Absorption of arginine-vasopressin and arginine-vasotocin in small intestine of the frog Rana temporaria

Experiments in vitro demonstrated a partial absorption of arginine-vasopressin (AVP) in the frog small intestine. Dynamics and efficiency of the nonapeptide absorption are studied with use of hydroosmotic method of recording of the osmotic permeability of the frog urinary bladder epithelium and immunoenzyme analysis. In the process of absorption there were preserved intactness of the hormone cyclic structure and its physiological activity, like in the case of the arginine-vasotocin (AVT) absorption. The AVP absorption increased at its administration into the gut with inhibitor of proteases. By methods of immunoelectron and immunofluorescent microscopy with use of polyclonal antibody to AVP, location of the label to the hormone was shown in the enterocyte cytoplasm. Thus, there was obtained a morphological evidence for the AVP absorption and transepithelial transfer in the frog small intestine. These data enlarge the concept of the poorly studied properties of the absorbing epithelium of the vertebrate intestine with respect to absorption of intact molecules of polypeptides.     

Immunocytochemical localization of vasopressin at its absorption by cells of rat small intestine

Morpho-physiological characteristics of the transport of cyclic nonapeptide arginine vasopressin (AVP) across the rat intestinal epithelium was studied in experiments in vitro. A partial absorption of physiologically active AVP was followed when filling the isolated intestinal lumen by hormone solution. By methods of immunoelectron and immunofluorescence confocal microscopy, using polyclonal anti-AVP antibodies, cytoplasmic localization of AVP label was shown in enterocytes. The AVP label was also observed in the intercellular space in the basal area of epithelium. No label was revealed in the intercellular junctions, and no predominant label accumulation was found in any cytoplasmic structures of the epithelial cells. The obtained results are considered as evidence for the transcellular pathway of partial AVP absorption in rat small intestine.     

Morphofunctional changes in the small intestine epithelium of the frog <Emphasis Type="Italic">Rana temporaria</Emphasis> in the course of hibernation

The structure and function of small intestine epithelium have been studied in overwintering frogs at various stages of hibernation. Experiments on the in vitro absorption of arginine vasotocin (AVT) have shown that the epithelium ability for absorption of this nonapeptide without hydrolysis persists during hibernation. However, there is a decline in AVT absorption observed in January–February and later on (as compared with that in October–December) which is most pronounced in March–April. Changes in the epithelial structure appear by midwinter and are growing towards spring. In April–May, enterocyte height decreased by 33%, microvilli length by 40%, and number of mitochondria by 57% relative to the values at the beginning of hibernation. The absence of signs of breakdown attests to the adaptive nature of the observed changes. The dynamics of the studied parameters reveals morphological and functional plasticity of small intestinal epithelium in Rana temporaria over the hibernation period.     

Renal clearance of absorbed intact GFP in the frog and rat intestine

Intestine absorption of intact green fluorescent protein (GFP) and its following accumulation in the renal proximal tubule cells after its intragastric administration have been established by confocal microscopy in the rat and frog. Reabsorbed GFP was revealed in the endosomes and lysosomes of the proximal tubule cells by the methods of GFP photooxidation and immunofluorescent microscopy. The GFP intestine absorption rate and GFP accumulation in the kidney were significantly higher in the frog than in the rat. No specific fluorescence was revealed in the liver and colon cells after the GFP intragastric administration. The data obtained indicate the ability of the small intestine in the frog and rat to absorb intact proteins and an important role of the kidney in exogenous protein metabolism.     

Evidence of basolateral water permeability regulation in amphibian urinary bladder

