Modulation of Neurotransmission

by arachidonic acid

ARA and its enzymically oxidized products are associated with many physiological processes in the brain including synaptic signaling, neuronal firing, neurotransmitter release neuronal gene expression, cerebral blood flow, and sleep/wake cycle. As stated above, cPLA2 releases ARA from glycerophospholipids. ARA is metabolized to prostaglandins, leukotrienes, and thromboxanes by cyclooxygenases, lipoxygenases, and epoxygenases [58]. ARA regulates glutamate uptake by astroglia. ARA also inhibits glutamate transport in several model systems. In the anterior uvea of the eye, another non-enzymically oxidized product of ARA, 8-isoprostane produces both excitatory and inhibitory effects on sympathetic neurotransmission in isolated mammalian iris ciliary bodies. Furthermore, stimulation of thromboxane receptors by isoprostane mediates the norepinephrine release from sympathetic nerves (4). In addition, other ARA containing endogenous molecules, such as virodhamine (O-arachidonylethanolamine), noladin, arachidonyldopamine, 2-arachidonylglycerol (2-AG) and arachidonylethanolamide (anandamide) have been reported to occur in brain. 2-AG and anandamide (Figure 3) are derived from the non-oxidative metabolism of ARA. 2-AG and anandamide are synthesized through two distinct pathways. Transfer of ARA from sn-1 position of 1,2-arachidonyl-PtdCho to the N-position of PtdEtn results in generation of 1-lyso-2-arachidonyl-PtdCho and N-arachidonyl-PtdEtn. This reaction is catalyzed by a Ca2+-dependent, membrane-associated N-acyltransferase. 1-Lyso-arachidonyl-PtdCho is converted to 2-AG by PLC and N-arachidonyl-PtdEtn is transformed into anandamide by N-acylphosphatidylethanolamine specific PLD (NAPE-PLD), a member of the metallo-P-lactamase family, which specifically hydrolyzes N-acylphosphatidylethanolamine among glycerophospholipids, and appears to be constitutively active [59-60] (Figure 4). The recombinant enzyme hydrolyzed various N-acylphosphatidylethanolamines, including the anandamide precursor N-arachidonoylphosphatidylethanolamine at similar rates, but is inactive with phosphatidylcholine and phosphatidylethanolamine. NAPE-PLD is expressed in hippocampus, cortex, thalamus, hypothalamus, but the intensity of immunostaining in these regions is weaker than in mossy fibers. It is suggested that NAPE-PLD is expressed by specific populations of neurons in the brain and targeted to axonal processes. NAEs generated by NAPE-PLD in axons may act as anterograde synaptic signaling molecules that regulate the activity of postsynaptic neurons [59-60].

Figure 3. Structures of structures of some cannabinoid receptor agonists. Noladin ether (a); 2-arachidonylglycerol (b); Anandamide (c); virodhamine (d); and docosatetraenoyl ethanolamide (e); Arachidonyl-2-'chloroethylamide (f).

An alternative pathway for the generation of 2-AG involves the hydrolysis of 1,2-arachidonyl-PtdCho by PLC, followed by the action of DAG-lipase on 1-acyl-2-arachidonylglycerol. Unlike traditional neurotransmitters, ARA-derived endocannabinoids are not stored in vesicles. They are generated from ARA-containing glycerophospholipids within the neural membranes. Two types of cannabinoid receptor (CB1 and CB2) have been reported to occur in mammalian tissues. The CB1 receptors are abundantly expressed in the brain, whereas CB2 receptors are limited to lymphoid organs. 2-AG and anadamide nonselectively bind to both CB1 and CB2 receptors and act as neurotransmitter or neuromodulators in brain, immune and cardiovascular systems. Both receptors inhibit cAMP formation via Gi/o proteins, and activate mitogen-activated-protein kinase [61].

