Cholesterol modulates function of connexin 43 gap junction channel via PKC pathway in H9c2 cells.

It has been shown that cholesterol modulates activity of protein kinase C (PKC), and PKC phosphorylates connexin 43 (Cx43) to regulate its function, respectively. However, it is not known whether cholesterol modulates function of Cx43 through regulating activity of PKC. In the present study, we demonstrated that cholesterol enrichment reduced the dye transfer ability of Cx43 in cultured H9c2 cells. Western blot analysis indicated that cholesterol enrichment enhanced the phosphorylated state of Cx43. Immunofluorescent images showed that cholesterol enrichment made the Cx43 distribution from condensed to diffused manner in the interface between the cells. In cholesterol enriched cells, PKC antagonists partially restored the dye transfer ability among the cells, downregulated the phosphorylation of Cx43 and redistributed Cx43 from the diffused manner to the condensed manner in the cell interface. In addition, reduction of cholesterol level suppressed PKC activity to phosphorylate Cx43 and restored Cx43 function in PKC agonist-treated cells. Furthermore, we demonstrated that cholesterol enrichment upregulated the phosphorylated state of Cx43 at Ser368, while PKC antagonists reversed the effect. Taken together, cholesterol level in the cells plays important roles in regulating Cx43 function through activation of the PKC signaling pathway.

Chlorpromazine confers neuroprotection against brain ischemia by activating BKCa channel.

Chlorpromazine (CPZ) is a well-known antipsychotic drug, still widely being used to treat symptoms of schizophrenia, psychotic depression and organic psychoses. We have previously reported that CPZ activates the BKCa (KCa1.1) channel at whole cell level. In the present study, we demonstrated that CPZ increased the single channel open probability of the BKCa channels without changing its single channel amplitude. As BKCa channel is one of the molecular targets of brain ischemia, we explored a possible new use of this old drug on ischemic brain injury. In middle cerebral artery occlusion (MCAO) focal cerebral ischemia, a single intraperitoneal injection of CPZ at several dosages (5mg/kg, 10mg/kg and 20mg/kg) could exert a significant neuroprotective effect on the brain damage in a dose- and time-dependent manner. Furthermore, blockade of BKCa channels abolished the neuroprotective effect of CPZ on MCAO, suggesting that the effect of CPZ is mediated by activation of the BKCa channel. These results demonstrate that CPZ could reduce focal cerebral ischemic damage through activating BKCa channels and merits exploration as a potential therapeutic agent for treating ischemic stroke.

Nicotine exerts neuroprotective effects against ß-amyloid-induced neurotoxicity in SH-SY5Y cells through the Erk1/2-p38-JNK-dependent signaling pathway.

Epidemiological data have indicated that smoking tobacco can decrease the risk of developing Alzheimer's disease (AD). Nicotine, a main component of tobacco, has been shown to have therapeutic effects in AD. The aim of the present study was to assess the neuroprotective effects of nicotine against toxicity induced by ß-amyloid (Aß) in relation to cell apoptosis, and to elucidate the role of the activation of the Erk1/2-p38-JNK pathway and the modulation of anti-apoptotic proteins in the nicotine-induced neuroprotective effects. We performed in vitro and in vivo experiments using SH-SY5Y cells and C57BL/6 mice, respectively. The effects of nicotine on cell apoptosis were determined by flow cytmetry and microscopic observation. The effects of nicotine on the expression of anti-apoptotic proteins were also determined by western blot analysis. Our results demonstrated that nicotine protected the SH-SY5Y cells against Aß25-35-induced toxicity by inhibiting apoptosis and upregulating the expression of anti-apoptotic proteins. As shown by our in vivo experiments, nicotine effectively ameliorated the impairment in spatial working memory induced by Aß25-35; this was confirmed by a Morris water maze navigation test and further supported by the upregulation of Bcl-2 in the hippocampus of Aß25-35-injected mice treated with nicotine. The phosphorylation of Erk1/2, p38 and JNK increased following treatment with nicotine in the SH-SY5Y cells, whereas caspase-3 activation was inhibited by treatment with nicotine prior to exposure to Aß25-35. Of note, these effects of nicotine against Aß25-35-induced damage were abolished by inhibitors of Erk1/2, p38 and JNK phosphorylation. These findings suggest that nicotine prevents Aß25-35-induced neurotoxicity through the inhibition of neuronal apoptosis, and may thus prove to be a potential preventive or therapeutic agent for AD.

Auditory sexual difference in the large odorous frog Odorrana graminea.

Acoustic communication is an important behavior in frog courtship. Male and female frogs of most species, except the concave-eared torrent frog Odorrana tormota, have largely similar audiograms. The large odorous frogs (Odorrana graminea) are sympatric with O. tormota, but have no ear canals. The difference in hearing between two sexes of the frog is unknown. We recorded auditory evoked near-field potentials and single-unit responses from the auditory midbrain (the torus semicircularis) to determine auditory frequency sensitivity and threshold. The results show that males have the upper frequency limit at 24 kHz and females have the upper limit at 16 kHz. The more sensitive frequency range is 3-15 kHz for males and 1-8 kHz for females. Males have the minimum threshold at 11 kHz (58 dB SPL), higher about 5 dB than that at 3 kHz for females. The best excitatory frequencies of single units are mostly between 3 and 5 kHz in females and at 7-8 kHz in males. The underlying mechanism of auditory sexual differences is discussed.

