Taste Receptor Membrane Research Articles

Selective Inhibitors of Bitter Taste

The development of a method to mask bitter taste has been actively sought after in pharmaceutical and food science. A lipoprotein (PA-LG) was found in the present study to be composed of phosphatidic acid (PA) and β-lactoglobulin (LG) was noted to selectively and reversibly suppress frog taste nerve response and human taste sensation to various bitter substances without affecting sweetness, saltiness and sourness. PA would thus appear importantly involved in inhibiting bitterness since PA-containing lipoproteins showed inhibitory action irrespective of protein species. As for the mechanism of bitter suppression, PA-LG would appear to mask the receptor site for bitterness on taste receptor membrane. Inhibitory action forward bitterness by PA itself without protein or other phospholipids in humans was examined. PA and phosphatidylinositol (PI) inhibited the bitterness of various bitter substances without affecting the taste sensation of other taste stimuli, although inhibitory activity was less than that of PA-LG. A lecithin fraction containing high content PA and PI was thus prepared from soybean lecithin on an industrial scale. The lecithin fraction showed inhibitory activity forward bitterness. This fraction and PA were thus concluded effective means for masking the bitter taste of drugs and food.

Receptor mechanisms of bitter substances

The receptor mechanism of bitter substances was discussed from the following points of views. (a) Both electrostatic and hydrophobic interactions of bitter substances with taste receptor membranes contribute to reception of bitter substances having a positive charge. (b) In the frog, the responses to bitter substances are easily adapted. The presence of Ca ion in the medium prolongs the responses. (c) Bitter substances elicit electrical responses in nongustatory cells such as neuroblastoma cells and olfactory cells, suggesting that bitter substances induce the response by nonreceptor-mediated mechanism. (d) There is also a possibility that receptors for some bitter substances are G-protein coupled. We cloned G-protein coupled receptors from bovine taste tissues. (e) A specific inhibitor of bitter taste has been desired in pharmaceutical and food sciences, but it has not been available. We found that a lipoprotein made of phosphatidic acid and β-lactoglobulin selectively inhibits the responses to bitter substances in the frog and humans. Binding of the lipoprotein to the receptor sites for bitter substances leads to suppression of the response.

Two types of sugar-binding protein in the labellum of the fly. Putative taste receptor molecules for sweetness.

Flies have taste cells specifically sensitive to sweetness. It has been suggested that the cells possess two types of receptor sites covering the receptive field of sweetness. By affinity electrophoresis with the site-specific inhibitory polysaccharides, two types of sugar-binding protein were isolated from the labellar extract of the blowfly. These proteins showed consistent sugar-binding specificities and affinities with the two types of receptor sites for sweetness, respectively. The dissociation constant of the protein-sugar complex varies 100-400 mM and the molecular weight of one type of the protein is 27,000, while that of the other is 31,000 or 32,000. Both proteins were water insoluble and were also detected in the isolated chemosensilla. Thus they are probably located on the taste receptor membrane, and the proteins are likely to act as the taste receptor molecules for sweetness in the fly.

Non-specific inhibition by amiloride of canine chorda tympani nerve responses to varius salts: do Na +-specific channels exist in canine taste receptor membranes?

In order to test the hypothesis that the taste response to NaCl is induced by entry of Na + through the apical membranes of taste cells, the effects of amiloride on the canine chorda tympani nerve responses to various stimuli were compared with those on the short-circuit current ( I sc) in the presence of salts in an in vitro preparation of the canine lingual epithelium. Application of amiloride to the tongue greatly inhibited the taste nerve responses to NaCl, LiCl, RbCl, KCl and NH 4Cl. There was no large difference in the amiloride inhibition among these salts. Amiloride also inhibited partly the responses to salts carrying impermeable cations such as choline + or glycineamide +. Amiloride shifted the dose-response curves for the salt taste responses to a higher concentration region without appreciable effects on the maximal responses, suggesting that amiloride inhibits the salt responses in a competitive manner. It was concluded that the effects of amiloride on the taste nerve responses observed in the present study were quite different from those on I sc. The present results favor a conclusion that in the dog, a competitive binding of amiloride carrying a positive charge to the receptor sites for the cations of the salt stimuli leads to inhibition of the salt responses.

