Secretogogue responses of leukotriene C4, D4: Comparison of potency in canine trachea

By the use of close arterial injection of leukotrienes into the circulation supplying the upper cervical canine trachea, it has been possible to assess the secretogogue effects of leukotriene C₄, and D₄ on mucus secretion. Both LTC₄ and LTD₄ increased mucus secretion over baseline levels by a statistically significant level (p = < 0.05). LTD₄ was more potent than C₄ with relative potencies of 2500, 320, 630, and 500 based on hillock formation (a measure of secretion) at 1, 2, 3, and 4 minutes after injection. The overall difference in potency in this animal model of mucus production was LTD₄ > C₄ by 1000-fold.     

Rapid invivo metabolism of Leukotriene C3 in the monkey, Macacairus

[5,6,8,9,11,12-³H₆] Leukotriene C₃ (5 μCi) was injected through a catheter into the right atrium of an anesthetized male monkey. Blood samples were drawn from the aorta via a second catheter. The concentration of tritium in blood decreased from 100 nCi/ml after 5 sec to 1 nCi/ml 15 min after injection, suggesting that leukotriene C₃ was rapidly eliminated from the circulation. Chromatographic analyses of radioactive material in blood collected before recirculation had occurred (15 sec after injection) demonstrated that 40% of the radioactive material had been converted into two less polar metabolites. These products had the same chromatographic properties as leukotrienes D₃ and E₃, respectively. The results indicate that leukotriene C₃ is rapidly transformed by monkey lung $\text{in}\text{vivo}$. Two minutes after injection, the component corresponding to leukotriene E₃ was the predominating metabolite in blood.     

Evidence for phospholipid in plasma membrane penicillinase of Bacilluslicheniformis749C

The plasma membrane-bound penicillinase of $\text{Bacillus}$$\text{licheniformis}$$\text{749}\text{C}$ has been purified. Amino acid analysis showed no significant differences in composition between the enzyme and exopenicillinase. Enzyme purified from cultures containing H₃³³PO₄ or [³H]-glycerol contained ³³P or [³H]-glycerol activity and treatment with 8 M urea, 0.2% sodium dodecyl sulfate at 80° C did not remove the ³H-activity from the enzyme protein. Trypsin readily cleaved the glycerol-containing moiety from the enzyme protein, forming enzyme with molecular weight and heat stability like that of the exoenzyme. Phospholipase D and C also produced enzyme resembling the exo-form.     

Effects of leukotrienes C4 and D4 on glycoprotein and lysozyme secretion by human bronchial mucosa

The effects of leukotrienes C₄ (LTC₄) and D₄ (LTD₄) on the secretion by human bronchial mucosa of [¹⁴C]glucosamine-labeled, trichloro-acetic acid/phosphotungstic acid-precipitable glycoprotein and lysozyme were evaluated . LTC₄ and LTD₄, in the concentration range of 0.16 to 1600 nM, induced a dose-related increase in the release of radiolabeled glycoprotein, but not of lysozyme. This secretagogue effect was selective for high molecular weight glycoproteins of about 2–5 × 10⁶ daltons, and the median effective concentrations (EC₅₀ of LTC₄ of 9.4 × 10⁻⁹ M and of LTD₄ of 2.44 × 10⁻⁸ M, indicate that these leukotrienes are approximately 100-fold more potent than the cholinergic agonist methacholine. Incubation of [¹⁴C]glucosamine-labeled bronchial mucosal explants with LTC₄ or LTD₄ for six sequential 15-min periods revealed a rapid, progressive decrement in glycoprotein release, compatible with stimulatory action on secretion rather than augmentation of the rate of glycoprotein synthesis. This interpretation is also consistent with the finding that the specific activity (ratio of bound radiolabel: protein content) of the macromolecular glycoprotein secreted by the explants is not changed with stimulation of release by the leukotrienes. Based upon the activity of synthetic leukotriene analogs, the specific C-6 chirality of the sulfidopeptide of LTD₄, the presence of a hydroxyl at C-5 and the presence of eiconsanoid carbons 9–20 were no importance for secretagogue activity. These findings contrast with the stereochemical requirements for the spasmogenic response to sulfidopeptide leukotrienes and suggest that leukotriene-induced secretion is not likely to be mediated via a specific receptor.     

