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Expression of functional GABA_A receptors in cholecystokinin-secreting gut neuroendocrine murine STC-1 cells

G. Glassmeier, K.-H. Herzig *, M. Höpfner, K. Lemmer, A. Jansen and H. Scherübl

Received 16 December 1997; accepted after revision 17 April 1998.

	ABSTRACT

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1. Gastrointestinal neuroendocrine (NE) cells synthesize, store and secrete -aminobutyric acid (GABA). Recently, an autocrine-paracrine function of GABA has been proposed for secretion from NE cells.

2. To search for functional GABA_A receptors in NE gut cells, we performed whole-cell and perforated-patch-clamp studies in the intestinal cholecystokinin (CCK)-secreting NE cell line STC-1.

3. Application of GABA evoked currents in STC-1 cells. These effects were mimicked by muscimol, an agonist of GABA_A receptors, and blocked by picrotoxin or bicuculline, antagonists of GABA_A receptors. The GABA- or muscimol-activated currents reversed near 0 mV, which under the recording conditions used was consistent with the activation of the GABA_A receptor-Cl^- channel complex.

4. In contrast to the effect on most neurons, GABA as well as muscimol led to a (reversible) depolarization of the membrane potential of STC-1 cells. Membrane depolarization in turn activated voltage-gated Ca²⁺ channels and increased intracellular Ca²⁺ concentrations in STC-1 cells.

5. In accordance with the observed membrane depolarization and activation of voltage-gated Ca²⁺ channels, both GABA and muscimol stimulated Ca²⁺-dependent CCK release. In contrast, bicuculline inhibited the GABA-induced secretion of CCK.

6. Using the reverse transcription-polymerase chain reaction (RT-PCR), mRNA of the GABA_A receptor subunits 2, 3, 5, 1, 3 and could be detected in STC-1 cells.

7. In summary, we have shown that the CCK-secreting gut NE cell line STC-1 expresses functional GABA_A receptors and that GABA stimulates CCK release. Thus, GABA is involved in the fine tuning of CCK secretion from the gut NE cell line STC-1.

	INTRODUCTION

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Cholecystokinin (CCK) is secreted from specialized neuroendocrine (NE) cells of the upper small intestine in response to ingestion of nutrients. CCK plays an important role in the regulation of digestion, in particular in gall bladder contraction, pancreatic secretion and bowel motility, and in satiety (Liddle, 1997). CCK-secreting cells are diffusely scattered in gut mucosa, which has prevented successful isolation. NE cells of the gut have been enriched (Liddle, 1997) but have not yet been purified for electrophysiological studies. Recently, the NE cell line STC-1 was derived from the murine gut and has been established as a model system for CCK secretion (Rindi et al. 1990). Despite being the best model available, STC-1 cells are still an imperfect model for I cells, in that they secrete not only CCK but also multiple other intestinal hormones (Rindi et al. 1990).

-Aminobutyric acid (GABA) is an important transmitter not only in the brain and spinal cord but also in neural and non-neural tissues outside the central nervous system (CNS) (Erdö & Wolff, 1990). Within the gut, GABA is a putative transmitter of myenteric neurons in a variety of mammals (Jessen, Hills, Dennison & Mirsky, 1983; Krantis, Kerr & Dennis, 1986; Bertrand & Galligan, 1992). Several studies have noted that gastrointestinal NE cells synthesize, store and release GABA (Williamson, Faulkner-Jones, Cram, Furness & Harrison, 1995; Ahnert-Hilger, Stadtbäumer, Strübing, Scherübl, Riecken & Wiedenmann, 1996). In perfused segments of rat stomach or colon, there is a high-affinity [³H]GABA uptake by NE cells (Jessen, Hills & Limbrick, 1988; Krantis, Tufts & Nichols, 1994). Moreover, GABA_A receptors have been identified in gastrin-secreting G cells and in insulin-secreting cells of the pancreas (Hales & Tyndale, 1994; von Blankenfeld et al. 1995).

GABA_A receptors belong to the superfamily of ligand-gated ion channels (Stephenson, 1995). Functional GABA_A receptors are hetero-oligomers that have been proposed to result from the assembly of five subunits. Five families of GABA_A receptor subunits have been identified by molecular cloning in the mammalian central nervous system (1-6, 1-3, 1-3, and ) (Stephenson, 1995; Gorrie et al. 1997).

