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The aim of the present work was to elucidate the role of cytosolic calcium ions, [Ca 2+ ] i , in the control of arachidonic acid release and metabolism. [Ca 2+ ] i was measured in resident peritoneal rat macrophages loaded with Fura2, and compared with the release of leukotriene B 4 (LTB 4 ) and prostaglandin l 2 (PGl 2 , assayed through its hydrolysis product 6-keto-PGF 1α ). The calcium ionophore A 23187 stimulated both an increase in [Ca 2+ ] i and the release of LTB 4 and 6-keto-PGF 1α ). On the contrary, zymosan and opsonized zymosan, while stimulating eicosanoid release to an extent only slightly lower than A 23187, did not affect [Ca 2+ ] i . Lipopolysaccharide stimulated 6-keto-PGF 1α , but not LTB 4 , release, without affecting [Ca 2+ ] i . In parallel experiments, macrophages were prelabelled with [ 3 H]arachidonic acid and the release of total 3 H-products was assayed and taken as an index of phospholipase activity. A 23187, zymosan and opsonized zymosan increased the release of 3 H-products in the presence of Ca 2+ . When extracellular Ca 2+ was removed, the ionophore-induced 3 H-products release was greatly blunted, while the release induced by zymosan was actually augmented. Our data indicate that a generalized [Ca 2+ ] i increase is not necessary for arachidonic acid release and metabolism in rat peritoneal macrophages.

RBL 2H3 cells, a model for mast cell function, sensitized with rat IgE, released histamine and peptidoleukotrienes (LT) in response to rabbit anti-rat IgE in a concentration-dependent manner. The calcium ionophore, A23187 also stimulated the release of both mediators but to a greater extent. The protein kinase C activator, 12-O-tetradecanoyl phorbol-13-acetate (TPA) failed to influence mediator release when added alone, but when added with either A23187 or anti-IgE, TPA significantly enhanced the release of both histamine and LT. The effects of anti-IgE, TPA and A23187 were completely inhibited by prior addition of the protein kinase C inhibitors staurosporine and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7) but not by N-(2-guanidinoethyl)-5-isoquinoline-sulfonamide dihydrochloride (HA1004), a compound which has similar potency to H7 as an inhibitor of some protein kinases but is less potent as a protein kinase C inhibitor. Although other explanations are possible, these results support the hypothesis that the release of histamine and leukotrienes from RBL 2H3 cells resulting from the cross bridging of the IgE receptors, is dependent on activation of protein kinase C.

The effects of adenosine (A) and the nonmetabolizable adenosine analogs, N-ethylcarboxamidoadenosine (NECA), L-phenylisopropyladenosine (L-PIA), D-PIA and 2-chloroadenosine (2CHA) were examined on the IgE-dependent mediator release from RBL-2H3 cells, a model for mast-cell function. Adenosine and the adenosine analogs failed to influence mediator release from cells, previously sensitized with monoclonal anti-TNP mouse immunoglobulin E (anti-TNP IgE), when added alone. When added prior to conjugated trinitrophenol-ovalbumin (TNP-OVA), adenosine and the adenosine analogs (10(-8)-10(-4) M) significantly potentiated the release of both histamine (marker for degranulation) and peptidoleukotrienes (LT) (marker for de novo synthesized mediators). The effects were concentration-dependent with the potency order being L-PIA greater than NECA greater than A greater than D-PIA, 2CHA. The stimulatory effect on both histamine and LT release were reversed by prior treatment of the cells with pertussis toxin but not by the purinoceptor antagonists, theophylline and 8-phenyltheophylline, nor adenosine uptake blockers. At higher concentrations (above 10(-5) M), adenosine and adenosine analogs were also inhibitory on LT but not on histamine release. This inhibition was more evident on pertussis-toxin-treated cells in which there was no effect of adenosine or adenosine analogs on histamine release, but a concentration-dependent inhibition of IgE-dependent LT release. These findings demonstrate that adenosine analogs have two distinct mechanisms on mediator release from RBL-2H3 cells; a stimulatory effect on both histamine and LT release, mediated via a pertussis-toxin-sensitive G protein and an inhibitory effect on LT release via a pertussis-toxin-insensitive pathway. An abstract of this work has been published.

The effect of bilateral vagal stimulation on aerosolized antigen-induced responses was examined in the sensitized, perfused guinea pig lung. Vagal stimulation in the sensitized, perfused lung resulted in bronchoconstriction (peak response 160 +/- 18% above baseline) that was unaffected by either atropine (1 microM), a muscarinic receptor antagonist, or CP 96,345 (1 microM), a NK-1 receptor antagonist, but was transiently augmented in the presence of physostigmine (1 microM), a cholinesterase inhibitor, through an atropine-sensitive mechanism. However, SR 48968 (1 microM), a NK-2 receptor antagonist, and SR 48968 + CP 96,345 reduced by approximately 50 and 90%, respectively, vagally mediated increases in intratracheal pressure in the perfused lung. Simultaneous challenge with vagal stimulation and aerosolized antigen in the sensitized perfused lung resulted in a significant (p0.01) increase in intratracheal pressure (Pi), pulmonary arterial pressure (Ppa), and lung weight (LW) compared with either vagal stimulation or aerosolized antigen alone. Increases in Pi, Ppa, and LW in response to vagal stimulation + aerosolized antigen were associated with elevated venous effluent concentrations of thromboxane A2 (TXA2), prostacyclin, leukotriene C4, and histamine. Vagally mediated potentiation of aerosolized antigen-induced increases in Pi, Ppa, and LW was unaffected by atropine or CP 96,345 but was inhibited by the NK-2 receptor antagonist, SR 48968. These data suggest that vagally mediated (predominantly NK-2) potentiation of aerosolized antigen-induced increases in Pi, Ppa, and LW is characterized by elevated venous effluent concentrations of eicosanoids and histamine.

