The Effect of Carbacyclin, a Prostaglandin Analogue, on Adenylate Cyclase Activity in Platelet Membranes

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Volume 165, number 2 FEBS 1118 January 1984

The effect of carbacyclin, a prostaglandin analogue, on adenylate cyclase activity in platelet membranes

Janet M. Stein and B.R. Martin

University of Cambridge, Department of Biochemistry, Tennis Court Road, Cambridge CB2 lQW, England

Received 4 November 1983

The effect of carbacyclin, a chemically stable analogue of prostacyclin, on the activity of adenylate cyclase in platelet membranes was measured, and compared with the effect of PGEr. When GTP was added in concentrations up to 10 PM the activation of adenylate cyclase by carbacyclin was increased, whereas higher concentrations of GTP were inhibitory. The addition of a non-hydrolysable analogue of GDP, guanosine 5’-[fi-thioldiphosphate (GDP[PS]) resulted in a dose-dependent inhibition of adenylate cyclase activation by carbacyclin; this inhibition was relieved by adding increased amounts of GTP.

Carbacyclin PGE, Adenylate cyclase Platelet membrane GTP GDPM

1. INTRODUCTION 2. MATERIALS AND METHODS

Agents which raise cyclic AMP levels in platelets Carbacyclin synthesized as in [5], was kindly also inhibit platelet aggregation. This is consistent supplied by Dr B.J.R. Whittle, Wellcome Research with the potent anti-aggregatory action of ‘pros- Laboratories (Langley Court, Beckenham, Kent) tacyclin (PGI2) a prostaglandin produced by the and PGEi was purchased from Sigma (Poole, vascular wall which activates adenylate cyclase and Dorset). Stock solutions of carbacyclin and of raises cyclic AMP levels in platelets [1,2]. Com- PGEi were made up in absolute ethanol and stored parisons have been carried out on the action of at - 20°C. When required they were diluted with PGIz and PGEi on inhibition of platelet aggrega- water, added to the adenylate cyclase assay tion, on changes in cyclic AMP levels [1,2] and on cocktail at pH 7.4, and kept in ice until the assay adenylate cyclase activity [2] and it seems likely was started. GDP[PS] was purchased from Boehr- that PGIz may act on the same receptor as PGEi inger (Lewes, East Sussex). in platelets [3]. Membranes were prepared from platelet concen- The experimental use of PGIz is complicated by trates as in [6]. As reported, the dose-response its instability at physiological temperature and pH. curves to PGEi for adenylate cyclase activity were The development of carbacyclin, a chemically similar for membranes from platelet concentrates stable analogue of prostacyclin has recently led to and from fresh human platelets. Adenylate cyclase detailed studies on the mechanism of inhibition of activity was assayed as in [7]. The assay contained platelet aggregation, both in vivo and in vitro [4]. 25 mM Tris-HCl buffer (pH 7.4), 0.1 mM ATP, 1 We have here examined the action of carbacyclin &i [QY~‘P]ATP, 0.1 mM cyclic AMP, 10 mM on adenylate cyclase activation in platelet mem- MgC12, 1 mM dithiothreitol, 5 mM phosphocre- branes and the role of GTP in this activation. A atine and 5 units of creatine kinase. Platelet mem- comparison has been made with the action of branes (20-40 pg protein) were incubated for 10 PGEi in this system. min at 37°C in the assay (vol. 0.1 ml). The activity of adenylate cyclase was linear for at least 10 min Abbrevlarioiir GDP[flS], guanosine 5 ’ -[@-thio]diphos- under all conditions. phate Protein was determined as in [8].

Published by Elsevier Science Publishers B. V. 290 001457593/84/$3.00 0 1984 Federationof European BiochemicalSocieties Volume 165, number 2 FEBSLETTERS January 1984

3. RESULTS

A comparison of the dose-response relation of adenylate cyclase in platelet membranes to carbacyclin and to PGEr in the presence of GTP (10 &Q is shown in fig.1. The dose-response curves were very similar, with the curve for carbacyciin slightty to the left of that for PGEI. The maximal levels of adenylate cyclase activity were the same for both carbacyclin and PGEI. Fig.2 shows the ef- 2 “, fect of adding increasing amounts of GTP to ” J 50 I platelet membranes incubated with carbacyclin (1 m E PM). The adenylate cyclase activity increased as 8 the concentration of added GTP was increased, 4 0 I 4t ’ i i with maximal activity observed at 10 pM GTP. 0 to-9 lo’* lo-’ 10-6 105 10.4 Higher concentrations of GTP (100 PM and mM) Prostaglandm concentration CM) resulted in some inhibition. Fig. 1. Dose-response relation of platelet membranes to The effect of GTP on the stimulation of carbacydin and PGEI. Platelet membranes (22 pg pro- adenylate cyclase was further examined using tein/assay) were incubated for 10 min at 37’C with GTP (10 PM) and with PGEi {o) or carbacyclin (e). GDP[@], a non-hydrolysable analogue of GDP Adenylate cyclase activity was determined as described which competes with GTP and other guanine in section 2. Each point is the mean value from 3 nucleotides and thus inhibits adenylate cyclase [9J. separate incubations f SE.

