bluuebunny/crossref_metadata_2025 huggingface dataset:
Cyclic ADP-ribose and related compounds activate sheep skeletal sarcoplasmic reticulum Ca2+ release channel
Authors: Sitsapesan R. , Williams A. J.
Published: December 2017

It has been suggested that adenosine 5'-cyclic-diphosphoribose (cADPR) can activate only nonskeletal isoforms of the ryanodine-sensitive Ca2+ release channel. We now demonstrate that cADPR is an effective activator of sheep skeletal sarcoplasmic reticulum (SR) Ca2+ release channels incorporated into planar phospholipid bilayers in the presence of activating levels of cytosolic Ca2+. In addition, the precursor of cADPR, beta-NAD+, and the metabolite, adenosine diphosphoribose (ADP-ribose), also increase the open probability (Po) of skeletal SR Ca2+ release channels in micromolar concentrations. At low concentrations of cADPR (1 microM), the mechanism for the increase in Po is an increase in the frequency of channel openings with no increase in the duration of the open events. We also show that the effect of cADPR is dependent on luminal [Ca2+]. cADPR has no effect on Po when the luminal [Ca2+] is < 40 microM. However, at millimolar concentrations of luminal Ca2+, cADPR 1 and 10 microM) increases Po in the presence of activating cytosolic Ca2+.
Stimulation of Na(+)-K(+)-ATPase by thyrotropin in cultured thyroid follicular cells
Authors: Pressley T. A. , Higham S. C. , Joson L. A. , Mercer D. W.
Published: December 2017

Thyroid-stimulating hormone (TSH; thyrotropin) produces a pleiotropic response in the thyroid gland, accelerating nearly every aspect of metabolic turnover within the follicular epithelia. We examined the effects of TSH on expression of Na(+)-K(+)-ATPase in FRTL-5 cells, a cell line derived from rat thyroid. TSH (10 mU/ml) produced a nearly twofold increase in abundance of the mRNA encoding the catalytic alpha 1-subunit within 6 h of treatment. With the four mRNAs encoding the beta 1-subunit, TSH produced a striking increase in abundance, but this regulation was discoordinate, and some species increased more than others. Similar increases in mRNA abundance were elicited by activators of the adenosine 3',5'-cyclic monophosphate second messenger system. In contrast to the alpha 1- and beta 1-mRNAs, the abundance of the mRNA encoding the beta 2-subunit was unchanged with TSH after 6 h, indicating that the effects of thyrotropin were not universal or indiscriminate. Thyrotropin also caused a 76% increase in Na(+)-K(+)-ATPase activity and a 46% increase in pump-mediated transport after 48 h. These studies suggest that the changes in metabolic turnover initiated by TSH during hormone synthesis include upregulation of the N(+)-K+ pump.
Boar sperm plasma membrane Ca(2+)-selective channels in planar lipid bilayers
Authors: Tiwari-Woodruff S. K. , Cox T. C.
Published: December 2017

Entry of Ca2+ through Ca2+ channels is thought to trigger the acrosome reaction of spermatozoa during fertilization. Antagonists of the L-type Ca2+ channel are known to prevent the intracellular Ca2+ (Ca2+) increase and inhibit acrosomal exocytosis in mammalian sperm. Planar bilayer recordings were used to study Ca2- channels incorporated from partially purified boar sperm plasma membranes. With symmetrical 50 mM NaCl and 100 mM BaCl2 on the cis side, single-channel events consistent with Ba2+ flux from cis to trans were observed. These channels were activated by the dihydropyridine agonist (+/-)BAY K 8644 and blocked by the antagonist nitrendipine. Sperm Ca2- channels did not require depolarization for activation and did not inactivate. The (+/-)BAY K 8644 and (S-)BAY K 8644 enantiomers increased apparent open time in a dose-dependent [half-maximal activity constant (K0.5) = 0.9 and 0.3 microM, respectively] manner. Dihydropyridine antagonists nitrendipine (K0.5 = 0.5 microM) and (R+)BAY K 8644 (K0.5 = 2.8 microM) decreased apparent open times. The channels described in this report share some properties with brain, cardiac, and skeletal muscle t tubule Ca2+ channels and may be involved in increasing Cai2+ before the acrosome reaction.
Locomotive forces produced by single leukocytes in vivo and in vitro
Authors: Guilford W. H. , Lantz R. C. , Gore R. W.
Published: December 2017

We report here the first time-resolved measurements of the forces produced during the migration of single leukocytes in vivo and in vitro. Pulmonary macrophages from hamsters and mice, in vitro, and Nembutal (pentobarbital sodium)-anesthetized hamster neutrophils, in vivo, generated maximum locomotive forces ranging from 1.9 to 10.7 nN or tenths of microdynes. Force production was periodic and correlated with the length of the leading lamellipod but not with generalized cell ruffling. Although the extension of the leading lamella is critical to locomotive force generation, these direct measurements suggest that lamellar extension may not arise from the same contractile processes driving forward motion of the cell mass. Indeed, cell ruffling, lamellar extension, and locomotive force generation may be differentially controlled and have different origins. This technique may be extended to test numerous hypotheses of how these and other nonmuscle cells crawl.
Constitutively active mutant GS alpha (G225T) and null-mutant G alpha i-2 (G203T) induce primitive endoderm from stem cells
Authors: Gao P. , Watkins D. C. , Malbon C. C.
Published: December 2017

