Venodilator action of an organotransition-metal nitrosyl complex

Nitrovasodilators, such as nitroglycerin, cause endothelium-independent dilatation of arterial and capacitance vessels via the release of nitric oxide (NO). This study examined the venodilator effect of CpCr(NO)(2)Cl (organotransition-metal nitrosyl complex) relative to that of nitroglycerin in conscious, unrestrained rats. Organotransition-metal nitrosyl complexes have releasable NO directly attached to metal centres. The dose-response effects of CpCr(NO)(2)Cl and nitroglycerin on the mean arterial pressure and the mean circulatory filling pressure (index of the body venous tone) were obtained in rats continuously infused with either normal saline or noradrenaline. The results show that both CpCr(NO)(2)Cl and nitroglycerin reduced the mean arterial pressure in rats with normal or elevated vasomotor tone. However, maximum depressor response of CpCr(NO)(2)Cl was greater than that of nitroglycerin. In vehicle-treated rats, both compounds increased the mean circulatory filling pressure. In rats with elevated vasomotor tone through the infusion of noradrenaline, both agents reduced the mean circulatory filling pressure. These results show that CpCr(NO)(2)Cl is an efficacious depressor and venodilator agent.     

Modulation of prostaglandin biosynthesis by nitric oxide and nitric oxide donors

The biosynthesis and release of nitric oxide (NO) and prostaglandins (PGs) share a number of similarities. Two major forms of nitric-oxide synthase (NOS) and cyclooxygenase (COX) enzymes have been identified to date. Under normal circumstances, the constitutive isoforms of these enzymes (constitutive NOS and COX-1) are found in virtually all organs. Their presence accounts for the regulation of several important physiological effects (e.g. antiplatelet activity, vasodilation, and cytoprotection). On the other hand, in inflammatory setting, the inducible isoforms of these enzymes (inducible NOS and COX-2) are detected in a variety of cells, resulting in the production of large amounts of proinflammatory and cytotoxic NO and PGs. The release of NO and PGs by the inducible isoforms of NOS and COX has been associated with the pathological roles of these mediators in disease states as evidenced by the use of selective inhibitors. An important link between the NOS and COX pathways was made in 1993 by Salvemini and coworkers when they demonstrated that the enhanced release of PGs, which follows inflammatory mechanisms, was nearly entirely driven by NO. Such studies raised the possibility that COX enzymes represent important endogenous "receptor" targets for modulating the multifaceted roles of NO. Since then, numerous papers have been published extending the observation across various cellular systems and animal models of disease. Furthermore, other studies have highlighted the importance of such interaction in physiology as well as in the mechanism of action of drugs such as organic nitrates. More importantly, mechanistic studies of how NO switches on/off the PG/COX pathway have been undertaken and additional pathways through which NO modulates prostaglandin production unraveled. On the other hand, NO donors conjugated with COX inhibitors have recently found new interest in the understanding of NO/COX reciprocal interaction and potential clinical use. The purpose of this article is to cover the advances which have occurred over the years, and in particular, to summarize experimental data that outline how the discovery that NO modulates prostaglandin production has impacted and extended our understanding of these two systems in physiopathological events.     

Regulation of leukocyte function by nitric oxide donors: the effect of S-nitroso-thiol complexes

The aim of this study was to evaluate the effectiveness of a novel protein-bound S-nitroso-thiol, S-nitroso-albumin (S-NO-alb), in modulating neutrophil-endothelial cell adhesion, activation, and interactions. Due to the highly variable kinetics of NO release from the low-molecular-weight thiol adducts S-nitroso-N-acetylpenicillamine (SNAP) and S-nitroso-glutathione (GSNO), we expected S-NO-alb to be a more effective modulator of inflammatory interactions through its slow, steady, and prolonged release of NO. Human umbilical-vein endothelial cells (HUVECs) challenged with lipopolysaccharide (LPS) demonstrated upregulated adhesion of neutrophils that was significantly attenuated by pretreatment with S-NO-alb (1.0-100 microM) (p < .05), but not SNAP or GSNO. Pretreatment with S-NO-alb, SNAP, or GSNO attenuated tumor necrosis factor-alpha primed *O2- release from neutrophils and increased neutrophil cGMP accumulation. On a molar basis, S-NO-alb expressed a 10-fold greater potency than SNAP or GSNO at modulating these effects. Kinetics studies confirmed the relative stability of spontaneous NO release from S-NO-alb compared with highly variable kinetic profiles of SNAP and GSNO. Our results demonstrate that S-NO-alb more effectively modulates endothelial-cell and neutrophil immunoinflammatory responses versus its related low-molecular-weight thiol complexes.     

