Life Sciences, Vol. Printed in the USA

51, pp. PL 201-205

Pergamon P r e s s

PHARMACOLOGY Accelerated

LETTERS

Communication

A N T I A L L E R G I C MECHANISMS OF B E T A - A D R E N E R G I C STIMULANTS IN RATS Naoki Inagaki, Toru Miura, Hiroichi Nagai and Akihide Koda Department of Pharmacology, Gifu Pharmaceutical University 5-6-1, Mitahorahigashi, Gifu 502, Japan (Submitted July 28, 1992; accepted August i0, 1992; received in final form September 15, 1992)

Abstract. Antiallergic mechanisms of beta-adrenergic stimulants were investigated in rats. Isoproterenol administered intravenously inhibited IgE antibody-mediated homologous passive cutaneous anaphylaxis (PCA) and histamine-induced cutaneous reaction (HCR) elicited at the same time in the same rats significantly. The inhibition of PCA was more potent than that of HCR, suggesting that PCA is inhibited by at least 2 mechanisms. One is the inhibition of vascular permeability increase. In vivo histamine release in the rat peritoneal cavity caused by intravenous antigen was inhibited by the intravenous administration of isoproterenol or salbutamol dosedependently. On the contrary, when the histamine release in the peritoneal cavity was caused by intraperitoneal antigen, isoproterenol or salbutamol administered simultaneously with antigen failed to inhibit the reaction. Furthermore, antigen-induced histamine release from sensitized peritoneal exudate cells in vitro was not inhibited by isoproterenol or salbutamol. These results indicate that the primary target of beta-adrenergic stimulants is the vascular endothelium, and that the direct inhibition of chemical mediator release from mast cells does not play an important role for the inhibition of PCA and in vivo histamine release in the peritoneal cavity in rats. Beta-adrenergic stimulants therefore may prevent intravenously administered antigen from activating sensitized mast cells through affecting endothelial cells.

Introduction Beta-adrenergic stimulants are important drugs for treatment of asthma as bronchodilators (1,2). These agents have a potent relaxing effect on contracted smooth muscle accompanied by an increase in intracellular cyclic AMP levels (3). It is reported that these agents also exhibit an inhibitory effect of chemical mediator release from mast cells and basophils through increasing intracellular cyclic AMP levels (4,5). However, it is difficult to demonstrate a potent inhibition of chemical mediator release in rats (6-10). In 1982, Marquardt and Wasserman demonstrated that the histamine release from rat peritoneal mast cells was not inhibited by beta-adrenergic stimulants in spite of the increased intracellular cyclic AMP levels (7). Previously we indicated that beta-adrenergic stimulants inhibit IgE antibody-mediated homologous passive cutaneous anaphylaxis (PCA) and histamine-induced cutaneous reaction (HCR) in rats, and that the inhibition of vascular permeability increase contributes to the PCA inhibition partly (11). In the present study, mechanisms involved in the PCA inhibition in rats were further investigated. Materials and Methods Male Wistar rats weighing 200-250 g obtained from Japan SLC Inc. (Hamamatsu, Japan) were used throughout. Dinitrophenyl-conjugated Ascaris suum extract (DNP-As) and bovine serum albumin (DNP-BSA) were prepared according to the method described by Eisen et al. (12). Rat antiDNP-As serum was prepared according to the method described by Tada and Okumura (13). The 0024-3205/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd All rights reserved.

PL-202

Antiallergic

Action

of B e t a - s t i m u l a n t s

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51, No.

21,

1992

PCA titer of the antiserum preparation was 1:512. Isoproterenol (hydrochloride) and salbutamol (hemisulfate) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). PCA and HCR were elicited at the same time in the same rats (14). Rats were sensitized with an intradermal injection of 0.1 ml of 30-fold diluted anti-DNP-As serum into the shaved back skin. Forty-eight hours after the sensitization, 0.1 ml of 10 -5 g/ml of histamine (dihydrochloride, Nacalai Tesque, Kyoto, Japan) was injected intradermally into the back skin. Immediately after the injection of histamine, 1 ml of 0.5 % of Evans blue saline solution containing 1 mg of DNP-BSA was injected intravenously. Thirty minutes after the antigenic challenge, rats were sacrificed and the reaction sites on the back skin were excised. Isoproterenol was administered intravenously just before eliciting the reactions according to the previous report (11). The reactions were evaluated by determining the extravasated dye according to the method described by Katayama et al. (15). In vivo histamine release in the rat peritoneal cavity was examined in 2 ways. Rats were sensitized with an intraperitoneal injection of 2 ml of 20-fold diluted anti-DNP-As serum and histamine release was evoked 48 hours after the sensitization. 1) Histamine release was caused by an intravenous injection of 1 mg of DNP-As. Two minutes later, rats were sacrificed and 10 ml of icecold Tyrode solution was injected into the peritoneal cavity. Drugs were administered intravenously just before the antigen injection. 2) Histamine release was caused by an intraperitoneal injection of 10 ml of warmed Tyrode solution (37°C) containing 10-5 g/ml of DNP-As. Five minutes later, rats were sacrificed. Drugs were given simultaneously with the antigen. The peritoneal cavity was opened and the Tyrode solution was recovered using a pipette. After centrifugation, concentration of histamine in the supernatant was measured. In vitro histamine release from peritoneal exudate cells was examined as follows. Rats were sensitized similarly as mentioned above. Forty-eight hours later, peritoneal exudate cells were recovered using Tyrode solution containing 5 units/ml of heparin. Cells were washed 2 times with Tyrode solution and finally suspended in Tyrode solution at a concentration of 1-2 x 105 mast cells/ml. Histamine release was initiated by adding DNP-As at a final concentration of 10 -5 g/ml. After incubation with the antigen at 37 °C for 15 minutes, the reaction was terminated. Drugs were added 5 minutes before the antigen challenge (8). The reaction mixture was centrifuged, and the concentration of histamine in the supernatant was measured.

Released histamine was measured by a post-column derivatization method on an automated histamine analyzing system (Tosoh Co., Ltd., Tokyo, Japan) (16). In brief, histamine was separated by an HPLC column (TSKgel SP-2SW, 150 x 4.6 mm, Tosoh) using a mobile phase of 0.25 M KH2PO 4. Then histamine was coupled with o-phthalaldehyde, and determined on a spectrofluorometer. Results were expressed as the mean value and the standard error. Multiple comparisons were made to examine the statistical significance. When uniform variance of data was identified by Bartlett's analysis (P

Antiallergic mechanisms of beta-adrenergic stimulants in rats.

Antiallergic mechanisms of beta-adrenergic stimulants were investigated in rats. Isoproterenol administered intravenously inhibited IgE antibody-media...
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