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Abstract

  1. Top of page
  2. Abstract
  3. methods
  4. results
  5. discussion
  6. Acknowledgements
  • 1
    Functional interactions between nicotinic and P2X receptors in submucosal neurons were investigated. Whole-cell currents induced by ACh (IACh) and ATP (IATP) were blocked by hexamethonium and PPADS), respectively. Currents induced by simultaneous application of the two transmitters (IACh+ATP) were only as large as the current induced by the most effective of these substances. This current occlusion indicates that activation of nicotinic and P2X channels is not independent.
  • 2
    Kinetic parameters of IACh+ATP indicate that they are carried through channels activated by either substance. In agreement with this interpretation, both IACh and IATP amplitudes were decreased when ATP and ACh were applied simultaneously, whereas no cross-desensitization was observed when nicotinic and P2X receptors were desensitized individually.
  • 3
    Current occlusion was observed at membrane potentials of −60 and +10 mV, when IACh and IATP were inward. However, when these currents were outward (at +40 mV), current occlusion was not observed. Current occlusion was still observed at +40 mV in experiments in which the reversal potential of these currents had been adjusted to more positive values.
  • 4
    Current occlusion occurred as soon as currents were detected (< 5 ms), was still present in the absence of Ca2+, Na+ or Mg2+, and after adding staurosporine, genistein, K-252a, or N-ethylmaleimide to the pipette solution. Similar observations were noted after substituting α,β-methylene ATP for ATP, or GTP for GTP-γ-S in the pipette and in experiments carried out at 36, 23 and 9 °C.
  • 5
    We propose that nicotinic and P2X channels are in functional clusters of at least two, and that the influx of ions through one activates (through allosteric interactions) a mechanism that inhibits the other channel.

ACh and ATP play a role as fast neurotransmitters in peripheral synapses (Nishi & North, 1973; Buckley & Caulfield, 1992; Evans et al. 1992; Silinsky & Gerzanich, 1993; Zhou & Galligan, 1996; Burnstock, 1997). These neurotransmitters activate specific cation channels in the postsynaptic membrane by acting at pharmacologically distinct receptors, named nicotinic and P2X receptors, respectively. Such receptors, like other ligand-gated ion channels, are thought to be formed by multisubunit complexes, and several of their different subunits have already been cloned. Each subunit of the nicotinic receptor is believed to have four transmembrane domains, whereas those of P2X receptors appear to cross the membrane only twice (Brake et al. 1994; Surprenant et al. 1995; Lindström, 1996; Lindström et al. 1996; North, 1996; Burnstock, 1997).

How these subunits organize themselves to form ion channels in native membrane is not completely understood (see Bean, 1992). Recently, Nakazawa (1994a) suggested that there is an overlap of P2X and nicotinic channels, and that ATP could open a subpopulation of nicotinic channels by binding to P2X receptors in rat sympathetic neurons. This ‘channel overlap’ hypothesis was based on the findings that channel activation by ATP and ACh is not independent. Indeed, the cationic current activated by ACh (IACh; which was selectively blocked by hexamethonium) occluded the smaller cationic current induced by ATP (IATP; which was selectively blocked by suramin), in rat sympathetic neurons. Similar observations have been reported in rat phaeochromocytoma PC12 cells by Nakazawa et al. (1991).

In the present study our aim was to functionally characterize a putative inhibitory interaction between P2X and nicotinic receptors in submucosal neurons. Our findings do not support the channel overlap hypothesis but rather indicate that activation of nicotinic and P2X receptors open two different channel populations. These two channels, however, negatively modulate each other when they are simultaneously activated. This inhibitory interaction occurs as soon as whole-cell currents are detected (within a few milliseconds), are triggered by ion influx through the channels, and do not require Ca2+, Na+, Mg2+, G-proteins or protein phosphorylation, suggesting that they might be mediated by direct interaction between these receptors.

methods

  1. Top of page
  2. Abstract
  3. methods
  4. results
  5. discussion
  6. Acknowledgements

Young guinea-pigs (150–300 g), either male or female, were stunned and immediately exsanguinated by severing major neck blood vessels. A segment of small intestine (jejunum; about 5 cm in length) was removed, placed in modified Krebs solution and opened longitudinally. The mucosa was removed and the submucosal layer (submucosal preparation) was dissected from the underlying layers of smooth muscle. Modified Krebs solution was composed of (mm): 126 NaCl, 1.2 NaH2PO4, 1.2 MgCl2, 2.5 CaCl2, 5 KCl, 25 NaHCO3, 11 glucose; gassed with 95 % O2 and 5 % CO2.

