J. Alanko

Modulation of arachidonic acid metabolism by phenols: Relation to positions of hydroxyl groups and peroxyl radical scavenging properties

We have shown earlier that catecholamines have opposite regulative effects on prostaglandin (PG)E2 and leukotriene (LT)B4 formation with a receptor-independent mechanism in human polymorphonuclear leukocytes (PMNs) and whole blood. To shed further light on the mechanisms involved and structure-action relationship, we tested the effects of phenols (catechol, hydroquinone, phenol, and resorcinol) on the synthesis of PGE2 and LTB4 in human A23187-stimulated PMNs. To study the mechanism of how phenols influence PGE2 and LTB4 synthesis, their peroxyl radical-scavenging properties were analyzed. In general, low concentrations of phenols stimulated (catechol > hydroquinone >> phenol) and high concentrations inhibited (resorcinol > catechol > hydroquinone > phenol) PGE2 formation. Resorcinol was different from the other phenols: It did not stimulate PGE2 synthesis at all, but it was effective inhibitor at high concentrations. Phenols had only an inhibitory effect on LTB4 formation (catechol = hydroquinone >> phenol > resorcinol). The order of both stochiometric factors and reactivities of phenols for scavenging peroxyl radicals was catechol > hydroquinone > resorcinol >> phenol. According to these results, phenols having hydroxyl groups in ortho- or paraposition have the greatest stimulative effect on PGE2 synthesis, the highest inhibitory action on LTB4 synthesis, and are good antioxidants. Resorcinol, having hydroxyl groups in the metaposition, behaves differently. It neither stimulates PGE2 nor inhibits LTB4 formation, but it is the most potent inhibitor of PGE2 formation. In spite of resorcinols two hydroxyl groups, it mimics as an antioxidant phenol more than catechol and hydroquinone.

Effects of antirheumatic drugs on leukotriene B4 and prostanoid synthesis in human polymorphonuclear leukocytes in vitro

The effects ofd-penicillamine, sodium aurothiomalate, indomethacin, timegadine and tolfenamic acid on the lipoxygenase and cyclo-oxygenase pathways of arachidonic acid metabolism were studied in human polymorphonuclear leukocytes (PMNs)in vitro. In short-term incubations,d-penicillamine and aurothiomalate did not affect leukotriene B4 (LTB4), prostaglandin E2 (PGE2) or thromboxane B2 (TXB2) production. Each of the three non-steroidal anti-inflammatory drugs (NSAIDs) used were potent inhibitors of prostanoid synthesis. In higher concentrations they also reduced LTB4 production; timegadine and tolfenamic acid were effective in concentrations comparable to those measured in plasma during drug therapy, whereas indomethacin was needed in ten times higher concentrations. The different effects of NSAIDs on 5-lipoxygenase activity may be of importance in their therapeutic actions as well as in the appearance of some side-effects, e.g. gastric irritation and “aspirin-induced” asthma.

Orally administered tolfenamic acid inhibits leukotriene synthesis in isolated human peripheral polymorphonuclear leukocytes

Special interest has been focused on the development of dual inhibitors of the cyclo-oxygenase and lipoxygenase pathways of arachidonic acid metabolism. In contrast to other classic NSAIDs, some fenamates in clinically achievable concentrations have been shown to inhibit synthesis of 5-lipoxygenase productsin vitro. In the present work, we studied the effect of orally administered tolfenamic acid (600 mg) on Ca ionophore A 23187-induced leukotriene synthesis in isolated human polymorphonuclear leukocytes. Leukotriene production was reduced in all 14 subjects studied, the mean inhibition of LTB4 synthesis being 16±3% and that of LTC4 33±7%. The inhibition correlated positively with serum tolfenamic acid concentrations. We suggest that inhibition of leukotriene synthesis is an additional mechanism of the anti-inflammatory, antimigraine and antidysmenorrhoeic effects of tolfenamic acid, and a possible explanation for its rare gastric and bronchoconstrictive side-effects.