Hormone secretion from electrically excitable neuronal or NE cells is coupled to changes in ion channel activity and thus depends on the membrane potential. Classical GABA_A receptors are coupled to their intrinsic Cl^- channels to regulate the Cl^- movement across the cell membrane (Bormann, 1988). Depending on the Cl^- gradient of a particular neuronal or NE cell type, activation of GABA_A receptors leads to either hyperpolarization or depolarization. In the present study, we have characterized functional GABA_A receptors in intestinal CCK-secreting cells by electrophysiological, fluorometric and cell biology techniques.

	METHODS

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Cell culture

The STC-1 cell line was derived from an intestinal NE tumour that developed in mice that were double transgenic for two oncogenes expressed from the insulin promoter (Rindi et al. 1990). The cell line was kindly provided by Dr Douglas Hanahan (University of California, San Francisco, CA, USA). Cells were maintained at 37°C in Dulbecco's modified Eagle's medium (DMEM) supplemented with 15 % horse serum, 2·5 % fetal calf serum (FCS), 1 % L-glutamine, 100 U ml^-1 penicillin and 100 µg ml^-1 streptomycin under 95 % air-5 % CO₂.

Reverse transcription-polymerase chain reaction (RT-PCR)

Total RNA from STC-1 cells and from mouse brain was purified using a commercial RNA isolation kit (PURE script, Biozym, Oldendorf, Germany). RNAs were treated with 2 U RQ1 DNase (µg RNA)^-1 (Promega, Mannheim, Germany) for 1 h at 40°C in order to eliminate any possible genomic DNA contamination. First-strand cDNA was generated by reverse transcription (RT) of RNA using SuperscriptII RNase H Reverse Transcriptase (Gibco, Eggenstein, Germany) according to the manufacturer's instructions. Briefly, 3 µg RNA was reverse transcribed using 200 U M-MLV reverse transcriptase in a final volume of 20 µl reaction buffer containing 100 pmol random hexamer primer (Boehringer Mannheim, Germany) and 500 µM of each deoxynucleoside 5'-triphosphate (dNTP, Promega). Control experiments were carried out by replacing M-MLV reverse transcriptase or mRNA with diethyl pyrocarbonate (DEPC)-treated water. An aliquot of 1·5 µl cDNA was used for PCR. Amplification of subunit-specific cDNA sequences encoding GABA_A receptor subunits was performed using subunit-specific primer sequences as shown in Table 1. The specificity of the primers used was confirmed by the NCBI (National Center for Biotechnology Information, Bethesda, MD, USA) BLASTN 2.0 search program (Altschul, Madden, Schaffer, Zhang, Miller & Lipman, 1997).

Table 1. GABA_A receptor subunit-selective RT-PCR primers

Subunit	Primer sequence	RT-PCR product	Reference/accession number
1	5'-AGC TAT ACC CCT AAC TTA GCC AGG-3'	305 bp	Borboni et al. 1994
	5'-AGA AAG CGA TTC TCA GTG CAG AGG-3'
2	5'-TCT CTC CCA AGT GTC ATT CTG-3'	584 bp	Drescher et al. 1993
	5'-GCC CAA AAG TAA CCA AGT CTA-3'
3	5'-CGG CTT TTG GAT GGC TAT GAT-3'	737 bp	Drescher et al. 1993
	5'-ATG GTG AGA ACA GTG GTG ACA-3'
5	5'-GGG CTC TTG GAT GGC TA-C GAC-3'	561 bp	Drescher et al. 1993 *
	5'-TGT GCT GGT GCT GAT GTT CTC-3'		Acc. no. L08485 (Knoll et al. 1993)
6	5'-TAC AAA GGA AGA TGG GCT ATT-3'	439 bp	Drescher et al. 1993
	5'-ACG ATG GGC AAA GTC AGA GAG-3'
1	5'-ACA GTA CAA AAT CGA GAG AGT -CTG-3'	664 bp	Tyndale et al. 1994 *
	5'-TCC ACC TTC TT-A GAC ACC ATC TTG-3'		Acc. no. X14767 (Schofield, Pritchett, Sontheimer, Kettenmann & Seeburg, 1989)
2	5'--CGA GAT GGC CAC ATC -CGA AGC AGT-3'	318 bp	Borboni et al. 1994 *
	5'-TCA -CGG -AAG GCT G-TA GTT TAG TTC-3'		Acc. no. U14419 (Kamatchi et al. 1995)
3	5'-GAA ATG AAT GAG GTT GCA GGC AGC-3'	356 bp	Borboni et al. 1994
	5'-CAG GCA GGG TAA TAT TTC ACT CAG-3'
1	5'-TTG TAT CAG TTT GCC TTT GTA-3'	601 bp	Drescher et al. 1993
	5'-GTA TGT GTA TTC GCC CTT CTC-3'
2	5'-TCA CAA GCC AAA AGT CAG ATG-3'	918 bp	Drescher et al. 1993
	5'-AGC AAA CAG ATA CAA AGA GAT-3'
	5'-TGA GGA ACG CCA T-CG TCC T-TT TCT-3'	334 bp	Borboni et al. 1994 *
	5'-ACC ACC -TCA CGT GGT A-T-G TGT A-CA-3'		Acc. no. M60596 (Sommer, Poustka, Spurr & Seeburg, 1990)

