bluuebunny/crossref_metadata_2025 huggingface dataset:
Chronic ethanol feeding in a high-fat diet decreases insulin-stimulated glucose transport in rat adipocytes
Authors: Wilkes J. J. , DeForrest L. L. , Nagy L. E.
Published: December 2017

Ethanol consumption has been associated with glucose intolerance and insulin resistance and is suggested to be an independent risk factor in the development of non-insulin-dependent diabetes mellitus. We have investigated the long-term effects of ethanol consumption on insulin-regulated glucose transport in rat adipocytes. Male Wistar rats were fed a high-fat liquid diet containing 35% ethanol (ethanol fed) or a control diet that isocalorically substituted maltose dextrin for ethanol (ad libitum). A third group was pair fed the control diet. Basal rates of 2-deoxyglucose uptake were similar in adipocytes from all three groups. Treatment with insulin increased 2-deoxyglucose uptake in ad libitum- and pair-fed rats but did not stimulate uptake in ethanol-fed rats. Similarly, although okadaic acid increased 2-deoxyglucose uptake in pair-fed rats, it had no effect in ethanol-fed rats. GLUT-1 quantity was greater in pair-fed and ethanol-fed rats compared with ad libitum controls. GLUT-4 was decreased in ethanol-fed compared with pair-fed rats but was not different from ad libitum controls. In ad libitum- and pair-fed rats, insulin increased the translocation of GLUT-4 to the cell surface by 2.0-fold. In contrast, translocation of GLUT-4 was not observed after insulin stimulation of ethanol-fed rats, paralleling the loss of insulin-stimulated glucose uptake. In ethanol-fed rats, GLUT-4 protein quantity was negatively associated with increased Gs alpha protein and isoproterenol-stimulated adenosine 3',5'-cyclic monophosphate production. These data suggest that loss of insulin-stimulated glucose uptake in rat adipocytes after chronic ethanol feeding is at least partially due to decreased movement of GLUT-4 to the cell surface after insulin stimulation.
Enrichment of bile with tauroursodeoxycholic acid and biliary cholesterol saturation in hamsters
Authors: Handelsman B. , Bonorris G. , Marks J. W. , Schoenfield L. J.
Published: December 2017

Three groups of golden Syrian hamsters were fed equimolar amounts of taurine-conjugated ursodeoxycholic acid (TUDCA) or unconjugated ursodeoxycholic acid (UDCA) with or without excess taurine for 2 wk. They also received a lithogenic diet composed of standard rodent chow containing ethynylestradiol and increased cholesterol. Bile was obtained from the gallbladder after ketamine anesthesia and analyzed for biliary lipids. The percentage of biliary UDCA was higher with TUDCA (38.5 +/- 3.7) than with UDCA plus taurine (26.5 +/- 2.0, P less than 0.01). The glycine-to-taurine ratio of biliary UDCA conjugates was lower with TUDCA (0.9 +/- 0.1) than with UDCA plus taurine (2.1 +/- 0.2, P less than 0.01) and was highest with UDCA without taurine (4.1 +/- 0.1, P less than 0.01). Biliary cholesterol (molar percentage) and the cholesterol saturation indices with or without correction for UDCA-rich bile were significantly lower with TUDCA than with unconjugated UDCA with or without added taurine. In conclusion, administration for 2 wk of TUDCA, compared with an equimolar amount of unconjugated UDCA plus taurine, produced in the bile of hamsters a higher percentage of UDCA, a lower glycine-to-taurine ratio of UDCA conjugates, and a lower saturation index before and after adjustment for UDCA-rich bile.
Postnatal development of intestinal secretin in rats and guinea pigs
Authors: Paquette T. L. , Shulman D. F. , Alpers D. H. , Jaffe B. M.
Published: December 2017

The amounts and concentrations of secretin in extracts of rat and guinea pig small intestine were measured with a porcine secretin radioimmunoassay. Immunoactivity in the extracts comigrated with porcine secretin in sodium dodecyl sulfate acrylamide gel electrophoresis. In the small intestines of adult rats and newborn and adult guinea pigs, there was a marked proximal-to-distal gradient in secretin concentration; newborn rats exhibited approximately equal proximal and distal secretin concentrations. Concentrations were as follows: most proximal newborn or adult guinea pig segments, 80 ng/mg prot; most distal newborn or adult guinea pig segments, 2 ng/mg prot; most proximal adult rat segments, 50 ng/mg prot; most distal adult rat segments, 8 ng/mg prot; and newborn rat intestine, 11 ng/mg prot. In either rats or guinea pigs, the secretin concentration in the proximal small intestine fell between days 2 and 6 postpartum to levels 3- and 10-fold below their respective newborn values. It is consistent with the relatively mature state of the newborn guinea pig digestive tract that it displays the proximal-to-distal gradient of secretin that is characteristic of both adult guinea pigs and rats. In the proximal intestine of both species, intestinal growth outpaces the accumulation of secretin.
Myoelectric effects of vasoactive intestinal peptide on rabbit small intestine
Authors: Sninsky C. A. , Wolfe M. M. , Martin J. L. , Howe B. A. , O'Dorisio T. M. , McGuigan J. E. , Mathias J. R.
Published: December 2017

