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Biomedical subjects

Y Sarne

Publications and source records attributed to Y Sarne.

At least 19 recordsLinked to original sources

Are cannabinoid drugs neurotoxic or neuroprotective?

Chronic exposure to cannabinoids was shown to induce long lasting impairment of learning and memory, which was accompanied by morphological damage to the brain. On the other hand, several studies have shown that cannabinoids can protect from various brain traumas. This enigmatic dualism is explained herein by a comprehensive hypothesis, which is based on our recent in vitro studies and on pharmacokinetic in vivo considerations. The hypothesis predicts that low concentrations of cannabinoids will be neurotoxic while high concentrations of the drugs will protect from neuronal damage, and suggests that chronic administration of cannabinoids will induce neuronal death, while their acute administration will protect the brain. We further propose straight forward experiments, both in vivo (animal models for brain damage) and in vitro (cell death in neuronal cultures) to verify this hypothesis. The outcome of these experiments may have practical applications when considering the use of cannabinoids as therapeutic agents and in evaluating the consequences of their use as recreational drugs.

Apoptosis↗

Divers pathways mediate delta-opioid receptor down regulation within the same cell.

Various mechanisms have been proposed for opioid receptor down regulation in different experimental preparations. The present study was aimed to test whether distinct mechanisms can mediate opioid receptor down regulation within the same cell. For this purpose we transfected HEK-293 cells with rat delta-opioid receptor (DOR). We exposed the cells to the opioid agonist etorphine in the absence or presence of various pharmacological agents and measured the binding of the opioid ligand [(3)H]diprenorphine to either isolated cell membranes or whole cells. We found that internalization of the receptors into the cell was mediated by clathrin coated pits and that the internalized receptors were degraded either in lysosomes or by proteosomes. Down regulation involved phosphorylation and at least two different kinases, a tyrosine kinase (TK) and MAPK kinase (MEK), mediated DOR down regulation in parallel routes. G-protein-coupled receptor kinase (GRK) was found to have only a minor role in DOR down regulation in HEK-293 cells. On the other hand, in N18TG2 cells that endogenously express delta-opioid receptors, GRK was the predominant kinase mediating DOR down regulation, with only a minor role for TK and MEK. We conclude that down regulation can take place via divers pathways within the same cell, and that in different cells down regulation is mediated by different mechanisms, depending on the kinase profile of the cells and the compartmentalization of the receptors within the cells.

Acetylcysteine↗

Opioid and cannabinoid receptors share a common pool of GTP-binding proteins in cotransfected cells, but not in cells which endogenously coexpress the receptors.

1. Opioid (mu, delta, kappa) and cannabinoid (CB1, CB2) receptors are coupled mainly to Gi/Go GTP-binding proteins. The goal of the present study was to determine whether different subtypes of opioid and cannabinoid receptors, when coexpressed in the same cell, share a common reservoir, or utilize different pools, of G proteins. 2. The stimulation of [35S]GTPgammaS binding by selective opioid and cannabinoid agonists was tested in transiently transfected COS-7 cells, as well as in neuroblastoma cell lines. In COS-7 cells, cotransfection of mu- and delta-opioid receptors led to stimulation of [35S]GTPgammaS binding by either mu-selective (DAMGO) or delta-selective (DPDPE) agonists. The combined effect of the two agonists was similar to the effect of either DAMGO or DPDPE alone, suggesting the activation of a common G-protein reservoir by the two receptor subtypes. 3. The same phenomenon was observed when COS-7 cells were cotransfected with CB1 cannabinoid receptors and either mu- or delta-opioid receptors. 4. On the other hand, in N18TG2 neuroblastoma cells, which endogenously coexpress CB1 and delta-opioid receptors, as well as in SK-N-SH neuroblastoma cells, which coexpress mu- and delta-opioid receptors, the combined effects of the various agonists (the selective cannabinoid DALN and the selective opioids DPDPE and DAMGO) were additive, implying the activation of different pools of G proteins by each receptor subtype. 5. These results suggest a fundamental difference between native and artificially transfected cells regarding the compartmentalization of receptors and GTP-binding proteins.

