bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of arginine vasopressin and atriopeptin on glial cell volume measured as 3-MG space
Authors: Latzkovits L. , Cserr H. F. , Park J. T. , Patlak C. S. , Pettigrew K. D. , Rimanoczy A.
Published: December 2017

This study evaluates the hypothesis that arginine vasopressin (AVP) and atriopeptin, peptide hormones synthesized and released within the brain, are regulators of brain cell volume using cultured astroglial cells derived from newborn rats. Cell water content, regarded as volume, was measured in defined, serum-free medium as the 3-O-methylglucose (3-MG) space. Initial experiments established conditions such that glucose, which competes with 3-MG for the glucose carrier, would not interfere with the measurement of the 3-MG space. AVP increased the 3-MG space of glial cells by an average of 25% between 30 and 120 min of exposure, whereas atriopeptin decreased it by 32%. The 3-MG space remained close to normal after coadministration of both peptides. The AVP-dependent increase in 3-MG space was blocked both by the V1 antagonist d(CH2)5Tyr(Me)AVP (Manning compound) and by the cotransport inhibitor, bumetanide. Results are consistent with a role for AVP and atriopeptin in the homeostasis of atroglial cell volume.
Hypoxia induces lymphocyte adhesion to human mesenchymal cells via an LFA-1-dependent mechanism
Authors: Ginis I. , Mentzer S. J. , Faller D. V.
Published: December 2017

We and others have previously reported that mesenchymal cells, including endothelial and muscle cells, sense oxygen tension and respond in a specific way during exposure to hypoxic environment. We have examined the interactions of human muscle and endothelial cells, which have been exposed to hypoxic environments, with T and B lymphoid cell lines and peripheral blood lymphocytes (PBL), not subjected to hypoxia. The adhesion of B lymphocyte cell line (JY) and the adhesion of T lymphocyte cell line (Jurkat) to muscle cell monolayers that had been incubated at PO2 of 50 Torr for 3 h increased more than four- and twofold, respectively. Hypoxia appears to upregulate a saturable muscle cell-associated adhesion mechanism, which is capable of withstanding distraction forces greater than 45 g, and is inhibitable by LFA-1-specific monoclonal antibodies (MAbs). Hypoxia also induced a reciprocal decrease in lymphocyte-muscle cell adhesion mechanisms inhibitable by VCAM-1- or VLA-4-specific MAbs. Cultured human endothelial cells when subjected to hypoxic conditions also increased their adhesion for lymphoid cells and cell lines. This induction of adhesion could again be attenuated by anti-LFA-1, but not by anti-ICAM-1 MAb, suggesting that hypoxia activates an adhesion molecule on human mesenchymal cells that is likely to be a new ligand for LFA-1. This report is the first demonstration of a direct induction of cell adhesion mechanisms by hypoxic environments.
Receptor externalization determines sustained contractile responses to endothelin-1 in the rat aorta
Authors: Marsault R. , Feolde E. , Frelin C.
Published: December 2017

The role of receptor internalization and recycling in the vasoconstrictor action of endothelin-1 (ET-1) is investigated using a combination of biochemical and physiological experiments. The binding of 125I-ET-1 to cultured aortic myocytes is first defined. Binding is rapidly followed by an internalization of the peptide. Part of the receptor sites then slowly reappears at the cell surface via a cycloheximide-insensitive mechanism. Evidence that externalizing receptors are functional and can trigger contractions is presented. Finally, the actions of cyclo[D-Trp-D-Asp-Pro-D-Val-Leu] (BQ-123), an antagonist of ETA receptors, are investigated. BQ-123 prevents 125I-ET-1 binding to aortic myocytes (dissociation constant, 10 nM). It prevents the constricting action of ET-1 but not that of angiotensin II. BQ-123 also relaxes almost completely aortic strips that have been precontracted by ET-1 irrespective of the time of its addition. It is concluded that a recycling of internalized ET-1 receptors occurs in ET-1-treated aortic myocytes. This process amplifies the action of the peptide and is probably responsible for the unique contractile action of ET-1.
Molecular adaptation of vascular endothelial cells to oxidative stress
Authors: Lu D. , Maulik N. , Moraru I. I. , Kreutzer D. L. , Das D. K.
Published: December 2017

Cellular organisms respond at the cellular and molecular level when confronted with sudden changes in environment, and molecular adaptation represents the ability of the cells to acclimate themselves to their new environment. In this study we examined the response of bovine vascular endothelial cells (VEC) to the oxidative stress by exposing the cultured cells to two different concentrations of H2O2, 0.04 or 0.08 mM, for 18-24 h. H2O2-exposed VEC displayed good viability (85-90% for 0.04 mM H2O2; 75-80% for 0.08 mM H2O2) and exhibited normal morphology. H2O2 treatment of the VEC was associated with the expression of a number of new proteins, as demonstrated by two-dimensional gel electrophoresis of total cell lysate. Cells exposed to 0.04 mM H2O2 expressed 25 new proteins, whereas 19 newly expressed proteins were detected when the cells were exposed to 0.08 mM H2O2. Western blot analysis of H2O2-treated VEC using specific antibodies to heat-shock proteins (HSP) identified one of these proteins as a member of the HSP 70 family. In addition, H2O2 induced an increase in antioxidative enzyme activities in the VEC, including superoxide dismutase, catalase, and glutathione peroxidase. Moreover, these changes were a truly adaptive phenomenon because challenging the VEC with brief exposure to toxic levels of H2O2 (1 mM for 30 min) showed increased viability (by Trypan blue exclusion test) and decreased injury (by lactate dehydrogenase supernatant-to-cellular ratio determination) in adapted cells (preexposed to 0.04 or 0.08 mM H2O2) compared with control cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Degradation of spectrin and ankyrin in the ischemic rat kidney
Authors: Doctor R. B. , Bennett V. , Mandel L. J.
Published: December 2017

