Here we investigate candidate intracellular mediators PI3K, Src, ß-arrestin and Ca2+ which may govern the ErbB1 transactivation pathway induced by clozapine. Methods: Primary murine frontal cortical neurons were exposed to wortmannin (PI3K inhibitor), monodansylcadaverine (MDC) (receptor endocytosis inhibitor) and BAPT-AM (Ca2+ inhibitor), and the effect on clozapine mediated ErbB1-ERK and Src phosphorylation examined by immunoelectrophoresis to assess mediator involvement. Results: Administration of wortmannin caused significant dose-dependent inhibition of ERK1/2 phosphorylation in the presence of clozapine (ERK1: clozapine 100±22% vs clozapine + 10 uM wortmannin 34±6%, p=0.0136).
In relation to ß-arrestin, MDC did not decrease clozapine induced ERK1/2 phosphorylation but rather elevated pERK1/2 levels (ERK2: clozapine 100±28% vs clozapine + 50 uM MDC 197±24%, p=0.0307). Similarly BAPTA-AM did not have a significant inhibitory effect on ERK activation following clozapine treatment of cells. Parallel detection of Src phosphorylation indicated a time disconnect with ERK phosphorylation. Conclusions: Clozapine modulates the PI3K/ Akt pathway, with aberrant signalling via ErbB4 documented in schizophrenia and Src a key component in the convergent ErbB1-dependent ERK signalling mechanism described. Therefore deficient ErbB1 signalling in schizophrenia may be augmented by clozapine with inverse action at ErbB4 receptors.