Endothelin receptor A-specific stimulation of glomerular inflammation and injury in a streptozotocin-induced rat model of diabetes.

AIMS/HYPOTHESIS: Activation of endothelin receptor-A (ET(A)) increases glomerular permeability to albumin (P(alb)) and elevates pro-inflammatory markers in hyperglycaemic rats. METHODS: Male Sprague-Dawley rats were given streptozotocin (n = 32) or saline (sham; n = 32). Half of the animals in each group received the ET(A)-selective antagonist, ABT-627 (atrasentan; orally), beginning immediately after hyperglycaemia was confirmed. Glomeruli were isolated by sieving techniques and P(alb) determined from the change in glomerular volume induced by oncotic gradients of albumin. Glomerular nephrin levels were assessed by immunofluorescence, whereas urinary nephrin was measured by immunoassay. RESULTS: At 3 and 6 weeks after streptozotocin injection, proteinuria was significantly increased compared with sham controls and significantly reduced by ABT-627 treatment. P(alb) was also increased at 3 and 6 weeks post-streptozotocin. ABT-627 had no effect on P(alb) or protein excretion in sham control rats. In glomeruli isolated from hyperglycaemic rats, incubation with BQ-123, a selective ET(A) antagonist, reduced P(alb), whereas BQ-788, a selective endothelin receptor-B antagonist had no effect (n = 6 rats per group, 5-8 glomeruli per rat). Glomerular and plasma content of soluble intercellular adhesion molecule-1 and monocyte chemoattractant protein-1 were significantly increased 6 weeks after streptozotocin (ELISA). ABT-627 attenuated these increases. After 6 weeks of hyperglycaemia, glomerular nephrin content was decreased with a concurrent increase in urinary nephrin excretion. ABT-627 prevented glomerular nephrin loss in hyperglycaemic rats (n = 5-8 rats per group; eight groups). CONCLUSIONS/INTERPRETATION: These observations support the hypothesis that endothelin-1, via the ET(A) receptor, directly increases P(alb), possibly via nephrin loss, as well as early inflammation in the hyperglycaemic rat.

Natriuretic response to renal medullary endothelin B receptor activation is impaired in Dahl-salt sensitive rats on a high-fat diet.

Renal medullary endothelin B receptors (ET(B)) mediate sodium excretion and blood pressure (BP) control. Several animal models of hypertension have impaired renal medullary ET(B) function. We found that 4-week high-caloric diet elevated systolic BP in Dahl salt-sensitive (Dahl S) rats (126+/-2 vs. 143+/-3 mm Hg, p<0.05). We hypothesized that renal medullary ET(B) function is dysfunctional in DS rats fed a high-caloric diet. We compared the diuretic and natriuretic response to intramedullary infusion of ET(B) agonist sarafotoxin 6c (S6c) in DS rats fed either a normal or high-caloric diet for 4 weeks. Urine was collected during intramedullary infusion of saline for baseline collection followed by intramedullary infusion of either saline or S6c. We first examined the ET(B) function in DS rats fed a normal diet. S6c increased urine flow (2.7+/-0.3 microl/min during baseline vs. 5.1+/-0.6 microl/min after S6c; p<0.05; n=5) and sodium excretion (0.28+/-0.05 vs. 0.81+/-0.17 micromol/min; p<0.05), suggesting that DS rats have renal medullary ET(B) function. However, DS rats fed a high-caloric diet displayed a significant increase in urine flow (2.7+/-0.4 vs. 4.2+/-0.4 microl/min, baseline vs. S6c infusion, respectively; p<0.05, n=6), but no significant change in sodium excretion in response to S6c (0.32+/-0.06 vs. 0.45+/-0.10 micromol/min). These data demonstrate that renal medullary ET(B) function is impaired in DS rats fed a high-caloric diet, which may be contributed to the elevation of blood pressure during high-caloric feeding in this model.

Production of goats by somatic cell nuclear transfer.

