Fractal analysis and ionic dependence of endocytotic membrane activity of human breast cancer cells

The endocytic membrane activities of two human breast cancer cell lines (MDA-MB-231 and MCF-7) of strong and weak metastatic potential, respectively, were studied in a comparative approach. Uptake of horseradish peroxidase was used to follow endocytosis. Dependence on ionic conditions and voltage-gated sodium channel (VGSC) activity were characterized. Fractal methods were used to analyze quantitative differences in vesicular patterning. Digital quantification showed that MDA-MB-231 cells took up more tracer (i.e., were more endocytic) than MCF-7 cells. For the former, uptake was totally dependent on extracellular Na⁺ and partially dependent on extracellular and intracellular Ca²⁺ and protein kinase activity. Analyzing the generalized fractal dimension (D _q ) and its Legendre transform f(α) revealed that under control conditions, all multifractal parameters determined had values greater for MDA-MB-231 compared with MCF-7 cells, consistent with endocytic/vesicular activity being more developed in the strongly metastatic cells. All fractal parameters studied were sensitive to the VGSC blocker tetrodotoxin (TTX). Some of the parameters had a “simple” dependence on VGSC activity, if present, whereby pretreatment with TTX reduced the values for the MDA-MB-231 cells and eliminated the differences between the two cell lines. For other parameters, however, there was a “complex” dependence on VGSC activity. The possible physical/physiological meaning of the mathematical parameters studied and the nature of involvement of VGSC activity in control of endocytosis/secretion are discussed.     

Contribution of functional voltage-gated Na+ channel expression to cell behaviors involved in the metastatic cascade in rat prostate cancer: II. Secretory membrane activity

The secretory membrane activities of two rat prostate cancer cell lines of markedly different metastatic potential, and corresponding electrophysiological characteristics, were studied in a comparative approach. In particular, voltage-gated Na(+) channels (VGSCs) were expressed in the strongly metastatic MAT-LyLu but not in the closely related, but weakly metastatic, AT-2 cells. Uptake and release of the non-cytotoxic marker horseradish peroxidase (HRP) were used as indices of general endocytotic and exocytotic membrane activity, respectively. The amount of tracer present in a given experimental condition was quantified by light microscopic digital imaging. The uptake of HRP was an active process, abolished completely by incubating the cells at low temperature (5 degrees C) and suppressed by disrupting the cytoskeleton. Interestingly, the extent of HRP uptake into the strongly metastatic MAT-LyLu cells was almost twice that into the weakly metastatic AT-2 cells. Vesicular uptake of HRP occurred in a fast followed by a slow phase; these appeared to correspond to cytoplasmic and perinuclear pools, respectively. Importantly, the overall quantitative difference in the uptake disappeared in the presence of 1 microM tetrodotoxin which significantly reduced the uptake of HRP into the MAT-LyLu cells. There was no effect on the AT-2 cells, consistent with functional VGSC expression occurring selectively in the former. A similar effect was observed in Na(+)-free medium. The uptake was partially dependent upon extracellular Ca(2+) but was not affected by raising the extracellular K(+) concentration. We suggest that functional VGSC expression could potentiate prostate cancer cells' metastatic ability by enhancing their secretory membrane activity.     

Patterning of endocytic vesicles and its control by voltage-gated Na<Superscript>+</Superscript> channel activity in rat prostate cancer cells: fractal analyses

