bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of nitric oxide synthase isoforms and detection of nitric oxide in rat placenta
Authors: Takizawa Tatsuya , Yoshikawa Hiroshi , Yamada Miho , Morita Hidetoshi
Published: May 2013

Nitric oxide (NO) production in the rat placenta was monitored and quantified by electron paramagnetic resonance (EPR) spectroscopy with hemoglobin and an Fe- N-(dithiocarboxy)sarcosine (DTCS) complex as NO-trapping reagents. Expression of nitric oxide synthase (NOS) isoforms was also examined by quantitative RT-PCR analysis. The EPR spectrum of the placenta with hemoglobin trapping showed a three-line hyperfine structure ( g = 2.008 and a = 1.66-mT). The EPR signal was diminished after the placenta was homogenized or the NOS inhibitor l-NAME was administered to pregnant rats. Therefore, the specific signal was definitely identified as being derived from endogenous NO spin-trapped by hemoglobin, and the EPR spectrum showed that the NO adduct existed as a pentacoordinate α-NO heme species. The EPR spectrum of the placenta with Fe-DTCS trapping showed a triplet signal ( g = 2.038) derived from an NO-Fe-DTCS complex. The height of the triplet signal did not vary significantly with gestational stage during the last few days of gestation. At the gestational stages examined, the level of NOS II mRNA expression was significantly higher than that of NOS III mRNA. NOS II expression in term ( day 21.5) placenta was significantly increased compared with that in preterm ( day 19.5) placenta ( P < 0.01, n = 4 or 5). These results suggest that NOS II is the predominant producer of NO in the placenta and that NOS II-generated NO plays significant roles in the maintenance of placental functions immediately before birth.
High K m of oxidative phosphorylation for ADP in skinned muscle fibers: where does it stem from?
Authors: Kongas Olav , Yuen Tai L. , Wagner Marijke J. , van Beek Johannes H. G. M. , Krab Klaas
Published: May 2013

Mitochondria in saponin-skinned cardiac fiber bundles were reported to have an order of magnitude lower apparent affinity to ADP than isolated mitochondria. Although ADP was measured outside the bundles, it was thought that the low affinity was not caused by diffusion gradients because of relatively short diffusion distances. Here we test the hypothesis that considerable ADP diffusion gradients exist and can be diminished by increasing the intrafiber ADP production rate. We increased the ADP-producing activity in rat heart skinned fiber bundles by incubating with 100 IU/ml yeast hexokinase and glucose. Consequently, we observed a significant decrease of the apparent Michaelis constant ( K m) to ADP of the respiration rate of bundles from 216 ± 59 to 50 ± 9 μM. Fitting the results with a mathematical model, we estimated the K m of mitochondria in the bundles to be 25 μM. We conclude that the affinity to ADP of in situ mitochondria in heart is of the same order of magnitude as that of isolated mitochondria.
Effects of osmotic swelling on voltage-gated calcium channel currents in rat anterior pituitary cells
Authors: Ben-Tabou De-Leon Shlomo , Blotnick Edna , Nussinovitch Itzhak
Published: May 2013

Decrease in extracellular osmolarity ([Os]e) results in stimulation of hormone secretion from pituitary cells. Different mechanisms can account for this stimulation of hormone secretion. In this study we examined the possibility that hyposmolarity directly modulates voltage-gated calcium influx in pituitary cells. The effects of hyposmolarity on L-type ( IL) and T-type ( IT) calcium currents in pituitary cells were investigated by using two hyposmotic stimuli, moderate (18-22% decrease in [Os]e) and strong (31-32% decrease in [Os]e). Exposure to moderate hyposmotic stimuli resulted in three response types in IL (a decrease, a biphasic effect, and an increase in IL) and in increase in IT. Exposure to strong hyposmotic stimuli resulted only in increases in both IL and IT. Similarly, in intact pituitary cells (perforated patch method), exposure to either moderate or strong hyposmotic stimuli resulted only in increases in both IL and IT. Thus it appears that the main effect of decrease in [Os]e is increase in calcium channel currents. This increase was differential ( IL were more sensitive than IT) and voltage independent. In addition, we show that these hyposmotic effects cannot be explained by activation of an anionic conductance or by an increase in cell membrane surface area. In conclusion, this study shows that hyposmotic swelling of pituitary cells can directly modulate voltage-gated calcium influx. This hyposmotic modulation of IL and IT may contribute to the previously reported hyposmotic stimulation of hormone secretion. The mechanisms underlying these hyposmotic effects and their possible physiological relevance are discussed.
Regulation of membrane-associated iPLA2 activity by a novel PKC isoform in ventricular myocytes
Authors: Steer Sarah A. , Wirsig Karin C. , Creer Michael H. , Ford David A. , McHowat Jane
Published: May 2013

Thrombin stimulation of rabbit ventricular myocytes increases membrane-associated, Ca2+-independent phospholipase A2 (iPLA2) activity, resulting in accelerated hydrolysis of membrane plasmalogen phospholipids and increased production of arachidonic acid and lysoplasmenylcholine. This study was designed to investigate the signal transduction pathways involved in activation of membrane-associated iPLA2. Incubation of isolated membrane fractions suspended in Ca2+-free buffer with thrombin or phorbol 12-myristate 13-acetate resulted in a two- to threefold increase in iPLA2 activity. Prior treatment with the PKC inhibitor GF-109203X blocked iPLA2 activation by thrombin. These data suggest that a novel PKC isoform present in the membrane fraction modulates iPLA2 activity. Immunoblot analysis revealed a significant portion of PKC-ε present in the membrane fraction, but no other membrane-associated novel PKC isoform was detected by this method. These data indicate that activation of membrane-associated iPLA2 is mediated by a membrane-associated novel PKC isoform in thrombin-stimulated rabbit ventricular myocytes.
Global/temporal gene expression in diaphragm and hindlimb muscles of dystrophin-deficient (mdx) mice
Authors: Rouger Karl , Le Cunff Martine , Steenman Marja , Potier Marie-Claude , Gibelin Nathalie , Dechesne Claude A. , Leger Jean J.
Published: May 2013

