Salinity-dependent changes in Na+/K+-ATPase content of mitochondria-rich cells contribute to differences in thermal tolerance of Mozambique tilapia

The Mozambique tilapia (Oreochromis mossambicus) is prone to osmoregulatory disturbances when faced with fluctuating ambient temperatures. To investigate the underlying causes of this phenomenon, freshwater (FW)- and seawater (SW)-acclimated tilapia were transferred to 15, 25, or 35°C for 2 weeks, and along with typically used indicators of osmoregulatory status [plasma osmolality and branchial and intestinal specific Na⁺, K⁺-ATPase (NKA) activity], we used tissue microarrays (TMA) and laser-scanning cytometry (LSC) to characterize the effects of temperature acclimation. Tissue microarrays were stained with fluorescently labeled anti-Na⁺, K⁺-ATPase antibodies that allowed for the quantification of NKA abundance per unit area within individual branchial mitochondria-rich cells (MRCs) as well as sections of renal tissue. Mitochondria-rich cell counts and estimates of size were carried out for each treatment by the detection of DASPMI fluorescence. The combined analyses showed that SW fish have larger but fewer MRCs that contain more NKA per unit area. After a 2-week acclimation to 15°C tilapia experienced osmotic imbalances in both FW and SW that were likely due to low NKA activity. SW-acclimated fish compensated for the low activity by increasing MRC size and subsequently the concentration of NKA within MRCs. Although there were no signs of osmotic stress in FW-acclimated tilapia at 25°C, there was an increased NKA capacity that was most likely mediated by a higher MRC count. We conclude on the basis of the different responses to temperature acclimation that salinity-induced changes in the NKA concentration of MRCs alter thermal tolerance limits of tilapia.     

Responses of gill mitochondria-rich cells in Mozambique tilapia exposed to acidic environments (pH 4.0) in combination with different salinities

On exposure to hyposmotic acidic water, teleost fish suffer from decreases in blood osmolality and pH, and consequently activate osmoregulatory and acid-base regulatory mechanisms to restore disturbed ion and acid-base balances. In Mozambique tilapia Oreochromis mossambicus exposed to acidic (pH 4.0) or neutral (pH 7.4-7.7) freshwater in combination with 0mM or 50mM NaCl, we examined functional and morphological changes in gill mitochondria-rich (MR) cells. We assessed gene expression of Na(+)/H(+) exchanger-3 (NHE3), Na(+)/Cl(-) cotransporter (NCC), vacuolar-type H(+)-ATPase (V-ATPase) and Na(+)/HCO(3)(-) cotransporter-1 (NBC1) in the gills. The mRNA expression of NHE3 and NCC in tilapia gills were higher in acidic freshwater than in that supplemented with 50mM NaCl, while there was no significant difference in mRNA levels of V-ATPase and NBC1. In addition, immunocytochemical observations showed that apical-NHE3 MR cells were enlarged, and frequently formed multicellular complexes with developed deep apical openings in acidic freshwater with 0mM and 50mM NaCl. These findings suggest that gill MR cells respond to external salinity and pH treatments, by parallel manipulation of osmoregulatory and acid-base regulatory mechanisms.     

Differential responses in gills of euryhaline tilapia, Oreochromis mossambicus, to various hyperosmotic shocks

Euryhaline tilapia (Oreochromis mossambicus) survived in brackish water (BW; 20‰) but died in seawater (SW; 35‰) within 6 h when transferred directly from fresh water (FW). The purpose of this study was to clarify responses in gills of FW tilapia to various hyperosmotic shocks induced by BW or SW. In FW-acclimated tilapia, scanning electron micrographs of gills revealed three subtypes of MR cell apical surfaces: wavy-convex (subtype I), shallow-basin (subtype II), and deep-hole (subtype III). Density of apical surfaces of mitochondrion-rich (MR) cell in gills of the BW-transfer tilapia decreased significantly within 3 h post-transfer due to disappearance of subtype I cells, but increased from 48 h post-transfer because of increasing density of subtype III cells. SW-transfer individuals, however, showed decreased density of MR cell openings after 1 h post-transfer because subtype I MR cell disappeared. On the other hand, relative branchial Na⁺/K⁺-ATPase (NKA) α1-subunit mRNA levels, protein abundance, and NKA activity of the BW-transfer group increased significantly at 6, 12, and 12 h post-transfer, respectively. In the SW-transfer group, relative mRNA and protein abundance of gill NKA α1-subunit did not change while NKA activity declined before dying in 5 h. Upon SW transfer, dramatic increases (nearly 2-fold) of plasma osmolality, [Na⁺], and [Cl⁻] were found prior to death. For the BW-transfer group, plasma osmolality was eventually controlled by 96 h post-transfer by enhancement of NKA expression and subtype III MR cell. The success or failure of NKA activation from gene to functional protein as well as the development of specific SW subtype in gills were crucial for the survival of euryhaline tilapia to various hyperosmotic shocks.     

