The CT response profiles for acidic stimuli are composed of a phasic and a tonic response. While several factors may be involved in determining the overall CT response profile to acidic stimuli, the data presented in this study demonstrate that the phasic and tonic parts of the CT response are largely determined by the entry of acid equivalents across the apical membrane and the exit of H+ across the basolateral membrane of TRCs via the NHE-1, respectively. The H+ conductive pathway in the apical membrane and the basolateral NHE-1 activity are regulated differentially by second messengers (cAMP or Ca2+) and pharmacological agents (zoniporide), and thus can be studied as separate entities.
In polarized TRCs, stimulating the apical membrane with acidic stimuli induced sustained decreases in pHi (Lyall et al. 2001, 2002a,b). Thus both strong acids and weak organic acids gain entry into TRCs across the apical cell membrane and induce a decrease in pHi. Weak organic acids permeate the apical membrane as neutral molecules, and strong acids via an H+ entry pathway that is both amiloride- and Ca2+-insensitive, but is activated by cAMP (Lyall et al. 2001, 2002a,b; DeSimone et al. 2001b). During acid stimulation, a decrease in TRC pHi, rather than a decrease in pHo, is the stimulus intensity variable that correlates specifically with increased CT taste nerve activity. Since inhibiting acid-induced TRC acidification also inhibits the acid-evoked CT response (Lyall et al. 2001, 2002b), it indicates that a decrease in TRC pHi is the proximate stimulus for sour taste.
Role of basolateral NHE-1 in TRC pHi regulation
Our pH- and Na+-imaging studies (Vinnikova et al. 2004) demonstrated that in TRCs, basolateral NHE-1 is functional in the nominal absence of CO2/HCO3−. At the physiological pH, NHE-1 activity is low. Thus, under control conditions, i.e. in the absence of an acid stimulus, TRCs regulate their pHi by maintaining a balance between the generation of intracellular H+ due to metabolic activity and the exit of intracellular acid equivalents from the cells, in part, by the basolateral NHE-1 (Vinnikova et al. 2004). At constant pHo, it is activated by intracellular acidification and by the Na+ concentration in the basolateral compartment. However, changes in pHo have an opposite effect on the basolateral NHE-1. An increase in basolateral pH enhanced, and a decrease in basolateral pH attenuated, the NHE-1 activity. Here, we demonstrate that NHE-1 activity is inhibited by zoniporide, a novel specific blocker of NHE-1 (cf. Figs 1–5). Cariporide (HOE 642), another specific blocker of NHE-1 activity, and amiloride, a non-specific blocker of NHEs, also inhibit NHE-1 activity in TRCs (Vinnikova et al. 2004).
Similar to the case in other tissues (Josette & Pouysségur, 1995; Ritter et al. 2001), in TRCs the basolateral NHE-1 activity is regulated by [Ca2+]i. Treating the basolateral membrane of polarized TRCs with ionomycin, a Ca2+ ionophore, increased [Ca2+]i, alkalinized resting pHi, and increased the spontaneous recovery of pHi from an NH4Cl pulse. Both the ionomycin-induced alkalinization and the increase in pHi recovery rate were blocked by zoniporide (Fig. 5).
Role of basolateral NHE-1 during acid stimulation
The topical lingual application of zoniporide enhanced the CT response to CO2 and acetic acid by 57% and 111%, respectively, relative to control (Fig. 6B). In contrast, HCl responses were not affected by zoniporide. The fact that zoniporide only enhanced the CT responses to CO2 and acetic acid, but not to HCl, is related to the pH of the acidic stimuli. The tongue was stimulated with CO2 solutions at the physiological pH of 7.4, and with acetic acid solutions that were adjusted to pH 6.1. In contrast, 20mm HCl stimulating solutions were at a pH of 1.7. We have previously shown that a decrease in pHo inhibits NHE-1 activity (Vinnikova et al. 2004). Similar dependence of NHE activity on pHo has been reported in other cells (Vaughan-Jones & Wu, 1990). Upon acidification of the basolateral compartment, protons bind to the external H+ binding site on the NHE-1 and inhibit its activity. Similarly, a decrease in apical pH inhibits the NHE-1 activity; however, the mechanism of inhibition is more complex, and most likely involves changes in cell volume and/or changes in cytoskeleton of the cell (Vinnikova et al. 2004). Thus, it follows that, during HCl stimulation at pHo 1.7, the basolateral NHE-1 activity is already inhibited and zoniporide treatment does not produce additional inhibition of the exchanger. In contrast, at pH 7.4 or 6.1, the basolateral NHE-1 still retains its basal activity, which can be blocked by zoniporide, eliciting increased CT responses to CO2 and acetic acid stimulation relative to control.
