Dimethylsphingosine increases cytosolic calcium and intracellular pH in human T lymphocytes

N,N-Dimethyl-D-erythro-sphingosine (DMS) is the N-methyl derivative of sphingosine; both are activators of sphingosine-dependent protein kinases. The aim of this work was to study the effect of DMS on cytosolic calcium and intracellular pH (pHi) in human T lymphocytes. The variations of calcium and pH were determined by fluorescence digital imaging using Fura-2-AM and BCECF-AM, respectively. DMS increased both pHi and Ca(2+)-cytoslic in human T lymphocytes. These effects were dose-dependent. This drug induced a fast increase in pHi and a release of calcium from different intracellular calcium pools than thapsigargin. DMS also induced a Ca(2+)-influx different from the store-operated calcium channels, since drug effect was not modified by 30 microM SKF 96365. The influx of calcium induced by DMS was completely blocked by preincubation in the presence of nickel, or lanthanum, while the increase in pHi was no affected. However, the presence of cadmium reduced but does not block Ca(2+)-influx. The inhibition of G-protein by 100 ng/mL pertussis toxin, and the inhibition of tyrosine kinases by genistein significantly reduced the cytosolic calcium increase induced by DMS by an inhibition of both, release of calcium from intracellular pools and influx from extracellular medium. The inhibition of pools emptiness by these drugs was related with the inhibition that they induce in the DMS cytosolic alcalinization. In summary, DMS increases pHi and as consequence releases calcium from intracellular pools, and it increases calcium-influx through a channel different from store-operated channel (SOC). Both cytosolic calcium and pHi increase are modulated by G-proteins and tyrosine kinases.     

Counteracting effects of NADPH oxidase and the Na+/Ca2+ exchanger on membrane repolarisation and store-operated uptake of Ca2+ by chemoattractant-activated human neutrophils

This study was designed to investigate the possible involvement of NADPH oxidase and the Na(+)/Ca(2+) exchanger in regulating membrane repolarisation and store-operated uptake of Ca(2+) by FMLP (1 microM)-activated human neutrophils. Diphenyleneiodonium chloride (DPI, 5-10 microM) and KB-R7943 (2.5-10 microM), inhibitors of NADPH oxidase and the reverse mode of the Na(+)/Ca(2+) exchanger respectively, were used as pharmacological probes. Transmembrane fluxes of Ca(2+), K(+) and Na(+) were determined radiometrically, while alterations in membrane potential and cytosolic Ca(2+) were evaluated using spectrofluorimetric procedures. DPI, added to the cells at the time of maximum FMLP-activated membrane depolarisation, accelerated the rates of both membrane repolarisation and influx of Ca(2+), while KB-R7943 effectively antagonised these processes. SKF 96365 (10 microM), an antagonist of store-operated Ca(2+) channels, abolished the influx of Ca(2+) into FMLP-activated neutrophils, but had no effects on membrane repolarisation, suggesting that the Na(+)/Ca(2+) exchanger is primarily involved in mediating membrane repolarisation, thereby facilitating uptake of Ca(2+) via store-operated channels. These observations are compatible with prominent negative and positive regulatory roles for NADPH oxidase and the Na(+)/Ca(2+) exchanger respectively in regulating the rates of membrane repolarisation and store-operated uptake of Ca(2+) by chemoattractant-activated neutrophils.     

Investigation into the relationship between calyculin A-mediated potentiation of NADPH oxidase activity and inhibition of store-operated uptake of calcium by human neutrophils

