Glucocorticoids and prostaglandin synthesis: we cannot see the wood for the trees.

Prostaglandins Leukotrienes and Essential 0 Lon,qnman Group UK Ltd 1992

Fatty

Acids

(1992) 45, 85-112

Review

Glucocorticoids and Fkostaglandin Synthesis: We Cannot See the Wood for the Trees D. Duval* and M. Freyss-Beguin Laboratoire de Pharmacologic, (Reprint requests to DD).

Faculti de Mt?decine, 1.56 rue de Vaugirard,

INTRODUCTION

arachidonic acid, the major fatty acid precursor of eicosanoids, are important for the understanding of the effects of glucocorticoids. Firstly, it was demonstrated long ago that only the free form of the fatty acid can be converted into prostaglandins (1, 2). Second, it is generally held that availability of free arachidonate is the rate limiting step in the biosynthesis of eicosanoids (3, 4). Since most of the cell arachidonate is found esterified in position 2 of membrane phospholipids (4, 5), it is generally accepted that arachidonate release is mediated through an activation of either phospholipase A2 or the combined action of phospholipase C and diglyceride lipase (6-8). A new pathway of arachidonate liberation mediated by phospholipase D has also been described recently (9, 10).

For almost 40 years, hydrocortisone and synthetic glucocorticoids have constituted an important class of antiinflammatory agents, since they represent the only drugs capable of abolishing all the symptoms of acute inflammatory reactions. Given their therapeutic importance, outstanding efforts have been made to improve our understanding of the mechanisms of action of glucocorticoids and to minimize their side effects. Until recently, the effects of corticosteroids were poorly understood but in the early 1980s a unifying concept of their mode of action slowly emerged, which is now generally accepted. According to this theory, glucocorticoids act by inhibiting the formation of prostaglandins and other derivatives of arachidonic acid. These effects appear to be mediated through the induction of lipocortins, a family of phospholipase inhibitory proteins. Although lipocortins have been purified and their genes cloned and sequenced, their role in the antiinflammatory action of corticosteroids has recently been questioned. In the present work, we have thus to review critically the material attempted withstanding the lipocortin scheme in order to determine whether or not they are valid and explain the overall spectrum of antinflammatory actions of glucocorticoids.

Glucocorticoids and prostaglandin synthesis: an historical account

Shortly after the demonstration that aspirin and other non steroidal antiinflammatory drugs inhibit prostaglandin synthesis (ll-13), it was realized that antiinflammatory steroids were also able to block eicosanoid formation (14-18). Despite these demonstrations, the mode of action of glucocorticoids remained elusive and it was believed that steroids act via membrane stabilization (19, 20). Between 1975 and 1980, two sets of parallel experiments progressively shed light on the mechanism of steroid action. Firstly, it was shown that glucocorticoids act upstream in the biosynthesis of prostaglandins. Several groups have indeed described that glucocorticoids are able to decrease the release of arachidonic acid from cellular phospholipid stores (21, 22). In addition, the inhibitory action of glucocorticoids on prostaglandin synthesis can be abolished

Discovery and features of the lipocortin family Background

Several

to the metabolism of arachidonic acid

characteristics

of

the

metabolism

*Present address: D. Duval, Centre Cyceron. 14021 Caen Cedex, France

75015 Paris, France

of

BP 5027,

85

86

Prostaglandins Leukotrienes

and Essential Fatty Acids

by addition of exogenous arachidonate (21, 23, 24). The demonstration of a blockade of the synthesis of both lipoxygenase and cyclooxygenase products, was taken as an argument in favor of an action of glucocorticoids at the level of arachidonate release (25-27). Secondly, the inhibitory effect of glucocorticoids on prostaglandin formation was shown to represent a typical steroid action, i.e. to be mediated through specific receptor occupancy and to require macromolecular synthesis (28, 29). The action of steroids on prostaglandin formation was first described as being specific for glucocorticoids since a good correlation exists between the affinity of a given molecule for glucocorticoid receptors and its action on PG synthesis (30-32). This concept was consolidated by the finding that the action of glucocorticoids on PG formation can be blocked by a specific receptor antagonist, RU 486 (33). Finally, it was shown that the effect of glucocorticoids on PG synthesis can be abolished by inhibitors of RNA and protein synthesis (30, 32, 34). In order to unify this body of information, it was proposed that glucocorticoids act at the level of phospholipase, either by inhibiting the synthesis of phospholipase A2 or by inducing the secretion of a phospholipase inhibitor. Soon after this suggestion, Hirata et al (35), Blackwell et al (36) and RussoMarie and Duval (37) described the presence of proteins able to inhibit arachidonate release and PG synthesis in the supernatants of dexamethasonetreated cells. Later on, these various proteins (lipomodulin, macrocortin, renocortin) were shown to constitute a discrete entity which was named lipocortin (38, 39). Characteristics of lipocortins

The discovery of steroid-induced proteins sharing possibly some of the antiinflammatory actions of glucocorticoids has raised a considerable amount of interest. In the present paper, we will only summarize briefly the major properties of lipocortins. For more complete reviews see (40-43). Huang and coworkers (44) first demonstrated the existence in human placenta of two different lipocortins which present approximately 50% sequence homology and soon the existence of a family of lipocortins was progressively recognized (45,46). Using monoclonal antibodies directed against lipocortins, or nucleotide probes, several groups have shown that lipocortins are widely distributed in different types of cells and organisms and may represent up to 1% of the total cell proteins (44, 47). The ability of purified or recombinant lipocortin preparations to inhibit phospholipase activity in vitro has been repeatedly confirmed in different systems (48-51). It was also shown that lipocortins may exert other antiinflammatory actions such as inhibi-

tion of prostacyclin and thromboxane synthesis (52, 53)) decrease in platelet agregating factor formation (54), and inhibition of 02 generation (55). In addition, support has been provided for a role of lipocortins in other biological processes: platelet activation (56), inhibition of blood coagulation (57), protection against coronary failure (58), and induction of terminal differentiation in human squamous carcinoma cells (59). Although the exact nature of the interaction between lipocortins and phospholipases remains to be defined (60) it seems that phosphorylation represents a key event in the regulation of its antiphospholipase activity (61-64). Lipocortins belong to the superfamily of the calcium-binding proteins, annexins

Questions have recently been raised concerning the physiological roles of lipocortin. Davidson and coworkers (65) first presented evidence that lipocortins did not inhibit phospholipase A2 through a direct interaction with the enzyme, as previously proposed by Hirata (35, 61). In vitro experiments indeed indicate that lipocortins bind phospholipids and may thus inhibit phospholipase by sequestering its substrate. The demonstration by Aarsman and coworkers (66) that lipocortin-induced inhibition of pancreatic phospholipase Az could be overcome by increasing amounts of Escherichia coli phospholipids, also supports the hypothesis of a substrate depletion mechanism. Similar results were obtained by Machoczek et al (67) when studying the effect of lipocortin I and II on phosphoinositide-specific phospholipase. It remains to be determined whether the phospholipase inhibitory activity of lipocortins observed in vitro reflects its endogenous action in intact cells. In recent years, the existence of a new family of calcium-binding proteins, with characteristics different from those of the EF hand calcium-regulated proteins, has progressively been recognized. These proteins were first identified and isolated on the basis of their calcium-dependent binding to membrane phospholipids and of their ability to promote liposome aggregation. Because of their calciumdependent interaction with the cytoskeleton and, in particular, with actin, two of these proteins were named calpactins. Related proteins such as chromobindins. calcimedins, calelectrins, endonexins have been detected in numerous tissues (68-70). The determination of the amino acid sequences of these calcium-binding proteins soon revealed two salient findings: a) The presence in several of these calciumbinding proteins of repeating units which contain a consensus sequence of 17 residues forming the endonexin fold (71, 72). b) The existence of a strong sequence homology between calpactins, lipocortins and related proteins.

