[EFFECT OF DIFFERENT PROTEINS ON THE REABSORPTION OF YELLOW FLUORESCENT PROTEIN IN THE KIDNEY OF BROWN FROG RANA TEMPORARIA].

Protein reabsorption in the proximal tubules (PT) of the frog kidney was studied by the methods of immunohistochemistry, fluorescent and confocal microscopy. Yellow fluorescent protein (YFP) was introduced in combination with other proteins. Reabsorption of YFP introduced simultaneously with ly- sozyme or green fluorescent protein (GFP) did not differ from the result of YFP injection only. Previous lysozyme injection did not change YFP absorption in contrast to YFP uptake reduced after GFP pretreat- ment. Lysozyme loading for 4 days resulted in a significant reduction in YFP absorption. The results show that receptor-mediated endocytosis in the frog kidney depends on the molecular nature of absorbable ligands, conditions of their competitive absorption and lysosomal accumulation in epithelial PT cells.

[Reabsorption of yellow fluorescent protein in the Rana temporaria kidney by receptor-mediated endocytosis].

The absorption of yellow fluorescent protein (YFP) and the expression of the endocytic receptors, megalin and cubilin, were investigated in the renal proximal tubules (PT) in frogs Rana temporaria after parenteral YFP injections. The methods of confocal microscopy and immunohistochemistry were used. The dynamics of YFP absorption was analyzed 2 h after injection. The logarithmic time dependence of the accumulation of YFP-containing endocytic vesicles in PT cells and the completion of absorption process 90-120 min after injection were shown. Unlike substantial megalin and cubilin expression 15-30 min after YFP introduction, immunolabeled endocytic receptors were not detected in PT cells after 2 h. The re-injection of YFP led to the appearance of apical endocytic vesicles containing megalin or cubilin colocalized with YFP. At the same time, the decrease of YFP uptake associated with reduction in the number of receptor-containing vesicles was demonstrated, suggesting a failure of megalin and cubilin expression. The decrease of absorption capacity of PT cells after YFP re-injection was similar to that found previously under conditions of the competitive absorption of green fluorescent protein (GFP) and YFP injected in different sequences. The data are the further demonstration of the proposed mechanism limiting the tubular protein absorption in the frog kidney and suggest the involvement of megalin and cubilin in uptake and vesicular transport of YFP.

Absorption capacity of renal proximal tubular cells studied by combined injections of YFP and GFP in Rana temporaria L.

The capacity for protein reabsorption in the renal proximal tubule (PT) was studied in Rana temporaria frogs by separate, simultaneous and sequential introduction of yellow fluorescent protein (YFP) and green fluorescent protein (GFP). The uptake patterns of YFP and GFP in PT epithelial cells were investigated 15-120min after their bolus intravenous and intraperitoneal injection. As shown by confocal microscopy, the tubular uptake of YFP and GFP was time- and dose-dependent. These proteins are absorbed in similar way and can be accumulated in the same endocytic vesicles after their combined injections. When GFP was injected 30 and 90min before YFP, and vice versa, the number of vesicles with pre-injected protein increased and the percentage of vesicles with colocalized GFP and YFP reduced. At the same time, the uptake rate of a protein injected later progressively and significantly decreased. Subcellular localization of endocytic receptors, megalin and cubilin, in renal PT cells after intravenous YFP introduction were revealed by immunofluorescent microscopy. Colocalization of internalized YFP with megalin or cubilin in the endocytic vesicles was demonstrated. The data suggest the possibility of protein uptake by receptor-mediated endocytosis and the existence of a mechanism limiting the protein absorption rate in wintering frogs.

[Morpho-functional changes in small intestine epithelium of frog Rana temporaria during hibernation].

