bluuebunny/crossref_metadata_2025 huggingface dataset:
Peroxide-induced membrane blebbing in endothelial cells associated with glutathione oxidation but not apoptosis
Authors: van Gorp Roosje M. A. , Broers Jos L. V. , Reutelingsperger Chris P. M. , Bronnenberg Nancy M. H. J. , Hornstra Gerard , van Dam-Mieras Maria C. E. , Heemskerk Johan W. M.
Published: December 2017

Cells under oxidative stress induced by peroxides undergo functional and morphological changes, which often resemble those observed during apoptosis. Peroxides, however, also cause the oxidation of intracellular reduced glutathione (GSH). We investigated the relation between these peroxide-induced effects by using human umbilical vein endothelial cells (HUVEC) and two HUVEC-derived cell lines, ECRF24 and ECV304. With HUVEC, tert-butyl hydroperoxide ( tBH) or hydrogen peroxide application in the presence of serum induced, in a dose-dependent way, reorganization of the actin cytoskeleton, membrane blebbing, and nuclear condensation. These processes were accompanied by transient oxidation of GSH. With ECRF24 cells, this treatment resulted in less blebbing and a shorter period of GSH oxidation. However, repeated tBH addition increased the number of blebbing cells and prolonged the period of GSH oxidation. ECV304 cells were even more resistant to peroxide-induced bleb formation and GSH oxidation. Inhibition of glutathione reductase activity potentiated the peroxide-induced blebbing response in HUVEC and ECRF24 cells, but not in ECV304 cells. Neither membrane blebbing nor nuclear condensation in any of these cell types was due to apoptosis, as evidenced by the absence of surface expression of phosphatidylserine or fragmentation of DNA, even after prolonged incubations with tBH, although high tBH concentrations lead to nonapoptotic death. We conclude that, in endothelial cells, peroxide-induced cytoskeletal reorganization and bleb formation correlate with the degree of GSH oxidation but do not represent an early stage of the apoptotic process.
Calcium dependence of C-type natriuretic peptide-formed fast K+ channel
Authors: Kourie Joseph I.
Published: December 2017

The lipid bilayer technique was used to characterize the Ca2+ dependence of a fast K+ channel formed by a synthetic 17-amino acid segment [ OaCNP-39-(1–17)] of a 39-amino acid C-type natriuretic peptide ( OaCNP-39) found in platypus ( Ornithorhynchus anatinus) venom ( OaV). The OaCNP-39-(1–17)-formed K+ channel was reversibly dependent on 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid-buffered cis (cytoplasmic) Ca2+ concentration ([Ca2+] cis ). The channel was fully active when [Ca2+] cis was >10−4 M and trans (luminal) Ca2+ concentration was 1.0 mM, but not at low [Ca2+] cis . The open probability of single channels increased from zero at 1 × 10−6 M cisCa2+ to 0.73 ± 0.17 ( n = 22) at 10−3 M cisCa2+. Channel openings to the maximum conductance of 38 pS were rapidly and reversibly activated when [Ca2+] cis , but not transCa2+ concentration ( n = 5), was increased to >5 × 10−4 M ( n = 14). Channel openings to the submaximal conductance of 10.5 pS were dominant at ≥5 × 10−4 M Ca2+. K+ channels did not open when cisMg2+ or Sr2+ concentrations were increased from zero to 10−3 M or when [Ca2+] cis was maintained at 10−6 M ( n = 3 and 2). The Hill coefficient and the inhibition constant were 1 and 0.8 × 10−4 M cisCa2+, respectively. This dependence of the channel on high [Ca2+] cis suggests that it may become active under 1) physiological conditions where Ca2+ levels are high, e.g., during cardiac and skeletal muscle contractions, and 2) pathological conditions that lead to a Ca2+ overload, e.g., ischemic heart and muscle fatigue. The channel could modify a cascade of physiological functions that are dependent on the Ca2+-activated K+ channels, e.g., vasodilation and salt secretion.
Cholecystokinin induction ofmob-1 chemokine expression in pancreatic acinar cells requires NF-κB activation
Authors: Han Bing , Logsdon Craig D.
Published: December 2017

Inflammatory mediators are involved in the early phase of acute pancreatitis, but the cellular mechanisms responsible for their generation within pancreatic cells are unknown. We examined the role of nuclear factor-κB (NF-κB) in cholecystokinin octapeptide (CCK-8)-induced mob-1 chemokine expression in pancreatic acinar cells in vitro. Supraphysiological, but not physiological, concentrations of CCK-8 increased inhibitory κB (IκB-α) degradation, NF-κB activation, and mob-1 gene expression in isolated pancreatic acinar cells. CCK-8-induced IκB-α degradation was maximal within 1 h. Expression of mob-1 was maximal within 2 h. Neither bombesin nor carbachol significantly increased mob-1 mRNA or induced IκB-α degradation. Thus the concentration, time, and secretagogue dependence of mob-1 gene expression and IκB-α degradation were similar. Inhibition of NF-κB with pharmacological agents or by adenovirus-mediated expression of the inhibitory protein IκB-α also inhibited mob-1 gene expression. These data indicate that the NF-κB signaling pathway is required for CCK-8-mediated induction of mob-1 chemokine expression in pancreatic acinar cells. This supports the hypothesis that NF-κB signaling is of central importance in the initiation of acute pancreatitis.
Multiple functional P2X and P2Y receptors in the luminal and basolateral membranes of pancreatic duct cells
Authors: Luo Xiang , Zheng Weizhong , Yan Ming , Lee Min Goo , Muallem Shmuel
Published: December 2017

