What Can Psychophysical Studies with Sweetness Inhibitors Teach Us

Chapter 12

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What Can Psychophysical Studies with Sweetness Inhibitors Teach Us about Taste? Veronica Galindo-Cuspinera and Paul A. S. Breslin Monell Chemical Senses Center, 3500 Market Street, Philadelphia, PA 19104

In this chapter we demonstrate how perceptual studies with inhibitors help guide molecular studies toward an understanding o f taste mechanisms. We illustrate this point through the use of sweet taste inhibitors in three different perceptual paradigms: water-taste induction, enhancement o f differential sensitivity (decreasing Weber's fraction), and cross-modal inhibition. We show how perceptual studies might complement molecular/functional studies to develop more complete understandings of taste physiology.

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The phenomenon of'Water-Taste : gustatory after-images The perception of a sensory quality, such as sweetness, is based upon specific patterns of activity in modality-specific sensory cortex, usually initiated by the activation o f receptors in the periphery. After-images can be useful for revealing these underlying qualitative codes, such as the red-green/blue-yellow opponency mechanisms unveiled by color after-images. In the gustatory system after-images are labeled 'water-tastes', as water becomes the neutral substrate for the 'after-image', and can appear sweet, sour, bitter or salty depending on the perceived quality of the previously tasted chemical and its chemical structure [1,2]. In general, the phenomenon of 'water-taste' has received little attention, and there have been no clear explanations o f this phenomenon. The term 'Water-taste' refers to the taste elicited by water after a chemical solution is rinsed from the mouth. One theory posits that water-tastes are adaptation phenomena, wherein adaptation to one taste solution causes the water presented

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© 2008 American Chemical Society In Sweetness and Sweeteners; Weerasinghe, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.


171 subsequently to act as a taste stimulus [3, 4]. According to this hypothesis, pure water will stimulate a taste when the normal pattern of neural activity is altered in just the subset of neurons utilized in common with the adapting stimulus; hence, these water rinses produce an atypical pattern of activity which may be perceived as taste. However, there is little physiological or molecular data at present that accounts for this theory. A n alternative hypothesis is that the removal of the stimulus when rinsing generates a receptor-based, positive, offresponse in taste receptor cells, ultimately inducing a gustatory perception [5, 6]. By studying the interaction that compounds, which elicit sweet water-taste, have with the T1R2-T1R3 sweet receptor, we have shown that the phenomenon of sweet 'water-taste' is directly related to sweet taste inhibition and that the perceived sweetness from water is the result of releasing the receptor from an inhibitory state [7].

Na-Saccharin: sweetener or sweetness inhibitor? Saccharin was discovered in 1879 at Johns Hopkins University by Remsen and Fahlberg and is the first artificial sweetener (figure 1). Ο

Figure L Sodium Saccharin

Throughout the 1970s, saccharin was used as a low-calorie sweetener in the United States, and today it continues to be important for a wide range of lowcalorie and sugar-free foods and beverages. Na-saccharin is a peculiar compound because when it is tasted at low concentrations a characteristic sweet taste is perceived, accompanied by a low level of bitterness; however, when the concentration of saccharin increases the sweetness perception diminishes. A t high concentrations, Na-Saccharin curiously elicits little sweetness and tastes mostly bitter to many observers (Figure 2a). A potential explanation for the low sweetness perceived at high concentrations is a mixture-suppression effect caused by the high bitterness elicited by strong Na-saccharin. This explanation is unlikely, as there is no correlation between perceived bitterness and sweetness at any concentration (figure 2a - inset). In addition, high concentrations of Nasaccharin elicit a strong sweet water-taste when they are rinsed from the mouth (Figure 2b). The apparent ability of Na-saccharin to inhibit its own sweet taste at

In Sweetness and Sweeteners; Weerasinghe, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.


Figure 2. .a) Concentration- intensity curve for Na-Saccharin (second tasting), black line (a) indicates bitterness, dark gray line(+) sweeteness. n=14subjects. Inset shows the correlation of bitterness with sweetness for Na-saccharin. b) Water-taste intensity measured after exposure to different concentrations of Na-Saccharin. Adapted by permission from Macmillan Publishers Ltd: Nature [7], copyright 2006. (See color insert in this chapter.)



