Calcitonin Gene-Related Peptide Reduces Taste-Evoked ATP Secretion from Mouse Taste Buds

Abstract

Immunoelectron microscopy revealed that peripheral afferent nerve fibers innervating taste buds contain calcitonin gene-related peptide (CGRP), which may be as an efferent transmitter released from peripheral axon terminals. In this report, we determined the targets of CGRP within taste buds and studied what effect CGRP exerts on taste bud function. We isolated mouse taste buds and taste cells, conducted functional imaging using Fura-2, and used cellular biosensors to monitor taste-evoked transmitter release. The findings showed that a subset of Presynaptic (Type III) taste cells (53%) responded to 0.1 μm CGRP with an increase in intracellular Ca(2+). In contrast, Receptor (Type II) taste cells rarely (4%) responded to 0.1 μm CGRP. Using pharmacological tools, the actions of CGRP were probed and elucidated by the CGRP receptor antagonist CGRP(8-37). We demonstrated that this effect of CGRP was dependent on phospholipase C activation and was prevented by the inhibitor U73122. Moreover, applying CGRP caused taste buds to secrete serotonin (5-HT), a Presynaptic (Type III) cell transmitter, but not ATP, a Receptor (Type II) cell transmitter. Further, our previous studies showed that 5-HT released from Presynaptic (Type III) cells provides negative paracrine feedback onto Receptor (Type II) cells by activating 5-HT1A receptors, and reducing ATP secretion. Our data showed that CGRP-evoked 5-HT release reduced taste-evoked ATP secretion. The findings are consistent with a role for CGRP as an inhibitory transmitter that shapes peripheral taste signals via serotonergic signaling during processing gustatory information in taste buds.

Significance statement: The taste sensation is initiated with a highly complex set of interactions between a variety of cells located within the taste buds before signal propagation to the brain. Afferent signals from the oral cavity are carried to the brain in chemosensory fibers that contribute to chemesthesis, the general chemical sensitivity of the mucus membranes in the oronasal cavities and being perceived as pungency, irritation, or heat. This is a study of a fundamental question in neurobiology: how are signals processed in sensory end organs, taste buds? More specifically, taste-modifying interactions, via transmitters, between gustatory and chemosensory afferents inside taste buds will help explain how a coherent output is formed before being transmitted to the brain.

Keywords: ATP; CGRP; Ca2+ imaging; biosensors; serotonin; taste.

Figure 1.

CGRP immunohistochemistry in mouse vallate papillae. A, Schematic drawing of a lingual slice containing the vallate papilla. Red box represents approximate position of the taste buds in B. B, A Nomarski optics image was merged with an immunofluorescent confocal micrograph for this micrograph (CGRP/DIC). Numerous CGRP-immunoreactive nerve fibers forming a dense network (large arrows) are seen in the connective tissue core of the papilla. Fine CGRP-immunoreactive varicose nerve fibers (small arrows) running within, or in close association with, taste buds (areas of dashed lines) and coursing through the entire thickness of the epithelium, are clearly seen. The optical thickness (z-stack) of the confocal image is 13 μm. T, Trench. Scale bar, 50 μm. C, Ultrastructure of synapses in taste buds. C1, Synapses between a Presynaptic (Type III) cells and non–CGRP-immunoreactive nerve terminals (NT). The Presynaptic (Type III) cell (III) has an ovoid nucleus with deep invagination (★) as well as possesses many dense-cored vesicles (arrowheads), accumulation of clear vesicles (small arrows), and mitochondria at areas of membrane specialization (large arrow). C2, C3, Based on the intense electron-dense precipitate distributed diffusely throughout the cytoplasm of nerve processes, electron micrographs represent CGRP-immunoreactive nerve terminals in apparent contact with Presynaptic (Type III) cells. Inset, Higher magnification of the triangle in C2. The accumulation of clear (small arrows) and dense-cored vesicles (arrowhead) at areas of membrane specialization (large arrows) in C3 is seen. Scale bar, 500 nm.

Figure 2.