Methods for dissociating and culturing enteric neurons have been described elsewhere (Barajas-López et al. 1996b). Briefly, the submucosal preparation was dissociated using sequential enzymatic treatments, first with a papain solution (0.01 ml ml−1; activated with 0.4 mg ml−1 of L-cysteine) and later with a collagenase (1 mg ml−1) plus dispase (4 mg ml−1) solution. After washing out these enzymes, submucosal 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−1 penicillin, 10 μg ml−1 streptomycin and 15 mm glucose.

Whole-cell currents were recorded from short-term (2–40 h) primary cultures of submucosal neurons. Membrane currents were recorded using an Axopatch 1D amplifier. Patch pipettes were made as previously described (Barajas-López et al. 1996b) and had resistances between 1 and 3 MΩ. Sixty to seventy percent of the series resistance was compensated in about one-third of the experiments reported here. Series resistance compensation, however, did not affect the lack of additivity of IATP and IACh (see Results), so in most cases no compensation was made for this factor. Except when otherwise mentioned the holding potential was −60 mV and currents were induced by a maximal concentration (1 mm) of ATP or ACh. The standard solutions used, unless otherwise mentioned, have the following composition, inside the pipette (mm): 160 caesium glutamate, 10 EGTA, 5 Hepes, 10 NaCl, 3 ATPMg, and 0.1 GTP; external solution (mm): 160 NaCl, 2 CaCl, 11 glucose, 5 Hepes and 3 CsCl. 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 GΩ. Whole-cell currents were recorded on a PC using AxoTape software (Axon Instruments). Analysis of these currents was performed on a Macintosh computer using AxoGraph software (Axon Instruments). Membrane potentials were corrected for the liquid junction potentials (pipette 11 mV negative). The recording chamber was continuously superfused with external solution at approximately 2 ml min−1. Rapid changes in the external solution were made by using an eight-barrelled device (Barajas-López et al. 1994). The external application of experimental substances was achieved by abruptly changing the tube delivering the external solution in front of the cell being recorded for a tube delivering the same solution plus the drug(s). Substances were washed out by switching back to the delivery tube containing only the external solution and by flushing of the bath. External solutions were delivered by gravity. Except where otherwise mentioned, experiments were performed at room temperature (∼23°C).

Staurosporine and K-252a were supplied by Kamiya (Thousand Oaks, CA, USA). Genistein, ACh and pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) were purchased from Research Biomedical Inc. (Natick, MA, USA). All other substances were purchase from Sigma (St Louis, MO, USA). 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 ±s.e.m. and the number of cells used as n. Student'spaired t test was used to evaluate differences between mean values obtained from the same cells and Student's unpaired 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

  1. Top of page
  2. Abstract
  3. methods
  4. results
  5. discussion
  6. Acknowledgements

General properties of the whole-cell currents induced by ACh and ATP

Whole-cell currents activated by ACh (IACh) and ATP (IATP) in enteric neurons have been characterized previously (Barajas-López et al. 1994, 1996a, c; Zhou & Galligan, 1996, 1997). These currents share several properties. For instance, they are known to be mediated by activation of non-specific cationic channels, and their current-voltage relationships display a strong inward rectification. The slope conductances of single channels activated by these transmitters have also been reported to be very similar; 25 and 15–22 pS for nicotinic and P2X receptors, respectively (Barajas-López et al. 1994, 1996a; Zhou & Galligan, 1996, 1997).

About 97 % (n= 202) and 88 % (n= 183), of a total of 208 neurons, responded to ACh and ATP, respectively. Concentration-response curves were obtained for these transmitters in submucosal neurons and analysed as reported previously (Barajas-López et al. 1996a). The EC50 values for ACh and ATP were 54 and 53 μm, whereas the Hill coefficient values were 1.84 ± 0.04 (significantly larger than unity; P < 0.01) and 1.11 ± 0.13, respectively. The currents induced by maximal concentrations of ACh and ATP (1 mm) had mean amplitudes of −2363 ± 122 and −2188 ± 111 pA, respectively, but had variable amplitudes in different cells, ranging from only a few picoamps up to 8 nA. The amplitudes of IACh and IATP were independent of each other. Indeed, in a few cells (17 out of 152) only one of these currents could be observed.

Inward currents induced by ACh or ATP (1 mm) were specifically and significantly (P < 0.001) inhibited by hexamethonium (300 μm) and PPADS (30 μm), respectively. The mean amplitude of IACh was −2199 ± 887 and −2559 ± 1190 pA (n= 7) before and in the presence of PPADS, respectively, whereas IATP mean values were −1449 ± 302 and −72 ± 24 pA (n= 10). Before and in the presence of hexamethonium, IACh values were −2972 ± 602 and −203 ± 86 pA (n= 14), respectively, whereas IATP values were −2082 ± 287 and −1973 ± 489 pA (n= 5).

