Characterization of rat glomerular thromboxane A2 receptors: comparison to rat platelets

doi:10.1016/0922-4106(92)90144-K

European Journal of Pharmacology: Molecular Pharmacology

Volume 227, Issue 1, 1 September 1992, Pages 71-78

https://doi.org/10.1016/0922-4106(92)90144-K Get rights and content

Abstract

This study was designed to characterize rat glomerular thromboxane A₂ (TxA₂) receptors and compare them to rat platelet TxA₂ receptors. The radioligand binding characteristics of the receptors were characterized using [¹²⁵I][1S-(1α,2β(5Z),3α-(1E,3R^∗),4α]-7-[3-(3-hydroxy-4-(4′-iodophenoxy)-l-butenyl)-7-oxabiocyclo-[2.2.1]heptan-2yl]-5-heptenoic acid ([¹²⁵I]BOP), a TxA₂ agonist. Equilibrium binding with [¹²⁵I]BOP, as well as competitive binding assays between [¹²⁵I]BOP and 13-azapinane TxA₂ receptors antagonists, were performed in rat glomerular membranes (RGM) and washed rat platelets (WRP). [¹²⁵I]BOP identified a single class of TxA₂ receptor sites in glomerular membrane with a K_d of 318±55 pM and a B_max of 260±62 fmol/mg protein (n = 14). [¹²⁵I]BOP was displaced by the TxA₂ agonist 15S-hydroxy-11α,9α(epoxymethano)-prosta-5Z,13E-dienoic acid (U-46,619) (IC₅₀ = 22±6 nM,n = 3), the antagonist SQ-29,548 (IC₅₀ = 41±7 nM, n = 4), and stereoselectively by the antagonists (−)-9-chlorobenzyl-6-fluoro-1,2,3,4-tetrahydrocarbazol-1-yl acetic acid (L-657,926) (IC₅₀ = 0.27±0.04 nM, n = 3) and (+)-9-chlorobenzyl-6-fluoro-1,2,3,4-tetrahydrocarbazol-l-yl acetic acid (L-657,926) (IC₅₀ = 124±0 nM,n = 2). The ability of six 13-azapinane TxA₂ antagonists to compete with [¹²⁵I]BOP was evaluated. The rank orders for the 13-azapinances showed no significant correlation between RGM and WRP. Phosphoinositide hydrolysis in whole glomeruli was stimulated with I-BOP (EC₅₀ = 1.99±0.43 nM, n = 4) and stereoselectively blocked by L-657,925 (IC₅₀ = 27.4±5.5 nM,n = 4) and L-65 (IC₅₀ = 811±116 nM, n = 3). These results indicate that functional TxA₂ receptors are present in glomeruli and that different subtypes of TxA₂ receptors exist in rat glomeruli and platelets.

