Comparative ultrastructural investigations of the uterine epithelium in the viviparous Salamandra atra Laur. and the ovoviviparous Salamandra salamandra (L.) (Amphibia,Urodela)

The uterine epithelium of the viviparous Salamandra atra and the ovoviviparous Salamandra salamandra was studied in non pregnant and ovulating females and in females during different stages of pregnancy. The epithelium of both species is organized in a monolayer. The epithelial cells are characterized by a moderate secretory activity, a variable amount of apical granules which include PAS-positive material and by some apical and basal exo- or endocytotic vesicles. Adjacent cells are joined by junctional complexes. The lateral surfaces form a tortuous boundary with adjoining cells which suggest that the epithelium is involved in transport. Sporadic light cells possess highly variable cytoplasmic inclusions and are not joined with neighbouring cells. Possibly they represent migratory cells. The entire epithelium, except for a small cranial portion of the uterus in S. atra, undergoes no remarkable morphological changes during the different physiological stages examined except that flattened cells seem to be more numerous in pregnant females. The results are discussed with regard to the possible supply of the developing young by the mother.     

Intercellular junctions in the uterine epithelium of Salamandra salamandra (L.) (Amphibia,Urodela)

Summary Intercellular junctions in the uterine epithelium of the ovoviviparous urodele Salamandra salamandra were studied in pregnant and non-pregnant females by freeze-fracture technique. Junctional complexes consist of zonulae occludentes (tight junctions) and numerous maculae adhaerentes (desmosomes); z. adhaerentes and nexuses (gap junctions) could not be identified. Tight junctions are of the flexible type exhibiting loosely interconnected fibrils. The fibrillary network appears stretched more often in pregnant females possibly due to the mechanical stress of pregnancy. The structure and occurrence of the junctions identified, especially that of the tight junctions, is discussed with regard to the functions of the uterus during pregnancy.Zusammenfassung Mit Hilfe der Gefrierbruchtechnik wurden im Uterus-epithel trächtiger und nichtträchtiger Feuersalamanderweibchen (Salamandra salamandra) Zonulae occludentes und Maculae adhaerentes, jedoch keine Z. adhaerentes sowie Nexus identifiziert. Die Z. occludentes sind flexibel. Ihr fibrilläres Netzwerk ist bei trächtigen Weibchen öfter gestreckt; das ist möglicherweise auf die stärkere Ausdehnung des Uterusgewebes während der Trächtigkeit zurückzuführen. Das Vorkommen der verschiedenen Kontakt-strukturen, namentlich das der Z. occludentes, wird im Hinblick auf die Funktionen des Uterus während der Trächtigkeit diskutiert.We are indebted to Mrs. K. Ott for excellent technical assistance and to Miss Dr. U. Beigel for linguistic help     

The innervation of the uterus in Salamandra salamandra (L.) (Amphibia,Urodela)

The innervation of the uterus in the ovoviviparous urodele Salamandra salamandra was studied. In whole mount preparations of the thin-walled uterus of pregnant females, a dense adrenergic network was demonstrated using a modified glyoxylic acid fluorescence technique. Based on vesicle type and cytochemical reactivity after chromate/dichromate fixation for electron microscopy at least two types of neural process were distinguished and classified as adrenergic and cholinergic. Both types are preferentially situated above or between the smooth muscles of the uterine tissue. Adjacent to the muscles in the walls of arterioles mainly adrenergic fibers are seen. Using high performance liquid chromatography with electrochemical detection in tissue homogenates of uterus a considerable amount of noradrenaline could be identified. The significance of the dense innervation is discussed with respect to the function of the uterus during pregnancy and birth.     

Scanning electron microscopy of the oviduct of Salamandra salamandra (L.) (Amphibia, Urodela)

The oviduct of non-pregnant females of the ovoviviparous salamander, Salamandra salamandra, was examined using SEM-techniques. In the luminal epithelium polygonal ciliated cells were found along the entire surface of the oviduct, except the uterus, and non-ciliated cells with a varying number of short or long microvilli. The ciliated cells occur in the most anterior portion of the oviduct, the pars recta; they are sparsely distributed in the p. convoluta I, but abundant in the p. convoluta II and III. Non-ciliated cells comprise several small gland cells, restricted to the p. convoluta I, II, III, and undifferentiated cells both provided with microvilli, but difficult to be discerned from their surface appearance. The p. convoluta I, II, III is characterized by three types of secretory cells forming tubular glands, each type confined to a given zone. The secretory cells have slender microvilli at their surfaces. In freeze-cracked glands details of their secretory products can be visualized. The findings are compared to previously published TEM-investigations and discussed with regard to some functions of the oviduct during reproduction.     

