Localization of Soluble Guanylate Cyclase Activity in the Guinea Pig Cochlea Suggests Involvement in Regulation of Blood Flow and Supporting Cell Physiology

Abstract

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 co-chlear 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.

Keywords

soluble guanylate cyclase cochlea immunohistochemistry NADPH-diaphorase cGMP pericytes supporting cells blood flow

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) (Arnold et al. 1977). sGC then produces cGMP, which can regulate cell physiology through activation of cGMP-dependent protein kinases. Well-established roles for this pathway include neuronal signaling and control of blood flow (Bredt and Snyder 1992; Knowles and Moncada 1992).

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 (Bredt et al. 1990; Dellacorte et al. 1995). In olfactory cilia, increases in cGMP in response to odorant stimulation are blocked by NOS inhibitors, a finding that implies a direct role for this pathway in olfactory transduction (Breer et al. 1992; Breer and Shepherd 1993). NOS may also be important in the development or regeneration of the olfactory epithelium (Roskams et al. 1994). In the visual system, NO synthase (Sagar 1986; Yamamoto et al. 1993), sGC (Ahmad and Barnstable 1993), and NO-mediated increases in cGMP (Zeevalk and Nicklas 1994) have been demonstrated in the retina. The NO/cGMP pathway has also been implicated in the establishment of neuronal connections to the developing retina (Wu et al. 1994). In contrast, in the auditory system this intriguing pathway has not been thoroughly examined.

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 (Fessenden et al. 1994). Localization of NOS using NADPH-diaphorase histochemistry indicates a distribution limited to spiral ganglion cells, blood vessels in the lateral wall tissues, and nerve fibers innervating inner and outer hair cells (Fessenden et al. 1994; Zdanski et al. 1994). Physiological effects attributed to NO include changes in cochlear blood flow, compound action potential, and endocochlear potential (Brechtelsbauer et al. 1994; Chen et al. 1995). A recent report suggests that sodium nitroprusside (SNP), an NO donor, suppressed cochlear evoked potentials, perhaps through toxic effects on afferent fibers underlying inner hair cells (Kong et al. 1996). However, interpretation of these observations lacks a mechanistic foundation because little is known about the downstream enzymes in the pathway that mediate the actions of NO. In particular, the characterization of soluble guanylate cyclase is of vital importance because this enzyme is the best characterized receptor of NO.

Soluble guanylate cyclase is a heterodimeric, cytosolic protein (Kamisaki et al. 1986). Studies of the purified enzyme indicate that each sGC heterodimer contains two heme prosthetic groups that bind NO, resulting in activation of the purified enzyme up to 400-fold (Stone and Marletta 1995; Stone et al. 1995). In the cochlea, the presence of cGMP in the sensory neuroepithelium and lateral wall tissues has been documented (Guth and Stockwell 1977; Thalmann et al. 1979), as well as cytosolic cGMP formation (Zubin et al. 1995). Particulate guanylate cyclases are also present in the cochlea (Meyer zum Gottesberge et al. 1991; Furuta et al. 1995), but these enzymes are activated by peptide hormones and are therefore distinct from the NO-stimulated soluble cyclase. NO-stimulated sGC activity has not been determined. Therefore, the goal of the present study was to measure NO-stimulated sGC activity and to localize sGC in the guinea pig cochlea.

Materials and Methods

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 (Fessenden et al. 1994). Briefly, inner ear tissues were isolated by microdissection in Hanks' balanced salt solution (HBSS; Gibco-BRL, Gaithersburg, MD) supplemented with 1 mM isobutylmethylxanthine (IBMX), a phosphodiesterase inhibitor. Auditory nerve, tissues of the lateral wall (from all turns), and cochlear neuroepithelium (from all turns) were placed in separate microhomogenizers (Kontes Life Sciences; Vineland, NJ), each containing 300 μl of dissection buffer, and briefly pelleted at 10,000 × g for 30 sec. The medium was then replaced with 100 μl of homogenization buffer consisting of 100 mM triethanolamine-HCl (TEA), 5 mM MnCl₂, 1.25 mM IBMX, 10 μg/ml leupeptin, 10 μg/ml pepstatin A, 1 mM PMSF, pH 7.4. Tissues were homogenized twice on ice and then centrifuged at 10,000 × g for 10 min at 4C. A “particulate fraction” was obtained by resuspending the resulting pellet in 100 μl of homogenization buffer. A “cytosolic fraction” was obtained by centrifuging the supernatant fraction at 100,000 × g for 1 hr at 4C.

