FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2058974065> ?p ?o ?g. }

• W2058974065 endingPage "35650" @default.
• W2058974065 startingPage "35644" @default.
• W2058974065 abstract "Outer hair cell electromotility is crucial for the proper function of the cochlear amplifier, the active process that enhances sensitivity and frequency discrimination of the mammalian ear. Previous work (Kalinec, F., Zhang, M., Urrutia, R., and Kalinec, G. (2000) J. Biol. Chem. 275, 28000–28005) has suggested a role for Rho GTPases in the regulation of outer hair cell electromotility, although the signaling pathways mediated by these enzymes remain to be established. Here we have investigated the cellular and molecular mechanisms underlying the homeostatic regulation of the electromotile response of guinea pig outer hair cells. Our findings defined a ROCK-mediated signaling cascade that continuously modulates outer hair cell electromotility by selectively targeting the cytoskeleton. A distinct ROCK-independent pathway functions as a fast resetting mechanism for this system. Neither pathway affects the function of prestin, the unique molecular motor of outer hair cells. These results extend our understanding of a basic mechanism of both normal human hearing and deafness, revealing the key role of the cytoskeleton in the regulation of outer hair cell electromotility and suggesting ROCK as a molecular target for modulating the function of the cochlear amplifier. Outer hair cell electromotility is crucial for the proper function of the cochlear amplifier, the active process that enhances sensitivity and frequency discrimination of the mammalian ear. Previous work (Kalinec, F., Zhang, M., Urrutia, R., and Kalinec, G. (2000) J. Biol. Chem. 275, 28000–28005) has suggested a role for Rho GTPases in the regulation of outer hair cell electromotility, although the signaling pathways mediated by these enzymes remain to be established. Here we have investigated the cellular and molecular mechanisms underlying the homeostatic regulation of the electromotile response of guinea pig outer hair cells. Our findings defined a ROCK-mediated signaling cascade that continuously modulates outer hair cell electromotility by selectively targeting the cytoskeleton. A distinct ROCK-independent pathway functions as a fast resetting mechanism for this system. Neither pathway affects the function of prestin, the unique molecular motor of outer hair cells. These results extend our understanding of a basic mechanism of both normal human hearing and deafness, revealing the key role of the cytoskeleton in the regulation of outer hair cell electromotility and suggesting ROCK as a molecular target for modulating the function of the cochlear amplifier. Cochlear outer hair cells (OHCs) 1The abbreviations used are: OHCs, outer hair cells; ACh, acetylcholine; LPA, lysophosphatidic acid; dn, dominant-negative.1The abbreviations used are: OHCs, outer hair cells; ACh, acetylcholine; LPA, lysophosphatidic acid; dn, dominant-negative. undergo reversible changes in length when electrically stimulated. This electromotile response results from a membrane-based force generator mechanism associated with conformational changes and rearrangement of a voltage-sensitive integral membrane protein (1Kalinec F. Holley M.C. Iwasa K. Lim D.J. Kachar B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8671-8675Crossref PubMed Scopus (214) Google Scholar). The OHC motor protein has been recently identified as a novel anion transporter (SLC26A5-prestin) (2Zheng J. Shen W. He D.Z.Z. Long K.B. Madison L.D. Dallos P. Nature. 2000; 405: 149-155Crossref PubMed Scopus (990) Google Scholar), with voltage sensitivity conferred by the intracellular anions chloride and bicarbonate (Ref. 3Oliver D. He D.Z.Z. Klöcker N. Ludwig J. Schulte U. Waldegger W. Ruppersberg J.P. Dallos P. Fakler B. Science. 2001; 292: 2340-2343Crossref PubMed Scopus (362) Google Scholar; reviewed in Ref. 4Dallos P. Fakler B. Nat. Rev. Mol. Cell Biol. 2002; 3: 104-111Crossref PubMed Scopus (240) Google Scholar). The vectorial component to the forces generated in the OHC plasma membrane is provided by the cortical cytoskeleton, an actin-spectrin meshwork placed immediately underneath the plasma membrane and connected to it through thousands of 25-nm-long rod-like structures (pillars) (5Holley M.C. Kalinec F. Kachar B. J. Cell Sci. 1992; 102: 569-580PubMed Google Scholar, 6Holley M.C. Dallos P. Popper A.N. Fay R.R. The Cochlea. Vol. 8. Springer-Verlag Inc., New, York1996: 386-434Google Scholar). It has been recently demonstrated that OHC electromotility is necessary for the correct work of the cochlear amplifier, the active process that enhances sensitivity and frequency discrimination of the mammalian ear (7Liberman M.C. Gao J. He D.Z.Z. Wu X. Jia S. Zuo J. Nature. 2002; 419: 300-304Crossref PubMed Scopus (682) Google Scholar). Cochlear amplification is maximal at low signal levels (∼0–5-db sound pressure level), decreases proportionally to increases in signal intensity, and becomes inhibited at sounds of ∼80 db (4Dallos P. Fakler B. Nat. Rev. Mol. Cell Biol. 2002; 3: 104-111Crossref PubMed Scopus (240) Google Scholar, 8Ruggero M.A. Curr. Opin. Neurobiol. 1992; 2: 449-456Crossref PubMed Scopus (207) Google Scholar). This “automatic gain control” is crucial for extending the dynamic range of the auditory system because it provides the cochlea with the ability to amplify very faint sounds and to process the loudest without suffering permanent damage (9Patuzzi R. Dallos P. Popper A.N. Fay R.R. The Cochlea. Vol. 8. Springer-Verlag New York Inc., New, York1996: 186-257Google Scholar). The direct association between cochlear amplification and OHC electromotility suggests that this gain control may be provided by the modulation of the OHC electromotile response. OHCs must undergo continuous mechanical changes to optimize the sensitivity and frequency selectivity of the cochlea (10Murugasu E. Russell I.J. Audit. Neurosci. 