The sensitive period for light and temperature regulation of sporangiophore development inPhycomyces

Light and temperature markedly influence sporangiophore development inPhycomyces blakesleeanus. Under normal conditions in the dark, low temperature drastically stimulates the production of dwarf sporangiophores (microphorogenesis) and inhibits that of giant sporangiophores (macrophorogenesis). These effects of low temperature could still be observed if applied only for a short period before sporangiophore initiation. Continuous white illumination strongly inhibits microphorogenesis and slightly stimulates macrophorogenesis. Short exposures to white light noticeably inhibit microphorogenesis and stimulate macrophorogenesis when given to mycelia grown for between 90 and 160 h at 14° C or 150 h or more at 10° C. These results indicate the existence in the mycelium of developmental stages for the regulation of sporangiophorogenesis by environmental signals.     

荔枝霜疫霉的研究

对华南的荔枝霜疫霉(Peronophythora litchii Chen ex Ko et al)的形态和营养特性进行了研究,并和新模式种进行了比较。发现此菌孢囊梗的生长是一种有限-无限生长类型,或称之为多级有限生长。即孢囊梗上的小分枝大多数是有限生长的,在其顶端同时形成孢子囊。但有时在同一孢囊梗上有的小分枝会继续生长,形成二级、三级甚至四级孢囊梗。在营养要求上与疫霉无大差别,能在天然和合成培养基上旺盛生长,需要硫胺素,ca~(++)和有机二元酸,能利用NH_4~+ 或No_3~-为其氮源,并能利用淀粉为其碳源,菌体匀浆中测出淀粉酶活性。根据孢囊梗的独特生长方式,我们认为完全有理由承认这菌是一个新属,并可成为新科,霜疫霉科。本文中已将霜疫霉科作了修改描述。孢囊梗已被修改为:孢囊梗多级有限生长。无疑这菌是腐霉科和霜霉科的中间类型。在营养类型与有性器官上和疫霉相近,而其孢子囊的形成和霜霉相似。但其孢囊梗的多级有限生长方式则和这两科都不相同。     

The Effects of Calcium and the Ionophore A23I87 on Modulation, Nitrogen Fixation and Growth of Soybeans

Tryptophan synthase in Phycomyces blakesleeanus. Part II: Activity of tryptophan synthase in Phycomyces blakesleeanus depending on the light and the content of zinc ions in the culture medium Five-day-old cultures of Phycomyces blakesleeanus show notice-able differences in the phenotype, depending on the culture conditions (permanent light, permanent dark, zinc deficiency, zinc sufficiency) and related to the distribution of tryptophan synthase activity between mycelium and sporangiophores. Permanent light and the presence of zinc ions in the medium during culturing have an antagonistic influence on the tryptophan synthase. The activity of the enzyme is being reduced in the sporangiophores and increased in the mycelium by the influence of light, while zinc ions in the culture medium increase the activity in the sporangiophores at simultaneous reduction in the mycelium. The importance of tryptophan synthase and tryptophan for the development of the fungus in relation to the metabolism of indole acetic acid is discussed.     

Glutamate Receptor Agonists Stimulate Nitric Oxide Synthase in Primary Cultures of Cerebellar Granule Cells

The glutamate receptor agonist N-methyl-D-aspartate (NMDA) stimulated a rapid, extracellular Ca(2+)-dependent conversion of [3H]arginine to [3H]citrulline in primary cultures of cerebellar granule cells, indicating receptor-mediated activation of nitric oxide (NO) synthase. The NMDA-induced formation of [3H]citrulline reached a plateau within 10 min. Subsequent addition of unlabeled L-arginine resulted in the disappearance of 3H from the citrulline pool, indicating a persistent activation of NO synthase after NMDA receptor stimulation. Glutamate, NMDA, and kainate, but not quisqualate, stimulated both the conversion of [3H]arginine to [3H]citrulline and cyclic GMP accumulation in a dose-dependent manner. Glutamate and NMDA showed similar potencies for the stimulation of [3H]citrulline formation and cyclic GMP synthesis, respectively, whereas kainate was more potent at inducing cyclic GMP accumulation than at stimulating [3H]citrulline formation. Both the [3H]arginine to [3H]citrulline conversion and cyclic GMP synthesis stimulated by NMDA were inhibited by the NMDA receptor antagonist MK-801 and by the inhibitors of NO synthase, NG-monomethyl-L-arginine (MeArg) and NG-nitro-L-arginine (NOArg). However, MeArg, in contrast to NOArg, also potently inhibited [3H]arginine uptake. Kainate (300 microM) stimulated 45Ca2+ influx to the same extent as 100 microM NMDA, but stimulated [3H]citrulline formation to a much lesser extent, which suggests that NO synthase is localized in subcellular compartments where the Ca2+ concentration is regulated mainly by the NMDA receptor.     

