bluuebunny/crossref_metadata_2025 huggingface dataset:
Endothelin-1 decreases glutamate uptake in primary cultured rat astrocytes
Authors: Leonova Julia , Thorlin Thorleif , Åberg N. David , Eriksson Peter S. , Rönnbäck Lars , Hansson Elisabeth
Published: December 2017

Endothelin-1 (ET-1) is a potent vasoconstrictor peptide that is also known to induce a wide spectrum of biological responses in nonvascular tissue. In this study, we found that ET-1 (100 nM) inhibited the glutamate uptake in cultured astrocytes expressing the glutamate/aspartate transporter (GLAST); astrocytes did not express the glutamate transporter-1 (GLT-1). The V maxand the K m of the glutamate uptake were reduced by 57% and 47%, respectively. Application of the ETA and ETB receptor antagonists BQ-123 and BQ-788 partly inhibited the ET-1-evoked decrease in the glutamate uptake, whereas the nonspecific ET receptor antagonist bosentan completely inhibited this decrease. Incubation of the cultures with pertussis toxin abolished the effect of ET-1 on the uptake. The ET-1-induced decrease in the glutamate uptake was independent of extracellular free Ca2+concentration, whereas the intracellular Ca2+ antagonists thapsigargin and 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester abolished the effect of ET-1 on the glutamate uptake. Incubation with the protein kinase C (PKC) antagonist staurosporine, but not with the fatty acid-binding protein bovine serum albumin, prevented the ET-1-induced decrease in the glutamate uptake. These results suggest that ET-1 impairs the high-affinity glutamate uptake in cultured astrocytes through a G protein-coupled mechanism, involving PKC and changes in intracellular Ca2+.
Human endothelial cell response to gram-negative lipopolysaccharide assessed with cDNA microarrays
Authors: Zhao Baiteng , Bowden Robert A. , Stavchansky Salomon A. , Bowman Phillip D.
Published: December 2017

To assess the feasibility of using cDNA microarrays to understand the response of endothelial cells to lipopolysaccharide (LPS) and to evaluate potentially beneficial agents in treatment of septic shock, human umbilical vein endothelial cells were exposed to Escherichia coli LPS for 1, 4, 7, 12, or 24 h. Total RNA was isolated and reverse-transcribed into33P-labeled cDNA probes that were hybridized to human GeneFilter microarrays containing ∼4,000 genes. The mRNA levels of several genes known to respond to LPS changed after stimulation. In addition, a number of genes not previously implicated in the response of endothelial cells to LPS also appeared to be altered in expression. Nuclear factor-κB (NF-κB) was shown to play an important role in regulating genes identified from the microarray studies. Pretreatment of endothelial cells with a specific NF-κB translocation inhibitor eliminated most of the alterations in gene expression. Quantitative RT-PCR results independently confirmed the microarray results for monocyte chemotactic protein-1 and interleukin-8, and enzyme-linked immunosorbent assays demonstrated that augmented transcription was followed by translation and secretion.
Endogenous protein phosphatase 1 runs down gap junctional communication of rat ventricular myocytes
Authors: Duthe Fabien , Plaisance Isabelle , Sarrouilhe Denis , Hervé Jean Claude
Published: December 2017

Gap junctional channels are essential for normal cardiac impulse propagation. In ventricular myocytes of newborn rats, channel opening requires the presence of ATP to allow protein kinase activities; otherwise, channels are rapidly deactivated by the action of endogenous protein phosphatases (PPs). The lack of influence of Mg2+ and of selective PP2B inhibition is not in favor of the involvements of Mg2+-dependent PP2C and PP2B, respectively, in the loss of channel activity. Okadaic acid (1 μM) and calyculin A (100 nM), both inhibitors of PP1 and PP2A activities, significantly retarded the loss of channel activity. However, a better preservation was obtained in the presence of selective PP1 inhibitors heparin (100 μg/ml) or protein phosphatase inhibitor 2 (I2; 100 nM). Conversely, the stimulation of endogenous PP1 activity by p-nitrophenyl phosphate, in the presence of ATP, led to a progressive fading of junctional currents unless I2 was simultaneously added. Together, these results suggest that a basal phosphorylation-dephosphorylation turnover regulates gap junctional communication which is rapidly deactivated by PP1 activity when the phosphorylation pathway is hindered.
Ca2+ channels activated by endothelin-1 in CHO cells expressing endothelin-A or endothelin-B receptors
Authors: Kawanabe Yoshifumi , Okamoto Yasuo , Enoki Taijiro , Hashimoto Nobuo , Masaki Tomoh
Published: December 2017

We compared the Ca2+ channels activated by endothelin-1 (ET-1) in Chinese hamster ovary (CHO) cells stably expressing endothelin type A (ETA) or endothelin type B (ETB) receptors using the Ca2+ channel blockers LOE-908 and SK&F-96365. In both CHO-ETA and CHO-ETB, ET-1 at 0.1 nM activated the Ca2+-permeable nonselective cation channel-1 (NSCC-1), which was sensitive to LOE-908 and resistant to SK&F-96365. ET-1 at 1 nM activated NSCC-2 in addition to NSCC-1; NSCC-2 was sensitive to both LOE-908 and SK&F-96365. ET-1 at 10 nM activated the same channels as 1 nM ET-1 in both cell types, but in CHO-ETA, it additionally activated the store-operated Ca2+ channel (SOCC), which was resistant to LOE-908 and sensitive to SK&F-96365. Up to 1 nM ET-1, the level of the formation of inositol phosphates (IPs) was low and similar in both cell types, but, at 10 nM ET-1, it was far greater in CHO-ETA than in CHO-ETB. These results show that, in CHO-ETAand CHO-ETB, ET-1 up to 10 nM activated the same Ca2+ entry channels: 0.1 nM ET-1 activated NSCC-1, and ET-1 ≥ 1 nM activated NSCC-1 and NSCC-2. Notably, in CHO-ETA, 10 nM ET-1 activated SOCCs because of the higher formation of IPs.
Influence of overload on phenotypic remodeling in regenerated skeletal muscle
Authors: Bigard A. X. , Zoll J. , Ribera F. , Mateo P. , Sanchez H. , Serrurier B. , Ventura-Clapier R.
Published: December 2017

