bluuebunny/crossref_metadata_2025 huggingface dataset:
Affinity labeling the bovine gallbladder cholecystokinin receptor using a battery of probes
Authors: Schjoldager B. , Powers S. P. , Miller L. J.
Published: December 2017

Although the gallbladder was the first recognized target of the peptide hormone cholecystokinin (CCK) and is a physiologically important target, only one preliminary report of the biochemical characterization of this receptor exists. Recently, a series of molecular probes for the affinity labeling of different domains of the pancreatic CCK receptor have been developed. In this work we report the application of several of those probes toward the biochemical characterization of the bovine gallbladder muscularis receptor. These include "long" (125I-Bolton-Hunter-CCK-33) and "short" (125I-D-Tyr-Gly-[Nle28,31)CCK-(26-33)]) probes chemically cross-linkable through their amino-terminal amino groups and monofunctional probes with their photolabile moieties at their amino terminus (2-diazo-3,3,3-trifluoropropionyl-125I-D-Tyr-Gly-[(Nle28,31) CCK-(26-33)]) and carboxyl terminus (125I-D-Tyr-Gly-[(Nle28,31,pNO2-Phe33)CCK-(26-33)]), that span the receptor-binding region. Each of these bound specifically and saturably to a preparation enriched in plasma membranes from bovine gallbladder muscularis (mean inhibitor constants: 5.2, 1.1, 0.8, and 1.8 nM, respectively). A major relative molecular weight (Mr) 70,000-85,000 band was specifically and reproducibly labeled with the appropriate apparent affinity by each of the probes, whereas labeling of minor bands of Mr 40,000-50,000, Mr 92,000, Mr 120,000, and Mr 200,000 was dependent on cross-linker type or concentration. These observations support the identification of the Mr 70,000-85,000 protein as the bovine gallbladder CCK-binding subunit and, since this is a different size from the pancreatic CCK-binding subunit, provide biochemical evidence for molecular heterogeneity of peripheral CCK receptors.
Differential localization of 5-HT1 receptors on myenteric and submucosal neurons
Authors: Galligan J. J. , Surprenant A. , Tonini M. , North R. A.
Published: December 2017

Intracellular recordings were made from guinea pig enteric neurons, and the effects of 5-hydroxytryptamine (5-HT) and the 5-HT1 selective agonists 5-carboxyamidotryptamine (5-CT) and 8-hydroxy-2-(n-dipropylamino)tetralin (DPAT) were studied on membrane potential and synaptic potentials. Most myenteric AH neurons were hyperpolarized when these agonists were applied by superfusion; this hyperpolarization was due to an increase in potassium conductance. Membrane hyperpolarizations to 5-HT, 5-CT, or DPAT were never observed in submucous neurons. Fast nicotinic excitatory postsynaptic potentials (EPSPs) and slow EPSPs recorded from S neurons in the myenteric plexus were suppressed by 5-HT, 5-CT, and DPAT; slow EPSPs in myenteric AH neurons were also inhibited by these agonists. Fast and slow EPSPs recorded from submucous S neurons were not affected by 5-CT or DPAT. However, slow EPSPs recorded from submucous AH neurons were readily blocked by 5-CT and DPAT. The results indicate that 5-HT1 receptors are located on the cell bodies of myenteric but not submucosal neurons. The nerve terminals that release the mediator or mediators of fast and slow synaptic potentials in myenteric neurons also have 5-HT1 receptors and presumably arise from other myenteric neurons; the nerve terminals responsible for the slow EPSP to AH neurons seem to be the only elements of the submucous plexus that express 5-HT1 receptors.
Control mechanisms of sphincter of Oddi contraction rate in the opossum
Authors: Suzuki T. , Dodds W. J. , Sarna S. K. , Hogan W. J. , Komorowski R. A. , Itoh Z.
Published: December 2017

Our aim in this study is to determine whether extrinsic autonomic nerves regulated spike-burst rate in the opossum sphincter of Oddi (SO) during fasting or after feeding. We implanted electrodes on the distal SO, proximal SO, gastric antrum, duodenum, and jejunum of 20 animals. A cut transection and reanastomosis was done at different levels of the SO to interrupt putative extrinsic or intrinsic nerves, or the SO was painted with phenol to impair extrinsic nerves. Like controls, animals with a cut and reanastomosis at the proximal SO or at the SO-duodenal junction showed a normal fasting pattern of cyclic changes in SO spike-burst rate. In contrast, animals treated by a distal SO cut or phenol treatment at the distal SO lost the normal cyclic pattern of SO spike bursts and had a constant rate of approximately 4/min. A cut through the middle SO uncoupled the spike bursts in the proximal and distal SO. After feeding, all animals developed an SO spike-burst rate of 5-6/min that lasted for several hours. We conclude that the normal fasting pattern of SO spike bursts is regulated by extrinsic nerves that ascend cephalad along the sphincter segment, whereas the sustained increase in SO spike-burst rate after feeding is at least in part hormonal.
Methodological errors in radioisotope flux measurements
Authors: Egnor R. W. , Vaccarezza S. G. , Charney A. N.
Published: December 2017