1995; 1: 139-150Google Scholar, 11Russell I.J. Cody A.R. Richardson G.P. Hear. Res. 1986; 22: 199-216Crossref PubMed Scopus (171) Google Scholar, 12Russell I.J. Kossl M. J. Physiol. (Lond.). 1991; 435: 493-511Crossref Scopus (29) Google Scholar, 13Russell I.J. Kossl M. Philos. Trans. R. Soc. Lond. B Biol. Sci. 1992; 336: 317-324Crossref PubMed Scopus (30) Google Scholar). We used the term “mechanical homeostasis” for the process of constant adjustment of control parameters that automatically bring OHCs near the optimal working point for each condition. However, the cochlea may be challenged by variations of 12 orders of magnitude in sound intensity in fractions of 1 s. Therefore, in addition to the homeostatic mechanism of continued regulation, OHCs may require a “reset” switch for fast inhibition of signal amplification to protect the cochlea from sudden bursts of high intensity noise. Although little is known about the nature of the homeostatic control, a strong inhibitory effect on the gain of the cochlear amplifier has been associated with acetylcholine (ACh) released by efferent terminals innervating OHCs (14Murugasu E. Russell I.J. J. Neurosci. 1996; 16: 325-332Crossref PubMed Google Scholar, 15Dallos P. He D.Z.Z. Lin X. Sziklai I. Mehta S. Evans B.N. J. Neurosci. 1997; 17: 2212-2226Crossref PubMed Google Scholar, 16Kalinec F. Zhang M. Urrutia R. Kalinec G. J. Biol. Chem. 2000; 275: 28000-28005Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Recent experimental evidence suggests a role for Rho GTPases in the regulation of OHC electromotility (16Kalinec F. Zhang M. Urrutia R. Kalinec G. J. Biol. Chem. 2000; 275: 28000-28005Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). However, the molecular and cellular mechanisms controlled by these enzymes remain to be determined. We have focused our attention on RhoA and its downstream targets ROCK, known to regulate dynamic reorganization of cytoskeletal proteins (17Amano M. Fukata Y. Kaibuchi K. Exp. Cell Res. 2000; 261: 44-51Crossref PubMed Scopus (452) Google Scholar), and adducin, a cytoskeletal protein that promotes actin-spectrin binding and that caps the fast-growing ends of actin filaments (18Matsuoka Y. Li X. Bennet V. Cell Mol. Life Sci. 2000; 57: 884-895Crossref PubMed Scopus (249) Google Scholar, 19Fukata Y. Oshiro N. Kaibuchi K. Biophys. Chem. 1999; 82: 139-147Crossref PubMed Scopus (28) Google Scholar). In this study, we show that the cellular mechanism of homeostatic control of OHC electromotility involves Rho-mediated cytoskeletal changes without affecting the prestin motors. In addition, we demonstrate that the molecular machinery underlying this phenomenon requires the activation of ROCK-dependent and ROCK-independent pathways. These results extend our understanding of the mechanisms by which Rho GTPases regulate OHC electromotility, reveal a key role for the cytoskeleton in this phenomenon, and suggest that ROCK may serve as a molecular target for modulating the function of the cochlear amplifier. Immunolabeling—Guinea pigs (200–300 g) were euthanized following the procedures and protocols approved by the Institutional Animal Care and Use Committee. For plastic sections, guinea pig cochleae were fixed in 4% paraformaldehyde (Electron Microscopy Sciences, Fort Washington, PA), decalcified in 8% EDTA for 4 weeks, and embedded in celloidin following standard procedures. Sections were labeled with biotinylated secondary antibodies that react with alkaline phosphatase-conjugated streptavidin molecules (Dako LSAB+System AP, Dako Corp., Carpinteria, CA). For confocal observation, whole-mount preparations and isolated OHCs were processed as described previously (16Kalinec F. Zhang M. Urrutia R. Kalinec G. J. Biol. Chem. 2000; 275: 28000-28005Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Anti-ROCK I (C-19), anti-ROCK II (C-20), anti-α-adducin (C-15), anti-phospho-Thr445 adducin, and anti-phospho-Ser726 adducin (Santa Cruz Biotechnology, Santa Cruz, CA) were used as primary antibodies. Blocking peptides were used in pre-adsorption (negative control) experiments. Samples were observed with Zeiss Axiovert 135 and LSM-410 laser confocal microscopes with a ×40 C-Apo (numerical aperature of 1.2) objective. Western Blotting—For Western blot analyses, total cell homogenates from guinea pig cochlea, lung, and brain were homogenized and lysed in 50 mm Tris buffer solution (pH 7.4) containing 1% Nonidet P-40, 2 mm EDTA, 100 mm NaCl, 1 mm vanadate, 0.1 m phenylmethylsulfonyl fluoride (10 μl/ml), and 10 mg/ml aprotinin (3 μl/ml). Samples were then mixed with loading buffer (0.04 g of SDS, 0.002 g of bromphenol blue, 1 ml of β-mercaptoethanol, 4 g of sucrose, and 10 ml of stacking buffer (all from Sigma)), boiled for 5 min and centrifuged. The supernatant was stored at –20 °C. Cochlear and positive control samples were separated by SDS-PAGE (30 μg of protein/lane), transferred to nitrocellulose membranes, and incubated with the primary antibodies. The reaction was detected by enhanced chemiluminescence using a peroxidase-labeled secondary antibody (Amersham Biosciences, Buckinghamshire, UK). Competition studies were performed with the antibodies and blocking peptides mentioned above. Adducin Phosphorylation—Guinea pig cochleae (n = 18) were opened in Leibovitz L-15 medium (Invitrogen), and the spiral was exposed by removing the bony shell. Six samples were preincubated for 30 min with the ROCK inhibitor Y-27632 (10 μm; Upstate Biotechnology, Inc., Lake Placid, NY), which is known to be incorporated into cells by carrier-mediated facilitated diffusion (20Ishizaki T. Uehata M. Tamechika I. Keel J. Nonomura K. Maekawa M. Narumiya S. Mol. Pharmacol. 