The endocochlear potential in experimental renal insufficiency

Endocochlear Potential (EP) was measured in male Wistar rats under control conditions and renal failure produced by means of bilateral nephrectomy. Results show a statistically significant (p less than 0.01) decrease in EP measured in animals with renal failure. This finding is in accordance with the decrease in Na-K ATPase activity found in the cochlea and other structures in human and experimental kidney insufficiency.     

Cyclic stretch of alveolar epithelial cells alters cytoskeletal micromechanics

Cytoplasmic transport of large molecules such as plasmid DNA (pDNA) has been shown to increase when cells are subjected to mild levels of cyclic stretch for brief periods. In the case of pDNA, this is in part due to the increased active transport of pDNA along stabilized, acetylated microtubules in the cytoplasm, whose levels are increased in response to stretch. It also has been shown that disruption of the dense actin network leads to increased pDNA and macromolecule diffusion as well. We hypothesize that stretch not only increases active transport of pDNA but also, similar to actin disrupting drugs, decreases cytoplasmic stiffness leading to a less restive pathway for macromolecules to diffuse. To test this we used particle tracking microrheology to measure cytoplasmic mechanics. We conclude that while cyclic stretch transiently decreases cytoplasmic stiffness and increases diffusivity, stretch‐independent modulation of the levels of acetylated, stable microtubules has no effect on cytoplasmic stiffness. Furthermore, stretching cells that have maximally acetylated microtubules increases cytoplasmic trafficking of pDNA, without increasing levels of acetylated microtubules. These findings suggest that stretch‐enhanced gene transfer may occur by two independent mechanisms: increased levels of acetylated microtubules for directed active transport, and reduced cytoplasmic stiffness for increased diffusion. Biotechnol. Bioeng. 2011;108: 446–453. © 2010 Wiley Periodicals, Inc.     

Gastric type H+,K+-ATPase in the cochlear lateral wall is critically involved in formation of the endocochlear potential

Cochlear endolymph has a highly positive potential of approximately +80 mV known as the endocochlear potential (EP). The EP is essential for hearing and is maintained by K⁺ circulation from perilymph to endolymph through the cochlear lateral wall. Various K⁺ transport apparatuses such as the Na⁺,K⁺-ATPase, the Na⁺-K⁺-2Cl^– cotransporter, and the K⁺ channels Kir4.1 and KCNQ1/KCNE1 are expressed in the lateral wall and are known to play indispensable roles in cochlear K⁺ circulation. The gastric type of the H⁺,K⁺-ATPase was also shown to be expressed in the cochlear lateral wall (Lecain E, Robert JC, Thomas A, and Tran Ba Huy P. Hear Res 149: 147–154, 2000), but its functional role has not been well studied. In this study we examined the precise localization of H⁺,K⁺-ATPase in the cochlea and its involvement in formation of EP. RT-PCR analysis showed that the cochlea expressed mRNAs of gastric ₁-, but not colonic ₂-, and -subunits of H⁺,K⁺-ATPase. Immunolabeling of an antibody specific to the ₁ subunit was detected in type II, IV, and V fibrocytes distributed in the spiral ligament of the lateral wall and in the spiral limbus. Strong immunoreactivity was also found in the stria vascularis. Immunoelectron microscopic examination exhibited that the H⁺,K⁺-ATPase was localized exclusively at the basolateral site of strial marginal cells. Application of Sch-28080, a specific inhibitor of gastric H⁺,K⁺-ATPase, to the spiral ligament as well as to the stria vascularis caused prominent reduction of EP. These results may imply that the H⁺,K⁺-ATPase in the cochlear lateral wall is crucial for K⁺ circulation and thus plays a critical role in generation of EP. hydrogen, potassium-adenosine triphosphatase; stria vascularis; spiral ligament     

Generation of the endocochlear potential: a biophysical model

The generation and maintenance of the endocochlear potential (EP) by the stria vascularis is essential for proper function of the cochlea. We present a mathematical model that captures the critical biophysical interactions between the distinct cellular layers that generate the EP. By describing the relationship between the K⁺ concentration in the intrastrial space and the intermediate cell transmembrane potential, we rationalize the presence of a large intermediate cell K⁺ conductance and predict that the intrastrial [K⁺] is ∼4 mM at steady state. The model also predicts that the stria vascularis is capable of buffering the EP against external perturbations in a manner modulated by changes in intrastrial [K⁺], thus facilitating hearing sensitivity across the broad dynamic range of the auditory system.     

