No implantation in an extra-uterine pregnancy of a placentotrophic reptile.

Placentation is a common feature of live-bearing reptiles and mammals. Placentae are variable between species and can be classified by the extent that embryonic tissue breaches (invades) the uterus. Non-invasive placentation in eutherians is maternally imposed as extra-uterine embryos of species with epitheliochorial placentation will readily invade non-uterine tissues. This study documents the first observation of an extra-uterine pregnancy in a reptile; Pseudemoia entrecasteauxii, which in-utero exhibits non-invasive epitheliochorial placentation. The extra-uterine embryo did not invade maternal tissue suggesting fundamental differences between the nature and evolution of placentation in P. entrecasteauxii and eutherian mammals.

In vivo proton (H1) magnetic resonance spectroscopy for cervical carcinoma.

Proton magnetic resonance spectroscopy (MRS) may be a useful tool in both the initial diagnosis of cervical carcinoma and the subsequent surveillance after radiation therapy, particularly when other standard diagnostic methods are inconclusive. Single voxel magnetic resonance (MR) spectral data were acquired from 8 normal volunteers, 16 patients with cervical cancer before radiation therapy, and 18 patients with cervical cancer after radiation therapy using an external pelvic coil at a 1.5-T on a Signa system. The presence or absence of various resonances within each spectrum was evaluated for similarities within each patient group and for spectral differences between groups. Resonances corresponding to lipid and creatine dominated the spectrum for the eight normal volunteers without detection of a choline resonance. Spectra from 16 pretreatment patients with biopsy-proven cervical cancer revealed strong resonances at a chemical shift of 3.25 ppm corresponding to choline. Data acquired from the 18 posttreatment setting studies was variable, but often correlated well with the clinical findings. Biopsy confirmation was obtained in seven patients. H1 MRS of the cervix using a noninvasive pelvic coil consistently demonstrates reproducible spectral differences between normal and neoplastic cervical tissue in vivo. However, signal is still poor for minimal disease recurrence. Further study is needed at intervals before, during, and after definitive irradiation with biopsy confirmation to validate the accuracy of MRS in distinguishing persistence or recurrence of disease from necrosis and fibrosis.

Implications for isoform-selective inhibitor design derived from the binding mode of bulky isothioureas to the heme domain of endothelial nitric-oxide synthase.

Nitric oxide produced by nitric-oxide synthase (NOS) is not only involved in a wide range of physiological functions but also in a variety of pathological conditions. Isoform-selective NOS inhibitors are highly desirable to regulate the NO production of one isoform beneficial to normal physiological functions from the uncontrolled NO production of another isoform that accompanies certain pathological states. Crystal structures of the heme domain of the three NOS isoforms have revealed a very high degree of similarity in the immediate vicinity of the heme active site illustrating the challenge of isoform-selective inhibitor design. Isothioureas are potent NOS inhibitors, and the structures of the endothelial NOS heme domain complexed with isothioureas bearing small S-alkyl substituents have been determined (Li, H., Raman, C.S., Martásek, P., Král, V., Masters, B.S.S., and Poulos, T.L. (2000) J. Inorg. Biochem. 81, 133--139). In the present communication, the binding mode of larger bisisothioureas complexed to the endothelial NOS heme domain has been determined. These structures afford a structural rationale for the known inhibitory activities. In addition, these structures provide clues on how to exploit the longer inhibitor substituents that extend out of the active site pocket for isoform-selective inhibitor design.

Chronic myocardial hypoxia increases nitric oxide synthase and decreases caveolin-3.

