Article Figures & Data
Figures
- Fig. 1.
Denatonium increased intracellular calcium levels. A, Light image of an isolated taste cell. Scale bar, 20 μm. B, Fluorescence image of the same cell showing the apical tip of the taste cell labeled with FITC-WGA. The labeled region is shown in white in this pseudocolor image. C, Calcium images of the same taste cell loaded with the Ca2+-sensitive dye fura-2. The pseudocolor scale of [Ca2+]i is shown on theright. a, [Ca2+]i before and b, immediately after application of 5 mm denatonium benzoate. The increase in [Ca2+]i begins at the apical tip. c, [Ca2+]i 30 sec after application of denatonium benzoate. [Ca2+]iincreases over entire cell. d, [Ca2+]i after washing the cells for 2 min with APS. [Ca2+]i returns to resting level.D, Typical time course of denatonium-induced calcium responses. Denatonium benzoate (5 mm) was applied during the periods labeled DN. Note that the denatonium-induced responses are similar after repeated application of denatonium.
- Fig. 2.
Time courses of changes in [Ca2+]i. Measurements of [Ca2+]i in individual cells using fura-2. Denatonium benzoate (5 mm) was applied during the periods labeled DN. A, The denatonium-induced calcium response was present in Ca2+-free extracellular solution. B, Thapsigargin (1 μm) abolished the denatonium-induced calcium response. C, The denatonium-induced calcium response was not affected by ryanodine (10 μm). D, The denatonium-induced calcium response was abolished by the PLC inhibitor U73122 (5 μm).
- Fig. 3.
Denatonium-induced changes in [Ca2+]i depended on intracellular stores. This graph shows maximum denatonium-induced changes in [Ca2+]i expressed as a percentage of resting [Ca2+]i. Cells were tested twice, before (hatched bars) and during or after (open bars) the treatments indicated, as illustrated in Figure 2[Ca2+-free bath solution (0 Ca; n = 17), thapsigargin (n = 34), ryanodine (n = 12), and U73122 (n = 18)]. Control cells were tested twice, as illustrated in Figure1D (n = 16), the first and second stimulations indicated by the hatched andopen bars, respectively.
- Fig. 4.
Electrophysiological response of a mudpuppy taste cell to denatonium. The bath contained TTX to block inward Na+ currents. A, Whole-cell recording under control conditions and in response to 1 mm denatonium benzoate (DN). The cell was held at −80 mV, and the membrane was stepped to +20 mV to elicit outward current. Leak and linear capacitative currents were subtracted from the record by computer. B, Current–voltage relationship of the denatonium response in the same cell, as shown in A. Notice that denatonium increased outward currents at most voltages (solid circles).
- Fig. 5.
Time course of the electrophysiological response to denatonium. The cell was held at −80 mV, and the membrane was stepped to +20 mV for 175 msec every 3 sec. Data were digitized at 125 Hz and plotted with Axoscope software. TTX was present in the bath solution to block Na+ currents. Note that 1 mmdenatonium elicited an increase in outward current, which is similar in kinetics to the [Ca2+]i response shown in Figures 1 and 2.
- Fig. 6.
Effect of GDP-β-S on the denatonium-induced outward current. This cell was held at −80 mV, and the membrane was stepped to +80 mV. TTX was present in the bath solution to block Na+ currents. A, After 6 min of whole-cell recording and B, after 15 min of whole-cell recording. Note that the GDP-β-S (1 mm in pipette solution) abolished the denatonium response, suggesting that the response was G-protein-dependent.
- Fig. 7.
Denatonium-induced changes in [Ca2+]i did not depend on intracellular cAMP. This graph shows maximum denatonium-induced changes in [Ca2+]i expressed as a percentage of resting [Ca2+]i. Control cells are those illustrated in Figure 3. PTX cells represent two groups of cells: a control group incubated overnight in APS (hatched bar,n = 13) and a treatment group incubated overnight in PTX (open bar, n = 24). Other cells were tested twice before (hatched bars) and during or after (open bars) the treatments indicated: IBMX (n = 17); cell permeant cyclic nucleotides (cNMP: db-cAMP, n = 4; db-cGMP, n = 6; 8-cpt-cAMP + db-cGMP, n = 3), and SQ22536 (n = 3).
