Abstract
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.
Footnotes
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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.
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DOI: 10.1124/jpet.104.066274.
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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.
- Received January 28, 2004.
- Accepted April 1, 2004.
- The American Society for Pharmacology and Experimental Therapeutics
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