The times required to reach the half-maximal current were 148 ± 11 and 108 ± 10 ms for the inward IACh and IATP, respectively. These currents usually reached their peak within the following second. After agonist removal from the external solution, both currents decayed rapidly, the decay being well fitted by a single exponential function (τ= 230 ± 22 and 260 ± 38 ms, respectively; n= 13). After reaching maximal amplitude, the currents decreased despite the continuous presence of the transmitters, indicating tachyphylaxis. This desensitization occurred with different kinetics; IACh desensitized faster than IATP (see below).

In experiments carried out on the same submucosal neurons, the reversal potential of the currents induced by ATP was always more positive (P < 0.001; n= 8) than that of the currents induced by ACh: +23 ± 2.5 vs.+ 19 ± 2.3 mV, respectively. This would suggest that nicotinic and P2X channels have different ion permeability. At membrane potentials more positive than the reversal potential, outward currents were recorded when ACh or ATP were added to the bath. The amplitude of these outward currents increased very little by further depolarization of the membrane, as described before (Barajas-López et al. 1994).

The following data indicate that outward currents induced by ACh and ATP are carried through nicotinic and P2X channels, similar to those activated at negative potentials. First, I-V relationships and the reversal potentials of IACh and IATP are shifted to the left by decreasing the concentration of Na+ outside the cell (Barajas-López et al. 1994) or to the right by decreasing its concentration inside the cell (see below). Second, outward currents had the same kinetics as inward currents (Fig. 1). Thus, in five analysed experiments carried out at +40 mV, the times to reach the half-maximal outward current were 161 ± 27 and 138 ± 33 ms for IACh and IATP, respectively. These times were not significantly different for the inward currents recorded at 0 mV, with mean values of 159 ± 25 and 140 ± 31 ms, respectively. Similar desensitization kinetics were also observed for outward and inward currents recorded in the same neurons at membrane potentials of +40 and 0 mV (Fig. 1). These were approximately equidistant membrane potentials from the reversal potential for these currents. Currents induced by ACh at these potentials were well fitted by the sum of three exponentials, whereas the currents induced by ATP were well fitted by the sum of two exponentials. The τ values of these exponentials were τ1= 0.9 ± 0.3, τ2= 6.4 ± 1.3 and τ3= 89 ± 54, and τ1= 0.9 ± 0.2, τ2= 7.2 ± 1.7 and τ3= 108 ± 51 s, respectively, for outward and inward currents induced by ACh (n= 4). The τ values for IATP were τ1= 5.3 ± 1.9 and τ2= 49 ± 27, and τ1= 4.2 ± 1.4 and τ2= 64 ± 44 s, respectively (n= 4). Third, outward and inward currents have similar pharmacological properties. Thus, inward and outward currents were also induced by the application of nicotine (n= 3) or ATP-γ-S (n= 5). Outward currents induced by ATP (1 mm) were also significantly (P < 0.05; n= 4) blocked by 30 μm PPADS, with mean values of 441 ± 126 and 24 ± 3 pA before and in the presence of PPADS, respectively. The outward currents induced by ACh (1 mm) were also significantly (P < 0.05; n= 4) blocked by 1 mm hexamethonium, with mean values of 109 ± 25 and 56 ± 13 pA before and in the presence of hexamethonium, respectively. Thus, hexamethonium blocked about 50 % of the outward currents but inhibited about 90 % of the inward currents (see above). This voltage-dependent effect of hexamethonium has been employed to support the idea that this drug blocks nicotinic channels by entering into the open pores (Ascher et al. 1979; Bertrand et al. 1990).

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Figure 1. Whole-cell outward and inward currents induced by either ATP or ACh show similar desensitization kinetics

Outward and inward currents induced by ATP (A) and ACh (B), and recorded at approximately equidistant membrane potentials from the reversal potential for these currents. Both agonists were applied as indicated by bars. Currents of panels A and B are from two different submucosal neurons. Vertical calibration bar labels indicate outward (upper label) and inward currents (lower label).

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Whole-cell currents induced by ACh and ATP occluded each other

In the experiments described above, we clearly demonstrated that IACh and IATP are mediated by activation of pharmacologically distinct receptors in submucosal neurons. We next investigated whether these currents are mediated by independent populations of channels. If IATP and IACh 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 ACh or ATP, as well as the simultaneous application of both agonists (IACh+ATP), in the same submucosal neurons. We found that the addition of individual currents (IATP+IACh=Iexpected) was significantly larger than IACh+ATP, revealing an occlusion between IACh and IATP (Fig. 2; n= 17). IACh+ATP was usually as large as the current induced by the most effective of these transmitters. For instance, if IATP was larger than IACh, then IATP+ACh had about the same magnitude as IATP (e.g. Fig. 2A). In only one of these seventeen experiments was Iexpected very similar to IACh+ATP.

