Endothelin-I induces gene expression through stimulation of endothelin type a receptors in normal rat kidney cells

RNA blots of total cellular RNA isolated from quiescent and endothelin (ET-1)-stimulated normal rat kidney (NRK) cells demonstrated that ET-1 induced the expression of c-jun, jun B, and c-fos mRNA in a time-dependent manner with maximal expression of mRNA by 1 hr after the addition of ET-1. Five hundred picomolal ET-1 was sufficient to induce maximal mRNA expression. These data agreed with saturation experiments which demonstrated that maximal binding of [¹²⁵I]ET-1 was achieved at concentrations greater than 100 pM. The K_d and B_max values for [¹²⁵I]ET-1 binding to NRK membranes were 20.5 pM and 22.2 fmol/mg protein, respectively. Competition experiments for the binding of [¹²⁵I]ET-1 to NRK membranes demonstrated that ET-1 was a more potent inhibitor (K_i = 0.047 nM) than ET-3 (K_i = 10.8 nM). No specific binding of [¹²⁵I]ET-3 (40 or 500 pM) to NRK membranes could be observed. The expression of c-jun, jun B, and c-fos mRNA was inhibited by the endothelin type A receptor (ET)-selective antagonist, BQ-123. Thus, these data demonstrate that ET-1 mediates the expression of immediate response gene mRNA in NRK cells via the ET_A receptor. ET-1 stimulation of NRK cells also upregulated EGF receptors, providing a possible mechanism for ET-1 complementation of epidermal growth factor (EGF) mitogenicity in NRK cells. © 1995 Wiley-Liss, Inc.     

c-Src tyrosine kinase mediates high glucose-induced endothelin-1 expression

Endothelin-1 (ET-1) plays an important role in the pathophysiology of diabetes-associated cardiovascular disorders. The molecular mechanisms leading to ET-1 upregulation in diabetes are not entirely defined. c-Src tyrosine kinase regulates important pathophysiological aspects of vascular response to insults. In this study, we aimed to elucidate whether high glucose-activated c-Src signaling plays a role in the regulation of ET-1 expression. Human endothelial cells EAhy926 (ECs) were exposed to normal or high levels of glucose for 24 h. Male C57BL/6J mice were rendered diabetic with streptozotocin and then treated with a specific c-Src inhibitor (Src I1) or c-Src siRNA. Real-time PCR, Western blot, and ELISA, were used to investigate ET-1 regulation. The c-Src activity and expression were selectively downregulated by pharmacological inhibition and siRNA-mediated gene silencing, respectively. High glucose dose-dependently up-regulated c-Src phosphorylation and ET-1 gene and protein expression levels in human ECs. Chemical inhibition or silencing of c-Src significantly decreased the high-glucose augmented ET-1 expression in cultured ECs. In vivo studies showed significant elevations in the aortic ET-1 mRNA expression and plasma ET-1 concentration in diabetic mice compared to non-diabetic animals. Treatment with Src I1, as well as in vivo silencing of c-Src, significantly reduced the upregulated ET-1 expression in diabetic mice. These data provide new insights into the regulation of ET-1 expression in endothelial cells in diabetes. Pharmacological targeting of c-Src activity and/or expression may represent a potential therapeutic strategy to reduce ET-1 level and to counteract diabetes-induced deleterious vascular effects.     

Shrinkage insensitivity of NKCC1 in myosin II-depleted cytoplasts from Ehrlich ascites tumor cells

Protein phosphorylation/dephosphorylation and cytoskeletal reorganization regulate the Na⁺-K⁺-2Cl^– cotransporter (NKCC1) during osmotic shrinkage; however, the mechanisms involved are unclear. We show that in cytoplasts, plasma membrane vesicles detached from Ehrlich ascites tumor cells (EATC) by cytochalasin treatment, NKCC1 activity evaluated as bumetanide-sensitive ⁸⁶Rb influx was increased compared with the basal level in intact cells yet could not be further increased by osmotic shrinkage. Accordingly, cytoplasts exhibited no regulatory volume increase after shrinkage. In cytoplasts, cortical F-actin organization was disrupted, and myosin II, which in shrunken EATC translocates to the cortical region, was absent. Moreover, NKCC1 activity was essentially insensitive to the myosin light chain kinase (MLCK) inhibitor ML-7, a potent blocker of shrinkage-induced NKCC1 activity in intact EATC. Cytoplast NKCC1 activity was potentiated by the Ser/Thr protein phosphatase inhibitor calyculin A, partially inhibited by the protein kinase A inhibitor H89, and blocked by the broad protein kinase inhibitor staurosporine. Cytoplasts exhibited increased protein levels of NKCC1, Ste20-related proline- and alanine-rich kinase (SPAK), and oxidative stress response kinase 1, yet they lacked the shrinkage-induced plasma membrane translocation of SPAK observed in intact cells. The basal phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) was increased in cytoplasts compared with intact cells, yet in contrast to the substantial activation in shrunken intact cells, p38 MAPK could not be further activated by shrinkage of the cytoplasts. Together these findings indicate that shrinkage activation of NKCC1 in EATC is dependent on the cortical F-actin network, myosin II, and MLCK. F-actin; Na⁺-K⁺-2Cl^– cotransporter; myosin light chain kinase; protein kinase A     