* The published rat DNA primer sequences were adapted to mouse DNA sequences in the case of the primers for 2 and , and adapted to human sequences in the case of the primers for 5 and 1 by single base changes. The single base changes were performed according to the indicated Genbank accession numbers (Genbank of the National Library of Medicine, Bethesda, MD, USA), and have been underlined.

The primers for amplification of cDNA from the subunits 1-3, 6, 3, 1 and 2 were selected as described previously (Drescher et al. 1993; Borboni, Porzio, Fusco, Sesti, Lauro & Marlier, 1994) (Table 1). The forward and reverse primers for 2, 3, 6, 2, 3, 2 and were authentic mouse sequences. The primers for 1 and 1, which were designed for amplification of rat cDNA, also hybridized with mouse cDNA. The primers corresponding to the subunits 5 (Drescher et al. 1993) and 1 (Tyndale, Hales, Olsen & Tobin, 1994), which we modified for amplification of human cDNA, also hybridized with mouse cDNA (for the changes made see Table 1).

Amplification of cDNA was performed in 50 µl 10 mM Tris-HCl buffer (pH 9·0) containing 50 mM KCl, 0·1 % Triton X-100, 1 mM MgCl₂, 200 µM of each dNTP, 1·25 U Taq polymerase (Promega) and 2 µM of each primer (Pharmacia, Freiburg, Germany). The samples were overlaid with 35 µl mineral oil. For amplification, a UNO Thermocycler (Biometra, Göttingen, Germany) was used.

Following an initial denaturation step for 5 min at 95°C, the samples were maintained at 85°C while adding 1·25 U Taq DNA polymerase. Amplification consisted of thirty-eight cycles with 45 s denaturation at 94°C, 45 s annealing at 60°C (1-3, 5, 2-3, ) or at 55°C (6, 2), or 1 min at 50°C (1, 1), and extending for 2 min at 72°C. PCR products were visualized in ethidium bromide-stained 1·8 % agarose gels and the bands were compared with a 123 bp DNA ladder (Gibco). Amplification resulted in single bands of the expected size. Control amplifications were performed with control RT samples as templates.

Electrophysiological recordings

Voltage-clamp recordings were obtained using the whole-cell mode of the patch-clamp technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981). Small coverslips with adherent cells were placed into a recording chamber (0·3 ml) mounted on an inverted microscope (Axiovert 100, Zeiss). Cells were continuously perfused at room temperature (22-24°C) with a standard external solution which comprised (mM): 130 NaCl, 5·4 KCl, 1 CaCl₂, 1 MgCl₂, 10 glucose, and 10 Hepes sodium salt (pH adjusted to 7·3). Patch pipettes were pulled from borosilicate capillary glass (Hilgenberg, Malsfeld, Germany) using a vertical puller in a two-stage process. The pipettes had an initial resistance of 7-10 M when filled with a solution containing (mM): 130 CsCl, 4 MgCl₂, 3 ATP, 10 EGTA, and 10 Hepes sodium salt (pH 7·3). Seal resistances > 10 G were routinely obtained by applying gentle suction to the pipettes. Membrane rupture was monitored electrically as an increase in capacitance. Pipette capacitance, membrane capacitance and series resistance were electronically compensated to achieve minimal capacitive currents (Glassmeier et al. 1997). Cell responses were recorded by an Axopatch 200A amplifier (Axon Instruments). A 486 computer in combination with ISO 2 patch-clamp software (MFK, Niedernhausen, Germany) was used for data acquisition and analysis. Whole-cell currents were filtered at 10 kHz and 1 kHz (Ca²⁺ currents). Ca²⁺ channel currents were corrected for linear leakage and capacitive components by the P/4 procedure before being digitized and stored by the computer at a sampling rate of 5 kHz. To isolate inward currents through voltage-gated Ca²⁺ channels, Ba²⁺ was used as charge carrier in a Na⁺- and K⁺-free solution consisting of (mM): 120 NMDG, 10·8 BaCl₂, 5·4 CsCl, 1 MgCl₂, 10 glucose, and 10 Hepes acid, adjusted to pH 7·3 with HCl.