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Figure 2. Whole-cell inward currents induced by ACh (IACh) and ATP (IATP) in submucosal neurons are not additive, revealing a current occlusion

A shows recordings from one neuron of a typical experiment and B shows the mean values of seventeen experiments. Currents were induced by application of either ACh (1 mm) or ATP (1 mm) and by the simultaneous application of both agonists (IACh+ATP). IACh and IATP were recorded 5 min before and 5 min after IACh+ATP. For the outer two bars, the downward error bar indicates s.e.m. for each individual current and the upward error bar s.e.m. for the expected currents (Iexpected=IACh+IATP). The mean IACh+ATP was significantly lower (***P < 0.001) than Iexpected. C, onset of the currents recorded from another submucosal neuron showing that IACh+ATP is smaller than Iexpected from the onset of these currents. The expected current shown in C is a graph representing the addition of IACh and IATP. Whole-cell currents were measured at a holding potential of −60 mV.

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Figure 2C shows the onset of IACh, IATP, IACh+ATP and Iexpected in a typical experiment from ten experiments analysed. As is shown in this figure, IACh+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 less than 5 ms.

Since desensitization kinetics of IACh, IATP and IACh+ATP appeared to be different during short term applications of these agonists (usually < 10 s), we decided to analyse this with relatively long applications (∼3 min). Exponential fits were performed using the data from the current peak to the ‘steady-state’ component (end of the agonist application). We found that IACh desensitization was well fitted by the sum of three exponential functions, whereas IATP desensitization was better fitted by the sum of two exponential functions (Fig. 3). IACh+ATP desensitized faster than IATP but more slowly than IACh. Thus, IACh+ATP desensitization was, like that of IACh, better fitted by the sum of three exponential functions; the τ value of the first exponential was, however, significantly slower (Fig. 3B; P < 0.05; n= 7) than the τ value of the first exponential for IACh desensitization. The τ values of the last two exponentials of IACh+ATP desensitization were no different than the values of the corresponding τ values of IACh desensitization or than the two τ values of IATP desensitization (Fig. 3B).

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Figure 3. Desensitization kinetics of currents induced by simultaneous application of ACh and ATP (IACh+ATP) cannot be explained by the desensitization kinetics of the currents induced by application of ACh (IACh) or ATP (IATP) alone

A, representative recordings from a submucosal neuron of IACh, IATP and IACh+ATP (grey current traces). The desensitization of IACh+ATP and IACh were better fitted by the sum of three exponential functions (continuous black lines), whereas IATP desensitization was better fitted by the sum of two exponential functions. Note that IACh+ATP desensitizes faster than IATP but slower than IACh. B, bars represent the mean ±s.e.m. values of the τ values of these exponential functions. The first exponential (τ1) of IACh was significantly smaller (*P < 0.05) than τ1 of IACh+ATP. τ values of the second and third exponentials of these currents were not different, however. The τ values of the second and third exponentials of IACh+ATP desensitization were also not different from those of the first and second exponentials of IATP. Exponential fits were performed using the data from the current peak to the ‘steady-state’ component. In these experiments agonists were applied for approximately 3 min and the holding potential was −60 mV.

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The following observations indicate that current occlusion is due to a postreceptor mechanism. In fourteen cells with very low response (< 100 pA; Fig. 4A) or no initial response to ATP, and in another six cells, in which IATP had been previously blocked with PPADS, IACh+ATP had the same amplitude and desensitization kinetics as IACh alone. Similarly, in three cells with very low response or no initial response to ACh and in those in which IACh was blocked with hexamethonium (n= 6), IACh+ATP had the same amplitude and desensitization kinetics as IATP alone (Fig. 4B). Furthermore, Fig. 4C and D shows IACh and IATP before (control currents) and during continuous application of the other agonist. The second application of ACh or ATP was made after the response of the other agonist had decayed by about 80 % of its peak amplitude. Currents induced by this second application had similar kinetics and almost the same amplitude as control currents. Control IACh and IATP had amplitudes of −2742 ± 428 and −1984 ± 163 pA, respectively. In the presence of the other agonist their amplitudes were −2234 ± 190 pA (n= 4) and −1884 ± 252 pA (n= 4). Finally (see below), IACh+ATP kinetics was changed to look like IACh kinetics when P2X channels were blocked with PPADS, and was made to resemble IATP kinetics by adding hexamethonium.

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Figure 4. Inhibitory interactions between nicotinic 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 ACh. Note that ATP did not modify either the amplitude or the kinetics of the current induced by ACh (IACh). Similar results were obtained when IATP was blocked with a P2X receptor antagonist (60 μm PPADS; not shown). B, currents induced by ACh, ATP and ACh + ATP in the presence of hexamethonium (1 mm). Note that ACh did not modify the kinetics of IATP. Similar results were obtained when IACh was blocked with a nicotinic receptor antagonist (not shown). IACh (C) and IATP (D) were recorded 5 min before (left recordings) and during continuous application of the other agonist. Note that currents induced by this second application have similar kinetics and amplitude to control currents. These experiments were repeated in four neurons with similar results. The four sets of currents were recorded in four different submucosal neurons and were taken at a holding potential of −60 mV.

