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Am J Physiol Heart Circ Physiol 289: H212-H219, 2005. First published February 11, 2005; doi:10.1152/ajpheart.01210.2004
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Modulation of cerebral microvascular permeability by endothelial nicotinic acetylcholine receptors

Brian T. Hawkins,1 Richard D. Egleton,2 and Thomas P. Davis1,2

1Program in Neuroscience and 2Department of Medical Pharmacology, University of Arizona College of Medicine, Tucson, Arizona

Submitted 2 December 2004 ; accepted in final form 4 February 2005


   ABSTRACT
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 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Nicotine increases the permeability of the blood-brain barrier in vivo. This implies a possible role for nicotinic acetylcholine receptors in the regulation of cerebral microvascular permeability. Expression of nicotinic acetylcholine receptor subunits in cerebral microvessels was investigated with immunofluorescence microscopy. Positive immunoreactivity was found for receptor subunits {alpha}3, {alpha}5, {alpha}7, and {beta}2, but not subunits {alpha}4, {beta}3, or {beta}4. Blood-brain barrier permeability was assessed via in situ brain perfusion with [14C]sucrose. Nicotine increased the rate of sucrose entry into the brain from 0.3 ± 0.1 to 1.1 ± 0.2 µl·g–1·min–1, as previously described. This nicotine-induced increase in blood-brain barrier permeability was significantly attenuated by both the blood-brain barrier-permeant nicotinic antagonist mecamylamine and the blood-brain barrier-impermeant nicotinic antagonist hexamethonium to 0.5 ± 0.2 and 0.3 ± 0.2 µl·g–1·min–1, respectively. These data suggest that nicotinic acetylcholine receptors expressed on the cerebral microvascular endothelium mediate nicotine-induced changes in blood-brain barrier permeability.

blood-brain barrier; mecamylamine; hexamethonium; nicotine; immunofluorescence microscopy

NICOTINE BINDS WITH HIGH AFFINITY to various forms of the nicotinic acetylcholine receptor (nAChR), where it acts as an agonist (40). As such, nAChRs are likely mediators of the effects of nicotine throughout the body (29, 38), although non-receptor-mediated effects of nicotine have been observed (55). Recently, we demonstrated that nicotine increases the permeability of the blood-brain barrier (BBB) via a redistribution of tight junction proteins in cerebral microvessels (26). These cytostructural and functional effects of nicotine on the BBB indicate a role for nAChRs in the regulation of cerebral microvascular permeability. It is not immediately clear, however, how nicotine could lead to changes in the neurovascular unit.

nAChRs are found throughout the brain, on both neurons and astrocytes (25). Given the extensive communication that occurs between the microvascular endothelium and the parenchyma (6, 8, 14, 60), it is possible that central nAChRs could mediate the release of a vasoactive factor or factors that would affect BBB structure and function, such as VEGF (11) or bradykinin (7). Some evidence suggests that nAChRs might be expressed on cerebral microvessels (25, 32). Functional nicotinic receptors have been characterized in peripheral endothelial cells (42, 47) and epithelial cells (57) and are thought to be involved in the regulation of cell shape and barrier integrity in surface-lining tissues (15).

nAChR subunits are expressed in cultured brain endothelial cells, and nicotine-induced hyperpermeability in this model system is attenuated by the {alpha}7 nAChR antagonist {alpha}-bungarotoxin (1). It is therefore possible that nAChRs expressed on the abluminal or luminal endothelial membranes could mediate a direct effect of nicotine on cerebral endothelial cells. However, care must be taken in extrapolating expression studies in cell culture to the native tissue. For example, in vitro studies originally indicated that cerebral endothelial cells did not express N-methyl-D-aspartate (NMDA) receptors (48), but subsequent studies in isolated microvessels indicated that NMDA receptors are expressed at the BBB (54). Furthermore, expression of receptor subunit proteins does not necessarily imply the assembly of functional receptors (56). Thus the expression and function of nAChRs at the BBB need to be examined in a whole animal model.

In this study, expression of neuronal-type nAChR subunits ({alpha}3, {alpha}4, {alpha}5, {alpha}7, {beta}2, {beta}3, and {beta}4) was investigated in isolated cerebral microvessels by immunofluorescence microscopy. Assessment of BBB permeability was performed by in situ brain perfusion with the paracellular permeability marker [14C]sucrose (30, 50) after nicotine treatment with and without nicotinic antagonists. The nicotinic antagonist mecamylamine, which acts at most neuronal-type nAChRs and crosses the BBB (58), was coadministered with nicotine to establish whether the effect of nicotine on BBB permeability (26) is nAChR mediated. Hexamethonium, a nonspecific nAChR antagonist that does not cross the BBB under normal circumstances (45), was coadministered to determine whether the effect of nicotine on BBB permeability is mediated by central nAChRs.


   MATERIALS AND METHODS
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 MATERIALS AND METHODS
RESULTS
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Animals, radioisotopes, antibodies, and chemicals. All animal protocols used in this study were approved by the University of Arizona Institutional Animal Care and Use Committee and conform to National Institutes of Health (NIH) guidelines. Female Sprague-Dawley rats were obtained from Harlan (Indianapolis, IN), housed under standard 12:12-h light-dark conditions, and given food and water ad libitum throughout the study.

