EFFECTS OF LOCAL ANESTHETICS ON FROG TASTE CELL RESPONSES

Transcription

1 Jap. J. Physiol., 25, , 1975 EFFECTS OF LOCAL ANESTHETICS ON FROG TASTE CELL RESPONSES Norio AKAIKE and Masayasu SATO Department of Physiology, Kumamoto University Medical School, Kumamoto 860, Japan Abstract Immediately after application of procaine or lidocaine to the surface of the tongue a transient response occurs in the glossopharyngeal nerve, while a depolarization of a few mv associated with an increase in the input resistance was observed in taste cells. When applied to the tongue for a long time, procaine and lidocaine depressed responses in the glossopharyngeal nerve to chemical stimulation of the frog tongue. The depressant effect is strongest on quinine response, next on responses to salts, and weakest on response to acid. Under the action of the local anesthetics both the magnitude of depolarization and the amount of input resistance change in taste cells, produced by stimulation by various chemicals, are small compared with those observed in normal cells. The action of anesthetics on the depolarization in taste cells is also strongest on quinine response and weakest on acid response. The results indicate that the local anesthetics exert a depressant action on taste cells by inhibiting changes in the membrane conductance and generation of depolarization, which are produced by chemical stimuli and are responsible for impulse initiation at sensory nerve terminals. It has sometimes been stated in the past that a solution of cocaine painted on the tongue will abolish taste and buccal sensations in the order of pain, bitter, sweet, saline, sour and touch (MONCRIEF, 1951). However, electrophysiological investigations, carried out so far on the anesthetic action of cocaine and procaine on taste receptors, yielded inconsistent results. For example, KUSANO (1959) demonstrated a depressant effect of cocaine on taste receptors in the isolated frog tongue by examining responses in the glossopharyngeal nerve to various chemical stimuli. OZEKI and NOMA (1972) also reported that application of 0.5% procaine to the frog tongue for half an hour decreased the magnitude of the glossopharyngeal nerve response to 1M NaCl to less than 5% of the control value. However, the latter authors showed that 0.1% procaine applied to the tongue of Received for publication March 11, 1975

2 586 N. AKAIKE and M. SATO rat produced little change in the chorda tympani nerve responses to 0.1M NaCl and 0.02M quinine, although it decreased the response to HCl and increased the response to sucrose. On the other hand, results quite different from the above were obtained by TATEDA and BEIDLER (1964) by recording potential changes with a glass microelectrode thrust inside a fungiform papilla of rat; they demonstrated that 1% cocaine elicited a positive deflection of the steady potential of taste cells and that under the effect of cocaine responses to NaCl reversed their polarity. Recently intracellular recording of membrane potential changes in a single taste cell of a frog has been made successfully, and slow depolarizations associated with the membrane conductance changes have been demonstrated during stimulation of taste cells by some chemicals (AKAIKE et al., 1973; SATO, 1969, 1972; SATO and BEIDLER, 1973). The depolarizations have been considered to be receptor potentials in taste cells, which initiate impulses at sensory nerve terminals, and the conductance changes to reflect the receptive process at the taste cell membrane. Therefore, in the experiments described below the effects of procaine and lidocaine on the electrical responses of taste cells in fungiform papillae of the frog to various chemical stimuli were investigated. Also their effects on the glossopharyngeal nerve responses were examined for comparison. In addition, the effects of tetrodotoxin (TTX) on nerve responses and taste cell responses to chemical stimuli were reexamined, because OZEKI and NOMA (1972) reported that it suppressed glossopharyngeal nerve responses but not taste cell responses. MATERIALS AND METHODS All the experiments were carried out at room temperature, about 17 Ž, with bullfrogs (Rana catesbeiana) anesthetized by intraperitoneal injection of a 20% urethane solution. Experimental procedures were essentially the same as those described in a previous paper (AKAIKE et al., 1973). To prevent muscular movement of the tongue, the hypoglossal nerves of both sides were cut. The tongue was pulled out and fixed with pins to the bottom of a Perspex chamber. The chamber had an inlet and outlet through which 0.01M NaCl solution (adapting solution) flowed throughout an experiment. The membrane potential of a single cell was recorded by inserting a glass capillary microelectrode (filled with 3M KCl and having a resistance of 10 to 40Mƒ ) through the sensory disc of a papilla. As an indifferent electrode a 3M-KCl-agar electrode was placed in the adapting solution. Both electrodes were connected through salt bridges to the calomel electrodes which were led to a Wheatstone bridge circuit to record the potential from inside a cell. Potential changes were displayed on a dual beam oscilloscope and simultaneously recorded with a pen-writing recorder. In most of the experiments hyperpolarizing current pulses of 5 ~10-10 A and of 100msec in duration were passed at a rate of one per sec through a microelectrode inserted inside a taste cell, and the resulting electro-

