Strain difference in amiloride-sensitivity of salt-induced responses in mouse non-dissociated taste cells

Neuroscience Letters 277 (1999) 13±16 www.elsevier.com/locate/neulet

Strain difference in amiloride-sensitivity of salt-induced responses in mouse non-dissociated taste cells Takenori Miyamoto*, Rie Fujiyama, Yukio Okada, Toshihide Sato Department of Physiology, Nagasaki University School of Dentistry, 1-7-1 Sakamoto, Nagasaki 852-8588, Japan Received 26 August 1999; received in revised form 8 October 1999; accepted 8 October 1999

Abstract The chorda tympani nerve responses to NaCl in a mouse strain, C57BL/6 are known to be much more sensitive than those in BALB/c. We compared the NaCl-induced responses obtained from taste cells of the fungiform papillae in these two strains of mice. Amiloride inhibited, in the same degree, the responses induced by a bath-application of normal extracellular solution (NES) containing 140 mM NaCl in either taste cells of C57BL/6 and BALBIc mice. In contrast, amiloride inhibited 62% of responses induced by an apically applied 0.5 M NaCl in the C57BL/6 strain, but only 33% of responses in the BALB/c strain. These results suggest that the difference in amiloride-sensitivity between taste cells in both strains mainly derives from the difference in density of functional amiloride sensitive Na 1 channels at the apical receptive membrane but not at the basolateral membrane. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: NaCl; Salty taste; Transduction; Apical; Basolateral; C57BL/6; BALB/c

A diuretic, amiloride greatly suppresses the NaClinduced response of the chorda tympani nerve and taste cells of the fungiform papilla in the mammalian gustatory system [9,18]. Therefore, amiloride-sensitive Na 1 channels are thought to be the main contributor to the transduction of NaCl-induced response. Prominent differences between strains in the rat and the mouse exist in their various taste responses [14]. For salty taste, the presence of strain difference has been also reported in the magnitude of the chorda tympani nerve response to NaCl and its amiloride-sensitivity in the rat [1] and the mouse [15,16]. For example, NaCl-induced responses of the chorda tympani nerve in mouse strains, C57BL/6 and C3H were signi®cantly suppressed by amiloride, whereas those in DBA and BALB/c were not [15]. However, the strain difference in NaCl-induced responses of taste cells and their amiloride-inhibition has not been reported so far. In the present experiment, we examined NaCl-induced responses and those chemical modi®cation with non-dissociated taste cells using a combination of whole-cell clamp technique and localized chemical stimulation procedure to apply taste stimuli to the apical membrane of taste cells * Corresponding author. Tel.: 181-95-849-7638; fax: 181-95849-7639. E-mail address: [email protected] (T. Miyamoto)

[11]. Under this condition, we could compare the amiloride effect on the responses induced by an apically applied NaCl solution with that on responses induced by a bath application of NaCl-containing solution. We found that the strain difference of amiloride sensitivity between C57BL/6 and BALB/c was observed in the apically applied NaCl-induced responses, but not in the bath application-induced responses. The result suggests that the apically applied NaCl-induced responses are generated by different mechanism from the bath applicationinduced responses. The mice C57BL/6 and BALB/c strains, ranging in weight from 20,35 g were anesthetized by an intraperitoneal injection of pentobarbital (30 mg/kg) and killed by dislocating the cervical vertebra. The non-dissociated taste cells within a taste bud were prepared as described previously [11,12]. After the tongue was incubated in normal extracellular solution (NES) for 30 min at 268C, following the injection of 1.5,2.0 mg/ml elastase (Boehringer Mannheim) dissolved in NES, the epithelial sheet was peeled free from the rest of the tongue. Individual taste bud with an epithelial brim was obtained by sucking the fungiform papilla from its inside with a pipette after incubation in the divalent cation-free extracellular solution for 20 min at 48C and washing by NES. The experimental setting up for combination of wholecell clamp technique and localized chemical stimulation

0304-3940/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 9 9) 00 82 8- 9

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T. Miyamoto et al. / Neuroscience Letters 277 (1999) 13±16

Fig. 1. Effect of amiloride on the resting membrane current and NaCl-induced voltage response in amiloride-sensitive taste cells. (A) Effects of 5 mM amiloride and removal of Na 1 on the stationary current under voltage clamp at 280 mV. After an outward current was induced by 5 mM amiloride, more outward current was induced by Na 1-free solution. The cells were adapted to NES at the beginning of the recording. The recording was obtained from a taste cell of BALB/c mouse. (B) Effect of 5 and 50 mM amiloride on the apically applied 0.5 M NaCl-induced responses. The responses were suppressed by 5 and 50 mM amiloride in the same degree. The recording was obtained from a taste cell of C57BL/6 mouse. Inset shows con®guration for stimulation, recording and perfusion.