Glucocorticoids Tahoe 1 Lipocortin related proteins Lipocortin

Mass (kDa)

Lipocortin 1

38

Lipocortin II

Apparent identity P 35 chromobindin calpactin II

9

38

P 36 cnromobindin calpactin I protein I

Lipocortin III and IV

58

endonexin chromobindin 4 35 kDa-calcimedin p 32.5 calelectrin protein II

Lipocortin V

35

renocortin chromobindin 5 endonexin II anticoagulant protein PAP

Lipocortin VI

68

protein III P 68 chromobindin 20 67 kDa calelectrin 67 kDa calcimedin

Reproduced

8

with permission from Pepinsky et al, 1988 (73).

Lipocortin II appears homologue to p 36, the substrate of pp 60src, and to the heavy chain of calpactin I. Similarly, lipocortin I was identified as calpactin II and as p 35, the substrate of EGF receptor kinase (Table 1, 40-42, 44, 69, 73-76). The characteristics of these proteins (ubiquitous distribution, ability to bind to acidic phospholipids at the cytoplasmic face of the membrane in the presence of micromolar concentrations of calcium, interaction with fodrin and spectrin, phosphorylation by membrane receptor protein kinases), have led to the suggestion that they may be involved in the organization of the network underlying the plasma membrane, in stimulus-secretion coupling and likely as coupling agents between growth factor receptors and their cellular targets (77-80). More recently, Ross and coworkers showed the identity of lipocortin III with an enzyme involved in the metabolism of inositol phosphate, inositol l-2 cyclic phosphate 2 phospho-hydrolase (81). Thus, the specificity of the antiinflammatory action of lipocortins is questionnable (42, 74).

Although there are numerous experiments demonstrating in many different cell models that glucocorticoids inhibit, in vitro, arachidonic acid release and eicosanoid formation some of the published results do not fit entirely with the lipocortin model (18,2124, 27, 32, 33, 82-86).

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87

In vitro experiments Even in the experiments showing an inhibition of arachidonic acid liberation and prostaglandin synthesis, there are features of the action of glucocorticoids that remain not completely understood. Firstly glucocorticoids, even at concentrations sufficient to saturate their receptors, do not completely inhibit prostaglandin secretion. The percentage inhibition is highly variable from one cell type to another and according to the treatment schedule, but usually ranges between 50 and 80%. In addition, the inhibitory action of hydrocortisone or dexamethasone is often less important on the basal release that on that triggered by exogenous signals (22, 87, 88). Another striking observation is the demonstration that, in a given tissue or a given cell population, the effect of glucocorticoids may vary according to the arachidonate derivative considered (33, 89-93). We have demonstrated, for example, in cultures of phagocytic cells derived from the mouse thymic reticulum, that glucocorticoids strongly decrease the secretion of PGE:! and 6-keto-PGE,, but inhibit only moderately the synthesis of PGFza, this would seem to implicate an effect on synthases/isomerases (33). In addition, it has been demonstrated that the degree of the steroid-induced inhibition may vary greatly according to the stimulus used to trigger arachidonic acid release and prostaglandin secretion. Heiman and Crews (94, 95), showed that dexamethasone decreased the release of radiolabeled arachidonate from purified rat mast cells when challenged by ovalbumin, concanavalin A, anti-IgE antibodies but failed to do so after stimulation by compound 48/80 or ionophore A23187. Similar observations were reported by Dieter et al (96) in rat Kupfer cells, by Crutchley et al (24) in bovine endothelial cells and by De Nucci et al in guinea-pig lungs (97). Collado-Escobar et al (98) even described that dexamethasone reduces the responsiveness of RBL cells to antigen stimulation but enhances in the same cells the liberation of arachidonate after adenosine treatment. Several explanations have been proposed to account for these inconsistent actions of steroids. The first is the existence of different metabolic pools of either arachidonic acid or phospholipases which could be differently coupled to hormonal stimulation or to glucocorticoid regulation. Such compartmentalization of arachidonic acid has been described in various tissues’including macrophages, platelets, cerebral cortex and lungs (99-103). The existence of different phospholipases with potentially different steroid sensitivity has been demonstrated by Wightman et al, Erman and Raz and Ghiara and coworkers (104-107). An alternative explanation is the existence in the tissue or the cell population under investigation of different popu-

88

ProstaaIandins Leukotrienes

and Essential Fattv Acids

lations or subpopulations of cells with variable sensitivities to steroid treatment (33, 89, 90, 92, 108). A third point which should be kept in mind is the fact that the number of glucocorticoid receptors, as well as the sensitivity to steroid treatment, may vary greatly in a given cell population according to the pattern of cell proliferation and/or differentiation, (109-114), and its pathophysiological state (115). The preceding observations are not sufficient to explain some results that are in complete contradiction with the proposal of a lipocortin-mediated action of steroids. Indeed, several puzzling situations could be observed with regards to the effects of glucosteroids on prostaglandin synthesis and/or arachidonate liberation. For example, several groups have demonstrated that steroid treatment in vitro may induce rather than inhibit PG secretion in myeloid leukemia cells, resting fibroblasts, mast cells, rat glomeruli and human amnion cells (116122). Duval and coworkers have demonstrated in cells of the thymic reticulum or U937 cells grown in serum-free medium that dexamethasone significantly enhances the release of arachidonic acid from prelabeled cells (123, 124). In addition, it was

Table 2 -. Species

shown that steroids may decrease prostaglandin production without any inhibition or even with a stimulation of arachidonate liberation in various cell types (96, 125-130). The development of specific RNA probes and antibodies has allowed the simultaneous determination of the expression of lipocortins and the inhibition of PG secretion in steroid-treated cells. In several recent studies, it was clearly demonstrated that dexamethasone failed to significantly enhance lipocortin synthesis (129, 131-135). Mitchell and coworkers reported a stimulation of lipocortin I synthesis in human amnion cells treated by dexamethasone, but this effect was associated with a marked increase in PG synthesis (136). Although Piltch et al described a stimulation of lipocortin I synthesis simultaneous with an inhibition of PG formation in rat thymic epithelial cells, they also presented evidence that the steroid-induced reduction of eicosanoid synthesis was associated with a reduction of phospholipase A2 activity but independent of lipocortin (137). Furthermore, in contrast with what was described earlier (31,35, 47, 48), it now appears that lipocortins are essentially intracellular proteins (74, 132, 133, 135).

Effect of glucocorticoids on the in vivo synthesis of prostanoids Treatment duration (days)

Tissue and Metabolites analyzed

Rat Rat

3 14

Rat Rat Rat Rat

3-7 3-l 7 8h

U U P U U U Air pouch exudate

Rat Rat Rat Rat Guinea-pig Rabbit

7 4h 6h 24 h 3.5 h I

&F sponge exudate sponge exudate bile U

Rabbit Rabbit

7 7

U U

Rabbit Rabbit Rabbit Human Human

30-90 10 h 120 4-8 2

Human

4

Femoral tissue ovule P synovial fluid U erythema fluid U

Human Human Human

21 2 7

P nasal lavage fluid U

PGF,,-M: PGF,, metabolites PGE-M: PGE, metabolites significant increase VS control; significant decrease VS control. =: no significant alteration U: Urine P: plasma