In this study, we demonstrate the production of transgenic goats by nuclear transfer of fetal somatic cells. Donor karyoplasts were obtained from a primary fetal somatic cell line derived from a 40-day transgenic female fetus produced by artificial insemination of a nontransgenic adult female with semen from a transgenic male. Live offspring were produced with two nuclear transfer procedures. In one protocol, oocytes at the arrested metaphase II stage were enucleated, electrofused with donor somatic cells, and simultaneously activated. In the second protocol, activated in vivo oocytes were enucleated at the telophase II stage, electrofused with donor somatic cells, and simultaneously activated a second time to induce genome reactivation. Three healthy identical female offspring were born. Genotypic analyses confirmed that all cloned offspring were derived from the donor cell line. Analysis of the milk of one of the transgenic cloned animals showed high-level production of human antithrombin III, similar to the parental transgenic line.

Increased reactive oxygen species contributes to kidney injury in mineralocorticoid hypertensive rats.

Hypertension is associated with increased reactive oxygen species (ROS). Renal ROS production and their effects on renal function have never been investigated in mineralocorticoid hypertensive rats. In this study we hypothesized that increased ROS production in kidneys from deoxycorticosterone (DOCA)-salt rats contributes to adverse renal morphological changes and impaired renal function in DOCA-salt hypertensive rats. We also determined whether ROS-induced renal injury was dependent on blood pressure. DOCA-salt hypertensive rats exhibited a marked increase in blood pressure, renal ROS production, glomerular and tubular lesions, and microalbuminuria compared to sham rats. Treatment of DOCA-salt hypertensive rats with apocynin for 28 days resulted in attenuation of systolic blood pressure and improvement of renal morphology. Renal superoxide level in DOCA-salt rats was 215% of sham-operated rats and it was significantly decreased to 140% with apocynin treatment. Urinary protein level was decreased from 27 +/- 3 mg/day in DOCA-salt hypertensive rats to 9 +/- 2 mg/day. 28 days of Vitamin E treatment also reduced renal injury in regard to urinary protein level and renal morphology but had no effect on blood pressure in DOCA-salt rats. Increased urinary 8-isoprostane, a marker for oxidative stress, in DOCA-salt hypertensive rats (55 +/- 8 ng/day) was diminished by vitamin E treatment (24 +/- 6 ng/day). These data suggest that renal injury characteristic of mineralocorticoid hypertension is associated with oxidative stress and is partly independent of blood pressure.

Nitric oxide synthases in the cardiovascular system.

The endothelium-derived relaxing factor that mediates the endothelium-dependent vasodilatation first observed in 1980 has been identified as nitric oxide (NO). In addition to the endothelium, NO is formed in other cells such as neuronal cells of the brain (where it mediates synoptic plasticity), peripheral nonadrenergic noncholinergic (NANC) nerves (where it acts as an atypical neurotransmitter relaxing vascular and nonvascular smooth muscle), and various specialized epithelial cells. Other cell types such as macrophages and smooth muscle cells can be induced with bacterial endotoxin and/or cytokines to synthesize large amounts of the radical. At low concentrations, NO is an inter- and intracellular messenger molecule whose target enzyme is the soluble isoform of guanylyl cyclase. At high concentrations, the NO radical has cytostatic effects on parasitic microorganisms and tumor cells. In the vascular system, endothelium-derived NO is a physiologically significant vasodilator and inhibitor of platelet aggregation and adhesion. NANC nerve-derived NO may also contribute to vasodilatation. In addition, NO can prevent leukocyte adhesion to the endothelium by interfering with the adhesion molecule CD11/CD18, and NO has been shown to inhibit the proliferation of vascular smooth muscle cells. In sepsis and during cytokine therapy, a different NOS is induced in the vascular wall (presumably in smooth muscle cells) where it synthesizes large amounts of NO that contribute to the massive vasodilatation and shock.

Hypoxia activates nitric oxide synthase and stimulates nitric oxide production in porcine coronary resistance arteriolar endothelial cells.