Assays for sGC were conducted in a final volume of 50 μl of 100 mM TEA, 5 mM MnCl₂, 2 mM dithiothreitol (DTT), 100 μ GTP, 5 mM phosphocreatine (PC), 152 U/1 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 × 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 × 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 (de Vente et al. 1989). This antibody has been used extensively to map the distribution of sGC in such diverse tissues as kidney, retina, and aorta (Berkelmans et al. 1989). Guinea pigs were decapitated after CO₂ anesthesia and the bullae were isolated, opened, and placed in HBSS containing 1 mM IBMX. After opening of a hole in the apex, the cochleae were transferred into either HBSS/IBMX alone (control) or HBSS/IBMX supplemented with 1 mM DEA-NO and subsequently perfused through the round and oval windows with these solutions. The lateral wall tissues from the apical turns were dissected at this point and incubated separately. Fifteen min after the perfusion, all tissues were fixed with 4% paraformaldehyde to terminate the reaction. After a 1-hr fixation, the tissues were washed three times in PBS (10 mM Na₂HPO₄/NaH₂PO₄, 138 mM NaCl, and 2.7 mM KCl, pH 7.4). The modiolus with the neurosensory epithelium attached was isolated from the bulla and the tectorial membrane and the remaining lower turns of the lateral wall tissues were discarded. Modiolus and neurosensory epithelium were incubated together with the previously dissected apical turns of the lateral wall tissues for 1 hr at room temperature (RT) in 1 ml of blocking solution consisting of 5% normal goat serum (Vector Laboratories; Burlingame, CA) and 0.1% Triton X-100 in PBS. Tissues were washed three times in PBS and then incubated overnight at 4C in 100 μl of a 1:300 dilution of anti-cGMP antibody (de Vente et al. 1989). For control experiments, the antibody had been preadsorbed for 2 hr at RT with 20 μg of cGMP–thyroglobulin conjugate (de Vente et al. 1987). Tissues were washed three times in PBS and then incubated for 1 hr at RT in 500 μl of anti-rabbit fluorescein isothiocyanate (FITC)-conjugated secondary antibody (Vector Laboratories) diluted 1:200 in PBS. Tissues were washed three times in PBS and the neurosensory epithelium from all cochlear turns was dissected from the modiolus. The lateral wall tissues and neurosensory epithelium tissues were mounted as surface preparations in Vectashield fluorescence mounting medium (Vector Laboratories).

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-μ 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 × 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 × 60 oil immersion objective and brightfield illumination.

Results

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.

Figure 1

Activity of cochlear soluble guanylate cyclase. Guanylate cyclase activity was determined in cytosolic fractions as described in Materials and Methods. Rates of cGMP formation are shown in auditory nerve (AN), lateral wall tissues (LW), and cochlear neuroepithelium (NE). Fractions were either incubated with buffer alone (open bars), 1 mM DEA-NO (solid bars), or a 1-mM DEA-NO solution depleted of NO (stippled bars). Values are means ± SEM for 9–11 independent determinations, each performed in duplicate. ^∗, significantly different from incubation with buffer alone (p>0.02 by Student's two-tailed t-test).

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 (de Vente et al. 1989). Immunoreactivity was absent from unstimulated tissue (Figure 2A). Furthermore, preincubation of the primary antibody with a cGMP–thyroglobulin conjugate abolished cGMP immunoreactivity in an NO-stimulated aorta (Figure 2C).

NO-stimulated cGMP immunoreactivity was detected in pericytes both in the lateral wall tissues (surrounding small capillaries of the suprastrial and substrial spiral ligament; Figures 3A and 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.

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 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.

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 (Fessenden et al. 1994).

Figure 2

Localization of cGMP in guinea pig aorta. Immunocytochemistry was carried out as described in Materials and Methods and labeling was visualized by confocal microscopy. (A) Cross-section from unstimulated tissue. No significant immunoreactivity is observed in the smooth muscle layer. (B) Tissue stimulated with 1 mM DEA-NO shows cGMP immunoreactivity in the smooth muscle layers. (C) Preadsorption of the anti-cGMP antiserum with a cGMP-thyroglobulin conjugate abolishes cGMP immunoreactivity in an NO stimulated aorta. Bar = 25 μ.

Discussion

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.

Figure 3

Localization of cGMP in the cochlear vasculature. Immunocytochemistry was carried out as described in Materials and Methods. (A, B) Surface preparation of apical lateral wall tissues isolated from an NO-stimulated cochlea. Pericytes encircling capillaries in spiral ligament are labeled (arrows). A and B represent the same section, photographed at different focal planes. Note the extensive branching of pericytes that surround capillaries in B. (C) Outer spiral vessel from an upper turn of an NO-stimulated cochlea. cGMP immunoreactivity can be seen in pericytes encircling this vessel (arrow). (D) Cross-section of a small modiolar vessel from an upper turn of an NO-stimulated cochlea. cGMP immunoreactivity can be detected in smooth muscle cells surrounding the vessel (arrow). Bars = 25 μ.

