bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of angiogenic growth factor expression by hypoxia, transition metals, and chelating agents
Authors: Gleadle J. M. , Ebert B. L. , Firth J. D. , Ratcliffe P. J.
Published: December 2017

Recent work has indicated that oxygen-sensing mechanism(s) resembling those controlling erythropoietin production operate in many non-erythropoietin-producing cells. To pursue the implication that such a system might control other genes, we studied oxygen-regulated expression of mRNAs for vascular endothelial growth factor, platelet-derived growth factor (PDGF) A and B chains, placental growth factor (PLGF), and transforming growth factor in four different cell lines and compared the characteristics with those of erythropoietin regulation. Oxygen-regulated expression was demonstrated for each gene in at least one cell type. However, the response to hypoxia (1% oxygen) varied markedly, ranging from a 13-fold increase (PDGF-B in Hep G2 cells) to a 2-fold decrease (PLGF in the trophoblastic line BeWo). For each gene/cell combination, both the magnitude and direction of the response to hypoxia were mimicked by exposure to cobaltous ions or two different iron-chelating agents, desferrioxamine and hydroxypyridinones. These similarities with established characteristics of erythropoietin regulation indicate that a similar mechanism of oxygen sensing is operating on a variety of vascular growth factors, and they suggest that chelatable iron is closely involved in the mechanism.
Evidence for location of the CFTR in human placental apical membrane vesicles
Authors: Faller D. P. , Egan D. A. , Ryan M. P.
Published: December 2017

Ion transport (36Cl uptake) and immunochemical studies were undertaken to detect the cystic fibrosis transmembrane conductance regulator (CFTR) in apical membrane vesicles prepared from human placenta. 36Cl uptake into membrane vesicles was studied in the absence and presence of inwardly directed potassium gradients and valinomycin (Ko = Ki and Ko > Ki, where Ko is potassium concentration outside and Ki is potassium concentration inside the vesicles). The sensitivities of 36Cl uptake to the inhibitors 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), bumetanide, and diphenylamine-2-carboxylate were investigated. Each compound significantly inhibited uptake under both sets of conditions. Additional inhibition of 36Cl uptake was found when the compounds were added together, indicating that they were acting at least partly on different components of the 36Cl uptake. The DIDS- and bumetanide-insensitive component of transport was more selective for Cl than I. These findings suggested that this component may, at least in part, represent Cl transport via CFTR. Addition of adenosine 5'-O-(3-thiotriphosphate) (0.8 mM) led to a decrease in total 36Cl uptake but masked in the overall decrease was an increase in the DIDS- and bumetanide-insensitive component of 36Cl uptake. Western blot analysis of the apical membrane proteins with an antibody specific for a region of human CFTR detected a protein band of approximately 190 kDa. These ion transport and immunochemical studies provide evidence that CFTR is located in human placental apical membrane vesicles.
Regulation of whole cell currents by cytosolic cAMP, Ca2+, and Cl- in rat fetal distal lung epithelium
Authors: Nakahari T. , Marunaka Y.
Published: December 2017

The whole cell patch-clamp technique was used to study ionic conductances in fetal distal lung epithelial (FDLE) cells. In unstimulated FDLE cells, K+ conductances were detected in lowered intracellular Cl- concentration ([Cl-]i, < or = 50 mM). The whole cell currents of FDLE cells were increased by elevation of intracellular Ca2+ concentration ([Ca2+]i) or intracellular adenosine 3',5'-cyclic monophosphate (cAMP) concentration ([cAMP]i). The elevation of [Ca2+]i activated the K+ currents. The amiloride-blockable whole cell currents were activated by [cAMP]i of 1 mM with [Cl-]i of 20 mM and were more frequently detected in the pipette solution without ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) than with it (0.5 mM). When the [Cl-]i was fixed at 50 or 145 mM, however, the increase in these currents was not detected even with cAMP and without EGTA. The amiloride-blockable currents were detected in both the Na+ and K+ pipette solutions. Thus the increase in amiloride-blockable whole cell currents was due to the activation of nonselective cation channels. In FDLE cells treated with terbutaline, which is a beta 2-adrenergic receptor agonist, or forskolin, these currents were detected in the pipette solution containing 20 mM Cl- but were suppressed with time when the pipette solution contained 50 or 145 mM Cl-. It seems likely that maintenance of [Cl-]i at the lowered level is an important requirement for the FDLE cells to activate the amiloride-blockable whole cell currents. It is proposed that cellular mechanisms, such as cell shrinkage, exist to reduce the [Cl-]i in response to cAMP.
Utilization and preferred metabolic pathway of ketone bodies for lipid synthesis by isolated rat hepatoma cells
Authors: Hildebrandt L. A. , Spennetta T. , Elson C. , Shrago E.
Published: December 2017

Morris hepatoma 7777 cells freshly isolated from highly malignant tumors grown in the hindlimb of buffalo rats actively convert ketone bodies to cholesterol and fatty acids. On the basis of results obtained with (-)-hydroxycitrate, an inhibitor of the ATP citrate lyase enzyme, the metabolic pathway for acetoacetate conversion to lipids is exclusively cytoplasmic, whereas that for 3-hydroxybutyrate involves both extra- and intramitochondrial compartments. Subcellular distribution studies indicated accumulation and compartmentation of 3-hydroxybutyryl CoA primarily in the cytoplasm of hepatoma cells incubated with either ketone body. In contrast, the compartmentation of acetoacetyl CoA is dependent on whether the substrate is acetoacetate or 3-hydroxybutyrate. With acetoacetate, the acetoacetyl CoA is entirely cytoplasmic, whereas with 3-hydroxybutyrate, it is equally divided between the intra- and extramitochondrial compartments. The results are discussed in terms of the known and proposed metabolic pathways for lipid synthesis from ketone bodies, particularly that from 3-hydroxybutyrate.
Hepatic Na(+)-K(+)-ATPase enzyme activity correlates with polarized beta-subunit expression
Authors: Simon F. R. , Leffert H. L. , Ellisman M. , Iwahashi M. , Deerinck T. , Fortune J. , Morales D. , Dahl R. , Sutherland E.
Published: December 2017

