Messenger RNA expression and localization of xenin in the gastrointestinal tract in sheep.

The present study aimed to determine the primary sequence of ovine xenin and clarify the mRNA expression and peptide localization of xenin in the gastrointestinal tract in sheep. The colocalization of xenin and glucose-dependent insulinotropic polypeptide was also compared in the antrum and duodenum. Analysis of the nucleotide sequence of ovine xenin revealed a high degree (97.9%) of sequence homology of the sequence between sheep and cattle, and the amino acids sequence determined for ovine xenin coincided (100%) with that of other mammalian species. Real-time quantitative PCR for ovine xenin did not show regional difference in the mRNA expression ratio of xenin. In contrast to the real-time quantitative PCR results, anti-xenin positive cells were abundantly localized in the abomasal antrum (P < 0.01) and at a lesser amount in the duodenum, but no antixenin positive cells were observed in the other regions. Anti-xenin single-positive cells were in a majority in the abomasal antrum, whereas anti-xenin single-positive cells, and anti-GIP single-positive cells, and double-positive cells were even colocalized in the duodenum. These results suggest that abomasal antrum is a major source of xenin in the ovine gastrointestinal tract.

Polymeric immunoglobulin receptor expression and local immunoglobulin A production in bovine sublingual, submandibular and parotid salivary glands.

The submandibular and parotid glands are the main sources of immunoglobulins A (IgAs) in human and rat saliva. These glands express the polymeric immunoglobulin receptor (pIgR), which transports IgAs into saliva. The main source of IgAs in saliva and pIgR expression in salivary glands has not been well documented in cattle. Expressions of pIgR were determined in the major bovine salivary glands (sublingual, submandibular, and parotid) by RT-PCR for mRNA and by Western blot analysis and immunohistochemistry (IHC) using an anti-human pIgR antibody for protein. The protein detected with the antibody was identified by nano-liquid chromatography-quadrupole time of flight mass spectrometry. Additionally, the distribution of Ig-producing plasma cells was analyzed by IHC. RT-PCR showed that pIgR was expressed in the sublingual and submandibular glands, but not in the parotid gland. Higher protein levels were observed in sublingual glands than in submandibular glands by Western blot. By IHC, pIgR was mainly located on the apical side of the cytoplasmic membrane in the sublingual gland, whereas it was observed only on the basal side in the submandibular gland. The highest density of plasma cells expressing IgAs was observed in the sublingual gland. These results suggest that the sublingual gland plays an important role in first-line defence of the oral cavity in cattle in contrast to humans and rats.

Ghrelin stimulates gastric motility of the guinea pig through activation of a capsaicin-sensitive neural pathway: in vivo and in vitro functional studies.

BACKGROUND: Ghrelin stimulates gastric motility in rats, mice and humans. Although ghrelin and the ghrelin receptor are known to be expressed in the guinea-pig gastrointestinal tract, the effects of ghrelin on gastric motility have not been examined. Aim of the present study was to clarify the motor-stimulating action of ghrelin in the guinea-pig stomach. METHODS: Gastric motility was measured as intraluminal pressure changes using a balloon inserted in the stomach of urethane-anaesthetized guinea pigs. The effects of ghrelin on gastric muscle contraction and [(3)H]-efflux from [(3)H]-choline-loaded strips were investigated in vitro. KEY RESULTS: Ghrelin (0.3-30 microg kg(-1), i.v.) increased gastric motility in a dose-dependent manner but des-acyl ghrelin was ineffective. The action of ghrelin was completely inhibited by hexamethonium and D-Lys(3)-growth-hormone releasing peptide-6. Atropine partially decreased the stimulatory action of ghrelin. In capsaicin-pretreated guinea pigs, the ghrelin-induced response was markedly decreased. Ghrelin (1 micromol L(-1)) did not affect [(3)H]-efflux in non-stimulated preparations but significantly decreased electrical field stimulation (EFS)-induced [(3)H]-efflux. L-Nitro arginine methylester (L-NAME) attenuated the inhibition of [(3)H]-efflux by ghrelin. Ghrelin did not cause any mechanical changes in gastric strips. Electrical field stimulation caused relaxation of gastric strips, which changed to atropine-sensitive contraction in the presence of L-NAME. Relaxation induced by EFS was slightly potentiated, but the EFS-induced contraction was not affected by ghrelin. CONCLUSIONS & INFERENCES: Ghrelin stimulates gastric motility of the guinea pig through activation of capsaicin-sensitive vago-vagal reflex pathway including efferent cholinergic neurons. Peripheral ghrelin receptors on enteric nitrergic nerves might affect the ghrelin-induced gastric action by releasing nitric oxide.

