bluuebunny/crossref_metadata_2025 huggingface dataset:
Dietary protein paradox: decrease of amino acid availability induced by high-protein diets
Authors: Moundras C. , Remesy C. , Demigne C.
Published: December 2017

The aim of the present study was to evaluate the effect of changes in dietary protein level on overall availability of amino acids for tissues. For this purpose, rats were adapted to diets containing various concentrations of casein (7.5, 15, 30, and 60%) and were sampled either during the postprandial or postabsorptive period. In rats fed the protein-deficient diet, glucogenic amino acids (except threonine) tended to accumulate in plasma, liver, and muscles. In rats fed high-protein diets, the hepatic balance of glucogenic amino acids was markedly enhanced and their liver concentrations were consistently depressed. This response was the result of a marked induction of amino acid catabolism (a 45-fold increase of liver threonine-serine dehydratase activity was observed with the 60% casein diet). The muscle concentrations of threonine, serine, and glycine underwent changes parallel to plasma and liver concentrations, and a significant reduction of glutamine was observed. During the postabsorptive period, adaptation to high-protein diets resulted in a sustained catabolism of most glucogenic amino acids, which accentuated the drop in their concentrations (especially threonine) in all the compartments studied. The time course of metabolic adaptation from a 60 to a 15% casein diet has also been investigated. Adaptation of alanine and glutamine metabolism was rapid, whereas that of threonine, serine, and glycine was delayed and required 7-11 days. This was paralleled by a relatively slow decay of liver threonine-serine dehydratase (T-SDH) activity in contrast to the rapid adaptation of pyruvate kinase activity after refeeding a high-carbohydrate diet.(ABSTRACT TRUNCATED AT 250 WORDS)
Voltage-dependent calcium channel current in isolated gallbladder smooth muscle cells of guinea pig
Authors: Shimada T.
Published: December 2017

The voltage-dependent Ca2+ current was studied in enzymatically dispersed guinea pig gallbladder smooth muscle cells using the whole cell patch-clamp technique. Depolarizing voltage (V) steps induced an inward current (I) that was carried by Ca2+. The threshold potential was -40 to -30 mV, the maximal current was observed at +10 to +20 mV, and the reversal potential was around +80 mV. I-V curves obtained with holding potentials of -80 and -40 mV were not significantly different. This current had a high sensitivity to dihydropyridine drugs, and the Ba2+ or Sr2+ current was larger than the Ca2+ current. Activation was accelerated by increasing the membrane potential. In general, the time course of decay was well fitted by the sum of two exponentials, but consideration of a third (ultra-slow) decay component was also necessary when the current generated by a 2-s command pulse was analyzed. Superimposition of activation and inactivation curves showed the presence of a significant window current. Carbachol suppressed the Ca2+ current only when the pipette contained a low concentration of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. These results show that the L-type Ca2+ current is dominant in gallbladder smooth muscle cells and may contribute to excitation-contraction coupling.
pH regulation in perfused rat liver: respective role of Na(+)-H+ exchanger and Na(+)-HCO3- cotransport
Authors: Durand T. , Gallis J. L. , Masson S. , Cozzone P. J. , Canioni P.
Published: December 2017

Na(+)-H+ antiport and Na(+)-HCO3- symport are involved in intracellular pH (pHi) homeostasis in cultured hepatocytes. We have studied the occurrence of these transport systems in the intact rat liver by 31P nuclear magnetic resonance. Livers perfused with a Krebs medium (25 mM HCO3-, pH 7.4, 37 degrees C) displayed a cytosolic pH 7.18 +/- 0.05 (n = 32). In response to an acid load (35 mM isobutyric acid), pHi remained constant. The same result was obtained in the presence of 1 mM amiloride (with or without acid load), indicating that the amiloride-sensitive Na(+)-H+ exchanger is inactive at external physiological pH (pHe). Under systemic acidosis (6.5-7.0 pHe range), during the acid load, pHi decreased with increased external proton concentrations and became amiloride sensitive. The pHi set point for the activation of the Na(+)-H+ exchange is 7.0. In the absence of HCO3-, livers showed a constant acidic shift of pHi (0.2 pH unit) in the 6.5-7.5 pHe range. Perfusion with 1 mM stilbene derivative (4-acetamido-4'-isothiocyanostilbene-2-2' disulfonic acid) in the presence of HCO3- and at pHe 7.4 induced a dramatic pHi fall (delta pH 0.15), further accentuated during an acid load (delta pH 0.25). Our results suggest that 1) the symport is always involved in pH homeostasis over a large range of pH variation (6.5-7.5) and 2) the Na(+)-H+ exchanger is activated under systemic acidosis as soon as pHi reaches the set point value.
Genetic evidence for role of DPP IV in intestinal hydrolysis and assimilation of prolyl peptides
Authors: Tiruppathi C. , Miyamoto Y. , Ganapathy V. , Leibach F. H.
Published: December 2017

The functional role of dipeptidyl peptidase IV (DPP IV) in the intestinal hydrolysis and assimilation of prolyl peptides was investigated using Japan F344 rats, which genetically lack this enzyme. USA F344 rats possess normal activity of this enzyme and served as matched controls. Intestinal brush-border membranes from the control rats were able to hydrolyze several proline-containing peptides. The hydrolytic ability of the brush-border membranes from the Japan rats against these peptides was markedly low. The difference in the hydrolytic activities between the two groups of rats was solely due to the absence of DPP IV in the Japan rats. There was no difference in the growth rate between the two groups of rats fed a reference diet whose protein constituents were not rich in proline. When the protein source was changed to gliadin, a proline-rich protein, USA F344 rats maintained their body weight for a 4-wk period on this diet, whereas the Japan rats experienced a significant weight loss under similar conditions. In situ perfusion experiments in intact animals revealed that the ability of morphiceptin (a peptide primarily hydrolyzable by DPP IV), when administered into the intestinal lumen, to block the cholera toxin-induced water secretion was significantly greater in Japan F344 rats than in USA F344 rats, indicating the resistance of morphiceptin to hydrolytic breakdown in the intestinal lumen of the Japan rats. It is concluded that the intestinal DPP IV plays a significant role in the hydrolysis of prolyl peptides and assimilation of proline-rich proteins.
Immunophysiology of the gut: a research frontier for integrative studies of the common mucosal immune system
Authors: Castro G. A. , Arntzen C. J.
Published: December 2017

