Adrenergic mechanisms of the regulation of stages of chemical carcinogenesis: role of presynaptic receptors

A modifying effect of a catecholamine precursor (L-DOPA, 20 mg/kg, intraperitoneally) on various stages (initiation and promotion) of hepatocarcinogenesis, induced by N-nitrosodiethylamine (NDEA, 85 mg per 1 litre of tap water) was studied in chronic experiments on 150 male rats. L-DOPA administration prior to NDEA (influence on initiation) stimulated hepatocarcinogenesis considerably, while its administration after NDEA (influence on promotion) inhibited the realization of a carcinogenic effect. A statistically significant decrease in noradrenaline hypothalamus content was identified during early stages of chemically induced neoplastic transformation of hepatic cellular elements (stages of diffuse and focal proliferation). The results are discussed in terms of the regulatory role of the tone of the adrenergic autonomous nervous system component at the level of presynaptic inhibitory receptors in realization of the chemical carcinogenic effect.     

Regulatory pathways and uptake of L-DOPA by capillary cerebral endothelial cells, astrocytes, and neuronal cells

We examined the nature and regulation of the inward L-3,4-dihydroxyphenylalanine (L-DOPA) transporter in rat capillary cerebral endothelial (RBE4) cells, type 1 astrocytes (DI TNC1), and Neuro-2a neuroblastoma cells. In all three cell types, the inward transfer of L-DOPA was largely promoted through the 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid-sensitive and sodium-independent L-type amino acid transporter. Only in DI TNC1 cells was the effect of maneuvers that increase intracellular cAMP levels accompanied by increases in L-DOPA uptake. Also, only in DI TNC1 cells was the effect of the guanylyl cyclase inhibitor LY-83583 accompanied by a 65% increase in L-DOPA accumulation, whereas the nitric oxide donor sodium nitroprusside produced a 25% decrease in L-DOPA accumulation. In all three cell types, the Ca2+/calmodulin inhibitors calmidazolium and trifluoperazine inhibited L-DOPA uptake in a noncompetitive manner. Thapsigargin (1 and 3 microM) and A-23187 (1 and 3 microM) failed to alter L-DOPA accumulation in RBE4 and Neuro-2a cells but markedly increased L-DOPA uptake in DI TNC1 cells. We concluded that L-DOPA in RBE4, DI TNC1, and Neuro-2a cells is transported through the L-type amino acid transporter and appears to be under the control of Ca2+/calmodulin-mediated pathways. Astrocytes, however, are endowed with other processes that appear to regulate the accumulation of L-DOPA, responding positively to increases in intracellular Ca2+ and cAMP and to decreases in cGMP.     

Effect of L-dihydroxyphenylalanine on rat behavior and on catecholamine metabolism of the brain in rats with different degrees of emotional-behavioral reactivity

The effect of L-DOPA in combination with benserazide (Madopar), administered intraperitoneally on the rat behaviour and L-DOPA, DA, NE, DOPAC content in rat brain structures was studied depending on the level of the animals' emotional-behavioural reactivity. The results indicate that L-DOPA metabolism in striatum, n. accumbens and hypothalamus in intact animals with high emotional reactivity was the greatest. Administration of Madopar (50 mg/kg) induced significant behavioural disturbances in animals with less emotional-behavioural response patterns. In contrast, 125 mg/kg Madopar completely abolished individual differences in the rats' behaviour and DA, but not L-DOPA and DOPAC content. The correlation between behavioural and biochemical differences in two groups of animals is discussed in view of distinctions in L-DOPA and DA compartmentation process.     

Purification and characterization of L-DOPA decarboxylase from human kidney

L-DOPA decarboxylase has been purified to homogeneity from post mortem removed human kidneys. Homogeneity was examined by polyacrylamide gel electrophoresis (PAGE) analysis both in the presence and absence of SDS. The enzyme has a molecular weight of 100,000 daltons estimated by gel filtration and 50,000 daltons determined after SDS-PAGE. Human L-DOPA decarboxylase therefore is a dimer. Polyclonal antibodies produced against human L-DOPA decarboxylase react with the 50,000 daltons enzyme subunit after immuno-blotting and also precipitates enzyme activity. Activity against L-DOPA is partially inhibited by 5-hydroxytryptophan (5-HTP). The effect of various cations on L-DOPA decarboxylase activity has also been tested.     

