Stimulation of arachidonic acid release by guanine nucleotide in saponin-permeabilized neutrophils: evidence for involvement of GTP-binding protein in phospholipase A2 activation

Addition of a guanine nucleotide analog, guanosine 5'-O-(thiotriphosphate) (GTP gamma S)(1-100 microM) induced release of [3H]arachidonic acid from [3H]arachidonate-prelabeled rabbit neutrophils permeabilized with saponin. The chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced arachidonate release was enhanced by GTP gamma S, Ca2+, or their combination. Ca2+ alone (up to 100 microM) did not effectively stimulate lipid turnover. However, the combination of fMLP plus GTP gamma S elicited greater than additional effects in the presence of resting level of free Ca2+. The addition of 100 microM of GTP gamma S reduced the Ca2+ requirement for arachidonic acid liberation induced by fMLP. Pretreatment of neutrophils with pertussis toxin resulted in the abolition of arachidonate release and diacylglycerol formation. Neomycin (1 mM) caused no significant reduction of arachidonate release. In contrast, about 40% of GTP gamma S-induced arachidonate release was inhibited by a diacylglycerol lipase inhibitor, RHC 80267 (30 microM). These observations indicate that liberation of arachidonic acid is mediated by phospholipase A2 and also by phospholipase C/diacylglycerol lipase pathways. Fluoride, which bypasses the receptor and directly activates G proteins, induced arachidonic acid release and diacylglycerol formation. The fluoride-induced arachidonate release also appeared to be mediated by these two pathways. The loss of [3H]arachidonate was seen in phosphatidylinositol, phosphatidylcholine, and phosphatidylethanolamine. These data indicate that a G protein is involved between the binding of fMLP to its receptor and activation of phospholipase A2, and also that the arachidonic acid release is mediated by both phospholipase A2 and phospholipase C/diacylglycerol lipase.     

The inhibition of arachidonic acid metabolism in human platelets by RHC 80267, a diacylglycerol lipase inhibitor

The diacylglycerol lipase inhibitor, RHC 80267, 1,6-di(O-(carbamoyl)cyclohexanone oxime)hexane, was tested for its ability to block the release of arachidonic acid from human platelets. At a concentration (10 microM) reported to completely inhibit diacylglycerol lipase in fractions of broken platelets, RHC 80267 had no effect on diacylglycerol lipase activity or the release of arachidonic acid from washed human platelets stimulated with collagen. At a high concentration (250 microM), the compound inhibited the formation of arachidonyl-monoacylglycerol by 70% and the release of arachidonate by 60%. However, at this concentration RHC 80267 was found to inhibit cyclooxygenase activity, phospholipase C activity and the hydrolysis of phosphatidylcholine (PC) (presumably by inhibiting phospholipase A2). The phospholipase C inhibition was attributed to the inhibition of prostaglandin H2 formation, as it was alleviated by the addition of the endoperoxide analog, U-46619. PC hydrolysis was only partially restored with U-46619, suggesting that RHC 80267 directly alters phospholipase A2 activity. The inhibition of arachidonate release observed was accounted for by the inhibition of PC hydrolysis. We conclude that RHC 80267, because of its lack of specificity at concentrations needed to inhibit diacylglycerol lipase, is an unsuitable inhibitor for studying the release of arachidonic acid in intact human platelets.     

Tumor necrosis factor-alpha stimulates phosphatidylinositol breakdown by phospholipase C to coordinately increase the levels of diacylglycerol, free arachidonic acid and prostaglandins in an osteoblast (MC3T3-E1) cell line

