Influence of nitric oxide on morphine-induced conditioned place preference in the rat central amygdala

Effects of intra-central amygdala injections of L-arginine, a nitric oxide (NO) precursor, and N(G)-nitro-L-arginine methyl ester (L-NAME), a NO synthase (NOS) inhibitor, on morphine-induced conditioned place preference in rats were investigated by using an unbiased 3-day schedule of place conditioning design. Animals receiving once daily injections of morphine (0.5-7.5 mg/kg, subcutaneously, s.c.) or saline (1.0 ml/kg, s.c.) showed a significant place preference in a dose-dependent manner. The maximum response was observed with 5.0 mg/kg of the opioid. Co-administration of morphine (5.0 mg/kg) with L-arginine (0.3, 1.0 and 3.0 microg/rat), but not with L-NAME (0.3, 1.0 and 3.0 microg/rat), during the acquisition of morphine-induced conditioned place preference increased morphine-induced conditioned place preference. The response to L-arginine was blocked by L-NAME preadministration. L-arginine and L-NAME by themselves did not induce conditioned place preference. When L-arginine or L-NAME at 0.3-3.0 microg/rat was administered 1 min before conditioned place preference testing, L-arginine but not L-NAME caused an increase in the expression of morphine-induced conditioned place preference, the effect that was blocked by L-NAME preadministration. A dose of L-arginine (0.3 microg/rat), but not L-NAME, during expression of morphine-induced conditioned place preference produced an increase in locomotion compared with that in the control group. It may be concluded that an increase in the NO levels in the central amygdala may have an effect on the acquisition and expression of morphine-induced conditioned place preference.     

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.     

The effect of cyclosporin A on morphine tolerance and dependence: involvement of L-arginine/nitric oxide pathway

Cyclosporin A is known to decrease nitric oxide (NO) production in nervous tissues. The effects of systemic cyclosporine A on the induction and expression of morphine tolerance and dependence, acute morphine-induced antinociception, and the probable involvement of the L-arginine/nitric oxide pathway in these effects were assessed in mice. Cyclosporin A (20 mg/kg), N(G)-nitro-L-arginine methyl ester (L-NAME) (10 mg/kg) and a combination of the two at lower and per se non-effective doses (5 and 3 mg/kg, respectively) showed a similar pattern of action, inhibiting the induction of tolerance to morphine-induced antinociception and increasing the antinociception threshold in the expression phase of morphine tolerance. These agents also inhibited the expression of morphine dependence as assessed by naloxone-precipitated withdrawal signs, while having no effect on the induction of morphine dependence. L-Arginine, at a per se non-effective dose (60 mg/kg), inhibited the effects of Cyclosporin A. Moreover, acute administration of Cyclosporin A (20 mg/kg) or L-NAME (10 mg/kg) enhanced the antinociception induced by acute administration of morphine (5 mg/kg), while chronic pretreatment with Cyclosporin A (20 mg/kg) or L-NAME (10 mg/kg) for 2 days (twice daily) did not affect morphine-induced antinociception. The inducible nitric oxide synthase inhibitor, aminoguanidine (100 mg/kg), did not alter morphine antinociception, tolerance or dependence. In conclusion, decreasing NO production through constitutive nitric oxide synthase may be a mechanism through which cyclosporin A differentially modulates morphine tolerance, dependence and antinociception.     

Molsidomine attenuates N(omega)-nitro-L-argininemethylester-induced deficits in a memory task in the rat

The present study was designed to investigate the role of nitric oxide (NO) on recognition memory in the rat. For this purpose, the effects on memory exerted by post-training administration of the NO synthase (NOS) inhibitor N(omega)-nitro-L-argininemethylester (L-NAME) and the NO donor molsidomine were assessed by using the object recognition task. In a first dose-response study, L-NAME, at 30 but not at 10 mg/kg impaired the animals' performance, whereas at 60 mg/kg, it induced side-effects. Molsidomine, 4 mg/kg, antagonized the L-NAME-induced performance deficits. These results indicate that NO is involved in post-training memory processes.     

