Inhibited autophagic degradation of cytoplasm during compensatory growth of liver cells after partial hepatectomy.

The livers from 56 sham-operated and 56 partially hepatectomized male albino rats killed 4--81 h after operation were investigated by electron microscopic morphometry. Following partial hepatectomy, the principal changes in volume fractions in the hepatocellular cytoplasm were: decrease of glycogen and, to a lesser extent, of mitochondria together with considerable increase of fat droplets. The volume fraction of microbodies (= peroxisomes) showed no significant difference between control and regenerating liver. By evaluating large test fields of about 40,000 micrometers 2 sectioned cytoplasm per animal it could be demonstrated that the volume fraction and the numerical density of autophagic vacuoles (AV's) were significantly reduced after partial hepatectomy. The extent of this reduction depended on the postoperative time interval. AV's were reduced by 75% at day 0 (4--17 h p.o.), by 98% at day I (19--33 h p-o.), by 75% at day II (43--57 h p.o.), and still by 50% at day III (67--81 h p.o.). The different types of AV's, defined on the basis of the different cytoplasmic components enclosed, were reduced to similar extent during the respective time periods. The reduction of AV's seems to be specific for the regenerating organ since no significant differences in the volume fraction of AV's could be found in the proximal tubular cells of the kidney of partially hepatectomized animals when compared with those of sham-operated controls. The inhibition of intracellular autophagic degradation in regenerating liver has its biochemical equivalent, i.e. inhibited protein catabolism, and is interpreted as an important and adequate mechanism in effecting the shift from the physiological steady state between anabolism and catabolism to the positive balance which is required for the compensatory growth of the liver after partial hepatectomy.     

Cardiac hypertrophy in rats after supravalvular aortic constriction. I. Size and number of cardiomyocytes, endothelial and interstitial cells

The ascending aorta of 22 adult male Sprague-Dawley rats was constricted with a silver ring, and 25 animals were subjected to a sham-operation. The hearts, including the main arteries, were fixed by retrograde perfusion 3, 7, 14, 21 and 35 days after the operation. The cross-sectional area of the aorta was reduced by the constriction to an average of 20% of the values found after sham-operation. Twenty-one days after the constriction the weight of the left ventricular myocardium including the septum was increased 1.7-fold compared with controls. No further increase in weight was observed 35 days after the operation. The relative volumes of the tissue components remained largely constant in the subepicardial myocardium. In the subendocardial myocardium, however, the volume fraction of interstitial and, to a lesser extent, of endothelial tissue was significantly increased. Twenty-one days after constriction the estimated total volumes of the different myocardial components per left ventricle were increased 1.7-fold for heart muscle parenchyma, 1.8-fold for endothelial tissue, 2.9-fold for interstitial tissue, and 1.3-fold for capillary lumina compared with controls. At 35 days, only the interstitial tissue showed a further increase to 4.8-fold of control values. The mean cardiomyocyte volume was increased after aortic constriction in proportion to the increase in left ventricular weight, i.e. 1.7-fold over controls at 21 days. After 35 days its value was 29,500 +/- 790 micron 3 in rats subjected to aortic constriction compared with 16,800 +/- 640 micron 3 in controls. At this time the estimated number of cardiomyocytes per left ventricle showed no significant differences between experimental animals (2.9 X 10(7)) and controls (3.1 X 10(7)). Endothelial and interstitial cells were not only increased in average single cell volume (1.3-fold and 2.0-fold, respectively), but also in number per left ventricle (1.4-fold and 2.7-fold, respectively). Two-dimensional parameters indicated that during hypertrophy the capillary supply lagged behind the overall mass increase but achieved control levels on termination of hypertrophic growth at 35 days. These results show that even in pronounced hypertrophy the increase in mass of the myocardial parenchyma in the rat is due exclusively to an enlargement of cardiomyocytes (hypertrophy), whereas in endothelial and interstitial tissues enlargement of cells as well as increase in cell number (hyperplasia) also plays a role.     

The activity of the pyruvate dehydrogenase complex in heart and liver from mice during the development of obesity and insulin resistance.

