Different expression patterns of MMP-2 and MMP-9 in breast cancer

The role of nitric oxide (NO) in activation of cGMP is well established. It has been proposed that the ratio of cAMP to cGMP may be important in the regulation of preimplantation embryonic growth and differentiation. Therefore, we determined the ability of murine preimplantation embryos to produce NO. In addition, NO as an endogenous smooth muscle relaxant and vasodilator is a candidate for involvement in embryo implantation because this process requires increased vascular permeability and uterine quiescence at the sites of blastocyst apposition. Nitrite assays, an indirect measure of NO production, indicate that preimplantation murine embryos produce NO. This production was reversibly inhibited by culture of embryos in medium containing a nonspecific NO synthase (NOS) inhibitor (NG-nitro-L-arginine). Additionally, inhibition of normal development was observed in embryos cultured with NOS inhibitor. NO levels increased in culture medium when ovariectomized progesterone-treated animals were exposed to estrogen for 1 h in utero. Such hormonal treatment induces implantation. These data indicate that NO levels are regulated by estrogen and may be important in regulation of implantation. In addition, these data demonstrate for the first time that NO production appears to be required for normal embryonic development.     

Nitric oxide synthase activity and localization do not change in uterus and placenta during human parturition

Animal studies have suggested that nitric oxide, a smooth muscle relaxant, is a fundamental mediator in the initiation of parturition. The purpose of this study was to test the hypothesis that the onset of human labour is associated with a reduction in the activity of the enzyme nitric oxide synthase (NOS), within the uterus. Samples of myometrium, placenta, decidua and fetal membranes were collected during Caesarean section from 11 women before and 11 women after the onset of labour at term. Immunocytochemistry was used to localize each of the three isoforms of NOS (endothelial NOS, brain NOS, and inducible NOS) in each of these tissues and the intensity of staining was qualitatively assessed. NOS enzyme activity was determined in homogenates of frozen myometrium, placenta and fetal membranes (with attached decidua), by measuring conversion of radio-labelled L-arginine to L-citrulline. Each of the three isoforms of NOS was localized in each of the tissues. We found no difference in either the expression or enzyme activity of NOS in myometrium, placenta or fetal membranes before and during labour at term. These results suggest that, in contrast to animal studies, a decrease in NOS enzyme activity may not be involved in the onset of parturition at term in the human.     

Electron transfer and catalytic activity of nitric oxide synthases. Chimeric constructs of the neuronal, inducible, and endothelial isoforms

The nitric oxide synthases (NOS) are single polypeptides that encode a heme domain, a calmodulin binding motif, and a flavoprotein domain with sequence similarity to P450 reductase. Despite this basic structural similarity, the three major NOS isoforms differ significantly in their rates of .NO synthesis, cytochrome c reduction, and NADPH utilization and in the Ca2+ dependence of these rates. To assign the origin of these differences to specific protein domains, we constructed chimeras in which the reductase domains of endothelial and inducible NOS, respectively, were replaced by the reductase domain of neuronal NOS. The results with the chimeric proteins confirm the modular organization of the NOS polypeptide chain and demonstrate that (a) similar residues establish the necessary contacts between the reductase and heme domains in the three NOS isoforms, (b) the maximal rate of .NO synthesis is determined by the maximum intrinsic ability of the reductase domain to deliver electrons to the heme domain, (c) the Ca2+ independence of inducible NOS requires interactions of calmodulin with both the calmodulin binding motif and the flavoprotein domain, and (d) the effects of tetrahydrobiopterin and L-arginine on electron transfer rates are mediated exclusively by heme domain interactions.     

