- Email: [email protected]
Glycinamide, a glycine pro-drug, induces antinociception by intraperitoneal or oral ingestion in ovariectomized rats
Life Sciences 92 (2013) 576–581
Contents lists available at SciVerse ScienceDirect
Life Sciences journal homepage: www.elsevier.com/locate/lifescie
Glycinamide, a glycine pro-drug, induces antinociception by intraperitoneal or oral ingestion in ovariectomized rats C. Beyer a, B.K. Komisaruk b, O. González-Flores a, P. Gómora-Arrati a,⁎ a b
Centro de Investigación en Reproducción Animal, Universidad Autónoma de Tlaxcala-CINVESTAV, Apdo. 62, Tlaxcala, Mexico Department of Psychology, Rutgers, the State University of New Jersey, Newark 07102, USA
a r t i c l e
i n f o
Article history: Received 23 August 2012 Accepted 15 January 2013 Keywords: Glycinamide Glycine Tail flick test Tail shock test Antinociception Female rat
a b s t r a c t Aims: The effect of i.p. injection or oral ingestion of glycinamide, a glycine pro-drug, on two tests for nociception was assessed in ovariectomized Sprague Dawley rats. Main methods: To explore the potential analgesic effect of glycinamide the vocalization threshold to tail shock (VT) and the tail flick latency (TFL) were used. Glycinamide was administered both through the intraperitoneal route (doses 0, 25, 100, 400, 800 mg/kg) and through ad libitum oral ingestion of glycinamide solution (40 mg/ml) following a 24 h period of water deprivation. Key findings: Glycinamide exerted a significant analgesic effect on VT when injected i.p. at doses of 400 or 800 mg/kg. Analgesia occurred 10–20 min post-injection and persisted approx 45 min. At the high dose level, glycinamide exerted a weaker and more delayed effect on TFL than on the VT test. I.p. injection of 800 mg/kg glycinamide inhibited vocalizations induced by the application of suprathreshold tail shocks (30% above threshold) with a latency of approx 3 min and duration of approx 1 h. The volume of a glycinamide solution (40 mg/ml) ingested by rats deprived of water for 24 h was positively correlated with the degree of analgesia in the VT test. Values between 100 and 200 mg glycinamide exerted clear analgesic responses. Significance: Thus, glycinamide, either by systemic or oral routes, exerts a clear analgesic effect in the VT test of nociception and a much weaker action in the TFL test. This effect is probably due to the conversion of glycinamide to glycine in the brain. © 2013 Elsevier Inc. All rights reserved.
Introduction Glycine is the most abundant inhibitory neurotransmitter in the spinal cord (Béchade et al., 1994; Curtis et al., 1968; Legendre, 2001; Lynch and Callister, 2006; Zeilhofer et al., 2012). Blockage of strychnine-sensitive glycine receptors by strychnine in the spinal cord elicits allodynia, a condition in which normally innocuous tactile stimuli provoke intense aversive responses suggestive of pain (Beyer et al., 1985, 1988; Roberts et al., 1985; Sherman and Loomis, 1994, 1995; Yaksh, 1989). This finding suggests that low intensity cutaneous stimulation activates A-delta fibers, whose activity is modulated by a spinal strychnine-sensitive glycinergic pathway (D'Mello and Dickenson, 2008). Furthermore, blockage of glycine receptors by strychnine enhances neuropathic pain (Seltzer et al., 1991; Simpson and Huang, 1998; Yamamoto and Yaksh, 1993). Intrathecal administration of glycine or other amino acids (i.e., alanine, taurine), known to interact with the strychnine-sensitive glycine receptor, blocks the production of allodynia by strychnine (Beyer et al., 1988; Lim and ⁎ Corresponding author at: Centro de Investigación en Reproducción Animal, Plaza Hidalgo s/n, Panotla, Tlaxcala, Mex, CP. 90140, Mexico. Tel./fax: +52 2464621727. E-mail address: [email protected] (P. Gómora-Arrati). 0024-3205/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.lfs.2013.01.022
Lee, 2010). Glycinergic neurons may also block C-fiber-induced afference (Zhou et al., 2008). Intracerebroventricular or intrathecal injection of glycine produces analgesia in thermal and chemical nociception (Cheng et al., 2009; Haranishi et al., 2010; Simpson et al., 1997), evidence that glycine modulates pain responses to a variety of nociceptive stimuli. The potential therapeutic use of glycine as an analgesic has been hindered by its limited penetrability into the CNS (Battistin et al., 1971; Lajtha and Toth 1961; Oldendorf 1971; Seta et al. 1972), as a large amount of the amino acid is being required to produce significant levels in CNS (Toth and Lajtha, 1981). Some precursors, e.g. milacemide, readily penetrate into the CNS and elevate glycine concentrations in the brain (Cristophe et al., 1983; Doheny et al., 1996; Janssens de Varebeke et al., 1988). Milacemide is used as an antiepileptic drug and interferes with the production of allodynia by strychnine (Khandwala and Loomis, 1998). Milacemide is converted to glycine through glycinamide, which is the immediate precursor of glycine (Doheny et al., 1996; Janssens de Varebeke et al., 1988). Thus, glycinamide is more effective than milacemide in producing analgesia. In order to characterize the possible analgesic effects of glycinamide, in the present study, we measured the antinociceptive
C. Beyer et al. / Life Sciences 92 (2013) 576–581
577
dosage and time-course effects of glycinamide via two different routes of administration and on two different types of tests of analgesia.
Control rats (n = 6) received saline and experimental rats (n = 6) 800 mg/kg glycinamide.
Material and methods
Protocol 3
Animals
Effect of oral ingestion of glycinamide on VT Twenty three rats were deprived of water for 24 h. At the end of this period, VT was determined in all rats. Thereafter, for a period of 15 min, 7 of twenty three rats were given access to pure water, and the other rats to a glycinamide solution of 40 mg/ml. The volume, and thereby the quantity, of glycinamide ingested was registered for each rat. After this 15 min drinking period, the VT was determined at 10, 30, 60, 90, 120, 180 and 240 min latencies.
Measurement of vocalization threshold (VT) Ovx rats were placed inside a round Plexiglas arena (50 cm. diameter) with floor covered with sawdust and allowed to adapt for 5 min. A pair of electrodes were attached to the tail and connected to a DC stimulator (Coulbourn model E 13-51) via a commutator as described previously (González-Mariscal et al., 1992). This arrangement allowed the rat to move freely about the cage. Stimulus parameters: 1 ms pulses, 50 Hz, 300 ms train duration. VT was determined by increasing the current in steps of 100 μA until the rat vocalized and then decreasing the current also in steps of 100 μA until vocalization did not occur. The process was repeated three times and the inflection points thus obtained were averaged to determine the VT. Measurement of tail flick latency (TFL) TFL was determined with an IITC model 33 heat lamp-Analgesiometer (Landing, NJ, at 90% intensity). Rats were placed in a Plexiglas restrainer with the tail exposed to the radiant heat lamp. The TFLs were measured automatically by activation of a photocell upon tail withdrawal. A cutoff time of 15 s exposure to the stimulus was selected to avoid tissue damage. Experiment 1 Experimental procedures Effect of i.p. glycinamide on VT and TFL. A total of 45 ovx rats were used to assess the effect of various dosages of glycinamide. The following groups of ovx rats constituted at random received the following treatments via the i.p. route: group 1, saline (n= 10); group 2, 25 mg/kg glycinamide (n= 7); group 3, 100 mg/kg glycinamide (n= 8); group 4, 400 mg/kg glycinamide (n= 10); group 5, 800 mg/kg of glycinamide (n= 10). VT was determined immediately before injections and 10, 20, 30, 45, 60 and 90 min thereafter. Effect of glycinamide on TFL. A total of 24 ovx rats were used in this study. Groups were constituted at random and received the following treatments i.p.: group 6, saline (n= 8); group 7, 100 mg/kg glycinamide (n= 8); group 8, 800 mg/kg glycinamide (n= 8). Rats were tested immediately before injection and at 5, 15, 30, 45 and 60 min thereafter. Protocol 2 To determine more precisely the time course of the analgesic effect of glycinamide, 12 rats were subjected to the following procedure. The VT was initially determined as described above (Protocol 1). Before injections (saline or glycinamide) ten shocks set at 30% intensity above each rat's threshold to vocalize were delivered at 1 min intervals and the number of vocalizations thus elicited was registered. Shocks delivered one per min were reinitiated immediately after the injection and continued for 60 min. The number of vocalizations was registered.
