Brain Homogenate Preparations Research Articles

Protective effects of Buxus hyrcana against memory impairment and oxidative stress in a pentylenetetrazole-kindled epileptic rat model

Pentylenetetrazole (PTZ) is a chemical substance which is largely used for the induction of seizure and epilepsy in the animal model, and it can also, disrupts free radicals balance and cause oxidative stress in the body with a negative impact on memory and learning. In this study, the medicinal plant Buxus hyrcana, was used to evaluate its effect on oxidative stress and memory deficit caused by PTZ in the rats. Twenty-four rats were randomly allocated to 4 groups: control negative under treatment with PTZ (sub-threshold dose 35 mg/kg for one month), control positive under treatment with phenobarbital (PB-30 mg/kg), and two PTZ groups under treatment with B. hyrcana extract (BHE-300, and -600 mg/kg). Standard passive avoidance test was used to evaluate retention and recall (evaluation of memory and learning). After behavioral evaluation, rats were anesthetized, brains were removed, and following preparation of brain homogenates, oxidative stress was evaluated using specified methods. Step-through latency in passive avoidance test in the BHE receiving group was greater than PTZ group (p<0.001). In addition, brain lipid peroxidation malondialdehyde (MDA) and nitric oxide (NO) level in BHE-treated group was significantly lower (p<0.001) and the reduced glutathione (GSH) activity levels were significantly greater versus PTZ group (p<0.001). Our results showed that BHE could prevent memory deficit and ameliorate oxidative stress in the PTZ-kindled rats.

Protective effects of Buxus hyrcana against memory impairment and oxidative stress in a pentylenetetrazole-kindled epileptic rat model

Pentylenetetrazole (PTZ) is a chemical substance which is largely used for the induction of seizure and epilepsy in the animal model, and it can also, disrupts free radicals balance and cause oxidative stress in the body with a negative impact on memory and learning. In this study, the medicinal plant Buxus hyrcana, was used to evaluate its effect on oxidative stress and memory deficit caused by PTZ in the rats. Twenty-four rats were randomly allocated to 4 groups: control negative under treatment with PTZ (sub-threshold dose 35 mg/kg for one month), control positive under treatment with phenobarbital (PB-30 mg/kg), and two PTZ groups under treatment with B. hyrcana extract (BHE-300, and -600 mg/kg). Standard passive avoidance test was used to evaluate retention and recall (evaluation of memory and learning). After behavioral evaluation, rats were anesthetized, brains were removed, and following preparation of brain homogenates, oxidative stress was evaluated using specified methods. Step-through latency in passive avoidance test in the BHE receiving group was greater than PTZ group (p<0.001). In addition, brain lipid peroxidation malondialdehyde (MDA) and nitric oxide (NO) level in BHE-treated group was significantly lower (p<0.001) and the reduced glutathione (GSH) activity levels were significantly greater versus PTZ group (p<0.001). Our results showed that BHE could prevent memory deficit and ameliorate oxidative stress in the PTZ-kindled rats.

Protective effects of Buxus hyrcana against memory impairment and oxidative stress in a pentylenetetrazole-kindled epileptic rat model

Pentylenetetrazole (PTZ) is a chemical substance which is largely used for the induction of seizure and epilepsy in the animal model, and it can also, disrupts free radicals balance and cause oxidative stress in the body with a negative impact on memory and learning. In this study, the medicinal plant Buxus hyrcana, was used to evaluate its effect on oxidative stress and memory deficit caused by PTZ in the rats. Twenty-four rats were randomly allocated to 4 groups: control negative under treatment with PTZ (sub-threshold dose 35 mg/kg for one month), control positive under treatment with phenobarbital (PB-30 mg/kg), and two PTZ groups under treatment with B. hyrcana extract (BHE-300, and -600 mg/kg). Standard passive avoidance test was used to evaluate retention and recall (evaluation of memory and learning). After behavioral evaluation, rats were anesthetized, brains were removed, and following preparation of brain homogenates, oxidative stress was evaluated using specified methods. Step-through latency in passive avoidance test in the BHE receiving group was greater than PTZ group (p<0.001). In addition, brain lipid peroxidation malondialdehyde (MDA) and nitric oxide (NO) level in BHE-treated group was significantly lower (p<0.001) and the reduced glutathione (GSH) activity levels were significantly greater versus PTZ group (p<0.001). Our results showed that BHE could prevent memory deficit and ameliorate oxidative stress in the PTZ-kindled rats.

