Corrigendum to "Activation of Melanocortin Receptors MC1 and MC5 Attenuates Retinal Damage in Experimental Diabetic Retinopathy".

[This corrects the article DOI: 10.1155/2016/7368389.].

Activation of Melanocortin Receptors MC 1 and MC 5 Attenuates Retinal Damage in Experimental Diabetic Retinopathy.

We hypothesize that melanocortin receptors (MC) could activate tissue protective circuit in a model of streptozotocin- (STZ-) induced diabetic retinopathy (DR) in mice. At 12-16 weeks after diabetes induction, fluorescein angiography (FAG) revealed an approximate incidence of 80% microvascular changes, typical of DR, in the animals, without signs of vascular leakage. Occludin progressively decreased in the retina of mice developing retinopathy. qPCR of murine retina revealed expression of two MC receptors, Mc1r and Mc5r. The intravitreal injection (5 µL) of the selective MC1 small molecule agonist BMS-470539 (33 µmol) and the MC5 peptidomimetic agonist PG-901 (7.32 nM) elicited significant protection with regular course and caliber of retinal vessels, as quantified at weeks 12 and 16 after diabetes induction. Mouse retina homogenate settings indicated an augmented release of IL-1α, IL-1ß, IL-6, MIP-1α, MIP-2α, MIP-3α, and VEGF from diabetic compared to nondiabetic mice. Application of PG20N or AGRP and MC5 and MC1 antagonist, respectively, augmented the release of cytokines, while the agonists BMS-470539 and PG-901 almost restored normal pattern of these mediators back to nondiabetic values. Similar changes were quantified with respect to Ki-67 staining. Finally, application of MC3-MC4 agonist/antagonists resulted to be inactive with respect to all parameters under assessment.

Urocortin protects chondrocytes from NO-induced apoptosis: a future therapy for osteoarthritis?

Osteoarthritis (OA) is characterized by a loss of joint mobility and pain resulting from progressive destruction and loss of articular cartilage secondary to chondrocyte death and/ or senescence. Certain stimuli including nitric oxide (NO) and the pro-inflammatory cytokine tumor necrosis factor α (TNF-α have been implicated in this chondrocyte death and the subsequent accelerated damage to cartilage. In this study, we demonstrate that a corticotrophin releasing factor (CRF) family peptide, urocortin (Ucn), is produced by a human chondrocyte cell line, C-20/A4, and acts both as an endogenous survival signal and as a cytoprotective agent reducing the induction of apoptosis by NO but not TNF-α when added exogenously. Furthermore, treatment with the NO donor S-nitroso-N-acetyl-D-L-penicillamine upregulates chondrocyte Ucn expression, whereas treatment with TNF-α does not. The chondroprotective effects of Ucn are abolished by both specific ligand depletion (with an anti-Ucn antibody) and by CRF receptor blockade with the pan-CRFR antagonist α-helical CRH(9-41). CRFR expression was confirmed by reverse transcription-PCR with subsequent amplicon sequence analysis and demonstrates that C-20/A4 cells express both CRFR1 and CRFR2, specifically CRFR1α and CRFR2ß. Protein expression of these receptors was confirmed by western blotting. The presence of both Ucn and its receptors in these cells, coupled with the induction of Ucn by NO, suggests the existence of an endogenous autocrine/paracrine chondroprotective mechanism against stimuli inducing chondrocyte apoptosis via the intrinsic/mitochondrial pathway.

Melanocortin peptide therapy for the treatment of arthritic pathologies.

Arthritic pathologies are a major cause of morbidity within the western world, with rheumatoid arthritis affecting approximately 1% of adults. This review highlights the therapeutic potential of naturally occurring hormones and their peptides, in both arthritic models of disease and patients. The arthritides represent a group of closely related pathologies in which cytokines, joint destruction, and leukocytes play a causal role. Here we discuss the role of naturally occurring pro-opiomelanocortin (POMC)-derived melanocortin peptides (e.g., alpha melanocyte stimulating hormone [alpha-MSH]) and synthetic derivatives in these diseases. Melanocortins exhibit their biological efficacy by modulating proinflammatory cytokines and subsequent leukocyte extravasation. Their biological effects are mediated via seven transmembrane G-protein-coupled receptors, of which five have been cloned, identified, and termed MC1 to MC5. Adrenocorticotrophic hormone represents the parent molecule of the melanocortins; the first 13 amino acids of which (termed alpha-MSH) have been shown to be the most pharmacologically active region of the parent hormone. The melanocortin peptides have been shown to display potent anti-inflammatory effects in both animal models of disease and patients. The potential anti-inflammatory role for endogenous peptides in arthritic pathologies is in its infancy. The ability to inhibit leukocyte migration, release of cytokines, and induction of anti-inflammatory proteins appears to play an important role in affording protection in arthritic injury, and thus may lead to potential therapeutic targets.