Nitric oxide synthase (NOS) is believed to play an important role in protecting the myocardium against ischemia. Chronic hypoxia from birth increases NOS activity in the myocardium resulting in enhanced nitric oxide production and increased resistance to ischemia. We examined the effects of chronic hypoxia on NOS gene and protein expression and on NOS protein association with caveolin-3. Rabbits were raised from birth in a normoxic (F(I)O(2) = 0.21) or a hypoxic (F(I)O(2) = 0.12) environment for 9 d, and then the hearts were isolated. Ribonuclease protection assays revealed that chronic hypoxia did not alter NOS transcript levels for NOS1, NOS2, or NOS3. The most abundant transcript was NOS3. Western analysis revealed NOS3 was the only isoform detected. Immunoblots of NOS3 immunoprecipitates showed that chronic hypoxia increases NOS3 protein by 2.0 +/- 0.4-fold and decreases the amount of caveolin-3 that can be coprecipitated with NOS3 by 5.5 +/- 0.9-fold. Immunoblots of normoxic and hypoxic hearts showed that chronic hypoxia decreases the amount of caveolin-3 in heart homogenates by 2. 2 +/- 0.5-fold. These data suggest that a decrease in caveolin-3 plays a role in the mechanisms by which chronic hypoxia increases NOS3 activity in the myocardium.

Strategies to reduce side effects of interleukin-2: evaluation of the antihypotensive agent NG-monomethyl-L-arginine.

PURPOSE: The clinical utility of high-dose intravenous recombinant interleukin (IL)-2 therapy is limited by severe toxicity including hypotension, fever, chills, pulmonary edema, and oliguria Hypotension has been previously shown to result from excessive vascular relaxation due to overproduction of the endogenous vasodilator nitric oxide. Nitric oxide production can be decreased by administration of the competitive enzyme inhibitor NG-monomethyl-L-arginine (NMA). A clinical trial to investigate the dose-dependent effects of NMA on blood pressure was undertaken in patients with metastatic renal cell carcinoma. PATIENTS AND METHODS: Patients with metastatic renal cell carcinoma receiving a 5-day continuous infusion of IL-2 (18 million IU/m2/d) who developed hypotension were treated with increasing doses of NMA, ranging from 3 to 36 mg/kg. RESULTS: Twenty-three patients received a total of 61 courses of IL-2; 18 of these patients developed hypotension and received NMA. Antihypotensive activity was observed at all dose levels, and the duration of the effect varied directly with the dose of NMA. At the higher dose levels tested (12 to 36 mg/kg), increased pulmonary vascular resistance and decreased cardiac output were observed. Patients experiencing a significant decrease in cardiac output received dobutamine (2.5 to 10 microg/kg/min). Pulmonary capillary wedge pressure was unaffected by administration of NMA. One patient treated at 24 mg/kg (bolus) experienced a major motor seizure, but no neurologic disorders were observed in other patients treated with NMA doses of 24 to 36 mg/kg. No other adverse events involving hepatic, renal, or hematologic systems were attributed to NMA. Three patients received NMA by an initial bolus followed by a continuous infusion. Similar antihypotensive effects were noted, and these patients were able to complete a full 5-day course of IL-2. CONCLUSION: The antihypotensive effects of NMA appear to be optimal at a dose of 24 mg/kg, with maintenance doses of 8 mg/kg every 4 to 6 hours. At this dose level, blood pressure was restored, and IL-2-associated vasodilatation was fully reversed. Coincident with the reversal of hypotension, the state of high cardiac output was also reversed by NMA administration. These results suggest that NMA may be effective for alleviating the hypotensive effects of high-dose IL-2 therapy in cancer patients.

Biologic and pharmacologic regulation of mammalian glutathione synthesis.

