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NEUROPHARMACOLOGY
Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, Texas
Received June 7, 2005; accepted September 16, 2005.
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). Endothelial cells of the cerebromicrovasculature form tight junctions that provide a physical barrier between blood and the central nervous system that can regulate transport of ions into and out of the brain (Brightman and Reese, 1969). A properly functioning blood-brain barrier will maintain brain homeostasis of key ions such as Na+, K+, and Cl-, all of which are important for mitigating neuronal action potentials (Keep et al., 1993). Edema associated with ischemia may be caused by two mechanisms, either vasogenic or cellular (Brillault et al., 2002). Increase of intracerebral pressure due to edematous swelling leads to further ischemia and may result in death (MacGregor et al., 2003). During ischemia following brain trauma, neurons undergo cellular swelling due to the depolarizing action of GABAA receptors and a resulting influx of Cl- and K+ (Payne et al., 2003). This damage is referred to as cellular edema and occurs after vasogenic edema, which is observed as blood-brain barrier opening (Betz et al., 1994; Kawai et al., 1996).
Two transporters of the blood-brain barrier important in brain K+ ion homeostasis include Na,K,ATPase and the Na,K,2Cl-cotransporter (NKCC) (Abbruscato et al., 2004). Recently, we demonstrated that hypoxia/aglycemia in bovine brain microvessel endothelial cells (BBMECs) increases the activity of the NKCC by 91% and decreases the activity of Na,K,ATPase by 62% (Abbruscato et al., 2004). NKCC functions as a cation-chloride cotransporter that can be modulated by alterations of the extracellular and intracellular ion gradients. Two NKCC isoforms are believed to exist, including NKCC1 and NKCC2, both with unique distributions. NKCC2 is present mostly in the epithelial cells of the thick ascending limb of the Loop of Henle of the kidneys (Haas and Forbush, 2000). NKCC1 is more ubiquitously expressed in secretory epithelial and endothelial cells (Haas et al., 1995; Yerby et al., 1997). The activity of NKCC1 is important in brain extracellular ion homeostasis and is present not only in brain microvascular endothelial cells but also in neurons and astrocytes (O'Donnell et al., 1995; Payne et al., 2003; Lenart et al., 2004).
Previously, our lab has shown that stroke conditions mimicked in primary cultures of endothelial cells through 6 h of exposure to hypoxia/aglycemia raised K+ uptake due to increased actions of abluminal NKCC1 expressed at the blood-brain barrier. This alteration of NKCC1 abluminal activity suggests a protective function of the blood-brain barrier during traumatic brain events such as stroke by shuttling Na+, K+, and 2 Cl- away from the brain extracellular space (Abbruscato et al., 2004). A decrease in the amount of K+ and Cl- from brain extracellular fluid would be neuroprotective and reduce the ability of GABAA receptors to continually depolarize and cause cellular swelling (Payne et al., 2003).
Cigarette smoking has been associated with a greater incidence of stroke (Gill et al., 1989; Hawkins et al., 2002). Up to one-quarter of the 700,000 strokes in the United States can be attributed to cigarette smoking (Hankey, 1999). Smoking alone without any other contributing factors increases the risk of stroke by more than 1.5 times in men and women (Hawkins et al., 2002). Worsening of the outcome of stroke, which is the focus of this work, has been shown after chronic nicotine exposure in a focal middle cerebral artery occlusion model showing an increase in edema and infarct ratio (Wang et al., 1997). Also, we have shown that the normal increase of blood-brain barrier abluminal NKCC1 activity during hypoxia/aglycemia is hindered by the presence of nicotine (100 ng/ml) and cotinine (1000 ng/ml) (Abbruscato et al., 2004). Although nicotine has been shown to increase the incidence of stroke and negatively impact the outcome of stroke, the effects of nicotine in the presence of the more than 4000 chemicals present in cigarette smoke have not been explored in models of stroke (Brunnemann and Hoffmann, 1991).
