Premium
Characterization of the Phosphoinositide‐Linked Dopamine Receptor in a Mouse Hippocampal‐Neuroblastoma Hybrid Cell Line
Author(s) -
Jin LiQing,
Cai Guoping,
Wang HoauYan,
Smith Candyce,
Friedman Eitan
Publication year - 1998
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.1998.71051935.x
Subject(s) - pertussis toxin , receptor , dopaminergic , biology , microbiology and biotechnology , inositol phosphate , g protein , dopamine , cell culture , biochemistry , endocrinology , inositol , genetics
Previous studies have established that dopamine (DA) can stimulate phosphoinositide (PI) metabolism in the CNS and in the periphery. The present study summarizes our attempt to find a cell line that expresses this dopaminergic system. We describe that the stable clonal HN33.11 cell line, established by fusion of mouse hippocampal cells with neuroblastoma cells (N18TG2) that originate from A/J mouse, natively expresses the D 1 DA receptor system that couples to PI hydrolysis. In this cell line, 500 µ M DA or SKF38393 produced 43 and 75% increases in inositol phosphate (IP) accumulations, respectively. In contrast, noradrenaline or 5‐hydroxytryptamine did not affect IP accumulations. The formation of IP that was stimulated by DA or SKF38393 was selectively blocked by the D 1 DA receptor antagonist SCH23390 with IC 50 values of 13 and 16 µ M . This response was not mediated by the D 1A DA receptor and was cyclic AMP‐independent, as HN33.11 cells did not express this receptor, and DA or SKF38393 was unable to stimulate the formation of cyclic AMP. In Ca 2+ ‐free/100 µ M EGTA medium, basal IP level was reduced by 31.5%, but SKF38393‐stimulated PI hydrolysis was not affected. SKF38393‐stimulated IP accumulation was also not affected by pertussis toxin (PTX) treatment (200 ng/ml), suggesting that this dopaminergic response is mediated by PTX‐insensitive G proteins. Co‐immunoprecipitation studies indicated that in membranes of HN33.11 cells, D 1 ‐like binding sites are coupled to Gα q protein. Blockade of SKF38393‐induced PI hydrolysis with antiserum against phospholipase C (PLC) isozymes, performed in permeabilized cells, as well as co‐immunoprecipitation studies implicate PLCβ3 and PLCβ4 in this dopaminergically mediated PI hydrolysis cascade. The results indicate that HN33.11 cells express a D 1 ‐like DA receptor that couples to PLCβ3/4 via Gα q protein. These cells may therefore be a useful model system for investigating this receptor system.