Dynorphin A Elicits an Increase in Intracellular Calcium in Cultured Neurons Via a Non-Opioid, Non-NMDA Mechanism

Qingbo Tang,1 Ronald M. Lynch,2 Frank Porreca,1 and Josephine Lai1

 1Department of Pharmacology and  2Department of Physiology, University of Arizona Health Sciences Center, Tucson, AZ 85724


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Tang, Qingbo, Ronald M. Lynch, Frank Porreca, and Josephine Lai. Dynorphin A Elicits an Increase in Intracellular Calcium in Cultured Neurons Via a Non-Opioid, Non-NMDA Mechanism. J. Neurophysiol. 83: 2610-2615, 2000. The opioid peptide dynorphin A is known to elicit a number of pathological effects that may result from neuronal excitotoxicity. An up-regulation of this peptide has also been causally related to the dysesthesia associated with inflammation and nerve injury. These effects of dynorphin A are not mediated through opioid receptor activation but can be effectively blocked by pretreatment with N-methyl-D-aspartate (NMDA) receptor antagonists, thus implicating the excitatory amino acid system as a mediator of the actions of dynorphin A and/or its fragments. A direct interaction between dynorphin A and the NMDA receptors has been well established; however the physiological relevance of this interaction remains equivocal. This study examined whether dynorphin A elicits a neuronal excitatory effect that may underlie its activation of the NMDA receptors. Calcium imaging of individual cultured cortical neurons showed that the nonopioid peptide dynorphin A(2-17) induced a time- and dose-dependent increase in intracellular calcium. This excitatory effect of dynorphin A(2-17) was insensitive to (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine (MK-801) pretreatment in NMDA-responsive cells. Thus dynorphin A stimulates neuronal cells via a nonopioid, non-NMDA mechanism. This excitatory action of dynorphin A could modulate NMDA receptor activity in vivo by enhancing excitatory neurotransmitter release or by potentiating NMDA receptor function in a calcium-dependent manner. Further characterization of this novel site of action of dynorphin A may provide new insight into the underlying mechanisms of dynorphin excitotoxicity and its pathological role in neuropathy.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Exogenously applied dynorphin elicits a variety of effects that vary from neurotoxicity (Caudle and Isaac 1987) to inhibition of calcium potentials (Werz and Macdonald 1985). Some of these effects, including loss of tail-flick and persistent hindlimb paralysis, are equally well produced with (des-Tyrosyl)dynorphin [dynorphin A(2-17)], a major biotransformation product of dynorphin A, which has very low affinity for opioid receptors (Walker et al. 1982). This suggests that a number of neurotoxic effects of dynorphin A are not mediated by opioid receptors. The physiological implication of the nonopioid actions of dynorphin is particularly relevant to animal models of peripheral neuropathy as a consequence of inflammation or nerve injury. These conditions give rise to abnormal pain states, including hyperalgesia and allodynia, and are associated with an elevated level of spinal dynorphin (Drasci et al. 1991; Kajander et al. 1990). In spinal nerve ligation injury models, antiserum to dynorphin A has the same profile of actions as the N-methyl-D-aspartate (NMDA) receptor antagonist (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine (MK-801) in blocking thermal hyperalgesia and restoring the efficacy of morphine against allodynia (Nichols et al. 1997). Neither dynorphin A antiserum nor MK-801 has any effect on the baseline nociceptive threshold or morphine efficacy in control animals. These findings suggest that dynorphin A contributes to the hyperesthetic states, possibly through modulating the NMDA receptor function via a nonopioid mechanism when the endogenous level of dynorphin is abnormally elevated.

