Specificity of Calreticulin Transacetylase to acetoxy derivatives of benzofurans: Effect on the activation of platelet Nitric Oxide Synthase

Calreticulin Transacetylase (CRTAase) catalyzes the transfer of acetyl group(s) from polyphenolic acetates (PAs) to functional proteins, such as Glutathione S-transferase (GST), NADPH Cytochrome c reductase and Nitric Oxide Synthase (NOS) resulting in the modulation of biological activities. A comparison of the specificities of the acetoxy derivatives of coumarins, biscoumarins, chromones, flavones, isoflavones and xanthones has been carried out earlier by us with an aim to study the effect of nature and position of the acetoxy groups on the benzenoid ring and the position of the carbonyl group with respect to oxygen/nitrogen heteroatom for the catalytic activity of CRTAase. In this communication for the first time, we have studied the influence of differently substituted benzofurans on the CRTAase activity to study the effect of the replacement of pyran ring of coumarin with furan ring, presence of carbonyl at C-3, substitution of C-3 carbonyl group with acetoxy group and presence of various substituents (OAc/OH/Cl) on the benzenoid ring. It was observed that acetoxy derivatives of benzofurans lead to inhibition of ADP induced platelet aggregation by the activation of platelet Nitric Oxide Synthase catalyzed by CRTAase. Accordingly, the formation of NO in platelets by 3-oxo-2,3-dihydrobenzofuran-6,7-diyl diacetate (3a) was found to be comparable with that of model polyphenolic acetate (PA), 7,8-diacetoxy-4-methylcoumarin (DAMC).     

Specificities of Calreticulin Transacetylase to acetoxy derivatives of 3-alkyl-4-methylcoumarins: Effect on the activation of nitric oxide synthase

Calreticulin Transacetylase (CRTAase) catalyzes the transfer of acetyl groups from polyphenolic acetates (PAs) to the receptor proteins and modulates their biological activities. CRTAase was conveniently assayed by the irreversible inhibition of cytosolic glutathione S-transferase (GST) by the model acetoxycoumarin, 7,8-diacetoxy-4-methylcoumarin (DAMC). We have studied earlier, the influence of acetoxy groups on the benzenoid ring, the effect of reduction of double bond at C-3 and C-4 position, the effect of methyl/phenyl group at C-4, and the influence of position of carbonyl group with respect to oxygen heteroatom in the benzopyran nucleus, for the catalytic activity of CRTAase. In this communication, we have extended our previous work; wherein we studied the influence of an alkyl group (ethyl, hexyl and decyl) at the C-3 position of the acetoxy coumarins on the CRTAase activity. The substitution at C-3 position of coumarin nucleus resulted in the reduction of CRTAase activity and related effects. Accordingly the formation of NO in platelets by C-3 alkyl substituted acetoxy coumarins was found to be much less compared to the unsubstituted analogs. In addition the alkyl substitution at C-3 position exhibited the tendency to form radicals other than NO.     

Substrate specificity of acetoxy derivatives of coumarins and quinolones towards Calreticulin mediated transacetylation: investigations on antiplatelet function

Calreticulin transacetylase (CRTAase) is known to catalyze the transfer of acetyl group from polyphenolic acetates (PA) to certain receptor proteins (RP), thus modulating their activity. Herein, we studied for the first time the substrate specificity of CRTAase towards N-acetylamino derivatives of coumarins and quinolones. This study is endowed with antiplatelet action by virtue of causing CRTAase catalyzed activation of platelet Nitric Oxide Synthase (NOS) by way of acetylation leading to the inhibition of ADP/Arachidonic acid (AA)-dependent platelet aggregation. Among all the N-acetylamino/acetoxy coumarins and quinolones screened, 7-N-acetylamino-4-methylcoumarin (7-AAMC, 17) was found to be the superior substrate to platelet CRTAase and emerged as the most promising antiplatelet agent both in vitro and in vivo. Further it caused the inhibition of cyclooxygenase-1 (Cox-1) resulting in the down regulation of thromboxane A2 (TxA2), modulation of tissue factor and the inhibition of platelet aggregation. It was also found effective in the inhibition of LPS induced pro-thrombotic conditions.     

