Inhibitory actions of the phosphatidylinositol 3-kinase inhibitor LY294002 on the human Kv1.5 channel

Background and purpose:

Kv1.5 channels conduct the ultra-rapid delayed rectifier potassium current (I_Kur), and in humans, Kv1.5 channels are highly expressed in cardiac atria but are scarce in ventricles. Pharmacological blockade of human Kv1.5 (hKv1.5) has been regarded as effective for prevention and treatment of re-entry-based atrial tachyarrhythmias. Here we examined blockade of hKv1.5 channels by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, a well-known inhibitor of phosphatidylinositol 3-kinase (PI3K).

Experimental approach:

hKv1.5 channels were heterologously expressed in Chinese hamster ovary cells. Effects of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 on wild-type and mutant (T462C, H463C, T480A, R487V, A501V, I502A, I508A, L510A and V516A) hKv1.5 channels were examined by using the whole-cell patch-clamp method.

Key results:

{"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 rapidly and reversibly inhibited hKv1.5 current in a concentration-dependent manner (IC₅₀ of 7.9 µmol·L⁻¹). In contrast, wortmannin, a structurally distinct inhibitor of PI3K, had little inhibitory effect on hKv1.5 current. {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 block of hKv1.5 current developed with time during depolarizing voltage-clamp steps, and this blockade was also voltage-dependent with a steep increase over the voltage range for channel openings. The apparent binding (k₊₁) and unbinding (k₋₁) rate constants were calculated to be 1.6 µmol·L⁻¹⁻¹·s⁻¹ and 5.7 s⁻¹ respectively. Inhibition by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 was significantly reduced in several hKv1.5 mutant channels: T480A, R487V, I502A, I508A, L510A and V516A.

Conclusions and implications:

{"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 acts directly on hKv1.5 currents as an open channel blocker, independently of its effects on PI3K activity. Amino acid residues located in the pore region (Thr480, Arg487) and the S6 segment (Ile502, Ile508, Leu510, Val516) appear to constitute potential binding sites for {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002.     

The PI3K/Akt pathway mediates the protection of SO2 preconditioning against myocardial ischemia/reperfusion injury in rats

Aim:

To explore the mechanisms underlying the protection by SO₂ preconditioning against rat myocardial ischemia/reperfusion (I/R) injury.

Methods:

Male Wistar rats underwent 30-min left coronary artery ligation followed by 120-min reperfusion. An SO₂ donor (1 μmol/kg) was intravenously injected 10 min before the ischemia, while {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (0.3 mg/kg) was intravenously injected 30 min before the ischemia. Plasma activities of LDH and CK were measured with an automatic enzyme analyzer. Myocardial infarct size was detected using Evans-TTC method. The activities of caspase-3 and -9 in myocardium were assayed using a commercial kit, and the levels of p-Akt, Akt, PI3K and p-PI3K were examined with Western blotting.

Results:

Pretreatment with SO₂ significantly reduced the myocardial infarct size and plasma LDH and CK activities, as well as myocardial caspase-3 and -9 activities in the rats. Furthermore, the pretreatment significantly increased the expression levels of myocardial p-Akt and p-PI3K p85. Administration of the PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 blocked all the effects induced by SO₂ pretreatment.

Conclusion:

The results suggest that the PI3K/Akt pathway mediates the protective effects of SO₂ preconditioning against myocardial I/R injury in rats.     

Sodium tanshinone IIA sulfonate protects rat myocardium against ischemia-reperfusion injury via activation of PI3K/Akt/FOXO3A/Bim pathway

