Intracellular Nitric Oxide Concentration Research Articles

Chronic nitroglycerine administration reduces endothelial nitric oxide production in rabbit mesenteric resistance artery

We investigated whether 10 days' in vivo treatment with nitroglycerine (NTG) would inhibit nitric oxide production by the endothelial cells of resistance arteries ex vivo and, if so, what the underlying mechanism might be. ACh increased the intracellular nitric oxide concentration ([NO]i; estimated using the nitric oxide-sensitive fluorescent dye diaminofluorescein-2) within the endothelial cells of rabbit mesenteric resistance arteries. This effect was significantly smaller in arteries isolated from NTG-treated rabbits than in those from control rabbits. The reduction in endothelial [NO]i in NTG-treated rabbits was prevented when olmesartan (blocker of type 1 angiotensin II receptors (AT1Rs)) was coadministered in vivo with NTG and also when the superoxide scavenger manganese (III) tetrakis-(4-benzoic acid) porphyrin (Mn-TBAP), the protein kinase C (PKC) inhibitor GF109203X or L-arginine (with or without the active form of folate (5-methyltetrahydrofolate)) was incubated with the arteries in vitro. Endothelial cell superoxide production (estimated by ethidium fluorescence) was greatly increased in arteries from NTG-treated rabbits. This was normalized by in vivo coadministration of olmesartan with NTG and also by in vitro application of Mn-TBAP or GF109203X (but not of 5-methyltetrahydrofolate+L-arginine). ACh increased the intracellular Ca2+ concentration (estimated using the Ca2+-sensitive dye Fura 2) within endothelial cells, the increase being not significantly different between NTG-treated rabbits and control rabbits. We conclude that in NTG-treated rabbits, endothelial nitric oxide production in mesenteric resistance arteries is reduced, possibly through a reduction in the bioavailability of L-arginine via an action mediated by superoxide. Activation of the AT1R-PKC pathway may be involved in increasing superoxide production.

Flash photolysis of caged nitric oxide inhibits proximal tubular fluid reabsorption in free-flow nephron

A nonobstructing optical method was developed to measure proximal tubular fluid reabsorption in rat nephron at 0.25 Hz. The effects of uncaging luminal nitric oxide (NO) on proximal tubular reabsorption were investigated with this method. Proximal fluid reabsorption rate was calculated as the difference of tubular flow measured simultaneously at two locations (0.8-1.8 mm apart) along a convoluted proximal tubule. Tubular flow was estimated on the basis of the propagating velocity of fluorescent dextran pulses in the lumen. Changes in local tubular flow induced by intratubular perfusion were detected simultaneously along the proximal tubule, indicating that local tubular flow can be monitored in multiple sites along a tubule. The estimated tubular reabsorption rate was 5.52 +/- 0.38 nl.min(-1).mm(-1) (n = 20). Flash photolysis of luminal caged NO (potassium nitrosylpentachlororuthenate) was induced with a 30-Hz UV nitrogen-pulsed laser. Release of NO from caged NO into the proximal tubule was confirmed by monitoring intracellular NO concentration using a cell-permeant NO-sensitive fluorescent dye (DAF-FM). Emission of DAF-FM was proportional to the number of laser pulses used for uncaging. Photolysis of luminal caged NO induced a dose-dependent inhibition of proximal tubular reabsorption without activating tubuloglomerular feedback, whereas uncaging of intracellular cGMP in the proximal tubule decreased tubular flow. Coupling of this novel method to measure reabsorption with photolysis of caged signaling molecules provides a new paradigm to study tubular reabsorption with ambient tubular flow.

The platelet-endothelium interaction mediated by lectin-like oxidized low-density lipoprotein receptor-1 reduces the intracellular concentration of nitric oxide in endothelial cells

ObjectivesTo address the potential role of the endothelial lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) in the thrombotic system, in this study we first examined whether platelet interaction with LOX-1 generated reactive oxygen species (ROS) and superoxide (O2·−) and then investigated the relationship between the intracellular production of O2·− and the availability of nitric oxide (NO). BackgroundOxidative inactivation of NO is regarded as an important cause of its decreased biologic activity which may favor platelet-dependent arterial thrombosis. MethodsBovine aortic endothelial cells (BAECs) and Chinese hamster ovary-K1 cells stably expressing bovine LOX-1 (BLOX-1-CHO) were incubated at different times with human platelets. The ROS, O2·−, and NO were measured in cells by flow cytometry. ResultsThe incubation of BAECs and BLOX-1-CHO cells with human platelets induced a sharp and dose-dependent increase in intracellular concentration of ROS and O2·− (p from <0.01 to <0.001). The increase in intracellular concentration of O2·− was followed by a dose-dependent reduction in basal and bradykinin-induced intracellular NO concentration (p from <0.01 to <0.001). The increase in O2·− and the reduction of NO were inhibited by the presence of vitamin C and anti-LOX-1 monoclonal antibody (p < 0.001). ConclusionsThe results of this study show that one of the pathophysiologic consequences of platelet binding to LOX-1 may be the inactivation of NO through an increased cellular production of O2·−.

Nitric oxide inhibits capacitative Ca2+ entry and enhances endoplasmic reticulum Ca2+ uptake in bovine vascular endothelial cells.

