The global calcium responses we observed previously in response to T2R38 activation 10 were not observed, which was expected because denatonium does not activate T2R38 However, this result also suggests that the ciliated epithelial cells expressing T2R38 do not express denatonium-responsive T2Rs. Denatonium-responsive T2Rs are instead expressed in a different cell type. This is intriguing, as it suggests that denatonium-responsive T2Rs serve a unique and likely novel function from the T2R38 nitric oxide response observed in ciliated cells.
This result implicates signal propagation through gap junction communication but not through the release of extracellular purines. The duration of denatonium-induced calcium responses was reduced by either removal of extracellular calcium or application of triphenylphosphine oxide Supplemental Figure 3B , an inhibitor of the TRPM5 component of taste signaling Additionally, the inositol triphosphate receptor IP 3 R inhibitor xestospongin B significantly inhibited the denatonium-induced calcium response Supplemental Figure 3C.
Repeated stimulation with denatonium induced minimal tachyphylaxis Supplemental Figure 4 , suggesting that these cells recover their signaling capacity e. Calcium responses in sinonasal ALIs during stimulation with denatonium benzoate. A Representative trace showing dose-dependent calcium elevations in response to denatonium.
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B Dose response plot of data from experiments shown in A. Each red data point is the mean of results from 5 to 12 experiments. C Representative images showing propagation of calcium signal from single denatonium-responding cells compared to global calcium mobilization by ATP.
Arrows denote single denatonium-responsive cells. Images shown are representative of 5 to 12 experiments for each condition. The localized denatonium responses observed in sinonasal epithelial cells upper airway contrast with those in human bronchial epithelial cells lower airway , where denatonium responses are more global ref. To confirm that the sinonasal cultures reflected the in vivo response, we performed calcium imaging of human sinonasal explants. The resulting denatonium-induced calcium responses also appeared to emanate from distinct points of origin similar to those observed in sinonasal cultures Supplemental Figure 5B.
These cultures contain ciliated cells and goblet cells and likely contain SCCs, and our data suggest that they recapitulate the in vivo epithelial physiology. We found that primary enzymatically dissociated bronchial ciliated epithelial cells responded to denatonium Supplemental Figure 6A , but nasal ciliated epithelial cells did not, despite robust ATP-induced responses Supplemental Figure 6B. In the process of screening bitter compounds, we found that absinthin activated a similar pattern of calcium signaling to that of denatonium and that it originated from the same denatonium-responsive cells Supplemental Figure 7, A—D.
Impalement of the sinonasal denatonium-responsive cells and subsequent injection of Lucifer yellow biocytin in the presence of carbenoxolone confirmed that these cells are nonciliated, unipolar Supplemental Figure 7E , and morphologically similar to SCCs 11 , 12 and to cells observed via immunofluorescence for the denatonium and absinthin-responsive T2R47 Supplemental Figure 7F using an antibody validated against transfected HEK cells Supplemental Figure 8.
Like T2R47, we observed that immunofluorescence for the T1R2 subunit of the sweet receptor was localized to solitary nonciliated cells Supplemental Figure 7G. Costaining of T2R47 with the T1R3 subunit revealed coexpression in the same unipolar cell type Figure 2 , which is likely the same as the SCCs previously described in mice and similar in morphology to the denatonium-responsive cells Supplemental Figure 7. These cells also appeared to express the type III IP 3 R IP 3 R3 , an intracellular calcium release channel that is expressed in chemosensory cells and important for taste signaling 23 , Human sinonasal chemosensory cells express both bitter and sweet taste receptors as well as IP 3 R3.
Merge shows T1R3 and T2R47 signals. B T2R47 staining was reduced when T2R47 antibody was preincubated with antigenic blocking peptide 2 hours before staining. However, when experiments were performed with carbenoxolone to block gap junctions and isolate the denatonium-responsive cells, glucose inhibited both denatonium- and ATP-induced calcium signals Figure 3 , D and E within the denatonium-responsive cells but not in cells that did not respond to denatonium. Cells that were not denatonium-responsive exhibited glucose-independent purinergic responses.
Sweet receptor activation inhibits T2R-activated calcium signaling. Experiments in A — C were performed without carbenoxolone. Together, these results support that the denatonium-responsive cells in the lower airway are ciliated epithelial cells 9 , while the sinonasal denatonium-responsive cells are nonciliated cells that express both bitter and sweet receptors, strongly suggesting that these cells are sinonasal SCCs Murine nasal SCCs have been more thoroughly characterized than human SCCs 11 , 14 , and thus we also examined murine nasal septal cultures 29 to determine whether the response to denatonium mimicked human sinonasal cultures.
As expected, the denatonium-induced calcium response was absent in gustducin knockout mice and altered faster decay in TRPM5 knockout mice but remained intact in cultures derived from Myd88 knockout mice, suggesting that it is independent of TLR signaling Supplemental Figure 11, E—I and further supporting this response as specific to taste signaling. Prior work suggested that denatonium stimulates calcium-dependent increases in CBF in human bronchial cells 9 , and, thus, we sought to determine whether this is the case in sinonasal cells.
We hypothesize that this effect is from intracellular acidification secondary to permeation of benzoic acid. Direct imaging of intracellular pH suggested that 10 mM denatonium benzoate and 10 mM Na benzoate caused a similar level of intracellular acidification. In agreement with this hypothesis, neither denatonium saccharide, which lacks the benzoic acid moiety, nor the other T2R agonists absinthin, parthenolide, or amarogentin caused a decrease in CBF Supplemental Figure 12, J and K.
