bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of P2X7 nucleotide receptor function in human monocytes by extracellular ions and receptor density
Authors: Gudipaty Lalitha , Humphreys Benjamin D. , Buell Gary , Dubyak George R.
Published: December 2017

P2X receptors function as ATP-gated cation channels. The P2X7receptor subtype is distinguished from other P2X family members by a very low affinity for extracellular ATP (millimolar EC50) and its ability to trigger induction of nonselective pores on repeated or prolonged stimulation. Previous studies have indicated that certain P2X7 receptor-positive cell types, such as human blood monocytes and murine thymocytes, lack this pore-forming response. In the present study we compared pore formation in response to P2X7 receptor activation in human blood monocytes with that in macrophages derived from these monocytes by in vitro tissue culture. ATP induced nonselective pores in macrophages but not in freshly isolated monocytes when both cell types were identically stimulated in standard NaCl-based salines. However, ion substitution studies revealed that replacement of extracellular Na+ and Cl−with K+ and nonhalide anions strongly facilitated ATP-dependent pore formation in monocytes. These ionic conditions also resulted in increased agonist affinity, such that 30–100 μM ATP was sufficient for activation of nonselective pores by P2X7receptors. Comparison of P2X7 receptor expression in blood monocytes with that in macrophages indicated no differences in steady-state receptor mRNA levels but significant increases (up to 10-fold) in the amount of immunoreactive P2X7 receptor protein at the cell surface of macrophages. Thus ability of ATP to activate nonselective pores in cells that natively express P2X7 receptors can be modulated by receptor subunit density at the cell surface and ambient levels of extracellular Na+and Cl−. These mechanisms may prevent adventitious P2X7 receptor activation in monocytes until these proinflammatory leukocytes migrate to extravascular sites of tissue damage.
n-6 and n-3 polyunsaturated fatty acids differentially modulate oncogenic Ras activation in colonocytes
Authors: Collett Esther D. , Davidson Laurie A. , Fan Yang-Yi , Lupton Joanne R. , Chapkin Robert S.
Published: December 2017

Ras proteins are critical regulators of cell function, including growth, differentiation, and apoptosis, with membrane localization of the protein being a prerequisite for malignant transformation. We have recently demonstrated that feeding fish oil, compared with corn oil, decreases colonic Ras membrane localization and reduces tumor formation in rats injected with a colon carcinogen. Because the biological activity of Ras is regulated by posttranslational lipid attachment and its interaction with stimulatory lipids, we investigated whether docosahexaenoic acid (DHA), found in fish oil, compared with linoleic acid (LA), found in corn oil, alters Ras posttranslational processing, activation, and effector protein function in young adult mouse colon cells overexpressing H- ras (YAMC- ras). We show here that the major n-3 polyunsaturated fatty acid (PUFA) constituent of fish oil, DHA, compared with LA (an n-6 PUFA), reduces Ras localization to the plasma membrane without affecting posttranslational lipidation and lowers GTP binding and downstream p42/44ERK-dependent signaling. In view of the central role of oncogenic Ras in the development of colon cancer, the finding that n-3 and n-6 PUFA differentially modulate Ras activation may partly explain why dietary fish oil protects against colon cancer development.
Intracellular Ca2+ signaling in endothelial cells by the angiogenesis inhibitors endostatin and angiostatin
Authors: Jiang Lianwei , Jha Vivekanand , Dhanabal Mohanraj , Sukhatme Vikas P. , Alper Seth L.
Published: December 2017

Intracellular signaling mechanisms by the angiogenesis inhibitors endostatin and angiostatin remain poorly understood. We have found that endostatin (2 μg/ml) and angiostatin (5 μg/ml) elicited transient, approximately threefold increases in intracellular Ca2+ concentration ([Ca2+]i). Acute exposure to angiostatin or endostatin nearly abolished subsequent endothelial [Ca2+]i responses to carbachol or to thapsigargin; conversely, thapsigargin attenuated the Ca2+ signal elicited by endostatin. The phospholipase C inhibitor U-73122 and the inositol trisphosphate (IP3) receptor inhibitor xestospongin C both inhibited endostatin-induced elevation in [Ca2+]i, and endostatin rapidly elevated endothelial cell IP3 levels. Pertussis toxin and SB-220025 modestly inhibited the endostatin-induced Ca2+signal. Removal of extracellular Ca2+ inhibited the endostatin-induced rise in [Ca2+]i, as did a subset of Ca2+-entry inhibitors. Peak Ca2+responses to endostatin and angiostatin in endothelial cells exceeded those in epithelial cells and were minimal in NIH/3T3 cells. Overnight pretreatment of endothelial cells with endostatin reduced the subsequent acute elevation in [Ca2+]i in response to vascular endothelial growth factor or to fibroblast growth factor by ∼70%. Intracellular Ca2+ signaling may initiate or mediate some of the cellular actions of endostatin and angiostatin.
Evaluation of islet heme oxygenase-CO and nitric oxide synthase-NO pathways during acute endotoxemia
Authors: Henningsson Ragnar , Alm Per , Lundquist Ingmar
Published: December 2017