Reference

138 PGE.M 139 6-keto-PGF,, = PGF, PGE, 6-keto-PGF,, = PGE, PGF,, 140 PGE, 141 PGE, 142 PGE, = PGF,, = PGD, = I2 HETE = 6-keto-PGF la 143 PGE, SRS-A PGF,,-M PGE-M = 144 PGE$ 14.5 TXB, LTB, 146 PGE, 6-keto-PGF,, 147 PGE, LTD., = 148 PGE, P&!&-M TXB r 6-keto-PGF,, 149 PGF,,-M PGF,, PGE, = PGF, 150 PGE, = PGF-M = 151 PGF,, PGEIM TXB, -6-keto-PGF,, PGE 152 PGE; = PGF,, = 153 PGB 154 PGE, PGE, = TXBz = 6-keto-PGF,, 89 PGF,, PGE, = PGF,_ = 155 PGI$ PGF;: 155 PGI,M = PGE-M = PGF-M = TXB, = TXB,-M = 156 PGE, TXB, = PG$-M = 157 LTB, 158 TXA,-M = PGI, = PGE-M = 159

,

Glucocorticoids

In vivo studies Given the suggestions that glucocorticoids inhibit eicosanoid synthesis in vitro, numerous attempts have been made to test their effects on PG formation in vivo. Several authors have therefore measured plasma or urinary concentrations .of prostaglandins or their metabolites following systemic sterom treatment. As shown in Table 2, in most cases steroids caused only a minimal reduction in the urine or plasma concentrations of prostaglandins; in some studies there was a slight enhancement of these values. However, this lack of effect does not necessarily indicate that glucocorticoids do not inhibit PC synthesis in vivo. Indeed, the plasma or urinary concentration of prostaglandins is a reflection of numerous reactions of synthesis and degradation occuring in different tissues. Moreover, the capacity of eicosanoid synthesis and the response to steroid may vary greatly from one cell to another. Kobzablack et al (155) have shown that prednisolone administration in man fails to reduce the urinary excretion of PGF2, metabelites but reduces PGEz and PGF,, concentrations at the level of skin inflammatory lesions. Similar observations have been made in inflammatory exudates in rats (143, 146, 147). Sebaldt et al (159) showed in humans that glucocorticoids failed to inhibit prostanoid synthesis in vivo but strongly supressed TXA2 and leukotriene synthesis by alveolar macrophages ex vivo. Ex vivo experiments have been carried out to investigate the effect of chronic steroid treatment on the ability of isolated organs or cells to produce prostaglandins and/or lipoxygenase derivatives. These studies, however, led to controversial results. Blajchman et al (160) described a decreased bleeding time in hydrocortisone treated rabbits associated with a decrease in the prostacyclin formation by the vessel wall. Similarly, Axelrod (161) proposed that the permissive effects of glucocorticoids on vascular tone was due to their inhibitory action on prostacyclin formation. Thong et al (162) failed, however, to demonstrate any effect of prednisolone administration on bleeding time and platelet functions of normal humans. Rogers et al (163) were similarly unable to demonstrate an effect of in vivo glucocorticoid administration on the production of 6-keto-PGB, by rings of rat aorta. Although Ohuchi, Watanabe and Levine (143) and Griinfeld et al (164) described a decreased prostaglandin formation in a rat air pouch model or in rat renal papilla, others reported a striking stimulation of rat glomerular PGEz synthesis (120), an increased conversion of arachidonic acid into 6-ketoPGFi, by fetal lung cells (165, 166) in culture, or no effect of dexamethasone on the preovulation increase in PG production in rabbit follicles (153). Wallace showed that administration of dexametha-

and PG Synthesis

89

sone to Wistar rats led to striking gastric damage after 6 days of treatment. In tissue samples taken from the fundic region of treated animals there was no reduction of prostacyclin synthesis but a striking inhibition of LTC, production as compared to samples taken from untreated animals (93). In addition, Erman and Nasjletti (167) and Erman et al (168) described an enhancement of both the prostaglandin synthetase activity and the concentration of unesterified arachidonate in the renal medulla of dexamethasone-treated rats. An indirect way to investigate the effect of glucocorticoids on prostaglandin formation would be to compare the formation of PG in control and adrenalectomized animals. Flower et al (169) thus reported a significant increase in the formation of and LTB4 synthesis by TXB2, 6-keto-PGFi. leucocytes of adrenalectomized animals, as compared to sham-operated rats. Similarly, Vincent et al (170, 171) demonstrated an increased eicosanoid formation in the kidneys and peritoneal macrophages of adrenalectomized rats but a decreased PGD2 and 1ZHETE svnthesis by their spleen cells. It has also been known for some time that longterm treatment by steroids or Cushing’s disease is associated with alteration of lipid metabolism, particularly with a significant increase in plasma unesterified fatty acids (154, 172-178). These observations are therefore difficult to reconcile with the hypothesis that glucocorticoides inhibit in vivo prostaglandin formation through a reduction of arachidonate availability. Are the basic hypotheses firmly established? Technical aspects It is likely that some of the contradictory

results listed above can be partly explained by differences in the experimental procedures used by the various investigators. In macrophages or inflammatory cells, the importance and the nature of the arachidonate metabolites formed depend on many factors such as the species or the cell line studied, the status of the cell (resident, circulating, elicited . . .), the nature and the dose of the agent tested and the schedule of treatment (179-187). In addition, the local degradation of prostaglandins or leukotrienes has orten been neglected and may greatly alter the actual patterns of eicosanoid secretion (182, 188, 189). There are two major experimental drawbacks which have generally been underestimated or ignored. The first is the extensive use of radioactive arachidonate as a precursor for eicosanoid formation. It is now obvious that the metabolism of the exogenous tracer does not accurately reflect the fate of endogenous arachidonate except after incorporation times long enough to reach metabolic We have recently equilibrium (4, 190-194).

90



described in cultures of rat heart myocytes that, even after 24 h incubation in the presence of (14C)arachidonate, there is no production of radioactive prostanoids despite a significant synthesis of immunoreactive prostaglandins (195). It appears also difficult in tracer studies to determine accurately the intracellular source of the prostaglandin precursor since a marked redistribution of this tracer among lipid classes has been observed by several groups during kinetic studies (194, 196-198). The second experimental bias is the current way of estimating phospholipase activity. In most of the studies it is generally assumed that the medium accumulation of radioactive arachidonate is a reflection of the cell phospholipase activity. This assumption is however incorrect given the early demonstration that non-esterified fatty acids are rapidly reacylated into membrane phosphohpids (199, 200). Furthermore, the reacylation step appears critical in the control of fatty acid availability (201-205). Thus, ‘arachidonate release’ only corresponds to the actual equilibrium between acylation and reacylation. Likewise, this equilibrium is highly dependent on the experimental conditions used to follow arachidonate liberation and particularly on the presence of serum and/or albumin. This issue remains however, controversial since serum has been described to enhance or to inhibit arachidonate liberation and prostaglandin synthesis whereas albumin, which is able to trap fatty acids, generally enhances medium arachidonate accumulation (87, 206-211). Is fatty acid availabilitythe limiting step in prostaglandin synthesis? It is almost universally accepted that arachidonic acid can only be converted into prostaglandins from its free form and that the liberation of the precursor from lipid stores is the rate limiting step in the biosynthetic cascade. This claim is based on the observation that exogenous arachidonate generally enhances PG secretion (6, 24, 212-215), but the relationship between cellular free arachidonate levels and eicosanoid synthesis is far from being firmly established (216). Most of the experiments designed to determine the liberation of prostaglandins in relation with the release of unmetabolized arachidonate and/or the intracellular accumulation of free fatty acids have been performed with radioactive tracers. Although significant variations are observed according to the experimental models and the nature of the agents used to trigger prostanoid synthesis, the authors generally reported a simultaneous stimulation of arachidonate release and prostaglandin formation (21, 22, 208, 217-220). But, as noted many years ago by Flower and Blackwell (6), there is no direct correlation between the accumulation of the endo-

