bluuebunny/crossref_metadata_2025 huggingface dataset:
Monoclonal antibody localization of Na+-K+-ATPase in the exocrine pancreas and parotid of the dog
Authors: Smith Z. D. , Caplan M. J. , Forbush B. , Jamieson J. D.
Published: December 2017

A monoclonal antibody specific to the beta-subunit of the canine Na+-K+-ATPase has been characterized and used to directly localize the enzyme in thin frozen sections of dog pancreas and parotid. The antibody, 7-2M, recognizes only the beta-subunit of the sodium pump as determined by immunoprecipitation and immunoblot and is not directed against an oligosaccharide determinant. 7-2M immunolocalizes to the same cellular and subcellular domains of renal tubular cells as do other, previously characterized, antibodies directed to the alpha-subunit of the sodium pump. In the pancreas the preponderance of the Na+-K+-ATPase is found on the basolateral membranes of centroacinar and intralobular duct cells. Interlobular duct cells also express a large component of basolaterally located enzyme, although comparatively little pump is seen on acinar cells. In the parotid a large amount of Na+-K+-ATPase is seen on the striated duct cells, with high levels also noted on cells of the intercalated ducts and serous demilunes. Again the acinar cells show comparatively low levels of Na+-K+-ATPase. In no instance is Na+-K+-ATPase found on the apical membranes of pancreas or parotid cells. These data suggest that Na+-K+-ATPase, located on the basolateral plasmalemma of duct-derived cells, may be involved in water and electrolyte secretion from the pancreas and parotid.
Hormonal effects on development of the secretory apparatus of chief cells
Authors: Tseng C. C. , Schmidt K. L. , Johnson L. R.
Published: December 2017

We used light microscopy, transmission electron microscopy, and stereological point-counting methods to investigate the response of rat gastric chief cells to corticosterone and thyroxine during postnatal development. Administration of corticosterone or thyroxine to normal animals in the first two postnatal weeks increased relative numbers of chief cells in the fundic mucosa but did not change the absolute number of total cells in each gland. The chief cells displayed more rough endoplasmic reticulum, zymogen granules, and Golgi saccules in corticosterone- or thyroxine-injected rats than in normal control rats. When rats were either adrenalectomized or made hypothyroid using propylthiouracil, the number of chief cells dramatically decreased. Ultrastructural studies of chief cells from adrenalectomized or hypothyroid rats showed retarded differentiation as indicated by increased free ribosomes, less rough endoplasmic reticulum, fewer zymogen granules, and poorly developed Golgi apparatuses. In adrenalectomized animals, thyroxine replacement alone failed to induce the histological maturation of chief cells, but addition of corticosterone stimulated chief cell differentiation. In hypothyroid animals, either thyroxine or corticosterone replacement restored the differentiation of chief cells to normal levels. Our data indicate that 1) corticosterone is necessary for gastric chief cell maturation during postnatal development, 2) a decrease or loss in either corticosterone and/or thyroxine results in retarded chief cell differentiation and functional activity, and 3) the effect of thyroxine on chief cell development is secondary to an accompanying increase in serum corticosterone.
Inhibitory action of peptide YY on gastric acid secretion
Authors: Guo Y. S. , Fujimura M. , Lluis F. , Tsong Y. , Greeley G. H. , Thompson J. C.
Published: December 2017

The purpose of this study is to investigate the effect of peptide YY (PYY) on pentagastrin-, histamine-, and bethanechol-stimulated gastric acid secretion and the possible mechanisms by which PYY inhibits gastric acid secretion. Six mongrel dogs with chronic gastric and duodenal fistulas were given an intravenous infusion of pentagastrin (0.5 microgram . kg-1 . h-1), histamine (18 micrograms . kg-1 . h-1), or bethanechol (80 micrograms . kg-1 . h-1) either alone or simultaneously with intravenous PYY (100, 200, 400, pmol . kg-1 . h-1). PYY (100, 200, 400 pmol . kg-1 . h-1) inhibited pentagastrin-stimulated gastric acid secretion in a dose-dependent manner. PYY (400 pmol . kg-1 . h-1) did not depress bethanechol-stimulated gastric acid secretion. PYY (400 pmol . kg-1 . h-1) also failed to inhibit histamine-stimulated gastric acid secretion. Furthermore, PYY inhibit pentagastrin-stimulated gastric acid secretion in the face of atropine, vagotomy, or indomethacin treatment. These findings indicate that the inhibitory action of PYY on gastric acid secretion is in part independent of long and short cholinergic pathways. These findings also indicate that the inhibitory mechanism of PYY is independent of prostaglandin synthesis. Our findings are discussed in relation to previous reports regarding the effects of PYY on gastric acid secretion.
Coupling of guanine nucleotide inhibitory protein to somatostatin receptors on pancreatic acinar membranes
Authors: Sakamoto C. , Matozaki T. , Nagao M. , Baba S.
Published: December 2017

