A novel sodium-hydrogen exchanger isoform-1 inhibitor, zoniporide, reduces ischemic myocardial injury in vitro and in vivo.

The cardioprotective efficacy of zoniporide (CP-597,396), a novel, potent, and selective inhibitor of the sodium-hydrogen exchanger isoform 1 (NHE-1), was evaluated both in vitro and in vivo using rabbit models of myocardial ischemia-reperfusion injury. In these models, myocardial injury was elicited with 30 min of regional ischemia and 120 min of reperfusion. Zoniporide elicited a concentration-dependent reduction in infarct size (EC(50) of 0.25 nM) in the isolated heart (Langendorff) and reduced infarct size by 83% (50 nM). This compound was 2.5- to 20-fold more potent than either eniporide or cariporide (EC(50) of 0.69 and 5.11 nM, respectively), and reduced infarct size to a greater extent than eniporide (58% reduction in infarct size). In open-chest, anesthetized rabbits, zoniporide also elicited a dose-dependent reduction in infarct size (ED(50) of 0.45 mg/kg/h) and inhibited NHE-1-mediated platelet swelling (maximum inhibition 93%). Furthermore, zoniporide did not cause any in vivo hemodynamic (mean arterial pressure, heart rate, rate pressure product) changes. Zoniporide represents a novel class of potent NHE-1 inhibitors with potential utility for providing clinical cardioprotection.

Discovery of zoniporide: a potent and selective sodium-hydrogen exchanger type 1 (NHE-1) inhibitor with high aqueous solubility.

Zoniporide (CP-597,396) is a potent and selective inhibitor of NHE-1, which exhibits high aqueous solubility and acceptable pharmacokinetics for intravenous administration. The discovery, synthesis, activities, and rat and dog pharmacokinetics of this compound are presented. The potency and selectivity of zoniporide may be due to the conformation that the molecule adopts due to the presence of a cyclopropyl and a 5-quinolinyl substituent on the central pyrazole ring of the molecule.

Aldose reductase inhibition alone or combined with an adenosine A(3) agonist reduces ischemic myocardial injury.

This study investigated whether aldose reductase (AR) inhibition with zopolrestat, either alone or in combination with an adenosine A(3)-receptor agonist (CB-MECA), reduced myocardial ischemic injury in rabbit hearts subjected to 30 min of regional ischemia and 120 min of reperfusion. Zopolrestat reduced infarct size by up to 61%, both in vitro (2 nM to 1 microM; EC(50) = 24 nM) and in vivo (50 mg/kg). Zopolrestat reduced myocardial sorbitol concentration (index of AR activity) by >50% (control, 15.0 +/- 2.2 nmol/g; 200 nM zopolrestat, 6.7 +/- 1.3 nmol/g). A modestly cardioprotective concentration of CB-MECA (0.2 nM) allowed a 50-fold reduction in zopolrestat concentration while providing a similar reduction in infarct size (infarct area/area at risk: control, 62 +/- 2%; 1 microM zopolrestat, 24 +/- 5%; 20 nM zopolrestat plus 0.2 nM CB-MECA, 20 +/- 4%). In conclusion, AR inhibition is cardioprotective both in vitro and in vivo. Furthermore, combining zopolrestat with an A(3) agonist allows a reduction in the zopolrestat concentration while maintaining an equivalent degree of cardioprotection.

Relative importance of adenosine A1 and A3 receptors in mediating physiological or pharmacological protection from ischemic myocardial injury in the rabbit heart.