Effect of NHE-1 activation on CT responses to acid stimulation
The Ca2+-induced increase in NHE-1 activity has important consequences for both TRC pHi regulation in vitro and the CT responses during acid stimulation in vivo (Lyall et al. 2002a). Stimulating the apical membrane with HCl induced a sustained decrease in TRC pHi. However, upon NHE-1 stimulation with ionomycin, the HCl-induced decrease in TRC pHi was transient and demonstrated rapid recovery towards its baseline value (Lyall et al. 2002a). Consistent with these in vitro results, topical lingual application of ionomycin, did not alter the initial magnitude of the CT responses to HCl (the phasic part of the CT response) but greatly accelerated the adaptation phase (tonic phase) of the neural response (Figs 7 and 8). The data further demonstrate that these effects of ionomycin can be generalized to strong acids, as well as to weak organic acids, such as CO2 and acetic acid. The ionomycin-induced enhanced adaptation in the CT response was independent of pHo and the accompanying anion. Although, these data are sufficient to demonstrate that the activation of a pH recovery mechanism in TRCs results in the rapid adaptation of the neural response to acid stimulation, they do not, by themselves, prove that the basolateral NHE-1 is involved in this process.
The additional proof that it is indeed the activation of basolateral NHE-1 that brings about the neural adaptation comes from our studies with zoniporide. Under control conditions, topical lingual application of zoniporide, increased CT responses to CO2 and acetic acid. In addition, zoniporide pretreatment reversed the ionomycin-induced increase in the adaptation phase of the CT responses to HCl, CO2 and acetic acid, in a dose-dependent manner. At a zoniporide concentration of 500μm, the CT responses to all three acidic stimuli, following ionomycin treatment, were at or exceeded control level responses. Thus at zoniporide concentrations that cause complete inhibition of the basolateral NHE-1 activity, an increase in TRC [Ca2+]i failed to enhance its activity and abolished high [Ca2+]i-induced neural adaptation.
Recent studies indicate that acid responses occur in a subset of TRCs that participate in sour taste transduction. There are significant variations in the resting pHi values in individual ROIs within a taste bud. TRCs can be separated into two distinct populations based on their initial value of resting pHi. One subset of TRCs demonstrated a normal distribution with a mean pHi of around 7.2. The second subset demonstrated a skewed distribution with a mean pHi of around 7.45 (Vinnikova et al. 2004). It is suggested that TRCs having relatively more alkaline resting pHi values, and consequently lower buffer capacity (β1) values, will produce greater decreases in pHi when stimulated with acids.
In polarized taste bud preparations, stimulating with apical acetic acid or HCl at pH 3.0, produced significantly different regional changes in TRC pHi. At the same pH acetic acid produced a greater mean decrease in TRC pHi compared to HCl. The changes in pHi varied widely within different ROIs after treatment with the two acids. After HCl treatment 34.5% of ROIs responded with a decrease in pHi>0.3 pH unit. It is likely that TRCs in the ROIs that respond with the greatest decrease in pHi participate most in sour transduction. In contrast, after acetic acid treatment, 91.4% of ROIs responded with a decrease in pHi>0.3 pH unit (Lyall et al. 2001). The data suggest that HCl-induced CT responses are elicited by a subpopulation of TRCs contained in different ROIs within the taste bud that contain an apical H+ entry mechanism. This conclusion is further supported by the observations that cAMP increases H+ conductive entry across the apical membranes of TRCs (Fig. 10) and enhanced CT responses to HCl stimulation without affecting responses to acetic acid or CO2 (Fig. 9). In contrast, for acetic acid, the influx of acid equivalents into TRCs is augmented by a significant flow of unionized acetic acid that results in a greater CT response relative to HCl (Lyall et al. 2001).
Although stimulating the apical membrane with acid stimuli decreases pHi in a significant number of TRCs within the taste bud, only a subset of TRCs respond with a decrease in pHi and a concomitant increase in [Ca2+]i (Liu & Simon, 2001; Richter et al. 2003; Lyall et al. 2003). Since an increase in [Ca2+]i is a prerequisite for the release of neurotransmitter, it is suggested that this subset of TRCs participate in sour taste transduction within the taste bud. Thus, it is likely that an acid-induced increase in TRC [Ca2+]i subsequently activates the basolateral NHE-1 and brings about pHi recovery and serves as a source of neural adaptation.
In a subset of TRCs in which an acid-induced decrease in pHi is not accompanied by a concomitant increase in [Ca2+]i, changes in pHi may participate in mixture interactions and/or in modulating responses to other taste modalities. We have previously shown that in TRCs containing ENaC, changes in pHi modulate amiloride-sensitive apical Na+ entry and the rat CT responses to NaCl (Lyall et al. 2002b).
