Nitric Oxide Donors as Potential Drugs for the Treatment of Vascular Diseases Due to Endothelium Dysfunction.

Endothelial dysfunction and consequent vasoconstriction are a common condition in patients with hypertension and other cardiovascular diseases. Endothelial cells produce and release vasodilator substances that play a pivotal role in normal vascular tone. The mechanisms underlying endothelial dysfunction are multifactorial. However, enhanced reactive oxygen species (ROS) production and consequent vasoconstriction instead of endothelium-derived relaxant generation and consequent vasodilatation contribute to this dysfunction considerably. The main targets of the drugs that are currently used to treat vascular diseases concerning enzyme activities and protein functions that are impaired by endothelial nitric oxide synthase (eNOS) uncoupling and ROS production. Nitric oxide (NO) bioavailability can decrease due to deficient NO production by eNOS and/or NO release to vascular smooth muscle cells, which impairs endothelial function. Considering the NO cellular mechanisms, tackling the issue of eNOS uncoupling could avoid endothelial dysfunction: provision of the enzyme cofactor tetrahydrobiopterin (BH4) should elicit NO release from NO donors, to activate soluble guanylyl cyclase. This should increase cyclic guanosine-monophosphate (cGMP) generation and inhibit phosphodiesterases (especially PDE5) that selectively degrade cGMP. Consequently, protein kinase-G should be activated, and K+ channels should be phosphorylated and activated, which is crucial for cell membrane hyperpolarization and vasodilation and/or inhibition of ROS production. The present review summarizes the current concepts about the vascular cellular mechanisms that underlie endothelial dysfunction and which could be the target of drugs for the treatment of patients with cardiovascular disease.

Enzymatic hydrolysis of starch into sugars is influenced by microgel assembly.

The use of alginate and chitosan polymer in the immobilization of Aspergillus oryzae ATCC 3940 fungal crude enzyme extract (CEE) amylase was presented. The assembly results change in the application of optimal pH and temperature hydrolysis to convert starch to sugar. Bead arrangement in three microgel supports: the internal support phase (IP), the external support phase (EP), and the internal and external support phase (UP). The best results were obtained using IP and EP. Reusing beads evaluated the stability of immobilized enzymes on IP support, remained active and bound during three cycles of reuse. For free and immobilized (IP) activity showed pH ranged from 5.0 to 7.0; optimum thermal enzymatic greater activity at 45 °C. The method of building the microgel influencing sugar reduction, in a single-step way to immobilize crude fungal amylase extracts can be used in industry.

PLGA nano/microparticles loaded with cresyl violet as a tracer for drug delivery: Characterization and in-situ hyperspectral fluorescence and 2-photon localization.

Here we present the production, characterization and in-vivo assessment of cresyl violet-loaded biodegradable PLGA nano/microparticles (CV-NP and CV-MP). We demonstrate that the beneficial spectral characteristics of cresyl violet make it suitable as a tracer for particle-based drug delivery using both hyperspectral wide field and two-photon excited fluorescence microscopy. Particles were prepared using a cosolvent method, after which the physicochemical properties such as morphology, particle size, drug entrapment efficiency, drug loading and in vitro drug release behavior were measured in addition to spectroscopic properties, such as absorption, fluorescence and infrared spectra. The particles were then tested in an in vivo mouse model to assess their biodistribution characteristics. The location and integrity of particles after injection was determined using both hyperspectral fluorescence and two-photon microscopy within intact organs in situ. Our results show that cresyl violet is efficiently entrapped into PLGA particles, and that the particles are spherical in shape, ranging from 300 to 5070nm in diameter. Particle biodistribution in the mouse was found to depend on particle size, as expected. Cresyl violet is shown to be an ideal tracer to assess the properties PLGA particle-based drug delivery in combination with our novel multi-scale optical imaging techniques for in-situ particle localization.

In vitro cytotoxicity and phototoxicity of surface-modified gold nanoparticles associated with neutral red as a potential drug delivery system in phototherapy.