Although ischemic preconditioning (IP) in several species can be pharmacologically mimicked by selective adenosine A1 or A3 receptor agonists, it is currently unclear which receptor subtype (A1 and/or A3) is physiologically involved in mediating IP. To investigate this question, we determined (a) the affinity of adenosine for rabbit adenosine A1 and A3 receptors, and (b) the effects of selective rabbit A1 receptor blockade on IP and adenosine-mediated cardioprotection in a rabbit Langendorff model of myocardial ischemia-reperfusion injury. Adenosine was 19-fold selective for inhibition of N6-(4-amino-3-[125I]iodobenzyl)adenosine (125I-ABA) binding to recombinant rabbit A1 v rabbit A3 receptors (A1 Ki: 28 nm; A3 Ki 532 nm). Buffer-perfused rabbit hearts were exposed to 30 min regional ischemia and 120 min of reperfusion, and infarct size was measured by tetrazolium staining and normalized for area-at-risk (IA/AAR). Ischemic preconditioning (5 min global ischemia and 10 min reperfusion) or adenosine (20 micro M, 5 min) perfusion reduced infarct size (IA/AAR) to 17+/-3 and 14+/-2%, respectively (controls: 59+/-2%). Ischemic preconditioning and adenosine-mediated cardioprotection were completely blocked (57+/-2 and 61+/-4% IA/AAR, respectively) in the presence of a rabbit A1-selective concentration (50 nm) of the antagonist BWA1433 (rabbit A1 Ki: 3 nm; A3 Ki; 746 n m). Thus, whereas recent studies have demonstrated that selective A1 or A3 receptor agonists can both pharmacologically mimic IP, the results of the present study suggest that the adenosine-mediated component of IP in the isolated rabbit heart is preferentially mediated by adenosine A1 receptors, potentially due to adenosine's selectivity for this receptor subtype.

Selective activation of adenosine A3 receptors with N6-(3-chlorobenzyl)-5'-N-methylcarboxamidoadenosine (CB-MECA) provides cardioprotection via KATP channel activation.

OBJECTIVE: The aim of this study was to characterize the adenosine A3 receptor agonist, N6-(3-chlorobenzyl)-5'-N-methylcarboxamidoadenosine (CB-MECA), evaluate its ability to reduce myocardial ischemia/reperfusion injury and determine the role of KATP-channel activation in A3 receptor-mediated cardioprotection. METHODS: Binding affinities and adenylate cyclase inhibition were examined in CHO cells expressing rabbit recombinant adenosine A1 or A3 receptors. Infarct size (normalized for area-at-risk; % IA/AAR) was measured in buffer-perfused rabbit hearts exposed to 30-min regional ischemia and 120 min of reperfusion. RESULTS: CB-MECA was 100-fold selective for A3 vs. A1 receptors (A3 Ki: 1 nM; A1 Ki: 105 nM). Five-min perfusion with CB-MECA before ischemia/reperfusion elicited a concentration-dependent reduction in infarct size (EC50: 0.3 nM). The CB-MECA-dependent cardioprotection (control: 58 +/- 2; CB-MECA: 21 +/- 3% IA/AAR) was unchanged by an A1-selective concentration of the antagonist, BWA1433, but was completely prevented (P < 0.05) by a nonselective (A1/A3) concentration (55 +/- 6% IA/AAR). The KATP channel inhibitors, glibenclamide and 5-HD, had no effect on control infarct size, yet significantly (P < 0.05) blunted the CB-MECA-dependent cardioprotection (glibenclamide: 49 +/- 6; 5-HD: 58 +/- 4% IA/AAR). CONCLUSIONS: CB-MECA is a novel 100-fold A3 receptor-selective agonist which should prove useful for elucidating A3-dependent mechanisms in the rabbit heart. Selective stimulation of adenosine A3 receptors with CB-MECA reduces myocardial ischemia/reperfusion injury via a mechanism which involves activation of KATP channels.

Expression of multiple isoforms of nitric oxide synthase in normal and atherosclerotic vessels.

Atherosclerosis is associated with reduced endothelium-derived relaxing factor bioactivity. To determine whether this is due to decreased synthesis of nitric oxide synthase (NOS), we examined normal and atherosclerotic human vessels by in situ hybridization and immunocytochemistry by using probes specific for endothelial (ecNOS), inducible (iNOS), and neuronal (nNOS) NOS isoforms, ecNOS was detected in endothelial cells overlying normal human aortas, fatty streaks, and advanced atherosclerotic lesions. A comparison of the relative expression of ecNOS to von Willebrand factor on serial sections of normal and atherosclerotic vessels indicated that there was a decrease in the number of endothelial cells expressing ecNOS in advanced lesions. iNOS and nNOS were not detected in normal vessels, but widespread production of these isoforms was found in early and advanced lesions associated with macrophages, endothelial cells, and mesenchymal-appearing intimal cells. These data suggest that there is (1) a loss of ecNOS expression by endothelial cells over advanced atherosclerotic lesions and (2) a significant increase in overall NOS synthesis by other cell types in advanced lesions composed of the ecNOS, nNOS, and iNOS isoforms. We hypothesize that the increased expression of NOS and presumably NO in atherosclerotic plaques may be related to cell death and necrosis in these tissues.