The mdx mouse is a model for human Duchenne muscular dystrophy (DMD), an X-linked degenerative disease of skeletal muscle tissue characterized by the absence of the dystrophin protein. The mdx mice display a much milder phenotype than DMD patients. After the first week of life when all mdx muscles evolve like muscles of young DMD patients, mdx hindlimb muscles substantially compensate for the lack of dystrophin, whereas mdx diaphragm muscle becomes progressively affected by the disease. We used cDNA microarrays to compare the expression profile of 1,082 genes, previously selected by a subtractive method, in control and mdx hindlimb and diaphragm muscles at 12 time points over the first year of the mouse life. We determined that 1) the dystrophin gene defect induced marked expression remodeling of 112 genes encoding proteins implicated in diverse muscle cell functions and 2) two-thirds of the observed transcriptomal anomalies differed between adult mdx hindlimb and diaphragm muscles. Our results showed that neither mdx diaphram muscle nor mdx hindlimb muscles evolve entirely like the human DMD muscles. This finding should be taken under consideration for the interpretation of future experiments using mdx mice as a model for therapeutic assays.
The functional and physical relationship between the DRA bicarbonate transporter and carbonic anhydrase II
Authors: Sterling Deborah , Brown Nathan J. D. , Supuran Claudiu T. , Casey Joseph R.
Published: May 2013

COOH-terminal cytoplasmic tails of chloride/bicarbonate anion exchangers (AE) bind cytosolic carbonic anhydrase II (CAII) to form a bicarbonate transport metabolon, a membrane protein complex that accelerates transmembrane bicarbonate flux. To determine whether interaction with CAII affects the downregulated in adenoma (DRA) chloride/bicarbonate exchanger, anion exchange activity of DRA-transfected HEK-293 cells was monitored by following changes in intracellular pH associated with bicarbonate transport. DRA-mediated bicarbonate transport activity of 18 ± 1 mM H+ equivalents/min was inhibited 53 ± 2% by 100 mM of the CAII inhibitor, acetazolamide, but was unaffected by the membrane-impermeant carbonic anhydrase inhibitor, 1-[5-sulfamoyl-1,3,4-thiadiazol-2-yl-(aminosulfonyl-4-phenyl)]-2,6-dimethyl-4-phenyl-pyridinium perchlorate. Compared with AE1, the COOH-terminal tail of DRA interacted weakly with CAII. Overexpression of a functionally inactive CAII mutant, V143Y, reduced AE1 transport activity by 61 ± 4% without effect on DRA transport activity (105 ± 7% transport activity relative to DRA alone). We conclude that cytosolic CAII is required for full DRA-mediated bicarbonate transport. However, DRA differs from other bicarbonate transport proteins because its transport activity is not stimulated by direct interaction with CAII.
Depolarization-induced slow calcium transients activate early genes in skeletal muscle cells
Authors: Carrasco Maria Angélica , Riveros Nora , Rı́os Juan , Müller Marioly , Torres Francisco , Pineda Jorge , Lantadilla Soledad , Jaimovich Enrique
Published: May 2013

The signaling mechanisms by which skeletal muscle electrical activity leads to changes in gene expression remain largely undefined. We have reported that myotube depolarization induces calcium signals in the cytosol and nucleus via inositol 1,4,5-trisphosphate (IP3) and phosphorylation of both ERK1/2 and cAMP-response element-binding protein (CREB). We now describe the calcium dependence of P-CREB and P-ERK induction and of the increases in mRNA of the early genes c- fos, c- jun, and egr-1. Increased phosphorylation and early gene activation were maintained in the absence of extracellular calcium, while the increase in intracellular calcium induced by caffeine could mimic the depolarization stimulus. Depolarization performed either in the presence of the IP3 inhibitors 2-aminoethoxydiphenyl borate or xestospongin C or on cells loaded with BAPTA-AM, in which slow calcium signals were abolished, resulted in decreased activation of the early genes examined. Both early gene activation and CREB phosphorylation were inhibited by ERK phosphorylation blockade. These data suggest a role for calcium in the transcription-related events that follow membrane depolarization in muscle cells.
Inhibition of DNA replication by fish oil-treated cytoplasm is counteracted by fish oil-treated nuclear extract
Authors: Rex Sybille , Kukuruzinska Maria A. , Istfan Nawfal W.
Published: May 2013

We have recently noted that cells treated with fish oil and n–3-fatty acids show slower DNA replication rates than cells treated with a control emulsion or corn oil only. However, it is not clearly understood how such an effect is induced. Fish oil and its metabolites are known to have several modulating effects on signal transduction pathways. Alternatively, they may influence DNA replication by interacting directly with nuclear components. To investigate this problem in greater detail, we have studied the kinetics of DNA synthesis in a cell-free system derived from HeLa cells. Nuclei and cytosolic extract were isolated from cells synchronized in early S phase after treatment with control emulsion, corn oil, or fish oil, respectively. The nuclei were reconstituted with cytosolic extract and a reaction mixture containing bromodeoxyuridine (BrdU) triphosphate to label newly synthesized DNA. The rate of DNA synthesis was measured by bivariate DNA/BrdU analysis and flow cytometry. We show that fish oil-treated cytosol inhibits the elongation of newly synthesized DNA by ∼80% in control nuclei. However, nuclei treated with fish oil escape this inhibitory effect. We also show that addition of nuclear extract from fish oil-treated cells reverses the inhibitory effect seen in the reconstitution system of control nuclei and fish oil-treated cytosol. These results indicate that polyunsaturated fatty acids can modulate DNA synthesis through cytosolic as well as soluble nuclear factors.
Role of EP1 and EP4 PGE2subtype receptors in serum-induced 3T6 fibroblast cycle progression and proliferation
Authors: Sanchez Teresa , Moreno Juan Jose
Published: May 2013

