Chikai Sakurada

Characterization of nociceptive responses and spinal releases of nitric oxide metabolites and glutamate evoked by different concentrations of formalin in rats

&NA; A comparison was made of spontaneous nociceptive behaviors elicited by subcutaneous injection of formalin (0.5–10.0%) into the plantar or dorsal surface of the right hindpaw in rats. In the present study, we also examined the effect of paw formalin injection on the release of nitric oxide (NO) metabolites (nitrite/nitrate) and glutamate from the spinal cord in anesthetized rats using a dialysis probe placed in the lumbar subarachnoid space. Two distinct quantifiable behaviors indicative of pain were identified by formalin injected into both regions of the paw. There were no significant alterations in the number of flinches during the early and late phases induced by different regions of formalin injection. However, the early phase licking/biting activity evoked by formalin injection into the plantar surface of the paw was significantly higher than that evoked by formalin injected into the dorsal region. The maximum effect in the early and late phases was produced by 5.0% formalin injection into the dorsal and plantar paw. At a higher concentration (10.0%) of formalin, nociceptive behavioral responses were decreased except for the late phase flinching when injected into the dorsal paw. Injections of formalin (5.0%) into both regions of the paw evoked a biphasic spinal release of nitrite/nitrate with a significant increase during the early phase (0–10 min) and the late phase (30–80 or 90 min). A higher concentration of formalin (10.0%) failed to produce a clear‐cut release of nitrite/nitrate. A significant increase of glutamate was observed in the 0–10 min samples obtained after injection of formalin (5.0%) into the plantar and dorsal surface of the paw, whereas 0.5 and 10.0% formalin induced no substantial release. These results suggest that 5.0% formalin should be used when studying antinociceptive activity of NO‐ and N‐methyl‐D‐aspartate‐related compounds in the formalin test in rats. Formalin injection into the plantar surface of the paw might prove to be useful for evoking the licking/biting response, particularly in the early phase.

Differential antinociceptive effects induced by intrathecally administered endomorphin-1 and endomorphin-2 in the mouse

Two highly selective mu-opioid receptor agonists, endomorphin-1 and endomorphin-2, have been identified and postulated to be endogenous ligands for mu-opioid receptors. Intrathecal (i.t.) administration of endomorphin-1 and endomorphin-2 at doses from 0.039 to 5 nmol dose-dependently produced antinociception with the paw-withdrawal test. The paw-withdrawal inhibition rapidly reached its peak at 1 min, rapidly declined and returned to the pre-injection levels in 20 min. The inhibition of the paw-withdrawal responses to endomorphin-1 and endomorphin-2 at a dose of 5 nmol observed at 1 and 5 min after injection was blocked by pretreatment with a non-selective opioid receptor antagonist naloxone (1 mg/kg, s.c.). The antinociceptive effect of endomorphin-2 was more sensitive to the mu (1)-opioid receptor antagonist, naloxonazine than that of endomorphin-1. The endomorphin-2-induced paw-withdrawal inhibition at both 1 and 5 min after injection was blocked by pretreatment with kappa-opioid receptor antagonist nor-binaltorphimine (10 mg/kg, s.c.) or the delta(2)-opioid receptor antagonist naltriben (0.6 mg/kg, s.c.) but not the delta(1)-opioid receptor antagonist 7-benzylidine naltrexone (BNTX) (0.6 mg/kg s.c.). In contrast, the paw-withdrawal inhibition induced by endomorphin-1 observed at both 1 and 5 min after injection was not blocked by naloxonazine (35 mg/kg, s.c.), nor-binaltorphimine (10 mg/kg, s.c.), naltriben (0.6 mg/kg, s.c.) or BNTX (0.6 mg/kg s.c.). The endomorphin-2-induced paw-withdrawal inhibition was blocked by the pretreatment with an antiserum against dynorphin A-(1-17) or [Met(5)]enkephalin, but not by antiserum against dynorphin B-(1-13). Pretreatment with these antisera did not affect the endomorphin-1-induced paw-withdrawal inhibition. Our results indicate that endomorphin-2 given i.t. produces its antinociceptive effects via the stimulation of mu (1)-opioid receptors (naloxonazine-sensitive site) in the spinal cord. The antinociception induced by endomophin-2 contains additional components, which are mediated by the release of dynorphin A-(1-17) and [Met(5)]enkephalin which subsequently act on kappa-opioid receptors and delta(2)-opioid receptors to produce antinociception.

