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NEUROPHARMACOLOGY

Regulation of Blood-Brain Barrier Na,K,2Cl-Cotransporter through Phosphorylation during in Vitro Stroke Conditions and Nicotine Exposure

Department of Pharmaceutical Sciences, Texas Tech University Health Sciences Center School of Pharmacy, Amarillo, Texas

Received January 28, 2004; accepted April 1, 2004.

	Abstract

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Nicotine, a major constituent of tobacco smoke, has important effects on brain recovery after focal ischemia (Wang et al., 1997). The purpose of this work is to systematically test the effects of nicotine during stroke conditions on blood-brain barrier (BBB) potassium transport, protein expression of the Na,K,2Cl-cotransporter (NKCC), and cell signaling pathways that control NKCC activity at the BBB. Confluent bovine brain microvessel endothelial cells (BBMECs) were exposed to both a hypoxic/aglycemic (H/A) environment to model BBB function during stroke conditions and nicotine and cotinine (N/C) to model plasma levels seen in smokers. BBMECs exhibit both Na,K-ATPase and NKCC activity (60 and 34 nmol/min/g, respectively) that contribute to 98% of the K⁺ uptake in cultured endothelial cells. An adaptive up-regulation of NKCC activity was identified to occur on the basolateral surface of the BBB after in vitro stroke conditions. Twenty-four hours of N/C exposure, at doses equivalent to plasma levels of smokers, combined with 6 h of H/A, reduced NKCC protein expression and total NKCC activity (shown by bumetanide-sensitive ⁸⁶RB uptake) compared with 6 h of H/A alone (P < 0.01). Basolateral K⁺ transport was found to be modulated by nicotinic acetylcholine receptors expressed at the BBB. NKCC activity on the basolateral surface of the BBB is controlled by an ongoing phosphorylation/dephosphorylation processes. We have identified a potential mechanism in altered BBB response to stroke conditions with prior N/C exposure directly implicating damage to brain-to-blood K⁺ transport mediated at the BBB and perhaps neuronal recovery after stroke.

Brain K⁺ levels are regulated at the BBB mainly by two proteins, the Na,K-ATPase and the Na,K,2Cl-cotransporter (NKCC). The Na,K-ATPase has been proposed to be present at high density in the antiluminal membrane of the BBB (Betz et al., 1980). The NKCC has been shown to be expressed in bovine (O'Donnell et al., 1995) and rat brain (Vigne et al., 1994) endothelial cells, yet its luminal versus antiluminal localization is still unknown. It is possible that either of these proteins could be involved in brain-to-blood transport of K⁺.

Additionally, cigarette smoking has been associated with an increased risk for stroke (Gill et al., 1989; Hawkins et al., 2002). Nicotine, a major constituent of tobacco smoke, has important effects on the brain in stroke. Chronic nicotine administration to rats has been shown to enhance focal brain ischemic injury and infarct size in rats using the reversible (1-h) middle cerebral artery occlusion model (Wang et al., 1997). Furthermore, the same treatment has been shown to down-regulate the expression and function of Na,K-ATPase at the BBB (Wang et al., 1994) and to enhance brain edema in stroke, and compromises blood flow in the periphery of the ischemic core (Wang et al., 1997). The mechanism(s) by which either nicotine or smoke constituents aggravate brain edema and injury in stroke have not been identified.

When studying the effects of nicotine and the major metabolite cotinine on the BBB, one has to investigate the role of nAChRs expression at the BBB. nAChRs belong to a family of ionotropic receptor proteins and are comprised of five subunits that make up a functional receptor containing a central transmembrane cation channel (Lindstrom 2000). Upon agonist stimulation this cation channel facilitates the inward movement of Ca²⁺ and Na⁺. Several laboratories have determined that non-neuronal cells can express functional nAChRs (Conti-Fine et al., 2000). Human keratinocytes, bronchial epithelial, and aortic endothelial cells have all been shown to express functional nAChRs of various subtypes (Grando et al., 1995; Maus et al., 1998; Wang et al., 2001). [³H]Nicotine binding sites have even been found in preparations of intraparenchymal cerebral microvessels and larger pial vessels from human and pig brains (Kalaria et al., 1994). Data from our laboratory strongly suggests that BBMECs express the -3, -5, -7, -2, and -3 nAChR subunit proteins (Abbruscato et al., 2002). We were also successful at reversing the BBB effects of nicotine on BBB function with a classical antagonist of nAChRs (bungarotoxin) (Abbruscato et al., 2002). These findings suggest that nAChRs modulate cellular functions outside of synaptic transmission and could have a role in nicotinic effects on the BBB during ischemia. Non-neuronal cell nAChRs have even been suggested to be involved in tobacco toxicity in tegumental type tissues, such as epithelium and endothelium (Conti-Fine et al., 2000). In the present studies, we have investigated classical agonist and antagonists of nAChRs to mimic or reverse the effects of nicotine and cotinine (N/C) on BBB mediated K⁺ transport during in vitro stroke conditions.

