Acclimation to decompression sickness in rats.

Protection against decompression sickness (DCS) by acclimation to hyperbaric decompression has been hypothesized but never proven. We exposed rats to acclimation dives followed by a stressful "test" dive to determine whether acclimation occurred. Experiments were divided into two phases. Phase 1 rats were exposed to daily acclimation dives of hyperbaric air for 30 min followed by rapid decompression on one of the following regimens: 70 ft of seawater (fsw) for 9 days (L70), 70 fsw for 4 days (S70), 40 fsw for 9 days (L40), 40 fsw for 4 days (S40), or unpressurized sham exposure for 9 days (Control). On the day following the last exposure, all were subjected to a "test" dive (175 fsw, 60 min, rapid decompression). Both L70 and S70 rats had significantly lower incidences of DCS than Control rats (36% and 41% vs. 62%, respectively). DCS incidences for the other regimens were lower than in Control rats but without statistical significance. Phase 2 used the most protective regimen from phase 1 (L70); rats were exposed to L70 or a similar regimen with a less stressful staged decompression. Another group was exposed to a single acclimation dive (70 fsw/30 min) on the day before the test dive. We observed a nonsignificant trend for the rapidly decompressed L70 dives to be more protective than staged decompression dives (44% vs. 51% DCS incidence). The single acclimation dive regimen did not provide protection. We conclude that protection against DCS can be attained with acclimating exposures that do not themselves cause DCS. The deeper acclimation dive regimens (70 fsw) provided the most protection.

Endothelial-mediated regulation of cerebral microcirculation.

Vascular endothelial cells are important not only for maintaining homeostasis, but also in pathogenesis of vascular disorders. Cerebral capillary and microvascular endothelial cells play an active role in maintaining cerebral blood flow, microvascular tone and blood brain barrier functions. Factors produced and released by endothelial cells, other brain cells and circulating blood cells participate in these regulatory functions. In particular, endothelin-1 (ET-1) and nitric oxide (NO) are known to contribute to the functional vascular changes under pathological conditions (e.g., hypertension, arteriosclerosis, and stroke). This report describes the involvement of endothelial cell mediators in the post-ischemic hypoperfusion induced by brain ischemia and in vitro endothelial responses (Ca(2+) mobilization and cytoskeletal rearrangements) to ET-1 and its interactions with NO or 2-AG. The capacity of NO and endocannabinoids to counteract ET-1-induced cerebral capillary and microvascular endothelial responses indicates that they may actively participate in EC function and implicates them in physiological and pathophysiological conditions.

ET-1- and NO-mediated signal transduction pathway in human brain capillary endothelial cells.

Previous studies have demonstrated that functional interaction between endothelin (ET)-1 and nitric oxide (NO) involves changes in Ca(2+) mobilization and cytoskeleton in human brain microvascular endothelial cells. The focus of this investigation was to examine the possible existence of analogous interplay between these vasoactive substances and elucidate their signal transduction pathways in human brain capillary endothelial cells. The results indicate that ET-1-stimulated Ca(2+) mobilization in these cells is dose-dependently inhibited by NOR-1 (an NO donor). This inhibition was prevented by ODQ (an inhibitor of guanylyl cyclase) or Rp-8-CPT-cGMPS (an inhibitor of protein kinase G). Treatment of endothelial cells with 8-bromo-cGMP reduced ET-1-induced Ca(2+) mobilization in a manner similar to that observed with NOR-1 treatment. In addition, NOR-1 or cGMP reduced Ca(2+) mobilization induced by mastoparan (an activator of G protein), inositol 1,4,5-trisphosphate, or thapsigargin (an inhibitor of Ca(2+)-ATPase). Interestingly, alterations in endothelial cytoskeleton (actin and vimentin) were associated with these effects. The data indicate for the first time that the cGMP-dependent protein kinase colocalizes with actin. These changes were accompanied by altered levels of phosphorylated vasodilator-stimulated phosphoprotein, which were elevated in endothelial cells incubated with NOR-1 and significantly reduced by ODQ or Rp-8-CPT-cGMPS. The findings indicate a potential mechanism by which the functional interrelationship between ET-1 and NO plays a role in regulating capillary tone, microcirculation, and blood-brain barrier function.

