Differential increase in endocannabinoid levels at the plasma and intracellular membranes

Endocannabinoids (eCBs) modulate the activity of proteins expressed at the plasma and intracellular membranes. Nothing is known about the dynamic changes in eCB levels in these subcellular compartments. We leveraged the eCB sensor, GRAB_eCB2.0, to establish the stimulus-induced increases in the eCB, 2-arachidonoyl glycerol (2-AG), at the plasma and intracellular membranes of undifferentiated Neuro2a cells in culture. Activating G protein-coupled B₂ receptors with bradykinin increased 2-AG levels at both the plasma and intracellular membranes within ≈5 and ≈15 s, respectively. By contrast, the activation of G proteins by the small peptide mastoparan and the ensuing opening of plasma membrane calcium channels increased 2-AG levels in plasma membrane within ≈1-2 s and in intracellular membranes after ≈30 s. While both these stimuli-induced increases in 2-AG production involved canonical lipases, they required distinct sources of calcium. Thus, distinct stimuli differentially increase 2-AG levels at plasma and intracellular membranes via distinct molecular mechanisms.