Myoelectric recording techniques were used to study the motility of rabbit ileum during infusions of vasoactive intestinal peptide (VIP). VIP was infused intravenously at a rate of 300 pmol X kg-1 X h-1, and peripheral venous blood samples were obtained hourly for VIP assay. VIP was also infused intraluminally at a similar rate, and hourly portal vein blood samples were obtained for VIP assay. Alterations in motility were observed after both intravenous and intraluminal infusions of VIP. These alterations in motility consisted of the migrating action potential complex and repetitive bursts of action potentials. The VIP infusion rate used and the mean peripheral plasma VIP level of 267 +/- 29 pg/ml attained during intravenous VIP infusion were similar to those that induced intestinal secretion in other animal species. Portal venous VIP levels (93 +/- 21 pg/ml) were unchanged during the intraluminal infusion of VIP. These studies show that intravenous infusion of VIP causes alterations in motility of rabbit ileum. These alterations in motility with concomitant secretion of water and electrolytes may contribute to the diarrhea induced by VIP infusion. In addition, intraluminal infusion of VIP also induced alterations in myoelectric activity, which suggested that this peptide has a luminal effect as well as a hormonal effect.
Morphology and electrophysiology of guinea pig gastric mucosal repair in vitro
Authors: Rutten M. J. , Ito S.
Published: December 2017

Guinea pig gastric mucosae stripped of their outer muscle layers were studied in Ussing chambers for up to 14 h. Ten minutes after the mucosae were mounted in the chamber, the electrical parameters were low but continued to rise over 90 min until steady-state potential difference (PD), resistance (R), and short-circuit current (Isc) were recorded. Morphological analysis during the first 10 min of the tissue in the chamber revealed gaps in the epithelium due to damaged cells. However, tissues examined after 20 min in the chamber showed little evidence of epithelial discontinuity. Thereafter, the initial rise in the electrical parameters was noted. After steady-state attainment, the lumen was exposed to 1.25 M NaCl for 5 min and then changed back to 150 mM NaCl. Ten minutes after washout and return to control solutions, the PD, R, and Isc had fallen to low values. At 30 min after washout of the NaCl, the PD, R, and Isc began to increase and after 2 h were back to control values. Morphological analysis of mucosae fixed up to 10 min after exposure to 1.25 M NaCl showed extensive damage and exfoliation of surface cells. However, by 30 min the epithelium was restored and had very few discontinuities, which was then followed by the return of the electrical parameters. The conclusions from these studies are 1) guinea pig gastric mucosae exposed to hypertonic NaCl on the luminal side will primarily result in surface epithelial cell destruction with an immediate drop in the transepithelial electrical values; 2) after return to isotonic saline the damaged mucosa can repair itself within minutes, which then allows the reestablishment of the transepithelial electrical parameters by 2 h; and 3) the good viability and reproducibility of this preparation present a suitable mammalian model system for the study of factors of mucosal repair.
Migrating action-potential complexes in vitro in cholera-exposed rabbit ileum
Authors: Koch K. L. , Martin J. L. , Mathias J. R.
Published: December 2017

The objective of this study was to determine whether cholera-exposed rabbit ileum exhibits altered myoelectric activity in vitro, without central nervous system connections. Whole-cell lysate of Vibrio cholerae, 100 mg in 1 ml saline, was injected into the jejunum of New Zealand White rabbits. Segments of ileum were removed at 12 and 24 h after inoculation and studied in vitro using myoelectric recording techniques. Propagating ring contractions were visualized and corresponded to intense action-potential activity that propagated over consecutive electrode sites. This altered myoelectric activity was similar to the previously described migrating action-potential complex (MAPC) in vivo after infection of rabbit ileum with live V. cholerae, its wholecell lysate, or the purified enterotoxin choleragen, with one exception. All MAPC activity propagated aborally in the in vivo-infected loops; in contrast 26% of the MAPCs propagated retrograde in the in vitro loops. Control segments were injected with saline, and no in vitro MAPCs were observed. Thus, the MAPC stimulated by cholera toxin may be maintained by the enteric nervous system of the gut wall. Although a role for extrinsic nerves is not excluded, our observations suggest that the small intestine may work autonomously, independent of the central nervous system.
Prostaglandin synthesis by microsomes of circular and longitudinal gastrointestinal muscles
Authors: Sanders K. M. , Northrup T. E.
Published: December 2017

Experiments were performed to determine whether longitudinal and circular muscles from various regions of stomach and small bowel had the capacity to convert arachidonic acid (AA) to prostaglandins (PGs). PG production by the microsomal fractions of isolated muscles was assayed by determining the conversion of [14C]AA to 14C-labeled 6-keto-PGF1 alpha, PGF2 alpha, PGE2, PGD2, PGA2, and thromboxane B2. Individual PGs were identified by thin-layer chromatography. The metabolism of [14C]AA to [14C]PGs was linearly related to substrate concentration, enzyme concentration, and incubation time at 37 degrees C and was inhibited in a dose-dependent manner by indomethacin. Longitudinal and circular muscles from all tested regions (corpus, fundus, antrum, pylorus, duodenum, jejunum, and ileum) synthesized PGs. In all regions the major end products of AA metabolism were 6-keto-PGF1 alpha, PGE2, and PGF2 alpha. The data indicate that circular and longitudinal muscles from all regions of the stomach and small bowel contain the enzymatic apparatus necessary to convert AA into prostaglandins.
Effects of scorpion venom on electrolyte transport by rabbit ileum
Authors: Hubel K. A.
Published: December 2017