Effect of oral ingestion of glycinamide on TFL Twenty one rats were deprived of water for 24 h at the end of this period the TFL was determined. After testing rats were allowed to drink either pure water (n = 7) or a solution of glycinamide (40 mg/ml; n = 14) for 15 min. Following the drinking period, the rats were tested for tail flick latencies at 10, 30, 60 and 90 min. Statistical tests The effect of glycinamide treatments on VT and TFL tests was analyzed by ANOVA with subsequent pairwise comparisons. Blocks of 10 trials (10 shocks) were made in order to assess the temporal course of the effect of glycinamide. The effect of variable amounts of glycinamide orally ingested on nociception (VT and TFL) was assessed via a regression analysis and calculation of the correlation coefficient. Probability values of p ≤ 0.05 were considered significant. Results VT test Fig. 1 shows the effect of various doses of i.p. glycinamide on the vocalization threshold to tail shock. Administration of 400 and 800 mg/kg of glycinamide elicited a significant increase in the VT with a short latency (10 min). Maximal responses were obtained within 10 min and gradually declined thereafter. Values for 400 and 800 mg/kg were still significantly higher than saline values 45 min
% Change in VT (mean ± s.e.m.)
Sprague Dawley female rats (200–250 g) were used in this study. The rats were maintained in a dark-light cycle (14 h light: 10 h dark: lights off at 12:00). Four rats were housed per cage; they were fed with Purina rat pellets and water ad libitum. Rats were ovariectomized (ovx) under ether anesthesia and injected after surgery with i.m. penicillin (100 I. U.).
Saline (n=10)
25 mg (n=7)
400 mg (n=10)
800 mg (n=10)
100
100 mg (n=8)
***
80
*** 60
*** 40
*
*
20 0 10
20
30
45
60
90
Minutes Post injection Fig. 1. Changes in the VT following i.p. injection of saline or glycinamide: 25, 100, 400, and 800 mg/kg. Values are expressed as percent increases from control, (preinjection values). Saline and the lower doses of glycinamide failed to elicit significant effects throughout the duration of the test (90 min). Higher doses of glycinamide elicited significant analgesia, persisting for 45 min in the 800 mg/kg group. Unless otherwise specified, in the following figures, the Kruskal–Wallis test, when significant, was followed by the Mann–Whitney test. *= p b 0.05; *** = p b 0.001.
C. Beyer et al. / Life Sciences 92 (2013) 576–581
after i.p. injection. Control rats (saline) showed relatively stable VT values throughout the testing period. When responses were analyzed by measuring the area under the response curves in each rat (Fig. 2), a clear dose response relationship was noted. Glycinamide administration did not produce any observable behavioral effects on the rats. Rats remained healthy after the test during the following week. TFL All postinjection values in response to i.p. injection of saline were lower than the initial preinjection values (Fig. 3). Although values obtained with 100 mg/kg glycinamide dose were somewhat higher than saline values, no significant analgesia occurred. The higher dose of glycinamide (800 mg/kg) elicited a delayed significant response, though still a modest analgesia, i.e., 10–20% rise in comparison with the preinjection value. When TFL was analyzed by measuring the area under the response curves in each rat (Fig. 4), a dose response relationship was noted. Glycinamide administration did not produce observable behavioral effects on the rats at the dosages employed.
Saline (n=8)
% Changes in TFL (mean ± s.e.m.)
578
Glycinamide 100 mg/kg (n=8) Glycinamide 800 mg/kg (n=8)
30
*** 20
**
10 0 -10 -20 -30
5
15
30
45
60
Minutes post injection Fig. 3. Tail flick latency following i.p. injection of two doses of glycinamide (100 and 800 mg/kg). Saline injection elicited a moderate hyperalgesia, TFL decreasing at all times throughout the test. TFL values varied among subjects; significant analgesia occurred only at the 800 mg/kg dose ** = p b 0.01; *** = p b 0.001.
Time course of glycinamide analgesia
Oral ingestion of glycinamide Ingestion of glycinamide after 24 h of water deprivation varied considerably among rats. Regression analysis revealed a highly significant positive correlation between quantities of glycinamide ingested and percentages of change from control values in the VT group. Significant positive correlations in this group were found at 30 and 60 min after ingestion and persisted for 90 min. Fig. 6A shows data at 60 min post ingestion when the maximal correlation coefficients were observed in this group. By contrast, a weak though significant positive correlation was found in the TFL at 10 min post ingestion of glycinamide. For purposes of temporal display of the
% Change in VT (mean ± s.e.m.)
50
***
40
*
30
20
10
0 0
25
100
400
800
Glycinamide (mg/kg, i.p.)
changes, rats were split into two subgroups: rats ingesting between 75 and 125 mg (both VT and TFL groups) and those ingesting between 150 and 225 mg (VT) and 126 and 200 mg of glycinamide (TFL). As shown in Fig. 7 the temporal course of the effect of glycinamide differed between the VT and TFL groups. Statistical comparison with values obtained with the group that ingested only water revealed significant differences with the VT group (Fig. 7A). No significant differences between the control water group and the TFL group were observed. The apparent contradiction between this finding and that obtained with the regression analysis is most likely because in the latter analysis all rats were included, while in the former analysis only half of the rats were included.
Discussion Glycine is likely the most important modulator of nociceptive information at the level of the spinal cord (Béchade et al., 1994; Curtis et al., 1968; Legendre, 2001; Lynch and Callister, 2006; Zeilhofer et al., 2012). Glycine exerts an inhibitory effect on various pain modalities that originate from mechanical or visceral thermal nociceptors and are conveyed by A-delta and C fibers (Dickenson and Le Bars, 1987; Villanueva and Le Bars, 1995). Removal of this inhibitory tone by strychnine, a glycine antagonist, also leads to allodynia in which normally innocuous stimuli,
% Changes in TFL (mean ± s.e.m.)
Fig. 5 shows that the application of tail shocks 30% above baseline threshold values (i.e., “suprathreshold”) induced vocalizations in about 80% of cases when applied in saline-treated rats. Vocalizations were drastically reduced to less than 20% of responses within the first 2– 10 min when 800 mg/kg of glycinamide was injected. Values of vocalization responses were still significantly lower in glycinamide-treated rats than in saline-treated rats at 1 h postinjection. The analgesic effect of glycinamide was very rapid since failure to respond to the suprathreshold shocks in glycinamide-treated rats occurred in 7 of out 8 rats tested within the first 5 min postinjection.
20
**
15 10
*
5 0 -5 -10 -15 -20 0
100
800
Glycinamide (mg/kg, i.p.) Fig. 2. Integrated response (area under the response curves of VT) to glycinamide administration (25, 100, 400, and 800 mg/kg). Only the two highest doses of glycinamide produced a significant response. * = p b 0.05; *** = p b 0.00; comparison VS saline.
Fig. 4. Integrated response to glycinamide administration (100 and 800 mg/kg). Both doses of glycinamide produced a significant response. * = p b 0.05; *** = p b 0.01.
C. Beyer et al. / Life Sciences 92 (2013) 576–581
Glycinamide 800 mg/kg (n=6)
80 60 40
p<0.05 p<0.01
20 0 Pre
2-10
11-20 21-30 31-40 41-50 51-60
A % Change in VTTS (mean ± s.e.m.)
