Inhibition by dexamethasone of antigen-induced c-Jun N-terminal kinase activation in rat basophilic leukemia cells

Antigen stimulation of IgE-sensitized rat basophilic leukemia RBL-2H3 cells induced activation of c-Jun N-terminal kinase (JNK) within a few minutes with maximum activity attained 40 min later. The increase in JNK activity was accompanied with an increase in phosphorylation of c-Jun in the cells. The Ag-induced JNK activation was inhibited by the phosphatidylinositol 3-kinase inhibitors wortmannin (10-100 nM) and LY 294002 (100 microM) but not by the protein kinase C inhibitors calphostin C (1 and 3 microM) and Ro 31-8425 (1 and 3 microM). Pretreatment with dexamethasone (10 and 100 nM) for 18 h inhibited the Ag-induced increase in JNK activity in a concentration-dependent manner. At least 6 h of preincubation with dexamethasone was necessary to inhibit the Ag-induced JNK activation. The phosphorylation of c-Jun induced by the Ag stimulation was reduced by pretreatment with dexamethasone without reduction of the content of c-Jun protein. The Ag-induced activation of the JNK kinase kinase mitogen-activated protein kinase-extracellular signal-regulated kinase kinase-1 was also inhibited by pretreatment with dexamethasone at 10 and 100 nM. These findings indicate that dexamethasone reduces JNK protein level and inhibits the Ag-induced activation of JNK resulting in the inhibition of c-Jun phosphorylation.     

Thiamine diphosphatase in rat small intestine

TDPase is located mostly in the proximal portion of the small intestine and its activity, like that of ALPase, decreased markedly in thiamine deficiency. The decreased enzyme activities were restored after thiamine or vitamin D3. Kinetic and other studies of the purified enzyme indicated the identity of the two enzymes.     

Inhibition of vasoactive amine induced contractions of vascular smooth muscle by hydrogen peroxide in rabbit aorta

OBJECTIVE: The aims were to investigate the effects of H2O2 on arterial contractions induced by vasoactive amine agonists and a high concentration of potassium ions (high K+) in vitro and to explore the possible underlying mechanism(s) involved. METHODS: Isometric tension of rabbit isolated aortic strips was measured and the effects of pretreatment with H2O2 on contractions induced by phenylephrine and high K+ were compared. The effects of H2O2 on precontracted strips were determined in the presence and absence of the aortic endothelium and compared with those of acetylcholine. RESULTS: The tension developed in response to an agonist was expressed as a percentage of the contraction induced by high K+ (64.7 mM) superfusion. Pretreatment with 300 microM H2O2 reduced the mean phenylephrine (0.3 microM) induced contraction from 96.2(SEM 1.4) to 61.8(2.8)%; the effect was stable and reversed by washing out the H2O2. Hydrogen peroxide relaxed phenylphrine precontracted strips with and without endothelium but it showed no relaxant effect when the strips were precontracted by high K+, whereas acetylcholine (1 microM) induced transient relaxation of high K+ precontracted strips by 27.8(2.9)%. The relaxant effect of H2O2 was not affected by pretreatment with indomethacin (a cyclo-oxygenase inhibitor), desferrioxamine (a hydroxyl radical scavenger), or diphenylphenylenediamine (a lipophilic antioxidant). CONCLUSIONS: H2O2 inhibits vasoactive amine induced contractions of the vascular smooth muscle of rabbit aorta in vitro without affecting voltage dependent Ca2+ influx or contractile machinery. The mechanism responsible for its inhibitory effects may be related to impairments of the cellular signalling reactions initiated by the agonists.     

