bluuebunny/crossref_metadata_2025 huggingface dataset:
Neutral amino acid transport systems of microvillous membrane of human placenta
Authors: Johnson L. W. , Smith C. H.
Published: December 2017

Placental transport produces concentrations of amino acids in fetal blood greater than those of maternal blood. Competitive inhibition studies of zwitterionic amino acid transport in isolated vesicles from the microvillous (maternal facing) plasma membranes of syncytiotrophoblast defined three transport systems: 1) a sodium-dependent system that supports methylaminoisobutyric acid (MeAIB) transport and has the characteristics of an A system; 2) a sodium-independent system with a high affinity for leucine and other amino acids with branched or aromatic side chains; and 3) a sodium-independent system with a preference for alanine as a substrate. The two sodium-independent systems could be further discriminated by marked specificity for trans stimulation with alanine or with leucine. System ASC, known to be present in whole placenta, and the neutral brush-border or imino systems of other polarized epithelia were apparently absent. Kinetic characteristics of the A system make it the probable primary driving force for concentrative transfer of its substrate amino acids to the fetus. Characteristics of the high-affinity leucine system demonstrated that it is saturated by normal serum leucine concentrations. Regulation of either system has the potential to alter placental amino acid uptake and transfer to the fetus.
Regulation of shortening velocity by cross-bridge phosphorylation in smooth muscle
Authors: Hai C. M. , Murphy R. A.
Published: December 2017

We have proposed a model that incorporates a dephosphorylated "latch bridge" to explain the mechanics and energetics of smooth muscle. Cross-bridge phosphorylation is proposed as a prerequisite for cross-bridge attachment and rapid cycling. Features of the model are 1) myosin light chain kinase and phosphatase can act on both free and attached cross bridges, 2) dephosphorylation of an attached phosphorylated cross bridge produces a noncycling "latch bridge," and 3) latch bridges have a slow detachment rate. This model quantitatively predicts the latch state: stress maintenance with reduced phosphorylation, cross-bridge cycling rates, and ATP consumption. In this study, we adapted A. F. Huxley's formulation of crossbridge cycling (A. F. Huxley, Progr. Biophys. Mol. Biol. 7: 255-318, 1957) to the latch-bridge model to predict the relationship between isotonic shortening velocity and phosphorylation. The model successfully predicted the linear dependence of maximum shortening velocity at zero external load (V0) on phosphorylation, as well as the family of stress-velocity curves determined at different times during a contraction when phosphorylation values varied. The model implies that it is unnecessary to invoke an internal load or multiple regulatory mechanisms to explain regulation of V0 in smooth muscle.
Na+-Ca2+ exchange in regulation of contractility in canine cardiac Purkinje fibers
Authors: Sonn J. K. , Lee C. O.
Published: December 2017

To study Na+-Ca2+ exchange, intracellular Na+ activity (aiNa), twitch tension, and transmembrane potential were simultaneously measured in canine cardiac Purkinje fibers driven at a constant rate (1 Hz) in the absence and presence of strophanthidin (5 X 10(-7) M) at normal, low, and high extracellular [Na+] ([Na+]o) or [Ca2+] ([Ca2+]o). Intracellular Ca2+ activity (aiCa) of the fibers was also measured in a normal Tyrode solution. Reductions of [Na+]o by 20, 40, and 60% decreased the ratio of extracellular Na+ activity (aoNa) and aiNa in the steady state but steeply increased twitch tension. This finding is consistent with the view that a decrease in aoNa/aiNa increases intracellular Ca2+ through Na+-Ca2+ exchange. In further agreement with this view, a Na+-free solution virtually depleted intracellular Na+ and increased the resting tension of the fibers. The slope of the relation of the logs of twitch tension and aiNa that was determined at normal [Na+]o and [Ca2+]o may reflect the properties of the Na+-Ca2+ exchange. Slope of log tension-aiNa relationship decreased when reducing [Na+]o or increasing [Ca2+]o had decreased the level of aiNa. On the other hand, the slope increased when a rise in [Na+]o or a reduction in [Ca2+]o had increased the level of aiNa. These results indicate that as the aiNa level increased, slope of tension-aiNa relation increased, which suggests that Na+-Ca2+ exchange may depend on level of aiNa.(ABSTRACT TRUNCATED AT 250 WORDS)
Extracellular acidosis delays onset of cell death in ATP-depleted hepatocytes
Authors: Gores G. J. , Nieminen A. L. , Fleishman K. E. , Dawson T. L. , Herman B. , Lemasters J. J.
Published: December 2017

A fluorometric assay using propidium iodide total fluorescence was utilized to quantitate cell viability in hepatocyte suspensions continuously. For viable hepatocytes exposed to KCN, fluorescence was linearly proportional to lactate dehydrogenase release and to nuclear labeling by propidium iodide. In KCN-treated hepatocytes, iodoacetate eliminated the protective effect of the fed state and fructose against the onset of cell death. A model of cell death with KCN and iodoacetate was developed to mimic the ATP depletion of anoxia. This "chemical hypoxia" was used to investigate the role of pH in cell death. At pH 7.4, cell viability decreased to 10% after 120 min, whereas at pH 5.5-7.0, cell viability was 65-85%, nearly the same as normoxic cells. During chemical hypoxia under acidotic conditions, a return of pH to 7.4 resulted in a rapid acceleration of cell killing, a "pH paradox." Inhibition of Na+-H+ exchange increased survival, whereas promoting exchange of intracellular Cl- for extracellular HCO3- potentiated cell killing. Monensin, a Na+-H+ ionophore, potentiated cell killing at pH 7.4 but not at pH 6.2. The results show that extracellular acidosis markedly protects against cell killing after ATP depletion, an effect that appears mediated through cytoplasmic acidification.
Development of a new fluorescent angiotensin II probe
Authors: Fatigati V. , Peach M. J.
Published: December 2017

