Endocytosis in identified rat corticotrophs

We used the patch‐clamp technique, in conjunction with membrane capacitance measurement, fluorescence measurement of intracellular calcium concentration ([Ca 2+ ] i ), and flash photolysis of caged Ca 2+ to study exo‐ and endocytosis in identified rat corticotrophs. 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 [Ca 2+ ] i < 3 μ m and increased to ∼1.6 at [Ca 2+ ] i > 3 μ m . 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. ‘Slow’ endocytosis did not require sustained [Ca 2+ ] i elevation and its kinetics was only weakly [Ca 2+ ] i dependent. Our results suggest that ‘slow’ endocytosis involves a Ca 2+ sensor with a high Ca 2+ affinity (∼500 n m ). At high [Ca 2+ ] i (> 10 μ m ), the ‘slow’ endocytosis was frequently preceded by a ‘fast’ endocytosis that comprised multiple steps of rapid decrease in membrane capacitance. Neither calmodulin nor calcineurin appeared to be the Ca 2+ sensor for endocytosis because the two forms of endocytosis were not affected by the calmodulin inhibitor calmidazolium (500 μ m ) or the calcineurin inhibitors cyclosporin A (1 μ m ) and calcineurin autoinhibitory peptide (1 mg ml −1 ). Ba 2+ , a poor activator of calmodulin, could support both forms of endocytosis but slowed the kinetics of ‘slow’ endocytosis ∼2‐fold. 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. We suggest that the high Ca 2+ affinity of ‘slow’ endocytosis may be important for maintaining continuous cycles of exocytosis‐endocytosis during sustained adrenocorticotropin secretion in corticotrophs.