bluuebunny/crossref_metadata_2025 huggingface dataset:
Cathepsin L mediates intracellular ileal digestion of gastric intrinsic factor
Authors: Gordon M. M. , Howard T. , Becich M. J. , Alpers D. H.
Published: December 2017

Although acidic proteases of lysosomal origin are implicated in the degradation of intrinsic factor (IF) during cobalamin (cbl) transport across enterocytes and proximal renal tubule cell lines, the enzyme(s) involved in this process is not known. Recombinant (baculovirus-produced) rat 125I-labeled IF (125I-rIF), 43 kDa, added in vivo to the lumen of rat ileum was converted intracellularly to peptides of 33 and 26 kDa. In vitro rat 125I-rIF was degraded to peptides of 33 and 31 kDa by addition of cathepsin L; this conversion was fully inhibited by leupeptin. Western blot analysis using antiserum against denatured native rat IF identified additional cathepsin L degradation products in the 17- to 23-kDa range. In vitro the binding of cobalamin partially inhibited cathepsin L degradation of IF. Rat rIF produced from either insect (Sf9) or mammalian (CHO) cells and native rat IF were all degraded by cathepsin L, although the prominence of the various products differed in the recombinant preparations, being 33 and 36 kDa, respectively. Native rat IF was most sensitive to proteolysis, and no degradation products were identified. Rat 125I-rIF was taken up by LLC-PK1 cells, and 125I from degraded IF appeared abundantly on the basolateral side of cell monolayers by 1 h. The intracellular products of rat rIF in LLC-PK1 cells were the same size as those produced in vitro by the action of cathepsin L. Antiserum against a human kidney cDNA cathepsin L fusion protein easily demonstrated the protease in rat intestinal mucosa, as well as in all other tissues tested. These data suggest that cathepsin L is the protease responsible for the leupeptin-sensitive intracellular degradation of IF.
Solubilization of receptors for pancreatic polypeptide from rat liver membranes
Authors: Nguyen T. D. , Wolfe M. S. , Heintz G. G.
Published: December 2017

We have previously identified, on rat liver microsomes and plasma membranes, proteins that bind pancreatic polypeptide (PP) with high affinity and specificity and that may serve as receptors for a hepatic effect of PP (J. Biol. Chem. 267: 9416-9421, 1992). Further characterization of these proteins requires the solubilization of receptors with conserved ability to bind PP selectively and efficiently. In this report, using 6 mM of the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), we solubilized, from liver microsomes, receptors that bound PP with high affinity (dissociation constant 6.15 +/- 1.6 nM) and specificity (no interaction with the homologous peptides neuropeptide Y and peptide YY). Gel filtration chromatography showed different degrees of receptor aggregation related to different concentrations of CHAPS in the eluent. To characterize the structure of these solubilized receptors, the chemical cross-linker N-(5-azido-2-nitrobenzoyloxy)succinimide was used to covalently bind these receptors to radiolabeled PP, and the resulting PP-receptor complexes were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A radioactive band with an apparent molecular weight (M(r)) of 46,000 was detected that was inhibited by unlabeled PP with a half-maximal inhibitory concentration of approximately 10(-8) M. It most likely reflected a PP receptor with an estimated M(r) of 42,000, excluding the molecular weight of PP. The migration of this complex was not affected by the reducing agent dithiothreitol, suggesting the absence of disulfide bonding. The solubilization and identification of a bioactive hepatic PP receptor will allow further characterization and purification of this receptor and will lead to the clarification of the interaction between PP and the digestive system.
Stability of circadian and minor cycles of exocrine pancreatic secretion in atropine- and MK-329-infused rats
Authors: Maouyo D. , Guan D. , Rivard N. , Adelson J. W. , Morisset J.
Published: December 2017

We previously demonstrated the existence of a circadian rhythm of pancreatic secretion of fluid and digestive enzymes, which was superimposed on by a regular 1.84-h minor cycle of exocrine pancreatic secretion of fluid and total protein, amylase, and chymotrypsinogen (25). Direct control of these daily and hourly rhythms of pancreatic secretory function has not been addressed. Cholinergic and cholecystokinin (CCK)-associated influences on these two rhythms of exocrine pancreatic secretion were investigated in rats provided with pancreatic, biliary, duodenal, and jugular vein cannulas, allowing separate drainage of bile and pure pancreatic juice, as well as intravenous infusions of atropine sulfate and/or MK-329. Rats were kept in restraint cages under controlled temperature and humidity, with a regular 12-h light cycle, and divided into five groups. The first group of fed rats was constantly infused with 200 micrograms.kg-1.h-1 atropine, the second with 0.5 mg.kg-1.h-1 MK-329, and the third with both. In the group where both drugs were simultaneously infused, 500 micrograms.kg-1.h-1 atropine was intraperitoneally administered, whereas MK-329 was infused by intravenous cannula. Two groups consisted of fasted rats, of which one was also given atropine (100 micrograms.kg-1.h-1). Three-day experiments were performed separately with fed rats, and 2-day experiments were performed with fasted rats; atropine and/or MK-329 infusion, starting on day 2, was constant over 48 h in both fed and fasted rats.(ABSTRACT TRUNCATED AT 250 WORDS)
Induction of liver growth in normal mice by infusion of hepatocyte growth factor/scatter factor
Authors: Roos F. , Ryan A. M. , Chamow S. M. , Bennett G. L. , Schwall R. H.
Published: December 2017

