Mayumi Kajimura

Hypoxia-Sensitive Fluorescent Probes for in Vivo Real-Time Fluorescence Imaging of Acute Ischemia

Based on the findings that the azo functional group has excellent properties as the hypoxia-sensor moiety, we developed hypoxia-sensitive near-infrared fluorescent probes in which a large fluorescence increase is triggered by the cleavage of an azo bond. The probes were used for fluorescence imaging of hypoxic cells and real-time monitoring of ischemia in the liver and kidney of live mice.

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 H2S formed by cystathionine β-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 H2S 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.

Nonendothelial Source of Nitric Oxide in Arterioles But Not in Venules. Alternative Source Revealed In Vivo by Diaminofluorescein Microfluorography

Abstract— This study aimed to examine topographic distribution of microvascular NO generation in vivo. To this end, nitrosonium ion (NO+)–sensitive diaminofluorescein diacetate was superfused continuously on the rat mesentery and the fluorescence was visualized in the microvessels through laser confocal microfluorography. Two major sites exhibited a time-dependent elevation of the fluorescence: microvascular endothelia and mast cells. As judged by the fluorescence sensitivity to local application of different inhibitors of NO synthase (NOS), NO availability in arteriolar endothelium and mast cells appeared to be maintained mainly by NOS1, whereas that in venular endothelium greatly depends on NOS3. In venules, the magnitude of inhibitory responses elicited by the inhibitors was positively correlated with the density of leukocyte adhesion. NOS inhibitors significantly reduced, but did not eliminate, the NO+-associated fluorescence in arterioles, capillaries, and venules, suggesting alternative sources of NO in circulation for these microvessels. Immunohistochemistry for NOS isozymes revealed that NOS1 occurred not only in nerve fibers innervated to arterioles but also abundantly in mast cells. Laser flow cytometry of peritoneal cells in vitro revealed abundant expression of NOS1 in mast cells. Interestingly, NOS3 occurred in endothelia of capillaries and venules but not in those of distal arterioles with comparable diameters. These results suggest that the arterioles receive NO from nonendothelial origins involving NOS1 present in nerve terminals and mast cells, whereas venules depend on the endothelial NOS as a major source. Furthermore, nonenzymatic sources of NO from circulating reservoirs constitute a notable fraction throughout different classes of microvessels. The full text of this article is available at http://www.circresaha.org.

Cystathionine β-synthase as a carbon monoxide–sensitive regulator of bile excretion†

Carbon monoxide (CO) is a stress‐inducible gas generated by heme oxygenase (HO) eliciting adaptive responses against toxicants; however, mechanisms for its reception remain unknown. Serendipitous observation in metabolome analysis in CO‐overproducing livers suggested roles of cystathionine β‐synthase (CBS) that rate‐limits transsulfuration pathway and H2S generation, for the gas‐responsive receptor. Studies using recombinant CBS indicated that CO binds to the prosthetic heme, stabilizing 6‐coordinated CO‐Fe(II)‐histidine complex to block the activity, whereas nitric oxide (NO) forms 5‐coordinated structure without inhibiting it. The CO‐overproducing livers down‐regulated H2S to stimulate HCO3−‐dependent choleresis: these responses were attenuated by blocking HO or by donating H2S. Livers of heterozygous CBS knockout mice neither down‐regulated H2S nor exhibited the choleresis while overproducing CO. In the mouse model of estradiol‐induced cholestasis, CO overproduction by inducing HO‐1 significantly improved the bile output through stimulating HCO3− excretion; such a choleretic response did not occur in the knockout mice. Conclusion: Results collected from metabolome analyses suggested that CBS serves as a CO‐sensitive modulator of H2S to support biliary excretion, shedding light on a putative role of the enzyme for stress‐elicited adaptive response against bile‐dependent detoxification processes. (HEPATOLOGY 2009;49:141‐150.)

Paradoxical ATP Elevation in Ischemic Penumbra Revealed by Quantitative Imaging Mass Spectrometry

Local responses of energy metabolism during brain ischemia are too heterogeneous to decipher redox distribution between anoxic core and adjacent salvageable regions such as penumbra. Imaging mass spectrometry combined by capillary electrophoresis/mass spectrometry providing quantitative metabolomics revealed spatio-temporal changes in adenylates and NADH in a mouse middle-cerebral artery occlusion model. Unlike the core where ATP decreased, the penumbra displayed paradoxical elevation of ATP despite the constrained blood supply. It is noteworthy that the NADH elevation in the ischemic region is clearly demarcated by the ATP-depleting core. Results suggest that metabolism in ischemic penumbra does not respond passively to compromised circulation, but actively compensates energy charges.

Carbon Monoxide From Heme Oxygenase-2 Is a Tonic Regulator Against NO-Dependent Vasodilatation in the Adult Rat Cerebral Microcirculation

Although the brain generates NO and carbon monoxide (CO), it is unknown how these gases and their enzyme systems interact with each other to regulate cerebrovascular function. We examined whether CO produced by heme oxygenase (HO) modulates generation and action of constitutive NO in the rat pial microcirculation. Immunohistochemical analyses indicated that HO-2 occurred in neurons and arachnoid trabecular cells, where NO synthase 1 (NOS1) was detectable, and also in vascular endothelium–expressing NOS3, suggesting colocalization of CO- and NO-generating sites. Intravital microscopy using a closed cranial window preparation revealed that blockade of the HO activity by zinc protoporphyrin IX significantly dilates arterioles. This vasodilatation depended on local NOS activities and was abolished by CO supplementation, suggesting that the gas derived from HO-2 tonically regulates NO-mediated vasodilatory response. Bioimaging of NO by laser-confocal microfluorography of diaminofluorescein indicated detectable amounts of NO at the microvascular wall, the subdural mesothelial cells, and arachnoid trabecular cells, which express NOS in and around the pial microvasculature. On CO inhibition by the HO inhibitor, regional NO formation was augmented in these cells. Such a pattern of accelerated NO formation depended on NOS activities and was again attenuated by the local CO supplementation. Studies using cultured porcine aortic endothelial cells suggested that the inhibitory action of CO on NOS could result from the photo-reversible gas binding to the prosthetic heme. Collectively, CO derived from HO-2 appears to serve as a tonic vasoregulator antagonizing NO-mediated vasodilatation in the rat cerebral microcirculation.

