Stimulation of Fibroblast Proliferation by Insoluble Gadolinium Salts

The purpose of this study was to assess insoluble salts containing gadolinium (Gd³⁺) for effects on human dermal fibroblasts. Responses to insoluble Gd³⁺ salts were compared to responses seen with Gd³⁺ solubilized with organic chelators, as in the Gd³⁺-based contrast agents (GBCAs) used for magnetic resonance imaging. Insoluble particles of either Gd³⁺ phosphate or Gd³⁺ carbonate rapidly attached to the fibroblast cell surface and stimulated proliferation. Growth was observed at Gd³⁺ concentrations between 12.5 and 125 μM, with toxicity at higher concentrations. Such a narrow window did not characterize GBCA stimulation. Proliferation induced by insoluble Gd³⁺ salts was inhibited in the presence of antagonists of mitogen-activated protein kinase and phosphatidylinositol 3-kinase signaling pathways (similar to chelated Gd³⁺) but was not blocked by an antibody to the platelet-derived growth factor receptor (different from chelated Gd³⁺). Finally, high concentrations of the insoluble Gd³⁺ salts failed to prevent fibroblast lysis under low-Ca²⁺ conditions, while similar concentrations of chelated Gd³⁺ were effective. In conclusion, while insoluble Gd³⁺ salts are capable of stimulating fibroblast proliferation, one should be cautious in assuming that GBCA dechelation must occur in vivo to produce the profibrotic changes seen in association with GBCA exposure in the subset of renal failure patients that develop nephrogenic systemic fibrosis.     

味觉受体第一家族与味觉识别

哺乳动物味觉受体第一家族(taste receptor family 1 member,T1R)的发现提供了甜味与鲜味(氨基酸味)味觉识别与味觉概念一个重要的新视野。T1R包括T1R1、T1R2、T1R3三个成员。这些受体属于G蛋白偶联受体家族第3亚型,其中T1R2 T1R3以异二聚体形式共表达并参与甜味识别,而T1R1 T1R3也以异二聚体形式共表达并参与鲜味(氨基酸味)识别。对T1R的系列研究证明了味细胞对甜味和鲜味(氨基酸味)的选择性识别及其外周味觉编码的逻辑性。     

Activation of p42 Mitogen-Activated Protein Kinase by Glutamate Receptor Stimulation in Rat Primary Cortical Cultures

Abstract— Recent studies have identified at least two homologous mitogen-activated protein (MAP) kinases that are activated by phosphorylation of both tyrosine and threonine residues by an activator kinase. To help define the role of these MAP kinases in neuronal signalling, we have used primary cultures derived from fetal rat cortex to assess the regulation of their activity by agonist stimulation of glutamate receptors and by synaptic activity. Regulation was assayed by monitoring changes in both tyrosine phosphorylation on western blots and in vitro kinase activity toward a selective MAP kinase substrate peptide. In initial studies, we found that phorbol ester treatment increased tyrosine phosphorylation of p42 MAP kinase and stimulated MAP kinase activity. A similar response was elicited by three agonists of metabotropic glutamate receptors, i.e., trans -(±)-1-amino-1,3-cyclopentane dicarboxylic acid, quisqualate, and (2S,3S,4S)-α-(carboxycyclopropyl)glycine. MAP kinase activity and p42 MAP kinase tyrosine phosphorylation were also stimulated by the ionotropic glutamate receptor agonist, kainate, but not by N -methyl- d -aspartate. To examine regulation of MAP kinase by synaptic activity, cultures were treated with picrotoxin, an inhibitor of GABA_A receptor-mediated inhibition that enhances spontaneous excitatory synaptic activity. Treatment of cultures with picrotoxin elicited activation of MAP kinase. This response was blocked by tetrodotoxin, which suppresses synaptic activity. These results demonstrate that p42 MAP kinase is activated by glutamate receptor agonist stimulation and by endogenous synaptic activity.     