味覚およびきゅう覚受容膜

The present article deals with the current understandings of the characteristics of receptor sites for odorants and taste stimulants, mechanisms of discrimination of odor and taste, synergism, and the transduction of chemical information into electric signals. 1. Responses to odorants are seen not only in the olfactory system, but also in such non-olfactory systems as neuroblastoma cell and liposomes. The latter systems have no specific receptor proteins for odorants. Changes in lipid composition of liposomes lead to changes in specificity of the liposomes to odorants.The membrane composition of each olfactory cell is postulated to be different. Different odorants will produce different response profiles in each olfactory cell, with odor quality encoded in this response profile.2. The fact that trutle olfactory responses are independent of ion concentrations on the olfactory epithelium and the fact a large olfactory response appeared after complete elimination of carp olfactory cilia suggest that ionic permeability changes at the apical membrane of olfactory cells, including ciliary membranes, do not contribute to the ol-factory receptor potentials.3. Sweet substances and amino acids are recognized by specific receptor proteins on taste receptor membrances, while the basic lipid-protein structure of membrane is involved with reception of salts, acids, and bitter substances.4. Permeability of the taste receptor membrane to ions does not contribute to the taste receptor potential elicited by most stimuli. Stimulus-induced changes in phase-boundary potential leads to depolarization of the apical membrane of taste cells. Depolarization of the apical membranes activates voltage-dependent Ca channels at the synaptic area, entry of Ca ion, and release of neurotransmitter (norepinephrine).

Ionic basis of receptor potential of frog taste cells induced by acid stimuli.

1. The ionic mechanism underlying the receptor potential in frog taste cells induced by acid stimuli was studied with single microelectrodes by replacing superficial and interstitial fluids of the tongue with modified saline solutions. 2. The removal of Na+, Ca2+ and Cl- from the normal interstitial fluid did not affect the receptor potential induced by acid stimuli. Interstitial 100 mM-K+ saline did not affect the acid response. 3. The receptor potential was reduced greatly when Ca2+ was removed from the superficial saline, but was increased when the Ca2+ concentration was elevated. The removal of superficial Cl- did not affect the receptor potential. The receptor potential elicited by superficial Ca2+-free saline was partly due to Na+. Li+, K+, NH4+ or choline + substituted for Na+ in producing the receptor potential. The amiloride-sensitive Na+ channel on the receptor membrane did not contribute to the receptor potential. With pure water adaptation of the tongue surface, the mean magnitude of the acid response was 35% of the control. 4. The receptor potential was unaffected by superficial tetrodotoxin (TTX) but was blocked by superficial Ca2+ antagonists such as Co2+ and Cd2+. Sr2+ substituted for Ca2+ in generating the receptor potential. 5. The receptor potentials observed under various concentrations of superficial Ca2+ became smaller when Na+ was present in the superficial fluid, indicating a competition between Ca2+ and Na+. 6. It is concluded that a large portion of the receptor potential induced by acid stimuli is produced by cations passing through a tastant-gated Ca2+ channel on the taste receptor membrane. Both divalent (Ca2+, Sr2+) and monovalent (Na+, Li+, K+, NH4+, choline+) cations can pass through the Ca2+ channel. The other mechanism responsible for the remaining part of the receptor potential is discussed.

Ionic mechanism of generation of receptor potential in response to quinine in frog taste cell

The ionic mechanism of generation of the receptor potential in a frog taste cell elicited by quinine-HCl (Q-HCl) was studied with an intracellular recording technique by replacing the superficial and interstitial fluids of the tongue with various saline solutions. The taste cells whose receptor membranes were adapted to normal saline and deionized water generated depolarizing receptor potentials at Q-HCl concentrations higher than 2 and 0.01 mM, respectively. The input resistance of taste cell during Q-HCl stimulation scarcely changed. The receptor potential did not change even when the membrane potential level was broadly changed. The magnitude of the receptor potential was increased by reducing the concentration of superficial Cl − on the taste receptor membrane, but was independent to the concentration of superficial Na +. Injection of Cl − into a taste cell increased the receptor potential to 170%. The magnitude of receptor potential was decreased to 20–30% by removing interstitial Na + or Cl − or both surrounding the basolateral membrane of taste cell. Furosemide (1 mM) added to the interstitial fluid decreased the receptor potential to 15%, while interstitial ouabain (0.1 mM) and superficial SITS (0.1 mM) did not influence it. From these results, we conclude: (1) an electroneutral Na +/Cl − cotransport occurs through the basolateral membrane of a taste cell in the resting state, so that Cl − accumulates inside the cell. (2) Q-HCl stimulation induces the active secretion of Cl − across the taste receptor membrane, resulting in a depolarizing receptor potential.