Because our in vitro data show that NKCC is the primary protein involved in removal of K⁺ from the ischemic brain, we also have investigated cell signaling pathways involved in altered BBB K⁺ transport via the cotransporter. In brain endothelial cells, it has been shown that protein kinase C (PKC) inhibition with staurosporine reduces basal activity of NKCC (Vigne et al., 1994). Also, PKC is believed to be the main operative mechanism involved in NKCC activity because this ion transport protein has been shown to have 10 putative sites for phosphorylation by PKC (Yerby et al., 1997). Additionally, calyculin A, an inhibitor of protein phosphatases, has been shown to stimulate NKCC activity (Vigne et al., 1994; Sun and O'Donnell, 1996). We have investigated phosphorylation and dephosphorylation pathways as a potential regulatory mechanism of the Na,K,2Cl-cotranport protein during H/A and/or N/C exposure.

Because 48 h of hypoxia has been shown to decrease the function of the BBB Na,K,-ATPase and increase the function of the BBB Na,K,2Cl-cotransporter (Kawai et al., 1996) and 14 days of nicotine exposure decreases the BBB expression of the Na,K,-ATPase (Wang et al., 1996), we decided to investigate the combined effects of H/A and N/C exposure on the function and expression of the two major K⁺ transport proteins believed to be present at the BBB. We have evaluated the effects of in vitro stroke conditions combined with N/C exposure on 1) the ability of the BBB to maintain expression polarity of NKCC, 2) activation of nAChRs expressed on endothelial cells of the BBB, and 3) cell signaling pathways involved in altered NKCC function at the BBB.

	Materials and Methods

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Cell Culturing. The isolation of BBMECs was performed using fresh bovine brains as described previously (Audus and Borchardt, 1986, 1987). Cells were seeded at a cell density of 50,000 cells/cm² onto 12-well Transwell plates (0.4-µm pore size). Prior experiments (O'Donnell et al., 1995) have shown that adding C6 conditioned medium to preconfluent endothelial cells provides the most robust Na,K,2Cl-cotransporter activity. Differentiation between luminal and abluminal endothelial surfaces was made based upon addition of C6-conditioned medium to the abluminal chamber 48 h preconfluence.

C6 cells were obtained from (American Type Cell Collection, Rockville, MD) #CC1-107 and were cultured in Ham's F-10 with 10% fetal bovine serum. Astrocyte conditioned media (ACM) was prepared by seeding C6 astrocytes at 40,000 cells/cm² and culturing to confluence and then refeeding with fresh growth media for 48 h. The resultant ACM was passed through a 0.22-µm sterile filter. After 10 days of growth, BBMECs were exposed to conditioned media (applied to the abluminal well) that consisted of a mixture of 45% fresh BBMEC growth media, 45% ACM, and 10% fetal bovine serum for 48 h. BBMECs were continuously exposed to media with an osmolality of 280 ± 5 mOsM/l as determined using an automated digital osmometer (Precision Systems Inc., Natick, MA). At day 12, confluent monolayers of BBMECs were used for measurement of K⁺ uptake or Western blot analysis (both described below).

H/A Treatment. Confluent monolayers of BBMECs were exposed to H/A conditions by adding RPMI 1640 (without L-glucose) bubbled with 95% N₂, 5% CO₂ at 3 l/min for 5 min. Hypoxia was induced by placing the cells in a humidified, sealed incubator chamber (Billups-Rothenberg, Del Ma, CA) at 37°C that had been flushed with 95% N₂, 5% CO₂. The concentration of oxygen in the atmosphere was maintained at 0%, and the PO₂ in the media was below 25 mm Hg. This H/A exposure described above has been used previously to study alterations in BBB properties (Abbruscato and Davis 1999a,b; Abbruscato et al., 2002).

Nicotine/Cotinine Treatment. Nicotine (100 ng/ml culture media) and cotinine (1000 ng/ml culture media) were added to the luminal (upper chamber) 12, 24, and 48 h before ⁸⁶Rb uptake experiments. These doses were chosen to model plasma levels seen in human smokers (Henningfield et al., 1993; Clarke et al., 1994). Levels of nicotine and cotinine were verified by HPLC analysis after a 48-h incubation and shown be >85% intact (data not shown). HPLC identification of nicotine and cotinine in the culture media after 24 h was accomplished by using the following method. Culture media (500 µl) was dispensed into a 1.5-ml centrifuge tube containing 10 µl of 2-phenylimidazole (15 ng/µl), 20 µl of 5% antifoam/phenol red solution, 50 µl of 30% NH₄OH, and 500 µl of dichloroethane and mixed by gentle inversion for 1 min. The solution was centrifuged for 15 min using a Beckman Microfuge (setting 12). The top layer was then discarded, and 400 µl of the clear bottom layer was placed into a 1.5-ml tube and evaporated under N₂ gas until dry. HPLC buffer (250 µl) was added to the tube. A standard curve was generated using culture media extractions of 0, 12.5, 25, 50, 100, and 200 ng/ml nicotine and cotinine and 150 ng of 2-phenylimidazole as the internal standard.