Aim： To investigate the mechanisms underlying the protective effects of sodium tanshinone IIA sulfonate （STS） in an ischemia- reperfusion （I/R）-induced rat myocardial injury model. Methods： Male SD rats were iv injected with STS, STS＋LY294002, or saline （NS） for 15 d. Then the hearts were subjected to 30 min of global ischemia followed by 2 h of reperfusion. Cardiac function, infarction size and area at risk were assessed. Cell apoptosis was evaluated with TUNEL staining, DNA laddering and measuring caspase-3 activity. In addition, isolated cardiomyocytes of neonatal rats were pretreated with the above drugs, then exposed to H202 （200 μmol/L） for I h. Cell apoptosis was detected using flow cytometric assay. The levels of p-Akt, p-FOXO3A and Bim were examined with immunoblotting. Results： Compared to NS group, administration of STS （20 mg/kg） significantly reduced myocardial infarct size （40.28%＋5.36% in STS group vs 59.52%±7.28% in NS group）, and improved the myocardial function as demonstrated by the increased values of dp/dt LVDP and coronary flow at different reperfusion time stages. Furthermore, STS significantly decreased the rate of apoptotic cells （15.11%±3.71% in STS group vs 38.21%±7.83% in NS group）, and reduced caspase-3 activity to nearly a quarter of that in NS group. Moreover, STS significantly increased the phosphorylation of Akt and its downstream target FOXO3A, and decreased the expression of pro-apoptotic gene Bim. Co-treatment with the PI3K inhibitor LY294002 （40 mg/kg） partially countered the protective effects induced by STS treatment. In isolated cardiomyocytes, STS exerted similar protective effects as shown in the ex vivo I/R model. Conclusion： STS pretreatment reduces infarct size and improves cardiac function in an I/R-induced rat myocardial injury model via activation of Akt/FOXO3A/Bim-mediated signal pathway.     

Over-production of nitric oxide by oxidative stress-induced activation of the TGF-β1/PI3K/Akt pathway in mesangial cells cultured in high glucose

Aim:

To investigate whether NO over-production in rat mesangial cells cultured in high glucose (HG) is related to activation of the TGF-β1/PI3K/Akt pathway.

Methods:

Rat mesangial cells line (HBZY-1) was exposed to HG (24.44 mmol/L) or H₂O₂ (10 μmol/L) for 16 h. NO release was quantified using the Griess assay. The TGF-β1 level was measured using ELISA. The protein expression of p-Akt, t-Akt, Bim, and iNOS was examined by Western blotting. The mRNA levels of TGF-β1 and Bim were measured using RT-PCR. The cell proliferation rate was estimated using a BrdU incorporation assay.

Results:

Treatment of the cells with HG, H₂O₂, or TGF-β1 (5 ng/mL) significantly increased the NO level that was substantially inhibited by co-treatment with the NADPH oxidase inhibitor diphenylene iodonium (DPI), TGF-β1 inhibitor SB431542, or PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. Both HG and H₂O₂ significantly increased the protein and mRNA levels of TGF-β1 in the cells, and HG-induced increases of TGF-β1 protein and mRNA were blocked by co-treatment with DPI. Furthermore, the treatment with HG or H₂O₂ significantly increased the expression of phosphorylated Akt and iNOS and cell proliferation rate, which was blocked by co-treatment with DPI, SB431542, or {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. Moreover, the treatment with HG or H₂O₂ significantly inhibited Bim protein and mRNA expression, which was reversed by co-treatment with DPI, SB431542, or {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002.

Conclusion:

The results demonstrate that high glucose causes oxidative stress and NO over-production in rat mesangial cells in vitro via decreasing Bim and increasing iNOS, which are at least partially mediated by the TGF-β1/PI3K/Akt pathway.     

LKB1 gene inactivation does not sensitize non-small cell lung cancer cells to mTOR inhibitors in vitro

Aim:

Previous study has shown that endometrial cancers with LKB1 inactivation are highly responsive to mTOR inhibitors. In this study we examined the effect of LKB1 gene status on mTOR inhibitor responses in non-small cell lung cancer (NSCLC) cells.

Methods:

Lung cancer cell lines Calu-1, H460, H1299, H1792, and A549 were treated with the mTOR inhibitors rapamycin or everolimus (RAD001). The mTOR activity was evaluated by measuring the phosphorylation of 4EBP1 and S6K, the two primary mTOR substrates. Cells proliferation was measured by MTS or sulforhodamine B assays.

Results:

The basal level of mTOR activity in LKB1 mutant A549 and H460 cells was significantly higher than that in LKB1 wild-type Calu-1 and H1792 cells. However, the LKB1 mutant A549 and H460 cells were not more sensitive to the mTOR inhibitors than the LKB1 wild-type Calu-1 and H1792 cells. Moreover, knockdown of LKB1 gene in H1299 cells did not increase the sensitivity to the mTOR inhibitors. Treatment with rapamycin or RAD001 significantly increased the phosphorylation of AKT in both LKB1 wild-type and LKB1 mutant NSCLC cells, which was attenuated by the PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. Furthermore, RAD001 combined with {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 markedly enhanced the growth inhibition on LKB1 wild-type H1792 cells and LKB1 mutant A549 cells.

