Endocytosis in identified rat corticotrophs.
1. 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. 2. Exocytosis stimulated by depolarization pulses was typically followed by a 'slow' endocytosis that retrieved the membrane with a time constant of approximately 6 s. The efficiency (the endocytosis/exocytosis amplitude ratio) of 'slow' endocytosis was approximately 1.2 at [Ca(2+)](i) < 3 microM and increased to approximately 1.6 at [Ca(2+)](i) > 3 microM. 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 [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 (approximately 500 nM). 5. At high [Ca(2+)](i) (> 10 microM), 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 Ca(2+) sensor for endocytosis because the two forms of endocytosis were not affected by the calmodulin inhibitor calmidazolium (500 microM) or the calcineurin inhibitors cyclosporin A (1 microM) 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 approximately 2-fold. 7. Non-hydrolysable analogues of GTP (GDP-beta-S) and ATP (ATP-gamma-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 Ca(2+) affinity of 'slow' endocytosis may be important for maintaining continuous cycles of exocytosis-endocytosis during sustained adrenocorticotropin secretion in corticotrophs.
Lee
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Department of Pharmacology, 9-70 Medical Science Building, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.
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D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000324:Adrenocorticotropic Hormone / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D000242:Cyclic AMP / Q000494:pharmacology; D004594:Electrophysiology; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D008564:Membrane Potentials / Q000502:physiology; D008954:Models, Biological; D018408:Patch-Clamp Techniques; D010902:Pituitary Gland / Q000166:cytology* / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley
C022571:adenosine 5'-O-(3-thiotriphosphate); D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000255:Adenosine Triphosphate; D000324:Adrenocorticotropic Hormone; D000242:Cyclic AMP; D002118:Calcium