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Although the gasotransmitter hydrogen sulfide (H 2 S) is well known for its vasodilatory effects, H 2 S also exhibits vasoconstricting properties. Herein, it is demonstrated that administration of H 2 S as intravenous sodium sulfide (Na 2 S) increased blood pressure in sheep and rats, and this effect persisted after H 2 S has disappeared from the blood. Inhibition of the L-type calcium channel (LTCC) diminished the hypertensive effects. Incubation of Na 2 S with whole blood, red blood cells, methemoglobin, or oxyhemoglobin produced a hypertensive product of H 2 S, which is not hydrogen thioperoxide, metHb-SH - complexes, per-/poly- sulfides, or thiolsulfate, but rather a labile intermediate. One-electron oxidation of H 2 S by oxyhemoglobin generated its redox cousin, sulfhydryl radical (HS • ). Consistent with the role of HS • as the hypertensive intermediate, scavenging HS • inhibited Na 2 S-induced vasoconstriction and activation of LTCCs. In conclusion, H 2 S causes vasoconstriction that is dependent on the activation of LTCCs and generation of HS • by oxyhemoglobin., (© 2024 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

Background: The administration of epinephrine after severe refractory hypotension, shock, or cardiac arrest restores systemic blood flow and major vessel perfusion but may worsen cerebral microvascular perfusion and oxygen delivery through vasoconstriction. The authors hypothesized that epinephrine induces significant microvascular constriction in the brain, with increased severity after repetitive dosing and in the aged brain, eventually leading to tissue hypoxia., Methods: The authors investigated the effects of intravenous epinephrine administration in healthy young and aged C57Bl/6 mice on cerebral microvascular blood flow and oxygen delivery using multimodal in vivo imaging, including functional photoacoustic microscopy, brain tissue oxygen sensing, and follow-up histologic assessment., Results: The authors report three main findings. First, after epinephrine administration, microvessels exhibited severe immediate vasoconstriction (57 ± 6% of baseline at 6 min, P < 0.0001, n = 6) that outlasted the concurrent increase in arterial blood pressure, while larger vessels demonstrated an initial increase in flow (108 ± 6% of baseline at 6 min, P = 0.02, n = 6). Second, oxyhemoglobin decreased significantly within cerebral vessels with a more pronounced effect in smaller vessels (microvessels to 69 ± 8% of baseline at 6 min, P < 0.0001, n = 6). Third, oxyhemoglobin desaturation did not indicate brain hypoxia; on the contrary, brain tissue oxygen increased after epinephrine application (from 31 ± 11 mmHg at baseline to 56 ± 12 mmHg, 80% increase, P = 0.01, n = 12). In the aged brains, microvascular constriction was less prominent yet slower to recover compared to young brains, but tissue oxygenation was increased, confirming relative hyperoxia., Conclusions: Intravenous application of epinephrine induced marked cerebral microvascular constriction, intravascular hemoglobin desaturation, and paradoxically, an increase in brain tissue oxygen levels, likely due to reduced transit time heterogeneity., (Copyright © 2023 American Society of Anesthesiologists. All Rights Reserved.)

Increased focus has been placed on the role of histone deacetylase inhibitors as crucial players in subarachnoid hemorrhage (SAH) progression. Therefore, this study was designed to expand the understanding of SAH by exploring the downstream mechanism of the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) in SAH. The expression of TDP-43 in patients with SAH and rat models of SAH was measured. Then, western blot analysis, immunofluorescence staining, and transmission electron microscope were used to investigate the in vitro effect of TDP-43 on a neuronal cell model of SAH established by oxyhemoglobin treatment. Immunofluorescence staining and coimmunoprecipitation assays were conducted to explore the relationship among histone deacetylase 1 (HDAC1), heat shock protein 70 (HSP70), and TDP-43. Furthermore, the in vivo effect of HDAC1 on SAH was investigated in rat models of SAH established by endovascular perforation. High expression of TDP-43 in the cerebrospinal fluid of patients with SAH and brain tissues of rat models of SAH was observed, and TDP-43 accumulation in the cytoplasm and the formation of inclusion bodies were responsible for axonal damage, abnormal nuclear membrane morphology, and apoptosis in neurons. TDP-43 degradation was promoted by the HDAC1 inhibitor SAHA via the acetylation of HSP70, alleviating SAH, and this effect was verified in vivo in rat models. In conclusion, SAHA relieved axonal damage and neurological dysfunction after SAH via the HSP70 acetylation-induced degradation of TDP-43, highlighting a novel therapeutic target for SAH., (© 2022. The Author(s).)

Subarachnoid hemorrhage (SAH), as one of the most severe hemorrhagic strokes, is closely related to neuronal damage. Neurogenesis is a promising therapy, however, reliable targets are currently lacking. Increasing evidence has indicated that CD24 is associated with the growth of hippocampal neurons and the regulation of neural stem/precursor cell proliferation. To investigate the potential effect of CD24 in astrocytes on neuron growth in the hippocampus, we used a Transwell co-culture system of hippocampal astrocytes and neurons, and oxyhemoglobin (OxyHb) was added to the culture medium to mimic SAH in vitro. A specific lentivirus was used to knock down CD24 expression in astrocytes, which was verified by western blot, quantitative real-time polymerase chain reaction, and immunofluorescent staining. Astrocyte activation, neurite elongation, neuronal apoptosis, and cell viability were also assessed. We first determined the augmented expression level of CD24 in hippocampal astrocytes after SAH. A similar result was observed in cultured astrocytes exposed to OxyHb, and a corresponding change in SHP2/ERK was also noticed. CD24 in astrocytes was then downregulated by the lentivirus, which led to the impairment of axons and dendrites on the co-cultured neurons. Aggravated neuronal apoptosis was induced by the CD24 downregulation in astrocytes, which might be a result of a lower level of brain derived neurotrophic factor (BDNF). In conclusion, the knock-down of CD24 in astrocytes suppressed hippocampal neuron growth, in which the SHP2-ERK signaling pathway and BNDF were possibly involved., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Early brain injury (EBI) is a major cause of adverse outcomes following subarachnoid hemorrhage (SAH). There is evidence that mesenchymal stem cells (MSCs) - derived exosomes are involved in the repair of SAH. Exosomes were extracted from human umbilical cord mesenchymal stem cells (hubMSCs) and identified. OxyHb treated PC12 cells were transfected with exosomes alone or together with miR-26b-5p inhibitor. Hub-MSCs derived exosomes promote cell proliferation, inhibit apoptosis and reduce inflammatory mediator expression. Transfection of miR-26b-5p inhibitor abolished the promoting effect of exosomes on the proliferation of PC12 cells, as well as the inhibitory effect on cell apoptosis. In addition, methionine adenosyltransferase II alpha (MAT2A) was one target gene of miR-26b-5p. OxyHb treated PC12 cells were transfected with exosomes alone or together with pcDNA-MAT2A and observed that the promoting effect of exosomes on PC12 cell proliferation was abolished by pcDNA-MAT2A, which was the same as the effect of miR-26b-5p inhibitor. OxyHb treated PC cells incubated with exosomes were transfected with miR-26b-5p inhibitor alone or together with si-MAT2A, respectively, and it was observed that exosomes decreased the phosphorylation levels of p38 MAPK and STAT3 proteins, inhibited cell apoptosis and inflammatory mediator expression, and miR-26b-5p inhibitor abrogated the effects of exosomes, while transfection of si-MAT2A reversed the effects of miR-26b-5p inhibitor. Moreover, injection of miR-26b-5p inhibitor resulted in increased MAT2A and pathway protein expression, increased inflammatory mediators, and aggravated neurological symptoms in the brain tissues of SAH rats., (Copyright © 2021 Elsevier Inc. All rights reserved.)

RNA-binding protein fox-1 homolog 1 (Rbfox-1), an RNA-binding protein in neurons, is thought to be associated with many neurological diseases. To date, the mechanism on which Rbfox-1 worsens secondary cell death in ICH remains poorly understood. In this study, we aimed to explore the role of Rbfox-1 in intracerebral hemorrhage (ICH)-induced secondary brain injury (SBI) and to identify its underlying mechanisms. We found that the expression of Rbfox-1 in neurons was significantly increased after ICH, which was accompanied by increases in the binding of Rbfox-1 to Ca 2+ /calmodulin-dependent protein kinase II (CaMKIIα) mRNA and the protein level of CaMKIIα. In addition, when exposed to exogenous upregulation or downregulation of Rbfox-1, the protein level of CaMKIIα showed a concomitant trend in brain tissue, which further suggested that CaMKIIα is a downstream-target protein of Rbfox-1. The upregulation of both proteins caused intracellular-Ca 2+ overload and neuronal degeneration, which exacerbated brain damage. Furthermore, we found that Rbfox-1 promoted the expression of CaMKIIα via blocking the binding of micro-RNA-124 to CaMKIIα mRNA. Thus, Rbfox-1 is expected to be a promising therapeutic target for SBI after ICH.

