bluuebunny/crossref_metadata_2025 huggingface dataset:
Zinc status affects p53, gadd45, and c-fos expression and caspase-3 activity in human bronchial epithelial cells
Authors: Fanzo Jessica C. , Reaves Scott K. , Cui Libin , Zhu Lei , Wu John Y. J. , Wang Yi Ran , Lei K. Y.
Published: December 2017

This study was designed to examine the influence of zinc depletion and supplementation on the expression of p53 gene, target genes of p53, and caspase-3 activity in normal human bronchial epithelial (NHBE) cells. A serum-free, low-zinc medium containing 0.4 μmol/l of zinc [zinc deficient (ZD)] was used to deplete cellular zinc over one passage. In addition, cells were cultured for one passage in media containing 4.0 μmol/l of zinc [zinc normal (ZN)], which represents normal culture concentrations (Clonetics); 16 μmol/l of zinc [zinc adequate (ZA)], which represents normal human plasma zinc levels; or 32 μmol/l of zinc [zinc supplemented (ZS)], which represents the high end of plasma zinc levels attainable by oral supplementation in humans. Compared with ZN cells, cellular zinc levels were 76% lower in ZD cells but 3.5-fold and 6-fold higher in ZA and ZS cells, respectively. Abundances of p53 mRNA and nuclear p53 protein were elevated in treatment groups compared with controls (ZN). For p53mRNA abundance, the highest increase (3-fold) was observed in ZD cells. In contrast, the highest increase (17-fold) in p53 nuclear protein levels was detected in ZS cells. Moreover, gadd45mRNA abundance was moderately elevated in ZD and ZA cells and was not altered in ZS cells compared with ZN cells. Furthermore, the only alteration in c-fos mRNA and caspase-3 activity was the twofold increase and the 25% reduction, respectively, detected in ZS compared with ZN cells. Thus p53, gadd45, and c-fos and caspase-3 activity appeared to be modulated by cellular zinc status in NHBE cells.
ANG II type 1 receptor downregulation does not require receptor endocytosis or G protein coupling
Authors: Modrall J. Gregory , Nanamori Masakatsu , Sadoshima Junichi , Barnhart Douglas C. , Stanley James C. , Neubig Richard R.
Published: December 2017

ANG II type 1 (AT1) receptors respond to sustained exposure to ANG II by undergoing downregulation of absolute receptor numbers. It has been assumed previously that downregulation involves endocytosis. The present study hypothesized that AT1 receptor downregulation occurs independently of receptor endocytosis or G protein coupling. Mutant AT1 receptors with carboxy-terminal deletions internalized <5% of radioligand compared with 65% for wild-type AT1 receptors. The truncated AT1 receptors retained the ability to undergo downregulation. These data suggest the existence of an alternative pathway to AT1 receptor degradation that does not require endocytosis, per se. Point mutations in either the second transmembrane region or second intracellular loop impaired G protein (Gq) coupling. These receptors exhibited a biphasic pattern of downregulation. The earliest phase of downregulation (0–2 h) was independent of coupling to Gq, but no additional downregulation was observed after 2 h of ANG II exposure in the receptors with impaired coupling to Gq. These data suggest that coupling to Gq is required for the later phase (2–24 h) of AT1 receptor downregulation.
CFTR modulates programmed cell death by decreasing intracellular pH in Chinese hamster lung fibroblasts
Authors: Barrière Hervé , Poujeol Chantal , Tauc Michel , Blasi Jean Michel , Counillon Laurent , Poujeol Philippe
Published: December 2017

To study the potential influence of cystic fibrosis conductance regulator (CFTR) on intracellular pH regulation during apoptosis induction, we used PS120 Chinese hamster lung fibroblasts devoid of the Na+/H+ exchanger (NHE1 isoform) transfected with constructs, allowing the expression of CFTR and/or NHE1. Kinetics of lovastatin-induced apoptosis were measured by orcein staining, double staining with Hoechst-33258, propidium iodide, DNA fragmentation, and annexin V labeling. In PS120 control cells, the percentage of apoptotic cells after 40 h of lovastatin treatment was 23 ± 3%, whereas in PS120 CFTR-transfected cells, this percentage was 40 ± 4%. In PS120 NHE1 cells, the transfection with CFTR did not modify the percentage of apoptotic cells after 40 h (control: 19 ± 3%, n = 8; CFTR: 17 ± 1%, n = 8), indicating that blocking intracellular acidification by overexpressing the Na+/H+ exchanger inhibited the enhancement of apoptosis induced by CFTR. In all cell lines, the initial pH values were identical (pH = 7.46 ± 0.04, n = 9), and treatment with lovastatin led to intracellular acidification. However, the pH value after 40 h was lower in PS120 CFTR-transfected cells (pH = 6.85 ± 0.02, n= 10) than in PS120 cells (pH = 7.15 ± 0.03, n = 10). To further investigate the origin of this increased intracellular acidification observed in CFTR-transfected cells, the activity of the DIDS-inhibitable Cl−/HCO[Formula: see text] exchanger was studied. 8-Bromoadenosine 3′,5′-cyclic monophosphate incubation resulted in Cl−/HCO[Formula: see text] exchanger activation in PS120 CFTR-transfected cells but had no effect on PS120 cells. Together, our results suggest that CFTR can enhance apoptosis in Chinese hamster lung fibroblasts, probably due to the modulation of the Cl−/HCO[Formula: see text] exchanger, resulting in a more efficient intracellular acidification.
Timolol may inhibit aqueous humor secretion by cAMP-independent action on ciliary epithelial cells
Authors: McLaughlin Charles W. , Peart David , Purves Robert D. , Carré David A. , Peterson-Yantorno Kim , Mitchell Claire H. , Macknight Anthony D. C. , Civan Mortimer M.
Published: December 2017