Germination growth response of different plant species to the allelochemical L-3,4-dihydroxyphenylalanine (L-DOPA)

The aim of this study was to investigate the seed germination response of different plant families to L-3,4-dihydroxyphenylalanine (L-DOPA), one of the strongest allelochemicals in nature. Three types of responses in terms of colouration changes on filter paper were obtained; black and gray (Gramineae and Compositae), no change (Leguminosae, Brassicaceae, and Cucurbitaceae) and an obstructed-circle around the seeds with black colouration on the outer side of the circle (Hydrophyllaceae) when L-DOPA solution was applied during seed germination. Radicle growth in the Gramineae and Leguminosae families was inhibited less by a single treatment of L-DOPA solution (250 g/ml) than in the other families. However, continuous treatment with L-DOPA demonstrated that the Gramineae family was less affected in terms of the inhibition of radicle growth than the Leguminosae family. When more seeds were added to the L-DOPA solution less inhibition of radicle growth was observed in all plants tested. The EC₅₀ of L-DOPA for bluebell (Hydrophyllaceae), white clover (Leguminosae), and lettuce (Compositae) was approximately 200, 100, and 50 g/ml, respectively. However, in perennial ryegrass (Gramineae) no EC₅₀ was observed even at 250 g/ml L-DOPA. In the Gramineae family, addition of more seeds into the L-DOPA solution increased the colouration on the filter paper. These results demonstrated that each seed functions to oxidize or dissolve L-DOPA. In the Gramineae, Leguminosae, Compositae, and Hydrophyllaceae, increasing the number of seeds imbibed in the L-DOPA solution increased the rate of L-DOPA disappearance from the petri-dish. Of the Grammaceous plants tested, only perennial ryegrass, which showed fairly weak allelopathic activity, metabolised L-DOPA to dopamine. Although the relationships between the changes in colouration of the filter paper and the inhibition of radicle growth in these experiments are still unknown, there appears to be a strong response in each species to protect the cell from L-DOPA damage.     

The simple and rapid quantification method for L-3,4-dihydroxyphenylalanine (L-DOPA) from plant sprout using liquid chromatography-mass spectrometry

L-3,4-dihydroxyphenylalanine (L-DOPA) is one of the important secondary metabolites of plants and has been used for various purposes, such as in clinical treatment for Parkinson’s disease and dopamine-responsive dystonia. In plants, L-DOPA is a precursor of many alkaloids, catecholamines, and melanin; the L-DOPA synthesis pathway is similar to that in mammals. L-DOPA acts as an allelochemical, has an important role in several biological processes, such as stress response and metabolism, in plants. L-DOPA is widely used in the clinical treatment as well as a dietary supplement or psychotropic drug, understanding of biosynthesis of L-DOPA in plant could lead to a stable supply of L-DOPA. This paper describes an improved method for simple and rapid quantification of L-DOPA content using liquid chromatography-tandem mass spectrometry. The standard quantitative methods for L-DOPA require multiple purification steps or relatively large amounts of plant material. In our improved method, quantification of L-DOPA was possible with extract of one–two pieces of cotyledon without any partitioning or column for purification. The endogenous L-DOPA (approximately 4,000 µg g⁻¹ FW (fresh weight)) could be detected from the one pieces of cotyledon of the faba bean sprout using this method. This method was also effective for samples with low endogenous amounts of L-DOPA such as broccoli, Japanese white radish, pea, and red cabbage sprouts. Therefore, this improved method will allow to measurement of L-DOPA content easily and accurately from a small amount of plant tissue and contribute to understanding biosynthesis, catabolism, and transport of L-DOPA.     

