Hydrogen Gas Inhalation Attenuates Endothelial Glycocalyx Damage and Stabilizes Hemodynamics in a Rat Hemorrhagic Shock Model.

BACKGROUND: Hydrogen gas (H2) inhalation during hemorrhage stabilizes post-resuscitation hemodynamics, improving short-term survival in a rat hemorrhagic shock and resuscitation (HS/R) model. However, the underlying molecular mechanism of H2 in HS/R is unclear. Endothelial glycocalyx (EG) damage causes hemodynamic failure associated with HS/R. In this study, we tested the hypothesis that H2 alleviates oxidative stress by suppressing xanthine oxidoreductase (XOR) and/or preventing tumor necrosis factor-alfa (TNF-α)-mediated syndecan-1 shedding during EG damage. METHODS: HS/R was induced in rats by reducing mean arterial pressure (MAP) to 35 mm Hg for 60 min followed by resuscitation. Rats inhaled oxygen or H2 + oxygen after achieving shock either in the presence or absence of an XOR inhibitor (XOR-I) for both the groups. In a second test, rats received oxygen alone or antitumor necrosis factor (TNF)-α monoclonal antibody with oxygen or H2. Two hours after resuscitation, XOR activity, purine metabolites, cytokines, syndecan-1 were measured and survival rates were assessed 6 h after resuscitation. RESULTS: H2 and XOR-I both suppressed MAP reduction and improved survival rates. H2 did not affect XOR activity and the therapeutic effects of XOR-I and H2 were additive. H2 suppressed plasma TNF-α and syndecan-1 expression; however, no additional H2 therapeutic effect was observed in the presence of anti-TNF-α monoclonal antibody. CONCLUSIONS: H2 inhalation after shock stabilized hemodynamics and improved survival rates in an HS/R model independent of XOR. The therapeutic action of H2 was partially mediated by inhibition of TNF-α-dependent syndecan-1 shedding.

Quantitative imaging mass spectroscopy reveals roles of heme oxygenase-2 for protecting against transhemispheric diaschisis in the brain ischemia.

Carbon monoxide-generating heme oxygenase-2 is expressed in neurons and plays a crucial role for regulating hypoxic vasodilation through mechanisms unlocking carbon monoxide-dependent inhibition of H2S-generating cystathionine ß-synthase expressed in astrocytes. This study aims to examine whether heme oxygenase-2 plays a protective role in mice against stroke. Focal ischemia was induced by middle cerebral artery occlusion. Regional differences in metabolites among ipsilateral and contralateral hemispheres were analysed by quantitative imaging mass spectrometry equipped with an image-processing platform to optimize comparison of local metabolite contents among different animals. Under normoxia, blood flow velocity in precapillary arterioles were significantly elevated in heme oxygenase-2-null mice vs controls, while metabolic intermediates of central carbon metabolism and glutamate synthesis were elevated in the brain of heme oxygenase-2-null mice, suggesting greater metabolic demands to induce hyperemia in these mice. In response to focal ischemia, heme oxygenase-2-null mice exhibited greater regions of ischemic core that coincide with notable decreases in energy metabolism in the contralateral hemisphere as well as in penumbra. In conclusion, these findings suggest that heme oxygenase-2 is involved in mechanisms by which not only protects against compromised energy metabolism of the ipsilateral hemisphere but also ameliorates transhemispheric diaschisis of the contralateral hemisphere in ischemic brain.

Neuroprotective Role of Astroglia in Parkinson Disease by Reducing Oxidative Stress Through Dopamine-Induced Activation of Pentose-Phosphate Pathway.