Hepatocyte growth factor/scatter factor (HGF/SF) is a potent stimulator of DNA synthesis in a variety of epithelial cells, including hepatocytes, and has been implicated in liver regeneration. We show here that combining dextran sulfate with HGF/SF markedly increases the plasma concentrations of HGF/SF that are achieved during intraperitoneal infusion. Three days of administration of HGF/SF by this mechanism caused a dose-dependent increase in liver wet weight. Mitotic figures were rarely observed in control livers but were abundant in livers exposed to HGF/SF, and liver DNA content was elevated. Serum levels of triglycerides, cholesterol, total protein, and albumin were also dose dependently increased, whereas alkaline phosphatase was reduced. From these data we conclude 1) that combining HGF/SF with dextran sulfate provides a novel method for delivering HGF/SF in a continuous manner, 2) that HGF/SF can induce liver growth in an intact animal, and 3) that HGF/SF-induced liver enlargement is associated with changes in serum biochemistry.
Influence of normal aging on oral-pharyngeal and upper esophageal sphincter function during swallowing
Authors: Shaw D. W. , Cook I. J. , Gabb M. , Holloway R. H. , Simula M. E. , Panagopoulos V. , Dent J.
Published: December 2017

The influence of aging on oral-pharyngeal swallowing was assessed by simultaneous manometry and videoradiography in 14 nondysphagic elderly individuals (mean age 76 yr) and 11 healthy, young controls (mean age 21 yr). Sphincter opening was diminished significantly in the elderly (P = 0.0001), but trans-sphincteric bolus flow rates were preserved. The increased impedance to trans-sphincteric bolus flow from reduced sphincter opening in the aged was reflected in a significant increase in hypopharyngeal intrabolus pressure (P = 0.003). Oral transit time was significantly prolonged in the aged (P = 0.01). The timing of upper esophageal sphincter (UES) manometric relaxation and of opening was significantly delayed in the aged (P = 0.0001), and this delay was comparable in magnitude to the prolongation in oral transit. Coordination of UES relaxation and opening with midpharyngeal contraction was not significantly affected by age. Deglutitive hyolaryngeal motion was not affected by age but was delayed by a duration equivalent to the prolongation in oral transit. We conclude that normal aging prolongs the oral-pharyngeal swallow that impairs UES opening but does not influence pharyngo-sphincteric coordination.
Intrapancreatic zymogen activation and levels of ATP and glutathione during caerulein pancreatitis in rats
Authors: Luthen R. , Niederau C. , Grendell J. H.
Published: December 2017

Studies in acutely inflamed pancreatic tissue in humans and animals suggest that premature activation of proteases within the gland plays a key role in its pathophysiology. The present study aimed to detect such protease activation in relation to protease inhibition and to changes in the concentrations of the vital cellular compounds ATP and glutathione in pancreatic tissue during caerulein-induced pancreatitis in rats. Within 1 h after supramaximal stimulation by intraperitoneal caerulein injection, pancreatic tissue activities of enzymatically active trypsin and elastase showed significant increases, accompanied by a twofold increase in trypsin inhibitory capacity. Over the same time course pancreatic ATP and glutathione concentrations dropped to 38% and 47%, respectively, after 1 h and reached a nadir of 22% and 28%, respectively, after 4-8 h. Intrapancreatic trypsin activation in this model, despite increasing trypsin inhibitory capacity, indicates concealed liberation of even more protease or enzyme-inhibitor complex instability. It is hypothesized that early acinar glutathione depletion, in part due to diminished ATP, could play a role in the premature activation of digestive enzymes by impairment of the integrity of the cytoskeleton and cell organelles or lowered defense capabilities against oxidant stress, finally leading to acute pancreatitis.
Neurons in the dorsal motor nucleus of the vagus may integrate vagal and spinal information from the GI tract
Authors: Renehan W. E. , Zhang X. , Beierwaltes W. H. , Fogel R.
Published: December 2017

Previous investigations have provided evidence that the activity of parasympathetic efferent neurons in the dorsal motor nucleus of the vagus (DMNV) may be influenced by either vagal afferent or spinal input from the gastrointestinal (GI) tract. Many questions remain, however, regarding the nature of this input and its integration by the brain stem. The present study was designed to examine one important aspect of this issue: the potential contribution of the spinal input to the brain stem in the generation of the response properties of intestine-sensitive neurons in the DMNV. Using intracellular recording and labeling techniques in adult rats, we found that ascending spinal pathways were capable of conveying both low- and high-threshold visceral information to the DMNV. We also determined that the neurons in the nucleus of the solitary tract failed to respond to intestinal distention when the vagal afferents to the brain stem had been severed, suggesting that the spinal projections terminate directly on the DMNV neurons. These data lend support to the emerging hypothesis that the spinal afferents that accompany the abdominal splanchnics are capable of responding to both innocuous and noxious stimuli. The results also suggest that the neurons in the DMNV play a larger role in the integration of visceral sensory information than was previously realized.
Oxidation of glucose carbon entering the TCA cycle is reduced by glutamine in small intestine epithelial cells
Authors: Kight C. E. , Fleming S. E.
Published: December 2017

The influence of glutamine on glucose oxidation was assessed in epithelial cells isolated from the mucosa of the proximal, mid-, and distal small intestine of young, fed, male rats. Glucose oxidation declined along the length of the small intestine, with values from the mid- and distal segments representing approximately 55% and 40%, respectively, of the value from the proximal segment. A gradient along the small intestine was noted also in the influence of glutamine on glucose oxidation: glutamine suppressed glucose oxidation approximately 60% in the proximal small intestine, 39% in the mid-intestine, and 31% in the distal small intestine. Glutamine suppressed the oxidation of glucose carbon that entered the tricarboxylic acid (TCA) cycle; this was determined using CO2 ratios derived from acetate and glucose isotopes. In cells from the proximal segment, the probability that carbon entering the cycle would complete one full turn was reduced by glutamine from 0.77 to 0.28. The entry of glucose-derived pyruvate into the TCA cycle did not appear to be influenced by the presence of glutamine, however. Glutamine had no influence on the proportion of glucose metabolism that occurred via the pentose phosphate pathway (which averaged 5% or less), but reduced flux of carbon through pyruvate carboxylase relative to flux through pyruvate dehydrogenase from 40% to 9% in cells from the proximal segment. These data suggest that, in the presence of glutamine, the fate of pyruvate carbon (derived from glucose or elsewhere) entering the TCA cycle is altered from that of oxidation to anaplerosis and subsequent efflux of TCA cycle intermediates into newly synthesized compounds.
Role of nitric oxide in regulation of vascular resistance in postnatal intestine
Authors: Nankervis C. A. , Nowicki P. T.
Published: December 2017