2000; 57: 976-983PubMed Google Scholar). Next, ACh (Sigma) was added to six samples (three preincubated with Y-27632 and three untreated) at a final concentration of 100 μm. Simultaneously, LPA (Sigma) was added to six other samples (three preincubated with Y-27632 and three untreated) at a final concentration of 10 μm. After 10 min, all of the treated samples plus the six without any treatment (controls) were fixed in 3% paraformaldehyde and 0.1% glutaraldehyde and labeled as described above using either anti-phospho-Thr445 adducin or anti-phospho-Ser726 adducin as the primary antibody. In similar experiments, additional guinea pig cochleae were incubated with ACh and LPA for periods of 1, 2, 4, 6, and 8 min. Electromotility Measurements—OHCs were isolated by microdissection, suspended in Leibovitz L-15 medium in a perfusion chamber on an Axiovert 135 inverted microscope stage, and patch-clamped as previously described (16Kalinec F. Zhang M. Urrutia R. Kalinec G. J. Biol. Chem. 2000; 275: 28000-28005Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Patch pipettes were filled with internal perfusion buffer (120 mm KF, 20 mm KCl, 2 mm MgCl2, 0.5 mm EGTA, 2 mm MgATP, 0.1 mm NaGTP, and 10 mm HEPES) adjusted with Trizma (Tris base) to pH 7.4 (control) or with internal perfusion buffer plus glutathione S-transferase (100 μg/ml; Calbiochem), C3 (100 μg/ml; Cytoskeleton, Denver, CO), Y-27632 (10 μm), and dominant-negative (dn) Rac1 and dnCdc42 (100 μg/ml; kindly provided by Dr. J. Silvio Gutkind, NIDCR, National Institutes of Health) or combinations of these compounds at identical concentrations. After being patch-clamped, cells were permitted to stabilize under their new mechanical conditions for 8–10 min. In those experiments not involving ACh, cells were electrically stimulated with bursts of three depolarization (+50 mV)/hyperpolarization (–145 mV) cycles (3 Hz) to elicit electromotility. The cell response was recorded on videotape and analyzed off line. In those experiments involving ACh, Leibovitz L-15 medium (negative control) and ACh (100 μm) were delivered to the basolateral wall of the cells (∼0.15 μl/s) using a computer-controlled perfusion system (DAD-12, ALA Scientific Instruments, Westbury, NY) as previously described (16Kalinec F. Zhang M. Urrutia R. Kalinec G. J. Biol. Chem. 2000; 275: 28000-28005Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). The electrical and structural integrity of every OHC was continuously evaluated through the whole experiment. The osmolarity of every solution used in these experiments was controlled and adjusted with glucose to 300 ± 2 mosmol with a μOsmette 5004 freezing-point osmometer (Precision Systems Inc., Natick, MA). Experiments were fully recorded on videotape and analyzed off line. Changes in electromotile amplitude were measured with a resolution better than 0.1 μm as described (21Frolenkov G.I. Kalinec F. Tavartkiladze G.A. Kachar B. Biophys. J. 1997; 73: 1665-1672Abstract Full Text PDF PubMed Scopus (21) Google Scholar). A total of 245 cells were included in the experiments. Nonlinear Capacitance Measurements—Membrane capacitance was evaluated during electromotility experiments and before and after ACh perfusion. We followed the transient analysis of currents protocol originally developed by Santos-Sacchi (22Santos-Sacchi J. J. Neurosci. 1991; 11: 3096-3110Crossref PubMed Google Scholar). Briefly, OHCs were electrically stimulated with 15-mV stair steps from a holding potential of –145 to +50 mV. Capacitance function was fitted to the first derivative of a two-state Boltzmann function relating nonlinear charge to membrane voltage (23Santos-Sacchi J. Kakehata S. Takahashi S. J. Physiol. (Lond.). 1998; 510: 225-235Crossref Scopus (115) Google Scholar). Because membrane stress may affect capacitance measurements, special care was taken to diminish potential effects of uncontrolled intracellular pressure changes during evaluation of capacitance and electromotility. Data were statistically analyzed by analysis of variance techniques using StatView Version 4.1 and SuperAnova (Abacus Concepts, Inc., Berkeley, CA). Statistical significance was set at p ≤ 0.05. Expression of ROCK and Adducin in the Guinea Pig Cochlea—There are two closely related isoforms of ROCK, ROCK I (p160ROCK and ROK-β) and ROCK II (Rho kinase, Rho-associated kinase, and ROK-α) (17Amano M. Fukata Y. Kaibuchi K. Exp. Cell Res. 2000; 261: 44-51Crossref PubMed Scopus (452) Google Scholar). ROCK I is expressed at high levels in heart, kidney, skeletal muscle, pancreas, lung, and liver, but it is nearly absent in brain. In contrast, ROCK II is abundantly expressed in brain and weakly in lung (17Amano M. Fukata Y. Kaibuchi K. Exp. Cell Res. 2000; 261: 44-51Crossref PubMed Scopus (452) Google Scholar). In this study, we found that both isoforms are expressed in the guinea pig cochlea at levels that appear to be higher for ROCK I than for ROCK II (Fig. 1). Confocal microscopy of isolated cells demonstrated that both ROCK I and ROCK II immunolocalize primarily at the OHC cortex (Fig. 2). This localization is consistent with the previously reported distribution of RhoA in these cells (16Kalinec F. Zhang M. Urrutia R. Kalinec G. J. Biol. Chem. 2000; 275: 28000-28005Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Antibodies against α-adducin, a marker for the actin-spectrin cytoskeleton and a target of ROCK, showed that this protein is also expressed in guinea pig cochlea (Fig. 1) and labeled OHCs with a pattern similar to that of ROCK I and ROCK II (Fig. 2). Thus, these results suggest that, similar to their upstream activator and downstream target, ROCK kinases localize primarily either immediately adjacent to or at the areas involved in OHC electromotility.Fig. 2Immunolocalization of ROCK I, ROCK II, and α-adducin in guinea pig cochlea. Labeling of plastic-embedded guinea pig cochleae with anti-ROCK I (a), anti-ROCK II (b), and anti-adducin (c) antibodies suggested abundant expression of these proteins in OHCs (arrows). Confocal images of isolated OHCs (d–f) indicated that ROCK I, ROCK II, and adducin localized primarily at the cell cortex. As negative controls, isolated OHCs were incubated with antibodies preadsorbed with their corresponding blocking peptides (g–i). Scale bars = 50 μm (a–c) and 10 μm (d–i).