RAPID COMMUNICATION |
Correspondence to: James J. Crouch, Dept. of Pathology and Laboratory Medicine, Medicial Univ. of South Carolina, 171 Ashley Ave., Charleston, SC 29425.
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Summary |
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We mapped the cellular and subcellular distribution of the Na-K-Cl co-transporter (NKCC) in the adult gerbil inner ear by immunostaining with a monoclonal antibody (MAb T4) generated against human colon NKCC. Heavy immunolabeling was seen in the basolateral plasma membrane of marginal cells in the stria vascularis and dark cells in the vestibular system. Subpopulations of fibrocytes in the cochlear spiral ligament and limbus and underlying the vestibular neurosensory epithelium also stained with moderate to strong intensity, apparently along their entire plasmalemma. Because MAb T4 recognizes both the basolateral secretory (NKCC1) and the apical absorptive (NKCC2) isoforms of the co-transporter, we employed reverse transcription and the polymerase chain reaction (RT-PCR) to explore isoform diversity in inner ear tissues. Using NKCC1 and NKCC2 isoform-specific PCR primers based on mouse and human sequences, only transcripts for NKCC1 were detected in the gerbil inner ear. The presence of abundant NKCC1 in the basolateral plasmalemma of strial marginal and vestibular dark cells confirms conclusions drawn from pharmacological and physiological data. The co-expression of NKCC1 and Na,K-ATPase in highly specialized subpopulations of cochlear and vestibular fibrocytes provides further evidence for their role in recycling K+ leaked or effluxed through hair cells into perilymph back to endolymph, as postulated in current models of inner ear ion homeostasis. (J Histochem Cytochem 45:773-778, 1997)
Key Words: ion transport, cochlea, endocochlear potential, RT-PCR
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Introduction |
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It is firmly established that generation of large endolymph-perilymph Na+ and K+ gradients and the endocochlear potential (EP) is dependent on activity of the Na,K-ATPase pump expressed abundantly in the basolateral plasma membrane of strial marginal cells (for review see
Despite the large body of pharmacological and physiological data supporting its presence, the precise cellular distribution and isoform expression patterns of NKCC in the inner ear have yet to be firmly established. Immunocytochemical data presented in a recent unpublished abstract have provided preliminary evidence for the localization of NKCC in the basolateral membrane of strial marginal cells in the rabbit cochlea (
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Materials and Methods |
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Animals and Tissue Processing
Inner ears were obtained from 14 Mongolian gerbils (Meriones unguiculatus) of both genders between 1 and 12 months of age. The gerbils were born and raised in a quiet vivarium with a mean noise level of approximately 40 dBA (
Six gerbils between 6 and 12 months of age were anesthetized by IP injection of urethane (1.5 g/kg in dH2O) and then exsanguinated by transcardial perfusion with a warm 0.9% saline solution containing 0.1% sodium nitrite. For light microscopic (LM) studies, 50 ml of a fixative, consisting of 10% formalin, 0.9% NaCl, and 0.5% zinc dichromate was administered transcardially. The bullae were opened rapidly, the stapes removed, the round window perforated, and 0.5 ml of fixative was infused gently through the oval window. The inner ears were dissected free and immersed in fixative for a further 15 min, rinsed with saline, and decalcified by immersion in 0.12 M EDTA (pH 7.0) for 48-72 hr at room temperature (RT) with gentle stirring. The EDTA solution was changed daily. The decalcified inner ears were dehydrated in a graded series of ethanols (70% 2 hr; 80% 2 hr; 95% 2 hr; 100% three times for 1 hr), cleared in Histoclear (twice for 1 hr; National Diagnostics, Manville, NJ), and embedded in Paraplast Plus (twice for 1 hr at 58C; Curtin Matheson, Marietta, GA). Serial mid-modiolar sections were cut at 5 µm thickness and mounted on glass slides.
Four gerbils between 6 and 12 months of age were processed for electron microscopic (EM) studies according to the procedure described above, except that the fixative consisted of a 0.5% glutaraldehyde and 4% paraformaldehyde mixture in 0.1 M phosphate buffer at pH 7.2. After decalcification with EDTA as described above, the cochleas were sliced into half-turns and, along with pieces of vestibular tissue, were dehydrated through a graded series of 50, 70, 90, and 95% ethanols, and infiltrated into Lowicryl K4M at -20C as previously described (
Immunohistochemical Staining Procedures
An MAb (T4) raised against a fusion protein encompassing the carboxy-terminal 310 amino acids of the human colon NKCC was provided by Dr. Christian Lytle (Division of Biomedical Sciences, University of California, Riverside, CA). MAb T4 recognizes both the apical NKCC2 and basolateral NKCC1 isoforms of the Na-K-Cl co-transporter (
For LM studies, immunoperoxidase staining was performed as described previously (
Nonimmune mouse serum diluted 1:80,000 was substituted for the primary antibody as a methods control. Sections from composite blocks containing a variety of tissues from a wide range of gerbil organs were processed in parallel with the inner ears as positive and negative tissue staining controls.