HPLC analyses was performed with a 0.46 x 15-cm Inertsil ODS-2 column (MetaChem Technologies Inc., Torrance, CA) at a flow rate of 1.0 ml/min at 37°C for 7.5 min and then increased to 1.5 for an additional 8 min. All samples were autoinjected using a Waters WISP 710B autoinjector. Separations were achieved using an isocratic mobile phase containing (30 mM citric acid, 30 mM monobasic potassium phosphate, 3.65 g/l triethylamine, 0.6 g/l 1-heptanesulfonic acid, and 90 ml/l acetonitrile, pH 4.8). Nicotine, cotinine, and 2-phenylimidazole were detected by a Shimadzu SPD-6A UV spectrophotometric detector (259 nm), and peaks were integrated with a Hewlett Packard 3396A integrator.

Measurement of K⁺ Uptake. All experiments were performed on BBMECs exposed to ACM, because these culturing conditions have been shown to increase BBMEC Na,K,2Cl-cotransporter activity (O'Donnell et al., 1995). To begin experiments, BBMECs were preincubated with a HEPES buffered medium ± 2 µM ouabain or 20 µM bumetanide for 15 min. ⁸⁶Rb (0.2 mCi/well) was then added, and the cells were rotated at 37°C for 10 min. In previous studies, it has been demonstrated that Rb quantitatively substitutes for K⁺ in the Na,K,2Cl-cotransport system (Owen and Prastein, 1985). The assay was terminated by rapid triple washing with ice-cold HEPES-buffered medium. BBMECs were solubilized with 1% Triton X-100, and the radioactivity present in each extract was determined by a liquid scintillation counter. Protein content was determined by using the detergent-compatible BCA assay (Pierce Chemical, Rockford, IL). K⁺ uptake into BBMECs (expressed as nanomoles per milligram of protein per minute) was calculated from the ratio of ⁸⁶Rb uptake, and the K⁺ content in the incubation buffer (Kawai et al., 1996). Bumetanide-insensitive and ouabain-insensitive K⁺ uptake were considered Na,K,-ATPase and Na,K,2Cl-cotransporter activity, respectively. For experiments that segregated luminal versus abluminal activity, both the inhibitor and the ⁸⁶Rb were added to either the upper (measuring luminal activity) or lower (measuring abluminal activity) part of the Transwell. Negligible transport of inhibitors or ⁸⁶Rb occurred within the short time course of these experiments.

Second Messenger Experiments. Staurosporine, a general protein kinase inhibitor, was incubated at a concentration of 20 nM (Kurihara et al., 2002). genistein, a potent protein tyrosine kinase inhibitor was incubated at a concentration of 50 µM (Grando et al., 1995). PMA, an activator of protein kinase C, was incubated at 100 nM (Yang et al., 2001). Calyculin A, an inhibitor of protein phosphatases 1 and 2A (Ishihara et al., 1989), was incubated at a concentration of 50 nM. All inhibitors and activators were added during both the preincubation and ⁸⁶Rb uptake time periods.

Western Blotting. Protein was isolated from BBMECs using TRI REAGENT LS (Sigma-Aldrich, St. Louis, MO) at 0.4 ml/10² cm of culture surface. Protein pellets were then air dried and dissolved in 1% SDS. The protein concentration in each tube was determined using the BCA assay (Pierce Chemical). Exactly 30 µg of protein from each sample and molecular weight markers was separated using a gradient (4–20%), Tris-glycine polyacrylamide gel (Novex, San Diego, CA). The protein markers and samples were electrophoretically transferred to a polyvinylidene difluoride membrane (Amersham Biosciences Inc., Piscataway, NJ). The polyvinylidene difluoride membranes containing the protein samples were then incubated in a blocking buffer (5% nonfat dry milk) overnight.

The antibody used for detection of the Na,K,2Cl-cotransporter was a monoclonal antibody,T4, which was developed against the carboxy-terminal 310 aa of the human colonic N,K,2Cl-cotransporter. This antibody was obtained from the Developmental Studies Hybridoma Bank (Iowa City, IA). The membrane was incubated for 2 h with the T4 monoclonal antibody at a dilution of 1:200 TBS TW-20. The membrane was washed three times in TBS TW-20 and then incubated with anti-mouse IgG-horseradish peroxidase secondary antibody for 2 h at a dilution of 1:10,000 TBS TW-20.

