bluuebunny/crossref_metadata_2025 huggingface dataset:
Amino acids and Ca2+ stimulate different patterns of Ca2+ oscillations through the Ca2+-sensing receptor
Authors: Young Steven H. , Rozengurt Enrique
Published: May 2013

We determined the effect of aromatic amino acid stimulation of the human extracellular Ca2+-sensing receptor (CaR) on intracellular Ca2+ concentration ([Ca2+]i) in single HEK-293 cells. Addition of l-phenylalanine or l-tryptophan (at 5 mM) induced [Ca2+]i oscillations from a resting state that was quiescent at 1.8 mM extracellular Ca2+concentration ([Ca2+]e). Each [Ca2+]i peak returned to baseline values, and the average oscillation frequency was ∼1 min−1 at 37°C. Oscillations were not induced or sustained if the [Ca2+]e was reduced to 0.5 mM, even in the continued presence of amino acid. Average oscillation frequency in response to an increase in [Ca2+]e (from 1.8 to 2.5–5 mM) was much higher (∼4 min−1) than that induced by aromatic amino acids. Oscillations in response to [Ca2+]e were sinusoidal whereas those induced by amino acids were transient. Thus both amino acids and Ca2+, acting through the same CaR, produce oscillatory increases in [Ca2+]i, but the resultant oscillation pattern and frequency allow the cell to discriminate which agonist is bound to the receptor.
Regulation of the cardiac L-type Ca2+ channel by the actin-binding proteins α-actinin and dystrophin
Authors: Sadeghi Abbas , Doyle Andrew D. , Johnson Barry D.
Published: May 2013

The actin-binding proteins dystrophin and α-actinin are members of a family of actin-binding proteins that may link the cytoskeleton to membrane proteins such as ion channels. Previous work demonstrated that the activity of Ca2+ channels can be regulated by agents that disrupt or stabilize the cytoskeleton. In the present study, we employed immunohistochemical and electrophysiological techniques to investigate the potential regulation of cardiac L-type Ca2+channel activity by dystrophin and α-actinin in cardiac myocytes and in heterologous cells. Both actin-binding proteins were found to colocalize with the Ca2+ channel in mouse cardiac myocytes and to modulate channel function. Inactivation of the Ca2+channel in cardiac myocytes from mice lacking dystrophin ( mdx mice) was reduced compared with that in wild-type myocytes, voltage dependence of activation was shifted by 5 mV to more positive potentials, and stimulation by the β-adrenergic pathway and the dihydropyridine agonist BAY K 8644 was increased. Furthermore, heterologous coexpression of the Ca2+ channel with muscle, but not nonmuscle, forms of α-actinin was also found to reduce inactivation. As might be predicted from a reduction of Ca2+ channel inactivation, a prolonging of the mouse electrocardiogram QT was observed in mdx mice. These results suggest a combined role for dystrophin and α-actinin in regulating the activity of the cardiac L-type Ca2+ channel and a potential mechanism for cardiac dysfunction in Duchenne and Becker muscular dystrophies.
Microvessel formation from mouse aorta is stimulated in vitro by secreted VEGF and extracts from metanephroi
Authors: Akimoto Tetsu , Hammerman Marc R.
Published: May 2013

We have demonstrated that during culture under 5% O2, the addition of recombinant human VEGF or FGF2 to mouse embryonic aorta explants (thoracic level to lateral vessels supplying the mesonephros and metanephros) stimulates microvessel formation. Here we show that microvessel formation is also stimulated by addition to explants of supernatants obtained from metanephroi grown in serum-free organ culture or of metanephroi extracts. Supernatants and extracts from metanephroi grown under hypoxic conditions are more stimulatory than supernatants/extracts from metanephroi grown in room air. VEGF and FGF2 can be detected by using immunohistochemistry in developing nephrons in the cultured renal anlagen. Metanephroi supernatants contain more VEGF if renal anlagen are grown under hypoxic conditions than if they are grown in room air. Metanephros supernatant-stimulated microvessel formation is completely inhibited by soluble sFlt-1 fusion protein or anti-VEGF antibodies (αVEGF). Extract-stimulated microvessel formation is inhibited by αVEGF or anti-FGF2 antibodies, or both. We conclude that metanephroi produce growth factors including VEGF and FGF that enhance microvessel formation from embryonic thoracic aorta in vitro.
DNA damage-induced [Zn2+]i transients: correlation with cell cycle arrest and apoptosis in lymphoma cells
Authors: Smith Paul J. , Wiltshire Marie , Davies Sharon , Chin Suet-Feung , Campbell Anthony K. , Errington Rachel J.
Published: May 2013

Reactive changes in free intracellular zinc cation concentration ([Zn2+]i) were monitored, using the fluorescent probe Zinquin, in human lymphoma cells exposed to the DNA-damaging agent VP-16. Two-photon excitation microscopy showed that Zinquin-Zn2+ forms complexes in cytoplasmic vesicles. [Zn2+]iincreased in both p53wt (wild type) and p53mut(mutant) cells after exposure to low drug doses. In p53mutcells noncompetent for DNA damage-induced apoptosis, elevated [Zn2+]i was maintained at higher drug doses, unlike competent p53wt cells that showed a collapse of the transient before apoptosis. In p53wt cells, the [Zn2+]i rise paralleled an increase in p53 and bax-to-bcl-2 ratio but preceded an increase in p21WAF1, active cell cycle arrest in G2, or nuclear fragmentation. Reducing [Zn2+]i, using N, N, N′, N′-tetrakis(2-pyridylmethyl)ethylenediamine, caused rapid apoptosis in both p53wt and p53mut cells, although cotreatment with VP-16 exacerbated apoptosis only in p53wt cells. This may reflect changed thresholds for proapoptotic caspase-3 activation in competent cells. We conclude that the DNA damage-induced transient is p53-independent up to a damage threshold, beyond which competent cells reduce [Zn2+]i before apoptosis. Early stress responses in p53wt cells take place in an environment of enhanced Zn2+ availability.
Increased phosphorylation of myosin light chain associated with slow-to-fast transition in rat soleus
Authors: Bozzo Cyril , Stevens Laurence , Toniolo Luana , Mounier Yvonne , Reggiani Carlo
Published: May 2013