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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 +10 mV, when Iexpected had a mean amplitude of only −296 ± 82 pA (Fig. 5A). This value is only about one-tenth of the IACh amplitude recorded at −60 mV (Fig. 2A). Second, the amplitude of the currents recorded at −60 mV and induced by ACh or ATP could be almost doubled by hyperpolarizing the membrane to −90 mV (not shown; n= 4; see also Barajas-López et al. 1994). Third, using the same experimental conditions and in the same submucosal neurons, we were able to record voltage-activated calcium and sodium inward currents as large as −8000 pA, which in many cases were as large as Iexpected at −60 mV (−6357 ± 793 pA). This is despite the fact that the onset of sodium and calcium currents (few milliseconds) is faster than the onset of IACh and IATP (hundreds of milliseconds).

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Figure 5. Currents induced by ATP (IATP) and ACh (IACh) were additive when they were outward

Data shown are from experiments carried out at two holding potentials of +10 mV (A and B; n= 5) and +40 mV (C and D; n= 10). Two sets of recordings from two typical experiments are shown in A and C and the mean values of similar experiments are shown in B and D, respectively. Currents were induced by application of either ACh (1 mm) or ATP (1 mm) and by simultaneous application of both agonists (IACh+ATP). IACh and IATP were recorded 5 min before and 5 min after IACh+ATP. Error bars show half-s.e.m. In the outer two bars, the downward half-s.e.m. is for each individual current and the upward half-s.e.m. is for the expected currents (Iexpected=IACh+IATP). Just as it was at a membrane potential of −60 mV, at +10 mV IACh+ATP was significantly lower (P < 0.05) than Iexpected. At +40 mV, however, no difference was observed between these currents.

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We also investigated whether current occlusion was present at 36°C. In seven experiments carried out at this temperature, IACh+ATP (−3673 ± 703 pA) was still significantly (P < 0.05) lower than Iexpected (−5547 ± 1292 pA). These observations rule out the possibility that current occlusion was an artefact of recording at room temperature.

Current occlusion was also observed when nicotinic and P2X channels were activated with nicotine and ATP-γ-S

Currents induced by maximal concentrations of nicotine or ATP-γ-S had a mean amplitude of −1328 ± 194 and −957 ± 252 pA (n= 4), respectively. In these experiments, the addition of such individual currents was −2285 ± 235 pA, which was significantly larger than the current induced by the simultaneous application of nicotine and ATP-γ-S (−1459 ± 218 pA).

Currents induced by the simultaneous application of both agonists are carried through both nicotinic and P2X channels

In seven experiments analysed in which IATP (−2342 ± 427 pA) was significantly (P < 0.01) larger than IACh (−1140 ± 400 pA), IACh+ATP (−2458 ± 518 pA) was significantly (P < 0.01) larger than IACh but was not different from IATP. Similarly, in six neurons analysed in which IACh (−3432 ± 867 pA) was significantly (P < 0.01) larger than IATP (−1790 ± 652 pA), IACh+ATP (−3459 ± 752 pA) was significantly (P < 0.01) larger than IATP but was not different from IACh. Furthermore, as mentioned above (Fig. 3), IACh+ATP desensitization occurs with kinetics that cannot be explained by the desensitization kinetics of IACh or IATP alone, suggesting that IACh+ATP is carried through both nicotinic and P2X channels.

Observations from four cells, in which current occlusion had been shown to occur, are also in agreement with such an interpretation. An example of such experiments is shown in Fig. 6. Here, we show that the desensitization kinetics of IACh+ATP can be changed to resemble IACh kinetics by adding PPADS (60 μm) to the external solution, or to resemble IATP kinetics by adding hexamethonium (1000 μm).

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Figure 6. Whole-cell currents induced by simultaneous application of ACh and ATP (IACh+ATP) appear to be mediated by the opening of both nicotinic and P2X channels

A, currents induced by ACh (IACh) and ATP (IATP). B, IACh+ATP before (Control), in the presence of 1 mm hexamethonium (a nicotinic channel blocker) or in the presence of 60 μm PPADS (a P2X receptor antagonist). In the presence of hexamethonium IACh+ATP (B) had a similar amplitude and kinetics to the current induced by ATP alone (A), whereas in the presence of PPADS IACh+ATP (B) had a similar amplitude and kinetics to the current induced by ACh alone (A). All recordings are from the same submucosal neuron and were taken at a holding potential of −60 mV.

