CNS Adenosine A1 Receptors Are Altered After the Administration of Convulsant 3-Mercaptopropionic Acid and Cyclopentyladenosine: An Autoradiographic Study

Rat CNS adenosine A₁ receptors were studied by quantitative autoradiography after the administration of convulsant 3-mercaptopropionic acid (MP) and an adenosine analogue cyclopentyladenosine (CPA), using 2-chloro-N⁶-[cyclopentyl-2,3,4,5-³H adenosine]-([³H]CCPA) as radioactive ligand. Specific binding was quantified in hippocampus, cerebellum, cerebral cortex, thalamic nuclei, superior colliculus and striatum, and the highest densities were found in CA1, CA2, and CA3 hippocampus subareas and the lowest levels in superior colliculus and striatum. MP administration (150 mg/kg, i.p.) produced significant increases in [³H]CCPA binding in CA1 subarea at seizure (15%) and postseizure (21%) and in CA2 at seizure (15%) but a tendency to decrease in dentate gyrus. There was an increase in cerebellum at seizure (18%) but no significant changes in the other studied regions. CPA injection (2 mg/kg, i.p.) enhanced [³H]CCPA binding in CA1 and CA2 areas (17–18%) but not in CA3 area of the hippocampus. When CPA was administered before MP, which delayed seizure onset, an increase in [³H]CCPA binding in CA1 hippocampus subarea (19%) and cerebellum (28%) was also observed. Results showed that the administration of convulsant MP and adenosine analogue CPA exerts differential effects on adenosine A₁ receptors in CNS areas; hippocampus is the most affected area with all treatments, specially CA1 subarea, supporting an essential role in convulsant activity as well as in seizure prevention.     

Effects of an adenosine analogue administration on the striatal NMDA receptors in an experimental model of epilepsy

Specific [3H]-MK801 binding to rat NMDA receptors following the administration of the convulsant drug 3-mercaptopropionic acid (MP) and the adenosine analogue cyclopentyladenosine (CPA) was studied in striatal membrane fractions. MP administration (150 mg/kg, i.p.) caused an increase of 53% and 82% in [3H]-MK801 binding during seizure and the postseizure period respectively. Administration of CPA (2 mg/kg, i.p.) raised [3H]-MK801 binding by 72%. When CPA was administered 30 min before MP and rats sacrificed at seizure (CPA + MPc), an increase of 64%, was observed. Saturation results indicate that receptor sites increased their maximal binding capacity (Bmax) in all treatments while the apparent dissociation constant (Kd) remained unchanged. MP administration brought about an increase of 52% and 42% in [3H]-MK801 binding sites during seizure and postseizure respectively. Administration of CPA raised receptor density by 75%. When CPA was administered 30 min before MP and rats sacrificed at seizure (CPA + MPc), an increase of 62%, was observed. These results show that striatal NMDA receptors have a selective role in seizure activity in the basal ganglia and that the adenosine analogue administration may modify [3H]-MK801 binding in a way similar to that of the convulsant drug.     

Striatum Adenosine A2 Receptors Are Modified During Seizure: Effect of Cyclopentyladenosine Administration

Rat CNS adenosine A_2A receptors were studied after administration of the convulsant drug 3-mercaptopropionic acid (MP) and the adenosine analogue cyclopentyladenosine (CPA) by means of a quantitative autoradiographic method. Specific binding was quantified in striatum only. The highest density was found in caudate-putamen (2.50 fmol/mm²), followed by nuclei accumbens (1.85 fmol/mm²) and the lowest values in the olfactory tubercle (1.26 fmol/mm²). These differerences were statiscally significant. MP administration (150 mg/kg) caused significant increases (12–18%) in caudate-putamen and nuclei accumbens in both stages: seizure and postseizure and no changes in the olfactory tubercle. CPA administration (2 mg/kg) originated a rise of 16% in nuclei accumbens but no change in the other two regions. When CPA was injected 30 minutes before MP, an increase (18 to 45%) in caudate-putamen and nuclei accumbens at seizure and postseizure stages was observed. Saturation results, in striatal membrane fraction, indicate that receptor sites increased their maximal binding capacity (Bmax) while the apparent dissociation constant (Kd) remained unchanged. These results suggest the involvement of the adenosine A_2A receptors in convulsant activity and that CPA administration at the dose selected brings about a rise in neuronal excitability in this area.     