The amount of pyruvate dehydrogenase in the active form (PDHa) was increased 1.7-fold compared with controls in heart muscle of mice 1 week after induction of obesity with a single injection of gold-thioglucose. At 4 weeks post injection, the amount of PDHa was decreased to 32% of control, a value which was observed in later stages of the obesity syndrome. In contrast, liver PDHa was increased and remained at an increased activity during the development of obesity. Despite normal post-prandial serum insulin contents, liver membrane insulin-receptor numbers were decreased 1 week after gold-thioglucose injection, and there was no change in receptor affinity. The decrease in heart PDHa in the obese animals was reversed by a single dose of 2-tetradecylglycidic acid, but this inhibitor of mitochondrial fatty acid oxidation did not affect liver PDHa in these animals. These early and diverse changes in PDHa argue for a multifactorial aetiology in the development of the whole-body insulin resistance seen in older gold-thioglucose-treated obese animals.     

Ubiquinone analyses in fish tissues and in some marine invertebrates.

1. Ubiquinone contents were determined in species of marine invertebrates, and in heart, red and white muscle and liver of three species of fish. 2. Three different methods of determination were compared, based on spectrophotometry, reduction and a reaction with the dimethoxy groups of ubiquinone. 3. Using ubiquinone homologues 6-10 prepared from beef heart and commercially available microorganisms (SCP) as standards, ubiquinone 10 was found in all samples. In addition were found minor amounts of Q-9 in samples of saithe heart and red muscle. 4. Less than 10 mg/kg wet wt of ubiquinone was found in the samples of marine invertebrates and in white muscle and liver of the fish samples, with one exception: 40 mg/kg in a sample of mackerel liver. 5. Higher contents of ubiquinone were found in fish heart and red muscle tissues, ranging from 24 to 116 mg/kg wet wt. The ubiquinone contents were comparable in the two tissues. 6. A test on cellular fragments of red muscle tissue of saithe showed that the ubiquinone was concentrated in the mitochondria fraction.     

Effects of duration of fast and animal age on the gastrointestinal absorption of plutonium

The fraction of plutonium absorbed after oral administration of Pu(VI) to 24-h-fasted mice was 19 X 10(-4), 13-fold higher than in fed mice, 1.4 X 10(-4). We have investigated the relevance of the high gastrointestinal (GI) absorption value for the 24-h-fasted animals in setting drinking water standards for humans. When fasting was initiated at the beginning of the active phase of the mouse's daily activity cycle (when they would normally eat), plutonium GI absorption rose from 2.8 X 10(-4) at zero-time to a level typical of the 24-h-fasted mouse after only 2 h of fasting. In contrast, in mice allowed to eat for 4 h into their active phase prior to initiation of the fast (meal-fed mice), 8 h of fasting were required before GI absorption rose to a level similar to that of the 24-h-fasted mouse. The fraction of plutonium retained after gavage administration of Pu(VI) to 1-day-old rats was 74 X 10(-4), 70-fold higher than the value for fed adults. Retention after GI absorption in neonates remained 30- to 70-fold higher than in adults until weaning. One week after weaning, the fraction absorbed and retained by fed weanling rats was the same as that for fed adults, 1 X 10(-4). Drinking water standards for plutonium have been set based on GI absorption values for fed adult animals. The 10- to 100-fold increases in plutonium absorption in young and fasted animals reported by ourselves and others, and the rapid rise to fasted levels of absorption at the start of the animal's active phase, indicate that consideration should be given to elevated levels of plutonium absorption in young and fasted individuals.     

Organ and intracellular localization of short-chain acyl-CoA synthetases in rat and guinea-pig.

