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Endocytosis in identified rat corticotrophs

Andy K. Lee and Amy Tse

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--S) and ATP (ATP--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.

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