We investigated, by a combined in vivo and in vitro approach, the temporal changes of islet nitric oxide synthase (NOS)-derived nitric oxide (NO) and heme oxygenase (HO)-derived carbon monoxide (CO) production in relation to insulin and glucagon secretion during acute endotoxemia induced by lipopolysaccharide (LPS) in mice. Basal plasma glucagon, islet cAMP and cGMP content after in vitro incubation, the insulin response to glucose in vivo and in vitro, and the insulin and glucagon responses to the adenylate cyclase activator forskolin were greatly increased after LPS. Immunoblots demonstrated expression of inducible NOS (iNOS), inducible HO (HO-1), and an increased expression of constitutive HO (HO-2) in islet tissue. Immunocytochemistry revealed a marked expression of iNOS in many β-cells, but only in single α-cells after LPS. Moreover, biochemical analysis showed a time dependent and markedly increased production of NO and CO in these islets. Addition of a NOS inhibitor to such islets evoked a marked potentiation of glucose-stimulated insulin release. Finally, after incubation in vitro, a marked suppression of NO production by both exogenous CO and glucagon was observed in control islets. This effect occurred independently of a concomitant inhibition of guanylyl cyclase. We suggest that the impairing effect of increased production of islet NO on insulin secretion during acute endotoxemia is antagonized by increased activities of the islet cAMP and HO-CO systems, constituting important compensatory mechanisms against the noxious and diabetogenic actions of NO in endocrine pancreas.
Calcium-sensing receptor activation induces intracellular calcium oscillations
Authors: Breitwieser Gerda E. , Gama Lucio
Published: December 2017

Parathyroid hormone secretion is exquisitely sensitive to small changes in serum Ca2+concentration, and these responses are transduced via the Ca2+-sensing receptor (CaR). We utilized heterologous expression in HEK-293 cells to determine the effects of small, physiologically relevant perturbations in extracellular Ca2+ on CaR signaling via phosphatidylinositol-phospholipase C, using changes in fura 2 fluorescence to quantify intracellular Ca2+. Chronic exposure of CaR-transfected cells to Ca2+ in the range from 0.5 to 3 mM modulated the resting intracellular Ca2+concentration and the subsequent cellular responses to acute extracellular Ca2+ perturbations but had no effect on thapsigargin-sensitive Ca2+ stores. Modest, physiologically relevant increases in extracellular Ca2+concentration (0.5 mM increments) caused sustained (30–40 min) low-frequency oscillations of intracellular Ca2+ (∼45 s peak to peak interval). Oscillations were eliminated by 1 μM thapsigargin but were insensitive to protein kinase inhibitors (staurosporine, KN-93, or bisindolylmaleimide I). Staurosporine did increase the fraction of cells oscillating at a given extracellular Ca2+ concentration. Serum Ca2+ concentrations thus chronically regulate cells expressing CaR, and small perturbations in extracellular Ca2+ alter both resting intracellular Ca2+ as well as Ca2+ dynamics.
Human intestinal epithelial cell survival: differentiation state-specific control mechanisms
Authors: Gauthier Rémy , Harnois Charlène , Drolet Jean-François , Reed John C. , Vézina Anne , Vachon Pierre H.
Published: December 2017

To investigate whether human intestinal epithelial cell survival involves distinct control mechanisms depending on the state of differentiation, we analyzed the in vitro effects of insulin, pharmacological inhibitors of Fak, MEK/Erk, and PI3-K/Akt, and integrin (β1, β4)-blocking antibodies on the survival of the well-established human Caco-2 enterocyte-like and HIEC-6 cryptlike cell models. In addition, relative expression levels of six Bcl-2 homologs (Bcl-2, Bcl-XL, Mcl-1, Bax, Bak, and Bad) and activation levels of Fak, Erk-2, and Akt were analyzed. Herein, we report that 1) the enterocytic differentiation process results in the establishment of distinct profiles of Bcl-2 homolog expression levels, as well as p125Fak, p42Erk-2, and p57Aktactivated levels; 2) the inhibition of Fak, of the MEK/Erk pathway, or of PI3-K, have distinct impacts on enterocytic cell survival in undifferentiated (subconfluent Caco-2, confluent HIEC-6) and differentiated (30 days postconfluent Caco-2) cells; 3) exposure to insulin and the inhibition of Fak, MEK, and PI3-K resulted in differentiation state-distinct modulations in the expression of each Bcl-2 homolog analyzed; and 4) Fak, β1 and β4 integrins, as well as the MEK/Erk and PI3-K/Akt pathways, are distinctively involved in cell survival depending on the state of cell differentiation. Taken together, these data indicate that human intestinal epithelial cell survival is regulated according to differentiation state-specific control mechanisms.
Effect of vitamin supplementation on cytokine response and on muscle damage after strenuous exercise
Authors: Petersen Emil Wolsk , Ostrowski Kenneth , Ibfelt Tobias , Richelle Myriam , Offord Elizabeth , Halkjær-Kristensen Jens , Pedersen Bente Klarlund
Published: December 2017

The present double-blinded, placebo-controlled study investigated whether antioxidant vitamin supplementation was able to modulate the cytokine and lymphocyte responses after strenuous eccentric exercise. Furthermore, muscle enzyme release was examined to see whether antioxidant treatment could reduce muscle damage. Twenty male recreational runners randomly received either antioxidants (500 mg of vitamin C and 400 mg of vitamin E) or placebo for 14 days before and 7 days after a 5% downhill 90-min treadmill run at 75% V˙o 2 max. Although the supplemented group differed significantly with regard to plasma vitamin concentration before and after exercise when compared with the placebo group, the two groups showed identical exercise-induced changes in cytokine, muscle enzyme, and lymphocyte subpopulations. The plasma level of interleukin (IL)-6 and IL-1 receptor antagonist increased 20- and 3-fold after exercise. The plasma level of creatine kinase was increased sixfold the day after exercise. The concentrations of CD4+ memory T cells, CD8+ memory and naı̈ve T cells, and natural killer cells increased at the end of exercise. The total lymphocyte concentration was below prevalues in the postexercise period. In conclusion, the present study does not support the idea that exercise-induced inflammatory responses are induced by free oxygen radicals.
In situ calibration and [H+] sensitivity of the fluorescent Na+indicator SBFI
Authors: Diarra Abdoullah , Sheldon Claire , Church John
Published: December 2017

