Comparison of central and intraesophageal factors between gastroesophageal reflux disease (GERD) patients and those with GERD-related noncardiac chest pain.

Gastroesophageal reflux disease (GERD) causes a wide range of symptoms. Some patients present with typical symptoms such as heartburn and regurgitation and others with atypical symptoms such as chest pain. The mechanism responsible for the varying clinical presentation of GERD is still not fully elucidated. The aim of this study was to prospectively evaluate differences in central and local intraesophageal factors between patients with typical GERD symptoms and those with noncardiac chest pain (NCCP). Patients presenting with typical and atypical symptoms suspicious of GERD underwent upper endoscopy and 24-hour pH monitoring with four sensors, each positioned at a different esophageal level. All patients completed GERD symptom, Hospital Anxiety and Depression Scale, and Symptom Stress Rating questionnaires. From January 2006 to December 2009, 50 patients were recruited, 29 with typical symptoms, and 21 with NCCP. Patients with proven GERD and NCCP had higher proximal extension of acid during reflux episodes than patients with typical symptoms. They were found to be older, had a shorter history of symptom onset, worse anxiety scores, and more endoscopic findings compatible with gastritis. Proximal extension of acid during the reflux episodes in patients with GERD presenting with NCCP may play a role in symptom generation.

Specificity of expression of the muscle and brain dystrophin gene promoters in muscle and brain cells.

The gene that is defective in Duchenne and Becker muscular dystrophies is expressed in the muscle and brain. However, the 5' ends of the 14 kb mRNA in these tissues are derived from two different exons, indicating the involvement of at least two promoters in the regulation of the cell-type and developmental specificities of expression of this gene. In the study presented here, we used the polymerase chain reaction and RNAase protection methods and various cell cultures to investigate the specificities of expression of these promoters. The results indicate a very stringent control of expression of the two promoters. In cloned rat myogenic cells, only the muscle-type dystrophin transcript was detected, and its presence was correlated with the formation of multinucleated fibers. In neuronal cell cultures, the brain-type transcript was detected. However, glial cell cultures expressed the muscle transcript only. Some cell lines derived from brain cells expressed both isoforms.

Histidine-rich glycoprotein inhibits the antiangiogenic effect of thrombospondin-1.

Angiogenesis is critical for the growth and proliferation of tumors as well as for normal development. We now describe a novel role for histidine-rich glycoprotein (HRGP) in the modulation of angiogenesis. HRGP is a plasma protein that circulates in relatively high concentrations (1.5 microM), but has no known function in vivo. We have shown previously that HRGP binds with high affinity to thrombospondin-1 (TSP-1), a homotrimeric glycoprotein that is a potent inhibitor of angiogenesis. The antiangiogenic activity of TSP-1 is mediated by the binding of properdin-like type I repeats to the receptor CD36. We found that binding of HRGP to TSP-1 was similarly mediated by TSP type I repeats. HRGP colocalized with TSP-1 in the stroma of human breast cancer specimens, and this interaction masked the antiangiogenic epitope of TSP-1. In assays performed in vitro of endothelial cell migration and tube formation, and in vivo corneal angiogenesis assays, HRGP inhibited the antiangiogenic effect of TSP-1. These studies suggest that HRGP can modulate the antiangiogenic activity of TSP-1, and identify a potential mechanism of resistance to the antiangiogenic effect of TSP-1.

Activation of cultured vascular endothelial cells by antiphospholipid antibodies.

