bluuebunny/crossref_metadata_2025 huggingface dataset:
Rho kinase inhibitor HA-1077 prevents Rho-mediated myosin phosphatase inhibition in smooth muscle cells
Authors: Nagumo Hiromitsu , Sasaki Yasuharu , Ono Yoshitaka , Okamoto Hiroyuki , Seto Minoru , Takuwa Yoh
Published: December 2017

In smooth muscle, a Rho-regulated system of myosin phosphatase exists; however, it has yet to be established whether Rho kinase, one of the downstream effectors of Rho, mediates the regulation of myosin phosphatase activity in vivo. In the present study, we demonstrate in permeabilized vascular smooth muscle cells (SMCs) that the vasodilator 1-(5-isoquinolinesulfonyl)-homopiperazine (HA-1077), which we show to be a potent inhibitor of Rho kinase, dose dependently inhibits Rho-mediated enhancement of Ca2+-induced 20-kDa myosin light chain (MLC20) phosphorylation due to abrogating Rho-mediated inhibition of MLC20 dephosphorylation. By an immune complex phosphatase assay, we found that guanosine 5′- O-(3-thiotriphosphate) (GTPγS) stimulation of permeabilized SMCs caused a decrease in myosin phosphatase activity with an increase in the extent of phosphorylation of the 130-kDa myosin-binding regulatory subunit (MBS) of myosin phosphatase in a Rho-dependent manner. HA-1077 abolished both of the Rho-mediated events. Moreover, we observed that the pleckstrin homology/cystein-rich domain protein of Rho kinase, a dominant negative inhibitor of Rho kinase, inhibited GTPγS-induced phosphorylation of MBS. These results provide direct in vivo evidence that Rho kinase mediates inhibition of myosin phosphatase activity with resultant enhancement of MLC20phosphorylation in smooth muscle and reveal the usefulness of HA-1077 as a Rho kinase inhibitor.
CCK stimulates mob-1 expression and NF-κB activation via protein kinase C and intracellular Ca2+
Authors: Han Bing , Logsdon Craig D.
Published: December 2017

Supraphysiological concentrations of cholecystokinin (CCK) induce chemokine expression in rat pancreatic acini through the activation of the transcription factor NF-κB. In the current study, the intracellular signals involved in these pathophysiological effects of CCK were investigated. CCK induction of mob-1 expression in isolated rat pancreatic acini was blocked by the protein kinase C (PKC) inhibitors GF-109203X and Ro-32–0432 and by the intracellular Ca2+ chelator BAPTA. CCK induced NF-κB nuclear translocation, and DNA binding was also blocked by GF-109203X and BAPTA. Direct activation of PKC with TPA induced mob-1 chemokine expression and activated NF-κB DNA binding to a similar extent as did CCK. Increasing intracellular Ca2+using ionomycin had no effect on mob-1 mRNA levels or NF-κB activity. Both CCK and TPA treatments decreased inhibitory κB-α (IκB-α) levels, whereas ionomycin had no effect. However, the effects of TPA on IκB-α degradation were less complete than for CCK. In combination, TPA and ionomycin degraded IκB-α to a similar extent as CCK. Therefore, activation of NF-κB and mob-1 expression by supraphysiological CCK is likely mediated by both PKC activation and elevated intracellular Ca2+.
TNF-α potentiates the ion secretion induced by muscarinic receptor activation in HT29cl.19A cells
Authors: Oprins Judith C. J. , Meijer Helen P. , Groot Jack A.
Published: December 2017

Chronic gastrointestinal diseases such as ulcerative colitis and Crohn's disease are characterized by severe diarrhea. Mucosal biopsies of these patients show enhanced levels of cytokines, secreted by infiltrated inflammatory cells. In this study, we investigated the effect of the cytokine tumor necrosis factor-α (TNF-α) on ion secretion in human intestinal epithelial cells. The conventional microelectrode technique in the cell line HT29cl.19A was used, which allows for simultaneous measurements of transepithelial potential difference and intracellular potential difference across the apical membrane. Preincubation (2–78 h) with 10 ng/ml TNF-α did not change basal secretory activity. However, the secretory response to the muscarinic receptor agonist carbachol was strongly increased after exposure to TNF-α. Application of the protein kinase C (PKC) inhibitor GF 109203X (bisindolylmaleimide I) inhibited the response to carbachol as well as the TNF-α-potentiated response, indicating that PKC mediates the effect of carbachol in this cell line. Propranolol, a substance that inhibits the phospholipase D (PLD) pathway, strongly reduced the response to muscarinic stimulation and its potentiation by TNF-α. The results indicate that activation of PLD is involved in ion secretion induced by muscarinic receptor activation and that TNF-α can potentiate this pathway.
Stretch-induced activation of Ca2+-activated K+ channels in mouse skeletal muscle fibers
Authors: Mallouk Nora , Allard Bruno
Published: December 2017

High-conductance Ca2+-activated K+(KCa) channels were studied in mouse skeletal muscle fibers using the patch-clamp technique. In inside-out patches, application of negative pressure to the patch induced a dose-dependent and reversible activation of KCa channels. Stretch-induced increase in channel activity was found to be of the same magnitude in the presence and in the absence of Ca2+ in the pipette. The dose-response relationships between KCa channel activity and intracellular Ca2+ and between KCa channel activity and membrane potential revealed that voltage and Ca2+ sensitivity were not altered by membrane stretch. In cell-attached patches, in the presence of high external Ca2+ concentration, stretch-induced activation was also observed. We conclude that membrane stretch is a potential mode of regulation of skeletal muscle KCa channel activity and could be involved in the regulation of muscle excitability during contraction-relaxation cycles.
Phosphatidylcholine participates in the interaction between macrophages and lymphocytes
Authors: Nishiyama-Naruke Anita , Curi Rui
Published: December 2017