Background: We have previously verified the beneficial effects of exosomes from endothelial progenitor cells (EPC-EXs) in ischemic stroke. However, the effects of EPC-EXs in hemorrhagic stroke have not been investigated. Additionally, miR-137 is reported to regulate ferroptosis and to be involved in the neuroprotection against ischemic stroke. Hence, the present work explored the effects of miR-137-overexpressing EPC-EXs on apoptosis, mitochondrial dysfunction, and ferroptosis in oxyhemoglobin (oxyHb)-injured SH-SY5Y cells., Methods: The lentiviral miR-137 was transfected into EPCs and then the EPC-EXs were collected. RT-PCR was used to detect the miR-137 level in EPCs, EXs, and neurons. The uptake mechanisms of EPC-EXs in SH-SY5Y cells were explored by the co-incubation of Dynasore, Pitstop 2, Ly294002, and Genistein. After the transfection of different types of EPC-EXs, flow cytometry and expression of cytochrome c and cleaved caspase-3 were used to detect the apoptosis of oxyHb-injured neurons. Neuronal mitochondrial function was assessed by reactive oxygen species (ROS) level, mitochondrial membrane potential (MMP) depolarization, and cellular ATP content. Cell ferroptosis was measured by lipid peroxidation, iron overload, degradation of glutathione, and glutathione peroxidase 4. Additionally, recombinational PGE2 was used to detect if activation of COX2/PGE2 pathway could reverse the protection of miR-137 overexpression., Results: The present work showed (1) EPC-EXs could be taken in by SH-SY5Y cells via caveolin-/clathrin-mediated pathways and macropinocytosis; (2) miR-137 was decreased in neurons after oxyHb treatment, and EXs miR-137 could restore the miR-137 levels; (3) EXs miR-137 worked better than EXs in reducing the number of apoptotic neurons and pro-apoptotic protein expression after oxyHb treatment; (4) EXs miR-137 are more effective in improving the cellular MMP, ROS, and ATP level; (5) EXs miR-137 , but not EXs, protected oxyHb-treated SH-SY5Y cells against lipid peroxidation, iron overload, degradation of glutathione, and glutathione peroxidase 4; and (6) EXs miR-137 suppressed the expression of the COX2/PGE2 pathway, and activation of the pathway could partially reverse the neuroprotective effects of EXs miR-137 ., Conclusion: miR-137 overexpression boosts the neuroprotective effects of EPC-EXs against apoptosis and mitochondrial dysfunction in oxyHb-treated SH-SY5Y cells. Furthermore, EXs miR-137 rather than EXs can restore the decrease in miR-137 levels and inhibit ferroptosis, and the protection mechanism might involve the miR-137-COX2/PGE2 signaling pathway.

Recent evidence suggests that CC chemokine ligand 20 (CCL20) is upregulated after subarachnoid hemorrhage (SAH). Here, we investigated the functions of CCL20 in SAH injury and its underlying mechanisms of action. We found that CCL20 is upregulated in an SAH mouse model and in cultured primary microglia and neurons. CCL20-neutralizing antibody alleviated SAH-induced neurological deficits, decreased brain water content and neuronal apoptosis, and repressed microglial activation. We observed increased levels of CCL20, CC chemokine receptor 6 (CCR6), interleukin 1 beta (IL-1β), and tumor necrosis factor alpha (TNF-α), as well as of microglial activation in microglia treated with oxyhemoglobin (OxyHb). CCL20 or CCR6 knockdown reversed the effects of OxyHb on microglia. Conditioned medium from OxyHb-treated microglia induced neuronal apoptosis, while the percentage of apoptotic neurons in the conditioned medium from microglia transfected with CCL20 siRNA or CCR6 siRNA was decreased. We observed no decrease in OxyHb-induced apoptosis in CCL20-knockdown neurons. Conditioned medium from OxyHb-treated neurons led to microglial activation and induced CCR6, IL-1β and TNF-α expression, while CCL20 knockdown in neurons or CCR6 knockdown in microglia reversed those effects. Our results thus suggest CCL20 may be targeted to elicit therapeutic benefits after SAH injury.

Photodynamic therapy (PDT) is strongly O2 dependent. Therefore, its therapeutic effects are seriously hindered in hypoxic tumors. Red blood cells are responsible for delivering O2 in the blood. In this manuscript, biomimetic red blood cells (BRBCs) were exploited using a layer-by-layer assembly method, using Fe3O4@CuO, oxyhemoglobin (OxyHb), a photosensitizer and a photo-cross linked acrylate modified hyaluronic acid (HA) gel shell. The Fe3O4@CuO core has very high OxyHb loading efficiency (the adsorption capacity of Fe3O4@CuO for OxyHb is derived to be 0.99 mg mg-1) to ensure a sufficient O2 supply. OxyHb was protected well by the HA shell in order to avoid O2 release during the delivery process in blood before arrival at the tumor tissue. The HA shell protection can be eliminated in position at the tumor to trigger O2 release through hyaluronidase (HAase) triggered HA degradation. Furthermore, Fe3O4 in the nanosystem can provide magnetic field assisted tumor targeting and magnetic resonance imaging of the tumor. Therefore, this work presents a highly efficient all-in-one biomimetic nanomedicine approach to overcome hypoxia and achieve tumor targeting theranostics.

Background: Gap junctions are involved in the development of cerebral vasospasm (CVS) after subarachnoid hemorrhage (SAH). However, the specific roles and regulatory functions of related connexin isoforms remain unknown. The aim of this study was to investigate the importance of connexin 43 (Cx43) in CVS and determine whether Cx43 alterations are modulated via the protein kinase C (PKC) signaling transduction pathway., Methods: Oxyhemoglobin (OxyHb)-induced smooth muscle cells of basilar arterial and second-injection model in rat were used as CVS models in vitro and in vivo. In addition, dye transfer assays were used for gap junction-mediated intercellular communication (GJIC) observation in vitro and delayed cerebral ischemia (DCI) was observed in vivo by perfusion-weighted imaging (PWI) and intravital fluorescence microscopy., Results: Increase in Cx43 mediated the development of SAH-induced CVS was found in both in vitro and in vivo CVS models. Enhanced GJIC was observed in vitro CVS model, this effect and increased Cx43 were reversed by preincubation with specific PKC inhibitors (chelerythrine or GF 109203X). DCI was observed in vivo on day 7 after SAH. However, DCI was attenuated by pretreatment with Cx43 siRNA or PKC inhibitors, and the increased Cx43 expression in vivo was also reversed by Cx43 siRNA or PKC inhibitors., Conclusions: These data provide strong evidence that Cx43 plays an important role in CVS and indicate that changes in Cx43 expression may be mediated by the PKC pathway. The current findings suggest that Cx43 and the PKC pathway are novel targets for developing treatments for SAH-induced CVS.

Cardiovascular effects were reported to occur in humans and in animal models during transfusion with hemoglobin (Hb)-based oxygen therapeutics. The effects of Hb's iron redox states on cardiac parameters during hypoxia/reoxygenation are however poorly defined. We hypothesize that acute exposures to ferric Hb during hypoxia leads to cardiomyocyte injury and an impaired left ventricular response accompanied by cardiac mitochondrial bioenergetic dysfunction. Recovery of left ventricular functions in an isolated rat heart Langendorff perfusion system was observed following perfusion with ferrous but not with ferric Hb. Ferric Hb induced the development of heart lesions, and impairment of the respiratory chain complex activity. Under normoxia, a sharp decline in cardiac parameters was observed following co-perfusion of low (20 μM) and high (100 μM) ascorbic acid (Asc) with ferrous Hb. This trend continued with ferric Hb co-perfusion, but only at the higher concentration of Asc. These observations suggest that perfusion of the hypoxic heart with ferric Hb increases oxidative stress thereby resulting in cardiac dysfunction. Intervention with Asc to reduce ferric Hb may offer a strategy to control Hb toxicity; however, timing of administration, and dosage of Asc may require individual optimization to target specific redox forms of Hb., (Published by Elsevier Inc.)

Background: Peroxiredoxin (Prx) protein family have been reported as important damage-associated molecular patterns (DAMPs) in ischemic stroke. Since peroxiredoxin 2 (Prx2) is the third most abundant protein in erythrocytes and the second most protein in the cerebrospinal fluid in traumatic brain injury and subarachnoid hemorrhage (SAH) patients, we assessed the role of extracellular Prx2 in the context of SAH., Methods: We introduced a co-culture system of primary neurons and microglia. Prx2 was added to culture medium with oxyhemoglobin (OxyHb) to mimic SAH in vitro. Neuronal cell viability was assessed by lactate dehydrogenase (LDH) assay, and neuronal apoptosis was determined by TUNEL staining. Inflammatory factors in culture medium were measured by ELISA, and their mRNA levels in microglia were determined by qPCR. Toll-like receptor 4 knockout (TLR4-KO) mice were used to provide TLR4-KO microglia; ST-2825 was used to inhibit MyD88, and pyrrolidine dithiocarbamate (PDTC) was used to inhibit NF-κB. Related cellular signals were analyzed by Western blot. Furthermore, we detected the level of Prx2 in aneurysmal SAH patients' cerebrospinal fluids (CSF) and compared its relationship with Hunt-Hess grades., Results: Prx2 interacted with TLR4 on microglia after SAH and then activated microglia through TLR4/MyD88/NF-κB signaling pathway. Pro-inflammatory factors were expressed and released, eventually caused neuronal apoptosis. The levels of Prx2 in SAH patients positively correlated with Hunt-Hess grades., Conclusions: Extracellular Prx2 in CSF after SAH is a DAMP which resulted in microglial activation via TLR4/MyD88/NF-κB pathway and then neuronal apoptosis. Prx2 in patients' CSF may be a potential indicator of brain injury and prognosis.

