Functional coupling of the NK1 tachykinin receptor to phospholipase D in Chinese hamster ovary cells and astrocytoma cells

In [H-3]myristic acid-prelabeled Chinese hamster ovary cells stably expressing the rat NK1 tachykinin receptor, the selective NK1 agonist [Pro(9)]substance P ([Pro(9)]SP) time and concentration dependently stimulated the formation of [H-3]phosphatidylethanol in the presence of ethanol. This [Pro(9)]SP-induced activation of phospholipase D (PLD) was blocked by NK1 receptor antagonists and poorly or not mimicked by NK2 and NK3 agonists, respectively. In confirmation of previous observations, [Pro(9)]SP also stimulated the hydrolysis of phosphoinositides, the release of arachidonic acid, and the formation of cyclic AMP (cAMP). All these [Pro(9)]SP-evoked responses could be mimicked by aluminum fluoride, but they remained unaffected in cells pretreated with pertussis toxin, suggesting that a G(i)/G(o) protein is not involved in these different signaling pathways. The activation of PLD by [Pro(9)]SP was sensitive to external calcium and required an active protein kinase C because the inhibition of this kinase (Ro 31-8220) or its down-regulation (long-term treatment with a phorbol ester) abolished the response. In contrast, a cAMP-dependent process was not involved in the activation of PLD because the [Pro(9)]SP-evoked response was neither affected by Rp-8-bromoadenosine 3',5'-cyclic monophosphorothioate nor mimicked by cAMP-generating compounds (cholera toxin or forskolin) or by 8-bromo-cyclic AMP. A functional coupling of NK1 receptors to PLD was also demonstrated in the human astrocytoma cell line U 373 MG stimulated by SP or [Pro(9)]SP. These results suggest that PLD activation could be an additional signaling pathway involved in the mechanism of action of SP in target cells expressing NK1 receptors.