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BEHAVIORAL PHARMACOLOGY
Department of Anesthesiology, University of California, San Diego, La Jolla, California (X.-Y.H., C.S.H., A.H., B.F., T.L.Y.); and Department of Neuropharmacology, The Harold Dorris Neurological Institute, The Scripps Research Institute, La Jolla, California (K.K., Ü.L., T.B.)
Received August 7, 2003; accepted November 4, 2003.
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Abstract |
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Gal1–30 > galanin-like peptide1–24 Gal2–11 = Gal 3–29 (an inactive analog). Galanin1–29 and Gal1–30 are both high-affinity agonists to GalR1/R2, whereas Gal2–11 is a GalR2 receptor agonist. Our data suggest that i.t. galanin-produced antinociception is mediated by activation of GalR1 receptors. When comparing antinociceptive effects of i.t. Gal1–29 to morphine and to 2-amino-5-phosphonopentanoic acid (AP-5, an N-methyl-D-aspartate antagonist), Gal1–29 is of intermediate potency between these two analgesic agents based on the ED50 values. An isobolographic analysis showed synergy between Gal1–29 and morphine and between Gal1–29 and AP-5 on the second phase. Fixed ratio dose combinations of morphine and Gal1–29, or AP-5 and Gal1–29 produced significantly greater antinociception than predicted from simple additivity. In summary, the present findings reveal that 1) spinal galanin produces a reliable inhibition of formalin-induced facilitated nociceptive processing, an effect possibly mediated by GalR1 receptors; and 2) galanin potentiates i.t. morphine and AP-5-induced antinociception.
). About one-half of the galanin-positive terminals in the dorsal horn are in primary afferent fibers (Zhang et al., 1993, 1995a), and a considerable amount of galanin is present in a subpopulation of lamina II interneurons, which also contain GABA and enkephalins (Simmons et al., 1995; Zhang et al., 1995b). Three galanin receptor subtypes, named GalR1, GalR2, and GalR3, have been cloned, and belong to the superfamily of the G protein-coupled receptors (Branchek et al., 2000). Activation of either GalR1 or GalR3 produces hyperpolarization via Gi/o and inhibits adenylyl cyclase. GalR2 activation leads to stimulation of phospholipase C via Gq/11, producing calcium mobilization, diacylgycerol formation, and subsequent activation of protein kinase C (Branchek et al., 2000). All three receptor transcripts are present in DRG and spinal cord (Waters and Krause, 2000). GalR1 mRNA has been found in lamina II local neurons (Parker et al., 1995). The anatomical location of galanin and galanin receptors in DRG and spinal cord suggests that endogenous or exogenous galanin may participate in the regulation of spinal nociceptive transmission.
Spinal effects of galanin on nociception seem complex, because both facilitatory and inhibitory effects have been observed. Early studies showed that i.t. galanin at low doses (
1 nmol) elicits a facilitation of the flexor reflex in rats (Wiesenfeld-Hallin et al., 1988, 1989; Xu et al., 1990) and increases responsiveness to noxious stimulation (Cridland and Henry, 1988). Recent studies on transgenic mice null mutated for galanin peptide demonstrated that the lack of galanin expression attenuates both spontaneous and stimulation-induced pain behaviors after peripheral nerve injury and inflammation (Kerr et al., 2000, 2001). Conversely, expression of galanin is up-regulated in sensory neurons and spinal cord after nerve injury where hyperalgesia and allodynia are observed (Hokfelt et al., 1987). This suggests that the increased level of spinal galanin may contribute to pain behavior. Although there are no selective galanin receptor antagonists, the excitatory effect of galanin is apparently mediated by activation of the GalR2 receptor (Liu et al., 2001). An inhibitory action of i.t. galanin on spinal nociceptive processing is also reported. This effect usually requires a higher dose and is presumably mediated through activation of the GalR1 (Xu et al., 2000; Liu and Hokfelt, 2002). In agreement with the antinociceptive effects of spinal galanin, electrophysiological studies reveal that galanin attenuates C-fiber stimulation induced nociceptive reflexes and dorsal horn neuron hyperexcitability (Yanagisawa et al., 1986). There are apparently some interactions between galanin and opioids on spinal antinociception. Spinally administrated M15 and M35, two chimeric peptide-type putative galanin receptor antagonists, attenuate analgesic actions of intrathecal morphine and [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (Reimann et al., 1994), suggesting that endogenous galanin may participate in spinal opioid-induced analgesia. Zhang et al. (2000), however, reported that i.t. galanin induced a naloxone-reversible increase in paw withdrawal latency to thermal and mechanical stimulation, suggesting that galanin facilitates a release of endogenous opioids in spinal cord. Inhibition induced by morphine and an antagonist of cholecystokinin B receptor on spinal nociceptive reflexes is enhanced by galanin (Wiesenfeld-Hallin et al., 1990).
