Archs oral Bid. Vol. 36, No. 1I, pp. 805413, Printed in Grcst Britain. All rights reserved
1991
Copyright 0
0003~9969/91 $3.00 + 0.00 1991 Pcrgamon Press plc
ELECTROPHYSIOLOGICAL RESPONSES TO NON-EL:ECTROLYTES IN LINGUAL NERVE OF RAT AND IN LINGUAL EPITHELIA OF DOG SIDNEY
A. SIMON and ANN L. S~WMAN
Department of Neurobiology, Duke University Medical Center, Durham, NC 27710, U.S.A. (Accepted 23 May 1991) Sununary-Epithelial and neural mechanisms underlying the trigeminal chemoreception of non-electrolytes were investigated in whole-nerve recordings from lingual nerve and in Ussing-chamber studies of isolated lingual epithelia. The non-electrolytes included menthol, amyl acetate, phenethyl alcohol, toluene, methanol, ethanol, propanol, butanol, hexanol and octanol. They produced different lingual nerve responses: methanol and ethanol only increased ongoing activity; longer-chain alcohols initially increased but then suppressed activity below baseline; phenethyl alcohol and toluene only suppressed activity. Their threshold concentrations for lingual nerve responses, with the exception of menthol, were proportional to the octanol:water partition coefficients of the stimuli. The threshold concentration for menthol was significantly lower than predicted by this coefficient. Calculation of the free energy of transfer from the threshold concentrations for the n-alcohols suggests that they undergo partition into a hydrophobic environment lsuch as is found in lipid bilayers. Lanthanum chloride, which inhibited lingual nerve
responses to hydrophilic compounds, presumably by blocking their diffusion across tight junctions, did not inhibit responses to these non-electrolytes. At high concentrations, hexanol acted as an anaesthetic in that the lingual nerve no longer responded to thermal and chemical stimuli whereas ethanol, which only increased lingual nerve activity, did not inhibit those responses. Epithelial transport, as indicated by the short-circuit current (I,) measured across tongues bathed in symmetrical solutions of Krebs-Henseleit buffer, was reversibly inhibited by ethanol, hexanol, octanol, phenyl ethanol and menthol. The stimulus concentration necessary to inhibit 50% of the kc decreased with increasing octanol:water partition coefficient. Epithelial responses to menthol differed from responses to the n-alcohols, in that the I, initially increased before decreasing, whereas for the n-alcohols and phenethyl alcohol, Isc monotonically decreased. Decreasing transport across lingual epithelia may play a role in modulating lingual nerve responses. These data show that menthol behaves differently than the other compounds tested and are consistent with its interaction with specific receptors. Key words: lingual nerve, tongue, menthol, anaesthetics, transduction, epithelial transport.
INTRODUCTION The
mechanisms by which hydrophilic and hydrophobic molecules Ipenetrate the oral mucosa and affect the intra-epithelial endings of trigeminal nerves are incompletely understood. A study from this laboratory has shown that applying high concentrations of salts to the tongue stimulates lingual nerves (Sostman and Simon, 1991). It was also found that different salts produced distinctly different wholenerve responses and that these were reversibly attenuated by pre-incublation of the tongue with LaCl,. Lanthanum is an established inhibitor of diffusion of electrolytes across tight junctions (Machen, Erlij and Wooding, 1972; Holland, Zampighi and Simon, 1989) and, in rat lingual epithelia, does not elicit lingual nerve responses (Sostman and Simon, 1991). These findings welre interpreted as indicating that small, hydrophilic compounds penetrate into lingual epithelia by diffusing across tight junctions at the interface between the stratum comeum and stratum Abbreviations: I,, short-circuit current; V,, open-circuit potential; I&, transepithelial resistance; KH, KrebsHenseleit.
granulosum where they then interact directly with lingual nerve endings. Hydrophobic molecules, by contrast, are thought to penetrate oral epithelia, as they do other epithelia, by partitioning into and diffusing across plasma membranes (for reviews see Diamond and Wright, 1969; Siegel, 1984; Squier and Johnson, 1975). Measurements of non-electrolyte permeability across rat lingual epithelium (Mistretta, 1971) and the lingual frenulum of the dog (Siegel and Izutsu, 1981) showed that their permeability coefficients were directly proportional to their oil : water partition coefficients. There are few electrophysiological studies of the effects of non-electrolytes, and specifically of alcohols, on the trigeminal nerve. In single-fibre studies of cat lingual nerve, Hellekant (1965) showed that applying ethanol to the tongue increased the activity of cold fibres but did not change the activity in myelinated mechanoreceptors or specific nociceptors. The threshold concentration and the concentration that gave the strongest response were 3.3 and 4.9 M ethanol, respectively. Menthol stimulates cold fibres (but not warm fibres) of cat lingual nerves at micromolar 805
SIDNEY A. SIMON and
806
concentrations (Hensel and Zotterman 195la; Schafer, Braun and Isenberg, 1986). These data, together with structure-activity studies of menthol analogues (Eccles, 1990), suggest that menthol interacts with receptors, possibly calcium channels (Sidell, Verity and Nord, 1990), in cold fibres. At high concentrations, menthol acts as an anaesthetic in that it decreases cold and tactile sensations (Hensel and Zotterman, 195la). Dawson (1962) showed that applying amyl acetate and phenethyl alcohol, two classical trigeminal stimulants, to the cornea increased activity in slowconducting fibres of the long ciliary nerve of the frog; the response thresholds were about 4-10 mM, and as the concentration was increased their response amplitudes first increased and then decreased but remained above that of the spontaneous activity. In studies of ethmoid nerves in rat, Silver and his colleagues (Silver, 1990; Silver et al., 1986; Silver and Moulton, 1982) showed that the threshold concentrations of a series of n-alcohols, amyl acetate, toluene and phenethyl alcohol, decreased with increasing lipid or oil: water partition coefficients. When considering mechanisms of chemical stimulation of trigeminal nerves the epithelium is usually thought to act as a passive barrier that delays the onset of the nerve response (Cain, 1981). Our goals now were two-fold. The first was to determine the pathways by which non-electrolytes diffuse into lingual mucosa to elicit responses from lingual nerves; the second was to determine whether the lingual epithelium modulates lingual nerve responses. MATERIALS
AND METHODS
The salts used were reagent grade. Amiloride, ouabain, menthol, the normal alcohols, phenethyl alcohol, toluene and amyl acetate were obtained from Sigma Chemical Company (St Louis, MO, U.S.A.). These chemicals were used without further purification. Trigeminal recordings
Methods were described by Sostman and Simon (1991) and are recapitulated only briefly. SpragueDawley, female rats (200-400 g) were anaesthetized with pentobarbital sodium (50mg/kg, i.p.; supplemented as necessary) and, after tracheal cannula-
ANN L.