The effects of amiloride on electrolyte transport and acid-base balance in the larval salamander,Ambystoma tigrinum

Summary The responses of net and unidirectional fluxes of Na⁺ and acid-base balance to the drug amiloride were assessed during normocapnia and hypercapnia in larval salamanders, Ambystoma tigrinum. Isotope flux measurements demonstrated that 10^-4 M amiloride in the external medium inhibits Na⁺ influx during normocapnia and reverses the usual increase in influx of this ion during hypercapnia, causing a significant decrease instead. Measurements of blood-gas/acid-base balance conditions of artcrially cannulated salamanders demonstrated a significant metabolic acidosis in amiloridetreated animals that did not occur in untreated animals over the same period. the same concentration of amiloride also blocked the normal compensatory increase in [HCO _- ³ ] that follows a respiratory acidosis produced by a hypercapnic environment.Abbreviations IU international nnits - J _in influx - J _net net flux - PCO ₂ parial pressure of carbon dioxide     

The distribution of monosaccharides and hexosamines in the oviduct of Salamandra salamandra (L.) (Amphibia,Urodela)

• 1.1. Biochemical analysis of the different segments of the oviduct in the ovoviviparous salamander, Salamandra salamandra, reveals the monosaccharides glucose, galactose, fucose, mannose, ribose and the hexosamines glucosamine and mannosamine.
• 2.2. In segment 1 (pars recta) ribose and mannose are absent, and in segments 2 (p. convoluta I) and 5 (p. convoluta IV, uterus) mannose is not detectable; fucose is absent in the uterus. Segments 3 (p. convoluta II) and 4 (p. convoluta II) contain all sugars identified.
• 3.3. The main hexoses present in the glandular segments are galactose, fucose and glucose.

     

cAMP and sodium transport in the freshwater crayfish, Cherax destructor

Crayfish in which sodium absorption was maximally stimulated had elevated levels of both cAMP and Na⁺-K⁺-ATPase activity in gill tissue. The concentration of cAMP and activity of Na⁺-K⁺-ATPase in gill tissue were monitored following transfer of crayfish from water containing 125 mmol.l⁻¹ Na to Na-free media. Both parameters were significantly elevated within 10 min of transfer to Na-free media and [cAMP] peaked between 1 and 2 h before falling transiently to the control level at 3 h. A second peak of [cAMP] and a further rise in Na⁺-K⁺-ATPase activity were evident 6 h after transfer and elevated levels were then maintained. The pattern observed was consistent with the existence of two separate mechanisms for the control of sodium absorption both of which stimulated the activity of Na⁺-K⁺-ATPase via elevation of the intracellular concentration of cAMP. The initial response was very rapid (<10 min) but of brief duration (1–2 h) and this mechanism appeared to be sensitive to changes in external ion levels. The second mechanism exhibited a much longer response time (3–6 h) and duration and was likely to be sensitive to changes in internal ion concentrations.     

Inhibitory effects of choleretic hydroxyacetophenones on ileal bile acid transport in rats

The effects of the choleretic and cholesterol lowering compound, 2,4,6-trihydroxyacetophenone (THA) and its analog, 2,6-dihydroxyacetophenone (DHA), on ileal bile acid absorption were investigated in rats. THA inhibited taurocholate (TC) uptake into ileal brush-border membrane vesicles (BBMV), showing a maximum inhibition of 50%, whereas DHA completely inhibited TC uptake into ileal BBMV. THA exhibited competitive inhibition with a Ki of 9.88 mM, while DHA showed non-competitive inhibition with a Ki of 7.65 mM. Both total and ouabain-sensitive basolateral membrane (BLM) Na+-K+-ATPase activities, which are essential for maintenance of the Na+-gradient for bile acid transport, were inhibited by THA and DHA in a dose-dependent manner. The inhibition of BLM ATPase was uncompetitive with a Ki of 10.1 and 5.0 mM for THA and DHA, respectively. Administration of THA or DHA (400 micromol/kg) twice a day, to hypercholesterolemic rats for 3 weeks caused similar and marked reductions in plasma cholesterol to 60% of the cholesterol-fed controls. The data suggest that the inhibitory actions of THA and DHA on two essential components of ileal bile acid recycling to liver could, in part, contribute to the cholesterol lowering effect of the hydroxyacetophenone compounds. These effects on decreasing bile acid recycling, in combination with their potent choleretic effect, accelerating biliary excretion of bile acids, are responsible for the effective cholesterol lowering capacities of these compounds.     

