Endocytosis in identified rat corticotrophs

Authors


Corresponding author A. Tse: Department of Pharmacology, 9–70 Medical Science Building, University of Alberta, Edmonton, Alberta, Canada T6G 2H7. Email: amy.tse@ualberta.ca

Abstract

  • 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.

  • 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.

  • 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 [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).

  • 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.

  • 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.

  • 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 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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