Glioblastoma-secreted factors induce IGFBP7 and angiogenesis by modulating Smad-2-dependent TGF-beta signaling.

Insulin-like growth factor-binding protein 7 (IGFBP7) is a selective biomarker of glioblastoma (GBM) vessels, strongly expressed in tumor endothelial cells and vascular basement membrane. IGFBP7 gene regulation and its potential role in tumor angiogenesis remain unclear. Mechanisms of IGFBP7 induction and its angiogenic capacity were examined in human brain endothelial cells (HBECs) exposed to tumor-like conditions. HBEC treated with GBM cell (U87MG)-conditioned media (-CM) exhibited fourfold upregulation of IGFBP7 mRNA and protein compared to control cells. IGFBP7 gene regulation in HBEC was methylation independent. U87MG-CM analysed by enzyme-linked immunosorbent assay contained approximately 5 pM transforming growth factor (TGF)-beta1, a concentration sufficient to stimulate IGFBP7 in HBEC to similar levels as U87MG-CM. Both pan-TGF-beta-neutralizing antibody (1D11) and the TGF-beta1 receptor (activin receptor-like kinase 5, ALK5) antagonist, SB431542, blocked U87MG-CM-induced IGFBP7 expression in HBEC, indicating that TGF-beta1 is an important tumor-secreted effector capable of IGFBP7 induction in endothelial cells. HBEC exposed to either U87MG-CM or IGFBP7 protein exhibited increased capillary-like tube (CLT) formation in Matrigel. Both TGF-beta1- and U87MG-CM-induced Smad-2 phosphorylation and U87MG-CM-induced CLT formation in HBEC were inhibited by the ALK5 antagonist, SB431542. These data suggest that proangiogenic IGFBP7 may be induced in brain endothelial cells by TGF-betas secreted by GBM, most likely through TGF-beta1/ALK5/Smad-2 pathway.

Indomethacin and cyclosporin a inhibit in vitro ischemia-induced expression of ICAM-1 and chemokines in human brain endothelial cells.

Brain inflammation has been implicated in the development of brain edema and secondary brain damage in ischemia and trauma. Mechanisms involved in leukocyte infiltration across the blood-brain barrier are still unknown. In this study, we show that human cere-bromicrovascular endothelial cells (HCEC) subjected to a 4 h in vitro ischemia (hypoxia + glucose deprivation) followed by a 4-24 h recovery express elevated levels of ICAM-1, IL-8, and MCP-1 mRNAs (semi-quantitative RT-PCR) and secrete increased amounts of the immunoreactive chemokines IL-8 and MCP-1 (ELISA). The ischemia-induced expression of ICAM-1 in HCEC, and the expression/release of IL-8 and MCP-1 in HCEC were abolished by the non-steroid anti-inflammatory drug, indomethacin (100-300 microM). The immunosuppressant cyclosporin A (50 microM) partially reduced the ischemia-stimulated IL-8 and MCP-1 secretion by HCEC. Both indomethacin and cyclosporin A also inhibited the ischemia-induced neutrophil chemotaxis elicited by HCEC media. The study indicates that in vitro ischemia augments the expression of adhesion molecules and leukocyte chemoattractants at the site of the BBB. This ischemic pro-inflammatory activation of HCEC may constitute a key event in initiating post-ischemic inflammation, and it can be suppressed by the anti-inflammatory drugs, indomethacin and cyclosporin A.

Functional calcitonin gene-related peptide type 1 and adrenomedullin receptors in human trigeminal ganglia, brain vessels, and cerebromicrovascular or astroglial cells in culture.

