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M D Norenberg

Publications and source records attributed to M D Norenberg.

At least 19 recordsLinked to original sources
PubMed1995

Inhibition of growth factor-induced DNA synthesis in astrocytes by ligands of peripheral-type benzodiazepine receptors.

The effect of diazepam and specific ligands of peripheral-type benzodiazepine receptors (PBRs) on growth factor-induced DNA synthesis in quiescent cultures of rat astrocytes has been examined. It was found that diazepam inhibited the ability of basic fibroblast growth factor (bFGF) to stimulate [3H]thymidine incorporation; the IC50 was approximately 5 microM. Ro5-4864, a specific agonist of PBRs, also blocked bFGF-induced DNA synthesis. PK11195, which in some cases functions as an antagonist of PBRs, did not prevent the effect of Ro5-4864 on bFGF-induced DNA synthesis; rather, addition of PK11195 also inhibited bFGF-induced DNA synthesis. In addition, diazepam reduced the stimulation of DNA synthesis caused by epidermal growth factor (EGF) and platelet-derived growth factor (PDGF), polypeptide growth factors coupled to receptor tyrosine kinases, as well as thrombin, an activator of G protein-coupled receptors. These data suggest that ligands of PBRs may limit astrocyte mitosis, a phenomenon that occurs following CNS injury.

Animals↗
PubMed1995

Ontogeny of peripheral-type benzodiazepine receptors in cultured astrocytes and brain from rat.

Peripheral-type benzodiazepine receptors (PBRs) in brain are primarily localized within astroglial cells, and the existence of PBR subtypes have been suggested. In the present study the ontogeny of PBRs labeled with [3H]Ro5-4864 and [3H]PK 11195 in cultured astrocytes was compared to their postnatal in-vivo development. The density of [3H]Ro5-4864 binding sites in cultured astrocytes from rat cortex progressively increased from 1- to 3-week-old cultures and did not change further in 5- and 8-week-old cultures. The density of [3H]PK 11195 binding sites progressively increased from 1- to 5-week-old cultures. The density of [3H]PK 11195 binding sites exceeded the density of [3H]Ro5-4864 binding sites by 40-50% in 2-, 5- and 8-week-old cultures. The affinity of the PBR ligands for the receptor sites was increased by 3- to 4-fold from the first to the second week in cultures, and did not change thereafter. A similar developmental pattern of PBRs was observed in rat cortex, except that: first, the difference between the Bmax of [3H]PK 11195 and [3H]Ro5-4864 was already apparent in postnatal-week-1 and persisted with maturation; second, the high affinity of the ligands for the receptor sites was apparent from postnatal-week-1 and did not change with maturation. Age-related differences in the ratio between the density of PBRs in astrocytes and rat cortex were also observed. These results lead us to suggest that the development of PBRs in vivo during the first postnatal week is more rapid than the development of the receptors in vitro during the first week in culture. Subsequently, the increased ratio between the density of PBRs in cultured astrocytes and brain with maturation indicates the predominantly astrocytic localization of these receptors. The finding that the density of [3H]PK 11195 binding sites in cultured astrocytes and in rat brain cortex is usually 40-50% greater than the density of [3H]Ro5-4864 binding sites further supports the existence of PBR subtypes.

Age Factors↗
PubMed1994

Attenuation of ammonia toxicity in mice by PK 11195 and pregnenolone sulfate.

Ammonia and benzodiazepines are thought to be involved in the pathogenesis of hepatic encephalopathy. The present study was undertaken to evaluate the effect of various benzodiazepine-receptor ligands and neurosteroids on ammonia toxicity in mice. Administration of ammonium acetate (8-15 mmole/kg; i.p.) to Swiss Webster mice resulted in a dose-dependent increase in mortality. Pretreatment with the central benzodiazepine receptor agonist clonazepam or the antagonist Ro15-1788 (7 mg/kg each; i.p.) had no significant effect on the lethal response to 10 mmole/kg ammonium acetate. However, pretreatment with the putative antagonist of the peripheral-type benzodiazepine receptor, PK 11195 (10 mg/kg; i.p.), reduced mortality from 50 to 10%. Ro5-4864 (10 mg/kg; i.p.), an agonist of the peripheral-type benzodiazepine receptors, had no effect on ammonia toxicity. The neurosteroid, pregnenolone sulfate (20 mg/kg; i.p.) reduced mortality from 50 to 25%. The non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801 (2 mg/kg; i.p.), had no effect on the lethal response to ammonium acetate. The results from the present study suggest a role for peripheral-type benzodiazepine receptors and specific neurosteroids in the alleviation of ammonia toxicity in mice.

