Microsoft Word - IAA371BF

Certain flavonoids inhibit antigen-induced release of histamine from mast cells and basophils and also inhibit contraction of guinea pig ileum induced by histamine, acetylcholine, and PGE2. We examined the effect of one flavonoid, quercetin, on anaphylactic smooth muscle contraction of ileum from guinea pigs sensitized to egg albumin. Quercetin inhibited both the phasic and tonic components of anaphylactic contraction in a concentration-dependent fashion (IC50 approximately 10 M). Whether this is primarily an effect on mast cell mediator release or inhibition of mediator effects on smooth muscle has not been established. Correspondence to: Dr. M.J. Fanning, State University of New York at Buffalo, and the Buffalo General Hospital, 100 High Street, Buffalo, NY 14203 (USA) Quercetin, a pentahydroxyflavone, belongs to a class of ubiquitous plant compounds known as flavonoids [1] and is structurally related to the antiallergic drug cromolyn sodium. These compounds are of interest because of the antiallergic properties which have been ascribed to them. For example, the flavonoid d-catechin prevents anaphylaxis in sensitized guinea pigs challenged with horse serum [2]. Derivatives of phloretin inhibit anaphylactic histamine release from passively sensitized human lung tissue [3]. Quercetin inhibits ragweed antigen-induced histamine release from human basophils of subjects with hay fever [4, 5] and also from rat mast cells [6, 7]. Quercetin and certain other flavonoids inhibit guinea pig ileal smooth muscle contraction induced by the agonists histamine, acetylcholine, and PGE2 [8]. We examined the effect of quercetin, the most active flavonoid in the basophil system, on anaphylactic contraction of guinea pig ileal longitudinal smooth muscle. Hartley guinea pigs were anaphylactically sensitized by subcutaneous administration in the lower back of 1 ml of a suspension of 100 μg of egg albumin (5 times recrystallized, Calbiochem, La Jolla, Calif.) in complete Freund’s adjuvant (Difco Labs, Detroit, 1 Supported by an Asthma and Allergic Diseases Center Grant (AI 14198). Mich.) [9]. Anaphylactic sensitivity was present 3 weeks after injection. Hartley male albino guinea pigs weighing 300–500 g were stunned and exsanguinated and the terminal 25 cm of ileum was removed and placed in Tyrode’s solution at 37 °C. The longitudinal muscle was removed from the underlying circular muscle by a method similar to that described by Rang [10]. The muscle was cut into 2.5-cm strips that were individually suspended in 10-ml jacketed glass tissue baths containing Tyrode’s buffer maintained at 37 °C and gassed with 95% 02:5% CO2. The Tyrode’s solution was of the following composition (millimolar): NaCl 137, KC1 2.7, CaCl2 1.8, MgCl2 2.1, NaH2P04 0.42, NaHCC1⁄8 12.0, and glucose 5.0 and was adjusted with 107V sodium hydroxide to give a final bath pH of 7.2–7.35. The lower end of the D ow nl oa de d by : 54 .1 91 .4 0. 80 4 /1 9/ 20 17 6 :5 4: 09 A M muscle was fixed and the upper end was threaded to the lever of a force displacement transducer (Grass Instruments FT03C, Grass Medical Instruments, Quincy, Mass.) with a resting tension of 300 mg. Contractile responses were recorded isometrically with an ink writing polygraph (Grass Instruments, Model 5D). Tracings were recorded on curvilinear paper at 0.5 mm/s. Tissues were equilibrated in Tyrode’s solution for 60 min with changes every 15–20 min. Phasic contraction was defined as the point of initial maximal pen deflection 372 Fanning/Macander/Drzewiecki/Middleton E3⁄43⁄43⁄4 Phasic Tonic 50 5 10 20 Quercetin concentration, μM Fig. 1. Graphic representation of data shown in table I. 140í D ow nl oa de d by : 54 .1 91 .4 0. 80 4 /1 9/ 20 17 6 :5 4: 09 A M 120 100 60 & 40 20 50 5 10 20 Quercetin concentration, μM Fig. 2. Log concentration-response relationship of data in table I (for the phasic component of anaphylactic contraction). Table I. Effect of quercetin on anaphylactic smooth muscle contraction a Mean ± SEM. Six experiments. b p < 0.001 compared to control contraction (100%). and tonic contraction as the postphasic, sustained contraction (higher than resting tension) which was measured arbitrarily at 90 s. After equilibration in the tissue bath histamine-in-duced contractions were obtained with each muscle to insure that it was normally responsive. Occasional unresponsive tissues were discarded. Quercetin was dissolved in DMSO (Sigma Chemical Co., St. Louis, Mo.) and the maximum final bath concentration did not exceed 0.25%. DMSO at this concentration had neither a direct effect on the muscle nor on anaphylactic contractions. Quercetin was added to one bath to give final concentrations of 5, 10, 20, or 50 μM, and was left in contact with the muscle for 10 min before addition of antigen. Controls consisted of muscle strips of equal length taken from the same segment of ileum. 