Detergent Action Research Articles

Markers of Myocardial Damage

We read with interest the editorial by Collinson on the use of cardiac troponins T and I (cTnT, cTnI) as specific markers of myocardial damage (1). We agree with Collinson when he suggests that there were some inconsistencies in the data presented by Bodor et al. (2). We, too, were concerned by the use of a system optimized for the measurement of serum troponin T to measure this analyte in extracts of muscle tissue, since there are several dilution and matrix problems to address in this setting (3). Bodor et al. have previously published data measuring cTnI, using the same extraction method (4). We would suggest that, if the methods were as described in both of these publications, they appear to be fundamentally flawed. The authors omitted a vital step in the purification process, which renders their data highly questionable. They prepared two separate fractions, one for soluble cytosolic proteins and one for insoluble myofibrillar proteins. The myofibrillar fraction was then solublized with 8 mol/L urea. The detergent action of urea denatures the proteins of the myofibrillar complex, causing the components of the complex to dissociate. It is well documented that even low concentrations of urea interfere with the Lowry protein assay (5)(6). The kit manufacturer, Sigma, does not recommend the use of this assay in the presence of urea. However, Bodor et al. used this assay to determine total protein concentration in the presence of 8 mol/L urea. The consequent problem is illustrated in Fig. 1⇓ . In addition, 8 mol/L urea could seriously interfere with antibody binding in the immunoassay for cTnT and contribute to a substantial matrix effect. The myofibrillar fraction should have been desalted before any analytical measurements for either total protein, cTnI, or cTnT were performed. Several studies have been published in … bAuthor for correspondence: e-mail GBodor{at}dhha.org

Surfactants and the skin

The skin surface is the interface between us, the organism, and the outside world. When we clean the skin we remove not only the bacteria, dirt and grease which have accumulated, but also part of our natural barrier - the stratum corneum. Corneocytes, both singly and in clumps, are released from the skin surface by the action of detergents and mechanical stimulation. So too are the lipids and proteins which make up the intercorneocyte region of the stratum corneum. The analysis of the types and amounts of materials released by a standard scrub procedure may prove useful in the selection of surfactants with particular properties. Changes in the physical properties of the skin occur after washing. For example, changes in skin surface pH and transepidermal water loss (a sensitive index of barrier function) are easily demonstrable. Excessive exposure to surfactants results in repeated damage to the stratum corneum which can in turn lead to an irritant dermatitis. Individual susceptibility to irritant dermatitis varies and this may be demonstrated using a simple patch test technique. This test is a way of potentially increasing the sensitivity of human based assays such as the soap chamber test by preselection of subjects. Alternatively it may be possible to use measurements of function such as transepidermal water loss or laser Doppler blood flow as an index of damage rather than conventional cutaneous irritancy. These approaches may help in the search for the ideal of a non-irritant cleanser.

Effect of tauroursodeoxycholic acid on bile-acid-induced apoptosis and cytolysis in rat hepatocytes

Background/Aims: In cholestatic liver disease, bile acids may initiate or aggravate hepatocellular damage. Cellular necrosis and cell death may be due to detergent effects of bile acids, but apoptosis may also play a role. In cholestasis, the conditions determining either apoptotic or cytolytic cell death are still unclear. Primary rat hepatocytes in culture represent a suitable model to study bile-acid-induced liver damage. Methods: Glycochenodeoxycholic acid, a hydrophobic bile acid, was used to induce cell damage. Tauroursodeoxycholic acid, a hydrophilic bile acid, served as substrate to study possible protective effects of such compounds. To study the time and concentration dependency of bile-acid-induced cytolysis and apoptosis, morphologic alterations, hepatocellular enzyme release and nucleosomal DNA fragmentation were evaluated. Results: Bile-acid-induced cytolysis, as indicated by hepatocellular enzyme release and by morphologic signs of membrane destruction, increased with concentration and time. Addition of tauroursodeoxycholic acid to the incubation medium reduced cytolysis significantly, indicating a direct hepatoprotective effect of this bile acid against the detergent action of hydrophobilic bile acids. In contrast to cytolysis, apoptosis with DNA fragmentation was induced by low concentrations of glycochenodeoxycholic acid a few hours after incubation. Coincubation with tauro-ursodeoxycholic acid in equimolar concentrations significantly reduced apoptosis, indicating another direct hepatoprotective effect of tauroursodeoxycholic acid. Conclusions: It seems likely that in severe cholestasis, bile-acid-induced injury of hepatocytes is due mainly to cytolysis, whereas in moderately severe cholestasis apoptosis represents the predominant mechanism of bile acid toxicity. Tauroursodeoxycholic acid may reduce both bile-acid-induced apoptosis and cytolysis.