Melanin granules prevent the cytotoxic effects of L-DOPA on retinal pigment epithelial cells in vitro by regulation of NO and superoxide radicals

Inasmuch as the nitrogen cycle elicits the direct reduction of N2 to NH3 through enzymatic reactions, and inasmuch as L-DOPA (L-dihydroxyphentlalamine), a catecholamine, can be a source of nitric oxide (NO), it is possible that melanin granules in the eye affect the generation of NO, which causes damage to the retinal pigment epithelial (RPE) cells during the oxidation of L-DOPA. In order to confirm this possibility, we analyzed the correlations of NO generation, cell growth, and superoxide dismutase (SOD) activities in two types (melanotic and amelanotic) of bovine RPE cells following exposure to L-DOPA. NO generation from L-DOPA was determined using an NO detector that is reliant on redox currents. The concentration of NO was measured in terms of diffusion currents run between a working electrode and a counter electrode, both being set in culture medium placed in a Petri dish. For the assays, L-DOPA was added to the medium at various concentrations (5, 29.9, 79.4, 152.7 or 249 microM), and 6 min after addition, an NO-trapping agent 2,4-carboxyphenyl-4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide (carboxy-PTIO) was also added. The melanotic and amelanotic types of RPE cells were cultured separately in medium with L-DOPA under an atmosphere containing 20, 10 or 5% oxygen. Cell numbers were counted using a Coulter counter, and SOD activities were determined following incubation for 24, 48 or 72 hr using a modification of the luminol assay. The results obtained indicated that: (a) NO was produced from L-DOPA in a concentration-dependent manner and was trapped quantitatively by carboxy-PTIO; (b) the generation of NO was inhibited more markedly in the melanotic cell line than in the amelanotic one, suggesting an increased tolerance to L-DOPA-derived cytotoxicity in the former; and (c) the SOD activities were more affected by oxygen concentration in the melanotic cells than in the amelanotic ones. From these results, it is concluded that melanin granules in RPE cells have a role in preventing the cytotoxicity derived from L-DOPA and in regulating the generation of NO and superoxide radicals.     

Inducive effect of cresoquinone on microbiological transformation of L-tyrosine to 3,4 dihydroxy phenyl L-alanine by Aspergillus oryzae NG-11(P1)

The present work describes the inducive effect of cresoquinone on microbiological transformation of L-tyrosine to 3,4 dihydroxy phenyl L-alanine ( L-DOPA) by Aspergillus oryzae NG-11(P1). Mould mycelium was used for biochemical conversion of L-tyrosine to L-DOPA because tyrosinases, beta-carboxylases and tyrosine hydroxylases are intracellular enzymes. The maximum conversion of L-tyrosine to L-DOPA (0.428 mg/ml) was achieved after 60 min of biochemical reaction. To enhance the production of L-DOPA, cresoquinone was added to the reaction mixture. Best L-DOPA biosynthesis results were observed when the concentration of cresoquinone was 3.5 x 10(-6) M (1.686 mg/ml L-DOPA produced with 1.525 mg/ml consumption of L-tyrosine). Cresoquinone not only increased enzyme activity but also enhanced cell membrane permeability to facilitate secretion of enzymes into the reaction broth. Comparison of kinetic parameters revealed the ability of the mutant to yield L-DOPA [Y(p/x) [i.e., mg L-DOPA formed (mg cells formed)(-1)] =7.360+/-0.04]. When the culture grown on various cresoquinone levels was monitored for Q(p), Q(s) and q(p) [ Q(p): mg L-DOPA produced ml(-1) x h(-1); Q(s): mg substrate consumed ml(-1) x h(-1); q(p): mg L-DOPA formed (mg cells)(-1) h(-1)], there was significant enhancement ( P<0.025) of these variables.     

Dopaminergic denervation enhances susceptibility to hydroxyl radicals in rat neostriatum