INVOLVEMENT OF NITRIC OXIDE IN SPINALLY MEDIATED CAPSAICIN- AND GLUTAMATE-INDUCED BEHAVIOURAL RESPONSES IN THE MOUSE *

The intrathecal (i.t.) injection of capsaicin (0.1 nmol/mouse) through a lumbar puncture elicited scratching, biting and licking responses. Pretreatment with the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) (320 nmol), by i.t. injection, resulted in a significant inhibition of the behavioural response produced by i.t. capsaicin (0.1 nmol/mouse). Similar behavioural responses were induced by i.t. injections of NMDA (0.4 nmol), kainate (0.05 nmol) or AMPA (0.05 nmol), which were all inhibited by co-administration of L-NAME (20-80 nmol). L-Arginine (600 mg/kg, i.p.) but not D-arginine (600 mg/kg, i.p.) reversed the inhibitory effect of L-NAME on capsaicin-, NMDA-, kainate- and AMPA-induced behavioural response. Scratching, biting and licking responses induced by tachykinin receptor agonists, substance P, [Sar9,Met(O2)11]substance P, neurokinin A and neurokinin B were not affected by co-administration of L-NAME (40 and 80 nmol). These results suggest that spinal nitric oxide may play a significant role in mechanisms of the behavioural response to capsaicin, probably through the release of glutamate, but not tachykinins.

Differential antagonism of endomorphin-1 and endomorphin-2 spinal antinociception by naloxonazine and 3-methoxynaltrexone

To determine the role of spinal mu-opioid receptor subtypes in antinociception induced by intrathecal (i.t.) injection of endomorphin-1 and -2, we assessed the effects of beta-funaltrexamine (a selective mu-opioid receptor antagonist) naloxonazine (a selective antagonist at the mu(1)-opioid receptor) and a novel receptor antagonist (3-methoxynaltrexone) using the paw-withdrawal test. Antinociception of i.t. endomorphins and [D-Ala(2), MePhe(4), Gly(ol)(5)]enkephalin (DAMGO) was completely reversed by pretreatment with beta-funaltrexamine (40 mg/kg s.c.). Pretreatment with a variety of doses of i.t. or s.c. naloxonazine 24 h before testing antagonized the antinociception of endomorphin-1, -2 and DAMGO. Judging from the ID(50) values of naloxonazine, the antinociceptive effect of endomorphin-2 was more sensitive to naloxonazine than that of endomorphin-1 or DAMGO. The selective morphine-6beta-glucuronide antagonist, 3-methoxynaltrexone, which blocked endomorphin-2-induced antinociception at each dose (0.25 mg/kg s.c. or 2.5 ng i.t.) that was inactive against DAMGO, did not affect endomorphin-1-induced antinociception but shifted the dose-response curve of endomorphin-2 3-fold to the right. These findings may be interpreted as indicative of the existence of a novel mu-opioid receptor subtype in spinal sites, where antinociception of morphine-6beta-glucuronide and endomorphin-2 are antagonized by 3-methoxynaltrexone. The present results suggest that endomorphin-1 and endomorphin-2 may produce antinociception through different subtypes of mu-opioid receptor.

Differential effects of intraplantar capsazepine and ruthenium red on capsaicin-induced desensitization in mice

Intraplantar injection of capsaicin (1.6 microg/paw) into the mouse hindpaw produced an acute paw-licking/biting response. This study was designed (1) to investigate the antinociceptive effects of intraplantar administration of capsazepine, a competitive vanilloid receptor antagonist, and ruthenium red, a noncompetitive antagonist, in the nociceptive licking/biting response induced by intraplantar injection of capsaicin, and (2) to determine whether these compounds were able to prevent capsaicin-induced desensitization in mice. Both capsazepine and ruthenium red produced a dose-dependent reduction in the capsaicin-induced nociceptive response. In licking/biting response to intraplantar capsaicin, ruthenium red was more potent than capsazepine in producing antinociceptive activity as assayed by the capsaicin test. The first injection of capsaicin induced a profound desensitization to the second and third injections of capsaicin at the interval of 15 or 30 min. The capsaicin-induced desensitization was prevented dose-dependently by antinociceptive doses of capsazepine, whereas ruthenium red in doses exhibiting antinociceptive activity was without effect on capsaicin-induced desensitization. The present results suggest that both capsazepine and ruthenium red can produce a local peripheral antinociceptive action, which may be mediated by inhibiting the membrane ion channel activated by capsaicin. In addition, these data suggest that capsazepine may act in the mechanism clearly different from ruthenium red in the capsaicin-induced nociceptive desensitization.