genous precursor and the amounts of PG formed. A similar result was also observed by others (99, 101, 192, 221-223) thus suggesting that the availability of substrate is not the only prerequisite for prostaglandin biosynthesis. In some reports, there is even a clear discordance between the effect of a given stimulus on arachidonate release and PG secretion. Deykin et al (224) have shown that addition of serum albumin to the incubation medium enhances arachidonate release but inhibits HETE and TXBz formation in human platelets stimulated by thrombin. Similarly, Robinson and coworkers (225) isolated a factor from rheumatoid synovial tissue which simultaneously enhances PG synthesis and decreases both intracellular free arachidonate and arachidonate release when applied to prelabeled synovial cells. In experiments where steroids inhibit both arachidonate liberation and eicosanoid formation, the percentage inhibition is generally lower for arachidonate release than for PG secretion. In cultures of rat renomedullary cells, 24 h treatment by dexamethasone led to a 60-70% inhibition of PGE2 production but only to a 20-30% decrease in arachidonate release (37). Tam et al (226) described that the concentration of dexamethasone required to block PG formation in cultures of MC5-5 fibroblast was higher than that necessary to inhibit arachidonate liberation. Some studies have been carried out to determine by non-radioactive methods (e.g. gas chromatography), the effect of glucocorticoids on both the secretion of immunoreactive prostaglandins and the release of free arachidonate into the culture medium. Puustinen et al (227) have shown that prednisolone treatment markedly inhibits the formation of prostaglandins in cultures of human keratinocytes without affecting the distribution of cell arachidonate. In contrast, we described that dexamethasone decreases PGEi and 6-keto-PGF1, production in culture of rat heart myocytes and slightly, but significantly, enhances the amount of polyunsaturated fatty acids present in cell phospholipids, a result in favor of a steroid-induced decrease in phospholipid hydrolysis (128). These uncertainties can be partly explained by the fact that the amount of arachidonic acid transformed into oxidation products only represents a very small proportion of the cell arachidctnic acid content. In tracer experiments, the amounts of radioactive prostaglandins formed relative to the total cellular incorporation is quite low since only l-5% of the incorporated radioactivity is metabolized into cyclooxygenase or lipoxygenase derivatives (37, 87, 228, 229). This percentage increases to reach 30-40% in thrombin-stimulated platelets (207, 224)) but clearly more arachidonate is present in cellular phospholipids than is needed to support maximal PG secretion.

Glucocurticoids

Taken together, these results suggest that the critical factor for PG secretion is not the overall arachidonate generated but more likely the presence of arachidonate in a metabolic pool accessible to the stimulus studied and directly coupled to I metabolizing enzymes (198). Origin of the fatty acid precursor

As stated above, it is generally held that the fatty acid precursor of eicosanoid synthesis is released from cell phospholipid stores under the action of phospholipases (3, 4, 9, 10, 217, 220, 230-234). In many cases, however, the authors only followed the release of labeled arachidonate and metabolites in the culture supernatants without analyzing simultaneously the precursor distribution among cell lipids. In addition, only a few experiments have been carried out to determine the cellular origin of the prostaglandin precursor by techniques measuring accurately the changes in neutral lipid and phospholipid mass associated with cell stimulation and prostanoid secretion (e.g. mass spectrometry, gas chromatography (130, 194). Nevertheless, there is evidence suggesting a contribution of sources, other than membrane phospholipids, to arachidonate supply. Vahouny et al (235) have shown in rat adrenal cells that, following ACTH stimulation, there was a decrease in the mass and radioactivity of arachidonate bound to cholesteryl esters associated with an increased free arachidonate and PGEz secretion. These authors therefore suggested that the major source of the substrate required for PG synthesis is derived from hormone-dependent hydrolysis of cholesteryl esters. Pomerantz et al (236) showed that incubation of porcine aortic endothelial cells with HDL containing cholesteryl(14C)arachidonate stimulated endothelial release of labeled PGI2 and PGF,, whereas enrichment with 1-palmitoyl-2-arachidonoyl phosphatidyl-choline failed to increase the liberation of radioactive prostaglandins. Habenicht et al (237) showed that arachidonate-enriched LDL are taken up by LDL receptors and serve as a source for PGIz synthesis in libroblasts stimulated by PDGF. Several groups have determined that arachidonate and free fatty acids are taken up, esterified into tryglycerides and stored in cytoplasmic lipid bodies in macrophages and mast cells (238, 239). They suggested that these stocks play an important role in the metabolism of arachidonate. Several years ago, Haye et al, Lewis et al, Erman et al and Fujimoto et al have proposed that triglycerides may serve as a source of arachidonate for prostaglandin production in thyroid cells, adipocytes and kidney medulla cells (167, 240-242). In addition, there are many indications that free arachidonate present in the serum or released by other cell types may be transformed into eicosanoids without previous

and PG Svnthesis

91

incorporation into phosphohpids. Chandrahose et al (243), using 3T3 cells stimulated by thrombin and bradykinin, suggested that the true substrate for PG biosynthesis is the free fatty acid that is released into the external medium and that there is no apparent coupling between the phospholipase and PG synthetase systems. We recently proposed a similar hypothesis from results obtained in unstimulated mouse thymocytes (127). Work by Goldyne and coworkers (244, 245) in coculture of human T lymphocytes and monocytes indicated that arachidonate released from T-cells may be used as a substrate for PG biosynthesis by macrophages. This concept of a cooperation between cell types leading to arachidonate supply and metabolism has recently been reviewed by Lagarde et al (246) and applies not only to the transformation of arachidonic acid into primary prostaglandins or hydroxyacids but also to the further metabolism of these products (247,248). INTERMEDIATE

CONCLUSION

It is apparent from the preceding discussion that the effect of glucocorticoids on arachidonate metabolism and eicosanoid synthesis cannot be explained satisfactorily by the simple lipocortin model presented by Hirata et al, Flower and coworkers and Russo-Marie et al (31, 35, 37). The discrepancies observed may be due, in part, to experimental biases (use of radioactive tracer, presence of serum and serum albumin, failure to determine the metabolism of cellular arachidonate), but also to the fact that the basic principles on which the lipocortin theory is based (40, 249) have not always been verified experimentally. Furthermore, it appears that many of the biological activities of glucocorticoids, which may directly or indirectly affect arachidonate supply and prostaglandin synthesis, have been forgotten or neglected. In the next part of this review we will thus describe some of these potential actions of glucocorticoids on eicosanoid metabolism. Effects of glucocorticoids on lipid metabolism in relation to fatty acid availability and prostaglandin synthesis Effects on cyclooxygenase prostaglandin metabolism

synthesis. and/or

Given the demonstration that cyclooxygenase activity was a major target for non steroidal antiinflammatory drugs (NSAIDS), early studies have focused on this enzyme as a possible site of glucocorticoid effect. This hypothesis has been rapidly superseded by the lipocortin theory, but there are still indications that steroid treatment indeed affects cyclooxygenase activity. However, marked discrepancies exist between the available

