Adipose tissue hypoxia induces inflammatory M1 polarity of macrophages in an HIF-1α-dependent and HIF-1α-independent manner in obese mice.

AIMS/HYPOTHESIS: As obesity progresses, adipose tissue exhibits a hypoxic and inflammatory phenotype characterised by the infiltration of adipose tissue macrophages (ATMs). In this study, we examined how adipose tissue hypoxia is involved in the induction of the inflammatory M1 and anti-inflammatory M2 polarities of ATMs. METHODS: The hypoxic characteristics of ATMs were evaluated using flow cytometry after the injection of pimonidazole, a hypoxia probe, in normal-chow-fed or high-fat-fed mice. The expression of hypoxia-related and inflammation-related genes was then examined in M1/M2 ATMs and cultured macrophages. RESULTS: Pimonidazole uptake was greater in M1 ATMs than in M2 ATMs. This uptake was paralleled by the levels of inflammatory cytokines, such as TNF-α, IL-6 and IL-1ß. The expression level of hypoxia-related genes, as well as inflammation-related genes, was also higher in M1 ATMs than in M2 ATMs. The expression of Il6, Il1ß and Nos2 in cultured macrophages was increased by exposure to hypoxia in vitro but was markedly decreased by the gene deletion of Hif1a. In contrast, the expression of Tnf, another inflammatory cytokine gene, was neither increased by exposure to hypoxia nor affected by Hif1a deficiency. These results suggest that hypoxia induces the inflammatory phenotypes of macrophages via Hif1a-dependent and -independent mechanisms. On the other hand, the expression of inflammatory genes in cultured M2 macrophages treated with IL-4 responded poorly to hypoxia. CONCLUSIONS/INTERPRETATION: Adipose tissue hypoxia induces an inflammatory phenotype via Hif1a-dependent and Hif1a-independent mechanisms in M1 ATMs but not in M2 ATMs.

Allelic diversity of the MHC class II DRB genes in brown bears (Ursus arctos) and a comparison of DRB sequences within the family Ursidae.

The allelic diversity of the DRB locus in major histocompatibility complex (MHC) genes was analyzed in the brown bear (Ursus arctos) from the Hokkaido Island of Japan, Siberia, and Kodiak of Alaska. Nineteen alleles of the DRB exon 2 were identified from a total of 38 individuals of U. arctos and were highly polymorphic. Comparisons of non-synonymous and synonymous substitutions in the antigen-binding sites of deduced amino acid sequences indicated evidence for balancing selection on the bear DRB locus. The phylogenetic analysis of the DRB alleles among three genera (Ursus, Tremarctos, and Ailuropoda) in the family Ursidae revealed that DRB allelic lineages were not separated according to species. This strongly shows trans-species persistence of DRB alleles within the Ursidae.

Wiener filter-magnetoencephalography of visual cortical activity.

This paper reports a revised Wiener filter to resolve the inverse problem for magnetoencephalograms (MEGs) according to the structural and functional constraints based on magnetic resonance imaging (MRI) and functional magnetic resonance imaging (fMRI). Wiener filter-MEG imaging for half field stimulation with the chromatic stimulus resolved fast, slow and late responses in V1, V4 and the inferotemporal cortex, respectively. The time courses of these responses were roughly comparable with those reported by unit recording studies of the corresponding monkey visual cortical areas. Wiener filter-MEG imaging had comparable spatial resolution and better signal to noise ratio than fMRI. The background noise was robust in fMRI responses, but became virtually eliminated in Wiener filter responses. Wiener filter-MEG imaging with upper and lower quadrant field stimulation demonstrated V1 responses differentially distributed respectively in the lower and upper banks of the calcarine sulcus. These results demonstrate that responses in two cortical areas facing close to each other can be resolved by Wiener filter-MEG. The present method provides a way to image brain activities with millisecond- and millimeter-order spatiotemporal resolution.

Sensitivity to modulation of color distribution in multicolored textures.

We evaluated the discriminability of color distributions in square-element textures. Each texture contained 225 colors, represented by a distribution of color vectors in color space defined by the L-M and S-(L+M) axes. Each color distribution was systematically manipulated by modulating the distribution of the vector lengths sinusoidally as a function of the direction in the color space. The results showed that it is difficult to resolve a color distribution modulated in more than three cycles per 360 degrees in the chromatic direction. The difference in components along the cardinal axes is not a critical factor in the discrimination of the color distribution. An analysis using a line-element model suggested that the discrimination of the color distribution is mediated by multiple chromatic channels that are tuned to a variety of directions in the color space with a half-height-half-bandwidth of about 40 degrees in the chromatic direction.

