Inhibitory interactions between 5-HT3 and P2X channels in submucosal neurons

Carlos Barajas-López1, Luis M. Montaño2, and Rosa Espinosa-Luna1

1 Department of Anatomy and Cell Biology, Queen's University, Kingston, Ontario K7L 3N6, Canada; and 2 Instituto Nacional de Enfermedades Respiratorias and Departamento de Farmacología, Facultad de Medicina, Universidad Nacional Autónoma de Mexico, México D.F, México


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Inhibitory interactions between 5-HT subtype 3 (5-HT3) and P2X receptors were characterized using whole cell recording techniques. Currents induced by 5-HT (I5-HT) and ATP (IATP) were blocked by tropisetron (or ondansetron) and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid, respectively. Currents induced by 5-HT + ATP (I5-HT+ATP) were only as large as the current induced by the most effective transmitter, revealing current occlusion. Occlusion was observed at membrane potentials of -60 and 0 mV (for inward currents), but it was not present at +40 mV (for outward currents). Kinetic and pharmacological properties of I5-HT+ATP indicate that they are carried through 5-HT3 and P2X channels. Current occlusion occurred as fast as activation of I5-HT and IATP, was still present in the absence of Ca2+ or Mg2+, after adding staurosporine, genistein, K-252a, or N-ethylmaleimide to the pipette solution, after substituting ATP with proportional to ,beta -methylene ATP or GTP with GTP-gamma -S in the pipette, and was observed at 35°C, 23°C, and 8°C. These results are in agreement with a model that considers that 5-HT3 and P2X channels are in functional clusters and that these channels might directly inhibit each other.

autonomic neurons; enteric neurons; ligand-gated channels; ion channels; ATP; ATP receptors; P2X receptors; serotonin; 5-hydroxytryptamine; 5-hydroxytryptamine 3 channels; 5-hydroxytryptamine 3 receptors; fast neurotransmission


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

5-HT AND ATP are known to play a role as neurotransmitters (17, 35, 37, 38) by directly activating cationic channels in the postsynaptic membrane, which are named 5-HT3 and P2X receptors, respectively. 5-HT3 receptors are part of the nicotinic ligand-gated channel superfamily, and two different subunits have been cloned. Each of these subunits have four transmembrane domains (14, 29). Of the seven different P2X subunits that have been cloned, each subunit appears to cross the membrane only twice (10, 16, 24). Some of these subunits are able to form homomeric functional cation channels, e.g., P2X2 and 5-HT3A. Other ones appear to participate only in heteromeric channels, e.g., P2X6 and 5-HT3B (14, 27). Current models of 5-HT3 and P2X channels propose that these channels are formed by a combination of five and three subunits, respectively (14, 32). Recent experimental evidence indicates that the nicotinic acetylcholine (nACh) channels and P2X are not independent and that they can inhibit each other when they are simultaneously activated (6, 39). This inhibitory interaction is very fast and might be mediated by an allosteric interaction between nACh and P2X channels. A similar cross-inhibition between the P2X2 and nicotinic proportional to 3beta 4-receptor subtypes was observed when these were coexpressed in Xenopus oocytes (25). Similar interactions have been shown between P2X and the GABAA receptors in dorsal root ganglia (36), supporting the hypothesis that other members of the nicotinic receptor superfamily could be interacting with P2X receptors. In myenteric neurons, however, P2X channels appear to interact with nACh and not with other members of the nicotinic receptor superfamily present in these cells, such as the 5-HT3 and GABAA receptors (39), thus indicating that cross-interactions between these receptors could be tissue specific. To add to this complexity, other types of pharmacological interactions appear to exist between the serotoninergic and cholinergic systems, and it has been reported that 5-HT molecules can themselves block nACh channels in various cell types including submucosal neurons (6, 18).

Our aim in the present study was to investigate and functionally characterize the putative inhibitory interactions between 5-HT3 and P2X native receptors present in submucosal neurons (6, 15, 39). Our findings indicate that activation of 5-HT3 and P2X receptors opens two different channel populations. These two channels, however, negatively modulate each other when they are simultaneously activated. This inhibitory interaction occurs simultaneously with current activation and does not require Ca2+, Mg2+, G proteins, or protein phosphorylation, implying that they are mediated by direct interaction between these receptors. A preliminary report of some of these results has been described previously (3).


    MATERIALS AND METHODS
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INTRODUCTION
MATERIALS AND METHODS
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Young guinea pigs (150-200 g), either male or female, were killed by decapitation, and 5 cm of proximal jejunum were removed, placed in modified Krebs solution (in mM: 126 NaCl, 1.2 NaH2PO4, 1.2 MgCl2, 2.5 CaCl2, 5 KCl, 25 NaHCO3, and 11 glucose; gassed with 95% O2-5% CO2), and opened longitudinally. The mucosa of this intestinal segment was removed, and the thin submucosal layer (submucosal preparation) was dissected from the underlying layers of smooth muscle.

