bluuebunny/crossref_metadata_2025 huggingface dataset:
Glutathione redox cycle regulates nitric oxide-mediated glyceraldehyde-3-phosphate dehydrogenase inhibition
Authors: Padgett C. M. , Whorton A. R.
Published: December 2017

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been identified as a potential target for nitric oxide (NO)-mediated cellular toxicity. We have previously shown that NO inhibits GAPDH by S-nitrosylation of the active site cysteine residue, which is reversed by low-molecular-weight thiols. Because endothelial cells contain high concentrations of low-molecular-weight thiols, principally glutathione, we investigated the effect of NO on GAPDH activity in intact endothelial cells and the influence that cellular glutathione has on GAPDH inhibition. Our results show that incubation of cells with an exogenous NO-generating system resulted in inhibition of GAPDH activity. The mechanism for inhibition appears to involve reversible modification of GAPDH because addition of thiols to cell extracts restored activity. Furthermore, cells were able to completely recover GAPDH activity after removal of the NO-generating system. Recovery did not require de novo protein synthesis. Depletion of cellular glutathione levels by treatment of cells with buthionine sulfoximine resulted in greater NO-mediated GAPDH inhibition as well as a lesser ability to recover activity. Finally, disruption of the glutathione redox cycle with the glutathione reductase inhibitor, 1,3-bis(2-chloroethyl)-1-nitrosourea, increased the extent of NO-mediated GAPDH inhibition and decreased both the rate and degree of recovery of GAPDH-activity. These results suggest that the glutathione redox cycle plays an important role not only in regulating the extent of NO-mediated GAPDH inhibition but also in the ability of endothelial cells to recover from NO-mediated GAPDH inhibition.
Role of intracellular calcium and metabolites in low-frequency fatigue of mouse skeletal muscle
Authors: Chin E. R. , Balnave C. D. , Allen D. G.
Published: December 2017

We have examined the extent to which prolonged reductions in low-frequency force (i.e., low-frequency fatigue) result from increases in intracellular free Ca2+ concentration ([Ca2+]i) and alterations in muscle metabolites. Force and [Ca2+]i were measured in mammalian single muscle fibers in response to short, intermediate, and long series of tetani that elevated the [Ca2+]i-time integral to 5, 17, and 29 microM x s, respectively. Only the intermediate and long series resulted in prolonged (>60 x min) reductions in Ca2+ release and low-frequency fatigue. When fibers recovered from the long series of tetani without glucose, Ca2+ release was reduced to a greater extent and force was reduced at high and low frequencies. These findings indicate that the decrease in sarcoplasmic reticulum Ca2+ release associated with fatigue has at least two components: 1) a metabolic component, which, in the presence of glucose, recovers within 1 h, and 2) a component dependent on the elevation of the [Ca2+]i-time integral, which recovers more slowly. It is this Ca2+-dependent component that is primarily responsible for low-frequency fatigue.
EGF and TGF-beta regulate neutral endopeptidase expression in renal vascular smooth muscle cells
Authors: Tharaux P. L. , Stefanski A. , Ledoux S. , Soleilhac J. M. , Ardaillou R. , Dussaule J. C.
Published: December 2017

We recently reported that neutral endopeptidase (NEP) expression on renal vascular smooth muscle cells (VSMC) was downregulated in the presence of serum. Here we examine the role of epidermal growth factor (EGF) and transforming growth factor-beta 1 (TGF-beta) in this downregulation and the consequences of the changes in NEP activity on their mitogenic effects. EGF inhibited NEP activity, whereas TGF-beta was stimulatory. Expression of the enzyme was studied by measuring the binding of [125I]RB-104, a specific NEP inhibitor, and the fluorescence intensity of NEP-labeled cells. Both parameters were decreased by EGF and were increased by TGF-beta. NEP mRNA expression in EGF-treated cells was reduced after 48 h. In contrast, it was increased in TGF-beta-treated cells. Interestingly, NEP inhibition influenced the mitogenic effect of EGF. Indeed, thiorphan, an NEP inhibitor, and an anti-NEP antibody decreased EGF-dependent [3H]thymidine incorporation and cell proliferation by approximately 50%. TGF-beta had no effect on VSMC growth. These results indicate that EGF but not TGF-beta participates in the downregulatory potency of serum on NEP expression in VSMC. They also demonstrate that the full effect of EGF on VSMC proliferation depends on intact NEP activity.
Effects of pinacidil on K+ channels in human coronary artery vascular smooth muscle cells
Authors: Bychkov R. , Gollasch M. , Ried C. , Luft F. C. , Haller H.
Published: December 2017

We investigated pinacidil-activated K+ currents in vascular smooth muscle cells (VSMC) from human coronary arteries with the patch-clamp method. In 19 of 54 VSMC, pinacidil (1 and 20 microM) induced a large, nonrectifying, outward current [IK(ATP)] and increased voltage-dependent outward K+ currents [IK(Ca)] positive to voltages of -25 mV. The pinacidil-induced (1 microM) IK(ATP) was blocked by glibenclamide (3 microM) but was not affected by iberiotoxin (100-300 nM). Pinacidil activated up to 150 functionally active ATP-dependent K+ channels (KATP channels) per cell with a single-channel conductance of approximately 17 pS at physiological membrane potentials (between -80 and -30 mV) and K+ gradients (6 mM/130 mM). In 26 of 54 VSMC, on the other hand, pinacidil (1-20 microM) failed to induce IK(ATP) but increased IK(Ca). This current was completely blocked by iberiotoxin (100-300 nM) and tetraethylammonium (1 mM) but not by glibenclamide (3 microM). The single-channel conductance of the channel underlying IK(Ca) was approximately 150 +/- 16 pS between -10 and +30 mV, consistent with large-conductance, maxi Ca(2+)-activated, K+ channels (BKCa channels). We conclude that pinacidil is a nonselective K+ channel opener targeting KATP and BKCa channels. Furthermore, the conductance of KATP channels in human coronary arteries is likely to be small under physiological conditions.
Extracellular matrix-anchored serum amyloid A preferentially induces mast cell adhesion
Authors: Hershkoviz R. , Preciado-Patt L. , Lider O. , Fridkin M. , Dastych J. , Metcalfe D. D. , Mekori Y. A.
Published: December 2017