Selective adenosine A3 receptor stimulation reduces ischemic myocardial injury in the rabbit heart.

OBJECTIVE: The aim of this study was to determine whether selective activation of the adenosine A3 receptor reduces infarct size in a Langendorff model of myocardial ischemia-reperfusion injury. METHODS: Buffer-perfused rabbit hearts were exposed to 30 min regional ischemia and 120 min of reperfusion. Infarct size was measured by tetrazolium staining and normalized for area-at-risk (IA/AAR). RESULTS: Preconditioning by 5 min global ischemia and 10 min reperfusion reduced infarct size (IA/AAR) to 19 +/- 4% (controls: 67 +/- 5%). Replacing global ischemia with 5 min perfusion of the rabbit A3-selective agonist, IB-MECA (A3 Ki: 2 nM; A1 Ki: 30 nM) elicited a concentration-dependent reduction in infarct size; 50 nM IB-MECA reduced IA/AAR to 24 +/- 4%. The A1-selective agonist, R-PIA (25 nM) reduced IA/AAR to a similar extent (21 +/- 6%). However, while the cardioprotective effect of R-PIA was significantly inhibited (54 +/- 7% IA/AAR) by the rabbit A1-selective antagonist, BWA1433 (50 nM), the IB-MECA-dependent cardioprotection was unaffected (28 +/- 6% IA/AAR). A non-selective (A1 vs. A3) concentration of BWA1433 (5 microM) significantly attenuated the IB-MECA-dependent cardioprotection (61 +/- 7% IA/AAR). CONCLUSIONS: These data clearly demonstrate that selective A3 receptor activation provides cardioprotection from ischemia-reperfusion injury in the rabbit heart. Furthermore, the degree of A3-dependent cardioprotection is similar to that provided by A1 receptor stimulation or ischemic preconditioning.

Relationships between NADPH diaphorase staining and neuronal, endothelial, and inducible nitric oxide synthase and cytochrome P450 reductase immunoreactivities in guinea-pig tissues.

The presence of NADPH diaphorase staining was compared with the immunohistochemical localization of four NADPH-dependent enzymes-neuronal (type I), inducible (type II), and endothelial (type III) nitric oxide synthase (NOS) and cytochrome P450 reductase. Cell types that were immunoreactive for the NADPH-dependent enzymes were also stained for NADPH diaphorase, suggesting that endothelial and neuronal NOS and cytochrome P450 reductase all show NADPH diaphorase activity in formaldehyde-fixed tissue. However, in some tissues, the presence of NADPH diaphorase staining did not coincide with the presence of any of the NADPH-dependent enzymes we examined. In vascular endothelial cells, the punctate pattern of staining observed with NADPH diaphorase histochemistry was identical to that seen following immunohistochemistry using antibodies to endothelial NOS. In enteric and pancreatic neurons and in skeletal muscle, the presence of NADPH diaphorase staining correlated with the presence of neuronal NOS. In the liver, sebaceous glands of the skin, ciliated epithelium, and a subpopulation of the cells in the subserosal glands of the trachea, zona glomerulosa of the adrenal cortex, and epithelial cells of the lacrimal and salivary glands, the presence of NADPH diaphorase staining coincided with the presence of cytochrome P450 reductase immunoreactivity. In epithelial cells of the renal tubules and zona fasciculata and zona reticularis of the adrenal cortex, NADPH diaphorase staining was observed that did not coincide with the presence of any of the enzymes. Inducible NOS was not observed in any tissue. Thus, while tissues that demonstrate immunoreactivity for neuronal and endothelial NOS also stain positively for NADPH diaphorase activity, the presence of NADPH diaphorase staining does not reliably or specifically indicate the presence of one or more NOS isoforms.

Cloning, expression and pharmacological characterization of rabbit adenosine A1 and A3 receptors.