Current-clamp recordings were obtained using the perforated-patch technique (Akaike, 1995; Glassmeier et al. 1997). In these experiments, fresh nystatin (50 mg ml^-1, dissolved in DMSO) or gramicidin (20 mg ml^-1, dissolved in methanol) was diluted 500 times in the pipette solution containing (mM): 130 KCl, 1 CaCl₂, 2 MgCl₂, 10 EGTA, and 10 Hepes sodium salt, adjusted to pH 7·3. Data are represented as means ± S.D.

Fluorometric measurement of cytosolic Ca²⁺

Changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i) were monitored by measuring fura-2 fluorescence. STC-1 cells were rinsed with standard external solution consisting of (mM): 5·4 KCl, 130 NaCl, 1 CaCl₂, 1 MgCl₂, 10 glucose, and 10 Hepes sodium salt, pH 7·3. Cells were loaded with 5 µM fura-2 AM (Sigma) for 45 min at room temperature as described previously (Eckert, Scherübl, Petzelt, Raue & Ziegler, 1989). Thereafter, cells were washed three times with the buffer described above.

Fluorescence measurements in single cells were performed at room temperature with a digital imaging system (T.I.L.L. Photonics, München, Germany). Cells were visualized using an inverted microscope (Zeiss Axiovert 135). Fura-2 fluorescence was excited alternately at 340 nm and 380 nm with illumination provided by a 75 W xenon lamp. Cellular fluorescence was filtered through a 510 nm band-pass filter.

Images were digitized and analysed using FUCAL 5.12c software (T.I.L.L. Photonics). Ratio images were generated at 1·5 s intervals. To compensate for background noise, illumination of an area void of any cells was subtracted. For each cell, the ratio F₃₄₀/F₃₈₀, which is proportional to [Ca²⁺]_i, was averaged from pixels within manually outlined regions of interest (ROIs). Calibration of the fluorescence ratios was not done. The Ca²⁺-free bath solution contained 1 mM EGTA.

Nifedipine was obtained from Biomol (Hamburg, Germany). Bicuculline, picrotoxin, fura-2 AM and nystatin were purchased from Sigma and gramicidin from Fluka (Neu-Ulm, Germany). Isradipine was a gift from Sandoz (Basel, Switzerland).

Cholecystokinin secretion studies

For secretion studies, 1 × 10⁵ cells were seeded into six-well plates and cultured for several days until they reached 80-90 % confluence. Before the experiment cells were washed twice with buffer followed by the addition of the stimulus or control vehicle for 15 min. The incubation was stopped by placing the plates on ice. For CCK analysis, 125 µl of the medium from each well was taken immediately following the secretion experiment.

CCK release into the medium was assayed using a pancreatic acini bioassay system as described previously (Herzig et al. 1996; Liddle, 1997). Amylase release was measured using an autoanalyser (Eppendorf ACP 5040, Hamburg, Germany).

Results of the CCK secretion experiments are expressed as means ± S.E.M. CCK concentrations were analysed using Student's non-paired two-tailed t test. Statistical significance was set at P < 0·05.

	RESULTS

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RT-PCR of GABA_A receptor subunits

Based on the predicted sizes of the PCR amplification products, eleven GABA_A receptor subunit transcripts, namely 1-3, 5, 6, 1-3, 1, 2 and , were detected in mouse brain. In STC-1 cells, PCR products of the 2, 3, 5, 1, 3 and subunits were visibly amplified (Fig. 1). PCR products of 1, 6, 2, 1 and 2 subunits could not be detected in STC-1 cells under the conditions used. mRNAs of the 4 and 3 subunits were not evaluated.