Mast cells are known to accumulate in various inflammatory processes, some of which are known to be associated with increased local and systemic levels of acute-phase reactants such as serum amyloid A (SAA) or with amyloid deposition. The mechanism(s) by which mast cells are recruited to these sites, however, has not been fully elucidated. It has recently been shown that SAA interacts with extracellular matrix (ECM) components and thereby acts as a chemoattractant and regulator of immune cell migration. On the basis of these observations, we examined the effect of SAA on mast cell adhesion to ECM, an essential step in cellular transmigration. We could first demonstrate strong specific binding of recombinant human SAA (rSAA) to murine mast cells using flow cytometry. Moreover, radiolabeled rSAA was found to bind, in a saturable manner, to mast cells, reaching a binding affinity of 10(-8) M. When immobilized by preincubation with ECM, SAA or its proteolytically degraded amyloid A fragment (amino acid residues 2-82), which contains RGD-related adhesion motif but not the COOH-terminal portion of SAA (amino acid residues 77-104), induced the adhesion of resting mast cells to ECM or laminin. SAA and AA, in soluble or immobilized forms, did not activate mast cells to release mediators. Mast cell adhesion to the immobilized ECM-SAA complex appeared to occur through an integrin recognition, inasmuch as adhesion was calcium dependent and could be blocked by an RGD-containing peptide or by anti-CD29 monoclonal antibody. Genistein also inhibited adhesion, indicating that tyrosine kinase activity was involved. These data suggest that SAA bound to ECM may serve as an important inducer of mast cell adhesion, thus regulating mast cell recruitment and accumulation at these sites, which in turn could potentiate further pathology.
Energy turnover of vascular endothelial cells
Authors: Culic O. , Gruwel M. L. , Schrader J.
Published: December 2017

Two noninvasive methods, calorimetry and 31P nuclear magnetic resonance (NMR), were used to further define energy-consuming and energy-providing reactions in endothelial cells. With 31P-NMR, cellular ATP content was measured; with calorimetry, heat flux as a result of ATP turnover was measured. For these measurements, pig aortic endothelial cells were cultured on microcarrier beads and perfused in a column at constant flow rate. Pig aortic endothelial cells synthesize ATP mainly through glycolysis and, as determined by NMR, contain no phosphocreatine. In such a system, calorimetry-measured heat flux reflects rate of cellular ATP turnover. By use of inhibitors of ATP-dependent processes, the following changes in basal heat flux (231 +/- 65.5 microW/mg protein) were obtained: 18% for 2,3-butanedione monoxime (inhibitor of actomyosin-ATPase), 17% for wortmannin (inhibitor of myosin light chain kinase), 10% for cytochalasin D (inhibitor of actin polymerization), 23% for cycloheximide (inhibitor of protein synthesis), 11% for thapsigargin (inhibitor of endoplasmic reticulum Ca(2+)-ATPase), and 6% for bafilomycin A1 (inhibitor of lysosomal H(+)-ATPase). Cytochalasin D, 2,3-butanedione monoxime, wortmannin, and thapsigargin caused changes in F-actin distribution, as revealed by rhodamine-phalloidin cytochemistry. In a separate experimental series, when cells were perfused with a medium containing no glucose, heat flux decreased by 40% while cellular ATP remained unchanged. Inhibition of glycolysis with 2-deoxy-D-glucose decreased heat flux by 73%, and ATP was no longer visible with 31P-NMR. Despite this massive ATP depletion, which was maintained for 3 h, cells fully recovered heat flux and ATP when 2-deoxy-D-glucose was removed. The results, together with previously published data for Na(+)-K(+)-ATPase [M. L. H. Gruwel, C. Alves, and J. Schrader. Am. J. Physiol. 268 (Heart Circ. Physiol. 37): H351-H358, 1995], demonstrate that > 70% of total ATP-consuming processes of endothelial cells can be attributed to specific cellular processes. Actomyosin-ATPase (18%) and protein synthesis (23%) comprise the largest fraction. At least three-fourths of ATP synthesized is provided by glycolysis. Endothelial cells exhibit the remarkable ability to coordinate downregulation of ATP synthesis and consumption when glycolysis is inhibited.
Thrombin promotes endothelial cell alignment in Matrigel in vitro and angiogenesis in vivo
Authors: Haralabopoulos G. C. , Grant D. S. , Kleinman H. K. , Maragoudakis M. E.
Published: December 2017

We have tested the effect of thrombin on endothelial cell tube formation in vitro and angiogenesis in vivo. Thrombin induces the differentiation of endothelial cells into capillary structures in a dose-dependent fashion (0.1-0.3 units thrombin/ml) on Matrigel, a laminin-rich reconstituted basement membrane matrix. At higher thrombin concentrations (1.0 unit/ml), a suppression of tube formation is evident, probably due to downregulation (desensitization) of the thrombin receptor. D-Phe-Pro-Arg-CH2Cl-thrombin is without effect when used alone, but it abolishes the tube-promoting effect of thrombin when used in combination with thrombin, indicating the involvement of the catalytic site of thrombin. Activation of protein kinase C (PKC) seems to be the transduction mechanism involved in the stimulation of tube formation by thrombin. Ro-318220 (3 micrograms/ml), a specific inhibitor of PKC, completely abolishes the stimulatory effect of thrombin. In the in vivo Matrigel system of angiogenesis, there is a 10-fold increase in endothelial cell infiltration in response to thrombin. These results provide evidence for the angiogenesis-promoting effect of thrombin in vivo and the induction by thrombin of the angiogenic phenotype of endothelial cells in vitro in the absence of other cell types such as smooth muscle cells, pericytes, and inflammatory cells.
Basis of passive tension and stiffness in isolated rabbit myofibrils
Authors: Bartoo M. L. , Linke W. A. , Pollack G. H.
Published: December 2017