Scorpion venom, which depolarizes nerves, was used to obtain further evidence that intramural nerves affect ion transport by the rabbit ileum. Ileal epithelium, stripped of muscularis propria, was mounted in a flux chamber modified to permit electrical field stimulation (EFS) of the tissue. Response of the short-circuit current (Isc) to venom was most rapid on the serosal surface, and the response was eliminated by tetrodotoxin. Isc response was influenced by venom batch number and by factors within the tissue. Venom (10 micrograms/ml) and EFS each caused chloride secretion by reducing mucosal-to-serosal movement and by increasing serosal-to-mucosal movement. Sodium transport and residual ion fluxes did not change. In the presence of venom, EFS caused no further changes in ion transport, but tissues still responded to glucose and to aminophylline. The early peak of Isc was reduced about 40% by atropine, implying that acetylcholine, released by venom, stimulates muscarinic receptors. The blockade of the Isc response to venom with tetrodotoxin is further evidence that venom depolarizes intramural nerves and liberates transmitters that cause chloride secretion. The identity of the other transmitters is not known.
Basal-lateral and intracellular membrane populations of rat exorbital lacrimal gland
Authors: Mircheff A. K. , Conteas C. N. , Lu C. C. , Santiago N. A. , Gray G. M. , Lipson L. G.
Published: December 2017

With the goal of isolating and identifying plasma membrane vesicle populations from epithelial cells of the rat exorbital lacrimal gland, we have designed an analytical fractionation of homogenates of the gland parenchyma. This fractionation utilizes separation procedures based on three independent physical properties of subcellular particles: sedimentation coefficient, density, and density after interaction of membrane cholesterol with digitonin. A commonly accepted marker for basal-lateral membranes, Na-K-ATPase, is associated with at least two physically distinct membrane populations. One population can be identified as basal-lateral membrane fragments on the basis of its fractional and specific contents of Na-K-ATPase; it accounts for 50% of the total Na-K-ATPase activity, enriched 29-fold with respect to the initial homogenate. With these values we calculate that the sample of basal membranes has been purified 60-fold with respect to the initial homogenate. The remaining Na-K-ATPase activity appears to be associated, at three- to fivefold lower specific activities, with intracellular membrane populations. We speculate that these populations have been derived from the Golgi complex.
Role of protein phosphorylation in regulating rat submandibular mucin secretion
Authors: Quissell D. O. , Deisher L. M. , Barzen K. A.
Published: December 2017

The possible involvement of protein phosphorylation during beta-adrenergic receptor stimulation in rat submandibular gland was investigated in vitro using a dispersed cell preparation. (-)-Isoproterenol, a beta-adrenergic agonist, or dibutyryl cAMP stimulation was associated with an enhanced phosphorylation of three protein bands having apparent molecular weights of 34,000, 26,000, and 21,000, respectively. (-)-Propranolol, a beta-adrenergic antagonist, inhibited the phosphorylation of the three proteins during beta-adrenergic stimulation but not during dibutyryl cAMP stimulation. The three proteins were not fragments of a higher-molecular-weight protein. Subcellular fractionation using differential centrifugation, fractionation in an aqueous two-phase polymer system, and discontinuous sucrose gradient ultracentrifugation coupled with marker enzyme analysis indicated that all three proteins were enriched in the same subfractions: a heavy plasma membrane fraction and a fraction containing plasma membrane and Golgi membrane material. The extent of protein phosphorylation for all three proteins increased as a function of time and dose after beta-adrenergic stimulation. After 20 min of maximal beta-adrenergic stimulation, the addition of a beta-adrenergic blocker caused a time-dependent decrease in the 32P content of all three proteins. Pure cholinergic or pure alpha-adrenergic receptor stimulation had no effect on the 32P content of the three proteins. These data are consistent with a role for cAMP-mediated protein phosphorylation during mucin secretion from rat submandibular cells.
Arachidonic acid stimulates mucin secretion in prairie dog gallbladder
Authors: LaMont J. T. , Turner B. S. , DiBenedetto D. , Handin R. , Schafer A. I.
Published: December 2017

Mucin glycoprotein secretion from prairie dog gallbladder explants was studied in 24-h organ culture using [3H]glucosamine as a precursor. Indomethacin caused a reversible dose-dependent inhibition of both mucin release (50% inhibition between 10(-6) and 10(-5) M indomethacin) and 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha) release (66% inhibition at 10(-7) M). Sepharose 4B chromatography of secreted [3H]-glucosamine-labeled glycoproteins revealed that indomethacin inhibited release of a high-molecular (greater than 10(6) daltons) mucin-type glycoprotein. Addition of sodium arachidonate (10(-4) M) to organ culture medium caused an approximate two- to fivefold increase (P less than 0.02) in mucin release compared with control and an increase in the secretory component of total glycoprotein synthesis from 37.5% in control explants to 75.2% (P less than 0.01). The stimulatory effect of arachidonate on mucin secretion was blocked by indomethacin. We incubated explants for 1 h with [14C]arachidonate and studied prostaglandin and thromboxane products in medium by high-performance liquid chromatography. The only conversion product identified was 6-keto-PGF1 alpha, the stable breakdown product of prostacyclin. Release of gallbladder mucin is significantly inhibited by indomethacin and increased by arachidonate. Mucin secretion may be regulated in part by endogenous levels of prostacyclin, the major cyclooxygenase product in prairie dog gallbladder epithelium.
Inhibitory action of somatostatin on isolated gastric glands and parietal cells
Authors: Chew C. S.
Published: December 2017

