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Experiments were designed to study the interaction of rat peritoneal neutrophils with the vascular smooth muscle of the rat aorta. Rings of aorta, suspended in 10-ml organ chambers containing a physiologic salt solution, were precontracted with phenylephrine. Neutrophils (1 X 10(5) -4 X 10(7) cells/organ chamber) caused a cell number-dependent relaxation of the rat aorta that was augmented by superoxide dismutase (100 U/ml) or changing the oxygen content from 95 to 21%. The neutrophil-induced smooth muscle relaxation occurred in rings with and without endothelium and in rings precontracted with increasing concentrations of phenylephrine, prostaglandin F2 alpha or KCI. Catalase (1000 U/ml) and mannitol (1 X 10(-3) M) did not block the neutrophil-induced relaxation, whereas phenazine methosulfate (1 X 10(-5) M), hydroquinone (3 X 10(-5) M) and methylene blue (1 X 10(-5) M) reversed the neutrophil-induced relaxation. Pre-exposure of endothelium-rubbed rings to neutrophils (2 X 10(7) cells/organ chamber; 15 min) depressed the subsequent concentration-response curve to phenylephrine but augmented the relaxation induced by the phosphodiesterase inhibitor zaprinast (1 X 10(-5) M). The effluent from a column restraining the neutrophils induced a relaxation of endothelium-rubbed aortic rings that was prevented by methylene blue (1 X 10(-5) M). These results demonstrate that rat neutrophils release a factor that has a pharmacologic profile similar to that previously reported for the relaxing factor released from the vascular endothelium.

SK&F 88046, an imidodisulfamide initially reported as an end organ leukotriene (LT)D4 antagonist on guinea-pig lung parenchyma (Gleason et al., 1982), was studied further in order to elucidate its mechanism of action. The LTD4-induced contraction of the guinea-pig lung parenchyma, which occurs via both a direct action and an indirect, thromboxane (Tx)-dependent pathway (Weichman et al., 1982), was antagonized by SK&F 88046 and FPL 55712. SK&F 88046 was not acting on the lung parenchyma as a result of antagonism of cyclooxygenase or Tx synthetase, as SK&F 88046 did not block the LTD4-induced generation of TxB2 from the parenchyma. In contrast, the LTD4-induced contraction of the guinea-pig trachea, which is only directly mediated, was antagonized by FPL 55712 but not by SK&F 88046. However, SK&F 88046 did antagonize the contractions elicited by carbocyclic TxA2, a stable Tx analog, as well as those elicited by prostaglandins F2 alpha and D2, but not those elicited by histamine, carbachol or KCI. FPL 55712 weakly antagonized the actions of carbocyclic TxA2, but not the contractions induced by the other agonists. These results demonstrate that SK&F 88046 is an antagonist of the indirect, Tx-dependent component of LTD4 action in the guinea pig, presumably via antagonism of Tx on the end organ. These results provide additional evidence that LTD4 can exert its bronchoconstrictive actions via two mechanisms, a direct pathway and an indirect, Tx-dependent pathway.

Pemedolac [cis-1-ethyl-1,3,4,9-tetrahydro-4-(phenylmethyl)-pyrano [3,4-b]indole-1-acetic acid; AY-30,715] exhibited potent analgesic effects against chemically induced pain in rats and mice and against inflammatory pain in rats. In each of the animal models used the analgesic potency of pemedolac was defined by an ED50 of 2.0 mg/kg p.o. or less. Significant analgesic activity was detected in rats at 16 hr after administration of 1 mg/kg p.o. (paw pressure test) and at 10 hr after administration of 10 mg/kg p.o. to mice (p-phenylbenzoquinone writhing). Inasmuch as pemedolac was inactive in the hot plate and tail-flick tests; and its analgesic activity was not antagonized by naloxone (1 mg/kg s.c.), and tolerance did not develop upon multiple administration; this drug does not exert its analgesic effects through an opiate mechanism. Pemedolac differed from standard nonsteroidal anti-inflammatory drugs (NSAIDs) in that the doses which produced analgesia were much lower than those required for either anti-inflammatory or gastric irritant effects. In acute anti-inflammatory tests, pemedolac exhibited only weak activity as evidenced by an ED50 approximately 100 mg/kg p.o. in the carrageenan paw edema procedure. This demonstrates for pemedolac a separation of at least 50-fold between the acute analgesic and anti-inflammatory activities, which was greater than that observed with reference NSAIDs. The compound also had a low ulcerogenic liability with an acute UD50 = 107 mg/kg p.o. and a subacute UD50 estimated to be 140 mg/kg/day p.o. In contrast, the reference NSAIDS (piroxicam, indomethacin, naproxen and ibuprofen) exhibited similar dose-response relationships for the analgesic, anti-inflammatory and gastric irritant effects.(ABSTRACT TRUNCATED AT 250 WORDS)

