Intracellular signaling pathway of substance P-induced superoxide production in human neutrophils

Abstract

We examined the intracellular mechanisms of substance P-induced superoxide anion (O − 2) production in human neutrophils. Addition of substance P (30 μM) caused O − 2 production and biphasic increases in intracellular Ca 2+ concentrations ([Ca 2+] i) (early transient and subsequent sustained components) associated with the formation of inositol 1,4,5-trisphosphate (IP 3). O − 2 and [Ca 2+] i were assayed by using ferricytochrome C and fura 2-AM, respectively. These responses were abolished by tachykinin NK 1 receptor antagonists, [ d-Pro 9[spiro-γ-lactam],Leu 10,Trp 11]physalaemin-(1–11) (GR82334) or ( d-Arg 1, d-Trp 7,9,Leu 11]substance P (spantide), and an intracellular Ca 2+ chelator, 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid (BAPTA-AM). Inhibition of IP 3 formation by GTP-binding protein (G-protein) inactivators such as guanosine 5′- O-(2-thiodiphosphate) (GDPβS) and islet-activating protein (IAP), or a phospholipase C inhibitor, 1-[6-[[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]1 H-pyrrole-2,5-dione (U-73122), blocked the substance P-induced O − 2 production and biphasic increases in [Ca 2+] i. An IP 3 receptor antagonist, heparin, reduced both the substance P-induced O − 2 production and the transient increase in [Ca 2+] i without any significant effects on the sustained increase in [Ca 2+] i. Protein kinase C inhibitors, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) and calphostin C, only slightly suppressed O − 2 production, and abolished the sustained increase in [Ca 2+] i without any significant effects on the transient increase in [Ca 2+] i. A Ca 2+ entry blocker, nicardipine, completely inhibited the sustained increase in [Ca 2+] i without affecting O − 2 production and the transient increase in [Ca 2+] i. These results suggest that the tachykinin NK 1 receptor/G-protein-linked IP 3 formation with the resulting IP 3-induced transient increase in [Ca 2+] i is the main signal transduction pathway for substance P-stimulated O − 2 production in neutrophils.