% Vocalization to 10 tail shocks (30% above threshold)
Saline (n=6) 100
579
0 mg 75-125 mg 150-200 mg
50 40
**
30 20 10
*
** **
**
0 -10 -20 10
Minutes post injection
30
60
90
120
180
240
Minutes post ingestion
e.g., hair deflection, elicit intense pain-like responses (Beyer et al. 1985; 1988; Lim and Lee 2010; Sherman and Loomis 1995; Yamamoto and Yaksh 1993). Glycine has been shown to tonically inhibit afference from mechanoreceptors in the skin and hair follicles (Mitchell et al., 1993; Sandkühler et al., 1997). As could be anticipated, the intracerebroventricular or intrathecal administration of glycine, or some amino acids such as alanine or taurine that are capable of interacting with the glycine receptors, prevents strychnine-induced allodynia and exerts analgesic effect on mechanical and thermal tests of nociception (Beyer et al., 1988; Lim and Lee, 2010). Some strategies have been used to increase glycine concentrations in the central nervous system
% Change in VT
60
40
R=0.65, F=14.4, p<0.001
20
0
0
50
100
150
200
60 minutes post glycinamide (mg) ingestion
B 80
% Change in TFL
50 0 mg 75-125 mg 125-175 mg
40 30 20 10 0 -10
10
30
60
90
Minutes post ingestion Fig. 7. VT following water or glycinamide quantities orally consumed during the 10 min access period (ranges 75–125; n = 8, or 150–200; mg n = 8). Values are expressed as percentage increase from control, water-consumption values (A). (B), TFL following water or glycinamide orally consumed during the 10 min (ranges 75–125; n= 8, or 126–175; mg n = 7). *= p b 0.05; ** = p b 0.01.
A
-20
B % Change in TFL (mean ± s.e.m.)
Fig. 5. Effect of glycinamide (800 mg/kg., i.p.) on vocalization elicited by shocks 30% higher than threshold vocalization values. Points represent data pooled from 10 shocks, over 10 minute intervals. Note the short latencies of vocalization suppression and prolonged duration of the effect. Mann–Whitney U test. *= p b 0.05; ** = p b 0.01.
60 R=0.55, F=7.79, p<0.02
40 20 0 -20 0
50
100
150
10 minutes post glycinamide (mg) ingestion Fig. 6. Positive correlation between glycinamide (40 mg/ml) quantities orally consumed during 10 min and changes in the VT. The VT control was obtained immediately before drinking the solution after 24 h water deprivation (A). Tail flick latencies (B).
or to enhance its action at glycine receptors. Another approach has been the administration of milacemide, which is a glycine pro-drug that readily penetrates into the CNS where it is rapidly metabolized to glycinamide and subsequently to glycine, thereby significantly elevating their concentration in various brain regions (Cristophe et al., 1983; Semba et al., 1991; Semba and Patsalos, 1993). The findings in the present study provide evidence that systemic administration of glycinamide i.p., or its oral ad libitum ingestion, produced a significant and clear analgesic effect to the VT test. Our results suggest that glycinamide, either i.p. or orally ingested, is much more effective in counteracting pain elicited by electrical shocks in the tail than that resulting from radiant heat noxious stimulation. Noxious heat stimulation activates a different pathway than that conveying noxious cutaneous stimulation (Willis, 1984). Several results suggest that noxious heat stimulation is only partially modulated by glycinergic activity. Thus, blockage of glycine receptors by strychnine elicits allodynia to local tactile stimulation but has no effect on the TFL or hot plate latency (Yaksh, 1989). We conclude that glycinamide inhibits activity in the A-delta and/ or C-fiber pathways on the basis that electrical stimulation activates both A-delta and C fibers (Fox and Melzack, 1976; LaMotte and Campbell, 1982; Melzack, 1975; Wall and Woolf, 1984; Woolf and Wall, 1986). As glycinamide is metabolized to glycine, it is likely that its effect is due to its conversion to this amino acid (Cristophe et al., 1983; Doheny et al., 1996; Janssens de Varebeke et al., 1988). It is also possible that glycinamide per se may exert some effects on other neurotransmitters or receptors conducive to analgesia (Beyer et al.,
580
C. Beyer et al. / Life Sciences 92 (2013) 576–581
1985; Roberts et al., 1985). The general issue of an agent acting in its native form and/or as a metabolite raises the question of whether glycinamide induces analgesia only through its conversion to glycine, or whether it acts as glycinamide per se, and/or that its action is mediated by additional neurotransmitters. This question requires further study, e.g., by blocking glycinamide's metabolism to glycine. One of the problems with the systemic use of glycine or glycine pro-drugs for producing analgesia is that this amino acid is a co-agonist for the glycine binding site of NMDA receptors (Beyer et al., 1992). Consequently, increased glycine levels in the spinal cord may enhance nociceptive transmission by increasing NMDA receptor activity in nociceptor pathways (Christensen et al., 1998; Dohi et al., 2009; Morita et al., 2008). Indeed antagonists of the glycine NMDA receptors facilitate the analgesic effects of morphine and of vaginocervical stimulation (Beyer et al., 1992; Caba et al., 1998). Although i.p. or oral administration of glycinamide most likely increased the availability of glycine at both strychnine-sensitive glycine receptors and at the NMDA receptor environments, the antinociceptive response prevailed, as shown by the present results (Johnson and Ascher, 1987; Rao et al., 1990). This agrees with previous reports in which intrathecal infusion of glycine induced analgesia (Beyer et al., 1985; Cheng et al., 2009). Conclusion Glycinamide, a glycine pro-drug, exerts significant antinociceptive effects on the vocalization threshold-to-tail-shock test and the weaker action on the tail flick latency test, most likely by increasing glycine levels in the central nervous system. The magnitude, duration, and effectiveness of the oral administration route, of this antinociceptive effect of glycinamide, suggest its potential therapeutic use as an analgesic. Conflict of interest statement The authors declare no conflicts of interest.
Acknowledgments The authors gratefully acknowledge the technical assistance of Guadalupe Domínguez-López. This work was supported by CONACYT/ 134291. References Battistin L, Grynbaum A, Lajtha A. The uptake of various amino acids by the mouse brain in vivo. Brain Res 1971;29:85–99. Béchade C, Sur C, Triller A. The inhibitory neuronal glycine receptor. Bioessays 1994;16: 735–44. Beyer C, Roberts LA, Komisaruk BR. Hyperalgesia induced by altered glycinergic activity at the spinal cord. Life Sci 1985;37:875–82. Beyer C, Banas C, Gómora P, Komisaruk BR. Prevention of the convulsant and hyperalgesic action of strychnine by intrathecal glycine and related amino acids. Pharmacol Biochem Behav 1988;29:73–8. Beyer C, Komisaruk BR, López-Colomé AM, Caba M. Administration of AP5, a glutamate antagonist, unmasks glycine analgesic actions in the rat. Pharmacol Biochem Behav 1992;42:229–32. Caba M, Komisaruk BR, Beyer C. Analgesic synergism between AP5 (an NMDA receptor antagonist) and vagino cervical stimulation in the rat. Pharmacol Biochem Behav 1998;61:45–8. Cheng W, Yin Q, Cheng MY, Chen HS, Wang S, Feng T, et al. Intracerebroventricular or intrathecal injection of glycine produces analgesia in thermal nociception and chemical nociception via glycine receptors. Eur J Pharmacol 2009;614:44–9. Christensen D, Idänpään-Heikkilä JJ, Guilbaud G, Kayser V. The antinociceptive effect of combined systemic administration of morphine and the glycine/NMDA receptor antagonist, (+)-HA966 in a rat model of peripheral neuropathy. Br J Pharmacol 1998;125:1641–50. Cristophe J, Kutzner R, Nguyen-Bui H, Damien C, Chatelain P, Gillete L. Conversion of orally administered 2-n-pentylamidoacetamide into glycinamide and glycine in the rat brain. Life Sci 1983;33:333–41. Curtis DR, Hösli L, Johnston GA. A pharmacological study of the depression of spinal neurons by glycine and related amino acids. Exp Brain Res 1968;6:1-18.