Antioxidant properties of the vasoactive endocannabinoid, 2-arachidonoyl glycerol (2-AG).

Reactive oxygen species (ROS) were shown to play a role in altering blood-brain barrier (BBB) permeability and formation of brain edema induced by trauma and/or ischemia. 2-arachidonoyl glycerol (2-AG), a novel, potent vasodilatory and cytoprotective endocannabinoid has been implicated to act as an antioxidative agent. This study examines: 1) the possible 2-AG modulation of BBB injury and edema formation induced by closed head injury (CHI); and 2) comparable effects between 2-AG and 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TPL), a known antioxidant nitroxide on endothelial Ca2+ and cytoskeletal responses to H2O2 (ROS). 2-AG treatment reduced the CHI-induced increase in BBB permeability and brain edema. The endothelial H2O2-stimulated Ca2+ mobilization and cytoskeleton (vimentin) rearrangement was modified by either 2-AG or TPL. These findings provide evidence of 2-AG antioxidant activity and are consistent with the involvement of ROS in the pathomechanism of CHI-induced BBB injury and brain edema.

Examination of several potential mechanisms for the negative outcome in a clinical stroke trial of enlimomab, a murine anti-human intercellular adhesion molecule-1 antibody: a bedside-to-bench study.

BACKGROUND AND PURPOSE: Enlimomab, a murine monoclonal anti-human intercellular adhesion molecule (ICAM)-1 antibody, had a negative outcome in a multicenter acute-stroke trial. We did a bedside-to-bench study in standardized rat stroke models to explore mechanisms for these untoward results. METHODS: After focal brain ischemia in Wistar rats and spontaneously hypertensive rats (SHR), we administered murine anti-rat ICAM-1 antibody (1A29), subclass-matched murine immunoglobulin (IgG1), or vehicle intravenously. To examine whether rat anti-mouse antibodies were generated against the mouse protein and whether these were deleterious, we sensitized Wistar rats with 1A29 or vehicle 7 days before surgery. Infarct volume, tissue myeloperoxidase activity, neutrophil CD11b expression, and microvascular E-selectin, P-selectin, and ICAM-1 expression were examined 48 hours after surgery. Complement activation was serially assessed for 2 hours after a single injection of either 1A29 or vehicle. RESULTS: 1A29 treatment did not significantly reduce infarct size in either strain. 1A29 sensitization augmented infarct size and generated rat anti-mouse antibodies. Although 1A29 inhibited neutrophil trafficking shown by reduction in brain myeloperoxidase activity, circulating neutrophils were activated and displayed CD11b upregulation. Complement was activated in 1A29-sensitized Wistar rats and 1A29-treated SHR. E-selectin (SHR), endothelial P-selectin (Wistar and SHR), and ICAM-1 (SHR) were upregulated in animals treated with 1A29. CONCLUSIONS: Administration to rats of a murine antibody preparation against ICAM-1, 1A29, elicits the production of host antibodies against the protein, activation of circulating neutrophils, complement activation, and sustained microvascular activation. These observations provide several possible mechanisms for central nervous system-related clinical deterioration that occurred when Enlimomab was given in acute ischemic stroke.

Human brain capillary endothelium: 2-arachidonoglycerol (endocannabinoid) interacts with endothelin-1.

In brain, the regulatory mechanism of the endothelial reactivity to nitric oxide and endothelin-1 may involve Ca(2+), cytoskeleton, and vasodilator-stimulated phosphoprotein changes mediated by the cGMP/cGMP kinase system.(1) Endothelium of human brain capillaries or microvessels is used to examine the interplay of endothelin-1 with the putative vasorelaxant 2-arachidonoyl glycerol, an endogenous cannabimimetic derivative of arachidonic acid. This study demonstrates that 2-arachidonoyl glycerol counteracts Ca(2+) mobilization and cytoskeleton rearrangement induced by endothelin-1. This event is independent of nitric oxide, cyclooxygenase, and lipoxygenase and is mediated in part by cannabimimetic CB1 receptor, G protein, phosphoinositol signal transduction pathway, and Ca(2+)-activated K(+) channels. The induced rearrangements of cellular cytoskeleton (actin or vimentin) are partly prevented by inhibition of protein kinase C or high levels of potassium chloride. The 2-arachidonoyl glycerol-induced phosphorylation of vasodilator-stimulated phosphoprotein is mediated by cAMP. These findings suggest that 2-arachidonoyl glycerol may contribute to the regulation of cerebral capillary and microvascular function.