Conclusion:

LKB1 gene inactivation in NSCLC cells does not increase the sensitivity to the mTOR inhibitors. The negative feedback activation of AKT by mTOR inhibition may contribute to the resistance of NSCLC cells to mTOR inhibitors.     

Ginsenoside Rd promotes neurogenesis in rat brain after transient focal cerebral ischemia via activation of PI3K/Akt pathway

Aim:

To investigate the effects of ginsenoside Rd (Rd) on neurogenesis in rat brain after ischemia/reperfusion injury (IRI).

Methods:

Male SD rats were subjected to transient middle cerebral artery occlusion (MCAO) followed by reperfusion. The rats were injected with Rd (1, 2.5, and 5 mg·kg⁻¹·d⁻¹, ip) from d 1 to d 3 after MCAO, and with BrdU (50 mg·kg⁻¹·d⁻¹, ip) from d 3 to d 6, then sacrificed on 7 d. The infarct size and neurological scores were assessed. Neurogenesis in the brains was detected by BrdU, DCX, Nestin, and GFAP immunohistochemistry staining. PC12 cells subjected to OGD/reperfusion were used as an in vitro model of brain ischemia. VEGF and BDNF levels were assessed with ELISA, and Akt and ERK phosphorylation was measured using Western blotting.

Results:

Rd administration dose-dependently decreased the infarct size and neurological scores in the rats with IRI. The high dose of Rd 5 (mg·kg⁻¹·d⁻¹) significantly increased Akt phosphorylation in ipsilateral hemisphere, and markedly increased the number of BrdU/DCX and Nestin/GFAP double-positive cells in ischemic area, which was partially blocked by co-administration of the PI3 kinase inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. Treatment with Rd (25, 50, and 100 μmol/L) during reperfusion significantly increased the expression of VEGF and BDNF in PC12 cells with IRI. Furthermore, treatment with Rd dose-dependently increased the phosphorylation of Akt and ERK, and significantly decreased PC12 cell apoptosis, which were blocked by co-application of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002.

Conclusion:

Rd not only attenuates ischemia/reperfusion injury in rat brain, but also promotes neurogenesis via increasing VEGF and BDNF expression and activating the PI3K/Akt and ERK1/2 pathways.     

Effects of ionotropic glutamate receptor antagonists on rat dural artery diameter in an intravital microscopy model

Background and purpose:

During migraine, trigeminal nerves may release calcitonin gene-related peptide (CGRP), inducing cranial vasodilatation and central nociception; hence, trigeminal inhibition or blockade of craniovascular CGRP receptors may prevent this vasodilatation and abort migraine headache. Several preclinical studies have shown that glutamate receptor antagonists affect the pathophysiology of migraine. This study investigated whether antagonists of NMDA (ketamine and MK801), AMPA (GYKI52466) and kainate ({"type":"entrez-nucleotide","attrs":{"text":"LY466195","term_id":"1258058612","term_text":"LY466195"}}LY466195) glutamate receptors affected dural vasodilatation induced by α-CGRP, capsaicin and periarterial electrical stimulation in rats, using intravital microscopy.

Experimental approach:

Male Sprague-Dawley rats were anaesthetized and the overlying bone was thinned to visualize the dural artery. Then, vasodilator responses to exogenous (i.v. α-CGRP) and endogenous (released by i.v. capsaicin and periarterial electrical stimulation) CGRP were elicited in the absence or presence of the above antagonists.

Key results:

α-CGRP, capsaicin and periarterial electrical stimulation increased dural artery diameter. Ketamine and MK801 inhibited the vasodilator responses to capsaicin and electrical stimulation, while only ketamine attenuated those to α-CGRP. In contrast, GYKI52466 only attenuated the vasodilatation to exogenous α-CGRP, while {"type":"entrez-nucleotide","attrs":{"text":"LY466195","term_id":"1258058612","term_text":"LY466195"}}LY466195 did not affect the vasodilator responses to endogenous or exogenous CGRP.