Leukotriene A4-induced bronchoconstriction in the guinea pig in vivo

Administration of leukotriene A₄ (0.03 - 0.3 μg kg^?1 i.v.) to anesthetized spontaneously breathing guinea pigs produced pronounced changes in pulmonary resistance, dynamic compliance and blood pressure. The pulmonary responses were unaffected either by pretreatment with indomethacin or following desensitization to leukotriene B₄ but were significantly attenuated by the leukotriene D₄ receptor antagonist, FPL-55712. Following administration of leukotriene A₄ increased levels of leukotriene C₄-immunoreactive material were determined in the plasma and neutrophil accumulation was observed in the lung. It was concluded that leukotriene A₄ induced bronchoconstriction in the guinea pig either by acting directly on the leukotriene D₄ receptor site or more probably through efficient metabolism in the lung to peptido-lipid leukotrienes which in turn exerted direct bronchoconstrictor actions.     

The modulatory effects of leukotrienes C4 and D4 on methacholine- and substance P-induced salivary secretion in rats

Although certain prostaglandins have been found to be inhibitory to nerve-evoked salivary flow, little is known of the effects the leukotrienes on salivary secretion. It was the purpose of this investigation to examine the effects of leukotrienes C₄ (LTC₄) and D₄ (LtD₄) on salivary secretion in the rat, using methacholine or substance P to induce basal secretion, and to test whether or not the observed effects of these eicosanoids were receptor-mediated by using the leukotriene receptor blocker FPL-55712.Methacholine (3 × 10⁻⁴ M), or substance P (1 × 10⁻⁶ M) was infused intra-arterially to stimulate secretion and saliva was collected separately from the parotid gland and the submandibular gland of anesthetized rats. LTC₄ and LTD₄ (each at 1 × 10⁻⁹ to 1 × 10⁻⁶ M) were found to reduce methacholine- and substance P-induced salivary flow in a dose-related manner. Salivary protein concentration and amylase activity were not significantly altered by the leukotrienes; however, arginine-esterase activity, stimulated by substance P, was increased by both leukotrienes. FPL-55712 (1 × 10⁻⁸ M) was shown to reduced the inhibitory effects of LTC₄ and LTD₄, suggesting the involvement of leukotriene receptors for these agents in their action.     

OKY-1581: A selective inhibitor of thromboxane synthesis invivo and invitro

OKY-1581 is an effective inhibitor of thromboxane synthesis $\text{in}$$\text{vivo}$ and $\text{in}$$\text{vitro}$. The generation of thromboxane B₂ (TxB₂), prostaglandin E (PGE) and prostaglandin F (PGF) was measured following clotting and during platelet aggregation induced by collagen. The presence of OKY 1581 either $\text{in}$$\text{vivo}$ or $\text{in}$$\text{vitro}$ caused a reduction in TxB₂ generation during clotting and platelet aggregation with a concomitant increase in PGE and PGF. The effect could be observed two hours after oral or subcutaneous administration of 5 to 100 mg per rabbit and lasted for 24 to 48 hours. The reduction in TxB₂ was not accompanied by an inhibition of clotting or platelet aggregation. OKY-1581 appears to be a suitable agent for studying the role of TxB₂ in atherosclerosis.     

The synthesis of 13-cis-dl-erythro-15, 16-dihydroxyprostaglandins

Both pairs of $\text{dl}$-ll-desoxy- and $\text{dl}$-13-$\text{cis}$-$\text{erythro}$-15, 16-dihydroxyprostaglandins have been synthesized via 1,4-conjugate additions of an appropriately functionalized $\text{cis}$-vinyl cuprate to the requisite cyclopentenone. These prostaglandin analogs are considerably less potent than PGE₂ as gastric secretion inhibitors or as bronchodilators.     

Vasoconstrictor effects of leukotrienes C4 and D4 in the feline mesenteric vascular bed

The effects of leukotriene C₄ (LTC₄) and leukotriene D₄ (LTD₄) in the feline mesenteric vascular bed were investigated under conditions of controlled blood flow so that changes in perfusion pressure directly reflect changes in vascular resistance. Intra-arterial injections of LTC₄ and LTD₄ (0.3–3.0 μg) increased perfusion pressure in a dose-related fashion. Vasoconstrictor responses to LTC₄ and LTD₄ were similar to norepinephrine (NE) whereas mesenteric vasoconstrictor response to the thromboxane analog, U46619, was markedly greater than were responses to LTC₄ and LTD₄. Meclofenamate in a dose that greatly attenuated the systemic depressor response to arachidonic acid was without effect on vasoconstrictor responses to LTC₄ and LTD₄, NE and U46619 in the mesenteric vascular bed. The present data show that LTC₄ and LTD₄ possess significant vasoconstrictor activity in the feline mesenteric vascular bed. In addition, the present data suggest that products of the cyclooxygenase pathway do not mediate vasoconstrictor responses to LTC₄ and LTD₄ in the intestinal circulation of the cat.     