The anxiolytic and antidepressant effect of Buxus hyrcana in the pentylenetetrazole kindled rat

Pentylenetetrazole (PTZ) is a chemical substance which largely used for induction of seizure and epilepsy in the animal model, and it can also, disrupts free radicals balance and causes oxidative stress in the body with a negative impact on behavioral statuses like anxiety and depression. In this study, the medicinal plant Buxus hyrcana, was used to evaluate its effect on oxidative stress, anxiety and depression caused by PTZ in the rat. Twenty-four male rats were randomly allocated to 4 groups: control negative under treatment with PTZ (sub-threshold dose 35 mg/kg for one month), control positive under treatment with phenobarbital (PB-30 mg/kg), and two PTZ groups under treatment with B. hyrcana extract (BHE-300, and -600 mg/kg). For anxiety parameters, the elevated plus maze (EPM) was used. The forced swim test (FST) and rotarod test were employed to assess the antidepressant and balance potential, respectively. After behavioral evaluation, rats were anesthetized, brains were removed, and following preparation of brain homogenates, oxidative stress was evaluated using specified methods. BHE administered at the doses of 300, and 600 mg/kg, reduced immobility time in the FST exerting antidepressant-like activity. In the EPM test, BHE at the same doses, produced the anxiolytic-like effect. Also, the rats which received BHE had a significant improvement in rotarod test in contrast to control groups. In addition, brain catalase activity and superoxide dismutase level were significantly greater versus PTZ group BHE-300 treated PTZ group was significantly lower and. BHE could prevent anxiety and depression and ameliorate oxidative stress in PTZ-kindled rats.

Effects of Tanacetum polycephalum on passive avoidance learning and oxidative stress in epileptic model of memory impairment in the male Wistar rats

In the present study, the medicinal plant Tanacetum polycephalum, was used to evaluate its effect on oxidative stress and memory deficit caused by PTZ in the rats. Twenty-four rats were randomly categorized into four groups: control negative under treatment with PTZ (sub-threshold dose 35 mg kg−1 for 1 month), control positive under treatment with phenobarbital (PB 30 mg kg−1), and two PTZ groups under treatment with T. polycephalum extract (TPE 300, and 600 mg kg−1). Standard passive avoidance test was used to evaluate memory and learning. After behavioral evaluation, the rats were anesthetized and their brains were removed; following the preparation of brain homogenates, oxidative stress parameters were evaluated using specified methods. It was found that step-through latency in passive avoidance test in PTZ group receiving TPE was greater than that of PTZ group. In addition, brain lipid peroxidation (malondialdehyde) and nitric oxide level in TPE-treated group was significantly lower and glutathione activity and superoxide dismutase level were significantly greater in comparison to PTZ group. Our results showed that TPE could prevent memory deficit and ameliorate hippocampal oxidative stress in PTZ-kindled rats.

Preparation of Synaptoneurosomes for the Study of Glutamate Receptor Function.

The use of synaptoneurosomes (SN) enables the detection of synaptic activity including the assessment of glutamate receptor function. SN are normally prepared by filtration and centrifugation methods. Here we review the preparation of SN by Percoll density gradient methodology for downstream applications that assesses glutamate receptor function such as measuring de novo protein synthesis. Major procedural steps include preparation of discontinuous Percoll-sucrose density gradients, collection of brain tissue, preparation of brain homogenates, isolation of synaptoneurosome bands from the discontinuous Percoll-sucrose gradients, and radiolabeling SN proteins. De novo protein synthesis can be reproducibly measured in SN prepared by this method.

Controversial alkoxyl and peroxyl radical scavenging activity of the tryptophan metabolite 3-hydroxy-anthranilic acid.

