High-affinity [3H] kainic acid binding to brain membranes: a re-evaluation of ligand potency and selectivity

[3H]Kainic acid ([3H]KA) is a widely used tool for studying the KA class of excitatory amino acid receptors. [3H]KA of significantly higher specific activity has become available permitting use of radioligand concentrations below the dissociation constant (K(D)) of the high-affinity binding site. We employed low radioligand (0.05-0.2 nM) and receptor concentrations (0.01 nM) to gain new insights into the binding characteristics of the high-affinity KA binding site in a standard preparation of lyzed synaptosomal membranes from the cerebral cortex of male Sprague-Dawley rats. Under these conditions, KA binds to a single class of high-affinity sites with a K(D) of 1.0+/- 0.3 nM. The potencies of competing agents are considerably higher than published reports. Specifically, domoic acid, glutamate, and glutamine exhibit IC(50) values for displacing [3H]KA of 0.37+/-0.02, 94+/-13, and 1500+/-500 nM, respectively. Domoate (1 microM) was tested against a panel of 32 central nervous system binding sites and found to be inactive at each, indicating this toxin displays considerable selectivity. This study illustrates the remarkable potency of domoic acid and underlines the importance of performing radioligand binding studies at concentrations of constituents that permit characterization of high-affinity interactions.     

Tattoo sites and psoriasis

The Koebner phenomenon refers to the development of lesions in response to injury of previously uninvolved skin. It occurs in psoriasis and a number of other inflammatory diseases. We present a patient who developed a Koebner reaction at the site of a tattoo. Treatment with ustekinumab resulted in striking clearance of the psoriasis changes at the tattoo site.     

Blue foot: a second case of "tattoo blow-out" pigment spread successfully treated with the QS-Nd:YAG laser

The "tattoo blow-out" phenomenon occurs when tattoo pigments spread outside the border of a tattoo. It is thought to occur when ink is injected too deeply. A healthy 36-year-old female presented to a dermatologist with diffuse spread of tattoo pigment outside the original tattoo that occurred within one day of the placement of a professional tattoo on the dorsum of her foot. The patient was seeking treatment six weeks after the tattoo was placed because she thought the discoloration would improve or resolve on its own, but it worsened. Two punch biopsies were obtained for histology. The biopsy results confirmed granular black pigment consistent with a tattoo in the dermis and subcutaneous fat. The location of pigment was deeper than expected. Due to the success of the QS-Nd:YAG laser in a prior patient, the same treatment was recommended for this patient. The patient received nine laser sessions using the Q-switched laser at 1064 nm, 4 mm, 10 Hz, with gradually increasing energy from 4.5 to 6.0 J/cm2. The pigment outside of the original tattoo borders faded and is barely perceptible. It is important that physicians be made aware of tattoo complications so they can advise patients in regards to the associated risks.     

Autoradiographic localization of high-affinity [3H]kainic acid binding sites in the rat forebrain

Utilizing in vitro autoradiographic techniques, we have studied the distribution of high affinity [3H]kainic acid ([3H]KA) binding sites in intact sections of the rat forebrain. These sites have the same kinetic and pharmacological characteristics as the [3H]KA site described in tissue homogenates. Moderate to high levels of specific binding were observed in several discrete brain regions. These include lamina I, V and VI of the neo- and cingulate cortex, superficial layers of the pyriform cortex, striatum, external plexiform and granule cell layers of the olfactory bulb, olfactory tubercle, the stratum lucidum of CA3 of the hippocampus, molecular layer of the dentate gyrus, reticular nucleus of the thalamus, the hypothalamic median eminence, and the granule cell layer of the cerebellum. Low levels of specific binding were associated with other discrete regions such as the lateral septum, bed nucleus of the stria terminalis, medial geniculate, superficial layers of the superior colliculus, nuclei of the central grey, interpeduncular nucleus and the molecular layer of the cerebellum. Moderate uniform levels of specific binding were observed over the hypothalamus, zona incerta and the amygdala. One of the important factors in KA neurotoxicity seems to be the presence of KA receptors, and regions that are susceptible to the toxic effects of KA after local administration, such as the striatum, hippocampus, amygdala and pyriform cortex, have moderate to high levels of binding. Thus, these data provide a useful map for studying the relationship between receptor-mediated and seizure-induced neuronal damage following KA administration.     