This review highlights work that, within the past decade, transformed mucosal immunophysiology from a hypothetical concept to a fully recognized interdiscipline. The regulation of epithelial and smooth muscle functions by the mucosal immune system represents an exquisitely sensitive adaptation to local antigenic challenge. Furthermore, immunologic cells communicate with nerves via paracrine secretions to rapidly transduce antigenic signals into panmucosal changes in function. These local immunocyte-nerve interactions are modulated by the autonomic and central nervous systems. Because of the common mucosal immune system, antigen-induced changes similar to those occurring in the intestine and colon are predicted to occur in mucosa of all hollow organs. The drawing together of fields as diverse as medicine and agriculture underscores the scope of areas encompassed by immunophysiology. Newly acquired knowledge has positioned the field to advance rapidly in both basic and applied directions. Forces that will remodel the field in the next decade will be derived from public concerns about human health maintenance and the explosive and novel use of new research tools stemming from molecular biology. These forces will draw on and advance our knowledge in areas as diverse as vaccine development and prevention of allergic reactions to foods, bioengineered foods in particular.
Gastric myoelectric and motor activity in dogs with gastric dilatation-volvulus
Authors: Hall J. A. , Solie T. N. , Seim H. B. , Twedt D. C.
Published: December 2017

Electrical and contractile properties of the stomach were assessed in six adult dogs after recovery from surgical treatment for gastric dilatation-volvulus (GDV), a disorder characterized by delayed gastric emptying of the solid phase. Electrodes and strain-gauge force transducers were sutured to the serosa of the antrum and pylorus at the time of surgical intervention for GDV. Ten days after implantation, electrical and mechanical activities were recorded before and after a standardized meal. The analog FM tape recordings of the electrical and mechanical signals were converted to digital time series for analysis by computer. Recordings from dogs after GDV showed increased slow wave propagation velocity in both the fasting and the fed states compared with controls. In addition, the GDV dogs had atypical fasting state phase III activity fronts. We found no difference in gastric slow wave frequency, dysrhythmia, or electromechanical coupling between the two groups. These results indicate that delayed gastric emptying in this syndrome is associated with increased gastric slow wave propagation velocity.
Characterization of cytochrome P-450-dependent arachidonic acid metabolism in rabbit intestine
Authors: Macica C. , Balazy M. , Falck J. R. , Mioskowski C. , Carroll M. A.
Published: December 2017

We characterized cytochrome P-450-dependent arachidonate (P-450-AA) metabolism throughout the intestinal tract, since some metabolites derived via this pathway modify epithelial ion transport and regional blood flow. Microsomes (0.3 mg/ml) were prepared from each region of the intestines of anesthetized New Zealand White male rabbits and incubated with [14C]AA (7 microM) for 30 min at 37 degrees C. In the presence of NADPH (1 mM), ileal microsomes exhibited the greatest P-450-AA metabolism, whereas duodenal microsomes exhibited little or no activity. For jejunal, ileal, and cecal microsomes, AA metabolism was reduced in the absence of NADPH and by boiling microsomes, was unaffected by indomethacin (10 microM) and BW-755C (50 microM), but was significantly attenuated by the P-450 enzyme inhibitors, 7-ethoxyresorufin (1 microM) and SKF-525A (100 microM). However, colonic (ascending, transverse, and descending) microsomal activity was inhibited by both P-450 and lipoxygenase inhibitors. Analysis of ileal AA metabolites by high-pressure liquid chromatography and negative ion chemical ionization gas chromatography-mass spectrometry revealed products corresponding to monohydroxyeicosatetraenoic acids (HETEs). Semiquantitative analysis showed that 20-, 19-, 18-, 17-, and 16-HETEs were present in a ratio of 6.2:3.3:0.3:0.1:0.1, respectively. Furthermore, ileal P-450-HETEs dilated the isolated perfused mesenteric bed, as did 20-HETE, the predominant ileal AA metabolite. Because 20-HETE was also shown to affect epithelial ion transport, we suggest that P-450-AA metabolites may make important contributions to intestinal function
Na(+)-K(+)-ATPase gene expression in rat intestine and Caco-2 cells: response to thyroid hormone
Authors: Giannella R. A. , Orlowski J. , Jump M. L. , Lingrel J. B.
Published: December 2017

