Rosa Espinosa-Luna

Functional interactions between nicotinic and P2X channels in short‐term cultures of guinea‐pig submucosal neurons

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.

P2x‐purinoceptors of myenteric neurones from the guinea‐pig ileum and their unusual pharmacological properties

1 Whole‐cell and outside‐out patch clamp recordings were used to characterize the physiological and pharmacological properties of the P2x‐purinoceptors of myenteric neurones from the guinea‐pig ileum. 2 Adenosine 5′‐triphosphate (ATP) and analogues (1–3000μm) evoked a rapid inward current in > 90% of all recorded neurones. The reversal potential of this current was dependent on the extracellular sodium concentration, at +14 ± 1.9, 0 ± 1.6 and −12 ± 1 mV for 166, 83 and 42 mM of sodium, respectively. The fast activation and inactivation of this current occurred even when guanosine 5′‐triphosphate (GTP) was omitted from the pipette solution or substituted with an equimolar concentration of guanosine 5′‐o‐[2‐thiotriphosphate] (GTP‐γ‐S). Single channel currents were observed when these outside‐out membrane patches were exposed to ATP (10–30 μm). These channels have a unitary conductance of about 17 picosiemens. 3 The rank‐order of potency of the agonists used to induce the whole‐cell currents was: ATP‐γ‐S = ATP = 2‐methylthio‐ATP (2‐Me‐S‐ATP) > > α,β‐methylene ATP = β,γ‐methylene ATP; adenosine and uridine 5′‐triphosphate (UTP) (up to 1 mM) were inactive. 4 Pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS) (1–30μm) antagonized the effects of ATP (1 mM) with an IC50 of 4 μm. α,β‐Methylene ATP (100 μm) did not affect the ATP (30 μm)‐induced current. Cibacron Blue 3GA increased the ATP activated cationic current whereas Basilen Blue E‐3G had a very weak antagonistic effect (IC50 μ 3 mM). Suramin potentiated the currents induced by ATP through a mechanism that was independent of its inhibitory effect on ectonucleotidase activity, as suramin also potentiated the effect of α,β‐methylene ATP (an ATP analogue that is resistant to nucleotidases). 5 In conclusion, the myenteric P2x‐purinoceptor shares some properties with other purinoceptors in particular with the P2times4‐ and P2X6‐purinoceptors. This receptor has also some unusual pharmacological properties suggesting that myenteric neurones express a novel subtype of P2x‐purinoceptors. The properties of this receptor, however, might be a result of the combination of two or more of the homomeric purinoceptors so far characterized.

Melatonin modulates cholinergic transmission by blocking nicotinic channels in the guinea-pig submucous plexus

Melatonin, a hormone produced and released by the pineal gland is also synthesized by cells of the gastrointestinal wall, where it might be a local regulator of gut functions. In this study, we investigated the possible role of melatonin as a modulator of the enteric nervous system. Intracellular recordings were made in neurons of the submucosal plexus from the guinea-pig ileum to measure the melatonin effects on their electrophysiological properties. Melatonin did not alter the membrane potential, the membrane resistance and the noradrenergic inhibitory postsynaptic potentials. However, melatonin (30-3000 microM) reversibly decreased the amplitude of nicotinic excitatory postynaptic potentials (EPSPs) in a concentration-dependent manner (IC50 = 247 microM). These actions of melatonin were not modified by the presence of idazoxan and atropine indicating that they are not mediated by endogenous release of acetylcholine, noradrenaline, or by direct activation of alpha 2-adrenoceptors or muscarinic receptors. The superfusion of melatonin also blocked the nicotinic depolarizations induced by locally applied acetylcholine, indicating that at least part of its effects are postsynaptic. In voltage-clamp experiments, using the whole-cell configuration, melatonin also inhibited the nicotinic inward currents induced by acetylcholine (IACh) in a concentration-dependent manner (IC50 = 257 microM). Melatonin decreased the maximal IACh but did not affect the potency of acetylcholine to induce this current, indicating a noncompetitive antagonism. This effect was voltage-dependent. Our observations indicate that melatonin inhibits the fast EPSPs by directly and specifically blocking the nicotinic channels. The relative high concentrations of melatonin required to produce such an effect rules this out as one of its humoral actions. Such an effect, however, might be of physiological significance close to the cells that release melatonin in the gastrointestinal wall or in other organs.

Cross-inhibitory interactions between GABAA and P2X channels in myenteric neurones

Inhibitory interactions between GABAA[induced by γ‐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 (IGABA) or ATP (IATP) were inhibited by picrotoxin or pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid, respectively. Currents induced by GABA + ATP (IGABA+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 IGABA+ATP indicate that they are carried through both GABAA and P2X channels. ATP did not affect IGABA 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 IATP reversal potential. Similarly, GABA did not affect P2X‐mediated currents in neurones: (i) in which GABAA channels were not present; (ii) in the presence of picrotoxin, a GABAA channel blocker; (iii) after GABAA receptor desensitization or (iv) at the IGABA reversal potential. Current occlusion occurred as fast as current activation and it was still present in the absence of Ca2+, at 11 °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‐β‐S and after treating the cells with N‐ethylmaleimide. Taken together, all of these results are consistent with a model of cross‐inhibition between GABAA and P2X.