We examined several sources of error in isotopic flux measurements in a commonly used experimental model: the study of 22Na and 36Cl fluxes across rat ileal tissue mounted in the Ussing flux chamber. The experiment revealed three important sources of error: the absolute counts per minute, the difference in counts per minute between serial samples, and averaging of serial samples. By computer manipulation, we then applied hypothetical changes in the experimental protocol to generalize these findings and assess the effect and interaction of the absolute counts per minute, the sampling interval, and the counting time on the magnitude of the error. We found that the error of a flux measurement will vary inversely with the counting time and the difference between the consecutive sample counts per minute used in the flux calculations and will vary directly with the absolute counts per minute of each sample. Alteration of the "hot" side specific activity, the surface area of the tissue across which flux is measured and the sample volume have a smaller impact on measurement error. Experimental protocols should be designed with these methodological considerations in mind to minimize the error inherent in measuring isotope flux.
On-line computer analysis of human lower esophageal sphincter relaxation
Authors: Castell J. A. , Dalton C. B. , Castell D. O.
Published: December 2017

Lower esophageal sphincter (LES) relaxation is usually interpreted by a subjective, mostly qualitative analysis. We studied sphincter relaxation in humans using a computer program for on-line analysis that provides objective measures of gastric pressure, resting LES pressure, percent of relaxation, residual pressure during relaxation, duration of relaxation, and area of relaxation. The program was validated by comparing computer-analyzed values to mean values obtained from manual readings by five individuals. Excellent correlation was obtained for all standard parameters. The parameters of LES relaxation for both wet and dry swallows were similar using either a carefully placed single recording orifice or a Dent sleeve. The one exception was the duration of LES relaxation, which was significantly shorter with the sleeve. All relaxation parameters and peristaltic velocity were then studied in 10 volunteers during 5 dry and 5 wet swallows under base-line conditions and after both atropine (10 micrograms/kg iv) and bethanechol (40 micrograms/kg sc). These studies showed that LES relaxation is affected by type of swallow (dry vs. wet). Percent of relaxation may not be the best measure of relaxation because it is too dependent on resting pressure. Residual pressure is not dependent on base-line pressure and may better define relaxation. Duration of the relaxation is dependent on the velocity of the peristaltic wave.
Kinins as mediators of intestinal secretion
Authors: Gaginella T. S. , Kachur J. F.
Published: December 2017

Kinins are small peptides that have diverse biological actions. Concentrations of kinins in the nanomolar or subnanomolar range induce intestinal smooth muscle contraction and evoke mucosal electrolyte secretion. Hyperkininemia is associated with effects on gastrointestinal motility and intestinal mucosal inflammation. Bradykinin and kallidin are the predominant kinins with effects on the gastrointestinal tract of mammals. Bradykinin stimulates chloride ion secretion by the guinea pig and rabbit ileum, rabbit colon, rat colon and monolayers of human HCA-7 cells. Kinins directly or indirectly stimulate phospholipase A2 and phospholipase C. Cells in the lamina propria of the mucosa (e.g., fibroblasts, mast cells, leukocytes), by liberating cyclooxygenase and lipoxygenase metabolites of arachidonic acid, are involved in the kinin response; direct effects on epithelial cells cannot be ruled out, however. Antagonists now exist for kinin receptors. Based on studies with these antagonists in smooth muscle preparations, two subgroups of kinin receptor have been identified. The B2-type receptor appears to be responsible for both the contraction of ileal muscle and ileal secretion. Kinins are probably more important as pathophysiological rather than as physiological mediators. They may amplify the effect of inflammatory products that induce intestinal secretion. The precise involvement of kinins in clinical mucosal secretory states and diarrhea will require quantitative assessment of their levels during each phase of mucosal inflammation. Additional studies on the mechanism of action of kinins will be essential in designing therapy to mitigate the symptoms associated with mucosal inflammation.
Prostaglandins augment muscarinic responses in the rabbit cecum
Authors: Nakao K. , Kitamura K. , Kuriyama H.
Published: December 2017

Effects of prostaglandin E2, F2 alpha, and synthetic thromboxane A2 (PGE2, PGF2 alpha, and STA2, respectively) on electrical and mechanical responses of the rabbit cecum were investigated. Transmural electrical stimulation evoked an excitatory junction potential (EJP) and contraction, events that were inhibited by 1 microM atropine or 0.3 microM tetrodotoxin. Indomethacin (up to 30 microM) modified neither the membrane potential nor the muscle tone but did inhibit amplitudes of the EJP and the twitch contraction. In the presence of 30 microM indomethacin, PGE2 (below 1 nM) had no effect on the membrane potential or muscle tone, while PGE2 (above 10 nM) contracted the cecal tissues, without depolarization. PGE2 enhanced the twitch contraction and restored the EJP in the presence of 30 microM indomethacin. Acetylcholine (0.1-3 microM) depolarized the membrane, but in the presence of 30 microM indomethacin, this depolarization was inhibited. PGE2 (0.1-100 nM) prevented these inhibitory actions of indomethacin. PGF2 alpha (0.1-100 nM) had weaker actions than PGE2 while STA2 (0.1-100 nM) had no effect on muscarinic responses. Thus muscarinic responses are augmented by primary prostaglandins.
Sphincteric action of the diaphragm during a relaxed lower esophageal sphincter in humans
Authors: Mittal R. K. , Rochester D. F. , McCallum R. W.
Published: December 2017