We studied the effects of 10 wk of functional overload on the expression of myosin heavy chain (MHC), sarcoplasmic reticulum Ca2+-ATPase isoforms (SERCA), and the activity of several metabolic enzymes in sham and regenerated plantaris muscles. Overload was accomplished by bilateral surgical ablation of its synergists 4 wk after right plantaris muscles regenerated after myotoxic infiltration. The overload-induced muscle enlargement was slightly less in regenerated than in sham muscles [28% ( P < 0.005) and 43% ( P < 0.001), respectively]. Overload led to an increase in type I MHC expression ( P < 0.01) to a similar extent in sham and regenerated plantaris, while the expected shift from type IIb to type IIa MHC was less marked in regenerated than in sham plantaris. The overload-induced decrease in the expression of the fast SERCA isoform and in the activity of the M subunit of lactate dehydrogenase occurred to a similar extent in sham and regenerated plantaris [66% ( P < 0.01) and 27% ( P < 0.005), respectively]. In conclusion, the lesser responses of muscle mass and fast MHC composition of regenerated plantaris to mechanical overload suggest an alteration of the transcriptional, translational, and/or posttranslational control of gene expression in regenerated muscle.
Insulin increases plasma membrane content and reduces phosphorylation of Na+-K+ pump α1-subunit in HEK-293 cells
Authors: Sweeney Gary , Niu Wenyan , Canfield Victor A. , Levenson Robert , Klip Amira
Published: December 2017

Insulin stimulates K+ uptake and Na+ efflux via the Na+-K+ pump in kidney, skeletal muscle, and brain. The mechanism of insulin action in these tissues differs, in part, because of differences in the isoform complement of the catalytic α-subunit of the Na+-K+ pump. To analyze specifically the effect of insulin on the α1-isoform of the pump, we have studied human embryonic kidney (HEK)-293 cells stably transfected with the rat Na+-K+ pump α1-isoform tagged on its first exofacial loop with a hemagglutinin (HA) epitope. The plasma membrane content of α1-subunits was quantitated by binding a specific HA antibody to intact cells. Insulin rapidly increased the number of α1-subunits at the cell surface. This gain was sensitive to the phosphatidylinositol (PI) 3-kinase inhibitor wortmannin and to the protein kinase C (PKC) inhibitor bisindolylmaleimide. Furthermore, the insulin-stimulated gain in surface α-subunits correlated with an increase in the binding of an antibody that recognizes only the nonphosphorylated form of α1 (at serine-18). These results suggest that insulin regulates the Na+-K+ pump in HEK-293 cells, at least in part, by decreasing serine phosphorylation and increasing plasma membrane content of α1-subunits via a signaling pathway involving PI 3-kinase and PKC.
Determination of the enhancing action of HSP90 on neuronal nitric oxide synthase by EPR spectroscopy
Authors: Song Yao , Zweier Jay L. , Xia Yong
Published: December 2017

Recent studies showed that heat shock protein 90 (HSP90) enhances nitric oxide (NO) synthesis from endothelial and neuronal NO synthase (eNOS and nNOS, respectively). However, these findings were based on indirect NO measurements. Moreover, although our previous studies showed that the action of HSP90 involves increased Ca2+/calmodulin (Ca2+/CaM) binding, quantitative measurements of the effect of HSP90 on CaM binding to nNOS have been lacking. With electron paramagnetic resonance spectroscopy, we directly measured NO signals from purified nNOS. HSP90 augmented NO formation from nNOS in a dose-dependent manner. Tryptophan fluorescence-quenching measurements revealed that HSP90 markedly reduced the K d of CaM to nNOS (0.5 ± 0.1 nM vs. 9.4 ± 1.8 nM in the presence and absence of HSP90, P < 0.01). Ca2+ ionophore triggered strong NO production from nNOS-transfected cells, and this was significantly reduced by the HSP90 inhibitor geldanamycin. Thus these studies provide direct evidence demonstrating that HSP90 enhances nNOS catalytic function in vitro and in intact cells. The effect of HSP90 is mediated by the enhancement of CaM binding to nNOS.
Characteristics of EYFP-actin and visualization of actin dynamics during ATP depletion and repletion
Authors: Herget-Rosenthal Stefan , Hosford Melanie , Kribben Andreas , Atkinson Simon J. , Sandoval Ruben M. , Molitoris Bruce A.
Published: December 2017

