Receptor seeks ligand: on the way to cloning the molecular receptors for sweet and bitter taste.
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Animals↗
Biomedical subjects
B Lindemann
Publications and source records attributed to B Lindemann.
Differential expression of RNA and protein of the three pore-forming subunits of the amiloride-sensitive epithelial sodium channel in taste buds of the rat.
Salt taste signals from the rat anterior tongue are probably transduced via epithelial sodium channels (ENaCs) residing in the apical cellular pole of taste cells. The signals are blocked by mucosal amiloride in low microM concentrations. In contrast, the rat vallate papilla does not contribute to amiloride-blockable salt taste. Two approaches were used to probe for the three subunits of ENaC in the anterior and posterior tongue of the rats in sodium balance. (a) Immunohistochemistry with antibodies against ENaC subunits and against amiloride binding sites. In the anterior tongue, reactivity for alpha-, beta-, and gamma-subunits was present in taste buds and lingual epithelium. In the posterior tongue vallate papilla, reactivity for alpha-subunit and for amiloride binding sites was easily demonstrable, whereas that for beta-subunit and especially for gamma-subunit was weaker than in the anterior tongue. (b) RT-PCR techniques were used to probe for the presence of ENaC subunit mRNA. In isolated taste buds of the anterior tongue, mRNA of all three subunits was found, whereas in isolated taste buds of the vallate papilla only mRNA of the alpha-subunit was easily detectable. That of beta- and gamma-subunits was much less abundant. RNA of all three subunits was abundant only in taste buds of the anterior tongue. Therefore, subsets of elongated taste cells do express ENaC, but regional differences exist in the transcription and expression of subunits. The regional differences suggest that amiloride-sensitive salt taste, which requires all three subunits, is present in the anterior but not the posterior tongue of rats, as functional studies indicate.
Acid Sensing Ion Channels↗
Occurrence of ENaC subunit mRNA and immunocytochemistry of the channel subunits in taste buds of the rat vallate papilla.
Epithelial Na+ channels (ENaCs) are thought to mediate the amiloride-blockable salt taste. The rat vallate papilla does not contribute to amiloride-blockable salt taste, yet the presence of ENaC-mRNA in this tissue has been reported. Is ENaC actually contained in the taste cells, or is it merely present in the supporting lingual epithelium? To avoid contamination by ENaC contained in the lingual epithelium, we physically isolated taste buds from the vallate papilla and used mRNA purification followed by reverse transcriptase polymerase chain reaction (RT-PCR) to investigate the presence of ENaC-type message in the isolated buds. mRNA of alpha-, beta- and gamma-subunits was detected, the alpha-signal being the strongest. These results provide first molecular evidence for the presence of ENaC subunits in taste buds that were isolated from the posterior tongue and were free of epithelial contamination. In addition, we used immunohistochemistry to show ENaC-like reactivity in posterior tongue taste cells. Interestingly, the immunoreactivity was not predominantly apical but was intracellular and close to or at the basolateral membrane. The function of basolateral ENaC-type channels is unknown. Possibly, the channels are normally closed or of very low open probability in the resting state.
Animals↗
MDR1 in taste buds of rat vallate papilla: functional, immunohistochemical, and biochemical evidence.
Multidrug resistance P-glycoprotein (MDR1) is a membrane protein of 150-170 kDa that catalyzes the ATP-driven efflux of hydrophobic xenobiotics, including fluorescent dyes, from cells. Expressed in many epithelial tissues and in the endothelia of the blood-brain barrier, the MDR1 protein provides major routes of detoxification. We found that taste cells of the rat vallate papilla (VP; posterior tongue) had only a slow increase in fluorescence due to uptake of the hydrophobic dye calcein acetoxymethyl ester. However, the development of fluorescence was accelerated two- to threefold by substrates and/or inhibitors of MDR1, such as verapamil, tamoxifen, and cyclosporin A, and by addition of the transport-blocking antibody to MDR1, UIC2. Western blots of vallate tissue rich in taste buds with the MDR1-specific monoclonal antibodies C219 and C494 revealed an immunoreactive protein at approximately 170 kDa. In contrast, the lingual epithelium surrounding the VP showed a much weaker band with these antibodies. Furthermore, using the antibodies C494 and UIC2 with tissue sections, MDR1-like immunoreactivity was found in taste cells. These results show that MDR1 is present and functional in vallate taste cells of the rat. MDR1-related transport may achieve active elimination of xenobiotics from the sensory cells and thereby protect the peripheral taste organs from potentially harmful molecules contained in an animal's food.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Sodium taste.
