N-methyl-D-aspartate Receptor Ion-channel Complex Research Articles

Intrathecally Administered D-Cycloserine Produces Nociceptive Behavior Through the Activation of N-Methyl-D-aspartate Receptor Ion-Channel Complex Acting on the Glycine Recognition Site

Intrathecal (i.t.) administration of D-cycloserine (100 and 300 fmol), a partial agonist of the glycine recognition site on the N-methyl-D-aspartate (NMDA) receptor ion-channel complex, produced a behavioral response mainly consisting of biting and/or licking of the hindpaw and the tail along with slight hindlimb scratching directed toward the flank in mice, which peaked at 5 – 10 min and almost disappeared at 15 min after the injection. The behavior induced by D-cycloserine (300 fmol) was dose-dependently inhibited by an intraperitoneal injection of morphine (0.5 – 2 mg/kg), suggesting that the behavioral response is related to nociception. The nociceptive behavior was also dose-dependently inhibited by i.t. co-administration of 7-chlorokynurenic acid (0.25 – 4 nmol), a competitive antagonist of the glycine recognition site on the NMDA receptor ion-channel complex; D-(−)-2-amino-5-phosphonovaleric acid (62.5 – 500 pmol), a competitive NMDA receptor antagonist; MK-801 (62.5 – 500 pmol), an NMDA ion-channel blocker; ifenprodil (0.5 – 8 nmol); arcaine (31 – 125 pmol); and agmatine (0.1 – 10 pmol), all being antagonists of the polyamine recognition site on the NMDA receptor ion-channel complex. However, [D-Phe7,D-His9]-substance P(6 – 11), a specific antagonist for substance P (NK1) receptors, and MEN-10,376, a tachykinin NK2-receptor antagonist, had no effect on D-cycloserine-induced nociceptive behavior. These results in the mouse spinal cord suggest that D-cycloserine-induced nociceptive behavior is mediated through the activation of the NMDA receptor ion-channel complex by acting on the glycine recognition site and that it does not involve the tachykinin receptor mechanism.

Pronociceptive role of dynorphins in uninjured animals: N-ethylmaleimide-induced nociceptive behavior mediated through inhibition of dynorphin degradation

Intrathecal (i.t.) administration into mice of N-ethylmaleimide (NEM), a cysteine protease inhibitor, 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. The behavior induced by NEM was inhibited by the intraperitoneal injection of morphine. We have recently reported that dynorphin A and, more potently big dynorphin, consisting of dynorphins A and B, produce the same type of nociceptive response whereas dynorphin B does not [Tan-No K, Esashi A, Nakagawasai O, Niijima F, Tadano T, Sakurada C, Sakurada T, Bakalkin G, Terenius L, Kisara K. Intrathecally administered big dynorphin, a prodynorphin-derived peptide, produces nociceptive behavior through an N-methyl- d-aspartate receptor mechanism. Brain Res 2002;952:7–14]. The NEM-induced nociceptive behavior was inhibited by pretreatment with dynorphin A- or dynorphin B-antiserum and each antiserum also reduced the nociceptive effects of i.t.-injected synthetic big dynorphin. The characteristic NEM-evoked response was not observed in prodynorphin knockout mice. Naloxone, an opioid receptor antagonist, had no effects on the NEM-induced behavior. Ifenprodil, arcaine and agmatine, antagonists at the polyamine recognition site on the N-methyl- d-aspartate (NMDA) receptor ion-channel complex, and MK-801, an NMDA ion-channel blocker inhibited the NEM-induced effects. Ro25-6981, an antagonist of the NMDA receptor subtype containing NR2B subunit was not active. NEM completely inhibited degradation of dynorphin A by soluble and particulate fractions of mouse spinal cord. Collectively, the results demonstrate that endogenous prodynorphin-derived peptides are pronociceptive in uninjured animals, and required for the NEM-induced behavior. The NEM effects may be mediated through inhibition of the degradation of endogenous dynorphins, presumably big dynorphin that in turn activates the NMDA receptor ion-channel complex by acting on the polyamine recognition site.

