EDITORIAL FOCUS
Focus on "Rapid entry of bitter and sweet tastants into liposomes and taste cells: implications for signal transduction"

John A. DeSimone

Department of Physiology, Virginia Commonwealth University, Richmond, Virginia 23298-0551


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THE TASTE QUALITIES of sweet and bitter have powerful influences on food selection for humans and other species. Generally speaking, sweet-tasting substances are innately appetitive, whereas those that are bitter tasting are aversive (2, 10). It is, therefore, not surprising that sweet-tasting (and calorie-rich) carbohydrates, in their various forms, are highly preferred meals. The association of the appetitive sweet with high-caloric intake has obvious survival value. However, modern society affords us an abundance (perhaps an overabundance) of sweet-tasting foods, and, as every dieter can attest, the persistent present-day challenge is limiting their ingestion to avoid excessive caloric intake. The chance discovery that some compounds (with negligible caloric value) can stimulate a sweet taste response at concentrations far lower than sugars, has, not surprisingly, spawned an entire industry. Similarly, the survival value in an aversive reaction to most naturally occurring bitter-tasting compounds is clear, considering that among these are many potentially toxic substances (e.g., plant alkaloids). Interestingly, the pleasant systemic effects that humans have discovered in less than acutely lethal encounters with some alkaloids (e.g., nicotine, caffeine, and others) results in their acceptance despite the bitterness. The structural diversity in bitter-tasting compounds has long been noted and includes mineral salts, amines, and ammonium salts (including those used pharmacologically as ion channel blockers), some L-amino acids and peptides (including the peptide toxin mastoparan), saponins, local anesthetics, and others (13, 20, 32, 35). The same sort of diversity is observed among sweet-tasting compounds. Sucrose and other naturally occurring saccharides form a unique class of compounds that are intensely sweet only at concentrations high enough to be nutritionally beneficial (20, 35). Nonsaccharide sweet-tasting substances include inorganic salts (including some lead salts, in this case an undesirable property of a potentially harmful substance), sulfimides (e.g., saccharin), amino acids (e.g., glycine, L-alanine, and some D-amino acids), peptides (e.g., aspartame), proteins (e.g., thaumatin and monellin), and other synthetic compounds (20, 35). In short, a large variety of chemical structures end up producing mainly one of only two chemical sensations. From a physiological standpoint this suggests multiple input routes to a common sensory transduction process within a submodality, or multiple transduction cascades within a submodality, or both multiple inputs and multiple transduction cascades.

Cross adaptation psychophysical studies on humans (30) and cross adaptation electrophysiological studies in other species (15) within either the sweet- or bitter-tasting stimulus group have produced evidence generally favoring the existence of multiple parallel transduction pathways within a submodality. Interestingly, data indicating the existence of more than one peripheral sensory pathway were often interpreted as evidence in favor of multiple taste cell apical membrane receptor sites, although the existence of the latter had not been unequivocally demonstrated. However, the concept of taste receptor site took a quantum leap toward demonstrated fact with the discovery of a taste receptor cell-specific G protein alpha -subunit (alpha -gustducin) by McLaughlin et al. (23). This G protein alpha -subunit is >80% homologous to alpha -transducin, and, like the latter, activates a phosphodiesterase. Interestingly, rod transducin is also present in taste receptor cells, where it has been shown to be capable of activating taste-cell-specific phosphodiesterases when stimulated with the bitter-tasting compound denatonium (29). That bitter taste transduction might depend on phosphodiesterase activation was itself confirmation of Price's earlier observations (27). Knockout mice lacking the alpha -gustducin gene showed significantly reduced behavioral and neural responses to denatonium, reinforcing the idea that alpha -gustducin mediates a transduction cascade associated with the bitter submodality and that a reduction in cell cAMP levels is part of the process (18, 40). This transduction pathway, however, does not appear to be unique for this bitter-tasting stimulus. Earlier work had already shown that denatonium activates taste cell inositol trisphosphate (IP3) through a G protein-dependent mechanism sensitive to pertussis toxin (31, 34), resulting in the mobilization of intracellular Ca2+ (1). It appears, therefore, virtually certain that taste cell membrane receptor molecules exist and that they can initiate multiple G protein-mediated transduction cascades. A similar conclusion holds for the sweet submodality. Striem et al. (37, 39) showed that saccharides stimulate a G protein-dependent increase in adenylyl cyclase activity and cAMP production. Moreover, artificial sweeteners did not increase cAMP levels in isolated rat taste receptor cells but caused IP3 levels to increase and Ca2+ to be released from intracellular stores (4).

The recent cloning of two seven-transmembrane domain proteins expressed in the apical poles of taste receptor cells by Hoon et al. (14) and a probable glutamate taste receptor protein by Chaudhari et al. (7) have provided the first candidate taste receptor molecules. Although functionality remains to be determined, they would appear to fulfill many of the expected properties of membrane taste receptors, and it seems only a matter of time before functionality is demonstrated. Although it is tempting to say at this point "end of story," there remain a number of outstanding observations that strongly suggest that many sweet and bitter tastants can initiate the sensory transduction chain at various points, i.e., transduction may not always require the binding of these tastants to a specific membrane receptor protein. For bitter tastants the evidence for this was extensively reviewed and discussed by Spielman et al. (32). Many intensely bitter-tasting stimuli, e.g., quinine, tetraethylammonium, BaCl2, CsCl, and 4-aminopyridine are part of the cell physiologist's arsenal of K+ channel blockers. Each has been shown to depolarize taste receptor cells by blocking one or more K+ channel conductance (3, 17, 33), a mode of action that might function in situ. However, most of the K+ conductances in mammalian taste receptor cells are located on the basolateral side of the cells and, therefore, are not immediately in contact with taste stimuli (11, 41).

