Clozapine Down-regulates Expression Of TRPC1 MRNA In Astrocytes By 5-HT_2B Receptor-mediated EGF Receptor Activation And ERK_1/2 Phosphorylation

Objective:Schizophrenia is a common psychosis disorder whose symptoms include positive symptoms,negative symptoms,and cognitive disorders.Clozapine is the representative of atypical antipsychotics and has significant therapeutic effects on the schizophrenia.Clozapine acts on various receptors including 5-HT receptors.Studies have shown that loss of 5-HT_2BB receptor confers a broad spectrum of antipsychotic-sensitive schizophrenic-like behavior and psychopharmacological phenotype in mice,and these reports provide evidence for a role of 5-HT_2BB receptor in schizophrenia.Astrocytes express 5-HT_2BB receptors.5-HT_2BB receptors are GPCRs,and clozapine has high affinity for 5-HT_2BB receptors.GPCR activates epithelial growth factor receptor（EGFR）,which in turn activates MAPK signaling pathway and changes ERK_1/2/2 phosphorylation.ERK_1/2/2 activation leads to changes in different effector such as ion channels,gene expression,and cytoskeletal function.The TRPC subfamilies include seven subtypes.Astrocytes mainly express TRPC1.Both the deletion and the overexpression of TRPC1 affect the level of cytoplasmic Ca²⁺.Although there is no direct evidence of the role of TRPC in schizophrenia at present,its implications for neuronal development and underlying synaptic mechanisms suggest its potential effect in schizophrenia.This experiment explored the mechanism of clozapine down-regulating expression of TRPC1 mRNA.1.To investigate the effect of clozapine on ERK_1/2/2 phosphorylation,and to explore its mechanism.2.To determine the effect of clozapine on the expression of TRPC1 m RNA,and to explore its mechanism.3.To determine the effect of clozapine on the function of TRPC1 in astrocytes and its mechanism.Methods:In this experiment,primary cultured astrocytes of neonatal mouse cerebral cortex can express 5-HT_2BB receptor.ERK_1/2/2 phosphorylation in astrocytes was determined by Western blot.The expression of TRPC1 mRNA was determined by reverse transcription-polymerase chain reaction and agarose gel electrophoresis.The level of intracellular Ca²⁺was determined by Olympus IX71 live cell imaging fluorescence microscope.Astrocytes were treated by different concentrations of clozapineatdifferenttimes,toobservethetime-dependentand concentration-dependent of clozapine.Inhibitor U0126 was used to determine whether the change of TRPC1 mRNA expression induced by clozapine depends on ERK_1/2/2 phosphorylation signal transduction pathway,5-HT_2BB receptor inhibitor SB204741 and EGFR inhibitor AG1478 were used to identify whether clozapine causes EGFR activation mediated by 5-the HT_2BB receptor,which in turn causes ERK_1/2/2 phosphorylation.Results:1.In the concentration dependence,100 nM of clozapine induced a statistically significant increase of ERK_1/2/2 phosphorylation（P<0.05）.In the time course,a statistically significant increase of ERK_1/2/2 phosphorylation was seen after 20min（P<0.05）.The increase of ERK_1/2/2 phosphorylation induced by clozapine（P<0.05）was inhibited by SB204741,AG1478,and U0126.2.Clozapine decreased TRPC1mRNA expression in astrocytes.The down-regulation of the expression of TRPC1mRNA induced by clozapine（P<0.05）was inhibited by SB204741,AG1478,and U0126.3.Clozapine inhibited the increase of intracellular Ca²⁺concentration in astrocytes.Inhibitors SB204741,AG1478 and U0126 can significantly inhibit the intracellular Ca²⁺concentration increased of astrocytes induced by clozapine（P<0.05）.Conclusion:Clozapine causes a concentration-dependent and time-dependent increase in phosphorylation of ERK_1/2/2 in astrocytes.Clozapine leads to EGFR activation and ERK_1/2/2 phosphorylation by a direct 5-HT_2BB receptors action.Elevated phosphorylation of ERK_1/2/2 in astrocytes can further induce the down-regulation of TRPC1 mRNA expression and the down-regulation of TRPC1 function.