Expression of the Na(+)-K(+)-adenosinetriphosphatase (ATPase) gene family in rat intestinal epithelial cells was examined using RNA blot hybridization analyses. Rat intestinal epithelial cells express only the alpha 1- and beta 1-subunit mRNAs. A gradient in expression of alpha 1- and beta 1-subunit mRNA was seen along the villus-crypt unit in both jejunum and ileum, i.e., villus tip >> crypt cells. Regional differences in expression were observed along the intestine. alpha 1- and beta 1-subunit mRNA abundance was similar in jejunum, ileum, and colon while enzymatic activity was highest in the jejunum and lowest in the ileum. Administration of thyroid hormone to thyroidectomized rats increased the expression of alpha 1- and beta 1-subunit mRNAs in jejunum but not in colon. Hypothyroidism had no effect on subunit mRNA expression. The human intestinal cell line Caco-2 was also studied. These cells also expressed only the alpha 1- and beta 1-isoform mRNAs and demonstrated a developmental profile in both mRNA and enzymatic activity. Furthermore, in Caco-2 cells both alpha 1- and beta 1-mRNAs and Na(+)-K(+)-ATPase enzymatic activity were stimulated by thyroid hormone. Caco-2 cells transfected with 5' flanking regions of the human Na(+)-K(+)-ATPase beta 1-gene linked to the chloramphenicol acetyltransferase (CAT) reporter gene responded to 3,5,3'-triiodothyronine (T3) treatment with increased expression of CAT activity. This suggests that the 5' flanking region of the beta 1-gene contains a thyroid hormone response element and that T3 upregulation occurs at the transcriptional level.(ABSTRACT TRUNCATED AT 250 WORDS)
Effect of [(CH2NH)4,5]secretin on pancreatic exocrine secretion in guinea pigs and rats
Authors: Kim C. D. , Li P. , Lee K. Y. , Coy D. H. , Chey W. Y.
Published: December 2017

[psi 4,5]Secretin was shown to be a secretin receptor antagonist that inhibits secretin-stimulated increase in adenosine 3',5'-cyclic monophosphate in isolated pancreatic acini of the guinea pig. To determine whether it inhibits pancreatic exocrine secretion in vivo, we have studied the effect of [psi 4,5]secretin on the pancreatic secretion stimulated by secretin in anesthetized guinea pigs and rats. In basal state, [psi 4,5]secretin given intravenously for 2 or 3 h in varying doses of 1.6-32.7 nmol.kg-1.h-1 dose dependently increased pancreatic secretion of both fluid and bicarbonate during the 1st h, but it returned gradually to basal level within 2 or 3 h. On the other hand, [psi 4,5]secretin significantly inhibited the pancreatic secretion stimulated by either exogenous or endogenous secretin in a dose-related manner. The inhibitory effect of [psi 4,5]secretin in guinea pigs was greater than that in rats. However, it did not completely block the secretin-stimulated pancreatic secretion, whereas a rabbit antisecretin serum suppressed it completely. We conclude that 1) in the unstimulated state, [psi 4,5]secretin is a partial agonist of pancreatic exocrine secretion of both fluid and bicarbonate; and 2) when pancreatic secretion is stimulated by secretin, unlike an antisecretin serum, it is a partial inhibitor in intact guinea pigs and rats.
Cyclin and cyclin-dependent kinase 1 mRNA expression in models of regenerating liver and human liver diseases
Authors: Albrecht J. H. , Hoffman J. S. , Kren B. T. , Steer C. J.
Published: December 2017

There is compelling evidence that the eukaryotic cell cycle is controlled by a family of proteins called cyclins, which complex with cyclin-dependent kinases (CDK) to modulate key events during cell division. We have examined the regulation of these genes in models of experimental liver regeneration and their expression in human liver diseases. Seventy percent partial hepatectomy (PH) was performed on rats and normal BALB/c and athymic nude mice to determine patterns of cyclin and CDK1 mRNA expression. It has been previously shown by [3H]thymidine incorporation that athymic nude mice manifest impaired regeneration after PH. Our results demonstrate a sequential pattern of cyclin and CDK1 transcript expression in each of the models. Cyclin D1 was the most abundant mRNA steady-state transcript in the regenerating livers. CDK1 and cyclins associated with later stages of the cell cycle showed delayed and diminished expression in nude mice compared with normals. Nuclear run-off assays performed at key time points post-PH revealed little change in transcription rates, suggesting that steady-state mRNA expression of the cyclin genes is regulated primarily by posttranscriptional events. Human liver tissue from various acute and chronic hepatic diseases showed increased expression of cyclins A and D1. We conclude that the regenerating liver post-PH offers an excellent in vivo model for studying cyclin and CDK gene expression. Impaired regeneration in the nude mouse is associated with altered cyclin and CDK1 mRNA transcript expression. Furthermore, cyclins may eventually provide clinically relevant molecular markers of regenerative activity in human liver diseases.
A cloned CCK-A receptor transduces multiple signals in response to full and partial agonists
Authors: Yule D. I. , Tseng M. J. , Williams J. A. , Logdson C. D.
Published: December 2017

Chinese hamster ovary (CHO) cells, stably transfected with the cloned rat CCK-A receptor, were used to study signal transduction events initiated by cholecystokinin octapeptide (CCK-8) and the partial agonist JMV-180. In single CHO-CCK-A cells loaded with fura-2, superfusion of CCK-8 (10 pM-1 nM) resulted in an increase in intracellular Ca2+ concentration ([Ca2+]i). At CCK-8 concentrations < 100 pM, the signal consisted of [Ca2+]i oscillations. At higher concentrations, CCK-8 induced a typical biphasic response consisting of a large peak followed by a lower sustained plateau. Superfusion of JMV-180 also resulted in an increase in [Ca2+]i; in contrast to acinar cells this increase did not consist exclusively of [Ca2+]i oscillations. Both CCK-8 and JMV-180 increased polyphosphoinositide hydrolysis, although JMV-180 stimulated formation of only 10% as much [3H]inositol phosphates. [Ca2+]i signals stimulated by both CCK-8 and JMV-180 were blocked by the aminosteroid U-73122. CCK-8 (1-10 nM) increased formation of adenosine 3',5'-cyclic monophosphate (cAMP) and release of arachidonic acid in CHO-CCK cells. These increases were not mimicked by JMV-180 (10 microM). Furthermore, no cAMP formation or arachidonate release could be detected when cells were incubated with both JMV-180 and CCK-8. These data indicate that in CHO-CCK-A cells, unlike acinar cells, both CCK-8 and JMV-180 increase [Ca2+]i by similar mechanisms. However, the CCK-A receptor can differentially recognize and then activate discrete transduction pathways on binding of these two agonists.
Regulation of capillary exchange capacity in postnatal swine intestine
Authors: Nowicki P. T. , Miller C. E.
Published: December 2017

