Thapsigargin, a Ca2+-ATPase inhibitor, relaxes guinea pig tracheal smooth muscle by producing epithelium-dependent relaxing factors

A non-phorbol ester-type tumor promoter, thapsigargin has been reported to deplete Ca2+ stores in endothelial cells by inhibiting Ca2+-ATPase, which i n turn increases intracellular Ca2+ by mobilization of extracellular Ca2+, leading to activation of constitutive nitric oxide synthase (cNOS) and resu ltant generation of nitric oxide (NO). In the present study, to evaluate th e role of Ca2+ in the release of epithelium-dependent relaxing factor (EpDR F), we determined the effect of thapsigargin (10(-6) M) on the contraction evoked by exogenous Ca2+ or acetylcholine (10(-5) M) in epithelium-denuded or epithelium-intact smooth muscle from guinea pig trachea. The following r esults were obtained: (1) In epithelium-denuded smooth muscle, the contract ion evoked by exogenous Ca2+ in Ca2+-free solution or by acetylcholine (10( -5) M) in Ca2+-containing solution did not change within 20 min after thaps igargin application, but the contraction evoked by exogenous Ca2+ increased markedly after 120 min, indicating that thapsigargin had no effect on smoo th muscle itself within 20 min of application. The following experiments we re performed within 20 min of thapsigargin application. (2) In epithelium-i ntact smooth muscle, thapsigargin significantly suppressed the contraction evoked by acetylcholine, suggesting that thapsigargin stimulate the epithel ium to produce EpDRF. NG-nitro-L-arginine methylester (L-NAME) partly, but significantly, attenuated this inhibitory effect of thapsigargin. (3) In ep ithelium-denuded smooth muscle, atropine (10(-6) M) and L-NAME (10(-5) M) d id not change the contraction evoked by exogenous Ca2+ after application of thapsigargin, suggesting that thapsigargin did not stimulate acetylcholine and NO release from nerve terminals. These results suggest that thapsigarg in (10(-6) M) may stimulate EpDRF, including NO and other factor(s) by Ca2-dependent mechanisms. (C) 2000 Elsevier Science B.V. All rights reserved.