By examining the mechanical properties of isolated skeletal and cardiac myofibrils in calcium-free, ATP-containing solution, we attempted to separate the stiffness contribution of titin filaments from that of weakly bound cross bridges. Efforts to enhance weak cross-bridge binding by lowering ionic strength were met by clear contractile responses. Even at low temperature, myofibrils bathed in low-ionic-strength relaxing solution generated increased force and exhibited sarcomere shortening, apparently caused by active contraction. At normal ionic strength, myofibril stiffness, estimated from the force response to rapid sinusoidal oscillations, increased steadily with sarcomere extension up to a strain limit. No obvious stiffness contribution from weak cross bridges was detectable. Instead, the stiffness response, which was frequency dependent at all sarcomere lengths, was apparently generated by the viscoelastic titin filaments. During imposed stretch-hold ramps, both peak force/stiffness and the amount of subsequent stress relaxation increased with higher stretch rates, larger stretch amplitudes, and longer sarcomere lengths. We conclude that, for a truly relaxed myofibril, both passive force and dynamic stiffness principally reflect the intrinsic viscoelastic properties of the titin filaments.
Upregulation of COX-2 in cerebral microvascular endothelial cells by smooth muscle cell signals
Authors: Parfenova H. , Eidson T. H. , Leffler C. W.
Published: December 2017

Cyclooxygenase (COX) isoform expression, intracellular localization, and function in endothelial cells from the newborn pig cerebral microvessels were investigated using COX-1- and COX-2-specific antibodies and the COX-2 inhibitor NS-398. Cerebral microvessels, microvascular endothelium, and cultured endothelial cells constitutively express both COX-1 and COX-2. NS-398 inhibits 70-90% of endothelial prostanoid production. Endothelial cells grown in noncontact coculture with smooth muscle cells for 24-48 h demonstrate a stable induction of COX-2 protein and an NS-398-sensitive increase in prostanoid synthesis. The induction of endothelial COX in mixed cell coculture is accompanied by intracellular redistribution of COX-2. In cocultured endothelial cells, COX-2 is observed in the nucleus, nuclear envelope, and cytoplasm, compared with the mainly intranuclear localization of COX-2 in cells cultured separately. No changes were observed in COX-1 protein, localized in endothelial cell cytoplasm and the nuclear envelope. These results indicate that smooth muscle cells may modify endothelial function by upregulating COX-2, which is a major functional COX isoform in cerebral microvascular endothelial cells.
Regulation of mitogenesis by kinins in arterial smooth muscle cells
Authors: Dixon B. S. , Dennis M. J.
Published: December 2017

Recent evidence suggests that bradykinin (BK) plays a role in regulating neointimal formation after vascular injury. The present study examined the mechanism whereby BK regulates platelet-derived growth factor (PDGF) AB-induced mitogenesis in smooth muscle cells from rat mesenteric artery. BK, but not other activators of phosphoinositidase C (e.g., angiotensin II), inhibited PDGF-stimulated mitogenesis. The B1 receptor agonist des-Arg9-BK (DABK) was more potent than the B2 agonist BK; smaller BK fragments had no activity. In studies in which the B2 receptor antagonist HOE-140 inverted question markD-Arg0[Hyp3,beta-(2-thienyl)-Ala5,D-Tic7,Oic 8]BK inverted question mark and the B1 receptor antagonist DHOE [[D-Arg0,Hyp3,beta-(2-thienyl)-Ala5,D-Tic7,Oi c8,des-Arg9]BK] were used, both receptors independently mediated inhibition of PDGF-induced mitogenesis. There was no evidence for metabolism of BK to DABK. The rank potency for activating phosphoinositidase C and increasing intracellular Ca2+ (BK > DABK) was opposite that for inhibiting mitogenesis (DABK > BK). Inhibition of cyclooxygenase did not prevent the kinin-mediated inhibition. Kinetic analysis of the cell cycle effects of kinins on PDGF-stimulated mitogenesis revealed that continuous exposure to DABK or BK was inhibitory even when added shortly before the cells initiated DNA synthesis (S phase). However, short-term exposure (5-60 min) to DABK or BK was inhibitory only when added after exposure to PDGF. These data suggest that the B1 and B2 receptors potently inhibited PDGF-stimulated mitogenesis and proliferation by activating an alternative signal transduction cascade not involving phosphoinositidase C or prostaglandins. The inhibition occurred at a point late in progression of the cell cycle from G1 to S and was dependent on the presence of kinins after exposure to PDGF.
Different effects of raised [K+]o on membrane potential and contraction in mouse fast- and slow-twitch muscle
Authors: Cairns S. P. , Hing W. A. , Slack J. R. , Mills R. G. , Loiselle D. S.
Published: December 2017

