Mediator involvement in antigen-induced bronchospasm and microvascular leakage in the airways of ovalbumin sensitized Brown Norway rats

Abstract

1. To determine which mediators are involved in antigen-induced bronchospasm and microvascular leakage in the airways of ovalbumin sensitised Brown Norway rats we investigated the effect of a histamine H(1) receptor antagonist, mepyramine, a 5-HT receptor antagonist, methysergide, and a cys-leukotriene-1 receptor antagonist, montelukast. 2. Ovalbumin at 1 mg kg(-1) i.v. caused a significant increase in microvascular leakage in the airways and at 3 mg kg(-1) i.v. caused a significant increase in airways resistance. 3. Histamine (1 mg kg(-1) i.v.), 5-HT (0.1 mg kg(-1) i.v.) and leukotriene D(4) (LTD(4), 50 microg kg(-1) i.v.) caused a significant increase in microvascular leakage in the airways. 4. Mepyramine (1 mg kg(-1) i.v.), methysergide (0.1 mg kg(-1) i.v.), or montelukast (30 mg kg(-1) i.v.) inhibited histamine, 5-HT or LTD(4) -induced microvascular leakage respectively. 5. Methysergide (0.1 mg kg(-1) i.v.) reduced ovalbumin-induced microvascular leakage in the trachea and at 0.3 mg kg(-1) i.v. inhibited bronchospasm (38 and 58%, respectively). Montelukast (30 mg kg(-1) p.o.) reduced ovalbumin-induced microvascular leakage in airway tissue to basal levels (78%) and inhibited ovalbumin-induced bronchospasm (50%). Mepyramine (3 mg kg(-1) i.v.) had no effect on ovalbumin-induced leakage or bronchospasm. 6. A combination of all three compounds (mepyramine, methysergide and montelukast) reduced ovalbumin-induced microvascular leakage in airway tissue to basal levels (70 - 78%) and almost completely inhibited bronchospasm (92%). 7. Antigen-induced bronchospasm appears to equally involve the activation of 5-HT and cys-leukotriene-1 receptors whereas ovalbumin-induced microvascular leakage appears to be predominantly mediated by cys-leukotriene-1 receptors.

Figure 1

Effect of ovalbumin (0.1 – 3 mg kg⁻¹ i.v.) on microvascular leakage into the (a) trachea, (b) bronchi or (c) intra-pulmonary airways of antigen-sensitized Brown Norway rats. Rats were anaesthetised with isofluorane (3.5% in oxygen) and received Evans blue (20 mg kg⁻¹ i.v.), 1 min later they received ovalbumin or vehicle i.v. The rats were killed 15 min after ovalbumin or vehicle administration and tissues removed and placed in formamide for Evans blue dye extraction. Values are presented as data minus basal leak at time 0 and expressed as mean±s.e.mean of the concentration of Evans blue dye (ng mg⁻¹ of tissue). n=4.^*** P<0.001.

Figure 2

Effect of mepyramine (3 mg kg⁻¹ i.v.), methysergide (0.3 mg kg⁻¹ i.v), montelukast (30 mg kg⁻¹ p.o.) or a combination of all three on antigen-induced changes in airways resistance in antigen-sensitized Brown Norway rats. Rats were anaesthetized with sodium pentobarbitone (105 mg kg⁻¹ i.v.) and the trachea and oesophagus cannulated to facilitate the recording of airways resistance. Mepyramine or methysergide were administered 5 min (i.v.), or montelukast 90 min (p.o.), prior to vehicle (saline 0.9%) or ovalbumin (3 mg kg⁻¹ i.v). Changes in airways resistance (cmH₂ 0 m⁻¹ s⁻¹) were measured over a 10 min period and expressed as mean±s.e.mean. n=4. ^**P<0.01.

Figure 3

Effect of ovalbumin (1 mg kg⁻¹ i.v.), LTD₄ (0.001 – 0.05 mg kg⁻¹ i.v.), 5-HT (0.03 – 3 mg kg⁻¹ i.v.) or histamine (0.3 – 30 mg kg⁻¹ i.v.) on microvascular leakage into the (a) trachea, (b) bronchi or (c) intra-pulmonary airways of antigen-sensitized Brown Norway rats. Rats were anaesthetized with isofluorane (3.5% in oxygen) and received Evans blue (20 mg kg⁻¹ i.v.), 1 min later they received ovalbumin, LTD₄, 5-HT, histamine or vehicle i.v. The rats were killed 15 min after ovalbumin or mediator administration and tissues removed and placed in formamide for Evans blue dye extraction. Values are presented as data minus basal leak at time 0 and expressed as mean±s.e.mean of the concentration of Evans blue dye (ng mg⁻¹ of tissue). n=4. ^*P<0.05, ^**P<0.01, ^***P<0.001.

