Similar to chromaffin cells, ECL cells maintain a negative membrane potential of -50 mV and intracellular Ca2+ levels of 50 nM under resting conditions (14, 16-19). Therefore, Ca2+ and Ca2+ channels may be of critical importance for ECL cell homeostasis and histamine secretion.

In previous experiments, we employed patch-clamp techniques to detect K+ and Cl- channels in single ECL cells (14). These studies showed that ECL cells are electrically nonexcitable cells, as defined by the absence of voltage-gated Na+ channels, and have a resting potential of about -50 mV with depolarization-activated K+ channels and gastrin-induced Cl- currents (14). Additional voltage-activated Ca2+ currents could not be detected due to the interference with other membrane currents.

Several types of voltage-gated Ca2+ channels have been described in excitable and nonexcitable cells (1, 2). High-voltage-activated Ca2+ channels (HVACC) and low-voltage-activated Ca2+ channels are defined by their activation characteristics. High-voltage-activated channels can be further classified by their inactivation characteristics into L- or N-type channels, which show slow or fast inactivation, respectively (1, 2, 4, 6-8, 12). In analogy to chromaffin cells, which are known to express HVACC, such as L- and N-type channels, Ca2+ channels might also play a pivotal role in ECL cells. In the current study, we employed patch-clamp techniques to demonstrate the presence of voltage-activated Ca2+ channels in ECL cells. We have identified these channels by activation and inactivation kinetics, effects of Ba2+ and Ca2+ addition, and pharmacological characterization. Our results show that L-type and possibly additional N-type Ca2+ channels are present in ECL cells.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell isolation and primary culture. All reagents and buffer constituents were of analytical grade and were purchased from the indicated suppliers: pronase E (Boehringer Mannheim, Mannheim, Germany); N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES; Serva, Heidelberg, Germany); nycodenz (Accurate Chemical); bovine serum albumin (Serva); Cell-Tak (Becton-Dickinson, Heidelberg, Germany); fetal bovine serum (GIBCO, Eggenstein, Germany); and dithiothreitol, acridine orange, trypan blue, Dulbecco's modified Eagle's medium-F-12 medium, gentamicin, insulin, transferrin, sodium selenite, hydrocortisone, and rat gastrin-17 (Sigma, Munich, Germany).

Mucosal cells from the stomachs of female Sprague-Dawley rats (n = 5/experiment; body weight 180-200 g; Charles River, Sulzfeld, Germany) were isolated by pronase E digestion (1.3 mg/ml) using an everted sac technique (16). The resulting crude cell preparation was fractionated by counterflow elutriation using the JE 6B rotor (Beckman Instruments, Glenrothes, UK) run in a J2-21 M/E Beckman centrifuge. The original method and modifications (14, 17, 18) have been described previously. Briefly, 1 × 108 crude cells were loaded into a standard chamber at a flow rate of 16 ml/min and a rotor velocity of 2,300 revolutions/min. Small cells (diameter <10 mm) were collected at 23 ml/min and 2,000 revolutions/min. For further purification, 7 ml of this cell fraction were overlaid above two different layers of nycodenz (supplemented with 1.2 mM MgCl2, 10 mM HEPES, and 10 mg/ml bovine serum albumin, pH 7.4) diluted 1:1 or 1:2 with buffer (140 mM NaCl, 1.2 mM MgSO4, 1.0 mM CaCl2, 15 mM HEPES, 11 mM D-glucose, 10 mg/ml bovine serum albumin, and 0.5 mM dithiothreitol). After centrifugation (3 min acceleration, 6 min at 209 g, 3 min deceleration; Rotanta, Hettich, Tuttlingen, Germany), a volume of 2 ml was aspirated at the interface between the top and second layer of the step density gradient at 1.052 g/ml. The aliquot contained 75-80% ECL cells, as determined by acridine orange uptake. Cell viability (trypan blue exclusion) exceeded 95%. This enriched ECL cell fraction was then placed on sterilized coverslips precoated with Cell-Tak (diluted 1:1 with 0.5 M NaHCO3). Cells were cultured in Dulbecco's modified Eagle's medium-F-12 medium supplemented with 2 mg/ml bovine serum albumin, 5% fetal bovine serum, 10 mg/ml gentamicin, 5 mg/l insulin, 5 mg/l transferrin, 5 mg/l sodium selenite, 5 nM hydrocortisone, and 1 pM gastrin. After 48 h of culture, 90-95% of total adherent cells were identified as ECL cells by acridine orange uptake. Acridine orange is a weak base that is accumulated in acidic spaces, thereby undergoing a metachromatic shift of the dye. Because histamine in ECL cells is stored in acidic vesicles, ECL cells stained with acridine orange show a bright red fluorescence in cytoplasmatic vesicles (5, 16).

