Two forms of triggered endocytosis in regulated secretory cells

It is likely that during physiological stimulation, exocytosis in regulated secretory cells will be followed by a matched membrane retrieval to maintain a constant cell surface area. Changes in cell surface area during exocytosis and endocytosis can be monitored by patch-clamp recording of plasma membrane capacitance. In such recordings, increases in capacitance due to exocytosis can be readily detected. In whole-cell recordings, membrane retrieval by endocytosis has not always been observed, since a form of endocytosis that recovers exocytosed membrane shows rapid run-down during whole-cell dialysis (Burgoyne, 1995). In some instances, however, with strong stimulation or high intracellular Ca²⁺ levels, an alternative very rapid form of endocytosis has been observed in whole-cell recordings of chromaffin (Neher & Zucker, 1993; Artalejo et al. 1996) and other cell types (Thomas et al. 1994; Proks & Ashcroft, 1995) that recovers membrane in excess of that inserted during the burst of exocytosis. The relationship between ‘compensatory’ retrieval and this ‘excess’ retrieval has now been clarified in two papers on adrenal chromaffin cells (Smith & Neher, 1997; Engisch & Nowycky, 1998, in this issue of The Journal of Physiology) using perforated-patch recording in which compensatory retrieval is maintained. The results of these two studies are in agreement and suggest the presence of two distinct pathways for endocytosis in chromaffin and probably other secretory cells.

Stimulation of exocytosis in chromaffin cells by depolarization can be followed by a subsequent decrease in capacitance due to a relatively rapid endocytotic mechanism that can retrieve most or all of the exocytosed membrane within tens of seconds but does not usually reduce plasma membrane capacitance below pre-stimulus levels. This is the compensatory retrieval that is lost during whole-cell dialysis. Excess retrieval, in contrast, can lead to a faster and extensive endocytosis, with membrane capacitance being reduced to considerably below the pre-stimulus level. The two recent studies (Smith & Neher, 1997; Engisch & Nowycky, 1998) agree remarkably well on the measurements of initial rates and kinetics of these two endocytotic processes and the conditions under which they are triggered, and suggest that they are due to distinct mechanisms. The properties of compensatory and excess retrieval are compared in Table 1. The distinction between the two forms of endocytosis was also emphasized by the finding of Engisch & Nowycky (1998) that compensatory but not excess retrieval was inhibited by cyclosporin A, an inhibitor of the Ca²⁺-dependent phosphatase calcineurin, implicating this enzyme in a positive role in only compensatory retrieval.

Table 1.

Comparison ofcompensatory retrieval and excess retrieval in adrenal chromaffin cells

Property	Compensatory retrieval	Excess retrieval
Effect of whole-cell dialysis ab	Retrieval is lost	Retrieval persists
Amount of membrane retrieved ac	60–100 % of exocytosed membrane	Up to 17 % of cell surface
Stimulus required ac	Exocytosis (Ca2+entry increases rate)	Threshold > 70–90 pC of Ca2+ entry, [Ca2+]i > 50 μM
Relationship to amount of prior exocytosis ac	Directly proportional	None
Initial rate a	6 fF s (0.6 μm2 s−1)	240 fF s (24 μm2 s−1)
Time constant (τ) c	3–6 s	0.67 s
Effect of cyclosporin A c	Inhibited	No effect

Based on data in:

^aSmith & Neher (1997)

^bBurgoyne (1995)

^cEngisch & Nowycky (1998).

The potential role of the Ca²⁺-binding protein calmodulin in retrieval mechanisms is an important and debated issue. The work of Artalejo et al. (1996) has implicated calmodulin in excess retrieval. One of the arguments for an essential role of calmodulin is based on the observation that Ba²⁺ and Sr²⁺ support exocytosis but not excess retrieval, and calmodulin can bind Ca²⁺ but not Ba²⁺ or Sr²⁺. The problem arises in comparison of these data with those from pancreatic β-cells. First, an important point is that while compensatory and excess retrieval have been compared in detail in chromaffin cells, it is clear from published records that both mechanisms also operate in pituitary melanotrophs (Thomas et al. 1994), pancreatic β-cells (Proks & Ashcroft, 1995) and probably other cell types (see references in Engisch & Nowcycky, 1998). One of the major aspects that we have learnt about basic cell biological mechanisms in recent years is their highly conserved and almost universal basis. It is a very good bet, therefore, that compensatory and excess retrieval will operate in most, if not all, regulated secretory cell types and involve the same basic mechanisms. The surprise is that in pancreatic β-cells, Ba²⁺ and Sr²⁺ support both forms of retrieval, ruling out calmodulin as a major component in triggering either pathway (Proks & Ashcroft, 1995). This is clearly an aspect for further work and it would be of interest to know if compensatory retrieval is supported by Ba²⁺ and Sr²⁺ in chromaffin cells.

One bizarre aspect of the excess retrieval phenomenon is that an equivalent amount of membrane to that retrieved beyond baseline is later returned to the plasma membrane following excess retrieval (Artalejo et al. 1996; Engisch & Nowycky, 1998). This would be occuring when cytosolic Ca²⁺ had returned to resting levels. Is this due to some form of constitutive exocytosis that does not require elevated Ca²⁺? How does the cell regulate its surface area so precisely back to the pre-stimulus level? There are intriguing issues here that will require further investigation.

Finally, a major question is whether both forms of endocytosis are physiologically relevant following exocytosis. Compensatory retrieval is reliable and accurate in the retrieval of exocytosed membrane. It only occurs following exocytosis and does so at similar Ca²⁺ levels to those that trigger exocytosis. In contrast, excess retrieval is unreliable, is not related to prior exocytosis and requires extreme levels of Ca²⁺ entry and a very high concentration of cytosolic Ca²⁺. It seems likely, therefore, that the normal mechanism for the precise recycling of exocytosed membrane is compensatory retrieval. The function of excess retrieval and whether it actually occurs under physiological conditions are yet to be established.