Nicotine and Cotinine Modulate Cerebral Microvascular Permeability and Protein Expression of ZO-1 through Nicotinic Acetylcholine Receptors Expressed on Brain Endothelial Cells

doi:10.1002/jps.10256

Journal of Pharmaceutical Sciences

Volume 91, Issue 12, December 2002, Pages 2525-2538

https://doi.org/10.1002/jps.10256 Get rights and content

ABSTRACT:

The blood–brain barrier (BBB) adapts to a variety of pathological processes. Little is known about the effects of nicotine exposure on BBB function and the ability to adapt to stroke conditions. We have demonstrated, using a well-characterized in vitro BBB model, bovine brain microvessel endothelial cells (BBMEC) model, that nicotine and its major metabolite, cotinine, modulate BBB integrity by opening the paracellular route of solute entry into the brain. Additionally, nicotine and cotinine together increase the permeability change observed after 6 h of hypoxia/aglycemia, an in vitro model of stroke. This has important implications for how the BBB initially adapts to stroke in an environment that is previousely exposed to nicotine. Nicotine and cotinine exposure also resulted in reduced ZO-1 immunoreactivity (tight junctional protein) that occurred in a time-dependent manner. Interestingly, attenuation of bovine brain microvessel endothelial cell (BBMEC) ZO-1 protein expression was reversed using 10 nM BGT, an α7 nicotinic acetycholine receptor (nAChR) antagonist, suggesting that the effects of nicotine on BBMEC protein expression of ZO-1 protein are mediated by nAChR expressed on brain endothelial cells. In addition to α7, we found that BBMEC also contain positive immunoreactivity for the α3, α5, β2, β3 nAChR subunit. Both α7 and β2 nAChR subunit protein levels decreased with prior nicotine and cotinine exposure. These data provide evidence that nicotine and cotinine alter BBB permeability and tight junctional protein expression of ZO-1, thereby altering the BBB response to stroke conditions. These changes in brain endothelial cell paracellular permeability are believed to be associated with nicotine binding to nAChRs present at the BBB. © 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association

Section snippets

INTRODUCTION

The blood–brain barrier (BBB) is a system of brain capillaries that regulate the level of glucose, electrolytes, amino acids, iron, insulin, and other metabolites in the extracellular space of the brain. Any changes in basal permeability of the cerebral capillary endothelium (paracellular or transcellular) can exacerbate a variety of pathological processes.1., 2., 3. Changes in BBB permeability have been demonstrated after hypoxia/aglycemia used to model stroke.²^,³ Despite the fact that smoking

In Vitro Bovine Brain Microvessel Endothelial Cells (BBMEC) Model

BBMECs were isolated from fresh bovine brains and cryo-preserved, as previously described.25., 26., 27. The isolated cells were seeded at a cell density of 50,000 cells/cm². All endothelial cells used for these studies were primary cultured cells, which have been shown to maintain excellent BBB characteristics as well as a good in vitro to in vivo correlation of permeability.27., 28., 29., 30., 31. All in vitro experiments were conducted with the combinations of two separate isolates of BBMECs

BBMEC CYTOTOXICITY EXPERIMENTS

Trypan Blue Exclusion experiments were conducted with all the above exposures to nicotine and cotinine. At the end of drug exposure, BBMECs were incubated in medium containing 0.4% trypan blue for 1 h. To dissociate the cells, 0.05% trypsin and 0.53 mmol/L EDTA were added. Cell viability was determined using light microscopy and viability counted as cells excluding trypan blue.

MTT assays were conducted with all nicotine and cotinine exposures. 0.5 mg/mL of MTT reagent was added for 4 h, followed

In Vitro BBMEC Permeability of [¹⁴C] Sucrose after Nicotine and Cotinine Treatment

The effect of both nicotine (100 ng/mL) and cotinine (1000 ng/mL) together on the basal permeability of BBMEC monolayers to [¹⁴C]sucrose was studied. It was found that BBMEC permeability to [¹⁴C]sucrose was increased to a statistically significant level after 12-h exposure to the combination of nicotine (100–1000 ng/mL) and cotinine (1000–10,000 ng/mL) (Fig. 1). The resultant increase in [¹⁴C]sucrose permeability after 24 h of nicotine (100 ng/ml) and cotinine (1000 ng/ml) was significantly decreased (

