A Calcium-Receptor Agonist Induces Gustatory Neural Responses in Bullfrogs

Abstract

The effect of calcium-sensing receptor (CaR) agonists on frog gustatory responses was studied using glossopharyngeal nerve recording and whole-cell patch-clamp recording of isolated taste disc cells. Calcimimetic NPS R-467 dissolved in normal saline solution elicited a large transient response in the nerve. The less active enantiomer of the compound, NPS S-467 induced only a small neural response. The EC₅₀ for NPS R-467 was about 20 μM. Cross-adaptation experiments were performed to examine the effect of 30 μM NPS R-467 and 100 μM quinine on the gustatory neural response. The magnitude of the R-467-induced response after adaptation to quinine was approximately equal to that after adaptation to normal saline solution, indicating that the receptor site for NPS R-467 is different from the site for quinine. NPS R-467 (100 μM) also induced an inward current accompanied with conductance increase and large depolarization in two (13%) of 15 rod cells, and a sustained decrease in outward current and small depolarization in six (40%) other rod cells. NPS S-467 (100 μM) induced a sustained decrease in outward current and depolarization in five (50%) of 10 rod cells. Another calcimimetic cinacalcet (100 μM) induced an inward current accompanied with conductance increase in three (27%) of 11 rod cells. The results suggest that NPS R-467 induces neural responses through cell responses unrelated to a resting K⁺ conductance decrease.

Introduction

It has been well known that frog gustatory organs are very sensitive to oral Ca²⁺ concentration (Zotterman 1949). The Ca²⁺ response is inhibited by adding salts to the Ca²⁺ solution (Casella and Rapuzzi 1957; Nomura and Sakada 1965; Junge and Brodwick 1970; Kitada 1978). Studies show that the inhibitory effect of the salts might be due to cations. Kitada (1984) further found that treatment of the tongue surface with pronase potently inhibits the response to Ca²⁺, but to Na⁺. The selective inhibition by pronase treatment on the Ca²⁺ response suggests that the receptive site for Ca²⁺ in frog taste cells may be different from the site for Na⁺ and that the Ca²⁺ site may be a protein. Necturus taste cells also display depolarization in response to Ca²⁺ (Bigiani and Roper 1991). Ca²⁺ may inhibit resting K⁺ conductance, resulting in depolarization of the membrane potential in the taste cells.

Gustatory organs respond to each of the five basic stimuli (salty, sour, sweet, bitter and umami). Salty and sour tastants directly affect ion channels, while sweet, bitter and umami tastants bind to G protein-coupled receptors, initiating intracellular signalling cascades (Lindemann 2001; Gilbertson and Boughter 2003; Chandrashekar et al. 2006). Recent results suggest that tastant binding to the receptors activates the heterotrimeric G proteins (Gα_gust and Gα_i2) leading to the dissociation of the Gβγ subunits and subsequent stimulation of phospholipase Cβ2 (PLCβ2). Activation of PLCβ2 hydrolyses phosphatidylinositol 4,5-biphosphate to produce inositol 1,4,5-trisphospahte (IP₃) and diacylglycerol (DAG), and finally activates the transient receptor potential protein (TRIPM5). These signalling cascades suggest that sweet, bitter and umami tastants increase the conductance in taste cells; nevertheless, a conductance increase in native taste cells has hardly ever been recorded in response to those tastants. A recent bioinformatic study showed that Xenopus (frog) possess 49 intact bitter taste receptor (T2R) genes (Go 2006). However, the expression of T2R-like receptors in frogs does not imply that they are really involved in bitter taste transduction. Physiological studies would be required to evaluate the role of these receptor proteins.

In vertebrates, the extracellular Ca²⁺ concentration in circulating body fluids is detected by various tissues and organs using the extracellular Ca²⁺-sensing receptor (CaR) (Hofer and Brown 2003). The agonist binding to CaR is followed by the activation of various G-protein-coupled intracellular signalling pathways (PLC, PLA and MAPK). The CaR is expressed not only in the cells that maintain systemic external Ca²⁺ homeostasis (parathyroid cells and several cells in the kidney, etc), but also in other cells with an indefinite role. Several synthetic CaR agonists (calcimimetics) have been developed for the medical management of disorders in Ca²⁺ metabolism (Nemeth et al. 1998; Nemeth et al. 2004).

