bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of distinct cAMP- and cGMP-dependent pathways by relaxant agents in isolated gastric muscle cells
Authors: Jin J. G. , Murthy K. S. , Grider J. R. , Makhlouf G. M.
Published: December 2017

The mechanism of action of vasoactive intestinal peptide (VIP) was examined in isolated gastric and taenia coli muscle cells and compared with that of nitric oxide (NO), sodium nitroprusside (SNP), and isoproterenol. In gastric muscle cells, VIP stimulated NO production, increased adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) levels, and induced relaxation in a concentration-dependent fashion. The NO synthase inhibitor NG-nitro-L-arginine abolished NO and cGMP production and partly inhibited relaxation. The soluble guanylate cyclase inhibitor LY 83583 abolished cGMP production and partly inhibited relaxation. (R)-p-adenosine 3',5'-cyclic phosphorothioate [(R)-p-cAMPS], a preferential inhibitor of cAMP-dependent protein kinase (cAK), and KT5823, a preferential inhibitor of cGMP-dependent protein kinase (cGK), partly inhibited relaxation separately and abolished relaxation in combination. The pattern implied that VIP induced relaxation by activation of cAK and by NO-mediated stimulation of cGMP and activation of cGK. In taenia coli muscle cells, VIP did not increase NO production or cGMP levels: relaxation was accompanied by an increase in cAMP and was partly inhibited by (R)-p-cAMPS and KT5823 and abolished by a combination of both inhibitors. Isoproterenol increased only cAMP levels in both cell types, which induced relaxation by activating cAK at low concentrations of agonist and both cAK and cGK at high concentrations in a pattern identical to that observed with VIP in taenia coli muscle cells. SNP and NO increased only cGMP levels in both cell types, which induced relaxation by activating cGK only. We conclude that cAK and cGK can be activated separately and mediate relaxation independently.(ABSTRACT TRUNCATED AT 250 WORDS)
Distribution and abundance of neutral endopeptidase (EC 3.4.24.11) in the alimentary tract of the rat
Authors: Bunnett N. W. , Wu V. , Sternini C. , Klinger J. , Shimomaya E. , Payan D. , Kobayashi R. , Walsh J. H.
Published: December 2017

The distribution of neutral endopeptidase (NEP; EC 3.4.24.11) was examined in the alimentary tract of the rat. Immunoreactive NEP and NEP mRNA were localized to epithelial cells of the small intestine and to muscle cells in the stomach, small intestine, and colon by immunohistochemistry and in situ hybridization histochemistry. NEP antisera recognized a protein on Western blots of membranes from gastric, jejunal, and colonic mucosa and gastric muscle with an electrophoretic mobility identical to that of recombinant human NEP (approximately 95 kDa). An antisense cRNA probe to NEP hybridized to RNA of approximately 3.5 kb and approximately 6.5 kb, corresponding to the primary transcripts of rat NEP, on Northern blots of total RNA from the jejunal mucosa. NEP message was detected in mRNA from jejunal and colonic mucosa and gastric, jejunal, and colonic muscle using a ribonuclease protection assay. NEP enzymatic activity, assessed by DL-thiorphan-inhibitable degradation of glutaryl-Ala-Ala-Phe-4-methoxy-2-naphthylamine, was highest in homogenates of jejunal mucosa (868 +/- 98 pmol.h-1 x micrograms protein-1) and was between 49- and 413-fold lower in other gastrointestinal tissues. The cellular origin of NEP in the gastric and colonic mucosa could not be determined.
Prostanoid release by Kupffer cells upon hypoxia-reoxygenation: role of pHi and Cai2+
Authors: Gyenes M. , De Groot H.
Published: December 2017

Primary cultures of rat Kupffer cells liberated significant amounts of prostaglandin (PG) D2, PGE2, and thromboxane (measured as thromboxane B2) when exposed to reoxygenation after 4 h of hypoxia. After a delayed onset, prostanoids were released at high rates for at least 8 h and after that time 700 pmol PGD2, 280 pmol PGE2, and 200 pmol thromboxane per 10(6) cells had been liberated. Unlike prostanoid release, leukotriene B4 production in reoxygenated cell cultures was only twice as much as in aerobic controls. Superoxide dismutase and catalase had no effect on PGD2, PGE2, and thromboxane production, indicating that prostanoid formation was independent of reactive oxygen species generated extracellularly and of cell injury. On the other hand, diphenyliodonium, as well as amiloride, blocked hypoxia-reoxygenation-induced PGD2, PGE2, and thromboxane release. The elevated prostanoid synthesis was preceded by increases in intracellular pH (from 7.23 to 7.38) and in intracellular Ca2+ (from 55 nM to a maximum level of 807 nM). These observations suggest a participation of NADPH oxidase and a related Na(+)-H+ exchange in the enhanced prostanoid synthesis, probably through the induction of an increased intracellular Ca2+ concentration.
Kinetic analysis of rat parotid gland muscarinic receptors in vivo: comparison with brain and heart
Authors: Hiramatsu Y. , Kawai R. , Reba R. C. , Simon T. R. , Baum B. J. , Blasberg R. G.
Published: December 2017