Ligand Binding to CNS Muscarinic Receptor Is Transiently Modified by Convulsant 3-Mercaptopropionic Acid Administration

The administration of convulsant drugs has proven a powerful tool to study experimental epilepsy. We have already reported that the administration of convulsant 3-mercaptopropionic acid (mp) at 150 mg/kg enhances binding affinity of muscarinic antagonist [³H]quinuclidinyl benzilate ([³H]QNB) to certain rat CNS membranes during seizure and postseizure without affecting site number. Results obtained with a 100-mg/kg dose of mp have shown reversible increases in [³H]QNB binding to cerebellum and hippocampus, whereas a delayed response has been found in striatum. Neither a subconvulsant dose nor in vitro addition modifies binding. In order to evaluate preseizure, seizure as well as early (30 min) and late (24 h) postseizure stages, we employed a 50 mg/kg dose and tested [³H]QNB binding to CNS membranes. Changes in binding were as follows (in %): in cerebellum, +37, +86, and +40 at preseizure, seizure and early postseizure stages, respectively, but there was a decrease at late postseizure; in hippocampus, +27 at pre- and seizure stages, but a decrease at early and late postseizure. No changes were found in striatum or cerebral cortex membranes at any stage studied. Saturation curves analysed by Scatchard plots indicated that changes in [³H]QNB binding to cerebellar membranes are attributable to an increase in ligand affinity at seizure, followed by a decrease in binding site number at postseizure. A similar profile was observed for hippocampus except that the decrease in binding site number, though lower than at postseizure, was already evident at seizure stage. Results confirm a region-specific response to the convulsant and transient changes provide an example of neuronal plasticity.     

NMDA receptor function is enhanced in the hippocampus of aged rats

The density and functional activity of theN-methyl-D-aspartate (NMDA)-sensitive glutamate receptor was examined in various brain areas of 3-, 18- and 24-month-old rats. The total numbers of binding sites for the NMDA receptor antagonists [³H]CGP 39653 and [³H]MK 801 binding sites were decreased in the hippocampus, cerebral cortex and striatum of 18- and 24-month-old rats, relative to 3-month-old animals. In the hippocampus of 18-month-old rats, the reduced number of NMDA receptors was associated with an increased sensitivity of [³H]MK 801 binding to the stimulatory action of glycine and glutamate. Thus, 10 M glycine and 10 M glutamate increased [³H]MK 801 binding in the hippocampus of 18-month-old rats by 75 and 160%, respectively; in 3-month-old animals, the same concentration of these amino acids increased binding by 37 and 95%, respectively. The sensitivity of [³H]MK 801 binding to glycine and glutamate was not increased in the cerebral cortex and striatum of aged rats. Moreover, an increased efficacy of glycine and glutamate in stimulating the binding of [³H]MK 801 in the hippocampus was no longer apparent in the 24-month-old rats. The increased sensitivity of [³H]MK 801 binding to glycine and glutamate in the hippocampus of 18-month-old rats may reflect an increase in NMDA receptor activity to compensate for the decrease in receptor number.     

Seizure Activity Produces Differential Changes in Adenosine A1 Receptors within Rat Hippocampus

Specific ligand binding to rat hippocampal adenosine A₁ receptor after administration of the convulsant drug 3-mercaptopropionic acid (MP) was studied by means of a quantitative autoradiographic method. 2-Chloro-N6-[cyclopentyl-2,3,4,5-³H adenosine] ([³H]CCPA), a potent and selective A₁ receptor ligand, was selected for binding studies. MP administration (150 mg/kg, i.p.), at seizure, caused significant increases in the following CA1 layers: pyramidal (45%), radiatum (18%) and lacunosum molecular (35%); in CA2 area, a significant decrease in stratum oriens (36%) and an increase in stratum radiatum (14%) and lacunosum molecular (33%) layers was observed. In CA3 area a rise in pyramidal (40%) and radiatum layers (26%), as well as in hillus (97%) was found. At postseizure, changes were restricted to CA1, CA2 and CA3 pyramidal layers and to CA1 lacunosum molecular layer, with increases ranging from 22 to 50%. These results show that [³H]CCPA binding is modified diversely in intrahippocampal layers and areas, thus indicating their dissimilar role in seizure activity.     