Our previous studies demonstrated that propranolol, an inhibitor of phosphatidic acid phosphohydrolase (PAPase) (EC 3.1.3.4) blocks the IgE-dependent mediator release from a rat mast (RBL 2H3) cell line. To continue these studies, we examined the ability of propranolol to inhibit the IgE-dependent or ionomycin-mediated phosphoinositide hydrolysis and calcium mobilization in RBL 2H3 cells. RBL 2H3 cells, sensitized with mouse monoclonal anti-trinitrophenol IgE (anti-TNP IgE), were stimulated to release both histamine and peptidoleukotrienes (LT) in response to a suboptimal concentration of trinitrophenol-ovalbumin conjugate (TNP-OVA) or ionomycin. Preincubation of the cells with d,l-propranolol (300 microM) significantly (P less than 0.05) inhibited the effects of both TNP-OVA and ionomycin on histamine and LT release. There was no difference in potency for the different isomers of propranolol, indicating that these effects were not a consequence of an effect on beta 2-adrenergic receptors. TNP-OVA produced a rapid hydrolysis of phosphoinositides resulting in a time-dependent increase in mono- (IP1), di- (IP2), tri- (IP3), and total inositol phosphate production. Ionomycin also produced a rapid increase in total inositol phosphate production; however, this largely reflected an accumulation of IP1. Both secretagogues produced a rapid elevation in cytosolic free calcium ([Ca2+]i); however, the effect of ionomycin maximized within a much shorter time frame than the effect of TNP-OVA. The effects of TNP-OVA on phosphoinositide hydrolysis and increase in [Ca2+]i were inhibited by propranolol over exactly the same concentration range as the effects of this compound on TNP-OVA-stimulated mediator release. In contrast, propranolol had no effect on the increase in [Ca2+]i and phosphoinositide hydrolysis in response to ionomycin. Taken together, these results suggest that PAPase/phospholipase D (PLD) (EC 3.1.4.4) activation may be a prerequisite for both IgE-dependent and ionomycin-stimulated mediator release from RBL 2H3 cells. Although other explanations are possible, the data further suggest that receptor-mediated, but not ionophore-stimulated, phosphoinositide hydrolysis and [Ca2+]i in RBL 2H3 cells may be regulated by a propranolol-sensitive pathway involving possible activation of PAPase.

The effect of phospholipase A2 (Naja naja) PLA2) on mean arterial blood pressure and intratracheal pressure was examined in anesthetized guinea pigs. Intracheally administered PLA2 (1 to 10 U) produced acute, dose-dependent increases in mean arterial blood pressure and intracheal pressure. However, Intravenously administered PLA2 (doses as large as 1,000 U) did not alter monitored variables. Acute PLA2-induced morphologic alterations were characterized by airway constriction, airway/alveolar cell damage, and pulmonary sequestration of both leukocytes and platelets. PLA2-induced increases in both mean arterial blood pressure and intratracheal pressure were attenuated to varying degrees by pretreating intravenously with indomethacin (10 mg/kg), a cyclooxygenase inhibitor, and WEB 2086 (0.1 mg/kg), a platelet-activating factor antagonist. Both ICI 198,615 (1 mg/kg), a leukotriene D4, receptor antagonist given intravenously, and dexamethasone (50 mg/kg), a steroidal anti-inflammatory agent given intraperitoneally as a 2-day pretreatment, reduced PLA2-induced increases in intratracheal pressure. Pyrilamine (2 mg/kg), a histamine1-receptor antagonist given intravenously, did not modify PLA2-induced pathophysiologic responses. Guinea pigs exposed to aerosolized PLA2 (100 U/ml) exhibited evidence of increased bronchoalveolar lavage macrophage, leukocyte, and lymphocyte accumulation at 24 h post-PLA2. These studies suggest that in vivo PLA2-induced pathophysiologic changes in the guinea pig involve alterations in resident airway cell populations as well as sequestration and infiltration of inflammatory cells. Both eicosanoids and platelet-activating factor appear to contribute to these PLA2-induced pathophysiologic effects.

Rat basophilic leukemia (RBL 2H3) cells were passively sensitized by exposure to monoclonal anti-trinitrophenol mouse immunoglobulin E (anti-trinitrophenol IgE) (0.5 microgram/ml) and triggered by exposure to a sub-optimal concentration of trinitrophenol ovalbumin conjugate (5 ng/ml). At this concentration, trinitrophenol-ovalbumin increased histamine release from a basal rate of 4.8 +/- 0.5 to 28.5 +/- 4.6% and peptidoleukotrienes from less than 0.1 to 4.2 +/- 1.3 ng/10(6) cells in the activated cells. Ro 19-3704 and Ro 19-1400, platelet activating factor (PAF) antagonists which are structural analogs of PAF, potently inhibited both the IgE-dependent release of histamine (IC50 values of 3.0 and 3.6 microM, respectively) and LT release (IC50 values of 5.0 microM for both compounds) from the cells. These effects appeared to be independent to the ability of the compounds to act as PAF antagonists since PAF on its own had no effect on mediator release, and WEB 2086 and BN 52021, structurally distinct PAF antagonists, were relatively ineffective as inhibitors of mediator release. Ro 19-3704 and Ro 19-1400 were observed to be potent inhibitors of the soluble phospholipase A2 activity in synovial fluid from rheumatoid arthritic patients (IC50 values of 6.5 and 8.4 microM, respectively). In contrast, WEB 2086 and BN 52021 had no effect on this phospholipase A2. Ro 19-3704 significantly inhibited the IgE-dependent formation of inositol phosphates in RBL 2H3 cells (IC50 value of 7.0 microM). These data suggest that the mediator release inhibitory action of these compounds may be related to the ability of these compounds to inhibit phospholipase A2 and/or phospholipase C.

Ro 21-7634 was examined for oral antiallergic activity in two in vivo models commonly used to evaluate antiallergics. In the rat PCA test, this drug had an oral ID50 of 1.14 mg/kg and was found to be more potent than several other antiallergics including Disodium Cromoglycate (cromoglycate), Oxatomide, Doxanthrazole, Xanoxate, 2,6-bis (ethyoxyoxalylamino) pyridine, PRD-92-EA and M + B 22,948. In contrast to cromoglycate, Ro 21-7634 was found to be an orally active inhibitor of antigen-induced broncho-constriction in passively sensitized rats (ID50 = 0.2 mg/kg). In addition, Ro 21-7634 inhibited antigen-induced histamine release in an in vivo passive peritoneal anaphylaxis test system, following intraperitoneal administration. Ro 21-7634 demonstrated no end organ antagonism toward histamine, metacholine or serotonin in the guinea pig.