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Homeostatic Regulation of OHC Electromotility—Given the abundant expression of ROCK in OHCs, we hypothesized that ROCK inhibition might result in changes in the mechanical properties of these cells and consequently in their electromotile response. To test this hypothesis, we inhibited the function of ROCK and RhoA in isolated OHCs using either pharmacological or molecular approaches and investigated their effect on cell electromotility. To inhibit RhoA-mediated signaling pathways, we used C3 exoenzyme from Clostridium botulinum, which ADP-ribosylates RhoA (but not Rac and Cdc42) at Asn41, thereby blocking RhoA-mediated signals (24Aktories K. Trends Microbiol. 1997; 5: 282-288Abstract Full Text PDF PubMed Scopus (136) Google Scholar). ROCK activity was controlled with the synthetic compound Y-27632, which inhibits both ROCK I and ROCK II with high specificity by competing with ATP for binding to the kinases (20Ishizaki T. Uehata M. Tamechika I. Keel J. Nonomura K. Maekawa M. Narumiya S. Mol. Pharmacol. 2000; 57: 976-983PubMed Google Scholar). We investigated the electromotile response of OHCs to different concentrations of either C3 or Y-27632 (Fig. 3). Both C3 and Y-27632 affected the electromotile response of isolated guinea pig OHCs. For instance, Fig. 3 (upper panel) shows that OHC electromotile amplitude decreased progressively up to 70% of the control value (70.7 ± 5.7%; p ≤ 0.01) as the C3 concentration was increased up to 100 μg/ml. In contrast, 100 μm Y-27632 decreased electromotile amplitude up to 30% of the control value (31.1 ± 11.4%; p ≤ 0.001) (Fig. 3, lower panel). However, cells receiving 100 μm Y-27632 showed signals of deterioration and yielded more variable results (Fig. 3, lower panel). The cellular damage observed in this experiment likely reflects a toxic effect of the drug rather than the pharmacological effect that is usually observed at lower concentrations. Therefore, we used concentrations of 100 μg/ml for C3 and 10 μm for Y-27632 in all subsequent experiments. To address whether C3 and Y-27632 work cooperatively and/or synergistically, we subsequently measured electromotile amplitude in OHCs co-perfused with these two compounds. Fig. 4a shows that whereas the electromotile amplitude of untreated (control) cells was 3.5 ± 0.1% of the total cell length in these experiments, C3 (100 μg/ml) and Y-27632 (10 μm) decreased it to 2.6 ± 0.2% (p ≤ 0.01) and 2.7 ± 0.2% (p ≤ 0.01), respectively. Co-perfusion of C3 and Y-27632 decreased electromotile amplitude to 2.8 ± 0.2%, a value that was not significantly different from that observed with either C3 or Y-27632 alone (Fig. 4a). These results suggest that RhoA and ROCK work “in series” and that a RhoA/ROCK-mediated signaling cascade might be antagonizing a parallel pathway that decreases OHC electromotility. To investigate whether these results might be associated with the existence of a parallel pathway mediated by other small GTPases, we used previously characterized dominant-negative constructs of Rac1 (dnRac1) and Cdc42 (dnCdc42) (16Kalinec F. Zhang M. Urrutia R. Kalinec G. J. Biol. Chem. 2000; 275: 28000-28005Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). As shown in Fig. 4b, electromotile amplitude did not change when OHCs were co-perfused with dnRac1 + C3 or dnRac1 + C3 + Y-27632. In contrast, replacement of dnRac1 with dnCdc42 resulted in a significant reduction in OHC electromotile amplitude. Although these results appear to suggest a role for Cdc42 in the electromotile response of OHCs, Fig. 4 (compare a and b) provides a different interpretation. Co-perfusion with dnRac1 indeed abolished the effect of C3 and C3 + Y-27632, returning the amplitude of the OHC electromotile response to control values (from 2.6 ± 0.2 to 3.4 ± 0.1% for C3 (p ≤ 0.01) and from 2.7 ± 0.2 to 3.6 ± 0.2% for C3 + Y-27632 (p ≤ 0.01)). In contrast, dnCdc42 did not significantly change the response of OHCs to C3 or C3 + Y-27632 (from 2.6 ± 0.2 to 3.0 ± 0.1% and from 2.8 ± 0.2 to 2.6 ± 0.2%, respectively). These results indicate that Rac1 (but not Cdc42) might be associated with the pathway antagonized by the RhoA/ROCK-mediated signaling cascade. ACh-activated Signaling Pathway—Subsequently, we asked whether the RhoA/ROCK-mediated pathway could be the same ACh-activated pathway previously reported as regulating OHC electromotility (16Kalinec F. Zhang M. Urrutia R. Kalinec G. J. Biol. Chem. 2000; 275: 28000-28005Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). We found that external stimulation with ACh did not change the electromotile response of isolated OHCs internally perfused with C3. Whereas C3 alone decreased electromotile amplitude from 3.5 to 2.6% of the total cell length, ACh stimulation left the amplitude statistically unchanged (2.5 ± 0.1%) (Fig. 5). In contrast, the electromotile amplitude of OHCs internally perfused with the ROCK