To better understand the effects of cigarette smoke constituents on NKCC activity during hypoxia/aglycemia, nicotine/cotinine, nicotine-containing cigarette smoke extract (N-CSE), or nicotine-free cigarette smoke extract (NF-CSE) were administered to BBMECs during paradigms of normoxia, hypoxia/aglycemia, and hypoxia/aglycemia with reoxygenation. These paradigms were designed to mimic the effects of normal state, brain ischemia, and the recovery period after brain ischemia on the activity of blood-brain barrier endothelial cells to move K+ ions. Additionally, the effects of nicotine, N-CSE, and NF-CSE on an in vivo model were explored with a four-vessel occlusion model of global ischemia to estimate the acute effects of tobacco smoke chemicals on brain extracellular fluid K+ (Pulsinelli and Brierley, 1979).
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). First passage cells were cultured on 12-well transwell plates (0.4-µm pore size) that were pretreated with collagen and fibronectin at a cell density of 50,000 cells/cm2 and grown to confluence in 12 days. To achieve the greatest NKCC activity, a mixture of astrocyte-conditioned media was placed in the abluminal chamber 48 h preconfluence (O'Donnell et al., 1995; Abbruscato et al., 2004). BBMECs were treated with astrocyte-conditioned media consisting of 45% BBMEC growth media, 45% astrocyte media, and 10% fetal bovine serum. Astrocyte media was prepared by culturing C6 cells (no. CC1-107; American Type Culture Collection, Manassas, VA) in Ham's F-10 with 10% fetal bovine serum. The C6 cells were seeded at 40,000 cells/cm2 and grown to confluence in 75-ml flasks. Fresh media in the flasks were replaced every 48 h and sterile filtered through a 0.22-µm filter. Media for the BBMECs were maintained at 280 ± 10 mOsM/l (Abbruscato et al., 2004). [14C]Sucrose permeability and K+ uptake were measured on the BBMEC confluent monolayers on the 12th day.
Hypoxia/Aglycemia ± Reoxygenation. Experimental conditions for the confluent BBMEC monolayers included 6 h of hypoxia/aglycemia ± 12 h of reoxygenation. Aglycemic media was prepared with RPMI 1640 (without L-glucose) and bubbled for 5 min with 1% O2/99% N2. Hypoxic conditions were maintained by placing the confluent BBMEC monolayers in a custom hypoxia polymer glove box (Coy Laboratories, Grass Lake, MI) infused with 1% O2/99% N2 at 37°C. These methods have previously been used to evaluate effects of hypoxia/aglycemia on blood-brain barrier characteristics (Abbruscato and Davis, 1999; Abbruscato et al., 2004). Reoxygenation of the 6-h hypoxia/aglycemia-treated cells was achieved by replacing the aglycemic media with BBMEC growth media and kept in an incubator for 12 h with 95% room air and 5% CO2. The concentration of oxygen in the atmosphere was maintained at 1%, and the PO2 in the media was <25 mm Hg. Such hypoxia/aglycemia exposure as described above has been used previously to study alterations in blood-brain barrier properties (Abbruscato and Davis, 1999; Mark and Davis, 2002). Reoxygenation produced a rapid return to control PO2 levels in the medium within 5 min, as previously reported (Mark and Davis, 2002).
Smoke Constituents. BBMECs were treated in the luminal chamber 24 h before hypoxia/aglycemia ± reoxygenation with nicotine (100 ng/ml) and cotinine (1000 ng/ml), N-CSE, or NF-CSE. The concentrations of nicotine and cotinine were chosen to mimic plasma concentrations detected in heavy smokers (Henningfield et al., 1993).
