Biomedical subjects

B Mayer

Publications and source records attributed to B Mayer.

At least 73 records · Page 4Linked to original sources

PubMed2000

Inhibitory effects of aclarubicin on nitric oxide production in aortic smooth muscle cells and macrophages.

The effects of aclaruhicin (ACR), an anthracycline antibiotic, on inducible nitric oxide (NO) synthesis was investigated in rat aortic smooth muscle cells (RASMCs) and RAW macrophages. ACR at concentrations as low as 0.1 microM significantly inhibited NO production induced by interleukin-1beta in RASMCs. About 5- to 10-fold higher concentrations of ACR were required for inhibition of interferon-gamma and lipopolipopolysaccharide-induced NO production in RAW cells. When ACR was subsequently administered to inducible NO synthase (iNOS) induction, the NO production was barely suppressed in RASMCs. Moreover, ACR (up to 10 microM) lacked direct inhibitory effects on iNOS activity in homogenates of these cells. ACR (0.1 microM) inhibited the expression of iNOS protein and mRNA in RASMCs without concomitant cytotoxic effects. ACR (>0.5 microM)-induced inhibition of NO production in RAW cells was associated with substantial cytotoxic effects as shown by measurement of lactate dehydrogenase release. These results suggest that ACR is a potent inhibitor of iNOS induction in vascular smooth muscle, but inhibits iNOS induction in macrophage only at high cytotoxic

Aclarubicin↗

PubMed2000

Dityrosine formation outcompetes tyrosine nitration at low steady-state concentrations of peroxynitrite. Implications for tyrosine modification by nitric oxide/superoxide in vivo.

Formation of peroxynitrite from NO and O-(*2) is considered an important trigger for cellular tyrosine nitration under pathophysiological conditions. However, this view has been questioned by a recent report indicating that NO and O-(*2) generated simultaneously from (Z)-1-(N-[3-aminopropyl]-N-[4-(3-aminopropylammonio)butyl]-amino) diazen-1-ium-1,2-diolate] (SPER/NO) and hypoxanthine/xanthine oxidase, respectively, exhibit much lower nitrating efficiency than authentic peroxynitrite (Pfeiffer, S. and Mayer, B. (1998) J. Biol. Chem. 273, 27280-27285). The present study extends those earlier findings to several alternative NO/O-(*2)-generating systems and provides evidence that the apparent lack of tyrosine nitration by NO/O-(*2) is due to a pronounced decrease of nitration efficiency at low steady-state concentrations of authentic peroxynitrite. The decrease in the yields of 3-nitrotyrosine was accompanied by an increase in the recovery of dityrosine, showing that dimerization of tyrosine radicals outcompetes the nitration reaction at low peroxynitrite concentrations. The observed inverse dependence on peroxynitrite concentration of dityrosine formation and tyrosine nitration is predicted by a kinetic model assuming that radical formation by peroxynitrous acid homolysis results in the generation of tyrosyl radicals that either dimerize to yield dityrosine or combine with (*)NO(2) radical to form 3-nitrotyrosine. The present results demonstrate that very high fluxes (>2 microM/s) of NO/O-(*2) are required to render peroxynitrite an efficient trigger of tyrosine nitration and that dityrosine is a major product of tyrosine modification caused by low steady-state concentrations of peroxynitrite.

Dimerization↗

PubMed2000

Interaction of endothelial and neuronal nitric-oxide synthases with the bradykinin B2 receptor. Binding of an inhibitory peptide to the oxygenase domain blocks uncoupled NADPH oxidation.

