Use of a fluorescent dye to measure secretion from intact bovine anterior pituitary cells.
The fluorescent dye FM1-43 has been used to indicate membrane changes in individual bovine anterior pituitary cells exposed to secretory stimuli. After ten minutes incubation with FM1-43 (2 microM), cells showed three patterns of dye fluorescence: annular, partly filled and uniformly filled. FM1-43 fluorescence was increased in 61% of the cells by TRH (40 nM), a physiological stimulus for prolactin secretion, and in 89% of the cells by 60 mM external K+. The fluorescence also increased when cells incubated in the presence of quinpirole, a dopamine D2-receptor agonist which inhibits prolactin secretion, were exposed to raclopride, a D-2 antagonist. The increases in FM1-43 fluorescence caused by these treatments suggests that the dye acts as an indicator of secretion, possibly through incorporation into secretory vesicle membranes exposed on the cell surface during exocytosis. If the dye was washed away after loading, the fluorescence of partly and uniformly filled cells was retained and a rise in fluorescence could still be seen on stimulation by TRH. This suggests that some dye had been taken up by endocytosis and trapped in an intracellular compartment, which expanded through membrane recapture after TRH stimulation. FM1-43 could therefore be a useful probe for membrane cycling associated with secretory responses.
Stafford
S J
SJ
Department of Biochemistry, School of Medical Sciences, University of Bristol.
Shorte
S L
SL
Schofield
J G
JG
D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism / Q000502:physiology / Q000648:ultrastructure; D002478:Cells, Cultured; D015259:Dopamine Agents / Q000494:pharmacology; D018492:Dopamine Antagonists; D004705:Endocytosis / Q000502:physiology; D004873:Ergolines / Q000494:pharmacology; D005456:Fluorescent Dyes; D007091:Image Processing, Computer-Assisted; D008853:Microscopy / Q000379:methods; D010455:Peptides / Q000494:pharmacology; D010903:Pituitary Gland, Anterior / Q000166:cytology* / Q000378:metabolism*; D011188:Potassium / Q000494:pharmacology; D011388:Prolactin / Q000494:pharmacology; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D019257:Quinpirole; D020891:Raclopride; D012457:Salicylamides / Q000494:pharmacology; D013997:Time Factors
D015259:Dopamine Agents; D018492:Dopamine Antagonists; D004873:Ergolines; C073804:FM1 43; D005456:Fluorescent Dyes; D010455:Peptides; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D012457:Salicylamides; D019257:Quinpirole; D020891:Raclopride; D011388:Prolactin; D011188:Potassium