Bovine Pulmonary Artery Endothelial Research Articles

Partial-frequency-spectrum reconstruction algorithm of SIM with reduced raw images

In structured illumination microscopy (SIM), the imaging speed is expected to be increased to observe living cells. The conventional 2D SIM reconstruction algorithm (RA) requires nine raw images to reconstruct a super-resolution image. Here, we develop a partial-frequency-spectrum (PFS) reconstruction algorithm, based on the subtraction of frequency spectrum, which can reconstruct a super-resolution image by using six raw SIM images (two SIM images for each orientation). Our experiments of actin filament in bovine pulmonary artery endothelial (BPAE) cell imaging indicate that by the PFS algorithm, the frame rate increases. The PFS algorithm can resolve 120 nm in our experiment, which is equivalent to the reconstruction result of conventional 9-frame SIM. The PFS algorithm only requires the phase estimation of the three images. The reconstruction speed is about 5 times faster that of the conventional nine-images SIM method.

Single shot, three-dimensional fluorescence microscopy with a spatially rotating point spread function.

A wide-field fluorescence microscope with a double-helix point spread function (PSF) is constructed to obtain the specimen's three-dimensional distribution with a single snapshot. Spiral-phase-based computer-generated holograms (CGHs) are adopted to make the depth-of-field of the microscope adjustable. The impact of system aberrations on the double-helix PSF at high numerical aperture is analyzed to reveal the necessity of the aberration correction. A modified cepstrum-based reconstruction scheme is promoted in accordance with properties of the new double-helix PSF. The extended depth-of-field images and the corresponding depth maps for both a simulated sample and a tilted section slice of bovine pulmonary artery endothelial (BPAE) cells are recovered, respectively, verifying that the depth-of-field is properly extended and the depth of the specimen can be estimated at a precision of 23.4nm. This three-dimensional fluorescence microscope with a framerate-rank time resolution is suitable for studying the fast developing process of thin and sparsely distributed micron-scale cells in extended depth-of-field.

Virtual k-Space Modulation Optical Microscopy.

We report a novel superresolution microscopy approach for imaging fluorescence samples. The reported approach, termed virtual k-space modulation optical microscopy (VIKMOM), is able to improve the lateral resolution by a factor of 2, reduce the background level, improve the optical sectioning effect and correct for unknown optical aberrations. In the acquisition process of VIKMOM, we used a scanning confocal microscope setup with a 2D detector array to capture sample information at each scanned x-y position. In the recovery process of VIKMOM, we first modulated the captured data by virtual k-space coding and then employed a ptychography-inspired procedure to recover the sample information and correct for unknown optical aberrations. We demonstrated the performance of the reported approach by imaging fluorescent beads, fixed bovine pulmonary artery endothelial (BPAE) cells, and living human astrocytes (HA). As the VIKMOM approach is fully compatible with conventional confocal microscope setups, it may provide a turn-key solution for imaging biological samples with ∼100 nm lateral resolution, in two or three dimensions, with improved optical sectioning capabilities and aberration correcting.

Spatiotemporal control of degenerate multiphoton fluorescence microscopy with delay-tunable femtosecond pulse pairs

Selective excitation of a particular fluorophore in an ensemble of different fluorophores with overlapping fluorescence spectra is shown to be dependent on the time delay of femtosecond pulse pairs in multiphoton fluorescence microscopy. In particular, the two-photon fluorescence behavior of the Texas Red and DAPI dye pair inside Bovine Pulmonary Artery Endothelial (BPAE) cells depends strongly on the center wavelength of the laser, as well as the delay between two identical laser pulses in one-color femtosecond pulse-pair excitation scheme. Thus, we present a novel design concept using pairs of femtosecond pulses at different central wavelengths and tunable pulse separations for controlling the image contrast between two spatially and spectrally overlapping fluorophores. This femtosecond pulse-pair technique is unique in utilizing the variation of dye dynamics inside biological cells as a contrast mode in microscopy of different fluorophores.