The primary objective of the current study was to investigate possible relationships between calyculin A (CA)-mediated potentiation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and inhibition of store-operated uptake of Ca2+ by chemoattractant-activated human neutrophils. Treatment of neutrophils with 100 nM CA, but not at lower concentrations (12.5-50 nM), prior to the addition of the N-formylated chemotactic tripeptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) (1 microM), both potentiated and prolonged the activity of NADPH oxidase which was accompanied by exaggerated membrane depolarisation, delayed and attenuated membrane repolarisation, and inhibition of store-operated Ca2+ influx. Inclusion of diphenylene iodonium chloride (DPI, 10 microM), an inhibitor of NADPH oxidase, antagonised the effects of CA on NADPH oxidase activity and the membrane repolarisation responses of FMLP-activated neutrophils, but failed to restore store-operated influx of Ca2+. Similarly, CA also inhibited store-operated influx of Ca2+ into FMLP-activated neutrophils from a patient with chronic granulomatous disease, a primary immunodeficiency disorder characterised by the absence of a functional NADPH oxidase. CA also inhibited the store-operated influx of Ca2+ into control neutrophils treated with 1 microM thapsigargin, a selective inhibitor of the endomembrane Ca2+-ATPase, which does not activate NADPH oxidase. Taken together, these observations demonstrate that augmentation of NADPH oxidase activity is not primarily involved in CA-mediated inhibition of the store-operated influx of Ca2+ into activated human neutrophils.     

Stimulation of intracellular Ca2+ elevation in neutrophils by thiol-oxidizing phenylarsine oxide

Phenylarsine oxide (PAO), a trivalent arsenical compound, stimulated [Ca2+]i elevation in rat neutrophils in a Ca2+-containing medium but caused no appreciable response in a Ca2+-free medium. PAO also induced external Mn2+ entry, which was inhibited by N-acetyl-L-cysteine (NAC), but failed to elicit any appreciable Ba2+ and Sr2+ entry. Pretreatment of neutrophils with thiol-reducing agents including dithiothreitol (DTT), NAC, 2,3-dimercapto-1-propanol (DMP), 2,3-dimercaptopropane-1-sulfonic acid (DMPS) and tris-(2-carboxyethyl)phosphine (TCEP), all greatly inhibited PAO-induced [Ca2+]i elevation. Addition of Ni2+ or La3+ followed by PAO stimulation also attenuated the Ca2+ signals in a concentration-dependent manner. PAO had no significant effect on the production of reactive oxygen intermediates (ROI) and nitric oxide (NO) nor did it decrease cellular low molecular weight thiols levels. PAO-induced [Ca2+]i elevation was significantly inhibited by 1-[6-[17beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U-73122), the inhibitor of phospholipase C-coupled processes, genistein, a general tyrosine kinase inhibitor, phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, calyculin A, a cortical actin stabilizer, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY 294002), a phosphoinositide 3-kinase inhibitor, 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole (SKF-96365), and cis-N-(2-phenylcyclopentyl)azacyclotridec-1-en-2-amine (MDL-12,330A), the blockers of receptor-gated and store-operated Ca2+ channels, whereas there was no appreciable effect exerted by aristolochic acid, a phospholipase A2 inhibitor, 7-nitroindazole and N-(3-aminomethyl)benzylacetamidine (1400W), the blockers of NO synthase, and by suspension in a Na+-deprived medium. In contrast, 2-aminoethoxydiphenyl borane (2-APB), the blocker of IP3 receptor and Ca2+ influx, enhanced the PAO-induced response. PAO had no effect on the plasma membrane Ca2+-ATPase (PMCA) activity in the pharmacological isolated neutrophil preparation and the neutrophil membrane fractions. These results indicate that PAO stimulates [Ca2+]i rise in rat neutrophils mainly through the oxidation of vicinal thiol groups on the cell surface membrane to activation of a non-store operated Ca2+ entry (non-SOCE) without affecting the activity of PMCA and the plasmalemmal Na+/Ca2+ exchanger.     