Involvement of Spinal NMDA Receptors in Capsaicin-Induced Nociception

Intraplantar injection of capsaicin into the mouse hindpaw produced an acute nociceptive response. The involvement of N-methyl-D-aspartate (NMDA) receptors was examined by intrathecal administration of various excitatory amino acid (EAA) receptor antagonists. The selective and competitive NMDA receptor antagonists, D(-)-2-amino-5-phosphono-valeric acid (APV) and (+/-)-3-(2-carboxypiperazin-4-yl) propyl-1-phosphoric acid (CPP), were most potent in inhibiting the nociceptive response induced by capsaicin (ED50, 0.23 nmol and 0.12 nmol). The noncompetitive NMDA receptor antagonist dizocilpine (MK-801) and the non-NMDA antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) had similar effects on the capsaicin-induced nociception (ED50, 2.90 and 7.98 nmol), while ketamine and 7-chlorokynurenic acid were without effect. Ifenprodil, an antagonist at the receptor-coupled polyamine site, showed a significant reduction of the nociceptive response (ED50, 13.8 nmol). The inhibitory effects of APV, CPP, MK-801, and ifenprodil were reversed by co-administration of NMDA. Coadministration of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) or kainate resulted in a marked reduction of CNQX-induced antinociception. The present results suggest that the NMDA receptor plays a key role in spinal nociceptive processing as measured by the capsaicin test in mice. This nociceptive test may be useful for evaluating competitive NMDA antagonists.

Nociceptin-induced scratching, biting and licking in mice: involvement of spinal NK1 receptors

Intrathecal (i.t.) injection of nociceptin at small doses (fmol order) elicited a behavioural response consisting of scratching, biting and licking in conscious mice. Here we have examined the involvement of substance P‐containing neurons by using i.t. injection of tachykinin neurokinin (NK)1 receptor antagonists and substance P (SP) antiserum. Nociceptin‐induced behavioural response was evoked significantly 5–10 min after i.t. injection and reached a maximum at 10–15 min. Dose‐dependency of the induced response showed a bell‐shaped pattern from 0.375–30.0 fmol, and the maximum effect was observed at 3.0 fmol. The behavioural response elicited by nociceptin (3.0 fmol) was dose‐dependently inhibited by intraperitoneal (i.p.) administration of morphine. The NK1 receptor antagonists, CP‐96,345, CP‐99,994 and sendide, inhibited nociceptin‐induced behavioural response in a dose‐dependent manner. A significant antagonistic effect of [D‐Phe7, D‐His9]SP (6–11), a selective antagonist for SP receptors, was observed against nociceptin‐induced response. The NK2 receptor antagonist, MEN‐10376, had no effect on the response elicited by nociceptin. Pretreatment with SP antiserum resulted in a significant reduction of the response to nociceptin. No significant reduction of nociceptin‐induced response was detected in mice pretreated with NKA antiserum. The N‐methyl‐D‐aspartate (NMDA) receptor antagonists, dizocilpine (MK‐801) and D(−)‐2‐amino‐5‐phosphonovaleric acid (APV) (D‐APV), and L‐NG‐nitro arginine methyl ester (L‐NAME), a nitric oxide (NO) synthase inhibitor, failed to inhibit nociceptin‐induced behavioural response. The present results suggest that SP‐containing neurons in the mouse spinal cord may be involved in elicitation of scratching, biting and licking behaviour following i.t. injection of nociceptin.

Intrathecally administered big dynorphin, a prodynorphin-derived peptide, produces nociceptive behavior through an N-methyl-D-aspartate receptor mechanism.