Cigarettes (Marlboro filter cigarettes; Philip Morris Inc., Richmond, VA; and Quest no. 3, Vector Tobacco Inc., Research Triangle Park, NC) were obtained through commercial sources. The Marlboro filter contains 1.1 mg of nicotine/cigarette, and Quest no. 3 contains no more than 0.05 mg of nicotine/cigarette and is therefore regarded to be nicotine-free. Mainstream smoke was bubbled through 50 ml of acetone by a slight vacuum drawn repetitiously through a custom-manufactured apparatus. To simulate a smoker's smoking behavior, each cigarette was lighted and smoked for a total of 5 min with alternating increments of smoking (vacuum, 3 s) and rest (no vacuum, 30 s). A CSE-acetone solution was generated that was concentrated under vacuum. The residue was subsequently redissolved in 1.626 ml of propylene glycol/dimethyl sulfoxide (1:1). The products of the Marlboro and Quest cigarette extraction were designated as N-CSE and NF-CSE, respectively. The concentration of nicotine in N-CSE was approximated at 0.677 mg/ml for Marlboro cigarette extract assuming 1.1 mg of nicotine is present per cigarette. These methods have successfully been used in vitro to model exposure to cigarette smoke chemicals (Sherratt et al., 1988).
Na,K,2Cl-Cotransporter Activity. The abluminal uptake of K+ into BBMECs was performed through 86Rb uptake studies for 10 min at room temperature with or without 2 µM ouabain as described previously (Abbruscato et al., 2004). Earlier studies have clearly shown that Rb quantitatively substitutes for K+ in the NKCC system (Owen and Prastein, 1985). Ouabain-insensitive 86Rb uptake was considered to be Na,K,2Cl-cotransporter activity. Our group has also shown that this ouabain-insensitive 86Rb uptake is both Na+- and Cl--dependent, ensuring that we are measuring NKCC activity (Abbruscato et al., 2004).
BBMEC Permeability Coefficient Calculation. Apical-to-basolateral [14C]sucrose permeability across BBMECs was expressed as a permeability coefficient (PC) (centimeters per minute x 10-4). The 12-well transwell plates were allowed to equilibrate in the hypoxia glove box in HEPES-buffered transport medium for 15 min at 37°C. Permeation of [14C]sucrose was measured across BBMECs using an adaptation of described methods (Dehouck et al., 1992; Rose and Audus, 1998). The flux was determined by dividing the amount (in picomoles) of radioactive drug appearing in the receiver chamber by the time in minutes (sampling times were 15, 30, and 60 min). The apparent permeability coefficient was then calculated using the equation PC = flux/(A x Cdo), where flux is the slope of the line (picomoles of [14C]sucrose versus min), A is the area of the membrane (1.0 cm2), and Cdo is the initial donor concentration.
Smoke Constituent Exposure. The nicotine regimen (4.5 mg/kg/day) maintains nicotine and cotinine plasma concentrations (45.04 ± 20.33 and 269.15 ± 74.64 ng/ml, respectively, confirmed by HPLC analysis, data not shown) comparable with those observed in chronic and heavy smokers (two to three packs per day) (Murrin et al., 1987; Wang et al., 1997). We calculated doses for CSEs using the body weight of the rats to deliver an equivalent amount of nicotine equal to a 4.5 mg/kg/day dose. Vehicle, nicotine (N), N-CSE, and NF-CSE were administered i.p. for 100 min prior to the 20-min sampling period during reperfusion allowing for a 2-h experiment. Equivalent doses of N-CSE and NF-CSE were administered to rats. For in vitro experiments, we exposed cells to nicotine (100 ng/ml) and cotinine (1000 ng/ml) for 24 h and extrapolated equivalent doses for the N-CSE and NF-CSE groups.
Global Ischemia in the Rat. All studies were approved by the Institutional Animal Care and Use Committee of Texas Tech University Health Sciences Center and were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Transient global forebrain ischemia was performed to simulate cerebral ischemia, according to the four-vessel occlusion model (Pulsinelli and Brierley, 1979). Rats were anesthetized with a cocktail of ketamine HCL (78.3 mg/ml), xylazine (3.1 mg/ml), and acepromazine maleate (0.6 mg/ml) at a volume of 1 ml/kg before both common carotid arteries were isolated via a ventral midline cervical incision. An arterial clasp was placed around each common carotid artery without interrupting blood flow, and the incision was closed with a single suture. A second incision was made behind the occipital bone that overlies the two cervical vertebrae. The right and left alar foramen and both vertebral arteries were then electrocauterized and permanently occluded. These procedures result in consistent bilateral hemispheric ischemia with a high incidence of predictable brain damage in the hippocampus, neocortex, and striatum (Pulsinelli and Brierley, 1979).
