Phase I evaluation of intravenous recombinant human interleukin 12 in patients with advanced malignancies

Guanidines, amidines, S-alkylisothioureas, and other compounds containing the amidine function (-C(=NH)NH2) have been described as inhibitors of the generation of nitric oxide (NO) by NO synthase (NOS). Here we report on the inhibition of the activity of NOS isoforms by compounds in which the amidine function is attached to a nitrogen of 1,2-diazo heterocycles to form N-carboxamidines and related compounds. 1H-Pyrazole-1-carboxamidine HCl (PCA) inhibited the activity of purified inducible NOS (iNOS), endothelial NOS (eNOS), and neuronal NOS (nNOS) isoforms to a similar extent (IC50 = 0.2 microM). 3-Methyl-PCA and 4-methyl-PCA showed reduced potencies, but a preference for iNOS [IC50 = 5 and 2.4 microM, respectively; cf. N(G)-methyl-L-arginine (NMA) IC50 = 6 microM]. Inhibition of purified iNOS by PCAs could be reversed completely by excess L-arginine, while their inhibition of NO production by stimulated RAW macrophages could be reversed by transfer to a drug-free medium. This suggests a competitive mode of inhibition. PCA caused potent concentration-dependent inhibition of the acetylcholine-induced, endothelium-dependent relaxations of precontracted rat thoracic aorta (IC50 = 30 microM). 4-Methyl-PCA inhibited the relaxations only at > or = 300 microM. In contrast, 4-methyl-PCA was more effective than both PCA and NMA in restoring the ex vivo contractility of aortic rings taken from lipopolysaccharide-treated rats. PCA and NMA, but not 4-methyl-PCA, caused marked increases in mean arterial pressure when administered i.v. to anesthetized rats. In conclusion, PCA and related compounds caused potent inhibition of NOS. Substitution of the pyrazole ring reduced potency, but improved selectivity towards iNOS as exemplified by 4-methyl-PCA.     

The structure of HLA-DM, the peptide exchange catalyst that loads antigen onto class II MHC molecules during antigen presentation

Endothelial cells produce C-type natriuretic peptide (CNP), which has been proposed as an endothelium-derived hyperpolarizing factor. In porcine coronary arteries, we investigated the vasodilatory effects of CNP and compared them with endothelium-dependent relaxations and hyperpolarizations to bradykinin. Isolated epicardial porcine coronary arteries were studied in organ chambers, and concentration-response curves to CNP and bradykinin were obtained. Membrane potential was measured in endothelial cells and smooth muscle of intact porcine coronary arteries during stimulation with CNP or bradykinin. In precontracted porcine coronary arteries with or without endothelium, CNP (10[-10]-10[-6] M) evoked relaxations (maximum, 42 +/- 4%) smaller than those evoked by bradykinin (100 +/- 1%), blunted in preparations contracted by KCl instead of U46619 (9,11-dideoxy-11a,9a-epoxymethano-prostaglandin F2alpha; p < 0.05) and unaffected by inhibition of NO synthase (NS). CNP evoked hyperpolarization of vascular smooth muscle of similar magnitude in endothelium-intact (-4.4 +/- 1 mV) and endothelium-denuded (-4.6 +/- 1 mV) porcine coronary arteries. Bradykinin (10[-10]-10[-6] M) evoked concentration-dependent relaxations in preparations with endothelium only. Although atrial natriuretic peptide-receptor antagonist HS-142-1 (25 microM) slightly reduced the sensitivity to bradykinin (log shift at IC50, twofold; p < 0.05), it had no effect on the maximal response to bradykinin. Inhibition of NO synthase partially attenuated, whereas high potassium chloride (30 mM) markedly inhibited relaxations to bradykinin (p < 0.05). Hyperpolarization to bradykinin was much more pronounced than that to CNP (-17 +/- 3 mV; p < 0.05 vs. CNP) and was observed in endothelium-intact preparations only and unaffected by HS-142-1. In conclusion, in contrast to bradykinin, CNP induces endothelium-independent and weaker relaxation and hyperpolarization of coronary artery vascular smooth muscle, suggesting that CNP is an unlikely mediator of endothelium-dependent hyperpolarization of porcine coronary arteries.     

