Effect of inhibition of arachidonic acid metabolism on anaphylaxis of guinea pig lung strips

The effect of inhibitors of the cyclooxygenase and/or lipoxygenase pathways of arachidonic acid metabolism on the anaphylactic contraction of lung parenchymal strips from ovalbumin sensitized guinea pigs was studied. Indomethacin, a selective cyclooxygenase inhibitor, significantly enhanced the anaphylactic contraction at 10^–6 M with no effect at 10^–5 or 10^–4 M. By contrast, nordihydroguaiaretic acid (NDGA, 10^–5 and 10^–4 M), a lipoxygenase inhibitor, and 1-phenyl-3-pyrazolidinone (phenidone, 10^–4 M), an inhibitor of both the cyclooxygenase and lipoxygenase markedly inhibited the anaphylactic contractile response. The pattern of inhibition was similar to that produced by FPL 55712, an antagonist of slow reacting substance of anaphylaxis (SRS-A), and was characterized by a decrease in the maximum tension and in the duration of the contraction. The results suggest that inhibition of the lipoxygenase pathway reduced anaphylactic contraction of guinea pig lung strips possibly by inhibiting the synthesis and release of SRS-A.     

The effect of Ro 21-7634 on the antigen-induced production of bronchoconstrictive arachidonic acid metabolites in the guinea pig lung

Ro 21-7634 has previously been shown to inhibit histamine and SRS-A release from actively-sensitized guinea pig lung fragments upon antigen challenge. In the studies described herein, it was observed that Ro 21-7634 does not decrease SRS-A release but instead acts to inhibit the synthesis of this mediator. This was confirmed by studying SRS-A synthesisin vitro in rat peritoneal cells after challenge with ionophore A23187. In the peritoneal cell system, Ro 21-7634 exhibited an IC₅₀ of 500 M, in comparison with 5,8,11,14-eicosatetraynoic acid, phenidone and BW755C (IC₅₀'s of 2, 100, and 100 M, respectively). When studied at 10^–4 and 10^–3 M in perfused guinea pig lung, Ro 21-7634 inhibited antigen-induced thromboxane A₂ production by 68 and 96%, respectively. In this system, antigen is believed to induce thromboxane A₂ production through the release of histamine and SRS-A from lung tissue. These mediators then interact at receptor sites in the lung parenchyma to induce thromboxane A₂ synthesis. Ro 21-7634 could thus be inhibiting thromboxane A₂ production by preventing the release of histamine and synthesis of SRS-A in the perfused lung system. Such a mechanism is suggested by the fact that although Ro 21-7634 was effective in inhibiting antigen-induced thromboxane production, it was ineffective in inhibiting thromboxane A₂ production induced in the guinea pig lung system by the direct perfusion of histamine or SRS-A through the lung.     

Antagonism of the in vivo and in vitro effects of leukotriene D4 by SC-39070 in guinea pigs

Leukotriene D₄ (LTD₄) causes contractions of guinea pig isolated ilea, evokes pulmonary bronchoconstriction and induces lesions of the dermal vasculature. In the present study, we assessed the antagonism of these actions by SC-39070 compared to FPL-55712, a known LTD₄ receptor antagonist. In guinea pig isolated ileum preparations, SC-39070 displayed selective antagonism of LTD₄ with a pA₂=8.20±0.06 (S.E.) and a Schild plot slope of –1.20. Administered intravenously to artificially-respired guinea pigs one minute prior to the agonist, SC-39070 antagonized (p<0.05) the bronchoconstrictive effect of LTD₄ in a dose-dependent manner (0.5–10 mg/kg). At a dose of 2.0 mg/kg, i.v. this activity was retained through a 60 minute pretreatment interval. Similarly, after oral administration of SC-39070, there was a dose-dependent antagonism of the bronchoconstrictive activity of LTD₄ (MED₅₀=3.8 mg/kg). Antagonism of LTD₄-induced bronchoconstriction was evidenced after oral administration of SC-39070 within one hour of treatment and efficacy was retained as long as 20 hours after treatment at a dose of 10 mg/kg. Finally, intravenously administered SC-39070 blocked LTD₄-induced dermal permeability in guinea pigs with a minimum effective dose of 1.0 mg/kg. In each assay, the LTD₄ antagonism evidence after treatment with SC-39070 appeared to be equal to or greater than that observed after treatment with FPL-55712.     

