Basal Flux Research Articles

Cholinergic regulation of epithelial ion transport in the mammalian intestine

Acetylcholine (ACh) is critical in controlling epithelial ion transport and hence water movements for gut hydration. Here we review the mechanism of cholinergic control of epithelial ion transport across the mammalian intestine. The cholinergic nervous system affects basal ion flux and can evoke increased active ion transport events. Most studies rely on measuring increases in short-circuit current (ISC = active ion transport) evoked by adding ACh or cholinomimetics to intestinal tissue mounted in Ussing chambers. Despite subtle species and gut regional differences, most data indicate that, under normal circumstances, the effect of ACh on intestinal ion transport is mainly an increase in Cl- secretion due to interaction with epithelial M3 muscarinic ACh receptors (mAChRs) and, to a lesser extent, neuronal M1 mAChRs; however, AChR pharmacology has been plagued by a lack of good receptor subtype-selective compounds. Mice lacking M3 mAChRs display intact cholinergically-mediated intestinal ion transport, suggesting a possible compensatory mechanism. Inflamed tissues often display perturbations in the enteric cholinergic system and reduced intestinal ion transport responses to cholinomimetics. The mechanism(s) underlying this hyporesponsiveness are not fully defined. Inflammation-evoked loss of mAChR-mediated control of epithelial ion transport in the mouse reveals a role for neuronal nicotinic AChRs, representing a hitherto unappreciated braking system to limit ACh-evoked Cl- secretion. We suggest that: i) pharmacological analyses should be supported by the use of more selective compounds and supplemented with molecular biology techniques targeting specific ACh receptors and signalling molecules, and ii) assessment of ion transport in normal tissue must be complemented with investigations of tissues from patients or animals with intestinal disease to reveal control mechanisms that may go undetected by focusing on healthy tissue only.

Drug specificity and intestinal membrane localization of human organic cation transporters (OCT)

This study was performed to investigate which human organic cation transporter, hOCT1, hOCT2 or hOCT3, participates with regard to cation specificity and membrane localization in the intestinal absorption of orally available cationic drugs. Inhibition of N-[methyl- 3H]4-phenylpyridinium ([ 3H]MPP +) uptake by various compounds into Caco-2 cells and into cells (HEK-293 or CHO) that were stably transfected with hOCT1, hOCT2 or hOCT3 was compared. The uptake of [ 3H]MPP + into Caco-2 cells was inhibited by atropine, butylscopolamine, clonidine, diphenhydramine, etilefrine, quinine and ranitidine with IC 50 values between 6 μM and 4 mM. Transepithelial, apical to basal flux of [ 3H]MPP + across Caco-2 cell monolayers was also strongly inhibited by these compounds. The inhibitory potency of the cationic drugs and prototypical organic cations at Caco-2 cells correlated well with the inhibitory potency measured at CHO-hOCT3 cells but much less with that at HEK-hOCT1 and -hOCT2 cells. This is functional evidence for the predominant role of hOCT3. Etilefrine and atropine were specifically transported into CHO cells by hOCT3. In Caco-2 cells, the mRNA of all three hOCT and the proteins hOCT2 and hOCT3 were detected. More importantly, immunocytochemical analyses of human jejunum revealed for the first time that hOCT3 is localized to the brush border membrane whereas hOCT1 immunolabeling was mainly observed at the lateral membranes of the enterocytes.

Existence of weak solutions to a system of nonlinear partial differential equations modelling ice streams

This paper deals with the mathematical analysis of a nonlinear system of three differential equations of mixed type. It describes the generation of fast ice streams in ice sheets flowing along soft and deformable beds. The system involves a nonlinear parabolic PDE with a multivalued term in order to deal properly with a free boundary which is naturally associated to the problem of determining the basal water flux in a drainage system. The other two equations in the system are an ODE with a nonlocal (integral) term for the ice thickness, which accounts for mass conservation and a first order PDE describing the ice velocity of the system. We first consider an iterative decoupling procedure to the system equations to obtain the existence and uniqueness of solutions for the uncoupled problems. Then we prove the convergence of the iterative decoupling scheme to a bounded weak solution for the original system.