Synergism between staurosporine and drugs inducing endoplasmic reticulum stress

Drugs causing endoplasmic reticulum or mitochondrial dysfunction may trigger apoptosis in eukaryotic cells. The thiol reagent dithiothreitol (DTT) belongs to the first group whereas the protein kinases inhibitor staurosporine acts on mitochondria. Since the endoplasmic reticulum and the mitochondrial pathways of apoptosis may converge in common steps, we examined the possibility of synergism between these two drugs. Using the activation of caspase-3 as indicator of apoptosis, we found that in two cell lines, Jurkat and Mono-Mac 6, staurosporine and DTT elicited apoptosis with a different pattern: staurosporine acted rapidly and at nanomolar concentrations while DTT acted slowly and at higher concentrations (1mM). When staurosporine and DTT were combined, the proapoptotic action was increased. This was confirmed examining late apoptotic events such as the translocation of phosphatidylserine across the plasma membrane and the cleavage of the antiapoptotic protein Mcl-1. The use of subthreshold DTT concentrations and isobologram analysis demonstrated the synergic nature of the interaction. Tunicamycin, a drug that, like DTT, inhibits protein folding in the endoplasmic reticulum also increased the proapoptotic effect of staurosporine. In agreement with the interplay between the mitochondrial and the endoplasmic reticulum pathways it was found that both staurosporine and DTT induced cytochrome c release. Furthermore, 90min incubation with DTT did not induce caspase-4 activation while staurosporine alone or in combination with DTT stimulated caspase-4 activity. We conclude that staurosporine is more active in cells undergoing endoplasmic reticulum stress. This synergism may warrant evaluation to establish whether the anticancer activity of staurosporine is also enhanced.     

Curcumin induces the degradation of cyclin E expression through ubiquitin-dependent pathway and up-regulates cyclin-dependent kinase inhibitors p21 and p27 in multiple human tumor cell lines

Curcumin, a well-known chemopreventive agent, has been shown to suppress the proliferation of a wide variety of tumor cells through a mechanism that is not fully understood. Cyclin E, a proto-oncogene that is overexpressed in many human cancers, mediates the G(1) to S transition, is a potential target of curcumin. We demonstrate in this report a dose- and time-dependent down-regulation of expression of cyclin E by curcumin that correlates with the decrease in the proliferation of human prostate and breast cancer cells. The suppression of cyclin E expression was not cell type dependent as down-regulation occurred in estrogen-positive and -negative breast cancer cells, androgen-dependent and -independent prostate cancer cells, leukemia and lymphoma cells, head and neck carcinoma cells, and lung cancer cells. Curcumin-induced down-regulation of cyclin E was reversed by proteasome inhibitors, lactacystin and N-acetyl-L-leucyl-L-leucyl-L-norleucinal, suggesting the role of ubiquitin-dependent proteasomal pathway. We found that curcumin enhanced the expression of tumor cyclin-dependent kinase (CDK) inhibitors p21 and p27 as well as tumor suppressor protein p53 but suppressed the expression of retinoblastoma protein. Curcumin also induced the accumulation of the cells in G1 phase of the cell cycle. Overall, our results suggest that proteasome-mediated down-regulation of cyclin E and up-regulation of CDK inhibitors may contribute to the antiproliferative effects of curcumin against various tumors.     

The lathyrus toxin, beta-N-oxalyl-L-alpha,beta-diaminopropionic acid (ODAP), and homocysteic acid sensitize CA1 pyramidal neurons to cystine and L-2-amino-6-phosphonohexanoic acid

