ADP-induced rapid inward currents through Ca(2+)-permeable cation channels in mouse, rat and guinea-pig megakaryocytes: a patch-clamp study.

AUTOR(ES)

Kawa, K

RESUMO

1. The rapid inward currents in mouse megakaryocytes evoked by adenosine diphosphate (ADP), a ubiquitous platelet-activating substance, were studied. Time and current resolution were improved by using patch-clamp recording and an extracellular fast perfusion ("Y tube') technique. 2. Application of ADP (40 microM) to megakaryocytes immersed in physiological saline evoked rapid inward currents (80-340 pA at -42 mV). The cellular responses to a second ADP application were markedly reduced, but in the absence of external Ca2+, responses to repeated ADP application were maintained and did not deteriorate. 3. The ADP-induced current recorded in Ca(2+)-free external media showed short latency (less than 20 ms) and approximately exponential decay (time constant, 300-500 ms), which was independent of the holding potential and seemed to be caused mainly by receptor desensitization; it took over 5.5 min for complete recovery. 4. The ADP concentration response relationship of the megakaryocytes revealed that the half-maximal concentration and the Hill coefficient were 12.6 microM and 1.4, respectively. 5. An ion replacement experiment showed that the ADP-induced currents could be carried by Na+, Cs+ and K+, but not Cl-, and the cation channels were permeable to Ca2+, Ba2+ and Mg2+. 6. Neither Ca2+ chelators (10 mM EGTA and 10 mM BAPTA) nor hydrolysis-resistant guanine nucleotides (2 mM GDP-beta-S and 0.4 mM 5'-guanylylimidodiphosphate) in the internal saline affected the rapid responses to ADP, and ADP-induced currents were recorded in excised membrane patches, suggesting that the ADP receptor site and the molecular structure forming the cation channel are tightly coupled and/or parts of the same molecule. 7. In rat and guinea-pig megakaryocytes, ADP-induced rapid inward currents showed the same properties as in mouse megakaryocytes.

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