PPARγ agonists do not directly enhance basal or insulin-stimulated Na+ transport via the epithelial Na+ channel

Selective agonists of peroxisome proliferator-activated receptor gamma (PPARgamma) are anti-diabetic drugs that enhance cellular responsiveness to insulin. However, in some patients, fluid retention, plasma volume expansion, and edema have been observed. It is well established that insulin regulates Na(+) reabsorption via the epithelial sodium channel (ENaC) located in the distal tubule. Therefore, we hypothesized that these agonists may positively modulate insulin-stimulated ENaC activity leading to increased Na(+) reabsorption and fluid retention. Using electrophysiological techniques, dose-response curves for insulin-mediated Na(+) transport in the A6, M-1, and mpkCCD(cl4) cell lines were performed. Each line demonstrated hormone efficacy within physiological concentration ranges and, therefore, can be used to monitor clinically relevant effects of pharmacological agents which may affect electrolyte transport. Immunodetection and quantitative PCR analyses showed that each cell line expresses viable and functional PPARgamma receptors. Despite this finding, two PPARgamma agonists, pioglitazone and GW7845 did not directly enhance basal or insulin-stimulated Na(+) flux via ENaC, as shown by electrophysiological methodologies. These studies provide important results, which eliminate insulin-mediated ENaC activation as a candidate mechanism underlying the fluid retention observed with PPARgamma agonist use.

Microvascular dysfunction in women with polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) is associated with multiple cardiovascular risk factors and an increased prevalence of arterial dysfunction. However, microvascular dysfunction in PCOS has not been assessed. Subjects comprised 12 women with PCOS and 12 age-matched controls with normal ovaries. Microvascular function was assessed by observing forearm skin microvascular erythrocyte flux responses, to cumulative iontophoretic doses of 1% (w/v) acetylcholine (ACh) and 1% (w/v) sodium nitroprusside (SNP), using laser Doppler imaging. Basal microvascular perfusion was comparable in PCOS and controls. The increase in skin microvascular perfusion in response to ACh was however generally blunted in PCOS women (P = 0.018). Peak ACh-induced erythrocyte flux was also less (p < 0.04) in PCOS women (125.1 +/- 21.7, i.e. 5.3-fold basal flux) than in controls (200.8 +/- 28.5, i.e. 8.3-fold basal flux). Analysis of covariance indicated this effect was unrelated to differences in body mass index or serum testosterone but serum insulin may be a weak confounder. No differences were noted between the PCOS and control groups in their response to SNP. Despite its limited sample size studied, this is the first demonstration that women with PCOS exhibit microvascular endothelial dysfunction, indicated by an inhibited vasodilatory response to ACh.

Observations of Branched-Chain Amino Acid Administration in Humans

Since the in vitro study of Buse and Reid in 1975 showing a stimulatory effect of leucine upon rat muscle protein synthesis and reduction in proteolysis, a similar effect has been sought in humans. In 1978, Sherwin demonstrated in humans an improvement in N balance with infusion of leucine in obese subjects fasting to lose weight. A variety of subsequent studies have been performed in humans where leucine alone or the BCAAs have been administered in varying amounts and durations, and the effect upon protein metabolism has been measured. Measurements of changes in muscle amino acid metabolism were made by arteriovenous difference measurements and by biopsies. An anabolic effect of leucine and the branched-chain amino acids (BCAAs) on reduction of muscle protein breakdown was found in these studies, with no measured effect upon muscle protein synthesis. Later studies using stable isotope tracers to define both whole-body protein turnover and leg or arm protein metabolism have similarly concluded that leucine administration specifically induces a reduction in protein breakdown without increasing protein synthesis. This anabolic effect, produced through a reduction of protein breakdown in vivo in humans by leucine is contrary to in vitro studies of rat muscle where stimulation of protein synthesis, has been demonstrated by leucine. Likewise an increase in protein synthesis has also been demonstrated by insulin in rat muscle that is not seen in humans. Of the various studies administering BCAAs or leucine to humans for varying periods of time and amount, the results have been consistent. In addition, no untoward effects have been reported in any of these studies from infusion of the BCAAs at upward 3 times basal flux or 6 times normal dietary intake during the fed portion of the day.