Altered expression of heme oxygenase-1 in the livers of patients with portal hypertensive diseases.

This study was designed to determine changes in expression of heme oxygenase (HO)-1, the stress-inducible and carbon monoxide-producing enzyme, in normotensive and portal hypertensive human livers. GTS-1, a monoclonal antibody against rat HO-1 cross-reacted with the human HO-1 and blocked its enzyme activity, allowing us to examine the activity and localization of HO-1. In controls, approximately 50% of the total HO activity was from HO-1 as judged by the sensitivity to GTS-1, while the rest of activity was from other isozymes such as HO-2. HO-1 was expressed mainly in a subpopulation of Kupffer cells, and the expression in hepatic stellate cells, sinusoidal endothelial cells, and hepatocytes was little, if any. The HO-1 expression exhibited quite different pictures in the livers of portal hypertensive diseases. In cirrhotic livers, which undergo portal hypertension through increases in intrasinusoidal resistance and regenerative changes in the parenchyma, HO-1 occurred in a majority of Kupffer cells and was also observed in hepatocytes. Consequently, the total HO-1 activities became significantly greater in these tissues than those from normal individuals. By contrast, livers of idiopathic portal hypertension that are characterized by an increase in presinusoidal resistance displayed a significant decrease in the HO-1 expression in Kupffer cells, and its hepatocellular expression was not detectable. Although factors involved in altered HO-1 expression in these cells remain unknown, the results suggest that Kupffer cells could alter their expression of HO-1 in response to local hemodynamic changes associated with chronic portal hypertension in humans.

Cloning and characterization of the promoter of murine cytohesin-1 gene.

Mouse cytohesin-1 (CTH-1) is a guanine nucleotide exchange factor, specific for ADP ribosylation factors. The CTH-1 gene promoter was characterized by deletion mapping and mutational analysis. The region between -101 and -38 relative to the transcription start site showed the essential promoter activity when transfected into both NIH3T3 and COS7 cells. The nucleotides of the core promoter region contain three tandem GC boxes, which can offer potential binding sites for the ubiquitous transcription factor Sp-1 family. Mutational analysis revealed that these tandem GC boxes are indispensable for activation of the gene transcription.

Carbon monoxide overproduced by heme oxygenase-1 causes a reduction of vascular resistance in perfused rat liver.

This study aimed to examine whether livers overexpressing heme oxygenase (HO)-1 could alter the vascular resistance through the vasorelaxing action of carbon monoxide (CO). The relationship among HO-1 expression, CO generation, and the vascular resistance was assessed in perfused rat livers pretreated with hemin, an inducer of HO-1. At 18 h after the hemin treatment, livers displayed marked increases in HO-1 expression in hepatocytes and venous CO flux and a reduction of the basal resistance. The reduction of the resistance in hemin-treated livers was canceled by administration of oxyhemoglobin, a reagent trapping both CO and nitric oxide (NO), but not by methemoglobin, which captures NO but not CO. Liposome-encapsulated oxyhemoglobin, which cannot access the space of Disse, did not cause vasoconstriction. Furthermore, these livers became less sensitive to endothelin-1, a vasoconstrictive peptide, than the untreated controls through mechanisms involving CO. On the other hand, at 12 or 24 h after the treatment when the HO-1 induction was not accompanied by CO overproduction, neither a decrease in the basal resistance nor vascular hyporeactivity to endothelin-1 was observed. These results suggest that CO overproduced in the extrasinusoidal compartment is a determinant of the HO-1-mediated vasorelaxation in the liver.

Characterization of alpha 1-adrenoceptors expressed in a novel vascular smooth muscle cell line cloned from p53 knockout mice, P53LMAC01 (AC01) cells.

