Comparative biotransformation of morphine,codeine and pholcodine in rat hepatocytes: identification of a novel metabolite of pholcodine

1. Pholcodine (3-morpholinoethylmorphine), a semi-synthetic alkaloid, is widely used as an antitussive agent. 2. Norpholcodine [7,8-didehydro-4,5alpha-epoxy-3-(2-morpholinoethoxy)morphinan-6alpha-ol] (NP) and pholcodine-N-oxide [1(9a)-dehydro-(4aR,5S,7aR,9cS,12S)-4a,5,7a,8,9,9a-hexahydro-5-hydroxy-12-methyl-3-morpholinoethoxy-1H-8,9,c-(iminoethano)phenanthro[4,5-bcd] furan-12-oxide] (PNOX) were identified in incubations of pholcodine with freshly isolated rat hepatocytes by liquid chromatography/electrospray-mass spectrometry (LC/ESI-MS). 3. Synthesized NP and PNOX were characterized by mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. 4. N-oxidation was the major metabolic pathway for pholcodine, producing a previously unreported metabolite. 5. The metabolism of morphine and codeine was also determined using freshly isolated hepatocytes. 6. For morphine, 3-glucuronidation was the major metabolic pathway, whilst for codeine it was dealkylation (O- and N-). 7. Neither morphine nor its metabolites were metabolites of pholcodine. 8. This observation supports the hypothesis that the absence of analgesic activity with pholcodine may be due to less O-dealkylation in vivo. 9. Together with the slow biotransformation of pholcodine (k(met) = 0.021 microM min(-1)) in comparison with morphine (k(met) = 0.057 microM min(-1)) and codeine (k(met) = 0.112 microM min(-1)), the results obtained were consistent with its low addiction potential and suggest that its antitussive efficacy is mediated by the parent drug or one of its metabolites other than morphine.     

Stimulation of noradrenaline release by T-588, a cognitive enhancer, in PC12 cells

Previously, we reported that (R)-(-)-1-(benzo[b]thiophen-5-yl)-2-[2-(N,N- diethylamino)ethoxy] ethanol hydrochloride (T-588), a novel putative cognitive enhancer, stimulated noradrenaline (NA) release from rat cerebral cortical slices. In this study, we investigated the effects of T-588 compared to other secretagogues on NA release from PC12 cells. Addition of as little as 10 microM T-588 stimulated [3H]NA release in a dose-dependent and an extracellular Ca(2+)-independent manner from PC12 cells. Ten micromolar ionomycin-, 300 microM adenosine-5'-O-(gamma-thiotriphosphate)- and 10 microM forskolin-induced extracellular Ca(2+)-dependent [3H]-NA release was further enhanced by 30 microM T-588. Cytosolic synaptophysin and 25-kDa synaptosome-associated protein immunoreactivity was increased by addition of T-588 in a dose-dependent manner. Interestingly, increases in synaptic vesicle-related proteins triggered by T-588 had a 4-min lag time and were completely dependent on extracellular CaCl2. These findings suggest that T-588 stimulates NA release from PC12 cells in a Ca(2+)-independent manner. T-588 also induced the translocation of synaptic vesicles in a Ca(2+)-dependent manner.     

Effects of tributyltin chloride on L-DOPA-induced cytotoxicity in PC12 cells

Tributyltin chloride (TBTC) at concentrations of 0.5-1.0 microM inhibits dopamine biosynthesis in PC12 cells. In this study, the effects of TBTC on L-3,4-dihydroxyphenylalanine (L-DOPA)-induced cytotoxicity in PC12 cells were investigated. TBTC at concentrations up to 1.0 microM neither affected cell viability, nor induced apoptosis after 24 or 48 h in PC12 cells. However, TBTC at concentrations higher than 2.0 microM caused cytotoxicity through an apoptotic process. In addition, exposure of PC12 cells to non-cytotoxic (0.5 and 1.0 microM) or cytotoxic (2.0 microM) concentrations of TBTC in combination with L-DOPA (20, 50 and 100 microM) resulted in a significant increase in cell loss and the percentage of apoptotic cells after 24 or 48 h compared with TBTC or L-DOPA alone. The enhancing effects of TBTC on L-DOPA-induced cytotoxicity were concentration- and treatment time-dependent. These data demonstrate that TBTC enhances L-DOPA-induced cytotoxicity in PC 12 cells.     