Endothelial nitric-oxide synthase (type III) (eNOS) was reported to form an inhibitory complex with the bradykinin receptor B2 (B2R) from which the enzyme is released in an active form upon receptor activation (Ju, H., Venema, V. J., Marrero, M. B., and Venema, R. C. (1998) J. Biol. Chem. 273, 24025-24029). Using a synthetic peptide derived from the known inhibitory sequence of the B2R (residues 310-329) we studied the interaction of the receptor with purified eNOS and neuronal nitric-oxide synthase (type I) (nNOS). The peptide inhibited formation of L-citrulline by eNOS and nNOS with IC(50) values of 10.6 +/- 0.4 microM and 7.1 +/- 0.6 microM, respectively. Inhibition was not due to an interference of the peptide with L-arginine or tetrahydrobiopterin binding. The NADPH oxidase activity of nNOS measured in the absence of L-arginine was inhibited by the peptide with an IC(50) of 3.7 +/- 0.6 microM, but the cytochrome c reductase activity of the enzyme was much less susceptible to inhibition (IC(50) >0.1 mM). Steady-state absorbance spectra of nNOS recorded during uncoupled NADPH oxidation showed that the heme remained oxidized in the presence of the synthetic peptide consisting of amino acids 310-329 of the B2R, whereas the reduced oxyferrous heme complex was accumulated in its absence. These data suggest that binding of the B2R 310-329 peptide blocks flavin to heme electron transfer. Co-immunoprecipitation of B2R and nNOS from human embryonic kidney cells stably transfected with human nNOS suggests that the B2R may functionally interact with nNOS in vivo. This interaction of nNOS with the B2R may recruit the enzyme to allow for the effective coupling of bradykinin signaling to the nitric oxide pathway.

Animals↗

PubMed2000

Tetrahydrobiopterin improves endothelium-dependent vasodilation in chronic smokers : evidence for a dysfunctional nitric oxide synthase.

Conditions associated with impaired nitric oxide (NO) activity and accelerated atherosclerosis have been shown to be associated with a reduced bioavailability of tetrahydrobiopterin (BH4). We therefore hypothesized that BH4 supplementation may improve endothelial dysfunction of chronic smokers. Forearm blood flow (FBF) responses to the endothelium-dependent vasodilators acetylcholine (ACh; 0.75, 1.5, and 3.0 microg/100 mL tissue/min) or serotonin (5-HT; 0.7, 2.1, and 6.3 ng/100 mL tissue/min), to the inhibitor of endothelial nitric oxide synthase (NOS) N(G)-monomethyl-L-arginine (L-NMMA; 2, 4, and 8 micromol/min), and to the endothelium-independent vasodilator sodium nitroprusside (SNP; 0.1, 0.3, and 1.0 microg/100 mL tissue/min) were measured by venous occlusion plethysmography in controls and chronic smokers. Drugs were infused into the brachial artery, and FBF was measured before and during concomitant intra-arterial infusion of BH4, tetrahydroneopterin (NH4; another reduced pteridine), or the antioxidant vitamin C (6 and 18 mg/min). In control subjects, BH4 had no effect on FBF in response to ACh, 5-HT, and SNP. In contrast, in chronic smokers, the attenuated FBF responses to ACh and 5-HT were markedly improved by concomitant administration of BH4, whereas the vasodilator responses to SNP were not affected. L-NMMA-induced vasoconstriction was significantly reduced in smokers compared with controls, suggesting impaired basal NO bioactivity. BH4 improved L-NMMA responses in smokers while having no effect on L-NMMA responses in controls. Pretreatment with vitamin C abolished BH4 effects on ACh-dependent vasodilation. In vitro, NH4 scavenged superoxide created by the xanthine/xanthine oxidase reaction equipotent like BH4 but failed to modify ACh-induced changes in FBF in chronic smokers in vivo. These data support the concept that in addition to the free radical burden of cigarette smoke, a dysfunctional NOS III due to BH4 depletion may contribute at least in part to endothelial dysfunction in chronic smokers.

Acetylcholine↗

PubMed2000

[ACE gene polymorphism and cardiovascular diseases].