Recent studies have suggested that prostaglandin E2 (PGE2) subtype receptors (EP) are involved in cellular proliferation and tumor development. We studied the role of EP1 and EP4PGE2 subtype receptor antagonists AH-6809 and AH-23848B, respectively, in serum-induced 3T6 fibroblast proliferation. This was significantly reduced in a dose-dependent manner (IC50∼100 and ∼30 μM, respectively) to an almost complete inhibition, without any cytotoxic effect. However, the effect of each antagonist on 3T6 cell cycle progression clearly differed. Whereas the EP1 antagonist increased the G0/G1population, the EP4 antagonist brought about an accumulation of cells in early S phase. These effects were associated with a decrease in cyclin D and E levels in AH-6809-treated 3T6 cells and lower cyclin A levels in AH-23848B-treated fibroblasts with respect to control cells. The G0/G1 accumulation caused by AH-6809 seems to be intracellular Ca2+ concentration ([Ca2+]i) dependent, because a 6-h 1 μM thapsigargin treatment allowed G0/G1-arrested cells to enter S phase. Similarly, treatment with 20 μM forskolin for 6 h allowed S-phase and G2/M progression of AH-23848B-treated cells. This study shows that the inhibitory effect of the EP1 and EP4 antagonists on serum-induced 3T6 fibroblast growth is due to their effect at various levels of the cell cycle machinery, suggesting that PGE2 interaction with its different subtype receptors regulates progression through the cell cycle by modulating cAMP and [Ca2+]i.
Nerve activity-independent regulation of skeletal muscle atrophy: role of MyoD and myogenin in satellite cells and myonuclei
Authors: Hyatt Jon-Philippe K. , Roy Roland R. , Baldwin Kenneth M. , Edgerton V. Reggie
Published: May 2013

Electrical activity is thought to be the primary neural stimulus regulating muscle mass, expression of myogenic regulatory factor genes, and cellular activity within skeletal muscle. However, the relative contribution of neural influences that are activity-dependent and -independent in modulating these characteristics is unclear. Comparisons of denervation (no neural influence) and spinal cord isolation (SI, neural influence with minimal activity) after 3, 14, and 28 days of treatment were used to demonstrate whether there are neural influences on muscle that are activity independent. Furthermore, the effects of these manipulations were compared for a fast ankle extensor (medial gastrocnemius) and a fast ankle flexor (tibialis anterior). The mass of both muscles plateaued at ∼60% of control 2 wk after SI, whereas both muscles progressively atrophied to <25% of initial mass at this same time point after denervation. A rapid increase in myogenin and, to a lesser extent, MyoD mRNAs and proteins was observed in denervated and SI muscles: at the later time points, these myogenic regulatory factors remained elevated in denervated, but not in SI, muscles. This widespread neural activity-independent influence on MyoD and myogenin expression was observed in myonuclei and satellite cells and was not specific for fast or slow fiber phenotypes. Mitotic activity of satellite and connective tissue cells also was consistently lower in SI than in denervated muscles. These results demonstrate a neural effect independent of electrical activity that 1) helps preserve muscle mass, 2) regulates muscle-specific genes, and 3) potentially spares the satellite cell pool in inactive muscles.
Chloro(2,2′:6′,2"-terpyridine) platinum inhibition of the renal Na+,K+-ATPase
Authors: Ruddock Nancy T. , Arnett Krista L. , Wilson Betty Jo , Milanick Mark A.
Published: May 2013

Chloro(2,2′:6′,2"-terpyridine) platinum, a bulky, hydrophilic reagent, inhibited the renal sodium pump with a single exponential time course. K+ increased the rate constant of the reaction by about twofold; the K+concentration dependence was monotonic, with a half-maximal effect observed at 1 mM, consistent with K+ acting at a transport site. Na+, Mg2+, eosin, and vanadate did not significantly alter the rate of reaction. The results of proteolysis and mass spectrometer analysis were consistent with terpyridine platinum labeling of Cys452, Cys456, or Cys457. Because phenylarsine oxide reacts with vicinal cysteines and did not prevent terpyridine platinum modification, terpyridine platinum most likely modifies Cys452. This modification prevents ADP binding; interestingly, the analogous residue in sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) is on the exterior of the nucleotide-binding pocket. Thus it appears that the terpyridine platinum residue is more accessible in the presence of K+than in its absence and that terpyridine platinum modification prevents nucleotide binding.
Rme-1 regulates the recycling of the cystic fibrosis transmembrane conductance regulator
Authors: Picciano John A. , Ameen Nadia , Grant Barth D. , Bradbury Neil A.
Published: May 2013

Endocytic motifs in the carboxyl terminus of cystic fibrosis transmembrane conductance regulator (CFTR) direct internalization from the plasma membrane by clathrin-mediated endocytosis. However, the fate of such internalized CFTR has remained unknown. Internalized membrane proteins can be either targeted for degradation or recycled back to the plasma membrane. Using cell surface biotinylation and antibody uptake studies, we show that CFTR undergoes constitutive endocytosis and recycling back to the plasma membrane. Expression of dominant negative Rme-1 (a protein that regulates exit from the endosomal recycling compartment) in CFTR-expressing cells results in the expansion of recycling compartments. Transferrin, a marker for the endosomal recycling compartment, and CFTR accumulate in these enlarged recycling endosomes. Such accumulation leads to a loss of cell surface CFTR because it is prevented from being recycled back to the cell surface. In contrast, traffic of the low-density lipoprotein (LDL) is unaffected by the expression of dominant negative Rme-1. In addition, chimeras containing the extracellular domain of the transferrin receptor and the carboxyl terminal tail of CFTR also enter Rme-1-regulated recycling compartments and accumulate in these compartments containing dominant negative Rme-1, suggesting that in addition to endocytic signals, the carboxyl terminal tail of CFTR also contains intracellular traffic information.
Protein kinase Cα participates in activation of store-operated Ca2+ channels in human glomerular mesangial cells
Authors: Ma Rong , Kudlacek Patrick E. , Sansom Steven C.
Published: May 2013

