New physiological and pathophysiological aspects on the thromboxane A2–prostacyclin regulatory system

Biochimica et Biophysica Acta 1532 (2001) 1^14 www.bba-direct.com

Review

New physiological and pathophysiological aspects on the thromboxane A2 ^prostacyclin regulatory system Volker Ullrich a

a;

*, Ming-Hui Zou b , Markus Bachschmid

a

Mathematisch-Naturwissenschaftliche Sektion, Fachbereich Biologie, Faculty of Sciences, University of Konstanz, Fach X 911-Sonnenbu«hl, D-78457 Konstanz, Germany b Department of Medicine, Vascular Biology, University of Boston, Boston, MA 02118, USA Received 11 April 2000; received in revised form 25 April 2001; accepted 2 May 2001

1. Introduction Since the discovery of the prostaglandins (PGIs) by von Euler [1] this class of highly active mediators and physiological messengers has been a carrier for hope in the diagnosis and control of diseases, especially after Bergstro«m and Samuelsson [2^4] and van Dorp [5] elucidated the chemical structures of PGs as a family of lipid compounds derived from arachidonic acid, and Vane and coworkers [6] unraveled the inhibitory action of O-acetylsalicylic acid (aspirin) on their biosynthesis and physiological actions [7^10]. However, this ¢rst wave of excitement was followed by a phase of some disappointment on the estimated further pharmacological impact. To a large extent this was due to the complex network of regulation in which prostaglandins are participating. As a consequence not only pain or fever could be controlled but also stomach or kidney functions were a¡ected leading to the well-known adverse e¡ects of the nonsteroidal antiin£ammatory drugs (NSAIDs). Also, the more complex and important a network the less it will su¡er from the elimination of one component as so impressively exempli¢ed by many mice knockout experiments. Leaks in the network usually are masked through bypasses and redundant pathways.

* Corresponding author. Fax: +49-7531-884084; E-mail: [email protected]

One typical example in prostaglandin drug research was the development of thromboxane A2 (TxA2 ) synthase blockers by several drug companies. Although excellent inhibitors of the enzyme were found they proved to be largely ine¤cient because the receptor for TxA2 also responded to its metabolic precursor PG-endoperoxide (PGH2 ) [11,12]. We shall come back to this unexpected and so far unexplained phenomenon, since it leaves the question why a receptor would have evolved to display responses to two quite di¡erent chemical molecules. Such experiences brought into focus the existence and properties of PG receptors which then were explored and characterized with other surprising results, i.e., the multiplicity of such receptors, their up- and downregulation and their multiple coupling to intracellular signaling transduction pathways [13,14]. This has led to the development of speci¢c PG receptor antagonists, but it can be foreseen that the enthusiasm in this new ¢eld probably will be dampened by the wellknown, but always neglected, fact that PGs have tissue-speci¢c actions and can be coupled to di¡erent e¡ector systems. What would be advantageous to block in one organ could cause adverse reactions in others. For several years now the discovery of the prostaglandin-endoperoxide synthase isoform (cyclooxygenase-2) has newly stimulated the PG ¢eld and has resulted in a new generation of NSAIDs which target the pathophysiological pathways more selectively

1388-1981 / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 8 - 1 9 8 1 ( 0 1 ) 0 0 1 2 6 - 3

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and have brought the PGs again into focus. The rationale behind this is convincing and promising, but also here it applies that other functions of COX-2 may show up and that side e¡ects in speci¢c organs or in organ development may arise. A solution for this general problem is not at hand, except that more detailed knowledge of the physiological functions of each of the PGs is required and this separately for each organ. Moreover, the network in which PGs are incorporated extends beyond the class of PGs and eicosanoids and also involves other mediators of lipid, peptide or even inorganic nature. Such connections are found increasingly and one can foresee a network of mediators connecting the PG system so that in the next future we may enter into another phase in which the physiological and pathophysiological relevance of the PGs can be understood in a broader aspect of cell regulation. Our present contribution to this problem will focus on some new additions to the PG network, which we believe are highly relevant and open up the view on a re¢ned chemistry involved in the control of such important functions as hemodynamics, homeostasis, immune responses and growth. Its key players are thromboxane A2 (TxA2 ) and prostacyclin (PGI2 ), but the network also comprises nitric oxide (c NO), superoxide anions (c O3 2 ), peroxynitrite, endothelins, platelet activating factor (PAF) and eicosanoids of even still unknown nature. 2. Biosynthetic pathways for thromboxane A2 and prostacyclin A network for the control of physiological functions involves mechanisms of regulation, counter-regulation and feedback that also apply for PG synthesis and action. Human thinking very early has been busy with this dynamic equilibrium and Chinese philosophy and medicine is largely based on the Yin^ Yang principle that turned out to be an early description of the general regulation^counter-regulation phenomenon in biology. In the TxA2 ^PGI2 couple we can perfectly recognize such a Yin^Yang system. With only a few exceptions of the rule, TxA2 represents the activating principle and PGI2 the corresponding antagonist causing the conversion back to the resting non-activated state. The action of

