IP3-Independent Release of Ca2+ From Intracellular Stores: A Novel Mechanism for Transduction of Bitter Stimuli

Tatsuya Ogura and Sue C. Kinnamon

Department of Anatomy and Neurobiology, Colorado State University, Fort Collins, Colorado 80523; and Rocky Mountain Taste and Smell Center, University of Colorado Health Sciences Center, Denver, Colorado 80262


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Ogura, Tatsuya and Sue C. Kinnamon. IP3-Independent Release of Ca2+ From Intracellular Stores: A Novel Mechanism for Transduction of Bitter Stimuli. J. Neurophysiol. 82: 2657-2666, 1999. A variety of substances with different chemical structures elicits a bitter taste. Several different transduction mechanisms underlie detection of bitter tastants; however, these have been described in detail for only a few compounds. In addition, most studies have focused on mammalian taste cells, of which only a small subset is responsive to any particular bitter compound. In contrast, ~80% of the taste cells in the mudpuppy, Necturus maculosus, are bitter-responsive. In this study, we used Ca2+ imaging and giga-seal whole cell recording to compare the transduction of dextromethorphan (DEX), a bitter antitussive, with transduction of the well-studied bitter compound denatonium. Bath perfusion of DEX (2.5 mM) increased the intracellular Ca2+ level in most taste cells. The DEX-induced Ca2+ increase was inhibited by thapsigargin, an inhibitor of Ca2+ transport into intracellular stores, but not by U73122, an inhibitor of phospholipase C, or by ryanodine, an inhibitor of ryanodine-sensitive Ca2+ stores. Increasing intracellular cAMP levels with a cell-permeant cAMP analogue and a phosphodiesterase inhibitor enhanced the DEX-induced Ca2+ increase, which was inhibited partially by H89, a protein kinase A inhibitor. Electrophysiological measurements showed that DEX depolarized the membrane potential and inhibited voltage-gated Na+ and K+ currents in the presence of GDP-beta -S, a blocker of G-protein activation. DEX also inhibited voltage-gated Ca2+ channels. We suggest that DEX, like quinine, depolarizes taste cells by block of voltage-gated K channels, which are localized to the apical membrane in mudpuppy. In addition, DEX causes release of Ca2+ from intracellular stores by a phospholipase C-independent mechanism. We speculate that the membrane-permeant DEX may enter taste cells and interact directly with Ca2+ stores. Comparing transduction of DEX with that of denatonium, both compounds release Ca2+ from intracellular stores. However, denatonium requires activation of phospholipase C, and the mechanism results in a hyperpolarization rather than a depolarization of the membrane potential. These data support the hypothesis that single taste receptor cells can use multiple mechanisms for transducing the same bitter compound.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Many compounds with diverse structures elicit a bitter taste in humans (Belitz and Weiser 1985). This structural diversity implies the presence of multiple transduction mechanisms and/or multiple receptor proteins in taste receptor cells. Several mechanisms have been proposed, and even multiple mechanisms have been reported for single compounds. For example, quinine has been reported to stimulate inositol-1,4,5-triphosphate (IP3) production (Spielman et al. 1996), decrease intracellular cAMP (Ming et al. 1998) and directly block K+ channels (Cummings and Kinnamon 1992), but whether these mechanisms coexist in the same taste cells has not been determined. Accordingly, we have undertaken a study using a variety of methodologies in a single species to examine mechanisms of bitter transduction.

Several investigators have reported bitter transduction mechanisms involving activation of G proteins. The first involves a G-protein-mediated activation of phospholipase C (PLC), which results in increased intracellular levels of IP3. The IP3 elicits release of Ca2+ from intracellular stores; this is proposed to lead directly to neurotransmitter release (Akabas et al. 1988; Hwang et al. 1990; Ogura et al. 1997a; Spielman et al. 1994). Another mechanism involves a G-protein-mediated activation of phosphodiesterase (PDE), leading to a decrease in intracellular levels of cyclic nucleotides (Ruiz-Avila et al. 1995; Wong et al. 1996). The decrease in cyclic nucleotides may depolarize taste cells by relieving block of a direct cyclic nucleotide-blocked cation channel (Kolesnikov and Margolskee 1995). These two hypotheses are not mutually exclusive. Recent studies suggest that the bitter compound denatonium elicits both increases in IP3 and decreases in cAMP in the same tissue (Yan et al. 1999). However, whether these mechanisms occur in the same taste cells has not been determined. Activation of second messengers presumably requires the binding of bitter compounds to G-protein-coupled receptors. Recently, a putative bitter taste receptor protein was cloned (Hoon et al. 1999), but the protein has not been characterized functionally.