3-(1-imidazolylmethyl) indoles: potent and selective inhibitors of human blood platelet thromboxane synthetase

Several 3-(1-imidazolylmethyl) indoles were tested for inhibition of the microsomal enzymes which catalyse the biosynthesis of thromboxane A₂, prostaglandin I₂, and prostaglandin endoperoxides. These products were measured by bioassay to assess levels of enzyme activity. The highest activity against human blood platelet thromboxane A₂-synthetase was obtained with 2-cyclopropyl-3(1-imidazolylmethyl) indole (IC₅₀ 1×10^–10 M). This compound also exhibited the highest activity against pig aorta prostaglandin I₂-synthetase (IC₅₀ 8.4×10^–7 M). Of much more potential therapeutic interest, 2-isopropyl-3-(1-imidazolylmethyl) indole and 3-(1-imidazolylmethyl) indole showed almost complete selectivity against thromboxane A₂-synthetase. Both compounds exhibited IC₅₀'s of 2×10^–8 M against the latter enzyme but showed only weak effects (IC₅₀'s>10^–4 M) against prostaglandin I₂-synthetase and ram seminal vesicle PGH₂-synthetase.     

Pre- and postjunctional effects of histamine on the guinea pig urinary bladder: Evidence for heterogeneity in the H1-receptor population?

Histamine was tested on the guinea pig isolated urinary bladder to determine both the direct effect on the muscle and the influence on the contractions induced by field stimulation. Histamine (10^–7 –10^–3 M) caused contraction of the bladder and enhancement of the atropine-resistant response to field stimulation. These effects were sensitive to the H₁-receptor antagonist pyrilamine (pA₂=8.55 and 7.07, respectively). The H₂-antagonists cimetidine and famotidine were ineffective on both parameters. It is concluded that predominantly H₁-receptors are present in the guinea pig urinary bladder and that they are localized both on the muscle and on non-cholinergic nerve terminals. The significant difference between the pA₂ values for pyrilamine is likely to suggest a heterogeneity in the H₁-receptor population.     

Antispasmogenic and spasmolytic effects of verapamil and salbutamol, alone and in combination, on histamine-induced guinea pig tracheal contractionin vitro

The antispasmogenic and spasmolytic effects of the calcium entry blocker verapamil were examined on histamine-induced guinea pig tracheal contractionin vitro in comparison with the beta₂-adrenergic agonist salbutamol. The effects of verapamil were found to be about 1000 times weaker than those of salbutamol. The antispasmogenic IC₅₀ for verapamil was 1.99×10^–4 M compared to 7.94×10^–8 M for salbutamol. The spasmolytic EC₅₀ for verapamil was 3.37×10^–5 M and for salbutamol was 1.95×10^–8 M. The combined application of verapamil and salbutamol resulted in additive antispasmogenic and spasmolytic effects.     