Despite the popularity of Na+-binding benzofuran isophthalate (SBFI) to measure intracellular free Na+ concentrations ([Na+]i), the in situ calibration techniques described to date do not favor the straightforward determination of all of the constants required by the standard equation (Grynkiewicz G, Poenie M, and Tsien RY. J Biol Chem 260: 3440–3450, 1985) to convert the ratiometric signal into [Na+]. We describe a simple method in which SBFI ratio values obtained during a “full” in situ calibration are fit by a three-parameter hyperbolic equation; the apparent dissociation constant ( K d) of SBFI for Na+ can then be resolved by means of a three-parameter hyperbolic decay equation. We also developed and tested a “one-point” technique for calibrating SBFI ratios in which the ratio value obtained in a neuron at the end of an experiment during exposure to gramicidin D and 10 mM Na+is used as a normalization factor for ratios obtained during the experiment; each normalized ratio is converted to [Na+]i using a modification of the standard equation and parameters obtained from a full calibration. Finally, we extended the characterization of the pH dependence of SBFI in situ. Although the K d of SBFI for Na+ was relatively insensitive to changes in pH in the range 6.8–7.8, acidification resulted in an apparent decrease, and alkalinization in an apparent increase, in [Na+]i values. The magnitudes of the apparent changes in [Na+]ivaried with absolute [Na+]i, and a method was developed for correcting [Na+]i values measured with SBFI for changes in intracellular pH.
Leiomodin and tropomodulin in smooth muscle
Authors: Conley Catharine A.
Published: December 2017

Evidence is accumulating to suggest that actin filament remodeling is critical for smooth muscle contraction, which implicates actin filament ends as important sites for regulation of contraction. Tropomodulin (Tmod) and smooth muscle leiomodin (SM-Lmod) have been found in many tissues containing smooth muscle by protein immunoblot and immunofluorescence microscopy. Both proteins cofractionate with tropomyosin in the Triton-insoluble cytoskeleton of rabbit stomach smooth muscle and are solubilized by high salt. SM-Lmod binds muscle tropomyosin, a biochemical activity characteristic of Tmod proteins. SM-Lmod staining is present along the length of actin filaments in rat intestinal smooth muscle, while Tmod stains in a punctate pattern distinct from that of actin filaments or the dense body marker α-actinin. After smooth muscle is hypercontracted by treatment with 10 mM Ca2+, both SM-Lmod and Tmod are found near α-actinin at the periphery of actin-rich contraction bands. These data suggest that SM-Lmod is a novel component of the smooth muscle actin cytoskeleton and, furthermore, that the pointed ends of actin filaments in smooth muscle may be capped by Tmod in localized clusters.
Modulatory role of ERK MAPK-caldesmon pathway in PDGF- stimulated migration of cultured pulmonary artery SMCs
Authors: Yamboliev Ilia A. , Gerthoffer William T.
Published: December 2017

Extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases (MAPKs) phosphorylate caldesmon in vivo, but the function of caldesmon phosphorylation in smooth muscle physiology is controversial. We hypothesized that ERK MAPKs and caldesmon modulate chemotactic migration of cultured canine pulmonary artery smooth muscle cells (PASMCs). Platelet-derived growth factor (PDGF; 10 ng/ml) and endothelin-1 (ET-1; 100 nM) transiently activated ERK MAPKs: PDGF produced higher maximal and more potent activation of ERK MAPKs over 5 h. While both PDGF and ET-1 increased caldesmon phosphorylation, only PDGF stimulated migration of cultured cells (13 times over basal migration). At concentrations from 0.01 to 10 nM, ET-1 failed to enhance migration; 100 nM ET-1 produced only a slight increase (1.31 ± 0.18 times basal migration). ET-1 (100 nM) did not potentiate migration triggered by 0.5 or 3 ng/ml PDGF. The MEK1 inhibitor PD-98059 (50 μM) abolished the PDGF-stimulated phosphorylation of ERK MAPKs and caldesmon and reduced cell migration by 50%. We conclude that while ERK MAPK activity is not required to initiate migration, an ERK MAPK-caldesmon pathway may modulate later events necessary for PDGF-stimulated migration of cultured PASMCs.
Effects of a domain peptide of the ryanodine receptor on Ca2+ release in skinned skeletal muscle fibers
Authors: Lamb Graham D. , Posterino Giuseppe S. , Yamamoto Takeshi , Ikemoto Noriaki
Published: December 2017

Mutations in the central domain of the skeletal muscle ryanodine receptor (RyR) cause malignant hyperthermia (MH). A synthetic peptide (DP4) in this domain (Leu-2442–Pro-2477) produces enhanced ryanodine binding and sensitized Ca2+ release in isolated sarcoplasmic reticulum, similar to the properties in MH, possibly because the peptide disrupts the normal interdomain interactions that stabilize the closed state of the RyR (Yamamoto T, El-Hayek R, and Ikemoto N. J Biol Chem 275: 11618–11625, 2000). Here, DP4 was applied to mechanically skinned fibers of rat muscle that had the normal excitation-contraction coupling mechanism still functional to determine whether muscle fiber responsiveness was enhanced. DP4 (100 μM) substantially potentiated the Ca2+release and force response to caffeine (8 mM) and to low [Mg2+] (0.2 mM) in every fiber examined, with no significant effect on the properties of the contractile apparatus. DP4 also potentiated the response to submaximal depolarization of the transverse tubular system by ionic substitution. Importantly, DP4 did not significantly alter the size of the twitch response elicited by action potential stimulation. These results support the proposal that DP4 causes an MH-like aberration in RyR function and are consistent with the voltage sensor triggering Ca2+ release by destabilizing the closed state of the RyRs.
Transmembrane domain length determines intracellular membrane compartment localization of syntaxins 3, 4, and 5
Authors: Watson Robert T. , Pessin Jeffrey E.
Published: December 2017

