Effect of salicylates on histamine and L-histidine metabolism. Inhibition of imidazoleacetate phosphoribosyl transferase.

In man and other animals, urinary excretion of the histidine and histamine metabolite, imidazoleacetate, is increased and that of its conjugated metabolite, ribosylimidazoleacetate, decreased by salicylates. Imidazoleacetate has been reported to produce analgesia and narcosis. Its accumulation as a result of transferase inhibition could play a part in the therapeutic effects of salicylates. To determine the locus of salicylate action, we have investigated the effect of anti-inflammatory drugs on imidazoleacetate phosphoribosyl transferase, the enzyme that catalyzes the ATP-dependent conjugation of imidazoleacetate with phosphoribosylpyrophosphate. As little as 0.2 mM aspirin produced 50% inhibition of the rat liver transferase. In vivo, a 30% decrease in the urinary excretion of ribosylimidazoleacetate has been observed with plasma salicylate concentrations of 0.4 mM. The enzyme was also inhibited by sodium salicylate but not by salicylamide, sodium gentisate, aminopyrine, phenacetin, phenylbutazone, or indomethacin. The last four drugs have been shown previously not to alter the excretion of ribosylimidazoleacetate when administered in vivo. Since both the drug specificity and inhibitory concentrations are similar in vivo and in vitro, it seems probable that the effect of salicylates on imidazoleacetate conjugation results from inhibition of imidazoleacetate phosphoribosyl transferase.

Direct inhibition of the N-methyl-D-aspartate receptor channel by dopamine and (+)-SKF38393.

1. Dopamine is known to modulate glutamatergic synaptic transmission in the retina and in several brain regions by activating specific G-protein-coupled receptors. We have examined the possibility of a different type of mechanism for this modulation, one involving direct interaction of dopamine with ionotropic glutamate receptors. 2. Ionic currents induced by fast application of N-methyl-D-aspartate (NMDA) were recorded under whole-cell patch-clamp in cultured striatal, thalamic and hippocampal neurons of the rat and in retinal neurons of the chick. Dopamine at concentrations above 100 microM inhibited the NMDA response in all four neuron types, exhibiting an IC50 of 1.2 mM in hippocampal neurons. The time course of this inhibition was fast, developing in less than 100 ms. 3. The D1 receptor agonist (+)-SKF38393 mimicked the effect of dopamine, with an IC50 of 58.9 microM on the NMDA response, while the enantiomer (-)-SKF38393 was ineffective at 50 microM. However, the D1 antagonist R(+)-SCH23390 did not prevent the inhibitory effect of (+)-SKF38393. 4. The degree of inhibition by dopamine and (+)-SKF38393 depended on transmembrane voltage, increasing 2.7 times with a hyperpolarization of about 80 mV. The voltage-dependent block by dopamine was also observed in the presence of MgCl2 1 mM. 5. Single-channel recordings showed that the open times of NMDA-gated channels were shortened by (+)-SKF38393. 6. These data suggested that the site to which the drugs bound to produce the inhibitory effect was distinct from the classical D1-type dopamine receptor sites, possibly being located inside the NMDA channel pore. It is concluded that dopamine and (+)-SKF38393 are NMDA channel ligands.

Excitatory amino acid receptors mediate the glutamate-induced release of GABA synthesized from putrescine in cultured cells of embryonic avian retina.

