bluuebunny/crossref_metadata_2025 huggingface dataset:
Correlation between electrical and morphological properties of canine pyloric circular muscle
Authors: Vogalis F. , Ward S. M. , Sanders K. M.
Published: December 2017

Electrical slow waves decay in amplitude as they conduct from the myenteric to the submucosal regions of the circular muscle layer in the canine pyloric sphincter. We used the partitioned chamber method to study the passive and active properties of pyloric muscles, and we found that length constants of circular muscles of myenteric region were significantly longer than muscles near the submucosal surface. These data suggested differences in either membrane resistance, junctional resistance, or cytoplasmic resistance. The first parameter was evaluated by measuring time constants in intact tissues and single cells isolated from the submucosal and myenteric regions. Membrane time constants were not different in the two regions, nor were differences found in the input resistances of isolated cells. Morphological studies failed to demonstrate differences in cell diameters in the two regions suggesting that cytoplasmic resistances are similar. These findings suggest that the different cable properties in the two regions may be due to differences in electrical coupling. Morphological examination revealed similar numbers of gap junctions between cells in the two regions, but large differences were noted in the size of muscular bundles. Muscles of the myenteric region were arranged into large, tightly packed bundles, whereas muscles of the submucosal region consisted of small bundles with an extensive extracellular space filled with connective tissue. We suggest that the difference in cable properties may be due to differences in electrical coupling between bundles. These data suggest that submucosal muscles function more like a multiunit smooth muscle, whereas myenteric muscles behave as a single unit.
Dual pathways for agonist-stimulated arachidonic acid release in pancreatic acini: roles in secretion
Authors: Pandol S. J. , Hsu Y. L. , Kondratenko N. F. , Schoeffield-Payne M. S. , Steinbach J. H.
Published: December 2017

The present experiments were performed to determine pathways responsible for arachidonic acid release stimulated by cholecystokinin (CCK) and phorbol ester, 4 beta-phorbol 12-myristate 13-acetate (PMA), and the roles of pathways in the secretory response in dispersed acini from guinea pig pancreas. Both CCK-octapeptide (CCK-OP) and PMA increased intracellular arachidonic acid. To determine the source of released arachidonic acid, we measured the effects of PMA and CCK-OP on cellular 1,2-diacylglycerol and lysophosphatidylcholine (LPC) and of diglyceride lipase inhibitor RHC 80267 on [3H]arachidonic acid release. Both PMA and CCK-OP increased 1,2-diacylglycerol and LPC. RHC 80267 had no effect on LPC but inhibited the increase in [3H]arachidonic acid release with a concentration of CCK-OP that was maximal for enzyme secretion. The increase in [3H]arachidonic acid release with PMA or a supramaximal concentration of CCK-OP was not inhibited by RHC 80267. In parallel fashion, RHC 80267 inhibited amylase release caused by maximally effective concentrations of CCK-OP but not that caused by PMA or by supramaximally effective concentrations of CCK-OP. Arachidonic acid stimulated amylase release. Exogenous addition of phospholipase A2 caused increases in [3H]arachidonic acid release, LPC formation, and amylase release. The results indicate that there are at least two pathways responsible for the increase in free cellular arachidonic acid stimulated by pancreatic agonists. One is sequential action of phospholipase C and diglyceride lipase on phosphatidylinositol. The other is a phospholipase A action on phosphatidylcholine. The results also suggest a stimulatory role for both pathways in the secretory response.
Effect of omeprazole on gene expression in canine gastric parietal cells
Authors: Campbell V. W. , Yamada T.
Published: December 2017

Stimulation of gastric parietal cells by carbachol induces coordinate expression of the genes for two enzymes involved in the process of acid secretion, H(+)-K(+)-ATPase and carbonic anhydrase II (CA II). The basis of this coordinate expression was examined in experiments using parietal cells that had been pretreated with omeprazole. We observed a twofold increase in the steady-state mRNA levels of both H(+)-K(+)-ATPase and CA II after cells were treated with the inhibitor. The induction of CA II mRNA by carbachol followed the same kinetics in omeprazole-pretreated cells as in those that were not pretreated, suggesting that the induction of CA II gene expression by carbachol was not dependent on activation of the gastric H(+)-K(+)-ATPase. In addition, carbachol stimulation of omeprazole-pretreated cells resulted in an induction of one or more larger mRNA species that hybridized with the H(+)-K(+)-ATPase probe. The observation that carbachol-induced increases in steady-state levels of beta-actin mRNA in parietal cells could be inhibited by omeprazole pretreatment suggests a possible linkage between increased beta-actin gene expression and the process of acid secretion.
Transport of L-proline and alpha-methyl-D-glucoside by chicken proximal cecum during development
Authors: Moreto M. , Amat C. , Puchal A. , Buddington R. K. , Planas J. M.
Published: December 2017

