A novel RING finger protein, Znf179, modulates cell cycle exit and neuronal differentiation of P19 embryonal carcinoma cells.

Znf179 is a member of the RING finger protein family. During embryogenesis, Znf179 is expressed in a restricted manner in the brain, suggesting a potential role in nervous system development. In this report, we show that the expression of Znf179 is upregulated during P19 cell neuronal differentiation. Inhibition of Znf179 expression by RNA interference significantly attenuated neuronal differentiation of P19 cells and a primary culture of cerebellar granule cells. Using a microarray approach and subsequent functional annotation analysis, we identified differentially expressed genes in Znf179-knockdown cells and found that several genes are involved in development, cellular growth, and cell cycle control. Flow cytometric analyses revealed that the population of G0/G1 cells decreased in Znf179-knockdown cells. In agreement with the flow cytometric data, the number of BrdU-incorporated cells significantly increased in Znf179-knockdown cells. Moreover, in Znf179-knockdown cells, p35, a neuronal-specific Cdk5 activator that is known to activate Cdk5 and may affect the cell cycle, and p27, a cell cycle inhibitor, also decreased. Collectively, these results show that induction of the Znf179 gene may be associated with p35 expression and p27 protein accumulation, which lead to cell cycle arrest in the G0/G1 phase, and is critical for neuronal differentiation of P19 cells.

Activation of protein kinase A and atypical protein kinase C by A(2A) adenosine receptors antagonizes apoptosis due to serum deprivation in PC12 cells.

We found in the present study that stimulation of A(2A) adenosine receptors (A(2A)-R) prevents apoptosis in PC12 cells. This A(2A)-protective effect was blocked by protein kinase A (PKA) inhibitors and was not observed in a PKA-deficient PC12 variant. Stimulation of PKA also prevented apoptosis, suggesting that PKA is required for the protective effect of A(2A)-R. A general PKC inhibitor, but not down-regulation of conventional and novel PKCs, readily blocked the protective effect of A(2A)-R stimulation and PKA activation, suggesting that atypical PKCs (aPKCs) serve a critical role downstream of PKA. Consistent with this hypothesis, stimulation of A(2A)-R or PKA enhanced nuclear aPKC activity. In addition, the A(2A)-protective effect was blocked by a specific inhibitor of one aPKC, PKCzeta, whereas overexpression of a dominant-positive PKCzeta enhanced survival. In contrast, inhibitors of MAP kinase and phosphatidylinositol 3-kinase did not modulate the A(2A)-protective effect. Dominant-negative Akt also did not alter the A(2A)-protective effect, whereas it significantly reduced the protective action of nerve growth factor. Collectively, these data suggest that aPKCs can function downstream of PKA to mediate the A(2A)-R-promoted survival of PC12 cells. Furthermore, the results indicate that different extracellular stimuli can employ distinct signaling pathways to protect against apoptosis induced by the same insult.

Modulation of dopamine transporter activity by nicotinic acetylcholine receptors and membrane depolarization in rat pheochromocytoma PC12 cells.

To elucidate the regulation of the rat dopamine transporter (rDAT), we established several PC12 variants overexpressing the rDAT. Treating these cells with a nicotinic agonist (1,1-dimethyl-4-phenylpiperazinium iodide, 30 microM) depolarized the plasma membrane potential from -31 +/- 2 to 43 +/- 5 mV and inhibited rDAT activity significantly in a calcium- and protein kinase C-independent manner. Membrane depolarization by a high external K+ concentration or two K+ channel blockers (tetraethylammonium hydroxide and BaCl2) also resulted in a marked inhibition of rDAT activity. Such inhibition of dopamine uptake is due to a reduction in Vmax, with no marked effect on the Km for dopamine. The potency of cocaine in inhibiting dopamine uptake was not significantly altered, whereas that of amphetamine was slightly enhanced by membrane depolarization. Removing extracellular Ca2+ or blocking the voltage-sensitive L-type calcium channels using nifedipine did not exert any significant effect on the inhibition of rDAT activity by depolarization. These data confirm that calcium influx on depolarization is not required for inhibition of the rDAT. Collectively, our data suggest that rDAT activity can be altered by a neurotransmitter that modulates the membrane potential, thus suggesting an exquisite mechanism for the fine-tuning of dopamine levels in the synapse.

Nicotinamide attenuates methamphetamine-induced striatal dopamine depletion in rats.

The aim of the present study was to examine the effects of nicotinamide, a co-factor in the electron transport chain, on the relationship between methamphetamine (MA)-induced striatal dopamine (DA) depletion and energy metabolism change. Four injections of MA (10 mg/kg, i.p.) at 2 h intervals resulted in decreases of 51% and 23%, respectively, in striatal DA and adenosine 5'-triphosphate (ATP) levels 5 days later. Nicotinamide (500 mg/kg, i.p.) treatment prior to each MA injection attenuated the reductions of striatal DA and ATP contents. Nicotinamide had no long-term effects on striatal DA and ATP levels. These findings suggest that energy impairment might play a role in MA-induced DAergic neurotoxicity in the striatum.

Effects of acute and chronic morphine on DOPAC and glutamate at subcortical DA terminals in awake rats.