Parametric sensitivity study of operating and design variables in wellbore heat exchangers

A numerical study was conducted to evaluate the potential for using Wellbore Heat Exchangers (WBHX) to extract heat for use in electricity generation. Variables studied included operational parameters such as wellbore geometries, working fluid properties, circulation rates, and regional properties including basal heat flux and formation rock type. Energy extraction is strongly affected by fluid residence time, heat transfer contact area, and formation thermal properties. Water appears to be the most appropriate working fluid. The effects of tubing properties and casing lengths are of second-order. On the basis of a sensitivity study, a Best Case model was simulated, and results compared against the geothermal fluid requirements of existing power generation plants that use low-temperature geothermal fluids. Even assuming ideal work conversion to electricity, a WBHX cannot supply sufficient energy to generate 200 kWe at the onset of pseudo-steady-state (PSS) conditions. Using realistic conversion efficiencies it is unlikely that the system would be able to generate 50 kWe at the onset of PSS.

Modeling thermal history of subducting crust in Nankai Trough: Constraints from in situ sediment temperature and diagenetic reaction progress

Values of borehole temperature and %‐smectite in illite/smectite mixed‐layer clay provide important information about the basal heat flux history of the Nankai Trough subduction zone. Numerical simulations of sediment accumulation, heat conduction, and illite/smectite diagenesis permit comparisons of various thermal‐history scenarios for ODP Site 1173. In our preferred models, basal heat flux during the first 10 Ma of sedimentation was near typical values for conductively cooling crust. During the last 5 Ma, basal heat flux was higher than expected. The anomalous heat flux was likely influenced by both off‐axis volcanism and hydrothermal circulation.

Mechanism of VEGF-Induced Uterine Venous Hyperpermeability

Background: Transfer of molecular signals from veins to adjacent arteries is an established mechanism of vascular communication in the uterine circulation, which ultimately depends on venous permeability. This study tests the hypotheses that: (1) uterine veins are permeable to intravenous solutes, using a 3-kDa dextran tracer and (2) this permeability is enhanced in response to vascular endothelial growth factor (VEGF). Additionally, the involvement of nitric oxide (NO), calcium, and the phospholipase C-protein kinase C (PLC-PKC) cascade in VEGF-enhanced permeability were investigated, and the impact of pregnancy-induced uterine vascular remodeling on permeability was evaluated. Methods: Studies utilized fluorimetry to quantitate solute flux in isolated segments of rat uterine vein as a function of endothelial surface area and time under basal and VEGF-stimulated conditions. VEGF signaling was probed using NO synthase inhibition (L-NNA), calcium channel blockade (lanthanum chloride) and withdrawal (calcium-free solution), and inhibitors of the PLC-PKC cascade (U-73122 and chelerythrine chloride, respectively). Gestational effects were assessed using vessels from late pregnant (day 20) rats. Results: Basal flux (control) in nonpregnant animals was 26 ± 2.5 molecules/µm²/min × 1,000 and was increased significantly by VEGF in a concentration-dependent manner (1 nM ≈ 3.3-fold, 10 nM ≈ 4.6-fold). Inhibition of PLC, PKC, and calcium signaling, but not NO, attenuated the response to VEGF. Gestation significantly increased flux (78 ± 9.3 molecules/µm²/min × 1,000), and maintained responsiveness to VEGF. Conclusions: These results demonstrate uterine venous permeability to intermediate-sized solutes through a VEGF-sensitive pathway involving calcium and PLC-PKC, but not NO, and further substantiate a role for veno-arterial communication in uterine blood flow regulation during pregnancy.