Calcitonin gene-related peptide (CGRP) and adrenomedullin (ADM) are potent dilators of human brain arteries, and they have been implicated in the neurogenic inflammation underlying migraine headache and in the evolution of stroke, respectively. However, little is known about the presynaptic and postsynaptic distribution of their respective receptors in the human cerebrovascular bed and trigeminovascular system. In the current study, the expression of mRNA for ADM and the two cloned human CGRP1 receptors (identified here as A-CGRP1 receptors [Aiyar et al., 1996] and K-CGRP1 receptors) [Kapas and Clark, 1995] were evaluated in human brain vessels and trigeminal ganglia. Further, the ability of CGRP and ADM to activate adenylate cyclase in cerebromicrovascular and astroglial cell cultures was determined, and the receptors involved were characterized pharmacologically. Isolated human pial vessels, intracortical microvessels, and capillaries, as well as cultures of brain endothelial (EC), smooth muscle (SMC), and astroglial (AST) cells, all expressed mRNA for the two cloned CGRP1 receptors; however, message for the K-CGRP1 receptor was barely detectable in microvascular tissues and cells. In contrast, only isolated capillaries and cultured AST exhibited message for the ADM receptor. In human trigeminal ganglia, mRNA for ADM and the two CGRP1 receptors was systematically present. The CGRP dose-dependently increased (up to 50-fold) cAMP formation in cell cultures, an effect significantly blocked by 0.1 to 10 micromol/L of the CGRP1 receptor antagonist CGRP8-37. The ADM receptor agonist, ADM13-52 (1 micromol/L), similarly increased cAMP production in all cell types, and this response was virtually abolished by 1 micromol/L CGRP8-37. Low concentrations (1 to 10 micromol/L) of the ADM receptor antagonist ADM22-52 blocked the ADM13-52-induced cAMP formation in AST (26% at 10 micromol/L, P < 0.05), whereas they potentiated this response in brain EC and SMC (40% and 100%, P < 0.001, respectively). Even at a higher dose (50 micromol/L), ADM22-52 inhibited the ADM13-52 effect in vascular cells (45%) much less effectively than in AST (95%). These results indicate that both CGRP and ADM can affect human brain vessels through a CGRP1 receptor, and they further suggest the presence of functional ADM receptors in human astroglial cells.

Multiple microvascular and astroglial 5-hydroxytryptamine receptor subtypes in human brain: molecular and pharmacologic characterization.

Physiologic and anatomic evidence suggest that 5-hydroxytryptamine (5-HT) neurons regulate local cerebral blood flow and blood-brain barrier permeability. To evaluate the possibility that some of these effects occur directly on the blood vessels, molecular and/or pharmacologic approaches were used to assess the presence of 5-HT receptors in human brain microvascular fractions, endothelial and smooth muscle cell cultures, as well as in astroglial cells which intimately associate with intraparenchymal blood vessels. Isolated microvessels and capillaries consistently expressed messages for the h5-HT1B, h5-HT1D, 5-HT1F, 5-HT2A but not 5-HT7 receptors. When their distribution within the vessel wall was studied in more detail, it was found that capillary endothelial cells exhibited mRNA for the h5-HT1D and for the 5-HT7 receptors whereas microvascular smooth muscle cells, in addition to h5-HT1D and 5-HT7, also showed polymerase chain reaction products for h5-HT1B receptors. Expression of 5-HT1F and 5-HT2A receptor mRNAs was never detected in any of the microvascular cell cultures. In contrast, messages for all 5-HT receptors tested were detected in human brain astrocytes with a predominance of the 5-HT2A and 5-HT7 subtypes. In all cultures, sumatriptan inhibited (35-58%, P < .05) the forskolin-stimulated production of cyclic AMP, an effect blocked by the 5-HT1B/1D receptor antagonists GR127935 and GR55562. In contrast, 5-carboxamidotryptamine induced strong increases (> or = 400%, P < .005) in basal cyclic AMP levels that were abolished by mesulergine, a nonselective 5-HT7 receptor antagonist. Only astroglial cells showed a ketanserin-sensitive increase (177%, P < .05) in IP3 formation when exposed to 5-HT. These results show that specific populations of functional 5-HT receptors are differentially distributed within the various cellular compartments of the human cortical microvascular bed, and that human brain astroglial cells are endowed with multiple 5-HT receptors. These findings emphasize the complex interactions between brain serotonergic pathways and non-neuronal cells within the CNS and, further, they raise the possibility that some of these receptors may be activated by antimigraine compounds such as brain penetrant triptan derivatives.

Developmental regulation of glutamate transporters and glutamine synthetase activity in astrocyte cultures differentiated in vitro.