Effects of a water-soluble antitumor ether phosphonoinositide, D-myo-inositol 4-(hexadecyloxy)-3(S)-methoxybutanephosphonate (C4-PI), on inositol lipid metabolism in breast epithelial cancer cell lines

We have demonstrated previously that D-myo-inositol 4-(hexadecyloxy)-3(S)-methoxybutanephosphonate (C4-PI), an isosteric phosphonate analog of phosphatidylinositol developed to inhibit inositol lipid metabolism, was unable to inhibit phosphatidylinositol (PI) 3-kinase activity. We now report the effects of the compound on other aspects of inositol metabolism. We demonstrated that C4-PI inhibits the activity of purified recombinant PI-phospholipase C-beta (PLC-beta) at all concentrations tested; it enhanced the activity of PI-PLC-gamma and PI-PLC-delta at low concentrations (10 microM), while severely inhibiting their activities at higher concentrations. In the breast cancer cell lines MCF-7 (estrogen receptor positive) and MDA-MB-468 (estrogen receptor negative), C4-PI had no effect on the uptake of D-myo-inositol but severely inhibited its incorporation into PI. In spite of the drastic decrease in PI synthesis, C4-PI did not affect the levels of inositol incorporated into phosphatidylinositol 4,5-bisphosphate (PIP2) in the cells. In vitro assays showed that C4-PI inhibited PI synthase activity (inhibition of 35% at 50 microM) but had little effect on PI 4-kinase activity (inhibition of 13% at 150 microM). C4-PI inhibited the proliferation of MCF-7 and MDA-MB-468 cell lines with IC(50) values of 12 and 18 microM. Taken together, the results suggest that the accumulation of [3H]inositol in PIP2 in cells incubated with C4-PI may be due to the inhibition of PIP2 hydrolysis in the cells with no effect on its synthesis. The role of these C4-PI-induced effects in the mechanism of growth inhibition by C4-PI remains to be established.     

Potentiation of nerve growth factor-action by picrosides I and II, natural iridoids, in PC12D cells

Natural iridoid, picroside I (beta-D-glucopyranoside, 1a,1b,2,5a,6, 6a-hexahydro-6-hydroxy-1a-(hydroxymethyl)oxireno[4,5]cyclopenta[1, 2-c]pyran-2-yl, 6-(3-phenyl-2-propenoate)) or II (beta-D-glucopyranoside, 1a,1b,2,5a,6, 6a-hexahydro-6-[(4-hydroxy-3-methoxybenzoyl)oxy]-1a-(hydroxymethyl )ox ireno[4,5]cyclopenta[1,2-c]pyran-2-yl) alone did not exhibit neuritogenic activity, but caused a concentration-dependent (>0.1 microM) enhancement of nerve growth factor (NGF, 2 ng/ml)-induced neurite outgrowth from PC12D cells. The picroside-induced enhancing action of NGF was abolished by GF109203X (2-[1-(3-dimethylaminopropyl)-indol-3-yl]-3-(indol-3-yl)maleimide) (0.1 microM), a protein kinase C inhibitor. Furthermore, PD98059 (2-(2'-amino-3'-methoxyphenyl)-oxanaphthalen-4-one) (20 microM), a potent mitogen-activated protein (MAP) kinase kinase inhibitor, completely blocked the picroside-induced enhancement of neurite outgrowth in the presence of NGF (2 ng/ml), suggesting that picrosides activate the MAP kinase-dependent signaling pathway. Interestingly, no increase in the expression of phosphorylated MAP kinase was observed in picroside-treated (60 microM) PC12D cells in the presence of NGF (2 ng/ml). These results suggest that picroside I or II enhances NGF-induced neurite outgrowth from PC12D cells, probably by amplifying a down-stream step of MAP kinase in the NGF receptor-mediated intracellular MAP kinase-dependent signaling pathway.     

