Sodium/calcium exchangers selectively regulate calcium signaling in mouse taste receptor cells

Abstract

Taste cells use multiple signaling mechanisms to generate appropriate cellular responses to discrete taste stimuli. Some taste stimuli activate G protein coupled receptors (GPCRs) that cause calcium release from intracellular stores while other stimuli depolarize taste cells to cause calcium influx through voltage-gated calcium channels (VGCCs). While the signaling mechanisms that initiate calcium signals have been described in taste cells, the calcium clearance mechanisms (CCMs) that contribute to the termination of these signals have not been identified. In this study, we used calcium imaging to define the role of sodium-calcium exchangers (NCXs) in the termination of evoked calcium responses. We found that NCXs regulate the calcium signals that rely on calcium influx at the plasma membrane but do not significantly contribute to the calcium signals that depend on calcium release from internal stores. Our data indicate that this selective regulation of calcium signals by NCXs is due primarily to their location in the cell rather than to the differences in cytosolic calcium loads. This is the first report to define the physiological role for any of the CCMs utilized by taste cells to regulate their evoked calcium responses.

Fig. 1.

Sodium-calcium exchangers (NCXs) significantly regulate calcium influx through voltage-gated calcium channels (VGCCs). A: replacing external sodium with lithium (light gray column) caused a reversible increase in cytosolic calcium levels while a 10 s application of 50 mM KCl (arrow) depolarized the taste cells and opened VGCCs. The calcium influx through the VGCCs generated a large increase in cytosolic calcium that slowly returned to baseline levels. B: inhibiting NCX activity alone generated a small increase in cytosolic calcium that was significantly smaller than the peak amplitude of the calcium response due to opening VGCCs (triple asterisk, P < 0.001, 1-way ANOVA with Bonferroni's post hoc analysis). Opening VGCCs while NCXs were inhibited did not affect the amplitude of the evoked response (P = 0.78). Data were plotted as average percent increase (with SE) over baseline calcium values. C: inhibiting NCX activity significantly increased the duration of the calcium response due to opening VGCCs (triple asterisk, P < 0.001, paired Student's t-test). Duration was measured at half-peak height. D: the area under the curve for each condition was integrated and compared using the 1-way ANOVA with a Bonferroni's post hoc analysis. 50 mM KCl + lithium was significantly larger than either 50 mM KCl or lithium alone (triple asterisk, P < 0.001). The area of the lithium induced response was significantly smaller than either 50 mM KCl or 50 mM KCl + lithium (double asterisk, P < 0.01). E: the integrated area under the 50 mM KCl + lithium elevation was significantly larger than the combination of the integrated areas reported in D for the calcium elevations that occurred when 50 mM KCl and lithium were applied individually (triple asterisk, P < 0.001, paired Student's t-test). Adding the area of the 50 mM KCl-evoked calcium response (light gray column, labeled [S]) to the integrated area of the calcium response when NCXs were inhibited (dark gray column, labeled [X]) was only 79% of the integrated area of the calcium response that was generated when VGCCs were open while NCXs were inhibited (striped column, labeled [B]).

Fig. 2.

NCXs do not significantly regulate bitter-evoked calcium responses. A: replacing external sodium with lithium (light gray column labeled Li) caused a reversible increase in cytosolic calcium levels while a 30 s application of 10 mM denatonium benzoate (dark gray column labeled D) caused calcium release from internal stores. When bitter receptors were activated while NCXs were inhibited, the cytosolic calcium response increased. B: analysis of the average peak increases for denatonium, external lithium, and denatonium + external lithium revealed that denatonium + lithium was significantly larger than the amplitude of the responses to lithium or denatonium alone (ANOVA, triple asterisk, P < 0.001). C: the area under the curve for each condition was integrated and compared using the 1-way ANOVA with a Bonferroni's post hoc analysis. Denatonium alone generated a significantly smaller response than lithium alone or lithium + denatonium (double asterisk, P < 0.01). No differences between the areas of lithium or lithium + denatonium were found. D: the integrated area under the lithium + denatonium response (Li + Den, striped column, labeled [B]) was approximately equal to the combination of the integrated areas reported in C for the calcium signals that occurred when denatonium (light gray column, labeled [S]) and lithium (dark gray column, labeled [X]) were applied individually.

