Abstract
The possible presence of an unknown prostacyclin synthesis inhibitory substance has been reported in some strains of rats. We purified the inhibitory substance from the heart of spontaneously hypertensive rats by collecting active fractions after gel-filtration column chromatography and two steps of reverse-phase high performance liquid chromatography. The amino acid composition and automated gas-phase sequencing of the full-length substance and fragments cleaved by AspN indicated that the prostacyclin-inhibitory peptide was identical to coupling factor 6. Recombinant rat coupling factor 6, which was synthesized using a cleavable fusion protein strategy, attenuated base-line and bradykinin (10(-6) M)-induced prostacyclin synthesis and [3H]arachidonic acid (AA) release in human umbilical vein endothelial cells in a dose-dependent manner (10(-9)-10(-7) M). Exogenous AA- and prostaglandin H2-induced prostacyclin synthesis were unchanged even after treatment with 10(-7) M recombinant coupling factor 6. Base-line and bradykinin-induced [3H]AA release were suppressed by arachidonyltrifluoromethyl ketone, a relatively specific inhibitor of cytosolic phospholipase A2 at 40 microM, and simultaneous administration of coupling factor 6 showed no further effect. Neither oleyloxyethyl phosphorylcholine at 1 microM nor bromoenol lactone at 1 microM affected AA release. Preincubation (1 min) with 10(-7) M recombinant coupling factor 6 had no influence on adenosine diphosphate- and collagen-induced platelet aggregations. We conclude that coupling factor 6 possesses a novel function of prostacyclin synthesis inhibition in endothelial cells via suppression of Ca2+-dependent cytosolic phospholipase A2, although it is unclear whether coupling factor 6 functions in normal conditions or only in pathophysiological states.
Highlights
Prostacyclin, a potent vasodilator and the most potent endogenous inhibitor of platelet aggregation known, is synthesized from arachidonic acid (AA)1 by various stimuli in many types of cells, including vascular endothelial cells and smooth
We tried to isolate and identify the prostacyclin synthesis inhibitory substance(s) by using spontaneously hypertensive rats (SHR), which appear to be a suitable source for isolarats; OPC, oleyloxyethyl phosphorylcholine; BEL, bromoenol lactone; DMEM, Dulbecco’s modified Eagle’s medium
Purification and Determination of Prostacyclin Synthesis Inhibitory Peptide—As shown in Fig. 1a, 6-keto-PGF1␣ production was most strongly suppressed by the heart extract, and its suppression was abolished by pretreatment with trypsin and chymotrypsin
Summary
Materials—SHR (16 –20 weeks old) were obtained from Charles River, Japan. [3H]6-Keto-PGF1␣ and [3H]AA were purchased from NEN Life Science Products. The cells were washed twice with 700 l of serum-free DMEM and incubated with various concentrations of coupling factor 6 in the absence or presence of 10Ϫ6 M bradykinin in 250 l of serum-free DMEM for 30 min at 37 °C. The cells were labeled for 24 h with [3H]AA (0.25 Ci in 250 l of complete medium/well at 37 °C), washed twice with 700 l of serumfree DMEM, and preincubated for 10 min in the same medium This medium was removed, and the cells were incubated with various concentrations of coupling factor 6 in both the absence and presence of 10Ϫ6 M bradykinin in 250 l of serum-free DMEM at 37 °C.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