(RR)- and (SS)-quinuclidinyl iodobenzilate enantiomers [(RR)- and (SS)-IQNB, active and inert, respectively] have been synthesized for quantitative evaluation of muscarinic acetylcholine receptor (mAChR) binding. Pharmacokinetic approaches have not been used previously to assess in vivo IQNB binding in nonexcitable tissues. We have applied this method to examine mAChRs in rat parotid gland in comparison to those in brain and heart. Short-term infusion studies in vivo showed that the "instantaneous" reversible binding of (RR)- and (SS)-IQNB was high in the parotid (greater nonspecific binding potential), intermediate in the heart, and lowest in cortex and cerebellum. Long-term bolus injection experiments showed that the parotid gland mAChRs possessed a binding potential for receptor specific sites (380), which was intermediate between that of parietal cortex (930) and cerebellum (10) and greater than that of heart (165). In vitro binding to plasma membranes was generally consistent with the in vivo findings. In aggregate, these studies show that mAChRs can be evaluated in vivo in a nonexcitable tissue with the use of stereospecific ligands and a pharmacokinetic approach. The data suggest that IQNB, a mAChR antagonist, can identify characteristics of specific binding sites, which may reflect tissue differences.
Expression and processing of prohormones in nonendocrine cells
Authors: Dickinson C. J. , Takeuchi T. , Guo Y. J. , Stadler B. T. , Yamada T.
Published: December 2017

Pancreatic polypeptide (PP) and gastrin are initially synthesized as larger precursors that require posttranslational processing to produce the biologically active peptides. These steps include tryptic cleavage at paired basic residues, their subsequent removal by carboxypeptidase H, and formation of a carboxy-terminal amide moiety via the action of peptidyl-glycine alpha-amidating monooxygenase (PAM). Although these posttranslational processing reactions are presumed to occur primarily in the secretory granule of endocrine cells, nonendocrine cells that do not possess these structures nevertheless are able to posttranslationally process a wide variety of proteins destined for export. In these studies we sought to determine whether the mechanisms for prohormone processing are present in nonendocrine cell lines. We examined two fibroblast cell lines (psi-2, BHK) a hepatocyte cell line (Hepa), and an exocrine pancreatic cell line (AR42J). We used the pZIPneo(SVX) retroviral vector to express cDNA clones encoding human PP and gastrin in the nonendocrine cells. Transfected psi-2, BHK, and Hepa cells produced a precursor of PP that appeared to be secreted constitutively, with little remaining in intracellular stores. Almost no posttranslational processing of the PP precursor was evident in these cells. By contrast, AR42J cells were capable of expressing and storing fully processed and carboxy-terminally amidated PP and gastrin. These data support the notion that the sorting mechanisms in endocrine and exocrine cells are similar and that the posttranslational processing of peptide hormone precursors requires storage in secretory granules.(ABSTRACT TRUNCATED AT 250 WORDS)
Effect of intracellular acidification on colonic NaCl absorption
Authors: Dagher P. C. , Egnor R. W. , Charney A. N.
Published: December 2017

CO2 stimulates Na+ and Cl- absorption in rat distal colon. This is most likely due to intracellular generation of H+ and HCO3- and stimulation of apical Na(+)-H+ and Cl(-)-HCO3- exchangers. We examined whether intracellular acidification by means other than CO2 would also stimulate Na+ absorption. Stripped segments of distal colon from male Sprague-Dawley rats were studied under short-circuit conditions in Ussing chambers. Identically prepared tissues were used for intracellular pH (pHi) measurements with the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. When the Ringer PCO2 was increased from 20 to 34 mmHg, pHi decreased from 7.50 +/- 0.04 to 7.35 +/- 0.04 and net Na absorption increased from 2.4 +/- 0.7 to 3.7 +/- 0.7 mu eq.cm-2 x h-1. A similar degree of intracellular acidification was obtained with 2.6 microM nigericin, but no stimulation of Na+ absorption was seen. When Ringer-HCO3- concentration was reduced from 39 to 11 mM at constant PCO2 = 35 mmHg, pHi decreased from 7.55 +/- 0.02 to 7.11 +/- 0.02 with no effect on net Na+ absorption. A similar reduction in pHi in a CO2-HCO3(-)-free, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered Ringer also did not stimulate Na+ absorption. Methazolamide had no effect on steady-state pHi at any given PCO2 but caused marked reductions in net Na+ absorption (9.6 +/- 2.4 to 5.2 +/- 1.2 mu eq.cm-2 x h-1 at PCO2 = 70 mmHg). We conclude that Na+ absorption in rat distal colon is not stimulated by intracellular acidification per se but rather has an absolute requirement for CO2 and carbonic anhydrase activity.
Kainic acid injection into medullary raphe produces gastric lesions through the vagal system in rats
Authors: Okumura T. , Uehara A. , Taniguchi Y. , Watanabe Y. , Tsuji K. , Kitamori S. , Namiki M.
Published: December 2017

There is little evidence of a role of nuclei in the brain stem, other than the dorsal motor nucleus of the vagus nerve in the medulla oblongata, in terms of central mechanisms for gastric ulcer formation. Recent reports suggest that the medullary raphe nuclei may be involved in the central regulation of gastric functions such as gastric acid secretion and motility. In the present study, we examined whether neurons in the nucleus raphe obscurus (NRO) played a significant role in the formation of gastric ulcers with the use of Sprague-Dawley rats. First, we determined whether excitation of the medullary raphe resulted in the development of gastric lesions. Chemical stimulation of neurons in the NRO by kainic acid produced gastric erosions within 4 h in 24-h fasted rats. Saline tested under the same conditions did not modify the integrity of the gastric mucosa. It was also demonstrated that kainic acid injection outside the raphe obscurus boundaries failed to develop gastric lesions. Next, we examined the effects of the vagal system on the gastric lesion formation induced by stimulation of the raphe nucleus. Bilateral gastric branch vagotomies completely prevented the development of the raphe nucleus-provoked gastric mucosal damage. All these results suggest, for the first time, that excitation of neurons in the medullary raphe obscurus induces gastric ulceration through vagal stimulation.
Adenosine is a mediator of ethanol-induced gastric vasodilation in dogs
Authors: Wood J. G. , Darnell M. P. , Cheung L. Y.
Published: December 2017

