John A. DeSimone, Ph.D. - US grants

The major purpose of this project is to characterize in greater detail the active transepithelial ion currents which we have discovered in the dorsal lingual epithelium of several mammalian species. These ion fluxes arise in response to a variety of taste stimuli. The study extensively employs two in vitro electrophysiological methods and in vivo neurophysiological recording of gustatory responses. The first in vitro method is a novel adaptation of the Ussing technique which we have used to prove that the dorsal surface of the canine tongue actively transports ions. The active transport system is stimulated by hyperosmotic NaC1, a property not found in any other known transporting epithelium. It also responds to sugars (including sucrose) with increased inward transepithelial current. The response to sugars can be eliminated by the Na-channel blocker amiloride as can most of the hyperosmotic NaC1 response. The change in transepithelial current occurs over the same concentration range as the canine response to various tastants, including HC1. These transport properties are sufficiently unique so as to suggest a direct or indirect role for the transepithelial current in gustatory transduction. In order to gain a better understanding of these stimulus induced currents and their relationship to lingual epithelial morphology, we have begun to map the shape of the electric field outside the contours of individual papilla, while the tissue is open or short-circuited using a vibrating probe electrode. These studies should allow us to differentiate the role of taste buds in determining field magnitude and direction in the face of various taste stimuli. The neurophysiological studies on the rat are designed to allow us to correlate transepithelial events with gustatory neural events. This is done in two ways: by doing parallel in vivo and in vitro experiments in the same animal, and by making simultaneous transepithelial and neural recordings, in situ. These studies should provide a far clearer picture of peripheral events in gustatory transduction than is currently available. They suggest for the first time an ionic basis for the gustatory transduction of nonelectrolyte sugars. These studies should also lead to a clearer understanding of the role of active and passive ion transport in taste, the effects of hormones, and various metabolic factors which may govern food preferences and intake.

DESCRIPTION (Adapted from the Investigator's Abstract): Regulation of the acidity (pH) of internal fluids within a narrow range is essential for all animals. The taste system plays a role in guarding against excessive intake of acid in foods and beverages with hyperacidity evoking an intense sour taste than is innately aversive. The mechanisms by which taste receptor cells respond to acid (sour taste transduction) will be studied using taste buds and taste cells isolated from rodents. Preliminary experiments indicate that changes in extracellular pH (acid stimulation) are mirrored by proportionate changes in intracellular pH, suggesting that pH-sensing is an intracellular process. In addition, changes in intracellular calcium levels alter internal pH, indicating that calcium may be involved in the acid-sensing transduction pathway. This hypothesis will be tested using pH- and calcium-sensitive fluorescent dyes to follow acid induced changes in intracellular pH and possible subsequent changes in intracellular calcium. Changes in membrane conductance during acid stimulation will be assessed using patch-clamp recording from isolated taste cells to help establish the nature of the predicted proton transport pathway. Complimentary experiments will involve a preparation which permits recording from chorda tympani taste nerve fibers while voltage-clamping the receptor epithelium. This powerful technique will be useful in determining whether the acid-sensing portions of the cells are apical or basolateral. Taste responses to ammonium salts share many of the properties of the acid sensing system and will be examined in a separate series of experiments using similar techniques. The results of these studies will provide new insights into sour (acid) taste reception, which plays an important role in pH balance.

