The influence of radial RBC distribution, blood velocity profiles, and glycocalyx on coupled NO/O2 transport.

The purpose of this investigation was to study the effect of the presence of red blood cells (RBCs) in the plasma layer near the arteriole wall on nitric oxide (NO) and oxygen (O2) transport. To this end, we extended a coupled NO and O2 diffusion-reaction model in the arteriole, developed by our group, to include the effect of the presence of RBCs in the plasma layer and the effect of convection. Two blood flow velocity profiles (plug and parabolic) were tested. The average hematocrit in the bloodstream was assumed to be constant in the central core and decreasing to zero in the boundary layer next to the endothelial surface layer. The effect of the presence or absence of RBCs near the endothelium was studied while varying the endothelial surface layer and boundary layer thickness. With RBCs present in the boundary layer, the model predicts that 1) NO decreases significantly in the endothelium and vascular wall; 2) there is a very small increase in endothelial and vascular wall Po2; 3) scavenging of NO by hemoglobin decreases with increasing thickness of the boundary layer; 4) the shape of the velocity profile influences both NO and Po2 gradients in the bloodstream; and 5) the presence of RBCs in the boundary layer near the endothelium has a much larger effect on NO than on O2 transport.     

Alterations in ET-1, not nitric oxide,in 1-week-old lambs with increased pulmonary blood flow

Altered pulmonary vascular reactivity is a source of morbidity and mortality for children with congenital heart disease and increased pulmonary blood flow. Nitric oxide (NO) and endothelin (ET)-1 are important mediators of pulmonary vascular reactivity. We hypothesize that early alterations in endothelial function contribute to the altered vascular reactivity associated with congenital heart disease. The objective of this study was to characterize endothelial function in our lamb model of increased pulmonary blood flow at 1 wk of life. Eleven fetal lambs underwent in utero placement of an aortopulmonary vascular graft (shunt) and were studied 7 days after delivery. The pulmonary vasodilator response to both intravenous ACh (endothelium dependent) and inhaled NO (endothelium independent) was similar in shunted and control lambs. In addition, tissue NO(x), NO synthase (NOS) activity, and endothelial NOS protein levels were similar. Conversely, the vasodilator response to both ET-1 and 4Ala-ET-1 (an ET(B) receptor agonist) were attenuated in shunted lambs, and tissue ET-1 concentrations were increased (P < 0.05). Associated with these changes were an increase in ET-converting enzyme-1 protein and a decrease in ET(B) receptor protein levels (P < 0.05). These data demonstrate that increased pulmonary blood flow induces alterations in ET-1 signaling before NO signaling and suggest an early role for ET-1 in the altered vascular reactivity associated with increased pulmonary blood flow.     

Biphasic modulation of vascular nitric oxide catabolism by oxygen

Endothelium-derived nitric oxide (NO) plays an important role in the regulation of vascular tone. Lack of NO bioavailability can result in cardiovascular disease. NO bioavailability is determined by its rates of generation and catabolism; however, it is not known how the NO catabolism rate is regulated in the vascular wall under normoxic, hypoxic, and anaerobic conditions. To investigate NO catabolism under different oxygen concentrations, studies of NO and O2 consumption by the isolated rat aorta were performed using electrochemical sensors. Under normoxic conditions, the rate of NO consumption in solution was enhanced in the presence of the rat aorta. Under hypoxic conditions, NO consumption decreased in parallel with the O2 concentration. Like the inhibition of mitochondrial respiration by NO, the inhibitory effects of NO on aortic O2 consumption increased as O2 concentration decreased. Under anaerobic conditions, however, a paradoxical reacceleration of NO consumption occurred. This increased anaerobic NO consumption was inhibited by the cytochrome c oxidase inhibitor NaCN but not by the free iron chelator deferoxamine, the flavoprotein inhibitor diphenylene iodonium (10 microM), or superoxide dismutase (200 U/ml). The effect of O2 on the NO consumption could be reproduced by purified cytochrome c oxidase (CcO), implying that CcO is involved in aortic NO catabolism. This reduced NO catabolism at low O2 tensions supports the maintenance of effective NO levels in the vascular wall, reducing the resistance of blood vessels. The increased anaerobic NO catabolism may be important for removing excess NO accumulation in ischemic tissues.     