Nociceptive behavior induced by poly- l-lysine and other basic compounds involves the spinal NMDA receptors

We have previously shown that spermine, a basic polyamine, and big dynorphin, a basic polypeptide, induce nociceptive behavior if injected intrathecally (i.t.) in mice (see [Pain 86 (2000) 55–61] and [Brain Res. 952 (2002) 7–14]). This suggests that other basic molecules might have the same effects. Here, i.t. administration of poly- l-lysine (12 and 36 pg) to mice was found to produce the same characteristic behavioral response, biting and/or licking of the hindpaw and the tail along with slight hindlimb scratching directed toward the flank, which peaked at 0–10 min after injection. The behavior induced by poly- l-lysine (12 pg) was dose-dependently inhibited by intraperitoneal injection of morphine (0.25–4 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, (5 R,10 S)-(+)-5-methyl-10,11-dihydro-5 H-dibenzo[ a, d]cycloheptene-5,10-imine hydrogen maleate (MK-801) (0.0156–4 nmol), an NMDA ion-channel blocker, and ifenprodil (2–8 nmol), an antagonist of the polyamine recognition site and the NR2B-containing NMDA receptor subtype. On the other hand, 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, or MEN-10,376, a tachykinin NK2 receptor antagonist, had no effect. These results confirm the observations obtained with other basic molecules and suggest that the behavior induced by poly- l-lysine is mediated through the activation of the NMDA receptor ion-channel complex acting either on the polyamine recognition site or on the NR2B subunit.

Metabotropic glutamate receptor in C6BU-1 glioma cell has NMDA receptor-ion channel complex-like properties and interacts with serotonin2 receptor-stimulated signal transduction.

We found in cultured glioma (C6BU-1) cells that excitatory amino acids (EAAs) such as glutamate, N-methyl-D-aspartate (NMDA), aspartate, and metabotropic glutamate receptor agonist trans-(+/-)-1-amino-1,3-cyclopentanedicarboxylate caused an increase in the inositol 1,4,5-trisphosphate formation and the intracellular Ca2+ concentration ([Ca2+]i) in the absence of extracellular Mg2+ and Ca2+. Pertussis toxin treatment abolished this glutamate-induced [Ca2+]i increase. Various antagonists against NMDA receptor-ion channel complex, such as Mg2+, D-2-amino-5-phosphonovalerate (D-APV), HA-966, and MK-801, also inhibited the increase in [Ca2+]i induced by glutamate. These results indicate that these metabotropic EAA receptors coupled to pertussis toxin-susceptible GTP-binding protein and phospholipase C system in C6BU-1 glioma cells have the pharmacological properties of NMDA receptor-ion channel complexes. We also found that in the presence of Mg2+ these metabotropic receptors resemble the NMDA receptor-ion channel complex interacted with 5-hydroxytryptamine2 (5-HT2) receptor signaling. EAAs inhibited 5-HT2 receptor-mediated intracellular Ca2+ mobilization and inositol 1,4,5-trisphosphate formation in a concentration-dependent manner. The inhibitory effect of glutamate was reversed by various NMDA receptor antagonists (D-APV, MK-801, phencyclidine, and HA-966), but L-APV failed to block the inhibitory effect of glutamate. The same result was observed in the absence of extracellular Ca2+. In addition, this inhibitory effect on 5-HT2 receptor-mediated signal transduction was abolished by treatment of C6BU-1 cells with pertussis toxin, whereas 5-HT2 receptor-mediated [Ca2+]i increase was not abolished by pertussis toxin treatment. We can, therefore, conclude that the inhibitory effect of glutamate is not a result of the influx of Ca2+ through the ion channel and that it operates via metabotropic glutamate receptors, having NMDA receptor-ion channel complex-like properties and being coupled with pertussis toxin-sensitive GTP-binding protein and phospholipase C.

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-10,376, 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.

Intrathecal spermine and spermidine at high-doses induce antinociceptive effects in the mouse capsaicin test

The antinociceptive effects of intrathecally (i.t.) administered high doses of spermine and spermidine, endogenous polyamines, were examined in the mouse capsaicin test. Spermine (2.5-10 nmol) and spermidine (10 and 15 nmol), when administered i.t. 5 min before the injection of capsaicin solution (1600 ng) into the plantar surface of a hindpaw, produced a significant reduction of the nociceptive behavioral response. Ifenprodil (2 and 4 nmol), an antagonist of the polyamine recognition site on N-methyl-D-aspartate (NMDA) receptor ion-channel complex, co-administered with spermine or spermidine significantly inhibited antinociception induced by both polyamines. These results indicate that spermine- and spermidine-induced antinociception may be mediated via the polyamine recognition site on NMDA receptor ion-channel complex in the mouse spinal cord.