Effect of tissue specificity of brain soluble fractions on Na+, K+-ATPase activity

Previous evidence from this laboratory indicated that catecholamines and brain endogenous factors modulate Na⁺, K⁺-ATPase activity of the synaptosomal membranes. The filtration of a brain total soluble fraction through Sephadex G-50 permitted the separation of two fractions-peaks I and II-which stimulated and inhibited Na⁺, K⁺-ATPase, respectively (Rodríguez de Lores Arnaiz and Antonelli de Gomez de Lima, Neurochem. Res.11, 1986, 933). In order to study tissue specificity a rat kidney total soluble was fractionated in Sephadex G-50 and kidney peak I and II fractions were separated; as control, a total soluble fraction prepared from rat cerebral cortex was also processed. The UV absorbance profile of the kidney total soluble showed two zones and was similar to the profile of the brain total soluble. Synaptosomal membranes Na⁺, K⁺- and Mg²⁺-ATPases were stimulated 60–100% in the presence of kidney and cerebral cortex peak I; Na⁺, K⁺-ATPase was inhibited 35–65% by kidney peak II and 60–80% by brain peak II. Mg²⁺-ATPase activity was not modified by peak II fractions. ATPases activity of a kidney crude microsomal fraction was not modified by kidney peak I or brain peak II, and was slightly increased by kidney peak II or brain peak I. Kidney purified Na⁺, K⁺-ATPase was increased 16–20% by brain peak I and II fractions. These findings indicate that modulatory factors of ATPase activity are not exclusive to the brain. On the contrary, there might be tissue specificity with respect to the enzyme source.     

Characterization of Na+K+-ATPase in bovine sperm

Existing as a ubiquitous transmembrane protein, Na⁺K⁺-ATPase affects sperm fertility and capacitation through ion transport and a recently identified signaling function. Functional Na⁺K⁺-ATPase is a dimer of α and β subunits, each with isoforms (four and three, respectively). Since specific isoform pairings and locations may influence or indicate function, the objective of this study was to identify and localize subunits of Na⁺K⁺-ATPase in fresh bull sperm by immunoblotting and immunocytochemistry using antibodies against α1 and 3, and all β isoforms. Relative quantity of Na⁺K⁺-ATPase in head plasma membranes (HPM's) from sperm of different bulls was determined by densitometry of immunoblot bands, and compared to bovine kidney. Sperm and kidney specifically bound all antibodies at kDa equivalent to commercial controls, and to additional lower kDa bands in HPM. Immunofluorescence of intact sperm confirmed that all isoforms were present in the head region of sperm and that α3 was also uniformly distributed post-equatorially. Permeabilization exposing internal membranes typically resulted in an increase in fluorescence, indicating that some antibody binding sites were present on the inner surface of the HPM or the acrosomal membrane. Deglycosylation of β1 reduced the kDa of bands in sperm, rat brain and kidney, with the kDa of the deglycosylated bands differing among tissues. Two-dimensional blots of β1 revealed three distinct spots. Based on the unique quantity, location and structure Na⁺K⁺-ATPase subunits in sperm, we inferred that this protein has unique functions in sperm.     

Effect of water temperature,salinity, and their interaction on growth,plasma osmolality,and gill Na,K-ATPase activity in juvenile GIFT tilapia Oreochromis niloticus (L.)

