Voltage-gated Ca2+ channels and intracellular Ca2+ release regulate exocytosis in identified rat corticotrophs.
1. The patch clamp technique was used in conjunction with a fluorescent Ca2+ indicator (indo-1, or indo-1FF) to measure simultaneously cytosolic Ca2+ concentration ([Ca2+]i) and exocytosis (changes in membrane capacitance) in single, identified rat corticotrophs. 2. Exocytosis could be stimulated by extracellular Ca2+ entry (via voltage-gated Ca2+ channels). A train of depolarizations could exhaust the pool of readily releasable granules and the pool replenished with a time constant of 42 s (at 22-25 C). 3. Recordings from cells with 0.5 mM intracellular cAMP showed that the amplitude of the depolarization-triggered exocytosis, the Ca2+ sensitivity of exocytosis, as well as the rate of replenishment of the readily releasable pool, were similar to the controls. 4. Exocytosis could also be stimulated by intracellular Ca2+ release from the inositol 1,4, 5-trisphosphate (IP3)-sensitive store (via flash photolysis of caged IP3). At comparable [Ca2+]i, extracellular Ca2+ entry and intracellular Ca2+ release had similar efficacy in triggering exocytosis. 5. The rate of exocytosis triggered via depolarization or intracellular Ca2+ release was much faster than that triggered via uniform elevation of [Ca2+]i (Ca2+ dialysis or flash photolysis of caged Ca2+). 6. The above findings suggest that both intracellular Ca2+ release and voltage-gated extracellular Ca2+ entry generate a spatial Ca2+ gradient, such that the local [Ca2+] near the exocytic sites was approximately 3-fold higher than the mean cytosolic [Ca2+]. However, neither cAMP nor the spatial Ca2+ gradient generated during depolarization could account for the high efficacy of corticotropin-releasing hormone (CRH) in stimulating adrenocorticotropic hormone (ACTH) secretion from corticotrophs.
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Department of Pharmacology, 9-70 Medical Science Building, University of Alberta, Edmonton, Alberta, Canada T6G 2H7. amy.tse@ualberta.ca
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D000324:Adrenocorticotropic Hormone / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D015220:Calcium Channels / Q000378:metabolism*; D002478:Cells, Cultured; D003346:Corticotropin-Releasing Hormone / Q000378:metabolism / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D005456:Fluorescent Dyes; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007424:Intracellular Fluid / Q000378:metabolism*; D008297:Male; D018408:Patch-Clamp Techniques; D010903:Pituitary Gland, Anterior / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011930:Reaction Time / Q000187:drug effects
D015220:Calcium Channels; D005456:Fluorescent Dyes; D015544:Inositol 1,4,5-Trisphosphate; D000324:Adrenocorticotropic Hormone; D003346:Corticotropin-Releasing Hormone; D000242:Cyclic AMP; D002118:Calcium