A miniature laser speckle fluorescence sectioning microscope for cell imaging

We present a miniature fluorescence sectioning microscope which uses a diode-pumped solid-state (DPSS) laser as the light source and a fast translating diffuser to produce dynamically changing speckle patterns onto the back aperture of the objective to illuminate the sample. Optical sectioning, which originates from the statistical characteristics of laser speckles, is obtained by calculating the contrast of a series of fluorescence images. High contrast fluorescence sectioning images of bovine pulmonary artery endothelial (BPAE) cells are obtained. The image quality is similar to that of the images acquired by standard laser scanning confocal microscope (LSCM). Compared with LSCM, the laser speckle fluorescence microscope (LSFM) presented in this letter has many advantages, such as simple configurations, low cost, compact, and easy to operate, which makes it possible to have wide spread applications in biomedicine.

Application of maximum likelihood estimator in nano-scale optical path length measurement using spectral-domain optical coherence phase microscopy

Spectral-domain optical coherence phase microscopy (SD-OCPM) measures minute phase changes in transparent biological specimens using a common path interferometer and a spectrometer based optical coherence tomography system. The Fourier transform of the acquired interference spectrum in spectral-domain optical coherence tomography (SD-OCT) is complex and the phase is affected by contributions from inherent random noise. To reduce this phase noise, knowledge of the probability density function (PDF) of data becomes essential. In the present work, the intensity and phase PDFs of the complex interference signal are theoretically derived and the optical path length (OPL) PDF is experimentally validated. The full knowledge of the PDFs is exploited for optimal estimation (Maximum Likelihood estimation) of the intensity, phase, and signal-to-noise ratio (SNR) in SD-OCPM. Maximum likelihood (ML) estimates of the intensity, SNR, and OPL images are presented for two different scan modes using Bovine Pulmonary Artery Endothelial (BPAE) cells. To investigate the phase accuracy of SD-OCPM, we experimentally calculate and compare the cumulative distribution functions (CDFs) of the OPL standard deviation and the square root of the Cramér-Rao lower bound (1/ square root 2SNR ) over 100 BPAE images for two different scan modes. The correction to the OPL measurement by applying ML estimation to SD-OCPM for BPAE cells is demonstrated.

Hic-5 promotes endothelial cell migration to lysophosphatidic acid

Endothelial cell migration is critical for proper blood vessel development. Signals from growth factors and matrix proteins are integrated through focal adhesion proteins to alter cell migration. Hydrogen peroxide-inducible clone 5 (Hic-5), a paxillin family member, is enriched in the focal adhesions in bovine pulmonary artery endothelial (BPAE) cells, which migrate to lysophosphatidic acid (LPA) on denatured collagen. In this study, we investigate the role of Hic-5 in LPA-stimulated endothelial cell migration. LPA recruits Hic-5 to the focal adhesions and to the pseudopodia in BPAE cells plated on collagen, suggesting that recruitment of Hic-5 to focal adhesions is associated with endothelial cell migration. Knockdown of endogenous Hic-5 significantly decreases migration toward LPA, confirming involvement of Hic-5 in migration. To address the role of Hic-5 in endothelial cell migration, we exogenously expressed wild-type (WT) Hic-5 and green fluorescent protein Hic-5 C369A/C372A (LIM3 mutant) constructs in BPAE cells. WT Hic-5 expression increases chemotaxis of BPAE cells to LPA, whereas migration toward LPA of the green fluorescent protein Hic-5 C369A/C372A-expressing cells is similar to that shown in vector control cells. Additionally, ERK phosphorylation is enhanced in the presence of LPA in WT Hic-5 cells. A pharmacological inhibitor of MEK activity inhibits LPA-stimulated WT Hic-5 cell migration and ERK phosphorylation, suggesting Hic-5 enhances migration via MEK activation of ERK. Together, these studies indicate that Hic-5, a focal adhesion protein in endothelial cells, is recruited to the pseudopodia in the presence of LPA and enhances migration.

The C-terminal part of the R-domain, but not the PDZ binding motif, of CFTR is involved in interaction with Ca(2+)-activated Cl- channels.

