Functional expression of P2X1, P2X4 and P2X7 purinergic receptors in human monocyte-derived macrophages.

This study sought to examine the co-expression of the following purinergic receptor subunits: P2X1, P2X1del, P2X4, and P2X7 and characterize the P2X response in human monocyte-derived macrophages (MDMs). Single-cell RT-PCR shows the presence of P2X1, P2X1del, P2X4, and P2X7 mRNA in 40%, 5%, 20%, and 90% of human MDMs, respectively. Of the studied human MDMs, 25% co-expressed P2X1 and P2X7 mRNA; 5% co-expressed P2X4 and P2X7; and 15% co-expressed P2X1, P2X4, and P2X7 mRNA.

Physiological Concentrations of Zinc Have Dual Effects on P2X Myenteric Receptors of Guinea Pig.

We, hereby, characterize the pharmacological effects of physiological concentrations of Zinc on native myenteric P2X receptors from guinea-pig small intestine and on P2X2 isoforms present in most myenteric neurons. This is the first study describing opposite effects of Zinc on these P2X receptors. It was not possible to determine whether both effects were concentration dependent, yet the inhibitory effect was mediated by competitive antagonism and was concentration dependent.

Recreating Intestinal Peristalsis in the Petri Dish.

Here we describe a culture technique of cells dissociated from the external muscularis of the guinea pig small intestine, which allows us to maintain all the elements involved in the intestinal peristaltic reflex. After a few days in culture, these cells reorganize to form a small group of cells that permit the generation of pacemaker activity, spontaneous contractions, and the development of inhibitory and excitatory junction potentials in the petri dish, all elements involved in the peristaltic reflex. Therefore, these co-cultures are suitable to study the cellular and molecular aspects related to the development, maintenance, and modulation of motor intestinal functions.

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.

Heteromeric channels with different phenotypes are generated when coexpressing two P2X2 receptor isoforms.

To investigate if channels with different stoichiometry are formed from P2X2 receptor isoforms during their heterologous co-expression. The two-electrode voltage-clamp technique was used to measured ATP induced currents in Xenopus laevis oocytes. We used a mutant (P2X2-2bm) because its ATP sensitivity is lower than P2X2-2b receptors, which highlights the differences with its splice variant P2X2-1a.

Expression of P2X3 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.

P2X4 subunits are part of P2X native channels in murine myenteric neurons.

The aim of the present study was to investigate if P2X4 receptors are expressed in murine myenteric neurons and if these receptors contribute to form functional channels in the neuronal membrane by using molecular and electrophysiological techniques. The whole-cell recording technique was used to measure membrane currents induced by ATP (I(ATP)) in myenteric neurons. Compared with recombinant P2X4 receptor-channels (reported by others in a previous study), native myenteric P2X receptors have a relative lower sensitivity for ATP (EC₅₀=102 µM) and α,β methylene ATP (not effect at 30 or 100 µM).

Dibenzo[1,2,5]thiadiazepines are non-competitive GABAA receptor antagonists.

A new process for obtaining dibenzo[c,f][1,2,5]thiadiazepines (DBTDs) and their effects on GABA(A) receptors of guinea pig myenteric neurons are described. Synthesis of DBTD derivatives began with two commercial aromatic compounds. An azide group was obtained after two sequential reactions, and the central ring was closed via a nitrene to obtain the tricyclic sulfonamides (DBTDs).

P2X7 receptors contribute to the currents induced by ATP in guinea pig intestinal myenteric neurons.

The whole-cell configuration, several pharmacological tools, and single-cell RT-PCR were used to investigate the contribution of P2X7 subunits to the ATP-induced currents (I(ATP)) in guinea pig myenteric neurons. I(ATP) was recorded in the great majority of tested neurons. ATP concentration-response curve (0.

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.

Two suramin binding sites are present in guinea pig but only one in murine native P2X myenteric receptors.

Whole-cell patch clamp recordings were used to characterise the physiological and pharmacological properties of P2X receptors of mouse and guinea pig myenteric neurons from the small intestine. ATP application induced a rapid inward current in 95% of recorded neurons of both species when were voltage clamped at -60 mV. Concentration-response curves for ATP (1-3000 microM) yielded EC(50) values of 114 and 115 microM for mouse and guinea pig myenteric neurons, respectively, with a Hill coefficient value of 1.

Cross-talking between 5-HT3 and GABAA receptors in cultured myenteric neurons.

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.

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

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.

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

Inhibitory interactions between 5-HT subtype 3 (5-HT(3)) and P2X receptors were characterized using whole cell recording techniques. Currents induced by 5-HT (I(5-HT)) and ATP (I(ATP)) were blocked by tropisetron (or ondansetron) and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid, respectively. Currents induced by 5-HT + ATP (I(5-HT+ATP)) were only as large as the current induced by the most effective transmitter, revealing current occlusion.