The role of phosphatidylcholine molecules as mediator for the control of lymphocyte proliferation by macrophages was investigated. Phosphatidylcholine added to the culture medium inhibited the concanavalin A-stimulated lymphocyte proliferation in a concentration-dependent manner. The potency of this effect was dependent on the presence of arachidonic acid in the phosphatidylcholine molecules. The phosphatidylcholine transfer from macrophages to lymphocytes was then investigated. Macrophages incorporated phosphatidylcholine at a much higher rate than lymphocytes and exported phosphatidylcholine to the culture medium. When cocultured, a significant amount of phosphatidylcholine incorporated by macrophages was transferred to lymphocytes. To examine the possible physiological importance of the transfer process, the lymphocyte proliferation was measured in coculture conditions. Macrophages were treated with phosphatidylcholine and washed, and then these cells were cocultured with concanavalin A-stimulated lymphocytes. The effect observed in coculture was an inhibition of lymphocyte proliferation, which was also dependent on the molecular species of the phosphatidylcholine. Therefore, phosphatidylcholine may act as a mediator of the macrophage effect on lymphocyte proliferation.
Effects of β2-agonist clenbuterol on biochemical and contractile properties of unloaded soleus fibers of rat
Authors: Ricart-Firinga Carole , Stevens Laurence , Canu Marie-Helene , Nemirovskaya Tatiana L. , Mounier Yvonne
Published: December 2017

The effects of clenbuterol β2-agonist administration were investigated in normal and atrophied [15-day hindlimb-unloaded (HU)] rat soleus muscles. We showed that clenbuterol had a specific effect on muscle tissue, since it reduces soleus atrophy induced by HU. The study of Ca2+ activation properties of single skinned fibers revealed that clenbuterol partly prevented the decrease in maximal tension after HU, with a preferential effect on fast-twitch fibers. Clenbuterol improved the Ca2+ sensitivity in slow- and fast-twitch fibers by shifting the tension-pCa relationship toward lower Ca2+ concentrations, but this effect was more marked after HU than in normal conditions. Whole muscle electrophoresis indicated slow-to-fast transitions of the myosin heavy chain isoforms for unloaded and for clenbuterol-treated soleus. The coupling of the two latter conditions did not, however, increase these phenotypical transformations. Our findings indicated that clenbuterol had an anabolic action and a β2-adrenergic effect on muscle fibers and appeared to counteract some effects of unloading disuse conditions.
HSV-1 amplicon vectors are a highly efficient gene delivery system for skeletal muscle myoblasts and myotubes
Authors: Wang Y. , Fraefel C. , Protasi F. , Moore R. A. , Fessenden J. D. , Pessah I. N. , DiFrancesco A. , Breakefield X. , Allen P. D.
Published: December 2017

Analysis of RyR1 structure function in muscle cells is made difficult by the low (<5%) transfection efficiencies of myoblasts or myotubes using calcium phosphate or cationic lipid techniques. We inserted the full-length 15.3-kb RyR1 cDNA into a herpes simplex virus type 1 (HSV-1) amplicon vector, pHSVPrPUC between the ori/IE 4/5 promoter sequence and the HSV-1 DNA cleavage/packaging signal ( pac). pHSVGN and pHSVGRyR1, two amplicons that expressed green fluorescent protein, were used for fluorescence-activated cell sorter analysis of transduction efficiency. All amplicons were packaged into HSV-1 virus particles using a helper virus-free packaging system and yielded 106 transducing vector units/ml. HSVRyR1, HSVGRyR1, and HSVGN virions efficiently transduced mouse myoblasts and myotubes, expressing the desired product in 70–90% of the cells at multiplicity of infection 5. The transduced cells appeared healthy and RyR1 produced by this method was targeted properly and restored skeletal excitation-contraction coupling in dyspedic myotubes. The myotubes produced sufficient protein to allow single-channel analyses from as few as 10 100-mm dishes. In most cases this method could preclude the need for permanent transfectants for the study of RyR1 structure function.
Age- and tissue-specific induction of NHE3 by glucocorticoids in the rat small intestine
Authors: Kiela Pawel R. , Guner Yigit S. , Xu Hua , Collins James F. , Ghishan Fayez K.
Published: December 2017

Of the two known apical isoforms of the Na+/H+ exchanger (NHE) family, only the NHE3 gene is regulated by glucocorticoids. The aim of these studies was to investigate the mechanisms underlying the effects of methylprednisolone (MP) on expression of NHE3 in the proximal and distal small intestine of suckling and adult rats. Immunoblots showed that the glucocorticoid responsiveness in the proximal small intestine was greatest in suckling animals (NHE3/β-actin: 0.43 ± 0.09 control vs. 1.57 ± 0.15 MP; P < 0.001), and responsiveness decreased with age with no effect in adults (0.56 ± 0.14 vs. 0.64 ± 0.17). Distal small intestine was responsive only in adult rats (0.49 ± 0.13 vs. 1.65 ± 0.09; P < 0.001). This pattern was confirmed at the mRNA level and by 22Na+ uptake. Western blot and [3H]dexamethasone mesylate binding showed that the responsiveness of NHE3 to glucocorticoids is directly related to the expression of glucocorticoid receptor (GR) in the small intestine. These studies suggest that loss and gain of glucocorticoid responsiveness in the proximal and distal small intestine, respectively, are related to age- and segment-dependent expression of GR.
Potentiation of rat brain sodium channel currents by PKA inXenopus oocytes involves the I-II linker
Authors: Smith Raymond D. , Goldin Alan L.
Published: December 2017

