Presynaptic Nicotinic Receptors Stimulate Increases in Intraterminal Calcium of Chick Sympathetic Neurons in Culture

Abstract: Stimulation of chick sympathetic neurons in culture by the cholinergic agonists acetylcholine, nicotine, and 1,1-dimethyl-4-phenylpiperazinium (all at 10–1,000 µmol/L) induced concentration-dependent increases of free calcium levels measured by fura 2 fluorescence in neuronal processes. The response evoked by acetylcholine had both nicotinic and muscarinic components, whereas that induced by 1,1-dimethyl-4-phenylpiperazinium was purely nicotinic. Tetrodotoxin (0.3 µmol/L) blocked completely the increase of intraterminal free calcium level evoked by electrical stimulation. On the other hand, stimulation with 1,1-dimethyl-4-phenylpiperazinium still evoked 20–25% of the control response in the presence of tetrodotoxin. The concentration-response relationship of 1,1-dimethyl-4-phenylpiperazinium stimulation did not differ in the absence and in the presence of tetrodotoxin. The nicotinic antagonists d -tubocurarine (10 µmol/L) and mecamylamine (10 µmol/L), but not α-bungarotoxin (125 nmol/L), prevented the increase of intraterminal free calcium level evoked by 1,1-dimethyl-4-phenylpiperazinium (100 µmol/L) in the presence of tetrodotoxin. These observations indicate the presence of nicotinic receptors on neuronal processes that increase the intraterminal concentration of free calcium and probably modulate transmitter release. Their pharmacological properties are similar to those of nicotinic receptors located on neuronal cell bodies.     

Spinal Neurokinin NK1 Receptor Down-Regulation and Antinociception: Effects of Spinal NK1 Receptor Antisense Oligonucleotides and NK1 Receptor Occupancy

Abstract: To define the effects of antisense oligonucleotides on spinal neurokinin 1 (NK1) receptor function in nociceptive processing, several antisense oligonucleotides directed against the NK1 receptor mRNA were intrathecally injected into rats via an implanted catheter, and their effect on the behavioural response to formalin injected into the paw was assessed. We observed that there was no significant reduction of pain behaviour or immunostaining of spinal NK1 receptors after repeated daily intrathecal treatment with an antisense oligonucleotide. However, spinal application of substance P (SP) in the antisense oligonucleotide-treated animals resulted in a profound and long-lasting reduction in the behavioural response to formalin injection, and a parallel reduction in the NK1 receptor immunoreactivity normally observed in spinal dorsal horn. Intrathecal SP in the control groups, i.e., rats treated with an oligonucleotide containing four mismatched bases, the corresponding sense oligonucleotide, a mixture of the sense and the antisense oligonucleotides, in each case had no effect. The effects of SP were blocked by NK1 receptor antagonists and were not mimicked by NMDA. The mechanism underlying these effects is not clear. It may be due to partial degradation of the internalised receptors, which cannot be replaced by newly synthesised receptors because of the action of the NK1 antisense oligonucleotide.     

Regulation of Corticotropin-Releasing Factor Receptor Function in Human Y-79 Retinoblastoma Cells: Rapid and Reversible Homologous Desensitization but Prolonged Recovery

Abstract: Homologous receptor desensitization is an important regulatory response to continuous activation by agonist that involves the uncoupling of a receptor from its G protein. When human retinoblastoma Y-79 cells expressing corticotropin-releasing factor (CRF) receptors were preincubated with CRF for 10 min-4 h, a time-dependent reduction in both the peak and sensitivity of CRF-stimulated intracellular cyclic AMP (cAMP) accumulation developed with a t _1/2 of 38 min and an EC₅₀ of 6–7 n M CRF. CRF receptor desensitization was slowly reversible after a 4-h CRF preincubation with a t _1/2 of 13 h and a full restoration of cAMP responsiveness to CRF at 24 h following the removal of 10 n M CRF. Because the ability of vasoactive intestinal peptide, forskolin, or (−)-isoproterenol to stimulate cAMP accumulation was not diminished in Y-79 cells desensitized with 10 n M CRF, the observed desensitization was considered to be a specific homologous action of CRF. CRF receptor desensitization was markedly attenuated by CRF receptor antagonists, which alone did not produce any appreciable reduction in CRF-stimulated cAMP accumulation. Although recent reports have demonstrated a rapid decline in steady-state levels of CRF receptor type 1 (CRF-R1) mRNA in anterior pituitary cells during several hours of exposure to CRF, there was no observed reduction in CRF-R1 mRNA levels when Y-79 cells were preincubated with 10 n M CRF for 10 min-24 h despite a rapid time- and concentration-dependent loss of CRF receptors from the retinoblastoma cell surface.     