KCNJ10 (Kir4.1) potassium channel knockout abolishes endocochlear potential

Striavascularis of the cochlea generates the endocochlear potential andsecretes K⁺. K⁺ is the main charge carrier andthe endocochlear potential the main driving force for the sensorytransduction that leads to hearing. Stria vascularis consists of twobarriers, marginal cells that secrete potassium and basal cells thatare coupled via gap junctions to intermediate cells. Mice lacking theKCNJ10 (Kir4.1) K⁺ channel in strial intermediate cells didnot generate an endocochlear potential. Endolymph volume andK⁺ concentration ([K⁺]) were reduced. Thesestudies establish that the KCNJ10 K⁺ channel provides themolecular mechanism for generation of the endocochlear potential inconcert with other transport pathways that establish the[K⁺] difference across the channel. KCNJ10 is also alimiting pathway for K⁺ secretion.

     

Mechanism generating endocochlear potential: role played by intermediate cells in stria vascularis

The endocochlear DC potential (EP) is generated by the stria vascularis, and essential for the normal function of hair cells. Intermediate cells are melanocytes in the stria vascularis. To examine the contribution of the membrane potential of intermediate cells (E(m)) to the EP, a comparison was made between the effects of K(+) channel blockers on the E(m) and those on the EP. The E(m) of dissociated guinea pig intermediate cells was measured in the zero-current clamp mode of the whole-cell patch clamp configuration. The E(m) changed by 55.1 mV per 10-fold changes in extracellular K(+) concentration. Ba(2+), Cs(+), and quinine depressed the E(m) in a dose-dependent manner, whereas tetraethylammonium at 30 mM and 4-aminopyridine at 10 mM had no effect. The reduction of the E(m) by Ba(2+) and Cs(+) was enhanced by lowering the extracellular K(+) concentration from 3.6 mM to 1.2 mM. To examine the effect of the K(+) channel blockers on the EP, the EP of guinea pigs was maintained by vascular perfusion, and K(+) channel blockers were administered to the artificial blood. Ba(2+), Cs(+) and quinine depressed the EP in a dose-dependent manner, whereas tetraethylammonium at 30 mM and 4-aminopyridine at 10 mM did not change the EP. A 10-fold increase in the K(+) concentration in the artificial blood caused a minor decrease in the EP of only 10.6 mV. The changes in the EP were similar to those seen in the E(m) obtained at the lower extracellular K(+) concentration of 1.2 mM. On the basis of these results, we propose that the EP is critically dependent on the voltage jump across the plasma membrane of intermediate cells, and that K(+) concentration in the intercellular space in the stria vascularis may be actively controlled at a concentration lower than the plasma level.     

Deafness induced by Connexin 26 (GJB2) deficiency is not determined by endocochlear potential (EP) reduction but is associated with cochlear developmental disorders

Connexin 26 (Cx26, GJB2) mutations are the major cause of hereditary deafness and are responsible for >50% of nonsyndromic hearing loss. Mouse models show that Cx26 deficiency can cause congenital deafness with cochlear developmental disorders, hair cell degeneration, and the reduction of endocochlear potential (EP) and active cochlear amplification. However, the underlying deafness mechanism still remains undetermined. Our previous studies revealed that hair cell degeneration is not a primary cause of hearing loss. In this study we investigated the role of EP reduction in Cx26 deficiency-induced deafness. We found that the EP reduction is not associated with congenital deafness in Cx26 knockout (KO) mice. The threshold of auditory brainstem response (ABR) in Cx26 KO mice was even greater than 110 dB SPL, demonstrating complete hearing loss. However, the EP in Cx26 KO mice varied and not completely abolished. In some cases, the EP could still remain at higher levels (>70 mV). We further found that the deafness in Cx26 KO mice is associated with cochlear developmental disorders. Deletion of Cx26 in the cochlea before postnatal day 5 (P5) could cause congenital deafness. The cochlea had developmental disorders and the cochlear tunnel was not open. However, no congenital deafness was found when Cx26 was deleted after P5. The cochlea also displayed normal development and the cochlear tunnel was open normally. These data suggest that congenital deafness induced by Cx26 deficiency is not determined by EP reduction and may result from cochlear developmental disorders.     

