Biomedical subjects

H P Zenner

Publications and source records attributed to H P Zenner.

At least 19 recordsLinked to original sources

PubMed1995

Cell-specific expression of the alpha 9 n-ACh receptor subunit in auditory hair cells revealed by single-cell RT-PCR.

Single-cell reverse transcription polymerase chain reaction was carried out in three different cell types from the organ of Corti of the four-day old rat. For this purpose, pieces of the organ of Corti were mounted under a differential-interference contrast video microscope. Two different mounting configurations were used to allow imaging of cells from two almost orthogonal angles. This method afforded unequivocal recognition of various cell types in the vital tissue, and extraction of nucleus and cytoplasm of specified individual cells with a patch pipette. Messenger RNA encoding the alpha 9 acetylcholine (ACh) receptor subunit was detected and sequenced from individual outer hair cells and inner hair cells, but was not found in Deiters' cells. The identical Deiters' cells were positive for a P2x receptor subunit. This indicates cell-specific expression of the alpha 9 subunit in inner hair cells and outer hair cells and supports the hypothesis that this subunit contributes to calcium (Ca2+) permeable ionotropic ACh receptors (ACh-R). ACh-dependent Ca2+ concentration increase has been observed in both outer hair cells and inner hair cells.

Animals↗

PubMed1995

Subunit-specific inhibition of inward-rectifier K+ channels by quinidine.

Distinct inward-rectifier K+ channel subunits were expressed in Xenopus oocytes and tested for their sensitivity to the channel blocker quinidine. The 'strong' inward-rectifier K+ channel IRK1 was inhibited by quinidine with an EC50 of 0.7 mM, while the 'weak' rectifier channel ROMK1 was only moderately inhibited. ROMK1(N171D)-IRK1C-term chimeric channels, which carry both sites for strong rectification of IRK1 channels (the negatively charged D171 in the second transmembrane domain and the IRK1-C-terminus including E224), displayed strong rectification like IRK1, but showed weak sensitivity to quinidine-like ROMK1, suggesting independence of quinidine binding and rectification mechanisms. Moreover, BIR10 and BIR11, two strong rectifier subunits originally cloned from rat brain, exerted subunit-specific sensitivity to quinidine, being much higher for BIR11. Quinidine blockade of IRK1 was not voltage-dependent, but strongly dependent on the pH in the superfusate. These results strongly suggest a subunit-specific interaction of inward-rectifier K+ channels with neutral quinidine within membrane lipid bilayers.

Animals↗

PubMed1995

Subunit-dependent assembly of inward-rectifier K+ channels.

Inward-rectifier, G-protein-regulated and ATP-dependent K+ channels form a novel gene family of related proteins which share two transmembrane segments as a common structural feature. These K+ channels are only distantly related to the voltage-gated Shaker-type K+ channels comprising six transmembrane segments. Although the quaternary structure of voltage-gated K+ channels has been extensively studied in the past, little is known about subunit assembly of inward-rectifier K+ channels. Differential sensitivity of inward-rectifier K+ channels to voltage-dependent pore block by spermine was used to analyse subunit assembly. It is shown that inward-rectifier K+ channel proteins are composed of four subunits whose assembly obeys the rules of a binomial distribution. 'Strong' and 'mild' inward-rectifier K+ channel subunits (BIR10 and ROMK1) which are co-expressed in individual auditory hair cells form hetero-tetramers. Distribution of these hetero-tetramers, however, is not binomial. Hetero- and homo-oligomeric channels form with similar probabilities resulting in independent channel populations with distinct functional properties.

Adenosine Triphosphate↗

PubMed1995

Strong voltage-dependent inward rectification of inward rectifier K+ channels is caused by intracellular spermine.

