bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of vasoactive intestinal contractor on voltage-activated Ca2+ currents in feline parasympathetic neurons
Authors: Nishimura T. , Krier J. , Akasu T.
Published: December 2017

Intracellular current-clamp and single-electrode voltage-clamp techniques were used to study in vitro action potentials and the action of vasoactive intestinal contractor (VIC; 0.03-1 microM) on the high-voltage-activated Ca2+ currents (ICa) of neurons in feline colonic parasympathetic ganglia. In the current-clamp recording mode, action potential amplitude was depressed by cobalt (1 mM) and omega-conotoxin (300 nM) or in nominally Ca(2+)-free Krebs solutions. In the single-electrode voltage-clamp recording mode, the ICa was isolated by blocking the voltage-gated Na+ current with tetrodotoxin (1-3 microM) and by Krebs solutions containing a low Na+ concentration. The voltage-activated K+ currents were blocked by intracellular injection of cesium through a recording electrode filled with 2 M CsCl and external application of tetraethylammonium (30-50 mM) and barium (2 mM). The Ca(2+)-dependent Cl- current was blocked by replacement of Ca2+ (2 mM) with equimolar barium. Anomalous rectification was blocked by external application of 2 mM cesium. The ICa was evoked by depolarizing step commands more positive than -40 mV from holding potentials ranging between -80 and -60 mV. ICa was depressed by cobalt (1 mM), cadmium (100 microM), and omega-conotoxin (500 nM) but not by nifedipine (10 microM), nicardipine (10 microM), and verapamil (10 microM). BAY K 8644 (3-10 microM) also did not affect the ICa. VIC (0.1-1 microM), one of the endothelin (ET) isopeptides, caused an inward current followed by an outward current. The VIC-induced inward and outward currents were associated with an increase and decrease in membrane conductance, respectively. VIC also caused an initial depression followed by a long-lasting augmentation of the ICa. ET-1, ET-2, and ET-3 equally mimicked the action of VIC on both holding current and ICa. These data suggest that VIC activates a receptor-operated channel and modulates the omega-conotoxin-sensitive voltage-activated Ca2+ channels through ETB receptor subtypes of neurons in feline colonic parasympathetic ganglia.
Endocrine and amino acid regulation of liver macroautophagy and proteolytic function
Authors: Bergamini E. , Del Roso A. , Gori Z. , Masiello P. , Masini M. , Pollera M.
Published: December 2017

Regulation of liver macroautophagy and protein degradation by hormones and direct regulatory amino acids were studied in male 2-mo-old Sprague-Dawley albino rats with the use of the antilipolytic agent 3,5'-dimethylpyrazole (DMP; 12 mg/kg body wt ip) as a stimulatory agent. Injection of DMP decreased glutamine plasma levels and glutamine release from the perfused liver. Autophagic vacuoles were observed in the pericanalicular area of liver cells after 30 min. Levels and release of other regulatory amino acids did not exhibit any significant decrease but subsequently increased. Intraperitoneal administration of glutamine inhibited the proteolytic response. In conclusion, these studies demonstrate that in vivo induction and control of liver macroautophagy and protein degradation by the physiological mechanism (i.e., by shortage of nutrients) involve unbalanced and asynchronous changes in the levels of selected direct regulatory amino acids (i.e., a decrease in glutamine and a subsequent increase in leucine and tyrosine levels)
Pancreas growth, tyrosine kinase, PtdIns 3-kinase, and PLD involve high-affinity CCK-receptor occupation
Authors: Rivard N. , Rydzewska G. , Lods J. S. , Martinez J. , Morisset J.
Published: December 2017

Cholecystokinin (CCK), CCK octapeptide (CCK-8), and caerulein (Cae) are the most potent trophic factors in the pancreas when given exogenously or released from the intestine. Recent studies have suggested that this growth-promoting effect of CCK was initiated through the occupation of the CCKA receptor. This study was then undertaken to determine whether occupation of the high- or low-affinity CCKA receptor is involved in the growth process and to establish which transduction signals have been selectively activated. As an answer to the first question, rats were infused with CCK JMV-180, a CCKA high-affinity agonist, at doses of 50, 100, 150, and 300 micrograms.kg-1.h-1, or Cae (0.25 micrograms.kg-1.h-1) for 4 days. After rats were killed, their pancreatic weight and contents of protein, DNA, RNA, amylase, and chymotrypsinogen were estimated. To investigate the transduction signals, rats were infused for 30 min to 4 h with 300 micrograms.kg-1.h-1 JMV-180, or pancreatic acini were exposed in vitro to 1 microM JMV-180 for 5 or 30 min. After rats were killed, pancreases were used to monitor tyrosine kinase, phosphatidylinositol (PtdIns) 3-kinase, and phospholipase D (PLD) activities. The first set of experiments indicates that JMV-180 caused a dose-dependent effect on pancreas growth, with the 300 micrograms.kg-1 x h-1 dose giving the maximal effect comparable to that of Cae. Furthermore, JMV-180 induced concomitant early increases in tyrosine kinase and PLD activities both in vivo and in vitro and PtdIns 3-kinase in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)
Effect of brefeldin A on lymphatic triacylglycerol transport in the rat
Authors: Mansbach C. M. , Nevin P.
Published: December 2017

