SIMULTANEOUS MEASUREMENT OF [CA2+](I) AND SECRETION-COUPLED MEMBRANE TURNOVER, BY SINGLE-CELL FLUORESCENCE MICROSCOPY

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 mu M) 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 GH(3) rat pituitary cells, probably because they contain too few secretory granules to give a detectable increase. The dopamine D-2-receptor agonist quinpirole (10 mu M) 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 mu M) 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 stare, 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 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 Ca2+-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.