The action of somatostatin in vitro was characterized using glands and parietal cells isolated from rabbit gastric mucosa. In the presence of the reducing agent dithiothreitol, somatostatin was found to inhibit gastrin- and histamine-stimulated acid formation in glands as measured by [14C]aminopyrine (AP) accumulation and oxygen consumption, both measurements that appear to be reliable indexes of parietal cell acid formation. In glands the inhibition of the secretory response to gastrin was more potent (60-80%) than that to histamine (15-25%). The kinetics of somatostatin inhibition of responses to both agents were noncompetitive. The apparent IC50 for the partial somatostatin inhibition of histamine-stimulated AP accumulation was similar to that for gastrin (approx 3 X 10(-9) M) when maximum concentrations of histamine (10(-4) M) or gastrin (10(-7) M) were used. The inhibitory action of somatostatin appeared to be specific, inasmuch as this peptide had no significant effect on basal secretion or secretion stimulated by carbachol, dibutyryl cAMP, cholera toxin, or elevated extracellular K+. In purified parietal cell preparations, somatostatin inhibited histamine- but not gastrin-stimulated AP accumulation. Moreover, the inhibition of histamine-stimulated AP accumulation in parietal cells was more pronounced than in glands. These results suggest that somatostatin acts directly on parietal cells to inhibit histamine activation of H+ secretion. Somatostatin also acts indirectly to inhibit gastrin, perhaps by blocking the release of histamine from paracrine- or endocrinelike cells present in the glands.
Cause-and-effect relationship between motilin and migrating myoelectric complexes
Authors: Sarna S. , Chey W. Y. , Condon R. E. , Dodds W. J. , Myers T. , Chang T. M.
Published: December 2017

We investigated the cause-and-effect relationship between plasma motilin levels and migrating myoelectric complexes (MMCs). Each dog was implanted with a set of eight bipolar electrodes on the small intestine. Premature phase IIIs were initiated by morphine bolus injections. Plasma samples were assayed for motilin and gastrin. All spontaneous and morphine-initiated phase IIIs were associated with peaks of plasma motilin, which always occurred after phase IIIs had started in the proximal duodenum. The plasma motilin level decreased consistently during phase I and started to increase again only after phase II had started in the duodenum. Either a meal or somatostatin infusion disrupted MMC cycling, but morphine boluses overcame this disruption and initiated phase IIIs that propagated distally. The phase IIIs thus initiated were associated with peaks in plasma motilin levels. In contrast, bolus injections of motilin did not initiate phase IIIs during the fed state or during somatostatin infusion. Our findings suggest that endogenous motilin does not initiate spontaneous MMCs. Instead, MMC contractions release motilin. The physiological role of motilin, thus released, may be to act as an endocrine agent to coordinate secretory and motor events with the start of phase III activity in the upper small intestine.
Response of rat jejunum to angiotensin III: pharmacology and mechanism of action
Authors: Levens N. R.
Published: December 2017

At low doses angiotensin III (A III) stimulates jejunal fluid absorption in the pentobarbital sodium-anesthetized rat. In contrast, at high doses the hormone inhibits absorption and/or stimulates secretory processes. The stimulation of jejunal absorption in response to A III can be blocked by guanethidine, phentolamine, and prazosin but not by propranolol or yohimbine, suggesting that A III-increased intestinal absorption is secondary to the release of norepinephrine from sympathetic nerves in the jejunum and activation of postsynaptic alpha 1-adrenergic receptors. The A III inhibition of water absorption is not affected by adrenergic antagonists but can be reversed to a net stimulation of transfer after pretreatment of the animals with indomethacin. This suggests that at high doses A III stimulates intestinal prostaglandin biosynthesis. The A III analogue [Ile7]A III is devoid of agonist activity over a wide dose range and behaves as a potent antagonist of both the stimulatory and the inhibitory effects of the parent peptide on jejunal absorption. [Ile7]A III will be a useful tool for investigating the physiological role of angiotensin peptides in the control of intestinal absorption.
Potentiation of pepsinogen secretion from dispersed glands from rat stomach
Authors: Raufman J. P. , Kasbekar D. K. , Jensen R. T. , Gardner J. D.
Published: December 2017

In the present study we examined the actions of various agents alone and in combination on pepsinogen secretion from dispersed gastric glands prepared from rat stomach. Potentiation of pepsinogen secretion occurred with secretin or vasoactive intestinal peptide plus carbamylcholine or cholecystokinin. The pattern of action of secretin and vasoactive intestinal peptide could be reproduced by 8-bromo-cAMP, and the pattern of action of carbamylcholine and cholecystokinin could be reproduced by the calcium ionophore A23187. A phosphodiesterase inhibitor, isobutylmethylxanthine, increased pepsinogen secretion, increased the potency but not the efficacy of the action of secretin on pepsinogen secretion, and potentiated the action of carbamylcholine on pepsinogen secretion. These results suggest that, in dispersed gastric glands from rat stomach, secretagogue-induced pepsinogen secretion can be stimulated by two different mechanisms: one mechanism is mediated by cAMP and the other is mediated by changes in cellular calcium. Secretagogues whose actions are mediated by one mechanism potentiate the actions of those secretagogues whose actions are mediated by the other mechanism.
Nicotinamide uptake and metabolism by chick intestine
Authors: Schuette S. A. , Rose R. C.
Published: December 2017

Intestinal uptake and metabolism of nicotinamide (NAm) were studied in isolated epithelial cells and in isolated segments in situ at a physiological concentration of [14C]NAm (11.7 microM). [14C]NAm was rapidly taken up from the bathing medium and largely metabolized to [14C]NAD by the isolated cells. Total accumulation of 14C label was energy dependent and saturable at higher concentrations of NAm (148 and 351 microM). In contrast, the tissue content of NAm was unaffected by metabolic inhibitors, and the bathing media NAm rapidly equilibrated with intracellular space at all levels of NAm. NAm was converted directly to NAD via the intermediate nicotinamide mononucleotide (NMN); nicotinic acid was not an intermediate in this conversion and was a less efficient precursor of NAD. NAm absorption in vivo was substantial, 30.6% of dose after 10 min. Also, data on NAm entry into the mucosa and subsequent metabolism in vivo supported the in vitro observations. Exogenous NMN reduced NAm entry into the mucosal cells both in vivo and in vitro; the effect was specific to NAm. This is the only suggestion to date that NAm entry might proceed by some form of specialized transport process.
Monoclonal antibodies against gastric H+ + K+ ATPase
Authors: Smolka A. , Helander H. F. , Sachs G.
Published: December 2017

