Dopamine-induced apoptosis is mediated by oxidative stress and Is enhanced by cyanide in differentiated PC12 cells

Dopamine (DA) oxidation and the generation of reactive oxygen species (ROS) may contribute to the degeneration of dopaminergic neurons underlying various neurological conditions. The present study demonstrates that DA-induced cytotoxicity in differentiated PC12 cells is mediated by ROS and mitochondrial inhibition. Because cyanide induces parkinson-like symptoms and is an inhibitor of the antioxidant system and mitochondrial function, cells were treated with KCN to study DA toxicity in an impaired neuronal system. Differentiated PC12 cells were exposed to DA, KCN, or a combination of the two for 12-36 h. Lactate dehydrogenase (LDH) assays indicated that both DA (100-500 microM) and KCN (100-500 microM) induced a concentration- and time-dependent cell death and that their combination produced an increase in cytotoxicity. Apoptotic death, measured by Hoechst dye and TUNEL (terminal deoxynucleotidyltransferase dUTP nick end-labeling) staining, was also concentration- and time-dependent for DA and KCN. DA plus KCN produced an increase in apoptosis, indicating that KCN, and thus an impaired system, enhances DA-induced apoptosis. To study the mechanism(s) of DA toxicity, cells were pretreated with a series of compounds and incubated with DA (300 microM) and/or KCN (100 microM) for 24 h. Nomifensine, a DA reuptake inhibitor, rescued nearly 60-70% of the cells from DA- and DA plus KCN-induced apoptosis, suggesting that DA toxicity is in part mediated intracellularly. Pretreatment with antioxidants attenuated DA- and KCN-induced apoptosis, indicating the involvement of oxidative species. Furthermore, buthionine sulfoximine, an inhibitor of glutathione synthesis, increased the apoptotic response, which was reversed when cells were pretreated with antioxidants. DA and DA plus KCN produced a significant increase in intracellular oxidant generation, supporting the involvement of oxidative stress in DA-induced apoptosis. The nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester and the peroxynitrite scavenger uric acid blocked apoptosis and oxidant production, indicating involvement of nitric oxide. These results suggest that DA neurotoxicity is enhanced under the conditions induced by cyanide and involves both ROS and nitric oxide-mediated oxidative stress as an initiator of apoptosis.     

Trimethyltin Stimulates Protein Kinase C Translocation Through Receptor-Mediated Phospholipase C Activation in PC12 Cells

Abstract: Trimethyltin (TMT) is a potent neurotoxic compound that initiates a delayed neuronal cell death. Previously we have shown that TMT-induced cytotoxicity is associated with protein kinase C (PKC) translocation and activation. The present study investigates the mechanism underlying TMT-stimulated PKC translocation in PC12 cells. TMT exposure led to a rapid increase in intracellular levels of inositol 1,4,5-trisphosphate (IP₃), a product of phospholipase C (PLC). This was significantly decreased by pretreating cells with antagonists to either the cholinergic muscarinic receptor (atropine) or the glutamatergic metabotropic receptor [(+)-α-methyl-4-carboxyphenylglycine; (+)-MCPG]. Furthermore, the rise in IP₃ level was blocked by pretreating cells with a PLC inhibitor (U-73122) or by a combination of atropine and (+)-MCPG. This pretreatment also significantly decreased TMT-stimulated PKC translocation, indicating that TMT-mediated PKC translocation was related to PLC activation, presumably through formation of diacylglycerol, an endogenous activator of PKC and product of PLC. It is interesting that atropine and (+)-MCPG did not provide protection against TMT-induced cytotoxicity in these cells. However, these data suggest that TMT causes the release of cellular constituents that activate G protein-coupled receptors, ultimately leading to PKC translocation.     

Pharmacological inhibition of lysosomes activates the MTORC1 signaling pathway in chondrocytes in an autophagy-independent manner

Mechanistic target of rapamycin (serine/threonine kinase) complex 1 (MTORC1) is a protein-signaling complex at the fulcrum of anabolic and catabolic processes, which acts depending on wide-ranging environmental cues. It is generally accepted that lysosomes facilitate MTORC1 activation by generating an internal pool of amino acids. Amino acids activate MTORC1 by stimulating its translocation to the lysosomal membrane where it forms a super-complex involving the lysosomal-membrane-bound vacuolar-type H⁺-ATPase (v-ATPase) proton pump. This translocation and MTORC1 activation require functional lysosomes. Here we found that, in contrast to this well-accepted concept, in epiphyseal chondrocytes inhibition of lysosomal activity by v-ATPase inhibitors bafilomycin A₁ or concanamycin A potently activated MTORC1 signaling. The activity of MTORC1 was visualized by phosphorylated forms of RPS6 (ribosomal protein S6) and EIF4EBP1, 2 well-known downstream targets of MTORC1. Maximal RPS6 phosphorylation was observed at 48-h treatment and reached as high as a 12-fold increase (p < 0.018). This activation of MTORC1 was further confirmed in bone organ culture and promoted potent stimulation of longitudinal growth (p < 0.001). Importantly, the same effect was observed in ATG5 (autophagy-related 5)-deficient bones suggesting a macroautophagy-independent mechanism of MTORC1 inhibition by lysosomes. Thus, our data show that in epiphyseal chondrocytes lysosomes inhibit MTORC1 in a macroautophagy-independent manner and this inhibition likely depends on v-ATPase activity.     