173 high concentrations is very revealing, as it requires the sweetener to act as both an agonist as well as an antagonist on the same receptor, albeit at different conentration ranges. This surprising observation led to an investigation of the sweet water-taste phenomenon [7]. By screening compounds for sweet water-taste, we found that the wellknown sweet taste blocker "lactisole", despite not eliciting sweet taste on its own, produces a pronounced sweet 'water-taste' when it is rinsed from the mouth. This raised the question of whether there is a causal relationship between sweetness inhibition and sweet water-taste. Given the parallel effects elicited by both strong Na-saccharin and lactisole, we determined i f Na-saccharin might also be a general sweetener inhibitor. B y mixing high concentrations of N a saccharin with several chemically-diverse, intensity-matched sweeteners, we demonstrated the general inhibitory effect of Na-saccharin on these sweeteners (Figure 3a and b). We further demonstated that other compounds which demonstrate a sweet 'water-taste' (Acesulfame-K and M g S 0 ) are also sweetener inhibitors. Furthermore, both the inhibition effect of concentrated saccharin and the sweet water-taste phenomenon were demonstrated in vitro with the sweetener receptor h T A S l R 2 - h T A S l R 3 , which was heterologously expressed in immortalized human kidney cells (HEK293/ Gal6gust44). Thus, these perceptual phenomena are explained at the receptor level. 4

But how do we understand that Na-saccharin is both a sweetener and a sweetness inhibitor? This is possible i f the sweetener receptor has more than one binding site for Na-saccharin. The first is an orthostheric site with high affinity for Na-saccharin, and the second is an allosteric site with lower affinity. When the receptor is exposed to low concentrations of Na-saccharin, the molecules bind to the orthostheric site activating the receptor and consequently eliciting sweet taste perception. But at higher concentrations, Na-saccharin will additionly bind to the low affinity allosteric site, which happens to inhibit the receptor and block sweetness. It is the release of the molecules from the inhibitory allosteric site by water rinses that triggers the sweet 'water-taste' perception. In general, we propose that certain stimuli elicit sweet water-tastes because they are h T A S l R 2 - h T A S l R 3 allosteric inhibitors and their removal activates the receptor. This would occur due to the equilibrium forces of a twostate allosteric receptor resulting in a coordinated rebound to the activated state after most receptors had been locked in the inactive state by the inhibitor. Perceptual studies of water-tastes have, therefore, indicated that taste receptors have multiple states and can be activated and inhibited by a single molecule. Alternatively, adaptation effects might also explain the low level of sweeteness perceived with high Na-Saccharin concentrations. Taste adaptation is caused by exposure to a taste stimulus over some interval(s) (either short or long periods) and is manifested as the subsequent decrease in the perceived intensity of the stimulus or decresed activation by the stimulus. In comparison, inhibition also causes a decrease in the perceived intensity of the stimulus, but



Figure 3. Sweetness blocking effects of a)200 mM Na-Saccharin and b) 1 mM lactisole. Gray bars refer to the mean intensity of the pure sweeteners listed on the X axis (n = 14); black bars reflect the mean sweetness for mixtures of sweetener and blocker. The agonists tested were 300mM sucrose (Sue), 4mMNa-saccharin (Sacch-4), 17.5mMNa-cyclamate (Cyc.) and 3mM aspartame (Asp.). Error bars indicate s.e.m. Asterisks indicate significance at a=0.05 using repeated measures ANOVA. Adapted by permission from Macmillan Publishers Ltd: Nature [7], copyright 2006.



175 does so via a different molecular interaction with the taste receptor. To test this hypothesis directly, we designed an experiment where 14 subjects were repeatedly exposed to a mixture of sucrose and high Na-saccharin, after which the saccharin was removed from the solution to assess the level o f adaptation to sucrose. Interestingly, after the removal o f Na-saccharin from the mixture, sucrose was perceived quite strongly which indicates the lack o f adaptation despite multiple sucrose presentations immediately prior (Figure 4). The failure of sucrose to adapt after 10 consecutive exposures indicates that the sucrose response was likely inhibited by the Na-saccharin; but when Na-saccharin was removed, the receptor returned to the active state and was normally activated by sucrose. It should be noted that sucrose presented alone ten times in a similar way without Na-saccharin adapts strongly.

