Predicting response to cancer immunotherapy using noninvasive radiomic biomarkers.

INTRODUCTION: Immunotherapy is regarded as one of the major breakthroughs in cancer treatment. Despite its success, only a subset of patients responds-urging the quest for predictive biomarkers. We hypothesize that artificial intelligence (AI) algorithms can automatically quantify radiographic characteristics that are related to and may therefore act as noninvasive radiomic biomarkers for immunotherapy response. PATIENTS AND METHODS: In this study, we analyzed 1055 primary and metastatic lesions from 203 patients with advanced melanoma and non-small-cell lung cancer (NSCLC) undergoing anti-PD1 therapy. We carried out an AI-based characterization of each lesion on the pretreatment contrast-enhanced CT imaging data to develop and validate a noninvasive machine learning biomarker capable of distinguishing between immunotherapy responding and nonresponding. To define the biological basis of the radiographic biomarker, we carried out gene set enrichment analysis in an independent dataset of 262 NSCLC patients. RESULTS: The biomarker reached significant performance on NSCLC lesions (up to 0.83 AUC, P < 0.001) and borderline significant for melanoma lymph nodes (0.64 AUC, P = 0.05). Combining these lesion-wide predictions on a patient level, immunotherapy response could be predicted with an AUC of up to 0.76 for both cancer types (P < 0.001), resulting in a 1-year survival difference of 24% (P = 0.02). We found highly significant associations with pathways involved in mitosis, indicating a relationship between increased proliferative potential and preferential response to immunotherapy. CONCLUSIONS: These results indicate that radiographic characteristics of lesions on standard-of-care imaging may function as noninvasive biomarkers for response to immunotherapy, and may show utility for improved patient stratification in both neoadjuvant and palliative settings.

Anti-inflammatory effects of pioglitazone on iron-induced oxidative injury in the nigrostriatal dopaminergic system.

AIMS: Transition metals, oxidative stress and neuroinflammation have been proposed as part of a vicious cycle in central nervous system neurodegeneration. Our aim was to study the anti-inflammatory effect of pioglitazone, a peroxisome proliferative activated receptor-γ agonist, on iron-induced oxidative injury in rat brain. METHODS: Intranigral infusion of ferrous citrate (iron) was performed on anaesthetized rats. Pioglitazone (20 mg/kg) was orally administered. Oxidative injury was investigated by measuring lipid peroxidation in the substantia nigra (SN) and dopamine content in the striatum. Western blot assay and DNA fragmentation were employed to study the involvement of α-synuclein aggregation, neuroinflammation as well as activation of endoplasmic reticulum (ER) and mitochondrial pathways in iron-induced apoptosis. RESULTS: Intranigral infusion of iron time-dependently increased α-synuclein aggregation and haem oxygenase-1 levels. Furthermore, apoptosis was demonstrated by TUNEL-positive cells and DNA fragmentation in the iron-infused SN. Systemic pioglitazone was found to potentiate iron-induced elevation in nuclear peroxisome proliferative activated receptor-γ levels. However, pioglitazone inhibited iron-induced α-synuclein aggregation, elevations in interleukin-1ß and interleukin-6 mRNA levels as well as increases in oxygenase-1, cyclo-oxygenase II, nitric oxide synthase and ED-1 protein levels, an indicator of activated microglia. Moreover, iron-induced DNA laddering as well as activation of ER and mitochondrial pathways were attenuated by pioglitazone. In addition, pioglitazone decreased iron-induced elevation in lipid peroxidation in the infused SN and depletion in striatal dopamine level. CONCLUSIONS: Our results suggest that pioglitazone prevents iron-induced apoptosis via both ER and mitochondrial pathways. Furthermore, inhibition of α-synuclein aggregation and neuroinflammation may contribute to the pioglitazone-induced neuroprotection in central nervous system.

Anti-inflammatory effect of afatinib (an EGFR-TKI) on OGD-induced neuroinflammation.