The stability of a ketamine-morphine solution.

Recent advances in acute pain mechanisms and management have implicated the N-methyl D-aspartate receptor-ion channel complex in the development of postoperative hyperalgesia and acute opioid tolerance. N-methyl D-aspartate receptor antagonists such as ketamine have been used increasingly in clinical studies in an effort to minimize acute postoperative pain and reduce opioid requirements. A mixture of ketamine and an opioid administered in the same solution and syringe would be a practical and useful technique for postoperative epidural analgesia, continuous IV infusion, or patient-controlled IV analgesia. We investigated the stability of a morphine sulfate and racemic ketamine solution in saline at pH 5.5-7.5 over a period of 4 days. Our study demonstrates that the ketamine-morphine mixture at a clinically relevant concentration seems to be stable at room temperature, at a wide range of pH values, for at least 4 days. Small-dose ketamine is used with increasing frequency in the acute postoperative setting as an adjunct to traditional opioid analgesics. We show that a racemic ketamine and morphine solution at a clinically relevant concentration seems to be stable at room temperature at a wide range of pH values for at least 4 days.

MS-377, a selective sigma receptor ligand, indirectly blocks the action of PCP in the N-methyl-D-aspartate receptor ion-channel complex in primary cultured rat neuronal cells

MS-377 ((R)-(+)-1-(4-chlorophenyl)-3-[4-(2-methoxyethyl)piperazin-1-yl]methyl-2-pyrrolidinone L-tartrate) is a antipsychotic agent that binds to sigma-1 receptor. MS-377 showed anti-dopaminergic and anti-serotonergic activities and antagonistic action against phencyclidine (PCP)-induced behaviors in an animal model. These anti-psychotic activities of MS-377 are attributable to association with sigma-1 receptor. However, the mechanism by which the sigma-1 receptor ligands exact those numerous effects remains to be elucidated. In the present study, we evaluated the effect of MS-377 on N-methyl-D-aspartate (NMDA) receptor ion-channel complex in primary cultured rat neuronal cells. First, we examined the effect of MS-377 on NMDA-induced Ca 2+ influx with fura-2/AM loaded cells. MS-377 showed no effects on the basal Ca 2+ concentration and NMDA-induced Ca 2+ influx by itself. PCP and SKF-10047 reduced the NMDA-induced increase in intracellular Ca 2+ concentration. Pre-incubation of 1 μM MS-377 was found to significantly block the reduction by PCP or SKF-10047 of the NMDA-induced Ca 2+ influx. Second, the effect of MS-377 on [ 3H]MK-801 intact cell binding was examined. PCP, haloperidol and (+)-pentazocine inhibited [ 3H]MK-801 binding, although MS-377 showed no effect by itself. Pre-treatment of MS-377 markedly reversed the inhibition of [ 3H]MK-801 binding by PCP in a dose-dependent manner. These effects of MS-377 may depend on its affinity for the sigma-1 receptor, because MS-377 is a selective sigma-1 receptor ligand without any affinity for NMDA receptor ion-channel complex. These observations suggest that the MS-377 indirectly modulated the NMDA receptor ion-channel complex, and the anti-psychotic activities of MS-377, in part, are attributable to such on action via sigma-1 receptor.