TxA2 and PGI2 is primarily restricted to intercellular communication, i.e., to the transfer of information between two or more cell types within an organ. Originally described as `Gewebshormone' such intercellular messengers now are termed `autacoids'. In its simplest form the onset of autacoid action comprises its synthesis and release from a cell, the di¡usion through the interstitial space, which in cases of direct cell^cell contacts can be extremely short, and the activation of a corresponding receptor on the surface of the neighboring target cell (paracrine action). But also a cell can stimulate itself either by release of PGs and binding to its own surface receptors or to nuclear receptors (autocrine action). Examples for regulations by the TxA2 ^PGI2 system are found in the interaction of platelets and the vessel endothelium where TxA2 released from stimulated platelets causes PGI2 formation in the endothelium that again inhibits platelet activity [15]. Up to now the vessel system is considered the only organ system in which TxA2 ^PGI2 play a relevant physiological and pathophysiological role. This classical view is certainly still valid and since all organs are a¡ected by the regulation of blood supply and the essential repair mechanisms in the circulation and ubiquitous occurrence of the TxA2 ^PGI2 Yin^Yang regulatory system is evident. It is the aim of this review to highlight some recent progress and to propose a framework of regulations that are present not only in the circulatory system but in most organs and coordinate immunological defense mechanisms with speci¢c organ functions. The basis of such new regulations relies on some recent ¢ndings on biochemical properties of the TxA2 ^PGI2 system and will be outlined ¢rst. 2.1. Biosynthesis and catalytic mechanisms Concerning the release of TxA2 or PGI2 from their synthesizing cells, the basic mechanisms are known for a long time. A rise in intracellular Ca2‡ is considered the initial event leading to activation of phospholipase A2 (PLA2 ) and the liberation of arachidonic acid (AA) [16^18] (Fig. 1). Newer additions to this simple scheme involve different Ca2‡ -pools from which the in£ux of extracellular Ca2‡ has a major and the primary release from intracellular stores has a minor e¡ect on the activa-

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Fig. 1. The cyclooxygenase pathway.

tion of phospholipase A2 (PLA2 ). Phosphorylation is another pathway for PLA2 activation, but not yet fully understood [19,20]. Meanwhile more than ten PLA2 isoenzymes are known which possibly act on di¡erent phospholipid pools [20]. This has led to the concept that AA is not evenly distributed in the cell but may be compartmentalized through metabolism, reacylation or the presence of fatty acid binding proteins. Certainly PLA2 activity is the ¢rst step for PG biosynthesis and sometimes can limit the availability of PGs. However, the release of AA is not generally the rate-limiting step in PG formation as often assumed. Its output may largely exceed its requirement for PG synthesis and the potent reacylation capacity in most cells underlines this assumption. After AA release the subsequent cyclization step to 15-OH-prostaglandin-9,11-endoperoxide (PGH2 ) in the biosynthetic scheme is irreversible and therefore has the potential to be rate-limiting under most conditions [21^23]. New aspects concerning the synthesis of PGH2 by the corresponding PG synthase commonly referred to as cyclooxygenase (COX) deal with the existence of the before mentioned cytokine-inducible COX-2 and have already found pharmacological applications [24^26]. This breakthrough in PG research will have to wait for a ¢nal evaluation since COX2 seems to be present also under normal or developmental conditions, e.g., in kidney and brain (see Section 3), and hence a pharmacological intervention may also a¡ect PG synthesis under physiological conditions if positive COX-2 e¡ects would be blocked by speci¢c COX-2 inhibitors [21,22]. In brain the action of some antiin£ammatory drugs like acetaminophen are di¤cult to match

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with the properties of the known COX-1 and COX-2 isoenzymes and a third form has been postulated [27,28]. However, the experimental basis for COX-3 is quite poor especially since COX-inhibitors show unusual kinetic behavior and seem to trigger conformational changes during their actions that can explain changes in a¤nities [27,28]. Finally, the last step in the synthesis of TxA2 and PGI2 involves two isomerases which possess similar biochemical and mechanistic properties [29^32]. Both enzymes convert the same PG-endoperoxide (PGH2 ) by a heme-thiolate dependent mechanism, but its rearrangement leads to the two di¡erent products with the described antagonistic properties [33^35]. For the chemically interested reader a proposed mechanistic view of this fascinating catalysis is added in Fig. 2 and also will be a subject of reconsideration in the discussion about a potential regulation of both pathways (Fig. 2). In essence this scheme contains equivalent structures and intermediates from which the carbon-centered radicals are the most interesting since their oxidation by the ferryl-thiolate (or ferric-thiyl) center is essential and most characteristic for heme-thiolate (P450) enzymes [33^35]. TxA2 -synthase has a high molecular activity [33^35] and is able to convert PGH2 e¤ciently to TxA2 but it is another unsolved question is why simultaneously and in almost equimolar amounts the products heptadecatrienoic acid (HHT) and malondialdehyde (MDA) are formed in

Fig. 2. Proposed mechanisms for Tx- and PGI2 -synthase.

