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Bradley A Schulte - One of the best experts on this subject based on the ideXlab platform.

  • Identification of ClC-2 and CIC-K2 chloride channels in cultured rat type IV Spiral Ligament fibrocytes.
    Journal of the Association for Research in Otolaryngology : JARO, 2007
    Co-Authors: Fenghe Liang, Nancy Smythe, Bradley A Schulte
    Abstract:

    Voltage-gated chloride channels (ClCs) are important mediators of cellular ion homeostasis and volume regulation. In an earlier study, we used immunohistochemical, Western blot, and reverse transcriptase PCR (RT-PCR) approaches to identify ClC-K variants in types II, IV, and V fibrocytes of the rodent Spiral Ligament. We have now confirmed the expression of ClC-K2 in these cells by in situ hybridization. All three of these fibrocyte subtypes are thought to be involved in cochlear K+ recycling; thus, it is important to understand the precise mechanisms regulating their membrane conductance and the role played by ClCs in this process. In this study, we report the characterization of a secondary cell line derived from explants from the region of the rat Spiral Ligament underlying and inferior to the Spiral prominence. The cultured cells were immunopositive for vimentin, Na,K/ATPase, Na,K,Cl-cotransporter, carbonic anhydrase isozyme II, and creatine kinase isozyme BB, but not for cytokeratins or Ca/ATPase, an immunostaining profile indicative of the type IV subtype. Evaluation of the cultures by RT-PCR and Western blot analysis confirmed the presence of both ClC-2 and -K2. Whole-cell patch clamp recordings identified two biophysically distinct Cl− currents in the cultured cells. One, an inwardly rectifying Cl− current activated by hyperpolarization or decreasing extracellular pH corresponded with the properties of ClC-2. The other, a weak outwardly rectifying Cl− current regulated by extracellular pH, Cl−, and Ca2+ resembled the channel characteristics of ClC-K2 when expressed in Xenopus oocytes. These findings suggest that at least two functionally different chloride channels are involved in regulating membrane anion conductance in cultured type IV Spiral Ligament fibrocytes.

  • Inhibition of the calcium- and voltage-dependent big conductance potassium channel ameliorates cisplatin-induced apoptosis in Spiral Ligament fibrocytes of the cochlea.
    Neuroscience, 2005
    Co-Authors: F. Liang, Bradley A Schulte, Z. Shen
    Abstract:

    Abstract The role of calcium- and voltage-dependent big conductance potassium channels in regulating apoptosis was investigated in cultured type I Spiral Ligament fibrocytes. Incubation of type I Spiral Ligament fibrocytes derived from gerbil cochlea with cisplatin induced dose- and time-dependent apoptosis as demonstrated by annexin V conjugated to fluorescein isothiocyanate/prodidium iodide assays. The average voltage activation threshold of whole cell current was sharply shifted to −40 mV in the cisplatin-treated cells as compared with a value of 40 mV in control cells. The average whole-cell current of cisplatin-treated cells induced by a depolarization voltage step from −80 to −10 mV was increased significantly to 1.2±0.4 nA as compared with 0.08±0.1 nA in control cells. Coincubation with tetraethylammonium and cisplatin retained the whole cell current in the normal range (0.12±0.2 nA). The increment of cisplatin-induced whole-cell current was inhibited (97±5%) by a specific calcium- and voltage-dependent big conductance potassium channel blocker iberiotoxin. Consistent with this, co-incubation with tetraethylammonium significantly attenuated cisplatin-induced apoptosis in type I Spiral Ligament fibrocytes by more than 50%. We conclude that the activation of BK channels is an early event associated with cisplatin-induced apoptosis in type I Spiral Ligament fibrocytes. These findings also point to the calcium- and voltage-dependent big conductance potassium channels as a potential pharmacological target for manipulating cisplatin ototoxicity.

  • Identification and characterization of an L-type Cav1.2 channel in Spiral Ligament fibrocytes of gerbil inner ear.
    Brain research. Molecular brain research, 2004
    Co-Authors: Fenghe Liang, Bradley A Schulte, Cungui Mao, Debra J Hazen-martin, Zhijun Shen
    Abstract:

    Intracellular free Ca2+ levels are critical to the activity of BK channels in inner ear type I Spiral Ligament fibrocytes. However, the mechanisms for regulating intracellular Ca2+ levels in these cells are currently poorly understood. Using patch-clamp technique, we have identified a voltage-dependent L-type Ca2+ channel in type I Spiral Ligament fibrocytes cultured from gerbil inner ear. With 10 mM Ba2+ as the conductive cation, an inwardly rectifying current was elicited with little inactivation by membrane depolarization. The voltage activation threshold and the half-maximal voltage activation were -40 and -6 mV, respectively. This inward whole-cell current reached its peak at around 10 mV of membrane potential. The amplitude of the peak current varied among cells ranging from 50 to 274 pA with an average of 132.4 +/- 76.2 pA (n = 19); 10(-6) M nifedipine significantly inhibited the inward currents by 90.3 +/- 1.2% (n = 11). RT-PCR analysis revealed that cultured type I Spiral Ligament fibrocytes express the alpha1C isoform of the L-type Ca2+ channels encoded by the Cav1.2 gene. The expression of this channel in gerbil inner ear was confirmed by RT-PCR analysis using freshly isolated Spiral Ligament tissues. The Cav1.2 channel may function in conjunction with a previously identified intracellular Ca-ATPase (SERCA) to regulate intracellular free Ca2+ levels in type I Spiral Ligament fibrocytes, and thus modulate BK channel activity in these cells.