The purpose of this study was to assess the role of histamine, adenosine, and prostaglandins as mediators of ethanol-induced gastric vasodilation. In an ex vivo segment of canine stomach, vasodilation occurred within the first minute of replacing luminal saline with ethanol (40% vol/vol). Ethanol caused vascular resistance to progressively decrease by approximately 53% compared with control values. In other experiments, intra-arterial infusion of histamine (300 ng/ml) or adenosine (30 micrograms/ml) to the gastric segment produced similar degrees of vasodilation as observed with ethanol. The response to these vasodilators could be markedly attenuated with specific antagonists of these substances (histamine: pyrilamine plus cimetidine; adenosine: 8-phenyltheophylline). In our final experiments, indomethacin or histamine- or adenosine-receptor antagonists were given before application of topical ethanol. Indomethacin or histamine antagonists had no significant effect on the time course or magnitude of ethanol-induced vasodilation. In contrast, pretreatment with 8-phenyltheophylline significantly reduced changes in vascular resistance during exposure to luminal ethanol. These results suggest that locally released adenosine is an important mediator of ethanol-induced vasodilation in the canine stomach under these conditions.
Functional difference between SP and NKA: relaxation of gastric muscle by SP is mediated by VIP and NO
Authors: Jin J. G. , Misra S. , Grider J. R. , Makhlouf G. M.
Published: December 2017

The mechanism of action of endogenous tachykinins [substance P (SP) and neurokinin A and B (NKA and NKB)] and of receptor-specific tachykinin analogues (SP methyl ester (SPME), [beta-Ala8]NKA-(4-10), and senktide) was examined in circular muscle of guinea pig stomach. Cross-desensitization studies confirmed that SPME and SP interacted with NK-1 receptors, [beta-Ala8]NKA-(4-10) and NKA with NK-2 receptors, and senktide and NKB with NK-3 receptors. NK-1 and NK-3-receptor agonists induced relaxation and stimulated vasoactive intestinal peptide (VIP) release and nitric oxide (NO) production: tetrodotoxin abolished VIP release, NO production, and relaxation, converting the response to NK-1-receptor agonists to contraction; the NO synthase inhibitor NG-nitro-L-arginine (L-NNA) abolished NO production, partly inhibited VIP release (56-64%, P < 0.01), and abolished relaxation; the VIP antagonist VIP-(10-28) partly inhibited NO production (73-74%, P < 0.001) and relaxation (56-58%, P < 0.01); and atropine augmented relaxation by 28-35% (P < 0.01). The pattern of inhibition implied that: 1) relaxation was mediated by VIP and NO; 2) VIP release was partly dependent on NO production, since it was strongly inhibited by L-NNA; and 3) NO was largely produced by the action of VIP on muscle cells, since it was strongly inhibited by VIP-(10-28). NK-2-receptor agonists elicited only contraction that was not affected by tetrodotoxin; these agonists also inhibited VIP release, NO production, and relaxation induced by NK-1- and NK-3-receptor agonists.(ABSTRACT TRUNCATED AT 250 WORDS)
Exocrine interstitial insulin and somatostatin in the perfused dog pancreas
Authors: Nakagawa A. , Samols E. , Stagner J. I.
Published: December 2017

To determine whether the islet-acinar relationship is mediated microvascularly or by diffusion from islets, isolated dog pancreata were perfused anterogradely (n = 4) or retrogradely (n = 5). Interstitial fluid (ISF) was sampled utilizing a microdialysis technique. Three to five microdialysis probes were placed in the dorsal lobe of each pancreas. Insulin and somatostatin were measured in dialysate, as well as in perfusate, under both resting (5.6 mM glucose) and stimulated (12.2 mM glucose plus 20 mM arginine) conditions. During the stimulative period, retrograde ISF responses were markedly less than anterograde responses, whereas perfusate (intravascular) anterograde and retrograde responses were not different; ratios of the sum of increments above basal levels of ISF concentrations to the sum of increments of perfusate concentrations (sigma delta ISF/sigma delta perfusate) of insulin and somatostatin during anterograde vs. retrograde perfusion were 35.1 +/- 8.8 (SE; n = 9) vs. 13.8 +/- 2.8% (n = 10) and 37.1 +/- 14.2 (n = 7) vs. 8.5 +/- 4.2% (n = 9), respectively (both, P < 0.05). These data strongly suggest that these islet hormones may be delivered to the exocrine interstitial space via a directed microcirculation from the islet to the acinar tissue.
Asialoglycoprotein receptor mRNAs are expressed in most extrahepatic rat tissues during development
Authors: Mu J. Z. , Tang L. H. , Alpers D. H.
Published: December 2017

The expression of an asialoglycoprotein receptor, comprised of a major (R-1) and minor (R-2/3) component, was characterized in various rat tissues during postnatal development. Two R-1 mRNA subspecies were identified in the liver by Northern blots. The 1.4-kb form, which was the predominant subspecies in the liver, was detected in small amounts only in the stomach and kidney. The 1.7-kb mRNA, however, was present in all extrahepatic tissues examined. R-1 mRNA content in the liver increased by only 38% during the suckling period, but the extrahepatic R-1 mRNA had a dramatic biphasic developmental pattern with the highest levels at birth and after weaning. The R-2/3 mRNA was present as a single species on Northern blot in the liver and in a restricted number of extrahepatic tissues (salivary glands, ileum, and kidney) but was detected in most tissues by polymerase chain reaction. The R-2/3 mRNA content in the liver increased fourfold after birth but was consistently low in the other tissues. [57Co]cobalamin-haptocorrin binding activity was present in all extrahepatic tissues examined, although only at a level 2-5% of hepatic binding. Binding affinities of the adult kidney brush-border membranes (1.9 x 10(9) M-1) and the adult liver plasma membranes (2.4 x 10(9) M-1) were comparable. On Western blot, < 4% of adult liver R-1 content and a relatively larger amount of R-2/3 were detected in all four extrahepatic tissues examined. Thus the low binding activity in extrahepatic tissues correlates with the presence of both receptor subunits, which are closely related to or identical with the hepatic receptor subunits.
Distinct receptors for cholecystokinin and gastrin on canine fundic D-cells
Authors: DelValle J. , Chiba T. , Park J. , Yamada T.
Published: December 2017