To determine whether intrinsic regulation of capillary exchange capacity is age-dependent during early postnatal life, we measured the capillary filtration coefficient (Kf,c), an index of capillary exchange capacity, within isolated perfused intestine from 3- and 35-day-old swine at different arterial pressures. Kf,c was determined gravimetrically, by means of the flow-equilibration technique. During baseline perfusion at age-appropriate pressures, Kf,c averaged 0.203 +/- 0.020 vs. 0.361 +/- 0.040 ml.min-1 x mmHg-1 x 100 g in 35- vs. 3-day-old intestine. In older animals, a progressive rise in Kf,c was noted as arterial pressure was lowered: Kf,c increased 14, 35, and 98% above baseline when pressure was reduced 20, 40, and 60% below baseline, respectively. An inverse linear correlation between Kf,c and blood flow was demonstrated in this group. Kf,c also increased in 3-day-old intestine during progressive hypotension, but these changes were of less magnitude: Kf,c averaged 19, 17, and 23% above baseline when pressure was reduced 20, 40, and 60% below baseline, respectively. In contrast to the circumstance noted in older animals, the relationship between Kf,c and blood flow was not significant within 3-day-old intestine. Intraarterial infusion of 1 microgram/min of isoproterenol, an agent known to increase capillary exchange capacity, caused Kf,c to increase 80% within 3-day-old intestine group. We conclude that Kf,c increases to a greater extent in 35- than 3-day-old swine intestine in response to arterial pressure reduction. However, intestine from younger animals is capable of increasing capillary exchange capacity in response to stimuli other than arterial hypotension, as evidenced by its response to isoproterenol.
Relationship between muscarinic receptor occupancy and response in rat parotid acinar cells
Authors: Dai Y. , Baum B. J.
Published: December 2017

To determine whether spare muscarinic cholinergic receptors (mAChRs) exist in rat parotid acinar cells, we examined the effect of propylbenzilylcholine mustard (PBCM) on agonist (carbachol)-stimulated inositol trisphosphate (IP3) formation and on mAChR number, using l-[N-methyl-3H]scopolamine methyl chloride (NMS)-binding assays. Treatment with PBCM (1, 3, 10, 30, 50 nM) for 15 min caused a 5, 22, 60, 66, and 72% decrease, respectively, in maximal IP3 formation stimulated by carbachol as well as a large reduction in the potency of carbachol in eliciting this response. Using these data, equilibrium constants (Ka) for activation of the mAChRs by carbachol were calculated. These Ka values agreed well with Kd values of high-affinity mAChR binding sites determined from carbachol displacement of [3H]NMS binding in parotid acinar cells. Reduction in mAChR number after PBCM treatment was determined by Scatchard analysis of specific [3H]-NMS binding sites and compared with the expected reduction (q values) calculated from dose-response curves for carbachol-stimulated IP3 formation before and after PBCM treatment. PBCM (1, 3, 10, 30 nM) decreased mAChR maximal binding in cells 47.5, 68.9, 82.4, and 85.3%, respectively, which did agree with the approximately 38, 70, 90, and 92% decrease in receptor number expected from the calculated q values. Data demonstrate that PBCM irreversibly inactivates mAChRs in rat parotid cells, and the decrease in receptor number, measured directly from [3H]NMS binding or calculated from receptor theory, is greater than that observed for stimulated IP3 production. These results suggest that a modest (30-40%) population of spare receptors exists for mAChR-mediated IP3 production in rat parotid glands.
Both low- and high-affinity CCK receptor states mediate trophic effects on rat pancreatic acinar cells
Authors: Hoshi H. , Logsdon C. D.
Published: December 2017

Cholecystokinin (CCK) stimulates the growth of pancreatic acinar cells. However, the molecular mechanisms involved in this trophic action are unknown. CCK binds to both high- and low-affinity receptor states, and these two states appear to activate separate sets of intracellular messengers and have opposite effects on amylase release. JMV-180 is a CCK analogue that interacts in the rat with the high-affinity state as an agonist and the low-affinity state as an antagonist. In the current study, CCK octapeptide (CCK-8) and JMV-180 were tested for their ability to stimulate the growth of rat pancreatic acinar cells in primary culture. CCK-8 stimulated [3H]thymidine incorporation into DNA in a dose-dependent manner. Effects were observed with 0.3 nM, and maximal increases were seen at 3 nM CCK-8 (442 +/- 53% of control, n = 5, P < 0.01). JMV-180 also stimulated DNA synthesis. Effects were noted with 10 nM, and a maximal increase of 267 +/- 82% (n = 4, P < 0.01) of control was stimulated by 100 nM JMV-180. These data with JMV-180 indicate that the high-affinity receptor state for CCK is capable of stimulating DNA synthesis. However, within the same experiment the effects of CCK were always significantly greater than those of JMV-180. To test whether CCK has an additional effect through interactions with the low-affinity state, the effects of a combination of JMV-180 with a maximal dose of CCK-8 were examined. JMV-180 inhibited the maximal effect of CCK-8 in a dose-dependent manner with a maximal inhibition occurring with 1 microM JMV-180. The effects of the combination of 3 nM CCK-8 and 1 microM JMV-180 were no greater than those of JMV-180 alone. Taken together these data indicate that CCK-mediated increases in DNA synthesis in rat pancreatic acinar cells in vitro occur by interactions with both high- and low-affinity receptor states.
Temporal pattern of rat small intestinal gene expression with refeeding
Authors: Hodin R. A. , Graham J. R. , Meng S. , Upton M. P.
Published: December 2017