Oxidative stress plays an important role in the onset and progression of Parkinson disease. Although released dopamine at the synaptic terminal is mostly reabsorbed by dopaminergic neurons, some dopamine is presumably taken up by astroglia. This study examined the dopamine-induced astroglial protective function through the activation of the pentose-phosphate pathway (PPP) to reduce reactive oxygen species (ROS). In vitro experiments were performed using striatal neurons and cortical or striatal astroglia prepared from Sprague-Dawley rats or C57BL/6 mice. The rates of glucose phosphorylation in astroglia were evaluated using the [14C]deoxyglucose method. PPP activity was measured using [1-14C]glucose and [6-14C]glucose after acute (60 min) or chronic (15 hr) exposure to dopamine. ROS production was measured using 2',7'-dichlorodihydrofluorescein diacetate. The involvement of the Kelch-like ECH-associated protein 1 (Keap1) or nuclear factor-erythroid-2-related factor 2 (Nrf2) system was evaluated using Nrf2 gene knockout mice, immunohistochemistry, and quantitative reverse transcription polymerase chain reaction analysis for heme oxygenase-1. Acute exposure to dopamine elicited increases in astroglial glucose consumption with lactate release. PPP activity in astroglia was robustly enhanced independently of Na+-dependent monoamine transporters. In contrast, chronic exposure to dopamine induced moderate increases in PPP activity via the Keap1/Nrf2 system. ROS production from dopamine increased gradually over 12 hr. Dopamine induced neuronal cell damage that was prevented by coculturing with astroglia but not with Nrf2-deficient astroglia. Dopamine-enhanced astroglial PPP activity in both acute and chronic manners may possibly reduce neuronal oxidative stress.

Preclinical evaluation of heat-denatured [18F]FDG-labeled red blood cells for detecting splenic tissues with PET in rats.

INTRODUCTION: Heat-denatured 99mTc-labeled red blood cells (RBCs) are used for detecting splenic tissues with scintigraphy. The present study aimed to evaluate the feasibility of using heat-denatured [18F]fluorodeoxyglucose ([18F]FDG)-labeled RBCs in detecting splenic tissues using positron emission tomography (PET) in rats. METHODS: RBCs were washed with phosphate buffered saline, labeled with [18F]FDG at 38°C, and heat-denatured at 50°C for 15 min. In vitro stability was assessed by measuring extracellular radioactivity during the 0-180 min incubation at 37°C. Thin layer chromatography (TLC) of the extracellular fluid was performed. The autologous RBCs were intravenously injected in four rats and PET scanning was simultaneously performed for 30 min. Time-activity curves of several organs, including the spleen, were analyzed on the PET images. RESULTS: Labeling efficiency was 92%. Low levels of radioactivity were released from the labeled RBCs for 180 min. TLC revealed that 80% of the released radioactivity was due to [18F]FDG-6-phosphate. Whole body images showed strong uptake of heat-denatured [18F]FDG-labeled RBCs in the spleen soon after injection in all four rats. Time-activity curves revealed that the splenic uptake continued to increase for 30 min and the amount of radioactivity in the other organs, except the urinary bladder, decreased after the initial surge. CONCLUSIONS: Heat-denatured [18F]FDG-labeled RBCs are suitable spleen-specific agents for PET. This method is clinically relevant as an alternative for heat-denatured 99mTc-labeled RBC scintigraphy.

18F-FDG-labeled red blood cell PET for blood-pool imaging: preclinical evaluation in rats.

BACKGROUND: Red blood cells (RBCs) labeled with single-photon emitters have been clinically used for blood-pool imaging. Although some PET tracers have been introduced for blood-pool imaging, they have not yet been widely used. The present study investigated the feasibility of labeling RBCs with 18F-2-deoxy-2-fluoro-D-glucose (18F-FDG) for blood-pool imaging with PET. RBCs isolated from venous blood of rats were washed with glucose-free phosphate-buffered saline and labeled with 18F-FDG. To optimize labeling efficiency, the effects of glucose deprivation time and incubation (labeling) time with 18F-FDG were investigated. Post-labeling stability was assessed by calculating the release fraction of radioactivity and identifying the chemical forms of 18F in the released and intracellular components of 18F-FDG-labeled RBCs incubated in plasma. Just after intravenous injection of the optimized autologous 18F-FDG-labeled RBCs, dynamic PET scans were performed to evaluate in vivo imaging in normal rats and intraabdominal bleeding models (temporary and persistent bleeding). RESULTS: The optimal durations of glucose deprivation and incubation (labeling) with 18F-FDG were 60 and 30 min, respectively. As low as 10% of 18F was released as the form of 18F-FDG from 18F-FDG-labeled RBCs after a 60-min incubation. Dynamic PET images of normal rats showed strong persistence in the cardiovascular system for at least 120 min. In the intraabdominal bleeding models, 18F-FDG-labeled RBC PET visualized the extravascular blood clearly and revealed the dynamic changes of the extravascular radioactivity in the temporary and persistent bleeding. CONCLUSIONS: RBCs can be effectively labeled with 18F-FDG and used for blood-pool imaging with PET in rats.