Flow cytometric characteristics of subchloroplast particles prepared by the action of various detergents

Agranal thylakoid membranes from leaves of Phaseolus vulgaris L. were fragmented using seven distinct detergents: digitonin, Triton X-100, cetylpyridinium chloride, sodium dodecylsulfate, and Zwittergents 3-12, 3-14, and 3-16, differing in chemical composition and/or electric charges. Subchloroplast particles separated on a Percoll gradient were examined by flow cytometry to determine their size and shape. Vesicle size was also determined by a haematological analyzer, which produced comparable results. Individual green bands consisted of vesicles of fairly wide size distribution. Simple direct proportionality between the particle density and their size was not observed in any case, nevertheless, bigger particles were more abundant in fractions of higher density. Some vesicles had even a larger size than the original thylakoids. This might reflect a specific action of the detergents in low concentrations on agranal membranes, with incorporation of detergent molecules into vesicles. Inner structures of particles of the same size and density were not necessarily identical, but represented several populations, as was apparent from the side scatter analysis. Flow cytometric analysis can thus be used for the investigation of mechanisms of membrane fragmentation by detergents.

Potential involvement of type II phospholipase A2 in atherosclerosis

Potential involvement of type II phospholipase A2 in atherosclerosis

Hydrolysis of membrane-bound liver alkaline phosphatase by GPI-PLD requires bile salts.

Circulating liver plasma membrane fragments (LPMF) were purified from human serum by means of a monoclonal antileucine aminopeptidase antibody, AD-1. This was done by immunoaffinity chromatography or by incubating the sera with AD-1-coated nitrocellulose disks. Alkaline phosphatase (ALP, EC 3.1.3.1) is bound to these LPMF through a glycosylphosphatidylinositol (GPI) anchor and is referred to as membrane-bound liver ALP (Mem-LiALP). Low concentrations of Triton X-100 or high bile salt concentrations released GPI anchor-bearing LiALP (Anch-LiALP) from purified LPMF; once released, Anch-LiALP was slowly and progressively converted to hydrophilic dimeric LiALP [soluble LiALP (Sol-LiALP)], free from its GPI anchor. Low levels of GPI-specific phospholipase D (GPI-PLD) activity were measured in the pure LPMF. Apparently, this membrane-associated GPI-PLD was released by the action of detergents and contributed to the spontaneous conversion of Anch-LiALP to Sol-LiALP. In the absence of detergents, GPI-PLD had little effect on Mem-LiALP, both in purified form as well as in serum. In vitro, isolated Anch-LiALP was converted to Sol-LiALP by both GPI-specific phospholipase C and GPI-PLD. Sol-LiALP in serum, however, appeared to be the product of GPI-PLD activity only. Five- to tenfold higher concentrations of Triton X-100 were needed to release Anch-LiALP from LPMF in serum, compared with those required in a solution of purified LPMF. In serum, as well as in purified conditions, only a small range of detergent of bile salt concentrations permitted the conversion of Mem-LiALP to Sol-LiALP. A model is proposed for the release in the circulation of Mem-LiALP, Anch-LiALP, and Sol-LiALP, involving both LPMF-associated GPI-PLD and liver sinusoid bile salts.

Synergistic effect of ethyl-6-O-decanoyl glucoside and chlorhexidine in contact lens disinfection.

Contact lenses with adherent Serratia marcescens and Pseudomonas aeruginosa could not be sterilised either with polyaminopropyl biguanide or with chlorhexidine (CHX) commercial solutions meant for contact lens disinfection, even after treatment lasting 12 h. Ethyl-6-O-decanoyl glucoside (EDG) with chlorhexidine enhanced the killing significantly. The same was also found to be the case for dispersed cells of P. aeruginosa on cellulose acetate filters. EDG enhances the bactericidal effect of 0.00025 CHX by a mechanism not solely due to detergent action and is a potential adjunct to CHX in clinical applications.