We examined the characteristics of amino acid and sugar absorption by the proximal cecum (PC) of chickens during posthatch development. Rates of absorption of L-proline (Pro) and alpha-methyl-D-glucoside (MG) were measured at 2 days, 5 wk, and 13 wk after hatch with an in vitro everted-sleeve method. For each age, pieces of PC and midjejunum were incubated in solutions containing 0.1-50 mM Pro or MG, and the active and passive components of Pro and MG absorption were determined. Five conclusions may be stated. 1) There are two carrier-mediated transport systems for Pro in the PC: a higher capacity Na(+)-dependent system (Vmax between 1.6 and 3.2 nmol.mg-1.min-1), and a lower capacity Na(+)-independent system (Vmax 0.3-0.8 nmol.mg-1.min-1). 2) Whereas both Pro transport systems are present in the PC at 5 and 13 wk, only the Na(+)-dependent system was found at 2 days. Although rates of transport per milligram tissue by the Na(+)-dependent system fell during development, when rates were normalized to nominal surface area, Vmax was significantly higher in the 5-wk-old group than in the other groups. 3) MG transport is by a Na(+)-dependent system. Vmax values (nmol.mg-1.min-1) were 0.32 (2 days), less than 0.43 (5 wk), and = 0.55 (13 wk). These differences were not affected by normalization to surface area. 4) Because at physiological concentrations passive influx of Pro and MG would be negligible, absorption of amino acids and sugars by the PC would be dependent on the presence of carrier-mediated systems.(ABSTRACT TRUNCATED AT 250 WORDS)
Effect of a purified somatostatin monoclonal antibody and its Fab fragments on gastrin release
Authors: McIntosh C. H. , Tang C. L. , Malcolm A. J. , Ho M. , Kwok Y. N. , Brown J. C.
Published: December 2017

The effect of a purified somatostatin monoclonal antibody, SOMA 10, and its Fab fragments on gastrin release have been examined in the isolated perfused rat stomach. SOMA 10 was purified from ascites fluid by ammonium sulfate precipitation followed by hydroxylapatite (HAP) chromatography. This gave a preparation of greater than 90% purity, as assessed by fast affinity and size exclusion high-performance liquid chromatography (HPLC). HAP-purified SOMA 10 had a binding capacity of 760 mmol somatostatin/mol antibody, a dissociation constant of 2.2 nM, and recognized amino acid residues 5-12 of the somatostatin molecule. Fab fragments of SOMA 10 were prepared and purified by protein A affinity chromatography, and the purity assessed by HPLC and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Single passage perfusion of SOMA 10 (100 micrograms/ml) into the gastric vasculature caused a paradoxical decrease in basal gastrin release. However, recirculation of the antibody (20 micrograms/ml) caused an increase in cumulated gastrin release. Perfusion of Fab fragments of SOMA 10 (66 micrograms/ml) also increased gastrin secretion. These results support the suggestion that somatostatin exerts a tonic restraint on gastrin release and that penetration of the antibody to the site of somatostatin release is necessary for it to have an effect.
Neutrophil-derived oxidants modify CCK-OP-stimulated guinea pig gallbladder contraction in vitro
Authors: Moummi C. , Gullikson G. W. , Grisham M. B. , Gaginella T. S.
Published: December 2017

Neutrophil-derived oxidants such as hydrogen peroxide (H2O2), hypochlorous acid (HOCl), and monochloramine (NH2Cl) may contribute to gallbladder inflammation in cholecystitis. We examined the influence of oxidants on the biological activity of different agonists and antagonists of gallbladder smooth muscle function. The concentration-response curves for cholecystokinin-octapeptide (CCK-OP) and carbachol were examined before and after incubation of the tissues with NH2Cl (30 microM). The 50% effective concentration of CCK-OP was shifted from 0.5 +/- 0.09 nM (control) to 4 +/- 1.2 nM in the presence of NH2Cl. The effect of carbachol was not affected by NH2Cl. The contractile effect of CCK-OP (3 nM) was abolished by prior exposure to HOCl or NH2Cl. These actions were prevented by 60 microM glutathione. Oxidant-induced degradation of CCK-OP was confirmed by high-performance liquid chromatography and thin-layer chromatography. NH2Cl also significantly reduced the contractile response to neurokinin A, bradykinin, leukotriene D4, and phorbol 12,13-dibutyrate and the relaxant response to isoproterenol. Prior exposure of acetylcholine, histamine, serotonin, prostaglandin E2, vasoactive intestinal polypeptide, or calcitonin gene-related peptide to NH2Cl had no effect on their activity. The results indicate that NH2Cl generated during inflammation may decrease the biological activities of different agonists and antagonists of smooth muscle function.
Characterization and modulation of rat small intestinal brush-border membrane transbilayer fluidity
Authors: Dudeja P. K. , Wali R. K. , Harig J. M. , Brasitus T. A.
Published: December 2017

In the present experiments, selective quenching by trinitrophenyl groups as well as steady-state fluorescence polarization and differential polarized phase fluorescence techniques, using three different lipid soluble fluorophores, were used to directly examine the fluidity of the exofacial and cytofacial leaflets of rat small intestinal brush-border membranes. These studies revealed that the fluidity of the exofacial hemileaflet was greater than the cytofacial hemileaflet. Differences in the distribution of phosphatidylcholine and phosphatidylethanolamine, as assessed by phospholipase A2 treatment and trinitrophenylation of aminophospholipids, were, at least partially, responsible for the asymmetrical fluidity of the hemileaflets. Moreover, in vitro addition of benzyl alcohol (final concn 25 mM) preferentially fluidized the exofacial leaflet and concomitantly decreased leucine aminopeptidase activity but did not affect the activities of maltase, sucrase, alkaline phosphatase, or gamma-glutamyltranspeptidase. In vivo addition of the membrane-mobility agent 2-(2-methoxyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl)octanate] (A2C) (final concn 7.5 microM) preferentially fluidized the cytofacial leaflet and increased Na(+)-gradient-dependent D-glucose transport but not Na(+)-gradient-dependent L-leucine transport.
Electrophysiological properties of neurons in submucosal ganglia of guinea pig distal colon
Authors: Frieling T. , Cooke H. J. , Wood J. D.
Published: December 2017