Fractal methods were used to analyze quantitative differences in secretory membrane activities of two rat prostate cancer cell lines (Mat-LyLu and AT-2) of strong and weak metastatic potential, respectively. Each cells endocytic activity was determined by horseradish peroxidase uptake. Digital images of the patterns of vesicular staining were evaluated by multifractal analyses: generalized fractal dimension (D_q) and its Legendre transform f(), as well as partitioned iterated function system – semifractal (PIFS-SF) analysis. These approaches revealed consistently that, under control conditions, all multifractal parameters and PIFS-SF codes determined had values greater for Mat-LyLu compared with AT-2 cells. This would agree generally with the endocytic/vesicular activity of the strongly metastatic Mat-LyLu cells being more developed than the corresponding weakly metastatic AT-2 cells. All the parameters studied were sensitive to tetrodotoxin (TTX) pre-treatment of the cells, which blocked voltage-gated Na⁺ channels (VGSCs). Some of the parameters had a simple dependence on VGSC activity, whereby pre-treatment with TTX reduced the values for the MAT-LyLu cells and eliminated the differences between the two cell lines. For other parameters, however, there was a complex dependence on VGSC activity. The possible physical/physiological meaning of the mathematical parameters studied and the nature of involvement of VGSC activity in control of endocytosis/secretion are discussed.     

Apical and basolateral endocytosis in Madin-Darby canine kidney (MDCK) cells grown on nitrocellulose filters.

Madin-Darby canine kidney (MDCK) cells (strain I) grown on 0.45 micron pore size nitrocellulose filters formed monolayers which were highly polarized and had high transepithelial electrical resistance (greater than 3000 ohm X cm2). Morphometric analysis showed that the area of the basolateral surface domain was 7.6 times larger than that of the apical. The uptake of fluid-phase markers [3H]inulin and horseradish peroxidase (HRP) was studied from the apical and the basal side of the monolayer. Uptake of [3H]inulin was biphasic and the rate during the first 40 min corresponded to a fluid phase uptake of 20.5 X 10(-8) nl/min per cell from the basolateral side, and 1.0 X 10(-8) nl/min per cell from the apical side. Electron micrographs of the monolayers after HRP uptake showed that the marker was rapidly delivered into endosome-like vesicles and into multivesicular bodies. No labelling of the Golgi complex could be observed during 2 h of uptake. Evidence was obtained for the transport of fluid phase markers across the cell. HRP and fluorescein isothiocyanate-dextran crossed the monolayers in either direction at a rate corresponding to approximately 3 X 10(-8) nl of fluid/min/cell. Adding the transcytosis rate to the rate of fluid accumulation into the cell yielded a total basolateral endocytic rate which was 6-fold greater than the apical rate. When the uptake rates were normalized for membrane area the apical and basolateral endocytic rates were about equal per unit cell surface area.     

Influence of forskolin and carbachol on intestinal absorption of horseradish peroxidase in the goldfish (Carassius auratus)

The transepithelial route for mucosa-to-serosa transport of the tracer macromolecule horseradish peroxidase (HRP; MW 40 kDa) and modulation of this transport by forskolin and carbachol have been studied in vi-tro in stripped goldfish intestinal epithelium mounted in Ussing-type chambers. Uptake and transport have been investigated by measuring the HRP flux from the muco-sal to serosal sides by an enzymatic method and by visualising HRP reaction products in the mucosa with electron-microscopical techniques. Both the cholinergic agonist carbachol (which is thought to increase intracellular Ca²⁺ and activate protein kinase C activity) and forskolin (a direct activator of adenylylcyclase) affect the amount of enzymatically active HRP in the tissue. In control tissue, HRP product is found only within the epithelial cells, the transepithelial flux reaching a constant value of about 1.5 pmoles/cm² per h. Carbachol increases the amount of HRP product in the cells, but has no significant effect on the HRP flux compared with control values. Forskolin decreases the amount of HRP product in the cells; however, in the presence of forskolin, the lateral intercellular spaces become filled with HRP product. HRP is found in the lamina propria and the transepithelial protein flux increases more than 2.5-fold. In the presence of forskolin plus carbachol, the results are no different from the control. It is concluded that carbachol increases the endocytotic uptake of HRP, whereas forskolin inhibits the uptake but increases the paracellular permeability for HRP in goldfish intestine. Received: 10 July 1995 / Accepted: 4 February 1996     

Klotho attenuates isoproterenol-induced hypertrophic response in H9C2 cells by activating Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase and inhibiting the reverse mode of Na<Superscript>+</Superscript>/Ca<Superscript>2+</Superscript>-exchanger