3 LOCAL ANESTHETICS ON TASTE CELL 587 tonic potentials were recorded with the same electrode. In a few experiments responses in the glossopharyngeal nerve to chemical stimulation of the whole tongue were recorded with a pen-writing recorder connected to an amplifier and an integrator circuit (BEIDLER, 1963). In these experiments the proximal portion of the glossopharyngeal nerve was cut and suspended on a pair of silver wire electrodes to record potential changes. In order to stimulate a single fungiform papilla by chemical stimuli, each of the test solutions was applied through one of four thin polyethylene tubes with about 50ƒÊm in hole diameter at the tip. The end of each of these tubes was connected by a polyethylene tube to an injection syringe filled with a taste solution, and the other end was placed close to the papilla to be tested. During the intervals between stimulation, the papilla was constantly rinsed with the adapting solution. As test stimuli 0.1M NaCl, 0.1M KCl, 0.02M quinine hydrochloride and 1mm HCl were employed in the experiments where membrane potential changes in taste cells were examined. In addition, NaCl and CaCl2 solutions of varying concentrations were used in some of the experiments. For rinsing the stimuli 0.01M NaCl solution was employed, to which taste receptors had been adapted before stimulation. Quinine, HCl and sucrose were dissolved in 0.01M NaCl solution, while NaCl, KCl, and CaCl2 were dissolved in deionized water. In the experiments to record the glossopharyngeal nerve response 0.3M NaCl, 0.3M KCl, 1mM CaCl2, 1mM HCl, deionized water and 1mM quinine were used as test stimuli. In addition, repeated mechanical stimuli were applied to the surface of the tongue for about 20sec with a glass rod driven by electric pulses of 5msec in duration at a rate of 4/sec, and responses to these stimuli were recorded from the glossopharyngeal nerve. Procaine hydrochloride and lidocaine hydrochloride (xylocaine) were dissolved in 0.01M NaCl solution. Procaine concentrations employed were 0.01, 0.05, 0.1, and 0.5%(w/v), while lidocaine concentrations were 0.05 and 0.5%. Tetrodotoxin was dissolved in 0.01M NaCl at a concentration of 1.6 ~10-7g/ml. All the chemicals were of reagent grade. For examining the effects of the above anesthetics on taste cells, the following procedures were adopted. First, resting membrane potentials and responses to four kinds of stimuli applied successively were recorded from a cell in a fungiform papilla, which had been adapted to 0.01M NaCl solution. After each stimulation the surface of the papilla was rinsed with the adapting solution. Similar procedures were repeated on several taste cells, Next 0.01M NaCl solution containing one of the anesthetics was applied over the surface of the tongue for 15 min, and then a number of fungiform papilla cells were penetrated with a microelectrode and their responses to the test stimuli were examined successively. Further, recovery of responses in taste cells was examined after their adaptation to 0.01M NaCl solution. The input resistance of a taste cell was calculated from the magnitude of

4 588 N. AKAIKE and M. SATO electrotonic potentials in response to injected electrical pulses, and the relative magnitude of the membrane conductance during chemical stimulation of a cell was obtained by a ratio of the electrotonic potential magnitudes at rest and stimulated states. RESULTS Depression of glossopharyngeal nerve responses to gustatory stimuli by procaine Procaine depressed reversibly taste nerve responses to various chemical stimuli applied to the tongue. Figure 1 presents the results of an experiment, where the effect of 0.01% procaine on the glossopharyngeal nerve responses to taste and mechanical stimuli was examined. As shown in this figure, immediately after application of procaine solution to the tongue surface a transient response occurred. Application of the procaine solution to the tongue for 15min depressed slightly responses to salts, quinine and deionized water, and 60min after the application responses to all the taste stimuli were suppressed markedly, but tactile response was scarcely affected. Responses to taste stimuli recovered sufficiently after replacement of the procaine solution by normal adapting solution. Fig. 1. Effects of 0.01% procaine on the glossopharyngeal nerve responses to various stimuli applied to the tongue. Records in the uppermost row are control responses before application of procaine, those in the second and third rows were taken 15min and 60min after application of the procaine solution, respectively, while records in the lowest row were taken 30min after adaptation of taste cells to the normal adapting solution. The lefthand record in the second row is the response to application of the procaine solution. The test stimuli used are indicated at the top of the figure. The thick line underneath each record indicates the period of application of each test stimulus.