Dermal exposure to m-xylene leads to increasing oxidative species and low molecular weight DNA levels in rat skin.

Dermal absorption of organic solvents, such as m-xylene, can lead to skin inflammation and pathological changes within hours after exposure. This study detected oxidative species formation and low molecular weight (LMW) DNA in rat skin as potential indicators of m-xylene-induced skin injury. At 0, 1, 2, 4, and 6 h after the beginning of a 1-h exposure, skin samples were removed and analyzed for oxidative species formation and LMW DNA analysis. At 2 h, mean oxidative species levels increased significantly (P < 0.05) above unexposed samples. Significantly higher (P < 0.05) LMW DNA values were observed at 2, 4, and 6 h compared to unexposed controls. These results show that oxidative species formation and LMW DNA levels in the skin may serve as indicators for predicting safe exposure levels to m-xylene and other volatile organic solvents.     

A flavanone and a dihydrodibenzoxepin from Bauhinia variegata

Phytochemical analysis of the root bark of Bauhinia variegata Linn yielded a new flavanone, (2S)-5,7-dimethoxy-3',4'-methylenedioxyflavanone (1) and a new dihydrodibenzoxepin, 5,6-dihydro-1,7-dihydroxy-3,4-dimethoxy-2-methyldibenz [b,f]oxepin (2) together with three known flavonoids (3-5). The structures of the new compounds were determined on the basis of spectral studies.     

Effect of α, β momorcharin on viability,caspase activity,cytochrome c release and on cytosolic calcium levels in different cancer cell lines

A multitude of plants have been used extensively for the treatment of cancers throughout the world. The protein, α, β momorcharin has been extracted from the plant Momordica charantia (MC), and it possesses anti-cancer and anti-HIV properties similar to the crude water and methanol soluble extract of the plant. This study investigated the anti-cancer effects and the cellular mechanisms of action of α, β momocharin (200–800 μM) on 1321N1, Gos-3, U87-MG, Sk Mel, Corl-23 and Weri Rb-1 cancer cell lines compared to normal healthy L6 muscle cell line measuring cell viability using MTT assay kit, Caspase-3 and 9 activities, cytochrome c release and intracellular free calcium concentrations [Ca²⁺]_i. The results show that α, β momorcharin can evoke significant dose-dependent (P < 0.05; Student’s t test) decreases in the viability (increases in cell death) of 1321N1, Gos-3, U87-MG, Sk Mel, Corl-23 and Weri Rb-1 cancer cell lines compared to healthy L6 muscle cell line and untreated glioma cells. α, β momorcharin (800 μM) also evoked significant (P < 0.05) increases in caspase-3 and 9 activities and cytochrome c release. Similarly, α, β momorcharin elicited significant (P < 0.05) time-dependent elevation in [Ca²⁺]_i in all five glioma cell lines compared to untreated cells. Together, the results have demonstrated that α, β momorcharin can exert its anti-cancer effect on different cancer cell lines by intracellular processes involving an insult to the mitochondria resulting in cellular calcium over loading, apoptosis, cytochrome release and subsequently, cell death.     

Role of prolactin on Leydig, Sertoli and germ cellular neutral lipids in bonnet monkeys, Macaca radiata.

To elucidate the specific influence of prolactin on neutral lipids in Leydig, Sertoli and germ cell compartments of the testis in immature and mature monkeys, the present study was carried out by injecting ovine prolactin (oPRL) (1 mg/kg body weight/twice daily for 10 days ip), to both age groups. Similarly, bromocryptine (an ergot alkaloid which inhibits prolactin secretion) was given to other sets of immature and mature monkeys (1 mg/kg body weight/twice daily for 10 days ip) to induce hypoprolactinemia. It was observed that after oPRL administration the total lipid accumulated in the germ cells of immature and mature monkeys. Total lipid was markedly decreased in the Leydig cells of mature monkeys only. But no such influence of PRL was evident in the Leydig cells of immature monkeys, suggesting an age-dependent effect of PRL on the Leydig cells. The increase in total lipid in the germ cells following PRL treatment was contributed by mono, di- and triacyl glycerols and free cholesterol. However, an opposite effect of PRL was evident in the Leydig cells of mature monkeys, where the cholesterols and glyceride fractions registered a decrease. The reduced cholesterol fractions in the Leydig cells following PRL treatment suggests the utilization of cholesterol for steroidogenesis. Sertoli cells were found to be comparatively resistant to change in PRL status. Bromocryptine treatment brought about the opposite effect of PRL in almost all parameters studied in both immature and mature monkeys. In general, these findings with prolactin suggests that PRL has a specific and definite influence on testicular neutral lipids and the response of different cellular compartments was found to vary.