Purinergic receptors in the basolateral and luminal membranes of the pancreatic duct can act by a feedback mechanism to coordinate transport activity in the two membranes during ductal secretion. The goal of the present work was to identify and localize the functional P2 receptors (P2R) in the rat pancreatic duct. The lack of selective agonists and/or antagonists for any of the cloned P2R dictated the use of molecular and functional approaches to the characterization of ductal P2R. For the molecular studies, RNA was prepared from microdissected pancreatic intralobular ducts and was shown to be free of mRNA for amylase and endothelial nitric oxide synthase (markers for acinar and endothelial cells, respectively). A new procedure is described to obtain an enriched preparation of single duct cells suitable for electrophysiological studies. Localization of P2R was achieved by testing the effect of various P2R agonists on intracellular Ca2+ concentration ([Ca2+]i) of microperfused intralobular ducts. RT-PCR analysis suggested the expression of six subtypes of P2R in the pancreatic duct: three P2YR and three P2XR. Activation of Cl− current by various nucleotides and coupling of the receptors activated by these nucleotides to G proteins confirmed the expression of multiple P2R in duct cells. Measurement of [Ca2+]iin microperfused intralobular ducts suggested the expression of P2X1R, P2X4R, probably P2X7R, and as yet unidentified P2YR, possibly P2Y1R, in the basolateral membrane. Expression of P2Y2R, P2Y4R, and P2X7R was found in the luminal membrane. The unprecedented expression of such a variety of P2R in one cell type, many capable of activating Cl− channels, suggests that these receptors may have an important role in pancreatic duct cell function.
Regulation of the epithelial Na+ channel by intracellular Na+
Authors: Awayda Mouhamed S.
Published: December 2017

The hypothesis that the intracellular Na+ concentration ([Na+]i) is a regulator of the epithelial Na+ channel (ENaC) was tested with the Xenopus oocyte expression system by utilizing a dual-electrode voltage clamp. [Na+]iaveraged 48.1 ± 2.2 meq ( n = 27) and was estimated from the amiloride-sensitive reversal potential. [Na+]iwas increased by direct injection of 27.6 nl of 0.25 or 0.5 M Na2SO4. Within minutes of injection, [Na+]istabilized and remained elevated at 97.8 ± 6.5 meq ( n = 9) and 64.9 ± 4.4 ( n = 5) meq 30 min after the initial injection of 0.5 and 0.25 M Na2SO4, respectively. This increase of [Na+]icaused a biphasic inhibition of ENaC currents. In oocytes injected with 0.5 M Na2SO4( n = 9), a rapid decrease of inward amiloride-sensitive slope conductance ( g Na) to 0.681 ± 0.030 of control within the first 3 min and a secondary, slower decrease to 0.304 ± 0.043 of control at 30 min were observed. Similar but smaller inhibitions were also observed with the injection of 0.25 M Na2SO4. Injection of isotonic K2SO4(70 mM) or isotonic K2SO4made hypertonic with sucrose (70 mM K2SO4-1.2 M sucrose) was without effect. Injection of a 0.5 M concentration of either K2SO4, N-methyl-d-glucamine (NMDG) sulfate, or 0.75 M NMDG gluconate resulted in a much smaller initial inhibition (<14%) and little or no secondary decrease. Thus increases of [Na+]ihave multiple specific inhibitory effects on ENaC that can be temporally separated into a rapid phase that was complete within 2–3 min and a delayed slow phase that was observed between 5 and 30 min.
Nitric oxide acts independently of cGMP to modulate capacitative Ca2+ entry in mouse parotid acini
Authors: Watson Eileen L. , Jacobson Kerry L. , Singh Jean C. , Ott Sabrina M.
Published: December 2017

Carbachol- and thapsigargin-induced changes in cGMP accumulation were highly dependent on extracellular Ca2+ in mouse parotid acini. Inhibition of nitric oxide synthase (NOS) and soluble guanylate cyclase (sGC) resulted in complete inhibition of agonist-induced cGMP levels. NOS inhibitors reduced agonist-induced Ca2+ release and capacitative Ca2+ entry, whereas the inhibition of sGC had no effect. The effects of NOS inhibition were not reversed by 8-bromo-cGMP. The NO donor GEA-3162 increased cGMP levels blocked by the inhibition of sGC. GEA-3162-induced increases in Ca2+ release from ryanodine-sensitive stores and enhanced capacitative Ca2+ entry, both of which were unaffected by inhibitors of sGC but reduced by NOS inhibitors. Results support a role for NO, independent of cGMP, in agonist-mediated Ca2+release and Ca2+ entry. Data suggest that agonist-induced Ca2+influx activates a Ca2+-dependent NOS, leading to the production of NO and the release of Ca2+ from ryanodine-sensitive stores, providing a feedback loop by which store-depleted Ca2+ channels are activated.
Ammonium transport by the colonic H+-K+-ATPase expressed in Xenopusoocytes
Authors: Cougnon Marc , Bouyer Patrice , Jaisser Frédéric , Edelman Aleksander , Planelles Gabrielle
Published: December 2017

Functional expression of the rat colonic H+-K+-ATPase was obtained by coexpressing its catalytic α-subunit and the β1-subunit of the Na+-K+-ATPase in Xenopus laevis oocytes. We observed that, in oocytes expressing the rat colonic H+-K+-ATPase but not in control oocytes (expressing β1 alone), NH4Cl induced a decrease in86Rb uptake and the initial rate of intracellular acidification induced by extracellular NH4Cl was enhanced, consistent with [Formula: see text] influx via the colonic H+-K+-ATPase. In the absence of extracellular K+, only oocytes expressing the colonic H+-K+-ATPase were able to acidify an extracellular medium supplemented with NH4Cl. In the absence of extracellular K+ and in the presence of extracellular [Formula: see text], intracellular Na+ activity in oocytes expressing the colonic H+-K+-ATPase was lower than that in control oocytes. A kinetic analysis of86Rb uptake suggests that[Formula: see text] acts as a competitive inhibitor of the pump. Taken together, these results are consistent with[Formula: see text] competition for K+ on the external site of the colonic H+-K+-ATPase and with [Formula: see text] transport mediated by this pump.
Cell proliferation is associated with enhanced capacitative Ca2+ entry in human arterial myocytes
Authors: Golovina Vera A.
Published: December 2017

