Membrane-active Substances Research Articles

The CssRS two-component system of Bacillus subtilis contributes to teicoplanin and polymyxin B response.

CssRS is a two-component system that plays a pivotal role in mediating the secretion stress response in Bacillus subtilis. This system upregulates the synthesis of membrane-bound HtrA family proteases that cope with misfolded proteins that accumulate within the cell envelope as a result of overexpression or heat shock. Recent studies have shown the induction of CssRS-regulated genes in response to cell envelope stress. We investigated the induction of the CssRS-regulated htrA promoter in the presence of different cell wall- and membrane-active substances and observed induction of the CssRS-controlled genes by glycopeptides (vancomycin and teicoplanin), polymyxins B and E, certain β-lactams, and detergents. Teicoplanin was shown to elicit remarkably stronger induction than vancomycin and polymyxin B. Teicoplanin and polymyxin B induced the spxO gene expression in a CssRS-dependent fashion, resulting in increased activity of Spx, a master regulator of disulfide stress in Bacillus subtilis. The CssRS signaling pathway and Spx activity were demonstrated to be involved in Bacillus subtilis resistance to teicoplanin and polymyxin B.

Isobavachalcone as an Active Membrane Perturbing Agent and Inhibitor of ABCB1 Multidrug Transporter.

Isobavachalcone (IBC) is an active substance from the medicinal plant Psoralea corylifolia. This prenylated chalcone was reported to possess antioxidative, anti-inflammatory, antibacterial, and anticancer activities. Multidrug resistance (MDR) associated with the over-expression of the transporters of vast substrate specificity such as ABCB1 (P-glycoprotein) belongs to the main causes of cancer chemotherapy failure. The cytotoxic, MDR reversing, and ABCB1-inhibiting potency of isobavachalcone was studied in two cellular models: human colorectal adenocarcinoma HT29 cell line and its resistant counterpart HT29/Dx in which doxorubicin resistance was induced by prolonged drug treatment, and the variant of MDCK cells transfected with the human gene encoding ABCB1. Because MDR modulators are frequently membrane-active substances, the interaction of isobavachalcone with model phosphatidylcholine bilayers was studied by means of differential scanning calorimetry. Molecular modeling was employed to characterize the process of membrane permeation by isobavachalcone. IBC interacted with ABCB1 transporter, being a substrate and/or competitive inhibitor of ABCB1. Moreover, IBC intercalated into model membranes, significantly affecting the parameters of their main phospholipid phase transition. It was concluded that isobavachalcone interfered both with the lipid phase of cellular membrane and with ABCB1 transporter, and for this reason, its activity in MDR cancer cells was presumptively beneficial.

Linear Peptides-A Combinatorial Innovation in the Venom of Some Modern Spiders.

In the venom of spiders, linear peptides (LPs), also called cytolytical or antimicrobial peptides, represent a largely neglected group of mostly membrane active substances that contribute in some spider species considerably to the killing power of spider venom. By next-generation sequencing venom gland transcriptome analysis, we investigated 48 spider species from 23 spider families and detected LPs in 20 species, belonging to five spider families (Ctenidae, Lycosidae, Oxyopidae, Pisauridae, and Zodariidae). The structural diversity is extraordinary high in some species: the lynx spider Oxyopes heterophthalmus contains 62 and the lycosid Pardosa palustris 60 different LPs. In total, we identified 524 linear peptide structures and some of them are in lycosids identical on amino acid level. LPs are mainly encoded in complex precursor structures in which, after the signal peptide and propeptide, 13 or more LPs (Hogna radiata) are connected by linkers. Besides Cupiennius species, also in Oxyopidae, posttranslational modifications of some precursor structures result in the formation of two-chain peptides. It is obvious that complex precursor structures represent a very suitable and fast method to produce a high number and a high diversity of bioactive LPs as economically as possible. At least in Lycosidae, Oxyopidae, and in the genus Cupiennius, LPs reach very high Transcripts Per Kilobase Million values, indicating functional importance within the envenomation process.

