Histamine N-methyltransferase Modulates Human Bronchial Smooth Muscle Contraction

To elucidate the modulatory role of histamine-degrading enzymes in airway constrictor responses, human bronchial strips were studied under isometric conditions in vitro. Pretreatment of tissues with the histamine N-methyltransferase (HMT) inhibitor SKF 91488 specifically potentiated the contractile responses to histamine, causing a leftward displacement of the concentration response curves, whereas the diamine oxidase inhibitor aminoguanidine had no effect. This potentiation was attenuated by mechanical removal of the epithelium. The HMT activity was detected in the human bronchi, which was less in the epithelium-denuded tissues than the epithelium-intact tissues. These results suggest that HMT localized to the airway epithelium may play a protective role against histamine-mediated bronchoconstriction in humans.


Introduction
Histamine is released from mast cells and basophils by a variety of stimuli including antigen cross-linked IgE, and induces airway smooth muscle contraction, microvascular leakage and mucus production." Thus histamine probably plays a principal role in the pathogenesis of immediate asthmatic responses. In addition, because increased bronchial responsiveness to histamine is a well-characterized feature of asthma, bronchial provocation by histamine has routinely been used to facilitate its diagnosis.
It has been known that histamine can be metabolized by two major pathways in the body; 4'5 50 to 70% of histamine is metabolized by histamine Nmethyltransferase (HMT, EC 2.1.1.8), located in the small intestine, liver, kidney and leukocytes, into Nmethylhistamine, and the remaining 30 to 45% is metabolized by diamine oxidase (DAO, EC  and were recorded on a pen recorder (Nihon Kohden, WT-685G). The tissues were allowed to equilibrate for 60 min while they were washed with Krebs-Henseleit solution every 15 min, and the resting tension was adjusted to 1 g. A contractile response was determined as the difference between peak tension developed and resting tension. All experiments were conducted in the presence of indomethacin (3 x 10 -6 M) and ranitidine (6 x 10 -5 M) to avoid prostaglandin release and histamine tachyphylaxis, 8 respectively.
Effects of histamine-degrading enzyme inbibitors: Following the equilibration period, histamine was added to the chamber in a cumulative manner at concentrations ranging from 10 -s to 10 -3 M in halfmolar increments at 5 min intervals or 2 min after stable plateau was achieved, whichever was the longer period. After establishing the first concentration-response curves, tissues were washed with Krebs-Henseleit solution until the tension returned to the baseline level and, 60 min later, SKF 91488 (10 -4 M), an inhibitor of HMT, 9'1 or aminoguanidine (10 -4 M), an inhibitor of DAO, 11 was added. Twenty min later, the second concentration-response curves for histamine were generated in a similar manner. In a control experiment, two successive concentration-response curves for histamine were likewise generated without addition of an inhibitor.
To test whether the inhibition of HMT activity also alters the contractile responses to other spasmogenic agonists, the effect of SKF 91488 (10 -4 M) on the concentration-response curves for acetylcholine and KC1 were examined, with the same time sequence for the contractile responses to histamine.
At the end of these experiments, each bronchial strip was blotted on a gauze pad and weighed. Active tensions were normalized for tissue weight and expressed as grams tension per gram of tissue weight.
To characterize the concentration-response curves, the maximal contractile response (Emax) and the negative logarithm of molar concentration of agonist required to produce 50% of Emax (pD,) were determined by linear regression analysis.
To assess concentration-dependent effects of histamine-degrading enzyme inhibitors, contractile responses of bronchial strips to 10 -5 M histamine were determined in the absence and presence of either SKF 91488 or aminoguanidine (10 to 10 -3 M). In this experiment, after determining the first responses to histamine, tissues were washed, applied with a single concentration of either inhibitor and, 20 min later, the second responses to histamine were determined.
To assess whether the modulation of the contractile responses to histamine by SKF 91488 was associated with the epithelium, concentration-response curves for histamine were constructed in the absence and presence of SKF 91488 (10 .4

