Introduction

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Norepinephrine (NE) is a major neurotransmitter in the descending inhibition of nociceptive transmission at the spinal level (Fields and Basbaum, 1978). Activation of bulbospinal noradrenergic pathways by stimulation of the periaqueductal gray results in spinal NE release associated with inhibition of spinal cord dorsal horn responses to nociceptive stimuli (Cui et al., 1999). Similarly, systemically administered opioids produce antinociception through activation descending noradrenergic pathways, as evidenced by spinal release of NE from i.v. morphine in animals and humans (Bouaziz et al., 1996). Such antinociceptive effect is blocked by spinally administered noradrenergic antagonists (Tseng and Tang, 1989). Descending noradrenergic inhibition also is activated by noxious peripheral input because noxious nerve stimulation increases spinal NE release (Men and Matsui, 1994) and produces analgesia (Steinman et al., 1983). Finally, spinal administration of NE produces antinociception in animals by stimulation of ₂-adrenergic receptors (Howe et al., 1983) and spinal administration of ₂-adrenergic agonists produces analgesia in humans (Eisenach et al., 1996a).

Stimulation of presynaptic ₂-adrenergic receptors on noradrenergic nerves is thought classically to diminish NE release, both in the periphery and in the central nervous system (Langer et al., 1985). In spinal cord slices, NE release from electrical stimulation is inhibited by exposure to ₂-adrenergic agonists, consistent with this classical effect (Umeda et al., 1997). However, in vivo data demonstrate a paradoxical increase in spinal NE from ₂-adrenergic agonists (Klimscha et al., 1997), and a decrease in spinal NE from ₂-adrenergic antagonists when NE is stimulated by systemic morphine (Bouaziz et al., 1996) or noxious peripheral nerve stimulation (Eisenach et al., 1996b).

The paradoxical action of ₂-adrenergic receptors on spinal NE release may be explained in part by an interaction with nitric oxide (NO). Neuronal NO synthase is concentrated in the dorsal horn of the spinal cord (Terenghi et al., 1993), and NO synthesis is necessary for development of hypersensitivity states after peripheral tissue or nerve injury (Meller and Gebhart, 1993). In contrast to this pain-enhancing effect, NO also can participate in analgesia at the spinal level. Thus, intrathecal administration of ₂-adrenergic agonists and i.v. morphine stimulate spinal NO synthesis and produce analgesia, which is blocked by NO synthase inhibitors (Xu et al., 1996; Pan et al., 1998; Song et al., 1998). NO has been shown to react under physiologic conditions with NE to produce 6-nitro-norepinephrine (6-NO₂-NE) (de la Breteche et al., 1994). 6-NO₂-NE is present in mammalian brain tissue and stimulates NE release in brain tissue in vitro and in microdialysis experiments in vivo (Shintani et al., 1996). We recently demonstrated the presence in spinal cord tissue of 6-NO₂-NE, increased formation of 6-NO₂-NE from spinal injection of NE in vivo, and release of NE from spinal cord after exposure to 6-NO₂-NE from intrathecal injection of microdialysis delivery (A.C., submitted for publication).

The purpose of the current study was to examine the mechanisms by which 6-NO₂-NE induces NE release in the spinal cord. Because all noradrenergic innervation of the spinal cord is extrinsic (Roy et al., 1991), the effect most likely occurs locally on noradrenergic terminals. Previous studies in vivo and in spinal cord slices have demonstrated that 6-NO₂-NE induces NE release (A.C., submitted). These studies suggest a local effect, but do not exclude activation of a spinal circuit to stimulate heterotopic excitatory receptors on noradrenergic terminals. Therefore, in the current study, we used a synaptosomal preparation to directly investigate actions of 6-NO₂-NE on noradrenergic terminals, in the absence of intact local neuronal circuits. Three mechanisms were specifically tested: 1) a stimulation of guanylate cyclase by decomposition to NO or other free radicals that could activate this enzyme; 2) an interaction with the NE transporter to inhibit uptake; and 3) Na⁺- and Ca²⁺ dependence of release of NE.

