Establishment of variant PC12 subclones deficient in stimulation-secretion coupling.
Clonal rat pheochromocytoma (PC12) cells have been widely used to study the molecular mechanism of exocytosis. We have isolated variant PC12 subclones with deficiencies in stimulation-secretion coupling, by a single cell recloning, and investigated the defects. PC12-1G2 hardly released dopamine following high-K(+)-induced depolarization, but normal release was evoked by the Ca(2+)-ionophore, ionomycin. Fura-2 fluorometry indicated that a nicardipine-sensitive component of Ca(2+) influx was missing, suggesting that PC12-1G2 has defects in L-type Ca(2+) channel function. PC12-2B3 was not responsive to high-K(+)-induced depolarization and ionomycin, and voltage-dependent Ca(2+) entry was identical to that of the normal clone. Electron microscopy revealed that the number of vesicles adjacent or directly attached to the plasma membrane was decreased in PC12-2B3. The expression of presynaptic proteins was analyzed by immunoblotting using a panel of antibodies. Syntaxin 1, VAMP-2, SNAP-25, Munc18, Rab3C and Sec-6 were decreased compared to the control clone and that of synaptophysin was extremely low. PC12-D60 synthesized and released dopamine normally, but had almost lost its catecholamine-uptake activity. These results show that multiple PC12 cells variants are spontaneously generated, and that recloning can select PC12 subclones useful for the study of the molecular mechanisms of neurotransmitter release.
Shoji-Kasai
Y
Y
Mitsubishi Kasai Institute of Life Sciences, Tokyo, Japan. kasai@libra.ls.m-kagaku.co.jp
Morishima
M
M
Kuwahara
R
R
Kondo
S
S
Itakura
M
M
Takahashi
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002999:Clone Cells; D017931:DNA Primers / Q000235:genetics; D004298:Dopamine / Q000378:metabolism; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D015870:Gene Expression; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D008564:Membrane Potentials; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D018377:Neurotransmitter Agents / Q000378:metabolism*; D016716:PC12 Cells; D011188:Potassium / Q000494:pharmacology; D051381:Rats
D017931:DNA Primers; D007476:Ionophores; D009419:Nerve Tissue Proteins; D018377:Neurotransmitter Agents; D015759:Ionomycin; D011188:Potassium; D002118:Calcium; D004298:Dopamine