Despite the extensive amino acid homology between gastrin and cholecystokinin (CCK) at the biologically active carboxyl terminus, the receptors through which these peptides exert their action are heterogeneous. In previous studies, we have examined the biological activity of gastrin/CCK peptides on isolated canine fundic D-cells and observed that CCK is a more potent and efficacious stimulant of somatostatin release than gastrin. We performed the present studies to distinguish between distinct CCK (CCK-A subtype) and gastrin (CCK-B/gastrin subtype) receptors on canine D-cells. Consistent with this observation was our finding that the CCK-A receptor selective antagonist L-364,718 dose dependently (10(-11)-10(-7) M) inhibited CCK-mediated somatostatin release but at the same doses did not alter the effect of gastrin. CCK and gastrin exhibited similar potency in displacing bound 125I-labeled Leu15 gastrin-17 from D-cells. However, when 125I-CCK octapeptide (CCK-8) was used as the radioligand, a fraction of the bound label could not be displaced with gastrin, but this fraction was completely displaced with CCK-8. In D-cells pretreated with high concentrations of gastrin, L-364,718 was able to inhibit the gastrin-resistant fraction of 125I-CCK-8 binding, but the CCK-B/gastrin receptor selective antagonist (PD 134308) was unable to influence this fraction of binding in doses as high as 10(-6) M. These studies delineate the presence of distinct CCK-A and CCK-B/gastrin receptors on canine fundic D-cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Attenuation of endotoxin-induced intestinal microcirculatory damage by eicosapentanoic acid
Authors: Miura S. , Imaeda H. , Shiozaki H. , Kurose I. , Fukumura D. , Tashiro H. , Serizawa H. , Suematsu M. , Sekizuka E. , Tsuchiya M.
Published: December 2017

The major objective of this study is to investigate whether oral administration of eicosapentanoic acid (EPA) has any preventive effect on endotoxin-induced microcirculatory damage of rat small intestine. EPA in a daily dose of 300 mg/kg was orally given to male Wistar rats for 3 wk. Submucosal microvessels of the ileum were observed by intravital microscopy equipped with a high-speed video camera system after the intra-arterial infusion of endotoxin at a dose of 2 mg.kg-1.h-1. The number of sticking leukocytes was significantly increased at 30 min after the treatment of endotoxin especially along the smaller branch of intestinal venules. It reached the maximal plateau at 45 min after treatment. The pretreatment of EPA significantly attenuated the increase in sticking leukocytes induced by endotoxin. A platelet-activating factor (PAF) antagonist 2-[N-acetyl-N-(2-methoxy-3-octadecylcarbamoyloxy propoxycarbonyl) aminomethyl]-1-ethylpyridinium chloride (CV-6209) significantly prevented the increased leukocyte sticking to the same extent as EPA treatment. Thirty minutes after endotoxin infusion, red blood cell (RBC) velocity was significantly decreased in both arterioles and venules. RBC velocity appeared to be continuously decreased thereafter and reached its minimum value at approximately 60 min. EPA treatment was revealed to prevent the decrease in RBC velocity of microvessels induced by endotoxin. CV-6209 also significantly attenuated the decreased RBC velocity. The remarkable elevation of PAF content in the ileal mucosa as observed by endotoxin infusion was also significantly attenuated by administration of EPA.(ABSTRACT TRUNCATED AT 250 WORDS)
Inhibition of VIP-stimulated ion transport by a novel Y-receptor phenotype in rabbit distal colon
Authors: Ballantyne G. H. , Goldenring J. R. , Fleming F. X. , Rush S. , Flint J. S. , Fielding L. P. , Binder H. J. , Modlin I. M.
Published: December 2017

Neurocrine, endocrine, and paracrine regulators are critical to the control of colonic secretion. These studies have investigated the inhibition of vasoactive intestinal polypeptide (VIP)-stimulated ion transport by peptide YY (PYY) and other Y-class effectors in rabbit distal colonic mucosa mounted in Ussing chambers. PYY decreased basal short-circuit current (Isc) but did not significantly change either basal Na+ or Cl- flux. PYY inhibited VIP-stimulated increases in Isc by up to 86% and abolished VIP-induced Cl- secretion. PYY decreased VIP-generated increases in Isc by a tetrodotoxin-insensitive mechanism. PYY inhibited cholera toxin-stimulated as well as forskolin-stimulated increases in Isc but failed to alter stimulation by 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP). PYY decreased VIP-stimulated increases in tissue cAMP by 88% and forskolin-stimulated increases by 84%. PYY, neuropeptide Y (NPY), (Leu31,Pro34)-NPY, and pancreatic polypeptide (PP) all demonstrated potent inhibition of VIP-stimulated increases in Isc. PYY-(13-36) demonstrated little effect on VIP stimulation. Thus the rabbit distal colon possesses a novel Y-class receptor phenotype that demonstrates high affinity for all three PP-fold peptides, NPY, PYY, and PP.
Hepatocyte horseradish peroxidase uptake is saturable and inhibited by mannose-terminal glycoproteins
Authors: Yamaguchi Y. , Dalle-Molle E. , Hardison W. G.
Published: December 2017