The effects of (human recombinant) tumor necrosis factor-alpha on phosphatidylinositol breakdown, release of 1,2-diacylglycerols, mobilization of arachidonate from diacylglycerol and prostaglandin synthesis were examined in a model osteoblast cell line (MC3T3-E1). Tumor necrosis factor-alpha (10 nM) caused a specific (30%) decrease in the mass of phosphatidylinositol (and no other phospholipids) within 30 min of exposure. Tumor necrosis factor-alpha doubled the rate of incorporation of [32P]orthophosphoric acid into phosphatidylinositol, indicating that the turnover of inositol phosphate was enhanced, and increased the content of diacylglycerol in parallel with phosphatidylinositol breakdown. The cytokine (10-50 nM; 4 h) also promoted a specific release of 24-34% of the [3H]arachidonate from prelabeled phosphatidylinositol, a release of 80% of the 3H-fatty acid from the diacylglycerol pool, and a 30-fold increase in the synthesis of prostaglandin E2. The tumor necrosis factor-alpha induced liberation of [3H]arachidonate from diacylglycerol, cellular arachidonate release and the synthesis of prostaglandin E2 were each blocked by an inhibitor of diacylglycerol lipase, the compound RHC 80267 (30 microM). Therefore, we conclude that, in the MC3T3-E1 cell line, tumor necrosis factor-alpha activates a phosphatidylinositol-specific phospholipase C (phosphatidylinositol inositolphosphohydrolase; EC 3.1.4.3) to release diacylglycerol, and increases the metabolism of diacylglycerol to liberate arachidonate for prostaglandin synthesis.     

Differential sensitivity of arachidonic acid release and 1,2-diacylglycerol formation to pertussis toxin, GDP beta S and NaF in saponin-permeabilized human platelets: possible evidence for distinct GTP-binding proteins involving phospholipase C and A2 activation

Human platelets labeled with [3H]arachidonic acid and permeabilized with saponin produced [3H]1,2-diacylglycerol (DG) by phospholipase C and released [3H]arachidonate by phospholipase A2, when activated with thrombin. Thrombin-induced arachidonate liberation was almost completely inhibited with pretreatment of pertussis toxin (10 micrograms/ml), whereas DG formation was decreased by only 20-40% in the toxin-treated platelets. Although guanosine 5'-o-(2-thiodiphosphate) (GDP beta S) suppressed arachidonate release and DG production in a dose-dependent manner, the half maximal inhibition required less than 10 microM for arachidonate release but more than 100 microM for DG production. Moreover, the dose-response effects of NaF on arachidonate release and DG formation were different. These results indicate that arachidonate release and DG formation are differently affected by these agents acting on guanine nucleotide binding proteins (G-proteins), suggesting that the distinct G proteins modulate the activity of phospholipase C and phospholipase A2.     

Neutrophil beta-adrenergic receptor responses are potentiated by acute exposure to phorbol ester without changes in receptor distribution or coupling.

Exposure to the phorbol ester, phorbol 12-myristate, 13-acetate (PMA, 100nM) for 10 minutes enhanced cyclic AMP accumulation in human neutrophils under basal conditions and in response to the beta-adrenergic receptor agonist isoproterenol (ISO), 1 microM) and the adenylate cyclase activator forskolin (FSK, 10mM). Potentiation of responses to ISO by PMA was dose-dependent between 0.1 and 100nM PMA. The diacylglycerol analogue, 1-oleoyl-2-acetylglycerol (OAG) (50 microM) also elevated beta-receptor responses, but 4 beta-phorbol (100nM), lacking the capacity to activate PMA, was ineffective. Short-term exposure (12 seconds) to the peptide n-formylmethionine leucyl-phenylalanine (FMLP, 1 microM) also elevated neutrophil cyclic AMP accumulation. All potentiating effects of PMA on cyclic AMP production were inhibited by the protein kinase inhibitor 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine (H7). Elevation of cyclic AMP by FMLP was insensitive to H7. PMA had no apparent effect on beta-receptor agonist-affinity, distribution between cell-surface and internalised compartments, or the capacity of ISO to induce beta-receptor internalisation. Responses to FSK or ISO in terms of fold-stimulation of basal cyclic AMP accumulation in the presence of PMA were not elevated by PMA. These findings indicate that PMA exerts a potentiating effect on neutrophil adenylate cyclase responses through protein kinase C activation. FMLP elevation of neutrophil cyclic AMP in the absence of other stimuli, appears however, to be insensitive to protein kinase inhibition.     

