Lack of correlation between the central anti-nociceptive and peripheral anti-inflammatory effects of selective COX-2 inhibitor parecoxib.

Non-steroidal anti-inflammatory drugs (NSAIDs) are thought to exert their pharmacological actions by a common mechanism: inhibition of cyclooxygenase (COX)-mediated prostanoid synthesis. Yet, differences and dissociation between their analgesic and anti-inflammatory effects have not been related to this enzymatic mechanism but mainly to pharmacokinetic factors. Thus, we have compared the effects of an equieffective anti-inflammatory dose (6 mg/kg i.p.) of two NSAIDs with comparable spinal pharmacokinetics, ketoprofen (moderately preferential for COX-1) and parecoxib (selective COX-2), on the activation of spinal nociceptive neurons (measured as c-Fos expression) induced by carrageenan-induced acute inflammation in the rat paw. Both NSAIDs showed a similar anti-inflammatory effect when administered after carrageenan injection (post-treatment). Post-treatment with ketoprofen produced inhibition of c-Fos but parecoxib did not have any significant effect. In addition, parecoxib anti-inflammatory effect was greater than that of ketoprofen, when administered before carrageenan injection (pre-treatment). Paradoxically, pre-treatment with ketoprofen produced a greater inhibition of c-Fos expression than with parecoxib, in all lamina of ipsilateral dorsal horn of the lumbar spinal cord. This suggests that NSAIDs therapeutic profile is related to their selectivity for COX isoforms and COX-2 is involved in the initiation but not in the maintenance of nociceptive spinal activation, which depends on COX-1.

Antinociceptive activity of Syzygium jambos leaves extract on rats.

Syzygium jambos (L.) Alston (Myrtaceae) (syn Eugenia jambos) is a widespread medicinal plant traditionally used in sub-Saharan Africa to treat several diseases. The analgesic potential of leaf hydro-alcoholic extracts was assessed in rats. Hot plate and formalin tests were used to estimate cutaneous nociception whereas measurements of forelimb grip force were done to assess muscular nociception under normal and inflammatory conditions. In the hot plate test, Syzygium jambos extract produced a significant increase in the withdrawal response latencies in a dose-dependant manner (10-300 mg/kg i.p.) and with a maximal effect (analgesic efficacy) similar to that of morphine. The extract (100-300 mg/kg i.p.) significantly reduced pain scores in all the phases of the formalin test with an analgesic efficacy higher than that shown by diclofenac. Although the extract (300 mg/kg) did not alter grip force in intact rats, it reversed the reduction in grip force induced by bilateral injection carrageenan in the forelimb triceps. This analgesic effect of the extract on muscle hyperalgesia was not antagonized, but enhanced, by naloxone. Thus, the Syzygium jambos extract has remarkable analgesic effects on both cutaneous and deep muscle pain that is not mediated by opioid receptors.

Perfil dopaminérgico del compuesto 2-aminoindano N-aralquil sustituido / Profile dopaminergic of the compound 2-aminoindane substituted N-araquil

El sistema dopaminergico central está implicado en las diversas etiologías que involucran a patologías neuropsiquiátricas, tales como la enfermedad de Parkinson, la depresión y la esquizofrenia. Son numerosas las drogas dopaminérgicas utilizadas en el tratamiento de esas dolencias, sin embargo estas terapias causan serios efectos adversos. En este contexto, la génesis de nuevos y más eficientes agentes dopaminergicos, representa un vasto campo de investigación. En el presente trabajo se sintetizó el compuesto 3, concebido como un ligando dopaminérgico, y se evaluó el perfil de su acción dopaminérgica mediante administración central del compuesto y la determinanción de parámetros conductuales como el comportameinto estereotipado (roer) y la medición de la respuesta renal en ratas. Los resultados de la evaluación farmacológica muestran que el compuesto 3 bloquea significativamente la estereotipia inducida por apomorfina, e inhibe la diuresis y natriuresis inducida por la administración central de dopamina. Estos hallazgos sugieren que el compuesto 3 se comporta como un antagonista dopaminérgico, frente a la respuesta tanto conductuales como renales

Increased perception of post-ischemic paresthesias in depressed subjects.