Protein kinase C (PKC) plays an important role in activating store-operated Ca2+ channels (SOC) in human mesangial cells (MC). The present study was performed to determine the specific isoform(s) of conventional PKC involved in activating SOC in MC. Fura 2 fluorescence ratiometry showed that the thapsigargin-induced Ca2+ entry (equivalent to SOC) was significantly inhibited by 1 μM Gö-6976 (a specific PKCα and βI inhibitor) and PKCα antisense treatment (2.5 nM for 24–48 h). However, LY-379196 (PKCβ inhibitor) and 2,2′,3,3′,4,4′-hexahydroxy-1,1′-biphenyl-6,6′-dimethanoldimethyl ether (HBDDE; PKCα and γ inhibitor) failed to affect thapsigargin-evoked activation of SOC. Single-channel analysis in the cell-attached configuration revealed that Gö-6976 and PKCα antisense significantly depressed thapsigargin-induced activation of SOC. However, LY-379196 and HBDDE did not affect the SOC responses. In inside-out patches, application of purified PKCα or βI, but not βII or γ, significantly rescued SOC from postexcision rundown. Western blot analysis revealed that thapsigargin evoked a decrease in cytosolic expression with a corresponding increase in membrane expression of PKCα and γ. However, the translocation from cytosol to membranes was not detected for PKCβI or βII. These results suggest that PKCα participates in the intracellular signaling pathway for activating SOC upon release of intracellular stores of Ca2+.
Role of PKCα in feedback regulation of Na+transport in an electrically tight epithelium
Authors: Awayda Mouhamed S. , Platzer Justin D. , Reger Roxanne L. , Bengrine Abderrahmane
Published: May 2013

It has long been known that Na+ channels in electrically tight epithelia are regulated by homeostatic mechanisms that maintain a steady state and allow new levels of transport to be sustained in hormonally challenged cells. Little is known about the potential pathways involved in these processes. In addition to short-term effect, recent evidence also indicates the involvement of PKC in the long-term regulation of the epithelial Na+ channel (ENaC) at the protein level (40). To determine whether stimulation of ENaC involves feedback regulation of PKC levels, we utilized Western blot analysis to determine the distribution of PKC isoforms in polarized A6 epithelia. We found the presence of PKC isoforms in the conventional (α and γ), novel (δ, η, and ε), and atypical (ι, λ, and ζ) groups. Steady-state stimulation of Na+ transport with aldosterone was accompanied by a specific decrease of PKCα protein levels in both the cytoplasmic and membrane fractions. Similarly, overnight treatment with an uncharged amiloride analog (CDPC), a procedure that through feedback regulation causes a stimulation of Na+ transport, also decreased PKCα levels. These effects were additive, indicating separate mechanisms that converge at the level of PKCα. These effects were not accompanied by changes of PKCα mRNA levels as determined by Northern blot analysis. We propose that this may represent a novel regulatory feedback mechanism necessary for sustaining an increase of Na+ transport.
pH dependence and compartmentalization of zinc transported across plasma membrane of rat cortical neurons
Authors: Colvin Robert A.
Published: May 2013

In this study, Zn2+ transport in rat cortical neurons was characterized by successfully combining radioactive tracer experiments with spectrofluorometry and fluorescence microscopy. Cortical neurons showed a time-dependent and saturable transport of65Zn2+ with an apparent affinity of 15–20 μM. 65Zn2+ transport was pH dependent and was decreased by extracellular acidification and increased by intracellular acidification. Compartmentalization of newly transported Zn2+ was assessed with the Zn2+-selective fluorescent dye zinquin. Resting cortical neurons showed uniform punctate labeling that was found in cell processes and the soma, suggesting extrasynaptic compartmentalization of Zn2+. Depletion of intracellular Zn2+ with the membrane-permeant chelator N, N, N′, N′-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN) resulted in the complete loss of punctate zinquin labeling. After Zn2+ depletion, punctate zinquin labeling was rapidly restored when cells were placed in 30 μM Zn2+, pH 7.4. However, rapid restoration of punctate zinquin labeling was not observed when cells were placed in 30 μM Zn2+, pH 6.0. These data were confirmed in parallel 65Zn2+transport experiments.
p38 mitogen-activated protein kinase inhibits calcium-dependent chloride secretion in T84 colonic epithelial cells
Authors: Keely Stephen J. , Barrett Kim E.
Published: May 2013

