Cultured Rat Brain Neurons Research Articles

Influence of Hemantane on Changes in Ca2+ and Na+ Caused by Activation of NMDA Channels in Cultured Rat Brain Neurons

Influence of Hemantane on Changes in Ca2+ and Na+ Caused by Activation of NMDA Channels in Cultured Rat Brain Neurons

Extracellular tau induces microglial phagocytosis of living neurons in cell cultures.

Tau is a microtubule-associated protein, found at high levels in neurons, and itsaggregation is associated with neurodegeneration. Recently, it was found that tau can be actively secreted from neurons, but the effects of extracellular tau on neuronal viability are unclear. In this study, we investigated whether extracellular tau2N4R can cause neurotoxicity in primary cultures of rat brain neurons and glial cells. Cell cultures were examined for neuronal loss, death, and phosphatidylserine exposure, as well as for microglial phagocytosis by fluorescence microscopy. Aggregation of tau2N4R was assessed by atomic force microscopy. We found that extracellular addition of tau induced a gradual loss of neurons over 1-2days, without neuronal necrosis or apoptosis, but accompanied by proliferation of microglia in the neuronal-glial co-cultures. Tau addition caused exposure of the 'eat-me' signal phosphatidylserine on the surface of living neurons, and this was prevented by elimination of the microglia or by inhibition of neutral sphingomyelinase. Tau also increased the phagocytic activity of pure microglia, and this was blocked by inhibitors of neutral sphingomyelinase or protein kinase C. The neuronal loss induced by tau was prevented by inhibitors of neutral sphingomyelinase, protein kinase C or the phagocytic receptor MerTK, or by eliminating microglia from the cultures. The data suggest that extracellular tau induces primary phagocytosis of stressed neurons by activated microglia, and identifies multiple ways in which the neuronal loss induced by tau can be prevented.

OS7-2 Injury threshold for axonal transport by observation of β-amyloid precursor protein in cultured rat brain neurons exposed to impulsive strain(OS7: Injury Biomechanics I)

OS7-2 Injury threshold for axonal transport by observation of β-amyloid precursor protein in cultured rat brain neurons exposed to impulsive strain(OS7: Injury Biomechanics I)

Cyclin C and cyclin dependent kinases 1, 2 and 3 in thrombin-induced neuronal cell cycle progression and apoptosis

The extent to which neurons proceed into the cell cycle and the mechanisms whereby cell cycle re-entry leads to apoptosis vary in response to agonists. We previously showed upregulation of early G1 regulators in thrombin-treated neurons yet neurons did not proceed to S phase but to apoptosis. The objective of this study is to explore mechanisms which might prevent S phase entry and promote apoptosis in thrombin-treated neurons. Cultured rat brain neurons are exposed to thrombin (200 nM) for 30 min to 4.5 h and the expression of cyclin C, cyclin dependent kinases (cdk1, cdk2, cdk3, cdk8) and the cell cycle inhibitor p27 assessed. Our data show a simultaneous decrease of both cyclin C and cdk3 proteins soon after thrombin treatment. The decrease in cyclin C also correlates with decreases in cdk1 and cdk2, at both mRNA and protein levels. There is no change in expression of cdk8 or the cell cycle inhibitor p27 in response to thrombin treatment. These results suggest that decreases in G1-S regulators cyclin C and cdks 3, cdk2 and cdk1 in response to thrombin could make conditions unfavorable for S phase entry and favor neuronal apoptosis.

