Hemomicrocirculatory bed in the rabbit small intestine wall during ontogenesis

Hemomicrocirculatory bed has been studied in fragments of the small intestine wall, obtained from 30 rabbits of various age. During ontogenesis the number and caliber of all components of the hemomicrocirculatory bed increase, as well as density of the capillary network in connection with formation and growth of the small intestine membranes. At the age of two or three years involutive changes are observed in the blood bed of this organ.     

Microvillar iron-binding glycoproteins isolated from the rabbit small intestine.

Rabbit intestinal microvillus membranes possess high-affinity receptors for iron whose activity reflects homeostatic changes in mucosal iron transport. To isolate and characterize these membrane components, purified microvilli were radiolabelled with 59Fe(II) and solubilized in Triton X-100. 59Fe in 105000g supernatants co-eluted with a major broad protein peak (Mr approx. 100000) on gel-permeation chromatography and was rendered diffusible by Pronase digestion but not mild periodate degradation. Fluorescence studies with castor-bean lectin conjugates showed specific binding of this affinity probe exclusively to brush-border membranes in the intestinal epithelium. Affinity chromatography of solubilized membrane proteins showed binding to columns of immobilized lectin. Elution with D(+)-galactose released glycoprotein-bound 59Fe purified up to sevenfold over initial membrane extracts. The lectin bound up to 82% of protein-bound 59Fe. In contrast polyspecific antisera raised against rabbit microvilli in guinea-pigs precipitated less than 10% of solubilized radioactivity. Significantly more protein-bound 59Fe in detergent extracts of microvilli purified from bled animals interacted specifically with the lectin, suggesting that membrane glycoprotein receptors are involved in the homeostatic control of intestinal iron transport.     

Motilin receptors in rabbit stomach and small intestine

Motilin receptors in rabbit antral and duodenal smooth muscle tissue were characterized by direct binding technique using 125I-labeled porcine motilin as a tracer ligand. Binding at 30 degrees C was maximal at 90 min, was saturable and partially reversible. Displacement studies with natural porcine motilin, synthetic leucine-motilin or norleucine-motilin indicated a dissociation constant (Kd) of 1.1 +/- 0.3 nM and a maximal binding capacity (Bmax) of 42 +/- 10 fmol/mg protein. Binding was unaffected by glucagon, pancreatic polypeptide and somatostatin, but substance P interfered via an unknown mechanism. By density gradient centrifugation motilin receptors were shown to be present in plasma membranes. Binding could only be demonstrated in preparations from antrum and upper duodenum. These observations provide evidence for a localized target region for motilin in the gastrointestinal tract, and for a direct interaction of motilin with gastrointestinal smooth muscle tissue.     

Tyrosine phosphorylation following alterations in arteriolar intraluminal pressure and wall tension

Arterioles respond to increased transmural pressure with myogenic constriction. The present study investigated the role of tyrosine phosphorylation in myogenic activity. Cannulated segments of a rat cremaster arteriole were fixed under pressure, followed by incubation with fluorescein isothiocyanate (FITC)-conjugated anti-phosphotyrosine. Smooth muscle cell fluorescence intensity was measured with the use of confocal laser-scanning microscopy. Anti-phosphotyrosine fluorescence intensity in muscle cells of arterioles maintained at 100 mmHg was reduced by the tyrosine kinase inhibitor tyrphostin A47 (30 microM) and increased by the tyrosine phosphatase inhibitor pervanadate (100 microM). In time-course experiments, anti-phosphotyrosine fluorescence increased slowly (over 5 min) after an acute increase in intraluminal pressure, and was dissociated from myogenic contraction (within 1 min). In contrast, angiotensin II (0.1 microM) caused rapid constriction and increased tyrosine phosphorylation. Anti-phosphotyrosine fluorescence was also pressure dependent (10-100 mmHg). Abolition of myogenic activity, either through removal of extracellular Ca2+, or exposure to verapamil (5 microM) or forskolin (0.1 microM) caused a further increase in anti-phosphotyrosine fluorescence. We conclude that transmural pressure and/or wall tension in arterioles causes increased tyrosine phosphorylation; however, this is not involved in the acute phase of myogenic constriction but may be involved in later responses, such as sustained myogenic tone or mechanisms possibly related to growth.     

Identification of multiple isolated lymphoid follicles on the antimesenteric wall of the mouse small intestine.