Differential expression of HSC73 and HSP72 mRNA and proteins between young and adult gerbils after transient cerebral ischemia: relation to neuronal vulnerability.

This study presents a quantitative comparison of the time courses and regional distribution of both constitutive HSC73 and inducible HSP72 mRNA expression and their respective encoded proteins between young (3-week-old) and adult (3-month-old) gerbil hippocampus after transient global ischemia. The constitutive expression of HSC73 mRNA and protein in the hippocampus of the young sham-operated gerbils was significantly higher than in the adults. The HSC73 mRNA expression after ischemia in the CA1 layer of young gerbils was greater than in adult gerbils. HSC73 immunoreactivity was not significantly changed after ischemia-reperfusion in adult hippocampus, whereas it decreased in young gerbils. Ischemia-reperfusion led to induction of HSP72 mRNA expression throughout the hippocampus of both young and adult gerbils. HSP72 mRNA induction was more intense and sustained in the CA1 subfield of young gerbils; this was associated with a marked induction of HSP72 proteins and neuronal survival. The transient expression of HSP72 mRNA in the CA1 layer of adult gerbils was not associated with a subsequent synthesis of HSP72 protein but was linked to neuronal loss. Expression of HSP72 mRNA was shifted to an earlier period of reflow in CA3 and dentate gyrus (DG) subfields of young animals. These findings suggest that the induction of both HSP72 mRNA and proteins in the CA1 pyramidal neurons of young gerbils, as well as the higher constitutive expression of HSC73, may partially contribute to higher neuronal resistance of young animals to transient cerebral ischemia.

Adrenergic mediation of TNF alpha-stimulated ICAM-1 expression on human brain microvascular endothelial cells.

Adrenergic innervation derived from locus ceruleus has been implicated in regulating BBB permeability and inflammatory responses associated with neurological disorders. This report demonstrates that adrenergic agents attenuate the tumor necrosis factor-alpha (TNF alpha)-induced expression of intercellular adhesion molecule-1 (ICAM-1) on cerebral microvascular endothelial cells (HBMEC) derived from human brains. HBMEC were incubated with isoproterenol (1-10 microM) alone or in the presence of propranolol (10 microM) for 30 min followed by the addition of various concentrations of TNF alpha. ICAM-1 expression on cultured HBMEC was dose-dependently upregulated by TNF alpha. Incubation with isoproterenol significantly reduced levels of ICAM-1 expression indicating the possible involvement of adrenergic agents on ICAM-1 expression. Treatment with propranolol (beta 1/beta 2-adrenergic antagonist) and butoxamine (beta 2-adrenergic antagonist), but not atenolol (beta 1-adrenergic antagonist) reversed this inhibitory effect. Isoproterenol also dose-dependently stimulated cAMP production (assayed by RIA) by HBMEC; propranolol treatment abolished this effect. These data show that the beta 2-adrenergic receptor/cAMP pathway may be partly involved in TNF alpha-stimulated ICAM-1 expression and indicate the possible involvement of adrenergic mediation of capillary function including BBB integrity.

Endothelin-1 and nitric oxide affect human cerebromicrovascular endothelial responses and signal transduction.

Endothelium plays a central role in regulating the vascular tone, blood flow and blood brain barrier (BBB) permeability. The experiments presented here examine the mechanisms by which nitric oxide (NO) and endothelin-1 (ET-1) may be involved in these processes. The findings indicate that ET-1-stimulated [Ca2+]i accumulation occurs through activation of ETA receptor. The capacity of NO to affect this response was indicated by results showing: 1) a two-fold increase in ET-1-stimulated [Ca2+]i by L-NAME, the inhibitor of nitric oxide synthase, and 2) a dose-dependent decrease in [Ca2+]i accumulation by pretreatment with Nor-1 (NO donor). Abrogation of this Nor-1 effect by ODQ (an inhibitor of guanylyl cyclase) or Rp-8-pCPT-cGMPS (an inhibitor of protein kinase G) and inhibition of ET-1 stimulated intracellular Ca2+ accumulation by 8-bromo-cGMP (a permeable, analog of cGMP) substantiate the involvement of interplay between ET-1 and NO in [Ca2+]i accumulation in HBMEC. ET-1 treatment also increased thickness of F-actin cytoskeletal filaments in HBMEC. This effect was attenuated by pretreatment with NO; NO also rarefied F-actin filaments in control cultures. The findings support a linkage between NO and ET-1 in regulating microvascular tone, microcirculation and BBB permeability and indicate a role for cGMP/cGMP protein kinase system and cytoskeletal changes in responses of HBMEC.