An mRNA Encoding a Putative GABA-Gated Chloride Channel Is Expressed in the Human Cardiac Conduction System

Abstract: GABA-gated chloride channels are the main inhibitory neurotransmitter receptors in the CNS. Conserved domains among members of previously described GABA_A receptor subunits were used to design degenerate sense and antisense oligonucleotides. A PCR product from this amplification was used to isolate a full-length cDNA. The predicted protein has many of the features shared by other members of the ligand-gated ion channel family. This channel subunit has significant amino acid identity (25–40%) with members of GABA_A and GABA_C receptor subunits and thus may represent a new subfamily of the GABA receptor channel. Although we cannot rule out that this clone encodes a receptor for an unidentified ligand, it was termed GABA _χ. This gene is mainly expressed in placenta and in heart; however, placenta appears to express only an unspliced mRNA. In situ hybridization reveals that the GABA _χ subunit mRNA is present in the electrical conduction system of the human heart. Our results suggest that novel GABA receptors expressed outside of the CNS may regulate cardiac function.     

Pharmacological Characterization of Tachykinin Receptors Controlling Acetylcholine Release from Rat Striatum: An In Vivo Microdialysis Study

Abstract: The regulation of striatal cholinergic function by tachykinins was examined in urethane-anesthetized rats by using microdialysis. Substance P (0.01–1 µ M ), [Sar⁹,Met(O₂)¹¹]substance P (1–10 µ M ), septide (0.1–3 µ M ), neurokinin (NK) A (0.1–10 µ M ), and senktide (0.1–10 µ M ) produced concentration-dependent increases in striatal acetylcholine (ACh) release. Septide was the most potent agonist for inducing release of ACh, whereas the stimulating effect of senktide was less pronounced and more progressive in onset. The response to septide was prevented by intraperitoneal administration of the nonpeptide NK₁ antagonist SR 140333 (1–3 mg/kg) but not by the nonpeptide NK₂ receptor antagonist SR 48968, indicating that the effect was mediated specifically by NK₁ receptors. ACh release caused by NKA was reduced by SR 48968 (1–3 mg/kg) and slightly affected by SR 140333, indicating a principal role for NK₂ receptors in the peptide response. The similar efficacy of SR 140333 and SR 48968 in blocking substance P-induced ACh release suggested that the effect of this peptide involves the stimulation of both NK₁ and NK₂ receptors. Finally, our results indicate that the increase in striatal ACh release induced by the D₁ agonist (+)-SKF-38393 (3 µ M ) may be mediated indirectly through local release of NKA or substance P acting at NK₂ receptors.     

Development of efficient transient transfection systems for introducing antisense oligonucleotides into human epithelial skin cells

Systemic treatment with antisense oligonucleotides is confounded by the dual problems of potential cytotoxicity of antisense oligonucleotides and carrier molecules such as cationic lipids. Treatment of pathologic conditions affecting the skin may avoid these problems to a large degree due to local application. The success of antisense strategies has been limited by the poor uptake of the transfection reagent and inadequate intracellular compartmentalization. Human skin epithelial cells, therefore, are attractive experimental tools for testing both in vitro and in vivo antisense therapies. In the present study, we determined commercially available liposomes which reproducibly induced a nontoxic increase of oligonucleotide uptake in cultured SZ95 sebocytes and keratinocytes. The final protocol for SZ95 sebocytes was a 4-hour incubation with DOTAP in a 2:1 (w/w) lipid/oligonucleotide ratio in serum-free medium. The fluorescein-labeled (ATCG)(5) random oligonucleotide molecules were detected within the nucleus. The optimum transfection system for primary keratinocytes was poly-L-ornithine (12 microg/ml) in a medium without bovine pituitary extract over 4 hours. The uptake of the oligonucleotide increased in the presence of the polycation and oligonucleotide molecules were localized in the cytoplasm of keratinocytes. Oligonucleotide transfection with the help of cationic lipids did not affect the expression of androgen receptor and of the house-keeping gene beta-actin. Thus, cationic lipids are useful for delivery of antisense oligonucleotides into skin cells in vitro and may be used for topical application on animal and human skin.     