DISCUSSION

Studying the effects nicotine on the brain microvasculature necessitates the use of nicotine and its metabolites at a concentration equivalent to what is observed in a smoker. Russell et al.³⁶ has shown that nicotine concentration in blood for individual smokers averages 33 ng/mL. Additionally, it has been shown that approximately 72% of nicotine is metabolized via C-oxidation to cotinine, with a range of 55–92%.³⁷ Cotinine has also been measured in human plasma at a concentration approximately

ACKNOWLEDGEMENTS

This work was supported by ADCRC Grant #1–341, #5011, RO1NS 39592, and AACP NIP. The authors would like to acknowledge the Burroughs Wellcome Fund and the American Foundation for Pharmaceutical Education for their support.

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Citation Excerpt :
Anatomically, some studies showed that there are nerve terminals around the BBB, which may contain cholinergic and noradrenergic terminals, constituting neurovascular units (Banerjee and Bhat, 2007; Harik et al., 1981; Iijima, 1977; Karcsú et al., 1981; Lok et al., 2007; Zlokovic, 2008). And the activation of α7nAChR in brain endothelial cells, which were the components of the BBB, could reduce the permeability of the BBB (Abbruscato et al., 2002; Hawkins et al., 2005; Kimura et al., 2019). Besides, the electrical stimulation of LC and norepinephrine (NE) itself could increase the pinocytotic activity of endothelial cells, without any damage to the capillary walls (Harik et al., 1981; Sarmento et al., 1994, 1991).
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Research ArticlesNicotine and Cotinine Modulate Cerebral Microvascular Permeability and Protein Expression of ZO-1 through Nicotinic Acetylcholine Receptors Expressed on Brain Endothelial Cells

ABSTRACT:

Section snippets

INTRODUCTION

In Vitro Bovine Brain Microvessel Endothelial Cells (BBMEC) Model

BBMEC CYTOTOXICITY EXPERIMENTS

In Vitro BBMEC Permeability of [14C] Sucrose after Nicotine and Cotinine Treatment

DISCUSSION

ACKNOWLEDGEMENTS

Brain Res

Eur J Pharm

J Invest Dermatol

Brain Res

Dev Biol

Neurosci Lett

Int Rev Cytol

Exp Cell Res

Brain Res

Peptides

Drug Alcohol Depend

Toxicol Lett

Neuropharmacology

FEBS Lett

Brain Res

J Auton Nerv Syst

Electron microscopy of the blood–brain barrier in disease

Microsc Res Tech

Combination of hypoxia/aglycemia compromises in vitro blood–brain barrier integrity

J Pharmacol Exp Ther

The structures of neuronal nicotinic receptors

The nicotinic acetylcholine receptor: Structure and autoimmune pathology

Crit Rev Biochem Mol Biol

Properties of neuronal nicotinic acetylcholine receptors: Pharmacological characterization and modulation of synaptic function

J Pharmacol Exp Ther

Molecular structure of the nicotinic acetylcholine receptor

Cold Spring Harb Symp Quant Biol

Human and rodent epithelial cells express functional nicotinic acetylcholine receptors

Mol Pharmacol

Human bronchial eptithelial and endothelial cells express alpha-7 nicotinic acetylcholine receptors

Mol Pharmacol

Chronic nicotine treatment enhances focal ischemic brain injury and depletes free pool of brain microvascular tissue plasminogen activator in rats

J Cereb Blood Flow Metab

Nicotine raises the influx of permeable solutes across the rat blood–brain barrier with little or no capillary recruitment

J Cereb Blood Flow Metab

Junctions between intimately apposed cell membranes in the vertebrate brain

J Cell Biol

Research Articles
Nicotine and Cotinine Modulate Cerebral Microvascular Permeability and Protein Expression of ZO-1 through Nicotinic Acetylcholine Receptors Expressed on Brain Endothelial Cells

In Vitro BBMEC Permeability of [¹⁴C] Sucrose after Nicotine and Cotinine Treatment