Slow-waves in rat small intestine

Rat small intestine generates rhythmic slow-wave activity. The slow-waves are not eliminated by ouabain application or incubation in potassium free solution. Exposure to low sodium or calcium free solution decreases slow-wave activity. Incubation in sodium and calcium free solution eliminates activity. It is concluded that rat small intestinal slow-waves may not result from the same mechanism as in the cat.     

Class II antigen-associated invariant chain mRNA in mouse small intestine.

MHC class II antigen-associated invariant (Ii) chain mRNA appears in mouse small intestine during postnatal development. Ii chain cDNA hybridizes to RNA from epithelial sheets dissociated from the lamina propria with EDTA. Of several mouse organs tested, only bone marrow and spleen contain higher levels of Ii chain mRNA than small bowel. Ii chain mRNA is not detected in stomach, colon, duodenum, testis, liver, submandibular gland, or L-cell RNA; brain contains a cross-reactive but uncharacterized sequence. cDNA amplification using primers specific for both Ii31 and Ii41 chain mRNAs showed that both forms occur in small intestine. These results support the conclusion that regulation of the class II Ii chain gene is associated with the ontogeny of intestinal immunity.     

Isoenzymes of glutathione transferase in rat small intestine.

The role of plasminogen activators (PAs) as potential mediators of involution of the rat ventral prostate was investigated by using an approach involving the administration in vivo of anti-PA drugs. The prostates of castrated rats, which had been injected daily for 7 days with the anti-PA drugs 6-aminohexanoic acid, tranexamic acid, aprotinin and cortisol, were assayed for PA activity, weight and cell number. In the prostates from the castrated controls, there was a 10-fold increase in the mean PA activity and a 7-fold decrease in cell number relative to that of the non-castrated animals. Although this rise in enzyme activity could be decreased to some extent by all the drugs except aprotinin, only treatment with high doses of tranexamic acid or cortisol had a statistically significant effect. A similar pattern was observed with respect to the relative potency of the drugs in preventing the loss of prostatic weight and cell number after castration. The effects of cortisol were dose-dependent, with complete inhibition of both the rise in PA activity and cell loss occurring at a dose of about 15 mg/day. Since the concentration of the principal intranuclear androgen, dihydrotestosterone, was the same in the prostates from treated and untreated castrated rats, the effects of cortisol are not due to increased retention of this androgen. Rather, the high inverse correlation (r = 0.86) between the cellular concentration of PA activity and the cell population of the prostate implies that PAs are directly associated with prostatic involution and that cortisol, and to a lesser extent tranexamic acid, blocks the involution process through inhibition of PAs.     

EGTA induced fast potentials in rat small intestine.

Rat small intestine exhibits spontaneous slow-waves and spikes in normal solution. When treated with 0.3 – 0.7 mM EGTA, in calcium free solution, normal rhythmicity disappears and fast rhythmic potentials of duration intermediate to slow-waves and spikes appear. These induced fast potentials are absent in sodium free solution and are eliminated by verapamil, a calcium channel blocker. Application of EGTA to cat, guinea pig, mouse, and toad intestine did not yield fast potentials. The fast potentials appear to result from sodium entering through channels usually used by calcium. The fast potentials may be a phenomena exclusive to rat small intestine.     

Lactate formation by rat small intestine in vitro.

The formation of lactic acid by mucosal slices, rings and muscle from rat jejunum has been studied for periods of up to 8 min. Lactate output by mucosal slices incubated in the absence of glucose was characterised by two phases: a rapid, initial phase of release lasting about 1 min, followed by a much slower phase extending over the remainder of the incubation period. Glucose addition at 30 s initiated a second rapid phase of lactate release into the medium which was again followed by a slower rate of lactate output up to 8 min. The time course of lactate output suggested that there was a negative Pasteur effect in mucosal slices, which could not be reversed by the addition of ADP or glucose 6-phosphate. By contrast, the rate of lactate formation by rings and muscle from rat jejunum increased steadily over the incubation period, indicating a positive Pasteur effect. When Na+ in the incubating medium were replaced by K+, lactate formation by mucosal slices and rings was considerably reduced. Measurements of tissue lactate content before and during incubation revealed that about three-quarters of the lactate released by mucosal slices during the first 30 s of incubation was present initially in the tissue. After the first 30 s the tissue lactate remained constant both in the presence and absence of glucose so that the lactate released into the incubation medium is equivalent to the lactate formed by the slices. The role of the various tissue components of the small intestine in lactate formation is discussed in relation to sites of glucose entry.     