POSTMAN
tion, placed in a non-traumatic headholder. Body temperature was maintained at 3638°C with a heating pad. After removal of overlying tissue, the lingual nerve was exposed for about 5 mm distal to its exit from the foramen ovale, cut proximally, desheathed and placed over a silver-wire electrode. The nerve was then covered with Vaseline and mineral oil. An indifferent electrode was placed in nearby muscle. Neural activity was amplified with a differential amplifier (Grass P-15), monitored with an oscilloscope and stored on tape for later integration (Coulboum S76-01; time constant, 0.5 s) and display on a Gould chart recorder. Stimulus solutions were gravity fed to the rat’s tongue which had been insterted through a latex dam into the base of a Y-shaped glass holder. Flow rate was approx. 2.5 ml/s. To control for mechano- and thermoreceptor activity elicited by flow onset and thermal change at the tongue surface, stimulus sequences involved application of a 50-ml adapting stimulus of distilled water followed immediately by 50 ml of the test solution. Sequences of two successive 50-ml applications of distilled water provided rinsing and also served as additional controls for comparison with the test sequences. These were interspersed at least twice between test stimuli. Solutions were kept at 29.5-3O.O”C unless otherwise stated. The temperature at the tongue surface was monitored with a thermistor (Yellow Springs Instrument Co., U.S.A.) and care was taken to minimize temperature changes between adapting and test stimuli. Responses to 2.5 M NH&l were monitored as a standard for qualitative assessment because the response to this stimulus is very robust and reproducible (Sostman and Simon, 1991). Test stimulus solutions (see Table 1 for formulae) included an n-alcohol series-methanol (at concentrations of 6.2, 12.4, 18.5 and 24.7 M), ethanol (1, 4, 10, 13.8 M), propanol (0.5, 1, 2, 3, 3.6 M), butanol (0.1, 0.95 M), hexanol (1, 3, 10, 30 mM) and octanol (0.01, 0.1, 1, 4 mM; and three benzene ring-containing compounds-menthol (1, 10,100 PM, 1,lO mM), toluene (0.1, 1, 5 mM) and phenethyl alcohol (1, 10, lOOmM), and amyl acetate (1, 10, 20mM). For solutions of menthol, where the concentration was above the saturation concentration in water [2.58 mM (Hensel and Zotterman, 195la)], the menthol solubility was increased to 10 mM by dissolving
Table 1. Lingual nerve response thresholds and partition coefficients for some organic non-electrolytes Compound
Formula
Part. coelf. (octanol: water)
HpC-OH H,C-CH,-OH H,C-(CH,)Z-OH H,C-(CH&3-OH H,C-(CH@-OH HJ-(CH,)7-OH C,H,CH, C,d%o0 C,H,CH(OH)CH, CWH,(CH,),CH, “From Leo et al. (1971). threshold range: highest concentration at lowest concentration at which responses Methanol Ethanol Propanol Butanol Hexanol Octanol Toluene Menthol Phenyl ethanol Amy1 acetate
0.18 0.49 2.19 1.59 107.2 1412.5 631.0 1174.9 22.9 1348.9
Threshold rangeb 3.1-12.4 M 4.Ck15.0 M 054.0 M O.l-1.OM 3.CN0.0 mM 0.14.0 mM 0.2-5.0 mM l.O-lO.OpM 1.~100.0 mM 1.O-20.0 mM
which responses never recorded to always recorded.
807
Lingual responses to non-ekctrolytes it in water-ethanol (to 4M) solutions. At these concentrations ethanol does not elicit responses from rat lingual nerves (Sostman and Simon, 1991). Moreover, the response to 1.0 mM menthol in distilled water was indistinguishable from its response in 4 M ethanol. Lanthanum chloride (LaCl,, 5 mM) and amiloride hydrochloride (0.1 mlvl) were tested for their effects on suprathreshold responses to methanol (18.5 M), ethanol (13.8 M), butanol (0.95 M), octanol (4 mM), amyl acetate (10 mkl), phenethyl alcohol (0.1 M), toluene (5 mM) and menthol (1 mM). In these studies, 60-ml solutions of either 5 mM LaCl, or 0.1 mM amiloride were applied to the tongue for 30 s and the inhibitors were also included (at the same concentrations) with the test solution. Each test stimulus at each concentration studied was tested on at least three animals. Each animal was exposed to only one type of test stimulus. Observations were repeated to ensure response reproducibility although thi,s often necessitated waiting 10-25 min between stimuli to allow baseline and response recovery (especially with methanol, ethanol, toluene, phenethyl alcohol and amyl acetate). Threshold concentrations were defined as being between the highest stimulus concentration at which responses were not recorded in any animal and the lowest concentration at which consistent responses were recorded in all animals (not maximal amplitude, however). Consequently, quantification of the integrated responses wa,s not undertaken routinely because of the variation in response shapes and amplitudes. The effects of hexanol (60 mM) and ethanol (13.8 M) on trigemin,al nerve responses to 2.5 M KC1 and to cooling the tongue to 10°C with distilled water were tested. After obtaining control measurements and then rinsing with water, 50 ml of 60 mM hexanol (13.8 M ethanol) were passed over the dorsal surface of rat tongue. This was immediately followed by flowing 50 ml solutions of 2.5 M KC1 or distilled water at 10°C with 60 mM hexanol(13.8 M ethanol) over the tongue. Epithelial transport studies
Adult dogs of either sex were killed by intravenous injection of sodium pentobarbital(70 mg/kg) and the anterior two-thirds of their tongues were removed. An excised section of epithelium, from which the muscle layer had been dissected away, was placed in an Ussing chamber (area 3.1 cm’) between symmetrical solutions of a modified Krebs-Henseleit (KH) solution at 35 k 1°C. as described by Simon, Labarca and Robb (1989). The composition of the KH solution was: 118 mM NaCl, 6 mM KCl, 5.6 mM D-glucose, 1.2 mM MgSO,, 2.0 mM CaCl,, 25 mM NaHCO,, 1.3 mM NaH,PO,. When equilibrated with 95% Or5% CO1 the pH was 7.4 We chose to investigate dog tongues rather than rat tongues because larger pieces of dog tongues can be used, thus minimizing problems associated with edge damage (Fromter, 1986). Transport across dog and rat tongues is similar in that they both have amilorideand ouabain-inhibitable short-circuit currents (DeSimone et al., 1984; Simon, Robb and Schiffman, 1988). Preliminary experiments with rat tongues gave
similar results to those of dog tongue, only the data were much more variable. Epithelial responses to ethanol, phenethyl alcohol, hexanol, octanol and menthol were obtained by direct addition of concentrated solutions of these stimuli to the mucosal solutions. If the volume to be added was large, then a volume of the mucosal solution was first removed and replaced with one of the same volume having KH buffer and the test compound. Each experiment was repeated at least three times. Measurements of the short-circuit current (I,) and the open-circuit potential (V,) have been described by Simon et al. (1989). The I, is the net current when the external potential is clamped, using an external circuit, to zero volts. Platinum wires are used as current-passing electrodes. I, has a negative sign when cations flow from the mucosal to serosal solutions or when anions flow from the serosal to mucosal solutions. V, is the potential when I, = OA. V, is defined with respect to the mucosal solution. V, is measured with calomel electrodes in saturated KC1 solutions interfaced to l-2% agar bridges containing 0.15 M NaCl. The four electrodes are connected to a voltage-clamp circuit that compensates for the series resistance arising from the electrodes and the KH solutions. The transepithelial resistance (R,) is determined by dividing V, by I,. Traces of the short-circuit current were obtained on a chart recorder. These tracings were then digitized and representative ones are seen in Fig. 6. The coordinates of the time axis signifying 0 min represent the time when the I, approached its steady state after placement in the Ussing chamber. RESULTS
Trigeminal responses The following sections contain a comparison of the response thresholds of the lingual nerve and the hydrophobicity of the stimuli, and descriptions of the different responses elicited by the various stimuli and of the effects of two alcohols on the response to KC1 and to cooling. Response thresholds. Table 1 summarizes the octanol : water partition coefficients of the test stimuli and the range of response threshold established for each stimulus. Figure 1 shows the whole-nerve responses of one animal to menthol of increasing concentration and illustrates how the ranges of the response threshold were estimated. In this animal, 1 mM menthol did not elicit a response while 10 mM produced a small, transient decrease (possibily arising from a small temperature difference between the test solution and the tongue) followed by a consistent increase in activity, thus providing an estimate for the range of response threshold of 1-10pM (Table 1). Figure 2 shows the upper concentration limit of the threshold concentration plotted against the octanol : water partition coefficients of the stimuli. For these 10 nonelectrolytes, the threshold stimulus concentrations (C) correlated inversely with the partition coefficients (P). Excluding the outlying result for menthol, the relationship described by the straight line in Fig. 2 is: log C (mol) = - 0.946 log P + 0.60 (r = -0.96). For the n-alcohol series alone, the equation is: log C (mol) = - 1.04 log P + 0.67
808
SIDNEY A. SIMON
and ANN L.
menthol
&SThfAN
methanol (15.5 MI
’ PJ
. .._. _
.. . ... .... . elhanol (13.8 M)
‘OP
-
-_
propenol-sw&+x. ._ (4 w
b;laM;oI
,ed&_
_ ..-
1nlM
--P----
..,...-
h(2c& 10mM
h
A. . . ..-
._
. . .._...,...