We have examined underlying causes for observations made in hepatocytes in which catalytic subunits of Na(+)-K(+)-ATPase are found both in bile canalicular (apical) and sinusoidal (basolateral) membrane domains, whereas functional activity is associated preferentially with sinusoidal membrane sites. In a series of parallel studies, we determined by both light and electron microscopy that Na(+)-K(+)-ATPase alpha-subunits were localized to both membrane domains of hepatocytes. With the use of purified liver plasma membrane subfractions, ouabain inhibition curves demonstrated similar inhibition constants (inhibition constant 10(-5) M), and immunoblots using alpha 1-, alpha 2-, and alpha 3-polyclonal and monoclonal antibodies demonstrated antigenic sites predominantly for alpha 1 in both membrane fractions. Also, Northern blot hybridization analysis revealed only the alpha 1-isoform in hepatocytes. In contrast to the bipolar distribution of the alpha 1-subunit, the beta-subunit was identified only at the sinusoidal surface using fluorescence labeling with a monoclonal antibody. The beta 1-isoform was demonstrated by Northern blot analysis and was present predominantly at the sinusoidal domain by immunoblotting with polyclonal antibodies. In addition to the bipolar distribution of alpha 1, immunoblotting of liver plasma membrane subfractions demonstrated a symmetrical distribution of fodrin, ankyrin, actin, and E-cadherin at both domains. These results suggest that functionally competent alpha/beta-complexes form at the sinusoidal domain, whereas only alpha 1-subunits are present at the apical pole.
Abnormal polarization of EGF receptors and autocrine stimulation of cyst epithelial growth in human ADPKD
Authors: Du J. , Wilson P. D.
Published: December 2017

The underlying mechanism of the hyperproliferative response of human autosomal dominant polycystic kidney disease (ADPKD) epithelia was studied. Epidermal growth factor (EGF) protein is highly expressed in ADPKD cyst epithelia in vivo, and primary cultures are hyperesponsive to mitogenic stimulation by EGF in vitro. Doses of > 1 ng/ml EGF were highly mitogenic to ADPKD epithelia. 3H-labeled thymidine proliferation assays showed that cyst fluids and ADPKD epithelial cell-conditioned media also stimulated renal epithelial cell proliferation and contained EGF immunoreactivity (6, 30, and 37 kDa) as detected by Western blots. Radioimmunoassays detected mean levels of 2.87 and 1.4 ng/ml EGF in cyst fluids from early (proliferative) and end-stage ADPKD cysts, respectively. Scatchard analysis of 125I-labeled EGF binding to apical and basolateral membrane showed high-affinity binding to basolateral membranes of normal and ADPKD kidneys but additional unique high-affinity receptor binding to apical membranes of ADPKD but not normal kidneys. Cross-linking analysis and antiphosphotyrosine Western analysis demonstrated functionally active apical EGF receptors at 150-170 kDa. These results suggest mediation of cyst expansion via an autocrine loop involving EGF synthesis and processing by cyst epithelial cells, apical secretion into cyst lumens, and subsequent binding to and phosphorylation of apical membrane EGF receptors. These findings are consistent with a membrane protein polarization defect in ADPKD cyst epithelia.
Adenosine enhances nitric oxide production by vascular endothelial cells
Authors: Li J. M. , Fenton R. A. , Cutler B. S. , Dobson J. G.
Published: December 2017

Adenosine per se is a potent vasodilator of vascular smooth muscle. Endothelial cells modulate vascular tone via the release of nitric oxide (NO), which also elicits vasodilation. This study was undertaken to determine whether adenosine could directly stimulate endothelial cells to enhance NO production, which could subsequently reduce vascular tone. NO production was evaluated in porcine carotid artery endothelial cells (PCAEC) and human saphenous vein endothelial cells (HSVEC) seeded on multiwell plates, grown to confluence, and treated with adenosine for 1 h. The bathing medium was collected, and the NO production was determined as reflected by the formation of NO2- and NO3-. NO production by PCAEC was significantly increased by adenosine in a dose-dependent manner, whereas there was only an insignificant tendency for an increase by HSVEC. The addition of the NO synthase competitive inhibitor, NG-monomethyl-L-arginine (NMMA), or the adenosine receptor antagonist, theophylline, prevented the increase in NO production by adenosine. The results suggest that adenosine stimulates, by a receptor-mediated mechanism, the production of NO by arterial, but not by venous, endothelial cells.
IP3-activated Ca2+ channels in the plasma membrane of cultured vascular endothelial cells
Authors: Vaca L. , Kunze D. L.
Published: December 2017