Conclusions and implications:

Although GYKI52466 has not been tested clinically, our data suggest that it would not inhibit migraine via vascular mechanisms. Similarly, the antimigraine efficacy of {"type":"entrez-nucleotide","attrs":{"text":"LY466195","term_id":"1258058612","term_text":"LY466195"}}LY466195 seems unrelated to vascular CGRP-mediated pathways and/or receptors. In contrast, the cranial vascular effects of ketamine and MK801 may represent a therapeutic mechanism, although the same mechanism might contribute, peripherally, to cardiovascular side effects.     

α1-Adrenoceptor-mediated contraction of rat aorta is partly mediated via transactivation of the epidermal growth factor receptor

Background and purpose:

High level of plasma catecholamines is a risk factor for vascular diseases such as hypertension and atherosclerosis. Catecholamines induce hypertrophy of vascular smooth muscle through α₁-adrenoceptors, which in cell culture involves the transactivation of epidermal growth factor receptor (EGFR). We hypothesized that EGFR transactivation was also involved in contractions of rat aorta mediated by α₁-adrenoceptors.

Experimental approach:

Thoracic aorta was isolated from 12–14 week old male Wistar rats. In vitro aortic contractile responses to cumulative doses of phenylephrine were characterized in the absence and presence of the EGFR kinase inhibitors, AG1478 and DAPH, in intact and endothelium-denuded rings. Involvement of signal transduction pathways was investigated by using heparin and inhibitors of Src, matrix metalloproteinase (MMP), extracellular signal-regulated kinase (ERK)1/2 and phosphatidyl inositol 3-kinase (PI3K). Phosphorylation of EGFR and ERK1/2 was measured after short-term phenylephrine or EGF stimulation in aorta segments in the presence of AG1478 and the PI3K inhibitor, wortmannin.

Key results:

AG1478 and DAPH concentration dependently attenuated phenylephrine-induced contractile responses in intact or endothelium-denuded aortic rings. Inhibition of PI3K (wortmannin and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002) but not heparin or inhibitors of Src or MMP, prevented the effect of AG1478 on the responses to phenylephrine. Phenylephrine induced phosphorylation of EGFR, which was partially blocked by AG1478. Phenylephrine also increased phosphorylation of ERK1/2, time-dependently and was blocked by AG1478 and wortmannin.

Conclusions and implications:

Contractions of rat thoracic aorta mediated by α₁-adrenoceptors involved transactivation of EGFR, mediated via a PI3K and ERK1/2 dependent pathway.     

Rosiglitazone via upregulation of Akt/eNOS pathways attenuates dysfunction of endothelial progenitor cells,induced by advanced glycation end products

Background and purpose:

Advanced glycation end products (AGEs) and endothelial progenitor cells (EPCs) play key roles in pathogenesis of diabetes-related vascular complications. AGEs can induce dysfunction in EPCs. The peroxisome proliferator-activated receptor-gamma (PPARγ) agonists are widely used in the treatment of type 2 diabetes, and it remains unknown if they could attenuate EPC dysfunction induced by AGEs.

Experimental approach:

EPCs isolated from healthy adults were cultured with various concentrations of AGEs (0, 50, 100 and 200 mg·L⁻¹) with or without rosiglitazone (10 nM), antibody for the receptors for AGE-human serum albumin (anti-receptor for advanced glycation end products (RAGE); 50 µg·mL⁻¹), phosphatidylinositol-3-kinase (PI3K) inhibitor ({"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, 5 µM), nitric oxide (NO) synthase inhibitor (L-N^G-nitro-arginine methyl ester (L-NAME), 100 µM) or sodium nitroprusside (SNP, 25 µM). Proliferation, apoptosis, cell adhesion, migration and NO production in EPCs were assessed, and expressions of endothelial NO synthase (eNOS) and Akt were determined.

Key results:

Number, proliferation/migration capacities, eNOS and Akt phosphorylation as well as NO synthesized by EPCs were increased by rosiglitazone and reduced by AGEs. AGEs promoted while rosiglitazone reduced EPC apoptosis. The AGE-induced effects were significantly ameliorated by pre-incubation with rosiglitazone, RAGE antibody and SNP. The beneficial effects of rosiglitazone could be blocked by pretreatment with L-NAME and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002.

Conclusions and implications:

The PPARγ agonist rosiglitazone increased EPC function and attenuated EPC dysfunction induced by AGEs via upregulating the Akt-eNOS signal pathways of EPCs.     

