ARTICLE |
Correspondence to: Jochen Schacht, Kresge Hearing Res. Inst., U. of Michigan, 1301 E. Ann St., Ann Arbor, MI 48109-0506.
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Summary |
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Although the nitric oxide/cGMP pathway has many important roles in biology, studies of this system in the mammalian cochlea have focused on the first enzyme in the pathway, nitric oxide synthase (NOS). However, characterization of the NO receptor, soluble guanylate cyclase (sGC), is crucial to determine the cells targeted by NO and to develop rational hypotheses of the function of this pathway in auditory processing. In this study we characterized guinea pig cochlear sGC by determining its enzymatic activity and cellular localization. In cytosolic fractions of auditory nerve, lateral wall tissues, and cochlear neuroepithelium, addition of NO donors resulted in three- to 15-fold increases in cGMP formation. NO-stimulated sGC activity was not detected in particulate fractions. We also localized cochlear sGC activity through immunocytochemical detection of NO-stimulated cGMP. sGC activity was detected in Hensen's and Deiters' cells of the organ of Corti, as well as in vascular pericytes surrounding small capillaries in the lateral wall tissues and sensory neuroepithelium. sGC activity was not observed in sensory cells. Using NADPH-diaphorase histochemistry, NOS was localized to pillar cells and nerve fibers underlying hair cells. These results indicate that the NO/cGMP pathway may influence diverse elements of the auditory system, including cochlear blood flow and supporting cell physiology.(J Histochem Cytochem 45:1401-1408, 1997)
Key Words: soluble guanylate cyclase, cochlea, immunohistochemistry, NADPH-diaphorase, cGMP, pericytes, supporting cells, blood flow
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Introduction |
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The nitric oxide/cGMP pathway is a multienzyme signal transduction system with many important physiological roles. It is composed of three principal enzymes. Activation of nitric oxide synthase (NOS) results in the production of NO, a gas that stimulates soluble guanylate cyclase (sGC) (
The NO/cGMP pathway is emerging as an important component of sensory systems. In the olfactory system, NOS has been localized to the accessory olfactory bulb and the olfactory epithelium (
In the peripheral auditory system, studies of the NO/cGMP pathway have focused mainly on the first enzyme, NOS, which has been detected enzymatically in inner ear tissues (
Soluble guanylate cyclase is a heterodimeric, cytosolic protein (
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Materials and Methods |
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Except where noted, all chemicals were purchased from Sigma (St Louis, MO). Pigmented guinea pigs initially weighing 250-300 g (Murphy's Breeding Labs; Plainfield, NJ) were used in this study. All experimental protocols on animal use were approved by the University of Michigan Committee on Use and Care of Animals. Animal care was under the supervision of the University of Michigan's Unit for Laboratory Animal Medicine.
sGC Assay
Tissue was prepared for enzymatic assays as described previously (
Assays for sGC were conducted in a final volume of 50 µl of 100 mM TEA, 5 mM MnCl2, 2 mM dithiothreitol (DTT), 100 µM GTP, 5 mM phosphocreatine (PC), 152 U/l creatine kinase (CK), 1 mM IBMX, 8 µg/ml leupeptin, 8 µg/ml pepstatin A, 0.8 mM PMSF at pH 7.4. Forty-µl aliquots of each tissue fraction to be tested were first delivered into a 1.5 ml Eppendorf tube. Five µl of a co-factor solution consisting of 10 x concentrations of GTP, DTT, PC, and CK were then added to the tissue fractions. Fifteen sec later the reaction was started with 5 µl of either 10 mM diethylamine NONOate (DEA-NO; Cayman Chemical, Ann Arbor, MI), 10 mM SNP, 10 mM depleted DEA-NO (each dissolved in 100 mM TEA-HCl and 5 mM MnCl2, pH 7.4) or solvent alone (control). DEA-NO was depleted of NO by acidification to pH 4, bubbling with nitrogen for 1 hr, and readjustment of the pH to 7.4 using NaOH. After 30 min at 37C, assays were terminated by the addition of 50 µl of 12% trichloroacetic acid. Reaction tubes were centrifuged at 12,000 x g for 5 min and the supernatant fraction was extracted three times with 4 volumes (400 µl) of water-saturated diethyl ether. The organic phase (top layer) was discarded after each extraction. The aqueous layer was frozen in liquid nitrogen and lyophilized for 2-4 hr. Lyophilized samples were reconstituted in 1 ml of cGMP assay buffer and cGMP levels were measured using the Amersham cGMP RIA kit (Amersham; Arlington Heights, IL). Differences in cGMP levels with or without NO stimulation were compared. Protein concentrations were measured using the colloidal gold assay protocol (Integrated Separation Systems; Natick, MA).