307 Modulation by adenosine of acetylcholine release from electric organ synaptosomes of narke japonica

We investigated the relationship between tension development and the cytosolic free Ca2+ level ([Ca2+]i) on exposure of the endothelium-denuded isolated rat aorta to palmitoyl-l-α-lysophosphatidylcholine. Lysophosphatidylcholine concentration-dependently induced a gradual increase in [Ca2+]i. Application of 10−4 M lysophosphatidylcholine induced a large and sustained tonic increase in [Ca2+]i (the peak [Ca2+]i was 125.2±11.5% of the 80 mM K+-induced response) but only a small contraction (4.0±1.4% of the 80 mM K+ induced contraction). The sustained increase in [Ca2+]i was attenuated when extracellular Ca2+ was removed but it was unaffected by verapamil or 1-(5-isoquinolinesulphonyl)-2-methylpiperazine dihydrochloride (H-7). Digitonin also produced a gradual increase in [Ca2+]i but with a pronounced contraction. Triton X-100 (0.1%) produced a marked elevation in [Ca2+]i with no detectable contraction. Triton X-100, however, caused a rapid leakage of fura PE-3. Treatment with 10−4 M lysophosphatidylcholine for 1 or 2 h did not affect the contractile response induced by 80 mM K+ and this treatment did not release lactate dehydrogenase from the rat aorta. Treatment with lysophosphatidylcholine did not affect either the cyclic AMP level or the cyclic GMP level in endothelium-denuded aortic tissues. These results show that in the rat aorta lysophosphatidylcholine produces a large increase in [Ca2+]i (possibly in a non-contractile compartment) which does not induce contraction. Thus, the increase in [Ca2+]i induced by lysophosphatidylcholine (i) requires external Ca2+ but is not due to an increased Ca2+ influx through voltage-dependent L-type Ca2+ channels, (ii) is not primarily due to protein kinase C activation and (iii) is probably not due to a detergent action (like those of digitonin and triton X-100). The relative lack of a contractile response to lysophosphatidylcholine is not due to formation of cyclic AMP or cyclic GMP.

308 Effects of dopamine D 3 receptor agonist on neuronal activities in ventral tegmental area

We investigated the relationship between tension development and the cytosolic free Ca2+ level ([Ca2+]i) on exposure of the endothelium-denuded isolated rat aorta to palmitoyl-l-α-lysophosphatidylcholine. Lysophosphatidylcholine concentration-dependently induced a gradual increase in [Ca2+]i. Application of 10−4 M lysophosphatidylcholine induced a large and sustained tonic increase in [Ca2+]i (the peak [Ca2+]i was 125.2±11.5% of the 80 mM K+-induced response) but only a small contraction (4.0±1.4% of the 80 mM K+ induced contraction). The sustained increase in [Ca2+]i was attenuated when extracellular Ca2+ was removed but it was unaffected by verapamil or 1-(5-isoquinolinesulphonyl)-2-methylpiperazine dihydrochloride (H-7). Digitonin also produced a gradual increase in [Ca2+]i but with a pronounced contraction. Triton X-100 (0.1%) produced a marked elevation in [Ca2+]i with no detectable contraction. Triton X-100, however, caused a rapid leakage of fura PE-3. Treatment with 10−4 M lysophosphatidylcholine for 1 or 2 h did not affect the contractile response induced by 80 mM K+ and this treatment did not release lactate dehydrogenase from the rat aorta. Treatment with lysophosphatidylcholine did not affect either the cyclic AMP level or the cyclic GMP level in endothelium-denuded aortic tissues. These results show that in the rat aorta lysophosphatidylcholine produces a large increase in [Ca2+]i (possibly in a non-contractile compartment) which does not induce contraction. Thus, the increase in [Ca2+]i induced by lysophosphatidylcholine (i) requires external Ca2+ but is not due to an increased Ca2+ influx through voltage-dependent L-type Ca2+ channels, (ii) is not primarily due to protein kinase C activation and (iii) is probably not due to a detergent action (like those of digitonin and triton X-100). The relative lack of a contractile response to lysophosphatidylcholine is not due to formation of cyclic AMP or cyclic GMP.