In striated muscles myosin light chain (MLC)2 phosphorylation regulates calcium sensitivity and mediates sarcomere organization. Little is known about the changes in MLC2 phosphorylation in relation to skeletal muscle plasticity. We studied changes in MLC2 phosphorylation in rats receiving three treatment conditions causing slow-to-fast transitions: 1) atrophy induced by 14 days of hindlimb suspension (HS), 2) hypertrophy induced by 14 days of clenbuterol administration (CB), and 3) 14 days of combined treatment (CB-HS). Three variants of the slow (MLC2s) and two variants of the fast MLC2 (MLC2f) isoform were separated with two-dimensional electrophoresis and identified with monoclonal and polyclonal antibodies specific for MLC2; their relative proportions were densitometrically quantified. In control soleus muscle MLC2s predominated over MLC2f (91.4 ± 3.9% vs. 8.5 ± 3.9%) and was separated into two spots, the less acidic spot being 73.5 ± 4.3% of the total. All treatments caused a decrease of the less acidic unphosphorylated spot of MLC2s (CB: 64.1 ± 5.6%, HS: 62.4 ± 6.8%, CB-HS: 56.4 ± 4.4%), the appearance of a third more acidic variant of MLC2s (representing 3.9–5.9% of total MLC2s), an increase of MLC2f (CB: 30.9 ± 3.1%, HS: 23.9 ± 3.3%, CB-HS: 25.3 ± 3.9%), and the phosphorylation of a large fraction of MLC2f (CB: 30.4 ± 6.7%, HS: 28.7 ± 6.5%, CB-HS: 21.8 ± 2.1%). Treatment with alkaline phosphatase or with protein phosphatase 1 (PP1) removed the most acidic spots of both MLC2f and MLC2s. We conclude that in rat skeletal muscles an increase of MLC2 phosphorylation is associated with the slow-to-fast transition regardless of whether hypertrophy or atrophy develops.
TIMAP, a novel CAAX box protein regulated by TGF-β1 and expressed in endothelial cells
Authors: Cao Wangsen , Mattagajasingh Subhendra N. , Xu Hangxue , Kim Kwanghee , Fierlbeck Wolfgang , Deng Jie , Lowenstein Charles J. , Ballermann Barbara J.
Published: May 2013

Representational difference analysis of the glomerular endothelial cell response to transforming growth factor-β1 (TGF-β1) revealed a novel gene, TIMAP (TGF-β-inhibited membrane-associated protein), which contains 10 exons and maps to human chromosome 20.q11.22. By Northern blot, TIMAP mRNA is highly expressed in all cultured endothelial and hematopoietic cells. The frequency of the TIMAP SAGE tag is much greater in endothelial cell SAGE databases than in nonendothelial cells. Immunofluorescence studies of rat tissues show that anti-TIMAP antibodies localize to vascular endothelium. TGF-β1 represses TIMAP through a protein synthesis- and histone deacetylase-dependent process. The TIMAP protein contains five ankyrin repeats, a protein phosphatase-1 (PP1)-interacting domain, a COOH-terminal CAAX box, a domain arrangement similar to that of MYPT3, and a PP1 inhibitor. A green fluorescent protein-TIMAP fusion protein localized to the plasma membrane in a CAAX box-dependent fashion. Hence, TIMAP is a novel gene highly expressed in endothelial and hematopoietic cells and regulated by TGF-β1. On the basis of its domain structure, TIMAP may serve a signaling function, potentially through interaction with PP1.
Oxidant and antioxidant modulation of chloride channels expressed in human retinal pigment epithelium
Authors: Weng T. X. , Godley B. F. , Jin G. F. , Mangini N. J. , Kennedy B. G. , Yu A. S. L. , Wills N. K.
Published: May 2013

Retinal pigment epithelium (RPE) possesses regulated chloride channels that are crucial for transepithelial fluid and ion transport. At present, little is known about the molecular nature of chloride channels in human adult RPE (haRPE) or the effects of oxidative stress on membrane conductance properties. In the present study, we assessed ClC channel and cystic fibrosis transmembrane conductance regulator (CFTR) expression and membrane chloride conductance properties in haRPE cells. ClC-5, ClC-3, ClC-2, and CFTR mRNA expression was confirmed with RT-PCR analysis, and protein expression was detected with Western blot analysis and immunofluorescence microscopy. Whole cell recordings of primary cultures of haRPE showed an outwardly rectifying chloride current that was inhibited by the oxidant H2O2. The inhibitory effects of H2O2 were reduced in cultured human RPE cells that were incubated with precursors of glutathione synthesis or that were stably transfected to overexpress glutathione S-transferase. These findings indicate a possible role for ClC channels in haRPE cells and suggest possible redox modulation of human RPE chloride conductances.
Regulation of K-Cl cotransport during reticulocyte maturation and erythrocyte aging in normal and sickle erythrocytes
Authors: Bize Isabel , Taher Samara , Brugnara Carlo
Published: May 2013

The age/density-dependent decrease in K-Cl cotransport (KCC), PP1 and PP2A activities in normal and sickle human erythrocytes, and the effect of urea, a known KCC activator, were studied using discontinuous, isotonic gradients. In normal erythrocytes, the densest fraction (d ∼33.4 g/dl) has only about ∼5% of the KCC and 4% of the membrane (mb)-PP1 activities of the least-dense fraction (d ∼24.7 g/dl). In sickle and normal erythrocytes, density-dependent decreases for mb-PP1 activity were similar (d50% 28.1 ± 0.4 vs. 27.2 ± 0.2 g/dl, respectively), whereas those for KCC activity were not (d50% 31.4 ± 0.9 vs. 26.8 ± 0.3 g/dl, respectively, P = 0.004). Excluding the 10% least-dense cells, a very tight correlation exists between KCC and mb-PP1 activities in normal ( r2 = 0.995) and sickle erythrocytes ( r2 = 0.93), but at comparable mb-PP1 activities, KCC activity is higher in sickle erythrocytes, suggesting a defective, mb-PP1-independent KCC regulation. In normal, least-dense but not in densest cells, urea stimulates KCC (two- to fourfold) and moderately increases mb-PP1 (20–40%). Thus mb-PP1 appears to mediate part of urea-stimulated KCC activity.
TNF-α is a mitogen in skeletal muscle
Authors: Li Yi-Ping
Published: May 2013

