The tachykinins substance P (SP) and neurokinin A (NKA) are present in nociceptive sensory fibers expressing transient receptor potential cation channel, subfamily V, member 1 (TRPV1). These fibers are found extensively in and around the taste buds of several species. Tachykinins are released from nociceptive fibers by irritants such as capsaicin, the active compound found in chili peppers commonly associated with the sensation of spiciness. Using real-time Ca 2+-imaging on isolated taste cells, it was observed that SP induces Ca 2+ -responses in a subset of taste cells at concentrations in the low nanomolar range. These responses were reversibly inhibited by blocking the SP receptor NK-1R. NKA also induced Ca 2+-responses in a subset of taste cells, but only at concentrations in the high nanomolar range. These responses were only partially inhibited by blocking the NKA receptor NK-2R, and were also inhibited by blocking NK-1R indicating that NKA is only active in taste cells at concentrations that activate both receptors. In addition, it was determined that tachykinin signaling in taste cells requires Ca 2+-release from endoplasmic reticulum stores. RT-PCR analysis further confirmed that mouse taste buds express NK-1R and NK-2R. Using Ca 2+-imaging and single cell RT-PCR, it was determined that the majority of tachykinin-responsive taste cells were Type I (Glial-like) and umami-responsive Type II (Receptor) cells. Importantly, stimulating NK-1R had an additive effect on Ca 2+ responses evoked by umami stimuli in Type II (Receptor) cells. This data indicates that tachykinin release from nociceptive sensory fibers in and around taste buds may enhance umami and other taste modalities, providing a possible mechanism for the increased palatability of spicy foods.
Spices that contain capsaicin, such as chili powder, are commonly used to increase the palatability of food in certain cultures. Capsaicin, as well as high temperature, activates the transient receptor potential cation channel, subfamily V, member 1 (TRPV1), found on a subpopulation of sensory afferent nociceptive nerve fibers. TRPV1 is a receptor for painful heat sensation, which explains why capsaicin produces a burning sensation. However, it is not clear as to why a pungent compound such as capsaicin is commonly used and enjoyed in the foods of many cultures.
Substance P (SP) and neurokinin A (NKA) are excitatory peptides of the tachykinin family. They are found extensively in capsaicin-sensitive peripheral sensory fibers. A third member of the tachykinin family, Neurokinin B, is generally not expressed in peripheral sensory fibers. In response to activation of TRPV1 by capsaicin and other painful stimuli, sensory nerve fibers release SP and NKA at their peripheral terminals. Release of these tachykinins from peripheral fibers modulates gastrointestinal motility, genitourinary tract function, immune responses, and many other physiological processes.
Nerve fibers containing SP and NKA are present in and around taste buds of several species. Several studies have shown that SP can directly stimulate or modulate physiological responses in gustatory neurons of the rostral nucleus tractus solitarius and gustatory sensory ganglion. In addition, intraventricular injections of the neurokinin 3 receptor (NK-3R) agonist senktide decreased salt intake in rats. Wang et al. (1995) previously hypothesized that release of peptides such as SP from peripheral nociceptive fibers may modulate taste responses at the level of taste buds. Indeed, they demonstrated that direct stimulation of the lingual nerve, which projects the SP-containing fibers to the tongue, modulated responses of the chorda tymphani to salt solution. In a separate study, the same group found the SP receptor neurokinin 1 receptor (NK-1R) immunohistochemically localized in taste cells of the rat. However, to date no physiological studies to have been performed to determine if tachykinins can directly stimulate taste cells.
In this study, it is shown that the tachykinin receptors NK-1R and to a lesser extent the NKA selective neurokinin 2 receptor (NK-2R) are expressed in mouse taste buds. Activation of these receptors induced Ca 2+-responses in taste cells. NK-1R had a much larger role these Ca 2+-responses as compared to NK-2R. In addition, NK-1R-mediated Ca 2+ responses were due to release of Ca 2+ from intracellular stores. The majority of tachykinin- responsive taste cells were identified to be Type I (Glial-like) and umami-responsive Type II (Receptor) cells. In addition, activation of NK-1R had an additive effect on Ca 2+ responses to umami stimulus in taste Type II (Receptor) cells, suggesting that tachykinins may enhance the taste sensation of umami and other taste modalities.