5 LOCAL ANESTHETICS ON TASTE CELL 589 Fig. 2. Time course of depression of the glossopharyngeal nerve responses to various stimuli, applied to the tongue, during application of 0.01% procaine. The tongue was adapted to 0.01M NaCl first, next to the one with procaine for 60min and to 0.01M NaCl again. The time-course of the depression of the glossopharyngeal nerve responses by 0.01% procaine is demonstrated in Fig. 2. As shown in the figure, the magnitudes of responses to quinine and salts were reduced to less than half of the original magnitude 60min after application of procaine, while those to HCl and mechanical stimulus were affected very little. The depressant effect of procaine becomes stronger with increasing concentration. For example, 0.5% procaine, 15min after its application, virtually abolished responses to all the taste stimuli including response to 1M sucrose. In a few experiments glossopharyngeal nerve responses to 0.1%(0.0037M) and 0.5%(0.0183M) procaine solutions were recorded and compared with those to 0.1M NaCl and 0.02M quinine. The response to 0.5% procaine is much greater in amplitude than that to 0.1M NaCl and comparable with that to 0.02M quinine. Depolarizing effect of procaine and lidocaine on taste cells Both procaine and lidocaine produced depolarizations in the taste cell. The transient response of the glossopharyngeal nerve to application of procaine solution in Fig. 1 is probably produced by its depolarizing effect on the cell. Table 1 presents the mean magnitudes of depolarizations and relative conductances, induced by NaCl, quinine and procaine. As shown in the table, the depolarization produced by 0.5% procaine is nearly the same as that by 0.02M quinine, and the mean relative conductance magnitude is slightly decreased by these two stimuli although it is increased by 0.1M NaCl. After applying procaine solution to the tongue for a relatively long period the decrease in the relative conductance of a taste cell progressed gradually. This can be recognized in Fig. 3 by a much increased magnitude of the electrotonic potential

6 590 N. AKAIKE and M. SATO Table 1. Depolarizations and relative conductance changes produced by procaine, quinine, and NaCl. Each value represents the mean }S.D. determined on 5 cells. recorded 15min after application of 0.5% procaine. Table 2 shows the mean. ( }S.D.) values of the resting potential, the input resistance and the relative conductance of a number of taste cells adapted for 15-30min to 0.01M NaCl solution without and with procaine in three series of experiments. As shown in this table, 0.5% procaine decreased the relative conductance to about 60% of the original value. The large amount of the reduction in the relative conductance in Table 2 compared with that in Table 1 may be due to a longer time of exposure of cells to procaine in the experiments in Table 2. In Table 2 both 0.1 and 0.5% lidocaine also decreased the relative conductance to about 90% and yielded a depolarizing action on taste cells. Table 2. Effects of local anesthetics on resting potential (Er), input resistance (Re), and relative conductance magnitude (Gr) in taste cells. Numerals indicate the mean }S.D., and those inside parentheses represent the number of cells examined.

7 LOCAL ANESTHETICS ON TASTE CELL 591 Fig. 3. Depressant effects of procaine on responses of taste cells to 0.1M NaCl, 0.1M KC1, 0.02M quinine and 0.001M HCl. The top records are responses in the cell adapted to 0.01M NaCl, the middle ones were taken 15min after application of 0.01M NaCl containing 0.5% procaine, and the bottom records were obtained after readapting the cell to 0.01M NaCl. Recordings of potentials were made from three different cells. The sudden negative and positive shifts at the left and right of each trace indicate impalement of a cell with a microelectrode and its withdrawal from the cell, respectively. Effects of procaine and lidocciine on the depolarization and membrane conductance change produced by chemical stimuli in taste' cells The effect of 0.5% procaine on responses of a taste cell to the four kinds of stimuli is demonstrated in Fig. 3. As shown in the figure, after applying 0.01M NaCl solution containing 0.5% procaine to the tongue for 15min, depolarizations produced by NaCl, KCl, and HCl in the cell became very small and the response to quinine was almost abolished. However, the response almost recovered to the normal value after readapting the tongue to 0.01M NaCl solution. The results of the experiments carried out on cells each before, during and after application of 0.5% procaine to the tongue are summarized in Fig. 4. As shown in the upper figure, depolarizing responses to all the four kinds of stimuli were markedly reduced in magnitude, and response to quinine was virtually abolished. Response to HCl was affected by procaine less than those to other three stimuli. The relative conductance of a taste cell is markedly increased during stimulation by NaCl and KCl, but its changes are small during stimulation by HCl and quinine (AKAIKE et al., 1973). Therefore, the relative conductance values during stimulations by the four test stimuli before, during and after application of pro-