The ability of 1-[3-(cyclopentyloxy)-4-methoxyphenyl]ethanone (E)-O-(aminocarbonyl) oxime) (WAY-PDA-641) to inhibit cyclic AMP-metabolizing phosphodiesterases (PDEs) and to relax respiratory muscle was explored as part of a program to identify a PDE-IV inhibitor for potential use in the treatment of asthma. WAY-PDA-641 was identified as a preferential inhibitor of PDE-IV, possessing 36 times greater potency versus canine trachealis PDE-IV than PDE-III (IC50, 4.2 x 10(-7) M and 1.5 x 10(-5) M, respectively). The classification of WAY-PDA-641 as a preferential PDE-IV inhibitor was supported in radioligand binding studies, which demonstrated that 10 microM WAY-PDA-641 did not displace ligands from a large number of receptors, and in functional studies, which used isolated guinea pig tracheal rings. Under conditions in which tracheal rings were made sensitive to the relaxant effects of PDE-IV or PDE-III inhibitors, WAY-PDA-641 induced relaxation with IC50S of 2.6 x 10(-8) M (PDE-IV) and 3.2 x 10(-5) M (PDE-III). Moreover, PDE-IV inhibitory concentrations of WAY-PDA-641 significantly potentiated the relaxant effects of albuterol. WAY-PDA-641 reversed tracheal contractions induced by prostaglandin F2 alpha, leukotriene D4 or histamine in a biphasic manner consistent with its activity as a preferential PDE-IV inhibitor. The IC50S for reversal of each spasmogen were similar, which confirmed that WAY-PDA-641 is a functional antagonist of respiratory muscle contraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Endogenous phospholipase A2 (PLA2) specific activity (SA) (% hydrolysis/min/mg protein) in the rat pleural cavity, measured as the acyl-hydrolysis of [3H]-arachidonic acidE. coli substrate, was quantitated after induction of a reverse passive Arthus reaction (RPAR). PLA2 SA, derived when the rate of hydrolysis was linear (1–5 min), was 1.9, 1.4, 3.8 and 4.1% h/min/mg at 10 min, 2 h, 4 h and 24 h, respectively, after induction of the RPAR. This time course appeared to correlate, with the influx of mononuclear inflammatory cells, although the effect of plasma leakage on changes in exudate PLA2 SA could not be determined. Oral adminstration of antiinflammatory drugs signficantly inhibited pleural fluid exudation and inflammatory cell influx to varying degrees. However, whereas these drugs additionally reduced total exudate protein and PLA2, they had no effect on the concentration of either parameter, implying that pleural PLA2 may be passively linked to fluid and cell movement.