Figure 4. A test of sweetness adaptation versus sweetness inhibition as an explanationsfor NaSacharin's sweetness suppressing effects. Mean sweetness and bitterness of580 mM sucrose and a mixture of this sucrose with 200 mM NaSaccharin. Fourteen subjects tasted the solutions shown at 10 sec intervals (90 sec total) and rated sweetness on a general labeled magnitude scale (gLMS) without rinsing in between tastings. Gray bars indicate sucrose sweetness and black bars its bitterness. Triangles indicate the sweetness of the mixture and circles its bitterness. Error bas indicate standard eror of the mean (s.e.m.). Reprinted by permission from Macmillan Publishers Ltd: Nature [7], copyright 2006.


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Sensitivity increases with inhibition Following the logic that there are similarities between inhibition and adaptation, we next wished to determine whether inhibition affected the slope of the concentration-intensity curve of sucrose, hence the sensitivity to sucrose. This comparison is based on the common observation that adaptation to stimuli increases observers' sensitivity to changes in stimulus strength. We measured the psychophysical curve for sucrose alone and in mixture with 50 m M Nasaccharin, as an inhibitor. Fifteen people rated all stimuli on a g L M S scale in triplicate and showed that the presence of the sweetener inhibitor Na-saccharin causes a rightward shift of the psychophysical function of sucrose, demonstrating saccharin's inhibition of sweetness over the whole concentration range (figure 5a). We examined the changes in slope by plotting the data in loglog coordinates (figure 5b). The addition of saccharin, as expected, lowers the perceived sweetness intensity but at the same time increases the slope of the curve from 0.48 to approximately 0.85, which means sensitivity increased. To confirm this sensitivity shift independently* Weber fractions were obtained for 400 m M sucrose alone and in the presence of 50 m M Na-saccharin. We show that standard Weber fractions for sucrose differential thresholds are between 12 to 14% (figure 5c). When testing sucrose mixed with inhibitory consentrations of Na-saccharin the average weber fraction appears lower than for sucrose alone, although the mean difference between conditions was not significant. When analyzing individual responses, we find significant decreases in weber fractions for several people, indicating an increase in differential sensitivity. However, this trend was not consistent in other subjects. We believe that when Nasacharin is used as an inhibitor the strong bitterness stimulated at high concentrations, interferes with some subject's ability to differentiate solutions based solely on sweetness. These subjects rated the bitterness close to 'very strong' on the g L M S , which could explain their low differential sensitivity, while others rated the bitterness between moderate and strong. Sweet receptor inhibition might make sweet receptor cells more sensitive to changes in concentration because small increases in concentration can stimulate against a low level of background activity. More studies are needed to assess the impact of inhibition on differential sensitivity.

Intramodal interactions of sweet taste inhibitors. Lactisole. A broad spectrum sweetener inhibitor Lactisole (Na, p-methoxy-phenoxy-propionate, figure 7A), is a potent broad-acting sweetener inhibitor specific to humans and other primates and has no effect on rodents response to sweet taste. Lactisole, despite not eliciting



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Log [sucrose M] Figure 5. Concentration intensity curves of sucrose (control-top line) and mixtures of sucrose with 50mMNa-Saccharin (bottom line), a) normal space, intensity measured on a gLMS, b) log-log space, linear regression analysis. Error bars indicate SEM, « = 7 5 . (See color insert in this chapter.)



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Figure 6. a) Weber'sfractionsfor sucrose (black) and sucrose + Na-saccharin (gray). Standard concentration =400 mMsucrose, b) individual Weber's fractions for sucrose and sucrose (black) + Na-saccharin (gray). Error bars indicate SEMfor 3 replicates. (See color insert in this chapter.)