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Fig. 3. Gene structure of PGI2 - and Tx-synthase.

parallel to TxA2 [34,35]: Do they ful¢ll other signaling functions or are they unavoidable by-products of the proposed isomerization pathway in which the intermediate C-radical becomes a bifurcation point because of mechanistic reasons (Fig. 2)? In fact both may be the case. The isomerization of PGH2 by PGI2 -synthase forms PGI2 as a single product but this reaction is relatively slow in its turnover compared to TxA2 synthase, possibly through the deprotonation of the ionic intermediate as a rate-limiting step in the isomerization process [33]. This comparably slow turnover may be compensated, however, by the high amount of enzyme being present in the PGI2 synthesizing cells and tissues. 2.2. Localization Some properties of the TxA2 ^PGI2 system can be

understood only if the cells and compartments in which both mediators are synthesized are considered. A localization of PGI2 -synthase to the cell membrane of smooth muscle cells had been reported [34,35] and recent work by Spisi et al. (personal communication) strongly suggests an association of the endothelial enzyme with caveolae. Such compartments of the cell membrane are known to be involved in other signaling processes [36] like nitric oxide synthesis, and localization to this compartment would also facilitate the release of PGI2 to the extracellular space. On the other hand it poses a new problem of localization for COX and PLA2 relative to PGI2 -synthase. From antibody labeling PGI2 -synthase was proposed to be attached to the cytosolic side whereas the same technique suggests COX-1 on the luminal side of the vesicles [24^26] but the sidedness of caveolae and the endoplasmic vesicles has to be considered. The picture of `enzymatic coupling' between the

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two COX isoforms and prostaglandin synthesizing enzymes is a further complication and remains largely unclear. Some groups report that PGI2 synthesis is more related to COX-2 in macrophages in spite of its low e¤cacy in PGH2 supply [24^26], whereas other authors clearly demonstrate that in endothelial cells synthesis is strongly associated with COX-1 activity. Further investigations should establish the PG patterns and their relative associations with the two COX isoforms under disease or developmental state conditions. The same problem arises for TxA2 and its release from platelets. TxA2 -synthase is microsomal but this would also be in agreement with a localization to the `open canalicular system' from which a release would be facilitated. In the case of platelets TxA2 synthase activity is clearly coupled to COX-1 activity. 2.3. Transcriptional control of TxA2 and PGI2 biosynthesis The demand for TxA2 and PGI2 can either be instantaneous, on a medium range, or even on a long time range. Accordingly, the rate-limiting enzymes required are either present constitutively, can be synthesized via `early genes', or may change as part of a di¡erentiation program. The release of TxA2 from platelets and of PGI2 from the endothelium is typical examples for an immediate biosynthesis after cell stimulation. Both involve COX-1, which is considered to be constitutive but reports on shear stress-induced endothelial COX1 induction prove that this de¢nition does not hold in a strict sense [37,38]. If the situation requires a higher output a medium range regulation starts by induction of COX-2 within 0.5^4 h of stimulation that usually involves cytokines and their activation by the transcription factor NFU B [25]. Both enzymes have half-life times between 25 and 30 h and therefore are not participating in a medium range regulation but rather in a long-range expression of activity. This is typical for di¡erentiating cells and in the case of TxA2 -synthase for example activin A and vitamin D3 can take part in the induction [39,40]. The promoter region of PGI2 synthase is more complex and suggests even a broader importance such as in developmental processes (Fig. 3).

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3. Post-transcriptional and redox regulation Considering the half-life times of 25^30 h for both isomerases, a short-term regulation could involve post-translational processes but at least for TxA2 synthase no indications for such reactions exist. For PGI2 synthase the primary structure contains potential phosphorylation sites and an A-cyclase dependent phosphorylation has been observed, however, with little change in activity [41]. There are also sites for palmitoylation and myristoylation but again their signi¢cance has to be investigated. The association of PGI2 -synthase with caveolae certainly requires transport processes during which the interaction with the COX-1 or COX-2 enzymes or the arachidonic acid pools may change and re£ect on the PGI2 output. Indeed, the correlation between the COX-1/COX-2 content and PGI2 synthesis is poor as shown for the endothelium or macrophages so that additional factors must play a role. 3.1. The `peroxide tone' Even resting cells will have basal levels of arachidonic acid which could lead to uncontrolled synthesis of prostaglandins if COX-1/COX-2 would be continuously active. This is not the case due to the requirement of limiting level of peroxides which allows conversion of the ferric enzyme into a ferryl-tyrosyl radical state [25,26]. This so-called `peroxide tone' upon which the COX enzymes become activated is di¡erent for both isoenzymes (COX-1W21 nM; COX-2W2.3 nM) [25,26] and usually not reached in resting cells: Upon oxidative signals and in addition by high levels of arachidonic acid the activation of the enzymes overcomes the continuous reductive inactivation in the cell and then autocatalytically by generation of the 15-hydroperoxy-prostaglandin9,11-endoperoxide (PGG2 ) both enzymes rapidly gain activity. Under physiological conditions the level of the peroxide tone can be restricted, especially for COX-1, and likewise in endothelial cells the supply of peroxides can be limiting for the generation of PGs. One can speculate therefore that the level of peroxides also is controlled and that signals for PG formation also trigger the formation of peroxides. In endothelial cells superoxide release from NADPHoxidase could be such a triggering event and indeed

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agonists like angiotensin II involve also the activation of NADPH-oxidase activity [42]. From superoxide the dismutation yields hydrogen peroxide as a mediator of peroxide tone but more e¤cient is a recombination of O3 2 with NO to give peroxynitrite (PN): c