Studies were conducted to determine whether endothelial production of nitric oxide (NO) participates in the regulation of vascular resistance in postnatal swine intestine. In vivo, intra-arterial infusion of the arginine analogue NG-monomethyl-L-arginine (L-NMMA, 10(-4) M) increased intestinal vascular resistance 34% in 3-day-old animals and 9% in 35-day-old animals (P < 0.01); similar findings were noted during infusion of 10(-3) M L-NMMA. Mechanical augmentation of gut flow rate induced intestinal vasodilation in both age groups; L-NMMA eliminated this flow-induced dilation in intestine of 3- but not 35-day-old animals. In vitro, precontracted mesenteric artery rings from both age groups relaxed to a similar extent in response to the endothelium-independent nitrovasodilator sodium nitroprusside (SNP) and the calcium ionophore A-23187; the effect of A-23187, but not SNP, was eliminated by mechanical disruption of the endothelium. Acetylcholine (ACh) and substance P (SP), agents with vascular effects that are secondary to receptor-mediated activation of NO, caused greater relaxation of rings from younger than from older animals, and this effect was attenuated by L-NMMA or methylene blue. Unstimulated accumulation of guanosine 3',5'-cyclic monophosphate (cGMP) occurred to a similar extent in vessel segments from both groups. ACh and SP increased cGMP accumulation in segments from 3- but not from 35-day-old animals. We conclude that the NO-cGMP axis participates to a greater extent in regulation of intestinal vascular resistance in 3- than in 35-day-old swine.
Liver ischemia-reperfusion increases pulmonary permeability in rat: role of circulating xanthine oxidase
Authors: Weinbroum A. , Nielsen V. G. , Tan S. , Gelman S. , Matalon S. , Skinner K. A. , Bradley E. , Parks D. A.
Published: December 2017

Reactive oxygen species play an important role in pathogenesis of a variety of pathological processes, e.g., ischemia-reperfusion, acute viral infections, thermal injury, hepatic diseases, and acute lung injury. Xanthine oxidase (XO) may be a significant source of these cytotoxic oxygen species. We tested the hypothesis that hepatic ischemia-reperfusion releases xanthine dehydrogenase + XO (XDH + XO) into the circulation and that circulating XO damages isolated perfused lung. Isolated liver + lung preparation was perfused with Krebs-Henseleit buffer to minimize confounding effects of circulating neutrophils. In one group, livers were rendered globally ischemic for 2 h and then reperfused (I/R). In another group, livers were pretreated with allopurinol and perfused with buffer containing additional allopurinol (I/R + Allo). After 2 h of ischemia, an isolated lung was connected to liver, and liver + lung preparation was reperfused in series for 15 min. Liver reperfusion was terminated, and lung was recirculated with liver effluent for 45 min. Capillary filtration coefficient (ml.min-1.cmH2O-1.100 g lung dry wt-1) was 2.0 +/- 0.3 and 1.9 +/- 0.4 in control and I/R + Allo lungs, respectively, and 9.0 +/- 1.2 in I/R lungs (P < 0.001). Lung wet-to-dry weight ratio in control and I/R + Allo lungs was 8.6 +/- 0.3 and 9.1 +/- 0.5, respectively, and 14.9 +/- 1.1 in I/R lungs (P < 0.01). Control and I/R + Allo bronchoalveolar lavage protein content was < 1.0 mg/ml compared with 32.6 +/- 8.4 mg/ml in I/R group.(ABSTRACT TRUNCATED AT 250 WORDS)
Descending peristaltic reflex in the opossum esophagus
Authors: Paterson W. G. , Indrakrishnan B.
Published: December 2017

To test the hypothesis that the distension-induced esophageal peristaltic reflex involves a polysynaptic pathway, a triple-chamber organ bath was used to chemically isolate different regions of the opossum smooth muscle esophagus while leaving in continuity the intramural neuromuscular apparatus. Balloon distension in the oral chamber evoked membrane hyperpolarization in the aboral chamber, which was followed, on balloon deflation, by depolarization, spike burst, and circular smooth muscle contraction. This reflex was abolished by adding tetrodotoxin to any of the chambers. Addition of Ca(2+)-free 20 mM Mg2+ Krebs solution (to block synaptic transmission) to the intermediate chamber did not affect the descending peristaltic reflex. However, Ca(2+)-free 20 mM Mg2+ Krebs solution markedly attenuated the reflex when placed in either the oral or the aboral chamber. Furthermore, the nitric oxide synthase inhibitor N omega-nitro-L-arginine methyl ester, when placed in the aboral chamber, abolished the reflex. Other putative neurotransmitter antagonists were without effect when placed in any chamber. These studies suggest that the intramural neural pathway that mediates the descending peristaltic reflex in the opossum esophagus is not polysynaptic, but rather involves long descending neurons that depend on nitric oxide as a final mediator.
Transmitter interactions in rabbit internal anal sphincter
Authors: Knudsen M. A. , Glavind E. B. , Tottrup A.
Published: December 2017