The angiotensin converting enzyme (ACE) is an integral part of enzymatic cascades leading to generation of angiotensin II as well as degradation of bradykinin. For this reason, it represents an important part for the metabolism of 2 vasoactive peptides. Early in this decade, convincing experimental evidence demonstrated the induction of this enzyme in several pathophysiological conditions including myocardial infarction and left ventricular hypertrophy. In parallel, a deletion/insertion (D/I) polymorphism of the human ACE gene was discovered that was related to 14 to 50% of the interindividual variance of serum ACE activity. More recently, this polymorphism was implicated in the pathogenesis of a variety of cardiovascular disorders including myocardial infarction, left ventricular hypertrophy, hypertension as well as nephropathy.

Chromosome Deletion↗

PubMed2000

"Danger is lurking everywhere". the relation between anxiety and threat perception abnormalities in normal children.

The present study examined the relation between anxiety and depression and threat perception abnormalities. Children were exposed to stories describing social situations. Some of the stories were ambiguous (i.e., these stories contained information that could be interpreted as threatening) whereas other stories were non-threatening (i.e., these stories contained no obvious trace of threat). From children's responses to the stories, several threat perception indices were derived. Children's level of anxiety and depression were assessed by means of self-report questionnaires. Results indicated that high levels of anxiety were accompanied by a high frequency of threat perception, high ratings of threat, a high frequency of threatening interpretations, high levels of negative feelings and cognitions, and an early detection of threat. Interestingly, significant associations between anxiety and threat perception abnormalities were not only observed in response to ambiguous stories but also in relation to non-threatening scenarios. Furthermore, depression was also connected with threat perception distortions. Even when controlling for anxiety levels, depression remained significantly related to threat frequency, threat ratings, and threat thresholds.

Adolescent↗

PubMed2000

How serious are common childhood fears?

OBJECTIVE: The present study examined the clinical status of common childhood fears. METHOD: Fears of the 290 children aged 8 to 13 years were assessed and then their severity was explored by means of a structured diagnostic interview measuring anxiety disorders according to the criteria of the Diagnostic and Statistical Manual of Mental Disorders (DSM). RESULTS: Childhood fears reflect significant anxiety disorders in a substantial minority (i.e. 22.8%) of the children. CONCLUSIONS: In most children, childhood fears are part of the normal development. However, in some children, these fears reflect serious anxiety problems which interfere with daily routine.

Adolescent↗

PubMed2000

If you suffer from a headache, then you have a brain tumour: domain-specific reasoning 'bias' and hypochondriasis.

We explored whether hypochondriacal patients selectively search for threat-confirming information when asked to judge the validity of conditional rules in the context of general and health threats. We also explored several factors that may underly participants' information selection (e.g., believability of the rule). Hypochondriacal patients (n = 20) and healthy controls (n = 20) were presented with modified Wason Selection Tasks (WSTs). The WSTs contained safety rules and danger rules. In the context of general threat, both groups of participants adopted a verificationistic strategy in the case of danger rules and a Popperian strategy in the case of safety rules. Importantly, only hypochondriacal persons showed a similar threat-confirming reasoning pattern in the context of health threat. The latter finding contrasts with the earlier study of de Jong et al. (1998) [de Jong, P. J., Haenen, M.-A., Schmidt, A., & Mayer, B. (1998a). Hypochondriasis: the role of fear-confirming reasoning. Behaviour Research and Therapy, 36, 65-74; de Jong, P. J., Mayer, B., van der Hijden, B., Bögels, S., & van den Hout, M. (1998b). Better safe than sorry: reasoning with conditionals in the context of threat. Submitted for publication.] in which both hypochondriacs and healthy controls showed a threat-confirming strategy in the domain of health threat. The WSTs in that study however, contained a (unintended) worry manipulation ("after hearing this, you get worried"), which might have induced a reasoning strategy in controls that is normally restricted to hypochondriacs. Taken together, the present results sustain the idea that the perception of threat activates a better safe than sorry strategy. In the case of phobic threats, such a reasoning strategy immunizes against refutation of phobic convictions.

Adult↗

PubMed2000

Lipoprotein lipase gene polymorphism, cholesterol subfractions and myocardial infarction in large samples of the general population.