Originally thought of merely as structural support, recent evidence suggests that Hensen's and Deiters' cells actively modulate the transduction process (Dulon 1995). Excitability of these cells is suggested by the intriguing observation that neurons innervating the organ of Corti also synapse on supporting cells (Burgess and Nadol 1995). ATP, a potential cochlear neuromodulator, elicits robust increases in intracellular calcium in supporting cells via activation of purinergic receptors (Dulon et al. 1993). In Deiters' cells, calcium increases lead to motile responses (Dulon et al. 1994). Nothing is known, however, about mechanisms necessary to restore elevated calcium levels to normal.

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 Wang and Robinson 1997). This may occur via cGMP-dependent phosphorylation events resulting in inhibition of voltage-gated calcium channels (Blatter and Wier 1994; Meriney et al. 1994) and IP₃ formation (Hirata et al. 1990). In supporting cells of the cochlea, the NO/cGMP pathway would lower calcium levels that had been elevated, e.g., after stimulation with ATP. This is an intriguing hypothesis because the effectors of both calcium elevation (ATP) and attenuation (NO) would be released from the same source, i.e., neurons in the organ of Corti. The difference in kinetics of fast calcium increases vs delayed decreases (ATP-gated calcium channels vs a multistep NO/cGMP pathway) may allow very rapid and transient “calcium spikes” in supporting cells. Fluctuations in calcium levels could be communicated radially and longitudinally by gap junctions between these cells and could therefore have a pronounced influence on homeostatic and micromechanical processes of the basilar membrane.

Figure 4

Localization of cGMP and diaphorase reaction product in guinea pig neurosensory epithelium. Immunocytochemistry and NADPH-diaphorase reactions were carried out as described in Materials and Methods with tissues taken from the middle turns of the cochlea. (A) Brightfield image of surface preparation from organ of Corti. 1, 2, and 3, rows of outer hair cells; HCs, Hensen's cells. The apical heads of Deiters' cells lie between the outer hair cells (arrow). (B) Surface preparation from a cochlea in the same alignment as in A, pretreated with 1 mM DEA-NO. cGMP immunoreactivity can be seen in the tips of Deiters' cell phalangeal processes and in Hensen's cells. No immunoreactivity is present in outer hair cells. (C) Cross-section of an NO-stimulated organ of Corti. Immunoreactivity is found in Hensen's cells (arrowhead) and in Deiters' cells (arrows). No immunoreactivity is seen in outer hair cells. (D) NADPH-diaphorase localization of NOS. Inner and outer pillar cells (arrowheads) and nerve endings underneath outer hair cells (arrow) are labeled. Bars = 25 μ.

A second important yet independent site of action for the NO/cGMP pathway is the regulation of co-chlear blood flow through pericyte contractility. Pericytes are smooth muscle-like cells that extend fingerlike processes to encircle small capillaries. Contractions of these cells may modulate capillary permeability and blood vessel diameter (for review see Shepro and Morel 1993). Retinal pericytes grown in vitro undergo calcium-dependent contractions in response to vasoconstrictive hormones such as endothelin (Chakravarthy et al. 1992; Ramachandran et al. 1993) Conversely, exogenously applied NO relaxes precontracted cultured retinal pericytes and increases cellular cGMP. The involvement of sGC and cGMP in pericyte relaxation is further supported by the fact that the cGMP analogue 8-bromo-cGMP also relaxes precontracted pericytes (Haefliger et al. 1994). Consistent with the proposed overall mechanism, the target enzyme of cGMP, cGMP-dependent protein kinase, is also expressed in pericytes from many different tissues (Joyce et al. 1984).

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 (Hawkins 1971; Hawkins et al. 1972). Furthermore, changes in cochlear blood flow are paralleled by changes in capillary diameter in lateral wall tissue vessels (Quirk et al. 1992; Quirk and Seidman 1995). Recent studies of microvessels isolated from the lateral wall tissues indicate that vasoconstrictors, such as endothelin, do indeed contract cochlear capillaries (Sadanaga and Wangemann 1996) and that these contractions are reversed by NO (Wangemann, personal communication). Our demonstration of sGC activity in pericytes provides a molecular basis for these observations and suggests that changes in cochlear blood flow seen with application of modulators of the NO/cGMP pathway (Ohlsen et al. 1992; Brechtelsbauer et al. 1994) are mediated by pericytes.

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.

Footnotes

Acknowledgements

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.

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