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Actions of nitric oxide on the release of prostacyclin from bovine endothelial cells in culture
European Journal of Pharmacology, 151 (1988) 19-25
19
Elsevier EJP 50315
Actions of nitric oxide on the release of prostacyclin from bovine endothelial cells in culture M. G a b r i e l l a D o n i a, B r e n d a n J.R. W h i t t l e *, R i c h a r d M.J. P a l m e r a n d S a l v a d o r M o n c a d a Wellcome Research Laboratories, Beckenham, Kent BR3 3BS, U.K.
Received 4 February 1988, revised MS received 22 March 1988, accepted 29 March 1988
Endothelial cells release the potent vasodilator prostacyclin, as well as the highly labile endothelium-derived relaxing factor (EDRF) which mediates vascular relaxation induced by some vasodilators including acetylcholine and bradykinin. EDRF has recently been characterised as nitric oxide (NO). The effects of NO on prostacyclin release, measured as 6-keto-PGFl,,, from endothelial cells obtained from bovine thoracic aorta, have now been investigated. Incubation of endothelial cells in culture with bradykinin (10-100 nM) stimulated the release of 6-keto-PGFl~. Pre-incubation (0.5-2 min) with NO (13-130 /.tM) caused a significant dose-dependent inhibition of 6-keto-PGFl,, release, reaching a maximum of 29 + 4% inhibition. Pre-incubation with superoxide dismutase (30 units ml-1) which prevents the breakdown of NO, significantly augmented the degree of inhibition, as did the selective inhibitor of cyclic GMP phosphodiesterase, M & B 22948 (5 /~M), reaching 51 + 2% inhibition. The potentiation by M & B 22948 suggests that this inhibitory effect of high concentrations of NO is brought about by elevation of intracellular cyclic GMP levels following activation of guanylate cyclase. Whether endogenous NO is produced by endothelial cells under physiological conditions in sufficient quantities to modulate prostacyclin release remains to be established. Endothelial cells; Prostacyclin; 6-keto-PGFl~; Endothelium-derived relaxing factor (EDRF); Nitric oxide; Bradykinin
1. Introduction
Vascular tissue synthesizes the unstable arachidonate metabolite prostacyclin, which is a potent vasodilator and inhibitor of platelet aggregation (Moncada et al., 1976). Prostacyclin is released from endothelial cells by a variety of stimuli including bradykinin, arachidonic acid, thrombin, the ionophore A23187 and histamine (Weksler et al., 1978). Vascular endothelium also releases the highly labile moiety, endothelium-derived relaxing factor (EDRF), which mediates the vascular relaxation of some vasodilators including acetylcho-
l Present address: Institute of Human Physiology, Padova 35131, Italy. * To whom all correspondence should be addressed.
line and bradykinin (Furchgott and Zawadzki, 1980; Furchgott, 1983). This action is brought about by the stimulation of soluble guanylate cyclase with the subsequent increase in vascular g u a n o s i n e - 3 ' : 5'-cyclic m o n o p h o s p h a t e (cyclic G M P ) levels (Rapoport and Murad, 1983). Like prostacyclin, E D R F has been shown to inhibit platelet aggregation in vitro (Azuma et al., 1986; Furlong et al., 1987; Radomski et al., 1987a). However, in contrast to the anti-aggregating action of prostacyclin which is brought about by increases in intracellular cyclic A M P ( G o r m a n et al., 1977; Tateson et al., 1977), this effect of E D R F is likely to be elicited via the elevation of cyclic G M P levels (Radomski et al., 1987a). Recently, E D R F has been characterized as nitric oxide (NO; Palmer et al., 1987). N O exhibits a comparable stability and pharmacological pro-
0014-2999/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
20
file on vascular smooth muscle as E D R F (Palmer et al., 1987; Hutchinson et al., 1987; Furchgott, 1987). Furthermore, N O is released from endothelial cells in sufficient quantities to account for the biological actions of E D R F (Palmer et al., 1987). It has also been demonstrated that subthreshold doses of prostacyclin and of E D R F or authentic NO can synergistically interact to inhibit substantially platelet aggregation in vitro, and such potentiating interactions may reflect a physiological mechanism for maintaining platelet homeostasis at the vascular endothelium (Radomski et al., 1987b). The interaction between the synthesis of these two endothelium-derived mediators has now been investigated by determining the effects of exogenous N O on the release of endogenous prostacyclin, measured by radioimmunoassay as its stable chemical degradation product 6-keto-PGFa~, from bovine endothelial cells in culture.
2. Materials and methods 2.1. Endothelial cell culture
Fresh bovine thoracic aortas were obtained from an abattoir and the endothelial cells subsequently prepared by collagenase digestion as previously described (Gryglewski et al., 1986a). Cells were plated onto 12-well (24 mm diameter) tissue-culture plates (Linbro, Flow Labs.) at a density of 1.5 x 105 cells/well in Dulbecco's modified Eagle's medium (Gibco) containing 15% foetal calf serum (Flow Labs.) and an antibacterial mixture, penicillin (100 units ml-a), streptomycin (10 /~g m1-1) and gentamycin (50 /~g ml-1). These plates were incubated at 3 7 ° C in a humidified atmosphere containing 5% CO 2 for 4-6 days until the cells formed a confluent monolayer (cell count, 1-1.3 × 106/well). All the experiments were performed with cells from the first three passages. 2.2. Incubation conditions
To measure the release of 6-keto-PGFl~ by endothelial cell monolayers, the culture medium was aspirated and the monolayers were gently
rinsed twice with 1 ml of prewarmed (37°C) Hanks' balanced salt solution without phenol red (Gibco) containing CaC12 (2.3 mM) and adjusted to p H 7.4 with 20 mM HEPES buffer. After rinsing, Hanks' balanced salt solution was added to each well followed by the addition of the agents under investigation (final volume in the well, 1 ml). In studies on the actions of NO, aliquots (1-100 /~1) of a solution of authentic NO (0.1-3% in He-deoxygenated distilled water) stored in a glass vessel (Palmer et al., 1987) were removed with a gas-tight syringe and immediately added to the Hanks' balanced salt solution in the wells. Controls were always performed with the same volume of the He-deoxygenated distilled water, added to parallel wells. After 2 min incubation, the stimulatory agent, bradykinin (10-100 nM) was added and incubated for 2.5-10 min at 37 ° C. In preliminary studies, the inhibitory effects of NO following preincubation for either 0.5, 1 or 2 min were comparable and hence a 2 min incubation period was employed to allow a fully randomized experimental design to be utilized on each plate. The incubation medium was then aspirated and transferred into plastic Eppendorf tubes and frozen at - 2 0 ° C prior to determination of 6keto-PGFl~ levels. All experimental, procedures were performed in triplicate on each batch of cells. 2.3. Radioimmunoassay of 6-keto-PGF1
Radioimmunoassay was performed as described previously (Salmon, 1978) and the results were expressed as the release of 6-keto-PGFl~ (nM over 10 rain/106 cells in 1 ml) or as the % inhibition of release. Cross reactions with other prostaglandins were less than 3% control. Control studies with N O (130 /~M) and the other agents indicated that they did not interfere with the radioimmunoassay. 2.4. Materials
Authentic NO ( > 98.9% pure; British Oxygen Corporation) was prepared freshly as 0.1 and 3% ( v / v ) solutions in He-deoxygenated, distilled and deionized water, as described previously (Palmer et al., 1987). Bradykinin and superoxide dis-
21
mutase, obtained from Sigma Chemical Co. were prepared in 0.85% saline as a stock solution and diluted in Hanks' balanced salt solution. Stock solutions of M & B 22948 (2-O-propoxyphenyl-8azapurin-6-one; a gift from May & Baker) were prepared in triethanolamine (20% v/v); the final concentrations of triethanolamine in the endothelial cell incubation did not exceed 0.01%. Purified human haemoglobin was prepared according to the method of Paterson et al. (1976) and diluted in Hanks' balanced salt solution.