Brefeldin A (BFA) has been shown in in vitro studies to either collapse the Golgi into the endoplasmic reticulum (ER) or the peripheral organelles into the trans-Golgi network. Our goal was to determine the effect of BFA on intestinal lipid transport, since the Golgi is thought to play an important role in this process and simultaneously establish the effectiveness of BFA in an in vivo system. We infused rats intraduodenally with glyceryl tri-[3H]oleate at 135 mumol/h for 15 h and included BFA, 750 micrograms/h, during hours 4-7 of infusion. Mass and lipid disintegrations per minute output into the lymph fell to 9% of input rates at 8 h of infusion and returned to steady-state values at 12 h of infusion. Both chylomicron and very low-density lipoprotein output were severely affected by the BFA. Electron microscopy showed that the Golgi was collapsed into the ER. Mucosal triacylglycerol (TG) mass and disintegrations per minute were increased at 7 h of infusion in BFA infused rats vs. controls in the proximal half of the intestine. Lipid absorption, lipase activity, and mucosal TG synthesis were normal in the BFA-treated rats. We conclude that BFA works in vivo and in the intestine collapses the Golgi into the ER. As a consequence, lymphatic TG transport was severely affected.
Effect of histamine on rat gastric H(+)-K(+)-ATPase alpha-subunit expression
Authors: Tari A. , Yamamoto G. , Yonei Y. , Sumii M. , Sumii K. , Haruma K. , Kajiyama G. , Wu V. , Sachs G. , Walsh J. H.
Published: December 2017

The H(+)-K(+)-adenosinetriphosphatase (ATPase) is expressed in the parietal cell and is responsible for acid secretion by the stomach. Histamine binds to an H2 receptor and activates adenylate cyclase and intracellular calcium concentration ([Ca2+]i) elevation, stimulating acid secretion. It has been shown that omeprazole administered to rats increases serum gastrin and transiently increases the level of mRNA for the alpha-subunit of the pump, but this increase is blocked by the presence of the H2-receptor antagonist, famotidine [A. Tari, G. Yamamoto, K. Sumii, M. Sumii, Y. Takehara, K. Haruma, G. Kajiyama, V. Wu, G. Sachs, and J. H. Walsh. Am. J. Physiol. 265 (Gastrointest. Liver Physiol. 28): G752-G758, 1993]. These observations suggest that the release of histamine induced by gastrin is essential for the increase of the expression of mRNA induced by omeprazole. Infusion of histamine at 15 mumol.kg-1.h-1 i.v. for 1 h increased the alpha-subunit mRNA level by 144 +/- 2.4% and induced a stimulated morphological appearance of the parietal cell. These changes were inhibited completely by the competitive H2-receptor antagonist famotidine, which elevated gastric pH and serum gastrin. Famotidine also reduced the level of H(+)-K(+)-ATPase mRNA compared with control animals. No change in the expression of beta-actin mRNA was observed in any group of animals. These data provide direct evidence for histamine stimulation of H(+)-K(+)-ATPase alpha-subunit gene expression by activation of the H2 receptor.
Cloning and characterization of a growth factor-inducible cyclooxygenase gene from rat intestinal epithelial cells
Authors: DuBois R. N. , Tsujii M. , Bishop P. , Awad J. A. , Makita K. , Lanahan A.
Published: December 2017

Growth factors have been shown to play a role in intestinal epithelial growth regulation and transformation. Utilizing standard differential cloning techniques, we have isolated a growth factor-inducible gene (RS-2) from rat intestinal epithelial cells that has approximately 95% homology to the mouse mitogen-inducible cyclooxygenase (COX-2) at the amino acid level. This cDNA hybridizes to a approximately 4.5-kb mRNA from transforming growth factor (TGF)-alpha-stimulated rat intestinal epithelial (RIE-1) cells and is constitutively expressed in vivo in adult rat kidney and brain. Nuclear run-on experiments demonstrate that the increase of RS-2 mRNA after TGF-alpha stimulation is in part due to an increased transcription rate of the gene. The coding region for RS-2 was subcloned into a pCMV-2 expression vector, and the RS-2 protein was expressed in COS-1 cells. Microsomal fractions isolated from the COS-1 cells transfected with the RS-2 expression vector contained cyclooxygenase activity. In addition to the production of prostaglandins, the recombinant RS-2 protein also catalyzed the formation of three other eicosanoid products. In summary, we have cloned a mitogen-inducible cyclooxygenase gene from rat intestinal cells that is induced following growth factor stimulation.
Absorption of platelet-activating factor acetylhydrolase by rat intestine
Authors: Furukawa M. , Frenkel R. A. , Johnston J. M.
Published: December 2017

Platelet-activating factor acetylhydrolase (PAF-AH) is a 43-kDa protein that catalyzes the degradation and inactivation of this potent phospholipid mediator. PAF plays an important role in the pathogenesis of necrotizing enterocolitis (NEC), and the elevation of the plasma PAF-AH activity may be beneficial in the prevention of this disease. The activity of PAF-AH was transferred from the mucosal to the serosal fluid in intestinal sacs from neonatal rats. Translocation was highest on day 15, decreased by day 21, and disappeared by day 24. Greater transport of the enzyme was noted in the neonatal jejunum compared with duodenum or ileum. PAF-AH absorption in 15-day-old rats was decreased by the addition of inhibitors of energy production and by low temperature. We have, therefore, concluded that the enzyme transport is an energy-dependent process. It is suggested that PAF-AH found in milk may prove to be beneficial in the prevention of NEC by its translocation across the intestinal mucosa. The absorption of macromolecules in the neonate, in addition to providing a passive immunity, may also serve a protective role by inactivating certain proinflammatory agents, such as PAF.
Regulation of intracellular pH by immortalized human intrahepatic biliary epithelial cell lines
Authors: Grubman S. A. , Perrone R. D. , Lee D. W. , Murray S. L. , Rogers L. C. , Wolkoff L. I. , Mulberg A. E. , Cherington V. , Jefferson D. M.
Published: December 2017

