Hiroshi Ohinata

Involvement of nitric oxide in noradrenaline-induced increase in blood flow through brown adipose tissue

N omega-nitro-L-arginine methyl ester (L-NAME) (80 mg), a NO synthase blocker, completely abolished noradrenaline (NA)-induced increase in blood flow through brown adipose tissue (BAT) in the urethan-anesthetized rat. L-NAME suppressed NA-induced increase in blood flow dose-dependently. L-arginine (80 mg), but not D-arginine (80 mg), reversed the inhibitory effect of L-NAME (0.8 mg). NA-induced increase in BAT temperature was also decreased by L-NAME. These results suggest that nitric oxide mediates NA-induced increase in BAT blood flow.

Occurrence of nonlymphoid leukocytes that are not derived from blood islands in Xenopus laevis larvae

Previous immunohistochemical observations using the monoclonal antibody (XL-1) which recognizes all types of leukocytes in Xenopus laevis revealed the occurrence of XL-1+ cells in the mesenchyme throughout the early larval body, before the appearance of any lymphocytes. The present experiments were performed to determine whether these leukocytes originate, like lymphocytes and red blood cells (RBCs), in the ventral blood islands (VBI) or the dorsolateral plate (DLP). For tracing the derivation of cells, a specific staining by quinacrine to nuclei of X. laevis and Xenopus borealis hybrid (LB) cells was used to distinguish them from X. laevis (LL) cells. Orthotopic graftings of VBI tissue from st.22-23 LB embryos to the stage-matched LL embryos and examinations at st.44-45 before differentiation of the lymphocytes showed that the proportion of XL-1+ LB cells was always significantly lower than that of RBCs with the same marker in all experimental larvae. The head (LB)-body (LL) chimeras from st.22-23 embryos and culture of the head-portions as VBI- and DLP-free explants from st.14-23 embryos both demonstrated that a significant number of XL-1+ cells which had originated in the head portions had begun to differentiate by st.42-43. These results indicate that there is a significant population of larval nonlymphoid leukocytes (mostly macrophages) that do not originate from either the VBI or DLP region, and are distributed in the mesenchyme throughout the body.

Two types of non-selective cation channel opened by muscarinic stimulation with carbachol in bovine ciliary muscle cells

In the ciliary muscle, the tonic contraction requires a sustained influx of Ca2+ through the cell membrane. However, little has hitherto been known about the route(s) of Ca2+ influx in this tissue that lacks voltage‐gated Ca2+ channels. To identify ion channels as the Ca2+ entry pathway we studied the effects of carbachol (CCh) on freshly isolated bovine ciliary muscle cells by whole‐cell voltage clamp. Experiments were carried out using pipettes filled with K+‐free solution containing 100 mm caesium aspartate, 5 mm BAPTA and 180 μm GTP (pH 7.0; the intracellular free Ca2+ concentration, [Ca2+]i= 70 nm). CCh evoked an inward current showing polarity reversal at a holding potential near 0 mV. Analysis of the current noise distinguished two types of non‐selective cation channel (NSCCL and NSCCS) with widely different unitary conductances (35 pS and 100 fS). The ratios of the permeabilities to Li+, Na+, Cs+, Mg2+, Ca2+, Sr2+ and Ba2+, estimated by cation replacement procedures, were 0.9: 1.0: 1.5: 0.2: 0.3: 0.4: 0.5 for NSCCL, and 1.0: 1.0: 1.8: 2.5: 2.6: 3.2: 5.0 for NSCCS. NSCCS, but not NSCCL, was strongly inhibited by elevation of [Ca2+]i. Both NSCCL and NSCCS were dose‐dependently inhibited by 1–100 μm SKF96365, La3+ and Gd3+, which also inhibited the tonic component of the contraction produced in muscle bundles by CCh without markedly affecting the initial phasic component. NSCCL and/or NSCCS may serve as a major Ca2+ entry pathway required for sustained contraction of the bovine ciliary muscle. RT‐PCR experiments in the bovine ciliary muscle (whole tissue) detected mRNAs of several transient receptor potential (TRP) channel homologues (TRPC1, TRPC3, TRPC4 and TRPC6), which are now regarded as possible molecular candidates for receptor‐operated cation channels.