Glutamate plays an important role in brain development, physiological function, and neurodegeneration. Astrocytes control synaptic concentration of glutamate via the high affinity glutamate transporters, GLT-1 and GLAST, and the glutamate catabolizing enzyme, glutamine synthetase. In this study we show that astrocytes cultured from rat brain in various stages of development including embryonic (E18), postnatal (P1-P21) and mature (P50), show distinct patterns of GLT-1 and GLAST expression, glutamine synthetase activity, and phenotypic changes induced by dibutyryl-cyclic adenosine monophosphate. The transcripts for GLT-1 message were detectable in embryonic astrocytes only, whereas the GLAST message was highly expressed in E18 and P1-P4 astrocyte cultures, declined in P10-P21, and was undetectable in P50 astrocytes. Uptake of 3H-glutamate correlated well with GLAST expression in astrocyte cultures of all developmental stages. Glutamine synthetase activity significantly declined from high embryonic levels in P4 astrocytes and remained low throughout postnatal maturation. Exposure of astrocyte cultures to the differentiating agent, db-cAMP (250-500 microM; 6 days), resulted in a pronounced stellation, up-regulation of GLT-1 and GLAST in E18, and GLAST in P4 cultures, while it was ineffective in P10 astrocytes. By contrast, db-cAMP induced a more pronounced stimulation of glutamine synthetase activity (up to 10-fold above basal) in P10 than in E18 cultures (up to 2 times above basal). The differences in expression/inducibility of glutamate transporters and glutamine synthetase observed in astrocyte cultures derived from various stages of fetal and postnatal development suggest that astrocytes in vivo might also respond differently to environmental or injurious stimuli during development and maturation.

[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.

Functional acetylcholine muscarinic receptor subtypes in human brain microcirculation: identification and cellular localization.

Acetylcholine is an important regulator of local cerebral blood flow. There is, however, limited information available on the possible sites of action of this neurotransmitter on brain intraparenchymal microvessels. In this study, a combination of molecular and functional approaches was used to identify which of the five muscarinic acetylcholine receptors (mAChR) are present in human brain microvessels and their intimately associated astroglial cells. Microvessel and capillary fractions isolated from human cerebral cortex were found by reverse transcriptase-polymerase chain reaction to express m2, m3, and, occasionally, m1 and m5 receptor subtypes. To localize these receptors to a specific cellular compartment of the vessel wall, cultures of human brain microvascular endothelial and smooth muscle cells were used, together with cultured human brain astrocytes. Endothelial cells invariably expressed m2 and m5 receptors, and occasionally the m1 receptor; smooth muscle cells exhibited messages for all except the m4 mAChR subtypes, whereas messages for all five muscarinic receptors were identified in astrocytes. In all three cell types studied, acetylcholine induced a pirenzepine-sensitive increase (62% to 176%, P<0.05 to 0.01) in inositol trisphosphate, suggesting functional coupling of m1, m3, or m5 mAChR to a phospholipase C signaling cascade. Similarly, coupling of m2 or m4 mAChR to adenylate cyclase inhibition in endothelial cells and astrocytes, but not in smooth muscle cells, was demonstrated by the ability of carbachol to significantly reduce (44% to 50%, P<0.05 to 0.01) the forskolin-stimulated increase in cAMP levels. This effect was reversed by the mAChR antagonist AFDX 384. The results indicate that microvessels are able to respond to neurally released acetylcholine and that mAChR, distributed in different vascular and astroglial compartments, could regulate cortical perfusion and, possibly, blood-brain barrier permeability, functions that could become jeopardized in neurodegenerative disorders such as Alzheimer's disease.

Evidence that functional glutamate receptors are not expressed on rat or human cerebromicrovascular endothelial cells.

Excitatory amino acids can modify the tone of cerebral vessels and permeability of the blood-brain barrier (BBB) by acting directly on endothelial cells of cerebral vessels or indirectly by activating receptors expressed on other brain cells. In this study we examined whether rat or human cerebromicrovascular endothelial cells (CEC) express ionotropic and metabotropic glutamate receptors. Glutamate and the glutamate receptor agonists N-methyl-d-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA), and kainate failed to increase [Ca2+]i in either rat or human microvascular and capillary CEC but elicited robust responses in primary rat cortical neurons, as measured by fura-2 fluorescence. The absence of NMDA and AMPA receptors in rat and human CEC was further confirmed by the lack of immunocytochemical staining of cells by antibodies specific for the AMPA receptor subunits GluR1, GluR2/3, and GluR4 and the NMDA receptor subunits NR1, NR2A, and NR2B. We failed to detect mRNA expression of the AMPA receptor subunits GluR1 to GluR4 or the NMDA receptor subunits NR1(1XX); NR1(0XX), and NR2A to NR2C in both freshly isolated rat and human microvessels and cultured CEC using reverse transcriptase polymerase chain reaction (RT-PCR). Cultured rat CEC expressed mRNA for KA1 or KA2 and GluR5 subunits. Primary rat cortical neurons were found to express GluR1 to GluR3 and NR1, NR2A, and NR2B by both immunocytochemistry and RT-PCR and KA1, KA2, GluR5, GluR6, and GluR7 by RT-PCR. Moreover, the metabotropic glutamate receptor agonist 1-amino-cyclopentyl-1S, 3R-dicorboxylate (1S,3R-trans-ACPD), while eliciting both inositol trisphosphate and [Ca2+]i increases and inhibiting forskolin-stimulated cyclic AMP in cortical neurons, was unable to induce either of these responses in rat or human CEC. These results strongly suggest that both rat and human CEC do not express functional glutamate receptors. Therefore, excitatory amino acid-induced changes in the cerebral microvascular tone and BBB permeability must be affected indirectly, most likely by mediators released from the adjacent glutamate-responsive cells.