Angiotensin II (ANG II) was conjugated to polystyrene Latex fluorescent microspheres (0.5 or 0.05 micron diam) with carbodiimide. Biological activity of the ANG II-conjugated microspheres (ANG II microspheres) was assessed in dispersed hepatocytes. The 0.05 micron ANG II microspheres inhibited glucagon-stimulated adenosine 3',5'-cyclic monophosphate accumulation and stimulated phosphorylase activity in hepatocytes, whereas the 0.5 micron ANG II microspheres only stimulated phosphorylase activity. The biological activity of the ANG II microspheres was caused by the conjugated ANG II and not by "free" ANG II associated with the spheres or by trypsin-like activity of hepatocytes causing release of ANG II from the microspheres. Binding of 0.05 micron ANG II to hepatocytes was readily observed with fluorescence microscopy. Little, if any, binding was observed with microspheres without conjugated ANG II. This fluorescent preparation of ANG II may have great utility in the study of receptor behavior after binding in individual cells.
Heat production of rat anococcygeus muscle during isometric contraction
Authors: Walker J. S. , Wendt I. R. , Gibbs C. L.
Published: December 2017

Heat production, unloaded shortening velocity (Vus), and load-bearing capacity (LBC) were studied in the isolated rat anococcygeus muscle during isometric contractions at 27 degrees C. The relation between the total suprabasal heat produced and the stress-time integral for isometric contractions of various durations was curvilinear, demonstrating a decreasing slope as contractile duration increased. The rate of heat production at 600 s was approximately 68% of the peak value of 6.55 mW/g that occurred at 10 s. At the same time, force rose from a mean of 92 mN/mm2 at 10 s to a value of 140 mN/mm2 at 600 s. This produced a nearly threefold increase in the economy of force maintenance. The decline in the rate of heat production was accompanied by a decline in Vus from 0.56 Lo/s at 10 s to 0.28 Lo/s at 600 s, where Lo is the length for optimal force development. This suggests the fall in the rate of heat production was caused, at least in part, by a slowing of cross-bridge kinetics. The ratio of LBC to developed tension at 10 s was not significantly different from the ratio at 600 s, suggesting that the increase in tension was due to an increased number of attached cross bridges. The decline in heat production, therefore, appears contradictory, since an increased number of attached cross bridges would predict an increased rate of energy expenditure. The observations can be reconciled if either 1) the increase in force is caused by a progressive increase in the attachment time of a constant number of cross bridges that cycle at a lower frequency or 2) the decline in energy expenditure caused by the slowing of cross-bridge cycling is sufficient to mask the increase caused by the recruitment of additional cross bridges.
TRH and BAY K 8644 synergistically stimulate prolactin release but not 45Ca2+ uptake
Authors: Pachter J. A. , Law G. J. , Dannies P. S.
Published: December 2017

Thyrotropin-releasing hormone (TRH) (1 microM) and the Ca2+-channel agonist BAY K 8644 (1 microM) each induced transient increases in prolactin secretion from primary cultures of rat anterior pituitary cells in perifusion. When BAY K 8644 was added after a TRH-induced secretory peak, the additional effect of BAY K 8644 on prolactin release was approximately twofold greater over a 30-min period than the effect of BAY K 8644 on previously untreated cells. TRH and BAY K 8644 were also synergistic when added in the opposite order or simultaneously. Substitution of other agents for BAY K 8644 revealed that only high K+ (40 mM) was at least additive with TRH in stimulating prolactin secretion; treatment with TRH inhibited, rather than facilitated, subsequent stimulation of prolactin secretion by angiotensin II (100 nM) or the ionophore A23187 (20 microM). The cooperative effect was not specific for TRH because BAY K 8644 also acted synergistically with angiotensin II or 40 mM K+. In GH4C1 cells, in which TRH and BAY K 8644 were also synergistic in releasing prolactin, measurements with the fluorescent indicator indo-1 showed that TRH and BAY K 8644 could each elevate cytosolic Ca2+ above the level stimulated by the other. Unexpectedly, TRH was found to inhibit BAY K 8644-stimulated 45Ca2+ uptake in both GH4C1 and primary cultured cells. These results indicate that BAY K 8644 and TRH synergistically stimulate prolactin secretion by a mechanism other than a cooperative effect on the activity of dihydropyridine-sensitive Ca2+ channels.
Effects of membrane potential on electrical slow waves of canine proximal colon
Authors: Smith T. K. , Reed J. B. , Sanders K. M.
Published: December 2017

The effects of membrane potential on the waveforms and propagation of slow waves were tested using circular muscles of the canine colon. Studies were conducted with intracellular recording techniques on cross-sectional strips of canine proximal colon. Circular muscle cells near the submucosa generated slow waves that decayed in amplitude as they spread through the circular layer. The membrane potentials of cells were less negative as a function of distance from the submucosal border. Cells near the submucosa were depolarized with elevated external K+ and electrical pulses using the partitioned chamber technique. The waveforms of depolarized submucosal cells were compared with events recorded from cells in the bulk of the circular layer. The waveform changes caused by experimental depolarization were different from the changes in waveform that occur during propagation, suggesting the latter are due to a different mechanism than depolarization. The effects of the membrane potential on syncytial input resistance and length constant were also evaluated. The results of these studies are consistent with the hypothesis that slow-wave propagation across the circular layer in canine proximal colon occurs passively.
SR Ca loading in cardiac muscle preparations based on rapid-cooling contractures
Authors: Bers D. M.
Published: December 2017