Activation of K(+) channels and increased migration of differentiated intestinal epithelial cells after wounding

Earlymucosal restitution occurs by epithelial cell migration to resealsuperficial wounds after injury. Differentiated intestinal epithelialcells induced by forced expression of the Cdx2 gene migrateover the wounded edge much faster than undifferentiated parental cellsin an in vitro model. This study determined whether thesedifferentiated intestinal epithelial cells exhibit increased migrationby altering voltage-gated K⁺ (Kv) channel expression andcytosolic free Ca²⁺ concentration([Ca²⁺]_cyt). StableCdx2-transfected IEC-6 cells (IEC-Cdx2L1) with highly differentiated phenotype expressed higher basal levels of Kv1.1 andKv1.5 mRNAs and proteins than parental IEC-6 cells. Neither IEC-Cdx2L1cells nor parental IEC-6 cells expressed voltage-dependent Ca²⁺ channels. The increased expression of Kv channels indifferentiated IEC-Cdx2L1 cells was associated with an increase inwhole cell K⁺ currents, membrane hyperpolarization, and arise in [Ca²⁺]_cyt. The migration rates indifferentiated IEC-Cdx2L1 cells were about four times those of parentalIEC-6 cells. Inhibition of Kv channel expression by polyamine depletiondecreased [Ca²⁺]_cyt, reduced myosin stressfibers, and inhibited cell migration. Elevation of[Ca²⁺]_cyt by ionomycin promoted myosin IIstress fiber formation and increased cell migration. These resultssuggest that increased migration of differentiated intestinalepithelial cells is mediated, at least partially, by increasing Kvchannel activity and Ca²⁺ influx during restitution.

     

ET-1- and NO-mediated signal transduction pathway in human brain capillary endothelial cells

Previous studies have demonstratedthat functional interaction between endothelin (ET)-1 and nitric oxide(NO) involves changes in Ca²⁺ mobilization and cytoskeletonin human brain microvascular endothelial cells. The focus of thisinvestigation was to examine the possible existence of analogousinterplay between these vasoactive substances and elucidate theirsignal transduction pathways in human brain capillary endothelialcells. The results indicate that ET-1-stimulated Ca²⁺mobilization in these cells is dose-dependently inhibited by NOR-1 (anNO donor). This inhibition was prevented by ODQ (an inhibitor ofguanylyl cyclase) or Rp-8-CPT-cGMPS (an inhibitor of proteinkinase G). Treatment of endothelial cells with 8-bromo-cGMP reducedET-1-induced Ca²⁺ mobilization in a manner similar to thatobserved with NOR-1 treatment. In addition, NOR-1 or cGMP reducedCa²⁺ mobilization induced by mastoparan (an activator of Gprotein), inositol 1,4,5-trisphosphate, or thapsigargin (an inhibitorof Ca²⁺-ATPase). Interestingly, alterations in endothelialcytoskeleton (actin and vimentin) were associated with these effects.The data indicate for the first time that the cGMP-dependent proteinkinase colocalizes with actin. These changes were accompanied byaltered levels of phosphorylated vasodilator-stimulated phosphoprotein, which were elevated in endothelial cells incubated with NOR-1 andsignificantly reduced by ODQ or Rp-8-CPT-cGMPS. The findings indicate a potential mechanism by which the functionalinterrelationship between ET-1 and NO plays a role in regulatingcapillary tone, microcirculation, and blood-brain barrier function.