Intracellular recording methods were used in vitro to study the electrophysiological behavior of neurons in ganglia of the submucosal plexus in the distal colon of the guinea pig. The results revealed subpopulations of submucosal ganglion cells that corresponded to the AH/type 2, S/type 1, type 3, and type 4 subpopulations found elsewhere in the intestine. Electrical behavior of colonic submucosal neurons differed from the myenteric plexus of the colon, rectum, and stomach and the small intestinal submucosal plexus mainly in the relative proportions of the different subpopulations. Regional differences in this respect may be a reflection of functional specialization in the diverse regions of the alimentary canal.
Biliary-hepatic recycling of a xenobiotic: gallbladder absorption of methyl mercury
Authors: Dutczak W. J. , Clarkson T. W. , Ballatori N.
Published: December 2017

The role of the gallbladder in the disposition of methyl mercury was investigated in guinea pig, hamster, and macaque monkey. 203Hg-labeled methyl mercury or inorganic mercury (5 microM) and [14C]inulin were instilled into the in situ guinea pig or hamster gallbladder. After 2 h, only 27.6 +/- 7.0% of the methyl mercury remained in guinea pig gallbladder fluid as compared with 85.0 +/- 3.2% of the inorganic mercury and 90.7 +/- 4.5% of the [14C]-inulin. In the hamster, 42.5 +/- 4.5% of methyl mercury and 95% +/- 0.9% of inorganic mercury remained after 2 h. When the sulfhydryl-containing compounds L-cysteine, glutathione, and bovine serum albumin (20 microM) were added to the test solution, cysteine increased and albumin decreased absorption of methyl mercury. Ligation of guinea pig cystic artery decreased gallbladder fluid absorption from 72.7 +/- 8.6 to 26.5 +/- 9.8% over 2 h but did not alter methyl mercury absorption. Bile was also sampled from gallbladders of four monkeys exposed chronically to CH3HgCl and from three control monkeys. For one of the exposed and one of the control monkeys, bile was also collected from the common hepatic duct. In both methyl mercury-exposed and control monkeys, the concentration of methyl mercury in gallbladder bile was lower than in hepatic bile. In contrast, the concentration of inorganic mercury in gallbladder bile was four to seven times that of hepatic bile, suggesting that methyl mercury but not inorganic mercury was being reabsorbed. To assess the functional significance of methyl mercury reabsorption by the gallbladder, guinea pig cystic ducts were ligated, the animals were given CH3 203HgCl (10 mumol/kg iv), and body burden of 203Hg was measured over 16 days.(ABSTRACT TRUNCATED AT 250 WORDS)
Adrenergic modulation of reactive hyperemia in rat gut
Authors: Pawlik W. W. , Hottenstein O. D. , Jacobson E. D.
Published: December 2017

The hypothesis was tested that peripheral, adrenergic nerves modulate reactive hyperemia (RH) in the intestinal circulation. In anesthetized rats, anterior mesenteric arterial occlusion for 30-120 s elicited subsequent RH responses, including 63-118% increases in the velocity of arterial blood flow, even greater increases in conductance, and durations of 64-139 s. The longer the period of arterial occlusion, the greater the magnitude of RH. Electrical stimulation of postganglionic, sympathetic nerves reduced RH responses in a frequency-dependent manner. RH responses were enhanced by pretreatment with hexamethonium and phenoxybenzamine and were diminished by pretreatment with propranolol. Propranolol also prevented the enhanced RH responses caused by hexamethonium and phenoxybenzamine. Reserpine prevented the enhanced RH responses to hexamethonium, but bilateral adrenalectomy did not. These findings support the hypothesis that peripheral sympathetic nerves modulate RH in rat gut, with alpha-adrenergic receptors restricting and beta-adrenergic receptors enhancing the hyperemia.
Spontaneous variation of anal "resting" pressure in healthy humans
Authors: Enck P. , Eggers E. , Koletzko S. , Erckenbrecht J. F.
Published: December 2017

To investigate anal sphincter performance during sleep and after a meal, a two-channel micro-transducer probe was used for 12-h stationary recording of basal anal pressure overnight in eight healthy male volunteers. It was shown that the basal anal pressure ("resting" pressure) exhibits three distinct patterns of cyclic activity changes in all subjects: a long-term rhythm with a prominent decrease of pressure during which sleep was approximately circadian, an ultradian rhythm of approximately 20 to 40 min in length that was more prominent at night, and spontaneous relaxations of the sphincter tone occurring between 3 and 20 times per hour with the maximum frequency after breakfast. These data indicate that the anal sphincter is a dynamic structure not often at rest. Long-term anorectal manometry may be supplementary to short-term clinical evaluation of anal sphincter performance in healthy subjects as well as in patients with defecation disorders.
Isolation and function of a receptor for human lactoferrin in human fetal intestinal brush-border membranes
Authors: Kawakami H. , Lonnerdal B.
Published: December 2017

Iron absorption is known to be higher from human milk than from infant formula or bovine milk. The high bioavailability of human milk iron suggests that lactoferrin (Lf), the major iron-binding protein in human milk, may be a factor contributing to iron absorption in infants. We have isolated a human Lf receptor from solubilized human fetal intestinal brush-border membranes by affinity chromatography using immobilized human Lf. We also investigated the interaction of 125I-labeled human Lf and bovine Lf with brush-border membrane vesicles (BBMVs) from human small intestine using a rapid filtration technique. The molecular weight of the receptor was 110,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions and 37,000 under reducing conditions. Competitive binding studies demonstrated specific binding of human Lf. The binding was pH dependent, with an optimum between pH 6.5 and 7.5. Scatchard plot analysis indicated 4.3 x 10(14) binding sites/mg membrane protein with an affinity constant of 0.3 x 10(6) M-1 for human Lf. Both half-Lf and deglycosylated Lf bound to the receptor with an affinity similar to intact Lf. In contrast, little binding of bovine Lf or human transferrin to human BBMVs occurred. These results suggest that the brush-border membrane receptor for human Lf may be responsible for the high iron absorption from human milk.
Postoperative course after portacaval anastomosis in rats is determined by the portacaval pressure gradient
Authors: Coy D. L. , Srivastava A. , Gottstein J. , Butterworth R. F. , Blei A. T.
Published: December 2017

