We examined the intracellular mechanisms of substance P induced oxyradical production in rheumatoid synovial cells by the luminol-dependent chemiluminescence method. After stimulation with substance P (30 microM), single synovial A (macrophage-like) or B (fibroblast-like) cells released oxyradicals such as superoxide anions (O2-) and/or hypochlorous anions (OCl-) under a microscope equipped with an ultrasensitive photonic image intensifier. The substance P induced oxyradical production was blocked by a tachykinin NK1 (NK1) receptor antagonist, GR82334, GTP-binding protein (G-protein) inactivators, GDP beta S and islet-activating protein (IAP), and a phospholipase C (PLC) inhibitor, U-73122. Substance P (30 microM) also induced a transient increase in the intracellular Ca2+ concentration ([Ca2+]i) in both synovial A and B cells as measured by a Ca2+ indicator, fura 2, BAPTA-AM and an inositol-1,4-5-triphosphate (IP3) receptor antagonist, heparin, inhibited the substance P induced increase in [Ca2+]i, but they had no effects on oxyradical production. In contrast to the effects of BAPTA-AM and heparin, protein kinase C (PKC) inhibitors, H-7 and calphostin C, completely inhibited substance P induced oxyradical production without any significant effects on [Ca2+]i increase. These findings suggest that the NK1 receptor/PLC-linked diacylglycerol (DAG) formation with the resulting activation of PKC is the main signal transduction pathway for substance P stimulated oxyradical production in synovial cells.

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ABSTRACT-The sympathetic nervous system has been indicated to influence the severity of inflammatory disease including rheumatoid arthritis. In this study, we elucidated the effects of catecholamine on the synovial cell populations. Stimulation with epinephrine or norepinephrine for 1 -2 weeks dose-and time-dependently increased the number of synovial A (macrophage-like) cells but decreased that of B (fibroblast-like) cells. These responses in A and B cells were inhibited by the a 2-antagonist yohimbine, the G-protein inactivator pertussis toxin and the phospholipase C (PLC) inhibitor U-73122. Furthermore, the protein kinase C (PKC) inhibitor calphostin C and mitogen-activated protein (MAP) kinase inhibitors PD98059 and wortmannin also abolished the norepinephrine effects on A and B cell numbers. In A cells cloned from an A and B cell mixture, norepinephrine also increased the cell number. In immunoblotting and immunocytostaining analyses, among the PKC isozymes, only PKC b II immunoreactivity was observed in the cytoplasm of unstimulated A and B cells. After a 2-adrenoceptor stimulation, PKC b II immunoreactivity increased in the plasma membranes of both A and B cells with decreases in the cytoplasm. These findings indicated that a 2-adrenoceptor stimulation of type A and B synoviocytes produced an increase and a decrease in the respective cell number, probably through Gi-coupled PLC activation and the resulting stimulation of the PKC bII /MAP kinase.