EXPRESSION OF G-PROTEIN SUBTYPES IN CULTURED CEREBRAL ENDOTHELIAL-CELLS

This paper describes Western-blotting evidence for the presence of var ious guanine nucleotide binding proteins, G-proteins in cultured rat c erebral endothelial cells (CECs) and two immortalized cerebral endothe lial cell lines, RBE4 and GP8. By using specific antibodies raised aga inst known sequences of appropriate G-protein types that were previous ly characterized, we demonstrated the presence of G(s)alpha, G(i2)alph a, G(i3)alpha, G(q/11)alpha, G(o)a and G beta in cell lysates of prima ry cultures of CECs, and plasmamembranes of RBE4 and GP8 cells. The ap pearance of G(o)alpha proteins in CECs might be of special importance, since they were not detected in peripheral endothelial cells in previ ous studies. Isoproterenol and bradykinin displayed significant, dose- dependent stimulation of [S-35]GTP gamma S binding above basal values. This assay, reflecting the GDP-GTP exchange reaction on G alpha-subun its by receptor agonists, suggested that there were functional, G-prot ein coupled beta-adrenergic and bradfkinin receptors in these systems. No significant stimulation of [S-35]GTP gamma S binding was noted wit h serotonin under our experimental conditions. Since stimulation of [S -35]GTP gamma S binding by isoproterenol and bradykinin was additive, it was concluded that different G alpha proteins were activated by the se two ligands. In analogy to other systems, activation of G(s) is mos t likely by isoproterenol, while G(i) and/or Gq/11 proteins might be a ctivated by bradykinin receptors. The possible significance of the rec eptors and G-proteins detected is being discussed in the functioning o f cerebral endothelium, and thus the blood-brain barrier. (C) 1998 Els evier Science Ltd. All rights reserved.