吸毒者中纹身情况调查与康复治疗

目的··:调查吸毒者中的纹身情况 ,寻求有效康复治疗方法。方法··:采用自行设计《药物滥用调查表》对716例住院强制戒毒者进行调查 ,并进行心理康复治疗。结果··:716例吸毒者中纹身者127例 ,占17.74 % ,其中男100例 ,女27例 ,年龄最小16a ,最大43a ,有纹身时间1 -25a ,平均8.8a±s5.75a。三位一体方式 (集体心理治疗、认知行为治疗、行为治疗 )的康复治疗有一定效果 ;有35例 (27.56% )表示出所后要手术除掉纹身。结论··:吸毒者中有纹身的比例很高 ,这与社会适应问题有一定相关性 ,康复治疗有助于他们摆脱困境     

A test of the null equation for functional antagonism.

1 The quantitative model for functional antagonism and synergism has been tested by studying its ability to fit data obtained from the functional antagonism of (-)-isoprenaline by muscarinic agonists on guinea-pig isolated atria. 2 The general form of the null equation has been shown to fit the experimental curves satisfactorily. 3 Functional interaction between (-)-isoprenaline and muscarinic agonists on atria has been shown to be type I although there does seem to be a discrepancy between values of the functional affinity constants, KA1F and KA2F, estimated in two different ways. 4 The affinity constants, KA, of the muscarinic agonists for their receptors have been estimated by use of the selective irreversible antagonist propylbenzilylcholine mustard. The discrepancy between KAF (i.e. both KA1F and KA2F) and KA is small for pentyltrimethylammonium which is an agonist of low intrinsic efficacy. By contrast the discrepancy between KAF and KA is much greater for methylfurmethide and oxotremorine both of which have much higher intrinsic efficacies. These results are as predicted by the model. 5 It is suggested that the discrepancy between KA1F and KA2F may be due to the limited ability of the equation l/S omega = aI + bI/S alpha to describe quantitatively the relation between sequential stimuli. However, it is concluded that this complication need not interfere with the use of the model to study mechanisms and possible sites of functional interaction.     

对药物滥用人群文身现象的调查和思考

目的:调查药物滥用人群存在文身现象的基本情况.方法:采用自行设计的"药物滥用人群文身现象调查表"做匿名问卷调查,并对其中的20项调查结果做统计处理和分析.结果:文身现象在药物滥用人群当中普遍存在,并与这个人群综合素质低、思想意识不正确紧密相联.同时,药物滥用人群所采用的不安全文身方式和他们控制难度较大的文身场所不得不引起人们的关注.结论:为了更好地在药物滥用人群中开展心理和行为矫治工作、减少伤害,对这个群体的文身现象做基本的调查和了解是十分必要的.     

吸毒者文身流行病学调查

目的 :了解吸毒人群中文身的情况。方法 :使用自拟的《文身情况调查表》对武汉市公安局强制戒毒所的戒毒者进行文身情况问卷调查。结果 :共收回有效问卷 76 6份 ,发现有文身者 2 89例 ,占 37.7% ,其中男 2 32例 ,占80 .3% ,女 5 7例 ,占 19.7% ;文身的主要原因是“好奇、好玩”(42 .6 % )、“无聊”(2 2 .2 % )和“情爱”(2 1.8% )。文身者中有孤独感的占 5 0 .2 % ,独立人格的 2 4 .2 % ,厌倦人生的 2 3.5 %。文身者中 2 18例 (75 .4 % )有违法犯罪行为 ,显著高于非文身者 (40 .2 % ,P <0 .0 1) ;使用没有经过消毒的文身针具者占 70 .6 % (197 2 89)。结论 :吸毒者中的文身比例高 ,文身原因多样化 ,病态人格多 ,犯罪比率大 ,易感染传染性疾病     

Relationship between alpha 2-adrenergic receptor binding sites and the functional receptors inhibiting norepinephrine release in rat cerebral cortex