  • BK channels mediate the voltage-dependent outward current in type I Spiral Ligament fibrocytes.
    Hearing research, 2004
    Co-Authors: Zhijun Shen, Fenghe Liang, Debra J Hazen-martin, Bradley A Schulte
    Abstract:

    Recent experimental and clinical studies have provided considerable evidence to support the phenomenon of K(+) recycling in the mammalian cochlea. However, the precise cellular and molecular mechanisms underlying and regulating this process remain only partially understood. Here, we report that cultured type I Spiral Ligament fibrocytes (SLFs), a major component of the K(+) recycling pathway, have a dominant K(+) membrane conductance that is mediated by BK channels. The averaged half-maximal voltage-dependent membrane potential for the whole-cell currents was 70+/-1.2 mV at 1 nM intracellular free Ca(2+) and shifted to 38+/-0.2 mV at 20 microM intracellular free Ca(2+) (n=4-6). The reversal potential of whole-cell tail currents against different bath K(+) concentrations was 52 mV per decade (n=3-6). The sequence of relative ion permeability of the whole-cell conductance was K(+)>Rb(+)z.Gt;Cs(+)>Na(+) (n=5-17). The whole-cell currents were inhibited by extracellular tetraethylammonium and iberiotoxin (IbTx) with IC(50) values of 0.07 mM and 0.013 microM, respectively (n=3-7). The membrane potentials of type I SLFs measured with conventional zero-current whole-cell configuration were highly K(+)-selective and sensitive to IbTx (n=4-9). In addition, the BK channels in these cells exhibited voltage-dependent and incomplete inactivation properties and the recovery time was estimated to be approximately 6 s with repetitive voltage pulses from -70 to 80 mV (n=3). These data suggest that BK channels in type I SLFs play a major role in regulating the intracellular electrochemical gradient in the lateral wall syncytium responsible for facilitating the K(+) movement from perilymph to the stria vascularis.

  • BK channels mediate the voltage-dependent outward current in type I Spiral Ligament fibrocytes.
    Hearing Research, 2003
    Co-Authors: Zhijun Shen, Fenghe Liang, Debra J Hazen-martin, Bradley A Schulte
    Abstract:

    Abstract Recent experimental and clinical studies have provided considerable evidence to support the phenomenon of K + recycling in the mammalian cochlea. However, the precise cellular and molecular mechanisms underlying and regulating this process remain only partially understood. Here, we report that cultured type I Spiral Ligament fibrocytes (SLFs), a major component of the K + recycling pathway, have a dominant K + membrane conductance that is mediated by BK channels. The averaged half-maximal voltage-dependent membrane potential for the whole-cell currents was 70±1.2 mV at 1 nM intracellular free Ca 2+ and shifted to 38±0.2 mV at 20 μM intracellular free Ca 2+ ( n =4–6). The reversal potential of whole-cell tail currents against different bath K + concentrations was 52 mV per decade ( n =3–6). The sequence of relative ion permeability of the whole-cell conductance was K + >Rb + ≫Cs + >Na + ( n =5–17). The whole-cell currents were inhibited by extracellular tetraethylammonium and iberiotoxin (IbTx) with IC 50 values of 0.07 mM and 0.013 μM, respectively ( n =3–7). The membrane potentials of type I SLFs measured with conventional zero-current whole-cell configuration were highly K + -selective and sensitive to IbTx ( n =4–9). In addition, the BK channels in these cells exhibited voltage-dependent and incomplete inactivation properties and the recovery time was estimated to be ∼6 s with repetitive voltage pulses from −70 to 80 mV ( n =3). These data suggest that BK channels in type I SLFs play a major role in regulating the intracellular electrochemical gradient in the lateral wall syncytium responsible for facilitating the K + movement from perilymph to the stria vascularis.

Kiyokazu Ogita - One of the best experts on this subject based on the ideXlab platform.

  • Disruption of Gap Junction-Mediated Intercellular Communication in the Spiral Ligament Causes Hearing and Outer Hair Cell Loss in the Cochlea of Mice
    Biological & pharmaceutical bulletin, 2019
    Co-Authors: Norito Nishiyama, Taro Yamaguchi, Masanori Yoneyama, Yusuke Onaka, Kiyokazu Ogita
    Abstract:

    It is well-known that outer hair cell (OHC) loss occurs in the cochlea of animal models of permanent hearing loss induced by intense noise exposure. Our earlier studies demonstrated the production of hydroxynonenal and peroxynitrite, as well as the disruption of gap junction-mediated intercellular communication (GJIC), in the cochlear Spiral Ligament prior to noise-induced sudden hearing loss. The goal of the present study was to evaluate the mechanism underlying cochlear OHC loss after sudden hearing loss induced by intense noise exposure. In organ of Corti explant cultures from mice, no significant OHC loss was observed after in vitro exposure to 4-hydroxynonenal (a product of lipid peroxidation), H2O2, SIN-1 (peroxynitrite generator), and carbenoxolone (a gap junction inhibitor). Interestingly, in vivo intracochlear carbenoxolone injection through the posterior semicircular canal caused marked OHC and hearing loss, as well as the disruption of gap junction-mediated intercellular communication in the cochlear Spiral Ligament. However, no significant OHC loss was observed in vivo in animals treated with 4-hydroxynonenal and SIN-1. Taken together, our data suggest that disruption of GJIC in the cochlear lateral wall structures is an important cause of cochlear OHC loss in models of hearing loss, including those induced by noise.