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Figure 1. Agarose gel electrophoresis with PCR products of GABAA receptor subunits Amplification was performed with cDNA from mouse brain (B) as control, and from STC-1 cells (S). The primers used corresponded to the GABAA receptor subunits 1-3, 5, 6, 1-3, 1, 2 and . M, 123 bp DNA ladder.

Electrophysiology of GABA_A receptor Cl^- channels

The functional properties of GABA_A receptors were first investigated with the patch-clamp technique in the whole-cell mode. Inward currents were elicited by GABA or the GABA_A receptor agonist muscimol in 55 % of the STC-1 cells (24 out of 44 cells). With 100 µM GABA or 100 µM muscimol and with the membrane potential voltage clamped at -60 mV, the evoked current amplitudes ranged between 45 and 430 pA (186 ± 124 pA, n = 12; Fig. 2A). After a rapid activation the currents showed a gradual time-dependent inactivation. The reversal potential of GABA-activated whole-cell currents was calculated by digital subtraction of current traces that were elicited by 300 ms voltage ramps (from -100 to +60 mV) in either the presence or the absence of GABA (Fig. 3A). The current reversed close to the Cl^- equilibrium potential, which was 0 mV under the recording conditions used (n = 5). Additionally, Fig. 3B shows current traces recorded at different holding potentials (V_h). Application of 250 µM muscimol activated outward currents at positive holding potentials and inward currents at negative holding potentials. Muscimol-activated currents were not observed at the Cl^- equilibrium potential (V_h = 0 mV). As both the pipette solution and the bath solution contained the same Cl^- concentration, inward currents observed at negative membrane potentials reflected the outward movement of Cl^-.

The GABA- or muscimol-induced currents were reversibly reduced by the GABA_A receptor antagonists bicuculline (100 µM, n = 3; Fig. 2B) and picrotoxin (100 µM, n = 3; not shown). These pharmacological properties indicated that GABA significantly altered the membrane permeability to Cl^- through GABA_A receptor channels. Any significant contribution of an electrogenic GABA uptake mechanism to the recorded currents could be excluded by the observed Cl^- reversal potential of 0 mV.

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Figure 2. GABA- and muscimol-activated whole-cell currents in STC-1 cells A, application of 100 µM GABA evoked an inward current which was mimicked in amplitude and kinetics by an equivalent concentration of muscimol. B, pharmacological properties of the GABA-activated currents in STC-1 cells. The inward currents were evoked by application of 100 µM GABA and could be reversibly depressed by the simultaneous application of the GABAA receptor antagonist bicuculline (100 µM). The holding potential was -60 mV in both A and B.

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Figure 3. Current-voltage relationship of the GABA- or muscimol-activated currents in STC-1 cells A, currents elicited by a 300 ms voltage ramp from -100 to +60 mV in the absence or presence of 0·5 mM GABA. The reversal potential was close to 0 mV which corresponded to the Cl- equilibrium potential. B, dependence of muscimol-activated currents on the holding potential (Vh) in STC-1 cells. Outward currents were recorded at positive holding potentials (+80 mV) and inward currents at negative holding potentials (-80 mV). Muscimol-activated currents were not observed at a holding potential of 0 mV, the equilibrium potential for Cl- ions under the recording conditions used. Both the pipette and the bath solutions contained 140 mM Cl-.

The effects of GABA or muscimol on the membrane potential were studied by the nystatin or gramicidin perforated-patch technique in the current-clamp mode. The membrane potentials recorded shortly after establishing electrical access to the cytoplasm varied between -42 and -65 mV (mean, -53 ± 6 mV; n = 23). STC-1 cells displayed different patterns of spontaneous electrical activity (data not shown). Application of 50 µM GABA or 50 µM muscimol reversibly depolarized STC-1 cells to about -20 mV (-19 ± 2 mV, in 14 out of 23 cells; Fig. 4).

