COMPENSATORY AND EXCESS RETRIEVAL - 2 TYPES OF ENDOCYTOSIS FOLLOWING SINGLE-STEP DEPOLARIZATIONS IN BOVINE ADRENAL CHROMAFFIN CELLS

1. Endocytosis following exocytosis evoked by single step depolarizati ons was examined in bovine adrenal chromaffin cells using high resolut ion capacitance measurements in perforated-patch voltage clamp recordi ngs. 2. Endocytosis was detected as a smooth exponential decline in me mbrane capacitance to either the pre-stimulus level ('compensatory ret rieval') or far below the pre-stimulus level ('excess retrieval'). Dur ing excess retrieval, >10% of the cell surface could be internalized i n under 5 s. 3. Compensatory retrieval was equal in magnitude to stimu lus-evoked exocytosis for membrane additions >100 fF (about fifty larg e dense-cored vesicles). In contrast, excess retrieval surpassed both the stimulus-evoked exocytosis, and the initial capacitance level reco rded at the onset of phase-tracking measurements. Cell capacitance was not maintained at the level achieved by excess retrieval but slowly r eturned to pre-stimulus levels, even in the absence of stimulation. 4. A large percentage of capacitance increases <100 fF, usually evoked b y 40 ms depolarizations, were not accompanied by membrane retrieval. 5 . Compensatory retrieval could occur with any amount of Ca2+ entry, bu t excess retrieval was never triggered below a threshold Ca2+ current integral of 70 pC. 6. The kinetics of compensatory and excess retrieva l differed by an order of magnitude. Compensatory retrieval was usuall y fitted with a single exponential function that had a median time con stant of 5.7 s. Excess retrieval usually occurred with double exponent ial kinetics that had an extremely fast first time constant (median, 6 70 ms) and a second time constant indistinguishable from that of compe nsatory retrieval. 7. The speed of compensatory retrieval was Ca2+ dep endent: the largest mono-exponential time constants occurred for the s mallest 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-depe ndent phosphatase calcineurin. 8. CsA also reduced the proportion of r esponses with excess retrieval, but this action was caused by a shift in Ca2+ entry values below the threshold for activation. The lower tot al Ca2+ entry in the presence of CsA was due to an increase in the rat e of Ca2+ current inactivation rather than a reduction in peak amplitu de. 9. Our data suggest that compensatory and excess retrieval represe nt two independent, Ca2+-regulated mechanisms of rapid membrane intern alization in bovine adrenal chromaffin cells. Alternatively, there is a single membrane internalization mechanism that can switch between tw o distinct modes of behaviour.