Local expression of AP/AngIV/IRAP and effect of AngIV on glucose-induced epithelial transport in human jejunal mucosa.

BACKGROUND: Recently it was shown that the classic renin-angiotensin system (RAS) is locally expressed in small intestinal enterocytes and exerts autocrine control of glucose transport. The aim of this study was to investigate if key components for the Angiotensin III (AngIII) and IV (AngIV) formation enzymes and the AngIV receptor, insulin-regulated aminopeptidase (IRAP), are present in the healthy jejunal mucosa. A second aim was to investigate AngIV effects on glucose-induced mucosal transport in vitro. MATERIAL AND METHODS: Enteroscopy with mucosal biopsy sampling was performed in healthy volunteers. ELISA, Western blotting and immunohistochemistry were used to assess the protein levels and localization. The functional effect of AngIV was examined in Ussing chambers. RESULTS: The substrate Angiotensin II, the enzymes aminopeptidases-A, B, M as well as IRAP were detected in the jejunal mucosa. Immunohistochemistry localized the enzymes to the apical brush-border membrane whereas IRAP was localized in the subapical cytosolic compartment in the enterocyte. AngIV increased the glucose-induced electrogenic transport in vitro. CONCLUSION: The present study indicates the presence of substrates and enzymes necessary for AngIV formation as well as the receptor IRAP in the jejunal mucosa. The functional data suggest that AngIV regulates glucose uptake in the healthy human small intestine.

The muscular expression of RAS in patients with achalasia.

INTRODUCTION: Angiotensin II (AngII) elicits smooth muscle contractions via activation of AngII type 1 receptor (AT1R) in the intestinal wall and in sphincter regions in several species. Achalasia is a rare swallowing disorder and is characterized by a loss of the wave-like contraction that forces food through the oesophagus and a failure of the lower oesophageal sphincter to relax during swallowing. AIMS AND METHODS: The present study was undertaken to elucidate expression and distribution of a local renin-angiotensin system (RAS) in the muscular layer of distal normal human oesophagus as well as in patients with achalasia using western blot analysis, immunohistochemistry and polymerase chain reaction (PCR). RESULTS: AT1R, together with enzyme renin and cathepsin D expression were decreased in patients with achalasia. In contrast, the mast cells chymase, cathepsin G, neprilysin and the receptor for angiotensin 1-7 peptides, the MAS receptor, were increased in patients with achalasia. CONCLUSION: The results showed the existence of a local RAS in human oesophageal muscular layer. The enzymes responsible for AngII production are different and there has been a shift in receptor physiology from AT1R to MAS receptor in patients with achalasia. These changes in the RAS might play a significant role in the physiological motor control for patients with achalasia.

Bone mineral density and expression of vitamin D receptor-dependent calcium uptake mechanisms in the proximal small intestine after bariatric surgery.

BACKGROUND: Roux-en-Y gastric bypass may lead to impaired calcium uptake. Therefore, operation-specific effects of gastric bypass and vertical banded gastroplasty on bone mineral density (BMD) were examined in a randomized clinical trial. Bone resorption markers and mechanisms of decreased calcium uptake after gastric bypass were investigated using blood and endoscopic samples from two additional patient cohorts. METHODS: Total BMD and non-weight-bearing skull BMD were measured by dual-energy X-ray absorptiometry at baseline, and 1 and 6 years after gastric bypass or vertical banded gastroplasty in patients who were not receiving calcium supplements. Bone resorption markers in serum and calcium uptake mechanisms in jejunal mucosa biopsies were analysed after gastric bypass by proteomics including radioimmunoassay, gel electrophoresis and mass spectrometry. RESULTS: One year after surgery, weight loss was similar after gastric bypass and vertical banded gastroplasty. There was a moderate decrease in skull BMD after gastric bypass, but not after vertical banded gastroplasty (P < 0·001). Between 1 and 6 years after gastric bypass, skull BMD and total BMD continued to decrease (P = 0·001). C-terminal telopeptide levels in serum had increased twofold by 18 months after gastric bypass. Proteomic analysis of the jejunal mucosa revealed decreased levels of heat-shock protein 90ß, a co-activator of the vitamin D receptor, after gastric bypass. Despite increased vitamin D receptor levels, expression of the vitamin D receptor-regulated calcium transporter protein TRPV6 decreased. CONCLUSION: BMD decreases independently of weight after gastric bypass. Bone loss might be attributed to impaired calcium absorption caused by decreased activation of vitamin D-dependent calcium absorption mechanisms mediated by heat-shock protein 90ß and TRPV6.