Antisense Strategy Unravels a Dopamine Receptor Distinct from the D2 Subtype, Uncoupled with Adenylyl Cyclase, Inhibiting Prolactin Release from Rat Pituitary Cells

The antisense strategy was used to unravel the functional contribution of the mRNAs encoding dopamine (DA) receptors to the multiple transduction mechanisms operated by DA in rat pituitary cells. An antisense oligonucleotide was designed to recognize seven nucleotides upstream and 11 nucleotides downstream from the initiation translation codon of the mRNA that encodes the DA D2 receptor. Addition of the antisense oligonucleotide for 7 days to primary culture of rat pituitary cells resulted in a decreased expression of DA D2 receptor as shown by (a) the virtual disappearance of [³H]spiroperidol binding sites and (b) the marked reduction in the levels of both the long and the short splice variant of the D2 receptor mRNAs. After this treatment, the DA D2 receptor agonist bromocriptine lost its capability both to inhibit adenylyl cyclase activity and to reduce prolactin mRNA levels. On the contrary, the inhibition of prolactin release induced by bromocriptine was affected minimally by the antisense oligonucleotide treatment. These data indicate that (a) translation of the mRNA encoding DA D2 receptors results in receptors that are negatively coupled with adenylyl cyclase and functionally linked to inhibition of prolactin synthesis; and (b) the release of prolactin might be regulated, at least in part, by a DA receptor that is encoded by mRNA species distinct from those encoding the D2 receptor.     

Antisense oligonucleotide inhibition of hepatitis C virus gene expression in transformed hepatocytes.

Genetic and biochemical studies have provided convincing evidence that the 5' noncoding region (5' NCR) of hepatitis C virus (HCV) is highly conserved among viral isolates worldwide and that translation of HCV is directed by an internal ribosome entry site (IRES) located within the 5' NCR. We have investigated inhibition of HCV gene expression using antisense oligonucleotides complementary to the 5' NCR, translation initiation codon, and core protein coding sequences. Oligonucleotides were evaluated for activity after treatment of a human hepatocyte cell line expressing the HCV 5' NCR, core protein coding sequences, and the majority of the envelope gene (E1). More than 50 oligonucleotides were evaluated for inhibition of HCV RNA and protein expression. Two oligonucleotides, ISIS 6095, targeted to a stem-loop structure within the 5' NCR known to be important for IRES function, and ISIS 6547, targeted to sequences spanning the AUG used for initiation of HCV polyprotein translation, were found to be the most effective at inhibiting HCV gene expression. ISIS 6095 and 6547 caused concentration-dependent reductions in HCV RNA and protein levels, with 50% inhibitory concentrations of 0.1 to 0.2 microM. Reduction of RNA levels, and subsequently protein levels, by these phosphorothioate oligonucleotides was consistent with RNase H cleavage of RNA at the site of oligonucleotide hybridization. Chemically modified HCV antisense phosphodiester oligonucleotides were designed and evaluated for inhibition of core protein expression to identify oligonucleotides and HCV target sequences that do not require RNase H activity to inhibit expression. A uniformly modified 2'-methoxyethoxy phosphodiester antisense oligonucleotide complementary to the initiator AUG reduced HCV core protein levels as effectively as phosphorothioate oligonucleotide ISIS 6095 but without reducing HCV RNA levels. Results of our studies show that HCV gene expression is reduced by antisense oligonucleotides and demonstrate that it is feasible to design antisense oligonucleotide inhibitors of translation that do not require RNase H activation. The data demonstrate that chemically modified antisense oligonucleotides can be used as tools to identify important regulatory sequences and/or structures important for efficient translation of HCV.     

NMDA Receptor Subtype Selectivity: Eliprodil, Polyamine Spider Toxins, Dextromethorphan, and Desipramine Selectively Block NMDA-Evoked Striatal Acetylcholine but Not Spermidine Release

Abstract: NMDA receptor stimulation concomitantly increases the release of [¹⁴C]acetylcholine and [³H]spermidine from rat striatal slices in vitro. The NMDA-induced release of both acetylcholine and spermidine was blocked with equal potency by the NMDA channel blocker phencyclidine (0.1–10 µ M ). However, certain other channel blockers, including dextromethorphan (1–100 µ M ), which antagonized NMDA-evoked acetylcholine release without affecting NMDA-evoked spermidine release, and dextrorphan (1–100 µ M ) and memantine (1–100 µ M ), which block NMDA-evoked acetylcholine release more potently than NMDA-evoked spermidine release, showed greater selectivity of action. As previously shown for ifenprodil, eliprodil (SL82.0715; 1–100 µ M ) blocked NMDA-evoked acetylcholine but not spermidine release. This selectivity is also observed for other agents interacting with the polyamine site(s) on the NMDA receptor, including arcaine (1–1,000 µ M ), philanthotoxin₃₄₃, and argiotoxin₆₃₆ (10 µ M ) and was also noted for desipramine (1–100 µ M ). The NMDA-induced release of acetylcholine and spermidine is likely to be mediated by different native NMDA receptor subtypes, and several NMDA antagonists may be candidates for a selective action at a particular NMDA receptor subtype.     

