Non-additive interaction between nicotinic cholinergic and P2X purine receptors in guinea-pig enteric neurons in culture

Abstract

1. Acetylcholine (ACh)-activated currents and their interaction with ATP-activated currents were studied in primary cultures of myenteric neurons from guinea-pig small intestine using patch clamp techniques. Peak currents caused by co-application of ACh (1 mM) and ATP (300 microM) were 78 +/- 2 % of the sum of currents activated by each agonist alone (P < 0.05, n = 29). Reversal potentials measured during co-application of ACh and ATP did not differ from those measured during application of ACh or ATP alone. Addition of BAPTA (10 mM) to the pipette solution or replacement of extracellular Ca2+ with Na+ did not prevent occlusion. 2. Responses caused by co-application of 5-HT (300 microM), acting at 5-HT3 receptors, and ACh (3 mM) or ATP (1 mM) were additive (94 +/- 3 or 96 +/- 4 %, respectively, of the sum of currents activated by 5-HT and ACh or ATP alone; P > 0.05). Currents caused by GABA (1 mM), acting at GABAA receptors, and ACh (3 mM) or ATP (1 mM) were also additive (105 +/- 4 or 100 +/- 3 %, respectively, of the sum of currents activated by GABA and ACh or GABA and ATP applied separately; P > 0. 05). 3. Single channel currents caused by ACh and ATP in the same outside-out patches were less than additive (85 +/- 10 % of the predicted sum, P < 0.05). 4. P2X receptors and nicotinic cholinergic receptors (nAChRs) are linked in a mutually inhibitory manner in guinea-pig myenteric neurons. The functional interaction does not involve ligand binding sites, Ca2+-dependent mechanisms, a change in the driving force for Na+ or cytoplasmic signalling mechanisms.

Figure 1. ACh- and ATP-induced inward currents in myenteric neurons

A, whole-cell currents elicited by acetylcholine (ACh, 3 mm) and ATP (1 mm). Downward deflections are current responses to 10 mV, 200 ms hyperpolarizing voltage commands. B, currents caused by ACh are reversibly blocked by hexamethonium (100 μm); holding potential, −60 mV. C, concentration-response curves for the peak currents elicited by ACh and ATP (holding potential, −60 mV). Each point is the mean ± s.e.m. of responses obtained from 4–6 cells per point; data points were fitted using a logistic function (see Methods). Data are normalized to the maximum ACh or ATP response recorded from each cell. The EC₅₀ for ACh was 112 ± 7 μm (slope = 1.6 ± 0.1); the EC₅₀ for ATP was 34 μm (slope = 1.3 ± 0.3).

Figure 2. Occlusion of currents elicited by saturating concentrations of ACh (3 mm) and ATP (1 mm)

Aa, currents evoked by ACh and ATP are not additive. Combined agonist application causes a response smaller in amplitude (middle trace) than the predicted sum of the individual agonist responses obtained before agonist co-application (left traces). Amplitude of the individual agonist responses obtained after agonist co-application (right traces) is the same as that obtained before agonist co-application; holding potential, −60 mV. Ab, pooled data from experiments shown in Aa; data are the mean ± s.e.m. amplitude (n = 29 cells) of responses to ACh alone, ATP alone and ACh and ATP co-applied. * Significantly less than the predicted sum of the ACh and ATP response in each cell tested (P < 0.05). Ba, differences in the rise time of ACh- and ATP-induced currents do not account for lack of additivity. ACh and ATP responses were obtained sequentially; ATP responses desensitize slowly and ACh was applied during the steady-state peak of the ATP current. Bb, pooled data (n = 41 cells) from experiments shown in Ba; data are the mean ± s.e.m. amplitude of currents caused by ACh alone, ATP alone, the ACh current in the presence of a steady-state response to ATP and the peak amplitude of the current caused by ACh plus ATP. * Significantly different from the ACh current alone (ACh in presence of ATP) or significantly different from the predicted sum of the ACh- and ATP-induced currents (ACh + ATP).

Figure 3. Ratio of ACh current amplitudes in the presence and absence of ATP is negatively correlated with the amplitude of ATP currents in the same cell

The amplitude of the ACh-induced current recorded in the presence of ATP is smaller when the amplitude of the ATP current is large. Points (n = 41) are the ratio of ACh currents recorded before and in the presence of a maximum concentration of ATP (1 mm) versus the amplitude of the ATP current in the same cell. The line is a best fit straight line (slope = −0.89, r = 0.62, P < 0.00001).

Figure 4. Non-additivity of ACh- and ATP-induced currents persists after receptor desensitization

Aa, ATP (1 mm)-activated currents evoked in the absence (left) and presence of a desensitizing concentration of ACh (3 mm, right) applied for 150 s. The ATP response is smaller after nAChR desensitization; holding potential, −60 mV. Ab, pooled data from experiments shown in Aa; data are mean ±s.e.m. amplitude of ATP currents, ACh currents and the amplitude of the ATP current after nAChR desensitization. * Significantly different from ATP control (P < 0.05). Ba, ACh (3 mm)-evoked current evoked before and after P2X receptor desensitization caused by ATP (1 mm) applied for 150 s. The ACh response is smaller after P2X receptor desensitization. Bb, pooled data from experiments shown in Ba; data are the mean ±s.e.m. amplitude of ACh currents, ATP currents and the amplitude of the ACh current after P2X receptor desensitization. * Significantly different from ACh control (P < 0.05).