Analgesics↗

Potentiation of transmitter release from NMB human neuroblastoma cells by kappa-opioids is mediated by N-type voltage-dependent calcium channels.

The selective kappa-opioid agonist trans-(+/-)-3, 4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl) cyclohexyl] benzenacetamidemethansulfonate (U50,488) potentiates both basal and depolarization-evoked [3H]dopamine release from NMB cells. The potentiation of dopamine release by U50,488 is mediated by N-type voltage-dependent calcium channels since it is blocked by omega-conotoxin, and is resistant to pertussis toxin (PTX)-treatment. When the stimulation of release by U50,488 is blocked by the N-channel antagonist omega-conotoxin, an inhibitory effect on dopamine release is revealed, suggesting that stimulatory and inhibitory effects of U50,488 are exerted in parallel.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗

Independence of, and interactions between, cannabinoid and opioid signal transduction pathways in N18TG2 cells.

N18TG2 neuroblastoma cells co-express delta-opioid and CB1-cannabinoid receptors. Both receptors are negatively coupled to adenylyl cyclase through pertussis toxin-sensitive GTP-binding proteins. In the present study, we confirmed the independent activity of opioid and cannabinoid agonists, and investigated chronic interactions between the two signal transduction pathways in these cells. Opioid and cannabinoid agonists stimulated [35S]guanosine-5'-O-(3-thiotriphosphate) binding to N18TG2 membranes. When the opioid agonist etorphine and the cannabinoid agonist desacetyllevonantradol (DALN) were applied together, the stimulation was similar to the arithmetic sum of the two separate effects. This additivity existed even after partial ablation of the G-proteins reservoir with a low concentration of pertussis toxin, indicating that opioid and cannabinoid receptors activate different pools of G-proteins in N18TG2 cells. Chronic treatment of the cells with either opioid or cannabinoid agonists induced desensitization to the respective drug. In addition, asymmetric cross-desensitization was found: while long-term exposure to DALN induced homologous desensitization, and did not reduce the effect of etorphine, long-term exposure to etorphine attenuated the cannabinoid activation of G-proteins. Chronic exposure to either DALN or etorphine not only induced desensitization, but also elevated the basal activity of G-proteins in the exposed cells. The combination of the two drugs did not yield an additive activation, suggesting that chronic exposure of N18TG2 cultures to cannabinoid and opioid agonists modified a common responding element within the cells. This work presents the N18TG2 neuroblastoma as a suitable experimental model to study the molecular mechanism(s) underlying chronic interactions between opioid and cannabinoid drugs.

Adenylate Cyclase Toxin↗

Dissociation between the inhibitory and stimulatory effects of opioid peptides on cAMP formation in SK-N-SH neuroblastoma cells.

Opioid agonists either potentiate or suppress basal cAMP production in SK-N-SH cells. The inhibitory effect is mediated by PTX-sensitive GTP-binding proteins, while the stimulatory effect involves Ca++ entry and calmodulin activation. Both pathways can be activated simultaneously by opioid agonists. Low (nM) concentrations of either mu (DAMGO) or delta (DPDPE) selective opioids potentiate cAMP formation. At higher (100 nM) concentrations, however, a net suppression takes over; this suppression can be eliminated by PTX, and the underlying stimulatory effect is disclosed. Micromolar concentrations of either mu or delta selective agonists cross-activate the other (delta or mu) receptors, and augment the stimulatory pathway. The overall outcome (either stimulation or inhibition of cAMP production) is dependent on the balance between the two overlapping pathways, and can be modified by blocking either of the two opposing mechanisms.

Calcium↗

Reexamination of opioid stimulation of cGMP formation in cell lines of neuronal origin.