Almost all about citrulline in mammals

Summary. Citrulline (Cit, C₆H₁₃N₃O₃), which is a ubiquitous amino acid in mammals, is strongly related to arginine. Citrulline metabolism in mammals is divided into two fields: free citrulline and citrullinated proteins. Free citrulline metabolism involves three key enzymes: NO synthase (NOS) and ornithine carbamoyltransferase (OCT) which produce citrulline, and argininosuccinate synthetase (ASS) that converts it into argininosuccinate. The tissue distribution of these enzymes distinguishes three “orthogonal” metabolic pathways for citrulline. Firstly, in the liver, citrulline is locally synthesized by OCT and metabolized by ASS for urea production. Secondly, in most of the tissues producing NO, citrulline is recycled into arginine via ASS to increase arginine availability for NO production. Thirdly, citrulline is synthesized in the gut from glutamine (with OCT), released into the blood and converted back into arginine in the kidneys (by ASS); in this pathway, circulating citrulline is in fact a masked form of arginine to avoid liver captation. Each of these pathways has related pathologies and, even more interestingly, citrulline could potentially be used to monitor or treat some of these pathologies. Citrulline has long been administered in the treatment of inherited urea cycle disorders, and recent studies suggest that citrulline may be used to control the production of NO. Recently, citrulline was demonstrated as a potentially useful marker of short bowel function in a wide range of pathologies. One of the most promising research directions deals with the administration of citrulline as a more efficient alternative to arginine, especially against underlying splanchnic sequestration of amino acids. Protein citrullination results from post-translational modification of arginine; that occurs mainly in keratinization-related proteins and myelins, and insufficiencies in this citrullination occur in some auto-immune diseases such as rheumatoid arthritis, psoriasis or multiple sclerosis.     

Pressure probe study of the water relations of Phycomyces blakesleeanus sporangiophores

The physical characteristics which govern the water relations of the giant-celled sporangiophore of Phycomyces blakesleeanus were measured with the pressure probe technique and with nanoliter osmometry. These properties are important because they govern water uptake associated with cell growth and because they may influence expansion of the sporangiophore wall. Turgor pressure ranged from 1.1 to 6.6 bars (mean = 4.1 bars), and was the same for stage I and stage IV sporangiophores. Sporangiophore osmotic pressure averaged 11.5 bars. From the difference between cell osmotic pressure and turgor pressure, the average water potential of the sporangiophore was calculated to be about -7.4 bars. When sporangiophores were submerged under water, turgor remained nearly constant. We propose that the low cell turgor pressure is due to solutes in the cell wall solution, i.e., between the cuticle and the plasma membrane. Membrane hydraulic conductivity averaged 4.6 x 10(-6) cm s-1 bar-1, and was significantly greater in stage I sporangiophores than in stage IV sporangiophores. Contrary to previous reports, the sporangiophore is separated from the supporting mycelium by septa which prevent bulk volume flow between the two regions. The presence of a wall compartment between the cuticle and the plasma membrane results in anomalous osmosis during pressure clamp measurements. This behavior arises because of changes in solute concentration as water moves into or out of the wall compartment surrounding the sporangiophore. Theoretical analysis shows how the equations governing transient water flow are altered by the characteristics of the cell wall compartment.     

Interleukin 1beta increases arginine accumulation and activates the citrulline-NO cycle in rat pancreatic beta cells.

