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

The technical assistance of Ardithanne Boyle, Linda Graham and Pamela Burke is appreciated. I thank Dr. H. Ronald Kaback and Dr. Leonard D. Kohn for their suggestions of experiments relevant to transport questions. This research was supported in part by NIH research grant No. NS-08775 from NINCDS. 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 10mM L-alanine and removal of the L-alanine by washing. Enhancement occurred whether the preparation was stored frozen (?65°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 K_D 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 ^?2M) 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 expiain 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 ahigh ligand concenration.     

Specific inhibition of the binding of the taste stimulus, L-alanine, by sulphydryl reagents, in Ictalurus punctatus

1. Taste receptors for L-alanine and L-arginine in the channel catfish, Ictalurus punctatus, are differentially reactive to N-ethylmaleimide (NEM) and p-chloromercuribenzenesulphonic acid (pCMBS). 2. The binding of L-[3H]alanine by a sedimentable membrane fraction (Fraction P2) isolated from taste epithelium was inhibited by both NEM and pCMBS while the binding of L[3H]arginine was unaffected. 3. Inhibition of the binding of L-[3H]alanine by pCMBS was reversible with dithiothreitol (DTT). 4. NEM (10(-3) M) inhibited multi-unit neural responses to both 10(-4) M L-alanine and 10(-4) M L-arginine, while pCMBS had little effect on neural responses. 5. Pretreatment of intact taste epithelium before the preparation of Fraction P2 with NEM caused strong inhibition of L-[3H]alanine binding, while pretreatment with pCMBS caused weak inhibition. 6. The presence of L-alanine during the reaction of pCMBS or NEM with taste plasma membranes did not substantially protect against the inhibition of L-[3H]alanine binding.     

Biochemical studies of taste sensation--VIII. Partial characterization of alanine-binding taste receptor sites of catfish Ictalurus punctatus using mercurials, sulfhydryl reagents, trypsin and phospholipase C.

1. Taste receptors for L-alanine in the channel catfish Ictalurus punctatus have been partially characterized. The binding activity, which is localized to a sedimentable fraction (Fraction P2), was assayed with L-[3H]alanine as the ligand. 2. Addition of HgCl2 or p-mercuribenzoate to the assay at 0.1-1 mM markedly inhibited binding. The effect was not reversible and was unaffected by increased L-alanine in the binding assay. 3. The sulfhydryl reagents iodoacetate, 5,5'-dithiobis(2-nitrobenzoic acid), arsenite, and N-ethylmaleimide did not show appreciable inhibition of binding. The results suggest that the inhibitory effect of mercurials is not on specific sulfhydryl groups at alanine-binding sites. 4. Treatment of Fraction P2 with phospholipase C decreased binding activity and treatment with trypsin led to increased binding activity.     

Biochemical studies of taste sensation. IX. Enhancement of L-[3H]glutamate binding to bovine taste papillae by 5'-ribonucleotides

The interaction of the taste substance monosodium L-glutamate with taste receptors has been investigated. Binding of L-[3H]glutamate was measured to preparations of bovine circumvallate (taste) papillae (type I preparation) and to control tongue epithelial preparations (type II preparation) devoid of taste receptors. Binding is operationally defined using a membrane filtration assay. Substantially greater binding occurred to the type I preparation than to the type II preparation, and the binding to the type I preparation showed evidence of saturation. The apparent Kd of L-glutamate was estimated to be in the range of 20--30 mM. The unique taste effect of L-glutamate was considered to depend importantly on its demonstrated synergism in combination with certain 5'-ribonucleotides. A several-fold enhancement of binding of L-[3H]glutamate occurred in the presence of certain 5'-ribonucleotides. 5'-GMP, 5'-IMP and 5'-UMP each increased the binding of L-[3H]glutamate, while 5'-XMP, 5'-AMP and 5'-CMP did not. None of these nucleotides affected the lower level of binding to the type II preparation. Neither the free bases, adenine and guanine, their nucleosides nor their di- or triphosphonucleotides were effective in increasing L-[3H]glutamate binding to the type I preparation. The nucleotide specificity of the glutamate binding enhancement therefore shows a marked similarity with the nucleotide specificity in evoking the synergistic taste effect in humans.     

