The mechanism of motilin excitation of the canine small intestine

Close intraarterial injections of motilin to the small intestine of the anaesthetized dog produce prolonged phasic contractions. Tetrodotoxin infused intraarterially blocked field stimulated contractions and abolished the response to motilin as did treatment with a combination of hexamethonium and atropine. Atropine alone increased the dose of motilin required to induce responses. Hexamethonium alone similarly increased the dose of motilin required in the jejunum, but not for the ileum. These results suggest that motilin acts to contract small intestine by stimulation of intrinsic excitatory nerves, some of which are post-ganglionic cholinergic and some of which are not, but are activated by a pathway with a nicotinic synapse. The ED₅₀ for ileal contractions was greater than that for the jejunum and the time to reach maximum contractions longer suggesting a decreased responsiveness of the lower small intestine to motilin as compared to the upper gastro-intestinal tract. These results and the lesser quantity of immunoreactive motilin in the ileum than in the jejunum may explain the lack of relationship of the activity front of the migrating motor complex in the lower small intestine to venous motilin concentrations.     

Epithelioid cell cultures from rat small intestine. Characterization by morphologic and immunologic criteria

Rat small intestinal epithelial cell lines have been established in vitro and subcultured serially for periods up to 6 mo. These cells have an epithelioid morphology, grow as monolayers of closely opposed polygonal cells, and during the logarithmic phase of growth have a population doubling time of 19--22 h. Ultrastructural studies revealed the presence of microvilli, tight junctions, an extensive Golgi complex, and the presence of extracellular amorphous material similar in appearance to isolated basement membrane. These cells exhibit a number of features characteristic of normal cells in culture; namely, a normal rat diploid karyotype, strong density inhibition of growth, lack of growth in soft agar, and a low plating efficiency when seeded at low density. They did not produce tumors when injected in syngeneic animals. Immunochemical studies were performed to determine their origin using antisera prepared against rat small intestinal crypt cell plasma membrane, brush border membrane of villus cells and isolated sucrase-isomaltase complex. Antigenic determinants specific for small intestinal epithelial (crypt and villus) cells were demonstrated on the surface of the epithelioid cells, but they lacked immunological determinants specific for differentiated villus cells. An antiserum specifically staining extracellular material surrounding the cells cultured in vitro demonstrated cross-reactivity to basement membrane in rat intestinal frozen sections. It is concluded that the cultured epithelioid cells have features of undifferentiated small intestinal crypt cells.     

Phosphofructokinase D from the epithelial cells of rat small intestine.

Phosphofructokinase from the epithelial cells of rat small intestine was characterized with respect to isoenzyme type in a comparison of its properties with those of the skeletal-muscle, brain and major liver isoenzymes by using five different techniques, namely electrophoresis on cellulose acetate and in polyacrylamide gels, chromatography on DEAE-cellulose, (NH4)2SO4 precipitation and immunotitration. When precautions were taken to inhibit the formation of active proteolytic artifacts by the action of endogenous proteinases, each technique revealed that rat intestinal mucosa contains only a single form of phosphofructokinase. The mucosal isoenzyme was found to be very similar to, although not identical with, the major liver isoenzyme and to be quite distinct from the skeletal-muscle isoenzyme when studied by the techniques of cellulose acetate electrophoresis, chromatography on DEAE-cellulose and immunotitration, whereas the converse was true when studied by the techniques of (NH4)2SO4 precipitation and polyacrylamide-gel electrophoresis. The mucosal isoenzyme was distinct from the brain isoenzyme when studied by each of the five techniques. Tsai & Kemp [(1973) J. Biol. Chem. 248, 785-792] reported that animal tissues contain three principal isoenzymes of phosphofructokinase, type A found as the sole isoenzyme in skeletal muscle, type B found as the major isoenzyme in liver and type C found as a significant isoenzyme in brain. Phosphofructokinase from mucosa is distinct from each of these isoenzymes. Following the nomenclature of Tsai & Kemp (1973), the isoenzyme from the mucosa of rat intestinal epithelial cells is designated phosphofructokinase D. The mucosal and liver isoenzymes behave so similarly with respect to their charge and immunological characteristics, on which the typing of isoenzymes is conventionally based, that it is likely that some tissues reported to contain the liver isoenzyme contain instead the mucosal isoenzyme.     

Characterization of immunoreactive somatostatin released from rat nervous system in vitro.

