Studies of Receptors and Modulatory Mechanisms in Functional Responses To

From the National Institute of Environmental Medicine Karolinska Institutet, 171 77 Stockholm, Sweden

Studies of Receptors and Modulatory Mechanisms in Functional Responses to

Cysteinyl-leukotrienes in Smooth Muscle

Magnus Bäck, M.D.

Stockholm 2001

Magnus Bäck Experimental Asthma & Allergy Research Institute of Environmental Medicine Karolinska Institutet 171 77 Stockholm Sweden

Tel: +46 8 728 72 03 Fax: +46 8 300 619 E-mail: [email protected]

Abstract

Cysteinyl-leukotrienes, i.e. leukotriene (LT) C4, D4 and E4, are inflammatory mediators and potent airway- and vasoconstrictors. Two different cysteinyl- leukotriene receptors have been cloned, CysLT1 and CysLT2. The aim of this thesis was to explore the effects of cysteinyl-leukotrienes in smooth muscle containing tissues of mainly vascular or airway origin. In order to characterise the CysLT receptors as well as the modulatory mechanisms involved in cysteinyl-leukotriene- induced responses, contractions of isolated tissue preparations were studied in organ baths and mediator release and leukotriene metabolism were analysed by enzyme immunoassay and RP-HPLC, respectively. In the guinea-pig trachea, the CysLT1 receptor antagonist ICI 198,615 partially inhibited the contractions induced by LTD4, and abolished the LTE4-induced contractions. The contractions induced by LTC4 in the guinea-pig trachea and ileum were resistant to CysLT1 receptor antagonism and competitively inhibited by the dual CysLT1/CysLT2 receptor antagonist BAY u9773, supporting that LTC4 activates CysLT2 receptors in these preparations. In contrast, the contractions of the human and porcine pulmonary arterial smooth muscles were resistant also to BAY u9773, suggesting the presence of another CysLT receptor subtype. In human and porcine pulmonary arteries, cyclooxygenase products were the major modulators of cysteinyl-leukotriene responses. In the human pulmonary artery, LTC4 and LTD4 stimulated the release of prostacyclin and consequently, the contractions induced by the leukotrienes were significantly enhanced after cyclooxygense inhibition. In contrast, porcine pulmonary arterial preparations mainly generated contractile cyclooxygenase products in response to LTC4. Nitric oxide synthase inhibition unmasked contractions to LTC4 in porcine pulmonary veins, but had no effect on LTC4-induced contractions in porcine pulmonary arteries. A preferential regulation by nitric oxide in porcine pulmonary veins compared with arteries was also observed with noradrenaline, acetylcholine and bradykinin. The metabolism of LTC4 into LTD4 and subsequently into LTE4 may modulate the responses to LTC4 and LTD4 in the guinea-pig trachea. In addition to this metabolism, it was discovered that the guinea-pig trachea also formed LTC4 from LTD4. The latter alternative metabolic pathway changed the LTD4-induced CysLT1 response into a CysLT2 receptor response. In conclusion, the present thesis suggests the existence of a previously unrecognised receptor for cysteinyl-leukotrienes. In addition, it was demonstrated that cysteinyl-leukotriene responses in pulmonary vessels were regulated by the release of modulatory factors, of which cyclooxygenase products dominated in the arteries and nitric oxide was the main modulator in porcine pulmonary veins. Moreover, in tissues with a heterogeneous CysLT receptor population, the interconversion between LTC4 and LTD4 may represent a major modulatory mechanism by deciding which CysLT receptor is activated by the cysteinyl-leukotrienes.

ISBN 91-628-4846-1

This thesis is based on the following papers, which will be referred to in the text by their Roman numerals.

I. Bäck, M., Wikström Jonsson, E. & Dahlén, S.E. 1996. The cysteinyl-leukotriene antagonist BAY u9773 is a competitive antagonist of

leukotriene C4 in the guinea-pig ileum Eur J Pharmacol, 317, 107-113

II. Bäck, M., Norel, X., Walch, L., Gascard, J.P., Mazmanian, G., Dahlén, S.E. & Brink, C. 2000. Antagonist resistant contractions of the porcine pulmonary artery by cysteinyl- leukotrienes Eur J Pharmacol, 401, 381-388

III. Bäck, M., Norel, X., Walch, L., Gascard, J.P., de Montpreville, V., Dahlén, S.E. & Brink, C. 2000. Prostacyclin modulation of contractions of the human pulmonary artery by cysteinyl-leukotrienes Eur J Pharmacol, 401, 389-395

IV. Bäck, M., Walch, L., Norel, X., Gascard, J.P., Mazmanian, G. & Brink, C. 2002. Modulation of vascular tone and reactivity by nitric oxide in porcine pulmonary arteries and veins Acta Physiol Scand, 174, 9-15

V. Bäck, M., Kumlin, M., Cotgreave, I.A. & Dahlén, S.E. 2001.

An alternative pathway for metabolism of leukotriene D4: effects on contractions to cysteinyl-leukotrienes in the guinea-pig trachea Br J Pharmacol, 133, 1134-1144

CONTENTS 1. BACKGROUND...... 9 1.1. CYSTEINYL-LEUKOTRIENES...... 9 1.1.1. Introduction ...... 9 1.1.2. Formation and metabolism of cysteinyl-leukotrienes...... 9 1.1.3. Cysteinyl-leukotriene receptors...... 11 1.2. EFFECTS OF CYSTEINYL-LEUKOTRIENES ...... 13 1.2.1. Airways ...... 13 1.2.2. Cardiovascular system ...... 15 1.2.3. Other targets...... 18 1.2.4. Secondary released factors...... 19 2. AIMS OF THE THESIS...... 21 3. METHODS ...... 22 3.1. GENERAL...... 22 3.2. EXPERIMENTS...... 22 3.2.1. Organ bath experiments ...... 22 3.2.2. Enzyme immunoassay...... 22 3.2.3. RP-HPLC...... 22 3.2.4. Measurements of γ-GT activity...... 23 3.2.5. Experimental protocols...... 23 3.2.6. Ethical approval ...... 23 3.3. DATA ANALYSIS...... 23 3.3.1. Contractions and relaxations ...... 23 3.3.2. Pharmacological analysis ...... 23 3.3.3. Statistics...... 24 4. RESULTS & DISCUSSION ...... 26 4.1. CYSTEINYL-LEUKOTRIENE RECEPTORS...... 26 4.1.1. Specific aims...... 26

4.1.2. CysLT1 receptors ...... 26

4.1.3. CysLT2 receptors ...... 32 4.1.4. Other CysLT receptor subtypes? ...... 35 4.1.5. Classification of CysLT receptors ...... 39 4.1.6. Summary: Cysteinyl-leukotriene receptors ...... 39

4.2. MODULATION OF RESPONSES TO CYSTEINYL-LEUKOTRIENES IN THE GUINEA-PIG TRACHEA...... 41 4.2.1. Specific aims...... 41 4.2.2. Modulation by prostaglandins...... 41 4.2.3. Metabolic modulation of contractions to cysteinyl-leukotrienes...... 44 4.2.4. Summary: Modulation of cysteinyl-leukotriene responses in the guinea-pig trachea...... 48 4.3. MODULATION OF RESPONSES TO CYSTEINYL-LEUKOTRIENES AND OTHER AGONISTS IN HUMAN AND PORCINE PULMONARY VESSELS ...50 4.3.1. Specific aims...... 50 4.3.2. Cysteinyl-leukotriene responses in the pulmonary vascular bed ...... 50 4.3.3. Modulation of cysteinyl-leukotriene responses by prostaglandins...... 51 4.3.4. Modulation of cysteinyl-leukotriene responses by nitric oxide ...... 54 4.3.5. Modulation of responses to other agonists...... 54 4.3.6. Regulation of basal vascular tone ...... 56 4.3.7. Mechanisms involved in the differential modulation of vascular responses in porcine pulmonary arteries and veins...... 56 4.3.8. Metabolism of cysteinyl-leukotrienes in vascular preparations...... 57 4.3.9. Pulmonary hypertension...... 59 4.3.10. Summary: Modulation of cysteinyl-leukotriene responses in the pulmonary vasculature...... 60 5. CONCLUSIONS...... 61 5.1 CysLT RECEPTORS...... 61 5.2 MODULATORY MECHANISMS ...... 61 5.2.1. Release of modulatory factors ...... 61 5.2.2. Interconversion between the cysteinyl-leukotrienes...... 61 5.3. CURRENT ISSUES...... 61 6. REFERENCES...... 64

Abbreviations cAMP cyclic adenosine 3’,5’-monophosphate COX cyclooxygenase cGMP cyclic guanosine-3’,5’-monophosphate EGF epidermal growth factor

Emax maximal contraction γ-GT γ-glutamyl transpeptidase GP guinea-pig GPNA γ-glutamyl-P-nitoanilide GSH reduced glutathione GSSG oxidised glutathione ω L-NOARG N -nitro-L-arginine LT leukotriene IUPHAR International Union of Pharmacology PBS phosphate buffered saline PG prostaglandin PNA P-nitoanilide RP-HPLC reverse phase high performance liquid chromatography SRS-A slow reacting substance of anaphylaxis TX thromboxane

hydroperoxyeicosatetraenoic acid (5-HPETE). 1. BACKGROUND This reaction requires a 5-lipoxygenase 1.1. Cysteinyl-leukotrienes activating protein (FLAP), which, in addition 1.1.1. Introduction to the actual enzyme, is a target for inhibitors of cysteinyl-leukotriene formation. The Cysteinyl-leukotrienes, i.e. leukotriene (LT) unstable epoxide LTA4 is conjugated with C , D and E (Fig. 1), are inflammatory 4 4 4 glutathione (GSH) by LTC4 synthase, an mediators belonging to the group of enzyme belonging to the family of Membrane eicosanoids. The name leukotriene originates Associated Proteins in Eicosanoid and in that these compounds were first isolated Glutathione Synthesis (MAPEG), and the from leukocytes and that the molecular reaction yields LTC4. The biosynthesis of structure includes three conjugated double cysteinyl-leukotrienes has been reviewed by bonds, i.e. a triene (Samuelsson et al., 1987). Samuelsson and co-workers (1987). Leukotriene C4, D4 and E4 all contain cysteine (Fig. 1) and are therefore referred to as the OH cysteinyl-leukotrienes in order to distinguish COOH them from LTB4, which is a non-cysteine LTC4 C5H9 S containing dihydroxy-leukotriene formed by Cys Gly Glu the action of LTA4 hydrolase (Samuelsson et al., 1987). Before the structures of the cysteinyl- leukotrienes were known, a Slow Reacting γ -GT Substance of Anaphylaxis (SRS-A; Brocklehurst, 1960) was shown to be released from animal lungs after challenge with a γ-Glu snake venom (Feldberg & Kellaway, 1938). This substance induced slow contractile OH responses of guinea-pig intestinal COOH preparations and was later related to LTD 4 C5H9 S anaphylactic reactions and broncho- Cys Gly constriction (Brocklehurst, 1960; Chakravarty & Uvnäs, 1960). The identification of the cysteinyl-leukotrienes led to the conclusion that what had been referred to as SRS-A was dipeptidase actually a mixture of LTC4, LTD4 and LTE4 (Samuelsson et al., 1987).

Gly 1.1.2. Formation and metabolism of cysteinyl-leukotrienes OH COOH LTE 1.1.2.1. Chemistry 4 C5H9 S The cysteinyl-leukotrienes are derived Cys from arachidonic acid that is liberated from cell membrane phospholipids by the action of phospholipase A (PLA ). The next two steps Fig. 1. Cysteinyl-leukotrienes (LTC4, LTD4 and LTE4) 2 2 and their metabolism by γ-glutamyl transpeptidase (γ- in cysteinyl-leukotriene biosynthesis are GT) and a Cys-Gly dipeptidase. Glu-Cys-Gly catalysed by 5-lipoxygenase and result in the represents the glutathione (GSH, γ-glutamyl formation of LTA4 via the intermediate 5- cysteinylglycine) side-chain of LTC4.

LTC4 is thus a glutathionyl between the cysteinyl and glycyl residues in eicosatetraenoic acid (Fig. 1) and shares its the LTD4 side chain (Bernström & subsequent metabolism with GSH (Örning & Hammarström, 1981; Hammarström, 1981; Hammarström, 1980; Hammarström, 1981). Sok et al., 1981). The product is thus a The metabolism of LTC4 is catalysed by γ- leukotriene with a cysteinyl group at carbon glutamyl transpeptidase (γ-GT), a membrane number 6 and is referred to as LTE4 (Fig. 1; bound enzyme that cleaves off the γ-glutamyl Samuelsson et al., 1987). The enzymatic group of the GSH side chain of LTC4, thus conversion of LTD4 into LTE4 is inhibited by yielding LTD4 (Fig. 1; Hammarström, 1981). L-cysteine (Sok et al., 1981). The enzyme γ-GT has two sites, a γ-glutamyl Cysteinyl-leukotriene metabolism takes donor site and an acceptor site (Tate & place in a number of tissues including human Meister, 1985). The donor site has a broad lung (Kumlin & Dahlén, 1990) and in specificity allowing most γ-glutamyl addition, the enzymes catalysing the compounds to serve as donor substrates formation of LTE4 are present in human whereas the acceptor site has a restricted plasma (Köller et al., 1985). Elimination of stereospecificity in that only L-amino acids or cysteinyl-leukotrienes takes place both via the dipeptides with both amino acids in L- urinary and faecal routes (Örning et al., 1985; configuration can serve as acceptors (Tate & Maltby et al., 1990) and urinary LTE4 can be Meister, 1985). Examples of donors, in used as a measure of whole body production addition to GSH and LTC4, are oxidised of cysteinyl-leukotrienes (Maltby et al., glutathione (GSSG), S-conjugated 1990). glutathiones such as S-hexyl GSH and S- decyl GSH (Fig. 2) as well as γ-glutamyl-P- nitroanilide (GPNA), a commonly used Glu substrate in experimental characterisation of Cys Gly γ-GT (Aharony & Dobson, 1984; Pologe et S al., 1984; Silber et al., 1986). In addition, iso- forms of γ-GT that preferentially metabolise SH S LTC4 rather than GSH have been described Cys Gly Cys Gly (Carter et al., 1997; Heisterkamp et al., 1991) Glu Glu and will be discussed in Section 4.2.3. L- cysteine, L-glutamine, L-glycylglycine and L- GSH GSSG cystine are examples of acceptors (Tate & Meister, 1985) and in addition, as will be shown in this thesis (Section 4.2.3.), also C6H13 C10H21 LTD4 can act as an acceptor of the γ-glutamyl group, in this way yielding LTC4 S S (Hammarström, 1981; Anderson et al., 1982; Cys Gly Cys Gly γ Paper V). Three different groups of -GT Glu Glu inhibitors will be addressed in the present thesis, namely the reversible inhibitor L- S-hexyl GSH S-decyl GSH serine borate (Tate & Meister, 1978; Örning & Hammarström, 1980), the irreversible inhibitor acivicin (Stole et al., 1994) and the substrate competitors GSH (Hammarström, 1981) and S-hexyl GSH. Fig. 2. Compounds used as γ-glutamyl donors in this thesis (Paper V): reduced glutathione (GSH), oxidised

The metabolism of LTD4 involves a glutathione (GSSG), S-hexyl GSH and S-decyl GSH. dipeptidase that cleaves the peptide bond

1.1.2.2. Source of cysteinyl-leukotrienes LTC4 were no longer inhibited by FPL 55712 (Snyder et al., 1984; Snyder & Krell, 1984; The formation of LTA4 via the 5- Weichman & Tucker, 1985; Hand & lipoxygenase pathway takes place in myeloid Schwalm, 1987; Muccitelli et al., 1987). cells, e.g. leukocytes, macrophages, mast cells (Samuelsson et al., 1987). The next step in Although FPL 55712 apparently cysteinyl-leukotriene synthesis, conjugation antagonises the receptors for cysteinyl- leukotrienes, its effect has been reported not of LTA4 with GSH, can take place in endothelial cells, vascular smooth muscle to be highly specific (Chasin & Scott, 1978; cells, platelets, mast cells and macrophages as Krell et al., 1981a). However, a number of well as in eosinophilic granulocytes (Claesson specific and potent cysteinyl-leukotriene & Haeggström, 1988; Maclouf & Murphy, receptor antagonists were subsequently 1988; Lindgren & Edenius, 1993). These developed, of which some are listed in differential activities mean that in some cells Table 1. In the guinea-pig ileum and/or (e.g. eosinophils, macrophages and mast guinea-pig trachea these antagonists all share the characteristics of being potent inhibitors cells), LTC4 can be formed from arachidonic of the LTD4- and LTE4-induced contractions, acid, whereas in cells that have LTC4 whereas they either not at all, or only synthase but lack 5-lipoxygenase, LTA4 is marginally, inhibit the LTC4-induced needed as a substrate. The LTA4 formed in one cell type can be donated to another cell contractions (see Table 4 in Section 4.1.). Despite these findings, the hypothesis of type in order to form LTC4. This phenomenon is referred to as transcellular biosynthesis and separate LTC4 and LTD4/LTE4 receptors was has been reviewed by Lindgren and Edenius not found generally applicable since in (1993). The biosynthesis of leukotrienes takes isolated human bronchi (Buckner et al., 1986; place in for example the human lung Buckner et al., 1990) as well as guinea-pig vasculature as shown by the increased gall bladder (Falcone & Krell, 1992) and rat formation of cysteinyl-leukotriene after lung strip (Norman et al., 1994), the stimulation of isolated perfused and ventilated contractions to all three cysteinyl-leukotrienes human lungs with calcium ionophore (Kiss et are inhibited by this class of antagonists. al., 2000). Moreover, in some preparations, both LTC4- and LTD4-induced contractions are resistant to the antagonists (Snyder & Krell, 1986; 1.1.3. Cysteinyl-leukotriene receptors Cuthbert et al., 1991a; Gardiner et al., 1993). In addition, in the guinea-pig trachea some 1.1.3.1. Functional characteristics results of studies of both contractions (Krell et The notion of cysteinyl-leukotriene al., 1983; Hand et al., 1989) and agonist receptors originates in the acetophenon binding (Aharony et al., 1988) suggest that FPL 55712, which was developed as an LTD4 may interact with more than one antagonist of SRS-A (Augstein et al., 1973). distinct receptor. After the structure elucidation of SRS-A, the Based on the above-mentioned findings, effects of the cysteinyl-leukotrienes were the names CysLT1 and CysLT2 were tested in different functional assays, for introduced for the receptors being sensitive example guinea-pig trachea and ileum. In the (CysLT1) or resistant (CysLT2) to the class of initial studies of the guinea-pig trachea, it was antagonists that had been developed found that FPL 55712 inhibited the (Table 1), which consequently are referred to contractions to all cysteinyl-leukotrienes as CysLT1 receptor antagonists (Coleman et (Krell et al., 1981b; Jones et al., 1983). al., 1995). The major interest of antagonists of However, when the metabolism of LTC4 into the CysLT1 receptor is because of their LTD4 (see above) was inhibited by either L- serine borate or GSH, the contractions to

beneficial effects in the treatment of asthma (see Section 1.2.1.), and ICI 204,219

CODE NAME GENERIC NAME TRADE NAME REFERENCE

ICI 198,615 Snyder et al., 1987 ICI 204,219 zafirlukast Accolate Krell et al., 1990

L 649,923 Jones et al., 1986 MK 571/L 660,711 Jones et al., 1989  MK 0476/L 706,631 montelukast Singulair Jones et al., 1995 LY 171,883 tomelukast Fleisch et al., 1985

SKF 104,353 pobilukast Hay et al., 1987 ONO 1078 pranlukast Onon Obata et al., 1992

Table 1: CysLT1 receptor antagonists.

 receptor antagonist (Cuthbert et al., 1991b). (zafirlukast, Accolate ), MK 0476 Subsequent studies extended this observation  (montelukast, Singulair ), and ONO 1078 by showing competitive antagonism by (pranlukast, Onon) have been clinically BAY u9773 of both CysLT1 and CysLT2 introduced for this purpose (Table 1; Drazen receptors in human bronchi and pulmonary et al., 1999). veins (Labat et al., 1992) as well as in rat The first evidence for the existence of also lung, ferret spleen and sheep bronchi CysLT2 receptors in human tissues, were (Tudhope et al., 1994). provided by Labat and co-workers (1992), who showed that the LTC4- and LTD4- induced contractions of the human pulmonary 1.1.3.2. Molecular characteristics venous smooth muscle were resistant to the During the course of the project presented