Disruption of the actin cytoskeleton in proximal tubule cells is a key pathophysiological factor in acute renal failure. To investigate dynamic alterations of the actin cytoskeleton in live proximal tubule cells, LLC-PK10 cells were transfected with an enhanced yellow fluorescence protein (EYFP)-actin construct, and a clone with stable EYFP-actin expression was established. Confluent live cells were studied by confocal microscopy under physiological conditions or during ATP depletion of up to 60 min. Immunoblots of stable transfected LLC-PK10 cells confirmed the presence of EYFP-actin, accounting for 5% of total actin. EYFP-actin predominantly incorporated in stress fibers, i.e., cortical and microvillar actin as shown by excellent colocalization with Texas red phalloidin. Homogenous cytosolic distribution of EYFP-actin indicated colocalization with G-actin as well. Beyond previous findings, we observed differential subcellular disassembly of F-actin structures: stress fibers tagged with EYFP-actin underwent rapid and complete disruption, whereas cortical and microvillar actin disassembled at slower rates. In parallel, ATP depletion induced the formation of perinuclear EYFP-actin aggregates that colocalized with F-actin. During ATP depletion the G-actin fraction of EYFP-actin substantially decreased while endogenous and EYFP-F-actin increased. During intracellular ATP repletion, after 30 min of ATP depletion, there was a high degree of agreement between F-actin formation from EYFP-actin and endogenous actin. Our data indicate that EYFP-actin did not alter the characteristics of the endogenous actin cytoskeleton or the morphology of LLC-PK10 cells. Furthermore, EYFP-actin is a suitable probe to study the spatial and temporal dynamics of actin cytoskeleton alterations in live proximal tubule cells during ATP depletion and ATP repletion.
In vivo fluorescence measurement of Na+concentration in the pericryptal space of mouse descending colon
Authors: Thiagarajah J. R. , Jayaraman S. , Naftalin R. J. , Verkman A. S.
Published: December 2017

A method involving surgical exposure of the colonic mucosa, fluorescent dye addition, and confocal microscopy has been developed for monitoring colonic crypt function in vivo in mice. Na+ concentration in the extracellular pericryptal space of descending colon was measured using a low-affinity Na+-sensitive fluorescent indicator consisting of an Na+-sensitive chromophore (sodium red) and an Na+-insensitive chromophore (Bodipy-fl) immobilized on 200-nm-diameter polystyrene beads. The Na+ indicator beads accumulated in the pericryptal spaces surrounding the colonic crypts after a 1-h exposure of the colonic luminal surface to the bead suspension. Na+ concentration ([Na+]) in the pericryptal space was 491 ± 62 mM ( n = 4). After a 70-min exposure to amiloride (0.25 mM), pericryptal [Na+] was reduced to 152 ± 21 mM. Blockage of the crypt lumen with mineral oil droplets reduced pericryptal [Na+] to 204 ± 44 mM. Exposure of the colonic mucosa to FITC-dextran (4.5 kDa) led to rapid accumulation of the dye into the crypt lumen with a half time of 19.8 ± 1.0 s, which was increased to 77.9 ± 6.0 s after amiloride treatment. These results establish an in vivo fluorescence method to measure colonic crypt function and provide direct evidence for accumulation of a hypertonic absorbate in the pericryptal space of descending colon. The pericryptal space represents the first example of a hypertonic extracellular compartment in mammals that is not created by a countercurrent amplification mechanism.
Cerebral vascular endothelial heme oxygenase: expression, localization, and activation by glutamate
Authors: Parfenova Helena , Neff Robert A. , Alonso Julie S. , Shlopov Boris V. , Jamal Chaklader N. , Sarkisova Svetlana A. , Leffler Charles W.
Published: December 2017

Endogenous carbon monoxide (CO) contributes to vasodilator responses of cerebral microvessels in newborn pigs. We investigated the expression, intracellular localization, and activity of heme oxygenase (HO), the key enzyme in CO production, in quiescent cerebral microvascular endothelial cells (CMVEC) from newborn pigs. HO-1 and HO-2 isoforms were detected by RT-PCR, immunoblotting, and immunofluorescence. HO-1 and HO-2 are membrane-bound proteins that have a strong preference for the nuclear envelope and perinuclear area of the cytoplasm. Betamethasone (10−6 to 10−4 M for 48 h) was associated with upregulation of HO-2 protein by ∼50% and inhibition of Cox-2 but did not alter HO-1 or endothelial nitric oxide synthase expression in CMVEC. In vivo betamethasone treatment of newborn pigs (0.2 and 5.0 mg/kg im for 48 h) upregulated HO-2 in cerebral microvessels by 30–60%. HO activity as 14CO production from [14C]glycine-labeled endogenous heme was inhibited by chromium mesoporphyrin (10−6 to 10−4 M).l-Glutamate (0.3–1.0 mM) stimulated HO activity 1.5-fold. High-affinity specific binding sites forl-[3H]glutamate suggestive of the glutamate receptors were detected in CMVEC. Altogether, these data suggest that, in cerebral circulation of newborn pigs, endothelium-derived CO may contribute to basal vascular tone and to responses that involve glutamate receptor activation.
Role of glutamate transporters in the regulation of glutathione levels in human macrophages
Authors: Rimaniol Anne-Cécile , Mialocq Patricia , Clayette Pascal , Dormont Dominique , Gras Gabriel
Published: December 2017

Cysteine is the limiting precursor for glutathione synthesis. Because of its low bioavailability, cysteine is generally produced from cystine, which may be taken up through two different transporters. The cystine/glutamate antiporter (x[Formula: see text] system) transports extracellular cystine in exchange for intracellular glutamate. The XAG transport system takes up extracellular cystine, glutamate, and aspartate. Both are sensitive to competition between cystine and glutamate, and excess extracellular glutamate thus inhibits glutathione synthesis, a nonexcitotoxic mechanism for glutamate toxicity. We demonstrated previously that human macrophages express the glutamate transporters excitatory amino acid transporter (EAAT)1 and EAAT2 (which do not transport cystine, X[Formula: see text] system) and overcome competition for the use of cystine transporters. We now show that macrophages take up cystine through the x[Formula: see text] and not the XAG system. We also found that glutamate, although competing with cystine uptake, dose-dependently increases glutathione synthesis. We used inhibitors to demonstrate that this increase is mediated by EAATs. EAAT expression in macrophages thus leads to glutamate-dependent enhancement of glutathione synthesis by providing intracellular glutamate for direct insertion in glutathione and also for fueling the intracellular pool of glutamate and trans-stimulating the cystine/glutamate antiporter.
PPAR-γ ligands modulate effects of LPS in stimulated rat synovial fibroblasts
Authors: Simonin Marie-Agnès , Bordji Karim , Boyault Sandrine , Bianchi Arnaud , Gouze Elvire , Bécuwe Philippe , Dauça Michel , Netter Patrick , Terlain Bernard
Published: December 2017