The properties of alpha 2-adrenergic receptor binding sites and the receptors inhibiting [3H]norepinephrine (3H-NE) release from slices of cerebral cortex were compared. [3H]RX 781094, an alpha 2-adrenergic receptor antagonist radioligand, labeled a single class of binding sites in membranes at 37 degrees with the pharmacological specificity expected of alpha 2-adrenergic receptors. 5'-Guanylimidodiphosphate (Gpp(NH)p) and NaCl caused small increases in the potencies of antagonists at the 3H-RX binding sites but decreased the potencies of agonists 4-22-fold. Antagonists blocked the inhibition of potassium-evoked 3H-NE release caused by exogenous NE with potencies similar to those in competing for 3H-RX binding sites. Partial receptor inactivation with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) was used to determine whether there was a receptor reserve. EEDQ dose-dependently decreased both the density of 3H-RX binding sites and the maximal inhibition of 3H-NE release by different agonists. For most agonists, KA values calculated after the receptor inactivation did not differ significantly from EC50 values; however, for epinephrine a small receptor reserve was apparent. The proportion of 3H-RX binding sites lost was similar to the proportion of functional receptors lost after EEDQ treatment. The functional KA values for agonists correlated most closely with KD values for the low affinity binding state observed in the presence of Gpp(NH)p and NaCl. However, both epinephrine and NE still showed two-site binding curves under these conditions, and it was the high affinity subpopulation of sites observed in the presence of Gpp(NH)p and NaCl which resembled most closely the functional KA values. These data suggest that 3H-RX labels binding sites with properties similar to the alpha 2-adrenergic receptors inhibiting 3H-NE release in cerebral cortex. There is little or no receptor reserve for this effect, and there appears to be a binding state for the natural catecholamine agonists which has an affinity lower than that for mediating this functional response.     

Involvement of kainate receptors in the analgesic but not hypnotic effects induced by inhalation anesthetics

In the present study, the role of kainate (KA) receptors in hypnosis and analgesia induced by emulsified inhalation anesthetics was investigated. A mouse model of hypnosis and analgesia was established by an intraperitoneal injection of emulsified enflurane, isoflurane or sevoflurane. We intracerebroventricularly (icv) or intrathecally (it) administered KA, a KA receptor agonist to mice. The effects of the KA on the sleep time were observed using a hypnosis test, and the tail-withdrawal latency was analyzed using the tail-withdrawal test. In the hypnosis test, KA (2.5, 5 or 10 ng; icv administered) treatment had no distinctive effects on the sleep time of mice treated with emulsified inhalation anesthetics. In the tail-withdrawal test, KA (0.2, 0.4 or 0.8 ng; it administered) treatment significantly and dose-dependently decreased the tail-withdrawal latency of mice treated with emulsified anesthetics. These results suggested that KA receptors may modulate the analgesic but not hypnotic effects induced by emulsified enflurane, isoflurane or sevoflurane.     

Evidence for the presence of glutamatergic receptors in adult Schistosoma mansoni

Several studies have suggested that L-glutamate is a putative neurotransmitter in helminths. The present study investigated the presence of non-N-methyl-D-aspartate (NMDA) ionotropic receptors for glutamate in four subcellular fractions from adult male Schistosoma mansoni. Low-affinity (K(d)=221+/-80 nM) binding sites for [3H]kainic acid (KA) were detected in the heterogeneous (P(1)) fraction, which contains pieces of unbroken worm tissues, tegument, nuclei, and some vesicles. This binding was inhibited by classical glutamatergic ligands in the following order of potency: KA>L-glutamate>alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA)>quisqualate congruent with 6,7-dinitroquinoline-2,3-dione (DNQX). However, neither NMDA, a selective agonist for NMDA receptors, nor DL-threo-beta-hydroxyaspartate (THA) and 1-trans-pyrollidine-2-dicarboxylic acid (PDC), inhibitors of high-affinity glutamate transporters, modified [3H]KA binding to the P(1) fraction. In addition, no specific binding for 10nM [3H]AMPA was detected in any subcellular fraction from S. mansoni. These results suggested the presence of KA receptors in adult male worms. This is supported by the evidence that direct application of 10 microM KA to whole worms produced a corkscrew-like coiling of their bodies, modifying the motility of the worms. The KA-induced response, measured as a decrease of the body area, was time-dependent and reversible. PDC was ineffective at blocking the KA effects, indicating that KA does not depend on high-affinity glutamate transporters to reach its site of action. On the other hand, DNQX, the non-NMDA antagonist, was able to partially inhibit KA-induced responses. As a whole, the present data support the presence of a glutamatergic signaling pathway in this parasite.     

Differences in muscarinic receptor reserve for inhibition of adenylate cyclase and stimulation of phosphoinositide hydrolysis in chick heart cells

Carbachol is 100 times more potent for inhibiting cyclic AMP formation than for stimulating phosphoinositide (PI) hydrolysis in chick heart cells. To determine whether this reflects differences in agonist affinity of the receptor(s) coupled to the two responses, we measured these functional responses following removal of receptor reserve with propylbenzilycholine mustard (PrBCM). Conditions of PrBCM treatment that led to progressive loss of up to 95% of the [3H]-N-methylscopolamine-binding sites decreased the potency but not the maximal capacity of carbachol to inhibit cyclic AMP formation. In contrast, there was a marked decrease in the maximal PI response to carbachol. The KA for carbachol, calculated by measuring functional responses following receptor inactivation, was similar whether the cyclic AMP or the PI response was examined. These KA values (approximately 40 microM) were similar to the KD calculated by examining carbachol competition for [3H]-N-methylscopolamine-binding sites on the intact cell. PrBCM treatment also decreased the maximal effect of oxotremorine on cyclic AMP formation under conditions in which carbachol remained a full agonist for this response. We interpret our data as indicating that: there is much greater receptor reserve in the coupling of muscarinic receptors to adenylate cyclase than to PI hydrolysis; this, rather than differences in receptor affinity underlies the disparate dose-response relationships for the two responses; and differences in the effects of weak agonist on the two responses may also reflect differences in receptor reserve. We suggest that muscarinic receptors with the same affinity for carbachol interact with different efficiency with the transducers (Gi and Gx) that regulate adenylate cyclase and phospholipase C.     

脑内GABA能神经元对红藻氨酸中枢性心血管效应的影响

采用侧脑室微最注射法证明,谷氯酸受体哑型激动剂红藻氨酸(KA)可依剂量性地升高大鼠血压和加快心率。预先给予GABA合成抑制剂氮基脲140 mg/kg ip或GABA拮抗剂印防己毒1 mg/kg iv均可明显增强KA的作用;而GABA转氨酶抑制剂氨氧乙酸25μg/rat icv明显减弱KA的作用。结果表明,脑内GABA能神经元功能受抑,KA的中枢性心血管效应增强。从而推测,脑内GABA能神经元与谷氯酸能神经元之间相互制约,共同参与中枢性调节心血管的活动。     

A case of cutaneous diphtheria in New Zealand

We report the case of an adult male who contracted cutaneous diphtheria after receiving a tattoo in Samoa. The infection required hospital admission. The Regional Public Health Service conducted urgent contact tracing. We review the techniques employed in traditional tattooing and highlight the importance of considering C. diphtheriae as a causative organism in cutaneous infection acquired in the tropics.     

Effect of pyrethroids on [3H]kainic acid binding to mouse forebrain membranes

The effect of the pyrethroid insecticides, decamethrin, cis-permethrin, and trans-permethrin, was examined on the in vitro specific binding of [³H]kainic acid ([³H]KA) to receptor sites in mouse forebrain homogenates. All three pyrethroids gave rise to dose-dependent decreases in levels of specific binding. Decamethrin was the most potent in displacing bound [³H]KA giving rise to significant decreases at decamethrin concentrations of 10^?7m and above. Intracere-broventricular injections of the pyrethroids into unanesthetized mice gave rise to neurotoxic symptoms including hyperactivity, tremor, and tonic seizures. The order of potency of the three compounds in causing these symptoms of toxicity (decamethrin >cis-permethrin >trans-permethrin) was found to be the same as their order of potency in displacing bound [³H]KA from binding sites in the mouse brain. These preliminary results suggest that the neurotoxic actions of pyrethroids in mammals may be at least partially mediated via interactions with a kainic acid binding site.     

Contact allergic dermatitis to gold in a tattoo: a case report

The art of tattooing has increasing in recent decades. Allergic sensitivity to one of the pigments is the most frequent cause of dermatological reactions at the site of the tattoo. Gold is a new pigment used in tattooing, because of its bright yellow color and luster. Allergy to this metal is uncommon. To our knowledge, this is the first reported case of allergic contact dermatitis to gold in a tattoo.     

Determination of binding site residues responsible for the subunit selectivity of novel marine-derived compounds on kainate receptors

Dysiherbaine (DH) and related molecules are high-affinity, subunit-selective kainate receptor (KAR) ligands originally isolated from a marine sponge. To elucidate why DH, an agonist, and MSVIII-19, a competitive antagonist, bind selectively to glutamate receptor (GluR) 5 but not to the KA2 KAR subunit, we used molecular dynamics simulations to generate binding models that were tested experimentally in radioligand binding and electrophysiological assays. Three candidate sites, Val685, Leu735, and Ser741 in GluR5, corresponding to Ile669, Phe719, and Met725 in KA2, were predicted to underlie the distinct binding profiles of the marine toxins. Single or multiple reciprocal mutations introduced into the receptor subunits produced a variety of effects on binding affinity. Most notably, mutation of Met725 to serine in KA2 increased the affinity of DH by 350-fold; in contrast, mutation of one or more of the residues in GluR5 did not markedly alter DH binding. MSVIII-19 affinity for the KA2 subunit was significantly increased in multiple site mutants, and reciprocal mutations in the GluR5 subunit produced substantial (700-fold) reductions in MSVIII-19 affinity. Physiological characterization of the double- and triple-mutant subunits demonstrated altered functional behavior consistent with the changes in binding affinity. The results provide experimental support for the importance of these three ligand binding domain (LBD) residues and suggest steric hindrance in the KA2 subunit LBD is largely responsible for the very low affinity for the two compounds. In this study, we identified the molecular basis for subunit selectivity of these marine-derived molecules on KARs, which could facilitate the rational design of selective ligands with distinct pharmacological profiles.     

