In vivo [31P]NMR studies on the influence of age on rat brain hypoxia

In this paper the response of cerebral phosphate metabolism to mild hypoxia in young, medium and old rats has been studied via in-vivo [31P]nuclear magnetic resonance (NMR). It was found that the young adults (5-6 months) were more sensitive to this mild stress than either the mature adult (11-12 months) or senescent (23-24 months) rats even though the depth of hypoxia (paO2 = 45-55 mm Hg) was equal for all age groups. They displayed an earlier onset of acidosis, a greater fall in PCr and larger rise in Pi. This response is presumably an attempt to maintain adequate adenosine triphosphate (ATP) levels via anaerobic glycolysis. In contrast, mature adults and senescent adults appear to be able to maintain ATP levels by increasing mitochondrial rates. Acidosis is less severe as are drops in PCr and rises in Pi. Recovery is less complete for the young rats: Pi levels remain high while PCr and pHi levels stay low after normoxia has been reinstigated. All metabolite levels in the mature and senescent adults return to within 10% of control levels. All the data were analyzed and differences were found to be statistically significant. This study reveals that, contrary to popular belief, mature and old rats respond more favorably to reduced O2 than younger individuals. This is due to a more severe anaerobic acidosis in the latter age group. Speculations to explain this disparity are based on the fact that previous in-vitro studies involve systems that are totally or partially disconnected from the organism will not account for important feedback control present in an in-vivo system as studied here.     

Quantitation of high energy phosphate compounds and metabolic significance in the developing dog brain.

The phosphate metabolites, PCr, ATP, ADP and inorganic phosphate (Pi), were quantitated in the brain of the newborn, neonatal, juvenile and adult dog to investigate the potential control mechanisms responsible for increased ATP demands during development. The concentrations of PCr and Pi were measured in vivo by MRS using the enzymatic-measured ATP as the internal standard. Phosphocreatine values increased during development from 2.08 mmol/kg wet weight in the 0-2 day newborn to 5.11 mmol/kg wet weight in the adult brain and paralleled the increases in the total creatine pool (PCr + Cr) from 4.12 to 10.05 mmol/kg wet weight. Brain ATP concentrations increased approximately 40% during postnatal development; however, when expressed as intracellular concentration, no increase in ATP was apparent due to the age-dependent decrease in extracellular space. The Pi concentration, estimated by MRS, increased significantly during postnatal development with a range of 1.78 to 2.52 mmol/kg wet wt, then decreased to 1.97 mmol/kg wet weight at adulthood. In those developmental stages where total Pi was measured enzymatically on freeze-clamped tissue, the NMR visible Pi comprised about 48 to 93% of the total, with the highest percentage being visible in the newborn brain. The intracellular pH decreased from 7.21 in the newborn to 7.10 in the adult. With development, the free ADP concentration, calculated from the components of the creatine kinase equilibrium, ranged from 27 to 34 microM. These values are close to the apparent in vitro Km of ADP for oxidative phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Axonal transport of nucleosides, nucleotides and 4S RNA in the neonatal rat visual system

The axonal migration of RNA, the nucleoside uridine and its nucleotide derivates (NS/NT) were compared in neonatal and young adult rat optic axons. Tritiated uridine was injected into right eyes of developing (1- or 4-day-old) and young adult (40-day-old) rats which were sacrificed at times after injection ranging from 6 h to 20 days. Right and left lateral geniculates were removed and assayed for trichloroacetic acid soluble (NS/NT) and RNA radioactivity. Left minus right geniculate (L-RLG) radioactivity was used as an index of axonally migrating radioactivity. Results showed that uridine and its phosphorylated derivatives were transported along both neonatal and young adult rat optic axons. Greater than 90% of right geniculate (blood-borne) TCA soluble radioactivity was metabolized to volatile substances (probably 3H2O) by three days after injection, leaving approximately 3% of the neonatal and approximately 10% of the adult activity as [3H]NS/NT. In left geniculate fractions (containing transported material) approximately 15% and 40% of total TCA soluble radioactivity was present as [3H]NS/NT in neonates and adults, respectively. Thus, axonal NS/NT appears to be relatively protected from degradation when compared with blood-borne NS/NT. The amount of L-RLG [3H]RNA in the neonates was 10 times higher than in young adults. Peaks of neonatal [3H]RNA occurred at 5 and 10 days after birth, whether injections were made at 1 or 4 days of age indicating that this [3H]RNA may be linked to developmental events. Gel electrophoretic analysis of neonatal geniculate RNA indicated that a small portion of the [3H]RNA in the first peak represented axonally transported 4S RNA. The remainder of the L-RLG [3H]RNA in the neonates was probably due to a rapid and efficient incorporation of axonally transported [3H]NS/NT into extraaxonal geniculate RNA. In contrast, little or no axonal RNA transport could be demonstrated in the young adults.     