Cardiac hypertrophy plays a major role in heart failure and is related to patient morbidity and mortality. Calcium overloading is a main risk for cardiac hypertrophy, and Na⁺/K⁺-ATPase (NKA) has been found that it could not only regulate intracellular Na⁺ levels but also control the intracellular Ca²⁺ ([Ca²⁺]_i) level through Na⁺/Ca²⁺-exchanger (NCX). Recent studies have reported that klotho could affect [Ca²⁺]_i level. In this study, we aimed at exploring the role of klotho in improving isoproterenol-induced hypertrophic response of H9C2 cells. The H9C2 cells were randomly divided into control and isoproterenol (ISO) (10 μM) groups. Klotho protein (10 μg/ml) or NKAα2 siRNA was used to determine the changes in isoproterenol-induced hypertrophic response. The alterations of [Ca²⁺]_i level were measured by spectrofluorometry. Our results showed that H9C2 cells which were treated with isoproterenol presented a higher level of [Ca²⁺]_i and hypertrophic gene expression at 24 and 48 h compared with the control group. Moreover, the expressions of NKAα1 and NKAα2 were both increased in control and ISO groups after treating with klotho protein; meanwhile, the NKA activity was increased and NCX activity was decreased after treatment. Consistently, the [Ca²⁺]_i level and hypertrophic gene expression were decreased in ISO group after klotho protein treatment. However, these effects were both prevented by transfecting with NKAα2 siRNA. In conclusion, these findings demonstrated that klotho inhibits isoproterenol-induced hypertrophic response in H9C2 cells by activating NKA and inhibiting the reverse mode of NCX and this effect may be associated with the upregulation of NKAα2 expression.     

Nanosecond Electric Pulses: A Novel Stimulus for Triggering Ca<Superscript>2+</Superscript> Influx into Chromaffin Cells Via Voltage-Gated Ca<Superscript>2+</Superscript> Channels

Exposing bovine chromaffin cells to a single 5 ns, high-voltage (5 MV/m) electric pulse stimulates Ca²⁺ entry into the cells via L-type voltage-gated Ca²⁺ channels (VGCC), resulting in the release of catecholamine. In this study, fluorescence imaging was used to monitor nanosecond pulse-induced effects on intracellular Ca²⁺ level ([Ca²⁺]_i) to investigate the contribution of other types of VGCCs expressed in these cells in mediating Ca²⁺ entry. ω-Conotoxin GVIA and ω-agatoxin IVA, antagonists of N-type and P/Q-type VGCCs, respectively, reduced the magnitude of the rise in [Ca²⁺]_i elicited by a 5 ns pulse. ω-conotoxin MVIIC, which blocks N- and P/Q-type VGCCs, had a similar effect. Blocking L-, N-, and P\Q-type channels simultaneously with a cocktail of VGCC inhibitors abolished the pulse-induced [Ca²⁺]_i response of the cells, suggesting Ca²⁺ influx occurs only via VGCCs. Lowering extracellular K⁺ concentration from 5 to 2 mM or pulsing cells in Na⁺-free medium suppressed the pulse-induced rise in [Ca²⁺]_i in the majority of cells. Thus, both membrane potential and Na⁺ entry appear to play a role in the mechanism by which nanoelectropulses evoke Ca²⁺ influx. However, activation of voltage-gated Na⁺ channels (VGSC) is not involved since tetrodotoxin (TTX) failed to block the pulse-induced rise in [Ca²⁺]_i. These findings demonstrate that a single electric pulse of only 5 ns duration serves as a novel stimulus to open multiple types of VGCCs in chromaffin cells in a manner involving Na⁺ transport across the plasma membrane. Whether Na⁺ transport occurs via non-selective cation channels and/or through lipid nanopores remains to be determined.     

