Social isolation-induced epigenetic changes in midbrain of adult mice.

Social isolation and loneliness increase the risk of death as much as well-established risk factors for mortality such as cigarette smoking and alcohol consumption. The underlying molecular mechanisms are poorly understood. In the present study, 3 months old male C57BL/6 mice were socially isolated by individual housing for another 3 months. At the age of 6 months, epigenetic changes were analyzed in midbrain. Social isolation of male adult mice led to an increased global DNA methylation, which was associated with enhanced activity of DNA methyltransferase. Di- and trimethylation of global histone H3 lysine 4 (H3K4) were increased in midbrain of socially isolated mice, accompanied by enhanced H3K4 histone methyltransferase activity. In addition, social isolation of adult mice led to activation of histone acetyltransferases as well as of histone deacetylases (HDAC) resulting in a net enhancement of histone H3 lysine 9 (H3K9) acetylation. Gene-specific effects were observed for Hdac1, Hdac3 and the serotonin transporter Slc6a4. Social isolation led to an up-regulation of Hdac1 and Hdac3, associated with decreased DNA methylation in the CpG island of the respective genes. On the contrary, the Slc6a4 gene was down-regulated, which was associated with enhanced DNA methylation. Collectively, the results from the present study demonstrate for the first time that social isolation of adult mice leads to a wide range of global epigenetic changes and these effects may have profound impact on gene expression pattern and phenotype of the socially isolated animals.

PON3 is upregulated in cancer tissues and protects against mitochondrial superoxide-mediated cell death.

To achieve malignancy, cancer cells convert numerous signaling pathways, with evasion from cell death being a characteristic hallmark. The cell death machinery represents an anti-cancer target demanding constant identification of tumor-specific signaling molecules. Control of mitochondrial radical formation, particularly superoxide interconnects cell death signals with appropriate mechanistic execution. Superoxide is potentially damaging, but also triggers mitochondrial cytochrome c release. While paraoxonase (PON) enzymes are known to protect against cardiovascular diseases, recent data revealed that PON2 attenuated mitochondrial radical formation and execution of cell death. Another family member, PON3, is poorly investigated. Using various cell culture systems and knockout mice, here we addressed its potential role in cancer. PON3 is found overexpressed in various human tumors and diminishes mitochondrial superoxide formation. It directly interacts with coenzyme Q10 and presumably acts by sequestering ubisemiquinone, leading to enhanced cell death resistance. Localized to the endoplasmic reticulum (ER) and mitochondria, PON3 abrogates apoptosis in response to DNA damage or intrinsic but not extrinsic stimulation. Moreover, PON3 impaired ER stress-induced apoptotic MAPK signaling and CHOP induction. Therefore, our study reveals the mechanism underlying PON3's anti-oxidative effect and demonstrates a previously unanticipated function in tumor cell development. We suggest PONs represent a novel class of enzymes crucially controlling mitochondrial radical generation and cell death.

Beyond reduction of atherosclerosis: PON2 provides apoptosis resistance and stabilizes tumor cells.

Major contributors to atherosclerosis are oxidative damage and endoplasmic reticulum (ER) stress-induced apoptosis; both of which can be diminished by the anti-oxidative protein paraoxonase-2 (PON2). ER stress is also relevant to cancer and associated with anti-cancer treatment resistance. Hence, we addressed, for the first time, whether PON2 contributes to tumorigenesis and apoptotic escape. Intriguingly, we found that several human tumors upregulated PON2 and such overexpression provided resistance to different chemotherapeutics (imatinib, doxorubicine, staurosporine, or actinomycin) in cell culture models. This was reversed after PON2 knock-down. Remarkably, just deficiency of PON2 caused apoptosis of selective tumor cells per se, demonstrating a previously unanticipated oncogenic function. We found a dual mechanistic role. During ER stress, high PON2 levels lowered redox-triggered induction of pro-apoptotic CHOP particularly via the JNK pathway, which prevented mitochondrial cell death signaling. Apart from CHOP, PON2 also diminished intrinsic apoptosis as it prevented mitochondrial superoxide formation, cardiolipin peroxidation, cytochrome c release, and caspase activation. Ligand-stimulated apoptosis by TRAIL or TNFα remained unchanged. Finally, PON2 knock-down caused vast reactive oxygen species formation and stimulated JNK-triggered CHOP expression, but inhibition of JNK signaling did not prevent cell death, demonstrating the pleiotropic, dominating anti-oxidative effect of PON2. Therefore, targeting redox balance is powerful to induce selective tumor cell death and proposes PON2 as new putative anti-tumor candidate.