Emerging evidence suggests that tumor necrosis factor (TNF)-α plays a role in muscle repair. To determine whether TNF-α modulates satellite cell proliferation, the current study evaluated TNF-α effects on DNA synthesis in primary myoblasts and on satellite cell activation in adult mouse muscle. Exposure to recombinant TNF-α increased total DNA content in rat primary myoblasts dose-dependently over a 24-h period and increased the number of primary myoblasts incorporating 5-bromo-2′-deoxyuridine (BrdU) during a 30-min pulse labeling. Systemic injection of TNF-α stimulated BrdU incorporation by satellite cells in muscles of adult mice, whereas no BrdU was incorporated by satellite cells in control mice. TNF-α stimulated serum response factor (SRF) binding to the serum response element (SRE) present in the c- fos gene promoter and stimulated reporter gene expression controlled by the same element. Our data suggest that TNF-α activates satellite cells to enter the cell cycle and accelerates G1-to-S phase transition, and these actions may involve activation of early response genes via SRF.
Effect of ouabain on CFTR gene expression in human Calu-3 cells
Authors: Baudouin-Legros Maryvonne , Brouillard Franck , Tondelier Danielle , Hinzpeter Alexandre , Edelman Aleksander
Published: May 2013

We have previously shown that ouabain, which changes the electrochemical properties of cell membranes by inhibiting Na+,K+-ATPase, induces the expression of multidrug resistance (MDR-1) gene in several human cell lines. Because the expressions of the MDR-1 and CFTR (which encodes the cAMP-activated Cl− channel associated with cystic fibrosis) genes are physiologically regulated in opposing directions, we wanted to determine whether ouabain also decreases CFTR transcripts and subsequently to analyze its mechanism of action. We found that the submicromolar concentrations of ouabain that increase MDR-1 mRNAs decrease the CFTR transcripts with analogous time-dependency in human pulmonary Calu-3 cells. By altering or reproducing the ouabain-induced changes in intracellular ionic activities (decreasing in external Na+ or K+ or using Na+ ionophore), we show that the ouabain-induced regulations of both CFTR and MDR-1 transcripts depend on the Na+/K+ pump inhibition but that the decrease in CFTR mRNAs also proceeds from cytoplasm reactions simultaneously activated by ouabain. These data, which emphasize the complex mechanism of action of ouabain, suggest that changes in intracellular ionic activities modulate CFTR/MDR-1 gene expressions.
Single cysteine to tyrosine transition inactivates the growth inhibitory function of Piedmontese myostatin
Authors: Berry Carole , Thomas Mark , Langley Brett , Sharma Mridula , Kambadur Ravi
Published: May 2013

Myostatin, a member of the transforming growth factor-β superfamily, is a secreted growth factor that is proteolytically processed to give COOH-terminal mature myostatin and NH2-terminal latency-associated peptide in myoblasts. Piedmontese cattle are a heavy-muscled breed that express a mutated form of myostatin in which cysteine (313) is substituted with tyrosine. Here we have characterized the biology of this mutated Piedmontese myostatin. Northern and Western analyses indicate that there is increased expression of myostatin mRNA and precursor myostatin protein in the skeletal muscle of Piedmontese cattle. In contrast, a decrease in mature myostatin was observed in Piedmontese skeletal muscle. However, there is no detectable change in the circulatory levels of mature myostatin in Piedmontese cattle. Myoblast proliferation assay performed with normal and Piedmontese myostatin indicated that mature wild-type myostatin protein inhibited the proliferation of C2C12 myoblasts. Piedmontese myostatin, by contrast, failed to inhibit myoblast proliferation. In addition, when added in molar excess, Piedmontese myostatin acted as a potent “competitive inhibitor” molecule. These results indicate that, in Piedmontese myostatin, substitution of cysteine with tyrosine results in the distortion of the “cystine knot” structure and a loss of biological activity of the myostatin. This mutation also appears to affect either processing or stability of mature myostatin without altering the secretion of myostatin.
Isoenzyme-selective regulation of SERCA2 gene expression by protein kinase C in neonatal rat ventricular myocytes
Authors: Porter Michael J. , Heidkamp Maria C. , Scully Brian T. , Patel Nehu , Martin Jody L. , Samarel Allen M.
Published: May 2013

Patients with cardiac hypertrophy and heart failure display abnormally slowed myocardial relaxation, which is associated with downregulation of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2) gene expression. We previously showed that SERCA2 downregulation can be simulated in cultured neonatal rat ventricular myocytes (NRVM) by treatment with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA). However, NRVM express three different PMA-sensitive PKC isoenzymes (PKCα, PKCϵ, and PKCδ), which may be differentially regulated and have specific functions in the cardiomyocyte. Therefore, in this study we used adenoviral vectors encoding wild-type (wt) and kinase-defective, dominant negative (dn) mutant forms of PKCα, PKCϵ, and PKCδ to analyze their individual effects in regulating SERCA2 gene expression in NRVM. Overexpression of wtPKCϵ and wtPKCδ, but not wtPKCα, was sufficient to downregulate SERCA2 mRNA levels, as assessed by Northern blotting and quantitative, real-time RT-PCR (69 ± 7 and 61 ± 9% of control levels for wtPKCϵ and wtPKCδ, respectively; P < 0.05 for each adenovirus; n = 8 experiments). Conversely, overexpression of all three dnPKCs appeared to significantly increase SERCA2 mRNA levels (dnPKCδ > dnPKCϵ > dnPKCα). dnPKCδ overexpression produced the largest increase (2.8 ± 1.0-fold; n = 11 experiments). However, PMA treatment was still sufficient to downregulate SERCA2 mRNA levels despite overexpression of each dominant negative mutant. These data indicate that the novel PKC isoenzymes PKCϵ and PKCδ selectively regulate SERCA2 gene expression in cardiomyocytes but that neither PKC alone is necessary for this effect if the other novel PKC can be activated.
Secretory activation of basolateral membrane Cl−channels in guinea pig distal colonic crypts
Authors: Li Yingjun , Halm Susan Troutman , Halm Dan R.
Published: May 2013

