Na+-K+-Cl− cotransport system in brain capillary endothelial cells: Response to endothelin and hypoxia

Effect of endothelin-1 and chemically induced hypoxia on Na⁺−K⁺−Cl⁻ cotransport activity in cultured rat brain capillary endothelial cells was examined by using⁸⁶Rb⁺ as a tracer for K⁺; bumetanide-sensitive K⁺ uptake was defined as Na⁺−K⁺−Cl⁻ cotransport activity. Endothelin-1, phorbol 12-myristate 13-acetate (PMA), or thapsigargin increased Na⁺−K⁺−Cl⁻ cotransport activity. A protein kinase C inhibitor, bisindolylmaleimide, inhibited PMA- and endothelin-1- (but not thapsigargin-) induced Na⁺−K⁺−Cl⁻ cotransport activity, indicating the presence of both protein kinase C-dependent regulatory mechanisms and protein kinase C-independent mechanisms which involve intracellular Ca²⁺. Oligomycin, sodium azide, or antimycin A increased Na⁺−K⁺−Cl⁻ cotransport activity by 80–200%. Oligomycin-induced Na⁺−K⁺−Cl⁻ cotransport activity was reduced by an intracellular Ca²⁺ chelator (BAPTA/AM) but not affected by bisindolylmaleimide, suggesting the involvement of intracellular Ca²⁺, and not protein kinase C, in hypoxia-induced Na⁺−K⁺−Cl⁻ cotransport activity. Portions were presented at “27th Annual Meeting, The American Society for Neurochemistry” Philadelphia, Pennsylvania, March 2–6, 1996.     

Characterization of a murine thymic CD4+ T cell subset-TCRαβ+ 3G11-6C10- CD4+ CD8-thymocytes

The presence of a relatively mature CD4⁺ CD8⁻ (SP) T cell subset in mouse thymus has been demonstrated. Composing of 10% of total CD4SP thymocytes, this subset is defined by the absence of 3G11 and 6C10 expression with a phenotype of CD69^+/−, HSA^med/lo and heterogeneous for Qa-2 expression. The proliferation capability of TCRαβ⁺ 3Gl l⁻ 6C10⁻ CD4⁺ CD8⁻ thymocytes was high while using Con A stimulus. And Con A stimulation could result in secretion of IL4, IL-10, IL-6 and a little amount of IFNγ. IL-2 was barely detectable. This is distinct from typical Th0 type cytokines. The cells of this subset were NK1.1 negative, but strongly expressed GATA-3 mRNA. The results suggest that the CD4⁺ subset of 3G11⁻ 6C10⁻ NK1.1⁻ phenotype possesses immunocompetent cells with functions characteristic of Th2-like cytokines, which may indicate the cells at transitional status from Th0 to Th2, with a propensity to Th2. Project supported by the National Natural Science Foundation of China (Grant No. 39730410).     

Gustatory papillae and taste bud development and maintenance in the absence of TrkB ligands BDNF and NT-4

Taste buds and the peripheral nerves innervating them are two important components of the peripheral gustatory system. They require appropriate connections for the taste system to function. Neurotrophic factors play crucial roles in the innervation of peripheral sensory organs and tissues. Both brain-derived neurotrophic factor (BDNF) null-mutated and neurotrophin-4 (NT-4) null-mutated mice exhibit peripheral gustatory deficits. BDNF and NT-4 bind to a common high affinity tyrosine kinase receptor, TrkB (NTRK-2), and a common p75 neurotrophin receptor (NGFR). We are currently using a transgenic mouse model to study peripheral taste system development and innervation in the absence of both TrkB ligands. We show that taste cell progenitors express taste cell markers during early stages of taste bud development in both BDNF^−/−xNT-4^−/− and wild-type mice. At early embryonic stages, taste bud progenitors express Troma-1, Shh, and Sox2 in all mice. At later stages, lack of innervation becomes a prominent feature in BDNF^−/−xNT-4^−/− mice leading to a decreasing number of fungiform papillae and morphologically degenerating taste cells. A total loss of vallate taste cells also occurs in postnatal transgenic mice. Our data indicate an initial independence but a later permissive and essential role for innervation in taste bud development and maintenance. This work was supported by NIH-NIDCD R01-RDC007628.     

