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Quantal analysis of 5-hydroxytryptamine release from mouse pancreatic -cells

Paul A. Smith, Peter Proks and Frances M. Ashcroft

1. A combination of patch-clamp, amperometric and fluorimetric methods were used to investigate the Ca²⁺ dependence and kinetics of secretion from pancreatic -cells elicited by voltage-gated Ca²⁺ entry.

2. Whether measured by the change in cell capacitance or by amperometric detection of 5-hydroxytryptamine (5-HT) release, the voltage dependence of the amount of secretion mirrored that of both the peak Ca²⁺ current and Ca²⁺ entry.

3. The magnitude of secretion elicited by a single pulse could be entirely accounted for by a readily releasable pool of ~200 vesicles. Neither depression nor potentiation of release was observed with 0·1 Hz pulse trains.

4. Transient amperometric currents were detected, which occurred independently of each other and were attributed to the fusion of single vesicles.

5. The time course of the macroscopic amperometric current could be accurately reconstructed by convolution of the all-events latency distribution and the unitary amperometric current.

6. In response to membrane depolarisation, secretion was initiated with a variable latency: ~95 % of the first secretory events occurred at least 50 ms after the start of the voltage pulse (and Ca²⁺ influx). Secretion fell rapidly on membrane repolarisation, even though the average intracellular calcium concentration ([Ca²⁺]_i) was still elevated.

7. The [Ca²⁺] in the locality of the release site was estimated from the all-events latency distribution. [Ca²⁺] rose during a voltage pulse and secretion was elicited at > 0·4 µM and peaked at ~2-10 µM.

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