CysLT1 receptor antagonists ICI 198,615, in this thesis, the human CysLT1 and CysLT2 MK 571 and SKF 104,353. In addition, Ortiz receptors were cloned (Lynch et al., 1999; and co-workers (1995) extended these Sarau et al., 1999; Heise et al., 2000; observations by indicating the presence of Takasaki et al., 2000; Nothacker et al., 2000). both CysLT1 and CysLT2 receptors on the Binding of radiolabelled cysteinyl- endothelium of human pulmonary veins. leukotrienes to the first cloned human CysLT receptor was shown to be inhibited by By structural modification of LTE4, two CysLT receptor antagonists were developed, antagonists such as zafirlukast, montelukast BAY x7195 and BAY u9773 (Cuthbert et al., and pranlukast, and the receptor was thus classified as being the human CysLT 1991b; Abram et al., 1993). BAY x7195 was 1 receptor (Lynch et al., 1999; Sarau et al., shown to be a CysLT receptor antagonist, 1 1999). This classification was further inhibiting LTD -induced contractions in 4 supported by results of in situ hybridisation guinea-pig trachea and human bronchi but not showing receptor mRNA expression in in human pulmonary veins (Abram et al., human bronchial smooth muscle cells (Lynch 1993; Gorenne et al., 1995), whereas et al., 1999; Sarau et al., 1999). About one BAY u9773 had a broader activity than the year later a second CysLT receptor was previously described CysLT1 receptor cloned by three laboratories (Heise et al., antagonists. In fact, BAY u9773 was shown 2000; Takasaki et al., 2000; Nothacker et al., to be a competitive antagonist of both LTC4- 2000). This receptor exhibited similar and LTE4-induced contractions of the guinea- characteristics as the functionally defined pig trachea, i.e. a dual CysLT1 and CysLT2 CysLT2 receptor in that it was resistant to

CysLT1 receptor antagonists (Heise et al., A recent report has demonstrated a small 2000; Takasaki et al., 2000; Nothacker et al., residual contraction induced by LTD4 in 2000) but inhibitable by the dual human bronchi also in the absence of both CysLT1/CysLT2 receptor antagonist BAY intra- and extracellular calcium, and that u9773 (Heise et al., 2000; Nothacker et al., LTD4 induces contractions of cultured human 2000). airway smooth muscle cells without increasing intracellular calcium (Accomazzo

et al., 2001). In the latter report, it was 1.1.3.3. Transduction mechanisms suggested that the LTD4-induced contractions The CysLT receptors are 7-transmembrane of human bronchi may consist of both a G-protein coupled receptors (Lynch et al., calcium-dependent phase, responsible for the 1999; Sarau et al., 1999; Heise et al., 2000; onset of contraction, and a slower phase that Takasaki et al., 2000; Nothacker et al., 2000). involves the activation of a calcium- independent isoform of protein kinase C, The calcium mobilisation in CysLT1 and responsible for the maintenance of contractile CysLT2 receptor transfected cells is not inhibited by pertussis toxin, suggesting that tone (Accomazzo et al., 2001). In the guinea- pig trachea, the LTD4-induced contractions the G-protein involved is of the Gq/11 rather are only somewhat inhibited by nifedipinde or than the Gi/o class (Sarau et al., 1999; Heise et al., 2000). However there may be a difference verapamil, but effectively inhibited by SKF between transfected cells and cells with 96,396 (Weichman & Tucker, 1985; Cuthbert endogenously expressed receptors since in et al., 1994), supporting the involvement of differentiated U937 cells, a human monocyte receptor operated calcium channels in smooth muscle contractions induced by cysteinyl- leukaemia cell line, the LTD4-induced calcium increase is partially inhibited by leukotrienes. pertussis toxin (Crooke et al., 1990). In support of the suggestion that both pertussis toxin sensitive and insensitive G proteins are 1.2. Effects of cysteinyl-leukotrienes involved in CysLT receptor transduction 1.2.1. Airways (Crooke et al., 1990), the LTD -induced 4 1.2.1.1. Asthma reorganisation of the actin cytoskeleton in cultured human bronchial smooth muscle Asthma is a chronic inflammatory disease cells is sensitive to pertussis toxin (Saegusa et of the airways causing wheezing, al., 2001). breathlessness and cough associated with airflow obstruction. The basic alterations in The increased intracellular calcium asthma are bronchoconstriction, increased induced by LTD 4 in CysLT1 receptor mucus formation, oedema of the airway transfected cells is minimally affected by mucosa and increase in airway responsiveness removal of extracellular calcium, suggesting a to a variety of stimuli (Bousquet et al., 2000). release from intracellular stores (Sarau et al., The chronic inflammation of the airways in 1999), whereas in U937 cells most of the addition causes long-term effects, resulting in calcium is extracellularly derived (Crooke et structural changes of the airway wall al., 1990). In isolated human bronchi, the (Bousquet et al., 2000). Drugs modifying the contractions induced by LTD4 are cysteinyl-leukotriene pathway have beneficial significantly suppressed by an inhibitor of effects in the treatment of asthma (Drazen et receptor operated calcium channels (SKF al., 1999) and the actions of these 96,396; Gorenne et al., 1998) but not by inflammatory mediators in this disease are diltiazem or nifedipine, which are inhibitors described below. of voltage operated calcium channels (Bourdillat et al., 1987; Gorenne et al., 1998).

1.2.1.2. Bronchoconstriction formation (Bousquet et al., 2000). Cysteinyl- leukotrienes have been shown to induce The direct and potent contractile responses plasma exudation in guinea-pig airways (Hua induced by cysteinyl-leukotrienes in isolated et al., 1985) associated with an extravasation human bronchi was first demonstrated by of macromolecules (Persson et al., 1986). Dahlén and co-workers (1980), and confirmed These studies suggest a possible involvement in subsequent studies (Hanna et al., 1981; of cysteinyl-leukotrienes in the development Jones et al., 1982; Buckner et al., 1986). In of bronchial oedema. addition, inhaled cysteinyl-leukotrienes were shown to induce bronchoconstriction The increase in airway responsiveness to (Holroyde et al., 1981), being up to 10,000 methacholine induced by allergen in times more potent than histamine (Weiss et sensitised rats is inhibited by the CysLT1 al., 1982). Furthermore, the clinically receptor antagonist MK 571 (Wang et al., introduced leukotriene synthesis inhibitors 1993), suggesting that the bronchial hyper- and CysLT1 receptor antagonists have been responsiveness observed in asthmatics may be shown to significantly reduce induced by cysteinyl-leukotrienes. Prolonged bronchoconstriction in asthmatics (Dahlén et exposure to LTE4 has been reported to induce al., 1994; Drazen et al., 1999). hyperresponsiveness to histamine in both guinea-pig and human airways in vitro via a mechanism proposed to involve a facilitation 1.2.1.3. Mucus secretion of cholinergic neurotransmission due to Cysteinyl-leukotrienes have been shown to secondary released cyclooxygenase products increase bronchial mucus secretion in human (Lee et al., 1984; Jacques et al., 1991). In the airways in vitro using radiolabelled porcine trachea, LTC4 does not induce contractions, but enhances the contractions glucosamine as marker (Marom et al., 1982; + Coles et al., 1983). However, a recent study induced by charbachol or K depolarisation measuring a specific mucin gene product by increasing the responsiveness of the (MUC5AC) from human bronchi in vitro did contractile apparatus to calcium (Setoguchi et not observe an increased release after al., 2001). challenge with LTD4 (Labat et al., 1999). In patients suffering from chronic asthma, Although the exact role of cysteinyl- changes in the structure of the airway wall, leukotrienes in mucus secretion remains to be leading to a narrowing of the bronchial established, the notion of cysteinyl- lumen, is a common feature referred to as leukotrienes being potent mucus airway remodelling (Bousquet et al., 2000). secretagogues has received support from Experimental models have shown that 5- studies of cats and dogs in vivo (Peatfield et lipoxygenase inhibitors and CysLT1 receptor al., 1982; Johnson & McNee, 1983) as well as antagonists inhibit the allergen-induced guinea-pig trachea in vitro (Liu et al., 1998). increase in airway smooth muscle in rats In the guinea-pig trachea, the increased mucus (Wang et al., 1993; Salmon et al., 1999). In secretion induced by mucosal administration cultured human airway smooth muscle cells, of either LTD4 or antigen is inhibited by the LTD4 does not have any direct effects on CysLT1 receptor antagonists zafirlukast and mitogenesis, but it augments the DNA pranlukast (Liu et al., 1998). synthesis induced by epidermal growth factor (EGF) and induces reorganisation of the actin cytoskeleton, both of which is inhibited by the 1.2.1.4. Other airway effects CysLT1 receptor antagonist pranlukast Oedema of the bronchial mucosa is another (Panettieri et al., 1998; Saegusa et al., 2001). key finding in asthma, in addition to Taken together, these studies thus suggest a bronchoconstriction and increased mucus role of cysteinyl-leukotrienes also in airway

remodelling and that this effect may be an somewhat between the reports, the main additional target for anti-leukotriene drugs in conclusion is a biphasic pressor/depressor long-term treatment of asthma. response with a sustained hypotension (Drazen et al., 1980; Omini et al., 1981;

Dahlén, 1983; Hua et al., 1985). Likewise, in 1.2.2. Cardiovascular system monkeys and rats, a transient rise in arterial 1.2.2.1. Hemodynamic effects pressure has been reported, followed by a long-lasting hypotensive period (Smedegård Hypotension is a common feature in severe et al., 1982; Zukowska-Grojec et al., 1982; allergic reactions (Smith et al., 1980) and in Iacopino et al., 1983) whereas in other addition, cardiac changes such as arrhythmias species, such as sheep and pigs, cysteinyl- and ischemia have been observed during leukotrienes induce an increase in systemic anaphylactic shock (Bernreiter, 1959). The blood pressure (Ahmed et al., 1985; Olson & identification of SRS-A as the cysteinyl- Fleisher, 1989; Zellner et al., 1991). leukotrienes thus raised the suggestion that, in addition to the airways, also the Hemodynamic effects after systemic cardiovascular system may be a target for administration of cysteinyl-leukotriene are these inflammatory mediators. complex responses and may vary between different species and also depend on what In humans, LTC4 injected into the right doses of cysteinyl-leukotrienes that have been atrium results in a transient increase in cardiac used in the studies. Moreover, the responses output followed by a prolonged phase with may be further complicated by secondary cardiac output below initial (Kaijser, 1982). released factors. For example, the vascular These cardiac changes are associated with responses to cysteinyl-leukotrienes are closely similar variations in heart rate and forearm linked the cyclooxygenase and nitric oxide blood flow as well as a slight decrease in pathways (see Sections 1.2.4. and 4.3.) and in pulmonary and systemic blood pressures addition, cysteinyl-leukotrienes have been (Kaijser, 1982). Also after intravenous shown to increase the levels of circulating injection, LTC4 decreases the mean arterial cathecolamines in both rats (Zukowska- pressure and in addition, intracoronary Grojec et al., 1982) and humans (Vigorito et injection of LTD4 causes an increase in al., 1997). coronary vascular resistance (Vigorito et al., 1997). Another difficulty is to interpret from what action of the cysteinyl-leukotrienes the effects In guinea-pigs, both increase and decrease on blood pressure originate. In general, the of mean systemic arterial pressure have been changes in systemic blood pressure induced reported after injection of cysteinyl- by cysteinyl-leukotrienes reflect both a leukotrienes (Drazen et al., 1980; Omini et al., vasoconstriction (pressor response) and a 1981; Dahlén, 1983; Berkowitz et al., 1984; depression of cardiac function (depressor Hua et al., 1985;). Some of the apparent response). In humans (Kaijser, 1982), differences between those studies may refer to monkeys (Smedegård et al., 1982), rats that the use of anaesthesia may affect the (Iacopino et al., 1983) and pigs (Leffler et al., systemic cysteinyl-leukotriene-induced effects 1984; Olson & Fleisher, 1989) a decreased (Drazen et al., 1980). In addition, cardiac function is observed after cysteinyl- intravenously injected leukotrienes cause leukotriene injection. The decrease in cardiac changes in pulmonary function in guinea-pigs output induced by cysteinyl-leukotrienes may (Weichman et al., 1982; Dahlén, 1983), which be a result of increased vascular resistance, in turn may affect systemic responses if the dilatation of capacitance vessels leading to animals are not artificially ventilated. decreased venous return (Pawloski & Although the time-course of the blood Chapnick, 1993b) or an increased pressure changes in guinea-pigs differs

extravasation of plasma leading to a may affect cardiac function and suggesting a decreased circulating blood volume (Hua et possible role for specific CysLT2 receptor al., 1985). There are however observations antagonists and/or agonists in the treatment of supporting that cysteinyl-leukotrienes also cardiac diseases. In fact, 5-lipoxygenase have a direct effect on cardiac function. inhibitors and CysLT receptor antagonists have been shown to reduce infact-size and 1.2.2.2. Cardiac effects reperfusion arrhythmias in animal models of In sheep and pigs, cysteinyl-leukotrienes myocardial ischemia (Lepran & Lefer, 1985; induce coronary vasoconstriction, associated Hock et al., 1992) with ischemia and impaired left ventricular function (Michelassi et al., 1982; Fiedler et al., 1985). Increased coronary vascular 1.2.2.3. Pulmonary circulation resistance after intracoronary administration In the pulmonary circulation, cysteinyl- of LTD4 has also been observed in humans leukotrienes have been reported to induce a (Vigorito et al., 1997), and cysteinyl- pressor response in most species (Kadowitz & leukotrienes contract isolated atherosclerotic Hyman, 1984; Ahmed et al., 1985; Farrukh et human coronary arteries in vitro (Allen et al., al., 1986; Ohtaka et al., 1987; Olson & 1993). Likewise, in Langendorff-perfused Fleisher, 1989; Zellner et al., 1991). However, guinea-pig hearts, cysteinyl-leukotrienes in rats, LTD4 induces a pulmonary reduce coronary flow (Burke et al., 1982; hypotension (Iacopino et al., 1983) and in Letts & Piper, 1982) and in addition, a direct humans, a slight decrease in pulmonary artery effect on the myocardium has been suggested pressure has been reported after LTC4 to be part of the resulting impaired cardiac injection into the right atria (Kaijser, 1982). In function (Burke et al., 1982). However, monkeys, the pulmonary artery pressure studies of isolated guinea-pig cardiac muscle follows the changes in systemic blood preparations have generated conflicting data pressure, i.e. a transient rise, associated with concerning the direct effects of cysteinyl- increased vascular resistance followed by a leukotrienes, including no myocardial effects prolonged fall associated with a decrease in (Letts & Piper, 1982) as well as negative cardiac output (Smedegård et al., 1982) and a (Burke et al., 1982) and positive (Falcone et subsequent study showed that LTD4 induces al., 1991) inotropic effects. The cardiac contractions of pulmonary arteries derived effects of cysteinyl-leukotrienes may in from monkeys (Berkowitz et al., 1984). addition be dose-dependent since in Likewise, an increased pulmonary vascular Langendorff-perfused isolated rat hearts, low resistance has been reported in guinea-pigs concentrations of either LTC4 or LTD4 induce (Berkowitz et al., 1984), sheep (Ahmed et al., positive inotropic effects whereas higher 1985; Kadowitz & Hyman, 1984) and pigs concentrations induce negative inotropic (Leffler et al., 1984; Ohtaka et al., 1987; effects (Karmazyn & Moffat, 1990). In the Olson & Fleisher, 1989; Zellner et al., 1991). study reporting negative inotropic effects of Since the pig has been extensively studied cysteinyl-leukotrienes on isolated guinea-pig with respect to cysteinyl-leukotrienes in the myocardium, a reduced contractile force of cardiovascular system (Leffler et al., 1984; human atrial myocardium was also observed Fiedler et al., 1985; Galton & Piper, 1987; (Burke et al., 1982). Ohtaka et al., 1987; Olson & Fleisher, 1989; Interestingly, the recently cloned human Zellner et al., 1991) it was considered a CysLT2 receptor has been shown to be suitable species for the exploration of the expressed in the heart (Heise et al., 2000; effects of cysteinyl-leukotrienes on the Takasaki et al., 2000; Nothacker et al., 2000), pulmonary vasculature in the present project. further supporting the possibility that Interestingly, pulmonary venous resistance is circulating cysteinyl-leukotrienes directly increased by LTC4 in pigs in vivo, whereas

isolated porcine pulmonary veins have been leukotrienes (Berkowitz et al., 1984; Sakuma reported not to contract in response to LTC4 et al., 1987). in vitro (Ohtaka et al., 1987). Cysteinyl-leukotrienes not only contract 1.2.2.4. Isolated vessels isolated vessels but have also been reported to The first evidence of contractile effects of induce endothelium-dependent relaxations, cysteinyl-leukotrienes on isolated human first shown in canine renal and mesenteric vessels was provided by Hanna and co- arteries (Secrest et al., 1985) and workers (1981). These findings in pulmonary subsequently described in canine renal and vessels were examined in greater detail in splanchnic veins (Pawloski & Chapnick, subsequent studies (Schellenberg & Foster, 1991; Pawloski & Chapnick, 1993b) as well 1984; Bourdillat et al., 1987), with the as in guinea-pig aorta and pulmonary artery conclusion of a preferential vasoconstriction (Sakuma et al., 1987; Fedyna et al., 1990). In to cysteinyl-leukotrienes in human pulmonary addition, both systemic and pulmonary veins compared with pulmonary arteries. It vessels from humans exhibit endothelium- dependent relaxations to cysteinyl- was also shown that the LTD4-induced contractions of human pulmonary veins are leukotrienes (Allen et al., 1992; Ortiz et al., modulated by both relaxant and contractile 1995). factors released from the endothelium (Ortiz et al., 1995). Cysteinyl-leukotriene-induced 1.2.2.6. Microcirculation effects in the pulmonary vasculature have also been observed in studies of pulmonary vessels In the hamster cheek pouch derived from different animals, for example microcirculation, LTC4 and LTD4 are potent guinea-pigs where cysteinyl-leukotrienes constrictors of arterioles and in addition, induce contractions of pulmonary arteries induce a dose-dependent plasma leakage and (Hand et al., 1981; Berkowitz et al., 1984; extravasation of macromolecules from Fedyna et al., 1990) but only small postcapillary venules (Dahlén et al., 1981). contractions of pulmonary veins (Berkowitz Moreover, intravenous injection of cysteinyl- et al., 1984). Also rodent and porcine leukotrienes causes plasma extravasation pulmonary arteries have been reported to be throughout the body in guinea-pigs (Hua et more sensitive to cysteinyl-leukotrienes al., 1985) and LTD4 increases pulmonary compared with pulmonary veins (Maddox et vascular permeability in rabbits (Farrukh et al., 1985; Ohtaka et al., 1987). al., 1986).

Human systemic vessels also contract in Intradermal injection of either LTC4 or response to cysteinyl-leukotrienes in vitro, the LTD4 in humans leads to an increase in the saphenous vein and internal mammary artery microvascular cutaneous blood flow being the most studied, due to their use in associated with a local erythema and an coronary graft bypass surgery. The human increased venular permeability causing saphenous vein exhibits greater contractile wheal-formation (Bisgaard et al., 1982; Soter responses to cysteinyl-leukotrienes than the et al., 1983). The CysLT1 receptor antagonist internal mammary artery (Allen et al., 1994), zafirlukast inhibits this cutaneous reaction to suggesting that cysteinyl-leukotrienes a lesser extent compared with its inhibition of preferentially contract systemic veins bronchoconstriction (Dahlén et al., 1994). compared with arteries. This notion is Cysteinyl-leukotrienes may also be supported by findings in the guinea-pig where involved in the recruitment of leukocytes cysteinyl-leukotrienes induce contractions of from the circulation into a tissue (Laitinen et the inferior vena cava (Berkowitz et al., 1984; al., 1993; Salmon et al., 1999). The Rinkema et al., 1993) whereas the aorta is mechanism behind this recruitment has been only slightly contracted by cysteinyl- proposed to involve cysteinyl-leukotriene-

induced expression of the adhesion molecule function (5-lipoxygenase knockout mice) P-selectin on the surface of endothelial cells have no apparent neurological abnormalities (Pedersen et al., 1997), leading to increased (Funk, 1996). However, in support of a link adhesion of neutrophils. Interestingly, the between cysteinyl-leukotrienes and human surface expression of P-selectin induced by neurological function it has been reported that cysteinyl-leukotrienes in cultured human patients with GSH synthase defiency, who umbilical vein endothelial cells is not blocked have low levels cysteinyl-leukotrienes due to by the CysLT1 receptor antagonists SKF decreased formation of LTC4, also present 104,353, pranlukast or zafirlukast (Pedersen progressive neurological symptoms (Pace- et al., 1997). Asciak et al., 1986; Mayatepek et al., 1993; Mayatepek et al., 1994).