Guanine nucleotides and pertussis toxin were used to investigate whether somatostatin receptors interact with the guanine nucleotide inhibitory protein (Ni) on pancreatic acinar membranes in the rat. Guanine nucleotides reduced 125I-[Tyr1]somatostatin binding to acinar membranes up to 80%, with rank order of potency being 5'-guanylyl imidodiphosphate [Gpp(NH)p] greater than GTP greater than GDP greater than GMP. Scatchard analysis revealed that the decrease in somatostatin binding caused by Gpp(NH)p was due to the decrease in the maximum binding capacity without a significant change in the binding affinity. The inhibitory effect of Gpp(NH)p was partially abolished in the absence of Mg2+. When pancreatic acini were treated with 1 microgram/ml pertussis toxin for 4 h, subsequent 125I-[Tyr1]somatostatin binding to acinar membranes was reduced. Gpp(NH)p further decreased somatostatin binding to islet-activating protein (IAP)-treated acinar membranes. Pertussis toxin treatment also abolished the inhibitory effect of somatostatin on vasoactive intestinal peptide-stimulated increase in cellular content of adenosine 3',5'-cyclic monophosphate (cAMP) in the acini. Furthermore, exposure of acini to IAP caused ADP ribosylation of a membrane protein with Mr = 41,000 in parallel to the inhibition of cAMP accumulation in acini. The present results suggest, therefore, that 1) somatostatin probably functions in the pancreas to regulate adenylate cyclase enzyme system via Ni, 2) the extent of modification of Ni is correlated with the ability of somatostatin to inhibit cAMP accumulation in acini, and 3) guanine nucleotides also inhibit somatostatin binding to its receptor.
Responses of feline gastroesophageal junction to changes in abdominal pressure
Authors: Boyle J. T. , Altschuler S. M. , Nixon T. E. , Pack A. I. , Cohen S.
Published: December 2017

The responses of the lower esophageal sphincter (LES), stomach, and diaphragm and their contribution to changes in the high-pressure zone (HPZ) at the gastroesophageal junction were determined during extrinsic abdominal compression or intragastric balloon distension in anesthetized cats. Abdominal compression consistently induced an increase in intraluminal end-expiratory LES and gastric pressure (P less than 0.01). Changes in LES pressure significantly exceeded the changes in gastric pressure (P less than 0.01). In contrast, the LES response during gastric distension was variable in the group of animals despite a consistent volume-dependent increase in gastric pressure. Mean LES pressure for the group was unchanged, although 33% of individual animals exhibited a decrease in LES pressure during gastric distension. Both abdominal stimuli induced sustained inhibition of crural (P less than 0.01), but not costal, diaphragmatic electromyographic activity. Vagotomy affected the LES but not the gastric or diaphragmatic responses to both stimuli. In the group of animals, the combined effect of the changes in the three measured variables on the HPZ resulted in maintenance of the antireflux barrier during abdominal compression but a significant decrease in the barrier during gastric distension.
Muscarinic receptor subtypes mediating the mucosal response to neural stimulation of guinea pig ileum
Authors: Carey H. V. , Tien X. Y. , Wallace L. J. , Cooke H. J.
Published: December 2017

Muscarinic receptors involved in the secretory response evoked by electrical stimulation of submucosal neurons were investigated in muscle-stripped flat sheets of guinea pig ileum set up in flux chambers. Neural stimulation produced a biphasic increase in short-circuit current due to active chloride secretion. Atropine and 4-diphenylacetoxy-N-methylpiperadine methiodide (4-DAMP) (10(-7) M) were more potent inhibitors of the cholinergic phase of the response than was pirenzepine. Dose-dependent increases in base-line short-circuit current were evoked by carbachol and bethanechol; 4-hydroxy-2-butynyl trimethylammonium chloride (McN A343) produced a much smaller effect. Tetrodotoxin abolished the effects of McN A343 but did not alter the responses of carbachol and bethanechol. McN A343 significantly reduced the cholinergic phase of the neurally evoked response and caused a rightward shift of the carbachol dose-response curve. All muscarinic compounds inhibited [3H]quinuclidinyl benzilate binding to membranes from mucosal scrapings, with a rank order of potency of 4-DAMP greater than pirenzepine greater than McN A343 greater than carbachol greater than bethanechol. These results suggest that acetylcholine released from submucosal neurons mediates chloride secretion by interacting with muscarinic cholinergic receptors that display a high binding affinity for 4-DAMP. Activation of neural muscarinic receptors makes a relatively small contribution to the overall secretory response.
Distribution of beta-adrenoceptors associated with cAMP-generating system in cat colon
Authors: Taniyama K. , Kuno T. , Tanaka C.
Published: December 2017

The localization of beta-adrenoceptor associated with the adenosine 3',5'-cyclic monophosphate (cAMP)-generating system was determined by an assessment of the smooth muscle cell contractility and by assay of cAMP in individual layers of the cat colon. Isoproterenol inhibited the acetylcholine (ACh)-induced contractions of isolated longitudinal and circular muscle cells in a dose-dependent manner. The response of the longitudinal muscle cells to isoproterenol was double that seen in the circular muscle cells, and this inhibition was antagonized by sotalol. The basal content of cAMP was 3.42 pmol/mg protein in longitudinal muscle layers, 3.09 pmol/mg protein in circular muscle layers, and 2.75 pmol/mg protein in Auerbach's plexus. Isoproterenol produced a dose-dependent elevation of cAMP levels in longitudinal and circular muscle layers, which were antagonized by sotalol and potentiated by theophylline. By contrast, isoproterenol had no effect on cAMP in Auerbach's plexus. Phenylephrine did not alter the cAMP levels in any layer. Therefore, beta-adrenoceptors associated with the cAMP-generating system probably exist in the longitudinal and circular muscles of the cat colon.
Fructose prevents hypoxic cell death in liver
Authors: Anundi I. , King J. , Owen D. A. , Schneider H. , Lemasters J. J. , Thurman R. G.
Published: December 2017

