Federico Kalinec

A Cochlear Cell Line as an in vitro System for Drug Ototoxicity Screening

Aminoglycoside antibiotics, loop diuretics, antineoplastic agents and other commonly used pharmacological drugs are ototoxic. Understanding of the cellular and molecular mechanisms underlying drug ototoxicity, however, has been hampered by the limited availability of inner ear tissues and drug side effects on laboratory animals. Immortalized cell lines derived from the auditory sensory organ, sensitive to ototoxic drugs and growing in environments that can be systematically manipulated, would facilitate the research directed at elucidating these mechanisms. Such immortalized cell lines could also be used to discover novel therapeutic agents for preventing drug-induced sensorineural hearing loss. Here, we report a conditionally immortalized organ of Corti-derived epithelial cell line, which shows evidence of activation of apoptosis when exposed to known ototoxic drugs. This cell line may be an excellent in vitro system to investigate the cellular and molecular mechanisms involved in ototoxicity and for screening of the potential ototoxicity or otoprotective properties of new pharmacological drugs.

Inhibition of outer hair cell electromotility by sulfhydryl specific reagents

Mammalian outer hair cells can change length at acoustic frequencies when they are electrically stimulated. It was postulated that these length changes depend on electromechanical transduction based on voltage dependent conformational changes in a membrane motor protein. In this report, we describe the effect of various sulfhydryl (SH)-specific reagents on the OHC electromotility. p-Chloromercuriphenylsulfonate (pCMPS), in addition to other mercurials that can react with well-protected SH-groups in proteins, inhibits this electromechanical transduction process. In contrast, N-ethylmaleimide and diamide, SH-reagents that only react with exposed SH-groups, showed no effect. These results suggest that one or more reactive SH-groups are present in a functionally important and protected region of the electromechanical transduction protein. Such reactivity can be utilized to identify and characterize this novel membrane motor.

Immunolocalization of anion exchanger 2α in auditory sensory hair cells

We have previously reported the isolation from a guinea pig organ of Corti cDNA library of a cDNA clone that encodes a novel isoform of the anion exchanger 2 (AE2) protein (Negrini, Rivolta, Kalinec and Kachar, 1995. Cloning of an organ of Corti anion exchanger 2 isoform with a truncated C-terminal domain. Biophys. Acta, 1236, 207–211). The deduced protein, named AE2α, has a conserved cytoplasmic domain and a short membrane domain with only two membrane spanning regions, as opposed to the fourteen present in the conventional AE2. Now, we are showing the immunolocalization and preliminary characterization of this protein using an antipeptide antibody specific for this novel AE2 isoform. In Western blots, this antibody binds to an ∼89 kDa polypeptide that corresponds to a phosphorylated protein with serines as main phosphate acceptor residues. In immunofluorescence experiments, the antibody labels the stereocilia and the lateral wall of the outer hair cells and the stereocilia of the inner hair cells. Our results suggest that AE2α is a membrane-cytoskeletal linker in regions of the hair cell, where sensory transduction mechanisms take place.

Cloning of an organ of Corti anion exchanger 2 isoform with a truncated C-terminal domain☆

We have isolated a cDNA clone from a guinea pig organ of Corti library encoding a new isoform of the Anion Exchanger 2 (AE2) protein. This cDNA clone shows an 83 bp deletion in the region that encodes the membrane domain of AE2. Analysis of the overlapping regions of genomic and cDNA clones indicates that the missing portion does not correspond exactly to a constitutive exon. The alternate splicing process that generates this transcript involves internal donor and acceptor sites which introduces a shift in the open reading frame. The resulting polypeptide has a conserved cytoplasmic N-terminal domain but the membrane C-terminal domain has only two of the fourteen membrane spanning regions. An affinity-purified antipeptide antibody to the novel C-terminus detects an 89 kDa polypeptide which agrees with the molecular mass predicted from the cDNA.

Working with Auditory HEI-OC1 Cells

HEI-OC1 is one of the few mouse auditory cell lines available for research purposes. Originally proposed as an in vitro system for screening of ototoxic drugs, these cells have been used to investigate drug-activated apoptotic pathways, autophagy, senescence, mechanism of cell protection, inflammatory responses, cell differentiation, genetic and epigenetic effects of pharmacological drugs, effects of hypoxia, oxidative and endoplasmic reticulum stress, and expression of molecular channels and receptors. Among other several important markers of cochlear hair cells, HEI-OC1 cells endogenously express prestin, the paradigmatic motor protein of outer hair cells. Thus, they can be very useful to elucidate novel functional aspects of this important auditory protein. HEI-OC1 cells are very robust, and their culture usually does not present big complications. However, they require some special conditions such as avoiding the use of common anti-bacterial cocktails containing streptomycin or other antibiotics as well as incubation at 33 °C to stimulate cell proliferation and incubation at 39 °C to trigger cell differentiation. Here, we describe how to culture HEI-OC1 cells and how to use them in some typical assays, such as cell proliferation, viability, death, autophagy and senescence, as well as how to perform patch-clamp and non-linear capacitance measurements.