Yosef Sarne

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

Abstract1. Opioid (μ, δ, κ) and cannabinoid (CB1, CB2) receptors are coupled mainly toGi/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]GTPγS 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 μ- and δ-opioid receptors led to stimulation of [35S]GTPγS binding by either μ-selective (DAMGO) or δ-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 μ- or δ-opioid receptors.4. On the other hand, in N18TG2 neuroblastoma cells, which endogenously coexpress CB1 and δ-opioid receptors, as well as in SK-N-SH neuroblastoma cells, which coexpress μ- and δ-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 receptorsubtype.5. These results suggest a fundamental difference between native and artificially transfected cells regarding the compartmentalization of receptors and GTP-binding proteins.

Opiates Inhibit Acetylcholine Release from Torpedo Nerve Terminals by Blocking Ca2+ Influx

Abstract: In the present communication we report that Ca2+‐dependent acetylcholine release from K+‐depolarized Torpedo electric organ synaptosomes is inhibited by morphine, and that this effect is blocked by the opiate antagonist naloxone. This finding suggests that the purely cholinergic Torpedo electric organ neurons contain pre‐synaptic opiate receptors whose activation inhibits acetylcholine release. The mechanisms underlying this opiate inhibition were investigated by comparing the effects of morphine on acetylcholine release induced by K+ depolarization and by the Ca2+ ionophore A23187 and by examining the effect of morphine on 45Ca2+ influx into Torpedo nerve terminals. These experiments revealed that morphine inhibits 45Ca2+ influx into K+‐depolarized Torpedo synaptosomes and that this effect is blocked by naloxone. The effects of morphine on K+ depolarization‐mediated 45Ca2+ influx and on acetylcholine release have similar dose dependencies (half‐maximal inhibition at 0.5–1 μM), suggesting that opiate inhibition of release is due to blockage of the presynaptic voltage‐dependent Ca2+ channel. This conclusion is supported by the finding that morphine does not inhibit acetylcholine release when the Ca2+ channel is bypassed by introducing Ca2+ into the Torpedo nerve terminals via the Ca2+ ionophore.

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.

The cannabinoid agonist DALN positively modulates L-type voltage-dependent calcium-channels in N18TG2 neuroblastoma cells

The present study demonstrates a novel stimulatory effect of a cannabinoid agonist on calcium channels. DALN (1 nM) potentiated 45Ca(2+)-uptake by N18TG2 neuroblastoma cells, an effect that was abolished by the specific CB1 receptor antagonist SR141716A. The stimulation of 45Ca(2+)-uptake by DALN was resistant to pertussis toxin (PTX), suggesting that Gi/Go GTP-binding proteins did not mediate this effect. Furthermore, PTX unmasked a stimulatory effect of a high concentration of DALN (1 microM), which by itself failed to stimulate calcium uptake in naive cells. The stimulatory effect of DALN on calcium entry to the cells was blocked by nicardipine but not by omega-conotoxin GVIA, indicating the entry of calcium through L-type voltage-dependent calcium channels. Blocking cAMP-dependent protein kinase (PKA) by H-89 completely eliminated the elevation in calcium uptake, while blocking protein kinase C (PKC) by chelerythrine and calphostine-C only partially attenuated the stimulation. Blocking calmodulin by W-7 revealed a similar partial inhibition of the stimulatory effect of DALN. Hence, we suggest a cannabinoid-specific, PTX-insensitive, stimulatory effect on L-type voltage-dependent calcium channels, which is mediated by PKA and modulated by PKC and calmodulin.

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.

Footshock-induced analgesia: Its opioid nature depends on the strain of rat

Previous studies have indicated that stressful footshock can induce both opioid, naloxone-sensitive, and non-opioid, naloxone-insensitive forms of analgesia, depending on stimulation parameters used with 30 min of intermittent footshock (3 mA, 1 s on, 5 s off) producing opioid analgesia and 3 min of continuous shock (3 mA) producing non-opioid analgesia. Using a local strain of Charles River (CR)-derived rats we conducted a parametric investigation of footshock-induced analgesia applying both AC and DC scrambled shock ranging from 1 to 4 mA, continuous shock of 1, 3 and 5 min in duration and intermittent shock lasting 1, 3, 5, 10, 20, 30 and 80 min. All shock parameters produced potent analgesia. In no case did 10 mg/kg of naloxone block this analgesia. Varying the dose of the antagonist (0.1-10 mg/kg) and testing the animals at different points in the diurnal cycle did not result in the emergence of naloxone-sensitive anangesia. Based on the assumption that non-opioid systems may mask the activity of opioid analgesia systems, we attempted to either enhance opioid analgesia by: preventing enkephalin degradation by the use of D-phenylalanine; increasing the entry of blood-borne opioids into the brain by the use of DMSO; and the attenuation of non-opioid analgesia by the use of reserpine. In no case did a naloxone-sensitive component of analgesia emerge. To test whether the animals possess an intact opioid analgesia system, both electrical stimulation of, and injection of opiates into the periaqueductal gray (PAG) were examined. Both procedures produced analgesia which was reversed by naloxone.(ABSTRACT TRUNCATED AT 250 WORDS)