Increasing extracellular K+ concentration ([K+]o) from 4 to 7-14 mM reduced both tetanic force and resting membrane potential (Em) in isolated slow-twitch soleus and fast-twitch extensor digitorum longus (EDL) muscles of the mouse. The tetanic force-[K+]o relationships showed a greater force loss over 8-11 mM [K+]o in soleus than EDL, mainly because the Em was 2-3 mV less negative at each [K+]o in soleus. The tetanic force-resting Em relationships show that force was reduced in two phases: phase 1 (Em < -60 mV), a 20% force decline in which the relationships superimposed in soleus and EDL, and phase 2 (Em -60 to -55 mV), a marked force decline that was steeper in EDL than soleus. Additionally in phase 2, longer stimulation pulses restored tetanic force; the twitch force-stimulation strength relationship was shifted toward higher voltages; caffeine, a myoplasmic Ca2+ concentration elevator, increased maximum force; and twitch force fell abruptly. We suggest that 1) the K(+)-depressed force is due to reduced Ca2+ release resulting from an altered action potential profile (phase 1) and inexcitable fibers due to an increased action potential threshold (phase 2), and 2) K+ contributes to fatigue in both fast- and slow-twitch muscle when it causes depolarization to about -60 mV.
Molecular cloning of two rat GRK6 splice variants
Authors: Firsov D. , Elalouf J. M.
Published: December 2017

Desensitization of G protein-coupled receptors is frequently triggered by G protein-coupled receptor kinases (GRKs) that preferentially phosphorylate agonist-occupied receptors. In this study, two GRK6 splice variants were cloned from the rat kidney. One isoform (GRK6a) encodes a 576-amino acid protein that is virtually identical (98% identity) to human GRK6. The second isoform is similar except for a 2-base pair insert that constitutes part of an intron interrupting the 3'-end coding region. This new isoform (GRK6b, 589 amino acids) has therefore a specific COOH-terminal region. A reverse transcription-polymerase chain reaction assay designed to discriminate GRK6 splice variants demonstrated that GRK6b mRNA is widely distributed and expressed at much higher levels than GRK6a mRNA in most peripheral tissues. In contrast, GRK6a predominates in brain. Functional studies, performed with cytosol extracts from transfected Chinese hamster ovary cells, indicated that GRK6a and GRK6b both phosphorylate light-activated rhodopsin as well as a synthetic peptide. The identification of GRK6b extends the family of GRKs. Further studies will be required to establish the tissue and subcellular distribution of this protein and to delineate its physiological role.
Expression of smooth muscle myosin heavy chains and unloaded shortening in single smooth muscle cells
Authors: Meer Daniel P. , Eddinger Thomas J.
Published: December 2017

The functional significance of the variable expression of the smooth muscle myosin heavy chain (SM-MHC) tail isoforms, SM1 and SM2, was examined at the mRNA level (which correlates with the protein level) in individual permeabilized rabbit arterial smooth muscle cells (SMCs). The length of untethered single permeabilized SMCs was monitored during unloaded shortening in response to increased Ca2+ (pCa 6.0), histamine (1 μM), and phenylephrine (1 μM). Subsequent to contraction, the relative expression of SM1 and SM2 mRNAs from the same individual SMCs was determined by reverse transcription-polymerase chain reaction amplification and densitometric analysis. Correlational analyses between the SM2-to-SM1 ratio and unloaded shortening in saponin- and α-toxin-permeabilized SMCs ( n = 28) reveal no significant relationship between the SM-MHC tail isoform ratio and unloaded shortening velocity. The best correlations between SM2/SM1 and the contraction characteristics of untethered vascular SMCs were with the minimum length attained following contraction ( n = 20 and r = 0.72 for α-toxin, n = 8 and r = 0.78 for saponin). These results suggest that the primary effect of variable expression of the SM1 and SM2 SM-MHC tail isoforms is on the cell final length and not on shortening velocity.
CCK-B receptors produce similar signals but have opposite growth effects in CHO and Swiss 3T3 cells
Authors: Detjen Katharina , Yule David , Tseng Min-Jen , Williams John A. , Logsdon Craig D.
Published: December 2017

Rat cholecystokinin-B (CCK-B) receptors were transfected into Chinese hamster ovary (CHO)-K1 (CHO-CCK-B) and Swiss 3T3 (Swiss 3T3-CCK-B) cells, and the effects of receptor activation on cell proliferation and intracellular signaling were investigated. CCK octapeptide (CCK-8) treatment had no effect on cell growth in quiescent CHO-CCK-B cells but inhibited DNA synthesis, proliferation, and colony formation when the cells were grown in fetal bovine serum (FBS). In contrast, CCK-8 stimulated DNA synthesis in quiescent Swiss 3T3-CCK-B cells and had no effect when the cells were grown in FBS. These differences in growth responses were not due to differences in the level of receptor expression, as similar numbers of receptors were present in both cell types. To determine whether the different growth effects were due to differences in receptor coupling to common second messenger pathways, we investigated the effects of CCK-8 on several known intracellular signals. In both cell types, CCK-8 stimulated increases in intracellular Ca2+concentration and polyphosphoinositide hydrolysis with similar potencies and efficacies. CCK-8 also stimulated arachidonate release from both cell types, although the potency was higher in the CHO cells. Adenosine 3′,5′-cyclic monophosphate generation was observed at high agonist concentrations in both cell types and was much greater in cells with higher receptor density. In summary, receptor activation had opposite effects on growth parameters in CHO and Swiss 3T3 cells, but only minor differences were observed in the characteristics of CCK-B receptor coupling to specific second messengers in the two cell types. Thus cellular context is a principal determinant of the biological effects of CCK-B receptor activation, and differences in biological responses may occur independently of major differences in receptor coupling.
Retinoids regulate tight junctional resistance of cultured human cervical cells
Authors: Gorodeski George I. , Eckert Richard L. , Pal Dipika , Utian Wulf H. , Rorke Ellen A.
Published: December 2017