Cytosolic phospholipase A2 alpha inhibition prevents neuronal NMDA receptor-stimulated arachidonic acid mobilization and prostaglandin production but not subsequent cell death

Phospholipase A(2) (PLA(2)) enzymes encompass a superfamily of at least 13 extracellular and intracellular esterases that hydrolyze the sn-2 fatty acyl bonds of phospholipids to yield fatty acids and lysophospholipids. The purpose of this study was to characterize which phospholipase paralog regulates NMDA receptor-mediated arachidonic acid (AA) release. Using mixed cortical cell cultures containing both neurons and astrocytes, we found that [(3)H]-AA released into the extracellular medium following NMDA receptor stimulation (100 microM) increased with time and was completely prevented by the addition of the NMDA receptor antagonist MK-801 (10 microM) or by removal of extracellular Ca(2+). Neither diacylglycerol lipase inhibition (RHC-80267; 10 microM) nor selective inhibition of Ca(2+)-independent PLA(2) [bromoenol lactone (BEL); 10 microM] alone had an effect on NMDA receptor-stimulated release of [(3)H]-AA. Release was prevented by methyl arachidonyl fluorophosphonate (MAFP) (5 microM) and AACOCF(3) (1 microM), inhibitors of both cytosolic PLA(2) (cPLA(2)) and Ca(2+)-independent PLA(2) isozymes. This inhibition effectively translated to block of NMDA-induced prostaglandin (PG) production. An inhibitor of p38MAPK, SB 203580 (7.5 microM), also significantly reduced NMDA-induced PG production providing suggestive evidence for the role of cPLA(2)alpha. Its involvement in release was confirmed using cultures derived from mice deficient in cPLA(2)alpha, which failed to produce PGs in response to NMDA receptor stimulation. Interestingly, neither MAFP, AACOCF(3) nor cultures derived from cPLA(2)alpha null mutant animals showed any protection against NMDA-mediated neurotoxicity, indicating that inhibition of this enzyme may not be a viable protective strategy in disorders of the cortex involving over-activation of the NMDA receptor.     

The role of endogenously formed diacylglycerol in the propagation and termination of platelet activation. A biochemical and functional analysis using the novel diacylglycerol kinase inhibitor, R 59 949

The putative roles for the second messenger, diacylglycerol, were investigated in intact platelets using a novel diacylglycerol kinase inhibitor, R 59 949, or (3-[2-[4-[bis(4-fluorophenyl)methylene]-1-piperidinyl]ethyl]-2,3- dihydro-2-thioxo-4(1H)-quinazolinone). The compound inhibited the diacylglycerol kinase in a concentration-dependent manner (10(-8) to 10(-5) M) in isolated platelet membranes and in intact platelets. When platelets were stimulated with vasopressin in the presence of the compound, protein kinase C activity was markedly increased; the formation of inositol phosphates, the increase in intracellular Ca2+ and shape-change reaction were antagonized while the vasopressin-induced polyphosphoinositide synthesis was amplified, and this in a distinct inositolphospholipid pool. In the presence of R 59 949, vasopressin- as well as collagen-induced release reaction and aggregation was strongly increased, independently of the formation of arachidonate metabolites. It is concluded that diacylglycerol formed after receptor activation, likely by activating the protein kinase C, plays an important role in the propagation of platelet functional responses in casu aggregation and secretion and controls the termination of the primary receptor coupled responses.     

Multifactorial regulation of prostaglandin synthesis in preovulatory goldfish ovarian follicles.