Retigabine, a novel anti-convulsant, enhances activation of KCNQ2/Q3 potassium channels

Retigabine [N-(2-amino-4-[fluorobenzylamino]-phenyl) carbamic acid; D-23129] is a novel anticonvulsant, unrelated to currently available antiepileptic agents, with activity in a broad range of seizure models. In the present study, we sought to determine whether retigabine could enhance current through M-like currents in PC12 cells and KCNQ2/Q3 K(+) channels expressed in Chinese hamster ovary cells (CHO-KCNQ2/Q3). In differentiated PC12 cells, retigabine enhanced a linopirdine-sensitive current. The effect of retigabine was associated with a slowing of M-like tail current deactivation in these cells. Retigabine (0.1 to 10 microM) induced a potassium current and hyperpolarized CHO cells expressing KCNQ2/Q3 cells but not in wild-type cells. Retigabine-induced currents in CHO-KCNQ2/Q3 cells were inhibited by 60.6 +/- 11% (n = 4) by the KCNQ2/Q3 blocker, linopirdine (10 microM), and 82.7 +/- 5.4% (n = 4) by BaCl(2) (10 mM). The mechanism by which retigabine enhanced KCNQ2/Q3 currents involved large, drug-induced, leftward shifts in the voltage dependence of channel activation (-33.1 +/- 2.6 mV, n = 4, by 10 microM retigabine). Retigabine shifted the voltage dependence of channel activation with an EC(50) value of 1.6 +/- 0.3 microM (slope factor was 1.2 +/- 0.1, n = 4 to 5 cells per concentration). Retigabine (0.1 to 10 microM) also slowed the rate of channel deactivation, predominantly by increasing the contribution of a slowly deactivating tail current component. Our findings identify KCNQ2/Q3 channels as a molecular target for retigabine and suggest that activation of KCNQ2/Q3 channels may be responsible for at least some of the anticonvulsant activity of this agent.     

New structural analogues of Tubulozole induce apoptosis, [Ca2+]i modifications and cytoskeletal disorganization in glial (GL15) and neuronal-like (PC12) cell lines

The synthesis and the biological activity of (+/-)-cis- and (+/-)-trans-[4-[[2-(1,1'-biphenyl-4-yl)-2-(1H-imidazol-1-ylmethyl)-1, 3-dioxolan-4-yl]methylthio]phenyl]carbamic acid ethyl esters (2a and 2b) are discussed. They were designed as structural analogues of Tubulozole, a synthetic tubulin polymerisation inhibitor with antimitotic properties. Biological tests were carried out on PC12, a neuronal-like cell line derived from rat pheochromocytoma, and on GL15, a cell line derived from human glioblastoma. The exposure (from 5 to 20 h) of GL15 and PC12 cells to different concentrations (0.1-1000 microM; IC50 approximately 1 microM) of 2a or 2b resulted in a drastic decrease in the number of viable cells without an apparent effect on the cell distribution in the various phases of the cell cycle. Compound 2a or 2b (10 microM) induced cell death by activating apoptosis. This was correlated with the activation of an oscillating Ca(2+)-dependent mechanism which increased the intracellular calcium concentration ([Ca2+]i) via Ca(2+)-release from internal stores. Moreover, 2a (10 microM) also induced severe damage of cytoskeletal F-actin filaments after a 5 h incubation in GL15 cells. This was also observed but to a smaller extent, for 2b. Under the same experimental conditions, PC12 cells showed similar actin deregulation.     