The objective of the study was to determine the effect of retinoids on paracellular resistance across the cervical epithelium and the mechanisms involved. The experimental model was cultures of human CaSki cells on filters, which retain phenotypic characteristics of the endocervical epithelium. End points for paracellular resistance were measurements of transepithelial electrical resistance and fluxes of pyranine (a trisulfonic acid that traverses the epithelium via the intercellular space). Paracellular resistance was significantly increased in cells grown in retinoid-free medium; the effect could be blocked and reversed with all- trans-retinoic acid (tRA) and with agonists of RAR and RXR receptors but only partially with retinol. The effect of tRA was dose dependent and saturable, with a 50% effective concentration of 0.8 nM. The increases in paracellular resistance induced by vitamin A deficiency required longer incubation in retinoid-free medium than decreases in resistance induced by retinoic acid. tRA had only a minimal effect on paracellular resistance in cells maintained in regular medium. Retinoid-free medium increased and tRA decreased the relative cation mobility across CaSki cultures. Also the effects of tRA were nonadditive to those of cytochalasin D (which decreases tight junctional resistance) and additive to those of ionomycin (which decreases the resistance of the lateral intercellular space), suggesting that tRA modulates tight junctional resistance. It is concluded that vitamin A determines the degree of paracellular resistance across cervical cells by a mechanism that involves modulation of tight junctional resistance.
Ca2+ dependence and pharmacology of large-conductance K+ channels in nonlabor and labor human uterine myocytes
Authors: Khan Raheela N. , Smith Stephen K. , Morrison J. J. , Ashford Michael L. J.
Published: December 2017

Two populations, Ca2+-dependent (BKCa) and Ca2+-independent K+ (BK) channels of large conductance were identified in inside-out patches of nonlabor and labor freshly dispersed human pregnant myometrial cells, respectively. Cell-attached recordings from nonlabor myometrial cells frequently displayed BKCa channel openings characterized by a relatively low open-state probability, whereas similar recordings from labor tissue displayed either no channel openings or consistently high levels of channel activity that often exhibited clear, oscillatory activity. In inside-out patch recordings, Ba2+ (2–10 mM), 4-aminopyridine (0.1–1 mM), and Shaker B inactivating peptide (“ball peptide”) blocked the BKCa channel but were much less effective on BK channels. Application of tetraethylammonium to inside-out membrane patches reduced unitary current amplitude of BKCa and BK channels, with dissociation constants of 46 mM and 53 μM, respectively. Tetraethylammonium applied to outside-out patches decreased the unitary conductance of BKCa and BK channels, with dissociation constants of 423 and 395 μM, respectively. These results demonstrate that the properties of human myometrial large-conductance K+channels in myocytes isolated from laboring patients are significantly different from those isolated from nonlaboring patients.
Urea inhibits inducible nitric oxide synthase in macrophage cell line
Authors: Prabhakar Sharma S. , Zeballos Guillermo A. , Montoya-Zavala Martin , Leonard Claire
Published: December 2017

Macrophage dysfunction is considered an important contributory factor for increased propensity of infections in uremia. Because nitric oxide (NO) is believed to be an effector molecule of macrophage cytotoxicity, we propose that the dysfunction may be related to impaired NO synthesis. To verify this hypothesis, we evaluated macrophage NO synthesis in the presence of urea, a compound that accumulates in renal failure and is believed by some to be a uremic toxin. Macrophages (RAW 264.7 cells) were incubated with bacterial lipopolysaccharide to induce NO synthesis, whereas the test groups had various concentrations of urea in addition. NO synthesis was measured by assaying the supernatant for nitrites and nitrates by chemiluminescence. We observed that urea consistently produced a dose-dependent reversible inhibition of inducible NO production in macrophages, whereas parathormone, another toxin retained in uremia, had no such inhibitory effects. Further studies revealed that mRNA for inducible NO synthase was not inhibited by urea. We thus conclude that urea inhibits inducible NO synthesis in macrophages by a posttranscriptional mechanism and that this may be important in macrophage dysfunction of uremia.
Histone-induced damage of a mammalian epithelium: the role of protein and membrane structure
Authors: Kleine Teri J. , Lewis Peter N. , Lewis Simon A.
Published: December 2017

In a previous report [T. J. Kleine, A. Gladfelter, P. N. Lewis, and S. A. Lewis. Am. J. Physiol. 268 ( Cell Physiol. 37): C1114–C1125, 1995], we found that the cationic DNA-binding proteins histones H4, H1, and H5 caused a voltage-dependent increase in the transepithelial conductance in rabbit urinary bladder epithelium. In this study, results from lipid bilayer experiments suggest that histones H5-H1 and H4 form variably sized conductive units. Purified fragments of histones H4 and H5 were used to determine the role of histone tertiary structure in inducing conductance. Isolated COOH- and NH2-terminal tails of histone H4, which are random coils, were inactive, whereas the central α-helical domain induced a conductance increase. Although the activities of the central fragment and intact histone H4 were in many ways similar, the dose-response relationships suggest that the isolated central domain was much less potent than intact histone H4. This suggests than the NH2- and COOH-terminal tails are also important for histone H4 activity. For histone H5, the isolated globular central domain was inactive. Thus the random-coil NH2- and COOH-terminal tails are important for H5 activity as well. These results indicate that histone molecules interact directly with membrane phospholipids to form a channel and that protein tertiary structure and the degree of positive charge play an important role in this activity.
Effect of TNF-α on SMIT mRNA levels andmyo-inositol accumulation in cultured endothelial cells
Authors: Yorek Mark A. , Dunlap Joyce A. , Thomas Michael J. , Cammarata Patrick R. , Zhou Cheng , Lowe William L.
Published: December 2017