Molecular structural requirements for binding and activation of L-alanine taste receptors

Summary L-Alanine binds to and activates specific taste receptors ofIctalurus punctatus, the channel catfish. In order to determine the structural requirements for receptor binding and activation in this model system, a number of analogues of L-alanine were tested using a neurophysiological assay and a competitive ligand binding assay. These assays measured the ability of analogues to activate taste receptors and to displace L-[³H]alanine from L-alanine binding sites. Of those derivatives with modifications of the sidechain, L-serine, glycine,-chloro-L-alanine and 1-amino-cyclopropane-1-carboxylic acid were the most potent analogues with IC₅₀s similar to and neural responses slightly decremented from that of L-alanine. Derivatives containing branched sidechains or sidechains of otherwise increased volume were considerably less active. All modifications of the-carboxylic acid and the-amine, including amides, esters and various isosteres, led to substantial reduction in the analogues' ability to displace L-[³H]alanine and, in most cases, very weak stimulatory capability. However, L-lactic acid was a reasonably strong stimulus, but a poor competitor, suggesting that it acts at a different receptor site. Overall, these results indicate the importance of the charged amine and carboxylic acid groups for binding to and activation of the receptor for L-alanine. Moreover, modifications around the chiral center of L-alanine support the hypothesis that receptor binding and activation are separate processes in this model taste system.     

Biochemical studies of taste sensation--XII. Specificity of binding of taste ligands to a sedimentable fraction from catfish taste tissue

The specificity of amino acid binding sites in a sedimentable fraction prepared from catfish taste epithelium was examined. Using seven 3H-labeled amino acids as ligands and the unlabeled amino acids in binding competition assays, the presence of possibly three classes of amino acid binding sites was deduced. Site 1 binds L-THR, L-SER, L-ALA and possibly D-ALA and beta-ALA, Site 2 binds L-SER, L-ALA, GLY, D-ALA, and beta-ALA and Site 3 binds L-ARG and L-LYS. Additional evidence supporting the specificity of Site 2 was obtained from the specificity of enhancement of L-ALA binding. The results demonstrate the presence of some major classes of taste receptor sites, and provide a basis for understanding taste receptor specificity at the biochemical level.     

Biochemical studies of tast sensation. Binding of L-[3H]alanine to a sedimentable fraction from catfish barbel epithelium.

Large numbers of taste buds are distributed over the body surface of the channel catfish ictalurus punctatus, with the barbels having an especially high density. L-Alanine, as well as certain other amino acids, are taste stimuli in this animal. Epithelial tissue obtained by gentle scraping of the barbel surface was fractionated by differential centrifugation. A sedimentable fraction (P2) was prepared that was enriched in L[OH]alanine binding activity, the plasma membrane marker enzyme 5'-nucleotidase, and the mitochondrial marker succinate cytochrome c reductase, but not the microsomal marker NADH cytochrome c redu.ctase. Binding of L-[OH]alanine was measured using a Millipore filter method in which correction for non-specific binding was also determined. Time, temperature, and pH for measuring binding activity were established. At the optimal pH of 7.8, the KD for L-alanine is 4.8 X 10(-6) M. The first order dissociation rate constant at 6 degrees is 3.8 X 10(-4) s-1 and at 24 degrees it is 12.1 X 10(-4) s-1. The second order rate constant for association is between 10(2) and 10(3) M-1 S-1. Reversibility of the binding interaction was also demonstrates by the rapid displacement of bound L-[3H]alanine by a large excess of unlabeled L-alanine. That the binding does not represent incorporation into protein was confirmed by the lack of effect of puromycin. The amounts bound of several other chemostimulatory amino acids werealso determined.     

