Absorption of arginine-vasopressin and arginine-vasotocin in small intestine of the frog <Emphasis Type="Italic">Rana temporaria</Emphasis>

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-vasotocine (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.     

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.     

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.     

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.     

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.     

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.     

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.     

Ultrastructural modifications of frog urinary bladder epithelium under the influence of hypertonic media

Summary The frog urinary bladder undergoes a marked increase in its water permeability when incubated in hypertonic media. Many similarities are found between this effect and the hydrosmotic action of antidiuretic hormone. The ultrastructural modifications of the epithelium observed under the influence of serosal hypertonicity (the intercellular spaces are dilated while the tight junctions remain closed) lead us to assume that the pathways of water movement across the epithelium could be the same in this case and in hydrosmotic response to the hormone. In contrast, when the mucosal medium is made hypertonic, the ultrastructure is differently altered: the intercellular spaces are closed, the tight junctions show small vesicles and numerous large vacuoles appearing in the cytoplasm.     

Endocrine cells of mucosal epithelium in the distal gut of the grass frog Rana temporaria

The epithelium of the distal part of Rana temporaria intestine has been studied by light and electron microscopy. It has been shown that the number of agrent-affined endocrinocytes in the mucosa of the frog intestinal epithelium decreases gradually from the small bowel to the colon, and then sharply increased in the distal part of colon (cloaca). Four types of endocrinocytes have been identified: EC, D, L and type IV. In frogs examined in March, endocrinocytes of the colon mucosal epithelium demonstrate structural features characteristic of the state of functional strain. These features are most pronounced in EC-cells. This seems to be due to the state of awakening from hibernation, serotonin (produced by EC-cells) playing an essential role in this process.     

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.     

Hydroosmotic activities of arginine-vasopressins modified either in positions 1, 2 and 4 or at N-terminal extensions.

Vasopressin and its synthetic analogs were studied for their effect on transepithelial water flux in frog urinary bladder. As compared with AVP, 1-deamino-8-D-arginine vasopressin (dDAVP) was about 40 times less effective in stimulating osmotic water flow. The vasopressin analogs obtained by modification in positions 1 and 2 were: [1-(1-mercapto-4-tert-butylcyclohexaneacetic acid)] AVP (I); [1-(1-mercapto-4-methylcyclohexaneacetic acid)]AVP (II); [1-(1-mercapto-4-methylcyclohexaneacetic acid)-2-O-methyltyrosine]AVP (III); and those modified in position 4 were: [1-(1-mercaptocyclohexaneacetic acid)-4-arginine] AVP (IV); [1-(2-mercaptopropionic acid)-4-arginine]AVP (V). Any of the above analogs did not influence basal, but antagonized vasopressin-stimulated water flux. N-terminally extended analogs of AVP: Ala-AVP (VI); Ser-Ala-AVP (VII) and Thr-Ser-Ala-AVP (VIII) stimulated osmotic water flux to the same extent in concentration 200 times higher as that of AVP. We conclude from these studies that vasopressin analogs (I-V) competitively antagonize vasopressin-stimulated hydroosmotic activity in frog urinary bladder probably at the epithelial vasotocin V1 and/or V2 receptor site. N-terminal extension of the vasopressin molecule did not influence the capacity of AVP to induce V2 receptor-mediated action, even when used at higher concentrations.     

Immunohistochemical analysis of ZnT1, 4, 5, 6, and 7 in the mouse gastrointestinal tract.

Expression of five zinc transporters (ZnT1, 4, 5, 6, and 7) of the Slc30 family in the mouse gastrointestinal tract was studied by immunohistochemical analysis. Results demonstrated unique expression patterns, levels, and cellular localization among ZnT proteins in the mouse gastrointestinal tract with some overlapping. ZnT1 was abundantly expressed in the epithelium of the esophagus, duodenum of the small intestine, and cecum of the large intestine. ZnT4 was predominantly detected in the large intestine. ZnT5 was mainly expressed in the parietal cell of the stomach and in the absorptive epithelium of the duodenum and jejunum. ZnT6 was predominantly detected in the chief cell of the stomach, columnar epithelial cells of the jejunum, cecum, colon, and rectum. Lastly, ZnT7 was observed in all epithelia of the mouse gastrointestinal tract with the highest expression in the small intestine. Expression of ZnT proteins in the absorptive epithelial cell of the gastrointestinal tract suggests that ZnT proteins may play important roles in zinc absorption and endogenous zinc secretion.     