Stimulation of Neuropeptide Y Overflow in the Rat Paraventricular Nucleus by Corticotropin-Releasing Factor

Abstract: Neuropeptide Y (NPY) and corticotropin-releasing factor (CRF) are present at high concentrations in the hypothalamus where they mediate important endocrine and autonomic functions. Morphological and physiological studies have suggested an interaction between these peptides, and opposing actions of CRF and NPY have been reported on feeding and other behaviors. This study investigated the effect of CRF on NPY release in vivo, measured by push-pull techniques, in the anesthetized rat. Push-pull probes implanted into the paraventricular nucleus of the hypothalamus (PVN) were perfused with modified Ringer solution containing bovine serum albumin at 15 µl/min, and the perfusate was lyophilized prior to NPY radioimmunoassay. NPY overflow from the rat PVN was increased threefold by perfusion of a depolarizing concentration of potassium (50 mmol/L KCI). When CRF was administered into the PVN via the push-pull cannula at 1 or 5 µg/ml, dose-dependent increases in NPY overflow of two- and fivefold were observed ( p < 0.05). These increases were abolished by prior intracerebroventricular (i.c.v.) administration of the CRF antagonist [ d -Phe¹²,Nle^21,38,C^αMeLeu³²]CRF (12–41) at 1 or 5 µg/µl, respectively. NPY overflow returned promptly to resting levels following CRF administration. In contrast, when CRF was administered by i.c.v. bolus at a similar total dose (2 µg), no significant effect on NPY overflow was observed. These data provide in vivo evidence for an interaction between CRF and NPY at the level of the PVN.     

Antisense Oligonucleotide-Induced Reduction in 5-Hydroxytryptamine7 Receptors in the Rat Hypothalamus Without Alteration in Exploratory Behaviour or Neuroendocrine Function

Abstract: The effect of a 5-hydroxytryptamine₇ (5-HT₇) receptor-directed antisense oligonucleotide on rat behaviour and neuroendocrine function was investigated. Six days of intracerebroventricular 5-HT₇ antisense oligonucleotide treatment significantly reduced [³H]5-HT binding to hypothalamic 5-HT₇ receptors, whereas cortical 5-HT_2C density remained unchanged. In rats on a food-restricted diet, both antisense and mismatch oligonucleotides reduced food intake and body weight compared with that in vehicle-treated controls by day 4 of administration. 5-HT₇ antisense oligonucleotide administration did not affect exploratory or locomotor activity in photocell activity monitors on day 4 or elevated plus-maze behaviour on day 6 of intracerebroventricular treatment. 5-HT₇ antisense oligonucleotide did not affect plasma corticosterone or prolactin levels or 5-HT turnover in either 5-HT cell body or terminal areas. These data demonstrate that intracerebroventricular 5-HT₇ antisense oligonucleotide administration selectively reduced rat hypothalamic 5-HT₇ receptor density without affecting any of the biochemical or behavioural measures. The results suggest that this antisense protocol could be a valuable tool to investigate central 5-HT₇ receptor functions, and that this receptor is not critical for the control of neuroendocrine function or food intake.     

GRK3 mediates desensitization of CRF1 receptors: a potential mechanism regulating stress adaptation

Potential G protein-coupled receptor kinase (GRK) and protein kinase A (PKA) mediation of homologous desensitization of corticotropin-releasing factor type 1 (CRF1) receptors was investigated in human retinoblastoma Y-79 cells. Inhibition of PKA activity by PKI(5-22) or H-89 failed to attenuate homologous desensitization of CRF1 receptors, and direct activation of PKA by forskolin or dibutyryl cAMP failed to desensitize CRF-induced cAMP accumulation. However, treatment of permeabilized Y-79 cells with heparin, a nonselective GRK inhibitor, reduced homologous desensitization of CRF1 receptors by approximately 35%. Furthermore, Y-79 cell uptake of a GRK3 antisense oligonucleotide (ODN), but not of a random or mismatched ODN, reduced GRK3 mRNA expression by approximately 50% without altering GRK2 mRNA expression and inhibited homologous desensitization of CRF1 receptors by approximately 55%. Finally, Y-79 cells transfected with a GRK3 antisense cDNA construct exhibited an approximately 50% reduction in GRK3 protein expression and an ~65% reduction in homologous desensitization of CRF1 receptors. We conclude that GRK3 contributes importantly to the homologous desensitization of CRF1 receptors in Y-79 cells, a brain-derived cell line.     