To determine if greater amounts of hydroxyl radical (*OH) are formed by dopamine (DA) denervation and treatment with L-dihydroxyphenylalanine (L-DOPA), the neostriatum was DA denervated (99% reduction in DA content) by 6-hydroxydopamine treatment (134microg icv, desipramine pretreatment) of neonatal rats. At 10 weeks the peripherally restricted dopa decarboxylase inhibitor carbidopa (12.5mg/kg i.p.) was administered 30min before vehicle, L-DOPA (60mg/kg i.p.), or the known generator of reactive oxygen species, 6-hydroxydopa (6-OHDOPA) (60mg/kg i.p.); and this was followed 30min later (and 15 min before termination) by the spin trap, salicylic acid (8micromoles icv). By means of a high performance liquid chromatographic method with electrochemical detection, we found a 4-fold increase in the non-enzymatically formed spin trap product, 2,3-dihydroxybenzoic acid (2,3-DHBA), with neither L-DOPA nor 6-OHDOPA having an effect on 2,3-DHBA content of the neostriatum. Basal content of 2,5-DHBA, the enzymatically formed spin trap product, was 4-fold higher vs. 2,3-DHBA in the neostriatum of untreated rats, while L-DOPA and 6-OHDOPA each reduced formation of 2,5-DHBA. We conclude that DA innervation normally suppresses *OH formation, and that the antiparkinsonian drug L-DOPA has no effect (2,3-DHBA) or slightly reduces (2,5-DHBA) *OH formation in the neostriatum, probably by virtue of its bathing the system of newly formed *OH.     

A site-specific mutation of tyrosine hydroxylase reduces feedback inhibition by dopamine in genetically modified cells grafted in parkinsonian rats

Aromatic L-amino acid decarboxylase (AADC) is necessary for conversion of L-DOPA to dopamine. Therefore, AADC gene therapy has been proposed to enhance pharmacological or gene therapies delivering L-DOPA. However, addition of AADC to the grafts of genetically modified cells expressing tyrosine hydroxylase (TH) and GTP cyclohydrolase 1 (GCH1), which produce L-DOPA in parkinsonian rats, resulted in decreased production of L-DOPA and dopamine owing to feedback inhibition of TH by dopamine. End-product feedback inhibition has been shown to be mediated by the regulatory domain of TH, and site-specific mutation of serine 40 makes TH less susceptible to dopamine inhibition. Therefore, we investigated the efficacy of using TH with serine 40 mutated to leucine (mTH) in an ex vivo gene-therapy paradigm. Primary fibroblasts (PF) from Fischer 344 rats were transduced with retrovirus to express mTH or wild-type rat TH cDNA (wtTH). Both cell types were also transduced with GCH1 to provide the obligate TH cofactor, tetrahydrobiopterin. PF transfected with AADC were used as coculture and cografting partners. TH activities and L-DOPA production in culture were comparable between PFwtTHGC and PFmTHGC cells. In cocultures with PFAADC cells, PFmTHGC cells showed significant reduction in the inhibitory effect of dopamine compared with PFwtTHGC cells. In vivo microdialysis measurement showed that cografting PFAADC cells with PFmTHGC cells resulted in smaller decreases in L-DOPA and no reduction in dopamine levels compared with cografts of PFAADC cells with PFwtTHGC cells, which decreased both L-DOPA and dopamine levels. Maintenance of dopamine levels with lower levels of L-DOPA would result in more focused local delivery of dopamine and less potential side-effects arising from L-DOPA diffusion into other structures. These data support the hypothesis that mutation of serine 40 attenuates TH end-product inhibition in vivo and illustrates the importance of careful consideration of biochemical pathways and interactions between multiple genes in gene therapy.     

Molecular and morphological characterization of heterorhabditid entomopathogenic nematodes from the tropical rainforest in Brazil

Despite massive losses of primary forest, the Amazonian rainforest remains an extremely rich source of biodiversity. In recent years, entomopathogenic nematodes (EPNs) have been isolated from soil in various parts of the world and used successfully as biological control agents against numerous insect pests. Therefore, a sampling in the rainforest of Monte Negro, Rond?nia, Brazil was conducted with the aim of discovering new strains and/or species of EPNs for future development as biological control agents. From 156 soil samples taken at nine collecting sites, 19 isolates were obtained, all of them belonging to the genus Heterorhabditis. Four strains were subjected to detailed morphological and molecular evaluation. Based on morphometrics and internal transcribed spacer (ITS) sequence data, the strains LPP1, LPP2 and LPP4 were identified as Heterorhabditis indica, whereas LPP7 was considered Heterorhabditis baujardi. Comparative analysis of the ITS1 sequence of H. indica and H. baujardi isolates showed a polymorphic site for the restriction enzyme Tth 111 that could be used to distinguish the two species. Consequently, strains LPP1, LPP2, LPP3, LPP4, and LPP9 were identified as H. indica, whereas LPP5, LPP7, LPP8 and LPP10 were identified as H. baujardi.     