Lipid Scrambling Induced by Membrane-Active Substances

The functional roles of the lipid asymmetry of biomembranes are attracting increasing attention. This study characterizes the activity of surfactants to induce transmembrane flip-flop of lipids and thus “scramble” this asymmetry. Detergent-induced lipid scrambling of liposomes mimicking the charge asymmetry of bacterial membranes with 20 mol % of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol in the outer leaflet only was quantified by ζ-potential measurements for octaethylene glycol dodecyl ether (C12EO8), octyl glucoside (OG), and dodecyl maltoside. Membrane leakage was separately measured by the fluorescence lifetime-based calcein leakage assay and the onset of the membrane-to-micelle transition by isothermal titration calorimetry. Partition coefficients and partial molar areas were obtained as well. For the quickly membrane-permeant C12EO8 and OG, leakage proceeds at a rather sharp threshold content in the membrane, which is well below the onset of solubilization and little dependent on incubation time; it is accompanied by fast lipid scrambling. However, unlike leakage, flip-flop is a relaxation process that speeds up gradually from taking weeks in the detergent-free membrane to minutes or less in the leaking membrane. Hence, after 24 h of incubation, 10 mol % of C12EO8 or 50 mol % of OG in the membrane suffice for virtually complete lipid scrambling, whereas leakage remains below 10% for up to 14 mol % of C12EO8 and 88 mol % of OG. There is thus a concentration window in which lipid scrambling proceeds without leakage. This implies that lipid scrambling must be considered a possible mode of action of antimicrobial peptides and other membrane-active drugs or biomolecules. A related, detergent-based protocol for scrambling the lipid asymmetry of liposomes and maybe cells without compromising their overall integrity would be a very valuable tool to study functions of lipid asymmetry.

Lipid Scrambling of Asymmetric Liposomes Induced by Membrane Active Substances

Membrane leakage assays based on vesicles loaded with self quenching dyes have been widely used for quantifying the activity of membrane active substances that induce membrane pores or leaks. Time-resolved fluorescence decays of calcein-loaded liposomes allow a parallel quantification of the free and entrapped dye fractions and their effective local concentrations.1 Thus, different types and mechanisms of membrane leakage to aqueous solutes can be distinguished by this method. However, much less is known about another way of membrane permeabilization: the enhancement of lipid flip flop that causes a scrambling of the lipids between the leaflets. Since virtually all biological membranes show an asymmetric distribution of lipids between the two leaflets, such a scrambling should have massive impact on the cell even if it proceeds without leakage to aqueous solutes. In order to assess such activity, we utilized a recent protocol2 to produce asymmetric vesicles with anionic diacyl phosphatidyl glycerol (POPG) in the outer layer exclusively. Addition of lipid scrambling agents permits the POPG to flip to the inner leaflet, which can be monitored in terms of a less negative zeta potential of the liposomes. The study compares the activities of nonionic detergents including n-Dodecyl β-D-maltoside, n-Octylglucoside and C12EO8 to induce lipid scrambling and pore formation, respectively. References 1 H. Patel, C. Tscheka, H. Heerklotz; Soft Matter, 2009, 5, 2849-2851. 2 M. Markones, C. Drechsler, M. Kaiser, L. Kalie, H. Heerklotz, S. Fiedler; Langmuir. 2018, 34(5), 1999-2005.

Squalene lipotoxicity in a lipid droplet-less yeast mutant is linked to plasma membrane dysfunction.