The anti-fibrotic effects of epigallocatechin-3-gallate in bile duct-ligated cholestatic rats and human hepatic stellate LX-2 cells are mediated by the PI3K/Akt/Smad pathway

Aim:

(−)-Epigallocatechin-3-gallate (EGCG) is one of the most abundant polyphenols in green tea with strong antioxidant activity and various therapeutic effects. In this study, we investigated the anti-fibrotic effects of EGCG and underlying mechanisms in bile duct-ligated (BDL) rats and a liver fibrosis model in vitro.

Methods:

BDL rats were treated with EGCG (25 mg·kg⁻¹·d⁻¹, po) for 14 d, and then the serum, bile and liver samples were collected. Liver fibrosis was assessed by serum, urine and bile biochemistry analyses and morphological studies of liver tissues. TGF-β1-stimulated human hepatic stellate LX-2 cells were used as a liver fibrosis model in vitro. The expression of liver fibrogenic genes and signaling proteins in the PI3K/Akt/Smad pathway was examined using Western blotting and/or real-time PCR.

Results:

In BDL rats, EGCG treatment significantly ameliorates liver necrosis, inflammation and fibrosis, and suppressed expression of the genes associated with liver inflammation and fibrogenesis, including TNF-α, IL-1β, TGF-β1, MMP-9, α-SMA, and COL1A1. In LX-2 cells, application of EGCG (10, 25 μmol/L) dose-dependently suppressed TGF-β1-stimulated expression of COL1A1, MMP-2, MMP-9, TGF-β1, TIMP1, and α-SMA. Furthermore, EGCG significantly suppressed the phosphorylation of Smad2/3 and Akt in the livers of BDL rats and in TGF-β1-stimulated LX-2 cells. Application of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, a specific inhibitor of PI3K, produced similar effects as EGCG did in TGF-β1-stimulated LX-2 cells, but co-application of EGCG and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 did not produce additive effects.

Conclusion:

EGCG exerts anti-fibrotic effects in BDL rats and TGF-β1-stimulated LX-2 cells in vitro via inhibiting the PI3K/Akt/Smad pathway.     

Atorvastatin enhances neurite outgrowth in cortical neurons in vitro via up-regulating the Akt/mTOR and Akt/GSK-3β signaling pathways

Aim:

To investigate whether atorvastatin can promote formation of neurites in cultured cortical neurons and the signaling mechanisms responsible for this effect.

Methods:

Cultured rat cerebral cortical neurons were incubated with atorvastatin (0.05–10 μmol/L) for various lengths of time. For pharmacological experiments, inhibitors were added 30 min prior to addition of atorvastatin. Control cultures received a similar amount of DMSO. Following the treatment period, phase-contrast digital images were taken. Digital images of neurons were analyzed for total neurite branch length (TNBL), neurite number, terminal branch number, and soma area by SPOT Advanced Imaging software. After incubation with atorvastatin for 48 h, the levels of phosphorylated 3-phosphoinoside-dependent protein kinase-1 (PDK1), phospho-Akt, phosphorylated mammalian target of rapamycin (mTOR), phosphorylated 4E-binding protein 1 (4E-BP1), p70S6 kinase (p70S6K), and glycogen synthase kinase-3β (GSK-3β) in the cortical neurons were evaluated using Western blotting analyses.

Results:

Atorvastatin (0.05–10 μmol/L) resulted in dose-dependent increase in neurite number and length in these neurons. Pretreatment of the cortical neurons with phosphatidylinositol 3-kinase (PI3K) inhibitors {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (30 μmol/L) and wortmannin (5 μmol/L), Akt inhibitor tricribine (1 μmol/L) or mTOR inhibitor rapamycin (100 nmol/L) blocked the atorvastatin-induced increase in neurite outgrowth, suggesting that atorvastatin promoted neurite outgrowth via activating the PI3K/Akt/mTOR signaling pathway. Atorvastatin (10 μmol/L) significantly increased the levels of phosphorylated PDK1, Akt and mTOR in the cortical neurons, which were prevented by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (30 μmol/L). Moreover, atorvastatin (10 μmol/L) stimulated the phosphorylation of 4E-BP1 and p70S6K, the substrates of mTOR, in the cortical neurons. In addition, atorvastatin (10 μmol/L) significantly increased the phosphorylated GSK-3β level in the cortical neurons, which was prevented by both {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and tricribine.