Receptor sites for amino acids in the facial taste system of the channel catfish

1. Receptor sites for different amino acids in the facial taste system of the channel catfish, Ictalurus punctatus, were determined from in vivo electrophysiological cross-adaptation experiments. 2. Relatively independent receptor sites were indicated for L-proline, D-proline, D-arginine, L-histidine and L-lysine, as well as those previously reported for L-alanine, L-arginine and D-alanine. 3. The functional isolation of two nerve twigs that were more responsive to D-alanine than to L-alanine or to other test stimuli provided further evidence for the existence of D-alanine sites that are independent from those to L-alanine. 4. Under all cross-adaptation regimes, the taste responses to the majority of test stimuli were reduced. Various possible mechanisms accounting for this generalized reduction in action potential activity during adaptation are discussed.     

Selective interactions of lectins with amino acid taste receptor sites in the channel catfish

Five lectins of varying carbohydrate specificities, Dolichosbiflorus (DBA), jacalin, Phaseolus vulgaris (PHA), Pisum sativum(PSA) and Ricinus communis (RCA I), were used to extend characterizationof the glycoprotein nature of taste plasma membranes and todifferentially affect the binding of two taste stimuli, L-alanineand L-arginine, to their respective taste receptor sites inthe cutaneous taste system of the channel catfish (Ictaluruspunctatus). The binding of the taste stimulus L-arginine toa partial membrane fraction (P_–) from taste epitheliumwas inhibited by 68 and 74% by preincubation in the presenceof the unconjugated lectins PHA and RCA I respectively. A correspondinglevel of inhibition of L-alanine binding was seen in the presenceof RCA I (76%); however, PHA had little effect upon L-alaninebinding. DBA appeared to selectively inhibit L-alanine but notL-arginine binding (60 versus 8% respectively) while jacalinmoderately inhibited the binding of both stimuli to fractionP2. PSA had little effect upon the binding of either L-alanineor L-arginine (4 and 5% inhibition respectively). Inhibitionof taste receptor binding by all lectins was time- and dose-dependent,and was fully abolished by incubation in the presence of theappropriate hapten sugar. The biotinylated lectins DBA, jacalin,PHA, RCA I and concanavalin A (Con A) were used to identifythe glycoprotein components of the chemosensory plasma membranesafter polyacrylamide gel electrophoresis. As previously shown,numerous protein components were labeled by Con A. In contrast,only a few minor protein components were labeled by PHA, DBAand RCA I. This differential labeling of the taste membranesand the differential inhibition of receptor binding by lectinssuggest that they may prove useful as tools in the isolationand purification of taste receptor proteins.     

Structure/activity relationships in the arginine taste pathway of the channel catfish

To characterize the molecular/structural requirements for activationor antagonism of the arginine taste pathways in catfish, Ictaluruspunctatus, structure/activity studies were performed using integratedmultiunit responses and cross-adaptation. Of all the guanidinium-containingcompounds tested, only L-arginine, L--amino-ß-guanidinopropionic acid (L-AGPA) and L-arginine methyl and ethyl esterswere strong stimuli. Results of functional group substitutionsand modification of the L-arginine parent molecule indicatedthat: (i) stereospecificity was observed with D-arginine beinga much less effective stimulus than L-arginine; (ii) an L-aminogroup must be present and unblocked (-chloro-guanidino-N-valericacid and N-acetyl L-arginine were weak or inactive stimuli);(iii) a free carboxylic acid group was not necessary for activity;(iv) the distance between the anomeric carbon and the guanidiniumgroup was not critical (L-AGPA, having two methylene groupsless than L-arginine was a moderately strong stimulus as wasL-canavanine) and (v) modification or substitution of the guanidinumgroup by other basic groups including amine, methyl or dimethylamineor by an isosterc (ureido) resulted in loss of stimulatory ability.In general, those stimuli and analogs that were good cross-adaptersof L-arginine stimulation were also good competitors for L-[³H]argininebinding to a partial membrane fraction (P2) from catfish tasteepithelium. On the other hand, compounds that were poor cross-adaptingstimuli were also poor binding competitors. While D-argininewas a poor stimulus, it did cross-adapt L-arginine and competedwell with L-[³H]arginine for binding to fraction P2.     

A stimulus-activated conductance in isolated taste epithelial membranes.