The influence of rest periods on twitches and rapid-cooling contractures (RCCs) was examined in trabeculae from rabbit, rat, guinea pig, and frog ventricle and rabbit atrium. RCCs were used as a relative index of sarcoplasmic reticulum (SR) Ca content. After increasing rest duration, rabbit and guinea pig ventricles exhibit a decline of both twitch force and RCC force (rest decay). When stimulation is resumed, both twitches and RCCs recover to steady-state levels. The SR (and cells) in these tissues may lose Ca during quiescence and become reloaded with progressive stimulation. Rat ventricle and rabbit atrium exhibited an increase in both twitch and RCC tension as a function of rest duration (rest potentiation). Resumption of stimulation resulted in parallel declines of both twitch and RCC tension approaching steady state. Thus stimulation in rat ventricle and rabbit atrium may lead to a net Ca loss from the SR (and the cell) and quiescence may lead to replenishment of cellular Ca. This major difference in Ca metabolism in mammalian cardiac muscles might be due to a fundamental difference in SR properties or, alternatively, different sarcolemmal transport properties (e.g., action potential configuration, Na-pump). After long rest intervals in rabbit and guinea pig ventricle, RCCs return toward their steady-state value in considerably fewer beats than does twitch tension. This implies that something other than SR refilling is responsible for the slow phase of twitch recovery after rest. In rabbit ventricle increasing frequency or extracellular Ca concentration ([Ca]o) generally increases both twitch and RCC tension. However, decreasing [Ca]o (to 0.2 mM) does not decrease RCCs much despite a dramatic decline in twitch tension (suggesting low twitch tension despite a loaded SR). Rapid rewarming during an RCC usually results in a transient rise in tension (or rewarming "spike"), which is due to a warming-induced increase in myofilament Ca sensitivity. Differences in rewarming spikes among the tissues studied suggest differences in temperature effects on myofilament Ca sensitivity.
Insulin stimulates in vivo cell proliferation in white adipose tissue
Authors: Geloen A. , Collet A. J. , Guay G. , Bukowiecki L. J.
Published: December 2017

The effects of insulin on white adipose tissue hyperplasia have been studied by quantitative radioautography in streptozotocin diabetic rats. After 1 mo of diabetes, the majority of the adipocytes were extremely small (less than 15 microM) but contained several tiny triglyceride droplets (pauciadipose cells). At this point, the diabetic animals were infused with insulin (8.5 U.kg-1.day-1) delivered via osmotic minipumps for 0-8 days. Control and diabetic rats were pulse-injected with tritiated thymidine (4 h), and samples of parametrial white adipose tissue (PWAT) were collected for quantitative analysis of mitotic frequencies. One-day insulin treatment increased PWAT weight and adipocyte size without stimulating mitoses. However, after 4 days of insulin replacement, the total tissue labeling index increased greater than 120 times over control values. This marked enhancement of mitotic activity principally occurred in interstitial cells rather than in typical adipocytes, pauciadipose cells, or endothelial cells. After 8 days of insulin infusion, the mitotic activity significantly decreased. The results demonstrate that 1) insulin is able to stimulate cell proliferation in PWAT of adult diabetic rats, 2) it transiently stimulates proliferative activity in adipose tissue after a 2- to 3-day period of induction, 3) the increase in adipocyte size precedes the enhancement of mitotic activity, and 4) the effects of insulin were specific, as in the same rats, under the same experimental conditions, insulin did not increase the cell labeling in brown adipose tissue.
Effect of gentamicin on phospholipid metabolism in cultured rabbit proximal tubular cells
Authors: Ramsammy L. S. , Josepovitz C. , Lane B. , Kaloyanides G. J.
Published: December 2017

We examined the hypothesis that the accumulation of phospholipid in cells exposed to gentamicin is due to impaired degradation. Experiments were performed in rabbit proximal tubular cells grown in primary culture. Cells exposed to 10(-3) M gentamicin manifested myeloid body formation and a progressive increase in total phospholipid that by day 6 was 44% higher than that of control cells and reflected increases of phosphatidylinositol of 235%, phosphatidylcholine of 60%, phosphatidylethanolamine of 90%, and phosphatidylserine of 55% above control values. Gentamicin impaired the degradation of these phospholipids. The t1/2 of the phospholipid pool labeled with [3H]myoinositol increased 146% from 1.17 (control) to 2.88 days (gentamicin); the t1/2 of the [3H]choline pool increased 34% from 1.77 to 2.38 days; the t1/2 of the [3H]ethanolamine pool increased 57% from 3.14 to 4.93 days; the t1/2 of the [3H] serine pool increased 37% from 6.30 to 8.63 days. Exposure of cells to gentamicin for 2 days also stimulated increased incorporation of [3H]myoinositol (68%) and [3H]ethanolamine (59%) into phospholipid. The data are consistent with the hypothesis that gentamicin inhibits the activity of lysosomal phospholipases that results in the accumulation of phospholipid within the lysosome in the form of myeloid bodies. Increased phospholipid synthesis may represent a compensatory response to the impaired lysosomal degradation of phospholipid. We postulate that the preferential increase of phosphatidylinositol reflects the capacity of the polycationic gentamicin to interact electrostatically with the anionic phosphoinositides and inhibit their turnover.
Electrogenic Na+-ascorbate cotransport in cultured bovine pigmented ciliary epithelial cells
Authors: Helbig H. , Korbmacher C. , Wohlfarth J. , Berweck S. , Kuhner D. , Wiederholt M.
Published: December 2017