3-Hydroxy-anthranilic acid (3-OHAA), a tryptophan metabolite produced in the kynurenine pathway, is an efficient antioxidant towards peroxyl radicals (ROO) derived from the AAPH (2,2'-azobis(2-amidinopropane) dihydrochloride) thermolysis. However, self-reactions of ROO can give rise to alkoxyl radicals (RO), which could strongly affect the fate of scavenging reactions. In the present work, we studied the influence of RO in the scavenging activity of 3-OHAA in three different systems: i) Monitoring of the direct reaction between 3-OHAA and AAPH-derived free radicals (kinetic studies); ii) Evaluation of the protective effect of 3-OHAA on the AAPH-induced consumption of fluorescein; and, iii) Inhibition, given by 3-OHAA, of the AAPH-initiated lipid peroxidation of both, rat brain synaptosomes and homogenate preparations (assessed by chemiluminescence). For such purposes, the fraction of free radicals (f) trapped per 3-OHAA molecule was determined in each system. Kinetic results show that the oxidation of 3-OHAA follows a process dominated by ROO with a zero order kinetic limit in 3-OHAA, and a fraction (fri) equal to 0.88. From the induction times, elicited by 3-OHAA in the kinetic profiles of fluorescein consumption, a fraction (fT) of 0.28 was determined. 3-OHAA also generated induction times in the kinetic profiles of light emission during the AAPH-initiated lipid peroxidation of rat brain synaptosomes and homogenates. From such induction times, fractions of 0.61 and 0.63 were determined for rat brain synaptosomes (fsyn) and homogenates (fhom), respectively. These results show that during the incubation of 3-OHAA and AAPH, a low fraction of ROO self-reacts to generate RO. Nevertheless, when 3-OHAA is employed to protect particular targets, such as fluorescein, rat brain synaptosomes and homogenates, reactions of ROO and/or RO should be considered.

Effect of treadmill running on soluble beta-amyloid concentrations in brains of young Tg2576 mice

Background: Alzheimer’s disease and Parkinson’s disease are the two most common neurodegenerative diseases, which are characterized by amyloid-beta (A b) plaques and alpha-synuclein (a-syn) rich Lewy bodies, respectively. An overlap of these two pathologies is found in patients with Lewy Body Dementia (LBD). Postmortem brains of LBD patients display significant synuclein accumulation in dystrophic neurites decorating A b plaques. Furthermore, in vitro studies report co-aggregation of Ab and a-syn, yet the precise role of a-syn in amyloid plaque formation remains elusive. Methods: In order to investigate a putative interaction of a-syn with amyloid plaque formation in vivo, we crossed APPPS1 with (Thy1)-[A30P]a-syn transgenic mice. Immunhistochemical and biochemical analysis revealed that a-syn affects amyloid plaque formation in vivo, whereas overall APP levels remain unaffected by the additional a-syn expression. Results: To further study the effect of a-syn on A b deposition, we performed cerebral stereotactic injections of 1) brain homogenate from aged APPPS1 transgenic mice into APPPS1x[A30P]a-syn transgenic mice and 2) a mixed preparation of brain homogenates from APPPS1 and (Thy1)-[A30P]a-syn transgenic mice into APPPS1 transgenic mice. We observed reduced seeding of Ab plaques under both experimental conditions, implying an inhibitory effect of a-syn on Ab plaque formation. Conclusions: In summary, our data suggest that a-syn plays an important role in Ab deposition. This work deepens our understanding of the complex pathophysiology of LBD and many other, related neurodegenerative diseases.

