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Treatment with acetylcholine (ACh) of a b-escin-permeabilized intrapulmonary bronchial smooth muscle of the rat induced force when the Ca 2+ concentration was clamped at 1 mM. The AChinduced Ca 2+ sensitization of myo®laments was signi®cantly greater in antigen-induced airway hyperresponsive rats than in control rats. The ACh-induced Ca 2+ sensitization was completely blocked by treatment with Clostridium botulinum C3 exoenzyme, an inactivator of Rho family of proteins. Moreover, the protein level of RhoA in the intrapulmonary bronchi was signi®cantly increased in the airway hyperresponsive rats. Thus, increased airway smooth muscle contractility observed in asthmatics may be related to augmented agonist-induced, Rho-mediated Ca 2+ sensitization of myo®laments.

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We investigated the effect of lipopolysaccharide (LPS) on the induction of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in muscularis resident macrophages of rat intestine in situ. When the tissue was incubated with LPS for 4 h, mRNA levels of iNOS and COX-2 were increased. The majority of iNOS and COX-2 proteins appeared to be localized to the dense network of muscularis resident macrophages immunoreactive to ED2. LPS treatment also increased the production of nitric oxide (NO), PGE(2), and PGI(2). The increased expression of iNOS mRNA by LPS was suppressed by indomethacin but not by N(G)-monomethyl-L-arginine (L-NMMA). The increased expression of COX-2 mRNA by LPS was affected neither by indomethacin nor by L-NMMA. Muscle contractility stimulated by 3 microM carbachol was significantly inhibited in the LPS-treated muscle, which was restored by treatment of the tissue with L-NMMA, aminoguanidine, indomethacin, or NS-398. Together, these findings show that LPS increases iNOS expression and stimulates NO production in muscularis resident macrophages to inhibit smooth muscle contraction. LPS-induced iNOS gene expression may be mediated by autocrine regulation of PGs through the induction of COX-2 gene expression.

In the pulmonary artery isolated from 1-week hypoxiainduced pulmonary hypertensive rats, endothelial NO production stimulated by carbachol was decreased significantly in in situ visualization using diaminofluorescein-2 diacetate and also in cGMP content. This change was followed by the decrease in carbachol-induced endothelium-dependent relaxation. Protein expression of endothelial NO synthase (eNOS) and its regulatory proteins, caveolin-1 and heat shock protein 90, did not change in the hypoxic pulmonary artery, indicating that chronic hypoxia impairs eNOS activity at posttranslational level. In the hypoxic pulmonary artery, the increase in intracellular Ca 2؉ level stimulated by carbachol but not by ionomycin was reduced. We next focused on changes in Ca 2؉ sensitivity of the eNOS activation system. A morphological study revealed atrophy of endothelial cells and a peripheral condensation of eNOS in hypoxic endothelial cells preserving co-localization between eNOS and Golgi or plasma membranes. However, eNOS was tightly coupled with caveolin-1, and was dissociated from heat shock protein 90 or calmodulin in the hypoxic pulmonary artery in either the presence or absence of carbachol. Furthermore, eNOS Ser 1177 phosphorylation in both conditions significantly decreased without affecting Akt phosphorylation in the hypoxic artery. In conclusion, chronic hypoxia impairs endothelial Ca 2؉ metabolism and normal coupling between eNOS and caveolin-1 resulted in eNOS inactivity.Hypoxia occurs commonly in patients with cardiopulmonary disease, and in normal individuals at high altitude. Chronic hypoxia is regarded as one of the critical factors that cause sustained pulmonary hypertension and aggravate pathophysiological conditions, and various forms of pulmonary hypertension pose a significant medical problem. Although there are many studies investigating the role of Kv channel, endothelin-1, and serotonin as the pathogenesis of the hypoxic pulmonary vasoconstriction (1-3), the mechanism governing the development of this syndrome is unclear. Consequently, current options for effective prevention and therapy are limited.The vascular endothelium releases various vasoactive products such as NO, which contributes not only to the local regulation of vascular smooth muscle tone (4), but also to cell proliferation (5). Impairment of NO release is observed in pulmonary hypertension caused by chronic obstructive lung disease, cardiopulmonary bypass, and congestive heart failure (6 -8), and loss of NO production has long been considered to be a pathogenesis of pulmonary hypertension (9, 10). However, serious doubt has been raised, because endothelial NO synthase (eNOS) 1 protein expression has been shown to be somewhat up-regulated in pulmonary hypertensive patients (11,12), and also because eNOS enzyme activity in lung homogenate is increased despite the impairment of endothelium-dependent relaxation in hypoxia-induced pulmonary hypertensive rats (13). It is therefore possible that impairment of endothelial NO production is a resu...

Interleukin-1␤ (IL-1␤) is a proinflammatory cytokine that plays a central role in inflammatory bowel disease (IBD). In order to elucidate the mechanism of motility disorders frequently observed in IBD, we investigated the long term effects of IL-1␤ on rat ileal smooth muscle contractility by using an organ culture system. When ileal smooth muscle strips were cultured with IL-1␤ (10 ng/ml), contractions elicited by high K ؉ and carbachol were inhibited in a time-dependent manner. IL-1␤ more strongly inhibited the carbachol-induced contractions than high K ؉ with decreasing myosin light chain phosphorylation. In the ␣-toxin-permeabilized ileal muscle, carbachol with GTP or guanosine 5 -3-O-(thio)triphosphate increased the Ca 2؉ sensitivity of contractile elements, and this G protein-coupled Ca 2؉ sensitization was significantly reduced in the IL-1␤-treated ileum. Among the functional proteins involved in the smooth muscle Ca 2؉ sensitization, CPI-17 expression was significantly reduced after the culture with IL-1␤, whereas the expressions of RhoA, ROCK-I, ROCK-II, MYPT-1, myosin light chain kinase, and myosin phosphatase (PP1) were unchanged. The phosphorylation level of CPI-17 by carbachol was low in accordance with the decrease in CPI-17 expression due to IL-1␤ treatment. In contrast, constitutively phosphorylated MYPT-1 was also decreased in the IL-1␤-treated muscles. These results suggest that long term treatment with IL-1␤ decreases either CPI-17 expression or MYPT-1 phosphorylation, which may result in an increase in myosin phosphatase activity to reduce force generation. Based on these findings, we consider IL-1␤ to be an important mediator of gastrointestinal motility disorders in IBD, and CPI-17 and MYPT-1 are key molecules in the decreased smooth muscle contractility due to IL-1␤.

17 beta-Estradiol inhibits the contraction of coronary vascular smooth muscle mainly inhibiting Ca2+ influx without changing Ca2+ sensitivity of contractile elements. The Ca2+ channel blocker-like action of 17 beta-estradiol may explain at least a part of the antiatherosclerotic effect of estrogen.