Low brain intracellular free magnesium in mitochondrial cytopathies.

The authors studied, by in vivo phosphorus magnetic resonance spectroscopy (31P-MRS), the occipital lobes of 19 patients with mitochondrial cytopathies to clarify the functional relation between energy metabolism and concentration of cytosolic free magnesium. All patients displayed defective mitochondrial respiration with low phosphocreatine concentration [PCr] and high inorganic phosphate concentration [Pi] and [ADP]. Cytosolic free [Mg2+] and the readily available free energy (defined as the actual free energy released by the exoergonic reaction of ATP hydrolysis, i.e., deltaG(ATPhyd)) were abnormally low in all patients. Nine patients were treated with coenzyme Q10 (CoQ), which improved the efficiency of the respiratory chain, as shown by an increased [PCr], decreased [Pi] and [ADP], and increased availability of free energy (more negative value of deltaG(ATPhyd)). Treatment with CoQ also increased cytosolic free [Mg2+] in all treated patients. The authors findings demonstrate low brain free [Mg2+] in our patients and indicate that it resulted from failure of the respiratory chain. Free Mg2+ contributes to the absolute value of deltaG(ATPhyd). The results also are consistent with the view that cytosolic [Mg2+] is regulated in the intact brain cell to equilibrate, at least in part, any changes in rapidly available free energy.     

Effect of the immunosuppressant drug FK506 on neonatal cerebral mitochondrial function and energy metabolism after transient intrauterine ischemia in rats

Mitochondrial respiratory activities and energy metabolism were measured in neonatal rat brains to evaluate the influence of transient intrauterine ischemia on the near-term fetus and to assess the effect of the immunosuppressant drug FK506 treatment. Transient intrauterine ischemia was induced by 30 min of right uterine artery occlusion at 17 days of gestation in Wistar rats. The vehicle or 1.0 mg/kg of FK506 was administered after 1 h of recirculation. All of the pups were delivered by cesarean section at 21 days of gestation and samples of cerebral cortical tissue were obtained from pups at 1 h after birth. The mitochondrial respiration was measured polarographically in homogenates. For the analysis of ATP, ADP, and AMP, neonatal brains were frozen in situ and fluorometric enzymatic techniques were used. In the neonatal cortical tissue exposed to ischemia, mitochondrial respiratory activities and ATP concentrations decreased significantly to approximately 59 and 67% of those in normoxic controls, respectively. The deterioration of both mitochondrial respiratory activities and high-energy phosphates was prevented by FK506, given 1 h after the start of recirculation. The present results indicate that transient intrauterine ischemia is accompanied by mitochondrial dysfunction and cellular bioenergetic failure in the neonatal rat brain and suggest that treatment with FK506 prevents the deterioration, even when administered after the ischemic periods.     

Comparison of cerebrovascular response to hypoxic and carbon monoxide hypoxia in newborn and adult sheep