[Expression of A-kinase anchor protein 95, cyclin E2, and connexin 43 in lung cancer tissue, clinical significance of their expression, and their expression correlation].

OBJECTIVE: To study the expression of A-kinase anchor protein 95 (AKAP95), cyclin E(2), and connexin 43 (Cx43) in lung cancer tissue, the clinical significance of their expression, and the expression correlation among the three proteins. METHODS: Fifty-one samples of lung cancer tissue were examined by immunohistochemistry to measure the expression of AKAP95, cyclin E2, and Cx43. RESULTS: The positive rate of AKAP95 expression in lung cancer tissue was significantly higher than that in paracancerous tissue (82.35% vs 33.33%, P < 0.05); AKAP95 expression was associated with the cell differentiation and histopathological type of lung cancer (P < 0.05). The positive rate of cyclin E(2) expression in lung cancer tissue was significantly higher than that in paracancerous tissue (43.14% vs 13.33%, P < 0.05); cyclin E(2) expression was associated with the lymph node metastasis and histopathological type of lung cancer (P < 0.05). The positive rate of Cx43 expression in lung cancer tissue was lower than that in paracancerous tissue (60.78% vs 80.00%); Cx43 expression was associated with the cell differentiation, lymph node metastasis, and histopathological type of lung cancer (P < 0.05). There was correlation between each two of AKAP95 expression, cyclin E(2) expression, and Cx43 expression in lung cancer tissue. CONCLUSION: High expression of AKAP95 and cyclin E(2) plays an important role in the occurrence and development of lung cancer. AKAP95 expression is associated with the cell differentiation and histopathological type of lung cancer, and cyclin E2 expression is associated with lymph node metastasis and histopathological type. There is correlation between each two of AKAP95 expression, cyclin E(2) expression, and Cx43 expression in lung cancer tissue.

Nicotine stimulated bone marrow-derived dendritic cells could augment HBV specific CTL priming by activating PI3K-Akt pathway.

Our previous studies have revealed that nicotine-treated immature dendritic cells (imDCs) have anti-tumor effects in murine lymphoma models. The present study is to explore HBV-specific CTL priming and its cytolytic activities of nicotine-treated murine DCs, the mechanism of α7 nicotinic acetylcholine receptor (nAChR) up-regulation by nicotine and the efficiency of nicotine with other cytokines. To address these hypotheses, bone marrow-derived imDCs were stimulated by nicotine and expression of α7 nAChR was firstly determined by flow cytometry and Western blot. Then, DCs-dependent HBV-specific T cell proliferation and IL-12 secretion were secondly determined by BrdU cell proliferation assay and ELISA, respectively. The HBV-specific CTL priming and its activities were further explored by intraperitoneal transfer of nicotine treated imDCs. The mechanism of nicotine up-regulating α7 nAChR was finally explored by Western blot. The results showed that: first, the maximal activation of PI3K and Akt was reached at 30 and 60-120 min respectively after nicotine stimulation. Nicotine up-regulated the expression of α7 nAChR by activating PI3K-Akt pathway in murine DCs; secondly, nicotine stimulation could enhance DCs' ability of HBV-specific T cell proliferation and IL-12 secretion; thirdly, adoptive transfer of nicotine stimulated DCs could induce HBV specific CTL priming in vivo and those CTL had cytolytic activities; fourthly, nicotine had equal efficiencies to 2 ng/ml IFN-γ in DCs-mediated T cell proliferation. All these data presented here indicated that nicotine treated imDCs might be considered as a potential candidate for HBV immunotherapy.

Lipopolysaccharide and dose of nicotine determine the effects of nicotine on murine bone marrow-derived dendritic cells.

The reported effects of nicotine on dendritic cells (DCs) are controversial. To investigate the factors which determine the effects of nicotine on DCs, immature dendritic cells (imDCs) induced from murine bone marrow were treated with different doses of nicotine with or without lipopolysaccharides (LPS). The morphology and expression of the co-stimulatory molecules CD80, CD86, CD40 and CD54 were observed and determined by microscopy and flow cytometry, respectively. The results showed that, firstly, nicotine treatment promoted the development of DC precursors into imDCs with a semi-mature phenotype revealed by a higher expression of CD11c and more branched projections. Secondly, lower doses of nicotine (16.5 ng/ml), but not higher (200 µg/ml), up-regulated the expression of the co-stimulatory molecules CD80, CD40 and CD54 on imDCs. Co-administration of LPS and nicotine revealed differential effects on co-stimulatory molecule expression on imDCs. Thirdly and importantly, treatment with lower doses of nicotine (16.5 ng/ml) did not augment expression of the CD80, CD86, CD40 and CD54 molecules in mature DCs. Fourthly and interestingly, high doses of nicotine (more than 165 µg/ml) revealed pro-apoptotic activity but lower doses of nicotine (16.5-0.165 ng/ml) achieved an anti-apoptotic effect on imDCs. All data presented here indicate that the controversial effects of nicotine on DCs may be due to the LPS of the nicotinic environment and the dose of nicotine used.