Fluorometric [Ca²⁺]_i imaging

To monitor changes in [Ca²⁺]_i, STC-1 cells were loaded with membrane-permeant fura-2 AM. The fluorescence ratio F₃₄₀/F₃₈₀ that corresponds to [Ca²⁺]_i was determined by means of a rapid-scanning monochromator in combination with a CCD camera. STC-1 cells showed a marked variability in the pattern of spontaneous Ca²⁺ oscillations. Bath application of either 100 µM GABA or 100 µM muscimol increased [Ca²⁺]_i in 70 % (14 out of 20) of the cells tested; nearly all cells responded to (60 mM) K⁺ depolarization (Fig. 5A). The GABA-induced increase in [Ca²⁺]_i was reduced by the GABA_A receptor antagonist bicuculline (100 µM; data not shown) and totally abolished in Ca²⁺-free bath solution (Fig. 5B). Furthermore, the GABA-evoked [Ca²⁺]_i elevation was strongly reduced by blockade of L-type Ca²⁺ channels with 1 µM nifedipine (data not shown). The [Ca²⁺]_i rise occurring after GABA_A receptor activation in STC-1 cells thus involved Ca²⁺ influx through voltage-gated Ca²⁺ channels.

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Figure 4. Effects of GABA or muscimol on the membrane potential in STC-1 cells The cytoplasm was accessed by the nystatin method of the patch-clamp technique. Spontaneous action potentials were observed. GABA or muscimol induced a reversible membrane depolarization in STC-1 cells.

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Figure 5. Effects of GABA on [Ca2+]i in STC-1 cells The fluorescence ratio F340/F380 was determined in 1·5 s intervals and was plotted against time. A, GABA or high K+ were added as indicated. Superfusion of 100 µM GABA caused a transient increase in [Ca2+]i, similar to that evoked by depolarization with 60 mM extracellular KCl. The extracellular Ca2+ concentration was kept at 1 mM throughout. B, a transient increase in [Ca2+]i was elicited by GABA in standard bath solution containing 1 mM Ca2+. The basal [Ca2+]i decreased in Ca2+-free bath solution, and under this condition GABA had no effect on [Ca2+]i.

Voltage-gated Ca²⁺ channels

To study voltage-dependent Ca²⁺ channel currents in STC-1 cells, the standard bath solution was replaced by a Na⁺- and K⁺-free solution and Ba²⁺ (10·8 mM) was used as charge carrier. Under these conditions, inward currents were elicited in all cells (n = 12) in response to 100 ms pulses to various test potentials starting from a holding potential of -80 mV (Fig. 6A). The current-voltage relationship of the Ca²⁺ channel currents is shown in Fig. 6B. The current trace was recorded during a voltage ramp from -80 to +60 mV. The inward currents activated at an apparent threshold potential of about -40 mV, peaked around 0 mV, and reversed beyond +50 mV. The peak Ba²⁺ current varied between -80 and -320 pA (mean, -190 ± 75 pA; n = 12). As is typical for voltage-gated Ca²⁺ channels, they were completely blocked by Cd²⁺ (100 µM). Extracellular application of the dihydropyridines isradipine or nifedipine reversibly reduced the Ca²⁺ channel currents (Fig. 6A); 1 µM isradipine blocked 73 ± 14 % (n = 6) of the peak Ba²⁺ current, whereas 1 µM nifedipine blocked 62 ± 16 % (n = 4).

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Figure 6. Voltage-dependent Ca2+ channel currents in STC-1 cells A, effects of Ca2+ channel blockers on Ca2+ channel currents. The cell was held at -80 mV and inward currents were activated by test pulses to 0 mV. The dihydropyridine isradipine (1 µM) strongly reduced the amplitude of the inward current. Addition of 100 µM CdCl2 to the bath solution blocked the inward current completely. B, current-voltage relationship of the Ca2+ channel current. The current was activated by a 300 ms voltage ramp from -80 to +60 mV. The current activated at approximately -40 mV and peaked at 0 mV. The Na+- and K+-free bath solution contained 10·8 mM Ba2+ as charge carrier in A and B.

Cholecystokinin secretion

To further characterize the function of GABA_A receptors in STC-1 cells we investigated CCK release upon receptor activation. Under basal conditions STC-1 cells released 3·8 ± 0·46 pmol l^-1 CCK within 15 min (n = 10). The addition of muscimol caused a dose-dependent increase in CCK secretion (from 10 pM to 10 µM; n = 5). Stimulation with 10 pM muscimol did not increase CCK release, while 10 nM, 100 nM, 1 µM and 10 µM muscimol significantly stimulated CCK secretion to 5·0 ± 1·0, 9·0 ± 1·0, 13·4 ± 3·9 and 17·5 ± 3·2 pmol l^-1 CCK, respectively. As shown in Fig. 7, the stimulatory effects of even the highest doses of muscimol (1 µM, 10 µM) were blocked by the GABA_A receptor antagonist bicuculline (100 µM).