The β-adrenergic antagonist timolol reduces ciliary epithelial secretion in glaucomatous patients. Whether inhibition is mediated by reducing cAMP is unknown. Elemental composition of rabbit ciliary epithelium was studied by electron probe X-ray microanalysis. Volume of cultured bovine pigmented ciliary epithelial (PE) cells was measured by electronic cell sizing; Ca2+ activity and pH were monitored with fura 2 and 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, respectively. Timolol (10 μM) produced similar K and Cl losses from ciliary epithelia in HCO[Formula: see text]/CO2 solution but had no effect in HCO[Formula: see text]/CO2-free solution or in HCO[Formula: see text]/CO2 solution containing the carbonic anhydrase inhibitor acetazolamide. Inhibition of Na+/H+ exchange by dimethylamiloride in HCO[Formula: see text]/CO2 solution reduced Cl and K comparably to timolol. cAMP did not reverse timolol's effects. Timolol (100 nM, 10 μM) and levobunolol (10 μM) produced cAMP-independent inhibition of the regulatory volume increase (RVI) in PE cells and increased intracellular Ca2+ and pH. Increasing Ca2+ with ionomycin also blocked the RVI. The results document a previously unrecognized cAMP-independent transport effect of timolol. Inhibition of Cl−/HCO[Formula: see text] exchange may mediate timolol's inhibition of aqueous humor formation.
EGF receptor function is required in late G1 for cell cycle progression induced by bombesin and bradykinin
Authors: Santiskulvong Chintda , Sinnett-Smith James , Rozengurt Enrique
Published: December 2017

We examined the role of epidermal growth factor (EGF) receptor (EGFR) tyrosine kinase activation in G protein-coupled receptor (GPCR) agonist-induced mitogenesis in Swiss 3T3 and Rat-1 cells. Addition of EGFR tyrosine kinase inhibitors (e.g., tyrphostin AG-1478) abrogated bombesin-induced extracellular signal-regulated kinase (ERK) activation in Rat-1 cells but not in Swiss 3T3 cells, indicating the importance of cell context in determining the role of EGFR in ERK activation. In striking contrast, treatment with tyrphostin AG-1478 markedly (∼70%) inhibited DNA synthesis induced by bombesin in both Swiss 3T3 and Rat-1 cells. Similar inhibition of bombesin-induced DNA synthesis in Swiss 3T3 cells was obtained using four structurally different inhibitors of EGFR tyrosine kinase. Furthermore, kinetic analysis indicates that EGFR function is necessary for bombesin-induced mitogenesis in mid-late G1 in both Swiss 3T3 and Rat-1 cells. Our results indicate that EGFR kinase activity is necessary in mid-late G1 for promoting the accumulation of cyclins D1 and E and implicate EGFR function in the coupling of GPCR signaling to the activation of the cell cycle.
Proton leak and CFTR in regulation of Golgi pH in respiratory epithelial cells
Authors: Chandy Grischa , Grabe Michael , Moore Hsiao-Ping H. , Machen Terry E.
Published: December 2017

Work addressing whether cystic fibrosis transmembrane conductance regulator (CFTR) plays a role in regulating organelle pH has remained inconclusive. We engineered a pH-sensitive excitation ratiometric green fluorescent protein (pHERP) and targeted it to the Golgi with sialyltransferase (ST). As determined by ratiometric imaging of cells expressing ST-pHERP, Golgi pH (pHG) of HeLa cells was 6.4, while pHG of mutant (ΔF508) and wild-type CFTR-expressing (WT-CFTR) respiratory epithelia were 6.7–7.0. Comparison of genetically matched ΔF508 and WT-CFTR cells showed that the absence of CFTR statistically increased Golgi acidity by 0.2 pH units, though this small difference was unlikely to be physiologically important. Golgi pH was maintained by a H+ vacuolar (V)-ATPase countered by a H+leak, which was unaffected by CFTR. To estimate Golgi proton permeability ( P H+ ), we modeled transient changes in pHG induced by inhibiting the V-ATPase and by acidifying the cytosol. This analysis required knowing Golgi buffer capacity, which was pH dependent. Our in vivo estimate is that Golgi P H+ = 7.5 × 10−4 cm/s when pHG = 6.5, and surprisingly, P H+ decreased as pHG decreased.
Temperature dependence of cloned mammalian and salmonid cardiac Na+/Ca2+ exchanger isoforms
Authors: Elias Chadwick L. , Xue Xiao-Hua , Marshall Christian R. , Omelchenko Alexander , Hryshko Larry V. , Tibbits Glen F.
Published: December 2017

The cardiac Na+/Ca2+ exchanger (NCX), an important regulator of cytosolic Ca2+ concentration in contraction and relaxation, has been shown in trout heart sarcolemmal vesicles to have high activity at 7°C relative to its mammalian isoform. This unique property is likely due to differences in protein structure. In this study, outward NCX currents ( I NCX) of the wild-type trout (NCX-TR1.0) and canine (NCX 1.1) exchangers expressed in oocytes were measured to explore the potential contributions of regulatory vs. transport mechanisms to this observation. cRNA was transcribed in vitro from both wild-type cDNA and was injected into Xenopus oocytes. I NCX of NCX-TR1.0 and NCX1.1 were measured after 3–4 days over a temperature range of 7–30°C using the giant excised patch technique. The I NCX for both isoforms exhibited Na+-dependent inactivation and Ca2+-dependent positive regulation. The I NCX of NCX1.1 exhibited typical mammalian temperature sensitivities with Q10 values of 2.4 and 2.6 for peak and steady-state currents, respectively. However, the I NCX of NCX-TR1.0 was relatively temperature insensitive with Q10values of 1.2 and 1.1 for peak and steady-state currents, respectively. I NCX current decay was fit with a single exponential, and the resultant rate constant of inactivation (λ) was determined as a function of temperature. As expected, λ decreased monotonically with temperature for both isoforms. Although λ was significantly greater in NCX1.1 compared with NCX-TR1.0 at all temperatures, the effect of temperature on λ was not different between the two isoforms. These data suggest that the disparities in I NCX temperature dependence between these two exchanger isoforms are unlikely due to differences in their inactivation kinetics. In addition, similar differences in temperature dependence were observed in both isoforms after α-chymotrypsin treatment that renders the exchanger in a deregulated state. These data suggest that the differences in I NCX temperature dependence between the two isoforms are not due to potential disparities in either the I NCX regulatory mechanisms or structural differences in the cytoplasmic loop but are likely predicated on differences within the transmembrane segments.
Acute inhibition of brain-specific Na+/H+ exchanger isoform 5 by protein kinases A and C and cell shrinkage
Authors: Attaphitaya Surat , Nehrke Keith , Melvin James E.
Published: December 2017