The surface of gold nanoparticles (AuNP) was modified, improving their interaction with neutral red (NR), by using sodium thioglycolate (TGA) as a covering agent. The resulting NR-AuNPTGA system was evaluated as a potential drug delivery system for photodynamic therapy (PDT). The associations of NR with the gold nanoparticles were evaluated using UV-vis spectrometry and measurement of their zeta potential and size distribution. The toxicity and phototoxicity of NR, AuNPTGA and NR-AuNPTGA were evaluated in NIH-3T3 fibroblast and 4T1 tumor cell lines. The compounds NR and NR-AuNPTGA induced toxicity in 4T1 tumor cells and NIH-3T3 fibroblasts under visible light irradiation. Modification of the surface of AuNP with TGA prevented nanoparticle aggregation and allowed greater association with NR molecules than for naked AuNP. The photosensitizer (PS) characteristics were not affected by its association with the modified surface of the gold nanoparticles, leading to a reduction of cell viability in both cell lines assayed. This NR-AuNPTGA system is a promising drug delivery system for photodynamic cancer therapy.

Blood pressure variability provokes vascular ß-adrenoceptor desensitization in rats.

Spontaneous variation in blood pressure is defined as 'blood pressure variability' (BPV). Sinoaortic denervation (SAD) is characterized by BPV without sustained hypertension. In the present study, we investigated whether BPV could be related to vascular ß-adrenoceptor desensitization in rats. Three days after surgery (SAD and control), aortic rings were placed in an organ chamber and the relaxation stimulated by ß-adrenoceptor agonists, isoprenaline, terbutaline, BRL37344 and cyanopindolol was verified. The participation of intracellular nucleotides signaling pathways was also verified using forskolin, sodium nitroprusside and acetylcholine to induce relaxation. The effects of BPV on the increase in endothelial cytosolic Ca(2+) concentration stimulated by the ß2-adrenoceptor agonist was examined by confocal microscopy. In addition, the vascular expression of the ß2-adrenoceptor was also examined by immunohistochemistry. The results show that isoprenaline and terbutaline-induced relaxation was lower in the aortas of rats with BPV. Relaxation responses to other vasorelaxant compounds were similar in both groups of rats. Histological analysis revealed a lower level of ß2-adrenoceptor and confocal microscopy showed minor cytosolic Ca(2+) concentration in endothelial cells stimulated by the ß2-adrenoceptor agonist in rats with BPV. In conclusion, BPV leads to desensitization of the ß2-adrenoceptor, which could contribute to worse ß-adrenoceptor agonist-induced relaxation in isolated aortas.

Gold nanoparticle modifies nitric oxide release and vasodilation in rat aorta.

Nitric oxide (NO) plays an important role on several biological functions. Recently, it has been reported the possibility of modifying the NO release profile from the NO donors through its coupling to gold nanoparticles (AuNPs). Thus, AuNPs were synthesized and they were exposed to the NO donor ruthenium complex Cis-[Ru(bpy)2(NO)(4PySH)].(PF6)3 termed (Ru-4PySH)-forming AuNPs-{Ru-4PySH}n cluster. Our results indicate that AuNPs do not modify the maximum effect (ME) and potency (pD2) in the vasodilation induced by Ru-4PySH. Both complexes induce similar vascular relaxation in concentration-dependent way. However, the NO released from the complex AuNPs-{Ru-4PySH}n is lower than Ru-4PySH. Both complexes release only NO(0) specie, but AuNPs-{Ru-4PySH}n releases NO in constant way and exclusively in the extracellular medium. In time-course, Ru-4Py-SH was faster than AuNPs-{Ru-4PySH}n in inducing the maximum vasodilation. Inhibition of soluble guanylyl cyclase (sGC) abolished the vasodilation induced by Ru-4PYSH, but not by AuNPs-{Ru-4PySH}n. Non-selective potassium (K(+)) channel blocker TEA had no effect on the vasodilation induced by AuNPs-{Ru-4PySH}n, but it reduced the potency to Ru-4PySH. In conclusion, our results suggest that AuNPs can reduce the permeability of NO donor Ru-4PySH due to AuNPs-{Ru-4PySH}n cluster formation. AuNPs reduce NO release, but they do not impair the vasodilator effect induced by the NO donor. Ru-4PySH induces vasodilation by sGC and K(+) channels activation, while AuNPs-{Ru-4PySH}n activates mainly sGC. Taken together, these findings represent a new pharmacological strategy to control the NO release which could activate selective biological targets.

Nitric oxide generated by the compound RuBPY promotes the vascular smooth cell membrane hyperpolarization.

