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Pulmonary fibrosis is the result of abnormal processes of repair that occur after lung injury. Transforming growth factor (TGF)-beta is a key molecule in the progression of pulmonary fibrosis. Although clinical use of interferon (IFN)-beta did not improve survival in patients with idiopathic pulmonary fibrosis, because some preclinical studies have suggested that IFN-beta is a potent inhibitor of fibrogenesis, beneficial effects of IFN-beta have been expected. We therefore attempted to determine effects of IFN-beta and investigated the mechanism of action of IFN-beta in bleomycin-induced pulmonary fibrosis. Bleomycin at Day 0 and IFN-beta for 4 wk were administered intravenously to ICR mice. At 28 d after bleomycin injection, histologic and chemical analysis was performed for evaluation of effects of IFN-beta. Tissue distribution and amounts of TGF-beta1 and thrombospondin (TSP)-1/2 were analyzed. IFN-beta attenuated prolylhydroxylase activity, resulting in inhibition of pulmonary fibrosis. Bleomycin-induced increase in TGF-beta1 in epithelial cells and extracellular matrix was attenuated by IFN-beta. TSP-1/2 was limited in platelets of control mice, but was present in foamy cells in fibrotic regions induced by bleomycin. These findings suggest that the antifibrotic effect of IFN-beta is inhibition of TGF-beta and its activation via decrease in TSP-1/2 in lung tissue and change in location of TSP-1/2 from platelets to foamy cells.

Characteristics of Ca2+ release from stores were investigated in strips from ileum and portal vein and in isolated myocytes from ileum and urinary bladder of the guinea pig with use of caffeine and 9-methyl-7-bromoeudistomin D (MBED), a potent releaser of Ca2+ from skeletal muscle sarcoplasmic reticulum. In skinned strips, 1-30 mM caffeine elicited a transient contraction, but 10-300 microM MBED did not. Pretreatment with 100 microM MBED did not affect the subsequent caffeine-induced contraction. In single cells loaded with indo 1-acetoxymethyl ester, 10 mM caffeine increased cytoplasmic Ca2+ concentration, whereas 100 microM MBED elicited a small or no increase. Under whole cell clamp, spontaneous transient outward currents through Ca(2+)-dependent K+ (BK) channels were first enhanced and then suppressed by 30 microM MBED or 5 mM caffeine. The amplitude of Ca(2+)-dependent transient K+ current on depolarization was reduced by MBED and caffeine (50% inhibitory concentrations = 20 microM and 1 mM, respectively). Other currents and single BK channel activity were not significantly affected by MBED. The Ca2+ release from stores responsible for BK channel activation may be resolved from that for the activation of the contractile system by MBED in these smooth muscle cells.

The time course of two types of Ca(2+)-dependent currents were compared in single smooth muscle cells freshly isolated from guinea-pig trachea. When the pipette solution contained mainly 140 mM KCl, depolarization from -60 mV to 0 mV evoked an initial inward current followed by an outward current which consisted of transient (I(to)) and sustained components. In addition, a long-lasting inward tail current (Itail) was occasionally observed after the repolarization to -60 mV. Although I(to) often occurred repetitively during depolarization, the first I(to) reached the peak of approximately 50 ms after the start of depolarization and had the largest amplitude in most cells examined. The amplitude of Itail increased with the increase in depolarization period up to about 500 ms. Pharmacological analyses indicate that I(to) and Itail are Ca(2+)-dependent K+ and Cl- currents (IK-Ca and ICl-Ca), respectively, and suggest that not only Ca(2+)-influx through Ca2+ channels but also subsequent Ca2+ release from stores contributes to activate these currents. Spontaneous transient outward and inward currents, IK-Ca and ICl-Ca, respectively, were simultaneously recorded at -40 mV. In over 80% of the spontaneous current events, outward and inward currents coupled one to one and always occurred in this order. Puff-application of 10 mM caffeine also induced IK-Ca and ICl-Ca in this order at -40 mV. When caffeine was applied twice with various intervals, the current amplitude in the second application depended upon the period of the interval. The recovery of ICl-Ca during the interval was faster than that of IK-Ca. The results indicate that the activation and decay time courses of ICl-Ca are slower but its recovery is faster than those of IK-Ca.

