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K B Raja

Publications and source records attributed to K B Raja.

53 records · Page 3Linked to original sources
PubMed1991

Effects of urogastrone-epidermal growth factor on intestinal brush border enzymes and mitotic activity.

The wet weight of the stomach, small intestine, caecum, and colon were significantly reduced (p less than 0.001) in intravenously fed rats compared with orally fed controls. Human epidermal growth factor (urogastrone) reversed this atrophy. Detailed analysis of the small intestine showed a similar effect on intestinal crypt cell population, mitoses per crypt, and protein content. Brush border gamma glutamyltransferase and alpha glucosidase activities were reduced by up to 50% throughout the small intestine of the animals fed intravenously. The specific activities (mU/mg protein) were unchanged, as a concomitant decrease in the tissue weight and protein content also occurred. Intestinal brush border enzyme activities in the rats treated with urogastrone-epidermal growth factor were restored to those seen in the orally fed rats except for alpha glucosidase activity in the proximal gut. In addition, the specific activity of gamma glutamyltransferase was highly significantly increased (p less than 0.01) in all regions of the small intestine. Thus, although urogastrone administration prevents the decrease in brush border enzyme activity seen after the removal of luminal nutrition, the response seems to differ depending on the intestinal location, with the specific activities of some enzymes being higher than those seen in orally fed rats. Urogastrone-epidermal growth factor can thus significantly increase the functional ability of the intestine in addition to its trophic effects.

Animals↗
PubMed1990

Effects of turpentine-induced inflammation on the hypoxic stimulation of intestinal Fe3+ absorption in mice.

Chronic subcutaneous turpentine administration (weekly for 6 weeks) induced a mild normocytic anaemia in mice. In-vitro and in-vivo intestinal Fe3+ absorption parameters were, however, not significantly altered from values in saline-treated or untreated mice. Normal mice, when exposed to 3 days hypoxia demonstrated a 2-3-fold increase in iron absorption in vivo, mainly due to changes in the amount of iron transferred from the mucosa to the plasma and thence to the carcass. A 2-3-fold increase in Vmax was also observed in in-vitro uptake experiments using isolated duodenal fragments. In contrast, turpentine-treated animals, though demonstrating an enhanced in-vitro maximal uptake capacity, failed to elicit an adaptive response in vivo following hypoxic exposure. These findings suggest that a circulating (humoral) factor may be responsible for the inhibition in absorption in vivo in this turpentine-induced inflammatory model.

Anemia↗
PubMed1989

Membrane potential dependence of Fe(III) uptake by mouse duodenum.

Intestinal iron uptake by mouse duodenal fragments is inhibited in the absence of oxygen and glucose from the incubation medium and by a variety of metabolic inhibitors. The mechanism of energy coupling to iron uptake is, however, unclear. In vitro experiments using duodenal fragments showed Fe3+ uptake to be markedly inhibited, in a reversible fashion, by the replacement of incubation medium Na+ by K+. Addition of phloridzin to the medium failed to affect iron uptake, suggesting that the above effect was not a consequence of reduced glucose uptake. Substitution of Na+ by Rb+ also potently reduced duodenal iron uptake. Replacement of medium NaCl by either mannitol or choline chloride had no significant effect on Fe3+ uptake, thus excluding the possibility of the Fe3+ uptake process being Na+-dependent. Similar observations were made with duodenal fragments from animals with enhanced Fe3+ absorption, due to chronic hypoxia. Valinomycin (1-5 microM) increased the uptake of both glucose and Fe3+. Higher concentrations (22.5 microM) of the ionophore were inhibitory. In vivo studies (tied-off segments) using Rb+-containing medium confirmed the inhibitory effects of univalent cations on Fe3+ absorption. Enhanced absorption of Fe3+ was also demonstrable in vivo, with low concentrations of valinomycin and nigericin added to the luminal medium. These observations suggest that the Fe3+ uptake process may be dependent on the brush-border membrane potential.

Animals↗
PubMed1989

Effect of orally-administered epidermal growth factor on intestinal iron absorption and mucosal permeability.

A progressive increase in intestinal 59Fe3+ absorption was observed on oral feeding of mice with physiological doses of EGF/UGO. Maximal changes were apparent after 3d and appeared to be dose-dependent. In addition to a small increase in intestinal cell proliferation, as reflected by increased ornithine decarboxylase activity, EGF/UGO-feeding increased mucosal permeability (evaluated with [51Cr]-EDTA): the latter could account for the increase in iron absorption. Sialoadenectomy, to remove the major source of endogenous EGF/UGO, had no appreciable effect on the intestinal absorption of iron.