3O
z 20
_o
_= z 10
1.3
13
39
130 IJM
[NO]
2.5. Statistical evaluation Results are expressed as means + S.E. of (n) separate experiments, each performed in triplicate. Student's t-test for paired or unpaired data, where appropriate, was used to determine the significance of differences between means and P < 0.05 was taken as statistically significant.
3. Results
3.1. Release of 6-keto-PGFj~ The basal release of 6-keto-PGFl~ from the bovine endothelial cell monolayer over the 10 min incubation period was 10 + I nM (n = 14; 3.8 _ 0.6 ng ml-1). Studies on the time course of release of 6-keto-PGFl~ by bradykinin (10-100 nM) indicated that maximal release was achieved between 5 and 10 min of incubation, and thus a 10 min incubation period was used in all further studies. Addition of bradykinin (10, 30 and 100 nM) to the endothelial cell monolayer induced a significant (P < 0.01) concentration-related stimulation of 6keto-PGFl~ release to 45 + 6 nM (n = 5), 53 _ 3 nM (n = 11) and 5 9 _ 6 nM (n = 5) respectively, over this 10 min incubation period.
3.2. Effects of nitric oxide
Fig. 1. Inhibition of bradykinin (30 nM)-stimulated release of 6-keto-PGF1, ~ release from bovine endothelial cells in culture by pre-incubation (2 min) with nitric oxide (NO, 1.3-130 jaM). Results, expressed as % inhibition of control release, are shown as m e a n s + S . E , of 3-7 experiments per group, where statistically significant difference from control value is shown as * P < 0.05.
39 and 130 ~tM) prior to the addition of bradykinin (30 nM), induced a dose-related inhibition of 6-keto-PGFl~ release, as shown in fig. 1, reaching a maximum of 29 + 4% inhibition (n = 7, P < 0.01). Incubation with N O (130 /~M) significantly (P < 0.05) reduced the 6-keto-PGFl~ release induced by bradykinin at concentrations of 30 and 100 nM (fig. 2). Pre-incubation with N O (130 /~M) for shorter time periods (0.5, 1.0 and 1.5 min) gave a comparable degree of inhibition, whereas when
[ • Control ] c
60-
~NO
50-
T
(1301JM)
¢'- 4 0 a.
I
o
30-
/
20-
w
< M,I
IO-
I1 i 30
5 BRADYKININ
Incubation of the endothelial cell monolayer with NO (1.3/~M), 2 min prior to the addition of bradykinin (30 nM) had no significant effect on the release of 6-keto-PGFl~ (n = 3). However, incubation with higher concentrations of N O (13,
l
100 nM
Fig. 2. Inhibition of bradykinin (30 and 100 nM)-stimulated release of 6-keto-PGFla from bovine endothelial cells in culture by pre-incubation (2 min) with nitric oxide (NO, 130 JAM). Results, expressed as 6-keto-PGF1, , release (nM), are shown as m e a n s + S . E , of (n) experiments, where statistically significant difference from the control is shown as * P < 0.05.
22
the pre-incubation period with NO (130/~M) was extended to 5 min, no significant inhibition of bradykinin-stimulated release could be observed. A 2 min incubation with NO (130 /~M) had no significant effect on the basal release of 6-ketoPGF1, (9 _+ 2 nM, n = 7). Incubation of endothelial cell monolayers with haemoglobin (1 /~M), 1 min prior to addition of NO (39 /~M), abolished its inhibitory action on bradykinin-stimulated release (n = 3). However, pre-incubation with higher concentrations of haemoglobin (10 #M) did not significantly alter either basal or bradykinin (30 nM)-stimulated 6-ketoPGF1, release (table 1). 3.3. Effects of superoxide dismutase and M & B 22948 Pre-incubation of endothelial cell monolayers for 2 min with superoxide dismutase (30 units ml-1), prior to the addition of NO (39 and 130 #M) augmented the inhibitory effect on bradykinin-induced release of 6-keto-PGF1, (fig. 3). Superoxide dismutase (30 units m1-1) alone did not significantly inhibit the basal or bradykinin (30 nM)-stimulated release of 6-keto-PGFl~ (table 1). Incubation with M & B 22948 (5 /~M) for 2 min, likewise significantly potentiated the inhibitory actions of NO (39 and 130 /~M) on the bradykinin-stimulated release of 6-keto-PGFl~ (fig.