Intrathecal high-dose morphine induces spinally-mediated behavioral responses through NMDA receptors

Previous research has demonstrated that intrathecal i.t. morphine in a dose of 60.0 nmol into the spinal subarachnoid space of mice can evoke nociceptive behavioral responses consisting of a severe hindlimb scratching directed toward the flank followed by biting/licking of the hindpaw. The present study was undertaken to examine the involvement of spinal N-methyl- d-aspartate (NMDA) and opioid receptors on the behavioral responses evoked by high-dose i.t. morphine. Pretreatment with naloxone, an opioid receptor antagonist (1.0 and 4.0 mg/kg, s.c.), failed to reverse the morphine-evoked behavioral response, suggesting that the morphine effect is not mediated through the opioid receptors in the spinal cord. The morphine-induced behavior was dose-dependently inhibited by i.t. co-administration of the competitive NMDA receptor antagonists, d(−)-2-amino-5-phosphonovaleric acid ( d-APV) (6.25–50.0 pmol) and 3-((+)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) (3.125–25.0 pmol). The characteristic behavior was also reduced by co-administration of (5 R,10 S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[ a, d]cycloheptene-5,10-imine maleate (MK-801) (74.1–250 pmol), an NMDA ion-channel blocker. Ifenprodil, a competitive antagonist of the polyamine recognition site of NMDA receptor ion channel complex, produced a dose-related inhibitory effect on the behavioral response to i.t. morphine with less potency than the competitive and non-competitive anatgonists examined. High doses of (+)-HA-966, a glycine/NMDA antagonist, induced a dose-dependent inhibition of morphine-induced response. The effective dose of i.t. 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA receptor antagonist, needed to reduce the morphine-induced response, was approximately 10-fold greater than that of d-APV. These results suggest that spinal NMDA receptors, but not non-NMDA receptors, may be largely involved in elicitation of the behavioral episode following i.t. injection of morphine in mice.

Phencyclidine-induced discriminative stimulus is mediated via phencyclidine binding sites on the N-methyl- d-aspartate receptor-ion channel complex, not via sigma 1 receptors

The effects of several N-methyl- d-aspartate (NMDA) receptor- and sigma receptor-related compounds on the discriminative stimulus effects of phencyclidine (PCP) were examined in rats trained to discriminate PCP (1.5 mg/kg, i.p.) from saline under a two-lever fixed ratio 20 schedule of food reinforcement. PCP produced a dose-dependent increase in PCP-appropriate responding. A non-competitive NMDA receptor antagonist, dizocilpine (0.2 mg/kg, i.p.) and a putative sigma 1 receptor agonist, (+)-SKF-10047 (10 mg/kg, i.p.) fully substituted for PCP in every rat tested. Neither a competitive NMDA receptor antagonist, CGS-19755 (0.1–3 mg/kg, i.p.), sigma 1 receptor agonist, (+)-pentazocine (10–30 mg/kg, i.p.) nor dextromethorphan (10–20 mg/kg, i.p.) produced PCP-like discriminative stimulus effects. The discriminative stimulus effects of PCP (1.5 mg/kg, i.p.), dizocilpine (0.2 mg/kg, i.p.) and (+)-SKF-10047 (10 mg/kg, i.p.) were significantly attenuated by CGS-19755 (1 mg/kg, i.p.), but not by sigma 1 receptor antagonist BMY-14802 (10 mg/kg, i.p.) and NE-100 (5 mg/kg, i.p.). These results suggest that the discriminative stimulus effects of PCP are predominantly mediated via PCP binding sites on the NMDA receptor-ion channel complex, not via sigma 1 receptors. In addition, the PCP-like discriminative stimulus effects of (+)-SKF-10047 were demonstrated to be mediated via PCP binding sites.

Intrathecally administered spermine produces the scratching, biting and licking behaviour in mice