Development of immortalized human cerebromicrovascular endothelial cell line as an in vitro model of the human blood-brain barrier.

The objective of this study was to generate an immortal cell line representative of specialized human brain microvascular endothelia forming the blood-brain barrier (BBB) in vivo. Human capillary and microvascular endothelial cells (HCEC) were transfected with the plasmid pSV3-neo coding for the SV40 large T antigen and the neomycin gene. The neomycin-resistant transfected cells overcame proliferative senescence, and after a 6-8 wk period of crisis produced immortalization-competent cell colonies. Single-cell clones of near-diploid genotype were isolated from these colonies, propagated, and characterized. Immortalized HCEC (SV-HCEC) exhibited accelerated proliferation rates, but remained serum and anchorage dependent and retained the characteristic cobblestone morphology at confluence. SV-HCEC displayed a stable nuclear expression of SV40 large T antigen, lacked the invasiveness of transformed cells, and maintained major phenotypic properties of early passage control cells including expression of factor VIII-related antigen, uptake of acetylated low-density lipoprotein, binding of fluorescently labeled lectins, expression of transferrin receptor and transferrin receptor-mediated endocytosis, and high activities of the BBB-specific enzymes alkaline phosphatase and gamma-glutamyl transpeptidase. The diffusion of radiolabeled sucrose across SV-HCEC monolayers was fivefold lower than that observed with human lung microvascular endothelial cells. Furthermore, media conditioned by fetal human astrocytes increased the transendothelial electrical resistance of SV-HCEC monolayers by 2.5-fold. Therefore, this newly established human cell line expressing the specialized phenotype of BBB endothelium may serve as a readily available in vitro model for studying the properties of the human BBB.

AMPA receptor-mediated regulation of a Gi-protein in cortical neurons.

Excitatory synaptic transmission in the central nervous system is mediated primarily by the release of glutamate from presynaptic terminals onto postsynaptic channels gated by N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors. The myriad intracellular responses arising from the activation of the NMDA and AMPA receptors have previously been attributed to the flow of Ca2+ and/or Na+ through these ion channels. Here we report that the binding of the agonist AMPA to its receptor can generate intracellular signals that are independent of Ca2+ and Na+ in rat cortical neurons. In the absence of intracellular Ca2+ and Na+, AMPA, but not NMDA, brought about changes in a guanine-nucleotide-binding protein (Galpha[il]) that inhibited pertussis toxin-mediated ADP-ribosylation of the protein in an in vitro assay. This effect was observed in intact neurons treated with AMPA as well as in isolated membranes exposed to AMPA, and was also found in MIN6 cells, which express functional AMPA receptors but have no metabotropic glutamate receptors. AMPA also inhibited forskolin-stimulated activity of adenylate cyclase in neurons, demonstrating that Gi proteins were activated. Moreover, both Gbetagamma blockage and co-precipitation experiments demonstrated that the modulation of the Gi protein arose from the association of Galpha(il) with the glutamate receptor-1 (GluR1) subunit. These results suggest that, as well as acting as an ion channel, the AMPA receptor can exhibit metabotropic activity.

Stimulation of glutamate uptake and Na,K-ATPase activity in rat astrocytes exposed to ischemia-like insults.