Inward rectifier K+ channels mediate the K+ conductance at resting potential in many types of cell. Since these K+ channels do not pass outward currents (inward rectification) when the cell membrane is depolarized beyond a trigger threshold, they play an important role in controlling excitability. Both a highly voltage-dependent block by intracellular Mg2+ and an endogenous gating process are presently assumed to underly inward rectification. It is shown that strong voltage dependence of rectification found under physiological conditions is predominantly due to the effect of intracellular spermine. Physiological concentrations of free spermine mediate strong rectification of IRK1 inward rectifier K+ channels even in the absence of free Mg2+ and in IRK1 mutant channels that have no endogenous rectification.

Animals↗

PubMed1995

Electromotile responses and frequency tuning of isolated outer hair cells of the guinea pig cochlea.

Isolated outer hair cells (OHC) of the guinea pig cochlea were exposed to external alternating electric fields parallel to the longitudinal axis of the cells. This resulted in oscillations of the cells' length that were measured photoelectrically using a ratiometric light amplifier. At 5 Hz and elongations up to 300 nm, amplitude of the cell length during oscillation was a linear function of the amplitude of the sinusoidal electric field. When increasing the stimulus frequency up to 32 kHz, OHC length changes followed the stimulus cycle-by-cycle. Oscillations at frequencies above 32 kHz escaped the experimental approach by their small amplitudes and could not be excluded. The frequency dependence of the motile response measured at 5-12,000 Hz had low-pass filter characteristics in cells of the second, third and fourth turns of the cochlea. However, frequency tuning of the motile response was absent in each OHC and systematic differences between different turns were not observed.

Animals↗

PubMed1995

Acute hyperpolarization and elongation of cochlear outer hair cells on superfusion with cis-platinum.

The acute effects of cis-platinum on isolated cochlear outer hair cells (OHC) were investigated with whole-cell patch-clamps and measurements of cell length changes. Our findings demonstrated that cis-platinum reversibly induced a hyperpolarization and cellular elongation. These results suggest that the effects produced are the result of an interaction between cis-platinum and transduction channels in OHC. These acute effects are distinctly different from the chronic, irreversible ones that are followed by death of the OHC. The exact mechanism of these chronic effects remains unknown as yet.

Animals↗

PubMed1995

A threshold decrease for electrically stimulated motor responses of isolated aging outer hair cells from the pigmented guinea pig.

When outer hair cells are isolated from guinea pig cochleas and are placed in normal Hank's medium, they exhibit aging as a slow tonic reduction in length and increase in diameter. During this time the lateral subsurface cisternae become progressively vesiculated and the optical density of the border seen under phase-contrast microscopy decreases. A study of 65 outer hair cells was carried out using video imaging of this process. The base of each cell bonded to the Petri dish and the motility of the cuticular plate was recorded in two ways. To quantify the slow contraction of each preparation, the dimensions of the cell were measured from video replay. Displacements of the cuticular plate in response to an alternating electric field in line with the cell axis were also monitored using a video tracking technique. The amplitude of a 1 Hz stimulus required to cause a visually detectable motor response above baseline noise decreased as the cell degraded. Typically, fresh cylindrical cells exhibiting high optical contrast showed relatively small movements for field strengths up to 2 kVm-1. However, as the cell depolarized, the rigidity initially decreased and the cell could respond to field strengths down to 0.1 kVm-1 before cell death ultimately occurred. Such a threshold phenomenon in the isolated OHC has not been demonstrated directly until now. This result explains the variability of electromotility in aging in vitro preparations from the cochlea.

Animals↗

PubMed1995

Sound-induced displacement responses in the plane of the organ of Corti in the isolated guinea-pig cochlea.

