Ann F. Welton

An in vivo model for measuring antigen-induced SRS-A-mediated bronchoconstriction and plasma S RS- A levels in the guinea-pig

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.

Specific in vitro activation of Ca/Mg-ATPase by vitamin D-dependent rat renal calcium binding protein (calbindin D28K)

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.

Pentadienyl carboxamide derivatives as antagonists of platelet activating factor

A series of N-[4-(3-pyridinyl)butyl]-5,5-disubstituted-pentadienamides was prepared and evaluated for PAF-antagonist activity. Compounds were assayed in vitro in a PAF-binding assay employing washed, whole dog platelets as the receptor source and in vivo after intravenous or oral administration for their ability to prevent PAF-induced bronchoconstriction in guinea pigs. Criteria required for good oral activity in the latter model include an (E,-E)-5-phenyl-2,4-pentadienamide, a second phenyl or a four- or five-carbon alkyl moiety in the 5-position of the diene, and an (R)-[1-alkyl-4-(3-pyridinyl)butyl] substituent on the carboxamide nitrogen atom. The alkyl substituent on this side chain can be methyl, ethyl, or cyclopropyl. Two members of this series, [R-(E)]-5,5-bis(4-methoxy-phenyl)-N- [1-methyl-4-(3-pyridinyl)butyl]- 2,4-pentadienamide (31) and [R-(E,E)]-5-(4-methoxyphenyl)-N-[1-methyl-4- (3-pyridinyl)butyl]-2,4-decadienamide (58), were selected for further pharmacological evaluation. Both were found to be substantially longer acting after oral administration than the corresponding S enantiomers in the guinea pig bronchoconstriction assay. A second in vivo model used to evaluate PAF antagonists determines the ability of test compounds to decrease the area of skin wheals induced by an intradermal injection of PAF. In this model, using both rats and guinea pigs, compounds 31 and 58 were found to be as active as the reference PAF antagonist 3-[4-(2-chlorophenyl)-9-methyl-6H- 1-(4-morpholinyl)-1-propanone (45).

Regulatory role of adenosine in antigen-induced histamine release from the lung tissue of actively sensitized guinea pigs

Abstract 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.

Biological activities of a chemically synthesized form of leukotriene E4

A chemically synthesized form of leukotriene E4 (LTE4) has been studied for its ability to induce contractions in isolated guinea pig ilea, to induce vascular permeability changes in rat skin when injected intradermally, and to induce bronchoconstriction in guinea pigs after intravenous injection. The synthetic compound induced a contraction in the guinea pig ileum which was slower in developing than that induced by histamine but faster in developing than that induced by a crude preparation of SRS-A isolated from guinea pig lung. The compound was 70-fold more active than histamine on the guinea pig ileum (EC50 of 5 x 10(-9) and 3.5 x 10(-7) M, respectively). FPL 55712, a known SRS-A antagonist, exhibited the same potency in blocking the contractions elicited by the synthetic material as it did in blocking contractions produced by guinea pig SRS-A generated biologically (IC50 of 3.5 x 10(-8) M). The synthetic LTE4 induced a dose dependent increase in vascular permeability in the rat skin which was antagonized by the intravenous injection of FPL 55712 (ID50 of 1.2 mg/kg). The synthetic material was also a potent bronchoconstrictor in the guinea pig when injected intravenously. The bronchoconstriction, too, was antagonized by FPL 55712 when injected intravenously (ID50 of 0.2 mg/kg). In both the rat and guinea pig, FPL 55712 exhibited a short duration of action in vivo. The in vivo model systems discussed in this study, utilizing the synthetic form of LTE4 should be useful in the future evaluation of other SRS-A antagonists.

Relaxant activity of atriopeptins in isolated guinea pig airway and vascular smooth muscle

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.

Identification of specific binding sites for leukotriene C4 in membranes from human lung

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 degrees, 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 X 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 complete with 3H-LTC4 for its binding sites with an IC50 of 7.8 X 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 (IC50 S = 2.2 X 10(-6), 2.4 X 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.

Sodiumcalcium exchange in sarcolemmal vesicles from tracheal smooth muscle

Abstract 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.

In vitro studies on the mechanism of action of a new antiallergic, Ro 21-7634.

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.

Analogs of arachidonic acid methylated at C-7 and C-10 as inhibitors of leukotriene biosynthesis

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-dimethyl-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 delta 5-lipoxygenase. These compounds failed to exhibit significant activity in an in vivo model designed to detect inhibitors of antigen-induced, leukotriene-mediated bronchoconstriction in sensitized guinea pigs.