Membrane vesicles isolated from the cutaneous taste epithelium of the catfish were incorporated into phospholipid bilayers on the tips of patch pipettes. Voltage-dependent conductances were observed in approximately 50% of the bilayers and single-channel currents having conductances from 8 to greater than 250 pS were recorded. In 40% of the bilayers displaying no voltage-dependent conductances, micromolar concentrations of L-arginine, a potent stimulus for one class of catfish amino acid taste receptors, activated a nonselective cation conductance. The L-arginine-gated conductance was concentration-dependent, showing half-maximal activation in response to approximately 15 microM L-arginine. L-Arginine-activated channels had unitary conductances of 40-50 pS and reversed between -6 and +18 mV with pseudointracellular solution in the pipette and Ringer in the bath. L-Alanine, a potent stimulus for the other major class of catfish amino acid taste receptors, did not alter bilayer conductance. D-Arginine, which is a relatively ineffective taste stimulus for catfish but a good cross-adapter of the L-arginine-induced neural response, had no effect on bilayer conductance at concentrations below 200 microM. However, increasing concentrations of D-arginine from 1 to 100 microM progressively suppressed the L-arginine-activated conductance, suggesting that D-arginine competed for the L-arginine receptor, but did not activate the associated cation channel. This interpretation is consonant with recent biochemical binding studies in this system. These results suggest that L-arginine taste receptor proteins in the catfish are part of or closely coupled to cation-selective channels which are opened by L-arginine binding.     

Biochemical studies of taste sensation XI. Isolation,characterization and taste ligand binding activity of plasma membranes from catfish taste tissue

Plasma membranes were isolated from taste receptor-containing epithelium of the channel catfish, Ictalurus punctatus. The membranes were prepared by ultracentrifugation of a sedimentable fraction in sucrose, using either a discontinous density gradient or a continous linear density gradient. The plasma membranes were characterized by their increased content of 5′-nucleotidase and by electron microscopy, as well as by a greatly diminished content of NADH-cytochrome c reductase and succinate-cytochrome c reductase. The recovery of binding activity for taste ligands was low, because of the long time-period required for ultracentrifugation, but of the recovered activity 80% occurred in the plasma-membrane preparation. Binding of seven chemostimulatory amino acids was demonstrated and found to correspond reasonably well with earlier binding data obtained using a less pure sedimentable fraction. The data provide direct evidence supporting the long-standing hypothesis that taste receptor sites are localized to the plasma membranes.     

Enhancement of retronasal odors by taste

Psychophysical studies of interactions between retronasal olfaction and taste have focused most often on the enhancement of tastes by odors, which has been attributed primarily to a response bias (i.e., halo dumping). Based upon preliminary evidence that retronasal odors could also be enhanced by taste, the present study measured both forms of enhancement using appropriate response categories. In the first experiment, subjects rated taste ("sweet," "sour," "salty," and "bitter") and odor ("other") intensity for aqueous samples of 3 tastants (sucrose, NaCl, and citric acid) and 3 odorants (vanillin, citral, and furaneol), both alone and in taste-odor mixtures. The results showed that sucrose, but not the other taste stimuli, significantly increased the perceived intensity of all 3 odors. Enhancement of tastes by odors was inconsistent and generally weaker than enhancement of odors by sucrose. A second experiment used a flavored beverage and a custard dessert to test whether the findings from the first experiment would hold for the perception of actual foods. Adding sucrose significantly enhanced the intensity of "cherry" and "vanilla" flavors, whereas adding vanillin did not significantly enhance the intensity of sweetness. It is proposed that enhancement of retronasal odors by a sweet stimulus results from an adaptive sensory mechanism that serves to increase the salience of the flavor of nutritive foods.     

Subcellular localization of nerve growth factor receptors. Thirteen-day chick embryo brain.