Modulation of Sweet Taste by Umami Compounds via Sweet Taste Receptor Subunit hT1R2

Although the five basic taste qualities—sweet, sour, bitter, salty and umami—can be recognized by the respective gustatory system, interactions between these taste qualities are often experienced when food is consumed. Specifically, the umami taste has been investigated in terms of whether it enhances or reduces the other taste modalities. These studies, however, are based on individual perception and not on a molecular level. In this study we investigated umami-sweet taste interactions using umami compounds including monosodium glutamate (MSG), 5’-mononucleotides and glutamyl-dipeptides, glutamate-glutamate (Glu-Glu) and glutamate-aspartic acid (Glu-Asp), in human sweet taste receptor hT1R2/hT1R3-expressing cells. The sensitivity of sucrose to hT1R2/hT1R3 was significantly attenuated by MSG and umami active peptides but not by umami active nucleotides. Inhibition of sweet receptor activation by MSG and glutamyl peptides is obvious when sweet receptors are activated by sweeteners that target the extracellular domain (ECD) of T1R2, such as sucrose and acesulfame K, but not by cyclamate, which interact with the T1R3 transmembrane domain (TMD). Application of umami compounds with lactisole, inhibitory drugs that target T1R3, exerted a more severe inhibitory effect. The inhibition was also observed with F778A sweet receptor mutant, which have the defect in function of T1R3 TMD. These results suggest that umami peptides affect sweet taste receptors and this interaction prevents sweet receptor agonists from binding to the T1R2 ECD in an allosteric manner, not to the T1R3. This is the first report to define the interaction between umami and sweet taste receptors.     

Signatures of Natural Selection in a Primate Bitter Taste Receptor

Bitter taste receptors (TAS2Rs) enable animals to detect and avoid toxins in the environment, including noxious defense compounds produced by plants. This suggests that TAS2Rs are under complex pressures from natural selection. To investigate these pressures, we examined signatures of selection in the primate TAS2R38 gene. Whole-gene (1,002 bp) sequences from 40 species representing all major primate taxa uncovered extensive variation. Nucleotide substitutions occurred at 448 positions, resulting in 201 amino acid changes. Two single-nucleotide deletions, one three-nucleotide in-frame deletion, and one premature stop codon were also observed. The rate of non-synonymous substitution (ω = dN/dS), was high in TAS2R38 (ω = 0.60) compared to other genes, but significantly lower than expected under neutrality (P = 4.0 × 10(-9)), indicating that purifying selection has maintained the basic structure of the receptor. However, differences were present among receptor subregions. Non-synonymous rates were significantly lower than expected in transmembrane domains (ω = 0.55, P = 1.18 × 10(-12)) and internal loops (ω = 0.51, P = 7.04 × 10(-5)), but not external loops (ω = 1.16, P = 0.53), and evidence of positive selection was found in external loop 2, which exhibited a high rate (ω = 2.53) consistent with rapid shifts in ligand targeting. These patterns point to a history of rapid yet constrained change in bitter taste responses in the course of primate evolution.     

Bitter Taste Receptor Polymorphisms and Human Aging

Several studies have shown that genetic factors account for 25% of the variation in human life span. On the basis of published molecular, genetic and epidemiological data, we hypothesized that genetic polymorphisms of taste receptors, which modulate food preferences but are also expressed in a number of organs and regulate food absorption processing and metabolism, could modulate the aging process. Using a tagging approach, we investigated the possible associations between longevity and the common genetic variation at the three bitter taste receptor gene clusters on chromosomes 5, 7 and 12 in a population of 941 individuals ranging in age from 20 to 106 years from the South of Italy. We found that one polymorphism, rs978739, situated 212 bp upstream of the TAS2R16 gene, shows a statistically significant association (p = 0.001) with longevity. In particular, the frequency of A/A homozygotes increases gradually from 35% in subjects aged 20 to 70 up to 55% in centenarians. These data provide suggestive evidence on the possible correlation between human longevity and taste genetics.     

Involvement of the Calcium-sensing Receptor in Human Taste Perception

By human sensory analyses, we found that various extracellular calcium-sensing receptor (CaSR) agonists enhance sweet, salty, and umami tastes, although they have no taste themselves. These characteristics are known as “kokumi taste” and often appear in traditional Japanese cuisine. Although GSH is a typical kokumi taste substance (taste enhancer), its mode of action is poorly understood. Here, we demonstrate how the kokumi taste is enhanced by the CaSR, a close relative of the class C G-protein-coupled receptors T1R1, T1R2, and T1R3 (sweet and umami receptors). We identified a large number of CaSR agonist γ-glutamyl peptides, including GSH (γ-Glu-Cys-Gly) and γ-Glu-Val-Gly, and showed that these peptides elicit the kokumi taste. Further analyses revealed that some known CaSR agonists such as Ca²⁺, protamine, polylysine, l-histidine, and cinacalcet (a calcium-mimetic drug) also elicit the kokumi taste and that the CaSR-specific antagonist, NPS-2143, significantly suppresses the kokumi taste. This is the first report indicating a distinct function of the CaSR in human taste perception.     