Rats were allowed to recover from the surgery, and control parameters were monitored. Awake rats were restrained, the ventral neck suture removed, and the carotid clasps tightened to produce four-vessel occlusion. Carotid clasps were removed after 5 min of four-vessel occlusion, and restoration of carotid blood flow was verified by laser Doppler flowmetry. Animals were then subjected to in vivo brain microdialysis as described below.
In Vivo Brain Microdialysis. The methods for in vivo microdialysis have been described previously (Allen et al., 1995). Rats were cannulated via femoral vein with PE 50 or 10 tubing to enable peripheral i.v. 86Rb injection with a specific activity of 7.68 mCi/mg. Microdialysis probe implantation into the frontal cortex was conducted in anesthetized rats, the heads of which remained secured in a stereotaxic frame throughout the experiment. Probes were implanted in the frontal cortex according to published coordinates (Paxinos et al., 1980). Ketamine/xylazine was used as the anesthetic agent with supplements as needed to suppress moderate foot pinch. Body temperature was maintained at 37°C by a rectal probe connected to a feedback device and a heated blanket. Microdialysis samples were collected for 20 min starting immediately after four-vessel occlusion. A sample of blood was drawn from the femoral vein cannulae during reperfusion and centrifuged to determine the counts of 86Rb in plasma. The disintegrations per minute detected in the brain extracellular fluid (disintegrations per minute per milliliter) were then expressed as a ratio of the disintegrations per minute detected in the plasma (disintegrations per minute per milliliter) and termed RECF. Due to the possibility of declining plasma concentration of 86Rb over the course of the experiment, there may be a slight overestimation in RECF, yet the significance is not altered for an endpoint comparison. The timeline of the in vivo experimental paradigm is explained in Fig. 1.
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Hypoxia and Aglycemia with Reoxygenation. Combining 6 h of hypoxia/aglycemia with 12 h of reoxygenation showed a continued increase in abluminally mediated K+ uptake compared with control (p < 0.01). Both nicotine and cotinine and N-CSE significantly decreased (p < 0.01) the abluminal K+ uptake following 6 h of hypoxia/aglycemia and 12 h of reoxygenation (Fig. 4, left), whereas the NF-CSE was ineffective. Viability of the cellular monolayer is maintained following 6 h of hypoxia/aglycemia combined with 12 h of reoxygenation with a return in sucrose permeability values back to basal levels (no different from normoxic controls). Sucrose permeability was significantly increased only with the presence of nicotine and cotinine during hypoxia/aglycemia combined with reoxygenation. Thus, reoxygenation returns the sucrose permeability to the original normoxic control range for all experimental paradigms except for the samples exposed to nicotine and cotinine (Fig. 4, right).
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) (Fig. 5).
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). A model of abluminal NKCC activity at the blood-brain barrier that monitors movement of K+ from brain extracellular fluid to endothelial cell has previously been reported (Vigne et al., 1994). Conditions that increase NKCC activity include cell shrinkage, lowered intracellular Cl-, and hypoxia (Haas et al., 1995; Kawai et al., 1996; Lytle, 1997). We have recently shown that hypoxia increases abluminal NKCC activity at the blood-brain barrier through phosphorylation of NKCC by protein kinase C (PKC) (Abbruscato et al., 2004). The activity of NKCC at the abluminal (brain-facing) side of the blood-brain barrier may provide a protective function during ischemic assaults by facilitating brain-to-blood extrusion of accumulated brain extracellular fluid K+.