8 592 N. AKAIKE and M. SATO caine are indicated in the lower figure of Fig. 4. As shown in the figure, during stimulation by 0.1M NaCl and KCl the relative conductance value is about 1.5 in normal taste cells, but after procaine application the value is only 1.1. Also after procaine treatment the relative conductance during stimulation by HCl becomes smaller than before procaine action. During stimulation by quinine the relative conductance is decreased slightly in both normal and procainized cells, and the decrease is smaller in the latter than in the former. Lidocaine possesses a depressant action on taste cell responses similar to that produced by procaine. As shown in Fig. 4, 0.5% lidocaine markedly depressed depolarizing responses to NaCl, KCl, and quinine. Similar to the effect of procaine depression of HCl response by lidocaine is smaller than that of responses to other stimuli. The amount of the relative conductance during stimulation was also reduced by lidocaine, from about 1.5 to 1.1 during stimulation by NaCl and KCl, and from 1.1 to 1.05 during stimulation by HCl. Fig. 4. Mean magnitudes of receptor potentials (upper figure) and relative conductance magnitudes (lower figure), elicited by 0.1M NaCl, 0.1M KCl, 0.02M quinine, and 0.001M HCl in taste cells, adapted to 0.01M NaCl solution (empty blocks), to the one with 0.5 procaine for 15-40min (dotted blocks), and to that with 0.5% lidocaine (hatched blocks). Empty blocks at the left for each stimuli represent the magnitudes obtained before application of the anesthetics while those at the right indicate the magnitudes after recovery from anesthetic effect. The number of cells employed in each determination is indicated by a numeral inside parenthesis, and the vertical bar represents one S.D. The relative conductance was calculated as the ratio of the electrotonic potential magnitudes at rest and stimulated state in the cell.

9 LOCAL ANESTHETICS ON TASTE CELL 593 The effects of 0.1 and 0.5% procaine and 0.05% lidocaine on taste cell responses to varying concentrations of NaCl were examined further, and those of procaine solutions are demonstrated in Fig. 5. Both kinds of anesthetics depressed the depolarizing responses to all the stimuli, and the depressant effect of 0.05% lidocaine was almost similar to that exerted by 0.1% procaine. The relative conductance magnitude during stimulation by NaCl solutions was also reduced by both procaine and lidocaine:the decrease is more marked with higher concentration of NaCl and with higher concentration of procaine. Depression of responses of taste cells to CaCl2 solutions of varying concentrations by 0.5% procaine is shown also in Fig. 5, where the depolarization is greatly depressed but the decrease in the relative conductance magnitude by procaine is relatively small. Figure 6 presents summarized results on the effect of 0.5% procaine on depolarizations and input resistance during stimulation of cells by various chemicals, obtained from several series of experiments. In the figure depolarizations produced by various stimuli are plotted as ordinates against the input resistances during stimulations as abscissae. Generally a greater depolarization is associated with a smaller input resistance during activity in normal cells, although response Fig. 5. Mean magnitudes of receptor potentials (upper figure) and relative conductance magnitudes (lower figure), elicited by NaCl and CaCl2 solutions of varying concentrations in taste cells adapted to 0.01M NaCl, 0.1% procaine solution, 0.5% procaine solution. The number of cells employed in each determination is shown above the figure. Vertical bar represents one S.D.

