Receptor-induced Ca2+ signaling in cultured myenteric neurons

Pieter Vanden Berghe1, Jan Tack1, Antonius Andrioli1, Ludwig Missiaen2, and Jozef Janssens1

1 Center for Gastroenterological Research and 2 Department of Physiology, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We studied the effect of excitatory neurotransmitters (10-5 M) on the intracellular Ca2+ concentration ([Ca2+]i) of cultured myenteric neurons. ACh evoked a response in 48.6% of the neurons. This response consisted of a fast and a slow component, respectively mediated by nicotinic and muscarinic receptors, as revealed by specific agonists and antagonists. Substance P evoked a [Ca2+]i rise in 68.2% of the neurons, which was highly dependent on Ca2+ release from intracellular stores, since after thapsigargin (5 µM) pretreatment only 8% responded. The responses to serotonin, present in 90.7%, were completely blocked by ondansetron (10-5 M), a 5-HT3 receptor antagonist. Specific agonists of other serotonin receptors were not able to induce a [Ca2+]i rise. Removing extracellular Ca2+ abolished all serotonin and fast ACh responses, whereas substance P and slow ACh responses were more persistent. We conclude that ACh-induced signaling involves both nicotinic and muscarinic receptors responsible for a fast and a more delayed component, respectively. Substance P-induced signaling requires functional intracellular Ca2+ stores, and the 5-HT3 receptor mediates the serotonin-induced Ca2+ signaling in cultured myenteric neurons.

enteric nervous system; calcium channel; enteric neuron


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE MYENTERIC PLEXUS NEURONS control various functions of the gastrointestinal tract. Intracellular Ca2+ concentration ([Ca2+]i) can be used to monitor activation of these neurons (32). In culture, myenteric neurons form new networks that can be used as a model for the enteric nervous system. Focal electrical stimulation of the neuronal processes induces a transient [Ca2+]i rise in a subset of the cultured neurons. These [Ca2+]i changes require neuronal conduction and excitatory synaptic transmission (40). The constitutive elements of synaptic communication between neurons are the fast and slow excitatory postsynaptic potentials (EPSPs). In whole-mount myenteric plexus preparations, it has been established that the majority of fast EPSPs are due to the activation of nicotinic ACh receptors, allowing cations to flow through the receptor channel (38). However, fast synaptic events are not restricted to nicotinic receptors. The activation of the serotonin 5-HT3 receptor and the purinergic P2x receptor, as well as ligand-gated cation channels, induce similar brief depolarizations (3, 5, 8, 24, 34, 35).

At present, there is still debate on which neurotransmitters control slow EPSPs. The main candidates are substance P (SP) and serotonin (5-HT) (6, 17, 25, 42, 43). The activation of the neurokinin (NK)1 receptor by SP induces a slow depolarization quite similar to the electrically evoked slow EPSPs (10, 16). Although NK1 seems to be predominant in the enteric nervous system, there is also evidence that NK3 receptors are involved in neurotransmission (15, 31). The activation of the atypical 5-HT1p receptor induces a similar slow depolarization in several types of myenteric neurons (1, 7, 12, 23, 24, 35). Besides SP and 5-HT, it is also suggested that ACh mediates slow EPSPs by activating muscarinic receptors (26, 28).

We aimed to demonstrate the existence of these candidate neurotransmitters in the myenteric neuron cultures and to confirm that they mimic the [Ca2+]i changes evoked by electrical stimulation of interconnecting fibers in cultured myenteric networks (40). Furthermore, we wanted to explore in more detail the underlying mechanism of Ca2+ signaling in myenteric neurons.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Myenteric Neuron Cultures

Primary cultures of myenteric neurons were prepared from adult guinea pig small intestine according to previously described methods (11, 14). In brief, the longitudinal muscle and the adherent myenteric plexus (LMMP) were dissected from the circular muscle and the mucosa; the LMMP was digested in an enzymatic solution containing protease (1 mg/ml) and collagenase (1.25 mg/ml). After a 30-min incubation (37°C), the suspension was placed on ice and spun at 1,600 rpm. Ganglia were picked up and plated in Lab-tek culture dishes (GIBCO, Merelbeke, Belgium), in which they adhered to the cover-glass bottom. After a few days, neurons started growing in networklike structures reminiscent of the ganglionated plexus. The culture medium was changed every two days. Medium 199 enriched with 10% fetal bovine serum and 50 ng/ml 7s nerve growth factor (7sNGF) was used. Antibiotics (streptomycin, 100 µg/ml; penicillin, 100 U/ml; amphotericin B, 1 µg/ml; gentamicin, 50 µg/ml) were added to the medium, glucose concentration was elevated to 30 mM, and the Ca2+ concentration was adjusted to 2.5 mM by adding CaCl2. The culture chambers were kept in an incubator at 37°C and continuously gassed with 95% O2-5% CO2. Arabinose-C-furanoside (10 µM) was added to prevent the proliferation of dividing cells (glial cells, fibroblasts).