The cis-[Ru(bpy)(2)(py)NO(2)](PF(6)) specie (RuBPY) has been used as nitric oxide (NO) delivery agent. It is an NO reservoir and it is thermodynamically stable in aqueous solution. This study aimed to evaluate the NO specie generated by RuBPY as compared to NO released from sodium nitroprusside (SNP) and to study the cellular mechanisms specially focusing the activation of soluble guanylyl-cyclase (sGC), K(+) channels and the cell membrane hyperpolarization, which are the main targets for NO-inducing vascular relaxation. NO generated by RuBPY and the vascular smooth muscle cell (VSMC) membrane potential were measured by confocal microscopy. The cellular mechanisms of aorta relaxation were investigated using K(+) channel blockers and sGC inhibitor. NO released from RuBPY was higher than NO released from SNP. RuBPY released only radicalar NO(0) and SNP released both NO(-) and NO(0). The concentration-effect curves for RuBPY-induced relaxation was shifted to the right by inhibition of sGC with ODQ and by the non-selective blockade of K(+) channels with TEA. The simultaneous combination of ODQ and TEA abolished the vasorelaxation induced by RuBPY. The membrane potential measured by the sensitive dye 4-Di-ANNEPS demonstrated that RuBPY induces cell membrane hyperpolarization. Taken together, our results indicate that the large amount of NO(0) specie generated by RuBPY induces vasorelaxation due to activation of sGC, K(+) channels sensitive to TEA, and cell membrane hyperpolarization. These results indicate that NO(0) generated from RuBPY can also directly activate the K(+) channels in an independent way of sGC.

Prostacyclin, not only nitric oxide, is a mediator of the vasorelaxation induced by acetylcholine in aortas from rats submitted to cecal ligation and perforation (CLP).

Nitric oxide has been pointed out as the main agent involved in the vasodilatation, which is the major symptom of septic shock. However, there must be another mediator contributing to the circulatory failure observed in sepsis. This study aimed to investigate the endothelium-dependent relaxation induced by acetylcholine and the factors involved in this relaxation, using aortic rings isolated from rats submitted to cecal ligation and perforation (CLP), 2h after induction of sepsis, which characterizes the hyperdynamic phase of sepsis. Under inhibition of constitutive NO-synthases (cNOS), the relaxation induced by acetylcholine was greater in the aortic rings of rats submitted to CLP compared with sham-operated rat aortic rings. The cyclooxygenase inhibitor indomethacin normalized this response, and the concentration of the stable metabolite of prostacyclin in the aorta of CLP rats increased in basal conditions and after stimulation with acetylcholine. Acetylcholine-induced NO production was lower in the endothelial cells from the aorta of CLP rats compared with sham rat aorta, but the protein expression of the cNOS was not altered. Moreover, iNOS protein expression could not be detected. Therefore, prostacyclin, and not only nitric oxide, is a mediator of the vasorelaxation induced by acetylcholine in aortas from rats submitted to CLP.

Decreased number of caveolae in endothelial cells impairs the relaxation induced by acetylcholine in hypertensive rat aortas.

The present study was designed to investigate the contribution of endothelial cell caveolae to vascular relaxation in aortas from a normotensive (2K) and renal hypertensive (2K-1C) rat. For that purpose, concentration-effect curves to acetylcholine were constructed in 2K and 2K-1C intact endothelium aortic rings, in the absence or in the presence of the caveolae disassembler methyl-beta-ciclodextrin. The potency (pD(2)) and the maximum relaxant effect to acetylcholine were greater in 2K than in 2K-1C aortas. Methyl-beta-ciclodextrin reduced the pD(2) in 2K and the maximum relaxant effect in both 2K and 2K-1C. The quantification of the caveolae number by electronic microscopy has shown a larger number of caveolae in 2K than in 2K-1C endothelial cells, which was reduced by methyl-beta-ciclodextrin in both 2K and 2K-1C. The production of NO stimulated with acetylcholine was greater in 2K than in 2K-1C endothelial cells, and this effect was impaired by methyl-beta-ciclodextrin in both 2K and 2K-1C. The cytosolic Ca(2+) concentration ([Ca(2+)]c) was simultaneously measured in endothelial and smooth muscle cells stimulated with acetylcholine by confocal image of aortic slices. Acetylcholine produced a greater [Ca(2+)]c increase in 2K than in 2K-1C endothelial cells, which response was inhibited by methyl-beta-ciclodextrin only in 2K cells. In smooth muscle cells the reduction of [Ca(2+)]c was higher in 2K than in 2K-1C. This effect was inhibited by methyl-beta-ciclodextrin only in 2K cells. Taken together, our results suggest that the decreased number of caveolae in the endothelial cells from 2K-1C rat aortas is involved in the impaired effect of acetylcholine on [Ca(2+)]c and NO.