Substrate binding and calmodulin binding to endothelial nitric oxide synthase coregulate its enzymatic activity

Endothelial nitric oxide synthase (NOS) is a constitutively expressed flavin-containing heme protein that catalyzes the formation of NO from L-arginine, NADPH, and molecular oxygen. We purified bovine endothelial NOS from transfected embryonic kidney cells by conventional chromatographic techniques and characterized the activity of the detergent-solubilized enzyme. Endothelial NOS displays a much lower specific activity of NO synthesis (143 +/- 11 nmol NO/min/mg enzyme) than the constitutive neuronal NOS or inducible NOS isoforms. Like the neuronal isoform, endothelial NOS requires binding of Ca2+/calmodulin to achieve Vmax NO synthase activity; however, we observed a basal level of NO synthesis even when Ca2+/calmodulin was omitted and 0.5 mM EDTA was present in the assay solution. Moreover, endothelial NOS demonstrates a high-affinity bonding interaction with calmodulin such that the enzyme as purified has a NO synthase activity at about 80% of Vmax. We also observed a more than twofold increase in NADPH consumption by endothelial NOS when it was coupled to arginine oxygenation as opposed to when oxygen is activated in the absence of substrate. Substrate binding was also shown to stimulate heme reduction in the absence of added calmodulin. Thus, the enzymatic synthesis of NO from L-arginine by endothelial NOS appears to be partially regulated by binding of both calmodulin and substrate. These findings for endothelial NOS represent a significant departure from the enzymatic properties of the other constitutive NOS isoform, neuronal NOS, and we interpret this result in terms of the physiological implications.     

Calcium channel blockade enhances nitric oxide synthase expression by cultured endothelial cells

In a recent study, we found marked increases in nitric oxide (NO) production and endothelial and inducible NO synthase (eNOS and iNOS) expressions with calcium channel blockade in rats with chronic renal failure. This study was undertaken to determine whether enhanced NO production with calcium channel blockade is a direct effect of this therapy or a consequence of the associated hemodynamic and humoral changes. We tested the effects of a calcium channel blocker, felodipine (10(-5), 10(-6), and 10(-7) mol/L), on nitrate and nitrite (NOx) generation, Ca2+-dependent and -independent NOS activity, and eNOS and iNOS protein masses in proliferating and quiescent rat aortic endothelial cells in culture. Compared with vehicle alone, felodipine significantly increased NOx generation, Ca2+-dependent NOS activity, and eNOS protein mass in proliferating and quiescent endothelial cells. Felodipine did not modify the stimulatory action of 10% fetal calf serum on DNA synthesis (thymidine incorporation) and cell proliferation. Ca2+-independent NOS activity and iNOS protein expression were negligible and unaffected by calcium channel blockade. NOx production and NOS expression were greater in proliferating cells than in quiescent cells. Thus, calcium channel blockade upregulates endothelial NO production in vitro, confirming our previous in vivo study. This observation indicates that the reductions in cytosolic [Ca2+] and vasodilation with calcium channel blockade are not only due to inhibition of Ca2+ entry but also to an NO-cGMP mediated mechanism.     

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.     

Localization of nitric oxide synthases and nitric oxide production in the rat mammary gland

We investigated nitric oxide (NO) production and the presence of nitric oxide synthase (NOS) in the mammary gland by use of an organ culture system of rat mammary glands. Mammary glands were excised from the inguinal parts of female Wistar-MS rats primed by implantation with pellets of 17beta-estradiol and progesterone and were diced into approximately 3-mm cubes. Three of these cubes were cultured with 2 ml of 10% FCS/DMEM plus carboxy-PTIO (an NO scavenger, 100 microM) in the presence or absence of LPS (0.5 microgram/ml) for 2 days. The amount of NO produced spontaneously by the cultured mammary glands was relatively minute at the end of the 2-day culture period, and the NO production was significantly enhanced by the presence of LPS. This enhancement of NO production was completely eliminated by addition of hydrocortisone (3 microM), an inhibitor of inducible NOS (iNOS), to the incubation medium. Immunoblot analyses with specific antisera against NOS isoforms such as iNOS, endothelial NOS (eNOS), and brain NOS (bNOS) showed immunoreactive bands of iNOS (122 +/- 2 kD) and eNOS (152 +/- 3 kD) in extracts prepared from the mammary glands in the culture without LPS. The immunoreactive band of iNOS was highly intense after the treatment of mammary glands with LPS, whereas the corresponding eNOS immunoreactive band was faded. The immunohistochemical study of anti-iNOS antiserum on frozen sections of the cultured mammary glands showed that an immunoreactive substance with the antiserum was localized to the basal layer (composed of myoepithelial cells of alveoli and lactiferous ducts) of the mammary epithelia and to the endothelium of blood vessels that penetrated into the interstitium of the mammary glands. Histochemical staining for NADPH-diaphorase activity, which is identical to NOS, showed localization similar to that of iNOS in the mammary glands. Similar observations were noted in the immunohistochemistry of eNOS. In contrast, the immunoreactive signal with the bNOS antiserum was barely detected in the epithelial parts of alveoli and lactiferous ducts of the mammary glands. These observations demonstrate that three isoforms of NOS are present not only in the endothelium of blood vessels but also in the parenchymal cells (the glandular epithelium) of the rat mammary gland, such as epithelial cells and myoepithelial cells, and suggest that NO may have functional roles in the physiology of the mammary glands.     