We used a central composite rotatable experimental design and response surface methodology to evaluate the effects of temperature (18–37 °C), salinity (0–20‰), and their interaction on specific growth rate (SGR), feed efficiency (FE), plasma osmolality, and gill Na⁺, K⁺-ATPase activity in GIFT tilapia juveniles. The linear and quadratic effects of temperature and salinity on SGR, plasma osmolality, and gill Na⁺, K⁺-ATPase activity were statistically significant (P<0.05). The interactive effects of temperature and salinity on plasma osmolality were significant (P<0.05). In contrast, the interaction term was not significant for SGR, FE, and gill Na⁺, K⁺-ATPase activity ⁽P>0.05). The regression equations for SGR, FE, plasma osmolality, and gill Na⁺, K⁺-ATPase activity against the two factors of interest had coefficients of determination of 0.944, 0.984, 0.966, and 0.960, respectively (P<0.01). The optimal temperature/salinity combination was 28.9 °C/7.8‰ at which SGR (2.26% d¹) and FE (0.82) were highest. These values correspond to the optimal temperature/salinity combination (29.1 °C/7.5‰) and the lowest plasma osmolality (348.38 mOsmol kg⁻¹) and gill Na⁺, K⁺-ATPase activity (1.31 µmol Pi. h⁻¹ g⁻¹ protein), and resulted in an energy-saving effect on osmoregulation, which promoted growth.     

Juvenile hormone dependent phosphorylation of a 100 kDa polypeptide is mediated by protein kinase C in the follicle cells of Rhodnius prolixus

Summary

The addition of juvenile hormone I (JH I) to membrane preparations of the follicle cells from vitellogenic follicles of the insect Rhodnius prolixus causes a significant increase in the phosphorylation of a 100 kDa polypeptide; and ouabain, a specific inhibitor of Na⁺K⁺-ATPase, eliminates this effect. H-7 (1-(5-isoquinolinesulfonyl)-2-methylpiperazine), an inhibitor of protein kinase C (PKC), also eliminates the JH-dependent phosphorylation of this polypeptide. PDBU (phorbol-12, 13-dibutyrate), an activator of PKC, mimics the action of JH in increasing the phosphorylation of the 100 kDa polypeptide. Because these findings parallel the action of JH in causing the patency, the appearance of large spaces between the follicle cells through which vitellogenin gains access to the oocyte surface, they suggest that phosphorylation of one or more membrane proteins is a key event in the development of patency in response to JH. The 100 kDa polypeptide may represent the a-subunit of Na⁺K⁺-ATPase.     

Recognition of sodium- and potassium-dependent adenosine triphosphatase on mouse lymphoid cells by means of a monoclonal antibody

Summary Previous evidence has established the similarity between (Na⁺+K⁺)-ATPase (ATP phosphohydrolase, EC.3.6.1.3) and the antigen recognized by the rat antimouse monoclonal antibody anti-BSP-3. This antibody has been used for investigation of the surface expression and biochemical analysis of the enzyme in different mouse lymphoid populations. The BSP-3 determinant is found on almost all thymocytes and concanavalin A-induced thymocytes, to a lesser extent on bone marrow cells and also on a minor population of spleen cells. Spleen cells from athymic mice are negative. The (Na⁺+ K⁺)-ATPase purified from mouse thymus by affinity chromatography migrates in SDS-polyacrylamide gels in the form of two polypeptide chains of 105000 and 51000 daltons. Chains of the same molecular weight, fractionated on SDS-PAGE from microsomes of mouse thymuses, are shown to react with subunit-specific polyclonal antisera against ATPase in immunoblotting experiments. Immunoprecipitation with anti-BSP-3 from surface iodinated thymocytes yields only the small subunit. Comparison of the chains isolated from thymus and brain shows molecular weight differences in both subunits. These results, and variations in the reactivity pattern of the anti-BSP-3 antibody on several cell types, may indicate a possible heterogeneity of the (Na⁺+K⁺)ATPase expressed by various tissues and cells.     