Nitric oxide-independent effects of nitric oxide donors on energy metabolism in erythrocytes

In order to study the roles of nitric oxide (NO) in various biological events, several types of NO-releasing agents have been extensively used. Although both NO and its donors and/or their decomposed products may have biological activities, most of the cellular responses to these donors have been postulated to reflect NO-dependent events. Among the various NO donors, 1-hydroxy-2-oxo-3-(N-methyl-aminopropyl)-3-methyl-l-triazene (NOC7), 3-morpholinosydnonimine N-ethylcarbamide (SIN-1), S-nitrosoglutathione, S-nitrosocysteine (CysNO), and related nitrosothiols are commonly used agents. To investigate the biological activities of these donors and their decomposed products, we tested their effects on energy metabolism in erythrocytes. When incubated with freshly prepared erythrocytes, NOC7, Cys-NO, and their decomposed products, but not NO and its oxidized metabolites, nitrite and nitrate, decreased cellular ATP levels. Although SIN-1 generates both NO and superoxide radical thereby forming peroxynitrite (ONOO-), this donor had no appreciable effect on cellular ATP levels, even in the presence of superoxide dismutase. These results indicate that NOC7 and CysNO and/or their decomposed product(s), but not NO and its oxidized metabolites, are responsible for the decrease in cellular ATP levels. Thus, the effects of not only NO and its oxidized metabolites (NO2, NO3 ), but also NO donors and their decomposed products, should be taken into account when attempting to understand the mechanism of biological responses induced by NO donors.     

The effect of nitric oxide synthase blockers,nitric oxide donors,and anti-ovarian antibodies on murine oocytes

The effects of NO synthase (NOS) blockers, NO donors, and anti-ovarian antibodies (AOABs) on the amount of oocytes isolated from murine ovary and their meiotic maturation were studied. It was established that the NOS blockers inhibit the their meiotic maturation of oocytes, while NO donors influenced neither the number of oocytes nor their ability to meiotic maturation in diaestrus or oestrous cycle. The AOAB effect on the gametogenesis was dose-dependent: inhibition for large doses and activation for small doses. The NO/NOS ovarian system is involved into mechanism of the AOAB action on murine oocytes. It is suggested that a decrease in the ovarian NO production, as well as the AOAB-induced inhibition of the murine oogenesis, are factors influencing female infertility.     

In vitro cytotoxicity of glyco-S-nitrosothiols. a novel class of nitric oxide donors.

The cytotoxicities of the nitric oxide (NO) donors, S-nitroso-N-acetylpencillamine (SNAP) and three glyco-SNAPs, glucose-1-SNAP, glucose-2-SNAP, and fructose-1-SNAP, towards the human gingival epithelioid S-G cell line and three human carcinoma cell lines derived from tissues of the oral cavity were compared using the neutral red (NR) assay. In general, the glucose-SNAPs were more cytotoxic than SNAP, which, in turn, was more cytotoxic than fructose-1-SNAP. Further studies focused on the response of S-G cells to glucose-2-SNAP. The cytotoxicity of glucose-2-SNAP was attributed to NO, as glucose-2-SNAP (t1/2=20 h at 28 degrees C) aged for 4 days was nontoxic, toxicity was eliminated in the presence of hydroxocobalamin, a specific NO scavenger, and toxicity was not noted with glucose-2-AP (the parent compound used to construct glucose-2-SNAP). Exposure of cells to glucose-2-SNAP resulted in a lessening of the intracellular level of glutathione and cells pretreated with the glutathione-depleter, 1,3-bis-(chloroethyl)-1-nitrosourea, were more sensitive to a subsequent challenge with glucose-2-SNAP. Cytotoxicity of glucose-2-SNAP was lessened upon coexposure with the antioxidants, myricetin, N-acetyl-L-cysteine, and L-ascorbic acid. S-G cells exposed to glucose-2-SNAP exhibited bi- and multinucleation. Death of S-G cells exposed to glucose-2-SNAP apparently occurred by apoptosis, as demonstrated with fluorescence microscopy by the appearance of brightly stained, hypercondensed chromatin in spherical cells and of membrane blebbing and by the DNA-ladder of oligonucleosome-length fragments noted with gel electrophoresis. In comparison with other classes of NO donors the sequence of toxicity towards S-G cells was S-nitrosoglutathione>glucose-SNAPs>SNAP, sodium nitroprusside>spermine NONOate>DPTA NONOate>DETA NONOate>fructose-1-SNAP>SIN-1.     