Depletion of Ca2+ stores in the sarcoplasmic reticulum (SR) activates extracellular Ca2+ influx via capacitative Ca2+ entry (CCE). Here, CCE levels in proliferating and growth-arrested human pulmonary artery smooth muscle cells (PASMCs) were compared by digital imaging fluorescence microscopy. Resting cytosolic free Ca2+ concentration ([Ca2+]cyt) in proliferating PASMCs was twofold higher than that in growth-arrested cells. Cyclopiazonic acid (CPA; 10 μM), which inhibits SR Ca2+-ATPase and depletes inositol 1,4,5-trisphosphate-sensitive Ca2+ stores, transiently increased [Ca2+]cytin the absence of extracellular Ca2+. The addition of 1.8 mM Ca2+ to the extracellular solution in the presence of CPA induced large increases in [Ca2+]cyt, indicative of CCE. The CPA-induced SR Ca2+ release in proliferating PASMCs was twofold higher than that in growth-arrested cells, whereas the transient rise of [Ca2+]cytdue to CCE was fivefold greater in proliferating cells. CCE was insensitive to nifedipine but was significantly inhibited by 50 mM K+, which reduces the driving force for Ca2+ influx, and by 0.5 mM Ni2+, a putative blocker of store-operated Ca2+ channels. These data show that augmented CCE is associated with proliferation of human PASMCs and may be involved in stimulating and maintaining cell growth.
Intracellular Cl regulates Na-K-Cl cotransport activity in human trabecular meshwork cells
Authors: Putney Luanna K. , Vibat Cecile Rose T. , O’Donnell Martha E.
Published: December 2017

The trabecular meshwork (TM) of the eye plays a central role in modulating intraocular pressure by regulating aqueous humor outflow, although the mechanisms are largely unknown. We and others have shown previously that aqueous humor outflow facility is modulated by conditions that alter TM cell volume. We have also shown that the Na-K-Cl cotransport system is a primary regulator of TM cell volume and that its activity appears to be coordinated with net efflux pathways to maintain steady-state volume. However, the cellular mechanisms that regulate cotransport activity and cell volume in TM cells have yet to be elucidated. The present study was conducted to investigate the hypothesis that intracellular Cl concentration ([Cl]i) acts to regulate TM cell Na-K-Cl cotransport activity, as has been shown previously for some other cell types. We demonstrate here that the human TM cell Na-K-Cl cotransporter is highly sensitive to changes in [Cl]i. Our findings reveal a marked stimulation of Na-K-Cl cotransport activity, assessed as ouabain-insensitive, bumetanide-sensitive K influx, in TM cells following preincubation of cells with Cl-free medium as a means of reducing [Cl]i. In contrast, preincubation of cells with media containing elevated K concentrations as a means of increasing [Cl]i results in inhibition of Na-K-Cl cotransport activity. The effects of reducing [Cl]i, as well as elevating [Cl]i, on Na-K-Cl cotransport activity are concentration dependent. Furthermore, the stimulatory effect of reduced [Cl]i is additive with cell-shrinkage-induced stimulation of the cotransporter. Our studies also show that TM cell Na-K-Cl cotransport activity is altered by a variety of Cl channel modulators, presumably through changes in [Cl]i. These findings support the hypothesis that regulation of Na-K-Cl cotransport activity, and thus cell volume, by [Cl]i may participate in modulating outflow facility across the TM.
Intracellular pH shifts in cultured kidney (A6) cells: effects on apical Na+ transport
Authors: Zeiske Wolfgang , Smets Ilse , Ameloot Marcel , Steels Paul , van Driessche Willy
Published: December 2017

We report, for the epithelial Na+ channel (ENaC) in A6 cells, the modulation by cell pH (pHc) of the transepithelial Na+ current ( I Na), the current through the individual Na+channel ( i), the open Na+ channel density ( N o), and the kinetic parameters of the relationship between I Na and the apical Na+ concentration. The i and N o were evaluated from the Lorentzian I Na noise induced by the apical Na+ channel blocker 6-chloro-3,5-diaminopyrazine-2-carboxamide. pHc shifts were induced, under strict and volume-controlled experimental conditions, by apical/basolateral NH4Cl pulses or basolateral arrest of the Na+/H+exchanger (Na+ removal; block by ethylisopropylamiloride) and were measured with the pH-sensitive probe 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. The changes in pHc were positively correlated to changes in I Na and the apically dominated transepithelial conductance. The sole pHc-sensitive parameter underlying I Na was N o. Only the saturation value of the I Na kinetics was subject to changes in pHc. pHc-dependent changes in N o may be caused by influencing P o, the ENaC open probability, or/and the total channel number, N T = N o/ P o.
Extracellular ATP stimulates volume decrease inNecturus red blood cells
Authors: Light Douglas B. , Capes Tracy L. , Gronau Rachel T. , Adler Matthew R.
Published: December 2017