Antibodies used for specific nAChR subtype immunodetection (Research and Diagnostic Antibodies, Benicia, CA) were specific to either the amino- or carboxy-terminal regions (3 carboxy-terminal aa 496–503, 4 amino terminal aa 620–627, 5 carboxy terminal aa 460–468, 7 carboxy terminal aa 493–502, 2 carboxy-terminal aa 493–502, 3 carboxy-terminal 450–458, and 4 carboxy terminal aa 490–498). The above-mentioned primary antibodies were incubated at a dilution of 1:200 TBS TW-20 for 2 h. The membrane was washed three times in TBS TW-20 and then incubated with anti-rabbit IgG-HRP secondary antibody for 2 h at a dilution of 1:2000.

After washing three times with TBS TW-20, the NKCC and nAChR signal was detected by enhanced chemiluminescence (Amersham Biosciences Inc.).

Statistical Methods. For all experiments, the data are presented as the mean ± S.E.M. Statistical analysis of the data were done with the use of one-way analysis of variance (ANOVA) with Newman-Keuls multirange post hoc comparison of the means (Bruning and Kintz, 1977).

	Results

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K⁺ Uptake after H/A and/or N/C Exposure. Total BBMEC K⁺ transport (luminal and abluminal) consists of Na,K,-ATPase (60 nmol/min/mg) and NKCC (34 nmol/min/mg) activity (Fig. 1). The activity of these two ion transport proteins contributes to 98% to total BBMEC K⁺ uptake. In vitro stroke conditions (6 h of H/A) significantly reduced K⁺ uptake due to the Na,K-ATPase (P < 0.05) and significantly increased K⁺ uptake due to NKCC (P < 0.01). Twenty-four hour exposure to nicotine (100 ng/ml) and cotinine (1000 ng/ml) significantly reduced the amount of H/A-stimulated BBMEC K⁺ uptake due to NKCC (P < 0.01). Control experiments also were performed to show the effects of removing either Na⁺ or Cl^– from the preincubation and assay buffer on NKCC activity measured as ouabain-insensitive K⁺ influx. We replaced Na⁺ and Cl^– isotonically with choline and gluconate, respectively. In either case, negligible ouabain-insensitive K⁺ influx was observed, ensuring that we are measuring cotransporter activity (Fig. 2).

View larger version (18K): [in this window] [in a new window]   Fig. 1. Effects of H/A and/or N/C exposure on total K+ uptake (luminal and abluminal) into BBMECs cultured with ACM. Bumetanide (20 µM)-insensitive and ouabain (2 µM)-insensitive K+ uptake (using 86Rb as a replacement for K+) are expressed as Na,K-ATPase and Na,K,2Cl-cotransporter activity, respectively. Data represent mean ± S.E.M. of six independent determinations. **, P < 0.01 and *, P < 0.05 using one-way ANOVA and Newman-Keuls post hoc analysis.

View larger version (13K): [in this window] [in a new window]   Fig. 2. Na+ and Cl– dependence of BBMEC K+ uptake. Oubain (2 µM)-insensitive K+ uptake (using 86Rb as a replacement for K+) is expressed as Na,K,2Cl-cotransporter activity. Cells assayed in Na–- or Cl–-free media were both preincubated and assayed in HEPES-buffered minimal essential medium in which Na+ was isosmotically replaced with choline and Cl– was isosmotically replaced with gluconate. Data represent mean ± S.E.M. of six independent determinations. **, P < 0.01 using one-way ANOVA and Newman-Keuls post hoc analysis.

Experiments also were designed to separate luminal from abluminal NKCC activity (Fig. 3, A–C). K⁺ uptake due to NKCC was on average 54% greater on the abluminal side of the endothelial cell compared with the luminal side. Only abluminal BBMEC K⁺ was sensitive to either N/C exposure and/or H/A. These observations occur at all N/C exposure (12, 24, and 48 h).

View larger version (37K): [in this window] [in a new window]   Fig. 3. A to C, contribution of abluminal and luminal Na,K,2Cl-cotransporter to total K+ uptake into BBMECs after H/A and/or N/C exposure. Ouabain (2 µM)-insensitive K+ uptake is considered Na,K,2Cl-cotransporter activity. A to C represent contribution of the luminal and abluminal membranes to total BBMEC K+ uptake after 12, 24, and 48 h of nicotine (100 ng/ml) and cotinine (1000 ng/ml), respectively. Data represent mean ± S.E.M. of six independent determinations. **, P < 0.01 compared with normal conditions. , P < 0.01 compared with 6-h H/A treatment using one-way ANOVA and Newman-Keuls post hoc analysis.