Rat liver microsomes have previously been shown to contain hemoproteins having molecular weights of 53,000, 50,000, and 45,000. The 45,000-dalton hemoprotein, which is induced in rat liver microsomes by pretreatment of animals with phenobarbital, is resistant to proteolysis by trypsin. This characteristic was used to purify it from the other microsomal hemoproteins. In the procedure used, a sodium cholate-solubilized microsomal fraction from phenobarbital-pretreated rats was treated with trypsin and chromatographed on Sephadex G-100 to separate the hemoprotein from proteolytic degradation products. The hemoprotein thus isolated was homogenous on the basis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was identified spectrally as a cytochrome P-420 hemoprotein. This hemoprotein was free of cytochrome b, and NADPH-cytochrome c reductase activity. Antibody prepared against the protease-treated cytochrome P-420 hemoprotein will not cross-react with the 53,000. and 50,000-dalton hemoproteins. This was assessed by three criteria. First, immunoprecipitation studies were conducted with detergent-solubilized partially purified cytochrome P-450 preparations isolated from the liver microsomes of control and phenobarbital- and 3methylcholanthrene-pretreated rats. The antibody immunoprecipitated only the 45,000-dalton hemoprotein from these partially purified cytochrome P-450 preparations, each of which contains all three hemoproteins. Second, the antibody demonstrated specificity with regard to the microsomal hydroxylation reactions it would inhibit in a reconstituted hydroxylation system containing partially purified cytochrome P-450 (448) fractions isolated from the liver microsomes from phenobarbitalor 3-methylcholanthrene-pretreated rats. The antibody would inhibit benzphetamine-N-demethylation catalyzed by both cytochrome P-450 fractions but would not inhibit benzpyrene hydroxylation catalyzed by either. Third, agglutination and complement fixation assays were performed to assess the binding of the antibody to liver microsomes isolated from control and phenobarbital- or 3methylcholanthrene-pretreated rats. These studies demonstrated that the antibody binds preferentially to the liver microsomes isolated from phenobarbital-pretreated rats, in which the 45,000-dalton hemoprotein has been shown to be induced. It is hypothesized that there are very significant structural and catalytic differences among the cytochrome P-450 hemoproteins. In the presence of NADPH and oxygen, the mixed-function oxidase system of the rat liver endoplasmic reticulum (microsomes) catalyzes the hydroxylation of a wide variety of substrates including steroids, fatty acids, and xenobiotics such as drugs, carcinogens, and pesticides (2, 3). The mechanism by which this system catalyzes the hydroxylation of such a large

Members of a series of basic amide and ester derivatives of 2-substituted pyrido[2,1-b]quinazoline-8-carboxylic acids were prepared and evaluated for their ability to prevent slow-reacting substance of anaphylaxis (SRS-A) induced contractions of guinea pig ilea. The results indicate that the presence of a branched-chain alkyl group in the 2-position and a sterically demanding substituted aminoethyl carboxylate or carboxamide in the 8-position give optimal in vitro activity. The phenylpiperazine 25 was further found to block SRS-A-related symptomatology after intravenous administration in two animal models.

Synthesis of Stereisomeric Pinanthromboxane Derivatives and Evaluation of the Compounds as Platelet Aggregation Inhibitors Starting from the two enantiomeric myrtenols ((−)-1 and (+)-1; cf. Scheme 1), the synthesis of twelve stereoisomeric pinanthromboxane derivatives ((+)- and (−)-10, -11, -14, -15, -21 and -22) is described (cf. Schemes 1–4). Biological data from the evaluation as platelet aggregation inhibitors (cf. Table 6 and 7), thromboxane synthetase inhibitors (cf. Table 8) and from the assessment as antagonists of leukotriene E4 induced bronchoconstriction (cf. Table 9) are presented.

The effects of Ro 21-7634 and disodium cromoglycate (cromoglycate) on the in vitro release of mediators of anaphylaxis from rat peritoneal cells and guinea pig lung tissue were compared. Ro 21-7634 was 25 fold more potent than cromoglycate as an inhibitor of antigen-induced histamine release from passively sensitized (IgE) rat peritoneal cells. Ro 21-7634 was also the more potent inhibitor of both compound 48/80- and concanavalin A-induced histamine release from rat peritoneal cells. The two drugs shared the common properties of producing the same maximal level of inhibition in each of the above releasing systems and exhibiting a time and concentration dependent loss of inhibitory activity when added to the cells prior to the releasing agent. Neither drug inhibited ionophore A23187- or ionophore X537A-induced histamine release from these cells. Ro 21-7634 inhibited antigen-induced (IgG1) histamine and SRS-A release from actively sensitized guinea pig lung fragments, whereas cromoglycate did not. The results indicate that Ro 21-7634 and cromoglycate act through a common mechanism to inhibit allergic mediator release and that Ro 21-7634 is the more potent inhibitor.

Prophylactic treatment (p.o.) of rats with adjuvant-induced arthritis (AA) with two retinoid-like 2,4,6,8-nonatetraenoic acids (NTA), Ro 23-6457 and Ro 23-2895, significantly reduced hind paw swelling between days 10-23 and the level of plasma fibrinogen (MED approximately 25 mumoles/kg). When given therapeutically (75 mumoles/kg between day 21 and 28) either NTA arrested the progression of the disease (MED, 25-75 mumoles/kg). Unseparated and adherent cell (AC) depleted spleen cells from rats with AA (day 12-15) responded poorly to the T cell mitogen, Con A (2.5 micrograms/ml) and the B cell mitogen, LPS (10 micrograms/ml). The responses were partially restored (approximately 30% of normal responses) in AC-depleted (but not unseparated) spleen cells from Ro 23-6457 treated rats (75 and 250 mumoles/kg/day). These data demonstrate an immunomodulatory effect of Ro 23-6457 in the adjuvant rat which may contribute to its anti-inflammatory activity in AA.

FPL 55712 was investigated by the aerosol route of administration for efficacy at protecting against leukotriene-induced bronchoconstrictions in guinea pigs and for mediator release inhibitory activity in passively sensitized rats. In the studies to investigate leukotriene antagonism; anesthetized, spontaneously breathing guinea pigs were pretreated with propranolol and were exposed via tracheal cannula to aerosols generated by a Monaghan nebulizer. Subsequently, the animals were artificially ventilated and challenged with LTD4 or LTE4 (25 micrograms/kg, i.v.). FPL 55712 produced a concentration-dependent inhibition of LTD4 and LTE4-induced bronchoconstriction (IC50's 0.5% and 0.8%, respectively). Although the biologic half-life of FPL 55712, administered intravenously, was very short (1.7 minutes against LTD4 and 1.2 minutes against LTE4) after aerosol administration the biological half-life was surprisingly long (120 minutes against LTD4 and 90 minutes against LTE4). Aerosolized FPL 55712 also possessed weak antiallergic activity in comparison to disodium cromoglycate when measured as an inhibitor of IgE-mediated anaphylactic bronchoconstriction in rats (IC50's of 2.0% and 0.01%, respectively). Thus, these studies demonstrate that, when administered by aerosol, FPL 55712 is effective at protecting against leukotriene-induced bronchoconstrictions, exhibits a long duration of action and also possesses weak antiallergic activity.