Figure 5. Non-additivity of currents elicited by ACh and ATP in the absence and presence of intra- and extracellular Ca²⁺

A, ACh- but not ATP-evoked responses are reduced in the absence of extracellular calcium, suggesting that calcium is an important charge carrier for the nAChR. The Ca²⁺ concentration of normal Krebs solution (2.5 mm) was used as the 100% control current amplitude. Bars represent the mean of 5–7 cells. *Significantly different from control (P < 0.01). B, left bars, currents activated by 3 mm ACh (▪), 1 mm ATP (□) and their combination () with 2.5 mm Ca²⁺ in the extracellular solution and 1 mm EGTA and 10 mm BAPTA in the Ca²⁺-free pipette solution; * indicates that the amplitude of the current evoked by combined application of ACh and ATP was significantly less than the predicted sum of the individual currents. Right bars, amplitude of currents caused by ACh, ATP and co-application of ACh and ATP using a Ca²⁺-free extracellular solution and a Ca²⁺-free plus 10 mm BAPTA pipette solution; amplitude for ACh plus ATP was 80.1 ± 4 % (n = 10) of the sum of currents activated by ACh and ATP alone. * indicates that the amplitude of currents evoked by combined application of ACh and ATP were significantly less than the predicted sum of the individual currents.

Figure 6. Reversal potentials of currents caused by ACh, ATP and co-application of ACh and ATP

A, current-voltage relationship for currents activated by ACh and ACh plus ATP. Data were obtained using a voltage ramp which changed the holding potential of the neuron from −70 to 40 mV in 100 ms. Records show agonist-induced currents after subtraction of the resting current-voltage curve. B, current- voltage relationships for ATP-induced current and currents caused by co-application of ATP plus ACh. Reversal potentials for currents caused by agonist co-application were not different from those currents caused by separate agonist treatment. The voltage ramp changed the holding potential from −80 to 30 mV in 100 ms.

Figure 7. Currents caused by 5-hydroxytryptamine (5-HT) and ACh or ATP are additive

A, concentration-response curve for 5-HT. Responses were normalized to that caused by 100 μm 5-HT in each neuron (holding potential, −60 mV; data are the mean ±s.e.m. of 7 cells). Data were fitted using a logistic function (see Methods). The EC₅₀ for 5-HT was 8 ± 0.6 μm and the slope of the curve was 1.6 ± 0.2. B, 5-HT-activated currents were reversibly inhibited by ondansetron (1 μm). * Significantly different from control (P < 0.05, n = 3). C, inward currents were evoked by ACh (3 mm), 5-HT (0.3 mm) and ACh plus 5-HT, respectively. The amplitude current activated by ACh plus 5-HT (right) was equal to the sum of currents evoked by ACh and 5-HT applied individually. D, pooled data from experiments shown in C. Currents activated by 5-HT plus ACh (n = 10) or 5-HT plus ATP (n = 8) were 94 ± 3 or 96 ± 4 %, respectively, of the sum of currents activated by 5-HT and ACh (P > 0.05) or ATP (P > 0.05) alone.

Figure 8. Currents caused by ACh or ATP and γ-aminobutyric acid (GABA) are additive

A, GABA concentration-response curve. Responses were normalized to those caused by 300 μm GABA in each neuron (holding potential, −60 mV; data are mean ±s.e.m. of 3–6 cells). Data were fitted by a logistic function (see Methods). The EC₅₀ for GABA was 17 ± 2 μm; the slope of the curve was 1.2 ± 0.1. B, GABA-activated currents were blocked by bicuculline (10 μm, P < 0.05, n = 7). C, currents caused by ACh, GABA and ACh plus GABA (left to right). The amplitude of currents activated by ACh plus GABA (right) equalled the sum of currents evoked by ACh and GABA alone (left and middle). D, pooled data from experiments in C. Currents caused by co-application of ACh and GABA were 105 ± 4 % of the sum of the currents activated by GABA and ACh alone (P > 0.05). Currents caused by co-application of ATP and GABA were 100 ± 3 % of the sum of the currents activated by ATP and GABA alone (P > 0.05).

Figure 9. ACh- and ATP-activated currents in outside-out patches

A, single channel currents activated by ACh in an outside-out patch. Recordings were obtained at the indicated patch potentials; C indicates closed level. B, current-voltage relationship for single channel currents activated by ACh. Data are mean ± s.e.m. of recordings from patches. C (upper), ATP-induced currents evoked by application of ATP at the indicated time intervals after patch formation (patch potential, −60 mV). C (lower), records of nAChR currents in an outside-out patch in response to application of 1 mm ACh (continuous line above traces) at the indicated time intervals after patch formation (patch potential, −60 mV). D, time course of ACh (1 mm, ○) but not ATP (300 μm, □) current run-down. Data are expessed as a percentage of the charge transfer measured 2 min after patch formation.

Figure 10. Non-additivity of ACh- and ATP-activated currents in outside-out patches

A, records of currents caused by sequential application of ATP (1 mm), ACh (3 mm) and ATP and ACh alone to the same patch. Currents caused by ACh plus ATP were less than the predicted sum of currents activated by ATP and ACh alone. B, pooled data from experiments shown in A. Data are expressed as mean ± s.e.m. charge transfer during 1 s agonist application. * Significantly different from the predicted sum of currents activated by ATP and ACh applied individually. C, records of currents caused by sequential application of ACh (3 mm), ATP (1 mm) and ACh plus ATP obtained after currents caused by ACh had run down. Currents caused by ATP plus ACh were similar to those caused by ATP alone. D, pooled data from experiements in C. Data are expressed as mean ± s.e.m. total charge transfer during 1 s agonist application. Currents caused by ATP alone were not different from those caused by co-application of ATP plus ACh.