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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.

ABSTRACT-PhospholipaseA2 has recently been proposed as the effector enzyme involved in the receptor-mediated release of arachidonic acid (AA). Released AA and its metabolites have been demonstrated to play an important role in the regulation of cell functions.[3H]AA release from prelabeled PC12 cells was stimulated by a Ca ionophore such as ionomycin or A23187. Although ATP and its effective analogs, adenosine 5'-O-(3-thiotrisphosphate) (ATPrS), 2-methylthio ATP and 3'-O-(4-benzoyl)benzoyl ATP, did not stimulate [3H]AA release on their own, they did enhance Ca ionophore-stimulated[3H]AA release. The effect of ATP analogs was dose-dependent. ADP, UTP, GTP, ITP, a f3-methylene ATP, i37-methylene ATP and 8-bromo ATP showed no effect or very limited effect. The effect of ATPrS was antagonized by suramin, a putative P2Y receptor antagonist. The effective ATP analogs also increased [Ca 2+], (cytosolic free Ca2+ concentration) via Ca 2+ influx. However, the addition of 50 mM KCl or 10 PM bradykinin, which are well-known to increase [Ca 2+]; by different pathways, did not stimulate [3H]AA release, either with or without the Ca ionophore. The addition of phorbol 12-myristate 13-acetate, an activator of protein kinase C, showed no effect on [3H]AA release, either with or without the Ca ionophore. These data suggest that 1) ATP increased Ca ionophore-stimulated AA release via a P2Y-like ATP receptor, and that 2) the elevation of [Ca 2+]; by ATP does not quantitatively explain the ATP-stimulated AA release in PC12 cells.Keywords: ATP, Arachidonic acid, Intracellular calcium, PC12 cell Purine nucleotides and nucleosides have widespread and potent extracellular actions on a variety of excitable and non-excitable cells. Burnstock (1) proposed two types of receptors: the P1 receptor for adenosine, and the P2 receptor for ATP. Different subtypes of the P2 receptor have been defined classically as follows (1-3): P2x and P2Y receptor subclasses have been differentiated on the basis of relative potencies of the ATP analogs and selective antagonism. Thus, for the P2X receptor: aj3-methylene ATP = (3r-methylene ATP > ATP; P2Y receptor: 2-methylthio ATP >_ ATP > a~-methylene ATP = Prmethylene ATP. P2x receptors are preferentially located in vascular smooth muscle cells and mediate vasoconstriction. P2Y receptors are located on a variety of cell types and regulate cell functions via the activation of phospholipase C. Suramin and reactive blue 2 have been claimed to be specific antagonists to P2Y receptors, at least over a limited concentration range (4, 5). Since the receptors for ATP on platelets, mast cells, lymphocytes and fibroblasts do not seem to fit this subclassification, they have been termed P2T and P2z receptors, respectively (1-3). The P2T receptors found in platelets are unique in being activated by ADP rather than ATP. The actual agonist form for P2z receptors is thought to be the tetrabasic acid ATP4-(6), because Mg2+ addition increased the ED50 (dose required to achieve 50010 of maximal effect) value for ATP. The rank o...

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 G(o), 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 Ca(2+)-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 microM 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 alpha 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.

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The influence of activation of protein kinase C (PKC) and cyclic AMP on noradrenaline (NA) release in the neurosecretory rat pheochromocytoma PC12 cell line was investigated. External ATP induced [3H]NA release from prelabeled PC12 cells, in the presence of extracellular CaCl2. The potency order of ATP analogs was adenosine 5'-O-(gamma-thiotriphosphate) > or = ATP > 2-methylthio ATP > 2',3'-O-(4-benzoyl)benzoyl ATP. alpha,beta-Methylene ATP, beta gamma-methylene ATP, and 8-bromo ATP were inactive. Neither ADP, GTP, nor ITP was active. The addition of phorbol 12-myristate 13-acetate (PMA) or agents elevating the cyclic AMP content, such as vasoactive intestinal peptide (VIP) or an adenosine analog, also stimulated [3H]NA release. Not only high K(+)- but also ATP-stimulated [3H]NA release was enhanced by co-addition with PMA or agents elevating the cyclic AMP content. PMA and VIP had no effect on the cytosolic free Ca2+ concentration ([Ca2+]i) or on the ATP-stimulated [Ca2+]i rise, although both stimulatory effects on [3H]NA release were dependent on extracellular CaCl2. The addition of PMA stimulated [3H]NA release dose-dependently, and enhanced 300 microM (maximal dose) ATP-stimulated [3H]NA release without changing the affinity for ATP. The effect of PMA was inhibited by PKC inhibitors such as calphostin C and in PKC-depleted cells, and potentiated by elevation of cyclic AMP. These data suggest that the process of ATP-stimulated NA release, not ATP-stimulated Ca2+ influx, is regulated by the dual, PKC- and cyclic AMP-dependent mechanisms, positively and independently. Treatment with pertussis toxin had no effect on the ATP-stimulated [Ca2+]i rise or [3H]NA release.(ABSTRACT TRUNCATED AT 250 WORDS)