Squalene is a naturally occurring triterpene with wide industrial applications. Due to limited natural resources, production of this valuable lipid in yeast is of high commercial relevance. Typically low levels of squalene in yeast can be significantly increased by specific cultivation conditions or genetic modifications. Under normal conditions, excess squalene is stored in lipid droplets (LD), while in a Saccharomyces cerevisiae mutant unable to form LD it is distributed to cellular membranes. We present here the evidence that squalene accumulation in this LD-less mutant treated with squalene monooxygenase inhibitor terbinafine induces growth defects and loss of viability. We show that plasma membrane malfunction is involved in squalene toxicity. We have found that subinhibitory concentrations of terbinafine increased the sensitivity of LD-less mutant to several membrane-active substances. Furthermore, squalene accumulation in terbinafine-treated LD-less cells disturbed the maintenance of membrane potential and increased plasma membrane permeability to rhodamine 6G. LD-less cells treated with terbinafine showed also high sensitivity to osmotic stress. To confirm the causal relationship between squalene accumulation, loss of viability and impaired plasma membrane functions we treated LD-less cells simultaneously with terbinafine and squalene synthase inhibitor zaragozic acid. Reduction of squalene levels by zaragozic acid improved cell growth and viability and decreased plasma membrane permeability to rhodamine 6G in terbinafine-treated LD-less cells. Our results support the hypothesis that plasma membrane malfunction is involved in the mechanisms of squalene lipotoxicity in yeast cells with defective lipid storage.

Antagonistic effects of α-tocopherol and ursolic acid on model bacterial membranes

α-tocopherol (Toc), the most active component of vitamin E can exert antagonistic effects disabling the therapy of cancers and bacterial infections. Such antagonisms were observed also between Toc and bioactive pentacyclic triterpenes (PT) exhibiting anticancer and antibacterial properties. Both Toc and PT are water-insoluble membrane active substances. Thus, our idea was to emulate their interactions with model Escherichia coli membranes. E. coli inner membranes were selected for the experiments because their lipid composition is quite simple and well characterized and the two main components are phosphatidylethanolamine and phosphatidylglycerol. As a model of E. coli membranes we applied Langmuir monolayers formed by the E. coli total extract of polar lipids (Etotal) as well as by the main lipid components: phosphatidylethanolamine (POPE) and phosphatidylglycerol (ECPG). The antagonistic effects of ursolic acid (Urs) and Toc were investigated with the application of ternary Langmuir monolayers formed by Urs, Toc and one of the phospholipids POPE or ECPG. Our studies indicated that the affinities of Urs and Toc towards the POPE molecule are comparable; whereas there are profound differences in the interactions of Urs and Toc with ECPG. Thus, the model experiments prove that in the case of E. coli membrane, the differences in the interactions between Urs and Toc with the anionic bacterial phosphatidylglycerol can be the key factor responsible for the antagonistic effects observed between PT and Toc in vivo.

The Bile Acid-Sensitive Ion Channel (BASIC) Is Activated by Alterations of Its Membrane Environment

The bile acid-sensitive ion channel (BASIC) is a member of the DEG/ENaC family of ion channels. Channels of this family are characterized by a common structure, their physiological functions and modes of activation, however, are diverse. Rat BASIC is expressed in brain, liver and intestinal tract and activated by bile acids. The physiological function of BASIC and its mechanism of bile acid activation remain a puzzle. Here we addressed the question whether amphiphilic bile acids activate BASIC by directly binding to the channel or indirectly by altering the properties of the surrounding membrane. We show that membrane-active substances other than bile acids also affect the activity of BASIC and that activation by bile acids and other membrane-active substances is non-additive, suggesting that BASIC is sensitive for changes in its membrane environment. Furthermore based on results from chimeras between BASIC and ASIC1a, we show that the extracellular and the transmembrane domains are important for membrane sensitivity.

Enhanced γ-CGTase Production by Bacillus Macorous with Membrane Active Substances

Some selected surfactants and alcohols were examined for their effects on γ-cyclodextrin glucanotransferase (γ-CGTase) production by Bacillus macorous WSH02-06. Both Tween 60 and n-Propyl alcohol had the ability to increase γ-CGTase yield about 1.7-fold. The investigation of fatty acid composition demonstrated that the addition of Tween 60 greatly increased the ratio of unsaturated to saturated fatty acids in cell membrane lipids, while n-propyl alcohol interfered with the proportion among unsaturated fatty acids. Study of the γ-CGTase fermentation in 5-L fermentor revealed that the addition of n-propyl alcohol improved the specific production rate and the yield of γ-CGTase.

Submicellar complexes may initiate the fungicidal effects of cationic amphiphilic compounds on Candida albicans.