TABLE 1 Effect of pre-incubation with superoxide dismutase (30 units m l - ] ) , M & B 22948 (5 ~M) or haemoglobin (10 FM) on the release of 6-keto-PGFl= from endothelial cell monolayers under basal and bradykinin (30 nM)-stimulated conditions. Results are shown as m e a n s + S.E. of (n) values, where none of the treatment groups were significantly different from the corresponding control. 6-keto-PGFl, , ( n M )
Control Superoxide dismutase (30 units m1-1) M&B22948(5/.tM) Haemoglobin (10 ttM)
Basal
Bradykinin
1 0 + 1 (14)
5 3 + 3 (11)
9_+1 (5) 1 0 + 2 (3) 9 + 1 (3)
59+8 55+3 57+3
(3) (4) (3)
i
50 z
40
0 ~" 30 -r
z
20
10 9 NO
+ + SOD M&B
30
5
U m r 1 gM
NO
+ + SOD M&B
30 5 Uml " l g M
Fig. 3. Potentiation of N O (39 and 130 ~M)-induced inhibition of bradykinin (30 nM)-stimulated 6-oxo-PGFl, release from bovine endothelial cells in culture by pre-incubation with superoxide dismutase (SOD, 30 units ml - l ) or M & B 22948 (5 /xM). Results, expressed as % inhibition, are shown as means + S.E. of (n) experiments where statistically significant difference from the effects of N O alone is shown as * P < 0.05, * * P < 0.01.
3). However, M & B 22948 (5/~M) alone failed to inhibit basal or bradykinin (30 nM)-stimulated 6-keto-PGF1, release (table 1).
4. Discussion
Both prostacyclin and the labile moiety EDRF, now characterised as NO (Palmer et al., 1987) are endothelial cell-derived mediators that exert vasodilator and anti-aggregatory activity. Since these mediators may therefore interact to modulate vascular tone and local haemostasis, we have investigated whether NO can influence the biosynthesis of prostacyclin. These present findings indicate that at low concentrations, NO (1.3/~M) does not affect basal or bradykinin-stimulated release of prostacyclin from bovine thoracic aorta cell monolayers in culture, under the incubation conditions used. At higher concentrations, NO (13-130 /~M) produced a dose-related inhibition of bradykinin-stimulated prostacyclin release, as determined by the levels of 6-keto-PGFl~. This inhibitory effect of NO was augmented in the presence of superoxide dismutase, which is known to prolong the duration of action of both E D R F and exogenous NO (Gryg-
23 lewski et al., 1986b; Rubanyi and Vanhoutte, 1986; Palmer et al., 1987). However, this enhancement by superoxide dismutase was modest, probably reflecting that the breakdown of N O under these conditions was not simply due to the presence of superoxide anions in the incubation medium or released from the endothelial cells. Thus, NO reacts readily with oxygen to produce N O 2 which then forms nitrite and nitrate ions in aqueous solution (Palmer et al., 1987). The mechanism underlying such inhibition of prostacyclin release by high concentrations of NO is not known. It would appear to reflect the action of NO itself, or an equally unstable moiety, since on more prolonged incubation, this inhibitory activity was no longer detectable. This finding also indicates that the inhibitory action of NO was reversible and not due to non-specific disruption of the cell or the cascade of enzymes involved in prostacyclin synthesis. The ability of the selective inhibitor of cyclic G M P phosphodiesterase, M & B 22948, (Lugnier et al., 1986) to augment significantly the degree of inhibition induced by NO suggests that this action was brought about by an action on the guanylate cyclase-cyclic G M P system. Previous studies have shown that M & B 22948 increases cyclic G M P levels but not cyclic AMP levels in porcine aortic endothelial cells (Martin and White, 1987). Furthermore, M & B 22948 increases cyclic G M P levels and induces relaxation in rabbit aorta in which the endothelial cells are present (Martin et al., 1986), as well as augmenting the platelet anti-aggregating action of E D R F and exogenous NO (Radomski et al., 1987a). Intracellular cyclic G M P is elevated by E D R F in vascular tissue (Rapoport and Murad, 1983), and is thought to regulate vascular smooth muscle calcium levels by inhibiting calcium influx and its mobilisation from intracellular stores (Collins et al., 1986). NO, as well as the nitrovasodilators which are considered to act by releasing NO, stimulate the formation of cyclic G M P in vascular smooth muscle (Katsuki et al., 1977; Arnold et al., 1977; Ignarro et al., 1981) and may thus modulate the intracellular calcium levels. Since calcium is an essential pre-requisite for the biosynthesis of prostacyclin, such an action on intracellular calcium
levels within endothelial cells may contribute to the inhibitory action of high concentrations of NO observed in the present study. In contrast to the present suggestion, it has been proposed that increases in endothelial cell cyclic G M P levels are associated with the stimulation of prostacyclin biosynthesis by bradykinin (Brotherton, 1986). However, since other agents that stimulated cyclic G M P formation such as nitroprusside or atriopeptin II did not stimulate prostacyclin biosynthesis in the same study, it was concluded that cyclic G M P does not play a primary role in stimulating prostacyclin formation. Other studies with the nitrates, including nitroglycerin, have likewise failed to demonstrate any consistent effect on prostacyclin production by human vascular fragments and cultured umbilical vein endothelial cells (De Caterina et al., 1985) despite the suggestion of earlier experiments (Levin et al., 1978). Likewise, in the present study, low concentrations of NO, which would be sufficient to induce relaxation in isolated strips of vascular tissue (Hutchinson et al., 1987) did not affect endothelial cell prostacyclin synthesis. The observation that much higher concentrations of NO did induce a moderate inhibition of prostacyclin of up to 30% may therefore reflect a pharmacological action not achieved by nitrovasodilators, although this effect may be unrelated to any physiological modulation of mediator release. However, since N O is highly unstable, the final concentration of the active species to which the endothelial cells were exposed under the incubation conditions used, is not known. In our further studies to investigate the possible interaction between endogenous NO and prostacyclin release, endothelial cells were incubated with haemoglobin, which prevents the actions of E D R F and N O (Martin et al., 1985; Keilin and Hartree, 1937; Palmer et al., 1987). Haemoglobin, in sufficient concentrations to abolish the inhibitory effects of exogenous NO, failed to affect either basal or stimulated prostacyclin release. This suggests that modulation of prostacyclin release by any endogenous N O derived from these endothelial cells is insignificant. Recent studies with the E D R F inhibitor, methylene blue have demonstrated inhibition of prostacyclin formation by
24
endothelial cells, but this action was unrelated to any inhibition of soluble guanylate cyclase (Martin and Drazan, 1987) and hence to any effect of endogenous NO. It would be of interest, however, to correlate the concentration of exogenous NO present under the incubation conditions with those levels produced endogenously from endothelial cells under comparable experimental conditions, as well as to investigate the effects of exogenous prostacyclin on the release of NO. Such studies should further clarify any interaction between these two endothelium-derived mediators in relation to their respective biosynthesis and the physiological or pathological relevance of such interaction in the regulation of vascular tone and local platelet function.
