Evidence that activation of a common G-protein by receptors for leukotriene B4 and N-formylmethionyl-leucyl-phenylalanine in HL-60 cells occurs by different mechanisms.

Using cultured human umbilical vein endothelial cells, in which phosphatidylcholine (PC) is equally pulse-labelled by various eicosanoid precursor fatty acids (EPFAs), we have studied the remodelling of EPFAs among the phospholipid classes and subclasses with and without activation, and the relationship of this remodelling process to the selective release of arachidonic acid (AA) by phospholipase A2-mediated cell stimulation. When endothelial cells are pulse-incubated with radiolabelled EPFA for 15 min, greater than 80% of cell-associated radioactivity is present in phospholipids, among which greater than 60% is found in 1,2-diacyl-sn-glycero-3-phosphocholine (diacyl PC). After removing unincorporated radioactivity, reincubation of the pulse-labelled cells for up to 6 h results in progressive decrease in EPFA-labelled diacyl PC, increase in AA- or eicosapentaenoic acid (EPA)-labelled 1-O-alk-1-enyl-2-acyl-sn-glycero-3-phosphoethanolamine (plasmalogen PE) and increase only in AA-labelled 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkyl PC). This redistribution of radiolabelled phospholipids is not altered by the presence of excess non-radiolabelled EPFAs. When aspirin-treated EPFA-labelled endothelial cells are stimulated with ionophore A23187, a very selective release of AA is noted in comparison with eicosatrienoate (ETA) or EPA, accompanied by an equivalent decrease in AA-labelled diacyl PC and specific increase in AA-labelled plasmalogen PE and alkyl PC. These selective changes in AA radioactivity induced by A23187 are enhanced 2-fold by pretreating the AA-labelled cells with phorbol 12-myristate 13-acetate, which by itself induces no changes. The changes in radioactivity induced by A23187 without and with phorbol ester among the released AA, the diacyl PC and the plasmalogen PE are significantly correlated with each other. These results indicate that human endothelial cells incorporate EPFAs (AA, ETA, EPA) equally into diacyl PC but selectively release AA esterified into diacyl PC with specific remodelling into plasmalogen PE and alkyl PC.     

Neomycin induces high-affinity agonist binding of G-protein-coupled receptors

Neomycin, an inositol-phospholipid-binding aminoglycoside antibiotic, is known to interfere with signal transduction mechanisms involving phospholipase C as effector enzyme. In this study, we report that neomycin can also markedly influence agonist binding of G-protein-coupled receptors. In membranes of differentiated human leukemia cells (HL 60 cells), neomycin (0.1-10 mM) was found to induce high-affinity binding of the chemotactic tripeptide, N-formyl-methionylleucylphenylalanine (fMet-Leu-Phe), to its receptor sites in a manner similar to magnesium. Gentamycin and streptomycin, two other aminoglycoside antibiotics, were as potent and as effective as neomycin or magnesium in inducing high-affinity agonist receptor binding. Pretreatment of the cells with pertussis toxin reduced the effects of magnesium and neomycin on agonist receptor binding likewise. In contrast, magnesium but not neomycin largely enhanced the potency of guanine nucleotides, particularly of GTP and its analog, guanosine-5'-O-(3-thiotriphosphate), to reduce fMet-Leu-Phe receptor binding, while maximal inhibition of agonist receptor binding by guanine nucleotides was identical with magnesium and neomycin. Furthermore, neomycin could not replace magnesium in providing stimulation of HL 60 membrane high-affinity GTPase by fMet-Leu-Phe. In close agreement to these findings on the pertussis-toxin-sensitive Gi-protein-coupled formyl peptide receptors, neomycin in a manner similar to magnesium induced high-affinity agonist binding of Gs-protein-coupled beta-adrenoceptors. Similar to formyl peptide receptor binding, high-affinity binding of isoproterenol to beta-adrenoceptors in guinea pig lung membranes induced by magnesium and neomycin was inhibited by the GTP analog, guanosine-5'-O-(3-thiotriphosphate), to a similar maximal extent but with an about 100-fold higher potency in the presence of magnesium than in the presence of neomycin. The data presented thus indicate that neomycin and other aminoglycoside antibiotics can mimic the action of magnesium (or other divalent cations) in inducing high-affinity agonist binding of Gi- and Gs-protein-coupled receptors, but not in inducing subsequent G-protein activation by guanosine triphosphates. The data, furthermore, suggest that neomycin by this selective action will be a powerful tool to dissect the multiple sites of magnesium's action in the agonist receptor-G-protein interaction.     