Cerebral blood flow (CBF) responses to two types of isocapnic hypoxia, hypoxic hypoxia (HH) and carbon monoxide hypoxia (COH), were examined in seven unanesthetized adult sheep by the radiolabeled microsphere technique. Comparisons were made with newborn lambs (5-12 days old) previously studied under similar conditions. The arterial O2 content (CaO2) was reduced in a graded manner to 50-60% of the control value. During HH, CBF increased to maintain cerebral O2 delivery (CaO2 X CBF) in both adults and newborns; however, cerebral O2 uptake (CMRO2) did not change. Although CMRO2 was higher in newborns, the responses of CBF/CMRO2 to HH did not differ significantly in newborns and adults. In newborns, regional CBF showed that brainstem areas were particularly responsive to HH. In both age groups, CBF increased to a greater extent with COH than with HH for similar reductions in CaO2. This resulted in an increase in cerebral O2 delivery with COH. The degree to which COH differed from HH correlated with the magnitude of the leftward shift of the oxyhemoglobin dissociation curve that accompanies COH. In adults, CMRO2 fell by 16% with COH but was maintained in newborns. We conclude that maintenance of cerebral O2 delivery during acute, isocapnic HH is a property of CBF regulation common to both newborn and adult sheep. During COH, the position of the oxyhemoglobin dissociation curve is an additional factor that sets the level of O2 delivery. The fetal conditions of low CaO2 and a left-shifted oxyhemoglobin dissociation curve may have provided the newborn with a microcirculation better suited for maintaining CMRO2 during COH.     

The Response of Neonates to Intra-uterine Sounds

Playing prerecorded intra-uterine sounds to newborns reportedly soothes the babies. This study investigates this claim, particularly because of its promise for identifying neonates with severe hearing losses. 60 babies, up to eight days old, were observed individually under two conditions. In one, activity was rated on the Brazelton scale before and during presentation of intra-uterine sounds. In another, similar ratings were made but no sounds presented. The results indicate that intra-uterine sounds calm 90 per cent of babies who are fussy or crying but have no evident effect on babies who are awake but merely alert or who are slightly drowsy. These findings are discussed and improvements in procedure are suggested for use of intra-uterine sounds in routine screening for severe neonatal hearing loss.     

Platelet and platelet-derived microparticle surface factor V/Va binding in whole blood: differences between neonates and adults

Platelet-derived microparticles (PDMP) appear to play a major role in the generation of procoagulant activity. In this study, we describe a novel flow cytometric method that allows direct evaluation of the procoagulant activity of PDMP and platelets in the physiological milieu of whole blood. The percent PDMP generated in response to calcium ionophore A23187 and calcium was increased in preterm neonates (67.5+/-3.4%, mean +/- S.E.M., n = 8, p <0.05) and term neonates (67.2+/-2.7%, n = 7, p<0.05) compared with adults (49.5+/-3.4%, n = 13). However, in preterm neonates A23187/calcium-induced binding of factor V/Va to PDMP and platelets (22.8+/-5.6 fluorescence units) was markedly reduced (p <0.05) compared to term neonates (58.2+/-7.2) and adults (50.6+/-6.3). In preterm blood, A23187/calcium-induced binding of factor V/Va to PDMP and platelets returned to adult levels when: a) adult plasma, rather than autologous preterm neonatal plasma, was added; or b) factor V, but not factor VIII, was added to autologous preterm neonatal plasma. In summary: 1) We have developed a flow cytometric method for the direct detection of procoagulant PDMP and platelets in whole blood. 2) Compared to adults and term neonates, PDMP and platelets of preterm neonates bound markedly less factor V/Va (reflecting reduced procoagulant activity), because of a relative lack of factor V in preterm neonates. 3) This procoagulant defect in PDMP and platelets may contribute to the propensity of preterm neonates, but not term neonates, to intraventricular hemorrhage. 4) The percent PDMP does not necessarily reflect the degree of procoagulant activity of PDMP or platelets.     

Metabolic and nonmetabolic components of fatigue monitored with 31P-NMR

The goal of this study was to determine the roles of metabolic and nonmetabolic factors in muscle fatigue. Rat gastrocnemius muscles were fatigued by stimulation of the nerve (n = 6) or muscle (n = 4, after 2 days of denervation). ³¹Phosphorus nuclear magnetic resonance spectroscopy was used to measure levels of intracellular inorganic phosphate (Pi) and hydrogen ions (H⁺) (which are thought to inhibit contraction) and the high-energy phosphates, phosphocreatine (PCr), and ATP. For both indirect and direct stimulation, with fatigue to ≈60% initial tetanic force, [Pi] increased from ≈3.5 mmol/L to ≈20 mmol/L and [PCr] decreased from ≈27 mmol/L to ≈9 mmol/L. However, with continued fatigue to 25–35% initial tetanic force, neither [Pi] or [PCr] changed further. [ATP] and pH changed only slightly during fatigue. The results are consistent with early fatigue arising from metabolic inhibition of contraction; but later fatigue arising independent of metabolites, due to impaired activation beyond the neuromuscular junction. © 1994 John Wiley & Sons, Inc.     

