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1Department of Physiology, Virginia Commonwealth University; and 2Philip Morris USA, Richmond, Virginia
Submitted 31 March 2007; accepted in final form 29 June 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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) and in neurons of the nucleus of the solitary tract (Lemon and Smith 2005). Nicotine also elicited responses in single gustatory neurons from the insular cortex in monkeys (Scott et al. 1999). Nicotine is perceived as predominantly bitter by humans but its effects on other taste qualities are presently unknown. Two additional properties of nicotine suggest that it could be a potential modulator of both salty and sour taste.
First, nicotine has been shown to modulate the transient receptor potential vanilloid-1 receptor (TRPV1) (Liu et al. 2004). Accordingly, we tested the hypothesis that nicotine modulates the chorda tympani (CT) taste nerve responses to NaCl and other mineral salts by interacting with the amiloride- and benzamil (Bz)-insensitive TRPV1 variant salt taste receptor (TRPV1t) (Lyall et al. 2004b, 2005a,b,c; Simon and De Araujo 2005). This hypothesis is based on the observations that in rat and mouse fungiform taste receptor cells (TRCs) TRPV1t is a constitutively active nonselective cation channel present in the apical membrane of TRCs and is derived from the TRPV1 gene. All of the Bz-insensitive CT taste nerve responses to Na+ salts and part of the response to K+, NH4+, and Ca2+ salts are elicited by cation flux through TRPV1t (Lyall et al. 2004b, 2005a,b,c). However, TRPV1 knockout (KO) mice maintain normal salt detection performance, suggesting that there may be other amiloride- and Bz-insensitive salt transduction mechanisms in taste receptor fields other than the anterior tongue (Ruiz et al. 2006; Treesukosol et al. 2007). TRPV1t is activated by vanilloids: resiniferatoxin (RTX) and capsaicin (CAP) and by elevated temperatures (>38°C), and is inhibited by the TRPV1 antagonists [capsazepine and N-(3-methoxyphenyl)-4-chlorocinnamide (SB-366791)]. TRPV1t demonstrates many similarities with the cloned TRPV1 (Lyall et al. 2005c). Therefore it is expected that agonists that either activate or sensitize the TRPV1 channel, such as endocannabinoids, lipoxygenase metabolites of arachidonic acid, lipid derivatives, nicotine, ethanol, H+, and intracellular second messengers (Davis et al. 2002; Geppetti and Trevisani 2004; Gunthorpe et al. 2002; Liu et al. 2004; Lyall et al. 2004b, 2005a,b,c; Trevisani et al. 2002) will also have similar effects on TRPV1t. To test whether nicotine interacts specifically with TRPV1t, additional studies were conducted to determine whether nicotine also affected the amiloride- and Bz-sensitive epithelial Na+ channels (ENaCs) in the apical membrane of TRCs.
Second, in solution nicotine behaves as a base and has the potential to alter cell pH. Accordingly, we also tested the hypothesis that on entry into TRCs, nicotine alkalinizes resting intracellular pH (pHi) and inhibits CT responses to acidic stimuli. This hypothesis is based on the observations that the proximate stimulus for sour taste transduction is an acid-induced decrease in pHi in a subset of taste bud cells, the acid-sensing TRCs (DeSimone and Lyall 2006). PKD2L1, a polycystic-kidney-disease-like ion channel, expressed in a subset of TRCs is most likely involved in pH sensing (Huang et al. 2006; Ishimaru et al. 2006; LopezJimenez et al. 2006). In particular, the transduction mechanism for the phasic component of the CT response to acidic stimuli involves a decrease in pHi that results in TRC shrinkage through the change in F-actin to G-actin equilibrium. Cell shrinkage activates a flufenamic acid–sensitive nonspecific cation conductance in the basolateral membrane of TRCs that is responsible for the elicitation of the phasic response to acid stimulation (Lyall et al. 2006). Nicotine can potentially modulate one or more steps in this transduction pathway.
The interactions between nicotine and the amiloride-insensitive and amiloride-sensitive salt taste receptors were investigated by measurement of TRC Na+ ([Na+]i) and H+ (pHi) activity in polarized rat fungiform TRCs and by the CT taste nerve recordings (Lyall et al. 2005c). The CT responses were monitored in two animal models: a rat model and a TRPV1 KO mouse model (Caterina et al. 2000; Lyall et al. 2004b, 2005a,c). CT responses were recorded while the tongue was stimulated with nicotine alone and in mixtures with mineral salts in the absence and presence of specific agonists (RTX and elevated temperature) and antagonists (SB-366791) of TRPV1t (Lyall et al. 2004b, 2005a,c) and in the presence of Bz, a specific ENaC blocker. The interactions between nicotine and the acidic stimuli were investigated by the measurement of TRC pHi and cell volume in polarized rat fungiform TRCs and by the CT recordings while stimulating the tongue with HCl (Lyall et al. 2006). The results presented herein support the conclusion that nicotine modulates the CT responses to mineral salts by interacting with both TRPV1t and ENaC and it modulates CT responses to acidic stimuli by inducing intracellular alkalinization and cell swelling.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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). In vitro studies
pHI AND CELL VOLUME MEASUREMENTS IN POLARIZED FUNGIFORM TRCS.