Liposomes as model for taste cells: receptor sites for bitter substances including N-C=S substances and mechanism of membrane potential changes.

Various bitter substances were found to depolarize liposomes. The results obtained are as follows: (1) Changes in the membrane potential of azolectin liposomes in response to various bitter substances were monitored by measuring changes in the fluorescence intensity of 3,3'-dipropylthiocarbocyanine iodide [diS-C3(5)]. All the bitter substances examined increased the fluorescence intensity of the liposome-dye suspension, which indicates that the substances depolarize the liposomes. There existed a good correlation between the minimum concentrations of the bitter substances to depolarize the liposomes and the taste thresholds in humans. (2) The effects of changed lipid composition of liposomes on the responses to various bitter substances vary greatly among bitter substances, suggesting that the receptor sites for bitter substances are multiple. The responses to N-C=S substances and sucrose octaacetate especially greatly depended on the lipid composition; these compounds depolarized only liposomes having certain lipid composition, while no or hyperpolarizing responses to these compounds were observed in other liposomes examined. This suggested that the difference in "taster" and "nontaster" for these substances can be explained in terms of difference in the lipid composition of taste receptor membranes. (3) It was confirmed that the membrane potential of the planar lipid bilayer is changed in response to bitter substances. The membrane potential changes in the planar lipid bilayer as well as in liposomes in response to the bitter substances occurred under the condition that there is no ion gradient across the membranes. These results suggested that the membrane potential changes in response to bitter substances stem from the phase boundary potential changes induced by adsorption of the substances on the hydrophobic region of the membranes.

生体膜における化学センサーをモデルとした人工膜センサー

In biological systems, most typical chemical sensors are olfactory and taste organs. The present article deals with molecular mechanisms of transduction in these organs and artificial chemical sensor based on the mechanisms in the organs.The responses to odorants are seen not only in olfactory systems but also in non-olfactory systems such as neuroblastoma cells which must have no specific receptor proteins for odorants. The lipid bilayers also exhibit the membrane potential changes in response to various odorants similarly to olfactory cells. Changes in lipid composition of the membranes lead to changes in specificity of the membranes to odorants. Odor discrimination can be explained by postulating that the membrane composition of each olfactory cell is different from cell to cell.Sweet substances and amino acids are recognized by specific receptor proteins in taste receptor membranes, while specific proteins unique to taste cells are not concerned with reception of salts, acids and bitter substances. The membrane potential of lipid bilayers is changed inresponse to various bitter substances similarly to taste cells. The membrane potential changes are induced by the phase boundary potential changes.

Susceptibility of the developing rat gustatory system to the physiological effects of dietary sodium deprivation.

1. Multifibre responses were recorded from the chorda tympani nerve in rats fed either a NaCl-deficient diet or a NaCl-replete diet from 3 days post-conception to at least 28 days post-natal. Responses were also recorded in rats fed the NaCl-deficient diet during early development and then fed the NaCl-replete diet for 1-20 days beginning at 28 days post-natal, and in rats fed the NaCl-deficient diet only as adults. The epithelial sodium transport blocker, amiloride, was used to study the physiological effects of the diet on taste receptor membrane function and to characterize the events involved in recovery of function. 2. Responses to lingual application of sodium salts increased with increasing stimulus concentration; however, response magnitudes were reduced in rats fed the NaCl-deficient diet during early development compared to controls. Responses to non-sodium salts and non-salt stimuli were similar to controls. Amiloride was ineffective in suppressing taste responses to NaCl in deprived rats but effectively suppressed responses in controls by at least 50%. After early-deprived rats were fed a NaCl-replete diet, responses to sodium salts recovered to control levels within 15 days. There was a concomitant decrease in amiloride sensitivity during this period. 3. Rats fed the NaCl-deficient diet from early gestation through adulthood had responses similar to younger deprived rats in that sodium responses were lower than controls. However, rats deprived only as adults were similar to controls. 4. The peripheral gustatory system in developing rats is susceptible to the sodium content of the diet and is 'plastic' in that early effects can be reversed by restricting dietary sodium. Once dietary manipulations are instituted past a sensitive period, however, functional taste responses seem unaffected.

