Minocycline inhibits apoptotic cell death via attenuation of TNF-alpha expression following iNOS/NO induction by lipopolysaccharide in neuron/glia co-cultures

We attempted to ascertain the neuroprotective effects and mechanisms of minocycline in inflammatory-mediated neurotoxicity using primary neuron/glia co-cultures treated with lipopolysaccharide (LPS). Neuronal cell death was induced by treatment with LPS for 48 h, and the cell damage was assessed using lactate dehydrogenase (LDH) assays and by counting microtubule-associated protein-2 (MAP-2) positive cells. Through terminal transferase deoxyuridine triphosphate-biotin nick end labeling (TUNEL)-staining and by measuring caspase-3 activity, we found that LPS-induced neuronal cell death was mediated by apoptosis. We determined that pre-treatment with minocycline significantly inhibited LPS-induced neuronal cell death. In addition, LPS induced inducible nitric oxide synthase (iNOS) expression significantly, resulting in nitric oxide (NO) production within glial cells, but not in neurons. Both nitric oxide synthase (NOS) inhibitors (N(G)-monomethyl-L-arginine monoacetate (L-NMMA) and S-methylisothiourea sulfate (SMT)) and minocycline inhibited iNOS expression and NO release, and increased neuronal survival in neuron/glia co-cultures. Pre-treatment with minocycline significantly inhibited the rapid and extensive production of tumor necrosis factor-alpha (TNF-alpha) mediated by LPS in glial cells. We also determined that the signaling cascade of LPS-mediated iNOS induction and NO production was mediated by TNF-alpha by using neutralizing antibodies to TNF-alpha. Consequently, our results show that the neuroprotective effect of minocycline is associated with inhibition of iNOS induction and NO production in glial cells, which is mediated by the LPS-induced production of TNF-alpha.     

NO对金丝桃悬浮细胞生长及金丝桃素生物合成的促进作用研究

一氧化氮 (NO)是近年来发现的一种新型植物信号分子。以硝普钠 (Sodiumnitroprusside ,SNP)为一氧化氮 (NO)的供体 ,研究外源NO对金丝桃悬浮细胞生长及金丝桃素生物合成的影响。试验结果表明 ,金丝桃悬浮细胞在含 0 5和 15 0mmol LSNP的培养基中培养 2 0d后 ,细胞的干重分别为对照组的 140%和50% ;细胞中金丝桃素的含量分别为对照组的 98%和210%。试验结果表明 ,低浓度SNP处理有利于金丝桃悬浮细胞生长 ,而高浓度SNP可以促进金丝桃素的合成。在细胞培养初期 (0d)加入 0.5mmol LSNP并在指数生长后期 (14d)加入15.0mmol LSNP的金丝桃悬浮细胞在培养 2.5d后 ,细胞的干重和金丝桃素的含量分别为对照组的1.4和1.8倍 ,金丝桃素的产量达15.2mg/L ,比对照高3.2倍。SNP对金丝桃悬浮细胞生长及金丝桃素含量的影响可以被NO专一性淬灭剂CPITO(2-4-carboxyphenyl-4 ,4 ,5 ,5-tetramethylimidazoline-1-oxyl-3-oxide)所抑制,说明SNP是通过其分解产物NO影响细胞生长和金丝桃素的合成。试验结果同时表明,在15.0mmol/L的SNP处理下,金丝桃悬浮细胞中的苯丙氨酸解氨酶(PAL)的活性显著升高,推测NO可能通过触发金丝桃悬浮细胞的防卫反应,激活了细胞中金丝桃素的生物合成途径。     

Sodium Nitroprusside Induces Internalization of Muscarinic Receptors Stably Expressed in Chinese Hamster Ovary Cell Lines

