Nitric oxide regulates oxygen uptake and hydrogen peroxide release by the isolated beating rat heart

Isolated rat heart perfused with 1.5-7.5µM NO solutions or bradykinin, which activates endothelial NOsynthase, showed a dose-dependent decrease in myocardial O₂uptake from 3.2 ± 0.3 to 1.6 ± 0.1 (7.5 µM NO, n = 18,P < 0.05) and to 1.2 ± 0.1 µM O₂ · min¹ · gtissue¹ (10 µM bradykinin, n = 10,P < 0.05). Perfused NO concentrations correlated with aninduced release of hydrogen peroxide (H₂O₂) inthe effluent (r = 0.99, P < 0.01). NO markedlydecreased the O₂ uptake of isolated rat heart mitochondria(50% inhibition at 0.4 µM NO, r = 0.99,P < 0.001). Cytochrome spectra in NO-treated submitochondrial particles showed a double inhibition of electron transfer at cytochrome oxidase and between cytochrome b andcytochrome c, which accounts for the effects in O₂uptake and H₂O₂ release. Most NO was bound tomyoglobin; this fact is consistent with NO steady-state concentrationsof 0.1-0.3 µM, which affect mitochondria. In the intact heart,finely adjusted NO concentrations regulate mitochondrial O₂uptake and superoxide anion production (reflected byH₂O₂), which in turn contributes to thephysiological clearance of NO through peroxynitrite formation.

     

Lack of endothelial nitric oxide synthase decreases cardiomyocyte proliferation and delays cardiac maturation

We recently demonstrated that deficiency in endothelial nitric oxide synthase (eNOS) results in congenital septal defects and postnatal heart failure. The aim of this study was to investigate the role of eNOS in cardiomyocyte proliferation and maturation during postnatal development. Cultured eNOS knockout (eNOS^–/–) cardiomyocytes displayed fewer cells and lower bromodeoxyuridine (BrdU) incorporation in vitro compared with wild-type (WT) cardiomyocytes (P < 0.05). Treatment with the nitric oxide (NO) donor diethylenetriamine NONOate increased BrdU incorporation and cell counts in eNOS^–/– cardiomyocytes (P < 0.05). Inhibition of nitric oxide synthase activity using N^G-nitro-L-arginine methyl ester decreased the level of BrdU incorporation and cell counts in WT cardiomyocytes (P < 0.05). Vascular endothelial growth factor (VEGF) increased the level of BrdU incorporation in cultured WT cardiomyocytes in a dose- and time-dependent manner (P < 0.05). Conversely, VEGF did not alter BrdU incorporation in eNOS^–/– cardiomyocytes (P = not significant). Furthermore, deficiency in eNOS significantly decreased BrdU labeling indexes in neonatal hearts in vivo. Although WT hearts displayed a rapid decrease in atrial natriuretic peptide (ANP) expression in the first week of neonatal life, ANP expression in eNOS^–/– hearts remain elevated. Our study demonstrated that NO production from eNOS is necessary for postnatal cardiomyocyte proliferation and maturation, suggesting that eNOS plays an important role during postnatal heart development. proliferation; heart development     

Hypoxia induces permeability in brain microvessel endothelial cells via VEGF and NO

In this study, an in vitro model of the blood-brain barrier,consisting of porcine brain-derived microvascular endothelial cells(BMEC), was used to evaluate the mechanism of hypoxia-induced hyperpermeability. We show that hypoxia-induced permeability in BMECwas completely abolished by a neutralizing antibody to vascular endothelial growth factor (VEGF). In contrast, under normoxic conditions, addition of VEGF up to 100 ng/ml did not alter monolayer barrier function. Treatment with either hypoxia or VEGF under normoxicconditions induced a twofold increase in VEGF binding sites and VEGFreceptor 1 (Flt-1) mRNA expression in BMEC. Hypoxia-induced permeability also was prevented by the nitric oxide (NO) synthase inhibitor N^G-monomethyl-L-arginine,suggesting that NO is involved in hypoxia-induced permeability changes,which was confirmed by measurements of the cGMP level. During normoxia,treatment with VEGF (5 ng/ml) increased permeability as well as cGMPcontent in the presence of several antioxidants. These results suggestthat hypoxia-induced permeability in vitro is mediated by the VEGF/VEGFreceptor system in an autocrine manner and is essentially dependent onreducing conditions stabilizing the second messenger NO as the mediatorof changes in barrier function of BMEC.     

