REGULATION OF QUANTAL TRANSMITTER SECRETION BY ATP AND PROTEIN-KINASES AT DEVELOPING NEUROMUSCULAR SYNAPSES

The effects of endogenously released ATP on the maturation of developi ng neuromuscular synapses were investigated in Xenopus nerve-muscle co -cultures. The potentiating action of ATP (1 mM) on spontaneous acetyl choline release was inhibited by P2-purinoceptor antagonists suramin ( 0.3 mM) and reactive blue 2 (RB-2, 3 mu M) in day 1 cultures. Bath app lication of suramin (10 mu M) or RB-2 in day 1 cultures and prolonged treatment for 2 days dramatically decreased the amplitude of both spon taneous synaptic currents (SSCs) and evoked synaptic currents (ESCs) i n the same cultures on day 3. Chronic treatment with 8-cyclopentyltheo phylline (4 mu M) or 6-cyano-2,3-dihydroxy-7-nitroquinoxaline (CNQX, 1 0 mu M), P1-purinoceptor and glutamate receptor antagonists respective ly, did not exert such an inhibitory effect. Chronic treatment with su ramin or RB-2 for 2 days had no significant effect on the amplitude of either iontophoretic acetylcholine-induced whole-cell currents or sin gle acetylcholine channel measurements in 3-day-old cultured myocytes. In addition, prolonged treatment for 2 days with various kinase inhib itors such as H-8 (10 mu M), KN-62 (5 mu M) and H-7 (10 mu M) also dec reased the amplitudes of both spontaneous and evoked synaptic currents in natural synapses, but not those of iontophoretic acetylcholine-ind uced currents. Furthermore, suramin and these protein kinase inhibitor s also decreased the amplitude of spontaneous synaptic currents in man ipulated synapses of 'vacated' nerve terminals. The results suggest th at endogenously released ATP, acting in concert with various protein k inases, is involved in the maintenance and/or development of the quant um size of synaptic vesicles at embryonic neuromuscular synapses.