It is well known that arginine vasopressin (AVP) produces up to a 40-fold increase (0.1 to 4,0 μL/min·cm²) in net water flux across the amphibian urinary bladder under an osmotic gradient (mucosal side 10% hypotonic). No AVP effect is observed when the gradient is in the opposite direction (serosal hypotonic). Similar asymmetrical behavior to osmotic gradients occurs in the frog corneal epithelium. This rectification phenomenon has not been satisfactorily explained. We measured net water fluxes in bladder sacs and confirmed that AVP has no effect when the serosal bath is hypotonic. We reasoned that the ‘abnormal’ serosal osmolarity was inducing changes in membrane water permeability, the very parameter being measured. Thus, we studied the effect of solution osmolarity on diffusional water flow (J_dw) across the frog bladder using ³H₂O. As expected, AVP doubled J_dw (in either direction from 12 to 21 μL/min·cm²) when the serosal solution was iso-osmolar regardless of mucosal osmolarity. However, in the AVP-stimulated bladders, hypo-osmolarity of the serosal solution reduced J_dw by 42%, an effect that was reversible when normal osmolarity was re-established. Amphotericin B (instead of AVP) was used to irreversibly increase the permeability to water of the apical membrane. Under these conditions, basolateral hypotonicity also reversibly decreased J_dw by 32%, suggesting the basolateral membrane as the site where permeability is reduced. SEM and TEM of the tissue shows extreme swelling when it was exposed to serosal hypotonicity with or without AVP and typical surface morphology changes following hormone stimulation. We conclude that this swelling may initiate a signaling mechanism that reduces basolateral water permeability. These findings constitute evidence of basolateral water channel permeability regulation, which can also contribute to cell volume regulation.     

Inductive action of epithelium on differentiation of intestinal connective tissue of Xenopus laevis tadpoles during metamorphosis in vitro

The action of the epithelium on differentiation of connective tissue cells of Xenopus small intestine during metamorphosis was investigated by using culture and morphological techniques. Connective tissue fragments isolated from the small intestine at stage 57 were cultivated in the presence or absence of homologous epithelium. In the presence of the epithelium, metamorphic changes in the connective tissue were fully induced by hormones including thyroid hormone (T₃), as during spontaneous metamorphosis, whereas they were partially induced in the absence of the epithelium. Macrophage-like cells showing non-specific esterase activity in the connective tissue were much fewer in the absence of the epithelium than in the presence of it, and aggregates of fibroblasts possessing well-developed rough endoplasmic reticulum developed only in the presence of the epithelium. Just before the aggregation of the fibroblasts, the connective tissue close to the epithelium became intensely stained with concanavalin A (ConA) and wheat germ agglutinin (WGA). The present results indicate that the epithelium plays important roles in the differentiation of intestinal connective tissue cells, which in turn affect the epithelial transformation from larval to adult form during anuran metamorphosis. Thus, the tissue interaction between the epithelium and the connective tissue in the anuran small intestine is truly bidirectional.     

Electron microscopic study of colonic epithelial cells from the grass frog Rana temporaria under different intensity of water absorption

Three cell types have been revealed in the epithelium of the frog large intestine: granular, mitochondria-rich, and mucosal cells. Under a low water permeability (0.12 +/- 0.10 mkl/(min.cm2)) the distribution of intramembrane particles (IMP) in the apical cell membrane was the same as in the most cell plasma membranes studied with freeze-fracture method. Under rising osmotic permeability and water absorption (0.43 +/- 0.05 mkl/(min.cm2)) the IMP distribution did not change. In these conditions, the quantity of fusion sites between granule membranes and the apical membrane increased, and the intercellular spaces in basolateral epithelial region were diluted. A a low water permeability, in addition to usual microtubules, bundles of noncentrosomal microtubules with associated osmiophilic globules were revealed. A comparative analysis has been made of the present evidence and previously obtained data on the frog urinary bladder epithelium.     

The problem of sugar resorption in the small intestine: histochemical studies by means of the Gomori method]

The role of alkaline phosphatase during active absorption of sugars is experimented with Gomori's histochemical method. Experiments on empty intestine show that the surgical intervention does not alter the phosphate content of the wall of the small intestine. Absorption experiments with 10% and 5,4% (isotonic) glucose solutions demonstrate a marked decrease in the alkaline phosphatase content of the wall of the small intestine. In half of the cases only, absorption experiments with a 10% mannitol solution determine a small decrease in the alkaline phosphatase of the wall of the small intestine. In 90% of the cases, there are lesions of the intestinal epithelium.     