Free nitric oxide diffusion in the bronchial microcirculation

Theoretical mass transfer rates and concentration distributions were determined for transient diffusion of free nitric oxide (NO) generated in vivo from vascular endothelial cells. Our analytical framework is typical of the bronchial circulation in the human pulmonary system but is applicable to the microvascular circulation in general. We characterized mass transfer rates in terms of the fractional mass flux across a boundary relative to the total endothelial NO production rate. NO concentration in the tissue surrounding blood vessels was expressed in terms of fractional soluble guanylate cyclase (sGC) activity. Our results suggest that endothelium-derived free NO is capable of vascular smooth muscle dilation despite its rapid consumption by hemoglobin in blood. An optimal blood vessel radius of 20 microm was estimated for NO signaling. We hypothesize intermittent generation of endothelial NO as a possible mechanism for sGC activation in vascular smooth muscle. This mechanism enhances the efficacy of NO-modulated vascular smooth muscle dilation while minimizing NO losses to blood and surrounding tissue.     

The role of arterial smooth muscle in vasorelaxation

The concept of endothelium-derived relaxing factor (EDRF) implies that nitric oxide (NO) produced by NO synthase (NOS) in the endothelium in response to vasorelaxants such as acetylcholine (ACh) acts on the underlying vascular smooth muscle cells (VSMC) inducing vascular relaxation. The EDRF concept was derived from experiments on denuded blood vessel strips and, in frames of this concept, VSMC were regarded as passive recipients of NO from endothelial cells. However, it was later found that VSMC express NOS by themselves, but the principal question remained unanswered, is the NO generation by VSMC physiologically relevant? We hypothesized that the destruction of the vascular wall anatomical integrity by rubbing off the endothelial layer might increase vascular superoxides that, in turn, reduced the NO bioactivity as a relaxing factor. To test our hypothesis, we examined ACh-induced vasorelaxation under protection against oxidative stress and found that superoxide scavengers restored vasodilatory responses to ACh in endothelium-deprived blood vessels. These findings imply that VSMC can release NO in amounts sufficient to account for the vasorelaxatory response and challenge the concept of the obligatory role of endothelial cells in the relaxation of arterial smooth muscle.     

Endogenous nitric oxide is present in the exhaled air of rabbits, guinea pigs and humans.

The presence of nitric oxide (NO) in the exhaled air of humans and of anaesthetized rabbits and guinea pigs was demonstrated by chemiluminescence, diazotization and mass spectrometry. This NO is endogenously produced in the lung by an NO synthase, since its generation in guinea pigs and rabbits was inhibited by N omega-nitro-L-arginine methyl ester and NG-monomethyl-L-arginine, inhibitors of this enzyme. The effect of the inhibitors was reversed by the precursor of NO synthesis, L-arginine. Since NO is produced by normal vascular endothelium for the physiological regulation of blood flow and pressure and also by activated macrophages to contribute to non-specific immunity, our experiments suggest that NO may play both vascular regulatory and host defence roles in pulmonary physiology and pathophysiology.     

Luminal flow regulates NO and O2(-) along the nephron

Urinary flow is not constant but in fact highly variable, altering the mechanical forces (shear stress, stretch, and pressure) exerted on the epithelial cells of the nephron as well as solute delivery. Nitric oxide (NO) and superoxide (O(2)(-)) play important roles in various processes within the kidney. Reductions in NO and increases in O(2)(-) lead to abnormal NaCl and water absorption and hypertension. In the last few years, luminal flow has been shown to be a regulator of NO and O(2)(-) production along the nephron. Increases in luminal flow enhance fluid, Na, and bicarbonate transport in the proximal tubule. However, we know of no reports directly addressing flow regulation of NO and O(2)(-) in this segment. In the thick ascending limb, flow-stimulated NO and O(2)(-) formation has been extensively studied. Luminal flow stimulates NO production by nitric oxide synthase type 3 and its translocation to the apical membrane in medullary thick ascending limbs. These effects are mediated by flow-induced shear stress. In contrast, flow-induced stretch and NaCl delivery stimulate O(2)(-) production by NADPH oxidase in this segment. The interaction between flow-induced NO and O(2)(-) is complex and involves more than one simply scavenging the other. Flow-induced NO prevents flow from increasing O(2)(-) production via cGMP-dependent protein kinase in thick ascending limbs. In macula densa cells, shear stress increases NO production and this requires that the primary cilia be intact. The role of luminal flow in NO and O(2)(-) production in the distal tubule is not known. In cultured inner medullary collecting duct cells, shear stress enhances nitrite accumulation, a measure of NO production. Although much progress has been made on this subject in the last few years, there are still many unanswered questions.     

