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 measuremen ts of either epithelial calcium ion concentration ([Ca2+](i)) or sodiu m ion concentration ([Na+](i)) with the ciliary beat frequency (CBF) i n native ciliated epithelia using either Fura-2 (AM) or SBFI (AM) rati ometric fluorescence photon counting along with nonstationary laser li ght scattering. Studies were performed using native epithelial tissues obtained from ovine tracheae. The dynamic range of the laser Light-sc attering system was determined by a simulated light ''beating'' experi ment. The nonstationary CBF was demonstrated by the time-frequency ana lysis of the raw photon count sequences of backscattered heterodyne ph otons from cultured and native epithelia. Calibrations of calcium and sodium ion concentrations were performed using the respective Fura-2 a nd SBFI impermanent salts as well as in native epithelia. The cumulati ve responses of 10(-6), 10(-5), and 10(-4) M nifedipine on [Ca2+](i) t ogether 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 al so determined. Nifedipine decreased [Ca2+](i) but had no effect on CBF . Amiloride decreased [Na+](i) and CBF. Stimulation of CBF corresponde d with either an increase of [Na+](i) or an increase of [Ca2+](i). Dec reases of [Na+](i) or substantial decreases of [Ca2+](i) were associat ed with decreases in the CBF. These data demonstrate the utility of th is system for investigating the regulatory mechanisms of intracellular ions dynamics and the CBF in native epithelia. (C) 1998 Biomedical En gineering Society.