Resveratrol reduces endothelial oxidative stress by modulating the gene expression of superoxide dismutase 1 (SOD1), glutathione peroxidase 1 (GPx1) and NADPH oxidase subunit (Nox4).

Resveratrol, an important antioxidant found in grapes and wine, is likely to contribute to red wine's potential to prevent human cardiovascular disease. In addition to its known (direct) antioxidant effect, we have found that resveratrol also regulates the gene expression of pro-oxidative and anti-oxidative enzymes in human endothelial cells. NADPH oxidases (Nox) are the predominant producers of superoxide in the vasculature, whereas superoxide dismutase (SOD) and glutathione peroxidase 1 (GPx1) are the major enzymes responsible for the inactivation of superoxide and hydrogen peroxide, respectively. Incubation of human umbilical vein endothelial cells (HUVEC) and HUVEC-derived EA.hy 926 cells with resveratrol resulted in a concentration- and time-dependent downregulation of Nox4, the most abundant NADPH oxidase catalytic subunit (quantitative real-time RT-PCR). The same resveratrol regimen upregulated the mRNA expression of SOD1 and GPx1. The addition the protein levels of SOD1 and GPx1 were enhanced by resveratrol in a concentration-dependent manner (Western blot analyses). Pretreatment of EA.hy 926 cells with resveratrol completely abolished DMNQ-induced oxidative stress. Thus, the expressional suppression of pro-oxidative genes (such as NADPH oxidase) and induction of anti-oxidative enzymes (such as SOD1 and GPx1) might be an important component of the vascular protective effect of resveratrol.

Dexamethasone suppresses eNOS and CAT-1 and induces oxidative stress in mouse resistance arterioles.

Long-term treatment with glucocorticoids is associated with mild to moderate hypertension. We reported previously that downregulation of endothelial NO synthase (eNOS) expression and activity is likely to contribute to this increase in blood pressure. In the present study, we tested the effects of dexamethasone on the vasodilation of microvascular arterioles using implanted dorsal skin-fold chambers in anesthetized C57BL/6J mice. Experiments were performed on control mice or on mice treated with dexamethasone (0.1-3 mg/kg of body wt). Endothelium-dependent vasodilation in response to ACh (0.1-10 microM) was reduced by dexamethasone in a dose-dependent fashion. Comparable inhibition was seen in tissues superfused with 30 microM N(G)-nitro-L-arginine methyl ester. In contrast, endothelium-independent vasodilation in response to S-nitroso-N-acetyl-D,L-penicillamine (10 microM) was not influenced by either dexamethasone or N(G)-nitro-L-arginine methyl ester. Levels of eNOS mRNA in murine hearts and NO(2)(-)/NO(3)(-) in serum were suppressed by dexamethasone (down to 63 and 50% of control values, respectively, at 3 mg/kg of body wt) along with a reduction in eNOS protein to 85.6%. Dexamethasone also concentration dependently reduced the expression of the cationic amino acid transporter-1 in murine hearts and cultured endothelial cells. The suppression by dexamethasone of the ACh-induced vasodilation could be partially reversed by dietary L-arginine (50 mg/kg of body wt) and by dietary vitamin C (10 g/kg of diet). We conclude that suppression by dexamethasone of the endothelium-mediated microvascular vasodilation involves several mechanisms including 1) downregulation of eNOS, 2) downregulation of cationic amino acid transporter-1, and 3) generation of reactive oxygen species. The demonstration that L-arginine and vitamin C can partially offset the effects of dexamethasone on microvascular arterioles suggests the potential clinical usefulness of these agents for the reduction of glucocorticoid-induced hypertension.