cGMP Immunocytochemistry
Cochlear sGC activity was localized through the identification of NO-stimulated increases in cGMP in cochlear cells using antibodies raised against a cGMP-thyroglobulin conjugate (
For detection of cGMP labeling in cochlear cross sections, NO-stimulated cochleae were decalcified for 4 days at 4C in 5% ethylenediaminetetraacetic acid, 4% paraformaldehyde in PBS. Next, cochleae were cryoprotected in 30% sucrose in PBS for 4 hr at 4C and then frozen in Tissue-Tek mounting medium (Miles; Elkhart, IN) using a dry ice/methanol bath. Fifteen-µm sections obtained using a cryostat (Bright Instruments; Huntingdon, UK) were thaw-mounted onto slides precoated with a solution of 5 mg/ml gelatin, 0.5 mg/ml CrK(SO4)2 in water. Sections were subsequently processed for anti-cGMP immunocytochemistry as described above.
For detection of sGC activity in guinea pig aorta, the following procedure was used. The ascending aorta was isolated from guinea pig immediately after decapitation. The tissue was placed in HBSS/IBMX and cut in half with a clean razor blade. One section was transferred to a solution containing 1 mM DEA-NO in HBSS/IBMX and incubated for 15 min at RT. The other section remained in HBSS/IBMX during the 15-min incubation period and served as a negative (unstimulated) control. The tissues were then fixed in 4% paraformaldehyde for 1 hr at RT. Cryoprotection, sectioning, and anti-cGMP immunohistochemistry were carried out as described above.
Immunolabeling was detected using a Nikon fluorescent microscope and the appropriate filter cubes for visualization of FITC labeling. Sections were photographed using Kodak Ektachrome 160T film at 160 ASA. Alternatively, labeling was detected using a Bio-RAD 600 laser scanning confocal unit (Bio-RAD; Richmond, CA) attached to a Nikon Diaphot TMD inverted microscope with a x 60 oil immersion objective. Fluorescent images were digitally processed using Adobe Photoshop 3.0 for Macintosh.
NADPH-diaphorase Histochemistry
Cochlear cross-sections from fixed and decalcified cochleae were prepared as described above. For visualization of NADPH-diaphorase histochemistry, 200 µl of a solution containing 1 mM NADPH, 0.5 mM nitroblue tetrazolium, 0.2% Triton X-100, and 50 mM Tris-HCl, pH 8.0, was added to each slide. After a 2-hr incubation at 37C, slides were washed twice in PBS and then mounted using GVA mount (Zymed Laboratories; South San Francisco, CA). Sections were photographed as described above using a x 60 oil immersion objective and brightfield illumination.
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Results |
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Biochemical Activities
Basal levels of cGMP production in cochlear cytosols were 37.4, 55.6, and 53.3 pmol cGMP/min/mg protein in auditory nerve (AN), lateral wall tissues (LW), and cochlear neuroepithelium (NE), respectively. Addition of 1 mM DEA-NO (an NO donor) increased cGMP production 15-, eight-, and threefold in these tissues, respectively (Figure 1). Incubation of cochlear cytosols with a 1-mM DEA-NO solution depleted of NO did not significantly increase cGMP levels over basal values. A structurally unrelated NO donor, sodium nitroprusside (SNP, 1 mM), also stimulated cGMP formation in cochlear cytosols nine-, three-, and twofold in AN, LW, and NE. In contrast, incubation of cochlear particulate fractions with 1 mM DEA-NO did not significantly increase cGMP production.
