Chloride channel activity in human lung fibroblasts and myofibroblasts

It is well established that transforming growth factor (TGF)-beta stimulates human lung fibroblasts (HLF) to differentiate into myofibroblasts. We characterized lysophosphatidic acid (LPA)-activated Cl- channel current (I(Cl-LPA)) in cultured human lung fibroblasts and myofibroblasts and investigated the influence of I(Cl-LPA) on fibroblast-to-myofibroblast differentiation. We recorded I(Cl-LPA) using the amphotericin perforated-patch technique. We activated I(Cl-LPA) using LPA or sphingosine-1-phosphate. We determined phenotype by Western blotting and immunohistochemistry using an anti-alpha-smooth muscle actin (SMA) antibody. RT-PCR was performed to determine which phospholipid growth factor receptors are present in HLF. We found that HLF cultured in TGF-beta (myofibroblasts) had significantly elevated alpha-SMA levels and I(Cl-LPA) current density compared with control fibroblasts. I(Cl-LPA) activation was blocked by DIDS, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), and the LPA receptor-specific antagonist dioctyl-glycerol pyrophosphate (1 microM). DIDS and NPPB, in a dose-dependent manner, significantly reduced alpha-SMA levels in HLF stimulated with TGF-beta. These results demonstrate the receptor-mediated activation of I(Cl-LPA) by LPA and sphingosine-1-phosphate in cultured human lung myofibroblasts, with only minimal I(Cl-LPA) activity in fibroblasts. This Cl- channel activity appears to play a critical role in the differentiation of human lung fibroblasts to myofibroblasts.     

Nongenomic effects of an anti-idiotypic antibody as an estrogen mimetic in female human and rat osteoblasts

We investigated the early effects of the anti-idiotypic antibody (clone 1D₅), which recognized the estrogen receptor (ER), on cytosolic free calcium concentration ([Ca²⁺]i) and its long term effects on creatine kinase (CK) specific activity in female human and rat osteoblasts. These actions were compared to the known membrane and genomic effects of 17β estradiol (E₂). Like E₂, clone 1D₅ increased within 5 s [Ca²⁺]i in both cell types by two mechanisms: 1) Ca²⁺ influx through voltage-gated Ca²⁺ channels as shown by using EGTA, a chelator of extracellular Ca²⁺, and nifedipine, a Ca²⁺ channel blocker; 2) Ca^2+; mobilization from the endoplasmic reticulum as shown by using phospholipase C inhibitors, such as neomycin and U-73122, which involved a Pertussis toxin-sensitive G-protein. Clone 1D₅ and E₂ stimulated CK specific activity in human and rat osteoblasts with ten fold higher concentrations than those needed for the membrane effects (0.1 μg/ml and 10 pM, respectively). Both effects were gender-specific since testosterone and 5α-dihydotesterone were uneffective. Tamoxifen and Raloxifene, two estrogen nuclear antagonists, inhibited CK response to 1D₅ and E₂ and Ca²⁺ response to 1D₅, but not CA²⁺ response to E₂. By contrast, (Fab′)₂ dimer, a proteolytic fragment of 1D₅ with antagonist properties, inhibited both membrane and genomic effects of 1D₅ and E₂. In conclusion, these results imply that clone 1D₅ has an estrogen like activity both at the membrane and nuclear levels in female human and rat osteoblasts. 1D₅ must therefore interact with membrane binding sites, penetrate the cells, and reach the nuclear receptors by an as yet uncharacterized mechanism. J. Cell. Biochem. 65:53–66. © 1997 Wiley-Liss, Inc.     

Contractile activity and smooth muscle alpha-actin organization in thrombin-induced human lung myofibroblasts

Activated fibroblasts, or myofibroblasts, are crucial players in tissue remodeling, wound healing, and various fibrotic disorders, including interstitial lung fibrosis associated with scleroderma. Here we characterize the signaling pathways in normal lung fibroblasts exposed to thrombin as they acquire two of the main features of myofibroblasts: smooth muscle (SM) alpha-actin organization and collagen gel contraction. Our results show that the small G protein Rho is involved in lung myofibroblast differentiation. Thrombin induces Rho-35S-labeled guanosine 5'-O-(3-thiotriphosphate) binding in a dose-dependent manner. It potently stimulates Rho activity in vivo and initiates protein kinase C (PKC)-epsilon-Rho complex formation. Toxin B, which inactivates Rho by ADP ribosylation, inhibits thrombin-induced SM alpha-actin organization, collagen gel contraction, and PKC-epsilon-SM alpha-actin and PKC-epsilon-RhoA coimmunoprecipitation. However, it has no effect on PKC-epsilon activation or translocation of PKC-epsilon to the membrane. Overexpression of constitutively active PKC-epsilon and constitutively active RhoA induces collagen gel contraction or SM alpha-actin organization, whereas, individually, they do not perform these functions. We therefore conclude that the contractile activity of myofibroblasts induced by thrombin is mediated via PKC-epsilon- and RhoA-dependent pathways and that activation of both of these molecules is required. We postulate that PKC-epsilon-RhoA complex formation is an early event in thrombin activation of lung fibroblasts, followed by PKC-epsilon-SM alpha-actin coimmunoprecipitation, which leads to the PKC-epsilon-RhoA-SM alpha-actin ternary complex formation.     

