Compensatory and Excess Retrieval: Two Types of Endocytosis Following Single Step Depolarizations in Bovine Adrenal Chromaffin Cells

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  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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