In vitro gentamicin exposure alters caveolae protein profile in cochlear spiral ligament pericytes

Background

The aminoglycoside antibiotic gentamicin is an ototoxic drug and has been used experimentally to investigate cochlear damage induced by noise.We have investigated the changes in the protein profile associated with caveolae in gentamicin treated and untreated spiral ligament (SL) pericytes, specialized cells in the blood labyrinth barrier of the inner ear microvasculature. Pericytes from various microvascular beds express caveolae, protein and cholesterol rich microdomains, which can undergo endocytosis and transcytosis to transport small molecules in and out the cells. A different protein profile in transport-specialized caveolae may induce pathological changes affecting the integrity of the blood labyrinth barrier and ultimately contributing to hearing loss.

Method

Caveolae isolation from treated and untreated cells is achieved through ultracentrifugation of the lysates in discontinuous gradients. Mass spectrometry (LC-MS/MS) analysis identifies the proteins in the two groups. Proteins segregating with caveolae isolated from untreated SL pericytes are then compared to caveolae isolated from SL pericytes treated with the gentamicin for 24 h. Data are analyzed using bioinformatic tools.

Results

The caveolae proteome in gentamicin treated cells shows that 40% of total proteins are uniquely associated with caveolae during the treatment, and 15% of the proteins normally associated with caveolae in untreated cell are suppressed. Bioinformatic analysis of the data shows a decreased expression of proteins involved in genetic information processing, and an increase in proteins involved in metabolism, vesicular transport and signal transduction in gentamicin treated cells. Several Rab GTPases proteins, ubiquitous transporters, uniquely segregate with caveolae and are significantly enriched in gentamicin treated cells.

Conclusion

We report that gentamicin exposure modifies protein profile of caveolae from SL pericytes. We identified a pool of proteins which are uniquely segregating with caveolae during the treatment, mainly participating in metabolic and biosynthetic pathways, in transport pathways and in genetic information processing. Finally, we show for the first time proteins associated with caveolae SL pericytes linked to nonsyndromic hearing loss.

     

Conservation of endocochlear potential in mice with profound hearing loss induced by co-administration of kanamycin and furosemide

Research in mammalian hair cell regeneration is hampered by a lack of in vivo model of adult mouse inner ear injury. In the present study we investigated the effects of a combination of a single dose of aminoglycoside followed by a loop diuretic in adult mice. The auditory brainstem response threshold shift, extent and defining characteristics of the cochlear lesion were assessed and verified at different time points post-treatment. Our data indicated that this drug combination caused the rapid and extensive death of outer hair cells (OHCs). OHC death presented throughout the cochlea that commenced in the basal turn by 24 h and progressed apically. In contrast, inner hair cell (IHC) loss was delayed and mild. Terminal deoxynucleotidyl transferase dUTP nick end labelling-positive nuclei demonstrated that the majority of OHCs died via an apoptotic pathway. Auditory threshold shifts of up to 90 dB SPL indicated a profound hearing loss. In addition, the endocochlear potential (EP) in the drug-treated animals displayed a significant decline at 12 h post-treatment followed by recovery by 48 h post-treatment. Despite this recovery, there was a significant and progressive decrease in strial vascularis thickness, which was predominantly due to atrophy of marginal cells. The present study reproduced an adult mouse model of aminoglycoside-induced hearing loss. The mechanism underlying the recovered EP in the model with extensive hair cell death is discussed.     