The high level of ascorbic acid (AA) in the aqueous humor of many mammals suggests an active transport of AA across the double-layered ciliary epithelium from blood to aqueous humor. We used [14C]AA to study AA uptake in bovine pigmented ciliary epithelial cells in tissue culture. We observed a 40-fold intracellular accumulation of AA, which was dependent on extracellular Na+. With labeled dehydroascorbate (DHA, the oxidized form of the vitamin) in the medium, there was a 20-fold intracellular accumulation of the label. However, the time course of DHA uptake was different compared with AA uptake and was not Na+ dependent, suggesting different transport systems for AA and DHA. AA uptake was inhibited by 1 mM phloretin and in the presence of isoascorbate. Furthermore, AA uptake was markedly reduced when intracellular Na+ was elevated by preincubation with ouabain or amphotericin B. With increasing AA concentration, Na+-dependent AA uptake exhibited first-order saturation kinetics with half-maximal uptake at 76 microM AA. Na+ dependence of AA uptake revealed a sigmoidal curve of Na+-dependent AA uptake vs. Na+ concentration with a half-maximal AA uptake at 45.4 mM Na+. The slope of the Hill plot from these data was 1.94, suggesting a transport system translocating two or more Na+ for one AA. This stoichiometry implies electrogenicity of the transporter. We, therefore, measured membrane potentials using conventional microelectrodes. Addition of 200 microM AA resulted in a depolarization of the membrane voltage by 4.9 +/- 0.5 mV (n = 22), which was absent in Na+ free medium and was markedly reduced by phloretin.(ABSTRACT TRUNCATED AT 250 WORDS)
Cigarette smoking decreases interleukin 1 release by human alveolar macrophages
Authors: Brown G. P. , Iwamoto G. K. , Monick M. M. , Hunninghake G. W.
Published: December 2017

To determine whether alveolar macrophages from smokers have an abnormal interleukin 1 beta (IL-1) release, we obtained macrophages by bronchoalveolar lavage (BAL) of otherwise healthy volunteers in three groups: nonsmokers (NS; n = 11), light smokers (LS, less than 10 pack-yr smoking history; n = 4) and heavy smokers (HS, greater than 10 pack-yr smoking history; n = 9). After 24 h in culture, unstimulated macrophages (from each group) released negligible amounts of IL-1. Lipopolysaccharide (LPS) (1 micrograms/ml) caused release of 21.77 +/- 4.33 ng IL-1/10(6) cells at 24 h from NS macrophages; IL-1 release from HS macrophages was significantly decreased (5.52 +/- 1.66 ng/10(6) cells; P less than 0.05), whereas LS macrophages released intermediate amounts (15.07 +/- 6.15 ng/10(6) cells). Release of IL-1 from HS macrophages was also decreased after 48 and 72 h in culture and was observed over a wide range of concentrations of LPS. The decreased amount of IL-1 in HS macrophage supernatants appeared to be due to a defect in release of IL-1 from the cells and not due to a defect in production of the mediator, since total IL-1 (IL-1 present in the cell lysates plus that in the cell supernatants) was similar in the NS and HS groups. In addition, after 24 h in culture, LPS-stimulated HS macrophages released significantly less prostaglandin E2 (PGE2) (which can suppress IL-1 production) than did NS macrophages; in the presence of indomethacin, which abolished macrophage PGE2 release, no augmentation of LPS-stimulated IL-1 release was observed. Cell viability, as measured by lactate dehydrogenase release, was not different between HS and NS macrophages under any conditions. We conclude that there is a defect in release but not production of IL-1 from the alveolar macrophages of chronic smokers.
Endothelial cells from human umbilical vein secrete functional transcobalamin II
Authors: Quadros E. V. , Rothenberg S. P. , Jaffe E. A.
Published: December 2017

Transcobalamin II (TCII) is a cobalamin (Cbl) binding protein in the plasma that mediates the cellular uptake of Cbl. Although the synthesis of TCII by a variety of cultured mammalian cells and by some isolated perfused organs has been reported, no single tissue has been identified as the source of TCII in vivo. In this study, we demonstrate that cultured human umbilical vein endothelial cells secrete a protein that binds CN[57Co]Cbl, elutes from a Sephacryl S-200 column in the same position as TCII, and precipitates with an antiserum to purified human TCII. The biosynthesis of TCII by these cells was confirmed by demonstrating the incorporation of [35S]methionine into a nascent protein that immunoprecipitated with anti-TCII and which, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) had an Mr of 43,000, the same as human TCII. This secreted protein also had the functional properties of TCII because it facilitated the uptake of CN[57Co]Cbl by the same endothelial cells that secreted it as well as other cell lines that express the membrane receptor for TCII. We also present evidence that the venous endothelium could be the source of TCII in vivo by showing that an intact umbilical vein in an isolated umbilical cord, when perfused with medium containing [35S]methionine, secretes a radiolabeled nascent protein with the same immunoreactive and electrophoretic properties as human TCII. These studies demonstrate that the endothelial cell, which has been shown to secrete a number of plasma proteins, also synthesizes and secretes TCII both in vitro and as an intact endothelium in situ, and therefore, could be the source of circulating TCII in vivo.
Electrical pacemakers of canine proximal colon are functionally innervated by inhibitory motor neurons
Authors: Smith T. K. , Reed J. B. , Sanders K. M.
Published: December 2017