We have previously shown that Ca2+-dependent Cl−secretion across intestinal epithelial cells is limited by a signaling pathway involving transactivation of the epidermal growth factor receptor (EGFR) and activation of ERK mitogen-activated protein kinase (MAPK). Here, we have investigated a possible role for p38 MAPK in regulation of Ca2+-dependent Cl− secretion. Western blot analysis of T84 colonic epithelial cells revealed that the muscarinic agonist carbachol (CCh; 100 μM) stimulated phosphorylation and activation of p38 MAPK. The p38 inhibitor SB-203580 (10 μM) potentiated and prolonged short-circuit current ( I sc) responses to CCh across voltage-clamped T84 cells to 157.4 ± 6.9% of those in control cells ( n = 21; P < 0.001). CCh-induced p38 phosphorylation was attenuated by the EGFR inhibitor tyrphostin AG-1478 (0.1 nM–10 μM) and by the Src family kinase inhibitor PP2 (20 nM–2 μM). The effects of CCh on p38 phosphorylation were mimicked by thapsigargin (TG; 2 μM), which specifically elevates intracellular Ca2+, and were abolished by the Ca2+ chelator BAPTA-AM (20 μM), implying a role for intracellular Ca2+ in mediating p38 activation. SB-203580 (10 μM) potentiated I sc responses to TG to 172.4 ± 18.1% of those in control cells ( n= 18; P < 0.001). When cells were pretreated with SB-203580 and PD-98059 to simultaneously inhibit p38 and ERK MAPKs, respectively, I sc responses to TG and CCh were significantly greater than those observed with either inhibitor alone. We conclude that Ca2+-dependent agonists stimulate p38 MAPK in T84 cells by a mechanism involving intracellular Ca2+, Src family kinases, and the EGFR. CCh-stimulated p38 activation constitutes a similar, but distinct and complementary, antisecretory signaling pathway to that of ERK MAPK.
Age-related differences in Na+-dependent Ca2+ accumulation in rabbit hearts exposed to hypoxia and acidification
Authors: Anderson S. E. , Liu H. , Ho H. S. , Lewis E. J. , Cala P. M.
Published: May 2013

In this study, we test the hypothesis that in newborn hearts (as in adults) hypoxia and acidification stimulate increased Na+ uptake, in part via pH-regulatory Na+/H+ exchange. Resulting increases in intracellular Na+ (Nai) alter the force driving the Na+/Ca2+ exchanger and lead to increased intracellular Ca2+. NMR spectroscopy measured Nai and cytosolic Ca2+ concentration ([Ca2+]i) and pH (pHi) in isolated, Langendorff-perfused 4- to 7-day-old rabbit hearts. After Na+/K+ ATPase inhibition, hypoxic hearts gained Na+, whereas normoxic controls did not [19 ± 3.4 to 139 ± 14.6 vs. 22 ± 1.9 to 22 ± 2.5 (SE) meq/kg dry wt, respectively]. In normoxic hearts acidified using the NH4Cl prepulse, pHi fell rapidly and recovered, whereas Nai rose from 31 ± 18.2 to 117.7 ± 20.5 meq/kg dry wt. Both protocols caused increases in [Ca]i; however, [Ca]i increased less in newborn hearts than in adults ( P < 0.05). Increases in Nai and [Ca]i were inhibited by the Na+/H+ exchange inhibitor methylisobutylamiloride (MIA, 40 μM; P < 0.05), as well as by increasing perfusate osmolarity (+30 mosM) immediately before and during hypoxia ( P < 0.05). The data support the hypothesis that in newborn hearts, like adults, increases in Nai and [Ca]i during hypoxia and after normoxic acidification are in large part the result of increased uptake via Na+/H+ and Na+/Ca2+exchange, respectively. However, for similar hypoxia and acidification protocols, this increase in [Ca]i is less in newborn than adult hearts.
Regulation of volume-sensitive outwardly rectifying anion channels in pulmonary arterial smooth muscle cells by PKC
Authors: Zhong Juming , Wang Ge-Xin , Hatton William J. , Yamboliev Ilia A. , Walsh Michael P. , Hume Joseph R.
Published: May 2013

We tested the possible role of endogenous protein kinase C (PKC) in the regulation of native volume-sensitive organic osmolyte and anion channels (VSOACs) in acutely dispersed canine pulmonary artery smooth muscle cells (PASMC). Hypotonic cell swelling activated native volume-regulated Cl− currents ( I Cl.vol) which could be reversed by exposure to phorbol 12,13-dibutyrate (0.1 μM) or by hypertonic cell shrinkage. Under isotonic conditions, calphostin C (0.1 μM) or Ro-31–8425 (0.1 μM), inhibitors of both conventional and novel PKC isozymes, significantly activated I Cl.vol and prevented further modulation by subsequent hypotonic cell swelling. Bisindolylmaleimide (0.1 μM), a selective conventional PKC inhibitor, was without effect. Dialyzing acutely dispersed and cultured PASMC with εV1–2 (10 μM), a translocation inhibitory peptide derived from the V1 region of εPKC, activated I Cl.vol under isotonic conditions and prevented further modulation by cell volume changes. Dialyzing PASMC with βC2–2 (10 μM), a translocation inhibitory peptide derived from the C2 region of βPKC, had no detectable effect. Immunohistochemistry in cultured canine PASMC verified that hypotonic cell swelling is accompanied by translocation of εPKC from the vicinity of the membrane to cytoplasmic and perinuclear locations. These data suggest that membrane-bound εPKC controls the activation state of native VSOACs in canine PASMC under isotonic and anisotonic conditions.
Peroxisome proliferators compete and ameliorate Hcy-mediated endocardial endothelial cell activation
Authors: Hunt Matthew J. , Tyagi Suresh C.
Published: May 2013

