cathepsin-g and fura-2-am

We investigated the effect of cathepsin G, a serine protease in polymorphonuclear granulocytes, on the function of human lymphocytes. Cathepsin G increased the [3H]thymidine incorporation into human lymphocytes. This mitogenic activity was due to the proteolytic activity of cathepsin G. Both B and T cells showed increased [3H]thymidine incorporation, and this effect was more remarkable for T cells than for B cells. Among the T cell subsets, CD4+ T cells showed the increase in DNA synthesis, but CD8+ T cells did not. When human lymphocytes were stimulated with cathepsin G, intracellular free Ca2+ concentration ([Ca2+]i) increased in B and T cells, including CD4+ T cells and CD8+ T cells. The change in intracellular Ca2+ was due to Ca2+ influx and release of intracellular stores. Cathepsin G also induced the production of inositol 1,4,5-trisphosphate (IP3) in B cells, CD4+ T cells, and CD8+ T cells, leading to the release of Ca2+ from intracellular stores. Moreover, the stimulation with cathepsin G resulted in alkalization of the cytosol of B cells, CD4+ T cells, and CD8+ T cells as the result of Na+/H+ antiport activation. The change in intracellular Ca2+, production of IP3, and cytoplasmic alkalization in lymphocytes were due to its proteolytic activity. Cathepsin G released from granulocytes is considered to act on human lymphocytes in vivo and lead to the increase in DNA synthesis of B cells and CD4+ T cells through IP3 production, an increase in [Ca2+]i, and alkalization. However, these second messengers do not lead to the increase in DNA synthesis of CD8+ T cells.

Topics: Amino Acid Sequence; B-Lymphocytes; Calcium; Cathepsin G; Cathepsins; Cells, Cultured; Fura-2; Humans; Hydrogen-Ion Concentration; Inositol 1,4,5-Trisphosphate; Lymphocyte Activation; Molecular Sequence Data; Serine Endopeptidases; T-Lymphocytes; Terpenes; Thapsigargin

Cathepsin G, a serine protease released by polymorphonuclear-leucocyte azurophilic granules upon stimulation, activates human platelets, inducing an increase in intra-platelet Ca2+ concentration ([Ca2+]i) in a concentration-dependent manner (50-200 nM). The [Ca2+]i rises elicited by low (50-80 nM) cathepsin G concentrations in fura-2-loaded platelets showed a biphasic mode, with a first small peak followed by a greater and more prolonged Ca2+ transient. Higher (100-200 nM) cathepsin G concentrations induced a monophasic increase in intracellular Ca2+. Acetylsalicylic acid, nordihydroguaiaretic acid and ketanserin did not affect platelet activation by cathepsin G, whereas the ADP-scavenger system phosphocreatine/creatine kinase significantly decreased Ca2+ mobilization, platelet aggregation and 5-hydroxytryptamine secretion by cathepsin G. Preventing cathepsin G-induced platelet aggregation with the synthetic peptide RGDSP (Arg-Gly-Asp-Ser-Pro) did not significantly affect cathepsin G-induced Ca2+ transients. Ni2+ (4 mM), a bivalent-cation-channel inhibitor, decreased the cathepsin G-induced fluorescence rise by more than 90%. This effect was reversed by either decreasing Ni2+ or increasing cathepsin G concentration. Preventing Ca2+ influx across the plasma membrane with 4 mM-EGTA totally abolished Ca2+ transients. However, EGTA also strongly decreased catalytic activity of cathepsin G, which is essential for platelet activation. Evidence of a rapid and sustained bivalent-cation channel opening in the platelet membrane was obtained by adding Mn2+ to the platelet suspension 30 s or 3 min after cathepsin G. No accumulation of InsP3 could be detected when platelets were stimulated with cathepsin G. All these data indicate that cathepsin G induces a [Ca2+]i increase mainly through an influx across the plasma membrane. This massive Ca2+ entry is probably due to opening of receptor-operated channels and is amplified by endogenous ADP release.

Topics: Adult; Amino Acid Sequence; Blood Platelets; Calcium; Cathepsin G; Cathepsins; Cations, Divalent; Fluorescent Dyes; Fura-2; Humans; Intracellular Fluid; Kinetics; Manganese; Molecular Sequence Data; Neutrophils; Nickel; Phosphatidylinositols; Platelet Activation; Serine Endopeptidases