Differences in membrane ion transport between hepatocytes from the periportal and the pericentral areas of the liver lobule.

We studied the Na+/K+ pump, Na+/K+ ATPase activity, and oxygen consumption (QO2) in hepatocytes isolated from the periportal (PH) and pericentral (CH) regions of the liver lobule, to provide an insight into the functional properties of these cells. Na+/K+ pump activity was determined using 86Rb+ (a functional analog of K+) and ouabain, a specific inhibitor of this transport system. Our results indicate the the Na+/K+ pump and Na+/K+ ATPase activity are significantly lower in CH than in PH, although basal ouabain-sensitive (OS) QO2 was negligible in both of these cell preparations. However, OSQO2 was significantly lower in CH than in PH when the Na+/K+ pump was activated using the ionophore nystatin in a Na(+)-containing medium. These results indicate that the differences in membrane ion transport exist between hepatocytes from different locations of the liver lobule.

Effects of hypothermic perfusion on pulmonary circulation in isolated rabbit lung.

The effects of hypothermic perfusion have been studied by using different perfusates in 24 isolated rabbit lung preparations, divided into three groups: G1, perfused with blood (hematocrit of 10%) and G2 and G3, perfused with erythrocyte-free plasma plus 6% protein in saline. In both G1 and G2 groups left atrial pressures were kept below airway pressure (Zone II conditions), and in G3 it was higher than airway pressure (Zone III conditions). Perfusate flow, pulmonary artery pressure, pulmonary vascular resistance, left atrial pressure, fluid filtration rate, colloid osmotic pressure and temperature were not different (p > 0.1) between G1 and G2 at the beginning of the experiments. Lowering perfusate temperature from 38 degrees C to 28 degrees C produced a significant increase in pulmonary artery pressure and pulmonary vascular resistance in G1 but they decreased in G2 lungs (p < 0.05). Fluid filtration rate increased in both groups during hypothermia. These responses were not inhibited by an alpha-adrenergic receptor blocker or a pulmonary vasodilator. In G3 lungs no changes were observed. The differences in the hemodynamic effects of hypothermia observed in G1 and G2, both in Zone II conditions, could result from the differences in the vessel distention state obtained by each of the perfusate before initiating hypothermia. As perfusate viscosity increase with cold, a greater possibility of vessel distention in G2 lungs occurs. This explains the decrease in pulmonary artery pressure and pulmonary vascular resistance with cold in this group. The increase in fluid filtration rate observed with hypothermia in G1 and G2 may be due to increases in fluid exchange area.

Oxidative capacity distribution in skeletal muscle fibers of the rat.

To study the distribution of oxidative capacity in muscle fibers, mitochondrial volume density and the oxidative capacity of isolated mitochondria were evaluated. Mitochondria were isolated from the subsarcolemmal and interfibrillar areas of the soleus (a muscle largely made up of slow oxidative fibers) and the gastrocnemius medial head (a muscle largely made up of fast glycolytic fibers) of the rat, and their oxidative capacities were evaluated using NADH- and FADH-generating substrates. In the soleus muscle, the subsarcolemmal mitochondria showed a lower oxidative capacity than interfibrillar mitochondria when NADH-generating substrates were used. This difference was not observed when FADH-generating substrates were used. In the gastrocnemius, there were no differences in the oxidative capacity of the subsarcolemmal and the interfibrillar mitochondria. Additionally, citrate synthase activity was found to be lower in mitochondria isolated from the subsarcolemmal area of the soleus than in the other mitochondrial preparations. These findings indicate that the difference in oxidative capacity of the isolated mitochondria is not related to differences in the inner mitochondrial membranes. Mitochondrial volume density was evaluated using electron micrographs of the subsarcolemmal and interfibrillar areas of slow oxidative fibers from the soleus and fast glycolytic fibers from the gastrocnemius. In the slow oxidative fibers, mitochondrial volume density in the subsarcolemmal area was four times higher than in the interfibrillar area. In the fast glycolytic fibers, mitochondrial volume densities in the subsarcolemmal and interfibrillar areas did not differ from that of the interfibrillar area of the slow oxidative fibers. The oxidative capacity of the tissue, calculated by multiplying the mitochondrial oxidative capacities by the mitochondrial volume densities, was 2-4 times higher in the subsarcolemmal areas of the soleus fibers than in the other areas studied. This was true in spite of the fact that the oxidative capacity of the subsarcolemmal mitochondria of the slow oxidative fibers was lower than those of the other mitochondrial populations studied. These results indicate that the difference in oxidative capacity between slow oxidative fibers and fast glycolytic fibers is the result of the much greater mitochondrial volume density in the subsarcolemmal area of the slow oxidative fibers.

