Expression and localization of the Na+-H+ exchanger in the guinea pig cochlea

doi:10.1016/S0378-5955(98)00191-9

Hearing Research

Volume 128, Issues 1–2, February 1999, Pages 89-96

https://doi.org/10.1016/S0378-5955(98)00191-9 Get rights and content

Abstract

Physiological studies have shown that the Na⁺-H⁺ exchanger (NHE) is a major carrier protein regulating the intracellular pH in the cells of the cochlea. The presence of multiple forms of the exchanger has been demonstrated by the recent cloning of four mammalian NHEs, NHE-1 to NHE-4. Despite the structural similarity, these NHE isoforms differ in their tissue distribution, kinetic characteristics, and responses to external stimuli. The present study was undertaken to examine the expression and distribution of four NHE isoforms in the guinea pig cochlea. We used reverse transcription-polymerase chain reaction to assess the expression of NHE-1–4 isoforms and non-radioactive in situ hybridization to examine their localization. Although NHE-2, -3 and -4 isoform mRNAs could be detected in the cochlear tissue, the NHE-1 message was predominant. Cloned guinea pig NHE-1–4 partial cDNA fragments were highly homologous to the corresponding rat NHE isoforms. NHE-1 isoform mRNA was distributed in the hair cells, marginal cells, spiral ligament fibrocytes, spiral prominence cells and spiral ganglion cells. NHE-1 localized in a variety of cochlear cells would contribute to their differential function.

Introduction

The Na⁺-H⁺ exchanger (NHE) is a plasma membrane transporter which mediates the electroneutral exchange of extracellular Na⁺ for intracellular H⁺ (pH_i) in a one-to-one stoichiometry (Aronson, 1985), and is found in virtually all mammalian cells. The NHE plays an essential role in intracellular pH homeostasis, in particular recovery from acid load, cell volume regulation (Grinstein et al., 1992; MacLeod and Hamilton, 1996), transepithelial sodium absorption (Tse et al., 1992; Hoogerwerf et al., 1996), and cell proliferation in response to growth factors (Grinstein et al., 1989). Studies from our laboratory have established that a variety of cochlear tissues and cells of the guinea pig including the outer hair cells (Ikeda et al., 1992) and the stria vascularis cells (Ikeda et al., 1994) regulate pH_i via NHE. Five distinct NHEs, NHE-1–5, have been identified in a variety of tissues and found to share significant amino acid sequence identity. NHE-1, the first member of the NHE gene family to be cloned (Sardet et al., 1989), is the housekeeping isoform which probably serves a cell maintenance function and is distributed ubiquitously in both epithelial and nonepithelial cells. NHE-2, -3, -4 and -5, however, are expressed in a tissue-specific manner. NHE-2 mRNA is expressed in the stomach, intestine, colon, kidney, uterus and adrenal gland (Wang et al., 1993; Tse et al., 1993; Collins et al., 1993). NHE-3 mRNA, which is restricted to the kidney, stomach, and intestine (Tse et al., 1992; Orlowski et al., 1992; Brant et al., 1995), may be an epithelial cell-specific isoform. The NHE-4 message is widely distributed in various tissues, and is especially abundant in stomach (Orlowski et al., 1992). NHE-5, a novel member of the NHE family, has been partially cloned in human and is found in brain, testis, spleen and skeletal muscle (Klanke et al., 1995).

In previous physiological studies, we first demonstrated the presence of the NHE activity in the guinea pig cochlea (Ikeda et al., 1992). The H⁺ extrusion was shown to be the inward-directed Na⁺ chemical gradient and was sensitive to amiloride in the guinea pig outer hair cell. Similar observations were made in the guinea pig cochlear lateral wall epithelium (Ikeda et al., 1994), and in the gerbil vestibular transitional cells (Wangemann et al., 1993) and dark cells (Wangemann et al., 1996). Although the NHE sensitive to amiloride has been analyzed using physiological methods, NHE isoforms have not yet been determined in the cochlea.

The present study was designed to identify molecular forms of NHE in the guinea pig cochlea, which is the same species that was used in the physiological studies. We used reverse transcription-polymerase chain reaction (RT-PCR) to assess the expression of NHE-1–4 isoforms and non-radioactive in situ hybridization (ISH) to examine their localization.

Section snippets

RNA isolation

Young adult guinea pigs (200–250 g) were anesthetized by inhalation of diethylether and decapitated. The temporal bone was dissected and the bony capsule of the cochlea was picked away in sterile phosphate buffer (PB) under a stereomicroscope, then the whole cochlea was removed and prepared for RNA extraction. Polyadenylated RNA was isolated from cochleae by guanidinium isothiocyanate extraction and oligo(dT) cellulose separation using a commercially available kit (Quick Prep mRNA Extraction

RT-PCR amplification

A small amount of first strand cDNA derived from a single guinea pig cochlea was used as a template for PCR amplification. The initial 30-cycle amplification of guinea pig cochlea cDNA with outer primer sets yielded a single band of gpNHE1 at the predicted size on an agarose gel, however, for gpNHE2, gpNHE3, and gpNHE4, no band was observed (Fig. 2A). Since the amount of mRNAs extracted from the cochlea was expected to be small, we decided to run nested PCR with inner primer sets and the

Discussion

This study revealed that multiple isoforms of NHE were expressed in the guinea pig cochlea. It is difficult to obtain cDNA molecules from the cochlea by cDNA library screening, since the cochlear tissue is too small to possess enough mRNA. The RT-PCR method is a common technique to detect mRNA in the cochlea (Oshima et al., 1997a, Oshima et al., 1997b; Hidaka et al., 1996) because of its high sensitivity. Accordingly, we applied RT-PCR to obtain NHE molecules, and four isoforms of the NHE,

Acknowledgements

This study was supported by Grants-in-Aid (No. 08457447) to K. Ikeda, (No. 40241608) to T. Oshima, and (No. 08407054) to T. Takasaka from the Ministry of Education, Science and Culture of Japan.

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Expression and localization of the Na+-H+ exchanger in the guinea pig cochlea

Abstract

Introduction

Section snippets

RNA isolation

RT-PCR amplification

Discussion

Acknowledgements

Cell

Prog. Neurobiol.

Biochim. Biophys. Acta

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Genomics

Biochim. Biophys. Acta

Neuroscience

J. Biol. Chem.

J. Biol. Chem.

Hear. Res.

Biochem. Biophys. Res. Commun.

Dev. Biol.

Cell

Hear. Res.

Neurosci. Res.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Expression and localization of the Na⁺-H⁺ exchanger in the guinea pig cochlea