INTRACELLULAR SIGNALING PATHWAY OF SUBSTANCE-P-INDUCED SUPEROXIDE PRODUCTION IN HUMAN NEUTROPHILS

We examined the intracellular mechanisms of substance P-induced supero xide anion (O-2(-)) production in human neutrophils. Addition of subst ance P (30 mu M) caused O-2(-) production and biphasic increases in in tracellular Ca2+ concentrations ([Ca2+](i)) (early transient and subse quent sustained components) associated with the formation of inositol 1,4,5-trisphosphate (IP3), O-2(-) and [Ca2+](i) were assayed by using ferricytochrome C and fura 2-AM, respectively. These responses were ab olished by tachykinin NK1 receptor antagonists, [D-Pro(9)[spiro-gamma- lactam], Leu(10)Trp(11)]physalaemin-(1-11) (GR82334) or [D-Arg(1),D-Tr p(7,9)Leu(11)]substance P (spantide), and an intracellular Ca2+ chelat or, 1,2-bis(2 aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM ), Inhibition of IP3 formation by GTP-binding protein (G-protein) inac tivators such as guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) and i slet-activating protein (IAP), or a phospholipase C inhibitor, 1-[6-[[ 17 -3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl] 1H-pyrrole-2,5-d ione (U-73122), blocked the substance P-induced O-2(-) production and biphasic increases in [Ca2+](i). An IP3 receptor antagonist, heparin, reduced both the substance Protein kinase C inhibitors, 1-(5-isoquinol inesulfonyl)-2-methylpiperazine dihydrochloride (H-7) and calphostin C , only slightly suppressed O-2(-) production, and abolished the sustai ned increase in [Ca2+](i) without any significant effects on the trans ient increase in [Ca2+](i), A Ca2+ entry blocker, nicardipine, complet ely inhibited the sustained increase in [Ca2+](i) without affecting O- 2(-) production and the transient increase in [Ca2+](i), These results suggest that the tachykinin NK1 receptor/G-protein-linked IP3 formati on with the resulting IP3-induced transient increase in [Ca2+](i) is t he main signal transduction pathway for substance P-stimulated O-2(-) production in neutrophils.