Insulin recruits glucose transporter 4 (GLUT-4) vesicles from intracellular stores to the plasma membrane in muscle and adipose tissue by specific interactions between the vesicle membrane-soluble N-ethylmaleimide-sensitive factor attachment protein target receptor (SNARE) protein VAMP-2 and the target membrane SNARE protein syntaxin 4. Although GLUT-4 vesicle trafficking has been intensely studied, few have focused on the mechanism by which the SNAREs themselves localize to specific membrane compartments. We therefore set out to identify the molecular determinants for localizing several syntaxin isoforms, including syntaxins 3, 4, and 5, to their respective intracellular compartments (plasma membrane for syntaxins 3 and 4; cis-Golgi for syntaxin 5). Analysis of a series of deletion and chimeric syntaxin constructs revealed that the 17-amino acid transmembrane domain of syntaxin 5 was sufficient to direct the cis-Golgi localization of several heterologous reporter constructs. In contrast, the longer 25-amino acid transmembrane domain of syntaxin 3 was sufficient to localize reporter constructs to the plasma membrane. Furthermore, truncation of the syntaxin 3 transmembrane domain to 17 amino acids resulted in a complete conversion to cis-Golgi compartmentalization that was indistinguishable from syntaxin 5. These data support a model wherein short transmembrane domains (≤17 amino acids) direct the cis-Golgi localization of syntaxins, whereas long transmembrane domains (≥23 amino acids) direct plasma membrane localization.
KChAP/Kvβ1.2 interactions and their effects on cardiac Kv channel expression
Authors: Kuryshev Y. A. , Wible B. A. , Gudz T. I. , Ramirez A. N. , Brown A. M.
Published: December 2017

KChAP and voltage-dependent K+ (Kv) β-subunits are two different types of cytoplasmic proteins that interact with Kv channels. KChAP acts as a chaperone for Kv2.1 and Kv4.3 channels. It also binds to Kv1.x channels but, with the exception of Kv1.3, does not increase Kv1.x currents. Kvβ-subunits are assembled with Kv1.x channels; they exhibit “chaperone-like” behavior and change gating properties. In addition, KChAP and Kvβ-subunits interact with each other. Here we examine the consequences of this interaction on Kv currents in Xenopusoocytes injected with different combinations of cRNAs, including Kvβ1.2, KChAP, and either Kv1.4, Kv1.5, Kv2.1, or Kv4.3. We found that KChAP attenuated the depression of Kv1.5 currents produced by Kvβ1.2, and Kvβ1.2 eliminated the increase of Kv2.1 and Kv4.3 currents produced by KChAP. Both KChAP and Kvβ1.2 are expressed in cardiomyocytes, where Kv1.5 and Kv2.1 produce sustained outward currents and Kv4.3 and Kv1.4 generate transient outward currents. Because they interact, either KChAP or Kvβ1.2 may alter both sustained and transient cardiac Kv currents. The interaction of these two different classes of modulatory proteins may constitute a novel mechanism for regulating cardiac K+ currents.
Expression and characterization of the anion transporter homologue YNL275w in Saccharomyces cerevisiae
Authors: Zhao Rongmin , Reithmeier Reinhart A. F.
Published: December 2017

A search of the yeast Saccharomyces cerevisiae genome has revealed an open reading frame, YNL275w, which encodes a 576-amino acid protein that shows sequence similarity to the family of mammalian Cl−/HCO[Formula: see text] anion exchangers and Na+/HCO[Formula: see text] cotransporters. This yeast protein also has a very similar hydropathy profile to the mammalian HCO[Formula: see text] transporters, indicating a similar membrane topology and structure. A V5 epitope and His6-tagged version of Ynl275wp was expressed in yeast and was localized to the plasma membrane by subcellular fractionation and immunofluorescence labeling. The protein was purified by nickel affinity chromatography and was found not to be N-glycosylated. The protein's mobility on SDS-PAGE gels was not altered by treatment with N-glycanase F, α-mannosidase, or by mutation of each of the five consensus N-glycosylation sites. The protein did not bind to concanavalin A by lectin blotting or lectin affinity chromatography. The expressed protein bound specifically to a stilbene disulfonate inhibitor resin (SITS-Affi-Gel), and this binding could be competed by certain anions (HCO[Formula: see text], Cl−, NO[Formula: see text], and I−) but not by others (SO[Formula: see text] and PO[Formula: see text]). These results suggest that the yeast gene YNL275w encodes a nonglycosylated anion transport protein, localized to the plasma membrane.
Functional modeling of tight junctions in intestinal cell monolayers using polyethylene glycol oligomers
Authors: Watson C. J. , Rowland M. , Warhurst G.
Published: December 2017

Despite significant advances in the characterization of tight junction (TJ) proteins, little is known about how molecular changes relate to function due primarily to the limitations of conventional paracellular probes. To address this, the paracellular pathway in Caco-2 and T84 cell lines was profiled by measuring the permeabilities of 24 polyethylene glycols (PEG) of increasing molecular radius (3.5–7.4 Å) analyzed by mass spectrometry. When combined with a paracellular sieving model, these data provided quantitative descriptors of the pathway under control conditions and after exposure to TJ modulators. PEG profiles in both cell lines conformed to a biphasic process involving a restrictive pore (radius 4.3–4.5 Å) and a nonrestrictive component responsible for permeability of larger molecules. PEG profiling revealed significant differences between the effects of EGTA and sodium caprate (C10). The restrictive component of EGTA-treated cells lost all size discrimination due to an increase in pore radius. Sodium caprate had no effect on pore radius but increased permeability via a different mechanism possibly involving increased numbers of functional pores. PEG profiling provides a useful tool for probing the functional regulation of the paracellular route.
Dual trafficking of Slit3 to mitochondria and cell surface demonstrates novel localization for Slit protein
Authors: Little Melissa H. , Wilkinson Lorine , Brown Darren L. , Piper Michael , Yamada Toshiya , Stow Jennifer L.
Published: December 2017