[14C]sucrose was purchased from ICN Pharmaceuticals (specific activity 462 mCi/mol; Irvine, CA). Rabbit polyclonal antisera against nAChR subunits {alpha}3, {alpha}4, {alpha}5, {alpha}7, and {beta}2–4 and specific blocking peptides were purchased from Research and Diagnostics (Benicia, CA). Alexa Fluor 488-conjugated anti-rabbit IgG was purchased from Molecular Probes (Eugene, OR). All other reagents were purchased from Sigma (St. Louis, MO) unless otherwise stated.

Immunofluorescence of nAChRs in cerebral microvessels. Naive female Sprague-Dawley rats (240–300 g) were anesthetized with 1 ml/kg of rat cocktail (0.6 mg/ml acepromazine, 78.3 mg/ml ketamine, and 3.1 mg/ml xylazine) and decapitated. The brain was removed from the skull, the meninges and choroid plexuses were removed, and the brain stem and cerebellum were dissected away from the cerebral hemispheres. The hemispheres were homogenized in a fivefold volume of buffer (in mM: 103 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 KH2PO4, 1.2 MgSO4, 15 HEPES, 25 NaHCO3, 10 glucose, and 1 sodium pyruvate, with 10 g/l 64K dextran), suspended in an equal volume of 26% dextran, and centrifuged for 10 min at 5,800 g and 4°C. The pellet was resuspended and passed through a 100-µm mesh. The filtrate was centrifuged for 10 min at 1,500 rpm and 4°C, and the resulting pellet was resuspended in 0.25 ml of buffer, smeared onto microscope slides, and heat fixed at 95°C for 10 min. The vessels were fixed with 4% paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100 in PBS, and blocked for 1 h in 1% BSA in PBS. Slides were incubated with rabbit polyclonal antisera against nAChR subunits {alpha}3, {alpha}4, {alpha}5, {alpha}7, and {beta}2–4 diluted 1:10 in PBS with 1% normal goat serum for 30 min, rinsed with 1% BSA in PBS, and incubated with Alexa Fluor 488-conjugated anti-rabbit IgG (2 µg/ml) for 30 min. After placement and sealing of coverslips, digital photographs were taken with x100 oil-immersion objectives on a Nikon TE300 fluorescent microscope with a fluorescein filter. Both brightfield and fluorescence images were taken of each vessel in these experiments to confirm the presence or absence of positive immunoreactivity. Preincubation (1 h, 4°C) of antisera with specific blocking peptides (Table 1) for each subunit was used to test for antibody specificity.


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  		Table 1. Amino acid sequences of nAChR subunit blocking peptides

 
Positive antibody controls were performed in 20-µm-thick coronal slices taken from the hippocampal region. Slices were mounted onto gelatin-coated slides and stored at –80°C until use. After being brought to room temperature, slices were fixed with ice-cold 2% paraformaldehyde for 10 min. They were then washed twice for 10 min with SSC buffer (30 mM Na3C6H5O7, 300 mM NaCl, pH 7.0), with ice-cold 50–50 methanol-acetone for 7 min, and three times for 10 min with SSC + 0.05% Tween 20. Endogenous peroxidase activity was quenched with freshly prepared 1% H2O2 for 20 min before blocking with 1% BSA in SSC for 40 min. Slices were incubated overnight at 4°C in nAChR antisera diluted 1:10 in 1% BSA in SSC, rinsed three times for 10 min with SSC + 0.05% Tween 20, incubated 1 h in Alexa Fluor 488-conjugated anti-rabbit IgG (2 µg/ml in 1% BSA in SSC), and rinsed twice for 10 min with PBS before coverslip mounting.

Coadministration of nicotine with mecamylamine and hexamethonium. Female Sprague-Dawley rats (240–300 g) were implanted with osmotic pumps (Alzet 2ML2; Durect, Cupertino, CA) for continuous subcutaneous delivery of nicotine (4.5 mg·kg–1·day–1), nicotine and mecamylamine (4.5 mg·kg–1·day–1), nicotine and hexamethonium (3 mg·kg–1·day–1), or 0.9% saline for 7 days as previously described (26). Blood pressure (BP) and heart rate (HR) were measured with a noninvasive tail-cuff monitor (Columbus Instruments NIBP-8) before implantation and on day 7 of treatment. Animals were habituated to handling and the measurement apparatus during at least three separate sessions the week before implantation. Animals were placed in a holding tube for at least 5 min but no more than 15 min before the beginning of measurement. All measurements were performed between 1200 and 1500 to minimize the effect of any circadian variation in BP or HR. Measurements from the final preimplant session were used as baseline.