There is considerable evidence that somatostatin is released from nerve terminals throughout the central nervous system in response to presynaptic stimulation, thus suggesting a neuromodulator role for the peptide. We here report the partial characterization of immunoreactive somatostatin released from rat nervous system in vitro (hypothalamus, spinal cord and hypothalamic, cortical, thalamic and striatal synaptosomes). Serial dilutions of released somatostatin immunoreactivity showed parallelism with dilutions of synthetic somatostatin standard. Somatostatin immunoreactivity released from all tissue areas coeluted with synthetic tetradecapeptide on Sephadex G-25 (fine grade) gel chromatography; more than 85% of this immunoreactivity bound to Sepharose-anti-somatostatin-serum immunoaffinity columns. In addition, immunoreactive material released from hypothalamus, spinal cord and hypothalamic and cortical synaptosomes inhibited somatotropin (growth hormone, 'STH', 'GH') release from perifused anterior pituitary in a dose-related manner, indicating biological similarity to synthetic somatostatin.     

Ontogeny of glucagon-like immunoreactive peptides in rat intestine

The ontogeny of the intestinal glucagon-like peptides was investigated in rats between 16 days of gestation and 4 postnatal days. The intestinal content of glucagon-like immunoreactive (GLI) peptides increased from 0.09 +/- 0.02 pmol/nmol protein at 16-17 days to plateau at 2.8 +/- 0.4 pmol/nmol protein by 20 days of gestation (P less than 0.001). The apparent immunoreactive glucagon (IRGa) content of the gut ranged from 0.03 +/- 0.01 to 0.08 +/- 0.01 pmol/nmol protein. No developmental trends in IRGa peptide content were observed. Following gel filtration of intestines extracted from rats of 18 days of gestation or greater, two main GLI peptides were detected with apparent mol. wts. of 11-12 and 5-6 kDa. Significant peaks of GLI peptides were not detected following gel filtration of intestines extracted from 16- or 17-day fetuses, nor were peaks of IRGa found at any age. In conclusion, the fetal rat intestine undergoes maturational development between 17 and 19 days of gestation to produce the GLI peptides.     

Studies on the immunoglobulin-G Fc-fragment receptor from neonatal rat small intestine.

1. A method for preparing the small-intestinal brush-border membrane of neonatal rats is described in which enzymic methods are used to remove associated polysaccharide and cell nuclei. 2. 125I-labelled IgG (immunoglobulin G) and 125I-labelled IgG Fc fragment have high specific binding and low non-specific binding to brush borders prepared in this way. F(ab)'2 fragment however, does not bind, indicating the existence of a specific receptor for the Fc fragment of IgG. The receptor system is saturable, and the affinity (KA) for the binding of rat IgG was determined by both equilibrium and kinetic methods. 3. The binding of heterologous IgG species (human and bovine) was compared and demonstrated a close similarity between human IgG and rat IgG in their receptor affinities. 4. Kinetic results are presented that are consistent with previously proposed models of ligand-induced receptor aggregation.     

Characterization of the low mol. wt zinc-binding ligand from rat small intestine by comparison to the organic zinc-binding ligands

1. 65Zn complexes of picolinate (PA), citrate (CA), L-histidine (L-his), arachidonic acid (AA) or low mol. wt zinc-binding ligand from rat intestine (LMW-ZBL) gave 65Zn eluting peak fraction numbers of 53, 53, 56, 59 and 59 respectively, in a Sephadex G-75 column chromatography. 2. The 65Zn eluting peak fraction numbers with CA, L-his, PA, prostaglandin (PG)E2, AA, no ligand, arachidonate (AT) or LMW-ZBL were 49, 50, 54, 55, 58, 64, 75 and 76 respectively in a Sephadex G-25 column chromatography. 3. In a Sephadex G-15 column chromatography, the 65Zn eluting peak fraction numbers with CA, PGE2, AA, L-his, LMW-ZBL or PA were 49, 50, 51, 52, 52 and 55 respectively. 4. The LMW-ZBL in rat small intestine appears to be an AA-like substance.     

Stereospecificity of the metal.ATP complex in flavokinase from rat small intestine.

Transfer of the gamma-phosphoryl group of ATP to riboflavin is catalyzed by flavokinase, which prefers Zn(II), and is essential in the biosynthesis of the flavocoenzyme, FMN. To study the mechanism and steric disposition of ATP binding, adenosine 5'-O-(2-thiotriphosphate) (ATP beta S) and adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) were tested in comparison to ATP with representative divalent metal ions. Activities with 0.1 mM Zn(II) for 1 mM ATP thio analogues compared to ATP with flavokinase were 60% for the S-isomer of ATP beta S, 312% for the R-isomer of ATP beta S, and 14% for ATP gamma S. With Mg(II), flavokinase showed stereospecificity for the R-isomer of ATP beta S with a Vmax ratio, S/R = 0.125. With Cd(II), the enzyme showed preference for the S-isomer of ATP beta S with a Vmax ratio, S/R = 1.261. The Vmax ratio progressively increases from Mg(II) to Cd(II) in the order of affinity for sulfur. The ratios, (Vmax/Km)Mg/(Vmax/Km)Cd, for the diastereomers of ATP beta S were expectedly greater than 1 for one diastereomer (R = 6.597) and less than 1 for the other (S = 0.292). Activities with ATP gamma S were much lower than with ATP or ATP beta S. With Mg(II), the gamma analogue was a good substrate; however, with Cd(II), it was hardly active. Altogether these results indicate that flavokinase uses the lambda, beta, gamma-bidentate Zn.ATP as a substrate.     

