大鼠杏仁核内注射八肽胆囊收缩素（CCK—8）拮抗吗啡镇痛的研究

大鼠双侧杏仁核内注射ＣＣＫ－８１ｎｇ（１μｌ）,能明显降低皮下注射４ｍｇ／ｋｇ吗啡产生的镇痛作用,并在０．１－１ｎｇ范围内呈量效关系。分别向双侧仁核注射ＣＣＫ－Ａ受体拮抗剂Ｄｅｖａｚｅｐｉｄｅ５０ｎｇ能部分翻转,２００ｎｇ则完全翻转ＣＣＫ－８的抗吗啡镇痛作用,１０ｎｇ无效;而ＣＣＫ－Ｂ受体拮抗剂Ｌ－３６５,２６０在５－８ｎｇ时即可完全番转ＣＣＫ－８的抗吗啡镇痛作用。杏仁核注射２００ｎｇ的Ｄｅｖａｚ     

鞘内注射MK-801对炎性痛大鼠海马NOS表达及NO含量的影响

目的：观察鞘内给予N-甲基-D-天门冬氨酸（NMDA）受体拮抗剂MK-801对足底注射甲醛诱导的自发痛反应和海马一氧化氮合酶（NOS）表达及一氧化氮（N0）含量的影响,探讨炎性痛诱导海马NO产生增多的机制。方法：通过观察舔足反射时间反映大鼠自发痛程度;采用NADPH—d组织化学法测定大鼠海马NOS表达;硝酸还原酶法测定海马组织NO含量。结果：足底注射甲醛后动物即出现舔、咬、摇动注射侧脚掌等自发痛相关表现,预先鞘内注射MK-801可使大鼠第二时相自发病程度显著降低,但对第一时相痛反应程度无明显影响。注射甲醛后12h时,海马CA1、CA2～3区及DG区NOS阳性细胞数目、阳性细胞染色深度均显著增加,海马组织NO含量显著增加;预先鞘内注射MK-801,可使甲醛炎性痛大鼠海马各区NOS阳性细胞数目明显减少,阳性细胞染色深度明显变浅,海马NO含量明显降低。结论：鞘内注射MK-801可逆转甲醛炎性痛诱导的海马NOS表达及NO产生的增加,表明甲醛炎性痛诱导的海马NO产生增加主要是由于伤害性信息传入所引起。     

Effects of Electroconvulsive Stimuli and MK-801 on Neuropeptide Y,Neurokinin A,and Calcitonin Gene-Related Peptide in Rat Brain

Rats were pretreated with 0.9% NaCl, or 0.1 or 1.0 mg/kg MK-801, an anticonvulsant and a psychotomimetic drug, and 60 minutes later given ECS or sham ECS. After six sessions the animals were sacrificed and neuropeptide Y (NPY-), neurokinin A (NKA-), and calcitonin gene-related peptide (CGRP-) like immunoreactivity (-LI) measured with radioimmunoassays. ECS increased NPY-LI in frontal cortex, striatum, occipital cortex and hippocampus, and NKA-LI in occipital cortex and hippocampus. MK-801 increased CGRP in a dose-response manner in frontal cortex, and NKA-LI in occipital cortex. Although the higher MK-801 dose reduced seizure duration by 50%, the ECS induced NPY-LI increase in striatum, occipital cortex and hippocampus, and NKA-LI in occipital cortex was not diminished. In contrast, there was a parallel decrease in seizures and NPY-LI and NKA-LI changes in frontal cortex and hippocampus, respectively. Investigation of neuropeptides in brain may contribute to understanding of the mechanisms of action of antide-pressive and antipsychotic treatments and of psychotomimetic drugs.     

Stereotaxic Administration of 1-Methyl-4-Phenylpyridinium Ion (MPP+) Decreases Striatal Fructose 2,6-Bisphosphate in Rats

Abstract: The Stereotaxic administration of 1-methyl-4-phenylpyridinium ion (MPP⁺) into the neostriatum of male rats caused a lesion that resulted in a large dose-dependent loss of striatal fructose 2,6-bisphosphate; initial values were restored 5 days after the treatment. This effect was not protected by systemic administration of MK-801 or by nitroarginine. The content of hexose 6-phosphates and ATP was also reduced by MPP⁺ treatment, whereas lactate was increased. Biochemical and histological results suggested that MPP⁺ caused a nonselective cell death, followed by a pronounced astroglial response, parallel to fructose 2, 6-bisphosphate recovery. The Stereotaxic administration of rotenone showed a different time effect on fructose 2,6-bisphosphate cerebral content, with a significantly faster recovery. These results indicate that cerebral fructose 2,6-bisphosphate may be a sensitive metabolite related to brain damage caused by potent neurotoxins such as MPP⁺. On the other hand, they show that MPP⁺ acts in the brain through a quick, strong cytotoxic mechanism, which probably involves mechanisms other than mitochondria! chain blockage     