Evaporative water loss and uptake in juvenile and adult Salamandra salamandra (L.) (Amphibia: Urodela)

• 1.1. Both juveniles and adults of this rare salamander were studied.
• 2.2. The rate of evaporative water loss increased with temperature and at lower humidities.
• 3.3. At all four temperatures and three humidities studied, adults lost water at a lower rate than juveniles.
• 4.4. Aggregating juveniles reduced water loss especially at lower moisture.
• 5.5. The rate of water uptake was greater in juveniles than in adults.
• 6.6. Juveniles were capable of absorbing moisture from moist soil even at 40% saturated soil.

     

Further freeze-fracture studies on the uterine epithelium of Salamandra salamandra (L.) (Amphibia,Urodela) using the antibiotic filipin

Summary The apical portion of the uterine lining of the ovoviviparous fire salamander, Salamandra salamandra, was studied by the freeze-fracture technique in conjunction with the polyene antibiotic filipin. Filipin-sterol complexes were found in the luminal plasmalemma and in the membranes limiting the mucous secretory granules typical of this epithelium. In all females, but particularly in non-pregnant females, more or less discrete clusters of filipinsterol complexes were occasionally found overlying heavily affected secretory granules. The findings are discussed with regard to comparable results (Orci et al. 1980) based on the examination of collapsed and stretched urinary bladders of toads.We are indebted to Mrs. K. Ott for excellent technical assistance, to Miss E.S. MacLure for linguistic corrections and to Dr. J.E. Grady of the Upjohn Co., Kalamazoo, Michigan USA, for kindly providing the filipin     

Indications for a Phagocytic Capacity of Smooth Muscle Cells in the Testicular Wall of Salamandra salamandra (L.) (Amphibia,Urodela)

In smooth muscle cells forming parts of the testicular wall in the European fire salamander, Salamandra salamandra, phagosomes have been found which contain collagen fibres in different stages of decomposition. As a rule, these cells occur in the regressive glandular part of the testis.     

Trichromatic color vision in the salamander (Salamandra salamandra)

Spectral sensitivity functions were measured between 334 nm and 683 nm in Salamandra salamandra by utilizing two behavioral reactions: the negative phototactic response, and the prey catching behavior elicited by a moving worm dummy. The action spectrum of the negative phototactic response revealed 3 pronounced maxima: at 360–400 nm, at 520–540 nm, and at 600–640 nm. In the range around 450 nm, there was a reaction gap where sensitivity could not be measured. The action spectrum of the prey catching behavior was entirely different: maximal sensitivity was found at 500 nm and at 570 nm. Between 500 nm and 334 nm sensitivity decreased continuously for about 1 log unit (Fig. 6).Experiments under chromatic adaptation using the prey catching behavior indicate that the relatively high sensitivity in the ultraviolet range is not due to a separate ultraviolet photoreceptor, but is based on the responses of a photoreceptor maximally sensitive at about 500 nm.Color discrimination was tested by moving a colored worm dummy within a differently colored surround of equal subjective brightness. The salamanders were able to discriminate blue from green, and green from red (Fig. 10). The results can be explained by assuming a trichromatic color vision based on 3 photoreceptor types maximally sensitive around 450 nm, 500 nm and 570 nm (Fig. 12).     