Drosophila slit is a secreted protein involved in midline patterning. Three vertebrate orthologs of the fly slit gene, Slit1, 2, and 3, have been isolated. Each displays overlapping, but distinct, patterns of expression in the developing vertebrate central nervous system, implying conservation of function. However, vertebrate Slit genes are also expressed in nonneuronal tissues where their cellular locations and functions are unknown. In this study, we characterized the cellular distribution and processing of mammalian Slit3 gene product, the least evolutionarily conserved of the vertebrate Slit genes, in kidney epithelial cells, using both cellular fractionation and immunolabeling. Slit3, but not Slit2, was predominantly localized within the mitochondria. This localization was confirmed using immunoelectron microscopy in cell lines and in mouse kidney proximal tubule cells. In confluent epithelial monolayers, Slit3 was also transported to the cell surface. However, we found no evidence of Slit3 proteolytic processing similar to that seen for Slit2. We demonstrated that Slit3 contains an NH2-terminal mitochondrial localization signal that can direct a reporter green fluorescent protein to the mitochondria. The equivalent region from Slit1 cannot elicit mitochondrial targeting. We conclude that Slit3 protein is targeted to and localized at two distinct sites within epithelial cells: the mitochondria, and then, in more confluent cells, the cell surface. Targeting to both locations is driven by specific NH2-terminal sequences. This is the first examination of Slit protein localization in nonneuronal cells, and this study implies that Slit3 has potentially unique functions not shared by other Slit proteins.
Increased neuronal excitability and seizures in the Na+/H+ exchanger null mutant mouse
Authors: Gu Xiang Q. , Yao Hang , Haddad Gabriel G.
Published: December 2017

Mice lacking the Na+/H+ exchanger isoform 1 (NHE1) manifest neurological diseases that include ataxia, motor deficits, and a seizure disorder. The molecular basis for the phenotype has not been clear, and it has not been determined how the lack of NHE1 leads, in particular, to the seizure disorder. We have shown in this work that hippocampal CA1 neurons in mutant mice have a much higher excitability than in wild-type mice. This higher excitability is partly based on an upregulation of the Na+ current density (608.2 ± 123.2 pA/pF in NHE1 mutant vs. 334.7 ± 63.7 pA/pF in wild type in HCO[Formula: see text]/CO2). Alterations in Na+channel characteristics, including steady-state inactivation (shift of 18 mV in the depolarization direction in the mutant), recovery from inactivation (τh = 5.22 ± 0.49 ms in wild-type neurons and 2.20 ± 0.20 ms in mutant neurons), and deactivation (at −100 mV, τd = 1.75 ± 0.53 ms in mutant and 0.21 ± 0.05 ms in wild-type neurons) further enhance the differences in excitability between mutant and wild-type mice. Our investigation demonstrates the existence of an important functional interaction between the NHE1 protein and the voltage-sensitive Na+ channel. We hypothesize that the increased neuronal excitability and possibly the seizure disorder in mice lacking the NHE1 is due, at least in part, to changes in Na+ channel expression and/or regulation.
Gene and protein expressions of nitric oxide synthases in ischemia-reperfused peripheral nerve of the rat
Authors: Qi Wen-Ning , Yan Zuo-Qin , Whang Peter G. , Zhou Qi , Chen Long-En , Seaber Anthony V. , Stamler Jonathan S. , Urbaniak James R.
Published: December 2017

This study examined mRNA and protein expressions of neuronal (nNOS), inducible (iNOS), and endothelial nitric oxide synthases (eNOS) in peripheral nerve after ischemia-reperfusion (I/R). Sixty-six rats were divided into the ischemia only and I/R groups. One sciatic nerve of each animal was used as the experimental side and the opposite untreated nerve as the control. mRNA levels in the nerve were quantitatively measured by competitive PCR, and protein was determined by Western blotting and immunohistochemical staining. The results showed that, after ischemia (2 h), both nNOS and eNOS protein expressions decreased. After I/R (2 h of ischemia followed by 3 h of reperfusion), expression of both nNOS and eNOS mRNA and protein decreased further. In contrast, iNOS mRNA significantly increased after ischemia and was further upregulated (14-fold) after I/R, while iNOS protein was not detected. The results reveal the dynamic expression of individual NOS isoforms during the course of I/R injury. An understanding of this modulation on a cellular and molecular level may lead to understanding the mechanisms of I/R injury and to methods of ameliorating peripheral nerve injury.
Disruption of the actin cytoskeleton regulates cytokine-induced iNOS expression
Authors: Zeng Chenbo , Morrison Aubrey R.
Published: December 2017