Dickenson AH, Le Bars D. Lack of evidence for increased descending inhibition on the dorsal horn of the rat following periaqueductal grey morphine microinjections. Br J Pharmacol 1987;92:271–80. D'Mello R, Dickenson AH. Spinal cord mechanisms of pain. Br J Anaesth 2008;101: 8-16. Doheny M, Nagaki S, Patsalos PN. A microdialysis study of glycinamide, glycine and other amino acid neurotransmitters in rat frontal cortex and hippocampus after the administration of milacemide, a glycine pro-drug. Naunyn Schmiedebergs Arch Pharmacol 1996;354:157–63. Dohi T, Morita K, Kitoyama T, Motoyama M, Morioka N. Glycine transporter inhibitors as a novel drug discovery strategy for neuropathic pain. Pharmacol Ther 2009;123: 54–79. Fox EJ, Melzack R. Transcutaneous electrical stimulation and acupuncture: comparison of treatment for low-back pain. Pain 1976;2:141–8. González-Mariscal G, Gómora P, Caba M, Beyer C. Copulatory analgesia in male rats ensues from arousal, motor activity, and genital stimulation: blockage by manipulation and restraint. Physiol Behav 1992;51:775–81. Haranishi Y, Hara K, Terada T, Nakamura S, Sata T. The antinociceptive effect of intrathecal administration of glycine transporter-2 inhibitor ALX1393 in a rat acute pain model. Anesth Analg 2010;110:615–21. Janssens de Varebeke P, Cavalier R, David-Remacle M, Youdim MB. Formation of the neurotransmitter glycine from the anticonvulsant milacemide is mediated by brain monoamine oxidase B. J Neurochem 1988;50:1011–6. Johnson JW, Ascher P. Glycine potentiates the NMDA-response in cultured mouse brain neurons. Nature 1987;325:529–31. Khandwala H, Loomis CW. Milacemide, a glycine pro-drug, inhibits strychnine allodynia without affecting normal nociception in the rat. Pain 1998;77: 87–95. Lajtha A, Toth J. The brain barrier system–II. Uptake and transport of amino acids by the brain. J Neurochem 1961;8:216–25. LaMotte RH, Campbell JN. Comparison of responses of warm and nociceptive C-fiber afferents in monkey with human judgments of thermal pain. J Neurophysiol 1982;41: 509–28. Legendre P. The glycinergic inhibitory synapse. Cell Mol Life Sci 2001;58:760–93. Lim ES, Lee IO. Effect of intrathecal glycine and related amino acids on the allodynia and hyperalgesic action of strychnine or bicuculline in mice. Korean J Anesthesiol 2010;58:76–86. Lynch JW, Callister RJ. Glycine receptors: a new therapeutic target in pain pathways. Curr Opin Investig Drugs 2006;7:48–53. Melzack R. Prolonged relief of pain by brief, intense transcutaneous somatic stimulation. Pain 1975;1:357–73. Mitchell K, Spike RC, Todd AJ. An immunocytochemical study of glycine receptor and GABA in laminae I–III of rat spinal dorsal horn. J Neurosci 1993;13:2371–81. Morita K, Motoyama N, Kitayama T, Morioka N, Kifune K, Dohi T. Spinal antiallodynia action of glycine transporter inhibitors in neuropathic pain models in mice. J Pharmacol Exp Ther 2008;326:633–45. Oldendorf WH. Brain uptake of radiolabeled amino acids, amines, and hexoses after arterial injection. Am J Physiol 1971;221:1629–39. Rao TS, Cler JA, Emmett MR, Mick SJ, Lyengar S, Wood PL. Glycine, glycinamide and D-serine act as positive modulators of signal transduction at the N-methyl-D-Aspartate (NMDA) receptor in vivo: differential effects on mouse cerebellar cyclic guanosine monophosphate levels. Neuropharmacology 1990;29:1075–80. Roberts LA, Beyer C, Komisaruk BR. Strychnine antagonizes vaginal stimulation-produced analgesia at the spinal cord. Life Sci 1985;36:2017–23. Sandkühler J, Chen JG, Cheng G, Randić M. Low-frequency stimulation of afferent Adelta-fibers induces long-term depression at primary afferent synapses with substantia gelatinosa neurons in the rat. J Neurosci 1997;17:6483–91. Seltzer Z, Cohn S, Ginzburg R, Beilin B. Modulation of neuropathic pain behavior in rats by spinal disinhibition and NMDA receptor blockade of injury discharge. Pain 1991;45:69–75. Semba J, Patsalos PN. Milacemide effects on the temporal inter-relationship of amino acids and monoamine metabolites in rat cerebrospinal fluid. Eur J Pharmacol 1993;230:321–6. Semba J, Ratnaraj N, Patsalos PN. Simple and rapid micro-analytical procedures for the estimation of milacemide and its metabolite glycinamide in rat plasma and cerebrospinal fluid by high-performance liquid chromatography. J Chromatogr 1991;565: 357–62. Seta K, Sershen H, Lajtha A. Cerebral amino acid uptake in vivo in newborn mice. Brain Res 1972;47:415–25. Sherman SE, Loomis CW. Morphine insensitive allodynia is produced by intrathecal strychnine in the lightly anesthetized rat. Pain 1994;56:17–29. Sherman SE, Loomis CW. Strychnine-dependent allodynia in the urethane-anesthetized rat is segmentally distributed and prevented by intrathecal glycine and betaine. Can J Physiol Pharmacol 1995;73:1698–705. Simpson Jr RK, Huang W. Glycine receptor reduction within segmental gray matter in a rat model in neuropathic pain. Neurol Res 1998;20:161–8. Simpson Jr RK, Gondo M, Robertson CS, Goodman JC. Reduction in thermal hyperalgesia by intrathecal administration of glycine and related compounds. Neurochem Res 1997;22:75–9. Toth E, Lajtha A. Elevation of cerebral levels of non essential amino acids in vivo by administration of large doses. Neurochem Res 1981;6:1309–17. Villanueva L, Le Bars D. The activation of bulbo-spinal controls by peripheral nociceptive inputs: diffuse noxious inhibitory controls. Biol Res 1995;28:113–25. Wall PD, Woolf CJ. Muscle but not cutaneous C-afferent input produces prolonged increases in the excitability of the flexion reflex in the rat. J Physiol 1984;356: 443–58.
C. Beyer et al. / Life Sciences 92 (2013) 576–581 Willis WD. The pain system: the neural of nociceptive transmission in the mammalian nervous system. Series Ed. In: Gilderberg PL, editor. Basel; New York: Karger; 1984. Woolf CJ, Wall PD. Relative effectiveness of C primary afferent fibers of different origins in evoking a prolonged facilitation of the flexor reflex in the rat. J Neurosci 1986;6: 1433–42. Yaksh TL. Behavioral and autonomic correlates of tactile evoked allodynia produced by spinal glycine inhibition: effects of modulatory receptor systems and excitatory amino acid antagonists. Pain 1989;37:111–23.
581
Yamamoto T, Yaksh TL. Effects of intrathecal strychnine and bicuculline on nerve compression-induced thermal hyperalgesia and selective antagonism by MK-801. Pain 1993;54:79–84. Zeilhofer HU, Wildner H, Yévenes GE. Fast synaptic inhibition in spinal sensory processing and pain control. Physiol Rev 2012;92:193–235. Zhou HY, Zhang HM, Chen SR, Pan HL. Increased C-fiber nociceptive input potentiates inhibitory glycinergic transmission in the spinal dorsal horn. J Pharmacol Exp Ther 2008;324:1000–10.
