- Email: [email protected]
Cross-adapted sugar responses in the mouse taste cell
Comp. Biochem. Physiof. Vol. 92A, No. 2, pp. 181483, 1989 Printed in Great Britain
CROSS-ADAPTED
0300-9629189 $3.00 + 0.00 0 1989 Pcrgamon Press plc
SUGAR RESPONSES IN THE MOUSE TASTE CELL
KEIICHITONOSAKI*and MALAYAFUNAKC%HI Department
of Oral Physiology, School of Dentistry, Asahi University, 1851 Hozumi, Hozumi-cho, Motosu-gun, Gifu 501-02, Japan. Telephone: Japan 0.5832-6-6131 (Received 20 June 1988)
Abstract-l. Intracellular recordings of mouse taste cell responses were made using a glass micro-electrode filled with Procion yellow dye solution. 2. Six sugars (sucrose, maltose, lactose, glucose, galactose and fructose) produced the depolarization responses. 3. Gustatory cross adaptation between sugars was determined. When the taste cell was pre-adapted with one of the six sugars, the other five sugars, cross adapted, produced depolarization, hyperpolarixation or null responses. 4. From these observations, it is suggested that there are multiple sugar receptor sites on the receptor membrane of the mouse taste cell.
INTRODUCTION
Sugar has been found to elicit a sweet taste response in nearly all animal species and the mechanism of sugar taste response has suggested the presence of multiple sugar receptor sites on the taste cell. These results have been provided by data from whole nerve, single neuron and single receptor cell studies (Jakinovich and Goldstein, 1976; Jakinovich, 1976, 1985; Ninomiya et al., 1982; Tonosaki and Funakoshi, 1984a, b, c). However, gustatory cross adaptation between sugars has not been determined, but it would lend further support to the idea of multiple receptor sites (Beidler, 1954; Beidler and Tonosaki, 1985). It has not been previously reported that the mammalian taste cell responds to the sweeteners tested to cross adapt with intracellular recording. Taste cells of mouse fungiform papilla of the tongue were reported to respond to sucrose taste stimulus with de~larization, a~ompanied by a membrane resistance increase or null. We studied only sucrose depolarization responses since sucrose hyperpolarization responses were never recorded from dye staining cells of taste buds (Tonosaki and Funakoshi, 1984a, b), The source of such responses is uncertain. In the present experiments, we recorded sugar cross-adapted responses in the mouse taste oell using intracellular recording. We propose that there are multiple sugar receptor sites on the mouse taste cell receptor membrane. MATERIALS AND METHODS
Adult mice (Slc:ICR) (30-50 g) were anesthetized with Nembutal and tracheotomized. After the head was fixed in a holder, the tongue was gently extended with hooks onto a plastic plate. Tongues were adapted to constantly flowing *Author to whom all correspondence should be addressed.
distilled water (0.08 ml/set). Test solutions (sucrose: OS, 1.0 M; maltose: .O.S, 1.0 M; lactose: 0.5, 1.0 M; galactose: 0.5. l.OM: alucose: 0.5. l.OM: fNCtOSC: 0.5. l.OM) (0.0s ml/se;) I&iodically replaced’the distilled water rinse without interrupting fluid flow. Conventional intracellular recordings were made with a high impedance glass microelectrode filled with 6% aqueous solution of Procion yellow. An AgCl electrode placed into the musculature of the neck was used as an indifferent electrode. The membrane resistance change of the taste cell was recorded with a bridge circuit and negative, 100 msec current pulses in the order of 0.1 nA were. passed from the recording electrode into the cell. After recording the responses, Procion yellow was el~trophoretically injected into the cell from the recording microelectrode by passing a steady current of - 1 to - 3 nA for 2-5 min. If a stained cell was not observed in the preparation using a fluorescence microscope, the responses were not included in the analysis. The taste cells are quite small and it has been difficult to record receptor potential with intracellular electrodes. Injection of the dye into a responding cell served as a reliable indicator that the electrode tip was actually inside a cell.