Fatty-acid chain tilt angles and directions in dipalmitoyl phosphatidylcholine bilayers.

X-ray diffraction has been applied to determine the various tilt angles and directions (if any) which can be assumed by oriented gel phase multilayers of dipalmitoyl phosphatidylcholine (DPPC) as a function of hydration. We report for the first time that oriented DPPC multilayers with a repeat spacing (d-spacing) of 55.2A at 25 degrees C and 0% relative humidity (RH) have hydrocarbon chains tilted at an angle theta of 21.5 degrees with respect to the bilayer normal. In addition, the chains are tilted along one of the bisectors (omega = 0 degrees) of the hexagonal lattice (8 wide-angle maxima, 2 unique), a phase not previously reported in DPPC studies. At 100% RH, the chain tilt angle and d-spacing increased to approximately 29.0 degrees and 58.9A, respectively. Since at 100% RH only 4 wide-angle maxima are observed, we analyze the data on the assumption that the hydrocarbon chains may rotate independently of the hexagonal lattice (omega = 0-30 degrees), at a fixed chain tilt angle theta (Stamatoff, J.B., et al. 1979. Biophys. J. 25:253-262). The largest observed angle phi made by the wide-angle maxima with the equator is 29.5 degrees corresponding to a theta of approximately 32.6 degrees (omega avg. = 24 degrees) and the sample having a d-spacing of 64.0 A (excess water condition). Finally, theta remains relatively constant (approximately 21.5 degrees) up to a RH of approximately 45% and a d-spacing of 57.8A, after which, with increases in RH, theta increases to a maximum of 32.6 degrees.     

The effect of short chain fatty acids on transmural electrical potential across rat small intestine in vivo.

Short chain fatty acids suddenly produce a phasic increase in transmural electrical potential difference (PD) when placed in the lumen of rat small intestine in vivo. With concentrations of propionate ranging from 50 muM to 1000 muM the amplitude of the response in jejunum is about 5.5 mV. The concentration giving half this effect is about 20 muM. With 10 mM propionate the duration of the response is 3-5 min; after this, PD again equals the control value and the gut is refractory to further additions. Removing propionate from the mucosal surface produces no change in PD, but does restore responsiveness to subsequent exposure to short chain fatty acids. This effect is independent of a variety of other alterations in PD such as those caused by sugars, amino acids, bile salts, theophylline, prostaglandins, and ATP. Mechanism and significance of this surprisingly sensitive response remain obscure.     

AMP deaminases of rat small intestine

Phosphocellulose column chromatography revealed the existence of two forms of AMP deaminase both in whole tissue and in the intestinal epithelium. AMP deaminase I, which eluted from the column as a first activity peak, exhibited hyperbolic, nonregulatory kinetics. The substrate half-saturation constants were determined to be 0.3 and 0.7 mM at pH 6.5 and 7.2, respectively, and did not change in the presence of ATP, GTP and Pi. AMP deaminase II, which eluted from the column as a second activity peak, was strongly activated by ATP and inhibited by GTP and Pi. The S0.5 constants were 3.5 and 7.1 at pH 6.5 and 7.2, respectively. At pH 7.2 ATP (1 mM) S0.5 decreased to 2.5 mM and caused the sigmoidicity to shift to hyperbolic. The ATP half-activation constant was increased 9-fold in the presence of GTP and was not affected by Pi. Mg2+ significantly altered the effects exerted by nucleotides. The S0.5 value was lowered 10-fold in the presence of MgATP and 5-fold in the presence of MgATP, MgGTP and Pi. When MgATP was present, AMP deaminase II from rat small intestine was less susceptible to inhibition by GTP and Pi. A comparison of the kinetic properties of the enzyme, in particular the greater than 100% increase in Vmax observed in the presence of MgCl2 at low (1 mM) substrate concentration, indicates that MgATP is the true physiological activator. GuoPP[NH]P at low concentrations, in contrast to GTP, did not affect the enzyme and even activated it at concentrations above 0.2 mM. We postulate that AMP deaminase II may have a function similar to that of the rat liver enzyme. The significance of the existence of an additional, non-regulatory form of AMP deaminase in rat small intestine is discussed.     