A brief exposure of hippocampal slices to L-quisqualic acid (QUIS) sensitizes CA1 pyramidal neurons 30- to 250-fold to depolarization by certain excitatory amino acids analogues, e.g., L-2-amino-6-phosphonohexanoic acid (L-AP6), and by the endogenous compound, L-cystine. This phenomenon has been termed QUIS sensitization. A mechanism similar to that previously described for QUIS neurotoxicity has been proposed to describe QUIS sensitization. Specifically, QUIS has been shown to be sequestered into GABAergic interneurons by the System x(c)(-) and subsequently released by heteroexchange with cystine or L-AP6, resulting in activation of non-NMDA receptors. We now report two additional neurotoxins, the Lathyrus excitotoxin, beta-N-oxalyl-L-alpha,beta-diaminopropionic acid (ODAP), and the endogenous compound, L-homocysteic acid (HCA), sensitize CA1 hippocampal neurons >50-fold to L-AP6 and >10-fold to cystine in a manner similar to QUIS. While the cystine- or L-AP6-mediated depolarization can be inhibited by the non-NMDA receptor antagonist CNQX in ODAP- or QUIS-sensitized slices, the NMDA antagonist D-AP5 inhibits depolarization by cystine or L-AP6 in HCA-sensitized slices. Thus, HCA is the first identified NMDA agonist that induces phosphonate or cystine sensitization. Like QUIS sensitization, the sensitization evoked by either ODAP or HCA can be reversed by a subsequent exposure to 2 mM alpha-aminoadipic acid. Finally, we have demonstrated that there is a correlation between the potency of inducers for triggering phosphonate or cystine sensitivity and their affinities for System x(c)(-) and either the non-NMDA or NMDA receptor. Thus, the results of this study support our previous model of QUIS sensitization and have important implications for the mechanisms of neurotoxicity, neurolathyrism and hyperhomocystinemia.     

The effect of sesquiterpene lactones on the release of human neutrophil elastase

Sesquiterpene lactones (SLs) are natural products responsible for the anti-inflammatory activity of a variety of medicinal plants, mainly from the Asteraceae family. Here, we investigated whether they also influence the process of exocytosis of pro-inflammatory enzymes, such as the human neutrophil elastase (HNE). Altogether, eight structurally different SLs from the eudesmanolide, guaianolide, pseudoguaianolide, and germacranolide type were studied. Neutrophils were isolated from fresh human blood. After pre-incubation with different concentrations of the respective SL and cytochalasin B, the exocytosis of elastase was initiated either by platelet activating factor or N-formyl-methionyl-leucyl-phenylalanine. Inhibition of HNE release was measured by p-nitroaniline formation. The SLs exhibited an inhibitory effect on elastase release from neutrophils challenged either by platelet activating factor or N-formyl-methionyl-leucyl-phenylalanine. Concentration-response curves were recorded and the IC(50) values ranged from 2 to 30 microM. Studies on isolated HNE showed that a selective direct inhibition on HNE can be excluded. Interestingly, the inhibitory activity did not correlate with the number of alpha,beta-unsaturated carbonyl functions. The structure-activity relationship and the molecular mechanism are discussed.     

Accessibility of endothelial and inducible nitric oxide synthase to the intracellular citrulline-arginine regeneration pathway

This study investigates our hypothesis that argininosuccinate synthase (AS), the rate-limiting enzyme for arginine (L-arg) regeneration from citrulline (L-cit), plays a pivotal role in supplying L-arg to endothelial (eNOS), but not inducible (iNOS) nitric oxide synthase, for nitric oxide (NO) production. Transgenic rat blood-brain barrier (TR-BBB) endothelial cells were used as a model to elucidate the accessibility of the L-arg compartments for NOS isozymes. NO production via eNOS or iNOS, with or without alpha-methyl-DL-aspartic acid (MDLA), an AS inhibitor, was measured by a fluorometric method. NO production via eNOS was activated by the calcium ionophore A23187, while via iNOS was induced by cytokines. AS activity was assayed by the amount of argininosuccinate regenerated from radioactive aspartic acid from cell extracts. Upon increased AS activity (5.9-fold) in cells grown in L-arg-free/L-cit-supplemented medium, A23187-activated NO production also significantly increased, however cytokine-induced NO production was not detected. A23187-activated NO production was observed not only in L-arg containing medium, but also L-arg-free and L-arg-free/L-cit-supplemented medium, and was abolished by MDLA regardless of medium type. Cytokine-induced NO production was only observed in L-arg containing medium, not in L-arg-free or L-arg-free/L-cit-supplemented medium, and it was not inhibited by MDLA in the L-arg containing medium. Our results indicate that extracellular L-arg was the only L-arg pool for cytokine-induced NO production and intracellular L-arg regenerated from L-cit via AS pathway was the major L-arg pool for A23187-activated NO production in TR-BBB endothelial cells. Therefore, modulation of AS activity could be a promising strategy to selectively alter NO production via eNOS, but not iNOS.     