1. We pharmacologically studied the alpha 1-adrenoceptor (AR) subtype(s) involved in receptor-mediated signalling in a novel vascular smooth muscle cell line cloned from p53 knockout mice, P53LMAC01 (AC01) cells. 2. Radioligand binding studies with [125I]-HEAT showed the existence of a homogeneous population of binding site with an affinity (Kd value) of 0.4 nM and a maximum number of binding sites (Bmax) of 100 fmol mg-1 protein. Catecholamines competed for [125I]-HEAT binding stereospecifically and with the characteristic alpha 1-AR potency series. 3. Displacement curves for BMY-7378 and KMD-3213 best fitted a one-site model with a pKi value (-log10 (equilibrium inhibition constant)) of 6.06 and 7.07, respectively. 4. Reverse transcription-polymerase chain reaction (RT-PCR) assay detected alpha 1B- and alpha 1D-AR, but not alpha 1A-AR transcript. 5. Chlorethylclonidine (CEC) treatment nearly abolished (-)noradrenaline (NA) (10 microM)-induced inositol[1,4,5]trisphosphate (IP3) production, and BMY-7378 inhibited the response with a Ki value of 0.3 nM, which value was similar to that obtained in the cells expressing alpha 1D-AR. In both AC01 cells and cells expressing alpha 1D-AR, BMY-7378 protected alpha 1-ARs from CEC alkylation while it had little protective effect on CEC alkylation and NA-induced IP3 production in cells expressing alpha 1B-AR. 6. The results indicate that AC01 cells contain predominantly alpha 1B-ARs and a small population of alpha 1D-ARs; however, phosphoinositide (PI)/Ca2+ signalling is mainly mediated through the minor population of alpha 1D-ARs, rather than the alpha 1B-ARs.

Characterization of the mouse alpha1D-adrenergic receptor gene.

Alpha1-adrenergic receptors (alpha1-ARs) play critical roles in the regulation of a variety of physiological processes. Increasing evidence suggests that multiple receptor subtypes of alpha1-ARs regulate these physiological processes. Molecular cloning has identified three distinct cDNAs encoding alpha1-AR subtypes (alpha1A, alpha1B and alpha1D) that are structurally homologous. Among the alpha1-AR subtypes, the function of the alpha1D-AR remains unclear. In order to examine the physiological role of alpha1D-AR, we cloned and characterized a gene for the mouse alpha1D-AR. Using a mouse alpha1D-AR cDNA as a probe, we isolated the gene for the mouse alpha1D-AR from a mouse genomic library. The alpha1D-AR consists of two exons and an intron that interrupts the coding region of the putative sixth transmembrane domain. The 5'-flanking region of exon 1 contains neither a TATA box nor a CAAT box but is high in GC content and contains several Sp1 binding sites (GC boxes). This pattern is similar to promoters described for other members of alpha1-ARs. The untranslated region also contains putative cyclic AMP response elements. Isolation of this gene will allow further investigation, via gene knock-outs and deletion mutants, of the mechanisms of transcriptional regulation and a greater understanding of the physiological role of alpha1D-AR.

Structure and sequence of the mouse V1a and V1b vasopressin receptor genes.

The structural organization and 5'-flanking region of the mouse V1a and V1b vasopressin receptor genes were determined. The mouse V1a receptor gene was located within an 8-kb XbaI fragment, and the mouse V1b receptor gene was located within a 14-kb EcoRV fragment. Both genes were comprised of two coding exons that were separated by a 2.3-kb and a 8.0-kb intron, respectively, located before the respective seventh transmembrane domain of the receptor sequence. The availability of these genes would allow us to study the functional role of V1a and V1b receptors by disrupting the gene in mice.

Discontinuous total parenteral nutrition prevents postischemic mitochondrial dysfunction in rat liver.

Although discontinuous total parenteral nutrition (d-TPN) has recently been favored for clinical use over continuous total parenteral nutrition (c-TPN) to ameliorate liver dysfunction, mechanisms for the protection against postoperative liver dysfunction remain unknown. This study aimed to examine differences in mitochondrial function in d-TPN- and c-TPN-pretreated livers during ischemia-reperfusion. Rat livers pretreated with d-TPN or c-TPN were perfused with Krebs-Ringer buffer and were exposed to 25% low-flow hypoxia followed by reperfusion. Intrahepatic mitochondrial membrane potential (triangle up) and cell viability were assessed by dual-color digital microfluorography using rhodamine 123 (Rh123) and propidium iodide (PI), respectively. In response to hypoxia, livers pretreated with c-TPN, d-TPN, and an ordinary chow diet exhibited a significant triangle up reduction among the entire lobules. Upon reperfusion, the regional triangle up values further decreased in the c-TPN liver, whereas those in the d-TPN-treated or chow-treated livers displayed a rapid recovery toward the control levels. The severity of cell injury did not differ among the groups, showing that the reperfusion-induced triangle up drop in the c-TPN-pretreated liver is not a consequence of cell injury. Differences in the triangle up drop among the groups appear to occur irrespective of those in the glycogen storage, because the livers undergoing d-TPN display a marked triangle up recovery even when reperfused at the end of a fasted state. These results indicate that c-TPN, but not d-TPN, jeopardizes mitochondrial re-energization and suggest that a circadian pattern of the TPN serves as a potentially beneficial strategy to reduce the risk of postischemic mitochondrial dysfunction in the liver.