a OL 4- a 0 10’6 10’5 10-4 10.3 OL‘---ii- ’ J 0 10.8 IO” 10~6 10-s 10‘4 IO.3 GTP concentrataon CM)

GTP concentration (MI Fig.3. Effect of GDP[@S] and GTP on carbacyclin- Fig.2. Effect of increasing concentrations of GTP on stimulated adenylate cyclase activity. Platelet mem- carba~yclin-stimuIated adenylate cyclase activity. branes (20 gg protein/assay) were incubated for 10 min Platelet membranes (37 pg protein/assay) were in- at 37% with carbacyciin (0.1 FM) and GTP at the con- cubated for 10 min at 37’C with carbacyclin (1 FM) and centrations indicated. GDP[flS] concentrations were: GTP at the concentrations indicated. Basal activity in (0) none; (0) 10 pM; (0) 0.1 M; (H) 1 mM. Basal ac- the absence of any additions was 19 f 0.8 pmol. mg tivity (no additions) was 81.1 f 5.0 pmol. mg protein-’ -min-‘. Each point is the mean value from 3 protein-’ . min- ‘, Each point is the mean of 3 separate separate incubations + SE. incubations YZSE.

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Platelet membranes were incubated with car- gregation, and a more marked difference between bacyclin (0.1 PM) and the amount of added GTP the levels of cyclic AMP produced by PGI2 and and GDP[PS] systematically varied (fig.3). PGEi, but in which the adenylate cyclase activa- Whereas the addition of 10 FM GDPVS] had little tion was the same for both prostaglandins (21. effect on adenylate cyclase activity, the addition of We conclude that carbacyclin and PGEi activate 0.1 mM GDP[flS] and 1 mM GDP[&S] progressive- adenylate cyclase to a similar maximal extent in ly inhibited activation. platelet membranes, and that the effect is mediated by GTP. 4. DISCUSSION ACKNOWLEDGEMENTS The dose-response curves of adenylate cyclase to carbacyclin and to PGEi were very similar. We wish to thank Dr B.J.R. Whittle, Wellcome There was also a concentration-dependent effect of Research Laboratories, for a generous gift of car- GTP on the activation of adenylate cyclase by carbacyclin, and the East Anglian Regional Transfu- bacyclin. The role of GTP was further defined us- sion Service, Cambridge, for platelet concentrates. ing GDP[PS], which resulted in a dose-dependent The work was supported by a Project Grant from inhibition of the carbacyclin activation of the Medical Research Council. adenylate cyclase; this was comparable to the inhibitory effect of GDP[PS] on the activation of adenylate cyclase by PGEr [6]. REFERENCES Carbacyclin has been shown to be rather more potent (about 2-fold) than PGEr in the inhibition VI Tateson, J.E., Moncada, S. and Vane, J.R. (1977) Prostaglandins, 13, 389-397. of ADP-induced aggregation of human platelets in PI Gorman, R.R., Bunting, S. and Miller, O.V. (1977) vitro [4]. However, we here found little difference Prostaglandins 13, 377-388. between the dose-response curves of adenylate t31 Whittle, B.J.R., Moncada, S. and Vane, R.R. (1978) cyclase activated by carbacyclin and PGEr , and the Prostaglandins 16, 373-388. maximum stimulation was the same. It appears 141Whittle, B.J.R., Moncada, S., Whiting, F. and that the differential effects of carbacyclin and Vane, J.R. (1980) Prostaglandins 19, 605-627. PGEi on inhibition of platelet aggregation are not 151Morton, D.R., Bundy, G.L. and Nishizawa, E.E. related to different degrees of activation of (1979) in: Prostacyclin (Vane, J.R. and Bergstrom, adenylate cyclase by these compounds. We can S. eds) Raven Press, New York. speculate that there is a further point of regulation 161 Stein, J.M. and Martin, B.R. (1983) Biochem. J. 214, 231-234. of cyclic AMP levels, possibly by phospho- 171 Salomon, Y., Londos, C. and Rodbell, M. (1974) diesterase, and that in intact platelets different Anal. Biochem. 58, 541-548. levels of cyclic AMP may result from stimulation 181 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and by carbacyclin and PGEi. This effect has been Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. described in platelets stimulated by PGI2 and 191Eckstein, F., Cassel, D., Levkovitz, H., Lowe, M. PGEr, in which PGI2 had a much higher potency and Selinger, Z. (1979) J. Biol. Chem. 254, (lo-fold) than PGEi in inhibition of platelet ag- 9829-9834.

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