This study examined whether extracellular ATP stimulates regulatory volume decrease (RVD) in Necturus maculosus (mudpuppy) red blood cells (RBCs). The hemolytic index (a measure of osmotic fragility) decreased with extracellular ATP (50 μM). In contrast, the ATP scavenger hexokinase (2.5 U/ml, 1 mM glucose) increased osmotic fragility. In addition, the ATP-dependent K+ channel antagonist glibenclamide (100 μM) increased the hemolytic index, and this inhibition was reversed with ATP (50 μM). We also measured cell volume recovery in response to hypotonic shock electronically with a Coulter counter. Extracellular ATP (50 μM) enhanced cell volume decrease in a hypotonic (0.5×) Ringer solution. In contrast, hexokinase (2.5 U/ml) and apyrase (an ATP diphosphohydrolase, 2.5 U/ml) inhibited cell volume recovery. The inhibitory effect of hexokinase was reversed with the Ca2+ ionophore A-23187 (1 μM); it also was reversed with the cationophore gramicidin (5 μM in a choline-Ringer solution), indicating that ATP was linked to K+ efflux. In addition, glibenclamide (100 μM) and gadolinium (10 μM) inhibited cell volume decrease, and the effect of these agents was reversed with ATP (50 μM) and A-23187 (1 μM). Using the whole cell patch-clamp technique, we found that ATP (50 μM) stimulated a whole cell current under isosmotic conditions. In addition, apyrase (2.5 U/ml), glibenclamide (100 μM), and gadolinium (10 μM) inhibited whole cell currents that were activated during hypotonic swelling. The inhibitory effect of apyrase was reversed with the nonhydrolyzable analog adenosine 5′- O-(3-thiotriphosphate) (50 μM), and the effect of glibenclamide or gadolinium was reversed with ATP (50 μM). Finally, anionic whole cell currents were activated with hypotonic swelling when ATP was the only significant charge carrier, suggesting that increases in cell volume led to ATP efflux through a conductive pathway. Taken together, these results indicate that extracellular ATP stimulated cell volume decrease via a Ca2+-dependent step that led to K+ efflux.
Acidosis antagonizes intracellular calcium response to κ-opioid receptor stimulation in the rat heart
Authors: Pei Jian-Ming , Yu Xiao-Chun , Bian Jin-Song , Wong Tak-Ming
Published: December 2017

To study the effects of κ-opioid receptor stimulation on intracellular Ca2+ concentration ([Ca2+]i) homeostasis during extracellular acidosis, we determined the effects of κ-opioid receptor stimulation on [Ca2+]iresponses during extracellular acidosis in isolated single rat ventricular myocytes, by a spectrofluorometric method. U-50488H (10–30 μM), a selective κ-opioid receptor agonist, dose dependently decreased the electrically induced [Ca2+]itransient, which results from the influx of Ca2+ and the subsequent mobilization of Ca2+ from the sarcoplasmic reticulum (SR). U-50488H (30 μM) also increased the resting [Ca2+]iand inhibited the [Ca2+]itransient induced by caffeine, which mobilizes Ca2+ from the SR, indicating that the effects of the κ-opioid receptor agonist involved mobilization of Ca2+ from its intracellular pool into the cytoplasm. The Ca2+responses to 30 μM U-50488H were abolished by 5 μM nor-binaltorphimine, a selective κ-opioid receptor antagonist, indicating that the event was mediated by the κ-opioid receptor. The effects of the agonist on [Ca2+]iand the electrically induced [Ca2+]itransient were significantly attenuated when the extracellular pH (pHe) was lowered to 6.8, which itself reduced intracellular pH (pHi) and increased [Ca2+]i. The inhibitory effects of U-50488H were restored during extracellular acidosis in the presence of 10 μM ethylisopropyl amiloride, a potent Na+/H+exchange blocker, or 0.2 mM Ni2+, a putative Na+/Ca2+exchange blocker. The observations indicate that acidosis may antagonize the effects of κ-opioid receptor stimulation via Na+/H+and Na+/Ca2+exchanges. When glucose at 50 mM, known to activate the Na+/H+exchange, was added, both the resting [Ca2+]iand pHi increased. Interestingly, the effects of U-50488H on [Ca2+]iand the electrically induced [Ca2+]itransient during superfusion with glucose were significantly attenuated; this mimicked the responses during extracellular acidosis. When a high-Ca2+ (3 mM) solution was superfused, the resting [Ca2+]iincreased; the increase was abolished by 0.2 mM Ni2+, but the pHi remained unchanged. Like the responses to superfusion with high-concentration glucose and extracellular acidosis, the responses of the [Ca2+]iand electrically induced [Ca2+]itransients to 30 μM U-50488H were also significantly attenuated. Results from the present study demonstrated for the first time that extracellular acidosis antagonizes the effects of κ-opioid receptor stimulation on the mobilization of Ca2+ from SR. Activation of both Na+/H+and Na+/Ca2+exchanges, leading to an elevation of [Ca2+]i, may be responsible for the antagonistic action of extracellular acidosis against κ-opioid receptor stimulation.
Endotoxin-mediated nitric oxide synthesis inhibits IL-1β gene transcription in ANA-1 murine macrophages
Authors: Schroeder Rebecca A. , Cai Charles , Kuo Paul C.
Published: December 2017

On the basis of previous work demonstrating nitric oxide (NO)-mediated inhibition of nuclear factor-κB (NF-κB) DNA binding, we hypothesized that NO downregulates NF-κB-dependent interleukin-1β (IL-1β) production in an ANA-1 macrophage model of lipopolysaccharide (LPS) stimulation. In the presence of LPS (100 ng/ml), levels of IL-1β protein and mRNA were significantly upregulated with NO synthase inhibition. Using nuclear run-on analysis and transient transfection studies, IL-1β gene transcription and IL-1β promoter activity were also found to be increased with inhibition of NO production. Parallel transfection studies using an NF-κB long terminal repeat-reporter plasmid exhibited similar findings, suggesting an NO-mediated effect on NF-κB activity. Gel shift studies showed that LPS-associated NF-κB DNA binding was increased, both in the setting of NO synthase inhibition and in a reducing environment. Repletion of NO by addition of an S-nitrosothiol restored IL-1β protein synthesis, mRNA levels, gene transcription, promoter activity, and NF-κB DNA binding to levels noted in the presence of LPS alone. Our studies indicate that NO may regulate LPS-associated inflammation by downregulating IL-1β gene transcription through S-nitrosation of NF-κB.
Phosphoinositide 3-kinase is required for aldosterone-regulated sodium reabsorption
Authors: Blazer-Yost Bonnie L. , Păunescu Teodor G. , Helman Sandy I. , Lee Kimberly D. , Vlahos Chris J.
Published: December 2017

