title: Simultaneous measurement of [Ca2+]i and secretion-coupled membrane turnover, by single cell fluorescence microscopy.
abstract: Thyrotropin releasing hormone (TRH), which stimulates prolactin secretion, increases the fluorescence of cultured bovine anterior pituitary (bAP) cells in the presence of the non-permeant membrane indicator dye FM 1-43 [Stafford SJV. Shorte SL. Schofield JG. (1993) Use of a fluorescent dye to measure secretion from intact bovine anterior pituitary cells. Biosci. Rep., 13, 9-17]. FM 1-43 is non-fluorescent in aqueous solution but becomes fluorescent when incorporated into the plasma membrane. The membrane area accessible to FM 1-43 dye, and therefore cell fluorescence, increases during exocytosis as secretory granules fuse with the plasma membrane, and endocytosis as vesicles formed at the plasma-membrane fuse with intracellular organelle membranes. We have here measured changes in FM 1-43 uptake and the intracellular calcium concentration ([Ca2+]i) concurrently in the same cells on exposure to TRH, phorbol myristate acetate (PMA) or NH4Cl. TRH (0.1-10 microM) caused a transient increase in [Ca2+]i in 70-90% of bAP cells and in 60-90% of the responding cells also caused a sustained increased FM 1-43 fluorescence. TRH increased [Ca2+]i but did not affect FM 1-43 fluorescence in GH3 rat pituitary cells, probably because they contain too few secretory granules to give a detectable increase. The dopamine D2-receptor agonist quinpirole (10 microM) had little effect on the TRH-induced [Ca2+]i rise in bAP cells, but abolished the increase in FM 1-43 fluorescence. The phorbol ester PMA (0.3-3 microM) caused a small, transient increase in [Ca2+]i followed by a fall to levels lower than original resting levels in 40-60% of bAP cells and increased FM 1-43 uptake in cells showing these changes. Extracellular NH4Cl, which mobilises calcium from an ionomycin-insensitive calcium store, caused a transient [Ca2+]i increase in over 90% of the bAP-cells and increased FM 1-43 uptake in a subpopulation (> 50%) of these. The Na+/H+ ionophore monensin prevented the increase in FM 1-43 fluorescence but not the [Ca2+]i rise induced by TRH, prevented the increases in both FM 1-43 fluorescence and [Ca2+]i caused by NH4Cl, and reduced the number of cells showing a rise in FM 1-43 fluorescence in response to PMA from 64% to 34%. The Ca(2+)-ATPase inhibitor thapsigargin reduced the number of bAP cells displaying TRH-induced increases in [Ca2+]i and membrane-turnover from 74% to 18%, but did not affect the changes in [Ca2+]i or FM 1-43 fluorescence caused by PMA or NH4Cl. We discuss the relationships between the secretogogue-induced increases in FM 1-43 fluorescence and changes in intracellular [Ca2+]i under these conditions.
authors.affiliation: Department of Biochemistry, University of Bristol, UK.
authors.forename: S L
authors.initials: SL
authors.lastname: Shorte
authors.forename: S J
authors.initials: SJ
authors.lastname: Stafford
authors.forename: V J
authors.initials: VJ
authors.lastname: Collett
authors.forename: J G
authors.initials: JG
authors.lastname: Schofield
mesh_terms: D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000032:analysis*; D002417:Cattle; D002462:Cell Membrane / Q000502:physiology*; D002478:Cells, Cultured; D005456:Fluorescent Dyes; D008561:Membrane Fusion; D008856:Microscopy, Fluorescence; D010902:Pituitary Gland / Q000502:physiology* / Q000648:ultrastructure; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D051381:Rats
chemical_list: C073804:FM1 43; D005456:Fluorescent Dyes; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D002118:Calcium