Leukotriene B4, administered via intracerebroventricular injection, attenuates the antigen-induced asthmatic response in sensitized guinea pigs

BackgroundDespite intensive studies focused on the pathophysiology of asthmatic inflammation, little is known about how cross-talk between neuroendocrine and immune systems regulates the inflammatory response during an asthmatic attack. We recently showed corresponding changes of cytokines and leukotriene B4 (LTB4) in brain and lung tissues of antigen-challenged asthmatic rats. Here, we investigated how LTB4 interacts with the neuroendocrine-immune system in regulating antigen-induced asthmatic responses in sensitized guinea pigs.MethodsOvalbumin-sensitized guinea pigs were challenged by inhalation of antigen. Vehicle, LTB4 or U75302 (a selective LTB4 BLT1 receptor inhibitor) was given via intracerebroventricular injection (i.c.v.) 30 min before challenge. Airway contraction response was evaluated using Penh values before and after antigen challenge. The inflammatory response in lung tissue was evaluated 24 h after challenge. The LTB4 content of lung and brain homogenate preparations was detected by reversed phase high-performance liquid chromatography (RP-HPLC). Plasma levels of adrenocorticotropic hormone (ACTH) and corticosterone (CORT) were measured using ELISA kits.ResultsAntigen challenge impaired pulmonary function and increased inflammatory cell infiltration in lung tissue. These responses could be significantly suppressed by LTB4, 30 ng i.c.v., in ovalbumin-sensitized guinea pigs. LTB4 content of lung and brain homogenates from antigen-challenged guinea pigs was significantly increased. In addition, administration of LTB4 via i.c.v. markedly increased CORT and ACTH level in plasma before antigen challenge, and there were further increases in CORT and ACTH levels in plasma after antigen challenge. U75302, 100 ng i.c.v., completely blocked the effects of LTB4. In addition, U75302, 100 ng via i.c.v. injection, markedly decreased LTB4 content in lung homogenates, but not in brain homogenates.ConclusionsIncreased LTB4 levels in brain during asthmatic attacks down-regulates airway contraction response and inflammation through the BLT1 receptor. Stimulation of the hypothalamic-pituitary-adrenal axis by LTB4 may result in an increase in systemic glucocorticoids which, in turn, would feed back to suppress the asthmatic response.

The β‐amyloid protein of Alzheimer’s disease binds to membrane lipids but does not bind to the α7 nicotinic acetylcholine receptor

Accumulation of the amyloid protein (Abeta) in the brain is an important step in the pathogenesis of Alzheimer's disease. However, the mechanism by which Abeta exerts its neurotoxic effect is largely unknown. It has been suggested that the peptide can bind to the alpha7 nicotinic acetylcholine receptor (alpha7nAChR). In this study, we examined the binding of Abeta1-42 to endogenous and recombinantly expressed alpha7nAChRs. Abeta1-42 did neither inhibit the specific binding of alpha7nAChR ligands to rat brain homogenate or slice preparations, nor did it influence the activity of alpha7nAChRs expressed in Xenopus oocytes. Similarly, Abeta1-42 did not compete for alpha-bungarotoxin-binding sites on SH-SY5Y cells stably expressing alpha7nAChRs. The effect of the Abeta1-42 on tau phosphorylation was also examined. Although Abeta1-42 altered tau phosphorylation in alpha7nAChR-transfected SH-SY5Y cells, the effect of the peptide was unrelated to alpha7nAChR expression or activity. Binding studies using surface plasmon resonance indicated that the majority of the Abeta bound to membrane lipid, rather than to a protein component. Fluorescence anisotropy experiments indicated that Abeta may disrupt membrane lipid structure or fluidity. We conclude that the effects of Abeta are unlikely to be mediated by direct binding to the alpha7nAChR. Instead, we speculate that Abeta may exert its effects by altering the packing of lipids within the plasma membrane, which could, in turn, influence the function of a variety of receptors and channels on the cell surface.

Age-dependent effects of Aroclor 1254 R on calcium uptake by subcellular organelles in selected brain regions of rats