Intrathecal (i.t.) administration of spermine (0.1–10000 fmol), an endogenous polyamine, produced the behavioural response mainly consisting of biting and/or licking of the hindpaw along with a slight hindlimb scratching directed toward the flank in mice, which peaked at 5–15 min and almost disappeared at 30 min after an injection. The behaviour induced by spermine (10 pmol) was dose-dependently inhibited by intraperitoneal injection of morphine (0.125–0.5 mg/kg). The characteristic behaviour was also inhibited dose-dependently by i.t. co-administration of ifenprodil (62.5–4000 pmol), a competitive antagonist of the polyamine recognition site on N-methyl- d-aspartate (NMDA) receptor ion-channel complex, and d(−)-2-amino-5-phosphonovaleric acid ( d-APV) (0.5–2 nmol) and 3-((±)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) (7.8–500 pmol), the competitive NMDA receptor antagonists, and (5 R,10 S)-(+)-5-methyl-10,11-dihydro-5 H-dibenzo[ a,b]cycloheptene-5,10-imine hydrogen maleate (MK-801) (0.5–4 nmol), an NMDA ion-channel blocker, but not by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA receptor antagonist. Both (2 S,3 S)-[cis-2-(diphenylmethyl)- N-[(2-methoxyphenyl)-methyl]-1-azabicyclo[2.2.2]octane-3-amine] (CP-96,345), a non-peptidic neurokinin-1 (NK-1) receptor antagonist, and CP-96,344, its inactive 2 R,3 R enantiomer, inhibited spermine-induced behavioural response in a dose-dependent manner. However, [Tyr 6, d-Phe 7, d-His 9]-substance P(6–11) (sendide) and [ d-Phe 7, d-His 9]-substance P(6–11), the selective antagonists for NK-1 receptors, were without affecting spermine-induced behaviour. These results indicate that spermine-induced behaviour is mediated through the polyamine recognition site on NMDA receptor ion-channel complex without the involvement of substance P system in the mouse spinal cord.

Spermine dependent activation of the N-methyl- d-aspartate receptor and the effect of nitric oxide synthase inhibition during hypoxia in the cerebral cortex of newborn piglets

This study tests the hypothesis that brain tissue hypoxia results in modification of spermine-dependent activation of the cerebral N-methyl- d-aspartate (NMDA) receptor ion-channel in newborn piglet brains and that pretreatment with N ω-nitro- l-arginine (NNLA), an inhibitor of nitric oxide synthase, will reduce the hypoxia-induced modification of the spermine-dependent activation of the receptor. Piglets were assigned to one of four groups; normoxia or hypoxia with or without NNLA. The infusion of NNLA or vehicle lasted for 60 min while the animals were ventilated under either hypoxic or normoxic conditions. Cerebral tissue hypoxia was confirmed by measuring ATP and phosphocreatine (PCr) levels. P 2 membranes were isolated and 3 H -MK-801 binding was measured in the presence of spermine. Steady state 3 H -MK-801 binding in the presence of spermine, showed an increase in receptor affinity in both normoxic (47% of control) and hypoxic (42% of control) animals without change in receptor density. During hypoxia, the spermine-dependent increase in the maximal response of the 3 H -MK-801 binding correlated inversely with the ATP concentrations. NNLA pretreatment prior to hypoxia, resulted in a decrease in the slope of the regression line describing the relationship between cellular energy state (ATP) and percent change in maximal response to spermine compared with vehicle treated animals indicating attenuation of the response to hypoxia. We conclude that the spermine-dependent modification of the affinity of the NMDA receptor ion-channel as assessed by 3 H -MK-801 binding is similar in hypoxic and normoxic cortical tissue. NNLA administration reduces the hypoxia-induced spermine-dependent activation of the receptor indicating that nitric oxide mediates modification of the spermine site activation of the NMDA receptor ion-channel complex.

Inhibition by soman of NMDA-stimulated [ 3H]norepinephrine release from rat cortical slices, studies of non-cholinergic effect

Effects of soman, an irreversible cholinesterase (ChE) inhibitor, on [ 3H]norepinephrine (NE) release evoked by N-methyl- d-aspartate (NMDA) were studied in rat brain cortical slices. Soman inhibited NMDA-stimulated [ 3H]NE release in a concentration-dependent manner. This effect was neither reversed by atropine, an antagonist of the muscarinic receptor, nor by d-tubocurarine, an antagonist of the nicotinic receptor. Incubation of the slices with NMDA antagonists, AP5, MK-801, ketamine or magnesium, resulted in inhibitory effects on NMDA-stimulated [ 3H]NE release. Soman significantly shifted the inhibition curves downward and significant interactions between these chemicals and soman were observed. Glycine potentiated the release of [ 3H]NE stimulated by NMDA, and soman did not alter this effect of glycine. Soman also inhibited the release of [ 3H]NE evoked by K + in a concentration-dependent manner. NMDA-stimulated [ 3H]NE release was inhibited by tetrodotoxin (TTX), an antagonist of voltage-dependent sodium channels, and a significant interaction between soman and TTX was observed. The [ 3H]NE release induced by NMDA was dependent on extracellular calcium concentrations and was inhibited by nifedipine, a selective blocker of the L-type voltage-dependent calcium channels (VDCC), or cadmium, a non-specific blocker of VDCC. However, no significant interaction between the effects of soman and calcium, nifedipine, or cadmium was observed. Taken together, the results suggested that: (1) soman has a direct action at non-cholinergic sites; (2) soman may interfere with some of the regulatory sites of the NMDA receptor–ion channel complex; and (3) the voltage-dependent sodium channel, but not VDCC, may be a site of action for soman.

