title: Compensatory and excess retrieval: two types of endocytosis following single step depolarizations in bovine adrenal chromaffin cells.
abstract: 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 Ca(2+)- 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, Ca(2+)-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.
authors.affiliation: Department of Neurobiology and Anatomy, Allegheny University of the Health Sciences, Medical College of Pennsylvania, Hahnemann University, Philadelphia, PA 19129, USA. ENGISCH@auhs.edu
authors.forename: K L
authors.initials: KL
authors.lastname: Engisch
authors.forename: M C
authors.initials: MC
authors.lastname: Nowycky
mesh_terms: D000311:Adrenal Glands / Q000166:cytology / Q000502:physiology*; D000818:Animals; D065095:Calcineurin Inhibitors; D002118:Calcium / Q000378:metabolism / Q000502:physiology; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism / Q000502:physiology; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000502:physiology*; D016572:Cyclosporine / Q000494:pharmacology; D004558:Electric Stimulation; D004594:Electrophysiology; D004705:Endocytosis / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D008564:Membrane Potentials / Q000502:physiology; D018408:Patch-Clamp Techniques
chemical_list: D065095:Calcineurin Inhibitors; D004791:Enzyme Inhibitors; D016572:Cyclosporine; D002118:Calcium