Perfusion of livers from fasted rats with nitrogen-saturated buffer caused hepatocellular damage within 30 min. Lactate dehydrogenase (LDH) was released at maximal rates of approximately 300 U . g-1 . h-1 under these conditions, and virtually all cells in periportal and pericentral regions of the liver lobule were stained with trypan blue. Infusion of glucose, xylitol, sorbitol, or mannitol (20 mM) did not appreciably change the time course or extent of damage due to perfusion with nitrogen-saturated perfusate. However, fructose (20 mM) completely prevented damage assessed by LDH release, trypan blue uptake, and ultrastructural changes for at least 2 h of perfusion. Neither glucose, xylitol, sorbitol, nor mannitol (20 mM) increased lactate formation above basal levels during hypoxia. On the other hand, fructose (0.4-20 mM) caused a concentration-dependent increase in lactate formation that reached maximal rates of approximately 180 mumol . g-1 . h-1. The dose-dependent increase in glycolytic lactate production from fructose correlated well with cellular protection reflected by decreases in LDH release. ATP:ADP ratios were also increased from 0.4 to 1.8 in a dose-dependent manner by fructose. The results indicate that fructose protects the liver against hypoxic cell death by the glycolytic production of ATP in the absence of oxygen.
Evidence for carrier-mediated Cl-SO4 exchange in rabbit ileal basolateral membrane vesicles
Authors: Schron C. M. , Knickelbein R. G. , Aronson P. S. , Dobbins J. W.
Published: December 2017

In rabbit ileal basolateral membrane (BLM) vesicles, an outwardly directed Cl gradient ([Cl] in/out = 60/6 mM) stimulated the initial velocity of SO4 uptake compared with uptake in the absence of Cl. Under Cl gradient conditions, SO4 was transiently accumulated at a concentration twice that found at equilibrium ("overshoot"). Chloride gradient-stimulated SO4 uptake was markedly reduced by inhibitors of anion exchange (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid) and was saturable (SO4 Km = 0.302 +/- 0.064 mM; Vmax = 1.59 +/- 0.22 nmol SO4 . mg protein-1 . min-1). SO4 uptake by BLM vesicles was not stimulated by imposition of an inside-positive electrical potential, suggesting that the stimulation by a Cl gradient was not due to an induced electrical potential. Oxalate, nitrate, iodide, and bromide inhibited the initial velocity of Cl gradient-stimulated SO4 uptake, whereas phosphate, beta-hydroxybutyrate, lactate, and p-aminohippurate had no effect. When SO4 uptake by BLM vesicles was compared with that of brush-border membrane vesicles, Cl gradient-stimulated SO4 uptake was found predominantly in the BLM preparation. In conclusion, these findings provide evidence for a carrier on the ileal basolateral membrane that mediates Cl-SO4 exchange.
Tissue distribution and vitamin D dependence of IMCAL in the rat
Authors: Kowarski S. , Cowen L. A. , Takahashi M. T. , Schachter D.
Published: December 2017

Integral membrane calcium-binding protein (IMCAL) is a vitamin D-dependent integral membrane protein that binds calcium with relatively high affinity (J. Biol. Chem. 225: 10834-10840, 1980). Specific immunoassays for IMCAL utilizing rabbit polyclonal and mouse monoclonal antibodies were developed and applied to studies of its tissue distribution and regulation by vitamin D3 and dietary calcium in the rat. The results indicate that vitamin D-dependent, cross-reactive protein is present in small intestinal mucosa, cecal mucosa, bone, kidney, brain, testis, heart, lung, spleen, and skin. Rats maintained on a low- (0.02%) compared with & high- (2.0%) calcium diet had significantly higher content of IMCAL in duodenal mucosa, cecal mucosa, bone, kidney, brain, testis, and heart. Treatment of rats on the high-calcium diet with 1,25-dihydroxyvitamin D3 increased the IMCAL content of the duodenal mucosa, cecal mucosa, and kidney. The widespread tissue distribution of vitamin D-dependent IMCAL, its close correlation in intestinal mucosa with the calcium transport mechanism, and its occurrence in isolated preparations of enterocyte plasma membranes (microvillus and basolateral membranes) suggest that the protein is involved in the regulation of calcium flux in a number of cell types.
Vasoactive intestinal peptide stimulates protein phosphorylation in a colonic epithelial cell line
Authors: Cohn J. A.
Published: December 2017

The T84 colonic epithelial cell line was used to examine protein phosphorylation during neurohumoral stimulation of ion transport. T84 cell monolayers grown on collagen-coated filters were mounted in Ussing chambers to measure ion transport stimulated by vasoactive intestinal peptide. Maximal stimulation of active secretion occurred after 8-10 min of stimulation. Protein phosphorylation events accompanying stimulated secretion were detected using two-dimensional gel electrophoresis to resolve phosphoproteins from monolayers previously labeled using 32Pi. Within 8 min of exposure to vasoactive intestinal peptide, several phosphorylation events were detected, including a two- to fivefold increase in 32P incorporation into four soluble proteins with apparent molecular weights of 17,000, 18,000, 23,000, and 37,000. The same phosphorylation response occurs in monolayers stimulated by dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP), suggesting that cAMP mediates these intracellular events. This study indicates that changes in protein phosphorylation accompany the secretory action of vasoactive intestinal peptide and suggests that T84 cells offer a useful model for studying the possibility that such phosphorylation events regulate enterocyte ion transport.
Effects of serotonin on Na+-H+ exchange and intracellular calcium in isolated chicken enterocytes
Authors: Hirose R. , Chang E. B.
Published: December 2017