The receptive site for frog Ca²⁺ response has not been identified. It is interesting to examine whether the calcimimetics elicit frog nerve response. In the present study, we report that frog gustatory nerves and taste disc cells can respond to the agonists for CaR, but that calcimimetics-induced frog nerve response is due to an unidentified receptor, not the CaR.

Materials and Methods

Preparation

Adult bullfrogs (Rana catesbeiana Shaw) weighing 250–550 g were used for the experiments during April and December. The experiments were performed in accordance with the Guidelines for Animal Experimentation of Nagasaki University. The animals were anaesthetized with an intraperitoneal injection of 50% urethane saline solution at 3 g/kg body mass. To prevent spontaneous contraction of the tongue, the hypoglossal nerve and the hyoglossal muscle were cut bilaterally. The tongue was fully pulled from the mouth and its base was fixed with steel pins onto a silicone rubber plate in an experimental chamber. For the patch-clamp experiments, taste disc cells were isolated from the tongue of decapitated and pithed animals, as described before (Okada et al. 2001). The fungiform papillae were dissected from the tongue in nominal Ca²⁺-free saline and stored in Ca²⁺-free saline containing 2 mM EDTA for 10 min. The papillae were bathed in the same saline containing 10 mM L-cysteine and 10 U/ml papain (Sigma, St Lois, MO, USA) for 10–13 min. The papillae were then rinsed with normal saline, and individual cells were dissociated by gentle trituration in normal saline. Isolated taste disc cells showing a characteristic morphology were readily distinguished from the other cells and classified into rod and wing cells (Okada et al. 1996). Rod cells had one dendrite-like process while wing cells had two or three. Rod cells were used for the experiments, because rod cells, but not wing cells, may be true taste cells (Osculati and Sbarbati 1995).

Nerve Recording

The glossopharyngeal nerves from both sides were dissected from the surrounding connective tissues and cut near the hyoid bone. The nerves were placed over bipolar silver wire recording electrodes and immersed in liquid paraffin. The gustatory neural impulses were amplified, integrated with a time constant of 0.3 s, and recorded on a pen recorder. Peak amplitudes were used as a measure of the neural responses. The adapting and stimulating solutions were flowed on the tongue surface at a rate of 0.5 ml/s with a 10 ml syringe.

Cell Recording

Voltage-clamp recording was performed in whole-cell configuration (Hamill et al. 1981) using a CEZ 2300 patch-clamp amplifier (Nihon Kohden, Tokyo, Japan). The patch pipettes were pulled from Pyrex glass capillaries containing a fine filament (Summit Medical, Tokyo, Japan) with a two-stage puller (Narishige PD-5, Tokyo, Japan). The tips of the electrodes were heat-polished with a microforge (Narishige MF-80). The resistance of the resulting patch electrode was 5–10 MΩ when filled with internal solution. The formation of 5–20 GΩ seals between the patch pipette and the cell surface was facilitated by applying weak suction to the interior of the pipette. The patch membrane was broken by applying strong suction, resulting in a sudden increase in capacitance. Recordings were made from taste disc cells that had been allowed to settle on the bottom of a chamber placed on the stage of an inverted microscope (Olympus IMT-2, Tokyo, Japan). The recording pipette was positioned with a hydraulic micromanipulator (Narishige WR-88). The current signal was low-pass-filtered at 5 kHz, digitized at 125 kHz using a TL-1 interface (Axon Instruments, Union City, CA, USA), acquired at a sampling rate of 0.25–5 kHz using a computer running the pCLAMP 5.5 software (Axon Instruments), and stored on hard disk. The pCLAMP was also used to control the digital-analogue converter for the generation of the clamp protocol. The indifferent electrode was a chlorided silver wire. Capacitance and series resistance were compensated for, as appropriate. The whole-cell current–voltage (I/V) relationship was obtained from the current generated by the 167 mV/s voltage ramp from −100 to + 100 mV. Input resistance was calculated from the slope conductance generated by the voltage ramp from −100 to −50 mV.