The Role of H<Superscript>+</Superscript>/OH<Superscript>−</Superscript> Channels in the Salt Stress Response of <Emphasis Type="Italic">Chara australis</Emphasis>

We investigate the electrophysiological salt stress response of the salt-sensitive charophyte Chara australis as a function of time in saline artificial pond water (saline APW) containing 50 mM NaCl and 0.1 mM CaCl₂. The effects are due to an increase in Na⁺ concentration rather than an increase in Cl⁻ concentration or medium osmolarity. A previous paper (Shepherd et al. Plant Cell Environ 31:1575–1591, 2008) described the rise in the background conductance and inhibition of proton pumping in saline APW in the first 60 min. Here we investigate the shift of membrane potential difference (PD) to levels above −100 mV and the change of shape of the current–voltage (I/V) profiles to upwardly concave. Arguing from thermodynamics, the I/V characteristics can be modeled by channels that conduct H⁺ or OH⁻. OH⁻ was chosen, as H⁺ required an unrealistic increase in the number/permeability of the channels at higher pH levels. Prolonged exposure to saline APW stimulated opening of more OH⁻ channels. Recovery was still possible even at a PD near −50 mV, with partial return of proton pumping and a decrease in OH⁻ current following APW wash. Upon change of pH from 7 to 9, the response was consistent with previously observed I/V characteristics of OH⁻ channels. For a pH change to 6, the response was transient before channel closure but could still be modeled. The consequences of opening of H⁺ or OH⁻ channels while the cell is under salt stress are discussed.     

The Renal Cortical Na+/HCO3 − Cotransporter VI: The Effect of Chemical Modification in Cotransporter Activity

The Na⁺/HCO₃ ⁻ cotransporter is the main system that mediates bicarbonate removal out of the proximal tubule cell into the blood. We have previously partially purified this protein and showed that chemical modification of the α-amino groups by fluorescein isothiocyanate (FITC) inhibited the activity of the Na⁺/HCO₃ ⁻ cotransporter. The inhibition was prevented by the presence of Na and bicarbonate suggesting that this compound binds at or near the substrate transport sites of the cotransporter. We examined the effect of agents that modify the sulfhydryl group (dithiothreitol), carboxyl groups (n-n′dicyclohexyl carbodiimide) and tyrosine residues (p-nitrobenzene sulfonyl fluoride, n-acetyl imidazole and tetranitromethane) on the activity of the cotransporter to gain insight into the chemical residues which may be important for transport function. The sulfhydryl residues modifier, carboxyl group modifier, and tyrosine modifier significantly inhibited bicarbonate dependent ²²Na uptake in basolateral membranes by 50–70% without altering the ²²Na uptake in the presence of gluconate indicating that these agents directly affected the cotransporter without affecting diffusive sodium uptake. The effect of the tyrosine modifier n-acetylimidazole was not prevented by the presence of Na and bicarbonate suggesting that the tyrosine residues are not at the substrate binding sites. To determine the presence and role of glycosylation on the Na⁺/HCO₃ ⁻ cotransporter protein, we examined the effects of different glycosidases (endoglycosidase F and H, N-glycosidase F, O-glycanase) on the cotransporter activity. All glycosidases caused a significant 50–80% inhibition of cotransporter activity. These data demonstrate that N-glycosylation as well as O-glycosylation are important for the function of the Na⁺/HCO₃ ⁻ cotransporter protein. Taken together, these results suggest that chemical modifiers of tyrosine, carboxyl and sulfhydryl groups as well as glycosylation are important for expression of full functional activity of the cotransporter. Received: 8 October 1996/Revised: 23 January 1997     

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).     

