Effect of dopamine depletion on DARPP-32 protein in ischemic rat striatum.

AIM: To study the effects of dopamine depletion on the phosphorylation level, intracellular distribution, and mRNA expression of DARPP-32 in the ischemic striatum and to elucidate the mechanisms underlying the ischemic injury. METHODS: A complex model of SN lesioning with 6-OHDA to deplete dopamine and four vessels occlusion for inducing forebrain ischemia was constructed in rats. DARPP-32 was investigated with autoradiogram, immunohistochemistry and in situ hybridization. RESULTS: The [32P]phosphate incorporation of DARPP-32 was reduced in vitro following ischemia. However, the [32P]phosphate incorporation, the numbers of positive neurons, and mRNA expression of DARPP-32 were increased in SN lesioning plus ischemic rats with denervated striatum. CONCLUSION: Dopamine depletion reduced the DARPP-32 phosphorylation in vivo following ischemia, and protected DARPP-32 immunoreactivity and mRNA expression level against the reduction induced by ischemia.

Stimulation of dopamine receptors inhibited Ca2+-calmodulin-dependent protein kinase II activity in rat striatal slices.

AIM: To investigate the mechanism underlying dopaminergic neurotoxicity in the striatum during anoxia. METHODS: Using rat striatal slices as an in vitro model, the activity of Ca2+-calmodulin-dependent protein kinase II (CCDPKII) was examined by the method of substrate phosphorylation 32P-incorporation. RESULTS: Anoxia for 30 min greatly reduced CCDPKII activity by about 75 %. Reserpinization by repeated reserpine administration (1 mg . kg-1 . d-1 for 7 d, sc) preserved CCDPK II activity against the anoxia-induced decrease (about 40 % of control). The activity of CCDPKII was reduced significantly by exposure of rat striatal slices to micromolar concentrations of dopamine in the presence of extracellular Ca2+. Omission of Ca2+ in the incubation medium (with addition of 1 mmol/L egtazic acid) diminished the dopamine-induced decrease of the kinase activity. Application of apomorphine, a non-selective dopamine receptor agonist, produced a similar concentration-related decrease of CCDPKII activity. Exposure to SKF38393 (selective D1-like receptor agonist) or quinpirole (selective D2-like receptor agonist) also inhibited the kinase activity. The dopamine-induced decrease of CCDPKII activity was attenuated by preincubation with Sch-23390 (selective D1-like receptor antagonist) or domperidone (selective D2-like receptor antagonist). CONCLUSION: Dopamine is involved in the anoxia-induced inhibition of CCDPKII activity by activation of both D1-like and D2-like receptors and influx of Ca2+, which may contribute to dopamine-mediated striatal neuronal damage.

[Changes in the state of phosphorylation of DARPP-32 of gerbil striatum during ischemia].

Using the mongolian gerbil model of transient forebrain ischemia by bilateral carotid arteries occlusion, the levels of phosphorylation and the amount of DARPP-32 in striatum were measured by back-phosphorylation and Western-blotting assay in vitro. Transient forebrain ischemia had no effect on the immunoreaction of DARPP-32 in striatum, but the state of phosphorylation of DARPP-32 showed a significant change. The [32P] phosphate incorporation in DARPP-32 decreased after 2, 7 or 10 min of ischemia, but increased after a 5-min ischemia. In contrast, the result of back-phosphorylation in vivo was opposite to that obtained in vitro.

Dopamine-glutamate interaction in rat striatal slices: changes of CCDPK II, PKA, and LDH activity by receptor-mediated mechanisms.

AIM: To study the effects of dopamine (DA) and glutamate (Glu) and their receptor agonists/antagonists on Ca2+/calmodulin-dependent protein kinase II (CCDPK II), cyclic AMP-dependent protein kinase A (PKA) activities and the LDH release in rat striatal slices, and to examine the interaction between DA and Glu transmitter systems in striatum. METHODS: The activities of CCDPK II, PKA, and the release of LDH were determined with the 32P-incorporation and colorimetry respectively in rat striatal slices. RESULTS: (1) Exogenous DA, D1 receptor agonist SKF 38393 and D2 receptor agonist LY 171555 reduced CCDPK II activity in striatal slices; Glu also inhibited CCPPK II activity in a concentration-dependent manner. NMDA receptor antagonist MK-801 could antagonize the inhibitory effect of SKF 38393 and LY 171555 on the CCDPK II activity. D1 and D2 receptor antagonists SCH 23390 and spiperone could also antagonize the decrease of CCDPK II activity induced by Glu; (2) DA and SKF 38393 markedly increased PKA activity in striatal slices, which was reduced by MK-801; (3) DA and Glu increased the release of LDH from the striatal neurons in a concentration-dependent manner. MK-801 antagonized the increase of LDH induced by DA. Spiperone, rather than SCH 23390, could reduce the release of LDH from striatal neuron in the presence of Glu. CONCLUSION: The interaction between DA and Glu transmitter systems is found in the regulation of the CCDPK II and PKA activities and cell function in the striatum.

Relevance between striatal expression of Fos, proenkephalin mRNA, prodynorphin mRNA and rotation induced by l-stepholidine in 6-hydroxydopamine-lesioned rats.