2Ds Fig. 1. Lingual nerve: integrated whole-nerve responses from one animal elicited by the indicated concentrations of menthol. Stimulus application indicated by solid line. Dis-
tilled water application indicated by broken line. Amplifier gain is constant throughout. (r = - 0.97). The free energy of transfer, G, from the bathing solutions to the sites in lingual epithelia is given by G = - RT In C, where RT = 140 J/mol at 30°C (Cevc and Marsh, 1987). A least-squares fit of G versus the number of methylene groups in the n-alcohol series yields a free energy of transfer per CH, of 186 J/mol (r = 0.97). The threshold concentration for menthol is significantly below the concentration predicted by its octanol: water partition coefficient (Fig. 2). Response time course. Figures 3 and 4 show representative responses at suprathreshold concentrations to the n-alcohols and to the four other substances which elicited responses. Small deflections in some of the traces about 20 s before applying the test solutions (indicated by solid bar) represent thermoand mechanoreceptor responses to the onset of the adapting, distilled water stimulus (indicated by
Fig. 3. Lingual nerve: integrated whole nerve responses to the indicated concentrations of n-alcohols. Stimulus application indicated by solid bar. Distilled water application indicated by broken line. lines), as described by Sostman and Simon (1991). In addition, the onset of responses to methanol, ethanol, propanol and, less obviously, menthol, was associated with sharp, transient decreases in the integrated activity. These transient responses were synchronous with transient 2-3°C increases measured on the tongue surface. As similar transient responses occur upon increasing the tongue temperature by l-2°C (Sostman and Simon, 1991), they are presumably a consequence of the heat of dilution produced at the interface between the adapting water stimulus and the molar alcohol solution. The non-electrolyte responses shown in Figs 1, 3 and 4 differ in shape, polarity and time course. The shape of the response also depends on the nonelectrolyte concentration. Methanol and ethanol,
dotted
menthol (1 mM)
amyl
acetate _hlj41c__
(20 mM)
phenethyl (100
Menthol
l
alcohol
mM)
toluene (5 mW
Fig. 2. Lingual nerve threshold concentrations for the indicated stimuli plotted against their octanol:water partition coeIiicients, P. Data taken from Table 1. C,OH = nalcohols; AA = amyl acetate; PEA = phenethyl alcohol.
Fig. 4. Lingual nerve: integrated whole nerve responses to the indicated concentrations of menthol, amyl acetate, phenethyl alcohol and toluene. Stimulus application indicated by solid bar. Distilled water application indicated by broken line.
Lingual responses to nonelectrolytes
Efects of alcohols on responses to KC1 and cooling. The effects of 60 mM hexanol and 13.8 M ethanol on responses to 2.5 M KC1 and to distilled water at 10°C were investigated (Fig. 5). Pre-incubation of the tongue with 60 mM hexanol markedly inhibited responses to 2.5 M KC1 (in 60 M hexanol) and to 60 mM hexanol at 10°C. In contrast, pre-incubation with 13.8 M ethanol slightly diminished the responses to 2.5M KC1 (in 13.8M ethanol) and to 13.8 M ethanol at 10°C.
produced (after a delay) only increases in activity, even at the highest concentrations tested. The compounds containing a benzene ring, phenethyl alcohol and toluene, produced only decreases in activity. The responses to menthol (Fig. l), n-alcohols, and amyl acetate produced responses that depended on the nonelectrolyte concentration. For these stimuli, the responses at low concentrations showed only a slow increase in activity. At higher concentrations the responses were transient in that the activity initially increased, reached a maximum, and then decreased; sometimes to values below its spontaneous activity. For hexanol and octanol at high concentrations, the steady-state activity decreased to below baseline levels (Figs 4 and 5). The responses to most stimuli were reversible, although in many cases, water rinses of several minutes were required. The rise times of lthe responses also varied between stimuli and with concentration. Rise times to maximal response were 60-90 s after application of methanol and ethanol at even the highest concentrations. For the more hydrophobic compounds, the time to the maximal response was a few seconds (Figs 3 and 4). ‘The rise times decreased with increasing concentration, as illustrated for menthol in Fig. 1. Pre-incubation of the tongue with 5 mM LaCl, had no consistent effects on responses to methanol, ethanol, butanol, octanol, amyl acetate, phenethyl alcohol, toluene and menthol. Similarly, 0.1 mM amiloride did not alter the responses to these stimuli.
Isolated lingual epithelia In this section, the responses of isolated canine lingual epithelia to a subset of the non-electrolytes used in the lingual nerve study are described. The open-circuit potential, short-circuit current and transepithelial resistance measured across isolated canine lingual epithelia bathed in symmetrical solutions of KH buffer were 10.4 f 2.7 mV, - 14.7 + 3.8pA/cm2 and 850 &-147 ohm/cm’ (N = 30; mean + SE), respectively. These values are similar to those found previously (DeSimone et al., 1984; Simon and Verbrugge, 1990). The addition of ethanol, hexanol [Fig. 6(A)], octanol, phenethyl alcohol [Fig. 6(B)], and menthol [Fig. 6(C)] reversibly inhibited I, in a dose-dependent manner. The transepithelial resistance remained unchanged upon the addition of these compounds, meaning that the changes in V, paralleled the changes in I,. The short-circuit current was also inhibited 94% by 1.6 mM octanol when the dorsal surface was bathed in 0.3 M NaCl, 2 mM HEPES, pH 7.4 (not shown). The addition of
KCI
I control:
KCI
-
hexano’-KC’ ethanol-KC2
A -
-
104
contrcll:
1OG
hexanol-10oC
Ii20
k
809
Hz0
-
HpO
Fig. 5. Lingual nerve: integrated whole-nerve responses. Upper panel: lingual nerve was stimulated by 2.5 M KCl. After extensive washing with distilled water, a solution of 60 mM hexanol was flowed over the tongue, followed by a solution of 60 m&l hexanol and 2.5 M KCI. After extensive washing the protocol was repeated by flowing a solution of 13.8 M ethanol over the tongue in the absence and presence of 2.5 M KCl. Stimulus application indicated by solid bars. Lower panel: the same protocol as above except that the stimuhts is obtained by flowing distilled water at 10°C over the tongue. Stimulus application indicated by solid bars.
810
SIDNEYA. SIMONand ANN L. SOWMU
80r IOQ-
\
60 00 f
.
EW
E g
40-
=
’
&? zoOI
20
I
I
I
40
60
80
Time (min)
-0
D
-6
log
KH J
20 I10 -
KH Oo
I
I
I
20
40
60
Time (min)
OO
-2
0
2
(M)
Fig. 7. Isolated canine lingual epithelia. Percent inhibition of the short circuit current (Isc) versus the stimuli concentration. Tongues originally bathed in symmetrical solutions of Krebs-Henseleit buffer. 100% inhibition means that Isc=OpA.