Calreticulin Transacetylase mediated activation of human platelet nitric oxide synthase by acetyl group donor compounds

Polyphenols have attracted immense interest because of their diverse biological and pharmacological activities. Surprisingly, not much is documented about the biological activities of acetoxy derivatives of polyphenol called polyphenolic acetates (PA). In our previous reports, we have conclusively established the Calreticulin Transacetylase (CRTAase) catalyzed activation of neuronal nitric oxide synthase (nNOS) and tumor necrosis factor-α (TNF-α) induced nitric oxide synthase (iNOS) by PA. In the present work, specificity of CRTAase to various classes of PA was characterized in human platelet. The effect of PA, on platelet NOS and intracellular cyclic guanosine monophosphate (cGMP), and adenosine diphosphate (ADP)-induced platelet aggregation were studied in an elaborated manner. Platelet CRTAase exhibited differential specificities to polyphenolic acetates upon incubation with l-arginine leading to activation of NOS. The intraplatelet generation of NO was studied by flowcytometry using DCFH-DA. The differential specificities of CRTAase to PA were found to positively correlate with increased production of NO upon incubation of PRP with PA and l-arginine. Further, the inhibitory effect of l-NAME on PA induced NO formation in platelets substantiated the CRTAase catalyzed activation of NOS. The real-time RT-PCR profile of NOS isoforms confirmed the preponderance of eNOS over iNOS in human platelets on treatment with PA. Western blot analysis also reiterated the differential pattern of acetylation of eNOS by PA. PA were also found effective in increasing the intraplatelet cGMP levels and inhibiting ADP-induced platelet aggregation. It is worth mentioning that the effects of PA were found to be in tune with the specificities of platelet CRTAase to PA as the substrates.     

Calreticulin transacetylase mediated upregulation of thioredoxin by 7,8-diacetoxy-4-methylcoumarin enhances the antioxidant potential and the expression of vascular endothelial growth factor in peripheral blood mononuclear cells

Extensive research carried out in our group on polyphenolic acetates (PAs) substantiated the potential role of PAs in causing diverse biological and pharmacological actions. Our earlier investigations firmly established the calreticulin transacetylase (CRTAase) catalyzed activation of nitric oxide synthase (NOS) by PAs. In this report, we have studied the effect of 7,8-diacetoxy-4-methylcoumarin (DAMC, a model PA) and other acetoxy coumarins on the thioredoxin and VEGF expression in human peripheral blood mononuclear cells (PBMCs), with a view to substantiate our earlier observation that DAMC was a superb inducer of angiogenesis. Real time RT-PCR analysis revealed the enhanced expression of thioredoxin reductase (TRXR) and diminished expression of thioredoxin interacting protein (TRXIP) leading to the increased expression and activity of thioredoxin (TRX) in PBMCs due to the the action of DAMC. The fact that TRX activity of PBMCs was enhanced by various acetoxy coumarins in tune with their affinity to CRTAase as substrate, suggested the possible activation of TRX due to acetylation. The overexpression of thioredoxin was found to correlate with that of VEGF as proved by real time RT-PCR and VEGF -ELISA results, apart from the DAMC-caused enhanced production of NO acting as an inducer of VEGF. Moreover, the intracellular ROS levels were also found to be reduced drastically, by DAMC thus reducing the oxidative stress in cells. These observations strongly evidenced the crucial role of TRX in DAMC-induced tissue angiogenesis with the involvement of VEGF.     

