Cytoskeletal control of calcium absorption across the rat small intestine

1. The effect of colchicine, cytochalasin-B and procaine on calcium transport across the rat small intestine was investigated. The results obtained show the following: 2. Colchicine and cytochalasin-B at different concentrations inhibited significantly (P less than 0.001) calcium accumulation in rat intestinal cells, whereas procaine at different concentrations increased significantly (P less than 0.001) calcium accumulation in the rat small intestine. 3. Unidirectional influx of calcium across the rat small intestine was significantly inhibited (P less than 0.01) in the presence of colchicine and cytochalasin-B in the preincubation medium. Procaine, on the other hand, caused a significant increase (P less than 0.01) in the unidirectional influx of calcium across the rat intestinal cells. 4. The cell water content was not altered in the presence of the different drugs indicating that the changes in calcium transport across the rat intestinal cells are not due to alterations in the structure of the cell membrane.     

Cytoskeletal control of alanine transport across the rat and turtle small intestine

Procaine inhibited significantly (P less than 0.01) alanine accumulation in the rat intestinal strips in a concentration-dependent pattern, whereas it showed no effect on alanine uptake by the turtle intestinal cells. Colchicine and Vinca alkaloids at 5 X 10(-4) and 1.5 X 10(-6) M respectively caused a significant inhibition (P less than 0.01) of intracellular alanine concentration in the rat with no effect noticed in the turtle. Unidirectional influx of alanine across the brush border membrane of the rat was significantly (P less than 0.01) reduced in the presence of procaine, colchicine and vincristine in the preincubation medium. The same drugs did not show any effect on alanine influx into the turtle small intestine. Electron microscopy showed major structural alterations in the cytoskeletal organization of the turtle intestine in response to procaine, colchicine or vincristine treatment. It is proposed that microtubular system may participate in the overall transport mechanism of alanine across the small intestine.     

Effect of enzymatic treatment on calcium absorption by small intestine: a comparative study between rat and rabbit transport mechanisms

Calcium absorption by the small intestine of rat and rabbit reached steady state after 60 min of incubation with intracellular to extracellular ratio of 2.0. Trypsin and neuraminidase significantly inhibited (P less than 0.05) calcium accumulation in rat small intestine. These enzymes showed no significant effect (P greater than 0.05) on calcium transport across rabbit small intestine. The inhibitory action of trypsin and neuraminidase on calcium accumulation by the rat small intestine does not involve the influx of calcium into the intestinal cells.     

Effect of enzymatic digestion on phenylalanine transport across the small intestine

Phenylalanine accumulation in mucosal strips isolated from rat small intestine was significantly inhibited (P less than 0.01) after preincubation with trypsin, chymotrypsin, phospholipase D and neuraminidase. Unidirectional phenylalanine influx across the small intestine was significantly reduced (P less than 0.01) when the mucosal strips were preincubated with the above mentioned enzymes. Intestinal cell water and volume were not significantly changed (P greater than 0.6) when the intestinal tissues were preincubated with these enzymes.     

The kinetics of calcium influx into rat liver slices

The unidirectional influx of calcium across rat liver slices is a carrier-mediated process which displays saturation kinetics. The presence of Mg2+ in the incubation medium competitively inhibits calcium influx into rat liver slices. Metabolic inhibitors such as ouabain and 2,4-dinitrophenol at a concentration of 1 X 10(-4) and 2.5 X 10(-4) M respectively, inhibited significantly (P less than 0.001) calcium influx across the liver slices. Calcium influx is dependent on the presence of sodium in the extracellular medium, and is significantly reduced (P less than 0.01) when sodium concentration in the preincubation solution is reduced to zero.     