Functional and morphological damage of endothelium in rabbit ear artery following irradiation with cobalt60

1. The relaxant actions of acetylcholine and A23187 were examined in the rabbit central ear artery at different intervals following exposure to different doses of radiation with a cobalt60 unit. The artery was irradiated with a dose of 10 Gy, 20 Gy and 45 Gy. Radiation caused dose- and time-dependent impairment of the endothelium-dependent relaxations. The impaired endothelium-dependent relaxations occurred as early as 1 week postirradiation and persisted throughout the experimental period (10 weeks). 2. The endothelium-independent response to sodium nitroprusside was well preserved up to 6 weeks after irradiation. The contractile response to noradrenaline was unaltered by irradiation throughout the experimental period, but in contrast to control vessels, an increase in the sensitivity to noradrenaline in the presence of the nitric oxide synthase (NOS) inhibitor N(G)-nitro-L-arginine was not observed in the irradiated vessels. 3. The impaired endothelium-dependent relaxations in the irradiated vessels were not improved by pretreatment with the NOS substrate L-arginine, the cyclo-oxygenase inhibitor indomethacin or the free radical scavengers superoxide dismutase and catalase. 4. Scanning electron microscopy indicated morphologically intact endothelial cells within the first 4 weeks after irradiation. 5. Western blot analysis showed a significant decrease in the expression of endothelial NOS (eNOS) in the irradiated vessels. 6. These data indicate that endothelial cell function is specifically impaired in the irradiated vessels before morphological endothelial cell damage can be detected. This impairment may be related to diminished eNOS expression.     

Role of nitric oxide in rat nephrotoxic nephritis: comparison between inducible and constitutive nitric oxide synthase

Nitric oxide (NO), generated by inducible NO synthase (iNOS) in migrating macrophages, is increased in glomerulonephritis. This study investigates the effect of NO inhibition on rat nephrotoxic nephritis (NTN) to clarify the role of NO production in glomerular damage. NTN was induced in Sprague Dawley rats by an injection of an anti-glomerular basement membrane (GBM) antibody. Urinary nitrite excretion and nitrite release from kidney slices (5.47 +/- 1.19 versus 2.15 +/- 0.73 nmol/mg protein, NTN versus Control, P < 0.05) were increased in NTN on day 2. Glomerular macrophage infiltration and intercellular adhesion molecule (ICAM)-1 expression increased from day 2. iNOS expression was increased in interstitial macrophages. Glomerular endothelial cell NOS (ecNOS) expression evaluated by counting immunogold particles along GBM was suppressed (0.06 +/- 0.02 versus 0.35 +/- 0.04 gold/micron GBM, P < 0.0001). Glomerular damage developed progressively. NG-nitro-L-arginine methyl ester (L-NAME), which inhibits both iNOS and ecNOS and aminoguanidine (AG), a relatively selective inhibitor for iNOS, equally suppressed nitrite in urine and renal tissue. Glomerular ICAM-1 expression and macrophage infiltration were reduced by L-NAME, but not by AG. Expression of ecNOS was significantly increased by L-NAME (0.91 +/- 0.08, P < 0.0001 versus NTN), but slightly by AG (0.18 +/- 0.04). AG significantly and L-NAME slightly attenuated the glomerular damage at day 4. In conclusion, suppression of iNOS prevents glomerular damage in the early stage of NTN. Treatment by L-NAME reduces macrophage infiltration by suppression of ICAM-1 expression, which may be explained by an increase in ecNOS expression.     