The syntheses and biological activity of (all Z )-7,7-dimethyl-5-8,- 11,14-eicosatetraenoic acid, (all Z )-7,7,-dimethyl-5,8,11-eicosatrienoic acid, ( Z , Z -7,7-dimethyl-5,8-eicosadienoic acid, (all Z )-10,10-dimetyl- 5,8,11,14-eicosatetraenoic acid, (all Z -10,10-dimethyl-5,8,11-eicosatrienoic acid, and rac .-( Z , Z -15-hydroxy-7,7-dimethyl-5,8-eicosadienoic acid are described. These arachidonic acid analogs are all inhibitors of ionophore-induced SRS-A biosynthesis in rat peritoneal cells. Their mode of action may involve inhibition of phospholipase A2 rather than Δ5-lipoxygenase. These compounds failed to exhibit significant activity in an in vivo model designed to detect inhibitors of antigen-induced, leukotriene-mediated bronchoconstriction is sensitized guinea pigs.

Atriopeptins are circulating peptide hormones which are secreted by atrial tissue and act at the kidney. Because the atriopeptins survive passage through the pulmonary circulation, they also may be involved in the modulation of airway or pulmonary vascular smooth muscle tone. Using in vitro organ bath techniques, atriopeptins were found to induce potent concentration-dependent relaxation of isolated guinea pig trachea, and pulmonary artery with a rank order of potency: atriopeptin III greater than atriopeptin II greater than atriopeptin I. Atriopeptin-induced smooth muscle relaxation was observed to be a direct response since it was not mediated by activation of relaxant VIP receptors, beta-adrenergic receptors, or H2 receptors nor affected by cyclooxygenase inhibition or denuding of the vasculature or trachea of endothelial and epithelial cells. The time course of atriopeptin II-induced relaxation of the pulmonary artery was transient in contrast to the prolonged relaxations on the trachea. The transient relaxant responses of atriopeptin II on pulmonary artery were not due to metabolism of atriopeptin II to atriopeptin I by angiotensin-converting enzyme since pretreatment with captopril did not augment the response. These results seem to indicate that distinct atriopeptin receptors may exist in airway and pulmonary arterial smooth muscle and that activation of these relaxant receptors may play an important role in the regulation of pulmonary vascular and bronchomotor tone.

Adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) was solubilized from rat hepatic plasma membranes with Lubrol PX. The enzyme could be stabilized during solubilization by pretreating the membrane with glucagon and 5′-guanylimidodiphosphate (Gpp(MH)p) or Gpp(NH)p alone but not by glucagon alone or in the presence of GTP or other guanine nucleotides. The solubilized enzyme was purified 3–4-fold by molecular exclusion chromatography on Ultrogel AcA22; the presence both 0.01% Lubrol and 25% sucrose were required to maintain both the solubility as well as stability of the enzyme. The enzyme was purified from 70% of the other solubilized membrane proteins and 85% of the solubilized phospholipids, and was completely separated from 5′-nucleotidase. In addition, the enzyme was purified 2-fold with respect to non-specific nucleotide phosphohydrolase and pyrophosphohydrolase activities but not with respect to ‘specific’ GTPase activity. No perceptible change in molecular size of partially purified adenylate cyclase was observed after it had been pre-activated in its membrane-bound form with hormone and Gpp-(NH)p. The solubilized enzyme also retained its ability to be activated by the guanine nucleotide analogues Gpp(NH)p and Gp(CH2)pp with similar concentration and time-dependent characteristics displayed by the membrane-bound form of the enzyme. Attempts to further purify the solubilized enzyme by a variety of size- and charge-based chromatography techniques proved unsuccessful. As an explanation for this phenomenon we suggest that, after solubilization with Lubrol, the enzyme is probably but a fraction of the total protein in the complex. That is, judging from the highest specific activity obtained for the partially purified liver enzyme (1.2 nmol./min per mg) and assuming that its turnover number is that of the highly purified bacterial adenylate cyclase [54], the enzyme comprises less than 0.1% of the protein present in the adenylate cyclase-containing fraction from the Ultrogel column. The idea of determining the physical properties of Lubrol-solubilized adenylate cyclase becomes even more difficult when one considers that properties, such as Stokes radii on Ultrogel columns depend upon the concentrationof detergent used in the column elution buffer. For example, in the absence of detergent we have observed that Lubrol-solubilized adenylate cyclase aggregates and elutes in the void volume of the column. Thus, knowledge of the aggregation characteristics of the enzyme further suggests that caution must be used in assigning values to the intrinsic physical properties of the enzyme.

Adenosine inhibits guinea-pig lung adenylate cyclase (ATP pyrophosphatelyase (cyclizing), EC 4.6.1.1) through a ‘P’ type regulatory site. The inhibition is of a non-competitive type. Divalent cations which activate the enzyme (Mg2+ and Mn2+) and also those which inhibit (Ca2+) increase the inhibitory potency of ‘P’ site analogs at this site. Guanine nucleotides also increase the inhibitory potency at this regulatory site but this does not appear to be directly related to the ability of the guanine nucleotides to activate the enzyme. Other regulators of lung adenylate cyclase, epinephrine and isoproterenol, do not affect the adenosine inhibitory process when examined at physiological concentrations. These studies demonstrate that two types of ligand which regulate the catalytic activity of the lung adenylate cyclase (metal ions and guanine nucleotides) also have a role in regulating the inhibition of the enzyme by adenosine.

A series of substituted (E)-3-(4-oxo-4H-quinazolin-3-yl)-2-propenoic acids was prepared and evaluated in the rat passive cutaneous anaphylaxis (PCA) test for antiallergic activity. Alkoxy, alkylthio, and isopropyl substituents at the 6- or 8-positions provided highly potent compounds. Conversion to the Z isomer, reduction of the side chain double bond, or reduction of the quinazoline ring resulted in substantial loss of activity. Among the analogues that exhibited oral activity in the PCA test, (E)-3-[6-(methylthio)-4-oxo-4H-quinazolin-3-yl]-2-propenoic acid (5i) was the most potent.