We also have identified (Fig. 4) that nicotine alone (100 ng/ml for 24 h) reduced hypoxic/aglycemic induced Na,K,2Cl-cotransporter activity (P < 0.01) on the abluminal sides of BBMECs to a comparable level as seen with the combination of nicotine and cotinine for 24 h. Additionally, cotinine had no effect on H/A-induced abluminal Na,K,2Cl-cotransporter activity. We also tested different doses of N/C and determined that only a dose equivalent to plasma levels of smokers resulted in a significant decrease (P < 0.01) in H/A-induced NKCC activity and doses below that had no significant effects.

View larger version (24K): [in this window] [in a new window]   Fig. 4. Effects of nicotine or cotinine and subtherapeutic doses of N/C on H/A induction of abluminal Na,K,2Cl-cotransporter activity. Na,K,2Cl-cotransporter activity is expressed as ouabain (2 µM)-insensitive K+ uptake, using 86Rb as a replacement for K+. Data represent mean ± S.E.M. of six independent determinations. **, P < 0.01 significantly different from normal conditions using one-way ANOVA and Newman-Keuls post hoc analysis.

Western Blotting of NKCC and nAChR Subunits. The monoclonal antibody T4, which was developed against the carboxy-terminal 310 aa of the human colonic N,K,2Cl-cotransporter, recognized a protein in cultured BBMECs and rat kidney microsomes that was 145 kDa (Fig. 5). A statistically significant (P < 0.01) increase in immunoreactivity was observed in the 145-kDa protein band after 6-h H/A treatment. Similar to the ⁸⁶Rb⁸⁶ experiments, 24-h N/C exposure significantly (P < 0.01) attenuated the H/A induction of NKCC protein expression.

View larger version (19K): [in this window] [in a new window]   Fig. 5. Western blot analyses indicate alterations in NKCC expression in BBMECs exposed to N/C and/or H/A conditions. Exposures consisted of either 6-h H/A and/or nicotine (100 ng/ml) and cotinine (1000 ng/ml) for 24 h. The T4 monoclonal antibody is specific for the carboxy terminal 310 aa of the human colonic Na,K,2Cl-cotransporter. Exactly 30 µg of protein was loaded per well as determined by a BCA protein assay (Pierce Chemical). Protein sample and molecular weight markers were separated on a 4 to 20% SDS-polyacrylamide gradient gel. Six hours of H/A exposure significantly (**, P < 0.01) increased the percentage of relative content of NKCC compared with control, and the combination of 6-h H/A and 24-h N/C significantly (##, P < 0.01) reduced the expression of NKCC compared with 6-h H/A alone. n = 4 monolayers/treatment and statistical significance determined using one-way ANOVA and Newman-Keuls post hoc analysis. Inset, representative blot image showing mobility of the BBMEC Na,K,2Cl-cotransporter is 145 kDa, which is equivalent to that detected in rat kidney microsomes.

Western blot analysis also was used to determine the dose-response effects of N/C on nAChR subunit expression (Fig. 6). The polyclonal antibody recognized a protein in BBMECs that was 55 kDa for the 3, 5, 7, 2, and 3 nAChR subunit experiments. The 55-kDa protein band was not recognized when using the 4 and 4 subunit-specific antibody (data not shown). A dose-dependent decrease in nAChR subunit expression was observed after a 24-h N/C exposure with concentrations of 1 x 10^2–5 ng/ml for nicotine and 1 x 10^3–6 ng/ml for cotinine (lanes 3–6). For all subunits tested, lane 7 represents an additional exposure of BBMECs to nicotine (100 ng/ml) and cotinine (1000 ng/ml), a concentration equivalent to plasma levels in smokers (same as lane 3).

View larger version (31K): [in this window] [in a new window]   Fig. 6. Western blot analyses indicate alterations in nAChR subunit protein in BBMECs incubated with nicotine and cotinine. Exposures consisted of control (lane 1), 10 ng/ml nicotine and 100 ng/ml cotinine (lane 2), 100 ng/ml nicotine and 1000 ng/ml cotinine (lane 3), 1000 ng/ml nicotine and 10,000 ng/ml cotinine (lane 4), 10,000 ng/ml nicotine and 100,000 (lanes 5), 100,000 ng/ml nicotine and 1,000,000 ng/ml cotinine (lane 6), and plasma equivalents to smokers (100 ng/ml nicotine and 1000 ng/ml cotinine, lane 7). The specific nAChR subunit antibodies consistently recognized a protein in cultured BBMECs that was approximately 55 kDa for the 3, 5, 7, 2, and 3 nAChR subunit experiments. Exactly 30 µg of protein was loaded per well as determined by a BCA protein assay (Pierce Chemical). Protein sample and molecular weight markers were separated on a 4 to 20% SDS-polyacrylamide gradient gel. Statistically significant reductions in nAChR subunit expression were observed for all subunits detected, yet the N/C concentration required for this reduction varied. n = 4 to 6 monolayers/treatment and **, P < 0.01 and *, P < 0.05 compared with control using one-way ANOVA and Newman-Keuls post hoc analysis. Inset, representative blot images showing the mobility of all nAChR subunit proteins is 55 kDa, and this protein band was not recognized when using the 4 and 4 subunit-specific antibody (data not shown).

