Taste responses in mice lacking taste receptor subunit T1R1.

The T1R1 receptor subunit acts as an umami taste receptor in combination with its partner, T1R3. In addition, metabotropic glutamate receptors (brain and taste variants of mGluR1 and mGluR4) are thought to function as umami taste receptors. To elucidate the function of T1R1 and the contribution of mGluRs to umami taste detection in vivo, we used newly developed knock-out (T1R1(-/-)) mice, which lack the entire coding region of the Tas1r1 gene and express mCherry in T1R1-expressing cells. Gustatory nerve recordings demonstrated that T1R1(-/-) mice exhibited a serious deficit in inosine monophosphate-elicited synergy but substantial residual responses to glutamate alone in both chorda tympani and glossopharyngeal nerves. Interestingly, chorda tympani nerve responses to sweeteners were smaller in T1R1(-/-) mice. Taste cell recordings demonstrated that many mCherry-expressing taste cells in T1R1(+/-) mice responded to sweet and umami compounds, whereas those in T1R1(-/-) mice responded to sweet stimuli. The proportion of sweet-responsive cells was smaller in T1R1(-/-) than in T1R1(+/-) mice. Single-cell RT-PCR demonstrated that some single mCherry-expressing cells expressed all three T1R subunits. Chorda tympani and glossopharyngeal nerve responses to glutamate were significantly inhibited by addition of mGluR antagonists in both T1R1(-/-) and T1R1(+/-) mice. Conditioned taste aversion tests demonstrated that both T1R1(-/-) and T1R1(+/-) mice were equally capable of discriminating glutamate from other basic taste stimuli. Avoidance conditioned to glutamate was significantly reduced by addition of mGluR antagonists. These results suggest that T1R1-expressing cells mainly contribute to umami taste synergism and partly to sweet sensitivity and that mGluRs are involved in the detection of umami compounds.

Responses to apical and basolateral application of glutamate in mouse fungiform taste cells with action potentials.

In taste bud cells, glutamate may elicit two types of responses, as an umami tastant and as a neurotransmitter. Glutamate applied to apical membrane of taste cells would elicit taste responses whereas glutamate applied to basolateral membrane may act as a neurotransmitter. Using restricted stimulation to apical or basolateral membrane of taste cells, we examined responses of taste cells to glutamate stimulation, separately. Apical application of monosodium glutamate (MSG, 0.3 M) increased firing frequency in some of mouse fungiform taste cells that evoked action potentials. These cells were tested with other basic taste compounds, NaCl (salty), saccharin (sweet), HCl (sour), and quinine (bitter). MSG-sensitive taste cells could be classified into sweet-best (S-type), MSG-best (M-type), and NaCl or other electrolytes-best (N- or E/H-type) cells. Furthermore, S- and M-type could be classified into two sub-types according to the synergistic effect between MSG and inosine-5'-monophosphate (S1, M1 with synergism; S2, M2 without synergism). Basolateral application of glutamate (100 µM) had almost no effect on the mean spontaneous firing rates in taste cells. However, about 10% of taste cells tested showed transient increases in spontaneous firing rates (>mean + 2 standard deviation) after basolateral application of glutamate. These results suggest the existence of multiple types of umami-sensitive taste cells and the existence of glutamate receptor(s) on the basolateral membrane of a subset of taste cells.

Multiple receptor systems for glutamate detection in the taste organ.

L-Glutamate and 5'-ribonucleotides such as guanosine-5'-monophosphate (GMP) and inosine-5'-monophosphate (IMP) elicit a unique taste called 'umami' that is distinct from the tastes of sweet, salty, sour, and bitter. For umami, like sweet and bitter compounds, taste signaling is initiated by binding of tastants to G-protein-coupled receptors (GPCR) in taste bud cells. To date, several GPCRs for umami compounds have been identified in taste cells, including the heterodimer T1R1/T1R3, and truncated type 1 and 4 metabotropic glutamate receptors missing most of the N-terminal extracellular domain (taste-mGluR4 and truncated-mGluR1). Apparently contradictory data in T1R3 knock-out (KO) mouse models have been reported. One study showed that behavioral preference and taste nerve responses to umami stimuli in T1R3-KO mice were totally abolished, suggesting that T1R1/T1R3 is a sole receptor for umami taste. The other reported reduced but not abolished responses to umami in T1R3-KO mice, suggesting existence of multiple receptors for umami taste. In this paper, we summarized the data from recent studies that further addressed this issue by using different experimental techniques. Some of the studies provided additional evidence for the existence of umami receptor systems mediated by mGluR1 and mGluR4 in addition to T1R1/T1R3. It is proposed that the signal mediated by the pathway involving T1R1/T1R3 may play a different role from that derived from the mGluRs. The former occurs mainly in the anterior tongue, and plays a major role in preference behavior, whereas the latter occurs mainly in the posterior tongue and contributes to behavioral discrimination between umami and other taste compounds.

Determination of the antifungal agent voriconazole in human plasma using a simple column-switching high-performance liquid chromatography and its application to a pharmacokinetic study.

A simple column-switching high performance liquid chromatographic (HPLC) method that does not require any complicated pretreatment has been developed to determine voriconazole in human plasma samples. An internal standard (IS) and borate buffer (pH 9.0) were added to plasma samples, which were then injected directly into the column-switching HPLC system using MAYI-ODS as a pre-column. The calibration curve for voriconazole showed good linearity in the range of 0.2-10 mug/ml in human plasma. The mean RSD (%) value of intra-day (n=6) and inter-day (n=5) precision were less than 5.4% and 8.2%, respectively. This system could make more than three hundred successive, accurate measurements when a washing step with ammonium acetate solution was added. This method was successfully applied to measure the therapeutic voriconazole level in patients' plasma, and was used in a study of voriconazole pharmacokinetics after oral administration.

Recovery of two independent sweet taste systems during regeneration of the mouse chorda tympani nerve after nerve crush.

In rodents, section of the taste nerve results in degeneration of the taste buds. Following regeneration of the cut taste nerve, however, the taste buds reappear. This phenomenon can be used to study the functional reformation of the peripheral neural system responsible for sweet taste. In this study we examined the recovery of sweet responses by the chorda tympani (CT) nerve after nerve crush as well as inhibition of these responses by gurmarin (Gur), a sweet response inhibitor. After about 2 weeks of CT nerve regeneration, no significant response to any taste stimuli could be observed. At 3 weeks, responses to sweet stimuli reappeared but were not significantly inhibited by Gur. At 4 weeks, Gur inhibition of sweet responses reached statistically significant levels. Thus, the Gur-sensitive (GS) component of the sweet response reappeared about 1 week later than the Gur-insensitive (GI) component. Moreover, single CT fibers responsive to sucrose could be classified into distinct GS and GI groups at 4 weeks. After 5 weeks or more, responses to sweet compounds before and after treatment with Gur became indistinguishable from responses in the intact group. During regeneration, the GS and GI components of the sucrose response could be distinguished based on their concentration-dependent responses to sucrose. These results suggest that mice have two different sweet-reception systems, distinguishable by their sensitivity to Gur (the GS and GI systems). These two sweet-reception systems may be reconstituted independently during regeneration of the mouse CT nerve.