Ernst-Christian Witte

Irreversible inhibition of the TXA2/PGH2 receptor of human platelets by a photoaffinity ligand

In order to tag the TXA2/PGH2 receptor of human platelets, we synthesized azido-BSP (= 4-[2-(4-azido-benzenesulfonylamino)-ethyl]phenoxyacetic acid), a photolabile derivative of the specific TXA2/PGH2 receptor antagonist sulotroban (= BM 13.177). If protected from UV light, azido-BSP competitively inhibited the shape change of human washed platelets stimulated by the TXA2 mimetic U 46619. Schild analysis revealed a pA2 = 6.7 (apparent KD = 0.2 mumol/l). Irreversible inhibition of the U 46619-induced platelet activation was achieved by irradiating for 5 min with UV light of 254 nm a platelet suspension containing azido-BSP. After subsequent washing, the platelets were stimulated with U 46619, ADP or PAF. Under these conditions azido-BSP inhibited the shape change, aggregation and [3H]serotonin release induced by U 46619 but not the shape change induced by ADP or PAF. The concentrations of azido-BSP which blocked the U 46619-induced [3H]serotonin release and the aggregation were 0.5 mumol/l and 1.0 mumol/l, respectively, whereas even 50.0 mumol/l of azido-BSP only partially inhibited the U 46619-stimulated shape change. Obviously, increasing numbers of thromboxane receptors have to be blocked in order to inhibit the [3H]serotonin release, the aggregation and the shape change. Even at an azido-BSP concentration equal to 250 times the apparent dissociation constant, enough receptor sites remained active to allow U 46619 to induce the shape change. In sulotroban was added prior to irradiation, the blocking effect of azido-BSP decreased with increasing concentrations of sulotroban. These results show that azido-BSP is a specific and high affinity ligand of the TXA2/PGH2 receptor and that it covalently links to the receptor under irradiation. Azido-BSP is a new tool to identify and characterize the TXA2/PGH2 receptor.

Reduction of BM 15.766-induced 7-dehydrocholesterol accumulation by bezafibrate and mevinolin in rats

SummaryThe effects of mevinolin, a 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitor and bezafibrate, a modulator of lipoprotein metabolism, were measured on BM 15.766-induced 7-dehydrocholesterol (7-DHC) accumulation in liver and serum of rats. BM 15.766, an inhibitor of Δ7 sterol reductase, leads to an accumulation of 7-DHC, which can be used as a measure of cholesterol (CH) synthesis de novo. The investigations were carried out to evaluate the usefulness of this new non-isotopic in vivo method for testing compounds that affect directly and indirectly the CH-biosynthetic pathway.Mevinolin showed a dose-dependent reduction of BM 15.766-induced 7-DHC accumulation after a single oral dose. The dose range for reduction of 7-DHC in the liver of rats was comparable with that for serum CH-lowering in humans. Bezafibrate reduced the BM 15.766-induced 7-DHC accumulation in liver in a dose- and time-dependent manner. These findings agree with the reported reduced activity of HMG-CoA reductase and support the view, that bezafibrate reduces CH biosynthesis by modulation of lipoprotein metabolism. The 7-DHC levels in serum do rot reflect those in the liver and cannot be used as a measure of CH biosynthesis. The investigations show that BM 15.766-induced 7-DHC accumulation in liver of rats is an appropriate measure for CH de novo synthesis and can be used for testing compounds that interfere directly and indirectly with the CH-biosynthetic pathway. In contrast to previously described methods, no radiolabelled precursors are necessary.The extraction of 7-DHC and its photometric determination is, in comparison with separation and measurement of radioactive cholesterol, a relatively simple analytical procedure.