Discovery of MK-7655, a β-lactamase inhibitor for combination with Primaxin®

β-Lactamase inhibitors with a bicyclic urea core and a variety of heterocyclic side chains were prepared and evaluated as potential partners for combination with imipenem to overcome class A and C β-lactamase mediated antibiotic resistance. The piperidine analog 3 (MK-7655) inhibited both class A and C β-lactamases in vitro. It effectively restored imipenem’s activity against imipenem-resistant Pseudomonas and Klebsiella strains at clinically achievable concentrations. A combination of MK-7655 and Primaxin® is currently in phase II clinical trials for the treatment of Gram-negative bacterial infections.     

Effect of Noncompetitive Blockade of N-Methyl-d-Aspartate Receptors on the Neurochemical Sequelae of Experimental Brain Injury

Pharmacological inhibition of excitatory neurotransmission attenuates cell death in models of global and focal ischemia and hypoglycemia, and improves neurological outcome after experimental spinal cord injury. The present study examined the effects of the noncompetitive N-methyl-D-aspartate receptor blocker MK-801 on neurochemical sequelae following experimental fluid-percussion brain injury in the rat. Fifteen minutes after fluid-percussion brain injury (2.8 atmospheres), animals received either MK-801 (1 mg/kg, i.v.) or saline. MK-801 treatment significantly attenuated the development of focal brain edema at the site of injury 48 h after brain injury, significantly reduced the increase in tissue sodium, and prevented the localized decline in total tissue magnesium that was observed in injured tissue of saline-treated animals. Using phosphorus nuclear magnetic resonance spectroscopy, we also observed that MK-801 treatment improved brain metabolic status and promoted a significant recovery of intracellular free magnesium concentrations that fell precipitously after brain injury. These results suggest that excitatory amino acid neurotransmitters may be involved in the pathophysiological sequelae of traumatic brain injury and that noncompetitive N-methyl-D-aspartate receptor antagonists may effectively attenuate some of the potentially deleterious neurochemical sequelae of brain injury.     

Cooperative Modulation of [3H]MK-801 Binding to the N-Methyl-d-Aspartate Receptor-Ion Channel Complex by l-Glutamate, Glycine, and Polyamines

In extensively washed rat cortical membranes [3H](+)-5-methyl-10,11-dihydro-5 H-dibenzo [a,d]cyclohepten-5,10-imine ([3H]MK-801) labeled a homogeneous set of sites (Bmax = 1.86 pmol/mg protein) with relatively low affinity (KD = 45 nM). L-Glutamate, glycine, and spermidine produced concentration-dependent increases in specific [3H]MK-801 binding due to a reduction in the KD of the radioligand. In the presence of high concentrations of L-glutamate, glycine, or spermidine, the KD values for [3H]MK-801 were reduced to 11 nM, 18 nM, and 15 nM, respectively. Maximally effective concentrations of combinations of the three compounds further increased [3H]MK-801 binding affinity as follows: L-glutamate + glycine, KD = 6.2 nM; L-glutamate + spermidine, KD = 2.2 nM; glycine + spermidine, KD = 8.3 nM. High concentrations of spermidine did not inhibit either [3H]glycine orf [3H]3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid binding to the N-methyl-D-aspartate (NMDA) receptor complex. The concentration of L-glutamate required to produce half-maximal enhancement (EC50) of [3H]MK-801 binding was reduced from 218 nM to 52 nM in the presence of 30 microM glycine and to 41 nM in the presence of 50 microM spermidine. The EC50 value for glycine enhancement of [3H]MK-801 binding was 184 nM. This was lowered to 47 nM in the presence of L-glutamate and to 59 nM in the presence of spermidine. Spermidine enhanced [3H]MK-801 binding with an EC50 value of 19.4 microM which was significantly reduced by high concentrations of L-glutamate (EC50 = 3.9 microM) or glycine (EC50 = 6.2 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthetic Analogues of Conantokin-G: NMDA Antagonists Acting Through a Novel Polyamine-Coupled Site