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.     

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.     

Direct contractile effect of motilin on isolated smooth muscle cells of guinea pig small intestine.

We examined the direct effect of motilin on longitudinal and circular smooth muscle cells isolated from the guinea pig small intestine. In addition, the effects of 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxy-benzoate hydrochloride (TMB-8, an inhibitor of intracellular Ca(2+)-release), verapamil (a voltage-dependent Ca(2+)-channel blocker), and removal of extracellular Ca2+ were investigated to evaluate the role of intracellular Ca2+ stores and extracellular Ca2+ on the muscle contraction induced by motilin. The effects of atropine (a muscarinic receptor antagonist), spantide (a substance P receptor antagonist) and loxiglumide (a CCK-receptor antagonist) were also examined to determine whether the motilin-induced contraction was independent of those receptors. Motilin induced a contraction of the longitudinal and circular smooth muscle cells in a dose-dependent manner with the maximal effect attained after 30 seconds of incubation. The ED50 values were 0.3 nM and 0.05 nM, respectively. TMB-8 suppressed completely the motilin-induced contraction of both types of smooth muscle cells. Verapamil had only a slight suppressive effect. Removal of extracellular Ca2+ did not have any significant influence on motilin-induced contraction. The contractile response to motilin was not affected by atropine, spantide or loxiglumide. Our findings showed that:1) motilin has a direct contractile effect on both longitudinal and circular smooth muscle cells; 2) this contractile effect is not evoked via muscarinic, substance P or CCK receptors, and 3) the intracellular release of Ca2+ plays an important role in the contractile response to motilin on both types of smooth muscle cells.     

Characterization of an epithelioid cell line derived from rat small intestine: demonstration of cytokeratin filaments

A cell line (RIE-1) of epithelioid morphology was established from rat small intestine. RIE-1 cells possess the normal rat diploid number of chromosomes (42) up to at least passage 20. Both early (less than 10) and late (greater than 20) passage cells are non-tumourigenic and do not form colonies in soft agarose. RIE-1 cells bind two epithelial-specific monoclonal antibodies. In addition, the cells exhibit a network of cytoskeletal filaments which are labelled by an antiserum to keratin. These findings provide strong evidence for an epithelial origin of the RIE-1 cell line.     

Permeability properties of isolated enterocytes from rat small intestine

Metabolic and permeability properties of enterocytes isolated by treatment of rat small intestine with hyaluronidase or EDTA were compared. No significant difference was observed in the ability of the two types of cell to produce lactate from glucose. However, while cells obtained with hyaluronidase accumulate alpha-methylglucoside, cells obtained with EDTA were unable to accumulate the sugar above the medium concentrations. When resuspended in a medium designed to resemble the intracellular medium, potentiometric measurements showed that cells obtained with hyaluronidase released Ca2+ to the medium while cells obtained with EDTA accumulated it. Using 45Ca transport assays, this was shown to be an ATP-dependent process, the accumulated 45Ca being totally released by the addition of the ionophore A23187. When cells obtained with EDTA were resuspended in a medium containing concentrations of free Ca2+ higher that 10 microM, the uptake was partially inhibited by sodium orthovanadate and also by oligomycin and antimycin. At free Ca2+ concentrations lower than 1 microM, the accumulation was inhibited up to 87% by sodium orthovanadate while mitochondrial inhibitors inhibited only 5%. Thus, it appears that during their preparation cells obtained with hyaluronidase retain their integrity while cells obtained with EDTA become permeable to Ca2+ and other ions. The usefulness of both types of preparation in metabolic and transport studies is discussed.     

Fatty-acid chain elongation in rat small intestine.

Microsomal fractions from rat small intestine contain a fatty-acid chain-elongation activity. Cofactor requirements are similar to those of the liver microsomal system, but substrate specificity is different. The polyunsaturated arachidonic and timnodonic acids were elongated at very low rates. These results suggest that the relative contents of specific chain-elongation enzymes are different in liver and small intestine.