In the liver, horseradish peroxidase (HRP) is thought to be taken up via mannose receptor-mediated endocytosis by non-parenchymal cells (NPC) and via fluid-phase endocytosis by hepatocytes. When we attempted to inhibit NPC uptake of HRP with mannan in the whole perfused rat liver, > 80% of HRP uptake was eliminated. Liver cell fractionation revealed that mannan not only inhibited HRP uptake by NPC (91%) but also by hepatocytes (81%). In isolated hepatocytes, HRP uptake was linear over 60 min and saturable in the range of 0 to 200 mg/l (Vmax = 4.3 ng.mg protein-1.min-1; Km = 8.3 mg/l). Mannan inhibited uptake competitively (Ki = 2.0-2.5 mg/l). At high concentrations of HRP, a nonsaturable component of HRP uptake became evident (k = 2.8 pg.mg protein-1.min-1.mg HRP-1.l-1). Hepatocyte uptake of HRP was inhibited by other glycoproteins and glycopeptides with mannose-terminal groups, as well as by mannan, but not by asialofetuin (ASF) or bovine serum albumin. Hepatocyte uptake of 125I-labeled ASF, which is taken up via the asialoglycoprotein receptor, was saturable and not inhibited by mannan. HRP binding to hepatocytes, determined at 4 degrees C, was also inhibited by mannan. Quantification of contamination of the parenchymal cell fraction by NPC by cell counting and by pronase digestibility suggested our results could not be explained by contamination of hepatocytes by NPC. At concentrations used for most morphological studies (1,000-10,000 mg/l), fluid-phase endocytosis accounts for much of HRP uptake. However, at low concentrations, a saturable low-capacity mechanism is responsible for most HRP uptake by the hepatocyte.(ABSTRACT TRUNCATED AT 250 WORDS)
Glucagon induces biliary protein excretion in guinea pigs
Authors: Lenzen R. , Tavoloni N.
Published: December 2017

This study was done to determine glucagon's effect on protein biliary excretion in anesthetized, bile duct-cannulated guinea pigs. Glucagon (1.4 nmol.min-1.kg-1) induced choleresis and increased protein biliary concentration from 0.12 +/- 0.04 to 0.20 +/- 0.6 mg/ml and protein output from 22.8 +/- 3.8 to 54.5 +/- 16.1 micrograms.kg-1.min-1. Protein biliary excretion increased during the first 10 min of glucagon infusion and progressively declined thereafter. Biochemical analysis of biliary protein revealed that the increase could be accounted for primarily by an increase in the lysosomal enzymes acid phosphatase and beta-glucuronidase. Biliary excretion of the canalicular membrane enzymes 5'-nucleotidase and alkaline phosphatase only modestly increased, whereas that of [14C]sucrose, a marker of paracellular fluid transport, was unaffected. On the other hand, glucagon enhanced biliary entry of horseradish peroxidase in a fashion similar to that observed with total endogenous protein. These effects were mediated by the adenosine 3',5'-cyclic monophosphate (cAMP) system, since infusion of dibutyryl-cAMP at 0.5 mumol.kg-1.min-1 increased bile flow and biliary protein excretion in a time-dependent manner, as observed with glucagon. Glucagon's failure to sustain enhanced protein biliary output was not due to declining hepatic concentrations of cAMP or to depletion of hepatocellular lysosomal enzymes. These studies provide evidence that glucagon stimulates biliary excretion of protein in guinea pigs that can be accounted for by biliary discharge of enzyme originating from the canalicular membrane and, primarily, from the lysosomal compartment. Although the precise mechanism(s) underlying these effects remains to be elucidated, it is suggested that the increase in canalicular membrane enzyme excretion is due to glucagon's effect on exocytosis.(ABSTRACT TRUNCATED AT 250 WORDS)
Changes in colonic motility following abdominal irradiation in dogs
Authors: Summers R. W. , Hayek B.
Published: December 2017

The purpose of the study was to compare colonic motor patterns before and after a single abdominal dose of X-rays in dogs. Recordings were made from five serosally implanted strain gauges at equidistant intervals along the colon in seven dogs (2 dogs also had 2 jejunal electrodes and 1 had ileal electrodes). Control recordings were made for 3 h in the fasted state and daily for 2 wk after an absorbed X-ray dose of 938 cGy was delivered to the abdomen. The duration of migrating colonic motor complexes decreased from 7.2 +/- 0.5 to 3.9 +/- 0.4 min while the mean amplitude fell from 10.3 +/- 0.6 to 1.8 +/- 0.2 g (P < 0.05). The rate of nonmigrating colonic motor complex occurrence increased from 0.6 +/- 0.1 to 1.2 +/- 0.2 per hour (P < 0.05). Colonic giant migrating contractions were rarely observed during control recordings (2 in 80 h of recording). In contrast, repetitive clusters of giant contractions were observed 5-8 days after exposure in five of seven dogs (1.5/h) and were associated with restlessness, whining, and passage of diarrheal stools (sometimes bloody) with nearly every occurrence. The basic colonic motility patterns were less disrupted than were jejunal myoelectric patterns at the same irradiation dosage. However, the study demonstrates the important role of colonic giant migrating contractions in pathological diarrheal states such as irradiation injury.
Altered expression of sodium pump isoforms in the inflamed intestine of Trichinella spiralis-infected rats
Authors: Khan I. , Collins S. M.
Published: December 2017