procedure to apply taste stimuli to the apical membrane of taste cells was made as previously described [11,12]. As the background ¯ow was slowed down at the taste pore region due to the barrier of the epithelial brim, the stimulus concentration ejected by constant pressure was maintained for 10 s or longer (see `Inset' in Fig. 1). Whole-cell currents were measured with a patch-clamp ampli®er (List, EPC-7), and the current signals were ®ltered at 1 kHz, digitized at 125 kHz, and analyzed by a pCLAMP, Axograph (Axon Instruments) and Origin (Microcal) softwares. All the experiments were performed at 22±248C. NES contained (in mM): 140 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 glucose, 1 sodium pyruvate, 10 HEPES±Tris (pH 7.4). A high K 1 pipette solution (K 1 solution) contained (in mM): 120 KCl, 2 MgCl2, 1 CaCl2, 11 EGTA, 10 HEPESTris (pH 7.2). KCl in K 1 solution was replaced by 20 mM KCl and 100 mM K-gluconate in low Cl 2 and high K 1 intracellular solution (low Cl K 1 solution) and 120 mM CsCl in high Cs 1 intracellular solution (Cs 1 solution). A pipette solution containing 250 mg/ml amphotericin B (Sigma) was employed for obtaining perforated patches. In a Na 1-free extracellular solution (Na 1-free solution), N-methyl-d-glucamine 1 (NMDG 1) was substituted for Na 1. A salt stimulus, 0.5 M NaCl was dissolved in deionized water. A 10 mM stock solution of amiloride was

prepared in deionized water, and was diluted by any of bathing solutions immediately before use. In the present experiment, we recorded from a total of 83 taste cells the fungiform taste buds. As has been shown previously, taste cells obtained from the mouse fungiform papillae displayed voltage-gated Na 1 and K 1 currents [11]. Taste cells were adapted to NES or Na 1-free solution until the membrane potential or current became constant. The amiloride-sensitivity was evaluated by difference between the means of the maximum responses before and during application of amiloride (see below). A hyperpolarization of 5,40 mV from the zero current membrane potential (Vzero: 210,227 mV) was induced by amiloride added to the bath in some taste cells (amiloridesensitive cells) [12]. Replacement of NES with Na 1-free solution induced more hyperpolarization of 8±52 mV in these taste cells under the current clamp [12]. Under the voltage clamp at 280 mV, stationary inward currents were blocked by amiloride or Na 1-free solution in amiloride-sensitive cells, resulting in the generation of outward current of 6-113 pA (Fig. 1A). In other taste cells, amiloride barely affected the membrane potential under the current clamp (amiloride-insensitive cells) [12]. However, replacement of NES with Na 1-free solution induced hyperpolarization of 5±52 mV from Vzero (210,244 mV) under the current clamp [12] or the outward current of 5±98 pA under the voltage clamp at 280 mV (Fig. 2A). Some NaCl-induced responses were suppressed by amiloride (amiloride-sensitive responses) (Fig. 1B), but other responses were not affected by amiloride (amilorideinsensitive responses) (Fig. 2B). However, the suppression was not complete even by the application of 50 mM amilor-

Fig. 2. Effect of amiloride on resting membrane current and NaCl-induced response in amiloride-insensitive taste cells under current clamp. (A) Effects of 5 mM amiloride and removal of Na 1 on the membrane current. An outward current was induced by Na 1-free solution but not by 5 mM amiloride. The cells were adapted to NES at the beginning of the recording. The recording was obtained from a taste cell of C57BL/6 mouse. (B) No inhibitory effect of 5 mM amiloride on the apically applied 0.5 M NaCl-induced responses. The recording was obtained from a taste cells of C57BL/6 mouse. The same con®guration for stimulation, recording and perfusion as in Fig. 1.