Expression of nitric oxide synthase isoforms in bone and bone cell cultures

Recent work has shown that nitric oxide (NO) acts as an important mediator of the effects of proinflammatory cytokines and mechanical strain in bone. Although several bone-derived cells have been shown to produce NO in vitro, less is known about the isoforms of NO synthase (NOS), which are expressed in bone or their cellular distribution. Here we investigated the expression, cellular localization, and regulation of NOS mRNA and protein in cultured bone-derived cells and in bone tissue sections. We failed to detect inducible NOS (iNOS) protein in normal bone using immunohistochemical techniques, even though low levels of iNOS mRNA were detected by sensitive reverse transcribed polymerase chain reaction (RT-PCR) assays in RNA extracted from whole bone samples. Cytokine stimulation of bone-derived cells and bone explant cultures caused dramatic induction of iNOS mRNA and protein in osteoblasts and bone marrow macrophages, but no evidence of iNOS expression was seen in osteoclasts by immunohistochemistry or in situ hybridization. Endothelial NOS (ecNOS) mRNA was also detected by RT-PCR in whole bone, and immunohistochemical studies showed widespread ecNOS expression in bone marrow cells and trabecular lining cells in vivo. Related studies in vitro confirmed that ecNOS was expressed in cultured osteoblasts, stromal cells, and osteoclasts. Neuronal NOS mRNA was detected by RT-PCR in whole bone, but we were unable to detect nNOS protein in bone cells in vivo or in studies of cultured bone-derived cells in vitro. In summary, our data show that mRNAs for all three NOS isoforms are expressed in bone and provide evidence for differential expression and regulation of the enzymes in different cell types. These findings confirm the likely importance of the L-arginine-NO pathway as a physiological mediator of bone cell function and demonstrate that it may be possible to exert differential effects on osteoblast and osteoclast activity in vivo by differential targeting of constitutive and inducible NOS isoforms by selective NOS inhibitors.     

Cardiac manifestations of polymyositis. Apropos of 2 Senegalese cases]

The relaxations mediated by the activation of 5-HT receptors in the guinea pig proximal colon were investigated. Longitudinal strips were cut from the colon segment and placed into the bath. In the presence of atropine (0.2 microM), the relaxations were evoked by adding increasing concentrations of 5-HT (1-100 microM). Noncumulative concentration-response curves were established in the absence and presence of either 5-HT or nitric oxide synthase (NOS) antagonists. Selective 5-HT3 antagonists tropisetron (10 and 100 nM) and ondansetron (1 microM) inhibited the relaxations and shifted the concentration-response curves to the right. Similar effects were observed in the presence of the NOS inhibitor N(G)-nitro-L-arginine (3.2, 10, 32 microM) and partly reversed with L-arginine (100, 320 microM). N(G)-nitro-D-arginine, serving as a negative control, was ineffective. The relaxations were further inhibited in the presence of the soluble guanylate cyclase blocker methylene blue (10 microM) or NO scavenger hemoglobin (32 microM). These results suggest that the 5-HT3 receptor plays a role in neurogenic relaxations of guinea pig proximal colon, which are at least partly mediated via release of NO from nerve endings.     

2-Amino-4-methylpyridine as a potent inhibitor of inducible NO synthase activity in vitro and in vivo

1. The ability of 2-amino-4-methylpyridine to inhibit the catalytic activity of the inducible NO synthase (NOS II) enzyme was characterized in vitro and in vivo. 2. In vitro, 2-amino-4-methylpyridine inhibited NOS II activity derived from mouse RAW 264.7 cells with an IC50 of 6 nM. Enzyme kinetic studies indicated that inhibition is competitive with respect to arginine. 2-Amino-4-methylpyridine was less potent on human recombinant NOS II (IC50 = 40 nM) and was still less potent on human recombinant NOS I and NOS III (IC50 = 100 nM). NG-monomethyl-L-arginine (L-NMMA), N6-iminoethyl-L-lysine (L-NIL) and aminoguanidine were much weaker inhibitors of murine NOS II than 2-amino-4-methylpyridine but, unlike 2-amino-4-methylpyridine, retained similar activity on human recombinant NOS II. L-NMMA inhibited all three NOS isoforms with similar potency (IC50S 3-7 microM). In contrast, compared to activity on human recombinant NOS III, L-NIL displayed 10 x selectivity for murine NOS II and 11 x selectivity for human recombinant NOS II while aminoguanidine displayed 7.3 x selectivity for murine NOS II and 3.7 x selectivity for human recombinant NOS II. 3. Mouse RAW 264.7 macrophages produced high levels of nitrite when cultured overnight in the presence of lipopolysaccharide (LPS) and interferon-gamma. Addition of 2-amino-4-methylpyridine at the same time as the LPS and IFN-gamma, dose-dependently reduced the levels of nitrite (IC50 = 1.5 microM) without affecting the induction of NOS II protein. Increasing the extracellular concentration of arginine decreased the potency of 2-amino-4-methylpyridine but at concentrations up to 10 microM, 2-amino-4-methylpyridine did not inhibit the uptake of [3H]-arginine into the cell. Addition of 2-amino-4-methylpyridine after the enzyme was induced also dose-dependently inhibited nitrite production. Together, these data suggest that 2-amino-4-methylpyridine reduces cellular production of NO by competitive inhibition of the catalytic activity of NOS II, in agreement with results obtained from in vitro enzyme kinetic studies. 4. When infused i.v. in conscious unrestrained rats, 2-amino-4-methylpyridine inhibited the rise in plasma nitrate produced in response to intraperitoneal injection of LPS (ID50 = 0.009 mg kg-1 min-1). Larger doses of 2-amino-4-methylpyridine were required to raise mean arterial pressure in untreated conscious rats (ED50 = 0.060 mg kg-1 min-1) indicating 6.9 x selectivity for NOS II over NOS III in vivo. Under the same conditions, L-NMMA was nonselective while L-NIL and aminoguanidine displayed 5.2 x and 8.6 x selectivity respectively. All of these compounds caused significant increases in mean arterial pressure at doses above the ID50 for inhibition of NOS II activity in vivo. 5. 2-Amino-4-methylpyridine also inhibited LPS-induced elevation in plasma nitrate after either subcutaneous (ID50 = 0.3 mg kg-1) or oral (ID50 = 20.8 mg kg-1) administration. 6. These data indicate that 2-amino-4-methylpyridine is a potent inhibitor of NOS II activity in vitro and in vivo with a similar degree of isozyme selectivity to that of L-NIL and aminoguanidine in rodents.     