The regulation of sodium metabolism is achieved by the adaptive sodium-saving capacities in epithelia and by hormones acting within the brain to modulate salt appetite. Taste, especially in rodents, has a sodium-specific component that provides a guiding function for salt intake. The taste responsiveness is not invariable, however, and may be modified in the case of sodium need. This review discusses emerging functional aspects of the peripheral and central branch of the salt sensory pathway.
Animals↗
Chemoreception: tasting the sweet and the bitter.
Our understanding of the molecular basis of taste transduction has lagged behind that of other senses, but now a signalling protein-the G protein alpha subunit gustducin-has been shown to be taste-tissue specific and essential for both bitter and sweet tastes.
Animals↗
Changes in IP3 and cytosolic Ca2+ in response to sugars and non-sugar sweeteners in transduction of sweet taste in the rat.
1. The transduction pathways of sweet-sensitive cells in rat circumvallate (CV) taste buds were investigated with assays for inositol 1,4,5-trisphosphate (IP3) and with Ca2+ imaging. Stimulation with the non-sugar sweeteners SC-45647 and saccharin rapidly increased the cellular content of IP3 by 400 pmol (mg protein)-1, while sucrose had a much smaller effect on IP3. As shown previously, sucrose, but not saccharin, increased the content of cyclic adenosine monophosphate (cAMP) of this preparation. 2. Stimulation of isolated CV taste buds with SC-45647 increased the cytosolic Ca2+ concentration ([Ca2+]i) by 56.7 +/- 3.2 nM (n = 181). Due to the non-confocality of the measuring system, these concentrations are underestimates. The increase in [Ca2+]i did not require the presence of extracellular Ca2+, suggesting that the Ca2+ release was from intracellular stores. 3. Individual cells responding to the non-sugar sweeteners with Ca2+ release also responded to sucrose and to forskolin with an increase in [Ca2+]i. Such cells did not respond to the bitter tastant denatonium chloride. 4. Responses to sucrose were abolished by lowering the Ca2+ concentration of the stimulus solution, indicating Ca2+ uptake from the extracellular medium. 5. The responses of sweet-sensitive cells to forskolin were also abolished when Ca2+ ions were omitted from the stimulus solution. They were partially inhibited by the presence of Co2+, Ni2+, D600 (methoxyverapamil) and amiloride, indicating multiple pathways of Ca2+ uptake activated by cAMP. 6. In conclusion, a sweet-sensitive cell of the rat responds to sucrose with an increase in cAMP and Ca2+ uptake, but to non-sugar sweeteners with an increase in IP3 and Ca2+ release. The increase in [Ca2+]i, common to both pathways, is presumably required for synaptic exocytosis and for signal termination.
Amiloride↗
Blockade of epithelial Na+ channels by triamterenes - underlying mechanisms and molecular basis.