Little is known of the functional properties of the mammalian, brain-specific Na+/H+ exchanger isoform 5 (NHE5). Rat NHE5 was stably expressed in NHE-deficient PS120 cells, and its activity was characterized using the fluorescent pH-sensitive dye 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. NHE5 was insensitive to ethylisopropyl amiloride. The transport kinetics displayed a simple Michaelis-Menten relationship for extracellular Na+ (apparent K Na= 27 ± 5 mM) and a Hill coefficient near 3 for the intracellular proton concentration with a half-maximal activity at an intracellular pH of 6.93 ± 0.03. NHE5 activity was inhibited by acute exposure to 8-bromo-cAMP or forskolin (which increases intracellular cAMP by activating adenylate cyclase). The kinase inhibitor H-89 reversed this inhibition, suggesting that regulation by cAMP involves a protein kinase A (PKA)-dependent process. In contrast, 8-bromo-cGMP did not have a significant effect on activity. The protein kinase C (PKC) activator phorbol 12-myristrate 13-acetate inhibited NHE5, and the PKC antagonist chelerythrine chloride blunted this effect. Activity was also inhibited by hyperosmotic-induced cell shrinkage but was unaffected by a hyposmotic challenge. These results demonstrate that rat brain NHE5 is downregulated by activation of PKA and PKC and by cell shrinkage, important regulators of neuronal cell function.
Coupling strength between localized Ca2+transients and K+ channels is regulated by protein kinase C
Authors: Bayguinov Orline , Hagen Brian , Kenyon James L. , Sanders Kenton M.
Published: December 2017

Localized Ca2+ transients resulting from inositol trisphosphate (IP3)-dependent Ca2+ release couple to spontaneous transient outward currents (STOCs) in murine colonic myocytes. Confocal microscopy and whole cell patch-clamp techniques were used to investigate coupling between localized Ca2+ transients and STOCs. Colonic myocytes were loaded with fluo 3. Reduction in external Ca2+([Ca2+]o) reduced localized Ca2+transients but increased STOC amplitude and frequency. Simultaneous recordings of Ca2+ transients and STOCs showed increased coupling strength between Ca2+ transients and STOCs when [Ca2+]o was reduced. Gd3+ (10 μM) did not affect Ca2+ transients but increased STOC amplitude and frequency. Similarly, an inhibitor of Ca2+influx, 1–2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl-1H-imidazole (SKF-96365), increased STOC amplitude and frequency. A protein kinase C (PKC) inhibitor, GF-109203X, also increased the amplitude and frequency of STOCs but had no effect on Ca2+ transients. Phorbol 12-myristate 13-acetate (1 μM) reduced STOC amplitude and frequency but did not affect Ca2+ transients. 4α-Phorbol (1 μM) had no effect on STOCs or Ca2+ transients. Single channel studies indicated that large-conductance Ca2+-activated K+ (BK) channels were inhibited by a Ca2+-dependent PKC. In summary 1) Ca2+ release from IP3 receptor-operated stores activates Ca2+-activated K+ channels; 2) Ca2+ influx through nonselective cation channels facilitates activation of PKC; and 3) PKC reduces the Ca2+ sensitivity of BK channels, reducing the coupling strength between localized Ca2+ transients and BK channels.
Differential MAP kinase activation and Na+/H+exchanger phosphorylation by H2O2 in rat cardiac myocytes
Authors: Wei Shan , Rothstein Emily C. , Fliegel Larry , Dell'Italia Louis J. , Lucchesi Pamela A.
Published: December 2017

Bursts in reactive oxygen species production are important mediators of contractile dysfunction during ischemia-reperfusion injury. Cellular mechanisms that mediate reactive oxygen species-induced changes in cardiac myocyte function have not been fully characterized. In the present study, H2O2 (50 μM) decreased contractility of adult rat ventricular myocytes. H2O2 caused a concentration- and time-dependent activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), p38, and c-Jun NH2-terminal kinase (JNK) mitogen-activated protein (MAP) kinases in adult rat ventricular myocytes. H2O2(50 μM) caused transient activation of ERK1/2 and p38 MAP kinase that was detected as early as 5 min, was maximal at 20 min (9.6 ± 1.2- and 9.0 ± 1.6-fold, respectively, vs. control), and returned to baseline at 60 min. JNK activation occurred more slowly (1.6 ± 0.2-fold vs. control at 60 min) but was sustained at 3.5 h. The protein kinase C inhibitor chelerythrine completely blocked JNK activation and reduced ERK1/2 and p38 activation. The tyrosine kinase inhibitors genistein and PP-2 blocked JNK, but not ERK1/2 and p38, activation. H2O2-induced Na+/H+ exchanger phosphorylation was blocked by the MAP kinase kinase inhibitor U-0126 (5 μM). These results demonstrate that H2O2-induced activation of MAP kinases may contribute to cardiac myocyte dysfunction during ischemia-reperfusion.
Heterotypic gap junction channel formation between heteromeric and homomeric Cx40 and Cx43 connexons
Authors: Cottrell G. Trevor , Burt Janis M.
Published: December 2017