RESULTS A survey of a wide variety of sugar taste stimuli was performed. Sugar stimuli produced depolarizations and they were accompanied by membrane resistance increase or null. No membrane resistance decrease in sugar responses was observed. With this type of experiment, it was extremely difficult to record intracellular potential and to hold the electrode tip inside the taste cell for a long time. Figure 1 shows an example of the responses of one taste cell to six sugar stimuli. A tixed con~ntration of sugar (0.5 M) was applied to the tongue. In this Figure, each sugar stimulus produces depolarization responses; however each sugar stimulus produces a different magnitude of responses. This has been accomplished in the mouse (Tonosaki and Funakoshi, 1984b). Usually, sucrose, maltose or lactose taste response was larger than the other sugar responses. When the taste cell 181
KEIICHITONOSAKIand MASAYA FUNAKOSHI
182
MAL
GAL GLU FRU
Fig. 1. Taste cell responses in mouse tongue to six sugar taste stimuli, recorded intracellularly. All the records were obtained from a taste cell. The taste cell was adapted to distilled water at first, and afterwards pm-adapted with one of the six sugars (0.5 M) about 1 min, cross-adapted sugar (0.5 M) responses were successively recorded respectively. The left column shows the pre-adapted sugars. A fixed concentration of sugar (0.5 M) was applied to the tongue. The lowest trace indicated the successively stimulated diagram. When the cross-adapted sugar was applied to the tongue, distilled water flow on the tongue was stopped. Dotted lines between the sugar stimuli in the lowest line indicated approx. 20 set intervals. The last record to the sucrose response presented the control sucrose response which was a depolarization response and was accompanied by the membrane resistance increase. A train of negative current pulses of about 0. I nA and 100 msec duration was applied at 1 Hz. An increase in the negative pulse height in the bridge record indicates an increase in the membrane resistance. The vertical and horizontal bars on the right show
20 MR, 20 mV and 10 see, respectively. Abbreviations: SUC, sucrose; MAL, maltose; LAC. lactose; GAL. galactose; GLU, glucose; FRU, fructose. was pre-adapted with the one of the six sugars, one can see a variety of response profiles to other sugars. Whatever taste cell was pre-adapted with one of the six sugars (e.g. in Fig. 1), lactose produced depolarization response, while sucrose, maltose, galactose, glucose and fructose produced depolarization, hyperpolarization or null response. Fructose stimulus usually produced smaller amplitude of response than the other sugars; however, when pre-adapted fructose was cross adapted with the other five sugars, the other five sugars, cross adapted, produced the feeble depolarization responses. Similar results were obtained from five experiments. DISCUSSION
We found that the response profile to several sugars as different in each taste cell with intracellular recording studies (Tonosaki and Funakoshi, 1984b). The multi-sensitivity of the mammalian taste cells to the fundamental four basic taste stimuli was demonstrated by some investigators with intracellular recordings (Ozeki and Sato, 1972; Tonosaki and Funakoshi, 1984a). According to the recent sugar reception hypothesis, there are two major ideas. One is the idea of multi-receptor sites on the taste receptor membrane (Beidler, 1954; Jakinovich and Goldstein, 1976; Jakinovich, 1976; Beidler and Tonosaki, 1985), and the other is the idea of the single receptor site (Shallenberger and Acree, 1967). Sugar stimulus adsorption is believed to occur at the receptor membrane on the taste cell. But due to technical difficulties of inserting glass microelectrode into the mammalian taste cell, little is known about the mechanisms of sugar taste transduction (Tonosaki and Funakoshi, 1988). There are many chorda tympani whole (or single) nerve sugar responses were reported (for review, see Jakinovich and Sugarman, 1988). Thus, a chorda tympani nerve fiber has a lot of synapses among taste cells, so that it is extremely difficult to
speculate whether the sugar receptor site is single or multi. However, no one previously determined the mechanism of cross adaptation with single taste cell. In this paper, we studied the sweeteners tested cross adapt with intracellular recording technique. From these experiments, it seems possible to classify into the following three cases. The first example, when the cross-adapted sugar produced the depolarization membrane potentials, it can be speculated that the sugar has different receptor sites on the taste cell, or it occupied more receptor sites than the preadapted sugar. In the second example, when the cross-adapted sugar produced hyperpolarization membrane potential, it can be that the sugar occupied different receptor sites or it occupied fewer of the receptor sites than the pre-adapted sugar. In the third example, when the membrane potentials were no different before and after the cross adaptation, it seems that both sugars occupied the same receptor sites or they have different receptor sites but the total number of occupied receptor sites are similar between two sugars. If the taste cell has a single sugar receptor site, it is difficult to account for the magnitude of response of cross-adapted sugars on the pre-adapted fructose was always smaller than that of controls. The other typical example can be seen in Fig. 1. The response magnitude to fructose was smaller than that of other sugars. However, cross-adapted fructose responses did not produce reasonable hyperpolarization responses when the taste cell was pre-adapted with five other sugars. These results indicated the complicated response generation mechanisms among sugars and suggest that multiple sugar receptor sites exist on the taste receptor membrane. This may further support the hypothesis (Beidler and Tonosaki, 1985) that a given taste cell has many different types of sugar receptor sites on the taste receptor membrane.