3 NO ‡ c O3 2 ! OONO

k ˆ 6:7U109 M31 s31

This reaction is faster than the Cu,Zn^SOD catalyzed dismutation and generates the very active peroxynitrite (PN) known to e¤ciently provide the peroxide tone for COX-1. 3.2. The inhibition of prostacyclin synthase by peroxynitrite During studies on the peroxide tone in the endothelium our group had observed that PN caused inhibition of puri¢ed PGI2 synthase already at very low levels [43]. The underlying reaction has been identi¢ed as a nitration of an active site-located tyrosine residue [44] and model investigations revealed that hemoproteins in general and other P450 enzymes especially can cause such nitrations by the following mechanism (Fig. 4). For PGI2 synthase this reaction occurred already below 0.1 WM PN bolus additions indicating a possible physiological signi¢cance. Indeed, endothelial cells in atherosclerotic vessels [45] and after events of hypoxia/reperfusion [46] showed the presence of nitrated enzyme and concomitantly an inhibition of the enzyme with an impaired relaxation of the vessel after angiotensin II stimulation. In such experiments it was also found that the total COX activity was not altered and that other PGs, mostly PGE2 , were generated instead of PGI2 . Most interestingly, however, the impaired relaxation of the vessel was even converted to a sustained contraction resulting in a vasospasm [47]. A clue to the underlying mechanism was provided by the action of a TxA2 receptor antagonist that completely abrogated the vasospasm. This unexpected behavior can be explained by the dual activity of the TxA2 ^PGH2 receptor reacting equally well with TxA2 and PGH2 . Thus the remaining PGH2 after PGI2 synthase inhibition can convert the physiological relaxation e¡ects of PGI2 into a contracting action for smooth muscle. It is surprising that PGI2 -synthase is the only pro-

Fig. 4. Heme-thiolate catalysed tyrosine nitration by peroxynitrite.

tein nitrated at such low levels of PN. Other enzymes also can be found nitrated, but at more than 20-fold higher levels under which also sulfoxidations of methionine or oxidation of vicinal dithiols to disul¢des take place. It is not known, but can be expected, that such additional post-translational modi¢cations also function in cellular signaling. Such changes are reversible by corresponding reductases whereas only one report on a potential nitrotyrosine reversibility has appeared in literature [48]. 4. Receptors for thromboxane A2 and prostacyclin The unusual properties of the TxA2 ^PGH2 receptor had been unravelled after the very potent TxA2 synthase blockers developed by pharmaceutical companies had shown no inhibitory e¡ects on platelet aggregation. In the light of the inhibition of PGI2 synthase by PN the results now could be interpreted as a switch from PGI2 actions to that of the antagonist TxA2 since PGH2 acted in the same way as TxA2 in the vessel system. As a consequence TxA2 ^ PGH2 receptor agonists were developed and indeed turned out to be very e¡ective. This brought into focus the biochemistry and physiology of the PGI2 and TxA2 receptors. Prostanoid receptors belong to the family of sevenloop transmembrane spanning G-protein coupled re-

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ceptors (GPCRs). For PGI2 there is only one type known which is named IP-receptor (stands for Prostaglandin I2 -receptor), but from pharmacological studies with IP-receptor agonists and antagonists it is likely that another receptor for PGI2 should exist since cells containing no IP receptors still react on PGI2 . In contrast, TxA2 clearly has two receptor isoforms, called TPK (343 aa) and TPL (369 aa), which are expressed as splice variants. Those isoforms only di¡er in their C-terminal cytosolic tail region, which as a consequence mediates di¡erent G-protein coupling, e¡ector activation and is responsible for di¡erent answers in di¡erent cell types. Both isoforms increase inositol phosphates, but in the case of TPK cAMP levels are lowered whereas TPL does the opposite. Surprisingly both receptors can react not only with chemically di¡erent precursor of TxA2 , prostaglandin endoperoxide H2 (PGH2 ), but also with some isoprostanes derived from a free radical type of AA cyclizations [49]. Thus, the TP receptors can transduce the signal for oxidative stress at least in two ways. TP-receptors show a decreased signaling activity after a ¢rst stimulation (`downregulation'). Also there exists a downregulation by cAMP suggesting that IP-receptor activation by PGI2 is linked to an inactivation of TPK. Vice versa, a stimulation of the phosphatidyl inositol (PI) pathway downregulates the IP-receptor which is matching up with a tightly controlled Yin^Yang system. Except for binding to its A-cyclase coupled IPreceptor, PGI2 and its synthetic homologues also interact with the peroxisome proliferator activated receptor L/N (PPARL/N) which is widespread in adipocytes, ¢broblasts, uterus or in blood vessels [50] and belongs to the steroid hormone nuclear receptor superfamily. Its function is still unclear but may be related to development, di¡erentiation, stress or carcinogenesis. It would be interesting to clarify its role and to see whether TxA2 also counteracts such processes. 5. Organ speci¢city of TxA2 and PGI2 actions The new ¢ndings of conversion of PGI2 generating systems into those with antagonistic TxA2 analogous actions require a new concept of relating the PGI2 /