Calcium uptake in isolated brush-border vesicles from rat small intestine.

Ca2+ uptake in brush-border vesicles isolated from rat duodena was studied by a rapid-filtration technique. Ca2+ uptake showed saturation kinetics, was dependent on the pH and ionic strength of the medium and was independent of metabolic energy. Uptake activity was readily inhibited by Ruthenium Red, La3+, tetracaine, EGTA, choline chloride and Na+ or K+. The effect of variations in medium osmolarity on Ca2+ uptake and the ionophore A23187-induced efflux of the cation from preloaded vesicles indicated that the Ca2+-uptake process involved binding to membrane components, as well as transport into an osmotically active space. Scatchard-plot analyses of the binding data suggested at least two classes of Ca2+-binding sites. The high-affinity sites, Ka = (2.7 +/- 1.1) x 10(4) M-1 (mean +/- S.D.) bound 3.2 +/- 0.8 nmol of Ca2+/mg of protein, whereas the low-affinity sites (Ka = 60 +/- 6 M-1) bound 110 +/- 17 nmol of Ca2+/mg of protein. In the presence of 100 mM-NaCl, 1.7 and 53 nmol of Ca2+/mg of protein were bound to the high- and low-affinity sites respectively. Decreased Ca2+-uptake activity was observed in vesicles isolated from vitamin D-deficient as compared with vitamin D-replete animals and intraperitoneal administration of 1,25-dihydroxycholecalciferol to vitamin D-deficient rats 16 h before membrane isolation stimulated the initial rate of Ca2+ uptake significantly. The data indicated that Ca2+ entry and/or binding was passive and may involve a carrier-mediated Ca2+-uptake component that is associated with the brush-border membrane. Altering the electrochemical potential difference across the membrane by using anions of various permeability and selected ionophores appeared to increase primarily binding to the membrane rather than transport into the intravesicular space. Since there is considerable binding of Ca2+ to the vesicle interior, a comprehensive analysis of the transport properties of the brush-border membrane remains difficult at present.     

Calcium-dependence of sugar transport in rat small intestine

The involvement of Ca2+ in the theophylline action on sugar transport was investigated in isolated rat small intestinal mucosa. Theophylline significantly increased cell water free sugar accumulation and reduced mucosal to serosal sugar fluxes both in the presence and absence of calcium, but the effects of theophylline were significantly less in calcium free media. In theophylline untreated tissues, calcium-deprived bathing solutions decreased tissue galactose accumulation and increased mucosal to serosal sugar flux. The calcium-channel blocker verapamil produced similar effects on intestinal galactose transport to those induced by low extracellular calcium activity. RMI 12330A and the calmodulin antagonist trifluoperazine abolished the theophylline-effects on intestinal galactose transport. Both drugs also affected sugar transport in basal conditions. These studies suggest that calcium might modulate sugar permeability across the basolateral boundary of rat enterocytes, and that its effect may be mediated by calmodulin.     

Metabolism of zinc-binding ligands in rat small intestine

Studies were conducted to determine the effects of zinc deficiency and excess zinc intake on the relative⁶⁵Zn-binding activities of metallothionein (MT) and low-molecular-weight zinc-binding ligand (LMW-ZBL) in vitro and in vivo. Zinc-binding ligands of small intestine from four groups, each of five rats (normal, zinc-deficient, excess zinc injected, and excess zinc given orally), were separated by column chromatography on Sephadex G-75. The ratio of⁶⁵Zn binding activities of MT to LMW-ZBL (MT/LMW-ZBL) in zinc-deficient rats was decreased both in vitro and in vivo compared to the control. When excess zinc was administered orally,⁶⁵Zn-binding activity of MT was low in vitro and substantially increased in vivo. However, when excess zinc was injected intraperitoneally,⁶⁵Zn-binding activity of MT in vitro greatly increased, but⁶⁵Zn-binding activities of both MT and LMW-ZBL were significantly reduced in vivo as compared to the control. Based onA ₂₈₀ readings of isolated MT and densities of protein bands in disc gel electrophoresis, the⁶⁵Zn-binding activity of MT in vitro appeared to be proportional to the MT content. Hence, these data indicate that oral administration of excess zinc decreases MT whereas intraperitoneal injection of excess zinc stimulates its synthesis. Zinc deficiency has little to no effect on the intestinal MT metabolism. These results suggest that MT may be important in zinc secretion but not involved in zinc absorption; while LMW-ZBL participates both in zinc absorption and secretion.     

