Michael S. Goligorsky

In vivo targeting of inducible NO synthase with oligodeoxynucleotides protects rat kidney against ischemia.

Gene products of all three distinct nitric oxide synthases are present in the mammalian kidney. This mosaic topography of nitric oxide synthase (NOS) isoforms probably reflects distinct functional role played by each enzyme. While nitric oxide (NO) is cytotoxic to isolated renal tubules, inhibition of NO production in vivo invariably results in the aggravation of renal dysfunction in various models of acute renal failure. We reasoned that the existing ambiguity on the role of nitric oxide in acute renal failure is in part due to the lack of selective NOS inhibitors. Phosphorothioated derivatives of antisense oligodeoxynucleotides targeting a conserved sequence within the open reading frame of the cDNA encoding the inducible NOS (iNOS) were designed to produce a selective knock-down of this enzyme. In vivo use of these antisense constructs attenuated acute renal failure in rats subjected to renal ischemia. This effect was due, at least in part, to the rescue of tubular epithelium from lethal injury. Application of antisense constructs did not affect endothelial NOS, as evidenced by a spared NO release after the infusion of bradykinin during in vivo monitoring with an NO-selective microelectrode. In conclusion, the data provide direct evidence for the cytotoxic effects of NO produced via iNOS in the course of ischemic acute renal failure, and offer a novel method to selectively prevent the induction of this enzyme.

Presence of the μ3 Opiate Receptor in Endothelial Cells COUPLING TO NITRIC OXIDE PRODUCTION AND VASODILATION

Initial confinement of opiate receptors to the nervous system has recently been broadened to several other cell types. Based on the well established hypotensive effect of morphine, we hypothesized that endothelial cells may represent a target for this opiate substance. Endothelial cells (human arterial and rat microvascular) contain a high affinity, saturable opiate binding site presumed to mediate the morphine effects that is stereoselectively and characteristically antagonized by naloxone. This opiate alkaloid-specific binding site is insensitive to opioid peptides. It is, therefore, considered to be the same subtype of opiate receptor (designated μ3) used in the mediation of morphine in other cell types exhibiting the same binding profile. Experiments with endothelial cultures and the aortic ring of rats cultured in vitro demonstrate that morphine exerts direct modulatory control over the activities of endothelial cells, which leads to vasodilation. It induces the production of nitric oxide, a process that is sensitive to naloxone antagonism and nitric oxide synthase inhibition. In contrast with that of opiates, the administration of opioid peptides does not induce nitric oxide production by endothelial cells. In conclusion, the data presented above reveal a novel site of morphine action, endothelial cells, where a μ3 receptor is coupled to nitric oxide release and vasodilation.

Carbon monoxide induces vasodilation and nitric oxide release but suppresses endothelial NOS.