Glutathione (L-gamma-glutamyl-L-cysteinylglycine, GSH) is synthesized from its constituent amino acids by the sequential action of gamma-glutamylcysteine synthetase (gamma-GCS) and GSH synthetase. The intracellular GSH concentration, typically 1-8 mM, reflects a dynamic balance between the rate of GSH synthesis and the combined rate of GSH consumption within the cell and loss through efflux. The gamma-GCS reaction is rate limiting for GSH synthesis, and regulation of gamma-GCS expression and activity is critical for GSH homeostasis. Transcription of the gamma-GCS subunit genes is controlled by a variety of factors through mechanisms that are not yet fully elucidated. Glutathione synthesis is also modulated by the availability of gamma-GCS substrates, primarily L-cysteine, by feedback inhibition of gamma-GCS by GSH, and by covalent inhibition of gamma-GCS by phosphorylation or nitrosation. Because GSH plays a critical role in cellular defenses against electrophiles, oxidative stress and nitrosating species, pharmacologic manipulation of GSH synthesis has received much attention. Administration of L-cysteine precursors and other strategies allow GSH levels to be maintained under conditions that would otherwise result in GSH depletion and cytotoxicity. Conversely, inhibitors of gamma-GCS have been used to deplete GSH as a strategy for increasing the sensitivity of tumors and parasites to certain therapeutic interventions.

L-arginine binding to nitric-oxide synthase. The role of H-bonds to the nonreactive guanidinium nitrogens.

Nitric-oxide synthase (NOS) catalyzes the oxidation of L-arginine to nitric oxide and L-citrulline. Because overproduction of nitric oxide causes tissue damage in neurological, inflammatory, and autoimmune disorders, design of NOS inhibitors has received much attention. Most inhibitors described to date include a guanidine-like structural motif and interact with the guanidinium region of the L-arginine-binding site. We report here studies with L-arginine analogs having one or both terminal guanidinium nitrogens replaced by functionalities that preserve some, but not all, of the molecular interactions possible for the -NH(2), =NH, or =NH(2)(+) groups of L-arginine. Replacement groups include -NH-alkyl, -alkyl, =O, and =S. Binding of L-canavanine, an analog unable to form hydrogen bonds involving a N(5)-proton, was also examined. From our results and previous work, we infer the orientation of these compounds in the L-arginine-binding site and use IC(50) or K(i) values and optical difference spectra to quantitate their affinity relative to L-arginine. We find that the non-reactive guanidinium nitrogen of L-arginine binds in a pocket that is relatively intolerant of changes in the size or hydrogen bonding properties of the group bound. The individual H-bonds involved are, however, weaker than expected (<2 versus 3-6 kcal). These findings elucidate substrate binding forces in the NOS active site and identify an important constraint on NOS inhibitor design.

Adaptation to chronic hypoxia confers tolerance to subsequent myocardial ischemia by increased nitric oxide production.

Chronic exposure to hypoxia from birth increased the tolerance of the rabbit heart to subsequent ischemia compared with age-matched normoxic controls. The nitric oxide donor GSNO increased recovery of post-ischemic function in normoxic hearts to values not different from hypoxic controls, but had no effect on hypoxic hearts. The nitric oxide synthase inhibitors L-NAME and L-NMA abolished the cardioprotective effect of hypoxia. Message and catalytic activity for constitutive nitric oxide synthase as well as nitrite, nitrate, and cGMP levels were elevated in hypoxic hearts. Inducible nitric oxide synthase was not detected in normoxic or chronically hypoxic hearts. Increased tolerance to ischemia in rabbit hearts adapted to chronic hypoxia is associated with increased expression of constitutive nitric oxide synthase.

The enzymes of glutathione synthesis: gamma-glutamylcysteine synthetase.

The metabolite glutathione fulfills many important and chemically complex roles in protecting cellular components from the deleterious effects of toxic species. GSH combines with hydroxyl radical, peroxynitrite, and hydroperoxides, as well as reactive electrophiles, including activated phosphoramide mustard. This thiol-containing reductant also maintains so-called thiol-enzymes in their catalytically active form, and maintains vitamins C and E in their biologically active forms. The key step in glutathione synthesis, namely the ATP-dependent synthesis of gamma-glutamylcysteine, is the topic of this review. Details are presented on (a) the enzyme's purification and protein chemistry, (b) the successful cDNA cloning, and characterization of the genes responsible for the biosynthesis of this enzyme. After considering aspects of the role of overexpression of this synthetase in terms of cancer chemotherapy, attention is focused on post-translational regulation. The remainder of the review deals with the catalytic mechanism (including substrate specificity, reactions catalyzed, steady-state kinetics, and chemical mechanism) as well as the inhibition of the enzyme (via feedback inhibition, reaction with S-alkyl homocysteine sulfoximine inhibitors, the clinical use of buthionine sulfoximine with cancer patients, and inactivation by cystamine, chloroketones, and various nitric oxide donors).