The major functional roles of K channels in smooth muscle cells are as follows: (i) Keeping resting membrane potential (RMP). (ii) Induction of hyperpolarization (HP) in response to bioactive substances. (iii) Facilitation of action potential repolarization (APR). (iv) Induction of action potential afterhyperpolarization (AH). (v) Facilitation of slow wave repolarization. (vi) Inhibition of depolarization and/or action potential. Major background K channels responsible for RMP have not been identified yet, whereas it is been postulated that several K channels including Ca-dependent K channels and ATP-dependent K channels may co-contribute in part to RMP. Further activation of these channels by bioactive substances results in hyperpolarization and muscle relaxation. APR and AH in several types of smooth muscles are due to activation of mainly large conductance Ca-dependent K channels (BK) and additionally delayed rectifier K channels. Moreover, another type of delayed rectifier K channels and early inactivating K channels may also contribute to some of the functional roles shown above. Although activation of Ca-dependent Cl channels in response to agonists depolarizes the cell membrane in several types of smooth muscle cells, a single channel current has not been identified. Ca-dependent K and Cl currents can be regulated by intracellular Ca store sites, which may be exhausted after a large release induced by inositol 1, 4, 5-trisphosphate or caffeine. The Ca-pump activity in the store sites may also regulate indirectly but strongly the activity of the channels and consequently membrane excitability.

The characteristics of the cromakalim-induced membrane current were examined in single tracheal myocytes of the guinea-pig under voltage-clamp conditions. When K(+) concentrations in the pipette and bathing solutions were approximately 140 mM, cromakalim activated a membrane current (I(crom)) which was inward at -60 mV and reversed at -2 mV. I(crom) was blocked by 10 microM glibenclamide and potentiated when the ATP concentration in the pipette solution was decreased. The K(d) and Hill coefficient of glibenclamide for I(crom) block were 200 nM and 1.05, respectively. Application of the tyrosine kinase inhibitors, genistein and alpha-cyano-3-ethoxy-4-hydroxy-5-phenylthiomethylcinnamamid (ST638), reduced I(crom) in a concentration-dependent manner. Daidzein, which does not inhibit tyrosine kinase, was about 10 times less effective than genistein. Herbimycin A had no effect on I(crom). Internal application of these inhibitors from the pipette did not affect I(crom). In conclusion, cromakalim is a potent activator of the ATP-sensitive K(+) channel (K(ATP) channel) in guinea-pig tracheal myocytes. The inhibition of I(crom) by genistein and ST638 may be due to the direct block of the channel from outside.

Characteristics of caffeine-induced inward current (Icaf) and spontaneous transient inward current were examined in single smooth muscle cells isolated from guinea-pig trachea. When a pipette solution contained mainly CsCl, an application of 10 mM caffeine elicited transient Icaf at a holding potential of −60 mV. Spontaneous transient inward currents were recorded in about 15% of cells examined and were abolished by caffeine. Both were Cl− current activated by an increase in intracellular Ca2+ concentration (ICl-Ca). When 10 mM caffeine was puff-applied twice with various intervals, the amplitude of the second Icaf depended upon the period of the interval. The relationship between the amplitude and the interval represents the recovery time course of Icaf, which was significantly slowed by 30 μM cyclopiazonic acid. The Icaf was not significantly affected by addition of Cd2+. Removal of external Ca2+ did not affect the first Icaf but markedly reduced the second one, regardless of the presence of Cd2+. In conclusion, Icaf is evoked by activation of ICl-Ca via Ca2+ release. The recovery time course of Icaf indicates the refilling of Ca2+ storage sites by the cyclopiazonic acid-sensitive Ca2+ pump. The refilling at −60 mV depends strongly upon Ca2+ influx through the Cd2+-insensitive pathway. Spontaneous transient inward currents may be also due to ICl-Ca activated by spontaneous Ca2+ release from local storage sites.