All experimental procedures were approved by the University of Miami Animal Care and Use Committee. C57BL/6J adult mice, as well as transgenic mice expressing enhanced green fluorescent protein (GFP) under control of the PLCβ2 promoter (PLCβ2–GFP mice), and transgenic mice expressing GFP under the control of the GAD67 promoter (GAD67-GFP mice) were euthanized by exposure to 100% CO 2 until clinical death was achieved. Cervical dislocation was performed, and tongues were excised for further dissection.
The lingual epithelium containing vallate mouse papillae was removed from the tongue by injecting an enzyme mixture (1 mg ml−1 collagenase A (Roche, Indianapolis, IN), 2.5 mg ml−1 dispase II (Roche, Indianapolis, IN), 0.25 mg ml−1 Elastase (Worthington, Lakewood, NJ), and 0.5 mg ml−1 DNAse I (Sigma, St. Louis, MO)) directly under the epithelium surrounding the taste papillae. The peeled epithelium was re-incubated for 2 min in the above mentioned enzyme mixture, then for 5 min in Ca 2+/Mg 2+-free Tyrode solution. Taste buds were gently drawn into fire-polished micropipettes with suction, and either processed for RNA extraction or transferred to a glass coverslip for isolated taste cell preparation. For isolated taste cell preparations, taste buds were incubated for 10 min in 0.25% trypsin, then triturated 20 times with a fire-polished micropipette and transferred the isolated cells to a shallow recording chamber with a glass coverslip coated with Cell-Tak (BD Biosciences, San Jose, California). Isolated taste cells were then loaded with 5 µM fura-2 AM for 45 min. Taste cells were perfused with Tyrode solution (in mM: 140 NaCl, 5 KCl, 2 CaCl 2, 1 MgCl 2, 10 HEPES, 10 glucose, 10 sodium pyruvate, 5 NaHCO 3, pH 7.2–7.4, 310–320 mosmol/l). For experiments in nominal extracellular Ca 2+, MgCl 2 was substituted equimolar for CaCl 2.
RNA was isolated from isolated whole taste buds, isolated taste cells, and from pieces of non-taste lingual epithelium (enzymatically peeled). RNA was also isolated from mouse intestine and eye for positive controls. Total RNA was isolated using the Absolutely RNA nanoprep kit (Agilent Technologies, Santa Clara, CA). Any remaining DNA was eliminated with DNAse I digestion, and RNA was reverse-transcribed using Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA). One to two taste bud equivalents of cDNA were used for subsequent whole-taste bud PCR reactions. Table 1 lists the sequences and annealing temperatures of primers used in this study. For the whole-taste bud studies, amplification was for 30 cycles (β-actin), 35 cycles (PLC-β2), or 40 cycles (NK-1R, NK-2R, NK-3R). For single-cell RT-PCR, amplification was for 45 cycles for all primers used. RT-PCR was performed on an iCycler (Biorad, Hercules, CA). PCR products were run on a 2% agarose gel and examined it under UV light using a gel imager (Cell Biosciences, Inc., Santa Clara, CA.).
Isolated taste cells loaded with Fura-2 were viewed on Olympus Optical IX70 inverted microscope (Tokyo, Japan). Sequential fluorescent images were recorded at 10–20× magnification at a rate of 1 capture every 2 seconds using a band pass emission filter (510±80 nm) and with sequential excitation at 340 nm followed by 380 nm (F340/F380). Images were processed with Imaging Workbench v5 software (INDEC Biosystems, Mountain View, CA). F340/F380 ratios were converted to Ca 2+ concentration values using a Fura-2 calcium calibration buffer kit (Invitrogen, Carlsbad, California) as follows:
