Currents In Endothelial Cells Research Articles

Visceral adipose of obese mice inhibits endothelial inwardly rectifying K+ channels in a CD36-dependent fashion.

Obesity imposes deficits on adipose tissue and vascular endothelium, yet the role that distinct adipose depots play in mediating endothelial dysfunction in local arteries remains unresolved. We recently showed that obesity impairs endothelial Kir2.1 channels, mediators of nitric oxide production, in arteries of visceral adipose tissue (VAT), while Kir2.1 function in subcutaneous adipose tissue (SAT) endothelium remains intact. Therefore, we determined if VAT versus SAT from lean or diet-induced obese mice affected Kir2.1 channel function in vitro. We found that VAT from obese mice reduces Kir2.1 function without altering channel expression whereas AT from lean mice and SAT from obese mice had no effect on Kir2.1 function as compared to untreated control cells. As Kir2.1 is well known to be inhibited by fatty acid derivatives and obesity is strongly associated with elevated circulating fatty acids, we next tested the role of the fatty acid translocase CD36 in mediating VAT-induced Kir2.1 dysfunction. We found that the downregulation of CD36 restored Kir2.1 currents in endothelial cells exposed to VAT from obese mice. In addition, endothelial cells exposed to VAT from obese mice exhibited a significant increase in CD36-mediated fatty acid uptake. The importance of CD36 in obesity-induced endothelial dysfunction of VAT arteries was further supported in ex vivo pressure myography studies where CD36 ablation rescued the endothelium-dependent response to flow via restoring Kir2.1 and endothelial nitric oxide synthase function. These findings provide new insight into the role of VAT in mediating obesity-induced endothelial dysfunction and suggest a novel role for CD36 as a mediator of endothelial Kir2.1 impairment.NEW & NOTEWORTHY Our findings suggest a role for visceral adipose tissue (VAT) in the dysfunction of endothelial Kir2.1 in obesity. We further reveal a role for CD36 as a major contributor to VAT-mediated Kir2.1 and endothelial dysfunction, suggesting that CD36 offers a potential target for preventing the early development of obesity-associated cardiovascular disease.

Vasodilators activate the anion channel TMEM16A in endothelial cells to reduce blood pressure.

Systemic blood pressure is acutely controlled by total peripheral resistance as determined by the diameter of small arteries and arterioles, the contractility of which is regulated by endothelial cells lining the lumen of blood vessels. We investigated the physiological functions of the chloride (Cl-) channel TMEM16A in endothelial cells. TMEM16A channels generated calcium (Ca2+)-activated Cl- currents in endothelial cells from control (TMEM16Afl/fl) mice that were absent in those from mice with tamoxifen-inducible, endothelial cell-specific knockout of TMEM16A (TMEM16A ecKO). TMEM16A currents in endothelial cells were activated by the muscarinic receptor agonist acetylcholine and an agonist of the Ca2+ channel TRPV4, which localized in nanoscale proximity with TMEM16A as assessed by single-molecule localization imaging of endothelial cells. Acetylcholine stimulated TMEM16A currents by activating Ca2+ influx through surface TRPV4 channels without altering the nanoscale properties of TMEM16A and TRPV4 surface clusters or their colocalization. In pressurized arteries, activation of TMEM16A channels in endothelial cells induced by acetylcholine; TRPV4 channel stimulation; or intraluminal ATP, another vasodilator, produced hyperpolarization and dilation. Furthermore, deficiency of TMEM16A channels in endothelial cells resulted in increased systemic blood pressure in conscious mice. These data indicate that vasodilators stimulate TRPV4 channels, leading to Ca2+-dependent activation of nearby TMEM16A channels in endothelial cells to produce arterial hyperpolarization, vasodilation, and reduced blood pressure. Thus, TMEM16A is an anion channel in endothelial cells that regulates arterial contractility and blood pressure.

Abstract 11032: Peptide Lv Promotes the Trafficking and Membrane Insertion of Endothelial K Ca 31 Through the MEK1/ERK Pathway

Background: Peptide Lv is an endogenous secretory peptide that is upregulated in the eyes of patients with diabetic retinopathy, an ocular disease with pathological angiogenesis. Peptide Lv promotes vascular endothelial cell proliferation, migration, and sprouting, and blocking peptide Lv with a specific antibody, anti-Lv, dampens neovascularization, so peptide Lv is an angiogenic factor. However, the molecular mechanism of how peptide Lv promotes angiogenesis is not clear. We found that peptide Lv causes endothelial cell membrane hyperpolarization, a step that is essential in endothelium-dependent angiogenesis. Peptide Lv augments the protein expression and current densities of K Ca 3.1, an ion channel underlying the endothelial hyperpolarization. As ion channels require trafficking and insertion to the plasma membrane to be functional after they are expressed, we aimed to determine the downstream signaling of peptide Lv that is involved in the trafficking of K Ca 3.1 in endothelial cells. Hypothesis: Peptide Lv activates the MEK1/ERK signaling pathway to promote the trafficking and membrane insertion of K Ca 3.1 in endothelial cells. Blocking MEK1/ERK will prevent augmentation of K Ca 3.1 by peptide Lv. Methods: Cultured human umbilical vein endothelial cells (HUVECs) were treated with peptide Lv (500 ng/ml) or PBS (vehicle control) in the presence/absence of FR180402 (10 μM; an ERK inhibitor) or PD98059 (10 μM, a MEK1 inhibitor) followed by the patch-clamp electrophysiological recordings and biotinylation assays. Results: Blocking ERK activation attenuated the peptide Lv-meditated membrane hyperpolarization and increase of K Ca 3.1 currents in endothelial cells. Blocking ERK activation did not affect peptide Lv-elicited increases of K Ca 3.1 protein expression, but it reduced the level of membrane-bound K Ca 3.1 Conclusions: The MEK1/ERK signaling pathway mediates peptide Lv-elicited trafficking and membrane insertion of K Ca 3.1 but does not affect the increase of K Ca 3.1 expression by peptide Lv.

Extracellular l-arginine Enhances Relaxations Induced by Opening of Calcium-Activated SKCa Channels in Porcine Retinal Arteriole.

We investigated whether the substrate for nitric oxide (NO) production, extracellular l-arginine, contributes to relaxations induced by activating small (SKCa) conductance Ca2+-activated potassium channels. In endothelial cells, acetylcholine increased 3H-l-arginine uptake, while blocking the SKCa and the intermediate (IKCa) conductance Ca2+-activated potassium channels reduced l-arginine uptake. A blocker of the y+ transporter system, l-lysine also blocked 3H-l-arginine uptake. Immunostaining showed co-localization of endothelial NO synthase (eNOS), SKCa3, and the cationic amino acid transporter (CAT-1) protein of the y+ transporter system in the endothelium. An opener of SKCa channels, cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine (CyPPA) induced large currents in endothelial cells, and concentration-dependently relaxed porcine retinal arterioles. In the presence of l-arginine, concentration-response curves for CyPPA were leftward shifted, an effect unaltered in the presence of low sodium, but blocked by l-lysine in the retinal arterioles. Our findings suggest that SKCa channel activity regulates l-arginine uptake through the y+ transporter system, and we propose that in vasculature affected by endothelial dysfunction, l-arginine administration requires the targeting of additional mechanisms such as SKCa channels to restore endothelium-dependent vasodilatation.

Hypoxic stress upregulates Kir2.1 expression by a pathway including hypoxic-inducible factor-1α and dynamin2 in brain capillary endothelial cells.

Brain capillary endothelial cells (BCECs) play a central role in maintenance of blood-brain barrier (BBB) function and, therefore, are essential for central nervous system homeostasis and integrity. Although brain ischemia damages BCECs and causes disruption of BBB, the related influence of hypoxia on BCECs is not well understood. Hypoxic stress can upregulate functional expression of specific K+ currents in endothelial cells, e.g., Kir2.1 channels without any alterations in the mRNA level, in t-BBEC117, a cell line derived from bovine BCECs. The hyperpolarization of membrane potential due to Kir2.1 channel upregulation significantly facilitates cell proliferation. In the present study, the mechanisms underlying the hypoxia-induced Kir2.1 upregulation was examined. We emphasize the involvement of dynamin2, a protein known to be involved in a number of surface expression pathways. Hypoxic culture upregulated dynamin2 expression in t-BBEC117 cells. The inhibition of dynamin2 by Dynasore canceled hypoxia-induced upregulation of Kir2.1 currents by reducing surface expression. On the contrary, Kir2.1 currents and proteins in t-BBEC117 cultured under normoxia were increased by overexpression of dynamin2, but not by dominant-negative dynamin2. Molecular imaging based on bimolecular fluorescence complementation, double-immunostaining, and coimmunoprecipitation assays revealed that dynamin2 can directly bind to the Kir2.1 channel. Moreover, hypoxic culture downregulated hypoxic-inducible factor-1α (HIF-1α) expression. Knockdown of HIF-1α increased dynamin2 expression in t-BBEC117 cells, in both normoxic and hypoxic culture conditions. In summary, our results demonstrated that hypoxia downregulates HIF-1α, increases dynamin2 expression, and facilitates Kir2.1 surface expression, resulting in hyperpolarization of membrane potential and subsequent increase in Ca2+ influx in BCECs.

Impairment of Coronary Endothelial Function by Hypoxia-Reoxygenation Involves TRPC3 Inhibition-mediated KCa Channel Dysfunction: Implication in Ischemia-Reperfusion Injury

Despite increasing knowledge of the significance of calcium-activated potassium (KCa) and canonical transient receptor potential (TRPC) channels in endothelial physiology, no studies so far have investigated the link between these two distinct types of channels in the control of vascular tone in pathological conditions. We previously demonstrated that hypoxia-reoxygenation (H-R) inhibits endothelial KCa and TRPC3 channels in porcine coronary arteries (PCAs). The present study further investigated whether modulation of TRPC3 is involved in H-R-induced KCa channel inhibition and associated vasodilatory dysfunction using approaches of wire myography, whole-cell voltage-clamp, and coimmunoprecipitation. Pharmacological inhibition or siRNA silencing of TRPC3 significantly suppressed bradykinin-induced intermediate- and small-conductance KCa (IKCa and SKCa) currents in endothelial cells of PCAs (PCAECs). TRPC3 protein exists in physical association with neither IKCa nor SKCa. In H-R-exposed PCAECs, the response of IKCa and SKCa to bradykinin-stimulation and to TRPC3-inhibition was markedly weakened. Activation of TRPC3 channels restored H-R-suppressed KCa currents in association with an improved endothelium-derived hyperpolarizing factor (EDHF)-type vasorelaxation. We conclude that inhibition of TRPC3 channels contributes to H-R-induced suppression of KCa channel activity, which serves as a mechanism underlying coronary endothelial dysfunction in ischemia-reperfusion (I-R) injury and renders TRPC3 a potential target for endothelial protection in I-R conditions.

Hypercholesterolemia Induces Endothelial Dysfunction Through Impaired Kir2.1 Channel Activity Upstream of NO Production

Hypercholesterolemia‐induced endothelial dysfunction is largely attributed to a loss of nitric oxide production/release, however the underlying mechanisms are unclear. Our recent study demonstrated that endothelial inwardly‐rectifying K+ channels (Kir2.1) are essential for flow‐induced activation of eNOS, NO production and flow‐induced vasodilation (FIV) in murine mesenteric resistance arteries. Our group also showed earlier that Kir channels are strongly suppressed by the elevation of cellular cholesterol. Therefore, we hypothesize that under hypercholesterolemic conditions cholesterol‐induced inhibition of Kir2.1, the major mesenteric EC Kir channel, would result in endothelial dysfunction (i.e. reduced dilations to flow) ultimately by preventing NO production. To test this hypothesis, we first determined the impact of clinically relevant concentrations of LDL on Kir current in human aortic ECs. We show that at concentrations considered to be at risk for atherosclerosis and heart disease we observed decreased Kir current density (10.8 ± 1.0 pA/pF for Control vs. 5.1 ± 0.3 pA/pF for 150 mg/dL LDL vs. 2.5 ± 0.4 pA/pF for 250 mg/dL LDL), an effect significantly reversed by the presence of HDL (5.1 ± 0.7 pA/pF for 150 mg/dL LDL vs. 10.2 ± 2.3 pA/pF for 50 mg/dL HDL + LDL). We also show that while Kir currents in endothelial cells freshly‐isolated from mesenteric arteries of WT mice are clearly shear‐stress sensitive, sensitivity of the channels to shear is lost in dyslipidemic ApoE−/− mice. Furthermore, the loss of FIV in pressurized and preconstricted 1st order mesenteric arteries (100–150 μm lumen diameters) isolated from dyslipidemic ApoE−/− mice can be attributed to the loss of endothelial Kir function. Specifically, in WT mice exposure to an intralumenal shear stress of ~30 dyn/cm2 results in an average dilation of 93.1% of the maximal diameter while ApoE−/− mice exhibit dilations of only 36.4%. In Kir2.1‐deficient mice (Kir2.1+/−), maximal dilation is reached at 42.2% and no further decrease is observed in ApoE−/−/Kir2.1+/− double knock‐out mice suggesting that impaired Kir channels are responsible for the decrease in FIV in hypercholesterolemic conditions. Consistent with these observations, Ba2+, a Kir channel blocker, can reduce dilations to flow in arteries of WT mice but has little to no effect in arteries from ApoE−/− mice. Additionally, NO production in arteries of ApoE−/− and ApoE−/− × Kir2.1+/− mice exposed to internal flow for 30 minutes is decreased compared to WT arteries. Most importantly, we found that overexpressing Kir2.1 specifically in mesenteric endothelium using adenoviral transfection driven by the VE‐Cadherin promoter recovered both FMV and flow‐induced NO production in ApoE−/− or ApoE−/− × Kir2.1+/− arteries strongly confirming the role for EC Kir2.1 impairment in hypercholesterolemia.Support or Funding InformationNIH RO1 HL073965American Heart Association Postdoctoral Award 16POST27000011

Acute Ischemia Activates Distinct Ion Currents in Endothelial Cells and Pericytes of Capillaries in Guinea Pig Cochlear Lateral Wall

Capillary blood flow in spiral ligament (SL) and strial vascularis (SV) of the cochlear lateral wall are critical for cochlear health and to meet the high energy‐demand of auditory transduction, but the physio‐pathology of the local capillary cells remains poorly understood. Using acutely isolated capillary segments and whole‐cell recording techniques, we characterized the main membrane currents and the responses to acute ischemic treatment (AIT, hypercapnic pH 6.2, pO2=0 mmHg) of the endothelial cells (EC) and pericytes (PC) in the SL and SV. We found: 1) In physiological solutions, the PCs, but not ECs, showed a [K+]o‐regulated and Ba2+‐sensitive inward rectifier current (Kir). 2) ECs, but PCs rarely, showed a significant outward rectifier current, which was inhibited by 1 mM 4‐AP, 100 μM niflumic acid (NFA) but not TEA. 3) When recorded in situ of a vessel segment, both types of cells showed variable electrical coupling that was blocked by 30 μM 18β‐glycyrrhetinic acid or 100 μM 2‐APB. 4) AIT (3 – 24 min) caused a reversible and partially repeatable outward current at holding potential −20 mV in isolated ECs and PCs. The AIT‐activated net current had I‐V curves and kinetics which mostly resemble to those of Ca2+−activated Cl−channels: strong outward rectification with quick or slow time course to reach steady‐state within 0.5 – 2 s voltage steps. This AIT‐induced current was inhibited by niflumic acid and 1 mM TEA but not 4‐AP. 5) AIT (>;3 min) also inhibited gap junction coupling current. We conclude that the capillary EC and PC in the SV and SL express different K+‐channels, suggesting a role of PCs as the K+‐sensor for blood flow regulation, and the acute ischemia activates chloride channels and BK channels likely via elevation of cytosolic Ca2+ in both ECs and PCs. Supported by NIH grants DC004716 (ZGJ) and DC005983, DC 00105 (ALN).

Use of intermediate/small conductance calcium-activated potassium-channel activator for endothelial protection

Endothelial dysfunction occurs in hypoxia-related states such as ischemic heart disease or heart surgery. Intermediate- and small-conductance calcium-activated potassium channels (IKCa and SKCa) are closely related to endothelium-dependent hyperpolarizing factor-mediated endothelial function. However, the status of these KCa under hypoxia is unknown. We investigated whether endothelial dysfunction under hypoxic state is related to the alterations of IKCa and SKCa and whether use of IKCa/SKCa activator may protect endothelium from hypoxia-reoxygenation injury. Isometric tension measurement, patch-clamp technique, intracellular membrane potential recording, and molecular methods were used to study porcine coronary arteries and endothelial cells. Hypoxia-reoxygenation (60-30 minutes) decreased endothelium-dependent hyperpolarizing factor-mediated relaxation at normothermia in Krebs solution (43.3%±6.3% vs 82.3%±2.9%) and in St Thomas' Hospital cardioplegic solution (28.9%±1.8% vs 78.1%±3.0%) (P<.001) as well as at hypothermia in St Thomas' Hospital solution (43.1%±2.6%, P<.001). Hypoxia-reoxygenation markedly reduced endothelial IKCa (2.8±0.6 vs 6.9±0.6 pA/pF) and SKCa currents (1.5±0.3 vs 4.3±0.4 pA/pF) (P<.05) and downregulated endothelial IKCa expression. IKCa/SKCa activator 1-ethyl-2-benzimidazolinone enhanced K+ current in endothelial cells that was blunted by hypoxia. Further, 1-ethyl-2-benzimidazolinone restored (P<.001) endothelium-dependent hyperpolarizing factor-mediated relaxation with hyperpolarization recovered from 6.0±0.3 to 7.8±0.4 mV (P<.05). In porcine coronary arteries, hypoxia markedly reduced endothelial K+ currents related to IKCa and SKCa with downregulation of protein expression and endothelium-derived hyperpolarizing factor function. IKCa/SKCa activator may preserve endothelium-dependent hyperpolarizing factor-mediated relaxation with enhancement of K+ current in endothelial cells and cellular membrane potential hyperpolarization in smooth muscle cells and may become a new strategy to protect coronary endothelium in cardiac surgery or transplantation.