Enhancement of Ca++ uptake by lactose in the rat small intestine

In previous experiments, lactose was shown to increase the absorption of Ca++ by the small intestine of the rat and other mammals. To further investigate the mechanism of the lactose effect, Ca++ uptake was studied in everted gut sac preparations. Gut sacs from the rat ileum were preincubated with or without lactose for 45 minutes, and then the tissue uptake of 45Ca over the first 3 minutes was measured in the presence or absence of lactose. The presence of 160 mM lactose increased the initial rate of Ca++ uptake in the first minute by 64% compared to the NaCl control. The lactose effect was dependent on the presence of lactose in the preincubation medium only and not on the presence of lactose during the measurement of Ca++ uptake. Lactose increased Ca++ absorption when the Ca++ concentrations ranged from 0.1 to 10 mM. However, the magnitude of the enhancement was dependent on the lactose concentration and was reduced below 160 mM lactose. When Ca++ and lactose uptake during a 45 minute period was measured in parallel experiments, no evidence for the co-transport of lactose and Ca++ into the tissue was found. These and other data indicated that lactose is not interacting directly with Ca++ in solution but is interacting with the absorptive cells of the intestine to increase their permeability to Ca++.     

Oxyntomodulin reduces hydromineral transport through rat small intestine

Glicentin (GLIC) and oxyntomodulin (OXM) are released from the ileum and colon during digestion. Both hormones reduce fluid and proton secretion in the stomach. The luminal concentration of sodium and chloride underlying the nutrient absorption, the effect of OXM on electrolyte transport through the small intestine, was assessed in vivo using ligated loops and in vitro using Ussing chambers. In vivo, a zero transport state, estimated by the net water, chloride, and sodium fluxes, was observed when an 80 mM NaCl normoosmolar solution (274 mosm) was administered intraluminally. Active secretion was observed with hyperosmotic challenge (474 mosm). The amplitude of this active secretion increased 2.5- to 3-fold when an electrogenic challenge (NaCl 40 mM) was substituted to the hyperosmotic one. OXM (800 fmol/ml plasma) did not modify the basal transport in the duodenum or in the jejunum (t = 45 min). When active secretion was induced by the hyperosmotic challenge, OXM (200 fmol/ml plasma) had no effect on duodenal or jejunal transport (t = 50 min). When active secretion was induced by an electrogenic challenge, OXM (300 fmol/ml plasma) preferentially reduced the hydromineral transport in jejunum. In vitro, OXM also induced a reduction in the ion transport towards the jejunal lumen (EC50 = 20 pM), the amplitude of which depended upon the integrity of the tetrodotoxin-sensitive neurons. In conclusion, OXM was able to reduce the large secretion induced in rat jejunum in vivo by an electrogenic gradient. In vitro, the antisecretory effect of OXM was partly mediated by the neurons present in the intrajejunal wall.     

Cinnamate uptake by rat small intestine: transport kinetics and transepithelial transfer

Due to their ubiquitous occurrence in the plant kingdom, plant phenolics, including monomeric cinnamic acids, are ingested by man and animals in variable amounts with their natural diets. Recently, Na(+)-dependent saturable transport of cinnamic acid across the brush-border membrane of rat jejunum has been described. It was the aim of the present study to characterize this mechanism in more detail. We therefore determined the transport kinetics of mucosal uptake of radioactively labelled cinnamic acid under various conditions using a short-term mucosal uptake technique. In addition, the transfer of cinnamic acid across the jejunal wall was investigated using everted intestinal sacs. Investigations of the kinetics of cinnamic acid uptake by the mid-jejunal mucosa revealed the involvement of two transport components, a diffusive Na(+)-independent mechanism and a saturable Na(+)-dependent mechanism. The results obtained with everted sacs provided further evidence of the existence of an active Na+ gradient-driven transport of cinnamic acid across the intestinal epithelium. In the presence of Na+, a significant accumulation of cinnamate occurred inside the serosal compartment and this was strongly inhibited by serosal ouabain. A decrease in the extracellular pH stimulated mucosal cinnamate uptake by increasing the apparent affinity (1/km). This may be attributable to the involvement of a transmembrane H+ gradient in Na(+)-dependent cinnamate transport because the protonophore FCCP caused a significant reduction of cinnamate uptake only in the presence of Na+. The kinetics of cinnamate transport in the absence or presence of a surplus of either unlabelled cinnamate or unlabelled butyrate indicates a reduction in the apparent affinity of the Na(+)-dependent mechanism involved in cinnamate uptake. These results may be explained by a modification of the mechanism by the intracellular pH. Additionally, competitive inhibition of cinnamate uptake by substances structurally related to cinnamic acid may also be involved.     