Endothelin receptor antagonist preserves microvascular perfusion and reduces ischemic brain damage following permanent focal ischemia.

Synthesis and release of the potent vasoconstrictor peptide endothelin-1 (ET-1) increases following cerebral ischemia and has previously been shown to mediate the delayed hypoperfusion associated with transient global ischemia. In this study we assessed the impact of ET-1 on perfusion and infarct volume in a focal model of cerebral ischemia by use of the selective ET(A) receptor antagonist Ro 61-1790 (affinity for ET(A) receptor 1000 fold greater than ETB receptor). Control rats subjected to permanent middle cerebral artery occlusion (MCAO) showed extensive reductions in microvascular perfusion 4 h post-MCAO that were significantly attenuated by Ro 61-1790 pretreatment (10 mg/kg, i.v.). Ro 61-1790 concomitantly and significantly reduced the ischemic lesion volume in the same animals. This effect was maintained 24 h post-MCAO providing that the animals received additional i.v. injections of 5 mg/kg Ro 61-1790 at 5 h and 8 h after MCAO. These findings demonstrate that ET(A) receptor antagonism partially preserves tissue perfusion following focal ischemia and that this effect is associated with significant neuroprotection. The results also support the hypothesis that vasoactive mediators, and ET-1 in particular, are important contributors to the pathogenesis of cerebral ischemic injury.

[Pathophysiologic effects of nitric oxide (NO) and endothelin-1 in global cerebral ischemia in an animal model--an overview]. / Pathophysiologische Effekte von Stickoxid (NO) und Endothelin-1 bei globaler zerebraler Ischämie im Tiermodell--ein Uberblick.

These studies were performed in an attempt to clarify some of the pathophysiologic mechanisms which occur during and after global ischemia. Both nitric oxide and endothelin were demonstrated in gerbils to participate in responses to ischemia. It was shown that endogenous nitric oxide influences early postischemic reperfusion, systemic blood pressure and postischemic dopamine metabolism. Furthermore, the results indicated that nitric oxide played a role in dopamine release and that preischemic intracerebral nitric oxide formation significantly decreased ischemic dopamine release. In addition, ischemic release of endothelin-1 was detected; participation of nitric oxide in this release was observed. Further indication of functional interactions between nitric oxide and endothelin-1 in postischemic reperfusion were indicated by observations that endothelin-1 antagonists inhibited early hypoperfusion caused by Nitro-L-arginin and late hypoperfusion caused by endogenous endothelin-1. Nitric oxide was shown to decrease edema formation during the early postischemic period but contribute to edema formation during the late postischemic period. The findings indicate the importance of nitric oxide in stroke and ischemia.

Nitric oxide modulates endothelin 1-induced Ca2+ mobilization and cytoskeletal F-actin filaments in human cerebromicrovascular endothelial cells.

A functional interrelation between nitric oxide (NO), the endothelial-derived vasodilating factor, and endothelin 1 (ET-1), the potent vasoconstrictive peptide, was investigated in microvascular endothelium of human brain. Nor-1 dose-dependently decreased the ET-1-stimulated mobilization of Ca2+. This response was mimicked with cGMP and abrogated by inhibitors of guanylyl cyclase or cGMP-dependent protein kinase G. These findings indicate that NO and ET-1 interactions involved in modulation of intracellular Ca2+ are mediated by cGMP/protein kinase G. In addition, Nor-1-mediated effects were associated with rearrangements of cytoskeleton F-actin filaments. The results suggest mechanisms by which NO-ET-1 interactions may contribute to regulation of microvascular function.

