Distinct cellular expressions of creatine synthetic enzyme GAMT and creatine kinases uCK-Mi and CK-B suggest a novel neuron-glial relationship for brain energy homeostasis

The creatine/phosphocreatine shuttle system, as catalysed reversibly by creatine kinases, is thought to be essential for the storing and buffering of high phosphate-bound energy in tissues with high energy demand. In the present study, we aimed to clarify the cellular system of creatine biosynthesis and its energy metabolism in the mouse brain by immunohistochemistry for creatine biosynthetic enzyme S-adenosylmethionine:guanidinoacetate N-methyltransferase (GAMT), ubiquitous mitochondrial creatine kinase (uCK-Mi) and brain-type cytoplasmic creatine kinase (CK-B). GAMT was expressed highly in oligodendrocytes and olfactory ensheathing glia and moderately in astrocytes, whereas GAMT was very low in neurons and microglia. By contrast, uCK-Mi was expressed selectively in neurons and localized in their mitochondria in dendrites, cell bodies, axons and terminals. The distinct and almost complementary distribution of GAMT and uCK-Mi suggests that the creatine in neuronal mitochondria is derived not only from the circulation, but also from local glial cells associated with these neuronal elements. By contrast, CK-B was selective to astrocytes among glial populations, and was exclusive to inhibitory neurons among neuronal populations. Interestingly, these cells with high CK-B immunoreactivity are known to be highly resistant to acute energy loss, such as hypoxia and hypoglycemia. Considering that phosphocreatine generates ATP much faster than the processes of glycolysis and oxidative phosphorylation, the highly regulated cellular expressions of creatine biosynthetic and metabolic enzymes suggest that the creatine/phosphocreatine shuttle system plays a role in brain energy homeostasis through a novel neuron-glial relationship.     

Exocytotic release of creatine in rat brain

The guanidino compound creatine has been shown to occur throughout the brain affecting energy metabolism and mental performance and to act at central GABAA receptors as a partial agonist. Therefore, we examined the possibility that creatine may in fact represent a neuromodulator that is released in the brain in an action-potential dependent manner. To that end, we studied the uptake of [3H]creatine and its electrically evoked release from superfused rat brain slices as well as the evoked release of endogenously synthesized creatine. [3H]creatine was accumulated in neocortex slices in a Na+-dependent manner, consistent with the involvement of the Na+-dependent SLC6A8 creatine transporter. Most importantly, the electrically evoked release of [3H]creatine from neocortex slices (like that from caudate putamen and hippocampus slices) as well as the evoked release of endogenous (unlabeled) creatine was abolished when Ca2+ was omitted from the superfusion medium or in the presence of the Na+-channel blocker tetrodotoxin (TTX). Moreover, blockade of K+-channels by 4-aminopyridine (4-AP) strongly enhanced the electrically evoked release of [3H]creatine as well as that of endogenous creatine. These in vitro data indicate that creatine is not only synthesized and taken up by central neurons, but also released in an action-potential dependent (exocytotic) manner, providing strong evidence for its role as a neuromodulator in the brain.     

Dissociated expression of mitochondrial and cytosolic creatine kinases in the human brain: a new perspective on the role of creatine in brain energy metabolism

The phosphocreatine/creatine kinase (PCr/CK) system in the brain is defined by the expression of two CK isozymes: the cytosolic brain-type CK (BCK) and the ubiquitous mitochondrial CK (uMtCK). The system plays an important role in supporting cellular energy metabolism by buffering adenosine triphosphate (ATP) consumption and improving the flux of high-energy phosphoryls around the cell. This system is well defined in muscle tissue, but there have been few detailed studies of this system in the brain, especially in humans. Creatine is known to be important for neurologic function, and its loss from the brain during development can lead to mental retardation. This study provides the first detailed immunohistochemical study of the expression pattern of BCK and uMtCK in the human brain. A strikingly dissociated pattern of expression was found: uMtCK was found to be ubiquitously and exclusively expressed in neuronal populations, whereas BCK was dominantly expressed in astrocytes, with a low and selective expression in neurons. This pattern indicates that the two CK isozymes are not widely coexpressed in the human brain, but rather are selectively expressed depending on the cell type. These results suggest that the brain cells may use only certain properties of the PCr/CK system depending on their energetic requirements.     