Effect of alpha-methyl-p-tyrosine and dihydroxyphenylalanine on plasma corticosterone levels in rats pretreated with prednisolone acetate

The effects on hypothalamic-pituitary-adrenal function and on negative feed-back action of a synthetic adrenal steroid, prednisolone acetate, have been studied in female Wistar rats treated with alpha-methyl-p-tyrosine (alpha MpT) and L-dihydroxyphenylalanine (L-DOPA) administered for six consecutive days. The parameter for study was taken from the plasmatic changes of corticosterone levels. alpha-MpT injection determines an increase of plasmatic corticosterone values, while L-DOPA injection produces the opposite effect, decreasing corticosterone levels as compared to the saline control group. The simultaneous administration of prednisolone acetate 250 micrograms/kg weight s.c. for six consecutive days to alpha-MpT or L-DOPA determines the following effects: a) With alpha-MpT the negative feed-back action of prednisolone was not modified. b) With L-DOPA this action of the steroid is cancelled and the plasmatic corticosterone values are increased significantly. These changes have been related to modifications of the catecholamines content in the brain. The importance of the catecholamines on the activity of the hypothalamic-pituitary-adrenal axis is discussed under experimental condition, specially the effects of L-DOPA, which depends for its activity on the presence or absence of prednisolone.     

The effects of melatonin and L-DOPA on the diurnal rhythms of free amino acids content in the rat retina

The effects of melatonin and dopamine precursor L-3,4-dihydroxyphenylalanine (L-DOPA) intraperitoneal administration on the rhythms of free amino acids content in the retina of rats were studied. The authors found that the levels of those amino acids, which are protein constituents but not neurotransmitters in the rat retina, change diurnally with maximum at 3-6 h after light onset. Diurnal changes of Ala, Arg, Asn, Ile, Met, Ser, Trp, and Val content persisted in the retina of rats maintained at constant darkness. This fact confirms the true circadian nature of these rhythms. Constant lighting abolished diurnal changes of the content of all amino acids with the exception of Trp. Daytime but not nighttime administration of melatonin decreased the levels of Ala, Asn, Gln, Ile, Met, and Ser down to nocturnal values. Diurnal changes of amino acids content vanished in melatonin-injected rats. The effect of melatonin administration disappeared when the protein synthesis was inhibited by cycloheximide. The effect of intraperitoneal administration of L-DOPA on the levels of free amino acids was opposite the effect of melatonin administration. L-DOPA increased nocturnal levels of Gly, Thr, Trp, and Val but had no effect on the daytime amino acids content. As in the case of melatonin administration, significant diurnal changes of amino acid levels disappeared in L-DOPA-injected rats. The authors hypothesize that melatonin and dopamine can serve as zeitgebers-antagonists of amino acids content rhythms in the rat retina.     

Effect of chronic neuroleptic or L-DOPA administration on GABA levels in the rat substantia nigra

Male albino rats were administered daily with haloperidol, clozapine or L-DOPA and sacrificed 18 hours after the last dose of the drug. Acutely haloperidol (5mg/kg, i.p.) greatly lowered nigral GABA levels whereas after 167 daily doses the nigral GABA levels were not significantly different from controls, but were significantly increased as compared with the acutely treated animals. In contrast, acute L-DOPA (2 × 100mg, p.o.) greatly raised nigral GABA levels whereas after chronic L-DOPA (167 days) nigral GABA levels were not significantly different from controls and were significantly lower as compared with the animals receiving the acute treatment. Clozapine (20 mg/kg, i.p. either acutely or chronically) did not have as marked an effect on nigral GABA levels as did haloperidol. Of these various drug regimens only chronic L-DOPA significantly affected nigral GAD activity, producing a moderate decrease.     