OBJECTIVE: Genetic variants of the lipoprotein lipase gene have been associated with dyslipidemia and coronary artery disease. However, data have been inconsistent and are mainly based on selected predominantly male patient groups. METHODS: We evaluated the influence of the HindIII restriction fragment length polymorphism on lipid levels in the general population (1361 participants of a large population-based survey from Augsburg, Germany; 50% women) as well as the association of this polymorphism with the risk of myocardial infarction (MI; genotype frequencies in 1159 patients with documented MI under 60 years of age). RESULTS: In the population-based survey, a highly significant association between the frequent H2H2 genotype and unfavorable cholesterol subfraction levels was observed in men and in postmenopausal women whereas no significant association was observed in premenopausal women (uni- and multivariate analysis). Such unfavorable lipid levels in homozygotes for the H2 allele may be expected to be associated with a 19-25% increased risk to suffer from myocardial infarction (MI). Nevertheless, genotype and allele frequencies in the general population were not different from those in patients with previous MI (H2H2 genotype frequency 51.3% vs. 53.2%, respectively; P=0.63). CONCLUSION: This large study shows that the H2H2 genotype of the lipoprotein lipase gene polymorphism is associated with unfavorable lipid levels. Estrogen status may modulate this association in women. The effects of the genotype on lipid levels were apparently not strong enough to reveal a significant association with MI.

Adult↗

PubMed2000

Inhibition of endotoxin-induced vascular hyporeactivity by 4-amino-tetrahydrobiopterin.

The 4-amino analogue of tetrahydrobiopterin (4-ABH(4)) is a potent pterin-site inhibitor of nitric oxide synthases (NOS). Although 4-ABH(4) does not exhibit selectivity between purified NOS isoforms, a pronounced selectivity of the drug towards inducible NOS (iNOS) is apparent in intact cells. This work was carried out to investigate the potential iNOS selectivity of 4-ABH(4) in isolated pig pulmonary and coronary arteries. Endothelium-dependent relaxations of pig pulmonary and coronary artery strips to bradykinin or calcium ionophore A23187 were inhibited by 4-ABH(4) in a concentration-dependent manner. Half-maximal inhibition was observed at 60 - 65 microM (pulmonary artery) and 200 - 250 microM 4-ABH(4) (coronary artery). Pig coronary artery strips precontracted with 0.1 microM 9, 11-dideoxy-9, 11-methanoepoxy-prosta-glandin F(2alpha) (U46619) showed a time-dependent relaxation (monitored for up to 18 h) upon incubation with 1 microg ml(-1) lipopolysaccharide (LPS). Addition of 10 microM 4-ABH(4) 1 h after LPS led to a pronounced inhibition of the LPS-triggered relaxation, whereas the pterin antagonist had no effect when given> or =4 h after LPS. Incubation of pulmonary and coronary artery strips with 1 microg ml(-1) LPS attenuated contractile responses to norepinephrine (1 microM) and U46619 (0.1 microM). This hyporeactivity of the blood vessels to vasoconstrictor agents was inhibited by 4-ABH(4) in a concentration-dependent manner [IC(50)=17.5+/-5.9 microM (pulmonary artery) and 20.7+/-3 microM (coronary artery)]. The effect of 0.1 mM 4-ABH(4) was antagonized by coincubation with 0.1 mM sepiapterin, which is known to supply intracellular BH(4) via a salvage pathway. These results demonstrate that 4-ABH(4) is a fairly selective inhibitor of iNOS in an in vitro model of endotoxaemia, suggesting that this drug and/or related pterin-site NOS inhibitors may be useful to increase blood pressure in severe infections associated with a loss of vascular responsiveness to constrictor agents caused by endotoxin-triggered iNOS induction in the vasculature.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed2000

Novel mode of nitric oxide neurotransmission mediated via S-nitroso-cysteinyl-glycine.

