Barry M. Weichman

The effect of synthetic leukotrienes on tracheal microvascular permeability

The effect of synthetic leukotrienes (LT) C4, D4 and E4 on the permeability of the airway microvasculature to plasma albumin was quantitatively evaluated using an in situ guinea pig tracheal model. Vascular permeability was measured as extravascular albumin content by employing 125I-bovine serum albumin and, in order to correct for blood volume, 51Cr-erythrocytes were used. Intratracheal injection of synthetic LTC4, LTD4 and LTE4 (0.1-1000 ng) produced dose-dependent increases in tracheal extravascular albumin content. The leukotrienes were approximately 100-1000 fold more potent than histamine, although histamine did produce a greater maximal increase in extravascular albumin than the leukotrienes. Methacholine did not increase extravascular albumin content. The microvascular permeability effect of LTD4 was antagonized by FPL 55712 but not by mepyramine; conversely, the effect of histamine was antagonized by mepyramine and not by FPL 55712. Additionally, indomethacin did not alter the LTD4-induced increases in tracheal vascular permeability. These results suggest that the effect of LTD4 on tracheal microvascular permeability is directly mediated and is not the indirect result of cholinergic stimulation, histamine release or de novo synthesis of cyclooxygenase products.

Contraction of guinea pig uterus by synthetic leukotrienes

Synthetic leukotrienes (LT) C4 and D4 elicited concentration-dependent contractions of the guinea pig uterus between 10(-8)-10(-6)M, whereas LTE4 appeared 1000-fold weaker. The potencies of LTC4 and LTD4 were similar to that of acetylcholine and PGF2 alpha but weaker than that of PGE2. The maximal contractions elicited by LTC4 and LTD4 were 66.0 +/- 2.1% and 63.8 +/- 4.6% that elicited by acetylcholine. FPL 55712 (10(-5)M) antagonized the uterine contractile activity of LTD4, while meclofenamic acid at 10(-5)M but not at 10(-6)M also antagonized the LTD4-induced contraction. Radioimmunoassay of the uterine tissue bathing fluid following LTD4 indicated the variable presence of low concentrations of PGE2, PGF2 alpha and TxB2. These results demonstrate that LTC4 and LTD4 possess significant uterine contractile activity, which may only partially be mediated indirectly via prostaglandin products.

Investigation of leukotriene involvement in the vasopermeability response associated with guinea pig tracheal anaphylaxis: Comparison with cutaneous anaphylaxis

A direct comparison of the role of leukotrienes in mediating the increase in microvascular permeability associated with guinea pig tracheal and cutaneous anaphylaxis was obtained by simultaneous administration of inflammatory stimuli to both trachea and ear. The SRS-A antagonist, FPL 55712, reduced the increase in tracheal extravascular albumin content evoked by LTC4, LTD4, and LTE4 but failed to significantly reduce the tracheal microvascular permeability response associated with local anaphylaxis. Moreover, the inhibitory effect of the histamine H1-receptor antagonist, mepyramine, was not augmented by the additional presence of FPL 55712. In contrast to tracheal anaphylaxis, a distinct leukotriene component was indicated in cutaneous anaphylaxis since the mepyramine-FPL 55712 combination produced a greater inhibition than mepyramine alone. These results suggest that the degree of leukotriene involvement in anaphylaxis may vary between tissues.

Effect of inhibition of thromboxane production on the leukotriene D4-mediated bronchoconstriction in the guinea pig

Leukotriene D4 (LTD4) administered intravenously to anesthetized, spontaneously breathing guinea pigs elicited decreases in dynamic lung compliance (Cdyn) and airway conductance (GAW) with a maximal response achieved at 0.5 min. Simultaneously, plasma levels of the thromboxane metabolite, TxB2, and the prostacyclin metabolite, 6-keto-PGF1 alpha, increased 10-fold over pre-LTD4 levels. Pretreatment of the guinea pigs with meclofenamic acid delayed the onset of the LTD4-induced bronchoconstriction, antagonized the magnitude of the decreases in Cdyn and GAW, and blocked the increase in plasma TxB2 and 6-keto-PGF1 alpha levels. The thromboxane synthetase inhibitor, UK 37,248, suppressed the LTD4-induced bronchoconstriction, while it completely blocked TxB2 production without significantly affecting 6-keto-PGF1 alpha. The SRS-A end organ antagonist, FPL 55712, blocked both the LTD4-induced bronchoconstriction and the production of the arachidonic acid metabolites. These results suggest that thromboxane A2 plays an important role in mediating part of the bronchoconstriction elicited by intravenously administered LTD4 in the guinea pig.