Experimental Medium

Experiments were performed in a modified Krebs solution containing (in mM) 150 NaCl, 6 KCl, 1.5 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES (pH 7.38). The Ca2+-free solution contained 2.5 mM MgCl2 and 2 mM EDTA. The high-K+ medium contained 75 mM K+. Osmolarity was adjusted by lowering the Na+ concentration to 81 mM. The pH of the modified Krebs solution was checked after solving the agonists and adjusted when necessary.

[Ca2+]i Measurements

The neurons were loaded for 30-45 min at room temperature in a modified Krebs solution containing 5 µM indo 1-AM and 2.5 µM Pluronic F-127 (25% wt/wt). The AM is cleaved by esterases in the cytoplasm, and the free indo 1 molecule is trapped in the cell. On binding to Ca2+, the emission peak of the indo 1 molecule is shifted from 480 nm to 405 nm. The ratio of these two signals is directly proportional to the free [Ca2+]i. Experiments were performed at 22°C on a Bio-Rad MRC 1024 confocal microscope equipped with an Ar+ ion laser. Sample frequency depended on the number of "regions of interest" selected and ranged between 1.25 and 1.75 Hz. All time experiments were monitored using Laser-sharp software. The results were expressed as a ratio of emitted fluorescence. The amplitude of the signals and the peak duration, defined as the width at half-height, were analyzed and compared with a Student's t-test.

Immunocytochemistry

Cultured cells were fixed in a freshly prepared phosphate buffered (pH 8.0) 2%/0.2% paraformaldehyde/picric acid solution. The cells were permeabilized (2 h), and nonspecific binding sites were blocked by treating the cells for 1 h in a 0.1 M PBS solution containing 0.5% (wt/vol) Triton X-100 and 4% (vol/vol) goat serum. Primary antisera were diluted in this blocking solution, and preparations were exposed for ~20 h at room temperature. To prevent bacterial growth, NaN3 was added to the incubation solution in a 0.3% (wt/vol) concentration. After incubation in the primary antibody solution, dishes were rinsed 3 × 5 min in a 0.1 M PBS solution and incubated in the appropriate fluorescence-labeled secondary antibodies (2 h, 0.1 M PBS, 4% goat serum). The fluorophores were visualized under a Nikon microscope equipped with a fluorescence unit. For Cy3, filtercube Y-2EC (EX BP540-580, DM RK595, EM BA 600-660) was used. Filtercube UV-2EC (EX BP340-380, DM NDM400, EM BA 435-485) and B-2A (EX BP470-490, DM 505, EM BA 510/20) were used for the visualization of aminomethylcoumarin and FITC, respectively.

Chemicals

Medium 199 and antibiotics were from GIBCO; omega -conotoxin and 7sNGF were from Alomone Labs (Jerusalem, Israel); protease, collagenase, nifedipine, atropine, hexamethonium (C6), arabinose-C-furanoside, 5-HT, ACh, and SP were from Sigma (Bornem, Belgium); 5-HT receptor blockers were from RBI (Natick, MA); primary antibodies to 5-HT were from DAKO (Glostrup, Denmark); SP was from Fitzgerald (Concord, MA); neuron-specific enolase (NSE) was from Polysciences (Warrington, PA); choline acetyltransferase (ChAT) was from Michael Schemann (Hannover, Germany); secondary antibodies were from Jackson Labs (West Grove, PA); and indo 1-AM was from Molecular Probes (Leiden, The Netherlands). Cisapride, which was a gift from Janssen Pharmaceuticals (Beerse, Belgium), was solved in a tartaric acid stock solution.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Differential Properties of Neurons and Nonneuronal Cells in Culture

Morphology and immunocytochemistry. The myenteric neuron cultures were not completely void of other cell types such as glial cells and fibroblasts. The latter two cell types had flat cell bodies and stuck closely to the glass. On the contrary, neuronal cell bodies were rather thick and could easily be distinguished under phase-contrast optics. Subsequent immunocytochemical processing for NSE (data not shown) confirmed the morphological identification.

