Naosada Takizawa

Nitrosyl hemoglobin in blood of normoxic and hypoxic sheep during nitric oxide inhalation

During nitric oxide (NO) inhalation therapy, NO combines with deoxyhemoglobin to form nitrosyl hemoglobin (HbNO). We used electron spin resonance (ESR) spectroscopy to measure HbNO in arterial and mixed venous blood of normoxic and hypoxic sheep during NO inhalation. Our aim was to quantitatively measure HbNO levels in the blood during NO inhalation, because large amounts of HbNO reduce the oxygen capacity of blood, particularly in hypoxia. Another aim was to investigate the transfer of exogenous NO to the alpha-heme iron of hemoglobin. Thirteen sheep were anesthetized with pentobarbital sodium, and 60 parts per million (ppm) NO were administered for 1 h in the presence of normoxia and hypoxia. Two-way analysis of variance revealed that the HbNO level was dependent on the oxygen level (normoxia vs. hypoxia) and NO inhalation, and there was a significant negative correlation between the HbNO level and arterial O2 saturation (SaO2). Although the HbNO level increased during NO inhalation in hypoxia, the HbNO level at SaO2 > 60% was < 11 mumol/l monomer hemoglobin (0.11% of total 10 mmol/l monomer hemoglobin). The peak of the HbNO ESR spectrum in arterial blood is located in almost the same position in mixed venous blood with an asymmetric HbNO signal, indicating that the NO in beta-heme HbNO molecules had been transferred to alpha-heme molecules. The three-line hyperfine structure of HbNO on ESR spectra was distinct in venous blood in hypoxia during NO inhalation, indicating pentacoordinate alpha-NO heme formation in hypoxic blood. In conclusion, the amount of HbNO during 60 ppm NO inhalation did not considerably reduce the oxygen capacity of the blood even in the presence of hypoxia, and the NO of HbNO was transferred to the alpha-heme iron of hemoglobin, forming pentacoordinate alpha-NO heme in mixed venous blood in hypoxia.During nitric oxide (NO) inhalation therapy, NO combines with deoxyhemoglobin to form nitrosyl hemoglobin (HbNO). We used electron spin resonance (ESR) spectroscopy to measure HbNO in arterial and mixed venous blood of normoxic and hypoxic sheep during NO inhalation. Our aim was to quantitatively measure HbNO levels in the blood during NO inhalation, because large amounts of HbNO reduce the oxygen capacity of blood, particularly in hypoxia. Another aim was to investigate the transfer of exogenous NO to the α-heme iron of hemoglobin. Thirteen sheep were anesthetized with pentobarbital sodium, and 60 parts per million (ppm) NO were administered for 1 h in the presence of normoxia and hypoxia. Two-way analysis of variance revealed that the HbNO level was dependent on the oxygen level (normoxia vs. hypoxia) and NO inhalation, and there was a significant negative correlation between the HbNO level and arterial O2 saturation ([Formula: see text]). Although the HbNO level increased during NO inhalation in hypoxia, the HbNO level at[Formula: see text] >60% was <11 μmol/l monomer hemoglobin (0.11% of total 10 mmol/l monomer hemoglobin). The peak of the HbNO ESR spectrum in arterial blood is located in almost the same position in mixed venous blood with an asymmetric HbNO signal, indicating that the NO in β-heme HbNO molecules had been transferred to α-heme molecules. The three-line hyperfine structure of HbNO on ESR spectra was distinct in venous blood in hypoxia during NO inhalation, indicating pentacoordinate α-NO heme formation in hypoxic blood. In conclusion, the amount of HbNO during 60 ppm NO inhalation did not considerably reduce the oxygen capacity of the blood even in the presence of hypoxia, and the NO of HbNO was transferred to the α-heme iron of hemoglobin, forming pentacoordinate α-NO heme in mixed venous blood in hypoxia.

Imaging of Oxygen Transfer Among Microvessels of Rat Cremaster Muscle

Background—The proximity of capillaries, arterioles, and venules provides complex spatial relationships that lead to oxygen transfer among microvessels. Although a conceptual image of complex oxygen transfer among microvessels has been hypothesized, in vivo mapping of oxygen saturation (So2) levels in microvessels had never been performed. Methods and Results—The oxygen profile of the arterioles and venules of the rat cremaster muscle during normoxia and hypoxia was visualized by preparing pseudo-color images of So2 levels based on microspectrophotometry data obtained by using 3 different optical filters and a cooled CCD camera. The So2 images showed lower So2 levels in arterioles close to their walls, and the So2 levels in the paired venules showed higher So2 levels close to the arterioles. There were capillaries that crossed the microvessels whose So2 levels changed as they crossed the microvessels. The So2 levels were lower close to the vessel wall than in the centerline level of the microvessels, and the highest So2 levels in venules paralleling arterioles were skewed toward the arterial side. The So2 images showed that the So2 level in arterioles decreased after crossing venules, whereas the So2 level in venules increased after crossing arterioles. Conclusions—Visualization of intravascular So2 levels suggested that oxygen is transferred between paired microvessels and between crossing microvessels in rat cremaster muscle. The possibility that oxygen is transported from some arterioles to venules and tissue through adjacent capillaries is proposed.