Mitochondrial respiratory chain and creatine kinase activities in mdx mouse brain

In this study we investigated energy metabolism in the mdx mouse brain.To this end, prefrontal cortex, cerebellum, hippocampus, striatum, and cortex were analyzed. There was a decrease in Complex I but not in Complex II activity in all structures. There was an increase in Complex III activity in striatum and a decrease in Complex IV activity in prefrontal cortex and striatum. Mitochondrial creatine kinase activity was increased in hippocampus, prefrontal cortex, cortex, and striatum. Our results indicate that there is energy metabolism dysfunction in the mdx mouse brain. Muscle Nerve, 2010     

Immunohistochemical localization of creatine kinase-BB isoenzyme to astrocytes in human brain

Indirect immunoperoxidase labelling at the light microscope level using a specific antisera to brain type creatine kinase-BB isoenzyme has localized the protein to astrocytes in the white matter of human cerebrum. No specific staining of neurones or of other glial elements was detected.     

Continuous measurement of net potassium movements in rat brain cortex suspensions: Effects of glutamate, veratridine, creatine and other substances

Net K fluxes in in vitro suspensions of sliced rat brain cortex were studied by means of a K-sensitive electrode. When incubation was in 3 mM K, a net K efflux occurred. It could be resolved into two first-order rate constants: k1 = 0.486 min-1, and k2 = 0.0102 min-1, that originated from compartments that contained 18% and 82% of tissue K, respectively. k1 Was suppressed by tetrodotoxin (TTX), and k2 was increased 38-fold by veratridine. The latter effect was blocked by TTX, methylphenidate (1 mM), creatine (25 mM), apamin (50 nM), quinine (100 microM), verapamil (22 microM) or D-600 (38 microM). Net K loss was greatly increased by 1 mM ouabain, and enhanced by sodium azide plus iodoacetamide, but not by 0.1 M ethanol. Glutamate (5 mM) induced a considerable and rapid net uptake of K, while aspartate or N-methylaspartate increased K efflux.     

Immunohistochemical localization of creatine kinase BB-isoenzyme in human brain: Comparison with tubulin and astroprotein

The distribution of creatine kinase BB-isoenzyme in the human central nervous system (CNS) was investigated immunohistochemically and the findings were compared with the distributions of tubulin and astroprotein. Creatine kinase BB-isoenzyme existed both in neurons and astrocytes, and was universally distributed within the CNS. Tubulin was visualized only in the neuronal elements, namely perikarya, dendrites and axons, while astroprotein was exclusively visualized in astrocytes. A combination of immunohistochemistry for structural and soluble proteins may be a useful tool for the investigation of pathophysiological conditions within the CNS.     

Relation of taurine transport and brain edema in rats with simple hyperammonemia or liver failure

Taurine (Tau), an amino acid that abounds in brain, has been implicated in inhibitory neuromodulation and osmoregulation, the latter function being manifested by Tau release along with osmotically obligated water in response to brain tissue edema. A previous study (Hilgier and Olson: J. Neurochem. 62:197–204, 1994) had shown that simple hyperammonemia (HA) induced in rats by daily administration of ammonium acetate resulted in a decrease of both tissue specific gravity indicative of edema and Tau content, in basal ganglia (BG) but not in cerebral cortex (CC). By contrast, rats with hepatic encephalopathy (HE) following administration of a hepatotoxin, thioacetamide, were characterized by CC edema and an increased Tau content in both BG and CC. In the present study, we tested the following parameters that may potentially have affected Tau distribution in the two models: a) spontaneous, and stimulated (hypoosmolarity-induced) release of loaded [3H] Tau in vitro from CC and BG slices; b) blood Tau content; and c) uptake of [14C] Tau in vivo from blood to brain corrected for [3H] water passage—the so-called brain uptake index (BUI). The two edema-affected structures: BG in the HA model and CC in the HE model, showed increased spontaneous Tau release. Edema-associated spontaneous release of Tau may favor inhibitory neurotransmission contributing to the pathomechanism of HA or HE. Stimulated release, reflecting the ability of the tissue to reduce water content, was decreased in the BG from HA rats, in agreement with the postulated role of Tau in osmoregulation. Stimulated release was unchanged in CC of HE rats. Neither spontaneous nor stimulated release of Tau were affected in CC of HA rats or in BG of HE rats. HE, but not HA, was associated with elevated blood content and increased BUI for TAU, which in combination, contributed to the increase of Tau content in CC. The latter phenomenon adds to the list of metabolic changes distinguishing simple HA from toxic liver damage, reemphasizing the crucial role of factors other than ammonia in the pathomechanism of HE. © 1996 Wiley-Liss, Inc.     

Progestin receptors in the developing rat brain and pituitary

Repeated application of electrical stimulation to the amygdala at a frequency of 3 Hz resulted in the development of afterdischarge and behavioral seizures. The rate of low-frequency kindling was faster than that of kindling with conventional 60 Hz stimulation, but the form of the seizures kindled with the two frequencies of stimulation was identical. Low-frequency kindling was obtained only when pulses of sufficient duration and intensity were administered.     