The question of how dynorphin modulates NMDA receptor function has been addressed in many studies. In-vitro evidence supports a direct interaction between dynorphin A and NMDA receptors because dynorphin A modulates the binding of a number of NMDA receptor ligands including glutamate (Massardier and Hunt 1989), 2-amino-4-propyl-5-phosphono-3-pentanoic acid (CGP-39653) (Dumont and Lemaire 1994), and MK-801 (Shukla et al. 1992). Heterologous expression of the NMDA receptor complex results in a concurrent increase in the specific binding of [3H]MK-801 and [125I]dynorphin A(2-17) to the transfected cells (Tang et al. 1999). Electrophysiological evidence, however, shows that dynorphin A inhibits NMDA-mediated currents in isolated trigeminal neurons (Chen et al. 1995) and in Xenopus oocytes that express various NMDA receptor complexes (Brauneis et al. 1996). This inhibitory action of dynorphin A on NMDA receptors in vitro is not consistent with the apparent excitatory actions of dynorphin A observed in vivo. Other studies demonstrated that dynorphin A can activate NMDA receptors in vitro. Dynorphin A at low concentration potentiates a NMDA receptor-mediated synaptic current in the CA3 region of hippocampal slices that is not blocked by naloxone (Caudle et al. 1994) but can be blocked by antagonists that bind to the polyamine site of the NMDA receptors (Caudle and Dubner 1998). Dynorphin A also has a moderate stimulatory effect on NMDA receptor-mediated currents in isolated periaqueductal gray neurons (Lai et al. 1998). These findings, however, do not preclude the possibility of an indirect mechanism of dynorphin A on NMDA receptor activity.

Several nonopioid, indirect mechanisms of potentiating NMDA receptor activation by dynorphin A have been proposed. For example, dynorphin A may modulate NMDA receptor activation through regulating excitatory neurotransmitter release (Faden 1992; Skilling et al. 1992). This excitatory effect of dynorphin A may be elicited through a reduction in presynaptic inhibition of primary afferent input (Ristic and Isaac 1994). Robertson et al. (1987) observed that dynorphin A and dynorphin A(2-17) could inhibit the action of GABA in the substantia nigra, probably by a postsynaptic excitatory mechanism. Thus the underlying mechanism of dynorphin-mediated NMDA receptor activity remains to be established. The present study aimed to test the hypothesis that dynorphin A and its fragments enhance neuronal excitability through the NMDA receptor. We measured the increase in intracellular calcium ([Ca2+]i) in cultured cortical neuronal cells as an index of excitability and examined the nonopioid effect of dynorphin A by using the peptide dynorphin A(2-17), which does not bind to opioid receptors because of the absence of the N terminal tyrosyl moiety (Walker et al. 1982; see also RESULTS). Surprisingly, dynorphin A(2-17) alone produces an increase in intracellular calcium, which does not appear to be mediated by the NMDA receptor complex but rather through a nonopioid, non-NMDA mechanism.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Materials

Dynorphin A(2-17) (sequence GGFLRRIRPKLKWDNQ) was kindly provided by the National Institute on Drug Abuse (the peptide was synthesized by Multiple System Peptides Inc. with a purity of 98% and a protein content of 74%). Tissue culture media were from Life Technologies (Gaithersburg, MD). MK-801, NMDA, and all buffering reagents were from Sigma (St. Louis, MO). Fluo-3/AM was from Molecular Probes (Eugene, OR). Radioligands were from New England Nuclear (Boston, MA).

Preparation of cortical neuron primary cultures

Four day-old Sprague Dawley rats (male and female) were anesthetized with ether and killed by decapitation. Brains were taken out and immersed in ice-cold dissection buffer consisting of (in mM) 137 NaCl, 5 KCl, 0.2 Na2HPO4. 7H2O, 0.2 KH2PO4, 33 glucose, 44 sucrose, and 10 HEPES, pH 7.3). Brain cortex was excised and minced. Tissue was dispersed sequentially by flame-polished Pasteur pipettes of decreasing sizes. Cells and cell aggregates were then plated on polylysine-coated cover slips placed in 6-well titer plates (typically, cells from 1 rat were dispensed into 6 wells). Cultures were maintained in minimal essential medium (MEM) supplemented with 0.4% glucose, 10% fetal calf serum, 10% horse serum, and 10 mM KCl. The medium was changed on day three to a maintenance medium that had a similar composition as the plating medium except that the fetal calf serum was omitted.