Analysis of Slow Hyperpolarizing Potentials in Frog Taste Cells Induced by Glossopharyngeal Nerve Stimulation

Chemical Senses, 29,     

The Receptor Potential of the Taste Cell of the Rat

The electrical responses of the taste cell of the rat to chemical stimuli were studied by means of microelectrode techniques. Although large positive potential changes in the taste cell were usually elicited by taste stimuli, the response was a small negative potential change with respect to surrounding tissues if the microelectrode was thrust deeply into the taste bud. Both FeCl₃ and cocaine produced a positive change in the steady potential. If this new potential is larger than a certain equilibrium potential, reversal of the polarity of the potential change caused by a taste stimulus is observed. Gamma-aminobutyric acid and acetylcholine had no effect on the receptor steady potential nor on the receptor responses elicited by taste stimuli.     

Self-Inhibition in Amiloride-sensitive Sodium Channels in Taste Receptor Cells

Electrophysiological recording techniques were used to study the Na⁺ dependence of currents through amiloride-sensitive sodium channels (ASSCs) in rat taste cells from the fungiform and vallate papillae. Perforated patch voltage clamp recordings were made from isolated fungiform and vallate taste receptor cells (TRCs) and Na⁺ transport was measured across lingual epithelia containing fungiform or vallate taste buds in a modified Ussing chamber. In isolated fungiform TRCs that contain Na⁺ currents sensitive to the diuretic amiloride, Na⁺ ions inhibit their own influx through ASSCs, a process known as sodium self-inhibition. Due to the interaction between self-inhibition and the driving force for Na⁺ entry, self-inhibition is most evident in whole-cell recordings at Na⁺ concentrations from 50 to 75 mM. In amiloride-sensitive cells, the Na permeability is significantly higher in extracellular solutions containing 35 mM Na⁺ than in 70 or 140 mM Na⁺. Compared with the block by amiloride, the development of self-inhibition is slow, taking up to 15 s to become maximally inhibited. Approximately one third of fungiform TRCs and all vallate TRCs lack functional ASSCs. These amiloride-insensitive TRCs show no signs of self-inhibition, tying this phenomenon to the presence of ASSCs. The sulfhydryl reagent, p-hydroxymercuribenzoate (p-HMB; 200 μM), reversibly removed self-inhibition from amiloride-sensitive Na⁺ currents, apparently by modifying cysteine residues in the ASSC. Na⁺ currents in amiloride-insensitive TRCs were unaffected by p-HMB. In sodium transport studies in fungiform taste bud–containing lingual epithelia, ∼40% of the change in short-circuit current (I_sc) after addition of 500 mM NaCl to the mucosal chamber is amiloride sensitive (0.5 mM). p-HMB significantly enhanced mucosal NaCl-induced changes in these epithelia at mucosal Na⁺ concentrations of 50 mM and above. In contrast, the vallate-containing epithelia, which are insensitive to amiloride, showed no enhancement of I_sc during p-HMB treatment. These findings suggest that sodium self-inhibition is present in ASSCs in taste receptor cells where it may play a crucial role in performance of salt-sensitive pathways in taste tissue during sodium stimulation. This phenomenon may be important in the process of TRC adaptation, in the conservation of cellular resources during chronic sodium exposure, or in the gustatory response to water.     

Electrophysiological Studies of a Water Receptor Associated With the Taste Sensilla of the Blowfly

Electrophysiological evidence is given that water is the specific stimulus for a fourth sensory cell associated with the taste sensilla of the blowfly. Water elicited impulses from a single cell which responded in two distinct phases: an initial rapid rate of discharge followed by a lesser, sustained steady rate. The latter, in the case of sucrose solutions, was inhibited in direct proportion to the log of the osmotic pressure over a 10⁴ range of pressures. Other non-electrolytes inhibited, but the effect could not be simply correlated with parameters of the solutions. Electrolytes inhibited the water response more sharply and at lower concentrations. The inhibition in all cases was not dependent on impulses in the other sensory cells of the taste sensillum.     