Earlier reports from our laboratory have indicated that polychlorinated biphenyls (PCBs) affect signal transduction mechanisms in brain, including Ca 2+ homeostasis, phosphoinositol hydrolysis, and protein kinase C (PKC) translocation in mature neurons and adult brain homogenate preparations. Present studies were designed to investigate whether there were any brain region-, gender-, or age-dependent effects of PCBs on 45Ca 2+-uptake by two subcellular organelles, microsomes and mitochondria. We have studied in vitro effects of a widely studied commercial PCB mixture, Aroclor 1254 R, on 45Ca 2+-uptake by microsomes and mitochondria in cerebellum, frontal cortex and hippocampus of postnatal day (PND) 7, 21, and 90–120 (adult) male and female Long–Evans (LE)-rats. In general, microsomal and mitochondrial 45Ca 2+-uptake in selected brain regions increased with age; PND 7<PND 21≤adults. Among three brain regions, hippocampus had relatively lower microsomal 45Ca 2+-uptake than cerebellum and frontal cortex throughout the development. Mitochondrial 45Ca 2+-uptake was comparable in three brain regions of PND 7 and adult animals, but in PND 21 rats, the cerebellum had much higher activity than frontal cortex and hippocampus. No gender-related differences were seen in 45Ca 2+-uptake by either microsomes or mitochondria in selected brain regions throughout development. Inhibition of 45Ca 2+-uptake by Aroclor 1254 in a concentration-dependent manner was observed throughout the study. However, the degree of inhibition of microsomal 45Ca 2+-uptake in these brain regions by Aroclor 1254 increased with age, PND 7<PND 21≤adults (IC 50s=21–34, 8–20 and 10–14 μM, respectively). Brain region-specific differential sensitivity to Aroclor 1254 on the inhibition of microsomal 45Ca 2+-uptake was not seen in PND 7 and adult animals but in PND 21 rats, hippocampus was more sensitive than the other selected brain regions. There were no age-, gender- or brain region-specific differential effects of Aroclor 1254 on mitochondrial 45Ca 2+-uptake. These results indicate that a commercial PCB mixture, Aroclor 1254, inhibited 45Ca 2+-uptake by both microsomes and mitochondria uniformly in selected brain regions of males and females during development. However, the inhibition of microsomal 45Ca 2+-uptake by Aroclor 1254 increased with age. The age- and gender-related differential sensitivity to Aroclor 1254 may be attributed to the changes in calcium homeostasis in various brain regions during development.

Assessing the Role of ortho-Substitution on Polychlorinated Biphenyl Binding to Transthyretin, a Thyroxine Transport Protein

ortho-Substituted polychlorinated biphenyls (PCBs) make up a large part of the PCB residue found in the environment and human tissues. Our laboratory as well as others have demonstrated that ortho-substituted congeners exhibit important biological activities by aryl hydrocarbon (Ah) receptor-independent mechanisms, including changes in second messenger systems necessary for normal cell function and growth. Previous structure-activity relationship (SAR) studies on second messengers and transthyretin (TTR; prealbumin) binding focused little attention on the ortho-substituted PCBs. Disruption of thyroid hormone (TH) transport is one potentially important mechanism by which PCBs can alter TH homeostasis. A more systematic study of PCB binding to TTR, a major TH transport protein, was undertaken, in which the role of ortho-substitution was more thoroughly investigated. Results from this study indicated that the ortho-only substituted series showed significant binding activity and the relative affinities were 2,2′,6 > 2,2′ = 2,6 ⪢ 2 = 2,2′,6,6′. As anticipated on the basis of steric considerations, bromine was shown to be more active as an ortho-substituent where the relative affinity of 2,2′-Br was equivalent to 2,2′,6-Cl. The congener patterns (di-meta-substitution in one or both rings) most closely resembling the diiodophenolic ring of thyroxine (T4) showed the highest binding activity. Multiple ortho-substituents were shown to decrease binding activity in such patterns. Congener patterns (single meta-substitution in one or both rings) more closely resembling the monoiodophenolic ring of T3 showed significantly lower binding activity, consistent with the relatively low binding activity of T3 and smaller size of chlorine compared to iodine. The addition of ortho-substitution to such patterns gave variable results depending on the substituent relationship (adjacency or nonadjacency) to the pattern. Some patterns such as 2,2′,4,4′,5,5′ showed good binding activity and represent common congeners in the commercial Aroclor mixtures and in the environment. The binding potencies of ortho-PCBs to TTR may represent a signature SAR that predicts specific biologic/toxic effects. In this regard, the binding potencies were consistent with measured biological activities of these PCBs, including effects on cell dopamine content, Ca2+ homeostasis, and protein kinase C translocation in neuronal cells and brain homogenate preparations.