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.     

Agmatine exerts anticonvulsant effect in mice: modulation by alpha 2-adrenoceptors and nitric oxide

The effect of agmatine, an endogenous polyamine metabolite, on seizure susceptibility was investigated in mice. Acute intraperitoneal administration of agmatine (5, 10, 20, 40 mg/kg) had a significant and dose-dependent inhibitory effect on pentylenetetrazole (PTZ)-induced seizures. The peak of this anticonvulsant effect was 45 min after agmatine administration. We further investigated the possible involvement of the alpha(2)-adrenoceptors and L-arginine/NO pathway in this effect of agmatine. The alpha(2)-adrenoceptor antagonist, yohimbine (0.5-2 mg/kg), induced a dose-dependent blockade of the anticonvulsant effect of agmatine. The nitric oxide synthase (NOS) substrate, L-arginine (60 mg/kg), inhibited the anticonvulsant property of agmatine and this effect was significantly reversed by NOS inhibitor N(G)-nitro-L-arginine (L-NAME, 30 mg/kg), implying an NO-dependent mechanism for L-arginine effect. We further examined a possible additive effect between agmatine (1 or 5 mg/kg) and L-NAME (10 mg/kg). The combination of L-NAME (10 mg/kg) with agmatine (5 but not 1 mg/kg) induced a significantly higher level of seizure protection as compared with each drug alone. Moreover, a combination of lower doses of yohimbine (0.5 mg/kg) and L-arginine (30 mg/kg) also significantly decreased the anticonvulsant effect of agmatine. In conclusion, the present data suggest that agmatine may be of potential use in seizure treatment.     

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.     

Endogenous nitric oxide does not modulate mesenteric mast cell degranulation in rats

The inhibitory effects of endogenous nitric oxide could explain the decreased mesenteric mast cell degranulation after anaphylaxis in genetically hypertensive rats (SHR). SHR and normotensive rats (NT) were sensitized to ovalbumin and challenged 14 days later. Degranulation of mast cells was assessed in duodenum, mesentery and skin by increased microvascular permeability using extravasation of Evans blue dye (20mg/kg, i.v.), and in the mesentery also by light microscopy after staining with toluidine blue. Pretreatment with an inhibitor of nitric oxide synthesis, L-NAME (30 mg/kg, i.v.) did not change dye extravasation after immunological challenge or after compound 48/80 in mesentery of either SHR or NT. PCA was also defective in SHR. Pretreatment with L-NAME did not affect either the defective PCA in SHR or the normal PCA reaction in NT. Our results show that inhibition by endogenous nitric oxide is not the cause of the defective mast cell degranulation in the SHR nor did it modulate degranulation of mesenteric or skin mast cells in NT.     

l-Arginine and l-glutamine as immunonutrients and modulating agents for oxidative stress and toxicity induced by sodium nitrite in rats

Sodium nitrite (NaNO₂) is a flavoring, coloring and preservative agent in meat and fish products. The study aimed to evaluate the efficacy of l-arginine and l-glutamine supplementation as a potentially novel and useful strategy for the modulation of oxidative stress and toxicity induced by NaNO₂ in male rats. Rats were divided into six groups each of 10 rats and treated for 6 weeks: group 1 as normal control; group 2 fed standard diet containing 0.2% NaNO₂; group 3 and 4 fed the previous diet supplemented with 1% and 2% arginine, respectively; group 5 and 6 fed NaNO₂ diet supplemented with 1% and 2% glutamine, respectively. NaNO₂ treatment induced a significant increase in serum malondialdehyde, nitric oxide, arginase, glutathione-S-transferase activities, urea and creatinine as well as differential leucocytes%. However, a significant decrease was recorded in reduced glutathione, catalase activity, total protein, albumin and some hematological parameters as well as immunoglobulin G. On the other hand, arginine or glutamine showed a remarkable modulation of these abnormalities as indicated by reduction of malondialdehyde and improvement of the investigated antioxidant and hematological parameters. It can be concluded that arginine or glutamine supplementation may reduce oxidative stress and improve the hazard effects of NaNO₂.     