inhibitor Y-27632 was increased ∼22% by ACh, bringing the response back to control values (from 2.7 ± 0.2 to 3.4 ± 0.1% of the total cell length; p ≤ 0.025). Importantly, this ACh-induced significant increase was abolished by co-perfusion of Y-27632 and C3 (2.7 ± 0.2) (Fig. 5). Altogether, these results suggest that the signaling cascade activated by ACh would be mediated by RhoA, but not by ROCK. Therefore, it would not be the same pathway involved in the homeostatic control of OHC electromotility. Next, we wondered whether adducin could help us to confirm that the ACh-activated pathway is indeed ROCK-independent. It has been suggested that phosphorylation of adducin by ROCK at Thr445 (phospho-Thr445 adducin) facilitates the recruitment of spectrin to F-actin and promotes the assembly of the actin-spectrin cytoskeleton beneath the plasma membrane (25Fukata Y. Oshiro N. Kinoshita N. Kawano Y. Matsuoka Y. Bennett V. Matsuura Y. Kaibuchi K. J. Cell Biol. 1999; 145: 347-361Crossref PubMed Scopus (253) Google Scholar). On the other hand, protein kinase C-mediated phosphorylation of adducin at Ser726 (phospho-Ser726 adducin) would inhibit the recruitment of spectrin to F-actin, inducing the disassembly of the actin-spectrin meshwork (25Fukata Y. Oshiro N. Kinoshita N. Kawano Y. Matsuoka Y. Bennett V. Matsuura Y. Kaibuchi K. J. Cell Biol. 1999; 145: 347-361Crossref PubMed Scopus (253) Google Scholar). Using antibodies that label phosphorylated forms of this protein, we found a pool of both phospho-Thr445 adducin and phospho-Ser726 adducin in untreated isolated OHCs (Fig. 6, a and b). Importantly, preincubation of guinea pig cochleae with Y-27632 for 30 min inhibited adducin phosphorylation at both Thr445 and Ser726 (Fig. 6, c and d). To investigate whether adducin is involved in the ACh-activated pathway, guinea pig cochleae were incubated with ACh for 1, 2, 4, 6, 8, and 10 min, fixed, and labeled with anti-phospho-Thr445 adducin and anti-phospho-Ser726 adducin antibodies. We found that ACh did not significantly increase the amount of labeled adducin (Fig. 6, e–h), confirming that ACh activates a ROCK-independent pathway. As a positive control, we replaced ACh with LPA. LPA activates a signaling pathway involving Gα13, RhoGEF-115, and RhoA (34Neves, S., Ram, P., and Iyengar, R. (2002) Science's STKE http://stke.sciencemag.org/cqi/CM/CMP_8809.Google Scholar). Subsequently, RhoA activates ROCK and protein kinase C, resulting in the phosphorylation of adducin at Ser726 (via protein kinase C) and at Thr445 and Thr480 (via ROCK). In contrast to ACh, just 2 min of incubation with LPA was enough to greatly increase the amount of phospho-Thr445 adducin and phospho-Ser726 adducin (Fig. 6, i and j), and both LPA-induced responses were abolished by preincubation with Y-27632 (Fig. 6, k and l). Interestingly, we found stronger labeling of phospho-Thr445 adducin in the hair bundle than in the OHC body (Fig. 6a, inset). In contrast, labeling of phospho-Ser726 adducin was homogeneous (Fig. 6b). Because the actin-spectrin cytoskeleton is not a major component of the OHC hair bundle, these results suggest that adducin might be functioning there as an actin-capping protein (18Matsuoka Y. Li X. Bennet V. Cell Mol. Life Sci. 2000; 57: 884-895Crossref PubMed Scopus (249) Google Scholar), modulating the renewal of actin in the stereocilia (26Schneider M.E. Belyantseva I.A. Azevedo R.B. Kachar B. Nature. 2002; 418: 837Crossref PubMed Scopus (151) Google Scholar). Regulation of OHC Electromotility Is a Prestin-independent Process—Finally, we measured voltage-dependent nonlinear capacitance (Cm) in isolated OHCs to explore whether the observed variations in electromotile amplitude could be associated with changes in the performance of prestin. Cm results from the movement of electrical charges across the plasma membrane associated with conformational changes in the motor proteins (4Dallos P. Fakler B. Nat. Rev. Mol. Cell Biol. 2002; 3: 104-111Crossref PubMed Scopus (240) Google Scholar). In addition, a positional shift of the voltage peak value (V pk Cm) indicates the existence of indirect effects (i.e. changes in cell turgor) that modify the operating point of the motors (27Kakehata S. Santos-Sacchi J. Biophys. J. 1995; 68: 2190-2197Abstract Full Text PDF PubMed Scopus (156) Google Scholar, 28Wu M. Santos-Sacchi J. J. Membr. Biol. 1998; 166: 111-118Crossref PubMed Scopus (24) Google Scholar, 29Takahashi S. Santos-Sacchi J. J. Membr. Biol. 1999; 169: 199-207Crossref PubMed Scopus (11) Google Scholar, 30Takahashi S. Santos-Sacchi J. Pfluegers Arch. Eur. J. Physiol. 2001; 441: 506-513Crossref PubMed Scopus (24) Google Scholar). In a first series of experiments, we found that up to 30 min of incubation of isolated OHCs with Y-27632 did not induce significant changes in either Cm or V pk Cm (data not shown). Next, we measured Cm before and after external stimulation with ACh (Fig. 7). In agreement with a recent report by Frolenkov et al. (31Frolenkov G.I. Mammano F. Belyantseva I.A. Coling D. Kachar B. J. Neurosci. 