Recent evidence indicating formation of functional homomeric/heterotypic gap junction channels by connexin40 (Cx40) and connexin43 (Cx43) raises the question of whether data previously interpreted as support for heteromeric channel formation by these connexins might not instead reflect the activity of homomeric/heterotypic channels. To address this question and to further characterize the behavior of these channels, we used dual whole cell voltage-clamp techniques to examine the junctions formed between cells that express only Cx40 (Rin40) or Cx43 (Rin43) and compared the results with those obtained when either of these cell types was paired with cells that naturally express both connexins (A7r5 cells). Rin40/Rin43 cell pairs formed functional gap junctions that displayed a strongly asymmetric voltage-dependent gating response. Single-channel event amplitudes ranged between 34 and 150 pS, with 90- to 130-pS events predominating. A7r5/Rin43 and A7r5/Rin40 cell pairs had voltage-dependent gating responses that varied greatly, with most pairs demonstrating strong asymmetry. These cell pairs exhibited a variety of single-channel events that were not consistent with homomeric/homotypic Cx40 or Cx43 channels or homomeric/heterotypic Cx40/Cx43 channels. These data indicate that Cx40 and Cx43 form homomeric/heterotypic as well as heteromeric/heterotypic channels that display unique gating and conductance properties.
Micromolar Ca2+ from sparks activates Ca2+-sensitive K+ channels in rat cerebral artery smooth muscle
Authors: Pérez Guillermo J. , Bonev Adrian D. , Nelson Mark T.
Published: December 2017

The goal of the present study was to test the hypothesis that local Ca2+ release events (Ca2+ sparks) deliver high local Ca2+concentration to activate nearby Ca2+-sensitive K+ (BK) channels in the cell membrane of arterial smooth muscle cells. Ca2+ sparks and BK channels were examined in isolated myocytes from rat cerebral arteries with laser scanning confocal microscopy and patch-clamp techniques. BK channels had an apparent dissociation constant for Ca2+ of 19 μM and a Hill coefficient of 2.9 at −40 mV. At near-physiological intracellular Ca2+ concentration ([Ca2+]i; 100 nM) and membrane potential (−40 mV), the open probability of a single BK channel was low (1.2 × 10−6). A Ca2+spark increased BK channel activity to 18. Assuming that 1–100% of the BK channels are activated by a single Ca2+ spark, BK channel activity increases 6 × 105-fold to 6 × 103-fold, which corresponds to ∼30 μM to 4 μM spark Ca2+ concentration. 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid acetoxymethyl ester caused the disappearance of all Ca2+sparks while leaving the transient BK currents unchanged. Our results support the idea that Ca2+ spark sites are in close proximity to the BK channels and that local [Ca2+]i reaches micromolar levels to activate BK channels.
B16-BL6 melanoma cells release inhibitory factor(s) of active pump activity in isolated lymph vessels
Authors: Nakaya Kei , Mizuno Risuke , Ohhashi Toshio
Published: December 2017

We investigated whether supernatant cultured with melanoma cell lines B16-BL6 and K1735 or the Lewis lung carcinoma cell line (LLC) can regulate lymphatic pump activity with bioassay preparations isolated from murine iliac lymph vessels. B16-BL6 and LLC supernatants caused significant dilation of lymph microvessels with cessation of pump activity. B16-BL6 supernatant produced dose-related cessation of lymphatic pump activity. There was no significant tachyphylaxis in the supernatant-mediated inhibitory response of lymphatic pump activity. Pretreatment with 3 × 10−5 M N ω-nitro-l-arginine methyl ester (l-NAME) or 10−7 M or 10−6 M glibenclamide and 5 × 10−4 M 5-hydroxydecanoic acid caused significant reduction of supernatant-mediated inhibitory responses. Simultaneous treatment with 10−3 Ml-arginine and 3 × 10−5 Ml-NAME significantly lessened l-NAME-induced inhibition of the supernatant-mediated response, suggesting that endogenous nitric oxide (NO) plays important roles in supernatant-mediated inhibitory responses. Chemical treatment dialyzed substances of <1,000 molecular weight (MW), producing complete reduction of the supernatant-mediated response. In contrast, pretreatment with heating or digestion with protease had no significant effect on supernatant-mediated response. These findings suggest that B16-BL6 cells may release nonpeptide substance(s) of <1,000 MW, resulting in significant cessation of lymphatic pump activity via production and release of endogenous NO and activation of mitochondrial ATP-sensitive K+ channels.
Molecular mechanisms of iNOS induction by IL-1β and IFN-γ in rat aortic smooth muscle cells
Authors: Teng Xingwu , Zhang Hanfang , Snead Connie , Catravas John D.
Published: December 2017

In rat aortic smooth muscle cells (RASMC), interferon (IFN)-γ enhanced nitrite accumulation and type II nitric oxide synthase (iNOS) protein expression induced by interleukin (IL)-1β. IFN-γ alone had no effect on nitrite accumulation or iNOS protein. IL-1β, but not IFN-γ, induced nuclear factor (NF)-κB and CCAAT box/enhancer binding protein (C/EBP) nuclear binding. Conversely, IFN-γ, but not IL-1β, induced signal transducer and activator of transcription (STAT) 1 and interferon regulatory factor (IRF)-1 binding. In a −1.4-kb rat iNOS promoter segment, deletion of an IFN-γ-activated site (GAS) increased IL-1β-induced activity but inhibited IFN-γ-enhanced activity, suggesting a two-way effect of the GAS site on iNOS induction: enhancing induction through STAT1 activation and inhibiting induction through a non-IFN-γ-mediated mechanism. Deletion of both an IRF and a C/EBP site reduced the IL-1β-induced and the IFN-γ-enhanced activities. However, IRF site mutations decreased the IFN-γ-enhanced activity without affecting the IL-1β-induced activity. Insertion of two IRF sites increased the IFN-γ-enhanced, but not the IL-1β-induced, activity. Mutations of a reverse NF-κB site did not significantly change IFN-γ-enhanced activity. We conclude that in RASMC, NF-κB and C/EBP mediate the IL-1β-induced iNOS expression, whereas IRF-1 and STAT1 mediate the IFN-γ-enhanced iNOS induction.
Secretory effects of VIP on isolated perfused porcine pancreas.
Authors: Jensen S L , Fahrenkrug J , Holst J J , Nielsen O V , Schaffalitzky de Muckadell O B
Published: December 2017

