Mechanisms of spontaneous cytosolic Ca2+ transients in differentiated human neuronal cells

Authors


Correspondence: B. A. Wolf. E-mail: wolfb@mail.med.upenn.edu

Abstract

We have studied Ca2+ homeostasis in a unique model of human neurons, the NT2N cell, which differentiates from a human teratocarcinoma cell line, NTera2/C1.D1 by retinoic acid treatment. When perifused with Krebs–HEPES buffer containing 2.5 mm CaCl2, fura-2 loaded NT2N cells produced spontaneous cytosolic Ca2+ oscillations, or Ca2+ transients. These cytosolic Ca2+ transients were not blocked by antagonists of glutamate (6-cyano-7-nitroquinoxaline-2,3-dione and d(–)-2-amino-5-phosphonopentanoic acid) or muscarinic (atropine) receptors. Omission of extracellular Ca2+ completely abolished Ca2+ oscillations and decreased the average Ca2+ level from 106 ± 14 nm to 59 ± 8 nm. Addition of the L-type Ca2+ channel blocker nifedipine (1 or 10 μm) or of the N-type inhibitor ω-conotoxin GVIA (5 μm) significantly, although incompletely, suppressed Ca2+ oscillations, while ω-conotoxin MVIIC (5 μm), a selective antagonist of P- and Q-channels, had no effect. Ni2+, at 100 μm, a concentration selective for T-type channels, did not inhibit Ca2+ transients. Non-specific blockage of Ca2+ channels by higher concentrations of Ni2+ (2–5 mm) or Co2+ (1 mm) abolished Ca2+ oscillations completely. The endoplasmic reticulum Ca2+-ATPase inhibitor, thapsigargin (1 μm), slightly decreased Ca2+ oscillation frequency, and induced a small transitory increase in the average cytosolic Ca2+ concentration. The mRNAs of L- (α1D subunit) and N-type (α1B subunit) Ca2+ channel were present in NT2N cells, while that of a T-type Ca2+ channel (α1-subunit) was not present in the NT2N cells as shown by reverse transcription–polymerase chain reaction. In conclusion, NT2N neuronal cells generate cytosolic Ca2+ oscillations mainly by influx of extracellular Ca2+ through multiple channels, which include L- and N-type channels, and do not require activation of glutamate or muscarinic receptors.

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