Functional modulation of voltage-gated sodium channels affects the electrical excitability of neurons. Protein kinase A (PKA) can decrease sodium currents by phosphorylation at consensus sites in the cytoplasmic I-II linker. Once the sites are phosphorylated, however, additional PKA activity can increase sodium currents by an unknown mechanism. When the PKA sites were eliminated by substitutions of alanine for serine, peak sodium current amplitudes were increased by 20–80% when PKA was activated in Xenopus oocytes either by stimulation of a coexpressed β2-adrenergic receptor or by perfusion with reagents that increase cAMP. Potentiation required the I-II linker of the brain channel, in that a chimeric channel in which the brain linker was replaced with the comparable linker from the skeletal muscle channel did not demonstrate potentiation. Using a series of chimeric and deleted channels, we demonstrate that potentiation is not dependent on any single region of the linker and that the extent of potentiation varies depending on the total length and the residues throughout the linker. These data are consistent with the hypothesis that potentiation by PKA is an indirect process involving phosphorylation of an accessory protein that interacts with the I-II linker of the sodium channel.
Alternatively spliced isoforms of the rat eye sodium/calcium+potassium exchanger NCKX1
Authors: Poon Susan , Leach Stephen , Li Xiao-Fang , Tucker Joseph E. , Schnetkamp Paul P. M. , Lytton Jonathan
Published: December 2017

We have investigated the structure, function, and expression of the rat eye sodium/calcium+potassium exchanger NCKX1. The sequence of independent rat NCKX1 clones and the analysis of rat eye mRNA by RT-PCR revealed a region of alternative splicing that comprised four exons and encoded a stretch of 113 amino acids near the beginning of the large cytosolic loop. In comparison with other NCKX1 molecules and the rat NCKX2 protein, rat NCKX1 was highly conserved within the hydrophobic regions but was quite divergent in the two large hydrophilic loops. The only exception was the region of the cytosolic loop encoded by the second alternatively spliced exon, which was ∼60% identical. Similar to bovine, but different from human, rat NCKX1 possessed an acidic motif that was repeated 14 times in the cytoplasmic loop. Analysis of NCKX1 expression in different rat tissues by Northern blot revealed a very high level of expression of a 7-kb transcript in the eye but also lower levels of transcripts of various lengths in other tissues. The recombinant rat NCKX1 protein was tagged in the extracellular loop with the FLAG epitope and expressed in HEK-293 cells. Surface delivery and potassium-dependent sodium/calcium exchange activity were observed for each spliced variant.
Involvement of estrogen receptors α and β in the regulation of cervical permeability
Authors: Gorodeski George I. , Pal Dipika
Published: December 2017

Estrogen increases the permeability of cultured human cervical epithelia (Gorodeski, GI. Am J Physiol Cell Physiol 275: C888–C899, 1998), and the effect is blocked by the estrogen receptor modulators ICI-182780 and tamoxifen. The objective of the study was to determine involvement of estrogen receptor(s) in mediating the effects on permeability. In cultured human cervical epithelial cells estradiol binds to high-affinity, low-capacity sites, in a specific and saturable manner. Scatchard analysis revealed a single class of binding sites with a dissociation constant of 1.3 nM and binding activity of ∼0.5 pmol/mg DNA. Estradiol increased the density of estrogen-binding sites in a time- and dose-related manner (half time ≈ 4 h, and EC50≈ 1 nM). RT-PCR assays revealed the expression of mRNA for the estrogen receptor α (αER) and estrogen receptor β (βER). Removal of estrogen from the culture medium decreased and treatment with estrogen increased the expression of αER and βER mRNA. In cells not treated with estrogen, ICI-182780 and tamoxifen increased βER mRNA. In cells treated with estrogen, neither ICI-182780 nor tamoxifen had modulated significantly the increase in αER or βER mRNA. The transcription inhibitor actinomycin D blocked the estrogen-induced increase in permeability, and it abrogated the estradiol-induced increase in estrogen binding sites. These results suggest that the estrogen-dependent increase in cervical permeability is mediated by an αER-dependent increase in transcription.
Regulation of mitochondrial respiration in heart cells analyzed by reaction-diffusion model of energy transfer
Authors: Vendelin Marko , Kongas Olav , Saks Valdur
Published: December 2017

The purpose of this study is to investigate theoretically which intracellular factors may be important for regulation of mitochondrial respiration in working heart cells in vivo. We have developed a model that describes quantitatively the published experimental data on dependence of the rate of oxygen consumption and metabolic state of working isolated perfused rat heart on workload over its physiological range (Williamson JR, Ford G, Illingworth J, Safer B. Circ Res 38, Suppl I, I39–I51, 1976). Analysis of this model shows that for phosphocreatine, creatine, and ATP the equilibrium assumption is an acceptable approximation with respect to their diffusion in the intracellular bulk water phase. However, the ADP concentration changes in the contraction cycle in a nonequilibrium workload-dependent manner, showing the existence of the intracellular concentration gradients. The model shows that workload-dependent alteration of ADP concentration in the compartmentalized creatine kinase system may be taken, together with the changes in Piconcentration, to be among the major components of the metabolic feedback signal for regulation of respiration in muscle cells.
Pharmacology and modulation of KATP channels by protein kinase C and phosphatases in gallbladder smooth muscle
Authors: Firth T. A. , Mawe G. M. , Nelson M. T.
Published: December 2017

ATP-sensitive K+ (KATP) channels exhibit pharmacological diversity, which is critical for the development of novel therapeutic agents. We have characterized KATP channels in gallbladder smooth muscle to determine how their pharmacological properties compare to KATP channels in other types of smooth muscle. KATP currents were measured in myocytes isolated from gallbladder and mesenteric artery. The potencies of pinacidil, diazoxide, and glibenclamide were similar in gallbladder and vascular smooth muscle, suggesting that the regions of the channel conferring sensitivity to these agents are conserved among smooth muscle types. Activators of protein kinase C (PKC), however, were less effective at inhibiting KATP currents in myocytes from gallbladder than mesenteric artery. The phosphatase inhibitor okadaic acid increased the efficacy of PKC activators and revealed ongoing basal activation of KATP channels by protein kinase A in gallbladder. These results suggest that phosphatases and basal kinase activity play an important role in controlling KATP channel activity.
Localization of two distinct type III phosphatidylinositol 4-kinase enzyme mRNAs in the rat
Authors: Zólyomi Annamária , Zhao Xiaohang , Downing Gregory J. , Balla Tamas
Published: December 2017