  • Calpain inhibitor alleviates permanent hearing loss induced by intense noise by preventing disruption of gap junction-mediated intercellular communication in the cochlear Spiral Ligament
    European journal of pharmacology, 2017
    Co-Authors: Taro Yamaguchi, Masanori Yoneyama, Kiyokazu Ogita
    Abstract:

    Our previous studies demonstrated that intense noise-induced hearing loss might be at least in part due to an oxidative stress-induced decrease in the level of gap junction-composing protein connexins in the Spiral Ligament (SL) of the cochlear lateral wall structures in mice. Further, an in vivo exposure of mice to intense noise activates calpain in the cochlear SL. Based on these studies, we sought to determine whether a calpain inhibitor would prevent an intense noise exposure from causing hearing loss, disruption of gap junction-mediated intercellular communication (GJIC) in the SL. An exposure of mice to intense noise (8-Hz octave band noise, 110-dB sound pressure level, 1h) produced permanent hearing loss and cochlear hair cell death. The results of an ex vivo assay using gap-fluorescence recovery after photobleaching of dissected lateral wall structures revealed that the intense noise disrupted GJIC in the cochlear SL at day-7 post exposure. A prior intracochlear injection of the calpain inhibitor PD150606 significantly abolished this noise-induced hearing loss on days 5 and 7 post exposure. Similarly, PD150606 prevented noise-induced hair cell death and the GJIC disruption on day-7 post exposure. The intense noise temporarily enhanced the gene expression of calpain subtypes Capn1 and Capn2 immediately after exposure. Taken together, our data suggest that calpain inhibitor alleviated the noise-induced hearing loss, at least in part, by preventing disruption of GJIC in the cochlear SL. It possible that calpain inhibitors would be useful as a candidate of therapeutic drugs for sudden sensorineural hearing loss.

  • Disruption of Ion-Trafficking System in the Cochlear Spiral Ligament Prior to Permanent Hearing Loss Induced by Exposure to Intense Noise: Possible Involvement of 4-Hydroxy-2-Nonenal as a Mediator of
    2016
    Co-Authors: Oxidative Stress, Taro Yamaguchi, Masanori Yoneyama, Reiko Nagashima, Tatsuo Shiba, Kiyokazu Ogita
    Abstract:

    Noise-induced hearing loss is at least in part due to disruption of endocochlear potential, which is maintained by various K+ transport apparatuses including Na+, K+-ATPase and gap junction-mediated intercellular communication in the lateral wall structures. In this study, we examined the changes in the ion-trafficking-related proteins in the Spiral Ligament fibrocytes (SLFs) following in vivo acoustic overstimulation or in vitro exposure of cultured SLFs to 4-hydroxy-2-nonenal, which is a mediator of oxidative stress. Connexin (Cx)26 and Cx30 were ubiquitously expressed throughout the Spiral Ligament, whereas Na+, K+-ATPase a1 was predominantly detected in the stria vascularis and Spiral prominence (type 2 SLFs). One-hour exposure of mice to 8 kHz octave band noise at a 110 dB sound pressure level produced an immediate and prolonged decrease in the Cx26 expression level and in Na+, K+-ATPase activity, as well as a delayed decrease in Cx30 expression in the SLFs. The noise-induced hearing loss and decrease in the Cx26 protein level and Na+, K+-ATPase activity were abolished by a systemic treatment with a free radical-scavenging agent, 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl, or with a nitric oxide synthase inhibitor, Nv-nitro-L-arginine methyl ester hydrochloride. In vitro exposure of SLFs in primary culture to 4-hydroxy-2-nonenal produced a decrease in the protein levels of Cx26 and Na+, K+-ATPase a1, as well as Na+, K+-ATPase activity, and also resulted in dysfunction of the intercellular communication between the SLFs. Taken together, our data suggest that disruption of the ion-trafficking system in the cochlear SLFs is caused by the decrease in Cxs level and Na+, K+

  • Involvement of calpain in 4-hydroxynonenal-induced disruption of gap junction-mediated intercellular communication among fibrocytes in primary cultures derived from the cochlear Spiral Ligament.
    Journal of pharmacological sciences, 2015
    Co-Authors: Taro Yamaguchi, Masanori Yoneyama, Eiichi Hinoi, Kiyokazu Ogita
    Abstract:

    The endocochlear potential in the inner ear is essential for hearing ability, and maintained by various K(+) transport apparatuses including Na(+), K(+)-ATPase and gap junction-mediated intercellular communication (GJ-IC) in the lateral wall structures of the cochlea. Noise-induced hearing loss is known at least in part due to disruption of GJ-IC resulting from an oxidative stress-induced decrease in connexins (Cxs) level in the lateral wall structures. The purpose of this study was to investigate, using primary cultures of fibrocytes from the cochlear Spiral Ligament of mice, the mechanism underlying GJ-IC disruption induced by 4-hydroxynonenal (4-HNE), which is formed as a mediator of oxidative stress. An exposure to 4-HNE produced the following events: i.e., an increase in 4-HNE-adducted proteins; a decrease in the protein levels of Cx43, β-catenin, and Cx43/β-catenin complex along with intracellular translocation of this complex from the cell membrane to the cytoplasm; enhanced calpain-dependent degradation of endogenous α-fodrin; and disruption of GJ-IC. The 4-HNE-induced decrease in these protein levels and disruption of GJ-IC were most completely abolished by the calpain inhibitor PD150606. Taken together, our data suggest that 4-HNE disrupted GJ-IC through calpain-mediated degradation of Cx43 and β-catenin in primary cultures of fibrocytes derived from the cochlear Spiral Ligament.

  • Disruption of ion-trafficking system in the cochlear Spiral Ligament prior to permanent hearing loss induced by exposure to intense noise: possible involvement of 4-hydroxy-2-nonenal as a mediator of oxidative stress.
    PloS one, 2014
    Co-Authors: Taro Yamaguchi, Masanori Yoneyama, Reiko Nagashima, Tatsuo Shiba, Kiyokazu Ogita
    Abstract:

    Noise-induced hearing loss is at least in part due to disruption of endocochlear potential, which is maintained by various K+ transport apparatuses including Na+, K+-ATPase and gap junction-mediated intercellular communication in the lateral wall structures. In this study, we examined the changes in the ion-trafficking-related proteins in the Spiral Ligament fibrocytes (SLFs) following in vivo acoustic overstimulation or in vitro exposure of cultured SLFs to 4-hydroxy-2-nonenal, which is a mediator of oxidative stress. Connexin (Cx)26 and Cx30 were ubiquitously expressed throughout the Spiral Ligament, whereas Na+, K+-ATPase α1 was predominantly detected in the stria vascularis and Spiral prominence (type 2 SLFs). One-hour exposure of mice to 8 kHz octave band noise at a 110 dB sound pressure level produced an immediate and prolonged decrease in the Cx26 expression level and in Na+, K+-ATPase activity, as well as a delayed decrease in Cx30 expression in the SLFs. The noise-induced hearing loss and decrease in the Cx26 protein level and Na+, K+-ATPase activity were abolished by a systemic treatment with a free radical-scavenging agent, 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl, or with a nitric oxide synthase inhibitor, Nω-nitro-L-arginine methyl ester hydrochloride. In vitro exposure of SLFs in primary culture to 4-hydroxy-2-nonenal produced a decrease in the protein levels of Cx26 and Na+, K+-ATPase α1, as well as Na+, K+-ATPase activity, and also resulted in dysfunction of the intercellular communication between the SLFs. Taken together, our data suggest that disruption of the ion-trafficking system in the cochlear SLFs is caused by the decrease in Cxs level and Na+, K+-ATPase activity, and at least in part involved in permanent hearing loss induced by intense noise. Oxidative stress-mediated products might contribute to the decrease in Cxs content and Na+, K+-ATPase activity in the cochlear lateral wall structures.

G Mogi - One of the best experts on this subject based on the ideXlab platform.

  • Classification and culture of Spiral Ligament fibrocytes from mice.
    Hearing research, 2000
    Co-Authors: T Suko, K Yoshida, I Ichimiya, M Suzuki, G Mogi
    Abstract:

    In this study, we established an immunocytochemical strategy to classify the fibrocytes of the murine Spiral Ligament (SL), and SL cultures were characterized. Similar to those in other mammals, three different types of fibrocytes were identified. Type I fibrocytes, which are found lateral to the stria vascularis, showed positive immunoreactivity for caldesmon and S-100 protein and were not stained for sodium-potassium-adenosinetriphosphatase (Na-K-ATPase). Type II fibrocytes are located lateral to the Spiral prominence epithelium and suprastrial region, and they were distinguishable by their positive staining for Na-K-ATPase. Type III fibrocytes, which are found adjacent to bone in the inferior region of the SL, contained caldesmon but not S-100 or Na-K-ATPase. Secondary cultures from the SL were positive for caldesmon and S-100 and negative for Na-K-ATPase, suggesting that these cells were type I fibrocytes. The present immunocytochemical approach was useful for the classification of murine fibrocyte cultures, and these cultures may benefit future immunological studies of the inner ear because mice have been well characterized immunologically.