Goldfish preovulatory ovarian follicles (prior to germinal vesicle breakdown) were utilized for studies investigating the actions of activators of different signal transduction pathways on prostaglandin (PG) production. The protein kinase C (PKC) activators phorbol 12-myristate 13-acetate (PMA; 100-400 nM), 1-oleoyl-2-acetylglycerol (5 and 25 micrograms/ml), and 1,2-dioctanoylglycerol (10 and 50 micrograms/ml) stimulated PGE production; the inactive phorbol 4 alpha-phorbol didecanoate, which does not activate PKC, had no effect. Calcium ionophore A23187 (0.25-4.0 microM) stimulated PGE production and acted in a synergistic manner with activators of PKC. Although produced in lower amounts than PGE, PGF was stimulated by PMA and A23187. The direct activator of phospholipase A2, melittin (0.1-1.0 microM), stimulated a dose-related increase in PGE production, whereas chloroquine (100 microM), a putative inhibitor of phospholipase A2, blocked basal and PMA + A23187-stimulated PGE production. Several drugs known to elevate intracellular levels of cAMP including the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.1-1.0 mM), forskolin (10 microM), and dibutyryl cAMP (dbcAMP; 5 mM) attenuate PMA + A23187-stimulated PGE production. Melittin-stimulated production of PGE was inhibited by dbcAMP, suggesting that the action of cAMP was distal to the activation of phospholipase A2. In summary, these studies demonstrate that activation of PKC and elevation of intracellular calcium levels stimulate PG production, in part, through activation of phospholipase A2. The adenylate cyclase/cAMP signalling pathway is inhibitory to PG production by goldfish ovarian follicles.     

Inhibition by adenosine of reactive oxygen metabolite production by human polymorphonuclear leucocytes.

The stimulation of reactive oxygen metabolite production from human polymorphonuclear leucocytes by chemotactic peptide (fMet-Leu-Phe) was inhibited by adenosine with a K0.5 of 0.6 microM. Dipyridamole (0.1 microM), an inhibitor of adenosine uptake, did not prevent the effect of adenosine. Non-metabolizable analogues could substitute for adenosine in the potency order N-ethoxycarboxamideadenosine greater than 2-chloroadenosine greater than adenosine greater than L-N6-(phenylisopropyl)adenosine = D-N6-(phenylisopropyl)adenosine, which is characteristic of an A2 adenosine receptor. The effects of adenosine, 2-chloroadenosine and N-ethoxycarboxamideadenosine were reversed by 8-phenyltheophylline. When endocytosis was inhibited with cytochalasin B, cells were still susceptible to adenosine receptor agonists. 2-Chloroadenosine (10 microM) reduced the activation of respiration in response to chemotactic peptide from 3.3-fold to 1.4-fold. Activation of reactive oxygen metabolite production in response to latex beads was not reversed by adenosine or its analogues. It was concluded that adenosine acts at an A2 adenosine receptor to antagonize the activation of polymorphonuclear leucocytes by those stimuli, such as chemotactic peptide, which cause an increase in the intracellular free Ca2+ concentration.     

Involvement of phosphoinositide-3-kinase and phospholipase C transduction systems in the non-genomic action of progesterone in vascular tissue

We investigate the participation of tyrosine kinase, phosphatidylinositol-3-kinase, phospholipase C systems in the intracellular transduction pathways involved in the non-genomic stimulation of vasodilators compounds synthesis induced by progesterone (Pg). Using aortic strips isolated from female fertile Wistar rats, we showed that physiological concentrations of progesterone markedly increase prostacyclin synthesis in a very short time interval (45 s to 10 min) as well as nitric oxide release (5-30 min). The stimulatory action of progesterone on nitric oxide synthase (NOS) activity was maintained even in the presence of an antagonist of progesterone receptor, compound RU486. In contrast, in the presence of tyrosine kinase inhibitor (1 microM genistein) or phosphatidylinositol-3-kinase inhibitor (1 microM LY294002), the enhancement of nitric oxide elicited by 10-100 nM progesterone was completely suppressed. The steroid stimulates phopholipase C activity, inducing significant increase in diacylglycerol generation (5-15-min treatment). The presence of an inhibitor of protein kinase C (PKC) impaired the anti-aggregatory action of the hormone. Due to the fact that phospholipase C activation implies calcium mobilization, we investigate the role of changes in calcium fluxes on progesterone nitric oxide generation. We found that calcium influx from extracellular medium and calcium mobilization from internal pools was required. The present results suggest that, tyrosine kinase and phosphatidylinositol-3-kinase cascades are involved in progesterone nitric oxide synthase stimulation and that diacilglicerol/protein kinase C system may be relevant for physiological regulation of platelet aggregation process.     