Although it is clear that D-myo-inositol 1,4,5-trisphosphate (IP3) plays an important role in the activation of Ca2+ influx, the mechanisms by which this occurs remain controversial. In an attempt to determine the role of IP3 in the activation of Ca2+ influx, patch-clamp single-channel experiments in the cell-attached, inside-out, and outside-out configurations were performed on cultured bovine aortic endothelial cells (BAEC). The results presented indicate that both IP3 and intracellular Ca2+ can modulate the activity of a Ca(2+)-selective channel found in the plasma membrane of these cells. Addition of 10 microM IP3 increased channel open probability (P(o)) from a control value of 0.12 +/- 0.05 to 0.7 +/- 0.13 at a constant intracellular Ca2+ of 1 nM in excised inside-out patches. D-Myo-inositol 1,3,4,5-tetrakisphosphate at 50 microM was ineffective in altering channel P(o). Channel activity declined after approximately 2 min in the continuous presence of IP3. Three to four minutes after addition of IP3, channel P(o) was reduced from 0.7 +/- 0.2 to 0.2 +/- 0.1, indicating that an additional regulator might be required to maintain channel activity in excised patches. The channel was reversibly blocked by application of 1 microgram/ml heparin to the intracellular side of inside-out patches. This Ca(2+)-selective channel is indistinguishable from the depletion-activated Ca2+ channel we have previously described in BAEC.
Protein kinase C in cyclic stretch-induced nerve growth factor production by urinary tract smooth muscle cells
Authors: Persson K. , Sando J. J. , Tuttle J. B. , Steers W. D.
Published: December 2017

Cyclic stretch of cultured urinary tract smooth muscle cells has been used to mimic some of the events that occur with bladder obstruction. The stretch stimulus induces production of nerve growth factor (NGF), which has been implicated in changes in bladder innervation. Stretch-induced NGF production was blocked by actinomycin. Involvement of protein kinase C (PKC) in the stretch-induced NGF production is strongly suggested by the following observations. Phorbol ester activators of PKC mimicked the stretch response as did platelet-derived growth factor (PDGF), which acts, in part, through generation of endogenous diacylglycerols. Both stretch- and PDGF-induced NGF production were blocked by prolonged incubation with phorbol ester to downregulate PKC. Western blot analysis confirmed partial downregulation of the Ca(2+)-dependent PKC-alpha and PKC-beta 1 and near complete downregulation of the Ca(2+)-independent PKC isozymes delta, epsilon, and zeta. The involvement of PKC in transducing a physical stimulus (stretch) into a biochemical response (NGF production) has implications for novel types of therapeutic intervention in ailments such as bladder obstruction.
Role of cytochrome P-450 epoxygenase metabolites in EGF signaling in renal proximal tubule
Authors: Burns K. D. , Capdevila J. , Wei S. , Breyer M. D. , Homma T. , Harris R. C.
Published: December 2017

Epidermal growth factor (EGF) is a potent epithelial cell mitogen and induces eicosanoid production in many cell types. The present study examined signaling mechanisms for EGF in the renal proximal tubule, where high concentrations of cytochrome P-450 epoxygenase have been reported. In primary cultures of rabbit proximal tubule cells, EGF (30 nM) increased endogenous epoxyeicosatrienoic acid (EET) levels 5.3 +/- 1.4-fold within 10 min (n = 6). In these cells EGF-stimulated [3H]thymidine incorporation was significantly inhibited by the cytochrome P-450 inhibitors ketoconazole or clotrimazole but not by the cyclooxygenase inhibitor indomethacin. In fura 2-loaded proximal tubule cells, EGF caused a concentration-dependent increase in cytosolic Ca2+ concentration ([Ca2+]i), due to Ca2+ influx, which was inhibited by either ketoconazole or SKF-525A but not by indomethacin. Addition of 5,6-EET (0.5 microM) also induced Ca2+ influx in proximal tubule cells, whereas 8,9-11,12-, or 14,15-EET did not. In cells treated with bis(2-amino-5-methylphenoxy)ethane N,N,N',N'-tetraacetic acid tetraacetoxy-methyl ester to chelate [Ca2+]i, EGF-stimulated [3H]thymidine incorporation. These results indicate that EGF increases EET levels in proximal tubule and suggest that 5,6-EET or its metabolites may be a modulator of EGF-induced [Ca2+]i increases and involved in mitogenesis.
Myocardial adaptation to ischemia by oxidative stress induced by endotoxin
Authors: Maulik N. , Watanabe M. , Engelman D. , Engelman R. M. , Kagan V. E. , Kisin E. , Tyurin V. , Cordis G. A. , Das D. K.
Published: December 2017

In this study, we examined the effects of oxidative stress adaptation on myocardial ischemic reperfusion injury. Oxidative stress was induced by injecting endotoxin (0.5 mg/kg) into the rat. After 24 h, rats were killed, hearts were isolated, and the effects of ischemia-reperfusion were studied using an isolated working heart preparation. The development of oxidative stress was examined by assessing malonaldehyde production in the heart. The antioxidant defense system was studied by estimating antioxidant enzyme activities and ascorbate- as well as thiol-dependent antioxidant reserve. The results of our study indicated that endotoxin induced oxidative stress within 1 h of treatment; the stress was reduced progressively and steadily up to 24 h. The antioxidant enzymes superoxide dismutase, catalase, glutathione (GSH) peroxidase, and GSH reductase were lowered up to 2 h and then increased. Both thiol- and ascorbate-dependent antioxidant reserve were enhanced, but the enhancement of the former was only transitory. After 24 h, endotoxin provided adequate protection to the heart from the ischemic-reperfusion injury, as evidenced by improved left ventricular function and aortic flow. Our results suggest that the induction of oxidative stress by endotoxin-induced adaptive modification of the antioxidant defense in the heart, thereby reducing ischemic-reperfusion injury.
Expression of sarcoplasmic reticulum Ca2+ transport proteins in cold-acclimating ducklings
Authors: Dumonteil E. , Barre H. , Meissner G.
Published: December 2017