Comparison of responses to novel nitric oxide donors in the feline pulmonary vascular bed

Pulmonary vascular responses to the novel diazeniumdiolate nitric oxide (NO) donors diethylamine/NO, diethylenetriamine/NO, spermine/NO, sulfite/NO, and angeli's salt, were investigated and compared in the intact-chest cat. Under conditions of controlled blood flow, when tone in the pulmonary vascular bed had been raised to a high steady level, intralobar injections of diethylamine/NO (0.3–10 μg), diethylenetriamine/NO (10–30 μg), spermine/NO (10–30 μg), sulfite/NO (10–30 μg), and angeli's salt (10–30 μg) caused dose-related decreases in lobar arterial pressure without changing left atrial pressure. In terms of relative vasodilator activity in the pulmonary vascular bed, the dose of the compounds that decreased lobar arterial pressure 4 mm Hg (ED₄ mm Hg) was significantly lower for diethylamine/NO compared to S-nitroso-N-acetylpenicillamine which was significantly less than diethylenetriamine/NO, spermine/NO, sulfite/NO, and angeli's salt. The half-life of the vasodilator responses, as measured by 50% response recovery time, to diethylamine/NO, diethylenetriamine/NO, spermine/NO, sulfite/NO, and angeli's salt was similar for doses with similar magnitudes of vasodilation, while the half-life to S-nitroso-N-acetylpenicillamine was significantly less than the diazeniumdiolate NO donors. The present data demonstrate that the diazeniumdiolate NO donors diethylamine/NO, diethylenetriamine/NO, spermine/NO, sulfite/NO, and angeli's salt have potent but relatively short-lasting vasodilator activity in the pulmonary vascular bed of the cat.     

Systemic inflammatory response, bacterial translocation and nitric oxide donors

Abdominal aortic surgery is relatively common and is associated with considerable post-operative morbidity and death. The aortic cross-clamping (supra or infrarenal) necessary for the insertion of a vascular graft, often in circumstances of haemorrhagic shock (e.e. a ruptured aneurysm) elicits a Systemic Inflammatory Response Syndrome (SIRS) and an Ischaemia-Reperfusion syndrome (I-R), with affectation of many organs including the kidneys and the intestine. Experimentally, the exogenous use of nitric oxide donors has proved to be able to control the SIRS, minimising the damage due to I-R and protecting from renal dysfunction and BT. However, clinical experience in these situations is still limited. Here we review the current status and experience of the authors in the use of nitric oxide donors in the control of the SIRS induced by infrarenal, suprarenal aortic cross-clamping, with or without haemorrhagic shock; and the Bacterial Translocation phenomenon (BT) induced by aortic cross-clamping below the mesenteric artery with or without associated hemorrhaging.     

Water-soluble two-photon absorbing nitrosyl complex for light-activated therapy through nitric oxide release

A water-soluble nitrosyl complex with large two-photon absorption was synthesized by incorporating a two-photon absorbing chromophore with tetra(ethylene glycol) units, into the Roussin's red salt. The nitrosyl complex exhibits quenched emission due to energy transfer from the two-photon chromophore to the Roussin's red salt. The nitric oxide (NO) release induced by one- or two-photon irradiation was detected by EPR spectroscopy with a chemical probe, the Fe(II)- N-(dithiocarbamoyl)- N-methyl- d-glucamine (Fe-MGD) complex. Increased one- or two-photon excited fluorescence, with a concomitant photochemical release of NO, was observed upon one- or two-photon light irradiation. With the observed light-dependent cytotoxicity against cancer cells of the water-soluble nitrosyl complex, it was demonstrated that two-photon-functionalized nitrosyl complexes can be effective NO donors for light-activated treatment.     