The hyaluronan receptor for RHAMM in noradrenergic fibers contributes to axon growth capacity of locus coeruleus neurons in an intraocular transplant model

The hyaluronan receptor for hyaluronic acid-mediated motility (RHAMM) plays a role in cell migration and motility in many systems. Recent observations on the involvement of RHAMM in neurite motility in vitro suggest that it might also be important in axon outgrowth in situ. This was addressed directly by investigating both RHAMM expression in the rat CNS and the ability of anti-RHAMM reagents to interfere with tissue growth and axon outgrowth in intraocular brainstem transplants. By western blotting, anti-RHAMM antibody detected a RHAMM isoform of 75 000 mol. wt in both whole brain homogenate and synaptosome preparations, and a 65 000 mol. wt isoform in synaptosomes. Immunofluorescence of adult brain sections revealed RHAMM-like immunoreactivity in varicose fibers that were also positive for the noradrenergic marker dopamine- β-hydroxylase. Not all noradrenergic fibers contained RHAMM, nor was RHAMM detected in other monoaminergic fiber types. Lesions of noradrenergic fiber systems with β-halobenzylamine- N-(2-chloroethyl)- N-ethyl-2-bromobenzylamine (DSP-4) eliminated RHAMM-positive fibers, but noradrenergic axons that sprouted extensively after this treatment were strongly RHAMM-positive. To assess RHAMM's role in fiber outgrowth, fetal brainstem tissue containing noradrenergic neurons was grafted into the anterior chamber of the eye. Treatment of grafts with anti-RHAMM antibody caused significant inhibition of tissue growth and axon outgrowth, as did a peptide corresponding to a hyaluronan binding domain of RHAMM. These agents had no such effects on transplants containing serotonergic and dopaminergic neurons. These results suggest that RHAMM, an extracellular matrix receptor previously shown to contribute to migratory and contact behavior of cells, may also be important in the growth and/or regenerative capacity of central noradrenergic fibers originating from the locus coeruleus.

Peroxidation of brain lipids in vitro: Nitric oxide versus hydroxyl radicals

The pro-oxidant effects of hydroxyl radical (-OH, ferrous ammonium sulfate/Fe 2+) or nitric oxide (NO-, S-nitroso-N-acetylpenicillamine/SNAP) generating compounds were studied in rat brain homogenate preparations. Submicromolar concentrations of Fe 2+, but not SNAP (up to 100 μM), increased the formation of fluorescent products of malondialdehyde in cortical homogenates. In fact, iron-catalyzed brain lipid peroxidation was inhibited by SNAP (100 μM), but not by light-exposed SNAP or its degradation product penicillamine (100 μM). This study provides relevant evidence to suggest that submicromolar concentrations of Fe 2+ can potentiate lipid peroxidation in disrupted brain tissue. NO· released from SNAP did not stimulate, but rather inhibited brain lipid peroxidation. These results support the hypothesis that NO·, as opposed to ·OH radicals, is not a pro-oxidant but rather an antioxidant.

Design of potent and selective inhibitors of myo-inositol 1,4,5-trisphosphate 5-phosphatase.