Angiotensin IV induced contractions in human jejunal wall musculature in vitro.

Angiotensin II (AngII) has been reported to mediate contractile actions in rats and human jejunal wall musculature. However, except for one report showing the angiotensin IV (AngIV) contractile effects on the internal anal sphincter of rats, no data is available describing the action of AngIV on smooth muscle in human small intestine. The aim of this study was to investigate the expression and localization of the enzymes responsible to AngIV formation, as well as the receptor, and to elucidate the contractile function of AngIV in the muscular layer of human jejunum in vitro. Jejunal smooth muscle was taken from 23 patients undergoing Roux-en-Y gastric bypass surgery and was used to record isometric tension in vitro in response to AngIV alone and in the presence of losartan or PD123319. ELISA, western blot and immunohistochemistry were used to investigate the expression and localization of key components for AngIV formation: the enzymes aminopeptidases-A, B, M, and the AngIV receptor insulin-regulated aminopeptidase (IRAP). AngIV elicited concentration-dependent contraction in both longitudinal and circular smooth-muscle preparation. Presence of losartan abolished AngIV-induced contraction, but not PD123319. The main peptide AngII, as well as the enzymes aminopeptidases-A, B and M was detected in all muscle samples. Immunohistochemistry localized the enzymes and IRAP in the myenteric plexus between longitudinal and circular muscle layers. The present study indicates that all enzymes necessary for AngIV formation exist in human jejunal smooth muscle and that the contractile action elicited by AngIV is primarily mediated through the AngII type 1 receptor.

Intraluminal polyethylene glycol stabilizes tight junctions and improves intestinal preservation in the rat.

Rapidly progressing mucosal breakdown limits the intestinal preservation time below 10 h. Recent studies indicate that intraluminal solutions containing polyethylene glycol (PEG) alleviate preservation injury of intestines stored in UW-Viaspan. We investigated whether a low-sodium PEG solution is beneficial for intestines stored in histidine-tryptophane-ketoglutarate (HTK) preservation solution. Rat intestines used as control tissue (group 1) were perfused with HTK, groups 2 and 3 received either a customized PEG-3350 (group 2) or an electrolyte solution (group 3) intraluminally before cold storage. Tissue injury, brush-border maltase activity, zonula occludens-1 (ZO-1) and claudin-3 expression in the tight junctions (TJ) were analyzed after 8, 14 and 20 h. We measured epithelial resistance and permeability (Ussing chamber) after 8 and 14 h. Group 2 had superior morphology while maltase activity was similar in all groups. TJ proteins rapidly decreased and decolocalized in groups 1 3; these negative events were delayed in group 2, where colocalization persisted for about 14 h. Intestines in group 2 had higher epithelial resistance and lower permeability than the other groups. These results suggest that a customized PEG solution intraluminally reduces the intestinal preservation injury by improving several major epithelial characteristics without negatively affecting the brush-border enzymes or promoting edema.

Angiotensin II-induced contractions in human jejunal wall musculature in vitro.