Inositol lipid kinases generate polyphosphoinositides, important regulators of several cellular functions. We have recently cloned two distinct phosphatidylinositol (PI) 4-kinase enzymes, the 210-kDa PI4KIIIα and the 110-kDa PI4KIIIβ, from bovine tissues. In the present study, the distribution of mRNAs encoding these two enzymes was analyzed by in situ hybridization histochemistry in the rat. PI4KIIIα was found predominantly expressed in the brain, with low expression in peripheral tissues. PI4KIIIβ was more uniformly expressed being also present in various peripheral tissues. Within the brain, PI4KIIIβ showed highest expression in the gray matter, especially in neurons of the olfactory bulb and the hippocampus, but also gave a signal in the white matter indicating its presence in glia. PI4KIIIα was highly expressed in neurons, but lacked a signal in the white matter and the choroid plexus. Both enzymes showed expression in the pigment layer and nuclear layers as well as in the ganglion cells of the retina. In a 17-day-old rat fetus, PI4KIIIβ was found to be more widely distributed and PI4KIIIα was primarily expressed in neurons. These results indicate that PI4KIIIβ is more widely expressed than PI4KIIIα, and that the two enzymes are probably coexpressed in many neurons. Such expression pattern and the conservation of these two proteins during evolution suggest their nonredundant functions in mammalian cells.
pH heterogeneity at intracellular and extracellular plasma membrane sites in HT29-C1 cell monolayers
Authors: Maouyo Djikolngar , Chu Shaoyou , Montrose Marshall H.
Published: December 2017

In the colonic mucosa, short-chain fatty acids change intracellular pH (pHi) and extracellular pH (pHe). In this report, confocal microscopy and dual-emission ratio imaging of carboxyseminaphthorhodofluor-1 were used for direct evaluation of pHi and pHe in a simple model epithelium, HT29-C1 cells. Live cell imaging along the apical-to-basal axis of filter-grown cells allowed simultaneous measurement of pH in the aqueous environment near the apical membrane, the lateral membrane, and the basal membrane. Subapical cytoplasm reported the largest changes in pHi after isosmotic addition of 130 mM propionate or 30 mM NH4Cl. In resting cells and cells with an imposed acid load, lateral membranes had pHi values intermediate between the relatively acidic subapical region (pH 6.3–6.9) and the relatively alkaline basal pole of the cells (pH 7.4–7.1). Transcellular pHigradients were diminished or eliminated during an induced alkaline load. Propionate differentially altered pHe near the apical membrane, in lateral intracellular spaces between adjacent cells, and near the basal membrane. Luminal or serosal propionate caused alkalinization of the cis compartment (where propionate was added) but acidification of the trans compartment only in response to luminal propionate. Addition of NH4Cl produced qualitatively opposite pHe excursions. The microscopic values of pHi and pHe can explain a portion of the selective activation of polarized Na/H exchangers observed in HT29-C1 cells in the presence of transepithelial propionate gradients.
Apoptosis in human cultured trophoblasts is enhanced by hypoxia and diminished by epidermal growth factor
Authors: Levy Roni , Smith Steven D. , Chandler Kala , Sadovsky Yoel , Nelson D. Michael
Published: December 2017

Preeclampsia and fetal growth restriction are associated with placental hypoperfusion and villous hypoxia. The villous response to this environment includes diminished trophoblast differentiation and enhanced apoptosis. We tested the hypothesis that hypoxia induces apoptosis in cultured trophoblasts, and that epidermal growth factor (EGF), an enhancer of trophoblast differentiation, diminishes hypoxia-induced apoptosis. Trophoblasts isolated from placentas of term-uncomplicated human pregnancies were cultured up to 72 h in standard ([Formula: see text]= 120 mmHg) or hypoxic ([Formula: see text] < 15 mmHg) conditions. Exposure to hypoxia for 24 h markedly enhanced trophoblast apoptosis as determined by DNA laddering, internucleosomal in situ DNA fragmentation, and histomorphology, as well as by the reversibility of the apoptotic process with a caspase inhibitor. Apoptosis was accompanied by increased expression of p53 and Bax and decreased expression of Bcl-2. Addition of EGF to cultured trophoblasts or exposure of more differentiated trophoblasts to hypoxia significantly lowered the level of apoptosis. We conclude that hypoxia enhances apoptosis in cultured trophoblasts by a mechanism that involves an increase in p53 and Bax expression. EGF and enhancement of cell differentiation protect against hypoxic-induced apoptosis.
Ca2+ regulates fluid shear-induced cytoskeletal reorganization and gene expression in osteoblasts
Authors: Chen Neal X. , Ryder Kimberly D. , Pavalko Fredrick M. , Turner Charles H. , Burr David B. , Qiu Jinya , Duncan Randall L.
Published: December 2017