Muscarinic acetylcholine receptor-stimulated binding of guanosine 5'-O-(3-thiotriphosphate) to guanine-nucleotide-binding proteins in cardiac membranes

Receptor-regulated binding of the labeled GTP analog, guanosine 5'-O-(3-thiotriphosphate) ([35S]GTP[S]), to guanine-nucleotide-binding proteins (G-proteins) was studied in porcine atrial membranes enriched in muscarinic acetylcholine (mACh) receptors. Binding of [35S]GTP[S] to the membranes was not or only slightly affected by the cholinergic agonist, carbachol, unless a second nucleotide was simultaneously present in the binding assay. This additional nucleotide requirement was best fulfilled by GDP, being maximally effective at 0.1-1 microM. In contrast, the GDP analog, guanosine 5'-O-(2-thiodiphosphate), could not replace GDP in promoting carbachol-induced increase in [35S]GTP[S] binding. In addition to GDP, agonist-induced stimulation of [35S]GTP[S] binding to porcine atrial membranes required the presence of Mg2+, being half-maximally and maximally effective at about 30 microM and 300 microM, respectively. Addition of NaCl, which decreased control binding measured in the presence of GDP alone, had no effect on the maximal extent of agonist-stimulated binding, but reduced the potency of carbachol in stimulating [35S]GTP[S] binding. Under optimal conditions, carbachol increased the binding of [35S]GTP[S] without apparent lag phase up to about 2.5-fold, with half-maximal and maximal increase being observed at 5-10 microM and 100 microM, respectively. The agonist-induced stimulation was competitively antagonized by the mACh receptor antagonist, atropine. The number of GTP[S] binding sites under receptor control was two--three-fold higher than the number of mACh receptors in the porcine atrial membranes used. Pretreatment of the membranes with pertussis toxin under conditions leading to 95% ADP-ribosylation of the toxin-sensitive G-protein alpha-subunits markedly reduced agonist-stimulated [35S]GTP[S] binding, with, however, about 30% stimulation still remaining. The data presented indicate that agonist-stimulated binding of [35S]GTP[S] to G-proteins can be a sensitive assay for measuring receptor-regulated G-protein activation in native membranes and, furthermore, suggest that one agonist-activated mACh receptor can activate two or three cardiac G-proteins, being mainly members of the pertussis-toxin-sensitive G-proteins.     

ADP-ribosylation of platelet actin by botulinum C2 toxin

Botulinum C2 toxin is a microbial toxin which possesses ADP-ribosyltransferase activity. In human platelet cytosol a 43-kDa protein was ADP-ribosylated by botulinum C2 toxin. Labelling of the 43-kDa protein using [32P]NAD as substrate was reduced by unlabelled NAD and nicotinamide. The label was removed by treatment with snake venom phosphodiesterase. Half-maximal and maximal ADP-ribosylation occurred at 0.1 microgram/ml and 3 micrograms/ml botulinum C2 toxin, respectively. The Km value of the ADP-ribosylation reaction for NAD was about 1 microM. The peptide map of the ADP-ribosylated 43-kDa protein was almost identical with platelet actin. The ADP-ribosylated 43-kDa substrate protein bound to and was eluted from immobilized DNase I in a manner similar to G-actin. Trypsin treatment of platelet cytosol decreased subsequent ADP-ribosylation of the 43-kDa protein without occurrence of smaller labelled polypeptides. Purified platelet actin was also ADP-ribosylated by botulinum C2 toxin with similar characteristics found with actin in platelet cytosol. Phalloidin decreased the ADP-ribosylation of actin in platelet cytosol and of isolated platelet actin. Half-maximal and maximal, about 90%, reduction of actin ADP-ribosylation was observed at 0.4 microM and 10 microM phalloidin, respectively. ADP-ribosylation of purified actin, induced by botulinum C2I toxin, abolished the formation of the typical microfilament network. The data indicate that platelet G-actin but not F-actin is a substrate of botulinum C2 toxin and that this covalent modification largely affects the functional properties of actin.     

Stimulation and inhibition of rat basophilic leukemia cell adenylate cyclase by forskolin

The influence of the diterpene, forskolin, was studied on adenylate cyclase activity in membranes of rat basophilic leukemia cells. Forskolin increased basal adenylate cyclase activity maximally 2-fold at 100 microM. However, adenylate cyclase activity stimulated via the stimulatory guanine nucleotide-binding protein, Ns, by fluoride and the stable GTP analog, guanosine 5'-O-(3-thiotriphosphate), was inhibited by forskolin. Half-maximal and maximal inhibition occurred at about 1 and 10 microM forskolin, respectively. The inhibition occurred without an apparent lag phase, whereas the enzyme stimulation by forskolin was preceded by a considerable lag period. The inhibition was not affected by treating intact cells or membranes with pertussis toxin and proteolytic enzymes, respectively, which have been shown in other cell types to prevent adenylate cyclase inhibition mediated by the guanine nucleotide-binding regulatory component, Ni. The forskolin inhibition of the stable GTP analog-activated adenylate cyclase was impaired by increasing the Mg2+ concentration and was reversed into a stimulation by Mn2+. Under optimal inhibitory conditions, forskolin even decreased basal adenylate cyclase activity. Finally, forskolin largely reduced the apparent affinity of the rat basophilic leukemia cell adenylate cyclase for its substrate, MgATP, which reduction resulted in an apparent inhibition at low MgATP concentrations and a loss of the inhibition at higher MgATP concentrations. The data indicate that forskolin can cause both stimulation and inhibition of adenylate cyclase and, furthermore, they suggest that the inhibition may not be mediated by the Ni protein, but may be caused by a direct action of forskolin at the adenylate cyclase catalytic moiety.     