THE INTERACTION OF SOLUBLE HORSERADISH PEROXIDASE WITH MOUSE PERITONEAL MACROPHAGES IN VITRO

The in vitro interaction of soluble horseradish peroxidase (HRP) with homogeneous mono layers of mouse macrophages has been studied using sensitive biochemical and cytochemical techniques. The compartmentalization of HRP in extracellular and intracellular sites has been quantitatively evaluated. A significant fraction is bound to a serum-derived layer, which coats the surface of culture vessels and may be removed by appropriate washes. Macrophages interiorize HRP as a solute in pinocytic vesicles without appreciable binding of the glycoprotein to the plasma membrane. Uptake is directly proportional to the concentration of HRP in the culture medium. 1 x 10⁶ cells ingest 0.0025% of the administered load per hr over a wide range of concentrations. Cytochemically, all demonstrable HRP is sequestered within the endocytic vesicles and secondary lysosomes of the vacuolar apparatus. After uptake, the enzymatic activity of HRP is inactivated exponentially with a half-life of 7–9 hr, until enzyme is no longer detectable. When macrophages have pinocytosed trace-labeled HRP-¹²⁵I, cell-associated isotope disappears with a t ½ of 20–30 hr and they release monoiodotyrosine-¹²⁵I into the culture medium. We were unable to obtain evidence that significant amounts of HRP (>2%) can be exocytosed after uptake, can exist intact on the cell surface, or can be digested extracellularly. It is difficult to reconcile these observations with several of the postulated mechanisms whereby macrophages are thought to play a prominent role in the induction of an immune response.     

Single cell adhesion measuring apparatus (SCAMA): application to cancer cell lines of different metastatic potential and voltage-gated Na<Superscript>+</Superscript> channel expression

We have developed a simple yet effective apparatus, based upon negative pressure directed to the tip of a micro-pipette, to measure the adhesiveness of single cells. The “single cell adhesion measuring apparatus” (SCAMA) could differentiate between the adhesion of strongly versus weakly metastatic cancer cells as well as normal cells. Adhesion was quantified as “detachment negative pressure” (DNP) or “DNP relative to cell size” (DNPR) where a noticeable difference in cell size was apparent. Thus, for rat and human prostate and human breast cancer cell lines, adhesiveness (DNPR values) decreased in line with increased metastatic potential. Using the SCAMA, we investigated the effect of tetrodotoxin (TTX), a specific blocker of voltage-gated Na⁺ channels (VGSCs), on the adhesion of rat and human prostate cancer cell lines of markedly different metastatic potential. Following pretreatment with TTX (48 h with 1 μM), the adhesion values for the Mat-LyLu cells increased significantly 4.3-fold; there was no effect on the AT-2 cells. For the strongly metastatic PC-3M cells, TTX treatment caused a significant (∼30%) increase in adhesion. The adhesion of PNT2-C2 (“normal”) cells was not affected by the TTX pretreatment. The TTX-induced increase in the adhesiveness of the strongly metastatic cells was consistent with the functional VGSC expression in these cells and the proposed role of VGSC activity in metastatic cell behaviour. In conclusion, the SCAMA, which can be constructed easily and cheaply, offers a simple and effective method to characterise single-cell adhesion and its modulation.     

Factors Associated with Determination of Root <Superscript>22</Superscript>Na<Superscript>+</Superscript> Influx in the Salt Accumulation Halophyte <Emphasis Type="Italic">Suaeda maritima</Emphasis>

Salinity is an increasing problem for agricultural production worldwide. The result of low-affinity Na⁺ uptake is toxic to the cytoplasm of most crop plants. Nevertheless, the pathways for this low-affinity Na⁺ uptake are still uncertain. In this work we used ²²Na⁺ isotope tracing technology to investigate factors associated with determination of root ²²Na⁺ influx in the salt accumulation halophyte Suaeda maritima. We found that a 2 min of exposure to the ²²Na⁺ labeled uptake solution was optimal for determining ²²Na⁺ influx into excised roots of S. maritima and that 7 min of blotting is suitable in ²²Na⁺ influx experiments. ²²Na⁺ influx did not increase linearly with the increasing external Na⁺ concentration, in the range tested, of 2 to 300 mM NaCl. But root ²²Na⁺ influx and root Na⁺ concentration were well correlated. ²²Na⁺ influx into excised roots of S. maritima was not, however, well correlated with the plant size. All the above results indicated further that this ²²Na⁺ isotope influx procedure is a good method for quantify Na⁺ uptake rate by the roots of the salt accumulation halophyte.     