Activated epidermal growth factor receptor (EGFR) has been proposed in the pathophysiology of neurodegenerative diseases. In the present study, the anti-inflammatory effect of afatinib, an EGFR-tyrosine kinase inhibitor (EGFR-TKIs) was investigated using CTX-TNA2 cells and primary cultured astrocytes subjected to oxygen/glucose deprivation (OGD). We found that OGD induced EGFR phosphorylation and activated subsequent signaling pathways, including phosphorylation of AKT and extracellular signal-regulated kinases (ERK). Afatinib blocked OGD-induced phosphorylation of EGFR, AKT and ERK. At the same time, afatinib attenuated OGD-induced elevations in glial fibrillary acidic protein (a biomarker of activated astrocytes) and proliferating cell nuclear antigen expression (a cell proliferating biomarker) as well as hypoxia-induced migratory ability. Furthermore, afatinib decreased OGD-induced increases in cyclooxygenase-II and inducible nitric oxide synthase expression of the treated astrocytes as well as NO content in the culture medium. Moreover, afatinib attenuated OGD-induced caspase 1 activation (a biomarker of inflammasome activation) and interleukin-1ß levels (a pro-inflammatory cytokine). Collectively, afatinib could block OGD-induced EGFR activation and its downstream signaling pathways in astrocytes. Moreover, afatinib attenuated OGD-induced astrocyte activation, proliferation and inflammasome activation. These data support the involvement of EGFR activation in neuroinflammation. Furthermore, EGFR-TKIs may be promising in inhibiting neuroinflammation in the CNS neurodegenerative diseases.

Arsenite-induced cytotoxicity in dorsal root ganglion explants.

Peripheral neuropathy is common in people chronically overexposed to arsenic. We studied sodium arsenite (arsenite)-induced cytotoxicity in dorsal root ganglion (DRG) explants. Incubation with arsenite concentration- and time-dependently increased the expression of stress proteins, heat shock protein 70, and heme oxygenase-1 in DRG explants. Furthermore, apoptosis was involved in the arsenite-induced cytotoxicity in the treated DRG. Elevation in cytosolic cytochrome c levels and reduction in procaspase 3 levels suggested an involvement of the mitochondrial pathway in arsenite-induced apoptosis in this preparation. At the same time, increases in the activating transcription factor-4 and C/EBP homologous protein and reduction in procaspase 12 levels indicated activation of the endoplasmic reticulum (ER) pathway in the arsenite-induced cytotoxicity in DRG explants. Salubrinal (30 microM), an ER inhibitor, was found to attenuate arsenite-induced DNA fragmentation and reduction in procaspase 12 in DRG explants. Cytotoxic effects by arsenite, sodium arsenate (arsenate), monomethylarsonic acid (MMA), and dimethylarsinic acid (DMA) were compared, and the potency was as follows: arsenite >>> arsenate>MMA and DMA. Recombinant adenovirus vectors encoding glial-cell-derived neurotrophic factor (AdGDNF) genes allowed a stable delivery of GDNF genes to the infected cells in DRG explants. Applied in this manner, AdGDNF was found to inhibit arsenite-induced DNA fragmentation in DRG explants. Moreover, AdGDNF attenuated the arsenite-induced reduction in procaspases 3 and 12 levels. Taken together, our study demonstrates that arsenite is capable of inducing cytotoxicity in DRG explants. Both ER and mitochondria pathways are involved in the arsenite-induced apoptosis in DRG explants. Glial-cell-derived neurotrophic factor appears to be protective against arsenite-induced peripheral neuropathy.

Iron-generated hydroxyl radicals kill retinal cells in vivo: effect of ferulic acid.

Siderosis bulbi is vision threatening. An investigation into its mechanisms and management is crucial. Experimental siderosis was established by intravitreous administration of an iron particle (chronic) or FeSO(4) (acute). After siderosis, there was a significant dose-responsive reduction in eletroretinogram (a/b-wave) amplitude, and an increase in OH level, greater when caused by 24 mM FeSO(4) than that by 8 mM FeSO(4). Furthermore, the FeSO(4)-induced oxidative stress was significantly blunted by 100 microM ferulic acid (FA). Siderosis also resulted in an excessive glutamate release, increased [Ca(++)](i), and enhanced superoxide dismutase immunoreactivity. The latter finding was consistent with the Western blot result. Obvious disorganization including loss of photoreceptor outer segments and cholinergic amacrines together with a wide-spreading ferric distribution across the retina was present, which were related to the eletro-retinographic and pathologic dysfunctions. Furthermore, b-wave reduction and amacrine damage were respectively, significantly, dose-dependently, and clearly ameliorated by FA. Thus, siderosis stimulates oxidative stress, and possibly, subsequent excitotoxicity, and calcium influx, which explains why the retina is impaired electro-physiologically and pathologically. Importantly, FA protects iron toxicity perhaps by acting as a free radical scavenger. This provides an approach to the study and treatment of the iron-related disorders such as retained intraocular iron and Alzheimer disease.