Influence of agents that affect intracellular calcium regulation on progesterone secretion in small and large luteal cells of the sheep

Enriched fractions of small and large luteal cells were incubated for 2 h with 1 or 10 microM calcium ionophore, A23187: unstimulated secretion of progesterone and viability in small cells were not affected but these measures were decreased (P less than 0.01) for unstimulated large cells and were significantly correlated (P less than 0.05). This effect in large cells was independent of extracellular calcium. Therefore, incubations of the two cell types were made in the presence of increasing concentrations of a protein kinase C activator, phorbol 12-myristate 13-acetate (TPA). Secretion of progesterone and viability were not augmented in unstimulated small cells, but TPA prevented (P less than 0.05) the full stimulation of secretion of progesterone by LH. Secretion of progesterone in unstimulated large cells was inhibited (P less than 0.01) by TPA (100 nM and 10 microM), although viability was unaffected. The non-tumour promoting phorbol ester, 4 alpha-phorbol didecanoate, had no effect on large cells. Extracellular calcium was not required for the observed effect of TPA. Sphingosine, an agent inhibitory to protein kinase C activity, inhibited (P less than 0.01) secretion of progesterone in small and large cells, and also reduced (P less than 0.01) cell viability. These values were significantly correlated (P less than 0.05) in both cell types. The above observations suggest that protein kinase C may invoke negative regulation on progesterone production in unstimulated large and hormone-stimulated small luteal cells of sheep. Since sphingosine significantly reduced viability in small and large cells and ionophore selectively inhibited viability in large cells, the ability of these agents to influence calcium-mediated intracellular regulation of steroidogenesis is still uncertain.     

Glucose 1-phosphate increases active transport of calcium in intestine

Active calcium transport in intestine is essential for serum calcium homeostasis as well as for bone formation. It is well recognized that vitamin D is a major, if not sole, stimulator of intestinal calcium transport activity in mammals. Besides vitamin D, endogenous glucose 1-phosphate (G1P) affects calcium transport activity in some microorganisms. In this study, we investigated whether G1P affects intestinal calcium transport activity in mammals as well. Of several glycolytic intermediates, G1P was the sole sugar compound in stimulating intestinal calcium uptake in Caco-2 cells. G1P stimulated net calcium influx and expression of calbindin D9K protein in rat intestine, through an active transport mechanism. Calcium uptake in G1P-supplemented rats was greater than that in the control rats fed a diet containing adequate vitamin D3. Bone mineral density (BMD) of aged rat femoral metaphysis and diaphysis was also increased by feeding the G1P diet. G1P did not affect serum levels of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] at all. These results suggest that exogenously applied G1P stimulates active transport of calcium in intestine, independent of vitamin D, leading to an increase of BMD.     

Oxalate uptake in intestinal and renal brush-border membrane vesicles (BBMV) in vitamin B6-deficient rats

Acute, subclinical, and chronic pyridoxine deficiency did not modify the oxalate influx in rat intestinal BBMV but elevated the oxalate reabsorption by renal tubular cells. The Na+ and K+ ions did not affect oxalate uptake in either intestinal or renal BBMV. Although thiol group blocking agents did not affect intestinal uptake of oxalate they significantly altered oxalate translocation across the renal tubular cells. Following pyridoxine deficiency the rat kidneys appear to be more specific for inducing oxalate lithiasis as compared to oxalate influx through the intestine.     

Intestinal iodide secretion and its dependence upon mucosal I- permeability

Iodide secretion across different regions of rat small intestine has been investigated in vitro using the standard Wilson-Wiseman technique. Net I- secretion was observed along the entire small intestine, being significantly higher in the central region. Anaerobic conditions, ouabain (2 mM) and Na+ free Ringer solution prevented net I- secretion, whilst both theophylline (1 mM) and carbachol (0,1 mM) enhanced the observed basal intestinal I- secretion. Furthermore, Ca2+-deprived bathing solutions significantly reduced intestinal I- secretion. Epithelial I- uptake from both mucosal and serosal sides was measured by using a Ussing-type chamber technique. The initial rate of I- uptake across the mucosal membrane was significantly higher in the central region than in the proximal part of rat small intestine. No significant differences were observed in the rate of I- uptake from the serosal side. These studies suggest that mucosal I- permeability might determine the direction of net I- intestinal transport and that cytosolic Ca2+ may be a physiological regulator of intestinal I- transport.     