Characterization of 4-methyl-2-oxo-1,2-dihydroquinolin-6-yl acetate as an effective antiplatelet agent

We have studied earlier a membrane bound novel enzyme Acetoxy Drug: protein transacetylase identified as Calreticulin Transacetylase (CRTAase) that catalyzes the transfer of acetyl groups from polyphenolic acetates (PAs) to the receptor proteins and thus modulating their biological activities. In this communication, we have reported for the first time that acetoxy quinolones are endowed with antiplatelet action by virtue of causing CRTAase catalyzed activation of platelet Nitric Oxide Synthase (NOS) by way of acetylation leading to the inhibition of ADP/Arachidonic acid (AA)-dependent platelet aggregation. The correlation of specificity of platelet CRTAase to various analogues of acetoxy quinolones with intracellular NO and consequent effect on inhibition of platelet aggregation was considered crucial. Among acetoxy quinolones screened, 6-AQ (4-methyl-2-oxo-1,2-dihydroquinolin-6-yl acetate/6-acetoxyquinolin-2-one, 22) was found to be the superior substrate to platelet CRTAase and emerged as the most active entity to produce antiplatelet action both in vitro and in vivo. 6-AQ caused the inhibition of cyclooxygenase-1 (Cox-1) resulting in the down regulation of thromboxane A2 (TxA2) and the inhibition of platelet aggregation. Structural modification of acetoxy quinolones positively correlated with enhancement of intracellular NO and antiplatelet action.     

Calreticulin transacylase: Genesis,mechanism of action and biological applications

Our earlier investigations have identified a unique enzyme in the endoplasmic reticulum (ER) termed Acetoxy Drug: Protein Transacetylase (TAase) catalyzing the transfer of acetyl group from polyphenolic acetates (PA) to certain receptor proteins (RP). An elegant assay procedure for TAase was developed based on the inhibition of glutathione S-transferase (GST) due to acetylation by a model acetoxycoumarin, 7, 8-Diacetoxy-4-methylcoumarin (DAMC). TAase purified from various mammalian tissue microsomes to homogeneity exhibited a molecular weight (M.wt) of 55 kDa. Further, by N-terminal sequencing TAase was identified as Calreticulin (CR), a multifunctional Ca²⁺-binding protein in ER lumen. The identity of TAase with CR was evidenced by proteomics studies such as immunoreactivity with anti-CR antibody and mass spectrometry. This function of CR was termed Calreticulin transacetylase (CRTAase). CRTAase was also found to mediate the transfer of acetyl group from DAMC to RP such as NADPH Cytochrome c Reductase (CYPR) and Nitric Oxide Synthase (NOS). The autoacetylation of purified human placental CRTAase concomitant with the acetylation of RP by DAMC was observed. CRTAase activity was found to be inhibited by Ca²⁺. Our investigations on the individual domains (N, P and C) of CR from a nematode Haemonchus contortus revealed that the P-domain alone was found to possess CRTAase activity. Based on the observation that the autoacetylated CR was a stable intermediate in the CRTAase catalyzed protein acetylation by PA, a putative mechanism was proposed. Further, CRTAase was also found capable of transferring propionyl group from a propoxy derivative of polyphenol, 7,8-Dipropoxy-4-methylcoumarin (DPMC) to RP and concomitant autopropionylation of CR was encountered. Hence, CRTAase was assigned the general term Calreticulin Transacylase. Also, CRTAase was found to act upon the biological acyl group donors, acetyl CoA and propionyl CoA. CRTAase mediated modulation of specific functional proteins by way of acylation was exploited to elicit the biological applications of PA.     