Slc26a9—Anion Exchanger,Channel and Na<Superscript>+</Superscript> Transporter

The SLC26 gene family encodes anion transporters with diverse functional attributes: (a) anion exchanger, (b) anion sensor, and (c) anion conductance (likely channel). We have cloned and studied Slc26a9, a paralogue expressed mostly in lung and stomach. Immunohistochemistry shows that Slc26a9 is present at apical and intracellular membranes of lung and stomach epithelia. Using expression in Xenopus laevis oocytes and ion-sensitive microelectrodes, we discovered that Slc26a9 has a novel function not found in any other Slc26 proteins: cation coupling. Intracellular pH and voltage measurements show that Slc26a9 is a nCl⁻-HCO₃⁻ exchanger, suggesting roles in gastric HCl secretion or pulmonary HCO₃⁻ secretion; Na⁺ electrodes and uptakes reveal that Slc26a9 has a cation dependence. Single-channel measurements indicate that Slc26a9 displays discrete open and closed states. These experiments show that Slc26a9 has three discrete physiological modes: nCl⁻-HCO₃⁻ exchanger, Cl⁻ channel, and Na⁺-anion cotransporter. Thus, the Slc26a9 transporter channel is uniquely suited for dynamic and tissue-specific physiology or regulation in epithelial tissues. Min-Hwang Chang, Consuelo Plata, and Kambiz Zandi-Nejad have contributed equally to this work.     

Effects of NO2? and NO3? on the Fe(III)EDTA reduction in a chemical absorption–biological reduction integrated NOx removal system

The biological reduction of Fe(III) ethylenediaminetetraacetic acid (EDTA) is a key step for NO removal in a chemical absorption–biological reduction integrated process. Since typical flue gas contain oxygen, NO₂ ⁻ and NO₃ ⁻ would be present in the absorption solution after NO absorption. In this paper, the interaction of NO₂ ⁻, NO₃ ⁻, and Fe(III)EDTA reduction was investigated. The experimental results indicate that the Fe(III)EDTA reduction rate decrease with the increase of NO₂ ⁻ or NO₃ ⁻ addition. In the presence of 10 mM NO₂ ⁻ or NO₃ ⁻, the average reduction rate of Fe(III)EDTA during the first 6-h reaction was 0.076 and 0.17 mM h⁻¹, respectively, compared with 1.07 mM h⁻¹ in the absence of NO₂ ⁻ and NO₃ ⁻. Fe(III)EDTA and either NO₂ ⁻ or NO₃ ⁻ reduction occurred simultaneously. Interestingly, the reduction rate of NO₂ ⁻ or NO₃ ⁻ was enhanced in presence of Fe(III)EDTA. The inhibition patterns observed during the effect of NO₂ ⁻ and NO₃ ⁻ on the Fe(III)EDTA reduction experiments suggest that Escherichia coli can utilize NO₂ ⁻, NO₃ ⁻, and Fe(III)EDTA as terminal electron acceptors.     

Effect of salinity and its composition on the accumulation of selenium by alfalfa

Alfalfa (Medicago sativa L.) was grown in greenhouse sand culture to examine the effect of salinity composition and concentration on Se accumulation by plants. In a 2×2×4 factorial experiment, salinity was added as either C1⁻ or SO ₄ ²⁻ salts to the irrigating solution to achieve an electrical conductivity of 0.5, 1.5–3.0, or 6.0 dS m⁻¹. Selenium was added to the nutrient solution at a concentration of 0.25 or 1.0 mg Se(VI)I⁻¹. Following the third cutting, the roots were washed and all plant material analyzed for dry weight and Se. Plant biomass production decreased with additions of either Se or salinity, regardless of composition. In the presence of Se, the yield reduction was greater with Cl⁻ salinity than with SO ₄ ²⁻ salinity. Plant Se accumulation was reduced from 948 mg Se kg⁻¹ to 6 mg Se kg⁻¹ in the presence of SO ₄ ²⁻ salts (0.5 mmol SO ₄ ²⁻ l⁻¹ vs. 40 mmol SO ₄ ²⁻ l⁻¹) due to an apparent Se(VI) −SO ₄ ²⁻ antagonism. This Se−SO ₄ ²⁻ antagonism prevented accumulation of Se and reduced Se-induced toxicity. A lesser antagonistic effect on Se accumulation was observed between Cl⁻, and Se. A synergistic interaction between SO ₄ ²⁻ and Se(VI) increased plant S concentrations in the presence of the relatively low basal SO ₄ ²⁻ concentrations but not at the higher solution SO ₄ ²⁻ concentrations. In many areas, soil and water containing high Se concentrations also contain large amounts of SO ₄ ²⁻ . The occurrence of SO ₄ ²⁻ with Se reduces plant accumulation of Se(VI) and may lower the risk of Se overexposure to animals feeding on forage material grown in high Se−SO ₄ ²⁻ regions.     