Circulating antiphospholipid antibodies (aPL) are associated with a syndrome of thrombosis, recurrent fetal loss, and thrombocytopenia. We have demonstrated the activation of cultured human umbilical vein endothelial cells (HUVEC) by IgG from patients with anticardiolipin antibodies (aCL). Incubation of HUVEC for 4 h with purified IgG (100 micrograms/ml) from patients with high-titer aCL induced a 2.3-fold increase in monocyte adhesion over that seen in HUVEC incubated with IgG's from normal subjects. The effect of aCL was not attributable to LPS contamination, Fc receptors, or immune complexes. Monocyte adhesion was not induced when the aCL were added in serum-free media but was restored by the addition of purified beta 2GP1, previously described as a necessary cofactor for aCL reactivity. Purified rabbit polyclonal IgG raised against beta 2GP1 also induced monocyte adhesion when incubated with HUVEC. Preadsorption of patient serum with cardiolipin reduced monocyte adhesion by 60%. Immunofluorescent microscopy demonstrated that endothelial cells incubated with patient IgG expressed cell adhesion molecules, including E-selectin, vascular cell adhesion molecule-1, and intracellular adhesion molecule-1. These data support the hypothesis that aPL activate vascular endothelial cells, thereby leading to a pro-thrombotic state.

Alterations in pp60c-src accompany differentiation of neurons from rat embryo striatum.

Cultured neurons from rat embryo striatum were found to contain two structurally distinct forms of pp60c-src. The 60-kilodalton (kDa) form appeared similar to pp60c-src from cultured rat fibroblasts or astrocytes. The 61-kDa form was specific to neurons and differed in the NH2-terminal 18 kDa of the molecule. In undifferentiated neurons the predominant phosphorylated species of pp60c-src was the fibroblast form. Upon differentiation, a second phosphorylated form of pp60c-src was detected. This form had two or more additional sites of serine phosphorylation within the NH2-terminal 18-kDa region of the molecule, one of which was Ser-12. The specific protein-tyrosine kinase activity of the total pp60c-src population increased 14-fold, as measured by autophosphorylation, or 7-fold, as measured by phosphorylation of an exogenous substrate, as striatal neurons differentiated. This elevation in protein kinase activity occurred without a detectable decrease in Tyr-527 phosphorylation or increase in Tyr-416 phosphorylation. Our results support the idea that the expression of the neuron-specific form of pp60c-src and the increase in specific protein kinase activity may be important for neuronal differentiation.

Enhancement of hormone action by a phorbol ester and anti-tubulin alkaloids involves different mechanisms.

The tumor-promoting phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) enhanced 1-isoproterenol and prostaglandin E1 stimulated cyclic AMP formation in clones of mouse myeloid leukemic cells. The enhancement was found up to 3h after TPA treatment and had disappeared after 24h, indicating its reversibility. The effect of TPA was not inhibited by removal of extracellular Ca2+ or pre-treatment with the calcium ionophore A23187. This enhancement by TPA seems to involve a different pathway than enhancement of response to the same hormones after treatment with the anti-tubulin alkaloids colchicine or vinblastine, since a myeloid leukemic cell mutant clone that was non-responsive to the anti-tubulin alkaloids responded to TPA. Furthermore, combined treatment of colchicine-sensitive cells with TPA and colchicine showed an additive stimulating effect. The enhancement of cell response to hormones by TPA was found in myeloid leukemic cell clones that either were or were not induced to differentiate after treatment with TPA. This suggests that enhancement of the effect of these and possibly other hormones by TPA may be an initial step of TPA action, but that this enhancement is not sufficient to induce the wide repertoire of TPA effects including induction of differentiation.

Role of phospholipase A2 and prostaglandin E in growth and differentiation of myeloid leukemic cells.

Phospholipase A2 activity and prostaglandin E synthesis have been studied in different clones of myeloid leukemic cells, which differ in their competence to be induced to differentiate by the macrophage and granulocyte differentiation-inducing protein or the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA). Clones that could be induced to differentiate by this protein showed a higher basal phospholipase A2 activity than clones that could not be induced to differentiate by this protein inducer. Cell competence to be induced to differentiate by TPA did not show this correlation, and the clone with the least ability to respond to TPA showed the lowest number of binding sites for [20-3H]phorbol 12,13-dibutyrate. Differentiation induced by the protein was accompanied by a 7-14-fold increase in prostaglandin E synthesis, whereas differentiation induced by TPA did not show this increase. Externally added prostaglandin E1 did not induce differentiation but inhibited cell proliferation and the degree of inhibition in the different clones was related to the basal phospholipase A2 activity. The results indicate that increase of prostaglandin E synthesis was not an essential pre-requisite for differentiation, that prostaglandin E seems to be involved in the inhibition of cell proliferation in association with phospholipase A2, and that the differentiation-inducing protein and TPA can induce differentiation by different pathways. The amount of basal phospholipase A2 activity was also related to previously found differences in the ability of the clones to develop desensitization to beta-adrenergic hormones or prostaglandin E1.