Visualization of gaseous monoxide reception by soluble guanylate cyclase in the rat retina

Immunohistochemistry using novel monoclonal antibodies (mAbs) allowed us to uncover tissue activities of soluble guanylate cyclase (sGC) fine tuned by NO and CO. Upon NO and CO applications in vitro, purified sGC increased the affinity to mAb3221 by 100‐ and 10‐fold, respectively, but not to mAb28131. Immunohistochemistry for gas‐generating enzymes revealed that NO occurred in amacrine, bipolar, and Müllers glia cells (MGCs), whereas CO was derived mostly from heme oxygenase (HO)‐2 in MGCs. Basal sGC immunoreactivities in vivo to mAb3221 but not to mAb28131 were enhanced by injecting L‐arginine and attenuated by blocking NO synthases, suggesting the ability of the former mAb to sense NO. Comparison of mAb‐assisted immunohistochemistry suggested that sGC activities were enhanced by zinc protoporphyrin‐IX, an HO inhibitor, and repressed completely by blocking NO. However, suggested roles of CO played in situ varied among different retinal layers. In inner plexiform and inner nuclear layers located in the proximity of the cellular NO sources, CO serves as a simple inhibitor of local sGC, while playing roles in housekeeping sGC activation in external limiting membrane standing far from them. These results suggest that CO generated in MGCs is a diffusible gas mediator regulating sGC in both autocrine and paracrine manners.

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 13C6‐labeled glucose, the mouse brain was exposed to focused microwave irradiation, which can stop brain metabolism within 1 s. Quantification of metabolic intermediates containing 13C atoms revealed that a majority of the 13C6‐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 13C2‐labeled glutamate. On the other hand, the midbrain region was enriched with an intensive 13C6‐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.

Organ Design for Generation and Reception of CO: Lessons from the Liver

Carbon monoxide (CO) is synthesized in vivo by heme oxygenase. Although for many years CO had been regarded as potentially toxic waste, recent studies have indicated that it is a signaling molecule with important physiological functions. Nitric oxide (NO), another diatomic diffusible gas, is regarded as an established signaling molecule. Structural similarities between CO and NO have led many investigators to draw analogies between the two gaseous mediators. Whereas the NO signaling system has been well defined as to its receptor molecule, soluble guanylate cyclase, the CO system has been conceived to require further tuning with respect to identifying its receptor molecules and its downstream effectors. Furthermore, there has been little quantitative information to argue for a physiological role of CO in vivo. This review, therefore, focuses on recent developments on both physiologic and pathophysiologic roles of CO in the model of isolated perfused liver of rats where endogenous production of CO is actually estimated. This model has revealed that CO acts as an endogenous vasorelaxant in the liver and that effects of CO are at least in part cyclic GMP-dependent. It has also provided answers to many questions of hepatobiliary functions that had not been resolved because of the complexity introduced by the interplay between NO and CO.

Kupffer cells alter organic anion transport through multidrug resistance protein 2 in the post–cold ischemic rat liver

Although Kupffer cells (KCs) may play a crucial role in post–cold ischemic hepatocellular injury, their role in nonnecrotic graft dysfunction remains unknown. This study examined reveal the role of KC in post–cold ischemic liver grafts. Rat livers treated with or without liposome‐encapsulated dichloromethylene diphosphonate, a KC‐depleting reagent, were stored in University of Wisconsin (UW) solution at 4°C for 8 to 24 hours and reperfused while monitoring biliary output and constituents. The ability of hepatocytes to excrete bile was assessed through laser‐confocal microfluorography in situ. Cold ischemia‐reperfused grafts decreased their bile output significantly at 8 hours without any notable cell injury. This event coincided with impaired excretion of glutathione and bilirubin‐IXα (BR‐IXα), suggesting delayed transport of these organic anions. We examined whether intracellular relocalization of multidrug resistance protein‐2 (Mrp2) occurred. Kinetic analyses for biliary excretion of carboxyfluorescein, a fluoroprobe excreted through this transporter, revealed significant delay of dye excretion from hepatocytes into bile canaliculi. The KC‐depleting treatment significantly attenuated this decline in biliary anion transport mediated through Mrp2 in the 8‐hour cold ischemic grafts via redistribution of Mrp2 from the cytoplasm to the canalicular membrane. Furthermore, thromboxane A2 (TXA2) synthase in KC appeared involved as blocking this enzyme improved 5‐carboxyfluorescein excretion while cytoplasmic internalization of Mrp2 disappeared in the KC‐depleting grafts. In conclusion, KC activation is an important determinant of nonnecrotic hepatocellular dysfunction, jeopardizing homeostasis of the detoxification capacity and organic anion metabolism of the post–cold ischemic grafts. (HEPATOLOGY 2004;39;1099–1109.)