Citrate Ions Enhance Taste Responses to Amino Acids in the Largemouth Bass

K. Ogawa and J. Caprio

Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
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Ogawa, K. and J. Caprio. Citrate ions enhance taste responses to amino acids in the largemouth bass. The glossopharyngeal (IX) taste system of the largemouth bass, Micropterus salmoides, is highly selective to amino acids and is poorly responsive to trisodium citrate; however, IX taste responses to specific concentrations of L- and D-arginine and L-lysine but not L-proline were enhanced by citrate but not sodium ions. Binary mixtures of L-arginine (3 × 10-4 M and 10-3 M) or D-arginine (10-3 M) + trisodium citrate (10-3 M; pH 7-9) resulted in enhanced taste activity, whereas binary mixtures of higher concentrations (10-2 M and 10-1 M) of L- or D-arginine + 10-3 M trisodium citrate were not significantly different from the response to the amino acid alone. Under continuous adaptation to 10-3 M citrate, taste responses to L-arginine were also enhanced at the identical concentrations previously indicated, but responses to 10-2 M and 10-1 M L-arginine were significantly suppressed. Under continuous adaptation to 10-2 M L-arginine, taste responses to 10-2 M, 10-1 M, and 100 M citrate were significantly enhanced. Cellular concentrations of both citrate and amino acids in prey of the carnivorous largemouth bass are sufficient for this taste-enhancing effect to occur naturally during consummatory feeding behavior. Citrate acting as a calcium chelator is presented as a possible mechanism of action for the enhancement effect.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Citric acid, a six-carbon tricarboxylic hydroxy acid, is heavily used in the food and beverage industries as an acidulant, a compound that renders food more palatable, and serves a variety of other functions, such as pH buffer, preservative, synergist to antioxidants, melting and viscosity modifier, and a curing agent (Gardner 1972). Citric acid is also known to enhance the palatability of animal foods. For example, citric acid increased daily food consumption as an additive in horse feed (Betz and Lantner 1980) and in combination with phosphoric acid enhanced the flavor of cat food (Kealy 1975). In the herbivorous fish Tilapia zillii citric acid increased the palatability of a nonpreferred diet to a level equivalent to that of the most preferred feed (Adams et al. 1988). Although citric acid and its salt (trisodium citrate) are used to modify the flavor of foods and beverages, little is known concerning the specific food target(s) or mechanism(s) of its action. A recent report indicated that citrate enhanced the preference for sweet compounds and glycine in rats and that citrate enhanced taste cell responses to both saccharin and glycine (Gilbertson et al. 1997). Our study indicates that trisodium citrate specifically enhances the taste response of the glossopharyngeal nerve in the carnivorous largemouth bass, Micropterus salmoides, to particular amino acids. Although citric acid is often used as a representative sour stimulus (i.e., proton donor) in the study of vertebrate taste, the current enhancing effect is not due to an acidic effect on taste cells as gustatory enhancement occurred only with stimuli at pH 7-9. A portion of these results appeared in abstract form (Ogawa and Caprio 1995).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Largemouth bass, Micropterus salmoides (~17-25 cm in length), were obtained from a fish farm (Ken's Hatchery and Fish Farms; Alapaha, GA) and shipped overnight to Louisiana State University, where they were maintained in a 250-l fiberglass aquarium in aerated, charcoal-filtered tap water (artesian well water) at 25°C and tested within 3 wk of shipment. Before experimentation, the bass were immobilized with an intramuscular injection of Flaxedil (gallamine triethiodide; 0.4 mg/100 g body weight), wrapped in wet tissue paper, and positioned on a wax block in a Plexiglas container. Aerated artesian tap water at room temperature (21°C) containing the anesthetic MS-222 (ethyl-m-aminobenzoate methane sulfonic acid) at 0.05% continually bathed (~500 ml/min) the gills on one side of the animal throughout the experiments. Supplemental Flaxedil was administrated as required. The glossopharyngeal nerve (cranial nerve IX) was targeted for the electrophysiological taste recordings because scanning electron microscopy indicated a higher density of taste buds occurring on the ventral oral epithelium innervated by IX than on the lips or rostral palate innervated by the facial nerve (cranial nerve VII) (Ogawa and Caprio, unpublished). The dorsal portion of the operculum was surgically removed contralateral to the side receiving the respiratory water flow. An incision was made along the margin of the first gill arch parallel to the supporting cartilage to expose the glossopharyngeal (IX) nerve that innervates taste buds on the floor of the oral cavity. Once freed from adjacent tissue, the IX nerve was transected and desheathed, and its peripheral cut end was separated with fine forceps into small nerve bundles. To create a recording cavity, one or two stitches were made with a fine surgical needle in the tissue ventral to the incision. Slight tension was placed on the surgical thread to open the incision and to create a cavity. Muscle tissue previously surgically removed and collected was positioned along the periphery of the cavity to create a dam. The taste activity of the different IX nerve bundles to amino acid stimulation of the oral cavity was sampled to find one that was responsive to amino acid stimuli. Preliminary studies indicated better signal-to-noise taste responses when recording from a smaller nerve bundle than from the entire nerve. The end of the selected IX bundle was placed over a platinum hook electrode, and the recording chamber cavity was filled with halocarbon oil to prevent the nerve bundle from drying during the recording session. An insect pin placed into tissue adjacent to the gill arch served as the reference electrode, and the fish was grounded via a hypodermic needle embedded in the flank musculature. The neural activity was AC amplified, integrated (0.5 s rise time), monitored aurally, displayed on an oscilloscope and chart recorder, and recorded on videotape. The height of peak magnitude of the integrated taste response was quantified and reported as a percentage of the response to a standard (either 3 × 10-3 M or 10-1 M L-arginine for most analyses or 10-1 M D-arginine). Responses were recorded from 29 dissected IX nerve branches obtained from 26 fish. Gustatory "enhancement" occurred if the integrated taste response to the mixture of citrate and an amino acid was significantly greater than the sum of the responses to the components tested separately at the same concentrations as presented in the binary mixture. Gustatory "suppression" (i.e., response decrement) is therefore a significantly smaller taste response under the same conditions as stated previously. The effects of concentration and treatment on the taste response were analyzed as a two-factor factorial randomized block design (Proc Mixed, SAS 6.12; alpha  = 0.05). Fish were treated as random effects. The interaction between concentration and treatment was also tested, and when significant the effect of treatment was tested for significance at each concentration.