Aldosterone, a steroid hormone, regulates renal Na+ reabsorption and, therefore, plays an important role in the maintenance of salt and water balance. In a model renal epithelial cell line (A6) we have found that phosphoinositide 3-kinase (PI 3-kinase) activity is required for aldosterone-stimulated Na+reabsorption. Inhibition of PI 3-kinase by the specific inhibitor LY-294002 markedly reduces both basal and aldosterone-stimulated Na+ transport. Further, one of the products of PI 3-kinase, phosphatidylinositol 3,4,5-trisphosphate, is increased in response to aldosterone in intact A6 monolayers. This increase occurs just before the manifestation of the functional effect of the hormone and is also inhibited by LY-294002. With the use of blocker-induced noise analysis, it has been demonstrated that inhibition of phosphoinositide formation causes an inhibition of Na+ entry in both control and aldosterone-pretreated cultures by reducing the number of open functional epithelial Na+ channels (ENaCs) in the apical membrane of the A6 cells. These novel observations indicate that phosphoinositides are required for ENaC expression and suggest a mechanism for aldosterone regulation of channel function.
Transforming growth factor-β1 modulates the expression of vascular endothelial growth factor by osteoblasts
Authors: Saadeh Pierre B. , Mehrara Babak J. , Steinbrech Douglas S. , Dudziak Matthew E. , Greenwald Joshua A. , Luchs Jonathan S. , Spector Jason A. , Ueno Hikaru , Gittes George K. , Longaker Michael T.
Published: December 2017

Angiogenesis is essential to both normal and pathological bone physiology. Vascular endothelial growth factor (VEGF) has been implicated in angiogenesis, whereas transforming growth factor-β1 (TGF-β1) modulates bone differentiation, matrix formation, and cytokine expression. The purpose of this study was to investigate the relationship between TGF-β1 and VEGF expression in osteoblasts and osteoblast-like cells. Northern blot analysis revealed an early peak of VEGF mRNA (6-fold at 3 h) in fetal rat calvarial cells and MC3T3-E1 osteoblast-like cells after stimulation with TGF-β1 (2.5 ng/ml). The stability of VEGF mRNA in MC3T3-E1 cells was not increased after TGF-β1 treatment. Actinomycin D inhibited the TGF-β1-induced peak in VEGF mRNA, whereas cycloheximide did not. Blockade of TGF-β1 signal transduction via a dominant-negative receptor II adenovirus significantly decreased TGF-β1 induction of VEGF mRNA. Additionally, TGF-β1 induced a dose-dependent increase in VEGF protein expression by MC3T3-E1 cells ( P < 0.01). Dexamethasone similarly inhibited VEGF protein expression. Both TGF-β1 mRNA and VEGF mRNA were concurrently present in rat membranous bone, and both followed similar patterns of expression during rat mandibular fracture healing (mRNA and protein). In summary, TGF-β1-induced VEGF expression by osteoblasts and osteoblast-like cells is a dose-dependent event that may be intimately related to bone development and fracture healing.
Hypoxia modulates nitric oxide-induced regulation of NMDA receptor currents and neuronal cell death
Authors: Gbadegesin Muyiwa , Vicini Stefano , Hewett Sandra J. , Wink David A. , Espey Michael , Pluta Ryszard M. , Colton Carol A.
Published: December 2017

Nitric oxide (NO) released from a new chemical class of donors enhances N-methyl-d-aspartate (NMDA) channel activity. Using whole cell and single-channel patch-clamp techniques, we have shown that ( Z)-1-[ N-(3-ammoniopropyl)- N-( n-propyl)amino]-NO (PAPA-NO) and diethylamine NO, commonly termed NONOates, potentiate the glutamate-mediated response of recombinant rat NMDA receptors (NR1/NR2A) expressed in HEK-293 cells. The overall effect is an increase in both peak and steady-state whole cell currents induced by glutamate. Single-channel studies demonstrate a significant increase in open probability but no change in the mean single-channel open time or mean channel conductance. Reduction in oxygen levels increased and prolonged the PAPA-NO-induced change in both peak and steady-state glutamate currents in transfected HEK cells. PAPA-NO also enhanced cell death in primary cultures of rodent cortical neurons deprived of oxygen and glucose. This potentiation of neuronal injury was blocked by MK-801, indicating a critical involvement of NMDA receptor activation. The NO-induced increase in NMDA channel activity as well as NMDA receptor-mediated cell death provide firm evidence that NO modulates the NMDA channel in a manner consistent with both a physiological role under normoxic conditions and a pathophysiological role under hypoxic conditions.
Kinetics of cyclocreatine and Na+ cotransport in human breast cancer cells: mechanism of activity
Authors: Maril Nimrod , Degani Hadassa , Rushkin Edna , Sherry A. Dean , Cohn Mildred
Published: December 2017

The growth-inhibitory effect of cyclocreatine (CCr) and the kinetics of CCr and Na+ cotransport were investigated in MCF7 human breast cancer cells and its adriamycin-resistant subline with use of 31P- and23Na-NMR spectroscopy. The growth-inhibitory effect in the resistant line occurred at a lower CCr concentration and was more pronounced than in the wild-type line. This correlated with an ∼10-fold higher affinity of CCr to the transporter in the resistant line. The passive diffusion coefficient of CCr was also higher in the resistant line by three- to fourfold. The transport of CCr was accompanied by a rapid increase in intracellular Na+. This increase was found to depend on the rate of CCr transport and varied differently with CCr concentration in the two cell lines. It is proposed that the cotransport of CCr and Na+followed by increased Na+concentration, together with the accumulation of the highly charged phosphocyclocreatine, are responsible for cell swelling and death.
Phosphorylation-dependent stimulation of prostanoid synthesis by nigericin in cerebral endothelial cells
Authors: Parfenova Helena , Haffner John , Leffler Charles W.
Published: December 2017