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Fig. 3. Localized suppression of 0.5 M NaCl-induced responses by amiloride. In this taste cell, 5 mM amiloride barely affected the membrane potential in NES, whereas after adaptation to Na 1-free solution, the responses induced by the apically applied 0.5 M NaCl were suppressed by 50%. The recording was obtained from a taste cell of BALB/c mouse. The same con®guration for stimulation, recording and perfusion as in Fig. 1 other than K 1 solution in the patch pipette.

ide, which was the maximum dose for suppression, even in the amiloride-sensitive responses (Fig. 1B). When the perforated patch method was employed, we observed in a few taste cells that the resting membrane potential in NES was amiloride-insensitive, whereas 0.5 M NaCl-induced responses were amiloride-sensitive (Fig. 3). When 0.5 M NaCl was apically applied, 62% of the responses obtained from taste cells were amiloride-sensitive in C57BL/6 strain. In contrast, only 33% of the 0.5 M NaClinduced responses were amiloride-sensitive in BALB/c strain (Table 1; x 2 test; x 2 ˆ 16.86, P , 0:0001). However, amiloride inhibited, in the same degree, the responses induced by a bath-application of NES containing 140 mM NaCl in either taste cells of C57BL/6 and BALB/c mice. (Table 1; x 2 ˆ 3:48, P . 0:06). Removal of Na 1 induced hyperpolarizing responses in 94% and 95% of the taste cells of C57BL/6 (n ˆ 31) and BALB/c (n ˆ 20) strains (x 2 ˆ 4:57, P . 0:21), respectively, due to decreasing of Na 1-permeable conductances. However, less than 70% of the taste cells in both strains were sensitive to 5 or 50 mM amiloride (Table 1; x 2 ˆ 1:19, P . 0:55). The similar results have been reported in the rat taste cells in the fungiform papillae [3]. Our results show that difference of amiloride-sensitivity in responses induced by the apically applied 0.5 M NaCl exists between two strains of mouse, C57BL/6 and BALB/c, whereas the amiloride sensitivity of responses induced by the bath-application of NES does not show any difference between these two strains. Therefore, the strain difference in the amiloride sensitivity in the NaCl-induced responses may derive from the difference in the density of the functional amiloride-sensitive Na 1 channels at the apical receptive membrane, but not at the basolateral membrane. The result suggests that the normal responses elicited by NaCl are mainly due to an apical component of the inward current.

Localization of amiloride-sensitive epithelial Na 1 channels (ENaC) at the basolateral membrane as well as the apical receptive membrane has been demonstrated even in circumvallate papillae by several immunohistochemical and molecular biological studies [7,8,19,20]. However, amiloride never affected NaCl responses obtained from glossopharyngeal nerves [4] and taste cells in circumvallate papillae [3,5] in the rat. The functional ENaC is known to be a heteroorigomer, consisting of three subunits, a -, b - and g -subunits. a -Subunit is suf®cient to induce the channel activity, whereas ENaC activity is greatly enhanced by the coexpression of the three subunits [2]. Most recent studies have demonstrated that expression of b - and g -subunits in the circumvallate papillae of the rat is much weaker than those in the fungiform papillae, whereas a -subunit is expressed in the same degree in both papillae [7,8]. We observed that the membrane potential was amilorideinsensitive, but 0.5 M NaCl-induced responses were amiloride-sensitive in some taste cells using the perforated patch method, which prevents intracellular mechanisms from inactivating. This fact indicates the possibility that addiTable 1 Differential sensitivity of taste cells to amiloride between different mouse strains a Strain

C57BL/6 BALB/c a #1

NaCl stimulation

Apical Bath #3 Apical Bath

#2

Amiloride #1-sensitivity Sensitive

Insensitive

Total

10 (62%) 24 (52%) 5 (33%) 15 (65%)

6 (38%) 22 (48%) 10 (67%) 8 (35%)

16 46 15 23

Five or 50 mM amiloride. #2Apical: apically applied 0.5 M NaCl. #3Bath: bath-application of NES containing 140 mM NaCl.

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T. Miyamoto et al. / Neuroscience Letters 277 (1999) 13±16

tional and temporal expression of the functional ENaC at the taste cell membrane, especially at the apical receptive membrane is evoked by NaCl stimulation to the apical receptive membrane via activation of some intracellular mechanisms. We have reported that amiloride-insensitive non-selective cation channels at the apical membrane greatly contribute to salty taste transduction both in the frog [10,18] and in the mouse taste cells [13]. Therefore, non-selective cation channels may have Na 1-receptor coupled with G-protein, resulting in the additional and temporal activation of amiloride-sensitive Na 1 channels via unknown second-messenger. The number of functional amiloride channels in the taste cells was reported to be increased by vasopressin in the frog [17], by cAMP in the hamster [6] and by aldosterone in the rat [8]. We observed lower sensitivity to amiloride in BALB/c strain than in C57BL/6 strain. More taste cells in BALB/c strain possibly lack the internal mechanism necessary for additional activation of the functional ENaC than in C57BL/6.

