Identification and localization of pertussis toxin-sensitive GTP-binding proteins in bovine kidney glomeruli

The vascular tree and the mesangium in the glomerulus respond to various hormones, growth factors, and autonomic signals, leading to generation of second messengers and regulation of ion channels. Guanine nucleotide regulatory proteins (G proteins) mediate these effects in other systems. Glomerular G proteins were studied by immunoblotting and immunohistochemical techniques. Glomeruli were isolated from bovine kidney cortex by differential sieving. Glomerular proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and nitrocellulose transfers were immunoblotted with antibodies to G proteins. G_α,Common antiserum (P-960) recognized proteins with a molecular mass of 41 to 45 kDa. Antibodies against peptide sequences specific to G_iα and G_oα demonstrated G_iα,1/3 (molecular mass, 39 to 41 kDa), G_iα2 (molecular mass, 40 kDa), and G_oα (molecular mass, 39 kDa). Presence of these proteins was further confirmed by pertussis toxin-catalyzed ADP ribosylation of protein(s) with a molecular mass of 39 to 41 kDa in the glomeruli. Immunohistochemical staining of frozen sections from bovine kidney cortex revealed the presence of G_iα2 in capillary loop distribution in glomeruli and interstitium, but G_i,1/3 or G_o could not be demonstrated. The pattern of immunofluorescence with G_iα2 antiserum suggested localization of G_iα2 to the endothelium in glomerular and interstitial vasculature. The novel finding of G_o in glomeruli requires localization of G_o to specific cells and determination of its role in glomerular physiology. In conclusion, these studies demonstrate that bovine kidney glomeruli express a subunits of pertussis toxin-sensitive GTP-binding proteins G_i,1/3, G_i,2 and G_o. G_iα2 is localized to the vascular endothelium in glomeruli and interstitium, but G_i,1/3 could not be localized. Further work is required to determine the role of these proteins in mediating effects of neuronal stimuli, hormones, and cytokines on glomerular blood flow.