Conclusion:

These results suggest that activation of both the PI3K/Akt/mTOR and Akt/GSK-3β signaling pathways is responsible for the atorvastatin-induced neurite outgrowth in cultured cortical neurons.     

17β-Oestradiol inhibits doxorubicin-induced apoptosis via block of the volume-sensitive Cl(-) current in rabbit articular chondrocytes

BACKGROUND AND PURPOSE

Chondrocyte apoptosis contributes to disruption of cartilage integrity in osteoarthritis. Recent evidence suggested that the volume-sensitive organic osmolyte/anion channel [volume-sensitive (outwardly rectifying) Cl⁻ current (I_Cl,vol)] plays a functional role in the development of cell shrinkage associated with apoptosis (apoptotic volume decrease) in several cell types. In this study, we investigated the cellular effects of 17β-oestradiol on doxorubicin-induced apoptotic responses in rabbit articular chondrocytes.

EXPERIMENTAL APPROACH

Whole-cell membrane currents and cross-sectional area were measured from chondrocytes using a patch-clamp method and microscopic cell imaging, respectively. Caspase-3/7 activity was assayed as an index of apoptosis.

KEY RESULTS

Addition of doxorubicin (1 µM) to isosmotic bath solution rapidly activated the Cl⁻ current with properties similar to those of I_Cl,vol in chondrocytes. Doxorubicin also gradually decreased the cross-sectional area of chondrocytes, followed by enhanced caspase-3/7 activity; both of these responses were totally abolished by the I_Cl,vol blocker DCPIB (20 µM). Pretreatment of chondrocytes with 17β-oestradiol (1 nM) for short (approximately 10 min) and long (24 h) periods almost completely prevented the doxorubicin-induced activation of I_Cl,vol and subsequent elevation of caspase-3/7 activity. These effects of 17β-oestradiol were significantly attenuated by the oestrogen receptor blocker ICI 182780 (10 µM), as well as the phosphatidyl inositol-3-kinase (PI3K) inhibitors wortmannin (100 nM) and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (20 µM). Testosterone (10 nM) had no effect on the doxorubicin-induced Cl⁻ current.

CONCLUSIONS AND IMPLICATIONS

17β-Oestradiol prevents the doxorubicin-induced cell shrinkage mediated through activation of I_Cl,vol and subsequent induction of apoptosis signals, through a membrane receptor-dependent PI3K pathway in rabbit articular chondrocytes.     

Simvastatin induces a central hypotensive effect via Ras-mediated signalling to cause eNOS up-regulation

BACKGROUND AND PURPOSE

Clinical studies indicate that statins have a BP-lowering effect in hypercholesterolemic individuals with hypertension. Specifically, statins modulate BP through the up-regulation of endothelial NOS (eNOS) activation in the brain. However, the signalling mechanisms through which statins enhance eNOS activation remain unclear. Therefore, we examined the possible signalling pathways involved in statin-mediated BP regulation in the nucleus tractus solitarii (NTS).

EXPERIMENTAL APPROACH

To investigate the involvement of Ras and other signalling pathways in simvastatin-induced effects on BP, BP and renal sympathetic nerve activity (RSNA) were determined in spontaneously hypertensive rats (SHRs) before and after i.c.v. administration of simvastatin in the absence and presence of a Ras-specific inhibitor (farnesyl thiosalicylic acid, FTS), a geranylgeranyltransferase inhibitor (GGTI-2133), a PI3K inhibitor ({"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002) or a MAPK-ERK kinase (MEK) inhibitor (PD98059).

KEY RESULTS

FTS significantly attenuated the decrease in BP and increased NO evoked by simvastatin and reversed the decrease in basal RSNA induced by simvastatin. Immunoblotting and pharmacological studies showed that inhibition of Ras activity by FTS significantly abolished simvastatin-induced phosphorylation of ERK1/2, ribosomal protein S6 kinase (RSK), Akt and decreased eNOS phosphorylation. Likewise, administration of Akt and ERK1/2 signalling inhibitors, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and PD98059, attenuated the reduction in BP evoked by simvastatin. Furthermore, i.c.v. simvastatin decreased Rac1 activation and the number of ROS-positive cells in the NTS.