92



results. Several groups have described a stimulation of cyclooxygenase activity in various tissues such as bone marrow derived mast cells (91, 119), renal medullary cells (167) and 3T3 fibroblasts (117). This stimulation, however, was associated either with an inhibition of PG synthesis (250) or with a stimulation of PG secretion (91, 119, 167). In addition, Erman et al (120) reported that chronic treatment with dexamethasone also enhances the ability of rat renal microsomes to convert arachidonic acid into 5- and 1ZHETE. On the other hand, it was demonstrated that glucocorticoids may inhibit cyclooxygenase activity in different tissues. Moore and Hoult (251) in rat kidney and caecum, Hawkey in human rectal mucosa (252) and Goppelt-Struebe et al (253) in cultures of bone marrow macrophages, have described a significant reduction of cyclooxygenase activity associated with a reduction in prostaglandin secretion. Raz and coworkers, Fu et al, and Pash and Bailey (254-256) have recently described two experimental systems where the induction of cyclooxygenase activity either by IL-l in fibroblast cultures or by EGF and TGF-l3 in vascular smooth muscle recovering from aspirin inactivation is blocked by glucocorticoids. The effect of dexamethasone was associated with an inhibition of the synthesis of the cyclooxygenase mRNA (257) or a reduction of the translation of this mRNA (254). It remains to be determined whether the ability of glucocorticoids to inhibit or trigger cyclooxygenase activity reflects the specificity of its action on different target cells or whether it depends on the experimental conditions used (258). Thus, regulation of cyclooxygenase synthesis and activity may represent a major step in the control of eicosanoid synthesis by glucocorticoids (259). Another way of regulating plasma levels of prostaglandins would be an action of steroids on their metabolism and/or excretion. Moore and Hoult (251) described, several years ago, that prednisolone treatment enhances the capacity of rat kidney and caecum cytosols to degrade prostaglandins, whereas Nasjletti et al (139) showed a decrease in PG-15-hydroxydehydrogenase activity in kidney cytosol of rats treated with dexamethasone. However, in the lung cytosol of the same animals, the enzyme activity was not altered by steroid treatment. In experiments intended to study the mechanism of protection by glucocorticoids against arachidonate induced sudden death, Araki et al (260) described that the clearance of the injected arachidonate was more rapid in steroid-treated rabbits than in control animals, although the mechanism of this action was not determined. During the last few years, a new pathway of arachidonic acid transformation has been described, mainly in kidney and liver. This metabolism which

involves a cytochrome Pds,-,monoxygenase is now known as the epoxygenase pathway (261-266). To our knowledge, almost nothing is known concerning the influence of glucocorticoids on this epoxygenase pathway. However, given the established capacity of steroids to induce cytochrome PAMgene expression in liver (267, 268), it would be worth studying the regulation of the enzymes involved in the epoxygenase pathway by glucocorticoids. In this respect, it is interesting to note that dexamethasone has been recently described to regulate the expression of epoxyde hydrolase, another enzyme associated with the cytochrome Pd5a mixed function oxidase system (269). Effects of glucocorticoids on TG metabolism and plasma fatty acid availability In mammalian organisms, some tissues including intestine, adipose tissue and liver, have distinct and specialized functions in the transport and utilization of triglycerides. The major routes for the disposition of fatty acids, as free fatty acids or in an esterified form, are depicted in the Figure. It is obvious from this scheme that the amount of arachidonic acid available for acylation into cell phospholipids and/or transformation by various oxidative pathways is critically dependent upon the activity of several organs and reflects the complex interplay of numerous metabolic steps. In addition, glucocorticoids have been known for a long time to affect several of these steps (29, 175, 176, 270). The complete description of the wide spectrum of action steroids on lipid metabolism is far beyond the scope of this review, but we will briefly consider some of these effects to underline the complexity of the roles played by steroid hormones on arachidonate availability. Metabolism of adipose tissue and mobilization of triglycerides. The triacylglycerol stores in adipose tissue are continually undergoing lipolysis and reesterification. The result of these two processes determines the magnitude of the free fatty acid pool in adipose tissue, which in turn is one of the sources of free fatty acids circulating in the plasma. The work of Jeanrenaud and Renold (173) has clearly shown that incubation of rat adipose tissue in the presence of corticosterone led to a net stimulation of fatty acid release. This effect appears due to a reduced esterification associated with an enhanced lipolysis (176, 270). It was suggested that glucocorticoids mainly exert a permissive effect on the lipolytic action of adrenalin and other hormones (272), but the basis of such a permissive action remains unclear (273, 274). Effects of glucocorticoids on hepatic lipid metabolism. Liver represents the primary source of plasma triglycerides secreted in the form of very low

Glucocorticoidsand PG Svnthesis 93

Fig. 1 Overview of lipid metabolismin the whole animal (271) FFA: free fatty acids TG: Triglyceride VLDL: very low density lipoprotein

density lipoproteins; thus, extensive studies have been carried out to investigate the effects of hormones on hepatic TG formation. Together with insulin, glucocorticoids are one of the major factors controlling triacylglycerol metabolism in liver. Indeed, they stimulate the synthesis of TG and the secretion of very low density lipoproteins (174, 178, 275, 276). This effect was shown to be mainly due to a significant stimulation of the activity of phosphatidate phosphohydrolase in the presence of steroids (177,277). In addition, glucocorticoids may also promote TG accumulation by the liver (177). Effect of glucocorticoia’s on lipoprotein lipase activity. Triacylglycerols in chylomicrons or in VLDL

cannot be taken up by intact tissue but must first undergo hydrolysis by lipoprotein lipase, an enzyme abundant at the luminal surface of capillary endothelial cells. Lipoprotein lipase thus controls the rate and extent of TG fatty acid uptake by extrahepatic tissues. It is therefore striking to note that glucocorticoids markedly enhance lipoprotein lipase activity in rat lung, adipose tissue and heart and in human adipocytes (278-281). Effect of glucocorticoids on de novo fatty acid synthesis. De novo fatty acid synthesis in mammalian

tissues requires the sequential action of two enzyme systems, acetyl COA carboxylase and fatty acid synthetase. In 1975, Volpe and Marasa described a significant inhibition of both systems by steroids in rat adipose tissue and liver (282). This effect was clearly due to a reduced synthesis of the enzymes as determined by immunological studies. Seillan and coworkers (283) also described in isolated mouse thymocytes that dexamethasone reduced incorporation of [3H]-acetate into triglycerides and non esterified fatty acids. In addition, the results of de Gomez-Dumm et al (284) and of Marra et al (285) suggested that dexamethasone may selectively inhibit arachidonate biosynthesis via a reduction of Asdesaturase activity in isolated rat liver and hepatoma cultures. Given the relative quantitative importance of the different pathways involved in the regulation of fatty acid availability, the final consequence of a systemic glucocorticoid treatment in animals and in man will be a significant increase in the levels of plasma triglycerides (154, 177, 178), yet it is possible that inhibition of As-desaturase may alter the composition of plasma and membrane fatty acids. Indeed, Huang et al (286) showed that dexa-

94

Prostaelandins

Leukotrienes


methasone treatment induces a significant decrease in the percentage of long-chain unsaturated fatty acids among rat plasma and liver phospholipids. Cellular regulation of fatty acid metabolism In the preceding section, we have mainly considered the effect of glucosteroids on the supply of free fatty acids at the level of the whole organism, but steroids may also play a role at the cellular level. Glucocorticoia!v and the acylation-deacylation cycle. Although it is widely accepted that steroids act on prostaglandin synthesis through an inhibition of phospholipases, almost no studies have been carried out to measure directly the activities of the enzymes involved in the release of fatty acids from phospholipid stores. As already described above, this is probably due to the current methods of measuring phospholipase activity by the release in the medium of a radioactive tracer previously taken up by the cells. In addition, many uncertainties remain concerning the enzymes responsible for the liberation of arachidonic acid. At least three different pathways have been described in mammalian cells: phospholipase Az, phosphatidyl-inositol specific phospholipase C and phospholipase D (7-10, 194, 201, 231, 287-295). The route of arachidonate liberation may also vary according to the tissue tested, the nature of the triggering agent and the experimental conditions used (4, 192, 296). Furthermore, in a given cell, several phospholipases with the same specificity but different sensitivity to pH and calcium ions have been identified (104, 105,297, 298). Thus, the precise knowledge of the action of glucocorticoids on the concentration and expression of phospholipases will await the development of specific antibodies against purified enzymes and/or nucleotide probes (299-302). However, there are some elements suggesting that glucocorticoids may directly affect phospholipases activities or synthesis. Koehler et al (259) demonstrated that dexamethasone significantly inhibits phospholipase AZ activity, measured in cell homogenates by the release of arachidonate from an exogenous substrate, in U937 cells differentiated following tetradecanoyl phorbol acetate (TPA) treatment. Millanvoye-Van Brussel et al (128) reported in cultures of newborn rat heart cells that dexamethasane treatment significantly enhances the mass of phospholipid-bound fatty acids, probably via an inhibition of phospholipase activity. This effect was also associated with a marked inhibition of prostaglandin release. Berenstein et al (303) reported that dexamethasone treatment of rat basophilic leukemia cells inhibits not only the release of (14C)arachidonate triggered by A23187, considered as a Plase