This work demonstrated the constitutive expression of peroxisome proliferator-activated receptor (PPAR)-γ and PPAR-α in rat synovial fibroblasts at both mRNA and protein levels. A decrease in PPAR-γ expression induced by 10 μg/ml lipopolysaccharide (LPS) was observed, whereas PPAR-α mRNA expression was not modified. 15-Deoxy-Δ12,14-prostaglandin J2(15d-PGJ2) dose-dependently decreased LPS-induced cyclooxygenase (COX)-2 (−80%) and inducible nitric oxide synthase (iNOS) mRNA expression (−80%), whereas troglitazone (10 μM) only inhibited iNOS mRNA expression (−50%). 15d-PGJ2 decreased LPS-induced interleukin (IL)-1β (−25%) and tumor necrosis factor (TNF)-α (−40%) expression. Interestingly, troglitazone strongly decreased TNF-α expression (−50%) but had no significant effect on IL-1β expression. 15d-PGJ2 was able to inhibit DNA-binding activity of both nuclear factor (NF)-κB and AP-1. Troglitazone had no effect on NF-κB activation and was shown to increase LPS-induced AP-1 activation. 15d-PGJ2 and troglitazone modulated the expression of LPS-induced iNOS, COX-2, and proinflammatory cytokines differently. Indeed, troglitazone seems to specifically target TNF-α and iNOS pathways. These results offer new insights in regard to the anti-inflammatory potential of the PPAR-γ ligands and underline different mechanisms of action of 15d-PGJ2 and troglitazone in synovial fibroblasts.
Voxx: a PC-based, near real-time volume rendering system for biological microscopy
Authors: Clendenon Jeffrey L. , Phillips Carrie L. , Sandoval Ruben M. , Fang Shiaofen , Dunn Kenneth W.
Published: December 2017

Confocal and two-photon fluorescence microscopy have advanced the exploration of complex, three-dimensional biological structures at submicron resolution. We have developed a voxel-based three-dimensional (3-D) imaging program (Voxx) capable of near real-time rendering that runs on inexpensive personal computers. This low-cost interactive 3-D imaging system provides a powerful tool for analyzing complex structures in cells and tissues and encourages a more thorough exploration of complex biological image data.
Organ culture studies of rat antrum: evidence for an antral inhibitor of gastrin release.
Authors: Lichtenberger L M , Shorey J M , Trier J S
Published: December 2017

The regulation of gastrin release in rodent antral mucosal organ cultures was investigated. The tissue was well preserved morphologically and medium gastrin concentration increased steadily throughout a 24-h culture period. The effects of peptone and a bovine serum albumin digest on gastrin release were independently investigated. During the periods these agents were in contact with the tissue, medium gastrin concentration did not differ from those of control cultures. However, treatment cultures released a significantly greater amount of gastrin into the medium than did control cultures during the two posttreatment periods. Prolongation of the period of exposure to peptone did not alter this secretory pattern. The rise in medium gastrin concentration that followed the removal of peptone was directly related to the medium peptone concentration and was partially inhibited by readdition of peptone to cultured antral explants. These results suggest that substances which stimulate gastrin release in vivo may cause the accumulation of an antral inhibitor of gastrin release when exposed to rat antral mucosa in culture.
Interaction of caerulein and secretin on pancreatic size and composition in rat.
Authors: Solomon T E , Petersen H , Elashoff J , Grossman M I
Published: December 2017

Rats were given injections of caerulein, secretin, or a combination of these two peptides subcutaneously 3 times daily for 5, 10, or 15 days. Caerulein produced significant dose- and time-dependent increases in pancreatic weight and content of DNA, RNA, protein, amylase, and trypsinogen. Secretin produced significant increases in pancreatic weight and content of RNA and lipase after 15 days of treatment. After only 5 days of treatment with a combination of secretin plus caerulein, pancreatic weight and content of RNA and protein more than doubled, and trypsinogen content increased more than fivefold. Comparing the averages across the 5-, 10-, and 15-day values, increases in weight, protein, and trypsinogen with the combination of secretin plus caerulein were significantly greater than the sum of the effects of the peptides given singly. Using increase in DNA content as an index of hyperplasia and increases in the ratios of pancreatic weight, RNA content, and protein content to DNA content as indices of hypertrophy, we concluded that caerulein produced both hyperplasia and hypertrophy of rat pancreatic acinar cells. Secretin markedly augmented the hypertrophic action of caerulein but did not alter its hyperplastic action.
Amino acid transport in isolated hepatocytes after partial hepatectomy in the rat.
Authors: Le Cam A , Rey J F , Fehlmann M , Kitabgi P , Freychet P
Published: December 2017

The effect of partial (70%) hepatectomy on amino acid transport was investigated by measuring the uptake of the nonmetabolizable amino acid analogue alpha-amino-[1(-14C]isobutyric acid in hepatocytes freshly isolated from the liver remnant. In hepatocytes from partially hepatectomized rats, both the influx and the efflux of alpha-aminoisobutyric acid were increased. Quantitative analysis of the relationship between the initial rate of uptake and the substrate concentration indicated that a high-affinity component was responsible for the increased rate of amino acid entry in hepatocytes from partially hepatectomized rats. This component could be detected 90 min after operation, but its Vmax increased with time. The high-affinity component was strictly dependent on sodium and had the properties of a pure A system; its mode of energization partly involved cationic transmembrane gradients. The L system of amino acid transport was not affected after partial hepatectomy. The rapid emergence of a high-affinity amino acid transport system in the liver remnant following partial hepatectomy may play an important role in the regulation of liver regeneration.
Glucagon-like peptide 1 undergoes differential tissue-specific metabolism in the anesthetized pig
Authors: Deacon C. F. , Pridal L. , Klarskov L. , Olesen M. , Holst J. J.
Published: December 2017