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Figure 7. Effects of GABAA receptor modulation on CCK release from STC-1 cells Muscimol (Musc) increased CCK release in a dose-dependent manner. The stimulatory effects of muscimol were inhibited by the GABAA receptor antagonist bicuculline (Bic). Means ± S.E.M. are given.

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

An interacting network of positive and negative feedback loops regulates the fine tuning of hormone secretion from NE gut cells. CCK-releasing cells of the gut have not yet been successfully isolated for electrophysiological studies. STC-1 cells, a clonal murine NE cell line of the gut, serves as a first model system for studying CCK release in vitro. CCK secretion from STC-1 cells can be stimulated by bombesin, -adrenergics or blockers of ATP-sensitive K⁺ channels (Liddle, 1997), but inhibited by galanin (Chang, Chey, Coy & Chang, 1995), nitric oxide or Ca²⁺ channel blockers (Liddle, 1997).

In the present study, we demonstrate that STC-1 cells express functional GABA_A receptors which upon stimulation release CCK in a dose-dependent manner. Our PCR results show that STC-1 cells contain mRNA coding for the GABA_A receptor subunits 2, 3, 5, 1, 3 and , which are believed to form heteropentameric ion channels. Although many cell lines express mRNA of at least one of thirteen receptor subunits (Tyndale et al. 1994), only a few, especially neuronal cells or NE pancreatic cells, respond to GABA (Hales & Tyndale, 1994; von Blankenfeld et al. 1995). In accordance with previous reports on NE cell lines of the pancreas (Borboni et al. 1994; Tyndale et al. 1994; von Blankenfeld et al. 1995) we also found a distinctive pattern of mRNA expression of GABA_A receptor subunits in STC-1 cells. So far, mRNA of the 6 subunit has not been detected in NE cells (Borboni et al. 1994; Tyndale et al. 1994); neither did we detect it in STC-1 cells. To study STC-1 cells for functional GABA_A receptor Cl^- channels we performed patch-clamp experiments in the whole-cell configuration. As expected for the GABA_A receptor-Cl^- channel complex, the reversal potential of the GABA-induced currents was close to the Cl^- equilibrium potential. The GABA-induced currents were mimicked by muscimol, a specific GABA_A receptor agonist, but were depressed by the GABA_A receptor antagonists picrotoxin and bicuculline (Bormann, 1988). Interestingly, the sensitivity of STC-1 cells to GABA varied considerably from cell to cell. Thus, the amplitude of GABA-induced Cl^- currents differed among individual cells to a great extent. The variable sensitivity to GABA may reflect a heterogeneous density or affinity of the GABA_A receptors in STC-1 cells. Similar results had been reported for bovine adrenomedullary chromaffin cells by Peters, Lambert & Cottrell (1989).

By applying both the nystatin and the gramicidin methods of the patch-clamp technique in the current-clamp mode, we consistently obtained an excitatory response to GABA or muscimol in STC-1 cells. The observed membrane depolarization is in contrast to the widely reported hyperpolarizing and inhibitory effects of GABA in neurons (Bormann, 1988). In CCK-secreting neuronal cells, Abucham & Reichlin (1991) showed that GABA hyperpolarized the membrane potential and thereby inhibited CCK release in cortical cells of the brain. Excitatory actions of GABA have, however, already been described in rat lactotrophs (Lorsignol, Taupignon & Dufy, 1994), dendrites of rat hippocampal pyramidal neurons (Anderson, Dingledine, Gjerstad, Langmoen & Mosfeldt Laursen, 1980), nerve endings of rat pituitary (Zhang & Jackson, 1993), and astrocytes (Fraser, Duffy, Angelides, Peres-Velazquez, Kettenmann & MacVicar, 1995). The effect of GABA on the membrane potential depends on the Cl^- equilibrium potential of a particular cell type. With the perforated-patch recording technique, the electrical access to STC-1 cells was obtained with minimal dialysis of the cytoplasm and, especially for the recordings with the gramicidin method, avoided artificial changes in the intracellular Cl^- concentration (Akaike, 1995). We therefore conclude that the depolarizing activity of GABA in STC-1 cells is caused by a depolarized Cl^- reversal potential, a condition that results in Cl^- effux when GABA-gated channels open.