The effects of serotonin or 5-hydroxytryptamine (5-HT) on Na absorption and intracellular free Ca (Cai) in isolated chicken enterocytes was examined. The rate of initial 22Na uptake was inhibited by 50% after 90 s of stimulation with 5-HT (10(-5) M), an effect that was not additive with amiloride (10(-3) M) and was transient (less than 10 min). 5-HT similarly decreased intracellular pH (pHi) in cells loaded with the pH indicator carboxyfluorescein, an effect that was also transient, not additive with amiloride, and Na dependent. The ED50 of this effect was approximately 10(-8) M. 5-HT also stimulated a transient increase in Cai as determined by quin2 fluorescence. A maximal increase of 60 nM occurred significantly before the peak change in pHi, but the total duration of response was similar in each case. In the absence of extracellular Ca, the 5-HT effects on pHi and Cai still persisted. In cells loaded with the Ca-buffering agent MAPTAM, the 5-HT (10(-5) M) inhibitory effect of 22Na influx was partially inhibited. We conclude that 5-HT directly inhibits Na absorption by isolated enterocytes by releasing endogenous Ca, which subsequently causes an inhibition of amiloride-sensitive Na+-H+ exchange.
Developmental regulation of rat intrinsic factor mRNA
Authors: Dieckgraefe B. K. , Seetharam B. , Alpers D. H.
Published: December 2017

Using a full-length rat intrinsic factor (IF) cDNA clone, we have examined gene expression of IF in selected tissues from the adult rat and during postnatal development in the rat stomach. IF mRNA was expressed only in the rat stomach, and in situ hybridization revealed that the expression was limited to the chief cells at the base of the oxyntic glands. IF mRNA concentration in the stomach increased with postnatal age and reached a peak at the 30th day. Postnatally the amount of IF mRNA hybridized increased in both individual cells and in total number of cells. Administration of cortisol to, or adrenalectomy of, rats between 5 and 12 days of age resulted in increase or decrease of IF mRNA levels, respectively. Other hormones tested (pentagastrin, thyroxine, and triiodothyronine) had no significant effect. During the suckling period, IF bound well to the ileal receptor, despite lower than adult levels of activity and different glycosylation, as assessed by binding to concanavalin A-Sepharose beads. These results show that developmental expression of IF activity in rat is closely related to the expression of its mRNA and that corticosteroids play an important role in such an expression.
Structure and organization of electrical activity of canine distal colon
Authors: Ward S. M. , Keller R. G. , Sanders K. M.
Published: December 2017

The morphology and electrophysiology of the canine distal colon were studied to compare this region with the proximal colon. Many morphological characteristics were similar including the presence of interstitial cells at the submucosal surface of the circular layer. Muscle cells near the submucosal surface had resting membrane potentials (RMPs) of -79 +/- 1 mV, and slow waves were generated in this region. Slow waves had similar waveform characteristics to those of the proximal colon, but rapid oscillations were superimposed on slow waves of some preparations. RMPs and slow waves decreased with distance from the submucosal surface. The latter were not resolvable in the myenteric half of the circular layer. Cells at the myenteric border had RMPs of -49.5 +/- 2 mV and a higher frequency oscillation of 16 min-1. Acetylcholine increased slow-wave amplitude and duration and caused fast oscillations on the plateau phase of slow waves. Isolated circular myocytes were studied with the patch-clamp technique. Cells from the submucosal border displayed voltage-dependent inward and outward currents. With outward currents blocked, the inward current was composed of two components. Nifedipine (10(-6) M) blocked a portion of the inward current but left a substantial transient component. The effect of nifedipine correlated with its effects on tissues, suggesting that two components of Ca2+ current participate in slow waves. These studies describe numerous similarities in the structure and activity of the proximal and distal portions of the colon but also show some potentially important differences between these regions.
Involvement of nitric oxide synthetic pathway in inhibitory junction potentials in canine proximal colon
Authors: Dalziel H. H. , Thornbury K. D. , Ward S. M. , Sanders K. M.
Published: December 2017

Experiments were performed to determine the involvement of nitric oxide synthase in the generation of nonadrenergic, noncholinergic (NANC) inhibitory junction potentials (IJPs) in the canine proximal colon. Smooth muscle cells were impaled near the myenteric border between the circular and longitudinal layers. Cells exhibited rhythmic myenteric potential oscillations. IJPs were evoked with electrical field stimulation in the presence of drugs to block adrenergic and cholinergic neurotransmission. N omega-nitro-L-arginine, methyl ester (L-NAME), a nitric oxide synthase inhibitor, progressively reduced the amplitude of IJPs. The effect of L-NAME was reversed by L-Arg but not by the stereoisomer D-Arg. IJPs disrupt the regular pattern of myenteric potential oscillations. This effect was also blocked by L-NAME and reversed by L-Arg. These experiments suggest that a product of the nitric oxide synthetic pathway is involved in NANC neurotransmission in the canine proximal colon.
Effects of calcium-modifying agents on integrity of rabbit isolated gastric mucosal cells
Authors: Tepperman B. L. , Tan S. Y. , Whittle B. J.
Published: December 2017