Giardia lamblia: stimulation of growth by human intestinal mucus and epithelial cells in serumfree medium

Giardia lamblia trophozoites specifically colonize the upper human small intestine which is normally serumfree but have been grown in vitro only in medium supplemented with serum or serum fractions. Recently, we demonstrated that biliary lipids will support the growth of G. lamblia without added serum. Now, we report that human duodenal jejunal mucus stimulates growth of Giardia in medium with biliary lipids. Stimulation by mucus was enhanced by inclusion of chymotrypsin or crude pancreatic proteases. Coculture of trophozoites with human intestinal epithelial cells also promoted growth, especially in the presence of mucus and/or biliary lipids. With biliary lipids alone, the mean increase in cell number was 3.2 fold and in the presence of mucus 8 fold (P less than 0.01) in 24 serial subcultures. Our demonstration that human intestinal mucus and epithelial cells promote serumfree growth of G. lamblia may help to explain specific colonization of the small intestine by G. lamblia.     

Effect of size of Trichinella spiralis (Nematode) infections on glucose and ion transport in the rat intestine

An in vivo perfusion technique, using 3 intestinal loops representing the anterior, mid and posterior regions of the rat small intestine, was used to determine intestinal glucose uptake 5 days after infection with Trichinella spiralis. At high levels of infection (3,000 and 6,000 larvae/rat) net glucose absorption by the intestinal mucosa was significantly impaired in all regions of the small intestine when compared to uninfected controls. At low levels of infection (50 larvae/rat) glucose uptake by the mucosa was significantly enhanced in all 3 regions of the small intestine. Intermediate levels of infections (200-1,000 larvae/rat) also enhanced glucose uptake, but only in the anterior regions of the small intestine. When washings from the small intestine of rats infected with 50 larvae/rat were added to the perfusion fluid used on uninfected rats, glucose uptake was also significantly enhanced. These results suggest that at low levels of infection the intestinal lumen contains a metabolite which may affect the mucosal transport of glucose and the related fluxes of H2O, Na+, Cl-, and K+, in the rat intestine. Luminal [H+] and pCO2 decreased from the proximal to distal regions of the small intestine following perfusion; pO2 was significantly decreased in the proximal and distal regions.     

Influence of Prolactin and Calcium Gluconate Concentration on Permeation and Intestinal Absorption of Ca(II) Ions

The in vitro permeation and absorption of calcium ions across the small intestine were measured at different concentrations of calcium gluconate solutions (1.0, 10.0 and 20.0 mM) with or without prolactin. The calcium ions permeated through the small intestine from a donor environment to an acceptor environment that mimicked the conditions in the stomach to ileum segment of the digestive tract. The permeation and absorption of calcium were directly dependent on the calcium concentration of the solutions. At 10 and 20 mM permeation was significantly higher than that at 1.0 mM (p < 0.05). In the presence of prolactin both permeation and absorption increase considerably. At the lowest concentration (1.0 mM) simulating calcium deficiency, there was compensation by the small intestine, suggesting that such deficiency stimulates its mobilization from intestinal tissue. Prolactin enhances the calcium mobilization process even at sufficient calcium intakes. It is suggested that prolactin takes part in regulation of calcium homeostasis in the organism.     

In vitro transport of beta-lactoglobulin across the jejunum of lactose-fed rats

Changes in protein absorption through the intestinal mucosa occur with aging and might reflect modifications in enterocyte membrane characteristics. We have observed that bovine beta-lactoglobulin (beta-LG) was efficiently transported across the small intestine of adult rats in vitro and that 12-16% of the absorbed protein was recovered intact or as large hydrophobic peptides. Ten percent lactose-feeding resulted in decreased tissue conductance and significantly reduced (-58%, P less than 0.05) beta-LG transport across rat small intestinal mucosa. The amounts of beta-LG absorbed, either as amino acids and peptides or as intact protein, were reduced to the same extent. Therefore, the effect of lactose feeding might be related to a decrease in protein endocytosis at the brush-border level, rather than to reduced protein transport across tight junctions.     