For EM studies, ultrathin sections of cochlear half-turns and the utricle and ampulla were cut and picked up on formvar-carbon-coated nickel grids (Ted Pella; Redding, CA). Immunogold cytochemistry was performed following procedures described in detail elsewhere (
Reverse-transcription Polymerase Chain Reaction
Four 3-month-old Mongolian gerbils of either gender were anesthetized as described above and exsanguinated by trans-cardial perfusion with warm 0.9% saline solution. Inner ears and kidneys were rapidly removed, immersed in liquid nitrogen, and stored at -80C. Total RNA was obtained using acid guanidinium thiocyanate as described by
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Results |
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Immunocytochemistry
In paraffin sections of adult gerbil inner ear, MAb T4 produced an intense immunostaining reaction in the stria vascularis and a less intense but still strong reaction in Type II fibrocytes in the inferior portion of the spiral ligament (Figure 1). Type V fibrocytes in the suprastrial region and a subpopulation of fibrocytes in the limbus were also immunoreactive (Figure 1). At higher magnification, immunostaining was clearly restricted to the basolateral plasmalemma of the strial marginal cells, whereas the entire plasma membrane of the nonpolarized fibrocytes appeared to stain (Figure 2). In the vestibular system, strong immunoreactivity was present in the basolateral plasmalemma of dark cells and along the cell membrane of select fibrocytes underlying the neurosensory epithelium (Figure 3).
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For a positive tissue control, abundant NKCC was detected in the apical plasmalemma of cells lining the thick ascending limb segment of the gerbil nephron (Figure 4), in agreement with results in rabbit kidney (
At the ultrastructural level, gold spherules demonstrative of NKCC decorated the basolateral plasma membrane of strial marginal cells, whereas the apical plasmalemma was unreactive (Figure 5). Immunogold labeling of vestibular dark cells also was restricted to the basolateral plasma membrane (Figure 6).
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RT-PCR
Use of the NKCC1-specific PCR primers produced a 314-base pair (BP) PCR product in both the gerbil inner ear and kidney (Figure 7). The PCR products were cloned and confirmed by sequence analysis to be transcripts for NKCC1. In contrast, the NKCC2 specific PCR primers produced a 424-BP PCR product in gerbil kidney but not in the inner ear. This PCR product was cloned and identified by sequence analysis to be NKCC2.
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Discussion |
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The Na-K-Cl co-transporter (NKCC) is an integral membrane protein responsible for the coupled electroneutral transport of 1Na:1K:2Cl across the plasma membrane in a variety of animal cells (see
The localization of the secretory isoform of NKCC to the basolateral plasma membrane of strial marginal and vestibular dark cells in the gerbil inner ear confirms previous pharmacological and physiological evidence indicating the presence of NKCC in these sites (
Previous biochemical, cytochemical, and pharmacological studies have demonstrated the expression of Na,K-ATPase in strial marginal and vestibular dark cells and mapped its distribution to the basolateral plasma membrane (see
An intriguing finding was the strong immunoreactivity observed in subpopulations of fibrocytes in the cochlea and vestibular system. A comparison of the immunostaining pattern for NKCC1 with previous data mapping the distribution of the Na,K-ATPase pump and its isoforms in inner ear tissues (
Also of interest are other cell types in the inner ear that have previously been shown to express moderate to abundant levels of immunoreactive Na,K-ATPase but do not appear to co-express the NKCC. The Na,K-ATPase-rich cell membrane of spiral ganglion neurons and their afferent nerve endings underlying the inner hair cells represent the most striking example of this non-co-expression. Other sites expressing Na,K-ATPase but failing to stain with MAb T4 include the basolateral plasma membrane of epithelial cells lining Reissner's membrane and the outer sulcus as well as that of interdental cells. Apparently these cell types express NKCC at levels below the detection limit of methods employed here or utilize an alternate mechanism(s) for generating the intracellular Na+ levels necessary to drive Na,K-ATPase activity.
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Acknowledgments |
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Supported by research grants RO1 DC00713 and PO1 DC00422 from the National Institute on Deafness and Other Communication Disorders, National Institutes of Health and by American Heart Association Grant-In-Aid AHA 94015270. We thank Ms Leslie Harrelson and Ms Nancy Smythe for technical and editorial assistance.
Received for publication March 3, 1997; accepted March 18, 1997.
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