Modifications of phospholipid metabolism induced by chlorpromazine, desmethylimipramine and propranolol in C6 glioma cells

The effects of chlorpromazine (CPZ), desmethylimipramine (DMI) and propranolol (PRO) on phospholipid metabolism in C6 glioma cells were studied by following the incorporation of 32Pi, [U-14C]glycerol, [2-3H]glycerol and [1-14C]oleate into lipids. The drugs produced a dose-dependent increase in the incorporation of 32Pi and [U-14C]glycerol, but not of [1-14C] oleate, into total phospholipids, that reached a plateau at 200 microM CPZ and 500 microM DMI and PRO. The three drugs shifted the incorporation of precursors from neutral [phosphatidylcholine (PC) and phosphatidylethanolamine (PE)] to acidic phospholipids [phosphatidic acid (PA), phosphatidylinositol (PI), phosphatidylglycerol, phosphatidylinositol-4-phosphate (PIP) and phosphatidylinositol-4,5-bisphosphate (PIP2)] in a dose-dependent, qualitatively similar manner. The incorporation of [2-3H]glycerol into diacylglycerol was also depressed markedly by CPZ. Addition of 1 mM 1,2-dioleoylglycerol, 1-oleoyl-2-acetylglycerol or oleate only partially reversed the decrease in PC labeling caused by CPZ. 12-O-Tetradecanoylphorbol-13-acetate counteracted this effect of CPZ completely but greatly increased PC labeling even in the absence of the drug. Polyphosphoinositides rapidly incorporated 32Pi at early times reaching a plateau in about 40 min. The labeling rate of PI was not parallel to that of PIP or PIP2 and continued to increase even after the polyphosphoinositides had reached a plateau. CPZ increased PI labeling much more than that of PIP and PIP2. These data suggest that cationic amphiphilic drugs may act by inhibiting CTP:phosphocholine cytidylyltransferase, thus decreasing incorporation of precursors into PC and PE; inhibiting PA phosphohydrolase with increased formation of phosphatidyl-CMP, the intermediate for the synthesis of acidic phospholipids; and stimulating the inositol exchange reaction, forming a pool of PI that is not available for PIP and PIP2 synthesis.     

Effects of tri butyl tin acetate on dopamine biosynthesis and l-dopa-lnduced cytotoxicity in pc12 cells

The effects of tributyltin acetate (TBTA) on dopamine biosynthesis and L-3,4-dihydroxyphenylalanine (L-DOPA)-induced cytotoxicity in PC12 cells were examined. TBTA at concentrations of 0.1-0.2 microM inhibited dopamine biosynthesis by reducing tyrosine hydroxylase (TH) activity and TH gene expression in PC12 cells. TBTA at 0.1-0.4 microM also reduced L-DOPA (20-50 microM)-induced increases in dopamine content for 24 h in PC12 cells. TBTA at concentrations up to 0.3 microM did not affect cell viability. However, TBTA at concentrations higher than 0.4 microM caused apoptotic cytotoxicity. Exposure of PC12 cells to non-cytotoxic (0.1 and 0.2 microM) or cytotoxic (0.4 microM) concentrations of TBTA with L-DOPA (20, 50 and 100 microM) significantly increased the cell loss and the percentage of apoptotic cells after 24 or 48 h compared with TBTA or L-DOPA alone. These data suggest that TBTA inhibits dopamine biosynthesis and enhances L-DOPA-induced cytotoxicity in PC12 cells.     

Inhibition of transmitter release by TI233, a calmodulin antagonist, from clonal neural cells and a presumed site of action

Effects of TI233, a calmodulin antagonist, on transmitter release were studied using a clonal pheochromocytoma cell line (PC12h). TI233, at a concentration of 30 microM, completely suppressed the release of preloaded [3H]NE and [3H]DA. The 50% suppression dose was around 3 microM. TI233 did not inhibit the [3H]NE release evoked by the calcium ionophore A23187. Electrophysiological examinations using a clonal neuroblastoma x glioma hybrid cell line (NG108-15) revealed that TI233 blocked the voltage-sensitive calcium channel of the membrane in the same concentration range. Thus it was suggested that TI233 inhibited transmitter release from neuronal cells by blocking the entry of calcium to the cytoplasm.     

