title: Continuous on-line monitoring of secretion from rodent pituitary endocrine cells using fluorescent protein surrogate markers.
abstract: We have developed a system to use secreted fluorescent proteins (FPs) as surrogate markers for the continuous on-line monitoring of hormone release from perfused tissue slices. We have tested this system using GH-GFP transgenic rats with green fluorescent protein (GFP) targeted to the secretory vesicles (SVs) of pituitary growth hormone (GH) cells. Brief exposures of vibratome slices to GH secretagogues [GH-releasing hormone (GHRH), GH-releasing peptide-6 (GHRP-6)] or somatostatin caused changes in FP output that correlate with hormone secretion, subsequently measured in fractions of perfusate by radioimmunoassay. The temporal resolution of this method was capable of revealing differences in the kinetics of response to GHRH and GHRP-6 between wild-type and dwarf (dw/dw) rats harbouring the GH-GFP transgene. We further tested the utility of the system by generating transgenic mice with red FPs targeted to secretory vesicles (PRL-mRFP(sv)) and to the cytoplasm (PRL-DsRed(cyto)) of lactotrophs. Dopamine had no effect on the FP output from pituitary slices of PRL-DsRed(cyto) mice but inhibited output from those of PRL-mRFP(sv) animals, with a rebound increase of release after removal, which again correlated with hormone output measured in the perfusate by radioimmunoassay. The inhibition of monomeric RFP secretion by dopamine was dose-dependent, as was stimulation by low concentrations of oxytocin. The temporal resolution afforded by this method provides useful insight into the release kinetics from large populations of pituitary cells, and fills a temporo-spatial gap between single vesicle and single cell monitoring of exocytosis in milliseconds, and in vivo sampling studies of release into the bloodstream on a time scale of minutes.
authors.affiliation: Division of Molecular Neuroendocrinology, MRC National Institute for Medical Research, London, UK.
authors.forename: Z
authors.initials: Z
authors.lastname: He
authors.forename: M
authors.initials: M
authors.lastname: Fernandez-Fuente
authors.forename: M
authors.initials: M
authors.lastname: Strom
authors.forename: L
authors.initials: L
authors.lastname: Cheung
authors.forename: I C
authors.initials: IC
authors.lastname: Robinson
authors.forename: P
authors.initials: P
authors.lastname: Le Tissier
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000032:analysis / Q000378:metabolism; D058948:Cell Tracking / Q000295:instrumentation / Q000379:methods*; D016903:Drug Monitoring / Q000295:instrumentation / Q000379:methods*; D004392:Dwarfism / Q000378:metabolism / Q000473:pathology; D055098:Endocrine Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D005260:Female; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000378:metabolism* / Q000493:pharmacokinetics; D013007:Growth Hormone-Releasing Hormone / Q000494:pharmacology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D008822:Mice, Transgenic; D009842:Oligopeptides / Q000494:pharmacology; D009862:Online Systems; D010477:Perfusion; D051381:Rats; D055647:Rats, Transgenic; D013004:Somatostatin / Q000494:pharmacology; D052683:Somatotrophs / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology
chemical_list: D015415:Biomarkers; D009842:Oligopeptides; D049452:Green Fluorescent Proteins; C041048:growth hormone releasing hexapeptide; D013004:Somatostatin; D013007:Growth Hormone-Releasing Hormone