5-Hydroxytryptamine and atropine inhibit nicotinic receptors in submucosal neurons

The whole-cell recording technique was used to investigate the pharmacological properties of acetylcholine-activated ion channels of cultured submucosal neurons from guinea-pig small intestine. Acetylcholine induced whole-cell membrane currents (I(ACh)) in a concentration-dependent manner (EC(50)=79 microM). I(ACh) exhibited strong inward rectification, had a reversal potential of +19+/-2 mV (Na(+) outside, Cs(+) inside), was reversibly inhibited in a concentration-dependent manner by hexamethonium (EC(50)=5 microM) and atropine (EC(50)=1.6 microM), and was unaffected by alpha-bungarotoxin (30 nM). Atropine was less potent in inhibiting the currents induced by 30 microM acetylcholine than those induced by 1 mM acetylcholine. I(ACh) was mimicked by the current induced by nicotine (I(Nic); EC(50)=52 microM). I(Nic) was also blocked by atropine (EC(50)=1.7 microM) and hexamethonium (EC(50)=3.6 microM). 5-Hydroxytryptamine (5-HT) also inhibited I(ACh) in a concentration-dependent manner (EC(50)=180 microM) in the experiments carried out in the presence of a 5-HT(3) receptor antagonist. 5-HT had a similar inhibitory effect after the desensitization of 5-HT(3) receptors or in neurons with relative small 5-HT(3)-mediated currents. The inhibitory actions of hexamethonium, atropine, and 5-HT on I(ACh) were voltage-dependent. Thus, inhibition was significantly smaller for outward currents (recorded at +40 mV) than for inward currents (recorded at -60 mV). Our observations indicate that the I(ACh) of submucosal neurons are mediated by activation of nicotinic channels, which are blocked by atropine, 5-HT, and hexamethonium. The possibility that one of the 5-HT roles in the gastrointestinal tract might be to directly modulate nicotinic channels is discussed.

Retention of a new-defined intron changes pharmacology and kinetics of the full-length P2X2 receptor found in myenteric neurons of the guinea pig

P2X2 plays an important role in ATP signaling in guinea pig myenteric plexus. Here, we cloned and characterized three P2X2 isoforms expressed in myenteric neurons. RT/PCR was used to amplify the cDNA of P2X2 variants. These were expressed in Xenopus oocytes, and nucleotide-induced membrane currents were recorded with the two-electrode voltage clamp technique. Three P2X2 cDNAs were identified in myenteric single neurons, named P2X2-1, P2X2-2 and P2X2-4. Based on the analysis of the structural organization of these variants we predicted that P2X2-2 is the fully processed variant, which lead us to propose a new exon-intron arrangement of P2X2 receptor gene with 12 exons and 11 introns. In agreement with this new model, the intron 11 is retained in P2X2-1 and P2X2-4 variants by alternative splicing. Expression of P2X2-1, P2X2-2 and P2X2-4 were found in 92, 42 and 37%, respectively, out of 40 analyzed single neurons. P2X2-4 does not form functional channels, and homomeric channels formed by P2X2-1 and P2X2-2 have different pharmacological profile. Thus, the former receptor is more sensitive to ATP, BzATP, and PPADS, whereas, suramin inhibited both receptors in a biphasic- and monophasic-manner, respectively. α,β-meATP has very low efficacy on either channel. Furthermore, ionic currents mediated by P2X2-1 have slower desensitization than P2X2-2. These results indicate that P2X2-1 was the most common P2X2 transcript in myenteric neurons and displays significant phenotypical changes implicating that retention of the intron 11 plays a major role in ATP signaling in the intestinal myenteric plexus.

Selectivity of antagonists for the Cys-loop native receptors for ACh, 5-HT and GABA in guinea-pig myenteric neurons

The three most common Cys-loop receptors expressed by myenteric neurons are nACh, 5-HT3 and GABAA . To investigate the function of these proteins researchers have used channel inhibitors such as hexamethonium (antagonist of nACh receptors), ondansetron (antagonist of 5-HT3 receptors), picrotoxin and bicuculline (both antagonists of GABAA receptors). The aim of this study was to investigate the specificity of these inhibitors on Cys-loop receptors of primary cultured neurons obtained from the guinea-pig small intestine. The whole-cell configuration of the patch clamp techniques was used to record membrane currents induced by ACh (IACh ), 5-HT (I5-HT ) and GABA (IGABA ) in the absence and the presence of various concentrations of hexamethonium, ondansetron, picrotoxin or bicuculline. The three Cys-loop receptors present in enteric neurons are expressed independently and they do not cross-desensitized. Hexamethonium inhibited IACh without affecting I5-HT and IGABA . Ondansetron inhibited I5-HT and also IACh but did not affect IGABA . Picrotoxin and bicuculline inhibited I5-HT , IACh and IGABA with different potency, being the lowest potency on 5-HT3 receptors. All these inhibitory effects were concentration dependent and reversible. Our observations showed that except for hexamethonium, all other inhibitors used here show different degrees of selectivity, which has to be considered when these antagonists are used in experimental studies aimed to investigate the functions of these receptors. In particular, in tissues expressing nACh receptors because these are the targets of all other inhibitors used here. The low potency of picrotoxin and bicuculline to inhibit 5-HT3 receptors suggests that these receptors are heteromeric proteins.