The relationship between the cold-induced increase in sarcoplasmic reticulum (SR) Ca2+ transport proteins and the development of muscular nonshivering theermogenesis (NST) was investigated by determining the time course of expression of the sarcoplasmic or endoplasmic reticulum Ca(2+)-ATPase (SERCA), Ca2+ release channel, and calsequestrin in control and cold-acclimating ducklings. 45Ca2+ uptake and [3H]ryanodine binding measurements with skeletal muscle homogenates showed that a cold acclimation period of approximately 4 wk was required to observe a substantial increase in Ca(2+)-ATPase activity and Ca2+ release channel content, which correlates well with NST development Immunoblot analysis of muscle homogenates showed no differences in calsequestrin levels but revealed that the decrease in SERCA2a content was delayed in cold-acclimating birds and that the SERCA1 level was increased after 4 wk of cold acclimation. The persistence of SERCA2a may be related to shivering thermogenesis occurring preferentially in slow-twitch fibers. SERCA1 may account for most of the cold-induced increase in 45Ca2+ uptake activity, suggesting the preferential occurrence of a Ca(2+)-dependent NST in fast-twitch fibers.
Smooth muscle contractility is modulated by myosin tail-S2-LMM hinge region interaction
Authors: Cai S. , Ferguson D. G. , Martin A. F. , Paul R. J.
Published: December 2017

The functional significance of two major smooth muscle myosin isoforms, which differ in the nonenzymic COOH-terminal tail region, is not known. We report here that a 13-amino acid peptide, which mimics a region of the tail unique to the SM1 myosin isoform, inhibits contraction velocity in permeabilized smooth muscle. This peptide is shown to bind to the S2-light meromyosin (LMM) hinge region of myosin using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, photoaffinity labeling, and immunoelectron microscopy. Our results suggest that novel intermolecular contacts between the tail and S2-LMM hinge regions of adjacent myosin molecules in the thick filament may modulate contractility and provide a basis for distinct isoform function.
Endothall thioanhydride inhibits protein phosphatases-1 and -2A in vivo
Authors: Erdodi F. , Toth B. , Hirano K. , Hirano M. , Hartshorne D. J. , Gergely P.
Published: December 2017

The objective of this study was to relate the toxicity of several cantharidin-derivative pesticides with their abilities to inhibit protein phosphatases-1 (PP1) and -2A (PP2A). Cantharidin (CA), endothall, and endothall thioanhydride (ETA) inhibited the activity of PP1 and PP2A, and the potency sequence was CA > endothall > ETA in vitro. We determined the inhibitory potency of these pesticides on hepatic protein phosphatases by administration of the toxins into the portal vein of rats. The potency sequence of ETA > CA > endothall was established for the inhibition of PP1 and PP2A in vivo and shows close correlation with the sequence of relative toxicity. ETA predominantly targets PP1 for inhibition in liver, as revealed by assays specific for PP1 or PP2A. Studies using 3T3 fibroblasts showed that only ETA, but not CA or endothall, induced marked morphological changes. These effects included cell rounding and detachment as well as extensive reorganization of actin filaments and are characteristic for the cell-permeable phosphatase-inhibitory toxins. It is suggested that the in vivo effectiveness is related to enhanced uptake of ETA, because this is permeable across the plasmalemma.
Integrative effects of EGF on metabolism and proliferation in renal proximal tubular cells
Authors: Nowak G. , Schnellmann R. G.
Published: December 2017

This study examined the relationship between alterations in cellular metabolism and induction of proliferation in renal proximal tubular cells (RPTC) after epidermal growth factor (EGF) exposure. EGF treatment (10 ng/ml) of confluent RPTC cultures for 6 consecutive days increased monolayer DNA content 3.3-fold. EGF-stimulated proliferation of RPTC was preceded by a rapid (within 4 h) induction of glycolysis and a decrease in basal and ouabain-sensitive oxygen consumption (20 and 30%, respectively). EGF stimulated the pentose cycle by 58% and decreased gluconeogenesis by 48%. Supplementation of the culture medium with ribose-5-phosphate or ribose abolished the stimulation of glycolysis and the pentose cycle by EGF but had no effect on proliferation. These results show that EGF rapidly stimulates the pentose cycle, shifts glucose metabolism from gluconeogenesis to glycolysis, and decreases oxygen consumption before any increase in proliferation. The lack of an EGF effect on the pentose cycle and glycolysis in the presence of exogenous precursors for DNA synthesis suggests that the stimulation of these pathways before proliferation is due to increased demands for ribose for subsequent nucleic acid synthesis.
Regulation of endothelial cell nitric oxide synthase mRNA expression by shear stress
Authors: Uematsu M. , Ohara Y. , Navas J. P. , Nishida K. , Murphy T. J. , Alexander R. W. , Nerem R. M. , Harrison D. G.
Published: December 2017