Osteoblasts subjected to fluid shear increase the expression of the early response gene, c- fos, and the inducible isoform of cyclooxygenase, COX-2, two proteins linked to the anabolic response of bone to mechanical stimulation, in vivo. These increases in gene expression are dependent on shear-induced actin stress fiber formation. Here, we demonstrate that MC3T3-E1 osteoblast-like cells respond to shear with a rapid increase in intracellular Ca2+ concentration ([Ca2+]i) that we postulate is important to subsequent cellular responses to shear. To test this hypothesis, MC3T3-E1 cells were grown on glass slides coated with fibronectin and subjected to laminar fluid flow (12 dyn/cm2). Before application of shear, cells were treated with two Ca2+ channel inhibitors or various blockers of intracellular Ca2+ release for 0.5–1 h. Although gadolinium, a mechanosensitive channel blocker, significantly reduced the [Ca2+]i response, neither gadolinium nor nifedipine, an L-type channel Ca2+ channel blocker, were able to block shear-induced stress fiber formation and increase in c-fos and COX-2 in MC3T3-E1 cells. However, 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid-AM, an intracellular Ca2+ chelator, or thapsigargin, which empties intracellular Ca2+ stores, completely inhibited stress fiber formation and c-fos/COX-2 production in sheared osteoblasts. Neomycin or U-73122 inhibition of phospholipase C, which mediates d- myo-inositol 1,4,5-trisphosphate (IP3)-induced intracellular Ca2+ release, also completely suppressed actin reorganization and c-fos/COX-2 production. Pretreatment of MC3T3-E1 cells with U-73343, the inactive isoform of U-73122, did not inhibit these shear-induced responses. These results suggest that IP3-mediated intracellular Ca2+release is required for modulating flow-induced responses in MC3T3-E1 cells.
IP3 receptors, IP3 transients, and nucleus-associated Ca2+ signals in cultured skeletal muscle
Authors: Jaimovich Enrique , Reyes Roberto , Liberona Jose L. , Powell Jeanne A.
Published: December 2017

Inositol 1,4,5-trisphosphate (IP3) receptors (IP3R) and ryanodine receptors (RyR) were localized in cultured rodent muscle fractions by binding of radiolabeled ligands (IP3 and ryanodine), and IP3R were visualized in situ by fluorescence immunocytological techniques. Also explored was the effect of K+ depolarization on IP3 mass and Ca2+ transients studied using a radio-receptor displacement assay and fluorescence imaging of intracellular fluo 3. RyR were located in a microsomal fraction; IP3R were preferentially found in the nuclear fraction. Fluorescence associated with anti-IP3R antibody was found in the region of the nuclear envelope and in a striated pattern in the sarcoplasmic areas. An increase in external K+ affected membrane potential and produced an IP3 transient. Rat myotubes displayed a fast-propagating Ca2+ signal, corresponding to the excitation-contraction coupling transient and a much slower Ca2+ wave. Both signals were triggered by high external K+ and were independent of external Ca2+. Slow waves were associated with cell nuclei and were propagated leaving “glowing” nuclei behind. Different roles are proposed for at least two types of Ca2+ release channels, each mediating an intracellular signal in cultured skeletal muscle.
Skeletal muscle function and water permeability in aquaporin-4 deficient mice
Authors: Yang Baoxue , Verbavatz Jean-Marc , Song Yuanlin , Vetrivel L. , Manley Geoffrey , Kao Wei-Ming , Ma Tonghui , Verkman A. S.
Published: December 2017

It has been proposed that aquaporin-4 (AQP4), a water channel expressed at the plasmalemma of skeletal muscle cells, is important in normal muscle physiology and in the pathophysiology of Duchenne's muscular dystrophy. To test this hypothesis, muscle water permeability and function were compared in wild-type and AQP4 knockout mice. Immunofluorescence and freeze-fracture electron microscopy showed AQP4 protein expression in plasmalemma of fast-twitch skeletal muscle fibers of wild-type mice. Osmotic water permeability was measured in microdissected muscle fibers from the extensor digitorum longus (EDL) and fractionated membrane vesicles from EDL homogenates. With the use of spatial-filtering microscopy to measure osmotically induced volume changes in EDL fibers, half times ( t 1/2) for osmotic equilibration (7.5–8.5 s) were not affected by AQP4 deletion. Stopped-flow light-scattering measurements of osmotically induced volume changes in plasmalemma vesicles also showed no significant differences in water permeability. Similar water permeability, yet ∼90% decreased AQP4 protein expression was found in EDL from mdx mice that lack dystrophin. Skeletal muscle function was measured by force generation in isolated EDL, treadmill performance time, and in vivo muscle swelling in response to water intoxication. No differences were found in EDL force generation after electrical stimulation [42 ± 2 (wild-type) vs. 41 ± 2 (knockout) g/s], treadmill performance time (22 vs. 26 min; 29 m/min, 13° incline), or muscle swelling (2.8 vs. 2.9% increased water content at 90 min after intraperitoneal water infusion). Together these results provide evidence against a significant role of AQP4 in skeletal muscle physiology in mice.
In vivo regulation of the β-myosin heavy chain gene in soleus muscle of suspended and weight-bearing rats
Authors: Giger Julia M. , Haddad Fadia , Qin Anqi X. , Baldwin Kenneth M.
Published: December 2017

In the weight-bearing hindlimb soleus muscle of the rat, ∼90% of muscle fibers express the β-myosin heavy chain (β-MHC) isoform protein. Hindlimb suspension (HS) causes the MHC isoform population to shift from β toward the fast MHC isoforms. Our aim was to establish a model to test the hypothesis that this shift in expression is transcriptionally regulated through specific cis elements of the β-MHC promoter. With the use of a direct gene transfer approach, we determined the activity of different length β-MHC promoter fragments, linked to a firefly luciferase reporter gene, in soleus muscle of control and HS rats. In weight-bearing rats, the relative luciferase activity of the longest β-promoter fragment (−3500 bp) was threefold higher than the shorter promoter constructs, which suggests that an enhancer sequence is present in the upstream promoter region. After 1 wk of HS, the reporter activities of the −3500-, −914-, and −408-bp promoter constructs were significantly reduced (∼40%), compared with the control muscles. However, using the −215-bp construct, no differences in promoter activity were observed between HS and control muscles, which indicates that the response to HS in the rodent appears to be regulated within the −408 and −215 bp of the promoter.
Extracellular Cl− modulates shrinkage-induced activation of Na+/H+exchanger in rat mesangial cells
Authors: Miyata Yukio , Muto Shigeaki , Yanagiba Satoru , Asano Yasushi
Published: December 2017