Pharmacological Activation and Inhibition of BBMEC nAChRs. BBMECs are sensitive to classical agonists and antagonists of nAChRs. Figure 7 clearly demonstrates that epibatidine, a potent agonist for nAChR, mimics the actions of nicotine by attenuating the increased abluminal K⁺ uptake induced by 6-h H/A at concentrations of 1 nM (P < 0.05), 10 nM (P < 0.01), and 100 nM (P < 0.01). Additional experiments confirmed that the noncompetitive antagonist mecamylamine and competitive antagonist bungarotoxin reverse the effects of N/C on H/A induction of abluminal NKCC activity at concentrations 100 µM (P < 0.05) and 500 µM (P < 0.01) for mecamylamine and 1 nm (P < 0.05) and 10 nm (P < 0.01) for bungarotoxin.

View larger version (34K): [in this window] [in a new window]   Fig. 7. Effects of a nAChR agonist and antagonists on BBMEC K+ uptake after 6-h H/A. Na,K,2Cl-cotransporter activity is expressed as ouabain (2 µM)-insensitive K+ uptake, using 86Rb as a replacement for K+. Epibatidine acts as a potent nAChR agonist, mecamylamine is a noncompetitive antagonist, and bungarotoxin is a competitive antagonist. Data represent mean ± S.E.M. of eight independent determinations. **, P < 0.01 significantly different from normal conditions; ##, P < 0.01; and #, P < 0.05 significantly different from 6-h H/A. , P < 0.01 and , P < 0.05 significantly different from 6-h H/A and 24-h N/C, all using one-way ANOVA and Newman-Keuls post hoc analysis.

Second Messenger Signaling Controlling NKCC Activity. We studied the possible cell-signaling pathways responsible for N/C-induced down-regulation of abluminal Na,K,2Cl-cotransporter activity during stroke conditions. We have determined (Fig. 8) that both basal and stroke (6-h H/A)-induced Na,K,2Cl-cotransporter activity is controlled by protein kinase C, because 20 nM staurosporine inhibits and 100 nM PMA induces cotransporter activity compared with each respective control experiment. Interestingly, tyrosine kinase also controls basal Na,K,2Cl-cotransporter activity, but not stroke (6-h H/A)-induced activity, as evidenced by genistein inhibition only during normal conditions. Additionally, combining both insults (6-h H/A with 24 h of N/C) resulted in an expected decrease in abluminal Na,K,2Cl-cotransporter activity. This double insult was not inhibited by either 20 nM staurosporine or 50 nM genistein, suggesting that PKC or tyrosine kinase is not involved in nicotine induced attenuation of cotransporter activity during H/A induction. Yet, 50 nM calyculin A was found to prevent nicotine's effects of attenuating cotransporter activity during H/A. This suggests that nicotine effects are mediated through the protein phosphatase pathway and this mechanism could prevent PKC induction of Na,K,2Cl-cotransporter activity at the abluminal side of the BBB during stroke conditions.

View larger version (26K): [in this window] [in a new window]   Fig. 8. Regulation of abluminal Na,K,2Cl-cotransport by intracellular messengers. Staurosporine, a general protein kinase inhibitor, was incubated at a concentration of 20 nM. Genistein, a potent protein tyrosine kinase inhibitor, was incubated at a concentration of 50 µM. PMA, an activator of protein kinase C, was incubated at 100 nM. Calyculin A, an inhibitor of protein phosphatases 1 and 2A, was incubated at a concentration of 50 nM. All inhibitors and activators were added during both the preincubation and 86Rb uptake time periods. Data represent mean ± S.E.M. of six independent determinations. **, P < 0.01 and *, P < 0.05 compared with their respective control conditions using one-way ANOVA and Newman-Keuls post hoc analysis.