Biphasic changes in tissue partial pressure of oxygen closely related to localized neural activity in guinea pig auditory cortex.

An understanding of the local changes in cerebral oxygen content accompanying functional brain activation is critical for making a valid signal interpretation of hemodynamic-based functional brain imaging. However, spatiotemporal relations between changes in tissue partial pressure of oxygen (Po2) and induced neural activity remain incompletely understood. To characterize the local Po2 response to the given neural activity, the authors simultaneously measured tissue Po2 and neural activity in the identical region of guinea pig auditory cortex with an oxygen microelectrode (tip < 10 μm) and optical recording with voltage-sensitive dye (RH 795). In addition, a laser displacement gauge and a laser-Doppler flowmeter were used to monitor the spatial displacement and regional cerebral blood flow, respectively, in the Po2 measurement region. In the activated region, tissue Po2 initially decreased during the ∼3seconds after the onset of acoustic stimuli, and then increased during the next ∼5 seconds. Such biphasic changes are consistently found in cortical layers I to IV. In addition, amplitude of the biphasic change was closely related to detected peak height of the optical signal changes. The results suggest that the initial decrease in tissue Po2 is coupled to the induced neural activity and depends on response time of local increase in cerebral blood flow.

Successive depth variations in microvascular distribution of rat somatosensory cortex

Although hemodynamic-based functional brain imaging techniques are powerful tools to explore the brain functions noninvasively, hemodynamic-based signal is strongly affected by spatial configuration of microvessels. Understanding the quantitative relations between microvascular structure and functional activity is therefore significant to make a valid signal interpretation for the imaging techniques. In the present study, we evaluated depth profiles of microvascular distributions in rat somatosensory subfields (barrel field, forelimb region, trunk region and hindlimb region) and characterized depth variations in microvascular structures, such as locations, lengths and directions of microvessels, throughout the cortical layers (I-VI). To obtain the accurate microvascular structure, we made a customized casting method by using confocal laser scanning microscope. We observed that microvascular distribution successively varied throughout the cortical layers (I-VI) and that the maximum number density of microvessels was consistently found in middle layers (III-V). In addition, superficial layers had relatively long microvessels, almost perpendicular to the cortical surface, whereas middle layers had short microvessels propagating in all directions. These regional differences in microvascular structures were closely related to the somatosensory subfields, e.g., barrel field was the greatest number density of microvessels among the investigated subfields. Based on these observations, we compared microvascular profiles with previously reported distribution patterns of tissue partial pressure of oxygen (pO2). The results showed that tissue pO2 was correlated with microvascular distribution in some but not all of the subfields. This finding shows that detailed microvascular profiles are helpful to investigate causal relationships between microvascular structure and functional activities in cerebral cortex.

Dual responses of tissue partial pressure of oxygen after functional stimulation in rat somatosensory cortex

To compare the spatial heterogeneity of brain tissue partial pressure of oxygen (pO(2)) among local brain regions, we focused on functional and anatomical variations in rat somatosensory cortex. Tissue pO(2) was measured by using an oxygen microelectrode with high spatio-temporal resolution, and investigated in three somatosensory areas including hindlimb (HL), forelimb (FL), and trunk region (Tr). Their anatomical structures were determined with histological techniques (Nissl stain). In addition to the measurement of baseline tissue pO(2), we examined temporal shifts in tissue pO(2) distribution elicited by functional stimulation using the brushing stimulation to the hindlimb, forelimb, and trunk regions of the body. We observed that average tissue pO(2) in the Tr (14+/-10 Torr) was significantly lower than those in the HL (25+/-13 Torr) and FL (24+/-13 Torr). Such regional differences in tissue pO(2) were closely related to the cytoarchitectonic variations among these three areas. In addition, the functional stimulation enlarged the regional differences in the pO(2) depending on each somatosensory area; the pO(2) in the HL increased by 3.6+/-2.9% after the stimulation to hindlimb, whereas that in the Tr decreased by -2.9+/-2.5% after the stimulation to trunk region. Such dual responses of tissue pO(2) (i.e. increase or decrease) after the functional stimulation to the corresponding body regions may provide a criterion to clinically predict regions susceptible to tissue hypoxia, because considerable decrease in tissue pO(2) occurred in the Tr showing the lowest baseline pO(2).

Evidence for heteromorphic sex chromosomes in males of Rana tagoi and Rana sakuraii in Nishitama district of Tokyo (Anura: Ranidae).