In F9 teratocarcinoma stem cells, retinoic acid induces a primitive endoderm-like phenotype and a sharp decline in G alpha i-2, a response mimicked by expression of RNA antisense to G alpha i-2 in the absence of this morphogen (D. C. Watkins, G. L. Johnson, and C. C. Malbon. Science Wash. DC 258: 1373-1375, 1992). The role of the GS alpha/G alpha i-2 axis in cellular differentiation was explored. In the absence of retinoic acid, F9 stem cells stably expressing a constitutively active mutant of GS alpha (G225T) progressed to the primitive endoderm phenotype, as judged by morphological and differentiation markers, such as tissue plasminogen activator. Although elevated in cells expressing G225T GS alpha, adenosine 3',5'-cyclic monophosphate does not mimic retinoic acid action and alone fails to induce stem cells to primitive endoderm. In the absence of retinoic acid, expression of a null mutant of G alpha i-2 (G203T) also induced stem cells to primitive endoderm. These observations establish G proteins in the GS alpha/G alpha i-2 axis as a control point for regulating progression to primitive endoderm independent of adenylate cyclase, in the present study's model of early mouse development.
Intercellular calcium waves mediated by diffusion of inositol trisphosphate: a two-dimensional model
Authors: Sneyd J. , Wetton B. T. , Charles A. C. , Sanderson M. J.
Published: December 2017

In response to mechanical stimulation of a single cell, airway epithelial cells in culture exhibit a wave of increased intracellular free Ca2+ concentration that spreads from cell to cell over a limited distance through the culture. We present a detailed analysis of the intercellular wave in a two-dimensional sheet of cells. The model is based on the hypothesis that the wave is the result of diffusion of inositol trisphosphate (IP3) from the stimulated cell. The two-dimensional model agrees well with experimental data and makes the following quantitative predictions: as the distance from the stimulated cells increases, 1) the intercellular delay increases exponentially, 2) the intracellular wave speed decreases exponentially, and 3) the arrival time increases exponentially. Furthermore, 4) a proportion of the cells at the periphery of the response will exhibit waves of decreased amplitude, 5) the intercellular membrane permeability to IP3 must be approximately 2 microns/s or greater, and 6) the ratio of the maximum concentration of IP3 in the stimulated cell to the Km of the IP3 receptor (with respect to IP3) must be approximately 300 or greater. These predictions constitute a rigorous test of the hypothesis that the intercellular Ca2+ waves are mediated by IP3 diffusion.
Increases in [Ca2+]i mediated by the 92.5-kDa putative cell membrane receptor for HCMV gp86
Authors: Keay S. , Baldwin B. R. , Smith M. W. , Wasserman S. S. , Goldman W. F.
Published: December 2017

We determined that changes in intracellular Ca2+ concentration ([Ca2+]i) occur in human fibroblasts within the first hour of human cytomegalovirus (HCMV) infection when viral adsorption and fusion take place, and we investigated whether such changes also occur in response to monoclonal anti-idiotype antibodies (MAb2) that mimic HCMV gp86 and bind to a 92.5-kDa putative cell membrane receptor for gp86. Digitized image analysis of fura 2-loaded human embryonic lung fibroblasts indicated specific transient increases in [Ca2+]i beginning in some cells within the first 5 min of incubation with cross-linked MAb2 (70-750 nM, P < 0.01), which were similar in timing and intracellular distribution to those induced by HCMV. A primary source of Ca2+ appeared to be intracellular Ca2+ stores, since prior depletion of these stores with 30 nM thapsigargin inhibited the response (91.7 +/- 8.6%, P < 0.01); influx of Ca2+ from the extracellular medium was apparently necessary to maintain the intracellular Ca2+ stores. Transient increases in inositol trisphosphate (IP3) occurred in response to MAb2 (up to 3,329 +/- 84%, P < 0.001) or HCMV (92.8 +/- 19%, P < 0.01) during this same time period. These data suggest that the 92.5-kDa receptor for HCMV gp86 mediates an increase in IP3 and subsequent release of Ca2+ from intracellular stores.
Action of insulin on Na(+)-K(+)-ATPase and the Na(+)-K(+)-2Cl- cotransporter in 3T3-L1 adipocytes
Authors: Sargeant R. J. , Liu Z. , Klip A.
Published: December 2017

The Na(+)-K(+)-ATPase presents several different isoforms of its alpha- and beta-subunits. We detected alpha 1- and beta 1-mRNA transcripts and polypeptides in 3T3-L1 fibroblasts; during differentiation into adipocytes, alpha 1-mRNA decreased, alpha 2-mRNA was induced, beta 1-mRNA dropped to undetectable levels, and beta 2-mRNA was never expressed, suggesting that 3T3-L1 adipocytes may express an unidentified Na(+)-K(+)-ATPase beta-subunit isoform. Insulin rapidly increased ion pump activity [ouabain-sensitive 86Rb+(K+) uptake] in 3T3-L1 fibroblasts and adipocytes without changing the plasma membrane concentration of alpha 1- or alpha 2-subunits as determined by subcellular membrane fractionation and immunoblotting or by [3H]ouabain binding to intact cells. Monensin, which raises the concentration of intracellular Na+, increased Na(+)-K+ pump activity, and no further stimulation was achieved with insulin. The stimulation of the pump by insulin was reduced by bumetanide, an inhibitor of the Na(+)-K(+)-2Cl- cotransporter, and was prevented by omission of extracellular Cl-. Insulin increased both ouabain-sensitive and bumetanide-sensitive 86Rb+(K+) uptake. These results suggest that insulin activation of the Na(+)-K(+)-ATPase in 3T3-L1 adipocytes is mediated by an elevation in intracellular Na+ that is likely the consequence of Na(+)-K(+)-2Cl- cotransporter activation.
Expression and function of P-glycoprotein in a mouse kidney cell line
Authors: Ernest S. , Bello-Reuss E.
Published: December 2017