Connective tissue is involved in adult epithelial development of the small intestine during anuran metamorphosis in vitro

Summary The role of connective tissue in metamorphic changes of the small intestinal epithelium inXenopus laevis tadpoles was investigated by using organ culture techniques and electron microscopy. Tissue fragments isolated from various parts of the small intestine at stage 57 were cultivated. Larval cell death of the epithelium was induced by thyroid hormone in all fragments, whereas adult epithelial development was observed only in fragments isolated from the anterior intestinal region containing the typhlosole where most of the larval connective tissue was localized. The epithelium was then cultivated in recombination with homologous or heterologous non-epithelial components. The adult epithelium developed only in recombinants containing a thick connective tissue layer from the typhlosole. There was no regional difference in the developmental potency of the epithelium itself. In all explants where adult epithelium developed, the connective tissue increased in cell density just beneath the epithelium, which was rapidly proliferating and forming typical islets. At the same time, fibroblasts possessing well-developed rough endoplasmic reticulum differentiated close to epithelial cells and often made contact with them. These results indicate that the connective tissue originating from the typhlosole plays an important role in adult epithelial development of the anuran small intestine, probably via direct cell-to-cell contacts or some factor(s) synthesized by the fibroblasts.     

Somatostatin-immunoreactive cells in the gastrointestinal tract of the frog Rana esculenta

The morphology and topographic distribution of somatostatin-immunoreactive cells in the stomach and small intestine of the frog Rana esculenta were studied at the light-microscopic level by the use of the peroxidase-antiperoxidase method. Scattered immunostained cells occurred in all regions of the gastrointestinal tract investigated. In the small intestine, the number of these cells decreased gradually in the oral to anal direction, i.e. from the pyloric (antral) stomach to the entrance into the colon. Most of the immunostained cells possessed thick, short cytoplasmic processes, which did not display a preferential spatial orientation. Other somatostatin-immunoreactive cells, which were exclusively located in the small intestine, gave rise to a single long extension oriented toward the lumen. In both stomach and small intestine, a complete penetration of the epithelial surface by these processes of somatostatin-immunoreactive cells was observed only occasionally. The morphological features of the somatostatin-immunostained cells speak in favor of endocrine, paracrine, and possibly also intraluminal secretory functions of the enteroendocrine somatostatin system in frogs.Fellow of the Alexander von Humboldt Foundation, Bonn, Germany     

Geometric peculiarities of intestinal surface and efficiency of coupling between membrane hydrolysis and transport of nutrients

Kinetics of the membrane hydrolysis of maltose and of the absorption of the released glucose in the isolated loop of rat small intestine has been examined in a wide range of maltose concentrations (25–200 mM) under the conditions of chronic experiments. The processes studied were simulated by means of mathematical models using two approximations of the villous surface of intestinal epithelium: (i) smooth flat surface with adjoining pre-epithelial (“unstirred”) layer and (ii) folded surface with “unstirred” layer between the folds and partly above them. The results of modeling matched well the experimental data in the whole range of maltose concentrations only in the case of the folded surface. A model with this approximation predicts a closer coupling between maltose hydrolysis and absorption of released glucose as well as a lower glucose concentration in the intestinal lumen than in the case of a flat surface. We conclude that in order to evaluate correctly a relative role of various mechanisms of glucose transport across intestinal epithelium under normal conditions, one should take into account the pre-epithelial layer of the small intestine and geometric peculiarities of its epithelial surface.     

Allometry of paracellular absorption in birds

Water-soluble nutrients can be absorbed across the intestinal epithelium by transcellular and paracellular processes. Recent studies suggest that small birds (<180 g) have more extensive paracellular absorption of glucose than nonflying mammals. This may be a feature that compensates for a reduced small intestine size because small birds have smaller mass-corrected intestinal length than do nonflying mammals, but the difference diminishes in larger birds. We hypothesized that if this explanation were correct, there would be a negative correlation between paracellular absorption and body mass in birds and that larger birds would have paracellular absorption comparable to that of nonflying mammals. We tested this hypothesis, using consistent methodology, by measuring the extent of absorption of a series of inert carbohydrate probes in heavier bird species (>300 g) selected from diverse taxa: American coots, mallards, pheasants, and pigeons. Absorption of carbohydrate probes was inversely related to body mass in birds, and absorption of these probes in large birds (>500 g) was comparable to absorption measurements in nonflying mammals. Higher paracellular uptake in the smaller avian species may offer a physiologically inexpensive means of nutrient absorption to compensate for a reduced small intestine size but may make those species more vulnerable to toxicant absorption.     