Glucagon-like peptide 1 (GLP-1) metabolism was studied in halothane-anesthetized pigs (n = 7) using processing-independent (PI) and COOH-terminal (C) radioimmunoassays (RIA) and an enzyme-linked immunosorbent assay (ELISA) specific for biologically active GLP-1. Renal extraction of endogenous GLP-1 was detected by PI-RIA (33.1 +/- 13.3%) and C-RIA (16.0 +/- 6.3%) and by all assays during GLP-1 infusion (ELISA, 69.4 +/- 6.3%; PI-RIA, 32.6 +/- 7.3%; C-RIA, 43.7 +/- 3.4%), indicating substantial fragmentation. Hepatic and pulmonary degradation were undetectable under basal conditions, but exogenous GLP-1 elimination by the liver (43.6 +/- 8.9%) and lungs (10.1 +/- 3.2%) was measured by ELISA, suggesting primarily NH2-terminal degradation. Endogenous GLP-1 extraction by the hindleg was only detected by C-RIA (16.0 +/- 6.3%). During GLP-1 infusion, greater hindleg extraction was measured by ELISA (38.5 +/- 6.8%) and C-RIA (33.0 +/- 6.4%) than by PI-RIA (11.4 +/- 3.2%), indicating limited degradation at each terminus or more substantial COOH-terminal degradation. A shorter (P < 0.01) plasma half-life was revealed by ELISA (1.5 +/- 0.4 min) than by PI-RIA (4.5 +/- 0.6 min) or C-RIA (4.1 +/- 0.5 min). Metabolic clearance rates measured by PI-RIA (20.0 +/- 3.8 ml.min-1.kg-1) and C-RIA (15.5 +/- 1.6 ml.min-1.kg-1) were shorter (P < 0.01) than that measured by ELISA (106.8 +/- 14.7 ml.min-1.kg-1). Tissue-specific differential metabolism of GLP-1 occurs, and NH2-terminal degradation, rendering GLP-1 inactive, is particularly important in its clearance.
IGFBP-3 proteolysis by plasmin, thrombin, serum: heparin binding, IGF binding, and structure of fragments
Authors: Booth B. A. , Boes M. , Bar R. S.
Published: December 2017

Insulin-like growth factor binding protein (IGFBP)-3 was exposed to plasmin, thrombin, and pregnancy serum, substances normally present at the endothelial surface in enriched concentrations. The NH2-termini of the proteolytic fragments were sequenced, and their ability to bind insulin-like growth factor (IGF) and heparin was assessed by ligand blotting. Plasmin generated at least five fragments, three beginning at the NH2-terminus of IGFBP-3 and two with NH2-termini corresponding to middle portions of IGFBP-3. The dominant fragment bound both IGF and heparin while NH2-terminal fragments bound only IGF. Thrombin generated three and serum five easily identified fragments; the dominant fragments, beginning at midportions of IGFBP-3, retained IGF and heparin affinity, whereas the remaining fragments had differential affinities for IGF and heparin. We suggest that such fragments, when generated at the endothelia surface, have the potential to alter regional vascular concentrations of IGF and thus influence both IGF and endothelial function.
Hyperleptinemia: an early sign of juvenile obesity. Relations to body fat depots and insulin concentrations
Authors: Caprio S. , Tamborlane W. V. , Silver D. , Robinson C. , Leibel R. , McCarthy S. , Grozman A. , Belous A. , Maggs D. , Sherwin R. S.
Published: December 2017

Leptin, the OB gene product, is an adipocyte-derived circulating protein. In several rodent models of obesity, such as the db/db mice, fa/fa rats, and ventromedial hypothalamus-lesioned mice, as well as adult obese subjects, leptin mRNA expression and the circulating levels are elevated, suggesting resistance to its action. However, it is unknown whether the rise in leptin concentration occurs early in the natural evolution of human obesity or is a chronic adaptation to the obese state. Moreover, whether the distribution of body fat (i.e., visceral vs. subcutaneous abdominal fat) influences circulating leptin levels has not been assessed. We have determined in a group of obese and nonobese children and young adults whether leptin levels 1) are increased early in the development of obesity, 2) are related to a specific fat depot measured by magnetic resonance imaging, 3) vary during hyperinsulinemic, euglycemic, and hyperglycemic clamp studies, and 4) are different in males vs. females. In the basal state, leptin levels were elevated in obese children. Children and adults demonstrated a strong positive correlation between leptin concentrations and the subcutaneous fat depot (r = 0.84, P < 0.001). Surprisingly, a weaker correlation was found with visceral fat mass (r = 0.59, P = 0.001). Leptin levels remained unchanged under both euglycemic and hyperglycemic hyperinsulinemic conditions in both obese and nonobese subjects. A pronounced effect of gender on leptin levels was also observed. We conclude that, early in the development of juvenile obesity, leptin concentrations are elevated and are more closely linked to subcutaneous than visceral fat mass. Acute increases in insulin concentrations do not affect circulating leptin levels.
Cingulate cortex: a closer look at its gut-related functional topography
Authors: Lawal Adeyemi , Kern Mark , Sanjeevi Arthi , Hofmann Candy , Shaker Reza
Published: December 2017