Aggravation of L-DOPA-induced neurotoxicity by tetrahydropapaveroline in PC12 cells

Tetrahydropapaveroline (THP) is formed in Parkinsonian patients receiving L-DOPA therapy and is detected in the plasma and urine of these patients. In this study, we have investigated the effects of THP on L-DOPA-induced neurotoxicity in cultured rat adrenal pheochromocytoma, PC12 cells. Exposure of PC12 cells up to 10 microM THP or 20 microM L-DOPA after 24 or 48 hr, neither affected the cell viability determined by MTT assay, nor induced apoptosis by flow cytometry and TUNEL staining. However, at concentrations higher than 15 microM, THP showed cytotoxicity through an apoptotic process. In addition, THP at 5-15 microM for both incubation time points significantly enhanced L-DOPA-induced neurotoxicity (L-DOPA concentration, 50 microM). Exposure of PC12 cells to THP, L-DOPA and THP plus L-DOPA for 48 hr resulted in a marked increase in the cell loss and percentage of apoptotic cells compared with exposure for 24hr. The enhancing effects of THP on L-DOPA-induced neurotoxicity were concentration- and treated-time-dependent. THP, L-DOPA and THP plus L-DOPA produced a significant increase in intracellular reactive oxygen species generation and decrease in ATP levels, supporting the involvement of oxidative stress in THP- and L-DOPA-induced apoptosis. The antioxidant N-acetyl-L-cysteine strongly inhibited changes in apoptosis, decreases in cell viability and ROS generation induced by THP associated with L-DOPA. These results suggest that THP aggravates L-DOPA-induced oxidative neurotoxic and apoptotic effects in PC12 cells. Therefore, Parkinsonian patients treated with L-DOPA for long-term need to be monitored for the relationship between plasma concentration of THP and the symptoms of neurotoxicity.     

Effects of organophosphates and nerve growth factor on muscarinic receptor binding number in rat pheochromocytoma PC12 cells

Muscarinic receptor binding in PC12 cells is influenced by both nerve growth factor (NGF) and organophosphates. Treatment of PC12 cells with a single dose of NGF (50 ng, 7S NGF/ml) increased [3H]N-methylscopolamine ([3H]-NMS) binding sites approximately two-fold at 48 hr but did not change the Kd for this ligand. Exposure of PC12 cells to soman, 50 microM, decreased [3H]-NMS binding in both undifferentiated and NGF-treated cells; however, decreases in muscarinic binding induced by the organophosphate were only minimal after the first hour after treatment and were maximal at about 24 hr. Other organophosphates including sarin, tabun, and VX as well as the carbamate, pyridostigmine, also reduced [3H]-NMS binding in PC12 cells measured 24-48 hr after treatment. The order of potency of organophosphates in lowering [3H]-NMS binding was soman greater than sarin greater than VX greater than tabun greater than DFP. High amounts of VX (200 microM) but not the other organophosphates inhibited [3H]-NMS binding when added to cells during the course of binding assays. Decreases in muscarinic receptor binding induced by the organophosphates differed markedly from that produced by carbamylcholine, which decreased [3H]-NMS binding maximally 30 min after addition to the cells. Decreases in [3H]-NMS binding produced by carbamylcholine were antagonized by atropine, but reductions in muscarinic binding produced by the organophosphates were not reversed by atropine. Thus, decreases in muscarinic receptor binding induced in PC12 cells by organophosphates occur via a novel mechanism that does not involve agonist-induced receptor desensitization.     

Protective effect of lignophenol derivative from beech (Fagus crenata Blume) on copper- and zinc-mediated cell death in PC12 cells

Lignophenol, prepared using a phase-separation system, is a derivative of lignin, which is one of the components in the plant cell wall, and possesses high phenolic function, high stability and antioxidant properties. However, little is known about the beneficial effect of lignophenol. In this study, we investigated the protective effect of lignophenol from the beech tree (Fagus crenata Blume) on copper- and zinc-mediated apoptosis in PC12 cells by using DNA fragmentation and TUNEL assays. In DNA fragmentation assays, the DNA ladder patterns in the PC12 cells treated with 200 microM Cu and 200 microM Zn were enhanced, whereas the DNA ladder pattern was hardly observed in these cells treated with 20 mM lignophenol. In the TUNEL assay, TUNEL signals increased significantly in the untreated PC12 cells exposed to 200 microM Cu compared with the control. In contrast, the degree of apoptosis in the 20 mM lignophenol-treated cells was significantly lower than in the untreated cells, indicating that lignophenol inhibited Cu-induced apoptotic cell death in PC 12 cells. In the 200 microM Zn-exposed group, the degree of apoptosis in the 20 mM lignophenol-treated cells was also low compared with the untreated cells. In conclusion, these results suggest that lignophenol plays a role in protecting against Cu- and Zn-mediated PC12 apoptotic cell death.     