Structure and function of small intestinal epithelium were studied in overwintering frogs Rana temporaria at various stages of hibernation. In the process of testing of absorption of arginine vasotocin (AVT) in experiments in vitro it is established that at the period of hibernation there is preserved the capability of the epithelium for absorption of this nonapeptide without hydrolysis. However, as compared with October-December, in January-February and later, a decrease of the AVT absorption takes place, which is the most pronounced in March-April. Changes in epithelial structures appear by the middle of winter and are progressing by spring. In April-May, as compared with the beginning of hibernation, the height of enterocytes, the length of microvilli, and the number of microvilli decrease by 33 %, 40 %, and 57 %, respectively. The absence of features of destruction indicates an adaptive character of the observed changes. Dynamics of the studied parameters indicates morphological plasticity of the small intestine epithelium of R. temporaria at the period of hibernation.

[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.

Tubular GFP uptake pattern in the rat and frog kidneys.

The renal tubular uptake of green fluorescent protein (GFP) after its bolus intravenous injection was studied in both frogs and rats. GFP fluorescence in the proximal tubule (PT) was revealed by fluorescent and confocal microscopy. Granular GFP fluorescence was observed nearby in the apical membrane of PT cells featuring distribution over the cytoplasm. GFP was internalized into endosomes and lysosomes as determined by immunocytochemistry in frogs. The tubular uptake and accumulation of GFP were dose- and time-dependent in both rats and frogs. Intralymphatic sac injection of arginine vasotocin (AVT) decreased the uptake of GFP in hydrated frogs. A high negative correlation between the AVT dose and the uptake of GFP was revealed. The effect of AVT was inhibited by a V(1)-receptor antagonist. A noted decrease in the average number of fluorescent PT profiles per kidney section and their irregular distribution after AVT injections suggest that not all of the glomeruli or preglomerular vessels are equally responsive to AVT. GFP may serve as a good marker for tubular uptake and intracellular traffic in the amphibian kidney for use in in vivo studies.

[Renal filtration and reabsorption of GFP in frog Rana temporaria: 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 the proximal tubule cells 30 min after intravenous GFP injection. The GFP fluorescence was distributed predominantly in the apical part of the cytoplasm in the form of the 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 sac decreased significantly the GFP absorption. The effect of arginine vasotocin was inhibited by pretreatment a vasopressin V1-receptor antagonist. These results suggest that a decrease in the GFP absorption is due to a fall of the AVT-dependent glomerular filtration rate and consequently a decrease in the filtered GFP amount. The effect of arginine vasotocin on the GFP absorption seems to be mediated via the V1-like receptors of preglomerular blood vessels.

[Green fluorescent protein absorption and accumulation in the renal proximal tubule cells after its increased entry into blood].

The uptake of green fluorescent protein (GFP) by the proximal renal tubules was studied in the anaesthetized rats using laser confocal microscopy after GFP intravenous injection or administration into the small intestine lumen. The specific green fluorescence revealed in the proximal tubule cells after intravenous injection correlated with the logarithm of GFP dose injected intravenously (r = 0.96, p < 0.05). GFP fluorescence after its intravenous injection was higher than that one after GFP infusion into the small intestine (p < 0.05). Following the increase of injected GFP dose, the epitheliocyte cytoplasm, in addition to diffuse fluorescence, demonstrated large intensely fluorescent vesicles, that was confirmed by a graphical analysis. The reported changes in the intensity and pattern of specific fluorescence indicate the enhancement of GFP absorption by the cells of proximal tubules and GFP accumulation in the intracellular compartments during its increased entry into circulation.

Receptor-mediated endocytosis of lysozyme in renal proximal tubules of the frog Rana temporaria.