Voltage-operated Ca2+ channels. All cells were briefly depolarized with 75 mM K+, which opened the voltage-operated Ca2+ channels (VOCC) and led to a Ca2+ influx. A one-to-one correlation was observed between the cells with a high-K+-induced [Ca2+]i response and the neurons as identified morphologically and by NSE staining. Nonneuronal cells seemed to lack VOCCs, and therefore the high-K+-induced [Ca2+]i response (Fig. 1A) could be used as a physiological means to distinguish between the cells.


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Fig. 1.   Capacitative and voltage-dependent Ca2+ entry in nonneuronal cells and neurons. Ratio of 2 indo 1 emission wavelengths is plotted against time. Arrow represents addition of 75 mM K+ (10 s), activating voltage-dependent Ca2+ entry. A: in a 5-µM thapsigargin (Tg)-pretreated (1 h) nonneuronal cell (gray line), intracellular Ca2+ concentration ([Ca2+]i) drops when Ca2+ is omitted from extracellular medium. Restoring 1.5 mM extracellular Ca2+ concentration ([Ca2+]e) results in a [Ca2+]i overshoot, which is characteristic of capacitative Ca2+ entry. No capacitative Ca2+ entry is observed in a 5 µM Tg-pretreated (1 h) myenteric neuron (black line). Trace shown is typical of 10 nonneuronal cells and 109 neurons. [Ca2+]e is represented by horizontal line. B: effect of acute administration of 75 mM K+ (arrow, 10 s) and 5 µM Tg (horizontal bar) to cultured myenteric neurons; 60% of the cells display an oscillatory response (n = 48).

Capacitative Ca2+ entry. Thapsigargin (Tg) prevents the refilling of the intracellular Ca2+ stores by irreversibly blocking their ATP-dependent Ca2+ pump. The acute administration of 5 µM Tg resulted in a [Ca2+]i rise in 96% of the cultured myenteric neurons (n = 48), indicating that the stores were Tg sensitive. In 60% of the neurons, the responses had a typical oscillatory character (Fig. 1B). Tg-pretreated (5 µM, 1 h) neurons were then used to screen for the presence of capacitative Ca2+ entry, which allows the Ca2+ entry in cells with depleted Ca2+ stores. Tg-pretreated cells with capacitative Ca2+ entry display characteristic [Ca2+]i signaling. When the extracellular Ca2+ concentration ([Ca2+]e) is changed from 1.5 mM to 0 mM, a decrease in [Ca2+]i can be observed. When 1.5 mM Ca2+ is restored, the [Ca2+]i suddenly increases (37). In all nonneuronal Tg-treated cells (n = 25), the [Ca2+]i dropped below the baseline when 0 mM [Ca2+]e was administered and a [Ca2+]i overshoot could be observed after readjusting the [Ca2+]e to 1.5 mM (Fig. 1A). Myenteric neurons did not display this typical signaling. A small [Ca2+]i rise was only observed in 20% of the neurons. Because of the low amplitude and the lack of a preceding [Ca2+]i drop, these responses were judged not characteristic of capacitative Ca2+ entry. In the majority of the myenteric neurons (80%), no [Ca2+]i changes were observed (n = 109) (Fig. 1A).

Presence of the Candidate Neurotransmitters

Fixed culture cells were processed for ChAT, SP, and 5-HT immunoreactivity. The majority of the neurons expressed ChAT, the main enzyme in the synthesis of ACh, and could therefore be considered cholinergic (Fig. 2). Immunoreactivity for SP was readily observed in the majority of fibers in the network. Although 5-HT occurrence was more limited, some fibers were clearly 5-HT positive.


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Fig. 2.   Immunochemical staining of cultured myenteric neurons. Substance P (SP)-containing fibers (green) are abundantly present, whereas 5-HT-positive fibers (red) are more rare. Majority of neurons contain choline acetyltransferase (blue) and can therefore be considered cholinergic.