Recent research has shown that NKCC1 overstimulation may play a role in ischemic cell damage at the neuron by increasing Na+, K+, and Cl- influx (O'Donnell et al., 2004). Additionally, NKCC1 has been shown to be one of the ion cotransporters responsible for astrocyte cell swelling when brain extracellular fluid K+ concentrations are high (Lenart et al., 2004). The contribution of enhanced NKCC1 activity at the brain facing (abluminal surface) of the blood-brain barrier should also be considered since it has been suggested and demonstrated that NKCC1 activity increases during oxygen/glucose deprivation (Kawai et al., 1996; Abbruscato et al., 2004). Brain endothelial cells may contribute significantly to extracellular fluid removal of key ions locally at the neurovascular unit, which includes the site of endothelial cell, astrocytic, and neuronal connections in brain.
Tobacco smoking clearly has been shown to be a risk factor for stroke (Bonita et al., 1999). One-quarter of stroke incidences are associated with smoking (Hankey, 1999). The increased risk of stroke due to tobacco smoking is dose-dependent and increases from 3.57 to 4.65 times the risk compared with nonsmoking populations when a smoker increases smoke consumption from 5 to 15 cigarettes per day (Bonita et al., 1999). In addition to being a risk factor for brain ischemia, there is a growing body of evidence that suggests that nicotine alters blood-brain barrier permeability characteristics that have a direct influence on stroke outcome (Abbruscato et al., 2002, 2004). Animal studies have shown that chronic nicotine exposure worsened the outcome of stroke through increased edema as assessed in a murine middle cerebral artery occlusion model (Wang et al., 1997). During hypoxic conditions, the presence of nicotine attenuated abluminal protein expression and activity of the NKCC in BBMECs (Abbruscato et al., 2004). The present study set out to determine the fate of NKCC activity after in vitro and in vivo hypoxia coupled to reoxygenation or reperfusion. Our in vitro results clearly show that the blood-brain barrier regains structural integrity after periods of hypoxia/aglycemia coupled to reoxygenation, evidenced by return to basal [14C]sucrose permeability values. Yet, hypoxia/aglycemia coupled to reoxygenation resulted in NKCC abluminal activity that remained elevated. These results show that upon reoxygenation, the blood-brain barrier can maintain paracellular barrier integrity while still maintaining enhanced brain-to-blood K+ transport, possibly aiding in brain extracellular fluid spatial buffering. The addition of both nicotine and cotinine and N-CSE attenuated the normal increase of brain-facing NKCC activity during hypoxia/aglycemia and reoxygenation conditions. Interestingly, NF-CSE did not show an alteration of permeability or K+ uptake. This finding suggests that nicotine is the chemical present in cigarette smoke, which is responsible for the attenuation of enhanced abluminal NKCC activity both during hypoxia/aglycemia insult and hypoxia/aglycemia insult coupled to reoxygenation. Structural integrity of the blood-brain barrier is also altered in the presence of nicotine and cotinine as shown through increased [14C]sucrose permeability in all the models of normoxia, hypoxia/aglycemia, and hypoxia/aglycemia with reoxygenation. This alteration was not seen in the presence of N-CSE or NF-CSE. Although the permeability was not increased in the presence of N-CSE, the K+ uptake in hypoxia/aglycemia and hypoxia/aglycemia with reoxygenation was significantly decreased. This suggests that during reperfusion periods, the NKCC at the blood-brain barrier is able to maintain polarity selectivity that would aid in brain-to-blood ion movement, which is sensitive to nicotine. Previous experiments showed that cotinine alone did not alter permeability of the BBMECs to [14C]sucrose or alter 86Rb uptake of BBMECs mediated by the NKCC (Abbruscato et al., 2002, 2004). In recent studies, we have shown that the NKCC is activated through phosphorylation by PKC during hypoxic conditions. Nicotine attenuates abluminal NKCC expression during hypoxia/aglycemia, possibly through decreased activity of PKC and increased activity of phosphatases (Abbruscato et al., 2004). Future work will explore these cell signaling mechanisms during reoxygenation.