Several bitter compounds do not require G-protein-coupled receptors for transduction. In the mudpuppy, Necturus maculosus, quinine and CaCl2 directly block voltage-gated K+ channels that are located on the apical membrane of taste cells (Bigiani and Roper 1981; Cummings and Kinnamon 1992; Kinnamon and Roper 1988); K+-channel block leads to membrane depolarization and transmitter release. In frog, quinine activates secretion of intracellularly accumulated Cl- through Cl- pumps on the apical receptive membrane, resulting in membrane depolarization (Sato et al. 1994). Recent studies have shown membrane permeant bitter compounds can increase intracellular cGMP by direct inhibition of PDE (Rosenzweig et al. 1999), which may depolarize cells by opening cyclic nucleotide-gated cation channels (Misaka et al. 1997). Other receptor-independent events, such as changing the phase-boundary potential at the outer surface of the membrane, have been reported in response to lipophilic bitter compounds (Koyama and Kurihara 1972). It is not known if these nonspecific effects contribute to bitter transduction in taste cells.

One of the difficulties in revealing general mechanisms for bitter transduction is that they have resulted from studies using different bitter compounds, different animal species, and a variety of techniques. To investigate whether the same bitter compound can use multiple transduction mechanisms in the same taste cells, it is reasonable to compare data obtained in a single species and with similar techniques. The mudpuppy, N. maculosus, is an ideal model for studying bitter transduction (Bowerman and Kinnamon 1994; Kinnamon 1992; Ogura et al. 1997a). First, mudpuppy taste cells are large, easily isolated, and amenable to both Ca2+ imaging and patch-clamp recording. In addition, most mudpuppy taste cells are bitter sensitive (Ogura et al. 1997a). This situation is in sharp contrast to mammals, where only a small fraction of taste cells responds to any particular bitter stimulus (Bernhardt et al. 1996). Low responsivity in mammalian taste cells makes detailed pharmacological dissection of transduction extremely difficult.

Our previous studies on bitter transduction in mudpuppy taste cells have revealed two different transduction mechanisms for bitter compounds, one relying on release of Ca2+ from intracellular stores and the other involving direct block of apical K+ channels. Denatonium, an extremely bitter compound to humans, increased intracellular Ca2+ levels ([Ca2+]i) by an IP3-mediated release of Ca2+ from intracellular stores. The elevation in [Ca2+]i activated Ca2+-dependent K+ and Cl- currents, which hyperpolarized the membrane potential. Intracellular cAMP levels did not significantly modify the Ca2+ responses (Ogura et al. 1997a). In contrast, quinine (Kinnamon 1992) and CaCl2 (Bigiani and Roper 1991) depolarized mudpuppy taste cells by direct block of the apical K+ conductance. In the present report, we studied the transduction of dextromethorphan bromide (DEX) in mudpuppy taste receptor cells by means of using Ca2+ imaging and whole cell patch recording. DEX, an antitussive, is avoided by pigs in behavioral tests and causes a strong bitter taste in humans (at 8.1 mM) (Nelson and Sanregret 1997). To test whether DEX uses transduction mechanisms similar to either quinine or denatonium, we used recording procedures similar to those used previously (Ogura et al. 1997a). Some of these results have been published in abstract form (Ogura et al. 1997b, 1998).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Isolation of taste receptor cells

Mudpuppies (N. maculosus) were obtained from commercial sources and housed in large aquaria at 10°C. Minnows were provided weekly as a food source. Taste receptor cells were isolated as described previously (Kinnamon and Roper 1988; Ogura et al. 1997a). Briefly, mudpuppies were decapitated after anesthesia in ice-cold water, and the lingual epithelium was separated from the underlying connective tissue. The apical surface of the stripped epithelium was then incubated for 15 min in fluorescein-conjugated wheat germ agglutinin (Molecular Probes: 0.5 mg/ml in amphibian physiological saline, APS), so that mature taste cells could be distinguished from other cell types after isolation (Kinnamon et al. 1988). The epithelium then was incubated in APS containing collagenase (1 mg/ml; Sigma, Type1), albumin (1 mg/ml), and glucose (5 mM) until the epithelium could be separated gently from the underlying connective tissue, leaving the taste buds atop their connective tissue papillae. The taste buds were dissociated in Ca2+-free APS. Isolated taste cells were removed by gentle suction and plated onto recording chambers made with cover slips (for Ca2+ imaging) or glass slides (for whole cell recording), both coated with Cell-Tak (Collaborative research).

Intracellular calcium measurement

[Ca2+]i in isolated taste receptor cells was measured using the membrane-permeable Ca2+ -sensitive dye fura-2 AM as described previously (Ogura et al. 1997a). Briefly, cells were loaded with fura-2 AM (2 µM, Molecular Probes) in the presence of a dispersing reagent, Pluronic F-127 (final <0.02%, Molecular Probes) for 10 min, then washed with normal APS for 20 min. Images were acquired with an intensified CCD camera (IC100-ICCD, Paultek Imaging) through an oil-immersion objective lens (Fluor ×40, 1.3 NA, Nikon) of an inverted microscope (Diaphot TMD, Nikon). The video signal from the camera was captured using a frame grabber board (Quick Capture, Data Translation) on a Macintosh computer (Quadra 800, Apple Computer). For dual-wavelength ratiometric Ca2+-measurements, pairs of fluorescent images were recorded at 350- and 380-nm excitation using a filter wheel (EMPIX Imaging). A 510- to 580-nm band-pass filter (Chroma Technology) collected emitted light. Intracellular Ca2+ concentration was calculated in selected areas from the ratio of 350- and 380-nm images (Grynkiewicz et al. 1985), using the public domain NIH Image program (developed at the National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/). The calcium calibration kit II (C-3009, Molecular Probes) was used to obtain Ca2+ calibration curves. Time-course measurements were obtained by plotting the time course of [Ca2+]i averaged over most of the cell area excluding the edges of the cell. Generally, <10 s. was required for total exchange of solutions in the 200-µl chamber by superfusion.