Enhanced tissue responsiveness in colonic ion transport of cow's milk-sensitized guinea pigs

The effects of leukotriene D₄ (LTD₄) on ion transport were investigated in submucosa/mucosa colonic segments from guinea pigs sensitized to cow's milk and in age-matched, non-immune animals. Mediators released from mast cells in immune animals challenged with -lactoglobulin evoked an increase in short-circuit current that was reduced by SK&F 102922, a peptidoleukotriene antagonist. Serosal addition of LTD₄ (0.15–1 M) evoked a concentration-dependent, bumetanide-sensitive increase in short-circuit current which was greater in immune than non-immune controls. In the absence of ongoing neural activity, 1 M LTD₄ evoked an 8–20 A/cm² increase in short-circuit current which was increased 8–13-fold when ongoing neural activity was present. In tissues with ongoing activity, the response to 0.15 M LTD₄ was reduced by SK&F 102922, tetrodotoxin and atropine. LTD₄ enhanced the responsiveness of the tissue to carbachol by a factor of two, but did not affect responses of T₈₄ colonic epithelial cell monolayers to this agent. These results show enhanced secretory function for LTD₄ in animals with allergy to cow's milk. They suggest that the level of ongoing neural activity in the enteric neural microc ircuits is one of the major determinants of colonic secretory capacity.     

Leukotriene induction of TxB2 in cultured bovine aortic endothelial cells

The leukotrienes (LT) are potent mediators of inflammation, capable of inducing plasma leakage from postcapillary venules and vasoconstriction of terminal arterioles. Some microvascular effects may be attributable to LT stimulation of thromboxane (Tx) synthesis. Incubation of primary cultures of bovine aortic endothelial cells with buffer, LTB₄ (10^–8 M) or LTD₄ (10^–8 M), resulted in TxA₂ production in pg/10⁵ cells to the extent of: 67 ± 20, 571 ± 180, and 333 ± 60 respectively, as measured by radioimmunoassay of the stable metabolite TxB₂. Endothelial pretreatment with the LTD₄ receptor antagonist FPL55712 (10^–5 M) significantly blocked any LTB₄- or LTD₄-stimulated TxA₂ synthesis. Pretreatment with the TxA₂ synthetase inhibitor ketoconazole (10^–6 M) also prevented LTB₄ of LTD₄ stimulation of TxA₂. Preincubation with DMTU (10^–5 M), an hydroxyl radical scavenger, decreased LTB₄-induced release of TxA₂ (405 ± 40 and 366 ± 20, respectively). These findings suggest that LT may mediate their inflammatory actions in vascular beds by stimulation of Tx release from endothelial cells.     

A histamine-induced decrease of action potential duration in cardiac muscle mediated by H2 receptors

The effects of histamine (10^–7–10^–5 M) on the cardiac action potential have been studied in ventricular strips of guinea pig heart, electrically driven at a constant rate.A decrease of action potential was constantly observed at histamine concentrations above 10^–6 M.No significant variations of the other electrophysiological parameters were induced by the amine.The decrease in the duration of the action potential was blocked by burimamide at a concentration (10^–4 M) which induced only slight changes in action potential outline.It is suggested that the shortening of the repolarization phase induced by histamine is due to the H₂ receptor-mediated activation of the calcium inward current, which can increase the intracellular calcium concentration influencing the potassium permeability.     

The effects of heavy metal ions (Cd2+, Hg2+, Pb2+, Bi3+) on histamine release from human adenoidal and cutaneous mast cells

The influence of lead (Pb[CH₃COO]₂), mercury (HgCl₂), cadmium (CdSO₄) and bismuth (BiO[ClO₄]) on the spontaneous and stimulated histamine release from human adenoidal and cutaneous mast cells was tested in the concentration range 10^–8–10^–4 M. Lead displayed a bell shaped dose-response relationship in adenoidal mast cells with a maximum at 10^–6 M whereas in cutaneous cells only the spontaneous release was slightly enhanced at 10^–4 M. Mercury induced a presumably toxic histamine release in adenoidal and cutaneous mast cells at 10^–4 M. Cadmium increased the histamine release in adenoidal cells at 10^–4 M but in cutaneous cells only the stimulated release (10^–8–10^–5 M) was affected. Bismuth inhibited the histamine release at 10^–4 M in the adenoidal mast cells only. In conclusion, human adenoidal and cutaneous mast cells are affected differently by metal ions.     