Micromere Differentiation in the Sea Urchin Embryo: Two‐Dimensional Gel Electrophoretic Analysis of Newly Synthesized Proteins

A method for large‐scale culture of isolated blastomeres of sea urchin embryos in spinner flasks was developed. Micromeres and meso‐, macromeres isolated from sea urchin embryos at the 16‐cell stage were cultured by this method and the patterns of protein synthesis by their descendants were examined by two‐dimensional gel electrophoresis of [35S] methionine‐labeled proteins. Six distinct proteins with molecular weights of 140–kDa, 105–kDa, 43–kDa, 32–kDa, and 28–kDa (two components) were specifically synthesized by differentiating micromeres. Quantitative analysis of the two‐dimensional gel patterns demonstrated that all these proteins, except the 32–kDa protein, appeared at the time of ingression of primary mesenchyme cells (PMCs) in vivo, several hours earlier than the onset of spicule formation. The synthesis of 32–kDa protein was paralleled to active spicule formation and the uptake of Ca2+. Cell‐free translation products directed by poly (A)+ RNAs isolated from descendant cells of micromeres and meso‐, macromeres were compared by two‐dimensional gel electrophoresis. Several spots specific to the micromere lineage were detected. However, none of them comigrated with the proteins synthesized specifically by the cultured micromeres. The results suggest that the expression of these proteins specific to differentiating micromeres may involve post‐translational modification.

Contribution of Ventral Blood Island (VBI)‐Derived Cells to Postembryonic Liver Erythropoiesis in Xenopus laevis

The ventral blood islands (VBI) of Xenopus laevis embryos are known as the hemopoietic site where the initial erythropoiesis takes place at st. 28. To determine the site of postembryonic erythropoiesis, larvae were induced anemic by phenylhydrazine (PHZ) at st. 31 and 40, and the tissue distribution of regenerating erythrocytes was determined with an anti‐larval hemoglobin (LHb) monoclonal antibody. Three days after total anemia induction, the LHb+cells were detected first in the liver and the digestive tract, followed by the appearance of a few LHb+cells in the blood vessels. The lavae which had been hepatectomized and cardiectomized before the PHZ treatment showed a remarkable reduction in recovery of the LHb+cells. Induction of anemia in the chimeric individuals containing cytogenetically labelled VBI tissues demonstrated that the VBI‐derived cells contribute to the regenerating LHb+cells in all experimental individuals. These results suggest that the larval liver is the major site where the VBI‐derived hemopoietic cells reside and differentiate into erythrocytes.

Fasting-induced modifications of fatty acids composition in brown adipose tissue

Abstract 1. 1.|Effect of fasting for 72 h on fatty acid (FA) composition was studied in triglyceride and phospholipid fractions of rat interscapular brown adipose tissue (BAT) 2. 2.|Fasting decreased the body and BAT weights, and tissue DNA was significantly decreased by fasting. The tissue triglyceride and phospholipid levels were not affected by fasting 3. 3.|In triglyceride FA or BAT the extent of unsaturation was increased as assessed by mol% of polyunsaturated FA, the ratio of polyunsaturated FA to saturated FA and the unsaturation index in the fasted state 4. 4.|In phospholipid FA or BAT the extent of unsaturation also increased as assessed by the same indices for TG. 5. 5.|These results, especially in phospholipid FA of BAT, were in good agreement with the previous report in the cold-acclimated rats, suggesting that BAT function is, at least in part, stimulated during fasting.

Non-thermal stress-induced modifications of fatty acids profiles in rat brown adipose tissue