The effects of non-steroidal inhibitors of phospholipase A2 on leukotriene and histamine release from human and guinea-pig lung

The effects of chloroquine and mepacrine were determined on the release of slow reacting substances (leukotrienes) from lung fragments in vitro. These drugs have been shown in a variety of tissues to inhibit phospholipase A2, and thus to reduce the availability of arachidonate, which is a substrate for leukotriene biosynthesis. Leukotriene and histamine release from unsensitized human lung was stimulated by calcium ionophore A23187, and from actively sensitized guinea-pig lung, by ovalbumin. Chloroquine (10 microM and 100 microM) significantly inhibited leukotriene release in lung from both species, and at 100 microM also inhibited histamine release. Mepacrine (10 microM) inhibited leukotriene release in human lung and at 100 microM in guinea-pig lung. The effects of chloroquine (100 microM) on leukotriene release were counteracted by the presence of arachidonic acid (10 microM), which suggests that chloroquine had impaired the availability of arachidonate. It seems probable that chloroquine and mepacrine inhibit leukotriene release by inhibition of phospholipase A2 in lung.     

Does protein kinase C activation mediate thrombin-induced arachidonate release in human platelets?

Thrombin stimulated rapid formation of diacylglycerol, inositol 1,4,5-trisphosphate (IP3) and thromboxane B2 (TXB2) in human platelets. Formation of diacylglycerol and IP3 appeared to precede that of TXB2. Activation of protein kinase C by diacylglycerol combining with Ca+2 mobilization by IP3 has been implicated in mediating arachidonate release. However, addition of the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) to platelet suspension did not inhibit thrombin-stimulated arachidonate release and TXB2 synthesis, whereas addition of the Ca+2 antagonist, 3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester (TMB-8) or the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) abolished arachidonate release. The correlation of IP3 production with arachidonate release on increasing the concentrations of thrombin was further examined. IP3 production reached near maximum at 0.2 U/ml, whereas TXB2 synthesis continued to increase at 1 U/ml. These results suggest that protein kinase C activation may not mediate arachidonate release and that Ca+2 mobilization by IP3 may only partially account for arachidonate release in platelets stimulated with relatively high concentrations of thrombin.     

Stimulation of prostaglandin synthesis in WI-38 human lung fibroblasts following inhibition of phospholipid acylation by p-hydroxymercuribenzoate

The release of arachidonic acid and its metabolites, prostaglandin E2 and thromboxane A2, from WI-38 human lung fibroblasts was modulated by p-hydroxymercuribenzoate. Exposure to the inhibitor resulted in a dose-dependent decrease in [1-14C]arachidonic acid uptake and incorporation into phospholipids and neutral lipid pools. Activities of lung fibroblast arachidonyl-CoA synthetase and lysolecithin acyltransferase were inhibited by 100 microM p-hydroxymercuribenzoate. [14C]Arachidonic acid labelled fibroblasts exhibited an increased release of [14C]arachidonate and [14C]prostaglandin E2 of 54% and 112%, respectively, when exposed to 100 microM of inhibitor. The stimulatory effects of 8.0 microM delta 1-tetrahydrocannabinol on arachidonate release and prostaglandin E synthesis (Burstein, S., Hunter, S.A., Sedor, C. and Shulman, S. (1982) Biochem. Pharmacol. 31, 2361-2365) were modified by the inclusion of inhibiting agent, resulting in a 608% stimulation in arachidonic acid release, while prostaglandin E2 and thromboxane A2 synthesis increased 894% and 390%, respectively, over levels obtained by untreated cells. The levels of arachidonate metabolites were altered by inhibitor when compared to cells treated with cannabinoid alone. No significant inhibition by delta 1-tetrahydrocannabinol was found on arachidonic uptake in these cells. In unlabelled studies, p-hydroxymercuribenzoate resulted in a profound, dose-dependent stimulation of prostaglandin E synthesis of 1490% at 150 microM inhibitor concentration. These results provide evidence that free arachidonate is reincorporated via acylation, thereby implicating this pathway as a possible control mechanism for the synthesis of arachidonic acid metabolites.     

