Effects of gymnemic acid concentration and time since exposure on intensity of simple tastes: A test of the biphasic model for the action of gymnemic acid

At several intervals following exposure to gymnemic acid, subjectsjudged the sweetness, bitterness, saltiness, and sourness ofsimple taste stimuli. The experiment was expressly designedto test Kennedy and Halpern's (1980) biphasic model for theaction of gymnemic acid. The model predicts selective suppressionof sweet taste immediately following exposure to gymnemic acidbut nonselective disruption of tastes with the passage of time.The data show dramatic reductions in sweet taste which recoverwith time but no reductions in bitterness, saltiness, or sournessat any time following exposure to any of a wide range of gymnemicacid concentrations.     

A model system for receptor cell studies with the taste modifier, hodulcin

Hodulcin (from Hovenia dulcis leaves) is known to suppress selectivelysweet taste perception in humans. The effects of hodulcin onPhormia regina behavioral and taste receptor cell action potentialresponses to sucrose and receptor cell responses to NaCl weretested to determine the suitability of this fly for neurophysiologicalstudies with hodulcin. The same hodulcin concentration and temporalparameters that had been used in the human studies were usedfor the fly experiments, so that the human perceptual, fly behavioraland fly neurophysiological inhibition times and hodulcin selectivitycould be compared. Hodulcin reversibly suppressed fly behavioraland receptor cell responses to sucrose with an inhibition timesimilar to the inhibition time in humans. The effect in flieswas selective: hodulcin did not suppress receptor cell responsesto NaCl. These data indicate that P.regina is an appropriatemodel for receptor cell studies with hodulcin.     

Study of the effect of gymnemic acid on taste in hamster

Behavioral and electrophysiological experiments with 10 sweetenershave been made to test if gymnemic acid (GA) is able to blockthe response to sweet stimuli in single taste fibres of thechorda tympani proper nerve in hamsters. The hamster was chosenbecause earlier studies show that it is more sensitive to GAthan any other non-human species. Since GA has been shown toaffect the sweetness of many different substances, its effectswere studied on an array of sweeteners. To avoid, however, theinclusion of sweeteners unpalatable to the hamster, the hamsters'liking of this array was tested with two-bottle preference techniquefor –24 h. It was found that acesulfam-K, fructose, glucose,sucrose and xylitol were strongly liked, while the animals showedno preference for aspartame, D-tryptophane, sodium cyclamate,sodium saccharin and thaumatin over water. The summated nerveresponse of these stimuli was then recorded. It was found thatneither thaumatin nor aspartame elicited a response, while theother stimuli gave a good response. Finally, the sweetenerswhich were both preferred in the two-bottle tests and gave anerve response were used as taste stimuli in single fibre experimentstogether with sodium chloride, quinine hydrochloride, citricacid and saccharin. The single fibre recordings were made beforeand after application of 5 mg GA for 3 min on the tongue. Itwas found that GA did not cause any dramatic decrease or disappearanceof the responses to either the sweet or the non-sweet substances.The responses to the sweeteners, however, were more depressedthan those to the non-sweet stimuli.     

Effects of gymnemic acid on sweet taste perception in primates

Application of gymnemic acid (GA) on the tongue depresses thetaste of sucrose in man. This effect, as indicated by electrophysiologicalresponses, has been found to be absent in three nonhuman primatespecies. In the present behavioral study the effect of GA ontaste responses in 22 primate species, with two subspecies,and 12 human subjects has been investigated. In all the nonhumanprimates studied, including the Pongidae which are closely relatedto man, GA did not suppress the response to sucrose, only inman did GA have a depressing effect.     

A biphasic action of estradiol on estrogen and progesterone receptor expression in the lamb uterus

Regulation of the uterine expression of estrogen and progesterone receptors was studied in 20 three-month-old lambs that were not treated or treated with estradiol- 17beta. Determinations of receptors were performed by binding assays in the nuclear and cytosolic fractions, receptor mRNAs by solution hybridization, and estrogen receptor protein by an enzyme-immunoassay. Estradiol treatment decreased the receptor binding capacity of both receptors and the levels of immunoreactive estrogen receptor 12 h after injection in the absence of decreased receptor mRNAs, suggesting that the initial decrease is due to degradation of the proteins or that mRNAs are translated into new receptor proteins at a reduced rate. The mRNA levels increased after estradiol treatment suggesting that the replenishment phase consists of synthesis of new receptors rather than recycling of inactivated receptors.     