1.2.3. Other targets 1.2.3.1. Central nervous system 1.2.3.2. Gastrointestinal system Cysteinyl-leukotrienes are formed in brain In addition to airway and vascular smooth tissue from rats (Lindgren et al., 1984) and muscle, also other tissues contract in response humans (Simmet et al., 1988) and the human to cysteinyl-leukotrienes, for example isolated guinea-pig ileum and gall bladder (Dahlén et CysLT2 receptor have been demonstrated to be expressed in the brain (Heise et al., 2000; al., 1987; Gardiner et al., 1990; Falcone & Nothacker et al., 2000), suggesting a possible Krell, 1992). Moreover, the human CysLT1 role of cysteinyl-leukotrienes in the central receptor has been reported to be expressed in nervous system. A limited number of studies small intestine, colon, liver and pancreas have investigated the effects of cysteinyl- (Sarau et al., 1999). Taken together, these leukotrienes on isolated human intracerebral findings suggest that cysteinyl-leukotrienes arteries, reporting no effects (von Holst et al., may be inflammatory mediators also in the 1982) or small contractions (Tagari et al., gastrointestinal tract. However, trials using 1983a; Tagari et al., 1983b). In the rat brain, leukotriene synthesis inhibitors in the cysteinyl-leukotrienes have been treatment of inflammatory bowel disease have demonstrated to stimulate release of not shown any significant beneficial effects luteinising hormone (LH) from pituitary cells (Hawkey et al., 1997). (Hulting et al., 1985) and to induce a prolonged excitation of cerebellar purkinje 1.2.3.3. Immune system neurons (Palmer et al., 1981). The human CysLT1 and CysLT2 receptors In two reported cases of LTC4 synthase are expressed in spleen, lymph nodes and on deficiency, the patients presented neurological peripheral blood leukocytes (Lynch et al., symptoms, including muscular hypotonia and 1999; Sarau et al., 1999; Heise et al., 2000; psychomotor retardation, and died at the age Takasaki et al., 2000; Nothacker et al., 2000). of 6 months (Mayatepek & Flock, 1998; A recent report extended these observations Mayatepek et al., 1999), suggesting that by immunohistochemically showing the cysteinyl-leukotrienes may be involved in expression of the CysLT receptor on B- normal neurological development. However, 1 lymphocytes, eosinophils and monocytes, but no studies of DNA were performed in these not on T-cells or neutrophils (Figueroa et al., studies and it cannot be excluded that the 2001). children, in addition to LTC4 synthase deficiency, had a second disorder that led to the neurological problems. In fact, mice that lack cysteinyl-leukotriene formation after disruption of the 5-lipoxygenase-gene

1.2.4. Secondary released factors evaluating the role of cyclooxygenase modulation of cysteinyl-leukotriene responses 1.2.4.1. Cyclooxygenase products within the present thesis (Paper II, III, IV and In some cells, activation of CysLT Fig. 6). receptors leads to the release of various The product of the cyclooxygenase mediators that contribute to the biological enzyme is thus PGH , which is an unstable actions of the cysteinyl-leukotrienes. For 2 endoperoxide that can be converted into the example, some of the effects of cysteinyl- different prostaglandins. The enzyme leukotrienes in both respiratory and thromboxane synthase catalyses the formation cardiovascular systems have been shown to of thromboxane A (TXA ), which activates a be mediated via release of products of the 2 2 TP receptor (Narumiya et al., 1999) leading to cyclooxygenase pathway of arachidonic acid constriction of airway and vascular muscle metabolism (see Sections 4.2.2. and 4.3.3.). It (McKenniff et al., 1988). However, TXA is has been reported that LTD increases the 2 4 rapidly converted to the biologically inactive release of arachidonic acid in isolated cells thromboxane B (TXB ), and concentrations via a receptor driven mechanism involving 2 2 of the latter compound can be measured in RNA and protein synthesis, leading to the order to determine the release of TXA in formation of various metabolites (Crooke et 2 biological systems (see Paper II). The rapid al., 1990). Depending on what metabolites are breakdown of TXA also makes it difficult to formed, the response of the smooth muscle 2 use in experimental studies, and therefore the may be either contractile or relaxant. stable TP receptor agonist U 46 619 (9,11- Prostaglandin endoperoxide synthase or dideoxy-9α,11α-methanoepoxy prostaglandin PGH synthase (in this thesis referred to as F2α) was used in the present study (see cyclooxygenase) is a membrane bound Sections 4.1.5. and 4.3.3.). The synthesis of enzyme that catalyses a cyclooxygenation of TXA2 (measured as TXB2) after cysteinyl- arachidonic acid leading to the formation of leukotriene challenge has been reported in a prostaglandin G2 (PGG2), which in turn is number of tissues and cells, for example peroxidised into prostaglandin H2 (PGH2) by guinea-pig lungs (Omini et al., 1981; Dahlén the same enzyme (DeWitt, 1999; Narumiya et et al., 1983), pig heart (Fiedler et al., 1985) al., 1999). Two iso-forms of cyclooxygenase and rat peritoneal macrophages (Feuerstein et have been identified, called COX-1 and COX- al., 1981). 2 and specific inhibitors of the latter isoform have recently been introduced for clinical use The enzyme prostacyclin synthase converts

(for review, see DeWitt, 1999). While COX-1 PGH2 into prostacyclin (prostaglandin I2, is constitutively expressed in most tissues and PGI2), which via the IP receptor activates though to produce prostaglandins involved in adenylate cyclase leading to increased levels the physiologic functions, COX-2 is in of cAMP and consequently, smooth muscle general not expressed in normal tissues relaxation (Hadhazy et al., 1983; Haye- (DeWitt, 1999). However, the COX-2 enzyme Legrand et al., 1987; Narumiya et al., 1999). is upregulated after inflammatory stimuli, Prostacyclin is rapidly metabolised into 6- making it a suitable target for anti- keto prostaglandin F1α (6-keto PGF1α), and inflammatory treatment without affecting the measured concentrations of this metabolite physiological functions of prostaglandins correspond to the release of prostacyclin (see such as gastric mucosal protection (DeWitt, Papers II and III). Cysteinyl-leukotrienes have 1999). Indomethacin, one of the most often been shown to induce formation of used cyclooxygenase inhibitors in prostacyclin in cultured human umbilical vein experimental studies, non-selectively inhibits endothelial cells (Cramer et al., 1983; Pologe COX-1 and COX-2 (O'Neill et al., 1994) and et al., 1984) as well as in guinea-pig lungs was therefore used in all experiments (Omini et al., 1981).

Other prostaglandins that are formed from to a relaxation of the vascular smooth muscle PGH2 include prostaglandins E2, D2 and F2α (Arnold et al., 1977). (PGE2, PGD2 and PGF2α). These The notion of endothelial nitric oxide as a prostaglandins activate receptors denoted EP regulator of vascular tone and reactivity (EP1, EP2, EP3, EP4), DP and FP, respectively originates in the finding that acetylcholine (for review, see Narumiya et al., 1999). induced relaxations of isolated rabbit aortic preparations with an intact endothelium, but not in endothelium denuded preparations 1.2.4.2. NO (Furchgott & Zawadzki, 1980). This In endothelial cells, cysteinyl-leukotrienes “endothelium derived relaxing factor” may induce activation of the enzyme nitric (Furchgott & Zawadzki, 1980) was later oxide synthase leading to formation of nitric shown to be associated with an increase in oxide (Ignarro, 1990; Allen et al., 1992; cGMP in rat aorta (Rapoport & Murad, 1983) Pawloski & Chapnick, 1993b; Ortiz et al., and bovine pulmonary arteries and veins 1995). The nitric oxide released by the (Ignarro et al., 1987b), which led to the endothelial cells diffuses to the underlying identification of this factor as nitric oxide vascular muscle where it stimulates the (Ignarro et al., 1987a; Palmer et al., 1987; for enzyme soluble guanylate cyclase, which by review, see Ignarro, 1990). increasing intracellular levels of cGMP leads

obtained. In addition, the guinea-pig trachea 2. AIMS OF THE THESIS metabolises cysteinyl-leukotrienes (Snyder et The aim of this thesis was to explore the al., 1984) and was therefore considered to be effects of cysteinyl-leukotrienes in order to a suitable model for the study of metabolic search for novel receptors or functions for modulation of cysteinyl-leukotriene-induced cysteinyl-leukotrienes. Therefore, responses contractions. to cysteinyl-leukotrienes were characterised In addition to airway effects, cysteinyl- using tissue preparations that were set up in leukotrienes also have potent cardiovascular organ baths. When the work with this thesis effects and affect the pulmonary circulation in was initiated, the molecular structure of the many species (see Section 1.2.2.). The CysLT and CysLT receptors were not 1 2 responses to cysteinyl-leukotrienes in human known, and one primary aim was therefore to pulmonary veins have been characterised further explore the functional characteristics (Labat et al., 1992; Ortiz et al., 1995), but also of CysLT receptors. pulmonary arteries have been reported to In addition, pharmacological studies of contract to cysteinyl-leukotrienes (Hanna et whole tissue preparations allow correlating al., 1981; Schellenberg & Foster, 1984; receptor characterisation with a physiological Maddox et al., 1985; Bourdillat et al., 1987). function. Such studies of CysLT receptors However, no previous studies have assessed may hence help to understand the either the receptors or the modulatory physiological or pathophysiological role of mechanism involved in the cysteinyl- cysteinyl-leukotrienes in different tissues. leukotriene-induced contractions of human This offers an essential advantage compared pulmonary arteries. Therefore, this was set up with studies using molecular pharmacological as one primary goal of this thesis. However, methods or studies of ligand binding. since human tissue may be difficult to obtain, In order to further explore the a suitable model for the cysteinyl-leukotriene- physiological significance of CysLT receptor induced contractions of the human pulmonary function, also the modulatory mechanisms vessels would be useful. To address this involved in the responses must be assessed. In matter, the porcine pulmonary vasculature this thesis, modulation of contractile was examined in order to evaluate if these responses induced by cysteinyl-leukotrienes preparations could be used as models for was studied and classified as either human pulmonary vessels. interconversion between the agonists or The guinea-pig ileum was previously the release of modulatory factors, of which main preparation used for bioassay of SRS-A factors of the cyclooxygenase and nitric oxide and cysteinyl-leukotrienes (Dahlén et al., pathways were particularly studied. 1987). However, no previous studies have The following three classes of preparations investigated the receptor involved in were used: airways (guinea-pig trachea), contractile responses induced by LTC4, which pulmonary vessels (human and porcine are resistant to CysLT1 receptor antagonism pulmonary arteries and porcine pulmonary (Gardiner et al., 1990). Since the modulatory veins) and intestinal smooth muscle (guinea- factors involved in regulating responses in the pig ileum longitudinal muscle). guinea-pig ileum previously have been reported to be associated with the epithelium The guinea-pig trachea is one of the most (Krilis et al., 1983), the guinea-pig ileum commonly used preparations in airway longitudinal muscle preparation (which does pharmacology and although the functional not contain epithelium) was not further CysLT receptors have been extensively studied with regard to modulatory studied in this preparation, no definitive mechanisms. conclusion as to the receptor activation by the individual cysteinyl-leukotrienes has been

as cumulative administration of increasing 3. METHODS concentrations according to the method of van 3.1. General Rossum (1963). Maximum contractions were According to the Nomenclature Committee performed with histamine (100 µM; guinea- of the International Union of Pharmacology pig ileum, Paper I), noradrenaline (10 µM; (NC-IUPHAR), a functional assay is defined human and porcine pulmonary vessels, Paper as “a pharmacological test system in which a II-IV) or the combination of histamine (1 response can be firmly attributed to the mM), acetylcholine (1 mM) and KCl (40 mM; function of a defined receptor type or guinea-pig trachea, Paper V) in order to subtype” (Vanhoutte et al., 1998). In the obtain a reference contraction. present thesis, the responses studied are contractions and relaxations of smooth muscle containing preparations placed in organ baths 3.2.2. Enzyme immunoassay in order to characterise the CysLT receptors In order to determine the release of the as well as the mechanisms involved in these vasoactive cyclooxygenase products responses. In addition, the release of prostacyclin and thromboxane A2, modulatory factors was measured using supernatants were collected from the organ enzyme immunoassay and the metabolism of baths after challenge of the vascular cysteinyl-leukotrienes was assessed by preparations with cysteinyl-leukotrienes incubation of tissue with radiolabelled (Paper II and III). Measurements of the stable cysteinyl-leukotrienes that were separated on metabolites 6-keto PGF1α (prostacyclin) and reverse phase high performance liquid TXB2 (TXA2) were performed using enzyme chromatography (RP-HPLC). immunoassay (Stallergenes, France) as previously described in detail by Pradelles and co-workers (1985). Briefly, 50 µl samples 3.2. Experiments of the supernatants were added to 96-well 3.2.1. Organ bath experiments microtiter plates with mouse monoclonal anti- rabbit IgG attached to the wells. The The tissues were prepared as rings quantification is based on the competition of (vascular preparations), spirals (guinea-pig trachea, Constantine, 1965) or longitudinal free 6-keto PGF1α or TXB2 with the muscle (guinea-pig ileum, Rang, 1964) and respective acetylcholinesterase-linked set up in 5- or 10-ml organ baths containing prostanoid for binding to specific polyclonal Tyrode’s solution bubbled with carbogen gas rabbit antisera, forming complexes that then (5-6.5% CO in O ) and kept at a constant bind to the antibody in the well. The addition 2 2 of Ellman’s reagent allows measuring the temperature of 37 °C. Changes in smooth increase in absorbance at 412 nm, which is muscle tension, i.e. contractions and inversely proportional to the amount of 6-keto relaxations, were recorded via isometric force-displacement transducers connected to a PGF1α or TXB2 in the samples. polygraph and responses were either displayed via a chart-recorder or recorded 3.2.3. RP-HPLC with a computerised data acquisition software (IOX; EMKA Technologies, France). In order to establish the metabolism of Calculations of tension changes were made cysteinyl-leukotrienes in the guinea-pig manually from charts or, more recently, trachea and to evaluate the effects of different computer aided with a data analysis software conversion inhibitors (Paper V), guinea-pig (Datanalyst; EMKA Technologies, France). tracheal tissue was incubated in 0.5 ml phosphate buffered saline (PBS-buffer) or Cysteinyl-leukotrienes and other agonists PBS-buffer containing the different were added either as single concentrations or

conversion inhibitors. After a 30-min unpublished data are presented, all 3 3 incubation period, [ H]LTC4, [ H]LTD4 or experiments were carried out as described in 3 [ H]LTE4 was added (3.7 kBq, 1 pmol) and the reports of the related studies. Any after another 30 min the reaction was stopped additional information is indicated in the by the addition of 0.5 ml ethanol. The figure legends. supernatants were injected onto an RP-HPLC The receptor antagonists and enzyme column in order to separate the cysteinyl- inhibitors used and their actions are leukotrienes as previously described in detail summarised in Table 2. by Kumlin and Dahlén (1990). The retention- times for the individual cysteinyl-leukotrienes were determined with authentic standards and 3.2.6. Ethical approval the distribution of radioactivity in the fractions was determined by liquid The experiments in the present thesis were scintillation counting. approved by Stockholms norra djurförsöks- etiska nämnd (N 343/95, N 317/98), Etiska kommittén at Karolinska Hospital (KS 3.2.4. Measurements of γ-GT activity 87:176, KS 00:267) or Ministère des Affaires Measurements of γ-GT activity were Sociales et de l’Intégration and Ministère de performed using the synthetic γ-GT substrate l’Agriculture et de la Forêt du Gouvernement γ-glutamyl-P-nitroanilide (GPNA) according Français (arrêté du 25 février 1992). to the method described in detail by Silber and co-workers (1986). The experiments were 3.3. Data analysis γ carried out using purified porcine kidney - 3.3.1. Contractions and relaxations GT (100 U/l; from Sigma, MO, USA) dissolved in 0.5 ml PBS-buffer. After 30 min Contractions are expressed as percent of incubation in the absence (control) or the specified reference contraction in each presence of different concentrations of the study (see above) and relaxations in percent conversion inhibitors, 30 µl of the supernatant of the precontraction before the relaxant was transferred to a 96-well microtiter plate. agonist was added. The results of the The reaction solution had the following cumulative dosings are expressed as composition: GPNA (6 mM), glycylglycine concentration-response curves. (80 mM) and Tris (120 mM; all from Sigma, MO, USA) and 280 µl was added to each 3.3.2. Pharmacological analysis well. The catalysis of GPNA by γ-GT involves the transfer of the γ-glutamyl group In the cases where the concentration- to an acceptor (glycylglycine), yielding P- response curves reached a plateau, the nitroanilide (PNA). The formation of PNA maximal contraction (Emax), i.e. the produces a yellow colour and the increase in contraction induced by the highest absorbance, measured at 405 nm after 90 min, concentration of each agonist, was is correlated to the amount of PNA formed. determined. The half-maximum effective Results (Fig. 8) are expressed at percent of concentrations (EC50-values) were calculated inhibition of enzymatic formation of PNA by linear regression and the pD2-value was compared with control. calculated as the negative log of the EC50- value. In the guinea-pig ileum, the half-

maximum effective concentrations were 3.2.5. Experimental protocols estimated as the concentration of LTC4 Descriptions of the experimental protocols, inducing a contraction that was 25% of the the drugs used and other details are indicated maximal histamine-induced contraction in the individual papers. When previously (EC25-value), since LTC4 induced an Emax of

about 50% of histamine maximum in this The pA2-value was determined by Schild-plot preparation (Paper I). analysis as described in detail by Arunlakshana and Schild (1959). Briefly, the In order to characterise the results with the logarithm of (CR-1) was plotted against the CysLT receptor antagonists, pK - and pA - B 2 negative logarithm of the antagonist values were calculated as described below. concentrations and a regression line was fitted These values are estimates of the dissociation to all data points. The slope of this regression constant of the antagonist-receptor complex line must not be significantly different from 1 and represent an indirect measurement of the in order to confirm competitive antagonism antagonist’s affinity for its receptor (Tallarida and to allow estimation of the pA -value et al., 1979). The pK - and pA -values will 2 B 2 (Arunlakshana & Schild, 1959). An coincide if antagonism is competitive alternative method to determine if the (MacKay, 1978; Jenkinson et al., 1995), antagonism is competitive, used in Paper V, is which is assumed when the two values are to compare the pK -values obtained for each compared in this thesis. If two agonists act on B antagonist concentration. If the pK the same receptor, they can be expected to be B-values antagonised to the same degree by the same are not significantly different depending on antagonist concentrations, the slope of the antagonists, i.e. similar pKB- or pA2-values should be obtained. In contrast, if the values Schild-plot regression is not significantly are different, the agonists probably act on different from 1 and antagonism consequently different receptors. Moreover, different competitive (MacKay, 1978). In this case the preparations with similar receptors would be pA2 value can also be determined as the mean of all pKB-values (MacKay, 1978; Paper V). expected to give the same pKB- and pA2- values for a particular antagonist (Tallarida et al., 1979). 3.3.3. Statistics A concentration ratio (CR) was determined Statistical evaluation was performed using as the ratio of EC - or EC -values in the 50 25 either a Student’s t-test or a one- or two-way presence and in the absence of antagonist. analysis of variances (ANOVA) test followed The pK -value was calculated as the negative B by Dunnett’s test (multiple comparisons). A logarithm of the following equation: [B]/(CR- P-value of less than 0.05 was considered 1), where [B] is the concentration of the significant. antagonist and CR the concentration ratio.