Extraction of the adenylate cyclase-activating factor of bovine sperm and its identification as a trypsin-like protease

We previously reported the activation of adenylate cyclases from rat brain (Johnson, R. A., Awad, J. A., Jakobs, K. H., and Schultz, G., (1983) FEBS Lett. 152, 11-16) and from human platelets (Jakobs, K. H., Johnson, R. A., and Schultz, G. (1983) Biochim. Biophys. Acta 756, 369-375) by a factor derived from bovine sperm. In this report we describe the conditions for the extraction of the factor from bovine sperm and characteristics of its effects on adenylate cyclase which are consistent with its being a protease. The activating capacity of sperm particles was extracted from previously washed and frozen sperm into a 30,000 X g supernatant fraction by various salts, but not by the nonionic detergent Lubrol-PX. The amount of extracted factor: (a) was greatest with NH4HCO3 greater than NaCl greater than Na acetate; (b) was optimal with 0.5 M salt; (c) was not appreciably affected by the pH of the extraction buffer between pH 5.0 and 8.5; and (d) exhibited the greatest specific activity at the lower pH. The extracted sperm factor could be concentrated without loss by ultrafiltration on Amicon PM-10 membranes. The effect on adenylate cyclase of concentrated and desalted sperm extracted was inhibited 50% by various salts at 10 to 30 mM. The effects of the sperm factor to activate platelet adenylate cyclase, to block its inhibition via the alpha-adrenoceptor, and to block inhibition of stimulated forms of the enzyme by stable guanine nucleotides were prevented by protease inhibitors. A 50% reduction in the sperm factor's activation of platelet adenylate cyclase was caused by 30 nM soybean trypsin inhibitor, 30 nM alpha 2-macroglobulin, 300 nM leupeptin, 1 microM antipain, 15 microM aprotinin, and 100 microM benzamidine. Up to 3 mM phenylmethanesulfonyl fluoride was without effect on activation of the platelet cyclase by the sperm factor. The effects of the sperm factor persisted after its removal by the washing of pretreated platelet membranes and after its inactivation by the subsequent addition of leupeptin. The data strongly support the conclusion that the bovine sperm factor is a trypsin-like protease. alpha-Chymotrypsin, trypsin, and sperm acrosin were comparably effective in stimulating the platelet adenylate cyclase 5- to 8-fold, with concentrations eliciting maximal stimulation being: 200 ng trypsin/ml; 2 micrograms alpha-chymotrypsin/ml; and 2 micrograms acrosin/ml.(ABSTRACT TRUNCATED AT 400 WORDS)     

Determination of plasma bile acids by capillary gas-liquid chromatography-electron capture negative chemical ionization mass fragmentography

Combined capillary gas-liquid chromatography-electron capture negative chemical ionization mass spectrometry of pentafluorobenzyl ester-TMSi ether derivatives of bile acids and isotope dilution using deuterated internal standards are introduced as a sensitive and selective analysis technique for plasma bile acids. As a result of the high ionization efficiency of pentafluorobenzyl derivatives under electron capturing conditions and minimal fragmentation, the detection limit of this technique is low: 1 pg for each bile acid. The high sensitivity enabled the detection and quantitation of atypical bile acids in 200-microliters aliquots of plasma from fasting healthy adults as exemplified by trihydroxycoprostanic acid (0.002 +/- 0.001 mumol/l) and dihydroxycoprostanic acid (0.013 +/- 0.002 mumol/l).     

Hyperoxia-induced clonogenic killing of HeLa cells associated with respiratory failure and selective inactivation of Krebs cycle enzymes

Cellular intoxication by elevated concentrations of O2 may be considered as a model for accelerated cellular aging processes resulting from excessive free radical production by normal metabolic pathways. We describe here that exposure of HeLa cell cultures to 80% O2 for 2 days causes progressive growth inhibition and loss of reproductive capacity. This intoxication was correlated with inhibition of cellular O2 consumption and inactivation of 3 mitochondrial flavoproteins, i.e., partial inactivation of NADH and succinate dehydrogenases and total inactivation of alpha-ketoglutarate dehydrogenase. As alpha-ketoglutarate dehydrogenase controls the influx of glutamine/glutamate into the Krebs cycle, which is the major pathway for oxidative ATP generation in HeLa cells, the inactivation of alpha-ketoglutarate dehydrogenase was expectedly correlated with a net fall in glutamine/glutamate utilization. Furthermore, a simultaneous increase in glucose consumption and lactate production was observed, indicating that the cellular response to respiratory failure is to generate more ATP from glycolysis. In spite of this response, extensive depletion of ATP was observed. Thus, hyperoxia-induced growth inhibition and loss of clonogenicity seem to be due primarily to an impairment of mitochondrial energy metabolism resulting from inactivation of SH-group-containing flavoprotein enzymes localized at or near the inner mitochondrial membrane. These observations may be relevant for theories implicating loss of mitochondrial function as a prime factor in the aging process.