The submucosal preparation was dissociated using a sequential treatment with two enzymatic solutions, as described elsewhere (9); the first contained papain (0.01 ml/ml; activated with 0.4 mg/ml of L-cysteine), and the second contained collagenase (1 mg/ml) and dispase (4 mg/ml). After these solutions were washed out, the neurons were plated on rounded coverslips coated with sterile rat tail collagen. Culture medium was minimum essential medium 97.5%, containing 2.5% guinea pig serum, 2 mM L-glutamine, 10 U/ml penicillin, 10 µg/ml streptomycin, and 15 mM glucose.

ATP and 5-HT are known to modulate the potassium membrane conductance of enteric neurons via G protein-linked receptors (4, 12, 19). To isolate the ionic membrane currents mediated by activation of ligand-gated channels, the experiments were carried out in the presence of Cs+ (a potassium channel blocker) and currents were measured by the whole cell patch-clamp configuration. This recording technique prevents effects mediated by second messengers. ATP- and 5-HT-induced currents were recorded using short-term (2-40 h) primary cultures of submucosal neurons and the Axopatch 1D amplifier. Patch pipettes were made as previously described (9) and had resistances between 1 and 3 MOmega . Sixty to seventy percent of the series resistance were compensated in about one third of the experiments reported here. Series resistance compensation, however, did not affect the lack of additivity of the whole cell currents activated by (IATP) and 5-HT (I5-HT; see RESULTS), so, in most cases, no compensation was made for this factor. Except when otherwise mentioned, the holding potential was -60 mV. The standard solutions used, unless otherwise mentioned, had the following composition (in mM): inside the pipette: 160 Cs-gluconate, 10 EGTA, 5 HEPES, 10 NaCl, 3 ATPMg, and 0.1 GTP; external solution: 160 NaCl, 2 CaCl2, 11 glucose, 5 HEPES, and CsCl 3. The pH of all solutions was adjusted to 7.3-7.4 with either CsOH (pipette solutions) or NaOH (external solutions). With these standard solutions, the usual input resistance of the neurons ranged from 1 to 10 GOmega . Whole cell currents were recorded on a PC using Axotape software (Axon Instruments) and analyzed on a Macintosh computer using Axograph software (Axon Instruments). Membrane potentials were corrected for the liquid junction potential (pipette -11 mV). The recording chamber was continuously superfused with external solution at ~2 ml/min. Rapid changes in the external solution were made by using an eight-barrelled device (5). The external application of experimental substances was achieved by abruptly exchanging the tube delivering control external solution in front of the cell being recorded for a tube delivering the same solution plus the drug(s). Experimental substances were removed by returning back to the tube containing the control external solution. External solutions were delivered by gravity. Except where otherwise mentioned, experiments were performed at room temperature (~23°C).

Genistein, ACh, 5-HT, tropisetron (before called ICS-205,930), and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) were purchased from Research Biomedical (Natick, MA). Staurosporine and K-252a were supplied by Kamiya (Thousand Oaks, CA). Ondansetron was purchased from Glaxo Wellcome (Mississauga, ON, Canada). All other substances were purchased from Sigma (St. Louis, MO). The pH of the external solution containing ATP, used to induce the IATP, was always readjusted with NaOH. The addition of the other substances to the external solution did not alter its pH.

Results were expressed as means ± SE [number of cells (n)]. The paired Student's t-test was used to evaluate differences between mean values obtained from the same cells, and the unpaired Student's t-test was used for data obtained from different groups of cells. Two-tailed P values of 0.05 or less were considered statistically significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Pharmacological and electrophysiological properties of the whole cell currents induced by 5-HT and ATP. Electrophysiological and pharmacological properties of I5-HT and IATP in enteric neurons have been previously characterized (5, 7, 15, 38). Both currents are mediated by activation of nonspecific cationic channels, and their slope single-channel conductance have slightly different magnitudes: 9-15 and 15-22 pS for 5-HT3 (15) and P2X (5, 7, 38) receptors, respectively.

5-HT induced an inward current in all neurons tested (n = 140), whereas ATP caused an inward current in only ~88% (n = 123) of a total of 140 neurons. Concentration-response curves (Fig. 1A) were obtained for these transmitters in submucosal neurons and analyzed as previously reported (7). The EC50 values for 5-HT and ATP were 55 and 53 µM, whereas the Hill coefficient values were 1.0 ± 0.1 and 1.1 ± 0.1 (not significantly different than unity), respectively. The currents induced by maximal concentrations of 5-HT and ATP (1 mM) had an average amplitude of -1,899 ± 14 and -2,767 ± 250 pA, respectively, but had a variable amplitude in different cells ranging from only a few picoAmperes up to 8.8 nA. A larger variability was noticed in the amplitude of IATP than in I5-HT. The amplitude of these currents was independent of each other and in a few cells (14 of 140), only I5-HT was observed.