2-Nor-leukotriene analogs: Antagonists of the airway and vascular smooth muscle effects of leukotriene C4, D4 and E4

A structural analog of LTD4, 4R-hydroxy-5S-cysteinylglycyl-6Z-nonadecenoic acid (4R, 5S, 6Z-2-nor-LTD1) has been synthesized and pharmacologically characterized. It significantly antagonized the contractile action of LTD4, LTC4 and LTE4 in guinea pig airways. In addition, this compound antagonized the in vitro vasoconstrictive effects of LTD4 in the guinea pig pulmonary artery. The study of a series of structural analogs of 4R, 5S, 6Z-2-nor-LTD1 suggests that the spatial separation of the C-1 (eicosanoid) carboxyl relative to the hydroxyl is a critical determinant in LTD4 agonist/antagonist activity.

Differentiation of the mechanisms by which leukotrienes C4 and D4 elicit contraction of the guinea pig trachea.

The contractions elicited by leukotriene (LT) C4 and D4 in isolated guinea pig trachea were characterized under conditions in which LTC4 to LTD4 metabolism was blocked by the presence of 45 mM l-serine-borate complex (SB). The presence of SB caused a shift of the LTC4-concentration-response curve to the left by 7.5-fold, and blocked the bioconversion of LTC4 to LTD4 by the trachea as estimated by HPLC analysis of the LTs present in the tissue bath fluid. The potency of FPL 55712 as an antagonist of the LTC4-induced contractions in the presence of SB was 15-30-fold less than its potency as an antagonist of the LTD4-induced contractions. In contrast, another LT antagonist, SK&F 101132, equally antagonized the contractions elicited by LTC4 and LTD4 in either the presence or absence of SB. The differential antagonism of LTC4 and LTD4 implies the existence of multiple pharmacologic receptors for the LTs. The calcium channel entry blockers, nifedipine and verapamil, at concentrations as high as 10 microM, suppressed the maximal LTC4-induced contraction by no more than 20%, whereas the purported intracellular calcium antagonist, TMB-8, completely suppressed the LTC4 concentration-response curve in the presence of SB, a profile identical to that previously reported for LTD4. Thus, if multiple LT receptors exist, they appear to mobilize calcium in a qualitatively similar fashion following LT stimulation.

Propranolol potentiates leukotriene D4-induced bronchoconstriction and enhances antagonism by FPL 55712

Aerosol LTD4-induced bronchoconstriction in anesthetized, spontaneously breathing guinea pigs was potentiated by either pretreatment with propranolol or bilateral adrenalectomy, whereas bilateral vagotomy did not affect the LTD4 response. The dose-response curve describing LTD4-induced changes in dynamic lung compliance (CDYN) and pulmonary resistance (RL) [as reflective indices of bronchoconstriction] was shifted to the left by approximately 20-fold by propranolol. Against an equal degree of LTD4-induced bronchoconstriction, the leukotriene antagonist, FPL 55712, had an apparent 20-fold greater potency in propranolol-pretreated animals vis a vis saline-treated controls. The duration of action of aerosol FPL 55712 was similar in both propranolol-treated and saline-treated animals. These results demonstrate that aerosol LTD4-induced bronchoconstriction is modulated by an adrenergic compensatory bronchodilator mechanism that is apparently dependent upon the adrenals and independent of vagal influences. Inhibition of the effect of this reflex with propranolol also enhances the apparent potency of an aerosol leukotriene antagonist, FPL 55712, presumably reflecting a constant LTD4 to antagonist ratio in the saline-treated and propranolol-pretreated guinea pigs.