OBJECTIVE: Hypoxia significantly alters vascular tone in coronary resistance arterioles during prolonged ischemia, potentially through the modulation of endothelial cell metabolism as well as endothelial function. The objective of this study was to test the hypothesis that constitutive nitric oxide synthase (cNOS) is sensitive to oxygen tension and that hypoxia increases the activity of cNOS and nitric oxide production in the porcine coronary microcirculation. METHODS: Monocultures of porcine coronary resistance arteriolar endothelial cells (RAEC) were isolated and proven to be endothelium based upon morphology, binding of acetylated LDL, and factor VIII antigen positivity. Cells were exposed to either hypoxia (pO2 = 10 mmHg) or normoxia (pO2 = 160 mmHg) for varying periods of time. Nitric oxide production was directly measured using a chemiluminescence method, while cNOS enzyme activity was assayed using a fibroblast-report cell method. cNOS protein was quantitated by Western blot analysis using the H32 monoclonal antibody to the endothelial cell constitutive isoform of NOS. RESULTS: Hypoxia significantly augmented A23187-stimulated nitric oxide production [23.77 (1.73) vs 14.94 (0.66) nmol . micrograms-1 protein, hypoxia vs. normoxia respectively, n = 8, P < 0.01]. Using the fibroblast reporter cell assay, cNOS activity was increased in RAEC after exposure to hypoxia for 30, 120 and 240 min [normoxia control: 0.16 (0.04) fmol . microgram-1 protein; hypoxia: 30 min = 1.00 (0.19), 120 min = 1.08 (0.04), 240 min = 1.26 (0.07) fmol . micrograms-1 protein (n = 6, p < 0.01)]. Western blots showed a single band at 135 kDa that was increased in homogenates of cells previously exposed to hypoxia. CONCLUSIONS: These experiments demonstrated that the regulation of cNOS is sensitive to oxygen tension. Hypoxia significantly activated constitutive nitric oxide synthase in coronary resistance arteriolar endothelial cells, and this was translated to an increased production of nitric oxide.

ET-1 increases reactive oxygen species following hypoxia and high-salt diet in the mouse glomerulus.

AIM: This study was designed to determine whether ET-1 derived from endothelial cells contributes to oxidative stress in the glomerulus of mice subjected to a high-salt diet and/or hypoxia. METHODS: C57BL6/J control mice or vascular endothelial cell ET-1 knockout (VEET KO) mice were subjected to 3-h exposure to hypoxia (8% O2) and/or 2 weeks of high-salt diet (4% NaCl) prior to metabolic cage assessment of renal function and isolation of glomeruli for the determination of reactive oxygen species (ROS). RESULTS: In control mice, hypoxia significantly increased urinary protein excretion during the initial 24 h, but only in animals on a high-salt diet. Hypoxia increased glomerular ET-1 mRNA expression in control, but not in vascular endothelial cell ET-1 knockout (VEET KO) mice. Under normoxic conditions, mice on a high-salt diet had approx. 150% higher glomerular ET-1 mRNA expression compared with a normal-salt diet (P < 0.05). High-salt diet administration significantly increased glomerular ROS production in flox control, but not in glomeruli isolated from VEET KO mice. In C57BL6/J mice, the ETA receptor-selective antagonist, ABT-627, significantly attenuated the increase in glomerular ROS production produced by high-salt diet. In addition, chronic infusion of C57BL6/J mice with a subpressor dose of ET-1 (osmotic pumps) significantly increased the levels of glomerular ROS that were prevented by ETA antagonist treatment. CONCLUSION: These data suggest that both hypoxia and a high-salt diet increase glomerular ROS production via endothelial-derived ET-1-ETA receptor activation and provide a potential mechanism for ET-1-induced nephropathy.

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.

Ca2+/calmodulin-regulated nitric oxide synthases.