Ionizing non-fatal whole-body irradiation inhibits Ca2+-dependent K+ channels in endothelial cells of rat coronary artery: Possible contribution to depression of endothelium-dependent vascular relaxation

Purpose: The goal of this study was to evaluate the influence of ionizing irradiation on large conductance Ca2+-dependent potassium (BKCa) channels in rat coronary endothelial cells.Materials and methods: Rats were exposed to a 6 Gy dose from a cobalt60 source. Experimental design of this study comprised recording of contractile force using isolated rat aortic rings and whole-cell patch clamp techniques to study whole-cell potassium currents in isolated rat coronary artery endothelial cells.Results: It has been shown that outward potassium currents in endothelial cells 9 days after irradiation appear to be suppressed or even totally abolished. The reversal potential for these currents in irradiated cells was shifted to more positive values. Paxilline (500 nM), an inhibitor of BKCa channels, had no or only a negligible effect on irradiated cells. The experiments using isolated aortic rings demonstrated that both paxilline and irradiation significantly shifted the acetylcholine dependent concentration-relaxation response curve to the right. Irradiated tissues were insensitive to paxilline.Conclusion: The results suggest that non-fatal, whole-body γ-irradiation suppresses large conductance, calcium-activated potassium channels, which control the driving force for Ca2+ entry and therefore Ca2+ dependent nitric oxide (NO) synthesis in endothelial cells. This may contribute, in part, to radiation-induced endothelium dysfunction and an increase in arterial blood pressure.

Altered expression of small-conductance Ca2+-activated K+ (SK3) channels modulates arterial tone and blood pressure.

The endothelium is a critical regulator of vascular tone, and dysfunction of the endothelium contributes to numerous cardiovascular pathologies. Recent studies suggest that apamin-sensitive, small-conductance, Ca2+-activated K+ channels may play an important role in active endothelium-dependent vasodilations, and expression of these channels may be altered in disease states characterized by vascular dysfunction. Here, we used a transgenic mouse (SK3T/T) in which SK3 expression levels can be manipulated with dietary doxycycline (DOX) to test the hypothesis that the level of expression of the SK subunit, SK3, in endothelial cells alters arterial function and blood pressure. SK3 protein was elevated in small mesenteric arteries from SK3T/T mice compared with wild-type mice and was greatly suppressed by dietary DOX. SK3 was detected in the endothelium and not in the smooth muscle by immunohistochemistry. In whole-cell patch-clamp experiments, SK currents in endothelial cells from SK3T/T mice were almost completely suppressed by dietary DOX. In intact arteries, SK3 channels contributed to sustained hyperpolarization of the endothelial membrane potential, which was communicated to the arterial smooth muscle. Pressure- and phenylephrine-induced constrictions of SK3T/T arteries were substantially enhanced by treatment with apamin, suppression of SK3 expression with DOX, or removal of the endothelium. In addition, suppression of SK3 expression caused a pronounced and reversible elevation of blood pressure. These results indicate that endothelial SK3 channels exert a profound, tonic, hyperpolarizing influence in resistance arteries and suggest that the level of SK3 channel expression in endothelial cells is a fundamental determinant of vascular tone and blood pressure.

On the endothelial cell ISOC

Ca 2+ store depletion activates both Ca 2+ selective and non-selective currents in endothelial cells. Recently, considerable progress has been made in understanding the molecular make-up and regulation of an endothelial cell thapsigargin-activated Ca 2+ selective current, I SOC. Indeed, I SOC is a relatively small inward Ca 2+ current that exhibits an approximate +40 mV reversal potential and is strongly inwardly rectifying. This current is sensitive to organization of the actin-based cytoskeleton. Transient receptor potential (TRP) proteins 1 and 4 (TRPC1 and TRPC4, respectively) each contribute to the molecular basis of I SOC, although it is TRPC4 that appears to be tethered to the cytoskeleton through a dynamic interaction with protein 4.1. Activation of I SOC requires association between protein 4.1 and the actin-based cytoskeleton (mediated through spectrin), suggesting protein 4.1 mediates the physical communication between Ca 2+ store depletion and channel activation. Thus, at present findings indicate a TRPC4–protein 4.1 physical linkage regulates I SOC activation following Ca 2+ store depletion.

