Ora Keren

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 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.

Long-term cognitive deficits induced by a single, extremely low dose of tetrahydrocannabinol (THC): behavioral, pharmacological and biochemical studies in mice.

We have previously reported that an injection of a single, extremely low dose (0.001 mg/kg) of delta 9-tetrahydrocannabinal (THC, the major psychoactive ingredient of marijuana) to mice deteriorated their performance in the Morris water maze test 3 weeks later. In the present study we verify our original findings and show that the long-term cognitive deficits that are induced in mice by a low dose of THC are even more pronounced in another behavioral test-the water T-maze. This effect was abolished by the CB1 receptor antagonist SR141716A, indicating the involvement of CB1 receptors. In an attempt to find a biochemical correlate to these deleterious consequences of such a low dose of THC, we investigated its effect on the activation of extracellular signal-regulated kinase (ERK1/2) in the cerebellum and hippocampus of the mice, two brain regions that were shown to participate in spatial learning. A significant increase in ERK1/2 phosphorylation was found in the cerebellum of mice 24 h following the injection of 0.001 mg/kg THC. These findings lead to further studies into the neuronal mechanisms underlying the long-term deleterious effects of THC and should be taken into consideration when evaluating the therapeutic benefits of cannabinoid drugs.

Modulation of extracellular signal-regulated kinase (ERK) by opioid and cannabinoid receptors that are expressed in the same cell.

In the present study we investigated the signal transduction pathways leading to the activation of extracellular signal-regulated kinase (ERK) by opioid or cannabinoid drugs, when their receptors are coexpressed in the same cell-type. In N18TG2 neuroblastoma cells, the opioid agonist etorphine and the cannabinoid agonist CP-55940 induced the phosphorylation of ERK by a similar mechanism that involved activation of delta-opioid receptors or CB1 cannabinoid receptors coupled to Gi/Go proteins, matrix metalloproteases, vascular endothelial growth factor (VEGF) receptors and MAPK/ERK kinase (MEK). In HEK-293 cells, these two drugs induced the phosphorylation of ERK by separate mechanisms. While CP-55940 activated ERK by transactivation of VEGFRs, similar to its effect in N18TG2 cells, the opioid agonist etorphine activated ERK by a mechanism that did not involve transactivation of a receptor tyrosine kinase. Interestingly, the activation of ERK by etorphine was resistant to the inhibition of MEK, suggesting the possible existence of a novel, undescribed yet mechanism for the activation of ERK by opioids. This mechanism was found to be specific to etorphine, as activation of ERK by the micro-opioid receptor (MOR) agonist DAMGO ([D-Ala(2), N-Me-Phe(4), Gly(5)-ol] enkephalin) was mediated by MEK in these cells, suggesting that etorphine and DAMGO activate distinct, ligand-specific, conformations of MOR. The characterization of cannabinoid- and opioid-induced ERK activation in these two cell-lines enables future studies into possible interactions between these two groups of drugs at the level of MAPK signaling.

A single low dose of tetrahydrocannabinol induces long-term cognitive deficits

Delta(9)-Tetrahydrocannabinol (THC) was shown to exert either neuroprotective or neurotoxic effects. Based on our in vitro studies and on pharmacokinetic considerations, we have recently presented a hypothesis that explains this dual activity of THC. This explanation is based on the assumption that extremely low doses of cannabinoids are neurotoxic. The present study verifies this assumption and shows that a single injection of 0.001 mg/kg THC (3-4 orders of magnitude lower than conventional doses) significantly affected the performance of mice in the Morris water maze test 3 weeks later. The THC-injected mice showed both longer escape latencies and lower scores in the probe tests compared to their matched controls, indicating the induction of cognitive deficits.

Pre- and post-conditioning treatment with an ultra-low dose of Δ9-tetrahydrocannabinol (THC) protects against pentylenetetrazole (PTZ)-induced cognitive damage

Preconditioning, a phenomenon where a minor noxious stimulus protects from a subsequent more severe insult, and post-conditioning, where the protective intervention is applied following the insult, offer new insight into the neuronal mechanism(s) of neuroprotection and may provide new strategies for the prevention and treatment of brain damage. We have previously reported that a single administration of an extremely low dose of Δ(9)-tetrahydrocannabinol (THC; the psychoactive ingredient of marijuana) to mice induced minor long-lasting cognitive deficits. In the present study we examined the possibility that such a low dose of THC will protect the mice from more severe cognitive deficits induced by the epileptogenic drug pentylenetetrazole (PTZ). THC (0.002 mg/kg, a dose that is 3-4 orders of magnitude lower than the doses that induce the conventional effects of THC) was administered 1-7 days before, or 1-3 days after the injection of PTZ (60 mg/kg). The consequences of this treatment were studied 3-7 weeks later by various behavioral tests that evaluated different aspects of memory and learning. We found that a single administration of THC either before or after PTZ abolished the PTZ-induced long-lasting cognitive deficits. Biochemical studies indicated a concomitant reduction in phosphorylated-ERK (extracellular signal-regulated kinase) in the cerebella of mice 7 weeks following the injection of THC. Our results suggest that a pre- or post-conditioning treatment with extremely low doses of THC, several days before or after brain injury, may provide safe and effective long-term neuroprotection.

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.

Divers pathways mediate δ-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 δ-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 [3H]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 δ-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.

Humoral endorphin: A new endogenous factor with opiate-like activity

Abstract Humoral (H) endorphin, a novel endogenous opioid ligand detected in brain, blood and cerebrospinal fluid was tested in a series of opiate sensitive assays. H-endorphin displaced radiolabeled enkephalin from its specific bindings sites and inhibited the electrically evoked contraction of the guinea pig ileum and mouse vas deferens. When injected to unanesthesized animals, humoral endorphin induced analgesia in rats and mydriasis in mice. The activity of H-endorphin both in vitro and in vivo attests to its opioid nature. However, while its antinociceptive effect was blocked by naloxone, mydriasis induced by H-endorphin was resistant to the effect of the opiate antagonist. Similarly, intermediate concentrations of naloxone inhibited the effect of H-endorphin on the guinea pig ileum while its effect on the mouse vas deferens was completely refractory to naloxone. The physiological function of humoral endorphin in various naturally occuring states that show similar paradoxical interactions with naloxone is discussed.