Abstract: Conantokin-G (con-G) is a 17-amino-acid polypeptide that acts as an N-methyl-d -aspartate (NMDA) antagonist. This action has been attributed to a specific but noncompetitive inhibition of the positive modulatory effects of polyamines at NMDA receptors. Con-G possesses several unusual structural features, including five γ-carboxyglutamate (Gla) residues and a high degree of helicity in aqueous media. Previous structure-activity studies indicated that one or more Gla residues are necessary for NMDA antagonist activity. Con-G analogues were synthesized with alanine (Ala), serine (Ser), and phosphoserine substituted for Gla to assess the contribution of individual Gla residues to biological activity and secondary structure. Replacement of Gla in positions 3 and 4 resulted in polypeptides with markedly reduced and no NMDA antagonist actions, respectively. In contrast, Gla residues in positions 7, 10, and 14 are not required for NMDA antagonist actions because the potencies of con-G analogues containing Ser⁷, Ser¹⁰, Ala¹⁴, and Ser¹⁴ to inhibit spermine-stimulated [³H]MK-801 binding are similar to the parent peptide. Moreover, the Ala⁷ derivative of con-G was about fourfold more potent than the parent peptide both as an inhibitor of spermine-stimulated increases in [³H]MK-801 binding (IC₅₀ of ～45 nM) and in reducing NMDA-stimulated increases in cyclic GMP levels (IC₅₀ of ～77 nM) in cerebellar granule cell cultures. Although con-G and its analogues assumed mixtures of 3₁₀ and α-helices, no clear-cut relationship was evinced between the NMDA antagonist properties of these peptides and the degree of helicity they assumed in aqueous solutions. Together with the inability of con-G to affect 5,7-dichloro[³H]kynurenic acid, [³H]CGP-39653, and [³H]ifenprodil binding, these data are consistent with the hypothesis that this polypeptide acts at a unique, polyamine-associated site on NMDA receptors.     

Effects of Subchronic Clozapine and Haloperidol on Striatal Glutamatergic Synapses

Abstract: Subchronic treatment with haloperidol increases the number of asymmetric glutamate synapses associated with a perforated postsynaptic density in the striatum. To characterize these synaptic changes further, the effects of subchronic (28 days) administration of an atypical antipsychotic, clozapine (30 mg/kg, s.c.), or a typical antipsychotic, haloperidol (0.5 mg/kg, s.c.), on the binding of [³H]MK-801 to the NMDA receptor-linked ion channel complex and on the in situ hybridization of riboprobes for NMDAR2A and 2B subunits and splice variants of the NMDAR1 subunit were examined in striatal preparations from rats. The density of striatal glutamate immunogold labeling associated with nerve terminals of all asymmetric synapses and the immunoreactivity of those asymmetric synapses associated with a perforated postsynaptic density were also examined by electron microscopy. Subchronic neuroleptic administration had no effect on [³H]MK-801 binding to striatal membrane preparations. Both drugs increased glutamate immunogold labeling in nerve terminals of all asymmetric synapses, but only haloperidol increased the density of glutamate immunoreactivity within nerve terminals of asymmetric synapses containing a perforated postsynaptic density. Whereas subchronic administration of clozapine, but not haloperidol, resulted in a significant increase in the hybridization of a riboprobe that labels all splice variants of the NMDAR1 subunit, both drugs significantly decreased the abundance of NMDAR1 subunit mRNA containing a 63-base insert. Neither drug altered mRNA for the 2A subunit, but clozapine significantly increased hybridization of a probe for the 2B subunit. The data suggest that some neuroleptic effects may be mediated by glutamatergic systems and that typical and atypical antipsychotics can have varying effects on the density of glutamate in presynaptic terminals and on the expression of specific NMDA receptor splice variant mRNAs. Alternatively, NMDAR1 subunit splice variants may differentially respond to interactions with glutamate.     

Transfection of N-Methyl-d-Aspartate Receptors in a Nonneuronal Cell Line Leads to Cell Death

Abstract: Neurons grown in culture die when they are exposed to high concentrations (0.1–1 m M ) of the neurotransmitter l -glutamate. A similar phenomenon may occur in the mammalian brain during ischemia and other injuries that cause excessive glutamate release. Activation of N -methyl- d -aspartate (NMDA) receptors and the consequent Ca²⁺ influx are thought to play a critical role in the process of neuronal toxicity. Events subsequent to the Ca²⁺ influx are not well understood. We have discovered that nonneuronal kidney cells expressing NMDA receptors after DNA transfection undergo cell death unless they are protected by drugs that block the NMDA receptor ion channel. Furthermore, transfected cells expressing a mutated NMDA receptor that conducts less Ca²⁺ are less vulnerable to cell death. In addition, we find that even though several active forms of NMDA receptors can be synthesized in these cells after transfection with different cloned subunits, not all receptor types are equally toxic. These experiments suggest that Ca²⁺ influx through NMDA channels may be toxic to nonneuronal cells and that the NMDA receptor expression may be the major neuron-specific component of excitotoxicity.