This study investigates ischemia-induced degradation of the spectrin-based cytoskeleton in rat brain, heart, and kidney. Spectrin, in conjunction with ankyrin, structurally supports the plasma membrane and sequesters integral membrane proteins. After 60 and 120 min of ischemia, brain tissue displayed both spectrin and ankyrin breakdown. The spectrin fragmentation pattern is similar to previously reported ischemia-induced calpain I proteolysis of spectrin in N-methyl-D-aspartate receptor-containing neurons. Ischemic heart tissue displayed no spectrin or ankyrin degradation. Ischemic renal tissue showed minimal breakdown of spectrin but a major loss of ankyrin (25%/30 min of ischemia) that was essentially complete after 120 min of ischemia. Interestingly, this profound loss of ankyrin in the intact ischemic kidney was not mimicked in three renal cell lines (MDCK, LLC-PK1, and JTC cell lines) exposed to chemical anoxia. Immunocytochemistry showed ankyrin was concentrated in thick ascending limb (cTAL) cells and, although delayed by 30 min, was lost at the same rate as measured by immunoblot analysis. Spectrin and Na(+)-K(+)-ATPase, which complex with ankyrin, were essentially unaffected by ischemia. Ankyrin degradation in cTAL cells correlated with the loss of basal infolding organization. In conclusion, the spectrin-based cytoskeleton is differentially targeted by ischemia-induced degradative processes in different in vivo tissues.
Blockers of voltage-gated K channels inhibit proliferation of cultured brown fat cells
Authors: Pappone P. A. , Ortiz-Miranda S. I.
Published: December 2017

Cultured brown fat cells have both voltage- and Ca(2+)-activated potassium channels. We tested whether potassium channel activity is necessary for brown fat proliferation by growing adipocytes and preadipocytes from neonatal rat brown fat in the presence of potassium channel blockers. Whole cell patch-clamp experiments showed that verapamil, nifedipine, and quinine block the voltage-gated potassium current (IK,V) with micromolar affinity. Ca(2+)-activated currents (IK,NE) could be activated by micromolar intracellular Ca2+ concentrations and were blocked by nanomolar concentrations of apamin. Both IK,V and IK,NE are blocked by millimolar concentrations of tetraethylammonium (TEA). Under standard culture conditions, the number of cells showing the multilocular morphology characteristic of brown fat cells doubled in 3-5 days. Continuous exposure to 100 nM norepinephrine had no effect on this process. Cell proliferation was inhibited by TEA, quinine, or verapamil. The inhibition was dose dependent, with concentrations for half-block of cell proliferation similar to the Kd values for block of IK,V. Apamin, which selectively blocks IK,NE, had no effect on cell growth. These results suggest that functional voltage-gated potassium channels, but not Ca(2+)-activated potassium channels, may be necessary for the normal proliferation of brown fat cells in culture.
Involvement of stretch-activated ion channels in Ca2+ mobilization to mechanical stretch in endothelial cells
Authors: Naruse K. , Sokabe M.
Published: December 2017

Endothelial cells are subjected to shear stresses by blood flow, normal stresses by blood pressure, and stretch by vessel expansion. These forces are known to induce secretions of several vasoactive substances probably via internal calcium mobilization (R. F. Furchgott. Circ. Res. 53: 557-573, 1983; M. J. Peach, A. L. Loeb, H. A. Singer, and J. Saye. Hypertension Dallas 7, Suppl. I: I-94-I-100, 1985). Here we report that stretching cellular membranes increased intracellular Ca2+ concentration ([Ca2+]i) in human umbilical endothelial cells cultured on silicon membranes. Upon application of a stretch pulse (3-s duration), [Ca2+]i increased rapidly and decayed slowly. The following results suggest that this increase arises from Ca2+ entry through stretch-activated (SA) channels: 1) the Ca2+ response disappeared when extracellular Ca2+ was removed; 2) gadolinium (Gd3+), a blocker for cation-selective SA channels, blocked the response but nifedipine did not; and 3) externally applied Mn2+, which is known to permeate mechanosensitive channels but not Ca2+ channels, entered the intracellular space immediately after an application of mechanical stretch. The increase in [Ca2+]i was found to consist of at least two components: an initial fast component and a delayed slower component. Ryanodine inhibited the slow component. It is suggested that stretching the membrane primarily induced extracellular Ca2+ entry through SA channels followed by Ca2+ releases from intracellular Ca2+ stores.
Cytosolic free calcium regulation in response to acute changes in intracellular pH in vascular smooth muscle
Authors: Batlle D. C. , Peces R. , LaPointe M. S. , Ye M. , Daugirdas J. T.
Published: December 2017

This study examined the mechanisms whereby alterations of intracellular pH (pHi) impact on free cytosolic calcium (Cai2+) in cultured rat aortic vascular smooth muscle cells (VSMC) assayed in the presence of HCO3/CO2. Rapid cell alkalinization, effected by the exposure to NH4Cl or removal of CO2 from the superfusate, produced a rapid increase in Cai2+. The rise in Cai2+ was markedly diminished when sarcoplasmic reticulum (SR) Ca2+ stores had been depleted by prior exposure to arginine vasopressin (AVP) in Ca(2+)-free media or when SR release and reuptake of Ca2+ were blocked by the addition of 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8), but was unaffected by the removal of external Ca2+ or inhibition of Ca2+ entry using NiCl2. Cell acidification also resulted in a rapid increase in Cai2+. This Cai2+ increase was most apparent when pHi was very low (< 6.6) and was unaffected by removal of external Ca2+ or NiCl2 addition. Unlike the effect of cell alkalinization, the increase in Cai2+ associated with cell acidification was not prevented by pretreatment with AVP or TMB-8. We conclude that, in cultured VSMC, acute intracellular alkalinization and, to a lesser extent, acidification result in release of Ca2+ from internal stores. Alkalinization increases Cai2+ by promoting its release from a store which is AVP and TMB-8 sensitive, most likely the SR. Cell acidification increases Cai2+ from an intracellular store(s) that is neither AVP nor TMB-8 sensitive. The increase in Cai2+ produced by cell acidification may be explained on the basis of cell buffering such that, as cytosolic H+ increases, it displaces Cai2+ from internal buffers with similar affinities for Ca2+ and H+.
Enzyme activities and maturation in unstimulated and exogenous gonadotropin-stimulated human oocytes
Authors: Yazigi R. A. , Chi M. M. , Mastrogiannis D. S. , Strickler R. C. , Yang V. C. , Lowry O. H.
Published: December 2017

