Exocytosis in peptidergic nerve terminals exhibits two calcium- sensitive phases during pulsatile calcium entry

Abstract

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 neurohypophysis (NHP), secretion is elicited by patterned bursts of action potentials (APs). All parameters of the bursts are important to elicit efficient secretion, including AP frequency, AP broadening, burst duration, and interburst interval (Leng, 1988). We have studied Ca(2+)-secretion coupling of peptide-containing large dense-core vesicles (LDCV) in isolated rat NHP terminals. Ca2+ influx through voltage-gated Ca2+ channels was elicited and recorded by the whole-cell patch-clamp technique. Exocytosis was monitored on line with high temporal resolution by the capacitance detection technique (Neher and Marty, 1982). AP bursts were simulated by depolarizing pulse trains that mimic pulsatile submembrane Ca2+ elevations predicted for physiological stimuli. The characteristic capacitance response (delta Cm) to a train of depolarizing pulses was triphasic. It consisted of a threshold phase during which early pulses did not elicit secretion, a subsequent secretory phase during which Cm increases were coupled to depolarizing pulses, and a fatigued or inactivated state during which additional Ca2+ entry was ineffective. Both the threshold phase and secretory phase were correlated with the integrals of Ca2+ current. Ca2+ chelators affect both the threshold and secretory phase at submillimolar concentrations. Thus, a “shell” rather than “microdomain” model of Ca2+ elevation is appropriate for analyzing Ca(2+)-secretion coupling in NHP terminals (Nowycky and Pinter, 1993). We propose a two- step model, with a ca(2+)-dependent preparatory step followed by a final exocytotic step that is coupled to active Ca2+ influx. The results suggest that under physiological conditions, APs early in a burst prepare an NHP terminal for secretion, but later APs actually trigger exocytosis. Since NHP terminals do not possess 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), Ca(2+)-secretion coupling mechanisms may be heterologous even within a single class of vesicles.

κ-Opioid Receptor Activation Modulates Ca2+Currents and Secretion in Isolated Neuroendocrine Nerve Terminals

“…The properties of the immediate and slow C m responses measured here, as illustrated in Figure 3A, for example, were similar to those described for these peptidergic nerve endings in previous studies (Lim et al, 1990;Seward et al, 1995;Hsu and Jackson, 1996;Giovannucci and Stuenkel, 1997). In contrast to the transient ⌬C m , both the immediate jump and slowly increasing C m responses were markedly altered by manipulations that resulted in attenuation of depolarization-evoked C a 2ϩ entry.…”

“…These results support the conclusion that both the immediate and slow C m responses measured here are tightly coupled to elevations in [Ca 2ϩ ] i produced by influx through voltage-sensitive Ca 2ϩ channels. Nevertheless, the normalized amplitude of the immediate C m increase exceeded that of the slow C m response at all test potentials, suggesting that the initial component of exocytotic release may have a different requirement for triggering by elevations in [Ca 2ϩ ] i than the slow phase of release (Lim et al, 1990;Lindau et al, 1992;Seward et al, 1995;Hsu and Jackson, 1996;Giovannucci and Stuenkel, 1997).…”

“…Given the close correlation observed between the opioid-induced inhibition in evoked C a 2ϩ currents and in slow C m responses, what might account for the lack of correspondence between changes in C a 2ϩ currents and the immediate C m increases that constitute the initial secretory event? One possibility is that such an outcome might derive from f undamental differences in the dependency of the initiation and maintenance of vesicular exocytosis on increases in C a 2ϩ concentration within functionally distinct intraterminal domains (Lindau et al, 1992;Horrigan and Bookman, 1994;Hsu and Jackson, 1996; but see Seward et al, 1995). We hypothesized that if the immediate C m response reflects exocytosis of predocked vesicles (Lindau et al, 1992;Horrigan and Bookman, 1994), then activation of C a 2ϩ channels by depolarizations to the peak of the current-voltage curve may increase submembrane C a 2ϩ concentration within spatially restricted domains close to the release sites beyond the level of saturation for vesicle f usion.…”

Short-Term Changes in the Ca2+-Exocytosis Relationship during Repetitive Pulse Protocols in Bovine Adrenal Chromaffin Cells

“…The C m jumps evoked by individual pulses usually are nonuniform: typically, C m jumps increase initially and then decrease in amplitude (Lim et al, 1990;Thomas et al, 1990;Augustine and Neher, 1992;Ä mmäläet al, 1993;von Rüden and Neher, 1993;Horrigan and Bookman, 1994;Seward et al, 1995;Hsu and Jackson, 1996;Huang and Neher, 1996;Seward and Nowycky, 1996;Giovannucci and Stuenkel, 1997). By analogy with fast synapses, it has been suggested that C m jump amplitudes increase because of Ca 2ϩ accumulation during the beginning of a train (residual Ca 2ϩ hypothesis) (Katz and Miledi, 1968).…”

“…By analogy with fast synapses, it has been suggested that C m jump amplitudes increase because of Ca 2ϩ accumulation during the beginning of a train (residual Ca 2ϩ hypothesis) (Katz and Miledi, 1968). Ca 2ϩ accumulation may directly affect the exocytotic trigger (Heinemann et al, 1993), contribute to recruitment of additional pools with different Ca 2ϩ sensitivities (Horrigan and Bookman, 1994;Gillis et al, 1996;Giovannucci and Stuenkel, 1997), or fulfill "threshold" requirements for Ca 2ϩ (Ä mmälä et al, 1993;Seward et al, 1995;Seward and Nowycky, 1996;Giovannucci and Stuenkel, 1997). At fast synapses, diminished postsynaptic responses are commonly attributed to depletion of finite pools of releaseready vesicles, although postsynaptic receptor desensitization and Ca 2ϩ current inactivation complicate interpretation.…”