Calyculin-A-induced fast neurite retraction in nerve growth factor-differentiated rat pheochromocytoma (PC12) cells

Rat pheochromocytoma (PC12) cells were treated with nerve growth factor (NGF) for 3-4 days. They formed growth cones and extended neurites. Addition of the phosphatase inhibitor calyculin A (CL-A) caused a concentration-dependent complete retraction of neurites within 15 min. Retraction of growth cones started with the filopodia still present. The cell bodies acquired a grape-like shape opposite to the cell nucleus. These morphological changes were reversible. After washout of the inhibitor, the cell bodies recovered to normal shape within about 30-60 min while neurites started to grow again within 1 day. Okadaic acid (OA) which, compared to CL-A, is less potent as a PP-1 and equally potent as a PP-2A class inhibitor, caused neurite retraction only when added at more than a thousand-fold higher concentration than CL-A. Ca2+ levels within neurites and cell bodies remained stable and low during neurite retraction as measured with fura-2. However, cells treated with CL-A showed reduced activity of voltage-gated Ca2+ channels. The results suggest that the observed reversible changes in cell morphology occur at a constant low Ca2+ level and are most likely due to the inhibition of PP-1 class phosphatases.