L-Arginine inhibits vasopressin-stimulated mesangial cell Ca2+

L-Arginine (L-Arg) affects variousparameters that modulate the progression of renal disease. These samefactors [e.g., glomerular filtration rate, changes in mesangialcell (MC) tension, and production of NO] are all controlled atleast in part by changes in MC intracellular Ca²⁺concentration([Ca²⁺]_i). Wetherefore evaluated the effect of L-Arg on MC[Ca²⁺]_i. We found thatL-Arg inhibits the vasopressin-stimulated rise in MC[Ca²⁺]_i both in rat andmurine cell cultures. This effect does not appear to be due tometabolism of L-Arg to either NO or L-ornithine (L-Orn). Blocking the metabolism of L-Arg withN-monomethyl-L-arginine, an NOsynthase inhibitor, or with 20 mM L-valine(L-Val), an inhibitor of Orn formation,does not reverse the inhibition. However, other cationic amino acids,as well guanidine, the functional group ofL-Arg, all inhibit thevasopressin-stimulated rise in[Ca²⁺]_i,consistent with a structural basis for this effect. We conclude that1)L-Arg inhibitsvasopressin-stimulated murine and rat MC [Ca²⁺]_irise, 2) this inhibition is notmediated by metabolism of L-Arg to either NO or L-Orn, and3) the effect ofL-Arg is due to its cationicfunctional group, guanidine.

     

Prolonged hypoxia increases vascular endothelial growth factor mRNA and protein in adult mouse brain

Brain hypoxiainduces an increase in brain vascularity, presumably mediated byvascular endothelial growth factor (VEGF), but it is unclear whetherVEGF is required to maintain the increase. In these studies, brain VEGFmRNA and protein levels were measured in adult mice kept in hypobaricchambers at 0.5 atm for 0, 0.5, 1, 2, 4, 7, and 21 days. Hypoxia wasaccompanied by a transient increase of VEGF mRNA expression: twofold by0.5 day and a maximum of fivefold by 2 days; these were followed by adecrease at 4 days and a return to basal levels by 7-21 days. VEGFprotein expression induced by hypoxia was bimodal, initiallyparalleling VEGF mRNA. There was an initial small increase at 12 h thatreached a maximum by day 2, and, aftera transient decrease on day 4, theprotein expression increased again on day7 before it returned to normoxic levels after 21 days.Thus, despite continued hypoxia, both VEGF mRNA and protein levelsreturned to basal after 7 days. These data suggest a metabolicnegative-feedback system for VEGF expression during prolonged hypoxiain the brain.

     

Role of endothelial nitric oxide synthase in endothelial activation: insights from eNOS knockout endothelial cells

The objective of this study was to determine whether absence of endothelial nitric oxide synthase (eNOS) affects the expression of cell surface adhesion molecules in endothelial cells. Murine lung endothelial cells (MLECs) were prepared by immunomagnetic bead selection from wild-type and eNOS knockout mice. Wild-type cells expressed eNOS, but eNOS knockout cells did not. Expression of neuronal NOS and inducible NOS was not detectable in cells of either genotype. Upon stimulation, confluent wild-type MLECs produced significant amounts of NO compared with N-monomethyl-L-arginine-treated wild-type cells. eNOS knockout and wild-type cells showed no difference in the expression of E-selectin, P-selectin, intracellular adhesion molecule-1, and vascular cell adhesion molecule-1 as measured by flow cytometry on the surface of platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31)-positive cells. Both eNOS knockout and wild-type cells displayed the characteristics of resting endothelium. Adhesion studies in a parallel plate laminar flow chamber showed no difference in leukocyte-endothelial cell interactions between the two genotypes. Cytokine treatment induced endothelial cell adhesion molecule expression and increased leukocyte-endothelial cell interactions in both genotypes. We conclude that in resting murine endothelial cells, absence of endothelial production of NO by itself does not initiate endothelial cell activation or promote leukocyte-endothelial cell interactions. We propose that eNOS derived NO does not chronically suppress endothelial cell activation in an autocrine fashion but serves to counterbalance signals that mediate activation. vascular biology; atherosclerosis; mouse models     

