Sheila A. Hunter

Receptor mediation in cannabinoid stimulated arachidonic acid mobilization and anandamide synthesis.

Numerous reports have suggested that increased synthesis of eicosanoids is a significant effect of cannabinoids in several models including the human. To address the question of receptor mediation in this process we have carried out experiments using oligonucleotides that are antisense to the CB1 and to the CB2 receptors. We have synthesized sense, antisense and random oligonucleotide probes to test for receptor involvement in THC stimulation of arachidonic acid release in three cell lines of both central and peripheral origin. Treatment of N18 mouse neuroblastoma cells with the CB1 antisense probe, at two concentrations, resulted in a dramatic decrease of THC stimulated arachidonate release while treatment with antisense CB2 was less effective. Synthesis of the novel eicosanoid, anandamide, was also reduced by antisense CB1 but not by antisense CB2. Western blot analysis indicated a decreased level of CB1 in CB1 antisense treated cells. The CB1 antagonist, SR141716A, was effective in reducing the THC elevated levels of free arachidonate in these cells in agreement with the antisense data. In the macrophage line, RAW 264.7, we found that while the sense, the random and the CB1 antisense oligonucleotides were ineffective, the CB2 antisense probe gave significant reductions of the THC induced response. The CB2 probe was also effective in reducing the release of arachidonate in WI-38 human lung fibroblasts. These findings support the idea of a receptor mediated process for cannabinoid stimulation of eicosanoid synthesis.

Phospholipase participation in cannabinoid-induced release of free arachidonic acid

The exposure of cells in culture to cannabinoids results in a rapid and significant mobilization of phospholipid bound arachidonic acid. In vivo, this effect has been observed as a rise in eicosanoid tissue levels that may account for some of the pharmacological actions of delta 9-tetrahydrocannabinol (THC), the major psychoactive cannabinoid. Fluoroaluminate pretreatment of mouse peritoneal cells potently reduced the cannabinoid response, while promoting arachidonate release on its own, consistent with earlier observations that this effect may be a receptor/G-protein-mediated process. Further support for receptor mediation was the demonstration of saturable, high-affinity cannabinoid binding in these cells. THC potency was reduced in the presence of ethanol, and was accompanied by significant increases in phosphatidylethanol (PdEt) levels, a unique product of phospholipase D (PLD) activity. THC-dependent arachidonate release was reduced partially in similar amounts by either propranolol or wortmannin, further implicating PLD as a mediator of THC action. A central role for diacylglyceride (DAG), a secondary product of PLD metabolism, in this THC-induced process, both as a source of arachidonate and as a stimulator of protein kinase C (PKC), is supported by the data in this report. Cells exposed to phorbol ester for 18 hr prior to THC challenge became less responsive, indicating a possible role for PKC. The involvement of PKC further suggests participation by phospholipase A2 (PLA2) whose activity may be regulated by the former. Treatment of cells with interleukin-1 alpha, an agent known to elevate PLA2 levels, caused an increase in the THC response, supporting a role for this enzyme in the release reaction. Direct evidence, by immunoblotting, for the activation and phosphorylation of PLA2 by THC was also obtained. In summary, the evidence presented in this report indicates that THC-induced arachidonic acid release occurs through a series of events that are consistent with a receptor-mediated process involving the stimulation of one or more phospholipases.

Stimulation of prostaglandin synthesis in WI-38 human lung fibroblasts following inhibition of phospholipid acylation by p-hydroxymercuribenzoate