Effect of intravenous infusion of proglumide on ruminal motility in conscious sheep (Ovis aries).

The effects of intravenous infusion of proglumide on regular ruminal contractions were examined in conscious sheep using doses that inhibit pancreatic exocrine secretion. After a control period of 20 min, proglumide was infused intravenously for 40 min at a dose of 15, 30 or 60 micromol/kg per min and venous blood was collected. The intravenous infusion of proglumide significantly increased the frequency of ruminal contractions at 15 micromol/kg per min without altering the amplitude, while it significantly decreased the frequency and amplitude of ruminal contractions at 30 and 60 micromol/kg per min in a dose-dependent manner. Proglumide did not increase contractile activity of the omasum, abomasum and duodenum or the plasma concentration of immunoreactive cholecystokinin (CCK). Application of proglumide at 1-30 mmol/L inhibited bethanechol-induced contraction in both longitudinal and circular muscle strips of the dorsal sac of the rumen. These results suggest that proglumide at a low dose acts indirectly on the rumen as a CCK receptor antagonist to increase the frequency of contractions, whereas at higher doses it inhibits cholinergic-induced contraction of the ruminal muscles or acts as an agonist to inhibit contractions in sheep. Hence, proglumide at high doses seems unsuitable for research or therapeutic use as a CCK receptor blockade in sheep.

Effects of nitric oxide donor and nitric oxide synthase inhibitor on ruminal contractions in conscious sheep.

The present study was planned to evaluate a role of nitric oxide (NO) in the regulation of regular ruminal contractions in conscious sheep. Intravenous infusion of S-nitroso-acetyl-DL-penicillamine (SNAP) at doses of 3-30 nmol kg(-1) min(-1)for 30 minutes inhibited both the amplitude and frequency of ruminal contractions in a dose-dependent manner. However, intravenous infusion of Nomega-nitro-L-arginine-methyl ester (L-NAME) at doses of 0.3-3.0 micromol kg(-1) min(-1)did not alter the basal tone of intraruminal pressure and the amplitude of ruminal contractions. The frequency of contractions was slightly inhibited by L-NAME infusion at 1.0 micromol kg(-1)min(-1). The effects of L-NAME were abolished by simultaneous infusion of L -arginine at 30 micromol kg(-1) min(-1). These results suggest that exogenous NO can diminish the ruminal contractions, while endogenous NO is not involved in the regulatory mechanism of basal tone and regular phasic contractions of the rumen in healthy sheep.

Effect of nitric oxide synthase inhibitors on the temporal coordination of duodenal contractions and pancreatic exocrine secretion in sheep.

The present study evaluated the role of nitric oxide in the regulation of duodenal motility and pancreatic exocrine secretion in conscious sheep. Intravenous infusions of nitric oxide synthase inhibitors, Nomega-nitro-L-arginine-methyl ester (L-NAME) and Nomega-nitro-L-arginine, induced clusters of duodenal contractions like phase III of migrating motor complexes and simultaneously inhibited flow rate, bicarbonate ion and enzyme outputs of pancreatic juice. The effects of L-NAME were inhibited by simultaneous infusion of L-arginine, but not altered by adrenergic blockade using a combined infusion of phentolamine and propranolol. Inhibition of the pancreatic secretion occurred in coincidence with initiation of the duodenal contractions, while the pancreatic secretion was not inhibited when the premature duodenal contractions were abolished by the L-arginine infusion. The initiation of the cluster of duodenal contractions by L-NAME was not abolished by background infusion of atropine, whereas the amplitude of contractions was significantly inhibited by atropine. These results suggest that intrinsic nitric oxide plays a crucial role in the regulation of duodenal tone and maintenance of continuous secretion by the exocrine pancreas in sheep. These results also implied that inhibition of pancreatic exocrine secretion by the nitric oxide synthase inhibitor is presumably mediated in part through the contractile effect on the duodenum.

