Isolation and identification of lysophosphatidylcholines as endogenous modulators of thromboxane receptors.
Inhibition of thromboxane receptor radioligand binding to human platelet membranes has been employed as the basis for a radioreceptor assay designed to measure thromboxane receptor binding activity in samples of biological fluids. This method was used during phase 1 clinical evaluation of the thromboxane receptor antagonist SQ 30,741. Frequently, baseline plasma samples as well as plasma samples from placebo-treated subjects showed significant inhibition of radioligand binding in the radioreceptor assay, suggesting the presence of endogenous thromboxane receptor ligands. This receptor binding activity was stable and could be monitored in blood from normal volunteers using a modification of the radioreceptor assay. In order to identify the substance responsible for the observed activity, the activity present in pooled bovine blood was isolated and evaluated by a combination of FAB/MS, 1H-NMR, 13C-NMR and co-injection with reference standards on HPLC. Several endogenous thromboxane receptor ligands were identified as L-alpha-lysophosphatidylcholine (LPC) species. One major species, palmitoyl-LPC, contracted isolated rat aortic spirals, and these contractions could be delayed or prevented, but not reversed by the thromboxane receptor antagonist SQ 29,548. Palmitoyl-LPC slightly potentiated aortic contractions induced by the thromboxane receptor agonist, U-46,619, and diminished in a concentration-dependent manner the antagonism by SQ 29,548 of contractile responses to U-46,619. These findings are consistent with a potential for LPC species to bind and activate thromboxane receptors.
Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543-4000.
D W
DW
Phillipson
A A
AA
Tymiak
J G
JG
Tuttle
K S
KS
Hartl
T W
TW
Harper
M S
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Bolgar
G T
GT
Allen
M L
ML
Ogletree
D000818:Animals; D001013:Aorta, Thoracic / Q000187:drug effects / Q000378:metabolism; D002250:Carbon Radioisotopes; D002851:Chromatography, High Pressure Liquid; D004952:Esters / Q000097:blood / Q000494:pharmacology; D005227:Fatty Acids / Q000097:blood / Q000494:pharmacology / Q000502:physiology; D006801:Humans; D066298:In Vitro Techniques; D008244:Lysophosphatidylcholines / Q000097:blood* / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism; D011522:Protons; D011869:Radioligand Assay; D051381:Rats; D017207:Rats, Sprague-Dawley; D017482:Receptors, Thromboxane / Q000037:antagonists & inhibitors* / Q000502:physiology*; D016339:Spectrometry, Mass, Fast Atom Bombardment
D002250:Carbon Radioisotopes; D004952:Esters; D005227:Fatty Acids; D008244:Lysophosphatidylcholines; D011522:Protons; D017482:Receptors, Thromboxane; C006065:We 201