Sound-induced displacement responses in the plane of the organ of Corti were studied in the apical turn in the isolated temporal-bone preparation of the guinea-pig cochlea. Swept sinusoidal sound stimuli (100-500 Hz) were delivered closed-field to the external auditory meatus. The surface of the organ of Corti was continuously monitored using a CCD video camera. Displacement responses in the plane of the organ of Corti were determined by analyzing the change of the location of the cells (pixel-by-pixel) within the visual field of the microscope. Displacement responses followed the stimulus amplitude and were observable at Hensen's cells, three rows of outer hair cells and inner hair cells. The most prominent displacement responses were over the outer hair cells; the maximum amplitude was 0.6-1.7 microns at 100 dB SPL. Tuned displacement responses were found; the Q10 dB was 1.3 +/- 0.6. The best frequency was tonotopically organized, decreasing toward the apex with a space constant of 0.4-0.9 mm/oct. The motion was directed either strial-apically or strial-basally in a frequency dependent manner. With the aid of laser interferometric measurements of the transverse displacement, it was concluded that sound stimulation does not induce slow DC motion in the organ of Corti for the isolated temporal-bone preparation.

Acoustic Stimulation↗

PubMed1995

Caloric evoked motile responses of mammalian vestibular sensory cells.

Evokation of caloric nystagmus in microgravity argues against thermal convection as a sufficient explanation for a caloric nystagmic response. By contrast, caloric nystagmus observed in weightlessness suggests a gravity independent mechanism. Here we demonstrate that direct caloric stimulation of isolated living vestibular hair cells from the guinea pig evoked mechanical responses of the sensory cells. Heating of the mechanoreceptor cells from 37 degrees C to 42 degrees C resulted in elongation cooling of the cells from 37 degrees C to 30 degrees C induced shortening of vestibular hair cells. The observed mechanism might contribute to the caloric evokation of a nystagmus both on earth and in orbit.

Animals↗

PubMed1995

Benign supraclavicular tumorous lymphangiectasia--a new disease?

We describe an isolated recurrent non-inflammatory tumorous swelling of the supraclavicular fossa in four premenopausal women. Ultrasonography, magnetic resonance imaging and computer tomography of the neck each suggested an inhomogeneous mass consistent with "lymphangioma." In each patient the clinical course and histopathologic findings suggested that the swellings were due to chronic localized lymph stasis with subsequent lymphangiectasia, possibly initiated by intermittent obstruction of the juncture of the thoracic or right lymph duct with the internal jugular vein. Enlargement may have been hormonally triggered by estrogens as each woman was taking oral contraceptive pills at the onset of the disease. To characterize this unique entity, we have termed the disorder benign supraclavicular tumorous lymphangiectasia.

Adult↗

PubMed1995

[Comparison of methods for early detection of noise vulnerability of the inner ear. Amplitude reduction of otoacoustic emissions are most sensitive at submaximal noise impulse exposure].

Noise-induced temporary impairment of cochlear function was measured with several audiometric tests in order to evaluate which method best predicts a vulnerable cochlea. We tested 10 normally-hearing and 13 subjects who were positive for temporary threshold shifts (TTS). The latter were selected from 194 soldiers who demonstrated a TTS higher than 15 dB after regular training with firearms. Acoustic distortion products (DPOAE), click-evoked otoacoustic emissions (TEOAE), upper limit of hearing (ULH) and pure-tone and high-frequency audiometry were used to evaluate possible increased vulnerability of the cochlea. Tests were conducted at lower sound intensities (white noise, 90 dB SPL, 5 min; impact noise, 100 or 106 dB SPLs, 10 impulses/s, 5 min). Seventy per cent of the TTS-positive soldiers studied exhibited significant reductions of TEOAE amplitudes, whereas a stable emission was observed in all control subjects. DPOAE alterations were seen in 38% of the soldiers tested. These results indicate that TEOAE is the most sensitive, objective method for detecting a positive disturbance in cochlear function. Although the upper limit of hearing was also a very sensitive method, variability of this psychoacoustic method depended on the help and experience of the subjects being tested.

Adult↗

PubMed1995

[Detection of GABA(A) receptor mRNA in cochlear tissue. An in situ hybridization study].