Chlorpromazine-induced inhibition of catecholamine secretion by a differential blockade of nicotinic receptors and L-type Ca2+ channels in rat pheochromocytoma cells.

We investigated the effect of chlorpromazine (CPZ), a phenothiazine neuroleptic, on catecholamine secretion in rat pheochromocytoma (PC12) cells. CPZ inhibited [3H]norepinephrine ([3H]NE) secretion induced by 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), an agonist of nicotinic acetylcholine receptors (nAChRs) with an IC50 value of 1.0 +/- 0.2 microM. The DMPP-induced rise in cytosolic free Ca2+ concentration [Ca2+]i was inhibited by CPZ with an IC50 of 1.9 +/- 0.1 microM. The DMPP-induced increase in cytosolic free Na+ concentration [Na+]i was also inhibited by CPZ with a similar potency. Furthermore, the binding of [3H]nicotine to PC12 cells was inhibited by CPZ with an IC50 value of 2.7 +/- 0.6 microM, suggesting that the nAChRs themselves are inhibited by CPZ. In addition, both 70 mM K+-induced [3H]NE secretion and [Ca2+]i increase were inhibited by CPZ with IC50 of 7.9 +/- 1.1 and 6.2 +/- 0.3 microM, respectively. Experiments with Ca2+ channel antagonists suggest that L-type Ca2+ channels are mainly responsible for the inhibition. We conclude that CPZ inhibits catecholamine secretion by blocking nAChRs and L-type Ca2+ channels, with the former being more sensitive to CPZ.     

Prevention of cycloheximide-induced apoptosis in hepatocytes by adenosine and by caspase inhibitors

The mechanism by which cycloheximide induces apoptosis in isolated rat hepatocytes was studied. Cycloheximide (1-300 microM) induced apoptosis within 3-4 hr in the hepatocytes. Specific apoptotic characteristics such as blebbing, phosphatidyl serine (PS) exposure, chromatin condensation, and nuclear fragmentation were induced. Cycloheximide (CHX) dose dependently activated the caspase-3-like proteases, but not the caspase-1-like proteases. Pretreatment of the hepatocytes with 100 microM of the caspase inhibitors z-Val-Ala-DL-Asp-fluoromethylketone or Ac-Asp-Glu-Val-Asp-aldehyde completely abrogated the caspase activation and the apoptosis. Addition of adenosine (100 microM) reduced phosphatidyl serine exposure and other morphological characteristics of apoptosis by 50%; however, it did not prevent the activation of the caspases, suggesting that adenosine inhibited downstream of caspase activation. The adenosine receptor antagonist 8-[4-[[[[(2-aminoethyl)amino]-carbonyl]methyl]oxy]phenyl]-1,3-dipropylxa nthine abolished the capacity of adenosine to prevent apoptosis, indicating that prevention was receptor-mediated. During apoptosis, the mitochondrial membrane potential in apoptotic cells (cells with PS exposition) was decreased to 50-60% of the control value; in the population viable cells, however, the mitochondrial membrane potential remained stable. Prevention of apoptosis by the caspase inhibitor z-Val-Ala-DL-Asp-fluoromethylketone or adenosine prevented the decrease in mitochondrial membrane potential. In conclusion, CHX rapidly induces apoptosis in isolated rat hepatocytes, which is inhibited by adenosine at a relatively late step.     

