The endogenous cannabinoids (endocannabinoids) are lipid molecules that may mediate retrograde signaling at central synapses and other forms of short-range neuronal communication. The monoglyceride 2-arachidonoylglycerol (2-AG) meets several criteria of an endocannabinoid substance: ( i ) it activates cannabinoid receptors; ( ii ) it is produced by neurons in an activity-dependent manner; and ( iii ) it is rapidly eliminated. 2-AG inactivation is only partially understood, but it may occur by transport into cells and enzymatic hydrolysis. Here we tested the hypothesis that monoglyceride lipase (MGL), a serine hydrolase that converts monoglycerides to fatty acid and glycerol, participates in 2-AG inactivation. We cloned MGL by homology from a rat brain cDNA library. Its cDNA sequence encoded for a 303-aa protein with a calculated molecular weight of 33,367 daltons. Northern blot and in situ hybridization analyses revealed that MGL mRNA is heterogeneously expressed in the rat brain, with highest levels in regions where CB ¹ cannabinoid receptors are also present (hippocampus, cortex, anterior thalamus, and cerebellum). Immunohistochemical studies in the hippocampus showed that MGL distribution has striking laminar specificity, suggesting a presynaptic localization of the enzyme. Adenovirus-mediated transfer of MGL cDNA into rat cortical neurons increased MGL expression and attenuated N -methyl-D-aspartate/carbachol-induced 2-AG accumulation in these cells. No such effect was observed on the accumulation of anandamide, another endocannabinoid lipid. The results suggest that hydrolysis by means of MGL is a primary mechanism for 2-AG inactivation in intact neurons.

Mitochondrial Ca²⁺efflux is linked to numerous cellular activities and pathophysiological processes. Although it is established that an Na⁺-dependent mechanism mediates mitochondrial Ca²⁺efflux, the molecular identity of this transporter has remained elusive. Here we show that the Na⁺/Ca²⁺exchanger NCLX is enriched in mitochondria, where it is localized to the cristae. Employing Ca²⁺and Na⁺fluorescent imaging, we demonstrate that mitochondrial Na⁺-dependent Ca²⁺efflux is enhanced upon overexpression of NCLX, is reduced by silencing of NCLX expression by siRNA, and is fully rescued by the concomitant expression of heterologous NCLX. NCLX-mediated mitochondrial Ca²⁺transport was inhibited, moreover, by CGP-37157 and exhibited Li⁺dependence, both hallmarks of mitochondrial Na⁺-dependent Ca²⁺efflux. Finally, NCLX-mediated mitochondrial Ca²⁺exchange is blocked in cells expressing a catalytically inactive NCLX mutant. Taken together, our results converge to the conclusion that NCLX is the long-sought mitochondrial Na⁺/Ca²⁺exchanger.

Increasing evidence suggests that glutamate neurotoxicity is partly mediated by reactive oxygen species, formed as a consequence of several processes, including arachidonic acid metabolism and nitric oxide production. Here we used an oxidation-sensitive indicator, dihydrorhodamine 123, in combination with confocal microscopy, to examine the hypothesis that electron transport by neuronal mitochondria may be an important source of glutamate-induced reactive oxygen species (ROS). Exposure to NMDA, but not kainate, ionomycin, or elevated potassium stimulated oxygen radical production in cultured murine cortical neurons, demonstrated by oxidation of nonfluorescent dihydrorhodamine 123 to fluorescent rhodamine 123. Electron paramagnetic resonance spectroscopy studies using 5,5-dimethyl-1- pyrroline-N-oxide (DMPO) as a radical-trapping agent, also showed production of ROS by cortical neurons after NMDA but not kainate exposure. NMDA-induced ROS production depended on extracellular Ca2+, and was not affected by inhibitors of nitric oxide synthase or arachidonic acid metabolism. The increased production of ROS was blocked by inhibitors of mitochondrial electron transport, rotenone or antimycin, and mimicked by the electron transport uncoupler, carbonyl cyanide p-trifluoromethoxyphenylhydrazone. These data support the possibility that NMDA receptor-mediated, Ca(2+)-dependent uncoupling of neuronal mitochondrial electron transport may contribute to the oxidative stress initiated by glutamate exposure.

Apoptosis of mouse neocortical neurons induced by serum deprivation or by staurosporine was associated with an early enhancement of delayed rectifier ( I _K ) current and loss of total intracellular K ⁺ . This I _K augmentation was not seen in neurons undergoing excitotoxic necrosis or in older neurons resistant to staurosporine-induced apoptosis. Attenuating outward K ⁺ current with tetraethylammonium or elevated extracellular K ⁺ , but not blockers of Ca ²⁺ , Cl ⁻ , or other K ⁺ channels, reduced apoptosis, even if associated increases in intracellular Ca ²⁺ concentration were prevented. Furthermore, exposure to the K ⁺ ionophore valinomycin or the K ⁺ -channel opener cromakalim induced apoptosis. Enhanced K ⁺ efflux may mediate certain forms of neuronal apoptosis.