The postsynaptic actions of glutamate are rapidly terminated by high affinity glutamate uptake into glial cells. In this study we demonstrate the stimulation of both glutamate uptake and Na,K-ATPase activity in rat astrocyte cultures in response to sublethal ischemia-like insults. Primary cultures of neonatal rat cortical astrocytes were subjected to hypoxia, or to serum- and glucose-free medium, or to both conditions (ischemia). Cell death was assessed by propidium iodide staining of cell nuclei. To measure sodium pump activity and glutamate uptake, 3H-glutamate and 86Rb were both simultaneously added to the cell culture in the presence or absence of 2 mM ouabain. Na,K-ATPase activity was defined as ouabain-sensitive 86Rb uptake. Concomitant transient increases (2-3 times above control levels) of both Na,K-ATPase and glutamate transporter activities were observed in astrocytes after 4-24 h of hypoxia, 4 h of glucose deprivation, and 2-4 h of ischemia. A 24 h ischemia caused a profound loss of both activities in parallel with significant cell death. The addition of 5 mM glucose to the cells after 4 h ischemia prevented the loss of both sodium pump activity and glutamate uptake and rescued astrocytes from death observed at the end of 24 h ischemia. Reoxygenation after the 4 h ischemic event caused the selective inhibition of Na,K-ATPase activity. The observed increases in Na,K-ATPase activity and glutamate uptake in cultured astrocytes subjected to sublethal ischemia-like insults may model an important functional response of astrocytes in vivo by which they attempt to maintain ion and glutamate homeostasis under restricted energy and oxygen supply.

Glutamate uptake and Na,K-ATPase activity in rat astrocyte cultures exposed to ischemia.

In this study we demonstrate the stimulation of both glutamate uptake and Na,K-ATPase activity in rat astrocyte cultures in response to a sublethal ischemic insult in vitro. To measure sodium pump activity and glutamate uptake, 3H-glutamate and 86Rb were simultaneously added to the cultures in the presence or absence of 2 mM ouabain. Na,K-ATPase activity was defined as ouabain-sensitive 86Rb uptake. Cell death was assessed by exclusion of the vital dye, calcein-AM from cells. Concomitant transient increases (2-3 fold above control levels) in both Na,K-ATPase and glutamate transporter activities were observed in astrocytes after 2-4 hours of ischemia. By contrast, 24 hours of ischemia caused a profound loss of both activities which paralleled significant cell death. The addition of 5 mM glucose to the cells after 4 hours of ischemia prevented the loss of sodium pump activity and glutamate uptake, and rescued astrocytes from the lethal effects of 24 hours of ischemia.

Increase in surface expression of ICAM-1, VCAM-1 and E-selectin in human cerebromicrovascular endothelial cells subjected to ischemia-like insults.

Secondary ischemic brain injury has been shown to develop as a consequence of inflammation and vasogenic brain edema. In this study we show that inflammatory cytokines and simulated in vitro ischemia stimulate the surface expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and endothelial-leukocyte adhesion molecule-1 (E-selectin) in human cerebromicrovascular endothelial cells (HCEC) in culture. The levels of all three adhesion molecules were dramatically (3 to 10-fold) up-regulated by 4-24 hour exposure to the inflammatory cytokines. IL-1 beta (10-200 u/ml) or TNF alpha (50 200 u/ml), and by a 4 hour exposure to "simulated" in vitro ischemia, as determined by immunocytochemistry and ELISA. Following 24 hours of subsequent reperfusion, the expression of ICAM-1 and VCAM-1 was maintained at ischemia-induced levels, whereas E-selectin was no longer detectable. Both the cytokine- and ischemia-induced up-regulation of adhesion molecules were completely abolished by the transcriptional inhibitor, actinomycin D (10 micrograms/ml), and inhibited by the cycloxygenase (COX) inhibitor, indomethacin (300 microM). These findings implicate HCEC in the processes of leukocyte adhesion and recruitment in the brain during stroke in vivo.

Free radical-induced endothelial membrane dysfunction at the site of blood-brain barrier: relationship between lipid peroxidation, Na,K-ATPase activity, and 51Cr release.