Evidence for the stimulatory effect of resveratrol on Ca(2+)-activated K+ current in vascular endothelial cells.

Resveratrol, a natural phytoalexin compound, is present in grapes and wine, and it can produce vasorelaxation. However, little is known of its mechanisms of action on ionic currents in endothelial cells. The effect of resveratrol on Ca(2+)-activated K+ currents in an endothelial cell line (HUV-EC-C) originally derived from human umbilical vein was investigated with the aid of the patch-clamp technique. In the whole-cell configuration, resveratrol reversibly increased the amplitude of K+ outward currents. The increase in outward current caused by resveratrol was greatly inhibited by iberiotoxin (200 nM) or paxilline (1 microM), but not by glibenclamide (10 microM), tamoxifen (10 microM), or beta-bungarotoxin (200 nM). Thus, this outward current is believed to be Ca(2+)-activated K+ current (I K(Ca)). In the inside-out configuration, bath application of resveratrol (30 microM) caused no change in the single-channel conductance, but increased the activity of large-conductance Ca(2+)-activated K+ (BKCa) channels. Resveratrol enhanced the channel activity in a concentration-dependent manner. The EC50 value for resveratrol-induced channel activity was 20 microM. The resveratrol-stimulated increase in the channel activity was independent of internal Ca2+. Resveratrol (30 microM) also shifted the activation curve of BKCa channels to less positive membrane potentials. The change in the kinetic behavior of BKCa channels caused by resveratrol in these cells in due to an increase in mean open time and a decrease in mean closed time. In a pancreatic islet endothelial cell line (MS1), resveratrol (30 microM) also increased the activity of intermediate-conductance KCa channels. These results provide evidence that in addition to the presence of antioxidative activity, resveratrol can also stimulate KCa channels in endothelial cells. The direct stimulation of these KCa channels by resveratrol may be responsible for its effect on the functional activities of endothelial cells.

Inhibition of angiogenesis by blockers of volume-regulated anion channels

Osmotic cell swelling activates an outwardly rectifying Cl − current in endothelial cells that is mediated by volume-regulated anion channels (VRACs). In the past, we have shown that serum-induced proliferation of endothelial cells is arrested in the presence of compounds that potently block the endothelial VRACs. Here we report on the effects of four chemically distinct VRAC blockers [5-nitro-2-(3-phenylpropylamino)benzoic acid] (NPPB), mibefradil, tamoxifen, and clomiphene—on several models of experimental angiogenesis. Mibefradil (20 μM), NPPB (100 μM), tamoxifen (20 μM), and clomiphene (20 μM) inhibited tube formation by rat microvascular endothelial cells plated on matrigel by 42.9 ± 8.8%, 25.3 ± 10.4%, 32.2 ± 4.5%, and 20 ± 5.8%, respectively (p < 0.05). Additionally, NPPB (50–100 μM) and mibefradil (10–30 μM) significantly inhibited bFGF (10 ng/ml) + TNFα (2.5 ng/ml)-stimulated microvessel formation by human microvascular endothelial cells plated on fibrin by 30–70%. Furthermore, NPPB, mibefradil, and clomiphene concentration dependently inhibited spontaneous microvessel formation in the rat aorta-ring assay and vessel development in the chick chorioallantoic membrane assay. These results suggest that VRAC blockers are potent inhibitors of angiogenesis and thus might serve as therapeutic tools in tumor growth and other angiogenesis-dependent diseases.

Endothelial Cells Freshly Isolated from Resistance-Sized Pulmonary Arteries Possess a Unique K+ Current Profile

We have, for the first time, developed a reliable method for freshly isolating viable endothelial cells from resistance-sized rat pulmonary arteries. The endothelial origin of these cells was confirmed using indirect immunofluorescence, utilizing fluorescently labeled low-density lipoprotein. Biophysical and pharmacological patch-clamp experiments conducted under quasiphysiological cationic gradients revealed that these cells had a mean resting membrane potential of ∼−38 mV and displayed a delayed-rectifying K+ current. Immunohistochemical staining of rat lung cross-sections revealed an abundance of KV1.5 channel protein in pulmonary endothelium. This is the first report of a delayed-rectifying K+ current in endothelial cells of resistance-sized pulmonary arteries. Since changes in membrane potential associated with K+ channel activity affect release of vasoactive substances from endothelial cells, this finding has important implications for understanding the mechanisms underlying control of pulmonary vascular tone.