To examine the effect of hyperosmolality on Na+/H+ exchanger (NHE) activity in mesangial cells (MCs), we used a pH-sensitive dye, 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-AM, to measure intracellular pH (pHi) in a single MC from rat glomeruli. All the experiments were performed in CO2/[Formula: see text]-free HEPES solutions. Exposure of MCs to hyperosmotic HEPES solutions (500 mosmol/kgH2O) treated with mannitol caused cell alkalinization. The hyperosmolality-induced cell alkalinization was inhibited by 100 μM ethylisopropylamiloride, a specific NHE inhibitor, and was dependent on extracellular Na+. The hyperosmolality shifted the Na+-dependent acid extrusion rate vs. pHi by 0.15–0.3 pH units in the alkaline direction. Removal of extracellular Cl− by replacement with gluconate completely abolished the rate of cell alkalinization induced by hyperosmolality and inhibited the Na+-dependent acid extrusion rate, whereas, under isosmotic conditions, it caused no effect on Na+-dependent pHi recovery rate or Na+-dependent acid extrusion rate. The Cl−-dependent cell alkalinization rate under hyperosmotic conditions was partially inhibited by pretreatment with 5-nitro-2-(3-phenylpropylamino)benzoic acid, DIDS, and colchicine. We conclude: 1) in MCs, hyperosmolality activates NHE to cause cell alkalinization, 2) the acid extrusion rate via NHE is greater under hyperosmotic conditions than under isosmotic conditions at a wide range of pHi, 3) the NHE activation under hyperosmotic conditions, but not under isosmotic conditions, requires extracellular Cl−, and 4) the Cl−-dependent NHE activation under hyperosmotic conditions partly occurs via Cl− channel and microtubule-dependent processes.
Polarity of A2b adenosine receptor expression determines characteristics of receptor desensitization
Authors: Sitaraman Shanthi V. , Si-Tahar Mustapha , Merlin Didier , Strohmeier Gregg R. , Madara J. L.
Published: December 2017

It is not known if, in polarized cells, desensitization events can be influenced by the domain on which the receptor resides. Desensitization was induced by 5′-( N-ethylcarboxamido)adenosine (NECA) and was quantitated by measurement of short-circuit current ( I sc) in response to adenosine. NECA added to either the apical or basolateral compartments rapidly desensitized receptors on these respective domains. Although apical NECA had no effect on the basolateral receptor stimulation, basolateral NECA induced a complete desensitization of the apical receptor. We hypothesized that desensitization of apical receptor by basolateral desensitization could relate to a trafficking step in which A2b receptor is first targeted basolaterally upon synthesis and transported to the apical surface via vesicular transport/microtubules. Because desensitization is associated with downregulation of receptors, apical adenosine receptor can thus be affected by basolateral desensitization. Both low temperature and nocodazole inhibited I scinduced by apical and not basolateral adenosine. In conclusion: 1) a single receptor subtype, here modeled by the A2b receptor, differentially desensitizes based on the membrane domain on which it is expressed, 2) agonist exposure on one domain can result in desensitization of receptors on the opposite domain, 3) cross-domain desensitization can display strict polarity, and 4) receptor trafficking may play a role in the cross-desensitization process.
Effects of altered tonicity by sodium chloride onl-tryptophan binding to hepatic nuclei
Authors: Sidransky Herschel , Verney Ethel , Orenstein Jan
Published: December 2017

This study was concerned with the effects of NaCl administered in vivo or added in vitro to isolated nuclei on [3H]tryptophan binding to rat hepatic nuclei assayed in vitro. Hypertonic (10.7%) NaCl administered in vivo to rats caused at 10 min a marked decrease in in vitro binding (total and specific) of [3H]tryptophan to hepatic nuclei. In vitro incubation of isolated hepatic nuclei, but not of isolated nuclear envelopes, with added NaCl (particularly at 0.125 × 10−4 M and 0.25 × 10−4 M) revealed significant inhibition of [3H]tryptophan binding. However, isolated hepatic nuclear envelopes prepared after in vitro incubation of isolated nuclei with added NaCl did show inhibition of [3H]tryptophan binding (total and specific) compared with controls. Other salts (KCl, MgCl2, NaHCO3, NaC2H3O2, NaF, or Na2SO4), at similar concentrations to that of NaCl except for MgCl2, when added to isolated nuclei did not appreciably inhibit nuclear tryptophan binding. Kinetic studies of in vitro nuclear [3H]tryptophan binding in the presence of 0.125 × 10−4 M NaCl revealed that binding decreased at 0.5 h and continued to 2 h compared with nuclear [3H]tryptophan binding with controls (without NaCl addition). The results obtained in vivo in rats and those obtained in vitro with isolated hepatic nuclei revealed NaCl-induced inhibitory effects on [3H]tryptophan binding to hepatic nuclei. Although the inhibitory effects were similar under the two different experimental conditions, the mechanism for each may be different in that the NaCl concentration in hepatic cells after administration of NaCl in vivo was appreciably higher than the low levels added in vitro to the isolated hepatic nuclei.
Skeletal muscle gene transfer: regeneration-associated deregulation of fast troponin I fiber type specificity
Authors: Hallauer Patricia L. , Karpati George , Hastings Kenneth E. M.
Published: December 2017