Ammonia↗
PubMed1994

Ammonia stimulates the release of taurine from cultured astrocytes.

The effect of ammonia on the release of the neuroactive amino acids taurine (TAU), gamma-aminobutyric acid (GABA) and D-aspartate (D-ASP), an analog of L-glutamate (L-GLU), from cultured rat cortical astrocytes was studied. NH4Cl (1 and 5 mM) induced the release of TAU. TAU release was reduced when Na+ was removed, and was almost completely abolished when Cl- was omitted. In contrast, TAU basal release was enhanced upon removal of Na+ or Cl-. Ammonia inhibited the release of GABA and D-ASP. Ammonia-induced release of astroglial TAU may modify the neuronal excitability accompanying hyperammonemic conditions.

Ammonium Chloride↗
PubMed1994

Regulation of peripheral-type benzodiazepine receptors in cultured astrocytes by monoamine and amino acid neurotransmitters.

Exposure of primary cultured astrocytes for 3 days to 1 microM of either dopamine, serotonin or norepinephrine resulted in upregulation (25-34% increase in Bmax) of the peripheral-type benzodiazepine receptors (PBRs) labeled with [3H]Ro5-4864. A similar treatment with gamma-aminobutyric acid [GABA] caused a 2-fold increase in the affinity (Kd) of [3H]Ro5-4864. The monoamines tested and GABA had no effect on the binding parameters of [3H]PK 11195, another selective PBR ligand. The present study indicates that Ro5-4864 binding sites are susceptible to regulation by specific neurotransmitters and provides further evidence for the distinction between Ro5-4864 and PK 11195 binding sites of the PBRs in cultured astrocytes.

Animals↗
PubMed1994

Ammonia-induced upregulation of peripheral-type benzodiazepine receptors in cultured astrocytes labeled with [3H]PK 11195.

Evidence suggests that peripheral-type benzodiazepine receptors (PBRs) may play a role in hepatic encephalopathy (HE), a condition associated with increased levels of ammonia in brain. In the present study, the regulation of [3H]PK 11195-binding to PBRs in cultured rat astrocytes that had been previously exposed to NH4Cl was investigated. 24 h treatment of 21-28-day-old cultures with 2, 5 or 10 mM NH4Cl resulted in 25 +/- 3, 48 +/- 3 and 42 +/- 4% increase in the number of [3H]PK 11195-binding sites, respectively. No further change in [3H]PK 11195-binding was observed after exposure of astrocytes to 5 mM NH4Cl for 48 or 72 h. Ammonia treatment did not cause any significant alteration in the affinity of [3H]PK 11195 for PBRs. The present study demonstrates the susceptibility of the PK 11195-binding site of PBRs in cultured astrocytes to ammonia and suggests that increase in brain ammonia concentration causes a supersensitivity of PBRs.

Ammonium Chloride↗
PubMed1994

Astrocyte swelling in liver failure: role of glutamine and benzodiazepines.

We examined the effect of methionine sulfoximine (MSO) and peripheral benzodiazepine (BZD) ligands on ammonia-induced swelling of primary astrocyte cultures. Swelling was completely abolished by co-treatment with MSO, an inhibitor of glutamine synthetase. We also established that many of the effects caused by ammonia, including the reduction in K+ uptake, increase in Cl- uptake and reduction in myo-inositol uptake were diminished by co-treatment with MSO. Agonists of the peripheral-type BZD receptor aggravated ammonia-induced swelling, whereas a peripheral BZD receptor blocker, PK 11195, diminished the extent of swelling. Our findings implicate glutamine and the peripheral-type benzodiazepine receptor in the pathogenesis of the edema associated with fulminant hepatic failure.

Ammonia↗
PubMed1994

Synergistic activation of DNA synthesis in astrocytes by fibroblast growth factors and extracellular ATP.