A subset of Presynaptic (Type III) taste cells express RCP. Double immunostaining of isolated and fixed taste buds. Nomarski optics images showing the side (A) and top (B) views of individually isolated taste buds were merged with immunofluorescent confocal micrographs for these micrographs. RCP-immunoreactive (red) taste cells (arrowheads) and SNAP25-immunoreactive (green) Presynaptic (Type III) cells (large arrow) were revealed. Some taste cells exhibit double labeling, suggesting that these Presynaptic (Type III) cells express RCP (small arrows). The optical thicknesses (z-stack) of confocal images are 12 and 21 μm for A and B, respectively. Scale bar, 25 μm. C, Venn diagrams representing the relative proportion of taste bud cells that show RCP (RCP-ir) and/or SNAP25 (SNAP25-ir) immunoreactivity. D, A control taste bud was processed with the omission of the both primary antibodies. Note the absence of immunostaining in cells normally rich with RCP and/or SNAP25 immunoreactivity (arrowheads). Compare with Figure 2A, B. Scale bar, 25 μm.

Figure 3.

Presynaptic (Type III) taste cells respond to CGRP. A, Taste cells were isolated from vallate papillae and their responses to CGRP recorded by Ca²⁺ imaging. Traces represent Ca²⁺ recordings from an isolated Receptor (Type II) cell (top traces) and an isolated Presynaptic (Type III) cell (bottom traces). Representative traces of an identified Presynaptic (Type III) taste cell depolarized by KCl (50 mm) (↓, KCl) followed by stimulation with CGRP (0.1 μm) (↓, CGRP). In marked contrast, a Receptor (Type II) cell showed a robust taste-evoked response (↓, taste) but absence of the response to CGRP. As shown in all records in this figure, KCl stimulation typically elicited more robust responses than for CGRP. B, Venn diagrams representing the relative proportions of Receptor (Type II) and Presynaptic (Type III) taste cells that responded to CGRP.

Figure 4.

CGRP stimulates its receptors on Presynaptic (Type III) cells and induces Ca²⁺ release from intracellular stores. A, Presynaptic (Type III) cells were initially stimulated with KCl (50 mm) to verify their identity (data not shown). Bath application of CGRP (0.1 μm) elicits Ca²⁺ mobilization in the taste cells (↓, CGRP). CGRP-induced Ca²⁺ responses were reversibly inhibited by CGRP_8-37 (0.2 μm), a CGRP receptor antagonist (present throughout the shaded area). B, Summary of CGRP-elicited Ca²⁺ responses before, during, and after the presence of CGRP_8-37. Points indicate normalized peak taste cell responses. Blue symbols represent mean ± 95% CI. **p < 0.01, ****p < 0.0001 (paired Student's t test). N = 7. C, CGRP-elicited Ca²⁺ mobilization in an isolated Presynaptic (Type III) cell, identified by its responses to KCl, but not taste, was not affected when Ca²⁺ was eliminated from the bathing solution (0 Ca, present throughout the shaded area). These findings indicated that CGRP-elicited responses in Presynaptic (Type III) cells were generated by release of the intracellular Ca²⁺, consistent with the excitation of GPCR. D, Summary of experiments testing sources of CGRP-elicited Ca²⁺ mobilization, plotted as in C. ns, Not significant (paired Student's t test). N = 6.

Figure 5.

Intracellular Ca²⁺ release in Presynaptic (Type III) cells is via a PLC-mediated pathway. A, Presynaptic (Type III) cells were initially stimulated with KCl (50 mm) to verify its identity (data not shown). Bath application of CGRP (0.1 μm) elicits Ca²⁺ mobilization in the taste cell (↓, CGRP). Treating Presynaptic (Type III) cells with thapsigargin (1 μm), a SER Ca-ATPase inhibitor (present throughout the shaded area), irreversibly reduced Ca²⁺ responses evoked by CGRP, consistent with Ca²⁺ store release mechanisms for these stimuli. B, Summary of CGRP-elicited Ca²⁺ responses before, during, and after the presence of thapsigargin. Points indicate normalized peak taste cell responses. Blue symbols represent mean ± 95% CI. ***p < 0.001 (paired Student's t test). N = 4. ns, Not significant. C, CGRP-elicited Ca²⁺ mobilization in an isolated Presynaptic (Type III) cell, identified by its responses to KCl, but not taste, was blocked when U73122 (10 μm) (present throughout the shaded area) was present in the bathing solution. Consistent with the blockage of PLC-mediated cascade, these findings indicated that CGRP-elicited responses in Presynaptic (Type III) cells were due to Ca²⁺ release from intracellular stores. D, Summary of experiments plotted as in C. *p < 0.05, ***p < 0.001 (paired Student's t test). N = 4.

