Mast cell degranulation mediates bronchoconstriction via serotonin and not via renin release

Abstract

To verify the recently proposed concept that mast cell-derived renin facilitates angiotensin II-induced bronchoconstriction bronchial rings from male Sprague–Dawley rats were mounted in Mulvany myographs, and exposed to the mast cell degranulator compound 48/80 (300 µg/ml), angiotensin I, angiotensin II, bradykinin or serotonin (5-hydroxytryptamine, 5-HT), in the absence or presence of the renin inhibitor aliskiren (10 µmol/l), the ACE inhibitor captopril (10 µmol/l), the angiotensin II type 1 (AT₁) receptor blocker irbesartan (1 µmol/l), the mast cell stabilizer cromolyn (0.3 mmol/l), the 5-HT_2A/2C receptor antagonist ketanserin (0.1 µmol/l) or the α₁-adrenoceptor antagonist phentolamine (1 µmol/l). Bath fluid was collected to verify angiotensin generation. Bronchial tissue was homogenized to determine renin, angiotensinogen and serotonin content. Compound 48/80 contracted bronchi to 24 ± 4% of the KCl-induced contraction. Ketanserin fully abolished this effect, while cromolyn reduced the contraction to 16 ± 5%. Aliskiren, captopril, irbesartan and phentolamine did not affect this response, and the angiotensin I and II levels in the bath fluid after 48/80 exposure were below the detection limit. Angiotensin I and II equipotently contracted bronchi. Captopril shifted the angiotensin I curve ≈ 10-fold to the right, whereas irbesartan fully blocked the effect of angiotensin II. Bradykinin-induced constriction was shifted ≈ 100-fold to the left with captopril. Serotonin contracted bronchi, and ketanserin fully blocked this effect. Finally, bronchial tissue contained serotonin at micromolar levels, whereas renin and angiotensinogen were undetectable in this preparation. In conclusion, mast cell degranulation results in serotonin-induced bronchoconstriction, and is unlikely to involve renin-induced angiotensin generation.

Introduction

Mast cells have been proposed to synthesize and release renin (Silver et al., 2004). This observation was made in the human mastocytoma cell line HMC-1. However, subsequent studies in the mast cell line LAD2 and in primary mast cells isolated from mastocytosis patients did not confirm such synthesis (Krop et al., 2008, Krop et al., 2009), suggesting that renin synthesis is not a uniform property of mast cells. Recently, the mast cell degranulator compound 48/80, when applied to isolated rat bronchial rings, was shown to elicit bronchoconstriction (Veerappan et al., 2008). Since both a renin inhibitor, BILA2157, and the active metabolite of the angiotensin II type 1 (AT₁) receptor antagonist losartan, EXP3174, prevented this constriction, it was concluded that angiotensin generated by mast cell-derived renin mediated this phenomenon. Indeed, exogenous angiotensin II was capable of constricting isolated rat bronchi. Remarkably however, the level of exogenous angiotensin II that was required to mimic the compound 48/80-induced constrictor response amounted to ≈ 0.1 μmol/l. This is roughly 5 orders of magnitude above the plasma levels of angiotensins. Given the well-established kinetics of the renin × angiotensinogen reaction (Bohlender et al., 2000, Fisher and Hollenberg, 2005), where micromolar angiotensinogen levels in blood plasma result in picomolar circulating angiotensin I and II levels (Admiraal et al., 1993, Danser et al., 1992), the generation of submicromolar concentrations of angiotensin II would require substantial concentrations of angiotensinogen and renin. Moreover, mast cells contain a wide range of other mediators, like serotonin (5-hydroxytryptamine, 5-HT), which might also induce bronchoconstriction (Metcalfe, 2008). They also contain histamine, but rat bronchi (the preparation used to show renin-dependent bronchoconstriction following mast cell degranulation) are insensitive to histamine (Veerappan et al., 2008). Finally, although BILA2157 is a human renin inhibitor (Simoneau et al., 1999), it also blocks cathepsin D, and thus its antagonizing properties do not necessarily reflect rat renin inhibition. In addition, both losartan and its active metabolite EXP3174 have a wide range of non-AT₁ receptor-mediated effects (Caballero et al., 2000, Li et al., 1997, Liu et al., 1992, Lynch et al., 1999, Thomas et al., 1996).

Given these uncertainties, we set out to further investigate the compound 48/80-induced constriction of rat bronchi. Not only did we study the blocking effects of a renin inhibitor, aliskiren, an ACE inhibitor, captopril, and an AT₁ receptor blocker, irbesartan, on the constriction induced by compound 48/80 in rat bronchial rings, but we also verified angiotensin generation under these conditions, and determined the renin and angiotensinogen content of rat bronchi. Finally, we evaluated the possibility that compound 48/80 induces the release of alternative bronchial constrictors such as serotonin.