Animal behaviors used to assess antinociceptive effects of spinal galanin typically are measurement of paw escape latencies and thresholds to thermal and mechanical stimulation, respectively. In the present work, we undertook a series of experiments in rats using the automated formalin test device (Yaksh et al., 2001) to characterize 1) the antinociceptive effects of i.t. administration of galanin (i.e., Gal1–29) and its analogs, which have different affinity for GalR1 and GalR2 receptors; and 2) the synergy between Gal1–29 and two analgesic agents, i.e., morphine and AP-5, by the use of an isobolographic analysis.
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Materials and Methods |
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Intrathecal Implantation. Rats were implanted with a single-lumen intrathecal catheter (Yaksh and Rudy, 1976) for drug delivery. To place the catheter, anesthesia was induced with 4% isoflurane in a room air/oxygen mixture (1:1). The back of the head and neck were shaved, and the animals then were placed in a stereotaxic head-holder with the head flexed forward. Anesthesia was maintained with 2% isoflurane delivered by mask. A midline incision was made on the back of the neck. The muscles were freed at the attachment to the skull exposing the cisternal membrane. A small (
1-mm) puncture was made in the dura, and an 8.5-cm polyethylene-10 catheter was then inserted through the cisternal opening and carefully passed caudally into the intrathecal space terminating at the L1–3 spinal segments. The end of the catheter was tunneled subcutaneously over the front dorsal skull bones, flushed with 10 µl of saline, and then plugged with a short length of wire. The skin incision was then closed using 3.0 USP black braided silk suture. The rats were given 5 ml of lactated Ringer's solution subcutaneously and allowed to recover under a heat lamp. Rats showing motor weakness or signs of paresis upon recovery from anesthesia were euthanized immediately. Animals recovered for 5–7 days before the formalin test.
Intrathecal Drugs and Injection. All drugs were injected i.t. in a total volume of 10 µl followed by 10 µl of saline to flush the catheter. The following drugs were used in this study: galanin1–29 (Gal1–29, mol. wt. 3165), galanin1–30 (Gal1–30, mol. wt. 3157), galanin-like peptide1–24 (GALP1–24, mol. wt. 2500), galanin2–11 (Gal2–11, mol. wt. 1137), galanin3–29 (Gal3–29, mol. wt. 2920), galanin1–13-pro-bradykinin2–9 amide (M35, mol. wt. 2249); all were synthesized on solid phase with ter-butynylcarbonate chemistry, and the high-performance liquid chromatography-purified peptides were analyzed by mass spectrometry. A nonpeptide ligand for galanin receptor: Fmoc-cycleoxylalanine-Lys-amidomythylcoumarin (galnon, mol. wt. 677) was synthesized as described by Saar et al. (2002). Morphine (morphine sulfate, mol. wt. 669) was obtained from Merck Sharp & Dohme (West Point, PA), and ±-2-amino-5-phosphonopentanoic acid (AP-5, mol. wt. 197) was from Sigma/RBI (Natrick, MA). A single i.t. drug injection was given 10 min before the formalin paw injection. For the drug combinations, Gal1–29 was given at 20 min before, and morphine or AP-5 given at 10 min before the formalin paw injection. All the drug solutions except galnon, which was in 10% dimethyl sulfoxide, were made in physiological saline.
Formalin Test. To quantify formalin paw injection-induced flinching/licking behavior, an automated sensing system was used (Yaksh et al., 2001). Briefly, a soft metal band (10 mm in width and 27 mm in length, 0.5 g, C-shaped) was placed on one of the hind paws of a rat. After acclimation for 30 min, animals were gently restrained and 50 µl of 2.5% formalin solution was injected subcutaneously into the dorsal surface of the banded paw with a 30-gauge needle. Data collection was initiated after the animal was placed inside of the test chamber. Nociceptive behavior was quantified by automatically counting incidences of spontaneous flinching/shaking of the injected paw. The flinches were counted over 1-min intervals for 60 min. The animals were sacrificed with CO2 immediately after the test.