In situ brain perfusion. Animals were anesthetized as above and heparinized by intraperitoneal injection (10,000 U/kg). A ventral midline incision was made at the neck, and the common carotid arteries were exposed. The arteries were cannulated with silicone tubing, and the jugular veins were cut. The perfusion medium consisted of a mammalian Ringer solution [in mM: 117 NaCl, 4.7 KCl, 0.8 MgSO4, 24.8 NaHCO3, 1.2 KH2PO4, 2.5 CaCl2, and 10 D-glucose, with 39 g/l dextran (mol wt 70,000) and 10 g/l BSA, pH 7.4] with Evans blue that was oxygenated with 95% O2-5% CO2. The perfusate was passed via peristaltic pump through a heating coil (37°C) and a bubble trap. Once the desired perfusion pressure and rate were achieved (~100 mmHg and 3.1 ml/min), [14C]sucrose (10 µCi/20 ml Ringer) was infused with a slow-drive syringe pump (0.5 ml·min–1·hemisphere–1; model 22, Harvard Apparatus, South Natick, MA). The animal was perfused for 5, 10, or 20 min (n = 4–6 per time point per treatment), after which the animal was decapitated and the brain was removed. Samples of the radioactive perfusate were collected from each carotid cannula as a reference. The choroid plexuses were excised, the meninges were removed, and the cerebral hemispheres were sectioned and homogenized. Brain tissue and 100-µl samples of perfusate were prepared for liquid scintillation counting by incubation in 1 ml of tissue solubilizer (TS-2, Research Products, Mount Pleasant, IL) for 2 days. Before counting was started, 100 µl of 30% acetic acid and 4 ml of Budget-Solve Liquid Scintillation Cocktail (Research Products) were added, and the samples were measured for radioactivity (model LS 5000 TD Counter; Beckman Instruments, Fullerton, CA). Results are reported as the ratio (Rbr) of radioactivity in the brain (Cbrain) to that in the perfusate (Cperfusate):

(1)
A least-squares regression of Rbr vs. perfusion time was performed for each experimental and control group:

(2)
where VD is the initial volume of distribution of [14C]sucrose, Kin is the unidirectional transfer coefficient, and T is the perfusion time in minutes.

Data analysis. BP and HR measurements were compared by Student’s t-test (treatment vs. saline). Analysis of the regression coefficients VD and Kin was performed as previously described (26) according to the methods of Glantz (22). The standard error of the regression (sRbr·T) was computed from the standard deviations of Rbr and T (sRbr and sT, respectively):

(3)
where n is the number of all data points (Rbr, T) in each regression. This value was used to calculate the standard errors of VD and Kin (sVD and sKin):

(4)

(5)
For statistical comparison of Kin and VD between two treatment groups, the pooled estimate of variation around the regression lines () was determined by the following:

(6)
where subscripts 1 and 2 indicate the groups being compared. This value was used to calculate t for comparison of Kin and VD:

(7)

(8)
Significance (P < 0.05) was determined by comparison of the calculated value for t with the critical value of t for n1 + n2 – 4 degrees of freedom.


   RESULTS
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nAChR subunit expression in cerebral microvessels. Expression of nAChR subunit proteins in cerebral microvessels was visualized by immunofluorescence microscopy. Positive immunoreactivity was observed for nAChR subunits {alpha}3, {alpha}5, {alpha}7, and {beta}2, but not for {alpha}4, {beta}3, or {beta}4 (Fig. 1). To test for antibody specificity, antisera were preincubated with blocking peptides corresponding to an amino acid sequence specific to the corresponding subunit (Table 1). In all cases, preincubation of the primary antibody with the corresponding blocking peptide attenuated or abolished immunoreactivity. Furthermore, positive immunoreactivity was not observed on glial fragments visible on the slides. Positive antibody controls for subunits {alpha}4, {beta}3, and {beta}4 were performed on brain slices, and positive immunoreactivity in the hippocampus (25) was observed for all three (Fig. 2). Slides prepared without primary antibody did not stain microvessels or brain slices, indicating that nonspecific binding of the fluorescent-tagged secondary antibody was minimal (data not shown).



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  		Fig. 1. Expression of nicotinic acetylcholine receptor (nAChR) subunits in rat brain microvessels. Positive immunoreactivity was observed for nAChR subunits {alpha}3, {alpha}5, {alpha}7, and {beta}2. In all cases, preincubation of the primary antibody with the corresponding blocking peptide attenuated or abolished immunoreactivity. No immunoreactivity was observed in cerebral microvessels for nAChR subunits {alpha}4, {beta}3, or {beta}4. Second and fourth columns are brightfield images of the fields presented in the first and third columns, respectively. All images are x100 in oil immersion. Ab, antibody; BP, blocking peptide; ft, fluorescent; bf, brightfield. Scale bar = 1 µm.

 


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  		Fig. 2. Expression of nAChR subunits in hippocampal slices. Positive antibody controls for nAChR subunits {alpha}4 (A), {beta}3 (B), and {beta}4 (C) were performed in 20-µm coronal slices. Immunoreactivity for all subunits was observed in the hippocampus. Slices stained without primary antisera or with antisera preincubated with blocking peptides did not stain (data not shown). Magnification x20.

 
Attenuation of nicotinic BBB disruption by nicotinic antagonists. BBB permeability was assessed by in situ brain perfusion with multiple time-uptake analysis (Fig. 3). Nicotine treatment led to a significant (P < 0.01) 3.7-fold increase in Kin, from 0.3 ± 0.1 to 1.1 ± 0.2 µl·g–1·min–1 (Fig. 3), similar to previously reported results (26). Coadministration with both mecamylamine and hexamethonium significantly (P < 0.05) attenuated the effect of nicotine on Kin, which was 0.5 ± 0.2 and 0.3 ± 0.2 µl·g–1·min–1, respectively, in these groups (Fig. 3). Estimated VD ranged from 2.4 to 7.4 µl/g, which is comparable to previously published measurements (53). No significant differences in VD were found with any treatment. Neither mecamylamine nor hexamethonium had any effect on BP or HR when coadministered with nicotine (pretreatment mean BP 105 ± 4 and 99 ± 5 mmHg, posttreatment mean BP 104 ± 5 and 105 ± 4 mmHg, pretreatment mean HR 466 ± 15 and 466 ± 7 beats/min, posttreatment mean HR 490 ± 12 and 470 ± 18 beats/min for nicotine + mecamylamine and nicotine + hexamethonium groups, respectively).