Expression of the cystic fibrosis transmembrane conductance regulator (CFTR) inhibits Ca(2+)-activated Cl- channels (CaCC) by an unknown mechanism. This inhibition does not require CFTR activation (activity-independent inhibition), but is potentiated when CFTR is activated (activity-dependent inhibition). In this study, we evaluated, in endothelial cells, possible structural determinants for this interaction. Bovine pulmonary artery endothelium (CPAE) cells, which do not express CFTR, were transfected transiently with three hybrid CFTR constructs. The functional interaction between CaCC and CFTR was assessed using the patch-clamp technique in the whole-cell configuration. CaCC was stimulated by application of adenosine 5'-triphosphate (ATP) to the bath solution. CFTR currents were evoked by application of a forskolin/3-isobutyl-l-methylxanthine (IBMX) cocktail. The inhibitory effect of CFTR was conserved when the PDZ (PSD-95/Discs large/ZO-1) binding motif was deleted (CFTR-delta PDZ). In contrast, both the CFTR activity-independent and -dependent inhibition of CaCC were abolished when the C-terminal part of the regulatory (R)-domain of CFTR was deleted (CFTR-delta R780-830). The activity-dependent inhibition of CaCC, but not the activity-independent inhibition, could be rescued by introducing the multiple drug resistance (MDR)-1 mini-linker in place of the deletion (CFTR-delta R-linker). It is concluded that the C-terminal part of the R-domain is an important determinant for CFTR-CaCC interaction.

Intramitochondrial pyruvate attenuates hydrogen peroxide-induced apoptosis in bovine pulmonary artery endothelium.

In the hydrogen peroxide (H2O2) apoptosis model of the murine thymocyte, redox reactant and antioxidant pyruvate prevents programmed cell death. We tested the hypothesis that such protection was mediated, at least in part, via pyruvate handling by mitochondrial metabolism. Cultured bovine pulmonary artery endothelial cells were incubated for 30 min with 0.5 mM H2O2 in the absence and presence of 0.5 mM alpha-cyano-3-hydroxycinnamate, as a selective inhibitor of the mitochondrial pyruvate transporter. In controls H2O2 decreased cell viability by 30% within 24 h; this was associated with apoptosis-like bodies, nuclear condensation, and biochemical DNA damage consistent with programmed cell death. Pyruvate (0.1-20 mM) enhanced cell viability in a dose-dependent manner, with > or = 85% viable cells at > or = 3 mM and no DNA laddering, no positive nick-end labeling (TUNEL), and no detectable Annexin V or propidium iodide staining. In contrast, using > or = 5 mM L-lactate as a cytosolic reductant or acetate as a redox-neutral substrate, cell death increased to approximately 40%, which was associated with intense DNA laddering, positive TUNEL and Hoechst 33258 assays. Alpha-cyano-3-hydroxycinnamate alone did not significantly decrease endothelial viability but reduced viability from 85+/-3 to 71+/-4% (p = 0.023) in presence of 3 mM pyruvate plus H2O2; pathological cell morphology and DNA laddering under the same conditions suggested loss of pyruvate protection against apoptosis. Since alpha-cyano-3-hydroxycinnamate re-distributed medium pyruvate and L-lactate consistent with selective blockade of pyruvate uptake into the mitochondria, the findings support the hypothesis that pyruvate protection against H2O2 apoptosis is mediated in part via the mitochondrial matrix compartment. Possible mediators include anti-apoptotic bcl-2 and/or products of mitochondrial pyruvate metabolism such as citrate that affect metabolic regulation and anti-oxidant status in the cytoplasm.

INTERACTION OF NITRIC OXIDE AND TRANSFORMING GROWTH FACTOR-β1 INDUCED BY ANGIOTENSIN II AND MECHANICAL STRETCH IN RAT RENAL TUBULAR EPITHELIAL CELLS

Changes in intrarenal pressure accompanying unilateral ureteral obstruction can result in tubular mechanical stretch and mediator release from renal tubules. Therefore, we examined the synthesis of nitric oxide and transforming growth factor-beta (TGF-beta), and their interaction in rat renal epithelial cells (NRK-52E) exposed to either angiotensin II or mechanical stretch. NRK-52E were exposed to either angiotensin II or mechanical stretch. Nitrite and TGF-beta in the supernatant were assessed by the Greiss reaction and bioassay, respectively. The level of cell hypertrophy and intracellular TGF-beta protein was determined by flow cytometry. TGF-beta messenger RNA and inducible nitric oxide synthase protein were detected by reverse transcriptase polymerase chain reaction and Western blot, respectively. Angiotensin II stimulated TGF-beta1 and nitric oxide. The nitric oxide synthase inhibitor, N-nitro-L-arginine (L-NAME) or angiotensin II type I receptor blocker, losartan, inhibited nitric oxide and TGF-beta1 induced by angiotensin II. Flow cytometry showed that either L-NAME or losartan inhibited angiotensin II induced cell hypertrophy. TGF-beta1 inhibited iNOS protein and nitric oxide, whereas an anti-TGF-beta antibody enhanced iNOS. Mechanical stretch induced TGF-beta, inducible NOS protein and nitric oxide. However, TGF-beta expression was not affected by L-arginine or L-NAME when cells were exposed to mechanical stretch. These results demonstrate that nitric oxide is an intermediate in angiotensin II stimulated TGF-beta1 expression but not in stretch induced TGF-beta expression, and that TGF-beta1 is a negative regulator of nitric oxide in rat renal epithelial cells. The complex interaction of these cytokines may be a target for intervention in the fibrotic and apoptotic processes in the obstructed kidney.