Ultraviolet A1 radiation induces nitric oxide synthase-2 expression in human skin endothelial cells in the absence of proinflammatory cytokines.

Skin exposure to ultraviolet radiation from sunlight causes erythema and edema formation as well as inflammatory responses. As some of these ultraviolet-induced effects are potentially mediated by nitric oxide synthases, we examined the role of cytokines and ultraviolet A1 radiation (340-400 nm) on the expression of the nitric oxide synthase-2 in endothelia of normal human skin biopsies during short-term organ culture as well as expression and activity of the nitric oxide synthase-2 in in vitro cell cultures of human dermal endothelial cells. Both, cytokine challenge (interleukin-1beta + tumor necrosis factor-alpha + interferon-gamma) but also ultraviolet A1 exposure (50 J per cm2) in the absence of cytokines led to the expression of nitric oxide synthase-2 in human skin organ cultures as shown by immunohistochemistry. Moreover, exposing human dermal endothelial cell cultures to proinflammatory cytokines but also to ultraviolet A1 radiation (6-24 J per cm2) in the absence of cytokines resulted in significant nitric oxide synthase-2 mRNA and protein expression as well as enzyme activity. Ultraviolet A1 irradiation of cytokine activated cells led to further increases in nitric oxide synthase-2 mRNA, protein expression, and enzyme activity. Moreover, a reporter gene assay using a human nitric oxide synthase-2 promoter construct provide evidence that ultraviolet A1, in the absence of cytokines, induces nitric oxide synthase-2 expression and activity, as previously shown for cytokines. Thus, the results presented here demonstrate for the first time that in dermal endothelia of human skin ultraviolet A1 radiation alone represents a proinflammatory stimulus sufficient to initiate nitric oxide synthase-2 expression as well as activity comparable with the respective response seen in the presence of proinflammatory cytokines.

The transport activity of the human cationic amino acid transporter hCAT-1 is downregulated by activation of protein kinase C.

1. The human cationic amino acid transporter hCAT-1 contains several consensus sequences for phosphorylation by protein kinase C (PKC). This study investigates the effect of PKC activation on hCAT-1-mediated transport. 2. When expressed in Xenopus laevis oocytes, hCAT-1-mediated L-arginine transport was reduced to 44+/-3% after a 30 min treatment of the oocytes with 100 nM phorbol-12-myristate-13-acetate (PMA). 4 alpha-phorbol-12,13-didecanoate (4 alpha-PDD, 100 nM) had no effect. 3. In EA.hy926 endothelial cells, maximal inhibition of hCAT-1-mediated L-arginine transport (to 3 -- 11% of control) was observed after treatment of the cells with 100 nM PMA for 4 h. A 20 -- 30 h exposure of the cells to 100 nM PMA led to the recovery of the L-arginine uptake rate that was now resistant to a second application of PMA. Phorbol-12,13-dibutyrate had similar effects as PMA, whereas 4 alpha-PDD had no effect. One microM bisindolylmaleimide I reduced the PMA effect significantly. 4. Interestingly, a 4 h treatment with 100 nM PMA increased the expression of hCAT-1 mRNA 3 -- 5 fold. hCAT-1 protein levels were unchanged for up to 4 h after PMA treatment and then increased slightly between 8 -- 28 h. 5. It is concluded that PMA downregulates the intrinsic activity of hCAT-1 by a pathway involving protein kinase C.

Mechanisms underlying endothelial dysfunction in diabetes mellitus.