With the advent of new techniques of human in vitro fertilization (IVF), identifying parameters of oocyte quality to allow selection of those most likely to fertilize becomes crucial. Morphology of oocytes, which correlates positively with biological performance, is the currently utilized classification criterion. However, biological links between form and function are tenuous, and underlying mechanisms remain elusive. We investigated whether biochemical activation is quantitatively associated with the stages of maturation in ova obtained from patients undergoing gynecologic surgery during unstimulated cycles and women undergoing IVF after exogenous gonadotropin stimulation. Changes in selected enzymes from protein, lipid, and carbohydrate metabolism (hexokinase, phosphoglucomutase, glycogen synthetase, uridine diphosphoglucose pyrophosphorylase, glucose-6-phosphate dehydrogenase, cytosolic thiolase, beta-hydroxyacyl-CoA dehydrogenase, alanine aminotransferase, and aspartate aminotransferase) were determined simultaneously, in individual oocytes, utilizing a highly sensitive biochemical methodology. Several enzyme activities paralleled maturation grade and were higher in stimulated oocytes after correction for grade. These biochemical findings quantify metabolic and functional changes that increase as ova mature, possibly contributing to their reproductive performance.
Expression of vascular permeability factor/vascular endothelial growth factor in normal rat tissues
Authors: Monacci W. T. , Merrill M. J. , Oldfield E. H.
Published: December 2017

Vascular permeability factor (VPF)/vascular endothelial growth factor (VEGF) is a approximately 43-kDa secreted protein that has been shown in bioassays to induce endothelial proliferation, angiogenesis, and capillary hyperpermeability. VPF has been suggested to play an important role in the physiology of normal vasculature. To further elucidate the natural functions of VPF in vivo, the expression of VPF in normal tissues was examined using Northern blot analysis and in situ hybridization histochemistry. VPF mRNA is expressed in the brain, kidney, liver, lung, and spleen of the healthy adult rat. On Northern blots, the relative abundance of VPF mRNA observed in these tissues was highest in the lung and lowest in the spleen. As determined by in situ hybridization, the patterns of VPF expression are organ specific. Hybridization of an antisense VPF probe was concentrated in the cerebellar granule cell layer of the brain and in the glomeruli and tubules of the kidney. In the liver and lung, intense hybridization was observed homogeneously throughout both tissues, demonstrating that VPF mRNA is present in virtually every hepatocyte and pulmonary alveolar cell. Hybridization to the spleen was weaker and more diffuse. The widespread expression and organ-specific distribution of VPF mRNA in normal rat tissues supports the suggestion of an extensive role for this factor in the physiology of normal vasculature.
ATP-sensitive potassium channels in smooth muscle cells from guinea pig urinary bladder
Authors: Bonev A. D. , Nelson M. T.
Published: December 2017

We explored the possibility that ATP-sensitive potassium (KATP) channels exist in urinary bladder smooth muscle, since synthetic openers (e.g., lemakalim) of KATP channels in other tissues relax bladder smooth muscle. Unitary currents through single potassium channels and whole cell potassium currents were measured in smooth muscle cells isolated from the detrusor muscle of the guinea pig bladder. Lemakalim (10 microM) increased whole cell K+ currents by 50 pA at -80 mV with 60 mM external K+ when the cells were dialyzed with 0.1 mM ATP and 140 mM K+. Glibenclamide (10 microM), a sulfonylurea blocker of KATP channels in other tissues, inhibited the entire lemakalim-stimulated current as well as 19 pA of the steady-state K+ current. Glibenclamide-sensitive K+ currents were not dependent on voltage. Increasing intracellular ATP from 0.1 to 3.0 mM reduced the glibenclamide-sensitive K+ current in both the presence and absence of lemakalim by about fourfold. External barium (100 microM) which blocks KATP channels in skeletal muscle reduced KATP channel currents in bladder smooth muscle by 50% at -80 mV. Lemakalim (10 microM) increased the open-state probability of single K+ channels in outside-out patches (with 0.1 mM internal ATP) by sixfold. The single-channel conductance was approximately 7 pS at 0 mV with a physiological K+ gradient. This single-channel conductance was in accord with estimates of conductance made from noise analysis of the lemakalim-induced whole cell current. Glibenclamide inhibited these channels. The number of channels per cell was estimated to be approximately 425. We conclude that urinary bladder smooth muscle has KATP channels and that these channels can be opened by the K+ channel opening drug, lemakalim, and blocked by external glibenclamide and barium. We propose that modulation of these channels may regulate bladder contractility.
Effect of volume changes on a potassium current in rabbit corneal epithelial cells
Authors: Farrugia G. , Rae J.
Published: December 2017