(8) so-
-4
(concentration)
I
1
I
I
50
100
150
200
Time
menthol to the solution placed on the dorsum produced a distinctly different response from ethanol (there were six experiments with ethanol-in two the I, initially increased before decreasing whereas in four the I, decreased upon its addition), hexanol, octanol, phenethyl alcohol in that the I, transiently increased before decreasing to its steady-state [Fig. 6(C)]. The magnitude of both the increasing and decreasing phases of the I, increased with menthol concentration. The percentage inhibition of the steady-state I, for these five stimuli plotted against their concentration is shown in Fig. 7. For a given
bin)
Fig. 6. Isolated canine lingual epithelia. Tongues were bathed in symmetrical solutions of Krebs-Henseleit buffer (KH; arrows) and the short circuit current (1s~) was measured. At the times indicated by the arrows; (A) hexanol, (B) phenethyl alcohol (PEA), (C) menthol were added to the mucosal solution. The small increase in kc seen after
the decrease produced by hexanol is caused by its evaporation. The reversibility of the responses to hexanol and phenethyl alcohol is indicated by KH near the breaks in the traces. The break in the menthol response (C) occurred when the mucosal bathing solution was completely exchanged for one having KH and 1 mM menthol. Chamber area = 3.1 cm*.
-I
-1
0 log
1 2 P 0ctanol:water
3
4
Fig. 8. Plots of concentration necessary to inhibit 50% of short-circuit current across isolated canine lingual epithelia (open squares) and lingual nerve threshold concentrations (solid squares) versus stimuli’s octanol:water partition coefficient. Ethanol (CsOH), phenethyl alcohol (PEA), hexanol (CsOH), octanol (CsOH). The solid lines are least-square fits to the data. Menthol is included in the fit for the epithelial responses (r = -0.94) but excluded from the lingual nerve responses (r = -0.97).
Lingual responses to non-electrolytes
811
stimulus, the concentration necessary to inhibit I, by 50% decreased with increasing octanol: water partition coefficient (Fig. 8).
1974; Sostman and Simon, 1991), and the responses to menthol (Hensel and Zotterman, 1951a and Fig. 1). Thus, it is likely that some of the spontaneous activity of lingual nerve arises from cold fibres.
DISCUSSION
The interaction of anaesthetics with trigeminal jibres Many of the stimuli tested are classified as anaesthetics, and as such, inhibit the generation of action potentials when a critical concentration in nerve membranes is attained (Over-ton, 1902; Seeman, 1972). If lingual nerves interact with anaesthetics as do most nerves (e.g. squid axons, sciatic nerves), then the addition of anaesthetics should decrease the level of activity. Such behaviour is observed upon the application of phenethyl alcohol and toluene to the dorsal surface of the tongue (Fig. 4) and hence it is likely that these compounds anaesthetize lingual nerves. The pattern of lingual nerve activity obtained with phenethyl alcohol and toluene differs from responses to amyl acetate, menthol and octanol even though these compounds have larger octanol-water partition coefficients than phenethyl alcohol and toluene (Leo, Hansch and Elkins, 1971; Table 1). In this regard, whole-nerve recordings from the ethmoid and long ciliary nerves show that phenethyl alcohol (and toluene in the ethmoid nerve) produce transient increases in activity (Dawson, 1962; Silver, 1990). Why lingual nerves respond differently from other trigeminal branches to these stimuli is unknown. Also, threshold concentrations are higher in rat lingual nerves than they are in the ethmoid and long ciliary branches. One reason may be that the lingual epithelium is stratified and thicker than the cornea1 or olfactory mucosa and thus, for a given time after application of a stimulus, a smaller concentration will reach either the epithelial cells or the nerves. For all the other stimuli tested, the response profiles obtained in lingual nerve recordings are similar to those obtained in ethmoid and long ciliary nerves. Other non-polar compounds that decrease lingual nerve activity below its spontaneous level act as anaesthetics. Thus, decreasing lingual nerve activity to below baseline levels with 60mM hexanol rendered the nerve unresponsive to 2.5 M KC1 and to water at 10°C whereas 13.8 M ethanol, which only increased lingual nerve activity, did not inhibit the responses to these stimuli. Application of most non-polar stimuli to the dorsal surface of the tongue initially increases lingual nerve activity (Hellekant, 1965; Silver, 1987; Silver, 1990). In this regard, at least at low concentrations, these non-electrolytes do not behave as classic anaesthetics. This means that if these compounds interact directly with lingual nerves they are not only inhibiting sodium and potassium channels. The initial increase in lingual nerve activity could arise from the direct interaction of the stimuli with epithelial cells or with nerve terminals. In recordings from lingual fibres that terminate in epithelia it is difficult to distinguish between these two possibilities. The non-electrolyte concentrations required to alter the short-circuit current are similar to those required to activate or inhibit lingual nerves (Fig. 8) and therefore, using only this criterion, one cannot distinguish whether epithelial cells modulate lingual nerve responses.
Hydrophobic puthwu.vs into lingual epithelia Our findings suggest that hydrophobic molecules applied to the dorsal surface of lingual epithelia elicit responses from lingual nerves by partitioning into and diffusing across plasma membranes of epithelial and nerve cells. This conclusion is based on the following. (1) The correlation between threshold concentrations for lingual nerve responses and octanol:water partition coefficients (Fig. 2). (2) The magnitude of the free energy of transfer per methylene group of 186 J/m.01 implies that these compounds undergo partition in sites similar to those found in the hydrophobic regions of plasma membranes or lipid bilayers (Cevc and hdarsh, 1987). (3) That LaCI,, an inhibitor of lingual nerve responses to electrolytes (Sostman and Simon, 1991), did not inhibit responses to non-polar stimuli. In addition, the permeability of non-electrolytes across rat lingual epithelia is directly proportional to their oil : water partition coefficients (Mistretta, 1971) as is the inhibition of the short-circuit current measumd across canine lingual epithelia (Fig. 8). Permeability measurements across skin have shown that hydrophobic compounds diffuse by first partitioning into lamellar bodies in the stratum comeum and then into the plasma membranes of epithelial cells (Williams and Elias, 1987). As the permeability of non-electrolytes through both skin and .lingual epithelia is proportional to their oil :water . partition coefficients, and as both are classified as stratified squamous epithelia, we assume these two types of epithelia have similar pathways for nonpolar stimuli. In lingual epithelia these pathways include the lamellar bodies in the stratum comeum (Williams and Elias, 1987), and the plasma membranes of epithelial, taste and nerve cells. Partitioning of non-polar compounds into lamellar bodies should not affect the activity of trigeminal fibres as these lipoprotein complexes are in the extracellular space and do not contain ion-transporting proteins (Williams and Elias, 1987). Partitioning of hydrophobic compounds into taste cells and/or the special sensory fibres that form synapses with them should have little effect on responses recorded from lingual nerves because the number of trigeminal fibres in taste buds is very small compared with the total number of lingual nerves (Biedenbach, Beuerman and Brown, 1975; Kinnman and Aldskogius, 1988; Yamasaki, Kubota and Tohyama, 1985). Consequently, non-polar stimuli may influence trigeminal fibre activity either by altering transport across epithelial cells and/or by directly interacting with trigeminal fibres. Spontaneous activity of lingual trigeminal jbres The responses of ,whole lingual nerve are similar to those obtained from cold fibres in regard to: decreasing temperature (Poulos and Lende, 1970; Hensel and Zotterman, 1951b; Sostman and Simon, 1991 and Fig. 5), the additio:n of CaCl, (Hensel and Schafer,
SWNEYA. SIMONamd ANN L.