Stimulatory effects of nitric oxide donors on gastric acid secretion in isolated mouse stomach

We previously reported on the stimulatory role of endogenous nitric oxide (NO) in gastric acid secretion. In the present study, we investigated the effects of NO donors on acid secretion in isolated mouse stomach. Nitroprusside (100 μM–1 mM) inhibited the gastric acid secretion induced by histamine (500 μM) in a concentration-dependent manner. In addition, nitroprusside abolished the acid secretion induced by bethanechol (100 μM) and by electrical stimulation (10 Hz) of the vagus nerve. On the other hand, nitroprusside, 75 μM, which did not affect the acid secretion induced by histamine, itself elicited an increase in acid secretion. The acid secretion induced by 75 μM nitroprusside was inhibited by 10 μM famotidine, a histamine H₂ receptor antagonist. These results suggest that NO donors at high doses act on gastric parietal cells, resulting in inhibition of the stimulated acid secretion, and, at lower doses, facilitate histamine release from histamine-containing cells, leading to the increased acid secretion.     

烟酸与一氧化氮供体偶联化合物的合成

目的寻找更有效的抗动脉粥样硬化药物,合成烟酸与NO供体基团3-苯磺酰基-1,2,5-(口恶)二唑-2-氧化物杂化的备选化合物.方法将NO供体单元3,4-二苯磺酰基-1,2,5-(哑)二唑-2-氧化物与烟酸经桥连基团通过酯键或酰胺键结合形成目标化合物.结果与结论合成了9个新的目标化合物,其结构经质谱、红外光谱及核磁共振氢谱确证.     

The use of nitric oxide donors in pharmacological studies

A growing appreciation of the involvement of nitric oxide (NO) in numerous bioregulatory pathways has not only opened up new therapeutic avenues for organic nitrates and other NO donors but also led to an increased use of such compounds in pharmacological studies. By definition, all NO donors produce NO-related activity when applied to biological systems and are thus principally suited to either mimic an endogenous NO-related response or substitute for an endogenous NO deficiency. However, the pathways leading to enzymatic and/or non-enzymatic formation of NO differ greatly among individual compound classes, as do their chemical reactivities and kinetics of NO release. Moreover, since the reaction of NO with oxygen is a function of its concentration, the same absolute amounts of NO generated over different periods of time may lead to substantially different rates of NO_x formation and, consequently, to varying extents of side reactions, such as nitration and/or nitrosation of biomolecules. Matters are further complicated by compound-specific formation of by-products, which may arise during decomposition or metabolism, sometimes in amounts far exceeding those of NO. The term “NO donor” implies that the compound releases the active mediator, NO. Ultimately, this may be true for many different chemical classes of compound, since the principal NO-related species generated may be converted to NO, if not directly released as such. However, in a biological system, the redox form of nitrogen monoxide (NO⁺, NO· or NO^–) that is actually released makes a substantial difference to the NO donor’s reactivity towards other biomolecules, the profile of by-products, and the bioresponse. Such considerations are likely to account for much of the discrepancy in experimental results obtained using the same cell or tissue preparation but different NO mimetics. Thus, compound selection is not a trivial issue and the investigator should be aware of the key properties and differences between various NO donor classes in order to avoid misinterpretation of experimental results.     

Effect of nitric oxide donors and nitric oxide synthase inhibitors in neonatal rat endotoxic shock.

Previous studies have shown an increased mortality in response to endotoxin in 24-hr-old neonatal rats compared with older neonates and adults. This increased susceptibility may be related to increased nitric oxide (NO) and thromboxane (TxB2) production. Twenty-four-hour-old neonatal rat pups were given either N(G)-nitro-L-arginine methyl ester (L-NAME; a nonspecific NO synthase inhibitor), S-methylthioisourea (SMT; a specific NO synthase inhibitor), or molsidomine (a NO donor) subcutaneously prior to or after an LD50 of intracardiac endotoxin. Mortality was followed for 72 hr. There was no statistically significant difference in mortality between control animals and those pretreated with L-NAME, SMT, or molsidomine. A trend toward increased mortality with nonspecific NO synthase inhibition and decreased mortality with the NO donor was noted. Splenic cells were obtained for in vitro cytokine stimulation studies. In vitro adherent splenic cell stimulation studies confirmed an increase in NO production with NO donor pretreatment and decreased production of NO with NO synthase inhibition pretreatment. There was no difference in TxB2 production with either the NO synthase inhibitor or the NO donor. In conclusion, at the several doses employed, neither nonselective or selective NO synthase inhibitors nor NO donors prevented endotoxin-induced mortality in rat neonatal shock. Although these findings do not preclude possible involvement of NO in neonatal pathophysiology, increased NO production thus does not appear to be the primary determinant of the increased susceptibility of the neonatal rat to endotoxic shock.     