Design of isoform-selective inhibitors of nitric oxide synthase.

Nitric oxide synthase, the mammalian enzyme catalyzing the oxidation of L-arginine to L-citrulline and nitric oxide, is present in three isoforms that have distinct physiological roles. Overstimulation or overexpression of individual nitric oxide synthase isoforms plays a role in a wide range of disorders including septic shock, arthritis, diabetes, ischemia-reperfusion injury, pain and various neurodegenerative diseases. Animal studies and early clinical trials suggest that nitric oxide synthase inhibitors could be therapeutic in many of these disorders, but preservation of physiologically important nitric oxide synthase functions might require use of isoform-selective inhibitors. Within the past few years both amino acid and nonamino acid nitric oxide synthase inhibitors with pharmacologically useful isoform selectivity have been reported. Selectivity has been achieved on the basis of initial binding affinity and, for mechanism-based inactivators, on the basis of isoform-dependent catalytic activation; particularly interesting are N5-(1-imino-3-butenyl)-L-ornithine, ARL 17477, 1400W and S-(2-aminoethyl)isothiourea.

Expression and purification of human gamma-glutamylcysteine synthetase.

gamma-Glutamylcysteine synthetase (gamma-GCS) catalyzes the ATP-dependent ligation of L-glutamate and L-cysteine to form L-gamma-glutamyl-L-cysteine; this is the first and rate-limiting step in glutathione biosynthesis. Inhibitors of gamma-GCS such as buthionine sulfoximine are widely used as tools for elucidating glutathione metabolism in vivo and as pharmacological agents for reversing glutathione-based resistance to chemotherapy and radiation therapy in certain cancers. Although gamma-GCS is readily isolated from rat kidneys, future drug design efforts are better based on structure-activity relationships established with the human enzyme. We report here the coexpression in Escherichia coli BL21(DE3) of the human gamma-GCS catalytic (heavy) subunit and regulatory (light) subunit using pET-3d and pET-9d vectors, respectively. Intracellular assembly of the holoenzyme occurred without difficulty, and levels of expression were acceptable (approximately 32 mg holoenzyme/100 g cells). Recombinant human gamma-GCS was purified to homogeneity in an overall yield of 45% by ammonium sulfate fractionation followed by sequential chromatography on Q-Sepharose ion-exchange, Superdex 200 gel filtration and ATP-affinity resins. Trace amounts of E. coli gamma-GCS were removed by immunoaffinity chromatography. The specific activity of the isolated enzyme was >1500 units/mg, comparable to the best preparations from rat kidney. The Km values for L-glutamate, L-cysteine, L-gamma-aminobutyrate (an L-cysteine surrogate), and ATP are 1.8, 0.1, 1.3, and 0.4 mM, respectively. Recombinant human gamma-GCS, like native rat gamma-GCS, is feedback inhibited by glutathione and is potently inhibited by buthionine sulfoximine and cystamine.

N5-(1-Imino-3-butenyl)-L-ornithine. A neuronal isoform selective mechanism-based inactivator of nitric oxide synthase.