Intracellular pH modulates taste receptor cell volume and the phasic part of the chorda tympani response to acids

The relationship between cell volume and the neural response to acidic stimuli was investigated by simultaneous measurements of intracellular pH (pHi) and cell volume in polarized fungiform taste receptor cells (TRCs) using 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) in vitro and by rat chorda tympani (CT) nerve recordings in vivo. CT responses to HCl and CO2 were recorded in the presence of 1 M mannitol and specific probes for filamentous (F) actin (phalloidin) and monomeric (G) actin (cytochalasin B) under lingual voltage clamp. Acidic stimuli reversibly decrease TRC pHi and cell volume. In isolated TRCs F-actin and G-actin were labeled with rhodamine phalloidin and bovine pancreatic deoxyribonuclease-1 conjugated with Alexa Fluor 488, respectively. A decrease in pHi shifted the equilibrium from F-actin to G-actin. Treatment with phalloidin or cytochalasin B attenuated the magnitude of the pHi-induced decrease in TRC volume. The phasic part of the CT response to HCl or CO2 was significantly decreased by preshrinking TRCs with hypertonic mannitol and lingual application of 1.2 mM phalloidin or 20 microM cytochalasin B with no effect on the tonic part of the CT response. In TRCs first treated with cytochalasin B, the decrease in the magnitude of the phasic response to acidic stimuli was reversed by phalloidin treatment. The pHi-induced decrease in TRC volume induced a flufenamic acid-sensitive nonselective basolateral cation conductance. Channel activity was enhanced at positive lingual clamp voltages. Lingual application of flufenamic acid decreased the magnitude of the phasic part of the CT response to HCl and CO2. Flufenamic acid and hypertonic mannitol were additive in inhibiting the phasic response. We conclude that a decrease in pHi induces TRC shrinkage through its effect on the actin cytoskeleton and activates a flufenamic acid-sensitive basolateral cation conductance that is involved in eliciting the phasic part of the CT response to acidic stimuli.     

A model for ganglioside behaviour in cell membranes.

Gangliosides from beef brain have been spin-labeled using two different attaching groups and employed to investigate the physical nature of ganglioside behaviour in membranes. Results obtained using EPR spectroscopy indicate that, in phosphatidylcholine bilayers at physiological pH, ganglioside oligosaccharide chains are quite mobile and show a measurable tendency towards cooperative interaction amongst themselves. We suggest that the source of this interaction is the formation of H-bonds between sugar residues in adjacent ganglioside molecules. We present evidence that physiological (extracellular fluid) levels of Ca2+ and Mg2+ lead to cross-linking and condensing of ganglioside headgroups by complexing sialic acid carboxyl residues. Ganglioside headgroup interactions are not very sensitive to changes in the buffer ionic strength, suggesting that ionic interactions are of minor importance. We have found no measurable tendency for headgroup carbohydrate to penetrate hydrophobic regions of lipid bilayers. EPR spectroscopy was also used to follow the interaction of spin-labeled gangliosides with the glycoprotein, glycophorin, and with intact BHK cells. We suggest that these carbohydrate-based interactions should contribute significantly to the properties of the eucaryotic cell glycocalyx. We predict that laterally mobile carbohydrate-bearing components of cell surface will show a tendency to cluster about complex glycoprotein arrays, especially if the species involved bear accessible carboxylic acid functions.     

Lipid solubility and the sourness of acids: implications for models of the acid taste receptor

Taste threshold concentrations (or equi-sour concentrations)of organic acids are significantly related to their octanol/waterpartition coefficients. Since the latter appear to model aqueous/membranepartitioning in biological systems this suggests that absorptionof an acid into the taste cell membrane plays an important rolein the perception of the sour taste. ^*This work was performed whilst the author was Group R. &D. Chemist, Harp Lager Ltd., Manor Park, Alton, Hampshire, U.K.     

Analysis of ectatomin action on cell membranes.