Objective evaluation of Papanicolaou staining

Altered endothelium-dependent vasodilation has been observed in congestive heart failure (CHF), a disease characterized by a sustained adrenergic activation. The purpose of our study was to test the hypothesis that chronically elevated catecholamines influence the nitric oxide (NO) pathway in the human endothelium. Human umbilical vein endothelial cells (HUVEC) were exposed for 7 days to a concentration of noradrenaline (NA, 1 ng/mL) similar to that found in the blood of patients with CHF. Kinetics of endothelial constitutive NO synthase (ecNOS) and inducible NO synthase (iNOS) activity, measured by [3H]L-arginine to [3H]L-citrulline conversion, and protein expression of ecNOS and iNOS, assessed by Western blot analysis, were unaffected by chronic NA treatment. Furthermore, no changes in subcellular fraction-associated ecNOS were found; this indirectly shows that chronic NA did not cause phosphorylation of the enzyme. Moreover, [3H]L-arginine transport through the plasma membrane was conserved in chronically NA-treated cells. The data demonstrate that prolonged in vitro exposure to pathologic CHF-like NA does not affect the L-arginine: NO pathway in human endothelial cells.     

Flufenamic and tolfenamic acids and lemakalim relax guinea-pig isolated trachea by different mechanisms

The effects of K+ channel inhibitors on the relaxations induced by flufenamic and tolfenamic acids and lemakalim were examined in guinea-pig isolated trachea precontracted with prostaglandin F2alpha (PGF2alpha, 1 microM). Flufenamic and tolfenamic acids (0.1-33 microM) and lemakalim (0.01-33 microM) relaxed guinea-pig trachea in a concentration-dependent manner. Tetraethylammonium (0.5-2 mM), a nonspecific inhibitor of K+ channels, inhibited the relaxations induced by flufenamic and tolfenamic acids without affecting lemakalim-induced relaxation. Charybdotoxin (ChTX, 33-100 nM), an inhibitor of the large Ca2+-activated K+ channels (BK(Ca)), also inhibited the relaxations induced by flufenamic and tolfenamic acids without affecting lemakalim-induced relaxation. Glipizide (3.3-33 microM), an inhibitor of the ATP-sensitive K+ channels (K(ATP)) inhibited lemakalim-induced relaxation without affecting those induced by flufenamic and tolfenamic acids. Our results indicate that the relaxations of guinea-pig isolated trachea by flufenamic and tolfenamic acids are due to activation of BK(Ca). The relaxant mechanism of flufenamic and tolfenamic acids thus differs from that of lemakalim, an activator of K(ATP).     