Endothelial response to stress from exogenous Zn2+ resembles that of NO-mediated nitrosative stress, and is protected by MT-1 overexpression

While nitric oxide (NO)-mediated biological interactions have been intensively studied, the underlying mechanisms of nitrosative stress with resulting pathology remain unclear. Previous studies have demonstrated that NO exposure increases free zinc ions (Zn²⁺) within cells. However, the resulting effects on endothelial cell survival have not been adequately resolved. Thus the purpose of this study was to investigate the role of altered zinc homeostasis on endothelial cell survival. Initially, we confirmed the previously observed significant increase in free Zn²⁺ with a subsequent induction of apoptosis in our pulmonary artery endothelial cells (PAECs) exposed to the NO donor N-[2-aminoethyl]-N-[2-hydroxy-2-nitrosohydrazino]-1,2-ethylenediamine. However, NO has many effects upon cell function and we wanted to specifically evaluate the effects mediated by zinc. To accomplish this we utilized the direct addition of zinc chloride (ZnCl₂) to PAEC. We observed that Zn²⁺-exposed PAECs exhibited a dose-dependent increase in superoxide (O₂^–·) generation that was localized to the mitochondria. Furthermore, we found Zn²⁺-exposed PAECs exhibited a significant reduction in mitochondrial membrane potential, loss of cardiolipin from the inner leaflet, caspase activation, and significant increases in TdT-mediated dUTP nick end labeling-positive cells. Furthermore, using an adenoviral construct for the overexpression of the Zn²⁺-binding protein, metallothionein-1 (MT-1), we found either MT-1 overexpression or coincubation with a Zn²⁺-selective chelator, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylene-diamide, in PAECs significantly protected the mitochondria from both NO and Zn²⁺-mediated disruption and induction of apoptosis and cell death. In summary, our results indicate that a loss of Zn²⁺ homeostasis produces mitochondrial dysfunction, increased oxidative stress, and apoptotic cell death. We propose that regulation of Zn²⁺ levels may represent a potential therapeutic target for disease associated with both nitrosative and oxidative stress. reactive nitrogen species; apoptosis mitochondrial dysfunction     

Taurine prevents high-glucose-induced human vascular endothelial cell apoptosis

Elevated blood glucose in uncontrolled diabetes is causallycorrelated with diabetic microangiopathy. Hyperglycemia-triggered accelerated endothelial cell apoptosis is a critical event in theprocess of diabetes-associated microvascular disease. The conditionallysemiessential amino acid taurine has been previously shown to protectagainst human endothelial cell apoptosis. Therefore, this study wasdesigned to investigate the role of taurine in the prevention ofhigh-glucose-mediated cell apoptosis in human umbilical veinendothelial cells (HUVEC) and the mechanisms involved. Exposure ofHUVEC to 30 mM glucose for 48 h (short-term) and 14 days (long-term)resulted in a significant increase in apoptosis, compared with normalglucose (5.5 mM; P < 0.05).High-glucose-induced DNA fragmentation preferentially occurred in the Sphase cells. Mannitol (as osmotic control) at 30 mM failed to induceHUVEC apoptosis. Taurine prevented high-glucose-induced HUVECapoptosis, which correlates with taurine attenuation ofhigh-glucose-mediated increased intracellular reactive oxygen species(ROS) formation and elevated intracellularCa²⁺ concentration([Ca²⁺]_i)level. Antioxidants, DMSO, N-acetylcysteine, and glutathione, only partly attenuated high-glucose-inducedHUVEC apoptosis. Glucose at 30 mM did not cause HUVEC necrosis.However, both glucose and mannitol at 60 mM caused HUVEC necrosis asrepresented by increased lactate dehydrogenase release and cell lysis.Taurine failed to prevent hyperosmolarity-induced cell necrosis. Theseresults demonstrate that taurine attenuates hyperglycemia-induced HUVECapoptosis through ROS inhibition and[Ca²⁺]_istabilization and suggest that taurine may exert a beneficial effect inpreventing diabetes-associated microangiopathy.

