Astrocytes from Na(+)-K(+)-Cl(-) cotransporter-null mice exhibit absence of swelling and decrease in EAA release

We reported previously that inhibition ofNa⁺-K⁺-Cl cotransporter isoform 1 (NKCC1) by bumetanide abolishes high extracellular K⁺concentration ([K⁺]_o)-induced swelling andintracellular Cl accumulation in rat cortical astrocytes.In this report, we extended our study by using cortical astrocytes fromNKCC1-deficient (NKCC1^/) mice. NKCC1 protein andactivity were absent in NKCC1^/ astrocytes.[K⁺]_o of 75 mM increased NKCC1 activityapproximately fourfold in NKCC1^+/+ cells (P < 0.05) but had no effect in NKCC1^/ astrocytes.Intracellular Cl was increased by 70% inNKCC1^+/+ astrocytes under 75 mM[K⁺]_o (P < 0.05) butremained unchanged in NKCC1^/ astrocytes. Baselineintracellular Na⁺ concentration([Na⁺]_i) in NKCC1^+/+ astrocyteswas 19.0 ± 0.5 mM, compared with 16.9 ± 0.3 mM[Na⁺]_i in NKCC1^/ astrocytes(P < 0.05). Relative cell volume ofNKCC1^+/+ astrocytes increased by 13 ± 2% in 75 mM[K⁺]_o, compared with a value of 1.0 ± 0.5% in NKCC1^/ astrocytes (P < 0.05).Regulatory volume increase after hypertonic shrinkage was completelyimpaired in NKCC1^/ astrocytes.High-[K⁺]_o-induced ¹⁴C-labeledD-aspartate release was reduced by ~30% inNKCC1^/ astrocytes. Our study suggests that stimulationof NKCC1 is required for high-[K⁺]_o-inducedswelling, which contributes to glutamate release from astrocytes underhigh [K⁺]_o.

     

AMPA-mediated excitotoxicity in oligodendrocytes: role for Na(+)-K(+)-Cl(-) co-transport and reversal of Na(+)/Ca(2+) exchanger

We investigated the role of Na(+)-K(+)-Cl(-) co-transporter isoform 1 (NKCC1) and reversal of Na(+)/Ca(2+) exchanger (NCX(rev)) in glutamate-mediated excitotoxicity in oligodendrocytes obtained from rat spinal cords (postnatal day 6-8). An immunocytochemical characterization showed that these cultures express NKCC1 and Na(+)/Ca(2+) exchanger isoforms 1, 2, and 3 (NCX1, NCX2, NCX3). Exposing the cultures to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) plus cyclothiazide (CTZ) led to a transient rise in intracellular (), which was followed by a sustained overload, NKCC1 phosphorylation, and a NKCC1-mediated Na(+) influx. In the presence of a specific AMPA receptor inhibitor 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX), the AMPA/CTZ failed to elicit any changes in . The AMPA/CTZ-induced sustained rise led to mitochondrial Ca(2+) accumulation, release of cytochrome c from mitochondria, and cell death. The AMPA/CTZ-elicited increase, mitochondrial damage, and cell death were significantly reduced by inhibiting NKCC1 or NCX(rev). These data suggest that in cultured oligodendrocytes, activation of AMPA receptors leads to NKCC1 phosphorylation that enhances NKCC1-mediated Na(+) influx. The latter triggers NCX(rev) and NCX(rev)-mediated overload and compromises mitochondrial function and cellular viability.     