Trisodium citrate and individual amino acids (Table 1) were purchased from Sigma Chemical (St. Louis, MO) and were of the highest quality available. Stimulus solutions were prepared weekly at 10-1 M in charcoal-filtered tap water, stored in glass bottles at 4°C, diluted to the desired experimental concentrations daily, and tested at room temperature (21°C). Stimulus solutions were added to a 0.5-ml Teflon loop of a manual sample injection valve (model No. 1106, Omnifit USA; Atlantic Beach, NY) and injected into the water flow (12 ml/min) directed to the floor of the oral cavity. The maximum stimulus concentration delivered was 75% of the concentration injected as determined by photodensitometry of dye solutions. The undiluted concentrations of the stimuli are reported in the text. Interstimulus intervals were >= 3 min. For experiments reported in Fig. 3, pH of the test solutions was adjusted with either NaOH or HCl.


                              
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Table 1. RSE of amino acids


    RESULTS
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INTRODUCTION
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DISCUSSION
REFERENCES

The glossopharyngeal (IXth) taste system in the largemouth bass, which innervates taste buds on the floor of the oral cavity, responds moderately but is highly selective to the basic amino acids L-arginine and L-lysine (Table 1). However, when presented with 10-3 M trisodium citrate, itself nonstimulatory, as a component in a binary mixture with L- or D-arginine, the taste response was significantly enhanced in comparison with the response to arginine alone (Fig. 1A). Other tested citrate concentrations in a binary mixture with L-arginine resulted in either no significant effect (10-5 M and 10-4 M citrate) or in a significant response suppression (10-2 M and 10-1 M citrate) (Fig. 1B). Citrate (10-3 M) enhanced the taste activity to 10-2 M L-lysine but not to other tested concentrations and had no significant effect on the response to 10-3 M, 10-2 M, or 10-1 M L-proline (Fig. 2).



