TY - JOUR
T1 - Immortalized Murine Striatal Neuronal Cell Lines Expressing Dopamine Receptors and Cholinergic Properties
AU - Wainwright, Mark S.
AU - Perry, Bruce D.
AU - Won, Lisa A.
AU - O'Malley, Karen L.
AU - Wang, Wen Yi
AU - Ehrlich, Michelle E.
AU - Heller, Alfred
PY - 1995/1
Y1 - 1995/1
N2 - Immortalized hybrid cells were generated by somatic cell fusion of 18-d-old embryonic corpus striatum of the mouse strain C57BL/6J with the N18TG2 neuroblastoma. One of the cell populations obtained was treated with a combination of 1 mM n-butyric acid and 10 μM SKF 38393 (a specific D1 agonist), and a surviving cell population (E1X) was subcloned. Twenty-seven monoclonal cell lines were obtained and screened for the expression of striatal-specific characteristics including γ-aminobutyric acid (GABA), choline acetyltransferase (ChAT), acetylcholine (ACh), mRNA for specific dopamine receptors, and dopamine- and adenosine 3′:5′-monophosphate-regulated phosphoprotein, Mr 32,000 (DARPP-32), and functional D1 and D2 dopamine receptors. Neither the parent hybrid cell population (E1X) nor any of the monoclonal cell lines examined expressed GABA levels significantly different than that of the N18TG2 parent neuroblastoma cells (1.36 ± 0.07 μg/mg protein). The range of ChAT activity in the monoclonal hybrid cell lines was 5.5 ± 0.3 to 921.3 ± 97.4 pmol/min/mg protein. Two of the cell lines expressing ChAT activity (X52 and X58) contained ACh (49.64 ± 4.23 and 1.78 ± 0.07 ng/mg protein, respectively). The neuronal origin of four of the monoclonal hybrid lines was shown by their immunoreactivity, following differentiation with 10 μM forskolin, to neurofilament protein, a neuron-specific marker. The monoclonal hybrid cell lines, but not the N18TG2 neuroblastoma, were shown to express an array of D1, D2, and D5 receptor mRNA as well as DARPP-32 mRNA. Two monoclonal cell lines expressed D1 receptor binding sites (X57, 29.2 ± 4.5 fmol/mg protein and X62, 43.8 ± 6.8 fmol/mg protein) which mediated the stimulation of adenylate cyclase activity. One cell line, X58, expressed only D2 dopamine receptors (80.9 ± 9.8 fmol/mg protein) which were negatively coupled to adenylate cyclase activity. These findings suggest that the immortalized monoclonal hybrid cell lines are of neuronal origin and have incorporated elements of the medium spiny and cholinergic neurons of the developing striatum.
AB - Immortalized hybrid cells were generated by somatic cell fusion of 18-d-old embryonic corpus striatum of the mouse strain C57BL/6J with the N18TG2 neuroblastoma. One of the cell populations obtained was treated with a combination of 1 mM n-butyric acid and 10 μM SKF 38393 (a specific D1 agonist), and a surviving cell population (E1X) was subcloned. Twenty-seven monoclonal cell lines were obtained and screened for the expression of striatal-specific characteristics including γ-aminobutyric acid (GABA), choline acetyltransferase (ChAT), acetylcholine (ACh), mRNA for specific dopamine receptors, and dopamine- and adenosine 3′:5′-monophosphate-regulated phosphoprotein, Mr 32,000 (DARPP-32), and functional D1 and D2 dopamine receptors. Neither the parent hybrid cell population (E1X) nor any of the monoclonal cell lines examined expressed GABA levels significantly different than that of the N18TG2 parent neuroblastoma cells (1.36 ± 0.07 μg/mg protein). The range of ChAT activity in the monoclonal hybrid cell lines was 5.5 ± 0.3 to 921.3 ± 97.4 pmol/min/mg protein. Two of the cell lines expressing ChAT activity (X52 and X58) contained ACh (49.64 ± 4.23 and 1.78 ± 0.07 ng/mg protein, respectively). The neuronal origin of four of the monoclonal hybrid lines was shown by their immunoreactivity, following differentiation with 10 μM forskolin, to neurofilament protein, a neuron-specific marker. The monoclonal hybrid cell lines, but not the N18TG2 neuroblastoma, were shown to express an array of D1, D2, and D5 receptor mRNA as well as DARPP-32 mRNA. Two monoclonal cell lines expressed D1 receptor binding sites (X57, 29.2 ± 4.5 fmol/mg protein and X62, 43.8 ± 6.8 fmol/mg protein) which mediated the stimulation of adenylate cyclase activity. One cell line, X58, expressed only D2 dopamine receptors (80.9 ± 9.8 fmol/mg protein) which were negatively coupled to adenylate cyclase activity. These findings suggest that the immortalized monoclonal hybrid cell lines are of neuronal origin and have incorporated elements of the medium spiny and cholinergic neurons of the developing striatum.
KW - ACh
KW - ChAT
KW - Corpus striatum
KW - DARPP-32
KW - Dopamine receptors
KW - Neuronal cell lines
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UR - http://www.scopus.com/inward/citedby.url?scp=0028896896&partnerID=8YFLogxK
M3 - Article
C2 - 7823171
AN - SCOPUS:0028896896
SN - 0270-6474
VL - 15
SP - 676
EP - 688
JO - Journal of Neuroscience
JF - Journal of Neuroscience
IS - 1 I
ER -