Cells were bathed in normal APS until the resting intracellular calcium level was stable. The bath then was perfused with APS containing dextromethorphan hydrobromide (DEX, 2.5 mM, Sigma and gift from the Procter and Gamble) or denatonium benzoate (2.5 mM, Sigma). Washing with normal APS followed until the intracellular calcium again reached prestimulus levels. Other treatments included: Ca2+-free APS, ryanodine (100 µM in APS for 5-10 min, Calbiochem), thapsigargin (1 µM in APS for 12-15 min, Sigma), U73122 (5 µM in APS for 5-10 min, Calbiochem), H89 (10 µM in APS for 15-20 min, Calbiochem), and a mixture of isobutyl methoxyxanthine (IBMX, 100 µM, Sigma) and 8-chlorophenylthio-cAMP (8-cpt-cAMP, 1 mM, Sigma) in APS for 3 min.

Each taste cell was considered to respond to DEX if it experienced an increase in [Ca2+]i that was >2 SD above the mean resting level. The effects of drug treatments on the DEX response were assessed using Student's t-tests. Statistical values are presented as mean [Ca2+]i ± SE.

Giga-seal whole cell recording

Membrane currents were measured using giga-seal whole cell recording (Hamill et al. 1981). Electrodes were made from microhematocrit capillary tubes (American Scientific Products, McGaw Park, IL) pulled on a two-stage vertical puller (PB-7, Narishige). When filled with intracellular saline, electrode resistance ranged from 5 to 7 MOmega . Cells were viewed at a magnification of ×400 using a Nikon Diaphot inverted microscope fitted with Hoffman optics. Seals of 1-10 GOmega were obtained by gentle suction, and entry into the cell was achieved by delivery of a short depolarizing pulse to the pipette. Whole cell currents were measured at room temperature using an Axopatch 200B patch-clamp amplifier (Axon Instruments). Signals were filtered at 5 kHz and recorded digitally at 100 µs. Data were stored using a laboratory computer (11/23, Digital Equipment Corporation) equipped with a Cheshire data interface and Basic 23 software (Indec Systems). All voltage commands were computer generated. Unless otherwise noted, leak and linear capacitative currents were subtracted from all records by computer. Series resistance, which was typically <10 MOmega , was not compensated. Gravity-fed stimuli were bath-applied to the 0.5-ml recording chamber. To prevent loss of the seal and to prevent perfusion artifacts during whole cell recording, the perfusion rate was lowered to 2-3 ml/min.

After membrane capacitance was compensated electronically, membrane currents were recorded in APS in response to depolarizing commands from a holding potential of -80 mV. The bath then was perfused with DEX (2.5 mM), and membrane currents were measured again. Finally, DEX was washed out of the bath with normal APS and the currents measured again. To record the time course of the DEX effect, only two voltage steps were used: one for inducing inward current and the other for inducing outward current. For some recordings, the tip of the pipette was filled with the normal pipette solution but back-filled with a solution containing 1 mM GDP-beta -S (Sigma) to inhibit activation of G proteins.

Solutions

Normal APS contained (in mM) 112 NaCl, 2 KCl, 8 CaCl2, and 3 HEPES, buffered to pH 7.2 with NaOH. Ca2+-free APS contained either 1 mM BAPTA (for cell isolation) or 1 mM EGTA (for Ca2+ imaging) without CaCl2 in normal APS. Patch pipette solution contained (in mM) 114 KCl, 2 NaCl, 0.09 CaCl2, 2 MgCl2, 1 BAPTA, 1 ATP, 0.4 GTP, and 10 HEPES, buffered to pH 7.2 with KOH. For recording Ba2+ currents, 30 mM NaCl was replaced with 20 mM BaCl2, 10 mM TEA Cl, and 0.001 mM TTX were added in APS, and KCl was replaced by CsCl in patch pipette solution.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