Integrin-dependent leukocyte adhesion involves geranylgeranylated protein(s)

Integrin-dependent leukocyte adhesion is modulated by alterations in receptor affinity or by post-receptor events. Pretreatment of Jurkat T-cells with the 3-hydroxymethylglutaryl-coenzyme A reductase inhibitor, lovastatin, markedly reduced (IC(50) approximately 1-2 microM) alpha(4)beta(1)-dependent adhesion to fibronectin (FN) stimulated by phorbol 12-myristate 13-acetate (PMA) which modulates post-receptor events. In contrast, lovastatin did not inhibit Jurkat cell adhesion to FN induced by the beta(1) integrin-activating monoclonal antibody (mAb) 8A2, which directly modulates beta(1) integrin affinity. Similarly, pretreatment of U937 cells with lovastatin inhibited PMA-stimulated, but not mAb 8A2-stimulated, alpha(6)beta(1)-dependent leukocyte adhesion to laminin. The inhibition of lovastatin on PMA-stimulated leukocyte adhesion was not mediated by mitogen-activated protein kinase or phosphatidylinositol 3-kinase pathway. The inhibitory effect of lovastatin on PMA-stimulated leukocyte adhesion was reversed by co-incubation with geranylgeraniol, but not with farnesol, with concurrent reversal of the inhibition of protein prenylation as shown by protein RhoA geranylgeranylation. The selective inhibition of protein geranylgeranylation by the specific protein geranylgeranyltransferase-I inhibitor, GGTI-298, blocked PMA-stimulated leukocyte adhesion but not mAb 8A2-induced leukocyte adhesion. The protein farnesyltransferase inhibitor, FTI-277, had no effect on leukocyte adhesion induced by either stimulus. These results demonstrate that protein geranylgeranylation, but not farnesylation, is required for integrin-dependent post-receptor events in leukocyte adhesion.     

Attenuation of sn-1,2-diacylglycerol second messengers by diacylglycerol kinase. Inhibition by diacylglycerol analogs in vitro and in human platelets

Diacylglycerol kinase is though to play a central role in the metabolism of diacylglycerol second messengers in agonist-stimulated cells. A series of diacylglycerol analogs were tested for their ability to act as substrates or inhibitors of diacylglycerol kinase with the goal of determining the substrate specificity of the enzyme, and of discovering inhibitors. Screening of these compounds was performed using a partially purified diacylglycerol kinase from pig brain. Modified assays for this enzyme using co-sonicated mixtures of diacylglycerol and anionic phospholipids were developed. This enzyme was found to be quite specific for sn-1,2-diacylglycerol (KM 24 microM for dioctanoyl-glycerol). Among the analogs investigated, only 1,2-dioctanoyl-2-amino-1,3-propanediol was utilized at a significant rate. Two analogs, dioctanoylethylene glycol (KI 58 microM) and 1-monooleoylglycerol (KI 91 microM), were potent inhibitors in vitro. These compounds were tested for effects on diacylglycerol formation and metabolism in thrombin-stimulated human platelets. Dioctanoylethylene glycol inhibited diacylglycerol phosphorylation in platelets (70-100% at 100 microM) leading to a longer-lived diacylglycerol signal. This compound may be a useful tool for studies of diacylglycerol kinase in other cell types. 1-Monooleoylglycerol treatment elevated diacylglycerol levels up to 4-fold in unstimulated platelets and up to 10-fold in thrombin-stimulated platelets. The implications with regard to the pathways of diacylglycerol metabolism in human platelets are discussed.     

