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  • br Experimental procedures br Conflicts of interests


    Experimental procedures
    Conflicts of interests
    Introduction Here we investigate differences in growth properties and short-term morphological changes in response to ET-1 in Chinese hamster ovary (CHO) cells stably and separately expressing ETA or ETB receptors. A stably transfected CHO system is advantageous for ET-1 growth studies in that these cells do not express endogenous ET-1 receptors [1], [2]. Thus growth effects of each receptor can be characterized by expressing each separately and at a constant level. This system is also effective since many cell types which express endogenous ET-1 receptors, such as human vascular smooth muscle cells, have very slow growth characteristics, and/or may show fluctuation (or complete absence) in ET-1 receptor expression under certain conditions (cell stress, passage number, etc.) [3], [4], [5], [6]. Previous studies using stably cDNA transfected CHO cells expressing either ETA or ETB receptors have shown them to similarly promote phosphatidylinositol hydrolysis, arachidonic bcl-2 inhibitor release from lipid stores and signals through a number of kinases [1], [7]. However, while ETA and ETB receptors share a number of signaling pathways, signaling differences have been reported. One such is the production of cAMP. While ETA stimulates cAMP accumulation through Gs alpha, ETB does not [1]. Clearly, other as yet unreported signaling differences exist.
    Materials and methods
    Discussion ET-1 activation of the ETB receptor leads to a cellular growth reduction. This phenomenon was detected via end-point cell counting, real-time impedance-based detection of cell proliferation, cell cycle analysis, and by determining the rate of cellular [3H]-thymidine incorporation. Cell counting and real-time impedance measurements illustrated the effect of ET-1 on CHO ETB cell division, but did not provide information as to where in the cell cycle this takes place. We therefore arrested the cells in late G1 phase and then released them to allow for synchronized progress into S phase. Since late G1 registers negligible [3H]-thymidine incorporation which then takes place in S phase, this approach provided us with a precise and readily measurable assay to study cell proliferation. Also flow cytometric analysis with propidium iodide DNA staining revealed that ET-1 delays cell cycle progression through S phase, but does not delay transit from G2/M to G1. Exposure to ET-1 in the CHO ETB led to lower [3H]-thymidine incorporation compared to the CHO ETB control without treatment. As Fig. 2A shows, ET-1 treated CHO ETB had a slower velocity and longer duration S phase compared to control. This effect was detected within 2h of ET-1 addition. A previous study looking at activated hepatic stellate cells which express predominately endogenous ETB receptors also show growth inhibition due to ET-1 [10]. However, in those cells ET-1 inhibition occurred only in the presence of serum or platelet-derived growth factor BB. One concern about ET-1 induced growth inhibition in our study was that it occurred in the presence of serum. Given that serum contains many growth factors, hormones, and other compounds that could be interacting with ET-1, we further tested whether this phenomenon occurred with low (0.5%) or no serum. We found that ET-1 induced growth reduction in CHO ETB was serum-independent (data not shown). In all cases, 10%, 0.5% and 0% serum, ET-1 reduced the growth rate of CHO ETB. This means that ET-1 is not interacting with serum factors to inhibit growth. The ET-1 induced morphological changes clearly show contrast between the actions of the ETA and ETB receptors. These changes were detected using a cell-substrate impedance-based method which has previously been verified to detect morphological changes due to GPCR activation [8]. ET-1 receptors, like other GPCRs, activate small Rho GTPases which control actin cytoskeleton organization [11], [12]. In this study, ET-1 induced bcl-2 inhibitor a contracted short-term morphological change in CHO ETA and a more expanded short-term morphological change in CHO ETB. The amount of ET-1 induced expansion in CHO ETB cells was dose-dependent.