Functional Characterization of Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> Exchangers in Primary Cultures of Prairie Dog Gallbladder

Gallbladder Na⁺ absorption is linked to gallstone formation in prairie dogs. We previously reported Na⁺/H⁺ exchanger (NHE1-3) expression in native gallbladder tissues. Here we report the functional characterization of NHE1, NHE2 and NHE3 in primary cultures of prairie dog gallbladder epithelial cells (GBECs). Immunohistochemical studies showed that GBECs grown to confluency are homogeneous epithelial cells of gastrointestinal origin. Electron microscopic analysis of GBECs demonstrated that the cells form polarized monolayers characterized by tight junctions and apical microvilli. GBECs grown on Snapwells exhibited polarity and developed transepithelial short-circuit current, I_sc, (11.6 ± 0.5 µA · cm^–2), potential differences, V_t (2.1 ± 0.2 mV), and resistance, R_t (169 ± 12 · cm²). NHE activity in GBECs assessed by measuring dimethylamiloride-inhibitable ²²Na⁺ uptake under a H⁺ gradient was the same whether grown on permeable Snapwells or plastic wells. The basal rate of ²²Na⁺ uptake was 21.4 ± 1.3 nmol · mg prot^–1 · min^–1, of which 9.5 ± 0.7 (~45%) was mediated through apically-restricted NHE. Selective inhibition with HOE-694 revealed that NHE1, NHE2 and NHE3 accounted for ~6%, ~66% and ~28% of GBECs total NHE activity, respectively. GBECs exhibited saturable NHE kinetics (V_max 9.2 ± 0.3 nmol · mg prot^–1 · min^–1; K_m 11.4 ± 1.4 mM Na⁺). Expression of NHE1, NHE2 and NHE3 mRNAs was confirmed by RT-PCR analysis. These results demonstrate that the primary cultures of GBECs exhibit Na⁺ transport characteristics similar to native gallbladder tissues, suggesting that these cells can be used as a tool for studying the mechanisms of gallbladder ion transport both under physiologic conditions and during gallstone formation.     

Ethanol exerts differential effects on high affinity choline uptake in neuron-enriched cultures from 8-day-old chick embryo cerebral hemispheres

Neuronal-enriched cultures were prepared from 8-day-old chick embryo cerebral hemispheres and exposed to ethanol (50 mM) from day 4 to 8 in culture. At day 8, both control and ethanol-treated cultures were processed for [³H]choline uptake in situ. Uptake was performed on cultures containing either Na⁺-plus or Na⁺-free (Li⁺) HEPES buffer. Total choline uptake as well as Na⁺-dependent and Na⁺-independent choline uptake were calculated. The K_m and V_max were calculated using the Lineweaver-Burke analysis. Our analysis of the data revealed that ethanol-treated cultures exhibited two values for V_max, one similar to that found in control cultures and one significantly lower than controls. No differences were observed in K_m values between control and ethanol-treated cultures. We interpret the low V_max to represent a population of cholinergic neurons which have been arrested at an immature stage as a result of ethanol insult.     