Na,K-ATPase activity, membrane lipid peroxidation (TBARM), and membrane 'leakiness' for small molecules were examined in rat cerebromicrovascular endothelial cells (RCEC) following exposure to hydrogen peroxide and xanthine/xanthine oxidase. Whereas short-term (15-30 min) exposure to either oxidant decreased ouabain-sensitive 86Rb uptake and increased TBARM in a concentration-dependent fashion, significant release of 51Cr (30-40%) from cells was observed only after one hour exposure to the oxidants. By comparison, much longer exposure times (i.e., 4 hours) were needed to induce significant lactate dehydrogenase release from oxidant-treated cells. The oxidant-evoked decrease in Na,K-ATPase activity and increases in TBARM and RCEC 'permeability' were abolished in the presence of the steroid antioxidants U-74500A and U-74389G (5-20 microM). Reduced glutathione (4 mM) partially attenuated oxidant-induced changes, whereas ascorbic acid (2 mM) and the disulfide bond-protecting agent, dithiothreitol (1 mM), were ineffective. These results suggest that the oxidant-induced loss of Na,K-ATPase activity in RCEC results primarily from changes in membrane lipids, and implicate both the inhibition of Na,K-ATPase and membrane lipid peroxidation in the mechanism responsible for the delayed free radical-induced increase in RCEC membrane 'permeability'.

The role of intracellular calcium and protein kinase C in endothelin-stimulated proliferation of rat type I astrocytes.

The increased expression of immunoreactive endothelin-1 (ET-1) in reactive astrocytes and its mitogenic effects on astrocytes and glioma cell lines, have implicated endothelins in the development of reactive gliosis. In this study, an increase in DNA synthesis in rat type I astrocytes was observed after cultures were transiently exposed to ET-1 for 15 min, suggesting that early signal transduction events are essential and sufficient for the propagation of the ET-1-induced mitogenic signal. Prompt increases in inositol triphosphate (IP3) formation and [Ca2+]i were observed upon the addition of ET-1 to these cells. The ET-1-evoked increase in [Ca2+]i consisted of an initial peak which was preserved in Ca(2+)-free medium, and a sustained phase which was abolished in Ca(2+)-free medium and partly attenuated by nifedipine. ET-1 also increased the activity of membrane-associated protein kinase C (PKC) and induced the in vivo phosphorylation of the 85 kD MARCKS protein, an endogenous PKC-specific substrate. The ET-1-evoked increases in DNA synthesis, IP3, [Ca2+]i, membrane PKC, and 85 kD MARCKS protein phosphorylation in rat cortical astrocytes were prevented by either the selective endothelin ETA receptor antagonist, BQ-123, or the phospholipase C (PLC)-specific inhibitor, U-73122. However, the inhibition of PKC activity did not affect ET-1-induced DNA synthesis in rat cortical astrocytes. These results suggest that ET-1-induced IP3 and/or [CA2+]i responses, but not the activation of PKC, are essential for the growth-factor like actions of ET-1 in rat cortical astrocytes.

Evidence for the role of protein kinase C in astrocyte-induced proliferation of rat cerebromicrovascular endothelial cells.

The proliferation of cerebral endothelial cells is a crucial step in neural angiogenesis and is a process responsive to changes in the surrounding environment. Serum-free medium conditioned by rat cortical astrocytes was found to accelerate DNA synthesis, induce transient activation of protein kinase C (PKC), and increase the endogenous phosphorylation of the PKC-specific substrate, the 85 kDa MARCKS protein, in rat cerebromicrovascular endothelial cells (RCEC). The stimulatory factor(s) in astrocyte conditioned media (ACM) were heat- and trypsin-sensitive and found to have an apparent molecular weight greater than 10 kDa. The potent PKC activator, 12-O-tetradecanoyl phorbol 13-acetate (TPA), also stimulated RCEC proliferation, whereas the inhibition of PKC by staurosporine caused a concomitant loss in ACM-induced PKC translocation, MARCKS protein phosphorylation and DNA synthesis. These findings implicate PKC activation as a critical early event in cerebral endothelial cell proliferation triggered by astrocyte-derived mitogen(s).

Signal transduction and Ca2+ uptake activated by endothelins in rat brain endothelial cells.