Ectatomin (m = 7928 Da) is a toxic component from the Ectatomma tuberculatum ant venom containing two homologous polypeptide chains (37 and 34 residues) linked to each other by a disulfide bond. In aqueous solution it forms a four alpha-helix bundle. At concentrations of 0.05-0.1 microm, ectatomin forms channels in cellular and artificial bilayer membranes. Immunochemical analysis of the intracellular distribution of ectatomin showed that the toxin gets efficiently inserted into the plasma membrane at a concentration of 5 x 10-7 m and does not penetrate inside the cell. The effect of ectatomin on cardiac L-type calcium current was studied. Calcium currents (ICa) in isolated rat cardiac ventricular myocytes were measured using the whole-cell perforated patch-clamp technique. It was shown that ectatomin at concentrations of 0.01-10 nm inhibited ICa after a latency of few seconds. ICa was decreased twofold by 10 nm ectatomin. However, the most prominent effect of ectatomin was observed after stimulation of ICa by isoproterenol, an agonist of beta-adrenoreceptors, or forskolin, a stimulator of adenylate cyclase. At a concentration of 1 nm, ectatomin abolished the isoproterenol- and forskolin-sensitive components of ICa. The inhibitory effect of ectatomin was partially reversed by subsequent application of 2 microm of forskolin, whereas subsequent isoproterenol application did not produce the same effect.     

A co-cultured skin model based on cell support membranes

Tissue engineering of skin based on collagen:PCL biocomposites using a designed co-culture system is reported. The collagen:PCL biocomposites having collagen:PCL (w/w) ratios of 1:4, 1:8, and 1:20 have been proven to be biocompatible materials to support both adult normal human epidermal Keratinocyte (NHEK) and mouse 3T3 fibroblast growth in cell culture, respectively, by Dai, Coombes, et al. in 2004. Films of collagen:PCL biocomposites were prepared using non-crosslinking method by impregnation of lyophilized collagen mats with PCL/dichloromethane solutions followed by solvent evaporation. To mimic the dermal/epidermal structure of skin, the 1:20 collagen:PCL biocomposites were selected for a feasibility study of a designed co-culture technique that would subsequently be used for preparing fibroblast/biocomposite/keratinocyte skin models. A 55.3% increase in cell number was measured in the designed co-culture system when fibroblasts were seeded on both sides of a biocomposite film compared with cell culture on one surface of the biocomposite in the feasibility study. The co-culture of human keratinocytes and 3T3 fibroblasts on each side of the membrane was therefore studied using the same co-culture system by growing keratinocytes on the top surface of membrane for 3 days and 3T3 fibroblasts underneath the membrane for 6 days. Scanning electron microscopy (SEM) and immunohistochemistry assay revealed good cell attachment and proliferation of both human keratinocytes and 3T3 fibroblasts with these two types of cells isolated well on each side of the membrane. Using a modified co-culture technique, a co-cultured skin model presenting a confluent epidermal sheet on one side of the biocomposite film and fibroblasts populated on the other side of the film was developed successfully in co-culture system for 28 days under investigations by SEM and immunohistochemistry assay. Thus, the design of a co-culture system based on 1:20 (w/w) collagen:PCL biocomposite membranes for preparation of a bi-layered skin model with differentiated epidermal sheet was proven in principle. The approach to skin modeling reported here may find application in tissue engineering and screening of new pharmaceuticals.     

A significant pool of amino acids is adsorbed on blood cell membranes

It is well known that the amino acids in the blood are distributed between the plasma and inside the cells. This study was conducted to determine whether amino acids can be located adsorbed on blood cell membranes. The amino acid concentration in the deproteinized haemolysed blood was higher than that in the fraction of blood after removal of the blood cell membranes by centrifugation. These results showed that a pool of amino acids representing 21.1% of the whole blood cell amino acids was adsorbed on the blood cell membranes of adult Wistar rats. The non-polar amino acids showed high adsorption on the membrane, whereas out of the polar amino acid group, only the non-ionic amino acids did adsorb.Bioquimica i Biologia Molecular. Dept. de Biologia Fonamental i Ciencies de la Salut.     