Roles of Volume-sensitive Cl<Superscript>−</Superscript> Channel in Cisplatin-induced Apoptosis in Human Epidermoid Cancer Cells

The anti-cancer drug cisplatin induces apoptosis by damaging DNA. Since a stilbene-derivative blocker of Cl⁻/HCO₃⁻ exchangers and Cl⁻ channels, SITS, is known to induce cisplatin resistance in a manner independent of intracellular pH and extracellular HCO₃⁻, we investigated the relation between cisplatin-induced apoptosis and Cl⁻ channel activity in human adenocarcinoma KB cells. A stilbene derivative, DIDS, reduced cisplatin-induced caspase-3 activation and cell death, which were detected over 18 h after treatment with cisplatin. DIDS was also found to reduce sensitivity of KB cells to 5-day exposure to cisplatin. Whole-cell patch-clamp recordings showed that KB cells functionally express volume-sensitive outwardly rectifying (VSOR) Cl⁻ channels which are activated by osmotic cell swelling and sensitive to DIDS. Pretreatment of the cells with cisplatin for 12 h augmented the magnitude of VSOR Cl⁻ current. Thus, it is concluded that cisplatin-induced cytotoxicity in KB cells is associated with augmented activity of a DIDS-sensitive VSOR Cl⁻ channel and that blockade of this channel is, at least in part, responsible for cisplatin resistance induced by a stilbene derivative.     

Optimizing environmental factors for large-scale multiplication of chrysanthemum (<Emphasis Type="Italic">Chrysanthemum grandiflorum</Emphasis>) in balloon-type bioreactor culture

Summary We have developed optimum culture conditions for the large-scale propagation of chrysanthemum in balloon-type bioreactors to achieve vigorous growth and quality. The effects of NH ₄ ⁺ /NO ₃ ⁻ ratio, air volume, air temperature, photosynthetic photo flux, and an inoculation density on the growth and quality of plantlets were investigated. The best production conditions were an NH ₄ ⁺ :NO ₃ ⁻ ratio of 20∶40 mM, air exchange of 0.1 vvm min⁻¹, air temperature 25°C, photosynthetic photo flux (PPF) at 100 μmol m⁻² s⁻¹, and an inoculation density of 40 nodes Chrysanthemum grandiflorum. Under each of these conditions, the maximum growth rate reached 279.0, 260,0, 20.0, 23.3, and 94.5 (g-fresh weight per plantlet d⁻¹), respectively, at 12 wk of culture. These results specify the key environmental factors that can be regulated to improve the quality and quantity of flowers and increase yield in large-scale bioreactor cultures of chrysanthemum.     

Regulation of Basolateral Cl− Channels in Airway Epithelial Cells: The Role of Nitric Oxide

The presence of basolateral Cl⁻ channels in airway epithelium has been reported in several studies, but little is known about their role in the regulation of anion secretion. The purpose of this study was to characterize regulation of these channels by nitric oxide (NO) in Calu-3 cells. Transepithelial measurements revealed that NO donors activated a basolateral Cl⁻ conductance sensitive to 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) and anthracene-9-carboxylic acid. Apical membrane permeabilization studies confirmed the basolateral localization of NO-activated Cl⁻ channels. Experiments using 8-bromo cyclic guanosine monophosphate (8Br-cGMP) and selective inhibitors of soluble guanylyl cyclase and inducible NO synthase (1H-[1, 2, 4] oxadiazolol-[4, 3-a] quinoxalin-1-one [ODQ] and 1400W [N-(3-Aminomethyl)benzyl)acetamidine], respectively) demonstrated that NO activated Cl⁻ channels via a cGMP-dependent pathway. Anion replacement and ³⁶Cl⁻ flux studies showed that NO affected both Cl⁻ and HCO ₃ ⁻ secretion. Two different types of Cl⁻ channels are known to be present in the basolateral membrane of epithelial cells: Zn²⁺-sensitive ClC-2 and DIDS-sensitive bestrophin channels. S-Nitrosoglutathione (GSNO) activated Cl⁻ conductance in the presence of Zn²⁺ ions, indicating that ClC-2 channel function was not affected by GSNO. In contrast, DIDS completely inhibited GSNO-activated Cl⁻ conductance. Bestrophin immunoprecipitation studies showed that under control conditions bestrophin channels were not phosphorylated but became phosphorylated after GSNO treatment. The presence of bestrophin in airway epithelia was confirmed using immunohistochemistry. We conclude that basolateral Cl⁻ channels play a major role in the NO-dependent regulation of anion secretion in Calu-3 cells.     