812
Let us first consider the responses of lingual epithelia to compounds that monotonically decrease the short-circuit current (e.g. hexanol). The current measured across tongues bathed in symmetrical solutions of Krebs-Henseleit buffer arises from a net influx of Na + and a net efflux of Cl - (DeSimone et al., 1984; Mierson et al., 1985). This current is completely inhibited by ouabain (DeSimone et al., 1984; Mierson et al., 1985) and thus is generated by Na,K-ATPase present in taste and epithelial cells. In hyperosmotic solutions of NaCl, the current can almost be entirely accounted for by a net Na + influx (DeSimone et al., 1984). Thus, non-polar compounds, such as hexanol and octanol, inhbit transport proteins associated with sodium influx. The one protein involved in sodium transport and present in both taste and epithelial cells is the Na,K-ATPase (Simon, Holland and Zampighi, 1991). Given the relatively small area of the anterior two-thirds of the tongue occupied by taste cells [
1991
Copyright 0
0003~9969/91 $3.00 + 0.00 1991 Pcrgamon Press plc
ELECTROPHYSIOLOGICAL RESPONSES TO NON-EL:ECTROLYTES IN LINGUAL NERVE OF RAT AND IN LINGUAL EPITHELIA OF DOG SIDNEY
A. SIMON and ANN L. S~WMAN
Department of Neurobiology, Duke University Medical Center, Durham, NC 27710, U.S.A. (Accepted 23 May 1991) Sununary-Epithelial and neural mechanisms underlying the trigeminal chemoreception of non-electrolytes were investigated in whole-nerve recordings from lingual nerve and in Ussing-chamber studies of isolated lingual epithelia. The non-electrolytes included menthol, amyl acetate, phenethyl alcohol, toluene, methanol, ethanol, propanol, butanol, hexanol and octanol. They produced different lingual nerve responses: methanol and ethanol only increased ongoing activity; longer-chain alcohols initially increased but then suppressed activity below baseline; phenethyl alcohol and toluene only suppressed activity. Their threshold concentrations for lingual nerve responses, with the exception of menthol, were proportional to the octanol:water partition coefficients of the stimuli. The threshold concentration for menthol was significantly lower than predicted by this coefficient. Calculation of the free energy of transfer from the threshold concentrations for the n-alcohols suggests that they undergo partition into a hydrophobic environment lsuch as is found in lipid bilayers. Lanthanum chloride, which inhibited lingual nerve
responses to hydrophilic compounds, presumably by blocking their diffusion across tight junctions, did not inhibit responses to these non-electrolytes. At high concentrations, hexanol acted as an anaesthetic in that the lingual nerve no longer responded to thermal and chemical stimuli whereas ethanol, which only increased lingual nerve activity, did not inhibit those responses. Epithelial transport, as indicated by the short-circuit current (I,) measured across tongues bathed in symmetrical solutions of Krebs-Henseleit buffer, was reversibly inhibited by ethanol, hexanol, octanol, phenyl ethanol and menthol. The stimulus concentration necessary to inhibit 50% of the kc decreased with increasing octanol:water partition coefficient. Epithelial responses to menthol differed from responses to the n-alcohols, in that the I, initially increased before decreasing, whereas for the n-alcohols and phenethyl alcohol, Isc monotonically decreased. Decreasing transport across lingual epithelia may play a role in modulating lingual nerve responses. These data show that menthol behaves differently than the other compounds tested and are consistent with its interaction with specific receptors. Key words: lingual nerve, tongue, menthol, anaesthetics, transduction, epithelial transport.
INTRODUCTION The
mechanisms by which hydrophilic and hydrophobic molecules Ipenetrate the oral mucosa and affect the intra-epithelial endings of trigeminal nerves are incompletely understood. A study from this laboratory has shown that applying high concentrations of salts to the tongue stimulates lingual nerves (Sostman and Simon, 1991). It was also found that different salts produced distinctly different wholenerve responses and that these were reversibly attenuated by pre-incublation of the tongue with LaCl,. Lanthanum is an established inhibitor of diffusion of electrolytes across tight junctions (Machen, Erlij and Wooding, 1972; Holland, Zampighi and Simon, 1989) and, in rat lingual epithelia, does not elicit lingual nerve responses (Sostman and Simon, 1991). These findings welre interpreted as indicating that small, hydrophilic compounds penetrate into lingual epithelia by diffusing across tight junctions at the interface between the stratum comeum and stratum Abbreviations: I,, short-circuit current; V,, open-circuit potential; I&, transepithelial resistance; KH, KrebsHenseleit.
granulosum where they then interact directly with lingual nerve endings. Hydrophobic molecules, by contrast, are thought to penetrate oral epithelia, as they do other epithelia, by partitioning into and diffusing across plasma membranes (for reviews see Diamond and Wright, 1969; Siegel, 1984; Squier and Johnson, 1975). Measurements of non-electrolyte permeability across rat lingual epithelium (Mistretta, 1971) and the lingual frenulum of the dog (Siegel and Izutsu, 1981) showed that their permeability coefficients were directly proportional to their oil : water partition coefficients. There are few electrophysiological studies of the effects of non-electrolytes, and specifically of alcohols, on the trigeminal nerve. In single-fibre studies of cat lingual nerve, Hellekant (1965) showed that applying ethanol to the tongue increased the activity of cold fibres but did not change the activity in myelinated mechanoreceptors or specific nociceptors. The threshold concentration and the concentration that gave the strongest response were 3.3 and 4.9 M ethanol, respectively. Menthol stimulates cold fibres (but not warm fibres) of cat lingual nerves at micromolar 805
SIDNEY A. SIMON and
806
concentrations (Hensel and Zotterman 195la; Schafer, Braun and Isenberg, 1986). These data, together with structure-activity studies of menthol analogues (Eccles, 1990), suggest that menthol interacts with receptors, possibly calcium channels (Sidell, Verity and Nord, 1990), in cold fibres. At high concentrations, menthol acts as an anaesthetic in that it decreases cold and tactile sensations (Hensel and Zotterman, 195la). Dawson (1962) showed that applying amyl acetate and phenethyl alcohol, two classical trigeminal stimulants, to the cornea increased activity in slowconducting fibres of the long ciliary nerve of the frog; the response thresholds were about 4-10 mM, and as the concentration was increased their response amplitudes first increased and then decreased but remained above that of the spontaneous activity. In studies of ethmoid nerves in rat, Silver and his colleagues (Silver, 1990; Silver et al., 1986; Silver and Moulton, 1982) showed that the threshold concentrations of a series of n-alcohols, amyl acetate, toluene and phenethyl alcohol, decreased with increasing lipid or oil: water partition coefficients. When considering mechanisms of chemical stimulation of trigeminal nerves the epithelium is usually thought to act as a passive barrier that delays the onset of the nerve response (Cain, 1981). Our goals now were two-fold. The first was to determine the pathways by which non-electrolytes diffuse into lingual mucosa to elicit responses from lingual nerves; the second was to determine whether the lingual epithelium modulates lingual nerve responses. MATERIALS
AND METHODS
The salts used were reagent grade. Amiloride, ouabain, menthol, the normal alcohols, phenethyl alcohol, toluene and amyl acetate were obtained from Sigma Chemical Company (St Louis, MO, U.S.A.). These chemicals were used without further purification. Trigeminal recordings
Methods were described by Sostman and Simon (1991) and are recapitulated only briefly. SpragueDawley, female rats (200-400 g) were anaesthetized with pentobarbital sodium (50mg/kg, i.p.; supplemented as necessary) and, after tracheal cannula-
ANN L.