We have produced continuous cell lines using retroviral transduction of SV40 large T antigen into human intrahepatic biliary epithelial (IBE) cells from three different normal individuals. These IBE cell lines grow in a hormone-supplemented medium in the presence of NIH/3T3 fibroblast coculture. These cells maintain their epithelial appearance and are positive for the biliary-specific markers cytokeratins 7 and 19 and gamma-glutamyl transpeptidase while being negative for the hepatocyte markers albumin and asialoglycoprotein receptor. To evaluate ion transport pathways in IBE cell lines, we utilized intracellular pH (pHi) measurements obtained using the intracellular fluorescent indicator 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. In the absence of HCO3(-)-CO2, an amiloride-sensitive Na(+)-H+ exchanger participated in the regulation of basal pHi. In the presence of HCO3(-)-CO2, a 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-sensitive, Na-, Cl-, and HCO3(-)-dependent acid extrusion mechanism accounted for approximately 60% of pHi recovery from acidic pHi; this mechanism is most consistent with the presence of a Na-dependent Cl-HCO3- exchanger (Na+HCO3(-)-Cl-H+). Under basal conditions, Cl- depletion revealed a DIDS-sensitive alkalinization consistent with a Na-independent Cl(-)-HCO3- exchanger. These model systems will allow the opportunity to study the normal mechanisms of IBE function and to study the pathobiology of IBE processes in disease states.
Enterocytes adhere preferentially to collagen IV in a differentially regulated divalent cation-dependent manner
Authors: Moore R. , Madara J. L. , MacLeod R. J.
Published: December 2017

Minor cell defects in epithelial sheets are a consequence of superficial injury in natural intestinal diseases. These defects are restored by migration of shouldering epithelial cells over the underlying matrix. Restitution of minor epithelial defects is facilitated by collagen IV (R. Moore, J. Madri, S. Carlson, and J. L. Madara. Gastroenterology 102: 119-130, 1992). Here we characterize enterocyte attachment to substrates and determine the role of divalent cations. Attachment and spreading of native intestinal epithelial cells (EC) isolated by mechanical vibration were greatest on collagen IV [compared with collagen I, collagen III, laminin, fibronectin, and plastic; P < 0.001]. Attachment was not inhibited by cycloheximide but was inhibited by cytochalasin D and by a functionally defined antibody to collagen IV. In fixed extracellular 1.25 mM Mg2+, EC attachment was nearly three times greater in nominally Ca(2+)-free medium compared with 5 mM Ca2+ (P < 0.001). Conversely, in fixed 1.25 mM Ca2+, cell attachment was three times greater in the presence of 2.5 mM Mg2+ compared with Mg(2+)-deplete media (P < 0.001). Last, in the presence of the intracellular Ca2+ chelator 1,2-bis(2-amino-phenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester, EC attachment was diminished by > 50% (P < 0.001). We speculate that local tissue concentrations of divalent cations may play an important role in restitution of intestinal epithelial wounds by mediating attachment of migrating EC to collagen IV.
Reduced glutathione modulates Ca(2+)-mediated damage to rabbit isolated gastric mucosal cells
Authors: Wong H. M. , Tepperman B. L.
Published: December 2017

In the present study, we have examined the role of reduced glutathione (GSH) in the modulation of Ca2+ ionophore A-23187-induced gastric mucosal cellular disruption. Experiments were conducted using cells isolated from rabbit oxyntic mucosa. Cell injury was assessed by release of the lysosomal enzyme acid phosphatase, by trypan blue dye uptake, and by examination of lipid peroxidation. Cellular GSH was assessed spectrophotometrically by measuring total soluble reduced thiol content. Ionophore A-23187 (3-25 microM) induced a concentration-dependent injury to mucosal cells and a concentration-dependent reduction in cellular GSH content. Removal of Ca2+ from the incubation medium abolished both the disruptive action and the GSH-lowering effect of A-23187. Gastric cellular GSH content was reduced significantly by preincubation of cells with an agent that depletes reduced sulfhydryls, diethyl maleate (DEM; 1 mM). Furthermore, DEM significantly augmented cellular disruption in response to A-23187. Similarly, incubation of cells with L-cysteine (1 mM), a stimulant of glutathione synthesis, increased mucosal cellular GSH content and decreased the cellular disruptive effect of A-23187. Exogenous addition of GSH to the cell suspension significantly reduced Ca2+ ionophore-induced cellular disruption and hastened the recovery of cellular free Ca2+ concentration ([Ca2+]) to baseline values. Similarly, exogenous GSH reduced cellular disruption produced by cyclopiazonic acid and thapsigargin, agents that increase intracellular [Ca2+] ([Ca2+]i) by inhibiting Ca2+ sequestration. These data suggest that a sustained increase in [Ca2+]i contributes to the pathogenesis of gastric mucosal cellular injury. This effect appears to be mediated by a reduction in the cellular content of GSH.
Membrane physical properties determine cardiac beta-adrenergic receptor function in cirrhotic rats
Authors: Ma Z. , Meddings J. B. , Lee S. S.
Published: December 2017

To elucidate the basic membrane mechanisms underlying cirrhotic cardiomyopathy, cardiac sarcolemmal plasma membrane physical properties, chemical composition, beta-adrenoceptor density, binding affinity, and isoproterenol-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) production were studied. Cirrhosis was induced by chronic bile duct ligation, while controls underwent a sham operation. The cardiac plasma membrane of cirrhotic rats was found to be more rigid than controls, primarily due to an increased cholesterol-to-phospholipid ratio. In cirrhotic animals, beta-adrenergic dysfunction was evident with a 21% decrease in beta-adrenoceptor density but no alteration in binding affinity. Despite the modest decrease in receptor number, beta-adrenoceptor-stimulated cAMP production was decreased by 37% in cirrhotic rats. When the membrane physical properties of the cirrhotic rats were restored to normal, by incubation with the fluidizing agent 2-(2-methoxy-ethoxy)ethyl 8-(cis-2-n-octylcyclopropyl)octanoate (A2C), isoproterenol-stimulated cAMP production also increased to levels indistinguishable from control animals. Restoration of membrane physical properties had no effect on either beta-adrenoceptor density or binding affinity. These results suggest that the increased rigidity of cardiomyocyte plasma membranes seen with cirrhosis is associated with decreased beta-adrenoceptor function. Moreover, restoring normal physical properties may result in restoration of beta-adrenoceptor-mediated contractile function.
Distribution of nitric oxide synthase activity in arterioles and venules of rat and human intestine
Authors: Nichols K. , Staines W. , Rubin S. , Krantis A.
Published: December 2017