     

ETB receptor activation leads to activation and phosphorylation of NHE3

In OKP cellsexpressing ET_B endothelinreceptors, activation ofNa⁺/H⁺antiporter activity by endothelin-1 (ET-1) was resistant to low concentrations of ethylisopropyl amiloride, indicating regulation ofNa⁺/H⁺exchanger isoform 3 (NHE3). ET-1 increased NHE3 phosphorylation incells expressing ET_B receptors butnot in cells expressing ET_Areceptors. Receptor specificity was not due to demonstrable differencesin receptor-specific activation of tyrosine phosphorylation pathways orinhibition of adenylyl cyclase. Phosphorylation was associated with adecrease in mobility on SDS-PAGE, which was reversed by treatingimmunoprecipitated NHE3 with alkaline phosphatase. Phosphorylation wasfirst seen at 5 min and was maximal at 15-30 min. Phosphorylationwas maximal with 10⁹ MET-1. Phosphorylation occurred on threonine and serine residues atmultiple sites. In summary, ET-1 induces NHE3 phosphorylation in OKPcells on multiple threonine and serine residues.ET_B receptor specificity, timecourse, and concentration dependence are all similar betweenET-1-induced increases in NHE3 activity and phosphorylation, suggestingthat phosphorylation plays a key role in activation.     

Rho activation in excitatory agonist-stimulated vascular smooth muscle

Small GTPase Rho and its downstream effector, Rho kinase, havebeen implicated in agonist-stimulated Ca²⁺ sensitization of20-kDa myosin light chain (MLC₂₀) phosphorylation andcontraction in smooth muscle. In the present study we demonstrated forthe first time that excitatory receptor agonists induce increases inamounts of an active GTP-bound form of RhoA, GTP-RhoA, in rabbit aorticsmooth muscle. Using a pull-down assay with a recombinant RhoA-bindingprotein, Rhotekin, we found that a thromboxane A₂ mimetic,U-46619, which induced a sustained contractile response, induced asustained rise in the amount of GTP-RhoA in a dose-dependent mannerwith an EC₅₀ value similar to that for the contractile response. U-46619-induced RhoA activation was thromboxaneA₂ receptor-mediated and reversible. Other agonistsincluding norepinephrine, serotonin, histamine, and endothelin-1 (ET-1)also stimulated RhoA, albeit to lesser extents than U-46619. Incontrast, ANG II and phorbol 12,13-dibutyrate failed to increaseGTP-RhoA. The tyrosine kinase inhibitor genistein substantiallyinhibited RhoA activation by these agonists, except for ET-1. Thusexcitatory agonists induce Rho activation in an agonist-specificmanner, which is thought to contribute to stimulation ofMLC₂₀ phosphorylation Ca²⁺ sensitivity.

     

Effects of nonselective cation channels and PI3K on endothelin-1-induced PYK2 tyrosine phosphorylation in C6 glioma cells

We recently demonstrated that endothelin-1 (ET-1) activates two types of Ca²⁺-permeable nonselective cation channels (designated NSCC-1 and NSCC-2) in C6 glioma cells. In the present study, we investigated the effects of NSCCs on the ET-1-induced proline-rich tyrosine kinase 2 (PYK2) phosphorylation in C6 glioma cells. In addition, we examined the effects of phosphoinositide 3-kinase (PI3K) on the ET-1-induced NSCCs activation and PYK2 phosphorylation. The PI3K inhibitors wortmannin and LY-294002 inhibited ET-1-induced Ca²⁺ influx through NSCC-2 but not NSCC-1. On the other hand, addition of these inhibitors after stimulation with ET-1 failed to suppress Ca²⁺ influx through NSCC-2. PYK2 phosphorylation was abolished by blocking Ca²⁺ influx through NSCCs. The PI3K inhibitors blocked the NSCC-2-dependent part of ET-1-induced PYK2 phosphorylation. These results indicate that 1) NSCC-2 is stimulated by ET-1 via a PI3K-dependent cascade, whereas NSCC-1 is stimulated via a PI3K-independent cascade; 2) PI3K seems to be required for the activation of the Ca²⁺ entry, but not for its maintenance; 3) Ca²⁺ influx through NSCC-1 and NSCC-2 plays an essential role in ET-1-induced PYK2 phosphorylation; and 4) PI3K is involved in the ET-1-induced PYK2 phosphorylation that depends on the Ca²⁺ influx through NSCC-2. endothelin; phosphoinositide 3-kinase; nonselective cation channel; proline-rich tyrosine kinase 2; glioma cell     

Voltage-insensitive Ca2+ channels and Ca2+/calmodulin-dependent protein kinases propagate signals from endothelin-1 receptors to the c-fos promoter.