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Fig. 1. A: paired integrated glossopharyngeal taste responses to 10-3 M trisodium citrate (Aa1) to 3 × 10-4 M L-arginine (Aa2) and to the binary mixture of 10-3 M trisodium citrate and 3 × 10-4 M L-arginine (Aa3); paired integrated glossopharyngeal taste responses to 10-3 M trisodium citrate (Ab1) to 3 × 10-4 M D-arginine (Ab2) and to the binary mixture of 10-3 M trisodium citrate and 3 × 10-4 M D-arginine (Ab3); paired responses are to indicate the repeatability of the responses. B: dose-response relation of the response to trisodium citrate (open circle ) and to the binary mixture of trisodium citrate and 3 × 10-4 M L-arginine (black-square); *: significantly greater taste response to the binary mixture than to the sum of the responses to 3 × 10-4 M L-arginine (=100) and citrate tested independently; **: significantly smaller taste response to the binary mixture than to the sum of the responses to 3 × 10-4 M L-arginine and citrate tested independently; bars, ±SD; mean control response to water + 1 SD is indicated by the line parallel to the abscissa; pH 9.0; n = 5 fish.



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Fig. 2. Dose-response relations of the integrated glossopharyngeal taste response to L-lysine alone (open circle ), the binary mixture of L-lysine and 10-3 M Na3 citrate (+ citrate; ), to L-proline alone (diamond ), and to the binary mixture of L-proline and 10-3 M Na3 citrate (+ citrate; black-diamond ); *: significantly greater response to the binary mixture than to the sum of the responses of the components tested individually; : response to 10-3 M Na3 citrate; pH 9.0; n = 3 fish.

The enhancement effect of trisodium citrate on the gustatory response to L-arginine was due to the citrate ion and not the sodium cation. A binary mixture of nonstimulatory 10-3 M trisodium citrate [2.4 ± 1.6% (SD) of the response to 10-1 M L-arginine; n = 7; pH 9.0] and 3 × 10-4 M L-arginine (26.3 ± 13.4%) dissolved in charcoal-filtered artesian tap water (cftw), which naturally contains 3 × 10-3 M NaCl, resulted in gustatory enhancement (53.0 ± 28.6%); however, no enhancement but a significant response decrement occurred in response to 3 × 10-4 M L-arginine dissolved in cftw to which an additional 3 × 10-3 M NaCl was added (16.9 ± 10.6%; repeated-measure analysis of variance, P < 0.05; n = 7 fish).

The enhancement effect of citrate on the amino acid taste response was pH dependent and was observed over the pH range of 7-9 (Fig. 3).



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Fig. 3. Effect of pH on the integrated glossopharyngeal taste response to charcoal-filtered tap water adjusted with either NaOH or HCl to pH 4-9, trisodium citrate (10-3 M), L-arginine (3 × 10-3 M), and to the binary mixture of trisodium citrate and arginine. *: significant enhancement of the taste response to the binary mixture of citrate and arginine compared with the response to 3 × 10-3 M L-arginine and citrate; the SD of the responses to citrate and the adjusted tap-water over the range of pH 9-4 was too small to indicate by bars; n = 3 fish.

The enhancing effect of 10-3 M citrate was dependent on the L-arginine concentration. Trisodium citrate (10-3 M) enhanced significantly the taste responses to 3 × 10-4 M and 10-3 M L-arginine, and this enhancement occurred whether the citrate was presented with the arginine as a brief bolus of binary mixture (Fig. 4; Table 2) or whether the taste buds innervated by IX were continuously adapted to citrate to which arginine was then presented (Fig. 5). Although 10-2 M and 10-1 M L-arginine in a binary mixture with 10-3 M citrate had no significant effect on the magnitude of the integrated taste response (Fig. 4), during continuous adaptation of the taste-receptive field to 10-3 M citrate, the responses to 10-2 M and 10-1 M L-arginine were significantly suppressed compared with the taste response to L-arginine alone (Fig. 5). Response enhancement also occurred when the taste-receptive field was continuously adapted to L-arginine, to which citrate was presented (Fig. 6). In this paradigm gustatory enhancement occurred at all tested concentrations of citrate >= 10-2 M.



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Fig. 4. Dose-response relation of the integrated glossopharyngeal taste response to L-arginine alone (circle) and to the binary mixture of L-arginine and 10-3 M Na3citrate (+ citrate, star). *: significant enhancement of the taste response to the binary mixture compared with the sum of the responses to the components tested individually; square, response to 10-3 M citrate; numbers adjacent to each response indicate the number of fish tested from a total of 10; pH 9.0


                              
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Table 2. Percent enhancement and suppression of the glossopharyngeal taste response



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Fig. 5. Dose-response relation of the integrated glossopharyngeal taste response to L-arginine alone (unadapted, circle) and to L-arginine during continuous presentation of 10-3 M trisodium citrate (adapted, star) to the taste-receptive field. *: significant enhancement of the taste response in comparison to the sum of the responses to arginine and trisodium citrate tested independently. **: significant inhibition of the taste response in comparison to the sum of the responses to the components tested independently; square, response to 10-3 M trisodium citrate; numbers adjacent to each response indicates the number of fish tested from a total of 7; pH 9.0.



