Protein kinase inhibition and the oestrogen-like relaxant effects of genistein on isolated rat aorta.

There is epidemiological evidence for a protective property of oestrogen on vasculature which may be linked to an ability to relax vascular smooth muscle [I]. 170 oestradiol was shown to relax rabbit aorta and coronary arteries [2,3] and dilate rat tail artery [4]. The biochemical target for this action is unclear, although effects on calcium flux have been implicated. Activation of protein kinase C increases vascular tone [5] and in isolated rat aorta, direct activation of protein kinase C by phorbol dibutyrate (PDBu) induces a tonic, prolonged contraction. We have demonstrated that the pharmacological properties of this response are altered dramatically by manipulation of extracellular calcium levels, and that both calcium dependent and independent contractions induced by phorbol dibutyrate are sensitive to relaxation by oestrogens; this and other studies led us to conclude that the relaxant effects of oestrogens on vasculature could operate independently of calcium related effects [6,7] and might be at least partially due to direct and selective interaction with protein kinase C isoforms [8]. Genistein is an isoflavonoid with oestrogen like activity, and has an established inhibitory activity on protein tyrosine k m e s [e.g. 91. Varying reports exist of inhibitory effects against protein kinase C [9,10,11]. It was therefore of interest to compare the pharmacological and biochemical profile of these two relaxants. Experiments on isolated rat aorta were carried out according to standard protocols as summarised below (Table 1). For protein kinase C separation, four brains from male Hooded Lister rats (Bradford Strain, 250 4008) were homogenised at 4°C in four volumes of 20mM Tris-CI pH 7.5 containing 10 mM EGTA, 5 mM EDTA, 1 mM dithioerythreitol, 1 mM benzamidine, 1 mM phenylmethylsulphonyl fluoride and 10 pg ml-' leupeptin. The homogenate was centrfiged at 15 000 g for 15 minutes, and the supernatant subjected to hydroxyapatite chromatography using a Gradifrac automated chromatography system (Pharrnacia). Proteins were eluted on a 20 400 mM, 100 ml phosphate gradient (pH 7.5) containing 5 mM EGTA , 2.5 mM EDTA, 1 mM dithioerythreitol, 1 mM P-mercaptoethanol and 1 mM benzamidine. 4 ml fractions were collected, and protein kinase C eluted throughout most of the gradient. Activity was assayed using 10 p1 aliquots of each fraction in a total volume of 60 p1 containing 3.3 pmoles ATPI 1000 nCi y "P ATP, 10 mg ml-' histone HIS, 0.2 mM MgC12, 0.2 mM CaCI2, 2 mM EGTA, 5 mM HEPES pH 7.5 and 0.33 mg m1-I phosphatidylserine (Lipid Products Inc., Surrey) in 0.15 'YO Triton X100 (final dilution in assay )(adapted from [ 121).