Human brain capillary endothelium: modulation of K+ efflux and K+, Ca2+ uptake by endothelin.

This report describes K+ efflux, K+ and Ca2+ uptake responses to endothelins (ET-1 and ET-3) in cultured endothelium derived from capillaries of human brain (HBEC). ET-1 dose dependently increased K+ efflux, K+ and Ca2+ uptake in these cells. ET-1 stimulated K+ efflux occurred prior to that of K+ uptake. ET-3 was ineffective. The main contributor to the ET-1 induced K+ uptake was ouabain but not bumetanide-sensitive (Na+-K+-ATPase and Na+-K+-Cl- cotransport activity, respectively). All tested paradigms of ET-1 effects in HBEC were inhibited by selective antagonist of ET(A) but not ET(B) receptors and inhibitors of phospholipase C and receptor-operated Ca2+ channels. Activation of protein kinase C (PKC) decreased whereas inhibition of PKC increased the ET-1 stimulated K+ efflux, K+ and Ca2+ uptake in HBEC. The results indicate that ET-1 affects the HBEC ionic transport systems through activation of ET(A) receptors linked to PLC and modulated by intracellular Ca2+ mobilization and PKC.

Increased endothelial expression of intercellular adhesion molecule-1 in symptomatic versus asymptomatic human carotid atherosclerotic plaque.

BACKGROUND AND PURPOSE: The mechanisms that cause carotid atherosclerotic plaque to become symptomatic remain unclear. Evidence suggests that mediators of inflammation are not only instrumental in the formation of plaque but may also be involved in the rapid progression of atheromatous lesions leading to plaque fissuring, endothelial injury, and intraluminal thrombosis. Our goal is to determine whether intercellular adhesion molecule-1 (ICAM-1), a known component of the inflammatory pathway, is preferentially expressed on symptomatic versus asymptomatic carotid plaques. METHODS: Carotid plaques from symptomatic (n = 25) and asymptomatic (n = 17) patients undergoing carotid endarterectomy with lesions involving >60% stenosis were snap-frozen at the time of surgery. Immunofluorescence studies were performed to measure the percentage of luminal endothelial surface that expressed ICAM-1. The relationships of stroke risk factors, white blood cell count, percent stenosis, and soluble ICAM-1 (sICAM-1) plasma levels to endothelial ICAM-1 expression were investigated. RESULTS: An increased expression of ICAM-1 was found in the high-grade regions of symptomatic (29.5%+/-2.4%, mean+/-SEM) versus asymptomatic (15.7%+/-2.7%, mean+/-SEM) plaques (P=0.002) and in the high-grade versus the low-grade region of symptomatic plaques (29.5+/-2.4, mean+/-SEM, versus 8.9+/-1.6; P<0.001). Plasma sICAM-1 levels were not predictive of symptomatic disease, and no significant correlation between risk factor exposure and endothelial ICAM-1 expression was found. CONCLUSIONS: An elevation in ICAM-1 expression in symptomatic versus asymptomatic plaque suggests that mediators of inflammation are involved in the conversion of carotid plaque to a symptomatic state. The data also suggest a differential expression of ICAM-1, with a greater expression found in the high-grade region than in the low-grade region of the plaque specimen.

Functional characterization of endothelin receptors on cultured brain capillary endothelial cells of the rat.