RECEPTOR ANTAGONISTS

Name Chemical name Function References

BAY u9773 6(R)-(4’-carboxyphenylthio)-5(S)-hydroxy- CysLT1/CysLT2 Cuthbert et al. 1991b 7(E),9(E),11(Z)14(Z)-eicosatetrenoic acid receptor antagonist

ICI 198,615 (1-((2-methoxy-4-(((phenylsulfonyl)amino) CysLT1 receptor Snyder et al., 1987 carbonyl)-phenyl)methyl)-1H-indazol-6-yl) antagonist carbamic acid cyclopentyl ester

ICI 204,219 4-(5-cyclopentyloxycarbonylamino-1-methyl CysLT1 receptor Krell et al., 1990 (zafirlukast) indol-3-ylmethyl)-3-methoxy-N-o-tolylsulfonyl antagonist benzamide

MK 571 (3-(-2(7-chloro-2-quinolinyl)ethenyl)phenyl)((3- CysLT1 receptor Jones et al. 1988 (dimethylamino-3-oxopropyl)thio)methyl)thio antagonist propanoic acid

SKF 104,353 2(S)-hydroxy-3(R)-((2-carboxyethyl)thio)-3-(8- CysLT1 receptor Hay et al., 1987 (pobilukast) phenyloctyl)phenyl)-propanoic acid antagonist

ENZYME INHIBITORS

Name Abbreviation Function References acivicin - irriversible γ-GT inhibitor Stole et al., 1994

L-cysteine L-cys inhibitor of LTD4 metabolism Sok et al., 1981

indomethacin INDO cyclooxygenase (COX) inhibitor O’Neill et al., 1993 (COX-1/COX-2 unselective) glutathione GSH inhibitor of LTC4 metabolism Hammarström, 1981 (substrate competition at γ-GT)

S-hexyl glutathione S-hexyl GSH inhibitor of LTC4 metabolism Paper V (substrate competition at γ-GT)

ω N –nitro-L-arginine L-NOARG nitric oxide synthase inhibitor Bina et al., 1998

L-serine borate SeBo reversible γ-GT inhibitor Tate & Meister, 1978

Table 2: Receptor antagonists and enzyme inhibitors used in pharmacological and biochemical studies in the present thesis

supported by the results in Paper V. The 4. RESULTS & DISCUSSION contractions to LTD4 were inhibited by the 4.1. Cysteinyl-leukotriene receptors three CysLT1 receptor antagonists ICI ± 4.1.1. Specific aims 198,615 (pA2=9.3 0.2), MK 571 (pK =9.2±0.3) and SKF 104,353 At the start of the work with this thesis, the B (pK =8.0±0.4) and LTE was inhibited by CysLT receptor was characterised as a B 4 2 ICI 198,615 (pK =9.4±0.2). Krell and co- functional CysLT receptor resistant to CysLT B 1 workers (1983) were the first to suggest the receptor antagonists (Coleman et al., 1995). presence of different subtypes of LTD - However, BAY u9773 had recently been 4 receptors in the guinea-pig trachea, based on shown to inhibit CysLT receptor antagonist 1 the finding of a bimodal distribution of pK - resistant cysteinyl-leukotriene responses B values for the inhibition of LTD -induced (Cuthbert et al., 1991b; Labat et al., 1992; 4 contractions by FPL 55712. Hand and co- Tudhope et al., 1994). The primary aim was workers (1989) extended this observation by therefore to study functional responses that demonstrating non-linear Schild-plots for a were resistant to CysLT receptor antagonists 1 number of different CysLT receptor in order to examine if the CysLT receptor 1 2 antagonists against LTD -induced represented a heterogeneous class of 4 contractions of the guinea-pig trachea. receptors. In this context, BAY u9773 was However, the use of inhibitors of cysteinyl- tested against LTC -induced contractions in 4 leukotriene metabolism in some (Hand et al., the guinea-pig ileum and trachea (Paper I and 1989), but not in other (Krell et al., 1983; V, respectively). In addition, since in the Snyder et al., 1987; Jones et al., 1989) studies human lung, CysLT receptors had been 2 of CysLT receptor antagonists in this context associated with vascular tissues (Labat et al., 1 makes the results difficult to compare and 1992; Ortiz et al., 1995), this thesis also interpret. As will be discussed in Section included investigations of two pulmonary 4.2.3. (Paper V), inhibiting the metabolism of vascular preparations that had not previously LTD is necessary when studying CysLT been studied with respect to cysteinyl- 4 receptor pharmacology in the guinea-pig leukotriene receptors, namely porcine and trachea. The suggestion of more than one human pulmonary arteries (Paper II and III, receptor for LTD4 is supported by the finding respectively). 3 that [ H]LTE4 binds to a subset of LTD4- In the guinea-pig trachea, it had been receptors in guinea-pig lung membranes proposed that LTD4 interacted with more than (Aharony et al., 1988). one receptor subtype (Krell et al., 1983; Hand The antagonism of LTD -induced et al., 1989), but no definite conclusion as to 4 contractions of guinea-pig trachea by these proposals had been obtained. Therefore, ICI 198,615 was dose-dependent for this part of the thesis also evaluates LTD4- induced contractions of the guinea-pig trachea antagonist-concentrations up to 10 nM (Paper in order to examine the possibility of V). However, at ICI 198,615-concentrations of 30 nM and higher, no further antagonism heterogeneity of the CysLT1 receptor. was observed, and a residual contraction to LTD4 was unmasked. This finding was in

4.1.2. CysLT1 receptors contrast to the LTE4-induced contractions, which could be abolished by ICI 198,615 4.1.2.1. LTD4-induced contractions of the (300 nM), supporting that there may be a guinea-pig trachea difference between how LTD4 and LTE4 The notion that the LTD4- and LTE4- interact with the CysLT1 receptor. induced contractions of the guinea-pig trachea Interestingly, the residual contraction to LTD4 are mediated via a CysLT1 receptor is was inhibited by BAY u9773, suggesting that

LTD4 activates both CysLT1 and CysLT2 (Lynch et al., 1999; Sarau et al., 1999; receptors, whereas LTE4-induced contractions Takasaki et al., 2000; Nothacker et al., 2000), were mediated solely by the CysLT1 receptor. with LTE4 being a partial agonist (Sarau et These results (Paper V) may offer an al., 1999; Takasaki et al., 2000). An explanation of the above-mentioned studies explanation for the differences in potency (Krell et al., 1983; Aharony et al., 1988; Hand between LTC4 and LTD4 in CysLT1 receptor et al., 1989) by suggesting that, in the guinea- transfected cells but not in human bronchi has pig trachea, the CysLT1 receptor represents a not been proposed. It is possible that in high affinity LTD4-receptor and the CysLT2 preparations with a high CysLT1 receptor receptor a low affinity LTD4-receptor. reserve, LTC4 may be equipotent with LTD4 despite its lower affinity for the receptor.

Anyhow, in contrast to results with the human 4.1.2.2 Comparison between guinea-pig and CysLT1 receptor on isolated bronchial human CysLT1 receptor preparations and on transfected cells, LTC4- induced contractions of the guinea-pig trachea The receptor activated by LTD4 and LTE4 in the guinea-pig trachea share the profile of were resistant to CysLT1 receptor antagonists the human bronchial CysLT receptor of being (Paper V). The rank orders of agonist potency inhibitable by CysLT1 receptor antagonists, at the human and guinea-pig CysLT1 but differs in that the CysLT1 receptor in receptors are summarised in Table 3. human bronchi is activated by all three Another difference between the human and cysteinyl-leukotrienes (Buckner et al., 1986; guinea-pig CysLT1 receptor is the sensitivity Buckner et al., 1990). The rank order of of the cysteinyl-leukotriene-induced agonist potency at the CysLT1 receptor in contractions to CysLT1 receptor antagonists. isolated human bronchial preparations varies Although the pA2 and pKB values that were somewhat between the limited numbers of obtained for ICI 198,615 and MK 571 against studies that have addressed this issue, being LTD4-induced contractions (Paper V) were either LTC4=LTD4>LTE4 (Muccitelli et al., similar to previous studies in the guinea-pig 1987; Labat et al., 1992; Björck, 1993) or trachea (Snyder et al., 1987; Jones et al., LTC4=LTD4=LTE4 (Buckner et al., 1986; 1989), they were about one log order lower Buckner et al., 1990). In addition, in studies than the values previously reported for the of cells transfected with the human CysLT1 same antagonists in human airways (Table 4). receptor, the rank order of potency is LTD4>LTC4>LTE4 for calcium mobilisation

Rank order of agonist potency

GPT CysLT1 Human bronchus hCysLT1 receptor

LTD4,LTE4>>LTC4* LTC4=LTD4>LTE4 LTD4>LTC4>LTE4

GPT CysLT2 Human pulmonary vein hCysLT2 receptor

LTC4>LTD4>>LTE4=BAY u9773 LTC4=LTD4>LTE4>BAY u9773 LTC4= LTD4>LTE4=BAY u9773

Table 3: The rank order of agonist potency at the CysLT receptors was estimated in the guinea pig trachea (GPT; Paper V). *The exact rank order of potency at the guinea pig tracheal CysLT1 receptor could not be determined since there are currently no selective CysLT2 receptor antagonists available (see text). Results are compared with previously published reports studying contractions of isolated human bronchus and pulmonary vein (Labat et al., 1992) or calcium mobilisation in cells transfected with the human CysLT receptors (hCysLT1 or hCysLT2 receptor; Nothacker et al., 2000).

FPL 55712 ICI 198,615 MK 571

LTC4 LTD4 LTE4 LTC4 LTD4 LTE4 LTC4 LTD4 LTE4

GP trachea 4.7 – 4.9 6.5 – 6.8 6.2 – 7.2 N.S. – 5 8.7 – 9.5 9.4 – 10.1 N.S. – 5.6 8.2 – 9.4 9.1 (5, 8) (3, 4 ,5, 8) (4, 5, 8) (2, 9, 10) (2, 9, 10, 23) (2, 9, 10) (2, 11, 23) (2, 11, 23) (11)

GP ileum 6.9 – 7.3 6.7 – 7.4 N.S. 9.2 – 11.5 10.5 10.4 (segment) (3, 12, 14) (12) (12) (12, 14) (11) (11)

GP ileum 6.8 N.S. 10.3 (muscle) (13) (1) (19)

GP gall 6.1 8.2 – 8.6 8.3 – 8.7 8.3 – 8.7 bladder (21) (17) (17) (17)

28 Rat Lung 7.6 – 7.9 7.5 7.4 7.8 7.5 - 7.6 7.3 (22, 23) (22, 23) (22) (22, 23) (22, 23) (22)

Human 5.8 - 6.4 5.8 - 6.3 8.5 – 9.8 8.2 – 9.2 8.3 - 8.6 8.5 - 8.8 bronchus (6) (6) (9, 18) (9, 18, 24) (18) (18)

Human pulm N.S. N.S. N.S. N.S. vein (18) (18) (18) (18)

Ferret N.S. N.S. N.S. – 5.6 N.S. N.S. - 4.7 N.S. spleen (20) (20) (20, 23) (20, 23) (20, 23) (20, 23)

Sheep N.S. N.S. N.S. N.S. bronchus (15, 23) (15, 23) (15, 23) (15, 23)

Sheep N.S. N.S. N.S. N.S. trachea (15, 25) (15, 25) (15) (15)

SKF 104,353 BAY u9773 Table 4: Antagonism of the contractile

responses of different preparations LTC4 LTD4 LTE4 LTC4 LTD4 LTE4 in functional assays expressed as

pA2- or pKB-values. N.S.= no GP trachea N.S. - 6.3 7.8 – 8.6 >8.9 6.8 – 7.3 7.4 7.7 significant inhibition. (2, 7, 23) (2, 7, 23) (7) (2, 16, 23) (23) (16) Values are from the following reports (numbers correspond to GP ileum N.S. 7.6 – 8.4 those indicated in parenthesis under (segment) (12) (12, 14) each value): 1: Paper I GP ileum 6.1 2: Paper V (muscle) (1) 3: Fleisch et al., 1982 4: Krell et al., 1983

5: Weichman & Tucker, 1985 GP gall 6.8 – 7.4 7.6 – 7.9 7.3 – 7.7 6: Buckner et al., 1986 bladder (17) (17) (17)

29 7: Hay et al., 1987 8: Muccitelli et al., 1987 Rat Lung 8.0 – 8.3 7.5 – 8.2 8.0 7.2 6.8 9: Snyder et al., 1987 (21, 22, 23) (22, 23) (22) (23) (23) 10: Hand et al., 1989 11: Jones et al., 1989 Human 8.4 7.0 – 8.2 >8.2 5.4 6.2 – 6.8 12: Gardiner et al., 1990 bronchus (7) (7, 18, 26) (7) (18) (18) 13: Gieske et al., 1990 14: Norman et al., 1990 HPV N.S. N.S. 5.8 – 6.7 6.5 – 6.8 15: Cuthbert et al., 1991a (18) (18) (18) (18) 16: Cuthbert et al., 1991b 17: Falcone & Krell, 1992

18: Labat et al., 1992

Ferret N.S. N.S. 6.9 6.8 19: Björck, 1993 spleen (20) (20) (23) (23) 20: Gardiner et al., 1993 21: Gardiner et al., 1994 Sheep N.S. N.S. 7.6 7.7 22: Norman et al., 1994 bronchus (15, 23) (15, 23) (23) (23) 23: Tudhope et al., 1994 24: Gorenne et al., 1995 Sheep N.S. N.S. 7.0 6.8 25: Wikström Jonsson, 1997 trachea (15) (15) (25) (25) 26: Panettieri et al., 1998

supporting the notion of different CysLT A simple explanation to the differences in 1 receptors. agonist and antagonist properties between human and guinea-pig CysLT1 receptors The findings compiled in Table 4 indicate would be to claim species-differences. that there are differences in rank order of However, in another guinea-pig preparation, antagonist potency between the different namely the guinea-pig gall bladder, cysteinyl- preparations, as well as a difference in rank leukotrienes induce contractions with a rank order of sensitivity to the different antagonists order of potency of LTC4=LTD4>LTE4 in each investigated tissue. For example, (Falcone & Krell, 1992), i.e. similar to what FPL 55712, ICI 198,615 and MK 571 inhibit most investigators have reported for human CysLT1 receptor mediated contractions with a bronchi (Muccitelli et al., 1987; Labat et al., rank order of sensitivity of guinea-pig ileum > 1992; Björck, 1993). In addition, in the guinea-pig trachea > guinea-pig gall bladder = guinea-pig gall bladder, the contractions to all human bronchus > rat lung, whereas for cysteinyl-leukotrienes are inhibited by SKF 104,353, the values reported are not so ICI 198,615 and SKF 104,353 (Falcone & different between these tissues (Table 4). In Krell, 1992), indicating that they all activate fact, the differences between the guinea-pig only CysLT1 receptors in that preparation. ileum and trachea in sensitivity to FPL 55712 Interestingly, the pKB-values obtained for (Table 4), have led to the suggestion that the ICI 198,619 in the guinea-pig gall bladder are CysLT1 receptor in the guinea-pig ileum may more close to those reported for human be different from that of the trachea (Fleisch bronchi than those obtained in the guinea-pig et al., 1982). Likewise, the rank orders of trachea (Paper V; Table 4). potency of the CysLT1 receptor antagonists in inhibiting LTD -induced contractions 4 (Table 4) are: 4.1.2.3. Subtypes of CysLT receptors 1 The current IUPHAR classification of Guinea-pig trachea and ileum: CysLT receptors thus support that the guinea- pig trachea, gallbladder and human bronchi ICI 198,615 = MK 571 > SKF 104,353 all contain CysLT1 receptors (Coleman et al., Human bronchi: 1995). A possible explanation of the differences in rank order of agonist potency ICI 198,615 = MK 571 = SKF 104,353 and the differences in sensitivity to CysLT1 Rat lung: receptor antagonists between the preparations may be that the CysLT1 receptor is a SKF 104,353 > ICI 198,615 = MK 571 heterogeneous class of CysLT receptor subtypes and that further subdivision may be necessary. Although a number of structurally These variations in rank order of different CysLT1 receptor antagonists equally antagonist potency may suggest that the inhibit cysteinyl-leukotriene binding to cells CysLT1 receptors are different between the expressing the human CysLT1 receptor preparations. Differential effects of CysLT1 (Lynch et al., 1999; Sarau et al., 1999) as well receptor antagonists have also been reported as cysteinyl-leukotriene-induced contractions within the same preparation, hence arguing of human bronchi (Buckner et al., 1986; against that variations in CysLT1 receptor Buckner et al., 1990; Labat et al., 1992; antagonist sensitivity are solely due to tissue Gorenne et al., 1995; Panettieri et al., 1998; differences. For example, the endothelium-

Table 4), differential effects between CysLT1 dependent relaxations induced by LTD4 in receptor antagonists have also been reported, canine renal arteries and veins are inhibited by ICI 198,615 but not by MK 571 (Pawloski

& Chapnick, 1993a). In the same study 4.1.2.4. Heterogeneous CysLT1/CysLT2 (Pawloski & Chapnick, 1993a), LY 171,883 receptor populations inhibited LTD4-induced relaxations of canine While a general species differences does renal veins but not arteries, whereas not seem to be a probable explanation for the L 649,923 inhibited arterial but not venous varying properties of CysLT1 receptors in relaxations. In addition, a previous study has different preparations, the findings discussed shown that the relaxations in canine renal in the previous paragraph thus suggest that the veins are sensitive to pranlukast (Pawloski & CysLT1 receptor may be a heterogenous class Chapnick, 1991). Similar observations have of receptor subtypes. Another possible been reported also in human tissues. In a hypothesis is that the expression of both study of human airway smooth muscle, the CysLT1 and CysLT2 receptors may change equal potency of zafirlukast, pranlukast and how agonists and antagonists interact with the

SKF 104,353 in inhibiting LTD4-induced receptors. In some of the preparations studied contractions was confirmed (Panettieri et al., in Table 4, both CysLT1 and CysLT2 1998). However, in the same study the receptors appear to be expressed, for example potentiating effect of LTD4 on EGF-induced in the guinea-pig ileum and trachea (Paper I DNA synthesis in human airway smooth and V). In contrast, human bronchi (Buckner muscle cells was abolished by either et al., 1986; Buckner et al., 1990) and guinea- pranlukast or a higher concentration of pig gall bladder (Falcone & Krell, 1992)

SKF 104,353, but unaffected by zafirlukast contain a homogenous CysLT1 receptor (Panettieri et al., 1998). population.

The notion that different CysLT1 receptor In fact, recent findings in CysLT receptor antagonists may be used in order to transfected cells, support that the human distinguish different subtypes of human CysLT1 receptor is the preferred target for CysLT1 receptors has also received support LTD4 and the human CysLT2 receptor is the from radioligand binding studies of human preferred target for LTC4 (Nothacker et al., 3 lung parenchyma membranes using [ H]- 2000). The latter observation may hence offer labelled leukotrienes as agonists (Capra et al., an explanation as to why LTC4-induced 1998b; Ravasi et al., 2000). In these contractions of the guinea-pig trachea were 3 3 preparations, [ H]LTC4 and [ H]LTD4 have resistant to CysLT1 receptor antagonists been proposed to interact with both high and (Paper V) although LTC4 would be expected low affinity binding-sites (Rovati et al., 1992; to be an agonist at this receptor. It can Capra et al., 1998a; Capra et al., 1998b) with therefore be anticipated that LTC4 may differential abilities of CysLT1 receptor activate the CysLT1 receptor only in the antagonists in displacing binding of either absence of CysLT2 receptors. Unfortunately, agonist. For example, (in the absence of S- there are presently no pharmacological tools decyl GSH, see Paper V), SKF 104,353 available to selectively block the CysLT2 3 3 displaces both [ H]LTC4 and [ H]LTD4 receptors in the guinea-pig trachea and this binding whereas zafirlukast and pranlukast hypothesis could therefore not be tested in the 3 displace only [ H]LTD4 binding (Ravasi et present study. al., 2000). Although it can not be excluded that binding sites for [3H]-labelled It has been reported that in human leukotrienes, in addition to CysLT receptors, saphenous veins the contractions induced by also may represent enzymes or transport LTD4 are inhibited by ICI 198,615, whereas proteins that binds cysteinyl-leukotrienes the LTC4-induced contractions (in the (Sun et al., 1986; Bannenberg et al., 1999), presence of acivicin in order to inhibit LTC4 the results of the binding studies may suggest metabolism, see Section 4.2.3.) are resistant to this antagonist (Allen et al., 1994). The the presence of different CysLT1 receptors in the human lung. latter observation is thus similar to results in

the guinea-pig ileum (Paper I) and trachea results between studies using this antagonist. (Paper V) and may support the hypothesis Since the results in both guinea-pig ileum proposed above, i.e. that co-expression of (Paper I) and trachea (Paper V) are within the CysLT1 and CysLT2 receptors changes the range of pA2/pKB-values described for BAY agonist preferences of the receptors. This u9773 by other investigators (Table 4), it assumption is reinforced by results of studies cannot be excluded that the CysLT2 receptor of cysteinyl-leukotriene-binding to human has similar properties in the two preparations. lung membranes that also have proposed human CysLT receptors that are preferentially activated by either LTC4 or LTD4 (Rovati et 4.1.3.2. The mechanism of antagonism by al., 1985; Rovati et al., 1992; Capra et al., BAY u9773 1998b; Ravasi et al., 2000). The findings in guinea-pig ileum (Paper I) and trachea (Paper V) suggest that BAY u9773 is a competitive antagonist at the 4.1.3. CysLT2 receptors CysLT2 receptor. However, since BAY u9773 4.1.3.1. LTC4-induced contractions of the is structurally related to LTE4 and a dual guinea-pig ileum and trachea CysLT1/CysLT2 receptor antagonist, the In both guinea-pig ileum and trachea, the question raised was if its antagonism at the CysLT2 receptor was dependent on interaction LTC4-induced contractions were resistant to with the CysLT1 receptor. In the guinea-pig CysLT1 receptor antagonism but inhibited by ileum, the combination of BAY u9773 with the dual CysLT1/CysLT2 receptor antagonist BAY u9773 (Paper I and V), supporting that the CysLT1 receptor antagonist ICI 198,615 did not alter the inhibition of the LTC4- LTC4 activates a CysLT2 receptor in these tissues. However, although the two different induced contraction by BAY u9773 (Paper I), tissues were studied under similar conditions, suggesting that the antagonistic effect of BAY u9773 was unrelated to activation of CysLT1 the pA2-values were somewhat different, 6.1 in the guinea-pig ileum and 6.8 in the guinea- receptors. pig trachea (Paper I and V). This is hence a BAY u9773 did not induce any significant reversed relationship compared with studies contractions of either the guinea-pig ileum using CysLT1 receptor antagonists, that have (Paper I) or trachea (Table 5, previously reported a higher potency of the antagonists in unpublished data). These findings are similar ileum compared with trachea (Table 4), and to a number of other functional smooth would suggest that also the CysLT2 receptor muscle assays (Tudhope et al., 1994; may be different between ileal and tracheal Wikström Jonsson, 1997) where BAY u9773 preparations. However, it cannot be excluded competitively inhibits CysLT1 and CysLT2 that the variation in pA2-values were due to receptor responses without having an methodological differences. For example, the agonistic activity. However, Labat and co- concentration-response curves for LTC4 were workers (1992) described the antagonism of different in the ileum compared with the CysLT2 responses by BAY u9773 as partial trachea, with pD2 values of 8.1 (Paper I) and agonism. Likewise, in cells expressing the 8.7 (Paper V), respectively and Emax of around human CysLT2 receptor, BAY u9773 has 90 % and 50 %, respectively. In addition, in been shown to be a partial agonist (see below) the guinea-pig trachea, half-log steps were and its antagonistic properties at this receptor used when establishing the concentration- proposed to be due to receptor desensitisation response curves as opposed to whole-log (Nothacker et al., 2000). In the latter study, steps in the ileum. Anyhow, data from BAY u9773 inhibited calcium mobilisation previous reports using BAY u9773 (Table 4) also in CysLT1 receptor transfected cells, but indicate that there are certain variations in without having any agonistic activity