Prostaglandins and neurotransmission at the guinea pig and rabbit urinary bladder

High concentrations of prostaglandins (PGE₁, PGE₂, or PGE₂) (2×10^–6 M) produced a slow contraction of longitudinal strips of detrusor muscle taken from the bladders of guinea pigs and rabbits. At a lower concentration (10^–6 M) prostaglandins enhanced contractions produced by field stimulation of nerves in guinea pig but not rabbit strips. The contractions were not affected by indomethacin. Contractions of guinea pig strips in response to acetylcholine at 10^–4 M were enhanced by prostaglandins and unaffected by indomethacin. Membrane potentials of smooth muscle cells recorded with micro electrodes, were unchanged up to 10^–6 M PGE₂. Above this the cells were depolarized with an increase in frequency of spontaneous action potentials. Synchronous recording of electrical and mechanical activity with the double sucrose gap indicated a decrease in amplitude of the evoked excitatory junction potential and action potential even when the contraction was enhanced in the presence of PGE₂. Responses to repeated stimulation at 10 Hz for 1 min were progressively depressed. This trend was slightly reduced by PGE₂ but unaffected by indomethacin. It is concluded that prostaglandins are not normally released by the nerves to the urinary bladder but are able to facilitate contraction in the guinea pig. This effect is probably on the excitatory-contraction coupling, possibly by mobilizing Ca²⁺. Some modification of transmitter release by the nerves may also occur.     

Effects of dithiothreitol on the isolated rabbit mesenteric artery

Rabbit mesenteric artery strips exposed to 10^–3 M dithiothreitol (DTT) were contracted with a series of concentrations of histamine and 2-pyridylethylamine (PEA). DTT exposure increased the sensitivity to histamine 100-fold but increased the sensitivity to PEA only 4-fold. DTT did not reduce dimaprit-induced relaxations, but reduced histamine-induced relaxations. Following a high concentration of histamine (10^–3 M), DTT itself produced a sustained, slowly developing contraction (29±6.8% of the maximal contraction) relaxed by 7×10^–6 M mepyramine but not by 10^–6 M phentolamine. Metiamide (3×10^–3 M) potentiated DTT-induced contractions (29±6.8 before, 57±7.5% after metiamide, as a percent of maximal contraction). Changing the bathing fluid and repeating DTT exposure slowly relaxed previously contracted strips. DTT did not prevent the increase in sensitivity of relaxant histamine receptors on exposure to cold. We conclude that DTT, in addition to potentiating histamine H₁-receptor responses, releases histamine presumably from non-mast cell pools when they are loaded with a high concentration of exogenous histamine.     

Antagonism of histamine and leukotrienes by azelastine in isolated guinea pig ileum

The effects of azelastine on histamine- and leukotriene C₄ and D₄ (LTC₄, LTD₄)-induced contractile responses in isolated guinea pig ileum were investigated. Following a 2-min contact with the ileum, azelastine produced competitive antagonism of histamine (pA₂=8.24). Following a 15-min contact, azelastine at 2.5×10^–9 M exerted competitive antagonism, but at higher concentrations (10, 40 and 160×10^–9M) it not only shifted histamine concentration-effect curves to the right but also suppressed its maximum. Thus, azelastine exerts a dual (competitive/noncompetitive) antagonism of histamine depending upon the concentration and duration of contact. Azelastine and FPL 55712 (a known LT receptor antagonist) produced concentration-dependent antagonism of LTC₄ and LTD₄. Azelastine and compound FPL 55712 also exerted concentration-dependent reversal (relaxation) of pre-existing LTC₄-induced contractions. In conclusion, the potent H₁-histamine and leukotriene receptor blocking activities of azelastine may contribute to its antiasthmatic/antiallergic activities.     

