Decrease in rat taste receptor cell intracellular pH is the proximate stimulus in sour taste transduction

Taste receptor cells (TRCs)respond to acid stimulation, initiating perception of sour taste.Paradoxically, the pH of weak acidic stimuli correlates poorly with theperception of their sourness. A fundamental issue surrounding sourtaste reception is the identity of the sour stimulus. We tested thehypothesis that acids induce sour taste perception by penetratingplasma membranes as H⁺ ions or as undissociated moleculesand decreasing the intracellular pH (pH_i) of TRCs. Our datasuggest that taste nerve responses to weak acids (acetic acid andCO₂) are independent of stimulus pH but strongly correlatewith the intracellular acidification of polarized TRCs. Taste nerveresponses to CO₂ were voltage sensitive and were blockedwith MK-417, a specific blocker of carbonic anhydrase. Strong acids(HCl) decrease pH_i in a subset of TRCs that contain apathway for H⁺ entry. Both the apical membrane and theparacellular shunt pathway restrict H⁺ entry such that alarge decrease in apical pH is translated into a relatively smallchange in TRC pH_i within the physiological range. Weconclude that a decrease in TRC pH_i is the proximate stimulus in rat sour taste transduction.

     

Amino Acid Content of Leaves in Mycorrhizal and Non-mycorrhizal Citrus Rootstocks

Nitrogen status was examined in leaves of sour orange and roughlemon citrus rootstocks grown in a low phosphorus sand inoculatedwith Glomus etunicatus, in sand amended with superphosphateat a rate of 2240 kg ha^–1, and in a sand control leftuntreated. Sour orange was 3.1- and 3.5-fold taller and roughlemon was 1.8- and 2.0-fold taller than the controls in theinoculated and phosphorus treatments, respectively. In the controls,leaf N was up to 2.5-fold higher than in the other treatments.Both total and free amino acids accumulated in leaves of bothrootstocks to higher levels in the control than in the othertreatments. Most total amino acids in the control were lowerthan in the treatments, with the exceptions of arginine (upto 12-fold increase), proline (up to 1.8-fold increase), lysine,and free ammonia. Twenty-two free amino acids, urea, and ammoniawere detected. Both rootstocks grown in control sand had significantincreases in citrulline, ornithine, lysine, histidine, arginineand ammonia. Levels of total and most free amino acids in theinoculated and phosphorus treatments were similar to one another.It is suggested that mineral deficiency caused by the absenceof G. etunicatus causes a reorganization of N-metabolism witha shift to a greater synthesis of ornithine cycle intermediates. Citrus aurantium L., Citrus limon (L.) Burm, amino acid content, citrus rootstocks, mycorrhiza, nitrogen metabolism     

Arginine metabolism by pumpkin seedlings. Separation of plant extracts by ion exchange resins

Arginine-U-¹⁴C was injected into the cotyledons of 7-day oldpumpkin seedlings. At most, 24% of the administered ¹⁴C wastransported to the axis tissue. The amounts of arginine incorporatedinto cotyledonary protein suggests that turnover was occurringat a rapid rate. Arginine was extensively metabolized, and after96 hr 50% of the administered ¹⁴C had been released as ¹⁴CO₂.The remaining label was primarily in unmetabolized arginine,protein or transported to the axis tissue with little labelin other amino acids. The results suggest that the carbon fromarginine is incorporated into protein or catabolized to CO₂while the carbon for new amino acid skeletons is derived fromsugar. A simple, reproducible method for the quantitative fractionationof plant extracts or hydrolysates of insoluble plant materialinto basic amino acids, acidic amino acids, neutral amino acids,organic acids and sugars was reported. (Received September 10, 1968; )     

Kinetic and Thermodynamic Aspects of Sodium-Coupled Amino Acid Transport by Marine Invertebrates

SYNOPSIS. Marine invertebrates absorb amino acids directly acrosstheir external body surfaces. This absorption process occursvia carrier-mediated transport systems which, recent evidencesuggests, may be sodium-dependent. Steady-state amino acid gradientsare maintained at levels exceeding 10³–10⁶ times thatof the external environment. Examination of the standing gradientsof total free amino acids, Na, and K in seven invertebratessuggests that an Na/amino acid cotransport model, or an Na/K/aminoacid cotransport model can account for amino acid gradientsof this magnitude. However, the Na : amino acid coupling coefficientsmust be 2 or 3 depending on factors such as membrane potentialand the intracellular Na activities. Evidence from studies ofL-alanine transport in the integument of the polychaete Glyceradibranchiata is presented showing that, for this case, the Na: alanine coupling coefficient is 3. It is concluded that themodels presented are plausible and readily testable explanationsfor the observed amino acid gradients in marine invertebrates.     