Estrogen and non‐genomic upregulation of voltage‐gated Na+ channel activity in MDA‐MB‐231 human breast cancer cells: Role in adhesion

External (but not internal) application of β‐estradiol (E2) increased the current amplitude of voltage‐gated Na⁺ channels (VGSCs) in MDA‐MB‐231 human breast cancer (BCa) cells. The G‐protein activator GTP‐γ‐S, by itself, also increased the VGSC current whilst the G‐protein inhibitor GDP‐β‐S decreased the effect of E2. Expression of GPR30 (a G‐protein‐coupled estrogen receptor) in MDA‐MB‐231 cells was confirmed by PCR, Western blot and immunocytochemistry. Importantly, G‐1, a specific agonist for GPR30, also increased the VGSC current amplitude in a dose‐dependent manner. Transfection and siRNA‐silencing of GPR30 expression resulted in corresponding changes in GPR30 protein expression but only internally, and the response to E2 was not affected. The protein kinase A inhibitor, PKI, abolished the effect of E2, whilst forskolin, an adenylate cyclase activator, by itself, increased VGSC activity. On the other hand, pre‐incubation of the MDA‐MB‐231 cells with brefeldin A (a trans‐Golgi protein trafficking inhibitor) had no effect on the E2‐induced increase in VGSC amplitude, indicating that such trafficking (‘externalisation’) of VGSC was not involved. Finally, acute application of E2 decreased cell adhesion whilst the specific VGSC blocker tetrodotoxin increased it. Co‐application of E2 and tetrodotoxin inhibited the effect of E2 on cell adhesion, suggesting that the effect of E2 was mainly through VGSC activity. Pre‐treatment of the cells with PKI abolished the effect of E2 on adhesion, consistent with the proposed role of PKA. Potential implications of the E2‐induced non‐genomic upregulation of VGSC activity for BCa progression are discussed. J. Cell. Physiol. 224: 527–539, 2010. © 2010 Wiley‐Liss, Inc.     

Cultured trout gill epithelia enriched in pavement cells or in mitochondria-rich cells provides insights into Na<Superscript>+</Superscript> and Ca<Superscript>2+</Superscript> transport

Summary  The lack of a suitable flat epithelial preparation isolated directly from the freshwater fish gill has led, in recent years, to the development of cultured gill epithelia on semipermeable supports. To date, their minimal capacity to actively transport ions has limited their utility as ionoregulatory models. The current study describes a new method of culturing gill epithelia consisting either of an enriched population of pavement (PV) cells or a mixed population of PV cells and mitochondria-rich (MR) cells from the gills of adult rainbow trout. Although the cell culture approach is similar to the double-seeded insert (DSI) technique described previously, it makes use of Percoll density centrifugation to first separate populations of PV and MR cells, which are then seeded on cell culture supports in varying proportions on successive days so as to produce preparations enriched in one or the other cell types. Based on rhodamine staining, the MR cell-rich epithelia exhibited a threefold higher enrichment of MR cells compared to traditional DSI preparations. In general, MR cell-rich epithelia developed extremely high transepithelial resistances (TER; >30 kΩ cm²) and positive transepithelial potentials (TEP) under symmetrical conditions (i.e., L15 medium on both apical and basolateral sides). Apical exposure of cell cultures to freshwater reduced TER and produced a negative TEP in all the epithelial preparations, although MR cell-rich epithelia maintained relatively high TER and negative TEP for over 2 d under these asymmetrical conditions. Measurement of unidirectional Na⁺ fluxes and application of the Ussing flux ratio criterion demonstrated active Na⁺ uptake in PV cell-rich and MR cell-rich epithelia under both symmetrical and asymmetrical conditions. In comparison, Ca²⁺ uptake and Na⁺/K⁺-ATPase activity were significantly elevated in MR cell-rich preparations relative to the traditional DSI or PV cell-rich cultures under symmetrical conditions. This new methodology enhances our ability to tailor cultured gill epithelia on semipermeable supports with different proportions of PV cells and MR cells, thereby illuminating the ionoregulatory functions of the two cell types.     