DEX increases [Ca2+]i in taste receptor cells

We measured DEX-induced changes in [Ca2+]i in isolated mudpuppy taste cells using calcium imaging with the Ca2+-sensitive fluorescent dye fura-2. Previously, we showed that >80% of mudpuppy taste cells respond to denatonium with an increase in [Ca2+]i (Ogura et al. 1997a). More than 90% of these denatonium-sensitive taste cells also responded to DEX with an increase in [Ca2+]i (67 of 72 cells); cells that did not respond to denatonium usually did not respond to DEX. Figure 1 compares the time course of responses to denatonium and DEX in the same taste cells. Responses to denatonium reached a peak rapidly and began declining, even in the continued presence of the stimulus. In contrast, responses to DEX were slower in most cells (Fig. 1, A-D and G) and did not decline until the stimulus was removed from the bath (Fig. 1, A, C, and D). A few cells, however, showed responses to DEX that resembled the kinetics of the denatonium response (Fig. 1F). An interesting feature of some DEX responses was a temporal increase in [Ca2+]i immediately after the wash-out of DEX; these OFF responses were larger than the ON response in some cells (Fig. 1G). OFF responses were not usually observed in response to denatonium. The peak [Ca2+]i elicited by 2.5 mM DEX was usually 20-80% above resting [Ca2+]i (43 ± 5%, n = 67). Repeated applications of DEX to the same cells resulted in similar increases in [Ca2+]i, as long as the [Ca2+]i was returned to resting levels after each wash (1st application, 53 ± 11% increase; 2nd application, 56 ± 13% increase, n = 11, see Fig. 2, control).



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Fig. 1. Time course of changes in [Ca2+]i. Measurements of [Ca2+]i in individual cells using fura-2. Dextromethorphan (DEX, 2.5 mM) and denatonium (DN, 2.5 mM) were applied during periods labeled DEX and DN, respectively. A: DEX- and DN-induced Ca2+ responses in the same taste cell. B: DEX-induced Ca2+ response was present in Ca2+-free extracellular solution. C: thapsigargin (1 µM) abolished the DEX- and the DN-induced Ca2+ responses. D: phospholipase C (PLC) inhibitor U73122 (5 µM) abolished the DN-induced Ca2+ response but not the DEX-induced response. E: ryanodine (100 µM) had no effect on the DEX-induced response. F: DEX failed to induce a Ca2+ response immediately after a DN-induced Ca2+ response. G: representative recording of a DEX-induced OFF response. In this cell, the OFF response was larger than the DEX-induced response. DN seldom induced OFF responses.



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Fig. 2. DEX-induced changes in [Ca2+]i depended on intracellular Ca2+ stores. Maximum DEX-induced changes in [Ca2+]i are expressed as a percentage of resting [Ca2+]i. Cells were tested twice, before () and during or after () the treatments indicated, as illustrated in Fig. 1: [Ca2+-free bath solution (0 Ca2+; n = 14), thapsigargin (n = 15), U73122 (n = 8), ryanodine (n = 14)]. Control cells were tested twice (n = 11); 1st and 2nd responses are indicated by  and , respectively. *, significant effect.

DEX releases Ca2+ from intracellular stores

Intracellular Ca2+ can increase by release from intracellular stores or by Ca2+ influx. To determine whether extracellular Ca2+ was required, we compared the DEX-induced increase in [Ca2+]i in normal and in Ca2+-free APS in the same cells. The DEX-induced increase in [Ca2+]i persisted even in Ca2+-free APS. The magnitude of the response, although slightly smaller, was not significantly different from in normal APS (Figs. 1B and 2; 0 Ca2+, paired 1-tailed Student's t-test = 0.62, df = 13, P = 0.27). These data suggest that intracellular stores are the primary source for the increase in [Ca2+]i. To investigate further the role of Ca2+ stores in the response, we used thapsigargin, a Ca2+-ATPase inhibitor. Thapsigargin inhibits the reloading of Ca2+ stores, resulting in their gradual depletion of Ca2+ (Meyer and Stryer 1990; Thastrup et al. 1990). Thapsigargin (1 µM) increased [Ca2+]i to a variable extent in all taste cells tested. This increase in [Ca2+]i was slow, as has been shown previously (Ogura et al. 1997a). After incubation with thapsigargin for 10-15 min, which should be sufficient for store depletion, neither 2.5 mM DEX nor 2.5 mM denatonium increased [Ca2+]i in all cells tested (Fig. 1C). The effect of thapsigargin on the response to DEX was statistically significant (paired t- test = 3.37, df = 14, P < 0.003, Fig. 2, thapsigargin). These data strongly suggest that DEX releases Ca2+ from intracellular stores. Because denatonium also releases Ca2+ from intracellular stores (Akabas et al. 1988; Hwang et al. 1990; Ogura et al. 1997a), we examined whether the mechanism of Ca2+ release in response to DEX is the same as the response to denatonium. These results are described in the following section.

Intracellular signaling pathway for DEX response

Two types of Ca2+ stores have been identified in many types of cells: one coupled to an IP3 receptor and the other coupled to a ryanodine receptor (Sharp et al. 1993; Simpson et al. 1995). To investigate whether ryanodine receptor-coupled Ca2+ stores are involved in the DEX response, we applied DEX in the presence of ryanodine, which inhibits Ca2+ release from the stores (Sutko et al. 1985). Ryanodine (100 µM for 5-10 min) had no effect on resting [Ca2+]i and did not inhibit the [Ca2+]i increase in response to DEX (Figs. 1E and 2; paired t-test = 0.88, df = 13, P = 0.20). Thus ryanodine receptor-coupled Ca2+ stores are not likely to be involved in the response to DEX.