Nongenomic stimulation of nitric oxide release by estrogen is mediated by estrogen receptor alpha localized in caveolae.

Acute administration of 17beta-estradiol (E(2)) exerts antiatherosclerotic effects in healthy postmenopausal women. The vasoprotective action of E(2) may be partly accounted for by a rapid increase in nitric oxide (NO) levels in endothelial cells (ECs). However, the signaling mechanisms producing this rise are unknown. In an attempt to address the short-term effect of E(2) on endothelial NO production, confluent bovine aortic endothelial cells (BAECs) were incubated in the absence or presence of E(2), and NO production was measured. Significant increases in NO levels were detected after only 5 min of E(2) exposure without a change in the protein levels of endothelial NO synthase (eNOS). This short-term effect of estrogen was significantly blunted by various ligands which decrease intracellular Ca(2+) concentration. Furthermore, plasma membrane-impermeable BSA-conjugated E(2) (E(2)BSA) stimulated endothelial NO release, indicating that in the current system the site of action of E(2) is on the plasma membrane rather than the classical nuclear receptor. The partial antagonist tamoxifen did not block E(2)-induced NO production; however, a pure estrogen receptor alpha (ERalpha) antagonist ICI 182,780 completely inhibited E(2)-stimulated NO release. The binding of E(2) to the membrane was confirmed using FITC-labeled E(2)BSA (E(2)BSA-FITC). Western blot analysis showed that plasmalemmal caveolae possess ERalpha in addition to well-known caveolae-associated proteins eNOS and caveolin. This study demonstrates that the nongenomic and short-term effect of E(2) on endothelial NO release is Ca(2+)-dependent and occurs via ERalpha localized in plasmalemmal caveolae.     

Cytoskeletal features of alveolar myofibroblasts and pericytes in normal human and rat lung.

Frozen or paraffin-embedded human and rat lung specimens were stained with antibodies against total actin, alpha-smooth muscle (SM) actin, vimentin, desmin, or gelsolin. Alveolar interstitial myofibroblasts [i.e., contractile interstitial cells (CIC)] were labeled by total actin antibody but not by alpha-SM actin antibody. They stained for vimentin and gelsolin and, in rat lungs, most of them for desmin. Pericytes located around venules at the junction of three alveolar septa were always positive for alpha-SM actin and never for desmin. Tissue samples were also immunostained by an alpha-SM actin antibody and studied by electron microscopy. With this technique we confirmed that cells, identified as pericytes on the basis of their location, were intensely labeled by alpha-SM actin antibodies, whereas alveolar myofibroblasts were not. We conclude that in the lung interstitium pericytes and alveolar myofibroblasts have distinct cytoskeletal features, alpha-SM actin antibody staining being a simple method to distinguish between them. Furthermore, it appears that alveolar myofibroblasts have a peculiar pattern of cytoskeletal protein composition which, in the rat, is similar to that previously described for stromal cells in uterine submucosa, liver sinusoids (Ito cells), or the core of intestinal villi.     

Nongenomic vasodilator action of progesterone on primate coronary arteries.

In the present investigation, we test the hypothesis that progesterone can rapidly relax, via a nongenomic mechanism, persistent flow occluding, agonist-activated coronary artery (CA) vasospasm, and hyperreactive vascular muscle cell (VMC) Ca(2+) responses in ovariectomized rhesus monkeys. CA vasospasm, induced by injection of 100 microM serotonin and 1 microM U-46619 (5-HT+U; 1 ml/30 s), resulted in a decrease in CA diameter (phi) from 1.8 +/- 0.2 to 0.3 +/- 0.1 mm at the site of focal constriction. Injection of 100 ng progesterone into the CA significantly relieved the severe vasoconstriction (1.3 +/- 0.2 mm) and reestablished distal flow in 3 min; the preconstriction phi was completely restored in 8.2 +/- 2.6 min (n = 6). Similarly, cell impermeant albumin-conjugated progesterone, but not albumin-conjugated 17 beta-estradiol, decreased 5-HT+U stimulated VMC Ca(2+) responses (250 +/- 34% of basal 30 min after stimulation) back to the prestimulation level (113 +/- 17% of basal) in 25 min (half time = 7 min). The presence of a rapid vasodilator action of progesterone in the primate CA and isolated VMC suggests its benefits in hormone replacement therapy may also include nongenomic vascular relaxant actions.     