Effect of acute stress on NTPDase and 5′-nucleotidase activities in brain synaptosomes in different stages of development

The aim of the present study was to examine the effect of acute restraint stress on rat brain synaptosomal plasma membrane (SPM) ecto-nucleotidase activities at specific stages of postnatal development (15-, 30-, 60- and 90-day-old rats) by measuring the rates of ATP, ADP and AMP hydrolysis 1, 24 and 72 h post-stress. At 1 h after stress NTPDase and ecto-5′-nucleotidase activities were decreased in rats aged up to 60 days old. In adult rats elevated enzyme activities were detected, which indicated the existence of different short-term stress responses during development. A similar pattern of ATP and ADP hydrolysis changes as well as the ATP/ADP ratio in all developmental stages indicated that NTPDase3 was acutely affected after stress. The long-term effect of acute stress on NTPDase activity differed during postnatal development. In juvenile animals (15 days old) NTPDase activity was not altered. However, in later developmental stages (30 and 60 days old rats) NTPDase activity decreased and persisted for 72 h post-stress. In adult rats only ATP hydrolysis was decreased after 24 h, indicating that ecto-ATPase was affected by stress. Ecto-5′-nucleotidase hydrolysing activity was decreased within 24 h in adult rats, while in 15- and 30-day old rats it decreased 72 h post-stress. At equivalent times in pubertal rats (60 days old) a slight activation of ecto-5′-nucleotidase was detected. Our results highlight the developmental-dependence of brain ecto-nucleotidase susceptibility to acute stress and the likely existence of different mechanisms involved in time-dependent ecto-nucleotidase activity modulation following stress exposure. Clearly there are differences in the response of the purinergic system to acute restraint stress between young and adult rats.     

脂质沉积性肌病骨骼肌磁共振31磷波谱的临床价值研究

目的探讨脂质沉积性肌病(LSM)患者骨骼肌磁共振31磷波谱(31P-MRS)改变特征,以及在LSM辅助诊断和疗效评价方面的临床价值。方法对12例LSM患者在治疗前后和11例对照者分别进行31P-MRS扫描,获取波谱图像,计算谱线中无机磷酸盐(Pi)、磷酸肌酸(PCr)及三磷酸腺苷(ATP)的峰下面积,记录Pi/ATP、PCr/ATP和Pi/PCr的比值,计算Pi、PCr、细胞内pH(pHint)、二磷酸腺苷(ADP)和磷酸化潜能(PP)的值,并比较LSM患者治疗前和对照组、LSM患者治疗前后上述31P-MRS指标的差异。结果 LSM患者治疗前的PCr、PCr/ATP和PP较对照组明显降低(P<0.05),Pi/PCr和ADP较对照组明显升高(P<0.05),Pi、Pi/ATP和pHint与对照组比较无明显差异(P>0.05);LSM患者治疗后的PCr、PCr/ATP和PP较治疗前明显升高(P<0.05),ADP较治疗前明显降低(P<0.05),Pi、Pi/ATP、Pi/PCr和pHint与治疗前比较无明显差异(P>0.05)。结论31P-MRS可无创性检测LSM患者肌肉组织的能量代谢变化,有利于LSM的辅助诊断,并可运用于LSM患者的疗效评价。     

31P magnetic resonance spectroscopy study of cerebral metabolism in developing dog brain and its relationship to neuronal function.

Cerebral metabolism and neuronal function of prefrontal brain cortex were studied in 6 dog litters from birth to 3 months of age. Noninvasive phosphorus magnetic resonance spectroscopy (31P-MRS) was used to observe longitudinal biochemical changes in the phosphorus compounds associated with cerebral metabolism. Neurological tests, examining reflex, motor and sensory nerve function, were performed in conjunction with the 31P-MRS study. During the neonatal period, exponential increases in PCr, Pi, and phophodiesters preceded neurological changes. Phosphomonoesters showed an exponential, nearly linear, decrease and PCr/Pi was maintained during the 3-month period. Developmental increases in high energy phosphates and the maintenance of PCr/Pi indicate that the increased energy demands of the developing animal are met by increased mitochondrial function (ATP turnover).     