Cultured retina cells from chick embryos took up [3H]putrescine and approx 10.8% of the incorporated amine was converted into [3H]GABA. The putrescine-derived GABA accumulated in a pool that was released in the medium at a rate corresponding to 3.66% of the total [3H]GABA in the cell at incubation intervals of 12 min. Treatment of cultures with L-glutamate (500 microM) promoted a 5-7 fold increase in the rate of [3H]GABA efflux which was totally independent on the presence of calcium ions in the superfusing medium. (+)-5-Methyl-10,11-dihydro-5h-Dibenzo(A,D)cyclohepten-5,10- Iminihydrogenmaleate (MK 801) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 100 microM, inhibited the glutamate evoked release of GABA by 78 and 73% respectively. N-methyl-D-aspartate (NMDA, 100 microM), elicited the release of putrescine-derived GABA only when magnesium ions were removed from the superfusing medium with 2 mM EGTA. In the presence of 1 mM MgCl2, NMDA was totally ineffective in inducing the release. As for glutamate, AMPA (R,S)-alpha-Amino-3-hydroxy-5-methyllisoxazole-4-propionicacid+ ++ hydrobromide (100 microM) also induced the release of GABA synthesized from putrescine. Our data show that putrescine is an important source of GABA in the embryonic CNS and that GABA synthesized from putrescine can be released in the extracellular space when cells are stimulated by L-glutamate through the activation of excitatory amino acid (EAA) receptors.

Aspartate as a selective NMDA receptor agonist in cultured cells from the avian retina.

Although glutamate is considered the natural neurotransmitter that mediates excitatory function in the CNS, other active natural compounds can also drive the functional activation of excitatory amino acid receptors (EAAR). L-aspartate is the most likely neurotransmitter to mimic the actions of glutamate. Here we show that L-aspartate promotes the release of GABA acting selectively on the NMDA receptor subtype. Retina cell cultures, when exposed to excitatory amino acids (EAA), release [3H] GABA previously incorporated by the cells. Both L-glutamate and L- and D-aspartate at 100 microM concentration, promote the release which can be mimicked by kainate and NMDA. While aspartate-induced release of [3H] GABA occurs in the presence of 1 mM Mg2+, NMDA (100 microM) promotes the release only when Mg2+ is omitted from the superfusing medium. However, in the absence of Mg2+ the efficacy of 1- and d-aspartate (100 microM) to activate [3H] GABA release increases by a factor of 2 when compared to the release observed in the presence of 1 mM Mg2+. NMDA and aspartate induced release of [3H] GABA is completely inhibited by 10 microM MK-801 and is not affected by CNQX (100 microM). In the presence of Mg2+, aspartate-induced release of [3H] GABA is also completely inhibited by MK-801 (10 microM) and is not significantly affected by CNQX (100 microM). The [3H] GABA release induced by kainate (100 microM) is fully inhibited by CNQX (100 microM) and is not affected by MK-801 (10 microM). Our results indicate that in the retina, l-aspartate modulates its excitatory function on a set of GABAergic cells via the selective activation of NMDA receptors. The fact that L- and D-aspartate (but not D-glutamate) induce the release of GABA even in the presence of Mg2+ suggests that the electrogenic uptake of aspartate is required to lower the affinity of the NMDA channel for Mg2+. The observation that D-glutamate (200 microM), which is not taken up by the cells, activates the efflux of GABA only when Mg2+ is omitted from the incubating medium, supports this possibility.

Transient coupling of NMDA receptor with ip3 production in cultured cells of the avian retina.

The mobilization of inositol triphosphate ip3 by N-methyl D-aspartate (NMDA) and kainate, two excitatory amino acid EAA receptor agonists, was studied in cultured chick retina cells as a function of culture differentiation. Kainate (EC50 = 30 microM) stimulated from 6 to 9-fold the production of [3H]ip3 between E8C3 (embryonic day 8 plus 3 days in vitro) and E8C13. The kainate response was blocked by CNQX (100 microM) by more than 80% until stage E8C9. MK-801, however, was totally ineffective in preventing the kainate induced ip3 generation. [3H]ip3 production evoked by NMDA was increased 4-fold above basal levels at E8C3. As cultures differentiated, [3H]ip3 production promoted by NMDA decreased to 2.5-fold at E8C6 to 1.6-fold the basal levels in cultures at later stages of differentiation. The removal of Mg2+ from the incubating medium at E8C3 increased the NMDA mediated [3H]ip3 production by 80%. However, at more differentiated stages of the cultures, when cells were not responsive to NMDA, removal of Mg2+ plus the addition of 1 mM glycine did not change the pattern of the response. Although NMDA mediated ip3 production is almost absent in more differentiated cultures, NMDA is able to induce [3H]GABA release in E8C3 and E8C13 cultures with characteristics that reflect typical NMDA receptor activation: it is highly potentiated by the absence of Mg2+ and by the presence of glycine. The NMDA induced production of [3H]ip3 at E8C3 was entirely blocked by MK-801 (100 microM) and APV (100 microM) but not by CNQX.(ABSTRACT TRUNCATED AT 250 WORDS)