Antisense oligonucleotide inhibition of encephalomyocarditis virus RNA translation

We report the inhibition of encephalomyocarditis virus (EMCV) RNA translation in cell-free rabbit reticulocyte lysates by antisense oligonucleotides (13-17-base oligomers) complementary to (a) the viral 5' non-translated region, (b) the AUG start codon and (c) the coding sequence. Our results demonstrate that the extent of translation inhibition is dependent on the region where the complementary oligonucleotides bind. Non-complementary and 3'-non-translated-region-specific oligonucleotides had no effect on translation. A significant degree of translation inhibition was obtained with oligonucleotides complementary to the viral 5' non-translated region and AUG initiation codon. Digestion of the oligonucleotide:RNA hybrid by RNase H did not significantly increase translation inhibition in the case of 5'-non-translated-region-specific and initiator-AUG-specific oligonucleotides; in contrast, RNase H digestion was necessary for inhibition by the coding-region-specific oligonucleotide. We propose that (a) 5'-non-translated-region-specific oligonucleotides inhibit translation by affecting the 40S ribosome binding and/or passage to the AUG start codon, (b) AUG-specific oligonucleotides inhibit translation initiation by inhibiting the formation of an active 80S ribosome and (c) the coding-region-specific oligonucleotide does not prevent protein synthesis because the translating 80S ribosome can dislodge the oligonucleotide from the EMCV RNA template.     

Properties of circular dumbbell RNA/DNA chimeric oligonucleotides containing antisense phosphodiester oligonucleotides.

We have designed a new class of oligonucleotides, "dumbbell RNA/DNA chimeric phosphodiesters", containing two alkyl loop structures with RNA/DNA base pairs (sense (RNA) and antisense (DNA) in the double helical stem. The reaction of nicked (NDRDON) and circular (CDRDON) dumbbell RNA/DNA chimeric oligonucleotides with RNaseH gave the corresponding antisense phosphodiester oligonucleotide together with the sense RNA cleavage products. The liberated antisense phosphodiester oligodeoxynucleotide was bound to the target 35mer RNA, which gave 35mer RNA cleavage products by treatment with RNaseH. The circular dumbbell RNA/DNA chimeric oligonucleotide showed more nuclease resistance than the linear antisense phosphodiester oligodeoxynucleotide(anti-ODN) and the nicked dumbbell RNA/DNA chimeric oligonucleotide.     

Stimulation of group I mGlu receptors in the ventrotegmental area enhances extracellular dopamine in the rat medial prefrontal cortex

Group I mGlu receptors have been implicated in the control of brain dopamine release. However, the receptor subtype involved and the precise site of action have not been determined. In this study we show that (R,S)3,5-dihydroxyphenylglycine (DHPG; 6 and 60 nmol ICV), a selective group I mGlu receptor agonist, raised extracellular dopamine respectively by 176% and 243% of basal values in the medial prefrontal cortex as assessed by in vivo microdialysis in conscious rats. (R,S)2-chloro-5-hydroxyphenylglycine (60 nmol ICV), a selective mGlu5 receptor agonist, raised extracellular dopamine by 396% of basal values. Intra-VTA DHPG (0.6–6 nmol) mimicked ICV injection whereas intracortical infusion (1–1000 µmol/L) had no effect. DHPG-induced rise of extracellular dopamine was reversed by tetrodotoxin and by the selective mGlu1 and mGlu5 receptor antagonists 7(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate (CPCCOEt) and 2-methyl-6-(phenylethynyl)pyridine (MPEP) either ICV or into the ventrotegmental area (VTA), suggesting that neuronal release and both mGlu1 and mGlu5 receptors were involved. These results support the existence of functional mGlu1 and mGlu5 receptors in the VTA regulating the release of dopamine in the medial prefrontal cortex.     