Flunarizine and lamotngine propnyiaxis effects on neuron-specific enolase,S-100,and brain-specific creatine kinase in a fetal rat model of hypoxic-ischemic brain damage

BACKGROUND:Calcium antagonists may act as neuroprotectants,diminishing the influx of calcium ions through voltage-sensitive calcium channels. When administered prophylactically,they display neuroprotective effects against hypoxic-ischemic brain damage in newborn rats.OBJECTIVE:To investigate the neuroprotective effects of flunarizine(FNZ),lamotrigine (LTG)and the combination of both drugs,on hypoxic-ischemic brain damage in fetal rats.DESIGN AND SETTING:This randomized,complete block design was performed at the Department of Pediatrics.Shenzhen Fourth People's Hospital,Guangdong Medical College.MATERIALS:Forty pregnant Wistar rats,at gestational day 20,were selected for the experiment and were randomly divided into FNZ,LTG,FNZ LTG,and model groups,with 10 rats in each group.METHODS:Rats in the FNZ.LTG,and FNZ LTG groups received intragastric injections of FNZ (0.5 mg/kg/d),LTG(10 mg/kg/d),and FNZ(0.5 mg/kg/d) LTG(10 mg/kg/d),respectively.Drugs were administered once a day for 3 days prior to induction of hypoxia-ischemia.Rats in the modeJ group were not administered any drugs.Three hours after the final administration,eight pregnant rats from each group underwent model establishment hypoxia-ischemia brain damage to the fetal rats.Cesareans were performed at 6,12,24,and 48 hours later;and 5 fetal rats were removed from each mother and kept warm.Twe fetuses without model establishment were removed by planned cesarean at the same time and served as controls.A total of 0.3 mL serum was collected from fetal rats at 6,12,24,and 48 hours,respectively,following birth.MAIN OUTCOME MEASURES:Serum protein concentrations of neuron-specific enolase and S-100 were measured by ELISA.Serum concentrations of brain-specific creatine kinase were measured using an electrogenerated chemiluminescence method.RESULTS:Serum concentrations of neuron-specific enolase,S-100,and brain-specific creatine kinase were significantly higher in the hypoxic-ischemic fetal rats.compared with the non-hypoxic-ischemic group.Serum concentrations of neuron-specific enolase,S-100,and brain-specific creatine kinase were significantly less in the FNZ,LTG,and FNZ LTG groups following ischemia,compared with the model group(P<0.01).However,these values wcrc significantly greater in the FNZ and LTG groups,compared with the FNZ LTG group,following ischemia(P<0.01).CONCLUSION:Preventive antenatal use of oral FNZ and LTG has positive neuroprotective effects on intrauterine hypoxic-ischemic brain damage.The combined effect of these two drugs is superior.     

Flunarizine and lamotrigine prophylaxis effects on neuron-specific enolase, S-100, and brain-specific creatine kinase in a fetal rat model of hypoxic-ischemic brain damage*

BACKGROUND： Calcium antagonists may act as neuroprotectants, diminishing the influx of calcium ions through voltage-sensitive calcium channels. When administered prophylactically, they display neuroprotective effects against hypoxic-ischemic brain damage in newborn rats.
OBJECTIVE： To investigate the neuroprotective effects of flunarizine （FNZ）, lamotrigine （LTG） and the combination of both drugs, on hypoxic-ischemic brain damage in fetal rats.
DESIGN AND SETTING： This randomized, complete block design was performed at the Department of Pediatrics, Shenzhen Fourth People＇s Hospital, Guangdong Medical College.
MATERIALS： Forty pregnant Wistar rats, at gestational day 20, were selected for the experiment and were randomly divided into FNZ, LTG, FNZ ＋ LTG, and model groups, with 10 rats in each group.
METHODS： Rats in the FNZ, LTG, and FNZ ＋ LTG groups received intragastric injections of FNZ （0.5 mg/kg/d）, LTG （10 mg/kg/d）, and FNZ （0.5 mg/kg/d） ＋ LTG （10 mg/kg/d）, respectively. Drugs were administered once a day for 3 days prior to induction of hypoxia-ischemia. Rats in the model group were not administered any drugs. Three hours after the final administration, eight pregnant rats from each group underwent model establishment hypoxia-ischemia brain damage to the fetal rats. Cesareans were performed at 6, 12, 24, and 48 hours later; and 5 fetal rats were removed from each mother and kept warm. Two fetuses without model establishment were removed by planned cesarean at the same time and served as controls. A total of 0.3 mL serum was collected from fetal rats at 6, 12, 24, and 48 hours, respectively, following birth.
MAIN OUTCOME MEASURES： Serum protein concentrations of neuron-specific enolase and S-100 were measured by ELISA. Serum concentrations of brain-specific creatine kinase were measured using an electrogenerated chemiluminescence method.
RESULTS： Serum concentrations of neuron-specific enolase, S-100, and brain-specific creatine kinase were significan     