Topographical organization of the dopamine-dependent adenylate cyclase of the chick embryo retina.

Retinas obtained from 9- and 16-day-old chick embryos and 5-day-old post-hatched chicken were analyzed with regard to the topographical distribution of the dopamine-stimulated adenylate cyclase system. The retinas were sectioned into 4 quadrants, namely ventroanterior, dorsoanterior, dorsoposterior and ventroposterior, taking the beak and the choroid fissure as references. Dopamine (0.1 mM) elicited the accumulation of cAMP in all 4 portions of tissue studied. However, when the ratio between the dopamine-stimulated vs the basal level of cAMP of 9-day-old embryo retina were compared in the different tissue portions, we observed that in the dorsoposterior quadrant the ratio was 15.5 while in the ventroanterior quadrant the ratio was approximately 6. The other two portions showed intermediate values. The same pattern was observed in retinas from 16-day-old embryos. However, the ratio between the dopamine-stimulated vs the basal cAMP levels were 4.6 and 3 in the dorsoposterior and ventroanterior quadrants respectively. Further, the retina responsiveness to dopamine from post-hatched chicken was evenly distributed in the tissue, showing a stimulated/basal cAMP ratio of approximately 2 in all 4 quadrants studied. Our results show that the dopamine-dependent cAMP accumulation of the chick retina is unevenly distributed in the embryonic tissue and tends to homogeneity as the tissue differentiates.

L-glutamate evoked release of GABA from cultured avian retina cells does not require glutamate receptor activation.

gamma-Aminobutyric acid (GABA) and L-glutamate are the major inhibitory and excitatory transmitters in the central nervous system. Recent evidence has indicated that L-glutamate may stimulate GABA release by a novel exchange mechanism (Nascimento and De Mello, J. Neurochem., 1985, 45: 1820-1827). Here we provide strong support for this hypothesis by showing that the L-glutamate-evoked release of [3H]GABA from cultured avian retina cells is not dependent on the activation of excitatory amino acid receptors. Retina cells were found to incorporate [3H]GABA into a pool that was released when cultures were treated with L-glutamate (100 microM). This release was unaffected when calcium ions were removed, but was prevented when NaCl was replaced by LiCl. D-Aspartate, which in tracer experiments was shown to be taken into cells by the same carrier as L-glutamate, was also able to evoke release of [3H]GABA, with the same requirement for NaCl. In addition, L-glutamate and D-aspartate uptake by retina cells was inhibited in more then 80% when the uptake was measured in the presence of LiCl. As opposed to GABA, the release of acetylcholine (ACh) promoted by L-glutamate showed characteristics of classical mechanisms of neurotransmitter release. Glutamate-induced efflux of ACh was Ca2+-dependent and was not affected when NaCl was replaced by LiCl. Also, D-aspartate was ineffective in eliciting the release of ACh. Even at high concentrations, antagonists of excitatory amino acid receptors were unable to diminish the glutamate-evoked release of [3H]GABA.(ABSTRACT TRUNCATED AT 250 WORDS)

Opposite roles of GABA and excitatory amino acids on the control of GAD expression in cultured retina cells.

