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Intracellular Ph Modulates Taste Receptor Cell Volume and The ARTICLE Intracellular pH Modulates Taste Receptor Cell Volume and the Phasic Part of the Chorda Tympani Response to Acids Vijay Lyall,1 Hampton Pasley,1 Tam-Hao T. Phan,1 Shobha Mummalaneni,1 Gerard L. Heck,1 Anna K. Vinnikova,2 and John A. DeSimone1 1Department of Physiology and 2Department of Internal Medicine, Virginia Commonwealth University, Richmond, VA 23298 The relationship between cell volume and the neural response to acidic stimuli was investigated by simultaneous measurements of intracellular pH (pHi) and cell volume in polarized fungiform taste receptor cells (TRCs) using 2’,7’-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) in vitro and by rat chorda tympani (CT) nerve recordings in vivo. CT responses to HCl and CO2 were recorded in the presence of 1 M mannitol and specific probes for filamentous (F) actin (phalloidin) and monomeric (G) actin (cytochalasin B) under lingual voltage clamp. Acidic stimuli reversibly decrease TRC pHi and cell volume. In isolated TRCs F-actin and G-actin were labeled with rhodamine phalloidin and bovine pancreatic deoxyribonuclease-1 conjugated with Alexa Fluor 488, respectively. A decrease in pHi shifted the equilibrium from F-actin to G-actin. Treatment with phalloidin or cytochalasin B attenuated the magnitude of the pHi-induced decrease in TRC volume. The phasic part of the CT response to HCl or CO2 was significantly decreased by preshrinking TRCs with hypertonic mannitol and lingual application of 1.2 mM phalloidin or 20 ␮M cytochalasin B with no effect on the tonic part of the CT response. In TRCs first treated with cytochalasin B, the decrease in the magnitude of the phasic response to acidic stimuli was reversed by phalloidin treatment. The pHi-induced decrease in TRC volume induced a flufenamic acid–sensitive nonselective basolateral cation conductance. Channel activity was enhanced at positive lingual clamp voltages. Lingual application of flufenamic acid decreased the magnitude of the phasic part of the CT response to HCl and CO2. Flufenamic acid and hypertonic mannitol were additive in inhibiting the phasic response. We conclude that a decrease in pHi induces TRC shrinkage through its effect on the actin cytoskeleton and activates a flufenamic acid–sensitive basolateral cation conductance that is involved in eliciting the phasic part of the CT response to acidic stimuli. INTRODUCTION Sour taste is a primary taste modality and is uniquely as- Recent studies indicate that the proximate signal for sociated with acidic stimuli. It evokes an innate rejec- sour taste transduction is an acid-induced decrease in tion response to extremely acidic or sour stimuli, limit- pHi in a subset of TRCs (Lyall et al., 2001). In the case ing the ad libitum ingestion of acid (Beauchamp et al., of strong acids, an apical adenosine 3’5’-cyclic mono- 1991; Scott and Plata-Salaman, 1991). In this regard, phosphate (cAMP)–sensitive, but Ca2ϩ-insensitive Hϩ along with the lungs and kidneys, which are the pri- conductance allows Hϩ entry and the subsequent de- The Journal of General Physiology mary organs for acid secretion, acid sensing taste recep- crease in TRC pHi (Lyall et al., 2002a; 2004a). However, tor cells (TRCs) in the oral cavity, by restricting the in- Hϩ-gated channels, such as the acid sensing ion chan- take of acid, function as part of a multi-organ system to nel (ASIC) in the apical membrane and both ASIC maintain acid–base homeostasis. Acids in aqueous solu- (Ugawa et al., 1998; Lin et al., 2002) and hyperpolariza- tion yield Hϩ, which can alter both the extracellular tion-activated channels (HCN) (Stevens et al., 2001) in (pHo) and intracellular pH (pHi) of cells. A large num- the basolateral membrane of TRCs, and TASK-2, a two ber of enzymatic reactions, channels, transporters, and pore domain Kϩ channel (Lin et al., 2004; Richter et intracellular signaling events are modulated by changes al., 2004a) may also play a role in sour taste transduc- in pHi. It is, therefore, a special challenge to identify the specific channels, transporters, and other intracel- Abbreviations used in this paper: BAPTA-AM, 1,2-bis(2-aminophe- lular signaling events in TRCs that sustain acid taste noxy)ethane-N,N,N’,N’-tetraacetic acid tetrakis-(acetoxymethyl es- transduction and ultimately the cortical events result- ter); BCECF, 2’,7’-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein; Bz, benzamil; cAMP, adenosine 3’5’-cyclic monophosphate; CPC, ing in sour taste sensation. In spite of this complexity, cetylpyridinium chloride; CT, chorda tympani; DAPI, 4’,6-diamidino- some progress has been made in understanding the un- 2-phenylin; F, filamentous; FIR, fluorescence intensity ratio; G, mono- derlying mechanisms involved in sour taste transduc- meric; NHE-1, Naϩ-Hϩ-exchanger-1; NPPB, 5-nitro-2-(3-phenylpropyl- tion at the level of TRCs. amino)-benzoate; ROI, region of interest; RVD, regulatory