The activation of signal transduction pathways by endothelin-1 or endothelin-3 were investigated in rat cerebromicrovascular endothelial cells. Endothelin-1 induced a rapid increase in inositol triphosphate (IP3) formation in these cells, whereas endothelin-3 was only moderately effective at high concentrations. Both endothelins also increased uptake of 45Ca2+ in these cells. Endothelin-1-induced IP3 formation or 45Ca2+ uptake were inhibited by endothelin ETA receptor antagonist BQ-123. Ryanodine, an inhibitor of intracellular Ca2+ mobilization, selectively endothelin-1-induced 45Ca2+ uptake, whereas nickel or suramin inhibited endothelin-3-induced 45Ca2+ uptake. The results indicate that endothelin-1 elevates 45Ca2+ uptake in rat brain endothelial cells by mechanisms coupled to the mobilization of intracellular Ca2+ stores. Both endothelin-1- and endothelin-3-induced 45Ca2+ uptake were inhibited by receptor operated Ca2+ channel blocker SK&F 96365, whereas they were insensitive to dihydropyridine derivatives nifedipine and nitrendipine. The release of arachidonic acid from rat brain endothelial cells observed in response to endothelin-1 was inhibited by ryanodine or SK&F 96365, implicating participation of both intra- and extra- cellular components of Ca2+ signaling in activating endothelial secretion of vasoactive substances.

Liposome-entrapped superoxide dismutase reduces ischemia/reperfusion 'oxidative stress' in gerbil brain.

Bilateral common carotid artery occlusion (15 min.) followed by two hours of recirculation reduced mitochondrial superoxide dismutase (SOD) and glutathione reductase (GR) activities, and increased susceptibility of mitochondrial membranes to in vitro lipid peroxidation in brain regions (i.e., cortex, striatum and hippocampus) of Mongolian gerbil. Intraperitoneal bolus injection (2 mg/kg b.w.) of liposome-entrapped CuZn superoxide dismutase (1-SOD) increased the endogenous SOD activity in normal brain tissue and, when given at the end of ischemia, counteracted both the ischemic reduction of endogenous SOD and the increased peroxidation of mitochondrial membranes. 1-SOD treatment was ineffective in reducing brain swelling, suggesting that superoxide radicals are not a main participant in the process of (post)ischemic brain edema formation.

Vasoactive peptides and prostaglandin D2 in human cerebromicrovascular endothelium.

Prostaglandin D2 (PGD2) is the major prostanoid formed among other prostanoids in cultured microvascular endothelium derived from human brain (HBEC). Angiotensin II, arginine vasopressin and endothelium-1 stimulated the production of PGD2 and PGF2 alpha in a concentration-dependent manner, and this effect was inhibited by their specific receptor antagonists or dexamethasone (inhibitor of phospholipase A2/cyclooxygenase II). Both the peptidergic-induced PGD2 and the exogenously added PGD2 were converted in HBEC to 9 alpha, 11 beta-PGF2, a potent vasoconstrictor. Exogenous PGD2 also dose-dependently enhanced the production of vasoconstrictive PGF2 alpha, thromboxane B2, vasodilatory prostaglandin PGE2, and cAMP in these cells. The PGD2 stimulated formation of the prostanoids was inhibited by acetylsalicylic acid or indomethacin (inhibitors of cyclooxygenase I) but not dexamethasone, demonstrating for the first time that PGD2 may contribute to the production of prostanoids in HBEC. These findings strongly suggest that PGD2 may play a pivotal role in the regulation of cerebromicrovascular function.

Interaction between histamine and adenosine in human cerebromicrovascular endothelial cells: modulation of second messengers.

This study demonstrates the presence of histamine H1 and H2 receptors and purinoreceptors A1 and A2 on endothelial cells derived from human brain microvessels (HBEC). Histamine induced formation of both inositol triphosphate (IP3) (EC50 = 10.2 +/- 0.9 microM) and cyclic adenosine monophosphate (cAMP) (EC50 = 5.2 +/- 0.9 microM) in HBEC in a concentration-dependent fashion. IP3 formation was inhibited by H1 receptor antagonists mepyramine maleate and chlorphenyramine, but not by H2 receptor antagonist cimetidine. Production of cAMP was efficiently inhibited by cimetidine. Selective A1 receptor agonists decreased, whereas A2 receptor agonists increased cAMP production in HBEC. When added together with histamine to HBEC cultures, both A1 and A2 receptor agonists diminished histamine-induced IP3 stimulation. This effect was reversed in the presence of specific A1 and A2 receptor antagonists, respectively. Marked augmentation of histamine-induced cAMP production by HBEC was observed in the presence of A2 agonist. This response was dependent on H1 receptors, since it was reduced in the presence of H1-receptor antagonist. It is suggested that interaction between histamine and adenosine modulating induction of second messengers in HBEC may influence endothelium-dependent responses of brain microvascular compartments.