Interleukin-1β (IL-1β) induces the inducible nitric oxide synthase (iNOS), resulting in the release of nitric oxide (NO) from glomerular mesangial cells. In this study, we demonstrated that disruption of F-actin formation by sequestration of G-actin with the toxin latrunculin B (LatB) dramatically potentiated IL-1β-induced iNOS protein expression in a dose-dependent manner. LatB by itself had little or no effect on iNOS expression. Staining of F-actin with nitrobenzoxadiazole (NBD)-phallacidin demonstrated that LatB significantly impaired F-actin stress fiber formation. Jasplakinolide (Jasp), which binds to and stabilizes F-actin, suppressed iNOS expression enhanced by LatB. These data strongly suggest that actin cytoskeletal dynamics regulates IL-1β-induced iNOS expression. We demonstrated that LatB decreases serum response factor (SRF) activity as determined by reporter gene assays, whereas Jasp increases SRF activity. The negative correlation between SRF activity and iNOS expression suggests a negative regulatory role for SRF in iNOS expression. Overexpression of a dominant negative mutant of SRF increases the IL-1β-induced iNOS expression, providing direct evidence that SRF inhibits iNOS expression.
17β-Estradiol normalizes immune responses in ovariectomized females after trauma-hemorrhage
Authors: Knöferl Markus W. , Jarrar Doraid , Angele Martin K. , Ayala Alfred , Schwacha Martin G. , Bland Kirby I. , Chaudry Irshad H.
Published: December 2017

Recent studies indicate that immune responses in proestrus females are maintained after trauma-hemorrhage but markedly depressed in ovariectomized females under such conditions. The current study tested the hypothesis that the decreased estrogen levels after ovariectomy are responsible for this immune depression. To study this hypothesis, ovariectomized female CBA/J mice were subjected to laparotomy (i.e., soft tissue trauma) and hemorrhagic shock (35 ± 5 mmHg for 90 min, then resuscitated) or sham operation. The mice received either 17β-estradiol (E2; 100 μg/25 g body wt) or vehicle subcutaneously during resuscitation. Immune cells were isolated 24 h thereafter. Splenocyte proliferation and interferon-γ, interleukin (IL)-2, and IL-3 release were significantly depressed after trauma-hemorrhage in vehicle-treated mice, whereas these functions were maintained in E2-treated mice. Peritoneal macrophage IL-1β and IL-6 release and splenic macrophage IL-6 and IL-12 release were also significantly depressed in vehicle-treated mice after trauma-hemorrhage, and release of these cytokines was restored by E2 treatment. In summary our findings indicate that the depressed splenic and peritoneal immune responses after trauma-hemorrhage can be normalized by a single dose of E2. Thus estrogen appears to be the causative factor in the maintenance of immunocompetence in females after trauma-hemorrhage, and its administration to ovariectomized or postmenopausal females should be helpful in preventing immune depression under such conditions.
All human Na+-K+-ATPase α-subunit isoforms have a similar affinity for cardiac glycosides
Authors: Wang Jiangnan , Velotta Jeffrey B. , McDonough Alicia A. , Farley Robert A.
Published: December 2017

Three α-subunit isoforms of the sodium pump, which is the receptor for cardiac glycosides, are expressed in human heart. The aim of this study was to determine whether these isoforms have distinct affinities for the cardiac glycoside ouabain. Equilibrium ouabain binding to membranes from a panel of different human tissues and cell lines derived from human tissues was compared by an F statistic to determine whether a single population of binding sites or two populations of sites with different affinities would better fit the data. For all tissues, the single-site model fit the data as well as the two-site model. The mean equilibrium dissociation constant ( K d) for all samples calculated using the single-site model was 18 ± 6 nM (mean ± SD). No difference in K d was found between nonfailing and failing human heart samples, although the maximum number of binding sites in failing heart was only ∼50% of the number of sites in nonfailing heart. Measurement of association rate constants and dissociation rate constants confirmed that the binding affinities of the different human α-isoforms are similar to each other, although calculated K d values were lower than those determined by equilibrium binding. These results indicate both that the affinity of all human α-subunit isoforms for ouabain is similar and that the increased sensitivity of failing human heart to cardiac glycosides is probably due to a reduction in the number of pumps in the heart rather than to a selective inhibition of a subset of pumps with different affinities for the drugs.
Hyperactive ENaC identifies hypertensive individuals amenable to amiloride therapy
Authors: Carter Artensie R. , Zhou Zhen Hong , Calhoun David A. , Bubien James K.
Published: December 2017

Pathophysiological features of both primary aldosteronism and pseudohyperaldosteronism are hyperactive amiloride-sensitive epithelial Na+ channels (ENaC) and refractory hypertension. Peripheral blood lymphocytes express ENaC, which functions and is regulated similarly to ENaC expressed by renal principal cells. Thus it was hypothesized that individuals with either of these hypertensive etiologies could be identified by assessment of the function and regulation of peripheral blood lymphocyte ENaC, by whole cell patch clamp. We also tested the hypothesis that specific inhibition of hyperactive ENaC with amiloride could ameliorate the hypertension. To test these hypotheses, we solicited blood samples from normotensive, controlled hypertensive, and refractory hypertensive individuals. Lymphocytes were examined electrophysiologically to determine whether ENaC was hyperactive. All positive findings were from refractory hypertensive individuals. Nine refractory hypertensive patients had amiloride added to their hypertensive therapy. Amiloride normalized the blood pressure of four subjects. These individuals all had hyperactive ENaC. Amiloride had no effect on individuals with normal ENaC. These findings suggest that whole-cell patch clamp of peripheral blood lymphocytes can be used to identify accurately and rapidly hypertensive individuals who will respond to amiloride therapy.
Cell-based assay for high-throughput quantitative screening of CFTR chloride transport agonists
Authors: Galietta Luis V. J. , Jayaraman Sujatha , Verkman A. S.
Published: December 2017