Annexin 1 and melanocortin peptide therapy for protection against ischaemic-reperfusion damage in the heart.

Cardiovascular disease is a major cause of mortality within the western world affecting 2.7 million British people. This review highlights the beneficial effects of naturally occurring hormones and their peptides, in myocardial ischaemic-injury (MI) models, a disease pathology in which cytokines and neutrophils play a causal role. Here we discuss two distinct classes of endogenous peptides: the steroid inducible annexin 1 and the melanocortin peptides. Annexin 1 and the melanocortins counteract the most important part of the host inflammatory response, namely, the process of leukocyte extravasation, as well as release of proinflammatory mediators. Their biological effects are mediated via the seven transmembrane G-protein-coupled receptors, the fMLP receptor family (or FPR), and the melanocortin receptors, respectively. Pharmacological analysis has demonstrated that the first 24 amino acids of the N-terminus (termed Ac2-26) are the most active region. Both exogenous annexin 1 and its peptides demonstrate cardioprotectiveness and continuing work is required to understand this annexin 1/FPR relationship fully. The melanocortin peptides are derived from a precursor molecule called the POMC protein. These peptides display potent anti-inflammatory effects in human and animal models of disease. In MI, the MC3R has been demonstrated to play an important role in mediating the protective effects of these peptides. The potential anti-inflammatory role for endogenous peptides in cardiac disease is in its infancy. The inhibition of cell migration and release of cytokines and other soluble mediators appears to play an important role in affording protection in ischaemic injury and thus may lead to potential therapeutic targets.

The annexin-1 knockout mouse: what it tells us about the inflammatory response.

The 37kDa protein annexin 1 (Anx-1; lipocortin 1) is a glucocorticoid-regulated protein that has been implicated in the regulation of phagocytosis, cell signalling and proliferation, and postulated to be a mediator of glucocorticoids action in inflammation and in the control of anterior pituitary hormone release. Immuno-neutralisation or antisense strategies support this hypothesis as they can reverse the effect of glucocorticoids in several systems. We recently generated a line of mice lacking the Anx-1 gene noting that some tissues taken from such animals exhibited an increased expression of several proteins including COX-2 and cPLA2. In models of experimental inflammation, Anx-1(-/-) mice exhibit an exaggerated response and a partial or complete resistance to the anti-inflammatory effects of glucocorticoids. Several other anomalies were noted including abnormal leukocyte adhesion molecule expression, an increased spontaneous migratory behaviour of PMN in Anx-1(-/-) mice and a resistance in Anx-1(-/-) macrophages to glucocorticoid inhibition of superoxide generation. This paper reviews these and other data in the light of the development of the 'second messenger' hypothesis of glucocorticoid action.

Involvement of the receptor for formylated peptides in the in vivo anti-migratory actions of annexin 1 and its mimetics.

An innovative avenue for anti-inflammatory therapy is inhibition of neutrophil extravasation by potentiating the action of endogenous anti-inflammatory mediators. The glucocorticoid-inducible protein annexin 1 and derived peptides are effective in inhibiting neutrophil extravasation. Here we tested the hypothesis that an interaction with the receptor for formylated peptide (FPR), so far reported only in vitro, could be the mechanism for this in vivo action. In a model of mouse peritonitis, FPR antagonists abrogated the anti-migratory effects of peptides Ac2-26 and Ac2-12, with a partial reduction in annexin 1 effects. A similar result was obtained in FPR (knock-out) KO mice. Binding of annexin 1 to circulating leukocytes was reduced (>50%) in FPR KO mice. In vitro, annexin binding to peritoneal macrophages was also markedly reduced in FPR KO mice. Finally, evidence of direct annexin 1 binding to murine FPR was obtained with HEK-293 cells transfected with the receptor. Overall, these results indicate a functional role for FPR in the anti-migratory effect of annexin 1 and derived peptides.

Natural and synthetic agonists of the melanocortin receptor type 3 possess anti-inflammatory properties.