Increased blood flow causes coordinated upregulation of arterial eNOS and biosynthesis of tetrahydrobiopterin

Shear stress, imposed on the vascular endothelium by circulating blood, critically sustains vascular synthesis of nitric oxide (NO). Endothelial NO synthase (eNOS) activity is determined by heat shock protein 90 (HSP90), caveolin-1, and the cofactor tetrahydrobiopterin (BH4). To determine whether increased blood flow concomitantly upregulates eNOS and GTP cyclohydrolase I (GTPCH I, the rate-limiting enzyme in BH4 biosynthesis), an aortocaval fistula model in the rat was employed wherein aortic blood flow is enhanced proximal but decreased distal to the fistula. Eight weeks after the creation of the aortocaval fistula, the proximal and distal aortic segments were harvested; sham-operated rats served as controls. Vasomotor function was assessed by isometric force recording. Expression of eNOS, HSP90, caveolin-1, Akt, phosphorylated eNOS (eNOS-Ser1177), and GTPCH I were determined by Western blot analysis. Biosynthesis of BH4 and GTPCH-I activity was examined by HPLC. In the aortic segments exposed to increased flow, contractions to KCl and phenylephrine were reduced, whereas endothelium-dependent relaxations were not affected compared with sham-operated or aortic segments with reduced blood flow. Expression of eNOS, caveolin-1, phosphorylated Akt, and eNOS-Ser1177 was enhanced in aortas exposed to increased blood flow. High flow augmented levels of cGMP and BH4 and increased expression of GTPCH I. In aggregate, these findings provide the first demonstration in vivo that coordinated vascular upregulation of eNOS, and GTPCH I accompanies increased blood flow. This induction of GTPCH I increases BH4 production, thereby optimizing the generation of NO by eNOS and thus the adaptive, vasorelaxant response required in sustaining increased blood flow.     

Renal cortical and medullary blood flow responses to L-NAME and ANG II in wild-type, nNOS null mutant, and eNOS null mutant mice

Experiments in wild-type (WT; C57BL/6J) mice, endothelial nitric oxide synthase null mutant [eNOS(-/-)] mice, and neuronal NOS null mutant [nNOS(-/-)] mice were performed to determine which NOS isoform regulates renal cortical and medullary blood flow under basal conditions and during the infusion of ANG II. Inhibition of NOS with N(omega)-nitro-l-arginine methyl ester (l-NAME; 50 mg/kg iv) in Inactin-anesthetized WT and nNOS(-/-) mice increased arterial blood pressure by 28-31 mmHg and significantly decreased blood flow in the renal cortex (18-24%) and the renal medulla (13-18%). In contrast, blood pressure and renal cortical and medullary blood flow were unaltered after l-NAME administration to eNOS(-/-) mice, indicating that NO derived from eNOS regulates baseline vascular resistance in mice. In subsequent experiments, intravenous ANG II (20 ng x kg(-1) x min(-1)) significantly decreased renal cortical blood flow (by 15-25%) in WT, eNOS(-/-), nNOS(-/-), and WT mice treated with l-NAME. The infusion of ANG II, however, led to a significant increase in medullary blood flow (12-15%) in WT and eNOS(-/-) mice. The increase in medullary blood flow following ANG II infusion was not observed in nNOS(-/-) mice, in WT or eNOS(-/-) mice pretreated with l-NAME, or in WT mice administered the nNOS inhibitor 5-(1-imino-3-butenyl)-l-ornithine (1 mg x kg(-1) x h(-1)). These data demonstrate that NO from eNOS regulates baseline blood flow in the mouse renal cortex and medulla, while NO produced by nNOS mediates an increase in medullary blood flow in response to ANG II.     

Renal medullary blood flow: its measurement and physiology

The vasculature of the mammalian renal medulla is complex, having neither discrete input nor output. There is also efficient countercurrent exchange between ascending and descending vasa recta in the vascular bundles. These considerations have hampered measurement of medullary blood flow since they impose pronounced constraints on methods used to assess flow. Three main strategies have been used: (i) indicator extraction; (ii) erythrocyte velocity tracking; and (iii) indicator dilution. These are discussed with respect to their assumptions, requirements, and limitations. There is a consensus that medullary blood flow is autoregulated, albeit over a narrower pressure range than is total renal blood flow. When normalized to gram tissue weight, medullary blood flow in the dog is similar to that in the rat, on the order of 1 to 1.5 mL X min-1 X g-1. This is considerably greater than estimated by the radioiodinated albumin uptake method which has severe conceptual and practical problems. From both theoretical and experimental evidence it seems that urinary concentrating ability is considerably less sensitive to changes in medullary blood flow than is often assumed.     