The aim of the present study was to investigate the relative importance of the different putative nonadrenergic noncholinergic (NANC) mediators and their interplay with cholinergic nerves in the rabbit internal anal sphincter (IAS). IAS preparations were mounted in organ baths for recording of isometric tension. Transmural field stimulation (TMS; 5-s trains; supramaximal voltage, 140-160 V; 0.4-ms impulse duration) was applied every 2 min with frequencies varying from 0.2 to 32 Hz. TMS induced frequency-dependent relaxations that amounted to 89.3 +/- 2.2% (n = 7). N omega-nitro-L-arginine (L-NNA; 10(-7)-10(-4) M; 8 Hz) reduced relaxations and this effect was partially inhibited by preincubation with L-arginine (10(-4) M). The effect of L-NNA was attenuated by atropine preincubation. Apamin (10(-6) M) shifted the frequency-response curve to the right but left maximal relaxations in response to TMS unaffected. In the presence of L-NNA (10(-4) M) and atropine (10(-6) M), the action (area between the frequency-response curve with or without a substance) of apamin was more pronounced, but, despite the presence of both L-NNA and apamin, some relaxation still remained. The frequency-response curve (control) was significantly shifted to the right by carbachol (10(-6) M). Concentration-response experiments showed that the response to exogenous nitric oxide (NO; 10(-7)-10(-4) M) was unaffected by carbachol (10(-6) M) preincubation, whereas responses to vasoactive intestinal polypeptide (VIP) and ATP were significantly reduced.(ABSTRACT TRUNCATED AT 250 WORDS)
Vitamin A-poor lipocytes: a novel desmin-negative lipocyte subpopulation, which can be activated to myofibroblasts
Authors: Ramm G. A. , Britton R. S. , O'Neill R. , Blaner W. S. , Bacon B. R.
Published: December 2017

Lipocytes have been classified as vitamin A-storing, desmin-positive cells. In hepatic fibrogenesis, lipocytes transform into myofibroblasts, which express alpha-smooth muscle actin (alpha-SMA) and produce increased amounts of collagen. We isolated a population of vitamin A-poor lipocytes (VAPL) from normal rat liver and examined the morphological and biochemical differences between VAPL and vitamin A-replete lipocytes (VARL). Desmin and alpha-SMA expression were determined by Western blot in quiescent cells and in cells activated by culture on uncoated plastic. Both cell types were alpha-SMA-negative; however, in contrast to VARL, freshly isolated VAPL did not contain desmin. Desmin expression was induced in VAPL on activation. With time in culture, both VAPL and VARL expressed alpha-SMA and produced collagen, indicative of transformation to myofibroblasts. Ferritin receptor expression was demonstrated in cultured VARL after 1 day and in VAPL after 5 days, indicating that this is an early marker of lipocyte activation. After 7 days, VARL and VAPL were indistinguishable in terms of desmin, ferritin receptor expression, and collagen production. This study demonstrates the first isolation and characterization of two distinct quiescent subpopulations of lipocytes from normal rat liver: desmin-negative VAPL and desmin-positive VARL. Both populations of cells can be activated to myofibroblasts, the phenotype associated with hepatic fibrogenesis.
Effect of 17-norleucine-VIP on gastroduodenal motility relative to serum VIP concentration and blockade of NOS
Authors: Holle G. E. , Steinbach E. , Wunsch E. , Holst J. J.
Published: December 2017

Mechanical and electrical activity in the antrum, pylorus, and duodenum was evaluated in the conscious dog, instrumented with seven strain gauges and five platinum electrodes. 17-Norleucine-vasoactive intestinal peptide (17-N-Leu-VIP) or 17-N-Leu-VIP plus NG-nitro-L-arginine methyl ester (L-NAME) was injected intra-arterially close to the pylorus to identify influences of nitric oxide (NO) on effects of VIP. VIP concentration was measured by radioimmunoassay in serum samples collected from the cubital and portal veins before and up to 2 h after VIP injection. VIP (0.004-0.006 mg.kg-1.10 min-1) abolished phasic contractions in the interdigestive state for 16.8 min and in the digestive state for 14.4 min, whereas whole serum VIP concentration rose above 42.4 +/- 13 pmol/l. Administration of L-NAME did not significantly influence the effects of VIP. Aftereffects of VIP, consisting of a reduced motility index, lasted 33 +/- 10.6 min in the interdigestive state and 44.5 +/- 42 min in the digestive state. This VIP aftereffect in the interdigestive state was shortened in time by the addition of L-NAME. The results overall suggest that NO release is a factor only in the aftereffects of VIP.
Halothane and octanol block Ca2+ oscillations in pancreatic acini by multiple mechanisms
Authors: Deutsch D. E. , Williams J. A. , Yule D. I.
Published: December 2017

This study has investigated halothane and octanol, reported inhibitors of gap junction permeability, for their effects on acinar cell intracellular Ca2+ concentration ([Ca2+]i) signaling. Halothane and octanol alone at maximal concentrations induced a sustained rise in [Ca2+]i of 23 +/- 4 and 29 +/- 5 nM, respectively. Cholecystokinin (CCK, 20 pM) induced [Ca2+]i oscillations in single acinar cells within the acinus to a peak of 275 +/- 17 nM, rising from a basal level of 55 +/- 3 nM. These oscillations were completely abolished by superfusion with both halothane (4 mM) and octanol (1 mM), concentrations that blocked the spread of Lucifer yellow from cell to cell within an acinus. Lower concentrations of octanol markedly reduced the oscillation frequency (0.2 and 0.5 mM octanol: reduction in oscillation frequency of 69 +/- 6 and 43 +/- 6%, respectively). These agents however, over the same concentration range, also exhibited similar inhibitory effects on [Ca2+]i oscillations in single cells dispersed from the acinus (reduction in oscillation frequency of 75 +/- 10 and 32 +/- 12% for 0.2 and 0.5 mM octanol, respectively), suggesting additional effects other than on gap junctions. Halothane inhibited inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] production in response to both 1 and 10 nM CCK (31 and 40% inhibition, respectively), possibly explaining its effects on [Ca2+]i oscillations, whereas octanol showed no significant inhibition. Octanol, unlike halothane, blocked Ins(1,4,5)P3-induced Ca2+ release from permeabilized acini, an effect that was most pronounced at a more physiological Ins(1,4,5)P3 concentration. Octanol did not affect Ins(1,4,5)P3 binding to Ins(1,4,5)P3 receptor preparation. In conclusion, although halothane and octanol block gap junction permeability in pancreatic acinar cells, these agents also affect Ins(1,4,5)P3 production and Ca2+ mobilization in response to agonist stimulation.
Molecular mechanisms of hepatic bile salt transport from sinusoidal blood into bile
Authors: Meier P. J.
Published: December 2017