Down regulation of enkephalin (delta) receptors. Demonstration in membrane-bound and solubilized receptors.

The pentapeptide leucine enkephalin induced down-regulation of enkephalin receptors in neuroblastoma-glioma NG108-15 hybrid cells in a reversible fashion, whereas the stable enkephalin analogue D-Ala2-Met-enkephalinamide (AMEA), and the potent opiate alkaloid, etorphine, had a prolonged effect. The opiate alkaloid, morphine, which has low affinity to delta-type enkephalin receptors of these cells did not induce down-regulation, whereas AMEA decreased the binding of both opiate agonists and antagonists but had no effect on the binding of the alpha 2-adrenergic ligand, [3H]yohimbine. From several experiments that were designed to remove the tightly bound AMEA, and from experiments with solubilized receptor we ruled out the possibility that the decreased binding capacity of enkephalin-treated cells reflects only receptor masking. The study suggests that down-regulation of enkephalin receptors that may also occur in vivo can account for some of the abnormal physiological responses of subjects treated chromically with opiates. However, since opiates from the morphine type can induce opiate tolerance in vivo, but not down-regulation of enkephalin receptors in the cultured cells, we suggest that down-regulation of delta-type opiate receptors may not be prerequisite for the development of the physiological tolerance/dependence on these alkaloids.

The antiangiogenic effect of thrombospondin-2 is mediated by CD36 and modulated by histidine-rich glycoprotein.

Thrombospondins-1 and -2 (TSP-1, TSP-2) are matricellular glycoproteins with potent antiangiogenic activity. We have previously shown that the antiangiogenic activity of TSP-1 is mediated by the interaction of the type I repeats (TSR) with the receptor CD36, although other domains of TSP-1 have also been implicated. We now show that the antiangiogenic activity of TSP-2, which contains three TSRs but, unlike TSP-1, lacks the capacity to activate TGF-beta, is similarly dependent on CD36. Using the corneal pocket assay we found that TSP-2 did not inhibit bFGF-induced angiogenesis in CD36 null mice. We then demonstrated that (125)[I]-TSP-2 bound to murine macrophages and that binding was diminished by 70% by anti-CD36 antibody or by using cells from CD36 null animals. Solid-phase binding studies revealed that (125)[I]-TSP-2 bound to CD36/glutathione-S-transferase (GST) fusion proteins encoding the region spanning amino acids 93-120, but not amino acids 298-439. This 93-120 amino acid region, previously identified as the TSP-1 binding site, is homologous to domains on other TSP binding proteins, such as LIMP-2 and histidine-rich glycoprotein (HRGP). Finally, we showed with an immunoabsorbent binding assay that TSP-2 bound HRGP with high affinity and that HRGP blocked the antiangiogenic activity of TSP-2, acting like a "decoy" receptor. These data suggest that modulation of the TSR/CD36 system may play an important role in the regulation of the angiogenic "switch," and may provide a target for therapeutic interventions.

Effect of naloxone on the neuropsychiatric symptoms of a woman with partial adrenal 21-hydroxylase deficiency.

A woman with a schizophreniform syndrome, drug-induced dyskinetic movements, and partial adrenocortical 21-hydroxylase deficiency was given short-term treatment with naloxone, which ameliorated the psychiatric symptoms and eliminated the dyskinetic movements.

Selective role of glutathione in protecting human neuronal cells from dopamine-induced apoptosis.