Administration, Oral↗
PubMed1989

Effect of exchange transfusion of reticulocytes on in vitro and in vivo intestinal iron (Fe3+) absorption in mice.

The effect of acute changes in erythron demands and in plasma-iron turnover (PIT) on the in vitro and in vivo absorption of 59Fe3+ was studied in the mouse. Hypoxic exposure for 20 h (a time at which intestinal iron absorption is markedly stimulated) induced reticulocytosis with a marked elevation in PIT. More prolonged exposure (3 d) further enhanced the plasma-iron clearance, even though the absorption of 59Fe was not further increased. Recipient mice, exchange transfused with whole blood from phenylhydrazine-treated animals, had a marked reticulocytosis and elevated PIT. However, in vivo studies exhibited only a small enhancement in intestinal 59Fe absorption. In vitro studies, in contrast, showed no changes in the kinetic parameters for duodenal Fe3+ uptake in similarly-transfused mice. Blood cells, rather than plasma, were responsible for the enhanced in vivo absorption in the transfused animals. These data indicate that acute changes in body iron demands and in PIT have only a small effect on iron absorption via a process independent of the adaptive increase in carrier-mediated uptake following chronic (3 d) hypoxia. This regulatory process, however, is inadequate to explain the adaptive changes seen during acute (20 h) hypoxic exposure.

Animals↗
PubMed1988

In vivo studies on the relationship between intestinal iron (Fe3+) absorption, hypoxia and erythropoiesis in the mouse.

The effect of hypoxia and changes in erythropoiesis on the absorption of 59Fe3+ from in situ tied-off duodenal segments was studied in the mouse. Hypoxia led to an increase in mucosal uptake within 6 h, whilst mucosal transfer was unaffected for about 20 h, suggesting independent regulation of these two processes. Hypoxia (3 d) stimulated erythropoiesis and resulted in a 2-3-fold increase in the total mucosal uptake of 59Fe. Conversely, hyperoxia (100% O2) caused a decrease in reticulocyte counts and the total mucosal uptake. The changes in the transfer of 59Fe from the mucosa to the body were more marked than changes in uptake in both hypoxia and hyperoxia. Mice subjected to subtotal nephrectomy showed a normal increase in the total mucosal uptake of 59Fe3+ following hypoxic exposure, despite the absence of any changes in the reticulocyte count. Obliteration of the erythroid tissue of animals by splenectomy and 89Sr treatment was accompanied by a marked decrease in the transfer of 59Fe from mucosa to the carcass. However, exposure of splenectomized 89Sr-treated mice to hypoxia resulted in an increase in the total mucosal uptake and carcass transfer of 59Fe, without any change in erythropoiesis. These results indicate that hypoxia enhances mucosal iron uptake by a mechanism which is independent of stimulated erythropoiesis, but that changes in the rate of erythropoiesis have an additional effect, particularly on the transfer phase of iron absorption.

Animals↗
PubMed1987

Comparison of 59Fe3+ uptake in vitro and in vivo by mouse duodenum.

Initial rates of 59Fe3+ uptake by mouse duodenal fragments (in vitro) and tied-off duodenal segments (in vivo) have been characterised for control and hypoxic animals. 59Fe3+ uptake by duodenal fragments was rapid, selective and dependent on medium Fe3+-nitrilotriacetate concentration. Most of the 59Fe3+ uptake (70-75%) occurred via the mucosal route and was dependent on the metabolic state of the tissue. Mucosal uptake showed an adaptive increase following exposure of animals to 3 days hypoxia; the enhancement was due to a 2-3-fold increase in Vmax app, without any significant changes in the Km app. Studies of upper small intestine transit times showed a mean residence time of 4-5 min for 59Fe-labelled mouse chow, emphasising the importance of initial uptake measurements. Time courses for in vivo total mucosal uptake exhibited linearity over a wide variety of absorption rates after correction for the permeation by intact metal-chelate complex. The corrected uptake showed a hyperbolic dependence on medium Fe3+-nitrilotriacetate concentration. Kinetic studies revealed a 2-3-fold increase in total mucosal uptake in hypoxia. Mucosa-to-carcass transfer of 59Fe was also markedly increased by chronic hypoxia. The in vitro system exhibits similar qualitative and quantitative kinetics for Fe3+ transport via the mucosal membrane to those obtained in vivo. The results observed in vitro are thus valid and provide a convenient method for further studies on Fe3+ transport in animals and in man.

Animals↗
PubMed1987

Effect of Ca2+ and Mg2+ on the uptake of Fe3+ by mouse intestinal mucosa.