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TxA2 Yin^Yang system to the intercellular network in which they are embedded. This network again is changing with the developmental state of a tissue, its immunologic response which re£ects on the participation of cytokine action and as a consequence the synthesis of COX-2 and inducible NO-synthase (NOS-2). As another main determinant the generation of superoxide comes into play since the peroxide tone and PN formation greatly in£uence the PGI2 / TxA2 system. Such dynamics in the system and the tissue-speci¢c actions of TxA2 and PGI2 make it dif¢cult to develop a general picture of this regulatory system. Rather organ-speci¢c actions have to be discussed, but in order to facilitate the understanding some common principles seem to emerge. First, the function of COX-2 should be addressed. Although its main characteristics is the inducibility by cytokines like IL-1, TNFK and IFNQ or even better by their combined action, the presence of COX-2 in normal kidney is casting some doubt on its role exclusively under in£ammatory conditions. Never-

Table 1 Thromboxane synthase content and activity in di¡erent human tissues Tissue

Immunostained cells

Lung

Alveolar macrophage Alveolar epithelial cells Bronchial epithelium Kup¡er cells Monocytic cells Mesangial cells Podocytes Macrophages Dendritic cells Macrophages Crypt epithelium Langerhans cells Macrophages Dendritic cells Hofbauer cells Endothelial cells Fibroblasts Leiomyocyte Monocytic cells Bronchial epithelium Dendritic cells Dendritic cells Macrophages Histiocytes

Liver Kidney

Spleen Tonsils Skin Thymus Placenta

Bronchi Umbilical cord Uterus Small intestine Connective tissue

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theless, there are only a few exceptions from the rule, that COX-2 can coordinate the in£ammatory response in most tissues. PGE2 has been suggested as a mediator of pain and fever whereas not much has been reported on COX-2 linked functions of TxA2 and PGI2 . When comparing, however, the immunohistochemical localization of PGI2 synthase and TxA2 synthase one observes a variety of localizations that in normal tissue do not seem to contribute to a release of both mediators (see Tables 1 and 2). Smooth muscle, mesangial cells or neurons are such cells that express high levels of PGI2 synthase but are silent in synthesis because of lack of COX activity. The same applies for some epithelial cells, like in the gall bladder duct, for TxA2 (unpublished). Those cells can, however, gain function after COX-2 under in£ammatory or other stress conditions. We therefore would like to put forward the hypothesis that stress of di¡erent origin via COX-2 induction can in non-endothelial and non-immune cells release PGI2 or TxA2 for tissue-speci¢c actions supporting the defense reaction. A second rather general observation concerns the induction of NOS-2 (iNOS) that usually occurs later but mostly together with COX-2 induction. Hence, the release of COX-2 dependent PGI2 is accompanied by rather high tissue levels of NO as a hallmark of in£ammation. Since in£ammation also involves superoxide formation from various sources one can consider this as a counter-regulation. With an excess of NO there is even a depression of PN formation since PN reacts with NO to give N2 O3 [51]. As a second hypothesis one can postulate therefore that the early phase of in£ammation is characterized by NOS-2 and COX-2 induction with PGI2 synthesis occurring in cells that already contain PGI2 synthase but no COX activity. The third general principle concerns the last and critical phase of in£ammation when superoxide production is further upregulated mainly by the action of leukocytes or other sources as discussed recently [52]. Higher levels of PN are the consequence and an excess of superoxide even will lead to cell death, either by apoptosis or necrosis. The action of PGI2 will again be abolished by PN and then PGH2 actions will prevail. The transition from healthy conditions to the severely diseased states like shock will be gradual but

Table 2 Occurrence of PGI2 -synthesizing cells Tissue

Synthesizing cells

Large arteries and veins

Endothelium Vascular smooth muscle Neurons Pyramid cells Tracheal epithelia Alveolar epithelia Bronchial smooth muscle Arterial endothelial cells Arterial smooth muscle cells Kup¡er cells Endothelial cells Stellate (Ito+) cells Epithelial cells Mesangial cells Endothelial cells Non-vascular smooth muscle Non-vascular smooth muscle Non-vascular smooth muscle Mucosa muscularis Epithelium Myometrium Stroma Luminal epithelium Glandular epithelium Longitudinal smooth muscle Circular smooth muscle Corpus lutetium Graa¢an follicle Follicle Myocytes Endothelial cells Vascular smooth muscle Adrenal cortical tissue Non-vascular smooth muscle

Brain Trachea Lungs

Liver

Kidney

Urinary bladder Gastrointestinal tract Stomach

Uterus

Ovary

Heart

Adrenal gland Gall bladder, prostate, ductus deferens Vesicular gland microsomes Corpora lutea

Non-vascular smooth muscle Luteal cells Non-luteal cells Vascular endothelial cells Vascular smooth muscle cells

the three states from proin£ammation to in£ammation and ¢nally to the non-compensated cell death are useful to de¢ne when the actions of PGI2 and TxA2 ^PGH2 in single organs are to be described. 5.1. The vascular system and the heart The control of hemodynamics and hemostasis is

