EXOCYTOSIS IN PEPTIDERGIC NERVE-TERMINALS EXHIBITS 2 CALCIUM-SENSITIVE PHASES DURING PULSATILE CALCIUM-ENTRY

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.