Our studies further suggest that cAMP and [Ca2+]i modulate CT responses to acids independently (Figs 9–11). Cyclic AMP enhanced the phasic part of the CT responses to HCl, but had no effect on the CT responses to CO2 and acetic acid. The observed voltage sensitivity of the post-cAMP CT responses to HCl suggest that cAMP activates an apical H+ conductance. However, cAMP does not affect the passive apical entry of the undissociated weak organic acids. In contrast, changes in [Ca2+]i modulate the tonic part of the CT response to both strong and weak organic acids. Since in the presence of cAMP, topical lingual application of ionomycin enhanced neural adaptation to HCl stimulation, we conclude that the effects of [Ca2+]i are independent of those due to cAMP. This is consistent with the concept that the sites of action of [Ca2+]i and cAMP are different, non-interacting cellular entities, i.e. the basolateral NHE1 and an apical H+ conducting pathway, respectively.
The main conclusions of the paper and the proposed mechanisms for the regulation of neural adaptation are summarized in a schematic diagram (Fig. 12). The apical cell membranes of TRCs contain acid entry mechanisms. In the case of fully dissociated strong acids, H+ ions enter across the apical membrane of TRCs via an amiloride- and Ca2+-insensitive, but cAMP-sensitive H+ pathway (Lyall et al. 2002a). Weak organic acids, such as acetic acid and dissolved CO2, enter across the apical membrane as neutral molecules and decrease pHi by generating intracellular acid equivalents. In the case of CO2, the dissolved gas is converted to H2CO3 by intracellular carbonic anhydrases, subsequently, yielding H++ HCO3−. However, there is some evidence that H+-gated channels, such as the acid-sensing ion channel (ASIC) in the apical membrane of TRCs (Ugawa et al. 1998; Lin et al. 2002), and both ASIC (Ugawa et al. 1998; Lin et al. 2002) and hyperpolarization-activated channels (HCN) (Stevens et al. 2001) in the basolateral membranes of TRCs may also play a role in sour taste transduction. These channels in the basolateral membrane could be activated if H+ ions can cross the tight junctions and decrease pHi in the basolateral compartment.
Figure 12. Proposed model for the mechanism of neural adaptation to acidic stimuli Our data suggest that the basolateral NHE-1 is involved in neural adaptation during sour taste transduction. Acidic stimuli applied to the lingual surface induce a decrease in TRC pHi as a result of the entry of acid equivalents across the apical cell membranes of TRCs. The apical membranes of TRCs exhibit an amiloride- and Ca2+-insensitive, cAMP-dependent H+ conductive pathway for strong acids (purple). In contrast, weak organic acids permeate across the apical membranes passively as undissociated molecules. The pHi recovery occurs, in part, due to the presence of zoniporide- and cariporide-sensitive basolateral NHE-1. Although NHE-3 is present in the apical membranes of TRCs, under our experimental conditions the NHE-3 seems to be quiescent, and does not participate in pHi regulation in TRCs (green). In the model the pathways for the apical entry and the basolateral exit of acid equivalents are shown in a single TRC. However, within a taste bud, TRCs are heterogeneous, and it is quite likely that not all of the above components are present in all cells. The favourable Na+ gradient for the Na+–H+ exchangers is maintained by the basolateral Na+–K+-ATPase. An increase in TRC [Ca2+]i induced by ionomycin, activates basolateral NHE-1, increasing the neural adaptation in CT responses to acidic stimuli. Zoniporide, a specific blocker of NHE-1, increased the magnitude of the CT responses to acidic stimulation under control conditions, and completely reversed the ionomycin-induced increase in neural adaptation to acidic stimuli. We conclude that during sour taste transduction the basolateral NHE-1 plays an important role in determining the adaptation (tonic) phase of the CT taste nerve responses to acidic stimuli.
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The H+ exit from the cells occurs in part, via the zoniporide-sensitive basolateral NHE-1. The accompanying Na+ ions exit TRCs via the basolateral ouabain-sensitive Na+–K+-ATPase. We have previously demonstrated the presence of mRNA transcripts for NHE-3 in TRCs and the specific binding of NHE-3 antibodies to the apical membranes of TRCs; however, under the experimental conditions examined so far, the apical NHE-3 appears to be quiescent and does not contribute to pHi regulation in TRCs (Vinnikova et al. 2004). Although the model depicts the presence of acid entry and exit pathways in a single TRC, it must be emphasized that within a taste bud, TRCs are heterogeneous, and it is likely that not all of the above mechanisms are present in all taste cells. Topical lingual application of ionomycin + CaCl2, increases TRC [Ca2+]i and results in the activation of basolateral NHE-1. The activation of NHE-1 results in an acid-induced decrease in pHi that is transient, i.e. recovers spontaneously, leading to a rapid adaptation in the CT responses to acid stimulation. However, topical lingual application of zoniporide, a specific NHE-1 blocker, reverses the observed adaptation in CT responses to acid stimulation. Taken together, the data indicate that in TRCs, a basolateral NHE-1 is involved in neural adaptation to acidic stimuli. Recent, studies (Liu & Simon, 2001; Richter et al. 2003; Lyall et al. 2003) suggest that in a subset of TRCs an acid-induced decrease in pHi is accompanied by an increase in TRC [Ca2+]i. It is likely that an increase in TRC [Ca2+]i activates basolateral NHE-1 and increases neural adaptation.