Pharmacological characterization of the N-methyl-d-aspartate (NMDA) receptor-channel in rodent and dog brain and rat spinal cord using [3H]MK-801 binding

The biochemical and pharmacological properties of [³H]MK-801 binding to the N-methyl-d-aspartate (NMDA) receptor-channel in homogenates of mouse, guinea pig and dog brain, dog cerebral cortex and rat spinal cord were determined using radioligand binding techniques. Specific [³H]MK-801 binding increased linearily with increasing tissue concentration and in general represented 80–93% of the total binding at 6–8 nM radioligand concentration. [³H]MK-801 interacted with brain and spinal homogenates with high affinity. The dissociation constants (K _d ) for all tissues studied were similar ranging between 7.9 and 11.9 nM, whereas the maximum number of binding sites (B_max) showed a wide, tissue-dependent range (0.1–6.75 pmol/mg protein). The rank order of tissue enrichment was found to be as follows: mouse brain>>dog cerebral cortex>>dog brain>> guinea pig brain>>rat spinal cord. Specific [³H]MK-801 binding in rodent and dog brain, dog cerebral cortex and rat spinal cord exhibited a similar pharmacological profile 9correlation coefficients=0.93–0.99). The rank order of potency of unlabelled compounds competing for [³H]MK-801 binding was: (+)MK-801>(–)MK-801>phencyclidine>(–)cyclazocine>>(+)cyclazocine ketamine>(+)N-allyl-N-normetazocine>(–)N-allyl-N-normetazocine>(–)pentazocine>(+)pentazocine. NMDA, Kainate, quisqualate and several other compounds failed to inhibit [³H]MK-801 binding at 100 M. In modulation studies conducted on extensively washed dog cortex membranes, Mg²⁺ ions stimulated [³H]MK-801 binding at 10 M-1 mM (EC₅₀=91.5 M) and then inhibited the binding from 1 mM to 10 mM (IC₅₀=3.1 mM). Glycine stimulated [³H]MK-801 binding at 30 nM-1 mM (EC₅₀=256 nM). In contrast, Zn²⁺ ions inhibited the binding of [³H]MK-801 binding site exhibited similar pharmacological and biochemical properties. These data appear to suggest that the pharmacological profile of the NMDA-receptor-channel is species and tissue independent.     

Up-regulation of Brain N-Methyl- -aspartate Receptors Following Multiple Intracerebro- ventricular Injections of [ -Pen, -Pen] Enkephalin and [ -Ala, Glu]deltorphin II in Mice

Bhargava, H. N., S. Kumar and J. T. Bian. Up-regulation of brain N-methyl- -aspartate receptors following multiple intracerebroventricular injections of [ -Pen², -Pen⁵]enkephalin and [ -Ala², Glu⁴]deltorphin II in mice. Peptides 18(10) 1609–1613, 1997.—The effects of chronic administration of [ -Pen², -Pen⁵]enkephalin and [ -Ala², Glu⁴]deltorphin II, the selective agonists of the δ₁- and δ₂-opioid receptors, on the binding of [³H]MK-801, a noncompetitive antagonist of the N-methyl- -aspartate receptor, were determined in several brain regions of the mouse. Male Swiss-Webster mice were injected intracerebroventricularly (i.c.v.) with [ -Pen², -Pen⁵]enkephalin or [ -Ala², Glu⁴]deltorphin II (20 μg/mouse) twice a day for 4 days. Vehicle injected mice served as controls. Previously we have shown that the above treatment results in the development of tolerance to their analgesic activity. The binding of [³H]MK-801 was determined in brain regions (cortex, midbrain, pons and medulla, hippocampus, striatum, hypothalamus and amygdala). At 5 nM concentration, the binding of [³H]MK-801 was increased in cerebral cortex, hippocampus, and pons and medulla of [ -Pen², -Pen⁵]enkephalin treated mice. In [ -Ala², Glu⁴]deltorphin II treated mice, the binding of [³H]MK-801 was increased in cerebral cortex and hippocampus. The changes in the binding were due to increases in the B_max value of [³H]MK-801. It is concluded that tolerance to δ₁- and δ₂-opioid receptor agonists is associated with up-regulation of brain N-methyl- -aspartate receptors, however, some brain areas affected differ with the two treatments. The results are consistent with the recent observation from this laboratory that N-methyl- -aspartate receptors antagonists block tolerance to the analgesic action of δ₁- and δ₂-opioid receptor agonists.     