     

Peptides inhibit selectin-mediated cell adhesion in vitro, and neutrophil influx into inflammatory sites in vivo

The selectins are cell adhesion molecules whose carbohydrate-bindingdomain (C-type lectin) is thought to be involved in leukocyteadhesion to activated vascular endothelium in the inflammatoryprocess. A series of peptides, based on a conserved region (⁴⁸YYWIGIRK⁵⁵-NH₂)of the lectin domain of E-, L- and P-selectins, were analysedfor their ability to block selectin-mediated cell adhesion invitro, and neutrophil infiltration into sites of inflammationin vivo. The peptides inhibited the adhesion of myeloid cellsto recombinant forms of E- and P-selectin. The adhesion of myeloidcells to human endothelial cells, stimulated to express E-selectin,was also inhibited by the peptides. Finally, the peptides blockedthe adhesion of lymphocytes, expressing L-selectin, to highendothelial venules in lymph nodes which contain the ligandfor L-selectin. A clear structure-activity relationship wasestablished when peptides of different amino acid chain lengthswere tested in these assays. Peptides lacking tyrosine residues(e.g. WIGIR-NH₂) at their amino terminus were poor inhibitorsof selectin-mediated cell adhesion in vitro. The peptides thatwere found to be inhibitors of cell adhesion in vitro were alsofound to inhibit (up to 70%) neutrophil infiltration into sitesof inflammation in a thioglycollate-induced peritonitis mousemodel system. They also significantly reduced (>50%) themigration of neutrophils into cytokine-treated skin. These resultsstrongly suggest that compounds based on these tyrosine-containing,selectin-derived peptides could be used as anti-inflammatorytherapeutic agents. inflammation neutrophils peptides selectins     

cGMP-mediated Ca2+ release from IP3-insensitive Ca2+ stores in smooth muscle

Recent studies on the role of nitric oxide (NO) ingastrointestinal smooth muscle have raised the possibility thatNO-stimulated cGMP could, in the absence of cGMP-dependent proteinkinase (PKG) activity, act as aCa²⁺-mobilizing messenger[K. S. Murthy, K.-M. Zhang, J.-G. Jin, J. T. Grider, and G. M. Makhlouf. Am. J. Physiol. 265 (Gastrointest. Liver Physiol. 28):G660-G671, 1993]. This notion was examined indispersed gastric smooth muscle cells with 8-bromo-cGMP (8-BrcGMP) andwith NO and vasoactive intestinal peptide (VIP), which stimulate endogenous cGMP. In muscle cells treated with cAMP-dependent protein kinase (PKA) and PKG inhibitors (H-89 and KT-5823), 8-BrcGMP (10 µM),NO (1 µM), and VIP (1 µM) stimulated⁴⁵Ca²⁺release (21 ± 3 to 30 ± 1% decrease in⁴⁵Ca²⁺cell content); Ca²⁺ releasestimulated by 8-BrcGMP was concentration dependent with anEC₅₀ of 0.4 ± 0.1 µM and athreshold of 10 nM. 8-BrcGMP and NO increased cytosolic freeCa²⁺ concentration([Ca²⁺]_i)and induced contraction; both responses were abolished after Ca²⁺ stores were depleted withthapsigargin. With VIP, which normally increases[Ca²⁺]_iby stimulating Ca²⁺ influx,treatment with PKA and PKG inhibitors caused a further increase in[Ca²⁺]_ithat reverted to control levels in cells pretreated with thapsigargin. Neither Ca²⁺ release norcontraction induced by cGMP and NO in permeabilized muscle cells wasaffected by heparin or ruthenium red.Ca²⁺ release induced by maximallyeffective concentrations of cGMP and inositol 1,4,5-trisphosphate(IP₃) was additive, independent of which agent was applied first. We conclude that, in the absence ofPKA and PKG activity, cGMP stimulatesCa²⁺ release from anIP₃-insensitive store and that itseffect is additive to that of IP₃.

