Haruko Oda

Inhibition of Inducible Nitric Oxide Synthase Expression by Endothelin in Rat Glial Cells Prepared from the Neonatal Rat Brain

Abstract: In primary cultured rat glial cells, a combination of inflammatory cytokines such as tumor necrosis factor‐α (TNF‐α) and interleukin‐1β (IL‐1β) stimulates production of nitrite via expression of the inducible form of nitric oxide synthase (iNOS). In these cells, simultaneous addition of endothelin (ET) decreased iNOS expression and nitrite accumulation induced by TNF‐α/IL‐1β. The inhibitory effect of ET on TNF‐α/IL‐1β‐stimulated iNOS expression appears to be mediated by ETB receptors, because (1) both ET‐1 and ET‐3 inhibited the effects of TNF‐α/IL‐1β on iNOS expression and nitrite accumulation, (2) a selective ETB receptor agonist, Suc‐[Glu9,Ala11,15]‐ET‐1 (8–21) (IRL1620), decreased the effects of TNF‐α/IL‐1β, and (3) a selective ETB receptor antagonist, N‐cis‐2,6‐dimethylpiperidinocarbonyl‐l‐γ‐methylleucyl‐d‐1‐methoxycarbonyltryptophanyl‐d‐norleucine, abolished the inhibitory effects of ETs and IRL1620. Incubation of glial cells with lipopolysaccharide (LPS) caused an increase in iNOS expression. Simultaneous addition of ET‐3 decreased the effects of LPS (10 and 100 ng/ml) on iNOS expression. Furthermore, cyclic AMP‐elevating agents (dibutyryl cyclic AMP and forskolin) inhibited TNF‐α/IL‐1β‐induced and LPS‐induced iNOS expression and nitrite accumulation. These findings suggest that ETs can decrease TNF‐α/IL‐1β‐induced and LPS‐induced iNOS expression via ETB receptors and that cyclic AMP may be involved in this process.

Endothelin enhances lipopolysaccharide-induced expression of inducible nitric oxide synthase in rat glial cells

Lipopolysaccharide is known to stimulate production of nitrite via expression of inducible nitric oxide (NO) synthase in not only macrophages but also glial cells. We found that in glial cell cultures lipopolysaccharide-stimulated inducible NO synthase expression and nitrite accumulation were synergistically enhanced by pretreatment with endothelin, whereas endothelin itself did not induce these responses. Pretreatment with endothelin-1, endothelin-3, and the selective endothelin type B (ETB) receptor agonist IRL 1620 caused the same effect with similar potencies, suggesting that the synergism was mediated via the endothelin ETB receptor. A protein kinase C inhibitor, calphostin C, suppressed endothelin-3-enhanced inducible NO synthase expression. Pretreatment with either endothelin-3 or phorbol ester enhanced lipopolysaccharide-induced production of tumor necrosis factor-alpha (TNF-alpha). Simultaneous addition of TNF-alpha increased lipopolysaccharide-stimulated inducible NO synthase expression. These results suggest that the increase in inducible NO synthase expression by endothelin was due to the elevated TNF-alpha production via protein kinase C. Our findings present the possibility that endothelin is implicated in neurotoxicity via enhancement of inducible NO synthase expression.

Pertussis toxin-insensitive effects of mastoparan, a wasp venom peptide, in PC 12 cells

Recent studies have shown that mastoparan, an amphiphilic peptide derived from wasp venom, modifies the secretion of neurotransmitters and hormones from a variety of cell types. Mastoparan interacts with heterotrimeric guanine nucleotide‐binding proteins (G proteins) such as Gi and Go, which are ADP‐ribosylated by pertussis toxin (PTX) and thereby uncoupled from receptors. Previously, some of the effects of mastoparan including secretion were reported to be modified selectively by PTX but not by cholera toxin (CTX). In the present study, we examined the influence of bacterial toxins on the effects of mastoparan in PC12 cells. Mastoparan stimulated [3H]noradrenaline (NA) release from prelabeled PC12 cells in the absence of CaCl2, although high K+ or ATP stimulated the release in a Ca2+‐dependent manner. Pretreatment with CTX, not PTX, for 24 h inhibited mastoparan‐stimulated [3H]NA release. Mastoparan inhibited forskolin‐stimulated cyclic AMP accumulation in a dose‐dependent manner, although mastoparan had no effect by itself. Pretreatment with PTX completely abolished the inhibitory effect of carbachol via Gi on cyclic AMP accumulation and partially reduced the effect of mastoparan. However, the inhibitory effect of 20 μM mastoparan was not modified by pretreatment with PTX. Thus, we investigated the effect of mastoparan on CTX‐catalyzed [32P]ADP‐ribosylation of proteins in PC12 cells. A subunit of CTX (CTX‐A) catalyzed [32P]ADP‐ribosylation of many proteins in the cytosolic fraction of PC12 cells. One of these was a 20 kDa protein, named ADP‐ribosylating factor (ARF). The addition of mastoparan to assay mixtures inhibited ADP‐ribosylation of many proteins including ARF and CTX‐A in the presence of the cytosolic fraction. In the absence of the cytosolic fraction, however, mastoparan slightly enhanced ADP‐ribosylation of bovine serum albumin and auto‐ADP‐ribosylation by CTX‐A. Mastoparan did not inhibit ADP‐ribosylation of the α subunit of Gs in the membrane fraction. These findings suggest that (1) mastoparan interacts with PTX‐insensitive and CTX‐sensitive factor(s) to stimulate NA release, and (2) mastoparan interacts with ARF inhibiting its activity to enhance the ADP‐ribosylation reaction by CTX. ARF may be an exocytosis‐linked G protein.