AIM: Angiotensin II is well known for its contractile effects on smooth muscle cells. This effect is also present in the gut previously shown in animal models. The aim of this study was to clarify expression and localization of angiotensin II receptors in the human small intestine and to explore the pharmacological profile of angiotensin II effects in vitro. METHODS: Strips of jejunal muscle wall from 32 patients undergoing bariatric surgery were used to record isometric tension in vitro in response to angiotensin II (10(-10)-10(-5) M) alone and in the presence of PD123319 (10(-7) M), losartan (10(-7) M), PD123319 (10(-7) M) and losartan (10(-7) M) in combination, tetrodotoxin (TTX) (10(-6) M), atropine (10(-6) M) and guanethidine (3 x 10(-6) M). Western blot, immunohistochemistry and RT-PCR were performed on corresponding muscle samples to identify expression and localization of key components of the renin-angiotensin system. RESULTS: Angiotensin II elicited concentration-dependent contraction in both longitudinal and circular jejunal muscle wall strips; neither TTX, atropine nor guanethidine affected this action. Losartan alone and in combination with PD123319 shifted the concentration-response curve to the right. Transcription of angiotensinogen, ACE and angiotensin II types 1 and 2 receptor RNA was detected in all patients. Immunohistochemistry detected angiotensin II type 1 receptors in the musculature; both angiotensin II types 1 and type 2 receptors were found in the myenteric plexus. CONCLUSION: This pharmacological analysis indicates that the contractile action elicited by angiotensin II on jejunal wall musculature is primarily mediated through the angiotensin II type 1 receptor located on the musculature.

Effects of dietary nitrate on oesophageal motor function and gastro-oesophageal acid exposure in healthy volunteers and reflux patients.

BACKGROUND/AIMS: High concentrations of nitric oxide (NO), derived from dietary nitrite in an acid environment, have been demonstrated in the gastric fundus and in the oesophagus. The aim of this study was to investigate whether luminal NO can influence oesophageal smooth muscle performance, lower oesophageal sphincter (LOS) function or gastric and oesophageal acid exposure. METHODS: Eleven healthy volunteers and 9 patients with chronic gastro-oesophageal reflux disease (GORD) received a diet deprived of nitrate/nitrite but supplemented with placebo or potassium nitrate for 4 days in a randomised order. On day 4 in each trial period, manometry was performed including a sleeve sensor registration of the LOS followed by a simultaneous 24-hour intra-gastric and oesophageal pH registration. RESULTS: Nitrate supplementation increased the proportion of effective peristalsis when analysed for the entire study population. No other significant effects of dietary nitrate were found on oesophageal motor variables, on the sphincter resting tone or on the number or duration of transient sphincter relaxations. No effect was found on either gastric acidity or gastro-oesophageal reflux variables. Major reflux symptoms were not influenced by nitrate administration. CONCLUSION: Dietary nitrate did not significantly affect oesophageal motor or LOS function, gastro-oesophageal acid reflux or reflux symptomatology either in healthy volunteers or in GORD patients.

Oesophageal intraluminal nitric oxide facilitates the acid-induced oesophago-salivary reflex.

BACKGROUND: The present study explores some aspects of the triggering of the acid-induced oesophago-salivary reflex. In addition to hydrogen ions, there are two acid-dependent molecules with messenger potential in the oesophageal lumen: CO2 and NO. The aim of this study was to clarify whether oesophageal NO and CO2 participate in the regulation of salivary neutralizing capacity in response to acid exposure. METHODS: Healthy volunteers received oesophageal acidification composed of HCl, with NO3-, or HCO3- or NO3- and HCO3- in combination. In a second series of experiments, the exposure period was divided into 2 separate 10-min events. Saliva volume and titratable buffering capacity were used to calculate alkaline secretion. RESULTS: Salivary alkaline secretion increased markedly following 20 min intraluminal exposure to HCl. The initial part of this response was 22% +/- 2.2% larger (P < 0.05) if NO3- was present. When HCO3- was added, or if NO3- and HCO3- were given simultaneously, the secretory response tended to be lower. The accumulated responses over 70 min to 2 short HCl exposures (10 min each separated by a 30 min 'rest') compared to one long one lasting 20 min were similar regardless of the presence of NO3-. CONCLUSION: The data suggest that oesophageal intraluminal NO facilitates initiation of the oesophago-salivary reflex. CO2 seems to have a negligible effect on alkaline salivation, and repeated stimulation does not influence the magnitude of the response over time.