Pacemaker activity in the canine proximal colon occurs at the submucosal and myenteric borders of the circular layer [Am. J. Physiol. 252 (Cell Physiol. 21): C215-C224 and C290-C299, 1987]. The present study investigated the neural regulation of rhythmic electrical activity. Spontaneous inhibitory junction potentials (IJPs) were observed in intracellular recordings from circular muscle cells near the myenteric border. The amplitudes of these events decayed with distance through the circular layer. Stimulation at the myenteric plexus surface evoked IJPs that mimicked the spontaneous events. Stimulation at the submucosal surface evoked IJPs in adjacent cells that were of shorter duration and of different waveform than myenteric IJPs. Amplitudes of IJPs evoked by stimulation near either surface decayed with distance from the site of stimulation. The decay functions for IJPs were essentially identical to the decay of spontaneous slow waves or myenteric potential oscillations. Spontaneous and evoked IJPs affected the amplitudes, durations, and patterns of ongoing rhythmic electrical activity. The data suggest that myenteric and submucosal pacemaker populations may be innervated by different populations of inhibitory nerve fibers. Innervation appears to be heterogeneous with dense populations of inhibitory nerve fibers predominantly located in the pacemaker regions. Neural regulations of pacemaker activity influences rhythmic electrical activity throughout the muscularis.
Dye-coupling compartments in the human eccrine sweat gland
Authors: Jones C. J. , Quinton P. M.
Published: December 2017

The dye-coupling status of secretory and reabsorptive cells in micro-dissected lengths of human eccrine sweat gland was investigated by means of intracellular microiontophoresis of the fluorescent naphthalimide dye Lucifer yellow CH (Mr 457), which passes through gap junctions. Cells of the reabsorptive duct exhibited complete dye coupling between the apical and basal layers of the epithelium. Conversely, cells of the secretory tubule exhibited selective dye coupling. Of the three cell types present, clear, dark, and myoepithelial, the dark cells were impaled and labeled almost exclusively in the present study. These cells were observed either as single cells or as dye-coupled groups of neighboring dark cells. In no instance were dark cells observed to be dye coupled to clear cells or to myoepithelial cells. Because myoepithelial cells are known to be dye coupled exclusively to neighboring myoepithelial cells, the remaining clear cells must either be uncoupled or show selective dye coupling to neighboring clear cells. The significance of these findings is considered with respect to the regulation and function of the different cell types present in the human eccrine sweat gland.
Rat lung lectin gene expression is regulated developmentally and by dexamethasone
Authors: Clerch L. B. , Whitney P. , Massaro D.
Published: December 2017

The cell-agglutinating activity of soluble beta-galactoside-binding proteins (lectins) is developmentally regulated in several mammalian organs. Little is known of the alterations in gene expression that underlie this developmental regulation. Rat lung contains a dimeric beta-galactoside-binding protein that exhibits a postnatal peak of hemagglutination activity caused in part by an increased rate of lectin synthesis. We now report rat lung lectin mRNA concentration increased to a peak at age 6 days; dexamethasone treatment aborted this increase. Southern blot analysis is compatible with the presence of more than one lectin gene. However, two lines of evidence indicate that we measured a single gene product: 1) only one lectin of subunit Mr 14,000 is present in rat lung (Biochemistry 27: 692-699, 1988), and 2) in Northern blot analysis of RNA, the lectin cDNA hybridized with only one mRNA species. Our present findings, taken with prior studies of lectin synthesis, indicate that the postnatal increase in lectin synthesis is mediated pretranslationally and by an increased efficiency of translation. Dexamethasone treatment impairs the increase of lectin mRNA concentration but increases translational efficiency.
Alanine transport systems in isolated basal plasma membrane of human placenta
Authors: Hoeltzli S. D. , Smith C. H.
Published: December 2017

Concentrative transfer of amino acids from mother to fetus is affected by transport across both microvillous (maternal-facing) and basal (fetal-facing) plasma membranes of the human placental syncytiotrophoblast. Isolated basal plasma membrane vesicles were used to elucidate transport systems for neutral amino acids across this membrane. The concentration dependence and inhibition of zero-trans-alanine uptake were studied and four pathways for alanine uptake were defined as follows: 1) a sodium-dependent system shared by methylaminoisobutyric acid, which has the characteristics of an A system; 2) a sodium-dependent system resistant to inhibition by methylaminoisobutyric acid, which has the characteristics of an ASC system; 3) a sodium-independent system which may resemble an L system; 4) nonsaturable uptake. The microvillous membrane of the syncytiotrophoblast possesses systems similar to 1 and 3, but system 2 is unique to the basal plasma membrane. Active and passive transport of amino acids across both microvillous and basal plasma membranes may contribute to trophoblast amino acid uptake and nutrition and to the transfer of amino acids to the fetus.
Protection of Ca pump of coronary artery against inactivation by superoxide radical
Authors: Grover A. K. , Samson S. E.
Published: December 2017

Superoxide radicals inactivate endoplasmic reticular (ER) Ca2+ pump in membranes isolated from smooth muscle of pig right coronary artery [Am. J. Physiol. 255 (Cell Physiol. 24): C297-C303, 1988]. We report on protective mechanisms against such inactivation. This tissue contained superoxide dismutase (SOD) and catalase. SOD was distributed primarily in cytosolic fraction, was cyanide sensitive, and was also present in mitochondrial fraction, and approximately 25% of this was cyanide insensitive. Catalase was distributed mainly in mitochondrial fraction and did not protect against inactivation of ER Ca2+ pump by superoxide radicals generated using xanthine plus xanthine oxidase. However, cytosolic fraction protected against this inactivation by two mechanisms: 1) DTT carried over from homogenization medium and 2) its intrinsic SOD content. Soluble fraction was concentrated, dialyzed to remove 1,4-dithiothreitol (DTT), lyophilized, and suspended in a small volume of DTT-free buffer. It still protected against superoxide inactivation of Ca2+ pump. On Sephacryl-300 gel chromatography, protecting activity comigrated with SOD. DTT protected against inactivation, but glutathione and cysteine protected only partially. Neither sulfhydryl agents nor SOD could reverse the inactivation process. Ca2+ pump activity was abolished by dithionitrobenzoate and p-chloromercuric benzoate. Superoxide may inactivate ER Ca2+ pump by irreversibly modifying key sulfhydryl group(s) on pump molecule and SOD in coronary artery smooth muscle may partially protect against this inactivation.
Intracellular Na+ regulation of Na+ pump sites in cultured vascular smooth muscle cells
Authors: Allen J. C. , Navran S. S. , Seidel C. L. , Dennison D. K. , Amann J. M. , Jemelka S. K.
Published: December 2017