Patch-clamp recordings from rabbit corneal epithelial cells have identified a large-conductance (167 pS in symmetrical 150 mM KCl) K channel that is the major contributor to the whole cell current (J. L. Rae and G. Farrugia. J. Membr. Biol. 129: 81-87, 1992). We report here on the regulation of this channel by changes in cellular osmolality and/or volume. Exchanging the bath solution with a hyposmotic (225 or 150 mosM) solution resulted in cellular swelling and selective activation of the K current (126 +/- 86 and 273 +/- 184% increase, respectively). Hyperosmotic solution changes (380 mosM) resulted in cell shrinkage and deactivation of the K current (44.2 +/- 21% decrease). Similar increases in the cell volume and whole cell current were observed on increasing (in perforated patch experiments) the chloride ion concentration (50 mM) in the pipette intracellular solution (127 +/- 63% increase). These changes were accompanied by marked shifts in the resting membrane voltage. We conclude that the K channels in these cells can respond to alteration in cellular osmolality or volume, resulting in changes in the whole cell current and resting voltage.
Thrombin promotes angiogenesis by a mechanism independent of fibrin formation
Authors: Tsopanoglou N. E. , Pipili-Synetos E. , Maragoudakis M. E.
Published: December 2017

The role of thrombin in angiogenesis was investigated in the chick chorioallantoic membrane (CAM) system. alpha-Thrombin promoted angiogenesis in a dose-dependent fashion and at 8.4 pmol/disk reached a maximum of 78% above the control. At a higher dose of alpha-thrombin (25 pmol/disk) the angiogenic effect declines and this can be explained by desensitization of the thrombin receptor. The promotion of angiogenesis by alpha-thrombin is specific as evidenced by the reversal of this effect by hirudin, which binds both the catalytic and the anion-binding exosite of thrombin or by heparin, which binds thrombin and accelerates its inactivation by antithrombin III. gamma-Thrombin, which is catalytically active but lacks the anion-binding exosite required for clotting activity, promotes angiogenesis in the CAM in the same fashion and to the same extent as alpha-thrombin, at doses up to 130 pmol/disk. Phenylalanyl-propyl-arginine chloromethyl ketone (P-PACK)-thrombin, the catalytically inactive analogue of alpha-thrombin that retains the anion-binding exosite, had no significant effect on angiogenesis in the CAM. When combined with alpha-thrombin, P-PACK-thrombin abolished the angiogenesis-promoting effect of alpha-thrombin. These results suggest that alpha-thrombin can promote angiogenesis in the CAM through interaction with its catalytic site without the requirement for fibrin formation.
M3 muscarinic acetylcholine receptor coupling to PLC in rat exorbital lacrimal acinar cells
Authors: Mauduit P. , Jammes H. , Rossignol B.
Published: December 2017

This study was designed to characterize the muscarinic acetylcholine receptor (mAChR) subtype present in rat exorbital lacrimal gland as well as its biochemical coupling. The nonselective muscarinic antagonist [N-methyl-3H]scopolamine ([3H]NMS) binds with high affinity to a homogeneous population of binding sites in both membranes [dissociation constant (Kd) = 82.3 +/- 3.2 pM] and acinar cell (Kd = 170.3 +/- 20 pM) preparations. Muscarinic antagonist inhibition of [3H]NMS binding is homogeneous with the following order of potency: atropine > or = 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) > pirenzepine > 11-([2-(diethylamino)-ethyl]-1-piperidinyl)-acetyl- 5,11-dihydro-6H-pirido[2,3-b]1,4,benzo diazepine-6-one (AFDX 116). Both the affinity of the selective antagonists 4-DAMP, pirenzepine, and AFDX 116 and Northern blot analysis of lacrimal gland mRNAs show a single mAChR population of the M3 subtype. Muscarinic agonist inhibition of [3H]NMS binding displays both high (approximately 20%)- and low-affinity sites (approximately 80%). Both the receptor occupancy and the stimulation by agonists or the inhibition by antagonists of the accumulation of [3H]inositol phosphate were examined under identical conditions with respect to tissue preparations (acinar cells) and buffer (Krebs-Ringer). Results demonstrate 1) the efficient coupling of the M3 mAChR subtype with the phosphatidylinositol (4,5))bisphosphate-specific phospholipase C activity and 2) that the efficacy of a muscarinic agonist is dependent on its structure. Lastly, comparison of the agonists affinity and potency to trigger the [3H]inositol phosphate accumulation suggests that the occupation of the high-affinity agonist binding state of the M3 mAChR was involved in the cellular response.
Modulation of glucose metabolism in macrophages by products of nitric oxide synthase
Authors: Albina J. E. , Mastrofrancesco B.
Published: December 2017

Nitric oxide (NO) is a product of L-arginine metabolism that suppresses cellular oxidative metabolism through the inhibition of tricarboxylic acid cycle and electron transport chain enzymes. The impact of NO synthase (NOS) activity on specific pathways of glucose metabolism in freshly harvested and overnight-cultured rat resident peritoneal macrophages, at rest and after stimulation with zymosan, was investigated using radiolabeled glucose. NOS activity was modulated through the L-arginine concentration in culture media and the use of its specific inhibitor, NG-monomethyl-L-arginine, and quantitated using radiolabeled L-arginine. Results demonstrated that NOS activity was associated with increased glucose disappearance, glycolysis, and hexose monophosphate shunt activity and, in line with the known inhibition of oxidative metabolism associated with the production of NO, with a decrease in the flux of glucose and butyrate carbon through the tricarboxylic acid cycle. In addition, the relative increase in glucose utilization that follows zymosan stimulation was enhanced by treatments that suppressed NOS activity. These results demonstrate that the characteristics of glucose metabolism by macrophages are, to a significant extent, determined by products of NOS.
Transduction of arginine vasopressin signal in skeletal myogenic cells
Authors: Teti A. , Naro F. , Molinaro M. , Adamo S.
Published: December 2017