Intracellular acidification in neurons induced by ammonium depends on KCC2 function

The Cl(-)-extruding neuron-specific K(+)-Cl(-) cotransporter KCC2, which establishes hyperpolarizing inhibition, can transport NH(4) (+) instead of K(+). It is, however, not clear whether KCC2 provides the only pathway for neuronal NH(4) (+) uptake. We therefore investigated NH(4) (+) uptake in cultured rat brain neurons. In neurons cultured for > 4 weeks, the response to NH(4)Cl applications (5 mM) consisted of an alkaline shift which reversed to an acid shift within seconds. Rebound acid shifts which followed brief applications of NH(4)Cl were blocked by furosemide (100 microM). They were rather insensitive to bumetanide (1 and 100 microM), in contrast to those induced in cultured glial cells. Rebound acid shifts persisted in the presence of 1 mM Ba(2+) and in Na(+)-free solution but were inhibited by extracellular K(+). In neurons with depolarizing GABA responses, indicating the absence of functional KCC2, applications of NH(4)Cl barely induced an acidosis. However, large rebound acid shifts occurred in neurons that had changed their GABA response from Ca(2+) increases to Ca(2+) decreases. Rebound acid shifts continued to increase even after the change in the GABA response had occurred and could be induced earlier in neurons transfected with KCC2 cDNA. We conclude that KCC2 provides the main pathway for fast neuronal NH(4) (+) uptake. Therefore, NH(4)Cl-induced rebound acid shifts can be used to indicate the development of KCC2 function. Further, the well known up-regulation of KCC2 function during development has the inevitable consequence of opening a major pathway for NH(4) (+) influx, which can be relevant under pathophysiological conditions.

Role of protein kinase Cβ in phorbol ester-induced c- fos gene expression in neurons of normotensive and spontaneously hypertensive rat brains

We have previously demonstrated that pressure application of the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) onto some neurons in the anterior hypothalamic area of rats increases neural activity in vivo and that this PKC activation-induced increase of neural activity is enhanced in spontaneously hypertensive rats (SHR), an animal model for genetic hypertension. Activation of PKC increases expression of the c- fos gene, an important transcription factor and proto-oncogene thought to be a marker of neural activity. To evaluate PKC isoforms responsible for neural activation, we examined which isoforms of PKC are involved in the PKC activation-induced c- fos gene expression in neuronal cultures of Wistar rat and spontaneously hypertensive rat (SHR) brains. PMA increased c- fos gene expression in neuronal cultures of Wistar rat brain and the PMA-induced c- fos gene expression was inhibited by the PKC inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7). The PKCα,β,γ activator thymeleatoxin also increased c- fos gene expression, while the PKCδ,ε activator ingenol did not affect it. In addition, the PMA-induced c- fos gene expression was inhibited by PKCβantisense oligonucleotides (AON) but not by PKCα and PKCγAONs. In SHR brain neuronal cultures, the PMA-induced c- fos gene expression was enhanced as compared with that of Wistar Kyoto rats (WKY), while basal c- fos gene expression was almost the same in both neuronal cultures. The enhancement of PMA-induced c- fos gene expression in SHR brain cultures was abolished by PKCβAON. These findings suggest that in rat brain neuronal cultures, PMA increases c- fos gene expression via activation of PKC and that PKCβisoforms are partly involved in the PMA-induced c- fos gene expression. In neuronal cultures of SHR brain, it appears that the PMA-induced c- fos gene expression is also enhanced via PKCβ.

Lithium protection from glutamate excitotoxicity: therapeutic implications

Long-term treatment with lithium, the primary drug used to treat bipolar mood disorder, robustly protects against glutamate-induced, NMDA receptor-mediated excitotoxicity in primary cultures of rat brain neurons. The neuroprotective effects of lithium are accompanied by a reduction in NMDA receptor-mediated calcium influx, up-regulation of anti-apoptotic Bcl-2, down-regulation of pro-apoptotic p53 and Bax, and activation of the cell survival signaling pathway. In addition, lithium antagonizes glutamate-induced activation of c-Jun-N-terminal kinase (JNK), p38 kinase and AP-1 binding, and suppresses glutamate-induced loss of phosphorylated CREB due to activation of protein phosphatase 1. Evidence has also been obtained that lithium induces BDNF and activates its receptor TrkB in cortical neurons and this BDNF/TrkB signaling activation is imperative for the neuroprotective effects of this drug. In a rat stroke model, post-insult treatments with therapeutic doses of lithium or valproic acid (VPA) markedly reduce ischemia-induced brain infarction and neurological deficits. These neuroprotective effects are associated with suppression of caspase-3 activation and induction of chaperone proteins. In a rat excitotoxicity model of Huntington's disease (HD) in which an excitotoxin was infused into the striatum, both long- and short-term pretreatment with lithium reduces excitotoxin-induced DNA damage, caspase-3 activation and striatal neuronal loss. This neuroprotection is associated with up-regulation of Bcl-2 in neurons. Lithium also induces cell proliferation near the injury site with a concomitant loss of proliferating cells in the subventricular zone. Some of these proliferating cells display neuronal or astroglial phenotypes. This finding corroborates our in vitro results showing that lithium stimulates the proliferation of neuroblasts in primary cultures of CNS neurons.