The involvement of leukotrienes (LTs) in antigen-induced airway hyper-reactivity (AHR) was characterized pharmacologically by using several 5-lipoxygenase (5-LO) inhibitors and LTD4 antagonists in guinea pigs. AHR was evidenced by consistent and significant increases in sensitivity to bronchoconstriction induced by i.v. methacholine in anesthetized and ventilated animals 24 hr after a single ovalbumin aerosol challenge, but maximum methacholine-induced bronchoconstriction did not increase. Animals were pretreated with maximum doses of WY-50,295 tromethamine (WY-50,295), LY-171,883, MK-886 or zileuton, based upon inhibition of antigen-induced LT-dependent bronchoconstriction. WY-50,295, having a long duration of action, was the only compound that prevented AHR when given once before antigen challenge. However, LY-171,883 and MK-886 prevented AHR when a second dose was given 4 hr after challenge. Zileuton, having a short duration of action, failed to prevent AHR when given before and after challenge. The prevention of AHR did not result from functional antagonism (bronchodilation) by any compound. In bronchoalveolar lavage studies, neither WY-50,295 nor MK-886 inhibited the influx of eosinophils into the airways 24 hr after antigen challenge. The results provide pharmacological evidence that LTs play an important role in the pathogenesis of antigen-induced AHR in guinea pigs. Furthermore, the effectiveness of 5-LO inhibitors and LTD4 antagonists in this model depends upon a long duration of drug action and appears to result from inhibition of a direct airway effect of LTs rather than inhibition of eosinophil influx into the airways.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunological challenge of sensitized fragmented cynomolgus monkey lung with anti-human IgE (immunoglobulin E) induced the appearance of slow reacting substance of anaphylaxis (SRS-A) in the tissue and evoked the release of SRS-A into the Tyrode's supernatant. The elevation of tissue or residual SRS-A levels was maximal 2 min after anti-IgE challenge and remained at that plateau for at least 60 min. The released SRS-A, first detectable 3 to 5 min after challenge, achieved a plateau by 10 min. Both the released and residual SRS-A were similarly inactivated by soybean lipoxidase and were antagonized by FPL 55712 on the guinea-pig ileum. Isoproterenol, 5, 8, 11, 14-eicosatraynoic acid and SK & F 64398 all inhibited the anti-IgE induced elevation in residual SRS-A and blocked SRS-A release. Indomethacin stimulated both the elevation of residual SRS-A and the amount released. Removal of the Tyrode's supernatant containing 209 +/- 73 U of released SRS-A/g of tissue 20 min after anti-IgE resulted in the release of an additional 239 +/- 69 U/g; residual SRS-A levels remained at the plateau level. Incubation of the Tyrode's supernatant from challenged, but not control, tissue with fresh lung tissue caused a 90 +/- 7% inhibition of SRS-A release from the fresh tissue. Leukotriene D4 at 5 to 50 ng/ml (concentrations relevant to SRS-A release) showed a concentration-dependent inhibition of SRS-A release, but no effect on histamine release. Leukotriene C4 at 5 to 50 ng/mg failed to significantly alter the amount of SRS-A release. However, at 150 ng/ml, significant inhibition was observed which, in part, may have been produced by metabolism to leukotriene D4. These results demonstrate a potential role for LTD in regulating the amount of SRS-A released from monkey lung.

Anti-human Ig (immunoglobulin) E induced the release of 2.84 +/- 0.33 ng/ml of immunoreactive leukotrienes (iLTs) from passively sensitized, fragmented rhesus monkey lung, whereas tissue not challenged with anti-IgE released 0.21 +/- 0.08 ng/ml of iLTs spontaneously. Whereas the preferential lipoxygenase inhibitor, nordihydroguaiaretic acid (100 microM), inhibited completely anti-IgE-induced release of iLTs, the calcium channel entry blockers, nifedipine and verapamil (1 and 10 microM), and the purported intracellular calcium antagonist, TMB-8 (100 microM), were without affect on iLT release. The cyclooxygenase inhibitor, indomethacin (5 microM), potentiated iLT release by an average 27.7%. Anti-IgE also induced a contraction of the sensitized monkey lung parenchyma, which was partially suppressed by the antihistamine, mepyramine (10 microM). Against the residual contraction elicited by anti-IgE in the presence of mepyramine, neither FPL 55712 (10 microM) nor verapamil (10 microM) significantly suppressed the contractile activity of anti-IgE. On the monkey lung parenchyma, LTD4 elicited a concentration-dependent contraction, which was antagonized by FPL 55712 (KB = 1 microM) and suppressed by 40 to 50% by verapamil (10 microM). On monkey tracheal rings, the contraction elicited by LTD4 (30 nM) was suppressed by an average 83% by FPL 55712 (10 microM), 47% by verapamil (1 microM) and 45% by TMB-8 (100 microM). In contrast, the KCI-induced contraction was suppressed completely by verapamil and suppressed 79% by TMB-8, suggesting that LTD4 does not elicit contraction of the monkey trachea simply via voltage sensitive calcium entry.(ABSTRACT TRUNCATED AT 250 WORDS)