The formation of nitric oxide donors from peroxynitrite.

1. Administration of peroxynitrite (ONOO-, 30-300 microM) caused relaxation of rabbit aortic strips superfused in series in a cascade. The compound responsible for this effect had a half-life greater than 20 s and could not therefore be either nitric oxide (NO) or ONOO- which have half-lives in the order of 1-2 s under these conditions. However the relaxation was inhibited by oxyhaemoglobin, suggesting the compound could be converted to NO in the vascular tissues or in the superfusate. 2. The products of the reactions between ONOO- and Krebs buffer containing 11 mM glucose, but not glucose-free Krebs buffer, caused relaxation of the bioassay tissues. These data suggest that stable NO donor(s) were formed from the reaction of ONOO- with glucose. We therefore prepared these NO donor(s) by the reaction of glucose solutions with ONOO- in order to characterize their ability to release NO. 3. These reaction product(s) caused relaxation in the cascade and inhibition of platelet aggregation. Both effects were dependent on the concentration of D-glucose, were equally effective if L-glucose was used as a reactant and were reversed by oxyhaemoglobin. 3. The products of the reaction between ONOO- and glucose or other biological molecules containing an alcohol functional group, such as fructose, glycerol, or glyceraldehyde, released NO in the presence of Cu2+and L-cysteine. 5. These results indicate that ONOO- reacts with sugars or other compounds containing an alcohol functional group(s) to form NO donors with the characteristics of organic nitrate/nitrites. This may represent a further detoxification pathway for ONOO- in vivo.     

Comparative effects of several nitric oxide donors on intracellular cyclic GMP levels in bovine chromaffin cells: correlation with nitric oxide production.

1. Sodium nitroprusside, S-nitroso-N-acetyl-D,L-penicillamine, Spermine NONOate and DEA NONOate raised cyclic GMP levels in bovine chromaffin cells in a time and concentration dependent manner with different potencies, the most potent being DEA/NO with an EC50 value of 0.38 +/- 0.02 microM. 2. Measurements of NO released from these donors revealed that DEA/NO decomposed with a half-life (t1/2) of 3.9 +/- 0.2 min. The t1/2 for SPER/NO was 37 +/- 3 min. SNAP decomposed more slowly (t1/2 = 37 +/- 4 h) and after 60 min the amount of NO produced corresponded to less than 2% of the total SNAP present. The rate of NO production from SNAP was increased by the presence of glutathione. 3. For DEA/NO and SPER/NO there was a clear correlation between nitric oxide production and cyclic GMP increases. Their threshold concentrations were 0.05 microM and maximal effective concentration between 2.5 and 5 microM. 4. For SNAP, threshold activation was seen at 1 microM, whereas full activation required a higher concentration (500-750 microM). The dose-response for SNAP increases in cyclic GMP was shifted nearly two orders of magnitude lower in the presence of glutathione. At higher concentrations an inhibition of cyclic GMP accumulation was found. This effect was not observed with either the nitric oxide-deficient SNAP analogue or other NO donors. 5. Although NO-donors are likely to be valuable for studying NO functions, their effective concentrations and the amount of NO released by them are very different and should be assessed in each system to ensure that physiological concentrations of NO are used.     