10 594 N. AKAIKE and M. SATO Fig. 6. Depressant effects of 0.5% procaine on depolarizations (ordinates) and input resistances (abscissae) during stimulations by varying chemicals. Different kinds of stimuli are shown by different symbols and the number of cells used in each determination is indicated by a numeral at the top of the figure. Numeral beside each symbol indicates the stimulus concentration (M). Two vertical dotted lines represent the input resistance in the resting cell before (left) and after application (right) of procaine. The data presented in this figure were collected from a number of series of experiments including those shown in Figs. 4 and 5. to quinine appears to be an exception. Two vertical dotted lines represent the input resistance in the resting cell before (left) and after application of procaine (right). After application of procaine the input resistance in the resting cell increased from 16.5Mƒ to 29Mƒ, and there was a depolarization of a few mv, though the latter is not indicated in the figure. Under the action of procaine, depolarizations produced by varying stimuli are reduced in magnitude and the depolarization-resistance relation shifted towards a smaller depolarization value along the y-axis and towards a higher resistance value along the x-axis. Thus the figure indicates clearly that procaine exerts a depressant action on taste cells by inhibiting a decrease in the membrane resistance or an increase in the conductance by chemical stimuli and by reducing the amount of depolarizations produced. Effects of TTX on taste cell responses As OZEKI and NOMA (1972) reported, TTX at a concentration of 1.6 ~10-7 g/ml was found in the present study to depress reversibly taste and tactile responses in the glossopharyngeal nerve of the frog. However, TTX at a concentration of 1.6 ~10-7g/ml had no significant effect on the depolarization of a taste cell produced by various chemical stimuli. Either the depolarization or the input resistance produced by test stimuli was scarcely affected by TTX.

11 LOCAL ANESTHETICS ON TASTE CELL 595 DISCUSSION The results of the present experiments clearly demonstrate that local anesthetics such as procaine and lidocaine reduce the amount of depolarization and at the same time decrease the amount of conductance change produced by chemical stimuli in taste cells, thereby raising the threshold concentration, and that the depressant action of local anesthetics is strongest on quinine response and weakest on acid response, while their effects on responses to salts are intermediate. Such differential action of the local anesthetics could also be observed in the glossopharyngeal nerve responses to varying chemicals and is in agreement with the classical observation on the human taste sensations, which are abolished by cocaine in the order, bitter, sweet, saline, and sour (MONCRIEF, 1951). In the present study NaCl and KCl produced depolarizations associated with an increase in the relative conductance in taste cells, and HCl and CaCl2 yielded depolarizations with a smaller amount of conductance increase, while the depolarization produced by quinine was accompanied by a decrease in the conductance. Such a fact is taken to indicate that the mechanism by which various chemicals interact with the receptor membrane at the tip of the taste cell is different according to the chemicals applied. Therefore, the differential anesthetic action of procaine and lidocaine on responses to various taste stimuli observed in the present experiments would be a consequence of the difference in the mechanism of interaction of chemicals with the receptor membrane. Both procaine and lidocaine were found in the present study to produce a depolarization of the membrane by a few mv and to induce gradually an increase in the input resistance in the resting taste cell. The magnitude of the glossopharyngeal nerve response and the amount of depolarization in a taste cell, elicited by procaine, are nearly the same as those produced by quinine of a similar molarity, respectively. In addition, both quinine and procaine reduced the relative conductance of the cell. Therefore, the effects of procaine on the taste cell are similar to those of quinine. Similarity of the action of procaine on taste receptors to that of quinine may be supported by the fact that procaine yields a slightly bitter taste in humans (MERCK INDEX, 1968). As described above, procaine and lidocaine suppress most strongly the stimulatory effect of quinine on taste receptors. This could probably be explained by assuming that both the local anesthetics and quinine affect the same receptive mechanism in the taste cell membrane. KOYAMA and KURIHARA (1972) reported that bitter compounds strongly interact with monolayers of lipids from bovine taste papillae and that there is a good correlation between the penetrating potency of the compounds into the lipid membrane and their taste thresholds. From these results they concluded that the initial event of bitter taste reception is penetration of bitter compounds into the lipid layer of the taste cell membrane. Since procaine is lipid-soluble and considered to penetrate inside the membrane (NARAHASHI et al., 1967) and its action