Comparative effects of etodolac,indomethacin, and benoxaprofen on icosanoid biosynthesis

Etodolac (Ultradol^®) is a new tetrahydropyrano-indole derivative which has shown pronounced analgesic and antiinflammatory properties. Its effects on the different pathways of metabolism of arachidonic acid have been studied and compared to the effects of indomethacin and benoxaprofen. All three drugs tested inhibited the cyclooxygenase activity of the parenchyma as assessed by inhibition of LTB₄- and LTD₄-induced contraction. However, the activity of human polymorphonuclear leukocyte 5-lipoxygenase and of human platelet 12-lipoxygenase was inhibited only by very high concentrations (1×10^–4 M and over) of these compounds, whereas nordihydroguaiaretic acid (NDGA) was active at concentrations 100 times lower. In the Schultz-Dale reaction of the guinea pig trachea, etodolac, benoxaprofen, and indomethacin produced increases of the myotropic contractile activity of the tissues sensitized to qvalbumin. Our results do not lend support to the hypothesis that the mechanism of action of these antiinflammatory drugs is linked to the inhibition of lipoxygenases.     

Ranitidine but not famotidine releases acetylcholine from the guinea pig myenteric plexus

The effects of the histamine H2-receptor antagonists ranitidine and famotidine on acetylcholine release have been studied in the guinea pig myenteric plexus longitudinal muscle preparation incubated with [³H]-choline. Ranitidine (3×10^–5–3×10^–4 M) dose-dependently increased the resting release of acetylcholine and that evoked by electrical stimulation. The effect was present only in strips perfused with 10^–5 M physostigmine. The effect of ranitidinc was inhibited by tetrodotoxin and hexamethonium. Famotidine (10^–5–3×10^–4 M) was totally ineffective in modifying both the resting release and that evoked by field stimulation. Ranitidine did not antagonize the inhibitory effect of oxotremorine, which specifically activates negative feedback mechanisms via presynaptic muscarinic receptors.     

Effect of BRL-35135 on LTB4-induced guinea pig eosinophil chemotaxis

The effect of an atypical -adrenoceptor agonist, BRL-35135 on leukotriene B₄-induced-guinea pig eosinophil chemotaxis was studied. BRL-35135 and SC-41930 (leukotriene B₄-antagonist) inhibited the chemotaxis in a concentration-dependent manner (IC₅₀=9.0×10^–6 and 2.6×10^–7 M, respectively). However, isoproterenol, fenoterol and another atypical -agonist, BRL-37344 had no effects. The inhibitory effect of BRL-35135 was not affected by (±)-propranolol (10^–4 M). In contrast, the nonselective -adrenoceptor antagonist, (–)-alprenolol (10^–4 M) dextrally shifted the inhibitory curve of BRL-35135. The response to BRL-35135 was antagonized in a competitive manner by (–)-alprenolol, with the slope of the Schild plot close to unity, and a pA ₂ value of 5.62. These findings suggest that guinea pig eosinophils possess an atypical receptor, which differs from either ₁, ₂ or atypical -adrenoceptor on guinea pig ileum, and through which eosinophil chemotaxis can be modulated by BRL-35135.     

Stimulation of release of prostaglandins and thromboxanes from isolated guinea pig lung cells by Bradykinin,f-Met-Leu-Phe,phorbol myristate,ionophore a23187, and leukotrienes

Guinea pig lungs were perfused for 15 min with a solution of protease type VII (0.05 mg/ml) and dispersed to yield a suspension of morphologically intact and metabolically active lung cells (over 250 × 10⁶ cells per animal). The viability of these cells assessed with the Trypan blue exclusion technique was more than 80%. Treatment of these mixed cells (1 × 10⁶ cells/ml) with chemicals such as phorbol myristate acetate (1 × 10^–9 to 1 × 10^–7 M), f-Met-Leu-Phe (5 × 10^–6 to 1 x 10^–4 M), and the calcium ionophore A23187 (3.1 × 10^–7 to 2.5 × 10^–6 M), and with autacoids such as bradykinin (1 × 10^–5 to 1 × 10^–4 M), leukotriene B₄ (1 × 10^–13 to 1 × 10^–7 M), and leukotriene D₄ (1 × 10^–10 to 1 × 10^–7 M) stimulated to a variable degree the release of prostaglandin E₂ and thromboxane B₂ (measured with a novel enzyme immunoassay). It is suggested that icosanoid release from the lungs is the result of direct chemical or hormonal stimulation of the cells and not a consequence of vascular changes. Studies are in progress to purify lung cell populations and characterize the cells responsible for the release of these icosanoids.This work was supported by the Medical Research Council of Canada (MT-7143).     