Inflammatory modulation mediated by microglial M1/M2 polarization is one of the main pathophysiological processes involved in early brain injury (EBI) after subarachnoid haemorrhage (SAH). Previous studies have shown that recombinant human erythropoietin (rhEPO) alleviates EBI following experimental SAH. However, the mechanisms of this beneficial effect are still poorly understood. Recent research has suggested that EPO shows anti-inflammatory properties. Therefore, we tried to analyse whether rhEPO administration influenced microglial M1/M2 polarization in early brain injury after SAH and to identify the underlying molecular mechanism of any such effect. We found that treatment with rhEPO markedly ameliorated SAH-induced EBI, as shown by reductions in brain cell apoptosis, neuronal necrosis, albumin exudation and brain edema. Moreover, the expression levels of p-JAK2 and p-STAT3 were significantly increased in the cortex after SAH induction and were further increased by EPO treatment; in addition, the p-JAK2 inhibitor AZD1480 impaired the protective effect of EPO against SAH-induced EBI in vivo. Furthermore, EPO promoted the polarization of microglia towards the protective M2 phenotype and alleviated inflammation. In cultured microglia under oxyhemoglobin (OxyHb) treatment, EPO up-regulated the expression of the EPO receptor (EPOR), which did not occur in response to OxyHb treatment alone, and EPO magnified OxyHb-induced increases in p-JAK2 and p-STAT3 and modulated OxyHb-challenged microglial polarization towards M2. Interestingly, the effect of EPO on microglia polarization was cancelled by EPOR knockdown or by p-JAK2 or p-STAT3 inhibition, suggesting a core role of the EPOR/JAK2/STAT3 pathway in modulating microglial function and phenotype. In conclusion, the therapeutic effect of rhEPO on the early brain injury after SAH may relate to its modulation of inflammatory response and microglia M1/M2 polarization, which may be mediated in part by the EPOR/JAK2/STAT3 signalling pathway. These results improved the understanding of the anti-inflammatory effect of EPO on microglia polarization, which might optimize the therapeutic modalities of EPO treatment with SAH., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Apoptosis plays a crucial role in the pathogenesis of early brain injury (EBI) following subarachnoid hemorrhage (SAH). However, the exact molecular mechanisms underlying neuronal apoptosis in EBI after SAH have not been fully elucidated. The present study showed that EBI induced significantly neuronal apoptosis activation of Ras/Raf/Erk signals in hippocampus after SAH. Intracisternal administration of PD98059, an inhibitor of Erk1/2, decreased the hippocampal neuronal apoptosis and alleviated the cognitive deficits induced by SAH. Interestingly, an increase in phosphorylation of p53 was paralleled with p-Erk, and PD98059 also blocked the level of p-p53. In primary cultures, oxyhemoglobin (OxyHb) treatment significantly increased p-Erk, p-p53, and apoptosis, which was used to mimic the pathological injury of SAH. Both p53 small interfering RNA (siRNA) and PD98059 reduced the OxyHb-induced apoptosis. Moreover, PD98059 significantly decreased the levels of p-Erk and p-p53; however, p53 siRNA had little effect on the level of p-Erk. Taken together, our study implicates that the Ras/Raf/Erk signals contribute to neuronal death through the phosphorylation of p53 in hippocampus after SAH and also suggests Erk/p53 as a potential target for clinical drug treatment of SAH.

Transient receptor potential channel 1/4 (TRPC1/4) are considered to be related to subarachnoid hemorrhage (SAH)-induced cerebral vasospasm. In this study, a SAH rat model was employed to study the roles of TRPC1/4 in the early brain injury (EBI) after SAH. Primary cultured hippocampal neurons were exposed to oxyhemoglobin to mimic SAH in vitro. The protein levels of TRPC1/4 increased and peaked at 5 days after SAH in rats. Inhibition of TRPC1/4 by SKF96365 aggravated SAH-induced EBI, such as cortical cell death (by TUNEL staining) and degenerating (by FJB staining). In addition, TRPC1/4 overexpression could increase calcineurin activity, while increased calcineurin activity could promote the dephosphorylation of N-methyl-D-aspartate receptor (NMDAR). Calcineurin antagonist FK506 could weaken the neuroprotection and the dephosphorylation of NMDAR induced by TRPC1/4 overexpression. Contrarily, calcineurin agonist chlorogenic acid inhibited SAH-induced EBI, even when siRNA intervention of TRPC1/4 was performed. Moreover, calcineurin also could lead to the nuclear transfer of nuclear factor of activated T cells (NFAT), which is a transcription factor promoting the expressions of TRPC1/4. TRPC1/4 could inhibit SAH-induced EBI by supressing the phosphorylation of NMDAR via calcineurin. TRPC1/4-induced calcineurin activation also could promote the nuclear transfer of NFAT, suggesting a positive feedback regulation of TRPC1/4 expressions.

Background: For managing major limb amputation, it is important to consider ischemic time and reperfusion injury by free radicals after the blood supply is reestablished. State of preservation during transplant surgery is crucial for the survival and function of the tissue, graft, or organ. In this study, we confirmed the effect of intermittent blood flow in rat ischemic hind limb and developed a new oxygenic preservation method using artificial oxygen carrying hemoglobin vesicles (HbVs)., Methods: We first compared a continuous ischemic model and an intermittent reflow model on rat hind limb. At postoperative day 7, hind limbs were evaluated. Next, we performed total amputation, normothermic preservation by perfusion with extracellular-trehalose-Kyoto (ETK) solution or HbV, and microsurgical replantation of the left hind limb. Venous efflux was analyzed, the amputated limb evaluated after 6 hr perfusion, and the replantation outcome of each model was compared., Results: In our early study, 24 hr continuous ischemic model necrotized, but intermittent reflow model almost survived except for partial necrosis at postoperative day 7. Scar tissue on the right limb showed myonecrosis and infiltration of inflammatory cells. Skeletal muscle on the right limb was structurally well maintained. Hemoglobin vesicle-treated limbs appeared to have much better oxygenation than ETK-treated limbs. Aerobic respiration remained in the amputated limb, gastrocnemius muscle was well maintained, and the overall replantation was successful in the limb preserved using HbV., Conclusion: These studies demonstrated that oxygenic preservation is effective for rat ischemic limb, suggesting that this method may be useful for other replantation and transplantation surgeries.

Objective: Nitric oxide (NO) production and Ca(2+) homeostasis are key determinants for the control of many cell functions. NO is known to be a mediator of Ca(2+) homeostasis in a highly complex and cell-specific manner and although Ca(2+) homeostasis has been explored in human oral cancer cells, the exact mechanisms are not completely understood. In this study we investigated the impact of exogenous NO on [Ca(2+)]c homeostasis in PE/CA-PJ15 cells., Design: Cells were treated with S-nitrosocysteine as NO-donor and the determinations of cytosolic Ca(2+) concentrations were performed using FURA-2 AM. Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) and oligomycin were used to challenge mitochondrial functionality, whereas thapsigargin (TG) and La(3+) were employed to perturb intracellular calcium levels., Results: NO derived from S-nitrosocysteine (CySNO) induced a dose-dependent reduction of cytosolic calcium [Ca(2+)]c whereas oxy-haemoglobin (oxyHb) completely counteracted this effect. Subsequently, we assessed possible relationships between NO and cellular structures responsible for Ca(2+) homeostasis. We found that uncoupling of mitochondrial respiration with carbonyl-cyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP) and oligomycin strongly reduced the effect of NO on [Ca(2+)]c. Moreover, we found that during this mitochondrial energetic deficit, the effect of NO on [Ca(2+)]c was also reduced in the presence of La(3+) or thapsigargin., Conclusions: NO induces a concentration-dependent [Ca(2+)]c reduction in PE/CA-PJ15 human oral cancer cells and potentiates mitochondrial Ca(2+) buffering in the presence of TG or La(3+). Further, we show that exogenous NO deregulates Ca(2+) homeostasis in PE/CA-PJ15 cells with fully energized mitochondria., (Copyright © 2014. Published by Elsevier Ltd.)

We aimed at evaluating the adequacy of the commonly employed compartmental model for quantitation of cerebral metabolic rate of oxygen (CMRO2) using (15)O-labeled oxygen ((15)O2) and positron emission tomography (PET). Sequential PET imaging was carried out on monkeys following slow bolus injection of blood samples containing (15)O2-oxyhemoglobin ((15)O2-Hb), (15)O-labeled water (H2(15)O), and C(15)O-labeled hemoglobin (C(15)O-Hb) into the internal carotid artery (ICA). Clearance slopes were assessed in the middle cerebral artery territory of the injected hemisphere. The time-activity curves were bi-exponential for both (15)O2-Hb and H2(15)O. Single exponential fitting to the early (5 to 40 seconds) and late (80 to 240 seconds) periods after the peak was performed and the (15)O2-Hb and H2(15)O results were compared. It was found that a significant difference between the clearance rates of the (15)O2-Hb and H2(15)O injections is unlikely, which supports the mathematical model that is widely used to describe the kinetics of (15)O2-Hb and H2(15)O in cerebral tissues and is the basis of recent approaches to simultaneously assess CMRO2 and cerebral blood flow in a single PET session. However, it should be noted that more data are necessary to unequivocally confirm the result.

Aligned fibers have been shown to facilitate cell migration in the direction of fiber alignment while oxygen (O2 )-carrying solutions improve the metabolism of cells in hypoxic culture. Therefore, U251 aggregate migration on poly(ε-caprolactone) (PCL)-aligned fibers was studied in cell culture media supplemented with the O2 storage and transport protein hemoglobin (Hb) obtained from bovine, earthworm and human sources at concentrations ranging from 0 to 5 g/L within a cell culture incubator exposed to O2 tensions ranging from 1 to 19% O2 . Individual cell migration was quantified using a wound healing assay. In addition, U251 cell aggregates were developed and aggregate dispersion/cell migration quantified on PCL-aligned fibers. The results of this work show that the presence of bovine or earthworm Hb improved individual cell viability at 1% O2 , while human Hb adversely affected cell viability at increasing Hb concentrations and decreasing O2 levels. The control data suggests that decreasing the O2 tension in the incubator from 5 to 1% O2 decreased aggregate dispersion on the PCL-aligned fibers. However, the addition of bovine Hb at 5% O2 significantly improved aggregate dispersion. At 19% O2 , Hb did not impact aggregate dispersion. Also at 1% O2 , aggregate dispersion appeared to increase in the presence of earthworm Hb, but only at the latter time points. Taken together, these results show that Hb-based O2 carriers can be utilized to improve O2 availability and the migration of glioma spheroids on nanofibers., (© 2014 American Institute of Chemical Engineers.)