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The following observations also support our interpretation that IACh+ATP is carried through both nicotinic and P2X channels. In these experiments, we measured the amplitude of both IACh and IATP before and immediately after (∼5 s) a long application of ACh, ATP or ACh + ATP. This long application lasted for at least 25 s or until the induced current had decreased approximately 80 % (usually within 1 min). Some typical recordings and the mean data from such experiments are shown in Fig. 7. We observed that nicotinic receptor desensitization with ACh decreased IACh whereas it did not affect IATP (Fig. 7A and B). Similarly, P2X receptor desensitization with ATP decreased IATP whereas it did not affect IACh (Fig. 7C and D). In other words, no cross-desensitization was observed between nicotinic and P2X receptors. When receptors were desensitized by the simultaneous application of ATP + ACh, both nicotinic- and P2X-mediated currents were significantly decreased (Fig. 7E–G).

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Figure 7. No cross-desensitization was observed between nicotinic and P2X receptors

Control IACh (A and D) and IATP (B and C) were recorded 5 min before (left recordings) and immediately after (∼5 s) a prolonged application of the other agonist. E and F, simultaneous application of both agonists desensitized both receptor populations. IACh (E) and IATP (F) recorded 5 min before (control currents; left recordings) and immediately after (∼5 s) a prolonged application of ACh + ATP. G, mean amplitude of IACh and IATP recorded after the prolonged application of ATP, ACh or ACh + ATP, as a percentage of control response. Error bars are s.e.m.A and B, C and D, and E and F are from three different submucosal neurons and the holding potential was −60 mV.

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Current occlusion was not observed for outward currents

Current occlusion was not observed at +40 mV (Fig. 5C). Thus, at this potential, IACh+ATP was very similar to Iexpected in five out of six experiments and the means of these currents were not different. At this membrane potential, IACh+ATP was significantly (P < 0.05) larger than the current induced by the most effective transmitter. We investigated whether the lack of current occlusion was due to membrane depolarization or to the fact that currents were outward at +40 mV. To study this we adjusted the reversal potential of these currents to a more positive value by replacing the Cs+ inside the pipette with N-methyl-D-glucamine and by decreasing Na+ from 10 mm to only 2 mm. During these experiments, Cs+ (3 mm) was also omitted from the external solution. With these experimental solutions, IACh (−59 ± 19 pA) and IATP (−237 ± 48 pA) were still inward at +40 mV and Iexpected (−296 ± 61 pA) was still significantly (P < 0.05; n= 16) larger than IACh+ATP (−244 ± 49 pA). These observations imply that the current occlusion is triggered by the inward movement of cations through nicotinic and P2X channels.

Ca2+, Na+ or Mg2+ are not required in the current occlusion

A simple explanation for the inward current occlusion might be that influx of Ca2+ and/or Na+ through one of these channels raises their intracellular concentration, thus inhibiting the neighbouring channels. The following data, however, rule out this possibility. First, inward current occlusion was still observed in the complete absence of Ca2+ in the extracellular and intracellular medium (Fig. 8). These experiments were carried out with the standard extracellular solution but containing no calcium and 50 μm EGTA, and with the standard intracellular solution plus 5 mm BAPTA. Furthermore, this occlusion and the amplitude of IATP and IACh were not different when Cs+ was replaced in the pipette by Na+. In the presence of high intracellular Na+, IATP and IACh had mean amplitudes of −2247 ± 533 and −2124 ± 690 pA, whereas Iexpected (−4371 ± 1116 pA) and IACh+ATP (−2824 ± 775 pA) were significantly different (P < 0.05; n= 6). Similarly, this current occlusion was still present when the external and internal Na+ was replaced by equimolar concentrations of Cs+. In the absence of intracellular and extracellular Na+, IACh and IATP had a mean amplitude −2234 ± 342 and −1456 ± 215 pA, whereas Iexpected (−3690 ± 418 pA) and IACh+ATP (−2673 ± 266 pA) were significantly different (P < 0.05; n= 6). 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= 8) or Na+ (n= 2). In these experiments Iexpected had a mean value of −5777 ± 837 pA, whereas IACh+ATP was significantly lower (−3977 ± 596 pA (P < 0.001)).

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Figure 8. Mean amplitude of inward currents induced by application of ACh (IACh), ATP (IATP) or ACh + ATP (IACh+ATP) in seven 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 mean IATP and IACh before application of ACh + ATP. This combined bar represents the expected current (Iexpected=IACh+IATP). The second bar represents IACh+ATP. In the combined bars, downward error bars show s.e.m. for IACh and IATP, while upward error bars show s.e.m. for IACh+ATP. Ca2+-free experiments were carried out in 0 Ca2+ plus 50 μm EGTA extracellular media and standard intracellular solution plus 5 mm BAPTA. In the α,β-methylene ATP (α,β-meATP) and GTP-γ-S experiments the pipette solution contained these substances instead of ATP and GTP, respectively. K-252a, staurosporine and genistein experiments were carried out using standard intracellular solution plus 10 μm of these agonists. During the N-ethylmaleimide (NEM) experiments, recorded neurons were pretreated for 10 min with standard extracellular solution plus 30 mm NEM. During these experiments, only one cell from each coverslip was recorded and coverslips were discarded after a neuron had been exposed to NEM. The holding potential was −60 mV in all these experiments.