The role of adenosine A1 and A3 receptors in mediating cardioprotection has been studied predominantly in rabbits, yet the pharmacological characteristics of rabbit adenosine A1 and A3 receptor subtypes are unknown. Thus, the rabbit adenosine A3 receptor was cloned and expressed, and its pharmacology was compared with that of cloned adenosine A1 receptors. Stable transfection of rabbit A1 or A3 cDNAs in Chinese hamster ovary-K1 cells resulted in high levels of expression of each of the receptors, as demonstrated by high-affinity binding of the A1/A3 adenosine receptor agonist N6-(4-amino-3-[125I]iodobenzyl)adenosine (125I-ABA). For both receptors, binding of 125I-ABA was inhibited by the GTP analog 5'-guanylimidodiphosphate, and forskolin-stimulated cyclic AMP accumulation was inhibited by the adenosine receptor agonist (R)-phenylisopropyladenosine. The rank orders of potency of adenosine receptor agonists for inhibition of 125I-ABA binding were as follows: rabbit A1, N6-cyclopentyladenosine = (R)-phenylisopropyladenosine > N-ethylcarboxamidoadenosine > or = I-ABA > or = N6-2-(4-aminophenyl) ethyladenosine > > N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide > N6-(4-amino-3-benzyl)adenosine; rabbit A3, N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide > or = I-ABA > > N-ethylcarboxamidoadenosine > N6-2-(4-aminophenyl) ethyladenosine = N6-cyclopentyladenosine = (R)-phenylisopropyladenosine > N6-(4-amino-3-benzyl)adenosine. The adenosine receptor antagonist rank orders were as follow: rabbit A1, 8-cyclopentyl-1,3-dipropylxanthine > 1,3- dipropyl-8-(4-acrylate)phenylxanthine > or = xanthine amine congener > > 8-(p-sulfophenyl)theophylline; rabbit A3, xanthine amine congener > 1,3-dipropyl-8-(4-acrylate)phenylxanthine > or = 8-cyclopentyl-1,3-dipropylxanthine > > 8-(p-sulfophenyl)theophylline. These observations confirm the identity of the expressed proteins as A1 and A3 receptors. The results will facilitate further in-depth studies of the roles of A1 and A3 receptors in adenosine-mediated cardioprotection in rabbits, which can now be based on the appropriate recombinant rabbit A1 and A3 receptor pharmacology.

Evidence for a role for both the adenosine A1 and A3 receptors in protection of isolated human atrial muscle against simulated ischaemia.

OBJECTIVE: Adenosine receptor activation has been implicated in the mechanism of ischaemic preconditioning protection. Evidence suggests adenosine A1 receptor involvement, and possibly A3 receptor involvement in the rabbit. This study investigated the roles of these receptors in human preconditioning. Human A1- and A3-selective compounds were chosen based on Ki values for inhibition of N6-(4-amino-3-[125I]iodobenzyl)adenosine (125I-ABA) binding to stably expressed recombinant human A1 and A3 receptors. Cyclopentyladenosine (CPA), a 194-fold selective A1 agonist, and iodobenzylmethylcarboxamidoadenosine (IBMECA), a 10-fold selective A3 agonist were used alone and in combination with dipropylcyclopentylxanthine (DPCPX) a 62-fold selective A1 antagonist. METHODS: Human atrial trabeculae were superfused with oxygenated Tyrode's solution. After stabilisation, muscles underwent one of 8 protocols (n = 6 per group), followed by 90 min of simulated ischaemia and 120 min of reoxygenation. The experimental endpoint was recovery of contractile function, presented as percentage baseline function. RESULTS: 5 nM CPA (52.2 +/- 3.1%), 30 nM IBMECA (49.7 +/- 3.8%) and preconditioning (55.3 +/- 2.5%) produced similar functional recoveries at 120 min of reoxygenation; significantly different to controls (27.7 +/- 1.0%; P < 0.05, ANOVA). When DPCPX (200 nM) was added prior to 5 nM CPA, protection was lost (31.8 +/- 0.9%), but when added prior to 30 nM IBMECA, muscles continued to be significantly protected (41.5 +/- 2.3%). CONCLUSIONS: In human atrium both A1 and A3 receptor stimulation appears to mimic ischaemic preconditioning. This may represent the first evidence for A3 receptor involvement in 'pharmacological' preconditioning of human myocardium.