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  		Fig. 3. Multiple time uptake in situ brain perfusion. Animals were treated with 0.9% saline (sal), nicotine (4.5 mg·kg–1·day–1; nic), nicotine and mecamylamine (4.5 mg·kg–1·day–1; nic + mec), or nicotine and hexamethonium (3 mg·kg–1·day–1; nic + hex) via subcutaneous infusion for 7 days. Rats were perfused with [14C]sucrose for 5, 10, or 20 min. Data are mean ± SE brain-to-perfusate radioactivity ratio (Rbr) values; n = 4–6 per time point.

 

   DISCUSSION
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 MATERIALS AND METHODS
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 DISCUSSION
GRANTS
 REFERENCES
 
In this study we investigated the expression of nAChRs by cerebral microvessels and the role that these receptors play in the BBB permeability increase by nicotine (26). Isolated cerebral microvessels were visualized by immunofluorescence microscopy (Fig. 1). These studies demonstrate that {alpha}3, {alpha}5, {alpha}7, and {beta}2, but not {alpha}4, {beta}3, or {beta}4 nAChR subunits are present in cerebral microvessels. These results are similar to what has been observed in cultured bovine brain endothelial cells, in which the expression of {alpha}3, {alpha}5, {alpha}7, {beta}2, and {beta}3 subunits has been previously described (1). The absence of {beta}3 in rat microvessels could be attributable to species differences in expression or antibody reactivity, or {beta}3 expression could be an artifact of cell culture. In cell culture, the effect of nicotine on permeability was blocked by {alpha}-bungarotoxin, a specific antagonist for {alpha}7 (homomeric) nAChR (1). Interestingly, subunits {alpha}3, {alpha}5, {beta}2, and {beta}4 are found in human aortic endothelial cells, where they form functional ion channels responsive to anatoxin and dihydro-{beta}-erythroidine, the classic agonist and antagonist, respectively, of {alpha}3-containing nAChRs (42). Again, these data may indicate species differences in patterns of nAChR subunit expression. Another possibility is that endothelial nAChR subunits are differentially expressed in different regions of the vasculature. For example, rat coronary microvessels contain mRNA encoding for subunits {alpha}2–5, {alpha}7, {beta}2, and {beta}4, but not {beta}3 (47). Striking heterogeneity of nAChR {alpha}-subunit expression has been observed in the arterial system of the rat as well (9, 10). Indeed, there may be heterogeneity of nAChR subunit expression within the brain microvasculature itself (21).

The pattern of staining for {alpha}3, {alpha}5, {alpha}7, and {beta}2 subunits was generally diffuse throughout the vessels. Discrete areas of immunoreactivity indicative of exclusive expression of nAChRs in microvascular pericytes, which cover 20–30% of the cerebral microvascular surface (19), were not observed, although expression of nAChR subunits by pericytes in addition to endothelial cells cannot be ruled out. Some clusters of intense staining were observed with {alpha}7 and {beta}2 subunits. Whether such apparent clustering is of functional significance is a matter of speculation at this point. At the neuromuscular junction, clustering of nAChRs is induced by the protein agrin released by motoneurons (31). It is not known whether similar signaling could occur in endothelial cells, as proteins involved in clustering of nAChRs in muscle cells (rapsyn, MuSK) have not been found in endothelial cells. However, agrin has recently been identified as a component of the extracellular matrix and is found throughout the cerebral microvasculature (36).

Several different combinations of the {alpha}3, {alpha}5, {alpha}7, and {beta}2 subunits could assemble into functional nAChRs. The homopentameric {alpha}7 nAChR is well-characterized (59) and expressed throughout the central nervous system (25), as well as being implicated in mediating the effects of nicotine on the BBB in vitro (1). {alpha}7 and {beta}2 Subunits are coexpressed in cholinergic neurons in the brain and have been shown to assemble into functional receptors (5, 34). {alpha}3{beta}2 Receptors are expressed in rabbit retina (33), where they may be involved in the generation of spontaneous waves of neural activity during visual system development (18). Studies in chromaffin cells of the adrenal medulla suggest that {alpha}5 subunits may colocalize with {alpha}3 and {beta}2 subunits, although assembled receptors composed of these specific subtypes have not been definitively characterized (16).

The role of nAChRs expressed at cerebral microvessels in nicotinic alteration of BBB function was examined by coadministration of the nicotinic antagonists mecamylamine and hexamethonium with nicotine. Both of these antagonists have low toxicity (58, 35). Although both have been used clinically as antihypertensive agents because of their ability to block ganglionic cholinergic transmission, no effect on BP or HR was observed in the present study when the antagonists were coadministered with nicotine.