The mechanism of protein reabsorption in the kidney of lower vertebrates remains insufficiently investigated in spite of raising interest to the amphibian and fish kidneys as a useful model for physiological and pathophysiological examinations. In the present study, we examined the renal tubular uptake and the internalization rote of lysozyme after its intravenous injection in the wintering frog Rana temporaria using immunohisto- and immunocytochemistry and specific markers for some endocytic compartments. The distinct expression of megalin and cubilin in the proximal tubule cells of lysozyme-injected frogs was revealed whereas kidney tissue of control animals showed no positive immunoreactivity. Lysozyme was detected in the apical endocytic compartment of the tubular cells and colocalized with clathrin 10 min after injection. After 20 min, lysozyme was located in the subapical compartment negative to clathrin (endosomes), and intracellular trafficking of lysozyme was coincided with the distribution of megalin and cubilin. However, internalized protein was retained in the endosomes and did not reach lysosomes within 30 min after treatment that may indicate the inhibition of intracellular trafficking in hibernating frogs. For the first time, we provided the evidence that lysozyme is filtered through the glomeruli and absorbed by receptor-mediated clathrin-dependent endocytosis in the frog proximal tubule cells. Thus, the protein uptake in the amphibian mesonephros is mediated by megalin and cubilin that confirms a critical role of endocytic receptors in the renal reabsorption of proteins in amphibians as in mammals.

An electrophysiological analysis of the hippocampal projections to the neurosecretory cells of the supraoptic nucleus of the hypothalamus and the neurons of the perinuclear zone of rats.

Impulse activity of antidromically identified neurosecretory cells, orthodromically activated neurons, and neurons not responding to this stimulation was recorded in the supraoptic area of the hypothalamus under conditions of the stimulation of the hypophyseal stalk. The reactions of all groups of cells to the stimulation of the ventral hippocampus were investigated; the number of responding cells came to 14, 59, and 46%, respectively. Short-latency excitatory reactions predominated substantially. It is conjectured that the influences of the hippocampus are most significant for the neurons of the perinuclear zone as compared with the neurosecretory cells of the supraoptic nucleus. Particular aspects of the morphofunctional organization of the supraoptic area are discussed, taking into account both the convergence of afferent inputs from the neurosecretory cells and the hippocampus on the orthodromically activated neurons and the features of the organization of the hippocampal projections to various groups of cells of the perinuclear zone of the supraoptic nucleus.

Electrophysiological analysis of the morphofunctional organization of the limbic control of magnocellular neurosecretory nuclei in the rat hypothalamus.

The effects of electrical stimulation of the ventral hippocampus, ventral subiculum, and corticomedial amygdala were used to obtain a general and comparative assessment of the organization of efferent outputs of limbic system structures to the magnocellular neurosecretory nuclei of the hypothalamus, i.e., antidromally identified neurosecretory cells and other groups of identified neurons in these nuclei and in the perinuclear zones. These studies showed that different efferent outputs of the hippocampal formation provide differential control of spike activity of neurosecretory cells in the supraoptic nucleus, with excitatory pathways from the ventral hippocampus and inhibitory pathways from the subiculum. The effects of the amygdala on neurosecretory cells of the paraventricular nucleus were shown to be excitatory, though they were less significant than the excitatory and inhibitory effects of the hippocampus. It was demonstrated that in general, the effect of limbic structures are addressed predominantly to cells which do not project to the posterior lobe of the hypophysis. Projections were mostly to interneurons, which, as convergence sites for excitatory influences both from limbic structures and neurosecretory cells, may thus be responsible for the involvement of the latter in integrative brain functions.

Electrophysiological investigation of the hippocampal projections to the neurosecretory cells of the supraoptic nucleus of the rat hypothalamus.

The impulse activity of antidromically identified neurosecretory cells of the supraoptic nucleus of the hypothalamus of rats in response to stimulation of the ventral hippocampus was investigated. Short-latency phasic excitation reactions were identified, and inhibition reactions were not found. The presence of excitatory synaptic inputs from the hippocampus to other neurons of the nucleus and of the perinuclear zone, which are predominant by comparison with analogous projections to the neurosecretory cells, was demonstrated. The features of limbic-hypothalamic relationships are discussed in the context of afferent control of the activity of the neurosecretory cells.

[Morphological studies of the neurons of the area of the hypothalamic supraoptic nucleus in rats in diffusion chambers]. / Morfologicheskoe issledovanie neironov oblasti supraopticheskogo iadra gipotalamusa krys v diffuzionnykh kamerakh.