Endothelin-1 (ET-1) triggers poorly understood nuclear signaling cascades that control gene expression, cell growth, and differentiation. To better understand how ET-1 regulates gene expression, we asked whether voltage-insensitive Ca2+ channels and Ca2+/calmodulin-dependent protein kinases (CaMKs) propagate signals from ET-1 receptors to the c-fos promoter in mesangial cells. Ca2+ influx through voltage-insensitive Ca2+ channels, one of the earliest postreceptor events in ET-1 signaling, mediated induction of c-fos mRNA and activation of the c-fos promoter by ET-1. A CaMK inhibitor (KN-93) blocked activation of the c-fos promoter by ET-1. Ectopic expression of CaMKII potentiated stimulation by ET-1, providing further evidence that CaMKs contribute to c-fos promoter activation by ET-1. The c-fos serum response element was necessary but not sufficient for CaMKII to activate the c-fos promoter. Activation of the c-fos promoter by ET-1 and CaMKII also required the FAP cis element, an AP-1-like sequence adjacent to the serum response element. Thus, voltage-insensitive Ca2+ channels and CaMKs apparently propagate ET-1 signals to the c-fos promoter that require multiple, interdependent cis elements. Moreover, these experiments suggest an important role for voltage-insensitive Ca2+ channels in nuclear signal transduction in nonexcitable cells.     

High-glucose-induced metallothionein expression in endothelial cells: an endothelin-mediated mechanism

Vascular endothelial cells areconstantly exposed to oxidative stress and must be protected byphysiological responses. In diabetes mellitus, endothelial cellpermeability is impaired and may be increased by high extracellularglucose concentrations. It has been postulated that metallothionein(MT) can protect endothelial cells from oxidative stress with itsincreased expression by cytokines, thrombin, and endothelin (ET)-1. Inthis study, we demonstrate that high glucose concentration can induceMT expression in endothelial cells through a distinct ET-dependentpathway. Exposure of human umbilical vein endothelial cells (HUVEC) toincreasing concentrations of glucose resulted in a rapid dose-dependentincrease in MT-2 and ET-1 mRNA expression. MT expression may be furtheraugmented with addition of ET-1. Preincubation of the cells with thespecific ET_B antagonist BQ-788 blocked MT-2 mRNA expressionmore effectively than the ET_A inhibitor TBC-11251. Highglucose also increased immunoreactive MT protein expression and inducedtranslocation of MT into the perinuclear area. Perinuclear localizationof MT was related to high-glucose-induced reorganization of F-actin filaments. These results demonstrate that an increase in extracellular glucose in HUVEC can lead to a rapid dose-dependent increase in MT-2mRNA expression and to perinuclear localization of MT protein withchanges to the cytoskeleton. These effects are mediated via the ETreceptor-dependent pathway.

     

Localized mechanical stress induces time-dependent actin cytoskeletal remodeling and stiffening in cultured airway smooth muscle cells

Mechanical stress (MS) causes cytoskeletal (CSK) and phenotypic changes in cells. Such changes in airway smooth muscle (ASM) cells might contribute to the pathophysiology of asthma. We have shown that periodic mechanical strain applied to cultured ASM cells alters the structure and expression of CSK proteins and increases cell stiffness and contractility (Smith PG, Moreno R, and Ikebe M. Am J Physiol Lung Cell Mol Physiol 272: L20–L27, 1997; and Smith PG, Deng L, Fredberg JJ, and Maksym GN. Am J Physiol Lung Cell Mol Physiol 285: L456–L463, 2003). However, the mechanically induced CSK changes, altered cell function, and their time courses are not well understood. Here we applied MS to the CSK by magnetically oscillating ferrimagnetic beads bound to the CSK. We quantified CSK remodeling by measuring actin accumulation at the sites of applied MS using fluorescence microscopy. We also measured CSK stiffness using optical magnetic twisting cytometry. We found that, during MS of up to 120 min, the percentage of beads associated with actin structures increased with time. At 60 min, 68.1 ± 1.6% of the beads were associated with actin structures compared with only 6.7 ± 2.8% before MS and 38.4 ± 5.5% in time-matched controls (P < 0.05). Similarly, CSK stiffness increased more than twofold in response to the MS compared with time-matched controls. These changes were more pronounced than observed with contractile stimulation by 80 mM KCl or 10^–4 M acetylcholine. Together, these findings imply that MS is a potent stimulus to enhance stiffness and contractility of ASM cells through CSK remodeling, which may have important implications in airway narrowing and dilation in asthma. mechanical stress; actin cytoskeleton; stiffness; airway smooth muscle cell; optical magnetic twisting cytometry; airway constriction and dilation; asthma     

