Research reportNicotine increases in vivo blood–brain barrier permeability and alters cerebral microvascular tight junction protein distribution
Introduction
The blood–brain barrier (BBB) is a vascular system that regulates the passage of materials between the peripheral circulation and the central nervous system (CNS). The BBB is essential for maintaining brain homeostasis and enabling proper neuronal function [32], [53] but makes the non-invasive delivery of therapeutics to the brain problematic [48]. Situated at the level of the cerebral microvascular endothelium [44], the BBB presents a diffusion barrier to most non-lipophillic molecules [33]. The barrier is established by a lack of fenestrations [13] and the presence of tight junctions (TJ) in the apical region of the interendothelial cleft. TJ are elaborate, interconnected membrane-spanning structures composed of transmembrane proteins linked via accessory proteins to the actin cytoskeleton [59] found in epithelial and endothelial barrier tissues throughout the body. Once thought to be static structures, TJ are in fact regulated in both physiological and pathological states [25], and changes in TJ protein expression and/or organization have been associated with altered permeability [4], [15], [27], [37], [46], [62].
Approximately 23% of people in the United States smoke cigarettes [5]. Cigarette smoking is associated with increased risk of cancer, lung disease, and cardiovascular disease including cerebrovascular disease [19]. Though cigarette smoke contains more than 4000 chemical constituents [22], the alkaloid nicotine merits special consideration with regard to vascular disease. In addition to being the primary mediator of the subjective and potentially addictive effects of tobacco [42], nicotine has profound effects on vascular tissues including stimulation of abnormal DNA synthesis in endothelial cells [58], increased expression of atherogenic genes [65], altered expression of signal transduction and transcription factor genes [66], and angiogenesis [20], [21].
The effect of nicotine on the BBB is controversial. One study found that acute nicotine at non-toxic concentrations had no effect on the permeability of large molecular weight markers at the BBB [49], though this finding did not rule out the possibility that nicotine may disrupt the BBB to passage of smaller molecules [31]. In subsequent chronic studies, non-toxic doses of nicotine altered both the expression and function of ion [2], [60] and glucose transporters [9], [10] in cerebral microvessels. Additionally, we have reported that nicotine and its major metabolite cotinine increase the permeability of an in vitro model of the BBB, and that this change is associated with diminished expression and altered distribution of the TJ protein ZO-1 mediated by endothelial nicotinic acetylcholine receptors [1].
In the current study, nicotine was infused via subcutaneous osmotic pumps into Sprague–Dawley rats in a dosing regimen designed to sustain plasma nicotine and cotinine concentrations comparable to those found in human smokers. Using modifications of the in situ brain perfusion method of Takasato [55], we investigated the effects of nicotine on in vivo BBB permeability. Expression and distribution of the TJ-associated proteins ZO-1, ZO-2, occludin, claudin-1, -3, and -5, and the cytoskeletal protein actin were examined using Western blot analysis and fluorescent immunohistochemistry.
Section snippets
Radioisotopes, antibodies, and chemicals
[14C]-Sucrose was purchased from ICN Pharmaceuticals (specific activity, 462 mCi·mol−1; Irvine, CA). Mouse monoclonal anti-actin was purchased from Sigma (St. Louis, MO). Rabbit polyclonal anti-ZO-1 and -2, rabbit polyclonal anti-claudin-1 and -3, mouse monoclonal anti-claudin-5 and anti-occludin were purchased from Zymed (San Francisco, CA). Conjugated anti-mouse and anti-rabbit IgG-horseradish peroxidase were obtained from Amersham (Springfield, IL). Alexafluor™ 488-conjugated phalloidin and
HPLC analysis of plasma nicotine and cotinine
To confirm the osmotic pump delivery of nicotine, plasma levels of nicotine and cotinine were measured by HPLC in blood samples collected from the tail vein prior to sacrifice. Nicotine eluted at a retention time of 4 min, cotinine at 5.5 min, and 2-phenylimidizole at 10 min. Nicotine and cotinine were not detected in naïve animals or in saline-treated controls. In the 1-day nicotine group, nicotine and cotinine plasma levels were 45.0±20.3 and 269.2±74.6 ng·ml−1 (n=3), respectively, and in the
Nicotine increases BBB permeability
In the current study, smoking was modeled by continuous subcutaneous administration of nicotine via an osmotic pump at 4.5 mg·kg−1·day−1. HPLC analysis confirmed that this regimen sustains nicotine and cotinine plasma levels comparable to those observed in chronic heavy smokers [39], [61]. This is a useful criterion for evaluating the relevance of a nicotine treatment in light of the observation that habitual smokers regulate their smoking behavior to sustain a certain level of nicotine in
Acknowledgements
This work was supported by NIH grants NS 39592, DA 11271, and Arizona Disease Control Research Commission contract 5011 to T.P.D, NS 046526 to T.J.A., DK 065003 to R.D.E., NRSA DA 06037 to J.D.H., and NRSA NS 43052 to R.C.B.
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