Incubation of endothelial cells in vitro with high concentrations of glucose activates protein kinase C (PKC) and increases nitric oxide synthase (NOS III) gene expression as well as superoxide production. The underlying mechanisms remain unknown. To address this issue in an in vivo model, diabetes was induced with streptozotocin in rats. Streptozotocin treatment led to endothelial dysfunction and increased vascular superoxide production, as assessed by lucigenin- and coelenterazine-derived chemiluminescence. The bioavailability of vascular nitric oxide (as measured by electron spin resonance) was reduced in diabetic aortas, although expression of endothelial NOS III (mRNA and protein) was markedly increased. NOS inhibition with N:(G)-nitro-L-arginine increased superoxide levels in control vessels but reduced them in diabetic vessels, identifying NOS as a superoxide source. Similarly, we found an activation of the NADPH oxidase and a 7-fold increase in gp91(phox) mRNA in diabetic vessels. In vitro PKC inhibition with chelerythrine reduced vascular superoxide in diabetic vessels, whereas it had no effect on superoxide levels in normal vessels. In vivo PKC inhibition with N:-benzoyl-staurosporine did not affect glucose levels in diabetic rats but prevented NOS III gene upregulation and NOS-mediated superoxide production, thereby restoring vascular nitric oxide bioavailability and endothelial function. The reduction of superoxide in vitro by chelerythrine and the normalization of NOS III gene expression and reduction of superoxide in vivo by N:-benzoyl-staurosporine point to a decisive role of PKC in mediating these phenomena and suggest a therapeutic potential of PKC inhibitors in the prevention or treatment of vascular complications of diabetes mellitus. The full text of this article is available at http://www.circresaha.org.

Immuno-electron microscopic localization of the alpha(1) and beta(1)-subunits of soluble guanylyl cyclase in the guinea pig organ of corti.

Guanylyl cyclases (GC) catalyze the formation of the intracellular signal molecule cyclic GMP from GTP. For some years it has been known that the heme-containing soluble guanylyl cyclase (sGC) is stimulated by NO and NO-containing compounds. The sGC enzyme consists of two subunits (alpha(1) and beta(1)). In the present study, the alpha(1) and beta(1)-subunits were identified in the guinea pig cochlea at the electron microscopic level using a post-embedding immuno-labeling procedure. Ultrathin sections of LR White embedded specimens were incubated with various concentrations of two rabbit polyclonal antibodies to the alpha(1)- and beta(1)-subunit, respectively. The immunoreactivity was visualized by a gold-labeled secondary antibody in an energy-filtering transmission electron microscope (EFTEM). Marked immunoreactivity for both antibodies was found in the inner and outer hair cells, with numerous gold particles at the border of the cuticular plates, associated with the cell nuclei or attached to electron-dense parts of the cytoplasm. In the pillar cells and apical Deiters cells, soluble guanylyl cyclase immunoreactivity was located at the rim of the cuticular plates and between the microtubuli bundles. Together with the recently identified nitric oxide synthase isoforms [Eur. Arch. Otorhinolaryngol. 254 (1997) 396; Eur. Arch. Otorhinolaryngol. 255 (1998) 483], the soluble guanylyl cyclase may be involved in signalling processes in the organ of Corti.

Amphotericin B severely affects expression and activity of the endothelial constitutive nitric oxide synthase involving altered mRNA stability.

The therapeutic use of the antifungal drug amphotericin B (AmB) is limited due to severe side effects like glomerular vasoconstriction and risk of renal failure during AmB administration. As nitric oxide (NO) has substantial functions in renal autoregulation, we have determined the effects of AmB on endothelial constitutive NO synthase (ecNOS) expression and activity in human and rat endothelial cell cultures. AmB used at concentrations of 0.6 to 1.25 microg ml(-1) led to increases in ecNOS mRNA and protein expression as well as NO production. This was the result of an increased ecNOS mRNA half-life. In contrast, incubation of cells with higher albeit subtoxic concentrations of AmB (2.5 - 5.0 microg ml(-1)) resulted in a decrease or respectively in completely abolished ecNOS mRNA and protein expression with a strongly reduced or inhibited ecNOS activity, due to a decrease of ecNOS mRNA half-life. None of the AmB concentrations affected promoter activity as found with a reporter gene construct stably transfected into ECV304 cells. Thus, our experiments show a concentration-dependent biphasic effect of AmB on expression and activity of ecNOS, an effect best explained by AmB influencing ecNOS mRNA stability. In view of the known renal accumulation of this drug the results reported here could help to elucidate its renal toxicity.