179 sweet taste per se, suppresses the sweet taste of most sugars, protein sweeteners, and other high potency sweeteners [8]. A n early study suggested that lactisole does not inhibit all sweeteners to the same degree [9]. However it is not clear i f the lack of inhibition observed in that study is due to the temporal properties of the sweeteners or the temporal dynamics of the inhibition. Recent in vitro studies have shown that lactisole binds specifically to the human T1R3 transmembrane domains causing inhibition of the T1R2-T1R3 receptor's response to sweeteners [10-12]. Jiang et al showed that the T M helices 3, 5, and 6 of h T l R 3 are involved in lactisole binding; by using chimeric and mutational studies they also identified Leu-7987.36 in T M helix 7 and Arg-790ex3 in extracellular loop 3 (which connects T M helices 6 and 7) as human-specific residues that affect responsiveness to lactisole [10]. Further research revealed that the exchange of valine 738 in the fifth transmembrane domain of rTaslr3 with an alanine is sufficient to confer lactisole sensitivity to the rat sweet taste receptor [12]. Given the broad effect that lactisole has on sweet taste and the specificity of its binding site, lactisole has become a useful tool to study taste modulation.

Figure 7. Chemical structures of a) lactisole and b) Monosodium glutamate MSG.

A molecular link between sweet and umami tastes. The T1R receptors comprise a family o f taste-specific class C G-protein coupled receptors, which mediate mammalian sweet and umami tastes. A glutamate or umami receptor (T1R1-T1R3) and a sweetener receptor (T1R2T1R3) share a common entity, the T1R3 G P C R [13-16]. Given that perception of both taste qualities likely involves the presence of the T1R3 receptor [5, 17], it is logical to think that compounds that bind to this monomer will have an effect in perception of both taste qualities. Using a psychophysical approach we tested this hypothesis. In this experiment we turned again to lactisole, which interacts specifically with the transmembrane domain of T1R3. Multiple concentrations of lactisole were tasted in mixtures with 20 and 100 m M monosodium glutamate ( M S G , figure 7) and the umami intensity ratings recorded. Our results showed a concentration dependent decrease in umami taste [18]. Lactisole also inhibited the perception of umami taste from M S G , albeit with less efficacy than it inhibits sweeteners, demonstrating that the shared



180 monomer (T1R3) allosterically moderates activation of T1R1 and T1R2 in humans. Given that the concentrations needed to inhibit umaminess were aprox. 16 times higher than those needed to inhibit sweet taste, we assessed the effect of lactisole on other taste modalities using the same concentrations. Exemplars of each taste quality were mixed with 16 m M lactisole. Subjects tasted these mixtures but found that lactisole inhibits only sweet and umami tastes (figure 8). The differences in the level of inhibition between the umami and sweet modalities suggest either potential changes in conformation due to interactions with the other receptor protein (figure 9), or different mechanism of modulation given that other compounds such as cyclamate which also bind to T1R3 does not have a significant effect on umami taste (unpublished observation). A distinctive characteristic of human umami taste is its powerful synergism derived from mixing 5'ribonucleotides with glutamate (figure 9 C, Ε & F) [19]. To test the effect of lactisole on the umami synergy of M S G with 5'ribonucleotides, increasing amounts o f lactisole were added to constant mixtures of 20 m M M S G plus 3mM of either IMP or G M P or to ribonucleotides alone. We found no significant effect of lactisole on the umami taste o f the synergized mixture with lactisole concentrations as high as 32 m M in human perceptual studies [18]. Based on the in vitro observation of M S G and 5' ribonucleotide synergy with h T l R l - h T l R 3 , i f we assume synergy occurs, at least in part, within the h T l R l - T l R 3 receptor, then our data suggest that 5'ribonucleotides bind to the T1R1 subunit but not the T1R2 subunit and alter the T1R1-T1R3 heterodimer, preventing lactisole from inhibiting umami taste [20]. Thus, we infer from lactisole's differential ability to inhibit both sweet

Figure 8. Effect of 16 mM lactisole (clear bars) on standard quality solutions (gray bars); 200 mM sucrose, 2.5e-2 mM quinine-HCl, 2 mM citric Acid, 100 mMNaCl and 100 mM monosodium glutamate. Data analysis: Repeated Measures ANOVA and Tukeypairwisepost-hoc comparisons (n=12). * Significant at a=0.05. W=weak intensity, BD=barely detectable intensity. Reprinted by permission from Oxford University Press: Chemical Senses, [18] copyright 2006.