Synaptic activity of frog retinal photoreceptors. A peroxidase uptake study

The uptake of horseradish peroxidase (HRP) into membranous structures, detectable by light and electron microscopy, is used here to monitor the synaptic activity of photoreceptors of isolated frog retinas maintained in the dark or under various illumination conditions. The major findings are: (a) Neurotransmission from photoreceptor terminals seems to involve the same types of endocytic membrane-retrieval processes that occur at other nerve terminals. Presumably, the endocytic processes compensate for exocytic events associated with neurotransmission. The retrieved membrane is "recycled" to form vesicles. Some of these accumulate near the synaptic ribbons, perhaps indicating reutilization for exocytosis. On the other hand, some retrieved membrane evidently is degraded via multivesicular bodies that appear to undergo "retrograde" transport from the receptor synapses to the myoid regions. (b) Photoreceptor terminals take up much HRP in the dark. Steady illumination markedly decreases uptake by rods. Uptake by cones is notably reduced only at illumination intensities higher than those that have maximal effects on rods. (c) The decrease in rod HRP uptake with light is reversible when retinas are allowed to adapt to the dark, if the light exposures used were at intensities that bleach very little visual pigment. Such "recovery" is not observed after light exposures that bleach a considerable amount of visual pigment. The cones recover their dark levels of HRP uptake even after light exposures that bleach considerable amounts of visual pigment. The changes in HRP uptake that we observe parallel expectations for photoreceptor synaptic neurotransmission derived from indirect physiological evidence.     

Effects of ouabain on proliferation of human endothelial cells correlate with Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase activity and intracellular ratio of Na<Superscript>+</Superscript> and K<Superscript>+</Superscript>

Side-by-side with inhibition of the Na⁺,K⁺-ATPase ouabain and other cardiotonic steroids (CTS) can affect cell functions by mechanisms other than regulation of the intracellular Na⁺ and K⁺ ratio ([Na⁺]_i/[K⁺]_i). Thus, we compared the doseand time-dependences of the effect of ouabain on intracellular [Na⁺]_i/[K⁺]_i ratio, Na⁺,K⁺-ATPase activity, and proliferation of human umbilical vein endothelial cells (HUVEC). Treatment of the cells with 1-3 nM ouabain for 24-72 h decreased the [Na⁺]_i/[K⁺]_i ratio and increased cell proliferation by 20-50%. We discovered that the same ouabain concentrations increased Na⁺,K⁺-ATPase activity by 25-30%, as measured by the rate of ⁸⁶Rb⁺ influx. Higher ouabain concentrations inhibited Na⁺,K⁺-ATPase, increased [Na⁺]_i/[K⁺]_i ratio, suppressed cell growth, and caused cell death. When cells were treated with low ouabain concentrations for 48 or 72 h, a negative correlation between [Na⁺]_i/[K⁺]_i ratio and cell growth activation was observed. In cells treated with high ouabain concentrations for 24 h, the [Na⁺]_i/[K⁺]_i ratio correlated positively with proliferation inhibition. These data demonstrate that inhibition of HUVEC proliferation at high CTS concentrations correlates with dissipation of the Na⁺ and K⁺ concentration gradients, whereas cell growth stimulation by low CTS doses results from activation of Na⁺,K⁺-ATPase and decrease in the [Na⁺]_i/[K⁺]_i ratio.     

Salt tolerance of barley: Relations between expression of isoforms of vacuolar Na<Superscript>+</Superscript>/H<Superscript>+</Superscript>-antiporter and <Superscript>22</Superscript>Na<Superscript>+</Superscript> accumulation