Solutions and Drugs

Normal saline solution consisted of (in mM): NaCl 115; KCl 2.5; CaCl₂ 1.8; Hepes 10; glucose 20. The pH of normal saline and standard K⁺ internal solutions was adjusted to 7.2 by Tris base. NPS R-467 and S-467 were kindly supplied by NPS Pharmaceuticals (Salt Lake City, UT, USA). NPS R-467 is a phenylalkylamine compound that acts as an allosteric agonist of the CaR (Nemeth et al. 1998). NPS S-467 is the less active enantiomer of NPS R-467 and was used as a control for non-specific effects. The drugs and quinine-HCl (100 μM) were dissolved in normal saline solution. Tablets of cinacalcet-HCl were purchased from Amgen (Luzern, Swizerland). For stock solution, cinacalcet (100 mM) was dissolved in dimethylsulphoxide (DMSO). Sample of the stock solution was added to normal saline solution to give the desired final concentration. NaCl (0.3 M) were dissolved in deionized water. The solution exchange was done by gravity flow. The standard K⁺ internal solution contained (in mM): KCl 100, CaCl₂ 0.1; MgCl₂ 2; EGTA 1; Hepes 10. In order to eliminate the outward K⁺ current, KCl was replaced with CsCl.

All experiments were carried out at room temperature (20–25°C).

Results

Neural Responses Elicited by NPS R-467

Figure 1A shows examples the gustatory neural responses elicited by 1–100 μM NPS R-467 dissolved in normal saline solution after the tongue was adapted to normal saline solution. The responses for R-467 were composed of large transient and small sustained responses. The magnitude of the R-467-induced responses increased with a dose-dependent manner of R-467 (Fig. 1B). The responses were stereo-selective and the R enantiomer was greatly more potent than the S enantiomer. The apparent EC₅₀ for NPS R-467 was about 20 μM. Thus, NPS R-467 dissolved in normal saline solution could induce a large neural response.

Fig. 1.

(A) Frog gustatory neural responses elicited by 1–100 μM NPS R-467, 10–100 μM NPS S-467 and 0.3 M NaCl after adaptation of the tongue to normal saline solution. All records were obtained from the same preparation. (B) Relationships between the concentrations of agonists and the amplitudes of the neural responses. The plot for NPS R-467 shows an apparent EC₅₀ of about 20 μM. The magnitudes of the responses are expressed as the value relative to the response elicited by 0.3 M NaCl. The values are mean ± S.E.M. and were obtained from 4 to 6 nerves except for 30 μM NPS R-467 (2 nerves)

Cross-adaptation experiments were performed to examine the effect of 30 μM NPS R-467 and 100 μM quinine (a bitter tastant) on the gustatory nerve response. The magnitude of R-467-induced gustatory neural response after adaptation to quinine was approximately equal to that after adaptation to normal saline solution (N = 2, Fig. 2), although the magnitude of the neural response was somewhat uneven. The quinine-induced response was slightly decreased by pre-adaptation to R-467, suggesting that R-467 may have multiple binding sites.

Fig. 2.

Cross-adaptation between 30 μM NPS R-467 and 100 μM quinine on frog gustatory neural response. The upper two records (A and B) are control responses and the lower two records are cross-adaptation responses. All records were obtained from the same preparation

NPS R-467-Induced Current in Frog Rod Cells

Under conventional whole-cell mode in a standard K⁺ internal solution, we analyzed the effect of 100 μM NPS R-467 on the membrane properties of 15 rod cells. The rod cells displayed resting potentials of −38 to −53 mV (−49.1 ± 2.1 mV, N = 15). The input resistance ranged from 3.6 to 8.8 GΩ (6.3 ± 0.5 GΩ, N = 15) and the membrane capacitance was 4.2–6.2 pF (5.0 ± 0.1 pF, N = 15). In response to 100 μM NPS R-467, only two (13%) of 15 rod cells displayed an inward current accompanied with conductance increase at a membrane potential of −50 mV (Fig. 3A and B). R-467 induced inward currents of −12 and −10 pA at −50 mV in the two cells, depolarized the resting potentials by 20 and 10 mV and decreased the input resistances to 21 and 53% of the controls. The conductance increase gradually became smaller in the presence of R-467, and the sustained outward current evoked by ramp voltage remained after the removal of R-467. Six other rod cells (40%) displayed a sustained reduction of outward K⁺ current at +50 mV (Fig. 3C and D). R-467 decreased the outward currents at +50 mV from 129 ± 28 pA to 45 ± 15 pA (P < 0.05, N = 6) and depolarized the resting potentials from −42.6 ± 1.4 mV to −39.3 ± 1.0 mV (P < 0.05, N = 6). Seven other rod cells did not show any response to NPS R-467. In five of other 10 rod cells, S-467 also decreased the outward currents at +50 mV from 80 ± 22 pA to 34 ± 12 pA (P < 0.05, N = 5) and depolarized the resting potentials from −50.6 ± 1.8 mV to −39.4 ± 2.6 mV (P < 0.05, N = 5) (Fig. 4). S-467 never induced an inward current accompanied with conductance increase.