Increased nitric oxide production in platelets from severe chronic renal failure patients

Nitric oxide (NO) production occurs through oxidation of the amino acid L-arginine by NO synthase (NOS). NO inhibits platelet activation by increasing the levels of cyclic guanosine monophosphate (cGMP), thus maintaining vascular homeostasis. Our group previously demonstrated (da Silva et al. 2005) an enhancement of the L-arginine-NO-cGMP pathway in platelets taken from chronic renal failure (CRF) patients on haemodialysis associated with reduced platelet aggregation. We investigate the platelet L-arginine-NO-cGMP pathway, platelet function, and inflammation from patients in CRF on conservative treatment. A total of 42 CRF patients and 42 controls (creatinine clearance = 27 ± 3 vs. 93 ± 1 mL per min per 1.73 m2, respectively) participated in this study. NOS activity and expression and cGMP concentration were measured in platelets. Platelet aggregation induced by collagen or ADP was evaluated and plasma levels of fibrinogen were determined by the Clauss method. A marked increase in basal NOS activity was seen in undialysed CRF patients compared with controls, accompanied by an elevation of fibrinogen plasma levels. There were no differences in expression of NOS and in cGMP levels. In this context, platelet aggregation was not affected. We provide the first evidence of increased intraplatelet NO biosynthesis in undialysed CRF patients, which can be an early marker of future haemostatic abnormalities during dialysis treatment.     

Stimulatory roles of nitric-oxide synthase 3 and guanylyl cyclase in platelet activation

Nitric oxide (NO) stimulates soluble guanylyl cyclase and, thus, enhances cyclic guanosine monophosphate (cGMP) levels. It is a currently prevailing concept that NO inhibits platelet activation. This concept, however, does not fully explain why platelet agonists stimulate NO production. Here we show that a major platelet NO synthase (NOS) isoform, NOS3, plays a stimulatory role in platelet secretion and aggregation induced by low doses of platelet agonists. Furthermore, we show that NOS3 promotes thrombosis in vivo. The stimulatory role of NOS is mediated by soluble guanylyl cyclase and results from a cGMP-dependent stimulation of platelet granule secretion. These findings delineate a novel signaling pathway in which agonists sequentially activate NOS3, elevate cGMP, and induce platelet secretion and aggregation. Our data also suggest that NO plays a biphasic role in platelet activation, a stimulatory role at low NO concentrations and an inhibitory role at high NO concentrations.     

Ovariectomy upregulates expression of estrogen receptors,NOS, and HSPs in porcine platelets

Platelets participate in normal and pathological thrombotic processes. Hormone replacement in postmenopausal women is associated with increase risk for thrombosis. However, little is known regarding how platelets are affected by hormonal status. Nitric oxide (NO) modulates platelet functions and is modulated by hormones. Therefore, the present study was designed to determine how loss of ovarian hormones changes expression of estrogen receptors and regulatory proteins for NO synthase (NOS) in platelets. Estrogen receptors (ER alpha and ER beta), NOS, heat shock proteins 70 and 90 (HSP70 and HSP90), caveolin-1, -2, and -3, calmodulin, NOS activity, and cGMP were analyzed in a lysate of platelets from gonadally intact and ovariectomized female pigs. Expression of ER beta and ER alpha receptors, endothelial NOS (eNOS), HSP70, and HSP90 increased with ovariectomy. NOS activity and cGMP also increased; calmodulin was unchanged. Caveolins were not detected. These results suggest that ovarian hormones influence expression of estrogen receptors and eNOS in platelets. Changes in estrogen receptors and NOS could affect platelet aggregation in response to hormone replacement.     

Two enzymatic sources of nitric oxide in different organs of apple plant

Nitric oxide production, nitric oxide synthase (NOS) and mitochondrial nitrite-reducing activities in roots, leaves and stems of different developmental stages were investigated, using potted 3-year-old apple (Malus domestica Borkh.) trees. The arginine-dependent NOS activity is sensitive to NOS inhibitor L-NAME and aminoguanidine (AG), with L-NAME being more effective than AG. Endogenous NO production, NOS and mitochondrial nitrite-reducing activities are predominately presented in young leaves and especially in young white roots and young stems. Root and stem mitochondria can reduce nitrite to nitric oxide at the expense of NADH, however, this mitochondrial nitrite-reducing activity is absent in leaves.     