P-glycoprotein (PGP), a transporter conferring multidrug resistance to cancer cells, is expressed in the kidney. C219 monoclonal antibody binding revealed PGP in proximal tubules and mesangium of mouse kidneys. A cell line (TKPTS) expressing PGP was developed from proximal tubules of the 8Tg(SV40E)Bri7 mouse. Northern blot analysis demonstrated a 5.0-kb message identified as mdr1 by ribonuclease protection assay. Cyclosporin A (CSA) at 0.15 and 10 microM increased cellular accumulation of verapamil (VRP) by 32 and 121%, respectively (P < 0.001). VRP at 5 microM increased steady-state cellular accumulation of CSA by 46% (P = 0.02). Basal-to-apical transport of the PGP substrate vinblastine was inhibited by VRP. Rhodamine-123 (R-123) influx was rapid and independent of PGP. R-123 efflux was inhibited by VRP and CSA. Inhibition of PGP transport by VRP, CSA, and PSC-833 decreased the 50% effective dose of adriamycin. The concomitant administration of VRP and CSA was not deleterious and coincided with preferential accumulation of VRP over CSA. Inhibition of PGP-mediated transport is demonstrated as a mechanism of renal cell toxicity.
Stimulation and binding of myocardial phospholipase C by phosphatidic acid
Authors: Henry R. A. , Boyce S. Y. , Kurz T. , Wolf R. A.
Published: December 2017

Exposure of adult ventricular myocytes to exogenous natural phosphatidic acid results in the production of inositol phosphates by unknown mechanism(s). We characterized stimulation of myocytic phosphoinositide-specific phospholipase C (PLC) by synthetic dioleoyl phosphatidic acid (PA) as a potential mechanism for modulation of inositol phosphate production. Our data demonstrate that exogenous PA, at 10(-8)-10(-5) M, caused a concentration-dependent increase in inositol 1,4,5-trisphosphate in adult rabbit ventricular myocytes. PA also caused a concentration-dependent increase in in vitro activity of myocytic PLC in the presence or absence of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). PLC-delta 1, the predominant isozyme of PLC expressed in adult rabbit ventricular myocytes, bound to liposomes of PA with high affinity in the presence of EGTA. The phosphomonoester group of PA was critical to in vitro stimulation of myocytic PLC activity and high-affinity binding of PLC-delta 1. We propose that binding of PLC-delta 1 to phosphatidic acid may be a novel mechanism for dynamic membrane association and modulation of PLC in adult ventricular myocytes.
Changes in lens connexin expression lead to increased gap junctional voltage dependence and conductance
Authors: Donaldson P. J. , Dong Y. , Roos M. , Green C. , Goodenough D. A. , Kistler J.
Published: December 2017

The differentiation of mouse lens epithelial cells into fiber cells is a useful model for studying the changes of the electrical properties of gap junction (cell-to-cell) channels that are induced by an alteration in connexin expression patterns. In this model, cuboidal lens epithelial cells differentiate into elongated fiber cells, and the expression of connexin43 (Cx43) in the epithelial cells is replaced with the production of high levels of Cx50 and Cx46 in the fiber cells. We now report a new procedure to isolate mouse lens fiber cell pairs suitable for double whole cell patch-clamp analysis. Analysis was also performed for fiberlike cell pairs differentiated from epithelial cells in culture. Voltage dependence and unitary conductance of fiber cell gap junction channels were determined and compared with the corresponding values previously measured for the channels joining lens epithelial cells and for lens connexin channels formed in Xenopus oocyte pairs. Our results support a differentiation-induced shift toward stronger gap junctional voltage dependence and larger unitary conductances in the fiber cells. Our data further reflect a balanced functional contribution of Cx50 and Cx46 in the fiber cell-to-cell channels rather than a predominance of a single connexin.
Effect of stretch on contraction and the Ca2+ transient in ferret ventricular muscles during hypoxia and acidosis
Authors: Hongo K. , White E. , Orchard C. H.
Published: December 2017

The effect of stretch on cardiac muscle contraction and the Ca2+ transient was studied during hypoxia and acidosis in isolated ferret ventricular muscles. In control conditions, a maintained stretch produced an immediate increase in tension followed by a slow increase in tension and the Ca2+ transient. A stretch between contractions (diastolic stretch) caused only a slow increase in tension and the Ca2+ transient, whereas a stretch during the period of contraction (systolic stretch) produced an immediate increase in tension followed by a small slow increase in tension and the Ca2+ transient. In hypoxia, the immediate percent increase in tension was the same as in control. However, the slow increase was smaller during all three types of stretch. In acidosis, the immediate percent increase in tension was larger than in control. The slow change was the same during maintained stretch. However, the slow increase in tension was smaller during diastolic stretch and larger during systolic stretch. Thus the stretch-dependent increase in contraction is inhibited during hypoxia and modulated by acidosis.
S-nitrosoglutathione reversibly inhibits GAPDH by S-nitrosylation
Authors: Padgett C. M. , Whorton A. R.
Published: December 2017

Nitric oxide (NO), produced by vascular endothelial cells, mediates both physiological and pathological responses. Although the molecular targets responsible for NO-mediated endothelial cell injury are not known, one candidate is the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In this study, we investigated the mechanism involved in NO-mediated GAPDH inhibition and found that S-nitrosoglutathione (GSNO) inhibited GAPDH activity in both purified enzyme preparations and endothelial cells. Furthermore, GSNO-mediated GAPDH inhibition occurred by modification of the active site cysteine residue in GAPDH, since increasing concentrations of the substrate, glyceraldehyde-3-phosphate, which interacts with the active site cysteine residue, protected GAPDH from inhibition by GSNO. Although under certain conditions both GSNO and the NO donor, sodium nitroprusside (SNP), led to the covalent NAD(+)-dependent modification of GAPDH, this putative ADP ribosylation was unlikely to be the primary mechanism for inhibition, since the stoichiometry was extremely low, and, in the case of GSNO, inhibition was completely reversed by thiol reagents. Furthermore, GSNO effectively S-nitrosylated GAPDH, and the extent of nitrosylation was linearly correlated with the degree of inhibition such that addition of 1 mole of NO per mole of GAPDH monomer was necessary to inhibit the enzyme. Consistent with this finding, GSNO-mediated GAPDH inhibition was reversible with low-molecular-weight thiols, and the reversal of inhibition correlated with the "denitrosylation" of GAPDH. These results suggest that endothelial GAPDH is a target for NO and that inhibition occurs principally by the reversible S-nitrosylation of the active site cysteine residue in GAPDH.
Regulation of Ca(2+)-dependent nitric oxide synthase in bovine aortic endothelial cells
Authors: Buckley B. J. , Mirza Z. , Whorton A. R.
Published: December 2017