A psychophysical assessment of sensory activity linked to unmyelinated and myelinated primary afferents was conducted by estimating the intensity of thermal and tactile post-ischemic paresthesias in 11 nontreated depressed subjects (Zung's index > or =50) and 19 controls. Blood flow in the dominant forearm was arrested until ischemic pain tolerance was reached. Ischemic pain and post-ischemic paresthesias were numerically rated. The duration of blood flow occlusion to the time of ischemic pain tolerance was similar in both groups. Thermal (warm/cool) and tactile (tingling) paresthesias were 96% and 57% more intense in depressed than in control subjects, respectively. Zung's depression scores were positively correlated with the tingling and thermal paresthesias. Ischemic pain intensity correlated positively with thermal paresthesias. These findings suggest that depression is associated with enhanced sensory paresthesias that are known to be predominately linked to unmyelinated afferent activity.

Substance P and calcitonin gene-related peptide increase IL-1 beta, IL-6 and TNF alpha secretion from human peripheral blood mononuclear cells.

We found that substance P (SP) and calcitonin gene-related peptide (CGRP) (0.3-1 microM) increased, in a concentration-dependent manner, the basal secretion of interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF alpha) from cultured lymphocyte-enriched mononuclear cells isolated from human peripheral blood. SP and CGRP (0.1 microM) synergistically increased basal TNF alpha secretion. Dynorphin A((1-17)) (0.1-1 microM) did not modify basal cytokine secretion. Lipopolysaccharide (10 ng/ml)-induced cytokine secretion and [(3)H]thymidine uptake were not altered by any neuropeptide (at 0.1 microM). Thus, SP and CGRP stimulate the production of pro-inflammatory cytokines from lymphocytes only at high concentrations, similar to those reached during tissue damage.

Long-lasting delayed hyperalgesia after subchronic swim stress.

Rats subjected to an inescapable subchronic stress, consisting of 10-20 min of forced swimming for 3 days, showed a thermal hyperalgesia and an enhanced nociceptive behavior to the subcutaneous administration of formalin 24 and 48 h, respectively, after the last swim session. Hyperalgesia to thermal and chemical stimulants was still present 8 and 9 days after the last swim session, respectively. Chemical, but not thermal, nociception was negatively correlated with the swim effort or struggle times during the last swim session. The serotonin-selective reuptake inhibitors clomipramine (2.5 mg/kg/day, i.p., started 3 or 7 days before stress) and fluoxetine (0.25 mg/kg/day, i.p., started 7 days before stress), or serotonin precursor tryptophan (3 mg/kg/day, i.p., 24 h before each swim stress) blocked the development of both the thermal and the chemical hyperalgesia and increased swim effort times compared to vehicle-treated rats. These treatments did not affect nociceptive responses in control rats subjected to sham swimming. These findings suggest that repeated stress can produce a long-lasting increase in pain sensitivity to both phasic or tonic noxious stimuli by diminishing central serotonin activity. This model may help elucidate the underlying neural mechanisms that mediate the effects of repeated stress on pain sensitivity and affective states.

Opposite modulation of capsaicin-evoked substance P release by glutamate receptors.

Substance P and glutamate are present in primary afferent C-fibers and play important roles in persistent inflammatory and neuropathic pain. In the present study, we have examined whether activation of different glutamate receptor subtypes modulates the release of substance P evoked by the C-fiber selective stimulant capsaicin (1 microM) from rat trigeminal nucleus slices. The selective NMDA glutamate receptor agonist L-CCG-IV (1-10 microM) enhanced capsaicin-evoked substance P release about 100%. This facilitatory effect was blocked by 0.3 microM MK-801, a selective NMDA receptor antagonist. The metabotropic glutamate receptor agonists L-AP4 (group III) and DHPG (group I) (30-100 microM) inhibited capsaicin-evoked substance P release by approximately 60%. These inhibitory effects were blocked by the selective metabotropic glutamate receptor antagonist (+/-)-MCPG (5 microM). On the other hand, AMPA and kainate (0.1-10 microM), did not significantly affect capsaicin-evoked substance P release. Thus, substance P release from non-myelinated primary afferents, and possibly nociception, may be under the functional antagonistic control of some metabotropic and ionotropic glutamate receptor subtypes.

Multiphasic morphine modulation of substance P release from capsaicin-sensitive primary afferent fibers.

Morphine produces a multiphasic modulation of K+-evoked substance P release from trigeminal slices and dorsal root ganglion neurons in culture. We now found that the C-fiber stimulant, capsaicin (1 microM), evoked release of substance P that was inhibited, enhanced and inhibited by 0.1 nM, 1 microM, and 10 microM morphine, respectively. This morphine's multiphasic effect was blocked by naloxone (100 nM). Neonatal treatment with capsaicin produced thermal hypoalgesia and abolished the multiphasic effect of morphine on substance P release evoked by 50 mM K+. These findings suggest that the multiphasic modulation of substance P release by morphine is dependent on C-type afferents and may be of relevance to nociception.