Dynamics of transpiration, sap flow and use of stored water in tropical forest canopy trees.

In large trees, the daily onset of transpiration causes water to be withdrawn from internal storage compartments, resulting in lags between changes in transpiration and sap flow at the base of the tree. We measured time courses of sap flow, hydraulic resistance, plant water potential and stomatal resistance in co-occurring tropical forest canopy trees with trunk diameters ranging from 0.34-0.98 m, to determine how total daily water use and daily reliance on stored water scaled with size. We also examined the effects of scale and tree hydraulic properties on apparent time constants for changes in transpiration and water flow in response to fluctuating environmental variables. Time constants for water movement were estimated from whole-tree hydraulic resistance (R) and capacitance (C) using an electric circuit analogy, and from rates of change in water movement through intact trees. Total daily water use and reliance on stored water were strongly correlated with trunk diameter, independent of species. Although total daily withdrawal of water from internal storage increased with tree size, its relative contribution to the daily water budget (approximately 10%) remained constant. Net withdrawal of water from storage ceased when upper branch water potential corresponded to the sapwood water potential (Psi(sw)) at which further withdrawal of water from sapwood would have caused Psi(sw) to decline precipitously. Stomatal coordination of vapor and liquid phase resistances played a key role in limiting stored water use to a nearly constant fraction of total daily water use. Time constants for changes in transpiration, estimated as the product of whole- tree R and C, were similar among individuals (~0.53 h), indicating that R and C co-varied with tree size in an inverse manner. Similarly, time constants estimated from rates of change in crown and basal sap flux were nearly identical among individuals and therefore independent of tree size and species.

Relationship between sympathetic reactivity and body weight loss in morbidly obese subjects.

To investigate the possible role of peripheral sympathetic activity in gastric bypass-induced body weight loss. In 42 morbidly obese patients (sex: 36 f/6 m; BMI: 46.0+/-0.7 kg/m(2)) undergoing a gastric bypass, the skin vasoconstrictor reflex in answer to a deep inspiration was measured by laser Doppler fluximetry. The extent of vasoconstriction, measured at the second finger of the left hand, was expressed as percent reduction of the basal blood flux (% vasoconstriction). Insulin sensitivity was assessed before surgery in a subset of patients (n=11), by the method of euglycemic, hyperinsulinemic clamp. Body weight and composition were evaluated before, and 3, 6 and 12 months after surgery. At the same time points, energy intake (kJ/day) was evaluated by means of both food record diary and alimentary anamnesis. The % vasoconstriction, which was significantly (P=0.01) greater in normoglycemic subjects than in diabetic ones, was also significantly (P=0.03) related to the extent of insulin sensitivity measured during the euglycemic clamp. The % vasoconstriction showed a significant (P>0.0001), positive correlation with weight reduction obtained between the 6th and 12th months following surgery; as a consequence, % vasoconstriction was significantly (P=0.0004) related to the overall body weight loss achieved during the year following the operation. These correlations remained significant in multiple regression analysis with adjustment for age, initial body weight, plasma glucose and insulin (P=0.0007 and 0.006, respectively). The % vasoconstriction was also significantly (P=0.0006), negatively related to energy intake measured 12 months after surgery. In conditions of stable body weight, the sympathetic nervous system (SNS) reactivity is influenced by the degree of insulin resistance. A high capacity to activate the SNS, measured before surgery, is associated with both a larger gastric bypass-induced weight loss and a lower energy intake, at the phase of weight stabilization.