Regulation of nitric oxide production in response to skeletal muscle activation

Nitric oxide (NO) is synthesized in normal muscle fibers by the neuronal (nNOS) and the endothelial (ecNOS) isoforms of nitric oxide synthase (NOS). NO contributes to the regulation of several processes such as excitation-contraction coupling and mitochondrial respiration. We assessed in this study whether NO production is regulated in response to an acute increase in muscle activation. Three groups of anesthetized, tracheostomized, spontaneously breathing rats were examined after an experimental period of 3 h. Group 1 served as a control (no loading), whereas groups 2 and 3 were exposed to moderate and severe inspiratory resistive loads, respectively, which elicited tracheal pressures of 30 and 70% of maximum, respectively. Ventilatory (diaphragm, intercostal, and transverse abdominis) and limb (gastrocnemius) muscles were excised at the end of the experimental period and examined for NOS activity and NOS protein expression. Neither submaximal nor maximum tracheal pressures were altered after 3 h of resistive loading. Diaphragmatic and intercostal muscle NOS activities declined significantly in response to moderate and severe loading, whereas those of transverse abdominis and gastrocnemius muscles remained unchanged. On the other hand, resistive loading had no significant effect on ventilatory and limb muscle NOS isoform expression. We propose that a contraction-induced decline in muscle NOS activity represents a compensatory mechanism through which muscle contractility and mitochondrial function are protected from the inhibitory influence of NO.     

Nitric oxide synthases in Kaposi's sarcoma are expressed predominantly by vessels and tissue macrophages

Kaposi's sarcoma (KS) is a tumor of presumed vascular origin frequently found in patients with AIDS. Recent data suggest that the development of KS is linked with the presence of a newly recognized herpesvirus, human herpesvirus type 8. Nitric oxide (NO), a messenger molecule with vasoactive, antitumor, and antimicrobial effects, is produced by three isoforms of nitric oxide synthases (NOS). In the present report, we investigated the expression of NOS isoforms in KS. By NADPH-diaphorase histochemistry, NOS activity was detectable in endothelia and CD45+ cells within KS lesions. Reactivity for endothelial NOS (eNOS) was found in blood vessel endothelia; however, eNOS reactivity was negative in KS spindle cells in 12 of 17 tumors, and moderately positive in the other 5 lesions. In contrast to KS, tumor cells in three hemangiomas and one angiosarcoma were strongly positive for eNOS. Inducible NOS (iNOS) was absent from KS tumor cells but was found regularly in CD45+, HLA-DR+ cells within the lesions. In five KS-derived spindle cell cultures, neither eNOS nor iNOS proteins were detectable. The sporadic expression of eNOS by KS spindle cells in vivo and the absence of eNOS protein from KS spindle cells in tissue cultures argue against the possibility that the cells are derived from blood vessel endothelia. The consistent expression of iNOS by CD45+, HLA-DR+ cells within KS lesions strongly suggests that leukocyte-derived NO participates in the pathology of this tumor.     