The release of arachidonic acid and its metabolites, prostaglandin E2 and thromboxane A2, from WI-38 human lung fibroblasts was modulated by p-hydroxymercuribenzoate. Exposure to the inhibitor resulted in a dose-dependent decrease in [1-14C]arachidonic acid uptake and incorporation into phospholipids and neutral lipid pools. Activities of lung fibroblast arachidonyl-CoA synthetase and lysolecithin acyltransferase were inhibited by 100 microM p-hydroxymercuribenzoate. [14C]Arachidonic acid labelled fibroblasts exhibited an increased release of [14C]arachidonate and [14C]prostaglandin E2 of 54% and 112%, respectively, when exposed to 100 microM of inhibitor. The stimulatory effects of 8.0 microM delta 1-tetrahydrocannabinol on arachidonate release and prostaglandin E synthesis (Burstein, S., Hunter, S.A., Sedor, C. and Shulman, S. (1982) Biochem. Pharmacol. 31, 2361-2365) were modified by the inclusion of inhibiting agent, resulting in a 608% stimulation in arachidonic acid release, while prostaglandin E2 and thromboxane A2 synthesis increased 894% and 390%, respectively, over levels obtained by untreated cells. The levels of arachidonate metabolites were altered by inhibitor when compared to cells treated with cannabinoid alone. No significant inhibition by delta 1-tetrahydrocannabinol was found on arachidonic uptake in these cells. In unlabelled studies, p-hydroxymercuribenzoate resulted in a profound, dose-dependent stimulation of prostaglandin E synthesis of 1490% at 150 microM inhibitor concentration. These results provide evidence that free arachidonate is reincorporated via acylation, thereby implicating this pathway as a possible control mechanism for the synthesis of arachidonic acid metabolites.

Prostaglandins and cannabis—IX: Stimulation of prostaglandin E2 synthesis in human lung fibroblasts by Δ1-tetrahydrocannabinol

Preliminary data [S. Burstein and S. A. Hunter, Biochem. Pharmac. 27, 1275 (1978)] showed that cannabinoids at levels of 1 microM or greater elevated the concentrations of prostaglandins in cell culture models. Further study [S. Burstein and S. A. Hunter, J. clin. Pharmac. 21, 240S (1981)] led to the suggestion that this effect was due to a stimulation of phospholipase A2 resulting in the release of free arachidonic acid which was then partly converted into the prostaglandin(s) normally synthesized by the particular target system. The present report gives detailed data on the cannabinoid-induced synthesis of prostaglandin E2 by te WI-38 fibroblast derived from human lung. The effect could be blocked by pretreatment with mepacrine, a phospholipase inhibitor, and aspirin, a cyclooxygenase inhibitor. These findings lend support to the hypothesis that some of the in vivo actions of the cannabinoids are due to modulations in prostaglandin synthesis at various tissue sites.

Prostaglandins and Cannabis—VIII. Elevation of Phospholipase A2 Activity by Cannabinoids in Whole Cells and Subcellular Preparations

Abstract: The previously reported release of arachidonic acid by THC has now been demonstrated in murine Leydig cells and WI‐38 human lung fibroblasts showing the generality of the effect. The release reaction could be antagonized by phospholipase A2 inhibitors such as quinacrine and quinine, suggesting that THC can stimulate the activity of this enzyme. Further evidence for this possibility was obtained by demonstrating the release effect on a subcellular fraction which contained the phospholipase A2 activity. The stimulation of this enzyme could have profound effects on prostaglandin synthesis and/or on the integrity of various membrane structures.

Cannabinoids and pain responses: a possible role for prostaglandins.

The principal metabolite of Δ1‐THC, Δ1‐THC‐7‐oic acid exhibits significant analgesic action in the mouse hot plate test. The parent Δ1‐THC has a similar effect when measured at later time points; however, 10 min after drug administration, a pronounced hyperalgesia is seen. This hyperalgesia can be inhibited by prior administration of either indomethacin or Δ1‐THC‐7‐oic acid, presumably because of their ability to inhibit eicosanoid synthesis. Administration of prostaglandin E2 (PGE2), at doses that were a small fraction of the Δ1‐THC given, resulted in a strong hyperalgesic response. Unlike Δ1‐THC, the metabolite does not produce a cataleptic state in the mouse, which eliminates this as a basis for the hot plate response. The evidence presented is consistent with a mechanism in which the metabolite inhibits eicosanoid synthesis whereas the parent drug elevates tissue levels of prostaglandins.— Burstein, S. H.; Hull, K.; Hunter, S. A.; Latham, V. Cannabinoids and pain responses: a possible role for prostaglandins. FASEB J. 2: 3022‐3026; 1988.