The effects of the natural and synthetic ligands for the melanocortin receptor type 3 (MC3-R) have been evaluated in a murine model of experimental gout. Systemic treatment of mice with gamma2-melanocyte-stimulating hormone (gamma2-MSH) and the synthetic agonist MTII inhibited accumulation of KC, interleukin-1 beta (IL-1beta), and PMN elicited by urate crystals in the peritoneal cavity. In vitro, macrophage (Mø) activation, determined as release of KC and IL-1beta, was inhibited by gamma2-MSH and MTII. The mixed MC3/4-R antagonist SHU9119 prevented the inhibitory actions of gamma2-MSH and MTII in vitro and in vivo, whereas the selective MC4-R antagonist HS024 was without effect. Western blotting also showed the presence of MC3-R protein on murine peritoneal Mø. Furthermore, agonism at the MC3-R evoked accumulation of cAMP within the Mø, which was inhibited by SHU9119. Thus, naturally occurring melanocortins, as well as the synthetic long-acting compound MTII, activate MC3-R on peritoneal Mø to inhibit the experimental inflammatory response.

Anti-inflammatory effects of a novel, potent inhibitor of poly (ADP-ribose) polymerase.

OBJECTIVE AND DESIGN: Oxygen- and nitrogen-derived free radicals and oxidants play an important role in the pathogenesis of various forms of inflammation. Recent work emphasizes the importance of oxidant-induced DNA strand breakage and activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) in the pathogenesis of various inflammatory diseases. We have recently demonstrated the efficacy of PJ34, a novel, potent phenanthridinone derivative PARP inhibitor, in rodent models of diabetic vascular dysfunction and stroke. Here we tested the efficacy of PARP inhibition in various models of local inflammation in rodents. MATERIALS AND METHODS: PJ34 (at doses of 0.03-30 mg/kg) was tested in rats and mice subjected to standard models of inflammation, with relevant parameters of inflammation measured using standard methods. RESULTS: PJ34 treatment (s.c, i.p. and i.v.) dose-dependently suppressed neutrophil infiltration and nitric oxide (but not KC and IL-1beta) production in peritonitis. In a model of systemic endotoxemia, PJ34 pretreatment significantly reduced plasma levels of TNF-alpha, IL-1beta and nitrite/nitrate (breakdown products of nitric oxide) production. PJ34 treatment (oral gavage) induced a significant suppression of the inflammatory response in dextran sulfate colitis, multiple low dose streptozotocin diabetes and cyclophosphamide-accelerated autoimmune diabetes in the non-obese diabetic mice, and reduced the degree of mononuclear cell infiltration into the iris in an endotoxin-induced uveitis model. Delaying the start of PJ34 administration in the colitis model conferred significant protective effects, while in the arthritis model the post-treatment paradigm lacked protective effects. CONCLUSIONS: PJ34 provides significant, dose-dependent, anti-inflammatory effects in a variety of local inflammation models. Some of its actions are maintained in the post-treatment regimen and/or after discontinuation of treatment. We conclude that PARP inhibition offers a powerful means for reducing the severity of various forms of local inflammatory responses.

Arthritic diseases: melanocortin type 3 receptor agonists as potential therapeutics.

Gouty arthritis is currently treated with drugs that have an array of side effects. Therefore, identification of novel endogenous targets for drug development may have beneficial properties ACTH4-10, a heptapeptide fragment derived from the hormone adrenocorticotrophin (ACTH) modulates the inflammatory response in a corticosterone-independent manner, via agonism at melanocortin type 3 receptors (MC3-R) expressed on peritoneal macrophages. MC3-R agonists inhibit cytokine formation and subsequent neutrophil migration, while antagonists abrogate these effects. Together, these data highlight MC3-R as a potential therapeutic target and suggest that small molecule agonists directed at MC3-R with more specific actions, may be potentially novel therapeutics for treating this pathology.

MC3-R as a novel target for antiinflammatory therapy.