NO is produced within peripheral blood vessels through the action of the differentially distributed constitutive and inducible NO synthase isoforms in the vessel wall. As in other sites in the periphery, NO exerts local vasodilatory actions in the gastrointestinal microvasculature and is proposed to play a role in enteric vasomotor regulation. Using NO synthase histochemistry and endothelial cell immunohistochemistry, we provide the first anatomic evidence of NO synthesis in both endothelial and smooth muscle cells of submucosal blood vessels in the rat and human intestine. The findings of this study indicate that 1) as in the periphery, both the endothelial and vascular smooth muscle cells of the microvessels irrigating the rat and human intestinal wall possess NO synthesis potential, 2) NO synthase activity is predominantly localized to discrete subcellular patches, and 3) the source of NO within the vascular wall, either intimal or medial, should be a consideration in future studies in terms of the relative contribution of these sources of vasomotor tone in the rat and human gut wall.
A volume-activated taurine channel in skate hepatocytes: membrane polarity and role of intracellular ATP
Authors: Ballatori N. , Simmons T. W. , Boyer J. L.
Published: December 2017

Osmoregulation in isolated hepatocytes and perfused livers of the little skate (Raja erinacea), an osmoconforming marine elasmobranch, is mediated in part by the uptake or release of the intracellular osmolyte taurine. To further characterize the efflux mechanism, [14C]taurine release and Na(+)-independent uptake were assessed after cell swelling in hypotonic media containing 0.1-100 mM taurine. Rate coefficients for [14C]taurine uptake (0.016 +/- 0.002 min-1) and efflux (0.015 +/- 0.003 min-1) were similar and independent of extracellular taurine concentration, indicating that a taurine-permeable channel mediates the release of this amino acid after cell swelling. Volume-activated taurine uptake and efflux were both blocked by pretreatment with the metabolic inhibitors 2,4-dinitrophenol, antimycin A, and KCN plus iodoacetate, by the sulfhydryl-reactive compound N-ethylmaleimide and the transport inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. [14C]taurine release via this channel was immediately blocked if isotonicity was restored or a membrane-permeable metabolic inhibitor (0.5 mM 2,4-dinitrophenol) was added at different times after a hypotonic stimulus. Similar, although delayed, effects were noted with antimycin A and KCN plus iodoacetate. When isolated perfused skate livers were exposed to hypotonic stimuli, all of the taurine was released into the sinusoidal circulation, but not into bile, an effect that was also blocked by restoring isotonicity. These findings demonstrate that taurine efflux from skate hepatocytes after cell swelling is mediated by a channel. This channel is localized to the basolateral membrane and appears to require the continual presence of intracellular ATP for its function.(ABSTRACT TRUNCATED AT 250 WORDS)
Diet affects hepatocyte membrane composition, fluidity, and taurocholate transport in suckling rats
Authors: Novak D. A. , Carver J. D. , Ananthanarayanan M. , Ray W.
Published: December 2017

We postulated that age-related changes in hepatocyte basolateral membrane lipid composition might contribute to the diminished Na(+)-dependent taurocholate transport noted in suckling animals. Basolateral membrane vesicles (BLMVs) were prepared from suckling rats (day 11) whose dams were fed diets predominant in lard (LBLMV), corn oil (COBLMV), or fish oil (FOBLMV). Fatty acid compositions of milk and BLMV differed significantly among the groups. Membrane cholesterol was higher in FOBLMV compared with the COBLMV and LBLMV groups; lipid phosphorus and the relative distribution of phospholipid classes were similar. Fluorescence anisotropy (1,6-diphenyl-1,3,5-hexatriene) was higher in FOBLMV (0.230) than in LBLMV (0.222) or COBLMV (0.217). Excited state lifetimes were similar in all groups. Na(+)-dependent taurocholate transport was increased at 5 and 20 s in LBLMV and COBLMV compared with FOBLMV. In vitro alteration of membrane cholesterol-fluidity did not alter taurocholate transport. In conclusion, although affected by alterations in diet, simple changes in membrane fluidity-cholesterol content do not affect Na(+)-dependent taurocholate transport.
Serotonin induces Cl- secretion in human jejunal mucosa in vitro via a nonneural pathway at a 5-HT4 receptor
Authors: Kellum J. M. , Budhoo M. R. , Siriwardena A. K. , Smith E. P. , Jebraili S. A.
Published: December 2017

We examined the effect of 5-hydroxytryptamine (5-HT) on Na+ and Cl- flux (J) and short-circuit current (Isc) in human jejunal mucosa. Segments of jejunum, taken at the time of gastric bypass surgery, were stripped of the seromuscular layers (and attached neural ganglia) and mounted as flat mucosal sheets in Ussing chambers under short-circuit conditions. 5-HT (0.1-100 microM) produced a concentration-dependent rise in Isc (mean effective concn = 2.5 microM). Using 22Na and 36Cl, we measured flux across control tissues and in those exposed to 5-HT. 5-HT decreased both net JNa and JCl and increased Isc (-1.1 +/- 0.6, -1.7 +/- 0.6, and 0.6 +/- 0.1 mueq.cm-2.h-1, respectively). Thus the 5-HT-induced rise in Isc could be accounted for by reduced net JNa and JCl. 5-HT induced a significant (P < 0.05) Cl- secretion (serosal-to-mucosal flux) when glucose was included in the buffer bathing the mucosal surface. Neither tetrodotoxin, the adrenergic receptor antagonists prazosin and propranolol, nor the cholinergic receptor antagonists atropine and hexamethonium inhibited the change (delta) in Isc induced by 5-HT. 5-Methoxytryptamine (5-MeOT) and zacopride, known 5-HT4 receptor agonists, induced significant delta Isc. The 5-HT receptor antagonists N-acetyl-5-hydroxytryptophyl-5-hydroxytryptamine (5-HT1P), ketanserin (5-HT2), and ICS-205-930 (preferential for 5-HT3 at 0.1 microM had no effect on delta Isc.(ABSTRACT TRUNCATED AT 250 WORDS)
Effect of protein derivatives on pancreatic secretion and release of secretin and CCK in rats
Authors: Shimizu K. , Shiratori K. , Watanabe S. , Takeuchi T. , Chang T. M. , Chey W. Y.
Published: December 2017