Synthesis and LTD4-antagonist activity of desamino-2-nor-leukotriene analogs

A series of desamino-2-nor-leukotriene analogs has been prepared by the reaction of various thiols with several methyl trans-4,5-epoxy-6Z-alkenoates, followed by deprotection. The products were assessed for their ability to antagonize the LTD4-induced contraction of the isolated guinea pig trachea. Several compounds displayed potent leukotriene antagonist activity, i.e., KB values in the sub-micromolar range, while only minimally affecting basal airway tone. The most potent analog, 4-hydroxy-5-(2-carboxyethylthio)-6Z-nonadecenoic acid, antagonized both LTD4- and LTE4-induced contractions of the trachea in an apparently competitive fashion. These agents possess increased potency relative to SK&F 101132, the first leukotriene analog identified as having LT-antagonist activity. Thus, these results demonstrate that deletion of the peptide amino group can produce leukotriene analogs which have minimal intrinsic contractile activity on the isolated guinea pig trachea, yet possess potent leukotriene-antagonistic effects.

Analysis of the antagonist profile of SK&F 88046 on guinea pig trachea

SK&F 88046 preferentially antagonized the contractions elicited by the functional thromboxane (Tx) A2 mimics and structural endoperoxide analogs, U-44069 and U-46619, on the guinea pig trachea. This concentration-dependent antagonism was described by pA2 values of 7.03 against U-46619 and 6.97 against U-44069; the slopes of both Schild plots were 0.9. Whereas SK&F 88046 did not antagonize the tracheal contractions elicited by leukotriene (LT) C4 or D4, carbachol or histamine, this agent did antagonize the contractions induced by carbocyclic thromboxane A2 (CTA2) and prostaglandin (PG) F2 alpha and D2. At 1 X 10(-5)M SK&F 88046, the antagonism was described by -log KB values of 5.9 for CTA2, 5.5 for PGF2 alpha, and 6.4 for PGD2. Thus, SK&F 88046 was 3 to 20-fold more potent an antagonist of U-44069 and U-46619, suggesting that SK&F 88046 may function primarily as a thromboxane/endoperoxide antagonist on the guinea pig trachea.

Leukotriene D4 elicits a non-sustained contraction of the guinea pig trachea in calcium-free buffer

The contraction of the isolated guinea pig trachea elicited by leukotriene D4 (LTD4) in Ca2+-free buffer (containing 10(-4) M EGTA) achieved a maximum at 6-8 min and relaxed back to baseline approximately 25 min after challenge with LTD4. In contrast, LTD4 elicited a sustained contraction in the presence of 1.8 mM calcium. This sustained contraction in the presence of calcium was reproduced upon repeated LTD4 challenge, whereas in Ca2+-free buffer, only one LTD4-induced contraction could be obtained. The amplitude of the LTD4-induced contraction in Ca2+-free buffer decreased in a time-dependent manner which was also dependent upon the concentration of LTD4. At 10(-7) and 10(-6) M LTD4, small contractions (11% and 20% of control, respectively) were measured after 30 min in Ca2+-free buffer, whereas 10(-8) M LTD4 elicited a contraction at 15 min but not after 30 min in Ca2+-free buffer. Whereas washing the trachea for 5 min with LaCl3 (1.8 mM) only partially suppressed the LTD4-induced contraction in the presence of calcium, the contraction elicited by LTD4 in Ca2+-free buffer was not affected by LaCl3. The LTD4-induced contraction in Ca2+-free buffer was not affected by verapamil (10(-6) M); in contrast, the putative intracellular calcium antagonist, TMB-8 (10(-4) M), blocked the LTD4-induced contraction. These results provide evidence that the release of an intracellular calcium store plays an important role in the initiation of the LTD4-induced contraction of the guinea pig trachea. In addition, these results suggest that an extracellular calcium source may account for a small part of the LTD4-induced contraction.