Regional distribution and plasma concentration of peptide YY in sheep.

Peptide YY (PYY)-positive cells are distributed in the mucosa of the ileum, cecum, colon, and rectum of sheep, but not in other layers of these regions. By radioimmunoassay, mucosal content of PYY in the ovine large intestine was much less than that in the rat intestine. The plasma concentration of immunoreactive PYY did not significantly fluctuate over a 48-h period in conscious sheep, even after ingestion of roughage and concentrate. Intraluminal nutrients into the ileum and i.v. CCK8 also did not raise the plasma level of PYY. Therefore, PYY seems unlikely to play a role as "ileal brake" in sheep.

Small intestinal UDP-glucuronosyltransferase sheUGT1A07: partial purification and cDNA cloning from sheep small intestine.

A phenol UDPglucuronosyltransferase (UGT) was partially purified, and the cDNA encoding the isoform was cloned and sequenced from sheep small intestine. The purified preparation containing a one major band (57 kDa) and one minor band (50 kDa) revealed high activities toward xenobiotics such as 1-naphthol (1-NA), 4-nitrophenol, and 4-methylumbelliferone. The preparation, however, had only little activity toward 4-hydroxybiphenyl and no activity toward bilirubin, suggesting that the preparation contains UGT1 isoforms. The NH2-terminal amino acid sequence of the major band was determined to be Gly-Lys-Leu-Leu-Val-Val-Pro-Met-Asp-Gly-Ser. A full-length UGT cDNA was obtained by reverse transcription-polymerase chain reaction with the degenerated 5'-primer from the NH2-terminal amino acid sequence of the purified major one and rapid amplification of cDNA ends from sheep small intestine. The cloned cDNA named sheUGT1A07 by amino acid similarity has a NH2-terminus sequence identical to that of the purified major one. Another phenol UGT cDNA named sheUGT1A6 was also cloned from sheep liver. sheUGT1A6 was expressed mainly in the liver, whereas sheUGT1A07 mRNA was expressed almost only in the alimentary organs, suggesting that sheUGT1A6 plays a role as a general drug metabolizing UGT isoform in the liver and sheUGT1A07 plays important role in the xenobiotics glucuronidation in the sheep small intestine.

Pituitary adenylate cyclase-activating polypeptide (PACAP) induces duodenal phasic contractions via the vagal cholinergic nerves in sheep.

This study examined the effect of intravenous infusion of pituitary adenylate cyclase-activating polypeptide (PACAP) on duodenal motility in sheep and the mechanism of the action of PACAP. The bilateral cervical vagus nerves were coiled with a cooling device under anesthesia. Duodenal motility was recorded by manometry in conscious animals. PACAP-27, PACAP-38 and vasoactive intestinal polypeptide (VIP) were infused intravenously at 3, 10, 30 and 100 pmol/kg in phase II of the duodenal migrating motor complexes (MMC). PACAP-27 induced a cluster of phasic contractions of the duodenum, while PACAP-38 only augmented the spontaneous contractions. The pattern of PACAP-27-induced contractions was different from that of phase III of MMC, and the contractions were followed by a quiescence period. VIP at only the highest dose induced phase III-like activity of the duodenum. Both the intravenous background infusion of atropine at 10 nmol/kg/min and the reversible cooling blockade of the bilateral cervical vagus nerves blocked the effect of PACAP-27. The application of PACAP-27 at concentrations ranging from 10 nM to 1 microM did not induce contractions of ovine duodenal smooth muscles. These results indicate that PACAP contracts the ovine duodenum via the vagal cholinergic efferent fibers, suggesting that PACAP acts on the central nervous system.

[Hyperbaric oxygen therapy for radiation-induced hemorrhagic cystitis].