DESCRIPTION: (Adapted from the Applicant's Abstract). Taste stimuli interact with taste-sensitive cells in taste buds, the majority of which are located in papillae on the upper surface of the tongue. Substances with appetitive tastes (e.g., sweet stimuli) are normally ingested; those that are aversive (e.g., bitter stimuli) are rejected, although these tendencies can be modified by experience. Some of the changes that occur in taste preferences appear to be age related and this has been well-documented in rodent models. Changes in taste sensitivity to sodium chloride with early postnatal development are especially clear. Some sensitivity to sodium chloride is present at birth, but much develops later, particularly when the animals are weaned. There are two modes of salt-taste sensing, which appear to reside in different regions of the taste bud. One sensor, on the apical microvilli of taste cells, makes direct contact with the mouth fluid. The other is sequestered on the basolateral membrane of the cells so that some stimuli must diffuse through the tight junctions separating the apical and basolateral domains of the cells to reach the receptor sites. Data suggest that access to these basolateral sites can be blocked by calcium, and that the tight junction permeability to taste stimuli changes during development. In vivo recordings of taste nerve responses, with the receptive field under voltage-clamp, will be used to screen for candidate stimuli of the basolateral receptor sites in developing and mature rats. In parallel, a newly developed in vitro technique will be used to make direct measurements of fluxes of various stimuli across the paracellular regions of single fungiform papillae. Both fluorescent dyes and ion selective microelectrodes will be used to measure fluxes of hydrogen ions due to several acids, various salts of sodium and potassium, calcium ions, and ammonium ion derivatives. Calcium is especially important because it is expected to reduce its own paracellular permeability as well as that of other stimuli. These experiments will determine the actual range of stimulating concentrations of stimuli on the basolateral side of the taste buds, which preliminary results indicate are far lower than the concentrations placed on the tongue. Methods will also be developed to determine the buffering properties of the lateral intercellular spaces for hydrogen and calcium.

DESCRIPTION (provided by applicant): Among the challenges that living systems encounter, few are as basic as the requirement that the acidity of both the intracellular and extracellular fluid compartments be maintained within narrow physiological limits. One important, and as yet only partially answered, question is: how do pH-sensing cells detect hyperacidity in the various fluid compartments being monitored? More specifically for taste receptor cells the question becomes: what is being detected and what are the cellular events resulting in excitation of the taste afferent nerves? It would be reasonable to assume that taste receptor cells would monitor the pH of potential foods or beverages (the extracellular pH). Surprising as it might seem that does not appear to be true. We have established that the proximal stimulus for sour response is the intracellular pH. This means that an acid must first enter the taste receptor cell before it can be detected. A major aim of this proposal is to research the various possible ways acids ma enter taste receptor cells. These include diffusion across cell membranes as neutral molecules (e.g. acetic acid), as gases (e.g. carbon dioxide), by electrodiffusion of hydrogen ions, and on special transporters (e.g. monocarboxylate transporters). The sensing cells themselves can only function within a narrow range of intracellular pH values, so a second issue is the determination of the pH regulatory mechanisms present in taste receptor cells and how their function may vary with intracellular pH. It is highly probable that a type 3 sodium-hydrogen exchanger is an important pH regulator in taste cells. This will be ascertained and wider probes initiated. Transduction ultimately depolarizes the taste cells and changes in intracellular calcium can be expected. These also will be probed. We will utilize two basic methods to carry out these studies. We will measure changes in intracellular pH, intracellular calcium, and membrane potential in the taste cells using fluorescent imaging method in a single fungiform papilla with epithelial tissue polarity preserved under voltage clamp conditions. These studies will be complemented by recordings from the chorda tympani with the lingual receptive field under voltage clamp. We have also observed that various bitter tasting substances cause the intracellular pH to become more alkaline. Preliminary studies show that this alkalinity is significantly reduced in the presence of GDP about S, a G-protein blocker. We will test the hypothesis that during bitter taste transduction taste cells become alkaline. For tastants that are not themselves bases (e.g. denatonium) w hypothesize that alkalinization occurs through the activation of a sodium-hydrogen exchanger or other pH regulatory mechanisms. We will test the hypothesis that blockers of pH regulatory mechanisms will also block increases in denatonium induced intracellular calcium. We have shown that the chorda tympani response to denatonium is voltage sensitive indicating that transduction involves modulation of a conductance. Having established a key role for intracellular pH in acid-sensing, these studies involving bitter-tasting compounds will determine if intracellular pH is also a key intermediate in bitter-taste transduction.