Proc. Natl. Acad. Sci. USA Vol. 96, pp. 9903–9908, August 1999 Neurobiology Blocking taste receptor activation of gustducin inhibits gustatory responses to bitter compounds DING MING*†,YUZO NINOMIYA‡, AND ROBERT F. MARGOLSKEE*§¶ §Howard Hughes Medical Institute, *Department of Physiology and Biophysics, The Mount Sinai School of Medicine, Box 1677, One Gustave L. Levy Place, New York, NY 10029; and ‡Department of Oral Physiology and Chemistry, Asahi University School of Dentistry, Hozumi, Motosu, Gifu 501-02, Japan Communicated by Sidney Udenfriend, Drew University, Madison, NJ, June 17, 1999 (received for review March 2, 1999) ABSTRACT Gustducin, a transducin-like guanine nucle- buffer lacking protease inhibitors, and further homogenized by otide-binding regulatory protein (G protein), and transducin 20 passages through a 25-gauge needle. Aliquots were either are expressed in taste receptor cells where they are thought to flash frozen or stored on ice until use. The concentration of mediate taste transduction. Gustducin and transducin are protein in the membrane preparations was determined by the activated in the presence of bovine taste membranes by several Peterson modification of the micro-Lowry method (7). Acti- compounds that humans perceive to be bitter. We have vation of transducin was based on the published trypsin monitored this activation with an in vitro assay to identify sensitivity procedure (6, 8). After the trypsin digestion, sam- compounds that inhibited taste receptor activation of trans- ples were diluted with Laemmli buffer (9) and separated by ducin by bitter tastants: AMP and chemically related com- SDS͞PAGE by using a 4–20% gel and Tris–glycine buffer. The pounds inhibited in vitro responses to several bitter com- separated polypeptides were transferred by electro-blotter to a pounds (e.g., denatonium, quinine, strychnine, and atropine). poly(vinylidene difluoride) membrane, which was blocked by AMP also inhibited behavioral and electrophysiological re- the addition of 5% BLOTTO [50 mM Tris⅐HCl, pH 7.4͞100 sponses of mice to bitter tastants, but not to NaCl, HCl, or mM NaCl͞5% nonfat dry milk], (30 min), then transducin sucrose. GMP, although chemically similar to AMP, inhibited peptides were visualized by binding of transducin antiserum neither the bitter-responsive taste receptor activation of trans- and horseradish peroxidase-labeled goat anti-rabbit secondary ducin nor the gustatory responses of mice to bitter com- antibody, followed by developing with bicinchoninic acid stain- pounds. AMP and certain related compounds may bind to ing reagents from Bio-Rad and exposure to x-ray film. bitter-responsive taste receptors or interfere with receptor-G All bitter tastant and buffer chemicals were of the highest protein coupling to serve as naturally occurring taste purity available and were purchased either from Sigma or modifiers. Boehringer Mannheim, unless otherwise noted. Rhodopsin was purified in the light as 6 M urea-washed bovine rod outer The taste of quinine and denatonium benzoate (perceived by segments by using published procedures (10). Bovine trans- humans as intensely bitter) is thought to be transduced via ducin heterotrimer was purified by standard procedures (11). seven transmembrane-helix receptors coupled to heterotri- The rabbit polyclonal antitransducin antibody was a kind gift meric guanine nucleotide-binding proteins (G proteins) (re- of Mel Simon and John Watson (California Institute of viewed in refs. 1 and 2). Gustducin, a transducin-like G Technology, Pasadena, CA). ͞ protein, and transducin itself are expressed in taste receptor Behavioral Assays. Multiple sets of male C57BL 6J mice ϭ cells (3, 4). Gustducin-null mice and transgenic mice express- from the Jackson Laboratory were tested. Each set (n 10) Ϯ ing a mutated form of gustducin that disrupts signal transduc- was tested with tastant AMP or GMP. Between tests, mice tion have dramatically diminished responsiveness to com- were provided with acidified water (pH 4.5) for about 2 wk. pounds that humans characterize as bitter or sweet, implicating Tested mice ranged in age from 8 to 20 wk. Mice were gustducin in the transduction of these taste qualities (refs. 5, 6; individually housed, provided with food ad libitum (Pico Lab L. Ruiz-Avila, G. T. Wong, and R.F.M., unpublished obser- Mouse Diet 20 no. 5058; PMI Feeds, St. Louis, MO) and vations). Gustducin and transducin can be activated in vitro by presented with distilled water in two sipper bottles for 48 h several bitter tastants in the presence of apparent taste recep- before testing. During each 48-h test period, a given concen- tor activities present in taste receptor cell membranes (4, 6). tration of tastant was provided in one sipper bottle, whereas We reasoned that this in vitro assay could be used to identify the other had distilled water. After 24 h, volumes consumed compounds that would function in vivo as bitter antagonists were recorded, the bottles refilled, and positions reversed (to and flavor modifiers. In the present work, we describe our