Function and role of voltage-gated sodium channel NaV1.7 expressed in aortic smooth muscle cells

Voltage-gated Na(+) channel currents (I(Na)) are expressed in several types of smooth muscle cells. The purpose of this study was to evaluate the expression of I(Na), its functional role, pathophysiology in cultured human (hASMCs) and rabbit aortic smooth muscle cells (rASMCs), and its association with vascular intimal hyperplasia. In whole cell voltage clamp, I(Na) was observed at potential positive to -40 mV, was blocked by tetrodotoxin (TTX), and replacing extracellular Na(+) with N-methyl-d-glucamine in cultured hASMCs. In contrast to native aorta, cultured hASMCs strongly expressed SCN9A encoding Na(V)1.7, as determined by quantitative RT-PCR. I(Na) was abolished by the treatment with SCN9A small-interfering (si)RNA (P < 0.01). TTX and SCN9A siRNA significantly inhibited cell migration (P < 0.01, respectively) and horseradish peroxidase uptake (P < 0.01, respectively). TTX also significantly reduced the secretion of matrix metalloproteinase-2 6 and 12 h after the treatment (P < 0.01 and P < 0.05, respectively). However, neither TTX nor siRNA had any effect on cell proliferation. L-type Ca(2+) channel current was recorded, and I(Na) was not observed in freshly isolated rASMCs, whereas TTX-sensitive I(Na) was recorded in cultured rASMCs. Quantitative RT-PCR and immunostaining for Na(V)1.7 revealed the prominent expression of SCN9A in cultured rASMCs and aorta 48 h after balloon injury but not in native aorta. In conclusion, these studies show that I(Na) is expressed in cultured and diseased conditions but not in normal aorta. The Na(V)1.7 plays an important role in cell migration, endocytosis, and secretion. Na(V)1.7 is also expressed in aorta after balloon injury, suggesting a potential role for Na(V)1.7 in the progression of intimal hyperplasia.     

Acute and chronic effects of eicosapentaenoic acid on voltage-gated sodium channel expressed in cultured human bronchial smooth muscle cells

This study investigated acute and chronic effects of eicosapentaenoic acid (EPA) on voltage-gated Na+ current (I(Na)) expressed in cultured human bronchial smooth muscle cells (hBSMCs). The whole-cell voltage clamp technique and quantitative real-time RT-PCR analysis were applied. The alterations in the fatty acid composition of phospholipids after treatment with EPA were also examined. Extracellular application of EPA produced a rapid and concentration-dependent suppression of tetrodotoxin-sensitive I(Na) with the half-maximal inhibitory concentration of 2 microM. After washing out EPA with albumin, I(Na) returned to the control level. Similar inhibitory effects were observed regarding other fatty acids (docosahexaenoic, arachidonic, stearic, and oleic acids), but EPA was the most potent inhibitor. The effect of EPA on I(Na) was not blocked by nordihydroguaiaretic acid and indometacin, and was accompanied by a significant shift of the steady-state inactivation curve to more negative potentials. In cells chronically treated with EPA, the EPA content of the cell lipid fraction (mol%) increased time-dependently, while arachidonic acid (AA) decreased, resulting in an increase of EPA to AA ratio. Then, the level of mRNA (SCN9A) encoding I(Na) decreased significantly. These results provide novel evidence that EPA not only rapidly inhibits I(Na), but also reduces the mRNA levels of the Na+ channel after cellular incorporation of EPA in cultured hBSMCs.     

Dexamethasone decreases phospholipase C beta1 isozyme expression in human vascular smooth muscle cells

The molecular characterization of the human PLC beta1 gene was just reported by Peruzzi et al. [Biochim. Biophys. Acta 1582 (2002) 46]. This prompted us to investigate the effects of dexamethasone on PLC beta1 expression in two types of human vascular smooth muscle cells--coronary artery smooth muscle cells (hCASMC) and aortic smooth muscle cells (hAoSMC), since glucocorticoids are known to affect the signaling pathways of Gprotein coupled receptors. Semi-quantitative RT-PCR was used to analyze mRNA expression and Western-blot for protein expression. Dexamethasone treatment in the two types of cells studied decreased (mRNA and protein) PLC beta1 isozyme expression. A rapid (2 h) fall in mRNA occurred in hCASMC after treatment, and hCASMC were more sensitive to dexamethasone (1 nM versus 100 nM) than hAoSMC. The major reduction (80%) was observed after 48 h of exposure in both VSMC. Treatment with mifeprisone, an antagonist of glucocorticoid receptors, blunted the dexamethasone effect on PLC beta1 mRNA and showed that this effect was mediated by glucocorticoids receptors.     