Nigericin decreases intracellular pH (pHi) and stimulates prostanoid (PG) synthesis in endothelial cells from cerebral microvessels of newborn pigs. Nigericin-induced PG production was abolished by protein tyrosine kinase (PTK) inhibitors and amplified by phorbol 12-myristate 13-acetate (PMA) or protein tyrosine phosphatase (PTP) inhibitors. Nigericin-induced PG production in PMA-primed cells was potentiated by PTP inhibitors and abrogated by PTK inhibitors. Phospholipase A2(PLA2) activity was stimulated by nigericin in a phosphorylation-dependent manner. Nigericin’s effects on PG production and PLA2activity were reproduced by ionomycin, which activates cytosolic PLA2(cPLA2). cPLA2 was immunodetected in endothelial cell lysates. We found no evidence that nigericin’s effects are mediated via mitogen-activated protein (MAP) kinase [extracellularly regulated kinase 1 (ERK1) and ERK2] activation: although nigericin stimulated detergent-soluble MAP kinase, its effects were not amplified by PMA or PTP inhibitors. Phosphorylation-dependent stimulation of PG synthesis was also observed when pHi was decreased by sodium propionate or a high level of CO2. Altogether, our data indicate that nigericin and decreased pHistimulate PG synthesis by a protein phosphorylation-dependent mechanism involving cross talk between pathways mediated by PTK and PTP and by protein kinase C; cPLA2 appears to be a key enzyme affected by nigericin and decreased pHi.
Volume-dependent taurine release from cultured astrocytes requires permissive [Ca2+]iand calmodulin
Authors: Mongin Alexander A. , Cai Zhaohui , Kimelberg Harold K.
Published: December 2017

Cell swelling results in regulatory activation of multiple conductive anion pathways permeable toward a broad spectrum of intracellular organic osmolytes. Here, we explore the involvement of extracellular and intracellular Ca2+ in volume-dependent [3H]taurine efflux from primary cultured astrocytes and compare the Ca2+ sensitivity of this efflux in slow (high K+ medium induced) and fast (hyposmotic medium induced) cell swelling. Neither Ca2+-free medium nor Ca2+-channel blockers prevented the volume-dependent [3H]taurine release. In contrast, loading cells with the membrane-permeable Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid (BAPTA)-AM suppressed [3H]taurine efflux by 65–70% and 25–30% under high-K+ and hyposmotic conditions, respectively. Fura 2 measurements confirmed that BAPTA-AM, but not Ca2+-free media, significantly reduced resting intracellular Ca2+concentration ([Ca2+]i). The calmodulin antagonists trifluoperazine and fluphenazine reversibly and irreversibly, respectively, inhibited the high-K+-induced [3H]taurine release, consistent with their known actions on calmodulin. In hyposmotic conditions, the effects were less pronounced. These data suggest that volume-dependent taurine release requires minimal basal [Ca2+]iand involves calmodulin-dependent step(s). Quantitative differences in Ca2+/calmodulin sensitivity of high-K+-induced and hyposmotic medium-induced taurine efflux are due to both the effects of the inhibitors on high-K+-induced cell swelling and their effects on transport systems and/or signaling mechanisms determining taurine efflux.
Correlation of oxidant-induced acute ATP depletion with delayed cell death in human neuroblastoma cells
Authors: Aito Henrikka , Aalto T. Kristiina , Raivio Kari O.
Published: December 2017

We correlated the adenine nucleotide (AN) levels and energy charge (EC) at the end of a transient oxidative exposure to the delayed death of neuronal cells. When wild-type (WT) or Bcl-2-overexpressing (BCL-2) human neuroblastoma cells (Paju) were exposed to 250 μM H2O2for 60 min, the EC of WT cells was unchanged, but that of BCL-2 cells decreased from 0.91 ± 0.03 to 0.67 ± 0.02. Depletion of ANs was significantly greater in BCL-2 (66.7 ± 2%) than in WT (38.8 ± 2%) cells. Proliferation of both lines decreased, averaging 63 ± 17% of control by 48 h. Exposure to 5 mM H2O2caused no further change in ANs in BCL-2 cells but in WT cells decreased the EC to 0.45 ± 0.08 and depleted ANs to 41 ± 9% of control; after 24 h, WT cells became pyknotic and showed DNA fragmentation but no chromatin condensation, whereas BCL-2 cells died by delayed necrosis. After 10 mM H2O2, EC dropped to 0.15 ± 0.1, and both lines were acutely killed. The EC after an oxidative insult correlated well with further growth of both cell lines. A significant decline in EC led to delayed death. Bcl-2 did not protect against the fall in EC or AN depletion, but, although survival was not improved, the mechanism of death appeared to be different.
Role of protein phosphatases in the regulation of human mast cell and basophil function
Authors: Peirce Matthew J. , Munday Michael R. , Peachell Peter T.
Published: December 2017

Many extracellular stimuli mediate physiological change in target cells by altering the phosphorylation state of proteins. These alterations result from the dynamic interplay of protein kinases, which mediate phosphorylations, and protein phosphatases, which catalyse dephosphorylations. The antigen-mediated aggregation of high-affinity receptors for IgE on mast cells and basophils triggers rapid changes in the phosphorylation of many proteins and culminates in the generation of inflammatory mediators involved in allergic inflammatory diseases such as asthma. Although protein kinases have an established role in this process, less is known about the involvement of protein phosphatases. This imbalance has been redressed in recent years by the availability of phosphatase inhibitors, such as okadaic acid, that facilitate investigations of the role of protein phosphatases in intact cells. Here we review a number of studies in which inhibitors of protein phosphatases have been used to shed light on the potential importance of these enzymes in the regulation of human mast cell and human basophil function.
Epidermal growth factor regulation in adult rat alveolar type II cells of amiloride-sensitive cation channels
Authors: Kemp P. J. , Borok Z. , Kim K. J. , Lubman R. L. , Danto S. I. , Crandall E. D.
Published: December 2017