Furthermore, we studied voltage-dependent Ca²⁺ channel currents in STC-1 cells. In accordance with previous reports (for review, see Liddle, 1997), STC-1 cells predominantly expressed dihydropyridine-sensitive L-type Ca²⁺ channels. The apparent threshold potential for the activation of voltage-gated Ca²⁺ channels was about -40 mV, a membrane potential trespassed by GABA_A receptor activation. Underlining this concept, GABA_A receptor activation led to a rise in [Ca²⁺]_i, which was comparable to that obtained by depolarizing STC-1 cells with 60 mM extracellular K⁺. The increase in [Ca²⁺]_i induced by GABA or muscimol could involve either Ca²⁺ influx through (voltage-gated) Ca²⁺ channels and/or Ca²⁺ release from intracellular stores. Our finding that the GABA- or muscimol-evoked increase in [Ca²⁺]_i was inhibited by the Ca²⁺ channel blocker nifedipine and totally abolished in Ca²⁺-free bath solution suggests the involvement of L-type Ca²⁺ channels. Both the GABA_A receptor antagonist bicuculline and the Ca²⁺ channel blocker nifedipine depressed the GABA-induced increase in [Ca²⁺]_i. Thus, activation of GABA_A receptors in STC-1 cells results in an efflux of Cl^- which depolarizes the cells beyond the threshold potential of voltage-dependent Ca²⁺ channels. In accordance with the electrophysiological and fluorometric findings, activation of GABA_A receptors by muscimol increased CCK secretion. Bicuculline, a blocker of GABA_A receptors, decreased CCK secretion from STC-1 cells. Stimulated CCK release could also be inhibited by the Ca²⁺ channel blocker diltiazem (Liddle, 1997).

Modulatory effects of GABA on hormone release have been described before for G and D cells of the stomach (Harty & Franklin, 1983), as well as for enterochromaffin cells (Racké, Schwörer & Reimann, 1995). Addition of GABA to rat antral mucosa fragments resulted in a dose-dependent and bicuculline-sensitive stimulation of gastrin release from G cells but in an inhibition of somatostatin release from D cells. Harty & Franklin (1986) suggested that GABA affected antral gastrin and somatostatin release through stimulation of antral postganglionic cholinergic neurons, but also discussed direct effects of GABA on G and D cells. Indeed, GABA_A receptors have recently been identified in G cells by immunocytochemistry (von Blankenfeld et al. 1995). The role of GABA for the in vivo secretion of gastrin or somatostatin has not yet been defined. Neither is it known how important the effects of GABA are on the in vivo CCK release compared with those of other agents.

In the enteric nervous system, an extensive distribution of GABAergic neurons and nerve fibres has been identified (Erdö & Wolff, 1990). GABA is known to stimulate cholinergic neurotransmission in the gut (Kaplita, Waters & Triggle, 1982). Recently, the involvement of GABA in the control of epithelial electrolyte transport has been demonstrated, which suggested a role for enteric GABAergic neurons in mucosal functions (MacNaughton, Pineau & Krantis, 1996). In our study we demonstrate for the first time that the activation of GABA_A receptors on intestinal NE cells stimulates CCK release. The physiological significance of these findings needs further investigation. As gastrointestinal NE cells have the capacity to take up GABA as well as synthesize, store and release it, GABA may well play a major role in the fine tuning of secretion from gut NE cells.

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Acknowledgements

We thank Sonja Niedrig and Gabriele Brunke for expert technical assistance and R. Marco Liehr for his comments on the manuscript. The project was supported by grants of the Mildred Scheel-Stiftung (10-1040-Sche I), the Sonnenfeld Stiftung and the Deutsche Forschungsgemeinschaft (DFG Sche 326/3-1, DFG He 1965/1-3). The study was presented in part at the 98th Annual Meeting of the American Gastroenterology Society, May 11-14th 1997, in Washington DC, USA.

This work is dedicated to Professor Ernst-Otto Riecken MD on the occasion of his 65th birthday.

Corresponding author

H. Scherübl: Abteilung Innere Medizin/Gastroenterologie, Universitätsklinikum Benjamin Franklin, Freie Universität Berlin, Hindenburgdamm 30, 12200 Berlin, Germany.

Email: hscher{at}zedat.fu-berlin.de

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