Role of sodium in intracellular calcium elevation and leukotriene B4 formation by receptor-mediated activation of human neutrophils

The role of Na(+) and Na(+) exchangers in intracellular Ca(2+) elevation and leukotriene B(4) (LTBs) formation was investigated in granulocyte macrophage colony-stimulating factor (GM-CSF)-primed, fMLP-stimulated human neutrophils. Isotonic substitution of extracellular Na(+) with N-methyl-D-glucamine(+) (NMDG(+)) resulted in over 85% inhibition of the LTBs generation observed (from 14.1+/-0.9pmol/10(6) neutrophils to 1.7+/-1.0pmol/10(6) neutrophils at 0.3 microM fMLP). Isotonic substitution of Na(+) with NMDG(+) also induced a significant inhibition of fMLP-induced rise in cytosolic Ca(2+) concentration ([Ca(2+)](i)) (from 2.17- to 0.78-fold increase over basal levels). Pretreatment with an inhibitor of the Na(+)/Ca(2+) exchanger (benzamil) did not inhibit either [Ca(2+)](i) rise or LTBs production, indicating that the observed effects of extracellular Na(+)-deprivation were unrelated to the Na(+)/Ca(2+) exchanger in receptor-mediated Ca(2+) influx, as previously hypothesized. LTBs production by thapsigargin-activated neutrophils was not affected by Na(+) depletion, but was totally abolished in the presence of EGTA, suggesting that store depletion-driven extracellular Ca(2+) influx is required for leukotriene synthesis and that this process is independent of Na(+)-deprivation. Exposure to Na(+)-free medium for the time of GM-CSF priming led to a significant decrease of intracellular pH values, suggesting a role of the Na(+)/H(+) exchanger in intracellular Na(+) depletion. Reducing the time of Na(+)-deprivation totally reversed the observed effect on LTBs production, resulting in enhanced, rather than inhibited, formation of LTBs. These results indicate that LTBs generation and [Ca(2+)](i) rise in human neutrophils primed by GM-CSF and stimulated with fMLP is dependent on intracellular Na(+) concentration, and, at variance with previously published results, unrelated to the Ca(2+) influx through the Na(+)/Ca(2+) exchanger.     

Evidence for the involvement of L-citrulline but not nitric oxide in the proconvulsant action of the precursor L-arginine on picrotoxin-induced convulsions in rats

To determine the role of the metabolites of L-arginine in its actions on picrotoxin-induced convulsions in rats, the concentrations of nitric oxide (NO) and L-citrulline were measured in the brain 30 and 60 min after the administration of L-arginine (1000 and 2000 mg/kg) or of N-nitro-L-arginine methyl ester (L-NAME, 30 mg/kg), an inhibitor of NO synthase. Animals treated similarly were challenged 30 and 60 min later with picrotoxin (5mg/kg), and the time of onset of myoclonus and clonic convulsions and the frequency of convulsions were determined. These parameters were also determined 30 and 60 min after administering L-arginine in L-NAME-pretreated (30 min) animals. Thirty minutes after the administration of L-arginine, the concentrations of both NO and L-citrulline were raised, the onset of myoclonus and clonic convulsions was delayed, and the frequency of convulsions was decreased, indicating the anticonvulsant property of L-arginine. A 60-min treatment of L-arginine produced a further increase in the concentration of L-citrulline but not that of NO and promoted the frequency of picrotoxin-induced convulsions. Pretreatment with L-NAME prevented L-arginine from raising the concentrations of both NO and L-citrulline; it also promoted the anticonvulsant actions and prevented the proconvulsant actions of L-arginine. These results lead to the conclusion that NO has no involvement in the time-dependent anti and proconvulsant actions of L-arginine on the picrotoxin convulsion model, and that L-citrulline seems to have a role in the proconvulsant action of L-arginine.     

