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.     

Influence of ethanol on glutathione level in the blood, liver and kidneys of Rana temporaria L. in the annual cycle

The effects of single doses (3 g/kg and 9 g/kg) of 35% ethanol, on the glutathione (GSH) contents of the blood, liver and kidneys of Rana temporaria L. were studied in the annual cycle. It was found that the single doses of ethanol generally caused a significant increase of GSH in the blood and liver of males and females of Rana temporaria L. in each period of the annual cycle as compared with the control values. In time, it was found that the same doses of ethanol caused a significant decrease in the GSH content of the kidneys of the male and female Rana temporaria L. during their active land life and a strong increase of this tripeptide during hibernation.     

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.     

Seasonal changes in the endocrine cells of the gastric epithelium of Rana temporaria

By means of the light and electron microscopy methods histotopography, quantity and ultramicroscopic organization of endocrine cells (EC) have been studied in gastric epithelium of the frog (Rana temporaria) during various periods of its life: hibernation, awakening, active life. At all the periods of life 8 types of EC are revealed. Depending on the season, certain changes both in their amount and ultrastructural organization are observed. Decreasing amount of EC is noted during hibernation and their increase in spring during awakening and in summer. Quantity of enterochromaffin cells is not subjected to seasonal fluctuations. This is explained by the fact that in hibernation these cells are also at their active functional state, since they produce serotonin necessary for maintaining deep hypothermia, specific for hibernating animals.     

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.     

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.     

蟾蜍冬眠期和排卵前后垂体中精氨酸催产素含量的变化

利用高效液相色谱结合荧光检测的技术,在pmol(10~(12)M)水平上,测定了不同时期单个蟾蜍脑垂体中精氨酸催产素(Arginine Vasotocin,AVT)的含量,发现排卵前期AVT含量最高,而排卵后则迅速下降;冬眠期AVT含量略高于排卵后期。AVT在蟾蜍动情、排卵的过程中可能起着重要的作用。     

Some pathomorphological bases for the disorders of the tissue amino acid pool in Salmonella infection]

Possible causes of disturbances of the amino acid pool of the blood serum and the tissue, as well as the presence of an association between these shifts and the changes in the ultrastructure of the striated columnar epithelium of the small intestine were studied. Investigations were conducted in 138 patients suffereing from salmonellosis and in 120 albino rats experimentally-infected with S. typhimurium culture. Free amino acids of the blood serum and the tissue were determined by paper chromatography and on the automatic amino acid analyzer; ultrastructure of the small intestinal mucosa was studied by electron microscopy. The height of salmonella infection was attended by dysproportional changes in the amino acid pool of the tissue and the blood caused by disturbance of absorption and intracellular transformation of amino acids. Ultrastructural changes in the microvilli of the absorbing cells of the small intestine underlied absorption disturbances: at the height of salmonella infection some of the microvilli of the striated columnar small intestine proved to show poor contours; reduction of mitochondrial cristae was observed in almost all the mitochondria.     

Changes in renal morphology and renin secretion in the golden-mantled ground squirrel (Spermophilus lateralis) during activity and hibernation

Summary Chronological changes in renal glomerular morphology and plasma renin activity were investigated during active and hibernating periods in the golden-mantled ground squirrel Spermophilus lateralis. The objective of this study was to determine whether the glomerular endothelium, visceral epithelium (podocytes), basement membrane, mesangial cells, proximal convoluted tubule cells and plasma renin activity exhibit measurable sequential differences between as well as within active and hibernating states at various time points. Limitations in the size of the experimental population prevented an evaluation of changes in these parameters during other important periods such as periodic arousal between hibernation bouts. In this study, glomerular endothelial pore number and epithelial filtration slit number significantly decreased by early hibernation when compared to those during summer activity, and then they increased back toward summer levels by late hibernation. In contrast, podocytic pedicel width along the glomerular basement membrane increased from summer activity to early hibernation, before significantly decreasing again by late hibernation. Mesangial cell and proximal convoluted tubule cell activity appeared increased during hibernation as compared to summer activity, whereas the width of the glomerular basement membrane showed no significant alterations throughout. Plasma renin activity significantly increased during early hibernation and mid-hibernation when compared to summer levels but had decreased by late hibernation toward summer values. The glomerular and plasma renin activity changes observed in this study clearly illustrate the drastic structural and functional adjustments which hibernating species make during torpor and also correlate well with the reported decrease in renal perfusion pressure and urine formation during hibernation. The observed morphological changes during hibernation do not appear to be temperature-dependent, because significant alterations in most of the parameters studied occurred during this period despite the fact that cold-room temperatures were kept constant throughout. The chronological approach to this study and its morphometric evaluation represent a pilot attempt at accurately documenting these changes during two critical states in the hibernator's cycle and may eventually lead to the characterization of these changes during the entire circannual cycle.     

