Implication of acidic lipids in 5-hydroxytryptamine receptor mechanisms

To establish the possible involvement of acidic lipids in 5-HT receptor mechanisms, we subjected whole rat brain synaptic plasma membranes to treatment with several kinds of lipid-modifying reagents and examined the [ 3H] 5-HT and [ 3H] spiperone binding properties of the membranes. [ 3H] 5-HT binding was decreased by treatment with Azure A, while [ 3H] spiperone binding was not altered. Similarly, prior treatment with arylsulphatase reduced the former binding, but had no effect on the latter binding. On the other hand, neither [ 3H] ligand binding was sensitive to phospholipases C and D. In contrast, prior treatment with phospholipase A 2 (unheated) drastically decreased the [ 3H] 5-HT binding and also affected the [ 3H] spiperone binding to some extent. Chelation of Ca 2+ by EGTA (5 mM) prior to incubation of membranes with the unheated phospholipase A 2 did not completely prevent the inhibitory effect of this enzyme on [ 3H] 5-HT binding, while in the heated enzyme (at 100°C for 10 min) EGTA exhibited this preventive effect perfectly. Furthermore, it was an interesting find that at least a low concentration of the heated phospholipase A 2 (0.01 U) had no effect on the [ 3H] spiperone binding, as contrasted with the case of [ 3H] 5-HT binding. In addition, the reduction of [ 3H] 5-HT binding capacity in membranes treated with phospholipase A 2 (heated and unheated) was restored only slightly by treatment with BSA (1 %). Scatchard analysis of the [ 3H] 5-HT binding showed that Azure A and phospholipase A 2 (heated) decreased the B max values with no significant alteration in the K D values, whereas arylsuphatase increased only the K D value. All these observations infer that certain acidic lipids may play a role as the recognition site(s) or modulator(s) of 5-HT 1 receptor molecules.