Latency of gustatory neural impulses initiated in frog tongue

Latencies of gustatory neural impulses evoked by stimulation of the bullfrog tongue with the 4 basic taste substances (NaCl, acetic acid, quinine-HCl (Q-HCl), sucrose), CaCl 2 and water were studied by recording antidromic impulses conducted to the fungiform papillae. Mean latencies of the impulses ranged from 58 to 107 ms when very strong stimuli, such as 2 M NaCl, 0.1 M acetic acid, 0.1 M Q-HCl, 2 M sucrose and 1 M CaCl 2, were applied. Mean latency in response to water was 2.41 s. The time required for arrival of an applied taste stimulus on the taste receptor membrane was a mean of 20.1 ms. The time required for antidromic conduction from the impulse initiation site to the recording site was a mean of 2.4 ms. Electrical stimulation of the fungiform papilla with a strong intensity produced the impulse with a long and fluctuating latency. The mean minimum latency of the fluctuating impulse, from which the conduction time was substracted, was 5.3 ms. Mechanical destruction of the taste disk situated at the top of the fungiform papilla resulted in a disappearance of the fluctuating impulse, suggesting that this was initiated synaptically via a depolarization of taste cells by electrical current. The minimum 5.3-ms latency was likely to be the time required from the onset of taste cell depolarization to the initiation of an impulse at the first node of Ranvier of myelinated gustatory fiber. These results indicate that the latencies of 58–107 ms by strong taste stimulation were composed of the 30- to 79-ms latency of taste cell receptor potential and the remaining 28 ms latency, which was the sum of the time of the stimulant diffusion, the time from taste cell depolarization to the first impulse and the time of impulse conduction.

Contribution of electrostatic and hydrophobic interactions of bitter substances with taste receptor membranes to generation of receptor potentials

The effects of changed ionic environments on the frog taste nerve responses to the bitter substances were examined. The responses to quinine and strychnine carrying a positive charge were suppressed by an increase in ionic strength of stimulating solutions. It was concluded that electrostatic interaction of these positive bitter substances with the receptor membranes greatly contributes to the adsorption of the substances on the membranes and that this interaction was suppressed by an increase in ionic strength. The responses to neutral bitter substances (caffeine and theophylline) were unchanged by an increase in salt concentration. The zeta potential of the mouse neuroblastoma (N-18 clone), which was depolarized by various bitter substances similarly to a taste cell, was measured in the presence of the bitter substances. The zeta potential was a little changed by quinine and practically unchanged by strychnine, caffeine and theophylline. The membrane fluidity of the N-18 cell monitored with 2-(9-anthroyloxy)stearic acid was changed in response to the bitter substances, while the fluidity monitored with 12-(9-anthroyloxy)stearic acid or 1,6-diphenyl-1,3,5-hexatriene was unchanged. This suggested that the bitter substances are adsorbed on the hydrophobic region near the surface and induce a conformational change at the region. The depolarization by the bitter substances seems to stem from changes in the “boundary potential” at the region near the surface within the membrane interior.

Selective suppression of judged sweetness by ziziphins

The effects of ziziphins and of control treatments upon judgments by human adults of the sweetness, sourness, bitterness, and saltiness of American apple cider or apple juice were measured with a category estimation method during repeated trials before, during (90 sec treatment duration only), and after, treatment. Sweetness was reduced after either a 10 sec or a 90 sec whole mouth treatment with ziziphins, but not after quinine sulfate or apple juice control treatment. No differences in after-treatment sourness, bitterness, or saltiness occurred between treatments. The reduction in sweetness was weak with 10 sec 3.5% W/V ziziphins treatment, but strong after 90 sec 0.88% W/V ziziphins treatment; duration of suppression was ca. 70 sec. The mechanism was identified as taste modification since adaptation, cross-adaptation, and mixture suppression were eliminated by control treatments and by post-treatment rests and rinse. Comparisons with known gumnemic acids effects suggest that net dissociation of ziziphins from taste receptor membranes and/or inactivation in the membrane may be much faster than with gymnemic acids.