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Fig. 6. Dose-response relation of the integrated glossopharyngeal taste response to trisodium citrate alone (preadaptation, star; post adaptation, diamond) and to trisodium citrate during adaptation of the taste-receptive field to 10-2 M L-arginine (square). *: significant enhancement of the taste response; pH 8.0; n = 5 fish.

The enhancement of the taste response to arginine by citrate is not stereospecific to the L-amino acid as a binary mixture of D-arginine and citrate also resulted in a significantly greater response magnitude than to D-arginine alone (Figs. 1A and 7). A significant enhancing effect of citrate on the D-arginine taste response occurred only with 10-3 M D-arginine. This same concentration of L-arginine with 10-3 M citrate resulted in taste enhancement in either the unadapted (Fig. 4) or adapted (Fig. 5) states (Table 2).



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Fig. 7. Dose-response relation of the integrated glossopharyngeal taste response to D-arginine alone (circle) and to the binary mixture of D-arginine and 10-3 M Na3 citrate (+ citrate, star). *: significant enhancement of the taste response to the binary mixture in comparison to the response to D-arginine plus control; square, response to 10-3 M Na3 citrate; pH 8.0; n = 2 fish.


    DISCUSSION
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INTRODUCTION
METHODS
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DISCUSSION
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This paper shows that the citrate anion enhances the glossopharyngeal taste response to specific amino acids in the largemouth bass. A previous study indicated that citric acid caused increased food consumption, bite size, and rate of feeding in the herbivorous fish Tilapia zillii (Adams et al. 1988). Citrate threshold for this enhancing effect in Tilapia was estimated to be between 10-3 M and 10-2 M, approximately one log unit higher in concentration than indicated here for the electrophysiological enhancement of citrate on the taste response to amino acids in the largemouth bass. However, the citrate effect in Tilapia may have in large part been a pH effect as 1) the citric acid solutions tested had the lowest pH of the several potential enhancers studied, 2) Tilapia showed a general feeding response to acidic substances, and 3) feeding enhancement by the various potential enhancer substances tested increased dramatically at pH values of <4.0. In this study, enhancement of IX taste activity in the largemouth bass was also pH sensitive, but the effect was not due to acidity of the test solutions as the synergistic effect occurred only between pH 7 and 9, pH values that are found naturally in pond water where the fish live (unpublished observations).

The enhancing effect of citrate in the IX taste system of the largemouth bass is selective for the more stimulatory amino acids. Citrate enhanced the taste response in the largemouth bass to L- and D-arginine and L-lysine but was ineffective in modifying the taste response to the less stimulatory amino acid L-proline. Although the percent enhancement of the taste response to both D-arginine and L-lysine was greater than that reported for L-arginine (Table 2), the absolute value of the integrated taste responses to all three amino acids was similar (compare Figs. 2, 4, and 7). In the channel catfish, citrate selectively enhanced the glossopharyngeal taste response to L-proline but had no effect on the taste response to either L-alanine or L-arginine (Davis and Caprio 1996). These results in both the largemouth bass and the channel catfish are consistent with the hypothesis that the target of the citrate action is the taste cell and either a specific amino acid receptor site or its immediate environment.