The effects of extracellular ATP and polypeptide growth factors on DNA synthesis in primary cultures of rat astrocytes have been examined. It was found that ATP acts synergistically with either acidic or basic fibroblast growth factor to stimulate DNA synthesis. The specificity of this effect was demonstrated by the inability of ATP to potentiate DNA synthesis induced by platelet-derived growth factor or epidermal growth factor. ATP appears to act via P2 purinergic receptors, because (a) it was more effective than adenosine and (b) the synergistic effect was observed with the hydrolysis-resistant P2 agonists, ADP beta S and ATP gamma S. The evidence suggests that extracellular ATP may be an important factor in regulating the extent of gliosis and, as such, may be involved in mechanisms of neural injury and repair.

Adenosine↗
PubMed1992

Role of calmodulin and protein kinase C in astrocytic cell volume regulation.

We investigated the role of Ca(2+)-dependent protein kinases in the regulation of astrocytic cell volume. Calmodulin (CaM) antagonists were used to inhibit CaM and thus Ca2+/CaM-dependent protein kinase. The effect of these inhibitors as well as activators and inhibitors of protein kinase C (PKC) on astrocytic volume was measured in response to hypoosmotic stress and under isoosmotic conditions. In conditions of hypoosmolarity, CaM antagonists had no effect on swelling, but inhibited the regulatory volume decrease. PKC activation facilitated the swelling induced by hypoosmotic stress. PKC inhibitors induced cell shrinkage and inhibited the initial phase of regulatory volume decrease, whereas PKC down-regulation caused pronounced swelling and partial inhibition of regulatory volume decrease. In isoosmotic conditions, CaM antagonists and PKC activation did not affect astrocytic volume, but PKC inhibitors caused shrinking and PKC down-regulation led to swelling of these cells. These studies indicate the importance of Ca(2+)-dependent protein kinases in the regulation of astrocytic cell volume.

Animals↗
PubMed1992

Involvement of second messengers and protein phosphorylation in astrocyte swelling.

In a hypoosmotic model of astrocyte swelling, we found that Ca2+ and intracellular signals such as diacylglycerol and inositol phosphate, as well as protein phosphorylation systems, are implicated in the generation and (or) modulation of volume regulatory processes. Cyclic AMP, which also has a significant effect on astrocyte volume regulation, in addition influences some of these second messengers.

Animals↗
PubMed1991

ATP-evoked calcium signal stimulates protein phosphorylation/dephosphorylation in astrocytes.

Extracellular adenosine 5'-triphosphate (ATP)-evoked increases in intracellular calcium and the consequent stimulation of calcium-mediated protein phosphorylation systems were investigated in primary cultures of rat cerebral cortical astrocytes. Measurement of calcium responses in fura-2-loaded astrocytes indicated that extracellular ATP stimulated a transient calcium peak followed by a sustained increase in intracellular calcium which declined to baseline when external calcium was removed, thereby indicating that ATP evokes mobilization of internal calcium as well as influx of external calcium. Protein phosphorylation studies revealed that application of extracellular ATP resulted in increased phosphorylation of 55 and 52 kDa proteins (4-fold and 2-fold, respectively) and decreased phosphorylation of 24 and 21 kDa proteins (approximately 50% for each protein). These effects were time- and dose-dependent. The changes in phosphate incorporation were (a) inhibited by lanthanum, (b) reduced when calcium was omitted from the bath and (c) mimicked by ionomycin, thus suggesting that the ATP-induced changes in protein phosphorylation were dependent on increased levels of intracellular calcium. Adenosine diphosphate (ADP) gave similar, but reduced, effects while adenosine and guanosine triphosphate (GTP) were ineffective, findings consistent with activation of P2 purinergic receptors. The 52 kDa protein co-migrated with glial fibrillary acidic protein. These results support the premise that calcium-dependent protein kinases and phosphatases are transducing elements for the calcium signal brought about by activation of P2 purinergic receptors in astrocytes. Since ATP is released from neurons and endothelial cells, this signal transduction mechanism may be an important component of neuronal- and endothelial-astrocytic communication.

Adenosine Triphosphate↗
PubMed1991

Peripheral-type benzodiazepines inhibit proliferation of astrocytes in culture.