Effects of morphine and naloxone on the levels of 3,4-dihydroxy-phenylacetic acid (DOPAC) and glutamate in the striatum and nucleus accumbens of awake rats were studied with in vivo microdialysis. Acute morphine (50 mg/kg, IP) treatment increased the levels of DOPAC and glutamate in the striatum and nucleus accumbens, but both decreased from the elevated levels when naloxone (10 mg/kg, IP) was given 2 h later. Chronic morphine treatment, twice daily for 5 days in incremental doses (5, 10, 20, 40 and 50 mg/kg, IP), increased the level of DOPAC but decreased that of glutamate in the striatum and nucleus accumbens. When naloxone was given 2 h later, the reverse of the above phenomena are found. After given repeated morphine treatment and experiencing naloxone-precipitated withdrawal, the rats with an intact cortex and the rats with ibotenic acid (5 microg/0.5 microl/2.5 min) lesions on the medial prefrontal cortex and sulcal cortex have similar alternations in the levels of DOPAC and glutamate in the striatum. However, in the nucleus accumbens, the level of DOPAC dropped more and the level of glutamate increased more in the intact rats than the lesioned rats during the withdrawal stage. These data suggested that the intact cortex ordinarily exerted an inhibitory role to influence the level of DOPAC in the striatum and nucleus accumbens during chronic morphine treatment. In conclusion, morphine seems to activate different pathways in dependent and non-dependent rats.

Amphetamine induces hydroxyl radical formation in the striatum of rats.

Amphetamine-induced hydroxyl radical formation in the striatum of rats was investigated in this study. With the utilization of the microdialysis and HPLC-ECD, the striatal dopamine (DA) release and the formation of 2,3-dihydroxybenzoic acid (2,3-DHBA), derived from the reaction of hydroxyl radicals (.OH) and salicylate in perfusion, were monitored and detected during desipramine and/or amphetamine (AMPH) administration. Our data revealed that after desipramine treatment AMPH injections not only amplified striatal DA release and 2,3-DHBA formation, but also intensified the stereotyped behaviors induced by AMPH. Furthermore, we discovered that alpha-methyl-para-tyrosine (alpha-MT) pretreatment prevented the onset of the above responses. In desipramine-treated rats, the tissue homogenization study demonstrated that a single dose of AMPH produced long-term depletion of striatal DA; this was not seen in saline-treated rats. Moreover, striatal DA depletion could be lessened by pretreatment with mannitol, a .OH scavenger. These results indicate that AMPH-induced striatal .OH formation might be DA-related in desipramine-treated rats, and suggest that .OH formation might be correlated with AMPH-induced neurodegeneration.

Morphine withdrawal and schedule-induced polydipsia.

Schedule-induced polydipsia (SIP) is the characterized behavior for reducing the heightened arousal in a schedule of intermittent feeding. In the present study, SIP rats received an incremental doses of morphine in repeated treatments on the first 5 days and were then challenged by naloxone on the 6th day. We examined the SIP performance during morphine dependence and withdrawal. The roles of the locus coeruleus (LC) and excitatory amino acid (EAA) pathways were examined by bilateral LC lesions and lateral ventricle kynurenic acid infusion. In each manipulation, the level of water intake was recorded as an index of SIP strength. Our results showed that morphine dependence reduced SIP strength, whereas withdrawal initially reduced but then elevated SIP strength. Such effects were attenuated by bilateral LC lesions or kynurenic acid administration. The implications of these results on morphine withdrawal reaction and SIP performance were discussed.

Evidence that 5-HT(2) Antagonism Elicits a 5-HT(3)-Mediated Increase in Dopamine Transmission.

Amperozide, a novel atypical antipsychotic drug with few extrapyramidal side effects, is a strong serotonin(2) (5-HT(2)) antagonist but has low affinity for dopamine receptors in vitro. The effect of amperozide on the dopaminergic synapse was studied with an in vivo microdialysis technique using anesthetized male Sprague-Dawley rats. Following implantation of dialysis probes into the striatum and nucleus accumbens (NuAc), amperozide was intravenously infused as six consecutive incremental doses (0.5, 0.5, 1.0, 2.0, 4.0 and 8.0 mg/kg) at intervals of 15 min. From the beginning of drug infusion, perfusates were collected in fractions every 30 min throughout a total period of 120 min. The samples were then immediately analyzed by high-performance liquid chromatography with electrochemical detection. Amperozide induced a dose-related elevation of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindolacetic acid (5-HIAA) levels in both areas. p-Chlorophenylalanine (pCPA) pretreatment abolished the production of 5-HIAA in both areas and attenuated the amperozide-induced rise of DOPAC but not of dopamine. After pretreatment with an intravenous 5-HT(3) antagonist, MDL 72222, the amperozide-induced changes in dopamine, DOPAC and 5-HIAA in both areas were lower than in the saline control group. Preliminary data showed that after pCPA pretreatment, incremental concentrations of the 5-HT(3) agonist 1-(m-chlorophenyl)-biguanide perfused via the probe also produced significant elevation of dopamine and DOPAC levels in these two areas. Taken together, these results suggest that amperozide may directly block 5-HT(2) receptors in the striatum and NuAc, thereby enhancing 5-HT transmission. The enhanced 5-HT transmission may activate postsynaptic 5-HT(3) receptors located on the dopaminergic terminals, leading to changes in dopamine transmission in these two areas. Copyright 1995 S. Karger AG, Basel