Ionic modulation of triethyllead neurotoxicity in cerebellar granule cell culture

While the molecular mechanism underlying triethyllead (TEL) neurotoxicity is unknown, we hypothesize that triethyllead mediates an accelerated Cl-/OH exchange across neuronal membranes leading to prolonged depolarization and neuronal cell injury. As a test of this hypothesis we have investigated the effect of external ion modulation on triethyllead neurotoxicity in cerebellar granule cell culture. Cultures were prepared from neonatal rats and used 10-20 days in vitro. Cytotoxicity was assessed by lactate dehydrogenase (LDH) release and trypan blue exclusion. A slow, dose-dependent (1-30 microM TEL) release of LDH occurred after a variable latent period dependent upon [TEL]. External replacement of [Cl-]e by Na isothionate dramatically shifted the dose response curve to the left reflecting an accelerated stimulation of LDH release, while replacement of extracellular [Na+]e with equimolar choline chloride had a minimal protective effect. Similarly, high [Mg2+]e or low [Ca2+]e did not protect or potentiate TEL cytotoxicity. The low [Cl-]e accelerated TEL cytotoxicity was dependent on medium pH: alkaline pH potentiated the cytotoxicity. Low [Cl-]e had no significant effect on culture ATP over 5 hrs. ATP reduction was markedly stimulated by TEL in low Cl- medium in contrast to the minimal decline in [ATP] in the control medium. The reduction of ATP in the low [Cl-]e medium occurred prior to LDH or trypan blue staining release confirming that such reduction in [ATP] was not secondary to cell damage. Substituting K sulfate or Na sulfate for the Cl(-)-free medium revealed marked loss of ATP without LDH release in control and TEL supplemented cultures. These observations provide supporting evidence for the role of an abnormal Cl- flux in mediating TEL-induced neurotoxic injury. Specifically, the membrane depolarization is proportional to the gradient imposed by Cl- efflux/OH influx, stimulated by low [Cl-]e. The rapid loss in ATP appeared early, was not a secondary reflection of neuronal damage but a result of a combination of increased ion flux at the plasma membrane, stimulation of Na+/K+ ATPase and direct TEL-induced inhibition of mitochondrial oxidative phosphorylation.     

Developmental changes in mitochondrial activity and energy metabolism in fetal and neonatal rat brain

Experiments were undertaken to investigate mitochondrial activity and energy metabolism in the developing rat brain from the late fetal stage to the neonatal stage. Samples of cerebral cortical tissue were obtained from fetuses at 14, 16, 18, and 20 days of gestation, and from pups at 1 h, 1 day and 7 days after birth. Mitochondrial respiration was measured polarographically using homogenates. Fetal and neonatal brains were frozen in situ and fluorometric enzymatic techniques were used for the analysis of ATP, ADP, AMP, and lactate. In the fetal brain, there was a gradual increase in stimulated (+ADP) and uncoupled respiratory rates using glutamate and malate as substrates, from 14 days to 20 days of gestation, together with a moderate increase in ATP concentration and in the sum total of adenine nucleotides, and a significant decrease in lactate. Since non-stimulated (-ADP) respiratory rates did not change with increasing gestational age, the respiratory control ratio appeared to increase over the same period. An increase in mitochondrial activity was more pronounced immediately after birth, together with a marked increase in ATP concentration and in the sum total of adenine nucleotides. The highest rate of mitochondrial respiration was observed in 1-hour-old pups. These results indicate that, in the rat brain, there is maturation of oxidative metabolism in mitochondria that is initiated in late gestation. Acceleration in mitochondrial respiration occurs immediately after birth in order to maintain high-energy phosphate levels, and this may be crucial for the successful outcome of the newborn.     