Melatonin Ameliorates Arsenite-Induced Neurotoxicity: Involvement of Autophagy and Mitochondria.

In the present study, the neuroprotective effect of melatonin on arsenite-induced neurotoxicity was investigated in rat primary cultured cortical neurons. Incubation of melatonin prevented arsenite-induced neuronal cell loss in a concentration-dependent manner. Furthermore, melatonin significantly attenuated arsenite-induced elevation in microtubule-associated protein light chain 3 (LC3)-II levels, a biomarker of autophagy. Our fluorescent staining assay showed that melatonin decreased arsenite-induced elevation of co-localized fluorescent puncta of monodansylcadaverine (a specific marker of autophagic vacuoles) and lysotracker red (a specific marker of lysosomes), indicating that melatonin is capable of inhibiting arsenite-induced autophagy and autolysosome formation. Because 3-methyladenine (an autophagic inhibitor) attenuated the arsenite-reduced α-synuclein levels (a protein essential for the neurite outgrowth and synaptic plasticity), melatonin via inhibiting autophagy attenuated the arsenite-reduced α-synuclein levels. At the same time, melatonin ameliorated the arsenite-induced reduction in growth associated protein 43 (a hallmark protein of neurite outgrowth) and discontinuous neurites of rat primary cultured cortical neurons. In addition, melatonin was found to prevent arsenite-induced decreases in cytochrome c oxidase levels (a biomarker of mitochondrial mass) and elevation in co-localized fluorescent puncta of autolysosomes and cytochrome c oxidase. Moreover, melatonin prevented arsenite-induced reduction in peroxisome proliferator-activated receptor gamma co-activator 1 α, a transcriptional co-activator of mitochondrial biosynthesis. Taken together, melatonin may exert its neuroprotective action via inhibiting arsenite-induced autophagy and enhancing mitochondrial biogenesis and thus restoring α-synuclein levels, neuronal integrity, and mitochondrial mass in rat primary cultured cortical neurons.

Long-term treatment of male F344 rats with deprenyl: assessment of effects on longevity, behavior, and brain function.

L-Deprenyl (selegiline) was chronically administered to male Fischer 344 rats via their drinking water beginning at 54 weeks of age (estimated daily dose: 0.5 mg/kg/day). Beginning at 84 weeks of age, the rats were behaviorally evaluated using a sensorimotor battery, a motor-learning task, and the Morris water maze. At 118 weeks of age, cerebellar noradrenergic function was evaluated in the surviving rats using in vivo electrochemistry. The rats were then sacrificed to measure brain monoamine oxidase activity and perform quantitative autoradiography to evaluate the effect of chronic deprenyl treatment on beta-adrenergic receptors in the cerebellum, alpha 2-adrenergic receptors several brain regions, and D1 and D2 dopamine receptors in the striatum. Deprenyl treatment reduced brain monoamine oxidase B activity by 85%, but had no effect on brain monoamine oxidase A. A clear effect of chronic deprenyl treatment upon longevity was not observed. Several measures of CNS function were altered in the deprenyl-treated animals: 1) spatial learning in the Morris water maze was improved; 2) electrochemical signals recorded following local application of NE were reduced, and the responsiveness to the reuptake blocker nomifensine was enhanced, in the cerebellum; 3) beta-adrenergic receptor binding affinity was increased in the cerebellum; 4) alpha 2-adrenergic receptor density was increased in the inferior colliculus; and 5) striatal D1 dopamine receptor density was reduced but binding affinity was enhanced. In contrast, chronic deprenyl treatment did not cause changes in: 1) sensorimotor function, as evaluated by balance beam, inclined screen, or wire hang tasks; 2) motor learning; 3) alpha 2-adrenergic receptor density in any region examined except for the inferior colliculus, or binding affinity in any region examined; or 4) striatal D2 dopamine receptor number or affinity. Thus, long-term oral administration of deprenyl extended the functional life span of rats with respect to cognitive, but not motor, performance.