Selective inhibition of protein kinase C, mitogen-activated protein kinase, and neutrophil activation in response to calcium pyrophosphate dihydrate crystals, formyl-methionyl-leucyl-phenylalanine, and phorbol ester by O-(chloroacetyl-carbamoyl) fumagillol (AGM-1470; TNP-470)

The effect of O-(chloroacetyl-carbamoyl) fumagillol (AGM-1470; TNP-470) was investigated on protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) activation in neutrophils stimulated by plasma-opsonized crystals of calcium pyrophosphate dihydrate (triclinic) [CPPD(T)], formyl-Met-Leu-Phe (fMLP), and phorbol 12-myristate 13-acetate (PMA). Neutrophil respiratory burst responses also were determined in AGM-1470-pretreated cells stimulated with the same agonists, using chemiluminescence and superoxide anion generation assays. AGM-1470 (5 microM) effectively inhibited PKC activation in cells treated with CPPD(T) crystals (50 mg/mL, 2 min) and fMLP (1 microM, 1 min), but had no effect on PMA-treated cells (0.5 microM, 5 min). AGM-1470 blocked MAPK activity completely and reduced neutrophil activation induced by fMLP and PMA but not by CPPD(T). The degree of inhibition of the respiratory burst plateaued at approximately 46+/-9 and 54+/-3% in fMLP- and PMA-treated cells, respectively. These data indicate that activation of neutrophil respiratory burst activity may be mediated through the MAPK pathway. AGM-1470 pretreatment did not inhibit CPPD(T) crystal- or fMLP-stimulated phosphatidylinositol 3-kinase (PI 3-kinase) activity. These findings, coupled with further observations that the PI 3-kinase inhibitor wortmannin (10 nM) inhibited fMLP- and CPPD(T) crystal-induced but not PMA-induced chemiluminescence, indicate that at least two distinct signaling pathways (mediated by PI 3-kinase or MAPK) lead to neutrophil respiratory burst responses. PKC may also be required in the MAPK-stimulated pathway. We propose that the inhibitory effect of AGM-1470 on the neutrophil respiratory burst may be due to its ability to inhibit PKC and MAPK activation.     

Salidroside protects PC12 cells from MPP-induced apoptosis via activation of the PI3K/Akt pathway

Oxidative stress plays an important role in the pathogenesis of Parkinson’s disease (PD). Salidroside (SAL), a phenylpropanoid glycoside isolated from Rhodiola rosea L., can exert potent antioxidant properties. In this study, we investigated the protective effects, and the possible mechanism of action, of SAL against 1-methyl-4-phenylpyridinium (MPP⁺)-induced cell damage in rat adrenal pheochromocytoma PC12 cells. Pretreatment of PC12 cells with SAL significantly reduced the ability of MPP⁺ to induce apoptosis in a dose and time-dependent manner. SAL significantly and dose-dependently inhibited MPP⁺-induced chromatin condensation and MPP⁺-induced release of lactate dehydrogenase by PC12 cells. SAL enhanced Akt phosphorylation in PC12 cells, and the protective effects of SAL against MPP⁺-induced apoptosis were abolished by LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K) phosphorylation. These findings suggest that SAL prevents MPP⁺-induced apoptosis in PC12 cells, at least in part through activation of the PI3K/Akt pathway.     

Comparative biotransformation of morphine,codeine and pholcodine in rat hepatocytes: identification of a novel metabolite of pholcodine

1. Pholcodine (3-morpholinoethylmorphine), a semi-synthetic alkaloid, is widely used as an antitussive agent. 2. Norpholcodine [7,8-didehydro-4,5 α -epoxy-3-(2-morpholinoethoxy)morphinan-6 α -ol] (NP) and pholcodine-N-oxide [1(9a)-dehydro-(4aR,5S,7a R,9c S,12S)-4a,5,7a,8,9,9a-hexahydro-5-hydroxy-12-methyl-3-morpholinoethoxy-1 H -8,9,c-(iminoethano)phenanthro[4,5-bcd] furan-12-oxide] (PNOX) were identified in incubations of pholcodine with freshly isolated rat hepatocytes by liquid chromatography/electrospray-mass spectrometry (LC/ESI-MS). 3. Synthesized NP and PNOX were characterized by mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. 4. N -oxidation was the major metabolic pathway for pholcodine, producing a previously unreported metabolite. 5. The metabolism of morphine and codeine was also determined using freshly isolated hepatocytes. 6. For morphine, 3-glucuronidation was the major metabolic pathway, whilst for codeine it was dealkylation (O - and N -). 7. Neither morphine nor its metabolites were metabolites of pholcodine. 8. This observation supports the hypothesis that the absence of analgesic activity with pholcodine may be due to less O -dealkylation in vivo. 9. Together with the slow biotransformation of pholcodine (k met = 0.021 µM?min -1) in comparison with morphine (k met = 0.057 µM?min -1) and codeine (k met = 0.112 µM?min -1), the results obtained were consistent with its low addiction potential and suggest that its antitussive efficacy is mediated by the parent drug or one of its metabolites other than morphine.     