Influence of bile salts on hepatic mdr2 P-glycoprotein expression

Mdr2 P-glycoprotein is expressed in the canalicular membrane of the mouse hepatocyte and is responsible for phospholipid secretion into bile. It is our hypothesis that it functions as a flippase in the translocation of phosphatidylcholine from the inner leaflet to the outer leaflet of the canalicular membrane. We have investigated the influence of different types of bile salts on the expression levels of mdr2 Pgp. Feeding mice a cholate-supplemented diet results in an increased mdr2 mRNA level, and this is accompanied by an increased biliary phospholipid secretion capacity. Cholate is a more hydrophobic bile salt than the main endogenous bile salt, muricholate. The induction of mdr2 gene expression and concomitant increase in phospholipid secretion are in line with the function of biliary phospholipids to inactivate the detergent action of hydrophobic bile salts.

Arachidonic acid and lysophosphatidylcholine modulate excitatory transmitter amino acid release from the rat cerebral cortex

The involvement of phospholipase A 2 (PLA 2) products in ischemia-evoked release of excitatory neurotransmitter amino acids (EAAs) from the cerebral cortex was studied in a four vessel occlusion rat model of cerebral ischemia/reperfusion. In comparison with untreated animals, arachidonic acid (AA; 5 × 10 −7 M to 5 × 10 −5 M) significantly reduced the ischemia-evoked efflux of glutamate and aspartate into cortical superfusates. Direct application of lysophosphatidylcholine (LysoPC; 55.4 μg/ml) to the cerebral cortex of non-ischemic animals resulted in a significant increase in glutamate levels. These results indicate that the immediate products of PLA 2 action on plasma membrane phospholipids can either enhance or inhibit excitotoxic amino acid release following cerebral ischemia. The effect of AA is likely to be a result of its ability to inhibit PLA 2; that of LysoPC, a consequence of its detergent action.

Conjugated bile salts regulate turnover of rat intestinal brush border membrane hydrolases

The mechanisms whereby the conjugated bile salts regulate the activities of the brush border membrane hydrolases and its physiological significance were investigated in rat small intestine, and comparisons were made with the action of pancreatic protease. Rat brush border membrane proteins were metabolically labeled with [35S]methionine, and isolated brush border membrane was incubated with taurocholate or pancreatic elastase. The activity of solubilized hydrolases was assayed and the molecular forms of the hydrolases were examined by SDS-PAGE. The activity and protein bands of alkaline phosphatase and sucrase-isomaltase were solubilized by taurocholate, while alkaline phosphatase was not solubilized by elastase. Solubilized sucrase-isomaltase molecules were proteolytically degraded by elastase, whereas the intact molecule of sucrase-isomaltase was solubilized by taurocholate. Next the physiological role of bile salts in brush border membrane hydrolase turnover were investigated using metabolic labeling of brush border membrane hydrolase and immunoprecipitation in biliary diversion rats. After three days of biliary diversion, a significant increase in alkaline phosphatase activity was observed. Although synthesis of alkaline phosphatase in biliary diversion rats was similar to that observed in control rats, biliary diversion rats showed 1.5-fold slower turnover of alkaline phosphatase when compared with control rats. These results suggest that conjugated bile salts in the intestinal lumen may cause a rapid turnover of brush border membrane hydrolases, which may be increased by the enhanced enzyme degradation. The mechanisms for the enhanced degradation appeared to be solubilization of hydrolases caused by the detergent activity of bile salts. Therefore, conjugated bile salts may play an important physiological role in the regulation of expression of the protease-resistant enzymes such as alkaline phosphatase.

Myometrial estrogen and progesterone receptor binding in pregnancy: Inhibition by the detergent action of phospholipids

We characterized the phospholipid inhibition of estradiol and progesterone binding to guinea-pig and human myometrial receptors. Of twelve compounds studied, phosphatidylinositol (PI), lysophosphatidic acid and lysophosphatidylcholine (lyso-PC) were the most active inhibitors (50% inhibition at 10(-5) M). Lyso-PC with fatty acid chain length C14:0 inhibited ligand binding both to estrogen receptor (ER) and progesterone receptor (PR), C16:0 only to PR and C18:0 neither to ER nor to PR. The lyso-derivates were more inhibitory than the parent compounds. The ionic detergent (sodium taurocholate) inhibited both ER and PR binding, but the non-ionic detergent (Triton X-100) only PR. Triton X-100 enhanced the PI-induced inhibition of ER binding by a factor of 10. PR was more sensitive to inhibition than ER in all cases. The type of inhibition was non-competitive. At term pregnancy, ligand binding to myometrial ER or PR was low or absent in humans, but moderate in the guinea-pig. Phospholipid extracts of human decidua and fetal membranes contained PI and phosphatidylserine rather than lyso-PC. The extract was a potent inhibitor of ligand binding to PR (50% inhibition at 10(-6) M phospholipid phosphorus), but not to ER. The physicochemical environment, modulated by phospholipids acting as detergents, may regulate sex steroid function also in vivo. This might have special significance for pregnancy maintenance.