Expression of P2X 3 and P2X 5 Myenteric Receptors Varies During the Intestinal Postnatal Development in the Guinea Pig

P2X3 receptor expression in various tissues appears to be modulated by age. In the present study, we used single cell RT-PCR to determine the number of P2X3 positive myenteric neurons at different stages of guinea pig postnatal development, and we tested if similar changes also occur to other myenteric P2X receptors. Moreover, we carried out whole-cell recordings using Patch Clamp techniques to determine possible changes in P2X receptors sensitivity to ATP and α,β-methylene ATP (α,β-meATP) between newborn and adult animals. Our data indicate that P2X3 subunit transcripts are present in a larger number of myenteric neurons from newborn guinea pigs whereas P2X5 mRNA is found more frequently in adults. Expression of P2X2 and P2X4 transcripts does not change during postnatal development. In newborn animals, virtually all neurons expressing P2X3 also expressed P2X2 transcripts. This is important because these two subunits are known to form heteromeric channels. ATP potency to activate P2X receptors in neurons of both newborn and adult animals was the same. α,β-meATP, a known P2X3 receptor agonist, induces only a marginal current despite the fact of the higher presence of P2X3 subunits in newborns. These findings imply that P2X3 subunits are mainly forming heteromeric, α,β-meATP insensitive channels perhaps because P2X3 contributes with only one subunit to the heterotrimers while the other subunits could be P2X2, P2X4, or P2X5.

Functional interactions between nicotinic and P2X receptors in celiac ganglia neurons

Here we characterized the cross-inhibitory interactions between nicotinic and P2X receptors of celiac neurons from the guinea pig by recording whole-cell currents induced by 1mM ACh (I(ACh)), 1mM ATP (I(ATP)) and by the simultaneous application of both agonists (I(ACh)(+ATP)). I(ACh) and I(ATP) were inhibited by hexamethonium (nicotinic channel blocker) and PPADS (P2X receptor antagonist), respectively. The amplitude of I(ACh)(+ATP) was equal to the current induced by the most effective agonist, indicating a current occlusion. Various observations indicate that I(ACh)(+ATP) is carried out through both nicotinic (nACh) and P2X channels: i) I(ACh)(+ATP) desensitisation kinetics were in between that of I(ACh) and I(ATP); ii) application of ATP+ACh, decreased I(ACh) and I(ATP), whereas no cross-desensitisation was observed between nACh and P2X receptors; iii) ATP did not affect I(ACh) in the presence of PPADS or after P2X receptor desensitisation; and iv) ACh did not affect I(ATP) when nACh channels were blocked with hexamethonium or after nACh receptor desensitisation. Current occlusion is not mediated by activation of metabotropic receptors as it is: i) voltage dependent (was not observed at + 5 mV); ii) present at low temperature (10 degrees C) and after inhibition of protein kinase activity (with staurosporine); and iii) absent at 30 microM ATP and 30 microM ACh (concentrations that should activate metabotropic receptors). In conclusion, current occlusion described here is similar to the previously reported myenteric neurons. This occlusion is likely the result of allosteric interactions between these receptors.

Two P2X1 receptor transcripts able to form functional channels are present in most human monocytes

To characterize the presence and general properties of P2X1 receptors in single human monocytes we used RT-PCR, flow cytometry, and the patch-clamp and the two-electrode voltage-clamp techniques. Most human monocytes expressed the canonical P2X1 (90%) and its splicing variant P2X1del (88%) mRNAs. P2X1 receptor immunoreactivity was also observed in 70% of these cells. Currents mediated by P2X1 (EC50=1.9±0.8µm) and P2X1del (EC50 >1000µm) channels, expressed in Xenopus leavis oocytes, have different ATP sensitivity and kinetics. Both currents mediated by P2X1 and P2X1del channels kept increasing during the continuous presence of high ATP concentrations. Currents mediated by the native P2X1 receptors in human monocytes showed an EC50=6.3±0.2µm. Currents have kinetics that resemble those observed for P2X1 and P2X1del receptors in oocytes. Our study is the first to demonstrate the expression of P2X1 transcript and its splicing variant P2X1del in most human monocytes. We also, for the first time, described functional homomeric P2X1del channels and demonstrated that currents mediated by P2X1 or P2X1del receptors, during heterologous expression, increased in amplitude when activated with high ATP concentrations in a similar fashion to those channels that increase their conductance under similar conditions, such as P2X7, P2X2, and P2X4 channels.