Fluorescence microscopy

Cortical cells were loaded with 6 µM Fluo-3/AM dissolved in MEM for 60 min at 37°C and then rinsed and incubated for an additional 30 min. Each cover slip was then mounted onto a temperature-controlled chamber (37°C), covered with 1 ml of bathing buffer consisting of (in mM) 10 HEPES, 25 glucose, 137 NaCl, 10 KCl, and 3 CaCl2, pH 7.4, and placed on the stage of an Olympus IMT-2 inverted microscope equipped with an Olympus 60 × 1.4NA objective. Fluorescence images were acquired using a Photometrics liquid-cooled charge-coupled device (CCD) camera coupled to the microscope with a 6.7× eyepiece. This camera had a linear response of up to 5 × 105 counts/image element. An electronic shutter under computer control was utilized to regulate exposure time from a 100-watt mercury lamp. Standard optics (Optical, Brattleboro, VT) for Fluo-3 included a 10-nm band-pass excitation filter centered at 480 nm and a 15-nm band-pass emission filter centered at 530 nm. The digitized output of the camera was stored on a microcomputer and analyzed using customized software on a Silicon Graphics IRIS 10/900. The fluorescence intensity within a single cell was quantified by acquiring sequential images with a constant exposure time. Exposure was set at 300 ms, which provided images with fluorescence intensities well above cell background [>500 integrated optical density (IOD)] without causing saturation of the imaging elements. For each cell, images were taken before, i.e., resting Ca2+, and after each drug administration (sample fluorescence). Drugs were administered directly to the bathing buffer in small volumes of concentrated stock to yield the desired final concentration. The maximal Fluo-3 fluorescence response for each cell was determined by depolarizing the cell with the addition of 40 mM KCl at the end of the experiment. The fluorescence intensity within a cell was expressed as the mean IOD averaged over the area of the cell body. Changes in intracellular calcium concentration were expressed as a percentage of the maximal fluorescence response induced by 40 mM KCl by the following equation: (sample - basal)/(maximal - basal) × 100%. This approach allowed for comparison of each cell response independent of differences in dye concentration. Statistical significance was determined at the 95% confidence level by unpaired t-test or by two-way analysis of variance (ANOVA).

Radioligand binding

The affinity of dynorphin A(1-17) and dynorphin A(2-17) for the three cloned human opioid receptor types was determined using clonal, stably transfected HN9.10 cells (Lee et al. 1990) that expressed µ, delta , or kappa  opioid receptors. Transfection, cell culture conditions, and membrane preparation were carried out as described in Lai et al. (1994). Ligand competition analysis was carried out using 10 concentrations of the dynorphin A peptides against [3H]U69,593-labeled kappa  receptors, [3H]DAMGO-labeled µ receptors, or [3H]pCl-DPDPE-labeled delta  receptors. Assay conditions were as described in Lai et al. (1994). Nonspecific binding was defined as the amount of radioligand bound in the presence of 10 µM naloxone. Reactions were terminated by rapid filtration through Whatman GF/B filters and washed with 3 × 4 ml ice-cold saline. Radioactivity was determined by liquid scintillation counting. Data were analyzed by GraphPad Prism. The IC50 values were converted to Ki values by the Cheng and Prusoff equation.


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Effect of dynorphin A(2-17) and NMDA on intracellular calcium concentration of cultured cortical neuronal cells

