Ribonucleotides differentially modulate oral glutamate detection thresholds.

The savory or umami taste of the amino acid glutamate is synergistically enhanced by the addition of the purines inosine 5'-monophosphate (IMP) and guanosine 5'-monophosphate (GMP) disodium salt. We hypothesized that the addition of purinergic ribonucleotides, along with the pyrimidine ribonucleotides, would decrease the absolute detection threshold of (increase sensitivity to) l-glutamic acid potassium salt (MPG). To test this, we measured both the absolute detection threshold of MPG alone and with a background level (3 mM) of 5 different 5'-ribonucleotides. The addition of the 3 purines IMP, GMP, and adenosine 5'-monophosphate (AMP) lowered the MPG threshold in all participants (P < 0.001), indicating they are positive modulators or enhancers of glutamate taste. The average detection threshold of MPG was 2.08 mM, and with the addition of IMP, the threshold was decreased by approximately 1.5 orders of magnitude to 0.046 mM. In contrast to the purines, the pyrimidines uridine 5'-monophosphate (UMP) and cytidine 5'-monophosphate (CMP) yielded different results. CMP reliably raised glutamate thresholds in 10 of 17 subjects, suggesting it is a negative modulator or diminisher of glutamate taste for them. The rank order of effects on increasing sensitivity to glutamate was IMP > GMP> AMP >> UMP// CMP. These data confirm that ribonucleotides are modulators of glutamate taste, with purines enhancing sensitivity and pyrimidines displaying variable and even negative modulatory effects. Our ability to detect the co-occurrence of glutamate and purines is meaningful as both are relatively high in evolutionarily important sources of nutrition, such as insects and fermented foods.

Probenecid inhibits the human bitter taste receptor TAS2R16 and suppresses bitter perception of salicin.

Bitter taste stimuli are detected by a diverse family of G protein-coupled receptors (GPCRs) expressed in gustatory cells. Each bitter taste receptor (TAS2R) responds to an array of compounds, many of which are toxic and can be found in nature. For example, human TAS2R16 (hTAS2R16) responds to ß-glucosides such as salicin, and hTAS2R38 responds to thiourea-containing molecules such as glucosinolates and phenylthiocarbamide (PTC). While many substances are known to activate TAS2Rs, only one inhibitor that specifically blocks bitter receptor activation has been described. Here, we describe a new inhibitor of bitter taste receptors, p-(dipropylsulfamoyl)benzoic acid (probenecid), that acts on a subset of TAS2Rs and inhibits through a novel, allosteric mechanism of action. Probenecid is an FDA-approved inhibitor of the Multidrug Resistance Protein 1 (MRP1) transporter and is clinically used to treat gout in humans. Probenecid is also commonly used to enhance cellular signals in GPCR calcium mobilization assays. We show that probenecid specifically inhibits the cellular response mediated by the bitter taste receptor hTAS2R16 and provide molecular and pharmacological evidence for direct interaction with this GPCR using a non-competitive (allosteric) mechanism. Through a comprehensive analysis of hTAS2R16 point mutants, we define amino acid residues involved in the probenecid interaction that result in decreased sensitivity to probenecid while maintaining normal responses to salicin. Probenecid inhibits hTAS2R16, hTAS2R38, and hTAS2R43, but does not inhibit the bitter receptor hTAS2R31 or non-TAS2R GPCRs. Additionally, structurally unrelated MRP1 inhibitors, such as indomethacin, fail to inhibit hTAS2R16 function. Finally, we demonstrate that the inhibitory activity of probenecid in cellular experiments translates to inhibition of bitter taste perception of salicin in humans. This work identifies probenecid as a pharmacological tool for understanding the cell biology of bitter taste and as a lead for the development of broad specificity bitter blockers to improve nutrition and medical compliance.

Individual differences in AMY1 gene copy number, salivary α-amylase levels, and the perception of oral starch.

BACKGROUND: The digestion of dietary starch in humans is initiated by salivary α-amylase, an endo-enzyme that hydrolyzes starch into maltose, maltotriose and larger oligosaccharides. Salivary amylase accounts for 40 to 50% of protein in human saliva and rapidly alters the physical properties of starch. Importantly, the quantity and enzymatic activity of salivary amylase show significant individual variation. However, linking variation in salivary amylase levels with the oral perception of starch has proven difficult. Furthermore, the relationship between copy number variations (CNVs) in the AMY1 gene, which influence salivary amylase levels, and starch viscosity perception has not been explored. PRINCIPAL FINDINGS: Here we demonstrate that saliva containing high levels of amylase has sufficient activity to rapidly hydrolyze a viscous starch solution in vitro. Furthermore, we show with time-intensity ratings, which track the digestion of starch during oral manipulation, that individuals with high amylase levels report faster and more significant decreases in perceived starch viscosity than people with low salivary amylase levels. Finally, we demonstrate that AMY1 CNVs predict an individual's amount and activity of salivary amylase and thereby, ultimately determine their perceived rate of oral starch viscosity thinning. CONCLUSIONS: By linking genetic variation and its consequent salivary enzymatic differences to the perceptual sequellae of these variations, we show that AMY1 copy number relates to salivary amylase concentration and enzymatic activity level, which, in turn, account for individual variation in the oral perception of starch viscosity. The profound individual differences in salivary amylase levels and salivary activity may contribute significantly to individual differences in dietary starch intake and, consequently, to overall nutritional status.

Perceptual variation in umami taste and polymorphisms in TAS1R taste receptor genes.

BACKGROUND: The TAS1R1 and TAS1R3 G protein-coupled receptors are believed to function in combination as a heteromeric glutamate taste receptor in humans. OBJECTIVE: We hypothesized that variations in the umami perception of glutamate would correlate with variations in the sequence of these 2 genes, if they contribute directly to umami taste. DESIGN: In this study, we first characterized the general sensitivity to glutamate in a sample population of 242 subjects. We performed these experiments by sequencing the coding regions of the genomic TAS1R1 and TAS1R3 genes in a separate set of 87 individuals who were tested repeatedly with monopotassium glutamate (MPG) solutions. Last, we tested the role of the candidate umami taste receptor hTAS1R1-hTAS1R3 in a functional expression assay. RESULTS: A subset of subjects displays extremes of sensitivity, and a battery of different psychophysical tests validated this observation. Statistical analysis showed that the rare T allele of single nucleotide polymorphism (SNP) R757C in TAS1R3 led to a doubling of umami ratings of 25 mmol MPG/L. Other suggestive SNPs of TAS1R3 include the A allele of A5T and the A allele of R247H, which both resulted in an approximate doubling of umami ratings of 200 mmol MPG/L. We confirmed the potential role of the human TAS1R1-TAS1R3 heteromer receptor in umami taste by recording responses, specifically to l-glutamate and inosine 5'-monophosphate (IMP) mixtures in a heterologous expression assay in HEK (human embryonic kidney) T cells. CONCLUSIONS: There is a reliable and valid variation in human umami taste of l-glutamate. Variations in perception of umami taste correlated with variations in the human TAS1R3 gene. The putative human taste receptor TAS1R1-TAS1R3 responds specifically to l-glutamate mixed with the ribonucleotide IMP. Thus, this receptor likely contributes to human umami taste perception.