Effect of chronic hypoxia on acetaminophen metabolism in the rat.

The effect of chronic hypoxia (10.5% O2 for 8-9 days) on acetaminophen metabolism was studied in vivo or in isolated cell or microsomal systems. Results from in vivo studies with oral administration of acetaminophen showed that in hypoxic rats, the plasma appearance of the drug was delayed and the plasma half-life was increased. Analyses of the area under the curve (AUCoral) showed that this value was higher in hypoxic rats, whereas the rate constants for elimination (kelim) and absorption (kabs) were lower in these animals. Formation of the glucuronide and sulfate conjugates was decreased significantly (P less than 0.05) in hypoxic animals. The calculated volume of distribution (Vd) after an intravenous dose was not different in either group but total clearance (CL) was 35% lower in hypoxic rats. Studies with isolated hepatocytes from both groups revealed that glucuronidation and sulfation were inhibited markedly at low O2 concentrations. The O2 concentrations required for half-maximal production (P50 values) of glucuronide (2.3 microM O2) and sulfate (1.8 microM O2) conjugates in cells from hypoxic animals were lower than for control cells (5.3 microM and 3.9 microM O2 for glucuronide and sulfate conjugates, respectively). Maximal rates of conjugation in cells from hypoxic rats were 60-70% of control rates. Similar decreases in microsomal UDP-glucuronosyltransferase and cytosolic sulfotransferase activities were found in livers of animals exposed to chronic hypoxia. These lower P50 values are consistent with a lower P50 for oxidation of mitochondrial cytochromes in hypoxic cells. In comparison, the P50 for glutathione conjugation (4.1 microM O2) was not statistically different from control (4.6 microM O2), but the maximal rate was 65% higher. The results show that chronic hypoxia causes a change of absorptive processes and decreased glucuronidation and sulfation reactions which affects the disposition of acetaminophen and potentially the disposition of a variety of other exogenous and endogenous compounds.

Defining the resistance to oxygen transfer in tissue hypoxia.

Studies of O2 supply in freshly isolated adult mammalian cells provide new insight into the factors that limit mitochondrial oxygenation in vivo. Of particular importance, mitochondria are present at high densities and often in apparent clusters, both of which contribute to local O2 gradients under hypoxic conditions. Current evidence indicates that the mitochondrial distribution is a component of the differentiated phenotype of adult mammalian cells and that specific motors and anchoring mechanisms are present to allow redistribution in response to developmental, physiological and pathological challenges. To compare the importance of resistance to O2 transfer under different conditions and at different sites along the supply path in vivo, a simple mathematical expression of relative resistance to O2 supply is introduced. Under various pathophysiological conditions, this resistance increases in specific regions of the pulmonary, circulatory or cellular supply path and results in O2 deficiency in the mitochondria. Regardless of cause, the relative resistance increases dramatically in the vicinity of mitochondrial clusters during hypoxia.

Bioavailability of dietary glutathione: effect on plasma concentration.

Plasma glutathione (GSH) concentration in rats increased from approximately 15 to 30 microM after administration of GSH either as a liquid bolus (30 mumol) or mixed (2.5-50 mg/g) in AIN-76 semisynthetic diet. GSH concentration was maximal at 90-120 min after GSH administration and remained high for over 3 h. Administration of the amino acid precursors of GSH had little or no effect on plasma GSH values, indicating that GSH catabolism and resynthesis do not account for the increased GSH concentration seen. Inhibition of GSH synthesis and degradation by L-buthionine-[S,R]-sulfoximine and acivicin showed that the increased plasma GSH came mostly from absorption of intact GSH instead of from its metabolism. Plasma protein-bound GSH also increased after GSH administration, with a time course similar to that observed for free plasma GSH. Thus dietary GSH can be absorbed intact and results in a substantial increase in blood plasma GSH. This indicates that oral supplementation may be useful to enhance tissue availability of GSH.