After three or four days in culture, many cortical cells were fluorescent after the cell culture was incubated with the cell-permeant form of the calcium-sensitive dye Fluo-3/AM (Fig. 1). These cells were clearly distinguishable from a subpopulation of cells by the difference in their basal fluorescence, presumably caused by a higher basal level of intracellular calcium concentration or a difference in the efficiency of dye loading. These cells also displayed a neuronal-like morphology. Most of these cells consisted of two or three long projections and exhibited a higher steady-state level of Fluo-3 fluorescence. Image analysis was performed on cells that exhibited these morphological characteristics and that could be visualized as discrete cells. Under the magnification used (~400×), a small number of cells (1-5) typically was visible within the field of view, which allowed the cells to be monitored simultaneously. Of all the cells sampled in this study, 45% (n = 179) responded to dynorphin A(2-17) and 76% (n = 30) responded to NMDA. Figure 2 shows a representative cell that responded to dynorphin A(2-17). Those cells that did not exhibit an increase in [Ca2+]i in response to either drug were excluded from further analysis. In responsive cells, 30 µM dynorphin A(2-17) or 300 µM NMDA produced a time-dependent increase in [Ca2+]i, reaching a plateau by ~1 min and remaining stable (Fig. 3). The stability was evident for up to 9 min (data not shown). The response induced by dynorphin A(2-17) and NMDA was dose-dependent (Fig. 4). The EC50 value of the NMDA-mediated increase in [Ca2+]i was 125 µM. On the other hand, the highest concentration of dynorphin A(2-17) used in the assay (100 µM) did not reach a maximal response. Pretreatment with 10 µM naloxone for 3 min before the addition of 100 µM dynorphin A(2-17) did not block the increase in [Ca2+]i in response to dynorphin A(2-17) [37 ± 6.4% by dynorphin A(2-17) alone and 33 ± 6.2% by dynorphin A(2-17) after pretreatment with naloxone]. Thus the stimulatory effect of dynorphin A(2-17) on [Ca2+]i even at high concentration was not mediated by opioid receptors. Radioligand competition analysis using dynorphin A(2-17) against opioid receptors kappa , µ, and delta  from stable transfected cells confirmed the low affinity of this peptide for the three cloned opioid receptor types (Ki values were >10 µM).



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Fig. 1. Fluorescence image of cortical cells that have been loaded with Fluo-3/AM. The fluorescence of Fluo-3 resides predominantly in the cell bodies. The contrast of the image is adjusted to illustrate the morphology of these cells with their long multiple projections.



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Fig. 2. Fluorescence image of a representative cell that was treated with dynorphin A(2-17). A: basal fluorescence. B: fluorescence 1 min after the addition of 30 µM dynorphin A(2-17).



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Fig. 3. Time course for the increase in [Ca2+]i induced by 30 µM dynorphin A(2-17) (A) or 300 µM N-methyl-D-aspartate (NMDA) (B). Data represent mean ± SE values from 9 or 10 cells.



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Fig. 4. Dose-effect of dynorphin A(2-17) (A) and NMDA (B) on the increase in [Ca2+]i. Data represent mean ± SE values from 4 to 6 cells. The fluorescence of the cell was sampled 1 min after the addition of each dose of drug.

Dynorphin A(2-17)-induced increase in [Ca2+]i is not mediated by NMDA receptors

To determine whether the increase in [Ca2+]i induced by dynorphin A(2-17) was mediated through NMDA receptor activation, the effect of MK-801 on the dynorphin A(2-17)-induced response was investigated. First, the effective concentration of MK-801 in blocking NMDA receptor-mediated [Ca2+]i was determined for our experimental system. Cells were observed for their response to 300 µM NMDA, to which 30 µM MK-801 was added 1 min after the application of NMDA. MK-801 decreased the elevated [Ca2+]i gradually, reaching basal [Ca2+]i by 2 min after the addition of MK-801 (Fig. 5A). When the experiment was repeated using 100 µM dynorphin A(2-17), the subsequent application of 30 µM MK-801 had no effect on the elevated [Ca2+]i produced by dynorphin A(2-17) (Fig. 5B). To show that the lack of effect of MK-801 on dynorphin A(2-17)-mediated [Ca2+]i was not simply due to the absence of NMDA receptors in the sampled cells, we took the alternative approach shown in Fig. 6. Cells were first determined by their responsiveness to NMDA by the addition of 300 µM NMDA. This response was blocked by the addition of 30 µM MK-801 with NMDA still present in the bathing buffer. It was found that subsequent application of 30 µM dynorphin A(2-17) to the bathing buffer produced an increase in [Ca2+]i in six of seven tested cells without any apparent delay in the time course (cf. Fig. 3A) or decrease in the amplitude of the signal to dynorphin A(2-17) (cf. Fig. 4A) when compared with that observed with dynorphin A(2-17) alone. When cells were tested for responsiveness to NMDA and dynorphin A(2-17), of the 21 cells which responded to NMDA, 15 also responded to dynorphin A(2-17). Thus a statistically significant number of sampled cells exhibited responsiveness to both compounds. Furthermore, blockade of NMDA receptors by MK-801 did not alter the prevalence of cells that responded to subsequent challenge with dynorphin A(2-17).