Arachidonic acid acts as an intracellular activator of NADPH-oxidase in Fc gamma receptor-mediated superoxide generation in macrophages

A protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), inhibited phorbol ester (12-o-tetradecanoylphorbol 13-acetate)-induced and Fc gamma receptor-mediated superoxide anion (O2-) generations in guinea pig macrophages, but the inhibitory effect on Fc gamma receptor-mediated O2- generation was only partial. Both O2- generations were inhibited extensively by a phospholipase A2 inhibitor, 4-p-bromophenacyl bromide (4-pBPB). It was confirmed in control experiments that H-7 and 4-pBPB had no direct inhibitory effect on NADPH-oxidase activity. Dose-dependent stimulation of O2- generation was induced by arachidonate in macrophages, and the arachidonate-induced O2- generation was not inhibited by H-7. Arachidonate could also induce NADPH-oxidase activation in a post-nuclear fraction obtained from unstimulated macrophages and this activation was not inhibited by H-7, indicating that protein kinase C activation was not involved in this cellfree system. These results support the hypothesis that the O2- generation induced by Fc gamma receptor stimulation is mainly mediated by arachidonic acid which is released by the action of phospholipase A2 activated by receptor stimulation. Arachidonic acid seems to be acting rather directly in activating the NADPH-oxidase system of macrophage membrane. Protein kinase C may have a significant role in Fc gamma receptor-mediated O2- generation but it is not obligatory, and protein kinase C seems to activate NADPH-oxidase rather indirectly, probably by inducing the arachidonic acid release.     

Inositol 1,4,5-trisphosphate transduction cascade in taste reception of the fleshfly, Boettcherisca peregrina

The role of an inositol 1,4,5-trisphosphate (IP3)-mediated transduction cascade in the response of taste receptor cells of the fleshfly Boettcherisca peregrina was investigated by using the following reagents: neomycin (an inhibitor of IP3 production), U73122 (an inhibitor of phospholipase C), adenophostin A (an agonist of the IP3-gated channel), IP3, ruthenium red (a blocker of the IP3-gated channel), and 2-aminoethoxydiphenylborate (2-APB; an antagonist of the IP3-gated channel). For introduction into the receptor cell, the reagents were mixed with a detergent, deoxycholate (DOC). After treatment with neomycin + DOC or U73122 + DOC, the response of the sugar receptor cell to sugars was depressed compared with responses after treatment with DOC alone. During the treatment of adenophostin A + DOC, the response of the sugar receptor cell was elicited. After treatment with IP3 + DOC, the response of the sugar receptor cell to sugars and to amino acids was apparently enhanced. When taste stimuli were administered in the presence of ruthenium red or 2-APB, the response of the sugar receptor cell to glucose were inhibited. The expression of genes for substances involved in the IP3 transduction cascade, such as G protein alpha subunit (dGqalpha), phospholipase C (norpA), and IP3 receptor (itpr), were examined in the taste receptor cell of the fruitfly Drosophila melanogaster by using the pox-neuro70 mutant (poxn70), which lacks taste receptor cells. The expressed levels of dGqalpha and itpr in the tarsus of poxn70 mutant flies were reduced compared with those of wild-type flies. These results suggest that the IP3 transduction cascade is involved in the response of the sugar receptor cell of the fly.     

Differentiation-linked activation of the respiratory burst in a monocytic cell line (U937) via Fc gamma RII. A study of activation pathways and their regulation.

