OBJECTIVETo investigate the effect of nifedipine on the non-selective inward current of cochlear Hensen cell induced by ATP in high concentrations.

METHODSThe organ of Corti was treated using enzyme, and then dissociated mechanically to isolate Hensen cells. The whole cell patch-clamp technique was used to record ion currents in Hensen cells which had integrated border, round shape and translucent intracellular cytoplasm. Drugs were delivered to the cell by a micro-manifold consisting made by three 100 microm diameter microtubules, including 0.1 mmol/L ATP, 1 mmol/L ATP, 10 mmol/L ATP, 0.1 mmol/L ATP + 0. 1 mmol/L suramin (purinergic antagonist), stimulation of extracellular fluid alone, 140 mmol/L CsCl (replace KCL in intracellular fluid) + 1 mmol/L ATP, 40 mmol/L TEA (blocker of potassium channel) + 1 mmol/L ATP, and 1 mmol/L ATP + 10 micromol/L nifedipine, respectively.

RESULTSWhen isolated Hensen cell was given 0.1 mmol/L (n = 10), 1 mmol/L (n = 10), 10 mmol/L( n = 6) ATP separately, an inward ion current could be recorded, which enhanced with increased ATP concentration and showed dose-dependence. Further study indicated that the inward ion current could be inhibited by 0.1 mmol/L suramin (n = 5), 140 mmol/L CsCl (n = 5) and 40 mmol/L TEA (n = 5). There was no ion current be recorded when the cell was stimulated with the extracellular fluid alone, neither inward nor outward. However, the inward ion current vanished and an outward ion current appeared instead, when 1 mmol/L ATP and 10 micromol/L nifedipine were given together (n = 5).

CONCLUSIONSAn inward current was evoked in isolated Hensen cell by ATP in high concentrations. This inward current seems to be associated closely with potassium channels without the participation of mechanical channels. Nifedipine can inhibit this inward current and induce an outward current, which is similar to the normal potassium current in isolated Hensen cell. It suggests that nifedipine have partly protective effect on the function of cochlea by inducing modulate of the potassium circle of cochlea in Hensen cell's tache.

Adenosine Triphosphate ; pharmacology ; Animals ; Cochlea ; cytology ; Female ; Guinea Pigs ; Labyrinth Supporting Cells ; drug effects ; metabolism ; Male ; Nifedipine ; pharmacology ; Patch-Clamp Techniques ; Potassium Channels, Inwardly Rectifying ; drug effects

BACKGROUND AND OBJECTIVES: The supporting cells in the organ of Corti help to maintain the structural integrity of the organ, but it has been suggested that they also actively participate in regulating sound transduction. The existence of neural control was implied by the finding of efferent synapses in Deiters' cells, and the fact that the intracellular Ca2+ concentration was increased by the application of neurotransmitters, such as ATP (adenosine triphosphate) and Ach (acetylcholine), resulting in movement of the phalangeal processes of the Deiters' cells. This study investigated the effects of neurotransmitters on the ion channels in Deiters' cells. MATERIALS AND METHOD: Deiters' cells were isolated from guinea pig organs of Corti using collagenase and pipettes. Whole-cell patch clamps were performed under an inverted microscope and the current was measured with pClamp 8.0.2 software. RESULTS: The resting membrane potential was -21.1+/-3.5 mV. ATP (100 microM) treatment depolarized the potential to -3.1+/-1.1 mV, while the same concentration of Ach had no effect on the resting potential. In the voltage-clamping condition, the holding potential was 0 mV, and then a -80 mV pre-pulse was applied for 500 ms, followed by step pulses from -140 to +10 mV. Under these conditions, 10 microM ATP increased the inward current from -14.9+/-1.9 to -163.5+/-14.9 pA/pF at the maximal stimulus of -140 mV (n=4). In the current-voltage curve, the reversal potential was around -20 mV. Neither Ach nor carbachol induced current responses. The co-application of suramin (30 microM) and ATP (10 microM) suppressed the ATP-induced currents by 50%, and 30 microM of PPADS (pyridoxal-phosphate- 6-azophenyl-2, 4-disulphonic acid) inhibited the current almost to the level of the control. The purinoceptor-agonist, alpha, beta-meATP (alpha, beta-methylene adenosine triphosphate), 30 microM increased the inward current from -16.2+/-2.9 to -27.7+/-3.8 pA/pF, which was much smaller than the ATP-induced change. CONCLUSION: ATP-gated purinergic receptors may play an important role in regulating sound transduction by inducing an inward current and depolarizing the Deiters' cell membrane.
Adenosine ; Adenosine Triphosphate ; Animals ; Carbachol ; Cell Membrane ; Cochlea* ; Collagenases ; Guinea Pigs* ; Guinea* ; Ion Channels* ; Labyrinth Supporting Cells ; Membrane Potentials ; Neurotransmitter Agents* ; Organ of Corti ; Receptors, Purinergic ; Suramin ; Synapses

