Platelet activating factor-induced increase in cytosolic calcium and transmembrane current in human macrophages

Platelet-activating factor (PAF) is synthesized and secreted by macrophages in response to inflammatory stimuli. When exogenously applied to human monocyte derived macrophages (HMDMs), PAF induces a rapid rise in cytosolic free calcium (Ca _i ) believed to be an early triggering event in macrophage activation. We investigated PAF-induced Ca²⁺ signaling in HMDMs using the calcium indicator Fura-2, combining single cell ratio fluorimetry and digital video imaging with whole-cell recording techniques. Application of PAF (20 ng/ml) to adherent macrophages induced transient increases in Ca, that were biphasic, consisting of an initial phase that could be observed in Ca²⁺-free solutions and a second phase that was critically dependent upon Ca²⁺ entry. When Mn²⁺ was applied to cells in the presence and absence of Ca²⁺, PAF increased the rate of Mn²⁺ entry rate only when Ca²⁺ was absent. PAF increased the rate of Ba²⁺ entry even when measured in the presence of external Ca²⁺. Ca²⁺ entry was reversibly inhibited in the presence of external La³⁺ (1 mm). Data obtained from simultaneous voltage-clamp/microfluorimetry experiments demonstrated the activation of a nonselective cation current which closely paralleled the rising phase of the Ca _i transient. We investigated whether the non-selective cation conductance provided for the bulk of the agonist-induced Ca²⁺ influx. Changes in Ca _i following removal of extracellular Ca²⁺ (Ca _o ) during the agonist-induced Ca _i response were not associated with changes in whole-cell current. The inability to detect whole-cell current changes correlated with a decrease in Ca _o suggests that the bulk of the Ca²⁺ influx was not through the nonselective conductance and either does not occur through a conductance pathway or occurs via a parallel pathway consisting of channels which are both low conductance and highly Ca²⁺ selective.