To determine whether homocysteine (Hcy)-mediated activation of endocardial endothelial (EE) cells is ameliorated by peroxisome proliferator-activated receptor (PPAR), we isolated EE cells from mouse endocardium. Matrix metalloproteinase (MMP) activity and intercellular adhesion molecule (ICAM)-1 in EE cells were measured in the presence and absence of Hcy, and ciprofibrate (CF; PPAR-α agonist) or 15-deoxy-Δ12,14-prostaglandin J2 (PGJ2; PPAR-γ agonist) by zymography and Western blot analyses, respectively. Results suggest that Hcy-mediated MMP activation and ICAM-1 expression are ameliorated by CF and PGJ2. To test the hypothesis that Hcy competes with other ligands for binding to PPARα and -γ, we prepared cardiac nuclear extracts. Extracts were loaded onto an Hcy-cellulose affinity column. Bound proteins were eluted with CF and PGJ2. To determine conformational changes in PPAR upon binding to Hcy, we measured PPAR fluorescence at 334 nm. Dose-dependent increase in PPAR fluorescence demonstrated a primary binding affinity of 0.32 ± 0.06 μM. There was dose-dependent quenching of PPAR fluorescence by fluorescamine-homocysteine (F-Hcy). PPAR-α fluorescence quenching was abrogated by the addition of CF but not by PGJ2. PPAR-γ fluorescence quenching was abrogated by the addition of PGJ2 but not by CF. These results suggest that Hcy competes with CF and PGJ2 for binding to PPAR-α and -γ, respectively, indicating a role of PPAR in amelioration of Hcy-mediated EE dysfunction.
Altered expression of skeletal muscle myosin isoforms in cancer cachexia
Authors: Diffee Gary M. , Kalfas Katherine , Al-Majid Sadeeka , McCarthy Donna O.
Published: May 2013

Cachexia is commonly seen in cancer and is characterized by severe muscle wasting, but little is known about the effect of cancer cachexia on expression of contractile protein isoforms such as myosin. Other causes of muscle atrophy shift expression of myosin isoforms toward increased fast (type II) isoform expression. We injected mice with murine C-26 adenocarcinoma cells, a tumor cell line that has been shown to cause muscle wasting. Mice were killed 21 days after tumor injection, and hindlimb muscles were removed. Myosin heavy chain (MHC) and myosin light chain (MLC) content was determined in muscle homogenates by SDS-PAGE. Body weight was significantly lower in tumor-bearing (T) mice. There was a significant decrease in muscle mass in all three muscles tested compared with control, with the largest decrease occurring in the soleus. Although no type IIb MHC was detected in the soleus samples from control mice, type IIb comprised 19% of the total MHC in T soleus. Type I MHC was significantly decreased in T vs. control soleus muscle. MHC isoform content was not significantly different from control in plantaris and gastrocnemius muscles. These data are the first to show a change in myosin isoform expression accompanying muscle atrophy during cancer cachexia.
Functional studies of the kidney of living animals using multicolor two-photon microscopy
Authors: Dunn Kenneth W. , Sandoval Ruben M. , Kelly Katherine J. , Dagher Pierre C. , Tanner George A. , Atkinson Simon J. , Bacallao Robert L. , Molitoris Bruce A.
Published: May 2013

Optical microscopy, when applied to living animals, provides a powerful means of studying cell biology in the most physiologically relevant setting. The ability of two-photon microscopy to collect optical sections deep into biological tissues has opened up the field of intravital microscopy to high-resolution studies of the brain, lens, skin, and tumors. Here we present examples of the way in which two-photon microscopy can be applied to intravital studies of kidney physiology. Because the kidney is easily externalized without compromising its function, microscopy can be used to evaluate various aspects of renal function in vivo. These include cell vitality and apoptosis, fluid transport, receptor-mediated endocytosis, blood flow, and leukocyte trafficking. Efficient two-photon excitation of multiple fluorophores permits comparison of multiple probes and simultaneous characterization of multiple parameters and yields spectral information that is crucial to the interpretation of images containing uncharacterized autofluorescence. The studies described here demonstrate the way in which two-photon microscopy can provide a level of resolution previously unattainable in intravital microscopy, enabling kinetic analyses and physiological studies of the organs of living animals with subcellular resolution.
Signal transduction of somatostatin in human B lymphoblasts
Authors: Rosskopf Dieter , Schürks Markus , Manthey Iris , Joisten Markus , Busch Stefan , Siffert Winfried
Published: May 2013

Somatostatin (SST) and somatostatin receptors (SSTR) are widely distributed in lymphoid tissues. Here, we report on the stimulatory effects of SST in Epstein-Barr virus-immortalized B lymphoblasts. By RT-PCR, we demonstrated the exclusive expression of the somatostatin receptor isoform 2A (SSTR2A) in B lymphoblasts. Addition of SST rapidly increased the cytosolic free calcium concentration [Ca2+]i maximally by about 200 nM, with an EC50 of 1.3 nM, and stimulated the formation of inositol phosphates. Furthermore, SST increased binding of guanosine 5′- O-(3-thiotriphosphate) by 50% above basal. These effects were partly inhibited by pertussis toxin (PTX), which indicates the involvement of PTX-sensitive G proteins. We provide further evidence that Gα16, a PTX-insensitive G protein confined to lymphohematopoietic cells, is involved in the otherwise unusual coupling of SSTR2A to phospholipase C activation. In addition, SST activated extracellular regulated kinases and induced a 3.5-fold stimulation of DNA synthesis and a 4.4-fold stimulation of B lymphoblast proliferation, which was accompanied by an enhanced immunoglobulin formation. Thus SST exerts a growth factor-like activity on human B lymphoblasts.
Channel-mediated water movement across enclosed or perfused mouse intrahepatic bile duct units
Authors: Gong Ai-Yu , Masyuk Anatoly I. , Splinter Patrick L. , Huebert Robert C. , Tietz Pamela S. , LaRusso Nicholas F.
Published: May 2013