(Nothacker et al., 2000). Taken together, fold higher IC50 than at the CysLT1 receptor these findings suggest a differential action of (Lynch et al., 1999; Sarau et al., 1999; Heise antagonism by BAY u9773, i.e. competitive et al., 2000). antagonism without agonist activity at Gieske and co-workers (1990) have CysLT1 and CysLT2 receptors in for example reported that the LTD -mimetic MDL 28,753 guinea-pig ileum (Paper I) and trachea (Paper 4 inhibits the LTC -induced contractions of the V) and at cloned human CysLT 4 1 receptors guinea-pig ileum longitudinal muscle. Since it (Nothacker et al., 2000), as well as partial was shown in Paper I that the LTC -induced agonism, as in human pulmonary venous 4 contractions of this preparation were mediated smooth muscle (Labat et al., 1992) and cloned by a CysLT receptor, it can thus be human CysLT 2 2 receptors (Nothacker et al., anticipated that MDL 28,753 would be a 2000). CysLT2 receptor antagonist. However, MDL 28,753 is a full LTD4-mimetic in the guinea- pig ileum, and the inhibition of LTC 4-induced BAY u9773 contractions was observed only in the 0.3 µM 1 µM 3 µM 10 µM presence of ICI 198,615 (Gieske et al., 1990). Interestingly, in the presence of ICI 198,615 (100 nM), a residual contraction to both MDL 4.2±2 % 4.8±1 % 3.1±1 % 5.0±1 % 28,753 and LTD4 was reported (Gieske et al., (n=8) (n=5) (n=7) (n=4) 1990), which, for the latter agonist, is

identical to results in the guinea-pig trachea Table 5: Changes in basal tone of guinea-pig tracheal (Paper V). In the guinea-pig trachea, the preparations during incubation with the residual contraction was inhibited by BAY CysLT1/CysLT2 receptor antagonist BAY u9773. Contractions (means±S.E.M) are expressed as percent u9773, indicating that LTD4 was a partial of a maximal contraction to histamine (1 mM), agonist at the CysLT2 receptor in this acetylcholine (1 mM) and KCl (40 mM; previously preparation (Paper V, see below). If it is unpublished data). hypothesised that, in the guinea-pig ileum longitudinal muscle, LTD4 and MDL 28,753 activate CysLT2 receptors as well, one 4.1.3.3. Other CysLT2 receptor antagonists possible explanation for the observations by The unselective CysLT1/CysLT2 receptor Gieske and co-workers (1990) may be that antagonists BAY u9773 is presently the only both LTD4 and MDL 28,753 in the presence pharmacological tool available for of ICI 198,615 inhibit LTC4-induced characterisation of CysLT2 receptors. contraction by partial agonism at the CysLT2 However, the use of BAY u9773 is limited receptor. since it is only commercially available at a very high price. 4.1.3.4. LTE4 as a CysLT receptor antagonist Some of the CysLT1 receptor antagonists have been shown to inhibit also CysLT2 In a number of different tissues, LTE4 have responses, but only at high concentrations been described to inhibit contractions induced where their specificity may be questionable. by LTC4 or LTD4 (Table 6). The mechanism For example, pranlukast antagonises the of this antagonism has in some tissues been LTC4-induced contractions of the guinea-pig characterised as partial agonism (Gardiner et trachea (in the presence of L-serine borate) al., 1990; Labat et al., 1992), whereas in other with a 100 fold lower potency than against tissues, LTE4 inhibits LTC4- and LTD4- LTD4-induced contractions (Obata et al., induced contractions although it induces no, 3 1992) and competes for [ H]LTD4 binding at or only very small, contractions of the the cloned CysLT2 receptor but with 1000-

preparations (Snyder & Krell, 1986; Tomioka effects of LTE4 against LTC4-induced et al., 1991; Gardiner et al., 1993). contractions in the latter study may have been due to an effect against LTD 4 being produced LTC4 LTD4 from the metabolism of LTC4. Pre-treatment

of guinea-pig ileum longitudinal muscle with

GP ileum [minor shift] pKP = 6.8 the TP-receptor agonist U 46 619 mimicked (Gardiner et al., 1990) (10 µM) (10 µM) the effect of LTE4, i.e. induced a similar degree of contraction but did not produce a Ferret trachea pKB = 6.3-6.5 pKB = 6.4-7.3 shift of the concentration-response curve for µ µ (Snyder & Krell, 1986) (10 & 1 M) (10 & 1 M) LTC4 (Paper I), supporting that LTE4 behaved

like any non-CysLT2 receptor agonist. Ferret spleen pKB = 5.8 pKB = 5.5 (Gardiner et al., 1993) (10 µM) (10 µM)

4.1.3.5. Rank order of agonist potency at the Sheep trachea [pKB = 6.9] pKB = 7.3 CysLT2 receptor (Tomioka et al., 1991) (1 µM) (0.1-1 µM) At the CysLT2 receptor on human Human pulm. vein pK = 6.6 pK = 6.3 P P pulmonary venous smooth muscle (Labat et al., 1992) (1 µM) (1 µM) preparations, the rank order of agonist

potency is LTC4=LTD4>LTE4 (Labat et al., Table 6: Previous reports demonstrating the inhibition of LTC - and LTD -induced contractions by LTE . 1992). In ferret trachea and spleen, two other 4 4 4 preparations with a homogenous CysLT Results are expressed as either pKB- or pKP-values 2 according to the original reports, where a pKP-value receptor population, a rank order of potency indicates inhibition by partial agonism. [ ] indicates of LTC4=LTD4>>LTE4 have been reported that experiments were carried out in the absence of (Snyder & Krell, 1986; Gardiner et al., 1993). inhibitors of LTC4 metabolism. In the guinea-pig trachea, the rank order of potency was LTC4=LTD4>LTE4 (Paper V), which is identical to previous reports in this In the guinea-pig ileum, LTE (10 µM) 4 tissue (Cuthbert et al., 1991b; Tudhope et al., induces a rightward shift of the LTD 4 1994) and in the guinea-pig ileum (Dahlén et concentration-response curve due to partial al., 1987; Gardiner et al., 1990). The latter agonism at the CysLT receptor (Gardiner et 1 preparations both contain a heterogeneous al., 1990; Table 6). In contrast, in this thesis, receptor population and the meaning of the LTC -induced contractions were not 4 estimates of rank order of potency can thus be inhibited by LTE (30 nM-1 µM; Paper I). 4 disputable. However, if it is hypothesised that Since the LTC -induced contractions of the 4 ICI 198,615 (300 nM) selectively inhibited guinea-pig ileum were shown to be mediated the whole population of CysLT receptors in via a CysLT receptor, inhibition by partial 1 2 the guinea-pig trachea, the cysteinyl- agonism is not expected for LTE (Paper I). 4 leukotriene-induced contractions in the However, in the study by Gardiner and co- presence of this antagonist concentration workers (1990), also a shift of the would be mediated solely via the CysLT concentration-response curve to LTC was 2 4 receptor (Paper V). In this case, the rank order observed, although this was minor compared of potency at the CysLT2-receptor in the with that observed for LTD4. A possible guinea-pig trachea was LTC4 (pD2=9.0±0.1) explanation as to the apparent differences in ± effects of LTE on LTC -induced contractions > LTD4 (pD2=7.7 0.2) >> LTE4 (no 4 4 µ between these two studies may be the use of contractions at concentrations up to 1 M) as is shown in Fig. 3 and Table 3. In addition, inhibitors of LTC4 metabolism in Paper I, but not in the study by Gardiner and co-workers LTD4 was a partial agonist at the CysLT2 ± (1990). It can thus not be excluded that the receptor (maximal contraction 48 4% compared with 86±6% for LTC4, Fig. 3).

antagonist ICI 198,615 (Wikström Jonsson et al., 1998), suggesting that the agonist activity of BAY u9773 may vary between guinea-pig and human tissues. In addition, since BAY u9773 induced negligible contractions of both the guinea-pig ileum and trachea, differences may also exist between different tissues within the same species. In the context of BAY u9773 being a selective agonist at the human CysLT2 receptor (Nothacker et al., 2000), it is noteworthy that Labat and co- workers (1992) have reported contractions to BAY u9773 in the human pulmonary vein. Moreover, those investigators also observed contractions to BAY u9773 in isolated human Fig. 3: Concentration response curves for LTC4 (in the bronchi (Labat et al., 1992). Since BAY µ presence of 100 M S-hexyl GSH and 5 mM L- u9773 have been reported not to be an agonist cysteine), LTD4 (in the presence of 5 mM L-cysteine) at the cloned human CysLT1 receptor and LTE4. All preparations were treated with the CysLT1 receptor antagonist ICI 198,615 (300 nM). (Nothacker et al., 2000), this may indicate the Contractions (means±S.E.M) are expressed as percent presence of also CysLT2 receptors in human of a maximal contraction to histamine (1 mM), bronchi. However, it has also been reported acetylcholine (1 mM) and KCl (40 mM), n=4-9 (data that BAY u9773 may activate TP-receptors from Paper V). (Wikström Jonsson et al., 1998), and such

receptors are present in human bronchi The rank order of agonist potency in cells (McKenniff et al., 1988). transfected with the human CysLT2 receptor is LTC4=LTD4>LTE4, with LTE4 being a partial agonist (Heise et al., 2000; Nothacker 4.1.4. Other CysLT receptor subtypes? et al., 2000; Takasaki et al., 2000), thus supporting functional results in human 4.1.4.1. Porcine pulmonary arteries pulmonary veins (Labat et al., 1992). Ohtaka and co-workers (1987) were the Interestingly, Nothacker and co-workers first to report that LTC4 and LTD4 contract (2000) recently described BAY u9773 as porcine pulmonary arteries, and those results being a selective agonist at the human CysLT2 were confirmed in Paper II. In endothelium receptor with the same potency and efficacy intact porcine pulmonary arteries, LTC4 was as LTE , but without any agonistic activity at 4 somewhat more potent than LTD4 and the the human CysLT1 receptor. This is in contractions to both agonists were only contrast to the results within the present thesis slightly inhibited by either the CysLT1 using guinea-pig ileum (Paper I) and trachea receptor antagonist zafirlukast (ICI 204,219) (Table 5), where pharmacologically a CysLT 2 or the dual CysLT1/CysLT2 receptor receptor was shown to be present, but no antagonist BAY u9773. In addition, after significant contractions to BAY u9773 were endothelium denudation, LTC4 and LTD4 observed. Estimates of the rank orders of were equipotent and the LTC4-induced agonist potency at the human and guinea-pig contractions were resistant to BAY u9773. CysLT receptors are summarised in Table 3. 2 The resistance to both CysLT1 and CysLT2 The guinea-pig lung parenchyma has receptor antagonism suggest that the receptor previously been shown to contract to BAY situated on the porcine pulmonary arterial u9773, but in that preparation the contractions smooth muscle was different from the above- are inhibited by the CysLT1 receptor

described CysLT1 and CysLT2 receptors observed only against LTC4-induced (Paper II). contractions (Paper II). Taken together, the In the guinea-pig lung parenchyma a data suggest that LTC4, but not LTD4, residual contraction has been observed after activated endothelial CysLT2 receptors, and a either CysLT receptor antagonism or schematic figure of the distribution of CysLT 1 receptors in porcine pulmonary arteries is combined CysLT /CysLT receptor 1 2 proposed in Fig 4. As will be discussed in antagonism by BAY u9773 (Tudhope et al., Section 4.3.8., these experiments were carried 1994; Wikström Jonsson et al., 1998). out in the absence of inhibitors of LTC However, in the lung parenchymal strip, 4 metabolism and it cannot be excluded that the several different target tissues contribute to activation of endothelial CysLT the contractile response (Brink et al., 1981) 1 receptors by may have been due to its metabolism and responses to cysteinyl-leukotrienes are in LTC4 into LTD (Fig. 4). part mediated by secondary released factors 4 (Dahlén et al., 1983). Therefore, the role of Endothelium different CysLT receptors in the guinea-pig CysLT1 lung parenchyma is difficult to assess and the LTD4 porcine pulmonary arterial smooth muscle LTC4 may represent a more appropriate model since CysLT2 in addition, as will be shown below, the Contractile factors results were similar to findings in the human CysLT? LTD lung. 4

The findings that both zafirlukast and BAY LTC4 u9773 slightly inhibited the LTC - and LTD - 4 4 Vascular smooth muscle induced contractions in endothelial intact preparations but were inactive in rubbed preparations also suggest that LTC4 and LTD4 activated CysLT receptors on the endothelium Fig. 4: Schematic presentation of CysLT receptors in the porcine porcine pulmonary artery and their that enhanced the contractions to these activation by LTC4 and LTD4. Dotted line indicates agonists, probably by the release of possible alternative mechanism for CysLT1 receptor contractile factors (see Section 4.3). In activation by LTC4. The scheme is based on data from addition, these findings indicate that the Paper II. endothelial receptors were of the CysLT1 type. However, there was a differential effect Incubation of porcine pulmonary arteries between zafirlukast and BAY u9773 against with BAY u9773 (3 µM) did not induce LTC4-induced contractions (Paper II). changes in basal tone that were significantly Whether this difference simply reflects the different from controls in either endothelium- differences in concentration and potency of intact or rubbed preparations (Fig. 5a and b, the antagonists or is due to the presence of previously unpublished data). Since BAY also CysLT2 receptors on the endothelium u9773 has been reported to be a selective remains to be established. In support of the agonist at the human CysLT2 receptor latter suggestion, it was observed that LTC4 (Nothacker et al., 2000) and to contract induced larger contractions than LTD4 in human pulmonary veins (Labat et al., 1992), endothelial intact preparations, whereas the these findings may support the notion of a two cysteinyl-leukotrienes were equipotent in non-CysLT2 receptor on the porcine rubbed preparations and that the difference pulmonary arterial smooth muscle. between zafirlukast and BAY u9773 was

Fig. 5: Changes in basal tone of isolated porcine (panels a and b) and human (panels c and d) pulmonary arterial preparations during a 30 min incubation period. Porcine pulmonary arterial preparations with (a) or without (b) endothelium were incubated in the absence (Control) or presence of either the CysLT1 receptor antagonist zafirlukast (ICI 204,619; 1 µM) or the CysLT1/CysLT2 receptor antagonist BAY u9773 (3 µM). Human pulmonary arterial preparations (endothelium intact, panels c and d) were incubated in the absence (Control) or presence of the cyclooxygenase inhibitor indomethacin (1.7 µM, INDO) and/or either the CysLT1 receptor antagonist MK 571 (1 µM) or BAY u9773 (3 µM). Contractions (means±S.E.M) are expressed as percent of a reference contraction to noradrenaline (10 µM), n=4-12 (previously unpublished data).

heterogeneity of CysLT receptors also within The rank order of potency this species. In addition, the latter findings (LTC =LTD >>LTE ; Paper II) in rubbed 4 4 4 confirm that the antagonists used indeed were porcine pulmonary arteries is similar to active compounds and that the resistance of results of previous studies of ferret spleen the contractions was not due to experimental (Gardiner et al., 1993), ferret trachea (Snyder factors. & Krell, 1986) and sheep trachea (Tomioka et al., 1991). However, although no or negligible contractions are observed after LTE 4 4.1.4.2. Human pulmonary arteries administration to the three latter preparations, LTE4 has been demonstrated to be able to The findings in the porcine pulmonary inhibit contractions induced by either LTC4 or artery thus suggest an additional subtype of LTD4 (Table 6), suggesting that LTE4 can CysLT receptors (Paper II). In order to interfere with a CysLT receptor without examine if this observation had relevance for inducing contractions. This is in contrast to human tissue, the study was continued using the findings in the porcine pulmonary artery human lung samples. The results (Paper III) where pre-treatment of rubbed preparations interestingly indicate that the human with LTE4 did not alter the LTC4-induced pulmonary artery exhibited a similar profile contractions (Paper II). These findings as the porcine pulmonary artery. suggest that LTE4 does not interfere with the In the human pulmonary artery, LTC4 and CysLT receptor present on the porcine LTD4 induced similar contractions (Paper III), pulmonary arterial smooth muscle neither as and Schellenberg and Foster (1984) have an agonist nor as an antagonist, further previously reported that LTE4 is inactive as supporting a difference from previously agonist in this preparation. The LTC4-induced described CysLT receptors. contractions of the human pulmonary artery Taken together, the findings in the porcine were resistant to the CysLT1 receptor pulmonary artery (Paper II) indicate that the antagonist MK 571 (Paper III). However, in cysteinyl-leukotriene receptor mediating contrast to human pulmonary veins (Labat et contractions of rubbed preparations is al., 1992), but in line with the findings in the different from the previously described porcine pulmonary artery (Paper II), the CysLT1 and CysLT2, based on the following LTC4-induced contractions were resistant also observations: to BAY u9773 (Paper III). In addition, incubation of human pulmonary arteries with The rank order of potency was BAY u9773 (3 µM) did not induce changes in LTC4=LTD4, whereas neither LTE4 basal tone that were significantly different µ µ (1 M) nor BAY u9773 (3 M) induced from untreated preparations both in absence any contractions. and presence of indomethacin (Fig. 5c and d,