Cell membrane-dependent chromatin condensation.

It is shown by means of flow cytometry that during several seconds after cell membrane damage by a non-ionic detergent in physiologically relevant buffer solution, the chromatin of mouse thymocyte nuclei undergoes a drastic decondensation, which is revealed by a sharp increase of binding of DNA-specific fluorochromes (olivomycin or propidium iodide) and of DNA accessibility to DNAse I digestion. A similar change is observed in dead cells. Roughly half of this decondensation can be prevented by lowering the pH of the outside medium to the level known to be inside the cells; the other half remains thus far unexplained (divalent cations and the difference between small anion species seem not to be involved). The approach is based on a novel observation that fixation by formaldehyde conserves chromatin structure before the action of detergent. Flow cytometric assay is proposed for monitoring these condensation/decondensation events in media of different composition. In addition, a new approach to viable/dead cell determination, which has the advantage of immediately fixing the cell state and preserving it for a reasonably long time, is proposed.

Lysophosphatidylcholine causes Ca 2+ influx, enhanced DNA synthesis and cytotoxicity in cultured vascular smooth muscle cells

The effects of lysophosphatidylcholine (LPC), a vasoactive phospholipid, on intracellular free calcium concentration ([Ca 2+]i), DNA synthesis and cytotoxicity of vascular smooth muscle cells (VSMC) were studied. LPC from 10 −7 to 10 −5 mol/l dose-dependently induced a sustained increase in [Ca 2+]i. In contrast to the response of [Ca 2+]i induced by angiotensin II, that induced by LPC was totally abolished when extracellular Ca 2+ was removed, was not affected by pretreatment of the cells with islet-activating protein, and was not desensitized by repeated addition. 8-( N,N-Diethylamino) octyl 3,4,5-trimethoxybenzoic acid (TMB-8), an inhibitor of Ca 2+ release from intracellular Ca 2+ stores, 1-(5-isoquinolinesulfonyl)-2-methylpiperadine dihydrochloride (H-7), an inhibitor of protein kinase C, KT5823, an inhibitor of protein kinase G, and Ca 2+ channel blockers failed to suppress the LPC-induced increase in [Ca 2+]i. LPC at 10 −5 mol/l caused significant stimulation of [ 3H]thymidine incorporation into VSMC, and at concentrations of 10 −5 mol/l and higher dose-dependently stimulated release of lactate dehydrogenase in cell culture supernatants. Moreover, digitonin mimicked the effects of LPC on [Ca 2+]i, and also caused similar effects to those of LPC on DNA synthesis and cytotoxicity in VSMC. These observations suggest that LPC causes both cell growth and cell injury of VSMC, at least partly, through its detergent action, causing membrane leakiness and resultant [Ca 2+]i overload in vitro, thus indicating the possible participation of LPC in atherosclerosis and/or injury of the vascular wall.

Introducing a new Element - Fluorine -Into the Liposomal Membrane

AbstractAmphiphiles with fluorinated hydrophobic tails constitute new, distinctively different components for membranes, liposomes, tubules and other self-aggregated supramolecular systems. Fluorinated liposomes (F-liposomes) can also be obtained from combinations of standard phospholipids with mixed fluorocarbon-hydrocarbon amphiphiles. The fluorinated moieties are considerably more hydrophobic than their hydrocarbon counterparts; they have also a larger cross section, are more rigid, and are lipophobic as well. As a result, fluorinated amphiphiles show enhanced propensity to self-assemble, lead to increased membrane ordering and stability, and their stacking creates a teflon-like repellent film within the liposomal membrane, which can significantly increase drug encapsulation stability. The fluorinated chains also impact on behavior in biological media and particle recognition, as exemplified by reduced hemolytic and detergent activity, prolonged intravascular persistence, or slower enzymatic hydrolysis...

Interaction of Helicobacter pylori and its fatty acids with parietal cells and gastric H+/K(+)-ATPase.

