Chem. 260, 10039- 10043. Evans, F.J., Parker, P.J., Olivier, A.R., Thomas, S., Ryves,. W.J., Evans, A.T., Gordge, P. and Sbarma, P. (1991). Biochem. Soc. Trans.
Biochemical Society Transactions ( 1 996) 24
Investigation of the role of protein kinase C inhibition in the cnkium independent relaxant effects of oestrogens on isolated rat aorta. HOSSEIN BABAEI, A. TUDOR EVANS, GRAHAM IRVING AND JANICE R. McCURRIE. Postgraduate Studies in Pharmacology, School of Pharmacy, University ofBradford. W. Yorks. BD7 IDP. Epidemiological tindings have identified protective properties of oestrogens on vasculature which may be linked to their abdity to relax vascular smooth muscle [I]. The biochemical target for this action is unclear, although effects on calcium flux have been implicated [2]. In isolated rat aorta, direct activation of protein kinme C by phorbol dibutyrate 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 semitive to relavaion 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 [3,4,], (Table I), and might be at least partially due to direct interaction with protein kinase C isofom. Therefore, in this study, we have compared the effects of oestrogens on protein kinase C activity from rat brain, which is a rich source of the enzyme, with actions on aorta taken h m the same rat strain. Four brains from male Hooded Lister rats (Bradford Strain, 250 400g) were homogenised at 4'C in four volumes of 2OmM Tris-CI pH 7.5 conhirung 10 mM EGTA, 5 mM EDTA, 1 mM dithioerythreitol, 1 mM benzamidme, 1 mM phenylmethylsulphonyl fluoride and 10 pg ml" leupeptin. The homogenate was centrifuged at 15 OOO g for 15 minutes, and the supernatant subjected to either DEAE 52 cellulose anion exchange or hydroxylapatite chromatography using a Gradiffac automated chromatography system (Pharmacia). In the former case, proteins were eluted on a 0 - 0.4 M, 100 ml NaCl gradient in 20 mM Tris -C1 containing 5 mM EGTA , 2.5 mM EDTA, 1 mM dithioerythreitol, 1 mM 0mercaptoethanol and 1 mM benzamidine. In the latter treatment, proteins were eluted with a 20 - 250 mM, 100 ml phosphate gradient in sodium p h p h a t e pH 7.5 containing the same additions. In each qx3e, 3 ml fractions were collected. Protein kinase C eluted as a single narrow peak in the 0.1 -0.2 M NaCl range from DEAE 52, and throughout most of the phosphate gradient from hydroxylapatite. Activity was assayed using 10 p1 aliquots of each fraction in a total volume of 60 pl containing 3.3 pmoles ATPI 1000 nCi y l2 P ATP, 10 mg ml - I histone IIIs, 0.2 mM MgCh, 0.2 mM CaC12, 2 mM EGTA, 5 mM HEPES pH 7.5 and 0.33 mg ml - I phosphatidylserine (Lipid Products Inc., Surrey) in 0.15 % Triton X-100(Mdilution in assay),(adapted from [5]).
The reaction was carried out at 30°C for ten minutes, initiated by addition of ATP, and terminated by addition of 25p1 10 % orthophosphoric acid and rapid spotting of a 20 p1 aliquot onto phosphocellulose paper strips (Whatman). The strips were washed extensively in 5 YOacetic acid and counted without scintillant by Cerenkov radiation. In assays where phorbol dibutyrate and oestrogens were tested, they were included in the micelle phospholipid mixture without use of a solvent vehicle. Oestrogens were found to possess weak inhibitory activity of the DEAE 52 resolved, pooled enzyme activity, when compared with their abdity to relax aorta precontracted with phorbol dibutyrate, i d in comparison to the selective protein kinase C inhibitor, bisindolylmaleimide (Table 1). Fig 1. Inhibition of Drotein kinase C activity in fractions from hvdroxylaDatite Drofile of rat brain suDematant by p- oestradiol.
I
1.
2. 3. 4.
Agent (pM) 17-p oestradiol(20) Bisindolylmnleimide (1)
?4relaxatioa (aorta) 100
81.2 (* 5.0)
%inhibition (brain PKC) 36 (i5.2) 86.4 (i12.4)
377s
5.
6.
I-PW
- .- - .-PD&JbEST]
Henderson, B.E. and Paganini-Hill, A. (1991) Arch. Intern. Med. 151,75-78. Gisclard, V., Miller, V.M. & Vanhoutte, P.M. (1 988) J. Pharmacol. Exp. Therap. 244, 19-22. Babaei, H., Irving, G. & McCurrie, J.R. (1995) Br. J. Pharmacol. 115, 152P Babaei, H., Evans, A.T., Irving, G. and McCurrie, J.R (1 996) Br. . I Pharmacol. 116, (in press). Hannun, Y . A., Loomis, C.R. and Bell, R.M. (1 985) J. Biol. Chem. 260, 10039-10043. Evans, F.J., Parker, P.J., Olivier, A.R., Thomas, S., Ryves, W.J., Evans, A.T., Gordge, P. and Sbarma, P. (1991) Biochem. Soc. Trans. 19, 397-402.