The effect of L-stepholidine, a novel extract of Chinese herb, on the acquisition, expression, maintenance, and re-acquisition of morphine conditioned place preference in rats

The effect of L-stepholidine (SPD), a novel alkaloid extract of the Chinese herb Stephania with partial dopamine D1 receptor agonistic and D2 receptor antagonistic dual actions, on morphine conditioned place preference (CPP) was studied. Daily injection of morphine (10 mg/kg, i.p.) for 6 days induced CPP in rats, and daily treatment with SPD at 10 or 20 mg/kg before morphine injection dose-dependently attenuated morphine-induced CPP. On the day following acquisition of morphine CPP, a single administration of SPD at 10 or 20 mg/kg failed to block the expression of CPP. However, daily administration of SPD at 20 mg/kg for 7 days attenuated the maintenance of CPP. Morphine-induced CPP extinguished after a 21-day saline training and then a single injection of morphine (3 mg/kg, i.p.) induced re-acquisition of morphine CPP; however, pretreatment with SPD at 10 or 20 mg/kg 30 min before morphine injection dose-dependently blocked morphine (3 mg/kg, i.p.)-induced re-acquisition of morphine CPP. Furthermore, our data indicate that SPD had no effect on food-induced CPP or state-dependent learning, suggesting that the observed effect of SPD does not result from an inhibition of general learning ability. These results demonstrate that SPD can inhibit acquisition, maintenance, and re-acquisition of morphine conditioned place preference and suggest its potential for treatment of opioid addiction.     

Effect of propionyl-l-carnitine,l-arginine and nicotinic acid on the efficacy of vardenafil in the treatment of erectile dysfunction in diabetes

ABSTRACT

Objective: The association of diabetes-related vascular damage and the role of metabolic factors in erectile dysfunction are well known in the literature. The compounds propionyl-l-carnitine (PLC), l-arginine (l-Arg) and nicotinic acid have numerous metabolic actions which have been reported to improve endothelial function. This study investigated the administration of the combination of these three compounds alone and in association with an inhibitor of 5-phosphodiesterase (5PDE), vardenafil, on endothelial function in diabetic patients with erectile dysfunction.

Methods: A total of 40 patients aged between 50 and 60 years with insulin-dependent diabetes (IDDM) for 3–4 years were selected from 509 patients presenting with erectile dysfunction. The patients were randomly subdivided into four groups of ten to be treated for 12 weeks. Group A was administered one sachet each day of test formulation containing PLC, l-Arg and nicotinic acid (Ezerex); group B with one 20 mg capsule of vardenafil (Levitra) twice a week; group C was treated with one sachet each day of the test formulation plus vardenafil 20 mg twice a week. Group D was administered placebo capsules twice weekly. Endothelial function was evaluated by examining flow-mediated dilation (FMD) and erectile function was estimated with the International Index of Erectile Function (IIEF5) questionnaire in all subjects.

Results: At the end of treatment group A showed an increment of 2 points in the IIEF5; group B showed an increment of 4 points; group C, the group which was administered all the treatments, showed an increment of 5 points, and group D, treated with placebo, showed no increment in the IIEF5.

Conclusion: Although there was a small number of subjects in this study the data suggest that the test formulation may improve the endothelial situation in diabetes. The test formulation together with vardenafil was better than the 5PDE inhibitor alone, but further studies are needed to confirm these findings.     