REGULATION OF INDUCIBLE NO SYNTHASE EXPRESSION BY ENDOTHELIN IN PRIMARY CULTURED GLIAL CELLS

Nitric oxide (NO), initially identified as an endothelium-derived relaxing factor, is a molecular mediator that has been implicated in many physiological and pathological processes. In primary cultured rat glial cells, a combination of inflammatory cytokines (tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta)) and bacterial lipopolysaccharide (LPS) stimulates production of nitrite via expression of the inducible form of nitric oxide synthase (iNOS). In these cells, simultaneous addition of endothelin (ET) markedly inhibited TNF-alpha/IL-1beta-induced and LPS-induced nitrite production and iNOS expression, although ET by itself had no effect. The inhibitory effect of ETs appears to be mediated by ET(B) receptors. Forskolin also inhibited the iNOS expression. By contrast, pretreatment with ET for 24 hours enhanced LPS-induced nitrite production and iNOS expression. This stimulatory effect of ETs was suppressed by calphostin C, a protein kinase C inhibitor, and pretreatment with phorbol ester enhanced LPS-induced iNOS expression. Our findings present the possibility that ET has dual effects on iNOS expression in glial cells.

Possible involvement of botulinum ADP-ribosyltransferase sensitive low molecular G-protein on 5-hydroxytryptamine (5-HT)-induced inositol phosphates formation in 5-HT2c cDNA transfected cells

To clarify the involvement of botulinum ADP-ribosyltransferase sensitive low molecular G-proteins in 5-hydroxytryptamine (5-HT)-induced stimulation of phosphatidylinositol turnover, we examined the effects of 5-HT on inositol phosphates formation in COS 7 cells transfected with 5-HT2c receptor cDNA, but did not in non-transfected or vector-transfected cells. A typical 5-HT2c receptor antagonist mianserin (0.3-3 microM) inhibited the 5-HT-induced inositol phosphates formation. Treatment with botulinum toxin D preparation (20 micrograms/ml, 8 h) that contained botulinum C3 ADP-ribosyltransferase, blocked the 5-HT-induced inositol phosphate formation, although botulinum toxin A preparation that did not contain the enzyme did not have an influence. These results support our previous findings suggesting that low molecular weight G-proteins ADP-ribosylated by botulinum ADP-ribosyltransferase are involved in phospholipase C activity.

Inhibition of noradrenaline release from PC12 cells by the long-term treatment with cholera toxin.

Guanine nucleotide-binding (G) proteins are required for intracellular vesicular transport and endocytosis. In this study, we investigated the effects of short-term (2 h) and long-term (24 h) treatment with cholera toxin (CTX), which ADP-ribosylates proteins having arginine residues such as the alpha subunit of Gs (G(s alpha)), on exocytosis from the neurosecretory rat pheochromocytoma PC 12 cell line. Short-term treatment with CTX stimulated the accumulation of cyclic AMP, and synergistically enhanced both extracellular Ca2+-dependent [3H]noradrenaline (NA) releases (induced by high K+ and ATP) and Ca2+-independent release (induced by mastoparan, a peptide in wasp venom). Long-term treatment with CTX for 24h inhibited Ca2+-dependent and -independent stimulated [3H]NA release. The inhibitory effect of long-term CTX treatment was not derived from a cyclic AMP-dependent system, because (1) H-89, an inhibitor of protein kinase A, had no effect on the inhibition induced by CTX, (2) the long-term treatment with forskolin did not show an inhibitory effect. [32P]ADP-ribosylation of G(s alpha) and its immunoreactivity with anti-G(s alpha) antiserum in the crude membrane fraction was inhibited in the long-term CTX-treated cells, but not in the long-term forskolin-treated cells. [32P]ADP-ribosylation of G(s alpha) in the membrane fraction of short-term CTX-treated cells was approximately 90% of the level in the control cells. These findings suggest that CTX stimulates [3H]NA release via a cyclic AMP-dependent system in the short-term, and that long-term CTX treatment inhibited its release, maybe via ADP-ribosylation of CTX-sensitive proteins such as G(s alpha).