The effects of Ca(2+)-modifying agents on the disruption of gastric isolated mucosal cells from the rabbit have been examined. Fundic mucosal cells were isolated and enriched by centrifugal elutriation, with cellular viability and disruption being assessed by trypan blue dye exclusion and by the release of lysosomal enzymes. The Ca2+ ionophore A23187 (3.125-50 microM) induced a concentration-dependent increase in enzyme marker release and decreased dye exclusion from the cells. Ionophore-induced enzyme release was reduced by removal of Ca2+ or by incubation with EDTA. Cells from the medium-sized fraction were found to be more sensitive to damage by A23187 than were larger-diameter cells. Enzyme release from these cells was also induced by the Ca(2+)-channel activator BAY K 8644 (1.5 microM). The Ca(2+)-channel antagonists, nifedipine, verapamil, and diltiazem (1 microM), abolished enzyme release in response to BAY K 8644 (1.5 microM) but did not affect A23187 (25 microM)-induced responses. Ethanol (5 and 8%) also induced a concentration-dependent increase in enzyme release and decrease in dye exclusion, but this effect was not dependent on the external Ca2+ concentration. However, threshold concentrations of A23187 (1.56 microM) substantially potentiated the cell damage induced by ethanol (5 or 8%), and this synergism was dependent on external Ca2+. These data suggest that agents that produce an inappropriate Ca2+ flux can disrupt or augment disruption of gastric mucosal cells and thus Ca2+ homeostasis is essential for maintenance of mucosal cell integrity.
Effect of hibernation and jejunal bypass on mucosal structure and function
Authors: Carey H. V. , Cooke H. J.
Published: December 2017

Intestinal mucosal structure and function may be regulated by systemic factors associated with oral feeding, as well as local responses initiated by contact of the mucosa with food. This study compared the relative effects of these factors in an animal model that undergoes seasonal long-term fasting. Jejunal bypass operations or sham surgeries were performed on active fed ground squirrels or on squirrels that subsequently ceased feeding and hibernated. Mucosal wet weight, protein content, villus height, and surface area were reduced in jejunal segments that had minimal exposure to luminal contents (bypassed segments of active squirrels and all segments of hibernators) compared with segments exposed to the luminal stream (incontinuity and sham segments of active squirrels). When normalized to mucosal weight, transepithelial absorption of 3-O-methylglucose and alanine-dependent sodium flux were greater in jejunal segments with minimal exposure to luminal nutrients. Altered structure in bypassed segments of active and hibernating squirrels paralleled changes in functional parameters despite the presence of different systemic factors in the two groups. Thus, in this animal model, contact of the mucosa with food, and not systemic factors associated with oral feeding, is the primary factor maintaining mucosal mass. The absence of mucosal contact with nutrients enhances specific absorptive function by mechanisms that have yet to be determined.
Hepatic transport of a fluorescent stearate derivative: electrochemical driving forces in intact rat liver
Authors: Fitz J. G. , Bass N. M. , Weisiger R. A.
Published: December 2017

We determined the effect of varying the transmembrane Na+ electrochemical gradient on extraction of a fluorescent derivative of stearate, 12-N-methyl-7-nitrobenzo-2-oxa-1,3,-diazol-amino stearate (NBD-stearate), by the isolated perfused rat liver. Membrane potential difference (PD) of individual hepatocytes and extraction of NBD-stearate were measured simultaneously under basal conditions and during changes in PD induced by perfusate ion substitutions. Under basal conditions, PD average -30 +/- 1 mV, and extraction of 10 microM NBD-stearate from 1% albumin solutions averaged 0.54 +/- 0.03. Fluorescence microscopy indicated that uptake exhibited a declining portal-to-central gradient in the presence but not absence of Na+. Substitution of nitrate for Cl- hyperpolarized PD to -59 mV and increased extraction to 131% of control values. Withdrawal of nitrate and substitution of gluconate for Cl- depolarized PD to -3 and -15 mV, respectively, and decreased extraction to 63 and 73% of control values. Substitution of choline for Na+ eliminated the out-to-in Na+ gradient, depolarized PD to -16 mV, and decreased extraction to 27% of control values, an effect greater than expected for membrane depolarization alone. Uptake of NBD-stearate was saturable and caused Na(+)-dependent membrane depolarization at higher concentrations (300 microM). These studies indicate that uptake of NBD-stearate occurs in large part by an efficient Na(+)-dependent mechanism compatible with electrogenic Na(+)-fatty acid cotransport.
Developmental expression of intestinal surfactant-like particles in rats
Authors: Eliakim R. , Becich M. J. , Green K. , Alpers D. H.
Published: December 2017

We have reported the appearance of surfactant-like particles enriched for intestinal alkaline phosphatase and phosphatidylcholine within enterocytes and in the lumen of adult fat-fed rat intestine. Because rat pulmonary surfactant decreases in abundance during the first postnatal days, we examined the developmental expression of these intestinal particles in suckling rats. Electron microscopy revealed abundant particles in 1-day-old rats within and surrounding the villus enterocytes, declining in frequency by day 14. Phosphatidylcholine content, alkaline phosphatase, sucrase-isomaltase, and lactase activity in particles peaked 1 day after birth, declining rapidly to adult levels by day 3 of life, except for sucrase, which peaked again after weaning. The postnatal developmental profile of the same brush-border-associated enzymes was totally different. Membrane fractions enriched for alkaline phosphatase and of similar density to rat surfactant-like particles were isolated from the small intestine of an amphibian (Xenopus laevis) and a fish (grass carp). Electron microscopy of the Xenopus membranes revealed unilamellar structures similar to the rat particles, but the carp membranes were of dissimilar morphology. We conclude that particles with surfactant-like properties in the rat intestine are ontogenically expressed like pulmonary surfactant; similar particles are evident only in animals with lungs.
Misoprostol attenuates acetic acid-induced increases in mucosal permeability and inflammation: role of blood flow
Authors: Yamada T. , Specian R. D. , Granger D. N. , Gaginella T. S. , Grisham M. B.
Published: December 2017