An increasingly complex picture has emerged in recent years regarding the bile salt transport polarity of hepatocytes. At the sinusoidal (or basolateral) plasma membrane two bile salt-transporting polypeptides have been cloned. The Na(+)-taurocholate-cotransporting polypeptide (Ntcp) can account for most, if not all, physiological properties of the Na(+)-dependent bile salt uptake function in mammalian hepatocytes. The cloned organic anion-transporting protein (Oatp1) can mediate Na(+)-independent transport of bile salts, sulfobromophthalein, estrogen conjugates, and a variety of other amphipathic cholephilic compounds. Hence, Oatp1 appears to correspond to the previously suggested basolateral multispecific bile sale transporter. Intracellular bile salt transport can be mediated by different pathways. Under basal bile salt flux conditions, conjugated trihydroxy bile salts bind to cytoplasmic binding proteins and reach the canalicular plasma membrane predominantly via cytoplasmic diffusion. More hydrophobic mono- and dihydroxy and high concentrations of trihydroxy bile salts associate with intracellular membrane-bound compartments, including transcytotic vesicles, endoplasmic reticulum (ER), and Golgi complex. A facilitated bile salt diffusion pathway has been demonstrated in the ER. The exact role of these and other (e.g., lysosomes, "tubulovesicular structures") organelles in overall vectorial transport of bile salts across hepatocytes is not yet known. Canalicular bile salt secretion is mediated by two ATP-dependent transport systems, one for monovalent bile salts and the second for divalent sulfated or glucuronidated bile salt conjugates. The latter is identical with the canalicular multispecific organic anion transporter, which also transports other divalent organic anions, such as glutathione S-conjugates. Potential dependent canalicular bile salt secretion has also been suggested to occur, but its exact mechanism and physiological significance remain unclear, since a potential driven bile salt uptake system has also been identified in the ER. Hypothetically, and similar to changes in cell volume, the intracellular potential could also play a role in the regulation of the number of bile salt carriers at the canalicular membrane and thereby indirectly influence the maximal canalicular bile salt transport capacity of hepatocytes.
Absorption of epidermal growth factor occurs through the gastrointestinal tract and oral cavity in adult rats
Authors: Purushotham K. R. , Offenmuller K. , Bui A. T. , Zelles T. , Blazsek J. , Schultz G. S. , Humphreys-Beher M. G.
Published: December 2017

Introduction of radiolabeled epidermal growth factor (125I-EGF) by gavage or sublingual confinement resulted in a time-dependent uptake and systemic organ dissemination in the adult rat. Intact EGF was recovered primarily from the tongue, parotid, and sublingual/submandibular glands after administration by sublingual lozenge, whereas gastrointestinal administration resulted in 125I-EGF recovery primarily from plasma, stomach, and lung. Recovered radiolabeled EGF retained the ability to bind to the EGF receptor. Sialoadenectomy caused an increase in 125I-EGF in most tissues by both routes of administration. Thus, in the adult rat, at least two pathways exist for the uptake and distribution for salivary gland-derived EGF present in saliva. With further analyses, sublingual absorbance of EGF may therefore provide a potential delivery route for therapeutic use of growth factor, which avoids the hepatic destruction of EGF after oral administration.
In vivo trafficking of nascent H(+)-K(+)-ATPase in rabbit parietal cells
Authors: Crothers J. M. , Chow D. C. , Scalley M. L. , Forte J. G.
Published: December 2017

Protein metabolic labeling in vivo was used to determine a time course for trafficking of nascent H(+)-K(+)-adenosinetriphosphatase (H(+)-K(+)-ATPase) from endoplasmic reticulum (ER) to mature tubulovesicles in parietal cells. Stomachs of cimetidine-treated rabbits were taken 15-90 min after injection of [35S]methionine/cysteine, and mucosal microsomes were fractionated on sucrose gradients for analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot, and autoradiography. After 15 min, labeled alpha-subunit peaked at approximately 1.14 g/ml, matching the distribution of the high-mannose beta-subunit precursor, "pre-beta." After 30 min, most labeled alpha-subunit was in a peak at approximately 1.10 g/ml, considered to be Golgi. By 90 min, most labeled alpha-subunit was in a light peak, at approximately 1.07 g/ml, aligned with the major peak of total H(+)-K(+)-ATPase previously characterized as mature tubulovesicles. From material enriched in pre-beta, alpha-subunit was coprecipitated with pre-beta by a terminal mannose-specific lectin, Galanthus nivalis agglutinin, in the same ratio as the mature alpha:beta ratio. Thus alpha- and beta-subunits associated early in the ER. This is the first use of protein metabolic labeling to study early trafficking of the H(+)-K(+)-ATPase in vivo. The techniques may be usefully applied to examining changes in H(+)-K(+)-ATPase synthetic rate in response to various pharmacological treatments and studying the divergent pathways for nascent H(+)-K(+)- and Na(+)-K(+)-ATPases.
Antioxidant enzymes in intramural nerves of the opossum esophagus
Authors: Thomas R. M. , Fang S. , Leichus L. S. , Oberley L. W. , Christensen J. , Murray J. A. , Ledlow A. , Conklin J. L.
Published: December 2017