Inhibition of small G proteins of the rho family by statins or clostridium difficile toxin B enhances cytokine-mediated induction of NO synthase II.

In order to investigate the involvement of Ras and/or Rho proteins in the induction of the inducible isoform of nitric oxide synthase (NOS II) we used HMG-CoA reductase inhibitors (statins) and Clostridium difficile toxin B (TcdB) as pharmacological tools. Statins indirectly inhibit small G proteins by preventing their essential farnesylation (Ras) and/or geranylgeranylation (Rho). In contrast, TcdB is a glucosyltransferase and inactivates Rho-proteins directly. Human A549/8- and DLD-1 cells as well as murine 3T3 fibroblasts were preincubated for 18 h with statins (1 - 100 microM) or TcdB (0.01-10 ng ml(-1)). Then NOS II expression was induced by cytokines. NOS II mRNA was measured after 4 - 8 h by RNase protection assay, and NO production were measured by the Griess assay after 24 h. Statins and TcdB markedly increased cytokine-induced NOS II mRNA expression and NO production. Statin-mediated enhancement of NOS II mRNA expression was reversed almost completely by cotreatment with mevalonate or geranylgeranylpyrophosphate. It was only slightly reduced by farnesylpyrophosphate. Therefore, small G proteins of the Rho family are likely to be involved in NOS II induction. In A549/8 cells stably transfected with a luciferase reporter gene under the control of a 16 kb fragment of the human NOS II promoter (pNOS2(16)Luc), statins produced only a small increase in cytokine-induced NOS II promoter activity. In contrast, statins had a considerable superinducing effect in DLD-1 cells stably transfected with pNOS2(16)Luc. In conclusion, our studies provide evidence that statins and TcdB potentiate cytokine-induced NOS II expression via inhibition of small G proteins of the Rho family. This in turn results in an enhanced NOS II promoter activity and/or a prolonged NOS II mRNA stability.

Effects of a nitrate-free interval on tolerance, vasoconstrictor sensitivity and vascular superoxide production.

OBJECTIVES: In the present study, we tested whether a nitrate-free interval is able to prevent increases in vascular superoxide (O2*-) and the development of hypersensitivity to vasoconstrictors and whether this may result in restoration of vascular nitroglycerin (NTG) sensitivity. BACKGROUND: Intermittent NTG-patch treatment (12 h patch on/patch-off) has been shown to increase ischemic periods in patients with stable coronary arteries, suggesting a rebound-like situation during the patch-off period. Recently, we demonstrated that long-term treatment with NTG induces tolerance, which was in part related to increases in vascular O2*- and increased vasoconstrictor sensitivity. METHODS: New Zealand white rabbits received a continuous application of NTG patches (0.4 mg/h) or an intermittent application of NTG patches (12 h patch on, 12 h patch off) for three days. Isometric tension studies were performed with aortic rings, and vascular O2*- was estimated using lucigenin-derived chemiluminescence (5 micromol/liter). Expression of the copper/zinc (Cu/Zn) superoxide dismutase (SOD) was assessed by Western blotting, and SOD activity was measured by autooxidation of 6-hydroxydopamine. RESULTS: Continuous treatment with NTG caused tolerance to NTG, cross-tolerance to the endothelium-dependent vasodilator acetylcholine, increased vascular O2*-, reduced Cu/Zn SOD expression and increased sensitivity to vasoconstrictors such as phenylephrine, serotonin and angiotensin II. On/off treatment with NTG improved tolerance, corrected endothelial dysfunction and decreased vascular O2*-. In addition the reduction in SOD expression was less pronounced, whereas increases in the sensitivity to vasoconstrictors such as phenylephrine and serotonin remained nearly unchanged. CONCLUSIONS: Enhanced vasoconstrictor sensitivity may explain, at least in part, the rebound phenomena observed in patients during a 12-h NTG patch-off period.

