Toshihiro Hidaka

Changes of membrane fluidity and Na+, K+-ATPase activity during cellular differentiation in the guinea pig epidermis

SummaryThe dynamic properties of plasma membrane in epidermal cells were determined by means of electron spin resonance using two kinds of doxyl stearic acid spin labeling agents: 5-DSA and 12-DSA. 5-DSA and 12-DSA are stearic acid analogues with a nitroxide radical ring at the 5th and 12th carbon positions, and these motions reflect molecular motion of lipid bilayer surrounding the hydrophilic region and the hydrophobic region, respectively. Guinea pig epidermal cells were separated into three regions of keratinocytes by Percoll density gradient centrifugation; the upper, middle, and lower epidermal cells. The order parameter S values for 5-DSA and 12-DSA incorporated into the isolated keratinocytes increased, suggesting a decrease in the plasma membrane fluidity, as cells approached the upper epidermal cell layer. The Na+, K+-ATPase activity as a plasma membrane-bound enzyme was determined in each epidermal cell region, and was found to decrease gradually as the cells approached the upper layer. Accordingly, the differentiation of epidermal cells in the keratinization process was found to be assiciated with a decrease in plasma membrane fluidity and with a decline of Na+, K+-ATPase activity.

Effects of vitamin E, vitamin B2 and selenite on DNA single strand breaks induced by sodium chromate (VI)

The effects of vitamin E, vitamin B2 and selenite on DNA single strand breaks induced by Na2CrO4 were examined by alkaline elution. Incubation of Chinese hamster V-79 cells with alpha-tocopherol succinate (vitamin E) for 24 h prior to exposure to Na2CrO4 resulted in a decrease of DNA breaks produced by this compound. However, similar pretreatment with riboflavin (vitamin B2) or Na2SeO3 resulted in an enhanced formation of breaks induced by Na2CrO4. Pretreatment with Na2SeO3 resulted in increased levels of glutathione in these cells while levels of glutathione remained the same with vitamin E or vitamin B2. These results suggest that Na2CrO4 induced DNA breaks appear to be mediated by the formation of free radicals and/or cellular reductive metabolism.

Reduction of chromium(VI) in Chinese hamster V-79 cells.

The cellular reduction of chromate(VI) was studied by electron spin resonance spectrometry. Incubation of Chinese hamster V-79 cells with Na2CrO4 resulted in the formation of both chromium(V) and chromium(III) complex in a manner dependent on time (30 min-2 h) and concentration (50–500 μM). Following removal of extracellular chromate, the level of chromium(V) complex decreased quickly during the first hour but more slowly for the next hour, whereas the level of chromium(III) remained unchanged, indicating that chromium(III) is the ultimate ion of this metal in cells. Alkaline elution studies demonstrated that treatment of cells with Na2CrO4 induced DNA single-strand breaks that decreased quickly and DNA-protein crosslinks that persisted for 2 h after removal of this metal. These results suggest that the cellular levels of chromium(V) and chromium(III) may be associated with the formation of DNA damage induced by chromium (VI).

Lipid peroxidation and radical formation in methyl linoleate following ultraviolet light exposure.

To gain information useful in understanding radical reactions of sunlight‐induced lipid peroxidation in the skin, methyl linoleate (ML) was used as a material for a study of electron spin resonance (ESR). The ESR spectrum of ML exposed to UV light was examined under aerobic and anaerobic conditions at low temperature. No ESR signal was detected from the unexposed ML, even at —150°C. However, exposure to UV resulted in the appearance of an ESR signal at —150°C. With the elevation of temperature from —150°C, the ESR signal became asymmetric in shape and the g value increased under aerobic conditions, but these alterations did not occur under anaerobic conditions. Lipid peroxidation through a radical reaction initiated by UV exposure was discussed.

Effects of diethyl maleate (DEM), a glutathione depletor, on prostaglandin synthesis in the isolated perfused spleen of rabbits.

To investigate the role of glutathione (GSH) on prostaglandin (PG) synthesis, isolated rabbit spleens were perfused with Tyrodes solution with or without the addition of diethyl maleate (DEM) in concentrations up to 1 mM. In the absence of DEM, PG synthesis was stimulated by the Ca2+ ionophore A23187 (20 nmole) or arachidonate (0.4 μmole). Prostaglandin (PG) E2 was a major product, accounting for 60–70% of the total cyclooxygenase products. Small amounts of PGF2α, 6-keto-PGF1α, PGD2 and thromboxane (Tx) B2 were also produced. When DEM was added to the perfusion medium, GSH content decreased dose-dependently with increasing DEM concentration. Lactate dehydrogenase activity was not detected in the venous effluent, indicating that DEM depleted intrasplenic GSH without causing any lysis of cellular membranes. A23187-induced production of PGs and of Tx was decreased with increasing concentrations of DEM up to 0.5 mM, whereas at 1.0 mM DEM, these products showed a tendency to increase as compared with levels at 0.5 mM DEM. However, this increase was only significant for TxB2, which returned to levels obtained in the absence of DEM. DEM 1 mM did not cause cell lysis, but it appears to perturb the cell membrane to a degree similar to that which occurs with stimulation of phospholipase A2. The small but significant increase of TxB2 with 1.0 mM DEM could be a result of decreased PGE2 isomerase activity. Perfusion with arachidonate gave virtually identical results: 1.0 mM DEM attenuated the production of all prostanoids except for TxB2 as compared with untreated controls. These results suggest that GSH contributes to the regulation and/or maintenance of PGs synthesis.