In vascular endothelial cells, elevation of cytosolic free calcium concentration ([Ca2+]i) causes activation of nitric oxide synthase (NOS) and release of nitric oxide (NO). The goal of the study was to characterize the interplay between [Ca2+]i and NO production in this cell type. Simultaneous measurements of [Ca2+]i and intracellular NO concentration ([NO]i) in cultured bovine vascular endothelial cells (CPAE cell line) with the fluorescent indicators fura-2 and DAF-2, respectively, revealed that Ca2+ influx following agonist-induced intracellular Ca2+ store depletion (capacitative Ca2+ entry, CCE) represents the preferential Ca2+ source for the activation of the Ca2+-calmodulin-dependent endothelial NOS (eNOS). Exposure to the NO donor sodium nitroprusside (SNP) showed that high NO levels suppressed CCE and had an inhibitory effect on Ca2+ extrusion by the plasmalemmal Ca2+-ATPase. This inhibitory effect on CCE was mimicked by the membrane-permeant cGMP analogue 8-bromo-cGMP, but was reversed by the NO scavenger haemoglobin and prevented by the inhibitor of the NO-sensitive guanylate cyclase ODQ. Brief exposure to SNP reduced the peak of ATP-induced Ca2+ release from the endoplasmic reticulum (ER) and accelerated Ca2+ reuptake into the ER. Prolonged incubation with SNP resulted in enhanced Ca2+ loading of the ER, as revealed by direct measurements of store content with the ER-entrapped low-affinity Ca2+ indicator mag-fura-2. The results suggest that in vascular endothelial cells, NO synthesis is under autoregulatory control that involves NO-dependent [Ca2+]i regulation. Via cGMP-dependent inhibition of CCE and acceleration of Ca2+ sequestration into the ER, NO can lower [Ca2+]i and therefore exert an autoregulatory negative feedback on its own Ca2+-dependent synthesis.

The Binding of Oxidized Low Density Lipoprotein (ox-LDL) to ox-LDL Receptor-1 Reduces the Intracellular Concentration of Nitric Oxide in Endothelial Cells through an Increased Production of Superoxide

Oxidized low density lipoprotein (ox-LDL) has been suggested to affect endothelium-dependent vascular tone through a decreased biological activity of endothelium-derived nitric oxide (NO). Oxidative inactivation of NO is regarded as an important cause of its decreased biological activity, and in this context superoxide (O(2)) is known to inactivate NO in a chemical reaction during which peroxynitrite is formed. In this study we examined the effect of ox-LDL on the intracellular NO concentration in bovine aortic endothelial cells and whether this effect is influenced by ox-LDL binding to the endothelial receptor lectin-like ox-LDL receptor-1 (LOX-1) through the formation of reactive oxygen species and in particular of O(2). ox-LDL induced a significant dose-dependent decrease in intracellular NO concentration both in basal and stimulated conditions after less than 1 min of incubation with bovine aortic endothelial cells (p < 0.01). In the same experimental conditions ox-LDL also induced O(2) generation (p < 0.001). In the presence of radical scavengers and anti-LOX-1 monoclonal antibody, O(2) formation induced by ox-LDL was reduced (p < 0.001) with a contemporary rise in intracellular NO concentration (p < 0.001). ox-LDL did not significantly modify the ability of endothelial nitric oxide synthase to metabolize l-arginine to l-citrulline. The results of this study show that one of the pathophysiological consequences of ox-LDL binding to LOX-1 may be the inactivation of NO through an increased cellular production of O(2).

A Kinetic Model for the Interaction of Nitric Oxide with a Mammalian Lipoxygenase

Nitric oxide (NO) has been known for a long while to act as an inactivator of the soybean lipoxygenase-1 and cyclooxygenase. More recently, NO was shown to interact also with a mammalian 15-lipoxygenase [Wiesner, R., Rathmann, J., Holzhütter, H. G., Stosser, R., Mader, K., Nolting, H. & Kühn, H. (1996) Nitric oxide oxidises ferrous mammalian lipoxygenases to a pre-activated ferric species, FEBS Lett. 389, 229-232]. In this paper we present a detailed kinetic analysis of the interaction of NO with the 15-lipoxygenase from reticulocytes. Time courses of hydroperoxide formation were monitored after an anaerobic incubation of the enzyme with varying concentrations of NO and for varying length of the incubation period. Owing to the presence of O2 during the enzymatic reaction, the inhibitory effect of NO declines in a time-dependent manner since NO is converted into non-reactive NO2. This is manifested as a lag phase of the time course. The lag phase becomes more pronounced if the enzyme is incubated over a fixed period (15 s) with increasing concentrations of NO. In contrast, increasing the length of the incubation period at fixed concentrations of NO diminishes the duration of the lag phase. These experimental findings can be accounted for by a kinetic model which assumes (a) fast reversible binding of NO to the inactive ferrous Fe(II) form of the enzyme and (b) slow irreversible conversion of the Fe(II)-NO complex into an activated ferric form of the enzyme which is susceptible to peroxide activation. The rate constants for these two different kinetic modes of NO action were estimated by fitting the solutions of the model equations to the experimental time courses. The dissociation constant of the Fe(II)-NO complex amounts to 2.5 microM. Computer simulations performed on the basis of the model indicate that the response of the lipoxygenase to increasing intracellular NO concentrations depend upon the available peroxide tone: at low peroxide concentration the enzyme is initially trapped in the inactive ferrous form and thus may be activated by NO via the activated ferric species. On the other hand, at high peroxide concentrations, a partial inhibition of the initially active enzyme is expected.