Data Analysis and Statistics. Antinociceptive ED50 values to the formalin test were calculated from the dose-response curves generated for galanin (Gal1–29), galanin analogs, morphine, and AP-5 alone or in combination based on the graded dose response method of Tallarida and Murray (1987). Combination of the two drugs (i.e., Gal1–29 + morphine, or Gal1–29 + AP-5) was obtained in a constant dose ratio based on the ED50 values of the single agents (1/2, 1/4, and 1/8 ED50). Drug synergism was analyzed by the conventional isobolographic analysis (Tallarida et al., 1989). Briefly, ED50 values of drugs alone were plotted, and a theoretical additive line was constructed on an isobologram. Experimental values from fixed ratio designed studies were also analyzed using linear regression, and ED50 values for each combination were determined and plotted on the isobologram for the comparison with the theoretical additive value. The Student's t test was used to determine significance of the difference between the theoretical additive point and experimental derived ED50 value. A P value less than 0.05 indicated that drugs produced a synergistic effect compared with either drug by itself. A total fraction value that reveals what portion of the single ED50 value was accounted for by the corresponding ED50 value for the combination was also calculated. Values less than 1 indicate a multiplicative interaction. Total fractions were calculated as described previously by Roerig and Fujimoto (1988):
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All the data are presented as mean ± S.E.M. For the data in Figs. 1, 2, 3, statistical significance was calculated using one-way analysis of variance (ANOVA) with multiple comparisons for independent measurement followed by Dunnett's test by using the Prism computer program. Differences were considered to be significant when the critical value reached a level of P < 0.05.
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). Using the automated flinch detecting system, the response count and distribution resembles that obtained with the manual counting system (Yaksh et al., 2001). Thus, the biphasic display of paw flinch behavior evoked by formalin paw injection (2.5%, 50 µl) has been used in the present study to evaluate the antinociceptive activity of i.t. galanin and its analogs.
Galanin and Analogs. In i.t. saline-treated rats, the total number of flinches after the formalin injection was 195 ± 19 in phase 1 (1–9 min) and 1094 ± 109 in phase 2 (10–60 min) of the response (n = 9; Fig. 1). These numbers are in the same range as that observed for naive rats (Yaksh et al., 2001). Intrathecal injection of either rat galanin Gal1–29 or the human galanin Gal1–30, both high-affinity agonists to GalR1 and GalR2 receptors (Ki = 1 nM), produced a dose-dependent (3–30 nmol) inhibition of the phase 2 flinching response; phase 2A (10–40 min) was preferentially affected (Figs. 1 and 2). In contrast, neither Gal1–29 nor Gal1–30 significantly altered flinching within phase 1 (Figs. 1 and 2). Gal1–29 or Gal1–30 at the highest dose, i.e., 30 nmol, which did not produce any sign of motor weakness or motor disfunction, markedly reduced the second phase response in comparison with the saline group (46–47% of saline group; n = 5–7; P < 0.05). Gal3–29, an N-terminally truncated inactive ligand (Ki > 1000 nM), given at the highest equivalent dose as Gal1–29 (30 nmol), did not significantly attenuate flinching behavior in either phase (n = 4; Fig. 3). GALP1–24 is a synthetic fragment of the recently discovered GALP (Ohtaki et al., 1999), which is a 60-amino acid-long peptide isolated from porcine hypothalamus containing an internal sequence (amino acids 9–21) identical to the N-terminal sequence of galanin. GALP has a similar affinity as Gal1–29 for GalR1 (IC50 of 2 nM), but a slightly higher affinity for GalR2 (IC50 of 0.2 nM). GALP1–24 is a likely peptidolytic cleavage product of GALP. In the present study, GALP1–24 at doses of 3 and 10 nmol had no effect (data not shown). Increased doses of GALP1–24 (30 nmol) produced 45% inhibition of the second phase (Fig. 3); however, this effect was not statistically significant. Gal2–11, with selective GalR2 agonist activity (IC50 of 1.8 nM for GalR2, and 879 nM for GalR1), at doses of 30 nmol, had little effect on the flinching response (n = 5; Fig. 3). The ED50 values (nanomoles) of galanin, based on the inhibition of phase 2 are Gal1–29 19 (95% C.I., 8–47) and Gal1–30 30 (95% C.I., 3–311). The estimated ED50 value of GALP1–24 is 48 (95% C.I., 5–458). Because neither Gal2–11 nor Gal3–29 at 30 nmol displayed a significant inhibition of the flinching behavior, the ED50 values of these two analogs, if there were any, would be estimated to be higher than 48 nmol. The rank order of potency of galanin homologs based on the ED50 values (or the estimated ED50 values) is Gal1–29 Gal1–30 > GALP1–24 Gal2–11 = Gal3–29. Previous work has shown that spinal administration of galanin, typically at low doses (1 nmol) also produces excitatory effects on nociception (see Introduction); however, we saw no facilitatory activities of either galanin or the peptide analogs at all examined doses including the low dose (3 nmol).
