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Compensatory and excess retrieval: two types of endocytosis following single step depolarizations in bovine adrenal chromaffin cells

Kathrin L. Engisch and Martha C. Nowycky

1. Endocytosis following exocytosis evoked by single step depolarizations was examined in bovine adrenal chromaffin cells using high resolution capacitance measurements in perforated-patch voltage clamp recordings.

2. Endocytosis was detected as a smooth exponential decline in membrane capacitance to either the pre-stimulus level ('compensatory retrieval') or far below the pre-stimulus level ('excess retrieval'). During excess retrieval, > 10 % of the cell surface could be internalized in under 5 s.

3. Compensatory retrieval was equal in magnitude to stimulus-evoked exocytosis for membrane additions > 100 fF (about fifty large dense-cored vesicles). In contrast, excess retrieval surpassed both the stimulus-evoked exocytosis, and the initial capacitance level recorded at the onset of phase-tracking measurements. Cell capacitance was not maintained at the level achieved by excess retrieval but slowly returned to pre-stimulus levels, even in the absence of stimulation.

4. A large percentage of capacitance increases < 100 fF, usually evoked by 40 ms depolarizations, were not accompanied by membrane retrieval.

5. Compensatory retrieval could occur with any amount of Ca²⁺ entry, but excess retrieval was never triggered below a threshold Ca²⁺ current integral of 70 pC.

6. The kinetics of compensatory and excess retrieval differed by an order of magnitude. Compensatory retrieval was usually fitted with a single exponential function that had a median time constant of 5·7 s. Excess retrieval usually occurred with double exponential kinetics that had an extremely fast first time constant (median, 670 ms) and a second time constant indistinguishable from that of compensatory retrieval.

7. The speed of compensatory retrieval was Ca²⁺ dependent: the largest mono-exponential time constants occurred for the smallest amounts of Ca²⁺ entry and decreased with increasing Ca²⁺ entry. The Ca²⁺ dependence of mono-exponential time constants was disrupted by cyclosporin A (CsA), an inhibitor of the Ca²⁺- and calmodulin-dependent phosphatase calcineurin.

8. CsA also reduced the proportion of responses with excess retrieval, but this action was caused by a shift in Ca²⁺ entry values below the threshold for activation. The lower total Ca²⁺ entry in the presence of CsA was due to an increase in the rate of Ca²⁺ current inactivation rather than a reduction in peak amplitude.

9. Our data suggest that compensatory and excess retrieval represent two independent, Ca²⁺-regulated mechanisms of rapid membrane internalization in bovine adrenal chromaffin cells. Alternatively, there is a single membrane internalization mechanism that can switch between two distinct modes of behaviour.

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