100 μg of egg albumin was added to the baths yielding a single maximal contraction. Both phasic and tonic responses were measured as millimeters of pen deflection and the effect of quercetin on anaphylactic contraction was expressed as percent of control contraction height obtained in the absence of quercetin. Statistical analysis consisted of the two-tailed t test and a p value of less than 0.05 was accepted as indicative of a significant difference between two sets of observations. Quercetin inhibited anaphylactic contraction of guinea pig ileum in a concentration-dependent fashion (table I). Phasic contractions were significantly less than control for all concentrations of D ow nl oa de d by : 54 .1 91 .4 0. 80 4 /1 9/ 20 17 6 :5 4: 09 A M quercetin except 5 μM. Tonic contractions were significantly less than control at concentrations of 20 and 50 μM quercetin. Linear regression analysis indicated an IC50 of approximately 10μM(fig. 1,2). Based on quercetin’s known effects on basophil [4, Quercetin Inhibits Anaphylaxis 373 5] and rat mast cell [6, 7] histamine release and agonist-induced smooth muscle contraction [8], it is perhaps not surprising that it was found to be a potent inhibitor of anaphylactic smooth muscle contraction. It is not clear whether this is due primarily to an effect on mediator release, mediator action, or both. However, two bits of evidence support the contention that inhibition of mediator release is the primary mode of action: (1) d-catechin prevented anaphylaxis in sensitized guinea pigs but did not inhibit histamine shock in the same animals [2], and (2) the concentration-response relationship in the present experiments is very similar to that found in the basophil histamine release experiments [4] but this could be fortuitous. Mobilization of Ca2+ and an increase in cytosolic Ca2+ concentration is required to trigger smooth muscle contraction [11] and it has been shown that the phasic and tonic components of contraction rely mainly on membrane-bound and extracellular Ca2+, respectively [12]. Therefore, quercetin must act in some fashion to reduce the availability of Ca2+ to the contractile machinery of smooth muscle cells but the precise locus (or loci) of action remains to be identified. Quercetin is known to have inhibitory effects on a number of enzyme systems including phospholipase A2, [13], catechol-O-methyltransferase, cyclic nucleo-tide phosphodiesterases, and adenosine triphospha-tases, amongst others [cf. 5]. Conceivably quercetin (and other flavonoids) may inhibit some crucial enzymes involved in cell activation due to different stimuli including anaphylactic and agonist-induced smooth muscle contraction. Foucard, T.; Strandberg, K.: Inhibition by derivatives of phlor-etin of anaphylactic histamine release from human lung tissue and of prostaglandin Frinduced bronchoconstriction. Int. Archs Allergy appl. Immun. 48: 132–142(1975). Middleton, E., Jr.; Drzewiecki, G.; KrishnaRao, D.: Quercetin: an inhibitor of antigen-induced human basophil histamine release. J. Immun. 127: 546–550(1981). Middleton, E., Jr.; Drzewiecki, G.: Effects of flavonoids and transitional metal cations on antigen-induced histamine release from human basophils. Biochem. Pharmac. 31: 1449–1453 (1982). Fewtrell, C.M.S.; Gomperts, B.D.: Effect of flavone inhibitors of transport ATPases on histamine secretion from rat mast cells. Nature, Lond. 265: 635–636(1977). Fewtrell, C.M.S.; Gomperts, B.O.: Quercetin: a novel inhibitor of Ca2+ influx and exocytosis in rat peritoneal mast cells. Bio-chim. biophys. Acta 499: 52–60(1977). Macander, P.J.; Middleton, E., Jr.: The effect of flavonoids on acetylcholine, prostaglandin and antigen-induced contraction in smooth muscle (in preparation, 1982). Levine, B.B.; Chang, H., Jr.; Vaz, N.M.: The production of hapten specific reaginic antibodies in the guinea pig. J. Immun. 106: 29–33(1971). Rang, H.P.: Stimulant actions of volatile ANA anesthetics on smooth muscle. Br. J. Pharmacol. 22: 356–365 (1964). D ow nl oa de d by : 54 .1 91 .4 0. 80 4 /1 9/ 20 17 6 :5 4: 09 A M Hurwitz, L.; Suria, A.: The link between agonist action and response in smooth muscle. A. Rev.Pharmacol. //: 303–326 (1971).Chang, K.-J.; Triggle, D.J.: Quantitative aspects of drug-receptor interactions. I. Ca2+ andcholinergic receptor activation in smooth muscle: a basic model for drug-receptor interactions. J.theor. Biol. 40: 125–154(1973).Lee, T.-P.; Matteliano, M.L.; Middleton, E., Jr.: Effect of quercetin on humanpolymorphonuclear leukocyte lysosomal enzyme release and phospholipid metabolism. Life Sci.(in press, 1982). ReferencesKunhau, J.: The flavonoids. A class of semi-essential food components: their role in humannutrition. Wld Rev. Nutr. Diet., vol. 24, pp. 117–191 (Karger, Basel 1976).Moss, J.N.; Beiler, J.M.; Martin, G.J.: Inhibition of anaphylaxis in guinea pigs by d-catechin.Science 112: 16(1950). Downloadedby: 54.191.40.80-4/19/20176:54:09AM