Biomedical subjects
Publications and source records attributed to R Simantov.
Because some evidence suggests that cocaine and GBR12935 bind to different sites, we utilized photoaffinity probes from both classes of compounds to see if they label the same protein. [125I]RTI-82 a cocaine analog, and [125I]DEEP, a GBR analog, labeled protein(s) showing the same molecular weight, a similar pharmacological profile and a similar sensitivity to neuraminidase.
The dopamine transporter from rat caudate-putamen was photolabeled with [125I]DEEP as previously described. Treatment of photolabeled membranes with neuraminidase and N-glycanase reduced the molecular weight of the [125I]DEEP photolabeled dopamine transporter complex, whereas treatment with alpha-mannosidase had no effect. The solubilized [125I]DEEP photolabeled dopamine transporter complex readily bound to wheat-germ agglutinin but not to concanavalin-A sepharose columns. These results suggest that the carbohydrate moiety of the dopamine transporter is N-linked and contains significant quantities of sialic acid but not high mannose residues. A DEEP binding protein was readily detectable in other brain regions including the nucleus accumbens and olfactory tubercle, but not in the prefrontal cortex, olfactory bulb or hypothalamus under similar conditions. The DEEP binding protein in the other brain regions was similar to that in the striatum.
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1. Aggregating fetal rat brain cells express a significant amount of proenkephalin A (PENK) mRNA, a selective radioimmunoassay shows that this mRNA is also translated into enkephalins. 2. Depolarization with potassium chloride (KCl) or veratridine increases the expression of PENK mRNA in a time-dependent fashion, with a maximal increase of sixfold. It is interesting, however, that depolarization of the same cultures with KCl has no effect on the expression of prodynorphin mRNA. 3. An increase in PENK mRNA levels has been also observed in cultures treated with 8-Br-cAMP, phorbol 12-myristate-13-acetate (TPA), or dexamethasone. 4. However, incubation of the cultures with the opioid agonist etorphine or the antagonist naltrexone did not alter PENK gene expression, suggesting that there is not feedback control of opioids on PENK biosynthesis in these cells. 5. The increase in PENK mRNA in depolarized and in TPA-dexamethasone-, or 8-Br-cAMP-treated cultures was not accompanied by a significant increase in the amount of free immunoreactive met-enkephalin. Fetal brain cell cultures are therefore a useful neuronal model system for studying the mechanism that regulated the expression of PENK mRNA.
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.
The developmental profile of opioid receptors was studied in rat and guinea pig striatum and hippocampus. The two brain regions show different receptor profiles during development, which are characteristic for each animal. Yet, both tissues and animal species share one common feature; the binding of the universal opioid ligand [3H]diprenorphine per milligram of protein is high at the early embryonic period, it decreases toward birth, and then gradually increases to the adult levels. This apparent transient expression of the receptors during the early developmental stage was manifested in the guinea pig as an actual decrease in the total receptor number. As an attempt to characterize the receptors involved in this process, the binding of the selective mu-opioid ligand [3H]Tyr-D-Ala-Gly-MePhe-NH(CH2)OH [( 3H]DAGO) was studied in striatal membranes of young (P1) and adult (P60) rats. Competition between [3H]DAGO and the delta-selective peptide Tyr-D-Pen-Gly-Phe-D-Pen (DPDPE) shows higher affinity of the delta opioid to P1 membranes than to P60 membranes, though the number of delta receptors in P1 membranes is very small. This observation is in line with a previous study suggesting that opioid receptors in embryonic striatum and hippocampus are less selective to various opioids than those of adult brain. An additional difference between adult and embryonic tissue was observed on Scatchard analysis of [3H]DAGO binding; striatum P60 membranes exhibit one binding site with a KD of 0.8 +/- 0.1 nM and Hill coefficient of 0.96, whereas striatum P1 membranes bind the peptide in an apparent cooperative fashion with an overall Hill coefficient of 1.30.(ABSTRACT TRUNCATED AT 250 WORDS)
The influence of chemical depolarization on the survival and differentiation of acetylcholinesterase (AChE)-containing neurons was examined in primary rat striatal cultures, maintained in different types of media (serum-free and serum-supplemented) and substrate (poly-ornithine and astrocyte monolayer). Chronic application of 5 microM veratridine resulted in a significant loss of neurites by AChE-positive cells, while a higher concentration (20 microM) reduced the number of stained cell bodies. These effects appeared to be selective with regard to AChE-positive cells, as indicated by morphological observations of the cells in the treated cultures and receptor binding measurements. Similarly, elevation of extracellular KCl levels (20-60 mM) produced a dose-dependent neurite loss by AChE-containing cells. Blockers of voltage-sensitive Ca2+ channels--verapamil (1 microM) and nifedipine (1 microM)--did not affect the veratridine-induced neurite loss, while tetrodotoxin (0.1 microM) had a partial effect. When cultures treated with 5 microM veratridine were allowed to recuperate for several days, the number of AChE-positive cells possessing neurites returned close to control values, thus indicating the reversibility of the effect of chemical depolarization. The possibility that chronic neuronal depolarization in the striatum might play a role in regulation of the neuronal processes outgrowth by AChE-containing cells is discussed.