     

Endogenous nitric oxide decreases xanthine oxidase-mediated neutrophil adherence: role of P-selectin

Terada, Lance S., John E. Repine, Dale Piermattei, andBrooks M. Hybertson. Endogenous nitric oxide decreases xanthine oxidase-mediated neutrophil adherence: role of P-selectin.J. Appl. Physiol. 82(3): 913-917, 1997.The oxygen radical-producing enzyme xanthine oxidase (XO) canpromote neutrophil adherence to endothelium. Recognizing that a balanceoften exists in inflammatory processes, we sought to determine whetherXO initiates antiadherent pathways. We found that bovine pulmonaryarterial endothelial cells (EC) exposed to XO released increasedamounts of nitrite into the media, reflecting an increased productionof nitric oxide (NO). When EC were subjected to shear stress, treatmentwith XO and/or the NO synthase inhibitorN-nitro-L-arginine(L-NNA) increased neutrophilrolling behavior and firm neutrophil adherence to EC in an additivefashion. Both rolling and adherent interactions were abolished bymonoclonal antibodies directed against P-selectin. In addition,treatment of EC with XO and/orL-NNA increased both surfaceexpression of P-selectin and release of von Willebrand factor intomedia. Finally, treatment of EC with the NO donor sodium nitroprussidedecreased XO-mediated neutrophil rolling and adherence. We concludethat XO stimulates EC to produce NO and that NO decreases theP-selectin-dependent neutrophil adhesion initiated by XO. Suchincreases in endogenous NO may constitute an importantnegative-feedback response to the acute proadhesive effects of XO.

     

Squalamine, a novel cationic steroid, specifically inhibits the brush-border Na+/H+ exchanger isoform NHE3

Squalamine, anendogenous molecule found in the liver and other tissues ofSqualus acanthias, hasantibiotic properties and causes changes in endothelial cell shape. Thelatter suggested that its potential targets might include transportproteins that control cell volume or cell shape. The effect of purifiedsqualamine was examined on clonedNa⁺/H⁺exchanger isoforms NHE1, NHE2, and NHE3 stably transfected in PS120fibroblasts. Squalamine (1-h pretreatment) decreased the maximalvelocity of rabbit NHE3 in a concentration-dependent manner (13, 47, and 57% inhibition with 3, 5, and 7 µg/ml, respectively) and alsoincreasedK'[H⁺]_i.Squalamine did not affect rabbit NHE1 or NHE2 function. The inhibitoryeffect of squalamine was 1) timedependent, with no effect of immediate addition and maximum effect with1 h of exposure, and 2) fullyreversible. Squalamine pretreatment of the ileum for 60 min inhibitedbrush-border membrane vesicleNa⁺/H⁺activity by 51%. Further investigation into the mechanism of squalamine's effects showed that squalamine required the COOH-terminal 76 amino acids of NHE3. Squalamine had no cytotoxic effect at theconcentrations studied, as indicated by monitoring lactate dehydrogenase release. These results indicate that squalamine 1) is a specific inhibitor of thebrush-border NHE isoform NHE3 and not NHE1 or NHE2,2) acts in a nontoxic and fullyreversible manner, and 3) has adelayed effect, indicating that it may influence brush-borderNa⁺/H⁺exchanger function indirectly, through an intracellular signaling pathway or by acting as an intracellular modulator.