We investigated the effect of intraduodenal administration of oligopeptide and a mixed amino acid solution, which contains the same amino acid composition as oligopeptide, on pancreatic exocrine secretion and the release of secretin and cholecystokinin (CCK). Anesthetized rats were prepared with pyloric ligation and cannulation of pancreatic duct and bile duct. Protein derivatives in three different doses (oligopeptide: 25, 100, and 400 mg/h; and mixed amino acid solution: 70, 140, and 280 mg/h, pH 7.0) were infused into the duodenum for 1 h. Pancreatic juice was collected, and plasma concentrations of secretin and CCK were measured by radioimmunoassay. In addition, the effect of intravenous injection of an antisecretin serum or a CCK antagonist, loxiglumide, on pancreatic secretion stimulated by oligopeptide or mixed amino acid solution was also studied. Oligopeptide produced a significant dose-related increase in pancreatic secretion including volume, HCO3-, amylase, and trypsin output, plasma secretin (r = 0.792, P < 0.001), and plasma CCK (r = 0.421, P < 0.01). Similarly, mixed amino acid solution produced a dose-related increase in pancreatic juice volume, HCO3-, amylase, and trypsin output. Compared with CCK, the percentage increase in plasma secretin was 7.3x and 2.8x higher in response to oligopeptide (400 mg/h) and mixed amino acid solution (280 mg/h), respectively. An antisecretin serum almost completely inhibited volume flow and HCO3- output stimulated by oligopeptide as well as mixed amino acid solution, but not amylase and trypsin output. In contrast, loxiglumide significantly suppressed amylase and trypsin output stimulated by protein derivatives, but did not affect volume flow or HCO3- output.(ABSTRACT TRUNCATED AT 250 WORDS)
Regulation of PI hydrolysis and cAMP formation by muscarinic M3 receptor in guinea pig gallbladder
Authors: Takahashi T. , Kurosawa S. , Owyang C.
Published: December 2017

Carbachol (10(-8)-10(-3) M) produced two distinct biochemical responses in the guinea pig gallbladder smooth muscle: simulation of phosphoinositide (PI) hydrolysis and inhibition of forskolin-mediated adenosine 3',5'-cyclic monophosphate (cAMP) formation in a dose-dependent manner. The mean effective dose (ED50) concentration (1.6 x 10(-5) M) of carbachol-mediated stimulation of PI hydrolysis was 145 times greater than the ED50 concentration (1.1 x 10(-7) M) of carbachol mediated inhibition of cAMP formation. The inhibitory effect of carbachol on cAMP formation was antagonized by the pretreatment of pertussis toxin. To determine whether these two biochemical responses were mediated by the same or different subtypes of muscarinic receptors, the relative potencies of muscarinic receptor antagonists were calculated by Schild analysis. The M3 muscarinic antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) exhibited inhibitory constant (Ki) values at 0.3 and 1.2 nM in antagonizing the stimulation of PI hydrolysis and the inhibition of cAMP formation, respectively. The corresponding Ki values for pirenzepine, a muscarinic M1 antagonist, were 11 and 130 nM. The corresponding Ki values for AF-DX 116, a muscarinic M2 antagonist, were 34 and 450 nM. Thus 4-DAMP was 37x and 108x more potent than pirenzepine in antagonizing the stimulation of PI hydrolysis and the inhibition of cAMP formation, respectively. In addition, compared with AF-DX 116, 4-DAMP was 113x and 375x more potent in reducing stimulation of PI hydrolysis and inhibition of cAMP formation. Cholecystokinin (CCK) octapeptide (10(-10)-(10-6) M) caused a significant increase of PI hydrolysis but had no inhibitory effects on cAMP formation evoked by forskolin (10(-5) M).(ABSTRACT TRUNCATED AT 250 WORDS)
Canine salivary secretion from the submaxillary glands before and during retching
Authors: Furukawa N. , Okada H.
Published: December 2017

To investigate the changes in salivary secretion associated with emesis, salivary secretion from the submaxillary gland and centrifugal discharge from the parasympathetic postganglionic nerve fibers from the submandibular ganglion were measured after emetic stimulation in chloralose-anesthetized and paralyzed dogs. In the basal condition, saliva flow and the basal frequencies of single unit discharges from the parasympathetic nerve were very low. Esophageal and gastric distension and lingual nerve afferent stimulation generally increased salivary secretion and nerve activity. Administration of apomorphine (0.2-0.3 mg/kg im) or intragastric infusion of copper sulfate (10%, 50 ml) elicited an excitation of parasympathetic nerve activity, and salivary secretion was facilitated in parallel to this nerve activity. The excitatory responses induced by emetic stimulation were suddenly depressed in correspondence with retching activities. This depression in nerve activity appeared 200-400 ms after the beginning of retching volleys of the phrenic nerve and continued during retching. These results indicate that the salivary center receives excitatory and inhibitory inputs from two different sources before and during retching activities, respectively.
Esophageal myoelectric responses to magnetic stimulation of the human cortex and the extracranial vagus nerve
Authors: Aziz Q. , Rothwell J. C. , Barlow J. , Hobson A. , Alani S. , Bancewicz J. , Thompson D. G.
Published: December 2017