Previously we have shown that hyperosmolarity increases Na+- myo-inositol cotransporter (SMIT) activity and mRNA levels in cultured endothelial cells. Because hyperosmolarity and cytokines, such as tumor necrosis factor-α (TNF-α), activate similar signal transduction pathways, we examined the effect of TNF-α on SMIT mRNA levels and myo-inositol accumulation. In contrast to the effect of hyperosmolarity, TNF-α caused a time- and concentration-dependent decrease in SMIT mRNA levels and myo-inositol accumulation. The effect of TNF-α on myo-inositol accumulation was found in large-vessel endothelial cells (derived from the aorta and pulmonary artery) and cerebral microvessel endothelial cells. In bovine aorta and bovine pulmonary artery endothelial cells, TNF-α activated nuclear factor (NF)-κB. TNF-α also increased ceramide levels, and C2-ceramide mimicked the effect of TNF-α on SMIT mRNA levels and myo-inositol accumulation in bovine aorta endothelial cells. Pyrrolidinedithiocarbamate, genistein, and 7-amino-1-chloro-3-tosylamido-2-hepatanone, compounds that can inhibit NF-κB activation, partially prevented the TNF-α-induced decrease in myo-inositol accumulation. The effect of TNF-α on myo-inositol accumulation was also partially prevented by the protein kinase C inhibitor calphostin C but not by staurosporine. These studies demonstrate that TNF-α causes a decrease in SMIT mRNA levels and myo-inositol accumulation in cultured endothelial cells, which may be related to the activation of NF-κB.
Transient regulation of c-fos, αB-crystallin, and hsp70 in muscle during recovery from contractile activity
Authors: Neufer P. Darrell , Ordway George A. , Williams R. Sanders
Published: December 2017

Endurance exercise training increases the oxidative capacity of skeletal muscles, reflecting the induction of genes encoding enzymes of intermediary metabolism. To test the hypothesis that changes in gene expression may be triggered specifically during recovery from contractile activity, we quantified c-fos, αB-crystallin, 70-kDa heat shock protein (hsp70), myoglobin, and citrate synthase RNA in rabbit tibialis anterior muscle during recovery from intermittent (8 h/day), low-frequency (10 Hz) motor nerve stimulation. Recovery from a single 8-h bout of stimulation was characterized by large (>10-fold) transient increases in c-fos, αB-crystallin, and hsp70 mRNA. Similar changes were noted during recovery after 7 or 14 days of stimulation (8 h/day). Myoglobin and citrate synthase mRNA were also induced during recovery, but the changes were of lesser magnitude (2- to 2.5-fold) and were observed only following repeated bouts of muscle activity (7th or 14th day) that promoted sustained (>24 h) increases in these transcripts. These findings indicate that recovery from exercise is associated with specific transient changes in the expression of immediate early and stress protein genes, suggesting that the products of these genes may have specific roles in the remodeling process evoked by repeated bouts of contractile activity.
Adenylyl cyclase is involved in desensitization and recovery of ATP-stimulated Cl−secretion in MDCK cells
Authors: Woo Jae Suk , Inoue Chiyoko N. , Hanaoka Kazushige , Schwiebert Erik M. , Guggino Sandra E. , Guggino William B.
Published: December 2017

We investigated the process of and recovery from desensitization of the P2 receptor-mediated stimulation of Cl− secretion in Madin-Darby canine kidney (MDCK) cell monolayers by assaying the response of short-circuit current ( I sc). When the cells were exposed to repeated 3-min challenges of ATP or UTP interspersed with 5-min washes, the response of I sc desensitized rapidly followed by spontaneous recovery. The pattern of inhibition by various channel blockers or enzyme inhibitors revealed that both the initial and recovered responses of I sc have the same ionic and signaling mechanisms. The desensitization and recovery processes were confined to the membrane exposed to the repeated challenges. When added during the desensitized phase, 8-bromoadenosine 3′,5′-cyclic monophosphate enhanced the ATP-stimulated I sc response, whereas it did not during the initial or recovered phases. ATP-induced increases of intracellular adenosine 3′,5′-cyclic monophosphate showed similar desensitization and recovery in parallel with the changes in the responses of I sc. The desensitization process was attenuated by pretreatment with cholera toxin or pertussis toxin. Taken together, our results suggest that the adenylyl cyclase system plays a role in the desensitization and recovery mechanism of the ATP-stimulated Cl− secretion in MDCK cells.
Thrombin activates a membrane-associated calcium-independent PLA2 in ventricular myocytes
Authors: McHowat Jane , Creer Michael H.
Published: December 2017

Activation of phospholipase A2(PLA2) and accumulation of lysophosphatidylcholine contribute importantly to arrhythmogenesis during acute myocardial ischemia. We examined thrombin stimulation of PLA2 activity in isolated ventricular myocytes. Basal and thrombin-stimulated cardiac myocyte PLA2 activity demonstrated a distinct preference for sn-1 ether-linked phospholipids with arachidonate esterified at the sn-2 position. The majority of PLA2 activity was calcium independent and membrane associated. Thrombin stimulation of membrane-associated PLA2 occurs in a time- and concentration-dependent fashion. An increase in PLA2 activity was also observed using the synthetic peptide SFLLRNPNDKYEPF (the tethered ligand generated by thrombin cleavage of its receptor). Bromoenol lactone, a selective inhibitor of calcium-independent PLA2, completely blocked thrombin-stimulated increases in PLA2 activity and arachidonic acid release. No significant inhibition of thrombin-induced PLA2 was observed following pretreatment with mepacrine or dibucaine. These data confirm the presence of high-affinity thrombin receptors on isolated cardiac myocytes and demonstrate the specific activation of a unique membrane-associated, calcium-independent PLA2 following thrombin receptor ligation.
Fourfold reduction of water permeability in inner medullary collecting duct of aquaporin-4 knockout mice
Authors: Chou C. L. , Ma Tonghui , Yang Baoxue , Knepper Mark A. , Verkman A. S.
Published: December 2017