Galnon, a nonpeptide agonist-like ligand of the galanin receptor with 1000 times lower affinity (Ki of 4.8 µM) than Gal1–29 for the GalR1 (Saar et al., 2002), given at a dose of 30 nmol i.t. did not alter the flinching response in either phase (n = 12; Fig. 3). A chimeric peptide of galanin1–13 and bradykinin2–9, M35, has a similar affinity for GalR1 and GalR2 as Gal1–29 (Ki of 1–10 nM) and has been shown to function as an antagonist/partial agonist (Xu et al., 2000). Intrathecal administration of M35 to rats at four doses (i.e., 0.3, 1, 3, and 10 nmol; n = 4–8) did not potentiate flinching response (data not shown), whereas the highest dose (i.e., 10 nmol) reduced the second phase flinching (M35, 549 ± 160; saline, 1021 ± 100; P < 0.05). Pretreatment with M35 at 0.3 to 1 nmol alone showed little effect on formalin-induced flinching and did not reverse the inhibitory effects of Gal1–29 or Gal1–30 (data not shown). No animal showed abnormal sensory or motor function after i.t. administration of galanin analogs, including galnon and M35, at all examined doses.
Interaction with Morphine or AP-5. Intrathecal injection of morphine (0.3–100 nmol; n = 22) or AP-5 (5.6–152 nmol; n = 16) resulted in a dose-dependent inhibition of second phase flinching behavior (Fig. 4; Table 1). AP-5 at the highest dose (152 nmol) also significantly reduced flinching within the first phase (data not shown). This result is in agreement with previous observations (Yamamoto and Yaksh, 1992; Yaksh et al., 2001). The relative potency of i.t. Gal1–29 compared with that of these two analgesic agents (ED50, in nanomoles, phase 2) is morphine (4) > Gal1–29 (19) > AP-5 (60) (Table 1). Synergistic antinociception between Gal1–29 and morphine or Gal1–29 and AP-5 in the formalin test was observed (Figs. 5 and 6; Table 1). Isobolographic analysis of the fixed dose combination of Gal1–29 and morphine (n = 11), or combination of Gal1–29 and AP-5 (n = 11) on the second phase, indicated that the interaction is synergistic. Figures 5B and 6B show the plots of the combination ED50 values in relation to the ED50 values of the drugs alone. In the series of dose combinations that were examined using the fixed dose ratios for morphine and Gal1–29 (1:4.8), or AP-5 and Gal1–29 (1:0.3), all the points fell below the line of additivity, and the differences are statistically significant (P < 0.05). The line of additivity reflects the theoretical dose combination required to produce 50% of the effect, assuming the agents interact in a linear manner. Total dose fractions (second phase) were calculated; 0.54 for the combination of morphine and Gal1–29 and 0.56 for AP-5 and Gal1–29. The values near 1 indicate an additive effect, and values less than 1 imply a multiplicative interaction. No motor weakness or sensory disfunction was seen in animals with the combination drug treatment.
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; Liu and Hokfelt, 2002) and confirm the antinociceptive actions of spinal galanin.
Galanin and Analogs. An aim of the present work was to assess the potential contribution of galanin receptor subtypes. Accordingly, we considered the dose-dependent inhibitory effects of several galanin analogs that have differential affinity for the GalR subtypes. Binding studies have shown that both Gal1–29 and Gal1–30 have high affinity for GalR1 and GalR2 receptors (Ki values about 1 nM), whereas Gal3–29 has approximately 1000-fold lower affinity for all three galanin receptors (Ki > 1000 nM). Consistent with the binding data, the present findings also revealed that i.t. Gal3–29 displayed no effect on formalin-induced nociception, in contrast to the marked antinociception produced by i.t. Gal1–29 and Gal1–30. It has been shown that GALP1–24 displaces 125I-galanin from galanin binding sites in mouse hippocampal membranes with high affinity (IC50 of 2 nM), similar to that of Gal1–29 (Ohtaki et al., 1999). However, the antinociceptive effect of GALP1–24 on the formalin test seems weak (estimated ED50 of 48 nmol). This suggests that galanin and GALP1–24 may have different GalR subtype specificity, as we know that GALP has a higher affinity for GalR2 (IC50 of 0.2 nM) than for GalR1 (IC50 of 4 nM) (Ohtaki et al., 1999). The galanin fragment Gal2–11, which is 500-fold more selective for GalR2 (IC50 of 1.76 nM) over GalR1 (IC50 of 879 nM) (Liu et al., 2001, and a similar observation from our laboratory), displayed no effect on formalin-evoked nociceptive response. This is in agreement with the finding that i.t. infusion of Gal2–11 in rats produced only hyperalgesia, and not analgesia (Liu et al., 2001). Together, our data support the assertion that the antinociceptive effect of i.t. galanin is mediated by activation of spinal GalR1, but not GalR2 receptors. We recognize the limitation of asserting this hypothesis regarding GalR1 versus GalR2 effects based on the lack of effect of a single drug (i.e., Gal2–11). However, given the marked and effective binding affinity of Gal2–11 for GalR2, the lack of effect of a dose of this agent equalmolar to the efficacious doses of Gal1–29 and Gal1–30 provides substantive support for our assertion. Development of additional GalR2 selective agonists will be required to confirm this hypothesis.
