Physiological stimuli evoke two forms of endocytosis in bovine chromaffin cells

Abstract

1. Exocytosis and endocytosis were measured following single, or trains of, simulated action potentials (sAP) in bovine adrenal chromaffin cells. Catecholamine secretion was measured by oxidative amperometry and cell membrane turnover was measured by voltage clamp cell capacitance measurements. 2. The sAPs evoked inward Na(+) and Ca(2+) currents that were statistically identical to those evoked by native action potential waveforms. On average, a single secretory granule underwent fusion following sAP stimulation. An equivalent amount of membrane was then quickly internalised (tau = 560 ms). 3. Stimulation with sAP trains revealed a biphasic relationship between cell firing rate and endocytic activity. At basal stimulus frequencies (single to 0.5 Hz) cells exhibited a robust membrane internalisation that then diminished as firing increased to intermediate levels (1.9 and 6 Hz). However at the higher stimulation rates (10 and 16 Hz) endocytic activity rebounded and was again able to effectively maintain cell surface near pre-stimulus levels. 4. Treatment with cyclosporin A and FK506, inhibitors of the phosphatase calcineurin, left endocytosis characteristics unaltered at the lower basal stimulus levels, but blocked the resurgence in endocytosis seen in control cells at higher sAP frequencies. 5. Based on these findings we propose that, under physiological electrical stimulation, chromaffin cells internalise membrane via two distinct pathways that are separable. One is prevalent at basal stimulus frequencies, is lessened with increased firing, and is insensitive to cyclosporin A and FK506. A second endocytic form is activated by increased firing frequencies, and is selectively blocked by cyclosporin A and FK506.

Dynamin I plays dual roles in the activity-dependent shift in exocytic mode in mouse adrenal chromaffin cells

“…Consistent with previous work [4,19,25], disrupting clathrinmediated endocytosis by blocking synaptojanin/endophilin binding has no effect on membrane retrieval in these conditions. Data presented here as well as provided in the literature, point to the major mode of exocytosis under light physiological electrical stimulation as a transient 'kiss and run' occurring through a stabilized Ω-figure.…”

“…Dynamin I activity has been implicated in membrane endocytosis under moderate stimulation in chromaffin cells independent of the actions of clathrin [4,23]. We set out to test if dynamin I function is required to support 'kiss and run' Ω-form exocytosis elicited under conditions that mimic native basal sympathetic tone [24,25]. Dynamin I function represents a core component for neuronal and neurosecretory function.…”

CaV1.3 as pacemaker channels in adrenal chromaffin cells: Specific role on exo- and endocytosis?

“…Square pulse depolarizations cause exocytosis followed by a decline in membrane capacitance, which can reach the pre-stimulus level (compensatory endocytosis) or fall even below (excess endocytosis). 73,74 Transition between these two modes appears to be regulated by intracellular Ca 2+ : the retrieval being accelerated and potentiated by increasing Ca 2+ levels. It is interesting that in chromaffin cells endocytosis is also supported by barium, 75 by the activation of kinase/phosphatasemediated pathways 73,76 and by additional pathways of vesicle the same experiment is repeated in the presence of 20 μM EGTA-AM, WT MCCs display their fast inactivating BK currents while the slowly inactivating channels are apparently lost.…”

Cytosolic organelles shape calcium signals and exo–endocytotic responses of chromaffin cells

“…For instance, compensatory and excess endocytosis represent two independent Ca 2+ -regulated mechanisms of rapid internalisation in bovine chromaffin cells [204,206]. The existence of these two Ca 2+ sensors is consistent with the fact that Ca 2+ and Ba 2+ support excessive membrane retrieval in bovine chromaffin cells [207].…”

“…Other studies have used action potential waveforms to correlate the stimulation frequency in bovine chromaffin cells with amperometric spike secretion in rat chromaffin cells [192] or with capacitance increase in bovine chromaffin cells [204]. In addition, depolarising pulses have been used in transgenic mice to study the role of exocytotic proteins on the kinetics of Cm [203].…”