Fluorescence and laser photon counting: measurements of epithelial [Ca2+]i or [Na+]i with ciliary beat frequency.
We describe a system we developed that enabled simultaneous measurements of either epithelial calcium ion concentration ([Ca2+]i) or sodium ion concentration ([Na+]i) with the ciliary beat frequency (CBF) in native ciliated epithelia using either Fura-2 (AM) or SBFI (AM) ratiometric fluorescence photon counting along with nonstationary laser light scattering. Studies were performed using native epithelial tissues obtained from ovine tracheae. The dynamic range of the laser light-scattering system was determined by a simulated light "beating" experiment. The nonstationary CBF was demonstrated by the time-frequency analysis of the raw photon count sequences of backscattered heterodyne photons from cultured and native epithelia. Calibrations of calcium and sodium ion concentrations were performed using the respective Fura-2 and SBFI impermanent salts as well as in native epithelia. The cumulative responses of 10(-6), 10(-5), and 10(-4) M nifedipine on [Ca2+]i together with the CBF as well as the cumulative responses of 10(-5), 10(-4), and 10(-3) M amiloride on [Na+]i together with the CBF were also determined. Nifedipine decreased [Ca2+]i but had no effect on CBF. Amiloride decreased [Na+]i and CBF. Stimulation of CBF corresponded with either an increase of [Na+]i or an increase of [Ca2+]i. Decreases of [Na+]i or substantial decreases of [Ca2+]i were associated with decreases in the CBF. These data demonstrate the utility of this system for investigating the regulatory mechanisms of intracellular ions dynamics and the CBF in native epithelia.
Mao
H
H
Department of Medicine and Bioengineering Program, University of Illinois at Chicago, 60612, USA.
Wong
L B
LB
D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D001572:Benzofurans; D001698:Biomedical Engineering; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002478:Cells, Cultured; D002923:Cilia / Q000187:drug effects / Q000502:physiology*; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D004988:Ethers, Cyclic; D005456:Fluorescent Dyes; D016257:Fura-2; D007424:Intracellular Fluid / Q000378:metabolism; D007834:Lasers; D009543:Nifedipine / Q000494:pharmacology; D017785:Photons; D012542:Scattering, Radiation; D012756:Sheep; D012964:Sodium / Q000378:metabolism*; D014132:Trachea / Q000166:cytology / Q000187:drug effects / Q000502:physiology
D001572:Benzofurans; D002121:Calcium Channel Blockers; D004988:Ethers, Cyclic; D005456:Fluorescent Dyes; C061665:sodium-binding benzofuran isophthalate; D000584:Amiloride; D012964:Sodium; D009543:Nifedipine; D002118:Calcium; D016257:Fura-2