To determine whether activation of PLC is involved in the DEX response, we used U73122, a PLC inhibitor (Salari et al. 1993; Thompson et al. 1991). Incubation with U73122 (5 µM for 10-15 min) completely inhibited the denatonium-induced response, as shown previously (Ogura et al. 1997a), but the response to DEX was unaffected (Figs. 1D and 2; paired t-test = 0.88, df = 7, P = 0.21). These results suggest that U73122-sensitive PLC is not involved in the response to DEX.

To determine if DEX and denatonium release Ca2+ from the same intracellular stores, we applied DEX immediately after stimulation with denatonium without an intervening wash. If DEX and denatonium release Ca2+ from different Ca2+ stores, the two Ca2+ responses should be mutually independent. However, as shown in Fig. 1F, DEX failed to induce a Ca2+ response when applied subsequent to denatonium. These data suggest that DEX and denatonium both release Ca2+ from the same IP3-coupled Ca2+ stores, but the DEX response apparently does not require activation of PLC.

OFF responses to DEX

An OFF response, involving a temporal increase in [Ca2+]i immediately after wash out of DEX, was observed in 45% (57 of 126 cells) of the taste cells that responded to DEX. Interestingly, we seldom observed OFF responses to denatonium. In some cells, the OFF response to DEX was larger than the DEX response itself (cf. Fig. 1G). The OFF response persisted in Ca2+-free saline (n = 10, data not shown), suggesting that the off response involves release of Ca2+ from intracellular stores. Electrophysiological OFF responses to taste stimuli have been described previously (Cummings et al. 1993; Tsunenari et al. 1996) but the mechanisms involved are not known. It is possible that electrophysiological off responses may result from the transient increases in [Ca2+]i due to release from intracellular stores.

Intracellular cAMP levels enhance DEX responses

A possible involvement of cAMP in the response to DEX was examined by increasing intracellular cyclic nucleotide concentrations because cAMP levels are modulated by some bitter compounds (Kinnamon and Margolskee 1996). Incubation with a mixture of IBMX (100 µM, a phosphodiesterase inhibitor) and 8-cpt-cAMP (1 mM, a cell permeant cAMP analogue) did not affect resting [Ca2+]i (Fig. 3). However, the DEX-induced increase in [Ca2+]i was enhanced under these conditions (Fig. 3). In the presence of IBMX and 8-cpt cAMP, the increase in [Ca2+]i reached peak levels faster than under control conditions, and the response began to decline in the continued presence of DEX. The increase in peak [Ca2+]i was significant (paired t-test = 3.3, df = 24, P < 0.002, Figs. 3 and 4, cAMP). Incubation with either IBMX (100 µM) or 8-cpt-cAMP (1 mM) alone did not enhance the DEX response (data not shown). The enhanced response persisted in Ca2+-free bath solution (paired t-test = 2.2, df = 3, P =0.12, Fig. 4, cAMP in 0 Ca2+), suggesting that enhanced portion of the Ca2+ response was due to release of Ca2+ from intracellular stores. The enhanced response was blocked partially after incubation in H89 (10 µM, a PKA inhibitor) in the presence of IBMX and 8-cpt-cAMP. The effect of H89 on the response to DEX was statistically significant (paired t-test = 3.6, df = 7, P < 0.005, Figs. 3B and 4, cAMP + H89). There was no effect of H89 itself on the DEX response (paired t-test = 0.37, df = 5, P = 0.36, Fig. 4, H89). These data suggest that DEX is unlikely to increase cAMP levels directly because incubation with the mixture of IBMX and 8-cpt-cAMP alone did not mimic the DEX response. Thus it is likely that cAMP levels (and PKA) are modulated by other regulatory mechanisms, which subsequently modulate the response to DEX.



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Fig. 3. Effect of increased cAMP level on DEX-induced Ca2+ response. A: mixture of isobutyl methoxyxanthine (IBMX; 100 µM) and 8-chlorophenylthio-cAMP (8-cpt-cAMP; 1 mM) enhanced DEX-induced Ca2+ responses. B: protein kinase A (PKA) inhibitor H89 (10 µM) partially suppressed the enhanced-response.



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Fig. 4. DEX-induced Ca2+ response is enhanced by PKA phosphorylation via increase in cAMP. This graph shows maximum DEX-induced changes in [Ca2+]i expressed as percentage of resting [Ca2+]i. Control cells are those illustrated in Fig. 1. Other cells were tested, before () and during () the treatments indicated: mixture of IBMX and 8-cpt-cAMP (cAMP; n = 25, see Fig. 3A); mixture of IBMX and 8-cpt-cAMP in Ca2+-free bath solution (cAMP in 0 Ca2+; n = 14); mixture of IBMX and 8-cpt-cAMP () followed by H89 with the mixture (; cAMP + H89; n = 8, see Fig. 3B); H89 only (H89; n = 6). *, significant effects, **, significant effect of H89 on the mixture compared with the mixture alone.