The mechanism of control of GAD expression by GABA and excitatory amino acids (EAAs) was studied in chick and rat retina cultures using immunohistochemical and PAGE-immunoblot detection of the enzyme, as well as by measuring enzyme activity. Aggregate cultures were prepared with retina cells obtained from chick embryos at embryonic days 8-9 (E8-E9). Organotypical cultures were also prepared with retinas from E14 chick embryos, post-hatched chicken and P21 rats. GABA (1-20 mM) fully prevented GAD expression in aggregate and organotypical cultures from chick embryo retinas. A substantial, but not complete, reduction of GAD was also observed in organotypical cultures of post-hatched chicken and P21 rats, in which both forms of the enzyme (GAD65 and 67) were affected. The GABA effect was not mimicked by THIP (100 microM), baclofen (100 microM) or CACA (300 microM), agonists of GABAa, b and c receptors, respectively. NNC-711, a potent inhibitor of GABA transporters, reduced by 50% the inhibition of GAD activity promoted by GABA. Aggregates exposed to GABA and treated with glutamate (5 mM) or kainate (100 microM) displayed an intense GAD-like immunoreactivity in many cell bodies, but not in neurite regions. Immunoblot analysis revealed that the increase in GAD-like immunoreactivity by EAA corresponded to a 67-kDa protein. However, GAD activity was not detected. Treatment of aggregates or retina homogenates with SNAP, a NO producing agent (but not its oxidized form), reduced GAD activity by more than 60% indicating that the lack of enzyme activity in GAD-like immunoreactive cells, could be due to NO production by EAA stimulation.

Atypical effect of dopamine in modulating the functional inhibition of NMDA receptors of cultured retina cells.

Cultured retina cells released accumulated [3H]GABA (gamma-aminobutyric acid) when stimulated by L-glutamate, N-methyl-D-aspartate (NMDA) and kainate. In the absence of Mg2+, dopamine at 200 microM (IC50 60 microM), inhibited in more than 50% the release of [3H]GABA induced by L-glutamate and NMDA, but not by kainate. This effect was not blocked by the D1-like dopamine receptor antagonist, R-(+)-7-chloro-8-hydroxy-3-methyl- -phenyl-2,3,4,5-tetrahydro- H-3-benzazepine hydrochloride (SCH 23390), neither by haloperidol nor spiroperidol (dopamine D2-like receptor antagonists). The dopamine D1-like receptor agonist R(+)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,diol hydrochloride (SKF 38393) at 50 microM, but not its enantiomer, also inhibited the release of [3H]GABA induced by NMDA, but not by kainate; an effect that was not prevented by the antagonists mentioned above. (+/-)-6-Chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepin e hydrobromide (SKF 812497) had no effect. Neither 8BrcAMP (5 mM) nor forskolin (10 microM) inhibited the release of [3H]GABA. Our results suggest that dopamine and (+)-SKF 38393 inhibit the glutamate and NMDA-evoked [3H]GABA release through mechanisms that seem not to involve known dopaminergic receptor systems.

Transient expression of an atypical D1-like dopamine receptor system during avian retina differentiation.