Vascular endothelium responds to Ca(2+)-mobilizing agonists by producing nitric oxide (NO), a potent vasodilator and inhibitor of platelet aggregation. Regulation of constitutively expressed endothelial NO synthase (eNOS) in intact cells is not well understood. We investigated the kinetics of NO formation in response to Ca(2+)-mobilizing agonists, the requirement for extracellular L-arginine, and the role of NO in regulating eNOS activity. When endothelial cells were stimulated with bradykinin and ATP in the presence of 100 microM L-arginine, we observed a rapid and transient rise in intracellular Ca2+ concentration ([Ca2+]i) from 50 +/- 8 nM to 698 +/- 74 and 637 +/- 53 nM, respectively, and a rapid and transient rise in NO production from a basal level of 37 pmol.min-1.mg protein-1 to 256 and 275 pmol.min-1.mg protein-1, respectively. When cells were stimulated with A-23187 or thapsigargin in the presence of 100 microM L-arginine, we observed a sustained increase in [Ca2+]i and a sustained increase in NO production. The rate of NO synthesis was linear over 30 min, rising above control levels of 7 pmol/min to 53 pmol/min for A-23187 and 62 pmol/min for thapsigargin. Thapsigargin stimulated NO production and [Ca2+]i with 50% effective concentration values of 0.01 and 0.05 microM, respectively. Ca(2+)-stimulated NO production was attenuated by the NO synthase inhibitor NG-monomethyl-L-arginine, the removal of extracellular L-arginine, and the Ca(2+)-chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. When we exposed cells to NO gas (3.1 mM for 15 min) and S-nitrosoglutathione (10 mM for 1 h) thapsigargin-stimulated NO production was decreased by 50%.(ABSTRACT TRUNCATED AT 250 WORDS)
Nuclear responses to depletion of mitochondrial DNA in human cells
Authors: Li K. , Neufer P. D. , Williams R. S.
Published: December 2017

The derivation of human cell lines devoid of mitochondrial (mt) DNA (rho 0) provides an opportunity to study nuclear responses to a chronic impairment of mitochondrial oxidative phosphorylation. Expression of several nuclear genes is induced in human rho 0 cells, including those encoding integral proteins of the mitochondrial inner membrane, intermediate filaments, and ribosomes. In contrast to conditions in which mitochondrial respiration is altered acutely, expression of heat shock proteins and immediate early genes is not induced. Mitochondria from rho 0 cells maintain a transmembrane electrochemical potential and are distributed within the cytoplasm of these cells in a manner indistinguishable from that of wild-type cells. We conclude that a chronic deficiency of mitochondrial oxidative phosphorylation produced by elimination of mtDNA is associated with a different pattern of gene induction than that provoked by other acute or subacute conditions that impair mitochondrial respiration or create energy demands in excess of mitochondrial respiratory capacity.
Gastrin stimulates expression of protooncogene c-myc through a process involving polyamines in IEC-6 cells
Authors: Wang J. Y. , Wang H. , Johnson L. R.
Published: December 2017

The current study tested the hypothesis that the protooncogene c-myc is involved in the mechanism by which gastrin modulates mucosal cell proliferation. Studies were conducted in the IEC-6 cell line, derived from rat small intestinal crypt cells. Administration of gastrin resulted in the rapid appearance of c-myc mRNA in IEC-6 cells. The increased expression of c-myc began 1 h and peaked 4 h after exposure to gastrin. Maximum increase in c-myc mRNA levels was 7.5-fold the normal value. When cellular protein synthesis was inhibited by addition of cycloheximide, gastrin superinduced c-myc mRNA levels. Gastrin also significantly increased the mRNA levels for ornithine decarboxylase (ODC), the rate-limiting enzyme for polyamine biosynthesis, enzyme activity, and intracellular polyamines in IEC-6 cells. Treatment with alpha-difluoromethylornithine (DFMO), a specific inhibitor of ODC, not only completely depleted intracellular polyamines but also significantly prevented the increased expression of c-myc in cells exposed to gastrin. These results show that 1) gastrin stimulates both polyamine biosynthesis and the expression of the c-myc protooncogene, and 2) depletion of intracellular polyamines by DFMO significantly prevented the increased expression of c-myc by gastrin.
Liddle's disease: abnormal regulation of amiloride-sensitive Na+ channels by beta-subunit mutation
Authors: Bubien J. K. , Ismailov I. I. , Berdiev B. K. , Cornwell T. , Lifton R. P. , Fuller C. M. , Achard J. M. , Benos D. J. , Warnock D. G.
Published: December 2017