We previously reported the development of reproducible techniques for isolating and perfusing intact intrahepatic bile duct units (IBDUs) from rats. Given the advantages of transgenic and knockout mice for exploring ductal bile formation, we report here the adaptation of those techniques to mice and their initial application to the study of water transport across mouse intrahepatic biliary epithelia. IBDUs were isolated from livers of normal mice by microdissection combined with enzymatic digestion. After culture, isolated IBDUs sealed to form intact, polarized compartments, and a microperfusion system employing those isolated IBDUs developed. A quantitative image analysis technique was used to observe a rapid increase of luminal area when sealed IBDUs were exposed to a series of inward osmotic gradients reflecting net water secretion; the choleretic agonists secretin and forskolin also induced water secretion into IBDUs. The increase of IBDU luminal area induced by inward osmotic gradients and choleretic agonists was reversibly inhibited by HgCl2, a water channel inhibitor. With the use of a quantitative epifluorescence technique in perfused mouse IBDUs, a high osmotic water permeability ( P f = 2.5–5.6 × 10−2 cm/s) was found in response to osmotic gradients, further supporting the presence of water channels. These findings suggest that, as in the rat, water transport across intrahepatic biliary epithelia in mice is water channel mediated.
Granulocyte colony-stimulating factor promotes adhesion of neutrophils
Authors: Chakraborty Arup , Hentzen Eric R. , Seo Scott M. , Smith C. Wayne
Published: May 2013

Granulocyte colony stimulating factor (G-CSF) is well known for its ability to drive the maturation and mobilization of neutrophils. G-CSF also appears to have the potential to activate functions of mature neutrophils, influencing recruitment at sites of inflammation and tissue injury. We investigated the ability of G-CSF to stimulate adhesion of isolated blood neutrophils. G-CSF induced significant adherence to intercellular adhesion molecule (ICAM)-1 that was both macrophage antigen-1 (Mac-1) and leukocyte function-associated antigen-1 dependent. The kinetics of G-CSF-stimulated adhesion to ICAM-1 peaked at 11 min without detectable surface upregulation of Mac-1. This was in marked contrast to chemokines, in which peak activation of adhesion is seen within 1 min of stimulation. In contrast to chemokine-induced adhesion, G-CSF stimulation was not inhibited by pertussis toxin. G-CSF also augmented the attachment of neutrophils to activated human umbilical vein endothelial cells (HUVEC) through specific effects on neutrophils, because HUVEC appear to lack functional G-CSF receptors.
Differential requirement for NF-κB-inducing kinase in the induction of NF-κB by IL-1β, TNF-α, and Fas
Authors: Russo Maria P. , Bennett Brydon L. , Manning Anthony M. , Brenner David A. , Jobin Christian
Published: May 2013

In this study, we examined the role of the nuclear factor-κB (NF-κB)-inducing kinase (NIK) in distinct signaling pathways leading to NF-κB activation. We show that a dominant-negative form of NIK (dnNIK) delivered by adenoviral (Ad5dnNIK) vector inhibits Fas-induced IκBα phosphorylation and NF-κB-dependent gene expression in HT-29 and HeLa cells. Interleukin (IL)-1β- and tumor necrosis factor-α (TNF-α)-induced NF-κB activation and κB-dependent gene expression are inhibited in HeLa cells but not in Ad5dnNIK-infected HT-29 cells. Moreover, Ad5dnNIK failed to sensitize HT-29 cells to TNF-α-induced apoptosis at an early time point. However, cytokine- and Fas-induced signals to NF-κB are finally integrated by the IκB kinase (IKK) complex, since IκBα phosphorylation, NF-κB DNA binding activity, and IL-8 gene expression were strongly inhibited in HT-29 and HeLa cells overexpressing dominant-negative IKKβ (Ad5dnIKKβ). Our findings support the concept that cytokine signaling to NF-κB is redundant at the level of NIK. In addition, this study demonstrates for the first time the critical role of NIK and IKKβ in Fas-induced NF-κB signaling cascade.
Phenotypic behavior of caveolin-3 R26Q, a mutant associated with hyperCKemia, distal myopathy, and rippling muscle disease
Authors: Sotgia Federica , Woodman Scott E. , Bonuccelli Gloria , Capozza Franco , Minetti Carlo , Scherer Philipp E. , Lisanti Michael P.
Published: May 2013

Four different phenotypes have been associated with CAV3 mutations: limb girdle muscular dystrophy-1C (LGMD-1C), rippling muscle disease (RMD), and distal myopathy (DM), as well as idiopathic and familial hyperCKemia (HCK). Detailed molecular characterization of two caveolin-3 mutations (P104L and ΔTFT), associated with LGMD-1C, shows them to impart a dominant-negative effect on wild-type caveolin-3, rendering it dysfunctional through sequestration in the Golgi complex. Interestingly, substitution of glutamine for arginine at amino acid position 26 (R26Q) of caveolin-3 is associated not only with RMD but also with DM and HCK. However, the phenotypic behavior of the caveolin-3 R26Q mutation has never been evaluated in cultured cells. Thus we characterized the cellular and molecular properties of the R26Q mutant protein to better understand how this mutation can manifest as such distinct disease phenotypes. Here, we show that the caveolin-3 R26Q mutant is mostly retained at the level of the Golgi complex. The caveolin-3 R26Q mutant formed oligomers of a much larger size than wild-type caveolin-3 and was excluded from caveolae-enriched membranes. However, caveolin-3 R26Q did not behave in a dominant-negative fashion when coexpressed with wild-type caveolin-3. Thus the R26Q mutation behaves differently from other caveolin-3 mutations (P104L and ΔTFT) that have been previously characterized. These data provide a possible explanation for the scope of the various disease phenotypes associated with the caveolin-3 R26Q mutation. We propose a haploinsufficiency model in which reduced levels of wild-type caveolin-3, although not rendered dysfunctional due to the caveolin-3 R26Q mutant protein, are insufficient for normal muscle cell function.
Ca2+-activated K+ channel inhibition by reactive oxygen species
Authors: Soto Marco A. , González Carlos , Lissi Eduardo , Vergara Cecilia , Latorre Ramón
Published: May 2013