1-O-acyl derivatives of glucose as non-penetrating inhibitors of glucose transport by hamster small intestine in vitro

1-O-acyl derivatives of glucose are not transported by the hamster small intestine in vitro. These derivatives, however, are potent inhibitors of the glucose transport system. 1-O-Decanoyl glucose is a competitive inhibitor of beta-methyl glucoside transport.     

beta-Glucosidase activity in the rat small intestine toward quercetin monoglucosides

In order to evaluate the positional specificity for a glucoside group in the hydrolysis of flavonoid glucosides in the rat small intestine, beta-glucosidase activity was measured with the quercetin monoglucosides, quercetin-3-O-beta-D-glucopyranoside (Q3G), quercetin-4'-O-beta-D-glucopyranoside (Q4'G) and quercetin-7-O-beta-D-glucopyranoside (Q7G), as well as with quercetin-3-O-rutinoside (rutin) and p-nitrophenyl-beta-D-glucopyranoside (NPG) by using the HPLC technique. Enzymes were prepared from rat small intestinal mucosa of the duodenum, jejunum and ileum, among which the enzyme activity of the jejunum was highest for all the glycosides tested. Q4'G was the richest substrate for a beta-glucosidase solution among these glycosides, while rutin and NPG were both poor substrates. This suggests that dietary flavonoid glucosides are primarily hydrolyzed and liberated aglycones in the jejunum.     

Mechanism of nitric oxide-induced contraction in the rat isolated small intestine

1. The contractile response to nitric oxide (NO) in ral ileal myenteric plexus-longitudinal muscle strips was pharmacologically analysed. 2. NO (10(-7) M) induced only contraction while 10(-6) M NO induced contraction followed by relaxation. Methylene blue (up to 10(-4) M) did not affect the NO-induced contractions but significantly reduced the relaxation evoked by 10(-6) M NO. Administration of 8-bromo-cyclic GMP (10(-6)-10(-4) M) only induced relaxation. 3. Sodium nitroprusside (SNP; 10(-7)-10(-5) M) induced concentration-dependent contractions per se; the contractile response to NO, administered within 10 min after SNP, was concentration-dependently reduced. The guanosine 3':5'-cyclic monophosphate (cyclic GMP) content of the tissues was not increased during contractions with 10(-8) M NO and 10(-6) M SNP; it was increased by a factor of 2 during contraction with 10(-7) M NO, and by a factor of 12 during relaxation with 3 x 10(-6) M NO. 4. The NO-induced contractions were not affected by ryanodine (3 x 10(-5) M) but were concentration-dependently reduced by nifedipine (10(-8)-10(-7) M) and apamin (3 x 10(-9)-3 x 10(-8) M). 5. These results suggest that cyclic GMP is not involved in the NO-induced contraction in the rat small intestine. The NO-induced contraction is related to extracellular Ca2+ influx through L-type Ca2+ channels, that might be activated in response to the closure of Ca(2+)-dependent K+ channels.     

Action of levorin on glucose transport in the rat small intestine

The technique of accumulating preparation of the mucosa and "turned out sac" was used to show that levorin, a polyenic antibiotic in a concentration of 10(-6) M, lowered the transport rate and accumulation of glucose by the epithelial cells of the rat thin intestine under conditions of oxygenation. Suppression of the glucose transport in the first stages resulted in partial inhibition of the transmembrane transfer. It is suggested that levorin suppression of the glucose transport through the erythrocyte apical membrane in the thin intestine is associated with a decrease in the electrochemical gradient of Na+.     