This report describes the effects of endothelins (ET-1 and ET-3) on ion transport systems expressed on cultured rat brain capillary endothelial cells (RBEC) and includes investigation of pharmacological properties of ET receptors, their reactivity and induction of signal transduction pathways. ET-1 stimulated IP3 formation and Ca2+ uptake with half-maximal effective concentrations (EC50) of 0.68 and 0.93 nM, respectively; the effects of ET-3 on these responses were much weaker. ET-1-stimulated IP3 formation and Ca2+ uptake were inhibited by an ETA antagonist (BQ123) and a phospholipase C (PLC) inhibitor (U73122), indicating the presence of ETA receptors coupled to PLC. ET-1 stimulated K+ efflux (through a quinine-sensitive mechanism) and K+ uptake (through both ouabain-sensitive and bumetanide-sensitive mechanisms) with EC50 of 0.59 and 0.68 nM, respectively. The potencies of ET-3 on these responses were considerably lower than those of ET-1. By contrast, ET-1 or ET-3 stimulated Na+ uptake with similarly high potencies (EC50 = 0.80 and 1.89 nM, respectively) through EIPA (a Na+/H+ exchange inhibitor)-sensitive mechanisms. ET-stimulated K+ efflux, K+ uptake and Na+ uptake activities were all inhibited by BQ123 (but not by BQ788), suggesting the involvement of ETA (and not ETB) receptors in all these responses. ET-1 stimulated K+ uptake and efflux were inhibited by either U73122 or an intracellular Ca2+ chelator, suggesting that these two responses were mediated via PLC. In contrast, ET stimulation of Na+ uptake was unaffected by PLC inhibition or intracellular Ca2+ chelation. These data suggest the presence of two distinct subtypes of ETA receptors on RBEC; one appears to be a typical ETA receptor which is coupled to PLC and has higher binding affinity for ET-1 than ET-3. The other (ETA-like) receptor is similarly activated by ET-1 and ET-3 with high potencies but is independent of PLC. This possibility was further confirmed by the [125I]ET-1 binding studies demonstrating the presence of high- and low-affinity ET-3 binding sites.

Immunologic tolerance to myelin basic protein decreases stroke size after transient focal cerebral ischemia.

Immune mechanisms contribute to cerebral ischemic injury. Therapeutic immunosuppressive options are limited due to systemic side effects. We attempted to achieve immunosuppression in the brain through oral tolerance to myelin basic protein (MBP). Lewis rats were fed low-dose bovine MBP or ovalbumin (1 mg, five times) before 3 h of middle cerebral artery occlusion (MCAO). A third group of animals was sensitized to MBP but did not survive the post-stroke period. Infarct size at 24 and 96 h after ischemia was significantly less in tolerized animals. Tolerance to MBP was confirmed in vivo by a decrease in delayed-type hypersensitivity to MBP. Systemic immune responses, characterized in vitro by spleen cell proliferation to Con A, lipopolysaccharide, and MBP, again confirmed antigen-specific immunologic tolerance. Immunohistochemistry revealed transforming growth factor beta1 production by T cells in the brains of tolerized but not control animals. Systemic transforming growth factor beta1 levels were equivalent in both groups. Corticosterone levels 24 h after surgery were elevated in all sham-operated animals and ischemic control animals but not in ischemic tolerized animals. These results demonstrate that antigen-specific modulation of the immune response decreases infarct size after focal cerebral ischemia and that sensitization to the same antigen may actually worsen outcome.

Functional properties of cultured endothelial cells derived from large microvessels of human brain.

This report describes the fractional separation of microvessels from human brain for establishment of segmentally derived endothelial cell (EC) cultures. The investigation comprised evaluation of media constituents and purity of the cell culture and focused on functional biochemical characterization of endothelium derived from large microvessels (EC) Cells contained endothelial marker factor VIII (von Willebrand antigen), secreted endothelin-1 (ET-1) and prostaglandins, and took up 86Rb+ as a measure of K+. Exogenous ET-1 stimulated phosphatidylinositol hydrolysis and K+ uptake; BQ-123 (selective ETA receptor antagonist) but not IRL-1038 or BQ-788 (selective ETB receptor antagonists) inhibited both. Ouabain (inhibitor of Na(+)-K(+)-ATPase) and bumetanide (inhibitor of Na(+)-K(+)-Cl- cotransport) reduced (74-80 and 20-40%, respectively) the ET-1-stimulated K+ uptake. Staurosporine [protein kinase C (PKC) inhibitor] selectively reduced Na(+)-K(+)-Cl- cotransport, whereas verapamil but not nifedipine (L-type voltage-dependent Ca2+ channel blockers) decreased Na(+)-K(+)-ATPase activity induced by ET-1. Phorbol 12-myristate 13-acetate (PMA; activator of PKC) stimulated K+ uptake, which was only decreased with bumetanide. N-ethylisopropylamiloride (inhibitor of Na+/H+ exchange) reduced the ET-1-stimulated but not the PMA-induced K+ uptake. Results indicate that phosphatidylinositol hydrolysis and ion transport systems in large microvascular EC are stimulated by ET-1 through activation of ETA receptors. The findings also suggest that the ET-1-stimulated Na(+)-K(+)-ATPase activity, in contrast to Na(+)-K(+)-Cl- cotransport, is not mediated by PKC. In addition, the data suggest a linkage between Na(+)-K(+)-ATPase activity and Na+/H+ exchange.