Recombinant adeno-associated virus serotype 2 effectively transduces primary rat brain astrocytes and microglia

Recombinant adeno-associated virus-2 (rAAV2) under control of the chicken beta actin promoter/truncated CMV enhancer (CBA) was investigated for its ability to transduce primary cultures of rat brain neurons, microglia and astrocytes. This vector was highly effective in all three cell types in heparin-sensitive manners (astrocytes, microglia and neurons transduced by >98%, 75%, and 95%, respectively). However, astrocytes co-cultured with neurons were not transduced. rAAV2/CBA is an important new method for genetic manipulation of brain cells, though this may be modulated by interactions among cell types.

Nicotinic Receptor Inhibition by Tetraponera Ant Alkaloids

1. Tetraponerines are a group of alkaloids occurring in the venoms of ants belonging to the genus Tetraponera. Eight compounds had been isolated and their structures elucidated, but their mechanisms of action had not yet been reported. We have studied the actions of several of these tetraponerines on vertebrate neuromuscular, ganglionic, and brain nicotinic acetylcholine receptors (nAChRs) using a variety of techniques including muscle contracture, cultured cell functional assays, neuronal patch clamping, and radioligand binding methods. 2. Potency for inhibition of the frog muscle carbachol-elicited contracture increased as the carbon 9 side chain alkyl group was increased in length to 10-12 carbons, then decreased when the chain was 18-carbons long. Potency differences between T-7 and T-8, which differ only in the stereochemistry of the carbon pentyl side chain were rather small. Quaternization of either N atom in a T-8 analog bearing a 10-carbon length alkyl substituent did not greatly affect potency for inhibition of the muscle response; thus the ionized form is an active form of this tetraponerine. 3. T-7 inhibited the nicotine-stimulated efflux of 86Rb from cultured PC12 cells, which primarily express alpha3-beta4 ganglionic type nicotinic receptors. T-8 blockade of BTX-sensitive and insensitive neuronal nAChRs, as studied by patchclamp recordings from cultured rat brain neurons, was also consistent with a noncompetitive type of inhibition. 4. T-7 displaced binding of the nAChR ion channel binding ligand thienylcyclophenidyl (TCP), an analog of PCP, to Torpedo neuromuscular type receptors. The affinity of the TCP binding site for T-7 did not depend upon the desensitization state of the receptor. 5. We conclude that the tetraponerines act at a site on nAChRs different from the ACh binding site which is probably located within the ion channel.

RNA interference suggests a primary role for monoacylglycerol lipase in the degradation of the endocannabinoid 2-arachidonoylglycerol.

The endogenous cannabinoid 2-arachidonoylglycerol (2-AG) is produced by neurons and other cells in a stimulus-dependent manner and undergoes rapid biological inactivation through transport into cells and catalytic hydrolysis. The enzymatic pathways responsible for 2-AG degradation are only partially understood. We have shown previously that overexpression of monoacylglycerol lipase (MGL), a cytosolic serine hydrolase that cleaves 1- and 2-monoacylglycerols to fatty acid and glycerol, reduces stimulus-dependent 2-AG accumulation in primary cultures of rat brain neurons. We report here that RNA interference-mediated silencing of MGL expression greatly enhances 2-AG accumulation in HeLa cells. After stimulation with the calcium ionophore ionomycin, 2-AG levels in MGL-silenced cells were comparable with those found in cells in which 2-AG degradation had been blocked using methyl arachidonyl fluorophosphonate, a nonselective inhibitor of 2-AG hydrolysis. The results indicate that MGL plays an important role in the degradation of endogenous 2-AG in intact HeLa cells. Furthermore, immunodepletion experiments show that MGL accounts for at least 50% of the total 2-AG-hydrolyzing activity in soluble fractions of rat brain, suggesting that this enzyme also contributes to 2-AG deactivation in the central nervous system.