Simultaneous measurement of cell volume changes and intracellular pH (pHi) were made using the pH-sensitive dye 2'-7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF, Molecular Probes, Eugene, OR) and recording at both the pH-sensitive and pH-insensitive (isosbestic) wavelengths (Lyall et al. 2006). Changes in pHi were monitored as variations in the fluorescence intensity ratio (FIR; F490/F440) of BCECF (Lyall et al. 2001). At the end of each experiment, the changes in TRC pHi were calibrated by bilateral perfusion of high K+ solutions containing 10 x 10–6 M nigericin adjusted to pH values between 6.5 and 8.0 (Table 1; HK). The relative changes in TRC volume were monitored at the isosbestic wavelength of 440 nm (F440). The fluorescence intensity at this wavelength is independent of pH and reflects the dye concentration inside the cell (Lyall et al. 2006).
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). Changes in TRC [Na+]i were monitored in the presence and absence of Bz or SB-366791. This was done to distinguish between the apical Na+ flux through the Bz-sensitive ENaCs and the Bz-insensitive TRPV1t in fungiform TRCs (Lyall et al. 2004b, 2005a,b). The relative changes in TRC [Na+]i were expressed as percentage change in fluorescence intensity (F490) of sodium green or as changes in FIR (F340/F380) relative to apical zero Na+ concentration. In individual taste buds, the data were presented as means ± SE of N, where N represents the number of regions of interest (ROIs) within the taste bud. The data were also presented as the means ± SE of n, where n represents the number of individual taste buds studied. Student's t-test was used to analyze the differences between sets of data. Because we are comparing the fluorescence intensity, FIR, and pHi values before and after nicotine treatment in the same polarized taste bud preparation, paired t-test was used to evaluate statistical significance. SOLUTIONS. Compositions of various solutions used in the in vitro experiments are given in Table 1.
In vivo studies
CT TASTE NERVE RECORDINGS.
Animals were housed in the Virginia Commonwealth University animal facility in accordance with institutional guidelines. All in vivo and in vitro animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Virginia Commonwealth University. Female Sprague–Dawley rats (150–200 g) were anesthetized by intraperitoneal injection of pentobarbital (60 mg/kg) and supplemental pentobarbital (20 mg/kg) was administered as necessary to maintain surgical anesthesia. The animal's corneal reflex and toe-pinch reflex were used to monitor the depth of surgical anesthesia. Body temperatures were maintained at 37 °C with a Deltaphase Isothermal PAD (Model 39 DP; Braintree Scientific, Braintree, MA). The left CT nerve was exposed laterally as it exited the tympanic bulla and placed onto a 32-gauge platinum/iridium wire electrode. The CT responses were recorded under zero current-clamp and voltage-clamp conditions referenced to the oral cavity (Lyall et al. 2005a,b).
The anterior lingual surface was stimulated with a rinse solution (R; 0.01 M KCl) and with NaCl solutions (0.01 M KCl + 0.1 M NaCl; N) containing (–)-nicotine hydrogen tartarate (Nic; 0–0.1 M). In some experiments both rinse (R) and NaCl solutions (N) with or without nicotine contained, in addition, 0.01 M 4-(2-hydroxyethyl)-piperazine-1-ethanesulfonic acid (HEPES) or 0.01 M Tris[hydroxymethyl]-aminomethane (Tris) and were adjusted to different pH values with 1 N NaOH or HCl. CT responses were recorded in the presence of benzamil (Bz; 5 x 10–6 M), a specific and potent blocker of the apical ENaC. CT responses were also recorded in the presence of the TRPV1 agonists, resiniferatoxin (RTX; 0.25 x 10–6 M), or elevated temperatures (23–55.5°C), and a TRPV1 antagonist, SB-366791 (0.25–1 x 10–6 M) (Lyall et al. 2004b, 2005a,c). All drugs were purchased from Sigma (St. Louis, MO). Typically, stimulus solutions remained on the tongue for 2 min. Control stimuli consisting of 0.3 M NaCl and 0.3 M NH4Cl, applied at the beginning and at the end of the experiment, were used to assess preparation stability. To compute the phasic or transient component of the response, we first obtained the area under the CT response curve from the onset of the stimulus to the onset of the tonic or time-invariant part of the response. To normalize this result, this area was divided by the area under the 0.3 M NH4Cl response curve over the final 30 s of the tonic response period. The normalized data were reported as means ± SE of the number of animals (N). Student's t-test was used to analyze the differences between sets of data. Because we are comparing the normalized CT responses before and after nicotine treatment in the same CT preparation, paired t-test was used to evaluate statistical significance.
To investigate the effect of temperature on the CT response to nicotine and to mixtures of nicotine + mineral salts, the lingual surface was superfused (8 ml/min) with salt solutions using syringe pumps and heating coils maintained between 23 and 55.5 °C as described previously (Lyall et al. 2004b, 2005a). CT responses were also monitored in wildtype (WT; C57BL/6J) and homozygous TRPV1 KO mice (B6.129S4-Trpv1tm1jul; The Jackson Laboratory, Bar Harbor, ME). Mice (30–40 g) were anesthetized by intraperitoneal (ip) injection of pentobarbital (30 mg/kg) and supplemental pentobarbital (10 mg/kg) was administered as necessary to maintain surgical anesthesia. The rest of the procedure was the same as described earlier for rats (Lyall et al. 2004b, 2005c). At the end of the experiment the animals were humanely killed by an ip overdose of pentobarbital (
195 mg/kg body weight for rats and 150 mg/kg body weight for mice).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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EFFECT OF NICOTINE ON TRC pHI AND VOLUME. Nicotine has two pK values: 6.16 and 10.96. Nicotine is a base and on entering TRCs it is expected to produce intracellular alkalinization. Accordingly, in a lingual epithelial preparation perfused on both sides with control solution (Table 1; C; pH 7.4) increasing nicotine concentration in the basolateral compartment (BLNic) in a stepwise manner from 0.0005 to 0.01 M produced a dose-dependent increase in TRC pHi (Fig. 1A). The relationship between basolateral nicotine concentration and TRC pHi was almost linear (pHi = 0.028 [Nic] + 7.24; r2 = 0.95; n = 8). In contrast, perfusing the apical membrane with 0.025 M nicotine at pH 7.4 produced only a small increase in TRC pHi over a 5-min period (Fig. 1B). The increase in pHi was not reversed on nicotine washout. Essentially similar results were obtained in two additional experiments. The results suggest that at physiological pH, the nicotine permeability of the TRC basolateral membrane is significantly greater than that of the apical membrane.