Comparison of the effect of cyclic AMP on the content and release of dopamine and 1-methyl-4-phenylpyridinium (Mpp+) in PC12 cells

1 The aim of this work was to investigate the effect of acute and chronic exposure of rat pheochromocytoma (PC12) cells to elevated cAMP, using forskolin, dibutyryl-cAMP (db-cAMP) or isobutylmethylxanthine (IBMX), on endogenous dopamine content and release and on [3H]-1-methyl-4-phenylpyridinium ([3H]-MPP+) uptake and release, under basal conditions and under KCl-stimulation. 2 Cultured PC12 cells synthetized and accumulated large amounts of dopamine, but not noradrenaline or adrenaline. The release of dopamine by the cells was markedly increased in response to 50 mM KCl. 3 Acute and chronic treatment of the cells with forskolin (30 microM), but not IBMX (100 microM), slightly increased the spontaneous release of dopamine and significantly decreased the release induced by 50 mM KCl. 4 Chronic treatment of the cells with forskolin (30 microM), but not IBMX (100 microM), markedly decreased the cellular content of dopamine. 5 Cultured PC12 cells removed and accumulated [3H]-MPP+, which, similarly to dopamine, was released by KCl. 6 Acute treatment of the cells with forskolin (30 microM) or db-cAMP (2.5 mM), but not IBMX (100 microM), slightly increased the spontaneous release, but did not affect KCl-induced release of [3H]-MPP+. On the other hand, chronic treatment of the cells with forskolin produced, on [3H]-MPP+, similar effects to those obtained for dopamine. 7 Acute and chronic treatment of the cells with reserpine (50 nM) produced similar results to those obtained with forskolin on either dopamine or [3H]-MPP+ handling. 8 In conclusion, cAMP, similarly to reserpine, increases the spontaneous release and decreases the KCl-induced release of [3H]-MPP+ and dopamine. This suggests that cAMP impairs the vesicular monoamine transporter.     

The nitric oxide donor NOC12 protects cultured astrocytes against apoptosis via a cGMP-dependent mechanism

We examined the effect of 3-ethyl-3-(ethylaminoethyl)-1-hydroxy-2-oxo-1-triazene (NOC12), a nitric oxide (NO) donor, on apoptosis in cultured astrocytes. Reperfusion after hydrogen peroxide (H2O2) exposure caused a decrease in cell viability, loss of mitochondrial membrane potential, caspase-3 activation, DNA ladder formation, and nuclear condensation. NOC12 at 10-100 microM significantly attenuated these apoptotic changes, while the NO donor at 1 mM caused cell injury and exacerbated the H202-induced cell injury. NOC12 increased intracellular cGMP levels in a dose dependent manner with the maximal effect at 100 microM. The protective effect of NOC12 was mimicked by the NO-independent guanylate cyclase activator 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole, and was attenuated by the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and the cGMP-dependent protein kinase inhibitor KT5823. ODQ and KT5823 did not block but rather exacerbated the cytotoxic effect of NOC12 at 1 mM. These findings demonstrate that lower concentrations of NOC12 inhibit the H2O2-induced apoptosis of astrocytes in a cGMP-dependent way, but higher concentrations of NOC12 show a toxic effect on astrocytes in a cGMP-independent way.