Abstract: We have characterized the internalization of muscarinic acetylcholine receptors induced by the nitric oxide (NO)-generating compound sodium nitroprusside. When Chinese hamster ovary cells, stably transfected with the human m4 muscarinic receptor subtype, were incubated for 1 h in the presence of 700 µ M sodium nitroprusside, the number of receptors measured in intact cells with the hydrophilic ligand N -[³H]methylscopolamine was reduced by 30%. The effect was dose dependent, beginning with a concentration of sodium nitroprusside as low as 45 µ M . Removal of sodium nitroprusside from the incubation medium did not result in a recovery of the binding sites. The phenomenon was temperature dependent and was blocked by the muscarinic antagonist atropine. No receptor diminution was detected when the number of binding sites was evaluated with the lipophilic antagonist [³H]quinuclidinyl benzilate. This indicates that sodium nitroprusside induces a redistribution of the muscarinic receptors between the plasma membrane and an internal compartment of the cell. Receptor loss was readily reversed by treatment with the sulfhydryl reducing agent diethyldithiocarbamate. Our data provide evidence that muscarinic receptors are internalized by sodium nitroprusside through the oxidation of sulfhydryl groups; they also suggest that NO could play a role in muscarinic receptor desensitization.     

Lipopolysaccharide (LPS) Stimulates the Production of Tumor Necrosis Factor (TNF)-α and Expression of Inducible Nitric Oxide Synthase (iNOS) by Osteoclasts (OCL) in Murine Bone Marrow Cell Culture

Osteoclasts (OCL) resorb bone. They are essential for the development of normal bones and the repair of impaired bones. The function of OCL is presumed to be supported by cytokines and other biological mediators, including tumor necrosis factor (TNF)-α and nitric oxide (NO). Bacterial lipopolysaccharide (LPS) is a potent inducer of TNF-α and inducible nitric oxide synthase (iNOS), which is the specific enzyme for synthesizing NO from L-arginine. To obtain direct evidence on LPS-induced TNF-α production and iNOS expression by OCL, OCL-enriched cultures were prepared by 7-day cocultures of bone marrow cells of adult BALB/c mice and osteoblastic cells (OBs) derived from calvaria of newborn BALB/c mice, and the generation of TNF-α and iNOS in OCL stimulated with LPS was examined immunocytochemically. When the cultured cells were stimulated with 100 ng/ml of LPS, OCL clearly showed TNF-α and iNOS expression. Without LPS-stimulation, no expression was observed. TNF activity in the culture supernatants of the OCL-enriched cultures in the presence of LPS was also detected by cytotoxic assay that used TNF-sensitive L929 cells. The dentin resorption activity of OCL was estimated by area and number of pits formed on dentin slices, which were covered by the OCL fraction and cultured in the presence or absence of LPS, sodium nitroprusside (SNP; a NO generating compound), N^G-monomethyl L-arginine acetate (L-NMMA; a competitive inhibitor of NO synthase (NOS)), or LPS plus L-NMMA. Pit formation was obviously inhibited in the presence of SNP and slightly inhibited in the presence of L-NMMA, but it was not affected in the presence of LPS or LPS plus L-NMMA. These findings indicate that OCL produces TNF and expresses iNOS in response to LPS, but the LPS-activation of OCL scarcely affects pit formation by them.     

Arginase II inhibited lipopolysaccharide-induced cell death by regulation of iNOS and Bcl-2 family proteins in macrophages

Arginase II catalyzes the conversion of arginine to urea and ornithine in many extrahepatic tissues. We investigated the protective role of arginase II on lipopolysaccharide-mediated apoptosis in the macrophage cells. Adenoviral gene transfer of full length of arginase II was performed in the murine macrophage cell line RAW264.7. The role of arginase II was investigated with cell viability, cytoplasmic histone-associated DNA fragmentation assay, arginase activity, nitric oxide production, and Western blot analysis. Arginase II is localized in mitochondria of macrophage cells, and the expression of arginase II was increased by lipopolysaccharide (LPS). LPS significantly increased cell death which was inhibited by AMT, a specific inducible nitric oxide synthase (iNOS) inhibitor. In contrast, LPS-induced cell death and nitric oxide production were increased by 2-boronoethyl-L-cysteine, a specific inhibitor of arginase. Adenoviral overexpression of arginase II significantly inhibited LPS-induced cell death and cytoplasmic histone-associated DNA fragmentation. LPS-induced iNOS expression and poly ADP-ribose polymerase cleavage were significantly suppressed by arginase II overexpression. Furthermore, arginase II overexpression resulted in a decrease in the Bax protein level and the reverse induction of Bcl-2 protein. Our data demonstrated that inhibition of NO production by arginase II may be due to arginine depletion as well as iNOS suppression though its reaction products. Moreover, arginase II plays a protective role of LPS-induced apoptosis in RAW264.7 cells.     