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 synthesis in vitro in rat peritoneal cells after challenge with ionophore A23187. In the peritoneal cell system, Ro 21-7634 exhibited an IC50 of 500 microM, in comparison with 5.8,11,14-eicosatetraynoic acid, phenidone and BW755C (IC50's of 2, 100, and 100 microM, 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.

The molecular weight of detergent-solubilized NADPH-cytochrome c reductase from rat liver microsomes has been estimated to be 79,000. The method used for this determination involves immunoprecipitation of deoxycholate-solubilized enzyme from 125 I-labeled microsomal proteins. The antibody was prepared against a purified preparation of Bromelain-solubilized enzyme (molecular weight 71,000). The immunoprecipitate was then subjected to SDS-polyacrylamide gel electrophoresis and the enzyme located by 125 I-gamma counting.

During enzymatic iodination of rat liver endoplasmic reticulum the peroxidation of endogenous lipids and the loss of cytochrome P450 has been observed. This lipid peroxidation can be inhibited by the inclusion of a low concentration of butylated hydroxytoluene in the iodination mixture. This results in increased incorporation of 125I− and stabilization of cytochrome P450.

Sodium dodecylsulfate-polyacrylamide gel electrophoresis and lactoperoxidase-catalyzed protein radio-iodination were used to compare the structure of the liver endoplasmic reticulum (microsomes) from control, phenobarbital-, and 3-methylcholantrene-pretreated rats. Prior to iodination, ribosomes and contaminating adsorbed cytoplasmic proteins were removed from the microsomal membrane by washing with 0.3 M sucrose containing 0.1 M sodium pyrophosphate, pH 7.5. Iodination of membranes was carried out in the presence of butylated hydroxytoluene to prevent the peroxidation of membrane lipids. The polyacrylamide gel protein and iodination profiles for control microsomes indicated that while many of the minor protein constituents of the membrane incorporated 125I, the major peak of 125I label corresponded to the migration position of major protein constituents having molecular weights of approx. 50 000. Treatment of 125I-labeled control microsomes with trypsin removed 125I from the membrane proteins confirming that lactoperoxidase was iodinating exterior proteins on the membrane. If membranes which were iodinated in the absence of butylated hydroxytoluene were similarly digested with trypsin, most membrane proteins were much more susceptible to trypsin digestion. This suggested that lipid peroxidation, occuring during iodination in the absence of the antioxidant, caused an extensive breakdown in the structure of the membrane. A comparison of the polyacrylamide gel patterns of the microsomes from control, phenobarbital-, and 3-methylcholantrene-pretreated rats demonstrated marked changes in both the protein and iodination patterns in the 50 000 molecular weight region of the gels. 3-Methylcholanthrene appeared to induce a protein of slightly higher molecular weight than 50 000 while phenobarbital induced a protein(s) of slightly lower molecular weight. Induction with both compounds increased the amount of 125I incorporated in the 50 000 molecular weight region of polyacrylamide gels indicating that the induced proteins have an exterior location on the microsomal membrane. The asssociation of the induced proteins with cytochrome P450, the terminal oxidase of the microsomal mixed-function oxidases, is discussed. A model is proposed for the mechanism of insertion of the induced proteins into the microsomal membrane.

A series of substituted (E)-3-(4-oxo-4H-quinazolin-3-yl)-2-propenoic acids was prepared and evaluated in the rat passive cutaneous anaphylaxis (PCA) test for antiallergic activity. Alkoxy, alkylthio, and isopropyl substituents at the 6- or 8-positions provided highly potent compounds. Conversion to the Z isomer, reduction of the side chain double bond, or reduction of the quinazoline ring resulted in substantial loss of activity. Among the analogues that exhibited oral activity in the PCA test, (E)-3-[6-(methylthio)-4-oxo-4H-quinazolin-3-yl]-2-propenoic acid (5i) was the most potent.

Leukotriene (LTC 4 ) is one of the components of Slow Reacting Substance of Anaphylaxis (SRS-A) and is a potent constrictor of guinea pig ilea. The contraction is likely to be a receptor-mediated process. Here we report the existence of specific binding sites for 3 H-LTC 4 in a crude membrane preparation from guinea pig ileal longitudinal muscle. At 4°C in the presence of 20 mM Serine-borate, binding increases linearly with protein concentration, reaches equilibrium in 10 minutes, and is reversible upon addition of 3 × 10 −5 M unlabelled LTC 4 . The dissociation curve is consistent with the existence of more than one class of binding site. Ca ++ and Mg ++ greatly enhance the binding of 3 H-LTC 4 at equilibrium. In the presence of 5mM CaCl 2 and MgCl 2 not only LTC 4 (IC 50 10 −7 M), but also LTD 4 (albeit with much lower affinity, IC 50 = 6 × 10 − 5M) and the SRS-A antagonist FPL 55712 (IC 50 = 10 −5 M) can compete with 3 H-LTC 4 for its binding sites. FPL 55712 only displaces 60–70% of the total amount bound, while LTC 4 displaces 90–95%. These studies indicate that multiple classes of binding sites exist for 3 H-LTC 4 in guinea pig ileal longitudinal muscle, and that at least part of these binding sites might be related to the ability of LTC 4 to contract guinea pig ilea.

Members of a series of basic amide and ester derivatives of 2-substituted pyrido[2,1-b]quinazoline-8-carboxylic acids were prepared and evaluated for their ability to prevent slow-reacting substance of anaphylaxis (SRS-A) induced contractions of guinea pig ilea. The results indicate that the presence of a branched-chain alkyl group in the 2-position and a sterically demanding substituted aminoethyl carboxylate or carboxamide in the 8-position give optimal in vitro activity. The phenylpiperazine 25 was further found to block SRS-A-related symptomatology after intravenous administration in two animal models.

Summary The vitamin D-dependent, calcium-binding protein from rat kidney, calbindin D28k (renal CaBP) specifically stimulates Ca,Mg-ATPase activity of human erythrocyte plasma membranes in a dose-dependent, calcium-sensitive manner. This stimulation was about two-fold compared to a three-fold stimulation by calmodulin. The effect was specific since other calcium-binding proteins and low molecular weight proteins did not stimulate Ca,Mg-ATPase activity. Renal CaBP did not stimulate cyclic nucleotide phosphodiesterase at concentrations greater than those which stimulated Ca,Mg-ATPase activity. This is the first report of a specific in vitro effect of renal CaBP on an enzyme system.