1. Homogenates of rat epididymal fat pad, heart, kidney, lactating mammary gland, liver, skeletal muscle and small intestinal mucosa have been partitioned into a particulate and supernatant fraction. With reliable marker enzymes for the mitochondrial matrix and the cytosol: propionyl-CoA carboxylase and pyruvate kinase, the distributions of the acyl-CoA synthetase activities measured at 1 and 10 mM C2, C3 and C4 over mitochondria and cytosol have been calculated. From these values an estimate was made of the K0.5 of the fatty acids. 2. A distinct fatty acid-activating enzyme was assumed to be present in one of the compartments when that fatty acid was activated with a K0.5 less than or equal to 1.5 mM in an amount of greater than 13% of the total cellular activity. Adipose tissue, gut, liver and mammary gland, all organs of a high lipogenetic capacity, contained a cytosolic acetyl-CoA synthetase. At 1 mM acetate 60, 31, 77 and 83% of the total cellular activities in these organs were cytosolic in nature, with activities of 0.021, 0.32, 0.37 and 1.16 mumol C2 activated per min per g wet weight, respectively. 3. Mitochondrial acetyl-CoA and butyryl-CoA synthetases were found in adipose tissue, gut, heart, kidney, mammary gland and muscle. They were absent in liver. Adipose tissue and liver contained a mitochondrial propionyl-CoA synthetase with activities at 1 mM C3 of 0.014 and 1.50 mumol C3 activated per min per g wet weight, respectively. 4. At 1 mM, C2 was activated with decreasing rates by kidney, heart, mammary gland and gut (7.6-1.0 mumol C2 activated per min per g wet weight). C3 (1 mM) activation was about equal (1.6-1.9 mumol C3 activated per min per g wet weight) in liver, kidney and heart. C4 (1 mM) was activated with decreasing rates by heart, liver, kidney and gut (4.0-0.5 mumol C4 activated per min per g wet weight) in the order given. 5. The influence of the isolation method and the diet on fatty acid activation in small intestinal mucosal scrapings have been studied. To demonstrate the existence of cytosolic acetyl-CoA synthetase in fed animals a pre-treatment of everted intestine by low amplitude vibration has been found essential. Also C16 activation was highly (95%) decreased in a non-pre-vibrated preparation. 24 h starvation lowered cytosolic C2 and total C16 activation by 90 and 80%, respectively. Refeeding of starved rats with a balanced fat-free diet, and not with sucrose only, gave the same cytosolic C2 and total C16 activation as normally fed rats. 6. In guienea-pig heart, kidney, liver and muscle about the same partitions have been found as in the respective rat organs. The acetate activation in liver was factor 6 lower. Acetate and butyrate activation in guinea-pig muscle was much higher (6 and 37 times, respectively).     

Effects of chronic streptozotocin-induced diabetes on the cardiac responses to milrinone

We have studied the effects of milrinone on various cardiac preparations obtained from 6-week streptozotocin diabetic rats. The basal rate of spontaneously beating right atrium from diabetics was significantly lower as compared with controls. Milrinone (5 X 10(-5) to 8 X 10(-4) M) produced a dose-dependent positive inotropic and positive chronotropic effect in left atrium and right atrium, respectively. The positive chronotropic response to milrinone was slightly increased in right atria from diabetic animals. In papillary muscle neither the maximum response nor the pD2 value of milrinone was altered significantly in diabetic animals. The pD2 values of milrinone in right atrium and left atrium were found to be significantly higher in diabetic preparations compared with controls. The data indicate that the responses to milrinone are either unchanged or enhanced in hearts from diabetic animals.     

Stimulatory (insulin-mimetic) and inhibitory (ouabain-like) action of vanadate on potassium uptake and cellular sodium and potassium in heart cells in culture

(1) The influence of vanadate (Na3VO4) on sodium and potassium uptake as well as on cellular ion contents of sodium and potassium has been studied in heart muscle and non-muscle cells obtained from various species. An ouabain-like inhibition of potassium uptake (up to 50%), combined with a decrease of cellular potassium (up to 20%) has been observed by vanadate (10(-4)-10(-3) M) in heart non-muscle cells obtained from neonatal guinea pigs and chick embryos. In heart muscle and non-muscle cells prepared from neonatal rats, as well as in Girardi human heart cells, a vanadate-induced stimulation of potassium uptake (up to 100%), combined with a rise in cellular potassium (up to 20%) and without significant alteration of cellular sodium, has been found. A slight increase of 22Na+ influx can be measured in rat heart muscle cells and in Girardi human heart cells in the presence of vanadate (10(-4)--10(-3) M). (2) In beating rat heart muscle cells in culture, detrimental effects of serum deprivation--concerning beating properties, potassium uptake and cellular potassium--can at least in part be overcome by addition of vanadate. Furthermore, this compound prevents ouabain-induced signs of toxicity (contractures) in these cells. (3) The stimulatory effects of vanadate on potassium can be mimicked by insulin (1-10 mU/ml). Furthermore, vanadate produces an insulin-like stimulation of 2-deoxy-D-glucose uptake in rat heart muscle and non-muscle cells as well as in Girardi human heart cells. (4) The experimental data demonstrate an ouabain-like inhibition as well as an insulin-mimetic stimulation of potassium-uptake in various heart cells. The reason for this antagonistic mode of action may be due to the different capabilities of the heart cell types to reduce vanadium in the V-valence state of vanadium in the IV-valence state, thereby favouring either ouabain-like inhibition (vanadium V) or insulin-mimetic stimulation (vanadium IV) of potassium transport.     

