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Thromboxane binding and signal transduction in rat glomerular mesangial cells
R F Spurney1, J J Onorato, F J Albers
1Department of Medicine, Duke University Medical Center, Durham, North Carolina.
The American Journal of Physiology
|February 1, 1993
Summary
This study characterizes Thromboxane A2 (TxA2) receptors in rat mesangial cells, revealing high-affinity binding and signal transduction. Findings identify specific binding sites and pathways activated by TxA2 agonists.
Area of Science:
- Nephrology
- Pharmacology
- Cell Biology
Background:
- Thromboxane A2 (TxA2) is known to stimulate glomerular mesangial cell contraction.
- However, the specific TxA2 receptors on these cells and their signaling pathways remained uncharacterized.
Purpose of the Study:
- To investigate and characterize Thromboxane A2 (TxA2) binding and associated signal transduction pathways in rat glomerular mesangial cells.
- To identify the presence and affinity of TxA2 receptors on mesangial cells.
Main Methods:
- Utilized a 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), for binding assays.
- Performed radioligand binding studies with [125I]BOP and analyzed data using Scatchard analysis.
- Investigated signal transduction by measuring phosphatidylinositol hydrolysis and protein kinase C substrate phosphorylation.
Main Results:
- Demonstrated saturable, displaceable, high-affinity binding of [125I]BOP to mesangial cells, with a Kd of 293 pM and Bmax of 33 fmol/mg protein.
- Confirmed specificity for TxA2, as other prostaglandins and TxA2 metabolites showed significantly lower binding affinity.
- Showed that IBOP stimulates phosphatidylinositol hydrolysis (EC50 = 229 pM) and enhances phosphorylation of an 80-kDa protein, indicating activation of signal transduction pathways.
Conclusions:
- Rat glomerular mesangial cells possess specific, high-affinity Thromboxane A2 (TxA2) receptors.
- Activation of these TxA2 receptors triggers signal transduction pathways, including phosphatidylinositol hydrolysis and protein kinase C activation.