We studied the secretion of insulin, glucagon, and the exocrine secretion of the isolated perfused porcine pancreas in response to vasoactive intestinal polypeptide (VIP) in concentrations ranging from 30 to 18,750 pmol/liter at various concentrations of glucose in the perfusion medium. VIP stimulated the insulin and glucagon secretion in a dose-dependent manner. The response pattern was critically dependent on the glucose concentration. In the presence of a glucose concentration of 7.5 mmol/liter, VIP enhanced insulin release without affecting glucagon release. Maximal insulin release was obtained at a VIP concentration of 3,750 pmol/liter. At a glucose concentration of 5.0 or 3.5 mmol/liter, VIP enhanced glucagon release but not insulin release. VIP stimulated the exocrine secretion in a secretin-like manner. The lowest concentration of VIP observed to increase pancreatic exocrine secretion was 30 pmol/liter, whereas the maximal pancreatic exocrine responses were not obtained.
Involvement of cGMP in LHRH-stimulated gonadotropin release.
Authors: Naor Z , Fawcett C P , McCann S M
Published: December 2017

Anterior pituitary content of cyclic AMP (cAMP) and cyclic GMP (cGMP) has been measured during stimulation of gonadotropin release by luteinizing-hormone-releasing hormone (LHRH) in vitro to gain more information concerning the relationship between the mechanism of action of LHRH and cyclic nucleotides. During the increased gonadotropin release obtained by incubation by hemipituitaries with LHRH (0.25--25 X 10(-9) M) for 180 min, the glands taken from both male and female rats exhibited increased cGMP content, whereas cAMP content rose only in those taken from male rats. The increase in cGMP content was observed after only 2 min in the presence of LHRH (5 X 10(-9) M) and prior to augmented gonadotropin release. The increase in cAMP content in the male glands was detectable only after 60 min of incubation. These results suggest that cGMP might be involved in the mechanism of action of LHRH.
Effect of prolonged anaerobiosis on 125I-insulin binding to rat soleus muscle: permissive effect of ATP.
Authors: Yu K T , Gould M K
Published: December 2017

The specific binding of 125I-insulin by rat soleus muscle was depressed when muscle ATP was depleted, either by prolonged anoxia or more rapidly with 2,4-dinitrophenol. Insulin binding was not eliminated in ATP-depleted muscle, but was reduced by 70--80%. Insulin binding by aerobic muscle could be resolved into two components; a high-affinity, low-capacity site (KD = 7.8 nM) and a low-affinity, high-capacity site (KD = 390 nM). The stimulatory effect of insulin on xylose uptake could be correlated with binding to the high-affinity site. These results indicate that there is some ATP-dependent process involved in the regulation of insulin binding by soleus muscle. It is suggested that this could be a phosphorylation-dephosphorylation system, acting either on the receptor itself or on some closely related membrane protein.
Role of pancreatic L-asparagine synthetase in homeostasis of L-asparagine.
Authors: Milman H A , Cooney D A , Young D M
Published: December 2017

L-Asparagine synthetase from mouse pancreas was found to be associated principally with the exocrine pancreas and to be dependent on the age of the animal, but not on gender, diet, or the presence of tumor under the conditions examined. The function of the pancreatic enzyme appears to be to supply L-asparagine for the synthesis of pancreatic proteins. This function is suggested by the high specific activity of L-asparagine in pancreatic proteins after intravenous treatment of BDF1 mice with L-[U-14C]asparatate. The pancreas is also able to function as a storage depot for L-asparagine under conditions in which the concentration of the amino acid in the blood is in excess. Unlike the liver, the pancreas is unable to add L-asparagine to the circulation when the concentration of the amide is below normal limits.
Effect of overexpressing GLUT-1 and GLUT-4 on insulin- and contraction-stimulated glucose transport in muscle
Authors: Johannsson E. , McCullagh K. J. , Han X. X. , Fernando P. K. , Jensen J. , Dahl H. A. , Bonen A.
Published: December 2017

To examine the effects of GLUT-1 on GLUT-4-dependent, insulin-stimulated, and contraction-stimulated 2-deoxy-D-glucose (2-DG) transport, we overexpressed GLUT-1 in metabolically heterogeneous skeletal muscles [red and white tibialis anterior (TA) and extensor digitorum longus (EDL)] via 7 days of chronic electrical stimulation. GLUT-1 was increased 1.6- to 16.4-fold (P < 0.05). Basal 2-DG transport was increased 1.7- to 3.0-fold (P < 0.05) and was equal to (red TA and EDL; P > 0.05) or exceeded insulin-stimulated 2-DG transport by 50% (white TA; P < 0.05) in the control muscles. GLUT-4 was concomitantly overexpressed (2.1- to 4.4-fold; P < 0.05). Insulin-stimulated 2-DG transport was increased 1.6- to 2.5-fold (P < 0.05). During muscle contractions, 2-DG transport increased 9- to 12-fold (P < 0.05) in control muscles, but this was reduced by approximately 25% (P < 0.05) in muscles overexpressing GLUT-1 and GLUT-4 (red TA and EDL). In contrast, in the experiment, white TA contraction-stimulated 2-DG transport was increased 1.7-fold (P < 0.05). Therefore, overexpression of GLUT-1, when GLUT-4 is also overexpressed, does not impair insulin-stimulated 2-DG transport, although contraction-stimulated transport may be reduced in some muscles.
Diurnal pattern of the interrelationships among leucine oxidation, urea production, and hydrolysis in humans
Authors: el-Khoury A. E. , Ajami A. M. , Fukagawa N. K. , Chapman T. E. , Young V. R.
Published: December 2017