Leukotriene D4 and E4 formation by plasma membrane bound enzymes

The homogenate of rat basophilic leukemia cells produces both the dihydroxy-leukotrienes and the peptido-leukotrienes (LT) C₄, D₄ and E₄. The enzymes responsible for the formation of LTA₄ and LTB₄ are in the soluble fraction while the enzymes for LTC₄, LTD₄ and LTE₄ are particulate (10, 000 × g pellet). Centrifugation of the 10, 000 × g pellet over a sucrose gradient resulted in two subfractions, a membrane fraction and a pellet (sucrose pellet.) The fractions were incubated with LTC₄, and the products were identified by bioassay, HPLC and UV spectra. The membrane fraction contained the enzymes γ-glutamyl transpeptidase and amino peptidase which convert LTC₄ to LTD₄ and LTD₄ to LTE₄, respectively. When incubated with LTC₄, the membrane fraction showed a dose dependent formation of LTD₄ and a time course which reached a plateau at 30 to 45 minutes. Addition of serine borate blocked the formation of LTD₄, and cysteine blocked LTE₄. We conclude that the γ-glutamyl transpeptidase and the amino peptidase which produce LTD₄ and LTE₄ respectively are plasma membrane bound.     

Possible involvement of thromboxane in bronchoconstrictive and hypertensive effects of LTC4 and LTD4 in guinea pigs

The actions of leukotriene (LT) C₄ and D₄ on the systemic arterial pressure and the insufflation pressure in guinea pigs and rabbits were examined. In guinea pigs, 0.3 – 3 nmole/kg of LTC₄ and 0.1 – 1.0 nmole/kg of LTD₄ administrated from left jugular vein caused dose-dependent increase of the airway resistance measured by the Konzett-Rössler method and a triphasic blood pressure response; an initial hypotension, a secondary hypertension and a third long-lasting hypotension. All of the hypertensive phase and 100 – 150% of the increase of the airway resistance by LTC₄ and LTD₄ were inhibited by a selective thromboxane synthetase inhibitor, OKY-1581 (10 mg/kg, i.v.) and only the hypertension was observed. Indomethacin (10 mg/kg, i.p.) also inhibited not only the airway resistance increase, but also the prolonged hypotension by LTC₄ and shortened the duration of the hypotension by LTD₄. It is suggested that thromboxane might be involved in bronchoconstriction and hypertensive effects by LTC₄ and LTD₄ and that hypotensive prostaglandin might be involved in the hypotensive phase after LTC₄ and LTD₄. In rabbits, the increse of the airway resistance by LTC₄ and LTD₄ (upto 100 nmole/kg, i.v.) was negligible and only the hypotension was observed.     

Invitro metabolism of aflatoxin B1 by rat liver nuclei

Rat liver nuclei were used to study the metabolism of aflatoxin B₁ (AFB₁). Nuclei were found to metabolize AFB₁ to aflatoxins M₁(AFM₁), Q₁(AFQ₁), and P₁(AFP₁); the formation of AFP₁ was relatively negligible. AFM₁ was preferentially formed when rats were pretreated with 3-methylcholanthrene (MC), whereas phenobarbital (PB) pretreatment enhanced the formation of both AFM₁ and AFQ₁. PB pretreatment also resulted in increased binding of AFB₁ metabolites to DNA and to other macromolecules in the nucleus. Following PB and MC pretreatment, induction specificities of the nuclear metabolism of AFB₁ were similar to those of the microsomal metabolism.     