  • Effect of proinflammatory cytokines on cultured Spiral Ligament fibrocytes.
    Hearing research, 1999
    Co-Authors: K Yoshida, I Ichimiya, M Suzuki, G Mogi
    Abstract:

    To clarify the effect of proinflammatory cytokines on Spiral Ligament (SL) fibrocytes, in vitro studies were performed using secondary cell cultures. Cultures from murine SL fibrocytes were stimulated by interleukin (IL)-1beta or tumor necrosis factor (TNF)-alpha, and secretion of various mediators was measured by enzyme-linked immunosorbent assay. After stimulation with the proinflammatory cytokines, IL-6, TNF-alpha, monocyte chemoattractant protein-1, KC, macrophage inflammatory protein-2, soluble intercellular adhesion molecule-1, and vascular endothelial growth factor levels were elevated. Secretion of these chemokines and other mediators could induce inflammatory cell movement, which would prolong the inflammatory response, leading to fibrocyte damage. Given that SL fibrocytes may play a role in cochlear fluid and ion homeostasis, such fibrocyte disruption could cause cochlear malfunction.

  • The influence of pneumococcal otitis media on the cochlear lateral wall.
    Hearing research, 1999
    Co-Authors: I Ichimiya, M Suzuki, T Hirano, G Mogi
    Abstract:

    The cochlear influence of otitis media was investigated in order to identify damaged regions causing cochlear malfunction. BALB/c mice were challenged with viable Streptococcus pneumoniae into the middle ear cavity and were killed 1 day to 1 month later for immunohistochemical analysis. Otitis media was induced in all of the animals, and some showed inflammatory cells in the cochlea. Although other changes were not obvious by hematoxylin and eosin staining, immunohistochemistry showed the presence of fibrinogen in the cochlea, mainly in the lower portion of the Spiral Ligament and in the Spiral limbus. Immunostaining for connexin 26 was decreased in the Spiral Ligament, accompanied by marked fibrinogen staining. Immunostaining for sodium-potassium-adenosine triphosphatase in the stria vascularis and in the type II fibrocytes of the Spiral Ligament was not affected obviously. The presence of fibrinogen in the cochlea suggests disruption of the blood-labyrinth barrier caused by the middle ear inflammation. Changes in connexin 26 staining suggest the possibility that the Spiral Ligament could be among the regions responsible for the cochlear malfunction.

  • Changes in immunostaining of inner ears after antigen challenge into the scala tympani.
    The Laryngoscope, 1998
    Co-Authors: I Ichimiya, T Hirano, Y Kurono, G Mogi
    Abstract:

    To study the mechanisms of immune responses and immune injuries in inner ears, labyrinthitis was induced by inoculation of keyhole limpet hemocyanin (KLH) into the scala tympani of systemically sensitized guinea pigs. Inner ears were then immunostained for KLH, immunoglobulin G (IgG), albumin, connexin26 (Cx26), and sodium-potassium adenosine triphosphate (Na,K-ATPase). Inflammatory cells containing KLH were observed in the scala tympani and in the collecting venule of the Spiral modiolar vein (SMV). Spiral Ligament, Spiral limbus, and blood vessels including the SMV were diffusely positive for IgG and albumin. Immunoreactivity for Cx26 and Na,K-ATPase was decreased compared with the normal ears in the fibrocytes of the Spiral Ligament. These results suggest that inflammatory cells and blood constituents could extravasate into the cochlea from blood vessels and that fibrocyte damage in the Spiral Ligament could cause cochlear dysfunction.

Zhijun Shen - One of the best experts on this subject based on the ideXlab platform.

  • Identification and characterization of an L-type Cav1.2 channel in Spiral Ligament fibrocytes of gerbil inner ear.
    Brain research. Molecular brain research, 2004
    Co-Authors: Fenghe Liang, Bradley A Schulte, Cungui Mao, Debra J Hazen-martin, Zhijun Shen
    Abstract:

    Intracellular free Ca2+ levels are critical to the activity of BK channels in inner ear type I Spiral Ligament fibrocytes. However, the mechanisms for regulating intracellular Ca2+ levels in these cells are currently poorly understood. Using patch-clamp technique, we have identified a voltage-dependent L-type Ca2+ channel in type I Spiral Ligament fibrocytes cultured from gerbil inner ear. With 10 mM Ba2+ as the conductive cation, an inwardly rectifying current was elicited with little inactivation by membrane depolarization. The voltage activation threshold and the half-maximal voltage activation were -40 and -6 mV, respectively. This inward whole-cell current reached its peak at around 10 mV of membrane potential. The amplitude of the peak current varied among cells ranging from 50 to 274 pA with an average of 132.4 +/- 76.2 pA (n = 19); 10(-6) M nifedipine significantly inhibited the inward currents by 90.3 +/- 1.2% (n = 11). RT-PCR analysis revealed that cultured type I Spiral Ligament fibrocytes express the alpha1C isoform of the L-type Ca2+ channels encoded by the Cav1.2 gene. The expression of this channel in gerbil inner ear was confirmed by RT-PCR analysis using freshly isolated Spiral Ligament tissues. The Cav1.2 channel may function in conjunction with a previously identified intracellular Ca-ATPase (SERCA) to regulate intracellular free Ca2+ levels in type I Spiral Ligament fibrocytes, and thus modulate BK channel activity in these cells.