Helicobacter pylori and the fatty acids produced by this organism were compared for their acid inhibitory activity in isolated parietal cells and their interaction with gastric H+/K(+)-ATPase. H pylori (intact organisms, sonicates, methanolic extracts, and extracts from culture medium) and the fatty acids cis 9,10-methyleneoctadecanoic acid and tetradecanoic acid inhibited at fairly high concentrations histamine- and dibutyryl cyclic adenosine monophosphate stimulated acid production in isolated parietal cells, dissipated (with a slow onset) the H+/K(+)-ATPase created H+ gradient in gastric membrane vesicles, and inhibited H+/K(+)-ATPase activity in a concentration dependent manner. The inhibitory potency of H pylori and the fatty acids in relation to H+/K(+)-ATPase depended on the amount of membrane protein. Bovine serum albumin prevented enzyme inhibition and proton dissipation from gastric vesicles. The data indicate that H pylori establishes its antisecretory action in parietal cells by blocking H+/K(+)-ATPase activity and also by a detergent action at the apical parietal cell membrane. The fatty acids cis 9,10-methyleneoctadecanoic acid and tetradecanoic acid are probably the acid inhibitory factors secreted by H pylori.

Use of ionic detergents for enterovirus recovery from waste water

Virus recovery from poliovirus-seeded waste water was attempted after treatment of the samples with ionic detergents in the presence of acoustic energy. Viral titers strongly fluctuated depending upon concentration and chemical structure of the detergent as well as upon the dissolved organic content of the aqueous samples. Supplementation of the cellular monolayers with an additional amount of cell culture medium or with a nonionic detergent in a subcytotoxic concentration 48 h after inoculation partly induced cytopathic effects in silent dilution steps. Since, in the presence of the same detergent, viral recovery rates varied with the type of waste water, titer-conditioning activity of detergents was suggested to depend upon their effective critical micellization concentration. Especially for virus recovery from concentrated waste water, treatment with strongly disruptive detergents such as sodium dodecylsulfate revealed to be much more efficient than with the less hydrophilic sodium N-laurylsarcosine, sodium glycodeoxycholate or lauryldimethylamine-oxide, whereas the opposite seemed to be the case if virus recovery from filtrate samples were to be optimized. The mechanisms by which viral particles become infectious for the cell appear to be triggered by general principles of equilibrium thermodynamics, which means that interactions between viral surface proteins and cellular receptor molecules seem to reflect the tendency of these reagents to assume the energetically most favored orientation.

Identification of a 130-kilodalton human biliary concanavalin a binding protein as aminopeptidase N

Background/Aims: Human gallbladder bile contains a group of nonmucin glycoproteins that binds to the lectin concanavalin A (con A) and has been reported to promote cholesterol monohydrate crystal nucleation, an event preceding the formation of gallstones. Several of these proteins, including a 130-kilodalton protein, have been isolated and shown to promote nucleation in vitro. The aim of this study was to identify this and other major biliary con A binding glycoproteins. Methods: Gallbladder bile was chromatographed on con A agarose, and the eluted proteins were electrophoresed, blotted, and subjected to amino-terminal sequence analysis. Results: The major con A binding proteins were identified as aminopeptidase N (a 130-kilodalton protein), α2 macroglobulin, hemopexin, immunoglobulin heavy chains, and the β chain of haptoglobin. After further purification, aminopeptidase N was found to be enzymatically active and to promote cholesterol crystallization at its approximate physiological concentration in bile. Conclusions: It is likely that aminopeptidase N is the previously characterized 130-kilodalton biliary crystallization promoting protein. Aminopeptidase N is probably released from the biliary canalicular membrane by the detergent activity of bile salts and may be one factor that promotes cholesterol crystallization in the gallbladder.

The production of nitric oxide in endothelial cells by amphiphiles

Lysophosphatidylcholine, an endogenous detergent is an endothelium-dependent smooth muscle relaxant, which acts through the release of nitric oxide. It is known to activate a number of membrane-bound enzymes. Because of the relationship between detergent action, relaxation of endothelium-intact rabbit aortic strips and the release of nitric oxide, we considered the possibility that other amphiphiles also produce nitric oxide from endothelial cells. We therefore investigated the effect of digitonin on relaxation of precontracted rabbit aortic strips and the release of nitric oxide from freshly harvested bovine endothelial cells as determined by chemiluminescence. We found that both digitonin and LPC release nitric oxide and that this process is inhibited by the NO synthase inhibitor Nω-Nitro-L-Arginine Methyl Ester (L-NNAME).