Chronic Atmospheric NO<Stack><Subscript>3</Subscript><Superscript>−</Superscript></Stack> Deposition Does Not Induce NO<Stack><Subscript>3</Subscript><Superscript>−</Superscript></Stack> Use by <Emphasis Type="Italic">Acer saccharum</Emphasis> Marsh.

The ability of an ecosystem to retain anthropogenic nitrogen (N) deposition is dependent upon plant and soil sinks for N, the strengths of which may be altered by chronic atmospheric N deposition. Sugar maple (Acer saccharum Marsh.), the dominant overstory tree in northern hardwood forests of the Lake States region, has a limited capacity to take up and assimilate NO₃⁻. However, it is uncertain whether long-term exposure to NO₃⁻ deposition might induce NO₃⁻ uptake by this ecologically important overstory tree. Here, we investigate whether 10 years of experimental NO₃⁻ deposition (30 kg N ha⁻¹ y⁻¹) could induce NO₃⁻ uptake and assimilation in overstory sugar maple (approximately 90 years old), which would enable this species to function as a direct sink for atmospheric NO₃⁻ deposition. Kinetic parameters for NH₄⁺ and NO₃⁻ uptake in fine roots, as well as leaf and root NO₃⁻ reductase activity, were measured under conditions of ambient and experimental NO₃⁻ deposition in four sugar maple-dominated stands spanning the geographic distribution of northern hardwood forests in the Upper Lake States. Chronic NO₃⁻ deposition did not alter the V _max or K _m for NO₃⁻ and NH₄⁺ uptake nor did it influence NO₃⁻ reductase activity in leaves and fine roots. Moreover, the mean V _max for NH₄⁺ uptake (5.15 μmol ¹⁵N g⁻¹ h⁻¹) was eight times greater than the V _max for NO₃⁻ uptake (0.63 μmol ¹⁵N g⁻¹ h⁻¹), indicating a much greater physiological capacity for NH₄⁺ uptake in this species. Additionally, NO₃⁻ reductase activity was lower than most values for woody plants previously reported in the literature, further indicating a low physiological potential for NO₃⁻ assimilation in sugar maple. Our results demonstrate that chronic NO₃⁻ deposition has not induced the physiological capacity for NO₃⁻ uptake and assimilation by sugar maple, making this dominant species an unlikely direct sink for anthropogenic NO₃⁻ deposition.     

Isolation and cultivation of murine hematopoietic stem cells and expression of hFIX mediated by recombinant lentiviral vectors in vitro

Hematopoietic stem cells (HSCs) are an attractive target for gene therapy, especially for inherited blood diseases. Moreover, recombinant lentiviral vectors are considered to be prospective in HSCs gene therapy for the high efficiency of infection. In this study, murine mononuclear cells (MNCs) were isolated from bone marrow and cultured in suspension, and then Lin⁻CD117⁺ HSCs were isolated by immunomagnetic beads. During culturing, cells and colonies increased in HSCs supplied with cytokines while no change was observed in the control group without cytokines. FUXW recombinant lentiviral vectors were produced by calcium phosphate-mediated transient cotransfection infected MNCs from ICR and C57 mice. The hFIX expressions were 41.7 ± 4.2 ng / mL and 34.5 ± 6.6 ng/mL in supernatant on 7d. The hFIX expressions of HSCs infected by FUXW recombinant lentiviral vectors were 46.6 ± 5.7 ng/mL (with cytokines) and 33.3 ± 4.8 ng/mL (without cytokines) in supernatant on 7d. Results indicate that recombinant lentiviral vectors can infect murine MNCs and Lin⁻CD117⁺ HSCs efficiently, and expression of the transgene can be improved when supplied with cytokines. __________ Translated from Journal of Fudan University (Natural Science), 2005, 44(4): 503–506 [译自: 复旦学报(自然科学版), 2005, 44(4): 503–506]     