The interactions of synthetic analogues of D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] with the Ins(1,4,5)P3 receptor in permeabilized SH-SY5Y cells and with two key metabolic enzymes, Ins(1,4,5)P(3)3-kinase from a supernatant preparation of rat brain homogenates and Ins(1,4,5)P(3)5-phosphatase from human erythrocyte ghosts, have been examined. L-chiro-Inositol 2,3,5-trisphosphorothioate [L-chiro-Ins(2,3,5)PS3], which we have previously identified as a partial agonist at the Ins(1,4,5)P3 receptor [Safrany, S. T., Wilcox, R. A., Liu, C., Dubreuil, D., Potter, B. V. L., & Nahorski S. R. (1993) Mol. Pharmacol. 43, 499-503], is identified as the most potent 5-phosphatase inhibitor yet described (inhibiting dephosphorylation of [3H]Ins(1,4,5)P3 with Ki = 230nM). L-chiro-Ins(2,3,5)PS3 was also found to be the most potent small-molecule inhibitor of 3-kinase (Ki = 820 nM). The properties of three novel, potent, and selective inhibitors of 5-phosphatase are described. L-myo-Inositol 1,4,5-trisphosphorothioate inhibited 5-phosphatase with Ki = 430 nM, showing 250-fold selectivity over 3-kinase (Ki = 108 microM); myo-inositol 1,3,5-trisphosphorothioate inhibited 5-phosphatase with 475-fold selectivity over 3-kinase (Ki = 520 nM and 247 microM, respectively). The most potent, selective inhibitor of 5-phosphatase was L-chiro-inositol 1,4,6-trisphosphorothioate [L-chiro-Ins(1,4,6)PS3]. L-chiro-Ins(1,4,6)PS3 inhibited 5-phosphatase with Ki = 300 nM and did not interact with the Ins(1,4,5)P3 receptor or 3-kinase at doses tested. These studies, therefore, identify a highly potent and selective inhibitor of 5-phosphatase, which should be considered the tool of choice when inhibiting this enzyme in a broken cell or cell-free system.

Purification and properties of carnitine acetyltransferase from human liver

Carnitine acetyltransferase was purified from the supernatant obtained after centrifugation of human liver homogenate to a final specific activity of 78.75 unit.mg-1 with acetyl-CoA as a substrate. Human carnitine acetyltransferase is a monomer of 60.5 kDa with maximum activity in the presence of propionyl-CoA and a pH optimum of 8.7. Apparent Km values for acetyl-CoA are three times lower than for decanoyl-CoA. Km values for L-carnitine in the presence of acetyl-CoA are six times lower than in the presence of decanoyl-CoA. Km values for acetylcarnitine are three times lower than for octanoylcarnitine. The polyclonal antibodies against human carnitine acetyltransferase recognize a 60.5-kDa peptide in the purified preparation of human liver and brain homogenates and in immunoblots of mitochondrial and peroxisomal fractions from human liver. Immunoprecipitation and SDS/PAGE analysis of 35S-labelled proteins produced by human fibroblasts indicate that mitochondrial carnitine acetyltransferase is synthesized as a precursor of 65 kDa. We also purified carnitine acetyltransferase from the pellet obtained after centrifugation of liver homogenate. The pellet was extracted by sonication in the presence of 0.5% Tween 20. The chromatographic procedures for the purification and the kinetic, physical and immunological properties of pellet-extracted carnitine acetyltransferase are similar to those of carnitine acetyltransferase purified from the supernatant of human liver homogenate.

Piperazine derivatives including the putative anxiolytic drugs, buspirone and ipsapirone, are agonists at 5-HT1A receptors negatively coupled with adenylate cyclase in hippocampal neurons.

Two putative anxiolytic drugs [ipsapirone (TVXQ 7821) and buspirone], structurally unrelated to benzodiazepines, have negligible ataxic and sedative side effects. These drugs are piperazine analogs which interact at 5-HT1 binding sites. It is demonstrated here that these drugs and two other piperazine derivatives, trifluoromethylphenylpiperazine (TFMPP) and m-chlorophenylpiperazine (mCPP), are agonists at 5-HT1A receptors, a subclass of the 5-HT1 receptor, mediating inhibition of forskolin (100 microM) stimulated adenylate cyclase in particulate fractions of guinea pig hippocampus as well as inhibition of the formation of cyclic AMP promoted by vasoactive intestinal polypeptide (0.1 microM) plus forskolin (1 microM) in mouse hippocampal neurons in primary culture. This study demonstrates that these piperazine based drugs act in both brain homogenate preparations and in intact neurons in a similar manner. The biochemical models described here may aid in the development of even more active drugs in this class.

Effects of quinupramine on the central monoamine uptake systems and involvement of pharmacokinetics in its pharmacological activities.