	Discussion

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We measured K⁺ uptake (using ⁸⁶RB⁺ as a tracer for K⁺) through a combination of both ouabain (2 µM)-sensitive K⁺ uptake (Na,K,ATPase) and bumetanide (20 µM)-sensitive K⁺ uptake (NKCC). Six hours of H/A, an in vitro model of stroke (Abbruscato and Davis 1999a,b), caused a 62% decrease in Na,K,ATPase activity and a 91% increase in NKCC activity (Fig. 1) These results suggest that increased BBB NKCC activity after H/A conditions may provide an additional mechanism for the removal of excess brain K⁺, specifically by the antiluminal NKCC, which shuttles K⁺ from the brain extracellular fluid to the blood. Similar results have been made in rat brain endothelial using longer periods of hypoxia without controlling glucose levels (24 h) (Kawai et al., 1996). We also determined the effects of incubating confluent BBMECs with nicotine (100 ng/ml) and cotinine (1000 ng/ml) on H/A induction of cotransporter activity. It was observed that N/C exposure, at levels equivalent to average plasma levels of smokers (Abbruscato et al., 2002), attenuated the H/A induction of NKCC function. These results are interesting in light of the fact that preexposure to N/C could effect the ability of the ischemic brain-to-efflux K⁺ ions back into the blood during the reestablishment of neuronal conduction. Additional experiments were conducted to determine the polarity (apical versus basolateral) expression of NKCC at the BBB. When BBMECs were cultured with ACM 48 h preconfluence, NKCC activity was determined to be greater on the abluminal side of the BBB. Abluminal NKCC was also significantly decreased with 12, 24, and 48 h N/C exposure combined with 6-h H/A exposure compared with 6-h H/A exposure alone (Fig. 3, A–C), whereas luminal NKCC activity was not changed. These experiments provide evidence that abluminal BBB K⁺ transport mechanisms during H/A are modulated N/C exposure, and this could influence ischemic brain K⁺ homeostasis in crucial times of neuronal recover after stroke.

More specific experiments also were designed to determine the independent effects of nicotine and cotinine on abluminal NKCC activity. We determined (Fig. 4) that nicotine alone (100 ng/ml for 24 h) reduced H/A-induced NKCC activity on the abluminal sides of BBMECs to a comparable level as seen with the combination of N/C for 24 h. Additionally, cotinine had no effect on H/A-induced NKCC activity. We also tested different doses of the combination of N/C and determined that only a dose equivalent to plasma levels of smokers resulted in a significant decrease in H/A-induced NKCC activity (100 ng/ml nicotine and 1000 ng/ml cotinine), and doses below that had no significant effects (Fig. 4). This suggests that drug therapies using nicotine at lower doses have no effects on BBB NKCC activity.

We observed a correlation when comparing ⁸⁶Rb⁸⁶ uptake data to the BBMEC protein expression of NKCC subjected to the same experimental paradigm described in Figs. 1 and 3. H/A conditions induced NKCC protein expression that was reversed with 24-h exposure to N/C (Fig. 5). Using the T4 monoclonal antibody specific for the carboxy-terminal portion (MET-902 to Ser-1212) of the human colonic T84 epithelial Na,K-2Cl-cotransporter, we see specific immunoreactivity with the fully glycosylated version of NKCC1 (145 kDa) and faint immunoreactivity for a low molecular weight, nonglycosylated form in both BBMECs and rat kidney control. These results confirm that the loss in NKCC function is most likely due to a reduction in total NKCC protein with the conditions tested in these studies.

An obvious target for nicotine action at the BBB is nAChRs expressed on endothelial cells of the cerebrovasculature. Previous studies have shown that BBMECs express -3, -5, -7, -2, and -3 nAChR subunit protein and do not express -4 and -4 nAChR subunit protein (Abbruscato et al., 2002). Additionally, N/C exposure was found to reduce -7 and -2 nAChR subunit protein expression in a time-dependent manner. In the present study, we determined the effects of exposing BBMECs to increasing doses of N/C on nAChR subunit protein expression. In all cases, we observed a dose-dependent decrease in -3, -5, -7 and -2, -3 nAChR subunit protein (Fig. 6). Interestingly, in all cases we observed this dose-dependent decrease in nAChR subunit protein expression occurred at a dose equivalent to plasma levels of smokers (100 ng/ml nicotine and 1000 ng/ml cotinine; Figure 6, lane 3). Future in vivo experiments are required to determine the effects of chronic exposure to N/C on brain microvascular nAChR expression.

N/C modulation of abluminal NKCC function during H/A was found to be both mimicked and reversed by classical agonist and antagonists of nAChRs. We incubated BBMECs with epibatidine, a potent natural agonist of nAChRs, and reversed the effect of H/A on abluminal NKCC activity to a level compared with the N/C exposure (Fig. 7). Additionally, we could antagonize the effects of N/C on H/A-induced NKCC activity with both bungarotoxin (competitive antagonist) and mecamylamine (noncompetitive antagonist), suggesting an nAChR-mediated response (Fig. 7). These experiments suggest that functional nAChRs are present on BBMECs, and they are activated at nicotine levels equivalent to smokers (0.1–1 µM).

