Estudo das propriedades biofísicas e farmacológicas do poro associado ao receptors P2X7 e poros de características similares. / Study of the biophysical and pharmacological properties of the pore associated with receiver P2X7 and pores of similar characteristics.

AUTOR(ES)
DATA DE PUBLICAÇÃO

2006

RESUMO

The P2X7 receptor (EC50= ~100μM) induces cell membrane permeabilization to molecules up to 900 Da when activated by high ATP concentrations. So far, the correlation between the opening of low conductance cationic channel (8 pS) formed after stimulation with low ATP concentrations (EC50= ~1 μM) and the opening of P2X7 related pore is unclear. To date, there are at least two hypotheses about this question: 1) the pore formation would be associated to gradual increase of permeability (dilation) of the cationic channels; 2) an intracellular second messenger wouldl be activated to act on P2X7 receptor itself or acting on a distinct pore. We do not know which protein is responsible for the pore formation, but there are descriptions of activities of other pores with biophysical characteristics similar to P2X7 pore that could be associated with the P2X7 related pore itself. In this context, we have investigated whether P2X7 related pore and channel are the same structure and if gap junctions hemichannels would be the P2X7 related pore. For this purpose, we used the patch clamp technique in 2BH4 cells and mice peritoneal macrophages, which express the P2X7 receptor. Using cell attached configuration, we have characterized biophysically and pharmacologically the pore opening in 2BH4 cells. The single currents recorded in these cells after application of 1mM ATP, showed conductance of 445 18pS, being blocked by P2X7 receptor antagonists. Using different patch clamp configurations, we demonstrated that the ~8pS ionic channel and the ~400pS pore activities are independently blocked by calmidazolium and 5-(N, Nhexamethylene) - amiloride (HMA), known channel and pore blockers, respectively. In addition, we have observed that, the pores opened in cell attached or whole cell condition only when the cytoplasmatic contents were preserved. Moreover, we have investigated if the calcium would be the second messenger involved in the P2X7 pore opening. Using decreased Ca2+ concentration in the pipette, in whole cell configuration, we observed reduction of P2X7 pore opening. This effect was reproduced by incubating the cells with Ca2+ chelator, BAPTA-AM, in cell attached configuration. Taking in account these results, we have investigated whether the massive increase in the intracellular calcium concentration in cells that express (2BH4) or do not express the P2X7 receptor (HEK-293 e IT45-RI) would induce P2X7 related pore opening. In 2BH4 cells, the stimulation with 10μM ionomycin using cell attached configuration induced activities of pore formation similar to the P2X7 pore presenting conductance of 421 14 pS. Importantly, HEK-293 cells also presented the pore formation (conductance of 415 10,4 pS) when stimulated with ionomycin. Thus, demonstrating that intracellular Ca2+ may activate the formation of a pore distinct from P2X7. Based on the fact that the new pore found presents a cut off similar to that of P2X7 pore as well as to the pore formed by the maitotoxin and hemichannels, we have investigated if hemichannels present in thymocytes have functional activities and if they could be the P2X7 pore itself. We observed that hemichannels activity is modulated by glicocorticoid application and is involved with thymocyte death. In addition, we concluded that the hemichannel opening is not related with the P2X7 pore activity. In according to our data, we suggest that there is, in the organism, a pore family biophysically related. Briefly, our data have indicated that these pores can be the same entity activated by different stimulus and distinct intracellular signaling pathways, however, presenting similar biophysical properties.

ASSUNTO(S)

receptors, purinergic p2 adenosine triphosphate biologia molecular receptores purinérgicos p2 electrophysiology eletrofisiologia trifosfato de adenosina

ACESSO AO ARTIGO

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