These agonists also failed to cause any CBF increase. This agrees with our data suggesting that sinonasal ciliated epithelial cells do not express functional denatonium- or absinthin-responsive T2Rs. It is not likely that the SCC-activated gap junction—propagated calcium response is properly localized to elevate CBF, a phenomenon similar to what we have previously observed when sinonasal epithelial cells are stimulated with histamine, an agonist that elevates calcium and stimulates fluid secretion but nonetheless has no effect on CBF Because we have shown that T2R38 detects gram-negative bacterial quorum—sensing molecules and activates innate immunity responses 10 , we hypothesized that the denatonium- and absinthin-responsive T2Rs likewise serve a role in innate immunity and thus began to examine other sinonasal epithelial innate immunity responses.
Although denatonium benzoate does not stimulate human sinonasal ALIs to produce bactericidal nitric oxide or reactive nitrogen species 10 or to secrete cytokines Supplemental Figure 13 , we found that denatonium does induce a bactericidal response. The solutions used in these experiments had no cell-independent effects on bacterial growth when tested alone, and there was no antibacterial activity when denatonium was added to PBS ASL after removal from the cells Supplemental Figure 14, A—C.
Together, these data suggest that this is a receptor- and calcium-dependent response and that the propagation of the T2R calcium signal to the surrounding cells is essential for antimicrobial secretion. Interestingly, inhibition of TRPM5 had no effect on bacterial killing Figure 4 C , suggesting that the observed antibacterial effects are dependent on intracellular calcium release. IP 3 R3 phosphorylation by PKA has been suggested to inhibit calcium signaling in some cells, including pancreatic acinar cells 34 , A similar mechanism may be operating here.
B Confirmation of bacterial kill by live-dead staining of planktonic Pseudomonas. Syto 9 and propidium iodide PI indicate live and permeabilized dead bacteria, respectively The images are representative of 5 experiments using ASL from 5 patients. Glucose inhibition was reversed by lactisole lact.
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Concentrations shown are in mM. These results follow the calcium data presented above and suggest that this response is unique to taste signaling mediated by a subset of T2Rs. Denatonium-stimulated ASL also effectively killed methicillin-resistant Staphylococcus aureus MRSA , Klebsiella pneumonia , and Staphylococcus epidermis , suggesting that the secretions are broad-spectrum antimicrobials Figure 4 E. These data strongly suggest that T2Rs have novel roles in sinonasal innate immunity beyond the role we previously demonstrated for T2R38 10 , strengthening the hypothesis that airway T2Rs represent a novel arm of innate immunity, functioning on a much faster time span than TLRs and Nod-like receptors 36 , and may be important therapeutic targets for upper respiratory infections.
Thus, we examined the denatonium-stimulated antimicrobial secretions in more detail. The secretion of bactericidal activity occurred within 10 minutes of denatonium stimulation, while bacterial killing a reduction in the number of CFUs occurred over a time course of approximately 2 hours of exposure to the secreted antimicrobials Figure 5 , A and B.
Sweet acceptance versus bitter resistance / by Peter Sacco
Notably, after stimulation and secretion of bactericidal compounds, the ALI cultures exhibited a refractory period before they could mount a second antibacterial secretion response, as ASL from cultures stimulated the day after a prior stimulation 1-day recovery did not kill Pseudomonas. Rather, cultures required a minimum of 3 days of recovery to restore bactericidal activity in the ASL Figure 5 C. This suggested that protein synthesis is required and that the secreted factors may be AMPs.
Supporting this, we found that NaCl inhibited the bactericidal activity in a dose-dependent fashion. Antibacterial activity was reduced with increasing [NaCl] Figure 6 C in a manner independent of any effects of NaCl on secretion described in the Methods. Increasing ionic strength prevents AMP function 38 , 39 , likely by shielding charge and preventing AMP interactions necessary for antibacterial effects.
Kinetics of the denatonium-induced antimicrobial response. Decreased CFU count equals a reduced number of viable bacteria. Human sinonasal ALI cultures secreted antimicrobials over the course of 5 to 10 minutes.
B ALI cultures were stimulated with denatonium for 30 minutes, and ASL was mixed with Pseudomonas for varying periods of time before dilution and spotting on LB plates. C Cultures were stimulated with 10 mM denatonium benzoate and then either stimulated the next day 1-day recover or given 3 or 6 days to recover. A Coomassie blue—stained gel showing a low-molecular-weight band in denatonium-stimulated ASL representative of 3 gels; 6 patients.
Anderson , H. Alimohammadi , and W.
Solitary chemoreceptor cells in the nasal cavity serve as sentinels of respiration. Danilova , J. Barrows , D. Bartel , A. Vigers , L. Stone , G. Hellekant , and S.
ATP signaling is crucial for communication from taste buds to gustatory nerves. Foster , S. Porrello , B. Purdue , H. Chan , A.
Sweet Acceptance Versus Bitter Resistance by Peter Sacco
Voigt , S. Frenzel , R. Hannan , K. Moritz , D. Simmons , P. Molenaar , et al. Expression, regulation and putative nutrient-sensing function of taste GPCRs in the heart. Blank , L. See Hoe , M. Behrens , W. Meyerhof , J. Peart , and W. Bitter taste receptor agonists elicit G-protein-dependent negative inotropy in the murine heart. Roura , and W. Extrasensory perception: odorant and taste receptors beyond the nose and mouth. Porrello , M.