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Fig. 1.   Whole cell inward currents induced by ATP (IATP) and serotonin (5-HT; I5-HT) are mediated by 2 different receptors. A: these 2 currents have similar concentration dependency. Symbols are mean ± SE values of the currents induced by 5-HT (n = 6-18) or ATP (n = 6-9). Sigmoidal lines were fitted with a 2-parameter logistic model. Relative currents were calculated assuming the effect of 1 mM as maximal. B: current-voltage relationships of I5-HT and IATP showed a more prominent inward rectification for the later current (n = 4). C: pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid [PPADS (30 µM); a P2X receptor antagonist] blocks IATP without affecting I5-HT. D: tropisetron (0.3 µM; a 5-HT3 receptor antagonist) blocks I5-HT without modifying IATP; similar observations were obtained with ondasetron, a more specific 5-HT3 receptor antagonist (not shown). In C and D, whole cell inward currents were measured from the same submucosal neurons, at a holding potential of -60 mV, and induced by a maximal concentration of 5-HT and ATP (1 mM). Currents were recorded before (control) and 5 min after starting the receptor antagonist superfusion. Bars represent the average current inhibition expressed as a percentage of control values, whereas SE are indicated by lines at top of these bars.

As shown in Fig. 1, C and D, I5-HT and IATP (1 mM) were significantly inhibited by tropisetron (0.3 µM) and PPADS (30 µM), respectively. Tropisetron did not affect IATP as PPADS did not alter I5-HT. Similar results were observed in three additional experiments when the specific 5-HT3 receptor antagonist ondansetron (0.3 µM) was used instead tropisetron (5-HT3 and 5-HT4 receptor antagonist) (20).

In eight experiments carried out on the same submucosal neurons, the reversal potential for IATP (23 ± 2.5 mV) was significantly more positive (P < 0.05) than for I5-HT (20 ± 2.3 mV). The average current-voltage relationships show a prominent inward rectification for IATP but not for I5-HT, as is illustrated in Fig. 1B (n = 4).

The I5-HT onset was clearly slower than IATP onset (Fig. 2C). In agreement with this, it was found that the time required to reach the half-maximal current was significantly different (P < 0.05) in 13 analyzed cells, which had average values of 124 ± 15 and 76 ± 12 ms for I5-HT and IATP, respectively (Fig. 2D). These currents usually reached their peak within the following second. After currents had reached maximal amplitude, they decreased despite the continuous presence of the transmitters, indicating tachyphylaxis. I5-HT desensitized faster than IATP. Furthermore, I5-HT desensitization was better fitted by the sum of three exponential functions, whereas IATP desensitization was better fitted by the sum of two exponential functions (Fig. 3, A and B). After agonist removal from the external solution, I5-HT decayed much more slowly than IATP (Fig. 4A). This decay was well fitted by a single exponential function for both currents, and their average tau  values were significantly different (P < 0.05; n = 8): 11.3 ± 4.4 and 0.35 ± 0.04 s for I5-HT and IATP, respectively.


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Fig. 2.   I5-HT and IATP in submucosal neurons are not additive, revealing a current occlusion. A: recordings from 1 neuron of a typical experiment; B: the average (bars) values of 16 experiments. Currents were induced by application of either 5-HT (1 mM) or ATP (1 mM) and by the simultaneous application of both agonists (I5-HT+ATP). I5-HT and IATP were recorded 5 min before and 5 min after I5-HT+ATP. In B, the first and third bars show I5-HT and IATP; the addition of these currents represents expected current (Iexpected = I5-HT+IATP). SE are shown as lines in the top of the bars for Iexpected and I5-HT+ATP (second bar). I5-HT+ATP is significantly lower than Iexpected. C: onset of the currents recorded from another submucosal neuron showing that I5-HT+ATP is smaller than Iexpected since the commencement of these currents. The expected current shown in C is a graph representing the addition of I5-HT and IATP. D: the mean half-onset time of I5-HT+ATP was significantly different than that of I5-HT and IATP. Whole cell currents were measured at a holding potential of -60 mV.



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Fig. 3.   Desensitization kinetics of I5-HT+ATP cannot be explained by the desensitization kinetics of I5-HT or IATP alone. A: representative recordings from a submucosal neuron of I5-HT, IATP, and I5-HT+ATP. The desensitization of I5-HT+ATP and I5-HT was best fitted by the sum of 3 exponential functions (thick grey lines), whereas IATP desensitization was best fitted by the sum of 2 exponential functions. Note that I5-HT+ATP desensitizes faster than IATP but slower than I5-HT. B: bars in the lower graphs represent the mean ± SE values of the tau  of these exponential functions. The exponential tau  of IATP were significantly larger than the correspondent tau  of I5-HT+ATP. The tau  of the first exponential (tau 1) of I5-HT was significantly smaller than the tau 1 of I5-HT+ATP. tau  Of the second and third exponentials of these currents were, however, not different. Exponential fits were performed using the data from the current peak to the "steady-state" component. In these experiments, agonists were applied for ~1-3 min, and the holding potential was -60 mV. NS, not significant.



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Fig. 4.   Decay kinetics of I5-HT+ATP can be explained by the decay kinetics of I5-HT or IATP alone. A: representative recordings from a submucosal neuron of I5-HT, IATP, and I5-HT+ATP. The decay of I5-HT+ATP was best fitted by the sum of 2 exponential functions (thick grey line), whereas I5-HT and IATP desensitization was best fitted by a single exponential function. Note that I5-HT+ATP desensitizes faster than IATP but slower than I5-HT. B: bars in these graphs represent the mean ± SE values of the tau  of these exponential functions. The tau 1 of IATP was not significantly different than tau 1 of I5-HT+ATP. The tau 1 of I5-HT was also not different than tau 2 of I5-HT+ATP. Exponential fits were performed using the data from a couple hundred milliseconds after removing the agonists to the steady-state component. In these experiments, the holding potential was -60 mV.