BACKGROUND: Radiation therapy has widely been used for cancers in the pelvis. Radiation cystitis, one of the late complications, presents often as hemorrhagic cystitis, which is refractory to the conventional therapy and may threaten the patient's life. We used hyperbaric oxygen therapy on patients with radiation cystitis to test its potential benifit. METHODS: Ten patients aged from 46 to 81 years with a mean of 62 years underwent one or more courses of hyperbaric oxygen therapy according to their symptoms, consisting of 20 sessions (3 to 5 sessions a week) at the Department of Hyperbaric Medicine, the University of the Ryukyus Hospital in the 9-year period from 1985 to 1994. They included 8 patients having a history of cervical cancer, one with external genital cancer and one with vaginal cancer. During the 75 min hyperbaric oxygen therapy patients received 100% oxygen at 2 absolute atmosphere pressure in the Multiplace Hyperbaric Chamber. RESULTS: Hematuria subsided and subjective symptoms including urinary frequency improved in seven patients. Cystoscopic findings including mucosal edema, redness, and capillary dilation were partially improved. The procedure subjectively and objectively palliated the 10 patients in a favorable manner. CONCLUSIONS: To date we have not armed any active procedure to control radiation-induced refractory hemorrhagic cystitis in terms of efficacy, invasiveness, and adverse effects. Therefore, in consideration of our clinical results, hyperbaric oxygen therapy appears to be useful for radiation cystitis.

Effects of proglumide on cholecystokinin-8-induced exocrine and endocrine pancreatic responses in conscious sheep.

The effects of cholecystokinin (CCK)-8, either alone or together with the CCK antagonist, proglumide on both exocrine and endocrine pancreatic responses were examined in conscious sheep. Intravenous infusions of CCK-8 (120 pmol/kg/min for 40 min) with vehicle (0 mumol/kg/min proglumide) significantly increased both amylase output in pancreatic juice and plasma insulin concentrations (P < 0.05). Concomitant infusions of proglumide (5-40 mumol/kg/min for 50 min) inhibited both amylase and insulin secretory responses induced by CCK-8 infusion. The antagonistic effects of proglumide occurred in a dose-dependent manner, and proglumide infusion at dose of 20 mumol/kg/min or above simultaneously inhibited CCK-8-induced amylase and insulin responses. In conclusion, although the type of receptor involved is not characterized at present, exogenously infused CCK-8 acts on B cells via a CCK-receptor-mediated mechanism and induces insulin secretion in sheep.

PGE2 inhibits glucose uptake in isolated villous epithelial cells of the ovine small intestine.

This study was designed to clarify whether PGE2 directly inhibits glucose absorption in villous cells. For this purpose, isolated villous epithelial cells of the ovine small intestine were used, and the uptake of 14C-labelled glucose in the absorptive epithelial cells was measured in the presence and absence of 0.2 mM phlorizin or 10 microM PGE2. In isolated villous epithelial cells of the small intestine in sheep, net glucose absorption was reduced by 68.2% in the presence of phlorizin. This result indicates that most of the glucose in ruminants is absorbed by the specific transporter SGLT1, which is sensitive to phlorizin. PGE2 decreased glucose absorption by 26.5% in isolated villous epithelial cells in sheep, although this glucose absorption did not change in the presence of phlorizin. This result clearly indicates the PGE2 inhibits the phlorizin-sensitive glucose absorptive process through direct action on the villous cells of ruminants.

Role of peptide YY in regulation of duodenal motility during the interdigestive period in sheep.

Temporal coordination between duodenal migrating myoelectric complexes (MMC) and pancreatic exocrine secretion, and the effects of porcine peptide YY (PYY) on gastroduodenal motility and pancreatic exocrine secretion were examined during the interdigestive period in conscious mature sheep. Fluid and enzyme secretions from the exocrine pancreas showed a periodic pattern corresponding to the phases of duodenal MMC, although these secretion rates were maintained at a high level during phase II in sheep. Intravenous continuous infusion of PYY at doses ranging from 50 to 200 pmol.kg-1.h-1 or intravenous bolus infusion of PYY at doses ranging from 50 to 200 pmol.kg-1 showed a tendency to prolong the first cycle of the duodenal MMC and significantly shorten the second cycle. However, there was almost no effect on ruminal contractions from the PYY administration. In the pancreatic exocrine secretion, PYY could inhibit only bicarbonate secretion at only the highest dose of 200 pmol.kg-1. These results imply that endogenous PYY may play a physiological role in the regulation of the duodenal MMC cycles in sheep but not in ruminal contractions. PYY seems unlikely to regulate the pancreatic exocrine secretion in normal sheep, because a supraphysiological dose of PYY was required to inhibit the pancreatic exocrine secretion.

Effects of oxytocin, arginine-vasopressin and lysine-vasopressin on insulin and glucagon secretion in sheep.