Drug discovery by high-throughput screening is a promising approach to develop new therapies for the most common lethal genetic disease, cystic fibrosis. Because disease-causing mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) protein produce epithelial cells with reduced or absent Cl− permeability, the goal of screening is to identify compounds that restore cell Cl− transport. We have developed a rapid, quantitative screening procedure for analysis of CFTR-mediated halide transport in cells with the use of a conventional fluorescence plate reader. Doubly transfected cell lines were generated that express wild-type or mutant CFTR together with a yellow fluorescent protein (YFP)-based halide sensor. CFTR function was assayed from the time course of cell fluorescence in response to extracellular addition of 100 mM I− followed by forskolin, resulting in decreased YFP fluorescence due to CFTR-mediated I− entry. Cell lines were chosen, and conditions were optimized to minimize basal halide transport to maximize assay sensitivity. In cells cultured on 96-well plastic dishes, the assay gave reproducible halide permeabilities from well to well and could reliably detect a 2% activation of CFTR-dependent halide transport produced by low concentrations of forskolin. Applications of the assay are shown, including comparative dose-dependent CFTR activation by genistein, apigenin, 8-cyclopentyl-1,3-dipropylxanthine, IBMX, 8-methoxypsoralen, and milrinone as well as activation of alternative Cl− channels. The fluorescence assay and cell lines should facilitate the screening of novel CFTR activators and the characterization of alternative Cl− channels and transporters.
ARCD-1, an apobec-1-related cytidine deaminase, exerts a dominant negative effect on C to U RNA editing
Authors: Anant Shrikant , Mukhopadhyay Debnath , Sankaranand Vakadappu , Kennedy Susan , Henderson Jeffrey O. , Davidson Nicholas O.
Published: December 2017

Mammalian apolipoprotein B (apoB) C to U RNA editing is catalyzed by a multicomponent holoenzyme containing a single catalytic subunit, apobec-1. We have characterized an apobec-1 homologue, ARCD-1, located on chromosome 6p21.1, and determined its role in apoB mRNA editing. ARCD-1 mRNA is ubiquitously expressed; phylogenetic analysis reveals it to be a distant member of the RNA editing family. Recombinant ARCD-1 demonstrates cytidine deaminase and apoB RNA binding activity but does not catalyze C to U RNA editing, either in vitro or in vivo. Although not competent itself to mediate deamination of apoB mRNA, ARCD-1 inhibits apobec-1-mediated C to U RNA editing. ARCD-1 interacts and heterodimerizes with both apobec-1 and apobec-1 complementation factor (ACF) and localizes to both the nucleus and cytoplasm of transfected cells. Together, the data suggest that ARCD-1 is a novel cytidine deaminase that interacts with apobec-1 and ACF to inhibit apoB mRNA editing, possibly through interaction with other protein components of the apoB RNA editing holoenzyme.
Role of JNK in hypertonic activation of Cl−-dependent Na+/H+ exchange in Xenopus oocytes
Authors: Goss Greg G. , Jiang Lianwei , Vandorpe David H. , Kieller Dawn , Chernova Marina N. , Robertson Marilyn , Alper Seth L.
Published: December 2017

In the course of studying the hypertonicity-activated ion transporters in Xenopus oocytes, we found that activation of endogenous oocyte Na+/H+ exchange activity (xoNHE) by hypertonic shrinkage required Cl−, with an EC50 for bath [Cl−] of ∼3 mM. This requirement for chloride was not supported by several nonhalide anions and was not shared by xoNHE activated by acid loading. Hypertonicity-activated xoNHE exhibited an unusual rank order of inhibitory potency among amiloride derivatives and was blocked by Cl− transport inhibitors. Chelation of intracellular Ca2+ by injection of EGTA blocked hypertonic activation of xoNHE, although many inhibitors of Ca2+-related signaling pathways were without inhibitory effect. Hypertonicity activated oocyte extracellular signal-regulated kinase 1/2 (ERK1/2), but inhibitors of neither ERK1/2 nor p38 prevented hypertonic activation of xoNHE. However, hypertonicity also stimulated a Cl−-dependent increase in c-Jun NH2-terminal kinase (JNK) activity. Inhibition of JNK activity prevented hypertonic activation of xoNHE but not activation by acid loading. We conclude that hypertonic activation of Na+/H+ exchange in Xenopusoocytes requires Cl− and is mediated by activation of JNK.
Two-way arginine transport in human endothelial cells: TNF-α stimulation is restricted to system y+
Authors: Sala Roberto , Rotoli Bianca Maria , Colla Emanuela , Visigalli Rossana , Parolari Alessandro , Bussolati Ovidio , Gazzola Gian C. , Dall'Asta Valeria
Published: December 2017

Human umbilical vein endothelial cells transport arginine through two Na+-independent systems. System y+L is insensitive to N-ethylmaleimide (NEM), inhibited byl-leucine in the presence of Na+, and referable to the expression of SLC7A6/y+LAT2, SLC7A7/y+LAT1, and SLC3A2/4F2hc. System y+ is referable to the expression of SLC7A1/CAT1 and SLC7A2/CAT2B. Tumor necrosis factor-α (TNF-α) and bacterial lipopolysaccharide induce a transient stimulation of arginine influx and efflux through system y+. Increased expression of SLC7A2/CAT2B is detectable from 3 h of treatment, while SLC7A1 expression is inhibited at later times of incubation. System y+L activity and expression remain unaltered. Nitric oxide synthase type 2 mRNA is not detected in the absence or presence of TNF-α, while the latter condition lowers nitric oxide synthase type 3 expression at the mRNA and the protein level. Nitrite accumulation is comparable in cytokine-treated and control cells up to 48 h of treatment. It is concluded that modulation of endothelial arginine transport by TNF-α or lipopolysaccharide occurs exclusively through changes in CAT2B and CAT1 expression and is dissociated from stimulation of nitric oxide production.
Conditional immortalization of growth factor-responsive cardiac endothelial cells from H-2Kb-tsA58 mice
Authors: Lidington Elaine A. , Rao Ravi M. , Marelli-Berg Federica M. , Jat Parmjit S. , Haskard Dorian O. , Mason Justin C.
Published: December 2017