  • BK channels mediate the voltage-dependent outward current in type I Spiral Ligament fibrocytes.
    Hearing research, 2004
    Co-Authors: Zhijun Shen, Fenghe Liang, Debra J Hazen-martin, Bradley A Schulte
    Abstract:

    Recent experimental and clinical studies have provided considerable evidence to support the phenomenon of K(+) recycling in the mammalian cochlea. However, the precise cellular and molecular mechanisms underlying and regulating this process remain only partially understood. Here, we report that cultured type I Spiral Ligament fibrocytes (SLFs), a major component of the K(+) recycling pathway, have a dominant K(+) membrane conductance that is mediated by BK channels. The averaged half-maximal voltage-dependent membrane potential for the whole-cell currents was 70+/-1.2 mV at 1 nM intracellular free Ca(2+) and shifted to 38+/-0.2 mV at 20 microM intracellular free Ca(2+) (n=4-6). The reversal potential of whole-cell tail currents against different bath K(+) concentrations was 52 mV per decade (n=3-6). The sequence of relative ion permeability of the whole-cell conductance was K(+)>Rb(+)z.Gt;Cs(+)>Na(+) (n=5-17). The whole-cell currents were inhibited by extracellular tetraethylammonium and iberiotoxin (IbTx) with IC(50) values of 0.07 mM and 0.013 microM, respectively (n=3-7). The membrane potentials of type I SLFs measured with conventional zero-current whole-cell configuration were highly K(+)-selective and sensitive to IbTx (n=4-9). In addition, the BK channels in these cells exhibited voltage-dependent and incomplete inactivation properties and the recovery time was estimated to be approximately 6 s with repetitive voltage pulses from -70 to 80 mV (n=3). These data suggest that BK channels in type I SLFs play a major role in regulating the intracellular electrochemical gradient in the lateral wall syncytium responsible for facilitating the K(+) movement from perilymph to the stria vascularis.

  • BK channels mediate the voltage-dependent outward current in type I Spiral Ligament fibrocytes.
    Hearing Research, 2003
    Co-Authors: Zhijun Shen, Fenghe Liang, Debra J Hazen-martin, Bradley A Schulte
    Abstract:

    Abstract Recent experimental and clinical studies have provided considerable evidence to support the phenomenon of K + recycling in the mammalian cochlea. However, the precise cellular and molecular mechanisms underlying and regulating this process remain only partially understood. Here, we report that cultured type I Spiral Ligament fibrocytes (SLFs), a major component of the K + recycling pathway, have a dominant K + membrane conductance that is mediated by BK channels. The averaged half-maximal voltage-dependent membrane potential for the whole-cell currents was 70±1.2 mV at 1 nM intracellular free Ca 2+ and shifted to 38±0.2 mV at 20 μM intracellular free Ca 2+ ( n =4–6). The reversal potential of whole-cell tail currents against different bath K + concentrations was 52 mV per decade ( n =3–6). The sequence of relative ion permeability of the whole-cell conductance was K + >Rb + ≫Cs + >Na + ( n =5–17). The whole-cell currents were inhibited by extracellular tetraethylammonium and iberiotoxin (IbTx) with IC 50 values of 0.07 mM and 0.013 μM, respectively ( n =3–7). The membrane potentials of type I SLFs measured with conventional zero-current whole-cell configuration were highly K + -selective and sensitive to IbTx ( n =4–9). In addition, the BK channels in these cells exhibited voltage-dependent and incomplete inactivation properties and the recovery time was estimated to be ∼6 s with repetitive voltage pulses from −70 to 80 mV ( n =3). These data suggest that BK channels in type I SLFs play a major role in regulating the intracellular electrochemical gradient in the lateral wall syncytium responsible for facilitating the K + movement from perilymph to the stria vascularis.

  • A voltage- and Ca2+-dependent big conductance K channel in cochlear Spiral Ligament fibrocytes
    Pflugers Archiv : European journal of physiology, 2003
    Co-Authors: Fenghe Liang, Bradley A Schulte, Debra J Hazen-martin, Samuel S Spicer, A. Niedzielski, Zhijun Shen
    Abstract:

    Evidence is accruing that Spiral Ligament fibrocytes (SLFs) play an important role in cochlear K+ homeostasis, but little direct physiological data is available to support this concept. Here we report the presence and characterization of a voltage- and Ca2+-dependent big-conductance K (BK) channel in type I SLFs cultured from the gerbil cochlea. A single-channel conductance of 298±5.6 pS (n=28) was measured under symmetrical K+. Membrane potentials for half-maximal open probability (P o) were −67, −45 and 85 mV with cytosolic free-Ca2+ levels of 0.7 mM, 10 μM and 1 μM, respectively (n=8–14). The Hill coefficient for Ca2+ affinity was 1.9 at a membrane potential of 60 mV (n=6). The BK channel showed very low activity (P o=0.0019, n=5) under normal physiological conditions, suggesting a low resting intracellular free [Ca2+]. Pharmacological results fit well with the profile of classic BK channels. The estimated half-maximal inhibitory concentration and Hill coefficient for tetraethylammonium were 0.086±0.021 mM and 0.99, respectively (n=4–9). In whole cell recordings, the voltage-activated outward K current was inhibited 85.7±4.5% (n=6) by 0.1 μM iberiotoxin. A steady-state kinetic model with two open and two closed stages best described the BK gating process (τo1 0.23±0.08 ms, τo2 1.40±0.32 ms; τc1 0.26±0.09 ms, τc2 3.10±1.2 ms; n=11). RT-PCR analyses revealed a splice variant of the BK channel α subunit in cultured type I SLFs and freshly isolated Spiral Ligament tissues. The BK channel is likely to play a major role in regulating the membrane potential of type I SLFs, which may in turn influence K+ recycling dynamics in the mammalian cochlea.