Oxidative capacity distribution in the cardiac myocytes of hypermetabolic rats.

To study the distribution of oxidative capacity in the cardiac myocyte in control and in hypermetabolic (hyperthyroid) rats, we evaluated mitochondrial volume density (Vv,mi) distribution by morphometry and oxidative capacity, cytochrome a + a3 concentration and protein yield of isolated subsarcolemmal and interfibrillar mitochondria by biochemical techniques. In control animals Vv,mi underneath the sarcolemma was higher than in the center of the myocytes and it decreased linearly with increasing distance from the capillaries. Interfibrillar mitochondria showed a greater oxidative capacity and a high concentration of cytochrome a + a3 than subsarcolemmal mitochondria. Values of Vv,mi and its distribution were not changed by the hypermetabolic condition. Oxidative capacity and cytochrome a + a3 concentration were higher in the interfibrillar mitochondria but not in the subsarcolemmal mitochondria of the hypermetabolic rats. The product of the oxidative capacity of the mitochondria times the Vv,mi indicated that the oxidative capacity of the interfibrillar zone is 30% higher than that of the subsarcolemmal zone. In the hypermetabolic rats, due to the increase in oxidative capacity of the interfibrillar mitochondria, the oxidative capacity of this zone was 80% higher than that of the subsarcolemmal zone.

Muscle capillarity in rats with increased blood oxygen affinity.

To investigate the effects of low P50Hb on muscle capillarity, rats were injected with Na-cyanate (50 mg/kg BW) every day for 2 or 4 weeks (experimental). Controls received injections of saline. After 2 weeks P50 was reduced in the experimental animals (20.4 vs 30.3 Torr). After 4 weeks experimental animals showed a 13% increase in hematocrit and Hb. PVO2 was lower in experimental than in control rats (28.3 +/- 1.0 vs 33.8 +/- 4.7 mm Hg). Fiber cross-sectional area (FCSA), capillary density (CD) and capillary-to-fiber ratio (C/F) were measured in sections treated with the ATPase technique. FCSA was smaller in experimental than in controls (3000 +/- 491 vs 4121 +/- 415 micron 2 for the soleus and 3633 +/- 236 vs 4251 +/- 194 micron 2 for the gastrocnemius) and CD was higher (729 +/- 99 vs 578 +/- 75 cap/mm2 in the soleus and 388 +/- 19 vs 323 +/- 14 cap/mm2 in the gastrocnemius) but C/F was not different. Diffusion distances for O2 measured by the closest individual method were not significantly different between the two groups. The higher CD in the experimental animals seems to be the result of their smaller FCSA and not the result of capillary proliferation. The volume of tissue served by a capillary was significantly reduced in the experimental animals. This could help the transfer of O2 to the muscle in animals with a low P50 Hb.

Fiber composition and capillarity in growing guinea pigs acclimated to cold and cold plus hypoxia.

The central portion of the medial head of the gastrocnemius of control (normoxic and normothermic), hypoxia-, cold-, and cold plus hypoxia-acclimated guinea pigs was analyzed for capillary supply and fiber composition to elucidate changes in capillarity induced by environmental stresses. The muscle was cut at midbelly, frozen, sectioned, and stained for myosin ATPase. Fiber cross-sectional areas; percentages of slow-twitch oxidative (SO), fast-twitch oxidative-glycolytic (FOG), and fast-twitch glycolytic (FG) fibers; and numbers of capillaries around each fiber type were measured. Growth rates of all four guinea pig groups were similar. Capillarity was not affected by acclimation to hypoxia. Cold and cold plus hypoxia acclimation led to increased numbers of capillaries around the fiber in all three fiber types. In addition, significant increases in the percentage of FOG fibers and concomitant decreases in the percentage of FG fibers compared to controls were found in cold and in cold plus hypoxia indicating that a transformation of fiber type from FG to FOG had occurred. The increase in FOGs at the expense of the FGs did not occur in the guinea pigs grown in a hypoxic environment. The increased total capillarity in those muscles studied was the result of more capillaries around all fiber types and was not due to simple transformation of fibers.