Effects of instrumental dead space reduction during weaning from synchronized ventilation in preterm infants.

OBJECTIVE: A majority of the modalities of synchronized ventilation in preterm infants require the use of flow sensors that can increase dead space and may adversely affect ventilator weaning. The objective of this study was to assess the effects of flow sensor dead space during synchronized intermittent mandatory ventilation (SIMV) weaning in preterm infants. STUDY DESIGN: Twelve preterm infants (gestational age 25+/-2 weeks, birth weight 705+/-158 g, age: 31+/-186 days, SIMV rate: 25+/-8 breaths min(-1), peak inspiratory pressure 18+/-2 cm H(2)O, positive end-expiratory pressure: 5+/-0.5 cm H(2)O, pressure support: 9+/-3 cm H(2)O, fraction of inspired oxygen: 34+/-6%) underwent two 2.5-h weaning periods during which SIMV rate was reduced twice by 5 breaths min(-1) at 30-min intervals as tolerated, with and without reduction of flow sensor dead space, in random sequence. A 30-min baseline was obtained before each weaning period. Dead space was reduced by flushing the flow sensor with a continuous gas leak flow in the endotracheal tube connector. RESULT: Transcutaneous CO(2) tension during SIMV weaning periods without and with reduced dead space did not differ from baseline, whereas total minute ventilation and tidal volume were lower during the SIMV weaning period with reduced dead space. Three infants did not tolerate SIMV weaning without while one infant did not tolerate weaning with reduced dead space. CONCLUSION: SIMV weaning elicited a compensatory rise in spontaneous ventilation. When flow sensor dead space was reduced during SIMV weaning, gas exchange was maintained with lower minute ventilation. Instrumental dead space imposes a ventilatory burden during SIMV weaning in small preterm infants.

Endothelium negatively modulates the vascular relaxation induced by nitric oxide donor, due to uncoupling NO synthase.

Nitrosyl ruthenium complexes have been characterized as nitric oxide (NO) donors that induce relaxation in the denuded rat aorta. There are some differences in their vascular relaxation mechanisms compared with sodium nitroprusside. This study investigates whether the endothelium could interfere with the [Ru(terpy)(bdq)NO](3+)-TERPY-induced vascular relaxation, by analyzing the maximal relaxation (Emax) and potency (pD(2)) of TERPY. Vascular reactivity experiments showed that the endothelium negatively modulates (pD(2): 6.17+/-0.07) the TERPY relaxation in intact rat aortic rings compared with the denuded rat aorta (pD(2): 6.65+/-0.07). This effect is abolished by a non-selective NO-synthase (NOS) inhibitor L-NAME (pD(2): 6.46+/-0.10), by the superoxide anion (O(2)(-)) scavenger TIRON (pD(2): 6.49+/-0.08), and by an NOS cofactor BH(4) (pD(2): 6.80+/-0.10). The selective dye for O(2)(-) (DHE) shows that TERPY enhances O(2)(-) concentration in isolated endothelial cells (intensity of fluorescence (IF):11258.00+/-317.75) compared with the basal concentration (IF: 7760.67+/-381.50), and this enhancement is blocked by L-NAME (IF: 8892.33+/-1074.41). Similar results were observed in vascular smooth muscle cells (concentration of superoxide after TERPY: 2.63+/-0.17% and after TERPY+L-NAME: -4.63+/-0.14%). Considering that TERPY could induce uncoupling NOS, thus producing O(2)(-), we have also investigated the involvement of prostanoids in the negative modulation of the endothelium. The non-selective cyclooxygenase (COX) inhibitor indomethacin and the selective tromboxane (TXA(2)) receptor antagonist SQ29548 reduce the effect of the endothelium on TERPY relaxation (pD(2) INDO: 6.80+/-0.17 and SQ29548: 6.85+/-0.15, respectively). However, a selective prostaglandin F(2alpha) receptor antagonist (AH6809) does not change the endothelium effect. Moreover, TERPY enhances the concentration of TXA(2) stable metabolite (TXB(2)), but this effect is blocked by L-NAME and TIRON. The present findings indicate that TERPY induces uncoupling of eNOS, enhancing O(2)(-) concentration. This enhancement in O(2)(-) concentration induces COX activation, producing TXA(2), which negatively modulates the rat aorta relaxation induced by the NO donor TERPY.