Direct gene transfer into skeletal muscle in vivo presents a convenient experimental approach for studies of adult muscle gene regulatory mechanisms, including fast vs. slow fiber type specificity. Previous studies have reported preferential expression of fast myosin heavy chain and slow myosin light chain and troponin I (TnIslow) gene constructs in muscles enriched in the appropriate fiber type. We now report a troponin I fast (TnIfast) direct gene transfer study. We injected into the mouse soleus muscle plasmid DNA or recombinant adenovirus carrying a TnIfast/ β-galactosidase (β-gal) reporter construct that had previously been shown to be expressed specifically in fast fibers in transgenic mice. Surprisingly, microscopic histochemical analysis 1 and 4 wk postinjection showed similar TnIfast/β-gal expression in fast and slow fibers. A low but significant level of muscle fiber segmental regeneration was evident in muscles 1 wk postinjection, and TnIfast/β-gal expression was preferentially targeted to regenerating fiber segments. This finding can explain why TnIfast constructs are deregulated with regard to fiber type specificity, whereas the myosin constructs previously studied are not. The involvement of regenerating fiber segments in transduction by plasmid DNA and recombinant adenoviruses injected into intact normal adult muscle is an unanticipated factor that should be taken into account in the planning and interpretation of direct gene transfer experiments.
Analysis of organ physiology in transgenic mice
Authors: Rao Shobha , Verkman A. S.
Published: December 2017

The increasing availability of transgenic mouse models of gene deletion and human disease has mandated the development of creative approaches to characterize mouse phenotype. The mouse presents unique challenges to phenotype analysis because of its small size, habits, and inability to verbalize clinical symptoms. This review describes strategies to study mouse organ physiology, focusing on the cardiovascular, pulmonary, renal, gastrointestinal, and neurobehavioral systems. General concerns about evaluating mouse phenotype studies are discussed. Monitoring and anesthesia methods are reviewed, with emphasis on the feasibility and limitations of noninvasive and invasive procedures to monitor physiological parameters, do cannulations, and perform surgical procedures. Examples of phenotype studies are cited to demonstrate the practical applications and limitations of the measurement methods. The repertoire of phenotype analysis methods reviewed here should be useful to investigators involved in or contemplating the use of mouse models.
Induction of apoptosis using sphingolipids activates a chloride current in Xenopus laevis oocytes
Authors: Souktani R. , Berdeaux A. , Ghaleh B. , Giudicelli J. F. , Guize L. , Le Heuzey J. Y. , Henry P.
Published: December 2017

The purpose of this study was to investigate whether the cell shrinkage that occurs during apoptosis could be explained by a change of the activity in ion transport pathways. We tested whether sphingolipids, which are potent pro-apoptotic compounds, can activate ionic currents in Xenopus laevis oocytes. Apoptosis was characterized in our model by a decrease in cell volume, a loss of cell viability, and DNA cleavage. Oocytes were studied using voltage-clamp after injection with N, N-dimethyl-d-erythrosphingosine (DMS) or d-sphingosine (DS). DMS and DS activated a fast-activating, slowly inactivating, outwardly rectifying current, similar to I Cl-swell, a swelling-induced chloride current. Lowering the extracellular chloride dramatically reduced the current, and the channel was more selective for thiocyanate and iodide (thiocyanate > iodide) than for chloride. The current was blocked by 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) and lanthanum but not by niflumic acid. Oocytes injected with a pseudosubstrate inhibitor of protein kinase C (PKC), PKC-(19–31), exhibited the same current. DMS-activated current was abolished by preexposure with phorbol myristate acetate. Our results suggest that induction of apoptosis in X. laevis oocytes, using sphingolipids or PKC inhibitors, activates a current similar to swelling-induced chloride current previously described in oocytes.
Interaction of D-600 with the transmembrane domain of the sarcoplasmic reticulum Ca2+-ATPase
Authors: Ortega Alicia , Becker V. M. , Alvarez R. , Lepock J. R. , Gonzalez-Serratos H.
Published: December 2017

Experiments were performed to determine whether the organic Ca2+ channel blocker D-600 (gallopamil), which penetrates into muscle cells, affects sarcoplasmic reticulum (SR) Ca2+ uptake by directly inhibiting the light SR Ca2+-ATPase. We have previously shown that at 10 μM, D-600 inhibits LSR ATP-dependent Ca2+ uptake by 50% but has no effect on ATPase activity (21). These data suggest that the SR Ca2+-ATPase might be a potential target for D-600. The ATPase activity of the enzyme is associated with its hydrophilic cytoplasmic domain, whereas Ca2+ binding and translocation are associated with the transmembrane domain (18). In the present experiments, we determined which of the two domains of the ATPase is affected by D-600. Thermal inactivation experiments using the SR Ca2+-ATPase demonstrated that D-600 decreased the thermal stability of Ca2+ transport but had no effect on the stability of ATPase activity. In addition, D-600 at a concentration of 160 μM did not have any leaking effect of Ca2+ on the Ca2+-loaded SR. Thermal denaturation profiles of SR membranes revealed that D-600 interacts directly with the transmembrane domain of the Ca2+-ATPase. No evidence for interaction with the nucleotide domain was obtained. We conclude that the Ca2+ blocker D-600 inhibits the SR Ca2+ pump specifically by interacting with the transmembrane Ca2+-binding domain of the Ca2+-ATPase.
Tenotomy decreases reporter protein synthesis via the 3′-untranslated region of the β-myosin heavy chain mRNA
Authors: Ashley William W. , Russell Brenda
Published: December 2017

