Intracellular signaling pathway of substance P-induced superoxide production in human neutrophils.
We examined the intracellular mechanisms of substance P-induced superoxide anion (O2-) production in human neutrophils. Addition of substance P (30 microM) caused O2- production and biphasic increases in intracellular Ca2+ concentrations ([Ca2+]i) (early transient and subsequent sustained components) associated with the formation of inositol 1,4,5-trisphosphate (IP3). O2- and [Ca2+]i were assayed by using ferricytochrome C and fura 2-AM, respectively. These responses were abolished by tachykinin NK1 receptor antagonists, [D- Pro9[spiro-gamma-lactam],Leu10,Trp11]physalaemin-(1-11) (GR82334) or [D-Arg1,D-Trp7,9,Leu11]substance P (spantide), and an intracellular Ca2+ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM). Inhibition of IP3 formation by GTP-binding protein (G-protein) inactivators such as guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) and islet-activating protein (IAP), or a phospholipase C inhibitor, 1-[6-[[17 beta-3-methoxyestra-1,3,5(10)- trien-17-yl]amino]hexyl]1 H-pyrrole-2,5-dione (U-73122), blocked the substance P-induced O2- production and biphasic increases in [Ca2+]i. An IP3 receptor antagonist, heparin, reduced both the substance P-induced O2- production and the transient increase in [Ca2+]i without any significant effects on the sustained increase in [Ca2+]i. Protein kinase C inhibitors, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) and calphostin C, only slightly suppressed O2- production, and abolished the sustained increase in [Ca2+]i without any significant effects on the transient increase in [Ca2+]i. A Ca2+ entry blocker, nicardipine, completely inhibited the sustained increase in [Ca2+]i without affecting O2- production and the transient increase in [Ca2+]i. These results suggest that the tachykinin NK1 receptor/G-protein-linked IP3 formation with the resulting IP3-induced transient increase in [Ca2+]i is the main signal transduction pathway for substance P-stimulated O2- production in neutrophils.
Department of Pharmacology, Kansai Medical University, Osaka, Japan.
T
T
Tanabe
H
H
Otani
L
L
Bao
Y
Y
Mikami
T
T
Yasukura
T
T
Ninomiya
R
R
Ogawa
C
C
Inagaki
D002118:Calcium / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000096:biosynthesis*; D064729:Neurokinin-1 Receptor Antagonists; D009504:Neutrophils / Q000187:drug effects* / Q000378:metabolism; D010803:Physalaemin / Q000031:analogs & derivatives / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D018040:Receptors, Neurokinin-1 / Q000187:drug effects*; D015398:Signal Transduction; D013373:Substance P / Q000031:analogs & derivatives / Q000494:pharmacology*; D013481:Superoxides / Q000378:metabolism*; D010738:Type C Phospholipases / Q000378:metabolism
D064729:Neurokinin-1 Receptor Antagonists; D018040:Receptors, Neurokinin-1; D013481:Superoxides; C079014:GR 82334; D010803:Physalaemin; D013373:Substance P; D015544:Inositol 1,4,5-Trisphosphate; C042867:spantide; D011493:Protein Kinase C; D010738:Type C Phospholipases; D002118:Calcium