Mechanisms underlying the vasorelaxant action of the pimarane ent-8(14),15-pimaradien-3beta-ol in the isolated rat aorta.

Pimarane-type diterpenes were described to exert antispasmodic and relaxant activities. Based on this observation we hypothesized that the diterpene ent-8(14),15-pimaradien-3beta-ol (PA-3beta-ol) induced vascular relaxation. With this purpose, the present work investigates the mechanisms involved in the vasorelaxant effect of the pimarane-type diterpene PA-3beta-ol. Vascular reactivity experiments, using standard muscle bath procedures, were performed in isolated aortic rings from male Wistar rats. Cytosolic calcium concentration ([Ca(2+)]c) was measured by confocal microscopy using the fluorescent probe Fluo-3AM. PA-3beta-ol (10, 50 and 100 micromol/l) inhibited phenylephrine and KCl-induced contraction in either endothelium-intact or denuded rat aortic rings. PA-3beta-ol also reduced CaCl(2)-induced contraction in Ca(2+)-free solution containing KCl (30 mmol/l) or phenylephrine (0.1 micromol/l). PA-3beta-ol (1-300 micromol/l) concentration dependently relaxed phenylephrine-pre-contracted rings with intact or denuded endothelium. The diterpene also relaxed KCl-pre-contracted rings with intact or denuded endothelium. Moreover, Ca(2+) mobilization study showed that PA-3beta-ol (100 micromol/l) and verapamil (1 micromol/l) inhibited the increase in Ca(2+)-concentration in smooth muscle and endothelial cells induced by phenylephrine (10 micromol/l) or KCl (60 mmol/l). Pre-incubation of intact or denuded aortic rings with N(G)-nitro-l-arginine methyl ester (L-NAME, 100 micromol/l) and 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 1 micromol/l) produced a rightward displacement of the PA-3beta-ol concentration-response curves. On the other hand, 7-nitroindazole (100 micromol/l), 1400 W (1 micromol/l), indomethacin (10 micromol/l) and tetraethylammonium (1 mmol/l) did not affect PA-3beta-ol-induced relaxation. Collectively, our results provide evidence that the effects elicited by PA-3beta-ol involve extracellular Ca(2+) influx blockade. Its effects are also partly mediated by the activation of NO-cGMP pathway.

Immunoreactivity for neuronal NOS and fluorescent indication of NO formation in the NTS of juvenile rats submitted to chronic intermittent hypoxia.

Exposure to chronic intermittent hypoxia (CIH) leads to significant autonomic and respiratory changes, similar to those observed in obstructive sleep apnea. The hypertension associated with CIH is due to sympathoexcitation triggered by long-term exposure to intermittent hypoxia. However, the mechanisms underlying these effects are unknown. Changes in central regulation of sympathetic activity may underlie CIH-induced hypertension. Since NO appears to be mainly sympathoinhibitory in the nucleus of the solitary tract (NTS), we hypothesized that CIH augments sympathetic activity, in part by reducing neuronal nitric oxide synthase (nNOS) expression and consequently nitric oxide (NO) production in this brain region. To test our hypothesis, juvenile male Wistar rats were exposed to CIH for 8 h/day for 10 days and sections of perfused brainstem were either stained to reveal nNOS-immunoreactivity or loaded with DAF 2-DA to label neurons containing NO. CIH rats showed a significant increase in mean arterial pressure and heart rate compared to controls. However, there was no significant difference in the distribution, staining intensity or numbers of nNOS-immunoreactive neurons in the NTS between experimental and control rats. We also found no significant change in NO content in the DAF 2-DA-loaded sections of NTS from CIH rats. Our data show that NO is not altered in the NTS of juvenile CIH rats, suggesting that nitrergic mechanisms, at least in the NTS, are unlikely to be involved in the sympathetic excitation that generates the hypertension observed after 10 days of CIH.

Fluorescent indication that nitric oxide formation in NTS neurons is modulated by glutamate and GABA.