Expression of inducible nitric oxide synthase in failing and non-failing human heart.

Recently, a significant activity of inducible nitric oxide synthase (iNOS) has been reported in biopsies from failing hearts due to idiopathic dilated cardiomyopathy (IDC). Thus, a potential pathophysiological role of iNOS in IDC has been stated. In order to investigate, whether iNOS expression is of pathophysiological relevance in human heart failure, we measured iNOS protein expression and cGMP content in left ventricular myocardium from non-failing and failing human hearts. Immunoblot analysis revealed iNOS protein expression in four out of six failing hearts from septic patients, whereas no iNOS-protein expression was detected in either non-failing human hearts (n = 6) or failing hearts due to IDC (n = 9), ischemic heart disease (IHD, n = 7), Becker muscular dystrophy (BMD, n = 2) and mitoxantrone-induced toxic cardiomyopathy TCM, n = 1). cGMP content was increased by 130% in septic hearts, whereas there was no cGMP increase in hearts with IDC. IHD and BMD compared to non-failing hearts. We conclude, that the induction of iNOS may play a role in contractile dysfunction observed in septic shock, but is unlikely to be of major pathophysiological importance in end-stage heart failure due to IDC, IHD, BMD and TCM.

Nitric oxide synthase isozymes antibodies.

Three isozymes of nitric oxide synthase (NOS) have been identified, cDNAs isolated and sequenced, and antibodies produced against each isozyme. Isozyme I (found primarily in central and peripheral neuronal cells), II (in cytokine-induced cells), and III (in endothelial cells) show less than 58% identity in the deduced amino acid sequences from humans. Many investigators have produced isozyme-specific antibodies and used these antibodies to locate these proteins in various cells and tissues. NOS-I is constitutively expressed, and the enzymatic activity is regulated by Ca2+ and calmodulin. The anti-NOS-I antibodies have allowed investigators to characterize non-adrenergic non-cholinergic neurons as nitrergic neurons, revealed NOS-I immunoreactivity in neurons and macula densa cells of the kidney and pancreatic islet cells, human skeletal muscle, and to demonstrate that various structures within the brain and spinal cord contain NOS-I. NOS-II is not regulated by Ca2+ and has been implicated in the pathophysiology of sepsis and autoimmune diseases. The anti-NOS-II antibodies have localized this isoform to infiltrating macrophages in pancreatic islets of diabetic rats, infiltrating macrophages and myocytes of a transplant heart model in rats, various cell types in bacterially and endotoxin-treated rats, alveolar macrophages in areas of inflammation in humans, and vascular smooth muscle cells of human atherosclerotic aneurysm. Isoform III is similar to NOS-I in that it is constitutively expressed and regulated by Ca2+ and calmodulin. Anti-NOS-III antibodies have found that this isoform is relatively specific for endothelial cells.

The nitric oxide synthase inhibitor, L-NG-monomethylarginine, reduces carrageenan-induced pleurisy in the rat.

Nitric oxide (NO) is a biological mediator that, when produced by the type II (inducible) nitric oxide synthase (NOS), has been implicated in the pathophysiology of inflammatory diseases. To examine this putative role of NO, pleural inflammation was elicited in rats by the intrapleural injection of carrageenan (1 mg). A pleural exudate and cellular influx developed, which peaked at 24 h and generally resolved by 72 h. The cellular influx was primarily composed of polymorphonuclear cells during the first 24 h, followed by macrophages during the subsequent 24 h. Inflammatory cell-associated NOS activity and pleural exudate nitrite (NO2-) + nitrate (NO3-) (NOx) also increased, peaking at 6 h and 24 h, respectively. Cell-associated NOS activity was calcium-independent, indicating the presence of the type II NOS isoform; NOS activity in the pleural cavity and polymorphonuclear cells influx were temporally correlated. Administration of L-NG-monomethylarginine (L-NMA) (200 mg/kg/day) attenuated the pleural exudation, cellular influx, pleural exudate NOx, and cell-associated NOS activity. The relative composition of the pleural cavity cellular infiltrate was not changed by L-NMA, indicating the influx of individual cell types were affected equally. L-Arginine (500 mg/kg/day) completely prevented the effects of L-NMA on pleural exudation and cellular influx and partially prevented the inhibition of pleural exudate NOx accumulation by L-NMA. These data implicate NO as a modulator of the pleural inflammatory response and support a future clinical role for NOS inhibitors in the treatment of inflammatory disease.

In vivo pharmacological evaluation of two novel type II (inducible) nitric oxide synthase inhibitors.

Selective type II (inducible) nitric oxide synthase (NOS) inhibitors have several potential therapeutic applications, including treatment of sepsis, diabetes, and autoimmune diseases. The ability of two novel, selective inhibitors of type II NOS, S-ethylisothiourea (EIT) and 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT), to inhibit type II NOS function in vivo was studied in lipopolysaccharide (LPS) treated rats. Type II NOS activity was assessed by measuring changes in plasma nitrite and nitrate concentrations ([NOx]). Both EIT and AMT elicited a dose-dependent and > 95% inhibition of the LPS-induced increase in plasma [NOx]. The ED50 values for EIT and AMT were 0.4 and 0.2 mg/kg, respectively. In addition, the administration of LPS and either NOS inhibitor resulted in a dose-dependent increase in animal mortality; neither compound was lethal when administered alone. Pretreatment with L-arginine (but not D-arginine) prevented the mortality, while not affecting the type II NOS-dependent NO production, suggesting the toxicity may be due to inhibition of one of the other NOS isoforms (endothelial or neuronal). Thus, although EIT and AMT are potent inhibitors of type II NOS function in vivo, type II NOS inhibitors of even greater selectivity may need to be developed for therapeutic applications.

Lipopolysaccharide-induced changes in plasma nitrite and nitrate concentrations in rats and mice: pharmacological evaluation of nitric oxide synthase inhibitors.

The benefits of nitric oxide synthase (NOS) inhibitors in the treatment of endotoxemia or sepsis presumably arise from inhibition of the type II (inducible) NOS. However, inasmuch as the effect of these inhibitors on NOS function in vivo is rarely assessed, NOS activity was evaluated in rats and mice by measuring changes in plasma nitrite and nitrate concentrations ([NOx]) after administration of lipopolysaccharide (LPS). In both species, [NOx] peaked at 20 hr, returning to base line by 48 to 72 hr. The ED50 values (dose that elicited a 50% inhibition of the LPS-dependent increase in [NOx] 6 hr after LPS administration) for L-NG-monomethylarginine acetate, L-NG-nitroarginine methyl ester and aminoguanidine (administered 3 hr after LPS) were 34, 21 and 19 mg/kg in the rat and 32, 5 and 4 mg/kg in the mouse. These compounds also decreased the survival of LPS-challenged animals, which in the case of L-NG-nitroarginine methyl ester was reversed by L-arginine. Dexamethasone (which prevents the induction of type II NOS) also inhibited the LPS-dependent increase in [NOx] with ED50 values of 0.05 mg/kg (rat) and 1 mg/kg (mouse), but did not lead to decreased survival. Thus, inhibition of the type I (neuronal) or type III (endothelial) NOS, rather than the type II isoform, may be a possible mechanism for the animal mortality. These models provide a simple and reproducible means for assessing the in vivo inhibition of type II NOS by various compounds.

Nitric oxide synthase activity is elevated in brain microvessels in Alzheimer's disease.

The cerebral microcirculation undergoes specific biochemical changes in Alzheimer's disease. In this study, we have compared the nitric oxide synthase (NOS) activity of brain microvessels isolated from Alzheimer and control brains. L-[3H]-citrulline, the stable co-product generated with nitric oxide (NO) from L-[3H]-arginine, was measured as an indicator of NOS activity. The results indicated a significant increase in NOS activity in microvessels isolated from Alzheimer brains. In addition, using antibodies to both the endothelial and inducible NOS isoforms, we demonstrated a significant increase in enzyme level in Alzheimer-derived vessels. Elevated vascular production of NO, a potentially neurotoxic mediator in the CNS, may contribute to the susceptibility of neurons to injury and cell death in Alzheimer's disease.