We describe the use of magnetic stimulation of the human motor cortex and the extracranial vagus nerve in the neck to study human esophageal electromyographic (EMG) responses. Diffuse cortical stimulation produced both an early and a late EMG response in the proximal esophagus with latencies of 10.9 +/- 0.5 and 43.4 +/- 4.3 ms, respectively. Increasing the stimulation intensity increased the amplitude and duration, but reduced the latency, of the early response (P < 0.01), whereas the late response remained unchanged. The early response, but not the late response, was facilitated when cortical stimulation was performed during valsalva maneuver. Maximal early response amplitudes were obtained by focal stimulation of either hemisphere 4-8 cm lateral to the vertex. Stimulation of the neck over the extracranial vagus nerve at the angle of the jaw also produced an early and a late EMG response in the proximal esophagus with latencies of 4.3 +/- 0.4 and 56.8 +/- 14.5 ms, respectively. Early responses were also recorded in the midesophagus and distal esophagus from either cortical or vagal stimulation, but amplitudes were smaller and rise times were longer, suggesting passive "volume conduction" from the proximal esophagus. Comparison of laryngeal, pharyngeal, and esophageal early EMG responses showed similar amplitudes and rise times, suggesting that the action potentials were generated locally at each site and that true esophageal responses were being detected.
Profile of IGF-binding proteins secreted by intestinal epithelial cells changes with differentiation
Authors: Oguchi S. , Walker W. A. , Sanderson I. R.
Published: December 2017

Previous reports have shown that gastrointestinal epithelial cells produce insulin-like growth factor-binding proteins (IGF-BP), which modulate the actions of IGF. This study aims to examine the relationship between differentiation and IGF-BP secretion by human intestinal epithelial cells and the effect of growth factors on their production. Caco-2 cells were cultured in serum-free media. IGF-BP secretion into the incubation media was analyzed by Western ligand blotting and immunoblotting. Caco-2 cells produced IGF-BP-2, IGF-BP-3, and IGF-BP-4. Secretion of IGF-BP-2 and IGF-BP-3 increased with differentiation, but IGF-BP-4 secretion diminished. The effect of exogenous growth factors on IGF-BP secretion was maximal at earlier stages of differentiation. IGF-I stimulated mainly IGF-BP-3 production, but epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) stimulated predominantly IGF-BP-4 secretion. Adding an anti-EGF receptor antibody to block autocrine TGF-alpha activity inhibited IGF-BP-4 production but stimulated IGF-BP-2 and IGF-BP-3. In conclusion, the profile of IGF-BP secretion changes with differentiation. IGF-I and EGF (or TGF-alpha) stimulate different types of IGF-BP, with autocrine TGF-alpha activity being a factor affecting IGF-BP production during differentiation.
Free concentrations of intracellular fluorescent anions determined by cytoplasmic dialysis of isolated hepatocytes
Authors: Weinman S. A. , Maglova L. M.
Published: December 2017

Intracellular organic ions exist in free solution bound to cytoplasmic proteins, partitioned within intracellular membranes, and enclosed in intracellular vesicles and organelles. The aim of this study was to develop a method for measurement of the free cytosolic concentration of organic ions. This was accomplished by measuring initial rates of diffusion between patch-clamp pipettes and cell cytoplasm and determining the null-point concentration of this process. Carboxydimethylfluorescein (CF) was used as a model compound. It readily diffused between cytoplasm and pipette, and there was a linear relationship between concentration in the pipette and equilibrium cell fluorescence. When cells previously loaded with CF were patched, intracellular fluorescence rapidly changed in a positive or a negative direction, depending on the concentration of CF in the pipette. The null point, defined as the concentration at which cells neither gained nor lost fluorescence, described the same relationship between free concentration and total cell fluorescence as that determined by direct loading of the cells to equilibrium. In hepatocytes preloaded with a fluorescent bile acid derivative, cholylglycylamidofluorescein (CGamF), by exposure (0.05 microM) for 30 min, the null point occurred at a CGamF concentration in the pipette of 0.6 microM. This value is 12 times greater than that in the bath. In conclusion, a new method is described that can measure free cytosolic concentrations of fluorescent molecules. It should prove useful in determining the intracellular location and state of transported organic ions.
Fasting enhances taurine transport by rat liver plasma membrane vesicles
Authors: Berkowitz D. , Hug P. , Sleight R. G. , Bucuvalas J. C.
Published: December 2017

Hepatic taurine stores are maintained by biosynthesis from the sulfur-containing amino acids, methionine and cysteine, and by uptake via a Na(+)- and Cl(-)-dependent transport system, which is specific for beta-amino acids. We hypothesized that liver stores of taurine are maintained by enhanced hepatic transport during fasting when dietary sources for taurine and its precursors are diminished. Liver plasma membrane vesicles, enriched for the basolateral domain, were prepared from adult male rats fasted for 72 h and from control rats. The maximum velocity for Na(+)-dependent taurine uptake was twofold greater for the fasted group compared with the control group (0.87 +/- 0.09 vs. 0.31 +/- 0.03 nmol.mg protein-1.min-1). The apparent Michaelis constant for taurine was also greater for fasted compared with control (154.0 +/- 0.5 vs. 80.0 +/- 2.0 microM). gamma-Aminobutyric acid, but not alanine or glutamine, abolished the effect of fasting on hepatic taurine transport. To determine the effect of fasting independent of changes in the lipid microenvironment, taurine uptake was measured in proteoliposomes reconstituted by inserting detergent-solubilized membrane proteins into asolectin vesicles. Taurine uptake by proteoliposomes reconstituted from membranes prepared from the fasted group was significantly greater than from the control group. We conclude that Na(+)-dependent taurine transport is enhanced in liver plasma membranes prepared from fasted rats. Our findings imply that enhanced taurine uptake with fasting is due to either an increased number of functional carriers or activation of existing transporters.
Apical acidification induces paracellular injury in canine gastric mucosal monolayers
Authors: Chen M. C. , Chang A. , Buhl T. , Tanner M. , Soll A. H.
Published: December 2017