We used polymerase chain reaction (PCR) and Northern analysis to examine the effect of Trichinella spiralis infection on mRNA expression of sodium pump isoforms in rat intestine. 28S RNA was coamplified as an internal standard, and the abundance of the isoformic mRNA was expressed relative to 28S RNA. As assessed by PCR and Northern analysis, the alpha 1- and beta 1-subunits were expressed in the jejunum and ileum of noninfected control rats. However, in the jejunum the levels were suppressed after 24 h of infection. The suppression became maximal by day 6 and was reversed to normal by day 23 postinfection (PI). In contrast, no suppression was observed in the noninflamed ileum of infected rats. Further analysis revealed that alpha 1-mRNA was suppressed in jejunal mucosa by 89% and in longitudinal muscle-myenteric plexus by 62% but was not suppressed in circular muscle. Western blot analysis revealed a 61% decrease in the alpha 1-subunit protein in the mucosa and a 62% decrease in longitudinal muscle, whereas there was no change in the alpha 1-protein in circular muscle on day 6 PI. In contrast, marked (89%) suppression of beta 1-mRNA was observed only in the mucosa; there was no change in either circular or longitudinal muscle on day 6 PI. These results indicate that T. spiralis infection in the rat is accompanied by a significant decrease in the alpha 1-subunit of the sodium pump in longitudinal muscle. This reduction could result from altered regulation involving pretranslational changes.(ABSTRACT TRUNCATED AT 250 WORDS)
Sequence, tissue distribution, and functional characterization of the rat fructose transporter GLUT5
Authors: Rand E. B. , Depaoli A. M. , Davidson N. O. , Bell G. I. , Burant C. F.
Published: December 2017

cDNA clones encoding rat GLUT5-small intestinal facilitative hexose transporter were isolated from a jejunum library by cross-hybridization with a human GLUT5 cDNA probe. The cDNA sequence indicates that rat GLUT5 is composed of 502 amino acids and has 81.5% amino acid identity and 87.3% similarity with the sequence of human GLUT5. Expression of synthetic rat GLUT5 mRNA in Xenopus oocytes showed that rat GLUT5 was able to mediate the uptake of fructose and, to a lesser extent, of glucose. RNA blotting studies showed that GLUT5 mRNA was present in rat small intestine, kidney, and brain. Although GLUT5 mRNA is expressed in human testis, adipose tissue, and skeletal muscle, it could not be detected by RNA blotting in these rat tissues. Developmental studies showed low levels of GLUT5 mRNA in rat small intestine and kidney during the prenatal period with a rapid induction of GLUT5 expression occurring postnatally. In situ hybridization studies of GLUT5 mRNA expression in the small intestine revealed differential expression along the crypt-villus axis with the highest levels of mRNA being in the midvillus region. In addition, there was quantitatively more GLUT5 mRNA detected in the proximal as opposed to the distal small intestine.
Actions of Helodermatidae venom peptides and mammalian glucagon-like peptides on gastric chief cells
Authors: Rai A. , Singh G. , Raffaniello R. , Eng J. , Raufman J. P.
Published: December 2017

The actions of peptides (helospectin I, helodermin, exendin-3, exendin-4) that have been isolated from the venoms of Helodermatidae lizards were examined using dispersed chief cells from guinea pig stomach. These actions were compared with those of mammalian glucagon-like peptides, particularly truncated glucagon-like peptide 1 (TGLP-1), a peptide that shares 53% homology with exendin-4. The Helodermatidae venom peptides and TGLP-1 caused a two- to threefold increase in chief cell adenosine 3',5'-cyclic monophosphate and pepsinogen secretion. Exendin-3 and exendin-4 were 100 times more potent than helospectin I and helodermin and 10 times more potent than TGLP-1. Helospectin I and helodermin, but not exendin-4 or TGLP-1, inhibited the binding of 125I-labeled vasoactive intestinal peptide (VIP) and 125I-secretin to dispersed chief cells. The actions of exendin-3, exendin-4, and TGLP-1, but not those of helospectin I, helodermin, VIP, or secretin, were progressively inhibited by increasing concentrations of an exendin-receptor antagonist, exendin-(9-39)-NH2. These data indicate that in gastric chief cells, whereas the actions of helospectin I and helodermin are mediated by interaction with high-affinity secretin (low-affinity VIP) receptors, the actions of exendin-3, exendin-4, and TGLP-1 are mediated by interaction with exendin receptors.
Tumor necrosis factor-alpha produces hepatocellular dysfunction despite normal cardiac output and hepatic microcirculation
Authors: Wang P. , Ayala A. , Ba Z. F. , Zhou M. , Perrin M. M. , Chaudry I. H.
Published: December 2017

Although plasma levels of tumor necrosis factor (TNF) are elevated and hepatocellular dysfunction occurs even in the early hyperdynamic stage of sepsis, the precise mechanism responsible for this dysfunction remains unknown. Although TNF at high doses produces circulatory failure, it is not known whether the dose of TNF that does not adversely affect hemodynamics alters hepatocellular function. To study this, recombinant murine TNF-alpha was infused intravenously (0.05 or 0.25 mg/kg) over 30 min in normal rats. At 1 and 4 h after infusion of TNF-alpha or an equivalent volume of saline, hepatocellular function [i.e., maximum velocity (Vmax) and Michaelis constant (Km)] was assessed using in vivo indocyanine green clearance without blood sampling. Additional parameters measured were as follows: cardiac output by dye dilution, hepatic microcirculation by laser Doppler flowmetry and colloidal carbon infusion, plasma TNF and interleukin-6 (IL-6) by cytokine-dependent cellular assays, and plasma glucose enzymatically. The results indicate that although infusion of 0.05 mg/kg TNF-alpha did not affect Vmax and Km, its infusion at 0.25 mg/kg produced a significant depression of hepatocellular function and markedly increased the synthesis and/or release of IL-6. TNF-alpha-induced hepatocellular dysfunction was not associated with any significant changes in hepatic microcirculation, plasma glucose, cardiac output, and other measured hemodynamic parameters. Thus hepatocellular dysfunction observed after TNF infusion may be due to the direct effect of this cytokine alone or in combination with IL-6.
Role of calcium fluxes in the action of glucagon on glucose metabolism in rat hepatocytes
Authors: Mine T. , Kojima I. , Ogata E.
Published: December 2017

