HRH4 activation regulates proliferation of colon cancer cell through a cAMP-dependent pathway. (A) Mock-Lovo and H4R-Lovo cells were exposed to 5mM clozapine for 10 min to 8 h. Intracellular cAMP levels were determined by cAMP Enzyme Immunoassay Kit as introduced in Material and Methods. Values are mean ± S.D. of two experiments performed in triplicate. (B) H4R-Lovo and Mock-Lovo cells were treated with 10-5M forskolin, forskolin together with clozapine or Rp-8-Br-cAMPS, and cell-cycle distributions were determined by propidium iodide flow cytometry analysis. *p < 0.05 vs. Control, cells without any treatment. (C) H4R-Lovo and Mock cells were treated with clozapine, clozapine together with FK or Rp-8-Br-cAMPS as in Fig.4B, and total cell lysates were examined by Western blotting.