Initial Fe3+ uptake rates by mouse intestinal fragments were determined in vitro. Uptake was dependent primarily on the Fe3+-nitrilotriacetate complex concentration. Addition of Ca2+ and Mg2+ to the incubation medium had only small effects on the Fe3+ uptake rate. Duodenal fragments from hypoxic animals showed enhanced uptake of Fe3+; this increase was more pronounced with a divalent cation-free medium. Ca2+ markedly diminished the Fe3+ uptake by mucosa from hypoxic mice; Mg2+ had no appreciable effect. Distal ileal fragments exhibited lower uptake rates compared to the duodenum, but were more sensitive to the effects of added Ca2+. The ileal fragments did not show an adaptive response of Fe3+ uptake to hypoxia. These results suggest the existence of more than one pathway for mucosal Fe3+ uptake. One pathway, sensitive to Ca2+ and not stimulated by hypoxia, may be present in the duodenum and ileum. A second pathway, inhibited by Ca2+ and exhibiting an adaptive response to hypoxia, occurs only in the duodenum. This latter pathway is more sensitive to the effects of metabolic inhibitors.

Animals↗
PubMed1987

In vitro measurement and adaptive response of Fe3+ uptake by mouse intestine.

In order to define the importance of the mucosal uptake step in the intestinal regulation of iron absorption, unidirectional uptake rates of Fe3+ from a nitrilotriacetic acid chelate were measured in duodenal fragments from mice using an in vitro technique. [57Co]-Cyanocobalamin was used as a marker of adherent incubation medium. Uptake showed saturation kinetics over the concentration range 18-450 microM. Uptake was increased in fragments from hypoxic, dietary iron-deficient and pregnant mice. The enhanced uptake was due to an increase in Vmaxapp. However, the modest increase in uptake rates in pregnancy and the gross changes observed in iron-deficiency make the hypoxic model the most convenient. The increase in uptake in hypoxic animals was located to the duodenal region and was not associated with changes in either total mucosal iron content or epithelial cell turnover. The rate of uptake of iron via the serosa did not change with hypoxia. This study implies that flux of Fe3+ across the brush border is subject to adaptive regulation. The hypoxic model is suitable for investigation into the regulation of iron homeostasis.

Adaptation, Physiological↗
PubMed1986

Studies on the role of transferrin and endocytosis in the uptake of Fe3+ from Fe-nitrilotriacetate by mouse duodenum.

Addition of iron-binding proteins (human serum transferrin, mouse serum transferrin, human lactoferrin) to the luminal fluid in tied-off segments of mouse intestine in vivo led to reduced 59Fe3+ absorption from 59Fe3+-nitrilotriacetate when compared to 59Fe3+-nitrilotriacetate alone. Assay of transferrin in luminal fluid from tied segments revealed only trace amounts of immunoreactivity. The levels of luminal transferrin are unaltered in chronic hypoxia where iron absorption is significantly enhanced. Studies in vitro revealed that NH4Cl, dansylcadavarine, para-chloromercuribenzoate and trinitrobenzenesulphonate have no effect on initial 59Fe3+ uptake rates from 59Fe3+-nitrilotriacetate, while N-ethylmaleimide (1 mM) caused a 40% inhibition. In vivo 59Fe3+ uptake was unaffected by preincubation of tied-off segments with colchicine (5 mM) for up to 2 h. These results suggest that receptor-mediated endocytosis of transferrin is not a significant mechanism in the uptake of luminal Fe3+ by mouse duodenum.

Animals↗
PubMed1986

Fe2+ uptake by mouse intestinal mucosa in vivo and by isolated intestinal brush-border membrane vesicles.

In vivo kinetics of mucosal uptake of luminal 59Fe2+ by tied segments of normal mouse duodenum are characterised by a Km of approx. 100 microM and a Vmax of approx. 9 pmol/min per mg wet weight of intestine. These values were determined at pH 7.25 in the presence of excess sodium ascorbate. Studies with luminal Fe2+ concentrations of 100 microM reveal: uptake is relatively independent of ascorbate: Fe ratio and luminal pH and uptake is potently inhibited by 1 mM Co2+ or Mn2+ and large luminal NaCl concentrations but not by Ca2+. 3 days of hypoxia (0.5 atmospheres) yields no significant increase in subsequent total mucosal uptake by in vivo tied segments while uptake is significantly reduced by semi-starvation. Quantitative comparison of in vivo mucosal uptake with subsequent determination of isolated brush-border membrane 59Fe2+ transport in individual mice reveals a positive correlation (P less than 0.01) between the two parameters. These results, in conjunction with studies of isolated mouse duodenal brush-border membrane (Simpson, R.J. and Peters, T.J. (1985) Biochim. Biophys. Acta, 814, 381-388 and (1986) Biochim. Biophys. Acta 856, 109-114) suggest that the Fe2+ transport properties of isolated brush-border membrane are quantitatively adequate to explain in vivo mucosal uptake in normal and hypoxic mice at Fe2+ concentrations up to 100 microM.