Effects of cannabinoids on the activities of mouse brain lipases

Cannabinoids were found to augment phospholipase activities and modify lipid levels of mouse brain synaptosomes, myelin and mitochondria. Delta-1-tetrahydrocannabinol (Δ1-THC) and several of its metabolites induced a dose-dependent (0.32–16 μM) stimulation of phospholipase A2 (PLA2) activity resulting in the increased release of free arachidonic acid from exogenous [1-14C]phosphatidylcholine (PC). The potencies of the cannabinoids in modulating PLA2 activity were approximately of the order: 7-OH-Δ1-THC > Δ1-THC > 7-oxo-Δ1-THC > Δ1-THC-7oic acid = 6α OH-Δ1-THC ≫ 6β-OH-Δ1-THC. The hydrolysis of phosphatidylinositol (PI) by synaptosomal phospholipase C (PLC) was enhanced significantly by Δ1-THC and promoted diacylglyceride levels by greater than 100 percent compared to control values. In contrast, arachidonate was the major product resulting from phospholipase activities of a 20,000g pellet. Synaptosomal diacylglyceride lipase activity was inhibited by Δ1-THC. [1-14C]Arachidonic acid was readily incorporated into subcellular membrane phospholipids and after exposure to cannabinoids led to diminished phosphoglyceride levels and concomitant increases in released neutral lipid products. These data suggest that cannabinoids control phospholipid turnover and metabolism in mouse brain preparations by the activation of phospholipases and, through this mechanism, may exert some of their effects.

Stimulation of anandamide biosynthesis in N-18TG2 neuroblastoma cells by δ9-tetrahydrocannabinol (THC)

A concentration-related stimulation of anandamide (arachidonylethanolamide) synthesis by delta 9-tetrahydrocannabinol (THC) was observed in N-18TG2 neuroblastoma cells. Anandamide was detected and measured using an approach in which [3H]arachidonic acid and [14C]ethanolamine were incorporated into the phospholipids of subconfluent monolayers of cells, and the radiolabeled products were analyzed by TLC following agonist exposure. Both precursors showed similar concentration-response relationships and time dependencies consistent with the production of a product containing both the ethanolamine and arachidonyl moieties. The radiolabeled product also migrated together with authentic anandamide on two-dimensional TLC, confirming its identity as arachidonylethanolamide. Approximately two-thirds of the observed synthesis could be inhibited by 1 microM wortmannin, an agent previously reported to inhibit THC-stimulated arachidonic acid release. These findings are in agreement with reports showing that THC can mobilize phospholipid bound arachidonic acid, leading to the production of other eicosanoids.

Elevation of brain prostaglandin E2 levels in rodents by Δ1-tetrahydrocannabinol

Abstract An isotopic dilution procedure using specific prostaglandin E 2 (PGE 2 ) brain receptors was utilized to determine the changes in brain PGE 2 levels subsequent to drug exposure. Delta-1-tetrahydrocannabinol (Δ 1 -THC) stimulated PGE 2 synthesis resulting in increased brain concentrations when compared with vehicle treated rats and mice. Indomethacin markedly inhibited the Δ 1 -THC elevated rise in PGE 2 levels presumably by inhibition of prostaglandin synthetase. The Δ 1 -THC-induced increase in PGE 2 brain levels was also suppressed by i.v. administered rabbit PGE 2 -antiserum. This suggests that one of the sites of Δ 1 -THC action is extracerebral and from here a portion of the released prostaglandins are transported to the brain. These results add further support to previous data that Δ 1 -THC given orally results in an increase in brain PGE 2 levels.

Prostaglandins and cannabis—XVI: Antagonism of δ1-tetrahydrocannabinol action by its metabolites

Prior exposure of cells in vitro to delta 1-tetrahydrocannabinol-7-oic acid (delta 1-THC-7-oic acid) reduced the degree of stimulation of prostaglandin synthesis incurred by subsequent treatment with delta 1-THC. The site of action of this inhibitory effect seemed to be on cyclooxygenase and not at the earlier step involving the phospholipase-mediated release of arachidonic acid. delta 1-THC-7-oic acid is a major metabolite of delta 1-THC and has no psychoactivity in humans. Our findings raise the possibility, however, that it may influence the in vivo activities of delta 1-THC by antagonizing its stimulatory action on cellular prostaglandin synthesis.