Nitric oxide synthase (NOS) catalyzes the NADPH- and O2-dependent conversion of L-arginine to nitric oxide (NO) and citrulline; three isoforms, the neuronal (nNOS), endothelial, and inducible, have been identified. Because overproduction of NO is known to contribute to several pathophysiological conditions, NOS inhibitors are of interest as potential therapeutic agents. Inhibitors that are potent, mechanism-based, and relatively selective for the NOS isoform causing pathology are of particular interest. In the present studies we report that vinyl-L-NIO (N5-(1-imino-3-butenyl)-L-ornithine; L-VNIO) binds to and inhibits nNOS in competition with L-arginine (Ki = 100 nM); binding is accompanied by a type I optical difference spectrum consistent with binding near the heme cofactor without interaction as a sixth axial heme ligand. Such binding is fully reversible. However, in the presence of NADPH and O2, L-VNIO irreversibly inactivates nNOS (kinact = 0.078 min-1; KI = 90 nM); inactivation is Ca2+/calmodulin-dependent. The cytochrome c reduction activity of the enzyme is not affected by such treatment, but the L-arginine-independent NADPH oxidase activity of nNOS is lost in parallel with the overall activity. Spectral analyses establish that the nNOS heme cofactor is lost or modified by L-VNIO-mediated mechanism-based inactivation of the enzyme. The inducible isoform of NOS is not inactivated by L-VNIO, and the endothelial isoform requires 20-fold higher concentrations to attain approximately 75% of the rate of inactivation seen with nNOS. Among the NOS inactivating L-arginine derivatives, L-VNIO is the most potent and nNOS-selective reported to date.

Characterization of 5-oxo-L-prolinase in normal and tumor tissues of humans and rats: a potential new target for biochemical modulation of glutathione.

5-Oxo-L-prolinase (5-OPase) is an enzyme of the gamma-glutamyl cycle involved in the synthesis and metabolism of glutathione (GSH), which is known to protect cells from the cytotoxic effects of chemotherapy and radiation. Previous studies on rats have shown that administration of the cysteine prodrug L-2-oxothiazolidine-4-carboxylate, a 5-oxo-L-proline analogue that is metabolized by 5-OPase, preferentially increases the GSH content of normal tissues while paradoxically decreasing it in the tumor and results in an enhanced in vivo tumor response to the anticancer drug melphalan. These observations initiated the present study of 5-OPase in experimental models and clinical specimens to investigate the potential role of this enzyme in the selective modulation of GSH in normal and tumor tissues. First, 5-OPase activity was measured in tissues of tumor-bearing rats, in the peripheral mononuclear cells of normal human subjects, and in surgically resected tumor and the adjacent normal tissues from patients. We found that the activity of 5-OPase in human kidney, liver, and lung is significantly lower than that found in rats. Second, we have raised a polyclonal IgG anti-5-OPase antibody by immunizing rabbits with purified 5-OPase from rat kidney. This antibody has very high affinity (shown by immunoprecipitation) and specificity (shown by Western blot) and cross-reacts with human 5-OPase (shown by Western blot and immunohistochemistry). It was then used to examine the distribution of 5-OPase in paired normal and neoplastic human specimens using Western blot and immunohistochemistry. Examination of paired normal and neoplastic tissues of stomach and lung revealed a significantly lower level of 5-OPase in tumor tissues than in the paired normal tissues. In colon tissues, there is no significant difference in 5-OPase level between the normal and tumor tissues. These findings could have implications for both carcinogenesis and therapy.

Measurements of total plasma nitrite and nitrate in pediatric patients with the systemic inflammatory response syndrome.