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Nicotine-induced alkalinization was accompanied by a decrease in F440, indicating cell swelling (Fig. 2A). Cell swelling will cause a decrease in dye concentration inside the cells and a decrease in F440. Nicotine-induced increase in pHi and a concomitant decrease in F440 were also observed when TRCs were perfused on both sides with 0 Na solution (Table 1; 0Na; pH 7.4), 0 Na-0 K solution (Table 1; 0Na-0K; pH 7.4), and 0 Na-0 K-0 Cl solution (Table 1; 0Na-0K-0Cl; pH 7.4) (data not shown). These results support the conclusion that nicotine-induced intracellular alkalinization is due to the entry of nicotine across the apical or basolateral membrane per se and does not involve the activation of Na+-, K+-, Cl–-, or HCO3–-dependent pH regulatory mechanisms. Because in the absence of Na+ TRC pHi becomes acidic (Lyall et al. 2004a), this suggests that the membrane permeability to nicotine is not modulated by changes in pHi. The results further suggest that nicotine-induced cell swelling is also the result of an increase in TRC pHi per se and does not involve secondary activation of Na+-, K+-, Cl–-, or HCO3–-dependent transport mechanisms (Lyall et al. 2006).
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, 2004b, 2005a). Figure 2B shows the effect of apical nicotine (APNic) on the Bz-insensitive Na+ flux across the apical membrane of TRCs. A lingual epithelial preparation loaded with SBFI was initially perfused on both sides with Na+-free solution (Table 1; 0Na; pH 7.4). Perfusing the apical membrane with control solution containing 0.15 M NaCl + 5 x 10–6 M Bz + 0.015 M nicotine (Table 1; C, pH 7.4) increased FIR (a–b). Subsequently perfusing the control solution with Bz but without nicotine decreased FIR (b–c). Nicotine also produced an increase in Bz-insensitive apical Na+ flux in isolated taste bud fragments in which the basolateral Na+-H+ exchanger-1 (NHE-1) was blocked by 10 x 10–6 M zoniporide (Lyall et al. 2004a) (data not shown). The nicotine-induced increase in the Bz-insensitive apical Na+ flux was blocked in the presence of 1 x 10–6 M SB-366791 (data not shown). Under these conditions no effect of nicotine was observed on the Bz-sensitive unilateral Na+ flux (data not shown). These results suggest that nicotine produces a greater unilateral Bz-insensitive apical Na+ flux relative to control through the TRPV1t cation channel (Lyall et al. 2005b).
Figure 2C shows the effect of basolateral nicotine (BLNic) on the Bz-sensitive Na+ flux across the apical membrane of TRCs. A lingual epithelial preparation loaded with Na-green was initially perfused on the apical side with Na+-free solution (Table 1; 0Na; pH 7.4) and on the basolateral side with control solution (Table 1; C; pH 7.4). Perfusing the apical membrane with control solution containing 0.15 M NaCl + 1 x 10–6 M SB-366791 reversibly increased F490 (a–b–c). SB-366791 inhibits Na+ flux through the Bz-insensitive pathway. Thus the increase in F490 represents the Na+ flux exclusively through ENaCs. Following this the basolateral membrane was perfused with control solution + 0.015 M nicotine (BLNic). In the continuous presence of basolateral nicotine, perfusing the apical membrane with control solution + 1 x 10–6 M SB-366791 produced a significantly greater increase in F490 relative to control (P < 0.01; n = 7; paired). The basolateral membrane of TRCs is readily permeable to nicotine (Fig. 1, A and C) and on entering TRCs it produced intracellular alkalinization resulting in the activation of apical ENaC (Lyall et al. 2002b). Taken together the results support the conclusion that nicotine produces differential effects on ENaC and TRPV1t depending on pHo and the cell membrane at which TRCs are exposed to nicotine.
In vivo studies
EFFECT OF NICOTINE ON THE CT RESPONSE TO 0.01 M KCL.
