Direct modulation of the outer mitochondrial membrane channel, voltage-dependent anion channel 1 (VDAC1) by cannabidiol: a novel mechanism for cannabinoid-induced cell death.

Cannabidiol (CBD) is a non-psychoactive plant cannabinoid that inhibits cell proliferation and induces cell death of cancer cells and activated immune cells. It is not an agonist of the classical CB1/CB2 cannabinoid receptors and the mechanism by which it functions is unknown. Here, we studied the effects of CBD on various mitochondrial functions in BV-2 microglial cells. Our findings indicate that CBD treatment leads to a biphasic increase in intracellular calcium levels and to changes in mitochondrial function and morphology leading to cell death. Density gradient fractionation analysis by mass spectrometry and western blotting showed colocalization of CBD with protein markers of mitochondria. Single-channel recordings of the outer-mitochondrial membrane protein, the voltage-dependent anion channel 1 (VDAC1) functioning in cell energy, metabolic homeostasis and apoptosis revealed that CBD markedly decreases channel conductance. Finally, using microscale thermophoresis, we showed a direct interaction between purified fluorescently labeled VDAC1 and CBD. Thus, VDAC1 seems to serve as a novel mitochondrial target for CBD. The inhibition of VDAC1 by CBD may be responsible for the immunosuppressive and anticancer effects of CBD.

The effects of NMDA receptor antagonists on acute morphine antinociception in mice.

Antagonists of the N-methyl- d-aspartate (NMDA) receptor complex inhibit the development of tolerance to antinociceptive effects of morphine and upon acute administration, influence morphine antinociceptive activity. The analysis of numerous studies investigating acute interaction between NMDA receptor antagonists and morphine in mice indicate a variety of procedural differences and reveal that these compounds may potentiate, attenuate and produce no effect on morphine antinociception. The conditions responsible for such conflicting experimental outcome of acute interaction remain unclear. It appears that the effects of NMDA receptor antagonists on morphine tolerance are not causally related to their acute effects on morphine antinociception.

Uncompetitive NMDA receptor antagonists potentiate morphine antinociception recorded from the tail but not from the hind paw in rats.

We investigated the effects of pretreatment with low-affinity, uncompetitive NMDA receptor antagonists on morphine-induced antinociception in rats using the same intensity of thermal stimulus applied to the tail and the paws. Similar baseline responses to thermal stimuli of the same intensity were recorded from tails and hind paws. However, morphine produced equal antinociception from the tail and hind paw when used at doses of 2.5 and 6 mg/kg, respectively. These doses were used in further experiments. Thirty minutes before morphine, rats were administered the NMDA receptor antagonists dextromethorphan (2.5--30 mg/kg), memantine (2.5--15 mg/kg) and MRZ 2/579 (1-amino-1,3,3,5,5-pentamethyl-cyclohexane HCl) (1.25--10 mg/kg). All three compounds significantly and dose-dependently potentiated morphine-induced antinociception recorded from the tail. However, none of these NMDA receptor antagonists affected morphine antinociception recorded from the paw. These findings suggest that low-affinity NMDA receptor antagonists modulate differently morphine antinociceptive activity recorded from the tail and hind paws.

Selective agonist of group II glutamate metabotropic receptors, LY354740, inhibits tolerance to analgesic effects of morphine in mice.