Autoradiographic Analysis of GABAA Receptors in μ-Opioid Receptor Knockout Mice

Anatomical evidence indicates that γ-aminobutyric acid (GABA)-ergic and opioidergic systems are closely linked and act on the same neurons. However, the regulatory mechanisms between GABAergic and opioidergic system have not been well characterized. In the present study, we investigated whether there are changes in GABA_A receptors in mice lacking μ-opioid receptor gene. The GABA_A receptor binding was carried out by autoradiography using [³H]-muscimol (GABA_A), [³H]-flunitrazepam (FNZ, native type 1 benzodiazepine) and [³⁵S]-t-butylbicyclophosphorothionate (TBPS, binding to GABA_A-gated chloride channels) in brain slices of wild type and μ-opioid receptor knockout mice. The binding of [³H]-FNZ in μ-opioid receptor knockout mice was significantly higher than that of the wild type controls in most of the cortex and hippocampal CA1 and CA2 formations. μ-Opioid receptor knockout mice show significantly lower binding of [³⁵S]-TBPS than that of the wild type mice in few of the cortical areas including ectorhinal cortex layers I, III, and V, but not in the hippocampus. There was no significant difference in binding of [³H]-muscimol between μ-opioid receptor knockout and wild type mice in the cortex and hippocampus. These data indicate that there are specific regional changes in GABA_A receptor binding sites in μ-opioid receptor knockout mice. These data also suggest that there are compensatory up-regulation of benzodiazepine binding site of GABA_A receptors in the cortex and hippocampus and down-regulation of GABA-gated chloride channel binding site of GABA_A receptors in the cortex of the μ-opioid receptor knockout mice.     

An electrophilic affinity ligand based on (+)-MK801 distinguishes PCP site 1 from PCP site 2

The electrophilic affinity ligand, (+)-3-isothiocyanato-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine hydrochloride {(+)-MK801-NCS} was characterized for its ability to acylate phencyclidine (PCP) and sigma binding sites in vivo. Initial studies, conducted with mouse brain membranes, characterized the binding sites labeled by [³H]1-[1-(2-thienyl)cyclohexyl]piperidine ([³H]TCP). The Kd values of [³H]TCP for PCP site 1 (MK801-sensitive) and PCP site 2 (MK801-insensitive) were 12 nM and 68 nM, with Bmax values of 1442 and 734 fmol/mg protein, respectively. Mice were sacrificed 18–24 hours following intracerebroventricular administration of the acylator. The administration of (+)-MK801-NCS increased [³H]TCP binding to site 2, but not to site. 1. Although (+)-MK801-NCS decreased [³H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d; cbcyclohepten-5,10-imine maleate ([³H](+)-MK801) binding to site 1, it had no effect on [³H]TCP binding to site 1. Viewed collectively with other published data, these data support the hypothesis that PCP sites 1 and 2 are distinct binding sites, and that [³H]TCP and [³H](+)-MK801 label different domains of the PCP binding site associated with the NMDA receptor.Abbreviations ((+)-MK801) (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine - ((+)-MK801-NCS) (+)-3-isothiocyanato-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine hydrochloride - (PCP) 1-(1-phencyclohexyl)piperidine - (TCP) 1-{1-(2-thienyl)cyclohexyl}piperidine - (DTG) (2-(tllyl)guanidine - (metaphit) (1-(1-(3-isothiocyanatophenyl)-cyclohexyl)piperidine) - (NMDA) N-methyl-D-aspartate - (HEPPSO) (N-[2-hydroxyethyl]piperazine-N-[2-hydroxypropanesulfoni c acid] - ((+)-MK801-NCS) (+)-5-methyl(3-isothiocyanatophenyl)-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine - (NMDA) N-methyl-D-aspartate Address reprint requests to Dr. Rothman, Phone (410)550-1487.FAX 410-550-2997     

Serotonin Modulation of Low-Affinity Ouabain Binding in Rat Brain Determined by Quantitative Autoradiography