Patch-clamp methods. Cells in short-term culture (3-4 days) were used for electrophysiological experiments, as described previously (10). The cells were placed in a transparent chamber and viewed with a Zeiss Axiovert inverted microscope. In addition to their high purity, ECL cells were visually distinguished by their small size (10 mm diameter) and form. All experiments were performed at 20°C. Previous studies have revealed a resting membrane potential of approximately -50 mV in ECL cells (14). Therefore, current clamp experiments to analyze membrane potentials were not performed in the present study.

Cells were measured in the whole cell method of the patch-clamp technique using standard methods (3). High seal resistances (>10 GOmega ) were easily obtained. Whole cell currents were measured using an EPC9 patch-clamp amplifier (List Electronics, Darmstadt, Germany), and data were analyzed using commercial software (pCLAMP; Axon Instruments). After whole cell patch clamping, ECL cells were allowed to recover for several minutes. For analysis, the currents were digitized with 10 kHz and low pass filtered at 2 kHz.

The Ca2+ currents were recorded by applying a series of depolarizing test pulses (with a duration of 200 ms) as indicated in Figs. 1-4. Only cells with an input resistance >10 GOmega were analyzed. The amplitude of the sustained current was measured at the end point of the 200-ms test pulse, immediately before the appearance of the capacitative transient. Leak currents were not subtracted because the amplitude of this current was not detectable under conditions of 10 GOmega input resistance. All experimental values are expressed as mean values ± SD.

Solutions. For the analysis of Ca2+ currents, ECL cells were bathed in the following extracellular solution (in mM): 120 choline chloride, 2 MgCl2, and 10 HEPES. CaCl2 or BaCl2 was added to this solution at concentrations as indicated in Table 1. Choline chloride was used as a substitute of Na+ in the extracellular solution. For the internal solution, the following composition was used (in mM): 130 CsCl, 20 tetraethylammonium chloride, 2 MgCl2, 10 ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid, and 10 HEPES. The pH of the solutions was adjusted to 7.3 with CsOH, and the osmolality was adjusted to 300 osmol/kgH2O with glucose. To reduce rundown of voltage-gated Ca2+ currents, 4 mM ATP was added to the internal solution shortly before each experiment.

                              
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Table 1.   Composition of solutions for external cations

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Voltage-gated Ca2+ currents were analyzed in 37 different ECL cells. The voltage-gated whole cell Ca2+ currents had small amplitudes (between -5 and -200 pA) at a holding potential of -60 mV and extracellular Ca2+ concentrations of 20 mM. Most of the cells showed inward currents in the presence of K+ and Na+ current blocking solutions (see MATERIALS AND METHODS). As shown in Fig. 1A for a single ECL cell, the inward current was activated at a test potential of around -20 mV at a holding potential of -80 mV. The current peaked at a test potential of +20 mV and decreased at higher potentials. Figure 1B shows the voltage dependence of the peak current (measured immediately) and the sustained current (measured after 200 ms). At a test potential between +50 and +60 mV, the current was reversed into a positive current (Fig. 1B). Using a holding potential of -80 mV, we found an average peak current of -52.2 ± 11.7 pA (n = 6) and a steady-state current of -22.8 ± 9.3 pA at a test potential of +20 mV and 20 mM extracellular Ca2+ (n = 6).