Monoclonal antibodies were prepared against a purified membrane fraction from hog gastric mucosa containing the H+ + K+ ATPase. On sodium dodecyl sulfate gels the molecular weight of this fraction corresponds to a single band of about 95,000. In contrast, on isoelectric focusing gels three groups of peptides are resolved with isoelectric points of 5.7, 6.2, and 8.5. One of the monoclonal antibodies (HK111) was shown to react selectively with the acidic peptide, whereas another antibody (HK113) reacted with the alkaline peptide, showing that the three peptides were antigenically distinct. Both monoclonal antibodies selectively labeled the parietal cell, and antibody HK111 labeled the tubulovesicles of the resting parietal cell and the microvilli of the secretory canaliculus of the secreting cell. This finding suggests translocation of membrane from the tubulovesicles to the secretory surface on stimulation.
Effects of inhibitors of cyclic nucleotide phosphodiesterase on actions of cholecystokinin, bombesin, and carbachol on pancreatic acini
Authors: Gardner J. D. , Sutliff V. E. , Walker M. D. , Jensen R. T.
Published: December 2017

In dispersed acini from guinea pig pancreas two inhibitors of cyclic nucleotide phosphodiesterase, Ro 20-1724 and 3-isobutyl-1-methylxanthine (IBMX), augmented the increase in amylase secretion caused by supramaximal concentrations of cholecystokinin but did not alter the stimulation of enzyme secretion caused by bombesin. The augmentations of the action of cholecystokinin caused by Ro 20-1724 or IBMX could be reproduced by 8-bromo-cAMP. When tested alone or with theophylline, cholecystokinin did not alter cAMP in pancreatic acini; however, with Ro 20-1724 or IBMX, concentrations of cholecystokinin that were supramaximal for stimulating amylase secretion caused a significant increase in cellular cAMP. These findings indicate that Ro 20-1724 and IBMX augment the action of cholecystokinin on enzyme secretion by inhibiting cyclic nucleotide phosphodiesterase and allowing a significant cholecystokinin-induced increase in cellular cAMP. IBMX but not Ro 20-1724 caused a parallel rightward shift in the dose-response curve for the stimulation of amylase secretion caused by carbachol. IBMX also caused a parallel rightward shift in the dose-response curve for the stimulation of outflux of 45Ca caused by carbachol. These results indicate that IBMX, but not Ro 20-1724, can function as a muscarinic cholinergic antagonist.
Receptor-mediated internalization and secretion of cholecystokinin into rat pancreatic duct fluid
Authors: Izzo R. S. , Praissman M.
Published: December 2017

We measured cholecystokinin (CCK) in pancreatic duct secretions (PDS) after infusion of different amounts of CCK-8 (the C-terminal octapeptide of cholecystokinin) into rats. Injection of 23, 46, and 92 ng of CCK-8 increased immunoreactive cholecystokinin in PDS by 3-, 13-, and 28-fold above basal levels within 30 min. Continuous intravenous infusion of CCK-8 (50 ng/min) into rats for 30 min, followed by a 30-min rest period, and then a final infusion of peptide for another 30 min increased CCK in PDS only during the final period by 12-fold to 500 pg/30 min. Neither gastrin nor oxidized CCK-8 were detected in PDS after intravenous infusion of each peptide. Administration of proglumide (ip), a CCK receptor antagonist, during continuous CCK infusion significantly reduced immunoreactive CCK levels in PDS to 2% of the control group (P less than or equal to 0.01). CCK was also rapidly internalized into dispersed pancreatic acinar cells in a temperature-dependent fashion, and this process was inhibited by proglumide. The above data suggest that CCK in PDS reflects a peptide-specific process that is receptor mediated. We propose that circulating cholecystokinin binds to specific receptors on pancreatic acinar cells, is internalized, and is then secreted into pancreatic duct fluid.
Mechanism for high PCO2 in gastric juice: roles of bicarbonate secretion and CO2 diffusion
Authors: Stevens M. H. , Thirlby R. C. , Feldman M.
Published: December 2017

The partial pressure of CO2 (PCO2) in gastric juice often exceeds the PCO2 of blood. CO2 in gastric juice may originate from blood and enter luminal fluid by diffusion, or CO2 may be produced in the lumen of the stomach from the reaction of HCO3- and H+. Because CO2 production from HCO3- is dependent on acid (low pH), we suppressed acid secretion with intravenous cimetidine to estimate to what extent appearance of CO2 in luminal fluid is due to production from HCO3-. When denervated fundic pouches of dogs were distended with saline, the PCO2 of the solution increased to the PCO2 of blood in approximately 20 min, with the initial rate of appearance of CO2 in the pouch solution only minimally affected by cimetidine. Thereafter, PCO2 of luminal fluid continued to increase to 50-60 mmHg in the absence of cimetidine, whereas PCO2 of luminal fluid remained approximately equal to that of blood when cimetidine was infused (P less than 0.001, cimetidine vs. control). The mean pH in the pouch solution remained between 6.3 and 6.9 during cimetidine infusion but decreased to 4.75 without cimetidine (P less than 0.001). In additional experiments, an acidic solution with high PCO2 (242 +/- 3 mmHg) was infused into the fundic pouches. PCO2 in luminal fluid decreased rather slowly toward plasma PCO2, requiring 240 min for luminal fluid PCO2 to decrease to 56 +/- 2 mmHg. Thus the permeability of the gastric mucosa to luminal CO2 was relatively low.(ABSTRACT TRUNCATED AT 250 WORDS)
Variations in rat mesenteric tissue thickness due to microvasculature
Authors: Barber B. J. , Oppenheimer J. , Zawieja D. C. , Zimmermann H. A.
Published: December 2017