     

VE-cadherin: adhesion at arm's length

VE-cadherin was first identified in the early 1990s and quickly emerged as an important endothelial cell adhesion molecule. The past decade of research has revealed key roles for VE-cadherin in vascular permeability and in the morphogenic events associated with vascular remodeling. The details of how VE-cadherin functions in adhesion became apparent with structure-function analysis of the cadherin extracellular domain and with the identification of the catenins, a series of cytoplasmic proteins that bind to the cadherin tail and mediate interactions between cadherins and the cytoskeleton. Whereas early work focused on the armadillo family proteins -catenin and plakoglobin, more recent investigations have identified p120-catenin (p120^ctn) and a related group of armadillo family members as key binding partners for the cadherin tail. Furthermore, a series of new studies indicate a key role for p120^ctn in regulating cadherin membrane trafficking in mammalian cells. These recent studies place p120^ctn at the hub of a cadherin-catenin regulatory mechanism that controls cadherin plasma membrane levels in cells of both epithelial and endothelial origin. endothelial cell; cytoskeleton; -catenin; p120^ctn; cell adhesion; vascular endothelial cadherin     

Determination of the enhancing action of HSP90 on neuronal nitric oxide synthase by EPR spectroscopy

Recent studies showed that heatshock protein 90 (HSP90) enhances nitric oxide (NO) synthesis fromendothelial and neuronal NO synthase (eNOS and nNOS, respectively).However, these findings were based on indirect NO measurements.Moreover, although our previous studies showed that the action of HSP90involves increased Ca²⁺/calmodulin (Ca²⁺/CaM)binding, quantitative measurements of the effect of HSP90 on CaMbinding to nNOS have been lacking. With electron paramagnetic resonancespectroscopy, we directly measured NO signals from purified nNOS. HSP90augmented NO formation from nNOS in a dose-dependent manner. Tryptophanfluorescence-quenching measurements revealed that HSP90 markedlyreduced the K_d of CaM to nNOS (0.5 ± 0.1 nM vs. 9.4 ± 1.8 nM in the presence and absence of HSP90,P < 0.01). Ca²⁺ ionophore triggered strongNO production from nNOS-transfected cells, and this was significantlyreduced by the HSP90 inhibitor geldanamycin. Thus these studies providedirect evidence demonstrating that HSP90 enhances nNOS catalyticfunction in vitro and in intact cells. The effect of HSP90 is mediatedby the enhancement of CaM binding to nNOS.

     

Reaction of nitric oxide with superoxide inhibits basolateral K+ channels in the rat CCD

We previously demonstrated that nitric oxide (NO) stimulates thebasolateral small-conductance K⁺channel (SK) via a cGMP-dependent pathway [M. Lu and W. H. Wang. Am. J. Physiol. 270 (Cell Physiol. 39): C1336-C1342,1996]. Because NO at high concentration has been shown to reactwith superoxide (O₂) to formperoxynitrite (OONO)[W. A. Pryor and G. L. Squadrito. Am. J. Physiol. 268 (Lung Cell. Mol.Physiol. 12): L699-L722, 1995 and M. S. Wolin.Microcirculation 3: 1-17,1996], we extended our study to examine, using patch-clamp technique, the effect of high concentrations of NO on SK in cortical collecting duct (CCD) of rat kidney. Addition of NO donors[100-200 µMS-nitroso-N-acetyl-penicillamine(SNAP) or sodium nitroprusside (SNP)] reduced channel activity,defined as the product of channel number and open probability, to 15 and 25% of the control value, respectively. The inhibitory effect ofNO was completely abolished in the presence of 10 mM Tiron, anintracellular scavenger of O₂. NOdonors, 10 µM SNAP or SNP, which stimulate channel activity undercontrol conditions, can also inhibit SK in the presence of anO₂ donor, pyrogallol, or in thepresence of an inhibitor of superoxide dismutase, diethyldithiocarbamic acid. The inhibitory effect of NO is still observed in the presence ofexogenous cGMP, suggesting that the NO-induced inhibition is not theresult of decreased cGMP production. We conclude that the inhibitoryeffect of NO on channel activity results from an interaction between NOand O₂.