The killing of Candida albicans by a series of amphiphilic quaternary ammonium compounds (QACs) with different hydrocarbon chain lengths was closely related to the binding of the compounds to the cells and damage of the cell membranes. The membrane damage was measured as the level of release of the UV-absorbing material into the medium in which the cells were suspended and as the level of uptake of propidium iodide in individual cells by flow cytometry. It was shown that of the compounds tested, hexadecyltrimethylammonium bromide (cetyltrimethylammonium bromide [CTAB]) bound most efficiently. Tetradecyl betainate chloride (B14), tetradecanoylcholine bromide (C14), tetradecyltrimethylammonium bromide (TTAB), and dodecyltrimethylammonium bromide (DTAB) followed and had declining degrees of binding efficiency. The proportion of CTAB bound was almost total at concentrations up to the critical micelle concentration (CMC) of the compound, whereas that of B14 was somewhat smaller. For the two remaining tetradecyl compounds (C14 and TTAB), still smaller proportions were bound at low concentrations, but the proportions rose disproportionally at increasing concentrations to a distinct maximum at concentrations of 0.2 to 0.5 times the CMC. We propose that interfacial micelle-like aggregates are formed at the cell surface as a step in the binding process. An analogous, but less conspicuous, maximum was seen for DTAB. Thus, great differences in the binding affinity of QACs with different hydrocarbon chains at different concentrations to C. albicans were observed. These differences were related to the CMC of the compound. In contrast, the binding of TTAB to Salmonella typhimurium 395 MS was almost total at low as well as high concentrations until saturation was attained, indicating fundamental differences between binding to the yeast and binding to gram-negative bacteria. The importance of lipid-type complexes or aggregates to the antifungal effect of membrane-active substances are discussed.

Spatial integration of local transmitter responses in motoneurones of the turtle spinal cord in vitro.

1. Integration of responses to local activation of transmitter receptors in the dendrites of motoneurones was investigated in a slice preparation of the turtle spinal cord. Membrane-active substances were applied from up to three independent iontophoresis electrodes during intracellular recording from the cell body. 2. Responses to glutamate could be evoked from dendrites closer than 20 microns from the tip of the glutamate electrode. The effects of other substances were more widespread. 3. In normal medium the configuration of a glutamate response was affected by time-dependent anomalous rectification. In the presence of muscarine the sum of glutamate responses from two different dendrites recruited a voltage-sensitive plateau potential. 4. The response to glutamate from one dendrite could be attenuated by local application of gamma-aminobutyric acid (GABA) without effects on soma conductance or glutamate responses from other dendrites. 5. The response to glutamate from one dendrite could be selectively enhanced by local application of tetraethylammonium (TEA) or N-methyl-D-aspartate (NMDA) without effects on soma conductance or glutamate responses from other dendrites. 6. NMDA could convert a tonic glutamate response from one dendrite into a phasic response without affecting the configuration of glutamate responses from other dendrites. 7. The effects of TEA and NMDA were facilitated by depolarization and reduced by hyperpolarization. 8. We conclude that the cable structure of motoneurones and the distribution of synapses and voltage-sensitive ion channels provide relative autonomy to non-linear synaptic processing and modulation in confined dendritic regions.

Anemonefish symbiosis: Vulnerability and resistance of fish to the toxin of the sea anemone

Protein toxins (20 kD molecular mass) causing lysis of human erythrocytes were isolated from sea anemones ( Heteractis magnifica, Madang, Papua New Guinea, and Entacmaea quadricolor, Red Sea), which host anemonefish ( Amphiprion sp.). These toxins are also ichthyotoxic. Freshwater and marine fish exposed to toxin concentrations of 0.5 μg/ml water were killed within 2 hr and exhibited extensive pathological alterations of the gill filaments. Amphiprion species, e.g. clarkii and percula, which live in the sea anemones Heteractis crispa and Stichodactyla mertensii, were highly vulnerable to the Heteractis magnifica toxin, whereas A. percula from the sea anemone H. magnifica proved to be toxin resistant. However, another species, A. perideraion also living in H. magnifica, was highly sensitive to the toxin. The two toxins exhibited cross-reactivity: Amphiprion, resistant to H. magnifica toxin, was also resistant to Entacmaea quadricolor toxin; but all fish were killed by other membrane-active substances such as gramicidin, saponin and latrunculin. The results of the study indicate that resistance to toxins secreted by the sea anemone has evolved in some Amphiprion species, but it is not an essential or a major factor in the anemonefish symbiosis. The skin's mucus layer seems to provide protection from nematocyst discharge.