Currents induced by 5-HT and ATP are not additive. The experiments described above demonstrated that I5-HT and IATP have completely different kinetics and that they are mediated by activation of pharmacologically distinct receptors. In the following experiments, we investigated whether these currents are mediated by independent populations of channels. If IATP and I5-HT are mediated by independent populations of channels, then the currents induced by a maximal concentration of these transmitters (when receptor occupancy is expected to be close to 100%) should be additive. We therefore measured the peak whole cell currents induced by maximal concentrations (1 mM) of 5-HT, ATP, or simultaneous application of both agonists (I5-HT+ATP) in the same neuron. We found that the addition of individual currents [IATP + I5-HT = expected current (Iexpected)] was significantly larger (P < 0.001) than I5-HT+ATP, revealing an occlusion between I5-HT and IATP (Figs. 2-4). In these experiments, I5-HT+ATP (-3,561 ± 369 pA) was not different than the current induced by the most effective of these transmitters (-3,294 ± 733 pA). For instance, if IATP was larger than I5-HT (which was the most common finding), then I5-HT+ATP had about the same magnitude as IATP (e.g., Fig. 2, A and C; see below). Indeed, in 10 analyzed experiments in which IATP (-3,090 ± 614 pA) was significantly (P < 0.01) larger than I5-HT (-1,166 ± 161 pA), I5-HT+ATP (-3,151 ± 587 pA) was significantly (P < 0.01) larger than I5-HT but was not different from IATP. Similarly, in five analyzed neurons in which I5-HT (-2,742 ± 636 pA) was significantly (P < 0.05) larger than IATP (-1,324 ± 270 pA), I5-HT+ATP (-2,796 ± 620 pA) was also significantly (P < 0.05) larger than IATP but was not different from I5-HT.

The current occlusion was also observed when 5-HT and ATP concentrations close to their EC50 (60 µM) were used. In these experiments, I5-HT+ATP (-2,127 ± 302 pA) had a significantly (P < 0.01) lower amplitude than Iexpected (-2,623 ± 324 pA). In such experiments, however, I5-HT+ATP (-2,127 ± 302 pA) was significantly (P < 0.01) larger than the current induced by the most effective of these transmitters (-1,445 ± 188 pA). This was qualitatively different than when maximal concentrations of 5-HT and ATP were used (see above) and thus indicates a lower level of current occlusion with concentrations of 60 µM. Therefore, in all the following experiments, we used a maximal concentration of these transmitters to better quantify this current occlusion.

Kinetics of the whole cell currents induced by simultaneous application of both agonists. Figure 2C shows the onset of I5-HT, IATP, I5-HT+ATP, and Iexpected of a typical experiment out of 13 analyzed neurons. As it is shown in Fig. 2, C and D, the average time required to reach the half-maximal amplitude of the currents induced by simultaneous application of both agonists was significantly different than that for I5-HT or IATP (P < 0.05). Figure 2C shows that I5-HT+ATP was smaller than Iexpected as soon as currents were detected. Because the sampling frequency during our recordings was 0.3-1 kHz, this current occlusion must be occurring in <5 ms.

Visual inspection of the recordings revealed that I5-HT+ATP desensitized faster than IATP but slower than I5-HT, as seen in Figs. 2A and 3A. To quantify this, in eight neurons, agonists were applied during relatively long periods (1-3 min), and exponential fits were performed using the data from the current peak to the "steady state" (end of the agonist application; Fig. 3A). As with I5-HT, desensitization of I5-HT+ATP was better fitted by the sum of three exponential functions. The tau  value of the first exponential (tau 1) was significantly different (P < 0.05) than the tau 1 value of I5-HT, whereas the average values of tau 2 and tau 3 of these two currents were not significantly different (Fig. 3B). However, the average values of tau 1 and tau 2 of I5-HT+ATP desensitization were significantly different (P < 0.001) than the corresponding tau  values of IATP desensitization (Fig. 3B). These observations indicate that I5-HT+ATP desensitization kinetics are different than those of IATP or I5-HT.

The decay of the I5-HT+ATP was quite different than that of either I5-HT or IATP (Fig. 4A). Indeed, the decay of I5-HT+ATP was well fitted by the sum of two exponential functions (Fig. 4B; n = 8). We tested the hypothesis that the first and second exponentials of I5-HT+ATP are the exponentials of IATP and I5-HT, respectively. The average tau  value of the first exponential (tau 1) was virtually the same as the tau 1 of IATP decay. The average tau  value of the second exponential (tau 2) of I5-HT+ATP decay was also not different than tau 1 of I5-HT decay (Fig. 4B), indicating that I5-HT+ATP are mediated by the opening of both P2X and 5-HT3 channels.