Arginine vasopressin (AVP) induced concentration-dependent (10(-9) to 10(-6) M) stimulation of inositol phosphate production and a biphasic increment of cytosolic free Ca2+ concentration ([Ca2+]i) in skeletal myogenic cells in culture. These effects were almost completely abolished when the cells were pretreated with the AVP antagonist [deamino-Pen1,Val4,D-Arg8]-vasopressin before stimulation with AVP, thus confirming a V1 receptor-mediated effect. Inositol 1,4,5-trisphosphate production was maximally stimulated within 2-3 s of treatment with AVP, immediately followed by release of Ca2+ from intracellular deposits. Both effects were inhibited by treatment with 12-O-tetradecanoyl phorbol 13-acetate (TPA). Such effect of TPA was reversed by the protein kinase C inhibitor staurosporine. Vasopressin also regulated the intracellular pH of responsive cells with mechanisms involving both Na+ and anion transport across the plasma membrane. However, unlike in other cell types, AVP stimulated the Na(+)-H+ antiport only simultaneously with a dramatic cell acidification or after treatment with TPA. Response to AVP was observed in L6 and L5 and, to a lesser extent, in chick embryo myogenic cells, regardless of the stage of differentiation (myoblast or myotube). Comparison of different subclones of the L6 cell line demonstrated that the responsiveness to AVP correlated positively with their myogenic potential.
Intracellular pH and Ca2+ homeostasis in the pH paradox of reperfusion injury to neonatal rat cardiac myocytes
Authors: Bond J. M. , Chacon E. , Herman B. , Lemasters J. J.
Published: December 2017

Ischemia is characterized by anoxia and a large decrease of tissue pH. After a critical period of ischemia, reperfusion precipitates irreversible injury. Previous work showed that reperfusion injury to cultured neonatal myocytes was precipitated by a rapid return to physiological pH, a "pH paradox" (Bond, J., B. Herman, and J. Lemasters. Biochem. Biophys. Res. Commun. 179: 798-803, 1991). The aim of this study was to measure intracellular pH (pHi) and cytosolic free Ca2+ during the pH paradox of reperfusion injury to cultured neonatal rat cardiac myocytes. pHi and free Ca2+ were measured by ratio imaging of 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein and fura 2 fluorescence. To simulate ATP depletion and acidosis of ischemia, myocytes were incubated with 20 mM 2-deoxyglucose plus 2.5 mM NaCN at pH 6.2. During simulated ischemia, pHi dropped to < 6.5 and subsequently remained constant. During this time, some blebbing but little hypercontraction occurred. After 3 or 4 h of simulated ischemia, inhibitors were removed and cells were incubated at pH 7.4 to simulate reperfusion. pHi began to increase, blebbing accelerated, and myocytes hypercontracted. As pHi increased, viability was lost. The same occurred if pH was increased but metabolic inhibitors were not removed. Monensin, a Na(+)-H+ ionophore, accelerated the increase of pH after reperfusion and hastened cell killing. Hypercontraction, blebbing, and loss of viability did not occur when inhibitors were removed at pH 6.2 or in the presence of dimethylamiloride, an inhibitor of Na(+)-H+ exchange. Protection was associated with maintenance of an acidotic pHi. Free Ca2+ progressively increased during simulated ischemia. After simulated reperfusion, free Ca2+ increased further.(ABSTRACT TRUNCATED AT 250 WORDS)
Signal transduction via P2-purinergic receptors for extracellular ATP and other nucleotides
Authors: Dubyak G. R. , el-Moatassim C.
Published: December 2017

Extracellular ATP, at micromolar concentrations, induces significant functional changes in a wide variety of cells and tissues. ATP can be released from the cytosol of damaged cells or from exocytotic vesicles and/or granules contained in many types of secretory cells. There are also efficient extracellular mechanisms for the rapid metabolism of released nucleotides by ecto-ATPases and 5'-nucleotidases. The diverse biological responses to ATP are mediated by a variety of cell surface receptors that are activated when ATP or other nucleotides are bound. The functionally identified nucleotide or P2-purinergic receptors include 1) ATP receptors that stimulate G protein-coupled effector enzymes and signaling cascades, including inositol phospholipid hydrolysis and the mobilization of intracellular Ca2+ stores; 2) ATP receptors that directly activate ligand-gated cation channels in the plasma membranes of many excitable cell types; 3) ATP receptors that, via the rapid induction of surface membrane channels and/or pores permeable to ions and endogenous metabolites, produce cytotoxic or activation responses in macrophages and other immune effector cells; and 4) ADP receptors that trigger rapid ion fluxes and aggregation responses in platelets. Current research in this area is directed toward the identification and structural characterization of these receptors by biochemical and molecular biological approaches.
Endothelial cell phospholipid distribution and phospholipase activity during acute and chronic hypoxia
Authors: Tretyakov A. V. , Farber H. W.
Published: December 2017

We have previously reported alterations in cyclooxygenase metabolism in cultured aortic and pulmonary arterial endothelial cells exposed to acute and chronic hypoxia. These alterations depended on the duration and degree of the hypoxic exposure, on the vascular bed from which the endothelial cells were derived, and possibly on the availability of arachidonic acid secondary to modifications in metabolic substrate, membrane phospholipids, and/or membrane phospholipase activity. To investigate this last point further, we have compared plasma membrane phospholipid distribution and phospholipase activity in cultured aortic and pulmonary arterial endothelial cells exposed to both acute and chronic hypoxia, using two different precursors (acetic acid and arachidonic acid) and three different membrane preparations (cell homogenates, partially purified plasma membranes, and highly purified plasma membranes). We found that exposure to acute and chronic hypoxia has profound and complicated effects on endothelial cell phospholipid composition and phospholipase activity and that these effects depend on the origin of the endothelial cells and the duration of hypoxia. Furthermore, we found that the alterations in endothelial cell phospholipid distribution in response to hypoxia depend on the purity of the plasma membrane preparation and the metabolic precursor used to study phospholipid metabolism. Finally, these studies suggested that alterations in phospholipids during hypoxia occurred to a greater extent in compartments of endothelial cells other than the plasma membranes and that the well-recognized tolerance of endothelial cells to hypoxia may be due, in part, to preservation of the integrity of their plasma membranes during exposure to acute and chronic hypoxia.
Calcium-activated K+ channels as modulators of human myometrial contractile activity
Authors: Anwer K. , Oberti C. , Perez G. J. , Perez-Reyes N. , McDougall J. K. , Monga M. , Sanborn B. M. , Stefani E. , Toro L.
Published: December 2017