We tested the hypothesis that the β-myosin heavy chain (β-MHC) 3′-untranslated region (UTR) mediates decreased protein expression after tenotomy of the rat soleus. We also tested the hypothesis that decreased protein expression is the result of RNA-protein interactions within the 3′-UTR. β-MHC was chosen for study because of its critical role in the function of postural muscles such as soleus. Adult rat soleus muscles were directly injected with luciferase (LUC) reporter constructs containing either the β-MHC or SV40 3′-UTR. After 48 h of tenotomy, there was no significant effect on LUC expression in the SV40 3′-UTR group. In the β-MHC 3′-UTR group, LUC expression was 37.3 ± 4% ( n = 5, P = 0.03) of that in sham controls. Gel mobility shift assays showed that a protein factor specifically interacts with the β-MHC 3′-UTR and that tenotomy significantly increases the level of this interaction (25 ± 7%, n = 5, P = 0.02). Thus the β-MHC 3′-UTR is directly involved in decreased protein expression that is probably due to increased RNA-protein binding within the UTR.
Signaling mechanisms regulating bombesin-mediated AP-1 gene induction in the human gastric cancer SIIA
Authors: Kim Hong Jin , Evers B. Mark , Litvak David A. , Hellmich Mark R. , Townsend Courtney M.
Published: December 2017

The hormone bombesin (BBS) and its mammalian equivalent gastrin-releasing peptide (GRP) act through specific GRP receptors (GRP-R) to affect multiple cellular functions in the gastrointestinal tract; the intracellular signaling pathways leading to these effects are not clearly defined. Previously, we demonstrated that the human gastric cancer SIIA possesses GRP-R and that BBS stimulates activator protein-1 (AP-1) gene expression. The purpose of our present study was to determine the signaling pathways leading to AP-1 induction in SIIA cells. A rapid induction of c- jun and jun-B gene expression was noted after BBS treatment; this effect was blocked by specific GRP-R antagonists, indicating that BBS is acting through the GRP-R. The signaling pathways leading to increased AP-1 gene expression were delineated using phorbol 12-myristate 13-acetate (PMA), which stimulates protein kinase C (PKC)-dependent pathways, by forskolin (FSK), which stimulates protein kinase A (PKA)-dependent pathways, and by the use of various protein kinase inhibitors. Treatment with PMA stimulated AP-1 gene expression and DNA binding activity similar to the effects noted with BBS; FSK stimulated jun-B expression but produced only minimal increases of c- jun mRNA and AP-1 binding activity. Pretreatment of SIIA cells with either H-7 or H-8 (primarily PKC inhibitors) inhibited the induction of c- jun and jun-B mRNAs in response to BBS, whereas H-89 (PKA inhibitor) exhibited only minimal effects. Pretreatment with tyrphostin-25, a protein tyrosine kinase (PTK) inhibitor, attenuated the BBS-mediated induction of c- jun and jun-B, but the effect was not as pronounced as with H-7. Collectively, our results demonstrate that BBS acts through its receptor to produce a rapid induction of both c- jun and jun-B mRNA and AP-1 DNA binding activity in the SIIA human gastric cancer. Moreover, this induction of AP-1, in response to BBS, is mediated through both PKC- and PTK-dependent signal transduction pathways with only minimal involvement of PKA.
VCAM-1-induced inwardly rectifying K+current enhances Ca2+ entry in human THP-1 monocytes
Authors: Colden-Stanfield Margaret , Scanlon Mary
Published: December 2017

Hyperpolarization in human leukemia THP-1 monocytes adherent to vascular cell adhesion molecule (VCAM)-1 is due to an induction of inwardly rectifying K+ currents ( I ir) (Colden-Stanfield M and Gallin EK, Am J Physiol Cell Physiol 275: C267–C277, 1998). We determined whether the VCAM-1-induced hyperpolarization is sufficient to augment the increase in intracellular free calcium ([Ca2+]i) produced by Ca2+ store depletion with thapsigargin (TG) and readdition of external CaCl2 in fura 2-loaded THP-1 monocytes. Whereas there was a 2.1-fold increase in [Ca2+]i in monocytes bound to glass for 5 h in response to TG and CaCl2addition, adherence to VCAM-1 produced a 5-fold increase in [Ca2+]i. Depolarization of monocytes adherent to VCAM-1 by I ir blockade or exposure to high [K+] abolished the enhancement of the peak [Ca2+]i response. In monocytes bound to glass, hyperpolarization of the membrane potential with valinomycin, a K+ ionophore, to the level of hyperpolarization seen in cells adherent to VCAM-1 produced similar changes in peak [Ca2+]i. Adherence of monocytes to E-selectin produced a similar peak [Ca2+]i to cells bound to glass. Thus monocyte adherence to the physiological substrate VCAM-1 produces a hyperpolarization that is sufficient to enhance Ca2+ entry and may impact Ca2+-dependent monocyte function.
Soluble P-selectin moderates complement-dependent reperfusion injury of ischemic skeletal muscle
Authors: Kyriakides Constantinos , Woodcock Sean A. , Wang Yong , Favuzza Joanne , Austen William G. , Kobzik Lester , Moore Francis D. , Valeri Robert C. , Shepro David , Hechtman Herbert B.
Published: December 2017

P-selectin is an adhesion molecule expressed on activated endothelial and platelet surfaces. The function of the short consensus repeats (SCRs) of P-selectin, homologous with the SCRs of complement regulatory proteins is largely unknown. In a model of murine hindlimb ischemia where local reperfusion injury is partly mediated by IgM natural antibody and classical complement pathway activation, we hypothesized that human soluble P-selectin (sP-sel) would moderate the complement component of the inflammatory response. Infusion of sP-sel supernatant or purified (p) sP-sel prepared from activated human platelets, reduced ischemic muscle vascular permeability by 48% and 43%, respectively, following reperfusion. Hindlimb immunohistochemistry demonstrated negligible C3 staining colocalized with IgM in these groups compared with intense staining in the untreated injured mice. In vitro studies of mouse serum complement hemolytic activity showed that psP-sel inhibited the classical but not alternative complement pathway. Flow cytometry demonstrated that psP-sel inhibited C1q adherence to sensitized red blood cells. From these data we conclude that sP-sel moderates skeletal muscle reperfusion injury by inhibition of the classical complement pathway.