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Thromboxane receptors in human kidney tissues
1999, Prostaglandins and Other Lipid Mediators
Thromboxane (TX) A₂ effects in the kidneys include contraction of glomerular mesangial cells and intrarenal vascular tissue. A kidney cDNA encoding a TX receptor expressed in rat renal glomeruli and rat renal arterial smooth muscle cells has been reported. However, TXA₂ receptors in human kidneys have not been documented. The purpose of this study was to identify and characterize TXA₂ receptors in glomeruli and intrarenal arteries isolated from human kidneys. Normal kidneys, not used for transplant because of technical reasons, were kept at −70°C and used for research purposes. The glomeruli and intrarenal arteries were isolated from renal cortical tissue by a mechanical sieving technique. The equilibrium dissociation constant and receptor number were determined by nonlinear analysis of binding inhibition data. The data were generated in radioreceptor assays using [¹²⁵I]-BOP, a stable analog of TXA₂. The dissociation constants (mean ± SEM) for binding of I-BOP to human glomeruli and intrarenal arterial membranes were 6.6 ± 1.1 nM (n = 7) and 20 ± 6 nM (n = 7), respectively (p < 0.05). The receptor number was 311 ± 91 fmol/mg protein (n = 7) in glomeruli and 74 ± 16 fmol/mg protein (n = 7) in intrarenal arterial membranes (p < 0.04). The order of specificity of TXA₂ analogs for [¹²⁵I]-BOP binding sites was similar in glomeruli and in arterial membranes and was I-BOP ≥ U46619 ≥ pinane TXA₂ ≥ carbocyclic TXA₂ ≥ PGH₂. These findings provide direct evidence for the presence of specific, high-affinity [¹²⁵I]-BOP binding sites in human renal glomeruli and extraglomerular vascular tissue. These data also indicate that the human binding sites have higher affinity for the TXA₂ agonist I-BOP than for PGH₂.
Cloning and characterization of an endogenous COS-7 cell thromboxane A<inf>2</inf> receptor
1998, Biochimica et Biophysica Acta - Molecular Cell Research
A cDNA for a thromboxane A₂ (TXA₂) receptor was cloned from an SV40 transformed African Green Monkey kidney cell line (COS-7). The sequence is 98% homologous with the isoform of the human TXA₂ receptor and has agonist and antagonist ligand binding characteristics that are not significantly different from the human receptor. Stimulation of the COS-7 cells with the TXA₂ receptor agonist, ONO 11113 resulted in a significant increase in cAMP formation that was blocked by a receptor antagonist. The results raise the question of the utility of the COS-7 cell line for studies of cloned and expressed TXA₂ receptor signalling mechanisms.
A genetic approach for studying the role of thromboxane A<inf>2</inf> in the kidney
1998, Kidney International, Supplement
A genetic approach for studying the role of thromboxane A₂in the kidney. Thromboxane A₂ (TxA₂) has been implicated in a number of processes in normal kidney physiology and as a mechanism for injury in renal disease. TxA₂ is a biologically active derivative of arachidonic acid and has potent vasoconstrictive and platelet-activation functions. Its actions are mediated by binding to specific G-protein-associated receptors designated TP receptors. There are at least two isoforms of the human TP receptor, and pharmacological evidence suggests TP receptor heterogeneity in other species. TP receptors are located in the renal cortex, and there may also be TxA₂ binding sites in the medulla. TP receptors are involved in some normal functions of the kidney, and there is considerable evidence that TP receptors may mediate renal damage in disease states. To assess directly the role of the TxA₂ in normal kidney function and in murine models of human disease, we have used gene targeting to eliminate expression of the TP receptor in mice.
Thromboxane a<inf>2</inf> interferes with a disposal process of aggregated protein in glomeruli
1997, Japanese Journal of Pharmacology
Immune complexes in glomeruli are involved in development of diverse glomerulonephritis. The disposal process of glomerular immune complexes has been unclarified. The present studies were undertaken to determine if thromboxane A₂ (TXA₂) is associated with the disposal of macromolecules in the glomeruli using mice injected with aggregated bovine serum albumin (a-BSA). A-BSA promptly accumulated in the glomeruli, the level reaching a plateau at 6 hr after the injection of a-BSA, and then decreased by 48 hr. The production of glomerular TXA₂, prostaglandin E₂ (PGE₂) and prostaglandin I₂ concomitantly increased with the decrease of a-BSA in the glomeruli. TXA₂ synthase inhibitors and TXA₂ receptor antagonists accelerated clearance of glomerular a-BSA without enhancing renal tissue blood flow. They did not affect a-BSA level in the plasma. In contrast, aminophylline, dopamine and mannitol significantly increased renal tissue blood flow, but did not decrease glomerular a-BSA. TXA₂ synthase inhibitors decreased TXA₂ production in the glomeruli. TXA₂ synthase inhibitors and TXA₂ receptor antagonists did not influence the generation of PGE₂. The TXA₂ analogue U-46619 significantly increased the accumulation of a-BSA in the glomeruli. We propose that TXA₂ interferes with the disposal process of aggregated protein in the glomeruli. We also postulate that interception of glomerular activity of TXA₂ may be an effective intervention for managing immune complex-mediated glomerulonephritis and glomerulosclerosis.
Structural determinants for agonist binding affinity to thromboxane/prostaglandin endoperoxide (TP) receptors. Analysis of chimeric rat/human TP receptors
1997, Journal of Biological Chemistry
The two most extensively characterized thromboxane/prostaglandin endoperoxide (TP) receptors, from human platelets and rat vascular smooth muscle, exhibit thromboxane agonist [15-(1α,2β(5Z),3α-(1E,3S),4α)]-7-[3-hydroxy-4-(p-iodophenoxy)-1-butenyl-7-oxabicycloheptenoic acid (I-BOP) binding affinities that differ by an order of magnitude, rat TP having the higher affinity. We utilized this difference in I-BOP affinity to identify structural determinants of TP receptor heterogeneity. No significant difference was found in the rank order of affinities for a series of thromboxane receptor ligands to bind to cloned human TPα versus rat TP, indicating that these represent species homologs, not distinct TP subtypes. Structural determinants for observed differences in I-BOP bindingK_d were localized by creating chimeric human/rat TP followed by mutational substitution of specific critical amino acids. Initially, seven chimeric receptors with splice sites in transmembranes 1, 2, 4, or 7 were constructed and expressed in HEK293 cells for analysis of ligand binding properties. Substitution of any part except the carboxyl tail of the human TP into the rat TP resulted in a receptor with I-BOP binding affinity intermediate between the two. Analysis of chimeras in which only the extracellular amino terminus and a portion of transmembrane 1 were switched localized the determinant of high affinity binding to the region between amino acids 3 and 40. Using this chimera, amino acids in the human portion (extracellular amino terminus and part of transmembrane 1) were replaced with analogous amino acids from rat TP to regain high affinity I-BOP binding. Only when amino acid Val³⁷ and either Val³⁶ or Ala⁴⁰ were reverted to their respective rat TP counterparts (Ala³⁶, Leu³⁷, and Gly⁴⁰, respectively) was high affinity I-BOP binding recovered. The mechanism for the increased I-BOP affinity may be the lengthening of the amino acid side chain at position 37, thus extending this group further into the putative I-BOP binding pocket, with compensatory shortening of side chains in spatially adjacent amino acids.
Thromboxane A<inf>2</inf> receptors
1995, Journal of Lipid Mediators and Cell Signalling