Mitochondrial nitric oxide synthase is constitutively active and is functionally upregulated in hypoxia

Nitric oxide is a potent modulator of mitochondrial respiration, ATP synthesis, and K_ATP channel activity. Recent studies show the presence of a potentionally new isoform of the nitric oxide synthase (NOS) enzyme in mitochondria, although doubts have emerged regarding the physiological relevance of mitochondrial NOS (mtNOS). The aim of the present study were to: (i) examine the existence and distribution of mtNOS in mouse tissues using three independent methods, (ii) characterize the cross-reaction of mtNOS with antibodies against the known isoforms of NOS, and (iii) investigate the effect of hypoxia on mtNOS activity. Nitric oxide synthase activity was measured in isolated brain and liver mitochondria using the arginine to citrulline conversion assay. Mitochondrial NOS activity in the brain was significantly higher than in the liver. The calmodulin inhibitor calmidazolium completely inhibited mtNOS activity. In animals previously subjected to hypoxia, mtNOS activity was significantly higher than in the normoxic controls. Antibodies against the endothelial (eNOS), but not the neuronal or inducible isoform of NOS, showed positive immunoblotting. Immunogold labeling of eNOS located the enzyme in the matrix and the inner membrane using electron microscopy. We conclude that mtNOS is a constitutively active eNOS-like isoform and is involved in altered mitochondrial regulation during hypoxia.     

一氧化氮合酶的遗传、生理研究进展

一氧化氮具有广泛的生理功能,哺乳动物体内的NO是由NO合酶(NOS)氧化L-精氨酸而合成的,合成后的NO迅速跨膜扩散释放,NO合成失调能介导多种疾病。催化NO生物合成的NOS有三种亚型:神经元型NOS(nNOS)、内皮型NOS(eNOS)和诱导型NOS(iNOS),目前,人的三型NOS已纯化并且已分子克隆成功,对一氧化氮合酶的遗传研究确认了NOS家族的基因结构和染色体定位。     

Mechanism of biochemical action of substituted benzopyran-2-ones. Part 8: Acetoxycoumarin: protein transacetylase specificity for aromatic nuclear acetoxy groups in proximity to the oxygen heteroatom

Our earlier work established a convenient assay procedure for acetoxycoumarin (AC): protein transacetylase (TA) by indirectly quantifying the activity of glutathione (GSH)-S-transferase (GST), the extent of inhibition of GST under the conditions of the assay represented TA activity. In this communication, we have probed the specificity for TA with respect to the number and position of acetoxy groups on the benzenoid as well as the pyranone rings of the coumarin system governing the efficient transfer of acetyl groups to the protein(s). For this purpose, coumarins bearing one acetoxy group, separately at C-3 or C-4 position and 4-methylcoumarins bearing single acetoxy group, separately at C-5, C-6 or C-7 position were synthesized and specificities to rat liver microsomal TA were examined. Negligible TA activity was discernible with 3-AC as the substrate, while the substrate efficiency of other AC were in the order 7-acetoxy-4-methylcoumarin (7 AMC)>6 AMC>5 AMC=5 ADMC=4 AC. To achieve a comparable level of GST inhibition which was proportional to the enzymatic transfer of acetyl groups to the protein (GST), the concentrations of 7-AMC, 6-AMC, 5-AMC and 4-AC were in the order 1:2:4:4, respectively. One diacetoxycoumarin, i.e., 7,8-diacetoxy-4-methylcoumarin (DAMC) was also examined and it was found to elicit maximum level of GST inhibition, nearly twice that observed with 7-AMC. These observations lead to the logical conclusion that a high degree of acetyl group transfer capability is conferred when the acetoxy group on the benzenoid ring of the coumarin system is in closer proximity to the oxygen heteroatom, i.e., when the acetoxy groups are at the C-7 and C-8 positions.     