Prereceptor mechanisms and technical artefacts as the origin for the current occlusion. The following experiments rule out that current occlusion is due to prereceptor mechanisms. In six analyzed cells with very low response or no initial response to ATP (-22 ± 16 pA; Fig. 5A), I5-HT+ATP (-2,147 ± 128 pA) had the same amplitude and similar kinetics as I5-HT alone (-2,274 ± 179 pA). Similarly, in another five cells, in which IATP (-44 ± 27 pA) had been previously blocked with PPADS (Fig. 5B), I5-HT+ATP (-1,253 ± 288 pA) had the same amplitude and similar kinetics as I5-HT (-1,103 ± 273 pA). In three cells with relatively low response to 5-HT (-304 ± 49 pA; Fig. 5C) I5-HT+ATP (-1,980 ± 298 pA) had the same amplitude and very similar desensitization kinetics as IATP (-1,875 ± 260 pA). Similarly, in another four cells in which I5-HT (-21 ± 18 pA) was blocked with tropisetron (0.3 µM; Fig. 5D), I5-HT+ATP (-1,900 ± 510 pA) had the same amplitude and similar kinetics as IATP (-2,045 ± 509 pA). Similar results were obtain in two neurons in which ondansetron [0.3 µM, a specific 5-HT3 receptor antagonist (20)] was used to blocked I5-HT.


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Fig. 5.   Inhibitory interactions between 5-HT3 and P2X receptors required the presence of functional channels. A: recordings from a neuron in which ATP induced only a small initial current (IATP), indicating few functional P2X channels in this cell, but with a prominent response to I5-HT. Note that ATP did not modify either the amplitude or the kinetics of the I5-HT. B: similar results were obtained when IATP was blocked with a P2X receptor antagonist (30 µM of PPADS). C: recordings from a neuron with a small I5-HT, indicating few functional 5-HT3 channels in this cell, but with a prominent IATP. Note that 5-HT did not modify either the amplitude or the kinetics of IATP. D: similar results were obtained when I5-HT was blocked with tropisetron (0.3 µM).

Figure 6A shows a typical recording of I5-HT before (control response was -1,262 ± 417 pA) and during the continuous application of ATP this maneuver did not block I5-HT, which reached a peak amplitude of -1,273 ± 374 pA (n = 4). Furthermore, the presence of 5-HT did not alter IATP (Fig. 6B), which had a peak amplitude of -2,264 ± 651 and -2,663 ± 940 before and in presence of 5-HT, respectively (n = 4).


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Fig. 6.   Inhibitory interactions between 5-HT3 and P2X receptors disappear after receptor desensitization. I5-HT (A) and IATP (B) were recorded 5 min before (left) and during continuous application of the other agonist. Note that currents induced by this second application have similar kinetics and amplitude to control currents. C: a similar experiment in which the nicotinic IACh was recorded before and then in the presence of 5-HT. As expected from previous information, 5-HT prevented IACh. These 3 sets of currents were recorded in 3 different submucosal neurons at a holding potential of -60 mV.

In a previous study, Barajas-Lopez et al. (8) showed that 5-HT blocks the current induced by ACh (IACh) and that this effect is likely mediated by a direct blockage of nicotinic channels by 5-HT. As positive control, we performed four experiments similar to those described above (Fig. 6, A and B), but in this case, we measured the effects of 5-HT on IACh. As expected, 5-HT blocked most IACh, which was -2,188 ± 528 and -315 ± 102 pA before and in the presence of 5-HT (P < 0.05; n = 4; Fig. 6C). Therefore, it is clear that 5-HT molecules do not block P2X channels in this preparation and that ATP molecules also do not block 5-HT3 channels.

Several observations rule out the possibility that current occlusion was due to a technical artefact of our recording system. First, current occlusion also occurs at 0 mV, when Iexpected had an average amplitude of only -654 ± 96 pA. This value is only about one-eighth of the Iexpected amplitude at -60 mV (Fig. 2B). At 0 mV, I5-HT+ATP (-504 ± 70 pA) was significantly (P < 0.05; n = 6) lower than Iexpected (-654 ± 96 pA). Second, the amplitude of the currents recorded at -60 mV and induced by 5-HT or ATP could almost be doubled by hyperpolarizing the membrane to -90 mV (n = 4). Third, our amplifier is capable of recording large and fast ionic currents in the same neurons and under exactly the same recording conditions. Thus we were able to record voltage-activated Ca2+ and Na+ inward currents as large as -8,000 pA (unpublished observations; see also Ref. 9) and that, in various cases, were larger than Iexpected at -60 mV (-5,309 ± 473 pA, range -1,918 to -9,932 pA). It is also important to stress that the onset of voltage-activated Ca2+ and Na+ currents is far faster (a few milliseconds) than that of I5-HT and IATP (hundreds of milliseconds). Fourth, experiments in which lack of voltage clamp was noticed were disregarded for the present analysis. Fifth, current occlusion was also present in experiments carried out at 35°C, ruling out the possibility that this occlusion was due to an artefact of recording at room temperature. Thus, in seven experiments carried out at 35°C, I5-HT+ATP (-2,827 ± 759 pA) was still significantly lower (P < 0.01; n = 7) than Iexpected (-4,036 ± 1,024 pA).