Cell-attached recordings revealed Cl− channel activity in basolateral membrane of guinea pig distal colonic crypts isolated from basement membrane. Outwardly rectified currents (gpClor) were apparent with a single-channel conductance (γ) of 29 pS at resting membrane electrical potential; another outward rectifier with γ of 24 pS was also observed (∼25% of gpClor). At a holding potential of −80 mV γ was 18 pS for bothgpClor currents, and at +80 mV γ was 67 and 40 pS, respectively. Identity as Cl− channels was confirmed in excised patches by changing bath ion composition. From reversal potentials, relative permeability of K+ over Cl− ( P K/ P Cl) was 0.07 ± 0.03, with relative permeability of Na+over Cl−( P Na/ P Cl) = 0.08 ± 0.04. A second type of Cl− channel was seen with linear current-voltage ( I-V) relations (gpClL), having subtypes with γ of 21, 13, and 8 pS. Epinephrine or forskolin increased the number of opengpClor and gpClL. Open probabilities ( P o) ofgpClor, gpClL21, andgpClL13 were voltage dependent in cell-attached patches, higher at more positive potentials. Kinetics ofgpClor were more rapid with epinephrine activation than with forskolin activation. Epinephrine increased P o at the resting membrane potential forgpClL13. Secretagogue activation of these Cl− channels may contribute to stimulation of electrogenic K+ secretion across colonic epithelium by increasing basolateral membrane Cl− conductance that permits Cl− exit after uptake via Na+-K+-2Cl− cotransport.
Alterations in Ca2+ cycling by lysoplasmenylcholine in adult rabbit ventricular myocytes
Authors: Liu Shi J. , Kennedy Richard H. , Creer Michael H. , McHowat Jane
Published: May 2013

We previously reported that lysoplasmenylcholine (LPlasC) altered the action potential (AP) and induced afterdepolarizations in rabbit ventricular myocytes. In this study, we investigated how LPlasC alters excitation-contraction coupling using edge-motion detection, fura-PE3 fluorescent indicator, and perforated and whole cell patch-clamp techniques. LPlasC increased contraction, myofilament Ca2+ sensitivity, systolic and diastolic free Ca2+ levels, and the magnitude of Ca2+ transients concomitant with increases in the maximum rates of shortening and relaxation of contraction and the rising and declining phases of Ca2+ transients. In some cells, LPlasC induced arrhythmias in a pattern consistent with early and delayed aftercontractions. LPlasC also augmented the caffeine-induced Ca2+ transient with a reduction in the decay rate. Furthermore, LPlasC enhanced L-type Ca2+ channel current ( I Ca,L) and outward currents. LPlasC-induced alterations in contraction and I Ca,L were paralleled by its effect on the AP. Thus these results suggest that LPlasC elicits distinct, potent positive inotropic, lusitropic, and arrhythmogenic effects, resulting from increases in Ca2+influx, Ca2+ sensitivity, sarcoplasmic reticular (SR) Ca2+ release and uptake, SR Ca2+ content, and probably reduction in sarcolemmal Na+/Ca2+exchange.
High glucose and endothelial cell growth: novel effects independent of autocrine TGF-β1 and hyperosmolarity
Authors: McGinn S. , Poronnik P. , King M. , Gallery E. D. M. , Pollock C. A.
Published: May 2013

Human endothelial cells were exposed to 5 mM glucose (control), 25 mM (high) glucose, or osmotic control for 72 h. TGF-β1 production, cell growth, death, and cell cycle progression, and the effects of TGF-β1 and TGF-β neutralization on these parameters were studied. High glucose and hyperosmolarity increased endothelial TGF-β1 secretion ( P < 0.0001) and bioactivity ( P < 0.0001). However, high glucose had a greater effect on reducing endothelial cell number ( P < 0.001) and increasing cellular protein content ( P < 0.001) than the osmotic control. TGF-β antibody only reversed the antiproliferative and hypertrophic effects of high glucose. High glucose altered cell cycle progression and cyclin-dependent kinase inhibitor expression independently of hyperosmolarity. High glucose increased endothelial cell apoptosis ( P < 0.01), whereas hyperosmolarity induced endothelial cell necrosis ( P < 0.001). TGF-β antibody did not reverse the apoptotic effects observed with high glucose. Exogenous TGF-β1 mimicked the increased S phase delay but not endoreduplication observed with high glucose. High glucose altered endothelial cell growth, apoptosis, and cell cycle progression. These growth effects occurred principally via a TGF-β1 autocrine pathway. In contrast, apoptosis and endoreduplication occurred independently of this cytokine and hyperosmolarity.
Arachidonic acid mediates dual effect of TNF-α on Ca2+ transients and contraction of adult rat cardiomyocytes
Authors: Amadou Aïssata , Nawrocki Artur , Best-Belpomme Martin , Pavoine Catherine , Pecker Françoise
Published: May 2013