Aquaporin (AQP)-3 and AQP4 water channels are expressed at the basolateral membrane of mammalian collecting duct epithelium. To determine the contribution of AQP4 to water permeability in the initial inner medullary collecting duct (IMCD), osmotic water permeability ( P f) was compared in isolated perfused IMCD segments from wild-type and AQP4 knockout mice. The AQP4 knockout mice were previously found to have normal gross appearance, survival, growth, and kidney morphology and a mild urinary concentrating defect (T. Ma, B. Yang, A. Gillespie, E. J. Carlson, C. J. Epstein, and A. S. Verkman. J. Clin. Invest. 100: 957–962, 1997). Transepithelial P f was measured in microdissected IMCDs after 18–48 h of water deprivation and in the presence of 0.1 nM arginine vasopressin (to make basolateral P f rate limiting). P fvalues (37°C; means ± SE in cm/s × 10−3) were 56.0 ± 8.5 for wild-type mice ( n = 5) and 13.1 ± 3.7 for knockout mice ( n = 6) ( P < 0.001). Northern blot analysis of kidney showed that transcript expression of AQP1, AQP2, AQP3, and AQP6 were not affected by AQP4 deletion. Immunoblot analysis indicated no differences in protein expression of AQP1, AQP2, or AQP3, and immunoperoxidase showed no differences in staining patterns. Coexpression of AQP3 and AQP4 in Xenopus laevis oocytes showed additive water permeabilities, suggesting that AQP4 deletion does not affect AQP3 function. These results indicate that AQP4 is responsible for the majority of basolateral membrane water movement in IMCD but that its deletion is associated with a very mild defect in urinary concentrating ability.
Control of Ca2+ wave propagation in mouse pancreatic acinar cells
Authors: Pfeiffer Fatima , Sternfeld Lutz , Schmid Andreas , Schulz Irene
Published: December 2017

We have investigated control mechanisms involved in the propagation of agonist-induced Ca2+ waves in isolated mouse pancreatic acinar cells. Using a confocal laser-scanning microscope, we were able to show that maximal stimulation of cells with acetylcholine (ACh, 500 nM) or bombesin (1 nM) caused an initial Ca2+ release of comparable amounts with both agonists at the luminal cell pole. Subsequent Ca2+ spreading to the basolateral membrane was faster with ACh (17.3 ± 5.4 μm/s) than with bombesin (8.0 ± 2.2 μm/s). The speed of bombesin-induced Ca2+ waves could be increased up to the speed of ACh-induced Ca2+waves by inhibition of protein kinase C (PKC). Activation of PKC significantly decreased the speed of ACh-induced Ca2+ waves but had only little effect on bombesin-evoked Ca2+waves. Within 3 s after stimulation, production of inositol 1,4,5-trisphosphate [Ins(1,4,5) P 3] was higher in the presence of ACh compared with bombesin, whereas bombesin induced higher levels of diacylglycerol (DAG) than ACh. These data suggest that the slower propagation speed of bombesin-induced Ca2+ waves is due to higher activation of PKC in the presence of bombesin compared with ACh. The higher increase in bombesin- compared with ACh-induced DAG production is probably due to activation of phospholipase D (PLD). Inhibition of the PLD-dependent DAG production by preincubation with 0.3% butanol led to an acceleration of the bombesin-induced Ca2+ wave. In further experiments, we could show that ruthenium red (100 μM), an inhibitor of Ca2+-induced Ca2+ release in skeletal muscle, also decreased the speed of ACh-induced Ca2+ waves. The effect of ruthenium red was not additive to the effect of PKC activation. From the data, we conclude that, following Ins(1,4,5) P 3-induced Ca2+ release in the luminal cell pole, secondary Ca2+ release from stores, which are located in series between the luminal and the basal plasma membrane, modifies Ca2+spreading toward the basolateral cell side by Ca2+-induced Ca2+ release. Activation of PKC leads to a reduction in Ca2+release from these stores and therefore could explain the slower propagation of Ca2+ waves in the presence of bombesin compared with ACh.
Cloning and functional studies of splice variants of the α-subunit of the amiloride-sensitive Na+ channel
Authors: Tucker J. Kevin , Tamba Kaichiro , Lee Young-Jae , Shen Li-Ling , Warnock David G. , Oh Youngsuk
Published: December 2017

The α-subunit of the amiloride-sensitive epithelial Na+ channel (αENaC) is critical in forming an ion conductive pore in the membrane. We have identified the wild-type and three splice variants of the human αENaC (hαENaC) from the human lung cell line H441, using RT-PCR. These splice variants contain various structures in the extracellular domain, resulting in premature truncation (hαENaCx), 19-amino acid deletion (hαENaC−19), and 22-amino acid insertion (hαENaC+22). Wild-type hαENaC and splice variants were functionally characterized in Xenopus oocytes by coexpression with hENaC β- and γ-subunits. Unlike wild-type hαENaC, undetectable or substantially reduced amiloride-sensitive currents were observed in oocytes expressing these splice variants. Wild-type hαENaC was the most abundantly expressed hαENaC mRNA species in all tissues in which its expression was detected. These findings indicate that the extracellular domain is important to generate structural and functional diversity of hαENaC and that alternative splicing may play a role in regulating hENaC activity.
Glutamine transport by the blood-brain barrier: a possible mechanism for nitrogen removal
Authors: Lee Wha-Joon , Hawkins Richard A. , Viña Juan R. , Peterson Darryl R.
Published: December 2017

Glutamine and glutamate transport activities were measured in isolated luminal and abluminal plasma membrane vesicles derived from bovine brain endothelial cells. Facilitative systems for glutamine and glutamate were almost exclusively located in luminal-enriched membranes. The facilitative glutamine carrier was neither sensitive to 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid inhibition nor did it participate in accelerated amino acid exchange; it therefore appeared to be distinct from the neutral amino acid transport system L1. Two Na-dependent glutamine transporters were found in abluminal-enriched membranes: systems A and N. System N accounted for ∼80% of Na-dependent glutamine transport at 100 μM. Abluminal-enriched membranes showed Na-dependent glutamate transport activity. The presence of 1) Na-dependent carriers capable of pumping glutamine and glutamate from brain into endothelial cells, 2) glutaminase within endothelial cells to hydrolyze glutamine to glutamate and ammonia, and 3) facilitative carriers for glutamine and glutamate at the luminal membrane may provide a mechanism for removing nitrogen and nitrogen-rich amino acids from brain.
Intracellular acidification induced by passive and active transport of ammonium ions in astrocytes
Authors: Nagaraja Tavarekere N. , Brookes Neville
Published: December 2017