Galnon is a low-molecular-weight galanin agonist-like ligand that can penetrate the blood-brain barrier and has potent anticonvulsant activity (Saar et al., 2002). The Ki value of galnon at the human GalR1 receptor is 4.8 µM. A recent study demonstrated that systemic galnon attenuated peripheral nerve injury-induced thermal hyperalgesia in rats and that the effect was blocked by spinal M35 (Wu et al., 2003). In the present study, administration of galnon directly into the intrathecal space at a dose of 30 nmol produced no inhibition of the formalin-induced flinching response. This discrepancy may suggest that the antihyperalgesic activity of systemic galnon could be due to either release of endogenous galanin or to an effect at GalRs outside the spinal cord.
Although M35 acts as partial agonist both in vitro and in vivo (Branchek et al., 2000; Xu et al., 2000), its value lies in its utility as a GalR-specific antagonist. M35 behaves as an antagonist of exogenous galanin action in several regions of central nervous system (Bartfai et al., 1991; Branchek et al., 2000). Previous work has shown that the spinal effect of galanin is antagonized by M35 and other chimeric peptides (e.g., M15 and M32) (Bartfai et al., 1991; Wiesenfeld-Hallin et al., 1992). In contrast, in the present study, M35 exerted an agonist-like action. M35 at 10 nmol significantly attenuated second phase flinches. However, low doses of M35 (0.3–1 nmol) displayed little or no agonist activity but did not reverse the antinociception of Gal1–29 or Gal1–30. These complex effects of M35 are not unexpected given that this molecule is a chimeric peptide with galanin1–13 as the N-terminal portion. This sequence defines the recognition of the ligand by galanin receptors and likely accounts for the agonist-like effect seen here. Although Wiesenfeld-Hallin and colleagues (1992) reported previously that intrathecal M35 blocks excitatory effects of low-dose spinal galanin, we did not observe substantial stimulatory activity of i.t. Gal1–29 or Gal1–30 at any of the given doses.
Potential Mechanisms of i.t. Galanin-Mediated Antinociception. The present findings support the assertion that at the spinal level an antinociceptive action is mediated by GalR1 activation. The presence of GalR1 mRNA in DRG and the spinal terminals of primary afferent fibers suggest a modulatory effect upon primary afferent terminal excitability. Previous work has shown that galanin may presynaptically inhibit release of glutamate in central nervous system (Zini et al., 1993), a finding consistent with the ability of GalR1 to block the opening of voltage-sensitive calcium channels (Parsons et al., 1998). In addition, GalR1 mRNA is present in spinal dorsal horn neurons. It is known that Gal1–29 opens K+ channels, leading to hyperpolarization of neurons (Ren et al., 2001). This suggests that postsynaptic action of galanin may be critical in abrogating the downstream events that lead to the spinal sensitization and hyperalgesia. Activation of spinal NMDA receptors is known to trigger a cascade of intracellular events, including activation of enzymes such as phospholipase A2 and nitric-oxide synthase that lead to the generation of prostanoids and nitric oxide. These intermediaries contribute to spinal sensitization that underlies the behaviorally defined hyperalgesia arising from peripheral tissue injury and inflammation (Yaksh et al., 1999). Consistent with a direct postsynaptic regulation by GalR1 of the spinal processing initiated by peripheral injury are the observations that galanin antagonizes substance P (SP)-induced neuronal hyperexcitability (Xu et al., 1990) and that i.t. Gal1–29 blocks spinal SP-evoked hyperalgesia and prostaglandin E2 release (Hua et al., 2002). These dual actions on neuronal excitability of afferent and secondary order neurons resembles the motif that has been described for several other agonists with analgesic properties, including the µ/
opioids and 
2 adrenergics acting at G protein-coupled receptors (Yaksh et al., 1999). Accordingly, the functional outcome is the suppression of the formalin evoked flinching response by intrathecal galanin.