Contents lists available at SciVerse ScienceDirect
Life Sciences journal homepage: www.elsevier.com/locate/lifescie
Glycinamide, a glycine pro-drug, induces antinociception by intraperitoneal or oral ingestion in ovariectomized rats C. Beyer a, B.K. Komisaruk b, O. González-Flores a, P. Gómora-Arrati a,⁎ a b
Centro de Investigación en Reproducción Animal, Universidad Autónoma de Tlaxcala-CINVESTAV, Apdo. 62, Tlaxcala, Mexico Department of Psychology, Rutgers, the State University of New Jersey, Newark 07102, USA
a r t i c l e
i n f o
Article history: Received 23 August 2012 Accepted 15 January 2013 Keywords: Glycinamide Glycine Tail flick test Tail shock test Antinociception Female rat
a b s t r a c t Aims: The effect of i.p. injection or oral ingestion of glycinamide, a glycine pro-drug, on two tests for nociception was assessed in ovariectomized Sprague Dawley rats. Main methods: To explore the potential analgesic effect of glycinamide the vocalization threshold to tail shock (VT) and the tail flick latency (TFL) were used. Glycinamide was administered both through the intraperitoneal route (doses 0, 25, 100, 400, 800 mg/kg) and through ad libitum oral ingestion of glycinamide solution (40 mg/ml) following a 24 h period of water deprivation. Key findings: Glycinamide exerted a significant analgesic effect on VT when injected i.p. at doses of 400 or 800 mg/kg. Analgesia occurred 10–20 min post-injection and persisted approx 45 min. At the high dose level, glycinamide exerted a weaker and more delayed effect on TFL than on the VT test. I.p. injection of 800 mg/kg glycinamide inhibited vocalizations induced by the application of suprathreshold tail shocks (30% above threshold) with a latency of approx 3 min and duration of approx 1 h. The volume of a glycinamide solution (40 mg/ml) ingested by rats deprived of water for 24 h was positively correlated with the degree of analgesia in the VT test. Values between 100 and 200 mg glycinamide exerted clear analgesic responses. Significance: Thus, glycinamide, either by systemic or oral routes, exerts a clear analgesic effect in the VT test of nociception and a much weaker action in the TFL test. This effect is probably due to the conversion of glycinamide to glycine in the brain. © 2013 Elsevier Inc. All rights reserved.
Introduction Glycine is the most abundant inhibitory neurotransmitter in the spinal cord (Béchade et al., 1994; Curtis et al., 1968; Legendre, 2001; Lynch and Callister, 2006; Zeilhofer et al., 2012). Blockage of strychnine-sensitive glycine receptors by strychnine in the spinal cord elicits allodynia, a condition in which normally innocuous tactile stimuli provoke intense aversive responses suggestive of pain (Beyer et al., 1985, 1988; Roberts et al., 1985; Sherman and Loomis, 1994, 1995; Yaksh, 1989). This finding suggests that low intensity cutaneous stimulation activates A-delta fibers, whose activity is modulated by a spinal strychnine-sensitive glycinergic pathway (D'Mello and Dickenson, 2008). Furthermore, blockage of glycine receptors by strychnine enhances neuropathic pain (Seltzer et al., 1991; Simpson and Huang, 1998; Yamamoto and Yaksh, 1993). Intrathecal administration of glycine or other amino acids (i.e., alanine, taurine), known to interact with the strychnine-sensitive glycine receptor, blocks the production of allodynia by strychnine (Beyer et al., 1988; Lim and ⁎ Corresponding author at: Centro de Investigación en Reproducción Animal, Plaza Hidalgo s/n, Panotla, Tlaxcala, Mex, CP. 90140, Mexico. Tel./fax: +52 2464621727. E-mail address: [email protected] (P. Gómora-Arrati). 0024-3205/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.lfs.2013.01.022
Lee, 2010). Glycinergic neurons may also block C-fiber-induced afference (Zhou et al., 2008). Intracerebroventricular or intrathecal injection of glycine produces analgesia in thermal and chemical nociception (Cheng et al., 2009; Haranishi et al., 2010; Simpson et al., 1997), evidence that glycine modulates pain responses to a variety of nociceptive stimuli. The potential therapeutic use of glycine as an analgesic has been hindered by its limited penetrability into the CNS (Battistin et al., 1971; Lajtha and Toth 1961; Oldendorf 1971; Seta et al. 1972), as a large amount of the amino acid is being required to produce significant levels in CNS (Toth and Lajtha, 1981). Some precursors, e.g. milacemide, readily penetrate into the CNS and elevate glycine concentrations in the brain (Cristophe et al., 1983; Doheny et al., 1996; Janssens de Varebeke et al., 1988). Milacemide is used as an antiepileptic drug and interferes with the production of allodynia by strychnine (Khandwala and Loomis, 1998). Milacemide is converted to glycine through glycinamide, which is the immediate precursor of glycine (Doheny et al., 1996; Janssens de Varebeke et al., 1988). Thus, glycinamide is more effective than milacemide in producing analgesia. In order to characterize the possible analgesic effects of glycinamide, in the present study, we measured the antinociceptive
C. Beyer et al. / Life Sciences 92 (2013) 576–581
577
dosage and time-course effects of glycinamide via two different routes of administration and on two different types of tests of analgesia.
Control rats (n = 6) received saline and experimental rats (n = 6) 800 mg/kg glycinamide.
Material and methods
Protocol 3
Animals
Effect of oral ingestion of glycinamide on VT Twenty three rats were deprived of water for 24 h. At the end of this period, VT was determined in all rats. Thereafter, for a period of 15 min, 7 of twenty three rats were given access to pure water, and the other rats to a glycinamide solution of 40 mg/ml. The volume, and thereby the quantity, of glycinamide ingested was registered for each rat. After this 15 min drinking period, the VT was determined at 10, 30, 60, 90, 120, 180 and 240 min latencies.
Measurement of vocalization threshold (VT) Ovx rats were placed inside a round Plexiglas arena (50 cm. diameter) with floor covered with sawdust and allowed to adapt for 5 min. A pair of electrodes were attached to the tail and connected to a DC stimulator (Coulbourn model E 13-51) via a commutator as described previously (González-Mariscal et al., 1992). This arrangement allowed the rat to move freely about the cage. Stimulus parameters: 1 ms pulses, 50 Hz, 300 ms train duration. VT was determined by increasing the current in steps of 100 μA until the rat vocalized and then decreasing the current also in steps of 100 μA until vocalization did not occur. The process was repeated three times and the inflection points thus obtained were averaged to determine the VT. Measurement of tail flick latency (TFL) TFL was determined with an IITC model 33 heat lamp-Analgesiometer (Landing, NJ, at 90% intensity). Rats were placed in a Plexiglas restrainer with the tail exposed to the radiant heat lamp. The TFLs were measured automatically by activation of a photocell upon tail withdrawal. A cutoff time of 15 s exposure to the stimulus was selected to avoid tissue damage. Experiment 1 Experimental procedures Effect of i.p. glycinamide on VT and TFL. A total of 45 ovx rats were used to assess the effect of various dosages of glycinamide. The following groups of ovx rats constituted at random received the following treatments via the i.p. route: group 1, saline (n= 10); group 2, 25 mg/kg glycinamide (n= 7); group 3, 100 mg/kg glycinamide (n= 8); group 4, 400 mg/kg glycinamide (n= 10); group 5, 800 mg/kg of glycinamide (n= 10). VT was determined immediately before injections and 10, 20, 30, 45, 60 and 90 min thereafter. Effect of glycinamide on TFL. A total of 24 ovx rats were used in this study. Groups were constituted at random and received the following treatments i.p.: group 6, saline (n= 8); group 7, 100 mg/kg glycinamide (n= 8); group 8, 800 mg/kg glycinamide (n= 8). Rats were tested immediately before injection and at 5, 15, 30, 45 and 60 min thereafter. Protocol 2 To determine more precisely the time course of the analgesic effect of glycinamide, 12 rats were subjected to the following procedure. The VT was initially determined as described above (Protocol 1). Before injections (saline or glycinamide) ten shocks set at 30% intensity above each rat's threshold to vocalize were delivered at 1 min intervals and the number of vocalizations thus elicited was registered. Shocks delivered one per min were reinitiated immediately after the injection and continued for 60 min. The number of vocalizations was registered.