Enzymatically dispersed cells from canine saphenous vein and femoral artery were grown in fetal calf serum and studied at day 0 (freshly dispersed) through confluence in primary culture. Intracellular Na levels (Nai), but not intracellular K (Ki), were increased after 24 h in culture and then decreased to a steady state by 4 days. Na+ pump site number [( 3H] ouabain binding) increased through day 3 and remained elevated. Nai was still elevated at 2 days when the Na+ pump site number began to increase. Total pump turnover (maximum ouabain-inhibited 86Rb uptake) reflected the increase in Na+ pump site number. These key events precede the observed increases in both protein production and cellular proliferation. If the same cells are maintained in defined medium, without fetal calf serum, Nai, Ki, and the number of [3H]ouabain binding sites do not change with time. These data are consistent with the suggestion that the initial mitogenic response of vascular smooth muscle cells to fetal calf serum involves an increased Na+ influx, and a Nai accumulation, caused by low Na+ pump density. The synthesis of new pump sites effects a decrease in the accumulated Nai, which may be related to cell proliferation.
Basolateral impalement of intestinal villus cells: electrophysiology of Cl- transport
Authors: White J. F. , Ellingsen D.
Published: December 2017

A method of dissecting the serosal muscle layers is described that transforms the villus of isolated Amphiuma small intestine into a flat sheet one cell layer thick, allowing rapid equilibration of the serosal medium with the basolateral membrane of the villus tip cells and direct access of the basal membrane to microelectrodes. The "villus sheet" preparation was used to examine the luminal and basolateral mechanisms of Cl- transport. The serosal membrane potential (Vs), measured with conventional microelectrodes, averaged -79.7 mV in tissues bathed in Cl- -free medium; the mucosal membrane potential (Vm) averaged -80.9 mV. Fractional resistance measured directly was 0.82 and 0.14 for the mucosal and serosal membranes, respectively. Elevation of bath [K] reduced Vm and Vs by 30.3 and 44.5 mV, respectively. Cl- (20 mM) added to the luminal medium reduced Vm by 23.9 mV and stimulated Cl- transport; luminal addition of furosemide then increased Vm by 5.6 mV and reduced Cl- transport. Addition of Cl- (20 mM) to the Cl- -free serosal fluid increased Vs 2.0 +/- 1.9 mV. On reducing the serosal [Cl] 10-fold Vs decreased 2.0 +/- 2.2 mV. These and other results indicate that basolateral Cl- exit is not over a conductive pathway. The villus sheet affords new opportunities for studying enterocyte function in the intact mucosa.
Rapid isolation of total RNA from small mammal and human skeletal muscle
Authors: Hood D. A. , Simoneau J. A.
Published: December 2017

The present study was designed to assess the efficacy and reliability of a method (Anal. Biochem. 162: 156-59, 1987) of total RNA isolation from small muscle samples. The RNA content of skeletal muscles in mouse, rabbit, guinea pig, rat, and human was also investigated. Enough RNA was extractable from muscle samples as small as 25 mg in weight to provide material for the analysis of specific mRNA sequences using RNA blot hybridization with a cDNA probe for a mitochondrially encoded subunit of cytochrome-c oxidase. The RNA was free of detectable levels of protein or DNA. Gel electrophoresis and cDNA probe hybridization indicated that the RNA was relatively undegraded. Significant differences (P less than 0.05) in RNA content were found among fiber types (type I fibers greater than type II fibers) and among the soleus muscles of various species (guinea pig greater than rat = mouse greater than rabbit). Human muscle possessed the lowest RNA content (350 ng/mg) among the species investigated. The results indicate that intact RNA can be rapidly and reliably isolated from small skeletal muscle, including samples typically obtained from human muscle biopsies. This technique should permit the analysis of gene expression in muscle subject to a variety of physiological treatments.
Effects of intracellular free Ca and rate of Ca influx on the Ca pump
Authors: Yang Y. C. , Yingst D. R.
Published: December 2017

The activity of the Ca pump of human red blood cells was studied in resealed ghosts as a function of intracellular free Ca (fCai). Resealed ghosts were made by the agarose column method to contain, in addition to other constituents, less than 0.1 microM fCai, 100 microM arsenazo III, and either 1 mM ATP plus an ATP regenerating system (active ghosts) or no added ATP and no regenerating system (passive ghosts). The rate of Ca influx into these ghosts was manipulated by suspending them in solutions containing various combinations of free Ca (1-30 microM) and the Ca ionophore A23187 (0.1-0.7 microM). Entering Ca increased the fCai and stimulated the pump in active ghosts. In passive ghosts, all the Ca movement could be described by a single rate constant. The activity of the Ca pump was calculated from the rate of net Ca uptake in the active ghosts, using the rate constant for passive Ca movement as determined in the passive ghosts. fCai and the rates of Ca transport in both active and passive ghosts were calculated from the absorbance of entrapped arsenazo III. In general, increasing fCai from 1 to 10 microM activated the pump. Higher fCai caused an inhibition compared with peak activity. The maximum rate of pumping was 80 microM/min. The major new finding is that the rate of active transport at a given fCai appeared to vary with the rate of fCai accumulation.(ABSTRACT TRUNCATED AT 250 WORDS)
pH effects on basolateral membrane ion conductances in gallbladder epithelium
Authors: Stoddard J. S. , Reuss L.
Published: December 2017

