Adrenergic differentiation and SSR2a receptor expression in CAD-cells cultured in serum-free medium.

The CAD cell line originates from catecholaminergic neurons in the central nervous system (CNS) of a simian virus large T antigen transgenic mouse. In the present study, we have immunohistochemically characterized the cell line after differentiation in serum-free medium, using immunofluorescence in combination with confocal laser scanning microscopy (CLSM), immunoblot, and ribonuclease protection assay (RPA). Tyrosine hydroxylase (TH)-, phenylethanolamine-N-methyltransferase (PNMT)-, neuropeptide Y (NPY)-, vesicular monoamine transporter subtype 2-, vasoactive intestinal peptide (VIP)-, somatostatin (SS)-, synaptophysin-, synaptic vesicle protein 2 (SV2)-, and growth-associated protein of 43 (GAP-43)-immunoreactivities (IRs) were present in the cells but not choline acetyltransferase and vesicular acetylcholine transporter. The immunoreactive substances were present in cell bodies in serum-containing medium (SCM), but after serum withdrawal (protein-free medium, PFM) these proteins and peptides were partially shifted into the long process and their varicosities. A few cells cultured in PFM were occasionally found with extremely high TH-immunoreactivity (IR) in cell bodies and processes. Growth-associated protein of 43-immunoreactivity was weak in SCM but was up-regulated (verified with immunoblot) in PFM and concentrated in varicosities along the processes and the distal tips of neurites. The somatostatin receptor subtype 2a (SSR(2(a))) was found in the cytoplasm and the plasma membrane of the CAD-cells. After serum deprivation, all three methods showed that SSR(2(a)) was up-regulated in the cells. Thus, the CAD cell line after differentiation may be suitable for studying dynamics of SSR(2(a)).