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Immunocytochemistry
Guinea pig aorta was used as a positive control tissue for sGC localization. The anti-cGMP antiserum labeled the smooth muscle layers in preparations stimulated with 1 mM DEA-NO (Figure 2B), a finding that is consistent with published reports (
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NO-stimulated cGMP immunoreactivity was detected in pericytes both in the lateral wall tissues (surrounding small capillaries of the suprastrial and substrial spiral ligament; Figure 3A and Figure 3B) and the organ of Corti (on the outer spiral vessel; Figure 3C). In addition, the smooth muscle layer of modiolar vessels (Figure 3D) was immunoreactive. No labeling was detected in the spiral ganglion cells of the modiolus.
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In the neuroepithelium, NO-stimulated increases in cGMP were observed in several distinct locations. In the organ of Corti, the Hensen's cells were strongly labeled, as were the Deiters' cells (Figure 4B and Figure 4C). This labeling was seen equally in all cochlear turns. In addition, immunoreactivity was detected in cells of the inner and outer sulcus region. No labeling of inner or outer hair cells was observed.
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In the lateral wall tissues and neurosensory epithelium, labeling could also be induced with 1 mM SNP. Use of this NO donor resulted in identical labeling patterns as were seen using 1 mM DEA-NO. No labeling was detected in unstimulated tissues or in stimulated tissues treated with a preadsorbed anti-cGMP antibody.
NADPH-diaphorase Labeling
The staining pattern for sGC contrasted with NADPH-diaphorase detection of NOS. In the organ of Corti, labeling was observed under the outer hair cells as well as in outer and inner pillar cells (Figure 4D), confirming and extending our previous observations (
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Discussion |
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The cellular distribution of sGC and NOS in the inner ear suggests the participation of the NO/cGMP pathway in cochlear homeostatic mechanisms through regulation of blood flow and supporting cell physiology. This notion is supported by the presence of sGC activity in pericytes as well as in Hensen's and Deiters' cells of the organ of Corti. Furthermore, these supporting cells are in close proximity to NOS, which is located in pillar cells and in nerve endings underlying outer hair cells. The lack of sGC activity in hair cells suggests that this pathway is not directly involved in auditory transduction.
Originally thought of merely as structural support, recent evidence suggests that Hensen's and Deiters' cells actively modulate the transduction process (
The NO/cGMP pathway may fulfill this crucial role in supporting cells. Downregulation of elevated calcium concentrations to basal levels is indeed a well-established function of this pathway in many cell types, including neurons (for review see
A second important yet independent site of action for the NO/cGMP pathway is the regulation of cochlear blood flow through pericyte contractility. Pericytes are smooth muscle-like cells that extend finger-like processes to encircle small capillaries. Contractions of these cells may modulate capillary permeability and blood vessel diameter (for review see
In the cochlea, pericyte contractility in vivo is strongly suggested by indirect evidence. Overstimulation of the cochlea with noise or treatment with quinine results in the blockage of capillaries in the lateral wall tissues and neuroepithelium, a phenomenon attributed to pericyte contraction (
In summary, this study delineates the intercellular connections of the NO/cGMP pathway in the cochlea and their potential contributions to cochlear homeostasis. The hypothesis derived from these data emphasizes the contribution of supporting cells to cochlear regulatory mechanisms. Furthermore, it proposes the vessels of the spiral ligament as major sites for control of cochlear blood flow.
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Acknowledgments |
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Supported by NIH NIDCD program project grants DC-00078 and DC-02982 and by training grant DC-00011.
We thank Dr Joseph E. Hawkins Jr. for helpful comments and discussions.
Received for publication March 4, 1997; accepted May 15, 1997.
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