DEX inhibits voltage-gated currents

We used giga-seal whole cell recording to examine the effect of DEX on membrane potential and voltage-dependent currents. Cells were voltage-clamped at a holding potential of -80 mV, and current was measured in response to voltage step pulses from -50 to +70 mV. A representative I-V relationship of peak inward current and steady outward current measured at 17.5 ms is plotted in Fig. 5A. Perfusion of DEX for 1 min completely blocked the transient inward Na+ current and significantly reduced outward K+ currents (Fig. 5A, right). The average inhibition of outward currents elicited by voltage steps to +40 mV was 47 ± 8.5% (n = 18). To examine the time course of the response, peak inward and steady state outward currents were monitored by voltage steps to -20 and +40 mV, respectively (Fig. 5B). These electrophysiological responses to DEX reached maximum levels more rapidly than responses in Ca2+-imaging experiments. In addition to Na+ and K+ currents, mudpuppy taste cells express a prominent, slowly inactivating Ca2+ current that is usually hidden in the large sustained outward current (Kinnamon and Roper 1988). Using Ba2+ as a charge carrier for Ca2+ channels, we measured Ba2+ currents elicited by voltage steps from -30 to +50 mV from a holding potential at -80 mV (Fig. 6A). The average inhibition of Ba2+ currents was 79 ± 6.1% (n = 5). To monitor the time course of the response, peak inward currents were elicited by a voltage step to +10 mV. DEX significantly reduced the Ba2+ currents. Compared with DEX-modulated inward and outward currents in normal saline, the reduction was slower in Ba2+ saline (Fig. 6B).



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Fig. 5. Electrophysiological responses to DEX. A, top: whole cell currents were elicited by voltage steps from a holding potential at -80 mV. Bottom: current-voltage relationships of DEX responses were plotted using the current records shown top. Note that DEX (2.5 mM) completely blocked peak inward currents and significantly reduced outward currents. B: time course of the electrophysiological response to DEX. Left: peak inward currents and peak outward currents elicited by voltage steps to -20 and +40 mV, respectively, were plotted. Holding potential was -80 mV. Right: current records at control (a), during DEX (b), and after wash (c). Corresponding data plots are labeled in the left panel.



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Fig. 6. Effect of DEX on voltage-gated Ca2+ current. A, top: Ca2+ currents were elicited by voltage steps from -30 to +50 mV from a holding potential at -80 mV. Ba2+ was the ionic carrier for the Ca2+ current. Bottom: current-voltage relationships of Ca2+ current were plotted using the current records shown top. B, top: Ca2+ current elicited by a voltage step to +10 mV from a holding potential of -80 mV, control (a) and during DEX stimulation (b). Note that DEX significantly reduced the Ca2+ current. Bottom: time course of responses to DEX. Records in bottom are from the same cells illustrated in top.

G proteins are not involved in the response to DEX

To examine whether G proteins are involved in the DEX-induced reduction of voltage-gated currents, we used a nonhydrolyzable GDP analogue, GDP-beta -S (1 mM) in the patch pipette solution. The effect of GDP-beta -S on peak inward and outward currents was monitored by voltage steps to -20 and +40 mV, respectively, from a holding potential of -80 mV (Fig. 7). Previously, we showed that the response to denatonium was inhibited completely by GDP-beta -S 15 min after establishment of the whole cell configuration (Ogura et al. 1997a). However, even after 17 min of whole cell recording, DEX continued to block inward and outward currents, similar to control responses. There was no significant difference between the amplitude of responses at 2 and at 17 min (paired t-test = 2.34, df = 2, P = 0.07). The time course of responses at 2 and 17 min was quite similar, suggesting that G proteins are not involved in the DEX-induced block of voltage-gated currents. Thus DEX apparently blocks voltage-gated channels directly.



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Fig. 7. Effects of GDP-beta -S on the DEX-induced inhibition of currents. Cell was held at -80 mV, and the membrane was stepped to -20 and +40 mV. Top left: control (a) and during DEX (b) records after 2 min of whole cell configuration. Top right: control (c) and during DEX (d) records after 17 min of whole cell recording. Bottom: time course of the electrophysiological response to DEX. Peak inward currents and peak outward currents elicited by voltage steps to -20 and +40 mV, respectively. Corresponding data plots from current records in top are labeled in bottom. Note that GDP-beta -S did not affect the current response to DEX, suggesting that the response was G-protein independent.

DEX depolarizes the membrane potential

Previously, we showed that another bitter compound, quinine, blocks voltage-gated outward currents, causing membrane depolarization in mudpuppy taste cells (Kinnamon and Roper 1988). In contrast, denatonium increases outward currents and hyperpolarizes the membrane potential (Ogura et al. 1997a). To determine the effect of DEX on membrane potential, we used giga-seal whole cell recording in current-clamp mode. As expected, DEX depolarized taste cells (Fig. 8). The time course was similar to the DEX-induced reduction of outward current. The amplitude of depolarization was 25-49 mV from the resting potential (resting potential: -73 ± 3.9 mV, after DEX: -27 ± 6.8 mV, n = 8).