Cosinor analysis of the seasonal changes in the cytoplasmic RNA level in the neurons of preoptic nucleus in Rana temporaria L. under the ultrasound energy

Authors studied the effects of single, total doses of ultrasound energy (frequency: 24.6 kHz; intensity: 0.35 W/cm2) on the cytoplasmic RNA level in the neurons of preoptic nucleus (PON) in Rana temporaria L., in the annual cycle. The cosinor analysis revealed that the applied ultrasound energy caused significant changes in the cytoplasmic RNA content in the PON neurons in Rana temporaria L. in the annual cycle. This was expressed by an increase in rhythm amplitude, except for the females of the second experimental group. Simultaneously, the rhythm acrophase in the animals exposed to ultrasound energy was shifted to later months (i.e. to the hibernation period), as compared to the control animals, where the acrophase occurred in August in females and in September in males (i.e. in their active land life period). In the females of the first experimental group the ultrasounds induced a disappearance of rhythmic changes in the cytoplasmic RNA of the PON neurons in the annual cycle.     

Opposite actions of different doses of arginine-vasotocin and 1-deamino-arginine-vasotocin on sodium ion transport in skin of the frog Rana temporaria

Active transport of sodium ions across the isolated abdominal skin of the frog Rana temporaria after application of arginine-vasotocin (AVT) and 1-deamino-arginine-vasotocin (1dAVT) was studied by measurement of the short-circuit current (SCC). The maximal increase in the SCC values (26 and 19 microA/cm2) was observed after addition of 10 nM AVT or 100 nM 1dAVT, respectively, to the frog skin basal surface. An increase of concentration of AVT to 100 nM and of IdAVT to 1 microM terminated the sodium transport in the frog skin. A preliminary addition of an antagonist of arginine-vasopressin V1a-receptors to the Ringer's solution at the frog skin basal surface led to a rise in the SCC values in response to administration of ineffective doses of AVT or 1dAVT. V2-receptor antagonists did not affect the frog skin reaction to administration of these doses of AVT or IdAVT.     

Gram-negative bacteria influence on the ability of the arginine-vasotocin to stimulate osmotic permeability of the frog urinary bladder epithelium

The influence of endogenous gram-negative bacteria colonizing the mucosal epithelium of frog Rana temporaria L. urinary bladders (FUB) on arginine-vasotocin AVT-stimulated osmotic water flow in isolated urinary bladders was investigated. 170 animals were examined and only 40% were contaminated with gram-negative bacteria (about 10(3)-10(6) CFU per hemibladder). Several Enterobacteriaceae species were identified (Hafnia alvei, 36.7%, E. coli, 32.3%, Serratia marcescens, 8.8%, Citrobacter freundii, 4.4% etc.). Basal osmotic water flow level was invariable in "clean" and contaminated FUB, whereas bacterial contamination resulted in considerable decrease in AVT-stimulated water flow ("clean": 2.53 +/- 0.13, n = 59, contaminated: 1.21 +/- 0.17 me/min/cm2, n = 38, p < 0.001, within first 15 min of incubation with 5 x 10(-10)M AVT). Gentamycin protection assay revealed predominantly adhesive forms of bacteria. Thus our data indicated that the presence of gram-negative bacteria colonizing the mucosal epithelium of the urinary bladder results in decreased adility of ADH to rise osmotic water permeability which in turn could impair body osmoregulation.     

A COMPARATIVE STUDY OF THE ULTRASTRUCTURE OF MICROVILLI IN THE EPITHELIUM OF SMALL AND LARGE INTESTINE OF MICE

A comparative analysis of the fine structure of the microvilli on jejunal and colonic epithelial cells of the mouse intestine has been made. The microvilli in these two locations demonstrate a remarkably similar fine structure with respect to the thickness of the plasma membrane, the extent of the filament-free zone, and the characteristics of the microfilaments situated within the microvillous core. Some of the core microfilaments appear to continue across the plasma membrane limiting the tip of the microvillus. The main difference between the microvilli of small intestine and colon is in the extent and organization of the surface coat. In the small intestine, in addition to the commonly observed thin surface "fuzz," occasional areas of the jejunal villus show a more conspicuous surface coat covering the tips of the microvilli. Evidence has been put forward which indicates that the surface coat is an integral part of the epithelial cells. In contrast to the jejunal epithelium, the colonic epithelium is endowed with a thicker surface coat. Variations in the organization of the surface coat at different levels of the colonic crypts have also been noted. The functional significance of these variations in the surface coat is discussed.     

Effects of microflora on the dimensions of enterocyte microvilli in the rat

The length and diameter of enterocyte microvilli at mid-villus position were measured on electron-micrographs. The duodenum, jejunum and ileum of axenic (germfree) and holoxenic (conventional) inbred rats fed the same diet have been studied. The microvilli were significantly shorter in all these intestinal regions when the microflora was present. The decrease in microvillus length (due to the presence of microflora), expressed as a percentage of the length in axenic rat, was 5% in the duodenum, 9% in the jejunum and 18% in the ileum. This was not true of microvillus diameter where only the values found for the ileum of axenic rat were significantly lower, the others not differing among themselves. In conclusion, the presence of a total microflora led to a decrease in the length of enterocyte microvilli. These results have been discussed in relation with studies on renewal and enzyme activity in the intestinal epithelium.     