Nitric oxide (NO) in NTS plays an important role in regulating autonomic function to the cardiovascular system. Using the fluorescent dye DAF-2 DA, we evaluated the NO concentration in NTS. Brainstem slices of rats were loaded with DAF-2 DA, washed, fixed in paraformaldehyde and examined under fluorescent light. In different experimental groups, NTS slices were pre-incubated with 1 mM l-NAME (a non-selective NOS inhibitor), 1 mM d-NAME (an inactive enantiomere of l-NAME), 1 mM kynurenic acid (a non-selective ionotropic receptors antagonist) or 20 microM bicuculline (a selective GABAA receptors antagonist) before and during DAF-2 DA loading. Images were acquired using a confocal microscope and the intensity of fluorescence was quantified in three antero-posterior NTS regions. In addition, slices previously loaded with DAF-2 DA were incubated with NeuN or GFAP antibody. A semi-quantitative analysis of the fluorescence intensity showed that the basal NO concentration was similar in all antero-posterior aspects of the NTS (rostral intermediate, 15.5 +/- 0.8 AU; caudal intermediate, 13.2 +/- 1.4 AU; caudal commissural, 13.8 +/- 1.4 AU, n = 10). In addition, the inhibition of NOS and the antagonism of glutamatergic receptors decreased the NO fluorescence in the NTS. On the other hand, d-NAME did not affect the NO fluorescence and the antagonism of GABAA receptors increased the NO fluorescence in the NTS. It is important to note that the fluorescence for NO was detected mainly in neurons. These data show that the fluorescence observed after NTS loading with DAF-2 DA is a result of NO present in the NTS and support the concept that NTS neurons have basal NO production which is modulated by l-glutamate and GABA.

Hypotensive effect of the nitrosyl ruthenium complex nitric oxide donor in renal hypertensive rats.

We have described a new compound (trans-[RuCl([15]aneN(4))NO](2+)), which in vitro releases NO by the action of a reducing agent such as catecholamines. We investigated the effect of this NO donor in lowering the mean arterial pressure (MAP) in severe and moderate renal hypertensive 2K-1C rats. MAP was measured before and after intravenous in bolus injection of the compound in conscious 2K-1C and normotensive (2K) rats. In the hypertensive rats (basal 196.70+/-8.70mmHg, n=5), the MAP was reduced in -34.25+/-13.50mmHg (P<0.05) 6h after administration of 10mmol/L/Kg of the compound in bolus. In normotensive rats the compound had no effect. We have also studied the effect of the injection of 0.1mmol/L/Kg in normotensive (basal 118.20+/-11.25mmHg, n=4), moderate (basal 160.90+/-2.30mmHg, n=6), and severe hypertensive rats (basal 202.46+/-16.74 mmHg, n=6). The compound at the dose of 0.1mmol/L/Kg did not have effect (P>0.05) on MAP of normotensive and moderate hypertensive rats. However, in the severe hypertensive rats (basal 202.46+/-16.70mmHg, n=6) there was a significant reduction on the MAP of -28.64+/-12.45mmHg. The NO donor reduced the MAP of all hypertensive rats in the dose of 10mmol/L/Kg and in the severe hypertensive rats at the dose of 0.1mmol/L/Kg. The compound was not cytotoxic to the rat aortic vascular smooth muscle cells in the concentration of 0.1mmol/L/Kg that produced the maximum relaxation.

Relaxation induced by calcium ionophore is impaired in carotid arteries from 2K-1C rats due to failed effect of nitric oxide on the smooth muscle cells.

Vascular endothelium generates nitric oxide (NO) in large vessels and induces relaxation of vascular smooth muscle cells (VSMC). The aim of this study was to evaluate the contribution of NO produced in the endothelial cells (EC) to the relaxation induced by the Ca2+ ionophore A23187 and whether this relaxation is impaired in renal hypertensive (2K-1C) rat arteries. Concentration-effect curves for A23187 were constructed in intact endothelium isolated carotid rings from 2K-1C and normotensive (2K) in the absence or in the presence of the extracellular NO scavenger haemoglobin or inhibitors of NO-synthase (NOS, L-NOARG), guanylyl-cyclase (GC, ODQ). In carotid rings loaded with Fluo-3AM, both EC and VSMC were simultaneously imaged by a confocal microscope and [Ca2+]c was derived from fluorescence intensities (IF). The maximal relaxation (ME) induced by A23187 was lower in 2K-1C than in 2K arteries. A23187-induced relaxation was abolished by haemoglobin and L-NOARG in both groups. ODQ reduced the ME to A23187 in 2K and abolished its relaxation in 2K-1C. A23187 increased [Ca2+]c in a similar way in 2K and 2K-1C EC, and decreased [Ca2+]c in VSMC, which effect was higher in 2K than in 2K-1C arteries. L-NOARG inhibited the effect of A23187 in VSMC from 2K and abolished it in 2K-1C rats. On the other hand, L-NOARG did not modify the effect of A23187 in EC from 2K and 2K-1C rats. The basal content of cGMP was higher in 2K than in 2K-1C arterial rings that was similarly increased by A23187. In conclusion, the Ca2+ ionophore A23187 increases Ca2+, activates NOS and NO production in the EC activating GC in VSMC and [Ca2+]c decrease. All these effects are higher in 2K, which contribute to the impaired relaxation to A23187 in 2K-1C rat arteries.

