Compensatory and excess retrieval: two types of endocytosis following single step depolarizations in bovine adrenal chromaffin cells.
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.
Engisch
K L
KL
Department of Neurobiology and Anatomy, Allegheny University of the Health Sciences, Medical College of Pennsylvania, Hahnemann University, Philadelphia, PA 19129, USA. ENGISCH@auhs.edu
Nowycky
M C
MC
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
D065095:Calcineurin Inhibitors; D004791:Enzyme Inhibitors; D016572:Cyclosporine; D002118:Calcium