Epidermal cell turnover across tight junctions based on Kelvin's tetrakaidecahedron cell shape.

In multicellular organisms, cells adopt various shapes, from flattened sheets of endothelium to dendritic neurons, that allow the cells to function effectively. Here, we elucidated the unique shape of cells in the cornified stratified epithelia of the mammalian epidermis that allows them to achieve homeostasis of the tight junction (TJ) barrier. Using intimate in vivo 3D imaging, we found that the basic shape of TJ-bearing cells is a flattened Kelvin's tetrakaidecahedron (f-TKD), an optimal shape for filling space. In vivo live imaging further elucidated the dynamic replacement of TJs on the edges of f-TKD cells that enables the TJ-bearing cells to translocate across the TJ barrier. We propose a spatiotemporal orchestration model of f-TKD cell turnover, where in the classic context of 'form follows function', cell shape provides a fundamental basis for the barrier homeostasis and physical strength of cornified stratified epithelia.

Visualization of in vivo metabolic flows reveals accelerated utilization of glucose and lactate in penumbra of ischemic heart.

Acute ischemia produces dynamic changes in labile metabolites. To capture snapshots of such acute metabolic changes, we utilized focused microwave treatment to fix metabolic flow in vivo in hearts of mice 10 min after ligation of the left anterior descending artery. The left ventricle was subdivided into short-axis serial slices and the metabolites were analyzed by capillary electrophoresis mass spectrometry and matrix-assisted laser desorption/ionization imaging mass spectrometry. These techniques allowed us to determine the fate of exogenously administered (13)C6-glucose and (13)C3-lactate. The penumbra regions, which are adjacent to the ischemic core, exhibited the greatest adenine nucleotide energy charge and an adenosine overflow extending from the ischemic core, which can cause ischemic hyperemia. Imaging analysis of metabolic pathway flows revealed that the penumbra executes accelerated glucose oxidation, with remaining lactate utilization for tricarboxylic acid cycle for energy compensation, suggesting unexpected metabolic interplays of the penumbra with the ischemic core and normoxic regions.

Cortical microcirculatory disturbance in the super acute phase of subarachnoid hemorrhage - In vivo analysis using two-photon laser scanning microscopy.

OBJECTIVE: Subarachnoid hemorrhage (SAH) causes cerebral ischemia and drastically worsens the clinical status at onset. However, the arterial flow is surprisingly well maintained on the cerebral surface. We investigated cortical microcirculatory changes in the super acute phase of SAH using two-photon laser scanning microscopy (TPLSM). METHODS: SAH was induced at the skull base in 10 mice using a prone endovascular perforation model. Before SAH, and 1, 2, 5, 10, 20, 30 and 60min after SAH, the cortical microcirculation was observed with TPLSM through a cranial window. Diameters of penetrating and precapillary arterioles were measured and red blood cell (RBC) velocities in precapillary arterioles were analyzed using a line-scan method after administration of Q-dot 655 nanocrystals. RESULTS: One minute after SAH, RBC velocity and flow in precapillary arterioles drastically decreased to <20% of the pre-SAH values, while penetrating and precapillary arterioles dilated significantly. Subsequently, the arterioles either dilated or constricted inconsistently for 60min with continual decreases in RBC velocity and flow in the arterioles, suggesting neurovascular dysfunction. CONCLUSION: SAH caused sudden worsening of the cortical arteriolar velocity and flow at onset. The neurovascular unit cannot function sufficiently to maintain cortical microcirculatory flow in the super acute phase of SAH.

Cystathionine ß-synthase and PGRMC1 as CO sensors.