Discovery of Trp-Nle-Tyr-Met as a novel agonist for human formyl peptide receptor-like 1

Formyl peptide receptor-like 1 (FPRL1) is a structural homologue of FPR, which binds chemotactic peptides as small as three amino acids (e.g., fMet-Leu-Phe, fMLF) and activates potent bactericidal functions in neutrophils. In comparison, FPRL1 ligands include peptides of 6-104 amino acids, such as Trp-Lys-Tyr-Met-Val-[d]Met (WKYMVm) and other synthetic peptides. To determine the core peptide sequence required for FPRL1 activation, we prepared various analogues based on WKYMVm and evaluated their bioactivities in an FPRL1-transfected cell line. Although substitution of d-Met(6) resulted in loss of activity, removal of Val(5) together with d-Met(6) produced a peptide that retained most of the bioactivities of the parent peptide. The resulting peptide, WKYM, represents a core structure for an FPRL1 ligand. Further substitution of Lys(2) with Nle slightly improved the potency of the tetrapeptide, which selectively activates FPRL1 over FPR. Based on these structure-activity relationship studies, we propose a model in which the modified tetrapeptide Trp-Nle-Tyr-Met (WNleYM) binds to FPRL1 through aromatic interactions involving the side chains of Trp(1) and Tyr(3), hydrophobic interaction of Nle(2), and the thio-based hydrogen bonding of Met(4), with the respective residues in FPRL1 which have not been identified. The identification of the core sequence of a potent peptide agonist provides a structural basis for future design of peptidomimetics as potential therapeutic agents for FPRL1-related disorders.     

The L-3,4-dihydroxyphenylalanine transporter in human and rat epithelial intestinal cells is a type 2 hetero amino acid exchanger

Information on the intestinal transport of L-3,4-dihydroxyphenylalanine (L-DOPA) is scarce. We present here the functional characteristics and regulation of the apical inward L-DOPA transport in two intestinal epithelial cell lines (human Caco-2 and rat IEC-6). The inward transfer of L-DOPA and L-leucine was promoted through an energy-driven system but with different sensitivity to extracellular Na(+) concentration: a minor component of L-leucine uptake (approximately 25%) was found to require extracellular Na(+) in comparison with L-DOPA transport which was Na(+)-independent. L-DOPA and L-leucine uptake was insensitive to N-(methylamino)-isobutyric acid, but competitively inhibited by 2-aminobicyclo(2,2,1)-heptane-2-carboxylic acid (BCH). L- and D-neutral amino acids, but not acidic and basic amino acids, markedly inhibited L-DOPA and [(14)C]L-leucine accumulation in both cell lines. The [(14)C]L-DOPA and [14C]L-leucine outward were markedly increased by L-leucine and BCH present in extracellular medium, but not by L-arginine. In both cell lines, L-DOPA transport was stimulated by acidic pH in comparison with [(14)C]L-leucine inward which was pH-independent. In conclusion, it is likely that system B(0) might be responsible for the Na(+)-dependent uptake of L-leucine in Caco-2 and IEC-6 cells, whereas sodium-independent uptake of L-leucine and L-DOPA may include system type 1 and type 2 L-amino acid transporter (LAT1 and LAT2), the activation of which results in trans-stimulation of substrates outward transfer.     

Role of nitric oxide in systemic effect of theophylline on mouse body temperature

In the present study, the interaction of nitric oxide synthase (NOS) inhibitors, L-NAME (N(G)-nitro-L-arginine methyl ester HCl) and L-NA (N(omega)-nitro-L-arginine), and its precursor, L-arginine (2-(S)-2-amino-5-[(aminoiminomethyl)amino] pentatonic acid), with theophylline on mouse body temperature was studied. Intraperitoneal (i.p.) injection of different doses of theophylline altered body temperature. Lower doses of theophylline (12.5 and 25 mg/kg) increased, but a higher dose (100 mg/kg) reduced, the animals' body temperature. The combination of L-arginine (20 and 40 mg/kg) with the highest dose of theophylline potentiated the hypothermic effect induced by the latter drug, while L-arginine by itself did not alter body temperature. L-NAME (10-80 mg/kg) or L-NA (10 mg/kg) plus a lower dose of theophylline (12.5 mg/kg) reduced the theophylline-induced hyperthermic response. L-NA (1, 5, and 10 mg/kg) in combination with the high dose of theophylline (100 mg/kg) also induced greater hypothermia. Both L-NAME and L-NA by themselves reduced body temperature. It is concluded that nitric oxide (NO) may be involved in the effects of theophylline on body temperature in mice.     