Fenghe Liang - One of the best experts on this subject based on the ideXlab platform.

  • Identification of ClC-2 and CIC-K2 chloride channels in cultured rat type IV Spiral Ligament fibrocytes.
    Journal of the Association for Research in Otolaryngology : JARO, 2007
    Co-Authors: Fenghe Liang, Nancy Smythe, Bradley A Schulte
    Abstract:

    Voltage-gated chloride channels (ClCs) are important mediators of cellular ion homeostasis and volume regulation. In an earlier study, we used immunohistochemical, Western blot, and reverse transcriptase PCR (RT-PCR) approaches to identify ClC-K variants in types II, IV, and V fibrocytes of the rodent Spiral Ligament. We have now confirmed the expression of ClC-K2 in these cells by in situ hybridization. All three of these fibrocyte subtypes are thought to be involved in cochlear K+ recycling; thus, it is important to understand the precise mechanisms regulating their membrane conductance and the role played by ClCs in this process. In this study, we report the characterization of a secondary cell line derived from explants from the region of the rat Spiral Ligament underlying and inferior to the Spiral prominence. The cultured cells were immunopositive for vimentin, Na,K/ATPase, Na,K,Cl-cotransporter, carbonic anhydrase isozyme II, and creatine kinase isozyme BB, but not for cytokeratins or Ca/ATPase, an immunostaining profile indicative of the type IV subtype. Evaluation of the cultures by RT-PCR and Western blot analysis confirmed the presence of both ClC-2 and -K2. Whole-cell patch clamp recordings identified two biophysically distinct Cl− currents in the cultured cells. One, an inwardly rectifying Cl− current activated by hyperpolarization or decreasing extracellular pH corresponded with the properties of ClC-2. The other, a weak outwardly rectifying Cl− current regulated by extracellular pH, Cl−, and Ca2+ resembled the channel characteristics of ClC-K2 when expressed in Xenopus oocytes. These findings suggest that at least two functionally different chloride channels are involved in regulating membrane anion conductance in cultured type IV Spiral Ligament fibrocytes.

  • Identification and characterization of an L-type Cav1.2 channel in Spiral Ligament fibrocytes of gerbil inner ear.
    Brain research. Molecular brain research, 2004
    Co-Authors: Fenghe Liang, Bradley A Schulte, Cungui Mao, Debra J Hazen-martin, Zhijun Shen
    Abstract:

    Intracellular free Ca2+ levels are critical to the activity of BK channels in inner ear type I Spiral Ligament fibrocytes. However, the mechanisms for regulating intracellular Ca2+ levels in these cells are currently poorly understood. Using patch-clamp technique, we have identified a voltage-dependent L-type Ca2+ channel in type I Spiral Ligament fibrocytes cultured from gerbil inner ear. With 10 mM Ba2+ as the conductive cation, an inwardly rectifying current was elicited with little inactivation by membrane depolarization. The voltage activation threshold and the half-maximal voltage activation were -40 and -6 mV, respectively. This inward whole-cell current reached its peak at around 10 mV of membrane potential. The amplitude of the peak current varied among cells ranging from 50 to 274 pA with an average of 132.4 +/- 76.2 pA (n = 19); 10(-6) M nifedipine significantly inhibited the inward currents by 90.3 +/- 1.2% (n = 11). RT-PCR analysis revealed that cultured type I Spiral Ligament fibrocytes express the alpha1C isoform of the L-type Ca2+ channels encoded by the Cav1.2 gene. The expression of this channel in gerbil inner ear was confirmed by RT-PCR analysis using freshly isolated Spiral Ligament tissues. The Cav1.2 channel may function in conjunction with a previously identified intracellular Ca-ATPase (SERCA) to regulate intracellular free Ca2+ levels in type I Spiral Ligament fibrocytes, and thus modulate BK channel activity in these cells.