Smoking cessation and nicotine substitution modulate eicosanoid synthesis ex vivo in man

The effects of smoking cessation with and without nicotine substitution on prostaglandin E2, leukotriene B4, leukotriene E4, and thromboxane B2 synthesis ex vivo in man were investigated. Blood samples were obtained from 20 healthy non-smoking controls and from 30 healthy smoking volunteers before and 3, 7 and 14 days after smoking cessation. Half of the smokers used nicotine chewing gum as a substitution therapy. Urinary cotinine and trans-3′-hydroxycotinine as well as thiocyanate excretions were used as indicators for the use of nicotine chewing gum and smoking, respectively. Prostaglandin E2, leukotriene E4, and thromboxane B2 were measured from whole blood after calcium ionophore A23187 stimulation by direct radioimmunoassay and leukotriene B4 by RP-HPLC.Prostaglandin E2 and thromboxane B2 syntheses were about three times and leukotriene B4 and E4 syntheses four times higher in smokers than in controls. Three days after smoking cessation without nicotine substitution, levels were lowered significantly to about 70%, 80%, 45% and 60% of the initial values; and after 14 days to 55%, 80%, 45% and 50%, respectively. In the nicotine substitution group no significant decreases were seen during the two-week follow-up.The increased level of eicosanoid synthesis detected in smokers in this ex vivo study may contribute to the harmful cardiovascular effects of smoking. Long-term nicotine substitution might diminish the beneficial effects of smoking cessation due to the possible stimulatory effects of nicotine and cotinine on eicosanoid synthesis even in vivo.

Catecholamines decrease leukotriene B4 and increase thromboxane B2 synthesis in A23187-stimulated human whole blood

Catecholamines (adrenaline, dopamine, isoprenaline, noradrenaline) and caffeic acid (catecholic compound without adrenergic receptor activity) decreased leukotriene (LT)B4 synthesis in A23187-stimulated human whole blood. Salbutamol, a non-catecholic beta 2-adrenergic agonist, did not influence LTB4 synthesis. Catecholamines stimulated thromboxane (TX)B2 synthesis with a concomitant inhibition of LTB4 synthesis; caffeic acid and salbutamol did not stimulate TXB2 synthesis. These results, obtained in A23187-stimulated whole blood, which also takes into account the complex interaction between different cell types, are similar to our previous results with polymorphonuclear leukocytes. Catecholamines show an opposite effect on lipoxygenase and cyclooxygenase pathways, which may give rise to a marked change in LT/TX ratio in physiological or pathological conditions where sufficient concentrations of catecholamines are present.

Catechol estrogens as inhibitors of leukotriene synthesis

Estrogens have a beneficial effect on atherosclerosis and osteoporosis after menopause, but their exact mechanism of action is still unknown. The aim of the present study was to investigate the effects of estradiol and its metabolites catechol estrogens on arachidonic acid metabolism in vitro. Estradiol had no effect on arachidonic acid metabolism up to 33 microM in A23187-stimulated human whole blood. All catechol estrogens (2-hydroxyestradiol, 2-hydroxyestrone, 4-hydroxyestradiol and 4-hydroxyestrone) had similar kinds of actions on arachidonic acid metabolism, being over ten times more potent inhibitors of leukotriene synthesis (IC50 values 0.044-0.16 microM) than thromboxane (IC50 values 0.99-2.1 microM) and prostaglandin E2 synthesis (IC50 values 0.84-5.5 microM). It is suggested that some of the protective actions of estrogens--e.g., on atherosclerosis and osteoporosis--may be related to the inhibition of leukotriene synthesis by catechol estrogens.