Studies were carried out to elucidate the molecular mechanisms underlying small intestinal epithelial growth. Adult rats were fasted for 4 days and then refed a chow diet for up to 48 h. Histological examination confirmed the sequential occurrence of mucosal atrophy and hyperplasia. Northern blot analyses of RNA derived from small intestinal mucosal scrapings revealed a striking pattern of alterations in the expression of two different categories of genes. There were very early increases in the expression of c-fos and c-jun, reflecting the mitogenic response to refeeding that occurs within the crypt compartment. Studies using the protein synthesis inhibitor cycloheximide suggest that c-fos and c-jun are part of the “immediate-early” response of the small intestine. At later time points after the refeeding stimulus, differential changes occurred in the expression of the brush-border enzymes, lactase, and intestinal alkaline phosphatase (IAP). Refeeding caused a decrease in lactase gene expression and an increase in the expression of the 3.0-kb IAP mRNA species, reflecting a return of the villus phenotype to the normal fed state. Thus we have demonstrated a complex and temporally related pattern of gene expression within the small intestinal epithelium upon refeeding. The results provide insight into the relationship between the processes of intestinal growth and differentiation.
Evidence that bile salts are important for iron absorption
Authors: Sanyal A. J. , Hirsch J. I. , Moore E. W.
Published: December 2017

The role of bile salts in intestinal Fe absorption has not previously been defined. We have recently shown that bile salts having cholanic ring 7 alpha-OH and/or 12 alpha-OH groups bind Fe2+ with high affinity at premicellar concentrations and also produce a two-to threefold enhancement in Fe2+ uptake from perfused intestinal segments in vivo. However, the physiological relevance of these observations for Fe2+ absorption was not known. To study this, we examined the effects of a single dose of taurocholate (TC) administered with 59Fe-FeSO4 in reversing iron malabsorption induced by bile duct ligation. Fe absorption from three 0.5-mg doses of 59Fe-FeSO4 was measured from five groups of six adult Sprague-Dawley rats each: group 1, before and after bile duct ligation and subsequent administration of 59Fe-FeSO4 + TC test dose; group 2, before and after bile duct ligation followed by a third dose of Fe alone; group 3, before and after bile duct ligation followed by a third dose of Fe+taurodehydrocholate (TDHC); group 4, sham laparotomy; and group 5, nonoperated controls where Fe absorption was measured from three doses of 59Fe-FeSO4 at the same time intervals as group 1. Absorption was measured, using whole body counting, after achievement of steady-state counts after each Fe dose. Mean percentage absorption from dose 1 (baseline) was 35.33 +/- 4.6% (SE). No significant differences between any groups were noted. In nonoperated controls (group 4), Fe absorption was virtually identical after all three doses. Also, hematocrit did not change in any of the 30 animals.(ABSTRACT TRUNCATED AT 250 WORDS)
Gastric smooth muscle cells possess two classes of endothelin receptors but only one alters contraction
Authors: Kitsukawa Y. , Gu Z. F. , Hildebrand P. , Jensen R. T.
Published: December 2017

Endothelin (ET)-like immunoreactivity and ET binding sites are widely distributed in the gastrointestinal tract, and ET causes contraction of stomach muscle strips. To determine whether ETs could interact with gastric smooth muscle cells directly and alter function, we measured binding of 125I-ET-1, 125I-ET-2, and 125I-ET-3 to dispersed gastric smooth muscle cells from guinea pig and their abilities to alter cell length. Each ligand bound in a time- and temperature-dependent manner, which was specific and saturable. Analysis of the dose-inhibition curves of both ET-1 and ET-3 for binding of each ligand indicated the presence of two classes of receptors, one class (ETA receptor) with a high affinity for ET-1 and ET-2 but a low affinity for ET-3, and the other (ETB receptor) with a high affinity for ET-1, ET-2, and ET-3. The ligands were rapidly internalized by both receptors; however, it was greater with ETA receptors. ET-1 stimulated muscle contraction (50% effective concentration approximately 2 nM), whereas ET-3 did not stimulate contraction or cause relaxation. These results demonstrate that gastric smooth muscle cells possess two classes of ET receptors. One type (ETA) has a high affinity for ET-1 and ET-2 and a low affinity for ET-3, and receptor occupation results in rapid ligand internalization and muscle contraction; the other type (ETB) has a high affinity for ET-1, ET-2, and ET-3, and receptor occupation results in a lesser degree of ligand internalization than the ETA receptor and does not alter contractile behavior.
Differential signal transduction pathways in cat lower esophageal sphincter tone and response to ACh
Authors: Biancani P. , Harnett K. M. , Sohn U. D. , Rhim B. Y. , Behar J. , Hillemeier C. , Bitar K. N.
Published: December 2017

