Role of L-type Ca2+ channels in neural stem/progenitor cell differentiation.
Ca(2+) influx through voltage-gated Ca(2+) channels, especially the L-type (Ca(v)1), activates downstream signaling to the nucleus that affects gene expression and, consequently, cell fate. We hypothesized that these Ca(2+) signals may also influence the neuronal differentiation of neural stem/progenitor cells (NSCs) derived from the brain cortex of postnatal mice. We first studied Ca(2+) transients induced by membrane depolarization in Fluo 4-AM-loaded NSCs using confocal microscopy. Undifferentiated cells (nestin(+)) exhibited no detectable Ca(2+) signals whereas, during 12 days of fetal bovine serum-induced differentiation, neurons (beta-III-tubulin(+)/MAP2(+)) displayed time-dependent increases in intracellular Ca(2+) transients, with DeltaF/F ratios ranging from 0.4 on day 3 to 3.3 on day 12. Patch-clamp experiments revealed similar correlation between NSC differentiation and macroscopic Ba(2+) current density. These currents were markedly reduced (-77%) by Ca(v)1 channel blockade with 5 microm nifedipine. To determine the influence of Ca(v)1-mediated Ca(2+) influx on NSC differentiation, cells were cultured in differentiative medium with either nifedipine (5 microm) or the L-channel activator Bay K 8644 (10 microm). The latter treatment significantly increased the percentage of beta-III-tubulin(+)/MAP2(+) cells whereas nifedipine produced opposite effects. Pretreatment with nifedipine also inhibited the functional maturation of neurons, which responded to membrane depolarization with weak Ca(2+) signals. Conversely, Bay K 8644 pretreatment significantly enhanced the percentage of responsive cells and the amplitudes of Ca(2+) transients. These data suggest that NSC differentiation is strongly correlated with the expression of voltage-gated Ca(2+) channels, especially the Ca(v)1, and that Ca(2+) influx through these channels plays a key role in promoting neuronal differentiation.
D'Ascenzo
Marcello
M
Institute of Human Physiology, Medical School, Catholic University S. Cuore, Largo F. Vito 1, I-00168 Rome, Italy.
Piacentini
Roberto
R
Casalbore
Patrizia
P
Budoni
Manuela
M
Pallini
Roberto
R
Azzena
Gian Battista
GB
Grassi
Claudio
C
D000814:Aniline Compounds / Q000378:metabolism; D000818:Animals; D000831:Animals, Newborn; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020746:Calcium Channels, L-Type / Q000502:physiology*; D020013:Calcium Signaling / Q000502:physiology; D002452:Cell Count / Q000379:methods; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology*; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D007150:Immunohistochemistry / Q000379:methods; D007381:Intermediate Filament Proteins / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism; D064231:Nestin; D009474:Neurons / Q000166:cytology*; D009543:Nifedipine / Q000494:pharmacology; D018408:Patch-Clamp Techniques / Q000379:methods; D011189:Potassium Chloride / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D053422:Receptor-Interacting Protein Serine-Threonine Kinases; D026941:Sodium Channel Blockers / Q000494:pharmacology; D013234:Stem Cells / Q000502:physiology*; D013779:Tetrodotoxin / Q000494:pharmacology; D013997:Time Factors; D014404:Tubulin / Q000378:metabolism; D047988:Tumor Necrosis Factor Receptor-Associated Peptides and Proteins / Q000378:metabolism; D014966:Xanthenes / Q000378:metabolism
D000814:Aniline Compounds; D002121:Calcium Channel Blockers; D020746:Calcium Channels, L-Type; C409648:Fluo 4; D005904:Glial Fibrillary Acidic Protein; D007381:Intermediate Filament Proteins; D008869:Microtubule-Associated Proteins; C079240:Mtap2 protein, mouse; C577484:NES protein, human; D009419:Nerve Tissue Proteins; C577485:Nes protein, mouse; D064231:Nestin; D026941:Sodium Channel Blockers; D014404:Tubulin; D047988:Tumor Necrosis Factor Receptor-Associated Peptides and Proteins; D014966:Xanthenes; D013779:Tetrodotoxin; D011189:Potassium Chloride; D017346:Protein Serine-Threonine Kinases; D053422:Receptor-Interacting Protein Serine-Threonine Kinases; C506251:Ripk1 protein, mouse; D009543:Nifedipine