Lovastatin maintains nitric oxide--but not EDHF-mediated endothelium-dependent relaxation in the hypercholesterolemic rabbit carotid artery

The endothelium contributes to the regulation of vascular tone by producing nitric oxide (NO) and the endothelium-derived hyperpolarising factor (EDHF). In hypercholesterolemia, endothelium-dependent relaxation is impaired but can be restored by treatment with lovastatin (LOVAS). We investigated the effects of LOVAS on NO and EDHF-mediated relaxation. Rabbits were fed 1% cholesterol diet for 4 weeks and 0.5%) cholesterol for the following 12 weeks (CHOL-group). The LOVAS group additionally received 10 mg of lovastatin over the last 12-week period. Experiments were performed in carotid artery rings. Relaxant responses to acetylcholine (ACh) were recorded in the presence of indomethacin. Nitro-L-arginine (NOARG, 100 microM) and potassium chloride (KCl, 35 mM) were used to differentiate between NO- and EDHF-mediated relaxations. Cholesterol impaired ACh-induced relaxations and this effect was prevented by LOVAS (control 100+/-1%, CHOL 81+/-6%, LOVAS 98+/-1%). In the presence of NOARG, relaxations to ACh were not different between the LOVAS and CHOL groups (control 78+/-4%, CHOL 64+/-6%, LOVAS 64+/-5%). When KCl was used, ACh-induced relaxations were similar in the LOVAS and control group (control 75+/-5%, CHOL 49+/-6%, LOVAS 76+/-2%). In arteries treated with NOARG and KCl together, no relaxations were observed. Relaxations of arteries from the control group were not affected by 18 h preincubation with lovastatin (10 microM). Lovastatin selectively maintains nitric oxide-mediated endothelium-dependent relaxation in hypercholesterolemic rabbit carotid arteries.     

Inhibition of alpha1-adrenoceptor-mediated contractions in isolated tail arteries and aorta of the rat by alpha2-adrenoceptor agonists

The effects of alpha2-adrenoceptor agonists, clonidine, tizanidine and UK-14304 on alpha1-adrenoceptor-mediated contractile responses were studied in isolated tail arteries and thoracic aorta of the rat. When applied during sustained contractile responses to almost maximum concentration (10 microM) of phenylephrine, clonidine (0.3 microM to 100 microM) produced concentration-dependent relaxations in both tissues. The maximum relaxation was smaller in tail arteries than in thoracic aorta. Clonidine up to 100 microM failed to relax both tissues precontracted with KCl (60 microM) or U-46619 (1 microM), a thromboxane mimetic. The clonidine-induced relaxation in tail arteries, was reversed by alpha2-adrenoceptor antagonists, yohimbine and idazoxane. Effects of the alpha2-adrenoceptor antagonists were concentration-dependent (0.1 microM to 1 microM), but not in a competitive manner. On the other hand, the relaxation in thoracic aorta was not significantly antagonized by these alpha2-adrenoceptor antagonists. Tizanidine and UK-14304 also relaxed both tail arteries and thoracic aorta precontracted with phenylephrine. The characteristics of the relaxation and their antagonism by yohimbine in both arteries were similar to those induce by clonidine. In tail arteries, NG-nitro-L-arginine, a nitric oxide synthase inhibitor, at a concentration that completely inhibited acetylcholine-induced relaxations did not significantly affect the relaxation induced by clonidine. In contrast, the relaxation of thoracic aorta in response to clonidine was partly reduced in the presence of NG-nitro-L-arginine. These results indicate that the alpha2-adrenoceptor agonists selectively inhibit the contractions induced by phenylephrine in both tissues. Regional differences in the modes of the inhibition by the alpha2-adrenoceptor agonists exist.     

