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.

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.

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.

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.

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.

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.

Effects of benzoic acid and its analogues on insulin and glucagon secretion in sheep.

The effects of benzoic acid and its analogues on insulin and glucagon secretion were investigated in conscious sheep. Intravenous injections of benzoic acid increased plasma insulin and glucagon concentrations in a dose-dependent manner between 39-1250 mumol/kg, with ED50s for increasing both hormones of about 625 mumol/kg. Various derivatives of benzoic acid (625 mumol/kg) were administered and structure-activity relationships were examined. A single carboxylic group was essential for stimulating insulin and glucagon secretion, since both hormone responses were abolished with compounds in which the carboxylic group was replaced by sulfonic or phosphoric groups, or in which another carboxylic element was introduced (phthalic acids). Most of the compounds which introduced other elements (amino and hydroxy groups, and halogens) onto the benzene ring had an altered stimulating activity. Thus the pancreatic endocrine system can recognize the chemical structure of benzoic acid and its derivatives in detail and induce insulin and glucagon secretion in sheep.

Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates exocrine pancreas in conscious preruminating calves.

The effects of new hypothalamic peptides, PACAP-27 and PACAP-38, and secretin and VIP on the interdigestive pancreatic secretion and duodenal myoelectric activity during the asecretory phase of the pancreatic interdigestive cycle, compared with the milk ingestion phase, were examined in five calves. Peptides were infused for 5 min into the external jugular vein (0, 3, 10, 30 and 100 pmol/kg body wt during the asecretory phase of the pancreatic interdigestive cycle, and the pancreatic secretory response was compared with that obtained during milk ingestion. Intravenous infusion of PACAP-27 caused dose-related stimulation of pancreatic juice flow and bicarbonate and protein output; this effect was identical to infusion of secretin. The effect of PACAP-38 was less pronounced, and that of VIP was the weakest. Pancreatic juice volume and bicarbonate responses during milk ingestion were similar to responses obtained with the highest doses of hypothalamic peptides and secretin, whereas postprandial protein secretion was much greater than the secretion stimulated with peptides. It was concluded that PACAP from the VIP/secretin family may stimulate pancreatic exocrine secretion in conscious calves and a part of the pancreatic response to food intake can be mediated by PACAP.

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.

Effects of intravenous infusions of cholecystokinin-8 and pentagastrin on plasma concentrations of insulin and glucagon in sheep.

The effects of cholecystokinin-8 (CCK-8) and pentagastrin on insulin and glucagon secretion were studied in conscious sheep. Intravenous infusions of CCK-8 (3 to 1000 pmol kg-1 min-1 for 30 minutes) induced a dose-dependent increase in plasma insulin, but did not alter plasma glucagon concentration. The threshold dose of CCK-8 for stimulation of insulin secretion was 10 to 30 pmol kg-1 min-1. Pentagastrin was infused intravenously at doses of 10 to 3000 pmol kg-1 min-1. The maximal dose of pentagastrin slightly stimulated insulin, but not glucagon, secretion. The insulin secretory activity of pentagastrin was only 1/300 that of CCK-8 on a molar basis. The threshold dose of CCK-8 for stimulation of insulin secretion was similar to that for exocrine pancreatic secretion obtained in earlier studies. In conclusion, CCK is a potential candidate as a physiological factor regulating insulin secretion in sheep.

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.

Plasma secretin fluctuates in phase with periodic pancreatic secretion and the duodenal migrating myoelectric complex in calves.

Plasma secretin and cholecystokinin (CCK) levels and the periodic secretions of the exocrine pancreas were studied simultaneously with the duodenal migrating myoelectric complexes (MMC) in six milk-fed calves which had been starved overnight. The experiments were performed first when the calves were 10 to 16 days old and subsequently when they were 36 to 45 days old. Plasma secretin and the secretion of pancreatic juice fluctuated periodically in phase with the duodenal MMC: plasma secretin, and the pancreatic secretion of water and protein were significantly higher during the phase of irregular spiking activity than during the phase of no spiking activity in both investigations. Plasma CCK did not change throughout the MMC. The intravenous infusion of secretin at 120 pmol kg-1 bodyweight for one hour markedly stimulated pancreatic secretion and prolonged the duodenal MMC cycle, but it did not abolish pancreatic and duodenal periodic activity.

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.

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.

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 vasoactive intestinal polypeptide (VIP) on the net movement of electrolytes and water and glucose absorption in the jejunal loop of sheep.

The effect of vasoactive intestinal polypeptide (VIP) on glucose absorption and the net movement of electrolytes and water in the jejunum of sheep was investigated using a Thiry-Vella loop. Intraluminal perfusion of glucose solution (10 mM) containing NaCl (149 mM) and PEG (1 mg/ml) was done at 1 ml/min and the outflow solution was collected every ten minutes. After a 30 min control period. VIP was infused into the jugular vein for 30 min at rates of 10, 30, 100, 300 and 1,000 pmol/kg/hr. In the control period, water, sodium, chloride and glucose were absorbed, while bicarbonate and potassium were secreted. VIP decreased water absorption at 10 and 30 pmol/kg/hr and converted to secretion at over 100 pmol/kg/hr in a dose-dependent manner. Sodium flux changed to secretion only at 1,000 pmol/kg/hr, but chloride flux remained absorptive even at the highest dose. Bicarbonate secretion was stimulated dose-dependently by VIP. Potassium secretion was also increased at all doses, though this response was not dose-dependent. The net glucose absorption was not altered by VIP at any dose. Our findings indicate that VIP stimulates the jejunal secretion of water, sodium, potassium and bicarbonate and that VIP does not inhibit glucose absorption when the secretion of luminal fluid is accelerated by VIP in the jejunal loop of sheep.

Effects of perfusion rate and glucose concentration on glucose absorption in intestinal thiry-vella loop in sheep.

The effects of the perfusion rate and glucose concentration in perfusate on glucose absorption in the intestinal Thiry-Vella loop of conscious sheep were studied. When 10 mM glucose was perfused at the rates of 0.5, 1, 2 and 4 ml/min, the absorption rates were 77.1 +/- 2.2, 54.9 +/- 2.3, 31.6 +/- 2.1 and 12.9 +/- 0.3%, respectively. When 5, 10, 20 and 40 mM glucose solutions were perfused at 1 ml/min, the absorption rates were 75.5 +/- 1.7, 52.2 +/- 2.2, 29.4 +/- 1.9 and 19.5 +/- 0.1%, respectively. We conclude that the perfusion of 10 mM glucose solution at 1 ml/min, in which about a 50% absorption rate was obtained, are the optimal conditions for the study of glucose absorption in the intestinal loop of sheep.

[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.

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.