Variability in experimental results have led to criticism regarding the validity of the rat after portacaval anastomosis (PCA) as a model of changes induced by portal-systemic shunting (PSS). A nonsuture technique using cyanoacrylate glue has been reported to yield a better experimental preparation. To investigate if variations in splanchnic hemodynamics could explain different outcomes after the procedure, male rats received either an end-to-side PCA or sham operations (16 rats each). The PCA was constructed using the "suture" or "glue" technique (8 rats each). Beginning on postoperative day 24 under methoxyflurane and ketamine anesthesia, pressures were recorded from the portal vein, inferior vena cava, and femoral artery. Blood flow to the splanchnic organs and the percent PSS were assessed using the microsphere technique. The rate of delivery of NH3 from the portal vein to the systemic circulation and the concentration of glutamine in the cerebrospinal fluid were determined. In PCA rats, weight gain was significantly impaired, and all animals had evidence of liver atrophy (in both suture and glue groups) when compared with sham animals; a trend toward greater weight gain was seen in glue rats. Portal vein inflow, PSS, NH3 delivery, and CSF glutamine were significantly increased in both PCA-suture and PCA-glue animals compared with sham rats, although no significant differences were seen between the two PCA techniques. When PCA rats from either technique were grouped according to the pressure gradient between portal vein and inferior vena cava, striking differences between animals were now evident.(ABSTRACT TRUNCATED AT 250 WORDS)
InsP3-dependent Ca2+ mobilization in circular but not longitudinal muscle cells of intestine
Authors: Murthy K. S. , Grider J. R. , Makhlouf G. M.
Published: December 2017

The presence and functional coupling of inositol 1,4,5-trisphosphate (InsP3) receptors was examined in permeabilized muscle cells and microsomes isolated separately from the circular and longitudinal muscle layers of guinea pig intestine. Specific binding of [3H]InsP3 occurred only in circular muscle cells and microsomes; binding to longitudinal muscle cells or microsomes was minimal or absent. Binding to circular muscle cell microsomes was rapid (steady state in 2 min), saturable, reversible, and temperature independent, and was inhibited in a concentration-dependent manner by unlabeled InsP3 (IC50 10 nM) and by the InsP3 receptor antagonist heparin (EC50 4 micrograms/ml); inhibition by InsP4 and InsP2 was minimal. Consistent with the preferential location of InsP3 receptors, InsP3 elicited concentration-dependent contraction (EC50 3 nM), 45Ca2+ efflux (EC50 55 nM), and increase in [Ca2+]i in circular muscle cells and 45Ca2+ efflux in microsomes; all these events were blocked by heparin (10 micrograms/ml). Although ATP-dependent Ca2+ stores were present in longitudinal muscle cells and microsomes, they were not sensitive to 1 microM InsP3, which was maximally effective in circular muscle cells; 100 microM InsP3 elicited partial Ca2+ release (30-50%) and contraction (47%) that could only be blocked by high concentrations of heparin (100 micrograms/ml). We conclude that specific, high-affinity, heparin-sensitive InsP3 receptors capable of mobilizing intracellular Ca2+ are present predominantly in circular muscle cells of the intestine.
Sucrase-isomaltase gene expression along crypt-villus axis of human small intestine is regulated at level of mRNA abundance
Authors: Traber P. G. , Yu L. , Wu G. D. , Judge T. A.
Published: December 2017

The mucosal lining of the small intestine is a complex epithelium that is continually renewed by division of a stem cell population located in intestinal crypts, migration of daughter cells along the villus, and, finally, extrusion of senescent cells into the lumen. The majority of cells in both crypt and villus cell compartments are enterocytes that acquire differentiated functions as they migrate out of the crypt. Sucrase-isomaltase (SI) is an enterocyte-specific, brush-border enzyme that has little activity in crypt cells and maximal activity in low and mid villus cells. The mechanism by which enterocytes acquire SI enzymatic activity as they move from crypt to villus is controversial. In this study we examined the distribution of SI mRNA along the crypt-villus axis of human small intestine using isolated epithelial cells and in situ hybridization. A complementary DNA to the 5' portion of the human SI mRNA was amplified and cloned using the polymerase chain reaction. Hybridization analysis of RNA extracted from human intestinal epithelial cells showed that the cloned cDNA recognized a single 6.5-kb mRNA. In situ hybridization of duodenal biopsy specimens was performed using a single-stranded RNA probe derived from this cDNA. This analysis showed that there was little SI mRNA in crypt cells and appearance of mRNA in enterocytes located at the crypt-villus junction. The mRNA levels were maximal in lower and mid villus cells with decreased levels noted in villus tip cells. These results are identical to those previously described in rat intestine and suggest that expression of the SI gene as enterocytes emerge from intestinal crypts is regulated primarily at the level of mRNA accumulation. Study of SI gene regulation may provide a useful model to investigate the mechanisms that regulate enterocyte-specific gene expression and intestinal differentiation.
Acetylcholine release from colonic submucous neurons associated with chloride secretion in the guinea pig
Authors: Javed N. H. , Cooke H. J.
Published: December 2017