volume decrease; RVI, regulatory volume increase; SANSCC, shrinkage-acti- vated nonselective cation channel; TRC, taste receptor cell; TRP, Correspondence to Vijay Lyall: [email protected] transient receptor potential; VGCC, voltage-gated Ca2ϩ channel. J. Gen. Physiol. © The Rockefeller University Press $8.00 15 Volume 127 Number 1 January 2006 15–34 http://www.jgp.org/cgi/doi/10.1085/jgp.200509384 tion. These channels in the basolateral membrane lated to the phasic part of the neural response to acid could be activated if Hϩ can cross tight junctions and stimulation? decrease pH in the basolateral compartment. However, In this study, we tested the hypothesis that a decrease recent studies suggest that in mice ASIC2 is not re- in pHi induces a decrease in TRC volume by altering quired for acid taste (Richter et al., 2004b). In contrast, the cell actin cytoskeleton leading to the activation of weak organic acids do not seem to have apical mem- one or more membrane conductances that are in- brane receptors. They enter TRCs across the apical volved in eliciting the phasic part of the CT response to membrane by passive diffusion as lipid soluble undisso- acidic stimuli. This hypothesis is based on our previous ciated neutral molecules. Once inside the cell they dis- studies in which hypertonic solutions of mannitol or ϩ sociate to generate H and decrease TRC pHi (Lyall cellobiose (Lyall et al., 1999) or ethanol (Lyall et al., et al., 2001). TRCs also contain 5-nitro-2-(3-phenylpro- 2005a,b) induced cell shrinkage and elicited a tran- pylamino)-benzoate (NPPB)-sensitive stretch-activated sient phasic CT response. This suggests that a decrease ClϪ channels (Gilbertson, 2002). The presence of an in cell volume is an intracellular signal for eliciting a NPPB-sensitive ClϪ channel activated by acid was dem- transient neural response. Second, since only a tran- onstrated in mouse taste cells (Miyamoto et al., 1998). sient phasic response was observed in ethanol solutions These results suggest a role for ClϪ channels in acid in the absence of permeable ions on the apical side, it taste transduction. In addition to ClϪ channels, there is would appear that cell depolarization involves changes also evidence that NPPB-insensitive poorly selective cat- in a basolateral membrane ion conductance or conduc- ionic conductance in the apical receptive membranes tances uniquely associated with transient phasic re- of mouse TRCs may also be involved in sour taste trans- sponses (Lyall et al., 2005a,b). To test this hypothesis, duction (Miyamoto et al., 1998). The above data sug- we investigated the relationship between TRC volume gest that sour taste transduction is mediated via multi- and the CT response to acidic stimuli by simultaneously ple pathways. Similarly, in the case of other acid-sensi- monitoring pHi and cell volume in polarized rat fun- tive cells, such as the central chemosensitive neurons, a giform TRCs loaded with 2’,7’-bis-(2-carboxyethyl)-5- multiple factors model has been proposed for acid sig- (and-6)-carboxyfluorescein (BCECF) using imaging naling (Putnam et al., 2004). techniques in vitro, and by rat CT taste nerve record- Recent studies in our laboratory have focused on ings in vivo. The results demonstrate that during acid identifying specific cellular mechanisms that determine taste transduction a decrease in TRC pHi induces cell the neural response profiles to acidic stimuli. Similar to shrinkage through its effect on the cell actin cytoskele- other taste stimuli, acidic stimuli elicit chorda tympani ton. A decrease in volume activates a flufenamic acid– (CT) responses that are composed of two components sensitive nonselective cation conductance in the baso- with distinct temporal characteristics: a rapid transient lateral membrane of TRCs that is involved in eliciting increase in the phasic neural response that slowly de- the phasic part of the CT response to acidic stimuli. clines to a quasi-steady state, defined as the tonic phase Some of the data have appeared in abstract form (De- of the neural response. Treating rat tongue with spe- Simone et al., 2005). cific membrane-permeable blockers of carbonic anhy- drases (MK-417 or MK-507) inhibited both the phasic MATERIALS AND METHODS and tonic components of the CT response to CO2 (Ly- all et al., 2001, 2002b). This indicates that an acid- In Vitro Studies induced decrease in TRC pHi is necessary to elicit both the phasic and the tonic components of the CT re- Simultaneous Measurement of TRC Volume and pHi Using sponse to acid stimulation. In a subset of TRCs a de- BCECF. The animals were housed in the Virginia Common- wealth University animal facility in accordance with institutional crease in pHi is followed by an increase in intracellular guidelines. All in vitro and in vivo animal protocols were ap- 2ϩ 2ϩ Ca ([Ca ]i) (Liu and Simon, 2001; Lyall et al., 2003; proved by the Institutional Animal Care and Use Commit- 2ϩ Richter et al., 2003).
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