Background: Cell-free hemoglobin (Hb) in the vasculature leads to vasoconstriction and injury. Proposed mechanisms have been based on nitric oxide (NO) scavenging by oxyhemoglobin (oxyHb) or processes mediated by oxidative reactions of methemoglobin (metHb). To clarify this, we tested the vascular effect and fate of oxyHb or metHb infusions., Study Design and Methods: Twenty beagles were challenged with 1-hour similar infusions of (200 μmol/L) metHb (n = 5), oxyHb (n = 5), albumin (n = 5), or saline (n = 5). Measurements were taken over 3 hours., Results: Infusions of the two pure Hb species resulted in increases in mean arterial blood pressure (MAP), systemic vascular resistance index, and NO consumption capacity of plasma (all p < 0.05) with the effects of oxyHb being greater than that from metHb (MAP; increase 0 to 3 hr; 27 ± 6% vs. 7 ± 2%, respectively; all p < 0.05). The significant vasoconstrictive response of metHb (vs. albumin and saline controls) was related to in vivo autoreduction of metHb to oxyHb, and the vasoactive Hb species that significantly correlated with MAP was always oxyHb, either from direct infusion or after in vivo reduction from metHb. Clearance of total Hb from plasma was faster after metHb than oxyHb infusion (p < 0.0001)., Conclusion: These findings indicate that greater NO consumption capacity makes oxyHb more vasoactive than metHb. Additionally, metHb is reduced to oxyHb after infusion and cleared faster or is less stable than oxyHb. Although we found no direct evidence that metHb itself is involved in acute vascular effects, in aggregate, these studies suggest that metHb is not inert and its mechanism of vasoconstriction is due to its delayed conversion to oxyHb by plasma-reducing agents., (© 2013 American Association of Blood Banks.)

Aim: A growing body of evidence indicates that the nuclear factor erythroid 2-related factor 2-antioxidant response element (Nrf2-ARE) pathway plays a protective role in many physiological stress processes such as inflammatory damage, oxidative stress, and the accumulation of toxic metabolites, which are all involved in the cerebral vasospasm following subarachnoid hemorrhage (SAH). We hypothesized that the Nrf2-ARE pathway might have a protective role in cerebral vasospasm following SAH., Materials and Methods: In our study, we investigate whether the oxyhemoglobin (OxyHb) can induce the activation of the Nrf2-ARE pathway in vascular smooth muscle cells (VSMCs), and evaluate the modulatory effects of sulforaphane (SUL) on OxyHb-induced inflammation in VSMCs., Results: As a result, both the protein level and the mRNA level of the nuclear Nrf2 were significantly increased, while the mRNA levels of two Nrf2-regulated gene products, both heme oxygenase-1 and NAD(P)H: quinone oxidoreductase-1, were also up-regulated in VSMCs induced with OxyHb. A marked increase of inflammatory cytokines such as IL-1β, IL-6 and TNF-α release was observed at 48 h after cells were treated with OxyHb. SUL enhanced the activity of the Nrf2-ARE pathway and suppressed cytokine release., Conclusions: Our results indicate that the Nrf2-ARE pathway was activated in OxyHb-induced VSMCs. SUL suppressed cytokine release via the activation of the Nrf2-ARE pathway in OxyHb-induced VSMCs.

Background: NO* is a key molecule involved in the regulation of cell survival, proliferation and differentiation in many cell types. In this study we investigated the contribution of NO* during the differentiation of human peripheral blood hemopoietic stem cells (CD34+HSCs) toward immunogenic dendritic cells (i-DCs)., Methods: We depleted autocrine NO* production, using NG-monomethyl-L-arginine monoacetate (L-NMMA) and paracrine NO', using oxy-hemoglobin (HbO2) as a NO* scavenger during in vitro differentiation of CD34+HSCs to i-DCs. We monitored the NO* level, cell proliferation, phenotype and differentiation potential., Results: We found that the depletion of paracrine or autocrine NO* correlated with (I) an active proliferation state at the end of differentiation, when control cells were not proliferating; (II) a significant reduction in the expression levels of differentiative markers (CD1a and HLA-DR) with a parallel high expression of the CD34 marker (III) with a retrieved clonogenic ability compared to control cells., Conclusions: On the whole, our data indicate that the depletion of NO* during the commitment stage blocks CD34+HSC differentiation into i-DCs and maintains an undifferentiated, highly proliferating cell population, indicating/revealing a novel role for NO* in the commitment of CD34+HSCs into i-DCs., General Significance: The essential finding of the present study is that NO*, produced in HSCs by NOS enzymes, may act as autocrine and paracrine effectors regulating the in vitro differentiation process of CD34+-HSCs toward i-DCs.

Potassium channels play an important role in the regulation of arterial tone, and decreased activity of these ion channels has been linked to pial artery vasospasm after subarachnoid hemorrhage (SAH). Our previous work has shown that acute application of a blood component, oxyhemoglobin, caused suppression of voltage-gated K(+) (K(V)) channels through heparin-binding epidermal growth factor-like growth factor (HB-EGF)-mediated activation of epidermal growth factor receptor (EGFR). Using patch clamp electrophysiology, we have now examined whether this pathway of K(V) channel suppression is activated in parenchymal arteriolar myocytes following long-term in vivo exposure to subarachnoid blood. We have found that K(V) currents, but not large conductance Ca(2+) activated or inwardly rectifying K(+) channel currents, were decreased in parenchymal arteriolar myocytes freshly isolated from day 5 SAH model rabbits. Interestingly, parenchymal arteriolar myocytes from control animals were more sensitive to exogenous HB-EGF (half-maximal inhibitory concentration [IC(50)] 0.2 ± 0.4 ng/ml) compared to pial arterial myocytes (IC(50) 2.4 ± 1.3 ng/ml). However, HB-EGF and oxyhemoglobin failed to decrease K(V) currents in parenchymal arteriolar myocytes from SAH animals, consistent with EGFR activation and K(V) current suppression by SAH. These data suggest that HB-EGF/EGFR pathway activation contributes to K(V) current suppression and enhanced parenchymal arteriolar constriction after SAH.

The purpose of the present study was to observe the proliferation of cerebral arterial smooth muscle cell (CASMC) induced by oxyhemoglobin (Oxyhb) and interfered by Adenovirus-mediate-PKGI (Ad-PKGI), and to investigate the potential regulative role of the PKGI gene in the molecule mechanism of cerebral vasospasm (CVS) after Subarachnoid hemorrhage (SAH). Tissue-sticking method was used for primary cultured rat CASMCs. Semi-quantitative reverse transcription and polymerase chain reaction (RT-PCR) and western blot were used to examine the PKGI mRNA and protein expressions after CASMC were transfected by Ad-PKG. The proliferation of CASMCs was determined by MTT assay and 3H-TdR incorporation. Ad-PKGI could be transfected into CASMCS and highly express. Oxyhemoglobin could stimulate the proliferation of CASMC; the value of 3H-TdR incorporation and the absorbance value of MTT increased and could block up after CASMC was transfected by Ad-PKG. The results suggested that the PKG signaling pathway might play an important role in CVS after SAH, and the PKG gene might be a target point of gene therapy.

Several pathologic conditions are associated with hemolysis, i.e. release of ferrous (Fe(II)) hemoglobin from red blood cells. Oxidation of cell-free hemoglobin produces (Fe(III)) methemoglobin. More extensive oxidation produces (Fe(III)/Fe(IV) O) ferryl hemoglobin. Both cell-free methemoglobin and ferryl hemoglobin are thought to contribute to the pathogenesis of hemolytic disorders. We show hereby that ferryl hemoglobin, but not hemoglobin or methemoglobin, acts as a potent proinflammatory agonist that induces vascular endothelial cells in vitro to rearrange the actin cytoskeleton, forming intercellular gaps and disrupting the integrity of the endothelial cell monolayer. Furthermore, ferryl hemoglobin induces the expression of proinflammatory genes in endothelial cells in vitro, e.g. E-selectin, Icam-1, and Vcam-1, through the activation of the nuclear factor kappaB family of transcription factors. This proinflammatory effect, which requires actin polymerization, involves the activation of the c-Jun N-terminal kinase and the p38 mitogen-activated protein kinase signal transduction pathways. When administered to naïve mice, ferryl hemoglobin induces the recruitment of polymorphonuclear cells, demonstrating that it acts as a proinflammatory agonist in vivo. In conclusion, oxidized hemoglobin, i.e. ferryl hemoglobin, acts as a proinflammatory agonist that targets vascular endothelial cells.