Long-term isoprenaline administration produces an increase in capillarity in the soleus muscle of the rat.

The effects of isoprenaline administration (300 micrograms/kg for 5 weeks) on rat soleus muscle capillarity and glycolytic and oxidative capacities were evaluated. The treatment resulted in ventricular hypertrophy. The activities of lactic dehydrogenase, pyruvate kinase, citrate synthase, and cytochrome c oxidase in soleus muscle homogenates were not different between control and isoprenaline-injected animals. Capillaries were visualized in muscle cross sections treated to demonstrate ATPase activity after acid preincubation. Capillary density was higher in the experimental (873 +/- 38 capillaries/mm2) than in the control (713 +/- 33 capillaries/mm2) animals. Capillary to fiber ratio was also higher in the experimental (2.47 +/- 0.10) than in control (2.09 +/- 0.08) animals, but fiber cross-sectional area was not changed by the treatment (2836 +/- 87 microns2 in controls and 2951 +/- 136 microns2 in experimental). A plot of capillary to fiber ratio vs. fiber cross-sectional area showed that at a given fiber cross-sectional area the value of capillary to fiber ratio of the treated animals was higher than that of the controls. This indicates that treatment resulted in the proliferation of microvessels. The results suggest that prolonged beta-adrenergic stimulation results in the development of new capillaries but that this is not accompanied by increases in the oxidative capacity of the soleus muscle of the rat.

Changes in soleus muscle capillarity, oxidative capacity and fiber composition in rats recovering from hyperthyroidism.

Soleus muscle oxidative capacity, fiber composition and capillarity were studied in rats that were injected with 300-400 micrograms/kg of triiodothyronine (T3) every other day for 28 days and then left to recover for 4, 8, 14, 21 or 28 days. The activities of cytochrome c oxidase and citrate synthase and the capacity to oxidize pyruvate plus malate of muscle homogenates were 40, 53 and 21% higher than control values at the end of T3 administration. The activity of citrate synthase and the capacity to oxidize pyruvate plus malate decreased rapidly after discontinuing T3 administration, reaching values below those of base line controls in 14 days, but were not different than controls at 21 days. The activity of cytochrome c oxidase decreased more slowly, reaching base line values 28 days after discontinuing treatment with T3. Fiber composition was studied in cross-sections treated to demonstrate ATPase activity after acid pre-incubation. Three fiber types were present in the soleus of control animals: Type 1 with a strong ATPase reaction (86%), type II a with a weak ATPase reaction (12%) and a small proportion of type IIc with intermediate ATPase reaction (2%). After 28 days of T3 administration the proportion of type I fibers was significantly reduced (74%) while that of IIc was higher (18%). Fiber composition returned to normal between 21 and 28 days after T3 treatment was discontinued. Muscle capillarity was increased by T3 administration. Values of capillary density or capillary to fiber ratio at a given fiber cross sectional area were approximately 30% higher than controls at the end of T3 treatment and remained high until 21 days after discontinuing T3.(ABSTRACT TRUNCATED AT 250 WORDS)

Capillarity, oxidative capacity and fibre composition of the soleus and gastrocnemius muscles of rats in hypothyroidism.

Muscle capillarity, mean and maximal diffusion distances and muscle fibre composition were evaluated in frozen sections stained for myosin ATPase of the soleus and the white area of the gastrocnemius medial head (gastrocnemius) of rats made hypothyroid by the injection of propylthiouracil (PTU) (50 mg kg-1) every day for 21 or 42 days. Oxygen consumption in the presence of excess ADP and Pi with pyruvate plus malate as substrates and the activity of cytochrome c oxidase were measured in muscle homogenates. Treatment with PTU decreased body oxygen consumption and the concentration of triiodothyronine in plasma. The capacity of the soleus and gastrocnemius muscles' homogenates to oxidize pyruvate plus malate and their cytochrome c oxidase activity were reduced after 21 or 42 days of treatment with PTU. Fibre composition in the soleus muscle was changed by treatment with PTU. There was a decrease in the proportion of type IIa or fast glycolytic oxidative fibres and an increase in type I or slow oxidative fibres. After 21 days of PTU administration there was also an increase in the proportion of fibres classified as IIc. The changes in fibre composition are believed to be the result of changes in the types of myosin synthesized by the fibres. Therefore, the fibres classified as IIc are, most probably, IIa fibres in the process of changing their myosin to that of the type I fibres. No changes in fibre composition were evident in the white area of the gastrocnemius medial head, an area made up of IIb or fast glycolytic fibres. The indices of capillarity: capillary density and capillary to fibre ratio, as well as mean and maximal diffusion distances from the capillaries, were not changed by the treatment with PTU in the muscles studied. The lack of changes in capillarity in spite of significant changes in oxidative capacity indicates that in skeletal muscle capillarity is not necessarily related to the oxidative capacity of the fibres.

