David Holtzman

In vitro effects of inorganic lead on isolated rat brain mitochondrial respiration

The effects of lead acetate on respiration in cerebral and cerebellar mitochondria from immature and adult rats were studied polarographically. With all substrates low lead concentrations produced an increase in respiration. Higher concentrations produced an inhibition of both this lead-induced respiration and ADP-dependent (State 3) respiration. Lead-induced respiration required inorganic phosphate and was inhibited by oligomycin, suggesting a coupling to oxidative phosphorylation. Inhibition of respiration was produced by much lower lead concentrations with NAD-linked citric acid cycle substrates than with succinate or α-glycerophosphate. In partially disrupted mitochondria, NAD-linked substrate oxidation was inhibited at lead concentrations which did not affect NADH oxidation. Thus, in brain mitochondria the NAD-linked dehydrogenases, located in the matrix space, were more sensitive to inhibition by lead than were inner membrane enzymes. All in vitro lead effects on mitochondrial respiration were comparable in cerebral and cerebellar mitochondria isolated from both immature and adult rats.

Respiration and cell volume of primary cultured cerebral astrocytes in media of various osmolarities

Respiration and cell volume of cerebral astrocytes from primary culture were measured in media of various osmolarities. Respiration was measured in 3T3 fibroblasts under similar conditions. Uncoupled respiration and respiration independent of oxidative phosphorylation were obtained by adding dinitrophenol and oligomycin, respectively. In NaCl media, dinitrophenol-stimulated respiration was inhibited at low and high osmolarities. With increasing osmolarity from 363 to 1185 mOsm, oligomycin-insensitive respiration increased and became the predominant respiratory component. The same respiratory changes in response to altered osmolarity were observed in 3T3 fibroblasts. In astrocytes, the oligomycin-insensitive respiration also increased in hyperosmolar sodium acetate media but was unchanged with increasing osmolarity in choline chloride or sucrose media. The increase in oligomycin-insensitive respiration in hyperosmolar NaCl media was blocked by amiloride, an inhibitor of passive Na+ movement. In contrast to amiloride, ouabain, an inhibitor of Na+, K+-ATPase, inhibited a constant amount of respiration with increasing NaCl concentration. The relationship of astrocyte volume to osmolarity was the same in hyper-osmolar media containing NaCl or sucrose. Cell volumes were greater in hypo-osmolar NaCl than in sucrose media. Our results suggest the presence of a Na+-dependent respiratory component in primary cultured cerebral astrocytes in media of increased osmolarity. This respiratory component is not coupled to oxidative phosphorylation or a Na+-K+-ATPase. It may be important in the proposed physiologic role of the astrocyte in maintaining brain extracellular water content and electrolyte concentrations.

Development of Resistance to Lead Encephalopathy During Maturation in the Rat Pup

The purpose of this study was to determine the maturational period during which the rat pup becomes resistant to the toxic effects of lead on the brain. Pups were fed lead, as lead acetate, by esophageal catheter for 14 days beginning at various ages between 14–24 days. The daily lead doses, which produced a hemorrhagic cerebellar encephalopathy in at least 50% of pups, were 400 /μg Pb/g body weight for animals fed from 14 days of age, 800,μg/g for animals fed from 16 days, and 1600 μg/g for animals fed from 18 days. In contrast, pups fed even higher lead doses beginning at 20 days showed only a patchy cerebellar edema by light microscopy while pups fed from 24 days had normal cerebellums by light microscopy. The encephalopathy lead doses in the younger pups resulted in the same cerebellar lead concentrations (about 30 μg/g protein) as the higher lead doses fed pups beginning at 20 or 24 days. When corrected for blood lead concentrations, the cerebellar lead concentrations were 20–25% higher in the encephalopathic compared to the older encephalopathy-resistant animals. This difference may be accounted for by cerebellar hemorrhages in the younger animals. Polarographic studies showed inhibition of respiration in cerebellar slices from animals fed lead from 14 days of age but not in animals fed from 20 or 24 days of age. Our results suggest that, during the en-cephalopathy-sensitive age period, a critical cerebellar concentration of lead is associated with the encephalopathy. Resistance to lead encephalopathy in older animals, with similar cerebellar lead concentrations, may be related to a capacity to sequester lead in new cellular locations away from its site of action on aerobic energy metabolism.

EFFECTS OF OSMOLAR CHANGES ON ISOLATED MITOCHONDRIA OF BRAIN AND LIVER

Abstract— The effects of altered osmolarity on respiration and fine structure were compared in isolated rat cerebral versus liver mitochondria.

Effects of Anticonvulsants on Hyperthermia‐Induced Seizures in the Rat Pup

Summary: We studied the effects of phenobarbital (PB), valproic acid (VPA), and phenytoin (PHT) on the electroencephalograms and behaviorally defined seizure threshold temperatures in rat pups exposed to ambient hyperthermia on the fifth day of life. Animals injected with 10, 20, or 40 μg PB/g body weight (GBW) or 80, 150, or 300 μg VPA/GBW daily from the second day of life showed seizure temperature thresholds that were about 1.0oC higher than those of controls. Pups injected with PHT had thresholds that were equal to or lower than those in controls despite blood levels greater than 20 μg/ml. Preictal rhythmic sharp wave activity appeared in VPA‐injected animals at temperatures below the threshold temperature. All experimental animals showed electrographic epileptiform activity at the threshold temperatures. We conclude that this animal model shows similar anticonvulsant efficacies to those found in human febrile seizures. This model may be useful in determining the efficacy of potentially useful anticonvulsant drugs and their mechanisms of action.