Heme oxygenase (HO) is a mono-oxygenase utilizing heme and molecular oxygen (O2) as substrates to generate biliverdin-IXα and carbon monoxide (CO). HO-1 is inducible under stress conditions, while HO-2 is constitutive. A balance between heme and CO was shown to regulate cell death and survival in many experimental models. However, direct molecular targets to which CO binds to regulate cellular functions remained to be fully examined. We have revealed novel roles of CO-responsive proteins, cystathionine ß-synthase (CBS) and progesterone receptor membrane component 1 (PGRMC1), in regulating cellular functions. CBS possesses a prosthetic heme that allows CO binding to inhibit the enzyme activity and to regulate H2S generation and/or protein arginine methylation. On the other hand, in response to heme accumulation in cells, PGRMC1 forms a stable dimer through stacking interactions of two protruding heme molecules. Heme-mediated PGRMC1 dimerization is necessary to interact with EGF receptor and cytochromes P450 that determine cell proliferation and xenobiotic metabolism. Furthermore, CO interferes with PGRMC1 dimerization by dissociating the heme stacking, and thus results in modulation of cell responses. This article reviews the intriguing functions of these two proteins in response to inducible and constitutive levels of CO with their pathophysiological implications.

A possible role of microglia-derived nitric oxide by lipopolysaccharide in activation of astroglial pentose-phosphate pathway via the Keap1/Nrf2 system.

BACKGROUND: Toll-like receptor 4 (TLR4) plays a pivotal role in the pathophysiology of stroke-induced inflammation. Both astroglia and microglia express TLR4, and endogenous ligands produced in the ischemic brain induce inflammatory responses. Reactive oxygen species (ROS), nitric oxide (NO), and inflammatory cytokines produced by TLR4 activation play harmful roles in neuronal damage after stroke. Although astroglia exhibit pro-inflammatory responses upon TLR4 stimulation by lipopolysaccharide (LPS), they may also play cytoprotective roles via the activation of the pentose phosphate pathway (PPP), reducing oxidative stress by glutathione peroxidase. We investigated the mechanisms by which astroglia reduce oxidative stress via the activation of PPP, using TLR4 stimulation and hypoxia in concert with microglia. METHODS: In vitro experiments were performed using cells prepared from Sprague-Dawley rats. Coexisting microglia in the astroglial culture were chemically eliminated using L-leucine methyl ester (LME). Cells were exposed to LPS (0.01 µg/mL) or hypoxia (1 % O2) for 12-15 h. PPP activity was measured using [1-(14)C]glucose and [6-(14)C]glucose. ROS and NO production were measured using 2',7'-dichlorodihydrofluorescein diacetate and diaminofluorescein-FM diacetate, respectively. The involvement of nuclear factor-erythroid-2-related factor 2 (Nrf2), a cardinal transcriptional factor under stress conditions that regulates glucose 6-phosphate dehydrogenase, the rate-limiting enzyme of PPP, was evaluated using immunohistochemistry. RESULTS: Cultured astroglia exposed to LPS elicited 20 % increases in PPP flux, and these actions of astroglia appeared to involve Nrf2. However, the chemical depletion of coexisting microglia eliminated both increases in PPP and astroglial nuclear translocation of Nrf2. LPS induced ROS and NO production in the astroglial culture containing microglia but not in the microglia-depleted astroglial culture. LPS enhanced astroglial ROS production after glutathione depletion. U0126, an upstream inhibitor of mitogen-activated protein kinase, eliminated LPS-induced NO production, whereas ROS production was unaffected. U0126 also eliminated LPS-induced PPP activation in astroglial-microglial culture, indicating that microglia-derived NO mediated astroglial PPP activation. Hypoxia induced astroglial PPP activation independent of the microglia-NO pathway. Elimination of ROS and NO production by sulforaphane, a natural Nrf2 activator, confirmed the astroglial protective mechanism. CONCLUSIONS: Astroglia in concert with microglia may play a cytoprotective role for countering oxidative stress in stroke.

Leukocyte plugging and cortical capillary flow after subarachnoid hemorrhage.