Nitric oxide (NO) may contribute to pancreatic beta cell damage during the development of type 1 diabetes. Its formation can be triggered by cytokines which induce the expression of the inducible form of nitric oxide synthase (iNOS) in pancreatic islets. In the iNOS-catalyzed reaction, arginine is converted into citrulline and NO. Cellular NO formation may be regulated by the availability of arginine. Arginine can be provided extracellularly, entering the cell mainly through the cationic amino acid transporter system y+CAT, and intracellularly, by protein degradation or synthesis from citrulline (the citrulline-NO cycle). This study demonstrates for the first time that the citrulline-NO cycle is induced in FACS-purified rat beta cells exposed to interleukin-1beta(IL-1beta) and that extracellular arginine or citrulline is required for NO production by beta cells. Moreover, the accumulation of arginine was higher in IL-1beta-treated beta cells than in control cells.beta cells expressed mRNAs for the two y+CAT transporters CAT-2A and CAT-2B with no change in transporter expression after exposure to IL-1beta. It is concluded that the activation of the citrulline-NO cycle and an increase in arginine accumulation may be adaptive responses in cytokine-exposed beta-cells to assure an adequate arginine supply for continuous NO production in the presence of low extracellular arginine levels which may prevail during insulitis.     

New photoresponses of phycomyces

The influence of light on asexual fruiting and mycelial growth of Phycomyces blakesleeanus Burgeff was studied by means of fruiting body counts and size measurements in cultures on solid media under varied incubation conditions. Five types of photoresponses were shown by ATCC Strain 8743a: (a) photoinduction of giant sporangiophores; (b) interference by light with an endogenous system that otherwise induces fruiting when the mycelium approaches the rim of the Petri dish; (c) inhibition of mycelial growth rates by light; (d) inhibition of dwarf sporangiophore induction by light; and (e) postponement by light of death in clones maintained by serial transfer at low temperature. A second strain, designated G5, showed responses comparable to (a), (b), and (d). The magnitudes of the responses were greatly affected by temperature of incubation and available nitrogen (asparagine) supply. The photoinduction of giant sporangiophores could be demonstrated with light of wavelengths between 380 and 480 nanometers but not with 520 nanometers or above. At 480 nanometers, light doses as small as 40 ergs per square centimeter were effective in inducing giant sporangiophores in strain 8743a.     

蓝光促进黑曲霉分生孢子发育和产糖化酶的研究

以黑暗为对照 ,研究了不同光质对黑曲霉产糖化酶及生长发育的影响。持续蓝光作用下 ,孢子萌发后菌丝较粗 ,菌丝细胞顶端膨大显著 ,菌丝细胞膜的通透性增加 ,残糖消耗快 ,孢子和孢子穗增大。在 3(4d时 ,蓝光下菌丝产糖化酶活力最高达 6 6 0 (30U mL ,比黑暗高出了 15. 4 % ,生物量增加了 4 9. 4 8% ,菌丝细胞可溶性蛋白含量提高了10 0. 5 6 % ,尤其是在开始产孢子的阶段 ,蓝光下黑曲霉产糖化酶活力、生物量有很大提高。研究表明 ,蓝光明显促进黑曲霉分生孢子发育和产孢阶段包括糖化酶在内的多种淀粉酶活力的迅速增加。     

Nitric oxide and L-arginine metabolism in a devascularized porcine model of acute liver failure

In acute liver failure (ALF), the hyperdynamic circulation is believed to be the result of overproduction of nitric oxide (NO) in the splanchnic circulation. However, it has been suggested that arginine concentrations (the substrate for NO) are believed to be decreased, limiting substrate availability for NO production. To characterize the metabolic fate of arginine in early-phase ALF, we systematically assessed its interorgan transport and metabolism and measured the endogenous NO synthase inhibitor asymmetric dimethylarginine (ADMA) in a porcine model of ALF. Female adult pigs (23-30 kg) were randomized to sham (N = 8) or hepatic devascularization ALF (N = 8) procedure for 6 h. We measured plasma arginine, citrulline, ornithine levels; arginase activity, NO, and ADMA. Whole body metabolic rates and interorgan flux measurements were calculated using stable isotope-labeled amino acids. Plasma arginine decreased >85% of the basal level at t = 6 h (P < 0.001), whereas citrulline and ornithine progressively increased in ALF (P < 0.001 and P < 0.001, vs. sham respectively). No difference was found between the groups in the whole body rate of appearance of arginine or NO. However, ALF showed a significant increase in de novo arginine synthesis (P < 0.05). Interorgan data showed citrulline net intestinal production and renal consumption that was related to net renal production of arginine and ornithine. Both plasma arginase activity and plasma ADMA levels significantly increased in ALF (P < 0.001). In this model of early-phase ALF, arginine deficiency or higher ADMA levels do not limit whole body NO production. Arginine deficiency is caused by arginase-related arginine clearance in which arginine production is stimulated de novo.     