1. The present study reexamines a previous notion on opioid stimulation of cyclic GMP (cGMP) formation and the retraction of the original findings. 2. The effect of opioid agonists on cGMP accumulation in two cell lines of neuronal origin was measured. The proportion of cGMP stimulation in NG108-15 neuroblastoma x glioma hybrid cells resembled the proportion of [Ca2+]in elevation by opioids in this culture. The failure of opioids to stimulate cGMP formation in SK-N-SH human neuroblastoma coincided with the lack of cGMP stimulation by other Ca2+ mobilizing agents in these cells. The nitric oxide donor nitroprusside elevated cGMP in both cell lines. 3. The implication of the opioid-Ca(2+)-NO-cGMP cellular pathway for opioid activity in vivo is discussed.

Animals↗

Opioids potentiate transmitter release from SK-N-SH human neuroblastoma cells by modulating N-type calcium channels.

Opioids induce dual (inhibitory and excitatory) regulation of depolarization-evoked [3H]dopamine release in SK-N-SH cells through either mu or delta receptors. The potentiation of dopamine release by opioid agonists is mediated by N-type voltage-dependent calcium channels and does not involve Gi/Go proteins. Removal of the excitatory opioid effect by blockade with omega-conotoxin, an N-channel antagonist, reveals the inhibitory effect of opioids on release, thus suggesting that both modulatory effects of opioids are exerted in parallel.

Brain Neoplasms↗

The stimulatory effect of opioids on cyclic AMP production in SK-N-SH cells is mediated by calcium ions.

The present study examines the stimulatory effect of opioids on adenosine 3':5'-cyclic monophosphate (cyclic AMP) production in the human neuroblastoma cell line SK-N-SH, and its dependence on calcium. We show that, in this culture, the mu-opioid selective agonist [D-Ala2, N-Me-Phe4, Gly5-ol]-Enkephalin stimulates cyclic AMP production by 30% in a naloxone-reversible manner. This stimulation is completely dependent on calcium and involves the activation of calcium/calmodulin since it is abolished in the presence of EGTA, calcium channel blockers or N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7). The results suggest that the activation of calcium/calmodulin dependent adenylyl cyclases by opioids in SK-N-SH cells is secondary to the induction of calcium influx and the consequent elevation of intracellular calcium level.

Calcium↗

Long-term regulation of opioid receptors in neuroblastoma and lymphoma cell lines.

Long-term regulation of opioid binding was studied in the human neuroblastoma NMB and in the murine lymphoma R1.1 and R1.EGO cell lines. Binding was down-regulated following prolonged exposure to opioid agonists and up-regulated following exposure to antagonist. Down-regulation was inhibited by the metabolic blocker sodium-azide and by the protein kinase H-7. Up-regulation was blocked by the protein and mRNA synthesis blockers cycloheximide, alpha-amanitin and actinomycin D. A significant difference was found between the response of neuronal and immune cells to ethanol exposure: while opioid binding in neuroblastoma culture underwent a pronounced (75%) up-regulation, no effect of ethanol on opioid receptors in lymphoma cultures was detected. The described cell lines present an excellent experimental model to study long-term regulation of opioid receptors in the nervous and immune systems and to elucidate the biological effects of chronic use of opiates and alcohol.

Diprenorphine↗

Determinants of the stimulatory opioid effect on intracellular calcium in SK-N-SH and NG108-15 neuroblastoma.

The opiate agonist etorphine elevated [Ca2+]i in two neuroblastoma cell lines. Fura-2 imaging of single cells revealed a small and variable calcium elevation in only 20% of cultures. Three factors were found to increase the probability (up to 70%) and the amplitude of the response to etorphine: (a) synchronization of the cultures; (b) differentiation of the cells; and (c) synergism with other stimulatory agents (carbachol in SK-N-SH and bradykinin in NG108-15 cells). The establishment of a reproducible experimental protocol may facilitate the study of the molecular mechanism(s) underlying the stimulatory activity of opiates.