Neuroprotective and Neurotrophic Actions of the Mood Stabilizer Lithium: Can It Be Used to Treat Neurodegenerative Diseases?

The mood stabilizing drug lithium has emerged as a robust neuroprotective agent in preventing apoptosis of neurons. Long-term treatment with lithium effectively protects primary cultures of rat brain neurons from glutamate-induced, NMDA receptor-mediated excitotoxicity. This neuroprotection is accompanied by an inhibition of NMDA-receptor-mediated calcium influx, upregulation of anti-apoptotic Bcl-2, downregulation of pro-apoptotic p53 and Bax, and activation of cell survival factors. Lithium treatment antagonizes glutamate-induced activation of c-Jun-N-terminal kinase (JNK), p38 kinase, and AP-1 binding, which has a major role in cytotoxicity, and suppresses glutamate-induced loss of phosphorylated cAMP responsive element binding protein (CREB). Lithium also induces the expression of brain-derived neurotrophic factor (BDNF) and subsequent activation TrkB, the receptor for BDNF, in cortical neurons. The activation of BDNF/TrkB signaling is essential for the neuroprotective effects of this drug. In addition, lithium stimulates the proliferation of neuroblasts in primary cultures of CNS neurons. Lithium also shows neuroprotective effects in rodent models of diseases. In a rat model of stroke, post-insult treatment with lithium or valproate, another mood stabilizer, at therapeutic doses markedly reduces brain infarction and neurological deficits. This neuroprotection is associated with suppression of caspase-3 activation and induction of chaperone proteins such as heat shock protein 70. In a rat model of Huntington's disease (HD) in which an excitotoxin is unilaterally infused into the striatum, both long- and short-term pretreatment with lithium reduces DNA damage, caspase-3 activation, and loss of striatal neurons. This neuroprotection is associated with upregulation of Bcl-2. Lithium also induces cell proliferation near the injury site with a concomitant loss of proliferating cells in the subventricular zone. Some of these proliferating cells display neuronal or astroglial phenotypes. These results corroborate our findings obtained in primary neuronal cultures. The neuroprotective and neurotrophic actions of lithium have profound clinical implications. In addition to its present use in bipolar patients, lithium could be used to treat acute brain injuries such as stroke and chronic progressive neurodegenerative diseases.

KCC2 mediates NH4+ uptake in cultured rat brain neurons.

Elevated levels of NH4+ in the brain impair neuronal function. We studied the effects of NH4+ on postsynaptic inhibition of cultured rat brain neurons using whole cell recording under nominally HCO3- -free conditions. Application of NH4+ shifted the reversal potentials for spontaneous inhibitory postsynaptic currents and currents elicited by dendritic GABA applications in a positive direction because [Cl-]i increased. The positive shift of the reversal potentials of GABA-induced Cl- currents was equal on equimolar elevation of [NH4+]o or [K+]o, respectively. The NH4+-induced increase in [Cl-]i was reversed by an inhibitor of cation-anion cotransport, furosemide (0.1 mM), but not by bumetanide (0.01 mM) or by replacement of [Na+]o by Li+. We conclude that neuron-specific K-Cl cotransporter (KCC2) transports NH4+ similar to K+. Despite this fact, the small increase of [NH4+]o during metabolic encephalopathies will barely elevate [Cl-]i. However, an impairment of neuronal function may result because KCC2 provides a pathway to accumulate NH4+, and thereby, a continuous acid load to neurons.

