CYTOPLASMIC CA2+ OSCILLATION IN RAT MEGAKARYOCYTES EVOKED BY A NOVEL TYPE OF PURINOCEPTOR

1. The responses of megakaryocytes isolated from rat bone marrow to ex ternally applied adenosine triphosphate (ATP) were investigated in the whole-cell mode by the use of nystatin perforated patch-clamp techniq ue. 2. ATP at 1-100 mum evoked periodic outward currents at a holding potential of - 40 mV. The reversal potential of the currents was close to K+ equilibrium potential (E(K)) and the K+ channel blockers such a s quinine and quinidine suppressed the currents, indicating that the o utward currents are predominantly carried by K+. 3. Since it has been reported that adenosine diphosphate (ADP) evoked monophasic K+ current using a conventional whole-cell recording, we compared the results ob tained by perforated and conventional patch-clamp techniques. The cruc ial difference between our results and previous results was due to the intracellular perfusion with internal solution containing a high conc entration of EGTA by which both current shape and concentration respon se were modified. 4. The membrane permeable Ca2+ chelator, ,2-bis(O-am inophenoxy)ethane-N,N,N',N'-tetraacetic acid (acetoxy methyl ester; BA PTA AM), inhibited the K+ current concentration dependently, suggestin g that ATP-induced oscillatory K+ currents are caused by changes in cy toplasmic free Ca2+ concentration ([Ca2+]i) . 5. With increasing ATP c oncentration, the frequency and the maximum amplitude of K+ current os cillation increased and the latency of current, which is the period re quired to activate the first K+ current after ATP application, decreas ed. 6. ADP, 2-methylthio-ATP and ATP-gamma-S could also evoke the peri odic K+ currents, but adenosine, uridine triphosphate (UTP) and alpha- beta-methylene adenosine 5'-triphosphate (AMP-CPP) failed. 2-Methylthi o-ATP was the most potent agonist; next was ADP which showed a 10-30 t imes stronger effect than ATP. Cross-desensitization was observed betw een ATP and ADP, but not between ATP or ADP and thrombin. 7. Extracell ular Ca2+ was not required for the ATP-induced K+ current activation, indicating that Ca2+ released from intracellular pools induced the osc illatory response. In addition, the agonist potency increased when ext racellular Ca2+ concentration ([Ca2+]o) decreased, suggesting that the principal agonists might be ATP4- and ADP3-. 8. The results suggest t he presence of a novel subtype of purinoceptor in the megakaryocyte pl asma membrane which induces cytoplasmic Ca2+ oscillation and evokes pe riodic K+ current flux.