BACKGROUND: It is believed that increased intracranial pressure immediately after subarachnoid hemorrhage (SAH) causes extensive brain ischemia and results in worsening clinical status. Arterial flow to the cerebral surfaces is clinically well maintained during clipping surgery regardless of the severity of the World Federation of Neurological Societies grade after SAH. To explore what kinds of changes occur in the cortical microcirculation, not at the cerebral surface, we examined cortical microcirculation after SAH using two-photon laser scanning microscopy (TPLSM). METHODS: SAH was induced in mice with an endovascular perforation model. Following continuous injection of rhodamine 6G, velocities of labeled platelets and leukocytes and unlabeled red blood cells (RBCs) were measured in the cortical capillaries 60 min after SAH with a line-scan method using TPLSM, and the data were compared to a sham group and P-selectin monoclonal antibody-treated group. RESULTS: Velocities of leukocytes, platelets, and RBCs in capillaries decreased significantly 60 min after SAH. Rolling and adherent leukocytes suddenly prevented other blood cells from flowing in the capillaries. Flowing blood cells also decreased significantly in each capillary after SAH. This no-reflow phenomenon induced by plugging leukocytes was often observed in the SAH group but not in the sham group. The decreased velocities of blood cells were reversed by pretreatment with the monoclonal antibody of P-selection, an adhesion molecule expressed on the surfaces of both endothelial cells and platelets. CONCLUSIONS: SAH caused sudden worsening of cortical microcirculation at the onset. Leukocyte plugging in capillaries is one of the reasons why cortical microcirculation is aggravated after SAH.

CO-CBS-H2 S Axis: From Vascular Mediator to Cancer Regulator.

CO is a gaseous mediator generated by HO. Our previous studies revealed that CO generated from inducible HO-1 or from constitutive HO-2 modulates function of different heme proteins or enzymes through binding to their prosthetic ferrous heme to alter their structures, regulating biological function of cells and organs. Such CO-directed target macromolecules include sGC and CBS. In the liver, CO serves as a sinusoidal dilator through its action on sGC in hepatic stellate cells, while the same gas accounts for vasoconstrictor that inhibits H2S generated by CO-sensitive CBS in astrocytes. Since molecular O2 is a substrate for HO, the latter mechanism contributes to hypoxic vasodilation in neurovascular units. We have recently uncovered that stress-inducible CO in and around cancer cells suppresses CBS to result in decreased methylation of PFKFB3, the enzyme regulating PFK-1, leading to a shift of glucose biotransformation from glycolysis toward pentose phosphate pathway; such a metabolic remodeling causes chemoresistance through increasing NADPH and reduced glutathione under stress conditions for cancer cells. This article reviews the intriguing networks of CO-sensitive metabolic regulatory mechanisms in microcirculation and cancer.

Therapeutic hypothermia achieves neuroprotection via a decrease in acetylcholine with a concurrent increase in carnitine in the neonatal hypoxia-ischemia.

Although therapeutic hypothermia is known to improve neurologic outcomes after perinatal cerebral hypoxia-ischemia, etiology remains unknown. To decipher the mechanisms whereby hypothermia regulates metabolic dynamics in different brain regions, we used a two-step approach: a metabolomics to target metabolic pathways responding to cooling, and a quantitative imaging mass spectrometry to reveal spatial alterations in targeted metabolites in the brain. Seven-day postnatal rats underwent the permanent ligation of the left common carotid artery followed by exposure to 8% O2 for 2.5 hours. The pups were returned to normoxic conditions at either 38 °C or 30 °C for 3 hours. The brain metabolic states were rapidly fixed using in situ freezing. The profiling of 107 metabolites showed that hypothermia diminishes the carbon biomass related to acetyl moieties, such as pyruvate and acetyl-CoA; conversely, it increases deacetylated metabolites, such as carnitine and choline. Quantitative imaging mass spectrometry demarcated that hypothermia diminishes the acetylcholine contents specifically in hippocampus and amygdala. Such decreases were associated with an inverse increase in carnitine in the same anatomic regions. These findings imply that hypothermia achieves its neuroprotective effects by mediating the cellular acetylation status through a coordinated suppression of acetyl-CoA, which resides in metabolic junctions of glycolysis, amino-acid catabolism, and ketolysis.