Nicotine significantly (P < 0.01) increased the uptake of [14C]sucrose into the brain (Fig. 4), as previously found (26). This effect was significantly (P < 0.05) attenuated by mecamylamine (Fig. 4), indicating that the functional effect of nicotine on the BBB is nAChR mediated. This is an important observation, as nicotine has been shown to interact directly with endothelial nitric oxide synthase (55), which could also lead to changes in BBB permeability (43, 46).



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  		Fig. 4. Attenuation of nicotinic effect on blood-brain barrier permeability by mecamylamine and hexamethonium. Data are mean ± SE unidirectional transfer coefficient (Kin) values. Nicotine (4.5 mg·kg–1·day–1) significantly increased the rate of sucrose uptake into the brain; this effect was attenuated by both mecamylamine (4.5 mg·kg–1·day–1) and hexamethonium (3 mg·kg–1·day–1), indicating that the permeability-increasing effect of nicotine is mediated by nAChRs likely outside the central nervous system. Significance was determined by the methods for comparing linear regressions described by Glantz (22). **P < 0.01 vs. saline control; {dagger}P < 0.05 vs. nicotine.

 
The effect of nicotine on BBB permeability was also significantly (P < 0.05) attenuated by hexamethonium (Fig. 4), further supporting the hypothesis that the effect is nAChR mediated. Furthermore, attenuation of this effect by hexamethonium strongly suggests that central nAChRs are not involved in the effects of nicotine on the BBB. Hexamethonium is a non-lipid-soluble quaternary ammonium compound that exists in a dicationic form at physiological pH. As a result, the entry of hexamethonium into the brain from the periphery is severely limited by the BBB and is highly dose dependent. Autoradiographic studies using a single intravenous bolus of [3H]hexamethonium at 2.18 mg/kg found that hexamethonium accumulated in the meninges and choroid plexus but did not penetrate into the parenchyma (24). A subsequent study found that a very small but significant fraction of [3H]hexamethonium did enter the brain after intravenous injection, but only at much higher (>10 mg/kg) doses (4). Furthermore, 95–100% of a single hexamethonium dose is excreted in the urine within 18 h of injection (39). In the present study, hexamethonium was delivered via subcutaneous infusion at a constant rate of 3 mg·kg–1·day–1, or 0.125 mg·kg–1·h–1. It is therefore highly unlikely that the plasma concentration of hexamethonium would ever become sufficiently high for a pharmacologically significant amount to accumulate in the brain.

It is possible that as nicotine increases BBB permeability to sucrose, passage of hexamethonium into the brain may be increased as well. However, hexamethonium injected directly into the cerebral ventricles is rapidly taken up from the cerebrospinal fluid by the choroid plexus and effluxed into the blood (52), meaning that any hexamethonium gaining access to the brain would not be expected to accumulate. Malin and colleagues (44) found that an acute withdrawal syndrome could be precipitated in animals chronically treated with nicotine by peripheral administration of mecamylamine. In a follow-up study, it was found that hexamethonium administered peripherally did not precipitate a withdrawal syndrome, but hexamethonium given intracerebroventricularly at a very low dose (~18 ng) did (45). In both of these studies, nicotine was administered by the same route used in our experiments (subcutaneous osmotic pump) for the same length of time (7 days), but at double the dose (9 mg·kg–1·day–1) (44, 45). The pharmacologically effective concentration of hexamethonium in the brain when it was administered centrally was estimated by the authors to be 195,000 times smaller than the concentration in the periphery after peripheral administration. It is therefore unlikely that nicotine increased brain entry of hexamethonium to any relevant extent in the present study.

These data support the hypothesis that nicotine increases the permeability of the BBB by acting on nAChRs outside the central nervous system. It is possible that this could be a ganglionic effect of nicotine; however, this is also unlikely, as hexamethonium blocks the BBB effect well below the ganglionic blocking intravenous dose of ~20 mg/kg in rats (51), and no changes in blood pressure were observed after treatment with hexamethonium or mecamylamine in this study. Thus the most likely mediators of the effect of nicotine on the BBB are nAChRs expressed on brain microvessels. Although the imaging techniques used in this study are inadequate to distinguish between luminal and abluminal expression of these proteins, on the basis of the pharmacological data described above, some—if not all—of these receptors are expressed on the luminal endothelial cell membrane.

This begs the question of what the physiological role of endothelial nAChRs might be in the brain. ACh has long been known to act on endothelial cells via muscarinic receptors to induce endothelium-dependent vasodilatation (20). Choline acetyltransferase, the enzyme responsible for ACh synthesis, is expressed in brain microvessels (49), and cerebral endothelial cells have been shown to synthesize ACh (3, 23). Coupled with the ubiquity of cholinesterases in the blood (41) that rapidly degrade any circulating ACh, it is possible that the endothelium itself may release ACh that acts as an autocrine factor (13) via nicotinic (as well as muscarinic) mechanisms.

It is not immediately obvious what this pathway may have to do with regulation of cerebral microvascular permeability. However, endothelial nAChRs in the periphery have been implicated in the regulation of angiogenesis (27, 28). VEGF, a potent angiogenic factor, also increases the permeability of the BBB (17). Furthermore, src-suppressed C-kinase substrate blocks cerebral angiogenesis in vivo and in vitro via suppressed expression of VEGF, increases tight junction proteins in endothelial cells, and decreases sucrose permeability (37). nAChR-mediated increases in microvascular permeability therefore might reflect an involvement of these receptors in some aspects of cerebral angiogenesis, in which nicotine acts as an agonist of an angiogenic pathway (28). Alternatively, nicotine-induced downregulation of endothelial nicotinic receptors, which has been observed in vitro (2), might impair normal cholinergic signaling necessary for the maintenance of barrier integrity (15). Another possibility is that nAChRs may be involved in regulating vesicle formation and transcytosis, the upregulation of which can be involved in BBB disruption (12).