Synergistic Actions of Galanin. Previous work has shown that activation of spinal opiate receptors or antagonism of NMDA receptors will attenuate spinal nociceptive processing and prevent development of a facilitated state by reducing injury-induced afferent input and diminishing the downstream cascade (Yaksh et al., 1999). Similar effects were seen in the present study. We sought to define the nature of the interaction between these receptors and the GalR1. The antinociceptive effects of Gal1–29 are of intermediate potency between those of morphine and AP-5 in the formalin model. Accordingly, we undertook a fixed ratio isobolographic analysis. Considering the interaction between Gal1–29 and morphine or between Gal1–29 and AP-5, a significant synergistic effect was observed between both classes of compounds (Figs. 5 and 6). This is likely to be the consequence of different sites of actions on a common cascade of nociceptive processing. GalR1 activation at presynaptic sites may speculatively reduce glutamate and SP release in the spinal cord, and at postsynaptic sites it may counteract the NMDA receptor-mediated Ca2+-dependent nitric oxide and prostanoid formation, which are thought to form the basis of NMDA-mediated nociception (Yaksh et al., 1999). Any of these effects, alone or in combination, correspond with the proposed mechanisms by which spinal opiate receptor activation or antagonism of the NMDA receptors are thought to alter nociceptive processing.
We wish to emphasize that synergistic action of galanin on spinal morphine and AP-5 is of potential clinical significance. Chronic treatment with opioid analgesics such as systemic or spinal morphine leads to tolerance and dependence, effects that limit their therapeutic efficacy. Although there are clinical studies showing that spinal delivery of an NMDA antagonist can diminish a major component of a postnerve injury pain state (Kristensen et al., 1992), the adverse cognitive effect of these agents is still of major concern (Max et al., 1995). It should be appreciated that low doses of the two drugs administrated simultaneously will produce enhanced analgesic effects with reduced adverse side effects. The potent antinociceptive synergy with no sign of additional side effects suggests GalR1 as a potential target for pain therapy.
In conclusion, although these data do not conclusively exclude a possible role of other GalRs, they do strongly support the hypothesis that spinal GalR1 mediates the inhibitory action of galanin on spinal nociceptive processing and that this antinociceptive effect is synergistic with that of both opiate receptor activation and NMDA receptor antagonism. Further definition of these actions will require development of more selective agonists and antagonists for GalR1 receptors.
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Footnotes |
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ABBREVIATIONS: DRG, dorsal root ganglia; Gal, galanin; GalR, galanin receptor; GALP, galanin-like peptide; AP-5, 2-amino-5-phosphonopentanoic acid; ANOVA, analysis of variance; C.I., confidence interval; NMDA, N-methyl-D-aspartate; SP, substance P.
1 Current address: Department of Neurochemistry and Neurotoxicology, University of Stockholm, SE-106 91, Stockholm, Sweden. 
Address correspondence to: Dr. Xiao-Ying Hua, Anesthesia Research Laboratory, 0818, Department of Anesthesiology, 9500 Gilman Dr., University of California, San Diego, La Jolla, CA 92103-0818. E-mail: xyhua{at}ucsd.edu
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References |
|---|
|
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|---|
Bartfai T, Bedecs K, Land T, Langel U, Bertorelli R, Girotti P, Consolo S, Xu XJ, Wiesenfeld-Hallin Z, Nilsson S, et al. (1991) M-15: high-affinity chimeric peptide that blocks the neuronal actions of galanin in the hippocampus, locus coeruleus and spinal cord. Proc Natl Acad Sci USA 88: 10961–10965.
Branchek TA, Smith KE, Gerald C, and Walker MW (2000) Galanin receptor subtypes. Trends Pharmacol Sci 21: 109–117.[CrossRef][Medline]
Cridland RA and Henry JL (1988) Effects of intrathecal administration of neuropeptides on a spinal nociceptive reflex in the rat: VIP, galanin, CGRP, TRH, somatostatin and angiotensin II. Neuropeptides 11: 23–32.[CrossRef][Medline]
Hokfelt T, Wiesenfeld-Hallin Z, Villar M, and Melander T (1987) Increase of galanin-like immunoreactivity in rat dorsal root ganglion cells after peripheral axotomy. Neurosci Lett 83: 217–220.[CrossRef][Medline]
Hua XY, Langel U, Bartfai T, and Yaksh TL (2002) Characterization of spinal antinociceptive activity of galanin and its analogues, in 10th World Congress on Pain, p 515, IASP Press, San Diego, CA.
