The mineralocorticoid receptor-p38MAPK-NFκB or ERK-Sp1 signal pathways mediate aldosterone-stimulated inflammatory and profibrotic responses in rat vascular smooth muscle cells.

AIM: To explore the signalling pathways involved in aldosterone-induced inflammation and fibrosis in rat vascular smooth muscle cells (VSMCs). METHODS: Using Western blotting and real-time RT-PCR, we investigated the effects of aldosterone on the expression of cyclooxygenase-2 (Cox-2) and IL-6, two important proinflammatory factors, and TGFß1, a critical profibrotic factor, in VSMCs. RESULTS: Aldosterone treatment significantly increased the expression of Cox-2 and IL-6 and activation of p38MAPK and NF-κB. The expression of both Cox-2 and IL-6 could be blocked by the mineralocorticoid receptor (MR) antagonist spironolactone and the p38MAPK inhibitor SB203580. Also, the rapid phosphorylation of p38MAPK could be suppressed by SB203580 but not by spironolactone, implicating in nongenomic effects of aldosterone. Similar to SB203580 and spironolactone, the NF-κB inhibitor α-p-tosyl-L-lysine chloromethyl ketone (TLCK) markedly attenuated expression of Cox-2, indicating that MR, p38MAPK and NF-κB are associated with aldosterone-induced inflammatory responses. Furthermore, aldosterone enhanced expression of TGFß1 in rat VSMCs. This result may be related to activation of the MR/ERK-Sp1 signalling pathway because PD98059, an ERK1/2 inhibitor, significantly blocked the rapid phosphorylation of ERK1/2 and function of Sp1 and led to reduced expression of TGFß1. Spironolactone was also shown to significantly inhibit TGFß1 and Sp1 expression but not ERK1/2 phosphorylation. CONCLUSION: These results suggest that aldosterone-induced inflammatory responses and fibrotic responses may be mediated by the MR/p38MAPK-NF-κB pathways and the MR/ERK-Sp1 pathways in VSMCs, respectively.

Inhibition of methamphetamine-induced apoptosis by the calcium channel blocker verapamil in rat cerebellar neurons.

OBJECTIVE: To study protective effects of verapamil on neurotoxicity induced by methamphetamine (MA) in rat cerebellar neuronal cells (R2 cells) and explore the possible mechanisms. METHODS: MA neurotoxicity to R2 cells was examined by MTT reduction and LDH leakage assays. Using flow cytometer and agarose gel electrophoresis, apoptosis induced by MA in R2 cells was observed. The effects of verapamil on neurotoxicity and apoptosis induced by MA in R2 cells were determined. RESULTS: (1) Incubation of the R2 cells with MA (0.25-3.00 mmol/L) for 48 h or 60 h concentration-dependently increased the LDH content in the extra-cellular bathing medium by 48.2%-89.3% and 62.5%-97.3%, respectively. Exposure to MA elicited the apoptotic DNA peak of the R2 cells (i.e. sub-G1-peak) in a concentration- and time-dependent manner by the use of flow cytometer. Agarose gel electrophoresis showed a typical DNA laddering pattern. (2) Verapamil (0.5-5.0 micromol/L) reduced the number of dead cells and the LDH content in the extra-cellular bathing medium, ameliorated morphological changes, and inhibited apoptosis induced by MA in R2 cells. CONCLUSION: These results suggest that verapamil has a neuroprotective effect on MA-induced apoptosis, and that L-type voltage dependent calcium channel may be involved in the neurotoxitic effect of MA.

Nerve growth factor protects R2 cells against neurotoxicity induced by methamphetamine.

It is well known that methamphetamine is a neurotoxic drug of abuse to cause cell death both in vitro and in vivo. In this study, the protective effect of nerve growth factor on methamphetamine-induced neurotoxicity was examined in R2 cells. Treatment of R2 cells with methamphetamine for 48 h induced apoptotic-like death as determined by cell viability assay, DNA fragmentation on agarose gel and flow cytometric analysis. Co-treatment of R2 cells with nerve growth factor (10-1000 U/ml) in the presence of methamphetamine increased cell viability, decreased the intensity of methamphetamine-induced DNA fragmentation and reduced the number of apoptotic cells with flow cytometry. These results indicate that nerve growth factor may be useful for the treatment of methamphetamine users.