Studies of microvascular, tissue and lymphatic transport using microphotometric and microfluorometric techniques are potentially subject to artifact due to variations in tissue specimen thickness. Absorbance techniques utilize the Lambert-Beer law in which A = log I0/I = act, where A is absorbance, I0 is incident light intensity, I is transmitted intensity, a is an absorbance coefficient, c is concentration of substance, and t is path length. If differences in t are known to be present, then inferences of changes in c from changes in A become suspect. In microfluorometry the amount of light gathered is proportional to the number of fluorochromes in the effective cuvette, which is determined by the microscope's numerical aperture and the sample thickness. If variations in thickness are known to occur, the effective cuvette volume may be changing; therefore, inferences of changes in fluorochrome concentration from changes in intensity become suspect. Existing data suggest that rat mesentery is 15-30 microns thick, but variation over a tissue region is unknown. Our goals are to determine thickness variation in avascular, fat-free mesenteric tissue regions; thickness variation near blood vessels; and average tissue thickness. Sprague-Dawley rats were anesthetized with Inactin. Mesenteric tissue from a loop of small intestine was draped over a platform for observation; thickness was measured with an oblique microscope and a microgravimetric technique. The average variation in avascular fat-free tissue was 1.1 micron/100-microns distance, and average thickness was 17.4 microns. There was a significant increase in thickness over the microvasculature.
Zymogen granule acidity is not required for stimulated pancreatic protein secretion
Authors: De Lisle R. C. , Williams J. A.
Published: December 2017

It has been demonstrated recently by acridine orange fluorescence that pancreatic zymogen granules are acidic in situ, with respect to the cytoplasm. To evaluate the relationship between the acidic intragranular pH and hormone-stimulated secretion, mouse pancreatic acini were treated with lysosomotropic agents to collapse the zymogen granule pH gradient. Methylamine, monensin, and chloroquine collapsed the granule pH gradient as evidenced by a disappearance of acridine orange fluorescence. Cholecystokinin octapeptide (CCK-8)-stimulated acinar amylase secretion was unaffected in the presence of up to 30 mM methylamine and slightly enhanced in the presence of 0.3-10 microM monensin or 3-300 microM chloroquine. Acini were also preincubated for 15 min before addition of either CCK-8 or bombesin with concentrations of the lysosomotropic agents that dissipated the granule acridine orange fluorescence within this time. With preincubation, basal amylase release was unaffected, while stimulated secretion was slightly enhanced by all three lysosomotropic agents. Monensin and methylamine caused vacuolization of Golgi and lysosomal membranes and inhibition of intracellular transport of newly synthesized proteins. Chloroquine affected lysosomes similarly but had little effect on Golgi membranes or on intracellular protein transport. We also demonstrate that parotid secretory granules are acidic in situ by the acridine orange technique. Thus acidified secretory granules may be a general feature of exocrine secretory granules, but the acid pH is not requisite for the final steps in protein secretion from isolated pancreatic acini.
Bile salt absorption in killifish intestine
Authors: Honkanen R. E. , Patton J. S.
Published: December 2017

Bile salt absorption was examined in vitro using entire small intestines from the killifish Fundulus heteroclitus. Intestines were everted over a glass rod and incubated in solutions containing 10 nM to 20 mM bile salts. After rinsing and correcting for the adherent fluid space, uptake rates and bile salt concentrations in the tissue were determined. The distal intestine contained a Na+-dependent active transport system for bile salt uptake with an apparent Vmax for taurocholate and cholate of 1.4 and 2.3 nmol.min-1.mg dry wt-1 (Km = 117 and 357 microM), respectively. At low concentrations (10 nM to 500 microM), absorption occurred almost exclusively (greater than 84%) in the distal intestine. However, at concentrations of 1 mM and above, bile salt absorption in the middle and proximal regions equaled that in the distal intestine. Thus, although an active transport system makes the distal intestine more efficient in absorbing bile salts, passive absorption appears to account for a significant amount of bile salt uptake at concentrations above the critical micellar concentration. The presence of oleic acid did not significantly affect bile salt uptake.
Biliary catabolism of glutathione and differential reabsorption of its amino acid constituents
Authors: Ballatori N. , Jacob R. , Barrett C. , Boyer J. L.
Published: December 2017

Biliary excretion of glutathione, free amino acids, and total amino acids (after acid hydrolysis) was measured in hepatic bile collected from guinea pigs, rabbits, and dogs anesthetized with pentobarbital sodium. In controls, the concentration of glutathione in bile was less than 20 microM in all three species. However, when hepatic gamma-glutamyltransferase activity was decreased by retrograde intrabiliary infusion of the irreversible inhibitor acivicin (AT-125; 20 mumol/kg), there was a marked increase in biliary glutathione excretion (in mumol glutathione equivalents.kg body wt-1.h-1) from 0.10 +/- 0.04 to 2.2 +/- 0.6 in guinea pigs, from 0.014 +/- 0.013 to 2.5 +/- 1.9 in rabbits, and from an undetectable level (less than 0.001) to 0.11 +/- 0.05 in dogs. Amino acid analysis of bile revealed that the concentration of glutathione's constituent amino acids (free glutamate, cystine, and glycine) in control bile samples from these three species were quite low and were not affected by AT-125. However, acid hydrolyzates of these same bile samples revealed an unusually high degree of amino acid conjugation. Glutamate (0.06-0.5 mM), cystine (0.2-1.1 mM), and glycine (1.7-2.8 mM) constituted the overwhelming majority of total amino acids in hydrolyzed bile from controls. After AT-125, concentrations of total glutamate and cystine were elevated in hydrolyzed bile, while concentrations of all other amino acids remained the same. Thus glutathione is avidly secreted into bile in the guinea pig, rabbit, and dog but is almost quantitatively broken down within the biliary tree. Subsequently, the glutamate and cysteine moieties derived from catabolism of glutathione must be partially reabsorbed either as peptides, conjugates, or free amino acids.(ABSTRACT TRUNCATED AT 250 WORDS)
Absorption and transport of milk calcium by infant rats
Authors: Blake H. H. , Henning S. J.
Published: December 2017