Cathepsin D and 2'',3''-cyclic nucleotide 3''-phosphohydrolase in developing human foetal brain

Three peaks of proteinases were observed with hemoglobin, bovine serum albumin and casein as substrates at the pH of 3.5, 6.5 and 8.5, in prenatal human cerebral cortex. Cathepsin D (EC 3.4.23.5) was the most prominent, with hemoglobin as the preferred substrate. The enzyme was partially purified by Concanavalin A - Sepharose affinity chromatography and the nature of the active site was assessed with proteinase inhibitors. Inhibitor studies showed that similar to pepstatin A, benzethonium chloride was also strongly inhibitory to the enzyme. The distribution of cathepsin D, a neuronal marker, and 2',3'-cyclic nucleotide 3'-phosphohydrolase (EC 3.1.4.37), a oligodendroglial marker in foetal brain regions with increasing gestation revealed that neurogenesis and gliogenesis occur concomitantly from earlier periods of gestation. Glial marker acquisition was particularly high in medulla and in spinal cord between 20 and 25 weeks of gestation.     

Ultrastructural differentiation and synaptogenesis in aggregating rotation cultures of rat cerebral cells

Cerebral cortices from fetal rats were dissociated into single cells by either trypsinization or mechanical sieving. Then the cells were allowed to form aggregates in rotation cultures. At 7, 14, 21, 28, and 35 days, aggregates were processed for electron microscopic study of morphological differentiation with special emphasis on synaptogenesis. Whether the tissue was initially dissociated by trypsin treatment or by mechanical sieving, the aggregating cultures did not exhibit any apparent differences in ultrastructural differentiation and synaptogenesis. At day 7, most neurons were immature and the extracellular space was large. Cell processes had not yet branched extensively but did contain numerous microtubules. A few immature synapses were observed. Astrocytes and oligodendrocytes displayed many of their typical cytological features. At day 14, dendritic spines had developed, some of which formed axodendritic spinous synapses. Multilayered myelin sheaths were tightly wrapped around axons. At day 21, synapses appeared mature and their number per unit area was maximal. The extracellular space had greatly decreased. At this age, asymmetrical synapses had increased approximately sixfold, whereas symmetrical synapses increased only fourfold when compared with the 7-day-old aggregates. Multifocal degeneration became apparent at 28 days and was accompanied by a significant decline in the number of synapses.     

Recirculation after cerebral ischemia. Simultaneous measurement of cerebral bloodflow, brain edema, cerebrovascular permeability and cortical EEG in the rat

The 4-vessel occlusion rat model of cerebral ischemia was modified to permit the simultaneous measurement of cerebral blood flow (hydrogen clearance), brain edema (specific gravity), cerebrovascular permeability (14C-AIB) and electrocardiogram. Surgery was performed in one stage in the anesthetised, paralysed and ventilated rat and severe hemispheric ischemia was produced in all animals. Electrode implantation did not alter cortical specific gravity or Ki for 14C-AIB. During 4-vessel occlusion mean cortical CBF was 5.8 +/- 1.4 ml-1 100 g-1 min. and this was associated with an isoelectric ECoG; 15 min of ischemia produced a significant reduction in mean cortical specific gravity (increase in brain edema). Following 15 min ischemia, 180 min of recirculation were permitted. Post-ischemic blood flow showed an immediate hyperemia (CBF = 202 +/- 12 ml-1 100 g-1 min.) followed by hypoperfusion (CBF = 58 +/- 8 ml-1 100 g-1 min). There was an early further decrease in cortical specific gravity. Further recirculation led to a significant increase in cortical specific gravity (resolution of brain edema). The transfer constant (Ki) for 14C-AIB was not altered at any stage in recirculation. This appears to be a model of pure cytotoxic edema until 180 min recirculation after 15 min cerebral ischemia. Recirculation permitted return of cortical electrical activity.     