We studied the effect of H2O2 on the gating behavior of large-conductance Ca2+-sensitive voltage-dependent K+ (KV,Ca) channels. We recorded potassium currents from single skeletal muscle channels incorporated into bilayers or using macropatches of Xenopus laevisoocytes membranes expressing the human Slowpoke(h Slo) α-subunit. Exposure of the intracellular side of KV,Ca channels to H2O2 (4–23 mM) leads to a time-dependent decrease of the open probability ( P o) without affecting the unitary conductance. H2O2 did not affect channel activity when added to the extracellular side. These results provide evidence for an intracellular site(s) of H2O2 action. Desferrioxamine (60 μM) and cysteine (1 mM) completely inhibited the effect of H2O2, indicating that the decrease in P o was mediated by hydroxyl radicals. The reducing agent dithiothreitol (DTT) could not fully reverse the effect of H2O2. However, DTT did completely reverse the decrease in P o induced by the oxidizing agent 5,5′-dithio-bis-(2-nitrobenzoic acid). The incomplete recovery of KV,Ca channel activity promoted by DTT suggests that H2O2 treatment must be modifying other amino acid residues, e.g., as methionine or tryptophan, besides cysteine. Noise analysis of macroscopic currents in Xenopus oocytes expressing h Slo channels showed that H2O2 induced a decrease in current mediated by a decrease both in the number of active channels and P o.
Differentiation in C2C12 myoblasts depends on the expression of endogenous IGFs and not serum depletion
Authors: Yoshiko Yuji , Hirao Keita , Maeda Norihiko
Published: May 2013

Myogenic differentiation in vitro has been usually viewed as being negatively controlled by serum mitogens. A depletion of critical serum components from medium has been considered to be essential for permanent withdrawal from the cell cycle and terminal differentiation of myoblasts. Removal of serum mitogens induces the expression of insulin-like growth factors (IGFs), whereas it inhibits that of basic fibroblast growth factor (bFGF) and transforming growth factor (TGF)-β in myoblasts. These responses of growth factors to medium conditioning seem to be well matched to their functions in proliferation/differentiation. In the present study, we showed that C2C12 myoblasts differentiated actively, even in mitogen-rich medium, and that this medium offered an advantage over mitogen-poor medium in terms of increasing differentiation. Our attention focused on endogenous growth factors, as described above, especially IGFs in mitogen-rich medium. During differentiation, IGF-I and IGF-II mRNA levels increased, but bFGF and TGF-β1 mRNAs decreased. Differentiation was commensurable with IGF mRNA levels and suppressed by antisense oligodeoxynucleotides and neutralizing monoclonal antibodies against IGFs. These results suggest that an autocrine/paracrine loop of IGFs, bFGF, and TGF-β1 is active in proliferating and differentiating C2C12 cells without a depletion of serum and that endogenous IGFs actively override the negative control of differentiation by serum mitogens.
Lanthanum is transported by the sodium/calcium exchanger and regulates its activity
Authors: Reeves John P. , Condrescu Madalina
Published: May 2013

La3+ uptake was measured in fura 2-loaded Chinese hamster ovary cells expressing the bovine cardiac Na+/Ca2+ exchanger (NCX1.1). La3+ was taken up by the cells after an initial lag phase of 50-60 s and achieved a steady state within 5-6 min. Neonatal cardiac myocytes accumulated La3+ in a similar manner. La3+ uptake was due to the activity of the exchanger, because no uptake was seen in nontransfected cells or in transfected cells that had been treated with gramicidin to remove cytosolic Na+. The low rate of La3+ uptake during the lag period resulted from insufficient cytosolic Ca2+ to activate the exchanger at its regulatory sites, as shown by the following observations. La3+ uptake occurred without a lag period in cells expressing a mutant of NCX1.1 that does not exhibit regulatory activation by cytosolic Ca2+. The rate of La3+ uptake by wild-type cells was increased, and the lag phase was reduced or eliminated, when the cytosolic Ca2+ concentration was increased before initiating La3+ uptake. La3+ could substitute for Ca2+ at very low concentrations to activate exchange activity. Thus preloading cells expressing NCX1.1 with a small quantity of La3+ increased the rate of exchange-mediated Ca2+ influx by 20-fold; in contrast, cytosolic La3+ partially inhibited Ca2+ uptake by the regulation-deficient mutant. With an estimated KD of 30 pM for the binding of La3+ to fura 2, we conclude that cytosolic La3+ activates exchange activity at picomolar concentrations. We speculatively suggest that endogenous trace metals might activate exchange activity under physiological conditions.
Spatial and temporal traction response in human airway smooth muscle cells
Authors: Tolić-Nørrelykke Iva Marija , Butler James P. , Chen Jianxin , Wang Ning
Published: May 2013

Tractions that cells exert on their substrates are essential in cell spreading, migration, and contraction. These tractions can be determined by plating the cells on a flexible gel and measuring the deformation of the gel by using fluorescent beads embedded just below the surface of the gel. In this article we describe the image correlation method (ICM) optimized for determining the displacement field of the gel under a contracting cell. For the calculation of the traction field from the displacement field we use the recently developed method of Fourier transform traction cytometry (FTTC). The ICM and FTTC methods are applied to human airway smooth muscle cells during stimulation with the contractile agonist histamine or the relaxing agonist isoproterenol. The overall intensity of the cell contraction (the median traction magnitude, the energy transferred from the cell to the gel, and the net contractile moment) increased after activation with histamine, and decreased after treatment with isoproterenol. Cells exhibited regional differences in the time course of traction during the treatment. Both temporal evolution and magnitude of traction increase induced by histamine varied markedly among different cell protrusions, whereas the nuclear region showed the smallest response. These results suggest that intracellular mediators of cell adhesion and contraction respond to contractile stimuli with different rates and intensities in different regions of the cell.