NO synthase (NOS) catalyzes the oxidation of L-arginine to L-citrulline and nitric oxide (NO) or a NO-releasing compound. At least three isoforms of NOS exist (types I-III). The activities of the type I isoform purified from brain and the type III isoform purified from endothelial cells are regulated by the intracellular free calcium concentration ([Ca2+]i) and the Ca(2+)-binding protein calmodulin. At resting [Ca2+]i, both isozymes are inactive; they become fully active at [Ca2+]i greater than or equal to 500 nM Ca2+. Longer lasting increases in [Ca2+]i may downregulate NO formation, for in vitro phosphorylation by Ca2+/calmodulin protein kinase II decreases the Vmax of NOS. Besides the conversion of L-arginine, type I NOS, Ca2+/calmodulin dependently, generates H2O2 and reduces cytochrome c/P450. Other redox activities, i.e. the reduction of nitroblue tetrazolium to diformazan (NADPH-diaphorase) or of quinoid-dihydrobiopterin to tetrahydrobiopterin, by NOS appear to be Ca2+/calmodulin-independent.

Genomic analysis and expression patterns reveal distinct genes for endothelial and brain nitric oxide synthase.

Constitutively active nitric oxide synthases (NOS) are a unique class of NADPH-dependent, calcium/calmodulin-dependent enzymes that catalyze the conversion of L-arginine to nitric oxide and L-citrulline. However, little is known about the molecular similarities or differences between the two prototypical constitutive NOS enzymes, endothelial NOS (ECNOS) and brain NOS (bNOS). The aims of this study were to begin characterizing the gene structure and tissue distribution of messenger RNAs (mRNAs) for ECNOS and bNOS and to examine the immunological resemblance of the proteins by Western blotting. Full-length complementary DNAs (cDNAs) encoding bovine ECNOS and rat bNOS hybridized, under high stringency, to different-sized fragments of endonuclease-digested bovine, rat, and human genomic DNA. In addition, more than one fragment was detected with both cDNAs, suggesting that ECNOS and bNOS genes contained multiple introns. Tissue distribution of ECNOS mRNA (4.4 kb) and bNOS mRNA (9.5 kb) in the rat was detected by Northern blotting. Patterns among tissue extracts were strikingly different, with ECNOS mRNA being most abundant in aorta, heart, lung, kidney, adrenal gland, spinal cord, and urogenital tissues and bNOS mRNA most prominent in brain regions, intestine, stomach, spinal cord, adrenal gland, and aorta. Interestingly, ECNOS cDNA detected two equally abundant RNA transcripts (4.4 and 4.0 kb) in most brain regions tested, suggesting an alternative splicing of the ECNOS pre-mRNA. Western blotting, using an ECNOS monoclonal antibody, recognized ECNOS protein from native bovine endothelial cells, cultured bovine endothelial cells, and COS cells transfected with ECNOS cDNA but did not recognize purified bNOS.(ABSTRACT TRUNCATED AT 250 WORDS)

Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions.

Three isozymes of nitric oxide (NO) synthase (EC 1.14.13.39) have been identified and the cDNAs for these enzymes isolated. In humans, isozymes I (in neuronal and epithelial cells), II (in cytokine-induced cells), and III (in endothelial cells) are encoded for by three different genes located on chromosomes 12, 17, and 7, respectively. The deduced amino acid sequences of the human isozymes show less than 59% identity. Across species, amino acid sequences for each isoform are well conserved (> 90% for isoforms I and III, > 80% for isoform II). All isoforms use L-arginine and molecular oxygen as substrates and require the cofactors NADPH, 6(R)-5,6,7,8-tetrahydrobiopterin, flavin adenine dinucleotide, and flavin mononucleotide. They all bind calmodulin and contain heme. Isoform I is constitutively present in central and peripheral neuronal cells and certain epithelial cells. Its activity is regulated by Ca2+ and calmodulin. Its functions include long-term regulation of synaptic transmission in the central nervous system, central regulation of blood pressure, smooth muscle relaxation, and vasodilation via peripheral nitrergic nerves. It has also been implicated in neuronal death in cerebrovascular stroke. Expression of isoform II of NO synthase can be induced with lipopolysaccharide and cytokines in a multitude of different cells. Based on sequencing data there is no evidence for more than one inducible isozyme at this time. NO synthase II is not regulated by Ca2+; it produces large amounts of NO that has cytostatic effects on parasitic target cells by inhibiting iron-containing enzymes and causing DNA fragmentation. Induced NO synthase II is involved in the pathophysiology of autoimmune diseases and septic shock. Isoform III of NO synthase has been found mostly in endothelial cells. It is constitutively expressed, but expression can be enhanced, eg, by shear stress. Its activity is regulated by Ca2+ and calmodulin. NO from endothelial cells keeps blood vessels dilated, prevents the adhesion of platelets and white cells, and probably inhibits vascular smooth muscle proliferation.

