The Ionic Basis of Chemotaxis. Separate Cation Requirements for Neutrophil Orientation and Locomotion in a Gradient of Chemotactic Peptide

The behavior of cells undergoing chemotaxis may be analyzed in terms of their orientation, a static characteristic, and of their locomotion. We have examined the extracellular divalent cation requirements for orientation and locomotion of rabbit polymorphonuclear leukocytes (neutrophils) in a gradient of the chemotactic peptide N-formyl-methionyl-leu-cyl-phenylalanine (F-Met-Leu-Phe) using the chemotaxis chamber recently developed by Zigmond. This chamber allows direct observation of cells attached to glass coverslips as they move up a gradient of chemotactic agent established across a 1-mm bridge. The orientation of neutrophils in the direction of the gradient was equally efficient whether cells and F-Met-Leu-Phe were suspended in merium supplemented with both Ca2+ and Mg2+ (complete medium), with Mg2+ but not Ca2+ (by simple omission of Ca2+ or by addition of EGTA), or with nonsupplemented medium (by omission of Ca2+ and Mg2+ or by addition of EDTA). These data confirm and extend Zigmond's earlier observation that exogenous divalent cations are not required for polymorphonuclear leukocyte orientation toward the chemotactic peptide. In contrast, cell locomotion, determined by linking the chemotaxis chamber to a time-lapse videocassette recorder and TV monitor, is markedly affected by the medium's content of divalent cations. Cells suspended in medium supplemented with Mg2+ but not calcium (by omission or chelation) or in nonsupplemented medium moved on the average 25% more rapidly than cells in complete Ca2+ and Mg2+ medium. Although the simple omission of Mg2+ does not prevent chemotaxis, chelation of Mg2+ in the medium completely abolishes leukocyte locomotion. Addition of varying concentrations of Mg2+ to the buffer in the presence of EDTA established that cell movement is fully restored by Mg2+ concentrations in the range of 3 X 10(-9) M, concentrations easily attained in the absence of added Mg2+. It was concluded that neither Ca2+ nor Mg2+ is needed for orientation in response to F-Met-Leu-Phe. However, low levels of exogenous Mg2+ but not Ca2+ are required for effective locomotion of neutrophils in the Zigmond changer. This result contrasts with data obtained in the Boyden chamber, where exogenous Ca2+ is considered essential for maximum chemotactic response.