Cerebral vasospasm remains a major cause of morbidity and mortality in patients with subarachnoid hemorrhage. Heme oxygenase-1 (HO-1) is an oxidative stress-inducible enzyme with multiple protective functions against vascular and neurological diseases, including delayed cerebral vasospasm. In the present study, intravenous administration (i.v.) of nicaraven (1 mg/kg/min, for 2 days after subarachnoid hemorrhage) ameliorated delayed cerebral vasospasm in rat subarachnoid hemorrhage models, marked synergistic induction of HO-1 protein (> 2.5-fold than 'subarachnoid hemorrhage with saline i.v.'), and elicited a rapid increase of cGMP accumulation in the basilar arteries. In the sham-operated rats, nicaraven could not induce HO-1 expression. Antisense HO-1 oligodeoxynucleotides abrogated this HO-1 induction and the antivasospastic effect of nicaraven. In vitro study using Hela cells, nicaraven enhanced the human HO-1 promoter (-4.5 kbp) activity, which was pre-activated with the blood component oxyhemoglobin to mimic the ability of subarachnoid hemorrhage. These results suggest that this enhanced HO-1 expression through a combination of pathological state and pharmacological agent could be an effective strategy to improve the prognosis of heme- and oxidative stress-induced diseases, such as delayed cerebral vasospasm.

CAS 1609 (compound 1) and CHF 2363 (compound 2) are two furoxan derivatives able to release nitric oxide (NO) under physiological conditions, and display typical NO-dependent vasodilator activity. The potential genotoxic effects of compound 1 and of the water-soluble analogue of CHF 2363 (compound 2a) were investigated. The results show that the two compounds induce genotoxic effects only at concentrations that significantly reduce cell viability. However, in the case of compound 1 this range of concentrations is one order of magnitude higher than the one leading to the beneficial effects, while in the case of compound 2a these ranges partially overlap. In both cases the release of NO plays a key role in the induction of the cytotoxic and genotoxic effects, since the non-NO-donating furazan analogues display a different toxicological profile, and since the effects were reduced in the presence of oxyhaemoglobin, a well-known NO-scavenger.

Background: Delayed cerebral ischemia due to cerebral vasospasm remains a major cause of morbidity and mortality following subarachnoid hemorrhage. Oxyhemoglobin (OxyHb) and vasoconstrictor prostanoids have been suggested as putative spasmogens. We have previously reported a synergistic vasoconstrictive action between thromboxane A(2) (TXA(2)) and OxyHb. In the present study we examine the effect of neomycin, a phospholipase C inhibitor, on the cerebral vasoconstriction induced by TXA(2) and OxyHb., Methods: Using an in vitro tissue bath method, we assess the effect of neomycin in a concentration-dependent manner, on isolated porcine basilar arteries constricted by U-46619 (TXA(2) analogue) and OxyHb., Results: The functional synergism between TXA(2) and OxyHb, leading to significant cerebral vasoconstriction, is attenuated in a dose-dependent manner by neomycin., Conclusion: Blockade of phospholipase C may provide an alternative strategy in the treatment of subarachnoid-hemorrhage-induced cerebral vasospasm., ((c) 2008 S. Karger AG, Basel.)

Voltage-dependent potassium (Kv) and calcium (VDCC) channels play an important role in the regulation of membrane potential and intracellular calcium concentration in cerebral artery myocytes. Recent evidence suggests VDCC activity is increased and Kv channel activity is decreased in cerebral arteries following subarachnoid hemorrhage (SAH), promoting enhanced constriction. We have examined the impact of the blood component oxyhemoglobin on Kv and VDCC function in small (100-200 microm) diameter cerebral arteries. Acute (10 min) exposure of oxyhemoglobin caused cerebral artery constriction and Kv current suppression that was abolished by tyrosine kinase inhibitors and a Kv channel blocker. Although short-term oxyhemoglobin application did not directly alter VDCC activity, five-day exposure to oxyhemoglobin was associated with enhanced expression of voltage-dependent calcium channels. This work suggests that acute and chronic effects of oxyhemoglobin act synergistically to promote membrane depolarization and increased VDCC activity in cerebral arteries. These actions of oxyhemoglobin may contribute to the development of cerebral vasospasm following aneurysmal subarachnoid hemorrhage.

Sinoaortic deafferentation (SAD) is characterized by arterial pressure lability, without sustained hypertension. Although SAD rats did not become hypertensive, their isolated aortas exhibit RCs. We have investigated whether these RCs are influenced by endothelium. Aortic rings were placed in an organ chamber, and the frequency and amplitude of the RCs were measured in SAD rat aortas with intact and denuded endothelium. Moreover, the participation of endothelial NO and cGMP pathways on the RCs were analyzed through the use of such drugs as L-NAME, acetylcholine, ODQ, L-arginine, and oxyhemoglobin. Indomethacin was used to evaluate the influence of prostanoids on the RCs. Under phenylephrine stimulus, 100% of SAD rat aortas presented RCs, with higher frequency and amplitude in denuded endothelium (8.5 +/- 0.50 cycles/2 min and 0.465 +/- 0.05 g, respectively) compared with intact endothelium (4.5 +/- 0.50 cycles/2 min and 0.311 +/- 0.04 g, respectively). In intact-endothelium aortas, L-NAME, ODQ, and oxyhemoglobin raised the frequency and amplitude of the RCs to values similar to those found in denuded endothelium. L-arginine and indomethacin did not alter the RCs. In conclusion, SAD rat aortas present rhythmic contractions, which are negatively modulated by endothelial NO. Our results indicate that endothelium-derived NO (and not prostanoids), acting through the cGMP pathway, has an inhibitory effect on the oscillations.

Background: Kupffer cells (liver macrophages) are a key initiator of inflammation following hepatic insults such as infection, ischemia/reperfusion, and rejection. Heme oxygenase 1 (HO-1) is protective against inflammatory injury. A hemoglobin-based oxygen carrier (HBOC) has been shown to prevent organ inflammation from hemorrhagic shock as well as induce HO-1 at the cellular level. Therefore, we hypothesize that HBOC can induce Kupffer cell HO-1 production., Methods: Mice administered 20% blood volume HBOC or saline intravenously were sacrificed at 0, 12, 24, 48 hours (n = 4-6/group). Hepatic protein underwent Western blotting for HO-1 and heat shock protein 72. Hepatic frozen sections underwent immunofluorescent staining for HO-1/CD68., Results: Following HBOC injection, hepatic HO-1 fold change peaked at 12 hours (7.3 +/- 0.8) (p < .01), remained increased at 24 hours (4.7 +/- 0.4) (p < .01), and returned to baseline by 48 hours. HSP72 expression was unaffected in all groups. Twleve-hour liver section immunostaining confirmed significant induction of HO-1 by HBOC. Double staining for HO-1 and CD68 identified Kupffer cells as the majority of cells expressing HO-1., Conclusion: HBOC induces hepatic HO-1 expression in Kupffer cells without heat shock protein response. These data provide the basis for further investigation into a clinical therapy to induce Kupffer cell HO-1 expression with the goal of attenuating the hepatic immunoresponse to various insults.

Objectives: Due to potential therapeutic value of vascular endothelial growth factor (VEGF) in coronary artery disease, the effect and mechanism of VEGF in human arteries used as coronary bypass grafts become important but not fully understood. VEGF-mediated endothelial regulation in vasorelaxation was studied in internal mammary artery (IMA) and radial artery (RA), compared with that of the classical agent-acetylcholine (ACh). The role of nitric oxide (NO), prostacyclin (PGI2), and endothelium-derived hyperpolarizing factor (EDHF) was investigated., Methods: VEGF- and ACh-induced responses were measured in RA and IMA with or without endothelium and in the absence or presence of inhibitors of nitric oxide synthase or prostacyclin. In addition, the VEGF-induced PGI2 was measured by enzyme immunoassay., Results: VEGF induced similar relaxation in RA (59.2+/-9.3%) and IMA (56.1+/-6.4%) that was significantly inhibited by N(omega)-nitro-L-arginine (L-NNA) plus oxyhemoglobin (HbO) (IMA: 24.9+/-4.3%, P=0.03 vs. RA: 25.0+/-8.6%, P=0.01) or by indomethacin (INDO) (IMA: 21.8+/-2.5%, P=0.000 vs. RA: 30.0+/-6.6%, P=0.04) with more inhibition in IMA than RA (P<0.05). In addition, the VEGF-induced PGI2 was significantly higher in IMA than RA (11.5+/-2.1 vs. 4.9+/-1.1 pg/ml/mg, P=0.002). INDO+L-NNA+HbO reduced the VEGF-induced relaxation to 20.8+/-4.6% in RA vs. 4.8+/-1.6% in IMA (P=0.01). In contrast, the maximal relaxation induced by ACh in RA (55.9+/-6.0%) and IMA (48.5+/-5.3%) was largely inhibited by L-NNA in IMA and RA (14.7+/-3.0%, P=0.000 vs. 15.2+/-3.2%, P=0.004) but little affected by INDO., Conclusions: VEGF induces similar relaxation in IMA and RA with significantly more PGI2-mediated relaxation and higher stimulated PGI2 level in IMA but more EDHF-mediated relaxation in RA. In comparison, ACh-induced relaxation mainly depends on NO. Thus, our study reveals a significant difference in the mechanism of the endothelium-dependent relaxation induced by VEGF and ACh.