Effect of oral ingestion of glycinamide on TFL Twenty one rats were deprived of water for 24 h at the end of this period the TFL was determined. After testing rats were allowed to drink either pure water (n = 7) or a solution of glycinamide (40 mg/ml; n = 14) for 15 min. Following the drinking period, the rats were tested for tail flick latencies at 10, 30, 60 and 90 min. Statistical tests The effect of glycinamide treatments on VT and TFL tests was analyzed by ANOVA with subsequent pairwise comparisons. Blocks of 10 trials (10 shocks) were made in order to assess the temporal course of the effect of glycinamide. The effect of variable amounts of glycinamide orally ingested on nociception (VT and TFL) was assessed via a regression analysis and calculation of the correlation coefficient. Probability values of p ≤ 0.05 were considered significant. Results VT test Fig. 1 shows the effect of various doses of i.p. glycinamide on the vocalization threshold to tail shock. Administration of 400 and 800 mg/kg of glycinamide elicited a significant increase in the VT with a short latency (10 min). Maximal responses were obtained within 10 min and gradually declined thereafter. Values for 400 and 800 mg/kg were still significantly higher than saline values 45 min
% Change in VT (mean ± s.e.m.)
Sprague Dawley female rats (200–250 g) were used in this study. The rats were maintained in a dark-light cycle (14 h light: 10 h dark: lights off at 12:00). Four rats were housed per cage; they were fed with Purina rat pellets and water ad libitum. Rats were ovariectomized (ovx) under ether anesthesia and injected after surgery with i.m. penicillin (100 I. U.).
Saline (n=10)
25 mg (n=7)
400 mg (n=10)
800 mg (n=10)
100
100 mg (n=8)
***
80
*** 60
*** 40
*
*
20 0 10
20
30
45
60
90
Minutes Post injection Fig. 1. Changes in the VT following i.p. injection of saline or glycinamide: 25, 100, 400, and 800 mg/kg. Values are expressed as percent increases from control, (preinjection values). Saline and the lower doses of glycinamide failed to elicit significant effects throughout the duration of the test (90 min). Higher doses of glycinamide elicited significant analgesia, persisting for 45 min in the 800 mg/kg group. Unless otherwise specified, in the following figures, the Kruskal–Wallis test, when significant, was followed by the Mann–Whitney test. *= p b 0.05; *** = p b 0.001.
C. Beyer et al. / Life Sciences 92 (2013) 576–581
after i.p. injection. Control rats (saline) showed relatively stable VT values throughout the testing period. When responses were analyzed by measuring the area under the response curves in each rat (Fig. 2), a clear dose response relationship was noted. Glycinamide administration did not produce any observable behavioral effects on the rats. Rats remained healthy after the test during the following week. TFL All postinjection values in response to i.p. injection of saline were lower than the initial preinjection values (Fig. 3). Although values obtained with 100 mg/kg glycinamide dose were somewhat higher than saline values, no significant analgesia occurred. The higher dose of glycinamide (800 mg/kg) elicited a delayed significant response, though still a modest analgesia, i.e., 10–20% rise in comparison with the preinjection value. When TFL was analyzed by measuring the area under the response curves in each rat (Fig. 4), a dose response relationship was noted. Glycinamide administration did not produce observable behavioral effects on the rats at the dosages employed.
Saline (n=8)
% Changes in TFL (mean ± s.e.m.)
578
Glycinamide 100 mg/kg (n=8) Glycinamide 800 mg/kg (n=8)
30
*** 20
**
10 0 -10 -20 -30
5
15
30
45
60
Minutes post injection Fig. 3. Tail flick latency following i.p. injection of two doses of glycinamide (100 and 800 mg/kg). Saline injection elicited a moderate hyperalgesia, TFL decreasing at all times throughout the test. TFL values varied among subjects; significant analgesia occurred only at the 800 mg/kg dose ** = p b 0.01; *** = p b 0.001.
Time course of glycinamide analgesia
Oral ingestion of glycinamide Ingestion of glycinamide after 24 h of water deprivation varied considerably among rats. Regression analysis revealed a highly significant positive correlation between quantities of glycinamide ingested and percentages of change from control values in the VT group. Significant positive correlations in this group were found at 30 and 60 min after ingestion and persisted for 90 min. Fig. 6A shows data at 60 min post ingestion when the maximal correlation coefficients were observed in this group. By contrast, a weak though significant positive correlation was found in the TFL at 10 min post ingestion of glycinamide. For purposes of temporal display of the
% Change in VT (mean ± s.e.m.)
50
***
40
*
30
20
10
0 0
25
100
400
800
Glycinamide (mg/kg, i.p.)
changes, rats were split into two subgroups: rats ingesting between 75 and 125 mg (both VT and TFL groups) and those ingesting between 150 and 225 mg (VT) and 126 and 200 mg of glycinamide (TFL). As shown in Fig. 7 the temporal course of the effect of glycinamide differed between the VT and TFL groups. Statistical comparison with values obtained with the group that ingested only water revealed significant differences with the VT group (Fig. 7A). No significant differences between the control water group and the TFL group were observed. The apparent contradiction between this finding and that obtained with the regression analysis is most likely because in the latter analysis all rats were included, while in the former analysis only half of the rats were included.
Discussion Glycine is likely the most important modulator of nociceptive information at the level of the spinal cord (Béchade et al., 1994; Curtis et al., 1968; Legendre, 2001; Lynch and Callister, 2006; Zeilhofer et al., 2012). Glycine exerts an inhibitory effect on various pain modalities that originate from mechanical or visceral thermal nociceptors and are conveyed by A-delta and C fibers (Dickenson and Le Bars, 1987; Villanueva and Le Bars, 1995). Removal of this inhibitory tone by strychnine, a glycine antagonist, also leads to allodynia in which normally innocuous stimuli,
% Changes in TFL (mean ± s.e.m.)
Fig. 5 shows that the application of tail shocks 30% above baseline threshold values (i.e., “suprathreshold”) induced vocalizations in about 80% of cases when applied in saline-treated rats. Vocalizations were drastically reduced to less than 20% of responses within the first 2– 10 min when 800 mg/kg of glycinamide was injected. Values of vocalization responses were still significantly lower in glycinamide-treated rats than in saline-treated rats at 1 h postinjection. The analgesic effect of glycinamide was very rapid since failure to respond to the suprathreshold shocks in glycinamide-treated rats occurred in 7 of out 8 rats tested within the first 5 min postinjection.
20
**
15 10
*
5 0 -5 -10 -15 -20 0
100
800
Glycinamide (mg/kg, i.p.) Fig. 2. Integrated response (area under the response curves of VT) to glycinamide administration (25, 100, 400, and 800 mg/kg). Only the two highest doses of glycinamide produced a significant response. * = p b 0.05; *** = p b 0.00; comparison VS saline.
Fig. 4. Integrated response to glycinamide administration (100 and 800 mg/kg). Both doses of glycinamide produced a significant response. * = p b 0.05; *** = p b 0.01.
C. Beyer et al. / Life Sciences 92 (2013) 576–581
Glycinamide 800 mg/kg (n=6)
80 60 40
p<0.05 p<0.01
20 0 Pre
2-10
11-20 21-30 31-40 41-50 51-60
A % Change in VTTS (mean ± s.e.m.)
