Endocytosis in identified rat corticotrophs

Abstract

1. We used the patch-clamp technique, in conjunction with membrane capacitance measurement, fluorescence measurement of intracellular calcium concentration ([Ca2+]i), and flash photolysis of caged Ca2+ to study exo- and endocytosis in identified rat corticotrophs. 2. Exocytosis stimulated by depolarization pulses was typically followed by a 'slow' endocytosis that retrieved the membrane with a time constant of ∼6 s. The efficiency (the endocytosis/exocytosis amplitude ratio) of 'slow' endocytosis was ∼1.2 at [Ca2+]i < 3 μM and increased to ∼1.6 at [Ca2+]i > 3 μM. 3. Whole-cell dialysis through a patch pipette did not affect the kinetics and the efficiency of 'slow' endocytosis, but the amplitude of exocytosis was reduced. 4. 'Slow' endocytosis did not require sustained [Ca2+]i elevation and its kinetics was only weakly [Ca2+]i dependent. Our results suggest that 'slow' endocytosis involves a Ca2+ sensor with a high Ca2+ affinity (∼500 nM). 5. At high [Ca2+]i (> 10 μM), the 'slow' endocytosis was frequently preceded by a 'fast' endocytosis that comprised multiple steps of rapid decrease in membrane capacitance. 6. Neither calmodulin nor calcineurin appeared to be the Ca2+ sensor for endocytosis because the two forms of endocytosis were not affected by the calmodulin inhibitor calmidazolium (500 μM) or the calcineurin inhibitors cyclosporin A (1 μM) and calcineurin autoinhibitory peptide (1 mg ml-1). Ba2+, a poor activator of calmodulin, could support both forms of endocytosis but slowed the kinetics of 'slow' endocytosis ∼2-fold. 7. Non-hydrolysable analogues of GTP (GDP-β-S) and ATP (ATP-γ-S) also failed to inhibit either form of endocytosis, indicating that neither GTP nor ATP was essential for endocytosis. 8. We suggest that the high Ca2+ affinity of 'slow' endocytosis may be important for maintaining continuous cycles of exocytosis-endocytosis during sustained adrenocorticotropin secretion in corticotrophs.