ABSTRACT
Title
Cochlear cells: P2X7 receptor is a candidate for regulation of glutamate release
Authors
M.C. Mazzotta1, S. Alloisio2, C. Cervetto1, R. Barbieri2, D. Frattaroli1, M. Marcoli1,3, G. Maura1,3,4, M. Nobile2
1Department of Experimental Medicine, Section of Pharmacology and Toxicology, University of Genoa, Genoa, Italy; 2Institute of Biophysics, National Research Council, Genoa, Italy; 3Center of Excellence for Biomedical Research, University of Genoa, Genoa, Italy; and 4National Institute of Neuroscience, INN, Turin,Italy
1Department of Experimental Medicine, Section of Pharmacology and Toxicology, University of Genoa, Genoa, Italy; 2Institute of Biophysics, National Research Council, Genoa, Italy; 3Center of Excellence for Biomedical Research, University of Genoa, Genoa, Italy; and 4National Institute of Neuroscience, INN, Turin,Italy
Abstract
Purinergic signalling represents an important paracrine and autocrine form of signalling in central and peripheral nervous system, including auditory system. Among ATP-activated purinergic receptors, the P2X7 receptor (P2X7R) is structurally and functionally divergent: it possesses a bifunctional activity as a cation channel or a non-selective pore permeable to large molecules like neurotransmitters, such as glutamate, and ATP itself. This study was aimed to detect the presence and functional activity of P2X7R in HEI-OC1, a conditionally immortalized epithelial cell line derived from the mouse organ of Corti, by means of immunocytochemistry, immunoblotting, and by measuring both intracellular calcium ([Ca2+]i) response and [3H]D-aspartate release. When HEI-OC1 cells are cultured in non-permissive conditions they further differentiate, representing a suitable in vitro system to investigate on cellular and molecular mechanisms involved in hearing physiology or ototoxicity.
Briefly, conditionally immortalized mouse auditory cells, HEI-OC1, kindly provided by Dr. Federico Kalinec (House Ear Institute, Los Angeles, CA, USA), were cultured under a permissive condition (33 °C and 10% CO2) in high-glucose Dulbecco’s Eagle’s medium DMEM containing 10% fetal bovine serum FBS and 50 U/ml gamma-interferon; after 2 weeks, cells were moved to non-permissive conditions (39 °C in DMEM plus 10% FBS and 5% CO2) and allowed to differentiate for up to 60 days. Experiments were performed in both proliferating and differentiated cells. Cultures were monitored on a daily basis by phase-contrast and video microscopy. The cells at 70–80% of confluence were used for single cell [Ca2+]i microfluorimetry by using the fluorescent Ca2+ indicator fura-2 AM, for YO-PRO1 uptake experiments by fluorescence assay and for evaluation of [3H]D-aspartate uptake and of [3H]D–aspartate release in superfused parallel chambers. P2X7R expression was verified by immunocytochemistry and Western blotting.
Immunocytochemistry results showed that P2X7R was expressed in both cell conditions, whereas immunoblot analysis demonstrated a higher level of protein expression in differentiated with respect to undifferentiated HEI-OC1 cells. The P2X7R agonist, BzATP, induced only in a low percentage of undifferentiated HEI-OC1 cells a Mg2+-sensitive [Ca2+]i response with an EC50 value of 375 and 184 μM in 1mM Mg2+ and Mg2+-free extracellular saline, respectively. In the same cells, at 300 μM concentration BzATP could also induce [3H]D-aspartate release, that was only partly sensitive to the selective P2X7R antagonist A-438079.
Conversely, BzATP stimulation of differentiated HEI-OC1 cells induced a [Ca2+]i response in 100% of the cells with an EC50 value of 137 and 71 μM in 1mM Mg2+ and Mg2+-free extracellular saline, respectively. [3H]D-aspartate was taken up in differentiated cells through Na+-dependent high affinity glutamate transporters, as indicated by the effectiveness of the non substrate inhibitor of glutamate transporters DL-TBOA. In differentiated cells, BzATP potently stimulated [3H]D-aspartate release (EC50 value = 12 μM) in an extracellular Mg2+-sensitive way; the P2X7R antagonist A-438079 abolished the releasing response to BzATP. The BzATP-evoked [3H]D-aspartate release was about 50% dependent on the presence of extracellular Ca2+, compatible with an exocytotic mode of efflux for the transmitter.
In conclusion, these results suggest that P2X7R can participate to the regulation of cochlear cell [Ca2+]i and glutamate release, providing evidence for a role of extracellular ATP, acting through P2X7R, in cochlea and auditory neurotransmission. Furthermore, our resultsconfirm that differentiated HEI-OC1 cells represent a good model of cochlear hair cells.
This work was supported by Linear SrL, Genoa (Italy)
Briefly, conditionally immortalized mouse auditory cells, HEI-OC1, kindly provided by Dr. Federico Kalinec (House Ear Institute, Los Angeles, CA, USA), were cultured under a permissive condition (33 °C and 10% CO2) in high-glucose Dulbecco’s Eagle’s medium DMEM containing 10% fetal bovine serum FBS and 50 U/ml gamma-interferon; after 2 weeks, cells were moved to non-permissive conditions (39 °C in DMEM plus 10% FBS and 5% CO2) and allowed to differentiate for up to 60 days. Experiments were performed in both proliferating and differentiated cells. Cultures were monitored on a daily basis by phase-contrast and video microscopy. The cells at 70–80% of confluence were used for single cell [Ca2+]i microfluorimetry by using the fluorescent Ca2+ indicator fura-2 AM, for YO-PRO1 uptake experiments by fluorescence assay and for evaluation of [3H]D-aspartate uptake and of [3H]D–aspartate release in superfused parallel chambers. P2X7R expression was verified by immunocytochemistry and Western blotting.
Immunocytochemistry results showed that P2X7R was expressed in both cell conditions, whereas immunoblot analysis demonstrated a higher level of protein expression in differentiated with respect to undifferentiated HEI-OC1 cells. The P2X7R agonist, BzATP, induced only in a low percentage of undifferentiated HEI-OC1 cells a Mg2+-sensitive [Ca2+]i response with an EC50 value of 375 and 184 μM in 1mM Mg2+ and Mg2+-free extracellular saline, respectively. In the same cells, at 300 μM concentration BzATP could also induce [3H]D-aspartate release, that was only partly sensitive to the selective P2X7R antagonist A-438079.
Conversely, BzATP stimulation of differentiated HEI-OC1 cells induced a [Ca2+]i response in 100% of the cells with an EC50 value of 137 and 71 μM in 1mM Mg2+ and Mg2+-free extracellular saline, respectively. [3H]D-aspartate was taken up in differentiated cells through Na+-dependent high affinity glutamate transporters, as indicated by the effectiveness of the non substrate inhibitor of glutamate transporters DL-TBOA. In differentiated cells, BzATP potently stimulated [3H]D-aspartate release (EC50 value = 12 μM) in an extracellular Mg2+-sensitive way; the P2X7R antagonist A-438079 abolished the releasing response to BzATP. The BzATP-evoked [3H]D-aspartate release was about 50% dependent on the presence of extracellular Ca2+, compatible with an exocytotic mode of efflux for the transmitter.
In conclusion, these results suggest that P2X7R can participate to the regulation of cochlear cell [Ca2+]i and glutamate release, providing evidence for a role of extracellular ATP, acting through P2X7R, in cochlea and auditory neurotransmission. Furthermore, our resultsconfirm that differentiated HEI-OC1 cells represent a good model of cochlear hair cells.
This work was supported by Linear SrL, Genoa (Italy)