Mechanisms of liquid flux across pulmonary alveolar epithelial cell monolayers

Summary Active transport of sodium by pulmonary alveolar epithelial cells (AEC) is believed to be an important component of edema clearance in the normal and injured lung. Data supporting this premise have come from measurements of sodium movement across AEC monolayers or from perfused lung model systems. However, direct measurement of fluid flux across AEC monolayers has not been reported. In the present work, AEC were studied with an experimental system for the measurement of fluid flux (Jv) across functionally intact cell monolayers. Primary adult rat type II alveolar epithelial cells were cultured on 0.8 μm nuleopore filters previously coated with gelatin and fibronectin. Intact monolayers were verified by high electrical resistance (> 1000 Θ) at 4–5 d of primary culture. At the same time interval, transmission electron microscopy revealed cells with type I cell-like morphology throughout the monolayer. These were characterized by both adherens and tight junctional attachments. Fluid flux across the monolayers was measured volumetrically over a period of 2 h in the presence of HEPES-buffered DMEM containing 3% fatty acid-free bovine serum albumin. Flux (Jv) was inhibited 39% by 1 × 10⁻⁴ M ouabain (P < 0.01) and 27% by 5 × 10⁻⁴ M amiloride (P < 0.05). These data support the concept that AEC Na⁺/K⁺-ATPase and Na⁺ transport systems are important determinants of AEC transepithelial fluid movement in vitro.     

Increases in transepithelial vectorial Na+ transport facilitates Na+-dependent L-DOPA transport in renal OK cells

The present study evaluated the hypothesis of whether increases in vectorial Na+ transport translate into facilitation of Na+-dependent L-DOPA uptake in cultured renal epithelial tubular cells. Increases in vectorial Na+ transport were obtained in opossum kidney (OK) cells engineered to overexpress Na+-K+-ATPase after transfection of wild type OK cells with the rodent Na+-K+-ATPase alpha1 subunit. The most impressive differences between wild type and transfected OK cells are that the latter overexpressed Na+-K+-ATPase accompanied by an increased activity of the transporter. Non-linear analysis of the saturation curve for l-DOPA uptake revealed a Vmax value (in nmol mg protein/6 min) of 62 and 80 in wild type and transfected cells, respectively. The uptake of a non-saturating concentration (0.25 microM) of [14C]-L-DOPA in OK-WT cells was not affected by Na+ removal, whereas in OK-alpha1 cells accumulation of [14C]-L-DOPA was clearly dependent on the presence of extracellular Na+. When Na+ was replaced by choline, the inhibitory profile of neutral l-amino acids, but not of basic and acidic amino acids, upon [14C]-L-DOPA uptake in both cell types, was significantly greater than that observed in the presence of extracellular Na+. It is concluded that enhanced ability of OK cells overexpressing Na+-K+-ATPase to translocate Na+ from the apical to the basal cell side correlates positively with their ability to accumulate L-DOPA, which is in agreement with the role of Na+ in taking up the precursor of renal dopamine.     

Ultrastructural and ultracytochemical studies of the gill epithelium in the larvae of Salamandra salamandra (Amphibia,Urodela)

Summary The morphology of Salamandra salamandra (Linné, 1758) larva gills is described by scanning and transmission electron microscopy. Three main cell types comprising the surface of the gill epithelium are described: (a) pavement cell, (b) ciliary cell and (c) mitochondria-rich cell (MRC). Two subtypes of MRC were distinguished by their ultrastructural characteristics: a fibrillar cell and a tubulovesicular cell. K^ü-dependent-nitrophenyl-phosphatase (K-NPPase) activity, indicative of Na^ü-K^ü-ATPase activity was confined to the basolateral membranes of the pavement cells. MRC were devoid of such activity. Possible relationships between structure and function of the different cell types are discussed.     

Ouabain resistance of the epithelial cell line (Ma104) is not due to lack of affinity of its pumps for the drug