Electrical field stimulation of submucous neurons in the guinea pig distal colon evokes an increase in chloride secretion sensitive to cholinergic blockade. This study was undertaken in the guinea pig to determine the feasibility of measuring acetylcholine (ACh) release simultaneously with ion transport in sheets of colonic submucosa/mucosa set up in flux chambers modified for perfusion of the submucosal surface. Release of [3H]ACh was determined in the absence of cholinesterase inhibitors as the stimulus-evoked outflow of 3H from preparations preloaded with [3H]choline. [3H]ACh released in response to electrical stimulation correlated with short-circuit current at frequencies from 0.5 to 10 Hz. At 5 and 10 Hz, the stimulus-evoked release of [3H]ACh decreased during subsequent stimulation periods. The stimulus-evoked increase in [3H]ACh was attenuated by tetrodotoxin. [3H]ACh release evoked at stimulus frequencies of 0.5-10 Hz was not altered by atropine despite a reduction in short-circuit current. This study illustrates the feasibility of measuring ACh release simultaneously with ion transport in flux chambers. The results provide new information on the response characteristics of colonic submucous neurons and provide direct evidence for regulation of chloride secretion by ACh.
Surface hydrophobicity of the intestinal tract
Authors: Mack D. R. , Neumann A. W. , Policova Z. , Sherman P. M.
Published: December 2017

To quantitate surface hydrophobicity of the intestine, we measured contact angles formed with water droplets in multiple regions of rabbit intestine at varying ages (suckling, weanling, and adult) and after dinitrochlorobenzene-induced colitis. Contact angles were measured using novel methods: axisymmetric drop-shape analysis-contact diameter for contact angles less than 90 degrees and axisymmetric dropshape analysis-maximum diameter for contact angles greater than 90 degrees. To determine whether mucus was present on the surface of intestine used, indirect immunofluorescence was performed using antibody specific to goblet cell mucin. To confirm that intestinal mucus could be responsible for the physical properties of surface mucosa, surface tensions of mucus prepared from distal ileum, distal colon, and inflamed distal colon of adult rabbits were measured by axisymmetric drop-shape analysis on pendant drops. Contact angles of adult small intestine [duodenum, 38.0 +/- 11.2 degrees (SD); jejunum, 44.0 +/- 22.9 degrees; ileum, 56.4 +/- 23.3 degrees] were less than proximal colon (93.2 +/- 6.7 degrees; P less than 0.05) and distal colon (86.4 +/- 24.2 degrees; P less than 0.05). Contact angles on proximal colon from suckling rabbits (53.2 +/- 8.4 degrees) were less than both weanling (93.2 +/- 23.3 degrees; P less than 0.05) and adult rabbits (93.2 +/- 6.7 degrees; P less than 0.05). Contact angles on inflamed adult distal colon (54.7 +/- 20.6 degrees) were decreased from values on normal distal colons (86.4 +/- 24.2 degrees). Indirect immunofluorescence demonstrated that mucin was present in both vacuoles of goblet cells and on the colonic surface.(ABSTRACT TRUNCATED AT 250 WORDS)
Vesicle acidification driven by a millionfold proton gradient: a model for acid influx through gastric cell membranes
Authors: Barreto J. , Lichtenberger L. M.
Published: December 2017

A delta pH 6 proton (internal pH 7.5, external pH 1.5) was imposed across the bilayer of egg phosphatidylcholine (PC) vesicles. The presence of the gradient was verified by the use of agents that predictably collapsed it. These agents included a detergent (Triton X-100), a pore-forming Na-H+ exchanger (nigericin), and weak acids capable of shuttling protons across the membrane (acetic and acetylsalicylic acids). The magnitude of the proton gradient and the rate of pH gradient collapse were determined by measuring pyranine fluorescence by use of an isosbestic point technique and calculating intravesicular pH from a calibration curve. Acid flux into the vesicles in the presence of chloride was measured directly by a simpler one-wavelength method. p-Xylene-bis-pyridinium bromide (DPX), a pyranine fluorescence quencher, was used to verify that the fluorescence signal originated from within the vesicles and that the observed rates of acidification were not an artifact due to pyranine leakage. Acid flux was found to be dependent on the ionic composition of the buffer. The presence of chloride ion in the external compartment caused a dramatic increase in the rate of acidification. It was demonstrated that the kinetics of acid flux into the vesicle were clearly controlled by the chloride-dependent mechanism at a relatively low chloride concentration of 2.5 mM. The model presented here is a simple and sensitive method for investigating the factors that influence acidification rates across membranes when acidification is driven by extremely large proton gradients, in the presence of chloride; conditions similar to those found in the stomach.
Effect of fluid perfusion and cleansing on canine colonic motor activity
Authors: Sarna S. K.
Published: December 2017

We investigated the effect of absorbable and nonabsorbable fluid perfusion and cleansing on colonic motor activity in eight intact conscious dogs. Each dog was instrumented with an indwelling catheter in the proximal colon and seven strain gauge transducers on the entire colon. After an overnight fast, a control recording was made for 3 h, followed by 3 h of perfusion and 3 additional h of postperfusion recording. Next day, a 3-h recording was made when the colon was empty. The colon exhibited normal migrating and nonmigrating motor complexes in the control uncleansed state. The perfusion of absorbable electrolyte or nonabsorbable Colyte solution immediately disrupted the migrating motor complexes and replaced them with almost continuous but irregular contractions at all recording sites. Both solutions significantly prolonged the mean and total duration per hour of contractile states in the proximal, middle, and distal colon. The dogs began to leak fluid stools in squirts approximately 40-80 min after the start of perfusion. This type of incontinence was not associated with any specific type of motor activity. Infrequently, giant migrating contractions occurred during perfusion and caused explosive diarrhea. The migrating motor complexes remained disrupted during the 3-h postperfusion period. However, on the next day, the empty colon exhibited normal migrating motor complexes. The frequency of giant migrating contractions during perfusion and in the empty colon was significantly greater than that in the normal uncleansed colon. The total duration per hour of colonic motor activity in the empty colon was also greater than that in the normal uncleansed colon. We conclude that excessive fluid in the colon significantly alters its motor pattern.(ABSTRACT TRUNCATED AT 250 WORDS)
Distribution of smg p25A and smg p21s, ras p21-like guanine nucleotide-binding proteins, in the rat stomach
Authors: Matsuda K. , Sakamoto C. , Nakano O. , Konda Y. , Matozaki T. , Wada K. , Kasuga M. , Mizoguchi A. , Kikuchi A. , Takai Y.
Published: December 2017