Expression of lipocortins in human bronchial epithelial cells: effects of IL-1beta , TNF-alpha, LPS and dexamethasone

In this study, we investigated the expression of lipocortin I and II (annexin I and I in the human bronchial epithelium, both in vivo and in vitro. A clear expression of lipocortin I and II protein was found in the epithelium in sections of bronchial tissue. In cultured human bronchial epithelial cells we demonstrated the expression of lipocortin I and II mRNA and protein using Northern blotting, FACScan analysis and ELISA. No induction of lipocortin I or II mRNA or protein was observed after incubation with dexamethasone. Stimulation of bronchial epithelial cells with IL-1beta, TNF-alpha or LPS for 24 h did not affect the lipocortin I or II mRNA or protein expression, although PGE(2) and 6-keto-PGF(1alpha) production was significantly increased. This IL-1beta- and LPS-mediated increase in eicosanoids could be reduced by dexamethasone, but was not accompanied by an increase in lipocortin I or II expression. In human bronchial epithelial cells this particular glucocorticoid action is not mediated through lipocortin I or II induction.     

Upregulation of RGS4 and downregulation of CPI-17 mediate inhibition of colonic muscle contraction by interleukin-1beta

The pro-inflammatory cytokine IL-1beta contributes to the reduced contractile responses of gut smooth muscle observed in both animal colitis models and human inflammatory bowel diseases. However, the mechanisms are not well understood. The effects of IL-1beta on the signaling targets mediating acetylcholine (ACh)-induced initial and sustained contraction were examined using rabbit colonic circular muscle strips and cultured muscle cells. The contraction was assessed through cell length decrease, myosin light chain (MLC(20)) phosphorylation, and activation of PLC-beta and Rho kinase. Expression levels of the signaling targets were determined by Western blot analysis and real-time RT-PCR. Short interfering RNAs (siRNAs) for regulator of G protein signaling 4 (RGS4) were used to silence endogenous RGS4 in muscle strips or cultured muscle cells. IL-1beta treatment of muscle strips inhibited both initial and sustained contraction and MLC(20) phosphorylation in isolated muscle cells. IL-1beta treatment increased RGS4 expression but had no effect on muscarinic receptor binding or Galpha(q) expression. In contrast, IL-1beta decreased the expression and phosphorylation of CPI-17 but had no effect on RhoA expression or ACh-induced Rho kinase activity. Upregulation of RGS4 and downregulation of CPI-17 by IL-1beta in muscle strips were corroborated in cultured muscle cells. Knockdown of RGS4 by siRNA in both muscle strips and cultured muscle cells blocked the inhibitory effect of IL-1beta on initial contraction and PLC-beta activation, whereas overexpression of RGS4 inhibited PLC-beta activation. These data suggest that IL-1beta upregulates RGS4 expression, resulting in the inhibition of initial contraction and downregulation of CPI-17 expression during sustained contraction in colonic smooth muscle.     

Analytical procedures for a cryptic messenger RNA that mediates translational control of prostaglandin synthase by glucocorticoids

Expression of the enzyme prostaglandin H synthase in cultured vascular smooth muscle cells required epidermal growth factor (EGF) and type beta transforming growth factor (TGF-beta) and was inhibited by cycloheximide but not actinomycin D. Preincubation with the glucocorticoid dexamethasone (0.5 microM) blocked the EGF-induced expression of prostaglandin H (PGH) synthase. Following dexamethasone addition, levels of hybridizable mRNA for PG synthase were reduced by over 90% within 1 h. After dexamethasone was removed, PG synthase mRNA recovered within 3 h by a process that was not inhibited by actinomycin D. These observations, together with other findings, suggested that the mRNA was being converted into some nonextractable and nontranslated form, probably by binding of a glucocorticoid-induced protein to the conserved 3' untranslated region. In order to investigate further the nature of this phenomenon, seven different literature procedures were evaluated for extracting and determining the PG synthase mRNA. Five of the seven procedures failed to detect hybridizable PG synthase mRNA in glucocorticoid-treated cells. Two procedures, however, recovered mRNA in both glucocorticoid-treated and control cells. A comparison of the protocols indicated that only those methods that incorporate a cationic detergent (sodium N-lauroylsarcosine), instead of anionic detergents in the lysis or homogenization buffers, successfully extract the glucocorticoid-suppressed PG synthase mRNA. Based upon these results two procedures are described, one that optimizes the extraction and determination of the glucocorticoid-suppressed (cryptic) form of the mRNA, and another which optimizes the analysis of normal mRNA without extracting the cryptic form. The results indicate that translational control of PG synthase by glucocorticoids is regulated by converting the mRNA into a cryptic form that is more firmly tissue bound than normal mRNA.     