Superoxide radical (O2-.) combines with nitric oxide (NO) to form peroxynitrite, thereby nullifying the biological activity of NO. Superoxide dismutase (SOD) prevents this reaction by converting O2-. to H2O2. We tested the hypotheses that the antioxidant enzymes catalase (CAT), Mn SOD, and Cu/Zn SOD are present in enteric neurons of the opossum esophagus, and that O2-. alters esophageal motor function. Immunostaining demonstrated CAT, Mn SOD, and Cu/Zn SOD immunoreactivity in interganglionic nerve bundles and ganglia of the myenteric and submucosal plexuses. Western blot analysis confirmed the presence of these enzymes in homogenates of esophageal muscularis propria, and enzyme assays demonstrated Cu/Zn SOD and Mn SOD activities of 262 and 73 U/mg protein, respectively. Both diethyldithiocarbamic acid, an inhibitor of Cu/Zn SOD, and xanthine (X) with xanthine oxidase (XO), which generate O2-., shortened the latency of the nerve-mediated contraction of circular esophageal muscle, the off response, by 20.2 and 23.4%, respectively. SOD alone did not affect the latency, but it inhibited the effect of X with XO on the latency. Antioxidant enzymes found in intramural esophageal nerves may play a role in regulating NO-mediated neuromuscular communication in the esophagus.
Insulin inhibits secretin-stimulated pancreatic bicarbonate output by a dose-dependent neurally mediated mechanism
Authors: Berry S. M. , Fink A. S.
Published: December 2017

Although previous reports suggest interactions between endocrine and exocrine pancreas, insulin's effect on pancreatic exocrine function remains unclear. Chronic pancreatic fistulas were created in six dogs with innervated INN and five dogs with denervated (DEN) pancreata; these animals were studied using the euglycemic, hyperinsulinemic clamp technique. After a 30-min unstimulated period, both groups received a 60-min 1.5 mU.kg-1.min-1 insulin (clamp) or vehicle (control) infusion. Intravenous secretin was then initiated at 16 ng.kg-1.h-1; the secretin dose was doubled every 30 min until 125 ng.kg-1.h-1 was achieved. These studies were then repeated in INN animals during infusion of a 10 micrograms.kg-1.h-1 atropine background. Finally, INN animals underwent a similar unstimulated period followed by a 1.25 mU.kg-1.min-1 insulin (clamp) or vehicle (control) infusion. after 60 min, a 64 ng.kg-1.h-1 secretin infusion was initiated. Insulin infusion was then increased by 0.25 mU.kg-1.min-1 at 30-min intervals until 2.0 mU.kg-1.min-1 was reached. Unstimulated (0-30 min) serum glucose and insulin levels and pancreatic bicarbonate and protein outputs did not differ between groups. Clamp (30-90 min) and stimulated (90-210 min) insulin were each significantly elevate in clamp groups (81.9 +/- 2.4 vs. 7.0 +/- 0.3 microU/ml, P < 0.001); glucose and bicarbonate were unchanged. Protein outputs during clamp (64 +/- 9 vs. 24 +/- 6 mg/10 min; P < 0.05) and secretin-stimulated (52 +/- 9 vs. 29 +/- 3 mg/10 min; P < 0.05) periods were diminished by atropine but were unaffected by insulin. Secretin-stimulated (90-210 min) bicarbonate output was diminished by insulin (0.03 +/- 0.01 vs. 0.31 +/- 0.05 meq/10 min; P < 0.003) in INN but not DEN animals; this effect was partially reversed by atropine. Dose-response studies demonstrated a threshold for insulin's inhibitory actions between 1.5 and 1.75 mU.kg-1.min-1. These data provide further evidence for exocrine-endocrine regulatory interactions and suggest that insulin may influence secretin's stimulation of ductal cell secretion by a mechanism that is at least partially cholinergic in character.
Role of endogenous 5-hydroxytryptamine in the regulation of gastric contractions by motilin in dogs
Authors: Haga N. , Mizumoto A. , Satoh M. , Mochiki E. , Mizusawa F. , Ohshima K. , Itoh Z.
Published: December 2017

It has been suggested that 5-hydroxytryptamine3 (5-HT3) receptors are involved in the control of phase III contractions in the stomach. We examined the effect of depletion of endogenous 5-HT by p-chlorophenylalanine (pCPA) on spontaneously and motilin-induced phase III contractions in conscious dogs, and the effect of 5,6-dihydroxytryptamine (5,6-DHT) in an isolated perfused dog stomach. Three-day treatment with pCPA significantly reduced plasma 5-HT concentration and 5-HT content in the stomach, and strongly suppressed the spontaneous and motilin-induced phase III contractions in the stomach. When spontaneous phase III contractions recovered in the stomach after a 3-day treatment, exogenous motilin induced typical phase III-like contractions, and the 5-HT content in the muscle layer was recovered to the normal pretreatment level. In the perfused stomach, 5,6-DHT decreased 5-HT content in the muscle layer alone and abolished motilin-induced contractions. In conclusion, endogenous 5-HT, probably in 5-HT neurons, plays an important role in the control of interdigestive phase III activity by motilin in the stomach.
Tumor necrosis factor mediation of NSAID-induced gastric damage: role of leukocyte adherence
Authors: Appleyard C. B. , McCafferty D. M. , Tigley A. W. , Swain M. G. , Wallace J. L.
Published: December 2017