Kinetics and Specificity of Amino Acid Uptake by the Duckweed Spirodela polyrhiza (L.) Schleiden

Borstlap, A. G, Meenks, J. L. D., van Eck, W. F. and Bicker,J. T. E. 1986. Kinetics and specificity of amino acid uptakeby the duckweed Spirodela polyrhiza (L.) Schleiden.—J.exp. Bot. 37: 1020–1035. Uptake of ¹⁴C-labelled amino acids by intact, axenically grownplants of Spirodela polyrhiza (L.) Schleiden was investigated.Experiments in which uptake was measured from the decrease inthe amino acid concentration in the medium, indicated that saturableuptake conforms to the sum of two Michaelis-Menten terms, possiblycorresponding with a high-affinity and a low-affinity system.Further experiments with L-leucine, L-glutamic acid, and L-lysine,in which uptake was measured by assaying the amount of ¹⁴ inthe plants, showed the presence of a non-saturable componentin addition to the dual saturable uptake. Uptake of L-glutamic acid precipitously declined between pH4?0 and 6? and that of L-leucine between pH 4?0 and 8? whereasL-lysine uptake was optimal at pH 6?0. No evidence was foundthat the apparent high-affinity and low-affinity systems respondeddifferently to changes in external pH or to the addition ofCCCP. The non-saturable uptake component was not affected bychanges in external pH or by adding CCCP, and might have beendue to free space uptake. Mutual inhibition of uptake was found between acidic and neutralamino acids (L-leucine, L-methionine, L-glutamic acid) and betweenbasic amino acids (L-lysine, L-ornithine). The basic amino acidshad no effect on the uptake of L-leucine, L-methionine and L-glutamicacid, although the uptake of basic amino acids was inhibitedby glutaminc acid and several neutral amino acids. It is suggested that the duckweed has a high-affinity transportsystem for neutral and acidic amino acids, and a distinct high-affinitysystem for basic amino acids. It is argued that the first systemtransports zwitterionic amino acids (z-system), and that thesecond system transports cationic amino acids(y⁺-system). Thespecificity of the low-affinity system is less certain, butthere is some evidence that it is similar to that of their high-affinitycounterparts. Key words: Kinetics, membrane transport, pH-dependency, transport systems, uptake isotherms     

The Regulation of Proton/Amino Acid Symport in Riccia fluitans L. by Cytosolic pH and Proton Pump Activity

In the aquatic liverwort Riccia fluitans the regulation of theplasma membrane H⁺/amino acid symport has been investigated.Cytosolic pH (pH_c), membrane potential (E_m) and membrane conductancehave been measured and related to transport data, (i) The releaseof [¹⁴C]amino acids is strongly stimulated by cytosolic acidification,induced by the external addition of acetic acid, a decreasein external K⁺, and in the change from light to dark. On average,a decrease in pHc of 0.5 to 0.6 units corresponded with a 4-foldstimulation in amino acid efflux. (ii) External pH changes havefar less effect on substrate transport than the cytosolic pHshifts of the same order. (iii) The inwardly directed positivecurrent, induced by amino acids, is severely inhibited by cytosolicacidification. (iv) Fusicoccin (FC) stimulates amino acid uptakewithout considerable change in proton motive force. (v) Whenthe proton motive force is kept constant, the uptake of aminoacids into Riccia thalli is much lower than when the pump isdeactivated. It is suggested that both the proton pump activityand cytosolic pH are the dominant factors in the regulationof the H⁺/amino acid symport across the plasma membrane of Ricciafluitans, and it is concluded that the proton motive force isnot a reliable quantity to predict and interpret transport kinetics. Key words: Amino acid, cytosolic pH, pH-sensitive electrode, proton motive force, regulation, Riccia fluitans     

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.     