The role of glutathione and other antioxidants in dopamine-induced apoptosis has been analyzed in cultures of the human neuronal cell line NMB. Apoptosis, induced by 0.1-0.3 mM dopamine, was blocked by glutathione in a dose- and time-dependent manner. This was observed by monitoring cell morphology, cell viability, and the release of the cytosolic enzyme lactate dehydrogenase into the culture medium. L-Cysteine and N-acetylcysteine had a similar effect in protecting against dopamine neurotoxicity, but at lower concentrations than glutathione. The dopamine-induced alteration in the cell cycle profile, detected by flow cytometry (FACS), and intranucleosomal DNA fragmentation, were both blocked by glutathione. Treatment of NMB cells with buthionine sulfoximine, an irreversible inhibitor of gamma-glutamylcysteine synthetase, increased the neurotoxic effect of, dopamine, suggesting that endogenous glutathione participates in reducing dopamine neurotoxicity. The relationship between glutathione and dopamine was further investigated by testing the effect of dopamine on the endogenous glutathione level. Dopamine decreased glutathione levels within 16-24 hr; however, this effect was preceded by a transient increase in the level of the tripeptide within the first 0.5-7 hr. Two other types of endogenous antioxidants, (+)-alpha-tocopherol (vitamin E) and ascorbic acid (vitamin C), were tested; vitamin E (at 1-100 microgram/ml) was inactive against dopamine toxicity, whereas vitamin C had no effect at 0.05-0.2 mM, but increased dopamine toxicity at 0.5-2 mM. The results indicate that glutathione has a selective role in protecting human neural cells from the toxic effect of dopamine. This study may contribute, therefore, to a better understanding of the mechanisms underling the excessive loss of dopaminergic neurons in neurodegenerative diseases, such as Parkinsonism, and in the aging process.

Carrier-mediated serotonin release induced by d-fenfluramine: studies with human neuroblastoma cells transfected with a rat serotonin transporter.

The NMB human neuronal cell line, transfected with a newly prepared plasmid expressing rat serotonin transporter (NMB-rSERT), shows specific [3H]5-HT uptake which is blocked by citalopram and fenfluramine (F) stereoisomers with IC50 values (1 nM. 0.5 microM (dF) and and 5 microM (IF), respectively) which are similar to those found in rat brain synaptosomes. d-Fenfluramine (0.5 and 10 microM) also stimulates tritium release from NMB-rSERT cells preloaded with [3H-]-5-HT. The d-fenfluramine-induced [3H-]5-HT release is blocked by 0.3 microM citalopram and is dependent on the density of SERT expressed per cell, but is not affected by removal of Ca++ ions from the incubation medium. Manipulation of the Na+ gradient across the plasma membrane (replacing 60 mM NaCl with an equimolar concentration of KCl or choline) also induced [3H-]5-HT release from NMB-rSERT cells, which was inhibited by 0.3 microM citalopram. These results, together with the finding that NMB-rSERT cells preloaded with 500 nM unlabelled 5-HT take up [3H-]d-fenfluramine, make NMB-rSERT cells a valuable tool for studying the transporter-mediated exchange release induced by amphetamine derivatives.

Dopamine-induced apoptosis in human neuronal cells: inhibition by nucleic acids antisense to the dopamine transporter.

Human neuroblastoma NMB cells take up [3H]dopamine in a selective manner indicating that dopamine transporters are responsible for this uptake. These cells were therefore used as a model to study dopamine neurotoxicity, and to elucidate the role of dopamine transporters in controlling cell death. Treatment with 0.05 0.4 mM dopamine changed cells' morphology within 4 h, accompanied by retraction of processes, shrinkage, apoptosis-like atrophy, accumulation of apoptotic particles, DNA fragmentation and cell death. Cycloheximide inhibited dopamine's effect suggesting that induction of apoptosis by dopamine was dependent upon protein synthesis. Dopamine cytotoxicity, monitored morphologically by flow cytometric analysis, and by lactate dehydrogenase released, was blocked by cocaine but not by the noradrenaline and serotonin uptake blockers desimipramine and imipramine, respectively. Attempting to inhibit dopamine transport and toxicity in a drug-free and highly selective way, three 18-mer dopamine transporter antisense phosphorothioate oligonucleotides (numbers 1, 2 and 3) and a new plasmid vector expressing the entire rat dopamine transporter complementary DNA in the antisense orientation were prepared and tested. Antisense phosphorothioate oligonucleotide 3 inhibited [3H]dopamine uptake in a time- and dose-dependent manner. Likewise, transient transfection of NMB cells with the plasmid expressing dopamine transporter complementary DNA in the antisense orientation partially blocked [3H]dopamine uptake. Antisense phosphorothioate oligonucleotide 3 also decreased, dose-dependently, the toxic effect of dopamine and 6-hydroxydopamine. Western blot analysis with newly prepared anti-human dopamine transporter antibodies showed that antisense phosphorothioate oligonucleotide 3 decreased the transporter protein level. These studies contribute to better understand the mechanism of dopamine-induced apoptosis and neurotoxicity.