The pH sensitivity of the basolateral membrane voltage of Necturus gallbladder epithelial cells (Vcs) was evaluated with conventional and pH-sensitive intracellular microelectrodes. Elevating solution CO2 from 1 to 5% (at constant [HCO3-] = 10 mM) caused a depolarization of Vcs from -76 +/- 3 to -60 +/- 2 mV and a decrease in intracellular pH (pHi) from 7.36 +/- 0.04 to 7.05 +/- 0.03. Serosal exposure to a 50 mM HCO3(-)-5% CO2 solution [at constant extracellular pH (pHo)] caused a similar cell acidification (delta pHi = 0.27), whereas at 3 min Vcs was unchanged. Exposure to 1 mM HCO3- (at constant CO2) depolarized Vcs from -77 +/- 2 to -56 +/- 2 mV and caused a small decrease in pHi (from 7.36 +/- 0.03 to 7.33 +/- 0.03). These results indicate that the observed depolarizations of Vcs are attributable to changes in pHo and not in pHi. Basolateral membrane potassium conductance (GK) congruent to chloride conductance (GCl) congruent to 0.50 mS/cm2 in 10 mM HCO3(-)-1% CO2 Ringer. The depolarization of Vcs caused by elevation of serosal [K+] in 50 mM HCO3(-)-5% CO2 was similar to that observed under control conditions. In contrast, the depolarization of Vcs elicited by elevating serosal [K+] was reduced by about two-thirds in 1 mM HCO3-, whereas the depolarization caused by reduction of serosal [Cl-] was increased twofold in 1 mM HCO3-, compared with control. Inasmuch as the apparent ratio of membrane resistances remained unchanged during serosal solution acidification, the most likely explanation for the observed decrease in Vcs is a reduction of basolateral K+ permeability concomitant with an increase in Cl- permeability.
Potential role of mitochondrial calcium metabolism during reperfusion injury
Authors: Vlessis A. A. , Mela-Riker L.
Published: December 2017

Ischemia-reperfusion injury has been associated with intracellular H2O2 and superoxide radical production from accumulated hypoxanthine (HX) and xanthine oxidase (XO). The effect of H2O2 and superoxide radical on mitochondrial Ca2+ efflux was characterized in isolated renal mitochondria using a HX-XO system. Mitochondria were suspended in buffered medium containing 200 microM HX. Extramitochondrial Ca2+ was monitored kinetically at 660-685 nm using the Ca2+ indicator arsenazo III. After preloading mitochondria with 18-25 nmol Ca2+/mg protein, addition of XO to the medium caused a rapid oxidation of mitochondrial NAD(P)H followed by Ca2+ release. Ca2+ efflux was attributed to mitochondrial metabolism of H2O2 because efflux could be prevented with catalase but not superoxide dismutase. The Ca2+ efflux rate (r = 0.995) and lag time to Ca2+ efflux (r = 0.987) both correlate well with the NAD(P)H oxidation rate. Exogenous ATP prevents Ca2+ efflux in a dose-dependent fashion (Km = 35 microM ATP) without affecting NAD(P)H oxidation; ATP plus oligomycin, however, had no effect. The protective effect of ATP on Ca2+ efflux was diminished by ruthenium red (RR). XO-induced Ca2+ efflux increased state 4 respiration 148% via a futile Ca2+ cycle involving the Ca2+ uniport. The increase in state 4 respiration could be reversed with RR (alpha less than 0.001) or ATP (alpha less than 0.01); ATP plus oligomycin, however, had no effect. The results are discussed in relation to the oxygen free radical theory of reperfusion injury.
Hypoxia stimulates endothelial cell angiotensin-converting enzyme antigen synthesis
Authors: King S. J. , Booyse F. M. , Lin P. H. , Traylor M. , Narkates A. J. , Oparil S.
Published: December 2017

Previous studies from our laboratory indicate that exposure of the rat to chronic normobaric hypoxia reduces stores of active angiotensin-converting enzyme (ACE) in the lung. This study assesses directly the effects of hypoxia on ACE synthesis in cultured porcine pulmonary artery endothelial cells. Confluent cultures were exposed to hypoxia [2.5% O2 at 1 atmosphere (atm)] in a triple gas incubator; controls were cultured in normoxic conditions. After 24-, 48-, and 72-h exposure to hypoxic or normoxic conditions, followed by incubation with [35S]methionine for an additional 24 h under the same conditions, newly synthesized radiolabeled ACE was quantitated. Radiolabeled ACE was isolated by an immunobead procedure using either anti-ACE (porcine lung) immunoglobin G (IgG) or nonimmune IgG. A single radiolabeled peak (150 kDa) with the same electrophoretic mobility as purified porcine lung ACE was observed. There was a significant time-dependent increase in endothelial cell ACE antigen synthesis without a concomitant change in either cell number or total trichloroacetic (TCA)-precipitable protein in hypoxic cells compared with normoxic controls. In contrast, ACE activity, assessed by conversion of 125I-labeled angiotensin I to 125I-labeled angiotensin II was unchanged in cultures exposed to hypoxia (2.5% O2). This suggests that an inactive form of ACE is synthesized by cultured pulmonary artery endothelial cells under hypoxic conditions.
Muscle transplantation between young and old rats: age of host determines recovery
Authors: Carlson B. M. , Faulkner J. A.
Published: December 2017