Coexistence of zinc and iron augmented oxidative injuries in the nigrostriatal dopaminergic system of SD rats.

Clinical studies have demonstrated an excess of transition metals, including zinc and iron, in the substantia nigra (SN) of Parkinson's patients. In the present study, the neurotoxic effect of zinc was investigated using iron as a positive control. Addition of zinc or iron to brain homogenates increased lipid peroxidation. Zinc was less potent than iron in inducing lipid peroxidation. Coincubation with desferrioxamine prevented zinc- and iron-induced lipid peroxidation. Furthermore, glutathione (GSH), S-nitroso-N-acetylpenicillamine, or melatonin inhibited zinc-induced lipid peroxidation. The oxidative effect of zinc was further investigated in anesthetized rats. Seven days after intranigral infusion of zinc, lipid peroxidation was elevated in the infused SN, and dopamine content and tyrosine hydroxylase-positive axons were decreased in the ipsilateral striatum. Zinc was less potent than iron in inducing neurodegeneration in vivo. L-Buthionine-[S,R]-sulfoximine pretreatment (i.c.v.), which depletes cellular GSH levels, enhanced zinc-induced oxidative injuries in the nigrostriatal dopaminergic system. Moreover, simultaneous infusion of zinc and iron appeared to augment oxidative injuries in rat brain. Taken together, our results demonstrate that intranigral infusion of zinc caused degeneration of the nigrostriatal dopaminergic system in rat brain. Furthermore, coexistence of zinc and iron augmented oxidative injuries in rat brain. These findings indicate that both zinc and iron contribute to the etiology of Parkinsonism.

Melatonin suppresses iron-induced neurodegeneration in rat brain.

The antioxidative action of melatonin on iron-induced neurodegeneration in the nigrostriatal dopaminergic system was evaluated in vivo. Intranigral infusion of iron chronically degenerated the dopaminergic transmission of the nigrostriatal system. An increase in lipid peroxidation in the infused substantia nigra and reductions in dopamine levels and dopaminergic terminals in the ipsilateral striatum were observed 7 d after iron infusion. Whereas local infusion of melatonin (60 microg/microl, 1 microl) alone did not alter dopaminergic transmission, coinfusion of melatonin with iron suppressed iron-induced oxidative damages. Systemic infusion of melatonin via osmotic pumps had no effect on iron-induced neurodegeneration. However, repetitive intraperitoneal injections of melatonin (10 mg/kg) prevented iron-induced oxidative injuries. The ratio of glutathione (GSH)/oxidized glutathione (GSSG) was moderately increased in the lesioned substantia nigra of the melatonin-treated rats compared to that of the lesioned group in control rats. The antioxidative effect of melatonin was verified in cortical homogenates. Melatonin dose-dependently suppressed autoxidation and iron-induced lipid peroxidation. Melatonin was as effective as GSH and was less effective than Trolox (a water-soluble analogue of vitamin E) in inhibiting iron-elevated lipid peroxidation of brain homogenates. Our data suggest that melatonin is capable of at least partially preventing the iron-induced neurodegeneration in the nigrostriatal dopaminergic system.

Striatal dopamine dynamics are altered following an intranigral infusion of iron in adult rats.

In vivo electrochemical detection was employed to examine iron-induced oxidative injury on dopamine dynamics in the nigrostriatal system of urethane-anesthetized rats. Seven days after an intranigral infusion of iron, oxidative stress was confirmed by an elevated lipid peroxidation in lesioned substantia nigra (SN). Local application of potassium (K+) evoked dopamine releases from the dopamine-containing nerve terminals in the striatum. Both amplitude and rate of clearance (Tc) of evoked dopamine releases were lower in striatum with lesioned SN; however, the time course parameters of dopamine release in the lesioned group were not different from those of the intact group, indicating a reduction in dopamine clearance. Indeed, iron-induced oxidative stress attenuated the effect of nomifensine, a high-affinity dopamine uptake blocker, on dopamine clearance. In striatum with intact SN, the amplitude and time course parameters of signals by exogenous dopamine were increased and Tc was decreased by nomifensine. In contrast, nomifensine did not significantly alter the dopamine signals of the lesioned group. Taken together, in addition to the increased lipid peroxidation in SN, our in vivo electrochemical data demonstrates that iron-induced oxidative injury attenuates K+-evoked dopamine release and dopamine uptake in the ipsilateral striatum. The diminished nomifensine effect implies a lack of high-affinity dopamine uptake sites.