We first investigated whether CT responses to 0.01 M KCl are modulated by nicotine in a dose-dependent fashion. A rat tongue was initially rinsed with 0.01 M KCl + 0.01 M HEPES (R; pH 6.1) and then with R containing varying concentrations of nicotine (0–0.1 M; pH 6.1). Figure 3A shows that in the presence of 0.01 M KCl (R) nicotine at 0.005, 0.01, and 0.015 M elicited phasic CT nerve response with a rapid onset and decay. However, the rate of return to a steady-state level varied with the nicotine concentration >0.015 M. This is illustrated in the inset above Fig. 3A using an expanded timescale for 0.005 and 0.05 M nicotine. The nicotine-induced variation in the phasic CT response (phasic 0.01 M KCl CT response/0.3 M NH4Cl CT response) in three animals is shown in Fig. 4A. The increased response seen at 0.025, 0.05, and 0.1 M nicotine is the result of the delayed return to steady-state levels at these concentrations. As seen in Fig. 3A, nicotine presented in R also produced tonic responses, starting at about 0.025 M concentration. The concentration dependence of the tonic response (0.01 M KCl/0.3 M NH4Cl) in three animals is shown in Fig. 4B (
, 0 SB). Tonic responses peaked at 0.05 M nicotine.
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showed that nicotine induced an increase in CAP-activated currents in cells with heterologously expressed TRPV1 cation channel, indicating that nicotine sensitizes the channel to activation by vanilloids. We hypothesize that nicotine either activates or serves as a modulator of TRPV1t. Figure 3A shows the effect of nicotine on the tonic CT response to 0.1 M NaCl + 5 x 10–6 M Bz + 0.01 M HEPES, pH 6.1 (N + Bz). The results show that nicotine enhanced the Bz-insensitive response to NaCl in a dose-dependent manner. The response to N + Bz + 0.015 M nicotine was 72% greater than the response to N + Bz alone. On the other hand, the response to N + Bz + 0.05 M nicotine was inhibited by 19% relative to N + Bz. In three animals nicotine produced a bell-shaped dose–response relationship on the Bz-insensitive CT response to 0.1 M NaCl (Fig. 4C; , 0 SB).
To investigate whether nicotine produces changes in the Bz-insensitive NaCl CT response by modulating the apical membrane conductance in TRCs, we monitored the sensitivity of the NaCl CT responses to the applied voltage across the receptive field. Figure 3C shows the CT response to 0.1 M NaCl + 5 x 10–6 M Bz in the absence (R + N + Bz) and presence of 0.015 M nicotine (R + N + Bz + Nic) as a function of the applied voltage across the receptive field at –60, 0, and +60 mV. As reported earlier (Lyall et al. 2004b, 2005a,c), the rat CT responses to 0.1 M NaCl + 5 x 10–6 M Bz were slightly enhanced at –60 mV and slightly suppressed at +60 mV. In the presence of 0.015 M nicotine (N + Bz + Nic), the same voltages exerted significantly larger effects on the response (Fig. 3C). The changes in the CT response at 0, –60, and +60 mV in the absence and presence of nicotine from three animals are summarized in Fig. 4D. In each case the slope of the relation between CT response and voltage was calculated as described previously (Lyall et al. 2004b). Nicotine (0.015 M) increased the slope from (6.37 ± 0.92) x 10–4 response units/mV () under control conditions to (13.39 ± 1.36) x 10–4 response units/mV (
; P < 0.05; n = 3; paired). This suggests that in nicotine-sensitive TRCs the increase in the Bz-insensitive NaCl CT response is due to an increase in the apical membrane conductance to Na+. This is consistent with the observation that in in vitro experiments nicotine increased the unilateral apical Na+ flux in polarized fungiform TRCs (Fig. 2).
MODULATORY EFFECT OF NICOTINE IS BLOCKED BY SB-366791.
To determine whether the effects of nicotine on the Bz-insensitive NaCl CT response are exerted through TRPV1t, we obtained the nicotine concentration versus the Bz-insensitive NaCl CT response function in the presence of SB-366791, a selective competitive inhibitor of both TRPV1 (Gunthorpe et al. 2004) and the TRPV1t (Lyall et al. 2004b, 2005a,b,c).
), the rightward shift of the nicotine concentration versus the tonic response function is increased. As reported previously (Lyall et al. 2004b), at this higher concentration of SB-366971, the constitutive activity of the TRPV1t cation channel is also partially blocked. In the presence of 0.5 x 10–6 M SB-366791, the CT response to 0.1 M NaCl + 5 x 10–6 M Bz + 0.015 M nicotine (R + N + Bz + Nic) was not different from baseline and there was no sensitivity of the NaCl CT responses to the applied voltage across the receptive field (data not shown). This suggests that SB-366791 not only inhibits the constitutively active Bz-insensitive apical membrane Na+ conductance but also the nicotine-induced increase in the Na+ conductance.
vs. ). However, on increasing SB-366791 to 0.25 x 10–6 M, the nicotine concentration versus tonic response function was shifted significantly to the right along the nicotine concentration axis (Fig. 4B; vs. or ).
EFFECT OF EXTERNAL pH ON THE CT RESPONSE TO NICOTINE IN 0.01 M KCl OR 0.1 M NaCl.
We have previously shown that changes in pHo modulate the effect of vanilloids on TRPV1t (Lyall et al. 2004b, 2005a). We hypothesize that the effect of nicotine on TRPV1t will also be modulated by changes in pHo. In these experiments we held the nicotine concentration at 0.015 M (the concentration that produces the maximal enhancement of the tonic response in the medium containing 0.01 M KCl + 0.1 M NaCl + 5 x 10–6 M Bz). Consistent with previous studies (Lyall et al. 2002b), in the absence of nicotine the Bz-insensitive NaCl CT responses were insensitive to pHo (Fig. 5A, inset). Nicotine (R + N + Bz + Nic) enhanced the Bz-insensitive NaCl CT response at all pH values relative to (R + N + Bz) alone and produced a bell-shaped tonic CT response function in the presence of 0.01 M KCl + 0.1 M NaCl + 5 x 10–6 M Bz at varying pHo values (Fig. 5A). The mean pHo versus CT response profile obtained in three animals is shown in the inset. In the presence of 0.01 M KCl (R), the response to 0.015 M nicotine was not significantly affected by changes in pHo (inset). The results support the conclusion that the effect of nicotine on TRPV1t in the presence of 0.1 M NaCl is modulated by changes in pHo and that nicotine enhances the activity of TRPV1t optimally at pH 6–7.