All previous studies of Ca absorption in the developing animal have used simple salts of Ca. The aims of the current study are 1) to determine the predominant form of Ca in rat milk and 2) to compare the absorption of milk Ca and CaCl2. In vivo-labeled rat milk was obtained by injecting lactating dams with 45CaCl2. Distribution of 45Ca in the cream, whey, and casein fractions was determined by differential centrifugation, the values being 0.3, 5, and 95%, respectively. To study Ca absorption, rats aged 14 and 28 days received either 45Ca-milk or 45CaCl2 by intragastric intubation. At 14 days, transport of milk Ca into the carcass was significantly slower than that of CaCl2, although by 6 h postintubation both had plateaued at approximately 92% of dose. At 28 days, the time course of transport was the same for the two forms of Ca, and the plateau was not significantly different from that at 14 days. In the younger animals, more Ca from milk than from CaCl2 was retained in gastrointestinal tissue. Quantitation of 45Ca in various segments of the gastrointestinal tract showed that the greater retention of milk Ca occurred in the stomach, the duodenum, and the distal jejunum. We conclude that milk Ca is efficiently absorbed by the suckling rat, possibly in a protein-bound form. The mechanism of the absorptive process awaits further investigation.
Increased responsiveness of jejunal longitudinal muscle in Trichinella-infected rats
Authors: Vermillion D. L. , Collins S. M.
Published: December 2017

We examined in vitro changes in contractility of jejunal longitudinal muscle strips in rats infected with the nematode parasite Trichinella spiralis. Length-passive tension relationships were unchanged. However, muscle from infected rats on days 5 and 6 postinfection (PI) generated maximal active tension induced by carbachol at significantly less stretch (39.9 +/- 1.0 and 34.3 +/- 6.3%, respectively) than control tissues (66.0 +/- 2.3%). In infected rats on day 5 PI, the maximum tension generated by carbachol (1.6 +/- 0.4 g/mm2) and by 5-hydroxytryptamine (5-HTP) (2.6 +/- 0.1 g/mm2) was significantly greater than in control tissue (0.5 +/- 0.2 g/mm2). On removal of calcium from the medium, responses of muscle from control and infected rats were reduced in a proportionate manner. The increased responsiveness to carbachol and 5-HTP was maximal by day 5 PI and was associated with a decrease in the ED50 value for 5-HTP but not for carbachol. All changes were reversed by 23 days PI. These results indicate that T. spiralis infection in the rat is associated with alterations in jejunal longitudinal smooth muscle function.
Evaluation of myosin light chain phosphorylation in isolated pancreatic acini
Authors: Burnham D. B. , Soling H. D. , Williams J. A.
Published: December 2017

The role of contractile proteins in secretory granule exocytosis was evaluated by determining whether myosin light chain phosphorylation was altered during stimulation of secretion in mouse pancreatic acini. Acinar myosin was purified by extraction into isosmotic sucrose solution containing 40 mM pyrophosphate followed by ammonium sulfate precipitation and Sepharose 4B-CL chromatography. Myosin was eluted as a single peak of K+-EDTA ATPase activity and was purified over 2,000-fold to a final ATPase specific activity of 0.96 mumol.min-1.mg protein-1. Three major myosin subunits of apparent Mr of 200,000, 20,000, and 17,000 were present in the purified myosin preparation. A fourth protein of Mr 21,000 was also present. Purification of myosin from 32P-labeled acini revealed the Mr 200,000, 21,000, and 20,000 proteins to be heavily labeled. The effect of cholecystokinin octapeptide (CCK-8) on myosin phosphorylation was studied after isolation of myosin from 32P-labeled acinar lysates by immunoprecipitation. Treatment of acini for 1-10 min with a concentration of CCK-8 that gives a maximal secretory response caused a 25-40% increase in light chain labeling. Treatment with a supramaximal CCK-8 concentration produced a 50-80% increase in light chain labeling. Phosphorylation of myosin heavy chain was not significantly affected by secretagogue treatment. These results indicate that stimulation of pancreatic acinar secretion is accompanied by an increase in myosin light chain phosphorylation.
Neural regulation of duodenal alkali secretion: effects of electrical field stimulation
Authors: Crampton J. R. , Gibbons L. G. , Rees W. D.
Published: December 2017

The role of transmitters released from enteric neurons in regulating bicarbonate secretion by the proximal duodenum has been studied using electrical field stimulation (EFS). Stripped duodenal mucosa from Rana catesbeiana was mounted as an intact tube over circular platinum electrodes, and luminal alkalinization was measured by pH stat titration before, during, and after EFS. Transmucosal potential difference (PD) was simultaneously measured before and after EFS by paired electrodes. Square-wave pulses 50 V, 2 ms in duration, at 10 Hz were delivered in trains of 0.5 s at 1 Hz for periods of 15 min after stable basal secretion. This resulted in a 63 +/- 27% increase in alkalinization that returned to basal values after cessation of the stimulus, without change in transmucosal PD. Serosal-to-lumen [3H]mannitol flux was not affected. Repetition of the stimulus resulted in similar responses for as long as the tissue remained viable. The response to EFS was abolished by tetrodotoxin (10(-6) M) and veratrine (0.1 mg/ml), indicating that intrinsic neurons were responsible for mediating the effect. In addition, the effect was blocked by serosal dinitrophenol (10(-4) M), indicating that the secretory response occurred by a metabolically dependent process. These results indicate that alkalinization by proximal duodenum may be controlled by neurotransmitter release from local enteric neurons.
Somatostatin inhibits VIP-stimulated amylase release from perifused guinea pig pancreatic acini
Authors: Singh P. , Asada I. , Owlia A. , Collins T. J. , Thompson J. C.
Published: December 2017