Lower esophageal sphincter (LES) basal tone and contraction in response to maximally effective doses (Emax) of acetylcholine (ACh) may be mediated by different intracellular transduction pathways. In the basal state resting tone, inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] formation and levels of diacylglycerol (DAG) (C. Hillemeier, K. N. Bitar, and P. Biancani, unpublished data) are higher in LES circular muscle than in esophageal muscle, which does not maintain tone. In vitro resting tone and spontaneously elevated formation of Ins(1,4,5)P3 in LES circular muscle strips decrease in a dose-dependent manner in response to the phospholipase C antagonist 1-[6-([(17-beta)-3-methoxyestra-1,3, 5(10)-trien-17-yl]amino)hexyl]-1H-pyrrole-2,5-dione (U-73122). Basal Ins(1,4,5)P3 formation, however, is submaximal, since it can be increased by cholinergic stimulation. These data suggest that LES tone is associated with partial activation of phospholipase C. We therefore tested submaximal doses of Ins(1,4,5)P3 and DAG in permeabilized LES muscle cells and found that they act synergistically; their interaction depends on calcium release and is mediated through a protein kinase C (PKC)-dependent pathway. In contrast, we have previously shown that contraction induced by Emax of ACh is mediated through calmodulin-dependent mechanisms (14). To investigate these differences, we tested high and low doses of ACh. Contraction induced by high doses of ACh was inhibited by calmodulin but not by PKC antagonists, as previously reported, but low ACh doses were preferentially inhibited by PKC antagonists. Similarly, low Ins(1,4,5)P3 concentrations activated a PKC-dependent pathway, whereas contraction induced by Emax of Ins(1,4,5)P3 was calmodulin dependent.(ABSTRACT TRUNCATED AT 250 WORDS)
Chief cells possess somatostatin receptors regulated by secretagogues acting through the calcium or cAMP pathway
Authors: Felley C. P. , O'Dorisio T. M. , Howe B. , Coy D. H. , Mantey S. A. , Pradhan T. K. , Sutliff V. E. , Jensen R. T.
Published: December 2017

Inhibition both in vivo and in vitro of pepsinogen secretion by somatostatin (SS) and the histological demonstration that fundic D-cells contain long cytoplasmic processes extending to chief cells suggest a possible direct effect of SS on chief cell function. The aim of the present study was to determine whether SS interacts directly with receptors on isolated gastric chief cells and, if so, how SS alters cell function. Binding of 125I-[Tyr11]SS14 to chief cells was saturable, time and temperature dependent, and was inhibited by both SS14 (Ki 1.6 nM) and SS28 (Ki 5.2 nM). SMS-201-995 was 1,300-fold less potent than SS14. Calcium-mobilizing secretagogues reduced binding of 125I-[Tyr11]SS14 with efficacies of cholecystokinin octapeptide (CCK-8) > carbachol > gastrin. Adenosine 3',5'-cyclic monophosphate (cAMP)-activating secretagogues also inhibited binding with efficacies of secretin > vasoactive intestinal polypeptide (VIP). 12-O-tetradecanoylphorbol 13-acetate (TPA) or A-23187 also decreased binding. Analyses demonstrated that CCK-8 and TPA were decreasing the affinity of SS receptors for 125I-[Tyr11]SS14 without affecting their binding capacity. Both SS14 and SS28 at a maximally effective concentration inhibited cAMP production caused by VIP or secretin (20-30%) but did not alter cytosolic calcium ([Ca2+]i), inositol phosphates, or pepsinogen release. We conclude that chief cells possess SS receptors with a high affinity for both SS14 and SS28 but low affinity for SMS-201-995 and thus resemble the SSB receptors described in the rat cerebral cortex. Although occupation of these receptors by SS has no effect on pepsinogen release induced by secretagogues acting through either the calcium or the cAMP pathway, SS receptor occupation is regulated by agents activating phospholipase C, adenylate cyclase, protein kinase C, and [Ca2]i.
Regulation of pancreatic tyrosine kinase and phosphatase activities by cholecystokinin and somatostatin
Authors: Rivard N. , Lebel D. , Laine J. , Morisset J.
Published: December 2017

Phosphorylation and dephosphorylation of proteins on tyrosyl residues are important reactions involved in cellular activities, namely, those associated with growth and differentiation. Although it is accepted that cholecystokinin (CCK) and somatostatin (SS) stimulate and inhibit pancreatic growth and secretion, the cellular mechanisms by which these two hormones trigger their stimulatory and inhibitory effects are not well known. It has recently been suggested that, in acinar cells, one of the early signals of SS would involve activation of a membrane tyrosine phosphatase, whereas the signal associated with CCK may involve stimulation of protein tyrosine phosphorylation. This study examines the effects of caerulein (Cae) and SMS-201-995 (SMS) on pancreatic growth, particulate and crude cytosolic tyrosine kinase (TRK), and phosphotyrosine phosphatase (PTase) activities. Rats infused intravenously with 0.05% bovine serum albumin (control), Cae (0.25 micrograms.kg-1.h-1), or SMS (5 micrograms.kg-1.h-1) were killed after 0.5, 1, 2, 3, 4, 8, 12, 24, and 48 h of infusion. The pancreas was excised, weighed, and evaluated for contents of DNA and protein and for TRK and PTase activities. The effects of subtotal pancreatectomy on TRK and PTase activities were also examined after 1, 2, 3, 4, and 7 days. In response to Cae, pancreatic growth was evident after 48 h and was accompanied by sustained increases in particulate TRK and particulate PTase. Increases in membrane PTase activities were localized on membranes of the zymogen granules. SMS treatment was associated with increases in pancreatic weight and protein as a result of inhibition of secretion.(ABSTRACT TRUNCATED AT 250 WORDS)
Primary culture of salamander intestinal epithelial cells from nests: whole cell Na+ currents induced by valine
Authors: White J. F.
Published: December 2017

