Thromboxane binding and signal transduction in rat glomerular mesangial cells.
Thromboxane A2 (TxA2) stimulates contraction of glomerular mesangial cells. However, mesangial cell TxA2 receptors have not been previously characterized. We therefore investigated TxA2 binding and TxA2-associated signal transduction pathways in rat glomerular mesangial cells using the specific thromboxane receptor agonist (1S-[1 alpha,2 beta(5Z),3 alpha-(1E,3S)4 alpha])-7-(3-[3-hydroxy-4-(p- iodophenoxy)-1-butenyl]7-oxabicyclo[2.2.1]hept-2-yl)-5-heptenoic acid (IBOP). In these cells, [125I]BOP binding was saturable, displaceable, and of high affinity. Scatchard analysis revealed a single class of binding sites with a dissociation constant (Kd) of 293 pM and a maximal density of binding sites (Bmax) of 33 fmol/mg protein. Specific binding was inhibited by the thromboxane agonist (15S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5Z,13E-dienoic acid (U-46619) [inhibitor dissociation constant (Ki) = 297 nM] and the TxA2 receptor antagonists SQ 29548 (Ki = 1 nM) and (1R-[1 alpha(Z),2 beta,3 beta,5 alpha])-(+)-7-(5-[(1,1'-biphenyl)- 4-yl-methoxy]-3-hydroxy-2-(1-piperidinyl)cyclopentyl]-4-heptenoic acid (GR 32191) (Ki = 92 nM). Binding was also highly specific for thromboxane because prostaglandin E2 (Ki = 16 microM) and the inactive thromboxane metabolite, TxB2 (Ki = 41 microM), were approximately 1,000-fold less potent at inhibiting binding. IBOP stimulated phosphatidylinositol hydrolysis with an effective concentration of drug that produces 50% of the maximal response of 229 pM, which correlated well with the equilibrium Kd and enhanced phosphorylation of an acidic 80-kDa protein substrate for protein kinase C.(ABSTRACT TRUNCATED AT 250 WORDS)
Spurney
R F
RF
Department of Medicine, Duke University Medical Center, Durham, North Carolina.
Onorato
J J
JJ
Albers
F J
FJ
Coffman
T M
TM
D000818:Animals; D001667:Binding, Competitive; D001713:Biphenyl Compounds / Q000378:metabolism / Q000494:pharmacology; D001643:Bridged Bicyclo Compounds / Q000378:metabolism; D019086:Bridged Bicyclo Compounds, Heterocyclic; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D005231:Fatty Acids, Unsaturated / Q000378:metabolism; D006538:Heptanoic Acids / Q000378:metabolism / Q000494:pharmacology; D006834:Hydrazines / Q000378:metabolism / Q000494:pharmacology; D007295:Inositol Phosphates / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D007678:Kidney Glomerulus / Q000166:cytology / Q000378:metabolism* / Q000502:physiology; D007700:Kinetics; D008565:Membrane Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction; D013931:Thromboxanes / Q000378:metabolism*
D001713:Biphenyl Compounds; D001643:Bridged Bicyclo Compounds; D019086:Bridged Bicyclo Compounds, Heterocyclic; D005231:Fatty Acids, Unsaturated; D006538:Heptanoic Acids; D006834:Hydrazines; D007295:Inositol Phosphates; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D011506:Proteins; D013931:Thromboxanes; C061750:7-(3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7-oxabicyclo(2.2.1)heptan-2-yl)-5-heptenoic acid; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; C045749:SQ 29548; D011493:Protein Kinase C; C060013:vapiprost