Acknowledgements We are grateful to N.A. Foxwell and M.J. Ashton for technical assistance.
References Arnold, W.P., C.K. Mittal, S.E. Katsuki and F. Murad, 1977, Nitric oxide activates guanylate cyclase and increases guanosine 3',5'-monophosphate levels in various tissue preparations, Proc. Natl. Acad. Sci. U.S.A. 74, 3202. Azuma, H., M. Ishikawa and S. Sekizaki, 1986, Endotheliumdependent inhibition of platelet aggregation, Br. J. Pharmacol., 88, 411. Brotherton, A.F.A., 1986, Induction of prostacyclin biosynthesis is closely associated with increased guanosine 3',5'-cyclic monophosphate accumulation in cultured human endothelium, J. Clin. Invest. 78, 1253. De Caterina, R., R. Dorso, B.S. Tack-Goldman and B.B. Weksler, 1985, Nitrates and endothelial prostacyclin production: studies in vitro, Circulation 71, 176. Collins, P., T.M. Griffith, A.H. Henderson and M.J. Lewis, 1986, Endothelium-derived relaxing factor alters calcium fluxes in rabbit aorta: a cyclic guanosine monophosphatemediated effect, J. Physiol. 381, 427. Furchgott, R.F., 1983, Role of endothelium in responses of vascular smooth muscle, Circ. Res. 53, 557. Furchgott, R.F., 1987, Studies on relaxation of rabbit aorta by sodium nitrite: the basis for the proposal that the acidactivatable inhibitory factor from bovine retractor penis is inorganic nitrite and the endothelium-derived relaxing factor is nitric oxide, in: Mechanisms of Vasodilation, Vol. IV., ed. P.M. Vanhoutte (Raven Press, New York) (in press). Furchgott, R.F. and J.V. Zawadzki, 1980, The obligatory role
of enthothelial cells in the relaxation of arterial smooth muscle by acetylcholine, Nature 228, 373. Furlong, B., A.H. Henderson, M.J. Lewis and J.A. Smith, 1987, Endothelium-derived relaxing factor inhibits in vitro platelet aggregation, Br. J. Pharmacol. 90, 687. Gorman, R.R., S. Bunting and O.V. Miller, 1977, Modulation of human platelet adenylate cyclase by prostacyclin (PGX), Prostaglandins 13, 377. Gryglewski, R.J., S. Moncada and R.M.J. Palmer, 1986a, Bioassay of prostacyclin and endothelium-derived relaxing factor (EDRF) from porcine aortic endothelial cells, Br. J. Pharmacol. 87, 685. Gryglewski, R.J., R.M.J. Palmer and S. Moncada, 1986b, Superoxide anion is involved in the breakdown of endothelium-derived vascular relaxing factor, Nature 320, 454. Hutchinson, P.A., R.M.J. Palmer and S. Moncada, 1987, Comparative pharmacology of EDRF and nitric oxide on vascular strips, European J. Pharmacol. 141,415. Ignarro, L.J., H. Lippton, J.C. Edwards, W.H. Baricos, A.L. Hyman, P.J. Kadowitz and C.A. Gruetter, 1981, Mechanism of vascular smooth muscle relaxation by organic nitrates, nitrites, nitroprusside and nitric oxide: Evidence for the involvement of S-nitrosothiols as active intermediates, J. Pharmacol. Exp. Ther. 218, 739. Katsuki, S., W. Arnold, C.K. Mittal and F. Murad, 1977. Stimulation of guanylate cyclase by sodium nitroprusside, nitroglycerin and nitric oxide in various tissues preparations and comparison to the effects of sodium azide and hydroxylamine, J. Cycl. Nucl. Prot. Phosphor. Res. 3, 23. Keilin, D. and E.F. Hartree, 1937, Reaction of nitric oxide with haemoglobin and methaemoglobin, Nature 139, 548. Levin, R.I., E.A. Jaffee, B.B. Weksler and K. Tack-Goldman, 1978, Nitroglycerin stimulates synthesis of prostacyclin by cultured human endothelial cells, J. Clin. Invest. 67, 762. Lugnier, C., P. Schoefter, A. Le Bec, E. Strouthou and J.C. Stoclet, 1986, Selective inhibition of cyclic nucleotide phosphodiesterases of human, bovine and rat aorta, Biochem. Pharmacol. 35, 1743. Martin, W. and K.M. Drazan, 1987, Methylene blue inhibits prostacyclin production by pig aortic endothelial cells, Br. J. Pharmacol. 92, 519P. Martin, W., R.F. Furchgott, C.M. Villani and D. Jothianandan, 1986, Phosphodiesterase inhibitors induce endothelium-dependent relaxation of rat and rabbit aorta by potentiating the effects of spontaneously released endothelium-derived relaxing factor, J. Pharmacol. Exp. Ther. 237, 539. Martin, W., G.M. Villani, D. Jothianandan and R.F. Furchgott, 1985, Selective blockade of endothelium-dependent and glyceryl trinitrate-induced ration by hemoglobin and by methylene blue in the rabbit aorta, J. Pharmacol. Exp. Tiler. 232, 708. Martin, W. and D.G. White, 1987, Pig aortic endothelial cells contain both soluble and particulate guanylate cyclase isoenzymes, Br. J. Pharmacol. 90, 18P. Moncada, S., R. Gryglewski, S. Bunting and J.R. Vane, 1976, An enzyme isolated from arteries transforms prostaglandin
25 endoperoxides to an unstable substance that inhibits platelet aggregation, Nature 263, 663. Palmer, R.M.J., A.G. Ferrige and S. Moncada, 1987, Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor, Nature 327, 524. Paterson, R.A., P.A.M. Eagles, D.A.B. Young and C.R. Beddell, 1976, Rapid preparation of large quantities of human haemoglobin with low phosphate content by counter-flow dialysis, Int. J. Biochem. 7, 117. Radomski, M.W., R.M.J. Palmer and S. Moncada, 1987a, Comparative pharmacology of endothelium-derived relaxing factor, nitric oxide and prostacyclin in platelets, Br. J. Pharmacol. 92, 181. Radomski, M.W., R.M.J. Palmer and S. Moncada, 1987b, The anti-aggregating properties of vascular endothelium: interactions between prostacyclin and nitric oxide, Br. J. Pharmacol. 92, 639.