Influence on antigen exposition of tumor cells by membrane active substances and viral infections.

HybridomaVol. 12, No. 5 Influence on Antigen Exposition of Tumor Cells by Membrane Active Substances and Viral InfectionsTHOMAS NESSELHUTTHOMAS NESSELHUTSearch for more papers by this authorPublished Online:24 Apr 2009https://doi.org/10.1089/hyb.1993.12.553AboutSectionsPDF/EPUB ToolsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail FiguresReferencesRelatedDetails Volume 12Issue 5Oct 1993 To cite this article:THOMAS NESSELHUT.Influence on Antigen Exposition of Tumor Cells by Membrane Active Substances and Viral Infections.Hybridoma.Oct 1993.553-557.http://doi.org/10.1089/hyb.1993.12.553Published in Volume: 12 Issue 5: April 24, 2009PDF download

Alteration of the L-type calcium current in guinea-pig single ventricular myocytes by heptaminol hydrochloride.

1. The effects of heptaminol on calcium current amplitude and characteristics were studied in single ventricular myocytes of guinea-pig by use of the whole cell configuration of the patch clamp technique. 2. A concentration-dependent decrease in ICa amplitude was observed. At heptaminol concentration as low as 10(-6) M, this effect was observed in only two cells (n = 6). At 10(-5) M the reduction of ICa was of 30 +/- 15% (n = 11). 3. The current recovery from inactivation at -40 mV holding potential (HP) seemed less sensitive to perfusion with heptaminol (greater than 10(-6) M). However, at -80 mV HP the overshoot of the recovery curve was decreased by heptaminol. 4. Both at -40 mV and -80 mV HP, heptaminol (10(-5) M) significantly increased the steady state inactivation of ICa. 5. As previously proposed by others to explain the effects of membrane active substances, the effects of heptaminol may result from alterations in cell membrane properties and possibly from an increase in intracellular free calcium ion concentration.

Transport and Excretion of L-Lysine in Corynebacterium glutamicum

Summary: L-Lysine transport in Corynebacterium glutamicum was investigated. The bacterium was shown to possess a highly specific, energy-dependent system of active lysine transport. The system transferred lysine into the cells and exchanged intra-and extracellular lysine. Mutations in the transport system did not lead to overproduction ofthe amino acid. Resting cells ofthe parent strain, or of its lysine-producer derivatives with a defective transport system, failed to excrete lysine into the medium. An efflux ofintracellular lysine could be induced by a hyperosmotic shock, and by different membrane-active substances. It has been suggested that C. glutamicum cells are equipped with channels (pores) for excreting lysine from the cytoplasm. These channels appeared to open in response to an increase in the intracellular lysine concentration. The channel permeability also depends on the membrane structure.

Search for new membrane-active substances: synthesis of tropan-3-ols with alkyl, alkenyl and alkenynyl groups at the bridgehead

The synthesis of tropan-3-ols with alkyl, alkenyl, and alkynyl groups at the bridgehead is described. The new compounds have been considered as new membrane-active substances that would serve as model compounds for histrionicotoxin (1). Application of the Noyori route to 2-substituted pyrroles was unsuccessful. In order to avoid difficulties in obtaining individual long-chain 4-keto aldehydes for the Robinson condensation of each new model compound a key tropane intermediate (8) was prepared instead. The first attempts to extend the chains of the mesyl ester of the diol (8b) and iodomethyltropanol (9b) by Grignard type coupling failed. Therefore we embarked on a Wittig reaction of the tropane-1-carbaldehyde (11) synthesized in high yield from the diol (8). Indeed, both pent-1-enyl- and pentyltropanols, (12) and (13) were obtained. Coupling with an acetylenic phosphorane led to the pent-1-enynyltropanol (14). A further new key compound, namely 3β-acetoxytropan-1-ylacetaldehyde (15) was also synthesized as a precursor to conjugated tropanyl enynes which would more closely resemble the natural product (1).

