Mediation of chemoattractant-induced changes in [Ca2+]i and cell shape, polarity, and locomotion by InsP3, DAG, and protein kinase C in newt eosinophils.
During chemotaxis large eosinophils from newts exhibit a gradient of [Ca2+]i from rear to front. The direction of the gradient changes on relocation of the chemoattractant source, suggesting that the Ca2+ signal may trigger the cytoskeletal reorganization required for cell reorientation during chemotaxis. The initial stimulatory effect of chemoattractant on [Ca2+]i and the opposite orientations of the intracellular Ca2+ gradient and the external stimulus gradient suggest that more than one chemoattractant-sensitive messenger pathway may be responsible for the generation of spatially graded Ca2+ signals. To identify these messengers, Ca2+ changes were measured in single live cells stimulated with spatially uniform chemoattractant. On stimulation spatially averaged [Ca2+]i increased rapidly from < or = 100 nM to > or = 400 nM and was accompanied by formation of lamellipods. Subsequently cells flattened, polarized and crawled, and [Ca2+]i fluctuated around a mean value of approximately 200 nM. The initial Ca2+ spike was insensitive acutely to removal of extracellular Ca2+ but was abolished by treatments expected to deplete internal Ca2+ stores and by blocking receptors for inositol-trisphosphate, indicating that it is produced by discharge of internal stores, at least some of which are sensitive to InsP3. Activators of protein kinase C (PKC) (diacyl glycerol and phorbol ester) induced flattening and lamellipod activity and suppressed the Ca2+ spike, while cells injected with PKC inhibitors (an inhibitory peptide and low concentrations of heparin-like compounds) produced an enhanced Ca2+ spike on stimulation. Although cell flattening and lamellipod activity were induced by chemoattractant when the normal Ca2+ response was blocked, cells failed to polarize and crawl, indicating that Ca2+ homeostasis is required for these processes. We conclude that InsP3 acting on Ca2+ stores and DAG acting via PKC regulate chemoattractant-induced changes in [Ca2+]i, which in turn control polarization and locomotion. We propose that differences in the spatial distributions of InsP3 and DAG resulting from their respective hydrophilic and lipophilic properties may change Ca2+ distribution in response to stimulus reorientation, enabling the cell to follow the stimulus.
Gilbert
S H
SH
Department of Physiology, University of Massachusetts Medical Center, Worcester 01605.
Perry
K
K
Fay
F S
FS
D000818:Animals; D002118:Calcium / Q000097:blood*; D015220:Calcium Channels / Q000378:metabolism; D016764:Cell Polarity / Q000187:drug effects; D048429:Cell Size / Q000187:drug effects; D002630:Chemotactic Factors / Q000494:pharmacology*; D002634:Chemotaxis, Leukocyte / Q000187:drug effects; D004075:Diglycerides / Q000494:pharmacology*; D004789:Enzyme Activation; D004804:Eosinophils / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D006706:Homeostasis; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007295:Inositol Phosphates / Q000494:pharmacology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000097:blood*; D018160:Receptors, Cytoplasmic and Nuclear / Q000037:antagonists & inhibitors / Q000378:metabolism; D012455:Salamandridae
D015220:Calcium Channels; D002630:Chemotactic Factors; D004075:Diglycerides; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007295:Inositol Phosphates; D018160:Receptors, Cytoplasmic and Nuclear; C058097:inositol 1,3,4-trisphosphate; D011493:Protein Kinase C; D002118:Calcium