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Figure 9. Human sweet (TlR2(blue)-TlR3 (purple)) and umami (TlRJ(dark grqy)-TlR3(medium gray)) taste heteromer receptor schematics: Inhibition of sucrose's (gray hexagon) sweet taste by the compound lactisole (small gray oval) (A & B); inhibition of monosodium glutamate 's (MSG) (gray pentagon) umami taste by lactisole (C & D); and modulatory effects of 5 'ribonucleotides, such as inosine monophosphate (IMP) (light gray star), on MSG binding and IMP's blockade of lactisole's inhibition (E & F). Reprinted by permission from Oxford University Press: Chemical Senses, [18] copyright 2006. (See color insert in this chapter.)


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(figure 9-A&B) and umami taste (figure 9-C&D) and from 5'ribonucleotide^ ability to block lactisole's inhibition of umami but not sweet taste that the identity of a receptor subunit and/or its activation by ligands can alter the conformation of the partner subunit and hence its ability to be activated or inhibited. The following schematic model is based on previous in vitro models and has been modified to accommodate our psychophysical data.

Conclusions We have used taste inhibition as a tool to demonstrate the utlity of combining perceptual & psychophysical studies for inferring the molecular mechanisms of several perception phenomena. First, we show that a sweet receptor agonist may simultaneously be an antagonist of sweet taste. This is illustrated with Na-saccharin, which both stimulates sweet taste at low concentrations and inhibits sweet taste at high concentrations. A parallel effect was also demonstrated with saccharin in vitro on heterologously expressed sweet taste receptors. We argue that the sweet receptor is a multi-state receptor that when released from its inhibited state will rebound to an activated state, acounting for the sweet water-taste that follows rinses. Similarly, in vitro responses when rinsing Na-saccharin away with water closely parallels the perceptual phenomenon. Second, we demonstrate that the inhibition of sucrose can increase the sensitivity to changes in concentration, much as adaptation is known to do. These data suggest that suprathreshold sensitivity to stimuli like sucrose is determined by the ability of the peripheral system to adjust responsiveness to ambient or background levels, so that changes in stimulus levels are perceived as large percent changes perceptually. This will result in the ability to detect physical intensity changes, even i f these changes are very small relative to background concentration levels. The observation of heightened sensitivity when an inhibitor is mixed with the agonist, indicates that this phenomenon may be explained at the receptor or cellular level, as opposed to higher in the signal processing pathway. Third, we demonstrate that the sweetener inhibitor lactisole, which binds to the T1R3 subunit and inhibits the T1R2-T1R3 sweetener receptor, also inhibits the savory taste of glutamate, albeit with low efficacy. In humans, this taste is believed to be transduced, in part, by the T1R1-T1R3 receptor, which shares the T1R3 subunit with the sweetener receptor. These data support the hypothesis that T1R1-T1R3 receptor plays a role in human savory taste, but appears only to convey part of the glutamate signal. Since the addition of 5' ribonucleotides to agonists does not affect lactisole's ability to inhibit the sweet taste of sugars but does prevent lactisole from inhibiting the savory taste of glutamate, we infer that the binding and activation of each T I R subunit by compounds may be allosterically modulated by the identity and state of its partner subunit. These three examples


183 of perceptual research with taste inhibitors, therefore, illustrate the utility of this approach for highlighting the underlying molecular mechanisms of chemosensory perception.


This research was funded in part by grant DC02995 from the N I H to P A S B .

References 1.