Subcellular Phototoxicity of Photofrin-II and Lutetium Texaphyrin in Cells In Vitro

. Three cell types including bovine pulmonary artery endothelium cells (CPAE), rat kangaroo kidney cells (PTK2), and human larynx epidermoid carcinoma cells (Hep-2) were used to study subcellular localisation and phototoxicity of Photofrin-II and lutetium texaphyrin (Lu Tex). Cells were examined for fluorescence after administration of the photosensitisers. Subcellular regions were exposed with a laser microbeam system that used an argon ion laser pumped dye laser generating a 630 nm for Photofrin-II and 730 nm for Lu Tex. Fluorescence detection suggests that the Photofrin-II is bound primarily to the mitochondria with some diffuse fluorescence in the rest of the cytoplasm. The fluorescence in Lu Tex treated cells appears to be localised to the lysosomes. The percentage of damaged cells following light exposure to the different subcellular regions after Photofrin-II or Lu Tex treatment demonstrates that the nuclear region was the most sensitive target followed by the perinuclear region and peripheral cytoplasm region.

Nitric oxide donors regulate nitric oxide synthase in bovine pulmonary artery endothelium.

This study examined the notion that exogenous generation of nitric oxide (NO) modulates NOS gene expression and activity. Bovine pulmonary artery endothelial cells (BPAEC) were treated with the NO donors, 1 mM SNAP (S-nitroso-N-acetylpenicillamine), 0.5 mM SNP (sodium nitroprusside) or 0.2 microM NONOate (spermine NONOate) in medium 199 containing 2% FBS. Controls included untreated cells and cells exposed to 1 mM NAP (N-acetyl-D-penicillamine). NOS activity was assessed using a fibroblast-reporter cell assay; intracellular Ca2+ concentrations were assessed by Fura-2 microfluorometry; and NO release was measured by chemiluminescence. Constitutive endothelial (e) and inducible (i) NOS gene and protein expression were examined by northern and western blot analysis, respectively. Two hours exposure to either SNAP or NONOate caused a significant elevation in NO release from the endothelial cells (SNAP = 51.4 +/- 5.9; NONOate = 23.8 +/- 4.2; control = 14.5 +/- 2.8 microM); but A23187 (3 microM)-stimulated NO release was attenuated when compared to controls. Treatment with either SNAP or NONOate for 2 h also resulted in a significant increase in NOS activity in endothelial homogenates (SNAP = 23.6 +/- 2.5; NONOate= 29.8 +/- 7.7; control = 14.5 +/- 2.5fmol cGMP/microg per 10(6) cells). Exposure to SNAP and SNP, but not NONOate, for 1 h caused an increase in intracellular calcium. Between 4 and 8 h, SNAP and NONOate caused a 2- to 3-fold increase in eNOS, but not iNOS, gene (P < 0.05) and protein expression. NAP had little effect on either eNOS gene expression, activity or NO production. Our data indicate that exogenous generation of NO leads to a biphasic response in BPAEC, an early increase in intracellular Ca2+, and increases in NOS activity and NO release followed by increased expression of the eNOS gene, but not the iNOS gene. We conclude that eNOS gene expression and activity are regulated by a positive-feedback regulatory action of exogenous NO.

Interaction between calcium-activated chloride channels and the cystic fibrosis transmembrane conductance regulator.