We have revealed that 100-200 clusters, filled with closely packed lymphocytes, can be found throughout the length of the antimesenteric wall of the mouse small intestine. They are composed of a large B cell area, including a germinal center, and epithelia overlying the clusters contain M cells. A large fraction of B cells displays B220+ CD19+ CD23+ IgM(low)IgD(high)CD5(-)Mac-1(-) phenotype, and the composition of IgA+ B cells is smaller but substantial. To our knowledge, these clusters are the first identification of isolated lymphoid follicles (ILF) in mouse small intestine. ILF can be first detected at 7 (BALB/c mice) and 25 (C57BL/6 mice) days after birth, and lymphoid clusters equivalent in terms of cellular mass to ILF are present in germfree, athymic nude, RAG-2(-/-), TCR-beta(-/-), and Ig mu-chain mutant (mu(-/-)) mice, although c-kit+ cells outnumber B220+ cells in germfree and athymic nude mice, and most lymphoid residents are c-kit+ B220(-) in RAG-2(-/-), TCR-beta(-/-), and mu(-/-) mice. ILF develop normally in the progeny of transplacentally manipulated Peyer's patch (PP)-deficient mice, and decreased numbers of conspicuously atrophied ILF are present in IL-7Ralpha(-/-) PP(null) mice. Neither ILF nor PP are detectable in lymphotoxin alpha(-/-) and aly/aly mice that retain well-developed cryptopatches (CP) and thymus-independent subsets of intraepithelial T cells, whereas ILF, PP, CP, and thymus-independent subsets of intraepithelial T cells disappear from common cytokine receptor gamma-chain mutant mice. These findings indicate that ILF, PP, and CP constitute three distinct organized gut-associated lymphoid tissues that reside in the lamina propria of the mouse small intestine.     

Close relationships between the cells of the immune system and the epithelial cells in the rat small intestine

Summary A possible contribution of the intestinal epithelium to the immune defence system was studied by electron microscopy in the rat small intestine. The cells of the immune system (CIS) such as lymphocytes, eosinophils and macrophages penetrate the basal lamina into the epithelium and make close relationships with the absorptive cells. At the points of close apposition, the two cell membranes run parallel at a regular distance of about 20 nm. On the other hand, about 5% of the intestinal absorptive cells also penetrate the basal lamina into the lamina propria via their basal protrusions and show a similar close association with CIS. The basal protrusions contain many microfilaments; this indicates that they are structures with a definite function rather than a simple hernia. These findings are discussed with respect to the transport of antigenic molecules and of intercellular communication between CIS and the intestinal epithelium.     

Vimentin-positive cells in the villus epithelium of the rabbit small intestine

In rabbit intestinal epithelium, vimentin intermediate filaments are selectively expressed in the M cells of follicle-associated epithelium (FAE). To find intestinal epithelial cells belonging to the M cell lineage, vimentin was detected immunohistochemically in the rabbit small and large intestines. Vimentin-positive columnar cells were scattered throughout the villus epithelium of the small intestine. In their cytoplasm, vimentin was located from the perinuclear region to the cell membrane touching intraepithelial lymphocytes. These cells had microvilli shorter than those of absorptive cells, and the alkaline phosphatase activity of the microvilli was markedly weaker than that of absorptive cell microvilli. Glycoconjugates on the surface of the microvilli were alcian blue positive and periodic acid-Schiff negative. The morphological and histochemical features of these vimentin-positive villus epithelial cells differed from those of adjacent absorptive cells and closely resembled those of the M cells in FAE covering Peyer's patches and solitary lymphatic nodules. These results suggest that the vimentin-positive cells in the villus epithelium belong to the M cell lineage.     

Absorption and metabolism of genistin in the isolated rat small intestine

Uptake and intestinal metabolism of physiologically active genistin were studied in an ex vivo intestinal perfusion model; luminally applied concentrations were 5.9, 12.0, and 23.8 micromol/l. The intestinal absorption of genistin was 14.9% (+/-2.3, n=9), irrespective of the amounts applied. The majority of the absorbed genistin appeared as genistein glucuronide (11.6%), also recovered as the main metabolite on the luminal side (19.5%). Minor amounts of genistin (1.3%) and genistein (1.9%) were found on the vascular side, whereas 15.4% of applied genistin was luminally cleaved to yield genistein. Sulfate derivatives of genistein or genistin were not observed.     

The effect of phosphoglycerides on the incorporation of cholesterol into isolated brush-border membranes from rabbit small intestine

The incorporation of cholesterol from bile salt micelles into brush-border membranes was found to be similar whether these originated from jejunum, ileum or whole small intestine. This incorporation, however, was appreciably lower in membranes obtained from duodenum. Studies pursued with membranes from whole small intestine revealed that dipalmitoyl or dioleoylphosphatidylcholine, when micellized together with the sterol and taurocholate markedly inhibited the incorporation. The didecanoyl and dilauroyl analogues of this lipid class were without significant effect. Preincubation of the membranes for 30 min at 37 degrees C with or without dipalmitoylphosphatidylcholine had no effect on cholesterol incorporation. Again in this case, suppression of sterol uptake could be seen only when phosphatidylcholine was admixed with the sterol. In contrast, dipalmitoyl and dilauroylphosphatidylethanolamines were found to be stimulatory rather than inhibitory. Addition of palmitic acid to the sterol-bile salt micelles had no effect on the uptake of cholesterol; however, this fatty acid could completely reverse the inhibition of cholesterol uptake by dipalmitoylphosphatidylcholine. The present study supports the conclusion that cholesterol incorporation into isolated brush-border membranes is governed largely by factors which affect the partitioning of the sterol out of the bile salt micelle.     