CONCLUSIONS AND IMPLICATIONS

Simvastatin modulates central BP control in the NTS of SHRs by increasing Ras-mediated activation of the PI3K-Akt and ERK1/2-RSK signalling pathways, which then up-regulates eNOS activation.     

Neuroprotection by donepezil against glutamate excitotoxicity involves stimulation of ��7 nicotinic receptors and internalization of NMDA receptors

BACKGROUND AND PURPOSE

Glutamate excitotoxicity may be involved in ischaemic injury to the CNS and some neurodegenerative diseases, such as Alzheimer''s disease. Donepezil, an acetylcholinesterase (AChE) inhibitor, exerts neuroprotective effects. Here we demonstrated a novel mechanism underlying the neuroprotection induced by donepezil.

EXPERIMENTAL APPROACH

Cell damage in primary rat neuron cultures was quantified by lactate dehydrogenase release. Morphological changes associated with neuroprotective effects of nicotine and AChE inhibitors were assessed by immunostaining. Cell surface levels of the glutamate receptor sub-units, NR1 and NR2A, were analyzed using biotinylation. Immunoblot was used to measure protein levels of cleaved caspase-3, total NR1, total NR2A and phosphorylated NR1. Immunoprecipitation was used to measure association of NR1 with the post-synaptic protein, PSD-95. Intracellular Ca²⁺ concentrations were measured with fura 2-acetoxymethylester. Caspase 3-like activity was measured using enzyme substrate, 7-amino-4-methylcoumarin (AMC)-DEVD.

KEY RESULTS

Levels of NR1, a core subunit of the NMDA receptor, on the cell surface were significantly reduced by donepexzil. In addition, glutamate-mediated Ca²⁺ entry was significantly attenuated by donepezil. Methyllycaconitine, an inhibitor of α7 nicotinic acetylcholine receptors (nAChR), inhibited the donepezil-induced attenuation of glutamate-mediated Ca²⁺ entry. {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, a phosphatidyl inositol 3-kinase (PI3K) inhibitor, had no effect on attenuation of glutamate-mediated Ca²⁺ entry induced by donepezil.

CONCLUSIONS AND IMPLICATIONS

Decreased glutamate toxicity through down-regulation of NMDA receptors, following stimulation of α7 nAChRs, could be another mechanism underlying neuroprotection by donepezil, in addition to up-regulating the PI3K-Akt cascade or defensive system.     

Protective effect of hydrogen sulphide against 6-OHDA-induced cell injury in SH-SY5Y cells involves PKC/PI3K/Akt pathway

BACKGROUND AND PURPOSE

Hydrogen sulphide (H₂S) is a novel neuromodulator. The present study aimed to investigate the protective effect of H₂S against cell injury induced by 6-hydroxydopamine (6-OHDA), a selective dopaminergic neurotoxin often used to establish a model of Parkinson''s disease for studying the underlying mechanisms of this condition.

EXPERIMENTAL APPROACH

Cell viability in SH-SY5Y cells was measured using MTT assay. Western blot analysis and pharmacological manipulation were employed to study the signalling mechanisms.

KEY RESULTS

Treatment of SH-SY5Y cells with 6-OHDA (50–200 µM) for 12 h decreased cell viability. Exogenous application of NaHS (an H₂S donor, 100–1000 µM) or overexpression of cystathionine β-synthase (a predominant enzyme to produce endogenous H₂S in SH-SY5Y cells) protected cells against 6-OHDA-induced cell apoptosis and death. Furthermore, NaHS reversed 6-OHDA-induced loss of tyrosine hydroxylase. Western blot analysis showed that NaHS reversed the down-regulation of PKCα, ε and Akt and the up-regulation of PKCδ in 6-OHDA-treated cells. Blockade of PKCα with Gö6976 (2 µM), PKCε with EAVSLKPT (200 µM) or PI3K with {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (20 µM) reduced the protective effects of H₂S. However, inhibition of PKCδ with rottlerin (5 µM) failed to affect 6-OHDA-induced cell injury. These data suggest that the protective effects of NaHS mainly resulted from activation of PKCα, ε and PI3K/Akt pathway. In addition, NaHS-induced Akt phosphorylation was significantly attenuated by Gö6976 and EAVSLKPT, suggesting that the activation of Akt by NaHS is PKCα, ε-dependent.

CONCLUSIONS AND IMPLICATIONS

H₂S protects SH-SY5Y cells against 6-OHDA-induced cell injury by activating the PKCα, ε/PI3K/Akt pathway.     