Sugar receptors in mouse taste cell Acknowledgements-This work was supported in part by Grant-in-Aid 62570848 for Scientific Research from the Ministry of Education, Science and Culture of Japan.
REFERENCES
Beidler L. M. (1954) A theory of taste stimulation. J. gen. Physiol. 38, 133-139. Beidler L. M. and Tonosaki K. (1985) Multiple sweet receptor sites and taste theory. In Taste, Olfaction and the Cent& Nervous Sysfem (Edited by Pfaff P. W.), pp. 47-64. Rockefeller University Press, New York. Jakinovich W. Jr and Goldstein I. J. (1976) Stimulation of the gerbil’s gustatory receptors by monosaccharides. Brain Res. 110, 491-504. Jakinovich W. Jr (1976) Stimulation of the gerbil’s gustatory receptors by disaccharides. Brain Res. 110, 481-490. Jakinovich W. Jr (1985) Sugar taste reception in the gerbil. In Taste, ~~a~~io~, and the Central Ne~o~ System (Edited by Pfaff D. W.), pp. 65-91. Rockefeller University Press, New York.
183
Jakinovich W. Jr and Sugarman D. (1988) Sugar taste reception in mammals. Chem. Sens. 13, 1331. Ninomiya Y., Tonosaki K. and Funakoshi M. (1982) Gustatory neuron response in the mouse. Brain Res. 244, 370-373. Ozeki M. and Sato M. (1972) Responses of gustatory cells in the tongue of the rat to stimuli representing the four taste qualities. Camp. Biochem. Physiol. PlA, 391-407. Shallenberger R. S. and Acme T. E. (1967) Molecular theory of sweet taste. Nature (Land.) 216, 480-482. Tonosaki K, and Funakoshi M. (1984a) Intracellular taste cell responses of mouse. Comp. Biochem. P~ysio~. 78A, 651-656. Tonosaki K. and Funakoshi M. (1984b) The mouse taste cell response to five sugar stimuli. Comp. Biochem. Physiol. 79A, 625-630. Tonosaki K. and Funakoshi M. (1984~) Effect of polarization of mouse taste cells. Gem. Sens. 9, 381-387. Tonosaki K. and Funakoshi M. (1988) Cyclic nueleotides may mediate taste transduction. Nature {Land.) 331, 354-356.
CROSS-ADAPTED
0300-9629189 $3.00 + 0.00 0 1989 Pcrgamon Press plc
SUGAR RESPONSES IN THE MOUSE TASTE CELL
KEIICHITONOSAKI*and MALAYAFUNAKC%HI Department
of Oral Physiology, School of Dentistry, Asahi University, 1851 Hozumi, Hozumi-cho, Motosu-gun, Gifu 501-02, Japan. Telephone: Japan 0.5832-6-6131 (Received 20 June 1988)
Abstract-l. Intracellular recordings of mouse taste cell responses were made using a glass micro-electrode filled with Procion yellow dye solution. 2. Six sugars (sucrose, maltose, lactose, glucose, galactose and fructose) produced the depolarization responses. 3. Gustatory cross adaptation between sugars was determined. When the taste cell was pre-adapted with one of the six sugars, the other five sugars, cross adapted, produced depolarization, hyperpolarixation or null responses. 4. From these observations, it is suggested that there are multiple sugar receptor sites on the receptor membrane of the mouse taste cell.
INTRODUCTION
Sugar has been found to elicit a sweet taste response in nearly all animal species and the mechanism of sugar taste response has suggested the presence of multiple sugar receptor sites on the taste cell. These results have been provided by data from whole nerve, single neuron and single receptor cell studies (Jakinovich and Goldstein, 1976; Jakinovich, 1976, 1985; Ninomiya et al., 1982; Tonosaki and Funakoshi, 1984a, b, c). However, gustatory cross adaptation between sugars has not been determined, but it would lend further support to the idea of multiple receptor sites (Beidler, 1954; Beidler and Tonosaki, 1985). It has not been previously reported that the mammalian taste cell responds to the sweeteners tested to cross adapt with intracellular recording. Taste cells of mouse fungiform papilla of the tongue were reported to respond to sucrose taste stimulus with de~larization, a~ompanied by a membrane resistance increase or null. We studied only sucrose depolarization responses since sucrose hyperpolarization responses were never recorded from dye staining cells of taste buds (Tonosaki and Funakoshi, 1984a, b), The source of such responses is uncertain. In the present experiments, we recorded sugar cross-adapted responses in the mouse taste oell using intracellular recording. We propose that there are multiple sugar receptor sites on the mouse taste cell receptor membrane. MATERIALS AND METHODS
Adult mice (Slc:ICR) (30-50 g) were anesthetized with Nembutal and tracheotomized. After the head was fixed in a holder, the tongue was gently extended with hooks onto a plastic plate. Tongues were adapted to constantly flowing *Author to whom all correspondence should be addressed.