The Effect of Different Levels of N Limitation on Sugars, Amino Acids, Growth and Biomass Partitioning in Broadbean (Vicia faba L.)

Broadbean plants (Vicia faba L.) were submitted to three differentlevels of steady state N limitation. Relative addition ratesof N were 0.06, 0.1 and 0.14d^-1. Plants were harvested at fiveevenly distributed times over a 2d period. Shoot growth correspondedwell with the imposed treatment. Root growth, relative to shootgrowth, was highest at the 0.06d^-1treatment. Diurnal patternsof soluble sugars, amino acids and starch were analysed. Onaverage, soluble sugar levels were highest in the plants ofthe 0.06d^-1treatment whereas average free amino acid levelswere highest in the 0.14d^-1treatment. Shoot growth increasedas the concentration of shoot amino acids increased. No suchcorrelation however could be found between root growth and freesugar levels in the root. Broadbean; Vicia faba L.; exponential addition; N limitation; free sugar; amino acids; diurnal cycle; functional equilibrium; starch     

Cloning and functional characterization of a new subtype of the amino acid transport system N

We have cloned a new subtype of theamino acid transport system N2 (SN2 or second subtype of system N) fromrat brain. Rat SN2 consists of 471 amino acids and belongs to therecently identified glutamine transporter gene family that consists ofsystem N and system A. Rat SN2 exhibits 63% identity with rat SN1. Italso shows considerable sequence identity (50-56%) with themembers of the amino acid transporter A subfamily. In the rat, SN2 mRNA is most abundant in the liver but is detectable in the brain, lung,stomach, kidney, testis, and spleen. When expressed in Xenopus laevis oocytes and in mammalian cells, rat SN2 mediatesNa⁺-dependent transport of several neutral amino acids,including glycine, asparagine, alanine, serine, glutamine, andhistidine. The transport process is electrogenic, Li⁺tolerant, and pH sensitive. The transport mechanism involves the influxof Na⁺ and amino acids coupled to the efflux ofH⁺, resulting in intracellular alkalization. Proline,-(methylamino)isobutyric acid, and anionic and cationic amino acidsare not recognized by rat SN2.

     

A kinetic study of the assimilation of 15N-labelled ammonium in rice seedling roots

The pathway of ammonium nitrogen assimilation, its incorporationinto amino acids and synthesis of protein was studied with theaid of nitrogen-15. The analysis of ¹⁵N involves the use ofoptical emission spectrometry. Kinetic analysis of nitrogen assimilation by the roots indicatesthat glutamine and glutamic acid were the primary products ofammonium assimilation. Possibly some of the amino acids, suchas aspartic acid and alanine received their amino nitrogen directlyfrom free ammonia in the roots. Amino groups were transformedinto other amino acids from these primary products, especiallyfrom glutamic acid through transamination. (Received April 1, 1974; )     

The Translocation of Sulphonamides in Higher Plants: III. ACETYLATION AND DEACETYLATION OF SULPHANILAMIDE IN BROAD BEANS AND WHEAT

The acetylation of both amino groups on sulphanilaniide hasbeen demon strated by paper chromatography of sap expressedfrom broad beans treated with suiphanilamide through the roots.Deacetylation of N¹- and N⁴ mides occurred in the leaves oftreated plants and the N⁴ compound was also acetylated to yieldthe N¹: N⁴-diacetylsulphanilamide. About 30 per cent. of the suiphanilamide entering broad-beanroots was acetylated in the roots, the maximum proportion ofthe N⁴-acetyl compound being reached after 7 days. Deacetylationis a more limited process but reaches a steady state after thesame time. Low levels of acetylation and deacetylation wererecorded in wheat. The amounts of suiphanilamide and N⁴ entering and accumulatingin the various tissues were calculated on a water-uptake basisarid these data show that the main site of acetylation is inthe roots and that deacetyla tion occurs to a limited extentin the roots but predominates in stems and leaves.     

