title: Mitochondrial membrane potential changes in osteoblasts treated with parathyroid hormone and estradiol.
abstract: This study assessed mitochondrial membrane potential changes in cultured osteoblasts treated with hormones known to regulate osteoblasts. A fluorescent carbocyanine dye, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine++ + iodide, also called JC-1, was used as a probe. JC-1 emits photons at 585 nm (orange-red) when the membrane potential in mitochondria is highly negative, but when the potential becomes reduced emission occurs at 527 nm (green). Osteoblasts were rinsed in serum-free medium for 5 min, then loaded with 1 x 10(-6) M JC-1 for 10 min. The distribution and intensity of JC-1 fluorescence were evaluated with a laser-scanning confocal microscope system. Hormone treatments included parathyroid hormone (PTH; 10(-8) M), 17beta-estradiol (10(-8) M), and thyroxine (T4; 10(-8) M). The potassium ionophore valinomycin (10(-6) M) was used as a control since it is known to disrupt the electrochemical gradient of mitochondria without interfering with the pH gradient. Valinomycin caused a profound, rapid increase (22.5% above untreated values) in the green/red ratio, which indicated a lowering of the mitochondrial membrane potential in all samples evaluated. PTH caused a less pronounced, but significant (7-14%), reduction in membrane potential in all cells examined. PTH is known to affect osteoblasts in a number of ways and is inhibitory to mitochondrial respiration; the results confirm this effect. For estradiol, half of the cells responded at a significant level, with a membrane potential reduction of 6 to 13% being recorded; the other half did not respond. Thyroxine did not alter mitochondrial membrane potential. Responses were detectable within 20 s for valinomycin, but occurred at a slower rate, over 200 to 300 s, following PTH and estradiol treatment. Responses to PTH and estradiol could be due to mitochondrial uptake of cytosolic Ca2+.
authors.affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park 16802, USA.
authors.forename: M B
authors.initials: MB
authors.lastname: Troyan
authors.forename: V R
authors.initials: VR
authors.lastname: Gilman
authors.forename: C V
authors.initials: CV
authors.lastname: Gay
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D002645:Chickens; D004958:Estradiol / Q000494:pharmacology*; D005456:Fluorescent Dyes; D007425:Intracellular Membranes / Q000502:physiology; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000502:physiology*; D010006:Osteoblasts / Q000187:drug effects / Q000502:physiology*; D010281:Parathyroid Hormone / Q000494:pharmacology*; D011188:Potassium / Q000502:physiology; D013974:Thyroxine / Q000494:pharmacology; D014634:Valinomycin / Q000494:pharmacology
chemical_list: D005456:Fluorescent Dyes; D010281:Parathyroid Hormone; D014634:Valinomycin; D004958:Estradiol; D013974:Thyroxine; D011188:Potassium