Specificity of receptors for leukotrienes A4, B4, C4, D4, E4 and histamine on the guinea-pig lung parenchyma. Effect of FPL-55712 and desensitization of the myotropic activity

The novel metabolites of arachidonic acid, leukotriene (LT) A₄, B₄, C₄, D₄ and E₄ have potent myotropic activity on guinea-pig lung parenchymal strip . The receptors responsible for their action were characterized using desensitization experiments and the selective SRS-A antagonist, FPL-55712. During the continuous infusion of LTB₄, the tissues became desensitized to LTB₄ but were still responsive to histamine, LTA₄, LTC₄, LTD₄ and LTE₄. When LTD₄ was infused continuously, the lung strips contracted to LTB₄ and histamine but were no longer responsive to LTA₄, LTC₄, LTD₄ and LTE₄. Furthermore, FPL-55712 (10 ng ml⁻¹− 10 ug ml⁻¹) produced dose-dependent inhibitions of LTA₄, LTC₄, LTD₄ and LTE₄ without inhibiting the contraction to LTB₄ and histamine. On the basis of these results, it appears that the guinea-pig lung parenchyma may have one type of receptor for LTB₄ and another for LTD₄; LTA₄, LTC₄ and LTE₄ probably act on the LTD₄ receptor.     

Bronchoconstrictor effects of leukotriene B4 in the guinea pig in vivo

Administration of leukotriene B₄ (LTB₄) to anesthetized spontaneously breathing guinea pigs either by the intravenous or aerosol route produced pronounced changes in pulmonary resistance and dynamic compliance. The effects were short lived and were completely abolished by pretreatment of animals with the cyclooxygenase inhibitor indomethacin. Histological examination of lungs following aerosol administration of LTB₄ showed a pronounced neutrophil infiltration. These results confirm previous $\text{in vitro}$ studies in which LTB₄ was shown to produce contractions on guinea pig parenchymal strips indirectly by releasing thromboxane A₂.     

The effect of leucotriene C4 and D4 on cutaneous blood flow in humans

Using a laser-Doppler-flowmeter the microvascular response to LTC₄ and LTD₄ was measured. Intradermal injection of 1 Ug LTC₄ and LTD₄ caused an increase in the microvascular cutaneous bloodflow. The increase in flow was equal to that caused by histamine in equimolar amounts. Blocking the triple-response did not change the response. The values measured after injection of histamine and leucotrienes were about 10–15 times the values found in undisturbed skin and represents probably a maximally dilated vascular bed. Injection of the leucotrienes caused a slight sensation of pain.     

Metabolism of two PGF2α analogues in primates: 15(S)-15-methyl-Δ4-cis-PGF1α and 16,16-dimethyl-Δ4-cis-PGF1α

The metabolism of the prostaglandin F_2α analogues, 15-methyl-Δ⁴-$\text{cis}$-PGF_1α and 16,16-dimethyl-Δ⁴-$\text{cis}$-PGF_1α, has been investigated in the cynomolgus monkey and the human female. The two analogues, tritium labelled in the 9β-position, were administered by intramuscular injections into the monkeys and by subcutaneous injections into the human. Excretion of tritium labelled products were followed in urine (in both species) and feces (in monkeys only) and several metabolites were identified by GC/MS. The analogues were found to be resistant to the 15-hydroxy dehydrogenase and furthermore the degradation by β-oxidation was delayed. About 13% of the given dose of 15-methyl-Δ⁴-$\text{cis}$-PGF_1α was excreted unchanged into urine and feces from the monkey. The corresponding figure for 16,16-dimethyl-Δ⁴-$\text{cis}$-PGF_1α was about 20%. In addition, a large part of the metabolites had the carbon skeleton intact and were only metabolized by ω-oxidation. The relative resistance to degradation of these two analogues is likely to be the basis for their prolonged pharmacological activity.     

Cyclooxygenase mediated airway response to leukotriene D4 in the cat

The effects of leukotriene D₄ (LTD₄) on pulmonary mechanics were investigated in anesthetized, paralyzed cats under conditions of controlled ventilation. Intravenous injections of LTD₄ in doses of 3, 10, and 30 μg caused significant increases in transpulmonary pressure (P_TP) and lung resistance (R_L) while decreasing dynamic compliance (C_dyn). LTD₄ also increased systemic arterial pressure (P_A0). The changes in P_TP, R_L, and C_dyn in response to LTD₄ were blocked by sodium meclofenamate, a cyclooxygenase inhibitor. However, there was no significant change in the increase in P_A0 following cyclooxygenase blockade. U 46619, a thromboxane mimic, was 30 to 100 times more potent than LTD₄ in increasing P_TP, R_L and decreasing C_dyn in the cat. These data show that LTD₄ has significant smooth muscle constrictor activity in central airways as well as peripheral portions of the feline lung. In addition, these data suggest that in the cat the actions of intravenously administered LTD₄ on lung mechanics are mediated by release of cyclooxygenase products while the systemic pressor effects are not dependent upon the integrity of the cyclooxygenase pathway.     