We describe an unconventional response of intracellular pH to NH4Cl in mouse cerebral astrocytes. Rapid alkalinization reversed abruptly to be replaced by an intense sustained acidification in the continued presence of NH4Cl. We hypothesize that high-velocity [Formula: see text] influx persisted after the distribution of ammonia attained steady state. From the initial rate of acidification elicited by 1 mM NH4Cl in bicarbonate-buffered solution, we estimate that [Formula: see text] entered at a velocity of at least 31.5 nmol ⋅ min−1 ⋅ mg protein−1. This rate increased with NH4Cl concentration, not saturating at up to 20 mM NH4Cl. Acidification was attenuated by raising or lowering extracellular K+ concentration. Ba2+ (50 μM) inhibited the acidification rate by 80.6%, suggesting inwardly rectifying K+ channels as the primary[Formula: see text] entry pathway. Acidification was 10-fold slower in rat hippocampal astrocytes, consistent with the difference reported for K+ flux in vitro. The combination of Ba2+ and bumetanide prevented net acidification by 1 mM NH4Cl, identifying the Na+-K+-2Cl−cotransporter as a second [Formula: see text] entry route.[Formula: see text] entry via K+ transport pathways could impact “buffering” of ammonia by astrocytes and could initiate the elevation of extracellular K+concentration and astrocyte swelling observed in acute hyperammonemia.
In vitro and in vivo evaluation of insulin-producing βTC6-F7 cells in microcapsules
Authors: Zhou D. , Sun A. M. , Li X. , Mamujee S. N. , Vacek I. , Georgiou J. , Wheeler M. B.
Published: December 2017

In the present study, the insulin secretory capacity of βTC6-F7 cells in microcapsules was evaluated. The cell mass within capsules was found to expand in a three-dimensional fashion, in contrast to cells seeded on plates that grew as a monolayer. In in vitro studies, both free and encapsulated cells were found to secrete insulin in the absence of glucose, at 13.6 ± 1.1 and 14.5 ± 0.9 ng ⋅ 106cells−1 ⋅ 60 min−1, respectively, with the response rising to a maximum of 26.0 ± 0.8 and 31 ± 2.3 ng ⋅ 106cells−1 ⋅ 60 min−1 in the presence of 16.8 mM glucose. Encapsulated cells were able to produce Ca2+ responses in the presence of KCl (50 mM) and BAY K 8644 (100 μM). In in vivo studies, intraperitoneal transplantation of 3.0 ×106 microencapsulated cells into mice ( n = 5) with streptozotocin-induced diabetes resulted in the restoration of normoglycemia up to 57 days. Insulin concentrations rose from 0.4 ± 0.1 ng/ml before the graft administration to 2.2 ± 0.8 ng/ml after the transplantation in the normoglycemic recipients. An oral glucose challenge in transplant recipients demonstrated a flat glucose response, suggesting extremely high glucose clearance rates. These data demonstrate the potential use of the immunoisolated β-cell lines for the treatment of diabetes.
Uptake and intracellular fate of l-DOPA in a human intestinal epithelial cell line: Caco-2
Authors: Vieira-Coelho M. A. , Soares-Da-Silva P.
Published: December 2017

The aim of the present study was to examine the kinetic characteristics of thel-3,4-dihydroxyphenylalanine (l-DOPA) transporter and the fate of newly formed dopamine in Caco-2 cells. In the presence of 50 μM benserazide (an inhibitor of aromaticl-amino acid decarboxylase), l-DOPA was rapidly accumulated in Caco-2 cells. At equilibrium (30 min of incubation) the intracellular l-DOPA concentration was 10.2 ± 0.1 μM at a medium concentration of 0.5 μM. In saturation experiments the accumulation of l-DOPA was saturable with a Michaelis-Menten constant ( K m) of 60 ± 10 μM and a maximal reaction velocity ( V max) of 6.6 ± 0.3 nmol ⋅ mg protein−1 ⋅ 6 min−1; at 4°C the amount of l-DOPA accumulated in the cells was nonsaturable. When cells were incubated with increasing concentrations of l-DOPA (10–100 μM) in the absence of benserazide, a substantial amount of thel-DOPA that was taken up was decarboxylated to dopamine, with an apparent K m of 27.2 μM. In experiments performed in cells cultured in polycarbonate filters, the accumulation of l-DOPA in the presence of benserazide was greater when the substrate was applied from the basolateral cell border than when it was applied from the apical cell border. In the absence of benserazide, l-DOPA applied from the basolateral cell border resulted in a nonlinear formation of dopamine ( K m = 43 ± 7 μM, V max = 23.7 ± 1.2 nmol ⋅ mg protein−1 ⋅ 6 min−1). The amount of dopamine leaving the cell through the apical cell border was lower than the amount that escaped through the basolateral cell border, and the process was saturable ( K m = 623 ± 238 μM, V max = 0.19 ± 0.02 nmol ⋅ mg protein−1 ⋅ 6 min−1). In conclusion, the data presented here show that Caco-2 cells are endowed with an efficient l-DOPA uptake system, and intracellularl-DOPA was found to be rapidly converted to dopamine, some of which diffuses out of the cell. The utilization of Caco-2 cells cultured on polycarbonate filters probably provides a better way to look at processes such as the outward transfer of intracellular molecules, namely, the outward transfer of newly formed dopamine.