Application of both agonists desensitized both 5-HT3 and P2X receptors. In the following experiments, we measured the amplitude of both I5-HT and IATP before and immediately after (~5 s) a long application of 5-HT, ATP, or 5-HT + ATP. This long application lasted for at least 25 s or until the induced current had desensitized ~80% (usually within 1 min). Some typical recordings and the average data from such experiments are shown in Fig. 7, A-G. We observed that P2X receptor desensitization with ATP decreased IATP significantly, whereas it did not affect I5-HT (Fig. 7, A and B). Similarly, 5-HT3 receptor desensitization with 5-HT decreased I5-HT significantly, whereas it did not affect IATP (Fig. 7, C and D). In other words, no cross-desensitization was observed between 5-HT3 and P2X receptors. When receptors were desensitized by the simultaneous application of ATP + 5-HT, both I5-HT and IATP were significantly decreased (Fig. 7, E-G). These observations coupled with the fact that I5-HT+ATP kinetics are different than the kinetics of I5-HT or IATP alone (Figs. 2C, 3, and 4) support the hypothesis that I5-HT+ATP is carried through both 5-HT3 and P2X channels.


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Fig. 7.   No cross-desensitization was observed between 5-HT3 and P2X receptors. Control IATP (A and B) and I5-HT (B and D) were recorded 5 min before (left) and immediately after (~5 s) a prolonged application of the other agonist. E and F: simultaneous application of both agonists desensitized both receptor populations. IATP (E) and I5-HT (F) recorded 5 min before (control currents; left) and immediately after (~5 s) a prolonged application of both agonists. G: average amplitude of I5-HT and IATP recorded after the prolonged application of ATP, 5-HT, or 5-HT + ATP as a percentage of control response. Lines on top of the bars are SE. Recordings taken at the holding potential of -60 mV.

Current occlusion was not observed for outward currents. Current occlusion was not observed at +40 mV (Fig. 8). Thus, at this potential, I5-HT+ATP was very similar to Iexpected in six experiments, and the averages of these currents were not significantly different. At this membrane potential, I5-HT+ATP (1,111 ± 298 pA) was significantly (P < 0.05) larger than the current induced by the most effective transmitter (896 ± 250 pA).


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Fig. 8.   IATP and I5-HT were additive when they were outward. A: outward currents recorded from a typical experiment at a holding potential of +40 mV. B: the average values of similar experiments (n = 6) as the 1 shown in A. Currents were induced by application of either 5-HT (1 mM) or ATP (1 mM) and by simultaneous application of both agonists (I5-HT+ATP). I5-HT and IATP were recorded 5 min before and 5 min after I5-HT+ATP. Bars and error lines on the top of the bars are as indicated in Fig. 2.

Role of Ca2+, Mg2+, and Cs+ in the current occlusion. P2X receptors are permeable to Ca2+, suggesting that the inward current occlusion might be mediated by a raise in the intracellular concentration of this ion. Against this hypothesis, however, the current occlusion was still observed in total absence of Ca2+ in the extracellular and intracellular media (Fig. 9). These experiments were carried out with the standard extracellular solution but containing no Ca2+ and 50 µM EGTA and with the standard intracellular solution + 5 mM BAPTA. The presence of Mg2+ inside the pipette was also not required for current occlusion, because it was still observed after this ion was replaced by lithium (n = 7). In these experiments, I5-HT+ATP (-2,315 ± 345 pA) was significantly lower (P < 0.01) than Iexpected (-3,433 ± 376 pA). The presence of Cs+ was also not required for current occlusion, because it was still observed after this ion was replaced with an equimolar concentration of Na+ (n = 3) in both the external and the pipette solutions. In these experiments I5-HT+ATP (-3,338 ± 628 pA) was significantly lower (P < 0.05) than Iexpected (-4,767 ± 933 pA).


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Fig. 9.   Calcium ions, protein phosphorylation, and G proteins are not required for current occlusion. Average amplitude of I5-HT, IATP, or I5-HT+ATP in 8 different experimental groups of submucosal neurons. Results for each group are represented by a pair of bars. The first bar of each pair is a combined bar and shows the average IATP and I5-HT before application of 5-HT + ATP. This combined bar, therefore, represents the expected current (Iexpected = I5-HT + IATP). The second bar represents I5-HT+ATP. Error lines on the top of the bars are SE for Iexpected and I5-HT+ATP. Ca2+-free experiments were carried out in 0-Ca2+ + 50 µM EGTA extracellular media and standard intracellular solution + 5 mM BAPTA. In the proportional to ,beta -methylene ATP and GTP-gamma -S experiments, the pipette solution contained these substances instead of ATP and GTP, respectively. K-252a (10 µM), staurosporine (5 µM), and genistein (10 µM) experiments were carried out using standard intracellular solution. During the N-ethylmaleimide (NEM) experiments, recorded neurons were pretreated for 10 min with standard extracellular solution + 30 µM NEM. During these experiments, only 1 cell from each coverslip was recorded, and coverslips were discarded after a neuron had been exposed to NEM. The recordings of the inset are from a typical experiment carried out at 8°C. The holding potential was -60 mV in all of these experiments.

