Role of Purinergic Receptors in Myeloid cells

In vitro studies of murine bone marrow-derived myeloid cells. Experimental design: We used an in vitro model of murine bone marrow-derived monocyte differentiation into myeloid mononuclear cells (Ryzhov S, Novitskiy SV, Goldstein AE, Biktasova A, Blackburn MR, Biaggioni I, Dikov MM, Feoktistov I. Adenosinergic regulation of the expansion and immunosuppressive activity of CD11b+Gr1+ cells. J Immunol 2011 December 1;187(11):6120-9) .We studied the effects of agonists and antagonist of purinergic receptors on monocyte differentiation into mature myeloid mononuclear cells. Phenotypic changes and cytokine/growth factors were analyzed. To determine the role of Gαq and Gαs pathways in the effects of A2B receptors, we suppressed Gαq protein function by overexpression of the regulator of G protein signaling (RGS)-2, a Gαq-specific GTPase accelerating protein. To mimic the A2B-dependent stimulation of Gαq, we expressed a constitutively active Gαq mutated at R183C. To ensure that only transfected cells are analyzed by flow cytometry, we cloned RGS2 constructs into pLVX-IRES-tdTomato bicistronic vector (Clontech) that allow the simultaneous expression of RGS2 and fluorescent tdTomato proteins separately but from the same RNA transcript. The same approach was used for transfection of constitutively active Gαq R183C. The following combinations of Gq/s activators/inhibitors were tested: 1) Inhibition of Gαq by overexpression of RGS2 followed by A2BRs stimulation with 1 µM NECA; 2) Activation of Gαs pathways by expression of the constitutively active Gαq followed by A2BRs stimulation with 1 µM NECA; Phenotypic changes in the expression of cell-surface markers after two-day differentiation of transfected cells are analyzed by gating for tdTomato fluorescence.