Liddle's disease is an autosomal dominant genetic disorder characterized by severe low renin hypertension ("pseudoaldosteronism") that has been genetically linked to a locus on chromosome 16 encoding the beta-subunit of an amiloride-sensitive Na+ channel (ASSC) (15). Peripheral blood lymphocytes (PBL) express ASSC that are functionally indistinguishable from those expressed by Na(+)-reabsorbing renal epithelial cells (3, 5). The amiloride-sensitive Na+ conductance in PBL from affected and unaffected individuals from the original Liddle's pedigree was examined using whole cell patch clamp. Typically, the basal Na+ currents in cells from affected individuals were maximally activated. Basal Na+ currents in cells from unaffected individuals were minimal and could be maximally activated by superfusion with 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP). Affected cells could not be further stimulated with CPT-cAMP. Superfusion with a supermaximal concentration of amiloride (2 microM) inhibited both the cAMP-activated Na+ conductance in unaffected cells and the constitutively activated inward conductance in affected cells. Cytosolic addition of a peptide identical to the terminal 10 amino acids of the truncated beta-subunit normalized the cAMP-mediated but not the pertussis toxin-induced regulation of the mutant ASSC. The findings show that lymphocyte ASSC are constitutively activated in affected individuals, that a mutation of the beta-subunit alters ASSC responsiveness to specific regulatory effectors, and that the cellular mechanism responsible for the pathophysiology of Liddle's disease is abnormal regulation of Na+ channel activity. These findings have important diagnostic and therapeutic implications and provide a cellular phenotype for the diagnosis of pseudoaldosteronism.
Role of contractile protein activation in the length-dependent modulation of tracheal smooth muscle force
Authors: Mehta D. , Wu M. F. , Gunst S. J.
Published: December 2017

The active isometric force developed by a muscle decreases at muscle lengths below an optimal length (Lo). However, when the length of an actively contracting muscle is abruptly decreased, a lower level of isometric force is reached during force redevelopment than when the contraction is initiated at the shorter length. This has been attributed to a deactivation of contractile proteins caused by shortening. In this study, intracellular Ca2+ and myosin light chain (MLC) phosphorylation were measured to assess the mechanisms for the modulation of isometric force caused by changing smooth muscle length before or during isometric contraction. The decline in isometric force between Lo and 0.5Lo was associated with decreases in MLC phosphorylation and intracellular Ca2+ during contractions elicited by acetylcholine or 60 mM KCl. Quick release of the muscle during contraction depressed force redevelopment at the shorter length but not MLC phosphorylation. We conclude that decreases in Ca(2+)-calmodulin-dependent MLC phosphorylation contribute significantly to the decline in isometric force at lengths below Lo, but the depression of contractility associated with the quick release of actively contracted smooth muscle is not caused by a shortening-induced deactivation of contractile proteins.
Osteoclasts express the B2 isoform of vacuolar H(+)-ATPase intracellularly and on their plasma membranes
Authors: Lee B. S. , Holliday L. S. , Ojikutu B. , Krits I. , Gluck S. L.
Published: December 2017

Osteoclasts express high levels of vacuolar H(+)-ATPase (V-ATPase) in their ruffled membranes, driving the secretion of H+ required for normal bone resorption. Previous reports have suggested that the B subunit of the osteoclast V-ATPase differs from those expressed in kidney and other tissues. In this study, B subunit isoform-specific antibodies and cDNA probes were used to examine which B subunit isoform is expressed in osteoclasts and osteoclast-like cells. Immunoblotting and RNA hybridization analysis were used to demonstrate that cells from an osteoclast-rich mouse bone marrow culture model express the B2 but not the B1 subunit isoform. Immunocytochemical staining of murine osteoclasts generated in vitro and of native rat osteoclasts in bone sections showed that the B2 but not the B1 isoform was expressed at high levels and was polarized to the ruffled membrane. Human marrow cultures and monocyte-derived macrophages, used as models for osteoclasts, also expressed the B2 but not the B1 subunit isoform. These results indicate that V-ATPases containing the B2 subunit isoform mediate osteoclast bone resorption.
Characterization and distribution of the neuronal glutamate transporter EAAC1 in rat brain
Authors: Velaz-Faircloth M. , McGraw T. S. , alandro M. S. , Fremeau R. T. , Kilberg M. S. , Anderson K. J.
Published: December 2017

The extracellular concentration of glutamate and other related excitatory amino acids (EAA) is regulated by the action of transporter proteins located on either presynaptic terminals or adjacent astroglial processes. Recent molecular advances have led to the cloning of three separate cDNAs encoding for Na(+)-dependent glutamate transporters; two are thought to be primarily glial in origin (GLAST and GLT-1) and the third (EAAC1) is localized to neurons in the brain and other nonneural tissues. An EAAC1 cDNA was initially cloned from rabbit small intestine (13). In this study, we report isolation and characterization of the homologous clone from rat brain. Northern blot hybridization revealed high levels of EAAC1 mRNA in rat brain and kidney and low levels in heart, lung, and skeletal muscle. Transient expression of EAAC1 in HeLa cells resulted in an increase in Na(+)-dependent high-affinity L-[3H]glutamate and D-[3H]aspartate transport. The pharmacological profile of EAAC1 was very similar to that reported for the rabbit and human EAAC1 homologues. Transport activity was potently inhibited by D- and L-threo-beta-hydroxyaspartate and L-trans-pyrrolodine-2,4-dicarboxylate. Dihydrokainate and L-alpha-aminoadipate did not inhibit transport at concentrations below 1 mM. Oligonucleotide cDNA probes (45-mer) were constructed and labeled with 35S-ATP for film- and emulsion-based in situ hybridization of rat brain. EAAC1 mRNA had the highest density in the cerebellar granule cell layer, hippocampus, superior colliculus, and neocortex. Sections that were emulsion-dipped and counterstained with cresyl violet revealed EAAC1 labeling localized exclusively over neuronal cell bodies, including some nonglutamatergic neurons such as spinal cord ventral horn cells.
Induction of heat shock protein 70 protects intestinal epithelial IEC-18 cells from oxidant and thermal injury
Authors: Musch M. W. , Ciancio M. J. , Sarge K. , Chang E. B.
Published: December 2017