Sequestration and metabolism of host cell arginine by the intraerythrocytic malaria parasite Plasmodium falciparum

Human erythrocytes have an active nitric oxide synthase, which converts arginine into citrulline and nitric oxide (NO). NO serves several important functions, including the maintenance of normal erythrocyte deformability, thereby ensuring efficient passage of the red blood cell through narrow microcapillaries. Here, we show that following invasion by the malaria parasite Plasmodium falciparum the arginine pool in the host erythrocyte compartment is sequestered and metabolized by the parasite. Arginine from the extracellular medium enters the infected cell via endogenous host cell transporters and is taken up by the intracellular parasite by a high‐affinity cationic amino acid transporter at the parasite surface. Within the parasite arginine is metabolized into citrulline and ornithine. The uptake and metabolism of arginine by the parasite deprive the erythrocyte of the substrate required for NO production and may contribute to the decreased deformability of infected erythrocytes.     

Sporulation of Plasmopara viticola: Differentiation and Light Regulation

Abstract: The development of grape downy mildew (Plasmopara viticola) was followed histologically during the entire latent period until the appearance of mature sporangia. Production of sporangiophores and sporangia was assessed using low-temperature scanning electron (LTSEM) and fluorescent light microscopy. Time-course studies using attached leaves of Vitis vinifera cv. Müller-Thurgau revealed that the production of sporangiophores and sporangia is a highly coordinated process and is completed within 7 h. As this differentiation is assumed to occur only in darkness, the influence of light was investigated. For this purpose, different light regimes were applied to infected leaf discs of V. vinifera cv. Müller-Thurgau. White light irradiation prevented formation of sporangia, although the growth of the mycelium was not affected. Many sporangiophores were observed that were abnormally shaped, i.e., short hyphae in clusters or thin, extremely elongated hyphae. For the formation of mature sporangia, a prolonged dark period was necessary. Light experiments suggest photosensitivity at the end of the latent period. A terminal white light irradiation caused an inhibitory effect, whereas a final phase of darkness promoted sporangium development. Different light qualities were tested, revealing an inhibition of sporangium development by blue light whereas neither red nor far-red light were effective.     

Acute renal response to LPS: impaired arginine production and inducible nitric oxide synthase activity

We have previously shown in rats that lipopolysaccharide (LPS) causes both decreased renal perfusion and kidney arginine production before nitric oxide (NO) synthesis, resulting in a >30% reduction in plasma arginine. To clarify the early phase effects of LPS, we asked the following two questions: 1) is the rapid change in renal arginine production after LPS simply the result of decreased substrate (i.e., citrulline) delivery to the kidney or due to impaired uptake and conversion and 2) is the systemic production of NO limited by plasma arginine availability after LPS? Arterial and renal vein plasma was sampled at 30-min intervals from anesthetized rats with or without citrulline or arginine (2 micromol.min(-1).kg(-1) iv) a dose with no effect on MAP, renal function, or NO production. Exogenous citrulline was quickly converted to arginine by the kidney, resulting in plasma levels similar to equimolar arginine infusion. Also, the increase in citrulline uptake resulted primarily from increased filtered load and reabsorption. In a separate series, citrulline was infused after LPS administration, verifying that citrulline uptake and conversion persists during impaired kidney function. Last, in rats given LPS, the elevation of plasma arginine had no discernable impact on mean arterial pressure, kidney function, or systemic NO production. This work demonstrates how arginine synthesis is normally "substrate limited" and explains how impaired kidney perfusion quickly results in decreased plasma arginine. However, contrary to in vitro studies, the significant reduction in extracellular arginine during the early phase response to LPS in vivo is not functionally rate limiting for NO production.     

Developmental stage-dependent response ofPilobolus crystallinus sporangiophores to gravitative and centrifugal stimulation

Gravitropic response of sporangiophores ofPilobolus crystallinus was studied by successive microscopic observation of the sporangiophores horizontally placed in the dark (red light) and by analysis of sporangiophore response to centrifugal stimulation. Negative tropism against the gravitative and also centrifugal stimulation was found only in mature sporangiophores after development of sporangium and after the resumption of elongation beneath the fully-developed subsporangial vescle, but there was no response in younger sporangiophores, implying that the gravitative perception system of the sporangiophores is dependent on their developmental stages.     