Endothelial nitric oxide synthase is myristylated.

The enzyme responsible for the synthesis of endothelium-derived relaxing factor and/or nitric oxide in the endothelium has been described as a particulate enzyme, whereas other isoforms of nitric oxide synthase are soluble enzymes. Here we are reporting that endothelial cells metabolically incorporate myristate (C14), but not palmitate (C16), into nitric oxide synthase. We are postulating that the endothelial-derived nitric oxide synthase is a particulate enzyme because of the fatty acid acylation of the protein which 'anchors' the enzyme into the membrane either directly or via another membrane-bound protein.

Cloned human brain nitric oxide synthase is highly expressed in skeletal muscle.

Complementary DNA clones corresponding to human brain nitric oxide (NO) synthase have been isolated. The deduced amino acid sequence revealed an overall identity with rat brain NO synthase of about 93% and contained all suggested consensus sites for binding of the co-factors. The cDNA transfected COS-1 cells showed significant NO synthase activity with the typical co-factor requirements. Unexpectedly, messenger RNA levels of this isoform of NO synthase was more abundant in human skeletal muscle than human brain. Moreover, we detected high NO synthase activity and the expressed protein in human skeletal muscle by Western blot analysis, indicating a possible novel function of NO in skeletal muscle.

Mapping of neural nitric oxide synthase in the rat suggests frequent co-localization with NADPH diaphorase but not with soluble guanylyl cyclase, and novel paraneural functions for nitrinergic signal transduction.

Nitric oxide synthases (NOS Types I-III) generate nitric oxide (NO), which in turn activates soluble guanylyl cyclase (GC-S). The distribution of this NO-mediated (nitrinergic) signal transduction pathway in the body is unclear. A polyclonal monospecific antibody to rat cerebellum NOS-I and a monoclonal antibody to rat lung GC-S were employed to localize the protein components of this pathway in different rat organs and tissues. We confirmed the localization of NOS-I in neurons of the central and peripheral nervous system, where NO may regulate cerebral blood flow and mediate long-term potentiation. GC-S was located in NOS-negative neurons, indicating that NO acts as an intercellular signal molecule or neurotransmitter. However, NOS-I was not confined to neurons but was widely distributed over several non-neural cell types and tissues. These included glia cells, macula densa of kidney, epithelial cells of lung, uterus, and stomach, and islets of Langerhans. Our findings suggest that NOS-I is the most widely distributed isoform of NOS and, in addition to its neural functions, regulates secretion and non-vascular smooth muscle function. With the exception of bone tissue, NADPH-diaphorase (NADPH-d) activity was generally co-localized with NOS-I immunoreactivity in both neural and non-neural cells, and is a suitable histochemical marker for NOS-I but not a selective neuronal marker.

Identification of the NO synthase isoforms expressed in human neutrophil granulocytes, megakaryocytes and platelets.