Mobilization of Reserves and Synthesis of Sterols and Triterpenes in Seedlings of Two Canarian Euphorbia Species

Seedlings from Euphorbia canariensis and Euphorbia lambii weregrown in the dark at 25 °C. Protein and triglyceride contentas well as levels of sugars and amino acids in the endospermwere determined during endosperm depletion. In the endospermof Euphorbia canariensis, relatively low levels of amino acids(up to 1 µmol.endosperm^–1) were found of which glutamine/glutamateaccounted for 40% at the stage of radicle emergence. High levelsof amino acids (up to 4 µmol.endosperm^–1) comparedwith sugars (up to 2 µmol sucrose.endosperm^–1) weredetected in the endosperm of Euphorbia lambii. Arginine wasthe main component (28 µmol%) of the amino acids in thistissue. In both species amino acid composition changed graduallyduring endosperm depletion. Cotyledons retained their ability to absorb a variety of watersoluble substrates after removal of the endosperm. ¹⁴C from[U-¹⁴C]sucrose was effectively incorporated into the triterpenesof the laticifers and to a lesser extent into the sterols ofthe seedling. The highest incorporation values were found inyoung seedlings about 2 d after the emergence of the radicle.Seedlings of this age also showed high incorporation rates of¹⁴C from labelled alanine, serine, threonine, valine, leucineand isoleucine into both triterpenols and sterols, but no generalconclusions about metabolic channelling in lipid synthesis couldbe made. Endosperm, Euphorbia canariensis L. Euphorbia lambii Svent., sterols, triterpenols, amino acids, laticifer, biosynthesis     

Effect of Potassium on Sucrose and Amino Acid Release from the Seed Coat of Developing Seeds of Pisum sativum

After removal of the embryo from developing seeds of Pisum sativum,the ‘empty’ ovules (seed coats without enclosedembryo) were filled with a solution (pH 5.5) containing mannitol(usually 400 mM) to which various salts were added. A solutioncontaining two isotopes ((a) [²H]-sucrose/[–¹⁴C]aminoisobutyricacid (AIB) or (b) [³H]valine/[₁₄C]asparagine mixture) was administeredto the plant via the petiole subtending the fruiting node, and[²H]solute and [¹⁴C]solute unloading from the seed coat wasmeasured, in pulse-labelling experiments of about 5 h. The presenceof 25 or 50 mM K⁺ in the ‘empty’ ovule enhancedthe release of sucrose from the seed coat particularly duringthe first hours of the experiment, but the stimulating effectof K⁺ on the release of labelled solutes derived from aminoacids was much smaller. The presence of 25 mM CaCl₂ did notaffect the release of sucrose or amino acids from the seed coat.The effect of K⁺ on sucrose and amino acid release is explainedas an inhibition of sucrose and amino acid resorption from theseed coat apoplast into seed coat cells, after unloading fromthe seed coat unloading sites. It is suggested that amino acidrelease is much less affected by K⁺ than sucrose release, becausefar less resorption of amino acids by seed coat parenchyma cellstakes place during amino acid transport into the seed coat cavity. Pisum sativum, pea, assimilate transport, assimilate unloading, seed-coat exudate, seed development, sucrose resorption, surgical treatment     

ORIGIN OF AMINO ACIDS CONSTITUTING CELLULAR PROTEIN IN GERMINATING CONIDIOSPORES OF ASPERGILLUS NIGER

Formation of pool amino acids in germinating spores of Aspergillusniger strain 1617 was investigated. The pool amino acids comprisedmainly glutamic acid and alanine. Small amounts of pyruvateand -ketoglutarate were found to increase almost in parallelwith the course of increase in the amount of free amino acidsup to the stage of onset of active protein synthesis. Asparticglutamictransaminase activity was exhibited even in dormant spores andit developed in response to the increase in cellular protein.Alanine-glutamic transaminase activity, on the other hand, waslacking in dormant spores and appeared at the stage of accumulationof amino acids preceding protein synthesis. It was revealed from the experiments with ³⁵S-labeled sporesthat the dormant spores of this fungus contain two unidentifiedsulfur substances, and the sulfur of these substances is incorporatedinto the sulfur amino acids of the protein synthesized in germinatingspores. ¹Present address: Institute of Applied Microbiology, Universityof Tokyo, Tokyo (Received September 11, 1959; )     