% Vocalization to 10 tail shocks (30% above threshold)
Saline (n=6) 100
579
0 mg 75-125 mg 150-200 mg
50 40
**
30 20 10
*
** **
**
0 -10 -20 10
Minutes post injection
30
60
90
120
180
240
Minutes post ingestion
e.g., hair deflection, elicit intense pain-like responses (Beyer et al. 1985; 1988; Lim and Lee 2010; Sherman and Loomis 1995; Yamamoto and Yaksh 1993). Glycine has been shown to tonically inhibit afference from mechanoreceptors in the skin and hair follicles (Mitchell et al., 1993; Sandkühler et al., 1997). As could be anticipated, the intracerebroventricular or intrathecal administration of glycine, or some amino acids such as alanine or taurine that are capable of interacting with the glycine receptors, prevents strychnine-induced allodynia and exerts analgesic effect on mechanical and thermal tests of nociception (Beyer et al., 1988; Lim and Lee, 2010). Some strategies have been used to increase glycine concentrations in the central nervous system
% Change in VT
60
40
R=0.65, F=14.4, p<0.001
20
0
0
50
100
150
200
60 minutes post glycinamide (mg) ingestion
B 80
% Change in TFL
50 0 mg 75-125 mg 125-175 mg
40 30 20 10 0 -10
10
30
60
90
Minutes post ingestion Fig. 7. VT following water or glycinamide quantities orally consumed during the 10 min access period (ranges 75–125; n = 8, or 150–200; mg n = 8). Values are expressed as percentage increase from control, water-consumption values (A). (B), TFL following water or glycinamide orally consumed during the 10 min (ranges 75–125; n= 8, or 126–175; mg n = 7). *= p b 0.05; ** = p b 0.01.
A
-20
B % Change in TFL (mean ± s.e.m.)
Fig. 5. Effect of glycinamide (800 mg/kg., i.p.) on vocalization elicited by shocks 30% higher than threshold vocalization values. Points represent data pooled from 10 shocks, over 10 minute intervals. Note the short latencies of vocalization suppression and prolonged duration of the effect. Mann–Whitney U test. *= p b 0.05; ** = p b 0.01.
60 R=0.55, F=7.79, p<0.02
40 20 0 -20 0
50
100
150
10 minutes post glycinamide (mg) ingestion Fig. 6. Positive correlation between glycinamide (40 mg/ml) quantities orally consumed during 10 min and changes in the VT. The VT control was obtained immediately before drinking the solution after 24 h water deprivation (A). Tail flick latencies (B).
or to enhance its action at glycine receptors. Another approach has been the administration of milacemide, which is a glycine pro-drug that readily penetrates into the CNS where it is rapidly metabolized to glycinamide and subsequently to glycine, thereby significantly elevating their concentration in various brain regions (Cristophe et al., 1983; Semba et al., 1991; Semba and Patsalos, 1993). The findings in the present study provide evidence that systemic administration of glycinamide i.p., or its oral ad libitum ingestion, produced a significant and clear analgesic effect to the VT test. Our results suggest that glycinamide, either i.p. or orally ingested, is much more effective in counteracting pain elicited by electrical shocks in the tail than that resulting from radiant heat noxious stimulation. Noxious heat stimulation activates a different pathway than that conveying noxious cutaneous stimulation (Willis, 1984). Several results suggest that noxious heat stimulation is only partially modulated by glycinergic activity. Thus, blockage of glycine receptors by strychnine elicits allodynia to local tactile stimulation but has no effect on the TFL or hot plate latency (Yaksh, 1989). We conclude that glycinamide inhibits activity in the A-delta and/ or C-fiber pathways on the basis that electrical stimulation activates both A-delta and C fibers (Fox and Melzack, 1976; LaMotte and Campbell, 1982; Melzack, 1975; Wall and Woolf, 1984; Woolf and Wall, 1986). As glycinamide is metabolized to glycine, it is likely that its effect is due to its conversion to this amino acid (Cristophe et al., 1983; Doheny et al., 1996; Janssens de Varebeke et al., 1988). It is also possible that glycinamide per se may exert some effects on other neurotransmitters or receptors conducive to analgesia (Beyer et al.,
580
C. Beyer et al. / Life Sciences 92 (2013) 576–581
1985; Roberts et al., 1985). The general issue of an agent acting in its native form and/or as a metabolite raises the question of whether glycinamide induces analgesia only through its conversion to glycine, or whether it acts as glycinamide per se, and/or that its action is mediated by additional neurotransmitters. This question requires further study, e.g., by blocking glycinamide's metabolism to glycine. One of the problems with the systemic use of glycine or glycine pro-drugs for producing analgesia is that this amino acid is a co-agonist for the glycine binding site of NMDA receptors (Beyer et al., 1992). Consequently, increased glycine levels in the spinal cord may enhance nociceptive transmission by increasing NMDA receptor activity in nociceptor pathways (Christensen et al., 1998; Dohi et al., 2009; Morita et al., 2008). Indeed antagonists of the glycine NMDA receptors facilitate the analgesic effects of morphine and of vaginocervical stimulation (Beyer et al., 1992; Caba et al., 1998). Although i.p. or oral administration of glycinamide most likely increased the availability of glycine at both strychnine-sensitive glycine receptors and at the NMDA receptor environments, the antinociceptive response prevailed, as shown by the present results (Johnson and Ascher, 1987; Rao et al., 1990). This agrees with previous reports in which intrathecal infusion of glycine induced analgesia (Beyer et al., 1985; Cheng et al., 2009). Conclusion Glycinamide, a glycine pro-drug, exerts significant antinociceptive effects on the vocalization threshold-to-tail-shock test and the weaker action on the tail flick latency test, most likely by increasing glycine levels in the central nervous system. The magnitude, duration, and effectiveness of the oral administration route, of this antinociceptive effect of glycinamide, suggest its potential therapeutic use as an analgesic. Conflict of interest statement The authors declare no conflicts of interest.
Acknowledgments The authors gratefully acknowledge the technical assistance of Guadalupe Domínguez-López. This work was supported by CONACYT/ 134291. References Battistin L, Grynbaum A, Lajtha A. The uptake of various amino acids by the mouse brain in vivo. Brain Res 1971;29:85–99. Béchade C, Sur C, Triller A. The inhibitory neuronal glycine receptor. Bioessays 1994;16: 735–44. Beyer C, Roberts LA, Komisaruk BR. Hyperalgesia induced by altered glycinergic activity at the spinal cord. Life Sci 1985;37:875–82. Beyer C, Banas C, Gómora P, Komisaruk BR. Prevention of the convulsant and hyperalgesic action of strychnine by intrathecal glycine and related amino acids. Pharmacol Biochem Behav 1988;29:73–8. Beyer C, Komisaruk BR, López-Colomé AM, Caba M. Administration of AP5, a glutamate antagonist, unmasks glycine analgesic actions in the rat. Pharmacol Biochem Behav 1992;42:229–32. Caba M, Komisaruk BR, Beyer C. Analgesic synergism between AP5 (an NMDA receptor antagonist) and vagino cervical stimulation in the rat. Pharmacol Biochem Behav 1998;61:45–8. Cheng W, Yin Q, Cheng MY, Chen HS, Wang S, Feng T, et al. Intracerebroventricular or intrathecal injection of glycine produces analgesia in thermal nociception and chemical nociception via glycine receptors. Eur J Pharmacol 2009;614:44–9. Christensen D, Idänpään-Heikkilä JJ, Guilbaud G, Kayser V. The antinociceptive effect of combined systemic administration of morphine and the glycine/NMDA receptor antagonist, (+)-HA966 in a rat model of peripheral neuropathy. Br J Pharmacol 1998;125:1641–50. Cristophe J, Kutzner R, Nguyen-Bui H, Damien C, Chatelain P, Gillete L. Conversion of orally administered 2-n-pentylamidoacetamide into glycinamide and glycine in the rat brain. Life Sci 1983;33:333–41. Curtis DR, Hösli L, Johnston GA. A pharmacological study of the depression of spinal neurons by glycine and related amino acids. Exp Brain Res 1968;6:1-18.