Large-area surface-enhanced Raman spectroscopy imaging of brain ischemia by gold nanoparticles grown on random nanoarrays of transparent boehmite.

Although SERS spectroscopy, which is sensitive to molecular vibration states, offers label-free visualization of molecules, identification of molecules and their reliable large-area imaging remains to be developed. Limitation comes from difficulties in fabricating a SERS-active substrate with homogeneity over a large area. Here, we overcome this hurdle by utilizing a self-assembled nanostructure of boehmite that is easily achieved by a hydrothermal preparation of aluminum as a template for subsequent gold (Au) deposition. This approach brought about random arrays of Au-nanostructures with a diameter of ∼125 nm and a spacing of <10 nm, ideal for the hot-spots formation. The substrate, which we named "gold nanocoral" (GNC) after its coral reef-like shape, exhibited a small variability of signal intensities (coefficient value <11.2%) in detecting rhodamine 6G molecule when 121 spots were measured over an area of 10 × 10 mm(2), confirming high uniformity. The transparent nature of boehmite enabled us to conduct the measurement from the back-side of the substrate as efficiently as that from the front-side. We then conducted tissue imaging using the mouse ischemic brain adhered on the GNC substrate. Through nontargeted construction of two-dimensional-Raman-intensity map using differential bands from two metabolically distinct regions, that is, ischemic core and contralateral-control areas, we found that mapping using the adenine ring vibration band at 736 cm(-1) clearly demarcated ischemic core where high-energy adenine phosphonucleotides were degraded as judged by imaging mass spectrometry. Such a detection capability makes the GNC-based SERS technology especially promising for revealing acute energy derangement of tissues.

Visualization and quantification of cerebral metabolic fluxes of glucose in awake mice.

Biotransformation of glucose in organs includes multiple pathways, while quantitative evaluation of percentages of its utilization for individual pathways and their spatial heterogeneity in vivo remain unknown. Imaging MS (IMS) and metabolomics combined with a focused microwave irradiation for rapidly fixing tissue metabolism allowed us to quantify and visualize metabolic fluxes of glucose-derived metabolites in the mouse brain in vivo. At 15 min after the intraperitoneal injection of (13) C6 -labeled glucose, the mouse brain was exposed to focused microwave irradiation, which can stop brain metabolism within 1 s. Quantification of metabolic intermediates containing (13) C atoms revealed that a majority of the (13) C6 -glucose was diverted into syntheses of glutamate, lactate, and uridine diphosphate (UDP)-glucose. IMS showed that regions rich in glutaminergic neurons exhibited a large signal of (13) C2 -labeled glutamate. On the other hand, the midbrain region was enriched with an intensive (13) C6 -labeled UDP-glucose signal, suggesting an active glycogen synthesis. Collectively, application of the current method makes it possible to examine the fluxes of glucose metabolism in a region-specific manner.

Cilostazol strengthens the endothelial barrier of postcapillary venules from the rat mesentery in situ.

OBJECTIVE: Although cilostazol, a phosphodiesterase 3 inhibitor, has been suggested to strengthen the endothelial barrier using cultured endothelial monolayers, its effect has not been tested in vivo. We, therefore, investigated effects of cilostazol on barrier properties of postcapillary venules of the rat in situ. METHODS: Cilostazol was administered to the rats through oral gavage at 4 hours before the measurements. The hydraulic permeability (L(p)) and the effective osmotic pressure (σΔπ), molecular sieving properties of microvascular walls, were estimated in single mesenteric postcapillary venules by a micro-occlusion technique, first during control perfusion and then in the presence of histamine. RESULTS: When the vessels were inflamed with histamine, cilostazol attenuated a transient increase in L(p) and prevented σΔπ from falling. Furthermore, it reduced baseline L(p) under a control state. CONCLUSION: Cilostazol appears to tighten the endothelial barrier in situ, at least in part by inhibiting the cAMP-degrading enzyme in the endothelium.

Gas biology: tiny molecules controlling metabolic systems.