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still considered a hallmark of TxA2 ^PGI2 action but as outlined in Section 3.2 the role of NO has added a new dimension to the regulatory network. No doubt that NO deserves this attention but it is now time to study potential interactions between the PG- and NO-systems and to di¡erentiate between their relative distribution within the various parts of the circulatory system. PGI2 is considered to be the major vasorelaxant of the larger vessels, especially the aorta, whereas the microcirculation seems to depend more on NO. In the endothelium a host of mediators, like thrombin, TxA2 , vasopressin, angiotensin II, endothelin or ADP can trigger the PI-response causing Ca2‡ -rise followed by NO-synthase and PLA2 activation. Alternatively, mechanical responses like shear stress or volume changes may a¡ect phosphorylation steps leading to the same e¡ect. In agreement with the postulated synergistic action of PGI2 and NO both mediators seem to be released in a cooperative manner [50]. Also, with respect to their actions on smooth muscle relaxation both e¡ectors behave similarly but through di¡erent intracellular second messengers. NO acts by stimulating guanylyl cyclase [53], whereas PGI2 through its receptor is coupled to cAMP production [54]. Looking at their e¡ects on the intraluminal side of the vessel both NO and PGI2 exert a strong antiaggregatory action on platelets but the anti-adherent action on polymorphonuclear leukocyte or monocytes may be stronger for PGI2 than for NO (S. Galkina, personal communication). In the overall picture NO and PGI2 have a similar pro¢le of action and both together guarantee not only relaxation of the vessel but also a tightly sealed endothelium with a strong inhibitory action on platelet and polymorphonuclear leukocyte adhesion and activation. As outlined above, this picture of the resting and inactivated state of the vessel with relaxed tone is changing dramatically when superoxide is generated in the endothelium. It is too early to ¢nally describe the sources of superoxide but a physiological and a pathophysiological condition can be envisioned. A short exposure of aortic vessels causes constriction and several reports show activation of endothelial NADPH-oxidase as part of the Ang-II response [42]. This brief burst is too low in O3 2 generation to cause nitration and inhibition of PGI2 synthase

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[43,44]. However, an event of hypoxia followed by reoxygenation results in a more sustained superoxide formation as measured by a pronounced nitration of PGI2 synthase leading to a vasospasm of the vessel segment [46]. Since this vasospasm is completely suppressed by a TxA2 ^PGH2 receptor antagonist although no TxA2 is formed [46] one can conclude that the antagonistic principle to PGI2 under ischemia/reperfusion is PGH2 instead of TxA2 [46]. We recently have shown this to apply also for LPS (endotoxin) treatment of aortic segments (M.H. Zou et al., unpublished results). This situation applies for a time window between 45 and 120 min when no new enzyme synthesis can be observed. However, after 2 h one has to take into account the induction of COX-2 after LPS and this starts PGI2 formation in smooth muscle to occur via the constitutively expressed PGI2 synthase. Inducible NOS-2 follows with some delay and characterizes the strongly vasorelaxing phase of a vessel under severe in£ammatory conditions [55]. Therefore, the high output of 6-keto-PGF1K , the stable degradation product of PGI2 , in situations like shock, would originate in large part from smooth muscle under conditions of COX-2 induction. This activity of PGI2 -synthase in smooth muscle could also become of importance in case of a denuded vessel or a damaged endothelium. Platelets would then adhere to the collagen matrix and would release PGH2 and TxA2 after stimulation of the collagen receptor. PGH2 according to the `stealing hypothesis' may then be converted to PGI2 by smooth muscle cells [15]. According to their mitogenic potentials PGH2 and TxA2 could initiate a proliferation of smooth muscle cells with the e¡ect of restenosis, e.g., in balloon-catheterized vessel. This process would be a subject of counter-regulation by PGI2 but also by NO. In e¡ect, it would depend on the TxA2 +PGH2 + O3 2 /PGI2 +NO ratio whether proliferation or the resting response prevails. With its mechanical activity the heart controls shear stress and blood pressure and hence important parameters for maintenance of the vessel tone. The coronary vessels heavily rely on the output of PGI2 after stimulation by agonists of the endothelium but under physiological conditions TxA2 may have no function in the heart. Changes occur after the onset of the atherosclerotic process which adds TxA2 syn-

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thesizing macrophages to the vessel wall or in the ¢nal stages platelets as the ultimate mediators of infarction. But even under the ischemic conditions of an infarcted heart there is counter-regulation by the cardiomyocytes which contain PGI2 -synthase in signi¢cant amounts [56]. Because of the large mass their contribution to PGI2 formation after COX-2 induction could be signi¢cant. Also, NO formation may play a role as an antagonist to O3 2 . Such events have not been thoroughly studied but are important for the interpretation of PGI2 /TxA2 actions under ischemia^reperfusion conditions. 5.2. Lung Because of its possible involvement in asthma or ARDS the in£uence of TxA2 and PGI2 on the respiratory tract has raised interest but the situation is complicated for several reasons. First, as can be observed for eicosanoids in general, the relative importance of mediators in organ functions varies with the species. Likewise, in rats TxA2 exhibits strong actions on alveolar and bronchial constriction whereas in humans leukotriene LTC4 plays a more dominant role [57]. Complications also arise from a di¡erent regulation by TxA2 ^PGI2 on the aveolar and the vessel side. The latter displays the usual properties of the endothelium, but in the isolated perfused lung model rat and mouse shows a large and strictly COX-2 dependent PGI2 release upon LPS pretreatment of the animal [58]. It is not clear, however, whether the endothelium or smooth muscle are the source. TxA2 in rat lungs is also upregulated by LPS [57] but since TxA synthase is abundant in alveolar macrophages and also sensitive to COX-2 inhibition these cells mainly contribute to its formation. This puts emphasis on the sampling from either a lavage or blood. The permeability for PGs between both sides may be low but also may change in diseased states. The contribution of epithelial cells which contain Tx-synthase is also an unknown factor, but certainly the quite rapid onset of COX-2 synthesis will also activate such probably silent cellular sites of TxA2 synthesis. According to unpublished results low levels of PGI2 synthase are found even in isolated alveolar macrophages and part of it is tyrosine-nitrated (S. Schwarz, diploma thesis). It will be interesting