The potential importance of inducible heat shock proteins (HSPs) in conferring protection to intestinal epithelial cells was investigated using IEC-18 cells. To establish optimal HSP induction, [35S]methionine and [35S]cysteine incorporation was assessed. Two 35S-labeled proteins of 70 and 90 kDa were preferentially synthesized. Western and Northern blot analyses confirmed induction of HSP70. This induction provided significant protection to the cells treated with the oxidant monochloramine or lethal heat (49 degrees C). To better establish the protective role of HSP70, cells were stably transfected with human HSP70 under control of the lac operator. When these cells were subjected to injury, they were protected by isopropylthiogalactoside-stimulated HSP70 induction under nonstress conditions. Additionally, the rate of protein synthesis (assessed by [3H]leucine incorporation) was protected. These results demonstrate that HSPs are preferentially and rapidly induced in IEC-18 cells and that induction of inducible HSP70 is important in promoting protection against cellular injury.
Generation of nitric oxide and superoxide during reperfusion after focal cerebral ischemia in rats
Authors: Kumura E. , Yoshimine T. , Iwatsuki K. I. , Yamanaka K. , Tanaka S. , Hayakawa T. , Shiga T. , Kosaka H.
Published: December 2017

We investigated the levels of nitrosyl hemoglobin (HbNO) in rat jugular blood by electron spin resonance (ESR) spectroscopy during and after middle cerebral artery occlusion. The levels of plasma nitric oxide (NO) end products, nitrate plus nitrate, were compared with the levels of HbNO. Small amounts of HbNO were detected in sham-operated rats (n=4) and those subjected to 2 h of occlusion (n=4), whereas nitrite plus nitrate was increased only in the latter (P<0.01; vs.sham). Upon reperfusion after 2 h of occlusion both HbNO and nitrite plus nitrate clearly increased after 15 min (n=4) and 30 min (n=6) reperfusion (P<0.01; vs.occlusion). Administration of superoxide dismutase (5 mg/kg) significantly increased HbNO (P<0.05) but not plasma nitrate plus nitrate (n=5). The increase in HbNO suppressed by administration of NG-nitro-L-arginine methyl ester (20mg/kg; n=4,P<0.01), and this suppression could be reversed by L-arginine (200 mg/kg) (n=4). The present study clearly showed that the L-arginine-NO synthase pathway was activated during reperfusion after focal cerebral ischemia and indicated the involvement of a reaction between NO and superoxide during early reperfusion.
Cloning of GRK2 cDNA from S49 murine lymphoma cells
Authors: Hughes R. J. , Anderson K. L. , Kiel D. , Insel P. A.
Published: December 2017

Beta-adrenergic receptor kinase is a member of the G protein-linked receptor kinase (GRK1) family that elicits receptor desensitization. We have cloned GRK2 from S49 mouse lymphoma cells. The nucleotide sequences of rat GRK2 and GRK3 were aligned and conserved primers chosen for use in reverse transcription-polymerase chain reaction (RT-PCR) of S49 mRNA. Direct sequencing of the PCR fragment provided a rapid means to identify the expression of the GRK2 but not the GRK3 transcript in these cells. Unique expression of GRK2 in S49 cells was confirmed by Western blotting. Three additional pairs of primers were chosen from the rat GRK2 sequence to amplify overlapping regions that together encompassed the entire coding sequence. After attempts to ligate the four fragments of S49 cell GRK2 cDNA by using PCR proved unsuccessful, the intact cDNA was assembled by digesting the PCR products in the region of the overlaps and ligating them in a single step into pBlue-script SK(+).
Mechanism of [Ca2+]i increase by extracellular ATP in isolated rabbit renal proximal tubules
Authors: Yamada H. , Seki G. , Taniguchi S. , Uwatoko S. , Suzuki K. , Kurokawa K.
Published: December 2017

The effects of extracellular ATP on cytosolic Ca2+ concentrations ([Ca2+]i) and cell membrane potentials (Vb) of rabbit renal proximal tubules were investigated using fura 2 and microelectrodes. ATP transiently increased [Ca2+]i without an apparent sustained phase, and the maximum effect was obtained at 10 microM. ADP, adenosine 5'-O-(3-thiotriphosphate), and 2-methylthioadenosine 5'-triphosphate were equally effective as ATP, whereas UTP, adenosine, and alpha beta-methyleneadenosine 5'-triphosphate were far less effective. The [Ca2+]i responses to ATP were strongly inhibited by reactive blue 2, a P2-purinergic receptor antagonist. The removal of extracellular Ca2+ as well as the addition of thapsigargin also markedly attenuated the responses to ATP. In addition, ATP had virtually no effect on Vb, except for the occasional small depolarization by 300 microM ATP. These results indicate that extracellular ATP increases [Ca2+]i through a P2-purinergic receptor and that this effect of ATP is dependent on both extracellular and intracellular Ca2+.
A mathematical model of cytosolic calcium dynamics in human umbilical vein endothelial cells
Authors: Wiesner T. F. , Berk B. C. , Nerem R. M.
Published: December 2017

Important among the responses of endothelial cells are cytosolic free calcium transients. These transients are mediated by several factors, including blood-borne agonists, extracellular calcium, and fluid-imposed shear forces. The transients are characterized by a rapid rise followed by a plateau phase. A base mathematical model is presented that reasonably reproduces the measured calcium transient in cultured human umbilical vein endothelial cells responding to thrombin. Kinetic equations for receptor activation and calcium mobilization comprise the model. A graded response of intracellular free calcium to increasing concentrations of agonist is predicted. Also predicted is the elevation of the peak value and the plateau level by steady nonspecific leak of calcium across the plasma membrane. The influences of capacitative calcium entry, calcium-induced calcium release, and buffering by cytosolic proteins are investigated parametrically. The model predicts significant depletion of cellular calcium in response to agonist stimulation.
TNF-alpha and IL-1 alpha induce mannose receptors and apoptosis in glomerular mesangial but not endothelial cells
Authors: Liu Z. H. , Striker G. E. , Stetler-Stevenson M. , Fukushima P. , Patel A. , Striker L. J.
Published: December 2017

