In airway smooth muscle (ASM), ATP induces a contraction associated with the increase of [Ca(2+)](i). Cytosolic Ca(2+) is extruded to the extracellular space by the Na(+)/Ca(2+) exchanger (NCX) in its normal mode. Some agonists activate the reverse mode of the NCX (NCX(REV)), inducing Ca(2+) entry. We investigated whether ATP, via P2X receptors, activates the NCX(REV) and whether the increment in [Ca(2+)](i) is used for contraction or for the sarcoplasmic reticulum (SR) refilling in guinea pig ASM. ATP contracted the ASM and this effect was blocked by indomethacin. Suramin and RB2 diminished the contraction induced by ATP; PPADS did not modify this response. In myocytes, ATP produces an increase in [Ca(2+)](i) not modified by indomethacin. In tracheal strips, using simultaneous measurements, ATP induced a biphasic change in [Ca(2+)](i), (a Ca(2+) peak followed by a plateau) accompanied by a contraction. Indomethacin or epithelium removal abolished this contraction, but not the Ca(2+) peak, whereas the plateau was decreased by indomethacin. In myocytes, the ATP-induced [Ca(2+)](i) increment was inhibited by suramin (~96%), PPADS (~40%), and RB2 (~57%). ATP augmented the NCX(REV) and this effect was abolished by SKF 96365 and TNP-ATP (P2X(1) and P2X(3) receptors antagonist). P2X(1) and P2X(3) receptors were corroborated by immunoblotting of ASM. NCX(REV) activation and ATP in the presence of RB2 favor the SR Ca(2+) refilling. In tracheal rings, successive ATP stimulations were reduced with KB-R7943. Therefore, ATP: (1) indirectly promotes muscle contraction via epithelial P2Y receptors and prostaglandins release; (2) increases the [Ca(2+)](i) through a prostaglandin-independent manner by activating P2X and P2Y receptors in smooth muscle; and (3) activates P2X(1) and P2X(3) receptors and the NCX(REV) which refills the SR.

In airway smooth muscle (ASM), adenosine 5'-triphosphate (ATP) induces a relaxation associated with prostaglandin production. We explored the role of K(+) currents (I (K)) in this relaxation. ATP relaxed the ASM, and this effect was abolished by indomethacin. Removal of airway epithelium slightly diminished the ATP-induced relaxation at lower concentration without modifying the responses to ATP at higher concentrations. ATPγS and UTP induced a concentration-dependent relaxation similar to ATP; α,β-methylene-ATP was inactive from 1 to 100 μM. Suramin or reactive blue 2 (RB2), P2Y receptor antagonists, did not modify the relaxation, but their combination significantly reduced this effect of ATP. The relaxation was also inhibited by N-ethylmaleimide (NEM; which uncouples G proteins). In myocytes, the ATP-induced I (K) increment was not modified by suramin or RB2 but the combination of both drugs abolished it. This increment in the I (K) was also completely nullified by NEM and SQ 22,536. 4-Amynopyridine or iberiotoxin diminished the ATP-induced I (K) increment, and the combination of both substances diminished ATP-induced relaxation. The presence of P2Y(2) and P2Y(4) receptors in smooth muscle was corroborated by Western blot and confocal images. In conclusion, ATP: (1) produces relaxation by inducing the production of bronchodilator prostaglandins in airway smooth muscle, most likely by acting on P2Y(4) and P2Y(2) receptors; (2) induces I (K) increment through activation of the delayed rectifier K(+) channels and the high-conductance Ca(2+)-dependent K(+) channels, therefore both channels are implicated in the ATP-induced relaxation; and (3) this I (K) increment is mediated by prostaglandin production which in turns increase cAMP signaling pathway.

Inhibitory interactions between GABA(A)[induced by gamma-aminobutyric acid (GABA)] and P2X [activated by adenosine 5'-triphosphate (ATP)] receptors of myenteric neurones from the guinea pig small intestine were characterized using whole-cell recordings. Currents induced by GABA (I(GABA)) or ATP (I(ATP)) were inhibited by picrotoxin or pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid, respectively. Currents induced by GABA + ATP (I(GABA+ATP)) were only as large as the current induced by the most effective transmitter, revealing current occlusion. This occlusion requires maximal activation of at least one of these receptors. Sequential applications of neurotransmitters, and kinetic and pharmacological properties of I(GABA+ATP) indicate that they are carried through both GABA(A) and P2X channels. ATP did not affect I(GABA) in neurones: (i) in which P2X channels were not present; (ii) after inhibiting P2X channels with Ca2+ (iii) in the presence of pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid, a P2X receptor antagonist; (iv) after P2X receptor desensitization or (v) at I(ATP) reversal potential. Similarly, GABA did not affect P2X-mediated currents in neurones: (i) in which GABA(A) channels were not present; (ii) in the presence of picrotoxin, a GABA(A) channel blocker; (iii) after GABA(A) receptor desensitization or (iv) at the I(GABA) reversal potential. Current occlusion occurred as fast as current activation and it was still present in the absence of Ca2+, at 11 degrees C, after adding to the pipette solution a cocktail of protein kinase inhibitors (staurosporine + genistein + K-252a), after substituting the GTP in the pipette with GDP-beta-S and after treating the cells with N-ethylmaleimide. Taken together, all of these results are consistent with a model of cross-inhibition between GABA(A) and P2X.

Our results suggested that 5-HT was involved in the development of AI-AHR to ACh in guinea-pigs. Specifically, 5-HT(2A), 5-HT(4) and 5-HT(7) receptors seem to be particularly involved in this phenomenon. Participation of 5-HT might probably be favoured by the enhancement of the PNECs 5-HT content observed after sensitization.

We recorded whole-cell ion currents induced by gamma-aminobutyric acid (I(GABA)) and serotonin (I(5-HT)) to investigate and characterize putative interactions between GABA(A) and 5-HT(3) receptors in myenteric neurons from the guinea pig small intestine. I(GABA) and I(5-HT) were inhibited by bicuculline and ondansetron, respectively. Currents induced by the simultaneous application of both, GABA and 5-HT (I(GABA+5-HT)) were significantly lower than the sum of I(GABA) and I(5-HT), indicating the existence of a current occlusion. Such an occlusion was observed when GABA(A) and 5-HT(3) receptors are virtually saturated. Kinetics, and pharmacological properties of I(GABA+5-HT) indicate that they are mediated by activation of both, GABA(A) and 5-HT(3) channels. GABA did not alter I(5-HT) in neurons without GABA(A) channels, in the presence of bicuculline (a GABA(A) receptor antagonist) or at the reversal potential for I(GABA). Similarly, 5-HT did not modify I(GABA) in neurons in which 5-HT(3) channels were absent, after inhibiting 5-HT(3) channels with ondansetron (a 5-HT(3) receptor antagonist) or at the reversal potential for I(5-HT). Current occlusion was observed as soon as GABA(A) and 5-HT(3) channels were being activated, in the absence of Ca(2+), at low temperature (11 degrees C), and after adding staurosporine (a protein kinase inhibitor) to the pipette solution. Our proposal is that GABA(A) and 5-HT(3) channels are organized in clusters and within these, both channels can cross-inhibit each other, likely by allosteric interactions between these proteins.