We investigated in six healthy adult men, who received an adequate intake of protein (1 g.kg-1.day-1), the relationship among urea production, excretion, and hydrolysis. At the end of a 6-day diet-adjustment period, subjects were studied using a 24-h continuous intravenous [1-13C]leucine and [15N,15N]urea tracer protocol (A. E. El-Khoury, N. K. Fukagawa, M. Sanchez, R. H. Tsay, R. E. Gleason, T. E. Chapman, and V. R. Young. Am. J. Clin. Nutr. 59: 1000-1011, 1994) to determine rates of irreversible protein nitrogen loss and urea kinetics. By combining leucine and urea kinetic data, we found a significant degree of urea hydrolysis over the 24-h period but no evidence to support the thesis that there is a net retention or "salvage" of the urea nitrogen liberated. Our measurements revealed little or no urea hydrolysis during the fed 12-h period of the 24-h tracer protocol but substantial hydrolysis during the 12-h fasting phase. Furthermore, a mass balance model and calculations (APPENDIX) indicated that nitrogen salvage, if any, is quantitatively indistinguishable from insensible nitrogen losses and aggregate estimation errors, accounting for no more than 5% of the nitrogen intake. We conclude that urea hydrolysis, via the intestinal microflora, although representing a component of the overall cycles of nitrogen flow within the body, does not contribute via a net retention of amino nitrogen to the maintenance of body nitrogen homeostasis in healthy adults consuming an adequate diet.
Inhibition of ovine gastric acid secretion by intraduodenal long-chain fatty acids
Authors: McLeay L. M. , Fitzgerald J. M.
Published: December 2017

Effects on ovine gastric function of procedures that increase intestinal unsaturated fatty acid content are unknown, and the present aim was to compare the effects of duodenal unsaturated and saturated fatty acids on gastric secretion in conscious sheep. During the maximal gastric secretory response to a meal, 10 ml gallbladder bile alone or with myristic, palmitic, and stearic acids and oleic, linoleic, and linolenic acids were infused into the duodenum at a rate of 5 g fatty acid . h-1 for 1 h. Compared with control 154 mM NaCl (100%), acid output was reduced to 4-7% of control with infusion of oleic, linoleic, and linolenic acids and myristic acids plus bile. Reductions in acid secretion persisted for up to 5 h from the end of infusion. In contrast, the infusion of palmitic and stearic acids with bile caused mean maximal reductions in acid output, respectively, to only 64 and 55% of control, and levels returned to control within 1 h of the end of infusion. Bile infusion alone caused no reduction in acid secretion. Under the conditions used, C18 unsaturated fatty acids and myristic acid were potent inhibitors of ovine gastric acid secretion. The lesser effects of palmitic and stearic acids were probably related to their reduced solubility and absorption.
Regulation of gastrin and somatostatin secretion by cholinergic and noncholinergic intramural neurons
Authors: Schubert M. L. , Bitar K. N. , Makhlouf G. M.
Published: December 2017

The regulation of gastrin and somatostatin secretion by intramural neurons was examined in the isolated vascularly perfused rat stomach. The optimal modalities of transmural electrical stimulation of the antrum were established to be 40 V and 10 Hz. Stimulation at increasing cycle durations (0.1-4 ms) caused increasing gastrin secretion that was progressively more resistant to atropine. The maximal gastrin response to 4-ms cycles was equal to the maximal response to methacholine. However, the response to methacholine was inhibited 70-90% by atropine, whereas the response to 4-ms cycles was inhibited by 15% only. Stimulation at all cycle durations caused a decrease in somatostatin secretion. Atropine converted the decrease to an increase, from which it was concluded that before atropine somatostatin secretion was the net result of cholinergic inhibition and noncholinergic stimulation of somatostatin. The results indicate that cholinergic and noncholinergic intramural neurons are predominantly but not exclusively activated by 0.1- and 4-ms cycles, respectively. The existence of distinct neurons was supported by results of stimulation of preganglionic vagal fibers with 0.2- and 4-ms cycles. The findings are consistent with a model according to which gastrin secretion is regulated by two interdependent intramural neurons: a cholinergic neuron that stimulates gastrin secretion indirectly by inhibition of somatostatin secretion and a noncholinergic neuron that stimulates gastrin secretion directly by release of a peptide stimulant, probably bombesin.
Factors influencing metabolism of arachidonic acid in guinea pig gastric mucosa
Authors: Ahlquist D. A. , Madson T. H. , Romero J. C. , Dozois R. R. , Malagelada J. R.
Published: December 2017

The metabolism of [14C]arachidonic acid in whole homogenates of gastric corpus mucosa in the guinea pig was characterized. Identity of labeled products was validated by comigration against standards in multiple chromatographic systems and by selective synthesis inhibition. At near-physiological conditions, homogenates converted arachidonic acid to the cyclooxygenase products prostaglandins F2, E2, and D2, 6-keto-prostaglandin F1 alpha, and thromboxane B2 (in descending order of magnitude) and to a lipoxygenase product cochromatographing with 12-hydroxyeicosatetraenoic acid. However, dramatic qualitative and quantitative changes in this product profile were noted with relatively small changes in incubation temperature, substrate concentration, and especially pH. Whole homogenates generated more prostaglandin and in different proportions than did the microsomal fraction from an equal mass of mucosa. No catabolism of prostaglandin products was observed in whole homogenates. Tissue preparation and storage also influenced prostaglandin synthesis activity. These in vitro observations may have important implications for future studies of prostaglandin generation in small gastric mucosal biopsies.
Fate of circulating isoamylases in the rabbit
Authors: Rosenblum J. L. , Niesen T. E. , Raab B. K. , Alpers D. H.
Published: December 2017

