Calcium and glucose uptake in rat small intestinal brush-border membrane vesicles. Modulation by exogenous hypercortisolism and 1.25-dihydroxyvitamin D-3

The effect of exogenous hypercortisolism and 1,25-dihydroxyvitamin D-3 on small-intestinal calcium and glucose transport in the rat was studied at the level of brush-border membrane vesicles generated from isolated villous cells by a freeze-thaw procedure. At 5 · 10^?5 M extravesicular calcium, initial uptake rates in vesicles prepared from triamcinolone-treated adult rats were decreased by 30% after 5 days. Since calcium ionophore A23187 virtually abolished the difference in calcium uptake, triamcinolone appeared to affect calcium channel density or activity rather than intravesicular binding capacity. Kinetic analysis showed that a decrease in V_max of a saturable calcium transport system could entirely account for the diminished rate of vesicular calcium uptake. Calcium transport rates could be partially restored by in vivo administration of 1,25-dihydroxyvitamin D-3 at a dosage which did not affect vesicular calcium uptake in control animals. Conversely, sodium-driven glucose accumulation in brush-border vesicles from triamcinolone-treated rats was stimulated by 50–70% after 36 h and appeared insensitive to vitamin D. A specific triamcinolone action on the glucose carrier itself rather than on the driving force of the sodium gradient was indicated by (i) a similar stimulation of glucose transport under equilibrium exchange conditions and (ii) an opposite effect of triamcinolone on sodium-driven alanine transport. The triamcinolone-induced changes in calcium and glucose uptake were not accompanied by a gross alteration of membrane integrity in vitro or by major alterations in vesicular protein composition, intravesicular glucose space and sucrase or alkaline phosphatase activity. The modification of vesicular transport properties is discussed in relation to the vitamin D-antagonized inhibition of intestinal calcium uptake and the stimulation of glucose absorption in response to supraphysiologic amounts of glucocorticoids observed in intact epithelium.     

Distribution of intrinsic nerve cell bodies and axons which take up aromatic amines and their precursors in the small intestine of the guinea-pig

Summary The sites of uptake, decarboxylation and retention of 1-dopa and the uptake and retention of dopamine and 6-hydroxytryptamine in the small intestine of the guinea-pig have been localised histochemically with a fluorescence technique for arylethylamines. In segments of ileum from untreated guinea-pigs only noradrenergic axons are fluorescent; these axons were eliminated by surgical denervation (crushing nerves running to the intestine through the mesentery) or by chemical denervation with 6-hydroxydopamine. In denervated segments of ileum, cell bodies and processes of intrinsic neurons become fluorescent after the injection of 1-dopa, dopamine or 6-hydroxytryptamine and the inhibition of monoamine oxidase, as do cells of Brunner's glands and Paneth cells. About 11% of the nerve cell bodies in the submucous plexus and 0.4% of those in the myenteric plexus become fluorescent. Varicose intrinsic axons which take up amines are found amongst the nerve cell bodies of the myenteric and submucous plexuses. They also ramify in the principal connections of the plexuses, in the tertiary strands of the myenteric plexus, in the deep muscular plexus and contribute sparse supplies of axons to arterioles in the submucosa and to the lamina propria of the mucosa. The axons are resistant to the degenerative actions of 6-hydroxydopamine.It is suggested that the intrinsic amine handling axons are more likely to utilise an indolamine related to 5-hydroxytryptamine than they are to utilise a catecholamine as a neurotransmitter.     

Establishment of regional differences in brush border enzymatic activities during the development of fetal mouse small intestine

Summary In order to study the establishment of regional differences in brush border enzymic activities during the development of fetal mouse small intestine we have followed (1) the differentiation of microvilli by morphometry, and (2) the developmental pattern of three brush border enzymes (lactase, glucoamylase and alkaline phosphatase). From day 16 to day 19 of gestation, the height of duodenal microvilli increases 2.4 times on the absorptive cells located near the tip of the villi. During the same period in the upper half of the duodenal villi, the number of microvilli per square m rises by a factor of 2.4 and the microvillous surface area increases by a factor of 5.2. The differentiation of ileal microvilli follows a similar pattern but they are always shorter and less numerous than those of the duodenum. Lactase activity appears at 18 days of gestation; the other two brush border enzymes are first detected at 16 days of gestation. Afterwards all three enzyme activities increase rapidly and a decreasing gradient of activity is established from the proximal to the distal segment of the small intestine. Hence, the structural development of the microvilli and the appearance of brush border enzyme activities occur simultaneously and a proximo-distal gradient is already established at 16 days of gestation.Supported by MRC of Canada research grant MA-6069Mr. D. Malka was supported by a studentship from the F.C.A.C.Dr. D. Ménard is a chercheur boursier du Conseil de la Recherche en Santé du Québec     

Ultrastructural identification of a new cell type — the N-cell as the source of neurotensin in the gut mucosa

The neurotensin-cell is identified immunohistochemically and ultrastructurally by differential counting of endocrine cells in the gut of a primate (Tupaia belangeri). Utilizing light microscopy, the EC-cells are identified by the Masson-Fontana silver stain; with the same method the neurotensin cells are not stained. The other endocrine cells have been quantified in the small intestine using the peroxidase-antiperoxidase stain with antisera against glucagon, somatostatin, cholecystokinin, gastrin, secretin, pancreatic polypeptide, gastric inhibitory peptide and neurotensin. In the ileal mucosa of Tupaia, the most frequent endocrine cell is the EC-cell followed by the glucagonoid cell, (L-cell). The immunoreactive neurotensin cell represents the third most frequent endocrine cell in this region. On the ultrastructural level, this third most frequent endocrine cell is a heretofore undescribed cell, the N-cell, containing electron dense secretory granules measuring 335 +/- 87 nm in diameter.     

