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M Gafni

Publications and source records attributed to M Gafni.

At least 19 recordsLinked to original sources
PubMed2001

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↗
PubMed1999

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↗
PubMed1998

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↗
PubMed1998

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↗
PubMed1998

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↗
PubMed1997

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↗
PubMed1996

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↗
PubMed1996

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↗
PubMed1995

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↗
PubMed1995

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↗
PubMed1993

The involvement of sodium ions in the positive inotropic effect of naloxone.

1. The opiate antagonist naloxone induces a positive inotropic effect in isolated cardiac muscles. 2. The response to naloxone is dependent on the presence of Na+ in the bathing solution, is proportional to the rate of electrical stimulation, and increased in the presence of veratridine. 3. Lowering [K+]o to 50% augments the response, while complete removal of K+ from the extracellular solution attenuates the response to naloxone. 4. Maximal concentration of naloxone decreases the inotropic effect of the cardiac glycoside ouabain. 5. The results indicate the involvement of intracellular sodium accumulation in the positive inotropic effect of naloxone, probably through the inhibition of the sarcolemmal Na(+)-K+ pump.

Animals↗
PubMed1993

Selective and interactive down-regulation of mu- and delta-opioid receptors in human neuroblastoma SK-N-SH cells.

Human neuroblastoma SK-N-SH cells, which contain both mu- and delta-opioid receptors, were grown under conditions that provided a mu:delta ratio of 1.5:1. Both receptors were down-regulated after 72 hr of exposure to 100 nM etorphine. Selective down-regulation was demonstrated using selective opioid agonists; the mu agonist Tyr-D-Ala2-Gly-(Me)Phe4-Gly-ol down-regulated mu- but not delta-opioid receptors, whereas prolonged exposure to the selective delta agonist D-Pen2,D-Pen5-enkephalin resulted in delta- but not mu-opioid receptor down-regulation. Morphine, which binds mu- as well as delta-opioid receptors, down-regulated both receptor subtypes. NG108-15 cells, which contain delta receptors exclusively, were also tested. NG108-15 cells did not exhibit delta-opioid receptor down-regulation when exposed to morphine. The discrepancy between the effect of chronic morphine treatment on delta receptors in SK-N-SH cells and in NG108-15 cells raised the question of whether the coexistence of mu receptors in the former allowed morphine to down-regulate delta receptors. The role of mu-opioid receptors in morphine-induced delta receptor down-regulation was studied by using the irreversible mu antagonist beta-funaltrexamine. Pretreatment of SK-N-SH cells with beta-funaltrexamine prevented down-regulation of delta receptors in response to chronic exposure to morphine but did not affect down-regulation of delta receptors in response to D-Pen2,D-Pen5-enkephalin. The experimental data indicate that morphine-induced delta-opioid receptor down-regulation is dependent on the presence of functional mu receptors in the same cell.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
PubMed1987

TSH binding proteins in rat and human serum.

When serum of hypothyroid rats was fractionated on a Sephadex G-100 column, most of the immunoreactive TSH was found as a front running peak, together with the high molecular weight serum proteins. Similarly, a rTSH preparation (10 mU), chromatographed in the presence of 1 ml of normal rat serum also migrated at the front, however, when a high load of TSH (4.4 U) was added to 1 ml of serum, two immunoreactive peaks were found, suggesting the saturation of the front running fraction. Immunoelectrophoresis and autoradiography of rat or human sera containing the respective 125I-labelled TSHs showed binding of the labelled TSH to IgG, alpha-2-macroglobulin, and to a third unidentified protein, migrating near the albumin line. In order to determine if the bound TSH is biologically active, the high molecular weight protein fraction was separated from hypothyroid rat serum by 45% ammonium sulphate precipitation. Immunoreactivity was determined by RIA and the biological activity was determined, in vitro, by the stimulation of 99Tc uptake by FRTL-5 cells. The 45% ammonium sulphate precipitate contained almost all of the immunoreactivity and the bioactivity of the TSH of whole serum. These results indicate that: a) The endogenous circulating TSH in the hypothyroid rat exists mainly in a protein-bound form and this protein-bound TSH contains most of the hormonal bioactivity of the serum. b) Exogenous TSH binds to serum proteins in euthyroid and hypothyroid rats and in humans. There are three protein fractions in these sera that bind TSH, one of which is an immunoglobulin. The occurrence of TSH binding immunoglobulins may involve autoimmune mechanisms.

Animals↗
PubMed1986

The depression of the auditory nerve-brain-stem evoked response in hypoxaemia--mechanism and site of effect.