The contractions induced by LTC4 were previously unpublished data). resistant to both CysLT1 and CysLT2 The concentration-response curves for receptor antagonism as well as to pre- LTC4 were significantly enhanced when the treatment with LTE4. LTC4-induced prostacyclin-release was It is not probable that the differences inhibited by indomethacin (see Section between the CysLT receptor on the porcine 4.3.3.). However, maximal contractions could pulmonary arterial smooth muscle and not be established since this would have previously described CysLT receptors are due demanded higher concentrations of to species-differences. The findings that both leukotriene, which were not available at the zafirlukast and BAY u9773 inhibited the time the experiments were carried out. The contractions in endothelial intact preparations non-sigmoidal shape of the concentration- (Paper II) suggest that there was response curves may limit the interpretation

of the results obtained with LTC4 in the CysLT2 receptor (i.e. resistant to CysLT1 human pulmonary artery. Further experiments receptor antagonists) may be a heterogeneous are needed in order to obtain the best group of receptors. In addition, the findings conditions for studies of cysteinyl- presented in Table 4 indicate a further leukotriene-induced contractions of the subdivision of also CysLT1 receptors. In the human pulmonary artery and to establish full initial preliminary report of the findings in concentration-response curves. However, the Paper II, the name CysLT3 was proposed for results obtained (Paper III) provide a first the receptor mediating contractions of the suggestion of a human CysLT receptor that porcine pulmonary arterial smooth muscle may be different from CysLT1 and CysLT2 (Bäck et al., 1999). The term CysLT3 has also receptors. been used in reviews of cysteinyl-leukotriene receptors when referring to receptors

mediating contractions resistant to 4.1.5. Classification of CysLT receptors BAY u9773 (Dahlén, 2000; Nicosia et al., According to the IUPHAR 2000). Another possibility is a subdivision of recommendations for nomenclature of CysLT2 receptors into CysLT2A and CysLT2B. receptors, a receptor type is defined as Such classification however requires the “nomenclature for a structurally and knowledge of the molecular structures in operationally distinct receptor in a given order to clarify what can be referred to as family” and a receptor subtype as receptor types and subtypes. “nomenclature for a receptor with strong structural homology to other types, but with distinct operational characteristics” 4.1.6. Summary: Cysteinyl-leukotriene (Vanhoutte et al., 1998). The CysLT receptors receptors together with the receptor(s) for LTB4 (BLT The finding that neither CysLT1 nor receptor) constitute the family of leukotriene CysLT2 receptor antagonism inhibited the receptors, and CysLT1 and CysLT2 are thus cysteinyl-leukotriene-induced contractions of CysLT receptor subtypes (Coleman et al., human and porcine pulmonary arteries 1995). suggests that BAY u9773 does not inhibit all The application of the classification of CysLT1 receptor antagonist resistant CysLT receptors is generally that a functional responses and that what is presently referred to as the CysLT2 receptor may in fact response resistant to CysLT1 receptor represent a heterogeneous group of receptors. antagonists is referred to as a CysLT2 receptor (Coleman et al., 1995). However, with the In the guinea-pig ileum longitudinal recent cloning of a second human CysLT muscle, the LTC4-induced contractions were receptor it is probable that the receptor resistant to CysLT1 receptor antagonism but described in those reports (Heise et al., 2000; inhibited by the dual CysLT1/CysLT2 receptor Nothacker et al., 2000; Takasaki et al., 2000) antagonist BAY u9773, indicating that LTC4 represents a distinct CysLT2 receptor. In activated CysLT2 receptors in this addition, there is only a weak homology (31- preparation. 38%) between the CysLT1 and CysLT2 receptors (Heise et al., 2000; Nothacker et al., In the guinea-pig trachea, the CysLT1 2000; Takasaki et al., 2000), suggesting that receptor antagonist ICI 198,615 (300 nM) they may be distinct receptor types rather than abolished the LTE4-induced contractions and subtypes. partially inhibited the LTD4-induced contractions, whereas the LTC4-induced The results obtained in porcine and human contractions were unaltered by this treatment. pulmonary arteries (Paper II and III) indicate These results suggest that LTC4 activated that what is currently referred to as the CysLT2 receptors and LTE4 CysLT1 receptors

and that LTD4 activated both CysLT1 and since the results indicate additional CysLT CysLT2 receptors. receptor subtypes or a further subdivision of the current CysLT receptor subtypes CysLT The current classification of leukotriene 1 and CysLT . receptors probably represents a simplification 2

(data not shown). A decrease of basal tone in 4.2. Modulation of responses to cysteinyl- guinea-pig tracheal preparations after leukotrienes in the guinea-pig trachea cyclooxygenase inhibition has been well 4.2.1. Specific aims established in previous reports (Orehek et al., The aim of this part of the thesis was to 1973; Braunstein et al., 1988) and indicates evaluate the modulatory mechanisms that endogenously formed contractile involved in the contractile responses to cyclooxygenase products are involved in the cysteinyl-leukotrienes in the guinea-pig regulation of basal tone. In addition, since trachea. Inhibitors of cysteinyl-leukotriene changes in basal tone have been shown to metabolism administered intravenously to alter the amplitude of contractile responses in guinea-pigs in vivo increase pulmonary the guinea-pig trachea (Braunstein et al., insufflation pressure in response to antigen 1988), basal tone was mechanically adjusted (Funayama et al., 1996), suggesting that the throughout the experiments in the present metabolic conversion of cysteinyl- study, ensuring that all preparations were leukotrienes may modulate their biological studied under the same preload (10 mN). This action. Therefore, the effects of cysteinyl- correction of basal tone thus excludes any leukotriene metabolism on the contractile indomethacin-induced influence on the length-tension relationship of the preparations responses to LTC4, LTD4 and LTE4 in isolated guinea-pig tracheal spiral and allows adequate comparisons between preparations were studied. controls and indomethacin-treated preparations. In addition, the effects of factors of the cyclooxygenase pathway in modulating Contractile responses in the guinea-pig cysteinyl-leukotrienes-induced contractions of trachea may be both inhibited and potentiated the guinea-pig trachea were examined. This by cyclooxygenase products since either question was primarily addressed as a pre- aspirin or indomethacin inhibits contractions study in order to obtain optimal conditions for induced by low concentrations of histamine or the study on the metabolic modulation of acetylcholine, whereas at higher contractions to cysteinyl-leukotrienes concentrations of the agonists, the (Paper V). Since most previous studies of the contractions are enhanced by the effects of cysteinyl-leukotrienes in isolated cyclooxygenase inhibitors (Orehek et al., guinea-pig tracheal preparations have been 1973). One possible explanation for this performed in the presence of indomethacin observation is that the predominantly released (Jones et al., 1983; Snyder et al., 1984; prostaglandin in the guinea-pig trachea, PGE2 Snyder & Krell, 1984; Jones et al., 1986; (Grodzinska et al., 1975; Braunstein et al., Hand & Schwalm, 1987; Snyder et al., 1987; 1988) can both relax and contract guinea-pig Hand et al., 1989; Jones et al., 1989; Buckner tracheal preparations under different et al., 1990; Krell et al., 1990; Cuthbert et al., conditions (Braunstein et al., 1988). Another 1991b; Obata et al., 1992; Jones et al., 1995), possibility is a release of both contractile and it was considered a principal issue to establish relaxant cyclooxygenase products, which the effect of this treatment in the present receives support from a report describing the experimental protocol. rank order of both spontaneous and stimulated prostanoid release from the guinea-pig trachea

as PGE2>PGF2α>prostacyclin>TXA2 (Burka 4.2.2. Modulation by prostaglandins et al., 1981). 4.2.2.1. Prostaglandins released from the Charette and co-workers (1995) have guinea-pig trachea shown that in the guinea-pig trachea, the COX-2 inhibitor NS-398 decreased basal tone Addition of indomethacin reduced basal and enhanced histamine-induced contractions tone of the guinea-pig tracheal preparations

to a similar degree as was observed with the guinea-pig, cysteinyl-leukotrienes indomethacin, suggesting that these effects stimulate the release of both were mediated by COX-2. This suggestion bronchoconstrictor and bronchodilator was supported by the observation with cyclooxygenase products in vivo. However, western blot analysis that COX-2, but not the results presented in Fig. 6 (previously COX-1, proteins were detected in tracheal unpublished data) show that in the presence of muscle and cartilage (Charette et al., 1995). indomethacin, the concentration-response There was however no detection of either curves for LTC4 and LTD4 in isolated guinea- COX-1 or COX-2 in tissues analysed directly pig tracheal preparations were not after removal from the animal (Charette et al., significantly different from those obtained in 1995), suggesting that dissection and organ absence of this cyclooxygenase inhibitor bath experiments may induce COX-2 in the (Table 7). guinea-pig trachea. Krell and co-workers (1981b) have previously reported that indomethacin enhances the contractions of guinea-pig 4.2.2.2. Effects on cysteinyl-leukotriene trachea induced by higher concentrations of contractions LTC4 whereas little or no effect on the lower The bronchoconstriction induced by portion of the LTC4 concentration-response intravenously injected cysteinyl-leukotrienes curves was observed. Likewise, another in guinea-pigs is inhibited by cyclooxygenase cyclooxygenase inhibitor, meclofenamic acid inhibition (Omini et al., 1981; Weichman et has been reported to enhance the maximal al., 1982; Dahlén, 1983), whereas after contractions to LTC4, LTD4 and LTE4 in the aerosol administration of cysteinyl- guinea-pig trachea, without altering their leukotrienes the bronchoconstrictor response EC50-values (Weichman et al., 1982). There is potentiated by cyclooxygenase inhibition are however some differences between those (Weichman et al., 1982; Dahlén, 1983). studies and the present. Taken together, these studies indicate that in

Fig. 6: Concentration response curves for LTC4 (a; in the presence of S-hexyl GSH and L-cysteine) and LTD4 (b; in the presence of L-cysteine) in the guinea-pig trachea in the absence (Control) or presence of the cyclooxygenase inhibitor indomethacin (10 µM, INDO). Experiments were performed according to the protocol described in detail in Paper V. Contractions (means±S.E.M) are expressed as percent of a maximal contraction to histamine (1 mM), acetylcholine (1 mM) and KCl (40 mM), n=3-5 (previously unpublished data, see also Table 7).

n Emax (%) Emax (g) pD2

LTC4 Control 4 89 ± 3 1.2 ± 0.2 8.6 ± 0.1 INDO 5 87 ± 2 1.2 ± 0.2 8.9 ± 0.1

LTD4 Control 3 57 ± 3 0.64 ± 0.17 8.8 ± 0.2

INDO 3 53 ± 1 0.85 ± 0.17 8.9 ± 0.3

Max Control 7 ± N.D. 1.2 0.2 INDO 8 1.4 ± 0.2 N.D.

Table 7: Maximal contractions (Emax) and pD2-values for LTC4 (in the presence of S-hexyl GSH and L-cysteine) and LTD4 (in the presence of L-cysteine) in the guinea-pig trachea in the absence (Control) or presence of the cyclooxygenase inhibitor indomethacin (10 µM, INDO). The maximal contraction induced by the combination of histamine (1 mM), acetylcholine (1 mM) and KCl (40 mM) is also shown (previously unpublished data, see also Fig. 6). There were no significant differences between controls and INDO in any of the parameters compared within the different agonists (Student's t-test). N.D.= not determined.

Firstly, in the previous reports, decrease in basal tone induced by experiments were carried out in the absence indomethacin in the present study (see above), of inhibitors of cysteinyl-leukotriene ensuring that differences in basal tone did not metabolism (Krell et al., 1981b; Weichman et affect the contractile responses of the guinea- al., 1982), whereas the results in Fig. 6 were pig trachea. obtained in the presence of either L-cysteine Watts and Cohen (1993) have shown that (LTD4) or the combination of L-cysteine with in the guinea-pig trachea, contractions S-hexyl GSH (LTC4). Secondly, the results induced by serotonin and KCl are enhanced presented in Fig. 6 and Table 7 are expressed with time until about 2.5 hours. However, in as percent of a maximal contraction to the presence of cyclooxygenase inhibitors, the histamine (1 mM), acetylcholine (1 mM) and time to obtain optimal contractility in the KCl (40 mM) performed at the end of the guinea-pig trachea decreases since experiment, which means that also the contractions induced by KCl are significantly reference contraction was obtained in the enhanced by indomethacin at time-points up presence of indomethacin in the treated to 2 hours, whereas the KCl-induced preparations. This is in contrast to the report contractions are unaffected by indomethacin by Krell and co-workers (1981b) where at later time-points (Watts & Cohen, 1993). responses are presented as percent of an initial The results of that study thus suggest that the charbachol-induced contraction in the absence guinea-pig trachea releases relaxant of indomethacin. However, in the present cyclooxygenase products that during the study, there were no significant differences in initial part of the experiments functionally absolute values between controls and inhibit contractions and that this release indomethacin-treated preparations regarding decreases with time, which causes an LTC4, LTD4 or maximal contractions improvement of contractile responses. When (Table 7), indicating that the lack of effect of studying the effects of cysteinyl-leukotrienes indomethacin on contractions of the guinea- in the guinea-pig trachea (Fig. 6; Paper V), a pig trachea (Fig. 6) was not due to an relatively long experimental protocol was enhanced maximal contraction masking the therefore chosen. An initial 90-min enhancement of responses to LTC4 or LTD4. equilibration-period was followed by A third difference is the correction for the histamine challenge (around 30 min) and then

another 60-min equilibration-period before combination of L-serine borate and L- the start of the 30-min treatment period (see cysteine, each of the three cysteinyl- Paper V for a detailed description of the leukotrienes had a specific profile (Fig. 7c; protocol). This means that the concentration- Paper V). response curves to cysteinyl-leukotrienes In human bronchi, L-serine borate has were commenced after a total incubation of been reported to slightly inhibit the LTC - about 3.5 hours, of which the indomethacin- 4 induced contractions (Buckner et al., 1986; treated preparations (Fig. 6) had been in Yamaguchi et al., 1992), but also no effect contact with indomethacin for about 2 hours. has been reported (Muccitelli et al., 1987). At these time-points, the contractions to KCl Likewise, L-cysteine does not alter the in the study by Watts and Cohen (1993) were concentration-response curves for LTD4 in identical between controls and indomethacin- human bronchi (Buckner et al., 1986; treated preparations. Bourdillat et al., 1987). It is possible that the The differences in effects of indomethacin inhibition of LTC4-induced contractions of on cysteinyl-leukotriene-induced contractions human bronchi observed by some between previous studies (Krell et al., 1981b; investigators (Buckner et al., 1986; Weichman et al., 1982) and the present Yamaguchi et al., 1992) might have been an (Fig. 6) may hence refer to experimental unspecific effect of L-serine borate on the factors such as the use of inhibitors of preparations, which has been reported for this cysteinyl-leukotriene metabolism, correction inhibitor in the guinea-pig ileum (Gardiner et for indomethacin-induced decrease in basal al., 1990). However, in contrast to findings in tone or the time-point of cysteinyl-leukotriene the guinea-pig trachea, L-serine borate does administration. Anyhow, the findings indicate not significantly influence the ability of the that under the present experimental CysLT1 receptor antagonists FPL 55712 conditions, indomethacin did not alter the (Buckner et al., 1986; Muccitelli et al., 1987) contractile responses induced by either LTC4 or pranlukast (Yamaguchi et al., 1992) to or LTD4 in the guinea-pig trachea. antagonise the LTC4-induced contractions of Accordingly, the experiments within the study human bronchi. Since LTC4 and LTD4 are presented in Paper V were performed in the equipotent in contracting the human bronchus absence of indomethacin. and apparently act on the same CysLT1 receptor (see Sections 1.1.3. and 4.1.2.), interconversion would not be expected to alter their functional responses in human bronchi 4.2.3. Metabolic modulation of contractions in vitro in the same way as in the guinea-pig to cysteinyl-leukotrienes trachea. 4.2.3.1. Metabolism of cysteinyl-leukotrienes The guinea-pig trachea metabolised 4.2.3.2. LTC4 metabolism and contractions exogenously added radiolabelled LTC4 and The metabolism of LTC4 caused a major LTD4, whereas LTE4 was not further change of the LTC4-induced contractions of metabolised (Fig. 7a; Paper V). These results the guinea-pig trachea (Paper V). Inhibition of support previous observations in the guinea- LTC4 metabolism with L-serine borate, pig trachea (Snyder et al., 1984) and are acivicin, GSH or S-hexyl GSH shifted the similar to findings in the human lung (Kumlin concentration-response curves almost one log & Dahlén, 1990). Under control conditions, order to the left and significantly enhanced LTC4, D4 and E4 induced similar contractions the maximal contractions induced by LTC4 of the guinea-pig trachea (Fig. 7b), but when (Paper V). metabolism was inhibited with the

Fig. 7: The metabolism of radiolabelled cysteinyl-leukotrienes (a) and concentration response curves for LTC4, LTD4 and LTE4 (b and c) in the guinea pig trachea. In panel a each bar is the 3 3 3 mean+S.E.M. of 4 observations, representing the metabolism of [ H]LTC4, [ H]LTD4 and [ H]LTE4 in the absence (open bars) and presence (black bars) of the combination of L-serine borate (45 mM) with L-cysteine (5 mM). Contractions (means±S.E.M) induced by LTC4, LTD4 and LTE4 were studied in the absence (b) or presence (c) of the combination of L-serine borate (45 mM) with L- cysteine (5 mM) and are expressed as percent of a maximal contraction to histamine (1 mM), acetylcholine (1 mM) and KCl (40 mM), n=4-12 (data from Paper V).

[3H]LTC metabolism was correlated to the In addition to the potentiation and 4 functional results (Paper V). enhancement of the LTC4-induced contractions (Paper V), inhibition of LTC4 Two LTC4-preferential iso-forms of γ-GT metabolism changes the character of the have been described, γ-GT rel in humans LTC4-induced response in the guinea-pig (Heisterkamp et al., 1991) and γ-glutamyl trachea from CysLT1 into CysLT2. This leukotrienease in mice (Carter et al., 1997). notion is based on the findings that CysLT1 These two isoforms share the profile of being receptor antagonists inhibit LTC -induced 4 able to metabolise LTC4 but not synthetic γ- contractions of the guinea-pig trachea in the GT substrates such as γ-glutamyl-P- absence, but not in the presence of inhibitors nitroanilide (GPNA; Heisterkamp et al., 1991; of LTC4 metabolism (see Section 1.1.3.). Carter et al., 1997). While γ-GT rel also Accordingly, after treatment with either S- metabolises GSH and GSSG (Heisterkamp et hexyl GSH or L-serine borate, the LTC - 4 al., 1991), γ-glutamyl leukotrienease has been induced contractions were resistant to CysLT 1 described not to metabolise either GSH or receptor antagonism, thus confirming that the GSSG, but to be able to metabolise S-decyl biochemically established inhibition of GSH (Carter et al., 1997).

Fig. 8: The metabolism of LTC4 in guinea pig trachea (a) and the metabolism of γ-glutamyl-P-nitroanilide (GPNA) by 3 porcine kidney γ-GT (b). In panel a, each bar represents the percent of unmetabolised [ H]LTC4 after 30 min incubation in the absence (Control) or presence of inhibitors of LTC4 metabolism, n=4-7 (data from Paper V). The data presented in panel b show the metabolism of the synthetic γ-GT substrate GPNA (6 mM) by porcine kidney γ-GT (100 U/l) in the presence of the acceptor molecule glycylglycine (80 mM), measured by increase in absorbence at 405 nm according to the method described in detail by Silber and co-workers (1986; see Methods). Each point is the mean of 3 measurements (previously unpublished data). * indicates a significant (P<0.05) difference compared with control.

4.2.3.3. LTD metabolism and contractions In an attempt to characterise the γ-GT in 4 the guinea-pig trachea according to the In the guinea-pig trachea, the metabolism 3 3 findings in humans and mice (Heisterkamp et of [ H]LTD4 into [ H]LTE4 was inhibited by al., 1991; Carter et al., 1997), the inhibition of L-cysteine, resulting in a potentiation of the LTC4 metabolism in the guinea-pig trachea concentration-response curves (Fig. 7; Paper was compared with the inhibition of the V), which supports previous findings (Sok et metabolism of GPNA by purified porcine al., 1981; Snyder et al., 1984). However, a 3 3 kidney γ-GT (previously unpublished data). formation of [ H]LTC4 from [ H]LTD4 was GSH inhibited LTC4 metabolism in the discovered in the presence of S-hexyl GSH guinea-pig trachea at 10 mM (Fig. 8a) but not (Paper V), a reaction not previously described lower concentrations (data not shown), which in lung tissue nor in smooth muscle is similar to its inhibition of GPNA preparations. In addition to S-hexyl GSH, also metabolism by purified porcine kidney γ-GT other γ-glutamyl donors, such as GSH, GSSG (Fig. 8b). S-hexyl GSH was equipotent with and S-decyl GSH induced formation of LTC4 GSH in inhibiting GPNA metabolism from LTD4 in the guinea-pig trachea γ whereas S-hexyl GSH inhibited LTC4 (Paper V). These donors are metabolised by - metabolism in the guinea-pig trachea at a 100- GT and the γ-glutamyl group that is cleaved times lower concentration compared with off by the enzyme is transferred to an GSH. Since S-hexyl GSH and GSH both acceptor (Tate & Meister, 1985) and if this inhibit LTC4 metabolism by substrate acceptor is LTD4, the reaction will yield LTC4 competition, these findings suggest that the γ- (Hammarström, 1981) as is shown in Fig. 9. GT in guinea-pig trachea may preferentially metabolise LTC4 and other S-conjugates of GSH. However, a more direct investigation is required in order to establish the γ-GT iso- OH enzymes in the guinea-pig trachea and there COOH LTC4 C H S are currently no tools available for such 5 9 Cys Gly characterisation Glu In further support of the suggestion of a C H difference between enzymes metabolising + 6 13 S GPNA and LTC4, it was found that acivicin, C6H13 Cys Gly previously described to be a potent and S rapidly acting γ-GT inhibitor (Stole et al., Cys Gly 1994), potently inhibited the metabolism of Glu GPNA by porcine kidney γ-GT (Fig. 8b) S-hexyl GSH - whereas a high concentration (1 mM) did not 3 completely block [ H]LTC4 metabolism in the OH guinea-pig trachea (Fig. 8a, Paper V). These LTD COOH observations hence indicate that acivicin may 4 C5H9 S be a less potent inhibitor of the LTC4- Cys Gly preferential iso-enzyme compared with its ability to inhibit γ-GT. A discriminative effect Fig. 9: The interconversion between LTC4 and LTD4 in of other γ-GT inhibitors on metabolism of the guinea pig trachea. S-hexyl GSH inhibited the GPNA and LTC has previously been metabolism of LTC4 into LTD4 and stimulated the 4 formation of LTC from LTD (Paper V). described in cultured human umbilical vein 4 4 endothelial cells (Pologe et al., 1984) and in rat peritoneal cells (Aharony & Dobson, 1984).