Carbon monoxide as a regulator of bile canalicular contractility in cultured rat hepatocytes.

This study aimed to examine the mechanism(s) by which carbon monoxide (CO), a product of heme oxygenase reaction, controls the contractility of bile canaliculus (BC) in hepatocytes. When BCs associated with the couplet cells in cultured rat hepatocyte suspension were observed using time-lapse video microscopy, they exhibited periodical contractions with a most-probable interval of 6 minutes under our experimental conditions. The addition of 1 micromol/L zinc protoporphyrin IX (ZnPP), a potent inhibitor of heme oxygenase, to the culture medium elicited a 40% shortening of the interval time together with an increase in intracellular calcium concentrations, while the same concentration of iron protoporphyrin IX did not induce such changes. The production of CO, which was 0.5 nmol/h/10(8) cells in the absence of ZnPP, diminished to less than 0.1 nmol/h/10(8) cells upon application of ZnPP. The ZnPP-elicited increases in both contractile frequency and intracellular calcium concentrations were attenuated by the addition of 1 micromol/L CO or 50 micromol/L 1,2-bis(2-aminophenoxy) ethane-tetraacetate, a calcium chelator. Clotrimazole or metyrapone, inhibitors of cytochrome P450-dependent monooxygenase activities, also attenuated the ZnPP-induced elevation of the contractile frequency. On the other hand, intracellular cyclic guanosine monophosphate (cGMP) contents were not altered significantly by the application of ZnPP or by CO. These results indicate that CO generated by heme oxygenase controls the BC function by changing intracellular calcium concentrations presumably through a mechanism involving the cytochrome P450 reaction.

Distribution of heme oxygenase isoforms in rat liver. Topographic basis for carbon monoxide-mediated microvascular relaxation.

Carbon monoxide (CO) derived from heme oxygenase has recently been shown to play a role in controlling hepatobiliary function, but intrahepatic distribution of the enzyme is unknown. We examined distribution of two kinds of the heme oxygenase isoforms (HO-1 and HO-2) in rat liver immunohistochemically using monoclonal antibodies. The results showed that distribution of the two isoforms had distinct topographic patterns: HO-1, an inducible isoform, was observed only in Kupffer cells, while HO-2, a constitutive form, distributed to parenchymal cells, but not to Kupffer cells. Both isoforms were undetectable in hepatic stellate cells and sinusoidal endothelial cells. Of the two isoforms, HO-2 in the parenchymal cell rather than HO-1 in the Kupffer cell, appears to play a major role in regulation of microvascular tone. In the perfused liver, administration of HbO2, a CO-trapping reagent that can diffuse across the fenestrated endothelium into the space of Disse, elicited a marked sinusoidal constriction, while administration of a liposome-encapsulated Hb that cannot enter the space had no effect on the microvascular tone. These results suggest that CO evolved by HO-2 in the parenchymal cells, and, released to the extrasinusoidal space, served as the physiological relaxant for hepatic sinusoids.

Endogenous carbon monoxide suppression stimulates bile acid-dependent biliary transport in perfused rat liver.

This study aimed to investigate whether carbon monoxide (CO), a product of heme oxygenase that degrades protoheme IX, serves as an endogenous modulator for biliary transport. To that end, effects of zinc protoporphyrin IX (ZnPP), a heme oxygenase inhibitor, on the biliary transport were tested in perfused rat liver. Perfusion of 1 microM ZnPP abolished detectable levels of CO in the venous perfusate and increased bile acid-dependent bile output accompanying an increased secretion of bile salts. The ZnPP-induced choleresis coincided with a reduction of tissue guanosine 3',5'-cyclic monophosphate (cGMP) levels and a decrease in vascular conductance. On administration of 2.5 microM CO, ZnPP-elicited choleresis, decreases in vascular conductance, and cGMP levels were all attenuated. Treatment with 1 microM 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) partly attenuated the ZnPP-induced choleresis in concert with repression of vascular conductance. Furthermore, treatment of the liver with methylene blue, a guanylate cyclase inhibitor, evoked a choleresis similar to that induced by ZnPP. Thus endogenous CO suppression stimulates the biliary transport in part through a cGMP-dependent mechanism.

Additive effect of luminance and color cues in generation of neon color spreading.