Figure 4. Pathway showing the generation of anandamide and 2-arachidonylglycerol in brain. Phosphatidylcholine (PtdCho); phosphatidylethanolamine (PtdEtn); protein kinase A (PKA); Cannabinoid receptorl (CB^ N-methyl-D-aspartate receptor (NMDA-R); N-acyltransferase (1); phospholipase C (2); Adenylyl cyclase (3); diacylglycerol lipase (4); N-acylphosphatidylethanolamine-specific phospholipase D (5); monoacylglycerol lipase (6); and amidase (7). Activation of receptors coupled to the phosphatidylinositol-specific phospholipase C and diacylglycerol lipase pathway leads to increases in 2-AG production.

Cannabinoid receptors may activate the extracellular signal-regulated kinase cascade through ceramide signaling. In addition, endocannabinoids also mediate their effects that are independent of cannabinoid receptors. Thus, in pharmacologically relevant concentrations, endocannabinoids modulate the functional properties of voltage-gated ion channels including

P/Q-type Ca2+ channels, Na+ channels and inwardly rectifying K+ channels, and ligand-gated ion channels such as 5-HT3, and nicotinic ACh receptors [62]. Furthermore, functional modulations of ion-transporting membrane proteins such as transient potential receptor-class channels, gap junctions, and neurotransmitter transporters by endocannabinoids have also been demonstrated. Although the molecular mechanisms associated with these effects are unclear, but it is likely that these direct actions of endocannabinoids may be due to their lipophilic structures. It is proposed that additional molecular targets for endocannabinoids may also exist and that these targets represent important sites for cannabinoids to alter either the excitability of the neurons or the response of the neuronal systems [62]. Anandamide mediated signal is deactivated through a two step process, whereby the lipid mediator is transported into cells by a presently uncharacterized entity, and then degraded by the membrane-bound enzyme fatty acid amide hydrolase to produce ethanolamine and ARA [6364].

A large body of preclinical data supports the view that either CB2-selective agonists or CB1 agonists acting at peripheral sites, or with limited CNS exposure, retard pain and neuroinflammation without side effects within the CNS [65]. CB1 receptors involve Gi/o protein participation and are coupled the signal transduction pathways in presynaptic nerve terminal. The G proteins are linked to the cannabinomimetic stimulation of MAP kinase and adenylate cyclase, thereby modulating the generation of cAMP. In the absence of cannabinoids, PKA phosphorylates potassium channel protein and decreases outward potassium current, whereas in the presence of cannabinoids, the phosphorylation of the potassium channel protein increases outward current. Furthermore, cannabinoids also mediate closing of sodium channels. These processes are closely associated with the regulation of neurotransmetter release [66]. In addition, anandamide also acts as a ligand for vanilloid or capsaicin receptor [67]. Direct modulation of NMDA receptor activity by arachidonylethanolamide has also been reported [68]. Cannabinoids also mediate emotional responses by interacting with and S-opioid receptors. These interactions may produce either anti- or pro-anxiety effects which can explain bi-directional action of cannabinoids on anxiety [69].

Endocannabinoids not only mediate retrograde signaling, but also modulate synaptic transmission in various regions of the brain. Depolarization-mediated elevation of intracellular Ca2+ concentration causes endocannabinoid-mediated suppression of excitatory/inhibitory synaptic transmission. Activation of G(q/11)-coupled receptors including group I metabotropic glutamate receptors (mGluRs) also produces endocannabinoid-mediated suppression of synaptic transmission [70]. In the hippocampus, CBi receptors are expressed on axon terminals of GABAergic inhibitory interneurons [71]. A well-known effect of cannabinoids is the impairment of cognitive processes, including short-term memory formation, by altering hippocampal and neocortical functions reflected in network activity. Acting on presynaptically located G protein-coupled receptors in the hippocampus, cannabinoids modulate the release of neurotransmitter molecules. Activation of CB1 receptors reduces GABA release from presynaptic terminals, thereby increasing the excitability of principal cells [71]. The molecular mechanism associated with the inhibition of GABA release is not fully understood. However, in brain endogenous cannabinoid, 2-AG is metabolized by COX-2, which oxygenates 2-AG to generate prostaglandin glyceryl esters