POSTMAN
tion, placed in a non-traumatic headholder. Body temperature was maintained at 3638°C with a heating pad. After removal of overlying tissue, the lingual nerve was exposed for about 5 mm distal to its exit from the foramen ovale, cut proximally, desheathed and placed over a silver-wire electrode. The nerve was then covered with Vaseline and mineral oil. An indifferent electrode was placed in nearby muscle. Neural activity was amplified with a differential amplifier (Grass P-15), monitored with an oscilloscope and stored on tape for later integration (Coulboum S76-01; time constant, 0.5 s) and display on a Gould chart recorder. Stimulus solutions were gravity fed to the rat’s tongue which had been insterted through a latex dam into the base of a Y-shaped glass holder. Flow rate was approx. 2.5 ml/s. To control for mechano- and thermoreceptor activity elicited by flow onset and thermal change at the tongue surface, stimulus sequences involved application of a 50-ml adapting stimulus of distilled water followed immediately by 50 ml of the test solution. Sequences of two successive 50-ml applications of distilled water provided rinsing and also served as additional controls for comparison with the test sequences. These were interspersed at least twice between test stimuli. Solutions were kept at 29.5-3O.O”C unless otherwise stated. The temperature at the tongue surface was monitored with a thermistor (Yellow Springs Instrument Co., U.S.A.) and care was taken to minimize temperature changes between adapting and test stimuli. Responses to 2.5 M NH&l were monitored as a standard for qualitative assessment because the response to this stimulus is very robust and reproducible (Sostman and Simon, 1991). Test stimulus solutions (see Table 1 for formulae) included an n-alcohol series-methanol (at concentrations of 6.2, 12.4, 18.5 and 24.7 M), ethanol (1, 4, 10, 13.8 M), propanol (0.5, 1, 2, 3, 3.6 M), butanol (0.1, 0.95 M), hexanol (1, 3, 10, 30 mM) and octanol (0.01, 0.1, 1, 4 mM; and three benzene ring-containing compounds-menthol (1, 10,100 PM, 1,lO mM), toluene (0.1, 1, 5 mM) and phenethyl alcohol (1, 10, lOOmM), and amyl acetate (1, 10, 20mM). For solutions of menthol, where the concentration was above the saturation concentration in water [2.58 mM (Hensel and Zotterman, 195la)], the menthol solubility was increased to 10 mM by dissolving
Table 1. Lingual nerve response thresholds and partition coefficients for some organic non-electrolytes Compound
Formula
Part. coelf. (octanol: water)
HpC-OH H,C-CH,-OH H,C-(CH,)Z-OH H,C-(CH&3-OH H,C-(CH@-OH HJ-(CH,)7-OH C,H,CH, C,d%o0 C,H,CH(OH)CH, CWH,(CH,),CH, “From Leo et al. (1971). threshold range: highest concentration at lowest concentration at which responses Methanol Ethanol Propanol Butanol Hexanol Octanol Toluene Menthol Phenyl ethanol Amy1 acetate
0.18 0.49 2.19 1.59 107.2 1412.5 631.0 1174.9 22.9 1348.9
Threshold rangeb 3.1-12.4 M 4.Ck15.0 M 054.0 M O.l-1.OM 3.CN0.0 mM 0.14.0 mM 0.2-5.0 mM l.O-lO.OpM 1.~100.0 mM 1.O-20.0 mM
which responses never recorded to always recorded.
807
Lingual responses to non-ekctrolytes it in water-ethanol (to 4M) solutions. At these concentrations ethanol does not elicit responses from rat lingual nerves (Sostman and Simon, 1991). Moreover, the response to 1.0 mM menthol in distilled water was indistinguishable from its response in 4 M ethanol. Lanthanum chloride (LaCl,, 5 mM) and amiloride hydrochloride (0.1 mlvl) were tested for their effects on suprathreshold responses to methanol (18.5 M), ethanol (13.8 M), butanol (0.95 M), octanol (4 mM), amyl acetate (10 mkl), phenethyl alcohol (0.1 M), toluene (5 mM) and menthol (1 mM). In these studies, 60-ml solutions of either 5 mM LaCl, or 0.1 mM amiloride were applied to the tongue for 30 s and the inhibitors were also included (at the same concentrations) with the test solution. Each test stimulus at each concentration studied was tested on at least three animals. Each animal was exposed to only one type of test stimulus. Observations were repeated to ensure response reproducibility although thi,s often necessitated waiting 10-25 min between stimuli to allow baseline and response recovery (especially with methanol, ethanol, toluene, phenethyl alcohol and amyl acetate). Threshold concentrations were defined as being between the highest stimulus concentration at which responses were not recorded in any animal and the lowest concentration at which consistent responses were recorded in all animals (not maximal amplitude, however). Consequently, quantification of the integrated responses wa,s not undertaken routinely because of the variation in response shapes and amplitudes. The effects of hexanol (60 mM) and ethanol (13.8 M) on trigemin,al nerve responses to 2.5 M KC1 and to cooling the tongue to 10°C with distilled water were tested. After obtaining control measurements and then rinsing with water, 50 ml of 60 mM hexanol (13.8 M ethanol) were passed over the dorsal surface of rat tongue. This was immediately followed by flowing 50 ml solutions of 2.5 M KC1 or distilled water at 10°C with 60 mM hexanol(13.8 M ethanol) over the tongue. Epithelial transport studies
Adult dogs of either sex were killed by intravenous injection of sodium pentobarbital(70 mg/kg) and the anterior two-thirds of their tongues were removed. An excised section of epithelium, from which the muscle layer had been dissected away, was placed in an Ussing chamber (area 3.1 cm’) between symmetrical solutions of a modified Krebs-Henseleit (KH) solution at 35 k 1°C. as described by Simon, Labarca and Robb (1989). The composition of the KH solution was: 118 mM NaCl, 6 mM KCl, 5.6 mM D-glucose, 1.2 mM MgSO,, 2.0 mM CaCl,, 25 mM NaHCO,, 1.3 mM NaH,PO,. When equilibrated with 95% Or5% CO1 the pH was 7.4 We chose to investigate dog tongues rather than rat tongues because larger pieces of dog tongues can be used, thus minimizing problems associated with edge damage (Fromter, 1986). Transport across dog and rat tongues is similar in that they both have amilorideand ouabain-inhibitable short-circuit currents (DeSimone et al., 1984; Simon, Robb and Schiffman, 1988). Preliminary experiments with rat tongues gave
similar results to those of dog tongue, only the data were much more variable. Epithelial responses to ethanol, phenethyl alcohol, hexanol, octanol and menthol were obtained by direct addition of concentrated solutions of these stimuli to the mucosal solutions. If the volume to be added was large, then a volume of the mucosal solution was first removed and replaced with one of the same volume having KH buffer and the test compound. Each experiment was repeated at least three times. Measurements of the short-circuit current (I,) and the open-circuit potential (V,) have been described by Simon et al. (1989). The I, is the net current when the external potential is clamped, using an external circuit, to zero volts. Platinum wires are used as current-passing electrodes. I, has a negative sign when cations flow from the mucosal to serosal solutions or when anions flow from the serosal to mucosal solutions. V, is the potential when I, = OA. V, is defined with respect to the mucosal solution. V, is measured with calomel electrodes in saturated KC1 solutions interfaced to l-2% agar bridges containing 0.15 M NaCl. The four electrodes are connected to a voltage-clamp circuit that compensates for the series resistance arising from the electrodes and the KH solutions. The transepithelial resistance (R,) is determined by dividing V, by I,. Traces of the short-circuit current were obtained on a chart recorder. These tracings were then digitized and representative ones are seen in Fig. 6. The coordinates of the time axis signifying 0 min represent the time when the I, approached its steady state after placement in the Ussing chamber. RESULTS
Trigeminal responses The following sections contain a comparison of the response thresholds of the lingual nerve and the hydrophobicity of the stimuli, and descriptions of the different responses elicited by the various stimuli and of the effects of two alcohols on the response to KC1 and to cooling. Response thresholds. Table 1 summarizes the octanol : water partition coefficients of the test stimuli and the range of response threshold established for each stimulus. Figure 1 shows the whole-nerve responses of one animal to menthol of increasing concentration and illustrates how the ranges of the response threshold were estimated. In this animal, 1 mM menthol did not elicit a response while 10 mM produced a small, transient decrease (possibily arising from a small temperature difference between the test solution and the tongue) followed by a consistent increase in activity, thus providing an estimate for the range of response threshold of 1-10pM (Table 1). Figure 2 shows the upper concentration limit of the threshold concentration plotted against the octanol : water partition coefficients of the stimuli. For these 10 nonelectrolytes, the threshold stimulus concentrations (C) correlated inversely with the partition coefficients (P). Excluding the outlying result for menthol, the relationship described by the straight line in Fig. 2 is: log C (mol) = - 0.946 log P + 0.60 (r = -0.96). For the n-alcohol series alone, the equation is: log C (mol) = - 1.04 log P + 0.67
808
SIDNEY A. SIMON
and ANN L.
