Stable Expression Of I₁ Imidazoline Receptors In CHO Cells And Study On The Functions Of The Receptors

Imidazoline receptors are a kind of new receptors that was recognized in 1980s, which was mainly divided into I1 and I2 subtypes. I1R participates in modulation of many physiological functions like modulation of blood pressure and secretion of insulin, furthermore, it was presumed to participate in the development of depression and Alzheimer's disease. Since there are no appropriate test models and high selective ligand, it's difficult to evaluate the biological functions and the signal transduction pathway of I1R, thus it's important to set up a proper cell model for I1R.Recombined I1R clone was transfected into the CHO cells by lipofectamine. Geneticin was used to select the cells stably expressed with I1R. [3H]-idazoxan and [3H]-clonidine were used to detect the expression of I1R. We used number 19 CHO cells expressed I1R (CHO-I1R-19) as the cell line for further studies.Saturation ligand binding test indicated that I1R in CHO-I1R-19 specifically binds with [3H]-idazoxan and [3H]-clonidine. Bmax for the two ligands were 713.3 ± 102 and 742 ± 17 fmol/mg protein, respectively. Kd value are 15.8±10.4 and 16.7 ± 3.7 nM separately. In whole cell binding test, we got the same results. Competitive ligand binding test showed that IR ligands including moxonidine, clonidine and idazoxan inhibited I1R binding with [3H]-clonidine, while a 2-receptor agonist noreadrenaline didn't. These results implicated the stable expression of I1R in CHO-I cell line.CHO-I and CHO cells exhibited same growth characteristic in DMEM with 10% new bovine serum. However, CHO-I showed stronger vitality in DMEM with 1% bovine serum than CHO cells. CHO-I also exhibited stronger ability withstanding the damage by glutamate. Agmatine, which is an endogenous ligand of I1R, stimulated the proliferation of CHO-I at the concentration of 0.1-5 μ M. This effect could be antagonized by efaroxan, an antagonist of I1R, further proved the participation of I1R in this process. These results inferred the possible role of I1R with growth and proliferation of CHO-I.[35S]-GTP Y S binding assay indicated that clonidine didn't stimulate the binding of G-protein, inferred that I1R might not be a G-protein coupled receptor. The concentration of calcium in CHO-I cells was determined by the methods of laser confocal microscope and flow cytometry. Agmatine, moxonidine and clonidine didn't influence the concentration of calcium in CHO-I cells. This result indicated stimulate I1R was not related to calcium signals.For the first time we stably expressed I1R in CHO cells and investigated the biological functions of this receptor.