Differential effects of natural polyphenols on neuronal survival in primary cultured central neurons against glutamate- and glucose deprivation-induced neuronal death

Neuronal injury in the central nervous system following ischemic insult is believed to result from glutamate toxicity and glucose deprivation. In this study, polyphenols isolated from Scutellaria baicalensis Georgi, including baicalin, baicalein, and wogonin, were investigated for their neuroprotective effects against glutamate/NMDA (Glu/NMDA) stimulation and glucose deprivation in primary cultured rat brain neurons. Cell death was accessed by lactate dehydrogenase (LDH) release assay for necrosis, and mitochondrial activity was accessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction activity assay. It was found that both baicalin and baicalein decreased LDH release of the cultured neurons after 24 h treatment, whereas wogonin profoundly increased LDH release after 2 h treatment and resulted in neuronal death after 24 h. Glu/NMDA treatment profoundly increased LDH release and moderately decreased MTT reduction activity in an NMDA receptor-dependent manner. Both baicalin and baicalein significantly reduced Glu/NMDA-increased LDH release, in which baicalein is much more potent than baicalin. Glu/NMDA-increased intracellular calcium was also significantly attenuated by baicalin and baicalein. Baicalin and baicalein did not affect glutamate receptor binding activity, but baicalein did moderately decrease Glu/NMDA-induced nitric oxide (NO) production. In the glucose deprivation (GD) study, baicalein but not baicalin showed significant protective effects on the GD-increased LDH release, without affecting the GD-induced NO production, in cultured rat brain neurons. These results suggest that baicalein is the most effective compound among three polyphenols tested in preventing neurotoxicity induced by both glutamate and GD, whereas baicalin was only effective in preventing glutamate toxicity. Wogonin might have a neurotoxic effect on the brain.

Identification of new thyroid hormone-regulated genes in rat brain neuronal cultures

As a first approach to study the molecular mechanisms that underlie the effects of thyroid hormones on the developing brain, we used a cDNA microarray technology to identify early thyroid hormone-regulated genes in brain neuronal cultures treated with tri-iodothyronine (T3) for 3 h. We identified three genes that were up-regulated by T3, basic transcription element-binding protein, nuclear pore glycoprotein and bone morphogenetic protein-4 and one that was down-regulated, the neuronal apoptosis-inducing gene. We confirmed that these genes were also regulated by the thyroid state in the developing brain. Our findings enrich our knowledge of signaling pathways regulated by thyroid hormones and open new avenues for studying the molecular mechanisms of thyroid hormones in the developing brain.

Neuroprotective effects of lithium in cultured cells and animal models of diseases

Lithium, the major drug used to treat manic depressive illness, robustly protects cultured rat brain neurons from glutamate excitotoxicity mediated by N-methyl-D-aspartate (NMDA) receptors. The lithium neuroprotection against glutamate excitotoxiciy is long-lasting, requires long-term pretreatment and occurs at therapeutic concentrations of this drug. The neuroprotective mcchanisms involve inactivation of NMDA receptors, decreased expression of pro-apoptotic proteins, p53 and Bax, enhanced expression of the cytoprotective protein, Bcl-2, and activation of the cell survival kinase, Akt. In addition, lithium pretreatment suppresses glutamate-induced loss of the activities of Akt, cyclic AMP-response element binding protein (CREB), c-Jun - N-terminal kinase (JNK) and p38 kinase. Lithium also reduces brain damage in animal models of neurodegenerative diseases in which excitotoxicity has been implicated. In the rat model of stroke using middle cerebral artery occlusion, lithium markedly reduces neurologic deficits and decreases brain infarct volume even when administered after the onset of ischemia. In a rat Huntington's disease model, lithium significantly reduces brain lesions resulting from intrastriatal infusion of quinolinic acid, an excitotoxin. Our results suggest that lithium might have utility in the treatment of neurodegenerative disorders in addition to its common use for the treatment of bipolar depressive patients.