Proenkephalin A (PEA) gene was found to be expressed in primary, secondary and tertiary cultures of rat fibroblasts. The 1.4 kb PEA mRNA was detected by Northern blot analysis. The same cultures do not express detectable amounts of proenkephalin B (prodynorphin) or (POMC) mRNAs. Acidic cell extracts were purified on a C18 octadecyl Amprep column and analysed with a specific methionine enkephalin radioimmunoassay to detect whether PEA mRNA is translated. A significant amount of enkephalin immunoreactivity (178-185 fmol/mg protein) was observed upon trypsin and carboxypeptidase B digestion of fibroblast cell extracts, whereas only 3-5% of this amount was free enkephalin. It is therefore indicated that the PEA mRNA expressed in fibroblasts is indeed translated to the proenkephalin precursor protein, but the cells accumulate only a small quantity of the processed pentapeptides. The implication of these observations to the possible developmental role of PEA in various non-neuronal cells, including mesodermal lineages, is discussed.
Previous studies about the differential effect of chronic membrane depolarization on the expression of mu and delta opioid receptors prompted us to investigate whether the same treatment regulates also the expression of kappa opioid receptors. Embryonic guinea pig brain cells that exhibit in culture a high density of kappa receptors were treated for different time periods with potassium chloride, and the number of these receptors was determined with the universal opioid ligand [3H]diprenorphine. In a second set of experiments the cultures were treated with the sodium channel activator veratridine and opioid receptors were determined with the selective kappa ligand [3H]U69,593. The results indicate that chronic membrane depolarization (for 3 or 6 days) decreases significantly the number of kappa receptors, with no effect on the affinity of the ligands. Taken together with our previous reports, it is suggested that neuronal activation has a selective and possibly opposite regulatory role on the expression of the 3 opioid receptors.
Rat and guinea pig fetal brain cell cultures and immunoblotting techniques were used to study the effect of receptor selective opioids on the level of the membrane-bound alpha i and alpha o GTP binding protein subunits. Incubation of rat hindbrain cultures with the mu selective peptide DAGO decreased the amount of both alpha proteins. The reduction observed was equivalent to 36% in alpha o and 41% in alpha i. On the other hand, incubation of rat forebrain cultures with this peptide had an opposite effect, increasing the alpha o and alpha i levels by 66% and 68%, respectively. This differential effect of the peptide on the G proteins at the two brain areas may reflect the selective interaction at the receptor level; DAGO induced a fast and effective receptor down-regulation (50% decrease in Bmax) in hindbrain but not in forebrain cultures. Moreover, delta and mu selective ligands differed in their effect, as indicated by the finding that the delta selective peptide DPDPE increased the amount of both alpha proteins in hindbrain cultures by 40%. Similar experiments conducted with guinea pig brain aggregate cultures indicated that the kappa selective agonist U50,488 decreased the amount of the membrane bound alpha i protein subunit by 56%. The results thus indicate that opioid agonists, interacting selectively with the three types of opioid receptors, induce a complex repertoire of changes in the immunoreactive levels of the membrane-bound alpha GTP binding protein subunits in various CNS structures.
A temperature-sensitive rat cerebellar cell line SC9 has been used to study the role of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) in glutamate cytotoxicity. GABA increases glutamate toxicity in a dose-dependent fashion, but NMDA and kainic acid were not toxic in the presence or absence of GABA. The specificity of this cytotoxicity was further indicated by the NMDA-selective antagonist 2-amino-7-phosphonoheptanoic acid (APV), which does not block glutamate effect. These observations, as well as binding experiments with 3H-glutamate, suggest that glutamate cytotoxicity in these cells depends on quisqualate-selective uptake sites of the amino acid. The study may open therefore a novel pathway for understanding the cytotoxic effect of excitatory amino acids in brain structures that are enriched with GABA and glutamate uptake sites.
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.
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The question whether chronic membrane depolarization regulates selectively the expression of different muscarinic cholinergic receptors has been studied in primary neuronal cultures prepared from two regions of embryonic rat brain. Chronic (3 or 6 days) but not acute (3 or 24 h) membrane depolarization of cultured septal neurons with 40 mM potassium chloride have increased the number of muscarinic receptors by 168 +/- 28%, but no such effect has been observed with hippocampal cultures. Displacement experiments with the M1 selective antagonist pirenzepine have shown that muscarinic receptors in the septum of adult rat have 3-5 times lower affinity to this compound than hippocampus receptors. The results suggest that chronic membrane depolarization selectively increases the number of presynaptic muscarinic receptors, expressed by septal neurons, whereas hippocampal neurons expressing postsynaptic receptors have no such response.