Complex contribution of the 3'-untranslated region to the expressional regulation of the human inducible nitric-oxide synthase gene. Involvement of the RNA-binding protein HuR.

Cytokine stimulation of human DLD-1 cells resulted in a marked expression of nitric-oxide synthase (NOS) II mRNA and protein accompanied by only a moderate increase in transcriptional activity. Also, there was a basal transcription of the NOS II gene, which did not result in measurable NOS II expression. The 3'-untranslated region (3'-UTR) of the NOS II mRNA contains four AUUUA motifs and one AUUUUA motif, known to destabilize the mRNAs of proto-oncogenes, nuclear transcription factors, and cytokines. Luciferase reporter gene constructs containing the NOS II 3'-UTR showed a significantly reduced luciferase activity. The embryonic lethal abnormal vision (ELAV)-like protein HuR was found to bind with high affinity to the adenylate/uridylate-rich elements of the NOS II 3'-UTR. Inhibition of HuR with antisense constructs reduced the cytokine-induced NOS II mRNA, whereas overexpression of HuR potentiated the cytokine-induced NOS II expression. This provides evidence that NOS II expression is regulated at the transcriptional and post-transcriptional level. Binding of HuR to the 3'-UTR of the NOS II mRNA seems to play an essential role in the stabilization of this mRNA.

Retinoic acid inhibits nitric oxide synthase-2 expression through the retinoic acid receptor-alpha.

Retinoids are multipotent modulators of cellular functions and suppress cytokine-induced production of nitric oxide (NO) in several cell types. We have explored the mechanisms by which retinoic acid (RA) regulates NO production in rat aortic smooth muscle cells (VSMC), which express NOS2 in response to proinflammatory cytokines. RA inhibited interleukin-1beta (IL-1beta)-induced NOS2 mRNA expression and NO production. These effects were attenuated by the retinoic acid receptor (RAR) antagonist CD3106, indicating that they were mediated through retinoic acid receptors (RARs). The synthetic retinoid agonists CD336 (which specifically binds RARalpha) and CD367 (which binds all RARs) but not agonists specific for RARbeta, RARgamma, or RXRs reduced IL-1beta-induced NOS2 expression and NO production. When transfecting VSMC with a 1570-bp NOS2 promoter fragment fused to a luciferase reporter gene, the NOS2 promoter activity was inhibited by RA. These results indicate that retinoids modulate NO production in VSMC via RARalpha, which inhibits the transcription of the NOS2 gene.

Nitric oxide in the pathogenesis of vascular disease.

Nitric oxide (NO) is synthesized by at least three distinct isoforms of NO synthase (NOS). Their substrate and cofactor requirements are very similar. All three isoforms have some implications, physiological or pathophysiological, in the cardiovascular system. The endothelial NOS III is physiologically important for vascular homeostasis, keeping the vasculature dilated, protecting the intima from platelet aggregates and leukocyte adhesion, and preventing smooth muscle proliferation. Central and peripheral neuronal NOS I may also contribute to blood pressure regulation. Vascular disease associated with hypercholesterolaemia, diabetes, and hypertension is characterized by endothelial dysfunction and reduced endothelium-mediated vasodilation. Oxidative stress and the inactivation of NO by superoxide anions play an important role in these disease states. Supplementation of the NOS substrate L-arginine can improve endothelial dysfunction in animals and man. Also, the addition of the NOS cofactor (6R)-5,6,7, 8-tetrahydrobiopterin improves endothelium-mediated vasodilation in certain disease states. In cerebrovascular stroke, neuronal NOS I and cytokine-inducible NOS II play a key role in neurodegeneration, whereas endothelial NOS III is important for maintaining cerebral blood flow and preventing neuronal injury. In sepsis, NOS II is induced in the vascular wall by bacterial endotoxin and/or cytokines. NOS II produces large amounts of NO, which is an important mediator of endotoxin-induced arteriolar vasodilatation, hypotension, and shock.