Enhanced utilization and altered metabolism of arginine in inflammatory macrophages caused by raised nitric oxide synthesis

Nitric oxide (NO) production was increased in macrophages during inflammation. Casein-elicitation of rodents causing a peritoneal inflammation offered a good model to study alterations in the metabolism of L-arginine, the precursor of NO synthesis. The utilization of L-arginine for NO production, arginase pathway and protein synthesis were studied by radioactive labeling and chromatographic separation. The expression of NO synthase and arginase was studied by Western blotting.Rat macrophages utilized more arginine than mouse macrophages (228+/-27 versus 71+/-12.8pmol per 10(6) macrophages). Arginine incorporation into proteins was low in both species (<15% of labeling). When NO synthesis was blocked, arginine was utilized at a lower general rate, but L-ornithine formation did not increase. The expression of enzymes utilizing arginine increased. NO production was raised mainly in rats (1162+/-84pmol citrulline per 10(6) cells) while in mice both arginase and NO synthase were active in elicited macrophages (677+/-85pmol ornithine and 456+/-48pmol citrulline per 10(6) cells).We concluded, that inflammation induced enhanced L-arginine utilization in rodent macrophages. The expressions and the activities of arginase and NO synthase as well as NO formation were increased in elicited macrophages. Specific blocking of NO synthesis did not result in the enhanced effectivity of the arginase pathway, rather was manifested in a general lower rate of arginine utilization. Different rodent species reacted differently to inflammation: in rats, high NO increase was found exclusively, while in mice the activation of the arginase pathway was also important.     

Reduced citrulline availability by OTC deficiency in mice is related to reduced nitric oxide production

The amino acid arginine is the sole precursor for nitric oxide (NO) synthesis. We recently demonstrated that an acute reduction of circulating arginine does not compromise basal or LPS-inducible NO production in mice. In the present study, we investigated the importance of citrulline availability in ornithine transcarbamoylase-deficient spf(ash) (OTCD) mice on NO production, using stable isotope techniques and C57BL6/J (wild-type) mice controls. Plasma amino acids and tracer-to-tracee ratios were measured by LC-MS. NO production was measured as the in vivo conversion of l-[guanidino-(15)N(2)]arginine to l-[guanidine-(15)N]citrulline; de novo arginine production was measured as conversion of l-[ureido-(13)C-5,5-(2)H(2)]citrulline to l-[guanidino-(13)C-5,5-(2)H(2)]arginine. Protein metabolism was measured using l-[ring-(2)H(5)]phenylalanine and l-[ring-(2)H(2)]tyrosine. OTC deficiency caused a reduction of systemic citrulline concentration and production to 30-50% (P < 0.001), reduced de novo arginine production (P < 0.05), reduced whole-body NO production to 50% (P < 0.005), and increased net protein breakdown by a factor of 2-4 (P < 0.001). NO production was twofold higher in female than in male OTCD mice in agreement with the X-linked location of the OTC gene. In response to LPS treatment (10 mg/kg ip), circulating arginine increased in all groups (P < 0.001), and NO production was no longer affected by the OTC deficiency due to increased net protein breakdown as a source for arginine. Our study shows that reduced citrulline availability is related to reduced basal NO production via reduced de novo arginine production. Under basal conditions this is probably cNOS-mediated NO production. When sufficient arginine is available after LPS stimulated net protein breakdown, NO production is unaffected by OTC deficiency.     

Mitochondria: Role of citrulline and arginine supplementation in MELAS syndrome

Mitochondria are found in all nucleated human cells and generate most of the cellular energy. Mitochondrial disorders result from dysfunctional mitochondria that are unable to generate sufficient ATP to meet the energy needs of various organs. Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome is a frequent maternally inherited mitochondrial disorder. There is growing evidence that nitric oxide (NO) deficiency occurs in MELAS syndrome and results in impaired blood perfusion that contributes significantly to several complications including stroke-like episodes, myopathy, and lactic acidosis. Both arginine and citrulline act as NO precursors and their administration results in increased NO production and hence can potentially have therapeutic utility in MELAS syndrome. Citrulline raises NO production to a greater extent than arginine, therefore, citrulline may have a better therapeutic effect. Controlled studies assessing the effects of arginine or citrulline supplementation on different clinical aspects of MELAS syndrome are needed.     