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Fig. 5. Effect of 30 µM (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine (MK-801) on the increase in [Ca2+]i induced by 300 µM NMDA (n = 8) (A) or by 100 µM dynorphin A(2-17) (n = 5) (B). *, values significantly different (P < 0.05) from the response induced by 300 µM NMDA at 1 min.



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Fig. 6. Effect of sequential application of 300 µM NMDA, 30 µM MK-801, and 30 µM dynorphin A(2-17) on [Ca2+]i in cortical cells (n = 6). *, values significantly different from the response induced by 300 µM NMDA at 1 min.

Dynorphin A(2-17) and NMDA have an additive effect in mediating increase in [Ca2+]i

Figure 7 shows the effect of dynorphin A(2-17) (100 µM) on the dose effect of an NMDA-mediated increase in [Ca2+]i. Administration of dynorphin A(2-17) elicited an increase of 20 ± 5% of the maximal [Ca2+]i response after 1 min, and this increase was further enhanced by the subsequent addition of an increasing concentration of NMDA. The mean difference in the two dose-response curves is 34%, which is comparable to the change induced by 100 µM dynorphin A(2-17) alone (see Fig. 4A). Thus these data suggest that dynorphin A(2-17) and NMDA have an additive effect on [Ca2+]i.



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Fig. 7. Dose-dependent increase in [Ca2+]i mediated by NMDA alone (n = 4, black-triangle) or by NMDA in the presence of 100 µM dynorphin A(2-17) (n = 6, ) in cortical cells. For the latter, dynorphin A(2-17) was added to the bath and the fluorescence of the cell was sampled 1 min before the addition of NMDA. Cells that did not produce an initial response to dynorphin A(2-17) were not used for further analysis. The fluorescence of the cell was sampled 1 min after the addition of each dose of NMDA.


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The major goal of this study was to determine whether dynorphin A potentiates neuronal excitability through an opioid-independent mechanism and whether these effects of dynorphin A are mediated through a direct activation of the NMDA receptors. Our findings demonstrate that dynorphin A(2-17) produces an increase in [Ca2+]i in cultured cortical cells and thus may have a direct effect on neuronal cell excitability. This excitatory effect of dynorphin A(2-17) is dose- and time-dependent and the magnitude of this response is comparable to that elicited by NMDA in these cultured cells. The maximal stimulation by NMDA and by dynorphin A(2-17) was robust, as reflected by a change in [Ca2+]i that was as much as 40% of the maximal [Ca2+]i under depolarizing conditions. Both the dynorphin A(2-17)-induced and NMDA-induced increases in [Ca2+]i remained stable for the duration of exposure to the drugs, showing little evidence of desensitization of the signal transduction mechanism. We found a statistically significant coincidence of responsiveness to dynorphin A(2-17) and NMDA in the cultured cortical cells (71%). Because NMDA receptors are primarily expressed in neuronal cells, this finding suggests that the observed responsiveness to dynorphin A(2-17) is most likely to be associated with neuronal cells in these cultures. This excitatory effect of dynorphin A(2-17) is not opioid receptor-mediated because this peptide exhibits very low affinity for opioid receptors (>10 µM) and its effect on [Ca2+]i is resistant to the opioid antagonist naloxone. Our data also demonstrate that dynorphin A(2-17) does not directly activate NMDA receptors in the cortical neurons because blockade of NMDA receptor-mediated [Ca2+]i by MK-801 has no effect on the excitatory action of dynorphin A(2-17). Thus we concluded that dynorphin A(2-17) stimulates an increase in [Ca2+]i via a non-opioid, non-NMDA receptor mechanism. As mentioned in the INTRODUCTION previous observations based on both radioligand binding and functional assays of the NMDA receptors in vitro show that dynorphin A interacts directly with NMDA receptors and can attenuate NMDA receptor-mediated current. Our experimental system did not preclude this possible action of dynorphin A(2-17) on the NMDA receptors in the cortical cells. However, the apparent additive effect of [Ca2+]i on the coadministration of NMDA and dynorphin A(2-17) seems to argue against an inhibitory effect of dynorphin A on the NMDA receptor-mediated calcium influx under the experimental conditions used in these assays.