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, 2005b). An increase in temperature produced a sharp increase in the CT response to 0.01 M KCl + 0.1 M NaCl + 5 x 10–6 M Bz (R + N + Bz) around 38°C and gave a maximum enhancement of the CT response around 42°C, and the response decreased at >42°C (Fig. 5B). Stimulating with 0.01 M KCl + 0.1 M NaCl + 5 x 10–6 M Bz + 0.01 M nicotine (R + N + Bz + Nic) enhanced the CT response at 23°C and at elevated temperatures (Fig. 5B). In three animals the temperature at which nicotine produced maximum increase in the CT responses was 42.2 ± 0.3°C, a value not different from R + N + Bz alone (41.9 ± 0.2°C). This is consistent with the idea that nicotine enhances the CT response to NaCl without affecting the temperature threshold of the TRPV1t cation channel.
NICOTINE–RTX INTERACTIONS WITH THE TRPV1T SALT TASTE RECEPTOR.
The Bz-insensitive NaCl CT responses are insensitive to pHo and adenosine triphosphate (ATP). However, in the presence of a subthreshold concentration of RTX, a TRPV1 and TRPV1t agonist, the Bz-insensitive NaCl CT responses become sensitive to both pHo and ATP (Lyall et al. 2004b, 2005a). Therefore we tested whether a subthreshold concentration (0.25 x 10–6 M) of RTX modulates the effects of nicotine and elevated temperature on the Bz-insensitive NaCl CT response.
Figure 5C shows that superfusing the tongue with 0.01 M KCl + 0.1 M NaCl + 5 x 10–6 M Bz + 0.25 x 10–6 M RTX at 23°C (R + N + Bz + RTX) produced a small increase in the Bz-insensitive NaCl CT response relative to 0.01 M KCl + 0.1 M NaCl + 5 x 10–6 M Bz (R + N + Bz). Subsequently, superfusing the tongue with 0.01 M KCl + 0.1 M NaCl + 5 x 10–6 M Bz + 0.25 x 10–6 M RTX + 0.015 M nicotine at 23°C (R + N + Bz + RTX + Nic) produced a CT response whose magnitude was significantly >0.01 M KCl + 0.1 M NaCl + 5 x 10–6 M Bz + 0.015 M nicotine at 23 °C (R + N + Bz + Nic). These results suggest that in the presence of a subthreshold concentration of RTX, nicotine produces a greater increase in the CT response relative to either RTX or nicotine alone.
We also monitored the sensitivity of the NaCl CT responses to ±60-mV applied voltage across the receptive field in the presence of RTX alone and RTX + Nic. The applied voltages exerted significantly larger effects on the 0.01 M KCl + 0.1 M NaCl + 5 x 10–6 M Bz + 0.25 x 10–6 M RTX + 0.015 M nicotine (R + N + Bz + RTX + Nic) CT response relative to 0.01 M KCl + 0.1 M NaCl + 5 x 10–6 M Bz + 0.015 M nicotine (R + N + Bz + Nic) (Figs. 3C and 4D). The mean data from three such experiments are also summarized in Fig. 4D. In the presence of 0.015 M nicotine + 0.25 x 10–6 M RTX, the slope of the response (21.53 ± 2.38) x 10–4 response units/mV () was greater relative to its value (8.09 ± 2.14) x 10–4 response units/mV in the presence of RTX alone (; P < 0.05; n = 3; paired). These results suggest that in the presence of 0.25 x 10–6 M RTX, nicotine produces a greater increase in the apical Bz-insensitive Na+ conductance relative to 0 RTX.
Next we tested the effect of RTX and nicotine on the temperature threshold of the TRPV1t cation channel in the presence of 0.1 M NaCl. The CT responses to 0.01 M KCl + 0.1 M NaCl + 5 x 10–6 M Bz (R + N + Bz) were monitored in the presence and absence of 0.01 M nicotine (R + N + Bz + Nic) and 0.25 x 10–6 M RTX (R + N + Bz + Nic + RTX), while the temperature of the solution was varied between 23 and 55.5 °C (Fig. 5C). The addition of 0.25 x 10–6 M RTX to 0.01 M nicotine solution enhanced the NaCl CT response and shifted the temperature curve to the left. The mean temperature at which the CT response gave the maximum response in the presence of 0.01 M KCl + 0.1 M NaCl + 5 x 10–6 M Bz + 0.015 M nicotine (R + N + Bz + Nic) and 0.01 M KCl + 0.1 M NaCl + 5 x 10–6 M Bz + 0.015 M nicotine + 0.25 x 10–6 M RTX (R + N + Bz + Nic + RTX) was 42.2 ± 0.3 and 39.3 ± 0.1 °C (n = 3; P < 0.05), respectively. The results suggest that nicotine enhances the CT response to NaCl without affecting the temperature threshold of the TRPV1t cation channel. In contrast, a mixture of nicotine and a subthreshold concentration of RTX enhanced the magnitude of the NaCl CT response and shifted the temperature curve to the left. The results further suggest that nicotine and RTX act on different sites on the TRPV1t cation channel.