Getting nano tattoos right—A checklist of legal and ethical hurdles for an emerging nanomedical technology

The nano tattoo represents a nascent technology designed to be implanted in the skin to provide continuous and reliable glucose detection for diabetics. Its potential benefits are compelling not only for its ability to prevent diabetic complications and decrease related social costs, but also for its ease of use and relative patient-user comfort. This Note aims to articulate a checklist of fundamental intellectual property, bioethical and system design issues that are appropriately considered in the pre-clinical, pre-commercialization phase of nano tattoo development. Early and regular consideration of these factors can increase the odds of a societally beneficial dissemination of this device by engaging relevant researcher, medical, patient-user and patient-advocate communities concerned with its appropriate application, as well as policymaking communities focused on effectively managing diabetes-related healthcare costs. The checklist of factors includes fundamental issues and is generally applicable to nanomedical inventions.From the Clinical EditorThis paper presents a comprehensive list of fundamental intellectual property, bioethical, and system design issues to be considered in the pre-commercialization phase of nanomedicine development, through the specific example of nano tattoo development. Nano tattoo is designed to be implanted in the skin to provide reliable glucose monitoring for diabetics, enabling enhanced prevention of complications and decreased socioeconomic costs.     

Pharmacological characterization of glutamatergic agonists and antagonists at recombinant human homomeric and heteromeric kainate receptors in vitro

An increasing body of evidence suggests that native kainate receptors form ion channels from homomeric and heteromeric combinations of five receptor subunits: GluR5, GluR6, GluR7, KA1 and KA2. We have examined the activity of agonists and antagonists at recombinant human kainate receptors expressed in HEK293 cells, using both whole-cell electrophysiological recording and 96-well plate fluo-3 based calcium microfluorimetry (FLIPR). Both homomeric (GluR5 and GluR6) and heteromeric (GluR5/6, GluR5/KA2 and GluR6/KA2) receptors were examined. Heteromeric receptor assemblies showed electrophysiological and pharmacological profiles which were distinct from homomeric channels. Several agonists, including AMPA, ATPA and (S)-5-iodowillardiine, and antagonists, including gamma-D-glutamylaminomethylsulphonic acid (GAMS) and the decahydroisoquinoline compounds LY293558, LY377770 and LY382884, were found to act at GluR5-containing channels while having no effect at GluR6 homomers. AMPA, ATPA and (S)-5-iodowillardiine did activate GluR6/KA2 heteromers, but only as partial agonists. Additionally, ATPA was shown to act as an antagonist at homomeric GluR6 receptors at high concentrations (IC50 approximately 2 mM). Kynurenic acid was also found to differentiate between GluR6 and GluR6/KA2 receptors, antagonizing glutamate at GluR6 (IC50 = 0.4 mM), while having no effect at GluR6/KA2 channels. The results of the current study provide a broad pharmacological characterization of both homomeric and heteromeric recombinant human kainate receptors, and identify which compounds are likely to be useful tools for studying these various receptor subtypes.     

Type II and III Taste Bud Cells Preferentially Expressed Kainate Glutamate Receptors in Rats

Glutamate-induced cobalt uptake reveals that non-NMDA glutamate receptors (GluRs) are present in rat taste bud cells. Previous studies involving glutamate induced cobalt staining suggest this uptake mainly occurs via kainate type GluRs. It is not known which of the 4 types of taste bud cells express subunits of kainate GluR. Circumvallate and foliate papillae of Sprague-Dawley rats (45~60 days old) were used to search for the mRNAs of subunits of non-NMDA GluRs using RT-PCR with specific primers for GluR1-7, KA1 and KA2. We also performed RT-PCR for GluR5, KA1, PLCβ2, and NCAM/SNAP 25 in isolated single cells from taste buds. Taste epithelium, including circumvallate or foliate papilla, express mRNAs of GluR5 and KA1. However, non-taste tongue epithelium expresses no subunits of non-NMDA GluRs. Isolated single cell RT-PCR reveals that the mRNAs of GluR5 and KA1 are preferentially expressed in Type II and Type III cells over Type I cells.