12 596 N. AKAIKE and M. SATO is similar to that of quinine, it is possible that procaine and lidocaine depress the ionic conductance in the taste receptor membrane by penetrating inside the membrane and dislocating the crystal lattice of the membrane structure. On the other hand, SINGER (1973) reported that local anesthetics such as dibucaine and tetracaine reduce both the surface charge and cation permeability of phosphatidylcholine liposomes. Therefore, there is a possibility that procaine and lidocaine produce a depolarization and conductance change by affecting the taste receptor membrane in a similar manner to that of dibucaine and tetracaine affecting the liposomes. Procaine is a member of a large group of chemically diverse substances which at certain concentrations reversibly depress the activity of the peripheral nerve with little or no effect on the resting membrane potential. Recent electrophysiological experiments using internally perfused squid axons indicate that the increase in permeability to sodium and potassium ions during activity is depressed by procaine and cocaine (TAYLOR, 1959; SHANES et al., 1959). FALK (1961) reported that the action of procaine on the muscle fiber is similar to that on the axon and that it produces a slow fall of the resting potential in muscle fibers, although the change is only of a few mv at low concentrations. The similarity of the effect of procaine on the muscle fiber to that of quinine and quinidine was also pointed out by FALK (1961), because they all reduce the membrane permeability to sodium and potassium ions. The fall in the resting potential can be attributed to a reduction in the resting permeability of the membrane to potassium ions because the resting membrane conductance in the squid axon is reduced significantly by cocaine and procaine (SHANES et al., 1959). In addition, DEGUCHI and NARAHASHI (1971) reported that the peak amplitudes of both sodium and potassium components of the end-plate current during transmitter action are suppressed by procaine. Such effects of local anesthetics on axons, muscle fibers and end-plates suggest that they would also depress the membrane permeability to potassium ions in the taste cell, thereby reducing the membrane potential. Since the effects of procaine to depress the glossopharyngeal nerve response and to increase the input resistance of a taste cell progress slowly with time, it is possible that procaine would affect not only the distal tip of the cell exposed to the surface of the tongue but also the cell body either by penetrating through the intercellular space or by crossing the cell membrane. OZEKI and NOMA (1972) reported that TTX suppresses the glossopharyngeal nerve response to chemical stimulation of the frog tongue but does not affect depolarizations produced by chemical stimuli in taste cells. This was confirmed in the present experiments. As they stated, it is taken to indicate that TTX and possibly other substances could pass from the surface of the frog tongue to the interior of the fungiform papilla through the intercellular space. The inability of TTX to affect responses in taste cells is attributed to its ineffectiveness in depressing the generation of depolarizations and the conductance change produced

13 LOCAL ANESTHETICS ON TASTE CELL 597 by chemicals in the taste cell membrane. This work was supported by grants Nos and from the Ministry of Education of Japan. REFERENCES AKAIKE, N., NOMA, A., and SATO, M.(1973) Frog taste cell response to chemical stimuli. Proc. Japan. Acad., 49: BEIDLER, L.M.(1963) Properties of chemoreceptors of tongue of rat. J. Neurophysiol., 16: DEGUCHI, T. and NARAHASHI, T.(1971) Effects of procaine on ionic conductances of end-plate membranes. J. Pharmacol. Exp. Ther., 176: FALK, G.(1961) Electrical activity of skeletal muscle. Its relation to the active state. In Biophysics of Physiological and Pharmacological Actions, ed. by SCHANES, A.M. Am. Ass. Adv. Sci., Washington, D.C., pp KOYAMA, N. and KURIHARA, K.(1972) Mechanism of bitter taste reception:interaction of bitter compounds with monolayers of lipid from bovine circumvallate papillae. Biochim. Biophys. Acta, 288: KUSANO, K.(1959) The influence of narcotics on the activity of gustatory receptors. Kumamoto Med. J., 12: MERCK INDEX, 8th ed.(1968) Merck&Co., Inc., Rahway. N.J., p.866. MONCRIEF, R.W.(1951) The Chemical Senses, 2nd ed., Leonard Hill Ltd., London, p.159. NARAHASHI, T., ANDERSON, N.C., and MOORE, J.W.(1967) Comparison of tetrodotoxin and procaine in internally perfused squid giant axons. J. Gen. Physiol., 50: OZEKI, M. and NOMA, A.(1972) The actions of tetrodotoxin, procaine, and acetylcholine on gustatory receptors in frog and rat. Jap. J. Physiol., 22: SHANES, A.M., FREYGANG, W.H., GRUNDFEST, H., and AMATINEK, E.(1959) Anesthetic and calcium action in the voltage clamped squid giant axon. J. Gen. Physiol., 42: SINGER, M.A.(1973) Interaction of local anesthetics and salicylate with phospholipid membranes. Can. J. Physiol. Pharmacol., 51: SATO, T.(1969) The response of frog taste cells (Rana nigromaculata and Rana catesbeiana). Experientia, 25: SATO, T.(1972) Multipe sensitivity of single taste cells of the frog tongue to four basic taste stimuli. J. Cell. Physoil., 80: SATO, T. and BEIDLER, L.M.(1973) Relation between receptor potential and resistance change in the frog taste cells. Brain Res., 53: TATEDA, H. and BEIDLER, L.M.(1964) The receptor potential of the taste cell of the rat. J. Gen. Physiol., 47: TAYLOR, R.(1959) Effect of procaine on electrical properties of squid axon membrane. Am. J. Physiol., 196: