Interactions between loreclezole,chlormethiazole and pentobarbitone at GABAA receptors: functional and binding studies

1. Interations were investigated between loreclezole, chlormethiazole and pentobarbitone as potentiators of depolarization responses mediated by γ-aminobutyric acid_A (GABA_A) receptors on afferent nerve terminals in the rat cuneate nucleus in vitro. These drugs were also compared as modulators of [³H]-flunitrazepam (FNZ) binding to synaptic membranes prepared from rat whole brain homogenate.
2. In rat cuneate nucleus slices, the drugs shifted muscimol log dose–response lines to the left in an approximately parallel fashion with the result that 200 μM chlormethiazole potentiated muscimol responses by 0.567±0.037 log unit (mean±s.e.mean, n=4) while loreclezole gave a maximal potentiation at 10 μM of only 0.121±0.037 (n=6) log unit and 0.071±0.039 (n=22) at 50 μM.
3. While 50 μM chlormethiazole and 30 μM pentobarbitone showed no significant interactions between each other when potentiating muscimol responses in combination, 50 μM loreclezole in combination with either chlormethiazole or pentobarbitone attenuated their potentiating effects, possibly by inducing desensitization of GABA_A receptors.
4. In the [³H]-FNZ binding studies on well-washed membranes, loreclezole enhanced binding to a maximum of 47.3±2.83% of control (mean±s.e.mean, n=3) at 300 μM. Scatchard analysis revealed no change in B_max but a decrease in K_D for [³H]-FNZ from 3.9±0.29 nM to 2.7±0.10 nM (mean±s.e.mean, n=4) in the presence of 100 μM loreclezole. In contrast, 100 μM chlormethiazole caused no potentiation. A small component of the enhancement by loreclezole could be blocked by 100 μM bicuculline and could also be blocked by 100 μM chlormethiazole. It seems likely that the effects on [³H]-FNZ binding are due predominantly to direct actions of the drugs on the GABA_A receptor and are separate from the GABA-potentiating effects.
5. The results indicate distinctly different profiles of action for loreclezole, chlormethiazole and pentobarbitone on GABA_A receptors.

     

Pharmacological characterization of type 1α metabotropic glutamate receptor-stimulated [35S]-GTPγS binding

1. The activation of G proteins by type 1α metabotropic glutamate receptors (mGluRs) in membranes from recombinant baby hamster kidney cells expressing the cloned rat mGluR1α receptor has been studied by use of a [³⁵S]-guanosine 5′-[γ-thio]triphosphate ([³⁵S]-GTPγS) binding assay.
2. L-Glutamate increased the rate of [³⁵S]-GTPγS binding in a concentration-dependent manner (−logEC₅₀ (M) 5.25±0.07), with an optimal (62.4±1.6%) increase over basal binding being observed following 60 min incubation at 30°C with 70 pM [³⁵S]-GTPγS, 1 μM GDP, 10 mM MgCl₂, 100 mM NaCl and 100 μg membrane protein ml⁻¹. The L-glutamate (100 μM)-stimulated increase in [³⁵S]-GTPγS binding was totally prevented in the presence of the group I mGluR antagonist (S)-4-carboxy-3-hydroxyphenylglycine (300 μM).
3. Quantitative analysis of the affinity and number of G proteins activated by a maximally effective concentration of L-glutamate revealed an equilibrium dissociation constant (K_D) for [³⁵S]-GTPγS binding of 0.76±0.20 nM and a maximal number of GTPγS-liganded G proteins (B_max) of 361±30 fmol mg⁻¹ protein.
4. Metabotropic glutamate receptor agonists, quisqualate (−logEC₅₀ (M) 6.74±0.06), 1S,3R-ACPD (4.64±0.08) and (S)-3,5-dihydroxyphenylglycine (5.16±0.23) also increased [³⁵S]-GTPγS binding in a concentration-dependent manner, with the latter two agents behaving as partial agonists.
5. (+)-α-Methylcarboxyphenylglycine (300 μM) caused a parallel rightward shift of the L-glutamate concentration-effect curve for [³⁵S]-GTPγS binding, allowing an antagonist equilibrium dissociation constant (K_D) of 34.0±7.8 μM to be calculated for this mGluR antagonist.
6. Pretreatment of BHK-mGluR1α cells with a concentration of pertussis toxin (PTX) shown to be maximally effective (100 ng ml⁻¹, 24 h) before membrane preparation resulted in a marked decrease in agonist-stimulated [³⁵S]-GTPγS binding (by 66.0±0.9%), and an altered concentration-effect relationship for agonist-stimulated [³⁵S]-GTPγS binding by the residual PTX-insensitive G-protein population.
7. The modulation of [³⁵S]-GTPγS binding by agonists and antagonists in membranes from recombinant cells provides an excellent system in which to study mGluR interactions with PTX-sensitive and -insensitive G proteins.

     

Interaction of class III antiarrhythmic drugs with muscarinic M2 and M3 receptors: radioligand binding and functional studies

We have recently reported that class III antiarrhythmic drugs inhibit the muscarinic acetylcholine (ACh) receptor-operated K⁺ current (I _K, ACh) in guinea-pig atrial cells by different molecular mechanisms. The data obtained from the patch-clamp study suggest that d,l-sotalol inhibits I _{K, ACh} by blocking the muscarinic receptors, whereas MS-551 inhibits the K⁺ current by blocking the muscarinic receptors and depressing the function of the K⁺ channel itself and/or the guanine nucleotide-binding protein (G protein). This study was undertaken to determine whether the class III antiarrhythmic drugs d,l-sotalol and MS-551 interact with the muscarinic receptors of cardiac and peripheral tissues. Both drugs inhibited concentration dependently the specific [³H]N-methylscopolamine ([³H]-NMS) binding to membrane preparations obtained from guinea-pig atria and submandibular glands. The competition curves of these drugs for [³H]-NMS binding to glandular membranes were monophasic, suggesting competition with [³H]-NMS at a single site. Although the competition curve of d,l-sotalol for [³H]-NMS binding to atrial membranes was monophasic, that of MS-551 was biphasic and showed high- and low-affinity states of binding. d,l-Sotalol showed slightly, but significantly, higher affinity for cardiac-type muscarinic receptors (M₂) than for glandular-type muscarinic receptors (M₃). The inhibition constant (K _i) for MS-551 in glandular membranes was also slightly greater than the high-affinity K _i value for the drug in atrial membranes. In guinea-pig left atria and ilea, d,l-sotalol shifted the concentration-response curves for the negative inotropic effect and the contracting effect of carbachol in a parallel manner. The slopes of Schild plot were not significantly different from unity, suggesting competitive antagonism, and the pA₂ for d,l-sotalol in left atria was slightly greater than that in ilea. MS-551 also shifted the concentration response curve for the negative inotropic effect of carbachol in atrial preparations to a greater extent than that for the contracting effect in ileal preparations, although MS-551 failed to show a pure competitive antagonism. These results suggest that both d,l-sotalol and MS-551 interact with cardiac M₂ and peripheral M₃ receptors, and that at high concentrations they exert anticholinergic activity in cardiac and peripheral tissues.     

The effects of d-cycloserine and MK-801 on the performance of rats in two spatial learning and memory tasks

The present study was undertaken to investigate the effects of modulation of the (NMDA) receptor on learning and memory. Thus, the performance of rats treated with d-cycloserine, a partial agonist at the glycine recognition site of the NMDA receptor complex, and MK-801, a noncompetitive NMDA receptor antagonist, either alone or concurrently were assessed in radial arm maze and water maze tasks. Administration of MK-801 (0.1 mg/kg, i.p.) impaired acquisition in the water maze (increased escape latency and distance) and working memory in the radial arm maze (increased re-entries) in rats. Moreover, in the radial arm maze, MK-801 disrupted locomotion (increased latencies and decreased arm entries per minute) and impaired the acquisition of reference memory (increased number of errors) performance of rats. d-Cycloserine (0.03, 0.3, 1.0, 3.0, 10 mg/kg, i.p.) had no effects on acquisition or memory performance of control or MK-801-treated rats in either of these tasks. However, d-cycloserine (0.03, 0.3, 3.0 mg/kg) reversed the MK-801-induced disruption in locomotion. Furthermore, 3.0 mg/kg d-cycloserine increased behavioral activity and also decreased the time needed to complete the task in control animals. To conclude, our results suggest that the consequences of NMDA receptor modulation on learning and memory processes and sensorimotor functions may be functionally different or have distinct anatomical locations.     

Morbus sacer in Africa: some religious aspects of epilepsy in traditional cultures

Epilepsy when manifested as grand mal seizure provokes strong and ambivalent feelings in those witnessing it. Terms such as morbus sacer, denoting both a sacred and demoniac condition, or folk names indicating divine punishment, have expressed these feelings in European societies from antiquity to the Middle Ages and beyond. An atmosphere of fear, shame and mysticism surrounds epilepsy even in our days in many non-Western and also in Western cultures. In the course of work and studies in Tanzania, where I organized the Mahenge Clinic for Epilepsy in 1960, and in other parts of Africa, I found that epilepsy is conceived of as an "African' affliction, a manifestation of supernatural forces that makes it difficult to reach epilepsy sufferers with modern medical treatment. Epilepsy is traditionally looked on as caused by ancestral spirits or attributed to possession by evil spirits. It is also thought to be due to witchcraft, and "poisoning," and often taken to be contagious. Epilepsy may, under Christian missionary teaching, have come to be considered as due to demoniac possession or divine punishment for sins, in accordance with biblical examples. In many parts of Africa, syncretic amalgamation of indigenous traditions with Judeo-Christian doctrines influenced popular attitudes toward epilepsy. We demonstrated that persistent efforts at health education in the context of organized treatment of epilepsy can result in a change of popular notions about epilepsy and consequently lead to significant improvement in the quality of life of epilepsy sufferers.     

Free amino acid pool in the brain of mice homozygous for the gene "dilute lethal"

The neurologic mutant "dilute lethal" (dl) mice, which reveal several neurologic and biochemical disturbances similar to human phenylketonuria, were used to investigate some aspects of amino acid disorder. We have studied the free amino pool in the brain of "dl" mice and of their control littermates as well as phenylalanine and tyrosine levels in brain and liver as a function of age and after phenylalamine overload. The tyrosine level decreased in brain and liver of affected mice whereas the phenylalanine/tyrosine ratio increased as a function of age. The significantly higher phenylalanine level and phenylalanine/tyrosine ratio in the liver of 20-day-old "dl" mice suggest a lower liver phenylalanine hydroxylase activity. After phenylalanine overload, the impairment of phenylalanine metabolism is predominant in the brain of "dl" mice, suggesting a disturbance in phenylalanine hydroxylation. A decrease in the level of several amino acids occurs in the brains of "dl" mice without or after phenylalanine overload; these facts might correspond to a disturbance in the transfer of amino acids to the brain and may lead to impairment in protein synthesis.     

水溶性药物聚丙交酯微球的研究

目的考察内相乙醇加入量及其他制备因素对聚丙交酯微球性质的影响。方法以水略溶性药物氟尿嘧啶和水溶性药物地基米松磷酸钠为模型药物，乳化／溶剂挥发法制备微球。维持其他制备条件不变，改变内相乙醇加入量、理论载药量、聚丙交酯浓度、溶剂挥发时间及聚丙交酯分子质量，考察微球载药量、包封率、粒径和释放的影响。结果随着内相乙醇加入量的增多，微球形成加快，微球的粒径减小，模型药物的载药量及包封率略有升高，但乙醇加入量太多时载药量及包封率均明显降低。内相聚丙交酯浓度越大，形成的微球粒径越大，载药量及包封率也愈大。理论载药量对微球粒径影响不大，地基米松磷酸钠的包封率随理论载药量的增加而减小，而氟尿嘧啶为理论载药量为15％时，包封率最大。溶剂挥发时间在0．5～3h，微球的性质无明显变化。分子质量愈小，微球的粒径愈小，载药量及包封率亦随之减小。结论内相中加入一定量乙醇可以提高药物的包封率，并减少有毒含氯溶剂的用量。     

高效液相色谱法测定右旋噻吗洛尔及其对映体含量

目的 :建立右旋噻吗洛尔含量测定、有关物质检查及对映体纯度检查方法。方法 :采用高效液相色谱法 ,以DiamonsilTM C18柱测定噻吗洛尔含量及有关物质 ;以CyclobondI 2 0 0 0 TM 柱检查右旋噻吗洛尔对映体纯度。结果 :噻吗洛尔与其中间体及降解产物之间分离良好 ;噻吗洛尔在 0～ 5 0 0mg·L- 1浓度范围内符合线性关系 ,日内、日间测定的相对标准偏差均小于 0 .6 % ;右旋噻吗洛尔与其左旋对映体可完全分离 (Rs>2 .4 )。结论 :建立的方法快速简便、准确可靠 ,可以用于右旋噻吗洛尔的质量控制     

应用循证医学观点对脑梗死急性期药物治疗的评价

目的 :以循证医学观点对脑梗死急性期各种药物治疗的临床应用进行评价。方法 :采用国内外文献综述方法。结果与结论 :对于脑梗死急性期应用尿激酶、rtPA静脉溶栓、尿激酶原动脉溶栓、阿司匹林等均有效。肝素、低分子肝素抗凝治疗对心源性脑栓塞、凝血性疾病、症状性脑动脉狭窄、症状性颅外段夹层动脉瘤及静脉血栓形成有效。降纤酶、甘油、神经节苷脂和常规应用甘露醇等尚未得出结论。而血液稀释疗法、皮质类固醇激素、氨茶碱和钙通道阻滞剂等均为阴性结果     

羊红膻根的化学成分研究

从羊红膻(Pimpinella thellungiana Wolff.)根中分得四个化合物,经理化常数测定和光谱数据解析证明为棕榈酸(Ⅰ)、4-丙烯基苯酚(Ⅱ)、松酯酚(Ⅲ)和新化合物2-甲基-2-羟基-5-甲氧基苯并[d]氢化呋喃-3-酮(Ⅳ)。