Structure-activity relationship of staurosporine analogs in regulating expression of endothelial nitric-oxide synthase gene.

In human umbilical vein endothelial cells and in human umbilical vein endothelial cell-derived EA.hy 926 cells, staurosporine (Stsp) and its glycosidic indolocarbazole analogs 7-hydroxystaurosporine (UCN-01) and 4'-N-benzoyl staurosporine (CGP 41251) enhanced nitric-oxide synthase (NOS) III mRNA expression (analyzed by RNase protection assay), protein expression (determined by Western blot), and activity [measured by rat fetal lung fibroblast (RFL-6) reporter cell assay] in a concentration- and time-dependent manner. In contrast, the bisindolylmaleimide analogs GF 109203X, Ro 31-8220 and Gö 6983 had no effect on NOS III expression, and Gö 6976, a methyl- and cyanoalkyl-substituted nonglycosidic indolocarbazole derivative of Stsp, even reduced NOS III expression in a concentration-dependent fashion. The up-regulation of NOS III expression by Stsp and analogs appears to be a transcriptional event because Stsp, 7-hydroxystaurosporine, and CGP 41251 enhanced the activity of a 1.6-kb human NOS III promoter fragment transiently transfected into EA.hy 926 endothelial cells. Stsp and analogs did not affect the stability of the NOS III mRNA. Stsp is known as a potent protein kinase (PK) inhibitor. Data obtained with other kinase inhibitors (and stimulators) indicated, however, that the effect of Stsp and analogs on NOS III expression was unrelated to the activities of PKC, PKA, PKG, or tyrosine kinase(s). Stsp analogs such as CGP 41251 also counteracted the NOS III mRNA-decreasing effect of tumor necrosis factor-alpha. These findings demonstrate that Stsp analogs represent a new class of compounds positively interacting with the transcription of the endothelial NOS III gene. Such compounds may prove useful in the prophylaxis and therapy of vascular disease.

Substrate supply for nitric-oxide synthase in macrophages and endothelial cells: role of cationic amino acid transporters.

The current study was designed to investigate the importance of cationic amino acid transporters (CATs) for the L-arginine supply to nitric oxide (NO) synthases in mouse J774A.1 macrophages and human EA.hy926 endothelial cells. CAT-1 was expressed in both cell types, whereas CAT-2B was only expressed in activated macrophages. Apparent K(M) values for transport of L-arginine in both cell types was consistent with the expression of the system y(+) carriers CAT-1 (and CAT-2B in macrophages). In addition, L-arginine transport was Na(+) independent and sensitive to trans-stimulation. A 2-h preincubation of activated macrophages in 2 mM L-lysine (which is exchanged for L-arginine by the CATs) reduced the intracellular L-arginine concentration from 2 mM to 160 microM. At the same time, nitric-oxide synthase (NOS) II activity was completely abolished. NOS II activity could be restored with extracellular L-arginine. No difference in NO production was seen between macrophages preincubated in L-arginine-containing buffer and incubated either with or without L-arginine during the 2-min NO assay. Incubation of endothelial cells in 2 mM L-lysine for up to 24 h decreased the intracellular L-arginine concentration from 3.5 mM to about 600 microM but did not reduce the NOS III activity. Our results suggest that both activated macrophages and endothelial cells have an L-arginine pool that is not freely exchangeable with the extracellular space. This pool seems to be accessible to NOS III in endothelial cells but not to NOS II in macrophages.

Effects of long-term nitroglycerin treatment on endothelial nitric oxide synthase (NOS III) gene expression, NOS III-mediated superoxide production, and vascular NO bioavailability.