大鼠小肠神经激肽A免疫反应（NKA－Ⅰ）物质分布特征的研究

本实验采用免疫细胞化学ＰＡＰ法,将切片与铺片技术相结合,全面研究了Ｗｉｓｔａｒ大鼠（ｎ＝２０）小肠神经激肽Ａ免疫（ＮＫＡ－Ｉ）阳性物质的分布特征。结果提示：ＮＫＡ－Ｉ神经元单个或多个散在分布于粘膜下和肌间的神经节内,粘膜下神经节处多见。ＮＫＡ－Ｉ神经纤维广泛分布于小肠各段的各层结构中。在小肠粘膜上皮和腺上皮中可见散在分布的ＮＫＡ－Ｉ强阳性细胞,推测为ＮＫＡ－Ｉ内分泌细胞。本文还对ＮＫＡ－Ｉ的形态学特点及生理功能进行了讨论。     

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.     

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.     

Effects of lysine-vasopressin (LVP) and 1-deamino-8-D-arginine-vasopressin (dDAVP) upon electrical potential, short-circuit current and transepithelial D.C. resistance of the frog skin

The synthetic analogue of vasopressin, 1-deamino-8-D-arginine-vasopressin (dDAVP), possesses a protracted antidiuretic activity while having practically no pressoric activity as compared to arginine-vasopressin (AVP) or lysine-vasopressin (LVP). The effects of LVP and dDAVP were studied on the frog skin (Rana temporaria) sodium transport as reflected by the short-circuit current (SCC) level, on an Ussing apparatus. The application two different equimolar doses of LVP or dDAVP (approx. 9.4 X 10(-8) mol X l-1 and 18.8 X 10(-8) mol X l-1 to the inner surface of the skin resulted in identical maximal increases of sodium transport. However, the maximum transport stimulation after the application of dDAVP was delayed by about 30 min as compared to the stimulation by LVP (P less than 0.01). In addition, a protracted recovery of SCC towards its original levels was observed in experiments with dDAVP application after the hormone removal (P less than 0.01). It is concluded that dDAVP stimulates Na+ transport through the frog skin despite its lacking pressoric activity. Thus, the natriferic activity of vasopressin is related to its antidiuretic rather than pressoric activity. Maximum increase in the sodium transport following dDAVP application was delayed and more protracted as compared to the effect of LVP.     

Gene Regulation by Thyroid Hormone During Amphibian Metamorphosis: Implications on the Role of Cell-Cell and Cell-Extracellular Matrix Interactions

SYNOPSIS. Amphibian metamorphosis is the developmental processinitiated by thyroid hormone which transforms a tadpole intoa frog. This transformation requires extensive remodeling ofalmost every tissue in the animal. One of the more well-studiedtadpole tissues that undergoes remodeling is the small intestine.This tissue requires a shortening in length as well as internalanatomical restructuring to function in the adult frog. Briefly,the tadpole epithelial cells undergo programmed cell death (orapoptosis) and are replaced by a layer of newly formed adultepithelium. About 20 thyroid hormone-regulated genes participatingin this intestinal remodeling have been identified. These genescan be divided into several groups based on the proposed functionsof their products. One of these groups contains several secretedand/or signaling molecules. Most prominent among these are theXenopus homologs of the hedgehog and stromelysin-3 genes. Basedon the expression profiles and cellular localization, hedgehogappears to be involved in adult epithelial morphogenesis. Stromelysin-3may participate in basal lamina modification which is potentiallyinvolved in the apoptosis of the larval epithelium and developmentof the adult epithelium. Here we will review in detail the potentialroles for these secreted factors as well as the proposed molecularmechanisms responsible for their physiological functions. Furthermore,we will examine the effect of these proteins on the extracellularenvironment and how this impacts upon cellular processes involvedin intestinal remodeling.