Regulation of Na(+)-K(+)-Cl(-) cotransporter in primary astrocytes by dibutyryl cAMP and high [K(+)](o)

In this study, we examined theNa⁺-K⁺-Cl cotransporter activityand expression in rat cortical astrocyte differentiation. Astrocyte differentiation was induced by dibutyryl cAMP (DBcAMP, 0.25 mM) for7 days, and cells changed from a polygonal to process-bearing morphology. Basal activity of the cotransporter was significantly increased in DBcAMP-treated astrocytes (P < 0.05).Expression of an ~161-kDa cotransporter protein was increased by 91%in the DBcAMP-treated astrocytes. Moreover, the specific[³H]bumetanide binding was increased by 67% in theDBcAMP-treated astrocytes. Inhibition of protein synthesis bycyclohexamide (2-3 µg/ml) significantly attenuated theDBcAMP-mediated upregulation of the cotransporter activity andexpression. The Na⁺-K⁺-Clcotransporter in astrocytes has been suggested to play a role inK⁺ uptake. In 75 mM extracellular K⁺concentration, the cotransporter-mediated K⁺ influx wasstimulated by 147% in nontreated cells and 79% in DBcAMP-treatedcells (P < 0.05). To study whether this highK⁺-induced stimulation of the cotransporter is attributedto membrane depolarization and Ca²⁺ influx, the role of theL-type voltage-dependent Ca²⁺ channel was investigated. Thehigh-K⁺-mediated stimulation of the cotransporter activitywas abolished in the presence of either 0.5 or 1.0 µM of the L-typechannel blocker nifedipine or Ca²⁺-free HEPES buffer. Arise in intracellular free Ca²⁺ in astrocytes was observedin high K⁺. These results provide the first evidence thatthe Na⁺-K⁺-Cl cotransporterprotein expression can be regulated selectively when intracellular cAMPis elevated. The study also demonstrates that the cotransporter inastrocytes is stimulated by high K⁺ in aCa²⁺-dependent manner.

     

Contribution of Na(+)-K(+)-Cl(-) cotransporter to high-[K(+)](o)- induced swelling and EAA release in astrocytes

We hypothesized that highextracellular K⁺ concentration([K⁺]_o)-mediated stimulation ofNa⁺-K⁺-Cl cotransporter isoform 1 (NKCC1) may result in a net gain of K⁺ and Cland thus lead to high-[K⁺]_o-induced swellingand glutamate release. In the current study, relative cell volumechanges were determined in astrocytes. Under 75 mM[K⁺]_o, astrocytes swelled by 20.2 ± 4.9%. This high-[K⁺]_o-mediated swelling wasabolished by the NKCC1 inhibitor bumetanide (10 µM, 1.0 ± 3.1%; P < 0.05). Intracellular³⁶Cl accumulation was increased from acontrol value of 0.39 ± 0.06 to 0.68 ± 0.05 µmol/mgprotein in response to 75 mM [K⁺]_o. Thisincrease was significantly reduced by bumetanide (P < 0.05). Basal intracellular Na⁺ concentration([Na⁺]_i) was reduced from 19.1 ± 0.8 to16.8 ± 1.9 mM by bumetanide (P < 0.05).[Na⁺]_i decreased to 8.4 ± 1.0 mM under75 mM [K⁺]_o and was further reduced to5.2 ± 1.7 mM by bumetanide. In addition, the recovery rate of[Na⁺]_i on return to 5.8 mM[K⁺]_o was decreased by 40% in the presenceof bumetanide (P < 0.05). Bumetanide inhibitedhigh-[K⁺]_o-induced ¹⁴C-labeledD-aspartate release by ~50% (P < 0.05).These results suggest that NKCC1 contributes tohigh-[K⁺]_o-induced astrocyte swelling andglutamate release.

     

Phosphorylation of the salivary Na(+)-K(+)-2Cl(-) cotransporter

Westudied the phosphorylation of the secretoryNa⁺-K⁺-2Cl cotransporter (NKCC1)in rat parotid acinar cells. We have previously shown that NKCC1activity in these cells is dramatically upregulated in response to-adrenergic stimulation and that this upregulation correlates withNKCC1 phosphorylation, possibly due to protein kinase A (PKA). We showhere that when ATP is added to purified acinar basolateral membranes(BLM), NKCC1 is phosphorylated as a result of membrane-associatedprotein kinase activity. Additional NKCC1 phosphorylation is seen whenPKA is added to BLMs, but our data indicate that this is due to aneffect of PKA on endogenous membrane kinase or phosphatase activities,rather than its direct phosphorylation of NKCC1. Also, phosphopeptidemapping demonstrates that these phosphorylations do not take place atthe site associated with the upregulation of NKCC1 by -adrenergicstimulation. However, this upregulatory phosphorylation can be mimickedby the addition of cAMP to permeabilized acini, and this effect can beblocked by a specific PKA inhibitor. These latter results provide good evidence that PKA is indeed involved in the upregulatoryphosphorylation of NKCC1 and suggest that an additional factor presentin the acinar cell but absent from isolated membranes is required to bring about the phosphorylation.