Bacterial chemotaxis: biochemistry of behavior in a single cell.

Bacterial chemotaxis is a primitive behavioral system that shows great promise for being amenable to a description of its molecular mechanism. In Gram-negatives like Escherichia coli, addition of amino acid attractant begins a series of events, starting with binding to certain intrinsic membrane proteins, the MCPs, and ending with a period of smooth swimming. Immediately, methyl-esterification of these MCPs begins and continues during this period. By contrast in the Gram-positive Bacillus subtilis, demethylation of MCPs occurs during the same period. At least two other mechanisms for mediating chemotaxis toward the attractants oxygen and phosphotransferase sugars exist in E. coli, and in these, changes in methylation of MCPs plays no role. Moreover, chemotaxis away from many repellents by B. subtilis appears to involve different mechanisms. Many of the repellents include drugs and toxicants, many of them man-made, so that chemoreceptors could not have specifically evolved; yet the bacteria are often exquisitely sensitive to them. Indeed, the B. subtilis membrane seems to act like a generalized antenna for noxious membrane-active substances.

Entrapping of the spin label tempocholine into human erythrocytes by resealing after hyposmolar stress. Comparison with haemolysis. The effects of some membrane-active substances

Human erythrocytes were subjected to a sudden hyposmolar stress by suspension in solutions of varying salt concentrations in the presence of the spin label tempocholine. The enlarged pores in the erythrocyte membranes produced by the influx of water, followed by stretching, allowed the passage of the spin label, so that a certain amount of tempocholine was entrapped when the erythrocytes spontaneously resealed with closing of the pores. The excess of spin label in the external solution was then reduced to a diamagnetic species by the addition of ascorbic acid. The positively charged tempocholine and the ascorbic add did not penetrate properly resealed erythrocytes, so that the electron spin resonance (ESR) signal from the entrapped spin label constituted a measure of the effective resealing of the pores and rifts in the membrane subsequent to hyposmolar stress. Some drug substances were found to influence the entrapping curves obtained when the amount of entrapped spin label was plotted against the osmolarity. Chlorpromazine, trifluoperazine, nicardipine, amperozide and haloperidol gave rise to a dose-dependent decrease of the entrapping of tempocholine, especially at low osmolarities. The exclusion of Ca 2+ and Mg 2+ ions from the solutions increased the action of chlorpromazine. The protective action against haemolysis brought about by a number of membrane-active substances at low concentrations [2] had its counterpart in the entrapping curve observed with chlorpromazine at 0.1 mM. It is suggested that the substances in this series exerted their action on the resealing process by interaction with the calmodulin system.

The efflux of spin label entrapped in human erythrocyte ghosts when suspended in hyposmolar solutions. The effect of chlorpromazine, trifluoperazine, nicardipine and some other membrane active substances

Human erythrocyte ghosts were loaded with the spin label tempocholine. Once entrapped in the ghosts, this spin label, carrying a positive charge, is not able to penetrate through intact ghost membranes. The ghosts were loaded with spin label to a concentration high enough to introduce exchange broadening of the electron spin resonance (ESR) signal with a relatively small signal amplitude. The efflux of the spin label brought about by hyposmolar stress was studied. The appearance of the label in the relatively large external volume gave rise to an increase of the ESR signal amplitude since the concentration of the spin label outside the ghosts was in the range in which exchange broadening can be excluded. The duration of the efflux following hyposmolar stress was less than half a minute. After this time, the ghosts resealed spontaneously and without restoration of the normal osmolarity. A number of membrane active substances were studied for possible influence on the efflux of spin label induced by hyposmolar stress. The drug sibstances chlorpromazine, trifluoperazine and nicardipine were found to increase the hyposmolar efflux of spin label. It was suggested that these substances, classified as calcium-antagonists and inhibitors of the calmodulin system, exert their action on the efflux of spin label by interaction with membrane proteins which maintain shape and tension of the erythrocytes.