The three subunits (alpha, beta, gamma) encoding for the rat epithelial Na+ channel (rENaC) were expressed in Xenopus oocytes, and the induced Na+ conductance was tested for its sensitivity to various triamterene derivatives. Triamterene blocked rENaC in a voltage-dependent manner, and was 100-fold less potent than amiloride at pH 7.5. At -90 mV and -40 mV, the IC50 values were 5 microM and 10 microM, respectively. The blockage by triamterene, which is a weak base with a pKa of 6.2, was dependent on the extracellular pH. The IC50 was 1 microM at pH 6.5 and only 17 microM at pH 8.5, suggesting that the protonated compound is more potent than the unprotonated one. According to a simple kinetic analysis, the apparent inhibition constants at -90 mV were 0.74 microM for the charged and 100.6 microM for the uncharged triamterene. The main metabolite of triamterene, p-hydroxytriamterene sulfuric acid ester, inhibited rENaC with an approximately twofold lower affinity. Derivatives of triamterene, in which the p-position of the phenylmoiety was substituted by acidic or basic residues, inhibited rENaC with IC50 values in the range of 0.1-20 microM. Acidic and basic triamterenes produced a rENaC blockade with a similar voltage and pH dependence as the parent compound, suggesting that the pteridinemoiety of triamterene is responsible for that characteristic. Expression of the rENaC alpha-subunit-deletion mutant, Delta278-283, which lacks a putative amiloride-binding site, induced a Na+ channel with a greatly reduced affinity for both triamterene and amiloride. In summary, rENaC is a molecular target for triamterene that binds to its binding site within the electrical field, preferably as a positively charged molecule in a voltage- and pH-dependent fashion. We propose that amiloride and triamterene bind to rENaC using very similar mechanisms.
Amiloride↗
Taste reception.
Recent research on cellular mechanisms of peripheral taste has defined transduction pathways involving membrane receptors, G proteins, second messengers, and ion channels. Receptors for organic tastants received much attention, because they provide the specificity of a response. Their future cloning will constitute a major advance. Taste transduction typically utilizes two or more pathways in parallel. For instance, sweet-sensitive taste cells of the rat appear to respond to sucrose with activation of adenylyl cyclase, followed by adenosine 3',5'-cyclic monophosphate (cAMP)-dependent membrane events and Ca2+ uptake. The same cells respond differently to some artificial sweeteners, i.e., with activation of phospholipase C (PLC) followed by inositol 1,4,5-trisphosphate (IP3)-dependent Ca2+ release from intracellular stores. Some bitter tastants block K+ channels or initiate the cascade receptor G1 protein, PLC, IP3, and Ca2+ release or the cascade receptor alpha-gustducin, phosphodiesterase (PDE), cAMP decrease, and opening of cAMP-blocked channels. Membrane-permeant bitter tastants may elicit a cellular response by interacting with G protein, PLC, or PDE of the above cascades. Salt taste is initiated by current flowing into the taste cell through cation channels located in the apical membrane, even though basolateral channels may also contribute (following salt diffusion through paracellular pathways). In rodents, the Na+-specific component of salt taste is typically mediated by apical amiloride-sensitive Na+ channels, but less specific and not amiloride-sensitive taste components exist in addition. Sour taste may in part be mediated by amiloride-sensitive Na+ channels conducting protons, but other mechanisms certainly contribute. Thus the transduction of taste cells generally comprises parallel pathways. Furthermore, the transduction pathways vary with the location of taste buds on the tongue and, of course, across species of animals. To identify these pathways, to understand how they are controlled and why they evolved to this complexity are major goals of present research.
Animals↗
Cyclic nucleotide-gated channels of rat olfactory receptor cells: divalent cations control the sensitivity to cAMP.