Capillarity and oxygen diffusion distances of the soleus muscle of guinea pigs and rats. Effects of hyperthyroidism.

The relationship between capillarity and oxidative capacity in the soleus muscle of rats and guinea pigs injected with triiodothyronine (T3) or with saline for up to 4 weeks was studied. The rats' soleus weight and FCSA were not affected by T3, but the guinea pigs that received T3 had smaller muscle weight and FCSA than the controls. The activities of cytochrome c oxidase and citrate synthase were significantly (41 and 65%) higher in the T3 than in the control rats. T3 administration did not affect the activities of these enzymes in the soleus of the guinea pigs. Capillary density (CD) was higher in T3 rats (892 +/- 80 vs 622 +/- 54 caps/mm2), and in T3 guinea pigs (1219 +/- 95 vs 739 +/- 142 caps/mm2). The higher CD in T3 rats was due to growth of new microvessels, while in the T3 guinea pigs it was due to a reduction in FCSA. Mean and maximal diffusion distances evaluated by the closest individual method were reduced by 2.02 and 3.37 microns in rats, and by 3.73 and 6.16 microns in guinea pigs. The magnitude of the reduction in diffusion distances brought about by the increased capillary density was partially offset by a concomitant change in the capillary arrangement from an ordered (hexagonal), towards a random distribution. These results seem to indicate that skeletal muscle capillarity is not necessarily determined by the oxidative capacity of the fibers.

The effect of hyperthyroidism on capillarity and oxidative capacity in rat soleus and gastrocnemius muscles.

Muscle fibre composition and capillarity were evaluated in frozen sections stained for myosin ATPase of the soleus and the white area of the medial head of the gastrocnemius of rats made hyperthyroid by injections of triiodothyronine (300-400 micrograms/kg body weight, every other day) for 2, 3 and 4 weeks. O2 consumption of homogenates of these muscles in the presence of excess inorganic phosphate (Pi) and ADP with pyruvate and malate as substrates was also measured. Increased oxidative capacity was observed in the soleus homogenates of hyperthyroid animals after 2 weeks of treatment while no changes were observed in the oxidative capacity of the homogenates of the white area of the medial head of the gastrocnemius, even after 4 weeks of treatment. Hyperthyroid animals showed a greater capillarity than controls in both muscles. In the soleus this was evident after 2 weeks of treatment while in the white area of the medial head of the gastrocnemius, it was evident only after 4 weeks of treatment. Fibre composition was affected in the soleus after 4 weeks of treatment. In control animals two fibre types were present in the soleus: slow-twitch oxidative fibres (s.o. or type I fibres) with a high ATPase activity after acid pre-incubation and fast-twitch glycolytic oxidative fibres (f.o.g. or type IIa fibres) with a low ATPase activity after acid pre-incubation. In the soleus of the hyperthyroid animals, a third fibre type with intermediate ATPase activity after acid pre-incubation was also present. This most probably represents a change in the type of myosin being synthesized by some fibres. No changes in fibre composition were observed in the white area of medial head of the gastrocnemius which was made up of only fast-twitch glycolytic fibres (f.g. or type IIb fibres). The changes in oxidative capacity and capillarity in the soleus preceded and did not seem to be related to the changes in the type of myosin being synthesized. The increased capillarity found in the white area of the medial head of the gastrocnemius of the hyperthyroid animals, in the absence of an increase in the oxidative capacity, indicates that the latter is not the only factor that determines capillarity in skeletal muscle.

Increased capillary supply in skeletal muscle of guinea pigs acclimated to cold.

