ATP-INDUCED CYTOPLASMIC [CA2+]] INCREASES IN ISOLATED COCHLEAR OUTER HAIR-CELLS - INVOLVED RECEPTOR AND CHANNEL MECHANISMS

Outer hair cells (OHC) of the mammalian cochlea are thought to preprocess the sound signal by active movements, which can be induced by electrical or chemical stimulation, e.g. depolarization evoked by high [K+] or increased cytoplasmic [Ca2+]. Extracellular ATP has been found to induce cytoplasmic [Ca2+] increases in OHC but involved mechanisms have not been elucidated. Cytoplasmic [Ca2+] was measured in non-enzymatically isolated single OHC using Fura-2 microspectrometry. Results, using ATP/derivatives and other P-2-purinergic receptor (P(2)R) ligands, as well as Ca2+-channel blockers and pertussis toxin, revealed several signal transduction; pathways that increase cytoplasmic [Ca2+] in OHC: a P-2-purinergic receptor (P(2)R) G-protein - effector (phospholipase C or an ion channel) system and a voltage-dependent Ca2+ channel. Agonist potency studies denote a pattern analogous to that found in skeletal muscle, i.e. ATP-alpha-S > ATP = 2-methyl-S-ATP much greater than ADP > alpha,beta-methylene-ATP, but no activation by ADP beta F or UTP, leaving a choice of P-2y or P(2z)R subtypes. The latter possibility gained strength from calculations showing that up to 8% of ATP may have formed the P(2z)R agonist ATP(4-) in the experimental medium. Experiments in Ca2+-free medium and with pertussis toxin revealed that the main Ca2+ source was intracellular. Pertussis toxin did not affect [Ca2+] increase induced by carbachoI. Acetylcholine, administered a few seconds before ATP, did not affect total cytoplasmic [Ca2+] increases. Induced cytoplasmic [Ca2+] increases were high enough (> 500 nM at 50 mu M ATP/derivatives) to hyperpolarize the OHC membrane by opening K+-channels and decreased little with time. Artifacts may have been caused by the sustained Ca2+ levels, e.g. activation of proteases by the high cytoplasmic [Ca2+]. Similar events in vivo may have pathological consequences.