Characterization of the sulfonylurea receptor on beta cell membranes

Specific, high affinity sulfonylurea receptors were characterized on membranes of an insulin-secreting hamster beta cell line (HIT cells). Saturable binding of the sulfonylurea, [3H]glyburide, was linear up to 0.8 mg/ml membrane protein. Scatchard analysis of equilibrium binding data at room temperature indicated the presence of a single class of saturable, high affinity binding sites with a Kd of 0.76 +/- 0.04 nM and a Bmax of 1.09 +/- 0.13 pmol/mg protein, n = 9. The insulin secretory potency of glyburide, glipizide, tolbutamide, tolazamide, and carboxytolbutamide was compared to the ability of these ligands to displace [3H]glyburide from the sulfonylurea receptor. Tolbutamide, tolazamide, and glipizide demonstrated reasonable agreement with ED50 values of 15 microM, 3 microM, and 30 nM and Ki values of 25.3 microM, 7.2 microM, and 45 nM, respectively. The inactive tolbutamide metabolite, carboxytolbutamide, at the highest concentration tested, only partially displaced [3H]glyburide from the receptor and was a very poor secretagogue. At 37 degrees C the affinity of [3H]glyburide binding, Kd = 2.0 nM, was similar to the ED50 of 5.5 nM when the free glyburide concentrations were corrected for binding of the drug to albumin. These studies suggest that sulfonylureas initiate their biologic effect through a high affinity, specific interaction with sulfonylurea receptors on the beta cell membrane.     

Glycocalyx of receptor cell membranes

Data are presented on the enzymes, glycosamines and glycolipidsof the premembranous calyx of the plasma membranes of photo-,chemo- and mechanoreceptor cells. Evidence that the glycocalyxcontributes to the morphogenesis of olfactory mucus and theotolithic membrane is summarized, as is evidence that it hasan auxiliary role in reception. It is hypothesized that theglycocalyx is involved in several steps in receptor processes.     

A mathematical model of chemoreception for odours and taste

We propose a mathematical model based on the occupation theory and on the hypothesis that, for a given stimulus, there exist two kinds of receptors. The receptors of the first kind react by a two-step process, first forming an intermediate inactive compound which is then changed into an active depolarizing form (this scheme was already used by Del Castillo & Katz, 1957). In the same way, the receptors of the second kind react by a two-step process, first forming an intermediate inactive compound which is then changed into an active hyperpolarizing form. The response is assumed to be proportional to the difference between the fraction of the active depolarizing compound and that of the active hyperpolarizing compound. The present paper deals only with the time course of the intensity of the response: in the first part, when a continuous flow of stimulus is applied and in the second part, when this continuous flow is removed. It does not deal with the quality and the discrimination of odours. The proposed mathematical model accounts for the depolarizing responses (which are the most frequent ones), the hyperpolarizing responses, the mixed responses reported by Patte et al. (1989), the off-responses reported by Takagi & Shibuya (1959) and for their variability, and the latent period in the olfactory response (Ottoson, 1974).     

The mobile receptor hypothesis for cell membrane receptor action

The mobile receptor hypothesis proposes that membrane receptors diffuse within the plane of the membrane and when occupied with ligand reversibly, associate with membrane enzymes or channels regulating their activity. Some of the implications and evidence for this model are presented.     

A taste receptor required for the caffeine response in vivo

Caffeine is a methylxanthine present in the coffee tree, tea plant, and other naturally occurring sources and is among the most commonly consumed drugs worldwide. Whereas the pharmacological action of caffeine has been studied extensively, relatively little is known concerning the molecular mechanism through which this substance is detected as a bitter compound. Unlike most tastants, which are detected through cell-surface G protein-coupled receptors, it has been proposed that caffeine and related methylxanthines activate taste-receptor cells through inhibition of a cyclic nucleotide phosphodiesterase (PDE) . Here, we show that the gustatory receptor Gr66a is expressed in the dendrites of Drosophila gustatory receptor neurons and is essential for the caffeine response. In a behavioral assay, the aversion to caffeine was specifically disrupted in flies missing Gr66a. Caffeine-induced action potentials were also eliminated, as was the response to theophylline, the methylxanthine in tea. The Gr66a mutant exhibited normal tastant-induced action potentials upon presentation of theobromine, a methylxanthine in cocoa. Given that theobromine and caffeine inhibit PDEs with equal potencies , these data further support the role of Gr66a rather than a PDE in mediating the caffeine response. Gr66a is the first gustatory receptor shown to be essential for caffeine-induced behavior and activity of gustatory receptor cells in vivo.