EFFECT OF NICOTINE ON SALT RESPONSES IN WT AND TRPV1 KO MICE. To confirm that nicotine produces its effect on the Bz-insensitive NaCl CT responses by modulating the activity of the TRPV1t, we monitored the effect of nicotine on the CT response to NaCl in WT and TRPV1 KO mice. In WT mice (Fig. 6A), stimulating the tongue with 0.01 M KCl + 0.1 M NaCl (R + N) produced a CT response and a significant part of the CT response was Bz-insensitive (0.01 M KCl + 0.1 M NaCl + 5 x 10–6 M Bz; R + N + Bz). Stimulating the tongue with 0.01 M KCl + 0.1 M NaCl + 5 x 10–6 M Bz + nicotine (Nic; 0–0.015 M) produced a dose-dependent increase in the CT response. The data from three WT mice are summarized in Fig. 6B and suggest that CT responses in WT mice are qualitatively similar to the responses observed in rats (Fig. 3) within the range of 0–0.015 M nicotine concentration.
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, 2005a,b,c). EFFECT OF NICOTINE-INDUCED INTRACELLULAR ALKALINIZATION AND CELL SWELLING ON THE CT RESPONSES TO NaCl AND HCl. Figure 7A shows the effect of nicotine on the Bz-sensitive and Bz-insensitive NaCl CT responses as a function of apical pH. At pH 6.5 stimulating the tongue with 0.01 M KCl + 0.1 M NaCl + 0.015 M nicotine (R + N + Nic6.5) produced a bigger CT response (e–f) relative to 0.01 M KCl + 0.1 M NaCl (R + N6.5; a–b). At pH 6.5 the increase in the NaCl CT response could be accounted for by the increase in the Bz-insensitive component of the NaCl CT response [(g–h) > (c–d)]. At pH 6.5, nicotine produced no effect on the Bz-sensitive component of the NaCl CT response [(f–g) = (b–c)]. At pH 9, NaCl (R + N9.0) produced a bigger CT response (i–j) relative to pH 6.5 (R + N6.5; a–b). The increase in the NaCl CT response could be accounted for by the increase in the Bz-sensitive component of the NaCl CT response [(j–k) > (b–c)]. At pH 9, superfusing the tongue with R + NaCl +0.015 M nicotine (N + Nic9.0), further enhanced the magnitude of the CT response [(m–n) > (i–j)]. The increase in the NaCl CT response could be accounted for by the increase in the Bz-sensitive component of the NaCl CT response [(n–o) > (j–k)]. At pH 9, nicotine produced no effect on the Bz-insensitive component of the NaCl CT response [(o–p) = (k–l)]. In three animals (Fig. 7C), R + NaCl at pH 9 and R + NaCl +0.015 M nicotine at pH 9 increased the Bz-sensitive NaCl CT response by 23.5 ± 4.5 and 57.6 ± 5.2%, respectively, relative to pH 6.5 (P < 0.01; n = 3). At pH 6.5, nicotine produced no significant increase in the Bz-sensitive NaCl response but specifically increased the Bz-insensitive component of the NaCl CT response (P < 0.01; paired; n = 3; Fig. 7C). These results suggest that at pH 6.5, nicotine specifically affects the Bz-insensitive part of the NaCl CT response and at pH 9, it affects only the Bz-sensitive ENaC component of the response.
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), a decrease in pHi of the acid-sensing TRCs is the proximate signal for the elicitation for both the phasic and tonic parts of the CT response. We further tested whether nicotine entry into TRCs and the subsequent increase in pHi, will interfere with the CT response to acidic stimuli. CT responses to HCl were monitored in the presence and absence of nicotine at pH values that facilitate apical nicotine entry into TRCs (Fig. 1D). Figure 7B shows that when the rinse solution (0.01 M KCl + 0.01 M HEPES) was maintained at pH 6 (R6), stimulating the tongue with 0.02 M HCl (HCl) elicited a fast phasic response (a) that slowly declined to the tonic response level (b). At a rinse pH of 6 (R6), the magnitude of the phasic response to two 0.02 M HCl stimulations is represented by (a–b) and (k–l) at the beginning and at the end of the CT record. At a rinse pH of 9 (0.01 M KCl + 0.01 M Tris; R9), the phasic CT response during two stimulations with 0.02 M HCl (c–d and e–>f) was smaller relative to at pH 6. In the final step, rinsing the tongue with rinse solution at pH 9 containing 0.015 M nicotine (R + Nic9) further inhibited the magnitude of the CT response during two stimulations with 0.02 M HCl (g–h and i–j). In contrast to the phasic response, the magnitude of the tonic part of the CT response to 0.02 M HCl was not altered when the rinse solution was at pH 9 or when the rinse solution contained, in addition, 0.015 M nicotine. Essentially similar results were obtained in two additional experiments. These results suggest that at alkaline pH nicotine specifically attenuates the magnitude of the phasic response to acidic stimulation. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Modulation of TRPV1t by nicotine
Results presented herein demonstrate that nicotine modulates CT responses to mineral salts. The effect of nicotine on the CT responses to mineral salts was observed even at low salt concentrations (0.01 M KCl). At low salt concentration increasing nicotine concentration increased the duration of the phasic response (Figs. 3A and 4A) and at higher concentrations elicited both phasic and tonic CT responses (Figs. 3A, inset and 4B). The increase in the duration of the phasic response and the appearance of the tonic response is related to the increase in the unilateral apical cation flux in the presence of nicotine (Fig. 2, B and C). The increase in the magnitude of the CT response to 0.01 M KCl in the presence of nicotine was dose dependent, indicating that the cation flux also varies with nicotine concentration.