Potent block of volume-activated chloride currents in endothelial cells by the uncharged form of quinine and quinidine.

1. The effects of quinine and quinidine on the volume-activated chloride current (ICl(vol)) in cultured endothelial cells from bovine pulmonary artery were studied by use of the whole-cell patch-clamp technique. 2. At pH 7.4 both quinine and quinidine induced a fast and reversible block of ICl(vol)) with Ki values of 20 +/- 4 microM and 30 +/- 10 microM, respectively. 3. The blocking efficiency of both drugs increased dramatically with increasing extracellular pH, indicating that the blockade is mediated by the uncharged form of quinine and quinidine. 4. These results suggest a hydrophobic interaction with high affinity between volume-activated chloride channels and uncharged quinine and quinidine within the membrane bilayer of endothelial cells.

Characterization of volume-activated chloride currents in endothelial cells from bovine pulmonary artery.

We have measured the kinetic and pharmacological properties of volume-activated Cl- currents (ICl, vol) in endothelial cells, and tried to correlate them with those of the already described volume-activated current ICln. Both conductances show a similar permeability sequence for monovalent anions, and they are blocked by extracellular ATP. In the present report, we demonstrate by Western blot and RT-PCR that cultured endothelial cells from bovine pulmonary artery (CPAE) contain pICln. The expression of this protein has been shown to be closely associated with the ICln current. ICl, vol showed however, in contrast with ICln, no striking inactivation at positive potentials. This property is also at variance with that of the volume-activated current related to MDR-1. Activation of ICl, vol at potentials more negative than -80 mV was not time dependent, which excludes a major contribution of a ClC-2 related current. The antiviral nucleoside analogue AZT (3'-azido-3'-deoxythymidine) inhibited ICl, vol by 21 +/- 2.7% (n = 10), at a concentration of 100 microM. Another antiviral drug, acyclovir (ACV, 9-[2-hydroxyethoxy) methyl]guanine) blocked ICl, vol by 27 +/- 6.2% at 100 microM (n = 11). Both blocking effects are much smaller than those reported for ICln. The phenol derivative gossypol, which blocks ICln-related currents, efficiently inhibited ICl, vol in CPAE cells (67 +/- 2.1% at 1 microM, n = 7, KI = 0.4 microns). The presence of pICln in CPAE cells and the similar qualitative pharmacological profile of ICl, vol and ICln support the hypothesis that pICln is a good molecular candidate for ICl, vol in endothelial cells. The discrepant kinetic properties may indicate that these time-dependent currents at high positive or negative potentials are not intrinsic properties of the channels, but are caused by time-dependent depletion/accumulation phenomena due to the large amplitudes of these currents.

Inhibition of volume‐activated chloride currents in endothelial cells by chromones

1. We have studied the effects of the reported chloride channel blocker, sodium cromoglycate, on volume-activated Cl- currents in endothelial cells from bovine pulmonary artery by means of the whole-cell patch clamp technique. Cl- currents were activated by challenging the cells with a hypotonic extracellular solution of 60% of the normal osmolarity. 2. Half maximal activation of the current at +95 mV occurred after exposure of the cells for 148 +/- 10 s (n = 6) to hypotonic solution (HTS). At the same membrane potential but in the presence of 100 microM sodium cromoglycate (disodium-1,3-bis (2'-carboxylate-chromone-5'-yloxy)-2-hydroxy-propane) activation was delayed (253 +/- 25 s, n = 6) and the maximal current amplitude was reduced to 63 +/- 7% of the control (n = 13). 3. In comparison, an equimolar concentration of NPPB (5-nitro-2(3-phenyl) propylamino-benzoic acid), another Cl- channel blocker, completely blocked the volume-activated current in less than 20 s. 4. Sodium cromoglycate, applied at the time when the HTS-induced current was completely activated, dose-dependently inhibited this current with a concentration for half maximal inhibition of 310 +/- 70 microM. Data for nedocromil sodium were not significantly different from those for sodium cromoglycate. 5. Sodium cromoglycate, loaded into the endothelial cells via the patch pipette in ruptured patches, resulted in a decline of the HTS activated current with a time course that was compatible with diffusion of the compound from the pipette into the cell. Intracellulary applied sodium cromoglycate was also more effective and at 50 microM caused a decrease in the amplitude of the current to 25 +/- 6% (n = 10) of the control current.6 It is concluded that blockade of volume-activated Cl- currents by extracellular sodium cromoglycatemay be due to an intracellular action following its permeation across the cell membrane.