Immunochemical detection of inducible NO synthase in human lung.

Type II (inducible) nitric oxide synthase (NOS) may play an important role in pulmonary pathophysiology, yet it remains controversial whether human tissues are capable of expressing this protein. Therefore, a polyclonal antibody (8196) was raised against type II NOS from induced RAW 264.7 macrophages and used to investigate the expression of this enzyme in human lung tissue. Anti-type II NOS antibody did not cross-react with either neuronal (type I) or endothelial (type III) constitutive NOS, whereas a 130-kDa protein was detected in cytosol from induced macrophages or liver removed from lipopolysaccharide (25 mg/kg)-treated rats. Cells or tissues that lacked NOS activity did not express immunoreactive proteins. Similarly, in grossly normal human lung tissue, no immunoreactivity was detected with the anti-type II NOS antibody. In contrast, strong immunoreactivity was detected in alveolar macrophages present in lung tissue from a patient with bronchiectasis and acute bronchopneumonia. These data demonstrate that human alveolar macrophages are able to express type II NOS and support a role for this enzyme in pulmonary inflammatory pathophysiology.

Identification of an endothelial-like type III NO synthase in LLC-PK1 kidney epithelial cells.

Porcine kidney tubular epithelial cells (LLC-PK1) produce nitric oxide or a related compound (e.g., a nitrosothiol) after stimulation with various agonists. We now report the identification and characterization of a constitutive, particulate nitric oxide (NO) synthase from LLC-PK1 cells. After partial purification on adenosine 2',5'-bisphosphate-Sepharose, the particulate NO synthase activity eluted anomalously from Superose 6 gel permeation columns near the total included volume, similar to that observed for the endothelial (type III) NO synthase. Substrate/cofactor requirements of the epithelial and endothelial NO synthases were identical, i.e., dependency on L-arginine, (6R)-5,6,7,8-tetrahydrobiopterin, FAD, calcium and calmodulin. The epithelial enzyme activity was inhibited by the arginine analogues, NG-methyl-L-arginine (100 microM) and NG-nitro-L-arginine (100 microM), as well as the calmodulin antagonists, trifluoperazine (100 microM) and calmidazolium (30 microM). Anti-type III (H32), but not anti-type I (brain, 6763-5) or anti-type II (macrophage, 8196) NO synthase antibodies, detected a single immunoreactive band in the LLC-PK1 particulate fraction of approximately 140 kDa by Western blot analysis. Finally, the presence of type III NO synthase mRNA in LLC-PK1 cells was demonstrated using the polymerase chain reaction. These data indicate that LLC-PK1 kidney epithelial cells contain type III NO synthase, which has been classically associated with the vascular endothelium.

Isoforms of nitric oxide synthase: functions in the cardiovascular system.

Various cell types, including endothelial cells, can synthesize nitric oxide (NO). Three different isoforms of NO synthase have been characterized, purified and cloned. Isozyme I is present in neuronal cells of the brain (where NO may mediate synaptic plasticity), in peripheral non-adrenergic non-cholinergic (NANC) neurons (where NO acts as an atypical neurotransmitter relaxing vascular and non-vascular smooth muscle), and in various specialized epithelial cells. Macrophages can be induced with bacterial endotoxin and/or cytokines to express isozyme II. The high concentrations of NO produced by this isoform have cytostatic effects on parasitic microorganisms and tumour cells. A similar isozyme can be induced in the vascular wall (presumably in smooth muscle cells) in sepsis and during cytokine therapy. The large amounts of NO produced by this enzyme contribute to the symptoms of septic shock, such as vasodilatation and microvascular endothelial damage. Endothelial cells contain isoform III of NO synthase which seems to be unique for this cell type. Endothelium-derived NO is a physiologically significant vasodilator and inhibitor of platelet aggregation and adhesion. In addition, vascular NO can prevent leukocyte adhesion to the endothelium by interfering with the adhesion molecule CD11/CD18, and NO has also been shown to inhibit the proliferation of vascular smooth muscle cells. Hence, NO represents a protective factor against vascular damage and probably atherogenesis.