S-nitroso-cysteinyl-glycine, a novel nitric oxide-adduct thiol compound, can be detected in the brain (2.3+/-0.6 pmol/mg protein), and released following stimulation of sensory afferents to the rat ventrobasal thalamus in vivo (resting conditions 17 nM; stimulation: 186 nM). Iontophoretic application of CysNOGly (20-80 nA) onto thalamic neurons in vivo resulted in enhancements of excitatory responses to either NMDA or AMPA (182+/-13.6% and 244+/-27.8% of control values, n = 15). CysNOGly enhanced responses to stimulation of vibrissal afferents to 132+/-2.2% (n = 7) of control values. In contrast, the dipeptide CysGly reduced responses of ventrobasal neurons to NMDA and AMPA (54+/-8.4% and 55+/-10.8% of control, n = 5). CysNOGly was also a potent activator of soluble guanylate cyclase in vitro. Moreover, we found that NMDA elevated CysNOGly levels in vitro and this stimulatory effect was reduced by inhibitors of the neuronal NO synthase and of the gamma-glutamyl transpeptidase, suggesting that production of NO and CysGly is a prelude to CysNOGly synthesis. These findings suggest that the nitrosothiol CysNOGly plays a role in synaptic transmission in the ventrobasal thalamus. We propose a novel synaptic buffering mechanism where S-nitroso-cysteinyl-glycine serves to restrict the locus of action of nitric oxide and so increase its local availability for target delivery. This could lead to a change in neuronal responses favouring sensory transmission similar to that seen in wakefulness or arousal in order to locally enhance transmission of persistent sensory stimuli.

Afferent Pathways↗

PubMed2000

Influence of the onion as an essential ingredient of the Mediterranean diet on arterial blood pressure and blood fluidity.

Mediterranean diet has got a favourable effect on life expectancy. One of the crucial components of the diet are onions. In an open and a randomized, placebo-controlled, double-blind, cross-over phase-I study a spontaneous pharmacological effect 5 h after administration of an onion-olive-oil maceration capsule formulation on arterial blood pressure could be demonstrated in apparently healthy subjects. In addition to a decrease in arterial blood pressure, a significant reduction in plasma viscosity and haematocrit were observed. These results are indicating a vasodilative effect of the onion-olive-oil-maceration product. The stickiness of the platelets was reduced. The effects were stronger in subjects with reduced blood fluidity compared to those subjects with normal rheological parameters.

Adolescent↗

PubMed2000

Comparative coronary anatomy in six twin pairs with coronary artery disease.

OBJECTIVE: To compare the coronary anatomy and the location of coronary lesions in monozygotic and dizygotic twin pairs affected with coronary artery disease. DESIGN: Coronary angiographic findings were compared with respect to the distribution of coronary blood supply and the location of coronary lesions. MAIN OUTCOME MEASURES: Distribution of coronary blood supply, diameters and length of proximal coronary arteries. RESULTS: The lumen diameters of the left main coronary artery, the left anterior descending coronary artery, the circumflex artery, and the right coronary artery, as well as the lengths of the left main coronary artery, showed similar variability within monozygotic and dizygotic twin pairs. With respect to the dominance pattern of coronary blood supply, two of three monozygotic twin pairs differed, while all dizygotic twin pairs were concordant. Concordant and discordant locations of stenoses were found with similar frequency in monozygotic and dizygotic twin pairs. CONCLUSIONS: The dominance pattern of coronary blood supply and the location of coronary lesions are not strictly hereditary. The high concordance of premature coronary artery disease found in monozygotic twin pairs may largely be related to factors that are independent of the macroanatomic distribution of the coronary blood supply.

Adult↗

PubMed2000

Evaluation of the aldosterone synthase (CYP11B2) gene polymorphism in patients with myocardial infarction.