     

L-type voltage-dependent Ca2+ channels in cerebral microvascular endothelial cells and ET-1 biosynthesis

We investigatedthe role of intracellular calcium concentration([Ca²⁺]_i) in endothelin-1 (ET-1) production,the effects of potential vasospastic agents on[Ca²⁺]_i, and the presence of L-typevoltage-dependent Ca²⁺ channels in cerebral microvascularendothelial cells. Primary cultures of endothelial cells isolated frompiglet cerebral microvessels were used. Confluent cells were exposed toeither the thromboxane receptor agonist U-46619 (1 µM),5-hydroxytryptamine (5-HT; 0.1 mM), or lysophosphatidic acid (LPA; 1 µM) alone or after pretreatment with the Ca²⁺-chelatingagent EDTA (100 mM), the L-type Ca²⁺ channel blockerverapamil (10 µM), or the antagonist of receptor-operated Ca²⁺ channel SKF-96365 HCl (10 µM) for 15 min. ET-1production increased from 1.2 (control) to 8.2 (U-46619), 4.9 (5-HT),or 3.9 (LPA) fmol/µg protein, respectively. Such elevated ET-1biosynthesis was attenuated by verapamil, EDTA, or SKF-96365 HCl. Toinvestigate the presence of L-type voltage-dependent Ca²⁺channels in endothelial cells, the [Ca²⁺]_isignal was determined fluorometrically by using fura 2-AM. Superfusionof confluent endothelial cells with U-46619, 5-HT, or LPA significantlyincreased [Ca²⁺]_i. Pretreatment ofendothelial cells with high K⁺ (60 mM) or nifedipine (4 µM) diminished increases in [Ca²⁺]_i inducedby the vasoactive agents. These results indicate that 1)elevated [Ca²⁺]_i signals are involved in ET-1biosynthesis induced by specific spasmogenic agents, 2) theincreases in [Ca²⁺]_i induced by thevasoactive agents tested involve receptor as well as L-typevoltage-dependent Ca²⁺ channels, and 3) primarycultures of cerebral microvascular endothelial cells express L-typevoltage-dependent Ca²⁺ channels.

     

Inhibition of nitric oxide synthase slows heart rate recovery from cholinergic activation

The role ofnitric oxide (NO) in the cholinergic regulation of heart rate(HR) recovery from an aspect of simulated exercise wasinvestigated in atria isolated from guinea pig to test the hypothesisthat NO may be involved in the cholinergic antagonism of the positivechronotropic response to adrenergic stimulation. Inhibition of NOsynthesis withN^G-monomethyl-L-arginine(L-NMMA, 100 µM) significantlyslowed the time course of the reduction in HR without affecting themagnitude of the response elicited by bath-applied ACh (100 nM) orvagal nerve stimulation (2 Hz). The half-times(t_1/2) of responses were 3.99 ± 0.41 s in control vs. 7.49 ± 0.68 s inL-NMMA(P < 0.05). This was dependent onprior adrenergic stimulation (norepinephrine, 1 µM). The effect ofL-NMMA was reversed byL-arginine (1 mM; t_1/2 4.62 ± 0.39 s). The calcium-channelantagonist nifedipine (0.2 µM) also slowed the kinetics of thereduction in HR caused by vagal nerve stimulation. However, thet_1/2 for the reduction in HR with antagonists (2 mM Cs⁺ and 1 µM ZD-7288) of thehyperpolarization-activated current were significantlyfaster compared with control. There was no additional effect ofL-NMMA orL-NMMA+L-arginineon vagal stimulation in groups treated with nifedipine,Cs⁺, or ZD-7288. Weconclude that NO contributes to the cholinergic antagonism of thepositive cardiac chronotropic effects of adrenergic stimulation byaccelerating the HR response to vagal stimulation. This may involve aninterplay between two pacemaking currents (L-type calcium channelcurrent and hyperpolarization-activated current). Whether NO modulatesthe vagal control of HR recovery from actual exercise remains to bedetermined.