Sources of intra-oesophageal nitric oxide production following intraluminal acid exposure.

BACKGROUND: The aim of the present study was to assess luminal nitric oxide (NO) levels in the oesophagus during baseline and acidic conditions and to clarify the sources of such oesophageal NO formation. METHODS: Healthy volunteers received an intra-oesophageal infusion of either HCl (100 mM) or NaCl (50 mM) on two separate study days. After a low nitrate diet, nitrate load or no dietary restrictions/pretreatment, direct intraluminal measurements of NO formation were performed using a tonometric technique. Endoscopy was performed and mucosal biopsies were taken and analysed by means of immunohistochemistry, Western blot and RT-PCR. RESULTS: No intra-oesophageal NO was detected during baseline conditions with pH neutrality. During the infusion of HCI the NO levels rose dramatically to around 12000 ppb. This high rate of NO formation fell by 95% following deviation of saliva. NO formation after an acute nitrate load was almost doubled during acid perfusion compared to control. Immunohistochemistry demonstrated distinct staining for iNOS in the oesophageal squamous epithelial cells, and Western blot and RT-PCR confirmed the presence of iNOS. CONCLUSION: Two sources exist for intra-oesophageal NO formation, both dependent on the luminal acidity: 1) chemical reduction of salivary nitrite, a mechanism related to dietary intake of nitrate, and 2) NO formation within the oesophageal mucosal epithelium by enzymatic degradation of L-arginine. In the latter case, the NO synthase has antigenic characteristics, indicating the inducible isoform, although a functional behaviour suggests an unconventional subtype.

Dynamic involvement of the inducible type of nitric oxide synthase in acid-induced duodenal mucosal alkaline secretion in the rat.

It has previously been shown that mucosal nitric oxide synthase (NOS) is involved in acid-induced duodenal mucosal alkaline secretion. The primary aim of the present study was to elucidate which isoform of NOS is responsible in rats. Immunohistochemistry showed that inducible NOS (iNOS) was constitutively expressed in villous epithelial cells. Exposing the duodenal mucosa to 10 mM HCl resulted in an increased duodenal mucosal alkaline secretion. This response was totally inhibited by intraluminal administration of a selective inhibitor of iNOS (L-N6-1-iminoethyl-lysine). One hour after the acid exposure, western blot technique showed a marked increase in mucosal iNOS expression. A second acid exposure resulted in a further stimulation of alkaline secretion. These data suggest that exposure of the duodenal mucosa to HCI initiates an increased mucosal alkaline secretion, via NO synthesis mediated by iNOS located in the epithelial cells of the villi. In addition, luminal acid stimulates expression of iNOS.

Effects of hypovolaemia on acid-induced duodenal mucosal damage in the rat.

Modest acute hypovolaemia in rats markedly decreases the duodenal mucosal alkaline secretion via neurohumoral links. The present study was undertaken to investigate if such a procedure influences the morphological changes observed following an acid challenge of the duodenal mucosa. Experiments were performed on anaesthetized male Sprague-Dawley rats. HCl (10 or 100 mM) was infused during 15 min into the duodenum via a luminally situated catheter. Time controls were compared with animals bled 10% of total blood volume. Mucosal damage was evaluated by light microscopic morphometry on transverse sections and by scanning electron microscopy of the luminal surface. Perfusion with either 10 mM or 100 mM HCl reduced villus length by about 30%. The villus area was unaffected by 10 mM HCl, but was reduced significantly by 10% by 100 mM HCl as compared with NaCl time controls. Hypovolaemia did not influence the morphometrical changes induced by 10 mM HCl but reduced significantly both villus length (-28%) and villus area (-10%) as compared with the unbled 100 mM HCl group. Scanning electrone microscopy (SEM)-based visual damage score was not influenced by the hypovolaemia procedure in any of the acidities. Morphological changes of the duodenal mucosa, induced by moderate intra-luminal acidity (10 mM HCl), is not influenced by hypovolaemia. However, at higher acidities (100 mM HCl) the hypovolaemia contributes to more severe mucosal damage.