Amylase purified from the pancreas and parotid gland of rabbits was labeled with 125I. Plasma disappearance was biexponential after bolus injection. Half-time of metabolic clearance was 97 and 95 min, respectively, for pancreatic and salivary 125I-amylase. About 20 and 30% of the injected pancreatic and salivary 125I-amylase, respectively, appeared intact in the urine within 24 h. Organs were assayed for radioactivity at specified times. No organ at any time contained more than 20% of injected radioactivity. At 24 h, more than 90% of radioactivity in organs was acid soluble. Bilateral nephrectomy significantly slowed plasma disappearance, although 90% hepatectomy did not. These studies in the rabbit indicate that 1) homologous isoamylases are cleared from plasma at rates comparable with those reported in primates; 2) in contrast to primates, urinary excretion of salivary amylase is greater than pancreatic amylase; and 3) within 24 h, virtually all of injected 125I-labeled isoamylases were catabolized or excreted.
Glutathione depletion inhibits amylase release in guinea pig pancreatic acini
Authors: Stenson W. F. , Lobos E. , Wedner H. J.
Published: December 2017

Isolated guinea pig pancreatic acini were specifically depleted of glutathione by treatment with 2-cyclohexene-1-one (2-CHX-1). Untreated acini contained 4.3 +/- 0.6 micrograms of glutathione per milligram protein. Incubation with 1 mM 2-CHX-1 for 5 min at 37 degrees C depleted glutathione to 17% of control values; 5 mM 2-CHX-1 depleted glutathione to less than 4% of control values. Incubation with 2-CHX-1 also impaired the ability of the isolated acini to secrete amylase in response to stimulation with carbachol and the ionophore A23187. The depletion of glutathione and the inhibition of amylase secretion by 2-CHX-1 were both dose dependent and time dependent. Incubation of acini with 2 mM 2-CHX-1 for 15 min at 37 degrees C reduced glutathione levels to 6.6% of control and reduced carbachol-stimulated amylase release to 63% of control. Higher doses of 2-CHX-1 or longer incubations resulted in greater depletion of glutathione and greater inhibition of carbachol-induced amylase release. These data indicate that specific depletion of glutathione impairs the ability of isolated acini to secrete amylase in response to physiological and pharmacologic stimuli and suggest that glutathione has a role in stimulus-secretion coupling in the exocrine pancreas.
Empirical strategy for analytical fractionation of epithelial cells
Authors: Mircheff A. K.
Published: December 2017

Despite the importance of cell fractionation methods in studies of epithelial transport mechanisms and of a variety of subcellular processes, current practices in cell fractionation have a number of shortcomings. Most cell fractionation studies depend on biochemical markers, but they provide little independent confirmation of the initial assumptions that markers are uniquely associated with particular subcellular structures and that they are uniformly distributed over the surfaces with which they are associated. Moreover, it is generally difficult and time-consuming to design new membrane isolation procedures. After reviewing the analytical nature of physical separation procedures, I suggest an empirical approach to cell fractionation that is both general and comprehensive. This approach uses physical separation procedures to generate spatial distributions of particles in which position is related to such physical properties as sedimentation coefficient, density, cholesterol content, surface charge, and coefficient of partitioning in aqueous polymer two-phase systems. Determination of the frequency distributions of biochemical markers permits detection of separate populations of particles even when the populations lack unique markers. This process requires no a priori assumptions about the subcellular localizations of particles.
Multiple 5-hydroxytryptamine receptors on feline ileum and ileocecal sphincter
Authors: Ouyang A. , Cohen S.
Published: December 2017

The effects of 5-hydroxytryptamine (5-HT) on the feline ileum and ileocecal sphincter (ICS) were studied in vivo using simultaneous myoelectric and pressure recordings. Both inhibitory and excitatory responses were seen in the ileum and ICS, with the inhibitory response being seen with lower doses of 5-HT (EDmax for inhibition was 5.0 micrograms/kg and EDmax for contractile response was 80.0 micrograms/kg). The inhibitory and excitatory responses were antagonized separately by different 5-HT antagonists. Methysergide inhibited the ICS relaxation response only, whereas cinanserin inhibited the ICS contractile response. Neither effect was inhibited by tetrodotoxin, suggesting that 5-HT acts directly on the smooth muscle at at least two separate receptors. In addition, methysergide alone resulted in a usually tonic increase (8.6 +/- 0.6 mmHg) in basal ICS pressure (P less than 0.05) without an ileal effect, and tetrodotoxin alone induced phasic ileal and ICS contractions (delta ICS = 7.3 +/- 1.5 mmHg). These data suggest the action of at least two inhibitory neurons tonically at the ICS.
Local modulation of intestinal ion transport by enteric neurons
Authors: Tapper E. J.
Published: December 2017