For receptor cell depolarization through K+ conductance blockage to be a factor in taste transduction, the conductances would have to be blocked from the cell cytoplasmic side or from the cell interstitial fluid compartment below the tight junctions. They are blocked from the cytoplasmic side when, for example, saccharides effect a G protein-mediated increase in cell cAMP, ultimately resulting in protein kinase A-mediated closure of K+ channels (8). Isolated taste receptor cells undergo sustained volume decrease when subjected to hyperosmotic concentrations of mannitol, suggesting relatively low membrane permeability for this and perhaps other saccharides (22). Sweet taste transduction involving saccharides, therefore, probably begins with specific cell surface receptors that are selective among saccharides, but specific membrane saccharide transporters, which could perform the same selectivity function, should not be completely ruled out. On the other hand, in taste receptor cells some hydrophilic tastants, such as urea, have the osmotic characteristics of solutes that are very membrane permeable (22). Moreover, many artificial sweeteners and bitter tastants are either amphipathic or can exist in both hydrophilic and lipophilic forms (usually depending on pH), so the possibility that they too enter taste receptor cells must be considered. Indeed, taste receptor cells readily absorb the nonionic forms of various fluorescent dyes (4, 21). Moreover, taste cells contain the multidrug resistance P-glycoprotein (MDR1) membrane protein, suggesting that some substances regularly enter taste cells in amounts that warrant a robust mechanism for their active removal (16).

Naim et al. (25) have already shown that saccharin, quinine, and other amphipathic tastants can directly activate transducin and a mixture of Gi/Go proteins in vitro. On this basis they proposed that direct activation of taste transduction could occur in some cases at the G protein level. This conclusion was supported by the fact that some amphipathic peptides do activate G proteins directly in other cell types. Perhaps the best-studied example is mastoparan, an amphipathic peptide in wasp venom, which activates G proteins directly in mast cells and must cross the cell membrane to reach its intracellular target (6, 24). Other agonists that can stimulate histamine secretion from mast cells by direct G protein activation are the neuropeptide substance P, bradykinin, and compound 48/80, a synthetic polyamide. It is interesting that bradykinin, substance P, and mastoparan all contain basic amino acids flanked by hydrophobic amino acids. Bradykinin is highly bitter tasting, as are synthetic short peptides containing similar arrangements of amino acids (32). The bitter-tasting local anesthetics procaine and tetracaine are also direct activators of G proteins (12). Further evidence supporting direct activation, from Naim and colleagues and others, is that artificial sweeteners stimulate G protein-dependent adenylyl cyclase activity in non-taste tissue (9, 38) and insulin secretion from isolated pancreatic islets (19). It is unlikely that muscle, liver, and pancreas contain taste receptor molecules for saccharin and other artificial sweeteners.

Key unanswered questions that remained are: Do tastants such as saccharin and quinine enter taste receptor cells and, if so, are the amount and rate of uptake in the time frame of taste responses? In the current article in focus by Peri et al. (Ref. 26, see page C17 in this issue), Naim and colleagues investigate the time course of uptake of saccharine, quinine, and the cyclic dipeptide (cyclo)Leu-Trp into rat circumvallate taste bud cells. The authors used the confocal microscope to monitor increasing intracellular concentrations of the tastants using their autofluorescence properties. The uptake was rapid and confined to the cytosol. The kinetics of uptake were determined by permeabilizing the cells following exposure to a tastant and then analyzing the cytosol using HPLC methods. The initial rates of uptake are clearly sufficiently fast to support the possibility of direct intracellular activation of transduction. In a parallel set of experiments Naim and colleagues studied the uptake of the tastants by liposomes. In each case the uptake was rapid and mechanistically consistent with a partitioning of the tastant into the lipid followed by diffusion into the vesicle interior. Cyclo(Leu-Trp) is uncharged at the pH values of the experiments and quinine is partially so, suggesting that these lipid-permeable forms are responsible for the membrane transit into taste receptor cells. However, saccharin is strongly anionic yet still transported, an observation meriting further investigation, especially in view of its very different taste quality. These studies, in so far as they suggest that the causal chemosensory stimulus may be intracellular concentration, complement recent work on cellular pH sensors, where it appears that intracellular receptor pH is the excitatory stimulus for acid taste receptors (21, 36), carotid body pH-sensing cells (5), and brain stem chemoreceptors (28). This study by Peri et al. (26) serves as a timely reminder that the multiple transduction pathways mediating taste sensation may themselves contain an additional level of diversity, i.e., different points of initiation. These may be 1) at a G protein-coupled receptor molecule, 2) at any of several intracellular loci, and 3) in some cases, no doubt, both of the above.


    FOOTNOTES

Address for reprint requests and other correspondence: J. A. DeSimone, Dept. of Physiology, Virginia Commonwealth Univ., Sanger Hall, Rm 3-002, 1101 E. Marshall St., Richmond, VA 23298-0551 (E-mail: jdesimon{at}hsc.vcu.edu).


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