Malaria severity and human nitric oxide synthase type 2 (NOS2) promoter haplotypes

Nitric oxide (NO) mediates host resistance to severe malaria and other infectious diseases. NO production and mononuclear cell expression of the NO producing enzyme-inducible nitric oxide synthase (NOS2) have been associated with protection from severe falciparum malaria. The purpose of this study was to identify single nucleotide polymorphisms (SNPs) and haplotypes in the NOS2 promoter, to identify associations of these haplotypes with malaria severity and to test the effects of these polymorphisms on promoter activity. We identified 34 SNPs in the proximal 7.3 kb region of the NOS2 promoter and inferred NOS2 promoter haplotypes based on genotyping 24 of these SNPs in a population of Tanzanian children with and without cerebral malaria. We identified 71 haplotypes; 24 of these haplotypes comprised 82% of the alleles. We determined whether NOS2 promoter haplotypes were associated with malaria severity in two groups of subjects from Dar es Salaam (N = 185 and N = 250) and in an inception cohort of children from Muheza-Tanga, Tanzania (N = 883). We did not find consistent associations of NOS2 promoter haplotypes with malaria severity or malarial anemia, although interpretation of these results was potentially limited by the sample size of each group. Furthermore, cytokine-induced NOS2 promoter activity determined using luciferase reporter constructs containing the proximal 7.3 kb region of the NOS2 promoter and the G-954C or C-1173T SNPs did not differ from NOS2 promoter constructs that lacked these polymorphisms. Taken together, these studies suggest that the relationship between NOS2 promoter polymorphisms and malaria severity is more complex than previously described.     

Phorbol ester mediated activation of inducible nitric oxide synthase results in platelet profilin nitration.

Nitric oxide is an important precursor for peroxynitrite production under in vivo conditions leading to cell injury and cell death. In platelets, a number of cytosolic and actin binding proteins were shown to be nitrated [K.M. Naseem, S.Y. Low, M. Sabetkar, N.J. Bradley, J. Khan, M. Jacobs, K.R. Bruckdorfer, The nitration of platelet cytosolic proteins during agonist-induced activation of platelets. FEBS Lett. 473 (1) (2000) 199-122 and M. Sabetkar, S.Y. Low, K.M. Naseem, K.R. Bruckdorfer, The nitration of proteins in platelets: significance in platelet function, Free Radic. Biol. Med. 33 (6) (2002) 728-736]. We investigated the possible mechanism that regulates profilin (an actin binding protein) nitration in platelets. Activation of bovine platelets with arachidonic acid, thrombin, and phorbol 12,13-dibutyrate resulted in nitration of profilin on tyrosine residue. In vivo profilin nitration showed a four- and eight-fold increase in the presence of thrombin and phorbol 12,13-dibutyrate, respectively. Analysis of nitroprofilin levels in the presence of NOS inhibitors (1400W and EGTA), indicated that profilin nitration in phorbol 12,13-dibutyrate treated platelets is mediated by inducible nitric oxide synthase. Phorbol ester treated platelets exhibited higher levels by inducible nitric oxide synthase (491% over control), while total nitric oxide synthase activity increased by 5% over control. Higher levels of peroxynitrite in platelets treated with phorbol 12,13-dibutyrate indicated that profilin nitration is mediated by peroxynitrite. Increase in phosphatidylinositol 3-kinase (PI 3-kinase) activity in platelets treated with thrombin and phorbol 12,13-dibutyrate indicates that nitration of platelet profilin could be mediated by PI 3-kinase. A decrease in the level of nitroprofilin in PDBu treated platelets in the presence of inducible nitric oxide synthase inhibitor, 1400W, was observed suggesting that profilin nitration is mediated by PI 3-kinase dependent activation of inducible nitric oxide synthase.     