The role of Ca(2+)-activated potassium (KCa) channels in the regulation of membrane potential, intracellular free calcium ([Ca2+]i) and contraction was investigated in uterine smooth muscle and myometrial cells. In an immortalized human myometrial cell line, oxytocin increased [Ca2+]i and [3H]inositol phosphate formation. Relaxin attenuated the oxytocin-induced increase in [Ca2+]i. In cell-attached patches, membrane depolarization activated a large-conductance KCa channel (179 +/- 4 pS). Iberiotoxin (IbTX), a potent blocker of "maxi" KCa channels (A. Galvez, G. Gimenez-Gallego, J. P. Reuben, L. Roy-Contanciin, P. Feigenbaum, G. J. Kaczorowski, and M. L. Garcia. J. Biol. Chem. 265: 11083-11090, 1990) produced long closed events (approximately 6 min) in these channels. In agreement with this blockage, IbTX depolarized the cells by 9.8 +/- 2.8 mV and caused a dose-dependent increase in [Ca2+]i with a half-maximal effective concentration of 0.79 nM. IbTX also caused phasic contractions in human myometrial strips and increased both the frequency and force of spontaneous contractions in estrogen-primed rat myometrial strips. Moreover, myometrial contractility was also affected by 1 mM tetraethylammonium, a concentration that blocks uterine smooth muscle KCa channels when applied to the extracellular side (G. J. Perez, L. Toro, S. D. Erulkar, and E. Stefani. Am. J. Obstet. Gynecol. 168: 652-660, 1993). These results strongly suggest that the large conductance KCa channels may actively participate in the control of human myometrial cell membrane potential and [Ca2+].
Cultured bovine corneal endothelial cells express CHIP28 water channels
Authors: Echevarria M. , Kuang K. , Iserovich P. , Li J. , Preston G. M. , Agre P. , Fischbarg J.
Published: December 2017

Cultured bovine corneal endothelial cells (CBCEC) transport fluid from the basal to the apical surface. In this study, we examined whether the plasma membranes of these cells have water channels. We cultured BCEC on glass plates and monitored the intensity of the light scattered (IS) by the cells. We determined the kinetic constant (k) of the change in IS on exposure to a 10% hypoosmotic challenge to calculate the osmotic permeability (Pf) of the plasma membrane. At 37 degrees C, we found values of k = 0.68 +/- 0.07 s-1 and Pf = 93.3 +/- 33 microns/S (n = 13). The sulfhydryl reagent p-chloromercuribenzenesulfonate (pCMBS; 1 mM) reduced Pf by 75%; 5 mM dithiothreitol reversed such inhibition. The activation energy (Ea) of Pf in the range 10-37 degrees C was 4.7 +/- 0.7 kcal/mol (n = 5). The high Pf, values, the inhibition by pCMBS, and the low Ea strongly suggest the presence of water channels. Therefore, we tested whether the injection of poly(A)+ RNA prepared from CBCEC into Xenopus laevis oocytes results in the expression of water channels. Four days after injection, we measured oocyte Pf values from the rate of volume increase on exposure to hypoosmotic medium. In control oocytes injected with 50 nl of water, Pf was 13.4 +/- 0.3 microns/S (n = 63). In oocytes injected with poly(A)+ RNA (50 ng/oocyte in 50 nl water), Pf was 40.9 +/- 1.6 microns/S (n = 72).(ABSTRACT TRUNCATED AT 250 WORDS)
Muscarinic inhibition of ATP-sensitive K+ channels by protein kinase C in urinary bladder smooth muscle
Authors: Bonev A. D. , Nelson M. T.
Published: December 2017

We explored the possibility that muscarinic receptor stimulation can inhibit ATP-sensitive K+ (KATP) channels in smooth muscle cells from guinea pig urinary bladder. Whole cell K+ currents were measured in smooth muscle cells isolated from the detrusor muscle of the guinea pig bladder. Stimulation of muscarinic receptors by carbachol (CCh; 10 microM) inhibited KATP currents by 60.7%. Guanosine 5'-O-(2-thiodiphosphate) in the pipette (internal) solution prevented the CCh-induced inhibition of KATP currents. Activators of protein kinase C (PKC), a diacylglycerol analogue, and phorbol 12-myristate 13-acetate inhibited KATP currents by 63.5 and 73.9%, respectively. Blockers of PKC (bisindolylmaleimide GF-109203X and calphostin C) greatly reduced CCh inhibition of KATP currents. We propose that muscarinic receptor stimulation inhibits KATP channels in smooth muscle cells from urinary bladder through activation of PKC.
Effects of orchiectomy on EDL muscle ultrastructure and energy-supplying enzymes in growing male guinea pigs
Authors: Beaune B. , Fellmann N. , Villie F. , Marcollet M. , Coudert J.
Published: December 2017

