TY - JOUR
T1 - Measurement of membrane potential and [Ca2+](i) in cell ensembles
T2 - Application to the study of glutamate taste in mice
AU - Hayashi, Yukako
AU - Zviman, M. Muz
AU - Brand, Joseph G.
AU - Teeter, John H.
AU - Restrepo, Diego
N1 - Funding Information:
This work was supported by National Institutes of Health grants DC001838 and DC00566, and by a grant from the Veterans Affairs Department to JGB.
Copyright:
Copyright 2017 Elsevier B.V., All rights reserved.
PY - 1996/8
Y1 - 1996/8
N2 - We have studied the spectral properties of the voltage-sensitive dye, 1- (3-sulfonatopropyl)-4-[β[2-(di-n-octylamino)-6-naphtyl]vinyl] pyridinium betaine (di-8-ANEPPS), and the Ca2+-sensitive dye, fura-2, in azolectin liposomes and in isolated taste buds from mouse. We find that the fluorescence excitation spectra of di-8-ANEPPS and fura-2 are largely nonoverlapping, allowing alternate ratio measurements of membrane potential and intracellular calcium ([Ca2+](i)). There is a small spillover of di-8- ANEPPS fluorescence at the excitation wavelengths used for fura-2 (340 and 360 nm). However, voltage-induced changes in the fluorescence of di-8- ANEPPS, excited at the fura-2 wavelengths, are small. In addition, di-8- ANEPPS fluorescence is localized to the membrane, whereas fura-2 fluorescence is distributed throughout the cytoplasm. Because of this, the effect of spillover of di-8-ANEPPS fluorescence in the [Ca2+](i) estimate is <1%, under the appropriate conditions. We have applied this method to study of the responses of multiple taste cells within isolated taste buds. We show that membrane potential and [Ca2+](i) can be measured alternately in isolated taste buds from mouse. Stimulation with glutamate and glutamate analogs indicates that taste cells express beth metabotropic and ionotropic receptors. The data suggest that the receptors responding to 2-amino-4- phosphonobutyrate (L-AP4), presumably metabotropic L-glutamate receptors, do not mediate excitatory glutamate taste responses.
AB - We have studied the spectral properties of the voltage-sensitive dye, 1- (3-sulfonatopropyl)-4-[β[2-(di-n-octylamino)-6-naphtyl]vinyl] pyridinium betaine (di-8-ANEPPS), and the Ca2+-sensitive dye, fura-2, in azolectin liposomes and in isolated taste buds from mouse. We find that the fluorescence excitation spectra of di-8-ANEPPS and fura-2 are largely nonoverlapping, allowing alternate ratio measurements of membrane potential and intracellular calcium ([Ca2+](i)). There is a small spillover of di-8- ANEPPS fluorescence at the excitation wavelengths used for fura-2 (340 and 360 nm). However, voltage-induced changes in the fluorescence of di-8- ANEPPS, excited at the fura-2 wavelengths, are small. In addition, di-8- ANEPPS fluorescence is localized to the membrane, whereas fura-2 fluorescence is distributed throughout the cytoplasm. Because of this, the effect of spillover of di-8-ANEPPS fluorescence in the [Ca2+](i) estimate is <1%, under the appropriate conditions. We have applied this method to study of the responses of multiple taste cells within isolated taste buds. We show that membrane potential and [Ca2+](i) can be measured alternately in isolated taste buds from mouse. Stimulation with glutamate and glutamate analogs indicates that taste cells express beth metabotropic and ionotropic receptors. The data suggest that the receptors responding to 2-amino-4- phosphonobutyrate (L-AP4), presumably metabotropic L-glutamate receptors, do not mediate excitatory glutamate taste responses.
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U2 - 10.1016/S0006-3495(96)79306-2
DO - 10.1016/S0006-3495(96)79306-2
M3 - Article
C2 - 8842242
AN - SCOPUS:0029740020
VL - 71
SP - 1057
EP - 1070
JO - Biophysical Journal
JF - Biophysical Journal
SN - 0006-3495
IS - 2
ER -