We have examined the direct effect of somatostatin (SRIF) on basal and stimulated amylase release from guinea pig pancreatic acini using the in vitro method of continuous perifusion. The optimal conditions of flow rate, chamber size, acinar cell volume per chamber, and period of secretagogue infusion were defined for the perifusion system. The kinetic profile of amylase release in response to cholecystokinin-octapeptide (CCK-8), vasoactive intestinal peptide (VIP), and SRIF was studied. Under optimal conditions, the acini were found to remain equally responsive to an ED50 dose of CCK-8 (0.5-0.8 nM) for 12 h of perifusion. The duration of amylase response to any given dose of CCK-8, given for the optimal period of 5 min, was 80-100 min. The total amylase released minus the basal release divided by 90 min (delta response) in response to the maximum effective (Maxeff) dose of CCK-8 (100 nM) was 14,667 +/- 1,433 U/l (amounting to a 10-fold increase compared with basal values). When compared with the amount of total delta amylase released in response to the Maxeff dose of CCK, the total amylase released in response to the Maxeff doses of SRIF (1 microM) and VIP (10 nM) was 10-21% and 51-59%, respectively. SRIF (100 nM) significantly decreased VIP- (0.1-1.0 nM) stimulated amylase release by 45-70% in the perifusion method of study but had no significant effect on the CCK-stimulated amylase release. This suggests that the perifusion method can be used for investigating the mechanism of SRIF-mediated inhibition of VIP effects on amylase release in an in vitro system.
Downregulation of protein kinase C in guinea pig pancreatic acini: effects on secretion
Authors: Sung C. K. , Hootman S. R. , Stuenkel E. L. , Kuroiwa C. , Williams J. A.
Published: December 2017

Pretreatment of guinea pig pancreatic acini with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induced a time- and concentration-dependent down-regulation of protein kinase C. In control acini almost all of the protein kinase C activity was present in a cytosolic fraction. Incubation with TPA initially shifted protein kinase C activity to a particular fraction which then disappeared over the following 24-h incubation with TPA. To study the role of protein kinase C in stimulus-secretion coupling, acini were pretreated with TPA and then amylase release was studied in response to various secretagogues. Preincubation of acini with TPA led to a time- and concentration-dependent decrease in TPA-stimulated amylase release that correlated with protein kinase C downregulation. Preincubation of acini with 1 microM TPA for 24 h, resulting in complete loss of protein kinase C activity, abolished the secretory effect of subsequently added TPA. By contrast, the secretory effects of cholecystokinin octapeptide (CCK-8) and carbamylcholine chloride (CCh) were only inhibited by 44 and 34%, respectively, and amylase release stimulated by the Ca2+ ionophore A23187 and an adenosine 3',5'-cyclic monophosphate-mediated agonist, vasoactive intestinal peptide, was unaffected. Dose-response curves for CCK-8- or CCh-stimulated amylase release in TPA-pretreated acini revealed attenuation of both maximal efficacy and sensitivity. However, the CCh-stimulated intracellular Ca2+ increase as determined by use of the fluorescent probe fura-2 was not affected by the long-term TPA pretreatment of acini. This study strongly suggests that both protein kinase C and intracellular Ca2+ play a significant role in CCK-8- and CCh-stimulated amylase release.
Nature of taurodehydrocholic acid uptake in rat hepatocytes
Authors: Hardison W. G. , Lowe P. J. , Gosink E.
Published: December 2017

We studied uptake into isolated rat hepatocytes of the bile acid analogue taurodehydrocholate (TDHC) over a concentration range of 2.5-4,000 microM. Uptake was mainly by a saturable sodium-dependent process with a Km of approximately 50 microM and a Vmax of 0.036 nmol.s-1.mg protein-1. A lesser sodium-independent process was evident but was linear in the range studied. Both processes were inhibited by incubation at 37 degrees C under nitrogen in the presence of 3 mM sodium cyanide or by incubation at 0 degrees C. A single transport site was suggested by the Eadie-Hofstee plot of TDHC uptake from 2.5 to 750 microM. TDHC was a weak competitive inhibitor of taurocholic acid (TCA) uptake (Ki = 236 microM) but was not itself taken up by the TCA transport site. TCA exhibited moderately potent mixed inhibition of TDHC uptake. Uptake of both compounds was strongly inhibited by bromosulfophthalein (BSP) and Rose Bengal, whereas 0.5 mM alanine uptake was not affected. BSP exhibited a complex pattern of inhibition of TDHC uptake: mixed partial inhibition. Degree of inhibition of both TDHC and TCA uptake did not increase as BSP concentrations were increased from 50 to 100 microM. BSP did not exert its inhibitory effects by alteration of membrane potential or sodium gradients; 50 microM BSP changed membrane potential less than 10% and sodium gradient not at all. The data indicate that despite close structural analogy between TDHC and TCA, the two compounds are taken up by different sodium-dependent mechanisms. Nonetheless, the similar qualitative and quantitative effects of BSP on their uptakes suggests the mechanisms are related.