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Role of protein phosphorylation and G-proteins in the current occlusion

Current occlusion was still observed after inhibiting protein phosphorylation by either replacing the ATP of the internal solution with its non-hydrolysable analogue α,β-methylene ATP or by adding to the internal solution either 10 μm genistein (a tyrosine kinase inhibitor; Liu et al. 1997), 10 μm K-252a or 5 μm staurosporine (non-specific protein kinase inhibitors; Kase et al. 1987; Fig. 8). Three micromolar staurosporine has been shown previously to inhibit the membrane depolarization induced by forskolin and phorbol esters in submucosal neurons (Barajas-López, 1993). Also, current occlusion remained unmodified when the temperature was lowered to 9°C, as expected if the mechanism for this occlusion is independent of the cellular metabolism. In three experiments carried out at this temperature, Iexpected (−2478 ± 369 pA) was still significantly (P < 0.05) larger than IACh+ATP (−1383 ± 141 pA).

On the other hand, current occlusion was still observed after superfusing N-ethylmaleimide (30 μm; known to uncouple receptors from G-proteins; Allende et al. 1991; Shapiro et al. 1994), or when GTP was replaced by GTP-γ-S in the internal solution (Fig. 8). These last two experimental manipulations have previously been shown to alter several responses known to be mediated by G-proteins in submucosal neurons (Barajas-López et al. 1996b).

Current occlusion is not observed for a low agonist-receptor occupancy

In these experiments IACh, IATP and IACh+ATP were induced with an agonist concentration of 10 μm; at this concentration, the nicotinic and P2X receptor occupancy is expected to be less than 0.2 in enteric neurons (Barajas-López et al. 1994, 1996a, c). The mean amplitude of these currents was −248 ± 99, −372 ± 124 and −687 ± 182 pA, respectively. In these experiments Iexpected (−620 ± 131 pA) was not different than IACh+ATP

discussion

  1. Top of page
  2. Abstract
  3. methods
  4. results
  5. discussion
  6. Acknowledgements

Our results indicate that in guinea-pig submucosal neurons, activation of nicotinic and P2X receptors with ACh and ATP, respectively, opens two different channel populations. Activation of these channels is, however, not independent and currents carried through them are occluded when they are simultaneously activated. This would imply that there are inhibitory interactions between these two populations of channels. Such interactions occur as soon as ionic currents are detected (less than 5 ms), are triggered by ion influx through the channels, and do not specifically require Ca2+, Na+, Mg2+, G-protein activation or protein phosphorylation, suggesting that they may be mediated by direct interaction between nicotinic and P2X channels.

The currents induced by either ACh (1 mm) or ATP (1 mm) were specifically blocked by hexamethonium and PPADS, respectively, confirming that these transmitters, despite the high agonist concentrations used here, can specifically activate pharmacologically distinct receptors in submucosal neurons. The fact that the amplitudes of these two currents are independent of each other in the recorded neurons implies that these ligand-gated channels can be expressed separately in these neurons.

Inward currents carried through nicotinic and P2X receptors are not additive when these channels are simultaneously and maximally activated, indicating that activation of these channels is not independent. A similar inhibitory interaction between nicotinic and P2X receptors in rat sympathetic neurons was reported by Nakazawa (1994a), who explained these observations by proposing a ‘channel overlap’ hypothesis (see Introduction). According to this hypothesis, a subpopulation of nicotinic receptors share the same channels with P2X receptors. Our results obtained at a holding potential of +40 mV do not support this hypothesis. Thus, at this potential the currents induced by simultaneous application of ATP + ACh are similar to the sum of the currents induced by individual applications of ATP or ACh. Therefore, IATP and IACh 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 nicotinic and P2X channels have different structures (Brake et al. 1994; Surprenant et al. 1995; Lindström, 1996; Lindström et al. 1996; North, 1996; Burnstock, 1997).