     

Macromolecular synthesis and stability in growth-arrested BALB/c-3T3 cells

Concentrations of methylglyoxal bis-(guanylhydrazone) (mGBG) that inhibited serum-stimulated BALB/c-3T3 cells in late G1 caused a marked inhibition of 3H-leucine incorporation during a 20-min incubation. No decrease was observed in the incorporation of 3H-uridine during a 20-min incubation; however, the amount of acid-insoluble 3H-uridine in mGBG-treated cultures was decreased when the incubation period was longer than 20 min. The amount of the decrease in the accumulation of incorporated 3H-uridine was directly proportional to the length of the incorporation time. Between 10 and 12 h after quiescent BALB/c-3T3 cells were serum-stimulated in mGBG no additional 3H-uridine was accumulated. The stability of the incorporated 3H-uridine, as determined by acid-insoluble radioactivity remaining after the addition of actinomycin D, was less in cells cultured in mGBG. Exogenous spermine or spermidine reversed the inhibition of 3H-uridine accumulation in acid-insoluble material produced by mGBG as well as the decrease in stability of the incorporated 3H-uridine in acid-insoluble material. The effects of mGBG on both the incorporation of 3H-uridine and the stability of the incorporated 3H-uridine can apparently be accounted for by an effect on ribosomal RNA.     

Intracellular acidification inhibits the proliferative response in BALB/c-3T3 cells

One of the earliest events to occur upon the addition of serum to quiescent cells is an increase in the intracellular pH (pHin). The relationship between this pH change and proliferation is not known. In the present study, we investigate the consequences of acidifying the cytosol using the weak acid, 5', 5"-dimethyl oxazolidine 2,4-dione (DMO). At a concentration of 50 mM, DMO inhibits the serum-induced increases in pHin, DNA synthesis, and cell number. This concentration of DMO is shown not to inhibit the steady-state rate of mitochondrial respiration and not to inhibit DNA synthesis in a pH-independent fashion. The effects of DMO treatments are also shown to be reversible, indicating that this compound is not cytotoxic. These observations indicate that DMO inhibits cell proliferation by lowering intracellular pH. One important event that must occur prior to the initiation of DNA synthesis is an elevated rate of protein synthesis. The rate of protein synthesis in situ is extremely pH sensitive. Addition of 50 mM DMO to serum-stimulated cultures reduces the rate of leucine incorporation to unstimulated levels. These observations suggest that cytoplasmic acidification may inhibit proliferation through its effects on protein synthesis.     

Cell cycle-dependent gene expression in V point-arrested BALB/c-3T3 cells

Density-arrested BALB/c-3T3 cells stimulated to proliferate in an amino acid-deficient medium arrest in mid-G₁ at a point termed the V point. Cells released from V point arrest require 6 hr to traverse late G₁ and enter S phase. As data presented here show that mRNA synthesis is needed for 2–3 hr after release of cells from the V point, after which inhibition of mRNA synthesis does not prevent entry into S phase, we used this mid-G₁ arrest protocol to analyze gene expression in late G₁. We found that although stimulation of cells in amino acid-deficient medium did not inhibit the induction of genes expressed in early G₁, genes normally expressed in late G₁ were expressed only after release from the V point. The expression of late G₁ genes in cells released from the V point was temporally similar, in respect to G₁ location, as was seen in stimulation of quiescent G_o cells. As this protocol effectively divides gene expression into early (pre-V point) and late (post-V point) categories, it should be useful in studies of growth factor-modulated events that regulate traverse of late G₁ and commitment to DNA synthesis. In addition, we used c-myb antisense oligonucleotides to show that c-myb expression, which occurs in late G₁, is required for BALB/c-3T3 fibroblasts to traverse late G₁ and initiate DNA synthesis. © 1993 Wiley-Liss, Inc.     

