Article Figures & Data
Figures
- Figure 1.
CHO cells stably expressing α1A receptors respond to bath-applied NE and can be used as NE biosensors. Cells were loaded with fura-2 and Ca2+ mobilization was measured. A , Concentration-response relationships for NE. Open circles indicate the mean ± SEM (N = 14 cells). B , NE biosensor responses evoked by 1 μm ATP to stimulate endogenous purinoceptors and 10 nm NE to excite transfected α1A receptors. Filled columns, control responses. Open columns, effect of prazosin (1 μm), an antagonist of α1A receptors. ATP and NE responses were measured in parallel, and responses were normalized to each agonist (in absence of prazosin) separately. As expected, prazosin selectively abolished the responses evoked by NE (***p < 0.0001). C , NE biosensor cells on their own do not respond to KCl depolarization or to stimulation with ATP or taste compounds. Trace shows mean ± SEM (gray bars) of Ca2+ responses from NE biosensor cells (N = 6) after incubating with 500 μm ATP to desensitize endogenous purinoceptors. NE, 10 nm; KCl, 50 mm; ATP, 10 μm; taste, mixture of cycloheximide (10 μm), saccharin (2 mm), SC45647 (0.1 mm), and denatonium (1 mm). Ordinate, calibration in units of F340/F380 in this and subsequent figures.
- Figure 2.
Stimulating taste buds evokes Ca-dependent release of norepinephrine. A , An NE biosensor cell positioned against a taste bud responds when the taste bud was depolarized by 50 mm KCl. Removing Ca2+ from the bathing medium (“0 Ca”) abolished stimulus-evoked NE release from the taste bud. B , Applying prazosin (“praz,” 1 μm) reversibly reduced NE biosensor responses evoked by depolarizing taste buds with KCl, verifying that biosensor responses originated from α1A receptors and thus confirming that NE was the transmitter being released. C , Taste stimuli (“taste,” cycloheximide, 10 μm; saccharin, 2 mm; SC45647, 0.1 mm; denatonium, 1 mm) and ATP (1 μm) also evoked NE release from taste buds. D , Prazosin blocked ATP-evoked biosensor responses, verifying that biosensor responses were generated by NE release and not elicited by activating endogenous biosensor cell purinoceptors. In all cases, endogenous biosensor cell purinoceptors were desensitized before the experiments, as described in Materials and Methods. Calibration: vertical, 0.5; horizontal, seconds, as marked.
- Figure 3.
Presynaptic (type III) taste cells, but not receptor (type II) cells, secrete NE. GAD-GFP mice were used to isolate and identify individual presynaptic taste cells; PLCβ2-GFP mice were used to identify receptor taste cells. A , Receptor cells do not secrete NE. The pair of traces shows simultaneous Ca2+ recordings in a receptor taste cell (Rec) and an apposed NE biosensor cell (NE bio). NE (10 nm) was applied first to verify biosensor sensitivity. Taste stimulation was a mixture, as in Figure 2. B , Presynaptic cells secrete NE. Stimulus-evoked responses are from the presynaptic cell shown in C2 (next). Stimulating with KCl (50 mm) or acetic acid (“HOAc,” 10 mm, pH 5.0) triggered Ca2+ responses in the presynaptic cell (Pre) and NE release (NE bio). As in A , 10 nm NE was applied initially to validate the NE biosensor. C , Fluorescence and interference contrast optics micrographs showing a NE biosensor (“NE-bio”) apposed to identified presynaptic cell (i.e., expressing GFP, “pre”). Traces in B were taken from the presynaptic cell shown in C 2. Note that debris attached to cells in C2 did not interfere with responses.
- Figure 4.
A subset of presynaptic (type III) taste cell corelease 5-HT and NE. Individual presynaptic taste cells were isolated and identified and tested for transmitter release with a dual biosensor, sensitive to either NE or 5-HT (NE- and 5-HT biosensor), as described in Materials and Methods. As in Figure 3, simultaneous recordings were made from a presynaptic cell and an apposed dual biosensor. A , KCl depolarization triggered Ca2+ responses in the presynaptic cell (pre; top) and evoked NE release (NE/5-HT bio; bottom). Mianserin (“mian,” 1 nm), a 5-HT2c receptor antagonist, reversibly blocked the dual biosensor cell responses, indicating that in this case, the presynaptic cell only released 5-HT. B , In other cases (4 of 13), mianserin or prazosin (“praz,” 1 μm) alone only partially blocked responses from the dual biosensor, but adding mianserin together with prazosin strongly inhibited dual biosensor responses. This finding indicates that in some cases, stimulating presynaptic taste cells coreleases 5-HT and NE.
- Figure 5.
Norepinephrine does not affect stimulus-evoked transmitter release (ATP, 5-HT) from receptor (type II) or presynaptic (type III) taste cells. A , Simultaneous recordings from an isolated taste receptor cell (top) and an apposed ATP-biosensor to monitor transmitter secretion during taste stimulation (Fig. 2). Taste-evoked responses in the receptor cell and ATP secretion were unaffected in the presence of even relatively high concentrations of NE (100 μm; shaded area). B , Summary of the effects of NE on taste receptor cells and taste-evoked ATP secretion. Error bars represent mean ± SEM of taste-evoked responses in receptor cells (gray bars) and in ATP biosensors (white bars) (N = 6 cells). Individual responses were normalized to the (pooled) average taste evoked responses for all experiments in this series. No significant effects were of applying NE. C , Similarly, comparable recordings taken from an isolated presynaptic taste cell, and an apposed 5-HT biosensor cell showed no effects of applying 100 μm NE. D , As in B , summary of data from eight cells showing the lack of effect of NE on KCl-evoked responses in presynaptic cells and in stimulus-evoked 5-HT release.