Maturation of Potassium‐Stimulated Respiration in Rat Cerebral Cortical Slices

Abstract: Maximal dinitrophenol‐stimulated respiration and K+‐stimulated respiration were measured polarographically in cerebral cortical slices taken from rats aged 2–60 days. Increasing K+ concentrations produced an increase in respiration in slices from animals aged 15 days and older, but not in slices from animals aged 10 days and younger. Dinitrophenol‐stimulated respiration, or the maximal respiratory capacity of the tissue, showed a similar increase between 10 and 15 days of age. At each age the maximal respiratory capacity was 6–8 ng at 0/mg protein min greater than the maximal K+‐stimulated respiration.

Factors influencing the growth and respiration of rat cerebral astrocytes in primary culture.

Cell densities and respiratory rates of astrocytes from neonatal rat brain grown in primary culture were determined after 20–30 days in vitro. Cells grown in flasks reached lower densities (μg DNA/cm2) and higher protein: DNA ratios than cells grown in petri dishes. Respiratory rates were lower for cells grown in flasks compared to cells grown in dishes. The pH of the medium in flasks fell below 6.9 between feedings while the pH of the medium in dishes remained at about 7.2. Cells grown in dishes with the medium pH adjusted to 6.8 also showed lower final cell densities, higher protein: DNA ratios, and lower respiratory rates, compared to cells grown under similar conditions at pH 7.5. Intermediate values of each parameter were found in cells grown at pH 7.5 for one week and then at 6.8 for 20 days. We conclude that the effects of ambient pH account for the differences in growth characteristics and respiratory rates of astrocytes grown in dishes versus those grown in flasks.

Microsomal Cytochromes in the Immature and Adult Rat Brain

Microsomes of liver and many other tissues contain two hemoproteins, cytochromes b5 and P450, which appear to serve in a mixed-function oxidase system active in the metabolism of steroids, certain drugs, and environmental chemicals (Matsubara et al., 1976; Oshino, 1978). Cytochrome b5 has been found in small quantities in microsomes from mature rabbit, guinea pig, and bovine brains (Inouye and Shinagawa, 1965). These investigators could find no spectrophotometric evidence for the presence of cytochrome P450. Recently, Cohn et al. (1977) and Sasame et al. (1977) reported the presence of a small quantity of P450 in mature rat brain microsomes. In the course of studies on the effects of inorganic lead feedings on cytochromes in the rat brain, we have found spectrophotometric evidence for the presence of both cytochromes b5 and P450. We now are reporting quantitative estimates of these cytochromes in brain microsomes isolated from normal immature and adult rats.

Absence of effects of lead feedings and growth-retardation on mitochondrial and microsomal cytochromes in the developing brain.

Abstract Heme-containing enzymes were measured by absorption spectroscopy in mitochondria and microsomes isolated from the brains of PbCO 3 -fed rat pups. Brain mitochondrial cytochromes also were measured in lead-fed adult rats and in growth-retarded pups. In normal animals, brain mitochondrial and microsomal cytochrome levels increased about twofold from 1 week of age to adulthood. In pups fed lead from birth, the resulting encephalopathy occurred most frequently in the first 2 weeks. Cerebellar mitochondrial cytochromes were decreased but only after 4 weeks of feedings. In pups fed lead from 2 weeks of age, the encephalopathy occurred after 2 weeks of feedings. There were no decreases in cerebral or cerebellar mitochondrial cytochromes. In both groups of pups, brain microsomal cytochromes tended to be higher in lead-fed than in control animals. Lead-feedings did not alter adult brain mitochondrial cytochrome levels. Brain mitochondrial levels also were unchanged in growth-retarded compared to normal rat pups. We conclude that the cytochrome concentrations of mitochondria and microsomes from both the immature and adult rat brain are relatively unaffected by factors such as inorganic lead exposure, known to influence pathways of heme breakdown and synthesis in other tissues. Second, changes in brain mitochondrial cytochrome contents are not associated with the development of lead encephalopathy in the rat pup.

In vitro cellular respiration at elevated temperatures in developing rat cerebral cortex

Cellular respiration in vitro was studied in cerebral cortical tissue from rats 2-60 days of age. Respiration was measured polarographically over the temperature range 34-44 degrees C in tissue slices in a basal condition; maximally stimulated by an uncoupler of oxidative phosphorylation, dinitrophenol; and inhibited by a blocker of mitochondrial oxidative phosphorylation, oligomycin. Basal respiration at 34 degrees C increased about 80% between 7 and 30 days of age. Oligomycin-insensitive respiration did not change with age. Dinitrophenol-stimulated respiration was unchanged from 2 to 10 days and then increased over 100% between 10 and 15 days of age. The Q10 for dinitrophenol-stimulated respiration increased from a value of 1 in tissue from rats 2-10 days of age to about 2 in tissue from rats 15 days and older. Our results confirm the previously reported maturational increases in basal respiration and in respiratory capacity in rat cerebral cortical tissue. The maturational increase in maximal respiratory capacity occurs in a short age interval coincident with a marked increase in the Q10 for the hyperthermic temperature range. Both these properties may be important in the increasing resistance to hyperthermia-induced seizures and their functional sequelae in the rat pup.