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Fig. 1.   Current-voltage relationship of voltage-activated Ca2+ currents in enterochromaffin-like (ECL) cells. A: top trace, schematic drawing of the pulse protocol with a holding potential of -80 mV and test potentials as indicated; bottom traces, voltage-gated Ca2+ currents with 20 mM Ca2+ added to the extracellular solution. B: current (in mV)-voltage (in pA) curve of the peak current (black-square) and the sustained current (square ) of the experiment shown in A.

Figure 2 shows the inactivation of the depolarization-activated currents at different holding potentials. These experiments were performed at constant test potentials. The experiment of Fig. 2A (performed with 20 mM extracellular Ca2+) demonstrated that the rate of inactivation depended on the holding potential. The rate of inactivation increased with the negativity of the holding potential. Inactivation reached nearly 50% at a holding potential of -100 mV during a 200-ms test pulse to +20 mV, and no inactivation occurred at a holding potential of -30 mV (Fig. 2B). The mean percentage of the inactivation at a holding potential of -80 mV was 52 ± 15% (n = 6).


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Fig. 2.   Time course and amplitude of Ca2+ currents at different holding potentials. A: top trace, schematic drawing of the pulse protocol with different holding potentials as indicated and a test potential of 20 mV at 20 mM extracellular Ca2+; bottom traces, Ca2+-activated currents with 20 mM Ca2+ added to the extracellular solution from different holding potentials as indicated. B: current-voltage relationship for the peak current (square ) and the sustained current (triangle ) of the experiment shown in A.

Further experiments were performed with Ba2+ added to the extracellular solution (Fig. 3). Ba2+ had a higher channel permeability, resulting in a higher peak current, and showed less inactivation at low holding potentials. In Fig. 3A, a single ECL cell was incubated with 20 mM Ca2+, washed, and subsequently incubated with 20 mM Ba2+. The ECL cell was activated by a test pulse of +80 mV from a holding potential of -60 to +20 mV with 20 mM Ba2+ or 20 mM Ca2+ in the same cell. The peak current with 20 mM extracellular Ca2+ was clearly smaller than the current that was activated with 20 mM extracellular Ba2+. Moreover, we observed a stronger inactivation at low holding potentials in the presence of 20 mM Ca2+. The Ba2+-induced current inactivated by only 8.2 ± 15% (n = 4) during a 200-ms test pulse. In Fig. 3B, the current-voltage relationship of the peak current of a representative ECL cell is shown; the ECL cell was infused with 20 mM Ba2+ or 20 mM Ca2+, and voltage was stepped from -60 mV to different test potentials (n = 3). The peak current was increased by substitution of Ca2+ with Ba2+.


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Fig. 3.   A: comparison of the permeability of Ca2+ and Ba2+ through Ca2+ channels in ECL cells. Top trace, schematic drawing of the pulse protocol; bottom traces, voltage-gated currents through Ca2+ channels with indicated concentrations of Ba2+ or Ca2+ added to the extracellular solution. B: current-voltage relationship of a single ECL cell with addition of either 20 mM Ca2+ (square ) or 20 mM Ba2+ (black-square, n = 3).

As shown in Fig. 4, we further studied pharmacological characteristics of voltage-gated Ca2+ currents in ECL cells. In these experiments, Ca2+ currents were activated by increasing the membrane potential from a holding potential between -60 and -30 mV to a test potential of +20 mV at 20 mM extracellular Ca2+. Two representative experiments are shown in Fig. 4, A and B. In Fig. 4A, the Ca2+ current was activated by changing the membrane potential from -60 mV to a test potential of +20 mV. The current was fully inhibited (almost 0 pA) by adding 0.2 mM verapamil to the extracellular solution. Identical results were obtained at a test potential between 0 and +40 mV (n = 3, data not shown). At 0.02 mM verapamil and conditions identical to Fig. 4A (holding potential -60 mV, test pulse to +20 mV), the inhibition was reduced to ~50% but was still effective (data not shown, n = 3). The verapamil effect was partly reversible in the cells tested (Fig. 4A, n = 4).