Shear stress enhances expression of Ca(2+)-calmodulin-sensitive endothelial cell nitric oxide synthase (ecNOS) mRNA and protein in bovine aortic endothelial cells (BAEC). The present studies were performed to investigate mechanisms responsible for regulation of ecNOS mRNA expression by shear stress and to determine if this induction of ecNOS mRNA is accompanied by an enhanced nitric oxide (NO) production. Shear stresses of 15 dyn/cm2 for 3-24 h resulted in a two- to threefold increase of ecNOS mRNA content quantified by Northern analysis in BAEC. Shear stresses (1.2-15 dyn/cm2) for 3 h resulted in an induction of ecNOS mRNA in a dose-dependent manner. In human aortic endothelial cells, shear stresses of 15 dyn/cm2 for 3 h also resulted in ecNOS mRNA induction. In BAEC, this induction in ecNOS mRNA was prevented by coincubation with actinomycin D (10 micrograms/ml). The K+ channel antagonist tetraethylammonium chloride (3 mM) prevented increase in ecNOS mRNA in response to shear stress. The ecNOS promotor contains putative binding domains for AP-1 complexes, potentially responsive to activation of protein kinase C (PKC). However, selective PKC inhibitor calphostin C (100 nM) did not inhibit ecNOS induction by shear stress. Finally, production of nitrogen oxides under both basal conditions and in response to the calcium ionophore A-23187 (1 microM) by BAEC exposed to shear stress was increased approximately twofold compared with cells not exposed to shear stress. These data suggest that ecNOS mRNA expression is regulated by K+ channel opening, but not by activation of PKC, and that shear not only enhances ecNOS mRNA expression but increases capacity of endothelial cells to release NO.
Water transport across mammalian cell membranes
Authors: Verkman A. S. , van Hoek A. N. , Ma T. , Frigeri A. , Skach W. R. , Mitra A. , Tamarappoo B. K. , Farinas J.
Published: December 2017

This review summarizes recent progress in water-transporting mechanisms across cell membranes. Modern biophysical concepts of water transport and new measurement strategies are evaluated. A family of water-transporting proteins (water channels, aquaporins) has been identified, consisting of small hydrophobic proteins expressed widely in epithelial and nonepithelial tissues. The functional properties, genetics, and cellular distributions of these proteins are summarized. The majority of molecular-level information about water-transporting mechanisms comes from studies on CHIP28, a 28-kDa glycoprotein that forms tetramers in membranes; each monomer contains six putative helical domains surrounding a central aqueous pathway and functions independently as a water-selective channel. Only mutations in the vasopressin-sensitive water channel have been shown to cause human disease (non-X-linked congenital nephrogenic diabetes insipidus); the physiological significance of other water channels remains unproven. One mercurial-insensitive water channel has been identified, which has the unique feature of multiple overlapping transcriptional units. Systems for expression of water channel proteins are described, including Xenopus oocytes, mammalian and insect cells, and bacteria. Further work should be directed at elucidation of the role of water channels in normal physiology and disease, molecular analysis of regulatory mechanisms, and water channel structure determination at atomic resolution.
Brefeldin A inhibition of apical Na+ channels in epithelia
Authors: Fisher R. S. , Grillo F. G. , Sariban-Sohraby S.
Published: December 2017

Brefeldin A (BFA) is used to probe trafficking of proteins through the central vacuolar system (CVS) in a variety of cells. Transepithelial Na+ transport by high-resistance epithelia, such as A6 cultured cells, is inhibited by BFA. Apical Na+ channels, as well as basolateral pumps and K+ channels, are complex proteins that probably traverse the CVS for routing to the plasma membrane. BFA (5 micrograms/ml) decreases transepithelial Na+ current near zero and increases resistance reversibly after 4 h. Longer exposures are toxic. When tissues were treated for 20 h with 0.2 microgram/ml BFA, Na+ transport also was reversibly inhibited. Using noise analysis, we found that BFA drastically reduced apical Na+ channel density. The increase in single channel current was consistent with cell hyperpolarization. After apical permeabilization with nystatin, changes in transepithelial current reflect changes in basolateral membrane transport. Transport at this membrane was inhibited by ouabain and cycloheximide, but not by BFA. After BFA, aldosterone was ineffective, suggesting that an intact CVS is required for stimulation by this hormone. Thus BFA inhibition of Na+ transport is localized at the apical membrane. Implications for channel turnover as a mechanism for regulating the Na+ transport rate are discussed.
Adenosine inhibits cytokine release and expression of adhesion molecules by activated human endothelial cells
Authors: Bouma M. G. , van den Wildenberg F. A. , Buurman W. A.
Published: December 2017

Ischemia induces excessive ATP catabolism with subsequent local release of its metabolite adenosine, an autacoid with anti-inflammatory properties. Because activation of the vascular endothelium is critical to the inflammatory host response during ischemia and reperfusion, the effects of adenosine on two major determinants of endothelial cell activation (i.e., the release of proinflammatory cytokines and the expression of adhesion molecules) were studied. Adenosine dose dependently inhibited the release of interleukin (IL)-6 and IL-8 by stimulated human umbilical vein endothelial cells (HUVEC). Expression of E-selectin and vascular cell adhesion molecule 1 (VCAM-1), but not intercellular adhesion molecule 1 (ICAM-1), by activated HUVEC was also reduced by adenosine. Inhibition of endogenous adenosine deaminase activity by erythro-9-(2-hydroxy-3-nonyl)adenine or 2'-deoxycoformycin strongly enhanced the inhibitory effects of exogenous adenosine on cytokine release and expression of E-selectin and VCAM-1. However, a clear role for specific adenosine receptors in the described inhibitory events could not be established. Together, these data imply that the vascular endothelium constitutes an important target for the anti-inflammatory actions of adenosine.
Cellular and molecular physiology of volume-sensitive anion channels
Authors: Strange K. , Emma F. , Jackson P. S.
Published: December 2017

Maintenance of a constant cell volume in the face of osmotic stress is an evolutionarily ancient homeostatic process. Over the last two decades physiologists have gained an impressive understanding of the "volume-sensitive" channels, cotransporters, exchangers, metabolic pathways, and genes that are responsible for modulating intracellular solute content and cell volume. This review focuses on one part of this story, the characteristics and osmoregulatory functions of volume-sensitive anion channels. Three distinct types of swelling-activated anion channels have been observed and studied extensively in animal cells. These channels include 1) ClC-2, which is a member of the ClC family of voltage-gated anion channels, 2) an outwardly rectifying intermediate conductance channel, and 3) a large-conductance or "maxi" channel. In addition to these three channels, several other less well-characterized anion channels have been observed. This review discusses the electrophysiological and molecular biological characteristics and regulation of these channels. The possible roles different types of anion channels might play in cell volume homeostasis are also discussed.
Modulation of cell membrane potential in cultured vascular endothelium
Authors: Vaca L. , Licea A. , Possani L. D.
Published: December 2017