Roles of accumulated endogenous nitric oxide synthase inhibitors, enhanced arginase activity, and attenuated nitric oxide synthase activity in endothelial cells for pulmonary hypertension in rats

Nitric oxide (NO) has been suggested to play a key role in the pathogenesis of pulmonary hypertension (PH). To determine which mechanism exists to affect NO production, we examined the concentration of endogenous nitric oxide synthase (NOS) inhibitors and their catabolizing enzyme dimethylarginine dimethylaminohydrolase (DDAH) activity and protein expression (DDAH1 and DDAH2) in pulmonary artery endothelial cells (PAECs) of rats given monocrotaline (MCT). We also measured NOS and arginase activities and NOS protein expression. Twenty-four days after MCT administration, PH and right ventricle (RV) hypertrophy were established. Endothelium-dependent, but not endothelium-independent, relaxation and cGMP production were significantly impaired in pulmonary artery specimens of MCT group. The constitutive NOS activity and protein expression in PAECs were significantly reduced in MCT group, whereas the arginase, which shares l-arginine as a common substrate with NOS, activity was significantly enhanced in PAECs of MCT group. The contents of monomethylarginine (MMA) and asymmetric dimethylarginine (ADMA), but not symmetric dimethylarginine (SDMA), were increased in PAECs of MCT group. The DDAH activity and DDAH1, but not DDAH2, protein expression were significantly reduced in PAECs of MCT group. These results suggest that the impairment of cGMP production as a marker of NO production is possibly due to the blunted endothelial NOS activity resulting from the downregulation of endothelial NOS protein, accumulation of endogenous NOS inhibitors, and accelerated arginase activity in PAECs of PH rats. The decreased overall DDAH activity accompanied by the downregulation of DDAH1 would bring about the accumulation of endogenous NOS inhibitors.     

高血压大鼠肾动脉内皮的一氧化氮合酶(NOS)活性与表面结构的改变

以还原型辅酶Ⅱ黄递酶（ＮＡＤＰＨｄ）组织化学技术及扫描电镜（ＳＥＭ）,对正常组与高血压组大鼠肾动脉内皮的一氧化氮合酶（ＮＯＳ）及表面结构进行了观察。结果表现：正常组肾动脉内皮细胞胞浆中可见蓝黑色斑块状的ＮＯＳ阳性反应产物,但在高血压组肾动脉内皮细胞胞浆中的ＮＯＳ反应明显减弱。扫描电镜显示高血压组肾动脉的部分内皮轮廓不清且凸凹不平,可见指压样印迹以及红细胞和单核细胞粘附。由此我们认为,高血压大鼠肾动脉内皮的结构和功能均发生了改变。     

Nitric oxide synthase inhibitors decrease human polymorphonuclear leukocyte luminol-dependent chemiluminescence

Nitric oxide synthase (NOS) inhibitors have been reported to modulate luminol-dependent chemiluminescence (CL) in rat macrophages, whereas the potent oxidant peroxynitrite (ONOO^-) was shown to react with luminol to yield CL in a cellfree system. We evaluated the role of the -arginine/NOS pathway in luminol CL by phorbol ester-activated human polymorpho-nuclear (PMN) leukocytes using the NOS inhibitors N^G-monomethyl- -arginine ( -NMMA) and N-iminoethyl- -omithine ( -NIO). Nitric oxide (·NO) release was determined by oxidation of oxymyoglobin. In addition, the effect of NOS inhibitors on superoxide anion O₂^-) production was measured. Luminol CL was notably diminished by -NMMA in a dose-dependent manner. Superoxide dismutase (SOD) also decreased luminol CL and -NMMA potentiated light emission decrease produced by SOD. Nitric oxide and O₂^·- production was significantly decreased by -NMMA; moreover, luminol-dependent CL but not O₂^·- production was attenuated by -NIO. These data suggest that products of catalytic activity of both ·NO synthase and NADPH oxidase are required to elicit maximal luminol CL in this system. These studies demonstrate that the NOS synthase pathway is involved in luminol CL by human PMN, and they suggest that ONOO would be an unrecognized mediator in this phenomenon.