GT in the presence of GSH (Anderson et al., The formation of LTC from LTD 4 4 1982; Bernström & Hammarström, 1982). changed the pharmacology of the LTD - 4 However, the results in Paper V indicate that induced contractions. In fact, in the presence no such transpeptidation of LTE occurred in of the γ-glutamyl donors, the responses to 4 the guinea-pig trachea, since there was no exogenously administered LTD4 displayed the 3 further metabolism of [ H]LTE4 under control characteristics of the LTC4-induced γ contractions (Paper V). Firstly, in the conditions nor in the presence of the - glutamyl donor S-hexyl GSH. This is further presence of L-cysteine, the LTD4-induced supported by the finding that the LTE4- contractions were inhibited by the CysLT1 receptor antagonist ICI 198,615 (Paper V), induced contractions of the guinea-pig trachea but after pre-treatment with the combination were not altered in the presence of S-hexyl GSH (Paper V). If LTE was metabolised into of L-cysteine with one of the γ-glutamyl 4 LTF , a leftward displacement of the donors, the LTD -induced contractions were 4 4 concentration-response curve would be resistant to ICI 198,615 (Paper V). Secondly, expected since LTF previously have been the maximal contraction (E ) induced by 4 max described to be somewhat less potent than LTD4 was increased after pre-treatment with γ LTE4 in inducing contractions of the guinea- the -glutamyl donors (Paper V) and thirdly, pig trachea (Jones et al., 1983). the LTD4-induced contractions displayed a significantly slower time-course in the presence of γ-glutamyl donors (Paper V). 4.2.4. Summary: Modulation of cysteinyl- These results were not significantly different leukotriene responses in the guinea-pig from those obtained with LTC4, thus trachea supporting that the biochemically detected formation of LTC4 from LTD4 also changed The cyclooxygenase inhibitor the functional responses into an LTC4- indomethacin decreased the basal tension of response. guinea-pig tracheal spiral preparations. In order to prevent an influence of this decrease This alternative pathway for metabolism of in basal tone on subsequent responses, basal LTD4 may modulate cysteinyl-leukotriene tone (10 mN) was re-established responses in any tissue expressing different mechanically. Using an experimental protocol CysLT receptors that are preferentially with a long incubation period before activated by either LTC4 or LTD4. challenge with cysteinyl-leukotrienes, Interestingly, LTC4 and LTD4 have been indomethacin did not alter the concentration- reported to activate separate receptors in response curves for either LTC4 or LTD4 in isolated human saphenous veins and in human the guinea-pig trachea (Fig. 6), suggesting lung membranes (Allen et al., 1992; Rovati et that prostaglandins did not modulate the al., 1992; Capra et al., 1998a; Capra et al., cysteinyl-leukotriene-induced contractions 1998b; Ravasi et al., 2000). Taken together, under these conditions. Therefore, subsequent these reports suggest that the alternative experiments were performed in the absence of metabolic pathway for LTD4 described in the indomethacin. guinea-pig trachea (Paper V) may also modulate cysteinyl-leukotriene responses in human tissues. In untreated preparations, LTC4, D4 and E4 induced similar contractions of the guinea- pig trachea, but in the presence of inhibitors 4.2.3.4. LTE 4 metabolism and contractions of their metabolism, the contractions induced Previous in vitro studies have shown that by the individual cysteinyl-leukotrienes had a γ-glutamyl LTE4 (LTF4) can be formed from specific profile (Paper V; Fig. 7). LTE4 after incubation with purified kidney γ-

The guinea-pig trachea metabolised activated a CysLT2 receptor, LTE4 a CysLT1 exogenously added radiolabelled LTC4 and receptor and LTD4 both CysLT1 and CysLT2 the results (Fig. 8) suggest that the guinea-pig receptors (see Section 4.1 and Paper V). trachea may have an isoform of γ-GT that is Accordingly, the metabolism of LTD4 into preferential for LTC4 and other S-conjugates LTE4 will push the LTD4-induced response of GSH. towards a pure CysLT1 receptor response (Fig. 10). In contrast, in the presence of a γ- Under control conditions, exogenously glutamyl containing compound (e.g. GSH, S- added LTD4 was metabolised into LTE4 by hexyl GSH etc., see Paper V), LTC4 will be the guinea-pig trachea, whereas in the formed from LTD4, and the response will thus presence of γ-glutamyl donors, LTD4 was be pushed towards a CysLT2 receptor converted into LTC4 (Paper V). In fact, these response (Fig. 10). two alternative pathways for metabolism of LTD4 may determine what receptor is activated in the guinea-pig trachea since LTC4

γ-GT + γ-glutamyl donor dipeptidase

+ + LTC4 LTD4 LTE4

CysLT2 CysLT1

Fig. 10: The metabolism of LTD4 and the activation of CysLT receptors in the guinea pig trachea.

contractions induced by cysteinyl- 4.3. Modulation of responses to cysteinyl- leukotrienes in human pulmonary arteries was leukotrienes and other agonists in human due to release of inhibitory endothelial and porcine pulmonary vessels factors. 4.3.1. Specific aims Also in isolated human systemic vessels, The aim of this part of the thesis was to venous preparations have been reported to be examine the contractile effects of cysteinyl- more sensitive to cysteinyl-leukotrienes than leukotrienes in pulmonary vascular arterial preparations (Allen et al., 1992; Allen preparations. In order to compare the et al., 1994; Cracowski et al., 1999; Stanke- modulations of cysteinyl-leukotriene-induced Labesque et al., 2000). However, the responses with those of other agonists, also preferential vasoconstriction of human vascular responses to noradrenaline, saphenous veins compared with internal acetylcholine and bradykinin were examined. mammary, femoral and gastroepiploic arteries In addition, as was found in the guinea-pig is not due to endothelial modulation since trachea (see Section 4.2.3. and Paper V), endothelium denudation (Allen et al., 1994; metabolism of cysteinyl-leukotrienes may Cracowski et al., 1999; Stanke-Labesque et modulate their responses. Therefore, the al., 2000) or pre-treatment with either effects of inhibitors cysteinyl-leukotriene indomethacin or a nitric oxide synthesis metabolism were assessed in preliminary inhibitor (Allen et al., 1994) fails to experiments. Since a similarity between significantly potentiate the cysteinyl- human and porcine pulmonary arteries was leukotriene-induced responses in these human observed at the level of cysteinyl-leukotriene systemic arterial preparations. receptors (see Section 4.1.3.), this part of the thesis is focused on human and porcine pulmonary vessels. 4.3.2.2. Porcine pulmonary vessels In the pig, the relationship between pulmonary arteries and veins was the reversed 4.3.2. Cysteinyl-leukotriene responses in the compared with human pulmonary vessels, i.e. pulmonary vascular bed the pulmonary arteries were more sensitive to 4.3.2.1. Human pulmonary vessels LTC4 and LTD4 than the pulmonary veins Previous reports have indicated that (Paper II and IV). In fact, the porcine cysteinyl-leukotrienes are potent constrictors pulmonary veins were practically insensitive of human pulmonary veins, whereas human to LTC4 (Paper IV), which has also been pulmonary arteries are only slightly observed in a previous study (Ohtaka et al., 1987). As will be discussed in greater detail contracted by either LTC4 or LTD4 (Schellenberg & Foster, 1984; Bourdillat et below, significant contractions to LTC4 were al., 1987). However, when the endothelium unmasked after the preparations were treated with a combination of cyclooxygense and had been removed, the contractions LTC4 and nitric oxide synthesis inhibitors (Paper IV). LTD4 in the human pulmonary artery were enhanced (Paper III) to a level similar to what When the modulatory factors had been has previously been described for human removed, the contractions to LTC4 were pulmonary veins (Schellenberg & Foster, similar in porcine pulmonary arteries and 1984; Bourdillat et al., 1987; Labat et al., veins (Paper IV) and, in addition, similar to 1992). These results suggest that the contractions of human pulmonary arteries mechanism behind the apparent small (Paper III).

4.3.3. Modulation of cysteinyl-leukotriene human pulmonary arteries. These findings responses by prostaglandins support a previous comparison of human and porcine pulmonary arteries using other 4.3.3.1. Isolated vessels agonists (Lawrence et al., 1998), suggesting In untreated endothelium-intact pulmonary that this difference in cyclooxygenase arterial preparations, LTC4 induced modulation may be general rather than LTC4- significantly greater contractions of porcine specific. preparations compared with human preparations (Fig. 11a; Paper II and III). The prostaglandin that was released after LTC challenge of human pulmonary arteries However, the LTC -induced contractions of 4 4 was thus relaxant (Fig. 11) and, according to a porcine pulmonary arteries were significantly previous report, human pulmonary arteries inhibited by the cyclooxygenase inhibitor can produce prostacyclin (Schellenberg et al., indomethacin (Paper II), whereas in human 1986). The relaxant effects of prostacyclin on pulmonary arteries a significant enhancement human pulmonary arteries are well known was observed after indomethacin pre- (Hadhazy et al., 1983; Haye-Legrand et al., treatment (Paper III). In fact, in the presence 1987) and in fact, prostanoid-induced of indomethacin, LTC induced similar 4 relaxations of human pulmonary arteries are contractions of porcine and human pulmonary mediated solely via an IP-receptor (Walch et arteries (Fig. 11b). Taken together, these al., 1999), i.e. the receptor for prostacyclin findings indicate that LTC stimulates the 4 (Narumiya et al., 1999). Moreover, cysteinyl- release of cyclooxygenase metabolites in both leukotrienes have previously been reported to porcine and human pulmonary arteries, but induce release of prostacyclin in cultured that while in porcine pulmonary arteries human umbilical vein endothelial cells vasoconstrictor prostanoids dominate, (Cramer et al., 1983; Pologe et al., 1984). vasorelaxant prostanoids are released from

Fig. 11: Concentration response curves for LTC4 in porcine and human pulmonary arterial preparations in the absence (a) or presence (b) of the cyclooxygenase inhibitor indomethacin (1.7 µM, INDO). Contractions (means±S.E.M) are expressed as percent of a reference contraction to noradrenaline (10 µM), n=5-10 (data from Paper II and III).

LTC -induced contractions remains to be The release of prostacyclin in the human 4 established. For example, exogenously pulmonary artery was confirmed by measurements of the stable metabolite 6-keto administered PGD2 and PGF2α have been reported to induce contractions of porcine PGF α and was found to correlate well with 1 pulmonary arteries (Greenberg et al., 1981). the functional tri-phasic response observed for both LTC4 and LTD4 (Paper III). After Interestingly, in contrast to the results administration of either LTC4 or LTD4 obtained with LTC4, the LTD4-induced (1 µM) an initial contractile response was contractions of porcine pulmonary arteries observed followed by a relaxation with a were not altered by indomethacin (Paper II). maximum around 2 min the challenge. This The latter finding is similar to a previous relaxant phase correlated with an increased study of isolated human saphenous veins release of prostacyclin, which was where indomethacin augments the significantly lower during the 18 following contractions to LTC4 whereas the LTD4- minutes of the LTC4- and LTD4-induced induced contractions are unchanged by this responses. When the prostacyclin release treatment (Allen et al., 1992). Taken together, subsided, the contractions to LTC4 and LTD4 these findings thus suggest that there may be (which are long lasting responses) continued, differences between how LTC4 and LTD4 which resulted in the third phase of the activate the cyclooxygenase pathway. response, i.e. a contraction. However, the In conclusion, cyclooxygenase products secreted prostacyclin was still enough to modulated the LTC4-induced responses of prevent a full cysteinyl-leukotriene both human and porcine pulmonary arteries. contraction (Paper III). However, the composition of the products On the basis of the observation that LTC4- formed in response to LTC4 may vary induced contractions of isolated porcine between the two preparations as is indicated pulmonary arteries were somewhat more in Fig. 12. inhibited by a thromboxane synthesis inhibitor than by indomethacin, Ohtaka and co-workers (1987) suggested that COX thromboxane A2 (TXA2) may be the LTC4 contractile factor which is released from Prostacyclin porcine pulmonary arteries during challenge + + + -- with LTC4. However, the findings in Paper II raise a doubt as to this suggestion since measurements of thromboxane B2 (TXB2) Human pulmonary arterial smooth muscle showed that the increase in this stable TXA2 metabolite was very small. Interestingly, COX porcine pulmonary arteries also released LTC4 prostacyclin in response to both LTC4 and Contractile factors Prostacyclin LTD4 (Paper II). Since prostacyclin is a + + + + + - vasorelaxant also in porcine pulmonary arterial preparations (Zellers et al., 1994), this finding suggests that a balance between Porcine pulmonary arterial smooth muscle different cyclooxygenase products, rather than one specific metabolite, regulates the contractions to cysteinyl-leukotrienes in Fig. 12: The regulation of cysteinyl-leukotriene- porcine pulmonary arteries. However, the induced contractions by cyclooxygenase (COX) in human and porcine pulmonary arteries. The figure is contractile prostanoid responsible for the based on data from Paper II and III. inhibitory effects of indomethacin on the

vascular effects in vitro and in vivo. For In porcine pulmonary veins, the negligible example, LTC4 is a potent aggregator of contractions induced by LTC4 were not changed after pre-treatment with porcine platelets (Letts et al., 1985), and indomethacin (Paper IV) and Bourdillat and platelet aggregation elicits release of TXA2 co-workers (1987) have previously reported (Hourani & Cusack, 1991). Therefore, the differential findings may depend on the that LTD -induced contractions of human 4 amount of platelets present during the pulmonary veins are not altered by investigations. cyclooxygenase inhibition. Taken together, these observations suggest that the cyclooxygenase pathway may preferentially modulate contractions to cysteinyl- leukotrienes in pulmonary arteries compared with pulmonary veins.

4.3.3.2. Comparison with in vivo studies Also in vivo studies of cysteinyl- leukotrienes have indicated that their responses in the pulmonary vascular bed may be related to secondary released cyclooxygenase products. In the pig, the LTC4-induced increase in pulmonary arterial pressure is inhibited by indomethacin (Olson & Fleisher, 1989). However, the finding that LTC4 did not contract porcine pulmonary veins (Paper IV; Ohtaka et al., 1987) is Fig. 13: Concentration response curves for the TP opposed by the previously reported in vivo receptor agonist U 46 619 in porcine pulmonary finding that in the pulmonary circulation of arterial and venous preparations. Cumulative the pig, LTC4 causes a 15-fold increase in administration of the agonist was performed according venous resistance compared with a 2.5-fold to the experimental protocols described in detail in ± increase in arterial resistance (Ohtaka et al., Paper II and IV. Contractions (means S.E.M) are expressed in gram, n=4 (previously unpublished data). 1987). Ohtaka and co-workers (1987) * indicates a significant difference (P<0.05, two way suggested that TXA2 from the porcine ANOVA) compared with porcine pulmonary artery. pulmonary artery may be transported downstream to the pulmonary veins and induce a venoconstriction. In support of that The exact mechanisms of cysteinyl- suggestion, the data presented in Fig. 13 leukotriene-induced responses in the (previously unpublished data) show that the pulmonary circulation and the relative TP-receptor agonist U 46 619 preferentially importance of direct vascular effects in these contracted porcine pulmonary veins compared responses remain to be established. In with arteries. As discussed above, the low general, cyclooxygenase product seem to TXB2-release detected from porcine mediate cysteinyl-leukotriene pressor pulmonary arterial preparations (see above, responses in the pulmonary circulation in pigs Paper II) raise a doubt as to how much TXA2 (Ohtaka et al., 1987; Olson & Fleisher, 1989) that is actually produced by the porcine as well as in other species, for example pulmonary arteries in vitro. However, it is guinea-pigs (Omini et al., 1981) and sheep possible that different cells account for the (Kadowitz & Hyman, 1984), whereas in the contribution of cyclooxygense modulation of cat, no effect of cyclooxygenase inhibition have been observed (Kadowitz & Hyman,

1984). In fact, in the study by Kadowitz and preferentially regulate cysteinyl-leukotriene Hyman (1984), the remaining pressor activity responses in pulmonary veins compared with of LTD4 in the ovine pulmonary circulation arteries. after cyclooxygenase inhibition was very similar to that in the untreated cat, suggesting that without cyclooxygenase products, 4.3.5. Modulation of responses to other cysteinyl-leukotrienes have only moderate agonists effects on the pulmonary circulation in vivo. 4.3.5.1. Contractile agonists

In line with the findings that LTC4 under 4.3.4. Modulation of cysteinyl-leukotriene control conditions contracted porcine responses by nitric oxide pulmonary arteries but not pulmonary veins (Paper II and IV), noradrenaline exhibited a Nitric oxide synthesis inhibition with L- similar profile (Paper IV). The latter results NOARG in combination with cyclooxygenase confirm a previous report (Joiner et al., 1975), inhibition with indomethacin did not modify but no studies have been performed in order the contractions induced by LTC in porcine 4 to elucidate the mechanisms involved in the pulmonary arteries compared with results lack of venous contractions to noradrenaline. obtained with indomethacin alone (Paper IV). In contrast, treatment of porcine pulmonary Indomethacin did not alter the veins with L-NOARG in combination with noradrenaline-induced contractions in either indomethacin unmasked a contractile arteries or veins, whereas L-NOARG response to LTC4 (Paper IV). These results significantly potentiated the contractile suggest that LTC4 stimulated the release of responses to noradrenaline in both vessels nitric oxide from porcine pulmonary veins (Paper IV). However, after treatment with that functionally inhibited the contractile either L-NOARG or indomethacin+L- effect of the leukotriene, and that significant NOARG, the contractions to noradrenaline contractions were unmasked only after nitric exhibited a markedly greater augmentation in oxide synthesis had been inhibited. Taken venous (about 4-fold) compared with arterial together, these observations indicate a (about 1.5-fold) preparations (Paper IV). preferential modulation of cysteinyl- These findings indicate that contractions leukotriene responses by nitric oxide in induced by noradrenaline and LTC4 are porcine pulmonary veins compared with similarly regulated in porcine pulmonary arteries. The LTD4-induced contractions of vessels, and support the notion of a human pulmonary veins have previously been preferential regulation by nitric oxide in reported to be enhanced after L-NOARG porcine pulmonary veins compared with treatment (Ortiz et al., 1995), suggesting that arteries (Paper IV). This is further supported also in human pulmonary veins cysteinyl- by a previous report that demonstrated that leukotriene responses are regulated by nitric porcine pulmonary venous, but not arterial, oxide. The effect of the combination of L- contractions induced by endothelin-1 are NOARG with indomethacin on the LTC4- potentiated by a nitric oxide synthesis induced contractions of human pulmonary inhibitor (Zellers et al., 1994). In contrast, arteries was evaluated in a limited number of contractions induced by noradrenaline and experiments, and the preliminary results endothelin-1 in human pulmonary vessels suggested no further enhancement of the may not be regulated by nitric oxide, since the amplitude compared with indomethacin alone combination of cyclooxygenase and nitric (Paper III). These results thus suggest a oxide synthase inhibition does not alter the similarity with results in the porcine maximal contractions induced by these pulmonary artery, indicating that also in agonists in isolated human pulmonary arteries human pulmonary vessels, nitric oxide may

or veins (Pussard et al., 1995; Holm & and support the notion of a dominant role of Franco-Cereceda, 1996). nitric oxide in porcine pulmonary veins. In contrast to acetylcholine, bradykinin induced similar relaxations of porcine 4.3.5.2. Endothelium-dependent relaxations pulmonary arterial and venous preparations Although endothelium-dependent (Fig. 14, previously unpublished data). relaxations to cysteinyl-leukotrienes were not Nevertheless, also these results support the experimentally addressed in the present notion of a dominant role of nitric oxide in project, it warrants some discussion in the pulmonary veins, since bradykinin-induced context of other agonists examined. In relaxations in the veins were completely endothelium intact human pulmonary vessels, abolished by L-NOARG whereas the arterial LTD4 induces relaxations that are greater in relaxations were not blocked by nitric oxide arteries compared with veins (Ortiz et al., synthesis inhibition and/or cyclooxygenase 1995). The exact mechanisms involved in inhibition (Fig. 14). These results are in line these relaxations have not been investigated. with a previous report where bradykinin- It is likely that the prostacyclin released from induced relaxations of porcine pulmonary human pulmonary arteries after challenge arteries were suggested to be mediated via an with either LTC4 or LTD4 (Paper III) endothelium derived hyperpolarizing factor contribute, but if also other mechanisms are (Félétou et al., 1995). Although the nature of involved remains to be established. such hyperpolarizing factor has been debated, In human pulmonary vessels, also two different mechanisms are currently acetylcholine has been shown to induce believed to be involved. The first possibility greater relaxations of arteries compared with is the release of a factor from the endothelium veins (Walch et al., 1997). However, the that hyperpolarizes the smooth muscle and arterial relaxations induced by acetylcholine this factor has been identified as 11,12- are markedly inhibited by indomethacin, epoxyeicosatrienoic acid, a cytochrome p450 whereas the venous relaxations are practically metabolite of arachidonic acid (Fisslthaler et unaltered by this treatment (Norel et al., 1996; al., 1999). The other possibility is that an Walch et al., 1998). In addition, human agonist hyperpolarizes the endothelial cells pulmonary arterial preparations release more and that this hyperpolarization is spread via prostacyclin compared with venous gap-junctions to the smooth muscle cells preparations in response to acetylcholine (Edwards et al., 2000).