The objectives of this study were 1) to quantify the effects of misoprostol (Miso; prostaglandin E1 analogue) on acetic acid-induced increases in mucosal permeability and inflammation; 2) to determine what effect acetic acid, Miso, or the combination of Miso plus acetic acid has on colonic blood flow; and 3) to assess whether the protective effect of Miso may be attributable to its vasodilatory properties. We found that intrarectal administration of acetic acid produced a 6.4-fold increase in colonic myeloperoxidase activity (an index of granulocyte infiltration), an 8.2-fold increase in mucosal permeability, a 1.6-fold increase in colonic weight, and a 6.8% decrease in body weight 48 h after enema. Miso pretreatment significantly attenuated the increases in colonic myeloperoxidase activity, mucosal permeability, and colon weight as well as prevented the loss of body weight. In a different series of experiments, we found that blood flow in the descending, transverse, and ascending colon increased 2.5- to 3.5-fold immediately after the acetic acid enema; however, it returned to control values at 1 and 4 h after enema. Miso pretreatment, followed by acetic acid, resulted in a further increase (2.5-fold) in blood flow in the descending colon 1 h after enema compared with acetic acid alone. This Miso-induced increase in blood flow at 1 h could not account for its protective effect inasmuch as colonic mucosal permeability (i.e., injury) in Miso-pretreated animals was not significantly different from values obtained in animals pretreated with vehicle and then given the enema.
Hepatic processing of recombinant human renin: mechanisms of uptake and degradation
Authors: Marks D. L. , Kost L. J. , Kuntz S. M. , Romero J. C. , LaRusso N. F.
Published: December 2017

Biologically active 125I-Bolton-Hunter-labeled recombinant human renin (BH-renin) was used to study hepatic processing of renin both in vivo in bile fistula rats and in vitro in isolated perfused rat livers. BH-renin was composed mainly (80%) of a form that bound to concanavalin A-agarose (CB-renin). Twenty minutes after femoral venous injection of CB-renin in vivo, 47% of injected radiolabel was present in liver. Hepatic uptake of CB-renin was inhibited in a dose-dependent manner by mannosylated bovine serum albumin (MBSA) and mannan, but was unaffected by asialofetuin and mannose 6-phosphate. MBSA also significantly inhibited the plasma disappearance of endogenous renin in kidney-ligated rats. Cell separation techniques and light microscopic autoradiography showed that CB-renin was preferentially cleared by hepatic nonparenchymal cells via the mannose receptor, but was also cleared by hepatocytes via an unidentified mechanism. Tissue fractionation demonstrated that after injection of CB-renin, radiolabel was concentrated in lysosome-enriched liver fractions. In the liver, CB-renin was rapidly degraded to trichloroacetic acid-soluble fragments, which accumulated in urine and bile. Leupeptin, an inhibitor of lysosomal proteases, decreased degradation of CB-renin by 60%; vinblastine and colchicine, microtubule binding agents, each inhibited CB-renin degradation by 40%. Our results show that the liver plays a major role in the regulation of plasma renin levels via clearance by the mannose receptor on nonparenchymal cells and subsequent degradation in lysosomes.
Functional subtyping of muscarinic receptors on canine esophageal mucosa
Authors: Lad R. , Donoff B. , Rangachari P. K.
Published: December 2017

Serosal addition of muscarinic agonists elicited rapid changes in electrical parameters across the isolated canine esophageal epithelium set up in vitro. Both carbachol and the M1-selective agonist, McNeil A343 (McN), increased transmucosal potential differences (PDs), decreased transmucosal resistances (R), and increased short-circuit currents (Isc). Carbachol was more potent and more effective than McN. Muscarinic antagonists were used to define the muscarinic receptor involved. The pA2 values obtained with Schild plots were as follows: atropine 9.14, 4-DAMP 8.98, AFDX-116 6.71, and pirenzepine 7.12. Low concentrations of pirenzepine (10(-8) M), produced a rightward shift in the dose-response curve to McN, without inhibiting responses to carbachol. Thus the receptor subtype is clearly not an M2. As in other glandular systems, M3 receptors are present. Whether M1 receptors also exist requires better definition of receptor densities-reserves in this tissue. Carbachol induced net secretion of Na and Cl and converted a predominantly absorptive tissue to a secretory one.
Ischemia-reperfusion-induced mucosal dysfunction: role of neutrophils
Authors: Kurtel H. , Fujimoto K. , Zimmerman B. J. , Granger D. N. , Tso P.
Published: December 2017

The ability of the small intestine to absorb and transport lipid into lymph is markedly reduced 24 h after a 10-min total occlusion of the superior mesenteric artery (SMA). The aim of this study was to define the role of neutrophils in the ischemia-reperfusion (I/R)-induced decrement in lipid absorption. A lipid test meal containing 40 mumol of radioactive triolein was infused intraduodenally at 3 ml/h for 8 h, and radioactive lipid output in lymph was monitored during lipid infusion in intestinal lymph fistula rats. Animals rendered neutropenic with antineutrophil serum (ANS) did not exhibit the reduction in lipid absorption and transport in lymph normally observed 24 h after I/R. This protective effect of ANS was specifically related to the reduction in the number of neutrophils in the intestinal mucosa. The amount of radioactive lipid detected in the liver of untreated rats was significantly higher than in control rats, suggesting an increased portal transport of infused radioactive lipid. Neutropenia reduced the liver lipid level toward the control value. The intestinal blood flow response to SMA occlusion was not altered by neutropenia. Our results suggest that neutrophils play an important role in the mucosal dysfunction associated with ischemia-reperfusion.
Nitric oxide and nitrosocysteine mimic nonadrenergic, noncholinergic hyperpolarization in canine proximal colon
Authors: Thornbury K. D. , Ward S. M. , Dalziel H. H. , Carl A. , Westfall D. P. , Sanders K. M.
Published: December 2017