Animals↗
PubMed1986

Relationship between erythropoiesis and the enhanced intestinal uptake of ferric iron in hypoxia in the mouse.

An in vitro technique was used to determine the unidirectional Fe3+ uptake rates in mouse duodenal fragments. In animals in which erythropoiesis had been stimulated by hypoxia, Fe3+ uptake by the duodenal fragments was enhanced due to an increase in Vappmax, However, when erythropoiesis was increased by injections of erythropoietin, intestinal Fe3+ uptake rates were unaffected. Mice subjected to sub-total nephrectomy showed an increased Vappmax for Fe3+ after exposure to hypoxia, despite the absence of an erythropoietic response. When the bone marrow was obliterated by treatment with 89Sr, a small increase in Vappmax for Fe3+ was found: permeability studies and morphometric analyses demonstrated no apparent irradiation damage to the duodenal mucosa of these animals. Exposure of 89Sr-treated mice to hypoxia caused a further significant increase in Vappmax. These results indicate that the increase in intestinal mucosal iron uptake which follows hypoxia is not mediated by erythropoietin or other factors associated with increased erythropoiesis.

Animals↗
PubMed1985

Fe3+ transport by brush-border membrane vesicles isolated from normal and hypoxic mouse duodenum and ileum.

Studies of 59Fe3+ uptake by brush-border membrane vesicles prepared from mouse duodenum have indicated that uptake represents transport across the brush-border membrane which is rate-limited by the membrane-transfer step (Simpson, R.J. and Peters, T.J. (1984) Biochim. Biophys. Acta 772, 220-226). Further studies presented here reveal that the uptake rate represents the net influx rate for Fe3+ and is independent of Na+ in the medium and of the method of vesicle preparation. Uptake by brush-border membrane vesicles prepared from mouse distal ileum also represents predominantly transport and is higher than that observed with duodenal brush-border membrane vesicles. Studies of the initial uptake rate by vesicles prepared from normal and hypoxic mouse intestine demonstrated an increase in Fe3+ transport in duodenal vesicles only.

Animals↗
PubMed1982

The concentration and subcellular localisation of zinc, magnesium and calcium in human polymorphonuclear leukocytes.

The calcium, magnesium and zinc content of human polymorphonuclear leukocyte homogenates and their subcellular fractions has been determined by atomic absorption spectrophotometry. Neutrophils were homogenised in sucrose medium and subjected to analytical subcellular fractionation. The sucrose density gradient fractions were assayed for zinc, magnesium and the principal organelle marker enzymes. Zinc was found to be largely soluble, with small amounts in the alkaline phosphatase-containing granules (phosphasomes). Magnesium had a multimodal distribution with a soluble component, and peaks corresponding to plasma membrane, specific granules and azurophils. The level of calcium, expressed per mg DNA, was significantly decreased in both chronic granulocytic leukaemia (CGL) and pregnancy. Leucocyte zinc was only significantly reduced in CGL. Leukocyte magnesium showed no significant variation within the three groups of subjects. No significant correlation was noted between the zinc, calcium or magnesium content and leukocyte alkaline phosphatase activity in any of the three groups.

Alkaline Phosphatase↗
PubMed1981

Subcellular localization and properties of alkaline inorganic pyrophosphatase in human polymorphonuclear leucocytes.

A new rapid assay for inorganic pyrophosphatase has been developed and the procedure optimised for measurement of the enzyme in human neutrophils. Kinetic studies showed that the activity was optimal at pH 8.0 and was activated by Mg2+. No neutral or acid pyrophosphatase was detected. Neutrophils were homogenised in isotonic sucrose and, after low speed centrifugation the intracellular localization of pyrophosphatase was determined by analytical subcellular fractionation with sucrose density gradient centrifugation. Pyrophosphatase was shown to have a dual localization to mitochondria and cytosol. No activity could be attributed to either the endoplasmic reticulum or alkaline phosphatase-containing granules (phosphasomes). Inhibitor studies clearly show that the cytosolic and mitochondrial pyrophosphatases are due to distinct enzymes. Neutrophils were isolated from control subjects, patients with chronic granulocytic leukaemia and subjects in the third trimester of pregnancy. The specific activity (mU/mg protein) of pyrophosphatase, in contrast to that of alkaline phosphatase was similar in the three groups. Levamisole, a potent inhibitor of alkaline phosphatase had no effect on pyrophosphatase activity, confirming that this activity is not attributable to neutrophil alkaline phosphatase.

Cytosol↗