Kerr BJ, Cafferty WB, Gupta YK, Bacon A, Wynick D, McMahon SB, and Thompson SW (2000) Galanin knockout mice reveal nociceptive deficits following peripheral nerve injury. Eur J Neurosci 12: 793–802.[CrossRef][Medline]
Kerr BJ, Gupta Y, Pope R, Thompson SW, Wynick D, and McMahon SB (2001) Endogenous galanin potentiates spinal nociceptive processing following inflammation. Pain 93: 267–277.[CrossRef][Medline]
Kristensen JD, Svensson B, and Gordh T Jr (1992) The NMDA-receptor antagonist CPP abolishes neurogenic `wind-up pain' after intrathecal administration in humans. Pain 51: 249–253.[CrossRef][Medline]
Liu HX, Brumovsky P, Schmidt R, Brown W, Payza K, Hodzic L, Pou C, Godbout C, and Hokfelt T (2001) Receptor subtype-specific pronociceptive and analgesic actions of galanin in the spinal cord: selective actions via GalR1 and GalR2 receptors. Proc Natl Acad Sci USA 98: 9960–9964.
Liu HX and Hokfelt T (2002) The participation of galanin in pain processing at the spinal level. Trends Pharmacol Sci 23: 468–474.[CrossRef][Medline]
Malmberg AB and Yaksh TL (1992) Antinociceptive actions of spinal nonsteroidal anti-inflammatory agents on the formalin test in the rat. J Pharmacol Exp Ther 263: 136–146.
Max MB, Byas-Smith MG, Gracely RH, and Bennett GJ (1995) Intravenous infusion of the NMDA antagonist, ketamine, in chronic posttraumatic pain with allodynia: a double-blind comparison to alfentanil and placebo. Clin Neuropharmacol 18: 360–368.[Medline]
Ohtaki T, Kumano S, Ishibashi Y, Ogi K, Matsui H, Harada M, Kitada C, Kurokawa T, Onda H, and Fujino M (1999) Isolation and cDNA cloning of a novel galanin-like peptide (GALP) from porcine hypothalamus. J Biol Chem 274: 37041–37045.
Parker EM, Izzarelli DG, Nowak HP, Mahle CD, Iben LG, Wang J, and Goldstein ME (1995) Cloning and characterization of the rat GALR1 galanin receptor from Rin14B insulinoma cells. Brain Res Mol Brain Res 34: 179–189.[Medline]
Parsons RL, Mulvaney JM, and Merriam LA (1998) Galanin activates an inwardly rectifying potassium conductance and inhibits a voltage-dependent calcium conductance in mudpuppy parasympathetic neurons. Ann NY Acad Sci 863: 156–169.[CrossRef][Medline]
Reimann W, Englberger W, Friderichs E, Selve N, and Wilffert B (1994) Spinal antinociception by morphine in rats is antagonised by galanin receptor antagonists. Naunyn-Schmiedeberg's Arch Pharmacol 350: 380–386.[Medline]
Ren J, Hu HZ, Starodub AM, and Wood JD (2001) Galanin suppresses calcium conductance and activates inwardly rectifying potassium channels in myenteric neurones from guinea-pig small intestine. Neurogastroenterol Motil 13: 247–254.[CrossRef][Medline]
Roerig SC and Fujimoto JM (1988) Morphine antinociception in different strains of mice: relationship of supraspinal-spinal multiplicative interaction to tolerance. J Pharmacol Exp Ther 247: 603–608.
Saar K, Mazarati AM, Mahlapuu R, Hallnemo G, Soomets U, Kilk K, Hellberg S, Pooga M, Tolf BR, Shi TS, et al. (2002) Anticonvulsant activity of a nonpeptide galanin receptor agonist. Proc Natl Acad Sci USA 99: 7136–7141.
Simmons DR, Spike RC, and Todd AJ (1995) Galanin is contained in GABAergic neurons in the rat spinal dorsal horn. Neurosci Lett 187: 119–122.[CrossRef][Medline]
Tallarida RJ and Murray RB (1987) Manual of Pharmacologic Calculations with Computer Programs, Springer, New York.
Tallarida RJ, Porreca F, and Cowan A (1989) Statistical analysis of drug-drug and site-site interactions with isobolograms. Life Sci 45: 947–961.[CrossRef][Medline]
Waters SM and Krause JE (2000) Distribution of galanin-1, -2, and -3 receptor messenger RNAs in central and peripheral rat tissues. Neuroscience 95: 265–271.[CrossRef][Medline]
Wiesenfeld-Hallin Z, Villar MJ, and Hokfelt T (1988) Intrathecal galanin at low doses increases spinal reflex excitability in rats more to thermal than mechanical stimuli. Exp Brain Res 71: 663–666.[CrossRef][Medline]
Wiesenfeld-Hallin Z, Xu XJ, Hughes J, Horwell DC, and Hokfelt T (1990) PD134308, a selective antagonist of cholecystokinin type B receptor, enhances the analgesic effect of morphine and synergistically interacts with intrathecal galanin to depress spinal nociceptive reflexes. Proc Natl Acad Sci USA 87: 7105–7109.