During severe hypoxaemia in the cat the ABR was depressed in 2 different patterns: if mean arterial blood pressure (MAP) was maintained then all other evoked potentials (EPs--somatosensory and visual) remained. If MAP was not maintained, all of these EPs were depressed. This study sought to document these different patterns of ABR depression and to ascertain their mechanisms. When MAP fell, the ABR loss began with the later waves and progressed to the earlier waves. These are signs of a central brain lesion. The hypoxaemia, detrimental to normal function of the cardiovascular system, leads to depression of MAP, to a fall in cerebral perfusion pressure and blood flow, to cerebral ischaemia and ABR loss. On the other hand, when MAP was maintained, severe hypoxaemia was accompanied by a depression of all of the ABR waves at the same time. The cochlear microphonic potential was also simultaneously depressed. These are signs of a peripheral, cochlear effect similar to the demonstrated depression of the positive endocochlear resting potential of the scala media and of the cochlear microphonic potential during hypoxaemia. This leads to interference with the cochlear transduction mechanism so that all of the auditory evoked potentials, including the ABR, are simultaneously depressed. These results lead to the suggestion that the ABR abnormalities seen in patients who suffered a hypoxic (anoxic) insult or an ischaemic episode (prolonged interpeak latencies, loss of later waves and finally all waves absent or only the first wave remaining) is always due to ischaemia even when the initial insult was hypoxic.

Animals↗
PubMed1985

The persistence of somatosensory and auditory pathway evoked potentials in severe hypoglycemia in the cat.

In a previous study it was shown that during severe insulin-induced hypoglycemia in rats and cats (0.38 mmol/l, i.e., 6.8 mg% and 0.8 mmol/l, i.e., 14 mg% respectively) with isoelectric EEG, the latency and amplitude of the auditory nerve-brain-stem evoked responses were not affected. In the present study on cats, the above evoked responses were complemented by recording in addition the cortical auditory evoked potential and the peripheral, brain-stem and cortical components of the somatosensory evoked potentials. Each of these evoked potentials remained in the presence of 0.75 mmol/l glucose in plasma. The persistence of the somatosensory cortical evoked potential was unexpected since two other groups have reported the disappearance of this potential during hypoglycemia. The types of neuronal activity which can still be recorded in severe hypoglycemia are probably generated by neuronal structures with lower metabolic demands such as axons and oligosynaptic pathways, surviving on the consumption of endogenous substrates with compensatory elevation of local cerebral blood flow.

Animals↗
PubMed1984

Persistence of auditory nerve response and absence of brain-stem response in severe cerebral ischaemia.

Cerebral ischaemia, in which the brain-stem components of the ABP were isoelectric, was accompanied by the paradoxical persistence of the compound action potential of the auditory nerve (wave 1). This ischaemia was induced in cats by reducing mean arterial blood pressure and elevating intracranial pressure, resulting in decreased cerebral perfusion pressure (CPP). This is unexpected since the inner ear is supplied by a branch of an intracranial artery. To study this phenomenon, CPP was manipulated and when average CPP was 13.5 mm Hg, only wave 1 remained. In 7 of 9 experiments, clamping of both common carotid arteries did not abolish wave 1. Experiments with radioactive tracers demonstrated a remaining residual blood flow through the inner ear. This remaining auditory nerve response is probably not due to a very low metabolism of the inner ear or to the cochlea being supplied by anastomoses from the middle ear, supplied by the external carotid artery. The residual cochlear blood flow and the persistent wave 1 can probably be explained in the following way: at low CPPs the smaller intracranial blood vessels collapse so that the brain tissue is not perfused, leading to loss of the brain-stem components of the ABP. However, flow still persists in the larger intracranial arteries. This blood preferentially flows to the cochlea since the intracochlear pressure is slightly below the intracranial pressure due to the presence of the oval and round windows. Thus a sufficient blood flow to the cochlea is maintained, with sparing of wave 1.

Animals↗
PubMed1983

Patterns of auditory nerve and brainstem-evoked responses (ABR) in different types of peripheral hearing loss.

Auditory brainstem response (ABR) recordings were made in different types of peripheral hearing loss (sensorineural, conductive, and mixed). Scatter diagrams were constructed by plotting different expressions of the hearing loss (audiometric and electrophysiologic) against the obtained wave latencies and for these two sets of variables, correlation coefficients and the parameters of the regression equation were calculated. Consideration is given to the possibility of diagnosing the type of peripheral hearing loss by correlating wave latencies and the electrophysiologic (ABR) threshold.

Adolescent↗