CHARACTERIZATION OF THE PHOSPHOINOSITIDE-LINKED DOPAMINE-RECEPTOR IN A MOUSE HIPPOCAMPAL-NEUROBLASTOMA HYBRID CELL-LINE

Previous studies have established that dopamine (DA) can stimulate pho sphoinositide (PI) metabolism in the CNS and in the periphery. The pre sent study summarizes our attempt to find a cell line that expresses t his dopaminergic system. We describe that the stable clonal HN33.11 ce ll line, established by fusion of mouse hippocampal cells with neurobl astoma cells (N18TG2) that originate from A/J mouse, natively expresse s the D-1 DA receptor system that couples to PI hydrolysis. In this ce ll line, 500 mu M DA or SKF38393 produced 43 and 75% increases in inos itol phosphate (IP) accumulations, respectively. In contrast, noradren aline or 5-hydroxytryptamine did not affect IP accumulations. The form ation of IP that was stimulated by DA or SKF38393 was selectively bloc ked by the D-1 DA receptor antagonist SCH23390 with lc,, values of 13 and 16 mu M. This response was not mediated by the D-1A DA receptor an d was cyclic AMP-independent, as HN33.11 cells did not express this re ceptor, and DA or SKF38393 was unable to stimulate the formation of cy clic AMP, In Ca2+-free/100 mu M EGTA medium, basal IP level was reduce d by 31.5%, but SKF38393-stimulated PI hydrolysis was not affected. SK F38393-stimulated IP accumulation was also not affected by pertussis t oxin (PTX) treatment (200 ng/ml), suggesting that this dopaminergic re sponse is mediated by PTX-insensitive G proteins. Co-immunoprecipitati on studies indicated that in membranes of HN33,11 cells, D-1-like bind ing sites are coupled to G alpha(q) protein. Blockade of SKF38393-indu ced PI hydrolysis with antiserum against phospholipase C (PLC) isozyme s, performed in permeabilized cells, as well as co-immunoprecipitation studies implicate PLC beta 3 and PLC beta 4 in this dopaminergically mediated PI hydrolysis cascade. The results indicate that HN33,11 cell s express a D-1-like DA receptor that couples to PLC beta 3/4 via G al pha(q), protein. These cells may therefore be a useful model system fo r investigating this receptor system.