Altered glial function causes neuronal death and increases neuronal susceptibility to 1-methyl-4-phenylpyridinium- and 6-hydroxydopamine-induced toxicity in astrocytic/ventral mesencephalic co-cultures

Altered glial function in the substantia nigra in Parkinson's disease may lead to the release of toxic substances that cause dopaminergic cell death or increase neuronal vulnerability to neurotoxins. To investigate this concept, we examined the effects of subjecting astrocytes to lipopolysaccharide (LPS)-induced activation alone or combined with L-buthionine-[S,R]-sulfoximine-induced glutathione depletion or inhibition of complex I activity by 1-methyl-4-phenylpyridinium (MPP+) on the viability of primary ventral mesencephalic neurones or susceptibility to MPP+ and 6-hydroxydopamine (6-OHDA) in co-cultures. LPS-activated astrocytes caused neuronal death in a time-dependent manner, but glutathione-depleted or complex I-inhibited astrocytes had no effect on neuronal viability. The neurotoxicity of LPS-activated astrocytes was inhibited by the inducible nitric oxide synthase inhibitor aminoguanidine, by the nitric oxide scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, and by reduced glutathione (GSH). MPP+-induced neuronal death was greater in ventral mesencephalic cultures previously cultured with LPS-activated, glutathione-depleted, or complex I-inhibited astrocytes compared with co-cultures containing normal astrocytes. The increased neuronal susceptibility to MPP+ caused by LPS-activated or complex I-inhibited astrocytes and glutathione-depleted astrocytes was inhibited by the NMDA/glutamate antagonist MK-801 and by GSH, respectively. Neuronal death caused by 6-OHDA was increased in ventral mesencephalic cultures previously cultured with LPS-activated and glutathione-depleted, but not complex I-inhibited astrocytes, compared with co-cultures containing normal astrocytes. Treatment of co-cultures with GSH prevented the increased neuronal susceptibility to 6-OHDA. These findings suggest that glial dysfunction may cause neuronal death or render neurones susceptible to toxic insults via a mechanism involving the release of free radicals and glutamate. Such a mechanism may play a role in the development or progression of nigrostriatal degeneration in Parkinson's disease.     

NO对银杏悬浮细胞生长及黄酮类物质合成的影响

以硝普钠(sodium nitroprusside,SNP)为一氧化氮(NO)的供体,向银杏悬浮细胞培养液中加入不同浓度的SNP,研究外源NO对银杏悬浮细胞生长状况、过氧化氢酶(CAT)活性、苯丙氨酸解氨酶(PAL)活性和黄酮类物质生物合成的影响.结果表明,低浓度SNP有利于银杏悬浮细胞生长,而高浓度SNP可以促进黄酮类物质的合成.银杏悬浮细胞在添加0.5和10 mmol/L SNP的培养基中培养16 d时,细胞干重分别为对照组的134%和73%;在添加10 mmol/L SNP的培养基中培养20 d时,细胞中黄酮类物质的含量为对照组的136%.同时,10 mmol/L SNP促进银杏悬浮细胞PAL和CAT活性显著升高.NO专一性淬灭剂c-PITO(carboxyl phenyltetramethylimidazoleoxide)抑制SNP对银杏悬浮细胞生长、CAT活性、PAL活性和黄酮类物质含量的促进作用,说明SNP是通过其分解产物NO影响细胞生长和黄酮类物质的合成.根据这些结果推测,NO可能通过触发银杏悬浮细胞的防卫反应,激活了细胞中黄酮类物质的生物合成途径.     