cAMP-gated channels were studied in inside-out membrane patches excised from the apical cellular pole of isolated olfactory receptor cells of the rat. In the absence of divalent cations the dose-response curve of activation of patch current by cAMP had a KM of 4.0 microM at -50 mV and of 2.5 microM at +50 mV. However, addition of 0.2 or 0.5 mM Ca2+ shifted the KM of cAMP reversibly to the higher cAMP concentrations of 33 or 90 microM, respectively, at -50 mV. Among divalent cations, the relative potency for inducing cAMP affinity shifts was: Ca2+ > Sr2+ > Mn2+ > Ba2+ > Mg2+, of which Mg2+ (up to 3 mM) did not shift the KM at all. This potency sequence corresponds closely to that required for the activation of calmodulin. However, the Ca(2+)-sensitivity is lower than expected for a calmodulin-mediated action. Brief (60 s) transient exposure to 3 mM Mg2+, in the absence of other divalent cations, had a protective effect in that following washout of Mg2+, subsequent exposure to 0.2 mM Ca2+ no longer caused affinity shifts. This protection effect did not occur in intact cells and was probably a consequence of patch excision, possibly representing ablation of a regulatory protein from the channel cyclic nucleotide binding site. Thus, the binding of divalent cations, probably via a regulatory protein, controls the sensitivity of the cAMP-gated channels to cAMP. The influx of Ca2+ through these channels during the odorant response may rise to a sufficiently high concentration at the intracellular membrane surface to contribute to the desensitization of the odorant-induced response. The results also indicate that divalent cation effects on cyclic nucleotide-gated channels may depend on the sequence of pre-exposure to other divalent cations.
Animals↗
Properties of cyclic nucleotide-gated channels mediating olfactory transduction. Activation, selectivity, and blockage.
Cyclic nucleotide-gated channels (cng channels) in the sensory membrane of olfactory receptor cells, activated after the odorant-induced increase of cytosolic cAMP concentration, conduct the receptor current that elicits electrical excitation of the receptor neurons. We investigated properties of cng channels from frog and rat using inside-out and outside-out membrane patches excised from isolated olfactory receptor cells. Channels were activated by cAMP and cGMP with activation constants of 2.5-4.0 microM for cAMP and 1.0-1.8 for cGMP. Hill coefficients of dose-response curves were 1.4-1.8, indicating cooperativity of ligand binding. Selectivity for monovalent alkali cations and the Na/Li mole-fraction behavior identified the channel as a nonselective cation channel, having a cation-binding site of high field strength in the pore. Cytosolic pH effects suggest the presence of an additional titratable group which, when protonated, inhibits the cAMP-induced current with an apparent pK of 5.0-5.2. The pH effects were not voltage dependent. Several blockers of Ca2+ channels also blocked olfactory cng channels. Amiloride, D 600, and diltiazem inhibited the cAMP-induced current from the cytosolic side. Inhibition constants were voltage dependent with values of, respectively, 0.1, 0.3, and 1 mM at -60 mV, and 0.03, 0.02, and 0.2 mM at +60 mV. Our results suggest functional similarity between frog and rat cng channels, as well as marked differences to cng channels from photoreceptors and other tissues.
Animals↗
Noninvasive recording of receptor cell action potentials and sustained currents from single taste buds maintained in the tongue: the response to mucosal NaCl and amiloride.
Apical membrane currents were recorded from the taste pore of single taste buds maintained in the tongue of the rat, using a novel approach. Under a dissection microscope, the 150-microns opening of a saline-filled glass pipette was positioned onto single fungiform papillae, while the mucosal surface outside the pipette was kept dry. Electrical responses of receptor cells to chemical stimuli, delivered from the pipette, were recorded through the pipette while the cells remained undamaged in their natural environment. We observed monophasic transient currents of 10-msec duration and 10-100 pA amplitude, apparently driven by action potentials arising spontaneously in the receptor cells. When perfusing the pipette with a solution of increased Na but unchanged Cl concentration, a stationary inward current (from pipette to taste cell) of 50-900 pA developed and the collective spike rate of the receptor cells increased. At a mucosal Na concentration of 250 mM, the maximal collective spike rate of a bud was in the range of 6-10 sec-1. In a phasic/tonic response, the high initial rate was followed by an adaptive decrease to 0.5-2 sec-1. Buds of pure phasic response were also observed. Amiloride (30 microM) present in the pipette solution reversibly and completely blocked the increase in spike rate induced by mucosal Na. Amiloride also decreased reversibly the stationary current which depended on the presence of mucosal Na (inhibition constant near 1 microM). During washout of amiloride, spike amplitudes were first small, then increased, but always remained smaller than the amiloride-blockable stationary current of the bud.(ABSTRACT TRUNCATED AT 250 WORDS)
Action Potentials↗
Current recording from sensory cilia of olfactory receptor cells in situ. I. The neuronal response to cyclic nucleotides.