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Fig. 8. Membrane potential response to DEX. Under current-clamp condition, DEX depolarized membrane potential.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we used Ca2+ imaging and giga-seal whole cell recording to examine the transduction mechanism of DEX in isolated mudpuppy taste cells. The results suggest that DEX depolarizes mudpuppy taste cells by direct block of an apically located K+ conductance. In addition, DEX causes a slow release of Ca2+ from IP3-sensitive Ca2+ stores by a PLC-independent mechanism (see Fig. 9 and Table 1). This latter mechanism is novel and may be due to the lipophilic DEX entering taste cells and interacting directly with the Ca2+ stores. Because many bitter compounds are lipophilic, this may represent a general mechanism for bitter transduction that may be associated with the persistent bitterness of some compounds. The transduction mechanism for DEX is distinct from that of denatonium (Ogura et al. 1997a) but has some similarity to that of quinine (Kinnamon and Roper 1988) in the same species. These data support the hypothesis that single taste receptor cells can use multiple mechanisms for transducing the same bitter compound (Spielman et al. 1992).



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Fig. 9. Proposed model for transduction of dextromethorphan and denatonium in mudpuppy taste receptor cells.


                              
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Table 1. Comparison of dextromethorphan and denatonium responses

A caveat in the interpretation of these data are that we have no direct behavioral evidence that DEX is bitter to mudpuppies. A previous behavioral study indicated that compounds that block the apical K+ conductance in taste cells are rejected when presented to mudpuppies in agar cubes (Bowerman and Kinnamon 1994). Given that DEX blocks the same conductance, it is likely that DEX would be rejected in a similar manner.

Ca2+-imaging data

Whereas denatonium transduction relies of a G-protein-coupled receptor-PLC-IP3 pathway (Ogura et al. 1997a), the response to DEX persists in the presence of the PLC inhibitor U73122. Thus the DEX response appears to be independent of IP3 formation. Yet, both compounds appear to release Ca2+ from the same intracellular Ca2+ stores. Release of Ca2+ from intracellular stores is involved in the transduction of many bitter compounds, including sucrose octaacetate, caffeine, strychnine, and denatonium, all of which increase IP3 levels in the taste tissue of rodents (Hwang et al. 1990; Spielman et al. 1994). Clearly, the transduction of DEX must involve an IP3-independent mechanism for Ca2+ release. Although we cannot rule out a mechanism involving a unique second-messenger pathway, we suggest that DEX may penetrate the membrane and release Ca2+ directly from intracellular stores. Several bitter compounds are lipophilic, and previous studies provided evidence for the plausibility of direct interaction with intracellular targets by quinine (Cummings and Kinnamon 1992) and caffeine (Koyama and Kurihara 1972; Rosenzweig et al. 1999). If DEX uses this mechanism, specific membrane-receptors for DEX would not be required. Such a mechanism could explain the relatively slow increase in [Ca2+]i and the insensitivity to U73122. It is not clear what effect the slow increase in [Ca2+]i has on transmitter release in taste cells. Presumably, transmitter is released initially as a result of the DEX-induced block of the apical K+ conductance. We hypothesize that the slow increase in [Ca2+]i that follows would prolong transmitter release, but further studies will be required to determine the Ca2+ dependence of vesicle release. Many lipophilic bitter compounds exhibit a bitter aftertaste, and a prolonged period of vesicle release may contribute to this phenomenon.

Effect of cAMP

Our data showed that increased cAMP levels enhance the [Ca2+]i increase in response to DEX by PKA phosphorylation. Similar modulatory effects of cAMP on Ca2+ release have been reported in hepatocytes and neuroblastoma cells (Bird et al. 1993; Wojcikiewicz and Luo 1998). The physiological relevance of the enhanced Ca2+ response in the presence of cAMP in taste cells is unclear. Because cAMP itself had no effect on intracellular Ca2+ levels, it is likely that other physiological processes modulate cAMP levels and that this modulation affects the DEX response. It has been shown that serotonin modulates voltage-dependent Ca2+ channels in mudpuppy taste cells via an increase in intracellular cAMP levels (Delay et al. 1997). Further experiments will be required to determine if the presence of serotonin affects Ca2+ responses in response to DEX stimulation.

In contrast to the present experiments, previous physiological studies suggest that a reduction in cAMP levels is associated with bitter transduction. In frog taste cells, an inward conductance induced by quinine is suppressed by membrane permeant cAMP analogs (Tsunenari et al. 1996), and the effect is antagonized by inclusion of transducin-derived peptides in the patch pipette solution (Kolesnikov and Margolskee 1995). These data in frog are considered to support a role for gustducin or transducin in bitter taste transduction. Quinine is believed to activate gustducin or transducin, which in turn activates PDE, reducing cAMP levels and relieving block of a cyclic nucleotide suppressible cation conductance (Kinnamon and Margolskee 1996). It is not known, however, if frog taste cells contain transducin or gustducin. In contrast, there is no evidence for a role of gustducin or transducin in mudpuppy taste cells. The denatonium Ca2+ response is unaffected by changes in intracellular cAMP (Ogura et al. 1997a), and transducin-derived peptides have no effect on membrane conductance (Kinnamon, unpublished data).

