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In the current study, we determined the functional significance of sodium-dependent/-independent glucose transporters at the neurovasculature during oxygen glucose deprivation (OGD). Confluent brain endothelial cells cocultured with astrocytes were exposed to varying degrees of in vitro stroke conditions. Glucose transporter (GLUT) 1 and sodium glucose cotransporter (SGLT) activity were investigated by luminal membrane uptake and transport studies using [3 H]D-glucose and also by [ 14 C]␣-methyl D-glucopyranoside (AMG), a specific, nonmetabolized substrate of SGLT. In vivo middle cerebral artery occlusion experiments were tested to determine whether blood-brain barrier (BBB) SGLT activity was induced during ischemia. Increases in luminal D-glucose and AMG uptake and transport were observed with in vitro stroke conditions. Specific inhibitor experiments suggest a combined role for both SGLT and GLUT1 at the BBB during OGD. A time-dependent increase in the uptake of AMG was also seen in mice exposed to permanent focal ischemia, and this increase was sensitive to the SGLT inhibitor, phlorizin. Infarct and edema ratio during ischemia were significantly decreased by the inhibition of this transporter. These results show that both GLUT1 and SGLT play a role at the BBB in the blood-to-brain transport of glucose during ischemic conditions, and inhibition of SGLT during stroke has the potential to improve stroke outcome. Pharmacological modulation of this novel BBB transporter could prove to be a brain vascular target in stroke.

Cigarette smoking is strongly implicated in the development of cardiovascular disorders. Recently identified nicotinium analogs may have therapeutic benefit as smoking cessation therapies but may have restricted entry into the central nervous system by the blood-brain barrier (BBB) due to their physicochemical properties. Using the in situ perfusion technique, lobeline, choline, and nicotinium analogs were evaluated for binding to the BBB choline transporter. Calculated apparent K i values for the choline transporter were 1., 393 M lobeline, and Ն1000 M Nmethylnicotinium iodide. Nicotine and N-methylpyridinium iodide, however, do not apparently interact with the BBB choline transporter. Given NONI's apparent K i value determined in this study and its ability to inhibit nicotine-evoked dopamine release from superfused rat brain slices, potential brain entry of NONI via the BBB choline transporter was evaluated. [3 H]NONI exhibited a BBB transfer coefficient value of ϳ1.6 ϫ 10 Ϫ3 ml/s/g and a K m of ϳ250 M. Unlabeled choline addition to the perfusion fluid reduced [ 3 H]NONI brain uptake. We hypothesize the N-n-octyl group on the pyridinium nitrogen of NONI facilitates brain entry via the BBB choline transporter. Thus, NONI may have utility as a smoking cessation agent, given its ability to inhibit nAChRs mediating nicotine-evoked dopamine release centrally, and to be distributed to brain via the BBB choline transporter.

Choline transport has been characterized by multiple mechanisms including the blood±brain barrier (BBB), and high-and low-af®nity systems. Each mechanism has unique locations and characteristics yet retain some similarities. Previous studies have demonstrated cationic competition by monovalent cations at the BBB and cation divalent manganese in the high-af®nity system. To evaluate the effects of divalent manganese inhibition as well as other cationic metals at the BBB choline transporter, brain choline uptake was evaluated in the presence of certain metals of interest in Fischer-344 rats using the in situ brain perfusion technique. Brain choline uptake was inhibited in the presence of Cd 21 (73^2%) and Mn 21 (44^6%), whereas no inhibition was observed with Cu 21 and Al 31 . Furthermore, it was found that manganese caused a reduction in brain choline uptake and signi®cant regional choline uptake inhibition in the frontal and parietal cortex, the hippocampus and the caudate putamen (45^3%, 68^18%, 58^9% and 46^15%, respectively). These results suggest that choline uptake into the CNS can be inhibited by divalent cationic metals and monovalent cations. In addition, the choline transporter may be a means by which manganese enters the brain.

Smoking tobacco, including cigarettes, has been associated with an increased incidence and relative risk for cerebral infarction in both men and women. Recently, we have shown that nicotine and cotinine attenuate abluminal (brain facing) K ϩ uptake mediated by the Na,K,2Cl-cotransporter (NKCC) in bovine brain microvessel endothelial cells (BBMECs) after hypoxic/ aglycemic exposure (stroke conditions). The purpose of the current study was to explore the effects of nicotine and tobacco smoke chemicals on K ϩ movement through the blood-brain barrier during both hypoxia/aglycemia and reoxygenation. BBMECs were exposed to nicotine/cotinine, nicotine-containing cigarette smoke extract (N-CSE), or nicotine-free cigarette smoke extract (NF-CSE) in quantities designed to mimic plasma concentrations of smokers. Stroke conditions were mimicked in vitro in BBMECs through 6 h of hypoxia/aglycemia with or without 12 h of reoxygenation, after which NKCCmediated K ϩ uptake and paracellular integrity were measured with 86 Rb and [ 14 C]sucrose, respectively. In addition, K ϩ concentrations in brain extracellular fluid were estimated in 86 Rb-injected rats that were administered nicotine, N-CSE, or NF-CSE and on whom global ischemia/reperfusion by in vivo four-vessel occlusion was performed. Both in vitro and in vivo paradigms showed nicotine, the major alkaloid present in tobacco smoke, to be the determining factor of an inhibited response of abluminal NKCC in BBMECs during and after stroke conditions. This was measured as a decrease in abluminal brain endothelial cell NKCC activity and as an increase in brain extracellular K ϩ concentration measured as the brain extracellular fluid 86 Rb/plasma ratio after in vivo four-vessel occlusion with reperfusion.

Specific PKC inhibitors or activators might be designed to individualize stroke therapies and improve health outcome for smokers by rebalancing ion transport into and out of the brain.

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