The [3H]leukotriene C4 ([3H]LTC4) and [3H]leukotriene D4 ([3H] LTD4) specific binding sites in guinea-pig lung membranes were characterized and correlated with smooth muscle contractile activities of a series of LTC-, D- and E-type analogs. [3H]LTC4 bound to the specific sites with high affinity (dissociation constant Kd = 15 +/- 5 nM), saturable capacity (maximum binding = 68 +/- 15 pmol/mg of membrane protein), stereoselectivity and specificity. The [3H]LTC4 specific binding sites were detected in the membranes isolated from leukotriene sensitive (e.g., lung and heart) or insensitive (e.g., brain and red blood cells) tissues. [3H] LTD4 also bound to specific sites with high affinity (Kd = 0.20 +/- 0.05 nM), low capacity (maximum binding = 1.1 +/- 0.2 pmol/mg of membrane protein) stereoselectivity and specificity. The [3H] LTD4 specific binding sites were detected in the membranes isolated from lung and trachea. [3H]LTC4 specific binding was inhibited by treatment of the membranes with the sulfhydryl alkylating agent N-ethylmaleimide. [3H]LTD4 specific binding was more sensitive to heat treatment and p-hydroxymercuribenzoate than the [3H]LTC4 specific binding. Radioligand competition activities of the LTD- and LTE-type analogs correlated well with the agonist and antagonist smooth muscle contractile activities. In contrast, the radioligand competition activity of the LTC-type analogs did not correlate with smooth muscle contractile activities. These results indicate that the [3H]LTC4 and [3H]LTD4 specific binding sites in guinea-pig lung membranes are chemically and physically distinct. The [3H]LTD4 specific binding sites represent physiologically and pharmacologically important receptors, and the smooth muscle contraction induced by LTD-, and possible LTE-, type analogs are mediated through the LTD4 receptors.

4R-hydroxy-5S-cysteinylglycine-6-Z-nonadecenoic acid (4R,5S, 6Z-2-nor-LTD1), a structural analog of leukotriene (LT) D4 (LTD4), significantly antagonized the pulmonary actions of LTD4 in several guinea pig models of LT-mediated bronchoconstriction and edema formation. In vitro, 4R,5S,6Z-2-nor-LTD1 (10(-5) and 10(-4) M) antagonized the LTD4-induced contraction of tracheal spirals and lung parenchymal strips. This antagonist action of 4R,5S,6Z-2-nor-LTD1 was specific for the LTs, in that LTC4- and LTE4-induced contractions of the trachea were also antagonized, whereas the contractions elicited by other spasmogens, e.g., histamine, carbachol, prostaglandin F2 alpha and KCl, were not antagonized. In vivo, the LTD4-induced bronchoconstriction in anesthetized, spontaneously breathing guinea pigs as reflected by decreases in dynamic lung compliance and airway conductance were attenuated significantly by a 1-min pretreatment with 4R,5S,6Z-2-nor-LTD1 at 5 mg/kg i.v. Similarly, the LTD4-induced increase in tracheal microvascular permeability, as assessed by extravasation of [125I]bovine serum albumin, was blocked by pretreatment with 4R,5S,6Z-2-nor-LTD1. These results provide the first demonstration that a structural analog of the peptidoleukotrienes can pharmacologically antagonize the potent actions of these LTs.

The directly mediated contractile activity of leukotriene (LT) D4 on isolated guinea-pig trachea and lung parenchyma was dependent upon the presence of calcium in the bathing buffer. Whereas 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8), a calcium antagonist believed to act intracellularly, completely antagonized the LTD4-induced contraction, the calcium channel entry blockers, nifedipine and verapamil, only partially inhibited LTD4 contractile activity; diltiazem was inactive. TMB-8, nifedipine and verapamil were more effective in blocking the contraction of the trachea elicited by KCl-induced membrane depolarization than the contraction induced by LTD4. Of the channel entry blockers, only nifedipine appeared capable of partially relaxing an established LTD4-contracted trachea, whereas TMB-8 almost completely reversed the LTD4 contraction. On the lung parenchyma, the LTD4-induced contraction was suppressed, but not abolished in Ca++-free buffer; this contraction was antagonized by meclofenamic acid, thus suggesting it could be due in part to the indirect thromboxane (Tx) A2-mediated pathway of LT action. In Ca++-free buffer, LTD4 was still capable of generating TxB2, although lower amounts were found when compared to Ca++-containing buffer. Incremental addition of calcium to the parenchyma in Ca++-free buffer containing LTD4 elicited greater than control LTD4-induced contraction and TxB2 generation. Neither the contraction of the parenchyma nor the generation of TxB2 was antagonized by nifedipine; conversely, TMB-8 blocked both completely. Thus, based upon the use of pharmacological antagonists of calcium, these results suggest that LTD4 contractile activity in the respiratory system is dependent upon calcium, with calcium of intracellular origin potentially of significance.