The effect of alcohol on zinc absorption

Two methods of intestinal perfusion are described and used to study the effecs of alcohol on zinc absorption in the rat small intestine. The first method used perfusion of the lumen of the rat small intestinein situ without interruption of the vascular supply. During perfusion with a zinc-containing medium (with and without alcohol), alcohol was found to have no effect on net zinc uptake from the lumen of the intestine. However, there were significantly higher serum zinc concentrations recorded in the rats perfused wih the zinc and alcohol, 28.8 μmol/L, when compared with a group perfused without alcohol, 19.1 μmol/L (P < 0.01). The second method used simultaneous perfusion of the lumen of the rat small intestine, with constant-rate perfusion of the vascular bed with an artificial blood supply. In this experiment with a zinc-containing medium, with and without alcohol, there was no difference noted in zinc absorption from the lumen of the intestine, or release into the artificial blood supply. Therefore, in conclusion, alcohol does not appear to directly influence zinc absorption by the mucosal cells of the small intestine.     

Intestinal contractions and migration behaviour in Hymenolepis diminuta.

The aim of this study was to determine if intestinal contractions were important in the migration behaviour of the rat tapeworm Hymenolepis diminuta. The objectives were to investigate the intestinal motility responses of the host to a meal which initiates worm migration, and the worms' responses to an artificial peristaltic contraction. A 1 g glucose meal elicited a significant orad migration by H. diminuta in the small intestine of the rat host when compared to water-fed controls (P less than 0.05). The glucose meal also significantly increased the transit rate, and thus, frequency of intestinal contractions in the small intestine of the rat, when compared to water-fed controls (P less than 0.05). Application of a circumintestinal ligature (6.3 g) (simulating an intestinal peristaltic contraction) resulted in significant worm migration when the ligature was applied in regions containing the worm's strobila as compared to controls where loose ligatures were tied in regions containing the strobila, or to controls where tight ligatures were tied ahead of the worm's strobila. These results suggest that H. diminuta migrates in an orad direction in response to the mechanical pressure produced by intestinal contractions induced by host feeding. It is concluded that contractions of the small intestine are an important cue in the migration behaviour of this cestode.     

Mast Cells and Gastrointestinal Dysmotility in the Cystic Fibrosis Mouse

Background

Cystic fibrosis (CF) has many effects on the gastrointestinal tract and a common problem in this disease is poor nutrition. In the CF mouse there is an innate immune response with a large influx of mast cells into the muscularis externa of the small intestine and gastrointestinal dysmotility. The aim of this study was to evaluate the potential role of mast cells in gastrointestinal dysmotility using the CF mouse (Cftr^tm1UNC, Cftr knockout).

Methodology

Wild type (WT) and CF mice were treated for 3 weeks with mast cell stabilizing drugs (ketotifen, cromolyn, doxantrazole) or were treated acutely with a mast cell activator (compound 48/80). Gastrointestinal transit was measured using gavage of a fluorescent tracer.

Results

In CF mice gastric emptying at 20 min post-gavage did not differ from WT, but was significantly less than in WT at 90 min post-gavage. Gastric emptying was significantly increased in WT mice by doxantrazole, but none of the mast cell stabilizers had any significant effect on gastric emptying in CF mice. Mast cell activation significantly enhanced gastric emptying in WT mice but not in CF mice. Small intestinal transit was significantly less in CF mice as compared to WT. Of the mast cell stabilizers, only doxantrazole significantly affected small intestinal transit in WT mice and none had any effect in CF mice. Mast cell activation resulted in a small but significant increase in small intestinal transit in CF mice but not WT mice.

Conclusions

The results indicate that mast cells are not involved in gastrointestinal dysmotility but their activation can stimulate small intestinal transit in cystic fibrosis.     

The effects of elevation and depletion of intracellular free calcium on progesterone and prostaglandin production by the primate corpus luteum.