Dickenson AH, Le Bars D. Lack of evidence for increased descending inhibition on the dorsal horn of the rat following periaqueductal grey morphine microinjections. Br J Pharmacol 1987;92:271–80. D'Mello R, Dickenson AH. Spinal cord mechanisms of pain. Br J Anaesth 2008;101: 8-16. Doheny M, Nagaki S, Patsalos PN. A microdialysis study of glycinamide, glycine and other amino acid neurotransmitters in rat frontal cortex and hippocampus after the administration of milacemide, a glycine pro-drug. Naunyn Schmiedebergs Arch Pharmacol 1996;354:157–63. Dohi T, Morita K, Kitoyama T, Motoyama M, Morioka N. Glycine transporter inhibitors as a novel drug discovery strategy for neuropathic pain. Pharmacol Ther 2009;123: 54–79. Fox EJ, Melzack R. Transcutaneous electrical stimulation and acupuncture: comparison of treatment for low-back pain. Pain 1976;2:141–8. González-Mariscal G, Gómora P, Caba M, Beyer C. Copulatory analgesia in male rats ensues from arousal, motor activity, and genital stimulation: blockage by manipulation and restraint. Physiol Behav 1992;51:775–81. Haranishi Y, Hara K, Terada T, Nakamura S, Sata T. The antinociceptive effect of intrathecal administration of glycine transporter-2 inhibitor ALX1393 in a rat acute pain model. Anesth Analg 2010;110:615–21. Janssens de Varebeke P, Cavalier R, David-Remacle M, Youdim MB. Formation of the neurotransmitter glycine from the anticonvulsant milacemide is mediated by brain monoamine oxidase B. J Neurochem 1988;50:1011–6. Johnson JW, Ascher P. Glycine potentiates the NMDA-response in cultured mouse brain neurons. Nature 1987;325:529–31. Khandwala H, Loomis CW. Milacemide, a glycine pro-drug, inhibits strychnine allodynia without affecting normal nociception in the rat. Pain 1998;77: 87–95. Lajtha A, Toth J. The brain barrier system–II. Uptake and transport of amino acids by the brain. J Neurochem 1961;8:216–25. LaMotte RH, Campbell JN. Comparison of responses of warm and nociceptive C-fiber afferents in monkey with human judgments of thermal pain. J Neurophysiol 1982;41: 509–28. Legendre P. The glycinergic inhibitory synapse. Cell Mol Life Sci 2001;58:760–93. Lim ES, Lee IO. Effect of intrathecal glycine and related amino acids on the allodynia and hyperalgesic action of strychnine or bicuculline in mice. Korean J Anesthesiol 2010;58:76–86. Lynch JW, Callister RJ. Glycine receptors: a new therapeutic target in pain pathways. Curr Opin Investig Drugs 2006;7:48–53. Melzack R. Prolonged relief of pain by brief, intense transcutaneous somatic stimulation. Pain 1975;1:357–73. Mitchell K, Spike RC, Todd AJ. An immunocytochemical study of glycine receptor and GABA in laminae I–III of rat spinal dorsal horn. J Neurosci 1993;13:2371–81. Morita K, Motoyama N, Kitayama T, Morioka N, Kifune K, Dohi T. Spinal antiallodynia action of glycine transporter inhibitors in neuropathic pain models in mice. J Pharmacol Exp Ther 2008;326:633–45. Oldendorf WH. Brain uptake of radiolabeled amino acids, amines, and hexoses after arterial injection. Am J Physiol 1971;221:1629–39. Rao TS, Cler JA, Emmett MR, Mick SJ, Lyengar S, Wood PL. Glycine, glycinamide and D-serine act as positive modulators of signal transduction at the N-methyl-D-Aspartate (NMDA) receptor in vivo: differential effects on mouse cerebellar cyclic guanosine monophosphate levels. Neuropharmacology 1990;29:1075–80. Roberts LA, Beyer C, Komisaruk BR. Strychnine antagonizes vaginal stimulation-produced analgesia at the spinal cord. Life Sci 1985;36:2017–23. Sandkühler J, Chen JG, Cheng G, Randić M. Low-frequency stimulation of afferent Adelta-fibers induces long-term depression at primary afferent synapses with substantia gelatinosa neurons in the rat. J Neurosci 1997;17:6483–91. Seltzer Z, Cohn S, Ginzburg R, Beilin B. Modulation of neuropathic pain behavior in rats by spinal disinhibition and NMDA receptor blockade of injury discharge. Pain 1991;45:69–75. Semba J, Patsalos PN. Milacemide effects on the temporal inter-relationship of amino acids and monoamine metabolites in rat cerebrospinal fluid. Eur J Pharmacol 1993;230:321–6. Semba J, Ratnaraj N, Patsalos PN. Simple and rapid micro-analytical procedures for the estimation of milacemide and its metabolite glycinamide in rat plasma and cerebrospinal fluid by high-performance liquid chromatography. J Chromatogr 1991;565: 357–62. Seta K, Sershen H, Lajtha A. Cerebral amino acid uptake in vivo in newborn mice. Brain Res 1972;47:415–25. Sherman SE, Loomis CW. Morphine insensitive allodynia is produced by intrathecal strychnine in the lightly anesthetized rat. Pain 1994;56:17–29. Sherman SE, Loomis CW. Strychnine-dependent allodynia in the urethane-anesthetized rat is segmentally distributed and prevented by intrathecal glycine and betaine. Can J Physiol Pharmacol 1995;73:1698–705. Simpson Jr RK, Huang W. Glycine receptor reduction within segmental gray matter in a rat model in neuropathic pain. Neurol Res 1998;20:161–8. Simpson Jr RK, Gondo M, Robertson CS, Goodman JC. Reduction in thermal hyperalgesia by intrathecal administration of glycine and related compounds. Neurochem Res 1997;22:75–9. Toth E, Lajtha A. Elevation of cerebral levels of non essential amino acids in vivo by administration of large doses. Neurochem Res 1981;6:1309–17. Villanueva L, Le Bars D. The activation of bulbo-spinal controls by peripheral nociceptive inputs: diffuse noxious inhibitory controls. Biol Res 1995;28:113–25. Wall PD, Woolf CJ. Muscle but not cutaneous C-afferent input produces prolonged increases in the excitability of the flexion reflex in the rat. J Physiol 1984;356: 443–58.
C. Beyer et al. / Life Sciences 92 (2013) 576–581 Willis WD. The pain system: the neural of nociceptive transmission in the mammalian nervous system. Series Ed. In: Gilderberg PL, editor. Basel; New York: Karger; 1984. Woolf CJ, Wall PD. Relative effectiveness of C primary afferent fibers of different origins in evoking a prolonged facilitation of the flexor reflex in the rat. J Neurosci 1986;6: 1433–42. Yaksh TL. Behavioral and autonomic correlates of tactile evoked allodynia produced by spinal glycine inhibition: effects of modulatory receptor systems and excitatory amino acid antagonists. Pain 1989;37:111–23.
581
Yamamoto T, Yaksh TL. Effects of intrathecal strychnine and bicuculline on nerve compression-induced thermal hyperalgesia and selective antagonism by MK-801. Pain 1993;54:79–84. Zeilhofer HU, Wildner H, Yévenes GE. Fast synaptic inhibition in spinal sensory processing and pain control. Physiol Rev 2012;92:193–235. Zhou HY, Zhang HM, Chen SR, Pan HL. Increased C-fiber nociceptive input potentiates inhibitory glycinergic transmission in the spinal dorsal horn. J Pharmacol Exp Ther 2008;324:1000–10.