The vascular effects of carbon monoxide (CO) resemble those of nitric oxide (NO), but it is unknown whether the two messengers converge or exhibit reciprocal feedback regulation. These questions were examined in microdissected perfused renal resistance arteries (RRA) studied using NO-sensitive microelectrodes. Perfusion of RRA with buffers containing increasing concentrations of CO resulted in a biphasic release of NO. The NO response peaked at 100 nM CO and then declined to virtually zero at 10 μM. When a series of 50-s pulses of 100 nM CO were applied repeatedly (150-s interval), the amplitude of consecutive NO responses was diminished. NO release from RRA showed dependence on l-arginine but notd-arginine, and the responses to CO were inhibited by pretreatment with N G-nitro-l-arginine methyl ester (l-NAME), an inhibitor of NO synthases (NOS). CO (100 nM) also suppressed NO release induced by 100 μM carbachol, a potent agonist for endothelial NOS (eNOS). RRA from rats in which endogenous CO production from inducible HO was elevated (cobalt chloride 12 h prior to study) also showed suppressed responses to carbachol. Furthermore, responses consistent with these findings were obtained in juxtamedullary afferent arterioles perfused in vitro, where the vasodilatory response to CO was biphasic and the response to acetylcholine was blunted. Collectively, these data suggest that the CO-induced NO release could be attributed to either stimulation of eNOS or to NO displacement from a cellular storage pool. To address this, direct in vitro measurements with an NO-selective electrode of NO production by recombinant eNOS revealed that CO dose-dependently inhibits NO synthesis. Together, the above data demonstrate that, whereas high levels of CO inhibit NOS activity and NO generation, lower concentrations of CO induce release of NO from a large intracellular pool and, therefore, may mimic the vascular effects of NO.The vascular effects of carbon monoxide (CO) resemble those of nitric oxide (NO), but it is unknown whether the two messengers converge or exhibit reciprocal feedback regulation. These questions were examined in microdissected perfused renal resistance arteries (RRA) studied using NO-sensitive microelectrodes. Perfusion of RRA with buffers containing increasing concentrations of CO resulted in a biphasic release of NO. The NO response peaked at 100 nM CO and then declined to virtually zero at 10 microM. When a series of 50-s pulses of 100 nM CO were applied repeatedly (150-s interval), the amplitude of consecutive NO responses was diminished. NO release from RRA showed dependence on L-arginine but not D-arginine, and the responses to CO were inhibited by pretreatment with NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthases (NOS). CO (100 nM) also suppressed NO release induced by 100 microM carbachol, a potent agonist for endothelial NOS (eNOS). RRA from rats in which endogenous CO production from inducible HO was elevated (cobalt chloride 12 h prior to study) also showed suppressed responses to carbachol. Furthermore, responses consistent with these findings were obtained in juxtamedullary afferent arterioles perfused in vitro, where the vasodilatory response to CO was biphasic and the response to acetylcholine was blunted. Collectively, these data suggest that the CO-induced NO release could be attributed to either stimulation of eNOS or to NO displacement from a cellular storage pool. To address this, direct in vitro measurements with an NO-selective electrode of NO production by recombinant eNOS revealed that CO dose-dependently inhibits NO synthesis. Together, the above data demonstrate that, whereas high levels of CO inhibit NOS activity and NO generation, lower concentrations of CO induce release of NO from a large intracellular pool and, therefore, may mimic the vascular effects of NO.

Cannabinoid Receptors Are Coupled to Nitric Oxide Release in Invertebrate Immunocytes, Microglia, and Human Monocytes*

The present study demonstrates that stereoselective binding sites for anandamide, a naturally occurring cannabinoid substance, can be found in invertebrate immunocytes and microglia. The anandamide-binding site is monophasic and of high affinity, exhibiting a Kd of 34.3 nM with a Bmax of 441 fmol/mg protein. These sites are highly selective, as demonstrated by the inability of other types of signaling molecules to displace [3H]anandamide. Furthermore, this binding site is coupled to nitric oxide release in the invertebrate tissues examined as well as in human monocytes. Interestingly, the cannabinoid-stimulated release of nitric oxide initiates cell rounding. Thus, these cannabinoid actions resemble those of opiate alkaloids. In this regard, we demonstrate that these signaling systems use the same effector system, i.e. nitric oxide release, but separate receptors. Last, the presence of a cannabinoid receptor in selected evolutionary diverse organisms indicates that this signaling system has been conserved for more than 500 million years.

Prevention and Reversal of Premature Endothelial Cell Senescence and Vasculopathy in Obesity-Induced Diabetes by Ebselen

Abstract— Although the accelerated atherosclerosis and premature aging of the cardiovascular system in patients with metabolic syndrome have been appreciated, the mechanisms of their development and potential therapeutic interventions remain unresolved. Our previous studies implicated advanced glycosylation end products in development of premature senescence preventable with a peroxynitrite scavenger, ebselen. Therefore, the effect of ebselen on endothelial senescence and vasculopathy in a model of metabolic syndrome—Zucker diabetic rats (ZDF)—was investigated. Ebselen decreased the abundance of 3-nitrotyrosine–modified proteins in ZDF rats. A 6-fold increase in the number of senescent endothelial cells in 22-week-old ZDF was prevented by ebselen. Development of vasculopathy, as collectively judged by the acetylcholine-induced vasorelaxation, NO production, angiogenic competence, and number of circulating microparticles, was almost completely prevented when ebselen was administered from 8 to 22 weeks and partially reversed when the treatment interval was 13 to 22 weeks. In conclusion, premature senescence of endothelial cells is progressively rampant in ZDF rats and is associated with the signs of severe vasculopathy. In addition, prevention of premature senescence of vascular endothelium through controlled decrease in nitrotyrosine formation was chronologically associated with the amelioration of vasculopathy, lending support to the idea of the pathogenetic role of premature senescence of endothelial cells in diabetic macrovasculopathy.