Neurotransmitters and [Ca2+]i Signaling

ACh-induced [Ca2+]i signaling. ACh induced a [Ca2+]i rise in a subset of the myenteric neurons in the network. The amplitude of the [Ca2+]i rise, as well as the number of responding cells, increased dose dependently. Application of 10-7 M ACh (n = 50) caused a detectable rise in 2.0% of the neurons, whereas 93.0% of the cells displayed a [Ca2+]i rise with 10-4 M ACh (n = 86) (Fig. 3A). The blockade of neuronal conduction by 10-6 M TTX did not alter the number of responding cells (n = 20). Selective nicotinic activation with 1,1-dimethyl-4-phenyl-piperazine (DMPP) evoked a [Ca2+]i rise in 28% (n = 68) and 93.5% (n = 154) of the neurons for 10-6 M and 10-5 M, respectively. Repeated application of ACh and DMPP revealed no significant reduction in the number of responding cells. The shape of the [Ca2+]i transient induced by DMPP and ACh clearly differed. Almost all responses to DMPP reached the maximum within 1-2 s (98.8%, n = 79), whereas ACh also evoked slow responses. This slow response subgroup, which was absent for DMPP, accounted for 9.8% (n = 81).


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Fig. 3.   Effect of ACh and 1,1-dimethyl-4-phenyl piperazine (DMPP) on [Ca2+]i signaling in cultured myenteric neurons. A: number of responding cells as a function of negative log of concentration of ACh (p[Ach], n > 50) and DMPP (p[DMPP], n > 68). B: responses of 2 neurons (black and gray lines represent 28 and 2 neurons, respectively) to 75 mM K+ (arrow, 10 s), 10-5 M ACh, and 10-5 M DMPP. Duration of agonist application is indicated with horizontal bars. [Ca2+]e is represented by full horizontal line.

The contribution of [Ca2+]e to the [Ca2+]i rise induced by ACh and DMPP was also studied. The number of responses to ACh was significantly reduced (12.3 vs. 92.4%) in the absence of extracellular Ca2+ (n = 65), whereas DMPP no longer evoked a response (n = 53). The responses to ACh that persisted in 0 mM [Ca2+]e had a much slower onset than in normal [Ca2+]e (Fig. 3B). This finding indicates that the [Ca2+]i rise mainly depended on the influx of extracellular Ca2+. The involvement of L- and N-type Ca2+ channels was studied using Ca2+-channel blockers (Fig. 4A). The number of responses to ACh was not significantly altered (n = 47, N.S., chi 2) either by nifedipine (10-6 M) or by omega -conotoxin (5 × 10-7 M). However, the amplitude of the ACh-induced [Ca2+]i rise was reduced to 76.2 ± 5.2% and 87.2 ± 5.3% by the blockers, respectively (n = 10) (Fig. 4B). Tg pretreatment had no significant effect on the number of responses to ACh (10-4 M) (97.8%) and to DMPP (10-5 M, 84.7%, n = 46).


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Fig. 4.   Effect of Ca2+-channel blockers nifedipine (10-6 M) and omega -conotoxin (5 × 10-7 M) on ACh (10-5 M)-induced [Ca2+]i signaling. A: neither nifedipine nor omega -conotoxin is able to abolish [Ca2+]i rise induced by ACh. Period of application is marked with horizontal bars. Tracing is typical of 10 neurons. B: effect of nifedipine (10-6 M) and omega -conotoxin (5 × 10-7 M) on amplitude of ACh response (n = 10). Closed bar represents amplitude of control response to ACh (set at 100%). Amplitudes (means ± SE) of ACh response in presence of nifedipine and omega -conotoxin are represented by gray and open bars, respectively (* P < 0.05 vs. control).

We assessed the role of muscarinic and nicotinic receptors in the initiation and maintenance of the [Ca2+]i rise. Atropine (a muscarinic blocker), pirenzepine and gallamine (selective M1 and M2 blockers, respectively), and the nicotinic blocker C6 were used. The number of responses to ACh was significantly decreased by atropine (10-5 M) (69.6% vs. 93%, chi 2, P < 0.001, n = 79) (Fig. 5A). Atropine also significantly reduced the duration of the response (15.7 ± 1.4 vs. 25.9 ± 1.2 s, P < 0.001, n = 45). In the presence of pirenzepine (10-6 M), only 7.2% of the neurons (n = 69) displayed a rise in [Ca2+]i, whereas gallamine (10-5 M) did not significantly affect the responses (n = 27). C6 significantly reduced the number of responses to ACh (14.3% vs. 52.7% and 15.8 vs. 93.1% for 10-5 M and 10-4 M, respectively) (Fig. 5A). In 54 cells, ACh and DMPP were consecutively applied in normal solution and in the presence of nicotinic and muscarinic antagonists. All cells responding to DMPP had a response to ACh, whereas few cells responded to ACh without responding to DMPP. In the majority of neurons, the ACh response in the presence of atropine mimicked the DMPP response (Fig. 5B). In addition, when atropine was administered during the ACh application, the [Ca2+]i immediately decreased to its basal level and no prolonged [Ca2+]i responses were observed (n = 20). Interestingly, the ACh responses that persisted in the presence of C6 often lacked the fast component.