Functional similarity of the P-2 form of primary sleep and hypobiosis in the frog Rana temporaria (based on data from luminescent microspectral analysis of the cytoplasm of neurosecretory neurons)

Using luminescent microspectral analysis of preparations stained by acridine orange, studies have been made on the ratio between single- and double-stranded parts in ribosomal RNA from the cytoplasm of neurosecretory neurones of the preoptic nucleus of the frog Rana temporaria. The animals were investigated in active period, during P-2 form of the primary sleep, in hypobiosis, and after injection of the active factor (peptide fraction with a molecular mass 1-10,000 Da) extracted from the small intestine of the ground squirrel Citellus undulatus during winter hibernation. It was shown that unlike actively awake frogs, animals from other experimental series exhibited similar changes, i.e. the decrease in the affinity of acridine orange to single- and double-stranded parts of rRNA and the decrease in the value of alpha which reflects the ratio of single- and double-stranded parts. It was also demonstrated that injection of the active factor from hibernating ground squirrels to frogs results in a condition which is rather similar to a natural resting form (P-2) of the primary sleep in cold-blooded vertebrates.     

Free amino acids in rat blood serum during A-hypovitaminosis]

A pool of free aminoacids of the blood serum has been studied in growing rats with a decreased retinol content in the liver tissue (to 5 g/g). The study shows a reliable drop in the concentrations of all essential aminoacids, except for phenylalanine. It is determined that the total amount of essential aminoacids decreases by 31%. At the same time concentrations of replaceable aminoacids increase, namely: glutamic acid and arginine--by 15.7%, ornithine and histidine--by 24%-45%, though proline concentration decreases abruptly by 31%. A disturbance in ultrastructural organization of microvilli in the apical membrane cells absorptive epithelium of the small intestine has been found. The results of the study confirm changes in a free aminoacid pool of the blood serum in growing rats and these changes occur first of all due to the disturbance of the absorption processes in the small intestine.     

Seasonal changes in morphology and function of the gastrointestinal tract of free-living alpine marmots (Marmota marmota)

The gastrointestinal tracts of 76 free-living alpine marmots ( Marmota marmota) shot during a population control program in Switzerland were collected and analysed for patterns of change in morphology and function over the period from emergence from hibernation in April to just before re-entry into hibernation in September. Between first emergence and mid-summer (July) the fresh tissue mass of the stomach increased by 105%, the small intestine by 259% (among the largest recorded for a mammal), caecum by 185%, proximal colon by 138%, and distal colon by 144%. Mitotic activity was greatest in the small intestine; the mitotic index was high (40%) compared with indexes in the stomach and hindgut (approximately 4%) even at emergence, and increased to approximately 60% by mid-summer. Microbial activity in the caecum was also significant at emergence. The stomach (length) and caecum (length and fresh mass) increased in response to ingested food earlier than did the small intestine. Between mid-summer and September there were decreases in small intestinal tissue mass and mitotic activity. It is concluded that the gastrointestinal tract of alpine marmots probably continues to function throughout hibernation at a low level, with a mid-winter trough as part of an endogenous circannual rhythm. However, after emergence in spring, increases in size and activity of the tract appear to be a response to ingested food rather than to an endogenous signal. The early signs of down-regulation of the small intestine before re-entry into hibernation, together with its delayed up-regulation in response to food in spring, are consistent with the high costs of maintaining this section of the digestive system.     

Immunochemical markers of embryonic lens differentiation in Rana temporaria. II. Immunohistochemical analysis of the manifestation and localization of individual classes of lens proteins]

Individual lens proteins were studied during development of Rana temporaria. Antisera to alpha-, beta-crystallins of chicks and gamma-crystallins of Rana ridibunda were used as immunochemical markers. Besides the main crystallins, a new antigen was found in the R. temporaria lens tentatively called alphabeta-crystallin. It appears to be characteristic only for the amphibian lens. Using the indirect method of fluorescent antibodies, it was shown that all the antigens under study appeared in the lens of the R. temporaria tadpoles within 1--2 days (at 20 degrees). The crystallins are found initially only in the developing lens fibers and later in the lens epithelium. It was established that the lens epithelium contained gamma-crystallins which appeared somewhat earlier than alpha- and beta-crystallins, but simultaneously with alphabeta-crystallin.     

New aspects in the study of the mechanism of intestinal absorption of thiamine in rats

Intestinal absorption of thiamine was studied in rats using such metabolic inhibitors as ouabain, sodium azide and theophylline. These substances changed certain biochemical parameters of the mucosa in the small intestine affecting the level of thiamine transport across the intestinal epithelium. It was found that the intestinal transport of thiamine at lower concentration was an active process depending on the activity of Na, K, Mg-ATPase in the intestinal microvilli, and on the activity of mitochondrial processes. The used metabolic inhibitors increased the intestinal diffusion of thiamine at its higher concentration suggesting that these inhibitors changed membrane permeability by affecting enterocyte homeostasis. At both studied concentrations thiamine was rapidly absorbed from the digestive tract reaching the state of saturation which suggested a carrier character of this transport.