2000; 20: 5940-5948Crossref PubMed Google Scholar), we did not detect any significant effect of ACh on untreated OHCs (Fig. 4b). Similar results were obtained in OHCs internally perfused with Y-27632 despite the significant increase in electromotile amplitude induced by ACh in these cells (Fig. 7c). Thus, we conclude that neither the pathways involved in mechanical homeostasis nor the one activated by ACh affects the performance of the motor proteins in a direct manner. An indirect effect on prestin mediated by changes in OHC turgor is also unlikely because we did not observe any shift in V pk Cm (Fig. 7, b and c). Because cell turgor is sensitive to ion flux and to changes in surface potential and membrane tension, a side effect of the treatment on these parameters may also be ruled out. In this study, we have provided evidence emphasizing the importance of prestin-independent processes in the control of OHC electromotility. We defined pathways that link distinct Rho GTPases to the remodeling of the OHC cytoskeleton. A ROCK-dependent pathway would be continuously adjusting the electromotile response of the OHC to optimize the sensitivity and frequency selectivity of the cochlea. A ROCK-independent pathway (activated by ACh) functions as a reset system to bypass the homeostatic mechanism and returns the cell to a mechanical equilibrium. These findings are integrated in the tentative model illustrated in Fig. 8. This model predicts that the gain of the OHC electromotile response is controlled by the RhoA/ROCK-regulated inhibition of a potent, constitutively active, amplitude-decreasing signaling cascade mediated by Rac1. On the other hand, the complete inhibition of the Rac1-mediated pathway would result in the largest electromotile response compatible with both the structural and functional parameters of the force generator mechanism and the mechanical load working on the system. This safety feature minimizes the risk of stereocilium damage and deafness associated with an uncontrolled increase in electromotile amplitude, thus protecting the auditory system against acoustic trauma. OHC Mechanical Homeostasis—The cochlear amplifier increases the dynamic range of the cochlea by enhancing the vibration of the basilar membrane. Continuous operation (responding and adapting to a fluctuating input) demands a feedback mechanism for automatic (homeostatic) control (10Murugasu E. Russell I.J. Audit. Neurosci. 1995; 1: 139-150Google Scholar, 11Russell I.J. Cody A.R. Richardson G.P. Hear. Res. 1986; 22: 199-216Crossref PubMed Scopus (171) Google Scholar, 12Russell I.J. Kossl M. J. Physiol. (Lond.). 1991; 435: 493-511Crossref Scopus (29) Google Scholar, 13Russell I.J. Kossl M. Philos. Trans. R. Soc. Lond. B Biol. Sci. 1992; 336: 317-324Crossref PubMed Scopus (30) Google Scholar). Demonstration that OHC electromotility is necessary to explain cochlear amplification (7Liberman M.C. Gao J. He D.Z.Z. Wu X. Jia S. Zuo J. Nature. 2002; 419: 300-304Crossref PubMed Scopus (682) Google Scholar) leads naturally to the association of homeostatic regulation of the cochlear amplifier with the parallel homeostatic regulation of the OHC electromotile response. The results summarized in Figs. 3 and 4a clearly indicate that C3 and Y-27632 (inhibitors of RhoA and ROCK, respectively) modify the electromotile response of untreated isolated OHCs. Note that the C3 and Y-27632 dose-response curves do not show a clear saturation in their effect on the OHC electromotile response (Fig. 3). Inhibition achieved with higher doses of these agents may be unreliable, however, as suggested by the toxic effect of 100 μm Y-27632 described above. Nonetheless, inhibition of ∼40% of OHC electromotility such as reported here significantly supports the participation of RhoA and ROCK in the regulation of this phenomenon. Because C3 and Y-27632 (used either alone or jointly) decreased OHC electromotile amplitude, we hypothesized that this effect could be mediated by the inhibition of a RhoA/ROCK-mediated signaling cascade regulating an amplitude-decreasing pathway (Fig. 8). The experiments with dnRac1 and dnCdc42 described in Fig. 4b support this hypothesis and suggest that this amplitude-decreasing pathway would be mediated by Rac1, but not Cdc42. What could be the structural target for these signaling pathways mediated by members of the Rho family of small GTPases that control OHC electromotility? Three potential molecular mechanisms may be envisioned. First, Rho-mediated signals could be directly targeting the membrane-embedded molecular motors. The conformational changes in the individual motor molecules or the number of them participating in the response, for instance, could be selectively altered. Second, Rho-mediated signals could be inducing cytoskeletal changes that modify the OHC mechanical response only by changing the mechanical load on the molecular motors. Finally, these potential Rho-mediated cytoskeletal changes could be influencing the performance of the membrane-embedded motors either directly or indirectly through changes in other parameters such as membrane tension. Our measurements of voltage-dependent nonlinear membrane capacitance, both during homeostatic regulation and when activated by ACh, clearly implicate the cytoskeleton as the only target for the RhoA-mediated signaling cascade. These results also suggest that a significant indirect effect on the performance of the membrane-embedded molecular motors (mediated by changes in membrane tension or ion flux, for instance) is unlikely. Adducin experiments provided indirect support to the hypothesis that the cytoskeleton is the essential structure involved in the homeostatic regulation of OHC electromotility. ROCK activation by RhoA induces phospho-Thr445 adducin and phospho-Thr480 adducin, promoting the recruitment of spectrin to actin filaments, the formation of the actin-spectrin cytoskeleton, and its connection to the plasma membrane via ERM (ezrin/radixin/moesin-protein family) proteins (25Fukata Y. Oshiro N. Kinoshita N. Kawano Y. Matsuoka Y. Bennett V. Matsuura Y. Kaibuchi K. J. Cell Biol. 1999; 145: 347-361Crossref PubMed Scopus (253) Google Scholar). In contrast, protein kinases C and A inhibit this activity and induce the disassembly of the actin-spectrin cytoskeleton by generating phospho-Ser726 adducin (18Matsuoka Y. Li X. Bennet V. Cell Mol. Life Sci. 2000; 57: 884-895Crossref PubMed