ATP-dependent calcium transport and its correlation with Ca2+-ATPase activity in basolateral plasma membranes of rat duodenum

Isolated basolateral plasmamembrane vesicles from rat duodenum epithelial cells exhibit ATP-dependent calcium-accumulation and Ca²⁺-dependent ATPase activity. Calcium accumulation stimulated by ATP is prevented by the calcium ionophore A23187, inhibited 80% by 0.1 mM orthovanadate but is not effected by oligomycin. Calcium accumulation is not observed with the substrate β-γ-(CH₂)-ATP, ADP and p-nitrophenyl phosphate. Kinetic studies reveal an apparent K_m of 0.2 μM Ca²⁺ and a V_max of 5.3 nmol Ca²⁺/min per mg protein for the ATP-dependent calcium-uptake system. Calmodulin and phenothiazines have no effect on calcium accumulation in freshly prepared membranes, but small effects are inducable after a wash with a 5 mM EGTA. The kinetic parameters of Ca²⁺-ATPase are: K_m = 0.25 μM Ca²⁺ and V_max = 19.2 nmol P_i/min per mg protein. Three techniques, osmotic shock, treatment with Triton X-100 or the channel-forming peptide alamethacin, reveal that about 40% of the vesicles are resealed. Assuming that half of the resealed vesicles have an inside-out orientation, the V_max of ATP-dependent calcium uptake amounts to 25 nmol Ca²⁺/min per mg protein and of the Ca²⁺-ATPase to 23 nmol P_i/min per mg protein. The close correlation between kinetic parameters of Ca²⁺-ATPase and ATP-dependent calcium-transport strongly suggests that both systems are expressions of a Ca²⁺-pump located in duodenal basolateral plasma membranes.     

Fast sampling,rapid filtration apparatus: Principal characteristics and validation from studies ofd-glucose transport in human jejunal brush-border membrane vesicles

Summary Kinetic data in (brush-border) membrane vesicles which rely on the validity of the initial rate assumption for their interpretation and depend on tracer flux studies using the rapid filtration technique for their experimental measurement have been limited to some extent by the absence of techniques that would allow for real-time data analysis. In this paper, we report on our successful design of a fast sampling, rapid filtration apparatus (FSRFA) which seems to fill up this technical gap since showing the following characteristics: (i) rapid injection (5 msec) and mixing (less than 100 msec) of small amounts of vesicles (10–40 l) with an incubation medium (0.2–1.0 ml); (ii) fast (20 to 80 msec depending on the sample volume) and multiple (up to 18 samples at a maximal rate of 4/sec) sampling of the uptake mixture followed by rapid quenching in the stop solution (approximately 5 msec) according to a predetermined time schedule (any time combination from 0.25 to 9999 sec); and (iii) fast, automated, and sampling-synchronized filtration and washings of the quenched uptake medium (only 15–20 sec are necessary for the first filtration followed by two washings and extra filtrations). As demonstrated using adult human jejunal brush-border membrane vesicles and Na⁺-d-glucose cotransport as models, the FSRFA accurately reproduces the manual aspects of the rapid filtration technique while allowing for very precise initial rate determinations. Moreover, the FSRFA has also been designed to provide as much versatility as possible and, in its present version, allows for a very precise control of the incubation temperature and also permits a few efflux protocols to be performed. Finally, its modular design, which separates the fast sampling unit from the rapid filtration device, should help in extending its use to fields other than transport measurement.     

Amiloride sensitivity of proton-conductive pathways in gastric and intestinal apical membrane vesicles