The present study explores the role of different ionic conductances in the regulation of membrane potential under resting conditions and after bradykinin (BK) or thapsigargin (TG) stimulation of cultured bovine aortic endothelial cells. Under resting conditions, the cell membrane potential observed was -62+/- 5 mV. The main conductance under these conditions is an inwardly rectifying potassium (IRK) channel. Application of 50 nM BK induced a transient hyperpolarization to -87 +/- 4 mV followed by sustained depolarization to -35 +/- 5 mV. The transient hyperpolarization was eliminated by 1 microM noxiustoxin, a blocker of calcium-activated postassium channels (K(Ca)). the sustained depolarization induced by BK was prevented by incubating the cells with the calcium channel blocker lanthanum. TG evoked a similar response in membrane potential, with the exception that the onset of the hyperpolarization was slower compared with BK. The results presented here indicate that the cell resting potential is maintained at -62 +/- 2 mV by the IRK channel. BK or TG stimulation induces a transient hyperpolarization of approximately -20 mV produced by activation of a KCa. This hyperpolarization is followed by a sustained depolarization produced by activation of a calcium-selective channel sensitive to lanthanum.
Phospholipase activation and secretion: evidence that PLA2, PLC, and PLD are not essential to exocytosis
Authors: Coorssen J. R.
Published: December 2017

Numerous studies have identified phospholipase metabolites as membrane fusogens, and phospholipase D (PLD) (J.R. Coorssen and R.J. Haslam. FEBS Lett. 316: 170-174, 1993), C (PLC), and A2 (PLA2) activities correlate with secretion. Do these enzymes have essential or modulatory roles? This study confirms that secretion does not require Ca2+ or PLC (Coorssen et al. Cell Regul. 1: 1027-1041, 1990). Arachidonic acid (AA), phosphatidic acid (PA) and analogues, exogenous metabolites of PLA2 and PLD, were tested in electropermeabilized human platelets. AA potentiated guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S)-induced secretion, and eicosanoids were not essential. Endogenous [3H]AA formation correlated with GTP gamma S-induced secretion, and phorbol 12-myristate 13-acetate (PMA) promoted these effects. Inhibitors were used to probe phospholipase influences on secretion. Only PLD inhibitors blocked secretion. However, PMA blocked inhibition of protein kinase C (PKC) and secretion by quercetin, suggesting that PA formed by PLD supports PKC activation and GTP gamma S-induced secretion. Thus PA analogues had no effect alone but enhanced GTP gamma S-induced PKC activity and secretion. Slower PLD activation compared with secretion also indicates a nonessential role. This is the first report of a Ca(2+)-independent PLA2 activity in human platelets, use of quercetin as a PLD inhibitor, and dissociation of PLA2, PLC, and PLD activities from secretion. No major phospholipase activities are essential to the final steps in exocytosis, but modulatory roles are indicated.
Subcellular distribution of sodium pump isoform subunits in mammalian cardiac myocytes
Authors: McDonough A. A. , Zhang Y. , Shin V. , Frank J. S.
Published: December 2017

The cardiac Na+ pump (Na+ -K+ -ATPase) provides the driving force for the Na+/Ca2+ exchanger, a determinant of intracellular Ca2+ stores. Three Na+ pump alpha-catalytic subunit isoforms are expressed in human heart, alpha1 and alpha2 are expressed in rat heart, and only alpha1 is expressed in guinea pig heart. The objective of this study was to determine whether there are isoform-specific patterns of expression in the transverse tubules (T tubules) vs. the peripheral sarcolemma. In adult rat cardiomyocytes, anti-alpha1-specific antibodies labeled the T tubules more intensely than the peripheral sarcolemma, in which labeling was patchy, the same pattern reported for distribution of the Na+/Ca2+ exchanger (J. S. Frank, G. Mottino, D. Reid, R. S. Molday, and K. D. Philipson, J. Cell Biol. 117: 337-345, 1992), whereas anti-alpha2- and anti-beta1-antibodies uniformly labeled T tubules and peripheral sarcolemma. In guinea pig cardiomyocytes, an anti-alpha-antibody against an extracellular epitope evenly labeled the peripheral sarcolemma and T tubules, and immunogold labeling demonstrated coincidence of alpha-subunits and intramembranous particles in sarcolemma. In summary, Na+ pumps are located in both peripheral sarcolemma and T tubules of cardiomyocytes expressing either multiple or single Na+ pump isoforms.
Osmotic regulation of Na-myo-inositol cotransporter mRNA level and activity in endothelial and neural cells
Authors: Wiese T. J. , Dunlap J. A. , Conner C. E. , Grzybowski J. A. , Lowe W. L. , Yorek M. A.
Published: December 2017