Correlated effects of guanabenz on single-neuron activity in the nucleus reticularis gigantocellularis, systemic arterial pressure and heart rate in the rat.

The simultaneous effects of guanabenz on the activity of single-neurons in the nucleus reticularis gigantocellularis of the medulla oblongata, systemic arterial pressure and heart rate were assessed in Sprague-Dawley rats, anesthetized with pentobarbital sodium (40 mg/kg, i.p.). Of the 35 arterial pressure-related neurons (neurons that temporally altered their spike rhythm subsequent to fluctuations in arterial pressure) which were evaluated in this reticular nucleus, 32 changed their discharge frequencies that exhibited a degree and time-course parallel to the hypotension promoted by the amino-guanidine compound (5, 10 or 20 micrograms/kg, i.v.). More importantly, these alterations neuronal activity preceded the occurrence of the induced vasodepression, signifying a causative relationship between the two events. Four of the 5 non-arterial pressure-related neurons in the gigantocellular reticular nucleus, on the other hand, manifested no basic modification in their spike frequencies in relation to the hypotension induced by guanabenz. These observations provided further support for the idea that the nucleus reticularis gigantocellularis participates actively in the cardiovascular suppressive actions of the aminoguanidine compound.

Optimization of culture conditions to enhance transfection of human CD34+ cells by electroporation.

The ability to culture CD34+ stem cells, while maintaining their pluripotency, is essential for manipulations such as gene transfection for therapeutic trials. Human peripheral blood (PB) CD34+ cells (> or = 90% purity) were cultured for up to 4 days in serum-free culture medium supplemented with thrombopoietin (TPO), stem cell factor (SCF), Flt-3 ligand (Flt-3L), with or without PIXY321 (IL-3/GM-CSF fusion protein) and human serum. The CD34 mean fluorescence intensity (MFI) and cell cycle status were evaluated daily using flow cytometry and hypotonic propidium iodide. Prior to culture (day 0), 97.0 +/- 0.9%, 1.9 +/- 0.3% and 1.0 +/- 0.6% of the selected CD34+ cells were in G0-G1, S-phase, or G2-M, respectively. After 2-4 days in culture with TPO/SCF/Flt-3L, there was an increase in the percent of cells in S-phase to 26.4 +/- 0.1% without significant loss of CD34 MFI. The addition of PIXY321 increased.the percentage of CD34+ cells in S-phase to 36.3 +/- 4.0%, but the CD34 MFI and numbers of CFU (colony-forming units) were significantly decreased at day 3 when cultured with PIXY321 or various recombinant cytokine combinations that included IL-3 and IL-6. There is an increase from day 0 to day 4 in the percentages of CD34+ with CD38-, HLA-DR-, and c-kit(low), but not Thy-1+ cells. Electroporation with EGFP reporter gene showed that 1-2 days of pre-stimulation in X-VIVO 10 supplemented with TPO/SCF/Flt-3L was necessary and sufficient for efficient transfection. Flow cytometry analysis demonstrated that 22% of the viable cells are CD34+/EGFP+ 48 h post electroporation. The introduced reporter gene appears to be stable as determined by EGFP+/LTC-IC (long-term colony-initiating cells), at 30-40 positive colonies (16 +/- 7%) per 1 x 10(5) electroporated CD34+ cells.

Lack of protective effect by intermittent hypoxia on MPTP-induced neurotoxicity in mice.

In the study we report here, several lines of evidence support the preventive action of intermittent hypoxia against oxidative injuries in CNS. Our in vitro data showed that autooxidation and iron-induced lipid peroxidation were attenuated in cortical homogenates of intermittent hypoxia-treated animals. Furthermore, our preliminary study found that iron induced oxidative injuries were abolished in rat brain after intermittent hypoxic treatment (paper submitted). Several antioxidative defensive systems improve in response to intermittent hypoxia. Since attenuation of autooxidation and iron-induced lipid peroxidation were observed in cortical homogenates of intermittent hypoxia-treated mice, the lack of prevention by intermittent hypoxia of MPTP-induced neurotoxicity may be due to the MPTP action that is not oxidative related. Together with our previous studies, in which several antioxidants were shown to successfully prevent oxidative injuries, our data here suggest that intermittent hypoxia may offer a potential treatment for preventing CNS degenerative diseases.