Fig. 3.

NCXs do not significantly contribute to sweet- or umami-evoked calcium responses. A: in sweet-sensitive taste cells, saccharin (2 mM, 30 s, dark gray striped column labeled Sac) caused an elevation in cytosolic calcium that was comparable to the calcium response due to inhibiting NCX activity (light gray striped column). Activating a saccharin response while NCXs were inhibited did not significantly increase the amplitude of the response (B, ANOVA, n = 14, P = 0.78). C: in umami-sensitive taste cells, application of monopotassium glutamate (MPG) for 30 s (20 mM, dark gray column) elevated cytosolic calcium. Replacement of external sodium with lithium (light gray striped column) elevated cytosolic calcium due to the inhibition of NCX activity. MPG in the presence of external lithium caused a larger increase in cytosolic calcium compared with either condition alone. D: analysis using 1-way ANOVA of the average peak increases for MPG, external lithium and MPG + external lithium found no significant differences in the response amplitudes (P = 0.33).

Fig. 4.

NCXs do not contribute to the termination of denatonium-evoked calcium responses in dual-responsive taste cells. A: comparison of the amplitude of the evoked calcium responses in type II taste cells (bitter sensitive and lack VGCCs, light gray columns) and dual-responsive taste cells (bitter sensitive and express VGCCs, dark gray columns) found that dual responsive taste cells generated significantly larger responses to denatonium compared with type II cells (Student's t-test, aterisk, P < 0.05). There were no differences in the amplitude of the lithium-induced calcium elevations (P = 0.86). In dual responsive taste cells, the amplitude of the denatonium + lithium response was significantly higher than the calcium response that was generated in type II taste cells under the same conditions (asterisk, P < 0.05). B: comparisons of the averaged integrated area for the type II (light gray columns) and dual-responsive taste cells (dark gray columns) in response to either denatonium, lithium external, or denatonium + lithium revealed no significant differences (P = 0.72). C: an example of lithium effects on a bitter-evoked calcium elevation in a taste cell that releases calcium from internal stores in response to bitter stimuli but does not express VGCCs. D: an example of the effect of inhibiting exchanger activity on the bitter-evoked calcium elevation in a dual responsive taste cell that releases calcium from internal stores in response to bitter stimuli and also expresses VGCCs.

Fig. 5.

NCXs significantly contribute to the termination of calcium elevations due to influx through VGCCs, regardless of calcium load. A: replacement of external sodium with lithium (light gray column) caused an elevation in cytosolic calcium, whereas a 10 s application of 20 mM KCl (arrow) slightly depolarized the taste cells to open VGCCs and generated a small calcium influx. This response was much larger when it was repeated while NCXs were inhibited. B: analysis using 1-way ANOVA of the average peak increases for 20 mM KCl, external lithium, and 20 mM KCl + external lithium revealed that the peak amplitude of the response for 20 mM KCl + lithium was significantly larger than the amplitude of the responses to 20 mM KCl or lithium alone (double asterisk, P < 0.01). C: inhibiting NCX activity significantly increased the duration of the calcium response due to opening VGCCs (triple asterisk P < 0.001, paired Student's t-test). Duration was measured at half-peak height. D: the area under the curve for each condition was integrated and compared using the 1-way ANOVA with Bonferroni's post hoc analysis. 20 mM KCl + lithium was significantly larger than either 20 mM KCl or lithium alone (triple asterisk, P < 0.001). E: the integrated area under the 20 mM KCl + lithium elevation was significantly larger than the combination of the integrated areas reported in D for the calcium elevations that occurred when 20 mM KCl and lithium were applied individually (double asterisk, P < 0.01, paired Student's t-test). Adding the area of the 20 mM KCl-evoked calcium response (light gray column, labeled [S]) to the integrated area of the calcium response when NCXs were inhibited (dark gray column, labeled [X]) was 60% of the integrated area of the calcium response that was generated when VGCCs were opened while NCXs were inhibited (striped column, labeled [B]).