Previous evidence suggests that nonadrenergic, noncholinergic (NANC) inhibitory neurotransmission in visceral muscles may be mediated by nitric oxide (NO). We have demonstrated that NO and the NO carrier S-nitrosocysteine can mimic the hyperpolarization in colonic muscle caused by nerve stimulation. The finding that S-nitrosocysteine breaks down fast enough to cause inhibitory junction potential (IJP)-like hyperpolarizations suggests that NO could be stored as a nitrosothiol in secretory vesicles in nerve terminals. Oxyhemoglobin blocked hyperpolarization responses to NO and S-nitrosocysteine and NANC IJPs. These findings suggest that NO is a biologically active transmitter substance in NANC inhibitory neurotransmission. NO enhanced the open probability of Ca(2+)-activated K+ channels in isolated colonic muscle cells. These channels may mediate the hyperpolarization response to NANC neurotransmission in colonic muscles.
Cholinergic dependence of gallbladder response to cholecystokinin in the guinea pig in vivo
Authors: Takahashi T. , May D. , Owyang C.
Published: December 2017

The physiological mechanism responsible for cholecystokinin (CCK)-induced gallbladder (GB) contraction is unclear. We investigated the relative roles of direct muscle stimulation and neural activation at physiological and supraphysiological levels of CCK-octapeptide (CCK-8) using an in vivo guinea pig model. GB pressure was measured by a pressure transducer inserted into the GB lumen. Infusion of CCK-8 (2.5-40 ng.kg-1.min-1) increased GB pressure in a dose-dependent fashion. Pretreatment with atropine or hexamethonium antagonized GB responses to low doses of CCK-8 (2.5-5 ng.kg-1.min-1) but had no effect on doses greater than 10 ng.kg-1.min-1. Bilateral truncal vagotomy also significantly reduced GB responses to low doses of CCK-8 (2.5-5 ng.kg-1.min-1) but did not affect responses to high doses (10-40 ng.kg-1.min-1). Atropine or hexamethonium had no further inhibitory effects on guinea pigs that had undergone truncal vagotomy. Fasted guinea pigs that were fed ad libitum produced a postprandial peak plasma CCK level of 7.8 +/- 1.8 pM. This level was most closely approximated by infusion of 5 ng.kg-1.min-1 of CCK-8 (8.4 +/- 2.6 pM). CCK-8 infusion at greater than or equal to 10 ng.kg-1.min-1 gave supraphysiological plasma CCK levels. These observations indicate that CCK stimulated GB contraction via both a neural and a direct smooth muscle effect. Doses of CCK-8 that produce physiological plasma CCK levels act via stimulation of presynaptic cholinergic neurons in a vagally mediated pathway, whereas doses of CCK-8 that produce supraphysiological CCK levels act directly on GB smooth muscle.(ABSTRACT TRUNCATED AT 250 WORDS)
Mechanism of changes in hepatic sinusoidal and biliary glutathione efflux with age in rats
Authors: Ookhtens M. , Maddatu T.
Published: December 2017

To delineate the kinetic mechanism(s) of declining sinusoidal reduced glutathione (GSH) efflux with age, we perfused livers of male rats ages approximately 1-1.5, approximately 2-3, and approximately 3.5-6 mo old and measured sinusoidal and biliary GSH and oxidized glutathione (GSSG) effluxes. Our results showed declining GSH transport to be solely due to a falling maximum transport rate (Vmax) and not an increasing Michaelis constant (Km)(Vmax = 24.2 +/- 2.95, 15.8 +/- 1.51, and 8.61 +/- 0.75 nmol. min-1.g-1; Km = 3.0 +/- 0.42, 2.6 +/- 0.31, and 2.6 +/- 0.43 mumol/g for the three age groups, respectively). Because hepatocyte membrane potential was earlier implicated as a driving force for GSH efflux and hepatocytes of female rats were reported to be less polarized than those of males, we likewise studied the kinetics of sinusoidal GSH efflux from livers of female rats of three age groups comparable to our males. Vmax in females tended to be lower than in males. This was more pronounced in the youngest group but was diminished in the older groups. Vmax was again the only parameter declining with age in the female livers, from 19.1 +/- 2.25 to 15.0 +/- 0.95 and 7.83 +/- 0.99 nmol.min-1.g-1, whereas Km remained unchanged at 3.0 +/- 0.45, 3.1 +/- 0.35, and 3.2 +/- 0.72 mumol/g, respectively. Age-dependent changes in GSH efflux were not due to a changing membrane potential. There was no appreciable change in the paracellular permeability with age either.(ABSTRACT TRUNCATED AT 250 WORDS)
Topography of the esophageal peristaltic pressure wave
Authors: Clouse R. E. , Staiano A.
Published: December 2017

Topographic plots constructed from conventional esophageal manometric waves were examined in 12 healthy volunteers to better understand spatial relationships of peristaltic contractions. Typical postdeglutitive waves were spatially interconnected using a computerized gridding and plotting system. Contour plots of the resultant peristaltic contraction revealed a consistent separation of a proximal pressure segment from the prominent distal contraction at 21.7 +/- 1.3% of the esophageal length. A second trough in the contraction sequence was found in 11 (91.7%) of the subjects at 64.0 +/- 2.7% of esophageal length separating the distal (smooth muscle) esophagus into two contraction segments. Similar topographic studies were also performed in eight symptomatic patients with frequent double-peaked waves. Poor alignment of the two distal contraction segments was observed in six (75%) of the eight patients. These topographic plots demonstrate previously unrecognized wave relationships and suggest that two neuromuscular contraction units can be detected in the distal smooth muscle esophagus. This technique should be useful in understanding altered relationships of these units under differing physiological conditions or with disease.
Effects of atropine on pancreatic response to bethanechol, cholecystokinin, and food intake in rats
Authors: O'Rourke M. F. , Reidelberger R. D. , Solomon T. E.
Published: December 2017