Two barley cultivars (Hordeum vulgare L., cvs. Elo and Belogorskii) differing in salt tolerance were used to study ²²Na⁺ uptake, expression of three isoforms of the Na⁺/H⁺ antiporter HvNHX1-3, and the cellular localization of these isoforms in the elongation zone of seedling roots. During short (1 h) incubation, seedling roots of both cultivars accumulated approximately equal quantities of ²²Na⁺. However, after 24-h incubation the content of ²²Na⁺ in roots of a salt-tolerant variety Elo was 40% lower than in roots of the susceptible variety Belogorskii. The content of ²²Na⁺ accumulated in shoots of cv. Elo after 24-h incubation was 6.5 times lower than in shoots of cv. Belogorskii and it was 4 times lower after the salt stress treatment. The cytochemical examination revealed that three proteins HvNHX1-3 are co-localized in the same cells of almost all root tissues; these proteins were present in the tonoplast and prevacuolar vesicles. Western blot analysis of HvNHX1-3 has shown that the content of isoforms in vacuolar membranes increased in response to salt stress in seedling roots and shoots of both cultivars, although the increase was more pronounced in the tolerant cultivar. The content of HvNHX1 in the seedlings increased in parallel with the enhanced expression of HvNHX1, whereas the increase in HvNHX2 and HvNHX3 protein content was accompanied by only slight changes in expression of respective genes. The results provide evidence that salt tolerance of barley depends on plant ability to restrict Na⁺ transport from the root to the shoot and relies on regulatory pathways of HvNHX1-3 expression in roots and shoots during salt stress.     

Dopamine and norepinephrine uptake and metabolism by astroglial cells in culture

Primary cultures of normal astroglia started from the cerebral hemispheres of neonatal rats took up dopamine (DA) and norepinephrine (NE) in the concentration range of 10^?7 to 10^?4M and metabolized each to their respective principal central nervous system products by the actions of both catechol-0-methyl transferase and monoamine oxidase. At 10^?7M, uptake of ³H labelled DA and NE was inhibited by omission of Na⁺, addition of ouabain or lowered temperatures. Uptake at 10^?4M was considerable but was Na⁺-independent. Only Na⁺-independent uptake was seen in primary cultures started from the meninges of neonatal rats. These data suggest that astroglial cells in the CNS have a high affinity uptake system for catecholamines, and such uptake is followed by catecholamine metabolism.     

Computational Flux Balance Analysis Predicts that Stimulation of Energy Metabolism in Astrocytes and their Metabolic Interactions with Neurons Depend on Uptake of K<Superscript>+</Superscript> Rather than Glutamate

Brain activity involves essential functional and metabolic interactions between neurons and astrocytes. The importance of astrocytic functions to neuronal signaling is supported by many experiments reporting high rates of energy consumption and oxidative metabolism in these glial cells. In the brain, almost all energy is consumed by the Na⁺/K⁺ ATPase, which hydrolyzes 1 ATP to move 3 Na⁺ outside and 2 K⁺ inside the cells. Astrocytes are commonly thought to be primarily involved in transmitter glutamate cycling, a mechanism that however only accounts for few % of brain energy utilization. In order to examine the participation of astrocytic energy metabolism in brain ion homeostasis, here we attempted to devise a simple stoichiometric relation linking glutamatergic neurotransmission to Na⁺ and K⁺ ionic currents. To this end, we took into account ion pumps and voltage/ligand-gated channels using the stoichiometry derived from available energy budget for neocortical signaling and incorporated this stoichiometric relation into a computational metabolic model of neuron-astrocyte interactions. We aimed at reproducing the experimental observations about rates of metabolic pathways obtained by ¹³C-NMR spectroscopy in rodent brain. When simulated data matched experiments as well as biophysical calculations, the stoichiometry for voltage/ligand-gated Na⁺ and K⁺ fluxes generated by neuronal activity was close to a 1:1 relationship, and specifically 63/58 Na⁺/K⁺ ions per glutamate released. We found that astrocytes are stimulated by the extracellular K⁺ exiting neurons in excess of the 3/2 Na⁺/K⁺ ratio underlying Na⁺/K⁺ ATPase-catalyzed reaction. Analysis of correlations between neuronal and astrocytic processes indicated that astrocytic K⁺ uptake, but not astrocytic Na⁺-coupled glutamate uptake, is instrumental for the establishment of neuron-astrocytic metabolic partnership. Our results emphasize the importance of K⁺ in stimulating the activation of astrocytes, which is relevant to the understanding of brain activity and energy metabolism at the cellular level.