Gq/G13 signaling by ET-1 in smooth muscle: MYPT1 phosphorylation via ETA and CPI-17 dephosphorylation via ETB

We analyzed the signaling pathways initiated by endothelin receptors ET_A and ET_B in intestinal circular and longitudinal smooth muscle cells. The response to endothelin-1 (ET-1) consisted of two phases in both cell types. The initial, transient phase of contraction and phosphorylation of 20-kDa myosin light chain (MLC₂₀) was mediated additively by ET_A and ET_B receptors and initiated by G_q-, Ca²⁺/calmodulin-dependent activation of MLC kinase. In contrast, the sustained phase was mediated selectively by ET_A receptors via a pathway involving sequential activation of G₁₃, RhoA, and Rho kinase, resulting in phosphorylation of MYPT1 at Thr⁶⁹⁶ and phosphorylation of MLC₂₀. Although PKC was activated, CPI-17 was not phosphorylated and hence did not contribute to inhibition of MLC phosphatase. The absence of CPI-17 phosphorylation by PKC reflected active dephosphorylation of CPI-17 by protein phosphatase 2A (PP2A). PP2A was activated via a pathway involving ET_B-dependent stimulation of p38 MAPK activity. CPI-17 phosphorylation was unmasked in the presence of the ET_B antagonist BQ-788, but not the ET_A antagonist BQ-123, and in the presence of a low concentration of okadaic acid, which selectively inactivates PP2A. The resultant phosphorylation of CPI-17 was blocked by bisindolylmaleimide, providing direct confirmation that it was PKC dependent. We conclude that the two phases of the intestinal smooth muscle response to ET-1 involve distinct receptors, G proteins, and signaling pathways. The sustained response is mediated via selective ET_A-dependent phosphorylation of MYPT1. In contrast, ET_B initiates an inhibitory pathway involving p38 MAPK-dependent activation of PP2A that causes dephosphorylation of CPI-17. endothelin receptor type A; endothelin receptor type B; myosin phosphatase targeting subunit     

Regulation of K+ current in human airway epithelial cells by exogenous and autocrine adenosine

The regulatory actions ofadenosine on ion channel function are mediated by four distinctmembrane receptors. The concentration of adenosine in the vicinity ofthese receptors is controlled, in part, by inwardly directed nucleosidetransport. The purpose of this study was to characterize the effects ofadenosine on ion channels in A549 cells and the role of nucleosidetransporters in this regulation. Ion replacement and pharmacologicalstudies showed that adenosine and an inhibitor of human equilibrative nucleoside transporter (hENT)-1, nitrobenzylthioinosine, activated K⁺ channels, most likely Ca²⁺-dependentintermediate-conductance K⁺ (I_K)channels. A₁ but not A₂ receptor antagonistsblocked the effects of adenosine. RT-PCR studies showed that A549 cellsexpressed mRNA for I_K-1 channels as well asA₁, A_2A, and A_2B but notA₃ receptors. Similarly, mRNA for equilibrative (hENT1 andhENT2) but not concentrative (hCNT1, hCNT2, and hCNT3) nucleosidetransporters was detected, a result confirmed in functional uptakestudies. These studies showed that adenosine controls the function ofK⁺ channels in A549 cells and that hENTs play a crucialrole in this process.

     

Cardiac myofibroblasts isolated from the site of myocardial infarction express endothelin de novo