	Experimental Procedures

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Synaptosome Preparation. After obtaining Animal Care and Use Committee approval, male Sprague-Dawley rats (250 g) were studied. After induction of anesthesia with 1.5 to 2.1% inhalational halothane, animals were sacrificed by decapitation, and the spinal cord was quickly removed and placed in aerated (95% O₂/5% CO₂) ice-cold modified Krebs-bicarbonate buffer containing 118 mM NaCl, 3.3 mM KCl, 1.2 mM MgSO₄, 1.25 mM CaCl₂, 1.2 mM KH₂PO₄, 25 mM NaHCO₃, 10 mM HEPES, 5 mM ascorbic acid, 11.5 mM glucose, 30 µM EDTA, and 10 µM pargyline. The dorsal half of the spinal cord was selected and homogenized in 8 ml of ice-cold 0.32 M sucrose. A crude synaptosomal pellet (P₂) was prepared by differential centrifugation at 2,000g followed by 20,000g (Lonart and Johnson 1995b).

[³H]NE Release. The crude P₂ pellet was resuspended into 4 ml of modified Krebs buffer, loaded with NE in a 50 nM final concentration containing 20% [³H]NE and incubated at 37°C for 5 min. The free NE was then removed by centrifugation at 15,000g for 10 min. The synaptosomal pellet was again suspended into 4.5 ml of modified Krebs buffer, and 150 µl of the suspension was aliquoted into each test tube with 850 µl of Krebs buffer containing 6-NO₂-NE at final concentrations ranging from 0 to 500 µM. The test tubes were then incubated for 10 min at 37°C in a 1-ml volume. At the end of incubation, the amount of [³H] remaining in synaptosomes was determined by rapid filtration through GF/C Glass fibers presoaked for 30 min or more in 0.1% (v/v) polyethylenimine to reduce nonspecific binding. This was followed by 4-ml washes (3×) with ice-cold buffer in which glucose was substituted for NaCl. The bound radioactivity was determined 24 h later by 1219 Rack Beta Scintillation Counter (LKB, Wallac Inc., Gaitherburg, MD) in Bio Safe II scintillation fluid. The [³H]NE release induced by 6-NO₂-NE was calculated from the amount of [³H]NE remaining in the synaptosome after vehicle (100 µl of buffer) compared with 6-NO₂-NE treatment. The influence of ionic composition and various antagonists on 6-NO₂-NE-induced NE release was determined in separate experiments comparing 6-NO₂-NE containing solutions alone or with various concentrations of antagonists or in solutions of differing ionic composition.

[³H]NE Uptake. The synaptosome P₂ pellet of one rat was suspended into 4.5 ml of modified Krebs buffer. One hundred and fifty microliters of the P₂ suspension was aliquoted into test tubes with 750 µl of the modified Krebs buffer containing 6-NO₂-NE in a final concentration of 0 to 500 µM. The test tubes were incubated in a 37°C water bath for 5 min, and then were added to 100 µl of a 500 nM NE mixture containing 100 nM [³H]NE. The test tubes were incubated in a 37°C water bath for an additional 5 min. [³H]NE uptake was determined by rapid filtration as described above. Values were corrected for nonspecific uptake, determined in the presence of 10 µM desipramine.

Materials. L-[2,5,6-³H]NE (62 Ci/mmol) was purchased from New England Nuclear (Wilmington, DE). LY83583, ODQ (1H-[1,2,4] oxadiazolo [4,3,a] quinoxalin-1-one) and 6-NO₂-NE were obtained from Research Biochemicals (Natick, MA). Bio Safe II scintillation cocktail was from Research Product International Corp (Mount Prospect, IL). MgSO₄, ascorbic acid, KCl, and glucose were from Fisher Scientific (Fairlawn, NJ). Hemoglobin, desipramine, nomifensine, and the remaining chemicals were from Sigma Chemical Co. (St. Louis, MO).

Data Analysis. All release experiments were performed in four sets in duplicate. The percentage of release of NE was calculated by dividing the loss of radioactivity in each sample against the basal radioactivity in the control sample without 6-NO₂-NE. Data are presented as means ± S.E. In NE uptake experiments, four or five sets of experiments were performed at different 6-NO₂-NE concentrations in duplicate with and without 10 µM desipramine. The percentage of uptake was calculated by dividing the radioactivity level in each sample against that in the control sample. Data were analyzed by one- or two-way ANOVA, with P < .05 considered significant.

