Physiological stimuli evoke two forms of endocytosis in bovine chromaffin cells.

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. The sAPs evoked inward Na+ and Ca2+ 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 (τ = 560 ms). 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. 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. 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. Over recent years, many studies have been completed which greatly improve the understanding of the biochemistry and kinetics of exocytosis-coupled endocytosis (De Camilli & Takei, 1996). In secretory cells, differing stimulation intensities can activate multiple endocytic mechanisms (Koenig & Ikeda, 1996; Cousin & Robinson, 2000). Biochemical studies have identified an array of intracellular proteins that are likely to be involved in the endocytic processes. Examples include cytoskeletal elements (Hamm-Alvarez & Sheetz, 1998), kinase/phosphatase enzymes (Turner et al. 1999), adaptor complexes (Robinson, 1994; Gonzalez-Gaitan & Jackle, 1997) and nucleotide binding proteins (De Camilli & Takei, 1996; Artalejo et al. 1997). Other studies have provided evidence that at least one route of membrane internalisation depends upon the activity of the protein phosphatase calcineurin (Bauerfeind et al. 1997; Marks & McMahon, 1998; Lai et al. 1999). Differing cell activity has been shown to play a modulatory role in the endocytic process. In experimental systems as diverse as the motor nerve terminal, pancreatic β-cells, pituitary cells and cultured hippocampal neurons, elevated activity enhances endocytosis (Ramaswami et al. 1994; Koenig & Ikeda, 1996; Klingauf et al. 1998; Lee & Tse, 2001). In the frog neuromuscular junction, increased cell firing results in a decreased endocytic response, but seems to be independent of cytosolic Ca2+ levels (Wu & Betz, 1996). In contrast, in several systems including pituitary neurohypophyseal terminals (Hsu & Jackson, 1996), brain synaptosomes (Cousin & Robinson, 2000) and retinal bipolar neurons (von Gersdorff & Matthews, 1994; Neves & Lagnado, 1999), heightened cell activity inhibits the endocytic response, perhaps acting through elevated Ca2+. Quantitative descriptions of multiple kinetic modes of endocytosis have been published for the neuroendocrine chromaffin cell system. Electrical square pulse stimuli, leading to subplasmalemmal Ca2+ elevations measuring up to a few tens of micromolar trigger exocytosis that is followed by graded membrane internalisation, so recovering a surface area roughly equal to that added immediately prior (Burgoyne, 1995; Smith & Neher, 1997; Engisch & Nowycky, 1998). More intense stimulation, leading to local [Ca2+] i measuring several tens of micromolar or higher causes cells to overinternalise membrane by retrieving much more membrane than added through the coupled exocytic event (Artalejo et al. 1995; 1996; Nucifora & Fox, 1999). These two retrieval modes have been shown to be the result of separate mechanisms and have been termed ‘compensatory’ and ‘excess’ endocytosis, respectively (Engisch & Nowycky, 1998). The control of transition between compensatory and excess endocytosis is commonly attributed to differences in Ca2+ affinity. Even within the compensatory endocytic mechanism, Ca2+ acts as a modulator, with increasing concentrations resulting in a more rapid kinetic and more complete retrieval (Engisch & Nowycky, 1998). Finally, endocytosis in chromaffin cells has also been shown to be modulated by divalent cations other than Ca2+ (Nucifora & Fox, 1998) as well as multiple kinase/phosphatase pathways (Artalejo et al. 1996; Engisch & Nowycky, 1998). However, given such a variety of endocytic behaviour, it is not yet clear which kinetic mode(s) or molecular cascade(s) is responsible for membrane recycling following physiological stimulation. Due to their accessibility for quantitative electrophysiological and electrochemical techniques, the chromaffin cell has proven a fertile system for the study of the highly convolved processes of stimulus-evoked exo- and endocytosis. From the literature, it appears likely that in chromaffin cells, Ca2+ influx either directly or indirectly provides a modulatory influence over the kinetics, and possibly affects mode transitions of exocytosis-coupled endocytosis. Previous studies of electrically evoked endocytosis utilised square pulse stimulation with pulse durations ranging from 10 ms (Engisch & Nowycky, 1998) up to 1 s (Smith & Betz, 1996). These pulses were commonly separated by long inter-pulse periods measuring several tens of seconds to minutes (Artalejo et al. 1995; Smith & Neher, 1997; Engisch & Nowycky, 1998). Such stimulation protocols are expected to cause influx of up to a few hundred picocoulombs Ca2+ charge, which is likely to lead to transient highly localised subplasmalemmal Ca2+ concentrations measuring several tens of micromolar. Within a few hundred microseconds these large Ca2+ gradients collapse to equilibrium within the cytosol (Chow et al. 1994; Monck et al. 1994) and are eventually cleared from the cell (Neher & Augustine, 1992; Xu et al. 1997). In essence, the square pulse protocol leads to brief, discrete periods of very high [Ca2+] i at the site of endocytosis. Action potential trains, on the other hand, form a very different Ca2+ profile from that seen for square pulse depolarisation. Due to their brief duration, Ca2+ influx is low. On average, a single action potential leads to the influx of approximately 1 pC Ca2+ charge (Giovannucci et al. 1999) (see also Figs 5 and ​and8).8). Action potential trains therefore do not lead to the transient, high calcium concentration gradients that are elicited by square pulse depolarisation, but rather lead to a sustained tonic elevation in [Ca2+] i whose magnitude depends on firing frequency. Given that endocytosis may be dependent upon, and modulated by, Ca2+, the differences in Ca2+ influx and clearance expected between square pulse and action potential stimulation are likely to result in different endocytic behaviours. Therefore, it is not certain to what extent our current understanding of endocytosis in chromaffin cells, which is based on square-pulse depolarisation, reflects physiological behaviour. Given the multiplicity of endocytic modes described in the literature, we wondered which, if any, of these kinetically or molecularly defined endocytic mechanisms were active in chromaffin cells following physiological action potential stimulation. We set out to determine which, if any, of the described endocytic modes are found to occur following physiological electrical stimulation, a condition that had not yet been measured. Figure 5 Cell capacitance and integrated amperometry vary with cell activity Figure 8 Cyclosporin A does not alter endocytosis by lowering Ca2+ influx In this study we employed the electrophysiological perforated patch clamp technique with concurrent electrochemical detection of catecholamine release to determine simultaneous exo- and endocytic activity. Simulated action potential voltage templates were developed to investigate exocytosis-coupled endocytosis at basal firing rates and under physiological electrical activation. Here we have reported for the first time that at low firing intensity, representing basal sympathetic activity (Brandt et al. 1976; Kidokoro, 1980; Wakade, 1981), chromaffin cells exhibited a robust and active membrane retrieval which we term ‘phase I’ endocytosis. Phase I endocytosis quickly diminished in response to increased cell firing. However, at the elevated frequencies, a second activity-enhanced form of endocytosis (termed phase II endocytosis) emerged. Cyclosporin A and FK506 selectively blocked phase II endocytosis.