1. Antagonists of glutamate N-methyl-D-aspartate (NMDA) subtype receptor inhibit the development of tolerance to the antinociceptive effects of opioids. Another way to inhibit the function of glutamate receptors is the stimulation of presynaptic metabotropic group II (mGluRII) receptors. Because LY354740 ((+)-2-aminobicyclo [3,1,0] hexane-2,6-dicarboxylic acid) is the first systemically active agonist of group II mGlu receptors, we investigated if this compound might inhibit the development of tolerance to antinociceptive effects of morphine and fentanyl. 2. As assessed by cumulative dose-response approach in the tail-flick test, administration of 10 mg kg(-1) morphine bid s.c. to male Albino Swiss mice for 6 days, right-shifted morphine dose-response curve by approximately 4 fold. In a separate group of mice, 12 injections of 0.04 mg kg(-1) of fentanyl over 3 days, right-shifted fentanyl dose-response curve by approximately 3.3 fold. 3. In experiment 1, LY354740 (1 and 10, but not 0.1 mg kg(-1)) as well as the reference compound, an uncompetitive NMDA receptor antagonist memantine (7.5 mg kg(-1)) inhibited the development of morphine tolerance. Neither LY354740 (10 mg kg(-1)) nor memantine (7.5 mg kg(-1)) affected the development of tolerance to fentanyl. In experiment 2, neither LY354740 (1 and 10 mg kg(-1)) nor memantine (7.5 mg kg(-1)) affected the tail-flick antinociceptive response, or the acute antinociceptive effect of morphine. 4. The present results are the first to suggest that the development of antinociceptive morphine tolerance may be inhibited by metabotropic group II glutamate agonist.

Clinically available NMDA receptor antagonists memantine and dextromethorphan reverse existing tolerance to the antinociceptive effects of morphine in mice.

The tail-flick test was used to investigate the effects of chronic administration of the N-methyl-D-aspartate (NMDA) receptor antagonists, dextromethorphan, memantine and MRZ 2/579, on the development and reversal of morphine tolerance in mice in three separate experiments. Experiment 1 investigated the effects of NMDA receptor antagonists on the development of tolerance. Morphine (10 mg/kg for 6 days, twice daily) produced a 5.9-fold rightward shift of the cumulative dose-response curves. Co-administration of dextromethorphan, memantine or MRZ 2/579 between tests 1 and 2 dose-dependently (5-10 mg/kg) inhibited the development of morphine tolerance. In experiment 2, in which the effects on the reversal were investigated, morphine-tolerant mice were treated b.i.d. for an additional 6 days (between tests 2 and 3) with vehicle+vehicle, NMDA receptor antagonist+vehicle, vehicle+morphine or NMDA receptor antagonist+morphine. Morphine-tolerant mice treated with vehicle+vehicle remained morphine tolerant, whereas this residual morphine tolerance was inhibited by administration of all three NMDA antagonists (each 10 mg/kg). Morphine-tolerant mice receiving vehicle+morphine injections demonstrated an unchanged degree of antinociceptive tolerance. In these mice, the co-administration of memantine and MRZ 2/579, but not dextromethorphan, resulted in the reversal of morphine tolerance. In experiment 3, memantine and MRZ 2/579 (10 mg/kg) inhibited the acute antinociceptive effect of morphine, but dextromethorphan did not. These data indicate that low-affinity, clinically available and/or therapeutically promising NMDA receptor antagonists may be used to inhibit ongoing morphine tolerance.

Clinically available NMDA antagonist, memantine, attenuates tolerance to analgesic effects of morphine in a mouse tail flick test.

Converging lines of evidence indicate that N-methyl-D-aspartate (NMDA) receptor antagonists attenuate the development of morphine tolerance tested in antinociception assays in rodents. The present study extends these findings to the effects of clinically available NMDA receptor antagonist, memantine. Male Albino Swiss mice were tested for analgesia using the tail-flick apparatus. Preliminary experiment was designed to find out the optimal dose of morphine and the number of injections that would produce tolerance to its analgesic effects. In the main experiment, during the development of tolerance period (6 days), mice received 10 mg/kg sc b.i.d. morphine injections in the animal room (non-associative tolerance). This treatment resulted in 5.8 fold rightward shift of morphine cumulative dose-response effect from 3.39 mg/kg on day 1 to 16.19 mg/kg on day 8 of the experiment. Memantine pretreatment (5 and 10 mg/kg, but not 2.5 mg/kg), given 30 min prior to each morphine dose during the development of tolerance period, inhibited the rightward shift of morphine cumulative dose-response curve. Thus, pretreatment with memantine at doses of 2.5, 5 and 10 mg/kg resulted in ED50 values of 12.13, 4.74 and 1.95 mg/kg, respectively, corresponding to 3.35, 1.02 and 0.94 fold changes. These data indicate that low affinity, clinically available NMDA receptor antagonist, memantine, may be used to inhibit the development of morphine tolerance.