The effects of a new tricyclic antidepressant drug, quinupramine, on the monoamine uptake of rat brain homogenate preparations were studied in comparison with imipramine. Pharmacokinetic studies on quinupramine and imipramine in plasma and brain were also performed in rats after a single oral administration. Quinupramine had few effects on the noradrenaline and serotonin uptake both in in vitro and ex vivo models. After the administration of [3H]quinupramine, the unchanged drug was estimated as 60-75% of the total radioactivity in the cerebral cortex. Imipramine and desipramine preferentially inhibited the uptake of serotonin and noradrenaline, respectively, in vitro. After the administration, imipramine showed a marked inhibitory effect on noradrenaline uptake. A considerable amount of desipramine but not imipramine could be detected in the brain and plasma after the administration of [3H]imipramine. These results demonstrate that 1) the antidepressant activity of quinupramine cannot be attributed to inhibition of monoamine uptake, 2) unchanged quinupramine penetrates into the CNS and affects some of the processes of neurotransmission and 3) the pharmacological activities of imipramine, when administered orally, may be attributed to desipramine, the metabolite formed.

Effects of Quinupramine on the Central Monoamine Uptake Systems and Involvement of Pharmacokinetics in Its Pharmacological Activities

The effects of a new tricyclic antidepressant drug, quinupramine, on the monoamine uptake of rat brain homogenate preparations were studied in comparison with imipramine. Pharmacokinetic studies on quinupramine and imipramine in plasma and brain were also performed in rats after a single oral administration. Quinupramine had few effects on the noradrenaline and serotonin uptake both in in vitro and ex vivo models. After the administration of [3H]quinupramine, the unchanged drug was estimated as 60-75% of the total radioactivity in the cerebral cortex. Imipramine and desipramine preferentially inhibited the uptake of serotonin and noradrenaline, respectively, in vitro. After the administration, imipramine showed a marked inhibitory effect on noradrenaline uptake. A considerable amount of desipramine but not imipramine could be detected in the brain and plasma after the adminisration of [3H]imipramine. These results demonstrate that 1) the antidepressant activity of quinupramine cannot be attributed to inhibition of monoamine uptake, 2) unchanged quinupramine penetrates into the CNS and affects some of the processes of neurotransmission and 3) the pharmacological activities of imipramine, when administered orally, may be attributed to desipramine, the metabolite formed.

Autoradiographic localization of tryptamine binding sites in the rat and dog central nervous system

Tryptamine, an endogenous trace amine, is currently postulated to be a neuromodulator or neurotransmitter in the mammalian CNS. High-affinity binding sites have been described for tryptamine in rat brain homogenate preparations. The present study further characterizes tryptamine binding throughout the CNS and delineates its distribution using in vitro receptor binding in conjunction with autoradiographic techniques. Saturation studies on 20-micron-thick brain sections suggest a single class of binding sites (Hill coefficient = 0.97 +/- 0.04) with a high affinity (KD = 4.79 +/- 1.55 nM). In competition studies, kynuramine and tetrahydrobetacarboline significantly inhibited H3-tryptamine binding while serotonin, dopamine, and phenylethylamine failed to significantly inhibit it. The most potent inhibitor of H3-tryptamine binding was tryptamine (KI = 4.19 +/- 2.13 nM). In rat brain sections processed for in vitro autoradiography, highest binding occurred in the following limbic structures: the accumbens nucleus, the amygdalohippocampal area, the lateral septal nucleus, the entorhinal cortex, and the anterior olfactory nucleus. At diencephalic levels, the highest binding was observed in the reuniens thalamic nucleus, the paraventricular thalamic nucleus, the medial habenular nucleus, the central medial thalamic nucleus, and the arcuate hypothalamic nucleus. In the midbrain of the rat, binding was most notable in the interpeduncular nucleus, the superficial layer of the superior colliculus, the periaqueductal gray, and the paranigral nucleus. In the lower brain stem of the dog, binding was evident in the external cuneate nucleus, the spinal trigeminal nucleus, and in the region of the solitary nucleus. Binding was also present in both the ventral and dorsal horns of the canine spinal cord.(ABSTRACT TRUNCATED AT 250 WORDS)