Sarcolemmal vesicles prepared by a new procedure from bovine tracheal smooth muscle were found to have a NaCa exchange activity that is significantly higher than that reported for different preparations from other types of smooth muscle. The exchange process system co-purified with 5′-nucleotidase, a plasma membrane marker enzyme, and was significantly enriched (over 100-fold) compared to mitochondria ( cytochrome -c oxidase) but only slightly enriched (4-fold) compared to sarcoplasmic reticulum ( NADPH-cytochrome -c reductase). The Na+ dependence of Ca2+ transport was demonstrated through both uptake and efflux procedures. The uptake profile with respect to Ca2+ was monotonic with a linear ν o vs. ν o · S −1 plot. The resultant K m of Ca2+ from the airway sarcolemmal vesicles (20 μM) was similar in magnitude to the K m of cardiac sarcolemmal vesicles (30 μM). Tracheal vesicles demonstrated a V max of 0.3–0.5 nmol · mg −1 · s − which is significantly higher than that reported in preparations from other smooth muscle types. Furthermore, two processes found to stimulate cardiac Na-Ca exchange, pretreatment with either a mixture of dithiothreitol and Fe2+ or with chymotrypsin, were ineffective on the tracheal smooth muscle. Thus, the Na-Ca exchanger identified in tracheal smooth muscle appears to be different from that observed in cardiac muscle, implying that regulation of this activity may also be different.

Adenosine potentiates the in vitro antigen-induced release of histamine from chopped lung tissue prepared from a guinea pig actively sensitized to egg albumin. By studying the activity of a variety of adenosine analogs, the potentiation process in lung has been characterized as occurring through an ‘R’ adenosine receptor site. Similar studies on ionophore A23187-induced release of histamine from purified rat peritoneal mast cells have demonstrated that an ‘R’ adenosine receptor site is also associated with adenosine potentiation in this system. Since adenosine inhibits adenylate cyclase activity in a homogenate of guinea pig lung, and since decreases in cyclic AMP levels are thought to be associated with mediator release, the regulatory site involved in the enzyme inhibition process was also characterized to determine if, in lung, the ability of adenosine to inhibit adenylate cyclase is associated with potentiation of mediator release. It was found, however, that inhibition of adenylate cyclase occurs through a ‘P’ site, suggesting that the two processes are not related phenomena. Theophylline blocks the effect of adenosine on the antigen-induced release of histamine from guinea pig lung. This is in accord with previous data demonstrating that theophylline is an ‘R’ site antagonist. This observation also supports the idea that part of the pharmacological action of theophylline in human bronchial asthma may be through inhibition of the ability of adenosine to potentiate mediator release.

Research carried out in numerous laboratories has led to the hypothesis that metabolites of the ∆5-lipoxygenase (5∆-L0) pathway (e.g., leukotrienes and 5-HETE) may play an important role in mediating a number of inflammatory diseases including asthma, inflammatory bowel disease, and diseases associated with cutaneous inflammation. The purpose of this chapter is to briefly review the rationale supporting this hypothesis and to present the results of some experimental studies with promising ∆5-L0 inhibitors and leukotriene antagonists which would support the clinical evaluation of these types of drugs in the three disease states.

1 Pharmacological modulation of antigen-induced anaphylaxis in actively sensitized guinea-pigs with intravenously administered indomethacin (10 mg/kg), pyrilamine (2.0 mg/kg) and propranolol (0.1 mg/kg) resulted in a delayed onset, slowly developing bronchoconstriction indicative of a slow-reacting substance of anaphylaxis (SRS-A) response. 2 Measurements of pulmonary mechanics on the drug-pretreated animals challenged with ovalbumin demonstrated a more prominent effect on dynamic compliance than resistance. This is consistent with the more potent effects of SRS-A on peripheral rather than central airways. 3 The slowly developing bronchoconstriction obtained after treatment with indomethacin, pyrilamine and propranolol was inhibited by the standard SRS-A antagonist, FPL 55712 and the SRS-A synthesis inhibitors, phenidone, BW 755C and nordihydroguaiaretic acid. 4 Plasma SRS-A levels were determined in guinea-pigs following antigen challenge. The appearance of SRS-A in the plasma preceded the onset of bronchoconstriction and SRS-A levels remained elevated throughout its development. Coincident with the inhibition of bronchoconstriction by the SRS-A synthesis inhibitor, phenidone, was a dose-dependent reduction in plasma SRS-A. The intravenous ED50 in each case was 4 mg/kg. 5 This model of antigen-induced SRS-A-mediated bronchoconstriction should prove useful for the in vivo evaluation and development of therapeutics which regulate the synthesis of SRS-A.

Leukotriene D4 (LTD4) and Platelet Activating Factor (PAF) are two important lipid mediators of allergy and inflammation which were defined structurally in the late 1970’s and early 1980’s, although these substances had been identified many years earlier based on their biological activities. The structure of LTD4 was elucidated from initial observations made during investigations ongoing in Samuelsson’s laboratory in 1979 (1). Subsequently it was demonstrated that LTD4 is one of a family of peptidoleukotriene metabolites of arachidonic acid formed by the sequential bioconversion of arachidonic acid via Δ5-lipoxygenase and LTC4 synthetase (a glutathione-S-transferase enzyme). Metabolism of arachidonic acid via this pathway leads to the formation of LTC4, LTD4, and LTE4 (the peptidoleukotrienes) (2). LTD4 was ultimately shown to be the major biologically active component of slow reacting substance of anaphylaxis (3), a substance originally described by Kellway and Trethewie in 1940 (4).

A series of compounds in which the 4-acetyl-3-hydroxy-2-propylphenoxy moiety of the standard SRS-A antagonist, FPL-55712, is linked by a polymethylene or a polyether chain to substituted (aryloxy)alkanoic acids was prepared. The compounds were evaluated for their ability to antagonize SRS-A-induced contractions of guinea pig ilea and LTE-induced bronchoconstriction in the guinea pig. The results showed that the compounds were all less potent than FPL-55712 in vitro, yet surprisingly, most were more potent by the inhalation route of administration. Some of the most potent analogues were selected for further pharmacological evaluation and, by inhalation, exhibited selective antagonism of leukotrienes as compared with PAF or histamine. In comparison to FPL-55712, compounds 28 and 37 were more potent against LTE (40- and 80-fold, respectively), LTD (4- and 3-fold, respectively), and LTC (27- and 20-fold, respectively) induced bronchoconstriction when tested by inhalation.