menthol
&SThfAN
methanol (15.5 MI
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--P----
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2Ds Fig. 1. Lingual nerve: integrated whole-nerve responses from one animal elicited by the indicated concentrations of menthol. Stimulus application indicated by solid line. Dis-
tilled water application indicated by broken line. Amplifier gain is constant throughout. (r = - 0.97). The free energy of transfer, G, from the bathing solutions to the sites in lingual epithelia is given by G = - RT In C, where RT = 140 J/mol at 30°C (Cevc and Marsh, 1987). A least-squares fit of G versus the number of methylene groups in the n-alcohol series yields a free energy of transfer per CH, of 186 J/mol (r = 0.97). The threshold concentration for menthol is significantly below the concentration predicted by its octanol: water partition coefficient (Fig. 2). Response time course. Figures 3 and 4 show representative responses at suprathreshold concentrations to the n-alcohols and to the four other substances which elicited responses. Small deflections in some of the traces about 20 s before applying the test solutions (indicated by solid bar) represent thermoand mechanoreceptor responses to the onset of the adapting, distilled water stimulus (indicated by
Fig. 3. Lingual nerve: integrated whole nerve responses to the indicated concentrations of n-alcohols. Stimulus application indicated by solid bar. Distilled water application indicated by broken line. lines), as described by Sostman and Simon (1991). In addition, the onset of responses to methanol, ethanol, propanol and, less obviously, menthol, was associated with sharp, transient decreases in the integrated activity. These transient responses were synchronous with transient 2-3°C increases measured on the tongue surface. As similar transient responses occur upon increasing the tongue temperature by l-2°C (Sostman and Simon, 1991), they are presumably a consequence of the heat of dilution produced at the interface between the adapting water stimulus and the molar alcohol solution. The non-electrolyte responses shown in Figs 1, 3 and 4 differ in shape, polarity and time course. The shape of the response also depends on the nonelectrolyte concentration. Methanol and ethanol,
dotted
menthol (1 mM)
amyl
acetate _hlj41c__
(20 mM)
phenethyl (100
Menthol
l
alcohol
mM)
toluene (5 mW
Fig. 2. Lingual nerve threshold concentrations for the indicated stimuli plotted against their octanol:water partition coeIiicients, P. Data taken from Table 1. C,OH = nalcohols; AA = amyl acetate; PEA = phenethyl alcohol.
Fig. 4. Lingual nerve: integrated whole nerve responses to the indicated concentrations of menthol, amyl acetate, phenethyl alcohol and toluene. Stimulus application indicated by solid bar. Distilled water application indicated by broken line.
Lingual responses to nonelectrolytes
Efects of alcohols on responses to KC1 and cooling. The effects of 60 mM hexanol and 13.8 M ethanol on responses to 2.5 M KC1 and to distilled water at 10°C were investigated (Fig. 5). Pre-incubation of the tongue with 60 mM hexanol markedly inhibited responses to 2.5 M KC1 (in 60 M hexanol) and to 60 mM hexanol at 10°C. In contrast, pre-incubation with 13.8 M ethanol slightly diminished the responses to 2.5M KC1 (in 13.8M ethanol) and to 13.8 M ethanol at 10°C.
produced (after a delay) only increases in activity, even at the highest concentrations tested. The compounds containing a benzene ring, phenethyl alcohol and toluene, produced only decreases in activity. The responses to menthol (Fig. l), n-alcohols, and amyl acetate produced responses that depended on the nonelectrolyte concentration. For these stimuli, the responses at low concentrations showed only a slow increase in activity. At higher concentrations the responses were transient in that the activity initially increased, reached a maximum, and then decreased; sometimes to values below its spontaneous activity. For hexanol and octanol at high concentrations, the steady-state activity decreased to below baseline levels (Figs 4 and 5). The responses to most stimuli were reversible, although in many cases, water rinses of several minutes were required. The rise times of lthe responses also varied between stimuli and with concentration. Rise times to maximal response were 60-90 s after application of methanol and ethanol at even the highest concentrations. For the more hydrophobic compounds, the time to the maximal response was a few seconds (Figs 3 and 4). ‘The rise times decreased with increasing concentration, as illustrated for menthol in Fig. 1. Pre-incubation of the tongue with 5 mM LaCl, had no consistent effects on responses to methanol, ethanol, butanol, octanol, amyl acetate, phenethyl alcohol, toluene and menthol. Similarly, 0.1 mM amiloride did not alter the responses to these stimuli.
Isolated lingual epithelia In this section, the responses of isolated canine lingual epithelia to a subset of the non-electrolytes used in the lingual nerve study are described. The open-circuit potential, short-circuit current and transepithelial resistance measured across isolated canine lingual epithelia bathed in symmetrical solutions of KH buffer were 10.4 f 2.7 mV, - 14.7 + 3.8pA/cm2 and 850 &-147 ohm/cm’ (N = 30; mean + SE), respectively. These values are similar to those found previously (DeSimone et al., 1984; Simon and Verbrugge, 1990). The addition of ethanol, hexanol [Fig. 6(A)], octanol, phenethyl alcohol [Fig. 6(B)], and menthol [Fig. 6(C)] reversibly inhibited I, in a dose-dependent manner. The transepithelial resistance remained unchanged upon the addition of these compounds, meaning that the changes in V, paralleled the changes in I,. The short-circuit current was also inhibited 94% by 1.6 mM octanol when the dorsal surface was bathed in 0.3 M NaCl, 2 mM HEPES, pH 7.4 (not shown). The addition of
KCI
I control:
KCI
-
hexano’-KC’ ethanol-KC2
A -
-
104
contrcll:
1OG
hexanol-10oC
Ii20
k
809
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-
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Fig. 5. Lingual nerve: integrated whole-nerve responses. Upper panel: lingual nerve was stimulated by 2.5 M KCl. After extensive washing with distilled water, a solution of 60 mM hexanol was flowed over the tongue, followed by a solution of 60 m&l hexanol and 2.5 M KCI. After extensive washing the protocol was repeated by flowing a solution of 13.8 M ethanol over the tongue in the absence and presence of 2.5 M KCl. Stimulus application indicated by solid bars. Lower panel: the same protocol as above except that the stimuhts is obtained by flowing distilled water at 10°C over the tongue. Stimulus application indicated by solid bars.
810
SIDNEYA. SIMONand ANN L. SOWMU
80r IOQ-
\
60 00 f
.
EW
E g
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=
’
&? zoOI
20
I
I
I
40
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KH Oo
I
I
I
20
40
60
Time (min)
OO
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(M)
Fig. 7. Isolated canine lingual epithelia. Percent inhibition of the short circuit current (Isc) versus the stimuli concentration. Tongues originally bathed in symmetrical solutions of Krebs-Henseleit buffer. 100% inhibition means that Isc=OpA.
(8) so-
-4
(concentration)
I
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I
I
50
100
150
200
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menthol to the solution placed on the dorsum produced a distinctly different response from ethanol (there were six experiments with ethanol-in two the I, initially increased before decreasing whereas in four the I, decreased upon its addition), hexanol, octanol, phenethyl alcohol in that the I, transiently increased before decreasing to its steady-state [Fig. 6(C)]. The magnitude of both the increasing and decreasing phases of the I, increased with menthol concentration. The percentage inhibition of the steady-state I, for these five stimuli plotted against their concentration is shown in Fig. 7. For a given
bin)
Fig. 6. Isolated canine lingual epithelia. Tongues were bathed in symmetrical solutions of Krebs-Henseleit buffer (KH; arrows) and the short circuit current (1s~) was measured. At the times indicated by the arrows; (A) hexanol, (B) phenethyl alcohol (PEA), (C) menthol were added to the mucosal solution. The small increase in kc seen after
the decrease produced by hexanol is caused by its evaporation. The reversibility of the responses to hexanol and phenethyl alcohol is indicated by KH near the breaks in the traces. The break in the menthol response (C) occurred when the mucosal bathing solution was completely exchanged for one having KH and 1 mM menthol. Chamber area = 3.1 cm*.