Lack of involvement of extracellular signal-regulated kinase (ERK) in the agonist-induced endothelial nitric oxide synthesis

In a recent paper, it was shown that stimulation of endothelial cells with bradykinin (BK) leads to phosphorylation of endothelial nitric oxide synthase (eNOS) mediated by extracellular signal-regulated kinase (ERK) (J. Biol. Chem. 275 (2000) 30707). Since in vitro phosphorylation by ERK reduced the catalytic activity of eNOS, it was suggested that this mechanism may be an important determinant of nitric oxide signalling in endothelial cells. To explore the physiological role of ERK as regulator of nitric oxide synthesis in intact cells, we measured the effects of the kinase inhibitor PD 98059 on BK- and ATP-induced nitric oxide formation in cultured endothelial cells and isolated vascular smooth muscle strips. PD 98059 completely inhibited ERK activation by BK and ATP in porcine aortic endothelial cells without affecting eNOS activation. Moreover, PD 98059 did not potentiate relaxation of isolated porcine pulmonary arteries to BK or ATP, indicating that ERK-catalysed eNOS phosphorylation does not contribute to the regulation of nitric oxide formation in intact cells or tissues.     

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.     

Recombinant arginine deiminase as a differential modulator of inducible (iNOS) and endothelial (eNOS) nitric oxide synthetase activity in cultured endothelial cells

Modulation of the extracellular level of arginine, substrate for nitric oxide synthetases, is a promising modality to alleviate certain pathological conditions where excess nitric oxide (NO) is produced. However, complications arise, as only preferential inhibition of the inducible nitric oxide synthetase (iNOS), but not endothelial nitric oxide synthetase (eNOS), is desired for the treatment of NO over-production. We investigated the effect of arginine deprivation mediated by a recombinant arginine deiminase (rADI) on the activity of iNOS and eNOS in an endothelial cell line, TR-BBB. Our results demonstrated that cytokine-induced NO production depends on the extracellular arginine as substrate. However, if sufficient citrulline is present in the medium, A23187-activated NO production by eNOS does not rely on extracellular arginine. Treatment with rADI can markedly inhibit cytokine-induced NO production via iNOS, but not A23187-activated NO production via eNOS. Our results also showed that the decrease of NO production by iNOS could be achieved by depleting arginine from the medium even under the conditions that would up-regulate iNOS expression. Thus, rADI appears to be a novel selective modulator of iNOS activity that may be a used as a tool in the study of pathological disorders where NO over-production plays a key role.     

Evaluation of an LC-MS/MS assay for 15N-nitrite for cellular studies of L-arginine action

The utility of an LC-MS/MS assay for nitrite determination in studying l-arginine (ARG) cellular action was examined in vitro. EA.hy926 human endothelial cells or cellular fractions (membrane and cytosol) were exposed to 0-500 μM of ¹⁵N₄-ARG for 2 h. ¹⁴N-nitrite and ¹⁵N-nitrite in the cell lysate and in the incubation medium were derivatized with 2,3-diaminonaphthalene (DAN) to form ¹⁴N- and ¹⁵N-naphthotriazole (i.e., ¹⁴N-NAT and ¹⁵N-NAT). Peak responses of ¹⁴N-NAT and ¹⁵N-NAT were analyzed by LC-MS/MS with 1H-naphth[2,3-d]imidazole as an internal standard. The calibration curves of DAN-derivatized ¹⁴N-NAT and ¹⁵N-NAT from ¹⁴N-nitrite and ¹⁵N-nitrite were linear. Intra- and inter-day variability of the quantification was below 14.2% in quality control samples. Following incubation of EA.hy926 cells with ¹⁵N₄-ARG, saturable increases of ¹⁵N-nitrite accumulation with increasing ¹⁵N₄-ARG exposure were observed clearly. This increase however could not be detected by the classical fluorescence method, nor were changes in ¹⁴N-nitrite level observed. When cellular fractions were exposed to ¹⁵N₄-ARG, ¹⁵N-nitrite formation was only observed in the membrane fragments. The sensitive and selective LC-MS/MS method reported here can be applied to quantify accumulated nitrite levels in human endothelial cells. The selectivity of this stable-isotope labeled LC-MS/MS method offers an advantage over other traditional methods for elucidating cellular ARG action when its stable isotope is employed as a substrate.     

L-arginine enhances muscle regeneration after experimental envenomation by B. jararacussu: a future for nitric oxide-based therapy?