Myo-inositol (MI) is an important factor in the synthesis of phosphoinositides, and as an osmolyte, MI contributes to the regulation of cell volume. In cells of renal origin, hypertonicity causes an increase in sodium-dependent MI transporter (SMIT) mRNA levels and MI transport. However, it is unknown whether changes in osmolarity regulate transport of MI in neural or endxsothelial cells. IN these studies, neural and endothelial cells were exposed to hyperosmotic medium for up to 48 h, and the effect on MI transport was determined. Transport of MI was maximally increased by exposing the cells to hyperosmotic medium for 24 h. Kinetic analysis of high-affinity MI transport demonstrated an increase in the apparent maximal velocity with no significant change in the apparent Km. The hyperosmotic induction of MI transport was blocked by the addition of cycloheximide, indicating a requirement for protein synthesis, and was associated with increased levels of SMIT mRNA. In contrast to the effect of hypertonicity, exposure of neural and endothelial cells to hypotonic conditions caused a decrease in SMIT mRNA levels and MI transport in endothelial cells. These studies demonstrate that, in extrarenal cell types, changes in osmolarity also regulate SMIT activity and mRNA levels.
Mechanical stimulation of organogenic cardiomyocyte growth in vitro
Authors: Vandenburgh H. H. , Solerssi R. , Shansky J. , Adams J. W. , Henderson S. A.
Published: December 2017

Adherent cultures of neonatal rat cardiomyocytes were subjected to progressive, unidirectional lengthening for 2-4 days in serum-containing medium. This mechanical stretch (25% increase in initial length each day) simulates the eccentric mechanical load placed on in vivo heart cells by increases in postnatal blood pressure and volume. The in vitro mechanical stimuli initiated a number of morphological alterations in the confluent cardiomyocyte population which were similar to those occurring during in vivo heart growth. These include cardiomyocyte organization into parallel arrays of rod-shaped cells, increased cardiomyocyte binucleation, and cardiomyocyte hypertrophy by longitudinal cell growth. Stretch stimulated DNA synthesis in the noncardiomyocyte population but not in the cardiomyocytes. Myosin heavy chain (MHC) content increased 62% over 4 days of stretch and included increased accumulation of both fetal beta-MHC and adult alpha-MHC isoforms. This new model of stretch-induced cardiomyocyte hypertrophy may assist in examining some of the complex mechanogenic growth processes that occur in the rapidly enlarging neonatal heart.
cGMP produced in response to ANP and CNP regulates proliferation and differentiation of osteoblastic cells
Authors: Hagiwara H. , Inoue A. , Yamaguchi A. , Yokose S. , Furuya M. , Tanaka S. , Hirose S.
Published: December 2017

The effects of natriuretic peptides on the proliferation and differentiation of osteoblast-like cells from rat calvariae were examined. Natriuretic peptides are physiological agonists that activate receptor guanylate cyclases, namely, natriuretic peptide receptor (NPR)-A and NPR-B. Exposure of cells to atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP) resulted in large increases in the rate of intracellular production of guanosine 3',5'-cyclic monophosphate (cGMP). Moreover, CNP-like immunoreactivity was detected in the conditioned medium from osteoblast-like cells, while ANP was undetectable. In cells exposed to natriuretic peptides, a dose-dependent reduction in the rate of DNA synthesis was observed. Natriuretic peptides also stimulated the activity of alkaline phosphatase (ALPase) and the expression of mRNA for ALPase and osteocalcin and the mineralization of nodules by the cultured cells. These results could be reproduced by treating cells with 8-bromo-cGMP. Endothelin-1, whose physiological functions are the opposite of those of natriuretic peptides, decreased the ALPase activity and the mineralization of nodules. In the present study, natriuretic peptides were demonstrated to promote bone formation via the action of cGMP in a signal-transduction pathway mediated by specific receptors in osteoblast-like cells.
Suppression of TCA cycle activity in the cardiac muscle cell by hydroperoxide-induced oxidant stress
Authors: Janero D. R. , Hreniuk D.
Published: December 2017

Excess H2O2 contributes to myocardial reperfusion injury. We detail the effect of H2O2-induced oxidant stress on the tricarboxylic acid (TCA) cycle in isolated heart muscle cells. Cardiomyocyte exposure to bolus H2O2 ( > 50 microM) acutely suppressed TCA cycle activity. Loss of cardiomyocyte TCA cycle function on cellular H2O2 exposure was supported by the rapid in situ inactivation of aconitase along with cardiomyocyte membrane peroxidation. Without peroxidation, the loss of aconitase catalysis was itself sufficient to jeopardize TCA cycle activity. Only H2O2 dismutation completely preserved both cardiomyocyte aconitase activity and TCA cycle flux during H2O2 overload. Restoration of aconitase catalysis after alleviation of the oxidant insult was prohibited by cell-permeable metal chelators, and TCA cycle flux could not be reestablished in peroxidized cells, even if aconitase activity had recovered. The characteristics of aconitase inactivation-reactivation observed are consistent with adverse redox changes to the enzyme's (Fe-S) cluster. These data demonstrate that specific aspects of the TCA cycle in heart muscle are sensitive to H2O2-induced oxidative stress and identify a peroxidative component of the injury process.
cAMP- and swelling-activated chloride conductance in rat hepatocytes
Authors: Meng X. J. , Weinman S. A.
Published: December 2017

