Regulation of secretory granule recruitment and exocytosis at rat neurohypophysial nerve endings

1. Time-resolved cell membrane capacitance (C-m) measurements were used in combination with fura-2 microfluorometry under whole-cell patch clamp recording to investigate the kinetics and Ca2+ sensitivity of exocytotic granule fusion evoked by depolarizing stimuli at single, isolated nerve endings of the rat neurohypophysis. 2. Single step depolarizations or trains of depolarizing pulses evoked voltage-dependent, inward Ca2+ currents (I-Ca) and induced both Ca2+-dependent and Ca2+-independent changes in C-m. Three distinct C-m responses were observed and were differentiated by their kinetics and Ca2+ sensitivity: a non-exocytotic transient (Delta C-m,C-t) and an exocytotic C-m 'jump' (Delta C-m,C-j) and a slower, often latent, exocytotic C-m rise (Delta C-m,C-s) that outlasted the depolarizing pulse stimulus . 3. The Delta C-m,C-t was characterized by a rapid, transient component observed in 70% of nerve endings and a voltage-activation relationship that preceded that of the I-Ca. The amplitude and kinetics of the Delta C-m,C-t were unaffected by I-Ca block by Cd2+, Ca2+ load reduction, or alterations in intracellular Ca2+ buffering. 4. In contrast to the Delta C-m,C-t, both the Delta C-m,C-J and Delta C-m,C-S were Ca2+ dependent as evidenced by their sensitivity to Cd2+ block of I-Ca, intraterminal application of 10 mM BAPTA and reduced [Ca2+](o) or replacement of Ca2+ as the charge carrier with Ba2+ 5. The Delta C-m,C-J was proportional to depolarization-evoked Ca2+ influx with initial exocytotic rate of approximately 350 granule fusions s(-1). The amplitude of the Delta C-m,C-J rose exponentially (tau = 40 ms) and approached an asymptote (15.5 fF) with longer duration depolarizations indicating the fusion from and depletion of an immediately releasable pool (IRP) estimated at nineteen docked and primed secretory granules. 6. The Delta C-m,C-S was induced by the application of repetitive long duration pulses and defined as the exocytosis of secretory granules from a readilgr releasable granule pool (RRP). The Delta C-m,C-8 response occurred only after exceeding a [Ca2+](i) threshold value and rose thereafter in proportion to Ca2+ influx with a mean initial secretory rate of 36 granule fusions s(-1). The mean latency for Delta C-m,C-S activation was 850 ms following the initiation of the step depolarizations. The Delta C-m,C-S response magnitude, reflecting the size of the RRP, was dependent on the resting [Ca2+](i) and the nerve ending size, and was depletable using repetitive depolarizations of long duration. 7. Recruitment into and release from the RRP and IRP were differentially sensitis e to changes in intraterminal Ca2+ buffering conditions. For example, introduction of 5 mns EGTA was shown to have no effect on the evoked TRP but significantly reduced the RRP. In comparison, diminishment of the endogenous Ca2+ buffering capacity of nerve endings by treatment with the mitochondrial Ca2+ uniporter blocker Ruthenium Red (10 mu M) potentiated the RRP size but had no significant effect on the IRP size. 8. The present study indicates that the Ca2+-dependent recruitment of and release from functionally distinct pools of peptide-containing secretory granules in combination with the [Ca2+](i) regulatory properties of neurohypophysial nerve endings may explain both the depletion of peptide release under prolonged stimulus and the potentiation of peptide release observed to occur during recurrent phasic action potential activity in this system.