Earlier studies have documented activation of the cingulate cortex during gut related sensory-motor function. However, topography of the cingulate cortex in relationship to various levels of visceromotor sensory stimuli and gender is not completely elucidated. The aim was to characterize and compare the activation topography of the cingulate cortex in response to 1) subliminal, 2) perceived rectal distensions, and 3) external anal sphincter contraction (EASC) in males and females. We studied 18 healthy volunteers (ages 18–35 yr; 10 women, 8 men) using functional MRI blood-oxygenation-level-dependent technique. We obtained 11 axial slices (voxel vol. 2.5–6.0 × 2.5 × 2.5 mm3) through the cingulate cortex during barostat-controlled subliminal, liminal, and supraliminal nonpainful rectal distensions as well as EASC. Overall, for viscerosensation, the anterior cingulate cortex exhibited significantly more numbers of activated cortical voxels for all levels of stimulations compared with the posterior cingulate cortex ( P < 0.05). In contrast, during EASC, activity in the posterior cingulate was larger than in the anterior cingulate cortex ( P < 0.05). Cingulate activation was similar during EASC in males and females ( P = 0.58), whereas there was a gender difference in anterior cingulate activation during liminal and supraliminal stimulations ( P < 0.05). In females, viscerosensory cortical activity response was stimulus-intensity dependent. Intestinal viscerosensation and EASC induce different patterns of cingulate cortical activation. There may be gender differences in cingulate cortical activation during viscerosensation. In contrast to male subjects, females exhibit increased activity in response to liminal nonpainful stimulation compared with subliminal stimulation suggesting differences in cognition-related recruitment.
ATP-sensitive K+ channels composed of Kir6.1 and SUR2B subunits in guinea pig gastric myocytes
Authors: Sim Jae Hoon , Yang Dong Ki , Kim Young Chul , Park Sung Jin , Kang Tong Mook , So Insuk , Kim Ki Whan
Published: December 2017

This study was designed to identify the single-channel properties and molecular entity of ATP-sensitive K+ (KATP) channels in guinea pig gastric myocytes with patch-clamp recording and RT-PCR. Pinacidil and diazoxide activated KATP currents in a glibenclamide-sensitive manner. The open probability of channels was enhanced by the application of 10 μM pinacidil from 0.085 ± 0.04 to 0.20 ± 0.05 ( n = 7) and was completely blocked by 10 μM glibenclamide. Single-channel conductance was 37.3 ± 2.5 pS ( n = 4) between −80 and −20 mV in symmetrical K+ gradient conditions. In inside-out mode, KATP channels showed no spontaneous openings and were activated by the application of nucleotide diphosphates to the cytoplasmic side. These single-channel properties are similar to those of the nucleotide diphosphate-dependent K+ channels in vascular smooth muscle, which are composed of Kir6.1 and sulfonylurea receptor (SUR)2B. RT-PCR demonstrated the presence of Kir6.1, Kir6.2, and SUR2B in guinea pig stomach smooth muscle cells. These results suggest that KATP channels in smooth muscle cells of the guinea pig stomach are composed of Kir6.1 and SUR2B.
Influence of sodium on major diffusion barriers to passive intestinal uptake
Authors: Thomson A. B.
Published: December 2017

Intestinal active transport processes have been shown to be sodium dependent, but the effect of varying the concentration of sodium in the luminal bulk phase on the passive uptake of lipids has not been established. An in vitro technique was used to measure the uptake of fatty acids and alcohols into the rabbit jejunum under conditions of stirring of the bulk phase selected to reduce the effective resistance of the unstirred water layer (UWL). Sodium withdrawal had no effect on the uptake of a homologous series of fatty alcohols, and thus the UWL was not influenced by varying bulk-phase sodium; in contrast, the uptake of a homologous series of short-, medium, and long-chain-length saturated fatty acids (FA) was progressively inhibited by reducing bulk-phase sodium from 145 to 0 meq/l. There was no difference in FA uptake between the use of magnesium chloride and mannitol for maintenance of isotonicity with sodium withdrawal. At each sodium concentration, the uptake of FA rose with increasing FA chain length. The relation between sodium concentration and incremental change in free energy (delta delta Fw leads to 1) was described by a sigmoid-shaped curve. The uptake of cholesterol was also inhibited by reducing sodium in the bulk phase. In summary, varying bulk-phase sodium has no effect on UWL, and the influence of sodium on the uptake of FA is mediated by altering delta delta Fw leads to 1 and therefore the physicochemical properties of the brush-border membrane.
Anion effects on fluid absorption from rat jejunum perfused in vivo
Authors: Humphreys M. H. , Chou L. Y.
Published: December 2017

Perfusion of rat jejunal segments in vivo with an isotonic, HCO3-free SO4-Ringer solution resulted in low rates of net sodium (JNanet) and water absorption. When the perfusion fluid was changed to one containing 25 mM Na2SO3, JNanet increased from 4.7 +/- 1.2 to 11.6 +/- 1.5 (SE) mumol X cm-1 X h-1 (P less than 0.001). This increased absorption was accompanied by comparable increases in chloride and water absorption, occurred without a detectable change in potential difference across the perfused segment, and was readily reversed on reinstitution of perfusion with SO4-Ringer. Perfusion with SO3-Ringer had no effect on electrolyte absorption from terminal segments of rat ileum. Addition of L-phenylalanine stimulated absorption from SO4-Ringer perfusate but not from SO3-Ringer perfusate. Addition of 25 mM NaHCO3 to SO4-Ringer perfusate caused parallel increases in JNanet and JHCO3net; when 25 mM NaHCO3 was added to SO4-Ringer perfusate that also contained 25 mM NaSCN, the same increase in JHCO3net occurred but was not associated with any increase in JNanet. These results indicate a potent effect of SO2-3 and HCO-3 to stimulate JNanet from rat jejunum but not from ileum. These anion effects on intestinal transport in vivo resemble their effects on ATPase activity of brush-border fractions from small intestine in vitro and raise the possibility that these effects on ion transport could be mediated through the changes in brush-border ATPase activity, which are brought about by exposure to these anions, although other explanations are also possible.
Effects of saturating metabolic uptake on space profiles and tracer kinetics
Authors: Goresky C. A. , Bach G. G. , Rose C. P.
Published: December 2017