Bromobenzene and p-bromophenol toxicity and covalent binding invivo

A hepatotoxic dose of bromobenzene (3 mmoles/kg) decreases hepatic glutathione concentration in rats by approximately 80% within 5 hr following ip injection. A major bromobenzene metabolite, p-bromophenol at a similar dose did not significantly alter hepatic glutathione levels compared to controls. Twenty four hr after administration, serum glutamate pyruvate transaminase (SGPT) levels were significantly increased by bromobenzene but not by p-bromophenol. After ¹⁴C-bromobenzene administration, a significant amount of covalently bound radiolabel was detected in liver, kidney and small intestine. A small amount of covalently bound radiolabel was also detected in the lung. After a similar dose of ¹⁴C-bromophenol, covalently bound radiolabel was found in liver (62% of the amount detected with ¹⁴C-bromobenzene) and smaller amounts were detected in kidney, small intestine and lung. These data are consistent with the view that the hepatotoxity and glutathione depleting ability of bromobenzene are mediated mainly by bromobenzene-3, 4-oxide rather than by chemically reactive metabolites of p-bromophenol derived from bromobenzene. Covalently bound radiolabel from ¹⁴C-bromobenzene, however, may be derived from both bromobenzene-3, 4-oxide and the nontoxic reactive metabolites of p-bromophenol.     

Synthesis and biological activities of leukotriene F4 and leukotriene F4 sulfone

Leukotriene F₄ (LTF₄ and LTF₄ sulfone have been synthesized and their biological activities determined in the guinea pig. LFT₄ displayed comparable activity to LTD₄ on guinea pig trachea and parenchyma but was less active on the ileum. When injected intravenously into the guinea pig, LTF₄ induced a bronchoconstriction (ED₅₀ 16 μg Kg⁻¹) which was blocked by indomethacin and FPL-55712 and was 50–100 X less potent than LTD₄ in this assay. LTF₄ sulfone was approximately 2–5 times less active than LTF₄ and . When injected into guinea pig skin with PGE₂ (100 ng); LTF₄ and LTF₄ sulfone (10–1000 ng) induced changes in vascular permeability. The order of potency in this assay was LTE₄ sulfone = LTD₄ = LTD₄ sulfone > LTE₄ > LTF₄ = LTF₄ sulfone.     

Pulmonary microcirculatory responses to leukotrienes B4, C4 and D4 in sheep

The pulmonary microvascular responses to leukotrienes B₄, C₄ and D₄ (total dosage of 4 μg/kg i.v.) were examined in acutely-prepared halothane anesthetized and awake sheep prepared with lung lymp fistulas. In anesthetized as well as unanesthetized sheep, LTB₄ caused a marked and transient decrease in the circulating leukocyte count. Pulmonary transvascular protein clearance (pulmonary lymph flow x lymph-to-plasma protein concentration ratio) increased transiently in awake sheep, suggesting a small increase in pulmonary vascular permeability. The mean pulmonary artery pressure (P ) also increased. In the acutely-prepared sheep, the LTB₄-induced pulmonary hemodynamic and lymph flow responses were damped. Leukotriene C₄ increased P to a greater extent in awake sheep than in anesthetized sheep, but did not significantly affect the pulmonary lymph flow rate (Q̇_lym) and lmph-to-plasma protein concentration (L/P) ration in either group. LTD₄ increased P and Q̇_lymp in both acute and awake sheep; Q̇_lym increased without a significant change in the L/P ratio. The LTD₄-induced rise in P occurred in association with an increase in plasma thromboxane B₂ (Txb₂) cocentration. The relativity small increase in Q̇_lym with LTD₄ suggests that the increase in the transvascular fluid filtration rate is the result of a rise in the pulmonary capillary hydrostatic pressure. In conclusion, LTB₄ induces a marked neutropenia, pulmonary hypertension, and may transiently increase lung vascular permeability. Both LTC₄ and LTD₄ cause a similar degree of pulmonary hypertension in awake sheep, but had different lymph flow responses which may be due to pulmonary vasoconstriction at different sites, i.e. greather pre-capillary constriction with LTC₄ because Q̇_lym did not change and greater post-capillary constriction with LTD₄ because Q̇ increased with the same rise in P .