A role for serotonin in the circadian system revealed by the distribution of serotonin transporter and light-induced Fos immunoreactivity in the suprachiasmatic nucleus and intergeniculate leaflet.

Components of the circadian system, the suprachiasmatic nucleus and the intergeniculate leaflet receive serotonin input from the raphe nuclei. Manipulations of serotonin neurotransmission disrupt cellular, electrophysiological, and behavioural responses of the circadian system to light, suggesting that serotonin plays a modulatory role in photic regulation of circadian rhythms. To study the relation between serotonin afferents and light-activated cells in the suprachiasmatic nucleus and intergeniculate leaflet, we used immunostaining for the serotonin transporter and for the transcription factor, Fos. Serotonin transporter, a plasma membrane protein located on serotonin neurons, regulates the amount of serotonin available for neurotransmission by re-accumulating released serotonin into presynaptic neurons; expression of Fos in the suprachiasmatic nucleus identifies light-activated cells involved in photic resetting of circadian clock phase. In the suprachiasmatic nucleus, immunostaining for serotonin transporter revealed a dense plexus of fibres concentrated primarily in the ventrolateral region. In the intergeniculate leaflet, serotonin transporter immunostaining identified vertically-oriented columns of fibres. Serotonin transporter immunostaining was abolished by pretreatment with the serotonin neurotoxin, 5,7-dihydroxytryptamine. Exposure to light for 30 min during the dark phase of the light cycle induced Fos expression in the ventrolateral suprachiasmatic nucleus and intergeniculate leaflet regions. In both structures the Fos-expressing cells were encircled by serotonin transporter-immunoreactive fibres often in close apposition to these cells. These results support the idea that serotonin activity plays a modulatory role in processing of photic information within the circadian system.

Effect of low-dose treatment with selegiline on dopamine transporter (DAT) expression and amphetamine-induced dopamine release in vivo.

1. Chronic treatment with low doses of the selective monoamine oxidase (MAO) type B inhibitors selegiline [(-)-deprenyl] and rasagiline, causes elevation in extracellular level of 3,4-dihydroxyphenylethylamine (dopamine) in the rat striatum in vivo (Lamensdorf et al., 1996). The present study was carried out to determine whether this effect of selegiline could be the result of an inhibition of the high-affinity dopamine neuronal transport process. 2. Changes in activity of the dopamine transporter (DAT) in vivo following selegiline treatment were evaluated indirectly by microdialysis technique in the rat, from the change in striatal dopamine extracellular concentration following systemic amphetamine administration (4 mg kg(-1), i.p.). Striatal levels of the DAT molecule were determined by immunoblotting. Uptake of [3H]-dopamine was determined in synaptosomes from selegiline-treated animals. 3. Amphetamine-induced increase in striatal extracellular dopamine level was attenuated by one day and by chronic (21 days) treatment with selegiline (0.25 mg kg(-1), s.c.). 4. Striatal levels of DAT were elevated after 1 and 21 days treatment with selegiline, but were not affected by clorgyline, rasagiline, nomifensine or amphetamine. 5. The increase in DAT expression, and attenuation of amphetamine-induced dopamine release, were not accompanied by a change in [3H]-dopamine uptake in synaptosomes of selegiline-treated animals. 6. The results suggest that a reversible inhibition of dopamine uptake occurs following chronic low dose selegiline treatment in vivo which may be mediated by an increase in endogenous MAO-B substrates such as 2-phenylethylamine, rather than by the inhibitor molecule or its metabolites. Increased DAT expression appears to be a special property of the selegiline molecule, since it occurs after one low dose of selegiline, and is not seen with other inhibitors of MAO-A or MAO-B. The new DAT molecules formed following selegiline treatment appear not to be functionally active.