Using Western blot and fluorescent immunocytochemistry, NOS III (or ecNOS) and NOS II (or iNOS), but no NOS I (or ncNOS), were identified in preparations of human platelets. Reverse-transcription polymerase chain reactions (RT-PCR) demonstrated NOS III mRNA, but no NOS II mRNA (which is short-lived) and no NOS I mRNA in platelets. Immunofluorescent staining of human bone marrow smears showed the presence of NOS III, but not NOS I in megakaryocytes. A subpopulation of megakaryocytes also expressed NOS II. In preparations of human neutrophils, immunocytochemistry demonstrated NOS I in all cells, whereas no NOS III was detected. The few NOS II positive cells were characterized as contaminating eosinophils. Similarly, in RT-PCR, transcripts for NOS I and NOS II, but not for NOS III, were identified. Thus, the constitutive NOS isoform in megakaryocytes and platelets is NOS III, whereas neutrophils express NOS I. Some megakaryocytes and eosinophils also express NOS II.

Characterization and localization of nitric oxide synthase in non-adrenergic non-cholinergic nerves from bovine retractor penis muscles.

1. Partially purified soluble nitric oxide (NO) synthase was isolated from the bovine retractor penis muscle (BRP), a tissue in which the inhibitory response to non-adrenergic non-cholinergic nerve (NANC) stimulation appears to be mediated by NO or NO-like material. 2. NO synthase from BRP used L-arginine as a substrate, required NADPH, tetrahydrobiopterin, and FAD as co-factors and was Ca2+/calmodulin-dependent. The activity of NO synthase was inhibited by NG-methyl-L-arginine and NG-nitro-L-arginine, and haemoglobin blocked the effect of NO formed by the enzyme. 3. On reducing SDS polyacrylamide gel electrophoresis the apparent molecular mass of NO synthase from BRP was 160 +/- 2 kDa, which is similar to that of the cerebellar NO synthase. Protein immunoblot and immunoprecipitation showed that NO synthase from BRP cross-reacted with the selective antiserum to neuronal NO synthase from rat cerebellum. 4. Immunohistochemistry using the same antiserum demonstrated that NO synthase in BRP was located exclusively within nerve fibres. Thus, autonomic nerves synthesizing the NANC neurotransmitter seem to contain an isoform of NO synthase which is similar to that from rat cerebellum.

Immunohistochemical localization of endothelial nitric oxide synthase in human villous and extravillous trophoblast populations and expression during syncytiotrophoblast formation in vitro.

We have examined the distribution of the endothelial isoform of nitric oxide synthase (eNOS) in villous and extravillous trophoblast populations by immunohistochemistry and have further studied expression of eNOS during differentiation of cytotrophoblast into syncytiotrophoblast in culture. In first trimester villous tissue, NADPH diaphorase activity and eNOS immunostaining were present in syncytiotrophoblast but not the progenitor cytotrophoblast layer. Extravillous trophoblast in the basal plate of the placenta was identified by anticytokeratin immunostaining and displayed NADPH diaphorase activity, but not eNOS immunostaining. Both amnion epithelial cells and chorion cytotrophoblast had NADPH diaphorase activity but no eNOS immunostaining, whereas eNOS immunostaining was seen in the fibroblast layer of amnion. Purified villous cytotrophoblast cells from term placentae aggregated and fused to form a syncytium with increasing time in culture as assessed by antidesmosomal protein and antinuclear antibody immunostaining. Following 24 h in culture, the majority of cells were still mononucleate cytotrophoblast which did not display eNOS immunostaining, whereas a few syncytial aggregates had formed which were both eNOS positive and hPL positive. By 3 to 5 days in culture, the majority of cells were present as syncytiotrophoblast. However, eNOS and hPL immunostaining was more diffuse and not all syncytial aggregates were positive. Of the trophoblast populations, only syncytiotrophoblast appears to express eNOS. Differentiation of cytotrophoblast into syncytiotrophoblast is associated with eNOS expression.

Immunohistochemical localization of nitric oxide synthase in the human placenta.