We studied the effects of involuntary and voluntary contraction of the diaphragm on esophagogastric junction (EGJ) pressure during esophageal distension in healthy human volunteers. The EGJ pressure was monitored using a Dent sleeve device. Along with the pressure we concurrently monitored diaphragm electromyogram (EMG) using intra-esophageal bipolar electrodes that were placed on the nonpressure sensing surface of the sleeve device. Graded esophageal distensions were performed by graded inflations of a 2-cm-diameter balloon that was positioned 7 cm above the EGJ. The graded esophageal distensions caused a graded increase in the amplitude of lower esophageal sphincter (LES) relaxation (end-expiratory EGJ pressure). In a majority of the subjects, esophageal distension had no effect on spontaneous inspiratory EGJ pressure increase and diaphragm EMG. During sustained LES relaxation of greater than 70% induced by sustained esophageal distention, graded voluntary contractions of the diaphragm induced proportional increases in the EGJ pressure and diaphragm EMG. The EGJ pressure and diaphragm EMG were similar during diaphragmatic contraction both before and during esophageal distension. During a maximal and sustained diaphragm contraction, esophageal distension had no effect on the EGJ pressure. We conclude that there are two distinct sphincteric mechanisms at the EGJ, the LES and crural diaphragm, and they respond differently to distension of the distal esophagus.
Effects of hCGRP I and II on gastric blood flow and acid secretion in anesthetized rabbits
Authors: Bauerfeind P. , Hof R. , Hof A. , Cucala M. , Siegrist S. , von Ritter C. , Fischer J. A. , Blum A. L.
Published: December 2017

Effects of intravenously administered human calcitonin gene-related peptides (hCGRP) I and II on regional blood flow and gastric acid secretion were examined in barbiturate-anesthetized rabbits. Blood flow was measured by injection of radioactively labeled microspheres at 0, 10, 20, 30, and 60 min. hCGRP I and II and vehicle were infused intravenously in five rabbits in rising doses of 0.01 (0-10th min), 0.03 (11-20th min), and 0.1 microgram.kg-1.min-1 (21-30th min). hCGRP I and II increased gastric blood flow dose dependently. Moreover, hCGRP I raised regional conductance (inverse of vascular resistance) in the stomach, duodenum, heart, brain, and skeletal muscle. As a result of the increased total peripheral conductance the mean arterial pressure was reduced, but the cardiac output remained unchanged. hCGRP II increased blood flow and conductance selectively in the stomach and the pancreas. The total peripheral conductance and mean arterial pressure remained unchanged. Apparently, hCGRP II exerts a more localized effect on the stomach than hCGRP I. hCGRP I and II did not affect basal gastric acid secretion. Pentagastrin-stimulated acid secretion was increased by 28% with hCGRP I (0.025 micrograms.kg-1.min-1) and decreased by 27% with hCGRP II (0.025 micrograms.kg-1.min-1). The inverse effect of hCGRP I and II and the parallel stimulation of blood flow brought about with hCGRP I and II indicate a different mode of action of the peptides on gastric blood flow and gastric acid secretion.
Dietary CHO and stimulation of carbohydrases along villus column of fasted rat jejunum
Authors: Morrill J. S. , Kwong L. K. , Sunshine P. , Briggs G. M. , Castillo R. O. , Tsuboi K. K.
Published: December 2017

Adult rats when fed a high carbohydrate diet of 70% sucrose or glucose for 24 h following a 4-day fast showed increased concentrations of intestinal sucrase-isomaltase (EC 3.2.1.48, EC 3.2.1.10) and maltase-glucoamylase (EC 3.2.1.20) but not lactase-phlorizin hydrolase (EC 3.2.1.23, EC 3.2.1.62). The concentration increases of these enzymes were accompanied by corresponding acceleration of their synthesis rates. Contrary to earlier studies by others, suggesting that upper villus cells in the fasted intestine are unresponsive to stimulation of sucrase activity by refeeding a high-sucrose diet, the concentration increases of both sucrase-isomaltase and maltase-glucoamylase were seen to occur in cells all along the length of the villus column. The earlier studies differed from the present study by basing enzyme assays relative to protein rather than the DNA content of villus cell fractions. We have shown that villus cells increase their protein content severalfold while migrating to villus tip, providing the basis for the difference between earlier and the present findings. Further evidence that stimulation of sucrase-isomaltase and maltase-glucoamylase by high carbohydrate is not restricted to the crypt and lower villus region was obtained by the finding that their synthesis rates appeared to be equally stimulated along the length of the villus column.
Reduction of 7-ketolithocholic acid by human liver enzyme preparations in vitro
Authors: Amuro Y. , Yamade W. , Kudo K. , Yamamoto T. , Hada T. , Higashino K.
Published: December 2017

The formation of chenodeoxycholic and ursodeoxycholic acids from 7-ketolithocholic acid by human liver preparations was examined in vitro. Liver preparations were incubated with 7-ketolithocholic acid at pH 5.5 in a sodium-potassium-phosphate buffer containing NADPH or NADH. The products formed were analyzed by gas chromatography and gas chromatography-mass spectrometry. Results showed that chenodeoxycholic and ursodeoxycholic acids could be formed from 7-ketolithocholic acid by human liver enzyme(s). The enzyme(s) required NADPH but not NADH as coenzyme and was localized largely in the microsomes. The conjugated 7-ketolithocholic acid, especially the taurine conjugated, was predominantly reduced to chenodeoxycholic acid, whereas the unconjugated 7-ketolithocholic acid was not reduced well to either chenodeoxycholic acid or ursodeoxycholic acid. Thus the reduction of 7-ketolithocholic acid by human liver enzyme(s) was found to be dependent on whether the substrate was conjugated or not.
Functional localization of specific receptors mediating gastrointestinal motor correlates of vomiting
Authors: Lang I. M. , Marvig J.
Published: December 2017