This study demonstrates that nAChRs are expressed in the cerebral microvasculature, that increased BBB permeability after nicotine treatment is a nAChR-mediated phenomenon, and that peripheral antagonism of nAChRs is sufficient to block the effect of nicotine on the BBB. Together, these data strongly suggest that nicotine increases the permeability of the BBB via interaction with endothelial nAChRs expressed at the lumen of the cerebral microvasculature. This implies a novel physiological role for endothelial nAChRs in the modulation of cerebral microvascular permeability. These findings present a potential mechanism by which chronic nicotine use could lead to a compromised neurovascular unit, and thus increase the risk and/or severity of certain neurological diseases. Another implication of these findings is that endothelial nAChRs may be potential targets for therapeutic modulation of the neurovascular unit. Nicotine or other nicotinic agonists might be useful for facilitating the entry of low-molecular-weight drugs into the brain. Alternatively, nicotinic antagonists may be able to attenuate pathological increases in BBB permeability following an acute ischemic attack or traumatic brain injury. This work represents a novel contribution to the understanding of how the neurovascular unit might be regulated and possibly manipulated under both physiological and pathophysiological conditions.


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 ABSTRACT
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This work was supported by Arizona Disease Control Research Commission Contract 5011 and National Institutes of Health Grants NS-42652, NS-039592, and DA-11271.


   ACKNOWLEDGMENTS
 
The authors thank Chane Rains and Marsha Penner for technical assistance with brain slice preparations.