Rapoport, R.M. and F. Murad, 1983, Endothelium-dependent nitrovasodilator-induced relaxation of vascular smooth muscle: role for cyclic GMP, J. Cycl. Nucl. Prot. Phosphor. Res. 9, 281. Rubanyi, G.M. and P.M. Vanhoutte, 1986, Superoxide anions and hyperoxia inactivate endothelium-derived relaxing factor, Am. J. Physiol. 250, H822. Salmon, J.A., 1978, A radioimmunoassay for 6-keto-prostaglandin FI~ , Prostaglandins 15, 383. Tateson, J.E., S. Moncada and J.R. Vane, 1977, Effects of prostacyclin (PGX) on cyclic AMP concentrations in human platelets, Prostaglandins 13, 389. Weksler, B.B., C.W. Ley and E.A. Jaffe, 1978, Stimulation of endothelial cell prostacyclin production by thrombin, trypsin, and ionophore A23187, J. Clin. Invest. 62, 923.
19
Elsevier EJP 50315
Actions of nitric oxide on the release of prostacyclin from bovine endothelial cells in culture M. G a b r i e l l a D o n i a, B r e n d a n J.R. W h i t t l e *, R i c h a r d M.J. P a l m e r a n d S a l v a d o r M o n c a d a Wellcome Research Laboratories, Beckenham, Kent BR3 3BS, U.K.
Received 4 February 1988, revised MS received 22 March 1988, accepted 29 March 1988
Endothelial cells release the potent vasodilator prostacyclin, as well as the highly labile endothelium-derived relaxing factor (EDRF) which mediates vascular relaxation induced by some vasodilators including acetylcholine and bradykinin. EDRF has recently been characterised as nitric oxide (NO). The effects of NO on prostacyclin release, measured as 6-keto-PGFl,,, from endothelial cells obtained from bovine thoracic aorta, have now been investigated. Incubation of endothelial cells in culture with bradykinin (10-100 nM) stimulated the release of 6-keto-PGFl~. Pre-incubation (0.5-2 min) with NO (13-130 /.tM) caused a significant dose-dependent inhibition of 6-keto-PGFl,, release, reaching a maximum of 29 + 4% inhibition. Pre-incubation with superoxide dismutase (30 units ml-1) which prevents the breakdown of NO, significantly augmented the degree of inhibition, as did the selective inhibitor of cyclic GMP phosphodiesterase, M & B 22948 (5 /~M), reaching 51 + 2% inhibition. The potentiation by M & B 22948 suggests that this inhibitory effect of high concentrations of NO is brought about by elevation of intracellular cyclic GMP levels following activation of guanylate cyclase. Whether endogenous NO is produced by endothelial cells under physiological conditions in sufficient quantities to modulate prostacyclin release remains to be established. Endothelial cells; Prostacyclin; 6-keto-PGFl~; Endothelium-derived relaxing factor (EDRF); Nitric oxide; Bradykinin
1. Introduction
Vascular tissue synthesizes the unstable arachidonate metabolite prostacyclin, which is a potent vasodilator and inhibitor of platelet aggregation (Moncada et al., 1976). Prostacyclin is released from endothelial cells by a variety of stimuli including bradykinin, arachidonic acid, thrombin, the ionophore A23187 and histamine (Weksler et al., 1978). Vascular endothelium also releases the highly labile moiety, endothelium-derived relaxing factor (EDRF), which mediates the vascular relaxation of some vasodilators including acetylcho-
l Present address: Institute of Human Physiology, Padova 35131, Italy. * To whom all correspondence should be addressed.
line and bradykinin (Furchgott and Zawadzki, 1980; Furchgott, 1983). This action is brought about by the stimulation of soluble guanylate cyclase with the subsequent increase in vascular g u a n o s i n e - 3 ' : 5'-cyclic m o n o p h o s p h a t e (cyclic G M P ) levels (Rapoport and Murad, 1983). Like prostacyclin, E D R F has been shown to inhibit platelet aggregation in vitro (Azuma et al., 1986; Furlong et al., 1987; Radomski et al., 1987a). However, in contrast to the anti-aggregating action of prostacyclin which is brought about by increases in intracellular cyclic A M P ( G o r m a n et al., 1977; Tateson et al., 1977), this effect of E D R F is likely to be elicited via the elevation of cyclic G M P levels (Radomski et al., 1987a). Recently, E D R F has been characterized as nitric oxide (NO; Palmer et al., 1987). N O exhibits a comparable stability and pharmacological pro-
0014-2999/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
20
file on vascular smooth muscle as E D R F (Palmer et al., 1987; Hutchinson et al., 1987; Furchgott, 1987). Furthermore, N O is released from endothelial cells in sufficient quantities to account for the biological actions of E D R F (Palmer et al., 1987). It has also been demonstrated that subthreshold doses of prostacyclin and of E D R F or authentic NO can synergistically interact to inhibit substantially platelet aggregation in vitro, and such potentiating interactions may reflect a physiological mechanism for maintaining platelet homeostasis at the vascular endothelium (Radomski et al., 1987b). The interaction between the synthesis of these two endothelium-derived mediators has now been investigated by determining the effects of exogenous N O on the release of endogenous prostacyclin, measured by radioimmunoassay as its stable chemical degradation product 6-keto-PGFa~, from bovine endothelial cells in culture.
