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Isolation and characterization by cell density adjustment of a PC12 pheochromocytoma variant with altered Ca2+
Abstract:
Rat PC12 pheochromocytoma cells loaded with the fluorescent Ca2+ dye fluo-3 or indo-1 and scanned fluorimetrically on a cell sorter apparatus showed a rapid cell density-dependent increase in free cytosolic calcium concentration [Ca2+]i when maintained in suspension cultures. Cell adhesion, measured under a defined set of conditions, was low when cells were seeded at 1.5 x 10(4) cells/ml but reached maximal levels after addition of A23187 calcium ionophore. A six to sevenfold increase in cell density mimicked the effect of the ionophore. Densities above 2 x 10(6) cells/ml caused a decrease in cell adhesion, which was further reduced by the addition of A23187. BAPTA, AM (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) and nifedipine (10 microM each), partially inhibited cell attachment (34% and 44% reduction), but at 0.25 microM and 1 microM, respectively, they enhanced attachment (46% and 67% increase). The data suggest that a certain permissive level of [Ca2+]i, attained by either increasing cell density or by the presence of a calcium ionophore, is sufficient for maximal cell adhesion. Above the permissive level, manipulation of [Ca2+]i either by altering cell density or by the addition of calcium blocking agents in high concentrations results in a significant reduction in cell adhesion. Based on these observations, we were able to isolate a biochemically and morphologically distinct cell population. The variant, designated PC12ds (density selected), differed substantially from the original cells. Most notable was a relatively lower content of free [Ca2+]i in the PC12ds cells, as independently assayed by using fluo-3 and indo-1 dyes. In addition, the variant cells exhibited a significantly diminished rate of 45Ca2+ uptake, most likely due to less efficient L-type voltage-dependent calcium (VDC) channels. Addition of several calcium channels agonists and antagonists suggested that PC12ds cells contained relatively more N-type VDC channels, possibly indicating a shift to a neuronal phenotype.