NMDA and Non-NMDA Ionotropic Glutamate Receptors Modulate Striatal Acetylcholine Release via Pre- and Postsynaptic Mechanisms

Abstract: The effects of NMDA and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) on endogenous acetylcholine release from rat striatal slices and synaptosomes were investigated. Both agonists (1–300 µ M ) facilitated acetylcholine release from slices in a dose-dependent manner. NMDA (100–300 µ M ) and AMPA (30–300 µ M ), however, subsequently inhibited acetylcholine release. NMDA (100 µ M )-induced facilitation was antagonized by 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) and dizocilpine (both 1–10 µ M ), whereas the 10 µ M AMPA effect was antagonized by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 1–30 µ M ). NMDA (100 µ M )-induced inhibition was counteracted by CPP, but not dizocilpine, and by the nitric oxide synthase inhibitor l -nitroarginine (1–100 µ M ). Tetrodotoxin (0.5 µ M ) prevented the facilitatory effect of 3 µ M NMDA and AMPA, but left unchanged that of 30 µ M NMDA and 100 µ M AMPA. Acetylcholine release from synaptosomes was stimulated by KCI (7.5–100 m M ) in a dose-dependent manner. NMDA and AMPA maximally potentiated the 20 m M KCl effect at 1 µ M and 0.01 µ M , but were ineffective at 100 µ M and 10 µ M , respectively. Inhibition of acetylcholine release was never found in synaptosomes. The effects of 1 µ M NMDA and 0.01 µ M AMPA were antagonized by CPP (0.0001–1 µ M ) or dizocilpine (0.0001–10 µ M ) and by CNQX (0.001–1 µ M ), respectively. These data suggest that glutamatergic control of striatal acetylcholine release is mediated via both pre- and post-synaptic NMDA and non-NMDA ionotropic receptors.     

Kainate-Evoked Release of Adenosine from the Hippocampus of the Anaesthetised Rat: Possible Involvement of Free Radicals

Abstract: Using microdialysis in the hippocampus of anaesthetised rats, the concentration of extracellular adenosine was estimated to be 0.8 µ M . Kainic acid (0.1–25 m M ) in the perfusate evoked a concentration-dependent release of adenosine with an EC₅₀ of 940 µ M . Two 5-min pulses of 1 m M kainic acid in the perfusate increased the dialysate levels with an S2/S1 ratio of 0.52 ± 0.03. Kainate-evoked release of adenosine was reduced significantly by 10 µ M tetrodotoxin and by a κ-receptor agonist, U50,488H (100 µ M ). The S2/S1 ratio was reduced by 4.5 µ M 6-cyano-7-nitroquinoxaline-2,3-dione, a non-NMDA receptor antagonist, but not by the NMDA receptor blockers (+)-MK-801 (dizocilpine; 100 µ M ) or (±)-2-amino-5-phosphonopentanoic acid (1 m M ), indicating a non-NMDA receptor-mediated process. The S2/S1 ratio was also reduced significantly by 10 m M ascorbic acid, 10 m M glutathione (a scavenger of hydroperoxides), and 1 m M oxypurinol (a xanthine oxidase inhibitor), indicating the possible involvement of free radicals. Neither the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine (100 µ M ) nor the A1 adenosine receptor agonist R (−)- N ⁶-(2-phenylisopropyl)adenosine (100 µ M ) affected release. Adenosine release evoked by kainic acid is therefore mediated by activation of non-NMDA receptors and may involve the propagation of action potentials and the production of free radicals.     

Increased reactive oxygen species production with antisense oligonucleotides directed against uncoupling protein 2 in murine endothelial cells.

Uncoupling protein 2 (UCP-2) belongs to the mitochondrial anion carrier family. It is ubiquitously expressed but is most abdundant in the reticuloendothelial system. In addition to uncoupling function, UCP-2 modulates the production of reactive oxygen species (ROS) by isolated mitochondria. Using an antisense oligonucleotide strategy, we investigated whether a defect in UCP-2 expression modulates ROS in intact endothelial cells. Murine endothelial cells (CRL 2181) pretreated by antisense oligonucleotides directed against UCP-2 mRNA exhibited a significant and specific increase in membrane potential and intracellular ROS level compared with control scrambled or anti-UCP-1 and -UCP-3 antisense oligonucleotides. These specific changes induced by UCP-2 antisense oligonucleotides were correlated with a rise in extracellular superoxide anion production and oxidative stress assessed by thiobarbituric acid reactive substance values. Taken together, these data suggest a role for UCP-2 in control of ROS production and subsequent oxidation of surrounding compounds mediating oxidative stress of endothelial cells. These data also support the notion that manipulations of UCP-2 at the genetic level could control ROS metabolism at the cellular level.