Multiple Human Taste Receptor Sites: A Molecular Modeling Approach

Numerous experimental data on the human peripheral taste systemsuggest the existence of multiple low-affinity and low-specificityreceptor sites which are responsible for the detection and thecomplete discrimination of a very large number of organic molecules.According to this hypothesis, a given molecule interacts withnumerous taste receptors and vice versa. Statistical analysisof taste intensities estimated by 58 human subjects for variousmolecules enables the calculation of taste intermolecular distances.For the present modeling study, we hypothesized that a shorttaste distance (i.e. taste similarity) between two distinctmolecules indicates that they bind with similar distributionsof affinities to the taste receptors, and hence display similarbinding motifs. In order to find common molecular binding motifsamong 14 selected organic tastants, hydrogen-bonding and hydrophobicinteraction properties were mapped onto their molecular surfaces.The 14 surfaces were then cut in 240 fragments, most of whichwere made up of 2–4 potentially interacting zones. A correspondenceindex was defined to measure the analogy between two optimallysuperimposed fragments. The 75 most representative fragmentswere all matched pairwise. Twelve distinct clusters of fragmentswere isolated from the 2775 calculated comparisons. These 12fragment types were used to calculate structural similaritydistances. We then performed a combinatorial analysis to identifywhich fragment combination best reconciled structural and tastedistances. We finally identified an optimal subset of sevenfragment types out of the 12, which significantly and best accountedfor the 91 pairwise taste distances between all 14 modeled tastants.These seven validated fragment types are therefore presentedas good candidates to be recognized by the same number of distincttaste receptor sites. Potential applications of these identifiedbinding motifs to tastant design are suggested. Chem. Senses21: 425–445, 1996.     

Positive Selection Drives the Evolution of Bat Bitter Taste Receptor Genes

Bitter taste reception is expected to be associated with dietary selection and to prevent animals from ingesting potentially harmful compounds. To investigate the genetic basis of bitter taste reception, we reconfirmed the bitter taste receptor (T2R) genes from cow (herbivore) and dog (carnivore) genome sequences and identified the T2R repertoire from the draft genome of the bat (insectivore) for the first time using an automatic data-mining method. We detected 28 bitter receptor genes from the bat genome, including 9 intact genes, 8 partial but putative functional genes, and 9 pseudogenes. In the phylogenetic analysis, most of the T2R genes from the three species intermingle across the tree, suggesting that some are conserved among mammals with different dietary preferences. Furthermore, one clade of bat-specific genes was detected, possibly implying that the insectivorous mammal could recognize some species-specific bitter tastants. Evolutionary analysis shows strong positive selection was imposed on this bat-specific cluster, indicating that positive selection drives the functional divergence and specialization of the bat bitter taste receptors to adapt diets to the external environment.     

Diet-Induced Obesity Reduces the Responsiveness of the Peripheral Taste Receptor Cells

Introduction

Obesity is a growing epidemic that causes many serious health related complications. While the causes of obesity are complex, there is conclusive evidence that overconsumption coupled with a sedentary lifestyle is the primary cause of this medical condition. Dietary consumption is controlled by appetite which is in turn regulated by multiple neuronal systems, including the taste system. However, the relationship between taste and obesity has not been well defined. Growing evidence suggests that taste perception in the brain is altered in obese animals and humans, however no studies have determined if there are altered taste responses in the peripheral taste receptor cells, which is the initiation site for the detection and perception of taste stimuli.

Methodology/Principal Findings

In this study, we used C57Bl/6 mice which readily become obese when placed on a high fat diet. After ten weeks on the high fat diet, we used calcium imaging to measure how taste-evoked calcium signals were affected in the obese mice. We found that significantly fewer taste receptor cells were responsive to some appetitive taste stimuli while the numbers of taste cells that were sensitive to aversive taste stimuli did not change. Properties of the taste-evoked calcium signals were also significantly altered in the obese mice. Behavioral analyses found that mice on the high fat diet had reduced ability to detect some taste stimuli compared to their littermate controls.

Conclusions/Significance

Our findings demonstrate that diet-induced obesity significantly influences peripheral taste receptor cell signals which likely leads to changes in the central taste system and may cause altered taste perception.     