Long-term nitroglycerin (NTG) treatment has been shown to be associated with cross-tolerance to endothelium-dependent vasodilators. It may involve increased production of reactive oxygen species (such as superoxide, O(2)(.-)) that rapidly inactivate the nitric oxide (NO) released from the endothelial cells. It remains to be elucidated, however, whether long-term treatment with NTG alters the activity and expression of the endothelial NO synthase (NOS III) and whether this enzyme can contribute to O(2)(.-) formation. We studied the influence of long-term NTG treatment on the expression of NOS III as assessed by RNase protection assay and Western blot. Tolerance was measured ex vivo in organ chamber experiments with rat aortic rings. O(2)(.-) and NO formation were quantified using lucigenin- and Cypridina luciferin analog-enhanced chemiluminescence as well as electron spin resonance (ESR) spectroscopy. Treatment of Wistar rats with NTG (Alzet osmotic minipumps, NTG concentration 10 microg x kg(-1) x min(-1)) for 3 days caused marked tolerance, cross-tolerance to the endothelium-dependent vasodilator acetylcholine, and a significant increase in O(2)(.-)-induced chemiluminescence. Tolerance was associated with a significant increase in NOS III mRNA to 236+/-28% and NOS III protein to 239+/-17%. In control vessels, the NOS inhibitor N(G)-nitro-L-arginine (L-NNA) increased the O(2)(.-)-mediated chemiluminescence, indicating that basal production of endothelium-derived NO depresses the baseline chemiluminescence signal. In the setting of tolerance, however, L-NNA decreased steady-state O(2)(.-) levels, indicating the involvement of NOS III in O(2)(.-) formation. Likewise, A23187-induced, NOS III-mediated O(2)(.-) production was more pronounced in tolerant than in control vessels. Vascular NO bioavailability as assessed with ESR spectroscopy using iron-thiocarbamate as a trap for NO was significantly reduced in tolerant vessels. Pretreatment of tolerant tissue in vitro with the protein kinase C (PKC) inhibitors reduced basal and stimulated NOS III-mediated O(2)(.-) production and partially reversed vascular tolerance. These findings suggest that NTG treatment increases the expression of a dysfunctional NOS III gene, leading to increased formation of O(2)(.-) and decreased vascular NO bioavailability. Normalization of NOS III-mediated O(2)(. -) production and improvement of tolerance with PKC inhibition suggests an important role for PKC isoforms in mediating vascular dysfunction caused by long-term NTG treatment.

Potential functional significance of brain-type and muscle-type nitric oxide synthase I expressed in adventitia and media of rat aorta.

Skeletal muscle and myocardium express microNOS I, an elongated splice variant of neuronal-type nitric oxide (NO) synthase (NOS I), and NOS III, endothelial-type NO synthase, respectively. This study was designed to elucidate whether vascular smooth muscle also contains a constitutively expressed NO synthase isoform. In the rat, microNOS I contains an insert of 102 nucleotides after nucleotide 2865 of the cDNA, yielding a protein of 164 kd. Reverse transcription-polymerase chain reaction with primers flanking this insert and with insert-specific primers indicated that endothelium-denuded rat aorta expresses both brain-type NOS I and microNOS I. RNase protection analyses with an antisense RNA probe overlapping the microNOS I insert detected significant amounts of NOS I mRNA and lesser amounts of microNOS I mRNA in endothelium-denuded aorta. Western blots using a specific polyclonal antibody recognizing NOS I and microNOS I showed a major band of the 160-kd NOS I and a lesser band of a slightly larger protein in endothelium-denuded aorta. Immunohistochemistry demonstrated low levels of NOS I/microNOS I immunoreactivity in the medial layer of rat aorta, whereas the endothelium expressed only NOS III immunoreactivity. When the adventitia also was removed, NOS I and microNOS I mRNA decreased markedly but remained detectable in the medial layer. In functional experiments with endothelium-denuded rat aortic rings (that contained no NOS III), contractions induced by KCl were markedly increased in the presence of the NOS inhibitor N(G)-nitro-L-arginine. These data demonstrate that 2 subforms of NOS I are expressed in nonendothelial components of rat aorta: NOS I and lesser amounts of microNOS I. Under certain conditions, this NOS I/microNOS I expression could serve as a backup system to the functionally predominant NOS III.