Na⁺, K⁺-pumps of most eukaryotic animal cells bind ouabain with high affinity, stop pumping, and consequently loose K⁺, detach from each other and from the substrate, and die. Lack of affinity for the drug results in ouabain resistance. In this work, we report that Ma104 cells (epithelial from Rhesus monkey kidney) have a novel form of ouabain-resistance: they bind the drug with high affinity (Km about 4×10^–8 m), they loose their K⁺ and stop proliferating but, in spite of these, up to 100% of the cells remain attached in 1.0 m ouabain, and 53% in 1.0 mm. When 4 days later ouabain is removed from the culture medium, cells regain K⁺ and resume proliferation. Strophanthidin, a drug that attaches less firmly than ouabain, produces a similar phenomenon, but allows a considerably faster recovery. This reversal may be associated to the fact that, while in ouabain-sensitive MDCK cells Na⁺, K⁺-ATPases blocked by the drug are retrieved from the plasma membrane, those in Ma104 cells remain at the cell-cell border, as if they were cell-cell attaching molecules. Cycloheximide (10 g/ml) and chloroquine (10 m) impair this recovery, suggesting that it also depends on the synthesis and insertion of a crucial protein component, that may be different from the pump itself. Therefore ouabain resistance of Ma104 cells is not due to a lack of affinity for the drug, but to a failure of its Na⁺, K⁺-ATPases to detach from the plasma membrane in spite of being blocked by ouabain.We wish to thank Dr. E. Rodríguez-Boulán for the generous supply of Ma104 cells, as well as acknowledge the generous economic support of the National Research Council (CONACYT) of Mexico. Confocal experiments were performed in the Confocal Microscopy Unit of the Physiology Department, CINVESTAV.     

The death of ouabain-treated renal epithelial cells: evidence against anoikis occurrence

The mechanisms of cell death signaling triggered by cardiotonic steroids are poorly understood. Based on massive detachment of ouabain-treated Madin-Darby canine kidney (MDCK) cells, it may be proposed that the cytotoxic action of these compounds is mediated by anoikis, i.e. a particular mode of death occurring in cells lacking cell-to-extracellular matrix interactions. We tested this hypothesis. Six hour incubation of MDCK cells with ouabain, marinobufagenin or K⁺-free medium almost completely blocked Na⁺,K⁺-ATPase, increased Na_i⁺ content by ∼10-fold and suppressed cell attachment to regular-plastic-plates by up to 5-fold. In contrast, the death of attached cells was observed after 24-h incubation with ouabain but not in the presence of marinobufagenin or K⁺-free medium. Cells treated with ouabain and undergoing anoikis on ultra-low attachment plates exhibited different cell volume behaviour, i.e. swelling and shrinkage, respectively. The pan-caspase inhibitor z-VAD.fmk and the protein kinase C activator PMA rescued MDCK cells from anoikis but did not influence the survival of ouabain-treated cells, whereas medium acidification from pH 7.2 to 6.7 almost completely abolished the cytotoxic action of ouabain, but did not significantly affect anoikis. Our results show that the Na _i⁺,K_i⁺-independent mode of MDCK cell death evoked by ouabain is not mediated by anoikis.     

Effect of taurine on the isolated retinal pigment epithelium of the frog: Electrophysiologic evidence for stimulation of an apical,electrogenic Na+−K+ pump

Summary The apical surface of the retinal pigment epithelium (RPE) faces the neural retina whereas its basal surface faces the choroid. Taurine, which is necessary for normal vision, is released from the retina following light exposure and is actively transported from retina to choroid by the RPE. In these experiments, we have studied the effects of taurine on the electrical properties of the isolated RPE of the bullfrog, with a particular focus on the effects of taurine on the apical Na⁺–K⁺ pump.Acute exposure of the apical, but not basal, membrane of the RPE to taurine decreased the normally apical positive transepithelial potential (TEP). This TEP decrease was generated by a depolarization of the RPE apical membrane and did not occur when the apical bath contained sodium-free medium. With continued taurine exposure, the initial TEP decrease was sometimes followed by a recovery of the TEP toward baseline. This recovery was abolished by strophanthidin or ouabain, indicating involvement of the apical Na⁺–K⁺ pump.To further explore the effects of taurine on the Na⁺–K⁺ pump, barium was used to block apical K⁺ conductance and unmask a stimulation of the pump that is produced by increasing apical [K⁺] ₀ . Under these conditions, increasing [K⁺] ₀ hyperpolarized the apical membrane and increased TEP. Taurine reversibly doubled these responses, but did not change total epithelial resistance or the ratio of apical-to-basal membrane resistance, and ouabain abolished these responses.Collectively, these findings indicate the presence of an electrogenic Na⁺/taurine cotransport mechanism in the apical membrane of the bullfrog RPE. They also provide direct evidence that taurine produces a sodium-dependent increase in electrogenic pumping by the apical Na⁺–K⁺ pump.