Inhibition of the low km cyclic AMP phosphodiesterase in intact canine trachealis by SK&F 94836: mechanical and biochemical responses.

The mechanical and biochemical responses of the canine trachealis to SK&F 94836 [2-cyano-1-methyl-3-[4-(4-methyl-6-oxo- 1,4,5,6-tetrahydropyridazine-3-yl)phenyl]guanidine], a selective inhibitor (ki = 1-3 microM) of the low km cyclic AMP (cAMP) phosphodiesterase, were assessed. Time course studies indicated that SK&F 94836-induced relaxation of trachealis strips contracted with 0.1 microM methacholine was accompanied by an activation of cAMP-dependent protein kinase (cAMP-PK). In subsequent experiments, trachealis strips were contracted with three concentrations of methacholine (0.1, 1.0 or 3.0 microM) or two concentrations of histamine (10 or 300 microM) before being relaxed by the cumulative addition of SK&F 94836. The relaxant response to SK&F 94836 (EC50 = 1-10 microM) decreased progressively as tissues were contracted with higher concentrations of methacholine. In parallel with its inhibitory effect on SK&F 94836-induced relaxation, methacholine suppressed the ability of SK&F 94836 to activate cAMP-PK. Interestingly, the inhibition of cAMP-PK activity was not accompanied by a significant inhibition of SK&F 94836-stimulated cAMP accumulation. Unlike the results with methacholine, the concentration of histamine used to contract tissues had no effect on SK&F 94836-induced relaxation or cAMP-PK activation. To determine the effect of SK&F 94836 on the mechanical and biochemical responses to the beta adrenoceptor agonist isoproterenol, tissues were first contracted with 3.0 microM methacholine and then incubated with 0, 0.3, 3.0 or 30 microM SK&F 94836 before being relaxed by the cumulative addition of isoproterenol. In these experiments, SK&F 94836 potentiated isoproterenol-induced relaxation, cAMP accumulation and cAMP-PK activation in a concentration-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Is the guinea pig trachea a good in vitro model of human large and central airways? Comparison on leukotriene-, methacholine-, histamine- and antigen-induced contractions.

To analyze comprehensively the relevance of the guinea pig trachea as a model of human large and central airways, the contractile effects of the peptidoleukotrienes (LTs), histamine, methacholine and antigen on guinea pig and human airways were compared in vitro. Although some differences were apparent, LTC4, LTD4, LTE4, histamine and methacholine had comparable EC50 values and elicited similar maximal responses in both guinea pig trachea and human bronchus (second-seventh generation). In the presence of l-serine borate (45 mM), LTC4 concentration-response curves were shifted significantly to the left in guinea pig trachea but not in human bronchus. Furthermore, the LT receptor antagonists (SK&F 102922 and FPL 55712) had similar potencies against LTC4- and LTD4-induced contractions of human bronchus, whereas, in the guinea pig trachea, they were much more effective antagonists of responses produced by LTD4 than those elicited by LTC4. These results provide further evidence that, unlike in human bronchus, LTC4- and LTD4-induced contractions in the guinea pig trachea are mediated via distinct leukotriene receptors. Ovalbumin-induced contractions of actively sensitized guinea pig tracheae exhibited the same profile as anti-immunoglobulin E-induced contractions of the passively sensitized human bronchus. Furthermore, antigen-induced contractions in both the guinea pig trachea and human bronchus possessed a similar sensitivity to inhibition by mepyramine (10 microM) and the LT antagonists (10 microM), added either alone or in combination. These results indicate that the isolated guinea pig trachea is a suitable model of human large and central airways.

Pharmacologic profile of SK&F 104353: a novel, potent and selective peptidoleukotriene receptor antagonist in guinea pig and human airways.

In this report, we describe the in vitro and in vivo pharmacologic profile of 2(S)-hydroxy-3(R)-[(2-carboxyethyl)thio]-3-[2-(8-phenyloctyl)pheny l]- propanoic acid (SK&F 104353) in guinea pig and human airways. In the isolated guinea pig trachea, SK&F 104353 was a potent, competitive antagonist of leukotriene (LT) D4-induced contractions (pA2 = 8.6). In contrast, SK&F 104353 produced little effect on LTC4 concentration-response curves under conditions where the bioconversion of LTC4 to LTD4 was inhibited. LTE4-induced contractions in guinea pig trachea were sensitive to inhibition by SK&F 104353 (pKB greater than 8.9). SK&F 104353 (10 microM) had no intrinsic contractile activity and was without effect on contractions produced by KCl, histamine, prostaglandin D2, platelet-activating factor or U-44069 in guinea pig trachea. Furthermore, unlike other purported LT antagonists, LT 171883 and FPL 55712, SK&F 104353 (30 microM) did not inhibit cyclic nucleotide phosphodiesterase activity measured in homogenates from canine tracheal smooth muscle. In the isolated human bronchus, SK&F 104353 produced concentration-dependent rightward shifts in LTD4 concentration-response curves and, unlike in guinea pig trachea, was an effective antagonist of LTC4-induced contractions with a pKB of 8.0 to 8.4. This provides further evidence that, in contrast to guinea pig airways, responses produced by LTC4 and LTD4 in human bronchus appear to be mediated via the same LT receptor population. SK&F 104353 was also an effective antagonist of LTE4-induced responses in human bronchus (pKB greater than 8.2).(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis and structure-activity relationship studies of a series of 5-aryl-4,6-dithianonanedioic acids and related compounds: a novel class of leukotriene antagonists.