	Results

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Incubation of synaptosomes with 6-NO₂-NE yielded a reproducible, concentration-dependent release of [³H]NE, as depicted in Fig. 1, which is a summary of all control experiments (n = 26). 6-NO₂-NE induced a significant NE release at 10 µM, with a maximal release of ~30% (EC₅₀ of 30 ± 8.2 µM; Fig. 1).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Concentration-dependent increase in [3H]NE release from preloaded spinal cord synaptosomes by incubation with 6-NO2-NE.

Two structurally dissimilar NE transporter inhibitors, desipramine and nomifensine, when included in the reaction mixture at 10 µM, produced a profound inhibition of 6-NO₂-NE induced NE release from spinal cord synaptosomes (Fig. 2). The concentration dependence of this inhibition was examined only for desipramine, which exhibited a complete blockade of the 6-NO₂-NE effect at concentrations 100 µM, and an IC₅₀ of 9.6 ± 2.6 µM (Fig. 3).

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Inhibition of [3H]NE release from spinal cord synaptosomes from 6-NO2-NE by 10 µM desipramine (left) or nomifensine (right). Synaptosomes were incubated with 6-NO2-NE alone () or with the NE transporter inhibitors ().

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Concentration-dependent inhibition of [3H]NE release from spinal cord synaptosomes from 6-NO2-NE, 50 µM, by desipramine.

The dependence of activation of guanylate cyclase on NE release from 6-NO₂-NE was investigated with two structurally dissimilar agents. Fractional NE release from 50 µM 6-NO₂-NE alone was 23 ± 0.9% in experiments with LY 85,583 and 23 ± 0.9% in experiments with ODQ. Neither LY 83,583 nor ODQ affects synaptosomal NE release alone, nor did they affect NE release induced by 6-NO2-NE (Fig. 4).

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Lack of inhibition of [3H]-norepinephrine release from spinal cord synaptosomes from 50 µM 6-nitro-norepinephrine by ODQ (left) or LY 83,583 (right). Synaptosomes were incubated with these inhibitors of guanylyl cyclase alone () or with 6-nitro-norepinephrine ().

The possibility that 6-NO2-NE could cause NE release first by inducing formation of NO or by decomposing to NO was tested by incubation with oxyhemoglobin, from 10⁶ to 10² M. Oxyhemoglobin had no effect alone on NE release (1.2 ± 3.4% fractional release at 10² M). Similarly, oxyhemoglobin did not reduce NE release from 6-NO2-NE, 50 µM (22 ± 2.7% fractional release from 6-NO2-NE alone, 23 ± 3.1% release in the presence of 10² M oxyhemoglobin).

A series of experiments was performed to determine the dependence of external Na⁺ and Ca²⁺ on 6-NO₂-NE-induced NE release. Removal of Ca²⁺ from the incubation buffer had no effect on NE release from 6-NO₂-NE, nor did removal of Ca²⁺ and chelation of any residual Ca²⁺ by addition of 1 mM ethylene glycol bis(-aininoethyl ether)-N,N,N',N',-tetraacetic acid (Fig. 5). In contrast, there was a clear dependence on external Na⁺ for the effect of 6-NO₂-NE. Replacement of NaCl with choline chloride significantly reduced the effect of 6-NO₂-NE, although 1 mM 6-NO₂-NE still caused some NE release (Fig. 6). Removal of the remaining Na⁺ in the buffer by substituting HEPES for NaHCO₃ completely abolished the effect of 6-NO₂-NE (Fig. 6).

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Lack of dependence on Ca2+ of [3H]NE release from 6-NO2-NE in spinal cord synaptosomes incubated in the presence of 1.25 mM external Ca2+ (), no added external Ca2+ (), or no added Ca2+ plus addition of the Ca2+ chelator ethylene glycol bis(-aininoethyl ether)-N,N,N',N',-tetraacetic acid (). No difference among groups.