We used primary monolayer cultures of enzyme-dispersed canine oxyntic mucosal cells mounted in Ussing chambers to characterize the apical barrier to H+. [3H]mannitol flux (MF) and [14C]inulin flux (IF) were used as size probes for tight junctions. Apical H+ produced a three-phase effect. In phase 1, as the apical pH was decreased from 7 to about 2.5, resistance (R) increased, but short-circuit current (Isc) did not change. In phase 2, an increased paracellular permeability developed at pH below 2.5-1.7, evidenced by decreased R and increased MF but not IF. Size sieving and monolayer integrity were preserved, and this paracellular leak was either fully reversed or stabilized by apical neutralization, depending on the duration of the paracellular leak. In phase 3, after sustained exposure to an apical pH below approximately 2, transepithelial integrity was lost; R decreased to fluid R, and both MF and IF increased. Basolateral acidification below pH 5.5 produced rapid monolayer disruption. Low concentrations of cytochalasin D (CD) decreased R and increased MF but not IF; apical acidification to pH 4 after CD increased R and decreased the MF, indicating reduced paracellular permeability by apical H+. Apical amiloride did not alter Isc; however, after 48 h of treatment with hydrocortisone and insulin, an amiloride-sensitive Isc component became evident. Our data indicate that the increase in R observed with apical acidification reflects decreased paracellular permeability and that the earliest injury with apical acidification is a selective paracellular leak.
Inhibition of ornithine decarboxylase does not prevent intestinal smooth muscle hyperplasia in the rat
Authors: Luck M. S. , Bass P.
Published: December 2017

The objective of this research is to examine the relationship between intestinal smooth muscle cell proliferation and polyamine metabolism. Proliferation of muscle is induced by serosal application of benzyldimethyltetradecylammonium chloride (BAC) to a segment of rat jejunum. This treatment destroys all of the longitudinal muscle, one-half of the circular muscle, and the myenteric and extrinsic nerves. The remaining muscle cells undergo mitosis and, by 15 days, the number of muscle cells is increased in both the longitudinal and circular muscle layers. Within 12 h after BAC treatment, there is an increase in the activity of ornithine decarboxylase (ODC) that returns to preinjury levels by 4 days. The smooth muscle content of the three polyamines putrescine, spermidine, and spermine is increased 6 h after injury, returning to preinjury levels within 1 day. DL-alpha-Difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, prevents the transient increases in both ODC activity and the three polyamines in the smooth muscle. In addition to preventing the transient increases in the three polyamines, DMFO has different effects on smooth muscle content of putrescine, spermidine, and spermine. Putrescine content is permanently depleted by DFMO. Spermidine and spermine content is initially decreased within 24 h by DFMO administration. In contrast to the persistent depletion of putrescine by DFMO, spermidine and spermine return to preinjury levels by 2 days. The maximal peak in DNA synthesis occurs 2 days after injury. In DFMO-administered animals, the maximal DNA synthesis occurs 5 days after injury. The increases in smooth muscle wet weight and DNA content 15 days after injury are not affected by DFMO.(ABSTRACT TRUNCATED AT 250 WORDS)
Evidence that hepatic triglycerides provide acylglycerides for synthesis of bile phosphatidylcholines
Authors: Patton G. M. , Fasulo J. M. , Robins S. J.
Published: December 2017

To determine the biochemical origin of bile phosphatidylcholines (PCs), rat liver perfusions with 16:1 fatty acid (FA) and [3H]glycerol were performed to generate novel radiolabeled bile and liver PCs and their hepatic glyceride precursors. Results showed total equilibration of bile and liver 16:1-16:1 PC when the specific activity of precursor glycerol-3-phosphate was kept constant. However, when the specific activity of glycerol-3-phosphate decreased during the labeling period and during a prolonged chase period with 17:1 FA and nonradiolabeled glycerol, the specific activity of bile 16:1-16:1 PC was appreciably higher than this same PC in the liver and during the chase period was even higher than its hepatic 16:1-16:1 acylglycerol precursors, phosphatidic acid and diglyceride. During the chase period with 17:1 FA, new radiolabeled 16:1-17:1 PC was formed, and again the specific activity of this PC in bile was greater than this PC and 16:1-17:1 phosphatidic acid and diglyceride in the liver. Only the specific activity of liver 16:1-16:1-(FA) triglyceride equaled or was high enough to support the formation of new bile 16:1-16:1 PC. These studies indicate that bile PCs do not directly derive from preexisting hepatic PCs or by de novo synthesis through phosphatidic acids and diglycerides, but likely originate by remodeling from a pool of hepatic triglycerides.
In vitro and in vivo regulation of hepatic mitogen-activated protein kinases in fetal rats
Authors: Boylan J. M. , Gruppuso P. A.
Published: December 2017

