Facilitation and delayed release at about 0 degree C at the frog neuromuscular junction: effects of calcium chelators, calcium transport inhibitors, and okadaic acid.

1. We studied two-pulse facilitation and delayed release at 0 degree C, because at low temperature facilitation is enhanced and extended whereas delayed release is increased. Our major goal was to test, by a number of approaches, the residual Ca2+ hypothesis of facilitation and delayed release. 2. As we increased the interval between pulses from 30 to 100-200 ms facilitation declined steeply. As we lengthened the interval further facilitation declined more slowly. In our entire series facilitation was still seen at 700 ms, in some preparations facilitation was apparent at 2 s. 3. We measured delayed release in preparations in which excitation-contraction was uncoupled. The decline in the rate of delayed release following the endplate potential (EPP) is similar to the decay of facilitation, both at 0 and 22 degrees C. 4. When we replaced the Ca2+ in the Ringer by Sr2+, facilitation persisted for a longer time, there was significant facilitation 2 s after an EPP. Delayed release also continued longer; the time courses for the decline of facilitation and delayed release were very similar. 5. We measured delayed release after EPPs triggered by electrotonic depolarization in isotonic CaCl2 solution or in Ringer in which the Na+ was replaced by methylamine (these solutions also contained 3,4-diaminopyridine). The time course of delayed release was very similar to that in Ringer. 6. We found that delayed release also facilitated, in the sense that the number of delayed releases, and the rate at which they were released, increased markedly after a second or third closely spaced EPP. The facilitation of delayed release and of EPPs were quantitatively similar. 7. We soaked preparations for 2 h in 200 microM bis-(aminophenoxy) ethane-tetraacetic acid (BAPTA/AM), a cell permeable Ca2+ chelator. In about one-half of these preparations facilitation was clearly diminished, judging from the EPPs evoked by a series of four to five stimuli at 30-ms intervals. The summed results from those preparations in which facilitation was reduced at 30 ms showed that it was also reduced at longer intervals. There was a comparable shortening in delayed release. Facilitation was significantly reduced when we pretreated with ethylene glycol bis-(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (EGTA/AM), another cell-permeable Ca2+ chelator. 8. It has been reported that in BAPTA loaded preparations facilitation during trains of EPPs transiently reappears after exposure to the ionophore X-537A, which presumably elevates intracellular [Ca2+].(ABSTRACT TRUNCATED AT 400 WORDS)