OBJECTIVES: The systemic inflammatory response syndrome (SIRS) is typified by the presence of fever, hemodynamic changes, and end organ dysfunction. Endothelial cell activation leads to overproduction of nitric oxide, which results in sustained vasodilation and hypotension. This study was undertaken to determine the sensitivity, specificity, and positive and negative predictive values of plasma nitrite/nitrate measurements in identifying patients with clinical characteristics of SIRS, as defined by criteria based on physician diagnosis. DESIGN: Prospective cohort study with consecutive sampling of patients. SETTING: Tertiary, multidisciplinary, pediatric intensive care unit (ICU) at Children's Hospital of Wisconsin. PATIENTS: Patients were divided into five groups. There were 16 pediatric controls undergoing elective surgery and 177 pediatric ICU patients without and 46 pediatric ICU patients with physician-diagnosed sepsis, septic shock, SIRS, or sepsis syndrome documented in the medical record (all considered physician-diagnosed sepsis). The 223 pediatric ICU patients included 195 pediatric ICU patients not meeting and 28 pediatric ICU patients meeting predetermined physiologic criteria for SIRS (considered criteria-based sepsis). INTERVENTIONS: Blood samples were obtained for quantitative nitrite/nitrate analysis at the time of admission to the pediatric ICU and daily until discharge. MEASUREMENTS AND MAIN RESULTS: Mean plasma nitrite/nitrate concentrations in the controls were 34.5 +/- 12 microM (95th percentile 54 microM). In pediatric ICU patients without and with physician-diagnosed sepsis, mean plasma nitrite/nitrate concentrations were 39 +/- 24 microM (p > .05 compared with controls) and 127 +/- 91 microM (p < .0001 compared with both controls and patients without physician-diagnosed sepsis), respectively. In pediatric ICU patients without and with criteria-based sepsis, the mean total plasma nitrite/nitrate concentrations were 56 +/- 59 microM (p = .008 compared with controls) and 80 +/- 64 microM (p = .003 compared with patients without criteria-based sepsis), respectively. The ability of plasma nitrite/nitrate > 54 microM to identify patients with physician-diagnosed sepsis is characterized as follows: 87% sensitivity, 77% specificity, 50% positive predictive value, and 96% negative predictive value. The ability of plasma nitrite/nitrate > 54 microM to identify patients with criteria-based sepsis is characterized as follows: 61% sensitivity, 68% specificity, 21% positive predictive value, and 92% negative predictive value. CONCLUSIONS: Clinical diagnosis of SIRS is strongly associated with increased total plasma nitrite/nitrate concentrations in pediatric patients in the pediatric ICU. Many patients with increased nitrite/nitrate concentrations have inflammation without having a clinical diagnosis of SIRS. Our data suggest that increased plasma nitrite/nitrate concentrations are the standard for identifying patients with inflammation in the pediatric ICU.

Inhibition of endothelial cell amino acid transport System y+ by arginine analogs that inhibit nitric oxide synthase.

A variety of N omega-monosubstituted L-arginine analogs are established inhibitors of nitric oxide synthase; in all cases, initial binding is competitive with the substrate L-arginine. The efficacy of such compounds in vivo will depend on their transport into the relevant nitric oxide synthase-containing cells; in fact, inhibition may actually be augmented if cellular uptake of L-arginine is also blocked by the analogs. Because vascular endothelial cells synthesize vasoactive nitric oxide under both physiological and pathophysiological conditions, we have performed inhibition analyses with novel arginine analogs to determine the substrate specificity of the primary L-arginine transport system. Na(+)-independent System y+, present in porcine pulmonary artery endothelial cells. As reported by others, no Na(+)-independent System bo,+ activity was detectable. For System y+. Dixon plots suggest competitive inhibition and apparent Ki values, which ranged between 0.1 and 0.8 mM, estimated for each inhibitor. Some influence of amino acid side chain structure could be detected, but in general, the data establish that this transport system accepts a broad range of arginine derivatives. Loading the cells with individual arginine analogs resulted in trans-stimulation of arginine uptake suggesting that they serve as substrates of System y+ as well as inhibitors. These results indicate that plasma membrane transport is unlikely to be a limiting factor in drug development for nitric oxide synthase inhibitors.

L-amino acid oxidase (LOX) modulation of melphalan activity against intracranial glioma.