Tumor necrosis factor (TNF)-α has a biphasic effect on heart contractility and stimulates phospholipase A2 (PLA2) in cardiomyocytes. Because arachidonic acid (AA) exerts a dual effect on intracellular Ca2+ concentration ([Ca2+]i) transients, we investigated the possible role of AA as a mediator of TNF-α on [Ca2+]i transients and contraction with electrically stimulated adult rat cardiac myocytes. At a low concentration (10 ng/ml) TNF-α produced a 40% increase in the amplitude of both [Ca2+]i transients and contraction within 40 min. At a high concentration (50 ng/ml) TNF-α evoked a biphasic effect comprising an initial positive effect peaking at 5 min, followed by a sustained negative effect leading to 50–40% decreases in [Ca2+]i transients and contraction after 30 min. Both the positive and negative effects of TNF-α were reproduced by AA and blocked by arachidonyltrifluoromethyl ketone (AACOCF3), an inhibitor of cytosolic PLA2. Lipoxygenase and cyclooxygenase inhibitors reproduced the high-dose effects of TNF-α and AA. The negative effects of TNF-α and AA were also reproduced by sphingosine and were abrogated by the ceramidase inhibitor n-oleoylethanolamine. These results point out the key role of the cytosolic PLA2/AA pathway in mediating the contractile effects of TNF-α.
Inhibition of apical Cl−/OH− exchange activity in Caco-2 cells by phorbol esters is mediated by PKCε
Authors: Saksena Seema , Gill Ravinder K. , Syed Irfan A. , Tyagi Sangeeta , Alrefai Waddah A. , Ramaswamy K. , Dudeja Pradeep K.
Published: May 2013

The present studies were undertaken to examine the possible regulation of apical membrane Cl−/OH− exchanger in Caco-2 cells by protein kinase C (PKC). The effect of the phorbol ester phorbol 12-myristate 13-acetate (PMA), an in vitro PKC agonist, on OH−gradient-driven 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS)-sensitive 36Cl uptake in Caco-2 cells was assessed. The results demonstrated that PMA decreased apical Cl−/OH− exchanger activity via phosphatidylinositol 3-kinase (PI3-kinase)-mediated activation of PKCε. The data consistent with these conclusions are as follows: 1) short-term treatment of cells for 1–2 h with PMA (100 nM) significantly decreased Cl−/OH−exchange activity compared with control (4α-PMA); 2) pretreatment of cells with specific PKC inhibitors chelerythrine chloride, calphostin C, and GF-109203X completely blocked the inhibition of Cl−/OH− exchange activity by PMA; 3) specific inhibitors for PKCε (Ro-318220) but not PKCα (Go-6976) significantly blocked the PMA-mediated inhibition; 4) specific PI3-kinase inhibitors wortmannin and LY-294002 significantly attenuated the inhibitory effect of PMA; and 5) PI3-kinase activators IRS-1 peptide and phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] mimicked the effects of PMA. These findings provide the first evidence for PKCε-mediated inhibition of Cl−/OH− exchange activity in Caco-2 cells and indicate the involvement of the PI3-kinase-mediated pathways in the regulation of Cl− absorption in intestinal epithelial cells.
The calcium-dependent chloride conductance mediator pCLCA1
Authors: Loewen Matthew E. , Gabriel Sherif E. , Forsyth George W.
Published: May 2013

The regulatory behavior, inhibitor sensitivity, and properties of the whole cell chloride conductance observed in cells expressing the cDNA coding for a chloride conductance mediator isoform of the CLCA gene family, pCLCA1, have been studied. Common C-kinase consensus phosphorylation sites between pCLCA1 and the closely related human isoform hCLCA1 are consistent with a role for calcium in channel activation. Both channels are activated rapidly on exposure to the calcium ionophore ionomycin. Direct involvement of calcium in the activation of pCLCA1 was supported by the finding that treatment with the intracellular calcium chelator 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid-AM reduced the rate of chloride efflux from NIH/3T3 cells expressing the pCLCA1 channel. No combination of A-kinase activators used was effective in activating chloride efflux via this channel despite the presence of a unique strong A-kinase consensus site in pCLCA1. Notable differences of pCLCA1 from the reported properties of CLCA family members include the failure of phorbol 12-myristate 13-acetate to activate chloride efflux in cells expressing pCLCA1 and a lack of inhibition of chloride efflux from these cells after treatment with DIDS or dithiothreitol. However, selected inhibitors of anionic conductance inhibited pCLCA1-dependent anion efflux. The electrogenic nature of the ionomycin-dependent efflux of chloride from cells expressing pCLCA1 was confirmed by detection of outwardly rectifying chloride current and inhibition of this current by chloride conductance inhibitors in a whole cell patch-clamp study.
Contribution of Na+-K+-Cl−cotransporter to high-[K+]o- induced swelling and EAA release in astrocytes
Authors: Su Gui , Kintner Douglas B. , Sun Dandan
Published: May 2013

We hypothesized that high extracellular K+ concentration ([K+]o)-mediated stimulation of Na+-K+-Cl− cotransporter isoform 1 (NKCC1) may result in a net gain of K+ and Cl−and thus lead to high-[K+]o-induced swelling and glutamate release. In the current study, relative cell volume changes were determined in astrocytes. Under 75 mM [K+]o, astrocytes swelled by 20.2 ± 4.9%. This high-[K+]o-mediated swelling was abolished by the NKCC1 inhibitor bumetanide (10 μM, 1.0 ± 3.1%; P < 0.05). Intracellular36Cl− accumulation was increased from a control value of 0.39 ± 0.06 to 0.68 ± 0.05 μmol/mg protein in response to 75 mM [K+]o. This increase was significantly reduced by bumetanide ( P < 0.05). Basal intracellular Na+ concentration ([Na+]i) was reduced from 19.1 ± 0.8 to 16.8 ± 1.9 mM by bumetanide ( P < 0.05). [Na+]i decreased to 8.4 ± 1.0 mM under 75 mM [K+]o and was further reduced to 5.2 ± 1.7 mM by bumetanide. In addition, the recovery rate of [Na+]i on return to 5.8 mM [K+]o was decreased by 40% in the presence of bumetanide ( P < 0.05). Bumetanide inhibited high-[K+]o-induced 14C-labeledd-aspartate release by ∼50% ( P < 0.05). These results suggest that NKCC1 contributes to high-[K+]o-induced astrocyte swelling and glutamate release.
Hypertonicity increases cAMP in PMN and blocks oxidative burst by PKA-dependent and -independent mechanisms
Authors: Orlic Tatjana , Loomis William H. , Shreve Amy , Namiki Sachiko , Junger Wolfgang G.
Published: May 2013