We investigated whether muscle fiber regeneration would be rescued by exogenous administration of l-arginine, the precursor of endogenous synthesis of nitric oxide. The right tibialis anterioris muscle of adult mice (n=20) was injected with 80 microg of venom. One group of mice (n=10) received drinking water containing l-arginine (3.75 mg/ml) and another group (n=10) did not receive any pharmacological treatment. Two months later, muscle regeneration was evaluated by counting the total number of muscle fibers. We found that in l-arginine-treated mice, muscle regeneration was significantly higher (p<0.05) than in saline-treated (2.230+/-478 muscle fibers versus 1.005+/-134, respectively) although the level of muscle fiber population of uninjured tibialis anterioris muscle (3.121+/-102) was not attained. These results show that muscle regeneration was significantly facilitated by l-arginine and suggest that pharmacological activators of the NO pathway may be potentially useful for improving muscle regeneration in human envenomation by B. jararacussu.     

The Role of nitric oxide in convulsions induced by lindane in rats

Lindane is an organochloride pesticide and scabicide. It evokes convulsions mainly trough the blockage of GABA_A receptors. Nitric oxide (NO), gaseous neurotransmitter, has contradictor role in epileptogenesis due to opposite effects of l-arginine, precursor of NO syntheses (NOS), and L-NAME (NOS inhibitor) observed in different epilepsy models. The aim of the current study was to determine the effects of NO on the behavioral and EEG characteristics of lindane-induced epilepsy in male Wistar albino rats.The administration of l-arginine (600, 800 and 1000 mg/kg, i.p.) in dose-dependent manner significantly increased convulsion incidence and severity and shortened latency time to first convulsion elicited by lower lindane dose (4 mg/kg, i.p.). On the contrary, pretreatment with L-NAME (500, 700 and 900 mg/kg, i.p.) decreased convulsion incidence and severity and prolonged latency time to convulsion following injection with a convulsive dose of lindane (8 mg/kg, i.p.). EEG analyses showed increase of number and duration of ictal periods in EEG of rats receiving l-arginine prior to lindane and decrease of this number in rats pretreated with L-NAME.These results support the conclusion that NO plays a role of endogenous convulsant in rat model of lindane seizures.     

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.     

Neuroprotectant effects of iso-osmolar D-mannitol to prevent Pacific ciguatoxin-1 induced alterations in neuronal excitability: a comparison with other osmotic agents and free radical scavengers

The basis for the neuroprotectant effect of D-mannitol in reducing the sensory neurological disturbances seen in ciguatera poisoning, is unclear. Pacific ciguatoxin-1 (P-CTX-1), at a concentration 10 nM, caused a statistically significant swelling of rat sensory dorsal root ganglia (DRG) neurons that was reversed by hyperosmolar 50 mM D-mannitol. However, using electron paramagnetic resonance (EPR) spectroscopy, it was found that P-CTX-1 failed to generate hydroxyl free radicals at concentrations of toxin that caused profound effects on neuronal excitability. Whole-cell patch-clamp recordings from DRG neurons revealed that both hyper- and iso-osmolar 50 mM D-mannitol prevented the membrane depolarisation and repetitive firing of action potentials induced by P-CTX-1. In addition, both hyper- and iso-osmolar 50 mM D-mannitol prevented the hyperpolarising shift in steady-state inactivation and the rise in leakage current through tetrodotoxin (TTX)-sensitive Na(v) channels, as well as the increased rate of recovery from inactivation of TTX-resistant Na(v) channels induced by P-CTX-1. D-Mannitol also reduced, but did not prevent, the inhibition of peak TTX-sensitive and TTX-resistant I(Na) amplitude by P-CTX-1. Additional experiments using hyper- and iso-osmolar D-sorbitol, hyperosmolar sucrose and the free radical scavenging agents Trolox and L-ascorbic acid showed that these agents, unlike D-mannitol, failed to prevent the effects of P-CTX-1 on spike electrogenesis and Na(v) channel gating. These selective actions of D-mannitol indicate that it does not act purely as an osmotic agent to reduce swelling of nerves, but involves a more complex action dependent on the Na(v) channel subtype, possibly to alter or reduce toxin association.