View larger version (26K): [in this window] [in a new window]   Fig. 6.   Dependence on Na+ of [3H]NE release from 6-NO2-NE in spinal cord synaptosomes incubated in the presence of 118 mM NaCl plus 25 mM NaHCO3 (), 118 mM choline chloride plus 25 mM NaHCO3 (), or no added Na+ ().

In addition to its ability to release NE, 6-NO₂-NE inhibited NE uptake into synaptosomes. This effect was present at 1 µM, yielded a complete blockade of NE uptake at concentrations of 6-NO₂-NE 100 µM, and exhibited an IC₅₀ of 8.3 ± 2.2 µM (Fig. 7).

View larger version (17K): [in this window] [in a new window]   Fig. 7.   Concentration-dependent inhibition of [3H]NE uptake by spinal cord synaptosomes by incubation with 6-NO2-NE.

	Discussion

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The bases for this study are the dual observations that spinal ₂-adrenergic receptor stimulation enhances, whereas ₂-adrenergic receptor blockade diminishes stimulated NE release (Bouaziz et al., 1996; Klimscha et al., 1997), and the presence of 6-NO₂-NE in normal spinal cord (A. Chiari, submitted). The positive feedback loop in the spinal cord of NE, acting on ₂-adrenergic receptors to further stimulate NE release is contrary to the classic function of autoinhibitory ₂-adrenergic receptors (Langer et al., 1985) and has been demonstrated to involve an NE  ₂-adrenergic receptor  acetylcholine release  NO synthesis cascade (Xu et al., 1997). Previous observations that 6-NO₂-NE stimulates NE release in brain (Shintani et al., 1996) and the presence of 6-NO2-NE and its NE releasing action in vivo in spinal cord (A. Chiari, submitted) support 6-NO₂-NE as a likely candidate in this positive feedback loop involving NO synthesis.

NO has long been recognized to stimulate neurotransmitter release, including NE release, in brain (Lonart and Johnson, 1995a,b), although the mechanisms by which it does so remain unclear. In some studies, NO donors enhance NE release only in the presence of thiol compounds (Satoh et al., 1996), whereas others observe NE release by NO donors alone as well as S-nitroso-thiol compounds (Lonart and Johnson, 1995a,b). These experimental conditions include isolated terminals, simplified cell monocultures, in vitro slices, and in vivo microdialysis. Following is a discussion of the current results and previous studies of the actions of 6-NO₂-NE in the context of these examinations of NO-induced NE release.

6-NO₂-NE does not interact with ₁-, ₂-, or -adrenoceptors in cortical membranes (Shintani et al., 1996), although it produces vasoconstriction in rat aorta that is partially blocked by the ₁-adrenergic antagonist prazosin (Nakaki et al., 1998). Two mechanisms have been proposed for the NE-releasing action of 6-NO₂-NE: catechol O-methyltransferase inhibition (IC₅₀ of 7.5 µM) and inhibition of NE reuptake (IC₅₀ of 31 µM in rat brain) (Shintani et al., 1996). The current study, demonstrating an IC₅₀ of 8.3 µM for inhibition of NE uptake in rat spinal cord, is consistent with these observations. As with any in vitro experiment, extrapolation of concentrations relevant to in vivo function of the proposed substance is difficult. Functional studies indicate that 6-NO2-NE is formed in spinal cord tissue and regulates NE release and its behavioral effects (antinociception) in vivo (A. Chiari, submitted).

Interpretation of the current results is complicated by the multiple mechanisms by which an agent may affect extrasynaptic neurotransmitter concentrations. It is unlikely that 6-NO₂-NE is mimicking the actions of NE itself. NE itself does not stimulate its own release in synaptosomal preparations and may reduce its own release by actions on presynaptic ₂-adrenergic receptors. Preliminary experiments in rat spinal cord synaptosomes demonstrate the existence of such ₂-adrenergic inhibitory receptors regulating NE release.

