Thromboxane binding and signal transduction in rat glomerular mesangial cells

Am J Physiol. 1993 Feb;264(2 Pt 2):F292-9. doi: 10.1152/ajprenal.1993.264.2.F292.

Abstract

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)

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Binding, Competitive
  • Biphenyl Compounds / metabolism
  • Biphenyl Compounds / pharmacology
  • Bridged Bicyclo Compounds / metabolism
  • Bridged Bicyclo Compounds, Heterocyclic*
  • Cells, Cultured
  • Dose-Response Relationship, Drug
  • Fatty Acids, Unsaturated / metabolism
  • Heptanoic Acids / metabolism
  • Heptanoic Acids / pharmacology
  • Hydrazines / metabolism
  • Hydrazines / pharmacology
  • Inositol Phosphates / metabolism
  • Intracellular Signaling Peptides and Proteins*
  • Kidney Glomerulus / cytology
  • Kidney Glomerulus / metabolism*
  • Kidney Glomerulus / physiology
  • Kinetics
  • Membrane Proteins*
  • Myristoylated Alanine-Rich C Kinase Substrate
  • Phosphorylation
  • Protein Kinase C / metabolism
  • Proteins / metabolism
  • Rats
  • Signal Transduction*
  • Thromboxanes / metabolism*

Substances

  • Biphenyl Compounds
  • Bridged Bicyclo Compounds
  • Bridged Bicyclo Compounds, Heterocyclic
  • Fatty Acids, Unsaturated
  • Heptanoic Acids
  • Hydrazines
  • Inositol Phosphates
  • Intracellular Signaling Peptides and Proteins
  • Membrane Proteins
  • Proteins
  • Thromboxanes
  • 7-(3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7-oxabicyclo(2.2.1)heptan-2-yl)-5-heptenoic acid
  • Myristoylated Alanine-Rich C Kinase Substrate
  • SQ 29548
  • Protein Kinase C
  • vapiprost