AIM: To study that l-stepholidine (SPD) regulates the expression of proenkephalin (PENK) and prodynorphin (PDYN) mRNA and Fos in the striatum after rotational test in the 6-hydroxydopamine (6-OHDA)-lesioned rats. METHODS: PENK and PDYN mRNA levels were examined with in situ hybridization, and Fos expression was detected with immunocytochemistry. The data were semi-quantified with image analyzer. RESULTS: (1) Following repeated SPD treatment, the rotation was kept on high activity in the 6-OHDA-lesioned rats. (2) SPD significantly elicited Fos expression in both sides of striatum, particularly in the denervated one. Repeated administration of SPD, Fos expression declined on both sides, particularly in the intact one. (3) In the denervated striatum of 6-OHDA-lesioned rats, the PENK mRNA level was extremely increased vs that in the intact striatum. This high level of PENK mRNA was significantly reduced by 7-d treatments of SPD. SPD also reduced the level of PENK mRNA in the intact striatum. However, the level of PDYN mRNA did not show significant change in both sides of striatum after denervation or SPD treatment. CONCLUSION: In the 6-OHDA-lesioned rats, the rotation induced by SPD was kept on a high activity, which was in pace with the inducement of Fos expression and the reduction of expression of PENK mRNA in the denervated striatum. But then the lesion and SPD treatment had no remarkable effect on the expression of PDYN mRNA.

Antagonistic effect of l-stepholidine on striatal ischemic injury in rat.

AIM: To elucidate the protection of l-stepholidine (SPD) on neuronal morphology and function against the striatal ischemic injury in rat. METHODS: The forebrain ishemia to Sprague Dawley rats was induced with four-vessel occlusion. Histological examination was performed on the dorsolateral striatum with cresylviolet stain. In striatal slices of rat as an in vitro ischemic model, the activity of calcium/calmodulin-dependent protein kinase II (CCDPK) and lactate dehydrogenase (LDH) was examined by the method of 32P-incorporation and colorimetry, respectively. RESULTS: In the SPD-treated groups, most of the neurons in the striatum kept the normal morphological appearance after 30-min ischemia followed by 6-h or 12-h reperfusion. The number of neurons was much more in SPD groups than that in vehicle group. The sparse and abnormal neurons were observed in the vehicle group. SPD attenuated the ischemic effect on the CCDPK activity in striatal slices. In addition, SPD inhibited the leakage of LDH from neurons induced by ischemia in incubated striatal slices. CONCLUSION: SPD protected striatal neurons against ischemic injury and antagonized the inhibitory action on CCDPK activity induced by ischemia. SPD reduced the leakage of LDH from striatal neurons induced by ischemia.

Negative regulation of a heterologous promoter by human cytomegalovirus immediate-early protein IE2.

The HCMV IE2 protein promiscuously activates transcription of many viral and cellular genes. IE2 also negatively autoregulates its own expression by binding to a strategically positioned IE2 binding site, called CRS, located immediately downstream of the TATA box of the HCMV major IE promoter. Here we show that IE2 is able to repress transcription driven by a heterologous promoter, RSV LTR. Repression of RSV LTR by IE2 is completely dependent of DNA sequences downstream of the TATA box of RSV LTR. A DNA sequence, 5'-CGATACAATAAACG-3', evidently matching the proposed CRS consensus sequence, is located between nucleotides -13 and +1 (relative to the transcription start site) of RSV LTR. Three lines of evidence support the notion that this RSV CRS element is involved in the IE2-mediated repression of RSV LTR. First, introduction of mutation to the RSV CRS element renders to the mutant RSV LTR resistance to IE2-mediated repression. Second, a mutant IE2 defective in DNA binding cannot downregulate transcription from RSV LTR. Third, IE2 specifically binds to the wild-type, but not the mutant, RSV CRS element in vitro. These data, in conjunction with previous works, demonstrate that IE2 can passively repress transcription of homologous and heterologous promoters that contain a CRS element.

Transcriptional regulation by p53. Functional interactions among multiple regulatory domains.

The tumor suppressor p53 protein possesses activities typical of eukaryotic transcriptional activators; p53 binds to specific DNA sequences and stimulates transcription of the target genes. By a series of deletion and domain-swapping studies, we report here that (i) p53 has two auxiliary domains, which have little effect on the DNA binding activity of its core domain but are capable of modulating its transactivation activity in a target site-dependent manner; (ii) p53 contains two cell-specific transcriptional inhibitory domains, I1 and I2, which are active in Saos-2 and HeLa cells but not in HepG2 and Hep3B cells; and (iii) I1 inhibits the activity of several structurally different activating regions. These results demonstrate that the apparent transcriptional activity of p53 is determined by collaborations among its regulatory domains, its target sites, and the cellular environment.

Tamoxifen stimulates human papillomavirus type 16 gene expression and cell proliferation in a cervical cancer cell line.

The widely adopted use of tamoxifen as a chemotherapeutic agent is primarily based on its inhibition of cancer cell growth. However, we report that tamoxifen at low concentrations (10(-9) and 10(-11) M) causes stimulation of cell proliferation in a cervical cancer cell line, SFR. The facts that SFR cells do not contain estrogen receptors and are estrogen nonresponsive imply the existence of an antiestrogen-specific binding protein and suggest that the effect of tamoxifen is possibly mediated through a pathway other than estrogen receptors. Tamoxifen at low concentrations stimulated human papillomavirus type 16 (HPV-16) gene transcription and E7 protein production. Levels of HPV-16 mRNA and E7 protein reached a peak at approximately 2-4 h after tamoxifen treatment, persisted for several hours, and subsequently decreased to their prestimulation levels by about 24 h after treatment. Our results indicate for the first time that tamoxifen stimulates cell proliferation of cervical cancer cells, and we suggest that the enhanced HPV-16 mRNA and E7 protein levels are probably responsible.