Inhibition of NE uptake by 6-NO₂-NE supports a role for the NE transporter in the action of 6-NO₂-NE, but this observation does not distinguish among the following possibilities: 1) direct action to inhibit transporter function, 2) transporter-mediated entry of 6-NO2-NE to its site of action intracellularly, and 3) dependence on reverse transport for NE release induced by 6-NO₂-NE.

The first possibility, a direct action to inhibit transporter function, is unlikely because NE release from 6-NO₂-NE is blocked, not enhanced by the pure transporter inhibitors nomifensine and desipramine. This is consistent with blockade of S-nitroso-thiol compound-induced NE release by NE transporter inhibitors in brain slices (Lonart and Johnson, 1995a,b) and PC12 cells in culture (Kaye et al., 1997), although NE release from free NO was not inhibited by desmethylimipramine in brain slices (Stout and Woodward, 1995).

The second possibility, competition for transporter-mediated intracellular entry between NE and 6-NO₂-NE, is supported by the Na⁺ dependence of 6-NO₂-NE-induced NE release. The uptake 1 mechanism for NE transport into synaptic terminals is sensitive to blockers such as nomifensine and desipramine and is dependent on the normal Na⁺ gradient across the cell membrane (Kanner, 1994). Only one previous study examined the Na⁺ dependence of NO-induced NE release, and documented a 25 to 50% reduction in this release in hippocampal slices when choline chloride was substituted for NaCl (Satoh et al., 1997). Similarly, we observed a 50% inhibition of NE release from 1 mM 6-NO₂-NE by substitution with choline chloride for NaCl, and a complete blockade when the remaining Na⁺ in the extracellular solution was eliminated.

The third possibility, dependence on reverse transport for NE release induced by 6-NO₂-NE, is supported by inhibition by nomifensine and desipramine. We propose that 6-NO₂-NE shares the same mechanism as amphetamine in stimulating NE release. Amphetamine enters monoaminergic terminals via specific monoamine transporters, and its monoamine-releasing effects are blocked by transporter inhibitors, similar to 6-NO₂-NE (Raiteri et al., 1979). Like 6-NO₂-NE, amphetamine is a weak base, and accumulation of amphetamine in vesicles disrupts the normal pH gradient that is responsible for concentrating monoamines within synaptic vesicles (Sulzer and Rayport, 1990; Sulzer et al., 1993). The resultant large increase in cytoplasmic concentrations of monoamines leads to release via reverse transport. This mechanism is Ca²⁺ independent, as observed for the NE-releasing effect of 6-NO₂-NE in the current study and the Ca²⁺-independent release of NE from NO donors previously observed in synaptosomes (Meffert et al., 1994) and in hippocampal slices (Satoh et al., 1996) (but see Lonart and Johnson, 1995a). Thus, the current studies suggest that the naturally occurring substance 6-NO₂-NE may be a candidate for an endogenous amphetamine-like substance in brain and in spinal cord.

An action of 6-NO₂-NE to release NO and stimulate guanylate cyclase activity in producing NE release is unlikely for several reasons. There is no strong reducing intracellular environment to cleave and reduce the nitro group on this compound, and scavenging for NO with oxyhemoglobin does not affect NE release from 6-NO₂-NE. The current study observed no inhibition of 6-NO₂-NE-induced NE release by compounds that inhibit guanylate cyclase activity. Similarly, others failed to observe blockade by guanylate cyclase inhibitors of NO donor-induced NE release in synaptosomes (Meffert et al., 1994) or in hippocampal slices (Lonart and Johnson, 1995a; Stout and Woodward, 1995; Satoh et al., 1996).

In summary, our previous studies document the presence of 6-NO₂-NE in spinal cord, its interaction with noradrenergic terminals to enhance antinociception, and its NE-releasing properties in vivo (A.C., submitted). This suggests that formation of 6-NO₂-NE and subsequent stimulation of NE release may underlie the paradoxical observation of increased spinal cord NE in the presence of ₂-adrenergic receptor stimulation and decreased spinal cord NE in the presence of ₂-adrenergic antagonists. The current results suggest that 6-NO₂-NE acts to release NE by entering noradrenergic terminals via the norepinephrine transporter, where it induces NE release by a Ca²⁺-independent manner, via reverse transport.