Differential effects of nonselective nitric oxide synthase (NOS) and selective inducible NOS inhibition on hepatic necrosis, apoptosis, ICAM-1 expression, and neutrophil accumulation during endotoxemia

The roles of nitric oxide derived from either the constitutive endothelial NO synthase (eNOS or NOS3) or the inducible NOS (iNOS or NOS2) in hepatic injury during endotoxemia remain controversial. To investigate this further, rats received a bolus of lipopolysaccharide (LPS) following implantation of osmotic pumps containing one of two nonselective NOS inhibitors (NMA or NAME), one of two inducible NOS inhibitors (NIL or AG), or saline. The inhibitors were infused continuously into the liver via the portal vein. Treatment of LPS-injected rats with NMA and NAME resulted in 106 and 227% increases, respectively, in circulating hepatic enzyme levels compared to LPS-treated control rats. In contrast, infusion of the iNOS-selective inhibitors had no effect on the LPS-induced hepatic necrosis. In rats receiving NAME, LPS induced greater neutrophil infiltration and ICAM-1 expression than in the LPS + saline group, whereas NIL infusion did not. The increased hepatic necrosis and PMN infiltration in the LPS + NAME group was partially prevented by a simultaneous infusion of a liver-selective NO donor. Inhibition of PMN accumulation using an anti-ICAM-1 antibody or by PMN depletion using vinblastine pretreatment, however, did not reverse the increased necrosis with NAME infusion during endotoxemia. In contrast to the assessment for necrosis, increased apoptosis was observed in the livers of LPS-treated rats receiving infusions of either NAME or NIL, but not with LPS alone. These data indicate that NO produced by eNOS may be adequate to prevent necrosis by a mechanism independent of PMN, while induced NO appears to prevent apoptosis.     

The versatile and complex enzymology of nitric oxide synthase

The biogenesis of nitric oxide is catalyzed by nitric oxide synthase (NOS) which forms L-citrulline and NO from L-arginine. Here we review the enzymology of NOS. We discuss its modular structure, its prosthetic groups and cofactors, and we provide a brief account of present knowledge regarding cellular targeting and regulation of the different isoforms. The various reactions which are catalyzed by NOS are reviewed, and an inventory of different inhibitor types is given. Special attention is paid to the role of the cofactor tetrahydrobiopterin (BH4) and of the dimeric structure, and to the possibility that the main product of NOS catalysis under some conditions may not be NO. Based on a number of recent observations, we postulate that neuronal NOS with one equivalent of BH4 per dimer (a state which may be physiologically relevant) catalyzes the concerted formation of peroxynitrite.     

NADPH diaphorase activity in mammalian retinas is modulated by the state of visual adaptation

NADPH diaphorase histochemistry is commonly used to identify cells containing nitric oxide synthase (NOS), the enzyme catalyzing the production of nitric oxide from L-arginine. NADPH diaphorase activity and NOS immunostaining was demonstrated in different cells of the vertebrate retina; photoreceptors, horizontal cells, amacrine cells, ganglion cells, and Müller cells. However, the physiological role of nitric oxide (NO) in the retina has yet to be elucidated. In this study, we tested the assumption that NADPH diaphorase activity in the retinas of rabbits and rats depended on the state of visual adaptation. In the rabbit, light adaptation enhanced NADPH diaphorase activity in amacrine cells and practically eliminated it in horizontal cells. Dark adaptation induced the opposite effects; the NADPH diaphorase activity was reduced in amacrine cells and enhanced in horizontal cells. Retinas from eyes that were injected intravitreally with L-glutamate exhibited a pattern of NADPH diaphorase activity that was similar to that seen in dark-adapted retinas. In rats, the NADPH diaphorase activity of amacrine and horizontal cells exhibited adaptation dependency similar to that of the rabbit retina. But, the most pronounced effect of dark adaptation in the rat's retina was an enhancement of NADPH diaphorase activity in Müller cells, especially of the endfoot region. Assuming that NADPH diaphorase activity is a marker for NOS, these findings suggest that NO production in the mammalian retina is modulated by the level of ambient illumination and support the notion that NO plays a physiological role in the retina.     

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.     

