Neurokinin 1 Receptor Mediates Membrane Blebbing in HEK293 Cells through a Rho/Rho-associated Coiled-coil Kinase-dependent Mechanism

Abstract

We have investigated the effect of neurokinin 1 receptor (NK1R) agonists on HEK293 cells transfected with the NK1R receptor. The NK1R receptor mediates dramatic shape changes that include contractions of the membrane cortex resulting in membrane bleb formation. We have found that the cell shape changes correlate with changes in electrical impedance measured in cellular monolayers. The shape and impedance changes were prevented after preincubation with NK1R antagonists aprepitant and L-73060. Although bleb formation usually heralds apoptotic cell death, we have found that NK1R-mediated cellular blebbing does not associate with apoptosis. Preincubation with a cell-permeable derivative of C3 transferase that blocks Rho or with the Rho-associated coiled-coil kinase inhibitor Y27632 completely prevented NK1R-induced shape and impedance changes. Blebbing was also completely inhibited by ML-9, a myosin light chain kinase inhibitor. Furthermore, the phospholipase C inhibitor U73,122 did not interfere with the effect of Substance P (SP) on cellular morphology and cellular impedance but completely blocked SP-induced intracellular calcium increase, indicating that the blebbing is a process independent of intracellular calcium elevations. Blebbing is a protein kinase C-independent process, since the nonselective protein kinase C inhibitor GF109203X did not interfere with SP-induced effects. Based on these results, we provide the first evidence that NK1R receptorligand interaction can cause apoptosis-independent cellular blebbing and that this process is mediated by the Rho/Rho-associated coiled-coil kinase pathway.

The NK-1 receptor is expressed in human melanoma and is involved in the antitumor action of the NK-1 receptor antagonist aprepitant on melanoma cell lines

“…[14][15][16]24 In a previous study, it has been reported that NK-1 receptors mediate shape changes in normal human embryonic kidney cells (HEK 293) and that these changes were prevented after preincubation with NK-1 receptor antagonists at 1 mM concentration. 36 We have previously reported that the IC 50 for the NK-1 receptor antagonist aprepitant against HEK 293 cells, was 490 mM (30). At 1 mM of aprepitant, we did not observe any antitumoral action on the tumor cells studied herein.…”

“…This is not associated with apoptosis, but it is a consequence of the Rho-activated ROCK system, leading to an increase in the phosphorylation of the myosin regulatory light chain. 36 Membrane blebbing is important in cell movement, cell spreading and in cancer cell invasion. 41 The same study reported that aprepitant and L-733060 blocked SP-induced rapid cellular shape change, including blebbing.…”

False positive reactivity of a substance P-antibody in the ectodermal/epithelial plug of the nose, ear, eye and perineum of the human and mouse fetuses

“…Therefore, we hypoth- esized that sc9758 cross-reacted with epithelium-related substances produced by the death process. Actually, SP seems to induce cell death by way of the NK-1 receptor (Castro-Obregón et al 2002;Santoni et al 2004;Meshki et al 2009; see also Introduction). We identified NK-1 receptor in most of the sc9758-positive sites.…”

“…Neuropeptide substance P (SP) and its receptor, neurokinin-1 receptor, were first identified by an in vitro study using cultures of rat embryonic nerve cells, and mediate an alternative, non-apoptotic form of programmed cell death (Castro-Obregón et al 2002). SP has killing activity against proliferating non-neuronal cells (Meshki et al 2009;D'Souza et al 2007;Santoni et al 2004). This type of cell death requires gene expression, displays non-apoptotic morphology, and is independent of caspase activation.…”

Paravertebral anesthesia: how substance P and the NK-1 receptor could be involved in regional block and breast cancer recurrence

“…NK-1 receptor antagonists induce cellular death by apoptosis [10,14]; (b) by inhibiting tumor neoangiogenesis [15]; and (c) by inhibiting the migration of tumoral cells (e.g., breast carcinoma cells) [9,16]. NK-1 receptor antagonists also block the mitogenic action exerted by SP on tumor cells.…”