The macrophage mannose receptor, a carbohydrate-binding membrane protein, mediates endocytosis and phagocytosis. This study was undertaken to determine whether mannose receptors were expressed in resting glomerular mesangial and endothelial cells and whether their level was affected by cytokines. Neither mannose receptor mRNA nor proteins were found in resting mesangial or endothelial cells. Mannose receptor mRNA was induced in a dose- and time-dependent manner in mesangial cells by interleukin-1 alpha (IL-1 alpha) or tumor necrosis factor-alpha (TNF-alpha) but not by platelet-derived growth factor-B or IL-6. Cell surface receptors were found by fluorescence-activated cell sorter analysis. Binding to stimulated mesangial cells was saturable and inhibited by excess mannose-bovine serum albumin (BSA) but not by galactose-BSA. TNF-alpha and IL-1 alpha also induced apoptosis in mesangial cells. Mannose receptor expression was not restricted to apoptotic stimulated mesangial cells. Neither agonist induced mannose receptor expression or apoptosis in endothelial cells. Because immunoglobulin A, M, and G contain mannose residues, immune aggregates may be removed from the mesangium through cytokine-induced mannose receptors.
Effect of low-protein diet-induced intrauterine growth retardation on rat placental amino acid transport
Authors: Malandro M. S. , Beveridge M. J. , Kilberg M. S. , Novak D. A.
Published: December 2017

Given the central role of the placenta in nutrient transport to the fetus, one might propose that maternal nutrition would have a regulatory effect on this nutrient delivery. We have examined the effect of a low-protein adequate-calorie diet on specific amino acid transport processes by the rat placenta. Maternal weight, fetal weight, and placental weight were all significantly reduced in dams fed a low-protein (5% casein), isocaloric diet when compared with dams pair-fed a control (20% casein) diet. Even though maternal serum amino acid levels were maintained in the low-protein animals, fetomaternal serum amino acid ratios were significantly reduced, suggesting a reduction in nutrient transfer to the fetus. Apical and basal membrane vesicles were isolated from the placental trophoblast and were used to examine the amino acid transport capacity of both maternal-facing and fetal-facing membranes, respectively. Na+-dependent neutral amino acid transport mediated by system A was decreased in both membrane preparations, while transport mediated by system ASC was unaffected. The Na+-dependent anionic amino acid uptake by system X(-)AG (EAAC1) was reduced on the basal membrane, while the Na+-independent component was similar between the low-protein and control diet-fed dams. Cationic amino acid uptake was also reduced on both membrane surfaces. A decreased steady-state mRNA content for EAAC1 and CAT1 (system y+) suggests that reduced synthesis of the transporter proteins is responsible for the decrease in transport activity. Taken together, these data support the hypothesis that maternal protein malnutrition affects nutrient delivery to the fetus by downregulation of specific amino acid transport proteins.
Perforated patch-clamp recording in cardiac myocytes using cation-selective ionophore gramicidin
Authors: Tajima Y. , Ono K. , Akaike N.
Published: December 2017

Whole cell currents were recorded in single myocytes dissociated from guinea pig ventricles by the patch-clamp technique. The addition of 0.1 mg/ml gramicidin D, a cation-selective ionophore, into the pipette solution induced a gradual spontaneous perforation of the patch membrane under a conventional cell-attached configuration. The access resistance, measured at approximately 12 min after formation of a gigaohm seal, was 9.2 +/- 1.5 M omega (n = 12). The perforated patch membrane exhibited ionic selectivity for various monovalent cations, with a relative order of Cs+ (1.11) > K+ (1.0) > Na+ (0.65) >> tris(hydroxymethyl)aminomethane+ (approximately 0) but was not permeable for Cl-. Under the gramicidin-perforated patch recording configuration, the cells showed the typical electrophysiological properties for ventricular cells reported previously. The intracellular Cl- concentration, estimated from the reversal potential of the catecholamine-induced Cl- current, was 36.3 +/- 2.9 mM (n = 17). We thus conclude that the gramicidin-perforated patch recording mode provides a useful tool for recording the ionic currents while maintaining the intracellular Cl- concentration.
Epidermal growth factor inhibits Ca(2+)-dependent Cl- transport in T84 human colonic epithelial cells
Authors: Uribe J. M. , Gelbmann C. M. , Traynor-Kaplan A. E. , Barrett K. E.
Published: December 2017

This study examined whether epidermal growth factor (EGF) inhibits Ca(2+)-dependent Cl- secretion by T84 cells. Basolateral EGF inhibited Cl- secretion induced by carbachol or thapsigargin, without blocking the rise in intracellular Ca2+. Studies have shown that carbachol renders T84 cells refractory to subsequent stimulation by thapsigargin, an effect ascribed to D-myo-inositol 3,4,5,6-tetrakisphosphate [D-Ins(3,4,5,6)P4]. EGF also increased DL-Ins(3,4,5,6)P4 to a maximum of 170% above control. However, despite the fact that EGF inhibited Cl- secretion at 1 min, DL-Ins(3,4,5,6)P4 was not elevated at this time point. EGF plus carbachol had a greater inhibitory effect on Cl- secretion than either alone, indicating the likely involvement of an additional inhibitory pathway activated by EGF. Staurosporine did not alter the ability of EGF to inhibit Cl- secretion or increase DL-Ins(3,4,5,6)P4. In contrast, genistein inhibited the rise in DL-Ins(3,4,5,6)P4 and partially reversed EGF's inhibitory effect on Cl- secretion. In conclusion, EGF and carbachol can both inhibit Cl- secretion via D-Ins(3,4,5,6)P4, whereas EGF also generates an additional inhibitory signal.
Absence of volume regulatory mechanisms contributes to the rapid activation of apoptosis in thymocytes
Authors: Bortner C. D. , Cidlowski J. A.
Published: December 2017