Peripheral-type benzodiazepine (BZD) receptors have been identified in brain and are predominantly localized to astrocytes. To determine their potential role in controlling astroglial proliferation, DNA synthesis, growth curves and mitotic index were investigated in primary astrocyte cultures which had been exposed to Ro5-4864 (a peripheral-type BZD ligand) and PK11195 (a peripheral-type BZD receptor antagonist). There was a dose-dependent inhibition of mitosis when two-week-old cells in culture were exposed to 50 nM, 500 nM, 1 microM and 10 microM Ro5-4864 for 24 h. Exposure of 5-, 8-, 12- and 15-day-old cultures to Ro5-4864 and PK11195 for 24 h did not affect growth rate and DNA synthesis; however, continuous exposure to 10 microM Ro5-4864 caused a persistent inhibition of cell growth and [3H]thymidine incorporation (P less than 0.05) while nanomolar concentrations did not cause any significant change. Concurrent administration of Ro5-4864 with PK11195 resulted in a partial reversal of Ro5-4864-induced inhibition in DNA synthesis and mitosis. These results indicate that peripheral-type BZDs are capable of inhibiting proliferation of astrocytes in culture.

Animals↗
PubMed1991

Aluminum chloride stimulates the release of endogenous glutamate, taurine and adenosine from cultured rat cortical astrocytes.

Primary astrocyte cultures derived from neonatal rat cerebral cortex were treated for 5 min with 0.5 mM or 5.0 mM AlCl3, and the incubation medium was analyzed by HPLC for the content of released glutamate (Glu), taurine (Tau), serine (Ser) and the nucleoside adenosine (Ade). At 0.5 mM, AlCl3 stimulated Tau release to about 170% of basal levels, but did not affect the release of the other compounds. Treatment with 5.0 mM AlCl3 enhanced the release of Tau, Glu and Ade, to 800%, 1000% and 250%, respectively, but decreased the release of Ser to 70% compared to basal levels. The enhanced release of these neuroactive compounds from astrocytes may contribute to changes in neural transmission known to accompany exposure to aluminum.

Adenosine↗
PubMed1991

Ammonia-induced astrocyte swelling in primary culture.

The effect of ammonia on water space of astrocytes in culture was determined as a means of studying the neurotoxicity of ammonia in fulminant hepatic failure (FHF). Treatment of primary astrocyte cultures obtained from neonatal rat cortices with 10 mM NH4Cl for 4 days resulted in a 29% increase in astrocytic water space, as measured by an isotopic method utilizing 3-O-methyl-[3H]-glucose. This effect was time- and dose-dependent. The ammonia-induced swelling was reversible as the water space in cultures treated with 10 mH NH4Cl for 3 days, and then returned to normal culture media for 1 day, was similar to control cultures. These findings suggest that elevated levels of ammonia lead to astrocyte swelling and may contribute to the brain edema in FHF.

3-O-Methylglucose↗
PubMed1991

Effect of methotrexate on glial fibrillary acidic protein content of astrocytes in primary culture.

We have recently reported that methotrexate (MTX) causes degenerative as well as reactive-like astroglial changes and alters the cell cycle kinetics of astrocytes in vitro. To further characterize the nature of the reactive-like changes that were noted by light and electron microscopy following MTX exposure, the glial fibrillary acidic protein (GFAP) content of astrocytes in culture was investigated by enzyme-linked immunosorbent assay, flow cytometry and double-immunofluorescent staining. An increase in GFAP content which did not correlate with drug dosage or DNA synthesis was noted in the MTX-treated cultures. It is postulated that this increase in GFAP content of astrocytes reflects an adaptive response to MTX-induced injury and partly explains the gliosis that is seen in methotrexate encephalopathy.

Animals↗
PubMed1990

The benzodiazepine receptor in cultured astrocytes from genetically epilepsy-prone rats.

Peripheral-type benzodiazepine (BZD) receptors were studied in cultured astrocytes derived from genetically epilepsy-prone and control rats. Scatchard analysis of the binding of [3H]Ro 5-4864 to astrocyte homogenates from epilepsy-prone rats showed 38% fewer BZD receptors (Bmax) as compared to controls. No significant change in affinity (Kd) was observed. These findings suggest that the astrocyte peripheral-type BZD receptor may be involved in some forms of epilepsy.

Animals↗