Scopus (249) Google Scholar, 32Matsuoka Y. Li X. Bennet V. J. Cell Biol. 1998; 142: 485-497Crossref PubMed Scopus (171) Google Scholar). The finding in untreated OHCs of phospho-Thr445 adducin and phospho-Ser726 adducin suggests that the OHC cortical cytoskeleton is dynamically regulated, with continuous assembly and disassembly of the actin-spectrin meshwork. Together, the experiments reported here indicate that the activation of a RhoA/ROCK-mediated pathway induces both adducin phosphorylation and regulation of OHC electromotility. These data also suggest that future research aimed at defining the role of adducin and adducin-associated proteins in the biology of OHCs will contribute significantly to the better understanding of OHC electromotility. In addition to promoting actin-spectrin association, adducin is known to act as an actin-capping protein (18Matsuoka Y. Li X. Bennet V. Cell Mol. Life Sci. 2000; 57: 884-895Crossref PubMed Scopus (249) Google Scholar). Like spectrin recruitment activities, adducin actin capping is activated by ROCK-mediated phosphorylation and is inhibited by protein kinase C-mediated phosphorylation (32Matsuoka Y. Li X. Bennet V. J. Cell Biol. 1998; 142: 485-497Crossref PubMed Scopus (171) Google Scholar). Kachar and co-workers (26Schneider M.E. Belyantseva I.A. Azevedo R.B. Kachar B. Nature. 2002; 418: 837Crossref PubMed Scopus (151) Google Scholar) described recently that the actin filaments forming the core of hair cell stereocilia are continuously remodeled. Complete remodeling is achieved every 48 h by addition of actin monomers to the stereocilium tips. The strong labeling of phospho-Thr445 adducin in the hair bundle of untreated OHCs (Fig. 6a, inset) suggests that actin renewal could be another homeostatic process regulated by a ROCK-mediated pathway involving adducin. Regulation of OHC Electromotility by ACh—The dynamic range of the mammalian cochlea is ∼120 db from the softest to the loudest sounds the cochlea can detect without permanent damage. This means that the cochlea can withstand sound pressures that are one million times greater in amplitude and therefore with 1012 more energy than sounds at the threshold of detection (9Patuzzi R. Dallos P. Popper A.N. Fay R.R. The Cochlea. Vol. 8. Springer-Verlag New York Inc., New, York1996: 186-257Google Scholar). It survives because the mechanical sensitivity and amplification are progressively reduced as the sound level increases. A self-regulated homeostatic response may be insufficient, however, to protect the cochlea from sudden bursts of high energy noise. ACh released by the efferent terminals innervating OHCs could be required to disengage this automatic gain control and to rapidly inhibit amplification in response to changes in the auditory input that may result in cochlear damage. The results summarized in Fig. 5 suggest that ACh influences OHC electromotility by activating a signaling pathway mediated by RhoA, but not by ROCK. Adducin experiments supported this hypothesis by showing that ACh did not increases ROCK-mediated phosphorylation at Thr445 (Fig. 6, e–h). Therefore, this ACh-activated pathway is different from those involved in OHC mechanical homeostasis. Because ACh reverses the decrease in electromotile amplitude induced by ROCK inhibition, we envision this ACh-activated pathway as a reset system able to move the cell quickly back to a mechanical steady state, ensuring the inhibition of the cochlear amplifier when necessary to protect the auditory system from acoustic trauma. If ACh would always increase OHC electromotile amplitude, why did we not detect any ACh-induced electromotile increase in untreated isolated OHCs? According to the proposed model (Fig. 8), the upper value for the ACh-induced increase in OHC electromotile amplitude should be equal to the biggest response compatible with structural and functional parameters of the force generator mechanism and the external and internal mechanical load working on it. Therefore, an isolated OHC (no external mechanical load) in complete mechanical equilibrium with the medium (lowest internal load) would respond to a given stimulus with the biggest possible electromotile amplitude for that condition. In consequence, the amplitude in the control group would be near the upper limit for the electromotile response under our experimental conditions, and it could not be further increased by ACh. Thus, the ACh effect would be evident only in isolated cells not in mechanical equilibrium or unable to reach it by a particular treatment. This was the response observed, for instance, in OHCs treated with the ROCK inhibitor Y-27632, for which the ACh effect was to bring the amplitude back to control values (Fig. 5). Conclusions—We have provided evidence that the small GTPase RhoA and its downstream target ROCK are crucial for the mechanical homeostasis and regulation of the electromotile response of cochlear OHCs. Our results support a model in which a RhoA/ROCK-mediated signaling cascade would be able to inhibit a Rac1-mediated parallel pathway that decreases OHC electromotile amplitude. Higher levels of inhibition would result in bigger OHC electromotile amplitude, providing a safe and reliable automatic control for the cochlear amplifier. In this model, ACh should be able to inhibit the cochlear amplifier by resetting OHC electromotile amplitude to values corresponding to mechanical equilibrium. OHC electromotility regulation would be associated with cytoskeleton reorganization, without changes in the performance of the membrane-embedded molecular motors. These results are an important step toward the elucidation of the mechanisms that regulate OHC electromotility and cochlear amplification." @default.