Neutrophil adherence to the vascular endothelium has been suggested to be a critical event in the pathogenesis of nonsteroidal anti-inflammatory drug (NSAID)-induced gastric damage. Recently, increased plasma levels of tumor necrosis factor-alpha (TNF-alpha), which can increase leukocyte adherence, have been reported after administration of indomethacin. This study was performed to determine the relationship between plasma TNF-alpha levels, leukocyte adherence, and NSAID-induced gastric injury. Administration of indomethacin to rats resulted in a significant elevation of plasma TNF-alpha levels within 30 min and the development of gastric erosions. Pretreatment with dexamethasone and prostaglandin E2 almost completely prevented gastric injury and abolished the rise in plasma TNF-alpha. Pentoxifylline dose dependently reduced both gastric damage and plasma TNF-alpha. Similar effects were observed with three other TNF-alpha synthesis inhibitors and with an anti-TNF-alpha antisera. Pentoxifylline also significantly reduced the extent of antral ulceration induced by naproxen. However, pentoxifylline did not significantly affect indomethacin-induced leukocyte adherence. These results suggest that TNF-alpha plays a critical role in the pathogenesis of NSAID-induced gastric injury, but this cytokine may not be responsible for NSAID-induced leukocyte adherence.
Action of sensory neurons in an experimental at colitis model of injury and repair
Authors: Reinshagen M. , Patel A. , Sottili M. , French S. , Sternini C. , Eysselein V. E.
Published: December 2017

The influence of sensory nerves on inflammation and healing was studied in a rat colitis model at different stages of inflammation. Studies were performed in animals with and without ablation of sensory nerves, which was achieved by pretreatment with the neurotoxin capsaicin. Colitis was induced by a rectal enema containing trinitrobenzenesulfonic acid (50 mg/kg) in 50% ethanol. Severity of inflammation was markedly increased 3 and 7 days after induction of colitis in the capsaicin-pretreated group compared with the vehicle group as determined by a macroscopic damage score (at 3 days, 12.0 +/- 0.7 vs. 7.5 +/- 1.5; at 7 days, 12.2 +/- 0.8 vs. 6.5 +/- 0.8; P < 0.05), by histology (ulceration score at 3 days, 82 +/- 12 vs. 40 +/- 11%; at 7 days, 92 +/- 5 vs. 46 +/- 13%; P < 0.05), and by myeloperoxidase activity (at 3 days, 133 +/- 30 vs. 42 +/- 14 U/mg protein; at 7 days, 76 +/- 11 vs. 39 +/- 11 U/mg protein; P < 0.05). There was no significant difference in the severity of colitis 14 and 21 days after induction of colitis between the capsaicin-pretreated group and the vehicle group. These data suggest that, in this model, sensory nerves have an important protective function in the acute and subacute phases of inflammation but do not seem to play a significant role in the later stages of chronic inflammation.
A critical role for nitric oxide in intestinal barrier function and dysfunction
Authors: Alican I. , Kubes P.
Published: December 2017

There is growing evidence that endogenous nitric oxide (NO) regulates mucosal barrier integrity under physiological conditions and counters the increase in mucosal permeability associated with acute pathophysiological states. The potential mechanisms of action for the protective effects of NO are discussed. These include maintenance of blood flow, inhibition of platelet and leukocyte adhesion and/or aggregation within the vasculature, modulation of mast cell reactivity, and scavenging of reactive oxygen metabolites such as superoxide. On the basis of the data presented, we conclude that both constitutive nitric oxide synthase (cNOS)-derived endogenous NO and exogenous NO (from NO donors) appear to reduce the sequelae of acute inflammation. The second section of this review summarizes the data germane to prolonged (chronic) inflammatory conditions associated with the overproduction of NO from the inducible form of NOS (iNOS). Some emphasis is placed on the role of NO in sepsis and inflammatory bowel disease (IBD), and data to suggest that NO, or more specifically a NO-derived mediator, is involved in these disorders are summarized. These studies are compared with recent publications suggesting that inhibition of NO synthesis with nonspecific inhibitors of NOS or selective iNOS inhibitors may not protect in models of sepsis or IBD. Overall, the review highlights the potential importance of the type of NOS enzyme involved in the particular inflammatory process being studied.
ATP-induced rise in apamin-sensitive Ca(2+)-dependent K+ conductance in adult rat hepatocytes
Authors: Yamashita Y. , Ogawa H. , Akaike N.
Published: December 2017

Exogenous ATP-induced transient outward currents (IATP) were investigated in isolated adult rat hepatocytes using conventional whole cell patch and nystatin perforated patch recording modes. The IATP increased in a sigmoidal fashion with an increase in ATP concentration, where the half-maximal concentration was 1.4 microM. The order of current potency was 2-methylthio-ATP > or = UTP = ATP > > alpha, beta-methylene-ATP. IATP was depressed in a concentration-dependent manner by suramin and apamin. IATP reversed its direction at the K+ equilibrium potential. IATP occurred easily in hepatocytes obtained from female rats weighing > 250 g. Removal of extracellular Ca2+ had no effect on the peak amplitude of IATP, but thapsigargin abolished it. Intracellular perfusion with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, heparin, guanosine 5'-O-(3-thiotriphosphate), or neomycin also abolished IATP. Pretreatment with pertussis toxin or calmodulin antagonists had no effect on IATP. It was concluded that ATP binding to both P2Y and P2U purinoceptors coupled to G protein may raise apaminsensitive Ca(2+)-dependent K+ conductance via a phospholipase C-inositol trisphosphate-Ca2+ signaling pathway.
Hep G2 cells: a model for studies on regulation of human cholesterol 7alpha-hydroxylase at the molecular level
Authors: Pandak W. M. , Stravitz R. T. , Lucas V. , Heuman D. M. , Chiang J. Y.
Published: December 2017