control for positional cues). Tastants were presented in as- initial results using this assay to identify such compounds. cending concentration. Preference ratios were calculated as the fraction of tastant consumed as a percentage of the total volume of liquid consumed. Mean preference ratios and MATERIALS AND METHODS Student’s t tests were calculated from total collected data. G Protein Activation Assays. Bovine (Bos primigenius) Nerve Recording. Glossopharyngeal nerve responses were tongues were collected fresh from a local slaughterhouse and recorded from male C57BL͞6J mice as previously described transported on ice to the laboratory. Circumvallate papillae (12). Each mouse was anesthetized with intraperitoneal injec- were hand dissected, frozen in liquid nitrogen, and stored at tion of sodium pentobarbital (40–50 mg͞kg) and maintained Ϫ80°C until use. The collected taste tissues were homoge- at a surgical level of anesthesia with supplemental injections of nized, particulates removed by centrifugation, and enriched the drug. The trachea was cannulated, and the mouse was then taste cell membranes collected as described (6). The pelleted fixed in the supine position with a head holder to allow membranes were rinsed twice, resuspended in homogenization Abbreviations: DEN, denatonium benzoate; QUI, quinine hydrochlo- The publication costs of this article were defrayed in part by page charge ride. †Present address: Nutrition and Consumer Sector, Monsanto, 800 payment. This article must therefore be hereby marked ‘‘advertisement’’ in North Lindbergh Boulevard, St. Louis, MO 63167. accordance with 18 U.S.C. §1734 solely to indicate this fact. ¶To whom reprint requests should be addressed. E-mail: margolskee@ PNAS is available online at www.pnas.org. msvax.mssm.edu. 9903 Downloaded by guest on September 24, 2021 9904 Neurobiology: Ming et al. Proc. Natl. Acad. Sci. USA 96 (1999) dissection of the glossopharyngeal nerve. The hypoglossal responses from stable recordings were used in the data anal- nerve was transected bilaterally to prevent inadvertent tongue ysis. In the analysis of whole nerve responses, the magnitudes movements. The right glossopharyngeal nerve was exposed by of the integrated response at 20, 25, 30, 35, and 40 s after removal of the digastric muscle and posterior horn of the hyoid stimulus onset were measured and averaged to generate tonic bone. The glossopharyngeal nerve was then dissected free responses: the tonic response represents the sustained nerve from underlying tissues and cut near its entrance to the response to continuous tastant stimulation of taste receptor posterior foramen lacerum. The entire nerve was placed on a cells. The relative tonic response for each stimulus was ob- silver wire electrode for whole nerve recording. An indifferent tained by normalization to the response from 0.1 M NH4Cl electrode was positioned nearby in the wound. Neural re- (the tonic response of NH4Cl was defined as 1.0). Student’s t sponses resulting from topical application of tastants to the test was used for statistical analysis. tongue were fed into an amplifier and displayed on an oscil- loscope screen. Whole nerve responses were integrated by RESULTS using an RMS-DC converter (Hendrick, Tallahassee, FL) with a time constant of 0.5 s. For chemical stimulation of the The active (GTP-bound) form of G proteins such as gustducin circumvallate and foliate papillae, an incision was made on and transducin can be distinguished from the inactive (GDP- each side of the animal’s face from the corner of the mouth to bound) form by limited trypsin digestion (13, 14). Using just above the angle of the jaw, the papillae were exposed, and transducin as a reporter in this in vitro assay, we identified their trenches opened via slight tension applied through a small compounds that inhibited gustatory responses to bitter com- suture sewn in the tip of the tongue. Tastant solutions were pounds. Taste membrane activation of transducin by the bitter delivered to the tongue by gravity flow, and flowed over the compounds denatonium benzoate (DEN) and quinine hydro- tongue for a controlled period. The stability of each prepara- chloride (QUI) was inhibited in a dose-dependent fashion by tion was monitored by the periodic application of 0.1 M AMP (Fig. 1 a and b). The inhibitory effect of AMP gener- NH4Cl. A recording was considered to be stable when the 0.1 alized to every bitter compound that activated transducin in MNH4Cl response magnitudes at the beginning and end of the presence of taste membranes: DEN, QUI, strychnine, each stimulation series deviated by no more than 15%. Only nicotine, atropine (Fig. 1c), sparteine, naringin, caffeic acid, FIG. 1. AMP inhibits activation of transducin by bitter stimuli in the presence of bovine taste receptor cell membranes. (a) Inactive (GDP-bound) transducin (rightmost lane) generates a 23 kDa fragment on digestion with trypsin. Active (GTP␥S-bound) transducin (second from right lane) activated by DEN plus taste membranes generates a 32-kDa fragment on treatment with trypsin.