The following observations rule out the possibility that the current occlusion reported here could be due to non-specific interactions between ATP and ACh molecules. First, in cells with no initial response to ATP or in which IATP had been previously blocked with PPADS, IACh+ATP has exactly the same amplitude and kinetics as the current induced by ACh alone. Second, nicotinic receptor desensitization also prevents any ACh effect on IATP and similarly, desensitization of P2X receptors prevents any ATP effect on IACh. Third, current occlusion was not observed for a low agonist-receptor occupancy. Fourth, in cells with no initial response to ACh or in which IACh had been blocked with hexamethonium, IACh+ATP has exactly the same amplitude and kinetics as IATP. It is also likely that this inhibitory interaction not only requires activation of receptors but might actually require channels to be open, since hexamethonium, a substance that is believed to be a nicotinic channel blocker (Ascher et al. 1979; Bertrand et al. 1990), prevents ACh effects on IATP.

At least four different observations indicate that IACh+ATP are carried through both nicotinic and P2X channels and not only through one population of these channels. (1) IACh+ATP desensitizes with different kinetics than IACh or IATP. Thus, IACh+ATP desensitizes faster than IATP but more slowly than IACh. (2) IACh+ATP desensitization kinetics are changed to resemble IACh kinetics when P2X receptors are blocked with PPADS, and can be made to resemble IATP kinetics by blocking nicotinic channels with hexamethonium. (3) When ATP and ACh are applied simultaneously, both nicotinic- and P2X-mediated currents are desensitized, whereas no cross-desensitization is observed when nicotinic 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, IACh+ATP amplitude is the same as that of the current induced by the most effective neurotransmitter, and is significantly larger than the weaker transmitter. Altogether, these observations imply that IACh+ATP is carried through both populations of channels and that inhibition between these channels is reciprocal.

The short latency for this current occlusion suggests that no second messenger is required for these inhibitory interactions. In agreement with such an interpretation, inhibition of G-proteins (with N-ethylmaleimide), functional modification of G-proteins (by replacing GTP with GTP-γ-S in the pipette solution) and inhibition of protein phosphorylation (with staurosporine, K-252 or genistein) do not modify the occlusion observed between IACh and IATP. Consistently, this occlusion is still present during experiments carried out at 9°C. In rat sympathetic neurons, Nakazawa (1994b) reported that occlusion between IACh and IATP was prevented by K-252a and by using a non-hydrolysable analogue in place of ATP in the pipette solution. In apparent contradiction, these procedures failed to prevent current occlusion in submucosal neurons of the guinea-pig (present study). Such inconsistency might be the result of a real difference between the two types of neurons used. However, these two studies also differ in that we used maximal concentrations of both agonists (1 mm), whereas Nakazawa (1994b) used submaximal concentrations (100 μm; see Nakazawa, 1994a). With submaximal agonist concentrations, currents might appear to be additive if a potentiatory effect was unmasked by a particular experimental procedure.

Nicotinic and P2X channels of neurons are permeable to Na+ and Ca2+, suggesting that their intracellular accumulation could mediate the inhibitory interaction between these channels. This hypothesis is supported by the fact that nicotinic and P2X receptors of enteric neurons are known to be permeable to these cations (Trouslard et al. 1993; Barajas-López et al. 1994, 1996a; Christofi et al. 1997). However, we have ruled out such a possibility, by showing that the interaction is not affected by the absence of Ca2+ or Na+ in the intracellular and extracellular solutions. Mg2+ are also not required because occlusion is still observed in the absence of these ions.

In conclusion, our results indicate that there is a very fast inhibitory interaction between nicotinic and P2X channels, which occurs only for inward currents. These interactions occur within just a few milliseconds, supporting the hypothesis that nicotinic and P2X channels are located very close to each other in the neuronal membrane, perhaps forming functional units constituted by at least one channel of each type. This hypothesis is an extension of the well-known phenomenon that transmitter-gated channels, like other ion channels, do not diffuse freely in the membrane but are clearly localized underneath the appropriate presynaptic terminal. Several proteins have been proposed to be involved in the clustering of ion channels at specific membrane sites, e.g. 43/rapsyn is believed to be responsible for the clustering of nicotinic channels (Sheng & Kim, 1996). The fact that such inhibitory interactions appear to occur only when nicotinic channels are permeable and only for inward currents suggests the existence of a sensory mechanism specific for inward ion movements. Therefore, we propose a model in which the detection of ion influx in one channel (e.g. nicotinic) would induce allosteric changes in the neighbouring channel (e.g. P2X) that would block it, so that two channels of a particular functional unit would never be open at the same time. The inhibitory effect on the second channel would disappear once the ion influx through the first channel was stopped. A functional significance of this inhibitory interaction between these ligand-gated channels would be the saving of cellular energy by limiting ion movements through the cellular membrane.

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Acknowledgements

  1. Top of page
  2. Abstract
  3. methods
  4. results
  5. discussion
  6. Acknowledgements

The authors thank Dr E. E. Daniel and M. D. Kawaja for helpful comments to this manuscript. This work was supported by the Medical Research Council of Canada (13491). C. B.-L. was supported by the Ontario Ministry of Health (Career Scientist Award no. 04500).