Angiotensin II type 2 receptor-mediated duodenal mucosal alkaline secretion in the rat.

The aims of this study were to elucidate the distribution of angiotensin receptors (AT(1) and AT(2)) in the duodenal wall and to investigate whether AT(2) receptors are involved in the regulation of duodenal mucosal alkaline secretion, which is of importance for the mucosal defense against gastric acid. Immunohistochemistry was used to locate AT(1) and AT(2) receptors in chloralose-anesthetized rats. Duodenal mucosal alkaline output was measured by use of in situ pH-stat titration. Immunohistochemistry demonstrated a distinct staining for both AT(1) and AT(2) receptors in the lamina propria of the villi and also for AT(1) receptors in the muscularis interna. When angiotensin II was infused in the presence of the AT(1) receptor antagonist losartan, mucosal alkaline secretion increased by ~50%. This response was inhibited by the AT(2) receptor antagonist PD-123319. The AT(2) receptor agonist CGP-42112A increased mucosal alkaline secretion by ~50%. This increase was absent in the presence of PD-123319 but not in the presence of losartan or the local anesthetic lidocaine. We conclude that angiotensin II stimulates duodenal mucosal alkaline secretion by activation of AT(2) receptors located in the duodenal mucosa/submucosa.

Tonometric assessment of jejunal mucosal nitric oxide formation in anaesthetized pigs.

Nitric oxide (NO) in the gut has attracted increasing interest as a regulatory factor for a wide variety of intestinal functions. This study was performed to evaluate some methodological aspects and jejunal sources for NO synthesis. Bench side evaluations and an animal model using chloralose-anaesthetized pigs were used. Immunohistochemistry was performed on samples from pig intestine and direct measurements of intestinal NO formation were performed using intraluminal tonometry. Tonometric measurements were quantitatively accurate and with high reproducibility. A substantial NO formation was assessed which was markedly inhibited by luminal administration of the non-selective NOS inhibitor L-NAME. Intravenous administration of L-NAME also reduced jejunal NO formation but to a lesser extent. Immunohistochemistry revealed staining for the inducible type of NOS in the mucosal surface epithelium whereas endothelial and neuronal subtypes were located in deeper layers of the jejunal wall. The study argues for that the source of jejunal NO production, as measured by intraluminal tonometry, is located in close proximity with the intestinal mucosa. The NOS in this compartment is predominantly of the inducible type.

Infiltration and lysis of tumour cell aggregates by adherent interleukin-2-activated natural killer cells is distinct from specific cytolysis.

Adoptively transferred activated natural killer (A-NK) cells infiltrate tumours in vivo. Two in vitro B16-F10 melanoma tumour models were used to study with fluorescence and electron microscopy the infiltration of adherent interleukin 2 (IL-2) A-NK cells: (1) substratum-bound sessile microtumours (MTs), and (2) three-dimensional cell growth on macroporous gelatinous microcarriers (Cultispheres). From 2 h and on increasing numbers of A-NK cells infiltrated the MTs regularly surrounded by a widened intercellular space. An IL-2-dependent disintegration of MTs began at 6-8 h resulting in a release of vital and dead cells. A-NK cell invasion into Cultispheres effectively displaced the melanoma cells from the highly convoluted substratum. Thus, A-NK cell infiltration had a protease-like effect on the tumour cell aggregates which might have a bearing on the interpretation of their cytolytic effect on target cells. Ultrastructural evidence was not obtained of specific A-NK/target conjugate formation or of granule-mediated target cell destruction in either model tumour.