The dual neuroprotective–neurotoxic profile of cannabinoid drugs

Extensive in vitro and in vivo studies have shown that cannabinoid drugs have neuroprotective properties and suggested that the endocannabinoid system may be involved in endogenous neuroprotective mechanisms. On the other hand, neurotoxic effects of cannabinoids in vitro and in vivo were also described. Several possible explanations for these dual, opposite effects of cannabinoids on cellular fate were suggested, and it is conceivable that various factors may determine the final outcome of the cannabinoid effect in vivo. In the current review, we focus on one of the possible reasons for the dual neuroprotective/neurotoxic effects of cannabinoids in vivo, namely, the opposite effects of low versus high doses of cannabinoids. While many studies reported neuroprotective effects of the conventional doses of cannabinoids in various experimental models for acute brain injuries, we have shown that a single administration of an extremely low dose of Δ9‐tetrahydrocannabinol (THC) (3–4 orders of magnitude lower than the conventional doses) to mice induced long‐lasting mild cognitive deficits that affected various aspects of memory and learning. These findings led to the idea that this low dose of THC, which induces minor damage to the brain, may activate preconditioning and/or postconditioning mechanisms and thus will protect the brain from more severe insults. Indeed, our recent findings support this assumption and show that a pre‐ or a postconditioning treatment with extremely low doses of THC, several days before or after brain injury, provides effective long‐term cognitive neuroprotection. The future therapeutical potential of these findings is discussed.

Multiple mechanisms of CB1 cannabinoid receptors regulation

Agonist-induced regulation of cannabinoid CB1 receptors was examined in HEK-293 cells transfected with CB1 receptors and in neuroblastoma N18TG2 cells that naturally express CB1 receptors. In HEK-293 cells, CB1 receptors internalization proceeded, in parallel, via clathrin-coated pits and caveolae. Simultaneous disruption of both pathways induced compensatory endocytic mechanism(s). In N18TG2 cells, endocytosis was not mediated by caveolae-like membrane domains. Heterologous, opioid-induced, downregulation of CB1 receptors was evident in HEK-293 but not N18TG2 cells. The data demonstrate the existence of multiple pathways of CB1 receptors regulation.

The Stimulatory Effect of Cannabinoids on Calcium Uptake Is Mediated by Gs GTP-Binding Proteins and cAMP Formation

Cannabinoids are neurodepressive drugs that convey their cellular action through Gi/o GTP-binding proteins which reduce cAMP formation and Ca2+ influx. However, a growing body of evidence indicates that the stimulatory effects of cannabinoids include the elevation in cAMP and cytosolic Ca2+ concentration. The present study expands our previous findings and demonstrates that, in N18TG2 neuroblastoma cells, the cannabinoid agonist desacetyllevonantradol (DALN) stimulates both cAMP formation and Ca2+ uptake. The stimulatory effect of DALN on cAMP formation was not eliminated by blocking Ca2+ entry to the cells, while its stimulatory effect on Ca2+ uptake was abolished by blocking cAMP-dependent protein kinase. Furthermore, elevating cAMP by forskolin stimulated calcium uptake, while elevating the intracellular Ca2+ concentration by ionomycin or KCl failed to stimulate cAMP formation. These findings suggest that cAMP production precedes the influx of Ca2+ in the cannabinoid stimulatory cascade. The stimulatory effect of DALN on calcium uptake resisted pertussis toxin treatment, and was completely blocked by introducing anti-Gs antibodies into the cells, indicating that the stimulatory activity of cannabinoids is mediated by Gs GTP-binding proteins. The relevance of the cellular stimulatory activity of DALN to the pharmacological profile of cannabinoid drugs is discussed.

Anti‐arrhythmic activities of opioid agonists and antagonists and their stereoisomers

1 A series of opioid agonists, antagonists and their (+)‐stereoisomers were tested for antiarrhythmic activity in the rat coronary artery occlusion model. 2 Naloxone (0.01–2 mg kg−1) significantly reduced the incidence and severity of cardiac arrhythmias, in accordance with previous published studies. 3 The non‐opioid stereoisomer, (+)‐naloxone, was equipotent with naloxone against occlusion‐induced arrhythmia. 4 Similar non‐stereospecific antiarrhythmic effects were induced by another opioid antagonist, Win 44,441–3 and its stereoisomer Win 44,441–2. 5 The opioid agonists, morphine and levorphanol, protected against occlusion‐induced arrhythmia as did the opioid antagonists, and the (+)‐stereoisomer, dextrorphan, was equipotent to levorphanol. 6 It is concluded that the antiarrhythmic effects of opioid drugs are not mediated by opioid receptors. A direct effect on ionic currents in cardiac muscle is suggested as the mechanism of opioid antiarrhythmic activity.