The gastrointestinal motor correlates of vomiting consist of two contractile events, 1) a giant retrogradely propagated contraction of the upper small intestine, the retrograde giant contraction (RGC) and 2) a series of post-RGC phasic contractions that occur primarily in the lower small intestine. The effects of cholinergic, dopaminergic, serotonergic, and opioid receptor antagonists and an opioid receptor agonist on vomiting and its gastrointestinal motor correlates initiated by apomorphine (APO), CuSO4, or cholecystokinin octapeptide (CCK-8) were determined in awake dogs. Atropine blocked the retrograde giant contraction only, and hexamethonium blocked all jejunoileal motor responses activated by APO, CuSO4, or CCK-8. Domperidone blocked all effects of APO only, whereas haloperidol, methysergide, 1-(1-naphthyl) piperazine, and fentanyl blocked or inhibited responses to both APO and CuSO4. None of the dopaminergic, serotonergic, or opioid receptor antagonists or the opioid receptor agonist affected the gastrointestinal motor responses to CCK-8. Cinanserin or Sch 23390 had no effect on any of the responses activated by APO, CuSO4, or CCK-8. These results suggested that D2 dopaminergic and 5-HT2 serotonergic receptors of the emetic central pattern generator mediate vomiting and its gastrointestinal motor correlates, whereas opioid receptors may mediate tonic inhibition of these responses. In addition, peripheral muscarinic or nicotinic cholinergic receptors but not peripheral 5-HT2, dopaminergic, or opioid receptors mediate the gastrointestinal motor correlates of vomiting.
Intraenterocyte distribution of absorbed lipid and effects of phosphatidylcholine
Authors: Parlier R. D. , Frase S. , Mansbach C. M.
Published: December 2017

The present studies were designed to investigate the distribution of absorbed lipid in intestinal mucosal cells and to identify the chylomicron precursor pool. Rats were infused intraduodenally with glyceryl tri[9,10(n)-3H]oleate (135 mumol/h); other rats were in addition infused with 9 mumol/h of phosphatidylcholine. After 5-h infusion the proximal one-half of intestine was removed and the mucosa obtained. It was found that 50% of the radioactivity in the whole homogenate pelleted on centrifugation at 75,000 g.min. The supernatant was further fractionated by high-speed centrifugation resulting in a floating lipid layer, a supernatant, and a microsomal pellet. The results showed that these subcellular fractions had a triacylglycerol specific activity 46-52% of the infusate's specific activity. Including phosphatidylcholine in the duodenal lipid infusion increased the triacylglycerol specific activity of all subcellular fractions (70%) resulting in a specific activity approaching that of the infusate, which would be expected of chylomicron triacylglycerol. These studies demonstrate 1) that considerable mucosal lipid is distributed into a low-speed pellet, 2) that mucosal triacylglycerol specific activity can be greatly increased by including phosphatidylcholine in a lipid infusion, and 3) that despite obtaining multiple subcellular fractions, the chylomicron precursor pool could not be clearly identified in the mucosa of control rats.
Dissociated effects of corticotropin-releasing factor on acid secretion and blood flow
Authors: Thiefin G. , Leung F. W. , Tache Y. , Guth P. H.
Published: December 2017

Peripherally administered corticotropin-releasing factor (CRF) inhibits gastric acid secretion in the rat via unknown mechanisms. Because intravenous CRF can produce marked systemic hypotension, the hypothesis that its antisecretory effect is due to a reduction in gastric mucosal blood flow (MBF) was tested. Corpus MBF was measured by hydrogen gas clearance in fasted, urethan-anesthetized rats. CRF (15 nmol.kg-1.h-1 iv) had no significant effect on MBF but significantly decreased systemic blood pressure (BP) by 21.8 +/- 3.4%. This dose of CRF significantly decreased acid secretion stimulated by 40 micrograms.kg-1.h-1 pentagastrin iv but did not decrease MBF despite a 30.3 +/- 2.3% decrease in BP. To avoid the confounding effect of systemic hypotension on MBF, close gastric intra-arterial infusions were performed. CRF (1.7 nmol/h ia) had no effect on BP and did not increase MBF. CRF (3.4 nmol/h ia) slightly decreased BP without modifying MBF. In contrast, histamine (200 micrograms/h ia) significantly increased MBF by 58 +/- 11%. We conclude that 1) the peripheral antisecretory effect of CRF is not related to a decrease in MBF, 2) the maintenance of MBF during CRF-induced hypotension is not due to a direct vasodilator effect of CRF on the gastric microvasculature.
Time-related changes in microsphere entrapment in canine jejunum
Authors: Prokopiw I. , Dinda P. K. , Beck I. T.
Published: December 2017

To assess the validity of repeated blood flow measurements using the microsphere technique, the apparent blood flows in the anatomic layers of the jejunum were determined from the entrapment of 9-, 11.5-, and 17-micron microspheres at 1.5, 15, 30, and 60 min after their injection. The entrapment of 17-micron spheres in the mucosa plus submucosa and in the muscularis propria remained similar at all times, but these spheres migrated (P less than 0.01) from the submucosa to the mucosa. By 1.5 min, 5 +/- 2% of 11.5-micron spheres had shunted, but no subsequent shunting was observed. No migration of 11.5-micron spheres from the mucosa, submucosa or the muscularis was observed. The shunting of 9-micron spheres from the whole wall increased from 19 +/- 4% at 1.5 min to 40 +/- 4% at 60 min (P less than 0.001). These data suggest that 17-micron spheres can only fractionate the blood flow of the whole wall into that of the mucosa plus submucosa and that of the muscularis propria, while 11.5-micron spheres may measure fractional flow to the submucosa separately. The continued washout of 9-micron microspheres precludes their use for repeated blood flow measurements.
Effect of CO2 on pHi in rabbit parietal, chief, and surface cells
Authors: Seidler U. , Carter K. , Ito S. , Silen W.
Published: December 2017