We have studied the distribution of the endothelial isoform of nitric oxide synthase (NOS) through the term human umbilical cord and placenta by immunohistochemistry. Histochemistry with the NADPH diaphorase substrate nitroblue tetrazolium (NBT) has also been used to establish if other isoforms of NOS may be present in these tissues. Positive immunofluorescence for endothelial NOS was found in umbilical cord artery and vein endothelium, although positive staining was only found in approximately 50% of veins. The endothelium of stem villous vessels dissected from beneath the chorionic plate was also intensely immunostained. In the terminal villi punctate immunostaining was found at the basal aspect and around nuclei of syncytiotrophoblast, but was absent from stroma and endothelium of terminal villous vessels. A positive histochemical stain for NBT was found in cord artery and vein endothelium and stem villous vessel endothelium. Intense diffuse staining with NBT was found in syncytiotrophoblast, but no other cell types in the terminal villi stained with NBT. The endothelial NOS isoform appears to be localized in the resistance vasculature of the placenta, but not in the capillary endothelium of terminal villi where there is no underlying smooth muscle. It may contribute to the 'endothelial' function of syncytiotrophoblast if secreted towards the intervillous space or alternatively fulfil other signal transduction roles. The pattern of staining with NBT was similar to that with endothelial NOS and suggests that other isoforms of NOS are not present in the placental unit.

Constitutive calcium-dependent isoform of nitric oxide synthase in the human placental villous vascular tree.

We have characterized the NO synthase enzyme in the villous vasculature of the human placenta as part of our ongoing studies of the regulation of NO synthesis in this circulation. NO synthase activity was determined by conversion of 3H L-arginine to 3H L-citrulline in cellular homogenate, cytosolic and particulate fractions. Optimal NO synthase activity was measured in all fractions in the presence of 1 mM NADPH, 10 microM tetrahydrobiopterin, 2 microM FAD, 100 microM free calcium and 50 U/ml calmodulin. The calmodulin inhibitor calmidazolium (50 microM) and FAD inhibitor diphenyliodonium chloride (1 microM) significantly reduced enzyme activity. The EC50 for calcium was 0.1 microM and Km for L-arginine 2.00 +/- 0.49 microM with Vmax 55.8 +/- 28.3 pmoles/mg protein/min. Enzyme activity was inhibited in both cytosolic and particulate fractions by ng-nitro-L-arginine and ng-monomethyl-L-arginine in a concentration-dependent manner (10(-8)-10(-4) M). A calcium-independent NO synthase activity was also determined, but only constituted between 5-6 per cent of total activity. On Western blotting, a single 135 kda species was identified in each fraction with a monoclonal antibody raised against bovine aortic endothelial NO synthase. The NO synthase enzyme of the villous vasculature appears to correspond to the type III calcium-calmodulin dependent endothelial isoform.

Exercise in sickle cell anemia: effect on inflammatory and vasoactive mediators.

The aim of this study was to determine the response of inflammatory and vasoactive mediators to 3 consecutive days of exercise in African-American women with and without sickle cell anemia (SCA). Circulating inflammatory mediators [C-reactive protein (CRP), interleukin-6 (IL-6), tumor necrosis factor alpha (TNFalpha)] were measured before, and vasoactive mediators [endothelin-1 (ET-1), nitric oxide metabolites (NOx)] before and after each exercise bout in ten subjects with SCA and ten controls. Exercise did not affect ET-1, IL-6 or CRP concentrations (p >.05). TNFalpha was higher in SCA than controls (p < or = .0005) at all times; however, the response pattern was similar for the groups: no change from day 1 to day 2, but a decrease from day 2 to day 3 (p < or = .05). NOx increased significantly after exercise (p < or = .0001) but returned to baseline by 24 h afterward. On the 3rd day, NOx increased after exercise in SCA but not in the controls (p < or = .05). In conclusion, exercise did not cause a harmful inflammatory response in these individuals with SCA. However, NOx increased after exercise on all 3 days in SCA but appeared attenuated after 2 days in controls.