Retinoic acid induction of calcium channel expression in human NT2N neurons.
Ca2+ channel expression and regulation of intracellular Ca2+ homeostasis were studied during retinoic acid (RA)-induced differentiation of the human teratocarcinoma cell line Ntera 2/C1.D1 (NT2- cells) into NT2N neurons, a unique model of human neurons in culture. The cytosolic Ca2+ level of undifferentiated NT2- cells was low (75 +/- 5 nM) and stable under basal conditions, and it was only marginally decreased (by 9%) upon removal of extracellular Ca2+. After 10 microM RA treatment, NT2- cells were irreversibly differentiated into a phenotype of neuron-like NT2N cells. Cytosolic Ca2+ level of NT2N neurons was higher (106 +/- 14 nM) than that of NT2- cells and spontaneously fluctuated (0.208 +/- 0.038 transients/min) under basal conditions. Although K+ increased 86Rb fluxes in both NT2- cells and NT2N neurons, it only increased cytosolic Ca2+ level in NT2N neurons. The K+-induced increase in cytosolic Ca2+ in NT2N neurons was antagonized by 0.1-10 microM nifedipine or verapamil, 5 microM omega-CgTx GVIA, but not by 1 microM omega-agatoxin IVA, 1 microM omega-agatoxin TK, 1 microM FTX-3.3, or 100 microM Ni+ implicating L- and N-type voltage-dependent Ca2+ channels. In L- and N-type channels, but not in P- and Q-types, mRNAs were expressed in NT2N neurons as well as NT2- cells. Quantitative analysis of L- and N-type Ca2+ protein levels showed major differences between NT2- cells and NT2N neurons. In NT2- cells, N-type Ca2+ channels were undetectable while L-type channels levels were fivefold lower compared to NT2N neurons. Our findings show that L- and N-type channels are expressed during differentiation of NT2- cells into neurons, and that these voltage-dependent Ca2+ channels have a major role in regulating intracellular Ca2+ homeostasis and neuronal excitability.
Gao
Z Y
ZY
Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, 19104, USA.
Xu
G
G
Stwora-Wojczyk
M M
MM
Matschinsky
F M
FM
Lee
V M
VM
Wolf
B A
BA
D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D017931:DNA Primers / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects; D006706:Homeostasis; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D009543:Nifedipine / Q000494:pharmacology; D010641:Phenotype; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014212:Tretinoin / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D014700:Verapamil / Q000494:pharmacology
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D017931:DNA Primers; D012333:RNA, Messenger; D014212:Tretinoin; D014700:Verapamil; D009543:Nifedipine; D002118:Calcium