Ratio of 5,6,7,8-tetrahydrobiopterin to 7,8-dihydrobiopterin in endothelial cells determines glucose-elicited changes in NO vs. superoxide production by eNOS

5,6,7,8-Tetrahydrobiopterin (BH(4)) is an essential cofactor of nitric oxide synthases (NOSs). Oxidation of BH(4), in the setting of diabetes and other chronic vasoinflammatory conditions, can cause cofactor insufficiency and uncoupling of endothelial NOS (eNOS), manifest by a switch from nitric oxide (NO) to superoxide production. Here we tested the hypothesis that eNOS uncoupling is not simply a consequence of BH(4) insufficiency, but rather results from a diminished ratio of BH(4) vs. its catalytically incompetent oxidation product, 7,8-dihydrobiopterin (BH(2)). In support of this hypothesis, [(3)H]BH(4) binding studies revealed that BH(4) and BH(2) bind eNOS with equal affinity (K(d) approximately 80 nM) and BH(2) can rapidly and efficiently replace BH(4) in preformed eNOS-BH(4) complexes. Whereas the total biopterin pool of murine endothelial cells (ECs) was unaffected by 48-h exposure to diabetic glucose levels (30 mM), BH(2) levels increased from undetectable to 40% of total biopterin. This BH(2) accumulation was associated with diminished calcium ionophore-evoked NO activity and accelerated superoxide production. Since superoxide production was suppressed by NOS inhibitor treatment, eNOS was implicated as a principal superoxide source. Importantly, BH(4) supplementation of ECs (in low and high glucose-containing media) revealed that calcium ionophore-evoked NO bioactivity correlates with intracellular BH(4):BH(2) and not absolute intracellular levels of BH(4). Reciprocally, superoxide production was found to negatively correlate with intracellular BH(4):BH(2). Hyperglycemia-associated BH(4) oxidation and NO insufficiency was recapitulated in vivo, in the Zucker diabetic fatty rat model of type 2 diabetes. Together, these findings implicate diminished intracellular BH(4):BH(2), rather than BH(4) depletion per se, as the molecular trigger for NO insufficiency in diabetes.

Permissive Role of Nitric Oxide in Endothelin-induced Migration of Endothelial Cells

Endothelin (ET) synthesis is enhanced at sites of ischemia or in injured vessels. The purpose of this study was to explore the possibility of autocrine stimulation of endothelial cell migration by members of the endothelin family. Experiments with microvascular endothelial cell transmigration in a Boyden chemotactic apparatus showed that endothelins 1 and 3, as well as a selective agonist of ETB receptor IRL-1620, equipotently stimulated migration. Endothelial cell migration was unaffected by the blockade of ETA receptor, but it was inhibited by ETB receptor antagonism. Based on our previous demonstration of signaling from the occupied ETB receptor to constitutive nitric oxide (NO) synthase (Tsukahara, H., Ende, H., Magazine, H. I., Bahou, W. F., and Goligorsky, M. S. (1994) J. Biol. Chem. 269, 21778-21785), we next examined the contribution of ET-stimulated NO production to endothelial cell migration. In three independent cellular systems, 1) migration and wound healing by microvascular endothelial cells, 2) wound healing by Chinese hamster ovary cells stably expressing ETB receptor with or without endothelial NO synthase, and 3) application of antisense oligodeoxynucleotides targeting endothelial NO synthase in human umbilical vein endothelial cells, an absolute requirement for the functional NO synthase in cell migration has been demonstrated. These findings establish the permissive role of NO synthesis in endothelin-stimulated migration of endothelial cells.