Expression of brain-type creatine kinase and ubiquitous mitochondrial creatine kinase in the fetal rat brain: Evidence for a nuclear energy shuttle

To test the hypothesis that embryonic brain cells utilize a creatine phosphate energy shuttle, we examined the pattern of creatine kinase (CK) isoform expression and localization in the fetal rat brain. Moderate levels of CK activity are present at embryonic day 14 (7 U/mg protein) and decrease slightly until 3 days postpartum followed by a rapid, fourfold up- regulation to adult levels by 1 month (18 U/mg protein). In parallel with changes in enzyme activity, there is a biphasic and coordinate pattern of expression of brain-type CK (BCK) and ubiquitous mitochondrial CK (uMtCK) determined by nondenaturing electrophoresis and immunoblot analysis. The localization of CK isoforms was examined by immunocytochemistry, and, during the fetal period, BCK and uMtCK immunoreactivity was detected throughout the central and peripheral nervous system, especially in neuroepithelial regions of the cerebral vesicles and spinal cord. In large cells within the olfactory neuroepithelium and ventral spinal cord, differential compartmentation of CK isoforms was evident, with BCK localized primarily in cell nuclei, whereas uMtCK immunoreactivity was present in the cell body (but not within nuclei). In olfactory bulb neuroepithelium, both isoforms were expressed in the middle zone of the germinal layer associated with DNA synthesis. In embryonic skeletal and cardiac muscle, which also express BCK, the same compartmentation of BCK was seen, with BCK localized primarily in the cell nucleus of cardiac and skeletal myoblasts. These results demonstrate a coordinate pattern of expression and compartmentation of BCK and uMtCK isoforms in the fetal brain that, in some cells, provides the anatomic basis for a nuclear energy shuttle. © 1995 Wiley-Liss, Inc.     

Effect of vitamin D Deficiency and 1,25-dihydroxyvitamin D3 on metabolism and D-glucose transport in rat cerebral cortex

We previously demonstrated that feeding rats Steenbock and Black's rickets-inducing diet produces remarkable changes in the metabolic pattern of the in testinal mucosa, kidney, and liver and in some membrane transport Systems of intestinal mucosa and kidney. 1,25-Dihydroxyvitamin D₃ administration to rachitic rats did not always prove to be effective in restoring normal values. We have now investigated the effect of 1,25-dihydroxyvitamin D₃ on the levels of some metabolites in rat cerebral cortex, on the activity of some enzymes, and on the transport of 2-deoxy-D-glucose and D-glucose in synaptosomes. Our experiments were carried out on three rat groups: control, rachitic, and rachitic treated with 1,25-dihydroxyvitamin D₃. The decrease in phosphorus content and the increase in citrate concentration observed in rachitic rat cerebral cortex were corrected by 1,25-dihydroxyvitamin D₃ treatment. The activity of acetylcholinesterase, glucose-6-phosphate dehydrogenase, and acyl phosphatase significantly increased in rachitic rat synaptosomes, as well as NAD⁺-dependent isocitrate dehydrogenase in cerebral cortex mitochondria; the administration of 1,25-dihydroxyvitamin D₃ to rachitic rats restored enzyme levels to normal. The transport of 2-deoxy-D-glucose and D-glucose in rachitic rat synaptosomes was lower than in the control group and returned to control values in consequence of 1,25-dihydroxyvitamin D₃ treatment. The results reported here support the hypothesis of a participation of 1,25-dihydroxyvitamin D₃ in some aspects of cerebral cortex metabolism.     

Effect of torasemide on intracranial pressure, mean systemic arterial pressure and cerebral perfusion pressure in experimental brain edema of the rat

The study was performed to establish whether a lipophilic loop diuretic, torasemide could modify intracranial pressure and cytotoxic brain edema. Brain edema was induced by water intoxication in nephrectomized rats. Following intravenous injection of 100 mg torasemide/kg body weight at 50, 60, 70, 90 and 120 min, a significant decrease of intracranial pressure was observed.     

Effect of gammahydroxybutyric acid on serotonin synthesis,concentration and metabolism in the developing rat brain

Summary 5-HT synthesis, levels and degradation were investigated in the whole brain and regional brain areas in 4,14, and 28 days old rats after administration of gammahydroxybutyric acid (GHBA). 5-HT synthesis was investigated by means of 5-HTP accumulation after decarboxylase inhibition by NSD 1015. 5-HTP accumulation increased in the 14 and 28 days old rats but decreased in the 4 days old animals 90 min after GHBA, 750 mg/kg. In the 28 days old rats a corresponding increase was also noted in the precursor amino acid tryptophan. Regional and whole brain 5-HT levels were not altered by GHBA treatment. Regional as well as whole brain levels of 5-HIAA increased in the 14 and 28 days old rats after GHBA administration.In conclusion, the present data indicate that GHBA increases the synthesis and degradation of 5-HT in adolescent rats. These effects of GHBA were not seen in the neonatal animals.