Existence and distribution of small guanine nucleotide binding proteins (G proteins) such as smg p25A, smg p21s, and ras p21s were examined in the rat gastric mucosa. By immunoblot analysis using the specific monoclonal antibodies against smg p25A and ras p21 and the anti-smg p21 antiserum, smg p25A and smg p21s were detected in the gastric mucosa, while ras p21s were not detected. In immunocytochemical studies, moderate fluorescence of smg p25A was homogenously seen in the cytoplasmic portions of chief cells and surface epithelial cells. smg p21 immunoreactivity was detected in glandular cells and submucosal muscle layer in the mucosa. On the other hand, the cells constituting gastric glands were unstained with the anti-ras p21 monoclonal antibody, RASK-4. These results suggest that smg p25A and smg p21s exist as in other tissues and might exert their specific actions in the rat gastric mucosa.
Measurement of hepatic perfusion in rats by laser Doppler flowmetry
Authors: Almond N. E. , Wheatley A. M.
Published: December 2017

Little is known about the performance of the laser Doppler flowmeter (LDF) with changes in flow characteristics in the hepatic microcirculation. Red blood cell (RBC) flux, as measured by LDF, is sensitive to alterations in RBC velocity and RBC concentration, and both parameters must be considered when evaluating the technique. In vitro, linearity of LDF signal with RBC velocity up to 4 mm/s (r greater than 0.99) and RBC volume fractions up to 1.5% were demonstrated (r greater than 0.98). At higher velocities and volume fractions, RBC flux was underestimated. In portally perfused rat liver, LDF output was linearly related to total liver blood flow (TLBF; r greater than 0.9) for perfusate hematocrits between 2.5 and 40%, although the slope varied between preparations. At constant TLBF (2 ml.min-1.g-1), the LDF output changed linearly with perfusate hematocrit up to 20% (r greater than 0.99) but underestimated the increase at 40%. These results suggest that the LDF responds linearly to velocity-mediated flow changes but that it may underestimate a change if mediated through alteration in tissue RBC concentration. With hepatic nerve stimulation, TLBF fell to 51 +/- 14% of prestimulation (P less than 0.001), whereas the LDF output and superficial flow measured by 85Kr clearance fell to 12 +/- 10 and 14 +/- 10% (both P less than 0.01 vs. TLBF), respectively, suggesting that the LDF may be used to follow rapid flow changes in the periphery of the liver.
Isolation of human colonic crypts for study of xenobiotics metabolism
Authors: Schwenk M. , Dechelotte P. , Riemenschneider T.
Published: December 2017

Methods allowing study of human colonic drug and carcinogen metabolism in intact epithelial preparations in vitro are needed. We describe an isolation method yielding intact colonic crypts with well-maintained ultrastructural integrity and good viability parameters. The crypt preparations obtained with this method were used to study phase I and II drug biotransformation. The colonic crypts demethylated aminopyrine and nitrosodimethylamine slowly, but the colonic carcinogen dimethylhydrazine at a higher rate. The phenolic compounds estrone, 17 beta-estradiol, and 1-naphthol were readily conjugated. In conclusion, the present method yields preparations well suited for further investigation of the role of human colonic biotransformation in carcinogenesis and drug pharmacokinetics.
A large conductance K(+)-selective channel of guinea pig villus enterocytes is Ca2+ independent
Authors: Mintenig G. M. , Monaghan A. S. , Sepulveda F. V.
Published: December 2017

The presence of K(+)-selective channels has been probed in enterocytes isolated from guinea pig small intestinal villi by the patch-clamp technique. A channel with a single-channel conductance of approximately 130 pS was observed in excised inside-out patches bathed in symmetrical K+. A change in the K+ concentration in the intracellular aspect of the membrane altered the current-voltage relationship as expected from the constant-field equation when it is assumed that K+ is the only permeant ion. A change in Cl- concentration was without effect. Neither the activity of the channel nor its conductance was altered by addition of ATP or Ba2+ to the intracellular side of the patches. Changes in the free Ca2+ concentration were also without effect. The channel's open probability showed no voltage dependence and appeared only occasionally active in cell-attached patches where it had a linear current-voltage relation. The K+ channel described, which cannot be readily classified in any of the known classes of K+ channels, might provide an exit pathway for K+ recycling in guinea pig villus enterocytes.
Prostaglandins and modulation of small bowel myoelectric activity
Authors: Frantzides C. T. , Lianos E. A. , Wittmann D. , Greenwood B. , Edmiston C. E.
Published: December 2017