Vascular and steady-state lengthwise concentrations evolving in tissue as a consequence of saturable uptake processes are explored when there is no effective barrier between blood and tissue, when there is one effective barrier (the liver), and when barriers are present at both capillary and cell membrane. Falling exponential profiles develop at low-input concentrations and grade over into much smaller linear decreases at high concentrations. Uptake behind an effective barrier produces a step-down in concentration. With no effective barriers, a tracer impulse propagates to the outflow in a delayed fashion, reversible binding to the enzymic removal mechanism increasing its space of distribution and delaying it further and irreversible uptake diminishing its area. As bulk concentration is increased, proportional tracer uptake diminishes and ultimately approaches zero. With barriers in the system, tracer output consists of an impulse damped by cell entry, followed by outflow recovery of material that has escaped cell uptake. Increase in bulk concentration is found to saturate the uptake mechanism, with a proportionate increase in the returning material.
Transmural fluxes of 5-hydroxytryptamine in guinea pig ileum
Authors: Cooke H. J. , Montakhab M. , Wade P. R. , Wood J. D.
Published: December 2017

Transmural movement of 5-hydroxytryptamine (5-HT) was studied in guinea pig small intestine in vitro in order to test the hypothesis that there is mucosal 5-HT barrier in this species. Segments of guinea pig ileum were mounted as flat sheets in flux chambers or were everted and perfused. Mucosal-to-serosal (Jm leads to s) and serosal-to-mucosal (Js leads to m) fluxes of 5-HT were measured in the absence of 5-HT gradients and under open- or short-circuited conditions. The results indicated that substantial transmural movement of 5-HT occurred in these preparations. Both Jm leads to s and Js leads to m were linear functions of the 5-HT concentration over a range of 1-30 microM and were not significantly different in the two directions. Addition of 2,4-dinitrophenol to both sides of the tissue reduced short-circuit current to zero and increased both tissue conductance and unidirectional 5-HT fluxes. These results suggested that the 5-HT fluxes across the guinea pig ileum occurred by passive mechanisms. Fluxes of 5-HT across preparations with the muscularis externa removed were not significantly different from fluxes across intact preparations. Mucosal-to-serosal 5-HT fluxes in everted perfused sacs were comparable with fluxes in the flat-sheet preparations. The data are not consistent with the hypothesis of a "tissue barrier" that functions to prevent 5-HT from reaching serotonergic receptors on enteric ganglion cells or enteroendocrine cells.
Adaptation of intestinal muscle in bypassed loops after jejunoileal bypass in rat
Authors: Nemeth P. R. , Kwee D. J. , Weisbrodt N. W.
Published: December 2017

The function and structure of intestinal smooth muscle in the bypassed intestine of rats with 70% intestinal bypass were compared with the function and structure of muscle from equivalent areas of intestine from transected and nontransected controls. Muscle function was assessed by evaluating changes in intestinal transit. At all times studied after operation, transit in transected controls was identical to that seen in nontransected controls. In the bypassed intestine at 3 days after operation, transit in fasted animals was significantly slower than intestinal transit in either control group. Over a period of 14-35 days, transit in the bypassed intestine of fasted animals returned toward control values. In fed animals, on the other hand, transit was delayed when measured at both 3 and 35 days after bypass operation. These findings demonstrate a persistent change in muscle function in bypassed intestine in response to the ingestion of a meal. No changes in intestinal structure were found when bypassed intestine was compared with tissue from transected controls. Thus there were no indications of mucosal or muscular atrophy in the bypassed intestine. The weights of the combined submucosal, muscular, and serosal layers in the study segments were increased at 35 days after either bypass or transection compared with tissue from nontransected controls. These changes appear to be a nonspecific result of operation.
Duodenal and ileal calcium absorption in the rat and effects of vitamin D
Authors: Pansu D. , Bellaton C. , Roche C. , Bronner F.
Published: December 2017

An in situ ligated loop procedure was applied to dissect transmural calcium transport in the intestine into two components, a saturable and a nonsaturable process. The existence of two such processes was confirmed in the duodenum, but ileal calcium transport was devoid of the saturable component. There was a small saturable component in the upper jejunum. The level of CaBP, the vitamin D-dependent cytosolic calcium-binding protein (Mr, approximately or equal to 9,000), corresponded to the magnitude of the saturable component. No CaBP was detected in the ileum. Vitamin D dependence of the saturable component was established by inducing it in the duodenum of vitamin D-deficient animals following intraperitoneal injection of 1,25-dihydroxyvitamin D3. In these same animals, conversely, the ileum did not respond to exogenous 1,25-dihydroxyvitamin D3. This confirms the absence in the ileum of the saturable component of transmural calcium movement and the fact that the nonsaturable component is not vitamin D dependent. Everted sac experiments also showed that duodenal sacs from vitamin D-replete or -repleted animals transported calcium against a chemical gradient, whereas ileal sacs did not. Vitamin D regulation of intestinal calcium absorption thus occurs only in the proximal intestine, even though calcium is absorbed down its chemical gradient all along the small intestine.
Effect of histamine on microvascular permeability in the rat stomach
Authors: Nagata H. , Guth P. H.
Published: December 2017