A series of 5-alkynyl- and 5-aryl-4,6-dithianonanedioic acids and related compounds has been prepared for evaluation of leukotriene antagonist activity. The alkynyl compounds were prepared by thioacetal exchange from the corresponding acetylenic acetals. The aryl derivatives were synthesized from the appropriate benzaldehydes, most of which were prepared by one of three general routes: Meyers' oxazolin method, a palladium coupling procedure, and a hydroxybenzaldehyde alkylation. The analogues were examined in vitro for their ability to antagonize an LTD4-induced contraction of isolated guinea pig tracheal smooth muscle and to compete with [3H]LTD4 for receptor sites on guinea pig lung membrane. A number of structure-activity relationships have emerged from this study. There is an optimal chain length of 10-12 atoms (or its equivalent) in the lipid tail and two methylenes in the polar region. In the aromatic series, the ortho- and meta-substituted compounds have comparable activity, whereas the para derivatives are inactive. Substitution in the aromatic ring and lipid tail is generally well tolerated, with the terminal phenyl (6) and acetylene (33) analogues having especially good activity. Conformational restriction of either the polar region or lipid tail produced compounds devoid of activity. A number of selected analogues were also evaluated in vivo as antagonists of LTD4-induced bronchoconstriction in the guinea pig. The data established these compounds as a novel class of leukotriene antagonists with potential utility for the treatment of asthma and other immediate hypersensitivity diseases.

Effects of SK&F 93944 (Temelastine), a potent histamine H1-receptor antagonist in pharmacologic and antigen-induced bronchoconstriction.

SK&F 93944, a previously reported non-sedating histamine H1-receptor antagonist, was evaluated for its ability to block pharmacologic-and antigen-induced bronchoconstriction. In the isolated guinea pig trachea, SK&F 93944 (10(-9)-10(-7) M) produced a concentration-dependent inhibition of contractions produced by histamine (pKB = 9.5). Another histamine antagonist, mepyramine (10(-8)-10(-6) M), was less potent (pKB = 8.5). SK&F 93944 (10(-8), 10(-7) M) also significantly depressed the rapid initial phase of antigen-induced contraction of the guinea pig trachea from animals actively sensitized to ovalbumin, while having no effect on the later, more protracted phase of the contractile response. In anesthetized mongrel dogs, selective inhibition of histamine (20 micrograms/kg, i.v.)-induced bronchoconstriction was achieved by SK&F 93944 in doses as low as 30 micrograms/kg, i.v. Terfenadine, a purportedly selective histamine H1-receptor antagonist, blocked both histamine and acetylcholine-induced bronchoconstriction at doses similar to SK&F 93944. In mongrel dogs natively allergic to Ascaris suum antigen, pretreatment with aerosols of either SK&F 93944 or mepyramine (1%; 50 tidal breaths) significantly inhibited bronchospasm elicited by increasing aerosol concentrations of antigen. Thus, SK&F 93944 is a highly potent, selective histamine H1-receptor antagonist which is efficacious vs. pharmacologic and antigen-induced bronchoconstriction.

Differential rank order of potency for antagonism of LTC4- and LTD4-induced contractions of guinea pig trachea.

In the presence of 1-serine-borate complex (SB), employed to prevent LTC4 to LTD4 metabolism, desamino-2-nor-LTE, [4-hydroxy-5-[(2,2-carboxyethyl)thio]-6-nonadecenoic acid; DN-LTE1] equally antagonized the contractions elicited by LTC4 (-log KB = 5.8 +/- 0.2) and LTD4 (-log KB = 5.5 +/- 0.4) on the isolated guinea pig trachea. In contrast, FPL 55712 preferentially antagonized the LTD4-induced contractions in the presence of SB with a -log KB = 6.2 +/- 0.2, whereas only weak antagonism of the LTC4-induced contractions was observed (-log KB = 4.9 +/- 0.2). Thus, the rank order of potency for antagonizing the LTC4-induced contractions was DN-LTE1 greater than FPL 55712, whereas the rank order for antagonizing LTD4 was FPL 55712 greater than DN-LTE1. On tissues pretreated with 10 microM FPL 55712 without SB, 10 microM DN-LTE1 antagonized the contractions elicited by LTC4 but not those elicited by LTD4. Thus, based upon the differential rank order of antagonism of the LTC4- and LTD4-induced contractions by these LT antagonists under two different experimental conditions, these results imply the existence of multiple LT receptors in the guinea pig trachea.

Effect of calcium antagonists on the biosynthesis and contractile effects of peptidoleukotrienes in rhesus 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)

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.

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.

Effect of calcium antagonists on leukotriene D4-induced contractions of the guinea-pig trachea and lung parenchyma.

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.

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.