Immunoreactive leukotriene B4 (iLTB4), detected in the pleural cavity following induction of a reverse passive Arthus reaction (RPAR), was inhibited by the mixed lipoxygenase-cyclooxygenase inhibitors, phenidone and BW 755C, but not by cyclooxygenase inhibitors or by chlorpheniramine or methysergide. Both iLTB4 production and the subsequent pleural inflammation were dependent upon the dose of BSA antigen employed to elicit the RPAR pleurisy. However, inasmuch as BW 755C and phenidone were not distinguished from the cyclooxygenase inhibitors in their effects on fluid accumulation and cellular infiltration in RPAR pleurisy, it is doubtful that LTB4 plays a functional role in this inflammation model.

Paw edema was produced in CD-1 mice by the injection of 0.3 μg of snake venom PLA2 (A.p. piscivorus D-49) into the hind paw. Edema peaked at 10 min, remained elevated until 60 min, and then declined slowly. The PLA2 inhibitors, luffariellolide and aristolochic acid, reduced the edema but only when coinjected with the PLA2. The histamine/serotonin antagonists were the most effective drug class against PLA2-induced paw edema. The PAF antagonists, CV-6202 (iv) and kadsurenone (coinjected) reduced the PLA2-induced edema, whereas high doses of the corticosteroids, dexamethasone and hydrocortisone, were also effective. NSAIDs only partially inhibited the paw edema. The LO/CO inhibitors yielded varying activities, with only BW755C and NDGA inhibiting the edema. These results suggest that PLA2 induces paw edema in the mouse via the action of several classes of inflammatory mediators.

At 10 min, 2 h and 4 h after induction of a reverse passive Arthus reaction (RPAR) in the pleural cavity of rats, pleural exudate was collected and analyzedin vitro for phospholipase A2 (PLA2) acyl hydrolytic activity. Inasmuch as exudate PLA2 was inhibited by addition of EDTA, the acyl hydrolytic activity appeared calcium-dependent. However, addition of exogenous calcium to the exudate had only a minor effect on the activity. Maximum hydrolysis in all exudates was observed over a pH range of 6–9 with a neutral optimum. The 4 h exudate PLA2 activity was almost fully inhibited by para-bromophenacyl bromide (pBPB), whereas the acyl hydrolytic activity in the 10 min and 2 h exudates was reduced by 44% and 46%, respectively, by 200 μM pBPB. Although the PLA2 in the 4 h exudate displayed an increased sensitivity to high exogenous calcium concentrations and to pBPB inactiontion, the basic properties of the exudate PLA2 at the three time points appeared similar.

WAY-121,520 inhibited human synovial fluid PLA2 (HSF-PLA2) (IC50=4 μM) using arachidonic acid-labeledE. coli as substrate. Further biochemical characterization of WAY-121,520 demonstrated potent inhibition of 5-lipoxygenase (5-LO) activity in the murine macrophage (LTC4, IC50=4nM) and rat PMN (LTB4, IC50=10 nM) and an ability to antagonize LTD4 binding to isolated guinea-pig trachea (pKB=6.0).In vivo anti-inflammatory activity was noted in murine TPA-induced (ED50=91 μg/ear) and arachidonic acid-induced (66% inhibition at 400 μg/ear) ear edema and in leukotriene-dependent antigen-induced bronchoconstriction in the guinea pig (73% inhibition at 50 mg/kg, p.o.). WAY-121,520 represents a novel series of indomethacin-based inhibitors of PLA2 with anti-inflammatory activity resulting from a combination of biochemical activities (inhibition of 5-LO and PLA2 and LTD4 antagonism). This agent may provide added therapeutic efficacy over more selective inhibitors.

The effect of selective PDE-I (vinpocetine), PDE-III (milrinone, CI-930), PDE-IV (rolipram, nitroquazone), and PDE-V (zaprinast) isozyme inhibitors on TNF-α and IL-1β production from LPS stimulated human monocytes was investigated. The PDE-IV inhibitors caused a concentration dependent inhibition of TNF-α production, but only partially inhibited IL-1β at high concentrations. High concentrations of the PDE-III inhibitors weakly inhibited TNF-α, but had no effect on IL-1β production. PDE-V inhibition was associated with an augmentation of cytokine secretion. Studies with combinations of PDE isozyme inhibitors indicated that PDE-III and PDE-V inhibitors modulate rolipram's suppression of TNF production in an additive manner. These data confirm that TNF-α and IL-1β production from LPS stimulated human monocytes are differentially regulated, and suggest that PDE-IV inhibitors have the potential to suppress TNF levels in man.