Figure 6.

CGRP induces serotonin release from taste buds. CHO/5-HT_2C cells (hereafter called 5-HT biosensors) were positioned against isolated taste buds to measure CGRP-elicited transmitter release. A, Micrograph of a biosensor cell abutted against an isolated taste bud in a living preparation. A Nomarski optics image and a fluorescence microscopy image were merged. B, Traces represent robust responses from the biosensor cell positioned against a taste bud when the taste bud was stimulated with 0.1 μm CGRP. CGRP_8-37 (0.2 μm) (present throughout the shaded area) reversibly reduced the biosensor responses evoked by stimulating taste buds repeatedly with CGRP. These results were consistent with that CGRP evoked serotonin release from taste buds. C, Summary of CGRP-evoked 5-HT release before, during, and after the presence of CGRP_8-37. Points indicate normalized peak biosensor responses. Blue symbols represent mean ± 95% CI. **p < 0.01, ***p < 0.001 (paired Student's t test). N = 6.

Figure 7.

CGRP inhibits ATP secretion from taste buds. CHO/ATP_P2X2/P2X3 cells (hereafter called ATP biosensors) were used to monitor taste-evoked ATP secretion from isolated taste buds. The extracellular 5-HT inhibits Receptor (Type II) cells (Huang et al., 2009). Whole taste buds were isolated from mouse vallate papillae to retain cell-to-cell communication between Receptor (Type II) and Presynaptic (Type III) cells. A, Traces represent robust responses from the biosensor positioned against an isolated taste bud when the taste bud was stimulated by a sweet-bitter taste mix (↓, taste; 1 mm sucralose, 0.1 mm SC45647, 10 μm cyclohexamide, 1 mm denatonium), indicating ATP secretion from the taste bud. Taste-evoked ATP secretion was inhibited by 0.1 μm CGRP (↓, taste + CGRP). Adding WAY100635 (0.01 μm), a 5-HT_1A receptor blocker, to the bath (present throughout the shaded area) rescued CGRP-inhibited ATP secretion, suggesting that CGRP plays as an inhibitory transmitter via the serotonergic signaling in taste buds. B, Summary of CGRP-inhibited taste-evoked ATP secretion before and during the presence of WAY100635. Points indicate normalized peak biosensor responses. Blue symbols represent mean ± 95% CI. *p < 0.05, ***p < 0.001 (paired Student's t test). N = 6.

Figure 8.

Schematic drawing represents the postulated scenario of CGRP, a putative efferent transmitter, in taste buds. Two distinct types of taste cells are shown. During taste stimulation, ATP activates gustatory afferent fibers that propagate taste signals (small arrows) to the brain (Finger et al., 2005; Huang et al., 2007; Jaber et al., 2014; Vandenbeuch et al., 2015). Chemesthetic stimulation activates sensory afferent fibers that propagate signals centrally (double-headed arrows) and have the ability to release the stored transmitter. CGRP may be directly released from the activated afferent axon branches (orange curved arrow). The activation of the CGRP receptors triggers Presynaptic (Type III) cells to elevate intracellular Ca²⁺ transients and to secrete 5-HT, which inhibits ATP release from Receptor (Type II) cells via serotonergic signaling pathways (black symbol) (Huang et al., 2009). Collectively, the peptidergic actions in taste buds could necessarily imply the complex interplay between taste cells and peripheral sensory neurons that becomes more important during the processing of the gustatory information.