To investigate the influence of androgens on muscle maturation during growth, guinea pigs were orchiectomized (CX) or sham operated (SO) at the end of weaning. Extensor digitorum longus (EDL) muscles were removed at weeks 4, 8, and 12 of postnatal development. According to their contractile and metabolic characteristics, four fiber types were distinguished, called I, IIa, IIbox, and IIbglyc. Muscle maturation consisted of a concomitant decrease in percentages of type IIa and IIbglyc fibers and increase in the percentage of IIbox fibers in both groups. At week 12, fiber distributions were not different between the two groups. Citrate synthase activity fell and phosphofructokinase and lactate dehydrogenase (LDH) activities rose from week 4 to week 12 and were the same in CX and SO. Muscular LDH subunits increased in SO and decreased in CX during this period. In conclusion, fiber type distribution and enzyme activities at puberty were not androgen dependent in guinea pig EDL muscle. Conversely, these hormones acted on LDH isozyme distribution through the enhancement of the most glycolytic LDH fractions.
Regulation of intracellular free Mg2+ and contraction in single adult mammalian cardiac myocytes
Authors: Silverman H. S. , Di Lisa F. , Hui R. C. , Miyata H. , Sollott S. J. , Hanford R. G. , Lakatta E. G. , Stern M. D.
Published: December 2017

Studies in isolated cardiac myocytes have increased our understanding of intracellular Ca2+ regulation. Because less is known about Mg2+ regulation, adult rat ventricular myocytes were loaded with the Mg(2+)-sensitive fluorescent probe mag-indo 1, and changes in intracellular Mg2+ concentration ([Mg2+]i) and cell length were examined under a variety of conditions. The fluorescent signal was calibrated intracellularly and found to differ slightly from that for the probe in solution. Roughly 40% of the signal was intramitochondrial; the remainder was localized in the cytosol. Basal [Mg2+]i averaged 1.02 +/- 0.03 mM (n = 53 cells). No change in [Mg2+]i was observed during a single electrically stimulated contraction, and only a minor increase was seen during rapid electrical stimulation, which was expected to raise intracellular Ca2+ concentration ([Ca2+]i) to approximately 1 microM. An acid shift in intracellular pH of approximately 1 pH unit was accompanied by a small change in [Mg2+]i (0.34 +/- 0.03 mM, n = 6, P < 0.05). No change in [Mg2+]i was observed when cells were superfused with 15 mM Mg2+, despite marked changes in contraction. [Mg2+]i more than doubled when cells were depleted of ATP by exposure to hypoxia or metabolic inhibitors. The increase in [Mg2+]i was abrupt and occurred at the time of the failure of contraction, plateauing as rigor contracture developed. Reoxygenation was accompanied by a gradual fall in [Mg2+]i in cells that recovered mechanical function, and in a subset of cells that underwent hypercontracture. Studies in cell suspensions confirmed that rapid cellular energy depletion was accompanied by increases in [Mg2+]i and parallel decreases in ATP. Thus [Mg2+]i was largely insensitive to changes in [Ca2+]i or pHi and extracellular [Mg2+] but was rapidly altered by changes in energy state in a manner that was related to specific changes in cell morphology and contractile function.
pH, morphology, and diffusion in lateral intercellular spaces of epithelial cell monolayers
Authors: Harris P. J. , Chatton J. Y. , Tran P. H. , Bungay P. M. , Spring K. R.
Published: December 2017

The lateral intercellular spaces (LIS) of reabsorptive epithelia are the site of the proposed local osmotic gradient responsible for transepithelial transport. We developed techniques for loading the LIS of living cultured renal cells (MDCK and LLC-PK1) with the fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF), visualizing LIS geometry, measuring pH, and determining the BCECF diffusion coefficient within the LIS. The LIS pH was remarkably constant and differed substantially from that of the superfusate in both the presence and absence of HCO3 or CO2. The LIS of MDCK cells had a pH of 7.66 +/- 0.04 in bicarbonate-free solutions of pH 7.0, 7.4, or 7.8. In bicarbonate-containing solutions, MDCK LIS pH was acidic to the superfusate by 0.3-0.4 units. In the absence of bicarbonate, the LIS of LLC-PK1 cells was markedly acidic (6.83 +/- 0.05), becoming alkaline by approximately 0.25 units in the presence of bicarbonate. Gradients in pH or dye concentration were not detected within the LIS. The diffusion coefficient of BCECF within the LIS was approximately equal to that seen in free solution.
The insulin receptor: structure, function, and signaling
Authors: Lee J. , Pilch P. F.
Published: December 2017

The insulin receptor is a member of the ligand-activated receptor and tyrosine kinase family of transmembrane signaling proteins that collectively are fundamentally important regulators of cell differentiation, growth, and metabolism. The insulin receptor has a number of unique physiological and biochemical properties that distinguish it from other members of this large well-studied receptor family. The main physiological role of the insulin receptor appears to be metabolic regulation, whereas all other receptor tyrosine kinases are engaged in regulating cell growth and/or differentiation. Receptor tyrosine kinases are allosterically regulated by their cognate ligands and function as dimers. In all cases but the insulin receptor (and 2 closely related receptors), these dimers are noncovalent, but insulin receptors are covalently maintained as functional dimers by disulfide bonds. The initial response to the ligand is receptor autophosphorylation for all receptor tyrosine kinases. In most cases, this results in receptor association of effector molecules that have unique recognition domains for phosphotyrosine residues and whose binding to these results in a biological response. For the insulin receptor, this does not occur; rather, it phosphorylates a large substrate protein that, in turn, engages effector molecules. Possible reasons for these differences are discussed in this review. The chemistry of insulin is very well characterized because of possible therapeutic interventions in diabetes using insulin derivatives. This has allowed the synthesis of many insulin derivatives, and we review our recent exploitation of one such derivative to understand the biochemistry of the interaction of this ligand with the receptor and to dissect the complicated steps of ligand-induced insulin receptor autophosphorylation. We note possible future directions in the study of the insulin receptor and its intracellular signaling pathway(s).
High-affinity [3H]PN200-110 and [3H]ryanodine binding to rabbit and frog skeletal muscle
Authors: Anderson K. , Cohn A. H. , Meissner G.
Published: December 2017