AZ-mediated pathway, but also the IgE- induced breakdown of phosphoinositides, which is essentially the reflection of phospholipase C activation. In contrast, DeGeorge et al (304) showed in acetylcholine stimulated C62B glioma cells that corticosteroids essentially blocks the phospholipase AT dependent pathway with little action on the phospholipase C-dependent products. More recently, Nakano et al (299,300) showed that dexamethasone inhibits the extracellular release of phospholipase A2 triggered in rat aortic smooth muscles by forskolin or TNF. This effect was due to a blocking of the transcription of the phospholipase mRNA. Platelelet activating factor (PAF) is produced by many different types of cell - macrophages, leucocytes, platelets, endothelial cells - and plays important roles in several pathophysiological conditions (305, 306). PAF is formed by a two step process in which l-G-alkyl-2 acyl-sn glycero 3 phosphocholine is cleaved into Lyso PAF by phospholipase A2 and then acylated by a specific CoA-dependent acyltransferase. Parente and Flower reported that glucocorticoids were able to inhibit Lyso PAF formation in rat macrophages (54), and Fradin et al (307) showed that dexamethasone treatment in vitro markedly reduces the formation of PAF in rat neutrophils stimulated by ionophore A23187 of fmlp. These authors proposed that the effect of glucocorticoids was mediated through the induction of lipocortin or lipocortin-like proteins. Benhamou et al also described an inhibition of PAF formation by dexamethasone in bone marrow derived mast cells (308). On the other hand, it has been clearly demonstrated that the accumulation of free fatty acids in the supernatant of cells triggered by a given stimulus does not really reflect phospholipase activity but rather a balance between the deacylation and the reacylation steps (201, 203, 204, 309). It was even proposed that acyltransferase activity represents the major enzyme controlling free arachidonate availability (203, 205). Little is known concerning the effect of glucocorticoids on the reacylation process. Duval et al (124) have shown in undifferentiated U937 cells that dexamethasone enhances arachidonate release via an inhibition of acyltranferase activity. Since this effect was abolished by addition of ATP, it appears that dexamethasone did not block the synthesis of the enzyme but rather acted indirectly through an inhibition of cellular ATP formation. ATP is indeed an important factor controlling the transfer of fatty acids towards phospholipids (199, 202, 310). A similar action of dexamethasone, leading to an intracellular accumulation of free fatty acids and to a relative decrease in the proportion of phospholipidbound arachidonate was also observed in murine thymocytes (127).

Glucocorticoids and PG Synthesis

Effect on acyl chains redistribution among cell lipidr.Acyl groups are rapidly taken up by cells and

mainly incorporated into phospholipids. However, within 24-48 h following incorporation, phospholipid fatty acids undergo active exchange either with phospholipids or with neutral lipids, mainly triglycerides but also cholesterol esters in some cells (197, 311-315). This redistribution which is stimulated during cell activation, is likely to be responsible for the existence of different metabolic pools of arachidonic acid (see above). Glucocorticoids are able to alter either the intracellular metabolism of triglycerides, leading to an accumulation of free fatty acids (124, 127, 168, 312, 316), or the remodeling of membrane phospholipids (128, 130, 227, 286). When measuring the mass of fatty acids present in rabbit endothelial cells treated by dexamethasone, Medow and coworkers (130) noted increased levels of saturated and mono unsaturated fatty acids together with a decrease in polyunsaturated fatty acids. These results are not in agreement with a generalized inhibition of arachidonate release as a mechanism for the dexamethasone-induced inhibition of PG synthesis seen in these cells. The mechanisms controlling acyl group transfer within cells are not yet fully understood but probably include acyltransferase/phospholipase AZ, transacylase and TG lipases (168, 313, 317). Furthermore, the remodeling process probably reflects the coordinated interplay of several different pathways (10, 3223. Further studies are therefore necessary to identify the ways whereby glucocorticoids control arachidonate transfer and availability within the different cell compartments. Nevertheless, these effects of glucocorticoids on fatty acid distribution among cellular lipids may explain why the steroids are able to modify membrane fluidity in various cells (323-326). Fatty acid binding proteins are relatively small proteins (13-15 kDa) capable of binding long-chain fatty acids and their coenzyme A and L carnitine esters, and are found in appreciable amounts in cells of mammalian tissues (327-329). These proteins are believed to play a role in the transcytosolic transport of fatty acids and esters and there are suggestions that FABP is a determinant of the flux through the fatty acid oxidation pathway (329). Cook and coworkers (330) recently demonstrated that glucocorticoids are able in 3T3-Ll preadipocytes to stimulate the expression of a gene, pAL 422, homologous to a fatty acid binding protein. This effect of glucocorticoids may represent one of the mechanisms by which they control fatty acid redistribution within cells. Glucocorticoids and biosynthesis of lung surfactank Glucocorticoids have been known for a long

time to control the synthesis of lung surfactant and

95

hence lung maturation at the end of gestation. The effect of steroids appears rather complex since they affect in a coordinated fashion the activity and/or the synthesis of various enzymes, choline-phosphate cytidylyl transferase , phosphatidic-acid phosphatase and fatty acid synthetase (331-335). In addition, glucocorticoids have been shown to regulate the expression of surfactant associated proteins SP-A, SP-B and SP-C (336-338). These proteins bind glycerophospholipids in the presence of calcium and, together with hydrophobic low molecular weight polypeptides, enhance the formation of surfactant surface film. We have compared the proposed sequences of these surfactant proteins to that of lipocortins, but these sequences bear very little homologies (C. Ged, personal communication). On the other hand, Tsai and coworkers (165) and Tsai (166) have shown that dexamethasone treatment of pregnant rats, enhances the conversion of ( 14C) arachidonate into prostanoids by homogenates of fetal lung without reducing the synthesis of immunoreactive prostacyclin in either fetal or maternal lung tissue. These results suggest that dexamethasone also accelerate the maturation of the enzymes involved in prostaglandin synthesis but the relationship between these two phenomenon remains to be established. Glucocorticoids control the activity and synthesis of many factors involved in the metabolism of arachidonic acid Effects mediated through the induction of inhibitors

In addition to lipocortins, many proteins and factors have been described to reduce the availability and/or transformation of arachidonic acid into eicosanoids. Although these factors have often been neglected or underestimated, they may play an important role in arachidonate metabolism. Furthermore, several of these factors appear to be induced by glucocorticoids. Pearson (339) extensively reviewed the various plasma factors regulating prostaglandin biosynthesis and catabolism and we will only briefly summarize the properties of some of these factors and the intluence of steroids on their synthesis. Albumin, As stated above, serum albumin is a major factor controlling fatty acid availability in cell cultures (123, 207, 209, 210, 224). By binding the arachidonate released from the cells, albumin reduces its reacylation and generally inhibits the formation of metabolites. In addition, albumin is able to catalyze prostaglandin catabolism (188, 340, 341). Thus, stimulation of albumin release which has been demonstrated in cultures of hepatocytes may be one of the mechanisms by which glucocorticoids control arachidonic acid transformation (342-345). Haptoglobin. Several authors have described the presence in plasma of subfractions able to inhibit