The subcellular location of the specific binding sites of nerve growth factor (NGF) as judged by binding of the 125I-labeled protein in 13-day chick embryo brain, has been examined. The homogenized tissue was separated into four fractions, P1, P2, P3, and S, by differential centrifugation. Fraction P2, which contained the majority of the specific 125I-NGF binding, was further separated by discontinuous sucrose density gradient centrifugation into three fractions. Fraction B contains many synaptosome-like structures, which, when derived from adult brain, result from the shearing off and resealing of synaptic terminals. This fraction contained 65% of the specific 125I-NGF binding of P2. Following hypoosmotic lysis by water, Fraction B was separated into seven fractions, O, D, E, F, G, H, and I, by discontinuous sucrose density gradient centrifugation. The specific 125I-NGF binding was localized with the denser fractions, G, H, and I, with about a 10-fold purification as compared to the original homogenate. However, only 65% of the binding of Fraction B was found in the sum of the tertiary fractions, indicating that some loss of specific binding accompanied the lysis. By means of marker enzymes and macromolecules, as well as electron microscopy, it was determined that the distribution of cellular components of embryonic tissue in this fractionation technique is very similar to that observed for adult brain tissue. Thus, the properties of the NGF receptors determined in whole brain, which are remarkably similar to those found in peripheral neurons, are the properties of the receptors that appear to be located in the developing synaptosomal structures.     

Ligand binding specificity of a neutral L-amino acid olfactory receptor

1. The ligand binding specificity of the L-[3H]alanine binding site was investigated in isolated cilia preparations from the olfactory epithelium of channel catfish (Ictalurus punctatus) by competitive binding experiments. 2. Approximately 45 amino acids, derivatives and enantiomers were tested for the ability to compete with radiolabeled L-alanine for common binding sites. 3. Acidic and basic L-amino acids and imino acids did not compete as effectively as L-alanine for the receptor, while long-chain neutral ligands were only partially effective inhibitors of L-alanine binding. 4. D-Alanine and L-alanine derivatives with substituted alpha-amino or carboxyl groups exhibited decreased ability to compete for the receptor, paralleling their lower neurophysiological potency. 5. In combination, the ligand binding results were consistent with previous electrophysiological data in catfish, and suggest the presence of an olfactory receptor site that selectively recognizes short-chain neutral amino acids.     

Amino acid receptor sites in the facial taste system of the sea catfish Arius felis

1. The amino acid sensitivity and specificity of the facial taste system of the marine catfish, Arius felis, is characterized electrophysiologically.
2. The facial taste system of Arius felis responded to all 28 amino acids tested, but was highly sensitive to only a few. In general, acidic amino acids and neutral amino acids with short side chains were more effective than imino, basic and neutral amino acids with long side chains.
3. A reciprocal cross-adaptation protocol used to characterize the receptor sites identified at least some relatively independent receptor sites for L-arginine, L-histidine, L-proline, L-alanine, glycine, D-alanine and L-glutamate.
4. Of the 7 amino acids that were indicated to have relatively independent receptor sites, the median electrophysiological threshold for L-alanine, the most stimulatory, and L-proline, the least stimulatory compounds, were 10 nM and 10,000 nM, respectively. The integrated facial taste response did not saturate at test amino acid concentrations up to 10 mM.
5. The generalized depression in responsiveness to test stimuli observed during amino acid adaptation is proposed to be a result of the co-distribution of sensitivity at the level of single taste cells rather than high cross-reactivity of the respective amino acid receptor sites for the test stimuli.

     

Interaction of leucine with the plasma membranes of catfish taste buds

The binding of L-[3H]leucine by the plasma membranes from the catfish Ictalurus nebulosus taste organ was studied. The two types of specific binding centers for amino acid with high (KD = 2,5 X 10(-10) M) and low (KD = 1,04 X 10(-9) M) affinities were found. Concentrations of high and low affinity centers were 11,85 nmol/mg and 26 nmol/mg respectively. The structural rearrangement of the surface layer of the chemoreceptor membrane was revealed by spin label technique using 5-doxylstearic acid at leucine concentrations 10(-4)-10(-9) M.     

Metabolism of inositol-1,4,5-trisphosphate in the taste organ of the channel catfish, Ictalurus punctatus.