There is a bell-shaped relationship between nicotine concentration and the CT response to 0.1 M NaCl (Figs. 3A and 4C). In our previous studies, a similar bell-shaped dose–response relation was observed with other TRPV1t agonists, such as RTX, CAP, cetylpyridinium chloride (CPC) (Lyall et al. 2004b), and ethanol (Lyall et al. 2005c). RTX and CAP produced maximum enhancement of the Bz-insensitive NaCl CT response at 1 x 10–6 and 40 x 10–6 M and completely inhibited the CT response at 10 x 10–6 and 200 x 10–6 M, respectively (Lyall et al. 2004b, 2005a). By comparison, nicotine is a far less potent modulator of TRPV1t because it caused a maximum enhancement in the Bz-insensitive NaCl CT response at 0.015 M and inhibited the response at 0.1 M by 80% relative to N + Bz alone (Fig. 4C). There are additional important quantitative differences between nicotine and other TRPV1t agonists. Nicotine induced a mean maximum enhancement of the CT response in the presence of 0.1 M NaCl + 5 x 10–6 M Bz of 72%. In contrast RTX, CAP, CPC, and ethanol increased the maximum enhancements in the NaCl + Bz CT response of >100%. Second, the maximum suppression of the CT response to 0.1 M NaCl + 5 x 10–6 M Bz by nicotine (19%) is less than that of the other agonists, which can suppress the response by as much as 100% (Lyall et al. 2004b, 2005a,c). Nonetheless, nicotine produces a concentration–response function that is qualitatively similar to that of a typical TRPV1t agonist.
Second, in the case of TRPV1, nicotine is not itself an agonist of the receptor, but it significantly sensitizes TRPV1 to the vanilloid, CAP (Liu et al. 2004). Thus nicotine has no effect on TRPV1 unless it is presented with capsaicin. In the case of the TRPV1t, nicotine itself is an agonist (Figs. 3A and 4C). This difference may be explained by the fact that, unlike TRPV1, which requires heat or a chemical agonist to become active, TRPV1t is constitutively active, so that nicotine modulates the receptor ion channel that is already in a conducting state.
Third, TRPV1 is activated by a decrease in pHo (Davis et al. 2002; Geppetti and Trevisani 2004; Gunthorpe et al. 2002). In contrast, in the absence of a TRPV1t agonist, the Bz-insensitive NaCl CT response is not affected by changes in pHo. Similarly, in the absence of an agonist, changes in pHo between 4.7 and 9.7 had no effect on the temperature threshold of the Bz-insensitive NaCl CT response. This indicates that the constitutively active TRPV1t is not affected by changes in pHo (Lyall et al. 2004b, 2005a). However, the CT responses activated by a TRPV1t agonist were strongly dependent on pHo. In our studies, CT response to NaCl + Bz + RTX was bell-shaped with the maximal response around pH 6 with lower responses observed both above and below pH 6 (Lyall et al. 2004b, 2005a). In addition, changing pHo from 6.0 to either 4.7 or 9.7 in the presence of RTX decreased the temperature threshold of the CT response (Lyall et al. 2004b, 2005a). Taken together, the data indicate that the TRPV1t cation channel in TRCs is affected by pHo only in the presence of an agonist (Lyall et al. 2004b, 2005a) such as nicotine (Fig. 5A). At constant pHo, changes in TRC pHi do not affect salt responses in the presence of RTX (Lyall et al. 2004b, 2005a). Therefore it is likely that H+ ions bind to an external site on the TRPV1t channel protein and modulate the affinity of nicotine and RTX to their respective intracellular binding sites on the channel protein.
Nicotine enhanced the NaCl CT response without a shift in the temperature threshold of the CT response (Fig. 5B). In this regard the effect of nicotine on TRPV1t is similar to that reported on the cloned TRPV1 receptor. Nicotine did not alter the threshold temperature of heat-activated currents in trigeminal ganglion neurons expressing TRPV1 (Liu et al. 2004).
SB-366791 inhibited the constitutive activity of TRPV1t and caused a rightward shift in the concentration–response to nicotine (Fig. 4C). Topical application of SB-366791 also inhibited RTX-, CAP-, temperature-, and ethanol-induced increases in Bz-insensitive NaCl CT responses (Lyall et al. 2004b, 2005a,c). Similarly, SB-366791 inhibits TRPV1 when activated by H+, elevated temperatures, CAP, and ethanol. SB-366791 produced similar rightward shift in concentration–response to CAP in hTRPV1-expressing cells (Gunthrope et al. 2004). This suggests that SB-366791 may also act as a competitive antagonist of TRPV1t cation channel. Because at present the detailed structure of TRPV1t is not known, it is not clear how SB-366791 competitively blocks the effects of nicotine, RTX, CAP, ethanol, and temperature on the receptor cation channel. However, based on the effect of SB-366791 on TRPV1 receptor channel two mechanisms of inhibition have been proposed. One possibility is that SB-366791 binds at the CAP-binding site on the TRPV1 receptor and affects the gating mechanism of the cation channel. SB-366791 can work not only as a true competitive antagonist but also as an inhibitor of the agonist properties of RTX, CAP, ethanol, and temperature by an allosteric effect on the cation channel (Gunthorpe et al. 2004). The second possibility is that SB-366791 is a pure allosteric effector of TRPV1t and produces an agonist-independent inhibition of the channel at an independent allosteric site (Gunthrope et al. 2004). However, the preceding two mechanisms are based on the assumption that all agonists produce effects on TRPV1 receptor by a common mechanism that involves gating of the receptor by shifting the temperature threshold of channel activation to lower temperatures. However, nicotine sensitizes both cloned TRPV1 and TRPV1t without a shift in the temperature threshold of the channel.