Wiesenfeld-Hallin Z, Xu XJ, Langel U, Bedecs K, Hokfelt T, and Bartfai T (1992) Galanin-mediated control of pain: enhanced role after nerve injury. Proc Natl Acad Sci USA 89: 3334–3337.
Wiesenfeld-Hallin Z, Xu XJ, Villar MJ, and Hokfelt T (1989) The effect of intrathecal galanin on the flexor reflex in rat: increased depression after sciatic nerve section. Neurosci Lett 105: 149–154.[CrossRef][Medline]
Wu WP, Hao JX, Lundstrom L, Wiesenfeld-Hallin Z, Langel U, Bartfai T, and Xu XJ (2003) Systemic galnon, a low-molecular weight galanin receptor agonist, reduces heat hyperalgesia in rats with nerve injury. Eur J Pharmacol 482: 133–137.[CrossRef][Medline]
Xu XJ, Hokfelt T, Bartfai T, and Wiesenfeld-Hallin Z (2000) Galanin and spinal nociceptive mechanisms: recent advances and therapeutic implications. Neuropeptides 34: 137–147.[CrossRef][Medline]
Xu XJ, Wiesenfeld-Hallin Z, Villar MJ, Fahrenkrug J, and Hokfelt T (1990) On the role of galanin, substance P and other neuropeptides in primary sensory neurons of the rat: studies on spinal reflex excitability and peripheral axotomy. Eur J Neurosci 2: 733–743.[CrossRef][Medline]
Yaksh TL, Hua XY, Kalcheva I, Nozaki-Taguchi N, and Marsala M (1999) The spinal biology in humans and animals of pain states generated by persistent small afferent input. Proc Natl Acad Sci USA 96: 7680–7686.
Yaksh TL, Ozaki G, McCumber D, Rathbun M, Svensson C, Malkmus S, and Yaksh MC (2001) An automated flinch detecting system for use in the formalin nociceptive bioassay. J Appl Physiol 90: 2386–2402.
Yaksh TL and Rudy TS (1976) Chronic catheterization of the spinal subarachnoid space. Physiol Behav 17: 1031–1036.[CrossRef][Medline]
Yamamoto T and Yaksh TL (1992) Comparison of the antinociceptive effects of pre- and posttreatment with intrathecal morphine and MK801, an NMDA antagonist, on the formalin test in the rat. Anesthesiology 77: 757–763.[Medline]
Yanagisawa M, Yagi N, Otsuka M, Yanaihara C, and Yanaihara N (1986) Inhibitory effects of galanin on the isolated spinal cord of the newborn rat. Neurosci Lett 70: 278–282.[CrossRef][Medline]
Zhang Q, Shi TJ, Ji RR, Zhang YZ, Sundler F, Hannibal J, Fahrenkrug J, Hokfelt T, and Zhang Y (1995a) Expression of pituitary adenylate cyclase-activating polypeptide in dorsal root ganglia following axotomy: time course and coexistence. Brain Res 705: 149–158.[CrossRef][Medline]
Zhang X, Ji RR, Nilsson S, Villar M, Ubink R, Ju G, Wiesenfeld-Hallin Z, and Hokfelt T (1995b) Neuropeptide Y and galanin binding sites in rat and monkey lumbar dorsal root ganglia and spinal cord and effect of peripheral axotomy. Eur J Neurosci 7: 367–380.[CrossRef][Medline]
Zhang X, Nicholas AP, and Hokfelt T (1993) Ultrastructural studies on peptides in the dorsal horn of the spinal cord–I. Co-existence of galanin with other peptides in primary afferents in normal rats. Neuroscience 57: 365–384.[CrossRef][Medline]
Zhang YP, Yu LC, and Lundeberg T (2000) An interaction of opioids and galanin in dorsal horn of the spinal cord in mononeuropathic rats. Regul Pept 86: 89–94.[Medline]
Zini S, Roisin MP, Langel U, Bartfai T, and Ben-Ari Y (1993) Galanin reduces release of endogenous excitatory amino acids in the rat hippocampus. Eur J Pharmacol 245: 1–7.[CrossRef][Medline]
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