In addition, we confirmed these in vitro results with an in vivo four-vessel occlusion model of global forebrain ischemia coupled with a period of reoxygenation, whereby reperfusion was established. In vivo experiments were designed to be short enough in duration (less than 3-6 h) to prevent the effects of blood-brain barrier breakdown (Betz et al., 1994). This paradigm eliminates damage associated with vasogenic edema that follows the ischemic incident. Previous work has shown that K+ is known to enter into the ventricular cerebral spinal fluid rapidly and diffuse from the cerebral spinal fluid into the brain tissue (Bradbury and Kleeman, 1967; Smith and Rapoport, 1986). Thus, animals were preloaded with 86Rb 40 min prior to four-vessel occlusion to allow sufficient time for 86Rb to reach steady state and enter into the brain extracellular fluid K+ pool. After four-vessel occlusion, we observed a significant increase of brain extracellular fluid K+ that was estimated by the ratio of 86Rb detected in the brain compared with that detected in plasma. Our data demonstrate that, even after only 5 min of global ischemia, increases in the brain K+ concentrations can be observed compared with control (p < 0.05). Animals subjected to four-vessel occlusion with nicotine or N-CSE showed a significantly larger increase (p < 0.05) in brain extracellular fluid K+ concentrations compared with control animals that underwent four-vessel occlusion alone. Also, brain extracellular fluid K+ values in NF-CSE animals were significantly decreased (p < 0.01) compared with the N-CSE-treated animals. Both our in vivo and in vitro results suggest that diminished NKCC abluminal activity at the blood-brain barrier may hinder the export of brain extracellular fluid K+ across the endothelial cell back into the blood. This work also provides convincing evidence that nicotine is most likely the chemical present in cigarette smoke responsible for altering NKCC activity and the movement of K+ out of the brain during global ischemia coupled to reperfusion.
We suggest that nicotine contributes to an alteration in abluminal NKCC activity at the blood-brain barrier during and after stroke conditions. In brain trauma, the presence of ion cotransporters may play an important role by removing the K+ ions that accumulate during an ischemic insult from brain extracellular fluid. The movement of K+ from brain extracellular fluid and astrocytic end feet to endothelial cells has previously been proposed (Vigne et al., 1994). In astrocytes, transporters such as Kir4.1 and aquaporin 4 have been shown to be abundantly expressed in astrocytic end feet (Higashi et al., 2001). The abundance of these transporters are thought to be responsible for the concept of potassium spatial buffering, which portends to transport the K+ from the brain extracellular fluid to the endothelial cells (Kofuji and Newman, 2004). Our research has shown that abluminal NKCC activity is increased during conditions of both hypoxia/aglycemia and hypoxia/aglycemia coupled to reoxygenation, both events that mimic stroke conditions and recovery in vitro. Most importantly, paracellular permeability returns to a normal restrictive condition upon reoxygenation that would allow for polarity of ion transporter action at the blood-brain barrier. Brain-to-blood movement of K+ could return brain extracellular fluid concentrations to 3 mM, which is imperative for proper neuronal function (Kofuji and Newman, 2004).
Smoking cigarettes has been known to increase the risk of many diseases including stroke. The alteration of NKCC activity is one mechanism that may contribute to an increase of brain extracellular fluid K+ during an ischemic assault in individuals using products containing nicotine. Down-regulation of blood-brain barrier abluminal NKCC activity during hypoxia/aglycemia in the presence of nicotine may exacerbate ischemic cellular damage. Recovery from ischemic damage may also be hampered due to the continuation of NKCC down-regulation following an ischemic assault. Together with an inhibition of the protective mechanism by which K+ is removed, brain extracellular fluid K+ could accumulate, leading to increased cellular edema in neurons and astrocytes and impaired neuronal conduction following stroke.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: NKCC, Na,K,2Cl-cotransporter; BBMEC, bovine brain microvessel endothelial cell; N-CSE, nicotine-containing cigarette smoke extract; NF-CSE, nicotine-free cigarette smoke extract; PC, permeability coefficient; N, nicotine; ANOVA, analysis of variance; PKC, protein kinase C; C, cotinine.
Address correspondence to: Dr. Thomas J. Abbruscato, School of Pharmacy, Texas Tech University Health Sciences Center, 1300 South Coulter, Amarillo, TX 79106. E-mail: thomas.abbruscato{at}ttuhsc.edu
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References |
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