[7]

[8]

[9] [10] [11] [12]

[13]

We thank Dr. Y. Ninomiya for his helpful comments. This work was supported in part by a grant from the Human Frontier Science Program Organization of France, Grants-in-Aid for Scienti®c Research from the Ministry of Education, Science, Sports and Culture of Japan (Nos. 06671862 and 08457491) and a Grant-in-Aid from the Salt Science Research Foundation. [1] Bernstein, I., Longley, A. and Taylor, E.M., Amiloride-sensitivity of the chorda tympani response to NaCl in Fisher-344 and Wister rats. Am. J. Physiol., 261 (1991) R329±R333. [2] Canessa, C.M., Schild, L., Buell, G., Thorens, B., Gautschi, I., Horisberger, J.-D. and Rossier, B.C., Amiloride-sensitive epithelial Na 1 channel is made of three homologous subunits. Nature, 367 (1994) 463±467. [3] Doolin, R.E. and Gilbertson, T.A., Distribution and characterization of functional amiloride-sensitive sodium channels in rat tongue. J. Gen. Physiol., 107 (1996) 545±554. [4] Formaker, B.K. and Hill, D.L., Lack of amiloride sensitivity in SHR and WKY glosso-pharyngeal taste responses to NaCl. Physiol. Behav., 50 (1991) 765±769. [5] Gilbertson, T.A. and Fontenot, D.T., Distribution of amiloride-sensitive sodium channels in the oral cavity of the hamster. Chem. Senses, 23 (1998) 495±499. [6] Gilbertson, T.A., Roper, S.D. and Kinnamon, S.C., Proton currents through amiloride-sensitive Na 1 channels in

[14]

[15] [16]

[17] [18] [19]

[20]

isolated hamster taste cells: enhancement by vasopressin and cAMP. Neuron, 10 (1993) 931±942. Kretz, O., Barby, P., Bock, R. and Lindemann, B., Differential expression of RNA and protein of the three pore-forming subunits of the amiloride-sensitive epithelial sodium channel in taste buds of the rat. J. Histochem. Cytochem., 47 (1999) 51±64. Lin, W., Finger, T.E., Rossier, B.C. and Kinnamon, S.C., Epithelial Na 1 channels subunits in rat taste cells: localization and regulation by aldosterone. J. Comp. Physiol., 405 (1999) 406±420. Lindemann, B., Taste reception. Physiol. Rev., 76 (1996) 719±766. Miyamoto, T., Okada, Y. and Sato, T., Ionic basis of saltinduced receptor potential in frog taste cells. Comp. Biochem. Physiol., 94A, (1989) 591±595. Miyamoto, T., Miyazaki, T., Okada, Y. and Sato, T., Wholecell recording from non-dissociated taste cells in mouse taste bud. J. Neurosci. Methods, 64 (1996) 245±252. Miyamoto, T., Fujiyama, R., Okada, Y. and Sato, T., Sour transduction involves activation of NPPB-sensitive conductance in mouse taste cells. J. Neurophysiol., 80 (1998) 1852± 1859. Miyamoto, T., Fujiyama, R., Okada, Y. and Sato, T., Salty and sour transduction. Multiple mechanisms and strain differences. Ann. N.Y. Acad. Sci., 855 (1998) 128±133. Ninomiya, Y. and Funakoshi, M., Genetic and neurobehavioral approaches to the taste receptor mechanism in mammals. In S.A. Simon and S.D. Roper (Eds.), Mechanisms of Taste Transduction, CRC Press, Boca Ranton, FL, 1993, pp. 253±272. Nimomiya, Y., Sako, N. and Funakoshi, M., Strain differences in amiloride inhibition of NaCl responses in mice, Mus musculus. J. Comp. Physiol. A, 166 (1989) 1±5. Ninomiya, Y., Fukami, Y., Yamazaki, K. and Beauchamp, G.K., Amiloride inhibition of chorda tympani responses to NaCl and its temperature dependency in mice. Brain Res., 708 (1996) 153±158. Okada, Y., Miyamoto, T. and Sato, T., Vasopressin increases frog gustatory neural responses elicited by NaCl and HCl. Comp. Biochem. Physiol., 100A, (1991) 693±696. Sato, T., Miyamoto, T. and Okada, Y., Comparison of gustatory transduction mechanisms in vertebrate taste cells. Zool. Sci., 11 (1994) 767±780. Simon, S.A., Holland, V.F., Benos, D.J. and Zampighi, G.A., Transcellular and pericellular pathways in lingual epithelia and their in¯uence in taste transduction. Microsc. Res. Tech., 26 (1993) 196±208. Stewart, R.E., Lasiter, P.S., Benos, D.J. and Hill, D.L., Immunohistochemical correlates of peripheral gustatory sensitivity to sodium and amiloride. Acta Anat., 153 (1995) 310± 319.