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Characterization of rat glomerular thromboxane A2 receptors: comparison to rat platelets

Abstract

Biochem. Biophys. Res. Commun.

Anal. Biochem.

Prostaglandins

Biochem. Pharmacol.

Eur. J. Pharmacol.

Biochim. Biophys. Acta

Biochem. Pharmacol.

J. Biol. Chem.

Comput. Programs Biomed.

Life Sci.

Anal. Biochem.

Biochim. Biophys. Acta

Kidney Int.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Biochim. Biophys. Acta

Eur. J. Pharmacol.

Antagonism of prostaglandin-mediated responses in platelets and vascular smooth muscle by 13-azaprostanoic acid analogs

Biochem. Pharmacol.

A highly sensitive radioreceptor assay for atrial natriuretic peptide in rat plasma

Am. J. Physiol.

Effects of administered thromboxanes on the intact, normal rat kidney

Renal Physiol. (Basel)

Angiotensin and thromboxane in genetically hypertensive rats: renal blood flow and receptor studies

Am. J. Physiol.

Distinct platelet thromboxane A2/prostaglandin H2 receptor subtypes: a radioligand binding study of human platelets

J. Clin. Invest.

Characterization of rat glomerular thromboxane A₂ receptors: comparison to rat platelets

Distinct platelet thromboxane A₂/prostaglandin H₂ receptor subtypes: a radioligand binding study of human platelets