1. The metabolism of inositol-1,4,5-trisphosphate was studied in the taste organ (barbel) of the channel catfish, Ictalurus punctatus. 2. Homogenates of epithelial barbel scrapings were incubated with [3H]-1,4,5-IP3, whose dephosphorylation or phosphorylation was assayed under first-order conditions by measuring the production of either [3H]-1,4-IP2 (representing the activity of IP3-5-phosphatase) or [3H]-1,3,4,5-IP4 (representing the activity of IP3-3-kinase). 3. Both enzymes were predominantly cytosolic, magnesium-dependent and maximally active at pH 6.4. For IP3-phosphatase, Km = 6 microM and Vmax = 10.5 nmol/min/mg. For IP3-kinase, Km = 0.23 microM and Vmax = 0.05 nmol/min/mg. 4. Neither enzyme was significantly affected by the presence of taste stimuli (amino acids), GTP gamma S, cAMP or phorbol esters. 5. In the presence of physiological levels of free calcium (0.05-12 microM) IP3-phosphatase was moderately activated whereas IP3-kinase was moderately inhibited. 6. IP3-phosphatase was moderately activated by Mn2+, unaffected by LiCl, and strongly inhibited by 2,3-diphosphoglycerate, Na-pyrophosphate, CdCl2, HgCl2, CuCl2, FeCl3 and ZnSO4 7. IP3-kinase was strongly activated by 2,3-diphosphoglycerate, Na-pyrophosphate, CdCl2, HgCl2, FeCl3 and LiCl and inhibited by ZnSO4 and Mn2+. 8. IP3-kinase was significantly activated in a calcium-dependent manner by exogenously-added phosphatidylcholine and sphingomyelin, and to a lesser extent by diacylglycerol. IP3-phosphatase was unaffected by exogenously-added lipids. 9. IP3-phosphatase may participate in taste transduction since calculations based on the first-order rate constant (6.9 sec-1) indicate that it is capable of dephosphorylating basal levels of IP3 with a half-life of 0.1 sec.     

The effect of temperature on the turnover of taste bud cells in catfish.

Renewal of taste bud cells on the barbels of channel catfish was studied. Groups of catfish, held in and acclimitized to 14 degrees C, 18 degrees C, 22 degrees C and 30 degrees C dechlorinated tap water were injected with [3H]thymidine (3.0 muCi/g body weight intraperitoneally). Barbels were sampled at various times after injection and prepared for light microscope autoradiography. Results show that epithelial cells surrounding the taste buds divide and some of their daughter cells migrate into the taste buds. The time at which 50% of the labelled cells have degenerated is taken as the average turnover time or average life span of the taste bud cells. The average life span as well as the time spent inside the taste buds is highly temperature-dependent. At 14 degrees C, 18 degrees C, 22 degrees C and 30 degrees C the average life span is on the order of 40, 30, 15 and 12 days respectively. Further studies indicate that both light and dark staining cells of the taste bud were labelled.     

Expression of rat liver Na+/L-alanine co-transport in Xenopus laevis oocytes. Effect of glucagon in vivo.

Poly(A)+ RNA (mRNA) isolated from rat liver was injected into Xenopus laevis oocytes, and expression of Na+/L-alanine transport was assayed by measuring Na(+)-dependent uptake of L-[3H]alanine. Expression of Na+/L-alanine transport was detected 3-7 days after mRNA injection, and was due to an increment of the Na(+)-dependent component. After injection of 40 ng of total mRNA, Na(+)-dependent uptake of L-alanine was 2.5-fold higher than in water-injected oocytes. In contrast with Na+/L-alanine transport by water-injected oocytes, expressed Na+/L-alanine transport was inhibited by N-methylaminoisobutyric acid, was inhibited by an extracellular pH of 6.5 and was saturated at approx. 1 mM-L-alanine. After sucrose-density-gradient fractionation, highest expression of Na+/L-alanine uptake was observed with mRNA of 1.9-2.5 kb in length. Compared with mRNA isolated from control rats, mRNA isolated from glucagon-treated rats showed a approx. 2-fold higher expression of Na+/L-alanine transport. The results demonstrate that both liver Na+/L-alanine transport systems (A and ASC) can be expressed in X. laevis oocytes. Furthermore, the data obtained with mRNA isolated from glucagon-treated rats suggest that glucagon regulates liver Na+/L-alanine transport (at least in part) via the availability of the corresponding mRNA.