Hypoxia modulates free radical formation in brain microvascular endothelium.

Although free radical species (ROS; i.e., .O2-. .OH.H2O2) among other mediators, may be involved in altering the blood-brain barrier (BBB), little is known about the endogenous ability of cerebromicrovascular endothelium to generate ROS. This study examines the capacity of rat endothelial cells (RBEC) to produce ROS in normoxia and hypoxia/reoxygenation. Cultured RBEC were exposed to an oxygen-depleted atmosphere (containing 95% N2 and 5% CO2) for 4 hr at 37 degrees C and air (10 min) at room temperature to simulate "ischemia/reperfusion". Nitroblue tetrazolium (NBT) reduction [formation of nitroblue formazan (NBF)] served as a marker for the production of ROS. The release of lactate dehydrogenase (LDH) and [3H]arachidonic acid (AA) was used to assess cellular integrity. RBEC exposed to hypoxia/reoxygenation produced up to 59% greater NBF formation than controls without affecting the LDH or AA release. The production of ROS was calcium-dependent and not affected by AA or its metabolites. The findings indicate that the RBEC can produce superoxide dismutase (SOD)-inhibitable ROS which are augmented by hypoxia/reoxygenation. It is suggested that in vivo cerebromicrovascular endothelium may contribute to the formation of ROS and play a role in ischemic brain edema.

The effect of endothelins on ion transport systems in cultured rat brain capillary endothelial cells.

Brain capillary endothelial cells regulate the movement of ions and water across the blood-brain barrier via specific ion transport systems. Disturbances in these ion transport systems are involved in the formation of ischemic brain edema. This study describes the effects of endothelins (i.e., ET-1 and ET-3) on ion transport systems in cultured rat brain capillary endothelial cells using 86Rb+ and 22Na+ as markers for K+ and Na+, respectively. ET-1 stimulated K+ uptake and efflux with EC50 values of 0.6 nM and 0.5 nM, respectively. The potencies of ET-3 on these responses were considerably lower. Both ET-1 and ET-3 stimulated Na+ uptake through a Na+/H+ exchange system with similar potencies (i.e., EC50 = 0.80 nM and 1.89 nM, respectively). ET-stimulated K+ uptake, K+ efflux, and Na+ uptake activities were all inhibited by BQ123 (selective ETA receptor antagonist). ET-1 stimulated K+ uptake and efflux, in contrast to Na+ uptake, were also reduced by protein kinase C inhibitors and by an intracellular Ca2+ chelator. The results suggest that ETs can affect the activities of ion and water transport at the blood-brain barrier through different signal transduction mechanisms.

The effect of nitric oxide inhibition on ischemic brain edema.

The involvement of nitric oxide (NO) in the development of ischemic cytotoxic edema was investigated by inhibiting nitric oxide synthase (NOS) activity with N omega-nitro-L-arginine (NLA). Bilateral carotid artery occlusion (15 min) alone or with release (15 and 60 min) served as a model for edema induction. NLA, N omega-nitro-D-arginine methyl ester (D-NAME) or Ringer's solution were administered 4 hr prior to ischemia or sham operation. Treatment with a stable nitroxide radical, 4-hydroxy-2,2, 6,6-tetramethylpiperidine-L-oxyl (TPL), was used to assess free radical involvement in edema. Accumulation of tissue water was evaluated by measuring specific gravity (SG) of brain cortex and histological examination. There was a greater reduction of cortical SG in early reperfusion (15 min) and a lesser decrease in SG (60 min later) in NLA-than in D-NAME- or Ringer's-treated gerbils. The NLA effect was confirmed by histological examination of the brain tissue. TPL treatment (pre- and postischemic) ameliorated the formation of edema to the same degree as NLA. The findings indicate a biphasic NLA modulation of cytotoxic edema most likely mediated through absence or presence of NO-derived free radicals.