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Fig. 5.   Involvement of muscarinic and nicotinic receptors in 10-4 M ACh-induced [Ca2+]i signaling. A: effect of atropine (10-5 M) and hexamethonium (10-4 M) on number of responding cells. Total number of cells is indicated on bars. * P < 0.05 vs. control (chi 2). B: effect on [Ca2+]i of 10-5 M ACh, 10-5 M DMPP, and 10-6 M atropine added as indicated by horizontal bars. Duration of responses is reflected in width at half-height, marked with double arrowheads. Trace is typical of 45 cells.

Substance P-induced [Ca2+]i signaling. SP induced a [Ca2+]i rise in a subset of the cultured myenteric neurons. Low concentrations (10-10 and 10-9 M) did not evoke a detectable [Ca2+]i rise (n = 43 and 63). The number of neurons responding to higher concentrations increased logarithmically (12.9% for 10-8 M, 17.6% for 10-7 M, 54.1% for 10-6 M, and 68.2% for 10-5 M, n = 85), reaching 85% of the cells for 10-4 M (n = 20) (Fig. 6A). The relative amplitude compared with the K+-induced response also increased (5.4 ± 1.3%, 7.6 ± 1.8%, 14.1 ± 3.0%, and 18.9 ± 2.9% for 10-8 M, 10-7 M, 10-6 M, 10-5 M, respectively, n = 7). TTX (10-6 M) did not alter the number of responses to SP (n = 39). In the absence of extracellular Ca2+, SP still induced responses in a number of cells; however, the number of responses to SP was significantly reduced (16.6 vs. 62.6%, P = 0.01). The amplitude of the remaining responses was reduced to 47.3 ± 12%. The L-type Ca2+-channel blocker nifedipine (10-6 M) did not alter either the number of responses or the amplitude of the response (58.3% and 93.6 ± 12%, respectively). Similarly, blockade of N-type Ca2+ channels by omega -conotoxin (5 × 10-7 M) did not significantly affect the number and the amplitude of the SP-induced [Ca2+]i rises (45.5% and 77 ± 16%, respectively).


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Fig. 6.   SP-induced [Ca2+]i signaling in myenteric neurons. A: number of responding neurons as a function of negative log of SP concentration (p[SP]; n > 20). B: number of cells responding to SP (10-6 and 10-5 M) after pretreatment with Tg (5 µM, 1 h, n = 50), ryanodine (Ry; 5 × 10-5 M, 45 min, n = 24), and pertussin toxin (PTX; 100 ng/ml, 24 h, n = 16). * P < 0.05 vs. control (chi 2).

To assess the role of the Ca2+ stores, 50 Tg-pretreated neurons were challenged with SP (10-6 and 10-5 M). The number of responses to SP was significantly (chi 2, P < 0.001) reduced in the pretreated neurons (6.0 vs. 54.1% and 8.0 vs. 68.2%, respectively) (Fig. 6B). To investigate whether ryanodine receptors (RyR) were involved, we pretreated the neurons with ryanodine (5 × 10-5 M), blocking the RyRs in the inactive state. No significant (chi 2, P = 0.05) difference in the number of responses to SP was observed (n = 24) (Fig. 6B). The 10 mM caffeine-induced [Ca2+]i rise, normally present in 80% of the neurons (n = 15), could only be seen in 18% of the pretreated cells (n = 11) (chi 2, P = 0.01), and their amplitude was significantly reduced, proving the efficacy of the ryanodine pretreatment. In three sets of experiments the neurons were pretreated with pertussin toxin (PTX, 100 ng/ml, 24 h) to check whether a PTX-sensitive G protein was involved. The number of responses to SP was not significantly altered (chi 2, P = 0.05) in the PTX-pretreated neurons (48.0 vs. 54.1% and 56.0 vs. 68.2% for 10-6 and 10-5 M SP, respectively) (Fig. 6B).

