Mast cell renin and a local renin-angiotensin system in the airway: role in bronchoconstriction

Abstract

We previously reported that mast cells express renin, the rate-limiting enzyme in the renin-angiotensin cascade. We have now assessed whether mast cell renin release triggers angiotensin formation in the airway. In isolated rat bronchial rings, mast cell degranulation released enzyme with angiotensin I-forming activity blocked by the selective renin inhibitor BILA2157. Local generation of angiotensin (ANG II) from mast cell renin elicited bronchial smooth muscle contraction mediated by ANG II type 1 receptors (AT(1)R). In a guinea pig model of immediate type hypersensitivity, anaphylactic mast cell degranulation in bronchial rings resulted in ANG II-mediated constriction. As in rat bronchial rings, bronchoconstriction (BC) was inhibited by a renin inhibitor, an AT(1)R blocker, and a mast cell stabilizer. Anaphylactic release of renin, histamine, and beta-hexosaminidase from mast cells was confirmed in the effluent from isolated, perfused guinea pig lung. To relate the significance of this finding to humans, mast cells were isolated from macroscopically normal human lung waste tissue specimens. Sequence analysis of human lung mast cell RNA showed 100% homology between human lung mast cell renin and kidney renin between exons 1 and 10. Furthermore, the renin protein expressed in lung mast cells was enzymatically active. Our results demonstrate the existence of an airway renin-angiotensin system triggered by release of mast-cell renin. The data show that locally produced ANG II is a critical factor governing BC, opening the possibility for novel therapeutic targets in the management of airway disease.

Fig. 1.

C48/80 elicits release of mast cell renin, triggering ANG II-induced contraction of rat bronchial rings. (A–G) Representative experimental traces of the C48/80-induced contractile responses in rat bronchial rings. (A, C, and E) Administration of C48/80 (300 μg/ml) elicits release of mast cell renin and induces smooth muscle contraction in bronchial rings (n = 12). The effect of C48/80 is attenuated by the mast cell stabilizer cromolyn (300 μM; n = 6) (B); the renin inhibitor BILA2157 (1 μM; n = 12) (D); and an AT₁R antagonist, EXP3174 (100 nM; n = 12) (F). y axis, Contractile responses to C48/80 were expressed as percentage of the response to K⁺ (80 mM). (G) Bar graph summarizing data from all experiments represented by traces in A–F. Bars indicate means ± SEM. **, Significantly different from own control; P < 0.01 by one-way ANOVA with Dunnett's procedure. W, wash.

Fig. 2.

Contractile responses of isolated rat bronchial rings to ANG II and methacholine. The administration of ANG II (open circles) and methacholine (solid circles) elicits a concentration-dependent contraction. Histamine (solid triangles; negative control) has no contractile effect. Contractile responses are expressed as percentage of the response to K⁺ (80 mM). Points are means (±SEM; n = 3).

Fig. 3.

Mast cells are closely apposed to smooth muscle cells expressing AT₁R in rat bronchus. (A–C) Rat bronchial mast cell colabeled with avidin-FITC (A) and an anti-renin Ab (B). Color overlay is shown in C with the nuclear stain, DAPI (blue). (Scale bar, 5 μm.) (D) Representative section of rat bronchus costained with avidin-rhodamine (to identify mast cells, red) and an anti-AT₁R Ab conjugated to Alexa Fluor 488. Smooth muscle cells stained positive for AT₁R (green). (Scale bar, 15 μm.) (E) Toluidine blue-stained mast cells adjacent to smooth muscle cells in rat bronchus. (Scale bar, 30 μm.)

Fig. 4.

Immediate hypersensitivity elicits the release of mast cell renin, resulting in ANG II-induced smooth muscle contraction in isolated guinea pig bronchial rings. Representative experimental traces of contractile responses measured in bronchial rings obtained from guinea pigs presensitized with rabbit anti-OA cytotropic IgG and challenged with OA (50 μg/ml) in the absence (control; n = 6) (A, C, and E) or presence of cromolyn (300 μM; n = 6) (B), BILA2157 (1 μM; n = 6) (D), or EXP3174 (100 nM; n = 6) (F). y axis, Contractile responses expressed as percentage of the response to K⁺ (80 mM). (G) Bar graph summarizing data from all experiments represented by traces in A–F. Bars indicate means ± SEM. *, **, Significantly different from control; P < 0.05 and 0.01, respectively, by one-way ANOVA with Dunnett's multiple-comparison test. W, wash.

Fig. 5.

Concentration–response curves for the contractile effect of ANG II on sensitized guinea pig bronchial rings. The administration of ANG II (control) elicits concentration-dependent contraction of isolated sensitized guinea pig bronchial rings. The effect of ANG II is progressively attenuated by EXP3174 as a function of its concentration (10–100 nM). Contractile responses are expressed as percentage of the response to K⁺ (80 mM). Points are means (± SEM; n = 4).

Fig. 6.

Mast cells isolated from human lung express renin. (A) Toluidine-blue stained mast cells isolated from human lung tissue. (Scale bar, 10 μm.) (B) Total RNA was extracted from isolated human lung mast cells and reverse transcribed. cDNA was amplified by PCR using specific primers for human renin gene (exons 4–7). A 100-bp DNA ladder (m) was run on the gel. (C) Mast cells isolated from human lung stained positive for renin protein with the monoclonal anti-renin Ab (1:200). (Scale bar, 10 μm.) (D) Measurement of renin activity [ANG I formed (in picograms per milliliter per hour)] in the lysate from isolated human lung mast cells (±SEM; n = 3).