We explored the effects of prostaglandins (PG) F2 alpha and E2 on the motor and myoelectric activity of the small intestine using closed intra-arterial injections in conscious chronically instrumented dogs. PGF2 alpha (0.125-5 micrograms) and PGE2 (1-10 micrograms) were injected via a T tube into a branch of the superior mesenteric artery perfusing a 15-cm segment of jejunum. Experiments were performed on four dogs in which the recording devices had been implanted above, below, and within the perfused segment. PGF2 alpha given during phase I of the migrating myoelectric complex cycle induced phasic contractions in the perfused segment of intestine in a dose-dependent manner. Atropine (50-100 micrograms), hexamethonium (15 mg), or TTX (10-15 micrograms) administered before the injection of PGF2 alpha failed to inhibit the effects of PGF2 alpha. In contrast pretreatment of the perfused segment with verapamil (2.5 mg) or PGE2 (1-5 micrograms) abolished the effects of PGF2 alpha. Moreover, PGE2 injected 5 min after the administration of PGF2 alpha inhibited the PGF2 alpha-induced contractions. Administration of PGE2 alone (3-10 micrograms) before the arrival of phase III activity in the perfused segment abolished phase III from this segment of intestine. Our studies indicate opposing effects of PGF2 alpha and PGE2 on small intestinal myoelectric and contractile activities. PGF2 alpha has a direct excitatory effect on the intestinal smooth muscle, which is calcium channel dependent but independent of intrinsic nerves. PGE2 has an inhibitory effect both on the spontaneous and PGF2 alpha-induced small intestinal myoelectric and contractile activity.
Distinction between chloride-dependent transport systems for taurine and beta-alanine in rabbit ileum
Authors: Munck L. K. , Munck B. G.
Published: December 2017

This study describes the influx of taurine and beta-alanine across the brush-border membrane of rabbit distal ileum. The kinetics of JmcTau [concentration at which half-maximal activation occurs (K1/2) = 41 microM and Jmax = 24 nmol.cm-2.h-1] are consistent with the kinetics of taurine uptake by jejunal brush-border vesicles. The taurine carrier differs from the beta-alanine carrier by being insensitive to leucine inhibition and by the jejunoileal variation of influx along the small intestine. The K1/2 for sodium and chloride activation of the beta-alanine carrier (48 and 8 mM, respectively) differ markedly from the values reported for the taurine carrier. In addition, taurine is not transported by the beta-alanine carrier. Thus the study demonstrates that the taurine and beta-alanine carriers are separate entities, and it adds to the imino and the taurine carriers the beta-alanine carrier as a third chloride-dependent intestinal transporter of amino acids.
Alpha 1-adrenergic and cholinergic agonists use separate signal transduction pathways in lacrimal gland
Authors: Hodges R. R. , Dicker D. M. , Rose P. E. , Dartt D. A.
Published: December 2017

The cellular transduction pathways used by alpha 1-adrenergic and cholinergic agonists were compared in isolated acini from rat exorbital lacrimal glands. Peroxidase secretion was the index of protein secretion. Inositol phosphates were measured by anion exchange chromatography, intracellular free Ca2+ concentration ([Ca2+]i) by fluorescence methods using fura-2, cellular adenosine 3',5'-cyclic monophosphate (cAMP) levels by protein binding radioassay, and protein kinase C (PKC) activity by [32P]ATP incorporation into exogenous substrate. Protein secretion stimulated by simultaneous addition of the alpha 1-adrenergic agonist phenylephrine and the cholinergic agonist carbachol was additive. Carbachol (10(-3) M) significantly increased the ratios of inositol phosphates to inositol during a 1- or 20-min incubation in contrast to phenylephrine (10(-5) to 10(-2) M), which did not. Phenylephrine (10(-3) M) significantly increased the [Ca2+]i by a maximum of 15 +/- 3 nM compared with carbachol (10(-4) M), which increased [Ca2+]i to a maximum of 90 +/- 14 nM. Phenylephrine (10(-4) M) did not increase cAMP levels. Phenylephrine (10(-5) to 10(-3) M) decreased cytosolic PKC activity in a concentration-dependent manner. Carbachol (10(-3) M) transiently caused a slight decrease in cytosolic PKC activity. Our results indicate that alpha 1-adrenergic and cholinergic agonists use separate and different pathways to stimulate the lacrimal gland.
Dietary regulation of pancreatic amylase in transgenic mice mediated by a 126-base pair DNA fragment
Authors: Schmid R. M. , Meisler M. H.
Published: December 2017

Expression of the mouse pancreatic amylase gene Amy-2.2 is increased approximately 10-fold in response to increasing the carbohydrate content of the diet from 9.6 to 74%. The DNA sequence mediating this response has been localized to the 5' flanking region of the amylase gene by analysis of hybrid constructs in transgenic mice. The results define a 127-base pair dietary response unit that includes two previously described regulatory elements, an insulin-responsive element and a pancreatic enhancer. Fragments containing these two elements alone fail to respond to diet, demonstrating a requirement for additional regulatory sequences. Another mouse amylase gene Amy-2.1 is only minimally responsive to insulin and to diet. The data are consistent with the hypothesis that the insulin-response element is necessary but not sufficient for regulation of amylase by dietary carbohydrate.
Capsaicin-induced hyperemia in the stomach: possible contribution of mast cells
Authors: Wallace J. L. , McKnight G. W. , Befus A. D.
Published: December 2017

