Monocytes migrate toward Ca²⁺ in a dose-dependent manner that is inhibitable by pretreatment with pertussis toxin, genistein, or herbimycin, and is potentiated by the selective CaR activator NPS R-467 and the chemokine MCP-1. (a) Transmigration assays were used to determine if Ca²⁺ was capable of inducing monocyte chemotaxis within a checkerboard analysis (upper panel). An input of 10⁴ monocytes was used, and the mean numbers of cells migrating in response to each Ca²⁺ gradient are shown for three independent experiments. A dose-dependent effect was seen, with maximal chemotaxis observed at 4.5 mM Ca²⁺, which was inhibitable by pretreatment with pertussis toxin (PTX) (lower panel). (b) Chemotaxis of monocytes toward a positive gradient of Ca²⁺ at 3 mM was also inhibitable by pretreatment with the tyrosine kinase inhibitors genistein (gen) or herbimycin (herb), but not by the PI-3 kinase inhibitor wortmannin (wort). (c) The role of the CaR in mediating the chemotactic response to elevated Ca²⁺ was supported by the greater potency of R-467 (agonist R) than of S-467 (agonist S) in stimulating chemotaxis. It should be noted that no specific antagonists or neutralizing antibodies for the CaR have been defined to date. P values represent comparison with 3-mM Ca²⁺ control using the Student’s t test. (d) Chemotaxis of PBMC obtained from CaR^–/– mice and CaR^+/+ mice also evaluated in response to 4.5 mM Ca²⁺ and to MCP-1 in the presence of 1.5 mM Ca²⁺. The percent migration of CaR^+/+ monocytes (black bars) and CaR^–/– monocytes (gray bars) are shown. The data from two separate experiments are shown (mean ± SEM). ^AP = 0.0481, Student’s t test. (e) Chemotaxis of CD14⁺ monocytes toward a positive concentration gradient of MCP-1 was also dependent upon and potentiated by Ca²⁺. Migration of monocytes in the presence of varying levels of Ca²⁺ with MCP-1 (black bars) or without MCP-1 (gray bars) is shown. The percentage of cells migrating in response to MCP-1 could be augmented by increased Ca²⁺ concentrations with peak added activity at 4.5 mM Ca²⁺. Data represent mean ± SEM of at least three independent experiments.