Synapse formation and LTP can be induced by released ATP phosphorylating extracellular domains of synaptic proteins

We have previously shown that spermine, a basic polyamine, and big dynorphin, a basic polypeptide, induce nociceptive behavior if injected intrathecally (i.t.) in mice (see [Pain 86 (2000) 55–61] and [Brain Res. 952 (2002) 7–14]). This suggests that other basic molecules might have the same effects. Here, i.t. administration of poly-l-lysine (12 and 36 pg) to mice was found to produce the same characteristic behavioral response, biting and/or licking of the hindpaw and the tail along with slight hindlimb scratching directed toward the flank, which peaked at 0–10 min after injection. The behavior induced by poly-l-lysine (12 pg) was dose-dependently inhibited by intraperitoneal injection of morphine (0.25–4 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, (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cycloheptene-5,10-imine hydrogen maleate (MK-801) (0.0156–4 nmol), an NMDA ion-channel blocker, and ifenprodil (2–8 nmol), an antagonist of the polyamine recognition site and the NR2B-containing NMDA receptor subtype. On the other hand, 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-Phe7, d-His9]-substance P (6-11), a specific antagonist for substance P (NK1) receptors, or MEN-10,376, a tachykinin NK2 receptor antagonist, had no effect. These results confirm the observations obtained with other basic molecules and suggest that the behavior induced by poly-l-lysine is mediated through the activation of the NMDA receptor ion-channel complex acting either on the polyamine recognition site or on the NR2B subunit.

Spermine Dependent Activation of the N-Methyl-D-Aspartate (NMDA) Receptor in the Cerebral Cortex of Newborn Piglets. † 1771

Previous studies have shown that maximal spermine-dependent activation of the NMDA receptor-ion channel complex is increased during hypoxia. The aim of the present study is to investigate the mechanism by which spermine activates the NMDA receptor. 3H-MK-801 binding was used to assess receptor affinity (Kd) and density (Bmax). Twelve piglets (<7 days old) were exposed for 60 min to either FiO2 0.21 (normoxia, n=7) or an FiO2 0.05-0.07 (PaO2≤ 20 mmHg, hypoxia, n=7). Brain tissue hypoxia was documented biochemically by a decrease in ATP and phosphocreatine. P2 membrane fractions were prepared from cerebral cortices and 3H-MK-801 binding was performed at 32°C in 200 μl of medium containing either buffer or 10 μM spermine plus 100 μM glutamate, 100 μM glycine, 10 mM HEPES/1 mM EDTA buffer (pH 7.0), increasing concentrations of 3H-MK-801(2.5 to 50 nM), and 75 μg protein. Saturation curves and Scatchard plots were constructed to determine the Bmax and Kd. Bmax was not significantly different among the 4 groups (1.5±0.3 pmol/mg protein in normoxia to 1.5±0.3 pmol/mg protein in normoxia+spermine, and from 1.3±0.5 pmol/mg protein in hypoxia to 1.3±0.5 pmol/mg protein in hypoxia+spermine). Kd (mean±SD) decreased from 5.4±0.7 nM in normoxia to 3.6±1.9 nM in normoxia+spermine, and from 6.4±0.7 nM in hypoxia to 3.1±1.2 nM in hypoxia+spermine (p<0.001). The results demonstrate that spermine increases the affinity of the NMDA receptor ion channel for MK-801 in both normoxia and hypoxia without changing the number of binding sites. We speculate that the spermine dependent activation of the NMDA receptor ion-channel occurs by alteration of the ion-channel's affinity through the glutamate recognition site and/or acts as a polycationic molecule to influence the micro environment of the channel. (Funded by MOD 6-FY94-0135, NIH #HD-20337)