Intact cultured retina cells from chick embryos at stage E9C5 (cultures initiated with retinae from 9-day old embryos followed by 5 days in culture), preincubated with 2 nM unlabelled SCH 23390 (R(+)-7- chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride) for 20 to 60 min at 37 degrees C and then washed 5 to 25 times (approximately 1.5 min/wash) with 2 ml SCH 23390-free medium, responded to dopamine with cAMP accumulation that corresponded to 30-50% of the dopamine-promoted cAMP accumulation observed in untreated cells or in cells exposed to the inactive isomer of SCH 23390. Therefore, 50 to 70% of the dopamine response of SCH 23390-pretreated cells was inhibited after extensive washings of the cultures. At E9C12 the fraction of the dopamine response that remained inhibited by SCH 23390 after the washings declined to 30% of the control cultures or the cultures exposed to the SCH 23390 enantiomer. Cultures at stage E9C5 treated with SCH 23390 followed by extensive washings as above and then used for measuring the number of [3H]-SCH 23390 specific binding sites revealed that 60% of the sites did not interact with the tritiated compound when compared to untreated cultures or to cultures preincubated with the inactive isomer of SCH 23390. When E9C12 cultures were subjected to the same experimental protocol less than 10% of D1-like sites did not interact with [3H]-SCH 23390 after the cells had been exposed to the unlabelled compound. Dissociated cells prepared from intact retinae obtained from 12-13-day old embryos also displayed a subpopulation of D1-like sites that interacted irreversibly with SCH 23390 in a stereospecific way. These sites corresponded to 25% of the total number of D1-like sites present in the retina at this developmental stage. In retina cells obtained from one-day old posthatched chicks these sites were no longer detected. These data show that cultured retina cells as well as cells obtained from retina developing in ovo display two populations of D1-like receptors. One interacts irreversibly with SCH 23390 and is present only in the undifferentiated tissue or in cells at the early stages of culture and the other has a lower affinity for SCH 23390 with which its interaction follows reversible kinetics. These sites are present throughout the differentiation stages studied.

Domoic acid induces neurotoxicity and ip3 mobilization in cultured cells of embryonic chick retina.

Domoic acid (DOM), 1 to 50 microM, a glutamate agonist responsible for several neurological effects such as loss of memory and confusion, induced the death of cultured neurons of chick embryonic retina, in a concentration- and Ca(2+)-dependent manner. This effect was blocked by 100 microM CNQX, a competitive antagonist of the non-NMDA receptor, but not by 10 microM MK-801, a non-competitive antagonist of the NMDA receptor. DOM also induced inositol triphosphate (ip3) accumulation 4 to 7 times above basal levels. This effect was also dependent on external Ca2+ and was entirely blocked by 100 microM CNQX, but not by 10 microM MK-801. These results suggest that DOM interaction with non-NMDA glutamate receptors mediates signal transduction with ip3 accumulation and cell death.

Extraoral implants for orbit rehabilitation: a comparison between one-stage and two-stage surgeries.

The aim of the study was to compare the osseointegration success rate and time for delivery of the prosthesis among cases treated by two-stage or one-stage surgery for orbit rehabilitation between 2003 and 2011. Forty-five patients were included, 31 males and 14 females; 22 patients had two-stage surgery and 23 patients had one-stage surgery. A total 138 implants were installed, 42 (30.4%) on previously irradiated bone. The implant survival rate was 96.4%, with a success rate of 99.0% among non-irradiated patients and 90.5% among irradiated patients. Two-stage patients received 74 implants with a survival rate of 94.6% (four implants lost); one-stage surgery patients received 64 implants with a survival rate of 98.4% (one implant lost). The median time interval between implant fixation and delivery of the prosthesis for the two-stage group was 9.6 months and for the one-stage group was 4.0 months (P < 0.001). The one-stage technique proved to be reliable and was associated with few risks and complications; the rate of successful osseointegration was similar to those reported in the literature. The one-stage technique should be considered a viable procedure that shortens the time to final rehabilitation and facilitates appropriate patient follow-up treatment.

Antiproliferative activity of anti-inflammatory drugs in two mammalian cell culture lines.

The nonsteroid anti-inflammatory drugs inhibited cell proliferation when added to rat hepatoma and human fibroblast cultures. The inhibition was reversible; normal growth resumed when the cultures were washed free of drug. Protein and nucleic acid synthesis, as measured by isotope incorporation was also reduced, although this reduction was probably a reflection of the decrease in cell numbers. An exception was that, in low concentration, the salicylate drugs, salicylamide, salicylic acid and aspirin, stimulated protein and nucleic acid synthesis, but in high concentrations (greater than 1 mM) they inhibited culture growth as well as protein and nucleic acid synthesis. Pharmacologically inactive derivatives, such as m-hydroxybenzoic acid and gentisic acid, were not inhibitory in concentrations up to 5 mM. The order of potency in inhibiting culture growth, meclofenamate greater than indomethacin greater than salicylamide greater than phenylbutazone greater than phenacetin greater than aspirin = salicylic acid, was similar to that reported for their anti-inflammator activity and their ability to inhibit prostaglandin synthesis. The antiproliferative activity of these drugs may, in part, account for their anti-inflammatory and toxic actions in vivo.