Functional calcium coupling with the human metabotropic glutamate receptor subtypes 2 and 4 by stable co-expression with a calcium pathway facilitating G-protein chimera in Chinese hamster ovary cells

The objective of the current study was to facilitate functional calcium assays, compatible with the fluorometric imaging plate reader platform, for the human metabotropic glutamate receptor (mGluR) subtypes 2 and 4, by co-expressing each receptor with a G-protein chimera comprising Galphaq with the C-terminal five amino acids replaced with those from Galphai3 (GqGi3). Transfection of GqGi3 into previously validated stable CHO cell lines expressing mGluR2 or mGluR4 allowed for the selection of new double transfectants in which application of L-glutamate and other mGluR agonists resulted in calcium coupling with a high signal:noise ratio (maximal changes in relative fluorescence units up to 20,000). The rank order of agonist potency for the stimulation of calcium mobilization in the mGluR2/GqGi3 stable cell line was LY354740>L-CCG-I=DCG-IV>L-glutamate>/=(2R,4R)-APDC>/=(1S,3R)-ACPD. In the mGluR4/GqGi3 stable cell line the rank order of agonist potency was L-AP4>L-SOP>/=ACPT-I=L-CCG-I>/=L-glutamate=(R,S)-PPG. By comparison, equivalent potency orders and a significant correlation in functional activities were observed when the same compounds were profiled in [35S]GTPgammaS binding assays for each mGluR subtype. These results validate the use of functional calcium assays, amenable to high-throughput applications on the fluorometric imaging plate reader, for the mGluR2 and mGluR4 subtypes when co-expressed in stable cell lines with the GqGi3 chimera.     

Subchronic oral toxicity assessment of N-acetyl-l-aspartic acid in rats

We investigated the systemic effects of subchronic dietary exposure to NAA in Sprague Dawley® rats. NAA was added to the diet at different concentrations to deliver target doses of 100, 250 and 500 mg/kg of body weight/day and was administered for 90 consecutive days. All rats (10/sex/group) survived until scheduled sacrifice. No diet-related differences in body weights, feed consumption and efficiency, clinical signs, or ophthalmologic findings were observed. No biologically significant differences or adverse effects were observed in functional observation battery (FOB) and motor activity evaluations, hematology, coagulation, clinical chemistry, urinalysis, organ weights, or gross pathology evaluations that were attributable to dietary exposure to NAA. Treatment-related increased incidence and degree of acinar cell hypertrophy in salivary glands was observed in both male and female rats in the high dose group. Because there was no evidence of injury or cytotoxicity to the salivary glands, this finding was not considered to be an adverse effect. Based on these results and the actual average doses consumed, the no-observed-adverse-effect-levels (NOAEL) for systemic toxicity from subchronic dietary exposure to NAA were 451.6 and 490.8 mg/kg of body weight/day for male and female Sprague Dawley® rats, respectively.     

S-carboxymethyl-l-cysteine and it (R/S)-S-oxides in beagle dog plasma and hepatic cytosol

Abstract

1. Incubation of beagle hepatic cytosol, under conditions promoting phenylalanine hydroxylase activity, led to the formation of the sulfoxide derivatives of S-carboxymethyl-l-cysteine, N-acetyl-S-carboxymethyl-l-cysteine, S-methyl-l-cysteine and N-acetyl-S-methyl-l-cysteine. Thiodiglycolic acid was not a substrate. Enzyme kinetic parameters (K_m, V_max) were derived indicating S-carboxymethyl-l-cysteine had the greatest clearance; no enantioselective preference was observed for this S-oxygenation reaction.