The effect of histamine on gastric microvascular permeability to macromolecules in the rat was studied using fluorescent in vivo microscopy. Histamine was applied topically to the serosal surface for study of the muscularis externa, to the submucosa, and to the superficial mucosa, and the area of leaks of a fluorescein-albumin conjugate from microvessels was quantitated. In the muscularis externa both histamine and an H1-agonist, but not an H2-agonist, caused dose-dependent leak of conjugate from venules. An H1-antagonist, but not an H2-antagonist, decreased the histamine-induced leak. In the submucosa histamine caused dose-dependent dilatation of arterioles but not leak of conjugate. In contrast, bradykinin caused both dose-dependent dilatation of arterioles and leak of conjugate from venules. In the superficial mucosa histamine did not cause any leak. In conclusion, topical histamine 1) increased microvascular permeability to macromolecules from venules in the muscularis externa via H1-receptors, 2) did not affect microvascular permeability in the submucosa (this may be due to lack of histamine receptors on the venules as bradykinin increased venular permeability), and 3) did not affect microvascular permeability in the superficial mucosa, but there might not have been adequate histamine backdiffusion.
Characterization of dogfish gastric mucosa under hyperbaric conditions
Authors: Kidder G. W.
Published: December 2017

Using hyperbaric conditions previously shown to give improved oxygen delivery to the chambered dogfish gastric mucosa, experiments were performed to further characterize this tissue under these conditions. Removal of Cl- depressed but did not abolish acid secretion, raised the transepithelial resistance, and caused the potential to become more negative with reference to the mucosal surface. Voltage clamping to -60 mV inhibited secretion significantly; voltage clamping to +60 mV sometimes gave a stimulation. There was no long time-constant transient response to current passage. Under hyperbaric conditions, addition of the normal 350 mM urea content of elasmobranch plasma stimulated secretion. A further increase in partial pressure of oxygen (PO2) above 1.9 atm had no effect on secretion. The current-voltage plot showed two breakpoints, one about -23 mV and a second (not always detectable) at +50 mV; the negative breakpoint was stable as PO2 changed from 0.9 to 1.9 atm. Many of these observations are different from those reported for normabaric tissue and closely resemble those in the isolated frog gastric mucosa.
Determinants of resting and passive intestinal vascular pressures in rat and rabbit
Authors: Bohlen H. G.
Published: December 2017

Microvascular pressures in the intestinal arteries, submucosal arterioles, and mucosal venules were measured in rats and rabbits at rest and during maximum dilation. From these data and Doppler velocimetry measurements of relative changes in whole organ blood flow on maximum dilation, it was possible to determine to what extent microvascular pressures at rest depend on the active control and passive hemodynamic characteristics of specific vascular segments. New Zealand White rabbits (2-3 kg body wt) had a mean arterial pressure of 70-75 mmHg. However, pressures in arterioles of both species became equivalent at the second order of arteriolar branching within the bowel wall, and pressures in the smallest mucosal venules were 13.7 +/- 0.6 (SE) mmHg in rabbits and 14.9 +/- 0.3 mmHg in rats. Maximum vasodilation to approximately 300% of the control blood flow increased mucosal venule pressures approximately 10 mmHg in rats compared with approximately 4 mmHg in rabbits. The increased mucosal venule pressure during vasodilation was primarily due to increased pressures within the submucosal small arterioles, which immediately precede the villus vasculature in both species. The increased blood flow during vasodilation was due primarily to a decreased resistance of the small arteries and large arterioles, even though pressures in these larger vessels changed only approximately 10%. This situation allows a major decrease in intestinal vascular resistance to substantially increase blood flow with a minimal increase in mucosal microvascular pressures.
Oxygen radicals stimulate guinea pig gallbladder glycoprotein secretion in vitro
Authors: Hale W. B. , Turner B. , LaMont J. T.
Published: December 2017

In several animal models of cholelithiasis, and in humans with gallstones, hypersecretion of gallbladder mucin is observed. This study was undertaken to determine the effect of oxygen radicals on guinea pig gallbladder glycoprotein secretion in organ culture. Mucosal explants were incubated with [3H]glucosamine hydrochloride to label glycoproteins, then exposed to oxygen radicals generated by chelated ferric iron and ascorbic acid. Marked stimulation of glycoprotein release was observed after a 30-min exposure to the oxygen radical-generating system, and the effect was inhibited by mannitol. The stimulatory effect of hydroxyl radical was not accompanied by leakage of intracellular lactate dehydrogenase. Parallel experiments with human granulocytes activated with f-Met-Leu-Phe and coincubated with gallbladder explants revealed similar results. These results indicate that oxygen radicals, especially the hydroxyl radical (OH.), are capable of stimulating rapid release of mucous-type glycoproteins from gallbladder epithelium.
GRP nerves in pig antrum: role of GRP in vagal control of gastrin secretion
Authors: Holst J. J. , Knuhtsen S. , Orskov C. , Skak-Nielsen T. , Poulsen S. S. , Jensen S. L. , Nielsen O. V.
Published: December 2017

We extracted gastrin-releasing peptide (GRP) and its C-terminal decapeptide corresponding to 6.4 and 6.8 pmol/g from pig antrum mucosa. By immunohistochemistry GRP was localized to mucosal, submucosal, and myenteric nerve fibers. A few nerve cell bodies were also identified. Using isolated perfused pig antrum with intact vagal innervation, we found concomitant, atropine-resistant release of GRP and gastrin during electrical stimulation of the vagal nerves. Intra-arterial GRP at 10(-11)-10(-10) mol/l caused up to fivefold, dose-dependent increases in gastrin secretion; higher doses were less effective and completely desensitized the gastrin cells for the lower doses. After desensitization, vagal stimulation no longer produced gastrin secretion. The substance P antagonist [D-Arg, D-Pro, D-Trp, Leu]-substance P, described as also antagonizing the actions of bombesin, decreased the gastrin response to GRP and abolished the effect of vagal stimulation. The available evidence strongly suggests that GRP nerves are responsible for the stimulatory vagal effects on gastrin secretion in the pig.