Androgens inhibit estrogen action in MCF-7 human breast cancer cells

We have observed that the estrogen-dependent augmentation of cytoplasmic progesterone receptor (PR_c) content in MCF-7 human breast cancer cells is completely inhibited in the presence of testosterone (T) or dihydrotestosterone (DHT)¹. This effect is neither a result of altered PR_c affinity for test ligand nor a result of the direct interaction of either androgen with PR_c. Furthermore, the antiestrogenic activity of DHT is blocked in the presence of the antiandrogen, cyproterone, indicating that it is mediated through the androgen receptor. Further investigations of this cell line may provide important insights into the effects of sex steroids upon the hormonal regulation of a variety of healthy and malignant human tissues.     

Role of estrogen receptor-alpha in pharmacogenetics of estrogen action

PURPOSE OF REVIEW: To summarize recent data regarding the role of estrogen receptor-alpha polymorphisms in determining the response to estrogen therapy or the risk of clinical cardiovascular events. RECENT FINDINGS: Recent clinical trials of hormone replacement therapy for cardiovascular disease have yielded surprisingly negative results, shifting clinical opinions from a position of presumed cardiovascular benefit to one of confirmed harm. Understanding why hormone replacement therapy has beneficial effects on intermediate risk markers for cardiovascular disease, but produces an increase in cardiovascular events, is an important public health question with the potential to elucidate fundamentally important aspects on atherogenesis, cardiovascular disease, and the biology of estrogen action. One question concerning the cardiovascular effects of hormone replacement therapy is whether genetic factors can substantially modify individual responses to estrogen treatment. New clinical trial evidence is emerging that links the presence of particular variants in the estrogen receptor to the response of HDL and other intermediate endpoints to hormone replacement therapy. SUMMARY: One or more common variants in estrogen receptor-alpha are associated with a differential response to hormone replacement therapy in several domains of estrogen action. However, the effect of these variants on the risk of clinical cardiovascular events in the setting of hormone replacement therapy is not yet known. Additional research focusing on the clinical impact of common variants in estrogen receptor-alpha, estrogen receptor-beta and the progesterone receptor promise to improve clinical decision-making concerning the use of hormone replacement therapy and other novel estrogen agonists.     

Apoptotic action of estrogen

Estrogen as a mitogen stimulates cell proliferation and prevents cell death in many cell types. In patients, estrogen is known to stimulate breast and uterus cancer development. Ironically, high doses of estrogen can induce regression of hormone-dependent breast cancer in postmenopausal women. The comprehensive mechanism by which estrogen induces tumor recession in breast cancer is still unknown, but activation of the Fas/FasL pathways plays a key role in this process. Laboratory studies show that the apoptotic action of estrogen is the major factor leading to cell number decreases in several cell types. The effects of estrogen are estrogen-receptor dependent. In this mini review, we will focus on the latest findings regarding estrogen apoptotic effects in several cell models, including breast cancer cells, and summarize the possible mechanisms involved in these estrogen mediated processes. New potential implications for the pharmacological control of breast cancer with estrogen in post-menopausal women are also discussed.     

Nongenomic action of progesterone in rat aorta: role of nitric oxide and prostaglandins

The mechanism of action of progesterone (Pg) on rat vascular tissue was investigated. We obtained evidence that 10-nM Pg inhibited platelet aggregation at 1-5 min. Previously, we reported that nitric oxide (NO) mediated this antiaggregatory effect. Rat aortic strips (RAS) NO synthase (NOS) activity in response to "in vitro" treatment with other sex steroids hormones was measured. The stimulatory action of Pg on NO production was specific for ovarian hormones and depends on sex. The effect was nongenomic since cycloheximide did not suppress the increment in NO induced by Pg. Finally, we demonstrated that Pg (5 min) increased prostacyclin release (42-182% above control) in a dose-dependent manner (1-100 nM). Indeed, indomethacin (10 microM) completely suppressed the increment in citrulline levels induced by the hormone. These results suggest that Pg exerts a direct nongenomic action on rat aortic metabolism, which involves NOS and cyclooxygenase (COX) activation and a cross-talk between NO- and prostacyclin (PGI(2))-dependent pathways.     