5-HT-induced [Ca2+]i signaling. The application of 10-6 M 5-HT induced a [Ca2+]i rise in 25.6% of the neurons (n = 122), and 90.7% responded when 10-5 M 5-HT was applied (n = 388). The relative amplitude of the response also increased dose dependently. The application of 5-HT induced a [Ca2+]i rise, with a relative amplitude of 9.1 ± 1.0% (10-6 M, n = 9) and of 45.3 ± 3.6% (10-5 M, n = 43) compared with the K+-induced response. No 5-HT-induced responses could be observed when Ca2+ was removed from the extracellular medium (n = 14). TTX did not alter the number of responses to 5-HT (n = 27). Nifedipine (10-6 M) and omega -conotoxin (5 × 10-7 M) did not alter the number of responses but significantly reduced the amplitude of the 5-HT-induced responses to 47.2 ± 5% (P < 0.01) and 87 ± 4.4% (P = 0.04), respectively. The response to 5-HT was also tested in Tg-pretreated (5 µM, 1 h) neurons. The number of responses to 5-HT was not significantly altered for 10-6 M (28.57% vs. 25.41%) (n = 28) and for 10-5 M (81.48% vs. 90.7%).

Several 5-HT receptors have been described in the gastrointestinal tract (9), and therefore the effect of several specific 5-HT receptor agonists and antagonists on the [Ca2+]i were studied. The 5-HT1a receptor agonist 8-hydroxy-2-(di-n-propylamino) tetralin at concentrations of 10-6 M (n = 35) and 10-5 M (50 neurons) did not induce a detectable rise in [Ca2+]i (Fig. 7A). Sumatriptan, a 5-HT1p-d receptor agonist (24, 41), failed to evoke a detectable rise in [Ca2+]i in the cultured neurons (Fig. 7A) either at 10-6 M (n = 67) or at 10-5 M (n = 104). During general 5-HT1-2 blockade by methiothepin (10-5 M), the number of responses to 5-HT was not significantly altered (from 90.7 to 84.9%, n = 106) (Fig. 7B). However, methiothepin significantly reduced the amplitude (to 47 ± 3.5%, n = 43) and the duration (to 47.6 ± 3.8%, n = 29) of the control 5-HT response (10-5 M), induced either before or after the methiothepin application. The number of responses to 5-HT (10-5 M) was significantly reduced (3.7 vs. 90.7%, n = 27) in the presence of the 5-HT3 blocker tropisetron (10-5 M) (data not shown). Since tropisetron applied in micromolar concentrations also blocks the 5-HT4 receptor, a more selective blocker, ondansetron (10-5 M), was used to demonstrate the importance of the 5-HT3 receptor. Only 2.4% of the neurons (n = 124) showed a residual response to 5-HT in the presence of ondansetron. The 5-HT response reappeared after washout in 88% of all neurons (n = 76), which was not significantly different from the original number of responses (Fig. 8A). In none of 30 neurons did the 5-HT4 agonist cisapride (10-5 M) induce a [Ca2+]i rise (n = 30). In line with this finding, the response to 5-HT was not abolished by the selective 5-HT4 antagonist DAU-6285 (10-5 M, n = 54) (Fig. 8B). The 5-HT4 receptor is positively coupled to adenylate cylase; therefore, direct stimulation of adenylate cyclase by forskolin was studied. No detectable changes in [Ca2+]i in cultured myenteric neurons were observed when 10-6 M forskolin was administered (n = 30) (data not shown).


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Fig. 7.   Role of serotonin (5-HT) receptors in control of 5-HT-induced [Ca2+]i signaling. Application of agonists and antagonists is marked with horizontal bars. Application of 75 mM K+ (10 s) is indicated by arrow. A: effect of 5-HT1p agonist sumatriptan (SUM; 10-5 M), 5-HT1a agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OHDPAT; 10-5 M), and 5-HT (10-5 M) on [Ca2+]i. Tracing is characteristic of 50 neurons. B: methiothepin (10-5 M), a nonselective 5-HT1 and 5-HT2 blocker, does not abolish 5-HT-induced [Ca2+]i response. Tracing is typical of 90 neurons.