The aim of the present study was to assess the role of calcium fluxes in the action of glucagon on glycogenolysis and gluconeogenesis in isolated rat hepatocytes. Calcium influx was blocked by two ways: by use of the compound tetramethrin and by reduction of extracellular calcium to 1 microM. The minimal concentration of tetramethrin that inhibited glucagon-mediated calcium entry was 7.5 x 10(-7) M. In the presence of 7.5 x 10(-7) M tetramethrin, glucagon-induced glycogenolysis was markedly attenuated when glucagon concentration was 10(-9) M or higher. In contrast, tetramethrin had no effect on glucogenolysis evoked by lower concentrations of glucagon. Similarly, tetramethrin greatly reduced gluconeogenesis induced by high concentrations of glucagon without affecting the effect of low concentrations of glucagon. The same results were obtained in the presence of 1 microM extracellular calcium. To abolish glucagon-induced elevation of cytoplasmic free calcium concentration, we heavily loaded quin2 into hepatocytes. In these cells, glycogenolysis evoked by low concentrations of glucagon was completely abolished. Glycogenolysis caused by high concentrations of glucagon was markedly inhibited. These results indicate that glucagon action on hepatic glucose metabolism is mediated by two different mechanisms, which depend on concentrations of glucagon.
Hepatic, renal, and total body galactose elimination in the pig
Authors: Winkler K. , Henriksen J. H. , Tygstrup N.
Published: December 2017

Galactose elimination capacity is used as a quantitative measure of liver function on the assumption that galactose elimination outside the liver is negligible or easily corrected for. The relationship between hepatic and extrahepatic removal of galactose was studied in anesthetized pigs during quasi-steady-state conditions by continuous infusion of galactose. The hepatic removal approximated a constant [maximal velocity = 585 +/- 41 mumol/min, Michaelis constant (Km) = 0.24 +/- 0.07 mmol/l, mean +/- SE, n = 20]. The renal removal was less than the amount filtered, showing maximal tubular reabsorption (Tm 178 +/- 3.0 mumol/min, Km 3.8 +/- 0.9 mmol/l, n = 20). Metabolic conversion of galactose in the kidney was not demonstrable. At all concentrations studied (0.4-5.8 mmol/l), total galactose elimination from the body exceeded the sum of hepatic and renal elimination by approximately 100 mumol/min, independent of the concentration. At blood concentrations usually used for clinical estimation of the galactose elimination capacity (approximately 4 mmol/l), hepatic removal in the pig accounted for 55% and renal removal for 30% of total removal; 15% of removal occurred in other organs. We conclude that estimation of the hepatic galactose elimination capacity from whole body elimination curves requires correction for renal removal of galactose.
Expression of rat intestinal L-lysine transport systems in isolated oocytes of Xenopus laevis
Authors: Harvey C. M. , Muzyka W. R. , Yao S. Y. , Cheeseman C. I. , Young J. D.
Published: December 2017

The mechanisms by which cationic amino acids are transported across the intestinal epithelium are poorly understood. We show that isolated stage VI oocytes of Xenopus laevis can express lysine transport activity, which is due to the microinjection of mRNA from rat small intestine. L-Lysine transport activity (0.2 mM, 20 degrees C) reaches 400 pmol.oocyte-1.h-1, compared with a typical endogenous rate of 85 pmol.oocyte-1.h-1. Na(+)-dependence and amino acid inhibition studies resolved the expressed transport activity into three components: 1) a Na(+)-dependent transport system that can be inhibited by leucine with high affinity and also by alanine; 2) a Na(+)-independent system that can be inhibited by leucine with high affinity when Na+ is present, but this affinity is reduced in its absence; 3) a Na(+)-independent system that is inhibited by leucine with high affinity only when Na+ is present. Peak arginine-inhibitable lysine influx was found in a mRNA size fraction of 1.5-2.25 (median 2.0) kb.
Peptide-regulated guanylate cyclase pathways in rat colon: in situ localization of GCA, GCC, and guanylin mRNA
Authors: Li Z. , Goy M. F.
Published: December 2017

Guanylate cyclases play a role in both physiological and pathological secretion in the mammalian intestine. Agents that raise guanosine 3',5'-cyclic monophosphate (cGMP) levels, such as atrial natriuretic peptide (ANP), guanylin (an endogenous intestinal peptide), or Escherichia coli heat-stable enterotoxin type a (STa; a bacterial toxin), enhance electrolyte secretion and the accumulation of luminal fluid. Although secretion in all parts of intestine is sensitive to changes in cGMP metabolism, an increasing body of evidence suggests that these responses are particularly important in proximal colon. To date, three peptide-sensitive membrane-bound guanylate cyclases [types A, B, and C (GCA, GCB, and GCC, respectively)] have been cloned from mammalian tissues. GCA responds to ANP, GCB to C-type natriuretic peptide, and GCC to guanylin and STa. Expression of these receptor/cyclase genes has not previously been investigated at the cellular level in the colon. Nucleotide probes specific for GCA, GCB, GCC, and guanylin were generated by polymerase chain reaction. These probes were used to evaluate colonic cyclase and guanylin mRNA expression in the rat. GCB mRNA is not detectable in this tissue either by in situ hybridization or by Northern blot analysis. In contrast, GCA, GCC, and guanylin mRNAs are all conspicuously expressed. With the in situ hybridization technique, GCA mRNA expression is seen in cells in the lamina propria. GCC mRNA expression is seen in epithelial cells throughout colonic crypts, and also, although at a slightly lower level, in cells of the surface epithelium.(ABSTRACT TRUNCATED AT 250 WORDS)
Calibration of albumin-fatty acid binding constants measured by heptane-water partition
Authors: Burczynski F. J. , Pond S. M. , Davis C. K. , Johnson L. P. , Weisiger R. A.
Published: December 2017