Background: The hypertension induced by haemoglobin-based oxygen carriers could be a result of different pharmacological and physicochemical factors., Objective: To investigate whether production of superoxide anion (O2*-) and release of endothelin could be the factors responsible., Methods: We studied the variation in mean arterial pressure (MAP) in guinea pigs by carrying out a 50% isovolaemic exchange transfusion with conjugated oxyhaemoglobin (non-oxidized form) or conjugated methaemoglobin (fully oxidized form) in the presence or absence of BQ-788 (5 nmol/l), an endothelin receptor type B (ETR-B) antagonist. At key timepoints of variation in MAP, the plasma concentrations of O2*- were measured. The presence of conjugated oxyhaemoglobin and increases in ETR-B concentrations inside the vascular wall were investigated in different vessels, using western blotting., Results: We found that the administration of conjugated oxyhaemoglobin induced a significant increase in MAP, whereas conjugated methaemoglobin had no significant haemodynamic effect. Pretreatment with BQ-788 attenuated the increase in MAP induced by conjugated oxyhaemoglobin. This haemoglobin induced the production of high concentrations of O2*- that declined towards control values after 120 min and decreased in the presence of BQ-788. Western blot analysis showed that the presence of conjugated oxyhaemoglobin inside the vascular wall was time-dependent and correlated with increased ETR-B., Conclusion: These results show that the release of O2*- during auto-oxidation of conjugated oxyhaemoglobin is associated with the observed increase in MAP, which may be a result of the vasoconstriction caused by an increase in activation of ETR-B. This activation may be caused by the massive release of endothelin induced by the production of O2*-.

Cerebral vasospasm following aneurysmal subarachnoid hemorrhage (SAH) has devastating consequences. Oxyhemoglobin (oxyhb) has been implicated in SAH-induced cerebral vasospasm as it causes cerebral artery constriction and increases tyrosine kinase activity. Voltage-dependent, Ca(2+)-selective and K(+)-selective ion channels play an important role in the regulation of cerebral artery diameter and represent potential targets of oxyhb. Here we provide novel evidence that oxyhb selectively decreases 4-aminopyridine sensitive, voltage-dependent K(+) channel (K(v)) currents by approximately 30% in myocytes isolated from rabbit cerebral arteries but did not directly alter the activity of voltage-dependent Ca(2+) channels or large conductance Ca(2+)-activated (BK) channels. A combination of tyrosine kinase inhibitors (tyrphostin AG1478, tyrphostin A23, tyrphostin A25, genistein) abolished both oxyhb-induced suppression of K(v) channel currents and oxyhb-induced constriction of isolated cerebral arteries. The K(v) channel blocker 4-aminopyridine also inhibited oxyhb-induced cerebral artery constriction. The observed oxyhb-induced decrease in K(v) channel activity could represent either channel block, or a decrease in K(v) channel density on the plasma membrane. To explore whether oxyhb altered trafficking of K(v) channels to the plasma membrane, we used an antibody generated against an extracellular epitope of K(v)1.5 channels. In the presence of oxyhb, staining of K(v)1.5 on the plasma membrane surface was markedly reduced. Furthermore, oxyhb caused a loss of spatial distinction between staining with K(v)1.5 and the general anti-phosphotyrosine antibody PY-102. We propose that oxyhb-induced suppression of K(v) currents occurs via a mechanism involving enhanced tyrosine kinase activity and channel endocytosis. This novel mechanism may contribute to oxyhb-induced cerebral artery constriction following SAH.

Background and Purpose: The present study investigated whether calcium-activated K+ channels are involved in acetylcholine-evoked nitric oxide (NO) release and relaxation., Experimental Approach: Simultaneous measurements of NO concentration and relaxation were performed in rat superior mesenteric artery and endothelial cell membrane potential and intracellular calcium ([Ca2+]i) were measured., Key Results: A combination of apamin plus charybotoxin, which are, respectively, blockers of small-conductance and of intermediate- and large-conductance Ca2+ -activated K channels abolished acetylcholine (10 microM)-evoked hyperpolarization of endothelial cell membrane potential. Acetylcholine-evoked NO release was reduced by 68% in high K+ (80 mM) and by 85% in the presence of apamin plus charybdotoxin. In noradrenaline-contracted arteries, asymmetric dimethylarginine (ADMA), an inhibitor of NO synthase inhibited acetylcholine-evoked NO release and relaxation. However, only further addition of oxyhaemoglobin or apamin plus charybdotoxin eliminated the residual acetylcholine-evoked NO release and relaxation. Removal of extracellular calcium or an inhibitor of calcium influx channels, SKF96365, abolished acetylcholine-evoked increase in NO concentration and [Ca2+]i. Cyclopiazonic acid (CPA, 30 microM), an inhibitor of sarcoplasmic Ca2+ -ATPase, caused a sustained NO release in the presence, but only a transient increase in the absence, of extracellular calcium. Incubation with apamin and charybdotoxin did not change acetylcholine or CPA-induced increases in [Ca2+]i, but inhibited the sustained NO release induced by CPA., Conclusions and Implications: Acetylcholine increases endothelial cell [Ca2+]i by release of stored calcium and calcium influx resulting in activation of apamin and charybdotoxin-sensitive K channels, hyperpolarization and release of NO in the rat superior mesenteric artery.

Uncontrolled diabetes mellitus (DM) adversely affects oocyte maturation and embryo development via mechanisms that are yet unclear. Nonetheless, DM may cause uncoupling of nitric oxide synthases (NOSs) with reduction in the bioavailability of nitric oxide (NO), which is critical to maintain oocyte viability and prevent aging. The current study investigates the role of NO-mediated signaling related to oocyte aging in diabetic and nondiabetic mice. Age-related alterations in the oocytes, including ooplasmic microtubule dynamics (OMD), cortical granule (CG) status, and zona pellucida (ZP) hardening as well as the integrity of the spindle/chromatin were studied using confocal microscopy. Oocytes obtained from diabetic mice exhibited accelerated aging compared to that from nondiabetic mice. Moreover, oocytes from diabetic animals were exquisitely sensitive to NOS and guanylate cyclase (GC) inhibitors (L-NAME, ODQ), which induced aging and relatively resistant to its delay by the cGMP derivative (8-Br-cGMP). Oocytes from nondiabetic control mice displayed similar sensitivity to L-NAME in older oocytes, although to a significantly lower extent than that of DM (P < 0.04-0.0001). Despite the differences in response between DM and nonDM mice, the activation of cGMP pathway is essential to maintain the integrity of oocytes and delay oocyte aging. These findings not only indicate the role of NO signaling in the prevention of oocyte aging but also suggest enhanced aging and NO insufficiency in oocytes from diabetic mice. A comprehensive model incorporating our current findings with NOS, GC, and G kinase cycles is presented.

Disulfiram (an alcohol-aversive drug) and related compounds are known to provoke several side effects involving behavioral and neurological complications. N,N-diethyldithiocarbamate (DDC) is considered as one of the main toxic species of disulfiram and acts as an inhibitor of superoxide dismutase. Since arachidonic acid (AA) formation is regulated by reactive oxygen species (ROS) and related to toxicity in neuronal cells, we investigated the effects of DDC on AA release and expression of the alpha type of cytosolic phospholipase A(2) (cPLA(2)alpha) in PC12 cells. Treatment with 80-120 microM DDC that causes a moderate increase in ROS levels without cell toxicity stimulated cPLA(2)alpha mRNA and its protein expression. The expression was mediated by extracellular-signal-regulated kinase (ERK1/2), one of the mitogen-activated protein kinases. Treatment with N(G) nitro-L-arginine methyl ester (an inhibitor of nitric oxide synthase, 1 mM) and oxy-hemoglobin (a scavenger of nitric oxide, 2 mg/mL) abolished the DDC-induced responses (ERK1/2 phosphorylation and cPLA(2)alpha expression). We also showed DDC-induced up-regulation of the mRNA expression of lipocortin 1, an inhibitor of PLA(2). Furthermore, DDC treatment of the cells enhanced Ca(2+)-ionophore-induced AA release in 30 min, although the effect was limited. Changes in AA metabolism in DDC-treated cells may have a potential role in mediating neurotoxic actions of disulfiram. In this study, we show the first to demonstrate the up-regulation of cPLA(2)alpha expression by DDC treatment in neuronal cells.

Objective: Increased nitric oxide (NO) production in sepsis precipitates microcirculatory dysfunction. We aimed (i) to determine if NO is the key water-soluble factor in the recently discovered sepsis-induced deficit in arteriolar conducted vasoconstriction, (ii) to identify which nitric oxide synthase (NOS) isoforms account for this deficit, and (iii) to examine the potential role of connexin37 (Cx37, a hypothesized signaling target of NO) in arteriolar conduction., Methods: Using intravital microscopy and the cecal ligation and perforation 24-h model of sepsis, arterioles in the cremaster muscle of male C57BL/6 wild-type (WT), iNOS-/-, eNOS-/-, nNOS-/- and Cx37-/- mice were locally stimulated with KCl to initiate conducted vasoconstriction. We used the ratio of conducted constriction (500 microm upstream) to local constriction as an index of conduction (CR500). NOS enzymatic activity and protein expression were determined in control and septic cremaster muscles., Results: Sepsis reduced CR500 in WT mice [from 0.77 +/- 0.05 to 0.20 +/- 0.02 (means +/- SE) independent of the site of stimulation along the arteriole], in iNOS-/- and eNOS-/- mice, but not in nNOS-/- mice. The nNOS inhibitor 7-nitroindazole or NO scavenger HbO2 restored CR500 in septic WT mice, but blockade of soluble guanylate cyclase had no effect. Sepsis increased cNOS (eNOS + nNOS) activity in WT mice (from 340 +/- 40 to 490 +/- 30 pmol/mg/h) and in eNOS-/-, but not in nNOS-/- mice (iNOS activity was negligible in all mice). Sepsis did not alter nNOS protein expression in WT mice. CR500 in non-septic Cx37-/- mice (0.15 +/- 0.1) was similar to that observed in septic WT mice., Conclusion: Increased nNOS activity and the resultant increased NO production in the septic mouse cremaster muscle are the key factors responsible for the deficit in conducted vasoconstriction along the arteriole. Deletion of Cx37 results in reduced CR500, which is consistent with the hypothesis that Cx37 in the arteriole could be a target of NO signaling.