Using the patch-clamp technique, we studied the effects of epidermal growth factor (EGF) on whole cell and single channel currents in adult rat alveolar epithelial type II cells in primary culture in the presence or absence of EGF for 48 h. In symmetrical sodium isethionate solutions, EGF exposure caused a significant increase in the type II cell whole cell conductance. Amiloride (10 μM) produced ∼20–30% inhibition of the whole cell conductance in both the presence and absence of EGF, such that EGF caused the magnitude of the amiloride-sensitive component to more than double. Northern analysis showed that α-, β- and γ-subunits of rat epithelial Na+ channel (rENaC) steady-state mRNA levels were all significantly decreased by EGF. At the single channel level, all active inside-out patches demonstrated only 25-pS channels that were amiloride sensitive and relatively nonselective for cations ([Formula: see text]/[Formula: see text]≈ 1.0:0.48). Although the biophysical characteristics (conductance, open-state probability, and selectivity) of the channels from EGF-treated and untreated cells were essentially identical, channel density was increased by EGF; the modal channel per patch was increased from 1 to 2. These findings indicate that EGF increases expression of nonselective, amiloride-sensitive cation channels in adult alveolar epithelial type II cells. The contribution of rENaC to the total EGF-dependent cation current under these conditions is quantitatively less important than that of the nonselective cation channels in these cells.
Leukoregulin upregulation of prostaglandin endoperoxide H synthase-2 expression in human orbital fibroblasts
Authors: Cao H. James , Smith Terry J.
Published: December 2017

Human orbital fibroblasts from patients with severe thyroid-associated ophthalmopathy are particularly susceptible to the actions of a variety of proinflammatory molecules. In this study, we demonstrate that the inductions of prostaglandin endoperoxide H synthase-2 (PGHS-2), interleukin (IL)-1α, and IL-1β by leukoregulin, a product of activated T lymphocytes, are far more robust in orbital fibroblasts than those observed in dermal fibroblasts. These actions of leukoregulin are mediated through an intermediate induction of IL-1α. In contrast, leukoregulin also induces IL-1-receptor antagonist (IL-1ra) expression in orbital fibroblasts, but this induction is considerably greater in dermal fibroblasts (2.3- vs. 8.5-fold). Interrupting the effects of IL-1α, either with a neutralizing antibody or with exogenous IL-1ra, can block the induction of PGHS-2 by leukoregulin. Leukoregulin increases PGHS-2 gene transcription in orbital fibroblasts but exerts the major effect on cyclooxygenase expression by enhancing the stability of mature PGHS-2 mRNA. The cytokine triggers nuclear translocation of nuclear factor-κB (NF-κB) p50/p50 homodimers and p50/p65 heterodimers, and an inhibitor of this transcriptional factor, pyrrolidinedithiocarbamate, can attenuate the PGHS-2 induction. Thus differential inducibility of the members of the IL-1 family of genes in orbital fibroblasts would appear to underlie, at least in part, the differences in PGHS-2 induction observed in orbital and dermal fibroblasts. NF-κB plays an important role in mediating the effects of leukoregulin on PGHS-2 expression.
Circadian regulation of uroguanylin and guanylin in the rat intestine
Authors: Scheving Lawrence A. , Jin Wen-Hui
Published: December 2017

Uroguanylin (UGN) and guanylin (GN) are the endogenous intestinal ligands for guanylyl cyclase C (GC-C). We examined the circadian expression of UGN, GN, and GC-C in the jejunum, ileum, and proximal colon of young adult rats by Northern blot analyses. These assays revealed that UGN is more abundant in the proximal small intestine, whereas GN and GC-C are more abundant in the proximal colon. mRNA levels showed significant circadian variation for UGN (3- to 18-fold peak/trough difference), GN (2.1- to 2.8-fold peak/trough difference), and GC-C (3- to 5-fold peak/trough difference). The maximal abundance occurred in the dark period for all three mRNAs, although peak UGN and GN expression occurred later in the dark period in the jejunum relative to the ileum and colon. Immunoblot analyses using monospecific polyclonal antibodies against UGN and GN prohormones confirmed the regional and circadian variation detected by Northern assays. Thus the expression of these genes is regulated not only by histological position but also by circadian time.
Matrix free Mg2+ and the regulation of mitochondrial volume
Authors: Jung Dennis W. , Brierley Gerald P.
Published: December 2017

Mitochondria must maintain volume homeostasis in order to carry out oxidative phosphorylation. It has been postulated that the concentration of free Mg2+([Mg2+]) serves as the sensor of matrix volume and regulates a K+-extruding K+/H+antiport (K. D. Garlid. J. Biol. Chem. 255: 11273–11279, 1980). To test this hypothesis, the fluorescent probe furaptra was used to monitor [Mg2+] and free Ca2+ concentration ([Ca2+]) in the matrix of isolated beef heart mitochondria, and K+/H+antiport activity was measured by passive swelling in potassium acetate. Concentrations that result in 50% inhibition of maximum activity of 92 μM matrix [Mg2+] and 2.2 μM [Ca2+] were determined for the K+/H+ antiport. Untreated mitochondria average 670 μM matrix [Mg2+], a value that would permit <1% of maximum K+/H+antiport activity. Hypotonic swelling results in large decreases in matrix [Mg2+], but swelling due to accumulation of acetate salts does not alter [Mg2+]. Swelling in phosphate salts decreases matrix [Mg2+], but not to levels that permit appreciable antiport activity. We conclude that 1) it is unlikely that matrix [Mg2+] serves as the mitochondrial volume sensor, 2) if K+/H+antiport functions as a volume control transporter, it is probably regulated by factors other than [Mg2+], and 3) alternative mechanisms for mitochondrial volume control should be considered.
Normalization of muscle plasmid uptake by Southern blot: application to SERCA1 promoter analysis
Authors: Mitchell-Felton Heather , Kandarian Susan C.
Published: December 2017