Principles of autonomic nervous system control of intestinal ion transport need to include the newer concepts of the enteric nervous system (ENS). Based on studies of nervous control of the myenteric plexus, it is likely that ENS control of intestinal transport occurs through local mechanisms. In vitro transport studies and a limited number of radioreceptor-binding studies in mucosal cells support the notion that putative neurotransmitters alter transport by acting directly with mucosal receptors. In vivo and in vitro studies cannot alone uncover the indirect transport effects that neurotransmitters may have when they interact with enteric neurons. Studies focused on uptake and release of neurotransmitters suggest that norepinephrine (NE)-induced absorption may be modulated by local NE presynaptic neuronal mechanisms. Endogenous NE release may be enhanced by nicotinic and angiotensin II agents but decreased by muscarinic and alpha-adrenergic agents or prostaglandins. Presynaptic neuronal mechanisms that modulate endogenous acetylcholine (ACh) release and ACh-induced secretion are less well defined. Intestinal transport may be controlled by negative feedback, interneuronal, or transsynaptic presynaptic mechanisms. We propose that transport is controlled by a balance between the principal neurotransmitters NE and ACh. These neurotransmitters may be modulated by neuroactive peptides located either in neurons or in enteroendocrine cells. Efferent neurons may modulate release of neuropeptides from enteroendocrine cells into the luminal or antiluminal sides of mucosal cells. Intestinal transport also may be controlled by luminal factors that cause neuropeptide release from enteroendocrine cells or by specialized luminal receptors acting on sensory afferent neurons and intrinsic neuronal reflexes. Therefore, local modulation of intestinal transport by the ENS represents a finely tuned neuronal system with complex interrelations similar to many found in the central nervous system.
Relation of gastric acid and pepsin secretion to serum gastrin levels in dogs given bombesin and gastrin-17
Authors: Hirschowitz B. I. , Molina E.
Published: December 2017

To quantitate bombesin stimulation of gastric acid and pepsin via release of gastrin, five gastric fistula dogs were given graded doses (60-1,250 pmol X kg-1 X h-1) of bombesin tetradecapeptide and 40-2,000 pmol X kg-1 X h-1 of synthetic gastrin-17 (G-17). Acid and pepsin output and serum gastrin were proportional to the dose of stimulant. The half-maximal dose of bombesin for gastrin release was 200 pmol X kg-1 X h-1. Bombesin-stimulated acid secretion related to serum gastrin concentrations was congruent with the G-17 curve, but with a maximum of only 62% of the G-17 maximum before declining by 27% despite higher serum gastrin levels. This suggested that bombesin stimulates acid secretion only via gastrin release and inhibits at higher doses by releasing another inhibitory peptide, most likely somatostatin, which is also released by bombesin. The same mechanism could apply to supramaximal inhibition of acid and pepsin seen with high doses of G-17. Because the pepsin curve related to serum gastrin was to the left of the G-17 curve, we concluded that another secretagogue released by bombesin acts synergistically with gastrin on pepsin secretion. Therefore, bombesin stimulates gastric secretion through gastrin release, but its effects are modified by peptides coreleased to a) increase pepsin output at low doses and b) limit the output of acid and pepsin to 50-60% of the G-17 maximum.
Comparison of effects of neurotensin and fat on pancreatic stimulation in dogs
Authors: Konturek S. J. , Jaworek J. , Cieszkowski M. , Pawlik W. , Kania J. , Bloom S. R.
Published: December 2017

In six conscious dogs with esophageal, gastric, and pancreatic fistulas, the effects of intravenous infusion of neurotensin and intraduodenal instillation of sodium oleate on gastric and pancreatic secretion were determined under basal conditions and after exogenous (secretin and cholecystokinin octapeptides) or endogenous stimulants (feeding and duodenal acidification). Neurotensin given intravenously in graded doses (1.5–200 pmol . kg-1 . min-1) to fasted dogs produced a dose-dependent stimulation of pancreatic bicarbonate and protein secretion reaching, respectively, about 18 and 100% of maximal responses to secretin and cholecystokinin octapeptide (CCK). Duodenal oleate in graded doses (0.5–16 mmol/h) resulted in a similar pattern of bicarbonate and protein secretion but increased plasma neurotensin only to about 10% of that achieved with infusion of exogenous neurotensin producing an equal rate of pancreatic secretion. Neurotensin, like oleate, potentiated the action of secretin and CCK on pancreatic bicarbonate and had additive effects on protein response to these secretagogues. Both neurotensin and oleate increased pancreatic response to liver extract meal kept in the stomach at constant pH (5.5) and the response to sham feeding but decreased the response to ordinary feeding, probably due to the inhibition of gastric acid secretion and reduction of duodenal acidification. Neurotensin given intra-arterially directly to the pancreas or to isolated intestinal segment increased dose dependently the blood flow and oxygen consumption without affecting general circulation. We conclude that 1) neurotensin mimics the pancreatic secretory effects of intestinal fat, 2) neurotensin may contribute in part to fat-induced stimulation of the pancreatic secretion, and 3) the secretory effects of neurotensin are accompanied by a marked stimulation of intestinal and pancreatic circulation and metabolism.
Quantitative determination of macromolecular transport rate across intestinal Peyer's patches
Authors: Keljo D. J. , Hamilton J. R.
Published: December 2017

We used horseradish peroxidase (HRP) (mol wt, 40,000) to compare in vitro, in Ussing chambers, the rates of protein transport across segments of piglet jejunum with and without Peyer's patches. The mean HRP transport rate across intestinal segments with a patch, 25.2 +/- 4.2 SE ng . min-1 . cm-2 (22 animals), was increased threefold (P less than 0.0005) compared with control (no patch) tissue, 7.9 +/- 1.0 ng . min-1 . cm-2 (n = 29). Neither rate showed saturation with increasing concentrations of HRP; both were inhibited 75–95% by a temperature drop from 37 to 15 degrees C. Transport across patch-containing tissue was inhibited 48 +/- 6% (n = 5, P less than 0.0025) by 1 mM NaF, but NaF had no consistent effect on the transport across tissue without Peyer's patches. We conclude that HRP transport is increased across Peyer's patches. This transport is dependent on metabolism and does not involve specific receptors. These findings support the concept that the Peyer's patch serves an antigen-sampling function in the gut.