The olfactory mucosa of the frog was isolated, folded (the outer, ciliated side faced outward), and separately superfused with Ringers solution on each side. A small number of sensory cilia (one to three) were pulled into the orifice of a patch pipette and current was recorded from them. Fast bipolar current transients, indicating the generation of action potentials by the receptor cells, were transmitted to the pipette, mainly through the ciliary capacitance. Basal activity was near 1.5 spikes s-1. Exposure of apical membrane areas outside of the pipette to permeant analogues of cyclic nucleotides, to forskolin, and to phosphodiesterase inhibitors resulted in a dose-dependent acceleration of spike rate of all cells investigated. Values of 10-20 s-1 were reached. These findings lend further support to the notion that cyclic nucleotides act as second messengers, which cause graded membrane depolarization and thereby a graded increase in spike rate. The stationary spike rate induced by forskolin was very regular, while phosphodiesterase inhibitors caused (in the same cell) an irregular pattern of bursts of spikes. The response of spike rate was phasic-tonic in the case of strong stimulation, even when elicited by inhibitors of phosphodiesterase or by analogues of cyclic nucleotides that are not broken down by the enzyme. Thus, one of the mechanisms contributing to desensitization appears to operate at the level of the nucleotide-induced ciliary conductance. However, desensitization at this level was slow and only partial, in contrast to results obtained with isolated, voltage-clamped receptor cells.
1-Methyl-3-isobutylxanthine↗
Current recording from sensory cilia of olfactory receptor cells in situ. II. Role of mucosal Na+, K+, and Ca2+ ions.
Action potential-driven current transients were recorded from sensory cilia and used to monitor the spike frequency generated by olfactory receptor neurons, which were maintained in their natural position in the sensory epithelium. Both basal and messenger-induced activities, as elicited with forskolin or cyclic nucleotides, were dependent on the presence of mucosal Na+. The spike rate decreased to approximately 20% when mucosal Na+ was lowered from 120 to 60 mM (replaced by N-methyl-D-glucamine+), without clear changes in amplitude and duration of the recorded action potential-driven transients. Mucosal Ca2+ and Mg2+ blocked spike discharge completely when increased from 1 to 10 mM in Ringer solution. Lowering mucosal Ca2+ below 1 mM increased the spike rate. These results can be explained by the presence of a cyclic nucleotide-dependent, Ca(2+)-sensitive cation conductance, which allows a depolarizing Na+ inward current to flow through the apical membrane of in situ receptor cells. A conductance with these properties, thought to provide the receptor current, was first described for isolated olfactory cells by Nakamura and Gold (1987. Nature (Lond.). 325:442-444). The forskolin-stimulated spike rate decreased when l-cis-diltiazem, a known blocker of the cyclic nucleotide-dependent receptor current, was added to the mucosal solution. Spike rate also decreased when the mucosal K+ concentration was lowered. Mucosal Ba2+ and 4-aminopyridine, presumably by means of cell depolarization, rapidly increased the spike rate. This suggests the presence of apical K+ channels that render the receptor cells sensitive to the K+ concentration of the olfactory mucus. With a slower time course, mucosal Ba2+ and 4-aminopyridine decreased the amplitude and caused rectification of the fast current transients (prolongation of action potentials). Abolishment of the apical Na+ current (by removal of mucosal Na+), as indicated by a strong decrease in spike rate, could be counteracted by adding 10 mM Ba2+ or 1 mM 4-aminopyridine to the mucosal solution, which re-established spiking. Similarly, blockage of the apical cation conductance with 10 mM Ca could be counteracted by adding 10 mM Ba2+ or by raising the mucosal K+ concentration. Thus mucosal concentrations of Na+, K+, and Ca2+ will jointly affect the sensitivity of odor detection.