Involvement and dual effects of nitric oxide in septic shock

Bacterial endotoxin (LPS) releases many mediators such as interleukins, tumour necrosis factor, oxygen free radicals, toxic eicosanoids, platelet activating factor, and nitric oxide (NO). LPS is a potent inducer of inducible nitric oxide synthase (iNOS). Large amounts of NO (made by iNOS) and peroxynitrite, among other factors, are responsible for the late phase of hypotension, vasoplegia, cellular suffocation, apoptosis, lactic acidosis and multiorgan failure in endotoxic shock. Indeed, experimental and clinical use of NOS inhibitors, which do not differentiate clearly between constitutive endothelial NOS (ceNOS) and iNOS, prevents LPS-induced hypotension. However, many detrimental effects of such NOS inhibitors are also reported, including increases in pulmonary resistance, decreases in cardiac output and organ perfusion, and even an increase in mortality of experimental animals. We believe that, in lungs, NO made by ceNOS plays a protective role against the pneumotoxic effects of LPS-released lipids such as thromboxane, leukotrienes and PAF. This is why selective iNOS inhibitors like aminoguanidine or thiourea derivatives might be preferred over nonselective NOS inhibitors for the treatment of septic shock. However, since iNOS-derived NO seems to have more than just a destructive action, the selective iNOS inhibition may be not as beneficial as expected. Accordingly, inhalation of NO gas or NO-donors in septic shock might be a complementary treatment to the use of NOS inhibitors.     

Estrogen upregulates endothelial constitutive nitric oxide synthase expression in human osteoblast-like cells

The protective effects of estrogen on the cardiovascular system are thought to be mediated, in part, by nitric oxide (NO). Estrogen also has protective effects on bone although the mechanisms of action have not been fully established. Since nitric oxide synthase (NOS) inhibitors have been found to abrogate the protective effect of estrogen on bone in ovariectomised rats, we studied the effects of 17beta-estradiol on NOS activity and NOS mRNA levels in cultured human osteoblast-like cells. 17beta-Estradiol stimulated NOS activity by approximately 2.0 fold and this effect was reversed by the calcium chelator, EGTA, and the NOS inhibitor, L-NMMA, implicating activation of a constitutive, calcium-dependent isoform. Further studies using RT/PCR indicated that only the endothelial nitric oxide synthase (ecNOS) isoform was expressed and RNase protection assays showed that 17beta-Estradiol treatment resulted in a 2.2 fold increase in ecNOS mRNA levels. These findings suggest that estrogen stimulates NOS activity in osteoblastic cells by activation of the ecNOS pathway, and taken together with previous data, is consistent with the possibility that NO may act as a mediator of estrogen actions on bone.     

Intrarenal haemodynamics and renal dysfunction in endotoxaemia: effects of nitric oxide synthase inhibition

1. This study investigated the effects of low dose endotoxin (lipopolysaccharide, LPS) on (i) systemic haemodynamics, (ii) renal blood flow (RBF), (iii) renal cortical and medullary perfusion and (iv) renal function in the anaesthetized rat. We have also investigated the effects of nitric oxide (NO) synthase (NOS) inhibition with NG-methyl-L-arginine (L-NMMA) on the alterations in systemic and renal haemodynamics and renal function caused by endotoxin. 2. Infusion of low dose LPS (1 mg kg-1 over 30 min, n = 6) caused a late fall in mean arterial blood pressure (MAP, at 5 and 6 h after LPS), but did not cause an early (at 1-4 h after LPS) hypotension. The pressor effect of noradrenaline (NA, 1 microgram kg-1, i.v.) was significantly reduced at 1 to 6 h after LPS (vascular hyporeactivity). Infusion of L-NMMA (50 micrograms kg-1 min-1 commencing 60 min before LPS and continued throughout the experiment, n = 7) abolished the delayed hypotension and significantly attenuated the vascular hyporeactivity to NA (at 2-6 h). 3. Infusion of LPS (1 mg kg-1 over 30 min, n = 6) caused a rapid (within 2 h) decline in renal function (measured by inulin clearance) in the absence of a significant fall in MAP or renal blood flow (RBF). L-NMMA (n = 7) attenuated the impairment in renal function caused by LPS so that the inulin clearance in LPS-rats treated with L-NMMA was significantly greater than in LPS-rats treated with vehicle (control) at 3-6 h after infusion of LPS. 4. Endotoxaemia also caused a significant reduction in renal cortical, but not medullary perfusion (measured as Laser Doppler flux). Infusion of L-NMMA caused a significant further fall in cortical perfusion and a significant fall in medullary perfusion in the absence of changes in RBF. 5. Infusion of LPS resulted in a progressive increase in the plasma levels of nitrite/nitrate (an indicator of the formation of NO), so that the plasma concentration of nitrite/nitrate was significantly higher than baseline at 150 to 330 min after LPS. Infusion of L-NMMA attenuated the rise in the plasma concentration of nitrite/nitrate (at 270 and 330 min, P < 0.05) caused by LPS. 6. Thus, the renal dysfunction caused by injection of low dose of endotoxin in the rat occurs in the absence of significant falls in blood pressure or total renal blood flow. Inhibition of NOS activity with L-NMMA attenuates the renal dysfunction caused by endotoxin (without improving intrarenal haemodynamics), suggesting that an overproduction of NO may contribute to the development of renal injury and dysfunction by causing direct cytotoxic effects.     