(PGE2-G) and prostaglandin ethanolamides [72]. PGE2-G is the main COX-2 oxidative metabolite of 2-AG. It increases the frequency of miniature inhibitory postsynaptic currents (mlPSCs). Similarly, PGD2-G, PGF2irG and PGD2-EA also increase the frequency of mlPSCs, but PGE2-EA and PGF2ft-EA have no effect. PGE2-G also enhances hippocampal glutamatergic synaptic transmission and mediates neuronal injury/death through caspase-3 activation. The actions of PGE2-G are not mediated via a cannabinoid receptor 1. This increase is not blocked by SR141716, a CB1 receptor antagonist suggesting other mechanisms are involved in this process. Detailed investigations indicate that PGE2-G mediates its effect through ERK, p38 mitogen-activated protein kinase, InsP3, and NF-kB signal transduction pathways. In addition, the PGE2-G-mediated neurotoxicity is attenuated by blockade of the NMDA receptors [72-73]. Thus, endocannabinoid-derived prostaglandin-mediate responses are different from the corresponding arachidonic acid-derived prostaglandin. The effects of endocannabinoid-derived prostaglandins are not mediated through known prostaglandin receptors. It is also shown that the inhibition of COX-2 activity decreases inhibitory synaptic activity and augments depolarization-mediated suppression of inhibition (DSI), whereas the increase in COX-2 augments the synaptic transmission and abolishes DSI. Collectively, these studies suggest that COX-2-derived oxidative metabolites of cannabinoid exert opposite effects to ARA-derived prostaglandin on inhibitory synaptic transmission, and alterations in COX-2 activity may have significant impact on endocannabinoid signaling in hippocampal synaptic activity [73].

Activation of 5-HT2A receptor facilitates the formation and release of 2-AG. This release of 2-AG partially depends on PtdIns-specific phospholipase C activation (Parrish, & Nichols, 2006). The production of DAG downstream of 5-HT2A receptor-mediated by phospholipase D or phosphatidylcholine-specific phospholipase C activation does not contribute to 2-AG formation in NIH3T3-5HT2A cells. DAG is hydrolyzed by diacylglycerol lipase to 2-AG, which may be further degraded by monoacylglycerol lipase to free ARA, a representative substrate of cyclooxygenase.

It remains to be seen whether or not DAG derived from the PLD-mediated hydrolysis of PtdCho can be converted to 2-AG or only inositol phosphoglyceride derived DAG is used for the synthesis of 2-AG. These studies support the view that there is a functional relationship between serotonin and endocannabinoid receptor. Thus, serotonin may act as regulators of endocannabinoid tone at excitatory synapses through the activation of phospholipase C-coupled G-protein coupled receptors [74].

Recently another arachidonic acid containing metabolite, arachidonoyl-L-serine (ARA-S) has been isolated from brain [75]. In contrast to anandamide, ARA-S very weakly interacts with cannabinoid CB1 and CB2 or vanilloid TRPV1 (transient receptor potential vanilloid 1) receptors. It produces endothelium-dependent vasodilation of rat isolated mesenteric arteries and abdominal aorta and stimulates phosphorylation of p44/42 mitogen-activated protein (MAP) kinase and protein kinase B/Akt in cultured endothelial cells [75]. ARA-S also suppresses LPS-mediated secretion of TNF-a in a murine macrophage cell line and in wildtype mice, as well as in mice deficient in CB1 or CB2 receptors. Many of these effects parallel to cannabidiol (Abn-CBD), a synthetic agonist of a putative novel cannabinoid-type receptor. Thus, ARA-S may be an endogenous agonist for this receptor [75]. In contrast, in non-neural cells, ARA-S directly activates large-conductance Ca2+- and voltage-activated K+ (BK(Ca)

channels). These interactions do not involve cannabinoid receptors or cytosolic factors, but depends on the presence of membrane cholesterol [76]. It is proposed that direct BK(Ca) channel activation probably contributes to the endothelium-independent component of ARA-S-mediated mesenteric vasorelaxation [76].

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