In vertebrate skeletal muscle, the voltage-dependent mechanism of sarcoplasmic reticulum (SR) Ca2+ release, commonly referred to as excitation-contraction (E-C) coupling, is mediated by the voltage-sensing dihydropyridine receptor (DHPR), which is believed to affect SR Ca2+ release through a physical interaction with the SR ryanodine receptor (RYR)/Ca2+ release channel. Scatchard analysis of ligand binding of [3H]PN200-110 to the DHPR and [3H]ryanodine to the RYR indicated the presence of high-affinity sites in muscle homogenates, with maximum binding (Bmax) values of 72 +/- 26 and 76 +/- 30 pmol/g wet wt for rabbit skeletal muscle, and 27 +/- 14 and 44 +/- 13 pmol/g wet wt for frog skeletal muscle, respectively. The Bmax values corresponded to a PN200-110-to-ryanodine binding ratio of 0.98 +/- 0.26 and 0.61 +/- 0.24 for rabbit and frog skeletal muscle, respectively, and were found by Student's t test to be significantly different (P < 0.02, n = 7). These results are compared with measurements with isolated rabbit skeletal muscle membrane fractions and discussed in relation to our current understanding of the mechanism of E-C coupling in skeletal muscle.
Intracellular pH recovery during respiratory acidosis in perfused hearts
Authors: Vandenberg J. I. , Metcalfe J. C. , Grace A. A.
Published: December 2017

Na(+)-H+ exchange and Na(+)-dependent HCO3- influx both contribute to recovery of intracellular pH (pHi) after an acidosis induced by using the NH4Cl prepulse technique in mammalian and avian cardiac tissue. We have investigated the relative contributions of these mechanisms to pHi recovery during respiratory acidosis in the Langendorff-perfused ferret heart with and without correction of extracellular pH (pHo). pHi was measured from the chemical shift of the exogenous 31P nuclear magnetic resonance pH indicator 2-deoxy-D-glucose 6-phosphate. Intrinsic intracellular buffering capacity, calculated from the change in intracellular HCO3- concentration after a change in CO2, was reduced from approximately 33 (no inhibitors of acid extrusion present) to 19 +/- 5 mM when H+ extrusion during the acid loading phase was inhibited. During respiratory acidosis (pHo approximately 6.95), the proton efflux rate (JH) calculated at pHi 6.85 was 0.30 +/- 0.04 mmol.l-1.min-1 (n = 9). When pHo was corrected by increasing external HCO3- concentration to 60 mM during respiratory acidosis (pHo approximately 7.33), JH was 1.11 +/- 0.11 mmol.l-1.min-1 (n = 7), and when pHo was partially corrected by the addition of 50 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid to the perfusion solution (pHo approximately 7.1), JH was 0.64 +/- 0.08 mmol.l-1.min-1 (n = 6). In all three groups Na(+)-H+ exchange and HCO3- influx each contributed approximately 50% to acid-equivalent efflux.(ABSTRACT TRUNCATED AT 250 WORDS)
Endothelial polyamine uptake: selective stimulation by L-arginine deprivation or polyamine depletion
Authors: Bogle R. G. , Mann G. E. , Pearson J. D. , Morgan D. M.
Published: December 2017

Uptake of putrescine and spermidine by cultured porcine aortic endothelial cells was time dependent and linear for 60 min. Transport, against a 5- to 10-fold concentration gradient, demonstrated both saturable and non-saturable components. Apparent concentration giving one-half maximal transport (Kt) values for putrescine and spermidine were 9 and 0.6 microM, respectively. Transport was reduced at 0 degrees C, suggesting that the process is energy requiring; inhibition by N-ethylmaleimide or p-chloromercuribenzoate suggested a requirement for sulfydryl groups. Transport of putrescine, but not spermidine, was partially activated by Na+. Spermidine and spermine did not inhibit putrescine uptake, and putrescine and spermine did not inhibit spermidine uptake, suggesting the presence of a separate transporter for each polyamine. Pretreatment with DL-2-difluoromethy-lornithine increased the uptake of putrescine but not spermidine. The endothelial cell putrescine transporter is thus sensitive to polyamine depletion, suggesting that transport from the extracellular space may be an important source of polyamines. L-Ornithine or L-arginine were not inhibitory, indicating that polyamine and cationic amino acid transport is mediated by independent systems. The sensitivity of putrescine transport to L-arginine but not to L-ornithine deprivation suggests that intracellular levels of arginine rather than ornithine regulate polyamine metabolism and transport in these cells. Thus factors that affect arginine utilization may also influence polyamine metabolism.
Length, force, and Ca(2+)-troponin C affinity in cardiac and slow skeletal muscle
Authors: Wang Y. P. , Fuchs F.
Published: December 2017

Troponin C occurs as two isoforms, one (sTnC) expressed in fast skeletal muscle and the other (cTnC) expressed in cardiac and slow skeletal muscle. On the basis of subunit exchange experiments it has been suggested that cTnC may play a specific role as a length-sensing molecule. In this study we have compared skinned fibers from bovine ventricle and slow rabbit soleus muscle with respect to the effects of force and sarcomere length on Ca2+ binding to troponin C. A double-isotope technique was used to measure Ca2+ binding concurrent with force generation. The phosphate analogue vanadate was used to regulate force independent of free Ca2+ concentration. To determine the effect of sarcomere length, muscle fibers were released from longer sarcomere length to shorter sarcomere length, and bound Ca2+ was determined either before or after the release. Reduction in force or length was associated with reduced binding of Ca2+ to cTnC in cardiac muscle, but no effect of these interventions was seen in soleus muscle. Thus the nature of the mechanical feedback on the regulatory Ca(2+)-binding sites appears to be a property of the myofilament system rather than the troponin C isoform.