A scanning electron-microscopic study of the rat thymus with special reference to cell types and migration of lymphocytes into the general circulation

Summary The three-dimensional structure of the rat thymus was studied by combined scanning and transmission electron microscopy. The thymus consists mainly of four types of cells: epithelial cells, lymphocytes, macrophages, and interdigitating cells (IDCs).The epithelial cells form a meshwork in the thymus parenchyma. Cortical epithelial cells are stellate in shape, while the medullary cells comprise two types: stellate and large vacuolated elements. A continuous single layer of epithelial cells separates the parenchyma from connective tissue formations of the capsule, septa and vessels. Surrounding the blood vessels, this epithelial sheath is continuous in the cortex, while it is partly interrupted in the medulla, suggesting that the blood-thymus barrier might function more completely in the cortex.Cortical lymphocytes are round and vary in size, whereas medullary lymphocytes are mainly small, although they vary considerably in surface morphology.Two types of large wandering cells, macrophages and IDCs, could be distinguished, as well as intermediate forms. IDCs sometimes embraced or contacted lymphocytes, suggesting their role in the differentiation of the latter cells.Perivascular channels were present around venules and some arterioles in the cortico-medullary region and in the medulla. A few lymphatic vessels were present in extended perivascular spaces.The present study suggests the possible existence of two routes of passage of lymphocytes into the general circulation. One is via the lymphatics, while the other is through the postcapillary venules into the blood circulation. Our SEM images give evidence that lymphocytes use an intracellular route, i.e., the endothelium of venules.     

一氧化氮相关的内皮功能障碍与高血压

动脉脉管系统在静息状态下处于收缩状态,具有一定的血管紧张度。血流增加时内皮细胞通过释放血管内皮舒张因子介导平滑肌舒张来维持正常的血压。当内皮依赖的舒张作用下降时,血流增加会导致局部或全身血压升高,最终引发高血压。内皮功能障碍是高血压的特征性异常变化之一,而一氧化氮(NO)-介导的舒张血管途径被认为对血压调节有重要作用。本文将对正常及高血压状态下NO相关的内皮功能做一综述。     

L-Arginine uptake affects nitric oxide production and blood flow in the renal medulla

Experiments were performed to determine whether L-arginine transport regulates nitric oxide (NO) production and hemodynamics in the renal medulla. The effects of renal medullary interstitial infusion of cationic amino acids, which compete with L-arginine for cellular uptake, on NO levels and blood flow in the medulla were examined in anesthetized rats. NO concentration in the renal inner medulla, measured with a microdialysis-oxyhemoglobin trapping technique, was significantly decreased by 26-44% and renal medullary blood flow, measured by laser Doppler flowmetry, was significantly reduced by 20-24% during the acute renal medullary interstitial infusion of L-ornithine, L-lysine, and L-homoarginine (1 micromol.kg(-1).min(-1) each; n = 6-8/group). In contrast, intramedullary infusion of L-arginine increased NO concentration and medullary blood flow. Flow cytometry experiments with 4-amino-5-methylamino-2',7'-difluorescein diacetate, a fluorophore reactive to intracellular NO, demonstrated that L-ornithine, L-lysine, and L-homoarginine decreased NO by 54-57% of control, whereas L-arginine increased NO by 21% in freshly isolated inner medullary cells (1 mmol/l each, n > 1,000 cells/experiment). The mRNA for the cationic amino acid transporter-1 was predominantly expressed in the inner medulla, and cationic amino acid transporter-1 protein was localized by immunohistochemistry to the collecting ducts and vasa recta in the inner medulla. These results suggest that L-arginine transport by cationic amino acid transport mechanisms is important in the production of NO and maintenance of blood flow in the renal medulla.     