Intrathecal (i.t.) administration of big dynorphin (1-10 fmol), a prodynorphin-derived peptide consisting of dynorphin A and dynorphin B, to mice produced a characteristic behavioral response, the biting and/or licking of the hindpaw and the tail along with slight hindlimb scratching directed toward the flank, which peaked at 5-15 min after an injection. Dynorphin A produced a similar response, though the doses required were higher (0.1-30 pmol) whereas dynorphin B was practically inactive even at 1000 pmol. The behavior induced by big dynorphin (3 fmol) was dose-dependently inhibited by intraperitoneal injection of morphine (0.125-2 mg/kg) and also dose-dependently, by i.t. co-administration of D(-)-2-amino-5-phosphonovaleric acid (D-APV) (1-4 nmol), a competitive N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801 (0.25-4 nmol), an NMDA ion-channel blocker, and ifenprodil (2-8 pmol), an inhibitor of the NMDA receptor ion-channel complex interacting with the NR2B subunit and the polyamine recognition site. On the other hand, naloxone, an opioid receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA glutamate receptor antagonist, 7-chlorokynurenic acid, a competitive antagonist of the glycine recognition site on the NMDA receptor ion-channel complex, [D-Phe(7),D-His(9)]-substance P(6-11), a specific antagonist for substance P (NK1) receptors, and MEN-10376, a tachykinin NK2 receptor antagonist, had no effect. These results suggest that big dynorphin-induced nociceptive behavior is mediated through the activation of the NMDA receptor ion-channel complex by acting on the NR2B subunit and/or the polyamine recognition site but not on the glycine recognition site, and does not involve opioid, non-NMDA glutamate receptor mechanisms or tachykinin receptors in the mouse spinal cord.

Degradation of endomorphin-2 at the supraspinal level in mice is initiated by dipeptidyl peptidase IV: an in vitro and in vivo study.

Endomorphin-2 (Tyr-Pro-Phe-PheNH(2)) was discovered as an endogenous ligand for the mu-opioid receptor. The physiological function of endomorphin-2 as a neurotransmitter or neuromodulator may cease through the rapid enzymatic process in the synapse of brain, as for other neuropeptides. The present study was conducted to examine the metabolism of endomorphin-2 by synaptic membranes prepared from mouse brain. Major metabolites were free tyrosine, free phenylalanine, Tyr-Pro and PheNH(2). Both the degradation of endomorphin-2 and the accumulation of major metabolites were inhibited by specific inhibitors of dipeptidyl peptidase IV, such as diprotin A and B. On the other hand, the accumulation of Phe-PheNH(2) and Pro-Phe-PheNH(2) was increased in the presence of bestatin, an aminopeptidase inhibitor, whereas that of free phenylalanine and PheNH(2) was decreased. Furthermore, purified dipeptidyl peptidase IV hydrolyzed endomorphin-2 at the cleavage site, Pro(2)-Phe(3) bond. Thus, degradation of endomorphin-2 by brain synaptic membranes seems to take place mainly through the cleavage of Pro(2)-Phe(3) bond by dipeptidyl peptidase IV, followed by release of free phenylalanine and PheNH(2) from the liberated fragment, Phe-PheNH(2) by aminopeptidase. We have also examined that the effect of diprotin A on the antinociception induced by intracerebroventricularly administered endomorphin-2 in the mouse paw withdrawal test. Diprotin A simultaneously injected with endomorphin-2 enhanced endomorphin-2-induced antinociception. These results indicate that dipeptidyl peptidase IV may be an important peptidase responsible for terminating endomorphin-2-induced antinociception at the supraspinal level in mice.

Evidence that N-terminal fragments of nociceptin modulate nociceptin-induced scratching, biting and licking in mice

The intrathecal (i.t.) injection of 3.0 fmol nociceptin (orphanin FQ) elicited scratching, biting and licking responses in mice. N-terminal fragments of nociceptin, nociceptin (1-7), nociceptin (1-9) and nociceptin (1-13), induced no characteristic behavioral response. When these N-terminal fragments of nociceptin were injected simultaneously with nociceptin, the behavioral response induced by nociceptin was reduced dose-dependently. Nociceptin (1-13) was much more potent than nociceptin (1-7) and nociceptin (1-9) and antagonized nociceptin-induced response at equimolar doses. No significant effects of the N-terminal fragments were observed against the scratching, biting and licking response elicited by i.t. administration of substance P or N-methyl-D-aspartate. These results suggest that N-terminal fragments formed endogenously in the spinal cord may have an antagonistic effect on nociceptin-induced behavioral responses.