Aorta and skeletal muscle NO synthase expression in experimental heart failure

Nitric oxide (NO), the free radical that accounts for the biological activity of endothelium-derived relaxing factor, is synthesized from L-arginine by NO synthase (NOS). There is evidence that NO availability is reduced in the peripheral vasculature of patients with congestive heart failure (CHF). The aim of this study was to investigate the expression of NOS in the descending aorta and in the skeletal muscles of rats subjected to heart failure. The alkaloid, monocrotaline, was used to induce pulmonary hypertension and cardiac failure in rats. The expression of both the constitutive (ecNOS) and the inducible (iNOS) isoforms of the enzyme was assessed by Western blot analysis. In CHF animals, the ecNOS location in the aorta is altered: the endothelial protein expression is substantially reduced (from 0.083 +/- 0.012 to 0.003 +/- 0.004 OD/microgram total proteins, P < 0.001) whereas the expression of ecNOS in the smooth muscle is increased (from 0.024 +/- 0.004 to 0.059 +/- 0.009 OD/ microgram total proteins, P < 0.01). The total aortic ecNOS is diminished in CHF respect to control animals (0.062 +/- 0.009 v 0.107 +/- 0.013 OD/microgram total proteins, P < 0.01). On the contrary, no difference in ecNOS protein expression was observed in the extensor digitorum longus and soleus muscles. Furthermore, iNOS was not detected in any of the tissues considered. In conclusion, experimental CHF causes a re-setting of the ecNOS protein expression in the descending aorta but not in skeletal muscles. The reduced abundance of ecNOS in the aortic endothelium is consistent with the impairment of the vasodilating function reported in patients with CHF.     

Inducible and endothelial nitric oxide synthase expression during development of transplant arteriosclerosis in rat aortic grafts

In the vascular system, distinct isoforms of nitric oxide synthase (NOS) generate nitric oxide (NO), which acts as a biological messenger. Its role in the development of transplant arteriosclerosis (TA) is still unclear. To investigate whether NO is involved in TA, we studied the expression of NOS isoforms, inducible NOS (iNOS) and endothelial NOS (eNOS), by immunohistochemistry and in situ hybridization during the first two post-transplantation months and their relation with cold ischemia (1 to 24 hours) and reperfusion injury using an aortic transplantation model in the rat. We found an increased iNOS expression in the intima and adventitia and a decreased expression in the media, whereas eNOS expression was not significantly altered during the development of TA. Co-localization studies suggested that iNOS-positive cells were vascular smooth muscle cells, monocyte-derived macrophages, and endothelial cells. Prolonged ischemic storage time resulted in an increase in eNOS expression in the neointima. In situ hybridization showed iNOS mRNA expression by vascular cells in the neointima and media. NO produced by iNOS and eNOS may be involved, at least in part, in the pathogenesis of TA in aortic grafts. Additional studies are needed to confirm the modulatory mechanism of NO during the development of TA.     

Vasoconstrictor responses via P2X-receptors are selectively antagonized by NF023 in rabbit isolated aorta and saphenous artery

