Kl. Engisch et Mc. Nowycky, COMPENSATORY AND EXCESS RETRIEVAL - 2 TYPES OF ENDOCYTOSIS FOLLOWING SINGLE-STEP DEPOLARIZATIONS IN BOVINE ADRENAL CHROMAFFIN CELLS, Journal of physiology, 506(3), 1998, pp. 591-608
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.