A common event that occurs during apoptosis is a loss of cell volume, but little information is available on its role in the cell death process. Lymphocytes undergo apoptosis in response to glucocorticoids and exhibit cell shrinkage, nuclear condensation, internucleosomal DNA fragmentation, and apoptotic body formation. Interestingly, only cells that exhibit a loss in cell volume degrade their DNA. To determine if physical shrinkage was sufficient to initiate apoptosis, S49 Neo lymphocytes were cultured in hypertonic medium. The normal osmolarity (approximately 300 mosM) of tissue culture medium was increased to either 550 or 800 mosM, using impermeant sugars such as mannitol and sucrose or NaCl. These hypertonic conditions led to a rapid killing of S49 Neo cells. Evaluation of the mode of cell death revealed that these hypertonic conditions resulted in apoptosis. Unlike glucocorticoid-induced cell death, hypertonically induced apoptosis did not require protein synthesis. When S49 Neo cells were cultured under hypotonic conditions, the cells swelled but apoptosis did not occur. Analysis of several cell types revealed that all lymphoid cells examined (S49 Neo, CEM-C7, primary thymocytes) undergo apoptosis in response to hypertonic conditions, whereas several other cell types (L cells, COS, HeLa, GH3) did not. Although these nonlymphoid cells showed a similar initial reduction in cell volume in response to hypertonic conditions, they subsequently maintained volume or regulated back to a near normal cell volume. These data indicate that thymic lymphoid cells have the machinery in place for rapid induction of apoptosis in response to physical shrinkage, whereas other cell types resist shrinkage-induced apoptosis by the activation of cell volume regulatory mechanisms.
Plasticity and stressor specificity of osmotic and heat shock responses of Gillichthys mirabilis gill cells
Authors: Kultz D.
Published: December 2017

Short-term effects of osmotic and heat shock on proteins of Gillichthys mirabilis gill cells were analyzed. The protein synthesis rate (PSR) of gill cells was influenced by hyperosmotic shock (335-->635 mosmol/kgH2O) and heat shock (25-->37 degrees C), but not by hyposmotic shock (335-->190 mosmol/kgH2O). Between 4 and 6 h after hyperosmotic shock, gill cell protein synthesis was inhibited relative to controls in serum-free medium but increased threefold over control values in medium supplemented with 10% serum. Serum-dependent stimulation of PSR was also observed after heat shock. By use of two-dimensional electrophoresis, 21 proteins, induced after hyperosmotic shock, 14 after hyposmotic shock, and 16 after heat shock were found. The osmotic shock response of gill cells was highly stressor specific because only five or three proteins that were induced after hyperosmotic or hyposmotic shock, respectively, were identical to proteins induced in response to heat shock. Heat shock protein 70 isoforms were only induced after heat shock, but not in response to osmotic shock. In gill and kidney epithelium, the transcription factor c-Jun was modified within 30 min after transfer of whole fish from 1,086 mosmol/kgH2O to 5 or 2,172 mosmol/kgH2O, but osmotic shock in vitro had no effect on c-Jun in isolated gill cells. Ion-substitution experiments revealed that the increase of PSR after hyperosmotic shock in serum-supplemented medium significantly depended on an elevation of extracellular Na+ concentration. These data provide evidence for the plasticity and stressor specificity of osmotic and heat shock responses of fish gill cells.
Iloprost activates KCa channels of vascular smooth muscle cells: role of cAMP-dependent protein kinase
Authors: Schubert R. , Serebryakov V. N. , Engel H. , Hopp H. H.
Published: December 2017

The patch-clamp technique was used to investigate the effect of iloprost on activity of calcium-activated potassium (KCa) channels of freshly isolated rat tail artery smooth muscle cells. In the whole cell configuration, outward current, determined largely by KCa channels, was enhanced 1.73 +/- 0.11-fold by 5 x 10(-7) M iloprost, 1.80 +/- 0.12-fold by 10(-4) M 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole-3', 5'-cyclic monophosphothioate (Sp-5,6-DCl-cBIMPS), a specific activator of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA), and 2.78 +/- 0.95-fold by 10 U/ml of the catalytic subunit of PKA + 10(-4) M MgATP, whereas the heat-inactivated catalytic subunit of PKA + MgATP was without effect. Iloprost at 5 x 10(-7) M increased this current 1.70 +/- 0.27-fold after pretreatment of cells with 10(-6) M okadaic acid, a specific phosphatase inhibitor, but did not alter this current after pretreatment of cells with 2 x 10(-4) M Rp-8-(4-chlorophenylthio)-adenosine-3',5'-cyclic monophosphorothioate, a specific PKA inhibitor. In the cell-attached configuration, activity of KCa channels was enhanced 2.48 +/- 0.44-fold by 5 x 10(-7) M iloprost and 2.09 +/- 0.07-fold by 10(-4) M Sp-5,6-DCl-cBIMPS. Iloprost at 5 x 10(-7) M did not alter intracellular calcium concentration in these cells measured using indo 1. In the inside-out configuration, activity of KCa channels was increased 87.12 +/- 45.04-fold by 10 U/ml of the catalytic subunit of PKA together with 10(-4) M MgATP, whereas no effect was observed after application of the catalytic subunit of PKA together with its regulatory subunit and MgATP, MgATP, cAMP, or the catalytic subunit of PKA alone also did not change KCa channel activity. Thus these results show that iloprost is able to activate KCa channels of freshly isolated rat tail artery smooth muscle cells and suggest that this effect is mediated by a PKA-induced phosphorylation of the channel.