Simultaneous measurements of exocytosis and intracellular calcium concentration with fluorescent indicators in single pituitary gonadotropes.
Previously, we established a method for the estimation of exocytosis in single gonadotropes using an impermeable fluorescent membrane probe, TMA-DPH. In this study, we have developed a method for the simultaneous measurement of exocytosis and intracellular free Ca2+ concentration ([Ca2+]i) by double-labeling with TMA-DPH and the intracellular Ca2+ probe, Fura-2/AM, using a fluorescence microscope with a 3-wavelength excitation and 2-wavelength emission system. We, therefore, clarified the relationship between spontaneous [Ca2+]i oscillation or gonadotropin releasing hormone (GnRH)-induced intracellular Ca2+ mobilization and exocytosis in gonadotropes. Under resting conditions, some gonadotropes showed various types of spontaneous [Ca2+]i oscillations, while others did not, but all showed basal exocytosis. Each [Ca2+]i peak oscillation did not cause Ca(2+)-regulated exocytosis, and even complete blockage of the [Ca2+]i increase by the intracellular Ca2+ chelator BAPTA/AM had no effect on basal exocytosis. Both GnRH-induced intracellular Ca2+ mobilization and regulated exocytosis showed a similar pattern of peaks and plateaus. Blockage of the [Ca2+]i increase by BAPTA/AM almost completely inhibited the GnRH-stimulated exocytosis. These results show that spontaneous [Ca2+]i oscillations under resting conditions are not linked to regulated or basal exocytosis, and that intracellular Ca2+ mobilization is essential for GnRH-stimulated exocytosis.
Department of Obstetrics and Gynecology, Osaka University Medical School, Japan.
N
N
Masumoto
K
K
Tasaka
J
J
Mizuki
K
K
Fukami
Y
Y
Ikebuchi
A
A
Miyake
D000818:Animals; D002118:Calcium / Q000032:analysis*; D002614:Chelating Agents / Q000494:pharmacology; D004161:Diphenylhexatriene / Q000031:analogs & derivatives*; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005089:Exocytosis / Q000187:drug effects; D005260:Female; D005456:Fluorescent Dyes; D005640:Follicle Stimulating Hormone / Q000378:metabolism; D016257:Fura-2 / Q000031:analogs & derivatives*; D007987:Gonadotropin-Releasing Hormone / Q000494:pharmacology; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D007986:Luteinizing Hormone / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods; D010903:Pituitary Gland, Anterior / Q000737:chemistry / Q000166:cytology* / Q000187:drug effects; D051381:Rats; D017208:Rats, Wistar
D002614:Chelating Agents; D005456:Fluorescent Dyes; D007476:Ionophores; C049925:fura-2-am; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004161:Diphenylhexatriene; D007987:Gonadotropin-Releasing Hormone; D004533:Egtazic Acid; D015759:Ionomycin; C033628:1-(4-(trimethylamino)phenyl)-6-phenylhexa-1,3,5-triene; D007986:Luteinizing Hormone; D005640:Follicle Stimulating Hormone; D002118:Calcium; D016257:Fura-2