Effects of noradrenaline and dopamine infusions on arachidonic acid metabolism in man

We infused noradrenaline (0.025 micrograms/kg/min for 60 min, n=7) and dopamine (3.0 micrograms/kg/min for 60 min, n=6) into healthy male volunteers to study the effects of these catecholamines on in vivo thromboxane A2, prostacyclin and leukotriene E4 production measured as urinary excretions of 11-dehydro-thromboxane (TX) B2, 2,3-dinor-6-keto-prostaglandin (PG) F1alpha and leukotriene (LT) E4, respectively. Plasma noradrenaline and dopamine concentrations were 2.9+/-0.3 and 233+/-17 nmol/l at the endo fo the noradrenaline and dopamine infusions, respectively. Noradrenaline decreased thromboxane production and increased leukotriene production almost two fold. It had hardly any effect on prostacyclin production. Dopamine had no significant effects on any of the variables, however, it had a tendency to increase prostacyclin and leukotriene production. The results indicate that noradrenaline is a more important modulator of arachidonic acid metabolism than dopamine in vivo.

Adrenaline infusion increases systemic prostacyclin production in man

We have previously shown that adrenaline infusion induces an almost twofold increase in systemic thromboxane synthesis, measured as urinary 11-dehydrothromboxane B2. The purpose of the present study was to investigate whether high levels of adrenaline, found e.g. in heavy physical exercise and myocardial infarction are involved in the regulation of prostacyclin synthesis. To this end the effect of adrenaline infusion (0.1 microgram/kg/min for 45 min and thereafter 0.2 microgram/kg/min for 15 min) on prostacyclin synthesis in healthy male volunteers was investigated. Adrenaline infusion produced an over twofold increase in systemic prostacyclin synthesis, measured as urinary 2,3-dinor-6-keto-prostaglandin F1 alpha. Our study demonstrates that high circulating levels of adrenaline are associated with increased formation of prostacyclin.

Eicosanoid production in rheumatoid synovitis

Leukotriene B4 (LTB4) synthesis in rheumatoid synovitis was studied using peripheral and synovial fluid polymorphonuclear leukocytes (PMNs) and rheumatic synovial lining cells. No differences were found in LTB4 synthesis between peripheral PMNs from healthy volunteers and rheumatoid arthritis patients. When peripheral and synovial PMNs from the same RA patient were compared, arachidonic acidinduced LTB4 synthesis in synovial fluid PMNs was increased 1.7–7.2 fold, whereas the response to Ca ionophore A23187 stimulation was similar. This suggests 5-lipoxygenase stimulating factor(s) in inflamed joints. Rheumatic synovial lining cells in a primary cell culture produced small amounts of LTB4, the concentrations being less than 0.1 per cent of those of prostaglandin E2 (PGE2). PGE2 synthesis in synovial cells was increased when arachidonic acid or interleukin-1 was added to the culture, whereas LTB4 production remained unaltered. The present results suggest that in inflamed joints LTB4 originates mainly from PMNs whereas synovial lining cells are the source for PGE2.

Effects of Trolox C and SIN-1 on arachidonic acid metabolism and on cyclic GMP formation in leukocytes.

The effects of Trolox C (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a vitamin E analogue, (60-900 microM) and SIN-1 (3-morpholino sydnonimine), a nitric oxide donor, (30-3000 microM) on arachidonic acid metabolism and on cyclic GMP formation in calcium ionophore A23187 (calcimycin)-stimulated human polymorphonuclear leukocytes were investigated. Trolox C elicited a dose dependent decrease in leukotriene B4 levels and increase in prostaglandin E2 levels but did not affect cyclic GMP levels. SIN-1 dose dependently inhibited leukotriene B4 and stimulated prostaglandin E2 and cyclic GMP formation. Dibutyryl cyclic GMP did not affect the formation of leukotriene B4 and prostaglandin E2. Trolox C (180 microM), which itself had no effect on cyclic GMP levels, enhanced the effect of SIN-1 (100 microM) on cyclic GMP levels more than 5-fold. The effects of SIN-1 on arachidonic acid metabolism seem to be independent of cyclic GMP and are probably due to nitric oxide. In this experimental model both Trolox C and SIN-1 have similar actions on the prostaglandin/leukotriene ratio, and Trolox C potentiates the SIN-1-induced increase in cyclic GMP levels.