Loss of SHP-1 phosphatase alters cytokine expression in the mouse hindbrain following cochlear ablation

Inflammatory cytokines in the central nervous system are largely modulated by glial cells and influence neuronal responses to CNS injury. The protein tyrosine phosphatase SHP-1, an intracellular regulator of many cytokine signaling pathways, has been implicated in mediating the activation of glia. There is a direct correlation between abnormally activated microglia and neuron loss within the SHP-1 deficient motheaten (me/me) mouse auditory brainstem after afferent injury. In order to determine whether loss of SHP-1 creates an aberrant cytokine environment driving the abnormal activation of me/me microglia, the expression of interleukin-4 (IL-4), interleukin-10 (IL-10), interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) was examined by enzyme-linked immunosorbent assay (ELISA). Normal uninjured me/me mice showed lower IL-10 but higher IL-1beta levels compared to wild-type. Following unilateral cochlear ablation, there is decreased expression of IL-4 and IL-10 in me/me brains compared to wild-type, but IL-1beta is significantly increased. These findings indicate that decreases in anti-inflammatory cytokines, in combination with increased expression of the pro-inflammatory cytokine IL-1beta, may initiate a robust inflammatory reaction within the me/me brain contributing to the neuronal degeneration in the deafferented me/me auditory brainstem. SHP-1 may therefore play a role in limiting CNS inflammation following injury and disease.     

The micromechanics versus the macromechanics of cortical bone--a comprehensive presentation

Secondary cortical bone is a complicated patchwork of structures which can be viewed as a hierarchy of four different orders. As far as the biomechanical properties of cortical bone are concerned, the lamellae is the most important of the four. The relative distribution of longitudinal lamellae (whose fiber bundles and crystallites have a longitudinal course and withstand loading by tension) with respect to transverse lamellae (whose fiber bundles and crystallites have a transverse course and withstand loading by compression) governs the mechanical properties of bone at macroscopic level both in normal and pathological conditions.     

Radio-frequency electromagnetic radiation alters the electric potential of Myriophyllum aquaticum

Electric signaling pathways are important for rapid and long-distance communication within a plant. Changes in the electric potential (EP) inside plants have been observed during the propagation of electric signals. Increasing radiofrequency electromagnetic radiation (EMR) in the environment raise the question about possible effects of EMR on the EP of plants. In the present experiment, we investigated the effect of 2, 2.5, 3.5, and 5.5 GHz EMR with a maximum field intensity of 23–25 V m^?1 on the EP in emergent Myriophyllum aquaticum plants. The 2 and 5.5 GHz exposures caused significant (16 and 13 %) decreases in the standard deviation of rapid fluctuations observed in the EP. The greatest change was caused by 2.5 GHz EMR (23 % increment), although it was not statistically significant. A recovery of the EP was only after 2.5 GHz EMR exposure. The temperature of the plants was not changed by the EMR exposure. These findings confirm the frequency-dependent non-thermal effects of EMR on the EP of plants.     

The group delay and suppression pattern of the cochlear microphonic potential recorded at the round window

Background

It is commonly assumed that the cochlear microphonic potential (CM) recorded from the round window (RW) is generated at the cochlear base. Based on this assumption, the low-frequency RW CM has been measured for evaluating the integrity of mechanoelectrical transduction of outer hair cells at the cochlear base and for studying sound propagation inside the cochlea. However, the group delay and the origin of the low-frequency RW CM have not been demonstrated experimentally.

Methodology/Principal Findings

This study quantified the intra-cochlear group delay of the RW CM by measuring RW CM and vibrations at the stapes and basilar membrane in gerbils. At low sound levels, the RW CM showed a significant group delay and a nonlinear growth at frequencies below 2 kHz. However, at high sound levels or at frequencies above 2 kHz, the RW CM magnitude increased proportionally with sound pressure, and the CM phase in respect to the stapes showed no significant group delay. After the local application of tetrodotoxin the RW CM below 2 kHz became linear and showed a negligible group delay. In contrast to RW CM phase, the BM vibration measured at location ∼2.5 mm from the base showed high sensitivity, sharp tuning, and nonlinearity with a frequency-dependent group delay. At low or intermediate sound levels, low-frequency RW CMs were suppressed by an additional tone near the probe-tone frequency while, at high sound levels, they were partially suppressed only at high frequencies.