A morphometric study of the inhibition of autophagic degradation during restorative growth of liver cells in rats re-fed after starvation.

In the parenchymal cells of the liver of adult male rats re-fed on the evening of the fifth day after a period of absolute starvation, a nearly complete absence of autophagic vacuoles (AV) has been found by the morphometric determination of the fractional cytoplasmic volume of AV. The mean value for that parameter increased only gradually during periods of re-feeding. The value was found to be in the range of the control values only on, or after, the fifth day of re-feeding. As in previous experiments, in the control animals the number of AV was again found to be dependent on a circadian rhythm with maxima during the light, and minima during the dark, periods. This rhythm reappeared in the period of re-feeding without a shift in phase. In the controls as well as in the re-fed animals the "segregated fraction" was highest for microbodies, intermediate for mitochondria and glycogen, but rather low for the remaining components of the cytoplasm. It is suggested that the long term inhibition of cellular autophagy, found in the present study, plays an important role in the restorative cellular growth of the liver during the recovery from the atrophy induced by starvation.     

Decreased cardiac lipoprotein lipase activity in rats treated chronically with adriamycin

Rats treated chronically with the anticancer agent adriamycin (1.5 mg/kg/week X 14 weeks) exhibited cardiac and renal lesions typical of anthracycline toxicity, and had serum hyperlipidemia characterized by 4 to 10 fold elevations in all lipoprotein classes. Heparin-releasable lipoprotein lipase activity measured in perfused heart preparations was decreased 69% in adriamycin-treated rats compared to saline-treated controls. Residual (non-heparin-releasable) activity was not significantly different after adriamycin treatment. The decrease in functional cardiac lipoprotein lipase may account, at least in part, for the serum hyperlipidemia observed in adriamycin-treated rats, and might play a role in the development of heart muscle disease.     

Tissue specific,sex and age--related differences in the 6-phosphogluconate dehydrogenase gene expression

Data presented in thid paper indicate that: (1) the age-related changes in 6-phosphogluconate dehydrogenase (6PGDH) activity depend on sex and tissue. No differences in the liver 6PGDH activity between young (1-month-old) males and females were found. In adult males, the activity was the same as in young animals but, in adult females, it reached the value twice as high as in the young. In adipose tissue (both white and brown) and kidney cortex, the enzyme activity decreased with age both in males and females. There were no differences between males and females 6PGDH activity in brain, heart and skeletal muscle. (2) The sex and age-related changes in the liver 6PGDH activity occur predominantly at the level of mRNA cellular concentration. (3) In the liver of ovariectomized rats decrease of 6PGDH activity and mRNA level was observed. Oestradiol administration to ovariectomized rats restored liver 6PGDH activity and liver 6PGDH mRNA levels to that observed in controls. No changes in 6PGDH activity and mRNA levels were found in white adipose tissue (WAT) of ovariectomized adult rats and in ovariectomized rats treated with oestradiol. (4) Oestradiol administration to males caused an increase of liver 6PGDH activity and mRNA levels to values observed in females, but was without an effect on WAT 6PGDH activity and mRNA level. (5) These results suggest that 6PGDH activity in different tissues is not regulated in coordinate fashion and that oestradiol plays an important role in the liver enzyme activity regulation.     

Inhibited autophagic degradation during ACTH-stimulated growth of rat adrenal zona fasciculata

Autophagic vacuoles (AVs) in adrenal parenchymal cells of the outer part of the zona fasciculata were investigated by means of electron microscopic morphometry in 40 adult male Sprague Dawley rats. Four test groups and four control groups received twice daily subcutaneous injections of ACTH (12.5 U/kg body weight) or physiological saline, respectively, for 3, 6, 9 and 12 days. At 12 days the total dry weight of the adrenals was increased in ACTH-treated animals from 17.2 to 71.0 mg (about fourfold) and the average volume of zona fasciculata cells from 1,500 to 5,100 microns3 (about threefold). The relatively larger increase in total mass reflects mitotic activity in the growing adrenal tissue. The calculated growth rate of total tissue expressed as the percentage increase in dry weight per day was 12% at day 3 and decreased to 4.6% at day 12 of ACTH stimulation. During the time interval investigated the cytoplasmic volume fraction of autophagic vacuoles was significantly reduced from 10 X 10(-5) in controls to 1 X 10(-5) in ACTH-treated animals (p less than 0.001). Assuming a half-life for AVs of 7.9 min (Papadopoulos and Pfeifer 1986), autophagic degradation of cytoplasm can be calculated as 1.3%/day in controls, but only 0.15%/day in ACTH-treated animals. Thus growth of 1.15%/day can be attributed to inhibition of autophagic degradation. Although this is only part of total growth, it is evident from the present results that anticatabolic growth induction is utilized nearly to its maximum in adrenocortical fasciculata cells under the influence of ACTH.     