Accumulation of lipid peroxidation products in cataractous lenses

A study was made of the content of lipid peroxidation products (LPP) in the lenses extracted during operations for cataract as well as in transparent human lenses. In a mature cataract, the elevated content of primary, secondary and end products of lipid peroxidation (diene and triene conjugates, Schiff bases) was revealed. The content of LPP was identical in different clinical patterns of a mature cataract (senile, traumatic, complicated), which points to the universal role of lipid peroxidation in lenticular opacity.     

The focal adhesion and nuclear targeting capacity of the LIM-containing lipoma-preferred partner (LPP) protein

Targeting of proteins to a particular cellular compartment is a critical determinant for proper functioning. LPP (LIM-containing lipoma-preferred partner) is a LIM domain protein that is localized at sites of cell adhesion and transiently in the nucleus. In various benign and malignant tumors, LPP is present in a mutant form, which permanently localizes the LIM domains in the nucleus. Here, we have investigated which regions in LPP target the protein to its subcellular locations. We found that the LIM domains are the main focal adhesion targeting elements and that the proline-rich region of LPP, which harbors binding sites for alpha-actinin and vasodilator-stimulated phosphoprotein (VASP), has a weak targeting capacity. All of the LIM domains of LPP cooperate in order to provide robust targeting to focal adhesions, and the linker between LIM domains 1 and 2 plays a pivotal role in this targeting. When overexpressed in the cytoplasm of cells, the LIM domains of LPP can deplete endogenous LPP and vinculin from focal adhesions. The proline-rich region of LPP contains targeting sites for focal adhesions and stress fibers that are distinct from the alpha-actinin and VASP binding sites, and the LPP LIM domains are dispensable for targeting LPP to the nucleus. Our studies have defined novel functional domains in the LPP protein.     

Plastidic phosphatidic acid phosphatases identified in a distinct subfamily of lipid phosphate phosphatases with prokaryotic origin

Plastidic phosphatidic acid phosphatase (PAP) dephosphorylates phosphatidic acid to yield diacylglycerol, which is a precursor for galactolipids, a primary and indispensable component of photosynthetic membranes. Despite its functional importance, the molecular characteristics and phylogenetic origin of plastidic PAP were unknown because no potential homologs have been found. Here, we report the isolation and characterization of plastidic PAPs in Arabidopsis that belong to a distinct lipid phosphate phosphatase (LPP) subfamily with prokaryotic origin. Because no homolog of mammalian LPP was found in cyanobacteria, we sought an LPP ortholog in a more primitive organism, Chlorobium tepidum, and its homologs in cyanobacteria. Arabidopsis had five homologs of cyanobacterial LPP, three of which (LPP gamma, LPP epsilon 1, and LPP epsilon 2) localized to chloroplasts. Complementation of yeast Delta dpp1 Delta lpp1 Delta pah1 by plastidic LPPs rescued the relevant phenotype in vitro and in vivo, suggesting that they function as PAPs. Of the three LPPs, LPP gamma activity best resembled the native activity. The three plastidic LPPs were differentially expressed both in green and nongreen tissues, with LPP gamma expressed the highest in shoots. A knock-out mutant for LPP gamma could not be obtained, although a lpp epsilon 1 lpp epsilon 2 double knock-out showed no significant changes in lipid composition. However, lpp gamma homozygous mutant was isolated only under ectopic overexpression of LPP gamma, suggesting that loss of LPP gamma may cause lethal effect on plant viability. Thus, in Arabidopsis, there are three isoforms of plastidic PAP that belong to a distinct subfamily of LPP, and LPP gamma may be the primary plastidic PAP.     