Recently it was demonstrated that treatment with a nonselective endothelin (ET) receptor antagonist significantly reduces myocardial infarct size, which suggests a major role for ET in tissue repair following myocardial infarction (MI). Tissue repair and remodeling found at the site of MI are mainly attributed to myofibroblasts (myoFbs), which are phenotypically transformed fibroblasts that express alpha-smooth muscle actin. It is unclear whether myoFbs generate ET peptides and consequentially regulate pathophysiological functions de novo through expression of the ET-1 precursor (prepro-ET-1), ET-converting enzyme-1 (ECE-1), a metalloprotease that is required to convert Big ET-1 to ET-1 and ET receptors. To address these intriguing questions, we used cultured myoFbs isolated from 4-wk-old MI scar tissue. In cultured cells, we found: 1) expression of mRNA for ET precursor gene (ppET1), ECE-1, and ETA and ETB receptors by semiquantitative RT-PCR; 2) phosphoramidon-sensitive ECE-1 activity, which converts Big ET-1 to biologically active peptide ET-1; 3) expression of ETA and ETB receptors; 4) elaboration of Big ET-1 and ET-1 peptides in myoFb culture media; and 5) upregulation of type I collagen gene expression and synthesis by ET, which was blocked by bosentan (a nonselective ETA- and ETB receptor blocker). These studies clearly indicated that myoFbs express and generate ET-1 and receptor-mediated modulation of type I collagen expression by ET-1. Locally generated ET-1 may contribute to tissue repair of the infarcted heart in an autocrine/paracrine manner.     

Effects of Selenium Supplementation on Expression of Endothelin-1 and Its Receptors in Pulmonary Microvascular Endothelial Cells from Chick Embryos

The objective of this study was to evaluate the effects of supplemental selenium (Se) on expression of endothelin-1 (ET-1) and its receptors in cultured chick embryos pulmonary microvascular endothelial cells (PMVECs). To accomplish this, PMVECs were treated in Se-deficient or Se-supplement (12, 24, 50, 100?ng/ml) culture medium for 48?h. Low Se medium was achieved by reducing serum concentrations and the essential growth factors were added. After the incubation, the effects of supplemental Se on ET-1 and its receptors gene expression were assessed by quantitative real-time PCR (qRT-PCR). Compared with the control group, our results showed that among the different concentrations of Se supplement, the levels of ET-1 gene expression treated with both the moderate Se doses (24, 50?ng/ml, P?<?0.01, P?<?0.01, respectively) and the high doses (100?ng/ml, P?<?0.05) were noticeably decreased, the low-dose group (12?ng/ml), which showed no changes. Meanwhile, Se supplement (24, 50, 100?ng/ml) was found to be effective in reducing the expression levels of ETA (P?<?0.01, P?<?0.05, P?<?0.05, respectively) in cultured PMVECs grown in low Se medium. However, there were no significant changes (P?>?0.05) in ETB mRNA levels during the cell proliferation. These observations indicated that Se may play both direct and indirect role in the regulation of ET-1 and its receptors gene expression and their production in avian PMVECs. Se supplement decreases in ET-1 and ETA production in Se-deficient PMVECs may partly explain the mechanism of the protective effects of the Se on the cardiovascular system.     

Differing temporal roles of Ca2+ and cAMP in nicotine-elicited elevation of tyrosine hydroxylase mRNA

The involvement of cAMP- andCa²⁺-mediated pathways in theactivation of tyrosine hydroxylase (TH) gene expression by nicotine wasexamined in PC-12 cells. ExtracellularCa²⁺ and elevations inintracellular Ca²⁺ concentration([Ca²⁺]_i)were required for nicotine to increase TH mRNA. The nicotine-elicited rapid rise in[Ca²⁺]_iwas inhibited by blockers of either L-type or N-type, and to a lesserextent P/Q-, but not T-type, voltage-gatedCa²⁺ channels. With continualnicotine treatment,[Ca²⁺]_ireturned to basal levels within 3-4 min. After a lag of~5-10 min, there was a smaller elevation in[Ca²⁺]_ithat persisted for 6 h and displayed different responsiveness toCa²⁺ channel blockers. This secondphase of elevated[Ca²⁺]_iwas blocked by an inhibitor of store-operatedCa²⁺ channels, consistent with theobserved generation of inositol trisphosphate.1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM (BAPTA-AM), when added before or 2 h after nicotine,prevented elevation of TH mRNA. Nicotine treatment significantly raised cAMP levels. Addition of the adenylyl cyclase inhibitor2',5'-dideoxyadenosine (DDA) prevented thenicotine-elicited phosphorylation of cAMP response element bindingprotein. DDA also blocked the elevation of TH mRNA only when addedafter the initial transient rise in [Ca²⁺]_iand not after 1 h. This study reveals that several temporal phases areinvolved in the induction of TH gene expression by nicotine, each ofthem with differing requirements forCa²⁺ and cAMP.

