neutrophils, the identification of new chemoattrac- tants and the characterization of their mechanisms of action are very much needed. For the identification of new bioactive molecules, various different types of molecular diversities can be used. Positional scanning synthetic peptide combinatorial library (PS-SPCL), which is an easy and powerful tool for identifying peptide sequences in certain biological reactions, was developed by Houghten et al. (Houghten et al., 1991). Many groups have used this method for various pur- poses, including the identification of human im- munodeficiency virus protease inhibitors, interleu- kin-8-specific antagonists, inhibitor for nuclear factor of activated T cells, and ligands for opioid receptors (Owens et al., 1991; Hayashi et al., 1995; Dooley et al., 1998; Aramburu et al., 1999). Further, we already identified several bioactive hexapeptide that stimulates superoxide anion production or arachidonic acid release by screening hexapeptide combinatorial libraries (Bae et al., 2001, 2003). Here, we adopted the PS-SPCL method to identify novel peptides that can stimulate a Ca2+ increase in human neutrophils. We found that the peptides Gly-Met-Met-Trp-Ala-Ile-CONH2 (GMMWAI), Met-Met-His-Trp-Ala-Met-CONH2 (MMHWAM), and Met-Met-His-Trp-Phe-Met-CONH2 (MMHWFM) can stimulate human neutrophils, resulting in intra- cellular Ca2+ increase. We also investigated the functional roles of the peptides and the target receptors of these three peptides. Results Identification of peptides that stimulate Ca2+ increase in human neutrophils A total of 114 peptide pools (around 47 million Novel neutrophil-activating peptides 131 peptides) from hexapeptide PS-SPCLs were screened to identify peptides that stimulate a Ca2+ increase in human neutrophils. As shown in Figure 1, we observed that each amino acid that was fixed at each position induced different levels of Ca2+ increase from the initial screening. The most active peptides at each position were as follows: Met (M) or Gly (G) in the 1st position, Met (M) in 2nd, His (H) or Met (M) in 3rd, Trp (W) in 4th, Ala (A) in 5th, and Met (M) or Ile (I) in 6th. Peptides-induced Ca2+ increase is mediated via G-proteins and PLC Based on the results of the initial screening of the peptide libraries, we synthesized three represen- tative hexapeptides (GMMWAI, MMHWAM, and MMHWFM) and confirmed that stimulation of neutrophils with various concentrations of these three peptides induced a Ca2+ increase in a con- centration-dependent manner with maximal activity of 0.5-5 μM (Figures 2A-2C). Intracellular Ca2+ increase can be induced by several different pathways. Firstly, the activation of some types of Ca2+ channels elicits intracellular Ca2+ increase in leukoyctic cells (Berridge, 1993; Burnashev, 1998; Zhu et al., 2010). Since we observed that the three novel peptides increased intracellular Ca2+ levels in human neutrophils, we examined the involvement of the cell surface Ca2+ channel. For this, we used several different Ca2+ channel-selective inhibitors. As shown in Figure 2D, MMHWAM-induced intracellular Ca2+ increas- es were not affected by preincubating human neutrophils with 1 μM nifedifine (voltage-sensitive L type Ca2+ channel inhibitor), 10 μM diltiazem (voltage-sensitive L type Ca2+ channel inhibitor), and 10 μM SK&F. These results indicate that Figure 1. Initial screening of PS- SPCLs for peptides stimulating in- tracellular Ca2+ increase in human neutrophils. Each panel shows the results obtained with the peptide pools with known amino acids at each of the six positions of the hexapeptide. The six positions were individually defined (O1, O2 etc.) by one of the 19 L-amino acids. The re- maining five positions consist of mixtures (X) of the 19 L-amino acids (except for cysteine). Human neu- trophils (1 ×106 cells/assay) were used for each assay. Fluorescence ratio (340/38) was monitored as de- scribed under Methods. The results represent one of three independent experiments.