REGULATION OF SECRETORY GRANULE RECRUITMENT AND EXOCYTOSIS AT RAT NEUROHYPOPHYSEAL NERVE-ENDINGS

1. Time-resolved cell membrane capacitance (C-m) measurements were use d in combination with fura-2 microfluorometry under whole-cell patch c lamp recording to investigate the kinetics and Ca2+ sensitivity of exo cytotic granule fusion evoked by depolarizing stimuli at single, isola ted nerve endings of the rat neurohypophysis. 2. Single step depolariz ations or trains of depolarizing pulses evoked voltage-dependent, inwa rd Ca2+ currents (I-Ca) and induced both Ca2+-dependent and Ca2+-indep endent changes in C-m. Three distinct C-m responses were observed and were differentiated by their kinetics and Ca2+ sensitivity: a non-exoc ytotic 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 tha t of the I-Ca. The amplitude and kinetics of the Delta C-m,C-t were un affected 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, bo th the Delta C-m,C-J and Delta C-m,C-S were Ca2+ dependent as evidence d by their sensitivity to Cd2+ block of I-Ca, intraterminal applicatio n of 10 mM BAPTA and reduced [Ca2+](o) or replacement of Ca2+ as the c harge carrier with Ba2+ 5. The Delta C-m,C-J was proportional to depol arization-evoked Ca2+ influx with initial exocytotic rate of approxima tely 350 granule fusions s(-1). The amplitude of the Delta C-m,C-J ros e exponentially (tau = 40 ms) and approached an asymptote (15.5 fF) wi th longer duration depolarizations indicating the fusion from and depl etion of an immediately releasable pool (IRP) estimated at nineteen do cked and primed secretory granules. 6. The Delta C-m,C-S was induced b y the application of repetitive long duration pulses and defined as th e 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+ i nflux 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 ma gnitude, reflecting the size of the RRP, was dependent on the resting [Ca2+](i) and the nerve ending size, and was depletable using repetiti ve depolarizations of long duration. 7. Recruitment into and release f rom the RRP and IRP were differentially sensitis e to changes in intra terminal 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+ bu ffering capacity of nerve endings by treatment with the mitochondrial Ca2+ uniporter blocker Ruthenium Red (10 mu M) potentiated the RRP siz e but had no significant effect on the IRP size. 8. The present study indicates that the Ca2+-dependent recruitment of and release from func tionally distinct pools of peptide-containing secretory granules in co mbination 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 t o occur during recurrent phasic action potential activity in this syst em.