Gamma-aminobutyric acid (GABA) and the GABAergic system play an important role in the efferent modulation of cochlear function. We examined surface preparations of guinea pig and mouse cochleae by in situ hybridization using radioactive labelled oligonucleotides for several subunits of the GABAA receptor. Frozen sections of rat and guinea pig brain (cortex, hippocampus, cerebellum) served as controls. In the mouse cochlea the mRNA of the alpha-1 and alpha-5, beta-1 and gamma-1 subunit were detected, while in guinea pig cochlea mRNA of the alpha-1, alpha-4, alpha-5, and gamma-1 subunit of the GABAA receptor were found. Positive signals were located in the regions of the outer hair cells and had a weaker intensity in the inner hair cells. In the brain sections the several subunits were detected in a variable distribution in the cerebellum, hippocampus and cortical regions. Rat specimens exhibited stronger signals than guinea pig brain sections. These investigations have extended previous results of immunocytochemical experiments from our laboratory demonstrating mRNA sequences of GABAA receptor subunits in the mammalian inner ear. Detection of these nucleotide sequences using surface preparations of the cochlea on a molecular level by in situ hybridization supports the importance of GABA as a cochlear neurotransmitter. Furthermore, it can be concluded that the mammalian cochlea is able to express a GABA-dependent neurotransmission system.

Animals↗

PubMed1994

A structural determinant of differential sensitivity of cloned inward rectifier K+ channels to intracellular spermine.

Large subtype-specific differences in the sensitivity of cloned inward-rectifier K+ channels of the IRK1, BIR10 and ROMK1 subtype to being blocked by intracellular spermine (SPM) are described. It is shown, by site-directed mutagenesis, that the four orders of magnitude larger SPM sensitivity of BIR10 channels compared to ROMK1 channels may be explained by a difference in a single amino acid in the putative transmembrane segment TMII. This residue, a negatively charged glutamate in BIR10, is homologous to the residue in IRK1 and ROMK1 which has previously been shown to change gating properties and Mg2+ sensitivity. Differential block by physiological SPM concentrations is suggested as a major functional difference between subtypes of inward-rectifier K+ channels.

Animals↗

PubMed1994

Frequency response of mature guinea-pig outer hair cells to stereociliary displacement.

Outer hair cells (OHC) were isolated from the apical two turns of the guinea-pig cochlea and their hair-bundle stimulated mechanically by a glass probe. In accordance with in vivo data (Dallos, 1985), the resting membrane potential was typically -64 mV (N = 200). The maximum amplitudes of the receptor potentials were between 0.4 and 5.2 mV peak-to-peak, with mean of 1.5 mV +/- 0.9 mV (N = 81). The sensitivity was 0.015 mV/nm or 2 mV/deg. The frequency response of the receptor potential followed a first order low-pass filter characteristic with a corner frequency of about 63 Hz. For frequencies up to at least 1.6 kHz, the frequency response of mechanoelectrical transduction was dominated by the electrical input impedance of the cell. The presence of a single time constant in the voltage response to stereociliary deflection implies that the frequency response of mechanoelectrical transduction far exceeds that of the electrical input impedance of the cell; its time constant must be faster than 100 microseconds. Under in vivo conditions, OHC should be capable of providing a sufficiently large receptor potential to supply enough energy for electromechanical feedback.

Acoustic Stimulation↗

PubMed1994

Short antisense oligonucleotide-mediated inhibition is strongly dependent on oligo length and concentration but almost independent of location of the target sequence.

The inhibitory effect of short antisense oligodeoxynucleotides (aODNs) on cRNA expression in Xenopus oocytes was measured using an electrophysiological assay based on subunit-specific block of cloned alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate receptors. The effect of both phosphorothioate-modified (PS) and phosphodiester (PO) aODNs was strongly length dependent with a half-maximal inhibition calculated for an oligo length of 7.6 nucleotides (nt) and 9.9 nt, respectively. More than 95% inhibition was mediated by a PS aODN of 12 nt and by PO aODNs > or = 15 nt. At a given length PS and PO aODNs showed differential dependence of their inhibitory effect on the injected aODN concentration (half-maximal inhibition at 18 ng/microliter for a PO 12-mer and at 0.19 ng/microliter for a PS 12-mer) and differential saturation behavior. The inhibitory effect of aODNs, even as short as 8 nt for PS oligomers, was highly sequence specific, but almost independent of the position of the respective target site on the cRNA (for PS 8-mers, > or = 70% expression inhibition throughout the tested target sites from the translation initiation to the 3'-untranslated region). Thus, short PS aODNs can be reliably used in order to specifically inhibit protein expression in experiments addressing physiological, molecular biological, and perhaps even therapeutical issues.