distilled water (0.08 ml/set). Test solutions (sucrose: OS, 1.0 M; maltose: .O.S, 1.0 M; lactose: 0.5, 1.0 M; galactose: 0.5. l.OM: alucose: 0.5. l.OM: fNCtOSC: 0.5. l.OM) (0.0s ml/se;) I&iodically replaced’the distilled water rinse without interrupting fluid flow. Conventional intracellular recordings were made with a high impedance glass microelectrode filled with 6% aqueous solution of Procion yellow. An AgCl electrode placed into the musculature of the neck was used as an indifferent electrode. The membrane resistance change of the taste cell was recorded with a bridge circuit and negative, 100 msec current pulses in the order of 0.1 nA were. passed from the recording electrode into the cell. After recording the responses, Procion yellow was el~trophoretically injected into the cell from the recording microelectrode by passing a steady current of - 1 to - 3 nA for 2-5 min. If a stained cell was not observed in the preparation using a fluorescence microscope, the responses were not included in the analysis. The taste cells are quite small and it has been difficult to record receptor potential with intracellular electrodes. Injection of the dye into a responding cell served as a reliable indicator that the electrode tip was actually inside a cell.
RESULTS A survey of a wide variety of sugar taste stimuli was performed. Sugar stimuli produced depolarizations and they were accompanied by membrane resistance increase or null. No membrane resistance decrease in sugar responses was observed. With this type of experiment, it was extremely difficult to record intracellular potential and to hold the electrode tip inside the taste cell for a long time. Figure 1 shows an example of the responses of one taste cell to six sugar stimuli. A tixed con~ntration of sugar (0.5 M) was applied to the tongue. In this Figure, each sugar stimulus produces depolarization responses; however each sugar stimulus produces a different magnitude of responses. This has been accomplished in the mouse (Tonosaki and Funakoshi, 1984b). Usually, sucrose, maltose or lactose taste response was larger than the other sugar responses. When the taste cell 181
KEIICHITONOSAKIand MASAYA FUNAKOSHI
182
MAL
GAL GLU FRU
Fig. 1. Taste cell responses in mouse tongue to six sugar taste stimuli, recorded intracellularly. All the records were obtained from a taste cell. The taste cell was adapted to distilled water at first, and afterwards pm-adapted with one of the six sugars (0.5 M) about 1 min, cross-adapted sugar (0.5 M) responses were successively recorded respectively. The left column shows the pre-adapted sugars. A fixed concentration of sugar (0.5 M) was applied to the tongue. The lowest trace indicated the successively stimulated diagram. When the cross-adapted sugar was applied to the tongue, distilled water flow on the tongue was stopped. Dotted lines between the sugar stimuli in the lowest line indicated approx. 20 set intervals. The last record to the sucrose response presented the control sucrose response which was a depolarization response and was accompanied by the membrane resistance increase. A train of negative current pulses of about 0. I nA and 100 msec duration was applied at 1 Hz. An increase in the negative pulse height in the bridge record indicates an increase in the membrane resistance. The vertical and horizontal bars on the right show
20 MR, 20 mV and 10 see, respectively. Abbreviations: SUC, sucrose; MAL, maltose; LAC. lactose; GAL. galactose; GLU, glucose; FRU, fructose. was pre-adapted with the one of the six sugars, one can see a variety of response profiles to other sugars. Whatever taste cell was pre-adapted with one of the six sugars (e.g. in Fig. 1), lactose produced depolarization response, while sucrose, maltose, galactose, glucose and fructose produced depolarization, hyperpolarization or null response. Fructose stimulus usually produced smaller amplitude of response than the other sugars; however, when pre-adapted fructose was cross adapted with the other five sugars, the other five sugars, cross adapted, produced the feeble depolarization responses. Similar results were obtained from five experiments. DISCUSSION