Lactate formation by rat small intestine in vitro

The formation of lactic acid by mucosal slices, rings and muscle from rat jejunum has been studied for periods of up to 8 min. Lactate output by mucosal slices incubated in the absence of glucose was characterised by two phases: a rapid, initial phase of release lasting about 1 min, followed by a much slower phase extending over the remainder of the incubation period. Glucose addition at 30 s initiated a second rapid phase of lactate release into the medium which was again followed by a slower rate of lactate output up to 8 min. The time course of lactate output suggested that there was a negative Pasteur effect in mucosal slices, which could not be reversed by the addition of ADP or glucose 6-phosphate. By contrast, the rate of lactate formation by rings and muscle from rat jejunum increased steadily over the incubation period, indicating a positive Pasteur effect. When Na⁺ in the incubating medium were replaced by K⁺, lactate formation by mucosal slices and rings was considerably reduced. Measurements of tissue lactate content before and during incubation revealed that about three-quarters of the lactate released by mucosal slices during the first 30 s of incubation was present initially in the tissue. After the first 30 s the tissue lactate remained constant both in the presence and absence of glucose so that the lactate released into the incubation medium is equivalent to the lactate formed by the slices. The role of the various tissue components of the small intestine in lactate formation is discussed in relation to sites of glucose entry.     

Immunofluorescent localization of transglutaminase in rat small intestine

The distribution of intestinal transglutaminase was investigated by immunofluorescence microscopy using rabbit anti-guinea pig transglutaminase immunoglobulin. Transglutaminase-related antigen was demonstrated principally in the cytoplasm of villous core interstitial cells with some activity in the brush border region of the villous epithelial cells. Implications for the pathogenesis of coeliac disease are discussed.     

Characterization of immunoreactive motilin from the rat small intestine.

Immunocytochemistry, radioimmunoassay, chromatography, and biological assay using a rabbit isolated duodenal muscle strip preparation were used in attempting to characterize motilin from the rat small intestine. Several different antisera and monoclonal antibodies directed against natural porcine motilin were used. A variety of fixation techniques using Bouin's, paraformaldehyde, and benzoquinone with different staining methods including, fluorescein-conjugated second antibody, peroxidase-antiperoxidase or peroxidase-conjugated second antibody techniques were used. All methods failed to detect immunoreactive motilin cells in the rat small intestine. The same antisera were used in radioimmunoassays for motilin to evaluate extracts of rat intestinal tissue. Two of these detected immunoreactive motilin in gut extracts, and these antisera showed a different distribution for the peptide. Samples containing immunoreactive motilin obtained from cation exchange chromatography on SP-Sephadex-G25 were concentrated and assayed for biological activity in a rabbit duodenal muscle strip preparation. Desensitization of duodenal tissue to porcine motilin could be demonstrated by pretreatment with this peptide. The biological activity of partially purified rat intestinal immunoreactive motilin was not prevented by pretreatment of the tissue with motilin. Further purification of this preparation on Bio-Gel P-10 yielded an immunoreactive motilin peak that co-eluted with natural porcine motilin. Rat intestinal immunoreactive motilin did not co-elute with natural porcine motilin following high pressure liquid chromatography on a Waters microBondapak C18 reversed-phase column using a linear gradient of water-acetonitrile (10-45%) over 30 min. Although of similar molecular size, rat motilin is probably structurally dissimilar to other mammalian motilins.     

Ammonia production by the small intestine of the rat.

1. Slices of duodenum and jejunum produce ammonia from glutamine in vitro. 2. Ammoniagenesis does not increase in response to acidosis or potassium deficiency, two conditions known to cause enhanced ammoniagenesis in the kidney. 3. Gut contains glutaminase 1 as well as gamma-glutamyl transpeptidase. 4. These enzymes do not show any increase during starvation.