Role of protein phosphorylation and G proteins in the current occlusion. ATP and 5-HT are also known to activate metabotropic receptors in enteric neurons (4, 12, 19), and their activation might lead to activation of G proteins, changes in second messengers, and protein phosphorylation, which could be responsible for the current occlusion observed here. The following observations, however, do not support this hypothesis.

Current occlusion was still observed after inhibiting protein phosphorylation by either lowering the temperature to 8°C, by replacing ATP of the internal solution with its less hydrolysable analog proportional to ,beta -methylene ATP, or by adding to the internal solution either 10 µM genistein [a tyrosine kinase inhibitor (31)], 10 µM K-252a, or 5 µM staurosporine [nonspecific protein kinase inhibitors (23); Fig. 9]. Staurosporine (3 µM) has been previously shown to inhibit the membrane depolarization induced by forskolin, phorbol esters, adenosine, and ATP in enteric neurons (2, 4).

Under the same experimental conditions used in the present study, Barajas-Lopez et al. (9) previously found that adenosine inhibits voltage-activated calcium currents by a G protein-mediated mechanism. Such an effect was prevented by the addition of N-ethylmaleimide [30 µM; known to uncouple receptors from G proteins (1, 34)]. In the present study, the same concentration of N-ethylmaleimide (30 µM) did not alter the occlusion between I5-HT and IATP (Fig. 9). Similarly, this occlusion was still observed after GTP was replaced with GTP-gamma -S in the internal solution (Fig. 9). This experimental maneuver altered the adenosine-induced effects on calcium currents (9).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This is the first demonstration that in guinea pig submucosal neurons, activation of 5-HT3 and P2X receptors/channels is not independent and ionic currents carried through them are occluded when they are simultaneously activated. This current occlusion is observed as soon as currents are activated, is voltage dependent, and does not require Ca2+, Mg2+, G protein activation, or protein phosphorylation. Currents induced by the simultaneous application of both agonists are carried through both 5-HT3 and P2X channels. Altogether, these observations suggest that this current occlusion is mediated by cross-inhibition and by a direct interaction between 5-HT3 and P2X receptors/channels. Similar interactions to the ones proposed here have been shown between nACh and P2X receptors (6, 25, 39), between GABAA and P2X receptors (22, 36), between dopamine (D2) and somatostatin (SSTR5) receptors (33), and between dopamine (D5) GABAA channels (28), suggesting that interactions between receptor proteins play and important role in neuronal signaling.

Activation of 5-HT3 and P2X receptors/channels is not independent. The whole cell currents induced by either 5-HT (1 mM) or ATP (1 mM), despite these relatively high concentrations, are mediated by pharmacologically distinct receptors. This is demonstrated by the specific inhibitory effect of tropisetron [or ondansetron; 5-HT3 receptor antagonists (20)] and PPADS [a P2X receptor antagonist (7)] on I5-HT and IATP, respectively. The fact that the amplitudes of these two currents are independent of each other in the recorded neurons indicates that these ligand-gated channels can be expressed independently in these neurons.

Activation of these channels is, however, not independent, as shown by the fact that inward currents carried through 5-HT3 and P2X receptors were not additive when maximally activated. A simple explanation of this current occlusion would be the existence of one single channel gated by both neurotransmitters. Our results obtained at a holding potential of +40 mV rule out this hypothesis. At this potential, the currents induced by simultaneous application of 5-HT + ATP are similar to the sum of the currents induced by individual applications of 5-HT or ATP. Therefore, IATP and I5-HT must be carried through different populations of channels; otherwise, they would not add at any potential. These results are in agreement with the fact that 5-HT3 and P2X currents have different reversal potentials, rectification properties, kinetics (present study), single-channel conductances (5, 7, 15, 38), and different molecular structures (10, 14, 16, 24, 29).

The unlikely possibility that the current occlusion was due to interactions between ATP and 5-HT molecules is ruled out by the following observations. First, in neurons in which IATP had been previously blocked with PPADS or with no initial response to ATP, I5-HT+ATP had virtually the same amplitude and kinetics as the current induced by 5-HT alone. Second, 5-HT3 receptor desensitization prevented any 5-HT effect on IATP and similarly, desensitization of P2X receptors also prevented any ATP effect on I5-HT. Third, in cells with no initial response to 5-HT or in which I5-HT had been blocked, I5-HT+ATP had virtually the same amplitude and kinetics as IATP. These observations also rule out the possibility that 5-HT blocks P2X channels or that ATP blocks 5-HT3 channels. An effect that was demonstrated before for 5-HT on nACh channels of submucosal neurons (8).