Indirect stimulation of protein synthesis by indoleacetic acid in the mung bean hypocotyl

No exact estimation of the amount of radioactive free aminoacids in the cells of the tissue with large size of apparentfree space was possible, since the exact size of the apparentfree space cannot be measured. Furthermore, estimation of thesize of the protein precursor pool, using the method of Hollemanand Key, was not possible in hypocotyl sections of mung bean(Phaseolus mungo L. cv. Black), because of the great differenceover the length of a section in the rate of the incorporationof leucine-¹⁴C into protein. Also, most of the radioactivityin the active pool disappeared within 10 min of the chase periodin the presence or absence of IAA, before the effect of IAAon protein synthesis was shown. Thus, neither can the pulse-chaseexperiment be used to study auxin-induced protein synthesis. IAA stimulated neither the formation of amino acids from acetate-¹⁴C,nor the incorporation of the newly formed amino acids into protein.However, IAA did stimulate both the uptake of sucrose-¹⁴C andpyruvate-¹⁴C into tissue and/or the formation of amino acidsfrom these substances, which resulted in stimulation of theincorporation of these radioactive amino acids into proteins.Enhancement effects of IAA on the rates of amino acid formationand the incorporation of amino acids into protein were of thesame magnitude. These results indicate that radioactive amino acids are spontaneouslyincorporated into proteins without any positive effect by IAA.Furthermore, IAA protects the degradation of some protein fractions.All diis evidence raises questions as to the validity of thehypothesis that auxin promotes protein synthesis. (Received July 17, 1972; )     

Evidence of ammonium assimilation via the glutamine synthetase-glutamate synthase system in rice seedling roots

When rice seedling roots were fed ¹⁵N-ammonium for 1 hr, theamide nitrogen of glutamine showed the highest ¹⁵N abundance.Moreover, glutamine amino, glutamic acid, aspartic acid andalanine showed higher ¹⁵N abundance than ammonium did. In roots whose GS activity was inhibited with MS, both the amountof ammonium and its ¹⁵N abundance were increased. In contrast,both the amount of all examined amino acids containing glutamicacid and their ¹⁵N abundance decreased in roots whose GS activitywas inhibited. From these results, it could be concluded thatthe first step of ammonium assimilation in rice seedling rootswas mainly glutamine synthesis by GS and the second was glutamicacid formation by the GOGAT system. The results of an experiment using ¹⁵N glutamine also supportedthis conclusion. (Received February 23, 1977; )     

The Significance of Transpirationally Derived Nitrogen in Protein Synthesis in Fruiting Plants of Pea (Pisum sativum L.)

Feeding of ¹⁵N-nitrate, ¹⁵N(amide)-L-glutamine, or ¹⁵N-L-glutamicacid to detached shoots of pea through the transpiration streamresults in the soluble and insoluble nitrogen of stem, leaves,and fruits becoming extensively enriched with isotopic nitrogen.The time course of labelling suggests that non-reproductiveparts are the principal centres of uptake and assimilation andthat from them translocation takes place to the developing seeds. Distribution patterns for ¹⁵N in free and protein-bound aminoacids of leaf and seed indicate that each labelled source donatesnitrogen to a wide range of amino compounds, with no evidenceof consistent differences in the manner in which each is assimilated.Alanine, glutamic acid, homoserine, and -aminobutyric acid,are the main recipients of ¹⁵N in the soluble fraction of theleaves, whilst in the insoluble fraction nitrogen of the aminoacids serine, glycine, alanine, threonine, glutamic acid + glutamine,and aspartic acid + asparagine achieves high specific labelling.Amino acids of the seeds are labelled more uniformly with ¹⁵N. A complementary ¹⁴C-labelling experiment on the translocationof photosynthetically fixed carbon from leaf to seed is describedand the labelling patterns obtained for amino acids in leaf,seed, and phloem exudate are discussed in relation to thosefor ¹⁵N.     