Fig. 3.

The effect of 100 μM NPS R-467 on the membrane properties of frog rod cells. The upper panels (A and C) show the pen recordings of the current signals at a holding potential of −50 mV. The lower panels (B and D) are plots of the whole-cell current/voltage (I/V) relationships produced by a voltage ramp (167 mV/s) from −100 to +100 mV. I/V relationships labelled a to d were obtained at the times indicated by the same letters on the pen recordings. The transient outward current deflections larger than 200 pA on the pen recording A are out of scale. The pipette contained standard K⁺ internal solution and the bath contained normal saline solution

Fig. 4.

The effect of 100 μM NPS S-467 on the membrane properties of frog rod cells. The upper panel (A) shows the pen recording of the current signal at a holding potential of −50 mV. The lower panel (B) is plots of the whole-cell current/voltage (I/V) relationships produced by a voltage ramp (167 mV/s) from −100 to +100 mV. I/V relationships labelled a and b were obtained at the times indicated by the same letters on the pen recording. The pipette contained standard K⁺ internal solution and the bath contained normal saline solution

Cinacalcet-Induced Current in Frog Rod Cells

Under conventional whole-cell mode in Cs⁺ internal solution, we analyzed the effect of 100 μM cinacalcet on the membrane properties of 11 rod cells. Although internal K⁺ was replaced with Cs⁺, the rod cells displayed small parabolic outward currents in the voltage range of −50 to +50 mV (Fig. 5B) and resting potentials of −31 to −57 mV (−48.7 ± 2.4 mV, N = 11). When 100 μM cinacalcet was added to the bath, the parabolic outward currents gradually decreased, and subsequently transient inward currents accompanied with conductance increase appeared (Fig. 5A and B). The outward currents recovered after the wash-out of cinacalcet. A transient inward current accompanied with conductance increase was seen in only three cells (−23 ± 3 pA at −50 mV, N = 3), while the inhibition of parabolic outward current was seen in all 11 cells.

Fig. 5.

The effect of 100 μM cinacalcet on the membrane properties of frog rod cells. The upper panel (A) shows the pen recording of the current signal at a holding potential of −50 mV. The lower panel (B) is plots of the whole-cell current/voltage (I/V) relationships produced by a voltage ramp (167 mV/s) from −100 to +100 mV. I/V relationships labelled a to d were obtained at the times indicated by the same letters on the pen recording. The pipette contained Cs⁺ internal solution and the bath contained normal saline solution

Discussion

Previous studies (Casella and Rapuzzi 1957; Nomura and Sakada 1965; Junge and Bordwick 1970; Kitada 1978) indicated that frog gustatory Ca²⁺ responses were greatly inhibited by the addition of NaCl to the Ca²⁺ solution. The addition of NaCl to the external solution also inhibits the activation of the CaR by external Ca²⁺ (Quinn et al. 1998). CaR exists in numerous regions of the brain (Yano et al. 2004). Extracellular high Ca²⁺ and CaR agonists activate nonselective cation channels in rat hippocampal and subfornical organ neurons (Ye et al. 1996; Washburn et al. 1999), resulting in the neural excitation. A receptor similar to the brain CaR may reside in frog taste disc cells, but the EC₅₀ (about 20 μM) of NPS R-467 for causing frog neural response was higher than that (1 μM) for increasing intracellular Ca²⁺ level in bovine parathyroid cells (Nemeth et al. 1998), suggesting that both receptors are not identical. A receptor protein for frog Ca²⁺ response has not yet been identified. A taste receptor for umami (L-glutamate) may possibly be related to one of brain metabotropic glutamate receptors (mGluRs) (Lindemann 2001). Although brain mGluR has a rather large amino-terminal domain, the taste umami receptor has a truncated amino-terminal domain, resulting in the higher glutamate concentrations for umami taste. Thus, the ligand affinity for taste receptors is relatively low.