We investigated the pH recovery mechanisms in rabbit parietal, chief, and surface cells during pH shifts induced by introduction or removal of exogenous CO2-HCO3-. Intracellular pH (pHi) was measured using the fluorescent dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescin (BCECF). Gastric cells were highly purified by density gradient centrifugation and elutriation. When cells suspended in N-2-hydroxyethylpiperazene-N'-2-ethanesulfonic acid (HEPES)-100% O2, extracellular pH (pHo) 7.4, were exposed to 24 mM HCO3- -5% CO2, pHo 7.4, all cells quickly acidified by 0.3-0.4 pH units. Almost complete pH-recovery occurred within 15 min. In parietal cells, 70% of this recovery was dependent on the presence of extracellular Na+ (Nao+) and was blocked by 1 mM amiloride. The Na+-independent recovery was blocked by intracellular Cl- depletion or by 0.4 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). In chief cells and surface cells no recovery occurred in the absence of NaO+, and 1 mM amiloride blocked pH recovery in Na+-containing buffer. On removal of HCO3- -CO2, the cells alkalinized, and subsequent pH recovery was fast, substantially extracellular Cl- (ClO-) and DIDS inhibitable in parietal cells but slow and ClO- -independent in chief and surface cells. These results suggest that during intracellular acidification the Na+-H+ exchanger is the major pH regulator in these three gastric cell types even in the presence of HCO3-. During alkalinization the Cl- -HCO3-(OH-) exchanger is the predominant pH recovery mechanism in parietal, but not in chief and surface cells. In parietal cells, this exchanger is also involved in recovery from acidification.
Identification and characterization of a pancreatic intrinsic factor in the dog
Authors: Batt R. M. , Horadagoda N. U. , McLean L. , Morton D. B. , Simpson K. W.
Published: December 2017

An intrinsic factor has been identified in the canine pancreas, and output and properties of this protein have been compared with those of gastric intrinsic factor in the dog. Mean concentrations of intrinsic factor and peak outputs per minute were approximately 5- to 10-fold higher in pure pancreatic juice after stimulation with secretin and cholecystokinin, respectively, than in pentagastrin-stimulated gastric juice. Purified gastric and pancreatic intrinsic factors had an identical molecular mass of 65 kDa, estimated by gel filtration on Sephacryl S-200, while sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated single bands corresponding to 53 kDa. Immunoblots showed that rabbit polyclonal antiserum to canine gastric intrinsic factor cross-reacted with canine pancreatic intrinsic factor. Gastric and pancreatic intrinsic factor-cyano[57Co]cobalamin complexes exhibited comparable association constants for ileal receptors in canine brush-border vesicles, while there was minimal binding to jejunal vesicles. These findings demonstrate that the canine pancreas is an important source of an intrinsic factor that closely resembles gastric intrinsic factor in the dog.
Neuropeptide Y augments adrenergic contractions at feline lower esophageal sphincter
Authors: Parkman H. P. , Reynolds J. C. , Ogorek C. P. , Kicsak K. M.
Published: December 2017

The purpose of this study was to determine the anatomic and physiological interactions between neuropeptide Y (NPY) and adrenergic transmitters at the feline lower esophageal sphincter (LES). Intraluminal pressures of the esophagus, LES, and gastric fundus were recorded in anesthetized cats. In a separate group of cats, gastroesophageal junctions were removed after locating the LES manometrically. Adjacent sections were stained with antibodies for NPY and tyrosine hydroxylase (TH). Indirect immunofluorescence revealed staining of TH- and NPY-like immunoreactive (LI) nerves in the circular muscle of the LES. Staining was also present for NPY-LI in longitudinal muscle, muscularis mucosa, periarterial nerves, and nerves and cell bodies of the myenteric plexus of the LES. In vivo, NPY produced a biphasic effect at the LES with an initial contraction (lasting approximately 30 s) followed by a relaxation (lasting approximately 3 min). NPY at the dose giving 50% of the maximum response (10(-6) g/kg) induced a contraction of 14.8 +/- 5.0 mmHg (P less than 0.05) followed by a decrease of basal LES pressure from 29.2 +/- 4.7 to 19.4 +/- 2.9 mmHg (P less than 0.01). Propranolol and phentolamine had no influence on either effect of NPY.(ABSTRACT TRUNCATED AT 250 WORDS)
Ultrastructural analysis of VIP internalization in rat beta- and acinar cells in situ
Authors: Anteunis A. , Astesano A. , Portha B. , Hejblum G. , Rosselin G.
Published: December 2017

We perfused the pancreas with 125I-labeled vasoactive intestinal peptide (VIP) to follow the concomitant distribution of radioactivity in beta- and acinar cells as a function of time. This distribution was quantitated by computer-assisted analysis of high-resolution video autoradiographs. Density labeling was expressed as normalized specific activity (disintegration density per volume density). Immediately after a 4-min perfusion of 125I-VIP, labeling in beta-cells was mainly concentrated on the cell surface and peripheral tubules and vesicles. After three 30-s pulses of 125I-VIP, separated by intervals of 3.5 min of buffer perfusion, lysosome-like structures were heavily labeled. When VIP internalization was prolonged, labeling was similar to that observed with the 4-min perfusion, indicating a high VIP disposal rate in the lysosome-like structures. In acinar cells, labeling persisted on the surface and the early vacuolar system. We conclude the following: 1) an active endocytotic system, linked to the transport and sorting of a neuromediator, is present in beta-cells; and 2) the differences between the distribution of labeling in acinar and beta-cells suggest that the regulation of VIP internalization is tissue specific.
Length-tension relationship of striated muscle of cat external anal sphincter
Authors: Krier J. , Meyer R. A. , Percy W. H.
Published: December 2017