Previous results showed that Na⁺/K⁺-ATPase may have a functional relationship with the neurotransmitter serotonin which activates the glial sodium pump in the rat brain. Both the reaction rate (V) of Na⁺/K⁺-ATPase activity and [³H]ouabain binding were significantly increased in the presence of serotonin. It is not known, however, which isoform is involved in the Na⁺/K⁺-ATPase response to serotonin and its regional distribution. Quantitative autoradiography of [³H]ouabain binding to rat brain slices was employed at different [³H]ouabain concentrations in order to gain information on both the distribution and the possible isoform involved. The results showed that 1500 nM [³H]ouabain binding was sensitive to serotonin 10^–3 M and significantly increased in the following brain regions: frontal cortex, areas CA1, CA2, and CA3 of the hippocampus, presubiculum, zona incerta, caudate putamen and the amygdaloid area, confirming and extending previous results. An effect of serotonin on brain but not kidney tissue at high, 1500 nM, and the lack of effect at low, 50 nM [³H]ouabain concentrations, strongly suggests the participation of the 2 isoform in the response of the pump to the neurotransmitter. Glial cells showed stimulation of ouabain binding by serotonin at ouabain concentrations above 350 nM. The present results open interesting questions related to the brain regions involved and the K⁺ handling by the glial 2 isoform of the pump.     

阻断NMDA受体可增强皮质酮对海马脑源性神经营养因子表达的抑制:cAMP反应元件结合蛋白的作用

高浓度的皮质酮可引起海马形态与功能的损伤,其中脑源性神经营养因子(brain-derived neurotrophic factor,BDNF) 表达的改变在海马形态与功能损伤中扮演重要角色。本实验的目的是观察单次皮下注射皮质酮后海马内BDNF-mRNA、前 体蛋白及成熟型蛋白表达的改变,并观察N-甲基-D-天冬氨酸(N-methyl-D-aspartate NMDA)受体阻滞剂MK801对皮质酮 作用的影响。实验结果显示,单次皮下注射皮质酮2 mg／kg,3 h后海马内BDNF mRNA、前体蛋白及成熟型蛋白的表达 均降低;MK801(0．1 mg／kg)对皮质酮的这一作用有增强效果。单独给予皮质酮或注射MK801 30 min后再给予皮质酮, 均能明显降低海马中cAMP反应元件结合蛋白(cAMP response element binding protein,CREB)的磷酸化水平,MK801与 皮质酮联用时CREB的磷酸化水平降低更为显著(与单独给予皮质酮相比,P<0．05)。实验结果提示,CREB磷酸化水平降 低可能是皮质酮引起海马BDNF表达减少的重要中间环节,阻断NMDA受体可加强皮质酮降低BDNF表达的效应。     

Differential alterations in ATP-supported calcium transport activities of sarcoplasmic reticulum and sarcolemma of aging myocardium