Hypertonic stress (HS) suppresses neutrophil (PMN) functions. We studied the underlying mechanism and found that HS rapidly (<1 min) increased intracellular cAMP levels by up to sevenfold. cAMP levels correlated with applied hypertonicity and the degree of neutrophil suppression. HS and cAMP-elevating drugs (forskolin and dibutyryl cAMP-acetoxymethyl ester) similarly suppressed extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase activation and superoxide formation in response to N-formylmethionyl-leucyl-phenylalanine (fMLP) stimulation. Inhibition of cAMP-dependent protein kinase A (PKA) with H-89 abrogated the suppressive effects of HS, restoring fMLP-induced ERK and p38 activation and superoxide formation. Inhibition of phosphodiesterase with 3-isobutyl-1-methylxanthine augmented cAMP accumulation and the suppressive effects of HS, while inhibition of adenylyl cyclase with MDL-12330A abolished these effects. These findings suggest that HS-activated cAMP/PKA signaling inhibits superoxide formation by intercepting fMLP-induced activation steps upstream of ERK and p38. In contrast to its effects in the presence of moderate hypertonicity levels (40 mM), H-89 was unable to rescue neutrophil functions from suppression by higher hypertonicity levels (100 mM), indicating that more severe HS suppresses neutrophils via secondary PKA-independent mechanisms.
Creatine uptake and creatine transporter expression among rat skeletal muscle fiber types
Authors: Brault Jeffrey J. , Terjung Ronald L.
Published: May 2013

Total creatine (Crtotal = phosphocreatine + creatine) concentrations differ substantially among mammalian skeletal muscle. Because the primary means to add Crtotal to muscle is uptake of creatine through the sodium-dependent creatine transporter (CrT), differences in creatine uptake and CrT expression could account for the variations in [Crtotal] among muscle fiber types. To test this hypothesis, hindlimbs of adult rats were perfused with 0.05–1 mM [14C]creatine for up to 90 min. Creatine uptake rates at 1 mM creatine were greatest in the soleus (140 ± 8.8 nmol · h−1 · g−1), less in the red gastrocnemius (117 ± 8.3), and least in the white gastrocnemius (97 ± 10.7). These rates were unaltered by time, insulin concentration, or increased perfusate sodium concentration. Conversely, creatine uptake rates were correspondingly decreased among fiber types by lower creatine and sodium concentrations. The CrT protein content by Western blot analysis was similarly greatest in the soleus, less in the red gastrocnemius, and least in the white gastrocnemius, whereas CrT mRNA was not different. Creatine uptake rates differ among skeletal muscle fiber sections in a manner reasonably assigned to the 58-kDa band of the CrT. Furthermore, creatine uptake rates scale inversely with creatine content, with the lowest uptake rate in the fiber type with the highest Crtotal and vice versa. This suggests that the creatine pool fractional turnover rate is not common across muscle phenotypes and, therefore, is differentially regulated.
Beneficial effect of taurine depletion on osmotic sodium and calcium loading during chemical hypoxia
Authors: Schaffer Stephen W. , Solodushko Viktoriya , Kakhniashvili David
Published: May 2013

Cellular sodium excess is cytotoxic because it increases both the intracellular osmotic load and intracellular calcium concentration ([Ca2+]i). Because sodium levels rise during hypoxia, it is thought to contribute to hypoxic injury. Thus the present study tested the hypothesis that taurine-linked reductions in [Na+]i reduce hypoxia-induced cell injury. Taurine depletion was achieved by exposing isolated neonatal cardiomyocytes to medium containing the taurine analog β-Alanine. As predicted, the β-Alanine-treated cell exhibited less hypoxia-induced necrosis and apoptosis than the control, as evidenced by less swelling, shrinkage, TdT-mediated dUTP nick end labeling staining, and accumulation of trypan blue. After 1 h of chemical hypoxia, [Na+]i was 3.5-fold greater in the control than the taurine-deficient cell. Although more taurine was lost from the control cell than from the β-Alanine-treated cell during hypoxia, the combined taurine and sodium osmotic load was lower in the β-Alanine-treated cell. Taurine deficiency also reduced the degree of hypoxia-induced calcium overload. Thus the observed resistance against hypoxia-induced necrosis and apoptosis is probably related to an improvement in sodium and calcium handling.
Modulation of cardiac PIP2 by cardioactive hormones and other physiologically relevant interventions
Authors: Nasuhoglu Cem , Feng Siyi , Mao Yanping , Shammat Imman , Yamamato Masaya , Earnest Svetlana , Lemmon Mark , Hilgemann Donald W.
Published: May 2013

Phosphatidylinositol 4,5-bisphosphate (PIP2) affects profoundly several cardiac ion channels and transporters, and studies of PIP2-sensitive currents in excised patches suggest that PIP2 can be synthesized and broken down within 30 s. To test when, and if, total phosphatidylinositol 4-phosphate (PIP) and PIP2 levels actually change in intact heart, we used a new, nonradioactive HPLC method to quantify anionic phospholipids. Total PIP and PIP2 levels (10–30 μmol/kg wet weight) do not change, or even increase, with activation of Gαq/phospholipase C (PLC)-dependent pathways by carbachol (50 μM), phenylephrine (50 μM), and endothelin-1 (0.3 μM). Adenosine (0.2 mM) and phorbol 12-myristate 13-acetate (1μM) both cause 30% reduction of PIP2 in ventricles, suggesting that diacylglycerol (DAG)-dependent mechanisms negatively regulate cardiac PIP2. PIP2, but not PIP, increases reversibly by 30% during electrical stimulation (2 Hz for 5 min) in guinea pig left atria; the increase is blocked by nickel (2 mM). Both PIP and PIP2 increase within 3 min in hypertonic solutions, roughly in proportion to osmolarity, and similar effects occur in multiple cell lines. Inhibitors of several volume-sensitive signaling mechanisms do not affect these responses, suggesting that PIP2 metabolism might be sensitive to membrane tension, per se.
Regulation of neuregulin/ErbB signaling by contractile activity in skeletal muscle
Authors: Lebrasseur Nathan K. , Coté Gregory M. , Miller Thomas A. , Fielding Roger A. , Sawyer Douglas B.
Published: May 2013