1. The effects of NF023, the symmetrical 3'-urea of 8-(benzamido)naphthalene-1,3,5-trisulphonic acid), and its parent compound suramin were investigated on vasoconstrictor responses to alpha, beta-methylene ATP in rabbit isolated saphenous artery and vasodilator responses to ATP in noradrenaline-precontracted rabbit isolated thoracic aorta. 2. In rabbit isolated saphenous artery, alpha, beta-methylene ATP-induced vasoconstrictor responses via P2X-receptors were concentration-dependently and competitively antagonised by NF023 (30-300 microM; pA2 = 5.69 +/- 0.04). Suramin (100-1000 microM) also competitively blocked vasoconstrictor responses to alpha, beta-methylene ATP, albeit with lower potency (pA2 = 4.79 +/- 0.05). In contrast, NF023 (100 microM) did not significantly affect contractile responses to noradrenaline or histamine in the saphenous artery. 3. In noradrenaline-precontracted rabbit isolated thoracic aorta preparations, ATP (3-3000 microM) concentration-dependently induced relaxations via endothelium-dependent or smooth muscle P2Y-receptor subtypes. NF023 (30-300 microM) failed to block relaxant responses to ATP at endothelium-dependent P2Y-receptors, whereas suramin (100-1000 microM) did antagonise endothelium-dependent vasodilator responses to ATP. Neither NF023 (100 microM) nor suramin (300 microM) influenced vasorelaxant responses to ATP via endothelium-independent P2Y-receptors. 4. In conclusion, this study outlines the selectivity of NF023 as an effective P2X-receptor antagonist in rabbit isolated blood vessels without affecting endothelium-dependent or endothelium-independent P2Y-receptor subtypes, adrenoceptors or histamine receptors.     

Role of inducible nitric oxide synthase in endotoxin-induced acute lung injury

The role of nitric oxide (NO) in lung injury remains unclear. Both beneficial and detrimental roles have been proposed. In this study, we used mutant mice lacking the inducible nitric oxide synthase (iNOS) to assess the role of this isoform in sepsis-associated lung injury. Wild-type and iNOS knockout mice were injected with either saline or Escherichia coli endotoxin (LPS) 25 mg/kg and killed 6, 12, and 24 h later. Lung injury was evaluated by measuring lactate dehydrogenase activity in the bronchoalveolar lavage, pulmonary wet/dry ratio, and immunostaining for nitrotyrosine formation. In the wild-type mice, LPS injection elicited more than a 3-fold rise in lactate dehydrogenase activity, a significant rise in lung wet/dry ratio and extensive nitrotyrosine staining in large airway and alveolar epithelium, macrophages, and pulmonary vascular cells. This was accompanied by induction of iNOS protein and increased lung nitric oxide synthase activity. By comparison, LPS injection in iNOS knockout mice elicited no iNOS induction and no significant changes in lung NOS activity, lactate dehydrogenase activity, lung wet/dry ratio, or pulmonary nitrotyrosine staining. These results indicate that mice deficient in iNOS gene are more resistant to LPS-induced acute lung injury than are wild-type mice.     

Adventitial expression of recombinant eNOS gene restores NO production in arteries without endothelium

The current study was designed to determine the effect of recombinant endothelial nitric oxide synthase (eNOS) gene expression on endothelium-dependent relaxations to bradykinin in isolated canine basilar, coronary, or femoral arteries. Arterial rings were exposed ex vivo (30 minutes at 37 degrees C) to an adenoviral vector encoding either the eNOS gene (AdCMVeNOS) or the beta-galactosidase reporter gene (AdCMVbeta-Gal). Twenty-four hours after transduction, transgene expression was evident mainly in the adventitia. Expression of recombinant proteins was much higher in basilar arteries than in coronary or femoral arteries. Rings of control, AdCMVbeta-Gal, and AdCMVeNOS arteries with and without endothelium were suspended for isometric tension recording. Levels of cGMP were measured by radioimmunoassay. In AdCMVeNOS basilar arteries with endothelium, relaxations to low concentrations of bradykinin (3 x 10(-11) to 10(-9) mol/L) were significantly augmented. In contrast, in coronary and femoral arteries with endothelium, AdCMVeNOS transduction did not affect relaxations to bradykinin. Removal of the endothelium abolished bradykinin-induced relaxations in control and AdCMVbeta-Gal basilar arteries. However, in basilar arteries transduced with AdCMVeNOS even when the endothelium was removed, stimulation with bradykinin (3 x 10(-11) to 10(-9) mol/L) caused relaxations as well as increases in cGMP production. The relaxations to bradykinin were completely blocked by an NOS inhibitor, NG-nitro-L-arginine methyl ester. Electron microscopic analysis revealed that recombinant eNOS protein was expressed in fibroblasts of the basilar artery adventitia. These results suggest that genetically modified adventitial fibroblasts may restore production of NO in cerebral arteries without endothelium. Our findings support a novel concept in vascular biology that fibroblasts in the adventitia may play a role in the regulation of vascular tone after successful transfer and expression of recombinant eNOS gene.