Left ventricular remodeling after myocardial infarction involves activation of the renin-angiotensin-aldosterone system. Recently, the biallelic -344T/C polymorphism of the aldosterone synthase gene was associated with increased aldosterone levels, arterial hypertension, diastolic dysfunction, and left ventricular dilatation. We hypothesized that this polymorphism may also affect left ventricular geometry and function after myocardial infarction. By using a standardized questionnaire, as well as anthropometric and echocardiographic measurements, we thus studied 606 patients (533 men and 73 women) who had a myocardial infarction before the age of 60 years. The aldosterone synthase gene polymorphism was analyzed after polymerase chain reaction amplification and restriction enzyme digestion. The results demonstrated that there was no association of the aldosterone synthase gene polymorphism with echocardiographically determined left ventricular dimensions, wall thicknesses, or indexes of systolic or diastolic function. Furthermore, anthropometric data, including blood pressure levels, were balanced between the different genotypes. Finally, the allele frequency was similar for patients with myocardial infarction and a sample group from the normal population (n=1675). The data indicate that the allele status of the aldosterone synthase gene polymorphism is not useful for the identification of patients with myocardial infarction who have impaired left ventricular function or unfavorable remodeling.

Alleles↗

PubMed2000

Nitric oxide-containing neurons in the bovine gut, with special reference to their relationship with VIP and galanin.

The presence and distribution of nicotinamide dinucleotide phosphate diaphorase (NADPH-d)-containing neurons have been studied by means of NADPH-d histochemistry in different regions of the adult cow gut, from the esophagus to the rectum. NADPH-d and nitric oxide synthase (NOS) were constantly recognized to be colocalized in the same neuron. The colocalization of vasoactive intestinal polypeptide (VIP) and galanin in such nitrergic neurons was also studied by means of combined histochemical and immunofluorescence techniques. NADPH-d-positive neurons were present along the myenteric plexus of the entire gut, and in the submucous plexus from the abomasum to the rectum. Notably, they formed two types of nerve networks in the submucous connective tissue of the jejunum-ileum. NADPH-d-positive innervation of the muscle layers occurred throughout the tract, and sometimes a clear correspondence was noted between the number of reactive fibres and the thickness of the muscle. Nitrergic fibres also occurred in the mucosa and often were in relation to glands and blood vessels. The nitrergic neurons varied in size, shape, and intensity of staining, and often their terminals were seen to surround unstained perikarya. Various types of neurons were recognized on the basis of their chemical content; one of them contained galanin, VIP and NOS simultaneously. The present results suggest that the nitrergic neurons of the bovine gastrointestinal tract play roles presumably for controlling the motility of the gut and the conduction of interneuronal impulses.

Animals↗

PubMed2000

Self-reported automaticity and irrationality in spider phobia.

Spider phobic women (n = 39) and nonfearful controls (n = 41) completed a 20-item questionnaire measuring the extent to which they experience their fear reactions to spiders as automatic and irrational. For the phobic sample, therapy outcome data were also collected. Results suggest that spider phobics tend to view their attitude to spiders as irrational and in this respect, they do not differ from control subjects. Furthermore, compared to control subjects, phobics more often perceive their responses to spiders as automatic, i.e., not under intentional control. Contrary to expectation, no robust correlation was found between automaticity and irrationality. Interestingly, automaticity was not related to treatment outcome, while irrationality to some extent predicted therapy outcome (i.e., the more phobics experienced their fear as irrational, the more they profited from exposure treatment).

Adult↗

PubMed1999

Characterization of recombinant human endothelial nitric-oxide synthase purified from the yeast Pichia pastoris.