Dexamethasone down-regulates the expression of endothelin receptors in vascular smooth muscle cells.

Steroid hormones have been shown to modulate a number of physiological processes in addition to their potent antiinflammatory effects. Endothelin (ET) is a newly discovered vasoconstrictor that is synthesized and released by endothelial cells and acts on adjacent vascular smooth muscle cells by interacting with specific cell surface receptors. Proinflammatory agents such as thrombin and transforming growth factor beta have been shown to up-regulate ET gene expression in vascular endothelial cells. We wondered whether the anti-inflammatory steroids might have any regulatory effect on the ET receptors present in the vascular smooth muscle cells. Rat vascular smooth muscle cells (A-10 cell line, ATCC.CRL 1476) were used as a model system to study the effects of glucocorticoids on ET receptor expression and function. These cells display high density and high affinity ET receptors that belong to the ETA subtype. Pretreatment of these cells with dexamethasone reduced the number of ET receptors by 50-60% without changing the affinity. Of the steroids tested, dexamethasone was most effective followed by prednisolone and hydrocortisone. Aldosterone, a mineralocorticoid, was 5000-fold less potent than dexamethasone. This effect of dexamethasone was dependent on the time of pretreatment and concentration of the steroid used. This down-regulation of ET receptors was also accompanied by an attenuated response to ET-1 in dexamethasone-pretreated cells. The inhibitory effect of dexamethasone was selective for ET receptors because the vasopressin-mediated response was unaffected. In addition, dexamethasone pretreatment of these cells resulted in 50-60% reduction in the steady-state level of ETA receptor mRNA as revealed by Northern analysis. These results suggest that glucocorticoid pretreatment of smooth muscle cells resulted in the down-regulation of the ETA receptor at the mRNA level.     

Corticosteroid inhibition of growth-related oncogene protein-alpha via mitogen-activated kinase phosphatase-1 in airway smooth muscle cells

Expression of the inflammatory chemokine, growth-related oncogene protein-alpha (GRO-alpha), from airway smooth muscle cells (ASMC) is regulated by pathways involving NF-kappaB and MAPK activation. We determined the effects of dexamethasone on GRO-alpha induced by IL-1beta or TNF-alpha with respect to the role of MAPK pathways and of MAPK phosphatase-1 (MKP-1). Human ASMC were studied in primary culture at confluence. Dexamethasone (10(-8)-10(-5) M) partially inhibited GRO-alpha expression and release induced by IL-1beta and TNF-alpha; this was associated with an inhibition of JNK, but not of p38 or ERK phosphorylation. Together with IL-1beta or TNF-alpha, dexamethasone rapidly induced mRNA and protein expression of MKP-1, which dephosphorylates MAPKs. Using MKP-1 small interfering RNA (siRNA) to block the expression of IL-1beta- and dexamethasone-induced MKP-1 by 50%, JNK phosphorylation was doubled. The inhibitory effect of dexamethasone on GRO-alpha release was partially reversed in ASMC treated with MKP-1 siRNA compared with those treated with scrambled siRNA. In contrast, overexpression of MKP-1 led to a reduction in IL-1beta-induced release of GRO-alpha, but the inhibitory effects of dexamethasone were preserved. Nuclear translocation of the glucocorticoid receptor was increased in ASMC exposed to dexamethasone and IL-1beta. Using chromatin immunoprecipitation assay, glucocorticoid receptor binding to the MKP-1 promoter was increased by IL-1beta and dexamethasone compared with either alone. Glucocorticoids and IL-1beta or TNF-alpha modulate GRO-alpha release partly through the inhibition of JNK pathway, resulting from an up-regulation of MKP-1 expression.     