A series of N-[4-(3-pyridinyl)butyl]-1,1'-biphenyl-4-carboxamides was prepared, and the compounds were evaluated for platelet-activating factor (PAF) antagonist activity in a binding assay employing washed, whole dog platelets and in vivo for their ability to inhibit PAF-induced bronchoconstriction in the guinea pig. The inclusion of a methyl group in the R configuration on the side-chain carbon adjacent to the carboxamide nitrogen atom of these derivatives resulted in a marked enhancement of potency in the binding assay for compounds unsubstituted in the biphenyl 2-position and, more importantly, in improved oral bioavailability. Previous work with related pyrido[2,1-b]-quinazoline-8-carboxamides suggests that the presence of such an alkyl group improves bioavailability by rendering the resulting compounds resistant to degradation by liver amidases. The most interesting compounds to emerge from this work are (R)-2-bromo-3',4'-dimethoxy-N-[1-methyl-4-(3-pyridinyl)butyl]-1,1'-bi phe nyl- 4-carboxamide (33) and (R)-2-butyl-3',4'-dimethoxy-N-[1-methyl-4-(3-pyridinyl)butyl]- 1,1'-biphenyl-4-carboxamide (40) each of which inhibits PAF-induced bronchoconstriction in the guinea pig by greater than 55%. 6 h after an oral dose of 50 mg/kg.

Rat liver plasma membranes were incubated with phospholipase A2 (purified from snake venom) or with filipin, a polyene antibiotic, followed by analysis of the binding of glucagon to receptors, effects of GTP on the glucagon-receptor complex, and the activity and responses of adenylate cyclase to glucagon + GTP, GTP, Gpp(NH)p, and F-. Phospholipase A2 treatment resulted in concomitant lossess of glucagon binding and of activation of cyclase by glucagon + GTP. Greater than 85% of maximal hydrolysis of membrane phospholipids was required before significant effects of phospholipase A2 on receptor binding and activity response to glucagon were observed. The stimulatory effects of Gpp(NH)p or F- remained essentially unaffected even at maximal hydrolysis of phospholipids, whereas the stimulatory effect of GTP was reduced. Detailed analysis of receptor binding indicates that phospholipase A2 treatment affected the affinity but not the number of glucagon receptors. The receptors remain sensitive to the effects of GTP on hormone binding. Filipin also caused marked reduction in activation by glucagon + GTP. However, in contrast to phospholipase A2 treatment, the binding of glucagon to receptors was unaffected. The effect of GTP on the binding process was also not affected. The most sensitive parameter of activity altered by filipin was stimulation by GTP or Gpp(NH)p; basal and fluoride-stimulated activities were least affected. It is concluded from these findings that phospholipase A2 and filipin, as was previously shown with phospholipase C, are valuable tools for differentially affecting the components involved in hormone, guanyl nucleotide, and fluoride action on hepatic adenylate cyclase.

Publisher Summary This chapter presents the knowledge of the cellular composition and anatomy, the physiological properties, and the biochemical mechanisms associated with normal lung function. This information provides the basis for describing the pathophysiology associated with various obstructive, restrictive, and vascular disease states of the lung. The diagnostic tests used to detect the lung diseases and the biochemical mechanisms associated with the therapeutic treatment of these diseases are described in the chapter. The lungs are innervated along the tracheobronchial tree by the autonomic nervous system. Afferent autonomic nerve fibers from “stretch receptors” in the alveoli, “irritant receptors” in the bronchi, bronchioles, and trachea, and “cough receptors” in the larynx transmit signals to the central nervous system via the vagus nerve. The lung is equipped with two arterial blood systems because of the unique function of the lung to supply oxygen to the circulatory system of the body. The primary function of the lung is to exchange oxygen and carbon dioxide between the gaseous atmosphere in which man lives and the blood that transmits oxygen to and removes carbon dioxide from the cells of the body.

Platelet activating factor (PAF; 1-o-alkyl-2-0-acetyl-sn-glycero-3-phosphocholine-3-phosphocholine) a potential mediator of anaphylaxis, stimulates secretion of mucin by explants of trachea from four separate rodent species (guinea pig, rat, rabbit, ferret) in organ culture. Enhanced secretion is not a result of cell damage or release of histamine by cells within the explants (e.g., platelets). It is inhibited by equimolar concentrations of the potent PAF-receptor antagonist, Ro 19-3704. PAF provokes production of immunoreactive peptidyl leukotrienes (ir-LTC4, LTD4, LTE4) within the explants. The stimulatory effect of PAF on mucin secretion is blocked by equimolar concentrations of nordihydroguiaretic acid (NDGA) a "mixed" inhibitor of both cyclo- and lipoxygenase pathways of arachidonic acid metabolism. Leukotrienes are localized within tracheobronchial epithelium by immunohistochemical staining, and physical removal of epithelium from explants inhibits production of leukotrienes in vitro under nonstimulated conditions and after exposure to PAF. In addition, the stimulatory effect of PAF on mucin secretion is not altered by FPL-55712, a receptor antagonist of LTD4. These results are consistent with the hypothesis that PAF stimulates secretion of mucin by activating biosynthesis of lipoxygenase products (e.g., peptidyl leukotrienes) within epithelial cells of the respiratory mucosa.