Caffeine‐evoked, calcium‐sensitive membrane currents in rabbit aortic endothelial cells

1. Single cell photometry and whole-cell patch clamp recording were used to study caffeine-induced intracellular Ca2+ signals and membrane currents, respectively, in endothelial cells freshly dissociated from rabbit aorta. 2. Caffeine (5 mM) evoked a transient increase in [Ca2+]i in fura-2-loaded endothelial cells. Pretreatment of cells with 10 microM ryanodine did not alter resting [Ca2+]i but irreversibly inhibited the caffeine-induced rise in [Ca2+]i. The caffeine-induced increase in [Ca2+]i was not attenuated by the removal of extracellular Ca2+ and did not stimulate the rate of Mn2+ quench of fura-2 fluorescence. 3. Bath application of caffeine evoked a dose- and voltage-dependent outward current. The rate of onset and amplitude of the caffeine-evoked outward current increased with higher caffeine concentrations and membrane depolarization. The relationship between caffeine-evoked current amplitude and membrane potential was non linear, suggesting that the channels underlying the current are voltage-sensitive. 4. In the absence of extracellular Ca2+, the amplitude of the caffeine-evoked outward current was reduced by approximately 50% but the duration of the current was prolonged compared to that observed in the presence of external Ca2+. Ca(2+)-free external solutions produced an unexpected increase in both the frequency and amplitude of spontaneous transient outward currents (STOCs). 5. Inclusion of heparin (10 micrograms ml-1) in the patch pipette abolished the acetylcholine (ACh)-induced outward current but failed to inhibit either STOCs or the caffeine-evoked outward current in native endothelial cells. In the absence of extracellular Ca2+, heparin did not affect either STOCs or the caffeine-induced outward current. 6. Externally applied tetraethylammonium ions (TEA, 3-10mM) reversibly inhibited unitary Ca2+-activated K+ currents and STOCs in endothelial cells but failed to inhibit completely the outward current evoked by 20 mM caffeine.7. Bath application of 0.1 mM zinc ion (Zn2+), a chloride channel blocker, did not affect unitary currents or STOCs but reduced the amplitude of the caffeine-evoked current by >75% compared to control. Replacement of extracellular NaCl with Na gluconate also reduced the amplitude of the caffeine-induced outward current. Bath application of 0.1 mM Zn2+ and 10 mM TEA completely blocked the caffeine-evoked outward current in endothelial cells.8. Caffeine-induced Ca2+ release from intracellular stores evokes a transient rise in [Ca2+1, which is correlated with a large, transient outward current. The ionic dependence and inhibition of the caffeine sensitive current by TEA and Zn2+ suggests that Ca2+-activated K+ and Cl- conductances contribute to the caffeine response in rabbit aortic endothelial cells.

CPA enhances Ca2+ entry in cultured bovine pulmonary arterial endothelial cells in an IP3-independent manner.

Studies of rat aorta revealed that cyclopiazonic acid (CPA), an inhibitor of the endoplasmic reticulum Ca2+ pump, released endothelium-derived relaxing factor (EDRF) and relaxed the muscle. We have used CPA to elucidate how this inhibitor of Ca2+ uptake into internal stores affects K+ channels and Ca2+ entrance in cultured bovine pulmonary endothelial cells using patch-clamp techniques. CPA increased a Ca(2+)-dependent outward K+ current for many minutes, presumably as a consequence of the unbalanced leakage of Ca2+ from internal stores and Ca2+ entrance across the cell membrane. An expected consequence of this activation of the outward current change is hyperpolarization of the cell membrane and increased driving force for Ca2+ entry. CPA activated the influx of extracellular Ca2+ through nonselective cation channels. Ca2+ influx through nonselective cation channels could help maintain intracellular Ca2+ concentration elevation and EDRF release. CPA also reduced the inwardly rectifying K+ current. Inositol 1,4,5-trisphosphate (IP3) in the patch pipette also produced an increase in outward K+ currents, which were Ca2+ dependent. After depletion of Ca2+ internal stores by CPA, the response to IP3 was abolished. Heparin in the patch pipette reduced the increase in outward currents induced by bradykinin, an agonist known to raise IP3 and to release Ca2+, but did not prevent CPA-induced increases in outward current. Thus CPA acts to elevate Ca(2+)-activated currents in endothelial cells by a mechanism independent of IP3-induced release, and this may lead to EDRF release both directly and as a consequence of Ca2+ entry through nonselective cation channels driven by an increased electrical gradient for Ca2+.