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. P. Davis, Dept. of Medical Pharmacology, Univ. of Arizona College of Medicine, 1501 N. Campbell Ave., Tucson, AZ 85724-5050 (E-mail: davistp{at}u.arizona.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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  1. Abbruscato TJ, Lopez SP, Mark KS, Hawkins BT, and Davis TP. Nicotine and cotinine modulate cerebral microvascular permeability and protein expression of ZO-1 through nicotinic acetylcholine receptors expressed on brain endothelial cells. J Pharm Sci 91: 2525–2538, 2002.
  2. Abbruscato TJ, Lopez SP, Roder K, and Paulson JR. Regulation of blood-brain barrier Na,K,2Cl-cotransporter through phosphorylation during in vitro stroke conditions and nicotine exposure. J Pharmacol Exp Ther 310: 459–468, 2004.
  3. Arneric SP, Honig MA, Milner TA, Greco S, Iadecola C, and Reis DJ. Neuronal and endothelial sites of acetylcholine synthesis and release associated with microvessels in rat cerebral cortex: ultrastructural and neurochemical studies. Brain Res 454: 11–30, 1988.
  4. Asghar K and Roth LJ. Entry and distribution of hexamethonium in the central nervous system. Biochem Pharmacol 20: 2787–2795, 1971.
  5. Azam L, Winzer-Serhan U, and Leslie FM. Co-expression of {alpha}7 and {beta}2 nicotinic acetylcholine receptor subunit mRNAs within rat brain cholinergic neurons. Neuroscience 119: 965–977, 2003.
  6. Ballabh P, Braun A, and Nedergaard M. The blood-brain barrier: an overview: structure, regulation, and clinical implications. Neurobiol Dis 16: 1–13, 2004.
  7. Borlongan CV and Emerich DF. Facilitation of drug entry into the CNS via transient permeation of blood brain barrier: laboratory and preliminary clinical evidence from bradykinin receptor agonist, Cereport. Brain Res Bull 60: 297–306, 2003.
  8. Braet K, Paemeleire K, D’Herde K, Sanderson MJ, and Leybaert L. Astrocyte-endothelial cell calcium signals conveyed by two signalling pathways. Eur J Neurosci 13: 79–91, 2001.
  9. Bruggmann D, Lips KS, Pfeil U, Haberberger RV, and Kummer W. Multiple nicotinic acetylcholine receptor {alpha}-subunits are expressed in the arterial system of the rat. Histochem Cell Biol 118: 441–447, 2002.
  10. Bruggmann D, Lips KS, Pfeil U, Haberberger RV, and Kummer W. Rat arteries contain multiple nicotinic acetylcholine receptor {alpha}-subunits. Life Sci 72: 2095–2099, 2003.
  11. Chodobski A, Chung I, Kozniewska E, Ivanenko T, Chang W, Harrington JF, Duncan JA, and Szmydynger-Chodobska J. Early neutrophilic expression of vascular endothelial growth factor after traumatic brain injury. Neuroscience 122: 853–867, 2003.
  12. Cipolla MJ, Crete R, Vitullo L, and Rix RD. Transcellular transport as a mechanism of blood-brain barrier disruption during stroke. Front Biosci 9: 777–785, 2004.
  13. Clancy RM and Abramson SB. Acetylcholine prevents intercellular adhesion molecule 1 (CD54)-induced focal adhesion complex assembly in endothelial cells via a nitric oxide-cGMP-dependent pathway. Arthritis Rheum 43: 2260–2264, 2000.
  14. Cohen Z, Bonvento G, Lacombe P, and Hamel E. Serotonin in the regulation of brain microcirculation. Prog Neurobiol 50: 335–362, 1996.
  15. Conti-Fine BM, Navaneetham D, Lei S, and Maus AD. Neuronal nicotinic receptors in non-neuronal cells: new mediators of tobacco toxicity? Eur J Pharmacol 393: 279–294, 2000.
  16. Di Angelantonio S, Matteoni C, Fabbretti E, and Nistri A. Molecular biology and electrophysiology of neuronal nicotinic receptors of rat chromaffin cells. Eur J Neurosci 17: 2313–2322, 2003.
  17. Dobrogowska DH, Lossinsky AS, Tarnawski M, and Vorbrodt AW. Increased blood-brain barrier permeability and endothelial abnormalities induced by vascular endothelial growth factor. J Neurocytol 27: 163–173, 1998.
  18. Feller MB. The role of nAChR-mediated spontaneous retinal activity in visual system development. J Neurobiol 53: 556–567, 2002.
  19. Frank RN, Dutta S, and Mancini MA. Pericyte coverage is greater in the retinal than in the cerebral capillaries of the rat. Invest Ophthalmol Vis Sci 28: 1086–1091, 1987.
  20. Furchgott RF and Zawadzki JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 288: 373–376, 1980.
  21. Ge S, Song L, and Pachter JS. Where is the blood-brain barrier... really? J Neurosci Res 79: 421–427, 2005.
  22. Glantz SA. Primer of Biostatistics. New York: McGraw-Hill, 2002.
  23. Gonzalez JL and Santo-Benito FF. Synthesis of acetylcholine by endothelial cells isolated from rat brain cortex capillaries. Brain Res 412: 148–150, 1987.
  24. Gosling JA and Lu TC. Uptake and distribution of some quaternary ammonium compounds in the central nervous system of the rat. J Pharmacol Exp Ther 167: 56–62, 1969.
  25. Graham AJ, Martin-Ruiz CM, Teaktong T, Ray MA, and Court JA. Human brain nicotinic receptors, their distribution and participation in neuropsychiatric disorders. Curr Drug Targets CNS Neurol Disord 1: 387–397, 2002.
  26. Hawkins BT, Abbruscato TJ, Egleton RD, Brown RC, Huber JD, Campos CR, and Davis TP. Nicotine increases in vivo blood-brain barrier permeability and alters cerebral microvascular tight junction protein distribution. Brain Res 1027: 48–58, 2004.
  27. Heeschen C, Jang JJ, Weis M, Pathak A, Kaji S, Hu RS, Tsao PS, Johnson FL, and Cooke JP. Nicotine stimulates angiogenesis and promotes tumor growth and atherosclerosis. Nat Med 7: 833–839, 2001.
  28. Heeschen C, Weis M, Aicher A, Dimmeler S, and Cooke JP. A novel angiogenic pathway mediated by non-neuronal nicotinic acetylcholine receptors. J Clin Invest 110: 527–536, 2002.
  29. Hogg RC, Raggenbass M, and Bertrand D. Nicotinic acetylcholine receptors: from structure to brain function. Rev Physiol Biochem Pharmacol 147: 1–46, 2003.
  30. Huber JD, Witt KA, Hom S, Egleton RD, Mark KS, and Davis TP. Inflammatory pain alters blood-brain barrier permeability and tight junctional protein expression. Am J Physiol Heart Circ Physiol 280: H1241–H1248, 2001.