Leukotriene C4 (LTC4), one of the major components of the slow-reacting substance of anaphylaxis (SRS-A), is a potent constrictor of bronchial smooth muscle in many species including humans. Here we report the identification and characterization of specific binding sites for LTC4 in membranes from human lung parenchyma. At 4°, 3H-LTC4 binding is specific, saturable Bmax = 32–41 pmoles mg prot.), rapid (equilibrium being attained within 15 min), reversible and of high affinity (Kd = 3.6−7 × 10−8 M). The binding sites are sensitive to heat and probably possess a protein moiety, being inactivated upon trypsinization. CaCl2 affects both the association and the dissociation rate and dose-dependently enhances the binding of 3H-LTC4 at equilibrium; maximal enhancement (4-fold) occurred at 10−2 M CaCl2. Unlabelled LTC4 is able to compete with 3H-LTC4 for its binding sites with an IC50 of 7.8 × 10−8 M. The addition of 10−2 M CaCl2 increases the potency of LTC4 in inhibiting the binding (2.2-fold); both the competition curves are monophasic, indicating the existence of a homogeneous class of binding sites. In the presence of CaCl2, LTD4, LTE4 and the SRS-A antagonist FPL 55712 can inhibit 3H-LTC4 specific binding, being, however, less potent than LTC4 ( ic 50 s = 2.2 × 10−6, 2.4 × 10−5 and 2.4 × 10−5 M, for LTD4, LTE4 and FPL 55712, respectively). FPL 55712 displayed a competitive mechanism; its affinity, however, was lower if absorption to glass was not prevented. The present studies indicate that specific binding sites for 3H-LTC4 exist in human lung parenchyma, and that a receptor-mediated process might be involved in the bronchoconstriction induced by LTC4.

Publisher Summary The purpose of this chapter is to describe the detection of hemoproteins in sodium dodecyl sulfate (SDS)-polyacrylamide gels. The chapter mentions an improved procedure for hemoprotein detection that facilitates photography and permits scanning of the stained gels. By modifying a conventional heme staining procedure for use with SDS gels, the chapter indicates the existence of three electrophoretically distinct hemoproteins in rat liver microsomes. Gels are electrophoresed in the dark at 0°-4°, to minimize the destruction of heme. Duplicate samples must be run in parallel and stained for protein to make final assignments of the heine-stained bands. Cytochrome P-450-catalyzed drug and steroid metabolism has been intensively investigated for more than a decade. Because the treatment of animals with a variety of compounds was known to cause marked alterations in the physical and catalytic properties of this mixed-function oxidase system, the existence of multiple forms of cytochrome P-450 is postulated.

A series of compounds in which the 4-acetyl-3-hydroxy-2-propylphenoxy moiety of the standard SRS-A antagonist, FPL-55712, is linked by a polymethylene or a polyether chain to substituted (aryloxy)alkanoic acids was prepared. The compounds were evaluated for their ability to antagonize SRS-A-induced contractions of guinea pig ilea and LTE-induced bronchoconstriction in the guinea pig. The results showed that the compounds were all less potent than FPL-55712 in vitro, yet surprisingly, most were more potent by the inhalation route of administration. Some of the most potent analogues were selected for further pharmacological evaluation and, by inhalation, exhibited selective antagonism of leukotrienes as compared with PAF or histamine. In comparison to FPL-55712, compounds 28 and 37 were more potent against LTE (40- and 80-fold, respectively), LTD (4- and 3-fold, respectively), and LTC (27- and 20-fold, respectively) induced bronchoconstriction when tested by inhalation.

Tiacrilast (Ro 22-3747) is an allergic mediator release inhibitor which has demonstrated potent oral activity in two IgE-mediated animal models of immediate hypersensitivity: the rat passive cutaneous anaphylaxis test (ID50 of 0.65 mg/kg) and a model in which anaphylactic bronchospasm is induced in passively sensitized rats (ID50 of 0.022 mg/kg). In addition to oral efficacy, in the latter model Ro 22-3747 was 23-fold more potent than cromoglycate by the aerosol (nebulization) route of administration.In vitro studies have confirmed that the mechanism of action of Ro 22-3747 in thein vivo models is through allergic mediator release inhibition since Ro 22-3747 was a potent inhibitor of antigen-induced (IgE-mediated) histamine release from passively sensitized rat peritoneal cellsin vitro (IC50 values of 0.25 and 1.5 μM for Ro 22-3747 and cromoglycate, respectively), and Ro 22-3747 did not display end organ antagonism to histamine, serotonin, or the leukotrienes. Clinical evaluations of Ro 22-3747 at a 350 mg oral dose have been conducted in patients with allergic asthma and allergic rhinitis. In a limited study in allergic asthmatics, Ro 22-3747 demonstrated significant inhibitory activity relative to placebo in reducing acute airway responses to inhaled pollen extracts in patients with ragweed sensitivity (measured by changes in log PD20 FEV1 and log PD35 SGaw). The activity seen, however, was less than that observed with cromoglycate (20 mg) administered by inhalation. In a pilot evaluation in patients with allergic rhinitis, Ro 22-3747 administered at a 350 mg oral dose exhibited activity significantly greater than placebo in terms of improvements in the objective measurement of changes in nasal airflow at 2 and 3 hrs after treatment and the subjective overall global evaluation made by the patients at the end of the treatment period. The preclinical pharmacology and initial clinical efficacy of Ro 22-3747 support the continued clinical evaluation of this drug for prophylactic use in the management of asthma and other allergic disorders.

Publisher Summary This chapter discusses the researches made during the past year on pulmonary and antiallergy agents. Clinical tests have proved that several leukotriene D 4 (LTD 4 ), the “first generation” compounds are not potent enough to fully test their role in treating asthmatics. More potent, orally-active compounds have recently been reported. LTB 4 is hypothesized to be a potential mediator of lung eosinophilia in asthmatics. For this reason, LTB 4 antagonists may be useful in treating asthma. Of special interest with regard to future antiasthmatic therapy are reports of new, orally-active, selective 5-LO inhibitors. ICI 207, 968 has been reported to be 200-times more potent an inhibitor of 5-LO than CO. The thromboxane (TXA 2 ) antagonist, GR 32191, has been reported to decrease allergen-induced bronchoconstriction in man. Phospholipases in the signal transduction mechanisms found in key cells are associated with the initiation of asthmatic disease. Two β 2 -adrenoceptor agonists, salmeterol, and formoterol have been evaluated clinically by inhalation. Recent progress in the cloning of the subunits of the human IgE receptor may allow the future development of high flux in vitro screens for non-peptide antagonists. In addition, histamine release inhibitory factors have been identified and may be novel tools in the development of mediator release inhibitors (MRI). Vasoactive intestinal peptide (VIP) has been postulated to be the peptide mediator of the non-adrenergic, non-cholinergic, inhibitory pathway. Thus, VIP may be an important endogenous bronchodilator in man. Pirenzepine, an orally-active specific M1 antagonist, increased lung volume in both large and small airways in asthmatics. These results indicated the functional role of M1 receptors in human airways and suggest the potential for the development of anticholinergic drugs based on pirenzepine.