• W2058974065 created "2016-06-24" @default.
• W2058974065 creator A5013280795 @default.
• W2058974065 creator A5051280662 @default.
• W2058974065 creator A5057817904 @default.
• W2058974065 creator A5080426319 @default.
• W2058974065 creator A5080733133 @default.
• W2058974065 date "2003-09-01" @default.
• W2058974065 modified "2023-10-11" @default.
• W2058974065 title "ROCK-dependent and ROCK-independent Control of Cochlear Outer Hair Cell Electromotility" @default.
• W2058974065 cites W1537491036 @default.
• W2058974065 cites W1554225425 @default.
• W2058974065 cites W1981531296 @default.
• W2058974065 cites W1986063005 @default.
• W2058974065 cites W1986733224 @default.
• W2058974065 cites W1988508433 @default.
• W2058974065 cites W1988926568 @default.
• W2058974065 cites W2001953889 @default.
• W2058974065 cites W2013030459 @default.
• W2058974065 cites W2017330689 @default.
• W2058974065 cites W2024137834 @default.
• W2058974065 cites W2028351974 @default.
• W2058974065 cites W2033670275 @default.
• W2058974065 cites W2033726591 @default.
• W2058974065 cites W2036708962 @default.
• W2058974065 cites W2038717281 @default.
• W2058974065 cites W2040181742 @default.
• W2058974065 cites W2047599628 @default.
• W2058974065 cites W2051035050 @default.
• W2058974065 cites W2064021058 @default.
• W2058974065 cites W2081439231 @default.
• W2058974065 cites W2098924164 @default.
• W2058974065 cites W2116007831 @default.
• W2058974065 cites W2160220435 @default.
• W2058974065 cites W2164820984 @default.
• W2058974065 cites W2165490569 @default.
• W2058974065 cites W2407777913 @default.
• W2058974065 cites W2770806897 @default.
• W2058974065 doi "https://doi.org/10.1074/jbc.m301668200" @default.
• W2058974065 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12837763" @default.
• W2058974065 hasPublicationYear "2003" @default.
• W2058974065 type Work @default.
• W2058974065 sameAs 2058974065 @default.
• W2058974065 citedByCount "34" @default.
• W2058974065 countsByYear W20589740652014 @default.
• W2058974065 countsByYear W20589740652015 @default.
• W2058974065 countsByYear W20589740652016 @default.
• W2058974065 countsByYear W20589740652017 @default.
• W2058974065 countsByYear W20589740652019 @default.
• W2058974065 countsByYear W20589740652020 @default.
• W2058974065 countsByYear W20589740652022 @default.
• W2058974065 crossrefType "journal-article" @default.
• W2058974065 hasAuthorship W2058974065A5013280795 @default.
• W2058974065 hasAuthorship W2058974065A5051280662 @default.
• W2058974065 hasAuthorship W2058974065A5057817904 @default.
• W2058974065 hasAuthorship W2058974065A5080426319 @default.
• W2058974065 hasAuthorship W2058974065A5080733133 @default.
• W2058974065 hasBestOaLocation W20589740651 @default.
• W2058974065 hasConcept C12554922 @default.
• W2058974065 hasConcept C127313418 @default.
• W2058974065 hasConcept C185592680 @default.
• W2058974065 hasConcept C2778585322 @default.
• W2058974065 hasConcept C2780130748 @default.
• W2058974065 hasConcept C2908762784 @default.
• W2058974065 hasConcept C548259974 @default.
• W2058974065 hasConcept C71924100 @default.
• W2058974065 hasConcept C86803240 @default.
• W2058974065 hasConceptScore W2058974065C12554922 @default.
• W2058974065 hasConceptScore W2058974065C127313418 @default.
• W2058974065 hasConceptScore W2058974065C185592680 @default.
• W2058974065 hasConceptScore W2058974065C2778585322 @default.
• W2058974065 hasConceptScore W2058974065C2780130748 @default.
• W2058974065 hasConceptScore W2058974065C2908762784 @default.
• W2058974065 hasConceptScore W2058974065C548259974 @default.
• W2058974065 hasConceptScore W2058974065C71924100 @default.
• W2058974065 hasConceptScore W2058974065C86803240 @default.
• W2058974065 hasIssue "37" @default.
• W2058974065 hasLocation W20589740651 @default.
• W2058974065 hasOpenAccess W2058974065 @default.
• W2058974065 hasPrimaryLocation W20589740651 @default.
• W2058974065 hasRelatedWork W1973062043 @default.
• W2058974065 hasRelatedWork W1976524596 @default.
• W2058974065 hasRelatedWork W2035595017 @default.
• W2058974065 hasRelatedWork W2081439231 @default.
• W2058974065 hasRelatedWork W2117727547 @default.
• W2058974065 hasRelatedWork W2460386992 @default.
• W2058974065 hasRelatedWork W2516115481 @default.
• W2058974065 hasRelatedWork W2728194919 @default.
• W2058974065 hasRelatedWork W3175455641 @default.
• W2058974065 hasRelatedWork W985531739 @default.
• W2058974065 hasVolume "278" @default.
• W2058974065 isParatext "false" @default.
• W2058974065 isRetracted "false" @default.
• W2058974065 magId "2058974065" @default.
• W2058974065 workType "article" @default.