A method of diffusion chambers was used for hypothalamic tissue culturing. Chambers containing fragments from the supraoptic nucleus region of young rats-donors were implanted to rats-recipients. The processes of cell differentiation and interneuron interactions were studied by light and scanning electron microscopy. Morphological peculiarities of the main neuron types of the culture investigated suggest that the large differentiated multi- and bipolar neurons are the neurosecretory cells of the supraoptic nucleus. In the culture investigated small multipolar neurons of this nucleus and perinuclear areas were also present. The results give the possibility to consider the method of diffusion chambers as a promising one for the investigation of neurosecretory hypothalamic regions.

[Immunocytochemical localization of vasopressin at its absorption by cells of rat small intestine]. / Immunotsitokhimicheskaia lokalizatsiia vazopressina pri ego vsasyvanii kletkami épiteliia tonkoi kishki krysy.

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.

[An electrophysiological analysis of the hippocampal projections to the neurosecretory cells of the hypothalamic supraoptic nucleus and to the neurons of the perinuclear zone in rats]. / Elektrofiziologicheskii analiz proektsii gippokampa k neirosekretornym kletkam supraopticheskogo iadra gipotalamusa i neironam perinuklearnoi zony u krys.

The impulse activity of the antidromically identified neurosecretory cells, orthodromically activated and non-responded neurons was recorded in the rat hypothalamic supraoptic area under the hypo-physeal stalk stimulation. Stimulation of the ventral hippocampus induced responses in 14, 59 and 46% of the corresponding cells, resp. The short-latency excitatory reactions were predominant. The greater importance of the hippocampal projections to the neurons of the perinuclear zone as compared with the supraoptic neurosecretory cells was revealed. Some aspects of the morphofunctional organisation of the supraoptic area were analysed. The convergence of the afferent inputs to the orthodromically activated neurons from the neurosecretory cells and the hippocampus and the peculiar organisation of the hippocampal projections to different cell groups of the perinuclear zone, were taken into consideration.

[Electrophysiologic study of hippocampal projections to the neurosecretory cells of the supraoptic nucleus in rats]. / Elektrofiziologicheskoe issledovanie proektsii gippokampa k neirosekretornym kletkam supraopticheskogo iadra gipotalamusa krys.

Impulse activity of antidromically identified neurosecretory cells of the hypothalamic supraoptic nucleus, evoked by stimulation of the ventral hippocampus, was studied. Short-latency excitatory phasic reactions were revealed and no inhibitory responses were found. The existence of excitatory synaptic inputs was found from the hippocampus to other neurons of the supraoptic nucleus and perinuclear zone. These inputs were predominant as compared with analogous projections to neurosecretory cells. Peculiarities of limbic-hypothalamic relationships are discussed in connection with the afferent control of the neurosecretory cell activity.

[Relation between epiphyseal-limbic-hypothalamic interrelations and regulation of gonadotropin secretion and lactation]. / Epifizarno-limbiko-gipotalamicheskie vzaimootnosheniia v sviazi s reguliatsiei sekretsii gonadotropinov i laktatsiei.

Functional activity of hypothalamic structures was studied in endocrinological and electrophysiological experiments as well as in vitro studies. Functional changes of limbic-hypothalamic relations and control mechanisms of gonadotropin secretion under the effect of pineal polypeptides and in relation to lactation, were analysed.

[Mechanism f kidney participation in maintaining osmotic and ion homeostasis in chronic renal failure]. / Mekhanizm uchastiia pochki v obespechenii osmoticheskogo i ionnogo gomeostaza pri khronicheskoi pochechnoi nedostatochnosti.

In examination of patients with chronic renal failure (CRF) at glomerular filtration rate below 30 ml/min and blood serum ion concentration within limits of normal values hyperosmia has been found. Under the natural regimen essential differences have been revealed neither in variation limits of renal excretion of ions nor osmotically active substances in CRF patients as compared with healthy controls. Diuresis correlated with renal excretion of osmotically active substances. It is shown that a decrease in reabsorption of osmotically active substances depends on secretion and excretion of prostaglandin E2. A suggestion is made about the role of prostaglandins in regulation of renal tubular function at terminal CRF stages.