Changes in expression of neuronal and glial glutamate transporters in rat hippocampus following kainate-induced seizure activity.

The expression of excitatory amino acid transporters (EAATs) in rat hippocampus was studied following kainic acid-induced seizure activity in vivo and in hippocampal slice cultures. Protein and mRNA levels of the glial (EAAT2) and neuronal (EAAT3) transporters were determined with affinity-purified antibodies and oligonucleotide probes, respectively. Kainate treatment decreased EAAT3 immunoreactivity in stratum lacunosum moleculare within 4 h of seizure onset. Upon pyramidal cell death (5 days after kainate treatment), EAAT3 immunoreactivity in stratum pyramidale of CA1 and in stratum lacunosum moleculare was almost completely eliminated. The rapid effect of kainate on EAAT3 expression was confirmed by in situ hybridization; EAAT3 mRNA levels were decreased in CA1 and CA3 regions within 4-8 h of seizure onset. Kainate treatment had an opposite effect on levels and expression of EAAT2. Developmental studies indicated that the rapid regulation of transporter expression was not observed in rats younger than 21 days, an observation congruent with previous reports regarding the resistance of young rats to kainate. In hippocampal organotypic cultures, which lack a major excitatory input from the entorhinal cortex, kainate produced a slow decrease in [3H]d-aspartate uptake. This study indicates that an early effect of kainate treatment consists of down-regulation of the neuronal transporter EAAT3 in restricted hippocampal regions, together with a modest increase in the expression of the glial transporter EAAT2. Differential regulation of neuronal and glial glutamate transporters may thus play a role in kainate-induced seizure, neurotoxicity and neuronal plasticity.

Heparinless cardiopulmonary bypass with active-site blocked factor IXa: a preliminary study on the dog.

OBJECTIVE: Cardiopulmonary bypass is a potent stimulus for activation of procoagulant pathways. Heparin, the traditional antithrombotic agent, however, is often associated with increased perioperative blood loss because of its multiple sites of action in the coagulation cascade and its antiplatelet and profibrinolytic effects. Furthermore, heparin-mediated immunologic reactions (that is, heparin-induced thrombocytopenia) may contraindicate its use. Cardiopulmonary bypass with a selective factor IXa inhibitor was tested to see whether it could effectively limit bypass circuit/intravascular space thrombosis while decreasing extravascular bleeding, thereby providing an alternative anticoagulant strategy when heparin may not be safely administered. METHODS: Active site-blocked factor IXa, a competitive inhibitor of the assembly of factor IXa into the factor X activation complex, was prepared by modification of the enzyme's active site by the use of dansyl glutamic acid-glycine-arginine-chlormethylketone. Twenty mongrel dogs (five were given standard heparin/protamine; 15 were given activated site-blocked factor IXa doses ranging from 300 to 600 microg/kg) underwent 1 hour of hypothermic cardiopulmonary bypass, and blood loss was monitored for 3 hours after the procedure. RESULTS: Use of activated site-blocked factor IXa as an anticoagulant in cardiopulmonary bypass limited fibrin deposition within the extracorporeal circuit as assessed by scanning electron microscopy, comparable with the antithrombotic effect seen with heparin. In contrast to heparin, effective antithrombotic doses of activated site-blocked factor IXa significantly diminished blood loss in the thoracic cavity and in an abdominal incisional bleeding model. CONCLUSION: These initial studies on the dog suggest that administration of activated site-blocked factor IXa may be an effective alternative anticoagulant strategy in cardiopulmonary bypass when heparin is contraindicated, affording inhibition of intravascular/extracorporeal circuit thrombosis with enhanced hemostasis in the surgical wound.

Bcl-2 and p53: role in dopamine-induced apoptosis and differentiation.