Phloem Loading and Metabolism of Xylem-Borne Amino Compounds in Fruiting Shoots of a Legume

Cut, fruiting shoots of Lupinus albus L. supplied with ¹⁴C-and ¹⁵N-labelled L-asparagine, L-glutamine, L-aspartic acid,or L-glutamic acid through the transpiration stream readilytransferred the labelled carbon and nitrogen of each compoundto pods and seeds of fruits. A time course of labelling of phloemsap collected from petioles and fruit tips following feedingof labelled asparagine indicated that xylem to phloem exchangein leaflets was an immediate and effective route of transferof the amide to fruits and that this and the loading onto phloemof additional asparagine from unlabelled pools of the amidein stems furnished a major source of the nitrogen for fruitfilling. Xylem to phloem exchange of nitrogen was accomplishedin different ways for each amino acid. The amide nitrogen ofasparagine was transferred mainly in the unmetabolized compound,the nitrogen of aspartate and glutamate largely in a wide rangeof amino acids synthesized in the leaf, and the amide nitrogenof glutamine was transferred in a manner intermediate betweenthese extremes. Glutamine and asparagine were the principalphloem solutes labelled with nitrogen from any of the suppliedcompounds, but the photosynthetically produced amino acids,glutamate, aspartate, serine, alanine, and valine also became¹⁵N-labelled in phloem. The main pathway for glutamine synthesisin vegetative parts of the shoot appeared to be by amidationof glutamate, but asparagine was not considered to be derivedsimilarly from aspartate. Leaflets metabolized glutamine morereadily than asparagine, but in each case the amide nitrogenwas used for synthesis of a variety of amino acids and the carbonwas recovered largely in non-amino compounds.     

Turgor Regulation in a Brackish Water Charophyte, Lamprothamnium succinctum III. Changes in Cytoplasmic Free Amino Acids and Sucrose Contents during Turgor Regulation

Cytoplasm and cell sap of Lamprothamnium succinctum were analyzedseparately for the contents of free amino acids and sucroseto find whether they contribute to turgor regulation. In thevacuole, both amino acids and sucrose were found to be minorcomponents contributing to the generation of osmotic pressure.Their amounts were almost insensitive to changes in externalosmotic pressure. In the cytoplasm, both amino acids and sucrosein the cytoplasm contributed about 20% to the osmotic pressure.Hypotonic treatment did not affect the contents of either, buthypertonic treatment, while not affecting the amino acid contents,caused a significant increase in sucrose content. The cytoplasmicsucrose content increased linearly with an increase in externalosmotic pressure, accounting for 40% of the increased osmoticpressure. ¹ Present address: Department of Biology, Osaka Medical College,Sawaragi-cho, Takatsuki, Osaka 569, Japan ² Present address: Department of Applied Physiology, NationalInstitute of Agrobiological Resources, Yatabe, Tsukuda, Ibaragi305, Japan (Received November 25, 1986; Accepted March 18, 1987)     

Tastes of salts and acids on circumvallate papillae and anterior tongue

In a psychophysical study with human subjects, three chloridesalts and three acids were tasted at two different isointenseconcentrations. Subjects profiled the taste sensations elicitedwhen the stimuli were applied to small regions of the anteriortongue and to individual circumvallate papillae. The resultsextended earlier findings showing systematic differences inthe responses to acids and salts as a function of the locusto which a stimulus is applied. All salts were perceived aspredominantly salty on the anterior tongue, and as predominantlysour or bitter on circumvallate papillae, although there wasa weak salty component in the response to lower concentrationsof salt on circumvallate papillae. Acids were perceived as souron circumvallate papillae, and as sour and salty on the anteriortongue. Cationic atomic weight was positively related to saltinesson both loci. However, while salts apparently stimulate bothbitter and sour receptors on both the anterior tongue and circumvallatepapillae, there was no systematic relationship between cationicatomic weight and the magnitude of the sour and bitter tasteselicited. It was concluded that the possibility of unequal distributionsof receptor types between fungiform and circumvallate regionsshould be taken into account when interpreting the results ofexperiments using whole-mouth stimulation with salts and acids. ¹This experiment was submitted as part of a dissertation bythe first author in partial fulfillment of the degree of Doctorof Philosophy at Brandeis University. The interpretations inthis paper are not to be construed as an official Departmentof the Army policy or position. ²Current address: Science and Advanced Technology Laboratory,US Army Natick Research and Development Laboratories, Natick,MA 01760.