Regulation of blood-brain barrier Na,K,2Cl-cotransporter through phosphorylation during in vitro stroke conditions and nicotine exposure

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 (RB)-R-86 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.