Radiochemical HPLC detection of arginine metabolism: measurement of nitric oxide synthesis and arginase activity in vascular tissue.

Nitric oxide (NO) plays a key role in vascular homeostasis. Accurate measurement of NO production by endothelial nitric oxide synthase (eNOS) is critical for the investigation of vascular disease mechanisms using genetically modified animal models. Previous assays of NO production measuring the conversion of arginine to citrulline have required homogenisation of tissue and reconstitution with cofactors including NADPH and tetrahydrobiopterin. However, the activity and regulation of NOS in vivo is critically dependant on tissue levels of these cofactors. Therefore, understanding eNOS regulation requires assays of NO production in intact vascular tissue that do not depend on the addition of exogenous cofactors and have sufficient sensitivity and specificity. We describe a novel technique, using radiochemical detection of arginine to citrulline conversion, to measure NO production within intact mouse aortas, without exogenous cofactors. We demonstrate the presence of arginase activity in mouse aortas which has the potential to confound this assay. Furthermore, we describe the use of N-hydroxy-nor-L-arginine (nor-NOHA) to inhibit arginase and permit specific detection of NO production in intact mouse tissue. Using this technique we demonstrate a 2.4-fold increase in NO production in aortas of transgenic mice overexpressing eNOS in the endothelium, and show that this technique has high specificity and high sensitivity for detection of in situ NO synthesis by eNOS in mouse vascular tissue. These results have important implications for the investigation of NOS regulation in cells and tissues.     

Caveolar localization of arginine regeneration enzymes, argininosuccinate synthase, and lyase, with endothelial nitric oxide synthase.

Although normal intracellular levels of arginine are well above the K(m), and should be sufficient to saturate nitric oxide synthase in vascular endothelial cells, nitric oxide production can, nonetheless, be stimulated by exogenous arginine. This phenomenon, termed the "arginine paradox," has suggested the existence of a separate pool of arginine directed to nitric oxide synthesis. In this study, we demonstrate that exogenous citrulline was as effective as exogenous arginine in stimulating nitric oxide production and that citrulline in the presence of excess intracellular and extracellular arginine further enhanced bradykinin stimulated endothelial nitric oxide production. The enhancement of nitric oxide production by exogenous citrulline could therefore be attributed to the capacity of vascular endothelial cells to efficiently regenerate arginine from citrulline. However, the regeneration of arginine did not affect the bulk intracellular arginine levels. This finding not only supports the proposal for a unique pool of arginine, but also suggested channeling of substrates that would require a functional association between nitric oxide production and arginine regeneration. To support this proposal, we showed that nitric oxide synthase, and the enzymes involved in arginine regeneration, argininosuccinate synthase and argininosuccinate lyase, cofractionated with plasmalemmal caveolae, a subcompartment of the plasma membrane. Overall, the results from this study strongly support the proposal for a separate pool of arginine for nitric oxide production that is defined by the cellular colocalization of enzymes involved in nitric oxide production and the regeneration of arginine.     

Argininosuccinate synthase expression is required to maintain nitric oxide production and cell viability in aortic endothelial cells

Although cellular levels of arginine greatly exceed the apparent K(m) for endothelial nitric-oxide synthase, current evidence suggests that the bulk of this arginine may not be available for nitric oxide (NO) production. We propose that arginine regeneration, that is the recycling of citrulline back to arginine, defines the essential source of arginine for NO production. To support this proposal, RNA interference analysis was used to selectively reduce the expression of argininosuccinate synthase (AS), because the only known metabolic role for AS in endothelial cells is in the regeneration of l-arginine from l-citrulline. Western blot analysis demonstrated a significant and dose-dependent reduction of AS protein as a result of AS small interfering RNA treatment with a corresponding diminished capacity to produce basal or stimulated levels of NO, despite saturating levels of arginine in the medium. Unanticipated, however, was the finding that the viability of AS small interfering RNA-treated endothelial cells was significantly decreased when compared with control cells. Trypan blue exclusion analysis suggested that the loss of viability was not because of necrosis. Two indicators, reduced expression of Bcl-2 and an increase in caspase activity, which correlated directly with reduced expression of AS, suggested that the loss of viability was because of apoptosis. The exposure of cells to an NO donor prevented apoptosis associated with reduced AS expression. Overall, these results demonstrate the essential role of AS for endothelial NO production and cell viability.