Bradykinin↗

Stimulatory effects of opioids on transmitter release and possible cellular mechanisms: overview and original results.

Opiates and opioid peptides carry out their regulatory effects mainly by inhibiting neuronal activity. At the cellular level, opioids block voltage-dependent calcium channels, activate potassium channels and inhibit adenylate cyclase, thus reducing neurotransmitter release. An increasing body of evidence indicates an additional opposite, stimulatory activity of opioids. The present review summarizes the potentiating effects of opioids on transmitter release and the possible cellular events underlying this potentiation: elevation of cytosolic calcium level (by either activating Ca2+ influx or mobilizing intracellular stores), blockage of K+ channels and stimulation of adenylate cyclase. Biochemical, pharmacological and molecular biology studies suggest several molecular mechanisms of the bimodal activity of opioids, including the coupling of opioid receptors to various GTP-binding proteins, the involvement of different subunits of these proteins, and the activation of several intracellular signal transduction pathways. Among the many experimental preparations used to study the bimodal opioid activity, the SK-N-SH neuroblastoma cell line is presented here as a suitable model for studying the complete chain of events leading from binding to receptors down to regulation of transmitter release, and for elucidating the molecular mechanism involved in the stimulatory effects of opioid agonists.

Adenylyl Cyclases↗

Multiple effects of opiates on intracellular calcium level and on calcium uptake in three neuronal cell lines.

The present study examines the modulation by opiates of intracellular calcium levels and calcium entry, using fura-2 imaging and 45Ca2+ uptake, in three neuronal cell lines. We show that opiates (10(-7)-10(-5) M morphine and 10(-9)-10(-7) M etorphine) exert both inhibitory and excitatory effects on KCl-induced elevation in intracellular calcium level in SK-N-SH, NG108-15 and NMB cell lines. In addition, opiates elevate basal (non KCl-stimulated) intracellular calcium level in all three cell cultures. 45Ca2+ uptake is augmented by opiates in SK-N-SH cells and this stimulatory effect is not blocked by pertussis toxin. In NMB cells, an additional inhibitory effect of opiates on basal calcium takes place: opiates reduce intracellular calcium level as measured by fura-2, and decrease calcium influx as detected by 45Ca2+ uptake. The heterogeneity in the opioid regulation of calcium could not be attributed to the type of opioid drug, neither to its concentration nor to the experimental conditions, since neighboring cells within the same culture responded differently.

Calcium↗

Imipramine binding to blood platelets and aggressive behavior in offenders, schizophrenics and normal volunteers.

The relationships between [3H]imipramine binding to blood platelets and manifestation of aggressive behavior was studied in three different groups of subjects. Arrested offenders displayed high imipramine binding compared to controls, with violent offenders exceeding their nonviolent counterparts. Hostile schizophrenic patients showed imipramine binding higher than nonhostile matched patients. In normal volunteers, on the other hand, no correlation between imipramine binding and scores of aggression was found. The results support the notion about the involvement of a neuronal serotonergic system in the regulation of aggressive behavior. The contribution of this biological factor is detectable in pathological manifestation of aggression but not in normal behavior.

Adolescent↗

Characterization of a triple opioid system in the human neuroblastoma NMB cell line.

The human neuroblastoma NMB cell line was found to contain the three types of opioid receptors (60% delta 25% kappa and 15% mu). The opioid receptors were negatively coupled to adenylyl-cyclase. Maximal reduction in cAMP content was achieved by selectively activating single receptor types, indicating the co-presence of the various opioid receptors in the same cells. The opioid receptors in NMB cells were up-regulated following prolonged exposure to the opioid antagonist naloxone and down-regulated following chronic treatment with the opioid agonist etorphine. Down-regulation was time-, dose- and temperature-dependent and was inhibited by colchicine and sodium azide. The NMB culture is presented as an excellent experimental model for studying the selective activation and regulation of the different opioid receptor types when they are co-expressed in the same neuron, as well as for studying interactions between the various opioid receptors.