Effect of Dephosphorylation on Spermine-Dependent Activation of N-Methyl-D-Aspartate (NMDA) Receptor in Normoxic Newborn Piglets. ♦ 343

Previous studies have shown that spermine increases the maximal activation of the NMDA receptor ionchannel complex in neuronal membranes of hypoxic newborn piglets. The present study tests the hypothesis that removal of phosphate will change spermine-dependent activation of the NMDA receptor in the newborn piglet brain. 3H-MK-801 binding was used to assess receptor affinity (Kd) and density (Bmax). P2 membranes were prepared from the cerebral cortices of six normoxic piglets and then incubated at 37°C with either 10 units alkaline phosphatase or buffer for 90 min at pH=9. Membranes were rewashed. 3H-MK-801 binding was performed at 32°C in 200μl of medium containing either buffer or 10μM spermine plus 100μM glutamate, 100μM glycine, 10mM HEPES/1mM EDTA buffer (pH 7.0), increasing concentrations of 3H-MK-801 (2.5 to 50nM), and 75μg protein. Saturation curves and Scatchard plots were constructed to determine the Bmax and Kd. Without spermine, the alkaline phosphatase treatment increased the affinity of the channel as indicated by a 33% decrease in Kd(15.64±5.89 without enzyme to 10.89±6.68 nM with enzyme). Similarly, with spermine, the alkaline phosphatase treatment also increased the affinity of the channel as indicated by a 33% decrease in Kd(18.92±10.15 without enzyme to 12.32±5.74 nM with enzyme). The results demonstrate that dephosphorylation changes the affinity of the receptor for MK-801. However, spermine-dependent activation of the NMDA receptor is not affected by dephosphorylation, indicating that the interaction at the polyamine site is phosphorylation independent. Since hypoxia decreases cellular energy metabolism and results in dephosphorylation, we speculate that dephosphorylation due to hypoxia is a potential mechanism for the NMDA receptor ionchannel modification observed in the brain of the newborn piglet.

Different binding affinities of NMDA receptor channel blockers in various brain regions—Indication of NMDA receptor heterogeneity

The N-methyl- d-aspartate (NMDA) receptor-channel complex exists in multiple forms which probably have different physiological and pharmacological properties. To further evaluate this concept of different NMDA receptor subtypes, receptor binding and autoradiographic techniques were used to study the phencyclidine (PCP) binding site of the NMDA receptor ion-channel complex. [ 3H]MK-801 was employed to characterize binding properties of (+)-MK-801, (-)-MK-801, phencyclidine (PCP), (±)-ketamine, amantadine (1-amino-adamantane) and memantine (3,5-dimethyl-1-amino-adamantane) in different brain regions. Saturation experiments on homogenized membranes revealed the existence of single classes of binding sites in cortex and cerebellum but with significant different affinities between these regions ( K D/Cortex = 4.59 nM, B max/Cortex = 0.836 pmol/mg protein; K D/Cereb = 25.99 nM, B max/Cereb = 0.573 pmol/mg protein) suggesting that the lower affinity in cerebellum indicates another population of NMDA receptor channels. In contrast, in striatum there was clear evidence for two binding sites ( K D/high = 1.43 nM, B max/high= 0.272 pmol/mg protein; K d/low = 12.15 nM, B max/low= 1.76 pmol/mg protein). Displacement studies (autoradiography and binding) revealed a lower affinity for unlabeled (+)-MK-801 in striatum which was clearly not the case for memantine. In cerebellar membranes there was a significant decrease in the affinity for both MK-801 enantiomers and PCP but not for the 1-amino-adamantanes. In contrast, all compounds showed lowered affinity in the dentate gyrus. These findings support NMDA receptor heterogeneity which may be of particular relevance for the development of subtype-selective drugs.