The antioxidative effect of carboxyfullerenes (C3/D3) on iron-induced oxidative injury in CNS.

Carboxyfullerenes, including two regioisomers C3 and D3, were investigated as antioxidants against iron-induced oxidative stress in vivo and in vitro. Both C3 and D3 dose-dependently inhibited autoxidation and iron-elevated lipid peroxidation in cortical homogenates. The antioxidative property of C3 was compared to Trolox (a water-soluble analogue of vitamin E) and glutathione. C3 was more effective than glutathione but was less effective than Trolox in inhibiting iron-induced elevation in lipid peroxidation. In urethane-anesthetized rats, intranigral infusion of iron degenerated the nigrostriatal dopaminergic system, including as elevation in lipid peroxidation in the infused substantia nigra (SN) and reductions in K(+)-evoked dopamine overflow and dopamine content in the ipsilateral striatum 7 days after the infusion. Local application of iron with C3 or D3 prevented iron-induced oxidative injuries. Our data suggest that carboxyfullerenes have a neuroprotective effect in preventing iron-induced oxidative injury in CNS.

Recovery by NO of the iron-attenuated dopamine dynamics in nigrostriatal system of rat brain.

In vivo electrochemical detection was employed to study the chronic effect of nitric oxide on iron-induced alterations in dopamine dynamics, including K+ -evoked dopamine overflows and the clearance of exogenous dopamine in rat striatum. Intranigral infusion of fresh S-nitroso-N-acetylpenicillamine (SNAP), a nitric oxide donor, did not alter either dopamine dynamics in the striatum or the lipid peroxidation in substantia nigra 7 days after the infusion, indicating that nitric oxide is not neurodestructive. By contrast, infusion of iron in substantia nigra chronically degenerated the nigrostriatal dopaminergic system. Co-infusion of iron and fresh SNAP, but not aged SNAP, prevented the iron-induced reductions in K+ -evoked dopamine overflows. Furthermore, the clearance of exogenous dopamine was attenuated in the striatum ipsilateral to the substantia nigra infused with iron. An improvement by fresh SNAP of iron-induced reduction in dopamine clearance was observed in rats co-infused with fresh SNAP and iron mixture compared to iron-lesioned group. Taken together, our in vivo electrochemical study suggests that nitric oxide does not alter dopamine dynamics in nigrostriatal dopaminergic system. Rather, nitric oxide appears to protect dopamine dynamics from iron-induced oxidative stress in rat brain.

Role of dopamine uptake in NMDA-modulated K(+)-evoked dopamine overflow in rat striatum: an in vivo electrochemical study.

An involvement of dopamine uptake in the N-methyl-D-aspartate (NMDA)-modulated dopaminergic transmission in rat striatum was studied using the technique of in vivo electrochemical detection. Microinjection of potassium (K+) evoked dopamine overflows from the dopamine-containing nerve terminals in the striatum. While application of NMDA did not evoke any dopamine overflow, co-application of NMDA and K+ induced larger dopamine overflows than those by K+ alone. Furthermore, dynamic analysis showed that the rate of clearance (Tc) was reduced by NMDA. Indeed, our uptake study demonstrated an NMDA-induced inhibition of dopamine clearance. The time course of electrochemical signals evoked by microinjection of exogenous dopamine was increased and Tc was reduced following NMDA application. In order to delineate the effects of NMDA on K(+)-evoked dopamine overflows and/or on dopamine uptake, nomifensine, a dopamine uptake inhibitor was used. Application of nomifensine potentiated K(+)-evoked dopamine overflows. Co-administration of NMDA further augmented dopamine overflows by the K+ and nomifensine mixture. Taken together, our data suggest that NMDA concomitantly potentiated dopamine overflows in response to depolarizing stimuli and attenuated dopamine uptake. The increment by NMDA of K(+)-evoked dopamine overflows may partially result from an attenuated dopamine uptake in rat striatum.

Dynamic analysis of ethanol effects on NMDA-evoked dopamine overflow in rat striatum.

