Ep. Seward et al., EXOCYTOSIS IN PEPTIDERGIC NERVE-TERMINALS EXHIBITS 2 CALCIUM-SENSITIVE PHASES DURING PULSATILE CALCIUM-ENTRY, The Journal of neuroscience, 15(5), 1995, pp. 3390-3399
The link between electrical activity, Ca2+ entry through voltage-gated
channels, and transmitter or hormone secretion is a central issue in
neurobiology. In peptidergic nerve terminals of the mammalian neurohyp
ophysis (NHP), secretion is elicited by patterned bursts of action pot
entials (APs), All parameters of the bursts are important to elicit ef
ficient secretion, including AP frequency, AP broadening, burst durati
on, and interburst interval (Leng, 1988). We have studied Ca2+-secreti
on coupling of peptide-containing large dense-core vesicles (LDCV) in
isolated rat NHP terminals, Ca2+ influx through voltage-gated Ca2+ cha
nnels was elicited and recorded by the whole-cell patch-clamp techniqu
e, Exocytosis was monitored on line with high temporal resolution by t
he capacitance detection technique (Neher and Marty, 1982), AP bursts
were simulated by depolarizing pulse trains that mimic pulsatile subme
mbrane Ca2+ elevations predicted for physiological stimuli. The charac
teristic capacitance response (Delta C-m) to a train of depolarizing p
ulses was triphasic, It consisted of a threshold phase during which ea
rly pulses did not elicit secretion, a subsequent secretory phase duri
ng which C-m increases were coupled to depolarizing pulses, and a fati
gued or inactivated state during which additional Ca2+ entry was ineff
ective. Both the threshold phase and secretory phase were correlated w
ith the integrals of Ca2+ current, Ca2+ chelators affect both the thre
shold and secretory phase at submillimolar concentrations. Thus, a ''s
hell'' rather than ''microdomain'' model of Ca2+ elevation is appropri
ate for analyzing Ca2+-secretion coupling in NHP terminals (Nowycky an
d Pinter, 1993). We propose a two-step model, with a Ca2+-dependent pr
eparatory step followed by a final exocytotic step that is coupled to
active Ca2+ influx. The results suggest that under physiological condi
tions, APs early in a burst prepare an NHP terminal for secretion, but
later APs actually trigger exocytosis. Since NHP terminals do not pos
sess a readily releasable pool of vesicles that require only a single
Ca2+ step for exocytosis as seen in chromaffin cells (Neher and Zucker
, 1993) and melanotrophs (Thomas et al., 1993a), Ca2+-secretion coupli
ng mechanisms may be heterologous even within a single class of vesicl
es.