Translocation of Na(+),K(+)-ATPase is induced by Rho small GTPase in renal epithelial cells

The distribution of transmembrane proteins is considered to be crucial for their activities because these proteins mediate the information coming from outside of cells. A small GTPase Rho participates in many cellular functions through its downstream effectors. In this study, we examined the effects of RhoA on the distribution of Na(+),K(+)-ATPase, one of the transmembrane proteins. In polarized renal epithelium, Na(+),K(+)-ATPase is known to be localized at the basolateral membrane. By microinjection of the constitutively active mutant of RhoA (RhoA(Val14)) into cultured renal epithelial cells, Na(+),K(+)-ATPase was translocated to the spike-like protrusions over the apical surfaces. Microinjection of the constitutively active mutant of other Rho family GTPases, Rac1 or Cdcd42, did not induce the translocation. The translocation induced by RhoA(Val14) was inhibited by treatment with Y-27632, a Rho-kinase specific inhibitor, or by coinjection of the dominant negative mutant of Rho-kinase. These results indicate that Rho and Rho-kinase are involved in the regulation of the localization of Na(+),K(+)-ATPase. We also found that Na(+),K(+)-ATPase seemed to be colocalized with ERM proteins phosphorylated at T567 (ezrin), T564 (radixin), and T558 (moesin) in cells microinjected with RhoA(Val14).     

Copper and the herbicide atrazine impair the stress response of the freshwater fish Prochilodus lineatus

In order to evaluate the effects of copper and atrazine on the stress response of the freshwater fish Prochilodus lineatus, juvenile fish were pre-exposed to copper (20 μg L(-1)) or atrazine (10 μg L(-1)) for 24 h and then submitted to air exposure for 3 min. Simultaneously fish kept in dechlorinated water for 24 h were subjected to air exposure and a non-stress group was not subjected to air stress or any contaminants. Animals were sampled immediately (t0) and after 1, 3 and 6 h of air exposure (t1, t3 and t6 respectively) for the analysis of plasma cortisol, glucose and Na(+), hepatic glycogen, branchial Na(+)/K(+)-ATPase (NKA), number of red blood cells per cubic millimeter of blood (RBC), hematocrit (Hct) and hemoglobin content (Hb). In fish pre-exposed to copper the stress response was inhibited, and at t1 and t3 both cortisol and glucose remained significantly lower compared to fish subjected to air stress only. In fish pre-exposed to atrazine there was no rise in cortisol, but there was an increase in plasma glucose, RBC, Hct and Hb at t0 and a return of these parameters to basal levels at t1, as they did not differ significantly in relation to non-stressed fish. Animals pre-exposed to either Cu or atrazine showed a significant reduction in NKA activity at t1 and t3, in relation to air stressed fish. These results clearly indicate that copper and atrazine impair cortisol stress response of P. lineatus and that fish subjected to a contaminant-induced stress, either by copper or atrazine, may not be able to respond to any additional stressors.     

Immunocytochemical characterization of two types of microvillar cells in rodent olfactory epithelium

Microvillar cells (MCs) have been identified in the olfactory epithelium of various mammalian species from rodents to humans. Studies on properties and functions of MCs to date have yielded partially controversial results, supporting alternatively an epithelial or a neuronal nature of these cells. In the present study, single and double immunolabeling investigations were carried out using antibodies against cytoskeletal and integral membrane proteins in order to further characterize MCs in rat and mouse olfactory epithelium. Application of antibodies against ankyrin (ANK), a protein that links integral membrane proteins to the submembrane cytoskeleton, led to intense labeling of the basolateral membranes of numerous cells with characteristic MC morphology. ANK-immunoreactive (ir) cells bore an apical tuft of -actin-ir microvilli, were filled with cytokeratin 18 (CK18)-ir filamentous network, and extended a basal process that appeared to end above the basal membrane. Immunoreactions for villin, an actin-crosslinking protein particularly prominently expressed in brush cells in the gastrointestinal and respiratory tract epithelia, and for the -subunit of sodium-potassium ATPase (Na⁺, K⁺-ATPase), revealed that ANK-ir MCs fall into two subpopulations. The less frequent type I MCs displayed villin immunoreactivity in their apical microvilli and underneath the basolateral membranes; the more numerous type II MCs were negative for villin but possessed intense basolateral immunoreactivity for Na⁺, K⁺-ATPase. Strong reactivity for the epithelial-type integral membrane protein of adherens junctions, E-Cadherin, was localized in basolateral membranes of both types of MCs. Our results support an epithelial nature of ANK-ir MCs in rat and mouse olfactory epithelium. Type I MCs strongly resemble brush cells in their immunocytochemical characteristics, namely, their ANK reactivity, CK18 reactivity, and villin reactivity. The intense Na⁺, K⁺-ATPase reactivity of type II MCs implicates these cells in transport processes.     