An outwardly rectifying Cl- conductance was identified in primary isolated rat hepatocytes, and the whole cell patch-clamp technique was used to characterize its properties and mechanisms of activation. With symmetrical Cl(-)-containing solutions on both sides and adenosine 3',5'-cyclic monophosphate (cAMP; 100 microM) in the pipette solution, a large outwardly rectifying conductance (1,014 +/- 153 pS/pF, n = 20) developed in all cells within 3 min. This cAMP-activated conductance was highly anion selective and slowly inactivated at voltages > 80 mV. It was completely inhibited by the anion channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (200 microM, n = 6) and partially inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (150 microM, n = 7). It displayed a halide selectivity of I- > Br- > Cl-. In the absence of cAMP, a functionally similar conductance was activated by cell swelling. Reduction of bath osmolality from 300 to 250 mosmol/kg increased membrane conductance from 64 +/- 16.4 to 487 +/- 23 pS/pF (n = 4). This swelling-activated conductance was also highly anion selective and had identical halide selectivity and blocker sensitivity as the cAMP-activated conductance. Although cell swelling was not necessary for cAMP activation, cell shrinkage with hyperosmotic bath (350 mosmol/kg), either before or after exposure to cAMP, inhibited the cAMP-activated conductance. By the determination of conductance as a function of bath osmolality in the presence and absence of cAMP, it was observed that cAMP shifted the osmotic set point for conductance activation without changing either the maximum or minimum conductance. In conclusion, both cAMP and cell swelling activate a large outwardly rectifying Cl- conductance in rat hepatocytes. Its ionic selectivity and sensitivity to channel blockers are identical to those seen for swelling-activated Cl- conductances in many cell types. The conductive properties are not those of cystic fibrosis transmembrane conductance regulator-mediated Cl- conductance. cAMP appears to activate this conductance by altering the volume set point of a swelling-activated channel.
Impaired cAMP-mediated gene expression and decreased cAMP response element binding protein in senescent cells
Authors: Chin J. H. , Okazaki M. , Frazier J. S. , Hu Z. W. , Hoffman B. B.
Published: December 2017

The capacity of various growth factors to induce c-fos expression is diminished with senescence. Because adenosine 3',5'-cyclic monophosphate (cAMP)-mediated responses are also blunted with aging, we wondered whether cAMP-induced c-fos gene expression might be impaired with senescence. Using IMR fibroblasts, we found that prostaglandin E1 (PGE1) and forskolin, stimulators of cAMP accumulation in young and senescent cells, increased abundance of c-fos and junB mRNA more in young than senescent cells. The abundance of the cAMP response element binding protein (CREB), a transcription factor which enhances gene expression when phosphorylated by protein kinase A, was markedly decreased in both whole cell and nuclear extracts of senescent cells, in both Western blotting and in gel retardation assays. Also, PGE1-induced phosphorylation of CREB by protein kinase A was markedly attenuated in senescent cells. There is a marked decrement in expression of CREB with senescence, and the results suggest the possibility that the diminished expression of CREB may contribute to altered cAMP-mediated regulation of gene expression with senescence.
Flickery block of single CFTR chloride channels by intracellular anions and osmolytes
Authors: Linsdell P. , Hanrahan J. W.
Published: December 2017

Cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation- and nucleotide-dependent chloride channel. Single CFTR currents recorded on cell show slight outward rectification, which has previously been suggested to be due to an asymmetrical chloride ion gradient or to a specific interaction between permeant intracellular anions and the channel. Using a single-channel recording from Chinese hamster ovary cells stably expressing CFTR, we have found that both the sparingly permeant anion glutamate and the impermeant anion gluconate cause a rapid, voltage-dependent block of CFTR channels when applied to the intracellular, but not the extracellular, face of excised patches. Both the affinity and the voltage dependence of block were affected by the extracellular chloride concentration in a manner consistent with chloride ions being able to repel these blocking ions from the pore. These results are discussed in terms of previous models of CFTR current outward rectification, and it is suggested that this rectification may result from a combination of asymmetrical chloride concentrations and voltage-dependent block of the channel by large cytoplasmic anions. In addition, we find that CFTR conductance is decreased by high concentrations of intracellular sucrose, sorbitol, and urea in a manner consistent with a rapid block of the channel by these molecules.
Isometric force and maximal shortening velocity of single muscle fibers from elite master runners
Authors: Widrick J. J. , Trappe S. W. , Blaser C. A. , Costill D. L. , Fitts R. H.
Published: December 2017

Single chemically permeabilized gastrocnemius fibers from six elite endurance-trained master runners (RUN group) and five age-matched sedentary controls (SED group) were mounted between a force transducer and a position motor, studied under conditions of maximal and submaximal Ca2+ activation, and subsequently electrophoresed on 5% polyacrylamide gels to determine myosin heavy chain (MHC) composition. For the SED group, peak isometric tension (Pzero) averaged 143 +/- 3, 156 +/- 4, and 170 +/- 4 kN/m2 and maximal shortening velocity (Vzero) averaged 0.43 +/- 0.01, 1.90 +/- 0.08, and 5.59 +/- 0.40 fiber lengths/s for fibers expressing type I, IIa, and IIx MHC, respectively (all comparisons, P < 0.05). Hill plot analysis of relative forces during submaximal Ca2+ activation indicated no SED vs. RUN differences in Ca2+ sensitivity or in the cooperativity of Ca2+ activation. However, at maximal Ca2+ activation, RUN type I and IIa fibers produced 15% less peak absolute force than SED fibers (P < 0.05). This reduction in fiber force was a direct result of the smaller diameter of the RUN fibers (P < 0.05), because Pzero, peak elastic modulus (Ezero), and Pzero/Ezero were not different between SED and RUN groups. RUN type I fibers also displayed a mean Vzero that was 19% higher than the average Vzero of the SED type I fibers (P < 0.05). In separate experiments, quantification of relative myosin light chain (MLC) isoform content revealed a 28% greater ratio of MLC3 to MLC2 in single type I fibers from the RUN group (P < 0.05), suggesting that the elevated Vzero of the RUN type I fibers was related to a greater expression of MLC3. In conclusion, the single fibers from the elite master runners displayed specific morphological and contractile properties that may enhance the performance of these athletes during prolonged muscular activity.