Human endothelial nitric-oxide synthase (eNOS) was expressed in the methylotrophic yeast Pichia pastoris, making use of the highly inducible alcohol oxidase promoter. The recombinant protein constituted approximately 3% of total protein and was largely soluble (>75%). About 1 mg of purified eNOS was obtained from 100-ml yeast cell cultures by affinity chromatography of crude cell supernatants. The purified enzyme had a V(max) of 192 +/- 18 nmol of L-citrulline x mg(-1) x min(-1), had a K(m) for L-arginine of 3.9 +/- 0.2 microM, and showed an absolute requirement for tetrahydrobiopterin (H(4)biopterin). NADPH oxidase activity was 136 +/- 9 and 342 +/- 24 nmol x mg(-1) x min(-1) in the absence and presence of 0.1 mM L-arginine, respectively, and not affected by H(4)biopterin. The protein contained 0.56 +/- 0.06 equivalents of FAD and 0.79 +/- 0.08 equivalents of FMN. On-line gel filtration/inductively coupled plasma mass spectrometry analysis confirmed that both iron (0.80 +/- 0.09 mol/subunit) and zinc (0.43 +/- 0.03 mol/subunit) were bound to the enzyme. Graphite furnace-atomic absorption spectroscopy yielded a value for bound iron of 0.84 +/- 0.04 mol/subunit. The absorbance of the enzyme at 398 nm implied a heme content of 0.85 +/- 0.09 mol/subunit, and the high pressure liquid chromatography heme assay gave an estimate of 0.71 +/- 0.02 mol heme/subunit. Gel permeation chromatography yielded one single peak with a Stokes radius of 6.62 +/- 0.7 nm, indicating that the native protein is dimeric. Upon low temperature gel electrophoresis the untreated protein appeared mainly as a monomer (88 +/- 3%), but pretreatment with H(4)biopterin and L-arginine led to a pronounced shift toward dimers (77 +/- 4%). Thus, in contrast to bovine eNOS (List, B. M., Klösch, B., Völker, C., Gorren, A. C. F., Sessa, W. C., Werner, E. R., Kukovetz, W. R., Schmidt, K., and Mayer, B. (1997) Biochem. J. 323, 159-165; Rodriguez-Crespo, I., Gerber, N. C., and Ortiz de Montellano, P. R. (1996) J. Biol. Chem. 271, 11462-11467), the human eNOS appears to be markedly stabilized by H(4)biopterin.

Animals↗

PubMed1999

Na(+)/Ca(2+) exchange facilitates Ca(2+)-dependent activation of endothelial nitric-oxide synthase.

Recent evidence suggests the expression of a Na(+)/Ca(2+) exchanger (NCX) in vascular endothelial cells. To elucidate the functional role of endothelial NCX, we studied Ca(2+) signaling and Ca(2+)-dependent activation of endothelial nitric-oxide synthase (eNOS) at normal, physiological Na(+) gradients and after loading of endothelial cells with Na(+) ions using the ionophore monensin. Monensin-induced Na(+) loading markedly reduced Ca(2+) entry and, thus, steady-state levels of intracellular free Ca(2+) ([Ca(2+)](i)) in thapsigargin-stimulated endothelial cells due to membrane depolarization. Despite this reduction of overall [Ca(2+)](i), Ca(2+)-dependent activation of eNOS was facilitated as indicated by a pronounced leftward shift of the Ca(2+) concentration response curve in monensin-treated cells. This facilitation of Ca(2+)-dependent activation of eNOS was strictly dependent on the presence of Na(+) ions during treatment of the cells with monensin. Na(+)-induced facilitation of eNOS activation was not due to a direct effect of Na(+) ions on the Ca(2+) sensitivity of the enzyme. Moreover, the effect of Na(+) was not related to Na(+) entry-induced membrane depolarization or suppression of Ca(2+) entry, since neither elevation of extracellular K(+) nor the Ca(2+) entry blocker 1-(beta-[3-(4-methoxyphenyl)-propoxy]-4-methoxyphenethyl)-1H-imidazol e hydrochloride (SK&F 96365) mimicked the effects of Na(+) loading. The effects of monensin were completely blocked by 3', 4'-dichlorobenzamil, a potent and selective inhibitor of NCX, whereas the structural analog amiloride, which barely affects Na(+)/Ca(2+) exchange, was ineffective. Consistent with a pivotal role of Na(+)/Ca(2+) exchange in Ca(2+)-dependent activation of eNOS, an NCX protein was detected in caveolin-rich membrane fractions containing both eNOS and caveolin-1. These results demonstrate for the first time a crucial role of cellular Na(+) gradients in regulation of eNOS activity and suggest that a tight functional interaction between endothelial NCX and eNOS may take place in caveolae.

Amiloride↗