Methods are described for isolating the cell nests, subepithelial clusters of germinative cells, from salamander intestinal mucosa and for growing the nests in culture into polarized monolayers of intestinal epithelial cells. Cells were viable in culture for up to 3 wk. The capacity of the monolayer cells to engage in membrane transport was evaluated using the patch-clamp technique in the whole cell mode. L-Valine (25 mM) induced an inward current in small intestinal cells of 25.8 +/- 5.7 pA and depolarized the cell membrane 14.5 +/- 1.6 mV. L-Alanine and L-phenylalanine were similarly effective, whereas D-valine was ineffective. The Km of the transporter for valine was 90 mM. Replacement of bath Na with tris(hydroxymethyl)aminomethane eliminated the inward current induced by valine. The basal (solute-independent) inward current was also reduced by Na+ replacement. Glucose did not induce a Na+ current. In contrast to the effect of valine on small intestinal cells, large intestinal cells were unresponsive to valine. It is concluded that the cultured small intestinal cells possess Na-amino acid but not Na-sugar cotransport. This profile of behavior is characteristic of undifferentiated small intestinal cells. Primary cultures of salamander small intestinal cells should be useful for studying enterocyte function and the developmental biology of the small intestinal mucosa.
Enrichment of rab11, a small GTP-binding protein, in gastric parietal cells
Authors: Goldenring J. R. , Soroka C. J. , Shen K. R. , Tang L. H. , Rodriguez W. , Vaughan H. D. , Stoch S. A. , Modlin I. M.
Published: December 2017

Parietal cell secretion of acid requires the coordinated fusion of H(+)-K(+)-adenosinetriphosphatase (ATPase)-containing tubulovesicles with a secretory canalicular target membrane. We have previously reported the presence of rab2 on parietal cell tubulovesicles (L. H. Tang, S. A. Stoch, I. M. Modlin, and J. R. Goldenring. Biochem. J. 285: 715-719, 1992). Since 60% of the small GTP-binding protein sequences obtained from parietal cells were > 95% homologous with human rab11 (J. R. Goldenring, K. R. Shen, H. D. Vaughan, and I.M. Modlin. J. Biol. Chem. 268: 18419-18422, 1993), we sought to study rab11 in gastric parietal cells. A complete rab11 sequence was obtained, and the deduced amino acid sequence of rabbit rab11 was identical to that for human. Rab11 mRNA was present throughout the gastrointestinal mucosa. mRNA for both rab11 and rab2 were enriched in isolated parietal cells compared with chief cells. A polyclonal antiserum against rab11 labeled a single 25-kDa band in isolated parietal cells. Immunostaining of rat fundic tissue demonstrated prominent staining of parietal cells. Rab11 staining cosegregated with alpha-H(+)-K(+)-ATPase staining in enriched preparations of rabbit parietal cell tubulovesicles. These results suggest that rab11 is enriched in parietal cells and is associated with intracellular tubulovesicles.
Molecular basis for somatostatin action: inhibition of c-fos expression and AP-1 binding
Authors: Todisco A. , Campbell V. , Dickinson C. J. , DelValle J. , Yamada T.
Published: December 2017

The mechanisms by which somatostatin exerts its widespread inhibitory actions have been investigated extensively but understood only partially. Recent studies have shown that somatostatin can inhibit gene transcription directly. In view of the critical importance of early response genes in induction of gene expression, we examined whether the action of somatostatin might be mediated by inhibition of early response genes. The products of some of these genes, such as c-fos and c-jun, are known to form a heterodimeric transcription factor complex (AP-1) that binds specifically to the consensus sequence TGAC(G)TCA. Accordingly, we examined the effects of somatostatin on c-fos gene expression and on the binding of the AP-1 complex to its specific DNA element using isolated gastric parietal cells and the GH3 pituitary cell line. In both parietal and GH3 cells, c-fos-specific mRNA was increased by agents known to act via both adenosine-3',5'-cyclic monophosphate and Ca(2+)-dependent signaling mechanisms, and octreotide significantly inhibited this response. Pertussis toxin pretreatment (200 ng/ml) reversed the inhibitory effect of octreotide. AP-1 binding activity, assessed by gel shift assays using a 32P-labeled AP-1 oligonucleotide probe, was stimulated by dibutyryl adenosine 3',5'-cyclic monophosphate and serum and inhibited by octreotide treatment. Our observations support the notion that the universal inhibitory action of somatostatin may be mediated by inhibition of expression of early response genes via a pertussis toxin-sensitive inhibitory pathway. This effect appears to lead to decreased binding of regulatory nuclear proteins to their specific DNA elements resulting, presumably, in diminished gene expression.
Copper transport in the Nagase analbuminemic rat
Authors: Vargas E. J. , Shoho A. R. , Linder M. C.
Published: December 2017

The copper binding components of serum and potential importance of albumin to copper transport were investigated in adult Nagase analbuminemic and Sprague-Dawley rats of both sexes. There was a sex difference in total plasma copper concentrations, which were 60 and 130% higher than in the parent strain, in male and female Nagase rats, respectively. The higher levels of plasma copper were accounted for by two- and threefold increases in ceruloplasmin, as measured by p-phenylenediamine oxidase activity and copper by atomic absorption after gel chromatography. Other nonalbumin plasma proteins were also present in higher concentrations. Albumin concentrations were one-four-thousandth that of Sprague-Dawley rats, at 15 micrograms/ml (determined by rocket immunoelectrophoresis and comparative Western blotting). The tissue distribution and rate of uptake of intravenously injected 67 Cu(II) were unaffected by the lack of circulating albumin. 67Cu entered the liver of Nagase rats at least as rapidly as in the parent strain and reemerged in the blood on ceruloplasmin at an accelerated rate. The initial binding of 67Cu(II) to plasma components was primarily to transcuprein compared with albumin in the case of Sprague-Dawley rats. In the Nagase rats, the rest of the 67Cu bound primarily to nonalbumin proteins with about the same size as albumin; in Sprague-Dawley rats, it bound primarily to transcuprein. Limited analyses of tissue copper confirmed previous reports showing no striking differences from the parent strain. We conclude that albumin is not critical to the normal distribution and metabolism of copper and that it may serve more as a reservoir for excess plasma copper than as a specific conduit for the delivery of this element to hepatocytes or other cells.
Rat parietal cell receptors for GLP-1-(7-36) amide: northern blot, cross-linking, and radioligand binding
Authors: Schmidtler J. , Dehne K. , Allescher H. D. , Schusdziarra V. , Classen M. , Holst J. J. , Polack A. , Schepp W.
Published: December 2017