  31. Huh KH and Fuhrer C. Clustering of nicotinic acetylcholine receptors: from the neuromuscular junction to interneuronal synapses. Mol Neurobiol 25: 79–112, 2002.
  32. Kalaria RN, Homayoun P, and Whitehouse PJ. Nicotinic cholinergic receptors associated with mammalian cerebral vessels. J Auton Nerv Syst 49, Suppl: S3–S7, 1994.
  33. Keyser KT, MacNeil MA, Dmitrieva N, Wang F, Masland RH, and Lindstrom JM. Amacrine, ganglion, and displaced amacrine cells in the rabbit retina express nicotinic acetylcholine receptors. Vis Neurosci 17: 743–752, 2000.
  34. Khiroug SS, Harkness PC, Lamb PW, Sudweeks SN, Khiroug L, Millar NS, and Yakel JL. Rat nicotinic ACh receptor {alpha}7 and {beta}2 subunits co-assemble to form functional heteromeric nicotinic receptor channels. J Physiol 540: 425–434, 2002.
  35. Kofke WA, Garman RH, Janosky J, and Rose ME. Opioid neurotoxicity: neuropathologic effects in rats of different fentanyl congeners and the effects of hexamethonium-induced normotension. Anesth Analg 83: 141–146, 1996.
  36. Kroger S and Schroder JE. Agrin in the developing CNS: new roles for a synapse organizer. News Physiol Sci 17: 207–212, 2002.
  37. Lee SW, Kim WJ, Choi YK, Song HS, Son MJ, Gelman IH, Kim YJ, and Kim KW. SSeCKS regulates angiogenesis and tight junction formation in blood-brain barrier. Nat Med 9: 900–906, 2003.
  38. Le Houezec J and Benowitz NL. Basic and clinical psychopharmacology of nicotine. Clin Chest Med 12: 681–699, 1991.
  39. Levine RR and Clark BC. Physiologic disposition of hexamethonium and chemically related compounds. Fed Proc 16: 317, 1957.
  40. Lindstrom J, Schoepfer R, and Whiting P. Molecular studies of the neuronal nicotinic acetylcholine receptor family. Mol Neurobiol 1: 281–337, 1987.
  41. Lotti M. Cholinesterase inhibition: complexities in interpretation. Clin Chem 41: 1814–1818, 1995.
  42. Macklin KD, Maus AD, Pereira EF, Albuquerque EX, and Conti-Fine BM. Human vascular endothelial cells express functional nicotinic acetylcholine receptors. J Pharmacol Exp Ther 287: 435–439, 1998.
  43. Madrigal JL, Moro MA, Lizasoain I, Lorenzo P, and Leza JC. Stress-induced increase in extracellular sucrose space in rats is mediated by nitric oxide. Brain Res 938: 87–91, 2002.
  44. Malin DH, Lake JR, Carter VA, Cunningham JS, Hebert KM, Conrad DL, and Wilson OB. The nicotinic antagonist mecamylamine precipitates nicotine abstinence syndrome in the rat. Psychopharmacology (Berl) 115: 180–184, 1994.
  45. Malin DH, Lake JR, Schopen CK, Kirk JW, Sailer EE, Lawless BA, Upchurch TP, Shenoi M, and Rajan N. Nicotine abstinence syndrome precipitated by central but not peripheral hexamethonium. Pharmacol Biochem Behav 58: 695–699, 1997.
  46. Mark KS, Burroughs AR, Brown RC, Huber JD, and Davis TP. Nitric oxide mediates hypoxia-induced changes in paracellular permeability of cerebral microvasculature. Am J Physiol Heart Circ Physiol 286: H174–H180, 2004.
  47. Moccia F, Frost C, Berra-Romani R, Tanzi F, and Adams DJ. Expression and function of neuronal nicotinic ACh receptors in rat microvascular endothelial cells. Am J Physiol Heart Circ Physiol 286: H486–H491, 2004.
  48. Morley P, Small DL, Murray CL, Mealing GA, Poulter MO, Durkin JP, and Stanimirovic DB. Evidence that functional glutamate receptors are not expressed on rat or human cerebromicrovascular endothelial cells. J Cereb Blood Flow Metab 18: 396–406, 1998.
  49. Parnavelas JG, Kelly W, and Burnstock G. Ultrastructural localization of choline acetyltransferase in vascular endothelial cells in rat brain. Nature 316: 724–725, 1985.
  50. Romero IA, Radewicz K, Jubin E, Michel CC, Greenwood J, Couraud PO, and Adamson P. Changes in cytoskeletal and tight junctional proteins correlate with decreased permeability induced by dexamethasone in cultured rat brain endothelial cells. Neurosci Lett 344: 112–116, 2003.
  51. Salmoiraghi GC, McCubbin JW, and Page IH. Effects of D-lysergic acid diethylamide and its brom derivative on cardiovascular responses to serotonin and on arterial pressure. J Pharmacol Exp Ther 119: 240–247, 1957.
  52. Schanker LS, Prockop LD, Schou J, and Sisodia P. Rapid efflux of some quaternary ammonium compounds from cerebrospinal fluid. Life Sci 10: 515–521, 1962.
  53. Smith QR, Ziylan YZ, Robinson PJ, and Rapoport SI. Kinetics and distribution volumes for tracers of different sizes in the brain plasma space. Brain Res 462: 1–9, 1988.
  54. St’astny F, Schwendt M, Lisy V, and Jezova D. Main subunits of ionotropic glutamate receptors are expressed in isolated rat brain microvessels. Neurol Res 24: 93–96, 2002.
  55. Tonnessen BH, Severson SR, Hurt RD, and Miller VM. Modulation of nitric-oxide synthase by nicotine. J Pharmacol Exp Ther 295: 601–606, 2000.
  56. Wanamaker CP, Christianson JC, and Green WN. Regulation of nicotinic acetylcholine receptor assembly. Ann NY Acad Sci 998: 66–80, 2003.
  57. Wang Y, Pereira EF, Maus AD, Ostlie NS, Navaneetham D, Lei S, Albuquerque EX, and Conti-Fine BM. Human bronchial epithelial and endothelial cells express {alpha}7 nicotinic acetylcholine receptors. Mol Pharmacol 60: 1201–1209, 2001.
  58. Young JM, Shytle RD, Sanberg PR, and George TP. Mecamylamine: new therapeutic uses and toxicity/risk profile. Clin Ther 23: 532–565, 2001.
  59. Zhao L, Kuo YP, George AA, Peng JH, Purandare MS, Schroeder KM, Lukas RJ, and Wu J. Functional properties of homomeric, human {alpha}7-nicotinic acetylcholine receptors heterologously expressed in the SH-EP1 human epithelial cell line. J Pharmacol Exp Ther 305: 1132–1141, 2003.
  60. Zonta M, Angulo MC, Gobbo S, Rosengarten B, Hossmann KA, Pozzan T, and Carmignoto G. Neuron-to-astrocyte signaling is central to the dynamic control of brain microcirculation. Nat Neurosci 6: 43–50, 2003.




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