-I
-1
0 log
1 2 P 0ctanol:water
3
4
Fig. 8. Plots of concentration necessary to inhibit 50% of short-circuit current across isolated canine lingual epithelia (open squares) and lingual nerve threshold concentrations (solid squares) versus stimuli’s octanol:water partition coefficient. Ethanol (CsOH), phenethyl alcohol (PEA), hexanol (CsOH), octanol (CsOH). The solid lines are least-square fits to the data. Menthol is included in the fit for the epithelial responses (r = -0.94) but excluded from the lingual nerve responses (r = -0.97).
Lingual responses to non-electrolytes
811
stimulus, the concentration necessary to inhibit I, by 50% decreased with increasing octanol: water partition coefficient (Fig. 8).
1974; Sostman and Simon, 1991), and the responses to menthol (Hensel and Zotterman, 1951a and Fig. 1). Thus, it is likely that some of the spontaneous activity of lingual nerve arises from cold fibres.
DISCUSSION
The interaction of anaesthetics with trigeminal jibres Many of the stimuli tested are classified as anaesthetics, and as such, inhibit the generation of action potentials when a critical concentration in nerve membranes is attained (Over-ton, 1902; Seeman, 1972). If lingual nerves interact with anaesthetics as do most nerves (e.g. squid axons, sciatic nerves), then the addition of anaesthetics should decrease the level of activity. Such behaviour is observed upon the application of phenethyl alcohol and toluene to the dorsal surface of the tongue (Fig. 4) and hence it is likely that these compounds anaesthetize lingual nerves. The pattern of lingual nerve activity obtained with phenethyl alcohol and toluene differs from responses to amyl acetate, menthol and octanol even though these compounds have larger octanol-water partition coefficients than phenethyl alcohol and toluene (Leo, Hansch and Elkins, 1971; Table 1). In this regard, whole-nerve recordings from the ethmoid and long ciliary nerves show that phenethyl alcohol (and toluene in the ethmoid nerve) produce transient increases in activity (Dawson, 1962; Silver, 1990). Why lingual nerves respond differently from other trigeminal branches to these stimuli is unknown. Also, threshold concentrations are higher in rat lingual nerves than they are in the ethmoid and long ciliary branches. One reason may be that the lingual epithelium is stratified and thicker than the cornea1 or olfactory mucosa and thus, for a given time after application of a stimulus, a smaller concentration will reach either the epithelial cells or the nerves. For all the other stimuli tested, the response profiles obtained in lingual nerve recordings are similar to those obtained in ethmoid and long ciliary nerves. Other non-polar compounds that decrease lingual nerve activity below its spontaneous level act as anaesthetics. Thus, decreasing lingual nerve activity to below baseline levels with 60mM hexanol rendered the nerve unresponsive to 2.5 M KC1 and to water at 10°C whereas 13.8 M ethanol, which only increased lingual nerve activity, did not inhibit the responses to these stimuli. Application of most non-polar stimuli to the dorsal surface of the tongue initially increases lingual nerve activity (Hellekant, 1965; Silver, 1987; Silver, 1990). In this regard, at least at low concentrations, these non-electrolytes do not behave as classic anaesthetics. This means that if these compounds interact directly with lingual nerves they are not only inhibiting sodium and potassium channels. The initial increase in lingual nerve activity could arise from the direct interaction of the stimuli with epithelial cells or with nerve terminals. In recordings from lingual fibres that terminate in epithelia it is difficult to distinguish between these two possibilities. The non-electrolyte concentrations required to alter the short-circuit current are similar to those required to activate or inhibit lingual nerves (Fig. 8) and therefore, using only this criterion, one cannot distinguish whether epithelial cells modulate lingual nerve responses.
Hydrophobic puthwu.vs into lingual epithelia Our findings suggest that hydrophobic molecules applied to the dorsal surface of lingual epithelia elicit responses from lingual nerves by partitioning into and diffusing across plasma membranes of epithelial and nerve cells. This conclusion is based on the following. (1) The correlation between threshold concentrations for lingual nerve responses and octanol:water partition coefficients (Fig. 2). (2) The magnitude of the free energy of transfer per methylene group of 186 J/m.01 implies that these compounds undergo partition in sites similar to those found in the hydrophobic regions of plasma membranes or lipid bilayers (Cevc and hdarsh, 1987). (3) That LaCI,, an inhibitor of lingual nerve responses to electrolytes (Sostman and Simon, 1991), did not inhibit responses to non-polar stimuli. In addition, the permeability of non-electrolytes across rat lingual epithelia is directly proportional to their oil : water partition coefficients (Mistretta, 1971) as is the inhibition of the short-circuit current measumd across canine lingual epithelia (Fig. 8). Permeability measurements across skin have shown that hydrophobic compounds diffuse by first partitioning into lamellar bodies in the stratum comeum and then into the plasma membranes of epithelial cells (Williams and Elias, 1987). As the permeability of non-electrolytes through both skin and .lingual epithelia is proportional to their oil :water . partition coefficients, and as both are classified as stratified squamous epithelia, we assume these two types of epithelia have similar pathways for nonpolar stimuli. In lingual epithelia these pathways include the lamellar bodies in the stratum comeum (Williams and Elias, 1987), and the plasma membranes of epithelial, taste and nerve cells. Partitioning of non-polar compounds into lamellar bodies should not affect the activity of trigeminal fibres as these lipoprotein complexes are in the extracellular space and do not contain ion-transporting proteins (Williams and Elias, 1987). Partitioning of hydrophobic compounds into taste cells and/or the special sensory fibres that form synapses with them should have little effect on responses recorded from lingual nerves because the number of trigeminal fibres in taste buds is very small compared with the total number of lingual nerves (Biedenbach, Beuerman and Brown, 1975; Kinnman and Aldskogius, 1988; Yamasaki, Kubota and Tohyama, 1985). Consequently, non-polar stimuli may influence trigeminal fibre activity either by altering transport across epithelial cells and/or by directly interacting with trigeminal fibres. Spontaneous activity of lingual trigeminal jbres The responses of ,whole lingual nerve are similar to those obtained from cold fibres in regard to: decreasing temperature (Poulos and Lende, 1970; Hensel and Zotterman, 1951b; Sostman and Simon, 1991 and Fig. 5), the additio:n of CaCl, (Hensel and Schafer,
SWNEYA. SIMONamd ANN L.
812
Let us first consider the responses of lingual epithelia to compounds that monotonically decrease the short-circuit current (e.g. hexanol). The current measured across tongues bathed in symmetrical solutions of Krebs-Henseleit buffer arises from a net influx of Na + and a net efflux of Cl - (DeSimone et al., 1984; Mierson et al., 1985). This current is completely inhibited by ouabain (DeSimone et al., 1984; Mierson et al., 1985) and thus is generated by Na,K-ATPase present in taste and epithelial cells. In hyperosmotic solutions of NaCl, the current can almost be entirely accounted for by a net Na + influx (DeSimone et al., 1984). Thus, non-polar compounds, such as hexanol and octanol, inhbit transport proteins associated with sodium influx. The one protein involved in sodium transport and present in both taste and epithelial cells is the Na,K-ATPase (Simon, Holland and Zampighi, 1991). Given the relatively small area of the anterior two-thirds of the tongue occupied by taste cells [