Nephropathy in Zucker Diabetic Fat Rat Is Associated with Oxidative and Nitrosative Stress: Prevention by Chronic Therapy with a Peroxynitrite Scavenger Ebselen

Zucker diabetic fat (ZDF) rats with the metabolic syndrome and hyperlipidemia develop focal and segmental sclerosis. The role of oxidative and nitrosative stress in the nephropathy in ZDF was studied. Renal histology, function, and immunohistologic and biochemical parameters of oxidative and nitrosative stress were evaluated at 8 and 22 wk of age in ZDF and Zucker lean (ZL) rats and after chronic treatment with ebselen, an antioxidant and peroxinitrite scavenger. At 8 wk, ZDF rats showed hyperglycemia, no proteinuria or nephropathy, but higher levels of dihydrobiopterin and 3-nitrotyrosine (3-NT)-modified proteins compared with age-matched ZL rats. At 22 wk, ZDF rats developed focal and segmental sclerosis, proteinuria, decreased creatinine clearance, and renal tissue levels of glutathione and tetrahydrobiopterin with further elevation in dihydrobiopterin and 3-NT-modified proteins, in contrast to age-matched ZL rats. Renal immunohistologic expression of lipid peroxidation products and 3-NT-modified proteins also increased in 22-wk-old ZDF but not in ZL rats. Chronic ebselen treatment of ZDF rats restored renal tissue levels of glutathione and tetrahydrobiopterin; prevented significant accumulation of dihydrobiopterin, lipid peroxidation products, and 3-NT-modified proteins; and ameliorated focal and segmental sclerosis, proteinuria, and fall in creatinine clearance without affecting mean BP, body weight, and blood glucose, compared with the untreated ZDF rats. Chronic ebselen therapy also ameliorated vasculopathy with lipid deposits and tubulointerstitial scarring, inflammation, and upregulated alpha-smooth muscle actin expression. These findings suggest that ZDF rats develop a progressive nephropathy with glomerular, vascular, and tubulointerstitial pathology. Oxidative and nitrosative stress predates the nephropathy, which is improved by peroxinitrite scavenger ebselen, and thus considered its cause and not consequence.

Bioinformatic Analysis of the Urine Proteome of Acute Allograft Rejection

The urinary proteome in health and disease attracts increasing attention because of the potential diagnostic and pathophysiologic biomarker information carried by specific excreted proteins or their constellations. This cross-sectional study aimed to analyze the urinary proteome in patients with biopsy-proven acute rejection (n = 23) compared with transplant recipients with stable graft function (n = 22) and healthy volunteers (n = 20) and to correlate this with clinical, morphologic, and laboratory data. Urine samples were preadsorbed on four different protein chip surfaces, and the protein composition was analyzed using a surface-enhanced laser desorption/ionization time-of-flight mass spectrometer platform. The data were analyzed using two independent approaches to sample classification. Patients who experienced acute rejection could be distinguished from stable patients with a sensitivity of 90.5 to 91.3% and a specificity of 77.2 to 83.3%, depending on the classifier used. Protein masses that were important in constructing the classification algorithms included those of mass 2003.0, 2802.6, 4756.3, 5872.4, 6990.6, 19,018.8, and 25,665.7 Da. Normal urine was distinguished from transplant urine using a protein marker of mass 78,531.2 Da with both a sensitivity and a specificity of 100%. In conclusion, (1) urine proteome in transplant recipients with stable graft function was significantly different from healthy control subjects, and (2) acute rejections were characterized by a constellation of excreted proteins. Analysis of the urinary proteome may expedite the noninvasive prediction of acute graft rejection, thus importantly assisting in establishing the diagnosis.

Workshop: Endothelial Cell Dysfunction Leading to Diabetic Nephropathy Focus on Nitric Oxide

Clinical manifestations of diabetic nephropathy are an expression of diabetic microangiopathy. This review revisits the previously proposed Steno hypothesis and advances our hypothesis that development of endothelial cell dysfunction represents a common pathophysiological pathway of diabetic complications. Specifically, the ability of glucose to scavenge nitric oxide is proposed as the initiation phase of endothelial dysfunction. Gradual accumulation of advanced glycated end products and induction of plasminogen activator inhibitor-1, resulting in the decreased expression of endothelial nitric oxide synthase and reduced generation of nitric oxide, are proposed to be pathophysiologically critical for the maintenance phase of endothelial dysfunction. The proposed conceptual shift toward the role of endothelial dysfunction in diabetic complications may provide new strategies for their prevention.