Although mouse endothelial cells (EC) may advance our understanding of endothelial function, primary EC remain difficult to isolate. We have established a murine cardiac endothelial cell line (MCEC-1) from transgenic mice harboring a temperature-sensitive simian virus 40 large TAg gene (tsA58 TAg) under H-2Kb class I promoter control. MCEC-1 cells were characterized by their ability to form tubes, Griffonia simplicifolia isolectin B4 binding, and CD31, intercellular adhesion molecule (ICAM)-2, and endoglin expression. MCEC-1 cells proliferated rapidly under permissive conditions [33°C with interferon (IFN)-γ], where the T antigen is active and transcription is activated by the presence of IFN-γ, whereas under nonpermissive conditions (38°C without IFN-γ) proliferation was reduced by 30-fold and the EC showed enhanced proliferation in response to growth factors. Expression of E- and P-selectin, ICAM-1, and vascular cell adhesion molecule-1 was upregulated by tumor necrosis factor-α and interleukin-1β, and MCEC-1 cells, in contrast to primary EC, were amenable to transfection by lipofection. This novel line will allow further study of the role of the endothelium in cardiovascular disease. Moreover, this technique will allow EC to be readily obtained from genetically modified mice backcrossed with H-2Kb-tsA58 mice.
Regulation of transcervical permeability by two distinct P2 purinergic receptor mechanisms
Authors: Gorodeski George I.
Published: December 2017

Micromolar concentrations of ATP stimulate biphasic change in transepithelial conductance across CaSki cultures, an acute increase (phase I response) followed by a slower decrease ( phase II response). Phase I and phase II responses involve two distinct calcium-dependent pathways, calcium mobilization and calcium influx. To test the hypothesis that phase I and phase II responses are mediated by distinct P2 purinergic receptors, changes in permeability were uncoupled by blocking calcium mobilization with 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid (BAPTA) or by lowering extracellular calcium, respectively. Under these conditions ATP EC50 was 25 μM for phase Iresponse and 2 μM for phase II response. The respective agonist profiles were ATP > UTP > adenosine 5′- O-(3-thiotriphosphate) (ATP-γS) = N 6-([6-aminohexyl]carbamoylmethyl)adenosine 5′-triphosphate (A8889) > GTP and UTP > ATP > GTP = A8889 > ATP-γS. Suramin blocked phase Iresponse and ATP-induced calcium mobilization, whereas pyridoxal phosphate-6-azophenyl-2′,4-disulfonic acid (PPADS) blocked phase II response and ATP-augmented calcium influx. ATP time course and pharmacological profiles for phase II response and augmented calcium influx were similar, with a time constant of 2 min and a saturable concentration-dependent effect (EC50 of 2–3 μM). RT-PCR experiments revealed expression of mRNA for both the P2Y2 and P2X4 receptors. These results suggest that the ATP-induced phase I and phase IIresponses are mediated by distinct P2 purinergic receptor mechanisms.
Role of insulin during exercise-induced glycogenesis in muscle: effect on cyclic AMP.
Authors: Ivy J L
Published: December 2017

Skeletal muscle cyclic AMP (cAMP) content and glycogen synthesis were investigated in male rats subjected to exhaustive exercise, alloxan diabetes, and combinations of these conditions. After an exhaustive swim or control treatment of wading, randomly selected animals were administered 500 mg glucose via stomach tube. Two hours after glucose administration, gastrocnemius glycogen levels rose from 1.31 to 10.67 mg/g wet wt in fatigued nondiabetics (FND), producing a 94% supercompensation above control values. Glycogen of fatigued diabetics (FD) increased from 0.88 to 4.21 mg/g wet wt during the first 2 hr after glucose administration and did not reach control values for 24 h. In conjunction with these glycogen changes, cAMP increased from 1.23 to 2.59 and 1.47 to 2.81 pmol/mg wet wt for FND and FD, respectively (P less than 0.05). No difference in cAMP levels between diabetics and nondiabetics was found. These in vivo data suggest that insulin may not be essential for muscle glycogen synthesis, but that after glycogen depletion it plays a prominent role in supercompensation. Also, this hormone's mechanism of action in skeletal muscle does not appear to be mediated through alteration in the tissue cAMP concentration.
Glucose delivery: a modulator of glucose uptake in contracting skeletal muscle.
Authors: Schultz T A , Lewis S B , Westbie D K , Wallin J D , Gerich J E
Published: December 2017

To evaluate glucose delivery and neuromuscular activity as modulators of glucose uptake in skeletal muscle, rat hindlimbs from pentobarbital-anesthetized rats were perfused in the presence of electrically stimulated muscular contractions. Glucose delivery was varied by altering non-cyclic perfusate flow. When flow was increased from 3 to 12 ml/min glucose disappearance increased from 1.1 +/- 0.2 to 4.0 +/- 0.2 mumol/min per 100 g rat (P less than 0.001). When glucose delivery was held constant, glucose disappearance was unaltered during muscular contractions. Insulin enhanced glucose disappearance, and its effect was unaltered during muscular contractions. Muscular contractions increased oxygen disappearance by two- to 3.5-fold and lactate appearance by 4.5- to fivefold (P less than 0.02). It was concluded that glucose delivery and insulin modulate glucose disappearance in isolated perfused rat hindlimbs. Electrical stimulation of muscle enhances lactate appearance and oxygen disappearance but does not alter glucose disappearance when flow is held constant. Because blood flow to muscle increases during exercise in vivo, increased glucose delivery may be a modulator of the augmented muscle glucose consumption observed under these conditions.