Figure 4

Effect of mepyramine (1 mg kg⁻¹ i.v.) on ovalbumin (1 mg kg⁻¹ i.v.), LTD₄ (0.05 mg kg⁻¹ i.v.), 5-HT (1 mg kg⁻¹ i.v.) or histamine (3 mg kg⁻¹ i.v.) induced microvascular leakage into the (a) trachea, (b) bronchi or (c) intra-pulmonary airways of antigen-sensitized Brown Norway rats. Rats were anaesthetized with isofluorane (3.5% in oxygen) and received Evans blue (20 mg kg⁻¹ i.v.), 1 min later they received ovalbumin, LTD₄, 5-HT, histamine or vehicle i.v. The rats were killed 15 min after ovalbumin or mediator administration and tissues removed and placed in formamide for Evans blue dye extraction. Values are presented as data minus basal leak at time 0 and expressed as mean±s.e.mean of the concentration of Evans blue dye (ng mg⁻¹ of tissue). n=4. ^*P<0.05, ^**P<0.01, ^***P<0.001 when compared to vehicle group. +++P<0.001 when compared to relevant control.

Figure 5

Effect of methysergide (0.1 mg kg⁻¹ i.v.) on ovalbumin (1 mg kg⁻¹ i.v.), LTD₄ (0.05 mg kg⁻¹ i.v.), 5-HT (1 mg kg⁻¹ i.v.) or histamine (3 mg kg⁻¹ i.v.) induced microvascular leakage into the (a) trachea, (b) bronchi or (c) intra-pulmonary airways of antigen-sensitized Brown Norway rats. Rats were anaesthetized with isofluorane (3.5% in oxygen) and received Evans blue (20 mg kg⁻¹ i.v.), 1 min later they received ovalbumin, LTD₄, 5-HT, histamine or vehicle i.v. The rats were killed 15 min after ovalbumin or mediator administration and tissues removed and placed in formamide for Evans blue dye extraction. Values are presented as data minus basal leak at time 0 and expressed as mean±s.e.mean of the concentration of Evans blue dye (ng mg⁻¹ of tissue). n=4. ^*P<0.05, ^**P<0.01, ^***P<0.001 when compared to vehicle group, +P<0.05, +++ P<0.001 when compared to relevant control.

Figure 6

Effect of montelukast (30 mg kg⁻¹ p.o.) on ovalbumin (1 mg kg⁻¹ i.v.), LTD₄ (0.05 mg kg⁻¹ i.v.), 5-HT (1 mg kg⁻¹ i.v.) or histamine (3 mg kg⁻¹ i.v.) induced microvascular leakage into the (a) trachea, (b) bronchi or (c) intra-pulmonary airways of antigen-sensitized Brown Norway rats. Rats were anaesthetized with isofluorane (3.5% in oxygen) and received Evans blue (20 mg kg⁻¹ i.v.), 1 min later they received ovalbumin LTD₄, 5-HT, histamine or vehicle i.v. The rats were killed 15 min after ovalbumin or mediator administration and tissues removed and placed in formamide for Evans blue dye extraction. Values are presented as data minus basal leak at time 0 and expressed as mean±s.e.mean of the concentration of Evans blue dye (ng mg⁻¹ of tissue). n=4. ^*P<0.05, ^**P<0.01, ^***P<0.001 when compared to vehicle group. ++ P<0.01, +++ P<0.001 when compared to relevant control.

Figure 7

Effect of mepyramine (1 mg kg⁻¹ i.v.), methysergide (0.1 mg kg⁻¹ i.v.), montelukast (30 mg kg⁻¹ p.o.) or a combination of all three on ovalbumin (1 mg kg⁻¹ i.v.) induced microvascular leakage into the (a) trachea, (b) bronchi or (c) intra-pulmonary airways of antigen-sensitized Brown Norway rats. Rats were anaesthetized with isofluorane (3.5% in oxygen) and received Evans blue (20 mg kg⁻¹ i.v.), 1 min later they received ovalbumin i.v. The rats were killed 15 min after ovalbumin administration and tissues removed and placed in formamide for Evans blue dye extraction. Values are presented as data minus basal leak at time 0 and expressed as mean±s.e.mean of the concentration of Evans blue dye (ng mg⁻¹ of tissue). n=4. ^**P<0.01, ^***P<0.001 when compared to vehicle/ovalbumin group.