In Necturus taste cells, it has been suggested that K⁺ channels work as receptors for sour and bitter tastes (Kinnamon and Roper 1988a, b; Bigiani and Roper 1991). Various compounds such as quinine, protons and Ba²⁺ all block the voltage-gated K⁺ channels. Since K⁺ channels in Necturus taste cells are localized to the apical membrane and maintain the resting membrane potential (Kinnamon et al. 1988), sour (protons) and bitter (quinine) stimuli may directly block the channels, inducing the depolarization of the taste cells. This mechanism may result in a lack of discrimination between sour and bitter taste in Necturus. In contrast, the frog neural responses elicited by protons and quinine are not inhibited by 10 mM Ba²⁺, which can block the outward K⁺ currents (Okada et al. 1994). NPS R-467 and S-467 also caused potent inhibition of outward K⁺ currents in basolateral side of isolated taste disc cells, but S-467 could not elicit the neural response. It is suggested that the transduction mechanism for R-467 does not have relevance to resting K⁺ conductance block.

In the rod cells accompanied with conductance increase, the conductance increase gradually became smaller in the presence of R-467 or cinacalcet, indicating a transient effect of calcimimetics on conductance increase. Furthermore, a sustained outward current remained after the removal of R-467. This suggests that R-467 induces two different conductances. We suppose that small transient inward currents are caused by a Ca²⁺-activated non-selective cation conductance (Okada et al. 1998), and that the sustained outward currents is due to a Ca²⁺-activated K⁺ conductance (Fujiyama et al. 1994). Similar results were observed in the saccharin-induced response in frog rod cells (Okada et al. 2001).

A diversity of pathways have been hypothesized for the signalling downstream of taste receptors. However, recent studies demonstrated that sweet, bitter and umami tastants use a common pathway (Chandrashekar et al. 2006). Tastant binding to the receptors activates the heterotrimeric G proteins triggering the dissociation of the Gβγ subunits and subsequent stimulation of phopholipase Cβ2 (PLCβ2). Activated PLCβ2 hydrolyses phosphatidylinositol-4,5-biphosphate to produce inositol-1,4,5-trisphophate (IP₃) and diacylglycerol (DAG). IP₃ releases Ca²⁺ from the internal store and the following rise in intracellular Ca²⁺ concentration activates the transient receptor potential protein that is a non-selective cation channel. We suppose that NPS R-467 elicits cell depolarization in a similar manner.

In the present study, only 13% of the rod cells displayed the R-467-induced inward current accompanied with conductance increase. A sweet tastant, saccharin (30 mM) also induces the inward current in 23% of the rod cells (Okada et al. 2001). Similarly, Bigiani et al. (1997) observed a transient inward current (about −20 pA at −85 mV) in only 4% of rat taste cells, and sustained conductance decrease (nominal appearance of an outward current of about 6 pA at −85 mV) in 56% of the cells in response to glutamate (umami). These results suggest that the G protein-coupled transduction machinery is expressed in a subset of taste cells. Frog rod cells are classified into type II and III. Type III cells possess voltage-gated Ca²⁺ channels, but type II cells do not (Suwabe and Kitada 2004). The rod cells in the present study displayed current–voltage (I–V) relationships that were observed in type II cells (data not shown). Mouse type II cells which contain taste receptors and phospholipase C (PLC) lack voltage-gated Ca²⁺ channels (Clapp et al. 2006). The classical synaptic transmission needs voltage-gated Ca²⁺ channels. The lack of voltage-gated Ca²⁺ channels in mouse and frog type II cells suggest that these cells may use an unknown signalling transmission to the gustatory nerve (Romanov et al. 2007).

The CaR is expressed abundantly throughout the gastrointestinal tract in mammals (Conigrave and Brown 2006) and may have a significant role as an L-amino acid sensor in the tract. CaR-like receptors are also expressed in the olfactory organs of the fish (Naito et al. 1998), and fish can detect the environmental Ca²⁺ concentration using these receptors (Hubbard et al. 2002).