96



prostaglandin synthesis and exhibiting antiinflammatory actions (339, 346-348). This endogenous inhibitor of PG synthesis (EiPS), which is augmented after steroid treatment or in situations associated with an increased level of circulating glucocorticoids, probably corresponds to haptoglobin (339). Haptoglobin synthesis was markedly stimulated by dexamethasone treatment in cultures of mouse hepatoma cells (349). In addition, several acute phase proteins with recognized antiinflammatory roles, orosomucoid, (Y~antitrypsin and ceruloplasmin are under steroid control (345, 350353). Inhibitors of phospholipase AZ, Several groups have described the presence in blood plasma, under basal conditions, of inhibitors of phospholipase AZ, distinct from lipocortins (354-356). Suwa and coworkers (357) purified from the peritoneal cavity of wistar rats two inhibitors of phospholipase A2 with no homology with lipocortins but very similar to complement component c3. On the other hand, extracellular phospholipase A2 activities have been detected in inflammatory sites and appear to play an important role in the development of inflammatory reactions (358, 359). It is therefore of significance to realize that glucocorticoids may either inhibit the release of phospholipase A2 (299, 300, 360) or stimulate the synthesis of inhibitors (361). Uteroglobin is a small molecular weight protein originally identified as being secreted by uterus but also present in extrauterine sites (362, 363). This protein, which is strongly induced by glucocorticoids in lung tissue (362, 364), is a potent inhibitor of phospholipase A2 (50, 363). However, uteroglobin shares sequence homology with lipocortin I and can be considered as a member of the annexin family (50). Giucocorticoids inhibit the synthesis on action of numerous triggers of arachidonic acid release

Numerous factors have been described to provoke the release of arachidonic acid and prostaglandins from organ fragments and tissue cultures. These include proteolytic enzymes, hormones and growth

Table 3 Steroid sensitive signals active on the arachidonate Bradykinin Angiotensin II ACTH

FGF EGF PDGF C% C,, Collagen Heat shock Proteins

Histamine

factors, vasoactive peptides, amines and cytokines. It is again striking to realize that glucocorticoids are able to inhibit the synthesis and/or activity of many of these factors and therefore control in an indirect fashion the activation of the arachidonate cascade (365-371). The various factors or mediators known to enhance the release of arachidonic acid and eicosanoids and whose synthesis and/or action is inhibited by glucocorticoids are given in Table 3. The mechanisms of the actions of steroids vary greatly from one mediator to another. Steroids may on some occasions enhance the synthesis of an inhibitor, as in the case of the proteases (372-375). In fact, there appears to be a complex regulation loop since prostaglandins also participate to the regulation of protease synthesis (376, 377). In many cases, glucocorticoids exert inhibitory effects on the transcription of the factors as in the case of Inf y IL-l, IL-2 and TNF (378-385). But, glucocorticoids may also block the release of mediators such as histamine (84, 92, 386). It has been suggested that glucocorticoid-induced inhibition of lipoxygenase metabolites may participate to the control of histamine release (387). The third mode of action of glucocorticoids is a reduction of the effect of mediators through a regulation of their receptors as in the case of EGF, IL-2 or Fc receptors (308, 388-390). The modulation of IgG Fc receptors by glucocorticoids may explain, in part, the influence of these steroids on the metabolism of phospholipids and the liberation of arachidonic acid. Indeed, Suzuki and coworkers (391, 392) have provided evidence suggesting that Fey receptors exert a phospholipase A2 activity in B lymphocytes and in macrophage lines. It has also been shown that Fc receptors are involved in the release of collagenase and lyzozyme by mouse peritoneal macrophages (393). The demonstration that free fatty acids and particularly polyunsaturated fatty acids are potent inhibitors of phospholipase activity and prostaglandin syntheses (394-397) suggests the existence of another indirect mechanism by which dexamethasone, through an elevation of the circulating level

cascade Collagenase Plasminogen activator Metalloprotease

PAF PUFA PGF,, TXA, Leukotrienes HETE

TNF IL-1 IL-2 INFy FcR -

FGF: Fibroblast growth factor; EGF: epithelial growth factor; PDGF: platelet-derived growth factor; C,,, C,: Complement components 3, and 5,; PAF: platelet activating factor; PUFA: polyunsaturated fatty acids; PGF,: prostaglandin Fti; TXA,: thromboxane A,; HETE: hydroxyeicosatetraenoic acids; TNF: tumor necrosis factor; IL-l: interleukin 1; IL-2: interleukin 2; InFy: interferon Fy; FcR: Fc receptor.

Glucocorticoidsand PG Svnrhcsis 97 of non esterified fatty acids, may exert a feedback inhibition on the mobilization and metabolism of cellular phospholipids. A similar proposal has been made recently by Garcia et al (398) for the regulation of PAF biosynthesis by the products of phospholipase AZ. Possible prospects for a steroid action Glucocorticoids

and calcium ions

Calcium is known as a major factor controlling the activity of membrane-bound phospholipases (4, 104, 105, 212, 234, 289, 296, 298, 399, 400). Therefore, regulation of calcium influx or of cytosolic calcium concentration might represent a way by which glucocorticoids modulate phospholipase activity. Several groups have suggested that glucocorticoids may in fact regulate cytosolic free calcium levels in different cells. Several years ago, Kaiser and Edelman (401) suggested that the lytic effects of glucocorticoids on rat thymic lymphocytes could be similar to those of ionophore A23187 (402, 403), and mediated via an effect of the steroids on membrane calcium permeability. Homo and Simon (404) indeed described an early effect of glucocorticoids on 45Calcium uptake by mouse thymocytes whereas McConkey et al (405) and Orrenius et al (406) demonstrated that thymocyte suicide in the presence of methylprednisolone is associated with an early, sustained, increase in cytosolic Ca++ concentration. Eilam and coworkers (407) showed that triamcinolone acetonide induced in rat calvaria cells in culture a significant increase in cytosolic calcium and also a marked alteration in the amount of mitochondria exchangeable calcium. Since elevation of cytosolic calcium would enhance rather than inhibit phospholipase activity, these observations are in contradiction with the customary inhibitory actions of glucocorticoids on arachidonate liberation and prostaglandin formation. However, it was observed in other cell types such as rat basophilic leukemia cells, B lymphocytes, pancreatic islets and rat intestine that glucocorticoids inhibit the calcium inflow and block the elevation of cytosolic free calcium induced by cell stimulation (408412). Further studies are necessary to determine the mechanisms of the action of steroids on membrane permeability and cytosolic calcium concentration. G proteins and phospholipases

In addition to their well known functions in the coupling between receptors and phosphoinositidespecific phospholipase C (413-417), several reports suggest that G proteins may also control phospholipase AZ activities. This hypothesis is based on experiments made in the presence of pertussis toxins, of GTPyS, a non hydrolysable analog of GTP, or of purified By, subunits of protein G (418-