To date five melanocortin receptors (MC-R) have been cloned, identified and shown to have a wide distribution throughout the body and likely many diverse functions. MC1-R, found on melanocytes, is involved in pigmentation, while MC2-R is the classic adrenocorticotropic (ACTH) receptor found on the adrenal cortex and adipocytes. MC3-R, MC4-R and MC5-R are in their infancy with regard to their characterization. MC4-R has generated wide interest for its involvement in obesity, whereas our own studies have indicated a role for MC3-R in experimental inflammation. An ACTH fragment unable to alter circulating corticosterone, ACTH-4-10, acts at murine MC3-R present on peritoneal macrophage to inhibit cytokine formation and subsequent neutrophil extravasation. These findings were confirmed using agonists with a higher degree of selectivity toward MC3-R, such as gamma-2-MSH and the synthetic mixed MC3/4-R agonist MTII. In vitro, all these agents were able to affect macrophage functions, including phagocytosis and production of the CXC chemokine KC. Besides using RT-PCR and cAMP formation assays, the involvement of MC3-R in the antiinflammatory actions of these melanocortins was validated with the antagonist SHU-9119. Together these experimental data support the notion that agonism at MC3-R can be used for the design of novel therapeutics for inflammatory conditions.

POMC gene-derived peptides activate melanocortin type 3 receptor on murine macrophages, suppress cytokine release, and inhibit neutrophil migration in acute experimental inflammation.

To investigate the relevance of adrenocorticotrophic hormone (ACTH) therapy in human gouty arthritis, we have tested the effect of several ACTH-related peptides in a murine model of experimental gout. Systemic treatment of mice with ACTH4-10 (MEHFRWG) (10-200 microgram s. c.) inhibited neutrophil accumulation without altering peripheral blood cell counts or circulating corticosterone levels. A similar effect was seen with alpha- and beta-melanocyte stimulating hormones (1-30 microgram s.c.). In vivo release of the chemokine KC-(detected in the lavage fluids before maximal influx of neutrophils) was significantly reduced (-50 to -60%) by ACTH4-10. Macrophage activation in vitro, determined as phagocytosis and KC release, was inhibited by ACTH and ACTH4-10 with approximate IC50 values of 30 nM and 100 microM, respectively. The melanocortin receptor type 3/4 antagonist SHU9119 prevented the inhibitory actions of ACTH4-10 both in vitro and in vivo. However, melanocortin type 3, but not type 4, receptor mRNA was detected in mouse peritoneal macrophages by RT-PCR. Therefore, we propose that activation of this receptor type by ACTH4-10 and related amino acid sequences attenuates KC release (and possibly production of other cytokines) from macrophages with consequent inhibition of the host inflammatory response, thus providing a notional anti-inflammatory mechanism for ACTH that is unrelated to stimulation of glucocorticoid release.

Molecular determinants of monosodium urate crystal-induced murine peritonitis: a role for endogenous mast cells and a distinct requirement for endothelial-derived selectins.

Injection of monosodium urate (MSU) crystals, the etiological cause of gouty arthritis, into murine peritoneal cavities produced an intense recruitment of polymorphonuclear leukocytes (PMN). After 3 mg MSU crystal injection, cell influx was maximal (approximately 10 x 10[6] cells per mouse) at 6 hr postinjection and sustained up to the 24 hr time-point. In mice depleted of mast cells by administration of compound 48/80 72 hr before challenge with MSU crystals a lower PMN influx was measured (58% reduction). The occurrence of endogenous mast cell activation, in the MSU response, was validated by the observation that MSU challenge reduced by more than 90% the number of intact mast cells recovered in the peritoneal washes. Pretreatment of mice with a histamine H1 antagonist (tripolidine; 0.5 mg/kg) or a platelet-activating factor receptor antagonist (WEB2086; 10 mg/kg) significantly reduced by 50 to 60% the number of PMN recovered from the peritoneal cavities. The molecular determinants of this process of leukocyte recruitment were also investigated. Treatment of mice with an anti-CD62P or anti-CD62E monoclonal antibody (mAb; 100 microg i.v.) produced a distinct inhibition of PMN recruitment measured at 6 hr, whereas only a combined administration of both monoclonal antibodies was effective in reducing by 60% the influx of PMN caused by the MSU crystals within 24 hr. In conclusion, these data highlight a role for endogenous mast cells and for endothelial-derived selectins in MSU crystal-induced PMN recruitment into the peritoneal cavity, and may be useful to dissect molecular mechanism(s) which may be operating in gouty arthritis.

Inhibition of poly (ADP-ribose) synthetase attenuates neutrophil recruitment and exerts antiinflammatory effects.