A morphometric study of cellular autophagy including diurnal variations in kidney tubules of normal rats

Cellular autophagy in convoluted tubules of kidney was studied in 24 rats, killed in pairs at constant time intervals during one diurnal cycle, by (a) morphometric evaluation of tubular cells by the point-counting method in randomly sampled micrographs, and (b) selective search for autophagic vacuoles (AV) directly on the electron microscopy screen. The total area of tubular cells recorded in the electron microscopy sections was 93 X 10(-4) mum2. Since the distal convoluted tubules, covering about 12% of the whole tubulocellular area, contained only 3-4% of all AV, they were omitted from the main calculations. The number of AV per area unit and the total amount of segregated material showed a distinct diurnal rhythm, synchronous for the different types of AV which were distinguished from each other according to their contents. The minimum was found during the night, the maximum during the day. This rhythm appears similar to that described elsewhere in liver cells. The mean segregated fractions were calculated from the relation of segregated to nonsegregated material in proximal convoluted tubular cells. The segregated fraction of the mitochondria was 4.4 X 10(-4). This value could account for the degradation of all mitochondria in a cell within 15 days, i.e., the upper limit of the lifetime of mitochondrial DNA in the cortex of the kidney, if one assumes that a mitochondrion is destroyed within 10 min after being segregated. The degregated fraction of microbodies was 11.7 X 10(-4). This suggests a shorter lifetime of these organelles. It is concluded that cellular autophagy plays a significant role in the turnover of cytoplasmic constituents, including the membranes of the endoplasmic reticulum.     

Amino acids augment muscle protein synthesis in neonatal pigs during acute endotoxemia by stimulating mTOR-dependent translation initiation

In skeletal muscle of adults, sepsis reduces protein synthesis by depressing translation initiation and induces resistance to branched-chain amino acid stimulation. Normal neonates maintain a high basal muscle protein synthesis rate that is sensitive to amino acid stimulation. In the present study, we determined the effect of amino acids on protein synthesis in skeletal muscle and other tissues in septic neonates. Overnight-fasted neonatal pigs were infused with endotoxin (LPS, 0 and 10 microg.kg(-1).h(-1)), whereas glucose and insulin were maintained at fasting levels; amino acids were clamped at fasting or fed levels. In the presence of fasting insulin and amino acids, LPS reduced protein synthesis in longissimus dorsi (LD) and gastrocnemius muscles and increased protein synthesis in the diaphragm, but had no effect in masseter and heart muscles. Increasing amino acids to fed levels accelerated muscle protein synthesis in LD, gastrocnemius, masseter, and diaphragm. LPS stimulated protein synthesis in liver, lung, spleen, pancreas, and kidney in fasted animals. Raising amino acids to fed levels increased protein synthesis in liver of controls, but not LPS-treated animals. The increase in muscle protein synthesis in response to amino acids was associated with increased mTOR, 4E-BP1, and S6K1 phosphorylation and eIF4G-eIF4E association in control and LPS-infused animals. These findings suggest that amino acids stimulate skeletal muscle protein synthesis during acute endotoxemia via mTOR-dependent ribosomal assembly despite reduced basal protein synthesis rates in neonatal pigs. However, provision of amino acids does not further enhance the LPS-induced increase in liver protein synthesis.     

Repopulation kinetics of rat rhabdomyosarcoma tumors following single and fractionated doses of low-LET and high-LET radiation