Modulation of alpha-ENaC and alpha1-Na+-K+-ATPase by cAMP and dexamethasone in alveolar epithelial cells

cAMP and dexamethasone are known to modulate Na+ transport in epithelial cells. We investigated whether dibutyryl cAMP (DBcAMP) and dexamethasone modulate the mRNA expression of two key elements of the Na+ transport system in isolated rat alveolar epithelial cells: alpha-, beta-, and gamma-subunits of the epithelial Na+ channel (ENaC) and the alpha1- and beta1-subunits of Na+-K+-ATPase. The cells were treated for up to 48 h with DBcAMP or dexamethasone to assess their long-term impact on the steady-state level of ENaC and Na+-K+-ATPase mRNA. DBcAMP induced a twofold transient increase of alpha-ENaC and alpha1-Na+-K+-ATPase mRNA that peaked after 8 h of treatment. It also upregulated beta- and gamma-ENaC mRNA but not beta1-Na+-K+-ATPase mRNA. Dexamethasone augmented alpha-ENaC mRNA expression 4.4-fold in cells treated for 24 h and also upregulated beta- and gamma-ENaC mRNA. There was a 1.6-fold increase at 8 h of beta1-Na+-K+-ATPase mRNA but no significant modulation of alpha1-Na+-K+-ATPase mRNA expression. Because DBcAMP and dexamethasone did not increase the stability of alpha-ENaC mRNA, we cloned 3.2 kb of the 5' sequences flanking the mouse alpha-ENaC gene to study the impact of DBcAMP and dexamethasone on alpha-ENaC promoter activity. The promoter was able to drive basal expression of the chloramphenicol acetyltransferase (CAT) reporter gene in A549 cells. Dexamethasone increased the activity of the promoter by a factor of 5.9. To complete the study, the physiological effects of DBcAMP and dexamethasone were investigated by measuring transepithelial current in treated and control cells. DBcAMP and dexamethasone modulated transepithelial current with a time course reminiscent of the profile observed for alpha-ENaC mRNA expression. DBcAMP had a greater impact on transepithelial current (2.5-fold increase at 8 h) than dexamethasone (1.8-fold increase at 24 h). These results suggest that modulation of alpha-ENaC and Na+-K+-ATPase gene expression is one of the mechanisms that regulates Na+ transport in alveolar epithelial cells.     

A mutation in telethonin alters Nav1.5 function

Excitable cells express a variety of ion channels that allow rapid exchange of ions with the extracellular space. Opening of Na(+) channels in excitable cells results in influx of Na(+) and cellular depolarization. The function of Na(v)1.5, an Na(+) channel expressed in the heart, brain, and gastrointestinal tract, is altered by interacting proteins. The pore-forming alpha-subunit of this channel is encoded by SCN5A. Genetic perturbations in SCN5A cause type 3 long QT syndrome and type 1 Brugada syndrome, two distinct heritable arrhythmia syndromes. Mutations in SCN5A are also associated with increased prevalence of gastrointestinal symptoms, suggesting that the Na(+) channel plays a role in normal gastrointestinal physiology and that alterations in its function may cause disease. We collected blood from patients with intestinal pseudo-obstruction (a disease associated with abnormal motility in the gut) and screened for mutations in SCN5A and ion channel-interacting proteins. A 42-year-old male patient was found to have a mutation in the gene TCAP, encoding for the small protein telethonin. Telethonin was found to be expressed in the human gastrointestinal smooth muscle, co-localized with Na(v)1.5, and co-immunoprecipitated with sodium channels. Expression of mutated telethonin, when co-expressed with SCN5A in HEK 293 cells, altered steady state activation kinetics of SCN5A, resulting in a doubling of the window current. These results suggest a new role for telethonin, namely that telethonin is a sodium channel-interacting protein. Also, mutations in telethonin can alter Na(v)1.5 kinetics and may play a role in intestinal pseudo-obstruction.     