Putative roles of neuregulin (NRG) and the ErbB receptors in skeletal muscle biology include myogenesis, ACh receptor expression, and glucose transport. To date, however, the physiological regulation of NRG/ErbB signaling has not been examined. We tested the hypothesis that contractile activity in vivo induces NRG/ErbB activation. Rat hindlimb muscle contraction was elicited with a single bout of electrical stimulation (RX) or treadmill running (EX). Western blot and immunofluorescence confirmed the expression of multiple NRG isoforms and the ErbB2, ErbB3, and ErbB4 receptors in adult skeletal muscle. Both RX and EX significantly increased phosphorylation of all NRG receptors. Furthermore, contraction induced a shift in the expression profile of NRG, consistent with proteolytic processing of a transmembrane isoform. Thus two distinct modes of exercise activated processing of NRG with concomitant stimulation of ErbB2, ErbB3, and ErbB4 signaling in vivo. To our knowledge, this is the first demonstration of physiological regulation of NRG/ErbB signaling in any organ and implicates this pathway in the metabolic and proliferative responses of skeletal muscle to exercise.
Effects of glucose on contractile function, [Ca2+]i, and glycogen in isolated mouse skeletal muscle
Authors: Helander Ingrid , Westerblad Håkan , Katz Abram
Published: May 2013

Extensor digitorum longus muscles were stimulated to contract to fatigue and allowed to recover for 2 h in the absence or presence of 5.5 or 11 mM extracellular glucose. This was followed by a second fatigue run, which ended when the absolute force was the same as at the end of the first run. During the first fatigue run, the fluorescence ratio for indo 1 increased [reflecting an increase in myoplasmic free Ca2+ concentration ([Ca2+]i)] during the initial tetani, peaking at ∼115% of the first tetanic value, followed by a continuous decrease to ∼90% at fatigue. During the first fatigue run, myofibrillar Ca2+ sensitivity was significantly decreased. During the second run, the number of tetani was 57 ± 6% of initial force in muscles that recovered in the absence of glucose and 110 ± 6 and 119 ± 2% of initial force in muscles that recovered in 5.5 and 11 mM glucose, respectively. Fluorescence ratios during the first, peak, and last tetani did not differ significantly between the first and second fatigue runs during any of the three conditions. Glycogen decreased by almost 50% during the first fatigue run and did not change further after recovery in the absence of glucose. After recovery in the presence of 5.5 and 11 mM glucose, glycogen increased 32 and 42% above the nonstimulated control value ( P < 0.01). These data demonstrate that extracellular glucose delays the decrease of tetanic force and [Ca2+]i during fatiguing stimulation and that glycogen supercompensation following contraction can occur in the absence of insulin.
An NAD(P)H oxidase regulates growth and transcription in melanoma cells
Authors: Brar Sukhdev S. , Kennedy Thomas P. , Sturrock Anne B. , Huecksteadt Thomas P. , Quinn Mark T. , Whorton A. Richard , Hoidal John R.
Published: May 2013

Malignant melanoma cells spontaneously generate reactive oxygen species (ROS) that promote constitutive activation of the transcription factor nuclear factor-κB (NF-κB). Although antioxidants and inhibitors of NAD(P)H oxidases significantly reduce constitutive NF-κB activation and suppress cell proliferation (11), the nature of the enzyme responsible for ROS production in melanoma cells has not been determined. To address this issue, we now have characterized the source of ROS production in melanoma cells. We report that ROS are generated by isolated, cytosol-free melanoma plasma membranes, with inhibition by NAD(P)H oxidase inhibitors. The p22 phox , gp91 phox , and p67 phox components of the human phagocyte NAD(P)H oxidase and the gp91 phox homolog NOX4 were demonstrated in melanomas by RT-PCR and sequencing, and protein product for both p22 phox and gp91 phox was detected in cell membranes by immunoassay. Normal human epidermal melanocytes expressed only p22 phox and NOX4. Melanoma proliferation was reduced by NAD(P)H oxidase inhibitors and by transfection of antisense but not sense oligonucleotides for p22 phox and NOX4. Also, the flavoprotein inhibitor diphenylene iodonium inhibited constitutive DNA binding of nuclear protein to the NF-κB and cAMP-response element consensus oligonucleotides, without affecting DNA binding activity to activator protein-1 or OCT-1. This suggests that ROS generated in autocrine fashion by an NAD(P)H oxidase may play a role in signaling malignant melanoma growth.
K+ depolarization induces RhoA kinase translocation to caveolae and Ca2+ sensitization of arterial muscle
Authors: Urban Nicole H. , Berg Krystina M. , Ratz Paul H.
Published: May 2013

KCl causes smooth muscle contraction by elevating intracellular free Ca2+, whereas receptor stimulation activates an additional mechanism, termed Ca2+ sensitization, that can involve activation of RhoA-associated kinase (ROK) and PKC. However, recent studies support the hypothesis that KCl may also increase Ca2+ sensitivity. Our data showed that the PKC inhibitor GF-109203X did not, whereas the ROK inhibitor Y-27632 did, inhibit KCl-induced tonic (5 min) force and myosin light chain (MLC) phosphorylation in rabbit artery. Y-27632 also inhibited BAY K 8644- and ionomycin-induced MLC phosphorylation and force but did not inhibit KCl-induced Ca2+ entry or peak (∼15 s) force. Moreover, KCl and BAY K 8644 nearly doubled the amount of ROK colocalized to caveolae at 30 s, a time that preceded inhibition of force by Y-27632. Colocalization was not inhibited by Y-27632 but was abolished by nifedipine and the calmodulin blocker trifluoperazine. These data support the hypothesis that KCl caused Ca2+ sensitization via ROK activation. We discuss a novel model for ROK activation involving translocation to caveolae that is dependent on Ca2+ entry and involves Ca2+-calmodulin activation.
The ClC-3 chloride channel promotes acidification of lysosomes in CHO-K1 and Huh-7 cells
Authors: Li Xinhua , Wang Ting , Zhao Zhifang , Weinman Steven A.
Published: May 2013