We investigated interactions between cystic fibrosis conductance regulator (CFTR) and endogenous Ca2+-activated Cl- channels (CaCC) in bovine pulmonary artery endothelium (CPAE). CPAE cells, which do not express CFTR, were transiently transfected with wild-type (WT) CFTR and the deletion mutant deltaF508 CFTR. Currents through CaCC were significantly reduced after expression of WT CFTR. This inhibition was increased by stimulation (isobutylmethylxanthine, forskolin) of CFTR in cells expressing WT CFTR. There were no such effects when deltaF508 mutant CFTR, which is retained in the endoplasmic reticulum, was expressed. It is concluded that CFTR and CaCC are functionally coupled probably through a direct channel-channel interaction.

PKCβI mediates the inhibition of P2Y receptor‐induced inositol phosphate formation in endothelial cells

1. Bovine pulmonary artery endothelium (CPAE) expresses phospholipase C (PLC)-linked P2Y1 and P2Y2 receptors, for them 2-methylthio-ATP (2MeSATP) and UTP are respective agonists. Here, we have investigated the particular protein kinase C (PKC) isoform(s) responsible for the inhibition of P2Y1 and P2Y2 receptor-evoked inositol phosphate (IP) formation by phorbol 12-myristate 13-acetate (PMA). 2. Although short-term (20 min) pretreatment of cells with PMA attenuated 2MeSATP- and UTP-induced phosphoinositide (PI) breakdown, this inhibition was lost after 15 h. Preincubation with PMA for 24 h, on the contrary, potentiated 2MeSATP and UTP responses. The IP formation stimulated by NaF was unaltered by PMA pretreatment. 3. Western blot analysis showed that treatment of CPAE with PMA resulted in a rapid translocation of PKC isoform betaI, epsilon and mu, but not lambda, from the cytosol to the membrane fraction. 4. Pretreatment of the selective PKC inhibitor Ro 31-8220 attenuated the inhibitory effect of PMA on IP formation. Go 6976 (an inhibitor of conventional PKCalpha, beta and gamma) and LY 379196 (a selective PKCbeta inhibitor) also dose-dependently inhibited the PMA-mediated desensitization. 5. Transfection of PKCbeta-specific antisense oligonucleotide reduced PKCbetaI protein level and inhibited PMA-mediated PI reduction. 6. RT - PCR analysis showed that PMA treatment for 4 - 24 h up-regulated P2Y1 and P2Y2 receptors at the mRNA levels. 7. These results suggest that PKCbetaI may exert a negative feedback regulation on endothelial P2Y1 and P2Y2 receptor-mediated PI turnover. The down-regulation of PKCbetaI and enhanced P2Y receptor expression together might contribute to the late PI enhancing effect of PMA.

5-(Pentafluorobenzoylamino)fluorescein: A Selective Substrate for the Determination of Glutathione Concentration and GlutathioneS-Transferase Activity

5-(Pentafluorobenzoylamino)fluorescein (PFB-F), a new thiol-reactive molecule was synthesized to improve the detection limits and specificity of the assays for glutathioneS-transferase (GST) activity and glutathione (GSH). A rapid assay method to measure GSH concentration or GST activity and the simultaneous analysis of multiple samples is possible because the glutathione adduct, GS-TFB-F, is separated from PFB-F by thin-layer chromatography (TLC) and can be quantitated by a fluorescence scanner. The detection limits for GSH and for GST activity using TLC were found to be as low as 10 pmol/μl and 1 ng/μl using equine liver GST, respectively. Determination of GSH concentration or GST activity in bovine pulmonary artery endothelial (BPAE) cell lysates gave a linear response for samples corresponding to 500–2500 cells. PFB-F could also measure GST activities of GST fusion proteins and prove to be a suitable substrate for determining the activities of human GST isozymes and other sources of mammalian GST. The selectivity of PFB-F with GSH was proven by comparing trace amount of the adducts that formed with cysteine and β-galactosidase to that formed with GSH. The HPLC profile of a reaction mixture where cell lysate was used in place of purified GST, also shows only two main peaks, corresponding to GS-TFB-F and unreacted PFB-F. The selectivity of PFB-F for GSH was further confirmed by exposing BPAE cells todl-buthionine-[S,R]-sulfoximine (BSO). Our results of GS-TFB-F determination indicate that 12-, 24-, or 36-h incubations with BSO caused 2-, 6-, or 7.6-fold reductions in GSH levels, respectively.