     

Rho-kinase-mediated Ca2+-independent contraction in rat embryo fibroblasts

Thus far, determining the relative contribution of Ca²⁺/calmodulin-dependent myosin light chain kinase (MLCK) and Ca²⁺-independent Rho-kinase pathways to myosin II activation and contraction has been difficult. In this study, we characterize the role of Rho-kinase in a rat embryo fibroblast cell line (REF-52), which contains no detectable MLCK. No endogenous MLCK could be detected in REF-52 cells by either Western or Northern blot analysis. In the presence or absence of Ca²⁺, thrombin or lysophosphatidic acid (LPA) increased RhoA activity and Rhokinase activity, correlating with isometric tension development and myosin II regulatory light chain (RLC) phosphorylation. Resting tension is associated with a basal phosphorylation of 0.31 ± 0.02 mol PO₄/mol RLC, whereas upon LPA or thrombin treatment myosin II RLC phosphorylation increases to 1.08 ± 0.05 and 0.82 ± 0.05 mol PO₄/mol RLC, respectively, within 2.5 min. Ca²⁺ chelation has minimal effect on the kinetics and magnitude of isometric tension development and RLC phosphorylation. Treatment of REF-52 cells with the Rho-kinase-specific inhibitor Y-27632 abolished thrombin- and LPA-stimulated contraction and RLC phosphorylation. These results suggest that Rho-kinase is sufficient to activate myosin II motor activity and contraction in REF-52 cells. myosin light chain kinase; RhoA; myosin II regulatory light chain phosphorylation     

Fiber type conversion alters inactivation of voltage-dependent sodium currents in murine C2C12 skeletal muscle cells

Each skeletal muscle of the body contains a unique composition of "fast" and "slow" muscle fibers, each of which is specialized for certain challenges. This composition is not static, and the muscle fibers are capable of adapting their molecular composition by altered gene expression (i.e., fiber type conversion). Whereas changes in the expression of contractile proteins and metabolic enzymes in the course of fiber type conversion are well described, little is known about possible adaptations in the electrophysiological properties of skeletal muscle cells. Such adaptations may involve changes in the expression and/or function of ion channels. In this study, we investigated the effects of fast-to-slow fiber type conversion on currents via voltage-gated Na⁺ channels in the C₂C₁₂ murine skeletal muscle cell line. Prolonged treatment of cells with 25 nM of the Ca²⁺ ionophore A-23187 caused a significant shift in myosin heavy chain isoform expression from the fast toward the slow isoform, indicating fast-to-slow fiber type conversion. Moreover, Na⁺ current inactivation was significantly altered. Slow inactivation less strongly inhibited the Na⁺ currents of fast-to-slow fiber type-converted cells. Compared with control cells, the Na⁺ currents of converted cells were more resistant to block by tetrodotoxin, suggesting enhanced relative expression of the cardiac Na⁺ channel isoform Na_v1.5 compared with the skeletal muscle isoform Na_v1.4. These results imply that fast-to-slow fiber type conversion of skeletal muscle cells involves functional adaptation of their electrophysiological properties. muscle plasticity; myosin heavy chain expression; sodium channel expression     

Ca2+ transients activate calcineurin/NFATc1 and initiate fast-to-slow transformation in a primary skeletal muscle culture

The calcineurin-mediated signal transduction via nuclear factor of activated T cells (NFATc1) is involved in upregulating slow myosin heavy chain (MHC) gene expression during fast-to-slow transformation of skeletal muscle cells. This study aims to investigate the Ca²⁺ signal necessary to activate the calcineurin-NFATc1 cascade in skeletal muscle. Electrostimulation of primary myocytes from rabbit for 24 h induced a distinct fast-to-slow transformation at the MHC mRNA level and a full activation of the calcineurin-NFATc1 pathway, although resting Ca²⁺ concentration ([Ca²⁺]_i) remained unaltered at 70 nM. During activation, the calcium transients of these myocytes reach a peak concentration of 500 nM. Although 70 nM [Ca²⁺]_i does not activate calcineurin-NFAT, we show by the use of Ca²⁺ ionophore that the system is fully activated when [Ca²⁺]_i is 150 nM in a sustained manner. We conclude that the calcineurin signal transduction pathway and the slow MHC gene in cultured skeletal muscle cells are activated by repetition of the rapid high-amplitude calcium transients that are associated with excitation-contraction coupling rather than by a sustained elevation of resting Ca²⁺ concentration. muscle plasticity; NFATc1; resting calcium concentration