Atropine was used to examine the role of cholinergic mechanisms in the pancreatic secretory response to food intake. Unanesthetized rats with gastric, jugular vein, bile-pancreatic, and duodenal cannulas were used; bile-pancreatic juice was recirculated. The maximal response to bethanechol (4 mg.kg-1.h-1) was similar to cholecystokinin (CCK)-8-induced maximal secretion. Atropine (25-200 micrograms.kg-1.h-1) markedly inhibited basal amylase output and caused dose-related inhibition of the incremental response to a maximal dose of bethanechol. Atropine (50 micrograms.kg-1.h-1) shifted the dose-response curve to bethanechol (1-32 mg.kg-1.h-1) to the right but did not alter maximal amylase output. L 364718 (0.5 mg/kg), a CCK receptor antagonist, had no effect on bethanechol-stimulated pancreatic secretion. Atropine (50 micrograms.kg-1.h-1) did not affect the incremental responses to low doses of CCK-8; the maximal response occurred at a higher CCK-8 dose because atropine decreased basal secretion. Atropine (50 or 200 micrograms.kg-1.h-1) did not decrease the amylase response to ingestion of a liquid meal. We conclude that 1) bethanechol is a full agonist for stimulation of pancreatic enzyme secretion and its effects are not mediated by CCK release; 2) atropine is a competitive antagonist of bethanechol-induced pancreatic secretion in vivo but does not directly affect responses to CCK-8; 3) cholinergic mechanisms do not mediate the pancreatic enzyme response to a liquid meal in rats.
Regional differences in gastrointestinal processing and absorption of epidermal growth factor in suckling rats
Authors: Rao R. K. , Koldovsky O. , Grimes J. , Williams C. , Davis T. P.
Published: December 2017

Gastrointestinal processing and absorption in vivo of 125I-labeled epidermal growth factor (125I-EGF) was studied in isolated stomach, jejunum, and ileum of 12-day-old suckling rats. Dose dependency and regional differences in the processing and absorption of EGF were determined. At 60 min after administration of 125I-EGF into the isolated gastric lumen, greater than 90% of radioactive material was recovered from gastric wall and lumen in both suckling and weanling rats. On the other hand, a considerable amount of EGF was absorbed (in immunoreactive and receptor active form) from isolated jejunum and ileum of suckling rats. The absorption of EGF from isolated jejunum and ileum increased linearly with the dose of EGF administered up to 1 microgram/rat; absorption of orogastrically administered EGF was also increased linearly with the dose of EGF administered (up to 5 micrograms/rat). Reverse-phase high-performance liquid chromatographic analysis of tissue extracts demonstrated that 125I-EGF is stable in gastric lumen but only partially stable in the jejunal lumen. Carboxy-terminally processed forms of EGF were detected in the luminal flushings of jejunum and ileum and in the wall of the stomach, jejunum, and ileum. 125I-des(48-53)EGF was found in ileal flushings and in the walls of the stomach, jejunum, and ileum, whereas 125I-des(53)EGF and 125I-des(49-53)EGF were detected in jejunal flushings. Receptor binding of 125I-des(53)EGF was significantly greater (by 57%) than that of intact 125I-EGF, but receptor binding of 125I-des(49-53)EGF and 125I-des(48-53)EGF was greatly diminished by 57 and 86%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Postinsertional processing of sucrase-alpha-dextrinase precursor to authentic subunits: multiple step cleavage by trypsin
Authors: Shapiro G. L. , Bulow S. D. , Conklin K. A. , Scheving L. A. , Gray G. M.
Published: December 2017

Sucrase-alpha-dextrinase, a hybrid digestive carbohydrase of the intestinal brush border, is initially synthesized and transported to the surface membrane as a single-chain glycoprotein, P, which is then cleaved to alpha- and beta-subunits, presumably by one or more pancreatic proteases. However, efforts to convert P under controlled conditions to authentic alpha and beta have been unsuccessful. Sucrase-dextrinase immunoprecipitates from rats intraintestinally labeled with [3H]leucine or [35S]methionine without presence of biliary-pancreatic secretions revealed only the 230-kDa P precursor. Restoration of intestinal flow converted the brush border P to the alpha- (140 kDa) and beta- (125 kDa) subunits. Biliary plus pancreatic secretions facilitated this postinsertional cleavage, but bile alone played no role in conversion. When isolated brush borders, prelabeled in vivo, were exposed to a mixture of pancreatic proteases at physiological concentrations, P was converted to authentic alpha and beta, but only trypsin was responsible for the conversion. Kinetic analysis in prelabeled isolated brush-border vesicles revealed the appearance of several intermediate species (205-145 kDa) produced either by endogenous membrane proteases or by trypsin itself. Reconstituted duodenal luminal contents yielded a fragmentation pattern identical to that produced by trypsin alone. Trypsin was necessary and sufficient for processing of the intermediate precursors to the final authentic alpha- and beta-subunits. Based on the alpha- to beta radioactivity ratio and the known amino acid composition of the subunits, differential cleavage occurred with relatively greater production of the beta-subunit (alpha-to-beta molar ratio = 0.77). The conversion of P to the alpha- and beta-units, rather than occurring in a single step after membrane insertion, is differentially catalyzed by trypsin trimming to unequal amounts of the subunits involving a complex series of cleavage steps.