Relationship between synoptic forcing and polynya formation in the Cosmonaut Sea: 2. Regional climate model simulations

In part I of this series [Arbetter et al., 2004], a relationship was found in the observations between synoptic atmospheric systems and polynya formation in the Cosmonaut Sea region of Antarctica. In this study, we use a regional coupled atmosphere‐sea ice model to investigate the role of atmospheric forcing of the polynyas in this area. The model successfully simulates both latent and sensible heat polynyas which are found in the region. In particular, a deep‐ocean polynya is formed coincident with the passage of an atmospheric synoptic scale system. The divergence in the sea ice is found to be highly correlated with the atmospheric divergence. We conclude that the initial formation of a polynya can be caused by the interaction of the atmospheric synoptic scale and the continental katabatic wind regimes. While we cannot directly evaluate the role of the ocean using the present model simulations, we performed experiments with different levels of prescribed basal heat flux. These experiments highlight a polynya which could be initiated by the winds and maintained by the oceanic heat. This leads to the concept of a wind‐driven sensible heat polynya, not typical of other deep‐ocean polynyas such as the Weddell polynya of the 1970s.

Detection of nitric oxide from pig trachea by a fluorescence method

A nitronyl nitroxide radical covalently linked to an organic fluorophore, pyrene, was used to detect nitric oxide (NO) from freshly excited tissues. This approach is based on the phenomenon of the intramolecular fluorescence quenching of the fluorophore fragment by the nitroxide. The pyrene-nitronyl (PN) reacts with NO to yield a pyrene-imino nitroxide radical (PI) and NO 2. Conversion of PN to PI is accompanied by changes in the electron paramagnetic resonance (EPR) spectrum from a five-line pattern (two equivalent N nuclei) into a seven-line pattern (two nonequivalent N nuclei). The transformation of the EPR signal is accompanied by an increase in the fluorescence intensity since the imino nitroxide radical is a weaker quencher than the nitronyl one. The results indicate that the fluorescence measurements enable detection of nanomolar concentrations of NO compared to a sensitivity threshold of only several micromolar for the EPR technique. The method was applied to the determination of NO and S-nitroso compounds in tissue from pig trachea epithelia. The measured basal flux of S-nitroso compounds obtained from the tissues was about 1.2 nmol/g × min, and NO-synthase stimulated by extracellular adenosine 5 ′-triphosphate produced NO flux of 0.9 nmol/g × min.

Heating of Stellar Chromospheres and Transition Regions

To explain the heating of stellar chromospheres and transition regions, two classes of heating mechanisms have been considered: dissipation of acoustic and magnetic waves generated in stellar convection zones; and dissipation of currents generated by photospheric motions of surface magnetic fields. The focus of this paper is on the wave heating mechanisms and on recent results which demonstrate that theoretical models of stellar chromospheres based on the wave heating can explain the “basal flux” and the observed Ca II emission in most stars but cannot account for the observed Mg II emission in active stars. The obtained results clearly show that the base of stellar chromospheres is heated by acoustic waves, the heating of the middle and upper chromospheric layers is dominated by magnetic waves associated with magnetic flux tubes, and that other non-wave heating mechanisms are required to explain the structure of the highest layers of stellar chromospheres and transition regions.

Capillary level imaging of local cerebral blood flow in bicuculline-induced epileptic foci