An in situ evidence for autocrine function of NO in the vasculature

The concept of endothelium derived relaxing factor (EDRF) implies that nitric oxide (NO) generated by NO synthase in the endothelium diffuses to the underlying vascular smooth muscle cells (VSMC) modulating thereby vascular tone. VSMC were regarded as passive recipients of NO from endothelial cells. However, this paradigm of a paracrine function of NO became currently subject to considerable debate. To address this issue, we examined the localization of enzymes engaged in l-arginine-NO-cGMP signaling in the rat blood vessels. Employing multiple immunocytochemical labeling complemented with signal amplification, electron microscopy, Western blotting, and RT-PCR, we found that NO synthase was differentially expressed in blood vessels depending on the blood vessel type. Moreover, the expression pattern of NO synthase in VSMC showed striking parallels with arginase and soluble guanylyl cyclase. Our findings challenge the commonly accepted view that the expression of NO synthase is restricted to vascular endothelial cells and lends further support to an alternative mechanism, by which constitutive local NOS expression in VSMC may modulate vascular functions in an endothelium-independent manner. Moreover, the co-expression of enzymes engaged in l-arginine-NO-cGMP signaling (NO synthase, arginase, and soluble guanylyl cyclase) in VSMC is indicative of an autocrine fashion of NO signaling in the vasculature in addition to the paracrine role of NO generated in the endothelium.     

Twenty-five years since the discovery of endothelium-derived relaxing factor (EDRF): does a dysfunctional endothelium contribute to the development of type 2 diabetes?

Twenty-five years ago, the discovery of endothelium-derived relaxing factor opened a door that revealed a new and exciting role for the endothelium in the regulation of blood flow and led to the discovery that nitric oxide (NO) multi-tasked as a novel cell-signalling molecule. During the next 25 years, our understanding of both the importance of the endothelium as well as NO has greatly expanded. No longer simply a barrier between the blood and vascular smooth muscle, the endothelium is now recognized as a complex tissue with heterogeneous properties. The endothelium is the source of not only NO but also numerous vasoactive molecules and signalling pathways, some of which are still not fully characterized such as the putative endothelium-derived relaxing factor. Dysfunction of the endothelium is a key risk factor for the development of macro- and microvascular disease and, by coincidence, the discovery that NO was generated in the endothelium corresponds approximately in time with the increased incidence of type 2 diabetes. Primarily linked to dietary and lifestyle changes, we are now facing a global pandemic of type 2 diabetes. Characterized by insulin resistance and hyperglycaemia, type 2 diabetes is increasingly being diagnosed in adolescents as well as children. Is there a link between dietary-related hyperglycaemic insults to the endothelium, blood flow changes, and the development of insulin resistance? This review explores the evidence for and against this hypothesis.     

Endothelial- and nitric oxide-dependent effects on oxidative metabolism of intact artery.

Oxidative metabolism and its possible modulation by nitric oxide (NO) was examined in endothelial-intact and endothelial-denuded segments of porcine carotid arteries. Endothelial-intact arteries displayed appropriate NO-mediated vasorelaxation to acetylcholine (ACh). Endothelial-denuded arteries demonstrated absent vasorelaxation to ACh stimulation and depressed contractile responsiveness to K(+) depolarization, which was normalized by inhibition of NO synthesis by N(omega)-nitro-L-arginine methylester (L-NAME). Confirmation that carotid arteries continued to produce NO despite removal of the endothelium was indicated by detection of NO metabolites in the incubation medium bathing the arteries. O(2) consumption and the oxidation of glucose and fatty acid were depressed in endothelial-denuded arteries. Depression of O(2) consumption and glucose oxidation was completely reversed by treatment with L-NAME. We conclude that endogenous NO produced by non-endothelial vascular cells depresses contractility, O(2) consumption, and oxidation of energy substrates in vascular smooth muscle. The endothelium may play a role in oxidative metabolism of vascular smooth muscle possibly by modulating the effects of NO produced by other cells of the vessel wall, or by other factors.     

Nitric oxide and atherosclerosis.

Endothelial dysfunction has been shown in a wide range of vascular disorders including atherosclerosis and related diseases. Here, we examine and address the complex relationship among nitric oxide (NO)-mediated pathways and atherogenesis. In view of the numerous pathophysiological actions of NO, abnormalities could potentially occur at many sites: (a) impairment of membrane receptors in the arterial wall that interact with agonists or physiological stimuli capable of generating NO; (b) reduced concentrations or impaired utilization of l-arginine; (c) reduction in concentration or activity both of inducible and endothelial NO synthase; (d) impaired release of NO from the atherosclerotic damaged endothelium; (e) impaired NO diffusion from endothelium to vascular smooth muscle cells followed by decreased sensitivity to its vasodilator action; (f) local enhanced degradation of NO by increased generation of free radicals and/or oxidation-sensitive mechanisms; and (g) impaired interaction of NO with guanylate cyclase and consequent limitation of cyclic GMP production. Therefore, one target for new drugs should be the preservation or restoration of NO-mediated signaling pathways in arteries. Such novel therapeutic strategies may include administration of l-arginine/antioxidants and gene-transfer approaches.