Dynorphin A increases substance P release from trigeminal primary afferent C-fibers.

Dynorphin A-(1-17) has been found to produce spinal antianalgesia and allodynia. Thus, we studied whether dynorphin A-(1-17) modulates substance P release evoked by the C-fiber-selective stimulant capsaicin (1 microM) from trigeminal nucleus caudalis slices. Very low concentrations of dynorphin A-(1-17) (0.01-0.1 nM) strongly facilitated capsaicin-evoked substance P release. This dynorphin A-(1-17) effect was not blocked by the opioid receptor antagonists naloxone (100 nM), beta-funaltrexamine (20 nM), naloxonazine (1 nM), nor-binaltorphimine (3 nM) and ICI 174,864 (N,N-dialyl-Tyr-Aib-Phe-Leu; 0.3 microM). Yet, the effect of dynorphin A-(1-17) was blocked by the NMDA receptor antagonist MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5-10-imine maleate; 0.3 microM). Neonatal treatment with capsaicin (50 mg/kg s.c.), which destroys substance P-containing primary afferents, abolished the excitatory effect of dynorphin A-(1-17) on K+-evoked substance P release. In conclusion, dynorphin A-(1-17) increases substance P release from C-fibers by the activation of NMDA receptors which supports the involvement of presynaptic mechanisms in dynorphin-induced antianalgesia and allodynia.

Reduced tolerance and cardiovascular response to ischemic pain in minor depression.

BACKGROUND: A paradoxical association between a higher prevalence of clinical pain and a reduced sensitivity to brief experimental pain seems to exist during depression. METHODS: We assessed the responses to sustained ischemic pain produced by a maximal effort tourniquet procedure in 32 controls and 11 individuals with minor depression (Zung autoscale > or =50). Stethoscopic blood pressures and heart rates were monitored throughout the procedure. RESULTS: Measures of pain threshold, and measures of pain intensity and pain unpleasantness during the ischemic procedure were similar in depressed and control subjects. Yet, the overall numerical ratings of ischemic pain during the procedure was 28% higher and pain tolerance was 44% lower in depressed compared to control subjects. Clinical pain complaints were reported by 91% of depressed but only by 41% of control subjects (P = 0.01). Sustained ischemic pain induced significant elevations of systolic and mean arterial blood pressures in controls but not in depressed subjects. LIMITATIONS: The main limitation of the present study was the preponderance of females in the depressed group. Yet, we did not find significant gender differences in the sensory-affective and autonomic responses to ischemic pain in our sample. CONCLUSIONS: These findings suggest alterations in the sensory and autonomic nervous systems during minor depression.

Structural model of Dex protein from Penicillium minioluteum and its implications in the mechanism of catalysis.

The DEX gene encodes an extracellular dextranase (EC 3.2.1.11); this enzyme hydrolyzes the alpha(1,6) glucosidic bond contained in dextran to release small isomaltosaccharides. Sequence analysis has revealed only one homologous sequence, CB-8 protein, from Arthrobacter sp., with 30% sequence identity. The secondary structure prediction for Dex was corroborated by circular dichroism measurements. To explore the possibility that Dex protein might adopt a fold similar to any known structure, we conducted a threading search of a three-dimensional structure database. This search revealed that the Dex sequence is compatible with the galactose oxidase/methanol dehydrogenase/sialidase fold. A structural model of Dex based on these results is physically and biologically plausible and leads to testable predictions, including the prediction that Asp246 and Glu299 might be catalytic residues. Also, according to this model the Dex enzyme has a mechanism of hydrolysis with net inversion of anomeric configuration.

Motor activity and quantitative autoradiographic analysis of muscarinic receptors in the brain of rats subjected to the forced swimming test.

A cholinergic dysfunction has been involved in the neurobiological mechanisms of stress and depression. In the present study, we determined the autoradiographic distribution of muscarinic cholinergic receptors in the brain of rats subjected to the forced swimming test for 15 days. Motor activity was automatically analyzed daily before swimming. In the forced swimming test group, both total horizontal activity and ambulatory movements exhibited a significant decrease, when the data from 1st and 15th day were compared. Neither the affinity of [3H]-quinuclidinyl benzilate nor the maximal number of receptors were affected by the forced swimming test in the caudate-putamen, cortex, and hippocampus. The distribution of [3H]-quinuclidinyl benzilate binding sites did not show significant differences in the 30 analyzed areas. Further analysis of muscarinic receptor subtypes after forced swimming test would be necessary to discard any cholinergic involvement.