to study whether activated macrophages when turning on TxA2 synthesis will turn o¡ PGI2 synthesis or even generate high enough NO and O3 2 concentrations to block PGI2 synthesis by PN in neighboring cells under pathophysiological conditions. Another function of the TxA2 ^PGI2 couple that may be of general importance can be observed in lung and is associated with edema formation and microvascular permeability. Pre-exposition of isolated perfused rat lungs to s 97% oxygen for 48 h, but not of normal lungs, respond to leukotriene D4 by TxA2 release and edema formation and since TxA2 analogs cause edema in lamb lung [59] a corresponding role of TxA2 can be presumed. On the other hand PGI2 had been found active in gap junction formation in the canine trachea [60] which suggests the extension of the Yin^Yang principle also to opening and closing of epithelial or endothelial barriers by TxA2 and PGI2 . 5.3. Kidney Next to the circulatory system and lung, kidney displays physiological and pathophysiological properties that are strongly in£uenced by PGs, especially also PGI2 and TxA2 [61]. Thus the situation is similar to lung, only that the blood supply to kidney also a¡ects the ¢ltration rate as a speci¢c function of this organ. Connected to this whole glomeruli generate PGI2 , TxA2 , but also PGE2 and PGF2K , although di¡erences in their relative rates of generation have been reported [61,62]. Analysis of the individual glomerular cell populations has provided insight into the intraglomerular localization of prostanoid synthesis. Mesangial cells are a particularly rich source of PGI2 -synthase but their COX activity is rather low unless they are stimulated by in£ammatory cytokines [63]. This potently stimulates COX-2 and NO-synthase-2 synthesis as a counterregulation to in£ammation. Interestingly, cytokineinducible COX-2 is already present under normal conditions in cells of the macula densa, the cortical thick ascending limb cells near the macula densa and medullary interstitial cells [64^68]. The nature of the COX metabolites produced in this special region has yet to be characterized. PGI2 coupled to COX-2 may also be involved in kidney development since knock-

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outs for the IP-receptor and for COX-2 show malformations [69]. TxA2 synthase can be detected in the randomly distributed monocytic interstitial cells but surprisingly, a marked immunostaining was detected in podocytes [70]. Whether under physiological conditions TxA2 is released from these cells is not known, but it is likely that after in£ammatory events this source could be activated and then would a¡ect the ¢ltration rate. A counteraction can be expected from mesangial cells by the TxA2 -mediated release of PGI2 in£ammatory stimuli as a consequence of induction of COX-2. In£amed kidney tissue abundantly contains immigrated monocytes with apparently upregulated TxA2 synthase. Following the many reports that the impairment of kidney ¢ltration is largely due to TxA2 action [62] one can conclude that kidney failure among other factors may involve enhanced TxA2 formation and a diminished PGI2 counter-regulation as a main determinant. This is in agreement with the reported bene¢cial e¡ects of TxA2 ^PGH2 receptor blockers in kidney diseases such as toxin-mediated acute tubular injury, hypertension, glomerular in£ammatory injury or urinary tract obstruction [62,71,72]. 5.4. Brain PGI2 -synthase mRNA could be detected in rat brain [73,74] and the expressed protein was identi¢ed by Western blots in rat, bovine, and human brain homogenates [73]. Immuno£uorescence staining with a PGI2 -synthase antibody established its occurrence in brain blood vessels and in the vessels of the plexus choroides [73^75]. 6-Keto-PGF1K and TxB2 levels are high in the cerebrospinal £uid [76]. PGI2 synthase also is localized to Purkinje cells of the cerebellum and pyramid cells of the cerebrum [72^75]. In contrast, isolated rat astrocytes were clearly negative, whereas for isolated neurons a signi¢cant content of the enzyme was con¢rmed. Investigations on the occurrence of COX-1 and COX-2 in brain had revealed on unusual expression of COX-2 in neurons of apparently normal tissues [77^79], which adds doubts to the current opinion that this enzyme is an immediate early gene and dependent on induction by cytokines. There is, however, no need to