426). Any effect of glucocorticoids on either the G protein family or on another effector in the signal transduction pathways may be important in the control of arachidonate release and transformation. Recent evidence suggests that dexamethasone indeed affects G protein expression or activity. Chang and Bourne (427) described in a rat pituitary elle line (GHs) that dexamethasone treatment caused a 5-fold increase in RNA encoding the ar chain of G, and a 2-fold increase in membrane bound g polypeptide. These authors proposed that induction of G protein synthesis may be one of the mechanisms responsible for the so called ‘permissive’ effect of glucocorticoids on the action of agents enhanching CAMP synthesis (273, 274, 428, 429). Recent work by Doucet et al also suggests that glucocorticoids affect G proteins synthesis or functions in isolated nephrons (430). Collado-escobar et al proposed that the potentiation by dexamethasone of the stimulation produced by adenosine in rat mast cells, was probably due to an altered coupling between receptors and G proteins (431). Grove et al (432) and Berenstein et al (303) demonstrated an effect of dexamethasone on phosphoinositide metabolism, but were unable to precisely identify the molecular site of steroid action. Na’/H+

antiport

One of the earliest responses elicited by addition of PDGF and other growth factors to quiescent cells is an increase in the fluxes of Na+, H+ and K+ across the plasma membrane. Cytoplasmic alcalinisation mediated through an amiloride-sensitive Na’/H+ antiport appears indeed to play a critical role in the onset of mitogenesis (433, 434). Several authors have proposed that Na+/H+ exchange may also regulate phospholipase A2 in human platelets or fibroblasts (43%437), although this hypothesis was recently questioned (438). Given the demonstration by Freiberg et al (439) that dexamethasone increased an amiloride-sensitive Na+/H+ exchange activity in brush border membranes isolated from rat proximal tubules, it would be interesting to test the effect of the steroid on the Na+/Hf antiport in other target cells. Another way by which glucocorticoids may alter transmembrane Na+/K+ gradients and thus, indirectly, phospholipase activity (438) would be through an action on Na+-K+ ATPase activity. Indeed, several groups have described an effect of glucocorticoids on sodium pump in various target tissues (440-443). Role of phospholipase-activating

proteins

Clark and coworkers (444) have isolated from murine smooth muscle cells, a protein of 28 kDa, antigenically related to melittin, a peptide found in

98

Prostarrlandins Leukotrienes


bee venom. This protein shares with melittin the ability to selectively activate a phosphatidylcholine specific phospholipase AZ. In addition, the synthesis of this phospholipase activating protein (PLAP) is enhanced by LTD4, TNF and IL-l (230, 445). It is thus possible that glucocorticoids regulate, in part, arachidonate liberation through either a direct inhibition of PLAP synthesis, or through an inhibition of IL-l, TNF and LTD4 formation. Clearly, additional investigations are required to study the effect of dexamethasone on PLAP expresssion and/or regulation. Recent experiments have also demonstrated the existence of FLAP, a Slipoxygenase activating protein of 18 kDa. This protein which strongly binds MK 866, a potent inhibitor of leukotriene synthesis, could play an important role in the transfer of arachidonic acid to Slipoxygenase or may interfere with the transfer of Slipoxygenase from the cytosol to the membrane (446, 447). It would be of interest to test the potential effect of glucocorticoids on FLAP synthesis and function.

CONCLUDING

REMARKS

At the beginning of this paper, several questions were raised concerning the influence of glucocorticoids on arachidonate metabolism and their mechanisms. In the light of the observations reported above, we wonder whether or not these questions have been answered satisfactorily. The first question was: do glucocorticoid inhibit prostaglandin synthesis? If we consider the results obtained in vitro, the answer is yes, in most cases. Indeed, there is only a small percentage of experimental situations where steroids increase prostaglandin formation or are ineffective. However, results from in vivo treatments are far less conclusive. It appears generally that glucocorticoids either do not affect on may even enhance plasma levels or urinary excretion of prostanoids. Yet it is possible to speculate that glucocorticoids may in some areas reduce prostaglandin formation but this hypothesis remains to be established in intact animals or in man. The second point was to determine whether glucocorticoids block prostanoid formation via an inhibition of phospholipase activity and arachidonate release. In spite of the numerous investigations performed to study this problem, it is still difficult to give a reliable response. Nevertheless several salient points can be drawn from the mass of data accumulated: 1. There is no straight forward correlation between the amount of free arachidonate available and the level of prostanoid formed within a given cell.

2. There are multiple ways by which glucocorticoids may directly or indirectly alter arachidonate synthesis, uptake and acylation, deacylation and metabolism. 3. There is, up to now, relatively little evidence for a direct inhibition of phopholipase by glucocorticoids. Thus, we believe that alteration of arachidonate liberation, mediated either through direct control of phospholipase expression or via the synthesis of inonly one of the hibitors, if any, represents multiple ways by which glucocorticoids control prostaglandin formation. The third question concerns the role of lipocortins in the steroid-induced inhibition of eicosanoid formation generally observed in vitro. Despite early suggestions that lipocortins are inducible in the presence of dexamethasone, recent data indicate that annexins are not induced by steroids. Furthermore, it appears that their effect on phospholipid deacylation may represent an artefact due to their ability to bind phospholipids. This suggests that lipocortins probably do not play a major role in the control by steroids of arachidonate transformation. Given their ubiquitous distribution in many species and cell types, their relative importance in the mass of cell proteins (up to 1% of the total proteins), the striking homology between the various members of the family and their highly conserved structure during evolution, together with their capacity to bind calcium ions as well as phospholipid molecules, it is very probable that annexins hold very important functions in the biology of the cell. But, these functions remain to be determined. The lipocortin theory is primarily based on the assumption that inhibition of eicosanoid synthesis constitutes the major antiinflammatory effect of glucocorticoids. This proposal however is not valid as emphasized by numerous reviews in the field (365, 366, 368-371, 448-453). It has been recognized increasingly that inhibition of cell to cell signals and mediators indeed represent a major clue in the antiinflammatory actions of glucosteroids, but arachidonate derivatives represent only one member of the vast family of inflammatory mediators which are affected by glucocorticoids (see Table 3). Furthermore, glucocorticoids also exert their antiinflammatory effects via other pathways including: 1. inhibition of energy sources uptake and utilization. 2. inhibition of cell proliferation and differentiation. 3. effect on cell traffic and recirculation. 4. reduction of vascular permeability, eventually through the induction of proteins/peptides controlling vascular permeability (vasoregulin, vasocortin).

Glucocorticoids

5. inhibition of protease and lysosomal enzyme release. 6. reduction of cytotoxic oxygen metabolite formation (367-369,448, 454-459). It is beyond the scope of this review to examine in details all the known effects of antiinflammatory steroids, but clearly these mechanisms are so diverse and complex that they could not be explained satisfactorily through the induction of a unique protein or protein family such as lipocortins. Even the action of glucocorticoids on arachidonate metabolism is still elusive in many cases and much work remains to be done towards the elucidation of their mechanisms. Acknowledgments The authors gratefully acknowledge the assistance and the comments of Margaret Morris, Cecile Ged, Bernadette Brun and Marie-ThCr&se Baptiste during the preparation of the manuscript.

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Forests: See the trees and the wood.

Genetics: HotNet2-see the wood for the trees.

Strenuous exercise and the heart: are we not seeing the wood for the trees?

[e-cigarette: who cannot see the wood for the tree? Letter on the article "Electronic cigarette: reliable and efficient?"].

HIV and cannot see.

Debate: This Society believes that in the last 25 years of back pain research we have failed to see the wood for the trees - Chaired by C.G. Greenough.

Wood and Trees.

[Early diagnosis of Alzheimer's disease: are we too close to the trees to see the forest?].

Addicted health care professionals: missing the wood for the trees?

Plant biology: Seeing the wood and the trees.

Wood and Trees do journal club.

Do rats see like we see?

The Patients We Have to See.

Seeing the wood for the trees: 20 years of the Cochrane collaboration.

Molecular control of wood formation in trees.

Cannot See the Flowers nor the Tree: Plants and the Pill.

Can't see the (bamboo) forest for the trees: examining bamboo's fit within international forestry institutions.

How we see electronic games.

We cannot keep ignoring the crisis in social care.

We cannot afford to lose the animal rights war.

Goal-Directed Fluid Therapy: What the Mind Does Not Know, the Eye Cannot See.

Stage 3 and what we see.

Lingual thyroid: can we 'wait and see'?

Do we see what we should see? Describing non-covalent interactions in protein structures including precision.