First-pass metabolism of 5-fluorouracil in the perfused rat small intestine

The first-pass intestinal metabolism of 5-fluorouracil (5-FU) was investigated by single-pass perfusion of the rat small intestine. At the low concentration of 0.06 mg/ml, the fraction of 5-FU absorbed into (i.e., appeared in) the mesenteric venous blood (Fa,b) was about 50% smaller than the fraction absorbed (disappeared) from the intestinal lumen (Fa), indicating the first-pass extraction of 5-FU in the intestinal mucosa. By addition of uracil (6 mg/ml), the Fa of 5-FU was reduced presumably by competition for the pyrimidine carrier at the process of intestinal uptake (entry into the mucosa). The Fa,b was also reduced, but to a lesser extent, resulting in insignificant first-pass extraction. These results suggest that the extraction of 5-FU in the absence of uracil is caused by metabolism and that uracil is a competitor for this pathway. When 5-FU concentration was raised from 0.06 to 0.6 mg/ml in the absence of uracil, the Fa was reduced by about 50%, consistent with the suggestion of the involvement of saturable uptake by the pyrimidine carrier, and thereafter remained unchanged at 6 mg/ml. However, since Fa,b was also reduced by a similar extent, the intestinal availability (FI=Fa,b/Fa) was unchanged at about 0.5, indicating that the intestinal first-pass extraction of 5-FU is independent of concentration with the extraction ratio (difference between unity and FI) of about 0.5 over the wide range of concentration from 0.06 to 6 mg/ml. Thus, the present study demonstrates that the significant first-pass metabolic extraction of 5-FU occurs in the mucosa of the small intestine, supporting our previous suggestion that 5-FU undergoes first-pass metabolism not only in the liver but also in the small intestine after oral administration. Considering that the oral bioavailability of 5-FU in the human (28%) is reportedly comparable with that in the rat (28%), it is likely that intestinal first-pass metabolism may be significant also in the human. Intestinal first-pass metabolism should be taken into account to explore more efficient and controlled oral 5-FU therapy.     

A diurnal rhythm in the absorption of glucose and water by isolated rat small intestine

1. Glucose and water absorption by isolated small intestine from rats which have had unrestricted access to food is 50-60% higher at night than during the daytime. 2. When the feeding time is restricted to 06.00-09.00 hr G.M.T. glucose and water absorption rates in the period from 3 to 7 hr after withdrawal of food are almost as high as the rates observed at night-time in the animals with unrestricted feeding. 3. These changes in absorption rates appear to be associated with feeding time and not with the pattern of illumination.     

The disruption of myofibre structures in rat skeletal muscle after forced lengthening contractions

Specific antibodies against structural proteins (actin, desmin, dystrophin, fibronectin) of muscle fibres were used to study the effect of forced lengthening contractions on muscle microarchitecture. Tibialis anterior (TA) muscle of male Wistar rats were subjected to 240 forced lengthening contractions. At consecutive time points (0, and 6 h, 2, 4, and 7 days) after stimulation, the TA muscle was excised for biochemical and histological assays. Beta-Glucuronidase activity, a quantitative indicator of muscle damage, showed increased values 2-7 days after the lengthening, peaking on day 4 (11.7-fold increase). A typical course of histopathological changes (myofibre swelling, necrosis and regeneration) was observed. In immunohistochemistry, the earliest abnormality observed was discontinuous dystrophin staining in some swollen fibres immediately after commencement of exercise, while at the same time no alterations occurred in the staining of the other antibodies studied. Six hours later, all the swollen fibres were uniformly desmin as well as dystrophin negative. The great majority, but not all, of the swollen fibres showed disorganized actin staining and intramyocellular localization of fibronectin. The early phase disruption of myofibre structures as measured in this study provides evidence of their central role following damage in skeletal muscle. These results suggest that the sequence of structural changes in the route to muscle fibre necrosis in injury induced by forced lengthening contraction originates in the disruption of the plasma membrane and the intermediate filament, which leads to disturbances in the myofibrillar system.     

Small intestinal dysmotility following abdominal irradiation in the rat small intestine

Abdominal symptoms such as diarrhoea, abdominal cramps and vomiting are common during and after abdominal radiotherapy for gynaecological and pelvic malignancy. It has recently been recognized that small intestinal dysmotility may contribute to these symptoms but the underlying mechanisms are unclear in part because of the technical difficulties inherent in performing studies in irradiated small intestine. The aim of the current study was to evaluate small intestinal motor activity using perfused micromanometric techniques in 6-8-cm segments of ileum during arterial perfusion with isotonic oxygenated fluorocarbon solution. Intestinal segments from six rats were studied 4 days after treatment with 10 Gy abdominal irradiation. Ileal segments from nine nonirradiated animals acted as controls. For each experiment the total number of pressure waves, high-amplitude (> 20 mmHg, long-duration > 6 sec) pressure waves, and long (> 20 associated) bursts of pressure waves were determined. Irradiation had no effect on the overall number of pressure waves, but increased high-amplitude long-duration (HALD) pressure waves (248 vs 7, P < 0.01). In control animals HALD waves were localized to a single recording site but after radiotherapy 74% of HALD waves were temporally associated with similar pressure waves in other manometric channels. Forty-seven per cent of associated HALD waves migrated aborally. Retrograde migration of HALD waves was seen in five segments following irradiation. Irradiation abolished bursts of > 20 pressure waves.     