Cannabidiol inhibits synaptic transmission in rat hippocampal cultures and slices via multiple receptor pathways

BACKGROUND AND PURPOSE

Cannabidiol (CBD) has emerged as an interesting compound with therapeutic potential in several CNS disorders. However, whether it can modulate synaptic activity in the CNS remains unclear. Here, we have investigated whether CBD modulates synaptic transmission in rat hippocampal cultures and acute slices.

EXPERIMENTAL APPROACH

The effect of CBD on synaptic transmission was examined in rat hippocampal cultures and acute slices using whole cell patch clamp and standard extracellular recordings respectively.

KEY RESULTS

Cannabidiol decreased synaptic activity in hippocampal cultures in a concentration-dependent and Pertussis toxin-sensitive manner. The effects of CBD in culture were significantly reduced in the presence of the cannabinoid receptor (CB₁) inverse agonist, {"type":"entrez-nucleotide","attrs":{"text":"LY320135","term_id":"1257555575","term_text":"LY320135"}}LY320135 but were unaffected by the 5-HT_1A receptor antagonist, WAY100135. In hippocampal slices, CBD inhibited basal synaptic transmission, an effect that was abolished by the proposed CB₁ receptor antagonist, AM251, in addition to {"type":"entrez-nucleotide","attrs":{"text":"LY320135","term_id":"1257555575","term_text":"LY320135"}}LY320135 and WAY100135.

CONCLUSIONS AND IMPLICATIONS

Cannabidiol reduces synaptic transmission in hippocampal in vitro preparations and we propose a role for both 5-HT_1A and CB₁ receptors in these CBD-mediated effects. These data offer some mechanistic insights into the effects of CBD and emphasize that further investigations into the actions of CBD in the CNS are required in order to elucidate the full therapeutic potential of CBD.     

PI3K/AKT/mTOR Signaling-Mediated Neuropeptide VGF in the Hippocampus of Mice Is Involved in the Rapid Onset Antidepressant-Like Effects of GLYX-13

Background:

VGF (nonacryonimic) and phosphatidylinositol 3-kinase (PI3K)/AKT (also known as protein kinase B, PKB)/mammalian target of rapamycin (mTOR) signaling play pivotal roles in depression. However, whether phosphatidylinositol 3-kinase/AKT/mTOR signaling-mediated VGF participates in rapid-acting antidepressant-like actions of GLYX-13 is unclear.

Methods:

Herein, we evaluated the effects of acute treatment of GLYX-13 (0.5, 5, and 10mg/kg, i.p.) in the forced swim test. In addition, we assessed whether the acute treatment with GLYX-13 reverses the depressive-like behaviors induced by chronic unpredictable mild stress. Furthermore, we determined whether the Vgf knockdown in hippocampus of mice blocks the effects of GLYX-13. Moreover, we also demonstrated the effects of intra-hippocampus infusion of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (10 nmol/side), a specific phosphatidylinositol 3-kinase inhibitor prior to the treatment of GLYX-13 in the forced swim test. Lastly, whether alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor and mTOR activation involves in the antidepressant-like effects of GLYX-13 was examined.

Results:

Our results shown that GLYX-13 dose-dependently reversed the depressive-like behaviors in forced swim test. Additionally, GLYX-13 significantly reversed the downregulation of phosphorylation of AKT, mTOR, and eukaryotic elongation factor 2 as well as VGF induced by chronic unpredictable mild stress in hippocampus. Further, Vgf knockdown in hippocampus of mice significantly blocked the rapid-acting antidepressant-like effects and upregulation on phosphatidylinositol 3-kinase/AKT/mTOR/VGF signaling of GLYX-13. Moreover, intra-hippocampus infusion of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 significantly abolished the antidepressant-like effects and upregulation on phosphatidylinositol 3-kinase/AKT/mTOR/VGF signaling of GLYX-13. Finally, antidepressant-like effects of GLYX-13 required AMPA receptor and mTOR activation, as evidenced by the ability of NBQX and rapamycin to block the effects of GLYX-13, respectively.

Conclusions:

Our results suggest that phosphatidylinositol 3-kinase/AKT/mTOR signaling-mediated VGF in hippocampus may be involved in the antidepressant-like effects of GLYX-13.