Most measurements of binding affinity of albumin for long-chain fatty acids are based on heptane-water partition. In this method, equilibrium partition of fatty acid between heptane and an albumin-containing buffer is calibrated using the partition ratio between heptane and buffer in the absence of protein. In the current study, we used a variety of techniques to examine potential problems with this approach. Hydrophobic impurities in commercial [3H]palmitate preparations were incompletely removed by standard purification techniques. These impurities contributed from 5% of the total radioactivity in the heptane phase at low albumin concentrations (5 microM) to 62% at higher albumin concentrations (500 microM), thus confounding determination of binding affinity. These were identified by gas chromatography/mass spectroscopy as radio-labeled glycerol monopalmitate and monostearate. When albumin was not present, the partition ratio was similar to values reported by others. However, our results varied by a factor of four (265-1,119) depending on how the solutions were prepared. Although a true equilibrium partition must not depend on starting conditions, the partition ratio after 24-72 h was > 2x as large when tracer [3H]palmitate was added to the heptane phase than when it was added to the aqueous phase. Results also depended on the relative volumes of heptane and buffer used, approaching a maximum of 1,445 +/- 112 for very low heptane/buffer volume ratios. Much of this variability was due to hydrophilic impurities in [3H]palmitate, which ranged from 0.2 to 1.2% in commercial lots down to 0.1-0.5% after alkaline ethanol extraction and < 0.05% after thin-layer chromatography (TLC).(ABSTRACT TRUNCATED AT 250 WORDS)
Human and rat intestinal plasma membrane calcium pump isoforms
Authors: Howard A. , Legon S. , Walters J. R.
Published: December 2017

The intestinal basolateral membrane Ca(2+)-transporting adenosinetriphosphatase is the energy-dependent step in the absorption of dietary Ca2+ by the vitamin D-dependent transcellular pathway. Multiple plasma membrane Ca(2+)-pump isoforms are produced from four genes (PMCA1 to 4) and alternative mRNA splicing. We have studied which isoforms are detectable in adult human and rat gastrointestinal tissues by polymerase chain reaction (PCR) amplification, sequencing, and blotting. PMCA1 was the predominant gene product amplified from human small intestinal mucosa, although a minor additional variant lacking the exon at splice site B was detected, which resembled that described for PMCA4. Of the variants described at site C, only the shortest transcript of PMCA1 was amplified; both previously described forms of PMCA4 were found, particularly in colon where PMCA4 predominated. From rat intestinal cDNA, mixed primer PCR amplified PMCA1 and a novel sequence, the rat PMCA4 homologue, which was expressed in many tissues including small intestinal muscle and colon. However, PMCA1 was overwhelmingly predominant in the mucosa of the small intestine, being most abundant in duodenum. These results suggest the involvement of the Ca(2+)-pump isoform PMCA1b in intestinal Ca2+ absorption.
Reversible disassembly of an intestinal epithelial monolayer by prolonged exposure to phorbol ester
Authors: Hecht G. , Robinson B. , Koutsouris A.
Published: December 2017

This article describes a model of reversible disassembly of a cultured human intestinal epithelial monolayer by prolonged exposure to the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). Prolonged phorbol ester exposure reduces protein kinase C (PKC) levels in numerous cell types including T84, as shown here. Under PKC-downregulated conditions, T84 monolayers, which simulate the highly organized structure of native intestinal crypt cells, become disassembled into 2 or 3 layers of rounded cells. Proliferation does not account for these morphological changes as assessed by thymidine incorporation studies. The effects of structural disorganization on epithelial barrier function was also examined. The permeability of disassembled monolayers was significantly greater than that of controls. Flux studies localized the permeability defect to the tight junction. PKC-associated alterations in the perijunctional ring of actin and myosin, one of the putative regulators of flow across the tight junction, were found to correlate with the observed functional changes. Most interesting was the fact that monolayer reassembly to the original columnar epithelial phenotype and reestablishment of barrier function occurred upon normalization of PKC levels. This model of reversible monolayer disassembly will allow investigation into the relationship between epithelial structure and function and examination of factors that govern monolayer formation.
Plasma membrane order parameter in periportal and perivenular hepatocytes isolated from ethanol-treated rats
Authors: Benedetti A. , Tangorra A. , Svegliati Baroni G. , Ferretti G. , Marucci L. , Jezequel M. , Orlandi F.
Published: December 2017

Hepatic ethanol (EtOH) metabolism has been assumed to involve hepatocytes differently, according to their location in the hepatic acinus. This study's aim was to gain information on plasma membrane (PM) order parameter in periportal (PP) and perivenular (PV) hepatocyte-enriched fractions isolated by a digitonin-collagenase perfusion technique from rats pair-fed for 6-8 wk liquid diets containing either EtOH or isocaloric carbohydrates. Fluorescence polarization (P) studies have been performed to measure PM order parameter of PP and PV hepatocytes cultured for 2-6 h on glass cover slips and labeled with 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH), a specific probe for PM of living cells. Fluorescence polarization and microscopy indicated that TMA-DPH is a suitable probe to study PM order parameter in subconfluent rat hepatocyte monolayers where it labeled, after a rapid incorporation, PM of cells. In pair-fed control rats, PM order parameter was lower in PP hepatocytes than in PV cells (P = 0.366 +/- 0.013 vs. 0.381 +/- 0.021, respectively, P < 0.02; n = 7). In EtOH-treated rats, these zonal differences tended to disappear (P = 0.419 +/- 0.012 in PP cells vs. 0.417 +/- 0.007 in PV cells; n = 7). In addition, the order parameter was significantly higher either in PP or PV hepatocytes compared with pair-fed control animals (P < 0.002 and 0.003, respectively). A 30-min culture of cells in the presence of 40-200 mM EtOH significantly decreased the PM order parameter of hepatocytes isolated from pair-fed control rats with respect to EtOH-treated animals both in PP and PV cells (P < 0.01 and 0.02, respectively; n = 4).(ABSTRACT TRUNCATED AT 250 WORDS)