In HEK-293 cells expressing TRPV1, CAP increased the outward currents at room temperature, and induced a leftward shift in the voltage-dependent activation curve in a dose-dependent manner (Voets et al. 2004). This suggests that TRPV1 agonists function as gating modifiers. Because the Bz-insensitive NaCl CT response is observed in the absence of any agonists and demonstrates significant voltage sensitivity but not voltage dependence (Fig. 3C), it suggests that TRPV1t is constitutively active at room temperature in the absence of agonists (Figs. 3–6). It follows that TRPV1t is active at resting TRC membrane potential. Nicotine not only increased the Bz-insensitive NaCl CT response but also increased the Bz-insensitive apical Na+ conductance. In addition, making the TRC potential more negative enhanced the CT response to 0.1 M NaCl (Figs. 3C and 4D). Taken together, these results suggest that RTX-, CAP-, ethanol-, and nicotine-induced activation of TRPV1t most likely does not involve a leftward shift in the voltage-dependent activation curve of the channel. It is likely that TRPV1t agonists increase salt responses by stabilizing the channel in the open state, and thereby further potentiating the Bz-insensitive Na+ flux across the apical membrane of TRCs. This results in the enhancement of the CT responses to mineral salts.
At present the exact mechanism of how nicotine modulates the activity of TRPV1 or TRPV1t is not known. However, several hypotheses have been put forward as possible mechanism of nicotine-induced sensitization of the TRPV1 channel (Liu et al. 2004). These include: nicotine-induced increase in the number of functional channels that increases the probability of the channel to remain in its open state (Hui et al. 2003; Kwak et al. 2000) or alterations of the cell membrane's mechanical properties that can in turn affect its binding to phosphatidylinositol-4,5-bisphosphate (PIP2) (Hui et al. 2003; Prescott and Julius 2003).
Nicotine also demonstrated interactions with RTX (Figs. 3C and 4D). In the presence of a subthreshold concentration of RTX (0.25 x 10–6 M) the nicotine-induced increase in the Bz-insensitive NaCl CT response was greater than that observed with RTX or nicotine alone. This increase in the response is related to the corresponding increase in the Bz-insensitive apical Na+ conductance in the presence of nicotine and RTX (Fig. 4D). In addition, in the presence of 0.25 x 10–6 M RTX, nicotine enhanced the CT response at room temperature and at elevated temperatures and shifted the temperature threshold of the channel to the left (Fig. 5C). These results suggest that the binding of RTX either to the intracellular binding site for CAP or to another ligand-independent allosteric site alters the conformation of the channel and sensitizes the channel to further stimulation with nicotine. The specificity of nicotine as a salt taste modulator is further demonstrated by the observation that TRPV1 KO mice, which lack the Bz-insensitive component of the NaCl CT response, also do not respond to nicotine (Fig. 6C), ethanol, RTX, and elevated temperatures (Lyall et al. 2005c). The data support the conclusion that nicotine produces its effect on salt responses by the amiloride- and Bz-insensitive TRPV1t cation channel in fungiform TRCs. However, the exact mechanism of how nicotine modulates the TRPV1t cation channel in TRCs remains to be established.
Possible consequences of nicotine-induced intracellular alkalinization and cell swelling in TRCs
Changes in TRC pHi and cell volume are involved in modulating salt taste (Lyall et al. 1995, 1999, 2002b) and acid taste transduction (Lyall et al. 2001, 2002a,b, 2006). Consistent with this our data indicate that at pH values >8, nicotine enters TRCs across the apical membrane, causing intracellular alkalinization and activation of ENaC. This results in an increase in Bz-sensitive NaCl CT response (Fig. 7, A and C). The pH of saliva secreted by the unstimulated human parotid gland ranges from 5.45 to 6.06. On stimulation the pH of parotid saliva becomes alkaline and increases to a maximum of 7.8 (Davenport 1982). It is likely that under these conditions the apical entry of nicotine into TRCs is enhanced. In a subset of TRCs an acid-induced decrease in pHi is the proximate signal for sour taste transduction (Lyall et al. 2001, 2002a,b, 2004a). In acid-sensing TRCs, a decrease in pHi may be sensed by PKD2L1 channel (Huang et al. 2006; Ishimaru et al. 2006; LopezJimenez et al. 2006). A decrease in TRC pHi is responsible for the elicitation of both the phasic and the tonic parts of the CT response to acid stimulation (Lyall et al. 2001, 2002b). The nicotine-induced intracellular alkalinization is accompanied by cell swelling (Fig. 2A). This results in a significant inhibition of the phasic part of the CT response to acids.
In addition, cell shrinkage activates Bz-sensitive ENaCs (Ji et al. 1998; Lyall et al. 1999, 2005c
) and (