Stimulation of the sodium transport across the frog skin by three N-terminally extended arginine-vasopressins

The standard Ussing method was used to electrophysiologically characterize the effects of three analogs of arginine-vasopressin (AVP) on the frog skin, a model Na-transporting epithelium. The analogs tested were N-terminally extended Arg8-vasopressins: Ala-AVP, Ser-Ala-AVP and Thr-Ser-Ala-AVP; synthetic Arg8-AVP was used as the reference agent. The vasopressins were applied to the basolateral side of the frog skin in concentrations ranging between 10(-8) to 10(-5) mol.l-1. All the three analogs increased both the short-circuit current (Isc) and the open-circuit transepithelial potential (Voc), and decreased the transepithelial d.c. resistance (Rt) similarly as did synthetic Arg8-AVP. The results show that N-terminal extension of the Arg8-AVP did not alter the natriferic properties of AVP.     

Impacts of Dietary Pleurotus eryngii Polysaccharide on Nutrient Digestion,Metabolism, and Immune Response of the Small Intestine and Colon—An iTRAQ‐Based Proteomic Analysis

Pleurotus eryngii polysaccharides have been shown to exert significant biological activities to the host. However, few studies have been conducted on its effects on gastrointestinal tract (GIT) health alteration. In the present study, small intestinal and colonic proteome alterations generated by dietary supplementation with a novel homogeneous P. eryngii polysaccharide (PEP) in C57BL/6 mice, based on the isobaric tag for relative and absolute quantification (iTRAQ) proteomics, are investigated. Compared to the control group, PEP supplementation result in a total of 113 and 194 significant differential proteins (DPs) in the small intestine and colon, respectively. Interestingly, DPs in small intestine are mainly related to the transport and biosynthetic process, along with the digestion and absorption pathway of nutrients, whereas the colonic DPs are significantly found participating in numerous metabolic processes. Moreover, the alterations of some DPs in small intestine and colon are speculated to correlate with the colonic microbiota structure and are involved in the regulation of host immune response. Subsequently, some critical DPs of small intestine and colon are selected and validated by Western blotting. The current research facilitated the generation of potential insights into the health benefit activities and functional mechanisms of polysaccharides from P. eryngii.     

Renal filtration and reabsorption of GFP in <Emphasis Type="Italic">Rana temporaria</Emphasis>: Effect of arginine-vasotocin

The renal tubular uptake of green fluorescent protein (GFP) in frog Rana temporaria was studied by laser confocal microscopy. The specific green fluorescence was revealed in cells of proximal tubules 30 min after intravenous GFP injection. The GFP fluorescence was located predominantly in the apical part of the cytoplasm in the form of intensively fluorescing vesicles. The GFP injections increased dose-dependently the GFP tubular uptake. This was confirmed by the quantitative assessment of intensity of the specific fluorescence, its relative vesicular density, and by correlation analysis. Preliminary administration of arginine-vasotocin into the dorsal lymphatic sack decreased significantly the GFP absorption. The effect of arginine-vasotocin was inhibited by pretreatment with an antagonist of vasopressin V_i-receptors. The results of this work together with literature data allow believing that a decrease in the GFP absorption in the frog kidney under effect of arginine-vasotocin is due to a fall of the AVT-dependent glomerular filtration rate and to a decrease in the input of protein into the lumen of tubules. The action of arginine-vasotocin seems to be mediated via the V_i-like receptors of preglomerular blood vessels.     

Responses of frog skin Na(+) and Cl(-) transport to guanylin

A possible role for the peptide hormone guanylin was investigated in frog skin (Rana pipiens) epithelium. Sodium and chloride fluxes in response to this peptide were evaluated in Ussing-type chambers. Net and unidirectional Na(+) fluxes were measured by using (22)Na(+) and atomic absorption analysis of total [Na(+)], whereas net Cl(-) fluxes were measured by using electrometric titration for [Cl(-)]. Mucosal application of guanylin (0.5-2.0 micromol/l) caused marked increases in serosal to mucosal net flux and efflux of Na(+). Serosal application of guanylin over the same dose range caused similar large increases in net serosal to mucosal (S-->M) Na(+) and Cl(-) flux as well as Na(+) efflux. Responses of Na(+) influx were small and inconsistent. When frog skin was bathed on the serosal side with Cl(-)-free Ringer's solution mucosal application of guanylin stimulated large efflux and S-->M net fluxes of Na(+). Serosal treatment yielded large Na(+) effluxes and S-->M Na(+) and Cl(-) net fluxes. When frog skin serosal surfaces were bathed with Na(+)- free Ringer's solution mucosal guanylin treatment had no effect but serosal treatment produced large S-->M Cl(-) net fluxes.     