Investigations into the Mechanism of Action of a Novel Nitric Oxide Generator on Cellular Respiration

Abstract: Nitric oxide may regulate cellular respiration by competition with oxygen at mitochondrial cytochrome oxidase. Using an astrocyte-derived cell line, we have compared the mechanism of action of the nitric oxide-generating compound Roussin's black salt with that of sodium nitroprusside on cellular oxygen consumption. Intense light exposure induced the release of large quantities of nitric oxide from both of the donor compounds. However, in room light only Roussin's black salt generated low levels of the radical. Simultaneous measurement of oxygen consumption and of nitric oxide production demonstrated that sodium nitroprusside only had inhibitory actions when exposed to intense light (nitric oxide release), whereas Roussin's black salt had inhibitory actions in room light. Extracellular haemoglobin did not prevent the inhibition of respiration rate induced by Roussin's black salt even though stimulation of nitric oxide release on light exposure was markedly reduced. Preincubation of cells with Roussin's black salt and subsequent measurement of levels of light-liberated nitric oxide demonstrated that the compound was rapidly internalised. The uptake of sodium nitroprusside was minimal. These data suggest that, in contrast to sodium nitroprusside, the cellular internalisation of Roussin's black salt allows site-directed nitric oxide release and very effective inhibition of cellular respiration.     

Endotoxin induces luteal cell apoptosis through the mitochondrial pathway

The effect of endotoxin (lipopolysacharide, LPS) exposure on luteal cells was studied using an in vitro cell culture system. Buffalo luteal cells were isolated from corpora lutea of the late luteal phase (days 14-16 post estrus) and exposed to various LPS doses (5, 10 and 100 microg/ml) for different time periods (6, 12, 18 or 24 h). The cultured cells were subsequently evaluated for oxidative stress (super oxide, nitric oxide, inducible nitric oxide synthase activity, reduced glutathione depletion and lipid peroxidation) and apoptotic markers (mitochondrial membrane potential, DNA fragmentation, apoptotic cells and cell viability). LPS exposure significantly increased the production of super oxide (P<0.05) and nitric oxide (P<0.01) and increased inducible nitric oxide synthase activity (P<0.01). LPS exposure further depleted reduced glutathione (P<0.05) levels and induced lipid peroxidation (P<0.05). LPS exposure also induced the loss of mitochondrial membrane potential (P<0.05), increased DNA fragmentation (P<0.01) and apoptosis (P<0.01) and decreased cell viability (P<0.01). LPS mediated apoptotic pathway in luteal cells was further characterized using a selected LPS dose (10 microg/ml). It was observed that LPS exposure induced mitochondrial translocation of proapoptotic protein Bax, increased the total Bad expression and down regulated the expression of antiapoptotic proteins Bcl2 and BclXL. LPS exposure further induced cytochrome c release and increased Caspase-9 (P<0.01) and Caspase-3 (P<0.01) activities. LPS exposure also inhibited luteal progesterone secretion (P<0.01). It was evident that the LPS mediated apoptotic effects could be prevented by the coincubation of luteal cells with mitochondrial permeability transition pore blocker Cyclosporine A, inducible nitric oxide synthase inhibitor N-[3-(aminomethyl)benzyl]acetamidine and oxidative stress scavenger N-acetyl cysteine. Our study clearly indicates that LPS induces oxidative stress mediated apoptosis in luteal cells through the mitochondrial pathway.     

Nitric oxide induces apoptosis in the fat body cell line IPLB-LdFB from the insect Lymantria dispar

