The fluorescent indicator fura-2 was used to measure cytoplasmic calcium in presynaptic terminals in the crayfish Procambarus clarkii under conditions that raise intracellular sodium to examine whether sodium can elevate intracellular calcium concentration ([Ca2+]i) or prolong its efflux and thus influence the magnitude and duration of posttetanic potentiation (PTP). Sodium was elevated in presynaptic terminals at rest by either (1) injection of sodium into the excitatory axon, (2) application of veratridine to open sodium channels, or (3) addition of ouabain to block Na/K exchange, with [Ca2+]i increasing by either 430, 400, or 180 nM, respectively. Intracellular calcium concentration increased only when external calcium was present, indicating that calcium influx occurred through Na/Ca exchange. Additionally, ouabain enhanced excitatory junctional potentials (EJPs) eightfold. Elevation of sodium using a high-frequency stimulation in zero-calcium Ringer's did not elevate [Ca2+]i during the train or immediately afterward when calcium-containing Ringer's was re- introduced. This indicates that a physiological sodium load does not release calcium from internal stores or reverse Na/Ca exchange to levels where [Ca2+]i accumulation is detectable. We examined the ability of sodium to interfere with calcium efflux from presynaptic terminals by loading boutons with both sodium and calcium or calcium alone using high-potassium depolarization. Elevation of internal sodium slowed calcium efflux from the terminal (12.3 min) compared to calcium removal without a sodium load (4.0 min). When sodium loading was increased during a tetanus by application of ouabain, the time constants for decay of EJP potentiation, 17.3 min, and for [Ca2+]i, 35 min, were longer than control values, 4.4 min and 5.8 min, respectively. In addition, using lithium to inhibit the efflux of calcium by Na/Ca exchange following a PTP-inducing train also lengthened the decay of [Ca2+]i to 15.7 min. Intracellular sodium accumulation in presynaptic terminals slows the efflux of calcium through Na/Ca exchange, and may therefore augment and prolong PTP.
SUMMARY1. Spontaneous and evoked transmitter release at the crayfish neuromuscular junction were potentiated in response to photolytic release of calcium from the 'caged' calcium compound DM-nitrophen, which had previously been injected into presynaptic terminals.2. The amount of calcium released from DM-nitrophen photolysis depends on the concentration of DM-nitrophen, its photoproducts, Ca2+, Mg2+, H+, ATP and the cell's native buffer. Since none of these are known in the crayfish terminal, the study was conducted in a qualitative fashion.3. Photolytic release of calcium from DM-nitrophen increased excitatory junctional potentials (EJPs) by a range of 2-31 times over control values and the miniature excitatory junctional potential (MEJP) frequency increased from resting values of 1-10 quanta/s to 3000-11000 quanta/s. 4. Extracellular calcium was not required for the light-evoked asynchronous release of transmitter. Calcium-bound DM-nitrophen previously pressure injected into crayfish presynaptic terminals increased the MEJP frequency from resting values of 1-8 quanta/s to 800-10000 quanta/s during photolysis in a calcium-free cobalt Ringer solution.5 R. M. MULKEY AND R. S. ZUCKER pulse, presumably due to the active extrusion of calcium from the presynaptic terminals.8. During photolysis of DM-nitrophen, the time courses of changes in EJP amplitude and MEJP frequency were different, indicating that the two measures of transmitter release were not linearly related.9. MEJP frequency and EJP amplitudes during DM-nitrophen photolysis were fitted to a 'non-linear summation model' in which photolytically released calcium sums with calcium entering during an action potential to evoke transmitter release with a calcium co-operativity of five.
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