Effect of glucagon on gastric acid secretion by the isolated fundus from immature rats

The effect of glucagon was studied on the isolated gastric fundus from immature rats in comparison with histamine. Glucagon (10^–7–3×10^–6 M) caused a concentration-dependent increase in acid output, being approximately 25 fold more potent than histamine (ED₅₀ values were 6.38×10^–7 M and 2.42×10^–5 M for glucagon and histamine, respectively). These compounds, however, did not differ in regard to the maximum response. The stimulatory effect of glucagon was not enhanced by pretreatment with 3×10^–8 M forskolin or 10^–7 M ICI 63197, a phosphodiesterase (PD) inhibitor. Conversely, both forskolin and ICI 63197 shifted to the left the concentration-response curve to histamine. The increase in acid secretion by glucagon was reduced by PGE₁ (10^–5 M) and PGE₂ (10^–5 M) but only PGE₂ inhibited the response to histamine. From these data it can be concluded that glucagon stimulated acid production in the stomach from immature rats, and this effect does not seem to involve the same adenylate cyclase activated by histamine.     

The effect of RG 12525 on leukotriene D4-mediated pulmonary responses in guinea pigs

RG 12525 (5-(2-[4-quinolin-2-yl)methoxy] phenoxymethyl)benzyl tetrazole) is under investigation as a specific inhibitor of leukotriene D₄ (LTD₄). The present studies examine the effect of orally administered RG 12525 on LTD₄ mediated pulmonary responses in three separate guinea pig models. The compound inhibited antigen-induced mortality in the systemic anaphylaxis model with an ED₅₀ (95% confidence interval)=2.2 (0.8–6.4) mg/kg. In this model, the activity half-life of RG 12525 was shown to be 6.5 hours and the compound offered significant protection within 15 minutes of administration. RG 12525 also protected against LTD₄-induced bronchoconstriction in a model measuring changes in pulmonary function with an ED₅₀=0.6 (0.4–1.0) mg/kg. The same level of activity was observed in a similar model which monitored changes in pulmonary function in response to exogenous antigen in actively-sensitized guinea pigs. Together, these data indicate that RG 12525 is a potent, orally active LTD₄ antagonist which possesses the requisite profile for potential clinical development.     

The effects of food additives and aflatoxin B1 on histamine release from human mast cells

The effects of the food additives tartrazine, biphenyl, sorbic acid and the mycotoxin contaminant aflatoxin B₁ were studied in mechanically isolated human adenoidal mast cells. Tartrazine inhibited the spontaneous histamine release in the concentration range of 10^–9 to 10^–5 M and the concanavalin A (Con A)-induced histamine release dose-dependently at 10^–11–10^–5 M [10.3%–31.6%]. Biphenyl [10^–9–10^–6 M] neither influenced the spontaneous nor the stimulated histamine release. Sorbic acid [10^–7–10^–4 M] slightly inhibited the Con A-induced release at the highest concentration tested. Aflatoxin B₁ [10^–10–10^–7 M] did not influence mediator release after a preincubation time of 5 min. Extension of the preincubation period inhibited the histamine release slightly. In summary, none of the tested substances enhanced histamine release from human adenoidal mast cells. Tartrazine even had an inhibitory effect.