The presence of immunoreactive inducible nitric oxide synthase molecules (ir-iNOS) is demonstrated in the Lymantria dispar IPLB-LdFB cell line. The maximum ir-iNOS inducibility is observed 18 h after incubation with sodium nitroprusside (SNP). The increase in NO provoked by SNP in turn induces apoptosis. However, this phenomenon is observed only after 48 h. The NOS-inhibitors N(omega)-nitro-L-arginine methyl ester (L-NAME) and N-[3-(aminomethyl)-benzyl]acetamide (1400W) were both unable to block the SNP-induced apoptosis at all the concentrations used. Incubation with SNP plus N-acetyl-L-cysteine (NAC) further augmented the percentage of cell death with respect to SNP used alone, and this process is seen earlier, i.e. after 24 h. Moreover, the induction of apoptosis in the presence of NAC is time- and concentration-dependent. The high percentage of cell death with SNP+NAC suggests that NAC forms S-nitrosothiols with NO, resulting in an increase in the bioavailability of NO. In conclusion, these findings show the existence of a close relationship between mammalian and invertebrate cells with regards to SNP and NAC induction and the related NO response.     

Crucial cytokine interactions in nitric oxide production induced by Mycoplasma arthritidis superantigen

Mycoplasma arthritidis causes autoimmune arthritis in rodents. It produces a superantigen (MAM) that simultaneously activates antigen presenting cells and T cells inducing nitric oxide and cytokine release. Nitric oxide is a key inducer and regulator of the immune system activation. Here, we investigated nitric oxide and cytokine production and interactions of these molecules in MAM-stimulated co-cultures of macrophages (J774A.1 cell line) with spleen lymphocytes. We found that: a) MAM-induced nitric oxide, interferon-gamma, membrane-associated tumor necrosis factor and interleukin-2 production in co-cultures of macrophages with lymphocytes from BALB/c and C3H/HePas but not from C57Bl/6 mice; b) production of nitric oxide was dependent on interferon-gamma whereas that of interferon-gamma was dependent on interleukin-2 and membrane-associated tumor necrosis factor; c) these cytokines up regulated MAM-induced nitric oxide production. Unraveling the mechanisms of cell activation induced by MAM might be helpful to design strategies to prevent immune system activation by superantigens and therefore in seeking amelioration of associated immunopathologies.     

Involvement of Bcl-2 Family and Caspase-3-Like Protease in NO-Mediated Neuronal Apoptosis

Abstract: To clarify mechanisms of neuronal death in the postischemic brain, we examined whether astrocytes exposed to hypoxia/reoxygenation exert a neurotoxic effect, using a coculture system. Neurons cocultured with astrocytes subjected to hypoxia/reoxygenation underwent apoptotic cell death, the effect enhanced by a combination of interleukin-1β with hypoxia. The synergistic neurotoxic activity of hypoxia and interleukin-1β was dependent on de novo expression of inducible nitric oxide synthase (iNOS) and on nitric oxide (NO) production in astrocytes. Further analysis to determine the neurotoxic mechanism revealed decreased Bcl-2 and increased Bax expression together with caspase-3 activation in cortical neurons cocultured with NO-producing astrocytes. Inhibition of NO production in astrocytes by N ^G-monomethyl- l -arginine, an inhibitor of NOS, significantly inhibited neuronal death together with changes in Bcl-2 and Bax protein levels and in caspase-3-like activity. Moreover, treatment of neurons with a bax antisense oligonucleotide inhibited the caspase-3-like activation and neuronal death induced by an NO donor, sodium nitroprusside. These data suggest that NO produced by astrocytes after hypoxic insult induces apoptotic death of neurons through mechanisms involving the caspase-3 activation after down-regulation of BCl-2 and up-regulation of Bax protein levels.     

Lipopolysaccharide induces relaxation in lung pericytes by an iNOS-independent mechanism