Nitric oxide plays an important role in the diurnal change of tuberoinfundibular dopaminergic neuronal activity and prolactin secretion in ovariectomized, estrogen/progesterone-treated rats

A significant diurnal change of tuberoinfundibular dopaminergic (TIDA) neuronal activity coincident with the estrogen (E2)-induced afternoon PRL surge has been reported in ovariectomized, E2-primed (OVX+E2) rats. Systemic injection of a nitric oxide (NO) synthase (NOS) inhibitor, N(G)-nitro-L-arginine (L-NA, 50 mg/kg, i.p. at 1000 and 1200 h), significantly blocked the diurnal changes of TIDA neuronal activity and PRL secretion at 1500 and 1700 h in OVX+E2 rats. Coadministration of L-arginine (300 mg/kg, i.p.) with L-NA completely prevented the effects of L-NA. Total nitrite/nitrate levels in the serum of L-NA- and L-NA+L-arginine-treated rats substantiated the effects of L-NA and L-arginine on NO production. Pretreatment of antisense oligodeoxynucleotide (ODN; 1 microg/3 microl; intracerebroventricularly at 48, 24, and 7 h before sacrifice) against the messenger RNA (mRNA) of constitutive NOS, i.e. neuronal NOS or endothelial NOS, was also effective in preventing the diurnal changes of TIDA neuronal activity and PRL surge at 1500 h. The same treatment of antisense ODN against the mRNA of inducible NOS, i.e. macrophage NOS, had no effect. Progesterone (P4) has been reported to advance and augment the diurnal changes of TIDA neuronal activity and the afternoon PRL surge, by 1 h, in both proestrous and OVX+E2 rats. We further showed that L-NA dose dependently (50 but not 5 mg/kg, i.p. at 1000 and 1200 h) blocked the effect of P4 on TIDA neurons and serum PRL at 1300 h, which effect could be negated by simultaneous administration of L-arginine (300 mg/kg, i.p.). Pretreatment with antisense ODNs against the mRNA of neuronal NOS or endothelial NOS, but not macrophage NOS, was also effective in preventing the P4's effect on TIDA neuronal activity and PRL secretion at 1300 h. In summary, NO may play a physiological role in the E2- and P4-regulated diurnal changes of TIDA neuronal activity and PRL secretion.     

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.     

Local injection of kainic acid causes widespread degeneration of NADPH-d neurons and induction of NADPH-d in neurons, endothelial cells and reactive astrocytes

Nitric oxide (NO), a diffusible gas, is a messenger molecule that mediates vascular dilatation and neural transmission. The enzyme nitric oxide synthase (NOS) present in neurons is activated by Ca2+ influx associated with activation of glutamate receptors. Cultured cortical neurons containing NOS are selectively vulnerable to injury by kainic acid (KA). However, the relationship between NOS neurons and excitotoxicity under in vivo conditions is not entirely clear. In the present study, we examined the time course and spatial distribution of changes in NOS neurons caused by an intracortical microinjection of KA in adult rats. NADPH-diaphorase (NADPH-d) histochemistry was used as a marker for NOS and the neuronal changes were correlated with changes in glial cells and endothelial cells. We demonstrated a rapid loss of NADPH-d neurons in the lesion center and degeneration of NADPH-d neurons and nerve terminals throughout ipsilateral cortex and hippocampus; the striatal neurons appeared to be unaffected. Subsequent to cortical neuronal degeneration, new NADPH-d activity appeared in proliferative reactive astrocytes and in endothelial cells at lesion periphery, and in neuronal groups at lesion periphery, in ipsilateral entorhinal cortex and bilateral hippocampus. These findings indicate that neurons expressing NADPH-d in cerebral cortex and hippocampus are selectively vulnerable to KA toxicity in vivo. The subsequent induction of NOS in neural and non-neural cells may be regarded as an adaptive response to the kainate-induced brain lesion.     

Memory-related changes of nitric oxide synthase activity and nitrite level in rat brain

The changes of nitric oxide synthase (NOS) activity and nitrite level in rat brain regions after spatial learning were investigated. NOS activity was assayed by conversion of [3H]L-arginine to [3H]L-citrulline, and a sensitive fluorometric assay for quantification of nitrite was used. Compared with sham-trained rats, NOS activity and nitrite level in hippocampus and cortex, and also the nitrite level in cerebellum, was elevated significantly one day after rats had learnt a water-rewarded spatial alteration task. These results suggest a spatial memory-related changes of endogenous NO in rat brain, and support the idea that NO participates in learning and memory processes.