     

Inhaled nitric oxide prevents left ventricular impairment during endotoxemia

We evaluated theeffect of long-term inhalation of nitric oxide (NO) on cardiaccontractility after endotoxemia by using the end-systolicelastance of the left ventricle (LV) as a load-independent contractility index. Chronic instrumentation in 12 pigs included implantation of two pairs of endocardial dimension transducers tomeasure LV volume and a micromanometer to measure LV pressure. One weeklater, the animals were divided into a control group (n = 6) or a NO group(n = 6). All animals receivedintravenous Escherichia coliendotoxin (10 µg · kg¹ · h¹)and equivalent lactated Ringer solution. NO inhalation (20 parts/million) was begun 30 min after the initiation of endotoxemia andwas continued for 24 h. In both groups, tachycardia, pulmonaryhypertension, and systemic hyperdynamic changes were noted. Theend-systolic elastance in the control group was significantly decreasedbeyond 7 h. NO inhalation maintained the end-systolic elastance atbaseline levels and prevented its impairment. These findings indicatethat NO exerts a protective effect on LV contractility in this model of endotoxemia.

     

Protective effects of intravascular pressure and nitric oxide in ischemic lung injury

Cessation of bloodflow during ischemia will decrease both distending and shearforces exerted on endothelium and may worsen ischemic lung injury bydecreasing production of nitric oxide (NO), which influences vascularbarrier function. We hypothesized that increased intravascular pressure(Piv) during ventilated ischemia might maintain NO productionby increasing endothelial stretch or shear forces, thereby attenuatingischemic lung injury. Injury was assessed by measuring the filtrationcoefficient(K_f) and theosmotic reflection coefficient for albumin(_alb) after 3 h of ventilated(95% O₂-5%CO₂; expiratory pressure 3 mmHg) ischemia. Lungs were flushed with physiological salt solution, and then Piv was adjusted to achieve High Piv (mean 6.7 ± 0.4 mmHg, n = 15) or Low Piv (mean0.83 ± 0.4 mmHg, n = 10).N^G-nitro-L-arginine methyl ester(L-NAME;10⁵ M,n = 10),N^G-nitro-D-argininemethyl ester (D-NAME;10⁵ M,n = 11), orL-NAME(10⁵M)+L-arginine (5 × 10⁴ M,n = 6) was added at the start ofischemia in three additional groups of lungs with High Piv.High Piv attenuated ischemic injury compared with Low Piv(_alb 0.67 ± 0.04 vs. 0.35 ± 0.04, P < 0.05). Theprotective effect of High Piv was abolished byL-NAME(_alb 0.37 ± 0.04, P < 0.05) but not byD-NAME(_alb 0.63 ± 0.07). The effects of L-NAME were overcomeby an excess of L-arginine(_alb 0.56 ± 0.05, P < 0.05).K_f did not differsignificantly among groups. These results suggest that Piv modulatesischemia-induced barrier dysfunction in the lung, and theseeffects may be mediated by NO.

     

Contrasting effects of NO and peroxynitrites on HSP70 expression and apoptosis in human monocytes

The free radicals nitric oxide(·NO) and superoxide (O₂·) react to formperoxynitrite (ONOO), a highly toxic oxidant species. Inthis study we investigated the respective effects of NO andONOO in monocytes from healthy human donors. Purifiedmonocytes were incubated for 6 or 16 h with a pure NO donor(S-nitroso-N-acetyl-DL-penicillamine, 0-2 mM), an ·NO/ONOO donor(3-morpholinosydnonimine chlorhydrate, 0-2 mM) with and withoutsuperoxide dismutase (200 IU/ml), or pure ONOO. Weprovide evidence that 3-morpholinosydnonimine chlorhydrate alonerepresents a strong stress to human monocytes leading to adose-dependent increase in heat shock protein-70 (HSP70) expression, mitochondrial membrane depolarization, and cell death by apoptosis andnecrosis. These phenomena were abolished by superoxide dismutase, suggesting that ONOO, but not ·NO, was responsible forthe observed effects. This observation was further strengthened by theabsence of a stress response in cells exposed toS-nitroso-N-acetyl-DL-penicillamine. Conversely, exposure of cells to ONOO alone also inducedmitochondrial membrane depolarization and cell death by apoptosis andnecrosis. Thus ONOO formation may well explain the toxiceffect generally attributed to ·NO.