HCO3 − Transport in a Mathematical Model of the Pancreatic Ductal Epithelium

We have used computer modeling to investigate how pancreatic duct cells can secrete a fluid containing near isotonic (∼140 mm) NaHCO₃. Experimental data suggest that NaHCO₃ secretion occurs in three steps: (i) accumulation of HCO⁻ ₃ across the basolateral membrane of the duct cell by Na(HCO₃) _n cotransporters, Na⁺/H⁺ exchangers and proton pumps; (ii) secretion of HCO⁻ ₃ across the luminal membrane on Cl⁻/HCO⁻ ₃ antiporters operating in parallel with Cl⁻ channels; and (iii) diffusion of Na⁺ through the paracellular pathway. Programming the currently available experimental data into our computer model shows that this mechanism for HCO⁻ ₃ secretion is deficient in one important respect. While it can produce a relatively large volume of a HCO⁻ ₃-rich fluid, it can only raise the luminal HCO⁻ ₃ concentration up to about 70 mm. To achieve secretion of 140 mm NaHCO₃ by the model it is necessary to: (i) reduce the conductive Cl⁻ permeability and increase the conductive HCO⁻ ₃ permeability of the luminal membrane of the duct cell, and (ii) reduce the activity of the luminal Cl⁻/HCO⁻ ₃ antiporters. Under these conditions most of the HCO⁻ ₃ is secreted via a conductive pathway. Based on our data, we propose that HCO⁻ ₃ secretion occurs mainly by the antiporter in duct segments near the acini (luminal HCO⁻ ₃ concentration up to ∼70 mm), but mainly via channels further down the ductal tree (raising luminal HCO⁻ ₃ to ∼140 mm). Received: 15 November 1999/Revised: 29 March 2000     

Evaluation of denitrification in an urban stream receiving wastewater effluent

Urban streams often contain elevated concentrations of nitrogen (N) which can be amplified in systems receiving effluent from wastewater treatment plants (WWTP). In this study, we evaluated the importance of denitrification in a stream draining urban Greensboro, NC, USA, using two approaches: (1) natural abundance of ¹⁵N–NO₃⁻ in conjunction with background NO₃⁻–N concentrations along a 7 km transect downstream of a WWTP; and (2) C₂H₂ block experiments at three sites and at three habitat types within each site. Overall lack of a longitudinal pattern of δ¹⁵N–NO₃⁻ and NO₃⁻–N, combined with high concentrations of NO₃⁻–N suggested that other factors were controlling NO₃⁻–N flux in the study transect. However, denitrification did appear to be significant along one portion of the transect. C₂H₂ block experiments showed that denitrification rates were much higher downstream of the WWTP compared to upstream, and showed that denitrification rates were highest in erosional and depositional areas downstream of the WWTP and in erosional areas upstream of the plant. Thus, the combination of the two methods for evaluating denitrification provided more insight into the spatial dynamics of denitrification activity than either approach alone. Denitrification appeared to be a significant sink for NO₃⁻–N upstream of the WWTP, but not downstream. Approximately 46% of the total NO₃⁻–N load was removed via denitrification in the upstream, urban section of the stream, while only 2.3% of NO₃⁻–N was lost downstream of the plant. This result suggests that controlling NO₃⁻–N loading from the plant could result in considerable improvement of downstream water quality.     

Characterization of Regulatory Volume Behavior by Fluorescence Quenching in Human Corneal Epithelial Cells