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Fig. 8.   Role of 5-HT receptors in control of 5-HT-induced [Ca2+]i signaling. Application of agonists and antagonists is marked with horizontal bars. Application of 75 mM K+ (10 s) is indicated by arrow. A: blockade of 5-HT-induced [Ca2+]i signaling by ondansetron (10-5 M), a 5-HT3 receptor blocker. Tracing is representative of 100 neurons. B: DAU-6285 (10-5 M), a specific 5-HT4 blocker, did not block 5-HT-induced signaling. Trace is typical of 30 neurons.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The results of this study add further to the understanding of how neurotransmitters induce a [Ca2+]i rise in myenteric neurons. Excitatory neurotransmitters (ACh, SP, and 5-HT) are endogenously present in the extensively branching networks of cultured myenteric neurons. The exogenous application of these neurotransmitters induced a [Ca2+]i rise via different mechanisms, mimicking the transient [Ca2+]i changes observed by electrically-induced synaptic transmission (40).

The basics of [Ca2+]i signaling in myenteric neurons are incompletely known. Previously, we demonstrated that several types of Ca2+ channels contribute to the high-K+-induced Ca2+ entry in cultured myenteric neurons (40). With respect to intracellular Ca2+ stores, inositol trisphosphate (IP3) as well as ryanodine-sensitive stores have been described (20). At present, the exact role of IP3 and RyR in myenteric neurons is poorly understood. A third possible Ca2+ source would be the entry of extracellular Ca2+ through capacitative Ca2+ entry. As far as intestinal cells are concerned, capacitative Ca2+ entry has been described for myocytes (39, 44) and enteric glial cells (29, 45). In line with other neuronal cells, we found no evidence for capacitative Ca2+ entry in myenteric neurons.

ACh, SP, and 5-HT induced a [Ca2+]i rise in a dose-dependently increasing number of cells. Kimball and Mulholland (19) reported a similar dose-dependent increase. This effect might be explained by the fact that, especially for the low concentrations, small responses might be lost in the noise of the signal, resulting in an underestimation of the number of responses. Another possible reason might be that secondary neurotransmitters were released, inducing a [Ca2+]i rise in other neurons. If present, this should be an action potential-independent mechanism, since TTX had no effect on the number of responses. Finally, agonists applied in high concentration may activate neurons with a limited receptor density.

Although Simeone et al. (33) reported that the M1 receptor controlled the ACh-induced [Ca2+]i signaling in myenteric neurons, our data show that both nicotinic and muscarinic receptors are involved. Selective activation of the nicotinic receptor, a ligand-gated cation channel, induced an increase in [Ca2+]i. It has been shown that DMPP activates a Ca2+ current through the receptor channel itself (38). Since in 0 mM [Ca2+]e no responses to DMPP persisted, we may conclude that the fast phase of the response was due to direct Ca2+ influx through the receptor channel. The muscarinic receptor apparently induced a prolonged [Ca2+]i elevation, since atropine significantly reduced the duration of the responses. Moreover, when atropine was administered together with ACh, the [Ca2+]i instantly dropped to its basal concentration, mimicking the nicotinic response. Only few responses persisted in the presence of C6, and these were all slow responses. The fact that ACh in the presence of atropine mimicked the shape of the DMPP response provides evidence for the hypothesized role of both receptors (Fig. 9). In line with data of Simeone et al. (33), we also found that it was indeed the muscarinic M1 receptor that was involved in the muscarinic [Ca2+]i signaling in myenteric neurons, whereas the M2 receptor did not contribute. Although the amplitude of the response was slightly reduced, the number of responses to ACh was not altered in the presence of an L- or N-type Ca2+-channel blocker. This suggests that VOCCs mainly contribute to the amplitude and maintenance of the elevated [Ca2+]i rather than to the initiation of the response. The activation of VOCCs was probably due to membrane depolarization induced by ACh. Involvement of N-type Ca2+ channels was also suggested by Takahashi et al. (36).


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Fig. 9.   Schematic overview of receptors involved in ACh-induced [Ca2+]i signaling. Nicotinic receptor causes a fast upstroke of response due to extracellular Ca2+ entry through receptor channel. Muscarinic receptor generates a slower, more prolonged [Ca2+]i rise. Stimulation with ACh results in a combined response.

We showed that SP induced a dose-dependent [Ca2+]i rise in myenteric neurons. Sarosi et al. (30) reported that this is mediated through an NK1 receptor. L-type and N-type Ca2+-channel blockade did not alter either the number of responses to SP or the amplitude of the signal. These data contrast with the findings of Sarosi et al. (30), who reported that [Ca2+]i signaling induced by SP was mainly due to influx through an L-type Ca2+ channel, whereas the intracellular stores were less important. However, the inhibition by Tg pretreatment suggests that the SP-induced [Ca2+]i rise did rely on Ca2+ release from intracellular Ca2+ stores. In addition, since the inactivation of RyR had no effect, we indirectly demonstrated that SP, also in myenteric neurons, activates an IP3-dependent pathway. Similar to other cell types (18), a PTX-insensitive G protein is involved in the signaling cascade induced by SP.