Karyotypes of the Tago brown frog Rana tagoi and stream Tago brown frog Rana sakuraii from a mountain region in the Nishitama district in Tokyo were examined by conventional Giemsa staining, C-banding and late replication (LR)-banding. Chromosome number was 2n = 26 in all cases. The 26 chromosomes consisted of five (1–5) pairs of large chromosomes and eight (6–13) pairs of small chromosomes. Chromosome 10 had a secondary constriction on the long arm. In all frogs, on chromosome pair 8, the XX/XY type sex chromosome was present. C-banding analysis indicated that, in R. sakuraii, neither the X nor Y chromosome possessed interstitial C-bands but each had centromere staining, while in R. tagoi, an interstitial C-band was present on the long arm of the X chromosome. The Y chromosome had no interstitial C-band. LR-banding analysis demonstrated the X and Y chromosomes to have a LR-band on the short arm and two LR-bands, each on the long arm, and the bands on both species to be essentially the same. Heteromorphic sex chromosomes in males of R. sakuraii and R. tagoi were identified for the first time in this study.

Intravascular inhomogeneous oxygen distribution in microvessels: theory

Cross-sectional oxygen distribution in microvessels in most previous studies has been assumed to be homogeneous. Recent studies using phosphorescence quenching microscopy or microspectrophotometry showed a decline in oxygen profile near the arterial wall. In this study we performed theoretical analysis of intravascular P(O(2)) and S(O(2)) profiles in arterioles by using a radial diffusion model with a constant oxygen efflux from the vascular lumen, taking intravascular flow distribution into account. Theoretical calculations indicated that radial oxygen diffusion and a laminar flow pattern would create inhomogeneous intravascular oxygen profile with a decline toward the arterial wall. As mean blood flow velocity became lower, the difference between the centerline oxygen level and the inner surface level became larger. In conclusion, it is suggested that oxygen efflux from the vascular lumen and less convective supply near the vascular wall create a decline in P(O(2)) as well as S(O(2)) toward the arterial wall.

Heat production during early development of frog egg

Abstract The production of heat in the fertilized eggs, during the early development of the Japanese pond frog, Rana brevipoda porosa, was measured using a pyroelectric detector constructed with a polyvinylidene fluoride film. One and 2 μW of heat production was detected during the cleavage periods in the embryogenesis of the 2-cell embryo and the 4-cell embryo, respectively. The heat production increased stepwise with the cleavage in the embryogenesis of each stage. In comparison with the heat production during the inter-cleavage periods, a larger amount was noted at the cleavage periods in the two embryonic stages.

Multiple molecular forms of cytochrome P-450SCC purified from bovine corpus luteum mitochondria

Cytochrome P-450 related to side-chain cleavage of cholesterol (P-450SCC) was isolated from bovine corpus luteum mitochondria in the form of its stable cholesterol complex. The isolation procedure included ammonium sulfate fractionation and chromatography on omega-aminohexyl-Sepharose (AH-Sepharose). Corpus luteum P-450SCC was resolved into one minor (AH-I) and two major (AH-II and AH-III) fractions by the chromatography. Results of re-chromatography suggested the possibility that AH-III Fraction was originally complexed with lipidic material. The two major fractions purified by the re-chromatography (AH-IIR and AH-IIIR Fractions) showed essentially a single band on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and their absorption spectra were indistinguishable from each other. Both fractions were further resolved into two major and some minor bands of P-450SCC by isoelectric focusing on polyacrylamide gel in the presence of a non-ionic detergent, as detected by protein staining, heme staining and immunoblot analysis with anti-bovine P-450SCC monoclonal antibody. Both AH-IIR and AH-IIIR Fractions were further resolved by high-performance liquid chromatography (HPLC) on SP-TSK gel column into two fractions, SP-I and SP-II. These fractions had the same N-terminal amino acid sequence, showed similar catalytic activity and resolved into one major and a few minor bands on isoelectric focusing on polyacrylamide gel. Much more heterogeneity was observed in purified P-450SCC preparations from bovine adrenal cortex mitochondria. These results indicated the presence of multiple molecular forms of corpus luteum P-450SCC as well as adrenal cortex P-450SCC. Computer simulation studies were carried out in order to analyze the mechanism of formation of multiple bands on isoelectric focusing. The multiple bands of corpus luteum P-450SCC could be explained by postulating the presence of two isozymes (or molecular forms) having a pair of sites each with or without a charged group.

Mouse strain variations in the magnitude of induction of liver DT-diaphorase and hereditary transmission of the trait

1. Strain variations among mice in terms of cytosolic DT-diaphorase activity were studied in liver, kidney, stomach and heart tissues with or without the administration of 3-tert-butyl-4-hydroxyanisole (BHA). 2. BHA induced DT-diaphorase activity in all strains examined, and the magnitude of induction varied depending on the strain and tissue. Among the 10 inbred strains tested, BALB/c and C57BL mice showed relatively large magnitudes of induction for liver DT-diaphorase, whereas C3H and CBA mice showed relatively small magnitudes. 3. Results of examinations of BALB/c-C3H-F1, -F2 and C57BL-CBA-F1 mice revealed that smaller magnitudes of induction of liver DT-diaphorase were inherited essentially as a dominant trait. The hereditary trait could be adequately explained by postulating two gene loci that regulate the magnitude of induction. 4. The possible significance of DT-diaphorase activity in chemical carcinogenesis was discussed.