Production of monoclonal antibody to deltorphin-I and its immunocytochemical application to adult rat brain and cultured rat brain neurons☆ ,1

A monoclonal anti-deltorphin-I antibody specifically recognizing its NH 2-terminal region was produced. In the adult rat brain sections, it recognized immunoreactive nerve fibers mainly in the bed nucleus of stria terminalis, central nucleus of amygdala, lateral hypothalamus, hippocampus, substantia nigra, periaqueductal gray and locus ceruleus. Occasionally, positive somata were localized in the bed nucleus of stria terminalis, central nucleus of amygdala, supraoptic and periventricular nuclei. In primarily cultured neurons from various brain regions of new-born rats, the antibody immunostained strongly neuronal somata and processes. The abundant DADTI-immunoreactive substance in the cultured neurons promises to provide an alternative pathway to search for the counterpart of deltorphins in mammals.

Characteristics of brain injury-derived neurotrophic peptide-binding sites on rat brain synaptosomes and neurons in culture

Brain injury-derived neurotrophic peptide is the fragmental 13-mer peptide of the novel neurotrophic factor which was extracted and purified from Sponge Gelform made of gelatin implanted at the mechanically-induced injury site in neonatal rat brains. Brain injury-derived neurotrophic peptide supports survival of septal cholinergic and mesencephalic dopaminergic neurons in culture, and rescues hippocampal neurons in culture from glutamate neurotoxicity. Here we studied the binding characteristics of brain injury-derived neurotrophic peptide to synaptosomes from normal adult rat brains and neurons in culture from neonatal rat brains. [ 125I]Asp-[Tyr 11]-brain injury-derived neurotrophic peptide binding to rat brain synaptosomes was specific and saturable. Equilibrium binding studies revealed that [ 125I]Asp-[Tyr 11]-brain injury-derived neurotrophic peptide bound to 1.1 pmol/mg protein with a K d (dissociation constant) of 0.17 μM in hippocampal synaptosomes and to 2.0 pmol/mg protein with a K d of 0.38 μM in septal synaptosomes. [ 125I]Asp-[Tyr 11]-brain injury-derived neurotrophic peptide could bind to a subpopulation of hippocampal neurons in culture from embryonic rat brains. Affinity cross-linking with the car☐yl-reactive cross-linking reagent I-ethyl-3-(3-dimethylaminopropyl)-carbodiimide-HCl and [ 125I]Asp-[Tyr 11]-brain injury-derived neurotrophic peptide produced radiolabeled bands corresponding to 100,000, 50,000 and 40,000 mol. wt molecules on hippocampal neurons in culture. These results suggest that the 13-mer sequence of brain injury-derived neurotrophic peptide plays a crucial role in expressing the neurotrophic properties of the factor.

Hypoxia/reoxygenation induces apoptosis through biphasic induction of protein synthesis in cultured rat brain neurons

To investigate biochemical events accounting for the outcome of central neurons following hypoxia/reoxygenation, cultured neurons from fetal rat forebrain were exposed to hypoxia (95% N 2/5% CO 2) for 6 h, and then reoxygenated for up to 96 h. Time-dependent changes in macromolecular biosynthesis were analysed by incorporation of [ 3H]uridine and [ 3H]leucine and were coupled to cell viability and lactate dehydrogenase leakage. Morphological features of necrosis and apoptosis were scored following nuclear incorporation of the fluorescent dye 4,6-diamidino-2-phenylindole. Hypoxia led to a 36% reduction of cell viability at the end of the reoxygenation period, while 23% of the neurons exhibited apoptosis. A biphasic increase in the rates of protein synthesis was measured 1 h after the onset of hypoxia (77% above controls) and by 48-h postreoxygenation (72%). The presence of cycloheximide during hypoxia inhibited both peaks of synthesis and prevented the development of apoptosis. Protein electrophoresis outlined specific alterations in constitutive proteins, and immunohistochemistry revealed an overexpression of the pro-apoptotic gene products Bax and ICE. Therefore, hypoxia followed by reoxygenation would trigger sequential changes in synthesis of specific proteins, leading to delayed and mainly apoptotic neuronal death.

