The prototypic formyl peptide fMet‐Leu‐Phe (fMLF) is an E. coli‐derived chemoattractant for neutrophils. fMLF is a potent agonist of human formyl peptide receptor 1 (FPR1) and induces bactericidal functions of human neutrophils at nanomolar concentrations. However, mouse neutrophil activation requires fMLF concentrations in the micromolar range. To determine whether the mouse FPR is a physiologically relevant receptor for the detection of invading bacteria, formyl peptides derived from S. aureus and L. monocytogenes were examined. It was found that a tetrapeptide (fMIFL) from S. aureus and a pentapeptide (fMIVIL) from L. monocytogenes are potent agonists for the activation of mouse neutrophils. Both fMIFL and fMIVIL induced chemotaxis at concentrations of 1–10 nM and superoxide production at concentrations of 10–100 nM. Additionally, fMIFL and fMIVIL induced greater calcium mobilization than fMLF in mouse neutrophils. Mouse FPR1‐transfected rat basophilic leukemia cells (RBL) also responded to these peptides with greater induction of calcium mobilization and phosphorylation of ERK1/2 compared to fMLF. In conclusion, the S. aureus and L. monocytogenes derived peptides are approximately 100‐fold more potent than fMLF for the mouse FPR1. Differential recognition of formyl peptides from different bacterial strains by mouse neutrophils may be important for the innate immunity to these microorganisms.

Ischemic injury induces actin cytoskeleton disruption and aggregation, but mechanisms affecting these changes remain unclear. To determine the role of actin-depolymerizing factor (ADF)/ cofilin participation in ischemic-induced actin cytoskeletal breakdown, we utilized porcine kidney cultured cells, LLC-PKA4.8, and adenovirus containing wild-type (wt), constitutively active, and inactive Xenopus ADF/cofilin linked to green fluorescence protein [XAC(wt)-GFP] in an ATP depletion model. High adenoviral infectivity (70%) in LLC-PKA4.8 cells resulted in linearly increasing XAC(wt)-GFP and phosphorylated (p)XAC(wt)-GFP (inactive) expression. ATP depletion rapidly induced dephosphorylation, and, therefore, activation, of endogenous pcofilin as well as pXAC(wt)-GFP in conjunction with the formation of fluorescent XAC(wt)-GFP/actin aggregates and rods. No significant actin cytoskeletal alterations occurred with short-term ATP depletion of LLC-PKA4.8 cells expressing GFP or the constitutively inactive mutant XAC(S3E)-GFP, but cells expressing the constitutively active mutant demonstrated nearly instantaneous actin disruption with aggregate and rod formation. Confocal image three-dimensional volume reconstructions of normal and ATP-depleted LLC-PKA4.8 cells demonstrated that 25 min of ATP depletion induced a rapid increase in XAC(wt)-GFP apical and basal signal in addition to XAC-GFP/actin aggregate formation. These data demonstrate XAC(wt)-GFP participates in ischemia-induced actin cytoskeletal alterations and determines the rate and extent of these ATP depletion-induced cellular alterations.

Ischemic-induced cell injury results in rapid duration-dependent actin-depolymerizing factor (ADF)/cofilin-mediated disruption of the apical microvilli microfilament cores. Because intestinal microvillar microfilaments are bound and stabilized in the terminal web by the actin-binding protein tropomyosin, we questioned whether a protective effect of tropomyosin localization to the terminal web of the proximal tubule microfilament cores is disrupted during ischemic injury. With tropomyosin-specific antibodies, we examined rat cortical sections under physiological conditions and following ischemic injury by confocal microscopy. In addition, Western blot analysis of cortical extracts and urine was undertaken. Our studies demonstrated the presence of tropomyosin isoforms in the proximal tubule microvillar terminal web under physiological conditions and their dissociation in response to 25 min of ischemic injury. This correlated with the excretion of tropomyosin-containing plasma membrane vesicles in urine from ischemic rats. In addition, we noted increased tropomyosin Triton X-100 solubility following ischemia in cortical extracts. These studies suggest tropomyosin binds to and stabilizes the microvillar microfilament core in the terminal web under physiological conditions. With the onset of ischemic injury, we propose that tropomyosin dissociates from the microfilament core providing access to microfilaments in the terminal web for F-actin binding, severing and depolymerizing actions of ADF/cofilin proteins.