Sigma ligands indirectly modulate the NMDA receptor-ion channel complex on intact neuronal cells via sigma 1 site

To investigate the modulatory effects of sigma ligands on the N-methyl-D-aspartate (NMDA) receptor-ion channel complex in vivo, we examined the intact cell binding of 3H-N-[1-(2-thienyl)cyclohexyl]piperidine (3H-TCP) to cultured neuronal cells prepared from fetal rat telencephalon. The 3H-TCP binding was saturable, reversible, and inhibited by a selective NMDA receptor antagonist, D-amino-5-phosphonovaleric acid. MII-limolar Mg2+ inhibited 3H-TCP binding both in the absence and presence of L-glutamate. 5-Methyl-10,11-dihydro-5H-dibenzo [a,d]cyclohepten-5,10-imine maleate (MK801) inhibited 3H-TCP intact cell binding in a competitive manner, while haloperidol inhibited it in a noncompetitive manner. The effect of the test drugs to inhibit 3H-TCP intact cell binding was in the order of dextromethorphan, haloperidol > (+/-)MK 801 > (+)pentazocine > (-)pentazocine > DTG > PCP > (+)-N-allylnormetazocine [(+)SKF 10047] > (+)3-(3-hydroxyphenyl)-N- (1-propyl)piperidine [(+)3-PPP] > (-)SKF 10047 > (-)3-PPP. The IC50 values of the six sigma ligands for 3H-TCP binding were closely correlated with the Ki values of the corresponding drugs for DTG site 1 in the guinea pig brain reported by Rothman et al. (1991). These findings suggest that the sigma ligand indirectly modulates the NMDA receptor ion channel complex, presumably through sigma 1 sites in vivo as well as in vitro.

Effects of chronic treatment with (+)- and (−)-nicotine on nicotinic acetylcholine receptors and N-methyl- d-aspartate receptors in rat brain

In this study, the effects of chronic treatment with (+)-nicotine on brain nicotinic acetylcholine receptors (nAChRs) and N-methyl- d-aspartate (NMDA) receptors, as well as on animal body weight were compared with those of chronic treatment with (−)-nicotine. Male Sprague-Dawley rats were s.c. injected with saline, (+)-nicotine (2.0 mg free base/kg b.w.) or (−)-nicotine (0.45 mg free base/kg b.w.) for 18 days. Brain nAChRs were investigated by (-)-[ 3H]nicotine binding. A significant increase in the high-affinity (−)-[ 3H]nicotine (5 nM)-binding sites was observed the cortex, hippocampus, midbrain and striatum but not in the cerebellum of the rats treated with either (+)- or (−)-nicotine. The displacement curves of (−)-[ 3H]nicotine/(−)-nicotine in the cortices of rats treated with either (+)- or (−)-nicotine showed only one population of high-affinity binding sites, whereas both high- and low-affinity binding sites were observed in the cortices of control animals. Brain NMDA receptors were studied by [ 3H]MK-801, which binds to the NMDA receptor-ion channel complex. A significant decrease in the B max, but not in the K D for [ 3H]MK-801 binding in the cortices of rats treated with either (+)- or (−)-nicotine was only detected under certain experimental conditions where the NMDA receptors seem not to be maximally activated. The body weight of the animals treated with (−)-nicotine was significantly lower than that of the control animals, whereas there was no difference in body weight between (+)-nicotine- and saline-treated animals. These results show that (+)-nicotine treatment has an effect on nAChRs in rat brains which is similar to that of (−)-nicotine treatment, but an effect on body weight which differs from (−)-nicotine, and also suggest that nicotinic agonists may interact with brain NMDA receptors in vivo.

Chronic prenatal and postnatal Pb 2+ exposure increases [ 3H]MK801 binding sites in adult rat forebrain

We have measured the binding of [ 3H]MK801 to the N-methyl-D-aspartate (NMDA) receptor-ion channel in membrane preparations from adult rat forebrain exposed to lead (Pb 2+) during gestation, lactation, and postweaning. Our results indicate a 30.9% increase in the number of [ 3H]MK801 binding sites in Pb 2+ exposed animals relative to controls. No significant changes in the affinity constant were observed. The level of blood Pb 2+ for which such changes were measured was 13.9±2.8 μg/dl. These results indicate that alterations in the NMDA receptor-ion channel complex are present at blood Pb 2+ levels which are environmentally relevant and suggest that chronic Pb 2+ exposure during development can influence the NMDA receptor complex in the adult rat brain.