Two membrane fractions, one enriched in sarcoplasmic reticulum and the other enriched in sarcolemma, were isolated from the myocardium of young (3–4-months-old) and aged (24–25-months old) rats. ATP-supported Ca²⁺ binding and accumulating activities as well as (Mg²⁺ + Ca²⁺)-ATPase activities of these membrane fractions were studied in an effort to determine the influence of age on the Ca²⁺ pump function of the two myocardial membrane systems. Sarcoplasmic reticulum from aged hearts showed significantly reduced (approx. 50%) rates of ATP-supported (oxalate-facilitated) Ca²⁺ accumulation compared to sarcoplasmic reticulum from young hearts; the amount of Ca²⁺ accumulated by this membrane of aged heart at steady state was also lower. On the other hand, sarcolemma from aged hearts displayed 2-fold higher rates of ATP-supported Ca²⁺ accumulation compared to sarcolemma from young hearts; at steady state, sarcolemma from aged hearts accumulated significantly higher amounts of Ca²⁺ than did sarcolemma from young hearts. Similar age-related differences were also observed in the ATP-dependent Ca²⁺ binding activities of the two membranes, determined in the absence of oxalate. The divergent age-associated changes in Ca²⁺ binding and accumulating activities of sarcoplasmic reticulum and sarcolemma were seen at varying Ca²⁺ concentrations (0.24–39.1 μM).With either membrane, kinetic analysis showed 2-fold age-related differences in the V values for ATP-supported Ca²⁺ accumulation (V (nmol Ca²⁺/mg protein per min): sarcoplasmic reticulum — young, 119 ± 8; aged, 59 ± 5; sarcolemma — young, 11 ± 2; aged, 21 ± 3); the concentrations of Ca²⁺ required for half-maximal velocities did not differ significantly with age (K_0.5 for Ca²⁺ (μM): sarcoplasmic reticulum — young, 2.5 ± 0.20; aged, 2.9 ± 0.25; sarcolemma — yount, 2.7 ± 0.25; aged, 3.2 ± 0.30). Kinetic parameters of ATP-dependent Ca²⁺ binding also indicated that the velocity of Ca²⁺ binding but not the concentration of Ca²⁺ required for half-maximal binding was altered due to aging. At identical Ca²⁺ concentrations, the combined Ca²⁺ accumulating activity of sarcoplasmic reticulum and sarcolemma from aged hearts was significantly lower (38–47%) than the combined Ca²⁺ accumulating activity of the two membranes from young hearts. No significant age-related differences were observed in the ATP-independent (passive) Ca²⁺ binding (or accumulation) by sarcoplasmic reticulum and sarcolemma, the (Mg²⁺ + Ca²⁺)-ATPase activities of these membranes, their polypeptide composition or relative purity. These results indicate that differential alterations occur in the ATP-supported Ca²⁺ pump activities of sarcoplasmic reticulum and sarcolemma in aging myocardium and such alterations may be due to age-associated changes in the efficacy of coupling ATP hydrolysis to Ca²⁺ transport. Further, the age-related increment in the Ca²⁺ pump activity of sarcolemma is inadequate to fully compensate for the diminished Ca²⁺ pump activity of sarcoplasmic reticulum. It is, therefore, suggested that deterioration of the Ca²⁺ pump function of sarcoplasmic reticulum may contribute to the increased relaxation time observed in aging heart.     

3-Mercaptopropionic Acid-Induced Repetitive Seizures Increase GluN2A Expression in Rat Hippocampus: A Potential Neuroprotective Role of Cyclopentyladenosine

The N-methyl-d-aspartate receptor (NMDAR) is involved in synaptic plasticity, learning, memory, and neurological diseases like epilepsy and it is the major mediator of excitotoxicity. Functional NMDARs in the mature brain are heteromeric complexes composed of different subunits: GluN1 and GluN2. There are four different GluN2 subunits (A–D) and each of them critically determines the pharmacological and electrophysiological properties of NMDARs. GluN1 is ubiquitously expressed in the central nervous system while the highest GluN2A expression is in the hippocampus. Adenosine, an endogenous anticonvulsant, is a neuromodulator with a critical role in the regulation of neuronal activity, mediating its effect on specific receptors, among which adenosine A₁ receptor is highly expressed in the hippocampus. In the present work hippocampal GluN2A expression after the convulsant drug 3-mercaptopropionic acid (MP) induced seizures and the effect of cyclopentyladenosine (CPA) given alone or prior to MP (CPA + MP) in an acute or repetitive experimental model was studied. CPA administered to rats for one or 4 days increases seizure threshold induced by MP. After one administration of MP, no significant difference in GluN2A expression was observed in CPA and CPA + MP by Western blot, although immunohistochemistry revealed an increase in CA2/3 area. However, repetitive MP administration during 4 days showed a significant increase of GluN2A expression, and the repetitive administration of CPA 30 min prior to MP caused a significant decrease of GluN2A expression with respect to MP treatment, returning to control levels. These results show that GluN2A subunit is involved in repetitive MP-induced seizures, while CPA administration displays a protective effect against it.     