The role of the phosphatidylinositol second messenger system in luteal regulation has not been extensively studied, particularly in the primate. The objectives of this study were (1) to further characterize the response of the primate CL to the calcium ionophore A23187, in terms of intracellular free calcium concentrations ([Ca2+]i) and progesterone (P) production; and (2) to assess the effects of depleting, as well as elevating, available calcium on luteal P and prostaglandin (PG) production. The response to A23187, in terms of [Ca2+]i, was measured by fura-2 fluorescence microscopy of single small and large luteal cells. A23187 significantly increased [Ca2+]i in both cell types (p less than 0.01). P production (basal and hCG-stimulated) by dispersed primate luteal cells incubated for various times (1-8 h) with and without A23187 was measured. Treatment with A23187 rapidly (within 1-2 h) attenuated (p less than 0.05) the time-dependent increase in basal and hCG-stimulated P production. Luteal P and PG production following treatment with the calcium ionophore, ionomycin, alone or in combination with additional CaCl2, was also monitored. Treatment with ionomycin (p less than 0.01) and CaCl2 (p less than 0.01) inhibited luteal P production. In contrast, treatment with ionomycin stimulated (p less than 0.01) luteal PG production. To determine the effects of Ca2+ depletion on luteal function, P and PG production by cells incubated for 2 and 8 h in the absence and presence of the Ca(2+)-chelator EGTA was measured. Luteal production of both P and PG was inhibited by 8-h treatment with EGTA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intestinal absorption of copper: influence of carbohydrates.

Macronutrients can modulate the intestinal absorption of trace elements by binding the metal or altering mucosal function. We investigated whether certain simple and complex carbohydrates modify copper (Cu) absorption, using an in vivo perfusion technique in the rat. Corn syrup solids, which contain a mixture of glucose polymers of diverse length, added at either 20 or 50 mosm/kg enhanced Cu absorption from a 31.5 microM (2 mg/liter) Cu solution (128 +/- 11 and 130 +/- 11 pmol/min x cm, respectively, vs 101 +/- 4 pmol/min x cm, P less than 0.05, in the absence of carbohydrate). This was concomitant with a stimulation of net water absorption (1.05 +/- 0.08 and 0.84 +/- 0.08 microliter/min x cm, respectively, vs 0.63 +/- 0.02 microliter/min x cm with no carbohydrate, P less than 0.05). Glucose, fructose, lactose, or sucrose had no influence on Cu absorption, although they altered water exchanges, an effect attributable to a reduction of the outflow component of fluid recirculation. Low concentrations of lactose resulted in a greater accumulation of Cu in the intestinal mucosa (8.75 +/- 0.71 micrograms/g vs 5.77 +/- 0.68 micrograms/g for controls, P less than 0.05). Hence, solutes that moderately stimulate mucosa-to-serosa fluid influx in a progressive manner, such as glucose polymers, may contribute to functionally increase Cu absorption. Conversely, conditions which tend to reduce water inflow or increase water outflow across the small intestinal mucosa, as may occur with high lactose diets or in cases of chronic diarrhea, may have negative effects.     

Studies on the effect of vitamin D on calcium absorption and transport

1. Mucosal cells of the small intestine obtained from rats deprived of vitamin D or given excessive amounts of the vitamin accumulated significantly more calcium than did cells from control animals. 2. Mucosal cells from vitamin D-deficient rats released less calcium than did cells from normal or hypervitaminotic D animals. 3. Studies in vivo showed that the transfer of (45)Ca from the intestine to the blood was delayed in vitamin D deficiency, but was accelerated in hypervitaminosis D. 4. The findings support the thesis that vitamin D is involved in the release of calcium rather than in its uptake by mucosal cells. 5. Further evidence is presented suggesting that uptake of calcium by intestinal mucosal cells at 0 degrees is primarily passive, whereas at 38 degrees uptake and release are effected by an active process that depends on energy derived from glycolytic activity.     

Changes in sialic acid and fucose contents of enterocytes across the crypt-villus axis in developing rat intestine

Alterations in sialic acid and fucose contents of different populations of epithelial cells have been studied in suckling and adult rat intestine. The progression of cells from crypt base to villus tip is associated with an increase in sialic acid and a decrease in fucose levels of the cells in adult rats. In suckling pups, sialic acid is uniformly distributed along the length of villi, and fucose is richly (P less than 0.01) present in cryptic cells compared to that at the villus tip. Adult-type changes in sialylation and fucosylation of enterocytes across the crypt-villus axis were precociously produced by cortisone administration to suckling pups. Thyroxine treatment was less effective in influencing the glycosylation process in rat intestine.