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Fig. 4.   Pharmacological characterization of the Ca2+ currents of ECL cells. A: block of Ca2+ currents of ECL cells by verapamil with concentrations as indicated. B: increase of Ca2+ currents by BAY K 8644 with concentrations as indicated (n = 5).

Figure 4B shows the effect of BAY K 8644 at a holding potential of -30 mV and a test pulse step to +20 mV. Similar results were observed at a holding potential of -60 mV. Ca2+ currents were increased by 33% when the well-known L-type Ca2+ channel activator BAY K 8644 (0.07 mM) was added to the extracellular solution (Fig. 4B, n = 3).

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Histamine in the gastric mucosa is an effective stimulant of acid secretion and is produced, stored, and released from fundic ECL cells, which are localized in close proximity to the parietal cells (11). In vitro studies using isolated, highly enriched ECL cells from rat tissue demonstrated that these cells have a negative membrane potential (14) and secrete histamine upon gastrin stimulation via a Ca2+-dependent process (16-18). We have previously described the physiological characteristics of the ECL cells using this model of single ECL cells, enabling us to study direct effects of added substances (14, 16-18). The electrophysiological features of ECL cells may parallel findings in chromaffin cells. Both cell types have a resting membrane potential of about -50 mV and maintain a constant intracellular Ca2+ concentration of 50 nM (5, 8, 14, 18). Chromaffin cells are known to express voltage-gated L-type and N-type Ca2+ channels (15). Therefore, we investigated the presence of these channels also in gastric ECL cells.

In the current study, we measured whole cell Ca2+ currents in ECL cells using patch-clamp techniques. Similar to previous experiments (14), whole cell currents were activated by depolarization steps with a minimal potential difference of 20 mV. In contrast to previous experiments, we blocked K+ and Na+ currents to investigate the role of other ions. In the absence of K+ and Na+, voltage-gated whole cell currents had small amplitudes between -5 and -200 pA at a holding potential of -80 mV. These currents were relatively small compared with previous studies that identified membrane currents of +400 to +800 pA in the presence of K+ (14). In previous studies, the current-voltage relationship of the depolarization-activated K+ current showed outward rectification (14). Our present findings, however, clearly indicate the presence of additional, voltage-gated ion channels in ECL cells, presumably Ca2+ channels.

Depolarization of ECL cells induced an inward current under the present experimental conditions at a test potential range of -20 to +40 mV. K+ was substituted by Cs+, and Na+ was replaced by choline; thus Ca2+ were the only extracellular cations capable of producing an inward current. Cations such as Ca2+ enter the plasma membrane after the electrochemical gradient. Under our experimental conditions, Ca2+ were the only cations in the extracellular space with a significant electrochemical, inward directed driving force. Cs+ were the only cations in the intracellular space that follow an outward directed electrochemical gradient. In the presence of K+ and Na+ blocking solutions, Ca2+ currents were detected as inward (=negative) currents in a potential range of -20 to +40 mV. At potentials between +50 and +60 mV shown in Fig. 1B, the current reversed to an outward current. The reversal of this current may reflect efflux of Cs+.

The current-voltage relationship of the inward current in single ECL cells corresponded to the kinetics of HVACC (Figs. 1 and 2). Low-voltage-activated Ca2+ currents such as T-type Ca2+ channels known to be activated at a more negative membrane potential (below -50 mV) were not detected. HVACC such as L- and N-type channels typically reveal activation of the inward current at a membrane potential of -30 to -20 mV (1, 2, 4-9). As shown in Fig. 1, A and B, the peak and sustained currents were activated at a membrane potential range between -20 and +40 mV and showed an inward (=negative) current. Maximal activation was observed at a membrane potential of +20 mV and 20 mM extracellular Ca2+. The current-voltage relationship therefore suggests that high-voltage-activated channels such as L- and N-type channels are present in ECL cells.