Abstract 1. 1.|The changes in fatty acids (FA) composition were studied in triglyceride and phospholipid fractions of interscapular brown adipose tissue (BAT) in cold-acelimated (5°C, 4 wk) (CA), repetitively immobilized (3 hr/day, 4 wk) (IM) and treadmill-running trained (T) (30 m/min, 30 min/day under 8° inclination, 4 wk) rats. 2. 2.|Fatty acids composition in triglyceride: in CA rats the saturated FA (SA) and polyunsaturated FA (PU) were higher, while monounsaturated FA (MU) was lower. In IM rats SA was lower, while MU and PU were higher. Unsaturation index (UI) and PU/SA were higher in IM rats. In T rats SA and MU were lower, while PU was higher, UI and PU/SA were higher in T rats. 3. 3.|Fatty acids composition in phospholipid: in CA rats SA and PU were higher, while MU was lower. UI and PU/SA were higher. In IM rats SA and MU were lower, while PU was higher. UI and PU/SA were higher. In T rats MU was lower, but no changes were observed in UI and PU/SA. 4. 4.|These findings have indicated that non-thermal stress such as immobilization caused similar changes in FA composition of rat BAT, especially an increase in the extent of unsaturation in FA of phospholipid, to those previously reported and presently confirmed in CA animals. On the other hand, exercise training did not affect the extent of unsaturation in FA of phospholipid as assessed by UI and PU/SA.

Oxidatively induced Cu for Mn exchange in protein phosphatase 1γ: A new method for active site analysis

Protein phosphatase 1gamma, a serine/threonine phosphatase, is a metalloprotein that coordinates two Mn(2+) in the active site when expressed in Escherichia coli in a buffer containing MnCl(2). Herein, we report on the oxidatively induced copper for manganese exchange in protein phosphatase 1gamma, thus enabling firm confirmation of the four histidine (His) amino acid residues (His66, His125, His173, and His248) involved in metal coordination. By exchanging manganese with copper the oxidation yields for the peptides increased dramatically, thus simplifying detection of the oxidized peptides and analysis of the oxidation sites within the oxidized peptides. We also found that when copper was added during the oxidation process a new metal coordination center was formed at cysteine 39, 105, 140, and 155.

Effect of sucrose-induced overfeeding on brown adipose tissue—With special reference to in vitro thermogenesis and fatty acids compositions

Effect of overfeeding induced by sucrose solution (32%) was investigated on in vitro oxygen consumption and fatty acids (FA) compositions of interscapular brown adipose tissue (BAT) in rat. Sucrose group (S) was fed standard diet with sucrose solution as drinking water. Food intake was greater by 10–24% in S as compared with that in standard diet control group (C). Colonic and skin temperatures were higher in S. The weight and DNA content of BAT were greater in S. In vitro basal and noradrenaline- or glucagon-stimulated oxygen consumption did not differ between the groups when expressed per DNA, while they were significantly greater in S in terms of whole tissue pad. In S BAT triglyceride (TG) and phospholipid (PL) levels as well as tissue contents were higher. In TG-FA of S saturated FA and monounsaturated FA were higher, while polyunsaturated FA were lower. In PL-FA of S monounsaturated FA were higher, while saturated FA and polyunsaturated FA were lower. These findings imply that sucrose-induced overfeeding increases BAT thermogenesis through tissue hyperplasia and higher PL-FA unsaturation as indicated by higher proportions of monounsaturates.

Brown Adipose Tissue and Nonshivering Thermogenesis in Stressful States

Adaptive transition of thermoregulatory thermogenesis from shivering to more efficient non-shivering thermogenesis in cold has been well documented. Nonshivering thermogenic mechanisms have been explored in the major nonshivering thermogenic organ, brown adipose tissue (BAT). Here we review our recent work on some regulatory elements (phospholipid fatty acids, nitric oxide, uncoupling protein, inhibitory central structure) of this tissue under various physiological conditions. The following points are suggested. (1) The docosahexaenoic acid (DHA) level in BAT phospholipids is closely associated with BAT in vitro thermogenesis; increase in DHA enhances, while decrease in DHA suppresses, thermogenesis of BAT. DHA is also involved in BAT hyperplasia leading to increased BAT thermogenesis. (2) The tonic inhibitory mechanism for BAT thermogenesis resides in the midbrain site in a hibernator, the hamster, as well as in the rat, a nonhibernator. (3) Nitric oxide (NO) produced by endothelial NO synthase in BAT is involved in an increase in BAT blood flow and possibly in BAT thermogenesis induced by the sympathetic outflow stimulated by the ventromedial hypothalamus (VMH). (4) Nonthermal stress of repetitive immobilization enhances BAT thermogenesis by increasing both capacity and activity of uncoupling protein 1 (UCP-1), leading to an acquisition of cross-adaptability to cold.