Bartoshuk, L.M. (1968). Water taste in man. Perception and Psychophysics 3, 69-72. 2. Bartoshuk, L.M. (1977). Water taste in Mammals. In Drinking Behavior, J.A.W.W. Weijnen and J. Mendelson, eds. (New Haven, C T : Plenum Publishing Co.), pp. 317-339. 3. McBurney, D.H., and Bartoshuk, L.M. (1971). Water taste in mammals. Paper presented at the fourth international symposium on olfaction and taste. 4. McBurney, D.H., and Bartoshuk, L.M. (1973). Interactions between stimuli with different taste qualities. Physiology and Behaviour 10, 1101-1106. 5. DuBois, G.E. (2004). Unraveling the biochemistry of sweet and umami tastes. Proc Natl Acad Sci 101, 13972-13973. 6. Dubois, G . (1999). Selective sweetness inhibitors and a biological mechanism for sweet water aftertaste. In Annual Meeting of the Association of Chemoreception Scientists vol. 21. pp. Abstract: Sarasota. 7. Galindo-Cuspinera, V . , Winnig, M . , Bufe, B . , Meyerhof, W., and Breslin, P.A.S. (2006). A TAS1R receptor-based explanation of sweet water-taste. Nature 441, 354-357. 8. Dubois, G.E. (1997). New insights on the coding of the sweet taste message in chemical structure. In Firmenich Jubilee Symposium 1895-1995, G . Salvadori, ed. (Carol Stream, IL: Allured Publishing Corp), pp. 32-95. 9. Schiffman, S.S., Booth, B.J., Sattely-Miller, E . A . , Graham, B . G . , and Gibes, K.M. (1999). Selective inhibition of sweetness by the sodium salt of (+/-)2-(4-methosyphenoxy)propanoic acid. Chemical Senses 24, 439-447. 10. Jiang, P., Cui, M., Zhao, B., Liu, Z., Snyder, L . A . , Benard, L.M.J., Osman, R., Margolskee, R.F., and Max, M. (2005). Lactisole Interacts with the Transmembrane Domains of Human T1R3 to Inhibit Sweet Taste. Journal of Biolgical Chemistry 280, 15238-15246. 11. X u , H., Staszewski, L., Tang, H., Adler, E., Zoller, M., and Li, X. (2004). Different functional roles of T1R subunits in the heteromeric taste receptors. Proc. Natl. Acad. Sci. 101, 14258-14263. 12. Winnig, M., Bufe, B., and Meyerhof, W. (2005). Valine 738 and lysine 735 in the fifth transmembrane domain of rTas1r3 mediate insensitivity towards lactisole of the rat sweet taste receptor. B M C Neuroscience 6, 1-7.



184 13. Damak, S., Rong, M., Yasumatsu, K . , Kokrashvili, Α., Varadarajan, V., Zou, S., Jiang, P., Ninomiya, Y . , and Margolskee, R.F. (2003). Detection of sweet and umami taste in the absence of taste receptor T1R3. Science 301, 850-853. 14. Zhao, G.Q., Zhang, Y., Hoon, M . A . , Chandrashekar, J., Erlenback, I., Ryba, N.J.P., and Zuker, C.S. (2003). The receptors for mammalian sweet and umami taste. Cell 115, 255-266. 15. Jiang, P., Ji, Q., Liu, Z., Snyder, L . A . , Benard, L.M.J., Margolskee, R.F., and Max, M . (2004). The Cysteine-rich Region of T1R3 Determines Responses to Intensely Sweet Proteins. Journal of Biological Chemistry 279, 45068-45075. 16. Nelson, G., Chandrashekar, J., Hoon, M . A . , Feng, L . , Zhao, G., Ryba, N.J.P., and Zucker, C.S. (2002). A n amino-acid taste receptor. Nature 416, 199-202. 17. L i X., Staszewski L . , X u H . , Durick K . , Zoller M., and E., A . (2002). Human receptors for sweet and umami taste. Proc Natl Acad Sci 99, 46924696. 18. Galindo Cuspinera, V . , and Breslin, P.A. (2006). The liaison of sweet and savory. Chemical Senses 31, 221-225. 19. Yamaguchi, S. (1967). The synergistic taste effect of M S G and disodium 5'inosinate. Journal of Food Science 32, 473-478. 20. Jensen, A . A . , and Spalding, T.A. (2004). Allosteric modulation of G-protein coupled receptors. European Journal of Pharmaceutical Sciences 21, 407420.


What Can Psychophysical Studies with Sweetness Inhibitors Teach Us

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