An in-depth understanding of the mechanisms underlying regulatory volume behavior in corneal epithelial cells has been in part hampered by the lack of adequate methodology for characterizing this phenomenon. Accordingly, we developed a novel approach to characterize time-dependent changes in relative cell volume induced by anisosmotic challenges in calcein-loaded SV40-immortalized human corneal epithelial (HCE) cells with a fluorescence microplate analyzer. During a hypertonic challenge, cells shrank rapidly, followed by a temperature-dependent regulatory volume increase (RVI), τ_c = 19 min. In contrast, a hypotonic challenge induced a rapid (τ_c = 2.5 min) regulatory volume decrease (RVD). Temperature decline from 37 to 24°C reduced RVI by 59%, but did not affect RVD. Bumetanide (50 μM), ouabain (1 mM), DIDS (1 mM), EIPA (100 μM), or Na⁺-free solution reduced the RVI by 60, 61, 39, 32, and 69%, respectively. K⁺, Cl⁻ channel and K⁺-Cl⁻ cotransporter (KCC) inhibition obtained with either 4-AP (1 mM), DIDS (1 mM), DIOA (100 μM), high K⁺ (20 mM) or Cl⁻-free solution, suppressed RVD by 42, 47, 34, 52 and 58%, respectively. KCC activity also affects steady-state cell volume, since its inhibition or stimulation induced relative volume alterations under isotonic conditions. Taken together, K⁺ and Cl⁻ channels in parallel with KCC activity are important mediators of RVD, whereas RVI is temperature-dependent and is essentially mediated by the Na⁺-K⁺-2Cl⁻ cotransporter (Na⁺-K⁺-2Cl⁻) and the Na⁺-K⁺ pump. Inhibition of K⁺ and Cl⁻ channels and KCC but not Na⁺-K⁺-2Cl⁻ affect steady-state cell volume under isotonic conditions. This is the first report that KCC activity is required for HCE cell volume regulation and maintenance of steady-state cell volume.     

Effect of an epoxy derivative of 2′5′-trioligoadenylate on human neuroblastoma cells

We studied the effect of an epoxy derivative of dephosphorylated 2′,5′-trioligoadenylate (5′,5′ApApAepoxy) resistive to the action of cellular phosphodiesterase on cells of human neuroblastoma IMR 32 cultured in vitro. Twenty-two hours after the addition of 5·10⁻⁶ M 2′,5′ApApAepoxy to the culture medium, the number of cells decreased by 20% (P < 0.05), while the content of protein in these cells increased, on average, by 52% (P < 0.01), as compared with the control. The activities of Na⁺,K⁺-and Ca²⁺, Mg²⁺-ATPases in a microsomal fraction obtained from cells cultured in the presence of 2′, 5′ ApApAepoxy decreased by 50% (P < 0.001) as compared with those in the control cells. Our data indicate that 2′,5′ApApAepoxy possess antiproliferative activity. According to our findings, the antiproliferative effect of 2′,5′ ApApAepoxy can, to a great extent, be explained by the fact that this oligoadenylate derivative significantly modulates the activities of Na⁺,K⁺-and Ca²⁺,Mg²⁺-ATPases. Neirofiziologiya/Neurophysiology, Vol. 38, No. 2, pp. 97–102, March–April, 2006.     

Effects of phorbol 12-myristate 13-acetate on potassium transport in the red blood cells of frog <Emphasis Type="Italic">Rana temporaria</Emphasis>

Phorbol 12-myristate 13-acetate (PMA), a stimulator of PKC, was examined for its influence on K⁺ (⁸⁶Rb) influx in the frog erythrocyte. PMA, 0.1 μM, was found to accelerate ouabain-sensitive K⁺ influx, which was suppressed by 73% with 1 mM amiloride, indicating secondary activation of the Na⁺–K⁺-pump due to stimulation of Na⁺ /H⁺ exchange. PMA-induced stimulation of the sodium pump was completely inhibited with 1 μM staurosporine and by ~50% with 20 μM chelerythrine. In contrast to Na⁺–K⁺-pump, an activity of Cl⁻-dependent K⁺ transport (K–Cl cotransport, KCC), calculated as the difference between K⁺ influxes in Cl⁻ and NO₃ ⁻-media, was substantially decreased under the influence of PMA. Staurosporine fully restored the PMA-induced inhibition of KCC, whereas chelerythrine did not exert any influence. Osmotic swelling of the frog erythrocytes was accompanied by approximately twofold stimulation of KCC. Swelling-activated KCC was inhibited by ~50 and ~83% in the presence of PMA and genistein, respectively, but not chelerythrine. Exposure of the frog erythrocytes to 5 mM fluoride (F⁻) also reduced the KCC activity in isotonic and hypotonic media, with maximal suppression of K⁺ influx in both media being observed upon simultaneous addition of PMA and F⁻. Furosemide and [(dihydronindenyl)oxy] alkanoic acid inhibited the K⁺ influx in both the media by ~50–60%. The results obtained show both the direct and indirect effects of PMA on the K⁺ transport in frog erythrocytes and a complicated picture of KCC regulation in frog erythrocytes with involvement of PKC, tyrosine kinase and protein phosphatase.