Upregulation of NMDA Receptors in Hippocampus and Cortex in the Pentylenetetrazol-Induced “Kindling” Model of Epilepsy

Kindling is a phenomenon of epileptogenesis, which has been widely used as an experimental model of temporal lobe epilepsy. At the present work we investigated the contribution of NMDA receptors in the Pentylenetetrazol-induced kindling model in the mouse brain, by using quantitative autoradiography and the radioactive ligands [³H]MK801 and [³H]L-glutamate (NMDA-sensitive component). One week after establishment of kindling, a small but significant increase in [³H]MK801 as well as NMDA-sensitive [³H]glutamate binding was seen, being restricted to the molecular layer (ML) of the dentate gyrus (DG) and the CA3 region of the hippocampus. These binding augmentations persisted one month after establishment of kindling. A significant increase of NMDA receptor binding was also observed in the cortex-somatosensory and temporal one week after acquisition of the kindled state. The upregulation of NMDA receptors seen in DG and CA3 region of the hippocampus could be associated with the kindling process of this model especially with its maintenance phase, since it persists at long term, is area-specific and consistent with electrophysiological data. The increase of NMDA receptors seen in the cortex of the kindled animals could underlie the hyperexcitability detected by electrophysiological studies in this area.     

The fate of carbon in pulse-labelled crops of barley and wheat

Wheat (cv. Gutha) and barley (cv. O'Connor) were grown as field crops on a shallow duplex soil (sand over clay) in Western Australia with their root systems contained within pvc columns. At four stages during growth, the shoots were pulse-labelled for 1.5h with¹⁴CO₂; immediately prior to labelling, the soil was isolated from the shoot atmosphere by pvc sheets. After labelling, the soil atmosphere was pumped through NaOH to trap respired CO₂ and after 2.5, 5, 7.5 and 24 h from the start of labelling, columns were destructively sampled to recover¹⁴C from the roots, soil and shoot.Both species showed similar patterns of¹⁴C distribution and changes in distribution through the growing season. During early tillering, 15–25% of the¹⁴C recovered after 24 h had been respired by the roots and rhizosphere, 17–27% was retained in the roots, 0.4–1.8% was recovered as water-soluble¹⁴C in the soil and the remainder (45–67%) was present in the shoot. These percentages changed during growth so that during grain filling only 2–3% of the¹⁴C recovered after 24 h was as respired CO₂, 2–6% was in the roots, 0.2% was in the soil and over 90% was in the shoot.The distribution of¹⁴C in components of the soil-plant system changed during the 24 h after labelling with the most rapid changes occurring generally during the first 7.5 h after labelling.Using growth measurements from adjacent plots, the amounts of C added to the soil were estimated for the whole season. Carbon input to the soil was about 48 gC m^–2 for wheat and 58 gC m^–2 for barley; the crops produced total shoot dry matter of 494 (wheat) and 735 g m^–2 (barley). Of the C input to the soil, 27.8% (wheat) and 40.3% (barley) was as respired C and only 3.3 (wheat) and 4.1% (barley) was collected as exudate (water-soluble material).     

Modulation of the levels of NMDA receptor subunit mRNA and the bindings of [3H]MK-801 in rat brain by chronic infusion of subtoxic dose of MK-801

The effects of continuous infusion of NMDA receptor antagonist MK-801 on the modulation of NMDA receptor subunits NR1, NR2A, NR2B, and NR2C were investigated by using in situ hybridization study. Differential assembly of NMDA receptor subunits determines their functional characteristics. Continuous intracerebroventricular (i.c.v.) infusion with MK-801 (1 pmol/10 l/h) for 7 days resulted in significant modulations in the NR1, NR2A, and NR2B mRNA levels without producing stereotypic motor syndromes. The levels of NR1 mRNA were significantly increased (9-20%) in the cerebral cortex, striatum, septum, and CA1 of hippocampus in MK-801-infused rats. The levels of NR2A mRNA were significantly decreased (11-16%) in the CA3 and dentate gyrus of hippocampus in MK-801-infused rats. In contrast to NR2A, NR2B subunit mRNA levels were increased (10-14%) in the cerebral cortex, caudate putamen, and thalamus. However, no changes of NR2C subunits in cerebellar granule layer were observed. Using quantitative ligand autoradiography, the binding of NMDA receptor ligand [³H]MK-801 was increased (12-25%) significantly in almost all brain regions except in the thalamus and cerebellum after 7 days infusion with MK-801. These results suggest that region-specific changes of NMDA receptor subunit mRNA and [³H]MK-801 binding are involved in the MK-801-infused adult rats.     