2. Materials and methods 2.1. Endothelial cell culture
Fresh bovine thoracic aortas were obtained from an abattoir and the endothelial cells subsequently prepared by collagenase digestion as previously described (Gryglewski et al., 1986a). Cells were plated onto 12-well (24 mm diameter) tissue-culture plates (Linbro, Flow Labs.) at a density of 1.5 x 105 cells/well in Dulbecco's modified Eagle's medium (Gibco) containing 15% foetal calf serum (Flow Labs.) and an antibacterial mixture, penicillin (100 units ml-a), streptomycin (10 /~g m1-1) and gentamycin (50 /~g ml-1). These plates were incubated at 3 7 ° C in a humidified atmosphere containing 5% CO 2 for 4-6 days until the cells formed a confluent monolayer (cell count, 1-1.3 × 106/well). All the experiments were performed with cells from the first three passages. 2.2. Incubation conditions
To measure the release of 6-keto-PGFl~ by endothelial cell monolayers, the culture medium was aspirated and the monolayers were gently
rinsed twice with 1 ml of prewarmed (37°C) Hanks' balanced salt solution without phenol red (Gibco) containing CaC12 (2.3 mM) and adjusted to p H 7.4 with 20 mM HEPES buffer. After rinsing, Hanks' balanced salt solution was added to each well followed by the addition of the agents under investigation (final volume in the well, 1 ml). In studies on the actions of NO, aliquots (1-100 /~1) of a solution of authentic NO (0.1-3% in He-deoxygenated distilled water) stored in a glass vessel (Palmer et al., 1987) were removed with a gas-tight syringe and immediately added to the Hanks' balanced salt solution in the wells. Controls were always performed with the same volume of the He-deoxygenated distilled water, added to parallel wells. After 2 min incubation, the stimulatory agent, bradykinin (10-100 nM) was added and incubated for 2.5-10 min at 37 ° C. In preliminary studies, the inhibitory effects of NO following preincubation for either 0.5, 1 or 2 min were comparable and hence a 2 min incubation period was employed to allow a fully randomized experimental design to be utilized on each plate. The incubation medium was then aspirated and transferred into plastic Eppendorf tubes and frozen at - 2 0 ° C prior to determination of 6keto-PGFl~ levels. All experimental, procedures were performed in triplicate on each batch of cells. 2.3. Radioimmunoassay of 6-keto-PGF1
Radioimmunoassay was performed as described previously (Salmon, 1978) and the results were expressed as the release of 6-keto-PGFl~ (nM over 10 rain/106 cells in 1 ml) or as the % inhibition of release. Cross reactions with other prostaglandins were less than 3% control. Control studies with N O (130 /~M) and the other agents indicated that they did not interfere with the radioimmunoassay. 2.4. Materials
Authentic NO ( > 98.9% pure; British Oxygen Corporation) was prepared freshly as 0.1 and 3% ( v / v ) solutions in He-deoxygenated, distilled and deionized water, as described previously (Palmer et al., 1987). Bradykinin and superoxide dis-
21
mutase, obtained from Sigma Chemical Co. were prepared in 0.85% saline as a stock solution and diluted in Hanks' balanced salt solution. Stock solutions of M & B 22948 (2-O-propoxyphenyl-8azapurin-6-one; a gift from May & Baker) were prepared in triethanolamine (20% v/v); the final concentrations of triethanolamine in the endothelial cell incubation did not exceed 0.01%. Purified human haemoglobin was prepared according to the method of Paterson et al. (1976) and diluted in Hanks' balanced salt solution.
3O
z 20
_o
_= z 10
1.3
13
39
130 IJM
[NO]
2.5. Statistical evaluation Results are expressed as means + S.E. of (n) separate experiments, each performed in triplicate. Student's t-test for paired or unpaired data, where appropriate, was used to determine the significance of differences between means and P < 0.05 was taken as statistically significant.
3. Results
3.1. Release of 6-keto-PGFj~ The basal release of 6-keto-PGFl~ from the bovine endothelial cell monolayer over the 10 min incubation period was 10 + I nM (n = 14; 3.8 _ 0.6 ng ml-1). Studies on the time course of release of 6-keto-PGFl~ by bradykinin (10-100 nM) indicated that maximal release was achieved between 5 and 10 min of incubation, and thus a 10 min incubation period was used in all further studies. Addition of bradykinin (10, 30 and 100 nM) to the endothelial cell monolayer induced a significant (P < 0.01) concentration-related stimulation of 6keto-PGFl~ release to 45 + 6 nM (n = 5), 53 _ 3 nM (n = 11) and 5 9 _ 6 nM (n = 5) respectively, over this 10 min incubation period.
3.2. Effects of nitric oxide
Fig. 1. Inhibition of bradykinin (30 nM)-stimulated release of 6-keto-PGF1, ~ release from bovine endothelial cells in culture by pre-incubation (2 min) with nitric oxide (NO, 1.3-130 jaM). Results, expressed as % inhibition of control release, are shown as m e a n s + S . E , of 3-7 experiments per group, where statistically significant difference from control value is shown as * P < 0.05.
39 and 130 ~tM) prior to the addition of bradykinin (30 nM), induced a dose-related inhibition of 6-keto-PGFl~ release, as shown in fig. 1, reaching a maximum of 29 + 4% inhibition (n = 7, P < 0.01). Incubation with N O (130 /~M) significantly (P < 0.05) reduced the 6-keto-PGFl~ release induced by bradykinin at concentrations of 30 and 100 nM (fig. 2). Pre-incubation with N O (130 /~M) for shorter time periods (0.5, 1.0 and 1.5 min) gave a comparable degree of inhibition, whereas when
[ • Control ] c
60-
~NO
50-
T
(1301JM)
¢'- 4 0 a.
I
o
30-
/
20-
w
< M,I
IO-
I1 i 30
5 BRADYKININ
Incubation of the endothelial cell monolayer with NO (1.3/~M), 2 min prior to the addition of bradykinin (30 nM) had no significant effect on the release of 6-keto-PGFl~ (n = 3). However, incubation with higher concentrations of N O (13,
l
100 nM
Fig. 2. Inhibition of bradykinin (30 and 100 nM)-stimulated release of 6-keto-PGFla from bovine endothelial cells in culture by pre-incubation (2 min) with nitric oxide (NO, 130 JAM). Results, expressed as 6-keto-PGF1, , release (nM), are shown as m e a n s + S . E , of (n) experiments, where statistically significant difference from the control is shown as * P < 0.05.
22
the pre-incubation period with NO (130/~M) was extended to 5 min, no significant inhibition of bradykinin-stimulated release could be observed. A 2 min incubation with NO (130 /~M) had no significant effect on the basal release of 6-ketoPGF1, (9 _+ 2 nM, n = 7). Incubation of endothelial cell monolayers with haemoglobin (1 /~M), 1 min prior to addition of NO (39 /~M), abolished its inhibitory action on bradykinin-stimulated release (n = 3). However, pre-incubation with higher concentrations of haemoglobin (10 #M) did not significantly alter either basal or bradykinin (30 nM)-stimulated 6-ketoPGF1, release (table 1). 3.3. Effects of superoxide dismutase and M & B 22948 Pre-incubation of endothelial cell monolayers for 2 min with superoxide dismutase (30 units ml-1), prior to the addition of NO (39 and 130 #M) augmented the inhibitory effect on bradykinin-induced release of 6-keto-PGF1, (fig. 3). Superoxide dismutase (30 units m1-1) alone did not significantly inhibit the basal or bradykinin (30 nM)-stimulated release of 6-keto-PGFl~ (table 1). Incubation with M & B 22948 (5 /~M) for 2 min, likewise significantly potentiated the inhibitory actions of NO (39 and 130 /~M) on the bradykinin-stimulated release of 6-keto-PGFl~ (fig.