Alterations of excitatory amino acid receptors in the brain of manganese-treated mice.

An excessive activation of excitatory amino acid (EAA) receptors has been associated with oxidative stress, which is considered the primary cause of manganese (Mn) poisoning neurotoxicity. Therefore, the EAA receptor distribution was analyzed by autoradiographic methods in several brain regions during Mn intoxication. We found that chronic treatment of mice with MnCl2 during 8 wk significantly alters the L-[3H]glutamate (L-[3H]Glu) binding to total glutamate (Glu) receptors, as well as to N-methyl-D-aspartate (NMDA) and quisqualate (QA) receptor subtypes. A generalized decrease of 16-24% of the L-[3H]Glu binding to total Glu receptors was found in all cortex, hippocampus, basal ganglia (except globus pallidus), and cerebellum. Saturation studies showed a significant reduction of the maximal number of receptors (Bmax) in Mn-treated mice, whereas the affinity (Kd) was not altered. L-[3H]Glu binding to NMDA sites was mainly decreased (10-21%) in a few cortical regions, basal ganglia (except globus pallidus), and hippocampus, whereas binding to QA receptor subtype was diminished (16-30%) in cortex, hippocampus, and cerebellum. The decrease of Glu receptor binding sites during Mn poisoning could reflect a receptor downregulation more than neuronal loss, since these reductions are moderate and diffuse. Thus, this down-regulation might mean a protection mechanism against an excitotoxic process associated with Mn toxicity.

Manganese poisoning reduces strychnine-insensitive glycine binding sites in the globus pallidus of the mouse brain.

Manganese (Mn) poisoning is characterized by central nervous system manifestations, including psychiatric disturbances and extrapyramidal disorders. This metal is thought to produce neuronal degeneration due to cytotoxic products originated by oxidative stress and through an indirect excitotoxic process. In previous studies, we have found a reduction in the density of N-methyl-D-aspartate (NMDA) recognition sites in some brain areas of Mn-treated mice. Due to the close relationship between NMDA sites and strychnine-insensitive glycine (Gly) modulatory sites in the NMDA receptor complex, the [3H]-glycine ([3H]-Gly) binding was analyzed by autoradiographic methods in the brain of mice treated with manganese chloride for 8 weeks. Among all analyzed areas, only the globus pallidus showed a significant reduction in [3H]-Gly binding (27-28%). The Gly binding decrease, focalized in the globus pallidus, could reflect a degeneration of structures containing strychnine-insensitive Gly receptors, since this area is the most frequently reported damaged brain region in Mn intoxication. However, it might also be due to a Gly receptor down-regulation to control NMDA complex activation during Mn poisoning.

Cloning and sequencing of a dextranase-encoding cDNA from Penicillium minioluteum.

A cDNA from Penicillium minioluteum HI-4 encoding a dextranase (1,6-alpha-glucan hydrolase, EC 3.2.1.11) was isolated and characterized. cDNA clones corresponding to genes expressed in dextran-induced cultures were identified by differential hybridization. Southern hybridization and restriction mapping analysis of selected clones revealed four different groups of cDNAs. The dextranase cDNA was identified after expressing a cDNA fragment from each of the isolated groups of cDNA clones in the Escherichia coli T7 system. The expression of a 2 kb cDNA fragment in E. coli led to the production of a 67 kDa protein which was recognized by an anti-dextranase polyclonal antibody. The cDNA contains 2109 bp plus a poly(A) tail, coding for a protein of 608 amino acids, including 20 N-terminal amino acid residues which might correspond to a signal peptide. There was 29% sequence identity between the P. minioluteum dextranase and the dextranase from Arthrobacter sp. CB-8.

Cloning of the Penicillium minioluteum gene encoding dextranase and its expression in Pichia pastoris.

The DEX gene encoding an extracellular dextranase was isolated from the genomic DNA library of Penicillium minioluteum by hybridization using the dextranase cDNA as a probe. Comparison of the gene and cDNA sequences revealed that the DEX gene does not contain introns. Amino acid sequences comparison of P. minioluteum dextranase with other reported dextranases reveals a significant homology (29% identity) with a dextranase from Arthrobacter sp. CB-8. The DEX gene fragment encoding a mature protein of 574 amino acids was expressed in the methylotrophic yeast Pichia pastoris by using the SUC2 gene signal sequence from Saccharomyces cerevisiae under control of the alcohol oxidase-1 (AOX1) promoter. Over 3.2 g/l of enzymatically active dextranase was secreted into the medium after induction by methanol. The yeast product was indistinguishable from the native enzyme in specific activity and the N-terminus of both proteins were identical.