高浓度地塞米松通过下调LMP-1表达抑制大鼠成骨细胞的分化

为探讨地塞米松（dexamethasone,DEX)抑制成骨细胞分化的机制,观察了不同浓度DEX对体外培养大鼠成骨细胞的碱性磷酸酶活性,骨钙素（osteocalcin,OC)合成,I型胶原蛋白表达的影响。并用RT-PCR方法检测了成骨细胞中LIM矿化蛋白1mRNA的表达量,结果显示：低浓度（10^-9mol/L）的DEX能增强碱性磷酸酶的活性、OC的分泌和I型胶原蛋白的表达;而高浓度（10^-7mol/L)的DEX对它们则起抑制作用,并下调成骨细胞正调节因子LMP-1mRNA的表达,上述结果表明,低浓度的DEX促进成骨细胞的分化;高浓度的DEX则抑制成骨细胞的分化,其抑制作用可能是通过下调LMP-1mRNA的表达而实现的。     

Glucocorticoid inhibition of vascular smooth muscle cell proliferation: influence of homologous extracellular matrix and serum mitogens

We examined the influence of glucocorticoid hormones on the proliferation of cultured adult bovine aortic smooth muscle cells (BASM) using both primary mass cultures and a cloned strain. Cloned BASM cells maintained on plastic culture dishes were inhibited by approximately 40% by dexamethasone treatment but showed no inhibition when grown of homologous extracellular matrix (ECM) coated dishes. Dexamethasone inhibited growth of primary cultures by 73% on plastic and by 45% on ECM. The inhibitory effect was specific for the glucocorticoids, dexamethasone, corticosterone, and cortisol and was not observed with progesterone, aldosterone, estradiol or 17-alpha OH progesterone. In cloned cells, the abolition of glucocorticoid inhibition by ECM was independent of seeding density and serum concentration. The inhibition on plastic was dependent on serum concentrations greater than 1% and resulted in both a slow rate of proliferation and a lower saturation density. A specific subset of peptides detected on two-dimensional gels was induced by glucocorticoids under growth inhibitory conditions but was not induced when the cells were grown on ECM. Primary cultures grown on ECM and exposed to Dulbecco's modified Eagle's Medium (DME) containing high density lipoprotein and transferrin grew at 40% of the rate observed for cultures exposed to DME with 10% serum. Both conditions showed growth inhibition of 70% in the presence of dexamethasone. The addition of epidermal and platelet-derived growth factors in DME containing high density lipoprotein and transferrin to cells grown on ECM resulted in growth rates comparable to that observed with cultures exposed to 10% serum and were inhibited 45% by dexamethasone. These results suggest that glucocorticoids inhibit smooth muscle proliferation by decreasing the sensitivity of the cells to mitogenic stimulation by high density lipoprotein when the cells are maintained on a homologous substrate.     

Effect of mibefradil on sodium and calcium currents

Interstitial cells of Cajal (ICC) generate the electrical slow wave. The ionic conductances that contribute to the slow wave appear to vary among species. In humans, a tetrodotoxin-resistant Na+ current (Na(V)1.5) encoded by SCN5A contributes to the rising phase of the slow wave, whereas T-type Ca2+ currents have been reported from cultured mouse intestine ICC and also from canine colonic ICC. Mibefradil has a higher affinity for T-type over L-type Ca2+ channels, and the drug has been used in the gastrointestinal tract to identify T-type currents. However, the selectivity of mibefradil for T-type Ca2+ channels over ICC and smooth muscle Na+ channels has not been clearly demonstrated. The aim of this study was to determine the effect of mibefradil on T-type and L-type Ca2+ and Na+ currents. Whole cell currents were recorded from HEK-293 cells coexpressing green fluorescent protein with either the rat brain T-type Ca2+ channel alpha(1)3.3b + beta(2), the human intestinal L-type Ca2+ channel subunits alpha(1C) + beta(2), or Na(V)1.5. Mibefradil significantly reduced expressed T-type Ca2+ current at concentrations > or = 0.1 microM (IC(50) = 0.29 microM), L-type Ca2+ current at > 1 microM (IC(50) = 2.7 microM), and Na+ current at > or = 0.3 microM (IC(50) = 0.98 microM). In conclusion, mibefradil inhibits the human intestinal tetrodotoxin-resistant Na+ channel at submicromolar concentrations. Caution must be used in the interpretation of the effects of mibefradil when several ion channel classes are coexpressed.