TABLE 1 Effect of pre-incubation with superoxide dismutase (30 units m l - ] ) , M & B 22948 (5 ~M) or haemoglobin (10 FM) on the release of 6-keto-PGFl= from endothelial cell monolayers under basal and bradykinin (30 nM)-stimulated conditions. Results are shown as m e a n s + S.E. of (n) values, where none of the treatment groups were significantly different from the corresponding control. 6-keto-PGFl, , ( n M )
Control Superoxide dismutase (30 units m1-1) M&B22948(5/.tM) Haemoglobin (10 ttM)
Basal
Bradykinin
1 0 + 1 (14)
5 3 + 3 (11)
9_+1 (5) 1 0 + 2 (3) 9 + 1 (3)
59+8 55+3 57+3
(3) (4) (3)
i
50 z
40
0 ~" 30 -r
z
20
10 9 NO
+ + SOD M&B
30
5
U m r 1 gM
NO
+ + SOD M&B
30 5 Uml " l g M
Fig. 3. Potentiation of N O (39 and 130 ~M)-induced inhibition of bradykinin (30 nM)-stimulated 6-oxo-PGFl, release from bovine endothelial cells in culture by pre-incubation with superoxide dismutase (SOD, 30 units ml - l ) or M & B 22948 (5 /xM). Results, expressed as % inhibition, are shown as means + S.E. of (n) experiments where statistically significant difference from the effects of N O alone is shown as * P < 0.05, * * P < 0.01.
3). However, M & B 22948 (5/~M) alone failed to inhibit basal or bradykinin (30 nM)-stimulated 6-keto-PGF1, release (table 1).
4. Discussion
Both prostacyclin and the labile moiety EDRF, now characterised as NO (Palmer et al., 1987) are endothelial cell-derived mediators that exert vasodilator and anti-aggregatory activity. Since these mediators may therefore interact to modulate vascular tone and local haemostasis, we have investigated whether NO can influence the biosynthesis of prostacyclin. These present findings indicate that at low concentrations, NO (1.3/~M) does not affect basal or bradykinin-stimulated release of prostacyclin from bovine thoracic aorta cell monolayers in culture, under the incubation conditions used. At higher concentrations, NO (13-130 /~M) produced a dose-related inhibition of bradykinin-stimulated prostacyclin release, as determined by the levels of 6-keto-PGFl~. This inhibitory effect of NO was augmented in the presence of superoxide dismutase, which is known to prolong the duration of action of both E D R F and exogenous NO (Gryg-
23 lewski et al., 1986b; Rubanyi and Vanhoutte, 1986; Palmer et al., 1987). However, this enhancement by superoxide dismutase was modest, probably reflecting that the breakdown of N O under these conditions was not simply due to the presence of superoxide anions in the incubation medium or released from the endothelial cells. Thus, NO reacts readily with oxygen to produce N O 2 which then forms nitrite and nitrate ions in aqueous solution (Palmer et al., 1987). The mechanism underlying such inhibition of prostacyclin release by high concentrations of NO is not known. It would appear to reflect the action of NO itself, or an equally unstable moiety, since on more prolonged incubation, this inhibitory activity was no longer detectable. This finding also indicates that the inhibitory action of NO was reversible and not due to non-specific disruption of the cell or the cascade of enzymes involved in prostacyclin synthesis. The ability of the selective inhibitor of cyclic G M P phosphodiesterase, M & B 22948, (Lugnier et al., 1986) to augment significantly the degree of inhibition induced by NO suggests that this action was brought about by an action on the guanylate cyclase-cyclic G M P system. Previous studies have shown that M & B 22948 increases cyclic G M P levels but not cyclic AMP levels in porcine aortic endothelial cells (Martin and White, 1987). Furthermore, M & B 22948 increases cyclic G M P levels and induces relaxation in rabbit aorta in which the endothelial cells are present (Martin et al., 1986), as well as augmenting the platelet anti-aggregating action of E D R F and exogenous NO (Radomski et al., 1987a). Intracellular cyclic G M P is elevated by E D R F in vascular tissue (Rapoport and Murad, 1983), and is thought to regulate vascular smooth muscle calcium levels by inhibiting calcium influx and its mobilisation from intracellular stores (Collins et al., 1986). NO, as well as the nitrovasodilators which are considered to act by releasing NO, stimulate the formation of cyclic G M P in vascular smooth muscle (Katsuki et al., 1977; Arnold et al., 1977; Ignarro et al., 1981) and may thus modulate the intracellular calcium levels. Since calcium is an essential pre-requisite for the biosynthesis of prostacyclin, such an action on intracellular calcium
levels within endothelial cells may contribute to the inhibitory action of high concentrations of NO observed in the present study. In contrast to the present suggestion, it has been proposed that increases in endothelial cell cyclic G M P levels are associated with the stimulation of prostacyclin biosynthesis by bradykinin (Brotherton, 1986). However, since other agents that stimulated cyclic G M P formation such as nitroprusside or atriopeptin II did not stimulate prostacyclin biosynthesis in the same study, it was concluded that cyclic G M P does not play a primary role in stimulating prostacyclin formation. Other studies with the nitrates, including nitroglycerin, have likewise failed to demonstrate any consistent effect on prostacyclin production by human vascular fragments and cultured umbilical vein endothelial cells (De Caterina et al., 1985) despite the suggestion of earlier experiments (Levin et al., 1978). Likewise, in the present study, low concentrations of NO, which would be sufficient to induce relaxation in isolated strips of vascular tissue (Hutchinson et al., 1987) did not affect endothelial cell prostacyclin synthesis. The observation that much higher concentrations of NO did induce a moderate inhibition of prostacyclin of up to 30% may therefore reflect a pharmacological action not achieved by nitrovasodilators, although this effect may be unrelated to any physiological modulation of mediator release. However, since N O is highly unstable, the final concentration of the active species to which the endothelial cells were exposed under the incubation conditions used, is not known. In our further studies to investigate the possible interaction between endogenous NO and prostacyclin release, endothelial cells were incubated with haemoglobin, which prevents the actions of E D R F and N O (Martin et al., 1985; Keilin and Hartree, 1937; Palmer et al., 1987). Haemoglobin, in sufficient concentrations to abolish the inhibitory effects of exogenous NO, failed to affect either basal or stimulated prostacyclin release. This suggests that modulation of prostacyclin release by any endogenous N O derived from these endothelial cells is insignificant. Recent studies with the E D R F inhibitor, methylene blue have demonstrated inhibition of prostacyclin formation by
24
endothelial cells, but this action was unrelated to any inhibition of soluble guanylate cyclase (Martin and Drazan, 1987) and hence to any effect of endogenous NO. It would be of interest, however, to correlate the concentration of exogenous NO present under the incubation conditions with those levels produced endogenously from endothelial cells under comparable experimental conditions, as well as to investigate the effects of exogenous prostacyclin on the release of NO. Such studies should further clarify any interaction between these two endothelium-derived mediators in relation to their respective biosynthesis and the physiological or pathological relevance of such interaction in the regulation of vascular tone and local platelet function.