Na+,K+-ATPase in developing fetal guinea pig brain and the effect of maternal hypoxia

Na⁺,K⁺-ATPase activity was determined in fetal guinea pig brain at 35, 40, 45, 50, 55, and 60 days of gestation. The activity remained at a constant level during the early periods (35–45 days) of gestation and increased significantly during 45–60 days. Following maternal hypoxia, the activity of Na⁺,K⁺-ATPase in the term (60 days) fetal brain was reduced by 50% whereas the preterm (50 days) brain activity was unaffected. Under identical hypoxic conditions, the enzymatic activity of adult brain was significantly reduced by 20%. Na⁺,K⁺-ATPase obtained from fetal brain (50 days of gestation) has both a low and a high affinity for ATP (K _m values =0.50 and 0.053 mM and correspondingV _max values =10.77 and 2.82 umoles Pi/mg protein/hr), whereas the enzyme in the adult brain has only a low affinity (K _m=1.67 mM andV _max=20.32 umoles Pi/mg protein/hr). The high and low affinity sites for ATP in the fetal brain suggests a mechanism essential for the maintenance of cellular ionic gradients at low concentrations of ATP and which would provide the fetal brain with a greater tolerance to hypoxia. The high sensitivity of Na⁺,K⁺-ATPase activity to hypoxia in guinea pig brain at term suggests that the cell membrane functions of the fetal brain may be more susceptible to hypoxia at term than it is earlier in gestation.     

Phospholemman expression is high in the newborn rabbit heart and declines with postnatal maturation

Phospholemman (PLM) is a small sarcolemmal protein that modulates the activities of Na(+)/K(+)-ATPase and the Na(+)/Ca(2+) exchanger (NCX), thus contributing to the maintenance of intracellular Na(+) and Ca(2+) homeostasis. We characterized the expression and subcellular localization of PLM, NCX, and the Na(+)/K(+)-ATPase alpha1-subunit during perinatal development. Western blotting demonstrates that PLM (15kDa), NCX (120kDa), and Na(+)/K(+)-ATPase alpha-1 (approximately 100kDa) proteins are all more than 2-fold higher in ventricular membrane fractions from newborn rabbit hearts (1-4-day old) compared to adult hearts. Our immunocytochemistry data demonstrate that PLM, NCX, and Na(+)/K(+)-ATPase are all expressed at the sarcolemma of newborn ventricular myocytes. Taken together, our data indicate that PLM, NCX, and Na(+)/K(+)-ATPase alpha-1 proteins have similar developmental expression patterns in rabbit ventricular myocardium. Thus, PLM may have an important regulatory role in maintaining cardiac Na(+) and Ca(2+) homeostasis during perinatal maturation.     

Induction of cell division in BALB/c-3T3 cells by phorbol myristate acetate or bovine serum: Effects of inhibitors of cyclic AMP phosphodiesterase and Na+−K+-ATPase

Summary Cell division is induced in stationary cultures of BALB/c-3T3 mouse embryo cells without renewal of medium by addition of the tumor promoter, phorbol myristate acetate (PMA), or bovine serum. The addition of dbcAMP (10⁻³ m) or other inhibitors of cAMP phosphodiesterase, papaverine (6.7×10⁻⁶ m), Persantin (5×10⁻⁵ m) or RO-20-1724 (10⁻⁴ m), prevents cell replication induced by PMA or serum. In contrast, ouabain (10⁻⁴ m) and N,N′-dicyclohexylcarbodiimide (10⁻⁵ m), inhibitors of Na⁺−K⁺-ATPase activity, block the PMA-stimulated effect but do not inhibit serum-stimulated cell division. Several stages in the cell cycle are sensitive to dbcAMP addition. One is early in the G₁ phase at the time of reinitiation of the cell cycle from a stationary (G₀) phase, a second is associated with the G₁-S transition, and a third with passage of cells from a post-S phase to mitosis. Based on observations of early morphological changes, responses of plasma membrane ezymes and effects of enzyme inhibitors, the stimulation of cell division in BALB/c-3T3 cells by PMA or serum appears to involve several membrane functions which may act in a cooperative manner. This work was supported by a USPHS Research Grant CA12503, and a Center Grant ES-00260 awarded to the Institute of Environmental Medicine. Mrs. Susan Kulina provided the consistent and excellent technical aid necessary to perform this work. Note added in proof: During the preparation and review of this paper, Boynton reported that PMA appears to sensitize BALB/c-3T3 cells to calcium ion which may play a critical role in the regulation of the DNA synthesis (36).     