Amino acid sequence of VIP, PHI and secretin from the rabbit small intestine

VIP, PHI and secretin were purified from rabbit small intestine throughout a maximum of 6 chromatographic steps. After elution on a reverse phase C18 column, the 3 peptides were separated on a Fractogel column using specific radioimmunoassays for detection. After cation exchange chromatography on Mono S, the final steps were performed using a reverse phase RP8-e column. For these steps, radioreceptor assays were utilized to detect VIP and PHI. We confirmed that the VIP sequence of rabbit was identical to that of porcine VIP. The PHI sequence was also found identical to that of porcine PHI. By contrast, rabbit secretin was highly original, differing from porcine secretin in having Leu, Arg and Leu-NH2 residues instead of Phe, Ser and Val-NH2 in, respectively, position 6, 16 and 27.     

Interaction between left ventricular wall motion and intraventricular flow propagation in acute and chronic ischemia

Myocardial ischemia has been associated with left ventricular (LV) postsystolic shortening. The combination of tissue Doppler imaging and high frame-rate acquisition of two-dimensional color flow makes it possible to study the interaction between LV wall motion and intraventricular flow propagation. The aim of this study was to examine in a clinical model the impact that acute myocardial ischemia and prior myocardial infarct might have on LV flow patterns and to explain the underlying mechanisms from the tissue Doppler data. LV flow propagation and tissue velocities during early diastole were studied in 18 healthy individuals, 17 patients with prior anterior myocardial infarct, and 16 patients before and during percutaneous coronary intervention (PCI) of the left anterior descending artery. Normal individuals had intraventricular flow propagation toward the apex during isovolumic relaxation. During this early diastolic time phase, myocardial velocities measured at mid- and apical septal segment were directed away from the apex. Before PCI, patients without myocardial infarction had similar findings as in normal individuals. In contrast, each patient with either prior myocardial infarction or PCI-induced acute ischemia had flow propagation opposite to normal individuals, and tissue velocities reversed toward the apex during early diastole. Reversal of early diastolic LV flow propagation in acute and chronic anterior myocardial ischemia reflects postsystolic shortening in the dyskinetic apical and septal myocardial segments.     

Relationship between transmural potential difference and smooth muscle slow waves and contractility in the rabbit small intestine in vitro

The relationship between transmural potential difference (PD) and smooth muscle electrical and mechanical activity was investigated in the rabbit ileum in vitro. Transmural PD was monitored using agar salt bridge electrodes connected via calomel half cells to an electrometer. Force displacement transducers recorded predominantly longitudinal smooth muscle activity. Concurrently, predominantly circular muscle activity was recorded at three sites using intraluminal pressure probes. At the same sites, suction electrodes monitored electrical activity of the smooth muscle. In all experiments, fluctuations in transmural PD were temporally linked to smooth muscle mechanical and electrical activity. The frequency of PD oscillations, electrical slow waves, and cyclic pressure changes were identical within each segment. Adrenaline abolished smooth muscle electrical spiking, all mechanical activity, and transmural fluctuations in PD. However, the slow waves were not abolished, though their frequency was increased. Phentolamine but not propranolol reversed the effects of adrenaline, thus slow wave frequency is influenced by alpha-adrenergic stimulation in the rabbit ileum. In conclusion, oscillations in transmural PD are unrelated to the ionic processes associated with the slow wave. However, they are in some way linked to smooth muscle contractile activity, possibly via an intrinsic neural mechanism as observed in the guinea pig.     

Effects of intraluminal administration of adrenoceptor agonists on transisthmic flow in the rabbit oviduct

By perfusing the isthmic segment of the rabbit oviduct in vivo at low hydrostatic pressures with Ringer's solution (+38 degrees C), the resistance of the isthmic segment could be measured quantitatively using a drop-recording system. Transisthmic flow, from mid-tube to the uterine end, was thus studied in 45 animals, 48 h after human chorionic gonadotropin (hCG) injection. Stimulation of alpha-adrenoceptors by administration of norepinephrine or phenylephrine to the perfusate resulted in an immediate reduction or cessation of the basal flow. The duration of this effect varied dose-dependently. The administration of beta-adrenoceptor agonists (isoprenaline, prenalterol or terbutaline) markedly increased transisthmic flow. The beta 2-adrenoceptor agonist terbutaline was considerably more potent than the other beta-agonists. Thus, it seems that the isthmic contraction caused by adrenergic luminal stimulation is mediated via alpha-adrenoceptors, while the isthmic dilation is mediated via beta-adrenoceptors, mainly of the beta 2-type.