We found that the response profile to several sugars as different in each taste cell with intracellular recording studies (Tonosaki and Funakoshi, 1984b). The multi-sensitivity of the mammalian taste cells to the fundamental four basic taste stimuli was demonstrated by some investigators with intracellular recordings (Ozeki and Sato, 1972; Tonosaki and Funakoshi, 1984a). According to the recent sugar reception hypothesis, there are two major ideas. One is the idea of multi-receptor sites on the taste receptor membrane (Beidler, 1954; Jakinovich and Goldstein, 1976; Jakinovich, 1976; Beidler and Tonosaki, 1985), and the other is the idea of the single receptor site (Shallenberger and Acree, 1967). Sugar stimulus adsorption is believed to occur at the receptor membrane on the taste cell. But due to technical difficulties of inserting glass microelectrode into the mammalian taste cell, little is known about the mechanisms of sugar taste transduction (Tonosaki and Funakoshi, 1988). There are many chorda tympani whole (or single) nerve sugar responses were reported (for review, see Jakinovich and Sugarman, 1988). Thus, a chorda tympani nerve fiber has a lot of synapses among taste cells, so that it is extremely difficult to
speculate whether the sugar receptor site is single or multi. However, no one previously determined the mechanism of cross adaptation with single taste cell. In this paper, we studied the sweeteners tested cross adapt with intracellular recording technique. From these experiments, it seems possible to classify into the following three cases. The first example, when the cross-adapted sugar produced the depolarization membrane potentials, it can be speculated that the sugar has different receptor sites on the taste cell, or it occupied more receptor sites than the preadapted sugar. In the second example, when the cross-adapted sugar produced hyperpolarization membrane potential, it can be that the sugar occupied different receptor sites or it occupied fewer of the receptor sites than the pre-adapted sugar. In the third example, when the membrane potentials were no different before and after the cross adaptation, it seems that both sugars occupied the same receptor sites or they have different receptor sites but the total number of occupied receptor sites are similar between two sugars. If the taste cell has a single sugar receptor site, it is difficult to account for the magnitude of response of cross-adapted sugars on the pre-adapted fructose was always smaller than that of controls. The other typical example can be seen in Fig. 1. The response magnitude to fructose was smaller than that of other sugars. However, cross-adapted fructose responses did not produce reasonable hyperpolarization responses when the taste cell was pre-adapted with five other sugars. These results indicated the complicated response generation mechanisms among sugars and suggest that multiple sugar receptor sites exist on the taste receptor membrane. This may further support the hypothesis (Beidler and Tonosaki, 1985) that a given taste cell has many different types of sugar receptor sites on the taste receptor membrane.
Sugar receptors in mouse taste cell Acknowledgements-This work was supported in part by Grant-in-Aid 62570848 for Scientific Research from the Ministry of Education, Science and Culture of Japan.
REFERENCES
Beidler L. M. (1954) A theory of taste stimulation. J. gen. Physiol. 38, 133-139. Beidler L. M. and Tonosaki K. (1985) Multiple sweet receptor sites and taste theory. In Taste, Olfaction and the Cent& Nervous Sysfem (Edited by Pfaff P. W.), pp. 47-64. Rockefeller University Press, New York. Jakinovich W. Jr and Goldstein I. J. (1976) Stimulation of the gerbil’s gustatory receptors by monosaccharides. Brain Res. 110, 491-504. Jakinovich W. Jr (1976) Stimulation of the gerbil’s gustatory receptors by disaccharides. Brain Res. 110, 481-490. Jakinovich W. Jr (1985) Sugar taste reception in the gerbil. In Taste, ~~a~~io~, and the Central Ne~o~ System (Edited by Pfaff D. W.), pp. 65-91. Rockefeller University Press, New York.
183
Jakinovich W. Jr and Sugarman D. (1988) Sugar taste reception in mammals. Chem. Sens. 13, 1331. Ninomiya Y., Tonosaki K. and Funakoshi M. (1982) Gustatory neuron response in the mouse. Brain Res. 244, 370-373. Ozeki M. and Sato M. (1972) Responses of gustatory cells in the tongue of the rat to stimuli representing the four taste qualities. Camp. Biochem. Physiol. PlA, 391-407. Shallenberger R. S. and Acme T. E. (1967) Molecular theory of sweet taste. Nature (Land.) 216, 480-482. Tonosaki K, and Funakoshi M. (1984a) Intracellular taste cell responses of mouse. Comp. Biochem. P~ysio~. 78A, 651-656. Tonosaki K. and Funakoshi M. (1984b) The mouse taste cell response to five sugar stimuli. Comp. Biochem. Physiol. 79A, 625-630. Tonosaki K. and Funakoshi M. (1984~) Effect of polarization of mouse taste cells. Gem. Sens. 9, 381-387. Tonosaki K. and Funakoshi M. (1988) Cyclic nueleotides may mediate taste transduction. Nature {Land.) 331, 354-356.