Effect of hyperbaric oxygenation on the Na+, K+-ATPase and membrane fluidity of cerebrocortical membranes after experimental subarachnoid hemorrhage

It is reported that CNS hemorrage causes membrane dysfunction and may exacerbate this damage as a result of secondary ischemia or hypoxia. Since hyperbaric oxygenation improves oxygen metabolism, it may reduce this membrane damage. The present study was conducted to reveal whether hyperbaric oxygenation influences membrane alteration after hemorrhage. Thirty minutes after subarachnoid hemorrhage induction, rats were treated with hyperbaric oxygenation 2 ATA for 1 hour. Rats were decapitated 2 hours after subarachnoid hemorrhage induction. Na⁺, K⁺-ATPase activity measurement, and spin-label studies were performed on crude synpatosomal membranes. Subarachnoid hemorrhage decreased Na⁺, K⁺-ATPase activity. Spin label studies showed that hydrophobic portions of near the membrane surface became more rigid and the mobility of the membrane protein labeled sulfhydryl groups decreased after subarachnoid hemorrhage. Hyperbaric oxygenation significantly ameliorated most of the subarachnoid hemorrhage induced alterations. We conclude that hyperbaric oxygenation may be a beneficial treatment for acute subarachnoid hemorrhage.     

Volume-dependent regulation of sodium and potassium fluxes in cultured vascular smooth muscle cells: Dependence on medium osmolality and regulation by signalling systems

Summary To identify ion transport systems involved in the maintenance of vascular smooth muscle cell volume the effects of incubation medium osmolality and ion transport inhibitors on the volume and ⁸⁶Rb and ²²Na transport in cultured smooth muscle cells from rat aorta (VSMC) have been studied. A decrease of medium osmolality from 605 to 180 mosm increased intracellular water volume from 0.6 to 1.3 l per 10⁶ cells. Under isosmotic conditions, cell volume was decreased by ouabain (by 10%, P< 0.005) but was not influenced by bumetanide, furosemide, EIPA and quinidine. These latter compounds were also ineffective in cell volume regulation under hypotonic buffer conditions. Under hyperosmotic conditions, cell volume was decreased by bumetanide (by 7%, P<0.05) and by ethylisopropyl amiloride (by 13%, P< 0.005). Ouabain-sensitive ⁸⁶Rb influx was decreased by 30–40% under hypoosmotic conditions. An increase in medium osmolality from 275 to 410 mosm resulted in an eightfold increase in bumetanide-inhibited ⁸⁶Rb influx and ⁸⁶Rb efflux. The (ouabain and bumetanide)-insensitive component of ⁸⁶Rb influx was not dependent on the osmolality of the incubation medium. However (ouabain and bumetanide)-insensitive ⁸⁶Rb efflux was increased by 1.5–2 fold in VSMC incubated in hypotonic medium. Ethylisopropyl amiloride-inhibited ²²Na influx was increased by sixfold following osmotic-shrinkage of VSMC. The data show that both Na⁺/H⁺ exchange and Na⁺/K⁺/2Cl^– cotransport may play a major role in the regulatory volume increase in VSMC. Basal and shrinkage-induced activities of Na⁺/K⁺/2Cl^– cotransport in VSMC were similarly sensitive to inhibition by either staurosporin, forskolin, R24571 or 2-nitro4-carboxyphenyl N,N-diphenylcarbomate (NCDC). In contrast basal and shrinkage-induced Na⁺/K⁺/2Cl^– cotransport were differentially inhibited by NaF (by 30 and 65%, respectively), suggesting an involvement of guanine nucleotide binding proteins in the volume-sensitive activity of this carrier. Neither staurosporin, forskolin, R24571 nor NCDC influenced shrinkage-induced Na⁺/H⁺ exchange activity. NaF increased Na⁺/H⁺ exchanger activity under both isosmotic and hyperosmotic conditions. These data demonstrate that different intracellular signalling mechanisms are involved in the volume-dependent activation of the Na⁺/K⁺/2Cl^– cotransporter and the Na⁺/H⁺ exchanger.The authors gratefully acknowledge the financial support of the Swiss National Foundation, grant No. 3.817.087. Bernadette Weber is thanked for preparing the figures.     