Transforming and carcinogenic potential of cadmium chloride in BALB/c-3T3 cells

A large number of workers are potentially exposed to cadmium during mining and processing. Therefore, there is a concern regarding the potential carcinogenic hazards of cadmium to exposed workers. Studies have been performed to determine if cadmium chloride (CdCl(2)) can induce morphological cell transformation, DNA from CdCl(2)-induced transformed cells can transform other mammalian cells, and the transformed cells induced by CdCl(2) can form tumors in nude mice. BALB/c-3T3 cells were treated with different concentrations of CdCl(2) for 72 h. The frequency of transformed foci from each treatment was determined after cells were cultured for 4 to 5 weeks. DNAs from five CdCl(2)-induced transformed cell lines were isolated and gene transfection assay was performed using NIH-3T3 cells. Non-transformed BALB/c-3T3 cells and cells from 10 transformed cell lines induced by CdCl(2) were injected into both axillary regions of nude mice. Mice were screened once a week for the appearance and size of tumors. CdCl(2) caused a statistically significant, concentration-related increase in the transformation frequency. DNA from all five CdCl(2)-induced transformed cell lines tested was found to induce varying degrees of transfection-mediated transformation in NIH-3T3 cells. All 10 CdCl(2)-induced transformed cell lines formed fibrosarcomas in nude mice within 39 days of inoculation. Within this time period, no tumors were found in nude mice injected with non-transformed BALB/c-3T3 cells. These results indicate that CdCl(2) is capable of inducing morphological cell transformation and that the transformed cells induced by CdCl(2) are potentially tumorigenic.     

Increases in cyclic AMP potentiate competence formation in BALB/c-3T3 cells

The ability of platelet-derived growth factor and fibroblast growth factor to stimulate the initiation of DNA synthesis in quiescent BALB/c-3T3 cells was enhanced by cholera toxin. However, the addition of cholera toxin to unsupplemented medium was not mitogenic, nor did cholera toxin increase the mitogenic potential of mediuum supplemented with platelet-poor plasma. The enhancement of serum-induced DNA synthesis by cholera toxin was due to a specific effect on competence formation and not plasma-controlled progression. Cholera toxin increased the rate of competence formation during a transient exposure of quiescent cells to platelet-derived growth factor; this rate was further increased by the addition of isobutylmethylxanthine, a cyclic nucleotide phosphodiesterase inhibitor. Intracellular cyclic AMP concentrations in quiescent BALB/c-3T3 cells were increased 2- to 3-fold after the addition of cholera toxin. The addition of cholera toxin plus 30 m?M isobutylmethylxanthine caused an even greater (7- to 8-fold) increase in the cellular levels of cyclic AMP. That these increases in cyclic AMP concentrations mediated at least part of the increased sensitivity of quiescent cells to competence factors was substantiated by the observation that 0.01 to 1 mM monobutrylcyclic AMP or 8-bromocyclic AMP also caused a concentration-dependent potentiation of competence formation in quiescent cells during a transient exposure to platelet-derived growth factor.     

Role of cholesterol in functional association between K(+)-Cl(-) cotransporter-3a and Na+,K(+)-ATPase

The conformation of amyloid-beta peptide (Aβ) determines if toxic aggregates are formed. The peptide structure by its turn depends on the environment and molecule-molecule interactions. We characterized the secondary structure of Aβ-(1-40) in surfactant solutions and interacting with monolayers. The peptide adopts β-sheet structure in solutions of ionic surfactants at sub-micelle concentrations and α-helix in the presence of ionic micelles. Uncharged micelles induce β-sheets. Aβ-(1-40) alters the critical micelle concentration value of the non-ionic surfactant, underlining hydrophobic interactions. At ionic monolayers the peptide forms β-sheets when its concentration at the surface is high enough. These results suggest that only electrostatic interactions of charged micelles that surround completely the peptide are able to induce non-aggregated α-helix structure.     