The active and passive length-tension curves of small strips of cat external anal sphincter (EAS) were examined in vitro. The striated muscle fibers were arranged perpendicular to the long axis of the longitudinal smooth muscle cells of the longitudinal layer of the anal canal. Histological examination indicated that the strips were comprised of striated muscle fibers oriented in the long axis of the strip. Electrical field stimulation elicited twitch and tetanus responses that were not altered by the administration of gallamine triethiodide (10(-6) to 10(-4) M), a neuromuscular blocking agent. At 37 degrees C the time from the onset of the twitch contraction to the development of peak force ranged from 30 to 37 ms, the time from peak force to one-half relaxation ranged from 20 to 25 ms. The maximum active twitch and tetanus tension (Po) averaged 0.23 and 0.90 kg/cm2, respectively. Active tension could be developed over a range of 0.6-1.4 optimum length (Lo). The passive length-tension curve showed that the muscle had significant passive tension at lengths below the Lo for tension development and a high passive tension at Lo (average of 44% of active isometric twitch tension, 12.2% of active isometric tetanus tension) and above Lo. We conclude that the passive length-tension curve for the EAS is stiffer than that of typical mammalian skeletal muscles. We suggest that this difference is related to the adaptation of the EAS to its sphincteric function as a component of a hollow organ.
Effects of methylene blue on rhythmic activity and membrane potential in the canine proximal colon
Authors: Sanders K. M. , Burke E. P. , Stevens R. J.
Published: December 2017

The hypothesis that methylene blue has a direct effect on colonic muscle cells was tested. Intracellular recordings were made from cross-sectional preparations of canine proximal colon. Cells through the circular layer were impaled and membrane potentials ranging from -81 mV at the submucosal surface to -46 mV at the myenteric border were recorded. Methylene blue (10(-5) M) depolarized cells near the submucosal border by an average of 43 mV and slow-wave activity ceased. The loss of slow-wave activity could be explained by the depolarizing effects of methylene blue rather than a specific effect of methylene blue on the pacemaker mechanism. Other experiments suggested that the depolarizing effects of methylene blue were not confined to muscle cells within the submucosal pacemaker region. Depolarization of cells was noted throughout the circular layer, but the magnitude of the depolarization decreased with distance from the submucosal border. After methylene blue, the gradient in resting potential across the circular layer was greatly reduced or abolished. The data suggest that methylene blue is not necessarily a specific probe for interstitial cells and has direct effects on smooth muscle cells in the canine proximal colon. This effect is similar to treatments that are known to block the electrogenic sodium pump.
Oxygen metabolites modulate prostaglandin E2 production by isolated gastric mucosal cells
Authors: Olson C. E. , Chen M. C. , Amirian D. A. , Soll A. H.
Published: December 2017

We previously found that the small cell fraction of isolated cells from canine gastric mucosa is a major producer of prostaglandin E2 (PGE2) and identified macrophages as the predominant cellular source. Prostaglandin-H synthase activity is dependent on the continuous presence of hydroperoxides. Because reactive oxygen metabolites may mediate mucosal injury in inflammatory or ischemic disease, we studied the release of PGE2 by isolated gastric cells during exposure to an oxygen metabolite-generating system, xanthine and xanthine oxidase. We found a concentration-dependent relationship between xanthine oxidase concentration and PGE2 production without cell lysis. The maximum PGE2 production stimulated by oxidants was equivalent to the maximum PGE2 response to bradykinin and A23187. The chief and parietal cell fractions produced very little PGE2 with xanthine oxidase concentrations that stimulated maximal PGE2 production in the small cell fraction. Uric acid did not stimulate PGE2 production. Catalase completely inhibited the response, while superoxide dismutase had a partial inhibitory effect. Hydrogen peroxide stimulated concentration-dependent PGE2 production with an ED50 of approximately 5 microM. We concluded that reactive oxygen metabolites stimulate PGE2 production by the small cell fraction of gastric mucosa.
Uptake and distribution of transferrin and iron in perfused, iron-deficient rat liver
Authors: Holmes J. M. , Morgan E. H.
Published: December 2017

Uptake of transferrin and iron by the rat liver was investigated by perfusion in vitro with 125I-59Fe-labeled rat transferrin and subcellular fractionation on sucrose density gradients. Most of the 125I-transferrin was located in a low-density vesicle fraction. The 59Fe was in three peaks, of lower, the same, and higher densities than the transferrin peak. Iron deficiency resulted in a large increase in transferrin and iron uptake into all subcellular fractions. When livers were perfused with increasing concentrations of transferrin the uptake into the different peaks of transferrin and iron increased in a curvilinear fashion, which indicated that uptake occurred by saturable and nonsaturable processes, both of which increased in iron deficiency. In contrast, the uptake of 131I-labeled rat serum albumin increased linearly with concentration, and there was no difference between control and iron-deficient livers. It is concluded that iron deficiency leads to an increase in the number of high-affinity transferrin receptors and receptor-mediated endocytosis of transferrin. It also increases a nonsaturable transferrin uptake process that is probably due to adsorptive, but selective, endocytosis of transferrin.
Identification and function of brain stem neurons regulating rat ileal water absorption
Authors: Martin K. , Kong T. H. , Renehan W. , Schurr A. , Dong W. , Zhang X. , Fogel R.
Published: December 2017

