Cytoplasmic Ca2+ oscillation in rat megakaryocytes evoked by a novel type of purinoceptor

1. The responses of megakaryocytes isolated from rat bone marrow to externally applied adenosine triphosphate (ATP) were investigated in the whole-cell mode by the use of nystatin perforated patch-clamp technique. 2. ATP at 1-100 microM evoked periodic outward currents at a holding potential of -40 mV. The reversal potential of the currents was close to K+ equilibrium potential (EK) and the K+ channel blockers such as quinine and quinidine suppressed the currents, indicating that the outward 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 obtained by perforated and conventional patch-clamp techniques. The crucial difference between our results and previous results was due to the intracellular perfusion with internal solution containing a high concentration of EGTA by which both current shape and concentration response were modified. 4. The membrane permeable Ca2+ chelator, 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (acetoxy methyl ester; BAPTA AM), inhibited the K+ current concentration dependently, suggesting that ATP-induced oscillatory K+ currents are caused by changes in cytoplasmic free Ca2+ concentration ([Ca2+]i). 5. With increasing ATP concentration, the frequency and the maximum amplitude of K+ current oscillation increased and the latency of current, which is the period required to activate the first K+ current after ATP application, decreased. 6. ADP, 2-methylthio-ATP and ATP-gamma-S could also evoke the periodic K+ currents, but adenosine, uridine triphosphate (UTP) and alpha-beta-methylene adenosine 5'-triphosphate (AMP-CPP) failed. 2-Methylthio-ATP was the most potent agonist; next was ADP which showed a 10-30 times stronger effect than ATP. Cross-desensitization was observed between ATP and ADP, but not between ATP or ADP and thrombin. 7. Extracellular Ca2+ was not required for the ATP-induced K+ current activation, indicating that Ca2+ released from intracellular pools induced the oscillatory response. In addition, the agonist potency increased when extracellular Ca2+ concentration ([Ca2+]o) decreased, suggesting that the principal agonists might be ATP4- and ADP3-. 8. The results suggest the presence of a novel subtype of purinoceptor in the megakaryocyte plasma membrane which induces cytoplasmic Ca2+ oscillation and evokes periodic K+ current flux.