Neurokinin 1 receptor mediates membrane blebbing in HEK293 cells through a Rho/Rho-associated coiled-coil kinase-dependent mechanism.
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 receptor-ligand interaction can cause apoptosis-independent cellular blebbing and that this process is mediated by the Rho/Rho-associated coiled-coil kinase pathway.
Meshki
John
J
Division of Allergy and Immunology, Joseph Stokes Jr. Research Institute, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, USA.
Douglas
Steven D
SD
Lai
Jian-Ping
JP
Schwartz
Lynnae
L
Kilpatrick
Laurie E
LE
Tuluc
Florin
F
D017209:Apoptosis; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D048430:Cell Shape; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D008854:Microscopy, Electron; D018994:Myosin Light Chains / Q000378:metabolism; D043263:Myosin-Light-Chain Phosphatase / Q000378:metabolism; D064729:Neurokinin-1 Receptor Antagonists; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D018040:Receptors, Neurokinin-1 / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D020741:rho GTP-Binding Proteins / Q000378:metabolism; D054460:rho-Associated Kinases / Q000378:metabolism*
D018994:Myosin Light Chains; D064729:Neurokinin-1 Receptor Antagonists; D047428:Protein Kinase Inhibitors; D018040:Receptors, Neurokinin-1; D054460:rho-Associated Kinases; D011493:Protein Kinase C; D043263:Myosin-Light-Chain Phosphatase; D020741:rho GTP-Binding Proteins; D002118:Calcium