Activation of the respiratory burst in the monocytic cell line U937 by cross-linking human 40-kDa FcR for IgG (Fc gamma RII) with the IgG1 mAb, CIKM5, is dependent on the maturation state of the cell. Addition of anti-Fc gamma RII to undifferentiated cells does not activate the respiratory burst but differentiation with human rIFN-gamma (200 U/ml) for 13 to 15 days results in maximal stimulation by this agonist, with half-maximal responses in cells incubated for 10 to 12 days. During maturation the development of responsiveness to cross-linking Fc gamma RII occurs later than the development of responsiveness to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (maximal responses at 7 to 9 days), or the chemotactic peptide FMLP (half-maximal responses at 7 to 9 days). The late development of maximal Fc gamma RII responses is not associated with either increased Fc gamma RII expression, enhanced calcium mobilization induced by anti-Fc gamma RII, changes in protein kinase C activity (PKC) or a switch in PKC isotype expression. Activation of the respiratory burst via Fc gamma RII may not be mediated by activation of PKC as the kinase inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride and N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride inhibited the Fc gamma RII response by less than 20% at concentrations which inhibit the 12-O-tetradecanoylphorbol-13-acetate-induced respiratory burst by more than 80%. IFN-gamma U937 cells did not metabolize incorporated arachidonate into eicosanoids when stimulated with anti-Fc gamma RII, suggesting that eicosanoids do not mediate activation of the respiratory burst, and this was confirmed by the lack of inhibition by the specific 5'-lipoxygenase and glutathione S-transferase inhibitor, piriprost, and the cyclo-oxygenase inhibitor, indomethacin. In addition there was no significant release of radiolabeled arachidonate in response to anti-Fc gamma RII. The response to anti-Fc gamma RII is inhibited by pertussis toxin, suggesting that signal transduction is via a GTP-binding protein. Agents that elevate intracellular cAMP increased the magnitude of the cAMP transients stimulated by anti-Fc gamma RII and also inhibited the respiratory burst. FMLP responses showed a similar pattern of sensitivity to this range of inhibitors, suggesting that both Fc gamma RII and FMLP receptor share common regulatory mechanisms. However, the termination of the respiratory burst activated via Fc gamma RII and FMLP receptor is independently regulated, in that after FMLP-induced activation there is no subsequent inhibition of the Fc gamma RII-mediated response and vice versa.     

Angiotensin II-mediated signal transduction events in cystic fibrosis pancreatic duct cells

Different signal transduction pathways, i.e. Ca2+- and cAMP-dependent, involved in mediating the effects of angiotensin II (AII) were investigated separately using the short-circuit current (Isc) technique and radioimmunoassay (RIA) in a cystic fibrosis pancreatic cell line (CFPAC-1) which exhibits defective cAMP-dependent but intact Ca2+-dependent anion secretion. The AII-induced Isc could be inhibited by the specific antagonist for AT1, losartan (1 microM), but not the antagonist for AT2, PD123177 (up to 10 microM). The AII-induced Isc was also reduced by the treatment of the cells with a Ca2+ chelator, BAPTA-AM (100 microM), indicating a dependence of the AII-induced anion secretion on the intracellular Ca2+. Treatment of the cells with pertussis toxin (0.1 microg/ml) or a phospholipase C (PLC) inhibitor, U73122 (5 microM), resulted in a substantial reduction in the AII-induced Isc indicating involvement of Gi and PLC in the Ca2+-dependent anion secretion. RIA measurements showed that AII stimulated an increase in cAMP production which could be reduced by losartan, pertussis toxin and U73122 but not BAPTA-AM. In addition, inhibitors of cyclooxygenase, indomethacin (10 microM) and piroxicam (10 microM), did not have any effect on the AII-induced cAMP production, excluding the involvement of prostaglandins. Our results suggest that both AII-stimulated cAMP and Ca2+-dependent responses are mediated by the AT1 receptor and Gi-coupled PLC pathway. However, the AII-stimulated cAMP production in CFPAC-1 cells is not dependent on Ca2+ or the formation of prostaglandins.     

Diacylglycerol provides arachidonic acid for lipoxygenase products that mediate angiotensin II-induced aldosterone synthesis

The lipoxygenase products of arachidonic acid metabolism have been shown to be important mediators of stimulus secretion coupling in various endocrine tissues. We have recently shown that the 12-lipoxygenase product, 12-hydroxyeicosatetraenoic acid plays a key role as a new specific mediator of angiotensin II-induced aldosterone secretion in the adrenal. In view of the several pathways by which cellular arachidonate can be generated and the important role of diacylglycerol in angiotensin II-responses, we studied the role of diacylglycerol as the source of arachidonic acid for 12-hydroxyeicosatetraenoic formation. Treatment of normal human adrenal glomerulosa cells with the selective diacylglycerol-lipase inhibitor, RHC 80267, resulted in a dose-dependent inhibition of angiotensin II-induced aldosterone as well as 12-hydroxyeicosatetraenoic formation. These results suggest that AA derived from diacylglycerol is the precursor of 12-hydroxyeicosatetraenoic involved in angiotensin II-induced aldosterone secretion. These results reveal a new second messenger role for diacylglycerol in addition to activation of protein kinase C.