The inducing NO-vasodilation by chemical reduction of coordinated nitrite ion in cis-[Ru(NO(2))L(bpy)(2)](+) complex.

The synthesis of [Ru(NO(2))L(bpy)(2)](+) (bpy = 2,2'-bipyridine and L = pyridine (py) and pyrazine (pz)) can be accomplished by addition of [Ru(NO)L(bpy)(2)](PF(6))(3) to aqueous solutions of physiological pH. The electrochemical processes of [Ru(NO(2))L(bpy)(2)](+) in aqueous solution were studied by cyclic voltammetry and differential pulse voltammetry. The anodic scan shows a peak around 1.00 V vs. Ag/AgCl attributed to the oxidation process centered on the metal ion. However, in the cathodic scan a second peak around -0.60 V vs. Ag/AgCl was observed and attributed to the reduction process centered on the nitrite ligand. The controlled reduction potential electrolysis at -0.80 V vs. Ag/AgCl shows NO release characteristics as judged by NO measurement with a NO-sensor. This assumption was confirmed by ESI/MS(+) and spectroelectrochemical experiment where cis-[Ru(bpy)(2)L(H(2)O)](2+) was obtained as a product of the reduction of cis-[Ru(II)(NO(2))L(bpy)(2)](+). The vasorelaxation observed in denuded aortic rings pre-contracted with 0.1 mumol L(-1) phenylephrine responded with relaxation in the presence of cis-[Ru(II)(NO(2))L(bpy)(2)](+). The potential of rat aorta cells to metabolize cis-[Ru(II)(NO(2))L(bpy)(2)](+) was also followed by confocal analysis. The obtained results suggest that NO release happens by reduction of cis-[Ru(II)(NO(2))L(bpy)(2)](+) inside the cell. The maximum vasorelaxation was achieved with 1 x 10(-5) mol L(-1) of cis-[Ru(II)(NO(2))L(bpy)(2)](+) complex.

Mechanical ventilatory support in preterm infants.

A large proportion of premature infants presents with acute respiratory failure after birth and require mechanical ventilatory support. In addition to conventional mechanical ventilation, an increasing number of these infants are currently supported by newer modes including synchronized, volume targeted and noninvasive mechanical ventilation. While these new modes have improved weaning from mechanical ventilation they have not had a consistent impact on respiratory outcome or other morbidities. This is a review of the different modes of invasive and noninvasive mechanical ventilation used to support premature infants with respiratory failure.

A novel mechanism of vascular relaxation induced by sodium nitroprusside in the isolated rat aorta.

Sodium nitroprusside (SNP) is an endothelium-independent relaxant agent and its effect is attributed to its direct action on the vascular smooth muscle (VSM). Endothelium modulates the vascular tone through the release of vasoactive agents, such as NO. The aim of this study was to investigate the contribution of the endothelium on SNP vasorelaxation, NO release and Ca2+ mobilization. Vascular reactivity experiments showed that endothelium potentiates the SNP-relaxation in rat aortic rings and this effect was abolished by l-NAME. SNP-relaxation in intact endothelium aorta was inhibited by NOS inhibitors for the constitutive isoforms (cNOS). Furthermore, endogenous NO is involved on the SNP-effect and this endogenous NO is released by cNOS. Moreover, Ca2+ mobilization study shows that l-NAME inhibited the reduction of Ca2+-concentration in VSM cells and reduced the increase in Ca2+-concentration in endothelial cells induced by SNP. This enhancement in Ca2+-concentration in the endothelial cells is due to a voltage-dependent Ca2+ channels activation. The present findings indicate that the relaxation and [Ca2+]i decrease induced by SNP in VSM cells is potentiated by endothelial production of NO by cNOS-activation in rat aorta.