Angiotensin II regulation of tyrosine hydroxylase gene expression in the neuronal cultures of normotensive and spontaneously hypertensive rats.

In the present study we investigated the regulation of tyrosine hydroxylase (TH) by angiotensin II (Ang II) in an attempt to provide cellular and molecular evidence that this hormone has increased neuromodulatory actions in the spontaneously hypertensive (SH) rat brain. Neuronal cells in primary culture from the hypothalamus-brain stem of both normotensive [Wistar-Kyoto (WKY)] and SH rats have been used. These cultures mimic in vivo situations. Ang II caused a time-dependent increase in TH activity in WKY rat brain neurons. A maximal increase of 2.5-fold was observed with 100 nM Ang II in an actinomycin- and cycloheximide-dependent process. In addition, Ang II caused a parallel increase in TH messenger RNA (mRNA) levels, with a maximal stimulation of 5-fold in 4 h by 100 nM Ang II in WKY rat brain neurons. The stimulation of TH mRNA was mediated by the AT1 receptor subtype, resulted from an increase in its transcription, and involved activation of phospholipase C and protein kinase C. Antisense oligonucleotide for c-fos attenuated Ang II stimulation of TH mRNA in a time- and dose-dependent fashion, indicating an involvement of c-fos as a putative third messenger in Ang II stimulation of TH. Ang II also caused stimulation of TH activity and its mRNA levels in neuronal cultures of SH rat brain by a mechanism similar to that observed for neuronal cultures of WKY rat brain, involving AT1 receptors, protein kinase C, and c-fos. However, the stimulation of TH activity and that of TH mRNA were approximately 30% and 80% higher, respectively, in the SH rat brain neurons than those in the WKY rat brain neurons. In vivo experiments have been carried out to validate the elevated response of TH gene expression to Ang II in SH rat brain neuronal cultures. Ang II stimulated both TH activity and TH mRNA levels in the hypothalami and brain stems of adult WKY and SH rats. The level of stimulation in the brain of the SH rat was significantly higher than that in the WKY rat. These observations are consistent with an increase in AT1, receptor gene expression and suggest that increased TH gene expression could be the cellular/molecular basis for the greater neuromodulatory action of Ang II in the SH rat brain.

Mitogen-activated Protein Kinases in Rat Brain Neuronal Cultures Are Activated by Angiotensin II Type 1 Receptors and Inhibited by Angiotensin II Type 2 Receptors

Neurons cultured from neonatal rat hypothalamus and brainstem contain many angiotensin II (Ang II) type 2 (AT2) receptors, and we previously determined that activation of these sites elicited a stimulation of serine/threonine phosphatase 2A (PP2A). Here, we have investigated the effects of Ang II on neuronal mitogen-activated protein (MAP) kinases, potential targets for PP2A. Using in-gel kinase assays and immunoprecipitation analyses we have shown that Ang II (10 nM-1 microM) elicits significant increases in p44(MAPK) (Erk1) and p42(MAPK) (Erk2) activities in cultured neurons, mediated via Ang II type 1 (AT1) receptors. This stimulatory effect of Ang II on Erk1 and Erk2 activities was potentiated by blockade of AT2 receptors with (S)-1-[4-(dimethylamino)-3-methylphenyl]methyl-5-(diphenylacetyl)- 4, 5,6,7-tetrahydro-1H-imidazo[4,5-C]pyridine-6-carboxylic acid (PD 123319, 1 microM). Furthermore, the AT2 receptor agonist N-alpha-nicotinoyl-Tyr-Lys-(N-alphaCBZ-Arg)-His-Pro-Ile-OH (CGP42112A) (10-50 nM) caused significant decreases in neuronal Erk1 and Erk2 activities, which were abolished by PD 123319 (1 microM) and by the PP2A inhibitor okadaic acid (3 nM). This indicates that AT1 and AT2 receptors have opposite actions on Erk1 and Erk2 activities in neonatal neurons. Since MAP kinases are involved in the regulation of growth/differentiation and apoptosis, our data may provide an intracellular basis for modulatory effects of Ang II receptors on these processes.