Topical application of capsaicin to the gastric mucosa results in marked hyperemia as a consequence of the release of vasoactive neuropeptides from sensory afferent neurons. Because many of these neuropeptides have the capacity to induce mast cell degranulation, we investigated the possible contribution of mast cells to capsaicin-induced hyperemia. Application of capsaicin to the gastric mucosa of normal rats resulted in a concentration-dependent increase in blood flow. In rats in which mastocytosis was induced by prior infection with Nippostrongylus brasiliensis, the hyperemic responses to capsaicin were significantly greater than in control rats. This augmented hyperemic response could be significantly attenuated by pretreatment with a histamine H1-receptor antagonist (pyrilamine) or with a mast cell stabilizer (doxantrazole). Depletion of mucosal mast cells through treatment with dexamethasone also significantly reduced the hyperemic response to capsaicin. Hyperemic response to capsaicin in normal rats and in rats with mucosal mastocytosis could be completely abolished by pretreatment with ruthenium red or prior ablation of the sensory afferent neurons with capsaicin. These results suggest that in rats with gastric mastocytosis, sensory neuron-dependent activation of mast cells contributes to the hyperemic response to topical capsaicin. These findings are therefore consistent with the hypothesis that there is communication between nerves and mast cells in the gastric mucosa, at least in rats previously infected with N. brasiliensis.
Influence of luminal nutrient composition on hemodynamics and oxygenation in developing intestine
Authors: Crissinger K. D. , Burney D. L.
Published: December 2017

Age-related differences in the intestinal hemodynamic and oxygenation responses to carbohydrate, protein, and lipid were studied in 1-day-, 3-day-, 2-wk-, and 1-mo-old piglets. A branch of the mesenteric vein draining an isolated loop of jejunoileum was used to measure intestinal blood flow, arteriovenous oxygen content difference, and venous and capillary pressure and to calculate oxygen uptake and vascular resistance. Fractionated intestinal flow was measured with radiolabeled microspheres. Measurements were made before and after luminal placement of either 5% glucose, 2.3% casec, or 5% corn oil. In 1-day-old animals, unlike all older age groups, total intestinal blood flow and vascular resistance were unchanged by any nutrient. Fractionated flow to the mucosa/submucosa levels did, however, increase in the intestine of 1-day-old piglets to a similar extent as that in older age groups. Placement of lipid or protein into the lumen led to increased oxygen uptake in all age groups, whereas carbohydrate absorption resulted in no increase in intestinal oxygen consumption in 1- and 3-day-old animals. In 1-day-olds, the increased oxygen consumption was achieved by enhanced oxygen extraction with no change in total blood flow, whereas all other groups demonstrated increases in blood flow and/or oxygen extraction. Compared with a mixed meal, oxygen consumption was not significantly greater for an individual nutrient component.
Characterization of opioid receptors in intestinal muscle cells by selective radioligands and receptor protection
Authors: Kuemmerle J. F. , Makhlouf G. M.
Published: December 2017

Opioid receptors were characterized on muscle cells isolated separately from the circular and longitudinal muscle layers of rabbit intestine. Selective radioligands for kappa- ([3H]U69,593), delta- ([3H][D-Pen2,5]enkephalin, DPDPE), and mu- ([3H][D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin, DAGO) opioid receptors were used in conjunction with a technique of receptor protection designed to enrich cells with a specific receptor type. Binding was observed only in cells from the circular muscle layer. Binding was rapid (peak within 2 min), temperature dependent, and concentration dependent. Dissociation constants (Kd) for high-affinity binding sites derived from saturation curves (1.1 +/- 0.3 nM for U69,593, 0.39 +/- 0.04 nM for DPDPE, and 1.9 +/- 0.3 nM for DAGO) were similar to Kd values derived from competition curves. In competition studies, the order of potency with which opioid ligands inhibited binding depended on the radioligand used: U69,593 (Kd 1.5 +/- 0.2 nM) inhibited preferentially the binding of [3H]U69,593, DPDPE (Kd 0.72 +/- 0.16 nM) the binding of [3H]DPDPE and DAGO (Kd 1.2 +/- 0.3 nM) the binding of [3H]DAGO. In each instance the other two ligands were 400-12,000 times less potent. In cells enriched with one receptor type, binding and contraction were observed only with the corresponding selective ligand. The potency of the ligand was slightly enhanced, whereas the potencies of the other two ligands were further reduced (greater than 10,000-fold). We conclude that distinct kappa-, delta-, and mu-opioid receptors are present on muscle cells of the circular but not longitudinal muscle layer of the intestine.
Carrier-mediated transport of tetrabromosulfonephthalein by rat liver plasma membrane vesicles
Authors: Torres A. M. , Rodriguez J. V. , Lunazzi G. C. , Tiribelli C.
Published: December 2017

To investigate the molecular requirements and mechanisms for the hepatic uptake of phthaleins, the transport of tetrabromosulfonephthalein (TBS) was investigated in basolateral rat liver plasma membrane vesicles. TBS uptake was electrogenic as greatly accelerated by the creation of a positive-inside membrane potential by the addition of valinomycin in the presence of an inwardly directed potassium gradient. No effect was observed when the ionophore was added in the presence of a sodium gradient. The transport occurred into an osmotic-sensitive space and was saturable with an apparent Michaelis constant of 5.32 +/- 0.56 microM and a maximal velocity of 9.23 +/- 0.25 nmol.s-1.mg protein-1 (mean +/- SD, n = 3 experiments). TBS uptake was directly related to the extra-vesicular pH, indicating the deprotonated quinoid negative-charged form of the dye as the transported species. In contrast, TBS uptake was inversely related to the intravesicular pH, suggesting that protonation inside the vesicles may act as an efficient trap in transport process. Addition of polyclonal monospecific anti-bilitranslocase antibody to liver vesicles specifically inhibited TBS uptake rate (3.27 +/- 0.17 vs. 5.82 +/- 0.61 nmol.s-1.mg protein-1, n = 3, P less than 0.001). These data indicate that TBS is electrogenically transported across the liver cell plasma membrane by bilitranslocase. They also indicate that the presence of a negative charged group on the benzenic ring of the ligand is important in accounting for the transport.