Background and purpose: Recent studies on blood vessels have provided evidence that testosterone may exert direct effects on smooth muscle. However, an acute effect on airway reactivity has not been shown yet. The aim of this study was to assess the direct effect of testosterone on the responsiveness of male adult rabbit airway smooth muscle (ASM), precontracted with 10 mM acetylcholine, 10mM carbachol or 80 mM KCl. Experimental approach: Contractility studies of rabbit tracheal smooth muscle were performed. Key results: Testosterone at concentrations of or above 1 nM had a significant relaxant effect on ASM precontracted with acetylcholine or carbachol, but did not affect ASM precontracted with KCl. The mechanical removal of airway epithelium as well as the inhibition of NO synthetase (by 100mM L-NAME) reduced the relaxation caused by testosterone. The effect of testosterone was not altered by impairing prostanoid synthesis (by 10mM indomethacin). The nitric oxide donor, sodium nitroprusside, had the same relaxant effect on ASM precontracted with either carbachol or KCl. Inhibitors of androgen receptors (10mM flutamide) or DNA transcription (100mM actinomycin D) did not alter the effect of testosterone. Prolonged incubation of ASM with 100 nM or 100 mM testosterone for 24 or 48 h did not alter their responsiveness to acetylcholine. BSAtestosterone (1pM to 100nM) relaxed significantly ASM precontracted with carbachol. The mechanical removal of airway epithelium abolished the relaxant effect of BSA-testosterone. Conclusions and implications: Testosterone relaxes precontracted ASM via an epithelium and NO-mediated way. This effect is mediated via a non-genomic pathway.

TO THE EDITOR: Recently, Massaro and Massaro (4) demonstrated a sexual dimorphism of alveolar size in male and female adult rats and mice and that estrogen receptor (ER)-␣ and ER-␤ are required for this sexual dimorphism.Our group recently showed that a similar phenomenon of sexual dimorphism presents in airway responsiveness to sex hormones in rabbits. We studied the direct effect of sex hormones (testosterone and 17␤-estradiol) on airway smooth muscle (ASM) at rest and precontracted with acetylcholine or carbachol. Testosterone induced a concentration-dependent (10 Ϫ12 M to 10 Ϫ4 M) contraction of ASM at rest and a concentration-dependent (10 Ϫ12 M to 10 Ϫ4 M) relaxation of ASM precontracted with 10 Ϫ5 M acetylcholine or carbachol (3). This action of testosterone was observed only in male rabbits, whereas testosterone had no effect in female rabbits. Mechanical removal of the airway epithelium abolished significantly (P Ͻ 0.05) the relaxing effect of testosterone (10 Ϫ7 M to 10 Ϫ4 M). In contrast to testosterone, 17␤-estradiol had an epithelium-independent relaxing effect on ASM from male rabbits precontracted with acetylcholine, only in the concentration of 10 Ϫ6 M (P ϭ 0.005). Similar results have been obtained in a previous study demonstrating that in male rabbits, ␤-estradiol caused an epithelial-independent relaxation of tracheal muscle strips precontracted with either acetylcholine or KCl (5). Indirect immunofluorescence with an anti-human androgen receptor (AR) monoclonal antibody revealed that ASM cells express AR. However, in culture of epitheliumintact ASM, the presence of testosterone (10 Ϫ8 M or 10 Ϫ4 M) for 24 or 48 h did not alter their responsiveness to acetylcholine. Besides, the testosterone effect was not influenced by the presence of the specific AR antagonist flutamide or the inhibitor of DNA transcription actinomycin D in the perfusing medium.Nevertheless, there are not many studies in airways. There are studies on vessels suggesting this phenomenon of sexual dimorphism. Many clinical and epidemiological studies show a bigger frequency of cardiovascular diseases in men than in pre-menopause women (1), whereas studies in rabbits demonstrated a different effect of testosterone in male and female animals in the development of athiromatic plaque (2).The results above suggest that in our experiments, sexual dimorphism was not due to classic steroid receptors. It remains to be investigated whether the action of testosterone on rabbit trachea is mediated by nonclassic membrane receptors that also exist in tracheal smooth muscle cells. REFERENCES 1. Barrett-Connor E. Sex differences in coronary heart diseases. Why are women so superior? The 1995 Ancel Keys Lecture. Circulation 95: 252-264, 1997. 2. Hanke H, Lenz C, Hess B, Spindler KD, Weidemann W. Effect of testosterone on plaque development and androgen receptor expression in the arterial vessel wall. Circulation 103: 1382-1385, 2001. 3. Kouloumenta V, Hatziefthimiou A, Paraskeva E, Gourgoulianis K, Molyvdas PA. Non-genomic effect of testosterone...

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