Lipopolysaccharide (LPS)-regulated contractility in pericytes may play an important role in mediating pulmonary microvascular fluid hemodynamics during inflammation and sepsis. LPS has been shown to regulate inducible nitric oxide (NO) synthase (iNOS) in various cell types, leading to NO generation, which is associated with vasodilatation. The purpose of this study was to test the hypothesis that LPS can regulate relaxation in lung pericytes and to determine whether this relaxation is mediated through the iNOS pathway. As predicted, LPS stimulated NO synthesis and reduced basal tension by 49% (P < 0.001). However, the NO synthase inhibitors N (omega)-nitro-L-arginine methyl ester, aminoguanidine, and N (omega)-monomethyl-L-arginine did not block the relaxation produced by LPS. In fact, aminoguanidine and N (omega)-monomethyl-L-arginine potentiated the LPS response. The possibility that NO might mediate either contraction or relaxation of the pericyte was further investigated through the use of NO donor compounds; however, neither sodium nitroprusside nor S-nitroso-N-acetylpenicillamine had any significant effect on pericyte contraction. The inhibitory effect of aminoguanidine on LPS-stimulated NO production was confirmed. This ability of LPS to inhibit contractility independent of iNOS was also demonstrated in lung pericytes derived from iNOS-deficient mice. This suggests the presence of an iNOS-independent but as yet undetermined pathway by which lung pericyte contractility is regulated.     

S100β Induces Neuronal Cell Death Through Nitric Oxide Release from Astrocytes

Abstract: The glial-derived neurotrophic protein S100β has been implicated in the development and maintenance of the nervous system. S100β has also been postulated to play a role in mechanisms of neuropathology because of its specific localization and selective overexpression in Alzheimer's disease. However, the exact relationship between S100β overexpression and neurodegeneration is unclear. Recent data have demonstrated that treatment of cultured rat astrocytes with high concentrations of S100β results in a potent activation of inducible nitric oxide synthase (iNOS) and a subsequent generation of nitric oxide (NO), which can lead to astrocytic cell death. To investigate whether S100β-induced NO release from astrocytes might influence neurons, we studied S100β effects on neuroblastoma B104 cells or primary hippocampal neurons co-cultured with astrocytes. We found that S100β treatment of astrocyte-neuron co-cultures resulted in neuronal cell death by both necrosis and apoptosis. Neuronal cell death induced by S100β required the presence of astrocytes and depended on activation of iNOS. Cell death correlated with the levels of NO and was blocked by a specific NOS inhibitor. Our data support the idea that overexpression of S100β may be an exacerbating factor in the neurodegeneration of Alzheimer's disease.     

Reactive Macrophages Increase Oxidative Stress and Alpha-Synuclein Nitration During Death of Dopaminergic Neuronal Cells in Co-Culture: Relevance to Parkinson’s Disease

Parkinson’s disease (PD) is characterized by progressive degeneration of dopaminergic neurons and a substantial decrease in the neurotransmitter dopamine in the nigro-striatal region of the brain. Increased markers of oxidative stress, activated microglias and elevated levels of pro-inflammatory cytokines have been identified in the brains of patients with PD. Although the precise mechanism of loss of neurons in PD remains unclear, these findings suggest that microglial activation may contribute directly to loss of dopaminergic neurons in PD patients. In the present study, we tested the hypothesis that activated microglia induces nitric oxide-dependent oxidative stress which subsequently causes death of dopaminergic neuronal cells in culture. We employed lipopolysaccharide (LPS) stimulated mouse macrophage cells (RAW 264.7) as a reactive microglial model and SH-SY5Y cells as a model for human dopaminergic neurons. LPS stimulation of macrophages led to increased production of nitric oxide in a time and dose dependent manner as well as subsequent generation of other reactive nitrogen species such as peroxynitrite anions. In co-culture conditions, reactive macrophages stimulated SH-SY5Y cell death characterized by increased peroxynitrite concentrations and nitration of alpha-synuclein within SH-SY5Y cells. Importantly 1400W, an inhibitor of the inducible nitric oxide synthase provided protection from cell death via decreasing the levels of nitrated alpha-synuclein. These results suggest that reactive microglias could induce oxidative stress in dopaminergic neurons and such oxidative stress may finally lead to nitration of alpha-synuclein and death of dopaminergic neurons in PD.     