Promotion of Cancer Cell Invasiveness and Metastasis Emergence Caused by Olfactory Receptor Stimulation

Olfactory receptors (ORs) are expressed in the olfactory epithelium, where they detect odorants, but also in other tissues with additional functions. Some ORs are even overexpressed in tumor cells. In this study, we identified ORs expressed in enterochromaffin tumor cells by RT-PCR, showing that single cells can co-express several ORs. Some of the receptors identified were already reported in other tumors, but they are orphan (without known ligand), as it is the case for most of the hundreds of human ORs. Thus, genes coding for human ORs with known ligands were transfected into these cells, expressing functional heterologous ORs. The in vitro stimulation of these cells by the corresponding OR odorant agonists promoted cell invasion of collagen gels. Using LNCaP prostate cancer cells, the stimulation of the PSGR (Prostate Specific G protein-coupled Receptor), an endogenously overexpressed OR, by β-ionone, its odorant agonist, resulted in the same phenotypic change. We also showed the involvement of a PI3 kinase γ dependent signaling pathway in this promotion of tumor cell invasiveness triggered by OR stimulation. Finally, after subcutaneous inoculation of LNCaP cells into NSG immunodeficient mice, the in vivo stimulation of these cells by the PSGR agonist β-ionone significantly enhanced metastasis emergence and spreading.     

Receptor Polymorphism and Genomic Structure Interact to Shape Bitter Taste Perception

The ability to taste bitterness evolved to safeguard most animals, including humans, against potentially toxic substances, thereby leading to food rejection. Nonetheless, bitter perception is subject to individual variations due to the presence of genetic functional polymorphisms in bitter taste receptor (TAS2R) genes, such as the long-known association between genetic polymorphisms in TAS2R38 and bitter taste perception of phenylthiocarbamide. Yet, due to overlaps in specificities across receptors, such associations with a single TAS2R locus are uncommon. Therefore, to investigate more complex associations, we examined taste responses to six structurally diverse compounds (absinthin, amarogentin, cascarillin, grosheimin, quassin, and quinine) in a sample of the Caucasian population. By sequencing all bitter receptor loci, inferring long-range haplotypes, mapping their effects on phenotype variation, and characterizing functionally causal allelic variants, we deciphered at the molecular level how a subjects’ genotype for the whole-family of TAS2R genes shapes variation in bitter taste perception. Within each haplotype block implicated in phenotypic variation, we provided evidence for at least one locus harboring functional polymorphic alleles, e.g. one locus for sensitivity to amarogentin, one of the most bitter natural compounds known, and two loci for sensitivity to grosheimin, one of the bitter compounds of artichoke. Our analyses revealed also, besides simple associations, complex associations of bitterness sensitivity across TAS2R loci. Indeed, even if several putative loci harbored both high- and low-sensitivity alleles, phenotypic variation depended on linkage between these alleles. When sensitive alleles for bitter compounds were maintained in the same linkage phase, genetically driven perceptual differences were obvious, e.g. for grosheimin. On the contrary, when sensitive alleles were in opposite phase, only weak genotype-phenotype associations were seen, e.g. for absinthin, the bitter principle of the beverage absinth. These findings illustrate the extent to which genetic influences on taste are complex, yet arise from both receptor activation patterns and linkage structure among receptor genes.     

Methylthioadenosine Reprograms Macrophage Activation through Adenosine Receptor Stimulation

Regulation of inflammation is necessary to balance sufficient pathogen clearance with excessive tissue damage. Central to regulating inflammation is the switch from a pro-inflammatory pathway to an anti-inflammatory pathway. Macrophages are well-positioned to initiate this switch, and as such are the target of multiple therapeutics. One such potential therapeutic is methylthioadenosine (MTA), which inhibits TNFα production following LPS stimulation. We found that MTA could block TNFα production by multiple TLR ligands. Further, it prevented surface expression of CD69 and CD86 and reduced NF-KB signaling. We then determined that the mechanism of this action by MTA is signaling through adenosine A2 receptors. A2 receptors and TLR receptors synergized to promote an anti-inflammatory phenotype, as MTA enhanced LPS tolerance. In contrast, IL-1β production and processing was not affected by MTA exposure. Taken together, these data demonstrate that MTA reprograms TLR activation pathways via adenosine receptors to promote resolution of inflammation.