Adenylyl Cyclases↗

Dual regulation by opioids of 3H-norepinephrine release in the human neuroblastoma cell-line SK-N-SH.

Depolarization-evoked 3H-norepinephrine release from SK-N-SH cells was found to be regulated by opioid ligands. Opioids exerted either inhibition or augmentation of 3H-norepinephrine release. Both effects were mediated by opioid receptors. In addition, a nonopioid inhibitory effect of opiates on release was observed. The SK-N-SH cell-line provides a suitable model for studying the various mechanisms underlying the opioid regulatory pathways within single cells.

Cell Line↗

Phe-Met-Arg-Phe-NH2 (FMRFa)-related peptides inhibit Na(+)-Ca2+ exchange in cardiac sarcolemma vesicles.

The molluscan cardioexcitatory tetrapeptide FMRF-amide (Phe-Met-Arg-Phe-NH2) and related peptides inhibit Na(+)-Ca2+ exchange in calf cardiac sarcolemma vesicles. FMRFa itself has a low inhibitory potency (IC50 = 750 microM) which completely resides in its COOH-terminal RFa portion. The physiologically active analog FLRFa is 10-fold more potent (IC50 = 60 microM). Two other substitutions of the Met2 in FMRFa, by either Ile or Lys increase inhibitory potency 7- and 50-fold, respectively. The inhibitory potency increases 300-500-fold if the NH2-terminal Phe1 in FMRFa is substituted by either Val or His (IC50 = 1-2 microM). The inhibitory activity of WnLRFa (IC50 = 40 microM) is lost when either the NH2-terminal amino group is acylated or the NH2-terminal Trp1 is deleted. These data suggest that the COOH-terminal portion is essential for the basic low potency inhibition of Na(+)-Ca2+ exchange, whereas the NH2-terminal portion is important for the potentiation of the inhibitory activity. Although the IC50 values of various peptides range widely (10(-6)-10(-3) M), all of them induce a complete inhibition. The dose-response pattern of the peptide-induced inhibition is identical for the Na(+)-Ca2+ exchange and its partial reaction, the Ca(2+)-Ca2+ exchange. The inhibitory effect is reversible and affects both Nai(or Cai)-dependent 45Ca uptake and Nao-dependent 45Ca efflux, suggesting that the bidirectional movements of ions are altered. A mild pretreatment of vesicles with trypsin augments the Na(+)-Ca2+ exchange 1.5-fold but diminishes the inhibitory potency 3-4-fold, suggesting that the inhibition is mediated by an extravesicular membrane protein. The characteristics of the peptide-induced inhibition resemble the effect of opiates on Na(+)-Ca2+ exchange. FLRFa and dextrorphan (a non-opioid stereoisomer of an opiate agonist) are mutually exclusive inhibitors, suggesting that they may bind to the same site. This putative site lacks the pharmacological properties of opiate receptors and may be located either on the Na(+)-Ca2+ exchanger or at its vicinity. Endogenous analogs of FMRFa may regulate intracellular calcium via Na(+)-Ca2+ exchange.

Amino Acid Sequence↗

Evidence for mu-, delta-, and kappa-opioid receptors in a human neuroblastoma cell line.

Human neuroblastoma cells were tested for the presence of opioid receptors. [3H]Diprenorphine binds to NMB cell membranes with a KD value of 0.46 +/- 0.13 nM and Bmax of 534 +/- 22 fmol/mg protein. The presence of mu, delta, and kappa opioid receptors was tested by displacing [3H]diprenorphine specific binding by the selective agonists DAMGO, DPDPE, and U50,488H, respectively. Using this procedure, the data suggest that the NMB neuroblastoma cells express the three opioid receptor types with the abundance of delta receptors (about 60%) and minor, yet substantial populations of mu and kappa receptors (about 20% each).

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