The basic secretagogue compound 48/80 activates G proteins indirectlyviastimulation of phospholipase D-lysophosphatidic acid receptor axis and 5-HT1Areceptors in rat brain sections

The basic secretagogues, such as compound 48/80 (c48/80) and mastoparans, are widely used histamine-releasing agents and their mechanism of action is commonly attributed to a direct, receptor-bypassing property to activate the Gi/o class of G proteins. We tested here whether c48/80 could directly stimulate [35S]guanosine-5′-[γ-thio]triphosphate ([35S]GTPγS) binding to rat brain sections in an attempt to visualize the entire signaling pool of Gi/o in its native neuroanatomical context. Instead of direct Gi/o activation, c48/80 (100 μg ml−1) from various suppliers stimulated brain phospholipase D (PLD) activity, leading to the generation of endogenous phospholipids capable of activating brain white matter-enriched, Gi/o-coupled lysophosphatidic acid (LPA) receptors. This response was sensitive to 1-butanol and was potently reversed by the LPA1/LPA3 receptor-selective antagonist Ki16425 (IC50 59±13 nM, mean±s.e.m.), and showed age-dependent decline, closely reflecting known developmental regulation of the PLD–LPA1 receptor axis in the CNS. In addition, c48/80 was found to modestly activate hippocampal 5-HT1A receptors in a pH-dependent and antagonist-sensitive manner. Consistent with the lack of direct Gi/o-activating properties in brain sections, c48/80 showed no activity in classical membrane [35S]GTPγS binding assays. Instead, c48/80 from one particular manufacturer elicited non-specific effect in these assays, therefore challenging the previous interpretations regarding the compound's ability to activate G proteins directly. We conclude that c48/80 is not a receptor-bypassing general G protein activator but rather activates PLD, leading to generation of endogenous LPA receptor-activating phospholipids. This property may also contribute to the compound's ability to release histamine from mast cells. British Journal of Pharmacology (2006) 147, 596–606. doi:10.1038/sj.bjp.0706671