The present study examines the feedback control governing human cholesterol 7alpha-hydroxylase mRNA expression in the human hepatoblastoma cell line, Hep G2. Glycochenodeoxycholate (GCDC) and glycodeoxycholate, hydrophobic bile salts, decreased cholesterol 7alpha-hydroxylase mRNA levels and bile acid synthesis in a concentration-dependent (76 +/- 8%, P<0.001, and 48 +/- 3%, P<0.01, respectively) and time-dependent manner. Cholesterol 7alpha-hydroxylase mRNA levels were repressed with a half-maximal inhibitory concentration of <12.5 microM by GCDC and a half-life of 30 min by 100 microM of the bile acid. The addition of actinomycin D (10 microgram/ml) alone or in combination with GCDC (100 microM) led to similar concentration-and time-dependent suppression of cholesterol 7alpha-hydroxylase mRNA. Glycocholate (100 microM), not internalized based on lack of uptake of a fluorescent cholate analogue, had no effect on cholesterol 7alpha-hydroxylase mRNA or total bile acid synthesis. In cultures transfected with a rat cholesterol 7alpha-hydroxylase promoter construct, reporter gene activity was decreased (31%, P<0.01) by GCDC (100 microM). Hep G2 cells maintain the intracellular machinery to express and rapidly regulate human cholesterol 7alpha-hydroxylase by hydrophobic bile acids. These data suggest that Hep G2 cells will support functional studies of the human cholesterol 7alpha-hydroxylase gene.
Anaphylactic intestinal response to milk proteins during malnutrition in guinea pigs
Authors: Darmon N. , Heyman M. , Candalh C. , Blaton M. A. , Desjeux J. F.
Published: December 2017

We investigated whether sensitization to cow's milk occurs during malnutrition and alters intestinal ion and macromolecular transport. Malnourished guinea pigs received a low-protein diet containing either 4% soy or 4% milk proteins, and well-nourished sensitized controls received 26% soy plus 4% milk proteins. To assess milk sensitization, we measured immunoglobulin (Ig) G and passive cutaneous anaphylactic (PCA) responses to beta-lactoglobulin (beta-Lg) and the intestinal anaphylaxis, reflected by the rise in short-circuit current (delta Isc) induced by beta-Lg in tissues mounted in Ussing chambers. To assess intestinal function, we measured ionic conductance and unidirectional fluxes of -14C-mannitol and -3H-horseradish peroxidase (HRP). In malnourished animals fed milk proteins, IgG, PCA, and delta Isc (beta-Lg) increased more than in well-nourished animals. Ionic conductance and mannitol permeability rose in both malnourished groups. Malnourished animals fed milk proteins also displayed enhanced permeability to HRP. These data suggest that increased paracellular permeability is due to malnutrition per se, whereas increased macromolecular transport seems to require both malnutrition and sensitization. They indicate that intestinal anaphylaxis in response to milk proteins is persistent and even enhanced during experimental malnutrition.
Rat intestinal and hepatic ferritin subunit expression during development and after dietary iron feeding
Authors: Yeh K. Y. , Alvarez-Hernandez X. , Glass J. , Yeh M.
Published: December 2017

Ferritin consists of 24 heavy (H) and light (L) subunits in varying proportions in different tissues and plays a significant role in iron metabolism. We studied rat ferritin subunit expression in the duodenum and liver during early life, when a cycle of iron depletion and repletion occurs. In both tissues, ferritin contents decreased to low levels from day 3 to day 12. The ferritin on day 3 had an H/L mRNA ration of 0.9 and an H/L subunit ratio of 0.6. The decrease of tissue ferritin levels, but not mRNA, on day 12 suggests translational repression consistent with iron depletion. In the duodenum, a twofold increase in both H and mRNA and subunit protein occurred on day 18. The subsequent increase of H mRNA was accompanied by a 50% decrease in L mRNA, resulting in the increase of H/L mRNA and subunit ratios to 7.9 and 9, respectively, by day 32. In contrast, liver H/L mRNA and subunit ratios were similar throughout development. The possibility that dietary iron regulates duodenal ferritin subunit expression was investigated. When day 12 rats were fed 6 ml of a milk formula containing 56 microgram/ml iron for 18 h, dietary iron increased the duodenal levels of L mRNA but not H mRNA. In contrast, hepatic H and L mRNA levels did not change. Dietary iron promoted greater increases in ferritin protein than mRNA in both tissues. Thus a shift from L-rich to H-rich ferritin isoforms occurs in the duodenum but not in the liver during neonatal development. This change is regulated at the pretranslational level and is independent of dietary iron.
Transport of an influenza virus vaccine formulation (iscom) in Caco-2 cells
Authors: Lazorova L. , Artursson P. , Engstrom A. , Sjolander A.
Published: December 2017

The influenza virus envelope glycoproteins hemagglutinin and neuraminidase were administered to the apical or basolateral sides of Caco-2 monolayers either as native protein micelles (mic-ag) or after incorporation into the orally active adjuvant formulation, immune stimulating complexes (iscoms) (isc-ag). Biotin-conjugated isc-ag were localized in intracellular vesicles as early as 2 min after administration to the apical side at 37 degrees C. Ten minutes after administration, both intracellular vesicles and intercellular spaces were labeled, and extracellular labeling was observed on the basolateral side of the cells, indicating that isc-ag were transported across the epithelium within 10 min of exposure. Transport of 125I-labeled isc-ag and mic-ag in the apical-to-basolateral and basolateral-to-apical directions across Caco-2 monolayers was comparable at 37 degrees C. Gel chromatography analysis revealed that only 0.55-3.1% of transported isc-ag and mic-ag had a molecular weight of > 5,000, while 21.0-42.3% was eluted at a position corresponding to peptides of approximately 10 amino acids. Although isc-ag and mic-ag were transported and degraded by Caco-2 monolayers in comparable amounts, only transported isc-ag induced a dose-dependent proliferative response in vitro of T cells primed with influenza virus antigen. High-performance gel chromatography and reverse-phase high-performance liquid chromatography indicated that transported antigenic isc-ag consisted of hydrophobic peptides with a molecular weight of < or = 3,000. These results indicate that antigens incorporated into the orally active adjuvant formulation iscom are degraded to antigenic peptides during transport across the intestinal epithelium.