Stimulatory effects of nitric oxide donors on histamine release in isolated rat gastric mucosal cells

We previously reported stimulatory effects of endogenous and exogenous nitric oxide (NO) on gastric acid secretion. In the present study, we investigated effects of NO donors on release of histamine, which is related to acid secretion, in isolated rat gastric mucosal cells. NO donors such as (+/-)-(E)-4-methyl-2-[(E)-hydroxyimino]-5-nitro-6-methoxy-3-hexanamide (NOR 1) and sodium nitroprusside significantly augmented the histamine release. It was inhibited by 2-(4-carboxyphenyl)-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-amide (carboxy-PTIO), a NO scavenger, and 6-(phenylamino)-5,8-quinolinedione (LY83583), a soluble guanylate cyclase inhibitor. Dibutyryl cyclic GMP also stimulated histamine release. These results suggest that NO donors act on cyclic GMP pathway in isolated gastric mucosal cells, resulting in facilitation of histamine release. NO may stimulate gastric acid secretion through histamine release from the histamine-containing cells, possibly enterochromaffin-like cells.     

Stimulatory effects of centrally injected nitric oxide donors on gastric acid secretion in anesthetized rats

The effects of centrally injected nitric oxide (NO) donors on gastric acid secretion were investigated in continuously perfused stomach of anesthetized rats. The lateral cerebroventricular (LV) injection of NOC5 (30 - 100 microg) and NOC12 (10 - 100 microg) dose-dependently stimulated gastric acid secretion. The LV injection of NOC18 (30 microg) also stimulated gastric acid secretion. The other type of NO donor, sodium nitroprusside (3 - 30 microg, LV), also dose-dependently stimulated gastric acid secretion. The effect of NOC5 at 100 microg was blocked by carboxy-PTIO, an NO scavenger, and by cervical vagotomy. Furthermore, NOC12 (30, 100 microg) dose-dependently stimulated gastric acid secretion in pylorus-ligated conscious rats. These results suggest that centrally injected NO donors stimulate gastric acid secretion in both conscious and anesthetized rats through vagus activation.     

Vascular smooth muscle relaxation mediated by nitric oxide donors: a comparison with acetylcholine, nitric oxide and nitroxyl ion

1. Vasorelaxant properties of three nitric oxide (NO) donor drugs (glyceryl trinitrate, sodium nitroprusside and spermine NONOate) in mouse aorta (phenylephrine pre-contracted) were compared with those of endothelium-derived NO (generated with acetylcholine), NO free radical (NO*; NO gas solution) and nitroxyl ion (NO(-); from Angeli's salt). 2. The soluble guanylate cyclase inhibitor, ODQ (1H-(1,2,4-)oxadiazolo(4,3-a)-quinoxalin-1-one; 0.3, 1 and 10 microM), concentration-dependently inhibited responses to all agents. 10 microM ODQ abolished responses to acetylcholine and glyceryl trinitrate, almost abolished responses to sodium nitroprusside but produced parallel shifts (to a higher concentration range; no depression in maxima) in the concentration-response curves for NO gas solution, Angeli's salt and spermine NONOate. 3. The NO* scavengers, carboxy-PTIO, (2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide; 100 microM) and hydroxocobalamin (100 microM), both inhibited responses to NO gas solution and to the three NO donor drugs, but not Angeli's salt. Hydroxocobalamin, but not carboxy-PTIO, also inhibited responses to acetylcholine. 4. The NO(-) inhibitor, L-cysteine (3 mM), inhibited responses to Angeli's salt, acetylcholine and the three NO donor drugs, but not NO gas solution. 5. The data suggest that, in mouse aorta, responses to all three NO donors involve (i) activation of soluble guanylate cyclase, but to differing degrees and (ii) generation of both NO* and NO(-). Glyceryl trinitrate and sodium nitroprusside, which generate NO following tissue bioactivation, have profiles resembling the profile of endothelium-derived NO more than that of exogenous NO. Spermine NONOate, which generates NO spontaneously outside the tissue, was the drug that most closely resembled (but was not identical to) exogenous NO.     

一氧化氮供体最新研究进展及应用前景

一氧化氮供体已经成为当今药物研究中的一大焦点,此文对近年来一氧化氮供体研究领域的最新研究成果作一综述,并展望了其在医疗上的应用前景。     

Current trends in the development of nitric oxide donors.

Nitric oxide (NO) is an important messenger molecule involved in many pathological and physiological processes within the mammalian body. Exogenous NO sources constitute a powerful way to supplement NO when the body can not generate enough for normal biological functions. In this article, general aspects on NO and NO donors are reviewed. Major focus is placed on recent developments of novel NO donors, NO releasing device(s) as well as innovative improvements to current NO donors. Finally, an outlook on future NO donor development is provided.