Cultures of aggregating fetal rat brain cells express mu, delta, and kappa opioid receptors. The potent and long-lasting opioid antagonist naltrexone was used to investigate whether different regulatory mechanisms are involved in the expression of the three receptor subtypes. In cultures treated for seven days, naltrexone increased dose-dependently the binding of 3H-diprenorphine to the three receptor subtypes, with the mu sites being affected at a lower concentration than the other two; A Scatchard analysis indicated that this receptor up-regulation was obtained by an increase in the BMax, with no significant change in the affinity of the ligand to the receptors. In contrast to these effects in cultures treated for 7 days, it was surprising to find that a 48 hr treatment with naltrexone had an apparent converse and subtype-specific influence; the antagonist decreased significantly the binding of 3H-diprenorphine to both mu and delta receptors but had no effect on kappa sites. Two other opioid antagonists, naloxone and levallorphan, had a similar effect. Further analysis of naltrexone's mode of action was obtained by studying its effect on the adenylate cyclase activity. Of several inducers of this enzyme, the beta-adrenergic inducer isoproterenol gave the highest increase in cyclic AMP. Naltrexone had no significant effect on the basal adenylate cyclase activity but it altered the pattern of cyclic AMP formation in isoproterenol-stimulated cultures. Overall, the results indicate that in addition to its classic antagonistic activity, naltrexone exhibits in fetal brain aggregates some properties associated with opiate agonists.
1. The possibility that a long-lasting neuronal activation regulates the expression of muscarinic cholinergic receptors was studied with three cultured neuronal cell lines. 2. Continuous depolarization of a subclone of the neuroblastoma-glioma NG108-15 hybrid cells with potassium chloride increased by 45-75% the number of cholinergic muscarinic receptors, monitored with 3H-QNB, whereas a short incubation with KCl for 10 min or 6 hr had no effect. 3. The calcium channel blocker verapamil increased the effect of KCl. 4. Two cell lines, named SC9 and WC5, that originate from the rat brain, also bind 3H-QNB. They were therefore used to test whether the effect of chronic depolarization is universal. Depolarized SC9 and WC5 cells, in the presence or absence of verapamil, did not show an increased 3H-QNB binding. 5. Muscarinic receptors of both SC9 and WC5 cells have a higher affinity to pirenzepine than the M-3 receptor subtype of the neuroblastoma-glioma cells, suggesting therefore that the two rat brain cell lines possess M-1 or M-2 receptors. 6. The physiological significance of this differential role of depolarization on the expression of different muscarinic receptors is discussed in the context of their postreceptor second messengers.
To determine whether the differences in development of acute tolerance to several morphine actions correlate with the mu receptor subtype mediating them, we have examined the appearance of acute tolerance to analgesia, respiratory depression, gastrointestinal transit, and hormone release in an intravenous morphine infusion model. Analgesia, a naloxonazine-sensitive mu1 action, peaked at 2 hr after initiation of the infusions. The log dose-response relationship of the infusion rate to peak tailflick latency was linear from 10 to 50 micrograms/kg/min. By 8 hr, the tailflick latencies declined nearly to baseline levels, implying the rapid development of tolerance. Tolerance to morphine-induced prolactin release, another mu1 action, also developed rapidly over 8 hr. In contrast two mu2 actions, respiratory depression measured with arterial blood gas, determinations and gastrointestinal transit, showed no significant tolerance over a similar 8 hr infusion. We also observed no tolerance to morphine-induced growth hormone release, a non-mu1 action, over the same period. Thus, these results demonstrate that mu1 actions develop tolerance in an infusion model far more rapidly than a number of naloxonazine-insensitive (non-mu1) ones and may help explain differences in the rate of tolerance development to morphine actions.
Expression of the three opioid receptor subtypes mu, delta and kappa in aggregating cell cultures prepared from embryonic guinea pig or rat brains was compared with the in vivo expression of the receptors in the brain of developing and adult animals of the same species. At the day of culturing, one third of the receptors in the brain of guinea pig embryos were of the kappa type. In culture, however, the aggregating brain cells acquired within 14 days a high percentage (75%) of kappa receptors. As only 28% of the receptors in the adult guinea pig brain are of this subtype, an attempt was made to further analyse the specificity of this developmental process. In guinea pig, the 2-fold increase in kappa receptors in culture was accompanied with a decline in both the percentage and the density (per protein) of mu and delta subtypes. In contrast, a marked increase in delta receptors was observed in rat whole brain, forebrain or hindbrain cultures. Thus, the developmental pattern of the three receptor subtypes in rat brain cultures, but not in guinea pig, was similar to that in vivo. These and additional experiments suggest that at the developmental stage taken to prepare the cultures, neurons expressing opioid receptors were already programmed in the rat but not in guinea pig brain.