Regulation of dopamine- and adenosine-dependent adenylate cyclase systems of chicken embryo retina cells in culture.

We have obtained evidence that receptor-stimulated adenylate cyclase activity [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] is regulated physiologically in both embryonic and mature neurons. In a series of experiments using cultured retina cells from chicken embryos, we found that dopamine-sensitive adenylate cyclase activity spontaneously desensitized as cultures differentiated. The cellular response to dopamine reached a maximum after 5 days in culture and then decreased to 40% during the next 5 days. This spontaneous desensitization appeared to be caused by functional dopaminergic transmission because it could be blocked by the dopamine antagonist haloperidol. The ability of added dopamine at 100 microM to cause near-complete desensitization is consistent with this conclusion. Pharmacologically induced desensitization required 31 hr for maximal effect and was half-maximal at 1-10 microM dopamine. Analogous desensitization of the adenosine-dependent adenylate cyclase system also was noted. When dopamine was removed from the medium of chronically treated cultures, cells resensitized to subsequent stimulation at a very slow rate. Resensitization likely depended on replacement of dopamine receptors because chronic dopamine treatment caused the disappearance of binding sites for the ligand [3H]spiroperidol. In a second series of experiments, using hatched animals, we found that similar regulation of dopamine receptor binding sites and activity could be elicited by manipulation of environmental light, a treatment thought to influence dopaminergic transmission. Retinas from animals in constant light had less specific [3H]spiroperidol binding (35 fmol/mg of protein) than did retinas from animals in constant darkness (66 fmol/mg of protein) and made less cAMP in response to added dopamine. Our results indicate that regulation of the dopamine receptor system begins early in development and continues to function in mature synapses.

Neurotransmitter release in aggregate cultures of chick embryo retina cells.

The study of neurotransmitter release using aggregate cell cultures has been limited by the fact that cell aggregates are obtained only when cells are maintained in suspension with rotatory agitation. In this report we describe a simple and easy method that uses aggregate cell cultures to study the release of neurotransmitters. The results demonstrate that this relatively simple technique can be of great value to address the problem of neurotransmitter release and study the mechanisms of action of natural and synthetic compounds on the differentiation of functional synapses in the CNS.

Inhibition of choline acetyltransferase by excitatory amino acids as a possible mechanism for cholinergic dysfunction in the central nervous system.

Choline acetyltransferase (ChAT) activity was reduced by more than 85% in cultured retina cells after 16 h treatment with 150 microM kainate (T(1/2) : 3.5 h). Glutamate, AMPA and quisqualate also inhibited the enzyme in equivalent proportion. Cell lesion measured by lactate dehydrogenase (LDH) release, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide - thiazolyl blue (MTT) reduction and microscopic observation was not detected even after 48 h with kainate. Other retina neurochemical markers were not affected by kainate and full recovery of the enzyme was achieved 9 days after kainate removal. Moreover, hemicolinium-3 sensitive choline uptake and hemicolinium-3 binding sites were maintained intact after kainate treatment. The immunoblot and immunohistochemical analysis of the enzyme revealed that ChAT molecules were maintained in cholinergic neurons. The use of antagonists showed that ionotropic and group 1 metabotropic receptors mediated the effect of glutamate on ChAT inhibition, in a calcium dependent manner. The quisqualate mediated ChAT inhibition and part of the kainate effect (30%) was prevented by 5 mM N(G)-nitro-L-arginine methyl ester (L-NAME). Veratridine (3 microM) also reduced ChAT by a Ca(2+) dependent, but glutamate independent mechanism and was prevented by 1 microM tetrodotoxin.