Acknowledgements We are grateful to N.A. Foxwell and M.J. Ashton for technical assistance.
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of enthothelial cells in the relaxation of arterial smooth muscle by acetylcholine, Nature 228, 373. Furlong, B., A.H. Henderson, M.J. Lewis and J.A. Smith, 1987, Endothelium-derived relaxing factor inhibits in vitro platelet aggregation, Br. J. Pharmacol. 90, 687. Gorman, R.R., S. Bunting and O.V. Miller, 1977, Modulation of human platelet adenylate cyclase by prostacyclin (PGX), Prostaglandins 13, 377. Gryglewski, R.J., S. Moncada and R.M.J. Palmer, 1986a, Bioassay of prostacyclin and endothelium-derived relaxing factor (EDRF) from porcine aortic endothelial cells, Br. J. Pharmacol. 87, 685. Gryglewski, R.J., R.M.J. Palmer and S. Moncada, 1986b, Superoxide anion is involved in the breakdown of endothelium-derived vascular relaxing factor, Nature 320, 454. Hutchinson, P.A., R.M.J. Palmer and S. Moncada, 1987, Comparative pharmacology of EDRF and nitric oxide on vascular strips, European J. Pharmacol. 141,415. Ignarro, L.J., H. Lippton, J.C. Edwards, W.H. Baricos, A.L. Hyman, P.J. Kadowitz and C.A. Gruetter, 1981, Mechanism of vascular smooth muscle relaxation by organic nitrates, nitrites, nitroprusside and nitric oxide: Evidence for the involvement of S-nitrosothiols as active intermediates, J. Pharmacol. Exp. Ther. 218, 739. Katsuki, S., W. Arnold, C.K. Mittal and F. Murad, 1977. Stimulation of guanylate cyclase by sodium nitroprusside, nitroglycerin and nitric oxide in various tissues preparations and comparison to the effects of sodium azide and hydroxylamine, J. Cycl. Nucl. Prot. Phosphor. Res. 3, 23. Keilin, D. and E.F. Hartree, 1937, Reaction of nitric oxide with haemoglobin and methaemoglobin, Nature 139, 548. Levin, R.I., E.A. Jaffee, B.B. Weksler and K. Tack-Goldman, 1978, Nitroglycerin stimulates synthesis of prostacyclin by cultured human endothelial cells, J. Clin. Invest. 67, 762. Lugnier, C., P. Schoefter, A. Le Bec, E. Strouthou and J.C. Stoclet, 1986, Selective inhibition of cyclic nucleotide phosphodiesterases of human, bovine and rat aorta, Biochem. Pharmacol. 35, 1743. Martin, W. and K.M. Drazan, 1987, Methylene blue inhibits prostacyclin production by pig aortic endothelial cells, Br. J. Pharmacol. 92, 519P. Martin, W., R.F. Furchgott, C.M. Villani and D. Jothianandan, 1986, Phosphodiesterase inhibitors induce endothelium-dependent relaxation of rat and rabbit aorta by potentiating the effects of spontaneously released endothelium-derived relaxing factor, J. Pharmacol. Exp. Ther. 237, 539. Martin, W., G.M. Villani, D. Jothianandan and R.F. Furchgott, 1985, Selective blockade of endothelium-dependent and glyceryl trinitrate-induced ration by hemoglobin and by methylene blue in the rabbit aorta, J. Pharmacol. Exp. Tiler. 232, 708. Martin, W. and D.G. White, 1987, Pig aortic endothelial cells contain both soluble and particulate guanylate cyclase isoenzymes, Br. J. Pharmacol. 90, 18P. Moncada, S., R. Gryglewski, S. Bunting and J.R. Vane, 1976, An enzyme isolated from arteries transforms prostaglandin
25 endoperoxides to an unstable substance that inhibits platelet aggregation, Nature 263, 663. Palmer, R.M.J., A.G. Ferrige and S. Moncada, 1987, Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor, Nature 327, 524. Paterson, R.A., P.A.M. Eagles, D.A.B. Young and C.R. Beddell, 1976, Rapid preparation of large quantities of human haemoglobin with low phosphate content by counter-flow dialysis, Int. J. Biochem. 7, 117. Radomski, M.W., R.M.J. Palmer and S. Moncada, 1987a, Comparative pharmacology of endothelium-derived relaxing factor, nitric oxide and prostacyclin in platelets, Br. J. Pharmacol. 92, 181. Radomski, M.W., R.M.J. Palmer and S. Moncada, 1987b, The anti-aggregating properties of vascular endothelium: interactions between prostacyclin and nitric oxide, Br. J. Pharmacol. 92, 639.
Rapoport, R.M. and F. Murad, 1983, Endothelium-dependent nitrovasodilator-induced relaxation of vascular smooth muscle: role for cyclic GMP, J. Cycl. Nucl. Prot. Phosphor. Res. 9, 281. Rubanyi, G.M. and P.M. Vanhoutte, 1986, Superoxide anions and hyperoxia inactivate endothelium-derived relaxing factor, Am. J. Physiol. 250, H822. Salmon, J.A., 1978, A radioimmunoassay for 6-keto-prostaglandin FI~ , Prostaglandins 15, 383. Tateson, J.E., S. Moncada and J.R. Vane, 1977, Effects of prostacyclin (PGX) on cyclic AMP concentrations in human platelets, Prostaglandins 13, 389. Weksler, B.B., C.W. Ley and E.A. Jaffe, 1978, Stimulation of endothelial cell prostacyclin production by thrombin, trypsin, and ionophore A23187, J. Clin. Invest. 62, 923.