Determination of intracellular pH of BALB/c-3T3 cells using the fluorescence of pyranine

In terms of accuracy and sensitivity, intracellularly trapped, pH-dependent fluorescent probes are appropriate to accurately measure intracellular pH. These probes are commonly introduced into living cells in esterified form, wherein the free acid is produced through enzymatic hydrolysis. The fluorescence characteristics of the ester and the free acid can differ markedly and spectral uncertainty can occur. We describe here the measurement of intracellular pH using 8-hydroxypyrene-1,3,6-trisulfonic acid (pyranine) that has been scrape-loaded into BALB/c-3T3 mouse cells. The excitation spectrum of pyranine is pH sensitive, with an isosbestic point at 415 nm and peaks at 405 and 465 nm which decrease and increase with pH, respectively. The 465/405 ratio can be used to monitor the pH, while the fluorescence at 415 nm indicates the total dye-dependent signal remaining. The scrape-loaded dye persists in cells for periods up to 6 h. We have calibrated this dye in situ using nigericin/high K+, and have found that the pKa of the dye in situ is 7.82, as compared to 7.68 in vitro. We have observed that the cells can slowly equilibrate their intracellular pH to near control levels when presented with either an acute alkaline or acid load.     

Epidermal growth factor (EGF) and somatomedin C regulate G1 progression in competent BALB/c-3T3 cells

The activity in platelet-poor plasma that allowed density-arrested BALB/c-3T3 cells rendered competent by a transient exposure to platelet-derived growth factor (PDGF) to traverse G1 and enter the S phase has been termed progression activity. Epidermal growth factor (EGF) and somatomedin C-supplemented medium was shown to be capable of replacing the progression activity of 5% platelet-poor plasma (PPP) for competent density-inhibited BALB/c-3T3 cells. Exposure of competent cells to medium supplemented with EGF and somatomedin C reduced the 12 h minimum G1 lag time found in plasma-supplemented medium by 2 h. It is suggested that the reduction in the minimum time required for progression through G1 is due to the availability of free, unbound somatomedin C. Complete G1 traverse required both EGF and somatomedin C; however, the traverse of the last 6 h of G1 and entry into the S phase required only somatomedin C. Though EGF and somatomedin C could replace the G1 phase progression activity of plasma, medium supplemented with EGF and somatomedin C did not support complete cell cycle traverse or growth of sparse cultures of BALB/c-3T3 cells.     

Dimethylsulfoxide modifies the sensitivity of BALB/c-3T3 cells to the activation of Na+/H+ exchange by phorbol esters

In a companion paper (Gillies et al.: J. Cell. Physiol. 139:124-129, 1989) we show that phorbol esters (PEs) are unable to stimulate Na+/H+ exchange in BALB/c-3T3 cells under a wide variety of conditions. The Na+/H+ exchangers of a number other cell types are also not responsive to PEs yet have been rendered responsive by treatment with agents such as dimethylsulfoxide (DMSO). We undertook the present study to evaluate whether or not the treatment of BALB/c-3T3 cells with DMSO will induce modifications in the sensitivity of these cells to activation by PEs. The present study indicates that a 3-5 day exposure of BALB/c-3T3 cells to 1.25% DMSO leads to changes in the sensitivity of these cells to the activation of Na+/H+ exchanger by PEs. These changes in sensitivity were apparent at day 3 and maximal at day 5. Non-tumor-promoting analogues of PEs do not activate Na+/H+ exchange, suggesting that the effect is mediated through kinase C. Sphingosine prevents PE-, but not serum-induced alkalinization. However, the half-time of the intracellular pH (pHin) response to serum was increased by sphingosine, suggesting that kinase C participates in, but is not required for, the serum induced activation. Since DMSO does not induce any apparent morphological change, the change in sensitivity of Na+/H+ exchange to PEs is not likely to be related to differentiation, but may be associated with structural changes in the Na+/H+ exchanger and/or changes in isoforms of kinase C which recognize the exchanger as a substrate.     