This study was undertaken to dynamically examine the effects of ethanol on the striatal dopaminergic transmission, in terms of N-methyl-D-aspartate (NMDA)-evoked dopamine release and dopamine uptake. In the striatum of urethane-anesthetized Sprague-Dawley rats, extracellular dopamine was measured using in vivo electrochemical detection coupled with a nafion-coated carbon fiber working electrode. Micro-ejection of NMDA evoked a transient dopamine release from the dopamine-containing nerve terminals in striatum. Local application of ethanol by pressure ejection did not elicit significant changes in spontaneous dopamine release. However, with ethanol pretreatment, the time course of NMDA-induced dopamine release was markedly prolonged while the magnitude and the rate of clearance were significantly reduced. These effects were compared to those of nomifensine, a dopamine uptake blocker. Nominfensine pretreatment was found to augment the time course of NMDA-evoked dopamine release analogous to those by ethanol pretreatment. Furthermore, pretreatment with ethanol did not increase the time course parameters of dopamine signals if dopamine releases were induced by co-application of NMDA and nominfensine. These data suggest that in addition to the attenuation of NMDA-evoked dopamine release, ethanol inhibits dopamine uptake in a similar fashion to that observed with nomifensine in situ in the striatum. Indeed, ethanol altered the uptake of exogenous dopamine from the extracellular space of striatal cortex. The time course of dopamine signals was prolonged and the rate of clearance was reduced after ethanol treatment. Taken together, our data demonstrate that ethanol simultaneously inhibits NMDA-evoked dopamine release and dopamine uptake in the striatum, suggesting the importance of the interplay between release and uptake in ethanol effects on striatal dopaminergic transmission.

Effects of ethanol and nomifensine on NE clearance in the cerebellum of young and aged Fischer 344 rats.

Rapid chronoamperometric recordings coupled with local application of drugs by pressure ejection were used to investigate the effects of nomifensine and ethanol (EtOH) on exogenous norepinephrine (NE) clearance in the cerebellum of young (5-month-old) and aged (24-26-month-old) male Fischer 344 rats. In the young rats, local nomifensine application prolonged exogenous NE clearance, indicating transporter mediated uptake inhibition. NE clearance was modestly but significantly prolonged in the aged rats as compared to the young rats, suggesting less efficient uptake. Consistent with this, there was little effect of nomifensine on NE clearance in the aged rats. In contrast to the effect of nomifensine, EtOH inhibited NE clearance in both young and aged rats. These data further support the hypothesis that one effect of EtOH in cerebellar NE systems is inhibition of NE uptake into NE-containing nerve terminals, and they also demonstrate that the effect of nomifensine on exogenous NE clearance in vivo in the cerebellum is altered by the aging process, while the effect of EtOH is not.

Pineal nitric oxide synthase: characteristics, adrenergic regulation and function.

Available studies indicate that the adrenergic stimulation of pineal cyclic GMP production involves stimulation of guanylyl cyclase activity by nitric oxide (NO) derived from arginine. This line of investigation was extended in the present study. Using a highly sensitive microassay, it was found that pineal NO synthase activity is present at levels approximately 30% of those in the cerebellum, that approximately 95% of enzyme activity is cytoplasmic, that the enzyme is Ca2+/calmodulin-dependent and that enzyme activity is inhibited by the arginine analog NG-nitro-L-arginine methyl ester (L-NAME). Norepinephrine treatment of intact glands in culture increased [3H]citrulline formation from [3H]arginine. This treatment also increased the formation of an NO-like compound, indicating that NO synthase activity in the intact gland is elevated by adrenergic stimulation. Studies on the effects of inhibition of NO synthase activity indicated that treatments known to inhibit NO synthase activity and the adrenergic stimulation of cyclic GMP accumulation did not inhibit adrenergic stimulation of pineal cyclic AMP, N-acetyltransferase activity or melatonin production. These observations support the hypothesis that NE stimulation of pineal cyclic GMP accumulation involves stimulation of a Ca2+/calmodulin-sensitive form of NO synthase, resulting in enhanced accumulation of NO; and, that although NO appears to play a role in the adrenergic stimulation of pineal cyclic GMP accumulation, it does not appear to play a critical role in the adrenergic stimulation of cyclic AMP, N-acetyltransferase activity or melatonin production.