The activity of NKCC protein is known to be modulated by pharmacological agents and conditions that change the phosphorylation state of the cotransporter. In general, conditions that promote phosphorylation of the NKCC activate the protein (Lytle and Forbush, 1992; Lytle and Forbush, 1996; Payne et al., 2001). We examined the modulation of BBMEC NKCC activity by reagents known to affect the phosphorylation state and activity of NKCC (Fig. 8). Our data support the hypothesis that basal NKCC activity on the abluminal side of BBB is regulated by protein kinases, because staurosporine pretreatment resulted in a statistically significant reduction in cotransporter activity (P < 0.05). Additionally, basal NKCC activity at the abluminal side of the BBB also is controlled by protein tyrosine kinases, because genistein reversed the normal activity of this cotransporter to statistically significant level (P < 0.05). Calyculin A, a protein phosphatase inhibitor known to lead to increased phosphorylation and activation of NKCC (Lytle and Forbush, 1996), markedly stimulated abluminal NKCC activity in BBMECs compared with control levels (P < 0.01). These results suggest that protein phosphatases must be involved in controlling the activity of NKCC on the abluminal side of the BBB during basal conditions. Interestingly, we also observed that H/A induction of abluminal NKCC activity in BBMECs was sensitive (P < 0.01) to treatment with 20 nM staurosporine, a general protein kinase inhibitor, but not sensitive to treatment with 50 µM genistein, an inhibitor of protein tyrosine kinase, suggesting that protein tyrosine kinase is not involved in H/A induction of abluminal NKCC activity. In all cases, PMA, a general PKC activator, augmented NKCC activity, suggesting a role for PKC. Because PMA does not stimulate all PKC isoforms and the inhibitors used in these studies block a broad range of kinases, future experiments will test the role of conventional, novel, and atypical PKC isoforms on regulation of NKCC activity.

When testing conditions of both H/A and N/C exposure, we observed that only 50 nM calyculin A was able to increase abluminal cotransporter activity (no effects by modulating protein kinase, PKC, or protein tyrosine kinase). These results suggest that nicotine effects are mediated through the protein phosphatase pathway, and this mechanism could prevent PKC induction of NKCC activity on the abluminal side of the BBB during stroke conditions (Fig. 9). Currently, our studies suggest that nAChR activation coupled to hypoxia/aglycemic (right side of schematic) could increase brain endothelial cell calcium levels to a point whereby the cytoplasmic pool of PKC is depleted (1). Additionally, nicotine also may activate protein phosphatase, which could either dephosphorylate PKC (2) and prevent translocation to the membrane for activity, or dephosphorylate the Na,K,2Cl-cotransporter directly (3), thus inhibiting its function. In the future, we plan to identify the specific PKC and protein phosphatase isoforms responsible for these effects and directly measure the phosphorylation state of BBMEC NKCC after stroke conditions with and without prior nicotine exposure. A better understanding of the mechanisms that regulate expression and activity of this key carrier protein after the above-mentioned insults potentially could lead to approaches that will protect the central nervous system from neurological damage associated with nicotine, smoke constituent, and/or ischemic insults. These experiments may provide important information about the long-term effects of nicotinic therapeutics that currently are under investigation for treatments of Alzheimer's and Parkinson's Disease.

View larger version (28K): [in this window] [in a new window]   Fig. 9. Cell signaling mechanisms involving ion transport during stroke with and without nicotine exposure. Our studies suggest that luminal nAChR activation coupled to hypoxia/aglycemic (right side of schematic) could increase brain endothelial cell calcium levels to a point whereby the cytoplasmic pool of PKC is depleted (1). Additionally, nicotine also may activate protein phosphatase, which could either dephosphorylate PKC (2) and prevent translocation to the membrane for activity or dephosphorylate the Na,K,2Cl-cotransporter directly (3), thus inhibiting its function.

We have determined that basal level activity of the NKCC is maintained by ongoing phosphorylation/dephosphorylation processes. Activation and inhibitions via phosphorylation may be dependent on the phosphorylation of AA sites on the cotransporter that result in either activation or inhibition of activity during H/A conditions or N/C exposure. Additionally, constitutively active kinases may exist that phosphorylate the cotransporter to maintain basal activity.

	Footnotes

 
This work was supported by the Texas American Heart Association Beginning Grant-in-Aid 0265220Y and National Institutes of Health R01 NS046526 (to T.J.A.). We also thank the Vascular Biology Research Center and Stroke Research Center at Texas Tech University Health Sciences Center for additional support.

DOI: 10.1124/jpet.104.066274.

ABBREVIATIONS: BBB, blood-brain barrier; NKCC, Na,K,2Cl-cotransporter; nAChR, nicotinic acetylcholine receptor; N/C, nicotine and cotinine; PKC, protein kinase C; H/A, hypoxia/aglycemia; ACM, astrocyte conditioned media; HPLC, high-performance liquid chromatography; PMA, phorbol 12-myristate 13-acetate; aa, amino acid(s); BBMEC, bovine brain microvessel endothelial cell; TBS TW-20, Tris-buffered saline Tween 20; ANOVA, analysis of variance.

Address correspondence to: Dr. Thomas J. Abbruscato, Texas Tech University Heath Sciences Center, School of Pharmacy, 1300 Coulter, Amarillo, TX 79106. E-mail: tja{at}ama.ttuhsc.edu

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