It has been recognized that gaseous molecules and their signaling cascades play a vital role in alterations of metabolic systems in physiologic and pathologic conditions. Contrary to this awareness, detailed mechanisms whereby gases exert their actions, in particular in vivo, have been unclear because of several reasons. Gaseous signaling involves diverse reactions with metal centers of metalloproteins and thiol modification of cysteine residues of proteins. Both the multiplicity of gas targets and the technical limitations in accessing local gas concentrations make dissection of exact actions of any gas mediator a challenge. However, a series of advanced technologies now offer ways to explore gas-responsive regulatory processes in vivo. Imaging mass spectrometry combined with quantitative metabolomics by capillary-electrophoresis/mass spectrometry reveals spatio-temporal profiles of many metabolites. Comparing the metabolic footprinting of murine samples with a targeted deletion of a specific gas-producing enzyme makes it possible to determine sites of actions of the gas. In this review, we intend to elaborate on the ideas how small gaseous molecules interact with metabolic systems to control organ functions such as cerebral vascular tone and energy metabolism in vivo.

Carbon monoxide: impact on remethylation/transsulfuration metabolism and its pathophysiologic implications.

Carbon monoxide (CO) is a gaseous product generated by heme oxygenase (HO), which oxidatively degrades heme. While the stress-inducible HO-1 has well been recognized as an anti-oxidative defense mechanism under stress conditions, recent studies suggest that cancer cells utilize the reaction for their survival. HO-2, the constitutive isozyme, also plays protective roles as a tonic regulator for neurovascular function. Although protective roles of the enzyme reaction and CO have extensively been studied, little information is available on the molecular mechanisms by which the gas exerts its biological actions. Recent studies using metabolomics revealed that CO inhibits cystathionine ß-synthase (CBS), which generates H(2)S, another gaseous mediator. The CO-dependent CBS inhibition may impact on the remethylation cycle and related metabolic pathways including the methionine salvage pathway and polyamine synthesis. This review focuses on the gas-responsive regulation of metabolic systems, particularly the remethylation and transsulfuration pathways, and their putative implications for cancer and ischemic diseases.

Hypoxic regulation of the cerebral microcirculation is mediated by a carbon monoxide-sensitive hydrogen sulfide pathway.

Enhancement of cerebral blood flow by hypoxia is critical for brain function, but signaling systems underlying its regulation have been unclear. We report a pathway mediating hypoxia-induced cerebral vasodilation in studies monitoring vascular disposition in cerebellar slices and in intact mouse brains using two-photon intravital laser scanning microscopy. In this cascade, hypoxia elicits cerebral vasodilation via the coordinate actions of H(2)S formed by cystathionine ß-synthase (CBS) and CO generated by heme oxygenase (HO)-2. Hypoxia diminishes CO generation by HO-2, an oxygen sensor. The constitutive CO physiologically inhibits CBS, and hypoxia leads to increased levels of H(2)S that mediate the vasodilation of precapillary arterioles. Mice with targeted deletion of HO-2 or CBS display impaired vascular responses to hypoxia. Thus, in intact adult brain cerebral cortex of HO-2-null mice, imaging mass spectrometry reveals an impaired ability to maintain ATP levels on hypoxia.

Matrix-Assisted Laser Desorption/Ionization (MALDI) Imaging Mass Spectrometry (IMS): A Challenge for Reliable Quantitative Analyses.

Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is capable of determining the distribution of hundreds of molecules at once directly from tissue sections. Since tissues are analyzed intact without homogenization, spatial relationships of molecules are preserved. The technology is, therefore, undoubtedly powerful to investigate the molecular complexity of biological processes. However, several technical refinements are essential for full exploitation of MALDI-IMS to dictate dynamics alteration of biomolecules in situ; these include ways to collect tissues, target-specific tissue pretreatment, matrix choice for efficient ionization, and matrix deposition method to improve imaging resolution. Furthermore, for MALDI-IMS to reach its full potential, quantitative property in the IMS should be strengthened. We review the challenges and new approaches for optimal imaging of proteins, lipids and metabolites, highlighting a novel quantitative IMS of energy metabolites in the recent literature.