Biochemical studies of taste sensation. VII. Enhancement of taste stimulus binding to a catfish taste receptor preparation by prior exposure to the stimulus

The taste receptor membrane fraction (Fraction P2) was prepared from a homogenate of the taste tissue of the channel catfish Ictalurus punctatus. This included the rostral, dorsal, and dorsolateral surfaces of the catfish in addition to those of the barbels. The yield of Fraction P2 is 4-7 mg protein from an individual fish, with a purification averaging 8- to 15-fold over that of the crude whole homogenate and essentially quantitative recovery of binding activity in Fraction P2. Treatment of Fraction P2 in vitro with a high concentration of the taste stimulus molecule L-alanine led to a several-fold enhancement of binding activity. Enhancement of the binding of 3H L-alanine was observed after treatment with unlabeled 10 mM L-alanine and removal of the L-alanine by washing. Enhancement occurred whether the preparation was stored frozen (-65 degrees C) for an extended period in the presence of the L-alanine, or merely exposed to it in the cold without freezing. D-Alanine enhanced the binding activity of 3H L-alanine to about 60% of the level induced by L-alanine. Nonspecific binding of 3H L-alanine was unaffected by the treatment. Scatchard analyses of saturation curves for binding of 3H L-alanine to freshly prepared Fraction P2 and to L-alanine-treated Fraction P2 revealed no change in the KD value, but a several-fold increase occurred in the amount bound. Binding activity is operationally defined. Because the enhancement observed here is reminiscent of an increase in transport due to a countertransport effect, further studies were carried out to examine whether the phenomenon reflects transport or true binding. The measured binding was not increased in the presence of Na+, indicating that it is not due to an Na+-coupled transport of L-alanine. When Fraction P2 was preloaded with L-alanine (10(-6)--10(-2) M) prior to assay, no stimulation of binding was observed; instead, binding decreased. This result is consistent with a true binding phenomenon but not with a carrier-mediated transport process to explain the enhancement phenomenon. Binding assays carried out over a range of osmolarities revealed decreased binding at high osmotic strengths, suggesting that a significant portion of the ligand might be contained in vesicles. It is postulated that "hidden" or "buried" receptor sites exist in the Fraction P2 as isolated, and that these are exposed upon perturbation of the membrane structure by a high ligand concentration.

Physicochemical studies of taste reception. I. Model membrane simulating taste receptor potential in response to stimuli of salts, acids and distilled water

A Millipore filter paper impregnanted with the lipids extracted from bovine tongue epithelium was used as a model for the taste receptor membrane and the membrane potentials arisen between two solutions across the membrane were measured under the presence of salts, acids and distilled water as taste stimuli. The changes in the membrane potential induced by these stimuli paralleled closely the taste receptor potentials observed intracellularly with living taste cells or taste never responses as shown in the following. 1. 1. Various salts carrying a common species of anion (NaCl, KCl, NH 4CL and CaCl 2) at a given concentration induced a different magnitude of potential deflections, while sodium salts carrying different species of anion (NaF, NaCl, NaBr and NaI) induced a practically identical potential deflection. The relationship between the magnitude of the potential deflection and the NaCl concentration followed the taste equation. The potential deflection induced by the application of a given concentration of NaCl was independent of pH in a wide range of pH between 3 and 12. 2. 2. A treatment of the membrane with FeCl 3 brought about a reversal of the polarity of the steady potential. An application of NaCl to the FeCl 3-treated membrane induced a variation of the potential deflection with a polarity opposite to that normally displayed. 3. 3. The relations between pH and the magnitude of the potential deflection induced by HCl and acetic acid closely resemble those derived from th taste nerve responses. 4. 4. The model membrane showed potential deflections corresponding to the “water response”; an application of distilled water to the membrane adapted with tap water, Ringer solution, CaCl 2 or NaCl solution brought about a potential deflection as if a high concentration of chemicals were applied to the membrane.