The central nervous system (CNS) regions regulating ileal water and ion absorption are unknown. We determined 1) the CNS origin of brain stem neurons that directly innervate the rat ileum, and 2) that these neurons influence intestinal water absorption. Horseradish peroxidase (HRP) was injected into the muscle layer of the rat ileum. The brains were examined for HRP reaction product (HRPRP) 3, 5, or 7 days later. Only cell bodies of the dorsal motor nucleus of the vagus (DMNV) were labeled. Unilateral cervical vagotomy prevented deposition in the ipsilateral DMNV. To determine whether the DMNV regulates ileal water absorption, electrical and chemical stimulation (30 microA, 4 Hz, 0.2 ms, and 300 pmol L-glutamate every 5 min, respectively) were used. Both the DMNV and the adjacent nucleus tractus solitarius (NTS) were stimulated, causing a reduction in water absorption. Bilateral vagotomy prevented the effect of bilateral electrical stimulation, but unilateral vagotomy did not prevent the decrease due to ipsilateral stimulation. These studies show that 1) the DMNV innervates the ileum, and 2) alteration of vagal efferent activity by stimulation of the DMNV and NTS reduces ileal water absorption.
Mucosal arachidonate metabolism and intestinal ischemia-reperfusion injury
Authors: Mangino M. J. , Anderson C. B. , Murphy M. K. , Brunt E. , Turk J.
Published: December 2017

Mucosal arachidonic acid metabolism was examined after 3 h of ischemia and 1 h of reperfusion in isolated ileal segments in the dog. The cyclooxygenase products thromboxane B2, 6-ketoprostaglandin F1 alpha, and prostaglandin E2 increased by 365%, 97%, and 158%, respectively, after ischemia and reperfusion but were not altered after 3 h of ischemia alone. The potent chemotactic lipoxygenase product leukotriene B4 (LTB4) increased by 687% after ischemia and reperfusion and was not affected by ischemia without reperfusion. In addition, tissue production of the thiol ether leukotrienes (LTC4, LTD4, and LTE4) increased threefold after ischemia and reperfusion. Quantitation of regionally isomeric hydroxy acids produced from arachidonate revealed a 300% increase in 12-hydroxyeicosatetraenoate (12-HETE) after intestinal ischemia and reperfusion without a change in other isomers (15-HETE and 5-HETE). Stereochemical analysis of 12-HETE demonstrated exclusive synthesis of the S-enantiomer. A significant and time-dependent decrease in intestinal blood flow also occurred during reperfusion. Administration of the dual cyclooxygenase-lipoxygenase synthesis inhibitor BW755C (1 mg/kg ia) did not alter time-dependent decreases in blood flow and failed to inhibit eicosanoid synthesis. Histologic examinations of intestinal samples revealed significant mucosal damage associated with ischemia alone and ischemia after reperfusion. This study indicates that intestinal ischemia-reperfusion injury is associated with dramatic alterations in mucosal production of vasoactive eicosanoids and with changes in blood flow that occur during reperfusion but not during ischemia alone. These events may be involved in the pathology characteristic of this injury.
Na-HCO3 cotransport in rabbit parietal cells
Authors: Townsley M. C. , Machen T. E.
Published: December 2017

Na- and HCO3-coupled recovery of intracellular pH (pHi) after acid loading was demonstrated in rabbit parietal cells. Intracellular pH of individual resting (i.e., cimetidine treated) parietal cells was continuously measured using microspectrofluorometry of the pH sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, acetoxymethylester. Parietal cells were acidified utilizing NH4Cl or by lowering external pH to 6.2. By means of either technique pHi decreased from 7.1 to approximately 6.6. In solutions containing 1 mM amiloride, which blocked Na-H exchange, pHi recovered only if Na and HCO3 were both present. This amiloride-resistant, Na- and HCO3-dependent pHi recovery was inhibited by 100 microM H2 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (reversibly) and occurred at equal rates in Cl-containing and Cl-free solutions. In NaCl solutions buffered with HCO3-CO2 and containing amiloride, after an acid load pHi recovers to pHi 7.0-7.1 solely through the activity of the Na-HCO3 cotransporter. Therefore, resting parietal cells appear to have a Na-HCO3 cotransporter that serves as a base loader under physiological conditions.
Volume-regulatory K+ fluxes in the isolated perfused rat liver: characterization by ion transport inhibitors
Authors: Haddad P. , Graf J.
Published: December 2017

Net hepatic release and uptake of K+ were examined in isolated perfused rat livers subjected to a 10-min period of hypotonic stress. Effluent Na+, K+, and Ca2+ activities were monitored throughout. Initiation and termination of hypotonic stress triggered sharp transient (less than 1 min) changes in effluent ion activities that indicated net water movement into and out of the liver, respectively. In addition, hypotonic stress caused a large transient net release of hepatic K+, whereas return to isotonicity triggered a transient net hepatic K+ uptake. The hypotonically induced K+ release was inhibited by 2 mM barium (95%) and by 1 mM quinine (60%). Net K+ influx, on the other hand, was inhibited by 1 mM ouabain (100%) and by 1 mM amiloride (50%). Osmotically induced K+ fluxes were not significantly affected by bicarbonate removal and were only partially inhibited by 0.1 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) or bumetanide. The results suggest that K+ conductance increases during hypotonic stress, whereas return to isotonicity induces a ouabain-sensitive K+ uptake partly because of increased Na+-H+ exchange. These mechanisms probably participate in regulatory volume decrease and regulatory volume increase, respectively.
Substance P: mechanism of action and receptor distribution at the feline ileocecal sphincter region
Authors: Rothstein R. D. , Johnson E. , Ouyang A.
Published: December 2017