Electroneutral sodium absorption and electrogenic anion secretion across murine small intestine are regulated in parallel

Electrolyte transport processes of small intestinal epithelia maintain a balance between hydration of the luminal contents and systemic fluid homeostasis. Under basal conditions, electroneutral Na(+) absorption mediated by Na(+)/H(+) exchanger 3 (NHE3) predominates; under stimulated conditions, increased anion secretion mediated by CFTR occurs concurrently with inhibition of Na(+) absorption. Homeostatic adjustments to diseases that chronically affect the activity of one transporter (e.g., cystic fibrosis) may include adaptations in the opposing transport process to prevent enterosystemic fluid imbalance. To test this hypothesis, we measured electrogenic anion secretion (indexed by the short-circuit current) across NHE3-null [NHE3(-)] murine small intestine and electroneutral Na(+) absorption (by radioisotopic flux analysis) across small intestine of mice with gene-targeted disruptions of the anion secretory pathway, i.e., CFTR-null [CFTR(-)] or Na(+)-K(+)-2Cl(-) cotransporter-null [NKCC1(-)]. Protein expression of NHE3 and CFTR in the intestinal epithelia was measured by immunoblotting. In NHE3(-), compared with wild-type small intestine, maximal and bumetanide-sensitive anion secretion following cAMP stimulation was significantly reduced, and there was a corresponding decrease in CFTR protein expression. In CFTR(-) and NKCC1(-) intestine, Na(+) absorption was significantly reduced compared with wild-type. NHE3 protein expression was decreased in the CFTR(-) intestine but was unchanged in the NKCC1(-) intestine, indicating that factors independent of expression also downregulate NHE3 activity. Together, these data support the concept that absorptive and secretory processes determining NaCl and water movement across the intestinal epithelium are regulated in parallel to maintain balance between the systemic fluid volume and hydration of the luminal contents.     

Structure and mucous histochemistry of the intestinal respiratory tract of the mud loach, Misgurnus anguillicaudatus (Cantor)

The straight intestinal tract of the mud loach Misgurnus anguillicaudatus was divided into an intestine and rectum which consisted of a mucosa (epithelial layer), lamina propria‐submucosa, muscularis and serosa. The intestine and rectum have shorter mucosal folds and a thinner wall. Extensive vascular capillary networks were present in the mucosa of the intestine and the rectum. The diffusion distance between the vascular capillaries and the lumen in the intestinal and rectal mucosal epithelium was about 11.2 μm (±1.12). The majority of the epithelial mucous cells contain acidic mucins although there are small amounts of a mixture of acidic and neutral mucins. The intestinal tract of M. anguillicaudatus is probably modified to suit its role of respiration for the deficient oxygen supply within their environment.     

Neurotrophin receptors and enteric neuronal development during metamorphosis in the amphibian<Emphasis Type="Italic"> Xenopus laevis</Emphasis>

During metamorphosis, the frog intestine goes through a dramatic shortening with extensive apoptosis and regeneration in the epithelial layer and connective tissue. Our aim was to study changes in the enteric nervous system represented by one inhibitory (vasoactive intestinal polypeptide; VIP) and one excitatory (substance P, neurokinin A; SP/NKA) nerve population and concomitant changes in neurotrophin receptor occurrence during this development in the gut of Xenopus laevis adults and tadpoles at different stages of metamorphosis (NF stages 57–66). Sections were incubated with antibodies against the neurotrophin Trk receptors and p75^NTR, and the neurotransmitters VIP and SP/NKA. Trk-immunoreactive nerves increased dramatically but transiently in number during early metamorphic climax. Nerves immunoreactive for p75^NTR were present throughout the gut, decreased in number in the middle intestine during climax, and increased in the large intestine during late metamorphosis. The percentage of VIP-immunoreactive nerves did not change during metamorphosis. SP/NKA-immunoreactive nerves were first apparent at NF stages 61–62 in the middle intestine and increased in the stomach and large intestine during metamorphosis. Endocrine cells expressing SP/NKA increased in number in stomach, proximal, and middle intestine during metamorphic climax. Thus, neurotrophin receptors are expressed transiently in neurons of the enteric nervous system during metamorphosis in Xenopus laevis and SP/NKA innervation is more abundant in the intestine of the postmetamorphic frog than in the tadpole.This study was supported by grants from the Swedish Research Council to S. Holmgren