Direct injection of plasmid DNA into muscle allows the study of promoters in a physiological environment. Because of the variability of reporter gene activity, attempts have been made to normalize activity to muscle plasmid uptake by coinjection of a second “control” plasmid whose reporter gene is constitutively expressed by a viral promoter. The purpose of this study was to evaluate the use of a control plasmid vs. Southern blot to normalize for differences in uptake of plasmids containing promoter fragments of the skeletal muscle-specific sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA1) gene. Results showed that the correlation of luciferase activity from control vs. SERCA1 plasmids is poor and that normalization by a virally driven control plasmid increased variability of SERCA1 luciferase activity. In several cases, the presence of a control plasmid inhibited SERCA1 reporter expression. When Southern blot analysis was used to normalize for differences in plasmid uptake there was less variability than with coinjection, and correlations between plasmid uptake and SERCA1 luciferase activity were better. Moreover, there were no inhibitory effects of a control plasmid allowing for optimization of injection conditions of the SERCA1 deletion constructs. The use of Southern analysis is suggested to determine whether plasmid uptake is differentially affected by physiological stimuli, muscle types, or plasmid sizes under study.
Effects of NO donors and synthase agonists on endothelial cell uptake of l-Arg and superoxide production
Authors: Ogonowski Alison A. , Kaesemeyer Wayne H. , Jin Liming , Ganapathy Vadivel , Leibach Fredrick H. , Caldwell R. William
Published: December 2017

It is commonly believed that the activity of NO synthase (NOS) solely controls NO production from its substrates, l-Arg and O2. The Michaelis-Menten constant ( K m) of NOS forl-Arg is in the micromolar range; cellular levels of l-Arg are much higher. However, evidence strongly suggests that cellular supply of l-Arg may become limiting and lead to reduced NO and increased superoxide anion ([Formula: see text]) formation, promoting cardiovascular dysfunction. Uptake ofl-Arg into cells occurs primarily (∼85%) through the actions of a Na+-independent, carrier-mediated transporter (system y+). We have examined the effects of NOS agonists (substance P, bradykinin, and ACh) and NO donors ( S-nitroso- N-acetyl-penicillamine and dipropylenetriamine NONOate) on transport ofl-Arg into bovine aortic endothelial cells (BAEC). Our results demonstrate that NOS agonists increase y+ transporter activity. A rapidly acting NO donor initially increasesl-Arg uptake; however, after longer exposure, l-Arg uptake is suppressed. Exposure of BAEC withoutl-Arg to substance P and a Ca2+ ionophore (A-23187) increased[Formula: see text] formation, which was blocked with concurrent presence ofl-Arg or the NOS antagonist N ω-nitro-l-arginine methyl ester. We conclude that factors including NO itself control y+ transport function and the production of NO and [Formula: see text].
Characterization of decorin mRNA in pregnant intrauterine tissues of the ewe and regulation by steroids
Authors: Wu Wen Xuan , Zhang Qi , Unno Nobuya , Derks Jan B. , Nathanielsz Peter W.
Published: December 2017

In this study, we characterized the changes in the extracellular matrix proteoglycan decorin in pregnant intrauterine tissues in late gestation and in association with labor and delivery in sheep. In addition, we examined the effects of estradiol and progesterone on regulation of decorin mRNA expression in myometrium from the nonpregnant ovariectomized sheep. Using suppression subtractive hybridization in combination with Northern blot analysis, we identified a significant increase in decorin mRNA in the pregnant sheep myometrium during labor. The abundance of decorin mRNA paralleled myometrial contractility. The increase in decorin mRNA during labor was only demonstrated in the myometrium; no increase was observed in the endometrium or fetal membranes. Estradiol upregulated decorin mRNA and may act as a potential stimulator responsible for the increased decorin in the myometrium during parturition. The ovine decorin cDNA spans 1288 nt, includes 1083 nt of coding sequence predicted to encode a protein of 360 amino acids, 119 nt of 5′-untranslated region (UTR) and 86 nt of 3′-UTR. Over the coding region, the protein shares 79–96% nt sequence identity and 73–94% identity in the deduced amino acid sequence with homologous mammalian sequences. Using cloned decorin cDNA, we observed that the fibroblasts are the predominant cell type in the pregnant sheep myometrium containing decorin mRNA. These data suggest that increased decorin synthesis participates in the matrix changes that may play a role in myometrial activation.
Retardation of cation channel deactivation by mitochondrial dysfunction in adrenal medullary cells
Authors: Inoue M. , Fujishiro N. , Imanaga I.
Published: December 2017

The mechanism for cyanide (CN) activation of a nonselective cation (NS) channel coupled with a muscarinic receptor in a guinea pig chromaffin cell was studied with the perforated-patch method. Bath application of a protein kinase inhibitor resulted in a dose-dependent inhibition of muscarine-induced current ( I M) but had no apparent effect on the CN-induced current ( I CN). On the other hand, production of I CN occluded muscarine activation of NS channels in an amplitude-dependent manner. Deactivation of I M after washout was retarded while I CN was also active, and the extent of the retardation increased with an increase in the relative production of I CN on muscarinic stimulation. Restoration of Na+ pump activity from CN suppression was conspicuously retarded below 19–20°C, and the apparent diminution of I M and I CN after washout was retarded in parallel with a decrease in temperature. The results suggest that CN activation of NS channels is due to suppression of deactivation of the channel.