The effects of the posterior-pituitary peptides oxytocin (OT), arginine-vasopressin (AVP) and lysine-vasopressin (LVP) on insulin and glucagon secretion were examined in adult sheep. Each peptide was injected intravenously at doses from 1 to 3000 pmol kg-1. All three peptides increased plasma insulin and glucagon concentrations, but their dose-response relationships revealed differences between them. The maximal insulin responses induced by OT and AVP were very similar, but the threshold and maximal doses of AVP for increasing plasma insulin were higher than those of OT. OT and AVP had the same activity for stimulating glucagon secretion in respect of the threshold and maximal doses and the maximal hormone response. LVP also increased plasma insulin and glucagon concentrations, but it had the weakest activity for stimulating both hormones. These results suggest that in sheep posterior-pituitary peptide may play a role in regulating nutrient metabolism by influencing pancreatic hormone secretion.

Responses of glucose absorption and fructose absorption to prostaglandin E2 in intestinal loops of sheep.

This study was designed to clarify whether the anti-absorptive action of prostaglandin E2 (PGE2) on glucose absorption was specific or non-specific to the glucose absorptive process, by investigating its effect on fructose absorption which has a different mechanism from that of glucose absorption. The effect of PGE2 on mucus secretion was also evaluated as one of the non-specific inhibiting factors. PGE2 significantly stimulated the secretion of water and mucus, and the absorptions of glucose and fructose were inhibited 49.3 per cent and 31.1 per cent respectively at the highest dose. However, fructose absorption was not inhibited by lower doses of PGE2, although the secretion of fluid and mucus was stimulated significantly and glucose absorption was inhibited significantly at the lower doses.

Effects of C-terminal fragments of cholecystokinin on plasma insulin and glucagon concentrations in sheep.

The effects of three C-terminal fragments of cholecystokinin (CCK) (CCK-8-sulphated form [SF], CCK-8-non-sulphated form [NSF] and CCK-4) on insulin and glucagon secretion were examined in sheep in vivo. Each CCK fragment was injected intravenously at a wide range of doses (1 pmol to 3 x 10(5) pmol kg-1). CCK-8(SF) had the lowest threshold dose (10 pmol kg-1) and a maximal response dose of 10(3) pmol kg-1 for increasing plasma insulin concentration; the respective threshold doses of CCK-8(NSF) and CCK-8 for increasing plasma insulin were 30 and 100 times greater than that of CCK-8(SF). A maximal insulin response was not obtained at the highest doses of CCK-8(NSF) or CCK-4 tested (3 x 10(3) and 3 x 10(5) pmol kg-1, respectively). These results indicate that CCK-A type receptors rather than CCK-B receptors may be involved in CCK-induced insulin secretion in sheep. None of the CCK fragments affected plasma glucagon concentration. The lack of a glucagon response to exogenous CCK-fragments may be one of the characteristics of the endocrine pancreatic responses of ruminant species.

Effect of L364718 on interdigestive pancreatic exocrine secretion and gastroduodenal motility in conscious sheep.

The present study examined roles of endogenous cholecystokinin (CCK) and CCK-A receptors in the regulation of pancreatic exocrine secretion and gastroduodenal motility in conscious sheep during interdigestive period. Interdigestive exocrine secretion of ovine pancreas changed cyclically corresponding with cycle of duodenal migrating myoelectric complexes (MMC). During second phase of the duodenal MMC, intravenous injection of L364,718 at 2.45 mumol kg-1 inhibited exogenous CCK-8-induced pancreatic exocrine secretion. Intravenous infusion of the antagonist at 2.45 mumol kg-1/5 min for 5 min also inhibited significantly the pancreatic enzyme secretion without CCK-stimulation to half of that in the control, but not the fluid and bicarbonate secretion. Atropine infusion (i.v.) at 72.0 nmol kg-1/5 min significantly inhibited not only enzyme but also fluid and bicarbonate secretion. Corresponding to the inhibition of the exocrine secretion, L364,718 induced premature phase III in duodenal electromyogram (EMG) in three of the five sheep. Omasal EMG was inhibited slightly but significantly by L364,718, however, neither regular ruminal contractions nor abomasal EMG were altered by L364,718. In contrast, the atropine infusion inhibited only amplitude of ruminal contractions. These results suggest that endogenous CCK contributes to the regulation of interdigestive pancreatic exocrine secretion, omasal contractions and duodenal MMC in the ovine gastrointestinal tract via CCK-A receptors.