Local hemodynamics of the cerebral cortex is the basis of modern functional imaging techniques, such as fMRIand PET. Despite the importance of local regulation of the blood flow, capillary level quantification of cerebral blood flow has been limited by the spatial resolution of functional imaging techniques and the depth penetration of conventional optical microscopy. Two-photon laser scanning microscopic imaging technique has the necessary spatial resolution and can image capillaries in the depth of the cortex. We have loaded the serum with fluorescein isothiocyanate dextran and quantified the flow of red blood cells (RBCs) in capillaries in layers 2/3 of the mouse somatosensory cortex in vivo. Basal capillary flux was quantified as approximately 28.9±13.6 RBCs/s ( n=50, mean±S.D.) under ketamine–xylazine anesthesia and 26.7±16.0 RBCs/s ( n=31) under urethane anesthesia. Focal interictal (epileptiform) activity was induced by local infusion of bicuculline methochloride in the cortex. We have observed that capillary blood flow increased as the cortical local field events developed into epileptiform in the vicinity of GABA receptor blockade (<300 μm from the administration site). Local blood flow in the interictal focus increased significantly (42.5±18.5RBCs/s, n=52) relative to the control conditions or to blood flow measured in capillaries at distant (>1 mm from the focus) sites from the epileptic focus (27.8±12.9 RBCs/s, n=30). These results show that hyper-synchronized neural activity is associated with increased capillary perfusion in a localized cortical area. This volume is significantly smaller than the currently available resolution of the fMRI signal.

Acoustic Heating Models for the Basal Flux Star Ceti Including Time-dependent Ionization: Results for Ca ii and Mg ii Emission

We present new calculations of chromospheric heating for τ Ceti (G8 V), a star exhibiting an extremely low level of chromospheric activity, thus also referred to as a basal flux star or a flat activity star. Our simulations consider energy deposition by acoustic shocks and also take into account time-dependent (i.e., noninstantaneous) ionization processes of hydrogen, magnesium, and calcium, allowing us to attain a new generation of chromospheric heating models previously obtained for the Sun. We consider both monochromatic waves and acoustic frequency spectra. The latter are calculated using new models of acoustic energy generation based on an extended Kolmogorov spectrum with a modified Gaussian frequency factor. Our models show that the theoretically deduced emergent Ca II and Mg II emission very much agree with observations, adding to the argument that the chromospheres of basal flux stars are predominantly heated by acoustic shocks.

Mapacalcine specifically blocks hypoxia-induced calcium influx in rat hepatocytes.

Post ischaemic cell calcium invasion has been described as one of the main causes of graft failure. Protective effects of calcium antagonists have been investigated but are not convincing and their mechanisms of action remain unclear. In this work we tested the protective effect of a new calcium inhibitor described to block a calcium current insensitive to all known calcium blockers. Specific mapacalcine receptors were first characterized on rat hepatocytes membranes using the 125I-labeled mapacalcine. 45Ca fluxes were then measured on cultured hepatocytes submitted (or not) to an hypoxic period. The action of mapacalcine was investigated on the ischaemia-induced calcium influx. We demonstrate here that: (a) there are specific receptors for mapacalcine in rat hepatocytes; (b) Mapacalcine is able to specifically block ischaemia-induced calcium influx with an IC50 of 0.3 micro m and does not significantly interact with the basal calcium flux. Our work demonstrates that the mapacalcine receptor is a cellular structure directly involved in the phenomenon of postischaemic cell invasion by calcium. Specific block of ischaemia-induced Ca2+ influx by mapacalcine suggests that the development of a panel of pharmacological drugs acting on this receptor could lead to the discovery of therapeutic agents able to protect cells against one of the events responsible for organ failure after transplantation or simply after an ischaemic period. Moreover, identification of the cellular protein which binds mapacalcine may become an important step in the research of mechanisms involved in postischaemic cell invasion by calcium.

The dynamics of submarine geothermal heat pipes

To better understand natural two‐phase hydrothermal systems, we have constructed one‐dimensional heat‐pipe solutions for NaCl‐H2O fluids and explored the effects of basal heat flux and permeability on their behavior. For seafloor conditions, saline brines form quickly at the base of the heat pipe; and in some cases halite is precipitated. NaCl‐H2O heat pipes may become liquid or vapor dominated but, in contrast to their pure‐water counterparts, often do not achieve steady state. When steady state solutions do exist, they are characterized either by broad, weak counter‐flow or by vigorous counter‐flow across a thin layer. The latter behavior may be analogous to that occurring in the Salton Sea Geothermal System, California.