The aim of the present study was to investigate the possible endogenous storage of photosensitive nitric oxide, and also to examine the relaxant effect of NO released from the compound by UV light irradiation. Aorta was isolated from rats and the endothelium was mechanically removed. Denuded aortic rings pre-contracted with prostaglandin F(2alpha) responded with relaxation to UV light irradiation. The first stimulation produced the greatest response that decreased until complete disappearance. After this, the addition of the compound in the absence of light did not produce any response. However, in the presence of UV light irradiation, the complex trans-[RuCl([15]aneN4)NO]2+ induced 100% relaxation. After incubation with the nitric oxide scavenger, oxyhaemoglobin, this relaxation was completely abolished. In PGF2(2alpha)-pre-contracted aortas, the time to reach maximum relaxation was only 50s. Taken together, these results suggest that preformed endogenous nitric oxide stores exist in the denuded rat aorta, and that they are sensitive to UV light. The photo-induction of the complex trans-[RuCl([15]aneN4NO]2+ induces complete aorta relaxation, which is due to release of nitric oxide in the extracellular medium.

The present study investigated whether activation of vasodilatory mechanisms masks the involvement of endothelin in hypoxic pulmonary vasoconstriction. Rat intrapulmonary arteries were mounted in microvascular myographs. In arteries with endothelium and contracted with phenylephrine, hypoxia, evoked by exchanging 5% CO2 in air for CO2 in N2, caused a transient contraction followed by a sustained contraction. Hypoxia evoked relaxation in preparations without endothelium. An inhibitor of ATP-sensitive K+ channels (KATP), glibenclamide (10 microM), blunted hypoxic relaxation in arteries without endothelium and enhanced the sustained hypoxic vasoconstriction in arteries with endothelium. Hypoxic contraction was more pronounced in endothelin compared with phenylephrine-contracted preparations in the absence, but not in the presence of glibenclamide. Antagonism of the endothelin ETA and ETB receptors with SB217242 or the combination of BQ123 and BQ788 inhibited endothelin and hypoxic contraction, but the latter only in the presence of glibenclamide. An inhibitor of nitric oxide (NO) synthase, N-nitro-L-arginine (100 microM), evoked contractions, which were left unaltered by SB217242 in hypoxic conditions. In conclusion, hypoxic contraction is mediated in part by an unknown endothelium-derived contractile factor and incubation with glibenclamide shows endothelin enhances hypoxic contraction in part through inhibition of KATP channels. Moreover, inhibition of NO formation in pulmonary arteries does not change endothelin receptor activation in severe hypoxia.

In erythrocytes, S-nitrosohemoglobin (SNO-Hb) arises from S-nitrosylation of oxygenated hemoglobin (Hb). It has been shown that SNO-Hb behaves as a nitric oxide (NO) donor at low oxygen tensions. This property, in combination with oxygen transport capacity, suggests that SNO-Hb may have unique potential to reoxygenate hypoxic tissues. The present study was designed to test the idea that the allosteric properties of SNO-Hb could be manipulated to enhance oxygen delivery in a hypoxic tumor. Using Laser Doppler flowmetry, we showed that SNO-Hb infusion to animals breathing 21% O2 reduced tumor perfusion without affecting blood pressure and heart rate. Raising the pO2 (100% O2) slowed the release of NO bioactivity from SNO-Hb (ie, prolonged the plasma half-life of the SNO in Hb), preserved tumor perfusion, and raised the blood pressure. In contrast, native Hb reduced both tumor perfusion and heart rate independently of the oxygen concentration of the inhaled gas, and did not elicit hypertensive effects. Window chamber (to image tumor arteriolar reactivity in vivo) and hemodynamic measurements indicated that the preservation of tissue perfusion by micromolar concentrations of SNO-Hb is a composite effect created by reduced peripheral vascular resistance and direct inhibition of the baroreceptor reflex, leading to increased blood pressure. Overall, these results indicate that the properties of SNO-Hb are attributable to allosteric control of NO release by oxygen in central as well as peripheral issues.

The aims of the present study were to investigate the pharmacological effects induced by Tityus serrulatus venom (TsV) and its fractions and to compare with the effects induced by pure alpha (TsTX-V) and beta (TsTX-I) toxins isolated from TsV on rat retractor penis muscle (RPM). TsV, fractions X, XI, XIIa, XIIb (0.01-100 microg/ml) and TsTX-V (1 nmol/l-10 micromol/l) induced concentration-dependent contractions. Prazosin and guanethidine or tetrodotoxin (TTX, 5 micromol/l, 30 min) completely abolished these contractions, suggesting complete dependence on sympathetic nerves. TsV or fractions X, XI, XIIa, XIIb (0.01- 100 microg/ml), TsTX-I and TsTX-V (1 nmol/l-10 micromol/l) induced concentration-dependent relaxations in the precontracted RPM. TTX or N(omega)-nitro-L-arginine methyl ester (L-NAME, 100 micromol/l, 30 min) completely abolished the relaxations. Our results suggest that most of TsV-derivated toxins induce contraction and relaxation on RPM by sympathetic and NANC nitrergic nerve stimulation. Noteworthy, TsTX-I only induces relaxation on RPM suggesting that this protein selectively acts on inhibitory nerves., (Copyright 2005 S. Karger AG, Basel.)

A number of rat islets were co-encapsulated in a diffusion chamber-type device, i.e., macrocapsule, with a thermoreversible polymeric extracellular matrix (ECM) and bioactive ingredient of cross-linked hemoglobin (Hb-C). The ECM was formed from an aqueous solution of N-isopropyl-acrylamide co-polymers with a small amount of acrylic acid, which exhibited unique sol-gel transition in a temperature range of 30-34 degrees C, without noticeable hysteresis. The incorporation of Hb-C in the islet macrocapsule showed a concentration-dependent effect on insulin secretion and viability of the entrapped islets. Insulin secretion stimulation by glucose and cell viability were more than doubled when compared with a control group (without Hb-C), at an optimum Hb-C concentration of 0.25 mM due to its unique oxygen transporting capacity. Furthermore, 0.25 mM Hb-C in the macrocapsule was able to support islet density up to 1000 islets/device in a 154 microl total volume without negative effects on islet functionality and viability. Hb-C incorporation is an effective strategy for a macrocapsule-type biohybrid artificial pancreas for Type-I diabetes treatment, which can be further developed to a rechargeable system by employing the thermoreversible ECM and designing a proper macrocapsule.

Prostaglandin E2 (PGE2) has been shown to dilate and constrict the systemic vascular beds, including cerebral vessels. The exact mechanism of PGE2-induced cerebral vasoconstriction, however, is less clarified. The authors' preliminary studies showed that PGE2 exclusively constricted the adult porcine basilar arteries. The present study, therefore, was designed to examine the receptor mechanisms involved in PGE2-induced constriction of large cerebral arteries in the adult pig. Results from an in vitro tissue-bath study indicated that PGE2 and its agonists 17-phenyl trinor PGE2 (17-PGE2), sulprostone (EP1/EP3 receptor agonists), and 11-deoxy-16,16-dimethyl PGE2 (11-PGE2, an EP2/EP3-receptor agonist) induced exclusive constriction, which was not affected by endothelium denudation or cold-storage denervation of perivascular nerves. The constriction induced by PGE2, 17-PGE2, and sulprostone, but not by potassium chloride, was blocked by SC-19220 (a selective EP1-receptor antagonist), AH-6809 (an EP1/EP2-receptor antagonist), and U-73122 and neomycin (phospholipase C inhibitors). AH-6809, however, did not affect 11-PGE2-induced contraction. These results suggest that the contraction was not mediated by the EP2-receptor, but was mediated by EP1- and EP3-receptors. Furthermore, EP1-receptor immunoreactivities were found across the entire medial smooth muscle layers, whereas EP3-receptor immunoreactivities were limited to the outer smooth muscle layer toward the adventitia. Western blotting also showed the presence of EP1- and EP3-receptor proteins in cultured primary cerebral vascular smooth muscle cells. In conclusion, PGE2 exclusively constricts the adult porcine large cerebral arteries. This constriction is mediated by phosphatidyl-inositol pathway via activation of EP1- and EP3-receptors located on the smooth muscle cells. These two receptor subtypes may play important roles in physiologic and pathophysiologic control of cerebral vascular tone.

The role of the vascular endothelial growth factors (VEGF) receptors (KDR and Flt-1) and their characteristics in VEGF-induced vasodilation in human vessels is unclear. This study investigated the in vitro vasorelaxant effects of KDR-selective (KDR-SM) and Flt-1-selective mutants (Flt-1-SM) in the human internal mammary artery (IMA). IMA segments (n = 183) taken from 48 patients were studied in organ baths. The cumulative concentration (-12 to -8 log10M)-relaxation curves were established for VEGF, KDR-SM, Flt-1-SM, and placenta growth factor (PlGF) in the absence or presence of indomethacin (INDO, 7 microM), N-nitro-L-arginine (L-NNA, 300 microM), L-NNA + oxyhemoglobin (HbO, 20 microM), or INDO + L-NNA + HbO. The VEGF-induced relaxation was abolished in endothelium-denuded IMA. In the endothelium-intact vessel rings, VEGF (63.2 +/- 3.9%) induced significantly more (P < 0.001) relaxation than Flt-1-SM (28.5 +/- 4.3%, 95% CI 18.1-51.3%), and PlGF (26.0 +/- 4.7%, 95% CI 17.6-56.8%). The maximal relaxation induced by KDR-SM (53.0 +/- 4.0%) was only slightly less than that by VEGF (P = 0.075) but significantly more than that by Flt-1-SM (P = 0.001, 95% CI 7.8-41.1%). Pretreatment of INDO or L-NNA + HbO significantly (P < 0.001) inhibited the relaxation by VEGF (21.2 +/- 3.9% or 23.3 +/- 4.3%) and KDR-SM (9.8 +/- 8.2% or 10.1 +/- 17.8%). INDO + L-NNA + HbO completely inhibited the relaxation by VEGF, KDR-SM, or Flt-1-SM. KDR may be the dominant receptor in mediating the VEGF-mediated relaxation, which is regulated by both PGI2 and nitric oxide but probably not by endothelium-derived hyperpolarizing factor, in the human IMA. This study gives insight into the characteristics of the VEGF-mediated vasodilation and provides a scientific basis for potential clinical application of VEGF/KDR-SM in ischemic heart disease.