As compared with age-matched controls, extensor digitorum longus (EDL) muscles autografted in young rats regenerated significantly greater mass (1.8 times) and developed greater maximum contractile force (2.6 times) than EDL muscles autografted in old rats. A cross-age transplantation study showed that the mass and maximum force of old muscles grafted into young hosts were not significantly different from those of young muscles grafted into the same young hosts. Conversely, young muscle grafted into old hosts regenerated no better than old muscles grafted into the same old hosts. We conclude 1) that chronological age alone is not a factor that limits the intrinsic ability of a muscle to regenerate and 2) that the poor regeneration of muscles in old animals is a function of the environment for regeneration provided by the old host.
Ouabain treatment of cardiac cells induces enhanced Na+-Ca2+ exchange activity
Authors: Vemuri R. , Longoni S. , Philipson K. D.
Published: December 2017

Inhibition of the cardiac Na+-K+-ATPase with cardiac glycosides causes a rise in internal Na+ and a subsequent increase in cellular Ca2+ due to the sarcolemmal Na+-Ca2+ exchange mechanism. We investigated the adaptation of cultured cardiac cells to prolonged elevation of internal Ca2+ after exposure to ouabain. Cultured neonatal rat heart cells were treated with 100 microM ouabain for 4-48 h. This ouabain concentration inhibited Na+-K+-ATPase activity by approximately 45% and caused modest cellular Ca2+ loading. We found that cells adapted to ouabain treatment by increasing the amount of sarcolemmal Na+-Ca2+ exchange activity by 50-90% over a 24-h period. Kinetic and immunological data indicate that the increase was due to increased numbers of functional exchangers. Neither total cellular nor total sarcolemmal protein content was affected by the ouabain treatment. These results may be relevant toward understanding the effects of therapeutic use of cardiac glycosides.
Inwardly rectifying K+ current in osteoclasts
Authors: Sims S. M. , Dixon S. J.
Published: December 2017

Membrane properties of freshly isolated rat osteoclasts were studied using the whole cell patch-clamp recording technique. The membrane potential could switch between two stable levels, approximately -70 and -15 mV. Voltage-clamp studies indicated that osteoclasts exhibited marked inward rectification, with hyperpolarizing voltage commands from -70 mV activating large inward currents. No voltage-dependent currents were observed in response to depolarization. An increase in external K+ concentration shifted the current-voltage relationship positive in a manner predicted for K+ current. Furthermore, barium and cesium reversibly suppressed the inward current. Thus the dominant current evident in osteoclasts was inwardly rectifying K+ current, resembling that found in a number of cell types, including cardiac and skeletal muscle and oocytes. The current-voltage relationship of osteoclasts was "N-shaped" and could intersect the zero-current level at three potentials, accounting for two stable membrane potentials. Switching of membrane potential between these two levels may regulate a number of the cellular processes involved in bone resorption.
cAMP delays disappearance of gap junctions between pairs of rat hepatocytes in primary culture
Authors: Saez J. C. , Gregory W. A. , Watanabe T. , Dermietzel R. , Hertzberg E. L. , Reid L. , Bennett M. V. , Spray D. C.
Published: December 2017

Freshly isolated adult rat hepatocytes were found to be coupled through gap junctions, but coupling decreased abruptly 5-8 h after plating the cells on plastic culture dishes in physiological saline containing insulin and fetal calf serum. Loss of intercellular coupling was associated with disappearance of 27-kDa gap junction protein and of gap junctions seen by electron microscopy or immunocytochemistry. This disappearance of coupling was delayed approximately 8 h by treatment of the cultures with membrane permeant adenosine 3',5'-cyclic monophosphate (cAMP) [but not guanosine 3',5'-cyclic monophosphate (cGMP)] derivatives. Levels of gap junction protein and anatomically identified junctions were also maintained by 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP). Level of mRNA encoding the gap junction protein was maintained longer in cells treated with 8-BrcAMP than in untreated cells, but 8-BrcAMP did not detectably increase the transcription rate. Thus prolongation of coupling must result at least partially from extension of the lifetime of gap junction mRNA, allowing translation of message and assembly of channels for a longer period after plating. Treatment of cells with mRNA or protein synthesis inhibitors (alpha-amanitin and cycloheximide) prolonged coupling to a similar extent as did treatment with 8-BrcAMP. alpha-Amanitin blocked transcription of gap junction mRNA, but levels of cytoplasmic mRNA encoding the 27-kDa gap junction protein were maintained, presumably by block of transcription of an mRNA degrading factor. The factor is probably a protein, since a similar effect on mRNA level was produced in cycloheximide-treated cells. Cells cultured in control medium were also observed to flatten as they became uncoupled, whereas cells cultured for as long as 16 h in elevated 8-BrcAMP remained round and well coupled. The correlation between shape and coupling strength was not obtained after treatment with the microtubule stabilizing agent, taxol, which maintained the spherical shape of the cells but did not delay the disappearance of dye coupling. Nocodazole, which blocks microtubule formation, also maintained the spherical shape of the cells but delayed the disappearance of dye coupling. In addition to gating by covalent modification or other mechanisms, hormones and drugs that alter the intracellular cAMP concentration may affect intercellular communication by changing the lifetime of the mRNA encoding the main gap junction protein, thereby decreasing or increasing its synthesis. In addition, cAMP may act by decreasing removal of junctions from appositional membranes.