Oxidative activation of guinea pig splenic cell guanylate cyclase activity by dehydroascorbate, ascorbate, fatty acid hydroperoxides and prostaglandin endoperoxides

The modulation of cyclic GMP metabolism by oxidative-reductive related processes was studied in intact guinea pig splenic cells and with respect to the activities of soluble and particulate guanylate cyclase. In intact cells the oxidant, dehydroascorbic acid (DHA), produced time and concentration dependent increases in cyclic GMP steady state levels. The thiol reductants, cysteine or DTT, decreased cellular cGMP levels 50 to 70%. Exposure of cells to sulfhydryl reactive reagents such as N-ethylmaleimide (NEM), p-hydroxymercuribenzoate, or 5,5′ dithiobis- [2-nitrobenzoic acid] or to the transition metal chelator, 4,7 -diphenyl-1,10 phenanthroline also caused striking lowering of cGMP levels. DHA-induced elevation of cGMP in intact cells persisted after removal of the oxidant and could be reversed upon reduction by DTT. Depression of cGMP steady state levels was induced by DTT which was also shown to persist after removal of the reductant and to be reversed by exposure of the cells to the oxidant, DHA. The effects produced by DHA, DTT, and NEM on soluble and particulate fractions of guanylate cyclase from these cells corresponded to the alterations each produced on cGMP steady state levels in the intact cells. DHA (0.1 to 10 m m) activated while DTT (0.1 to 5 m m) or NEM (0.5 to 5 m m) inhibited guanylate cyclase activity. DHA activation of the soluble enzyme was rapid and additive with the slower activation that occurred spontaneously in air; DTT could prevent or reverse DHA as well as spontaneous air activation. Activation or inhibition by DHA or DTT, respectively, of the particulate enzyme persisted after removal of the effectors but the stable effect produced by the oxidant could be reversed by the reductant and vice versa. Low concentrations of NEM (0.05–0.1 m m) which inhibited basal guanylate cyclase activity minimally (20–25%) completely abolished sensitivity to DHA-activation. Cells exposed to DHA, resuspended in DHA-free media and lysed, exhibited 3 to 4 fold enhanced activity of guanylate cyclase which was restored to the lower basal level of activity upon addition of DTT during the course of the cyclase reaction. Ascorbic acid has similar effects on cGMP metabolism in intact cells including the effect to produce a stable enhancement of guanylate cyclase activity which persists after cell disruption and is suppressed by DTT. However, unlike DHA, ascorbate has no effect on guanylate cyclase activity after cell disruption. Fatty acid hydroperoxides 12-OOH-20:4 (HPETE), 15-OOH-20:4 13-OOH-18:2, and PGE 2-OOH and the prostaglandin endoperoxides PGG 2 and PGH 2 were also shown to be effective activators of soluble (but not particulate) splenic cell guanylate cyclase activity. The hydroxy analogues of these fatty acids and the prostaglandins PGE 2 and PGF 2 α as well as arachidonic, linoleic, oleic and stearic acids had little or no effect. The stimulatory effects of the lipid hydroperoxides and endoperoxides were demonstrable in air or an argon atmosphere and were prevented and reversed by thiol reductants. Stimulation by both DHA and PGG 2 exhibited additivity with respect to promoting the rate at which activation proceeded to achieve a state of maximal catalysis. These results indicate that the soluble guanylate cyclase from splenic cells exhibits regulatory sites for hydrophobic and hydrophilic oxidants which can activate the enzyme by promoting a sulfhydryl-disulfide interconversion at these sites. It is suggested that the action of certain hormones and other membrane active substances may be linked to guanylate cyclase through specific redox systems. The components comprising the link, which remain to be identified, may differ depending upon the cell-type or specific effector at the cell plasma membrane.