Measurements were made of clonogenic cell survival in rat rhabdomyosarcoma tumors as a function of time following in situ irradiation with single or fractionated doses of 225-kVp X rays or with 557-MeV/u neon ions in the distal position of a 4-cm extended-peak ionization region. Single doses of 20 Gy of X rays or 7 Gy of peak neon ions reduced the initial surviving fraction to approximately 0.025 for each modality. Daily fractionated doses (four fractions in 3 days) of either peak neon ions (1.75 Gy per fraction) or X rays (6 Gy per fraction) achieved a cell survival of approximately 0.02-0.03 after the fourth dose of radiation. In the single-dose experiments, significant 5- and 10-fold decreases in the fraction of clonogenic cells were observed between the third and fourth days after irradiation with peak neon ions and X rays, respectively. After the sixth day postirradiation, the residual clonogenic cells exhibited a rapid burst of proliferation leading to doubling times for the surviving cell fractions of approximately 1.5 days. Radiation-induced growth delay was consistent with the cellular repopulation dynamics. In the fractionated-dose experiments with both radiation modalities, a large delayed decrease in cell survival was observed at 1-3 days after completion of the fractionated-dose schedule. Cellular repopulation was consistent with postirradiation tumor volume regression and regrowth for both radiation modalities. The extent of decrease in survival following the four-fraction radiation schedule was approximately two times greater in X-irradiated than in neon-ion-irradiated tumors that produced the same survival level immediately after the fourth dose. Mechanisms underlying the marked reduction in cell survival 3-4 days postirradiation are discussed, including the possible role of a toxic host cell response against the irradiated tumor cells.     

Hexose transport stimulation and membrane redistribution of glucose transporter isoforms in response to cholera toxin, dibutyryl cyclic AMP, and insulin in 3T3-L1 adipocytes

Exposure of 3T3-L1 adipocytes to 100 ng/ml of cholera toxin or 1 mM dibutyryl cyclic AMP caused a marked stimulation of deoxyglucose transport. A maximal increase of 10- to 15-fold was observed after 12-24 h of exposure, while 100 nM insulin elicited an increase of similar magnitude within 30 min. A short term exposure (4 h) of cells to cholera toxin or dibutyryl cyclic AMP resulted in a 3- to 4-fold increase in deoxyglucose transport which was associated with significant redistribution of both the HepG2/erythrocyte (GLUT1) and muscle/adipocyte (GLUT4) glucose transporters from low density microsomes to the plasma membrane fraction. Total cellular amounts of both transporter proteins remained constant. In contrast, cells exposed to cholera toxin or dibutyryl cyclic AMP for 12 h exhibited elevations in total cellular contents of GLUT1 (but not GLUT4) protein to about 1.5- and 2.5-fold above controls, respectively. Although such treatments of cells with cholera toxin (12 h) versus insulin (30 min) caused similar 10-fold enhancements of deoxyglucose transport, a striking discrepancy was observed with respect to the content of glucose transporter proteins in the plasma membrane fraction. While insulin elicited a 2.6-fold increase in the levels of GLUT4 protein in the plasma membrane fraction, cholera toxin increased the amount of this transporter by only 30%. Insulin or cholera toxin increased the levels of GLUT1 protein in the plasma membrane fraction equally (1.6-fold). Thus, a greater number of glucose transporters in the plasma membrane fraction is associated with transport stimulation by insulin compared to cholera toxin. We conclude that: 1) at early times (4 h) after the addition of cholera toxin or dibutyryl cyclic AMP to 3T3-L1 adipocytes, redistribution of glucose transporters to the plasma membrane appears to contribute to elevated deoxyglucose uptake rates, and 2) the stimulation of hexose uptake after prolonged treatment (12-18 h) of cells with cholera toxin may involve an additional increase in the intrinsic activity of one or both glucose transporter isoforms.     

Subcellular localization of vitamin D3 25-hydroxylase in human liver

Vitamin D3 25-hydroxylase activity was measured in subcellular and submitochondrial fractions of human liver. Quantitation of 25-hydroxyvitamin D3 was based on high performance liquid chromatography. Vitamin D3 25-hydroxylase activity was detected in the mitochondrial fraction only. The mitochondrial 25-hydroxylase activity was linear with time up to 60 min and with mitochondrial protein up to 1 mg/ml. An apparent Km value of about 10(-5) M was found. Substrate satuation level was not reached. In the presence of 2.4 X 10(-4) M vitamin D3, the rate of 25-hydroxyvitamin D3 formation was 0.19 nmol X mg of protein-1 X h-1 After fractionation of the mitochondria, 86% of the 25-hydroxylase activity was recovered in the mitoplast fraction. The outer membrane fraction was devoid of activity. It is concluded that human liver contains only one detectable vitamin D3 25-hydroxylase enzyme localized to the mitochondrial inner membrane.     