Alteration of catecholamine uptake in cerebral cortex from rats fed a saturated fat diet

The transport of [³H]norepinephrine into chopped cerebral cortex of neonates was changed by feeding pregnant rats with semisynthetic diets enriched in saturated fat (coconut oil) as compared to polyunsaturated fat (sunflower oil). There was a significant decrease in the uptake of norepinephrine in neonates from dams fed coconut oil compared to neonatal pups from animals fed sunflower oil. Differences were observed on days 7, 11, 24, and 37 but not in adults which had been maintained on the diet since birth. Kinetic analysis of norepinephrine uptake on day 7 neonates demonstrated a 5-fold increase in K_m and a 2-fold increas in V_max for the coconut oil fed group as compared to control or the sunflower oil fed rats. [³H]Dopamine uptake kinetics revealed no significant difference in day 7 neonates but a doubling of the K_m and V_max in day 37 rats and in adults fed coconut oil. Lipid analysis of cerebral cortex synaptosome membrane fractions revealed significant differences in the fatty acyl composition of the phospholipid. The results indicate that the norepinephrine and dopamine transport systems can be differentially affected by dietary lipid composition in the developing rat. It is suggested that the changes may be due to alterations in membranes fluidity in the local environment of the transport system.     

Experimental study on pathogenesis of neonatal hypoxic-ischemic encephalopathy. II. Energy metabolism in the brain

In a preceding paper, we showed that the exposure to hypoxia after unilateral carotid artery ligation of sucking rat on 7 days of age caused various pathological changes in the ipsilateral forebrain, which had close similarity to human perinatal hypoxic-ischemic encephalopathy. To evaluate the energy metabolism in various regions of the brain during hypoxia after unilateral carotid artery ligation, tissue pH was monitored in the cortex by direct insertion of an electrode. The contents of the lactate, pyruvate, ADP and ATP were also measured during hypoxia. The tissue pH in the cortex of ligated side declined rapidly after initiation of hypoxia as compared to non-ligated side. The contents of pyruvate and ADP showed no particular changes through various regions. Only in the cortex and hippocampus of ligated side, ATP was decreased, and elevation of lactate was more salient than in non-ligated side. In the striatum and thalamus, only elevation of lactate was indicated. However, the levels of lactate, pyruvate, ADP and ATP disclosed no significant difference between the ligated side and non-ligated side, although neuropathological changes were observed only in their ligated side. These results suggest that pathogenesis of the cerebral damage caused by hypoxic-ischemic condition may be different between the cerebral cortex and subcortical structures.     

Lipoic (thioctic) acid increases brain energy availability and skeletal muscle performance as shown by in vivo31P-MRS in a patient with mitochondrial cytopathy

A woman affected by chronic progressive external ophthalmoplegia and muscle mitochondrial DNA deletion was studied by phosphorus magnetic resonance spectroscopy (³¹P-MRS) prior to and after 1 and 7 months of treatment with oral lipoic acid. Before treatment a decreased phosphocreatine (PCr) content was found in the occipital lobes, accompanied by normal inorganic phosphate (Pi) level and cytosolic pH. Based on these findings, we found a high cytosolic adenosine diphosphate concentration [ADP] and high relative rate of energy metabolism together with a low phosphorylation potential. Muscle MRS showed an abnormal work-energy cost transfer function and a low rate of PCr recovery during the post-exercise period. All of these findings indicated a deficit of mitochondrial function in both brain and muscle. Treatment with 600 mg lipoic acid daily for 1 month resulted in a 55% increase of brain [PCr], 72% increase of phosphorylation potential, and a decrease of calculated [ADP] and rate of energy metabolism. After 7 months of treatment MRS data and mitochondrial function had improved further. Treatment with lipoate also led to a 64% increase in the initial slope of the work-energy cost transfer function in the working calf muscle and worsened the rate of PCr resynthesis during recovery. The patient reported subjective improvement of general conditions and muscle performance after therapy. Our results indicate that treatment with lipoate caused a relevant increase in levels of energy available in brain and skeletal muscle during exercise.     