Crystalline lens induction of lipid peroxidation

The level of lipid peroxidation products (LPP) was determined in the aqueous humor from the anterior chamber of patients with cataract and donor eyes. The content of LPP in senile cataract aqueous humor was shown to be significantly increased. To determine the possible mechanism of LPP increase in aqueous humor, human lenses at different stages of cataract as well as transparent human and rabbit lenses were incubated for 3 hours in 3.0 ml medium containing liposomes (0.5 mg/ml) prepared from phospholipids from the egg yolk and 0.14 M NaCl + 0.01 M TRIS-HCl buffer, pH 7.4). Corrections were made for phospholipid autooxidation. The level of LPP accumulation in the medium was determined by MDA assay. The rate of LPP production increased significantly in transparent lenses and in early senile cataract, as compared to controls and advanced (mature) cataracts. EDTA (1 mM), superoxide dismutase (114 u/sample), catalase (900 u/sample), chelated iron (III): Fe3+-ADP addition to the incubation medium depressed the level of LPP accumulation. This suggests the participation of Fe2+, O2-., H2O2 in the mechanism of LPP production in the lens. The induction of lipid peroxidation in the lens can be significant for leukotriene and prostaglandin synthesis in the eye.     

Inhibition of lipid phosphate phosphatase activity by VPC32183 suppresses the ability of diacylglycerol pyrophosphate to activate ERK(1/2) MAP kinases

The lipidic metabolite, diacylglycerol pyrophosphate (DGPP), in its dioctanoyl form (DGPP 8:0), has been described as an antagonist for mammalian lysophosphatidic acid (LPA) receptors LPA1 and LPA3. In this study we show that DGPP 8:0 does not antagonize LPA dependent activation of ERK(1/2) MAP kinases but strongly stimulated them in various mammalian cell lines. LPA and DGPP 8:0 stimulation of ERK(1/2) occurred through different pathways. The DGPP 8:0 effect appeared to be dependent on PKC, Raf and MEK but was insensitive to pertussis toxin and did not involve G protein activation. Finally we showed that DGPP 8:0 effect on ERK(1/2) was dependent on its dephosphorylation by a phosphatase activity sharing lipid phosphate phosphatase properties. The inhibition of this phosphatase activity by VPC32183, a previously characterized LPA receptor antagonist, blocked the DGPP 8:0 effect on ERK(1/2) activation. Moreover, down-regulation of lipid phosphate phosphatase 1 (LPP1) expression by RNA interference technique also reduced DGPP 8:0-induced ERK(1/2) activation. Consistently, over expression of LPP1 in HEK293 cells increases DGPP 8:0 hydrolysis and this increased activity was inhibited by VPC32183. In conclusion, DGPP 8:0 does not exert its effect by acting on a G protein coupled receptor, but through its dephosphorylation by LPP1, generating dioctanoyl phosphatidic acid which in turn activates PKC. These results suggest that LPP1 could have a positive regulatory function on cellular signaling processes such as ERK(1/2) activation.     

L-DOPA produced nitric oxide in the vitreous and caused greater vasodilation in the choroid and the ciliary body of melanotic rats than in those of amelanotic rats

The nitrogen cycle initiates direct reduction of N2 to NH3 by enzymatic reactions. We hypothesize that L-dihydroxyphenylalanine (L-DOPA), a catecholamine, could be a source of nitric oxide (NO). In order to determine whether L-DOPA generates NO and induces any biological change in the eye, we measured the generation of NO in vitro and in vivo, and investigated the histopathological changes caused by injection of L-DOPA into the vitreous of rats. We also hypothesized that melanin granules may affect the generation of NO during the metabolism of L-DOPA, since L-DOPA is a precursor of melanin in the brain and the eye. Therefore, we compared the effects of L-DOPA on the generation of NO between amelanotic and melanotic rats. NO was measured as diffusion currents by NO electrodes. In vitro, various concentrations of L-DOPA (5, 29.9, 79.4, 152.7, and 249 microM) were added to the medium. The inhibition of NO generation by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide (carboxy-PTIO) was tested. In vivo, NO generation in the vitreous of rats was measured and the eyes were enucleated under anesthesia after L-DOPA injection. The ocular tissues were subjected to histological examination. NO was produced from L-DOPA in a dose-dependent manner and was scavenged by carboxy-PTIO in vitro. NO in the vitreous of melanotic rats was generated from L-DOPA. Histological examination with hematoxylin-eosin staining revealed vasodilation in the ciliary vessels and the choroid after L-DOPA injection. Both effects were greater in melanotic rats than in amelanotic rats. The vasodilation may be attributable to NO as well as to superoxides, which can be regulated by the existence of melanin.