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abandon this view since already a brief exposure to hypoxic conditions could have been an appropriate stimulus for COX-2 induction [78,79]. Ischemia indeed would be a suitable indication for PGI2 formation to dilate surrounding vessels for increased blood £ow. Brain tissue seems to express two types of PGI2 -receptors [80,81] and the fact that they both are upregulated by an in£ammatory response supports a function of PGI2 in the defense against injury and infection. Alzheimer's disease could be a related pathophysiological situation since PGI2 synthesis also was found upregulated [82]. Comparing such ¢ndings with those from kidney, a similar picture emerges in which the kidney mesangium as well as neurons contain high levels of PGI2 -synthase, which become e¡ective only after COX-2 induction. TxA2 -synthase could clearly be detected in astrocytes [79], so that the Yin^Yang system seems to function also in brain, although its targets besides the vascular system are still to be de¢ned. 5.5. Other organs and cancer cells Surprisingly little is known about the TxA2 ^PGI2 system in liver. The hepatocyte seems to be not involved but Kup¡er cells exhibit TxA2 -synthase activity and stain heavily with a TxA2 -synthase antibody [83]. A double staining of liver tissue with a PGI2 synthase antibody showed some reaction also with Kup¡er cells and a clear positive staining of cells which were either endothelial cells or stellate (Ito-) cells (unpublished data). The levels of 6-keto-PGF1K were found to correlate positively with transplanted liver function and stable analogs of PGI2 improved ischemia-reperfusion damage in liver [84]. In contrast TxA2 analogs contract the vessel system until activation of glycogenolysis indicating hypoxic conditions [85,86]. Complement C5a can stimulate Kup¡er cells to release TxA2 which will activate endothelial cells through their TP receptor present in high amounts as judged from its mRNA [85,86]. Thus, only under in£ammatory or hypoxic conditions the TxA2 ^PGI2 system seems to come into play, which again suggests that similar to lung also in liver the induction of COX-2 may be the critical event for the release of substantial amounts of both prostanoids.

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Using the TxA2 -synthase antibody a positive staining was found in the Langerhans cells of skin (T. Rosenbach, V. Ullrich, unpublished) and the enzyme is expressed in all monocytic cells (e.g., interstitial cells) as well as in blood monocytes [70,87]. In£amed tissues heavily stained for TxA2 -synthase in immigrated monocytes [70,87]. Since such cells exhibit a more dense staining as circulating monocytes, an induction of TxA2 -synthase may have occurred. Indeed, inductions have been observed with activin A and vitamin D3 in culture [39,40] but such conditions may re£ect the onset of a di¡erentiation program. This may even apply for polymorphonuclear leukocytes since normal human PMN isolated from blood of healthy donors are devoid of the enzyme but under pathological conditions PMN may acquire TxA2 synthesis (unpublished). As a result of such immunohistochemical studies a localization of TxA2 -synthase was found in 70^80% of the tumor tissues investigated [88]. A correlation with proliferation could not be established but there may be connected with metastasis, as ¢rst proposed by Honn [89]. Studies with a TxA2 receptor blocker were successful in blocking metastasis in BL6 black mice. In agreement with the antagonistic action of PGI2 an antimetastatic potential of this prostanoid or its stable analogs would be likely and indeed has been established [90]. As a working hypothesis it appears that tumor cells make use of the endotheliumactivating potential of TxA2 and thus open up a promising therapeutic aspect to prevent invasion of tumor cells into tissue. In summary, physiological functions of TxA2 ^ PGI2 so far clearly involve the circulatory system. Kidney may also rely on speci¢c roles associated with development and glomerular ¢ltration. A similar function also may apply for the vessels of the plexus choroides in brain. Otherwise, TxA2 -synthase and PGI2 -synthase are present in a variety of cells, but with low and insu¤cient quantities of COX activity. This dramatically changes under the in£uence of in£ammatory stimuli or other stress situations and then under pathophysiological conditions all investigated organs will display TxA2 and PGI2 actions. In most cases TxA2 is released ¢rst and then counteracted by PGI2 .

6. Conclusions and outlook Summarizing the recent developments in the TxA2 ^PGI2 ¢eld the emerging role as a Yin^Yang regulatory couple certainly deserves attention especially because of the close association with states of disease. Future work should be concerned with the spacial or time-dependent separation of the physiological actions of both mediators. One means of separating their e¡ects could be by a di¡erent coupling to the COX-1 and COX-2 isozymes which also appears as a prerequisite for a rational use of COX-2 inhibitors. Very new and unexpected came the inactivation of PGI2 -synthase by tyrosine nitration but, together with the hitherto unexplained dual speci¢city of the TxA2 ^PGI2 receptor, this ¢nding provides a mechanistic basis for endothelial dysfunction seen under many conditions which require permeability changes and endothelial activation. Work on this aspect of PGI2 -synthase nitration is currently going on in our laboratory. Such investigations on physiological or pathophysiological regulation of TxA2 ^PGI2 action would be incomplete without knowing the network involving other regulators, like cytokines, growth factors or additional eicosanoids and lipid mediators. Pharmacological interventions should consider the whole network and this not only for the circulatory system but also for the main organs and even the not yet mentioned actions of TxA2 ^PGI2 in the eye, the skin, the spleen or the male and female reproductive system. Many results worth to be mentioned here are already present in literature but could not be included. They certainly will help to bring together more pieces of the puzzle which will emerge together with future studies and ¢nally will show a complex but highly e¤cient array of regulatory pathways. Acknowledgements Most of this work has been supported by grants of the DFG over the past 10 years, especially by the SFB 156 und Forschergruppe `Endogene Gewebszer-

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