Intermittent hypoxia protects cerebral mitochondrial function from calcium overload

Hypoxia leads to Ca²⁺ overload and results in mitochondrial uncoupling, decreased ATP synthesis, and neuronal death. Inhibition of mitochondrial Ca²⁺ overload protects mitochondrial function after hypoxia. The present study was aimed to investigate the effect of intermittent hypoxia on mitochondrial function and mitochondrial tolerance to Ca²⁺ overload. Wistar rats were divided into control and intermittent hypoxia (IH) groups. The IH group was subject to hypoxia for 4 h daily in a hypobaric cabin (5,000 m) for 7 days. Brain mitochondria were isolated on day 7 following hypoxia. The baseline mitochondrial functions, such as ST3, ST4, and respiratory control ratio (RCR = ST3/ST4), were measured using a Clark-type oxygen electrode. Mitochondrial adenine nucleotide concentrations were measured by HPLC. Mitochondrial membrane potential was determined by measuring rhodamine 123 (Rh-123) fluorescence in the absence and presence of high Ca²⁺ concentration (0.1 M), which simulates Ca²⁺ overload. Our results revealed that IH did not affect mitochondrial respiratory functions, but led to a reduction in AMP and an increase in ADP concentrations in mitochondria. Both control and IH groups demonstrated decreased mitochondrial membrane potential in the presence of high Ca²⁺ (0.1 M), while the IH group showed a relative higher mitochondrial membrane potential. These results indicated that the neuroprotective effect of intermittent hypoxia was resulted partly from preserving mitochondrial membrane potential, and increasing mitochondrial tolerance to high calcium levels. The increased ADP and decreased AMP in mitochondria following intermittent hypoxia may be a mechanism underlying this protection.     

Cerebral energy metabolism measured in vivo by 31P-NMR in middle cerebral artery occlusion in the cat--relation to severity of stroke

The energy metabolism of the brain has been measured in a middle cerebral artery (MCA) occlusion model in the cat utilizing 31P-nuclear magnetic resonance (NMR). 31P-NMR spectra were serially obtained during 2 h of ischemia and a subsequent 4-h recovery period. The ratio of creatine phosphate (PCr) to inorganic phosphate (Pi) (PCr/Pi) showed a precipitous decrease in parallel with changes in electroencephalographic (EEG) amplitude in severe strokes during ischemia as well as during recirculation. Animals with mild strokes, as determined by EEG criteria, exhibited a much smaller decrease in PCr/Pi during ischemia. In the severe strokes, there was a splitting and significant shift of the Pi peak immediately after occlusion. In addition, the shifted Pi peak rapidly increased and remained elevated throughout the study. In the mild strokes, Pi also increased, but not as markedly. Intracellular pH determination by chemical shift of the Pi peak revealed a decrease from 7.1 to 6.2-6.3 during ischemia and the subsequent recovery period in the animals with severe strokes, whereas the pH in the animals with mild strokes did not show a significant change. A gradual decrease in adenosine triphosphate (ATP) to 57-79% of the control was exhibited in severely stroked animals during both the ischemia and the recovery period, whereas there was no change in ATP in the mild stroked animals. These results suggest that the dynamic process of pathophysiological changes in an MCA occlusion model in the cat leads to significant differences in cerebral metabolism between animals with mild and severe strokes.     

PEPT2-mediated transport of 5-aminolevulinic acid and carnosine in astrocytes

5-aminolevulinic acid (ALA) and carnosine have important physiological and pathophysiological roles in the CNS. Both are substrates for the proton-coupled oligopeptide transporter PEPT2. The purpose of the current study was to determine the importance of PEPT2 in the uptake of ALA and carnosine in rat and mouse (PEPT2+/+ and PEPT2-/-) cultured neonatal astrocytes. Although neonatal astrocytes are known to express PEPT2, its quantitative importance in the transport of these compounds is not known. [14C]ALA uptake in neonatal rat astrocytes was inhibited by dipeptides, an alpha-amino containing cephalosporin (which is a PEPT2 substrate) but was not affected by a non-amino containing cephalosporin (which is not a PEPT2 substrate). Uptake was pH sensitive as expected from a proton-coupled transporter and was saturable (Vmax=715+/-29 pmol/mg/min, Km=606+/-14 microM). [3H]Carnosine uptake in neonatal rat astrocytes was inhibited by dipeptides but not by histidine (a substrate for the peptide/histidine transporters PHT1 and PHT2) and also showed saturable transport (Vmax=447+/-23 pmol/mg/min, Km=43+/-5.5 microM). Neonatal astrocytes from PEPT2-/- mice had a 62% reduction in [14C]ALA uptake and a 92% reduction in [3H]carnosine uptake compared to PEPT2+/+ mice. These results demonstrate that PEPT2 is the primary transporter responsible for the astrocytic uptake of ALA and carnosine.