Cerebromicrovascular endothelial cells are resistant to l-glutamate

Abstract

Cerebral microvascular endothelial cells (CMVECs) have recently been implicated as targets of excitotoxic injury by L-glutamate (L-glut) or N-methyl-Daspartate (NMDA) in vitro. However, high levels of L-glut do not compromise the function of the blood-brain barrier in vivo. We sought to determine whether primary cultures of rat and piglet CMVECs or cerebral microvascular pericytes (CMVPCs) are indeed sensitive to L-glut or NMDA. Viability was unaffected by 8-h exposure to 1-10 mM L-glut or NMDA in CMVECs or CMVPCs isolated from both species. Furthermore, neither 1 mM L-glut nor NMDA augmented cell death induced by 12-h oxygen-glucose deprivation in rat CMVECs or by 8-h medium withdrawal in CMVPCs. Additionally, transendothelial electrical resistance of rat CMVEC-astrocyte cocultures or piglet CMVEC cultures were not compromised by up to 24-h exposure to 1 mM L-glut or NMDA. The Ca 2ϩ ionophore calcimycin (5 M), but not L-glut (1 mM), increased intracellular Ca 2ϩ levels in rat CMVECs and CMVPCs assessed with fluo-4 AM fluorescence and confocal microscopy. CMVEC-dependent pial arteriolar vasodilation to hypercapnia and bradykinin was unaffected by intracarotid infusion of L-glut in anesthetized piglets by closed cranial window/intravital microscopy. We conclude that cerebral microvascular cells are insensitive and resistant to glutamatergic stimuli in accordance with their in vivo role as regulators of potentially neurotoxic amino acids across the blood-brain barrier.N-methyl-D-aspartate; blood-brain barrier; cerebrovascular reactivity; glutamate excitotoxicity; intracellular calcium L-GLUTAMATE (L-glut) is one of the most important excitatory amino acids in the central nervous system. Apart from its well-known neurotransmitter role, L-glut plays a critical role in ammonia removal from the brain. L-Glut transported from the blood plasma takes up ammonia and leaves as L-glutamine (12). This balanced L-glutamate/L-glutamine countertransport is regulated by cerebral microvascular endothelial cells (CMVECs) forming the blood-brain barrier (BBB). Furthermore, CMVECs can pump L-glut from the brain to the blood, and thus they provide a functional interface for the regulated bidirectional transport of L-glut (18, 39). Human blood plasma L-glut levels range between 20 and 200 M, and they may exceed 700 M after ingestion of high amounts of L-glut under experimental conditions (4, 41). These plasma L-glut concentrations are not neurotoxic in vivo, although these levels are excitotoxic to neurons in vitro, suggesting that CMVECs are resistant to high L-glut levels and prevent the passive transport of L-glut into the brain.Surprisingly, several recent reports have proposed that the L-glut levels that occur during cerebral ischemia can injure or kill CMVECs via excitotoxic mechanisms in vitro. CMVECs have been reported to possess various L-glut receptor subunits or L-glut binding sites in rat, piglet, or human CMVECs (1,5,27,34,36,45). High concentrations of L-glut (1-3 mM), at least in vitro, are claimed to result in N-met...

Increased Susceptibility to Amyloid-β Toxicity in Rat Brain Microvascular Endothelial Cells under Hyperglycemic Conditions

“…At day 3, the cell medium was replaced by fresh medium with different glucose concentrations and was then changed every 48 h. The cell culture medium consisted of DMEM supplemented with 20% fetal bovine plasma-derived serum, 2 mM glutamine, 1 ng/ml basic fibroblast growth factor, 50 μg/ml endothelial cell growth supplement, 100 μg/ml heparin, 5 μg/ml vitamin C, and antibiotics. We have previously demonstrated the purity of our cultures consisting of >95% BMECs, verified by positive immunohistochemistry for von Willebrand factor and by negative immunochemistry for glial fibrillary acidic protein (GFAP) and α-smooth muscle actin [16]. …”

Blood–Brain Barrier Pathophysiology in Traumatic Brain Injury

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A Fast One‐Step Extraction and UPLC–MS/MS Analysis for E2/D2 Series Prostaglandins and Isoprostanes

“…Keywords Prostaglandin Á Isoprostane Á Brain lipids Á Prostanoid analysis Á Ischemia Á Microwave irradiation Á Ultra high performance liquid chromatography Á Tandem mass spectrometry Abbreviations PG Prostaglandins iso-PG Isoprostanes PGE 2 9-oxo-11a,15S-dihydroxyprosta-5Z,13Edien-1-oic acid 11b-PGE 2 9-oxo-11b,15S-dihydroxyprosta-5Z,13Edien-1-oic acid 8iso-PGE 2 9-oxo-11a,15S-dihydroxy-(8b)-prosta-5Z,13E-dien-1-oic acid Ent-PGE 2 9-oxo-11b,15R-dihydroxy-(8b,12a)-prosta-5Z,13E-dien-1-oic acid 15(R)-PGE 2 9-oxo-11a,15R-dihydroxy-prosta-5Z,13Edien-1-oic acid 5trans-PGE 2 9-oxo-11a,15S-dihydroxy-prosta-5E,13Edien-1-oic acid PGE 2 -d 4 9-oxo-11a,15S-dihydroxy-prosta-5Z,13Edien-1-oic-3,3,4,4-d 4 acid PGD 2 9a,15S-dihydroxy-11-oxo-prosta-5Z,13Edien-1-oic acid 15(R)-PGD 2 9a,15R-dihydroxy-11-oxo-prosta-5Z,13Edien-1-oic acid D12PGD 2 9a,15S-dihydroxy-11-oxo-prosta-5Z,12Edien-1-oic acid PL Phospholipids PLA 2 Phospholipase A 2 sPLA 2 Secreated phospholipase A 2 20:4n- 6 Arachidonic acid UPLC Ultra high performance liquid chromatography Introduction Prostaglandins (PG) are critically important signaling molecules derived from polyunsaturated fatty acids. In the brain, PG regulate a variety of normal processes such as synaptic plasticity due to modulation of adrenergic, noradrenergic, glutamatergic neurotransmission and membrane excitability [1][2][3][4], and are critical for instantaneous regulation of cerebral blood flow in response on changes in neuronal activity [5][6][7]. PG also play a critical role in many pathophysiological conditions including inflammation, cancer, neurodegeneration, neuropsychiatric conditions, and central nervous system injury [8][9][10][11][12][13][14][15].…”