Plume capture by divergent plate motions: implications for the distribution of hotspots, geochemistry of mid-ocean ridge basalts, and estimates of the heat flux at the core–mantle boundary

The coexistence of stationary mantle plumes with plate-scale flow is problematic in geodynamics. We present results from laboratory experiments aimed at understanding the effects of an imposed large-scale circulation on thermal convection at high Rayleigh number (10 6≤Ra≤10 9) in a fluid with a temperature-dependent viscosity. In a large tank, a layer of corn syrup is heated from below while being stirred by large-scale flow due to the opposing motions of a pair of conveyor belts immersed in the syrup at the top of the tank. Three regimes are observed, depending on the ratio V of the imposed horizontal flow velocity to the rise velocity of plumes ascending from the hot boundary, and on the ratio λ of the viscosity of the interior fluid to the viscosity of the hottest fluid in contact with the bottom boundary. When V≪1 and λ≥1, large-scale circulation has a negligible effect on convection and the heat flux is due to the formation and rise of randomly spaced plumes. When V>10 and λ>100, plume formation is suppressed entirely, and the heat flux is carried by a sheet-like upwelling located in the center of the tank. At intermediate V, and depending on λ, established plume conduits are advected along the bottom boundary and ascending plumes are focused towards the central upwelling. Heat transfer across the layer occurs through a combination of ascending plumes and large-scale flow. Scaling analyses show that the bottom boundary layer thickness and, in turn, the basal heat flux q depend on the Peclet number, Pe, and λ. When λ>10, q∝Pe 1/2 and when λ→1, q∝(Pe λ) 1/3, consistent with classical scalings. When applied to the Earth, our results suggest that plate-driven mantle flow focuses ascending plumes towards upwellings in the central Pacific and Africa as well as into mid-ocean ridges. Furthermore, plumes may be captured by strong upwelling flow beneath fast-spreading ridges. This behavior may explain why hotspots are more abundant near slow-spreading ridges than fast-spreading ridges and may also explain some observed variations of mid-ocean ridge basalt (MORB) geochemistry with spreading rate. Moreover, our results suggest that a potentially significant fraction of the core heat flux is due to plumes that are drawn into upwelling flows beneath ridges and not observed as hotspots.

Endothelin contributes to basal vascular tone and endothelial dysfunction in human obesity and type 2 diabetes.

Endothelium-dependent vasodilation is impaired in clinical states of insulin resistance such as obesity and type 2 diabetes. Individuals who have hyperinsulinemic insulin resistance have relatively elevated circulating levels of endothelin (ET)-1, suggesting that ET-1 may be important in the endothelial dysfunction and alterations of vascular tone in these conditions. In 8 lean subjects, 12 nondiabetic obese subjects, and 8 subjects with type 2 diabetes, we measured basal and methacholine-stimulated rates of leg blood flow (LBF) and total serum nitrates (NOx) before and after the intrafemoral arterial administration of BQ123, a specific blocker of ET(A) receptors. BQ123 produced significant vasodilation in the obese and type 2 diabetic subjects (leg vascular resistance = mean arterial pressure/LBF fell by 34 and 36%; P < 0.005) but not in the lean subjects (13%; P = NS, P = 0.018 comparing all groups). ET(A) blockade did not change basal NOx flux (NOx*LBF). This suggests increased basal ET-1 constrictor tone among obese and type 2 diabetic subjects. BQ123 corrected the baseline defect in endothelium-dependent vasodilation seen in obese and type 2 diabetic subjects, suggesting an important contribution of ET-1 to endothelial dysfunction in these subjects. In contrast to basal conditions, stimulated NOx flux was augmented by BQ123 in obese and type 2 diabetic subjects but not in L subjects (P = 0.04), suggesting a combined effect of ET(A) blockade to reduce constrictor tone and augment dilator tone. Endothelin seems to contribute to endothelial dysfunction and the regulation of vascular tone in human obesity and type 2 diabetes.