We have studied the role of mitogen-activated protein (MAP) kinases in fetal hepatocyte growth in vitro and in vivo. With myelin basic protein (MBP) as the phosphate acceptor, kinase activity in cultured fetal hepatocyte lysates increased fourfold after exposure to transforming growth factor-alpha (TGF-alpha) for 10 min. This TGF-alpha-responsive MBP kinase activity was accounted for by five distinct MAP kinase isoforms detected by Western immunoblotting. All had negligible activity in cultured fetal hepatocytes under basal conditions. Treatment of fetal hepatocytes with hepatocyte growth factor led to activation of the predominant isoforms, relative molecular weight (M(r)) = 42,000 and 44,000 in a manner indistinguishable from TGF-alpha, whereas insulin had no effect. All five of the immunoreactive MAP kinases were present in both fetal and adult liver homogenates. The M(r) = 42,000 and 44,000 isoforms were only minimally activated in vivo. We conclude that the mitogen-independent growth exhibited by fetal hepatocytes in primary culture is not associated with tonic activation of the MAP kinase system. Our data support the possibility that fetal hepatic growth may be, in part, independent of the action of growth factors as mediated via the MAP kinase system.
Protective role of NO in hepatic microcirculatory dysfunction during endotoxemia
Authors: Nishida J. , McCuskey R. S. , McDonnell D. , Fox E. S.
Published: December 2017

Nitric oxide (NO) has been reported to have a protective function in attenuating hepatic injury during endotoxemia or sepsis. As a result, the role of NO in attenuating the hepatic microcirculatory alterations associated with endotoxemia was investigated in mice by in vivo microscopy. The livers were examined 2 h after intravenous injection of Escherichia coli 0111:B4 lipopolysaccharide (LPS) alone or in combination with inhibitors of the synthesis of NO, NG-nitro-L-arginine methyl ester or NG-monomethyl-L-arginine. In the animals treated with the combination of NO synthase inhibitors and LPS, leukocyte adherence was increased threefold above that in animals treated with LPS alone. This was accompanied by a 33% reduction in sinusoidal blood flow. Simultaneous administration of L-arginine, but not D-arginine, eliminated these microcirculatory disturbances. The results demonstrate that inhibition of LPS-stimulated NO production results in an early hepatic microvascular inflammatory response to a dose of endotoxin which by itself is scarcely inflammatory. This suggests that NO plays a significant role in stabilizing the hepatic microcirculation during endotoxemia, thereby helping to protect the liver from ischemia and leukocyte-induced oxidative injury.
Activation of somatostatin receptor subtype 2 inhibits acid secretion in rats
Authors: Lloyd K. C. , Wang J. , Aurang K. , Gronhed P. , Coy D. H. , Walsh J. H.
Published: December 2017

Somatostatin is a potent inhibitor of gastric acid secretion. Recently, at least five distinct somatostatin receptor subtypes (SSTR) have been characterized and evaluated using relatively selective peptide analogues of somatostatin. We sought to determine which somatostatin receptor subtypes are involved in peripheral regulation of gastric acid secretion. Fasted, male Sprague-Dawley rats were anesthetized and were implanted with a double-lumen cannula in the stomach. Acid secretion was measured in gastric samples collected every 10 min by backtitration to pH 7. After a 30-min basal period, a 2-h intravenous infusion of pentagastrin (24 micrograms.kg-1.h-1 i.v.) was started. During the second pentagastrin hour, a 1-h intravenous infusion of either vehicle (0.1% canine serum albumin in 0.9% saline) or somatostatin receptor agonists was begun. The somatostatin receptor agonists included peptides with relative specificity for SSTR1-5 (somatostatin-14; 10 nmol.kg-1.h-1); SSTR2, SSTR3, and SSTR5 [SMS-(201-995); 10 nmol.kg-1.h-1]; SSTR2 (1-1,000 nmol.kg-1.h-1); SSTR3 (10-1,000 nmol.kg-1.h-1); and SSTR5 (10-1,000 nmol.kg-1.h-1). The SSTR2 agonist decreased pentagastrin-stimulated acid secretion dose dependently, from 82 +/- 7% of maximum acid output at 1 nmol.kg-1.h-1 to 4 +/- 7% of maximum at 100 nmol.kg-1.h-1. At 10 nmol.kg-1.h-1, the SSTR2 agonist inhibited acid secretion (40 +/- 7% of maximum) similarly to somatostatin (37 +/- 4% of maximum) and SMS-(201-995) (31 +/- 4% of maximum). The SSTR2 agonist inhibited acid secretion approximately 10- to 100-fold more potently than either the SSTR3 or the SSTR5 agonist. These results indicate that somatostatin regulates gastric acid secretion by activation of SSTR2 receptors.
Effects of sex steroid hormones on gastric emptying and gastrointestinal transit in rats
Authors: Chen T. S. , Doong M. L. , Chang F. Y. , Lee S. D. , Wang P. S.
Published: December 2017

In vitro studies have shown that estrogen and progesterone can affect the contractile response and myoelectric activity of the gastrointestinal smooth muscle. The present study was designed to investigate the effect of sex steroid hormones on gastric emptying and gastrointestinal transit were assessed in rats 15 min after intragastric instillation of a test meal containing charcoal and 51Cr. Gastric emptying was determined by measuring the amount of labeled chromium contained in the small intestine as a percentage of the initial amount received. Gastrointestinal transit was evaluated by calculating both the geometric center of distribution of the radiolabeled marker and the charcoal transit in the intestine. The experimental animals included diestrus rats; ovariectomized rats treated with vehicle, estradiol, and/or progesterone; and normal male and orchiectomized rats treated with vehicle or testosterone. Female rats in diestrus had a slower gastric emptying and a lesser geometric center value than ovariectomized rats. Estradiol inhibited gastric emptying but did not affect gastrointestinal transit. Progesterone increased gastric emptying. Progesterone at lower dose (10 mg/kg) decreased the geometric center compared with higher doses (20 or 40 mg/kg) or vehicle controls. A mixture of estradiol (10 micrograms/kg) and progesterone (20 mg/kg) inhibited gastric emptying to a similar degree as estradiol (10 micrograms/kg) did. Testosterone had no influence on gastric emptying or gastrointestinal transit. These results suggest that estradiol and a mixture of estradiol and progesterone inhibit, whereas progesterone enhances, gastric emptying. Testosterone did not play a role in gastrointestinal motility.