(Walch et al., 1998), thus suggesting a In canine renal arteries, the LTD4-induced similarity between responses induced by endothelium-dependent relaxations are not acetylcholine and cysteinyl-leukotrienes in attenuated by nitric oxide synthase inhibitors the human pulmonary vasculature. in combination with indomethacin (Pawloski In porcine pulmonary vessels, venous & Chapnick, 1990), suggesting the possibility preparations relaxed to a greater extent in that an endothelium derived hyperpolarizing response to acetylcholine than did arterial factor may be involved also in cysteinyl- preparations (Paper IV). In addition, L- leukotriene responses. Likewise, neither NOARG completely abolished the relaxations cyclooxygenase nor nitric oxide synthesis in both types of vessels, indicating that the inhibitors block the endothelium-dependent acetylcholine-induced relaxations of porcine relaxations induced by LTC4 in human pulmonary vessels were mediated solely via saphenous veins, although the effect of the release of nitric oxide (Paper IV). combination of these to inhibitors was not Consequently, the results with acetylcholine examined in that study (Allen et al., 1992). are in line with the results obtained with the contractile agonists LTC4 and noradrenaline

Fig. 14: Relaxations induced by bradykinin in endothelium intact porcine pulmonary arterial (a) and venous (b) preparations after a 30 min incubation period in the absence (Control) or presence of either indomethacin (1.7 µM, INDO), Nω–nitro-L-arginine (100 µM; L-NOARG), or the combination INDO (1.7 µM) and L-NOARG (100 µM). Cumulative administration of the agonists was performed after a precontraction with histamine (10 µM) according to the experimental protocol described in detail in Paper IV. Relaxations are expressed as percent of the precontraction. Each point is the mean of n=2 (previously unpublished data).

4.3.6. Regulation of basal vascular tone 4.3.7. Mechanisms involved in the differential In both porcine pulmonary arteries and modulation of vascular responses in porcine veins, basal tension was increased after pulmonary arteries and veins addition of the nitric oxide synthesis inhibitor The results with LTC4, noradrenaline, L-NOARG with the latter preparation acetylcholine and bradykinin thus suggest a exhibiting larger contractions (Paper IV). preferential role of nitric oxide regulation in These findings support a previous study (Bina porcine pulmonary veins compared with et al., 1998) and extend the observation by arteries, regulating relaxations and showing that indomethacin did not affect contractions as well as basal tone (Paper IV). basal tone or further alter the effects of L- The findings in porcine pulmonary arteries NOARG in either arteries or veins (Paper IV). also indicate that LTC4 stimulates the release Together these results suggest that in the of prostacyclin in this preparation (Paper II). porcine pulmonary vasculature, a basal Since different enzymes and mechanisms release of nitric oxide, but not are involved in the formation and action of cyclooxygenase products, regulates basal nitric oxide, the question raised was from tone. In contrast, in the human pulmonary what level the observed differences in nitric artery, basal tone was significantly increased oxide regulation between arteries and veins by indomethacin (Fig. 5 in Section 4.1.4.), originated. The nitric oxide released from which is similar to previous studies in the arteries and veins leads to the formation of same preparation using either indomethacin cGMP via stimulation of soluble guanylate (Hadhazy et al., 1983; Ortiz et al., 1992) or cyclase (see Section 1.2.4.2.). The nitric oxide flurbiprofen (Lawrence et al., 1998). donor sodium nitroprusside (SNP) induced

similar relaxations of porcine pulmonary Prostacyclin activates the IP receptor, arteries and veins, indicating that there was no which stimulates adenylate cyclase and difference in guanylate cyclase stimulation by increases levels of cAMP (Narumiya et al., nitric oxide between arteries and veins (Paper 1999). Zellers and co-workers (1994) have IV). Neither was there any difference between previously reported similar maximal arteries and veins in sensitivity to the relaxant relaxations to prostacyclin in porcine effect of 8-bromo-cGMP, a cell membrane pulmonary arteries and veins and the data permeable cGMP analogue (Paper IV). presented in Fig. 15 (previously unpublished Together these findings suggest that the data) extend that observation by showing that observed differences in nitric oxide regulation the sensitivity to dibutyryl-cAMP was the of basal tone, relaxations and contractions same in arteries and veins. This latter between arteries and veins were due to greater observation is similar to what was observed amounts of nitric oxide being released from with 8-bromo-cGMP (Paper IV) and further the veins rather than a difference in sensitivity supports the notion that there may not be any to either nitric oxide or cGMP. This is in difference between arteries and veins at the concordance with a previous biochemical level of sensitivity of the smooth muscle cells study (Bina et al., 1998) that reported both a to intracellular cyclic nucleotides. greater nitric oxide release as well as a greater amount of constitutive nitric oxide synthase protein in porcine pulmonary veins than in 4.3.8. Metabolism of cysteinyl-leukotrienes arteries. in vascular preparations In the guinea-pig trachea, the interconversion between LTC4 and LTD4 and the metabolism of LTD4 into LTE4 represented major modulatory mechanisms (see Section 4.2.3. and Paper V). In the porcine pulmonary artery, LTC4 and LTD4 induced contractions whereas the preparations were insensitive to LTE4 (Paper II), suggesting that the metabolism into LTE4 would lead to an inactivation of the cysteinyl-leukotrienes in this preparation. However, Galton and Piper (1987) have previously reported a slower metabolism of LTD4 compared with LTC4 in chopped porcine pulmonary arteries and the present report extends those findings by incubating endothelium intact porcine pulmonary arterial preparations with radiolabelled LTD4. The results presented in Fig. 16 (previously unpublished data) indicate Fig. 15: Relaxations to dibutyryl-cAMP in porcine that there was only marginal metabolism of pulmonary arterial and venous preparations with the LTD4 when the tissues were studied as endothelium removed. Cumulative administration of endothelium intact vascular preparations. the agonists was performed after a precontraction with These findings are further supported by the histamine (10 µM) according to the experimental findings that treatment with L-cysteine, which protocol described in detail in Paper IV. Relaxations is an inhibitor of LTD metabolism (Paper V), (mean±S.E.M) are expressed as percent of the 4 precontraction, n=4 (previously unpublished data). did not alter the concentration-response curves to LTD4 (Fig. 17; Paper II).

3 3 Fig. 16: The metabolism of [ H]LTC4 and [ H]LTD4 by porcine pulmonary artery and chopped porcine lung, examined according to the experimental protocol described in detail in Paper V. Top panels show the distribution of radioactivity 3 3 3 for authentic standards of [ H]LTC4, [ H]LTD4 and [ H]LTE4. The results presented in the middle panels show that the 3 3 endothelium intact porcine pulmonary artery metabolised [ H]LTC4 into [ H]LTD4 whereas exogenously added 3 [ H]LTD4 showed less metabolic degradation. In contrast, after incubation of chopped porcine lung (lower panels) with 3 3 3 either [ H]LTC4 or [ H]LTD4, the distribution of radioactivity was identical to that for [ H]LTE4, suggesting a rapid cysteinyl-leukotriene metabolism into LTE4 in porcine lung tissue (each panel shows n=1, previously unpublished data).

(Paper V) did not cause any apparent changes of the concentration-response curves for LTC4 in this preparation (Paper II). However, L- serine borate have been described to have unspecific effects (Gardiner et al., 1990) and it cannot be excluded such effects may have masked a possible potentiation of the LTC4- induced contractions due to an inhibition of LTC4 metabolism. In any case, the data (Paper II) suggest that LTC4 and LTD4 activate the same receptor on the vascular smooth muscle and that this receptor mediates the major part of the contractions to LTC4 and LTD4. Therefore, an interconversion between LTC and LTD would not be expected to 4 4 cause any major changes in the overall Fig. 17: Concentration response curves for LTD4 in responses to these cysteinyl-leukotrienes in porcine pulmonary arterial preparations in the absence the porcine pulmonary artery in vitro. (Control) or presence of the inhibitor of LTD4 metabolism, L-cysteine (5 mM). Experiments were Bourdillat and co-workers (1987) have performed according to the protocol described in detail previously reported that the LTD -induced in Paper II. Contractions (means±S.E.M) are expressed 4 as percent of a reference contraction to noradrenaline contractions of human pulmonary arteries and (10 µM), n=3. veins are not altered by pre-treatment with L- cysteine and moreover, LTC -induced 4 contractions of human pulmonary veins are In fact, also in cultured human umbilical not altered by L-serine borate (Labat et al., vein endothelial cell, the conversion of LTD4 1992). Preliminary experiments using S-hexyl into LTE4 is considerably less compared with GSH as an inhibitor of LTC4-metabolism the conversion of LTC4 into LTD4 (Pologe et demonstrated no apparent effects of this al., 1984; Claesson & Haeggström, 1988), treatment on the LTC4-induced contractions suggesting that vascular tissues may exhibit a of the human pulmonary artery (data not minor metabolism of LTD4. This suggestion shown). However, in line with the findings in is further supported by similar findings in the chopped porcine lung, human lung tissue guinea-pig, using pulmonary artery (Fedyna metabolises cysteinyl-leukotrienes (Kumlin & et al., 1990), inferior vena cava (Rinkema et Dahlén, 1990) and in addition, cysteinyl- al., 1993) or cardiac tissue (Falcone et al., leukotriene metabolising enzymes are present 1991). in human plasma (Köller et al., 1985). In contrast the porcine pulmonary artery, Taken together these results indicate that the results obtained in chopped porcine lung cysteinyl-leukotriene metabolism may be an tissue indicate a rapid metabolism of both additional way of modulating responses to 3 3 [ H]LTC4 and [ H]LTD4 in this tissue, since cysteinyl-leukotrienes in the pulmonary 3 only [ H]LTE4 was detected after 30 min circulation, but that this modulatory incubation (Fig 16). These results thus mechanism seems to be difficult to study suggest that other cells in the porcine lung using isolated vessels. than those associated with the vasculature are responsible for the metabolism of LTD4. 4.3.9. Pulmonary hypertension Although the porcine pulmonary artery 3 exhibited some metabolism of [ H]LTC4 Stenmark and co-workers (1983) detected (Fig. 16), the γ-GT inhibitor L-serine borate elevated levels of cysteinyl-leukotrienes in

bronchoalveolar lavage fluid from infants pulmonary vessels, further supporting the with pulmonary hypertension, leading to the notion of a dominant role of nitric oxide in suggestion that cysteinyl-leukotrienes may be porcine pulmonary veins compared with involved in this disease, and a recent study arteries. demonstrated that cysteinyl-leukotriene are In both human and porcine pulmonary formed in the human pulmonary circulation arteries, cyclooxygenase products were the (Kiss et al., 2000). However, based on the major modulators of cysteinyl-leukotriene present in vitro findings in the human responses. However, while the contraction of pulmonary artery (Paper III), the question can the human pulmonary artery by LTC was be raised as to what role cysteinyl- 4 functionally inhibited by the release of leukotrienes play in a disease like pulmonary prostacyclin, porcine pulmonary arterial hypertension. Isolated human pulmonary preparations mainly generated contractile arteries were contracted by LTC and LTD 4 4 cyclooxygenase products in response to (Paper III), which supports the hypothesis that LTC . cysteinyl-leukotrienes may increase 4 pulmonary arterial pressure in humans. On the The insensitivity of the porcine other hand, LTC4 challenge led to the release pulmonary veins to LTC4 (Paper II) is of prostacyclin at concentrations sufficient to opposed by the increase in venous resistance functionally antagonise the pulmonary arterial that has been reported for LTC4 in vivo contractions (Paper III). Prostacyclin’s (Ohtaka et al., 1987). Taken together, the relaxant effect has a beneficial role in results (Paper IV; Fig. 13) indicate that these pulmonary hypertension and is used as contradictory findings may relate to release of treatment of this disease (Gaine & Rubin, nitric oxide from porcine pulmonary veins in 1998). In addition, LTC4 injection into vitro and/or to secondary release of TXA2 in humans leads to a slight decrease in vivo. pulmonary artery pressure (Kaijser, 1982). While a definitive role for cysteinyl- The relaxant responses to bradykinin in leukotrienes (constriction and/or dilatation) in porcine pulmonary arteries were not blocked pulmonary vessels under physiological by indomethacin and/or L-NOARG, conditions is difficult to establish, the data suggesting the involvement of an presented in Paper III suggest that the degree endothelium-dependent hyperpolarizing of cyclooxygenase activity and endothelial factor. However, the role of this/these function may have modulatory influences on mediator(s) in regulation of cysteinyl- the LTC responses in vivo. leukotriene-induced responses remains to be 4 established.

In line with the findings in the guinea-pig 4.3.10. Summary: Modulation of cysteinyl- trachea (Paper V), metabolising enzymes may leukotriene responses in the pulmonary alter responses to cysteinyl-leukotrienes also vasculature in the porcine and human pulmonary The results obtained in porcine pulmonary circulations, but do not seem to significantly vessels (Paper II and IV) and human alter the contractions induced by these pulmonary arteries (Paper II), taken together agonists in isolated pulmonary vessels. with previous studies of human pulmonary The results suggest that studying veins (Bourdillat et al., 1987; Ortiz et al., modulatory and indirect factors may be 1995), suggest that nitric oxide preferentially pertinent in order to elucidate the pathological modulates cysteinyl-leukotriene-induced and/or physiological role of cysteinyl- responses in pulmonary veins. A similar leukotrienes in the cardiovascular system. pattern was observed with noradrenaline, acetylcholine and bradykinin in porcine

cysteinyl-leukotrienes were modulated by 5. CONCLUSIONS cyclooxygenase products. However, while 5.1 CysLT receptors contractile prostanoids dominated in porcine The results presented in this thesis indicate pulmonary arteries, release of the relaxant that the current classification of cysteinyl- prostanoid prostacyclin represented a major modulatory mechanism in human pulmonary leukotriene receptors as CysLT1 and CysLT2, based on the sensitivity or resistance to arteries. In contrast, in the porcine pulmonary vein and guinea-pig trachea, contractions CysLT1 receptor antagonists, probably represents a simplification. The resistance of induced by the cysteinyl-leukotrienes cysteinyl-leukotriene-induced contractions of examined were not altered by indomethacin. human and porcine pulmonary arteries to both CysLT1 and CysLT2 receptor antagonism suggest the presence of additional receptors 5.2.1.2. Nitric oxide for cysteinyl-leukotrienes. Nitric oxide was a preferential regulator of basal vascular tone as well as of contractions In the guinea-pig ileum and trachea, the and relaxations in porcine pulmonary veins contractions induced by LTC were resistant 4 compared with arteries. In fact, contractions to CysLT receptor antagonism but inhibited 1 of porcine pulmonary veins in response to by the dual CysLT /CysLT receptor 1 2 LTC antagonist BAY u9773, supporting that LTC 4 were uncovered only after inhibition of 4 nitric oxide synthesis. activates a CysLT2 receptor in these preparations.

The contractions induced by LTD4 and 5.2.2. Interconversion between the cysteinyl- LTE4 in the guinea-pig trachea were inhibited leukotrienes by CysLT receptor antagonists, supporting 1 In the guinea-pig trachea, LTD was that both these leukotrienes activate CysLT 4 1 shown to have two alternative pathways of receptors in this preparation. However, metabolism. Metabolism of LTD by a systematic comparisons of these results with 4 dipeptidase yielded LTE and consequently previous studies of CysLT 4 1 receptor activation of the CysLT receptor. In the antagonists in different tissues revealed a 1 presence of a γ-glutamyl donor, LTD4 was pattern that may suggest that what is now γ referred to as the CysLT receptor, may in converted into LTC4 via the action of -GT, 1 leading to the formation of LTC and hence, fact represent a heterogeneous group of 4 activation of the CysLT receptor subtypes. 2 receptor. In tissues with a heterogeneous CysLT receptor In addition, in the presence of the CysLT1 population with preferential activation by receptor antagonist ICI 198,615, LTD4, but either LTC4 or LTD4 the metabolic not LTE4 activated CysLT2 receptors, interconversion between these agonists may suggesting that the expression of multiple represent a major modulatory mechanism CysLT receptors in a preparation may affect since it may decide which CysLT receptor is the receptor preference of the cysteinyl- activated by the cysteinyl-leukotrienes. leukotrienes.

5.3. Current issues 5.2 Modulatory mechanisms The results of this thesis thus raise the 5.2.1. Release of modulatory factors possibility of additional CysLT receptors. 5.2.1.1. Cyclooxygenase products However, further characterisation of this receptor is needed and identification of its In both human and porcine pulmonary molecular structure or the development of arteries, the contractions induced by the

selective CysLT2 receptor antagonists would The recent molecular characterisation of facilitate such studies. the human CysLT receptors will offer further possibilities for studies of CysLT receptors. The potent vasoconstrictor actions of the However, the initial molecular studies have cysteinyl-leukotrienes in human pulmonary revealed discrepancies between studies of arteries, characterised for the first time in the cells transfected with the human CysLT present thesis, warrant further exploration of receptors compared with functional studies of the role of cysteinyl-leukotrienes in the human tissues. These discrepancies draw pulmonary circulation. The finding that the attention to the need of functional studies CysLT receptors on human pulmonary using whole tissues, where the arteries and veins may be different opens up characterisation of response to cysteinyl- for possibilities to pharmacologically target leukotrienes can be studied taking into specifically the arterial or venous circulations. account receptor function as well as In the search for selective antagonists at the modulatory mechanisms. respective receptors, the porcine pulmonary artery may serve as a suitable model for the corresponding human vascular segment.

ACKNOWLEDGMENTS I would like to thank:

Professor Sven-Erik Dahlén for The members of the Laboratory of supervising this thesis project, generously Pulmonary Pharmacology (France); Xavier supporting my PhD-studentship and for Norel, Laurence Walch and Jean-Pierre providing excellent laboratory equipment and Gascard for collaboration on Paper II, III and facilities. I am also grateful for the possibility IV and for all advice on what to see in Paris to present the data of this thesis at congresses during my limited spare time. in Europe and the USA. Pellina Jansson at Scheelelaboratoriet, Professor Charles Brink, co-supervisor of Karolinska Institutet for help with providing this thesis project, for an excellent period in the experimental tissue for Paper V. the Laboratory of Pulmonary Pharmacology Associate professor Ian. A. Cotgreave at (Centre Chirurgical Marie Lannelongue, Biochemical Toxicology, Karolinska France) and for all interest and support of my Institutet for collaboration on Paper V. subsequent work. Associate professor Bertil Waldeck in The members of the Experimental Asthma Lund for help with the statistical and and Allergy Research group: Kiyoto Sakata pharmacological evaluation of the data in for an excellent collaboration in the organ Paper IV and V. bath lab, stimulating discussions and for travel company in England and Italy, Kerstin Associate professor Monica Nordberg at Ström for help in general, and with the HPLC the IMM Center of Education, Karolinska in particular, as well as for travel company in Institutet for arranging the curriculum for my Italy and Spain, Eva Wikström Jonsson for studies in environmental medicine. introducing me to the organ bath technique Sandrine, my family and my friends for all and for collaboration on Paper I, Christina support and encouragement. Sjöberg for general help and support, associate professor Maria Kumlin for collaboration on Paper V, and all other members of Experimental Asthma and The work within this thesis was supported by The Allergy Research who have been in the group Heart Lung Foundation, The Medical Research during part of the time of this thesis project, Council (Project n° 71X-9071), Vårdalstiftelsen, Margareta Andersson, Ingrid Delin, Ministère des Affaires Etrangères du Magdalena Gullriksson, Lilian Larsson, Gouvernement Français (C.I.E.S.), Svensk- Siobhàn O’Sullivan and Anita Sydbom. Franska Stiftelsen and Karolinska Institutet.

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