  • BK channels mediate the voltage-dependent outward current in type I Spiral Ligament fibrocytes.
    Hearing research, 2004
    Co-Authors: Zhijun Shen, Fenghe Liang, Debra J Hazen-martin, Bradley A Schulte
    Abstract:

    Recent experimental and clinical studies have provided considerable evidence to support the phenomenon of K(+) recycling in the mammalian cochlea. However, the precise cellular and molecular mechanisms underlying and regulating this process remain only partially understood. Here, we report that cultured type I Spiral Ligament fibrocytes (SLFs), a major component of the K(+) recycling pathway, have a dominant K(+) membrane conductance that is mediated by BK channels. The averaged half-maximal voltage-dependent membrane potential for the whole-cell currents was 70+/-1.2 mV at 1 nM intracellular free Ca(2+) and shifted to 38+/-0.2 mV at 20 microM intracellular free Ca(2+) (n=4-6). The reversal potential of whole-cell tail currents against different bath K(+) concentrations was 52 mV per decade (n=3-6). The sequence of relative ion permeability of the whole-cell conductance was K(+)>Rb(+)z.Gt;Cs(+)>Na(+) (n=5-17). The whole-cell currents were inhibited by extracellular tetraethylammonium and iberiotoxin (IbTx) with IC(50) values of 0.07 mM and 0.013 microM, respectively (n=3-7). The membrane potentials of type I SLFs measured with conventional zero-current whole-cell configuration were highly K(+)-selective and sensitive to IbTx (n=4-9). In addition, the BK channels in these cells exhibited voltage-dependent and incomplete inactivation properties and the recovery time was estimated to be approximately 6 s with repetitive voltage pulses from -70 to 80 mV (n=3). These data suggest that BK channels in type I SLFs play a major role in regulating the intracellular electrochemical gradient in the lateral wall syncytium responsible for facilitating the K(+) movement from perilymph to the stria vascularis.

  • BK channels mediate the voltage-dependent outward current in type I Spiral Ligament fibrocytes.
    Hearing Research, 2003
    Co-Authors: Zhijun Shen, Fenghe Liang, Debra J Hazen-martin, Bradley A Schulte
    Abstract:

    Abstract Recent experimental and clinical studies have provided considerable evidence to support the phenomenon of K + recycling in the mammalian cochlea. However, the precise cellular and molecular mechanisms underlying and regulating this process remain only partially understood. Here, we report that cultured type I Spiral Ligament fibrocytes (SLFs), a major component of the K + recycling pathway, have a dominant K + membrane conductance that is mediated by BK channels. The averaged half-maximal voltage-dependent membrane potential for the whole-cell currents was 70±1.2 mV at 1 nM intracellular free Ca 2+ and shifted to 38±0.2 mV at 20 μM intracellular free Ca 2+ ( n =4–6). The reversal potential of whole-cell tail currents against different bath K + concentrations was 52 mV per decade ( n =3–6). The sequence of relative ion permeability of the whole-cell conductance was K + >Rb + ≫Cs + >Na + ( n =5–17). The whole-cell currents were inhibited by extracellular tetraethylammonium and iberiotoxin (IbTx) with IC 50 values of 0.07 mM and 0.013 μM, respectively ( n =3–7). The membrane potentials of type I SLFs measured with conventional zero-current whole-cell configuration were highly K + -selective and sensitive to IbTx ( n =4–9). In addition, the BK channels in these cells exhibited voltage-dependent and incomplete inactivation properties and the recovery time was estimated to be ∼6 s with repetitive voltage pulses from −70 to 80 mV ( n =3). These data suggest that BK channels in type I SLFs play a major role in regulating the intracellular electrochemical gradient in the lateral wall syncytium responsible for facilitating the K + movement from perilymph to the stria vascularis.

  • A voltage- and Ca2+-dependent big conductance K channel in cochlear Spiral Ligament fibrocytes
    Pflugers Archiv : European journal of physiology, 2003
    Co-Authors: Fenghe Liang, Bradley A Schulte, Debra J Hazen-martin, Samuel S Spicer, A. Niedzielski, Zhijun Shen
    Abstract:

    Evidence is accruing that Spiral Ligament fibrocytes (SLFs) play an important role in cochlear K+ homeostasis, but little direct physiological data is available to support this concept. Here we report the presence and characterization of a voltage- and Ca2+-dependent big-conductance K (BK) channel in type I SLFs cultured from the gerbil cochlea. A single-channel conductance of 298±5.6 pS (n=28) was measured under symmetrical K+. Membrane potentials for half-maximal open probability (P o) were −67, −45 and 85 mV with cytosolic free-Ca2+ levels of 0.7 mM, 10 μM and 1 μM, respectively (n=8–14). The Hill coefficient for Ca2+ affinity was 1.9 at a membrane potential of 60 mV (n=6). The BK channel showed very low activity (P o=0.0019, n=5) under normal physiological conditions, suggesting a low resting intracellular free [Ca2+]. Pharmacological results fit well with the profile of classic BK channels. The estimated half-maximal inhibitory concentration and Hill coefficient for tetraethylammonium were 0.086±0.021 mM and 0.99, respectively (n=4–9). In whole cell recordings, the voltage-activated outward K current was inhibited 85.7±4.5% (n=6) by 0.1 μM iberiotoxin. A steady-state kinetic model with two open and two closed stages best described the BK gating process (τo1 0.23±0.08 ms, τo2 1.40±0.32 ms; τc1 0.26±0.09 ms, τc2 3.10±1.2 ms; n=11). RT-PCR analyses revealed a splice variant of the BK channel α subunit in cultured type I SLFs and freshly isolated Spiral Ligament tissues. The BK channel is likely to play a major role in regulating the membrane potential of type I SLFs, which may in turn influence K+ recycling dynamics in the mammalian cochlea.