PACAP stimulates pancreatic exocrine secretion via the vagal cholinergic nerves in sheep.

The present study evaluates the possible role of the vagus nerves in mediating the stimulatory effect of PACAP-27, PACAP-38 and VIP on the exocrine pancreas, especially on enzyme secretion which is atropine sensitive in sheep. The animals were equipped with two cannulae into the common bile duct, a duodenal cannula, and a ruminal cannula under anesthesia. The bilateral cervical vagus nerves were coiled with a cooling device. In conscious animals, the peptides were infused intravenously for 10 min at 10 pmol kg(-1)min(-1) in phase II of the duodenal migrating motor complexes and the same peptide infusion was repeated in the reversible cooling blockade of the vagus nerves. Increment in fluid secretion was not significantly altered by the vagal blockade in all the peptide infusions, while increment in bicarbonate ion by only PACAP-27 was inhibited by the vagal blockade. Increments in protein and amylase output decreased significantly to 32.0+/-5.0 and 23.2+/-2.6% in PACAP27, and to 26.1+/-7.7 and 20.8+/-6.4% in PACAP-38 in the vagal blockade, but the increments by VIP did not decrease. These results demonstrate that circulating PACAP stimulates pancreatic enzyme secretion via the vagal cholinergic preganglionic neurons in sheep, suggesting the central action of PACAP.

Effect of cholera toxin on glucose absorption and net movements of water and electrolytes in the intestinal loop of sheep.

This study was designed to evaluate the effect of cholera toxin on glucose absorption and net movement of water and electrolytes in the jejunal loop of sheep. Intraluminal perfusion was performed at the rate of 1 ml/min with isotonic 10 mM glucose solution. Osmolality was adjusted by adding NaCl, and the outflow solution was collected every 10 min. After a 30 min control period, cholera toxin was applied intraluminally for 30 min at doses of 30, 60, and 120 micrograms/loop. In the control period, water, sodium and chloride were absorbed, while potassium and bicarbonate were secreted. Cholera toxin reversed the net absorption of water, sodium and chloride to net secretions, and this secretory response to cholera toxin was dose-dependent. Bicarbonate secretion was stimulated dose-dependently by cholera toxin. Potassium secretion was also increased at all doses, though this response was not dose-dependent. The net glucose absorption was decreased dose-dependently by cholera toxin. In conclusion, these results indicate that cholera toxin stimulates water and electrolyte secretion, and inhibits glucose absorption in the jejunal loop of sheep.

Effect of pituitary adenylate cyclase-activating polypeptide on exocrine and endocrine secretion in the ovine pancreas.

The role of pituitary adenylate cyclase-activating polypeptide (PACAP) in the regulation of exocrine and endocrine pancreas was investigated in conscious sheep. Intravenous infusions of PACAP-27 and PACAP-38 (1, 3, and 10 pmol/kg/min) for 10 min during phase II of the duodenal migrating myoelectric complex accelerated pancreatic protein and amylase outputs dose-dependently. The responses in enzyme secretion to both PACAPs at the highest doses were inhibited significantly by atropine infusion (14.4 nmol/kg/min). Vasoactive intestinal polypeptide (VIP) at 3 pmol/kg/min significantly accelerated protein but not amylase outputs, although the response to the highest dose was not significantly influenced by atropine. PACAP-27 and VIP increased pancreatic juice flow and bicarbonate output dose-dependently; however, the responses to the highest dose were not altered significantly by atropine. On the other hand, intravenous injection of PACAP-38 (100 pmol/kg) did not influence basal plasma concentration of insulin, glucagon, and glucose. Moreover, PACAP-38 (1-100 pmol/kg) altered neither pancreatic endocrine response to intravenous infusion of glucose (20 mumol/kg/min) not that to n-butyric acid (33 mumol/kg/min). These results suggest that PACAP contributes to the regulation of exocrine secretion of the ovine pancreas but not to endocrine secretion. PACAP appears to accelerate pancreatic enzyme secretion mostly via the cholinergic nerves.