Results
Muscle contraction: As demonstrated in Fig. 1 The SKF 91488-induced potentiation of the contractile responses to 10 -5 M histamine was concentration dependent, with a threshold concentration and the maximal increase from the baseline value being   Fig. 2). In contrast to the effect on histamine-induced contraction, pretreatment of tissues with SKF 91488 (10 -4 M) had no effect on the contractile responses to other spasmogenic agonists, acetylcholine and KCl (Table 1).
In bronchial strips with the epithelium removed, SKF 91488 (10-4 M) still potentiated the contractile responses to histamine, the pD values being increased from 5.6 _+ 0.3 to 6.1 _+ 0.2 (p< 0.05, n 11, Tissue HMT activity: The activity of HMT in human bronchi determined by high-performance liquid chromatography was 920 _+ 230 pmol/mg protein/h (n 11) for the epithelium-intact tissues and 280 + 70 pmol/mg protein/h (n 12) for the tissues without epithelium. There was a significant difference between these values (p< 0.05, Fig. 4).

Discussion
The present in vitro studies demonstrate that HMT probably localized to human airway epithelium may play a protective role in the histamine-mediated bronchoconstriction through the degradation of histamine to its inactive metabolite. This notion is based on the following findings. First, pretreatment of bronchial strips with the HMT inhibitor SKF 914889,1 potentiated the histamine-induced contraction in a concentration dependent fashion without affecting contractile responses to other spasmogenic agonists including acetylcholine and KCl; secondly, the magnitude of the SKF-induced leftward displacement of histamine concentration-response curves was significantly less in bronchial strips with the epithelium mechanically removed than in the epithelium-intact tissues; and thirdly, the activity of HMT was measured by high-performance liquid chromatography using post-column derivatization with ophthalaldehyde and it was found that the HMT activity of human bronchial tissues was greatly decreased by removal of the epithelium.
It has been known that histamine is metabolized by two major enzymes, HMT and DAO, located on a variety of mammalian tissues and inflammatory cells. TM HMT catalyses methyl transfer from Sadenosyl-t-methionine to histamine to form Nmethylhistamine, which is further metabolized by monoamine oxidase to N-methylimidazole acetic acid, 15 and DAO also metabolizes histamine to Nmethylimidazole acetic acid. 16 In the present study, it was found that, in contrast to the effect of SKF 91488, aminoguanidine at concentrations sufficient to inhibit DAO activity 11 had little effect on the contractile responses to histamine. Therefore, histamine may be metabolized principally through the HMT pathway in human airways, as is also true in the brain. 17 The airway epithelium has been shown to inhibit bronchoconstrictor responses to a variety of stimuli by releasing epithelium-derived relaxing factor, which is not a product of arachidonic acid and is not nitric oxide, 18-2 and by metabolizing tachykinins with neutral endopeptidase. 2 Concerning the histamine metabolism, there are conflicting reports on guinea-pig tracheal epithelium. Lindstr6m et al. showed the aminoguanidine-induced potentiation of the contractile responses to histamine, but Ohrui et al. recently reported the lack of aminoguanidine's effect and showed the presence of HMT activity and its mRNA in the epithelium by in situ hybridization, and Sekizawa et al. reported a similar finding in antigen-induced bronchoconstriction in vivo. The present findings on human bronchi were in agreement with the latter two reports. Although DAO activity was not measured, the results of the muscle bath experiments suggest that the epithelial HMT activity is more important than DAO in limiting the biological actions of histamine in human airways. In tissues without epithelium, the, HMT activity was still present and the SKF 91488-induced potentiation of the contractile responses to histamine was small but still significant. These findings suggest that HMT localized to other cell types such as endothelial cells could also contribute to histamine degradation.
In conclusion, the histamine-degrading enzyme HMT in the airway epithelium may play a modulatory role in the bronchoconstriction in human airways through a metabolism of histamine. The authors therefore speculate that airway hyperresponsiveness to histamine in asthmatic subjects could be due, at least in part, to the epithelial damage-associated loss of HMT activity.