A cytotoxic cycle triggered by DNA single-strand breakage and poly (ADP-ribose) synthetase activation has been shown to contribute to the cellular injury during various forms of oxidant stress in vitro. The aim of this study was to investigate the role of poly (ADP-ribose) synthetase (PARS) in the process of neutrophil recruitment and in development of local and systemic inflammation. In pharmacological studies, PARS was inhibited by 3-aminobenzamide (10-20 mg/kg) in rats and mice. In other sets of studies, inflammatory responses in PARS-/- mice were compared with the responses in corresponding wild-type controls. Inhibition of PARS reduced neutrophil recruitment and reduced the extent of edema in zymosan- and carrageenan-triggered models of local inflammation. Moreover, inhibition of PARS prevented neutrophil recruitment, and reduced organ injury in rodent models of inflammation and multiple organ failure elicited by intraperitoneal injection of zymosan. Inhibition of PARS also reduced the extent of neutrophil emigration across murine mesenteric postcapillary venules. This reduction was due to an increased rate of adherent neutrophil detachment from the endothelium, promoting their reentry into the circulation. Taken together, our results demonstrate that PARS inhibition reduces local and systemic inflammation. Part of the antiinflammatory effects of PARS inhibition is due to reduced neutrophil recruitment, which may be related to maintained endothelial integrity.

Inhibition of neutrophil and monocyte recruitment by endogenous and exogenous lipocortin 1.

1. The role played by endogenous lipocortin 1 in the anti-migratory action exerted by dexamethasone (Dex) on monocyte recruitment in an in vivo model of acute inflammation was investigated by use of several neutralizing polyclonal antibodies raised against lipocortin 1 or a lipocortin 1-derived N-terminus peptide (peptide Ac2-26). The efficacy of peptide Ac2-26 in inhibiting monocyte and polymorphonuclear leucocyte (PMN) recruitment was also tested. 2. Intraperitoneal (i.p.) injection of zymosan A (1 mg) produced a time-dependent cell accumulation into mouse peritoneal cavities which followed a typical profile of acute inflammation: PMN influx was maximal at 4 h post-zymosan (between 15 and 20 x 10(6) cells per mouse), and this was followed by an accumulation of monocytes which peaked at the 24 h time-point (between 10 and 15 x 10(6) cells per mouse). 3. Dex administration to mice reduced zymosan-induced 4 h PMN infiltration and 24 h monocyte accumulation with similar efficacy: approximately 50% of inhibition of recruitment of both cell types was achieved at the dose of 30 micrograms per mouse (approximately 1 mg kg-1, subcutaneously (s.c.)). Maximal inhibitions of 64% and 67% on PMN and monocyte recruitment, respectively, were measured after a dose of 100 micrograms per mouse (approximately 3 mg kg-1, s.c.). 4. Dex (30 micrograms s.c.) inhibited monocyte (53%) and PMN (69%) accumulation in response to zymosan application in mice which had been treated with a non-immune sheep serum (50 microliters s.c.). In contrast, the steroid was no longer active in reducing cell accumulation in mice which had been passively immunized against full length human recombinant lipocortin 1 (serum LCS3), or against lipocortin 1 N-terminus peptide. 5. Treatment of mice with vinblastine (1 mg kg-1, intravenously (i.v.)) produced a remarkable leucopenia as assessed 24 h after administration. This was accompanied by a 60% reduction in 4 h-PMN influx, and by a 27% reduction in 24 h-monocyte accumulation, measured after zymosan administration. The inhibitory effect of Dex on monocyte recruitment was not significantly modified in vinblastine-treated mice, with 36% and 57% of inhibition calculated at the dose of 30 micrograms Dex, and 70% and 60% of inhibition at 100 micrograms Dex, in vehicle- and vinblastine-treated mice, respectively. 6. Treatment of mice with peptide Ac2-26 dose-dependently attenuated PMN influx at 4 h post-zymosan with a significant effect at 100 micrograms per mouse (45% of inhibition, n-9, P < 0.05) and a maximal effect of 61% inhibition at the highest dose tested of 200 micrograms s.c. (n = 14, P < 0.05). No effect of peptide Ac2-26 (200 micrograms s.c.) was seen on zymosan-induced 24 h monocyte recruitment. In contrast, administration of 200 micrograms peptide Ac2-26 every 6 h was effective in reducing the number of monocytes harvested from the inflamed peritoneal cavities at 24 h post-zymosan: 9.40 +/- 0.58 x 10(6) monocytes per mouse (n = 13) and 5.74 +/- 0.34 monocytes per mouse (n = 14) in vehicle- and peptide Ac2-26-treated mice, respectively (P < 0.05). 7. Finally, peptide Ac2-26 produced a concentration-dependent inhibition of the rate of phagocytosis of mouse resident peritoneal macrophages as measured by flow cytometry, with a maximal reduction of 34% at the highest concentration tested of 100 micrograms ml-1 (n = 8 experiments performed in duplicate; P < 0.05). 8. In conclusion, this study suggests that in vivo monocyte recruitment during acute inflammation is, at least in part, under the negative modulatory control of endogenous lipocortin 1 (as seen after administration of Dex by using the specific antisera) and exogenous lipocortin 1 mimetics (as observed with peptide Ac2-26. In addition to the neutrophil, we can now propose that the monocyte also can be a target for the in vivo anti-inflammatory action of lipocortin 1.