The intracellular Ca2+ stores do not play a crucial role in the control of the 5-HT-induced signaling. The 5-HT3 receptor mediates short-lived depolarizations in the myenteric neurons of several regions in the gut. We showed that this 5-HT receptor was essential for the 5-HT-induced [Ca2+]i signaling. Specific blockade abolished the [Ca2+]i response in almost all cells. Therefore, we can conclude that at least the onset of the [Ca2+]i rise was mediated by this receptor. Since the 5-HT3 receptor is a ligand-gated cation channel (5), we may expect that a direct influx through the receptor channel is responsible for the initiation of the response. Activation of VOCCs might lead to an additional influx of extracellular Ca2+, which might explain the reduced amplitude in the presence of nifedipine and omega -conotoxin.

We observed no [Ca2+]i changes in response to either 5-HT1a or 5-HT1p receptor activation. The absence of a 5-HT1a response is in line with the general hyperpolarizing action of this receptor. Surprisingly, the activation of the 5-HT1p receptor, which induces a depolarization in a subset of myenteric neurons, did not induce a [Ca2+]i rise in the cultured neurons. This might be due to the fact that the 5-HT1p-induced depolarization was too small to activate VOCCs or, alternatively, that the 5-HT1p receptor was not expressed in culture. We observed that 5-HT1-2 blockade with methiothepin suppressed the [Ca2+]i rise induced by 5-HT. Although 5-HT2a-c receptors are present in the gastrointestinal tract, these receptors seem to be located on smooth muscle cells (13, 21). The only evidence for neuronal 5-HT2-like receptors was found in the guinea pig colon (2). However, electrophysiological studies failed to demonstrate the presence of a 5-HT2-like receptor on guinea pig myenteric neurons. Alternatively, Crespi et al. (4) also described a nonspecific effect of methiothepin on an intracellular target common to the 5-HT and dopamine release mechanisms. Although in snail neurons, methiothepin inhibits the cAMP-dependent L-type Ca2+ channel phosphorylation (22), its mode of action in myenteric neurons remains to be further elucidated. 5-HT4 receptor antagonism did not influence the response to 5-HT. The 5-HT4 receptor is positively coupled to adenylate cyclase. Direct activation of the adenylate cyclase with forskolin depolarizes the membrane of AH/type II neurons both in the myenteric plexus in situ (27) and in culture (11). However, forskolin did not induce a [Ca2+]i rise in the cultured cells. This may imply that no [Ca2+]i rise is involved in the cAMP pathway, or, alternatively, that this pathway was absent in the cultures because of a phenotypic shift.

In summary, we studied in detail the Ca2+ signaling induced by three major excitatory neurotransmitters on cultured myenteric neurons of the small intestine. For each of the three neurotransmitters, we have investigated the mechanism by which the [Ca2+]i rise is induced. Both muscarinic and nicotinic receptors were involved in the control of the ACh-induced [Ca2+]i rise. Activation of nicotinic receptors, which are ligand-gated channels, was accompanied by Ca2+ influx generating a fast rise in [Ca2+]i. Activation of M1 receptors induces a prolonged [Ca2+]i rise. Release of Ca2+ from intracellular stores was essential in the SP-induced signaling, whereas 5-HT-induced Ca2+ signaling was activated by the 5-HT3 receptor, also a ligand-gated channel. It is clear that these data obtained from cultured neurons should be interpreted with caution, especially when extrapolating to in vivo situations. Studies on neurons in situ will be needed to assess the extent to which [Ca2+]i signaling was affected by the culturing procedure.


    ACKNOWLEDGEMENTS

The antibody for ChAT was kindly provided by Dr. Michael Schemann, Hannover, Germany.


    FOOTNOTES

Parts of this study were already published in abstract form in Neurogastroenterol Motil (10: 106 and 107, 1998) and Gastroenterology (114: A1187 and A1187, 1998).

The "Principles of Laboratory Animal Care" were followed as well as the specific national laws of the Ministerie van Landbouw, Belgium.

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: J. Tack, Center for Gastroenterological Research, Catholic Univ. Leuven, B-3000 Leuven, Belgium (E-mail: jan.tack{at}med.kuleuven.ac.be).

Received 27 September 1999; accepted in final form 6 January 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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