Conclusions/Significance

We conclude that the group delay of the RW CM provides no temporal information on the wave propagation inside the cochlea, and that significant group delay of low-frequency CMs results from the auditory nerve neurophonic potential. Suppression data demonstrate that the generation site of the low-frequency RW CM shifts from apex to base as the probe-tone level increases.     

Reduced P2x<Subscript>2</Subscript> receptor-mediated regulation of endocochlear potential in the ageing mouse cochlea

Extracellular adenosine triphosphate (ATP) has profound effects on the cochlea, including an effect on the regulation of the endocochlear potential (EP). Noise-induced release of ATP into the endolymph activates a shunt conductance mediated by P2X₂ receptors in tissues lining the endolymphatic compartment, which reduces the EP and, consequentially, hearing sensitivity. This may be a mechanism of adaptation or protection from high sound levels. As inaction of such a process could contribute to hearing loss, this study examined whether the action of ATP on EP changes with age and noise exposure in the mouse. The EP and the endolymphatic compartment resistance (CoPR) were measured in mice (CBA/CaJ) aged between 3 and 15 months. The EP and CoPR declined slightly with age with an associated small, but significant, reduction in auditory brainstem response thresholds. ATP (100–1,000 μM) microinjected into the endolymphatic compartment caused a dose-dependent decline in EP correlated to a similar decrease in CoPR. This was blocked by pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonate, consistent with a P2X₂ receptor-mediated shunt conductance. There was no substantial difference in the ATP response with age. Noise exposure (octave-band noise 80–100 decibels sound pressure level (dBSPL), 48 h) in young animals induced an upregulation of the P2X₂ receptor expression in the organ of Corti and spiral limbus, most noticeably with the 90-dB exposure. This did not occur in the aged animals except following exposure at 90 dBSPL. The EP response to ATP was muted in the noise-exposed aged animals except following the 90-dB exposure. These findings provide some evidence that the adaptive response of the cochlea to noise may be reduced in older animals, and it is speculated that this could increase their susceptibility to noise-induced injury.     

Exotic grass invasion alters potential rates of N fixation in Hawaiian woodlands

Exotic grass invasion promotes fire which drives the conversion of native woodlands to exotic grasslands in the seasonally dry submontane forests of the island of Hawai'i. We compared potential rates of N fixation in an unburned forest site and a converted grassland site using the acetylene reduction assay. In addition to measuring rates of N fixation on separate and mixed substrates in each site, we tested the effect of abiotic factors on rates of N fixation of specific substrates. We hypothesized that rates of N fixation would be higher in the converted grassland site. N fixation estimates were 4.9 kg N ha⁻¹ year⁻¹ for the unburned forest, and 0.10 kg N ha⁻¹ year⁻¹ for the grassland site, so our hypothesis was rejected. The N fixation in the unburned forest occurs mostly on the leaf litter of native woody species. These substrates are absent from the grassland site, except for wood debris which was not consumed during the fires. No nitrogenase activity was detected in the rhizosphere and litter of grasses, the rhizospheres of shrubs or in soil. Although wood debris is not a significant contributor to the N fixed in the unburned forest, it contributes the majority of N fixed in the grassland. The response of nitrogenase activity to varying conditions of moisture and temperature suggests that microclimatic differences between sites do not control differences in N fixation activity; rather, these differences are due to the abundance of N-fixing substrates. The substantial decrease in N fixation activity after the conversion from woodland to grassland implies that ecosystem-level rates of N accretion are decreased by fire in these sites so much that the N lost during volatilization due to fire is not replenished over the long term by N fixation. Received: 10 January 1997 / Accepted: 7 August 1997