Subacute treatment of rats with dexamethasone reduces ICAM-1 levels on circulating monocytes.

We report for the first time that in vivo treatment with dexamethasone (DEX) reduces levels of intercellular adhesion molecule-1 (ICAM-1) expression on rat circulating unstimulated monocytes (-55%) and peritoneal macrophages (-26%). This effect was present following sub-acute (5 days) treatment with a low dose (0.1 mg/kg per day), but not after single administration of a high dose (1 mg/kg, -2 h), of the steroid. Both acute and sub-acute treatment with DEX failed to modify either basal or up-regulated CD11b expression on peripheral blood monocytes and neutrophils, elastase release from neutrophils, and beta-glucuronidase release from cultured macrophages. The lack of alteration of CD11b expression on circulating leukocytes suggests that the effect of DEX on ICAM-1 expression is secondary to gene repression rather than a non-specific blockade of cell differentiation. These data promote the concept that different dose-regimens with glucocorticoids affect distinct molecular targets and indicate that clinically-related protocols of DEX may reveal new mechanism(s) of action.

Biphasic modulation of GABA release by nitric oxide in the hippocampus of freely moving rats in vivo.

The effect of altering hippocampal nitric oxide (NO) levels on basal and N-methyl-D-aspartate receptor-evoked release of GABA has been studied in freely moving rats. N-Methyl-D-aspartate (NMDA) increased extracellular GABA in a concentration-dependent manner. The nitric oxide synthase inhibitor L-nitro-arginine-methyl ester (L-NAME; 100 microM) increased basal GABA release, and also enhanced release of GABA evoked by NMDA (100 microM) compared with the same concentration of NMDA infused alone. 200 microM L-NAME increased basal dialysate GABA, but to a lesser extent than the 100 microM concentration of the drug, and the NMDA-induced release of GABA was decreased. 1.0 mM L-NAME significantly decreased basal extracellular GABA, while abolishing the NMDA-evoked release of the amino acid. The actions of L-NAME were not mimicked by its much less active isomer D-nitro-arginine-methyl ester. The NO donor S-nitroso-N-acetylpenicillamine decreased dialysate GABA at a 500 microM concentration but increased the extracellular level of the transmitter when infused at 1.0 and 2.0 mM concentrations. These data suggest that NO may mediate both excitatory and inhibitory functions in vivo.

Nitric oxide regulates excitatory amino acid release in a biphasic manner in freely moving rats.

The effect of altering hippocampal nitric oxide levels on basal and N-methyl-D-aspartate (NMDA) receptor-evoked release of glutamate and aspartate has been studied in freely moving rats. NMDA increased extracellular glutamate and aspartate in a concentration-dependent manner. The nitric oxide synthase inhibitor L-nitro-arginine-methyl ester (L-NAME; 100 microM) increased basal glutamate and aspartate release, and also enhanced release of these amino acids evoked by NMDA (100 microM) compared with the same concentration of NMDA infused alone. L-NAME at 200 microM increased basal dialysate glutamate, but not aspartate, to a lesser extent than the 100 microM concentration of the drug, and the NMDA-induced release of glutamate and aspartate was decreased. L-NAME at 1.0 mM did not significantly alter basal extracellular glutamate but significantly decreased dialysate aspartate, while abolishing the NMDA-evoked release of both amino acids. The actions of L-NAME were not mimicked by its much less active isomer D-nitro-arginine-methyl ester. The nitric oxide donor drug S-nitroso-N-penicillamine decreased dialysate glutamate and aspartate at a 500 microM concentration but increased the extracellular level of both amino acids when infused at 1.0 mM and 2.0 mM concentrations. These data suggest that nitric oxide may mediate both excitatory and inhibitory functions, according to the level of nitric oxide production in vivo.