OBJECTIVE: To explore whether the Ad5-atoh1/EGFP could transdifferent the supporting cells into the new hair cells in young adult guinea pigs cochlea in vivo. METHOD: Twelve healthy pigmented guinea pigs weighted 200-250 g were included in this experiment. 5 ul of Ad5-E1/E3 defected-atoh1/EGFP were infused into the scala media through a hole made on the lateral wall of the cochlea. Six of the 12 animal were killed 2 weeks after the infusion operation. The others were killed 4 weeks after the operation. The whole mount of the basal membranes were directly observed under the fluorescence microscope for the expression of the EGFP (enhance green fluorescent protein) or for the expression of the hair cellspecific marker and nuclear after staining with myosin VIIa rabbit polyclonal antibody and Dapi dye. RESULT: New cells with big nuclear, ellipse body and expressed with EGFP were found in the region near to the outmost row of the outer hair cells in 2 animal 2 weeks after the infusion. Moreover there were 3 animals with specific morphologic new cells in the location where ever been located by the outer hair cells and the region as 2 weeks animals 4 weeks after the infusion. Those cells were stained by myosin VIIa antibody. CONCLUSION Atoh1 gene could transdifferent some supporting cells in the basal membrane into hair cell like cells in young adult guinea pigs in vivo. These supporting cells locate in the region of outer hair cells and the basal membrane which do not belong to the region of outer hair cells.
Animals ; Basic Helix-Loop-Helix Transcription Factors ; genetics ; metabolism ; Cell Transdifferentiation ; genetics ; Cochlea ; cytology ; Ear, Inner ; Green Fluorescent Proteins ; genetics ; metabolism ; Guinea Pigs ; Hair Cells, Auditory ; cytology ; Labyrinth Supporting Cells ; cytology

BACKGROUND AND OBJECTIVES: The Deiters' cell is one of the supporting cells in the organ of Corti and is known to possibly regulate the signal transduction pathway in the organ of Corti. The signal transduction process can be modulated by ATP and acetylcholine, the so-called neurotransmitters, in Deiters' cells. Intracellular Ca2+ concentration can be also increased by these neurotransmitters and the control mechanism on the organ of Corti is highly suggested in Deiters' cells. Potassium ion (K+) is known to be important both in hair cells and supporting cells. Through K+ channel, the membrane potential may be controlled and the signal transduction pathway can be regulated. Furthermore, the motility of outer hair cell and the signal transduction from the apical stereocilia are considered to be regulated by this channel. The aim of this study is to record the K+ current in the isolated Deiters' cells from guinea pig cochlea. MATERIALS AND METHODS: Deiters' cells were isolated from the organ of Corti of guinea pig by using collagenase and a pipet. A whole cell patch clamp was performed under the inverted microscope and the current was measured with List-7 amplifier and pClamp 8.0.2 software. RESULTS: The resting membrane potential was -15.02+/-2.66 mV (n=6). When the cell membrane was hyperpolarized into -110 mV from the -40 mV holding potential, the peak current was -227+/-39.9 pA (n=15). After having depolarized to the maximum, (50 mV), the peak current was 7123+/-737 pA, and the reversal potentials of different external K+ concentration changed in the K+-dependent manner. About 80% of this current was inhibited by TEA. When K+ was substituted by Cs+, the peak current was 1788+/-231 pA at 50 mV step pulse. Activation curve of this outward current showed two different Vh (half activation voltage) and K (slope factor). CONCLUSION: Outward rectifying K+ channels exist in Deiters' cells and they can be inhibited by TEA and permeable to Cs+. More than two types of K+ current can exist and they may play a role in the recovery of membrane potential after depolarization,
Acetylcholine ; Adenosine Triphosphate ; Animals ; Cell Membrane ; Cochlea* ; Collagenases ; Guinea Pigs* ; Guinea* ; Hair ; Labyrinth Supporting Cells ; Membrane Potentials ; Neurotransmitter Agents ; Organ of Corti ; Patch-Clamp Techniques ; Potassium ; Potassium Channels ; Signal Transduction ; Stereocilia ; Tea