The purpose of this study was to determine the mechanism of action of substance P at the distal ileum, ileocecal sphincter (ICS), and proximal colon in the cat and to determine the localization of substance P receptors at these sites by autoradiography. Intraluminal pressures and myoelectric activity were recorded at the feline distal ileum, ICS, and colon. Substance P caused a tonic and phasic spike-dependent contractile response at all three sites. The antagonists propranolol, phentolamine, and naloxone did not affect the contractile response to substance P at the ileum, ICS, or colon. The ganglionic blocker trimethaphan camsylate potentiated the response to substance P at all three sites, P less than 0.05. Both atropine and tetrodotoxin reduced the response of substance P at the ileal site. At the ICS, atropine or tetrodotoxin reduced, but did not obliterate, the effect of substance P. Neither atropine nor tetrodotoxin reduced substance P-induced colonic contractions. By use of autoradiography, specific binding for substance P was determined to be present at all three sites with the greatest concentration of substance P receptors in the circular muscle layer. In conclusion, these studies suggest multiple sites of action of substance P. At the ileum, substance P causes contraction via a cholinergic pathway. At the ICS, substance P has an excitatory action through a cholinergic pathway and also at smooth muscle receptors. In the proximal colon, the excitatory action of substance P is via smooth muscle receptors. An inhibitory ganglionic pathway also exists at all three sites. Substance P receptors exist predominantly in the circular muscle region of the ileum, ICS, and the proximal colon.
Network thermodynamic model of rat lingual epithelium: effects of hyperosmotic NaCl
Authors: Fidelman M. L. , Mierson S.
Published: December 2017

A network thermodynamic model was developed to describe steady-state and transient ion flows (Na+, K+, and Cl-) and related electrical events in the rat lingual epithelium. The model, which uses only standard assumptions about topology and membrane ion transport processes from other tight epithelia, successfully simulates steady-state transepithelial electrical measurements seen in the in vitro rat dorsal lingual epithelium for a wide range (50-2,000 mM) of mucosal NaCl concentrations. It also simulates the intracellular depolarization and subsequent repolarization observed in situ in rat taste cells in response to a mucosal hyperosmotic NaCl stimulus. Because the intracellular potential has been identified with the receptor potential for gustation, the depolarization and repolarization may explain both the phasic and tonic components of taste nerve excitation in response to a salt stimulus. This study suggests that a better understanding of taste transduction may be gained by focusing more attention on the transport properties and mechanisms of lingual epithelia.
Gastric oxygen uptake during autoregulatory escape from sympathetic stimulation
Authors: Kiel J. W. , Shepherd A. P.
Published: December 2017

To assess the effects of sympathetic stimulation on gastric blood flow and oxygen utilization, the perivascular nerves were stimulated at 2, 4, 6, and 8 Hz in chambered segments of canine gastric corpus perfused at constant pressure. Spectrophotometric arteriovenous oxygen difference and electromagnetic blood flow were recorded continuously. Except at the lowest frequency of stimulation (2 Hz), total blood flow exhibited autoregulatory escape, i.e., blood flow decreased initially but then returned toward control. The fall in total blood flow at the onset of sympathetic stimulation was smaller at 2 Hz than at 4 Hz, but stimulation at 6 and 8 Hz caused no further reductions in total blood flow. However, at all frequencies, total blood flow escaped to the same steady-state value (approximately 17 ml.min-1.100 g-1). Although total blood flow was still less than control (approximately 25 ml.min-1.100 g-1), oxygen extraction increased proportionately so that oxygen consumption was not significantly less than control at any frequency of stimulation. We conclude that autoregulatory escape from sympathetic stimulation is mediated by local mechanisms acting to maintain tissue oxygenation in the stomach.
Theophylline inhibits transcellular Ca transport in intestine and Ca binding by CaBP
Authors: Pansu D. , Bellaton C. , Roche C. , Bronner F.
Published: December 2017

Theophylline, when added to the incubation medium of everted duodenal sacs prepared from rats on a low-calcium diet, was found to inhibit transcellular Ca transport in a concentration-dependent manner, with an inhibitor constant (Ki) of 10.8 mM theophylline. Neither the rate of cellular Ca entry, as evaluated with the aid of brush-border membrane vesicles, nor the rate of cellular Ca extrusion, assessed by measuring ATP-dependent Ca uptake of basolateral membrane vesicles, was significantly altered by the addition of theophylline to the uptake media. However, Ca-binding by calcium-binding protein (CaBP; calbindin D9k), Mr approximately 8,800) was depressed by theophylline in a concentration-dependent manner, with Ki = 3.2 mM theophylline. Theophylline had no effect on Ca binding by calmodulin and the theophylline-induced inhibition of transcellular calcium transport was independent of adenosine 3',5'-cyclic monophosphate levels. Theophylline also had no effect on paracellular Ca movement. Since the theophylline-induced inhibition of Ca-binding by CaBP paralleled the inhibition of transcellular Ca transport, it is concluded that CaBP functions in transcellular Ca transport via its ability to bind Ca.