L- and N-type Ca2+ channels show different inactivation kinetics and are characterized by slow inactivation (within minutes) or fast inactivation (within seconds), respectively (1, 2). Moreover, N-type Ca2+ channels are inactivated at more negative membrane potentials (-80 to -100 mV), whereas L-type channels remain open (2, 7). The experiments shown in Fig. 2, A and B, investigated the inactivation kinetics within 200 ms. At a holding potential of -30 mV, no inactivation of the current was observed. However, at more negative holding potentials, we observed ~50% inactivation of the inward currents at a holding potential of -100 mV (Fig. 2), typical for N-type channels (2, 4, 9, 13). Because N-type channels were inactivated by only 50%, it can be concluded that the remaining current was mediated by L-type channels. Therefore, our findings regarding the inactivation kinetics suggest that L-type and N-type channels are present in ECL cells.

Addition of Ba2+ (Fig. 3) instead of Ca2+ increased the inward current. Ba2+ has a greater permeability for Ca2+ channels than Ca2+ itself (13). It has been previously reported that Ba2+ is well permeable for L-type Ca2+ channels and decreases the inactivation rate, which is consistent with our present observations (4). After replacement of Ca2+ with Ba2+ during the experiment, the tail current decreased slightly (<10%), as shown in Fig. 3A. Because this experiment was performed with a single ECL cell infused continuously over 15-25 min with different solutions, it may be that the continuing clamp conditions finally interfered with ECL cell viability. Yet, the tail current was decreased by <10%, indicating that the integrity of the cell response was still viable.

Our present results provide direct pharmacological evidence for the presence of voltage-activated L-type Ca2+ channels in ECL cells. Because the activation and inactivation kinetics in our studies suggested that L- and N-type channels might be present, we performed additional experiments with hyperpolarized membrane potentials (-60 and -30 mV) in which N-type channels are less activated. Verapamil is a well-tested inhibitor of high-voltage-gated L-type Ca2+ channels (20). As shown in Fig. 4A, the membrane potential was stepped from a holding potential of -60 mV to a test pulse of +20 mV, achieving maximal currents and better comparisons. Verapamil blocked the Ca2+ currents of ECL cells almost completely.

Furthermore, the effect of the specific L-type Ca2+ channel activator BAY K 8644 was investigated on Ca2+ currents in ECL cells (Fig. 4B). The membrane potential was stepped from -30 to +20 mV. At this potential, no inactivation and thereby no activation of N-type channels can be observed, allowing specific activation of L-type Ca2+ channels by BAY K 8644. The Ca2+ current was increased by BAY K 8644, supporting the idea that ECL cells have Ca2+ channels that display properties of L-type Ca2+ currents.

Our current work strongly suggests the presence of voltage-gated L- and N-type Ca2+ channels in ECL cells. The physiological mechanism that leads to depolarization of HVACC in ECL cells is currently unknown. Voltage-gated Ca2+ channels were originally described in excitable cells, such as chromaffin cells of the adrenal glands (7). In chromaffin cells, acetylcholine stimulates epinephrine secretion via depolarization and activation of HVACC. Ca2+ entry through HVACC in this cell type, which is the triggering event for catecholamine secretion (15), in turn activates K+ currents (21). These currents inhibit the voltage-activated Ca2+ channels, decrease intracellular Ca2+ concentration, and finally restore the membrane potential in chromaffin cells. ECL cells, despite their great structural similarities to chromaffin cells, are electrically nonexcitable. It may be speculated that stimulation of ECL cells with gastrin would result in depolarization of ECL cells, which in turn could stimulate HVACC and/or which could stimulate Ca2+-activated outward-rectifier K+ currents, as anticipated in earlier studies (14, 21). Our current data, however, only demonstrate the presence of HVACC in ECL cells but do not allow conclusions on whether stimulation of ECL cells with gastrin finally leads to activation of HVACC.

    ACKNOWLEDGEMENTS

This work was supported by grants of the Deutsche Forschungsgemeinschaft (DFG PR 411/2-1 and P4 411/2-2) and by a grant from the Technical University of Munich (HSP II, 230-11/814/91).

    FOOTNOTES

Address for reprint requests: C. Prinz, II Medizinische Klinik, Technical Univ., Ismaningerstrasse 22, D-81675 Munich, Germany.

Received 12 July 1996; accepted in final form 14 October 1997.

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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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