Inhibition of early platelet-derived growth factor responses in BALB/c-3T3 cells by interferon

Stimulation of total inositol phosphate production, alteration of cytosolic free calcium [( Ca++]i), vinculin disruption from adhesion plaques, and DNA synthesis caused by PDGF were examined in normal and INF pretreated density arrested BALB/c-3T3 fibroblasts. In normal cells, PDGF caused an increase in total inositol phosphates, a rapid, transient increase in [Ca++]i, disappearance of vinculin from adhesion plaques, and stimulation of DNA synthesis. Pretreatment of cells with INF inhibited PDGF-stimulated increases in [Ca++]i, vinculin disruption from adhesion plaques, and DNA synthesis, but had no effect on PDGF-induced increase in total inositol phosphate levels. These findings suggest that INF prevents entry of quiescent BALB/c-3T3 cells into G1 by inhibiting PDGF-induced release of Ca++ from intracellular stores.     

Tumorigenicity of morphologically distinct transformed foci induced by 3-methylcholanthrene in BALB/c-3T3 cells

4 mm in diameter), invasiveness (smooth vs. invading margins) and other properties (piling vs. spread). In our previous report, we showed that cells from all five types grew in soft agar, transformed normal NIH 3T3 cells and formed foci on normal layer of BALB/c-3T3 cells. In this study, the neoplastic/tumorigenic potential of cells from the five different types of transformed foci was investigated in nude mice. About two million cells from each transformed focus were injected into 4-week-old nude mice. Non-transformed BALB/c-3T3 cells were used as control. The results of this study indicate that all the 45 athymic mice injected with different transformants developed tumors between 2 and 4 weeks after injection. Tumors were not observed in eight mice injected with non-transformed BALB/c-3T3 cells. All tumors were histopathologically confirmed fibrosarcomas. These findings indicate that all five morphologically different foci show tumorigenicity and that any foci of size > or =2 mm regardless of invasiveness and piling could be scored as positive during the cell transformation assay.     

Platelet-derived growth factor-induced alterations in vinculin and actin distribution in BALB/c-3T3 cells

Exposure of BALB/c-3T3 cells (clone A31) to platelet-derived growth factor (PDGF) results in a rapid time- and dose-dependent alteration in the distribution of vinculin and actin. PDGF treatment (6-50 ng/ml) causes vinculin to disappear from adhesion plaques (within 2.5 min after PDGF exposure) and is followed by an accumulation of vinculin in punctate spots in the perinuclear region of the cell. This alteration in vinculin distribution is followed by a disruption of actin-containing stress fibers (within 5 to 10 min after PDGF exposure). Vinculin reappears in adhesion plaques by 60 min after PDGF addition while stress fiber staining is nondetectable at this time. PDGF treatment had no effect on talin, vimentin, or microtubule distribution in BALB/c-3T3 cells; in addition, exposure of cells to 5% platelet-poor plasma (PPP), 0.1% PPP, 30 ng/ml epidermal growth factor (EGF), 30 ng/ml somatomedin C, or 10 microM insulin also had no effect on vinculin or actin distribution. Other competence-inducing factors (fibroblast growth factor, calcium phosphate, and choleragen) and tumor growth factor produced similar alterations in vinculin and actin distribution as did PDGF, though not to the same extent. PDGF treatment of cells for 60 min followed by exposure to EGF (0.1-30 ng/ml for as long as 8 h after PDGF removal), or 5% PPP resulted in the nontransient disappearance of vinculin staining within 10 min after EGF or PPP additions; PDGF followed by 0.1% PPP or 10 microM insulin had no effect. Treatment of cells with low doses of PDGF (3.25 ng/ml), which did not affect vinculin or actin organization in cells, followed by EGF (10 ng/ml), resulted in the disappearance of vinculin staining in adhesion plaques, thus demonstrating the synergistic nature of PDGF and EGF. These data suggest that PDGF-induced competence and stimulation of cell growth in quiescent fibroblasts are associated with specific rapid alterations in the cellular organization of vinculin and actin.