132 Exp. Mol. Med. Vol. 44(2), 130-137, 2012 Figure 2. Effects of peptides on Ca2+ increase in human neutrophils. Fura-2-loaded human neutrophils were stimulated with various concentrations of GMMWAI, MMHWAM, and MMHWFM. The change in 340 nm/380 nm was monitored. The peak level of the increase in Ca2+ was monitored. Data are presented as means ± S.E. of four independent experiments (A-C). Fura-2-loaded human neutrophils were stimulated with 5 μM MMHWAM in the ab- sence or presence of SK&F (10 μM), diltiazem (1 μM), nifidifin (1 μM), U-73122 (5 μM), U-73343 (5 μM), and 2A-PB (5 μM). The change in 340 nm/380 nm was monitored. The results are representative of three independent experiments (D, E). Human neutrophils were preincubated with or without 1 μg/ml of PTX for 4 h, after which fura-2 was loaded into the cells. Fura-2-loaded cells were stimulated with 5 μM MMHWAM. The peak level of the increase in Ca2+ was monitored. Data are presented as means ± S.E. of three independent experiments (F). *, P ＜ 0.05, compared with the value obtained from the vehicle control; #, P ＜ 0.05, significantly different from the -PTX control. MMHWAM increased Ca2+ concentration indepen- dent of the Ca2+ channel-dependent pathway in human neutrophils. Another pathway for intracellular Ca2+ increase is mediated by the activation of PLC (Noh et al., 1995; Rhee, 2001). To determine the role of PLC in the MMHWAM-induced Ca2+ increase, we pre- treated cells with a specific PLC inhibitor, U-73122, or with its inactive analogue, U-73343. As shown in Figure 2E, U-73122 but not U-73343 completely inhibited the MMHWAM-induced Ca2+ increase. 2-aminoethoxydiphenyl borate (2-APB), which is used to block IP3 receptor in cells (Maruyama et al., 1997), also completely inhibited the MMHWAM- induced Ca2+ increase in human neutrophils (Figure 2E). These results indicate that MMHWAM stimulated Ca2+ increase via PLC activation in human neutrophils. MMHWAM resulted in intracellular Ca2+ elevation not only in the presence of extracellular Ca2+ but also in the absence of extracellular Ca2+ (data not shown), supporting that the peptide induced Ca2+ increase via the activation of PLC in human neutrophils. We also examined the effect of PTX, a specific inhibitor of Gi/o type G proteins, on the peptides- induced Ca2+ increase. When human neutrophils were preincubated with 1 μg/ml of PTX prior to stimulation with MMHWAM, the peptides-induced Ca2+ increase was almost completely inhibited (Figure 2F). These results indicate that MMHWAM stimulated Ca2+ increase via PTX-sensitive G pro- teins. We also observed that the other two peptides (GMMWAI and MMHWFM) stimulated Ca2+ increase via Gi protein and PLC but not the Ca2+ channel (data not shown). Leukocyte-specific effects of the novel peptides The fact that GMMWAI, MMHWAM, and MMHWFM stimulated human neutrophils led us to examine the effects of the peptides on other leukocytes such as monocytes. Stimulation of monocytes with the three peptides resulted in Ca2+ increase (Figure 3). The three peptides also enhanced Ca2+ levels in monocytes with a similar concentration dependency as observed for the Ca2+ increase (Figure 3 and data not shown). Next, we examined the effects of GMMWAI, MMHWAM, and MMHWFM on intracellular Ca2+ release in several non-leukocytic cell lines. NIH3T3 (NIH Swiss mouse embryo fibroblast), 3Y1 (Rat embryo- nic fibroblast), 3T3L1 (preadipocyte), and PC12