| The mammalian organ of Corti is comprised of sensory (inner hair cells and outer hair cells) and supporting cells (e.g., Hensen, Deiter, Pillar cells). Hensen cells in the cochlea maintain the homeostasis of the organ of Corti and contribute to the electrical and micromechanical environment of the hair cells. The physiologic source of ATP in the organ of Corti remains open. Extracellular application of ATP at concentrations of 10 M evoked a rapidly activating inward current in all Hensen cells. The effects of ATP on electrophysiological properties in Hensen Cells from the guinea pig cochlea and the physiologic source of rise in [Ca2+]i aren't systematically investigated. Calcium ions are involved in noise induced hearing loss. Nifedipine can prophylaxis noise induced hearing loss partly. In the long term, nifedipine could ameliorate microcirculation of guinea-cochlea and block L-type calcium channel to prophylaxis noise induced hearing loss.My expriment intends to study ion channels and their properties in isolated Hensen cells and observe effects of ATP and nifedipine on ion currents through the ion channels.My expriment's objectives are (1)To probe the method of isolated Hensen cells for patch-clamp technique(2)To study potassium currents and their properties in isolated Hensen cells and observe effects of ATP on ion currents(3)To study an inward current evoked by ATP in isolated Hensen cells and observe effects of nifedipine on ion currents.Pigmented guinea pigs with a positive Preyer's reflex were killed. The cochleae were then taken out and the bony shell of the cochlea were gently removed. The organ of Corti were treated using enzyme, and then dissociated. Isolated Hensen cells were identified by their characteristic lipid inclusions, andthen settled on Poly-L-lysine coated coverslips. The whole cell patch-clamp was used to record ion currents in Hensen cells. Drugs were delivered to the cell by a micro-manifold consisting made by three 100 M diameter microtubules. The results were as follows:1 .the method of isolated Hensen cells for patch-clamp technique: The number of good-activity isolated Hensen cells was 12 + 3. Good-activity Hensen cells have integrated border, transparent birefringence phenomenon, anhydr-edema, translucent intracellular cytoplasm, round shape, dimension discrepancy, and no nucleus. They contain one or several round lipid droplet, which have integrated border, strong interception.2. The outward K+ currents of isolated Hensen cells:The outward K+ currents were delayed rectified potassium currents (7K), not rapidly inactive, 4-AP-sensitive potassium currents (IA). It can be blocked by CsCl (140mmol/L) and TEA can inhibit outward ion currents.3. In low concentrations (0.1 mol/L, 1 mol/L, 10 mol/L) , the amplitude of potassium current was reduced by ATP .4.An inward current evoked by ATP in high concentrations:In high concentrations (100 mol/L,1mmol/L,10mmol/L) , ATP evoked an inward dose-dependence current with. It was reversed by the purinergic antagonist (100 mol/L suramin) .5. Nifedipine inhibited inward current evoked by ATPIn high concentrations (1mmol/L) , ATP evoked an inward current ,which could be inhibited by nifedipine (10 mol/L) .We mainly discussed Hensen cells for patch clamp; Ion currents can be recorded by Axopachl-D patch clamp; The relation between outward potassium current and EP; potassium current reduced by ATP in low concentrations; an inward current evoked by ATP in high concentrations; The relation between theinward current inhibited by nifedipine and prophylaxising noise induced hearing loss.The results indicated that:(1)Isolated Hensen cells for patch clamp can be obtained by proper enzyme concentration and petri dish treated poly-lysine. It can meet the need of patch clamp technique for voltage-gated channel and chemically gated channel.(2)Outward potassium current of Hensen cell take part in potassium circle and balance in the cochlea;(3)The outward potassium current of Hensen cell take part... |
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