Type II and III Taste Bud Cells Preferentially Expressed Kainate Glutamate Receptors in Rats

Sang-Bok Lee; Cil-Han Lee; Se-Nyun Kim; Ki-Myung Chung; Young-Kyung Cho; Kyung-Nyun Kim

doi:10.4196/kjpp.2009.13.6.455

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Lee, Lee, Kim, Chung, Cho, and Kim: Type II and III Taste Bud Cells Preferentially Expressed Kainate Glutamate Receptors in Rats

Original Article

Korean J Physiol Pharmacol 2009;13(6):455-460.

Published online: 18 February 2009

DOI: https://doi.org/10.4196/kjpp.2009.13.6.455

Type II and III Taste Bud Cells Preferentially Expressed Kainate Glutamate Receptors in Rats

Sang-Bok Lee¹, Cil-Han Lee¹, Se-Nyun Kim², Ki-Myung Chung^1,³, Young-Kyung Cho^1,³, Kyung-Nyun Kim^1,^3,

¹Department of Physiology and Neuroscience, College of Dentistry, Gangneung-Wonju National University, Gangneung 210-702, Korea

²Department of Pharmacology and Mechanism, Research Institute, Oscotec Inc., Cheonan 331-831, Korea

³Research Institute of Oral Science, Gangneung-Wonju National University, Gangneung 210-702, Korea

Corresponding to: Kyung-Nyun Kim, Department of Physiology and Neuroscience, College of Dentistry, Gangneung-Wonju National University, 123, Chibyun-dong, Gangneung 210-702, Korea. (Tel) 82-33-640-2450, (Fax) 82-33-642-6410, (E-mail) knkim@gwnu.ac.kr

Received 2 November 2009 Revised 13 November 2009 Accepted 24 November 2009

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Glutamate-induced cobalt uptake reveals that non-NMDA glutamate receptors (GluRs) are present in rat taste bud cells. Previous studies involving glutamate induced cobalt staining suggest this uptake mainly occurs via kainate type GluRs. It is not known which of the 4 types of taste bud cells express subunits of kainate GluR. Circumvallate and foliate papillae of Sprague-Dawley rats (45∼60 days old) were used to search for the mRNAs of subunits of non-NMDA GluRs using RT-PCR with specific primers for GluR1–7, KA1 and KA2. We also performed RT-PCR for GluR5, KA1, PLCβ2, and NCAM/SNAP 25 in isolated single cells from taste buds. Taste epithelium, including circumvallate or foliate papilla, express mRNAs of GluR5 and KA1. However, non-taste tongue epithelium expresses no subunits of non-NMDA GluRs. Isolated single cell RT-PCR reveals that the mRNAs of GluR5 and KA1 are preferentially expressed in Type II and Type III cells over Type I cells.

Keywords: Taste bud, Kainate receptor, RT-PCR, Single isolated cell

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Fig. 1.

Results of tissue RT-PCR of mRNA obtained from tongue epithelium and hippocampus. N, non-taste epithelium; F, epithelium including foliate papilla; CV, epithelium including circumvallate papilla; H, hippocampus as positive control; –, no tissue as negative control; M, size marker.

Fig. 2.

Representative isolated single cell RT-PCR results. Data is from 15 cells obtained from a single rat. Data from lanes 11 and 15 were discarded because both PLCβ2 and SNAP25 are expressed. Two cells express PLCβ2, another 2 cells express SNAP 25, 6 cell express GluR5, and 2 cells express KA1. Even cells in lanes 11 and 15 do not express KA1. However, GAPDH is expressed in all lanes except the negative control.

Fig. 3.

Results of isolated single cell RT-PCR for taste bud cells. Each column represents data from an individual taste cell. Each row indicates expression of different mRNAs.

Table 1.

DNA sequences, annealing temperatures, and expected product sizes of specific primers of subunits of non-NMDA glutamate receptors used in RT-PCR

Name		Sequence	Annealing temperature (°C)	Expected product size (bp)
GluR1	Sense	GCT TCA TGG ACA TTG TA	40	623
	Antisense	ATC TCA AGT CGG TAG GAG TA
GluR2	Sense	ATT GTA GAC TAC GAT GAT TC	40	643
	Antisense	AAT AGT CAG CTT GTA CTT GA
GluR3	Sense	AAA CGA TAC TTG ATT GAC TG	40	655
	Antisense	GCT GAT TTG TTG ATC TGA GA
GluR4	Sense	CCA CTG CTA GAA GAG CTT GA	40	612
	Antisense	CAT ATC TTG AAT CAA GAC TA
GluR5	Sense	GCC CCT CTC ACC ATC ACA TAC	52	208
	Antisense	ACC TCG CAA TCA CAA ACA GTA
GluR6	Sense	TTC CTG AAT CCT CTC TCC CCT	52	260
	Antisense	CAC CAA ATG CCT CCC ACT ATC
GluR7	Sense	TGG AAC CCT ACC GCT ACT CG	52	356
	Antisense	CCG CAA GCC ACT GGT TTT GTT
KA1	Sense	AGC GTT ATG TCA TGC CCA GAC CAG	52	316
	Antisense	GGG GAG GAT CTG ACA CAT
KA2	Sense	TGC CCC GTG TCC TCA ACT CA	52	398
	Antisense	CAC CGA CAC CTC CTC AGA CT

Table 2.

DNA sequences, annealing temperatures and expected product sizes for specific primers used in RT-PCR to test specific markers for cell type in taste buds (positive control GAPDH)

Name		Sequence	Annealing temperature (°C)	Expected product size (bp)
PLCβ2	Sense	CTG GAG GCT GAA GTA AAG GAG	40	623
	Antisense	GCC CCT GCA TGT ATG TTA GG
NCAM	Sense	TTG TGG GCA TCC TCA TTG TC	52	643
	Antisense	TGT CAG TGG TGT GGT CTC GT
SNAP25	Sense	GGC AAT AAT CAG GAT GGA GTA G	58	612
	Antisense	AGA TTT AAC CAC TTC CCA GCA
GAPDH	Sense	TGG GGT GAT GCT GGT GCT GA	60	398
	Antisense	CGC CTG CTT CAC CAC CTT CT

Table 3.

Expression of GluR5, KA1, PLCβ2 and NCAM/SNAP25 in 146 isolated single taste bud cells from12 rats

	PLCβ2	NCAM/SNAP25	None	Total
GluR5	17	7	27	51
KA1	6	2	4	12
GluR5 & KA1	1	2	3	6
None	18	8	51	77
Total	42	19	85	146

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