Membrane responses of B-16 melanoma cells to single exposure to ultraviolet light

SummaryElectron spin resonance spectroscopy using the spin probe (5-, 12-and 16-deoxylstearic acid) was employed to analyze the changes in membrane fluidity in B-16 melanoma cells following UV-B exposure. The UV exposure resulted in the immediate accumulation of lipid peroxide, being accompanied by a change in membrane fluidity. The 12-DSA is the most sensitive to the changes in membrane organization caused by UV light. Na+,K+-ATPase activity was regulated by a change in membrane fluidity. Following UV exposure, the release of the prelabeled arachidonic acid from the cells was observed immediately. Ca2+-dependent calmodulin-dependent phospolipase A2-like activity was involved in the UV-stimulated arachidonic acid release from phospholipid.

Stimulation of prostaglandin synthesis by cholecystokinin in primary culture cells of bovine gallbladder muscle

Primary culture cells derived from bovine gallbladder muscles synthesize PGE2 as a major cyclooxygenase product with a trace amount of 6-keto-PGF1 alpha-like material. The synthesis of PGE2 and total cyclooxygenase products was enhanced in response to cholecystokinin (CCK). In the presence of indomethacin the synthesis of PG was inhibited and the release of arachidonic acid (AA) in response to CCK was enhanced. These data suggest that CCK may stimulate the release of AA, probably by activating phospholipase A2/C, from membrane phospholipids in the gallbladder muscle.

Increased platelet thromboxane synthesis in renal glomerular diseases

Washed platelets were prepared from healthy children and adults, and patients with renal glomerular diseases, and incubated with [1-14C] arachidonate to measure the generation capacities of thromboxane (Tx) A2 and 12-hydroxyeicosatetraenoate (12-HETE). Tx generation capacity of platelets was significantly higher in patients with chronic glomerulonephritis, purpura nephritis and lupus nephritis than in healthy control subjects. There was no significant increase in minimal change nephrotic syndrome. 12-HETE showed a decreasing tendency in the glomerular diseases, which was restored to normal level by in vitro addition of indomethacin. Such increased Tx generation capacity of platelets may cause abnormal enhancement of platelet functions and conceivably constitute an aggravating factor of glomerular and microvascular damage in the affected kidney.

Generation of leukotrienes and hydroxyeicosatetraenoic acids in peritoneal macrophages of tumor-bearing mice.

To examine the potential role of lipoxygenase products in the pathophysiology observed after experimental tumor implantation, we examined the generation of leukotrienes (LTs) and hydroxyeicosatetraenoic acids (HETEs) in peritoneal macrophages. C57BL/6 mice were given subcutaneous inoculations of B16 melanoma cells, and peritoneal macrophages were isolated various days after the inoculation. Macrophages were incubated for 1 h at 37 degrees C in serum-free RPM11640 containing 10 microM calcium ionophore A23187, 10 microM exogenous arachidonic acid (AA), 5 mM cysteine hydrochloride and 1 mM reduced glutathione. LTs and HETEs were separately extracted, passed through Sep-Pak cartridges, then identified and quantitated with a HPLC system using UV absorbance. The B16 melanoma-cell-treated/untreated macrophages were found to produce substantial amounts of 15-HETE, 12-HETE and 5-HETE and LTC4 by enzymatic mechanisms. Thus, when determined under various conditions, the production of HETEs was dependent on substrate-concentration, incubation-time and cell-number. The production of LTC4 was dependent on incubation-time and cell number but not substrate-concentration, indicating utilization of endogenous AA stores. Of these products, 12-HETE and LTC4 showed a significant increase on the fourth day after the tumor cell inoculation and returned to the control level by the 11th day after the same treatment. These results suggest that in vivo tumor cell implantation may induce a transient increase of 12-HETE and LTC4 production in macrophages.

Prevention of 4-pentenoic acid-induced liver injury in rats by 16,16-dimethyl PGE2

16,16-Dimethyl PGE2 (dmPGE2) is known to protect against cellular damage in various tissues. Histological and biochemical approaches were used to examine the effect of this prostaglandin on hepatocellular damage in an experimental Reyes syndrome model produced in rats by 4-pentenoic acid. Chronic intraperitoneal administration of 4-pentenoic acid induced an accumulation of fatty droplets throughout the hepatic lobules along with mitochondrial abnormalities including swelling, disappearance of christae, and heterogeneity of matrix. These abnormalities were more intense in the marginal zone and successively decreased nearer to the central vein. Such hepatic abnormalities were markedly reduced by the combined administration of dmPGE2 with 4-pentenoic acid. Biochemical examination confirmed that dmPGE2 was able to inhibit the accumulation of hepatic triglyceride seen after the treatment with 4-pentenoic acid alone. These results indicated that dmPGE2 can prevent characteristic hepatocellular damage in this experimental Reyes syndrome model, suggesting that the involvement of prostaglandins should be taken into account in discussing the etiology and management of this syndrome.