Affinities of muscarinic drugs for [3H]N-methylscopolamine (NMS) and [3H]oxotremorine (OXO) binding to a mixture of M1−M4 muscarinic receptors: Use of NMS/OXO-M ratios to group compounds into potential agonist,partial agonist,and antagonist classes

The relative affinities of various muscarinic drugs in the antagonist ([³H]N-methyl scopolamine ([³H]NMS)) and agonist ([³H]Oxotremorine-m ([³H]OXO-M)) binding assays using a mixture of tissues containing M₁–M₄ receptor subtypes have been determined. [³H]NMS bound with high affinity (K_d=25±5.9 pM; n=3) and to a high density (B_max=11.8±0.025 nmol/g wet weight) of muscarinic receptors. [³H]OXO-M appeared to bind to two binding sites with differing affinities (K_d1=2.5±0.1 nM; K_d2=9.0±4.9 M; n=4) and to a different population of binding sites (B_max1=5.0±0.26 nmol/g wet weight; B_max2=130±60 nmol/g wet weight). Well known antagonists exhibited high affinity for [³H]NMS binding but a lower affinity for [³H]OXO-M binding. The opposite was true for acetylcholine and other known agonists. However, pilocarpine and McN-A-343 had similar affinities for sites labeled by both radioligands. Using the ratios of antagonist-to-agonist binding affinities, it was possible to group compounds into apparently distinct full agonist (ratios of 180–665; e.g. carbachol, muscarine, OXO-M, OXO-S and arecoline), partial agonist (ratios of 14–132; e.g. McN-A-343, pilocarpine, aceclidine, bethanechol, OXA-22 and acetylcholine) and antagonist (ratios of 0.22–1.9; e.g. atropine, NMS, pirenzepine, methoctramine, 4-DAMP and p-fluorohexahydrosialo-difenidol) classes. These data suggest that the NMS/OXO-M affinity ratios using a mixture of M₁–M₄ muscarinic receptors may be a useful way to screen and group a large number of compounds into apparent agonist, partial agonist, and antagonist classes of cholinergic agents.     

MK801对大鼠脑缺血再灌注后海马CA1区c-fos基因表达及神经元凋亡的影响

目的:研究MK801对大鼠脑缺血再灌注后海马CA1区c-fos基因表达及神经元凋亡的影响。方法:将大鼠随机分为正常对照组、脑缺血再灌注模型组和脑缺血用药后再灌注组,用药组在再灌注前经腹腔注射MK801(0.5mg/kg);分别于再灌注后2、6、24和48 h,采用免疫组化法和Western印迹观察海马CA1区c-fos基因的表达情况。结果:c-fos基因的表达在脑缺血再灌注后2 h即开始增强,至再灌注后6 h达到高峰,24 h表达降低,至48 h又至高峰,但较再灌注6 h后较低。用药组海马CA1区c-fos基因的表达均较模型组的相应时间段降低,具有显著性差异(P<0.01)。结论:海马CA1区c-fos基因在脑缺血再灌注后表达升高,MK801可降低脑缺血再灌注后c-fos基因的表达,对缺血再灌注后的脑组织具有保护作用。     

Effect of L-arginine on Na+,K+-ATPase activity in rat aorta endothelium

The effect of L-arginine on the Na⁺,K⁺-ATPase activity in rat aorta endothelium was studied at its physiological concentrations in the range of 10^–6-10^–3 M. The enzyme activity was 35.5% increased by low concentrations of L-arginine (10^–5 M) and its activity was 32.3-37.1% decreased at the L-arginine concentrations of 10^–4-10^–3 M. A similar inhibition (by 34.5-42.8%) was also found in the presence of a NO-donor nitroglycerol (10^–4-10^–3 M). An optical isomer of L-arginine, D-arginine, at the concentrations of 10^–5 M also increased the enzyme activity by 37.1%, but its inhibiting effect was much less pronounced and was 15.7% at the D-arginine concentration of 10^–3 M. An inhibitor of NO-synthase, L-NAME (N^G-nitroarginine, methyl ester), failed to inhibit Na⁺,K⁺-ATPase. However, the presence of L-NAME abolished the inhibition of Na⁺,K⁺-ATPase by high concentrations of L-arginine. Thus, the effect of L-arginine on the endothelial Na⁺-pump depended on its concentration, and it is suggested that the enzyme inhibition by high concentrations of L-arginine should be associated with activation of the endogenous synthesis of NO.