S-Nitrosothiols have been suggested to be mediators of many nitric oxide-dependent processes, including apoptosis and vascular relaxation. Thiol nitrosation is a poorly understood process in vivo, and the mechanisms by which nitric oxide can be converted into a nitrosating agent have not been established. There is a discrepancy between the suggested biological roles of nitric oxide and its known chemical and physical properties. In this study, we have examined the formation of S-nitrosothiols in lipopolysaccharide-treated RAW 264.7 cells. This treatment generated 17.4 +/- 1.0 pmol/mg of protein (means +/- SE, n =27) of intracellular S-nitrosothiol that slowly decayed over several hours. S-Nitrosothiol formation depended on the formation of nitric oxide and not on the presence of nitrite. Extracellular thiols were nitrosated by cell-generated nitric oxide. Oxygenated ferrous hemoglobin inhibited the formation of S-nitrosothiol, indicating the nitrosation occurred more slowly than diffusion. We discuss several mechanisms for S-nitrosothiol formation and conclude that the nitrosation propensity of nitric oxide is a freely diffusible element that is not constrained within an individual cell and that both nitric oxide per se and nitric oxide-derived nitrosating agents are able to diffuse across cell membranes. To achieve intracellular localization of the nitrosation reaction, mechanisms must be invoked that do not involve the formation of nitric oxide as an intermediate.

Background: The effect of cold storage of porcine pulmonary microvessels in University of Wisconsin (UW) and Euro-Collins (EC) solutions on the cellular electrophysiologic properties remains unknown., Methods: The pulmonary microarteries (PA, 381.6 +/- 62.8 microm; n = 60) and microveins (PV, 360.8 +/- 54.5 microm; n = 60) were incubated with Krebs (control), UW, or EC solution at 4 degrees C for 4 hours in a myograph. The resting membrane potential and the endothelium-derived hyperpolarizing factor-mediated hyperpolarization to bradykinin (0.1 micromol/L) in the presence of inhibitors of nitric oxide and prostacyclin, N(omega)-nitro-l-arginine, hemoglobin, and indomethacin, in a single smooth muscle cell were directly measured., Results: The resting membrane potential (-60.8 +/- 1.3 mV in PA and -48.1 +/- 0.7 mV in PV, n = 6) was depolarized after exposure to UW solution (to -18.4 +/- 0.7 mV in PA and -13.6 +/- 0.8 mV in PV; n = 8; p < 0.001). The amplitude of endothelium-derived hyperpolarizing factor-mediated hyperpolarization to bradykinin was also decreased (from 7.4 +/- 0.7 mV to 2.6 +/- 0.7 mV in PA and from 4.6 +/- 0.5 mV to 0.9 +/- 0.4 mV in PV; p < 0.001). In comparison, EC depolarized the membrane potential to a lesser extent (to -28.3 +/- 0.9 mV in PA and to -21.3 +/- 0.8 mV in PV; n = 8; p < 0.001) and almost abolished the hyperpolarization to bradykinin. After washout, hyperpolarization was partially restored (UW, 4.9 +/- 0.7 mV in PA and 2.0 +/- 0.3 mV in PV. p < 0.01; EC, 2.3 +/- 0.5 mV in PA and 1.0 +/- 0.3 mV in PV. p < 0.01)., Conclusions: Cold storage of porcine PA and PV with UW or EC solution impairs the electrophysiologic properties (hyperpolarization) related to endothelium-smooth muscle interaction. The alteration is more profound with EC than UW solution and in veins than in arteries. The findings urge further studies on lung preservation solutions.

There is a long history of science seeking to develop artificial substitutes for body parts damaged by disease or trauma. While defective teeth and limbs are commonly replaced by imitations without major loss of functionality, the development of a substitute for red blood cells has proved elusive. There is a permanent shortage of donor blood in western societies. Nevertheless, despite whole blood transfusions carrying measurable risks due to immunogenicity and the transmission of blood-borne infectious diseases, red blood cells are still relatively inexpensive, well tolerated and widely available. Researchers seeking to develop products that are able to meet and perhaps exceed these criteria have responded to this difficult challenge by adopting many different approaches. Work has focussed on two classes of substances: modified haemoglobin solutions and perfluorocarbon emulsions. Other approaches include the creation of artificial red cells, where haemoglobin and supporting enzyme systems are encapsulated into liposomes. Haemoglobin is ideally suited to oxygen transport when encased by the red cell membrane; however, once removed, it rapidly dissociates into dimers and is cleared by the kidney. Therefore, it must be stabilised before it can be safely re-infused into humans. Modifications concomitantly alter the vascular half-life, oxygen affinity and hypertensive characteristics of raw haemoglobin, which can be sourced from outdated blood stores, genetically-engineered Escherichia coli or even bovine herds. In contrast, perfluorocarbons are entirely synthetic molecules that are capable of dissolving oxygen but biologically inert. Since they dissolve rather than bind oxygen, their capacity to serve as a blood substitute is determined principally by the oxygen pressure gradients in the lung and at the target tissue. Blood substitutes have important potential areas of clinical application including red cell replacement during surgery, emergency resuscitation of traumatic blood loss, oxygen therapeutic applications in radiography (oxygenation of tumour cells is beneficial to the effect of certain chemotherapeutic agents), other medical applications such as organ preservation, and finally to meet the requirements of patients who cannot receive donor blood because of religious beliefs. Given the elite athlete's historical propensity to experiment with novel doping strategies, it is likely that the burgeoning field of artificial oxygen carriers has already attracted their attention. Scientific data concerning the performance benefits associated with blood substitutes are virtually nonexistent; however, international sporting federations have been commendably proactive in adding this category to their banned substance lists. The current situation is vulnerable to exploitation by immoral athletes since there is still no accepted methodology to test for the presence of artificial oxygen carriers.

Neurogenic ATP and nitric oxide (NO) may play important roles in the physiological control of gastrointestinal motility. However, the interplay between purinergic and nitrergic neurons in mediating the inhibitory neurotransmission remains uncertain. This study investigated whether neurogenic NO modulates the purinergic transmission to circular smooth muscles of the hamster proximal colon. Electrical activity was recorded from circular muscle cells of the hamster proximal colon by using the microelectrode technique. Intramural nerve stimulation with a single pulse evoked a fast purinergic inhibitory junction potential (IJP) followed by a slow nitrergic IJP. The purinergic component of the second IJP evoked by paired stimulus pulses at pulse intervals between 1 and 3 s became smaller than that of the first IJP. This purinergic IJP depression could be observed at pulse intervals <3 s, but not at longer ones, and failed to occur in the presence of NO synthase inhibitor. Exogenous NO (0.3-1 microM), at which no hyperpolarization is produced, inhibited purinergic IJPs, without altering the nitrergic IJP and exogenously applied ATP-induced hyperpolarization. In the presence of both purinoceptor antagonist and nitric oxide synthase (NOS) inhibitor, intramural nerve stimulation with 5 pulses at 20 Hz evoked vasoactive intestinal peptide (VIP)-associated IJPs, suggesting that VIP component may be masked in the IJPs of the hamster proximal colon. Our results suggest that neurogenic NO may modulate the purinergic transmission to circular smooth muscles of the hamster proximal colon via a prejunctional mechanism. In addition, VIP may be involved in the neurotransmitter in the hamster proximal colon.

Although there is evidence that the Rho/Rho kinase pathway and protein kinase C (PKC) are involved in the development of cerebral vasospasm, the mechanism by which subarachnoid hemorrhage (SAH) activates these pathways is unclear. A large body of evidence points to oxyhemoglobin (OxyHb) as a major causative component of blood clot responsible for vasospasm. Therefore, the present studies were conducted to explore whether the Rho/Rho kinase and PKC may be involved in a sustained vasoconstriction induced by OxyHb in cerebral arteries. OxyHb evoked sustained vasoconstriction in the endothelium-denuded rabbit basilar arteries, which was reversed by the selective inhibitors of Rho kinase, Y-27632, and HA-1077, with the IC50 values of 0.26+/-0.02 and 0.74+/-0.1 micromol/L, respectively. In quiescent cerebrovascular smooth muscle (CVSM) cells, OxyHb induced Rho translocation, as assessed by immunoblotting, with a time course, which paralleled the contractile action of OxyHb. Rho translocation was also observed in intact arteries stimulated with OxyHb for 24 hours (219%) and 48 hours (160%). The increase in Rho translocation was fully inhibited by GGTI-297, an inhibitor of Rho prenylation. OxyHb also caused significant translocation of both PKCalpha and PKCepsilon (P<0.01), which was maximal at the time corresponding to maximal tension developed in response to OxyHb. Ro-32-0432, an inhibitor of PKC, attenuated vasoconstriction mediated by OxyHb in basilar artery. These results show, for the first time, that OxyHb-mediated signaling in CVSM utilizes the Rho/Rho kinase and PKC-based mechanisms.