The intestinal peptide hormone glucagon-like peptide-1 (GLP-1) (7-36) amide is a potent stimulus of H+ production in isolated rat parietal cells, suggesting the presence of specific GLP-1-receptors on this cell type. Our aim was to characterize these receptors. Enzymatically isolated rat gastric mucosal cells (F0) were fractionated by counterflow elutriation, resulting in five fractions (F1-F5) according to increasing cell diameter and parietal cell content (3, 5, 4, 27, 81%). Additional density gradient centrifugation of F4 yielded enriched chief cells (74%; parietal cells: 1%; F6), whereas density gradient centrifugation of F5 almost purified parietal cells (97%; chief cells: 1%; F7). Northern blot of total cellular RNA from F0-F7 with a probe specific for the GLP-1-(7-36) amide receptor revealed two RNA species of 2.7 and 3.6 kb. These messages were present to some extent in small cells (F1, F2), much more pronounced in F5, abundant in F7, barely detectable in F3 and F4, and absent from F6. Cross-linking of 125I-labeled GLP-1-(7-36) amide to parietal cell membranes revealed a single 59-kDa band that was abolished by unlabeled GLP-1-(7-36) amide. Throughout fractions F1-F7 specific binding of 125I-GLP-1-(7-36) amide was correlated with parietal cell content (r = 0.99; P < 0.01) and H+ production ([14C]aminopyrine accumulation) in response to GLP-1-(7-36) amide or histamine (r = 0.96; P < 0.01). Binding was maximal in purified parietal cells (F7), whereas almost no binding was detectable in enriched chief cells (F6). In F7, Scatchard analysis revealed a single class of high-affinity binding sites (KD = 2.8 +/- 0.6 x 10(-10) M, Bmax = 6.8 +/- 1.4 fmol/10(6) cells, 4,096 +/- 793 receptors/parietal cells). The following half-maximal inhibition values were found for GLP-1-(7-36) amide and (1-37) and (1-36) amide: 6.6 +/- 0.9 x 10(-10), 1.4 +/- 0.7 x 10(-7), and 2.6 +/- 0.4 x 10(-7) M, respectively. Pancreatic glucagon, GLP-2, and oxyntomodulin, products of the proglucagon gene, were 3-4 log units less potent displacers while gastric inhibitory peptide, vasoactive intestinal peptide, and secretin were ineffective. We conclude that rat parietal cells are equipped with specific high-affinity receptors for GLP-1-(7-36) amide, which, in addition, are present in as yet unidentified small cells (F1, F2) but not in chief cells.
Gastric mucosal repair and release of HCO3- after damage by 2 M NaCl in cat: role of systemic acid base status
Authors: Guttu K. , Grong K. , Svanes K. , Gronbech J. E.
Published: December 2017

To study the influence of acid base balance on gastric mucosal repair, NH4Cl or NaHCO3 was given intravenously to anesthetized cats after mucosal damage induced by intraluminal 2 M NaCl. Saline at pH 5 or 1 was perfused via an oral tube through the stomach lumen and evacuated via a pyloric tube to a chamber with pH and PCO2 electrodes. Luminal bicarbonate (HCO3-) was markedly increased early after damage in both acidotic and alkalotic animals. In alkalotic animals mucosal blood flow increased about twofold in response to mucosal damage, whereas the early hyperemic response was either completely attenuated or blunted in acidotic animals. HCO3- release was correlated to availability of HCO3- by blood in alkalotic animals with luminal pH 5. Alkalotic animals showed improved repair compared with acidotic animals, and mucosal restitution was correlated to availability of HCO3- by blood. We conclude that luminal leakage of HCO3- or plasma after mucosal damage depends on availability by blood and consumption of HCO3- within the mucosa and that blood borne HCO3- has a major influence on gastric mucosal repair.
Characterization of cloned rat liver Na(+)-bile acid cotransporter using peptide and fusion protein antibodies
Authors: Ananthanarayanan M. , Ng O. C. , Boyer J. L. , Suchy F. J.
Published: December 2017

A cDNA encoding a rat liver Na(+)-bile acid cotransporter (Ntcp) has recently been cloned (Hagenbuch, B., B. Steiger, M. Fouget, H. Lubbert, and P. J. Meier. Proc. Natl. Acad. Sci. USA 88: 10629, 1991) using expression cloning in Xenopus laevis oocytes. Although the open reading frame coded for a protein of 39 kDa, in vitro translation experiments produced a 35-kDa protein which increased to a product of 41 kDa after glycosylation by pancreatic microsomes. To more clearly characterize the native protein in rat liver, we have raised antipeptide and anti-fusion protein antibodies to the COOH-terminal part of the cloned transporter. On Western blot analysis both antisera but not preimmune serum specifically detected a protein of approximately 50 kDa in isolated rat liver basolateral plasma membranes (BLPM). The reactivity was abolished when the antiserum was preincubated with the synthetic alpha-337 peptide. Deglycosylation of BLPM with N-glycanase followed by antibody probing led to decrease of the molecular mass to 34.5 kDa, suggesting that the protein is N-glycosylated in vivo. Two-dimensional immunoblotting indicated that the Ntcp protein had an isoelectric point of approximately 6.0. The antibody did not react with any proteins in rat ileal and kidney cortex brush-border membranes, human liver basolateral plasma membranes, or rat hepatoma tissue culture cell homogenates. Immunofluorescence localization studies with both antibodies revealed specific staining of the sinusoidal membrane domain but not of intracellular or bile canalicular membranes. Moreover, there was no acinar gradient in the pattern of staining.(ABSTRACT TRUNCATED AT 250 WORDS)