The mechanism of the dynorphin A(2-17)-mediated increase in [Ca2+]i is clearly distinct from opioid receptor-mediated effects of dynorphin A. Much of the evidence that supports a modulatory role of dynorphin A on calcium conductances in neuronal cells shows that its effect is primarily inhibitory and is mediated by opioid receptors. For instance, dynorphin A inhibits high-threshold, voltage- dependent calcium currents in sensory neurons via kappa  and µ receptors (Moises et al. 1994). In the hippocampal CA3 region, dynorphin A attenuates calcium channel-dependent synaptic transmission at mossy fibers (Castillo et al. 1996). Dynorphin A also reduces calcium currents in acutely dissociated nodose ganglion neurons via a Gi/Go-coupled system in a naloxone-sensitive manner (Gross et al. 1990). Such effects of dynorphin A are consistent with the inhibitory role of opioid receptors in neurotransmission and neurotransmitter release, which is consistent with the physiological role of an endogenous opioid (Tiseo et al. 1989; Zachariou and Goldstein 1997). The excitatory actions of dynorphin A, on the other hand, are associated with elevated levels of dynorphin A in vivo, i.e.,through exogenous dynorphin A or elevated levels of the endogenous peptide associated with injuries to the nervous system. These actions of dynorphin A are far more complex, as illustrated by the excitotoxicity and neuropathic pain that have been causally linked to dynorphin overexpression and NMDA receptor activity, which is indicative of an excitatory mechanism by this peptide. Because this excitatory action is primarily associated with elevated levels of dynorphin A, such action may be caused by the exposure of other areas of the nervous system to the influence of dynorphin A and its fragments and/or because high concentrations of dynorphin A and its fragments increase the probability of their interaction with multiple non-opioid sites.

The novel site that mediates the effect of dynorphin A(2-17) on [Ca2+]i described here is a potential mechanism for such excitatory actions of dynorphin A in vivo. This mechanism could conceivably enhance neuronal excitability. More specifically, evidence has linked NMDA receptor activation to the excitatory effects of dynorphin A in vivo based on their sensitivity to MK-801. If this novel non-opioid, non-NMDA mechanism contributes to NMDA receptor activation, it ought to precede NMDA activity. One speculation that is consistent with previous findings is that the dynorphin A-induced increase in [Ca2+]i could potentiate excitatory amino acid release (Faden 1992; Skilling et al. 1992). More recently, Claude et al. (1999) showed that dynorphin A(2-17) augmented the capsaicin-evoked release of the calcitonin gene-related peptide (CGRP) in a spinal cord slice preparation, suggesting that dynorphin A may modulate excitatory primary afferent input. Another possible effect of the dynorphin A-mediated increase in [Ca2+]i is to potentiate the NMDA receptor through receptor phosphorylation by the calcium-dependent activation of protein kinase C. This has been shown to increase the probability of channel openings by reducing the voltage-dependent Mg2+ block of NMDA receptor channels (Chen and Huang 1992). It remains to be determined whether this novel excitatory effect of dynorphin A(2-17) observed in this in-vitro analysis has physiological and pathological relevance for dynorphin A in vivo. It is clear, however, that further characterization of this novel site of action of dynorphin A will address these issues. It may also provide new insight into the excitotoxic mechanisms and the role of dynorphin in peripheral nerve injury, as well as a potential new modality for the treatment of neuropathic pain.


    ACKNOWLEDGMENTS

The authors thank Dr. Shou-wu Ma for technical assistance. The cDNA for the µ, delta , and kappa  opioid receptors was kindly provided by Drs. Lei Yu, Brigitte Kieffer, and Erik Mansson, respectively.

This work was supported by National Institute on Drug Abuse Grants DA-11823 and DA-04248.


    FOOTNOTES

Address reprint requests to J. Lai.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 17 August 1999; accepted in final form 26 January 2000.


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