Invertase secretion in Hansenula polymorpha under the AOX1 promoter from Pichia pastoris.

A DNA fragment containing a transcription regulating region of the alcohol oxidase (AOX1) gene from the methylotrophic yeast Pichia pastoris was used in the construction of a vector for the expression of heterologous proteins in the methylotrophic yeast Hansenula polymorpha. We used this vector to clone the SUC2 gene from Saccharomyces cerevisiae into H. polymorpha yeast. The culture conditions for invertase production using a fed-batch culture were studied. More than 1.5 x 10(3) U/ml of biologically active invertase (1 g/l) were secreted to the cellular periplasmic space. The fermentative process was scaled up to 50 l. Invertase produced from H. polymorpha was glycosylated, but it contained significantly less carbohydrate than protein produced by S. cerevisiae. Using the Western-blot technique, it was observed that invertase secreted from H. polymorpha and invertase secreted from S. cerevisiae showed common antigenic determinants.

Morphine produces a biphasic modulation of substance P release from cultured dorsal root ganglion neurons.

We have previously reported that morphine produces a concentration-dependent multiphasic modulation (inhibitions and facilitations) of substance P (SP) release from trigeminal nucleus caudalis slices by activation of distinct populations of mu-, delta- and kappa-opioid receptors. In the present study, we have examined a wide range of morphine concentrations on K(+)-evoked SP release from dissociated rat dorsal root ganglion (DRG) neurons in culture. SP immunoreactivity was measured in the release buffer. Morphine produced a biphasic effect on K(+)-evoked SP release without affecting basal release. A concentration of 30 nM morphine facilitated SP release while a concentration of 1 microM suppressed release. Higher concentrations of morphine (10-30 microM) did not alter SP release. The facilitatory effect evoked by 30 nM morphine was abolished by opioid-receptor blockade with naloxone (30 nM) and the inhibitory effect produced by 1 microM morphine tended to be reversed. We conclude that an intact neuronal circuitry is not required for morphine to produce an opioid receptor mediated biphasic modulation of SP released from unmyelinated primary afferents. It is plausible that the dose-dependent biphasic effects of opioid agonists may also produce biphasic effects on nociception.

Autoreceptor presynaptic control of dopamine release from striatum is lost at early stages of manganese poisoning.

Manganese (Mn) poisoning in man produces an early psychotic disorder that is later followed by a Parkinson-like syndrome. Since alterations in the brain DA system are thought to be involved, we assessed the presynaptic autoreceptor regulation of K(+)-evoked 3H-DA release from superfused striatal slices of mice treated i.p. with 5 mg Mn/kg weight/day for 2 and 8 weeks. Mn poisoning did not change basal and evoked DA release. In controls, 1 microM apomorphine (APO), a D2-like DA receptor agonist, produced an inhibition of K(+)-evoked 3H-DA release that was blocked by the D2-like DA receptor antagonist, S(-)-sulpiride (1 microM). Yet, APO lost its capacity to inhibit the K(+)-evoked 3H-DA release after 2 weeks of Mn poisoning. After 8 weeks of Mn poisoning, APO was again able to reduce K(+)-evoked 3H-DA release. MK-801 (0.3 microM), a NMDA-glutamate receptor antagonist, could restore APO inhibitory control on DA release lost at week 2 of Mn poisoning. These findings suggest a NMDA-glutamate-receptor-mediated loss of autoreceptor presynaptic control of striatal DA release at early Mn poisoning.

[Spontaneous motor activity in mice overloaded with iron-dextran]. / Actividad motora espontánea en ratones sobrecargados con hierro-dextrano.

We studied the spontaneous motor activity and the levels of iron in brain (frontal cortex and striatum) and liver of mice overloaded with iron dextran. Two groups of mice injected intraperitoneally with two different doses of iron and the control group were used. We found a significant increase in the spontaneous motor activity only for the experimental group treated with the highest dose of iron. In frontal cortex and striatum the concentration of iron did not augment in the experimental groups. However, in liver we detected a highly significant rise in the iron content of both experimental groups. Our results suggest that the increase in the spontaneous motor activity observed in mice injected with the highest dose is not directly caused by changes in the iron concentration in the brain.