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.     

In search of synaptosomal Na+, K+-ATPase regulators

The arrival of the nerve impulse to the nerve endings leads to a series of events involving the entry of sodium and the exit of potassium. Restoration of ionic equilibria of sodium and potassium through the membrane is carried out by the sodium/potassium pump, that is the enzyme Na⁺,K⁺-ATPase. This is a particle-bound enzyme that concentrates in the nerve ending or synaptosomal membranes. The activity of Na⁺,K⁺-ATPase is essential for the maintenance of numerous reactions, as demonstrated in the isolated synaptosomes. This lends interest to the knowledge of the possible regulatory mechanisms of Na⁺,K⁺-ATPase activity in the synaptic region. The aim of this review is to summarize the results obtained in the author's laboratory, that refer to the effect of neurotransmitters and endogenous substances on Na⁺,K⁺-ATPase activity. Mention is also made of results in the field obtained in other laboratories. Evidence showing that brain Na⁺,K⁺-ATPase activity may be modified by certain neurotransmitters and insulin have been presented. The type of change produced by noradrenaline, dopamine, and serotonin on synaptosomal membrane Na⁺,K⁺-ATPase was found to depend on the presence or absence of a soluble brain fraction. The soluble brain fraction itself was able to stimulate or inhibit the enzyme, an effect that was dependent in turn on the time elapsed between preparation and use of the fraction. The filtration of soluble brain fraction through Sephadex G-50 allowed the separation of two active subfractions: peaks I and II. Peak I increased Na⁺,K⁺- and Mg²⁺-ATPases, and peak II inhibited Na⁺,K⁺-ATPase. Other membrane enzymes such as acetylcholinesterase and 5′-nucleotidase were unchanged by peaks I or II. In normotensive anesthetized rats, water and sodium excretion were not modified by peak I but were increased by peak II, thus resembling ouabain effects.³H-ouabain binding was unchanged by peak I but decreased by peak II in some areas of the CNS assayed by quantitative autoradiography and in synaptosomal membranes assayed by a filtration technique. The effects of peak I and II on Na⁺,K⁺-ATPase were reversed by catecholamines. The extent of Na⁺,K⁺-ATPase inhibition by peak II was dependent on K⁺ concentration, thus suggesting an interference with the K⁺ site of the enzyme. Peak II was able to induce the release of neurotransmitter stored in the synaptic vesicles in a way similar to ouabain. Taking into account that peak II inhibits only Na⁺,K⁺-ATPase, increases diuresis and natriuresis, blocks high affinity³H-ouabain binding, and induces neurotransmitter release, it is suggested that it contains an ouabain-like substance.     

Brain tryptophan uptake and sodium-potassium ATPase activity in long-term streptozotocin diabetic rats

Serum, liver and brain tryptophan concentrations and brain Na⁺K⁺-ATPase activity were studied in streptozotocin diabetic rats after an acute tryptophan load. Results show that tryptophan administration in the experimental diabetic group produces a generalized fall in tryptophan uptake in all the brain regions studied, though it does not increase serum and hepatic tryptophan concentrations. These parameters are normalized in insulin-treated diabetic rats. With regard to Na⁺K⁺-ATPase, diabetic animals showed a diminished and unchanged activity; whereas, the other two experimental groups showed a gradual decrease and a negative correlation with brain tryptophan uptake.     

Formation Mechanism of the Free Radical Product and Its Precursor by the Reaction of Dehydro-L-ascorbic Acid with Amino Acid

During the formation of radical A (2) and its precursor (tris(2-deoxy-2-L-ascorbyl)amine, 1) by the reaction of dehydroascorbic acid (DHA) with amino acid, ascorbic acid (AsA) and the reduced red pigment (3) were newly identified, in addition to scorbamic acid (SCA) and the red pigment (4), as intermediate products. The addition of AsA to the DHA-amino acid reaction, as well as to the DHA-SCA reaction, greatly increased the formation of 3 and 1. The reaction of AsA with 4 gave rapidly 3, followed by the gradual production of 1. From these results, a reaction pathway is proposed that 3 formed by the reduction of 4 with AsA is a key intermediate and its condensation with DHA followed by reduction with AsA might produce 2 and 1.