Influence of ischaemia-reperfusion injury on CD44 expression in rat small intestine

BACKGROUND: CD44 is an adhesion molecule expressed by neutrophils and lymphocytes which is involved in cell-cell and cell-matrix binding. In this study, the effect of ischaemia-reperfusion injury on CD44 messenger RNA (mRNA) and cell surface immunohistochemical expression of CD44 in the rat small intestine was evaluated. METHODS: Wistar rats (n=16) were randomized to either serve as controls (sham surgery) or to be subjected to a standardized ischaemia-reperfusion injury (suprarenal aorta occluded for 1 h followed by 1 h of reperfusion). Standardized segments of jejunum were harvested after ischaemia-reperfusion injury (ischaemic and reperfused samples) to measure the mucosal protein and DNA content, mRNA expression of CD44 and the immunohistochemical expression of CD44. RESULTS: Reperfusion significantly damaged the jejunal mucosa, e.g. mucosal protein content was lower after reperfusion compared with that in the control group (z=-2.31, P=0.02) and the ischaemic samples (z=-2.52, P=001). The expression of cell surface CD44 protein was also significantly decreased after ischaemic injury (z=-1.99, P=0.04); this coincided with a decrease in the amount of cytoplasmic CD44 mRNA within isolated enterocytes (z=-2.31, P=0.02). CONCLUSION: Ischaemia-reperfusion injury decreases the expression of CD44 within the jejunal mucosa. This may contribute to the failure of the gut barrier after such injury.     

Effect of hypothyroidism on glucose transport and metabolism in rat small intestine

A cell line (SMKT-R3) established from human renal cell carcinoma was characterized for the presence of sulfolipids and glycolipid sulfotransferases. Sulfolipids were found to constitute a large part of the acidic glycolipid fraction in SMKT-R3 cells. These findings were confirmed by metabolic labelling with 35S-sulfate. These sulfolipids were expressed at the surface of SMKT-R3 cells as ascertained by cytofluorometry using a monoclonal antibody directed to sulfolipids. Furthermore, markedly high activity levels of glycolipid sulfotransferases were observed in SMKT-R3 cells compared with other cell lines. These results suggest that the increased synthesis of sulfolipids in renal cell carcinoma tissue (Sakakibara et al., 1989. Cancer Res., 49, 335-339) is due to the elevation of the sulfotransferase activities of renal carcinoma cells themselves.     

Rhythmic contractions of isolated small arteries from rat: influence of the endothelium

Small arteries of the mesenteric arcade from Wistar rats display rhythmic oscillations superimposed on the tonic contractile response when exposed to submaximal doses of noradrenaline. We have previously shown that mechanical removal of the endothelium abolishes these oscillations. In the present study different methods to eliminate or modify the influence of the endothelium were used in order to further characterize the mechanisms behind rhythmic contractions in these vessels. Endothelium was removed either mechanically or chemically by perfusing the vessels with 0.3% CHAPS. The absence of functional endothelium enhanced noradrenaline sensitivity and simultaneously abolished oscillations in tension and membrane potential, but did not affect resting membrane potential. The rhythmic activity was also reduced or abolished by exposure to haemoglobin, methylene blue, LY83583 or L-NNA. Indomethacin and propranolol were without effect. Sodium nitroprusside or the permeant analogue of cyclic GMP, 8-bromo cyclic GMP, restored rhythmic activity in precontracted endothelium-denuded vessels. The data suggest that release of nitric oxide from the endothelium, and subsequent generation of cyclic GMP in the smooth muscle, activates oscillations in membrane potential and tension; the oscillator itself appears to be located within the smooth muscle cells.