Summary Passive proton permeability of gastrointestinal apical membrane vesicles was determined. The nature of the pathways for proton permeation was investigated using amiloride. The rate of proton permeation (k _H ⁺ was determined by addition of vesicles (pH _i = 6.5) to a pH 8.0 solution containing acridine orange. The rate of recovery of acridine orange fluorescence after quenching by the acidic vesicles ranged from 4 × 10^–3 (gastric parietal cell stimulation-associated vesicles; SAV) and 5 × 10^–3 (duodenal brush-border membrane vesicles; dBBMV) to 11 × 10+^–3 sec^–1 (ileal BBMV; iBBMV). Amiloride, 0.03 and 0.1 mm, significantly reduced the rate of proton permeation in dBBMV and iBBMV, but not gastric SAV. The decreases in k _H ⁺ were proportionately greater in iBBMV as compared with dBBMV. The presence of Na⁺/H⁺ exchange was demonstrated in both dBBMV and iBBMV by proton-driven (pH _i < pH _o ) ²²Na⁺ uptake. Evidence was also sought for the conductive nature of pathways for proton permeation. Intravesicular acidification, again determined by quenching of acridine orange fluorescence, was observed during imposition of K⁺-diffusion potential ([K⁺] _i [K⁺ _o ). In dBBMV and iBBMV, intravesicular acidification was enhanced in the presence of the K⁺-ionophore valinomycin, indicating that the native K⁺ permeability is rate limiting. In the presence of valinomycin, the K⁺-diffusion potential drove BBMV intravesicular acidification to levels close to the electrochemical potential. In gastric SAV, acidification was not limited by the K⁺ permeability. Valinomycin was without effect, but the K⁺/H⁺ ionophore nigericin enhanced acidification in gastric SAV, illustrating the low proton permeability of these membranes. Amiloride, 0.03–1 mm, resulted in concentration-dependent reductions of K⁺-diffusion potential-driven acidification in dBBMV and iBBMV but not in gastric SAV. These data demonstrate that proton permeation in the three membrane types is rheogenic. The sensitivity of the proton-conductive pathways in intestinal BBMV to high concentrations of amiloride correlated with the presence of the Na⁺/H⁺ antiport and indicates that this transmembrane protein may represent a pathway for proton permeation.We thank Ruth Briggs for assistance with the Na/H exchange experiments. This work was supported by a grant from the Medical Research Council (G8418056CA).     

Effects of starvation on valine and alanine transport across the intestinal mucosal border in sea bass,Dicentrarchus labrax

Summary kinetics of intestinal transport of l-alanine and l-valine (substrates of the A-system and the L-system, respectively, in mammals) across the brush-border membrane in sea bass, Dicentrarchus labrax, were studied on intact mucosa using a short-term uptake technique. When fish were starved for 4–8 weeks, total influx (mucosa-to-cell) of valine fell owing to disappearance or modification of the diffusion component. The maximum influx rate of saturable component increased but its affinity (reflected by the Michaelis constant) decreased. Alanine transport by Na⁺-dependent and diffusion pathways was unchanged after starvation Fasting also induced an almost 20% decrease in the length of intestinal microvilli.Abbreviations K _d diffusional constant - K _m Michaelis constant - V _max maximum influx rate     

Trichinella spiralis: comparison of stages in host intestine with those of an Arctic Trichinella sp

The intestinal phase of Trichinella spiralis and of Trichinella sp. isolated in the Arctic were compared in experimental animals. Reproductive capacity, pathogenicity, distribution, and persistence of adults in the small intestine, morphological measurements, and release of newborn larvae in vitro were examined. Numerous passages of 40 days each for T. spiralis and the Trichinella sp. isolate in mice did not affect reproductive capacity, distribution of adults in the small intestine, and size of worms. Reproductive capacity index for T. spiralis, I = 151.27 ± 27.30 was significantly higher compared to the Trichinella sp. isolate index, I = 63.46 ± 19.34. The Trichinella sp. isolate was more pathogenic to mice and wild rodents compared to T. spiralis during the intestinal phase. Both parasites were located in the anterior part of the small intestine but the position of T. spiralis adults (P = 17.08) differed from position of the Trichinella sp. isolate adults (P = 23.46) in the small intestine. Intestinal phase of T. spiralis was longer (20 days) compared to the Trichinella sp. isolate (15 days) but sex ratios (:♂) were similar for both parasites. T. spiralis females released significantly higher numbers of larvae in vitro/24 hr compared to the Trichinella sp. isolate. Release of larvae was continuous during the intestinal phase and the average fecundity for T. spiralis was 335 larvae/female and for the Trichinella sp. isolate 114 larvae/female. Adults of T. spiralis and the Trichinella sp. isolate were morphologically indistinguishable and did not differ in size. A comparative index for the intestinal phase is proposed for comparison of any Trichinella spp. isolates and standard T. spiralis.     

The relation between cell proliferation,differentiation and ultrastructural development in rat intestinal epithelium

Summary The ultrastructural development of the principal cells in rat small intestine was studied by morphometric analyses in relation to the exact cell position along crypt and villus. From the bottom to the tip of the crypt, a gradual increase occurred in absolute size of the total cell, the cytoplasm, the terminal web and of nearly all cell organelles. Also, the relative size of the cytoplasm, mitochondria, microvilli and endoplasmic reticulum increased during crypt cell differentiation. No sudden changes in ultrastructure were observed in the so-called critical decision zone, normally located halfway up the crypt where the proliferative activity ceases. At the crypt-villous junction a 1.4–3 fold increase in cell size, cytoplasm, terminal web and of most organelles was noted. Expansion of the proliferative cell compartment over the total length of the crypt as occurs during recovery after a low X-irradiation dose (72 h after 400 R) does not affect the normal development of cellular ultrastructure. These findings are discussed in relation to biochemical and cell kinetic data.