ClC-3 is a voltage-gated Cl−channel that is highly conserved and widely expressed, although its function, localization, and properties remain a matter of considerable debate. In this study, we have shown that heterologous expression of ClC-3 in either Chinese hamster ovary (CHO-K1) or human hepatoma (Huh-7) cells results in the formation of large, acidic vesicular structures within cells. Vesicle formation is prevented by bafilomycin, an inhibitor of the vacuolar ATPase, and is not induced by an E224A mutant of ClC-3 with altered channel activity. This demonstrates that vesicle formation requires both proton pumping and Cl−channel activity. Manipulation of the intracellular Cl−concentration demonstrated that the ClC-3-associated vesicles shrink and swell consistent with a highly Cl−-permeable membrane. The ClC-3 vesicles were identified as lysosomes based on their colocalization with the lysosome-associated proteins lamp-1, lamp-2, and cathepsin D and on their failure to colocalize with fluorescently labeled endosomes. We conclude that ClC-3 is an intracellular channel that conducts Cl− when it is present in intracellular vesicles. Its overexpression results in its appearance in enlarged lysosome-like structures where it contributes to acidification by charge neutralization.
Increase in epidermal growth factor receptor protein induced in osteoblastic cells after exposure to flow of culture media
Authors: Ogata Toshiko
Published: May 2013

To investigate how bone cells respond to mechanical stimuli, we subjected osteoblastic cells to fluid flow. We and others already reported that in a culture system of osteoblast-like cells, ERK1/2, Shc, and other proteins were tyrosine-phosphorylated by medium flow and the early response gene, egr-1 or c- fos mRNA, increased. These are the same as events found after stimulation by various growth factors. Moreover, because there were also reports suggesting that growth factor signaling is involved in the responses to mechanical stimuli, we examined the change in epidermal growth factor (EGF) receptor in the cells exposed to medium flow. The results demonstrated that EGF receptor protein increased after exposure to medium flow. This increase did not occur without serum in media, and the addition of EGF restored it. Furthermore, leupeptin blocked this increase. These results suggest that degradation of EGF-occupied EGF receptor by leupeptin-sensitive protease(s) in endosomes decreased with exposure to medium flow. This was presumed to participate, at least in part, in signaling of fluid flow.
Stretch-induced contractile differentiation of vascular smooth muscle: sensitivity to actin polymerization inhibitors
Authors: Zeidan Asad , Nordström Ina , Albinsson Sebastian , Malmqvist Ulf , Swärd Karl , Hellstrand Per
Published: May 2013

Signaling mechanisms for stretch-dependent growth and differentiation of vascular smooth muscle were investigated in mechanically loaded rat portal veins in organ culture. Stretch-dependent protein synthesis was found to depend on endogenous release of angiotensin II. Autoradiography after [35S]methionine incorporation revealed stretch-dependent synthesis of several proteins, of which SM22 and actin were particularly prominent. Inhibition of RhoA activity by cell-permeant C3 toxin increased tissue mechanical compliance and reduced stretch-dependent extracellular signal-regulated kinase (ERK)1/2 activation, growth, and synthesis of actin and SM22, suggesting a role of the actin cytoskeleton. In contrast, inhibition of Rho-associated kinase by Y-27632 did not reduce ERK1/2 phosphorylation or actin and SM22 synthesis and did not affect tissue mechanical compliance but still inhibited overall growth. The actin polymerization inhibitors latrunculin B and cytochalasin D both inhibited growth and caused increased tissue compliance. Whereas latrunculin B concentration-dependently reduced actin and SM22 synthesis, cytochalasin D did so at low (10−8 M) but not at high (10−6 M) concentration. The results show that stretch stabilizes the contractile smooth muscle phenotype. Stretch-dependent differentiation marker expression requires an intact cytoskeleton for stretch sensing, control of protein expression via the level of unpolymerized G-actin, or both.
Characterization of vectorial chloride transport pathways in the human pancreatic duct adenocarcinoma cell line HPAF
Authors: Fong Peying , Argent Barry E. , Guggino William B. , Gray Michael A.
Published: May 2013

Pancreatic duct cells express a Ca2+-activated Cl- conductance (CaCC), upregulation of which may be beneficial to patients with cystic fibrosis. Here, we report that HPAF, a human pancreatic ductal adenocarcinoma cell line that expresses CaCC, develops into a high-resistance, anion-secreting epithelium. Mucosal ATP (50 μM) caused a fourfold increase in short-circuit current ( I sc), a hyperpolarization of transepithelial potential difference (from -4.9 ± 0.73 to -8.5 ± 0.84 mV), and a fall in resistance to less than one-half of resting values. The effects of ATP were inhibited by mucosal niflumic acid (100 μM), implicating an apical CaCC in the response. RT-PCR indicated expression of hClC-2, hClC-3, and hClC-5, but surprisingly not hCLCA-1 or hCLCA-2. K+ channel activity was necessary to maintain the ATP-stimulated I sc. Using a pharmacological approach, we found evidence for two types of K+ channels in the mucosal and serosal membranes of HPAF cells, one activated by chlorzoxazone (500 μM) and sensitive to clotrimazole (30 μM), as well as one blocked by clofilium (100 μM) but not chromanol 293B (5 μM). RT-PCR indicated expression of the Ca2+-activated K+ channel KCNN4, as well as the acid-sensitive, four transmembrane domain, two pore K+ channel, KCNK5 (hTASK-2). Western blot analysis verified the expression of CLC channels, as well as KCNK5. We conclude that HPAF will be a useful model system for studying channels pertinent to anion secretion in human pancreatic duct cells.