The ATPase technique was used to visualize blood capillaries and to study fiber composition in 10-micrometer transverse sections of guinea pig gastrocnemius and soleus muscles. A control group of newborn, weanling, juvenile and adult male guinea pigs (GP) (BW = 89-1274 g) was studied in a 20-24 degrees C environment (22 degrees C GP) while 2-3 week old animals were exposed continuously to 5 degrees C for 2-18 weeks before sacrifice (5 degrees C GP) (BW = 239-1074 g). Body weight gain was not affected by cold exposure; however, the gastrocnemius and soleus muscles of the 5 degrees C GP grew at a slower rate than did the muscles of the 22 degrees C GP. The equations relating fiber cross sectional area (FCSA) and muscle weight (MW) were not different between the 22 degrees C GP and 5 degrees C GP for the soleus and gastrocnemius. Therefore, in both muscles at the same BW, FCSA was smaller in the 5 degrees C GP than in the 22 degrees C GP. In both of the two muscles of each group, capillary density (CD) decreased hyperbolically with increasing FCSA, while the capillary to fiber ratio (C/F) and the average number of capillaries around each fiber (CAF) increased linearly with increasing FCSA. The regression lines for CD, C/F and CAF versus FCSA for both muscles were parallel between groups, but at any FDSA, the CD, C/F and CAF were greater in the 5 degrees C GP than in the 22 degrees C GP. Percent fiber composition of the gastrocnemii of the 22 degrees C GP and 5 degrees C GP were not different; however, at the same FCSA each fiber type had a greater capillary supply in the 5 degrees C GP. The increased capillarity in the gastrocnemius and soleus muscles of the 5 degrees C GP suggests an improved capacity for oxygenation, a response which would correlate well with the increased oxygen utilization during prolonged cold exposure.

Pulmonary arterial hypertension in male and female chickens at 3300 m.

Two-hundred one-dayy-old male (M) and female (F) chickens were exposed to 3300 m (HA). Two-hundred control chickens were raised at sea level (SL). Chickens from both HA and SL were studied each week from the 3rd to the 7th week of age. Pulmonary arterial pressure (Ppa) was measured under local anesthesia in conscious animals. Hb, Hct and red blood cell counts (E) and the weights of the right ventricle (RV), left ventricle (LV) and septum (S) were obtained. HA chickens, both males and females, reached lower body weights than SL chickens. Ppa were 16 and 43 mm Hg (2.13 and 5.73 kPa) while RV/(RV + LV + S) values were 0.195 and 0.347 in SL and HA chickens, respectively. RV was 90% greater and LV 20% smaller in the HA chickens. S was not affected by altitude. Hb, Hct and E were higher in the HA birds. Sixteen HA M and 1 HA F died with signs of right heart insufficiency (RHI), however, M did not show greater values of Ppa or RV/(RV + LV + S) than F at SL or at HA. This indicates that the higher incidence of RHI in the M cannot be attributed to a higher responsiveness of their pulmonary vasculature to hypoxia.

Chronic hypoxia does not affect guinea pig skeletal muscle capillarity.

The soleus and gastrocnemius muscles of chronically hypoxic guinea pigs were analyzed for capillary supply and myoglobin concentration. Weanling male guinea pigs were exposed to a simulated altitude of 5,100 m and an average ambient temperature of 22% C for 2, 4, 6, 10 and 14 weeks (range of BW 244--965 g). The soleus and gastrocnemius-plantaris muscles of one leg were analyzed for myoglobin concentration while the soleus and medial head of the gastrocnemius of the contralateral leg were cut at the midpoint, frozen and sectioned in a cryostat. The myosin ATPase method was used to visualize fibers and capillaries. Values of muscle weight, fiber cross sectional area, capillary density, capillary to fiber ratio and the number of capillaries around the fiber were compared to the values of the same parameters from normoxic guinea pigs selected to match the average body weights of the hypoxic animals. The growth rates of the two groups were not different. No significant differences in the regression lines for the normoxic and hypoxic animals were found so that when the data were combined no significant differences in the normoxic lines were introduced by adding the values of the hypoxic animals. The myoglobin values were significantly higher only in the hypoxic soleus after 14 weeks of exposure.