Polyphenol amentoflavone affords neuroprotection against neonatal hypoxic-ischemic brain damage via multiple mechanisms

Flavonoids are naturally occurring polyphenolic compounds that have many biological properties, including antioxidative, anti-inflammatory and neuroprotective effects. Here, we report that amentoflavone significantly reduced cell death induced by staurosporine, etoposide and sodium nitroprusside in neuroblastoma SH-SY5Y cells. In post-natal day 7 rats, hypoxic-ischemic (H-I) brain damage induced by unilateral carotid ligation and hypoxia resulted in distinct features of neuronal cell death including apoptosis and necrosis. In this model, a systemic administration of amentoflavone (30 mg/kg) markedly reduced the H-I-induced brain tissue loss with a wide therapeutic time window up to 6 h after the onset of hypoxia. Amentoflavone blocked the activation of caspase 3, characteristic of apoptosis, and the proteolytic cleavage of its substrates following H-I injury. Amentoflavone also reduced the excitotoxic/necrotic cell death after H-I injury in vivo and after oxygen/glucose deprivation in mouse mixed cultures in vitro. Treatment of mouse microglial cells with amentoflavone resulted in a significant decrease in the lipopolysaccharide-induced production of nitric oxide and induction of inducible nitric oxide synthase and cyclo-oxygenase-2. Furthermore, amentoflavone decreased the inflammatory activation of microglia after H-I injury when assessed by the microglial-specific marker OX-42. These data demonstrate for the first time that amentoflavone strongly protects the neonatal brain from H-I injury by blocking multiple cellular events leading to brain damage.     

Keratinocytes control the pheo/eumelanin ratio in cultured normal human melanocytes

The pheo/eumelanin ratio of cultured normal human melanocytes is distinct from the ratio observed for the same cells in vivo where they are in close contact with keratinocytes. To study the possible involvement of keratinocytes in the control of melanogenesis, we compared quantitatively and qualitatively the melanin production in melanocyte mono-cultures, in melanocyte-keratinocyte co-cultures and in pigmented reconstructed epidermis. Pheomelanin and eumelanin contents were assessed by high-performance liquid chromatography with electrochemical and fluorometric detection of their specific degradation products and revealed striking differences in the presence of keratinocytes. In the absence of keratinocytes (melanocyte mono-cultures), we observed a very limited eumelanin production and a very high pheomelanin synthesis. The pheo/eumelanin ratio in mono-cultures could be slightly influenced by changing the composition of the culture medium, however, the very strong imbalance in favor of pheomelanin remained unchanged. An induction of eumelanin synthesis accompanied by an important reduction of pheomelanin formation was only observed in the presence of keratinocytes. The pheo/eumelanin ratio in melanocyte mono-culture dropped from 1043 down to about 25 in the presence of keratinocytes (co-cultures). The same observations were made when the melanocytes were integrated into a reconstructed human epidermis. Interestingly, under co-culture conditions resulting in only a partial contact between melanocytes and keratinocytes, the reduction of the pheo/eumelanin ratio were less pronounced. From these results we conclude that keratinocytes play an important role in the melanin production, affecting the melanogenic pathways.     

Nitric oxide inhibits calpain-mediated proteolysis of talin in skeletal muscle cells

We tested the hypothesis that nitric oxide caninhibit cytoskeletal breakdown in skeletal muscle cells by inhibitingcalpain cleavage of talin. The nitric oxide donor sodium nitroprusside prevented many of the effects of calcium ionophore onC₂C₁₂ muscle cells, including preventing talinproteolysis and release into the cytosol and reducing loss of vinculin,cell detachment, and loss of cellular protein. These results indicatethat nitric oxide inhibition of calpain protected the cells fromionophore-induced proteolysis. Calpain inhibitor I and a cell-permeablecalpastatin peptide also protected the cells from proteolysis,confirming that ionophore-induced proteolysis was primarily calpainmediated. The activity of m-calpain in a casein zymogram was inhibitedby sodium nitroprusside, and this inhibition was reversed bydithiothreitol. Previous incubation with the active site-targetedcalpain inhibitor I prevented most of the sodium nitroprusside-inducedinhibition of m-calpain activity. These data suggest that nitric oxideinhibited m-calpain activity via S-nitrosylation of the active sitecysteine. The results of this study indicate that nitric oxide produced endogenously by skeletal muscle and other cell types has the potential to inhibit m-calpain activity and cytoskeletal proteolysis.