TY - JOUR
T1 - Adrenergic differentiation and SSR2(a) receptor expression in CAD-cells cultured in serum-free medium
AU - Hashemi, Sayed Hossein
AU - Li, Jia Yi
AU - Faigle, Roland
AU - Dahlström, Annica
N1 - Funding Information:
The CAD-cells were a generous gift from Dr. James K.T. Wang via Dr. Peter F.T. Vaughn in Leeds, UK. We thank Dr. Markus Schindler for generous supply of the sheep-anti-SSR 2(a) . Also, we thank Dr. Keiko Funa for help with immunoblot. This study was supported by the Swedish MRC (2207), the Medical Faculty, Göteborg University, and funds (LUA) of the Sahlgren’s University Hospital.
PY - 2003/1
Y1 - 2003/1
N2 - 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 (SSR2(a)) was found in the cytoplasm and the plasma membrane of the CAD-cells. After serum deprivation, all three methods showed that SSR2(a) was up-regulated in the cells. Thus, the CAD cell line after differentiation may be suitable for studying dynamics of SSR2(a).
AB - 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 (SSR2(a)) was found in the cytoplasm and the plasma membrane of the CAD-cells. After serum deprivation, all three methods showed that SSR2(a) was up-regulated in the cells. Thus, the CAD cell line after differentiation may be suitable for studying dynamics of SSR2(a).
KW - Confocal microscopy
KW - GAP-43
KW - Immunoblot
KW - Immunocytochemistry
KW - RPA
KW - TH
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U2 - 10.1016/S0197-0186(02)00065-7
DO - 10.1016/S0197-0186(02)00065-7
M3 - Article
C2 - 12441163
AN - SCOPUS:0037207506
SN - 0197-0186
VL - 42
SP - 9
EP - 17
JO - Neurochemistry International
JF - Neurochemistry International
IS - 1
ER -