Btochemical Pharmacology, Vol. 40, No. 5, pp. 961-966, 1990. Printed in Great Britain. oo(M295y90 $3.00 + 0.00 @ l!Wl. Pergamon Press plc zyxwvu CATECHOLAMINES INHIBIT LEUKOTRIENE FORMATION AND DECREASE LEUKOTRIENE/PROSTAGLANDIN RATIO JOUKO PARANTAINEN,* JUHA ALANKO,~ EEVA MOILANEN,? TIMO METsA-I&TEL&t M. ZAINI ASMAW@and HEIKKI VAPAATALot *Leiras, Clinical Research, P.O. Box 325,OOlOl Helsinki, Finland; $School of Pharmaceutical Sciences, Universiti Sains Malaysia, Minden, 11800 Penang, Malaysia; and TDepartment of Biomedical Sciences, University of Tampere P.O. Box 607, 33101 Tampere 10, Finland (Receiued 10 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA October 1989; accepted 30 March 1990) Abstract-Adrenaline, noradrenaline, isoprenaline, and to a lesser extent dopamine inhibit the release of leukotriene (LT) B4 from calcium ionophore-stimulated human polymorphonuclear leukocytes, while the release of prostaglandin (PG) Ez is proportionally elevated. The inactivity of salbutamol, a non- catechol adrenergic &-receptor agonist, and the inability of propranolol to antagonize the effects of adrenaline, suggest the mediation through Preceptor independent mechanisms. Neither are cu-l- receptors involved, as prazosin, a specific antagonist, fails to inhibit the reaction. As the principles for biochemical regulation of LT- and PG-production are met by catecholamines in several tissues, the mechanism is considered to be of general physiological importance. Catecholamines may function as coenzymes/antioxidants which, by altering the redox state of the enzyme iron or heme, decrease the LT/ PG ratio thus protecting the organism against tissue anaphylaxis and other LT-related pathophysioiogy. Adrenaline, noradrenaline and dopamine stimulate biosynthesis of prostaglandins (PGs) acting as poss- ible “natural coenzymes” [l]. Caffeic acid, struc- turally related to catecholamines, is as active as adrenaline [2], and several other phenolic com- pounds have the same effect [3,4]. On the other hand, some compounds with catechol structure are known as potent inhibitors of leukotriene (LT) for- mation. These include 6,7-dihydroxycoumarin [S], some bioflavonoids [6], nordihydroguaiaretic acid [7,8], derivatives of caffeic acid [9] and indirectly also isoprenaline [lo]. Compounds with catechol structure thus seem to regulate the formation of LTs and PGs in diametrically opposite directions. This has not been studied previously in the same test system, however, and the effect of endogenous cat- echolamines on LT formation has not been estab- lished. We present evidence that catecholamines modify the formation of LTs and the LT/PG ratio, and thus possibly some pathophysiological reactions as reported preliminarily by us [ 111. MATERIALS AND METHODS zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFE Isolation of poly morphonuclear leukocytes (PMNs). Blood samples for PMNs were collected by venipuncture from healthy volunteers having abstained for at least 1 week from any drugs. PMNs were isolated with density gradient centrifugation. After isolation, 5 x lo6 PMNs (>98% purity and >98% viability) were preincubated in 465 PL of Dul- becco’s phosphate-buffered saline (components in g/L: CaClz 0.1, KC12 0.2, KH2P04 0.2, MgCl*6H20 0.1, NaCl 8.0, Na2HP04.7H20 2.16) for 15 min at 37”, and further 15 min with the test compound. The compounds were added in 25 PL of 0.9% NaCl solution. The final concentration range was 0.18 nM to 1800pM for adrenaline, dopamine, isoprenaline, noradrenaline and salbutamol; for caffeic acid 0.37 nM - 370 ,uM. Eicosanoid synthesis. Eicosanoid synthesis was triggered by Ca ionophore A23187 added in 10,uL of DMSO. The final concentrations of A23187 was 1 PM, incubation time was 10 min at 37”. HPLC apparatus. The HPLC system consisted of a dual piston LKB Model 2150 HPLC pump (LKB, Bromma, Sweden), a Rheodyne Model 7125 injector (Rheodyne, Cotati, CA, U.S.A.) equipped with a 200 PL sample loop, a C1s column (particle size 5 ,uM, length 20 cm, i.d. 3 mm; Chrompack, Mid- delburg. Netherlands), a Waters model Lambda- Max 480 LC Spectrophotometer (Waters Associates, Milford, MA, U.S.A) and a Hewlett-Packard Model 3390A integrator (Hewlett-Packard, Avondale, PA, U.S.A.). In the assay zyxwvutsrqponmlkjihgfedcbaZYXW of catecholamines a C1s column (particle size 5 pm, length 10 cm, i.d. 3 mm; Chrom- pack), a Bioanalytical Systems model LC-4 amper- ometric detector with glassy carbon electrode (Bioanalytical systems Inc., West Lafayette, IN, U.S.A.), and a LKB 2210 amplifier were used. For catecholamines the mobile phase was (per 1000 mL): 11.3 g Na-Ac*H*O, 6.8 g NaH2P04.H20, 1.2 g C+Na, 0.15 g EDTA, 10 mL 2M HCl, approx. 1 mL H2P04 for adjustment of pH to 4.85, and finally 27.5 mL acetonitrile. Determination. After pelleting the cells (10,000 g for 60 set), the samples were assayed for PGE;! with RIA, having negligible cross reactivity with other arachidonic acid metabolites [ 12,131. LTB4, 20-OH- LTB4 and 20-COOH-LTB, were assayed with HPLC [14,15]. For quantitation of LTB4 tetrahydro- furan: methanol: 0.1% aqueous EDTA:acetic acid: ammonia (25:20:55: O.l:O.l), pH adjusted to 5.5, and for 20-OH-LTB4 and 20-COOH-LTB4 methan- ol:water:acetic acid:ammonia (55:45:0.1:0.1) pH 961