The enhancing effect of citrate on amino acid taste responses in carnivorous teleosts observed in the laboratory likely occurs naturally in the aquatic habitat. Amino acids are naturally occurring in the environment emanating from all living and decaying organisms. Concentrations of amino acids can be rather high (up to a few hundred millimoles) in living tissue (Carr 1988; Carr et al. 1996). In addition, because of the citric acid cycle (i.e., Kreb's cycle) citrate concentrations in the hemolymph of a variety of potential food organisms (e.g., insects) for both catfish and bass exceed 10-3 M and can reach over 3 × 10-2 M (Wyatt 1961). Although citrate, a cellular metabolite during aerobic respiration, enhances gustatory neural activity (Davis and Caprio 1996; this report) and ingestion (Adams et al. 1988) in fish, lactic acid, a cellular by-product of anaerobic respiration, also increases ingestion in fish. Lactic acid enhanced the palatability of a synthetic white muscle extract of the jack mackeral (Trachurus japonicus) for young yellowtail (Seriola quinqueradiata) (Kohbara et al. 1993) and a synthetic mixture of squid muscle for plaice (Pleuronectes platessa) (Mackie 1982). Similar to citrate, lactic acid also occurs in tissues of fish and crustaceans in concentrations as high as 2 × 10-2 M to 7 × 10-2 M (Carr et al. 1996). Although the mechanism for the lactic acid effect is unknown, it is tempting to speculate that lactic acid like citrate may enhance the taste of specific amino acids present in the extracts.

Citrate also enhanced the taste responses of amino acids in mammals (Gilbertson et al. 1997). In 2- and 4-day two-bottle preference tests, citrate (10-3 M to 2.5 × 10-2 M; pH 7.0) significantly enhanced the preference of Sprague-Dawley rats for glycine [and also saccharin (5 × 10-4 M), sucrose (10-1 M), and the synthetic sweetener SC-4567 (5 × 10-5 M)] over control (noncitrate containing) solutions. Perforated patch-clamp recordings of isolated rat fungiform taste cells in current-clamp mode indicated that citrate (5 × 10-3 M) enhanced the frequency of action potentials in response to glycine (5 × 10-2 M) [and saccharin (2 × 10-2 M)], which confirmed that citrate has a direct effect on taste cells (Gilbertson et al. 1997). Because citrate alone produced small depolarizations of rat taste receptor cells that were insufficient to generate taste cell action potentials, it was proposed that the additive depolarizing effects of the citrate and a depolarizing taste stimulus summed to result in the enhanced action potential output.

The report of citrate effects in the rat taste system (Gilbertson et al. 1997) is consistent with the current results in the largemouth bass. These data collectively suggest that citrate might act as a calcium chelator at the surface of the taste cell microvilli and not as an effector at specific gustatory amino acid receptor sites. The citrate ion is a tricarboxylic anion that can bind calcium ions at the surface of taste cell microvilli reducing the surface potential and shifting sodium channel activation to more negative potential (Hille 1992). This chelating effect of the citrate ion on extracellular calcium ions is likely to cause taste cells of largemouth bass to be depolarized slightly as it did for rat taste cells (Gilbertson et al. 1997) and more ready to activate voltage-gated ion channels (Roper 1983) on effective taste cell stimulation. This hypothesis for the mechanism of action of citrate is consistent with the experimental observations in this report of pH dependence (Fig. 3) and the effects of citrate at both high arginine (Figs. 4, 5, and 7) and citrate (Fig. 1) concentrations. The lack of a taste-enhancing effect of citrate at pH values of <= 6.0 (Fig. 3) is consistent with the hydration of one of the carboxyl groups of the citrate ion whose dissociation constant at 25°C is 4.0 × 10-7 M. Also, the lack of a taste-enhancing effect at 10-2 M and 10-1M L- and D-arginine concentrations (Figs. 4 and 7) is consistent with the large number of positively charged arginine molecules neutralizing the negative charge of the carboxyl groups of the citrate ions. It is currently unknown why a suppression of taste responses to 10-2 M and 10-1 M L-arginine occurred during adaptation to 10-3 M citrate (Fig. 5) but did not occur during their testing in an unadapted preparation as a binary mixture (Fig. 4). It is possible, however, that the continuous presence of citrate as an adapting stimulus lowered the extracellular calcium sufficiently to have a modulating effect on the gustatory transduction mechanism for arginine.


    ACKNOWLEDGMENTS

We thank Dr. T. Gilbertson and two anonymous reviewers for critically reviewing this manuscript. We also thank R. Bouchard for assistance with the figures and Dr. E. Obata for partial financial support for K. Ogawa.

This work was supported by National Science Foundation Grant IBN-9221891.

Present address of K. Ogawa: Dept. of Otolaryngology, Kagoshima University Medical School, 8-35-1 Sakuragaoka, Kagoshima 890, Japan.


    FOOTNOTES

Address for reprint requests: J. Caprio, Dept. of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 26 January 1998; accepted in final form 15 December 1998.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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