Modulation of amino acid uptake by TGF-beta in lung myofibroblasts

Hormones such as insulin, growth factors, and cell stress stimulate system A amino acid transporter. Transforming growth factor-beta (TGF-beta) stimulates amino acid uptake thereby inducing cell proliferation, cellular hypertrophy, and matrix synthesis. Insulin appears to activate amino acid in smooth muscle cells via a phosphatidylinositol 3-kinase (PI3-kinase)-dependent pathway. We examine the effect and interaction of TGF-beta, insulin, and PI3-kinase activity on amino acid uptake in human lung myofibroblasts. TGF-beta treatment induced large increases in system A activity and a small delayed increase in the phosphorylation of protein kinase B, also termed phospho-Akt. In contrast, insulin induced small increases in system A activity and large increases in phospho-Akt levels. LY294002, a PI3-kinase inhibitor, blocked the TGF-beta-induced amino acid uptake only partially, but completely blocked TGF-beta-induced Akt phosphorylation. Moreover, the level of phospho-Smad3 was found to be high even when LY294002 blocked TGF-beta-induced phospho-Akt levels. Inhibition of PI3-kinase activity resulted in increase in Km, consistent with a major change in transporter activity without change in transporter number. The PI3-kinase inhibitor also did not change the amino acid transporter 2 (ATA2) mRNA levels. Taken together, these results suggest that TGF-beta induced Smad-3 and amino acid uptake through a PI3-kinase independent pathway.     

Parabronchial smooth muscle cells and alveolar myofibroblasts in lung development

Epithelial-mesenchymal interactions and extracellular matrix remodeling are key processes of embryonic lung development. Lung smooth muscle cells, which are derived from the mesenchyme, form a sheath around bronchi and blood vessels. During lung organogenesis, smooth muscle differentiation coincides with epithelial branching morphogenesis and closely follows developing airways spatially and temporally. The precise function of parabronchial smooth muscle (PBSM) cells in healthy adult lung remains unclear. However, PBSM may regulate epithelial branching morphogenesis during lung development by the induction of mechanical stress or through regulation of paracrine signaling pathways. Alveolar myofibroblasts are interstitial contractile cells that share features and may share an origin with smooth muscle cells. Alveolar myofibroblasts are essential for secondary septation, a process critical for the development of the gas-exchange region of the lung. Dysregulation of PBSM or alveolar myofibroblast development is thought to underlie the pathogenesis of many lung diseases, including bronchopulmonary dysplasia, asthma, and interstitial fibrosis. We review the current understanding of the regulation of PBSM and alveolar myofibroblast development, and discuss the role of PBSM in lung development. We specifically focus on the role of these cells in the context of fibroblast growth factor-10, sonic hedgehog, bone morphogenetic protein-4, retinoic acid, and Wnt signaling pathways in the regulation of lung branching morphogenesis.     

Thrombin inhibits migration of human hepatic myofibroblasts

Several lines of data recently pointed out a role of the serine proteinase thrombin in liver fibrogenesis, but its mechanism of action is unknown. The aim of this study was to evaluate the effect of thrombin on the migration of human liver myofibroblasts. We show here that thrombin inhibits both basal migration and platelet-derived growth factor (PDGF)-BB-induced migration of myofibroblasts. By using a thrombin antagonist, a protease-activated receptor (PAR)-1 mimetic peptide, and a PAR-1 antibody, we show that this effect is dependent on the catalytic activity of thrombin and on PAR-1 activation. Thrombin's effect on basal migration was dependent on cyclooxygenase 2 (COX-2) activation because it was blocked by the COX-2 inhibitors NS-398 and nimesulide, and pharmacological studies showed that it was relayed through prostaglandin E(2) and its EP(2) receptor. On the other hand, thrombin-induced inhibition of PDGF-BB-induced migration was not dependent on COX-2. We show that thrombin inhibits PDGF-induced Akt-1 phosphorylation. This effect was consecutive to inhibition of PDGF-beta receptor activation through active dephosphorylation. Thus thrombin, through two distinct mechanisms, inhibits both basal- and PDGF-BB-induced migration of human hepatic liver myofibroblasts. The fine tuning of myofibroblast migration may be one of the mechanisms used by thrombin to regulate liver fibrogenesis.     

皮质酮对高钾诱导PC12细胞内钙升高的非基因组调节作用

目的：分析皮质酮快速抑制高钾诱导PC12细胞内钙升高的机制。方法：使用荧光影像系统,实时检测细胞内钙变化。结果：①在预处理PC12细胞5min后,皮质酮可呈量－效关系抑制高钾诱导的PC12细胞内钙升高。②牛血清白蛋白耦联的皮质酮（B－BSA）可模拟皮质酮快速抑制高钾诱导PC12细胞内钙升高的作用,并呈现量－效关系。③糖皮质激素的细胞内受体拮抗剂RU38486对皮质酮快速抑制效应无明显拮抗作用。④蛋白合成抑制剂放线菌酮预处理细胞3h后,皮质酮对高钾诱导PC12内钙升高仍有较强抑制作用。结论：①皮质酮的作用点位于细胞膜上。②皮质酮的快速作用不直接依赖细胞内蛋白合成和不依赖细胞内糖皮质激素受体。③皮质酮对高钾诱导PC12内钙升高的快速抑制作用属非基因组作用。