Animals↗

PubMed1994

Acute gentamicin ototoxicity in cochlear outer hair cells of the guinea pig.

The acute effects of the aminoglycoside antibiotics gentamicin on isolated cochlear outer hair cells (OHC) was investigated by whole-cell patch-clamp and measurements of the intracellular potassium level by means of the potassium-sensitive dye PBFI. In addition, the accompanying length changes of OHC are described. It could be shown that gentamicin at different concentrations reversibly induces a hyperpolarization by about 5-10 mV, potassium outflow from the cytoplasm (by about 22 mM) and a cellular elongation (by about 10%). It is suggested that these effects are the result of an interaction between gentamicin and the cochlear transduction channels in OHC as suggested earlier. These acute effects are distinctly different from the chronic gentamicin effects which are based on the metabolization of the antibiotics to cause the death of the OHC by interaction with the phosphoinositide signalling cascade.

Animals↗

PubMed1994

Gamma-aminobutyric acidA-receptor messenger ribonucleic acid (alpha-1 subunit) detection by in situ hybridization.

The inhibitory neurotransmitter gamma-aminobutyric acid (GABA) is most likely involved in the efferent cochlear neurotransmission. In situ hybridization (ISH) results in specific annealing of a labelled nucleic acid probe to complementary sequences in fixed tissue and allows subsequent visualization of the location of the probe. We used the ISH technique to localize messenger ribonucleic acid (mRNA) sequences of the alpha-1 subunit of the GABAA receptor with an S-35 labeled oligonucleotide probe. Experiments were performed in rat and guinea pig brain sections and surface tissue preparations of the guinea pig cochlea. Positive signals were obtained for the alpha-1 probe in cortical and hippocampal regions of the rat brain and had weaker expression in the guinea pig brain. Alpha-1 subunit mRNA was localized in Purkinje cells and in stellate and basket cells of the stratum moleculare in the rat and guinea pig cerebellum. In surface tissue preparations of the guinea pig cochlea mRNA sequences of the alpha-1 subunit were detectable with high signal expression. Positive signals were seen on both sides of the tunnel of Corti, predominantly in the region of the outer hair cells. The results indicate expression of GABAA-receptor mRNA in cochlear tissue, supporting the importance of GABAA receptors in cochlear neurotransmission.

Animals↗

PubMed1994

Specific glutathione-SH inhibition of toxic effects of metabolized gentamicin on isolated guinea pig hair cells.

The present investigation has shown that only metabolized gentamicin (mG) but not native gentamicin (G) is cytotoxic for isolated guinea pig outer hair cells (OHC). Using FURA-2 fluorescence, both G and mG were found to reduce intracellular free Ca2+ concentrations in unstimulated OHC and inhibit increases in intracellular Ca2+ concentrations during K(+)-induced depolarization. Glutathione-SH (GSH), a detoxificating agent, did not interfere with G and mG effects on intracellular Ca2+ concentration. In non-stimulated OHC, mG but not G induced pathological OHC depolarization, indicating the opening of transduction channels to allow influx of K+ ions. GSH completely inhibited the lytic effect of mG. Electrophysiological investigations also revealed that GSH probably inhibits mG-induced pathological opening of transduction channels. These results suggest that GSH selectively inhibits mG-specific toxic effects on the guinea pig OHC, possibly by enzymatic detoxification.

Animals↗