2. Following oral administration of S-carboxymethyl-l-cysteine to beagle dogs, the parent substance and its sulfoxide were the only compounds identified in the plasma. Pharmacokinetic data have been obtained indicating that the small amount of sulfoxide formed persisted within the body for longer than the parent material, but that the majority of the ingested dose remained in the administered sulfide form.

3. The sulfide moiety within the muco-regulatory drug, S-carboxymethyl-l-cysteine, is thought to be vital as it acts as a free radical scavenger, resulting in the inactive sulfoxide. Additional extensive enyzme-mediated sulfoxidation would decrease the amount of active sulfide available. In the dog this appears to not be an issue, signalling possible exploitation for therapeutic benefit in treating airway disease.     

Inhibition of arachidonic acid release and cytosolic phospholipase A2 alpha activity by D-erythro-sphingosine

Sphingolipid metabolites such as sphingosine 1-phosphate (S1P) and ceramide can mediate many cellular events including apoptosis, stress responses and growth arrest. Although ceramide stimulates arachidonic acid metabolism in several cells, the effects of sphingosine and its endogenous analogs have not been established. We investigated the effects of D-erythro-sphingosine and its metabolites on arachidonic acid release in the two cells and on the activity of cytosolic phospholipase A2alpha. C2-Ceramide (N-acetyl-D-erythro-sphingosine, 100 microM) alone stimulated [3H]arachidonic acid release and enhanced the ionomycin-induced release from the prelabeled PC12 cells and L929 cells. In contrast, exogenous addition of D-erythro-sphingosine inhibited the responses in a concentration-dependent manner in the two cell lines. D-erythro-sphingosine, D-erythro-N,N-dimethylsphingosine (D-erythro-DMS) and D-erythro-dihydrosphingosine (D-erythro-DHS) significantly inhibited mastoparan-, but not Na3VO4-, stimulated arachidonic acid release in PC12 cells. D-erythro-S1P and DL-threo-DHS showed no effect on the responses. Production of prostaglandin F2alpha was also enhanced by C2-ceramide (20 microM) and suppressed by D-erythro-sphingosine (10 microM) in PC12 cells. An in vitro study revealed that D-erythro-sphingosine, D-erythro-DMS and D-erythro-DHS directly inhibited cytosolic phospholipase A2alpha activity. These findings suggest that ceramide and D-erythro-analogs of sphingosine have opposite effects on phospholipase A2 activity and thus regulate arachidonic acid release from cells.     

Pronounced hypothermic synergy between systemic baclofen and NOS inhibitor

Baclofen was administered to rats systemically (intraperitoneal, i.p.) by itself or with L-NAME. Baclofen (1-7.5 mg/kg, i.p.) evoked dose-dependent hypothermia. L-NAME (50 mg/kg, i.p.) was ineffective. For combined administration, L-NAME increased the relative potency of baclofen (F=10.77, p<0.05), indicating multiplicative interaction and synergism. The present data reveal a surprising and significant interaction between nitric oxide synthase (NOS) and baclofen-induced hypothermia.     

Preliminary Assessment of the Immune Response to Olea europaea Pollen Extracts Encapsulated into PLGA Microspheres

In this study, poly (d,l-lactide-co-glycolide) (PLGA) microspheres encapsulating Olea europaea pollen extracts were prepared by using the double emulsion (w/o/w) based on a solvent evaporation/extraction method. The resulting microspheres were 1.93 μm in size. The total allergen loading and surface-associated allergen were 8 and 0.64%, respectively. The release of the allergen from the microspheres showed a biphasic profile with an initial burst release followed by a sustained release phase. Finally, the polyacrylamide gel electrophoresis (SDS-PAGE) results showed that the encapsulation process does not affect the stability of the protein. We describe here some preliminary observations concerning the use of these microspheres as parenteral antigen delivery systems for immunization with O. europaea pollen extracts, in a small animal model, the mouse.