Several studies suggest a role for gustducin (and/or transducin) in bitter taste transduction in mammalian taste cells. Both G proteins are present in taste cells, and gustducin knockout mice are less sensitive to bitter compounds than control mice (Wong et al. 1996). These data are supported by biochemical measurements showing that several bitter compounds, including quinine and denatonium, activate gustducin and transducin in the presence of taste cell membranes (Ming et al. 1998; Ruiz-Avila et al. 1995). These same compounds also activate IP3 production in taste cells (Miwa et al. 1997; Spielman et al. 1996). The relative importance of these second messenger systems in bitter taste has not been examined.

OFF responses

We often observed OFF responses to DEX. Similar OFF responses to taste stimuli have been observed previously in electrophysiological recordings from taste receptor cells (Cummings et al. 1993; Tsunenari et al. 1996). It is possible that relief from a suppressive mechanism may be involved in the off response. We often recorded a decrease in [Ca2+]i in response to denatonium or DEX after inhibition of Ca2+ release with thapsigargin. Similar suppressive effects are proposed as a mechanism for off responses in olfactory receptor neurons (Kurahashi et al. 1994). Further studies on the suppressive mechanism in taste receptor cells may reveal a role for off responses in taste adaptation and taste coding mechanisms.

Electrophysiological data

Our electrophysiological data showed that DEX depolarized mudpuppy taste cells by blocking the voltage-dependent K+ conductance, which is also a resting conductance in these cells (Cummings and Kinnamon 1992). Similar results were obtained with quinine (Cummings and Kinnamon 1992; Kinnamon and Roper 1988) and CaCl2 (Bigiani and Roper 1991). In contrast, denatonium hyperpolarized the membrane, due to activation of Ca2+-dependent K+ channels (Cummings and Kinnamon 1992) and Cl- channels (Taylor and Roper 1994) due to the Ca2+ released from intracellular stores (Ogura et al. 1997a).

In brain, DEX has been shown to bind to Sigma receptors, which are G-protein-linked receptors that affect a variety of targets, including a resting K+ conductance (Su 1991). We do not believe that DEX is blocking K+ channels in taste cells by activating Sigma receptors, however, because the DEX-induced block of K+ channels in taste cells was unaffected by the G protein inhibitor GDP-beta -S.

DEX also blocked both inward Na+ and Ca2+ currents. Similar results were obtained by quinine, which blocked all voltage-gated conductances when bath-applied to mudpuppy taste cells (Kinnamon and Roper 1988) as well as to rat taste cells (Chen and Herness 1997; Ozeki 1971; Sato and Beidler 1983). Blockage of inward currents is not likely to interfere with transduction, however, because voltage- and Ca2+-dependent K+ channels are localized to the apical membrane in mudpuppy taste cells, whereas Na+ and Ca2+ channels are relatively evenly distributed on the taste cell membrane (Kinnamon et al. 1988). Thus bitter compounds would likely block the K+ conductance and depolarize the membrane potential before a substantial number of Na+ and Ca2+ channels would be blocked. Because both DEX and quinine are membrane permeant, however, prolonged stimulation may result in blockage of Na+ and Ca2+ channels due to interaction with basolateral channels. This could result in a prolonged inhibition of the taste cells and cause them to be refractory to taste stimulation. This type of inhibitory effect has been observed in afferent nerve recordings of catfish, where increased nerve firing to quinine is followed by complete suppression of the firing to all taste stimuli (Ogawa et al. 1997). Quinine suppression of taste responsivity also has been observed in mammals (Formaker et al. 1997). It is not known if prolonged DEX stimulation causes taste cells to be in a refractory state.

Our electrophysiological data for the DEX response show some similarity to that of quinine (Kinamon and Roper 1988) in the same species. It will be interesting to examine [Ca2+]i in response to quinine in mudpuppy taste cells. Currently, however, the autofluorescence of quinine prevents us from using conventional ratiometric measurement of [Ca2+]i with fura-2. Using a different Ca2+-sensitive dye that does not interact with the fluorescence of quinine would make it possible to compare responses to quinine and other bitter compounds in the same taste cells.


    ACKNOWLEDGMENTS

We thank Dr. Sandra L. Nelson for helpful discussions and Drs. Thomas Finger and Diego Restrepo for helpful comments on the manuscript.

This work was supported by National Institute of Deafness and Other Communication Disorders Grants DC-00244 and DC-00766 and by a grant from the Procter & Gamble Co.


    FOOTNOTES

Address for reprint requests: T. Ogura, Dept. of Anatomy and Neurobiology, Colorado State University, Fort Collins, CO 80523.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 26 May 1999; accepted in final form 12 July 1999.


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