A Novel Receptor for N-Arachidonoyl Dopamine Mediates Intracellular Signaling Events in Human T-Cells.

Abstract N-arachidonoyl dopamine (NADA) is a relatively new member of the expanding family of endocannabinoids. NADA is also able to activate the transient receptor potential (TRP) channel V1, which is permeable for calcium and sodium. TRPV1 is the receptor for capsaicin and is present in dorsal horn neurons but it is also functional in other cell types. In a recent report NADA has been shown to effectively inhibit the production of IL-2 and TNFα in human T cells by negatively regulating the activation of several transcription factors, such as NFκB, NF-AT, and AP-1, strongly suggesting that NADA may play important roles in regulating T cell function. However, the signaling mechanisms triggered by NADA are poorly studied in lymphocytes. In the present study we show that NADA activates specific intracellular signaling pathways in isolated human T cells and we tested the hypothesis that these effects are mediated by a cannabinoid receptor or/and the TRPV1 channel. NADA (1 to 100 μM) induced intracellular calcium increase in fura 2-loaded human T cells in a dose-dependent manner. The effect of NADA on free cytosolic calcium is biphasic: it initially triggers mobilization of calcium from the intracellular stores followed by a strong calcium influx lasting more than 15 minutes. In addition, we show that NADA can trigger robust phosphorylation of protein kinase C# as well as the phosphorylation of p44/42 MAP kinase with a time course similar to that observed for intracellular calcium mobilization. Arachidonic acid elicited no calcium increase in isolated T cells, although it caused calcium increase in isolated human granulocytes. Moreover, phenidone (100 μM), a 5-lipoxygenase inhibitor, did not inhibit the calcium response to NADA, demonstrating that the effect of NADA on T cells is not due to generation of arachidonic acid metabolites. Cannabinoid receptors have been reported to inhibit rather than activate calcium channels. However, the CB2 cannabinoid receptor has been linked to calcium mobilization by activation of PLC through the βγ subunit of Gi proteins. We tested the effects of arachidonyl cyclopropylamide, a potent CB1 agonist, and JWH015, a potent CB2 agonist, on isolated human T cells to determine if activation of cannabinoid receptors can explain NADA-induced calcium mobilization. Neither of these two cannabinoid agonists altered intracellular calcium levels when tested at concentrations up to 10 μM. Moreover, AM251 (10 μM), an antagonist at cannabinoid receptors, did not inhibit the effect of NADA on calcium increase, further supporting the view that the effect of NADA on intracellular calcium levels in T cells is not mediated by cannabinoid receptors. Although NADA has been shown in other cell types to cause calcium influx through TRPV1 channels, capsaicin, a classical TRPV1 agonist, failed to alter the intracellular calcium levels in T cells. In addition, capsazepine (100 μM), a TRPV1 antagonist, did not inhibit the response to NADA, strongly suggesting that TRPV1 does not mediate the effect of NADA on calcium influx in human T cells. Our data support the view that NADA may function as an important endogenous modulator on T cell function, activating a novel receptor that has yet to be identified and which is distinct from either cannabinoid receptors or the TRPV1 channel.