The effect of one year's swimming exercise on oxidant stress and antioxidant capacity in aged rats

The effect of exercise on oxidant stress and on alterations in antioxidant defense in elderly has been investigated extensively. However, the impact of regularly performed long-term physical activity starting from adulthood and prolonged up to the old age is not yet clear. We have investigated the changes in the activities of antioxidant enzymes - superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) - and lipid peroxidation in various tissues of rats which had performed (old-trained) or had not performed (old-control) regular swimming exercise for one year. These animals were compared with young-sedentary rats. Increased lipid peroxidation was observed with ageing in all tissues (heart, liver, kidney, striated muscle) and swimming had no additional effect on this elevation of lipid peroxidation. Heart and striated muscle SOD activites, and striated muscle CAT activity increased as a consequence of ageing, whereas kidney and liver CAT activities, as well as GPx activities in kidney, liver, lung and heart were significantly decreased compared to young controls. Lung and heart SOD, liver CAT activities as well as GPx activities in liver, lung and heart were increased significantly in rats which performed exercise during ageing, compared to the old-control group. These findings suggest that lifelong exercise can improve the antioxidant defense in many tissues without constituting any additional oxidant stress.     

Cholesterol synthesis in the perfused liver of pregnant hamsters

Pregnancy is a risk factor for the development of cholesterol gallstones. In pregnant women, biliary cholesterol saturation and secretion are increased. To investigate whether this was due to increased cholesterol synthesis, we studied hepatic cholesterol synthesis in Syrian Golden hamsters. Female controls and animals 10- to 14-days pregnant were studied. The studies were performed in the in situ perfused hamster liver. Cholesterol synthesis was determined by measuring the incorporation of 3H2O added to the perfusate into hepatic, perfusate, and bile cholesterol during a 90-min period. In both pregnant groups, bile flow decreased significantly, but biliary cholesterol concentration increased only in the 14-day pregnant group. The cholesterol synthesis rate averaged (mean +/- SD) 172 +/- 27, 127 +/- 37, and 552 +/- 79 nmol X hr-1 X g liver-1 in controls, 10-day, and 14-day pregnant animals, respectively. The 14-day pregnant animals secreted a markedly higher fraction (47.3 +/- 11.3 vs. 11.1 +/- 13.4%; P less than 0.01) of newly synthesized cholesterol into bile but not into perfusate. Chenodeoxycholate, but not cholate, synthesis rate was decreased in both pregnant groups. We conclude from our studies that hepatic cholesterol synthesis increases towards the end of pregnancy in the hamster and that more newly synthesized cholesterol is secreted into bile at that time. This could at least partially explain the increased biliary cholesterol saturation and secretion observed in women in the third trimenon, and explain pregnancy as a risk factor in the development of cholesterol gallstones.     

Hemodynamic effects of epinephrine: concentration-effect study in humans

The hemodynamic effects of three different infusion rates of epinephrine (25, 50, or 100 ng X kg-1 X min-1 for 14 min) were examined in 10 normal human subjects. Ejection fraction and changes in cardiac volumes were assessed by radionuclide ventriculography. Plasma epinephrine was increased to levels that spanned the normal physiological range (178 +/- 15, 259 +/- 24, and 484 +/- 69 pg/ml, respectively). Epinephrine infusions resulted in dose-dependent increases in heart rate (8 +/- 3, 12 +/- 2, and 17 +/- 1 beats/min, mean +/- SE) and systolic pressure (8 +/- 1, 18 +/- 2, and 30 +/- 6 mmHg). Although epinephrine infusions had minimal effects on end-diastolic volume, there were significant increases in stroke volume (+26 +/- 2, 31 +/- 4, and 40 +/- 4%), ejection fraction (+0.10 +/- 0.01, 0.14 +/- 0.02 and 0.16 +/- 0.03 ejection fraction units), and cardiac output (+41 +/- 4, 58 +/- 5, and 74 +/- 1%). These increases in left ventricular performance were associated with a decreased systemic vascular resistance (-31 +/- 3, -42 +/- 2, and -48 +/- 8%). Supine bicycle exercise resulted in similar plasma epinephrine levels (417 +/- 109 pg/ml) and similar changes in stroke volume, ejection fraction, and systemic vascular resistance but greater increases in heart rate and systolic blood pressure. Since infusion-associated hemodynamic changes occurred at plasma epinephrine levels commonly achieved during many types of physical and emotional stress, epinephrine release may have an important role in regulating systemic vascular resistance, stroke volume, and ejection fraction responses to stress in man.