These studies evaluated the efficacy of sequential pretreatment with L-amino acid oxidase (LOX) and LOX antiserum in the modulation of melphalan activity against intracranial glioma in athymic nude mice. LOX produced statistically significant (P < 0.01) depletion of the large neutral amino acids isoleucine, leucine, methionine, phenylalanine, tyrosine, and valine in murine plasma at doses of 100 and 200 micrograms administered intravenously. Polyclonal anti-LOX antibody was successfully produced in mice, rabbits, and goats subsequent to immunization with LOX. Staphylococcal protein A-purified rabbit anti-LOX serum inhibited approximately 50% of LOX activity in vitro relative to control samples. This antiserum was used in vivo to inactivate LOX after it had depleted the large neutral amino acids, thereby preventing LOX-mediated catabolism of melphalan. Inoculation of three mice with rabbit anti-LOX serum after the treatment with LOX (100 micrograms) reduced LOX activity by 100%, 89%, and 100% at 6 h compared with reductions of 80%, 59%, and 52% over the same period in animals receiving LOX alone. In three separate studies using groups of eight to ten mice bearing intracranial human glioma xenografts, pretreatment with LOX followed by anti-LOX serum increased the antitumor activity of melphalan as compared with treatments with melphalan plus LOX, melphalan plus anti-LOX serum, or melphalan alone.

Characterization of the mechanisms of busulfan resistance in a human glioblastoma multiforme xenograft.

Busulfan is an alkylating agent commonly used in the treatment of chronic myelogenous leukemia and in combination with cyclophosphamide in preparation for allogeneic bone marrow transplantation. Serial treatment of a childhood high-grade glioma xenograft (D-456 MG) with busulfan resulted in a busulfan-resistant xenograft, D-456 MG(BR). Cross-resistance to 1,3-bis(2-chloroethyl)-1-nitrosourea was seen but not resistance to cyclophosphamide or CPT-11. Cytoplasmic levels of glutathione in D-456 MG(BR) were approximately one-half those found in D-456 MG. This depletion could not be explained by levels of glutathione-S-transferase, or by amplification, rearrangement, or increased levels of transcript of gamma-glutamylcysteine synthetase. Furthermore, depletion of glutathione in D-456 MG did not alter busulfan activity. Quantitation of busulfan levels in D-456 MG and D-456 MG(BR) xenografts following treatment of mice at the dose lethal to 10% of the animals demonstrated that significantly lower levels of drug were achieved in D-456 MG(BR). These studies suggest that alterations in drug transport or metabolism of busulfan may play a role in the resistance of D-456 MG(BR) to this alkylator.

Design of nitric oxide synthase inhibitors and their use to reverse hypotension associated with cancer immunotherapy.

It is now just 10 years since it was first appreciated that NO is endogenously synthesized in mammals. In this period, two constitutive and one inducible isoform of NOS have been isolated, sequenced, and characterized with respect to their protein chemistry and catalytic mechanism. A wide variety of NOS inhibitors, most targeted to the arginine binding site in the oxygenase domain, have been synthesized and used to elucidate the physiological and pathophysiological roles of NO. It is now clear that NO is involved in signal transduction (e.g., in neurotransmission and blood pressure homeostasis), and that these roles are mediated by low concentrations of NO synthesized by nNOS or eNOS. The NO receptor is the heme cofactor of soluble isoform of guanylyl cyclase. Higher amounts of NO, typically but not always synthesized by iNOS, are often cytotoxic. At a minimum, high concentrations of NO derange the signal transduction pathways normally served by nNOS or eNOS. In addition, NO or its nitrosative products (RSNO, N2O3, or ONOO-) inhibit or damage cellular constituents, interfering with DNA synthesis, energy metabolism, and the structural integrity of the cell. Such cytotoxicity can be beneficial to the host if pathogens or tumor cells are destroyed, but is detrimental to the host if it results in inappropriate inflammation, hypotension, or immunosuppression. Therapeutic utility of NOS inhibitors has been demonstrated in sepsis and cytokine-induced hypotension; additional applications are being identified in a treatment of inflammatory and autoimmune disorders.