Animals↗
Single unit recording from olfactory cilia.
Sensory cilia from olfactory receptor cells can be pulled into a patch pipette located above the mucus layer of an olfactory mucosa. While the pipette does not form a tight electrical seal with the ciliary membrane, it nevertheless allows to record current transients driven by action potentials arising in the olfactory neuron. This method is an alternative to single-unit-recording with electrodes pushed into the mucosa and, in some respects, to patch clamp recordings from isolated olfactory cells. Its advantage is technical simplicity and minimal disturbance of the neuron from which signals are derived. Less than 5% of the chemosensitive apical surface of the neuron is covered by the pipette. The neuron remains in situ and its cilia remain covered with some mucus. (However, mucus is in part dissolved by the bathing solution). Odorant thresholds in the picomolar range were thus obtained.
Action Potentials↗
Membrane currents in taste cells of the rat fungiform papilla. Evidence for two types of Ca currents and inhibition of K currents by saccharin.
Taste buds were isolated from the fungiform papilla of the rat tongue and the receptor cells (TRCs) were patch clamped. Seals were obtained on the basolateral membrane of 281 TRCs, protruding from the intact taste buds or isolated by micro-dissection. In whole-cell configuration 72% of the cells had a TTX blockable transient Na inward current (mean peak amplitude 0.74 nA). All cells had outward K currents. Their activation was slower than for the Na current and a slow inactivation was also noticeable. The K currents were blocked by tetraethylammonium, Ba, and 4-aminopyridine, and were absent when the pipette contained Cs instead of K. With 100 mM Ba or 100 mM Ca in the bath, two types of inward current were observed. An L-type Ca current (ICaL) activated at -20 mV had a mean peak amplitude of 440 pA and inactivated very slowly. At 3 mM Ca the activation threshold of ICaL was near -40 mV. A transient T-type current (ICaT) activated at -50 mV had an average peak amplitude of 53 pA and inactivated with a time constant of 36 ms at -30 mV. ICaL was blocked more efficiently by Cd and D600 than ICaT. ICaT was blocked by 0.2 mM Ni and half blocked by 200 microM amiloride. In whole-cell voltage clamp, Na-saccharin caused (in 34% of 55 cells tested) a decrease in outward K currents by 21%, which may be expected to depolarize the TRCs. Also, Na-saccharin caused some taste cells to fire action potentials (on-cell, 7 out of 24 cells; whole-cell, 2 out of 38 cells responding to saccharin) of amplitudes sufficient to activate ICaL. Thus the action potentials will cause Ca inflow, which may trigger release of transmitter.
Action Potentials↗
Single chloride channels in colon mucosa and isolated colonic enterocytes of the rat.
Chloride channels from rat colonic enterocytes were studied using the patch-clamp technique. After removal of mucus, inside-out patches were excised from the apical membrane of intact epithelium located at the luminal surface. They contained spontaneously switching Cl- channels with a conductance of 35-40 pS. The channels were blocked reversibly by anthracene-9-carboxylic acid (1mM). In excised patches from single enterocytes, isolated by calcium removal, the Cl- channels were studied in more detail. The I-V relation was linear between +/- 80 mV. The selectivity was I- greater than Br- greater than Cl- = NO3- greater than F- = HCO3-. Thirty pS Cl- channels were also found on the basolateral membrane of crypts isolated by brief calcium removal. The I-V curve of these Cl- channels was also linear. The results provide direct evidence for the existence of Cl- channels in the apical membrane of surface cells in colonic mucosa. The properties of these channels are similar to those previously observed when incorporating membrane vesicles into planar lipid bilayers. Both results support the validity of the theoretical models describing intestinal secretion.
Animals↗
Perspectives of taste reception.
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Animals↗