A series of experiments were designed to examine how luminance and color cues influence the occurrence of neon color spreading for the Ehrenstein pattern plus the cross pattern. The proportion of "see" responses for color spreading was obtained for different combinations of the luminance and color of the pattern components. The following were obtained. (1) Increase in the luminance of the inducing pattern and/or the color difference between the cross and the inducing pattern raised the proportion of "see" responses for color spreading. This implies that luminance and color signals additively contribute to the generation of the color spreading, but in different ways. (2) The "iso-spreading contours" for the generation of color spreading were determined on the two-dimensional isoluminant plane composed of the L-M and S-(L+M) axes. The contours were approximately described by rotated quadratic or ellipse functions. The additive interaction within the chromatic systems [the L-M system and the S-(L+M) system] was less significant than that across the luminance and chromatic systems. (3) The pattern of the experimental results could not be explained straightforwardly by existing models.

Moving stimuli define the shape of stationary chromatic patterns.

A study is reported of phenomena involved in perceptually unified organisation of a stationary chromatic pattern and a moving black outline or dot pattern. When the corners of the outline pattern were temporally oscillated on a stationary chromatic square, the chromatic border appeared to follow the moving outline, as if captured by it. This capture effect was also observed with moving dots: the chromatic border was defined by an imaginary line connecting the moving dots. Both capture effects occur over a region that becomes wider with increasing velocity of the oscillation. These observations suggest that the visual system effectively uses information from moving features to define the shape of overlapping chromatic image regions.

Modulation of mitochondrion-mediated oxidative stress by nitric oxide in human placental trophoblastic cells.

Intracellular hydroperoxide generation in cultured human placental trophoblastic cells (HPTCs) was quantitatively monitored in the presence or absence of an NO synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME, 1 mM), by digital microfluorography with use of carboxydichlorofluorescein, a hydroperoxide-sensitive fluorogenic probe. In the absence of L-NAME, HPTCs displayed a time-dependent gradual elevation of the fluorescence, suggesting the ability to produce oxidants spontaneously. In the presence of L-NAME, however, the fluorescent response in these cells increased further; the oxidative impact elicited by L-NAME treatment for 30 min was equivalent to that induced by application of 230 microM tert-butyl hydroperoxide for 5 min. This oxidative process was completely blocked by rotenone, a reagent that interferes with electron entry into complex I of the mitochondrial respiratory chain. On the other hand, antimycin A, which blocks mitochondria at the distal site of the ubiquinone pool, potentiated the L-NAME-induced oxidative change. These findings suggest that constitutive levels of nitric oxide production contribute to regulation of mitochondrion-derived intracellular oxidant generation in HPTCs.

Carbon monoxide: an endogenous modulator of sinusoidal tone in the perfused rat liver.

Heme oxygenase is a heme-oxidizing enzyme which generates biliverdin and carbon monoxide (CO). The present study was designed to elucidate whether CO endogenously produced by this enzyme serves as an active vasorelaxant in the hepatic microcirculation. Microvasculature of the isolated perfused rat liver was visualized by dual-color digital microfluorography to alternately monitor sinusoidal lining and fat-storing Ito cells. In the control liver, the CO flux in the venous effluent ranged at 0.7 nmol/min per gram of liver. Administration of a heme oxygenase inhibitor zinc protoporphyrin IX (1 microM) eliminated the baseline CO generation, and the vascular resistance exhibited a 30% elevation concurrent with discrete patterns of constriction in sinusoids and reduction of the sinusoidal perfusion velocity. The major sites of the constriction corresponded to local sinusoidal segments colocalized with Ito cell which were identified by imaging their vitamin A autofluorescence. The increase in the vascular resistance and sinusoidal constriction were attenuated significantly by adding CO (1 microM) or a cGMP analogue 8-bromo-cGMP (1 microM) in the perfusate. From these findings, we propose that CO can function as an endogenous modulator of hepatic sinusoidal perfusion through a relaxing mechanism involving Ito cells.

Carbon monoxide as an endogenous modulator of hepatic vascular perfusion.

Carbon monoxide (CO) generated by heme oxygenase has recently been considered a neural messenger in brain. This observation prompted us to investigate whether CO participates in vascular regulation in the liver, another organ with high levels of heme oxygenase activity. In isolated perfused rat liver, submicromolar levels of CO were detectable in the effluent and were able to be suppressed by the administration of Zn protoporphyrin IX (1 microM), a potent inhibitor of heme oxygenase. Furthermore, zinc protoporphyrin IX (1 microM) promoted an increase in the perfusion pressure under the constant flow conditions. These changes were reversed by adding CO (2 microM) or a cGMP analogue 8-bromo-cGMP (1 microM) in the perfusate. The present findings indicate that CO can function as an endogenous modulator of vascular perfusion in the liver.