The fate of a neuron in the developing brain to multiply, differentiate, or die in an apoptotic manner depends on the expression of genes that are involved in regulating the cell cycle. Recent studies determined the involvement of several genes, including cyclin A and B2, in dopamine-induced apoptosis in cultured chick sympathetic neurons. Another gene that plays a role in apoptosis and differentiation of neurons, oligodendrocytes and PC12 cells is p53. It is also known that DNA damage increases p53 levels, triggering repair or apoptosis in response to moderate or severe damage, respectively. NMB cells express active and inducible forms of p53, thus being particularly suitable to analyze the role of this gene in dopamine-induced apoptosis and differentiation. The main observation of this work is that low concentrations of dopamine induce differentiation while high concentrations induce apoptosis, and that concentrations of dopamine that induce apoptosis increased p53 levels. There peak increase in p53 was within 3-6 h, before cell death. Thus, treatment with a high dopamine concentration may result in oxidation products and/or free radicals that heavily damage DNA, thus increasing p53 levels and initiating a cascade of events leading to apoptosis. Lower concentrations of dopamine apparently have a milder damaging effect on the DNA and induce growth arrest and differentiation. In various systems Bcl-2 inhibits cell death, being apoptotic or necrotic. Bcl-2, and other members of the family, such as Bax, are located downstream to p53 in the apoptotic pathway, and they contain negative or positive p53 response elements. Bcl-2 also protects cells by acting as antioxidant. Neuronal differentiation may be accompanied with an increase in Bcl-2, though it was suggested that the role of Bcl-2 in differentiation is less critical than in apoptosis. Herein, Bcl-2 was found to inhibit dopamine neurotoxicity. Whether the expression of Bcl-2 is regulated by different dopamine concentrations, or by dibutyryl-cAMP and DMSO, remains to be determined.

Selectivity in the control of opiate receptor density in the animal and in cultured fetal brain cells.

Two aspects of the mechanisms controlling down-regulation of opiate receptors were studied: 1. The possibility that morphine does not induce down-regulation of delta receptors is an observation confined to in vitro conditions was investigated by studying the regulation of receptors in neuroblastoma-glioma cells in diffusion chambers implanted in ICR mice peritonea. Injection of morphine for 9 days at a dose inducing opiate tolerance did not change the number of receptors in the chamber-implanted cells, whereas etorphine at a 1/100 dose had a profound effect. 2. Embryonic cells from rat forebrain or hindbrain were cultured with mu type opiate alkaloid (morphine) or peptide (morphiceptin) to further establish the selectivity of opiate action. A partial effect of morphiceptin but not morphine on the number of receptors in hindbrain aggregates was observed. Thus, conclusions derived from experiments with morphine may not be applicable to mu type peptides. The results suggest that the mammalian brain may contain sub-types of mu receptors. Alternatively, although interacting with a common mu receptor, morphine and mu opioid peptides may induce different regulatory mechanisms.

A rapid binding assay for solubilized dopamine transporters using [3H]WIN 35,428.

The cocaine analog [3H]WIN 35,428 was used to label digitonin-solubilized dopamine transporters from dog caudate nucleus. The assay consists of incubation of extracts with the ligand followed by separation of free from bound ligand by centrifugation after adding activated charcoal. Specific binding was observed in dog caudate but was absent in dog cerebellar extracts. Binding was linear with tissue, saturable, and of high affinity (Kd = 16 nM). In competition studies, soluble [3H]WIN 35,428 binding was inhibited strongly by mazindol, GBR 12909, and (-)-cocaine but only weakly by citalopram, desipramine, and (+)-cocaine; this is typical of binding to the dopamine transporter. Compared to assays using [3H]GBR 12935, (-)-cocaine was relatively more potent, suggesting that the cocaine and GBR 12935 binding sites are somewhat different. When soluble extract was chromatographed on a wheat germ agglutinin-Sepharose column, [3H]WIN 35,428 binding activity was eluted with N-acetylglucosamine in a manner similar to photoaffinity-labeled dopamine transporters.