Currents induced by the simultaneous application of both agonists are carried through both 5-HT3 and P2X channels. At least four different observations indicate that I5-HT+ATP is carried through both 5-HT3 and P2X channels and not only through one population of these channels. 1) I5-HT+ATP kinetics is clearly different than the kinetics of I5-HT or IATP alone. Thus the onset of I5-HT+ATP is faster than the onset of I5-HT but slower than IATP. I5-HT+ATP desensitizes slower than I5-HT but more rapidly than IATP. Furthermore, the decay of I5-HT+ATP resembles the decay of both I5-HT and IATP. 2) I5-HT+ATP desensitization kinetics resemble I5-HT when P2X receptors are inhibited with PPADS and resemble IATP when 5-HT3 receptors were inhibited. 3) When ATP + 5-HT are applied simultaneously, both 5-HT3 and P2X receptors are desensitized, whereas no cross-desensitization is observed when 5-HT3 and P2X receptors are desensitized individually. 4) In experiments in which one of the individual currents is significantly larger than the current induced by the other transmitter, I5-HT+ATP amplitude is the same as that of the current induced by the most effective neurotransmitter and is significantly larger than the weaker transmitter. These observations imply that I5-HT+ATP is carried through both 5-HT3 and P2X channels and that inhibition between these channels is reciprocal.

Cross-inhibition between 5-HT3 and P2X channels is mediated by a direct interaction between these receptors. Enteric neurons also express metabotropic ATP and 5-HT receptors, which are known to be linked to G proteins (4, 12, 19), second messengers, and protein phosphorylation. Several of our observations, however, indicate that activation of these receptors is not required for the current occlusion observed here. First, current occlusion occurs as soon as 5-HT3 and P2X channels are activated, indicating that this occlusion is as fast as the activation of these ligand-gated channels. Second, PPADS prevents the effects of ATP on the 5-HT3 channels (present study), but it does not block the slow depolarization mediated by P2Y receptors (4). Finally, tropisetron and ondansetron (5-HT3 receptor antagonists) prevented the effect of 5-HT on the P2X channels. Third, inhibition of G proteins (with N-ethylmaleimide), functional modification of G proteins (by replacing GTP with GTP-gamma -S in the pipette solution), and inhibition of protein phosphorylation (with staurosporine, K-252, or genistein) do not modify the occlusion observed between I5-HT and IATP. Fourth, this occlusion is still present during experiments carried out at 8°C or when ATP was substituted by its less hydrolysable analog alpha ,beta -methylene ATP.

5-HT3 and P2X neuronal channels might be Ca2+ permeable, suggesting that the intracellular accumulation of this cation could mediate the inhibitory interaction between these channels. This hypothesis is supported by the fact that 5-HT3 and P2X receptors of enteric neurons are known to be permeable to this cation (4, 12, 26). However, we have ruled out such a possibility by showing that the interaction is not affected by the absence of Ca2+ in the intracellular and extracellular solutions. Mg2+ is also not required because occlusion is still observed in the absence of this cation.

Functional implications for these channel interactions. At least some of the current experimental information suggests that inhibitory interactions between ligand-gated channels might be a widely used mechanism to limit the ionic currents through the cellular membrane. Thus a functional interaction such as the one demonstrated here has been shown to exist between P2X and nicotinic channels in enteric neurons (6, 39) and between P2X and other ligand-gated channels (GABAA) in enteric (22) and in root ganglion neurons (36). This does not appear to be always the case, because according to Zhou and Galligan (39), P2X receptors do not interact with GABAA or 5-HT3 receptors in myenteric neurons; however, see Ref. 22.

The role of P2X and the fact that ATP, the endogenous agonist for most P2X receptors, has been shown to be coreleased with various neurotransmitters, including GABA (21), ACh, and noradrenaline (11), suggests the hypothesis that the inhibitory interactions between ligand-gated channels might play an important modulator role for the synaptic transmission. In the submucosal plexus, fast synaptic potentials mediated by 5-HT and ATP have not yet been reported, and, therefore, the role of these interactions in the synaptic transmission of this plexus remains speculative. In other cells, e.g., mast cells (13, 30), ATP and 5-HT can be coreleased, suggesting that the interactions between P2X receptors and 5-HT could play a role in some paracrine effects of these substances. These interactions will limit the excitatory effect when these substances are simultaneously release; particularly, when they reach maximal concentrations. A more general functional significance of the inhibitory interaction between ligand-gated channels would be the saving of cellular energy by limiting ion movements through the cellular membrane.

In conclusion, our results indicate that there is a very fast inhibitory interaction between 5-HT3 and P2X channels. These interactions occur as fast as the activation of 5-HT3 and P2X channels, supporting the hypothesis that these receptors are located very close to each other in the neuronal membrane, perhaps forming functional units constituted by at least one channel of each type. A general functional significance of these interactions would be to limit the expenditure of cellular energy by limiting ion movements through the cellular membrane, which might decrease the likelihood of neurotransmitter excitotoxicity. The putative role in synaptic transmission of these interactions would require direct demonstration.


    ACKNOWLEDGEMENTS

This work was supported by the Canadian Institutes of Health Research (MOP 36438). C. Barajas-López was supported by the Ontario Ministry of Health (Career Scientist Award #04500).


    FOOTNOTES

Address for reprint requests and other correspondence: C. Barajas-López, Botterell Hall, Ninth Floor, Queen's Univ., Kingston, Ontario, Canada K7L 3N6 (E-mail: barajasc{at}meds.queensu.ca).

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. Section 1734 solely to indicate this fact.

June 5, 2002;10.1152/ajpgi.00054.2002

Received 11 February 2002; accepted in final form 8 May 2002.


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