REGULATION OF EXTRACELLULAR MATRIX PROTEINS BY TRANSFORMING GROWTH FACTOR β1IN CULTURED PULMONARY ENDOTHELIAL CELLS

Transforming growth factor beta-1 (TGF-β1), which is present in lung tissue, has been suggested to play a role in modulating vascular cell function in vivo. The action of TGF-β1in vivo, especially at the local site of application to connective tissue, is anabolic and leads to pulmonary fibrosis and angiogenesis, strongly indicating that TGF-β may have practical applications in repair of tissue injury caused by burns, trauma, or surgery. In the present study, we have used cultured bovine pulmonary artery endothelial (BPAE) cells as a model system. Expression of various proteins, including SPARC (secreted protein acidic and rich in cysteines), type IV procollagen and fibronectin (FN) was examined by radiolabeling the cells with [3H]proline, immunoprecipitation with specific antibodies, and Northern blot analyses by using specific cDNA probes. Cultured cells were labeled with [3H]proline for 24h in either the absence or in the presence of TGF-β1(0–20ng/ml). Incorporation of radioactivity was observed in a concentration-dependent manner, maximal at 5ng/ml. Northern blot hybridization demonstrated that TGF-β1(5ng/ml) treatment of BPAE cells caused an increase in steady-state levels of mRNA for SPARC (75%) and FN, thus, indicating a positive modulation. To determine whether TGF-β1has an effect on cell proliferation in the cultures, the incorporation of [3H]thymidine into cellular nuclei was used to measure DNA replication; 5ng/ml had an inhibitory effect (42%) on cell proliferation. Protein production by TGF-β1, surprisingly, showed decrease in SPARC levels (42%) and in contrast increased levels of FN (86%) and type IV procollagen (334%). Our results indicate that on exposure to TGF-β1, cultured BPAE cells differentially change expression of extracellular matrix proteins which may be important in remodeling of tissue and healing at sites of injury.

TNF-α Injury to Pulmonary Artery Endothelium is not Blocked by Steroids. † 1510

Glucocorticoids have anti-inflammatory properties and are beneficial in treating pulmonary edema associated with chronic lung disease and infection. We have shown that steroids affect bovine pulmonary artery endothelium by decreasing permeability to albumin. To test the hypothesis that steroids would block inflammatory mediated endothelial injury, we measured permeation of radiolabeled albumin across endothelial monolayers in the presence of the cytokine TNF-α. Monolayers were incubated in Dulbecco's modified Eagle medium and had a 24 hr exposure to 20 ng/mL TNF with (n=29) and without (n=31) pretreatment with 1 μM dexamethasone or with 24 hr exposure to dex alone(n=105). Control monolayers (n=108) were not exposed to TNF or dex. The figure shows permeability p as a function of treatment. Monolayers exposed to TNF were significantly more permeable to albumin than controls (p<0.0001, rank sum test), and pretreatment with dexamethasone did not prevent the injury. Monolayers exposed to dex alone were significantly less permeable than controls (p<0.0001). We conclude that while dex is effective in decreasing endothelial permeability, dex does not block the increased endothelial permeability caused by the inflammatory mediator TNF-α in this preparation.

Suppression of angiotensin-converting enzyme expression and activity by shear stress.

Shear stress caused by the frictional forces of a fluid moving over a cell monolayer is an important regulator of gene expression. In this study, we investigated the effect of shear stress on angiotensin-converting enzyme (ACE) expression and promoter activity in vitro and on local vascular ACE activity in vivo. ACE activity measured in bovine pulmonary artery endothelial (BPAE) cells was reduced by 49.5% after exposure to a shear stress of 20 dyne/cm2 for 18 hours. Short-term shearing (2 hours) elevated ACE activity in BPAE cells, whereas long-term shearing produced a time-dependent reduction in ACE activity by 23.3%, 33.5%, and 48.9% at 8, 12, and 18 hours, respectively. Northern blot analysis revealed that shear stress (20 dyne/cm2 for 18 hours) significantly reduced ACE mRNA expression by 82%. To determine the mechanism of ACE activity and message reduction, the effect of shear on transcriptionally related events was determined in a rabbit aortic endothelial cell line (W3LUC) stably transfected with 1.3 kb of a rat ACE promoter/luciferase construct. Different shear stress magnitudes (5 to 20 dyne/cm2) caused suppression of luciferase activity by an average of 40.7%. ACE promoter activity was suppressed by 2 hours of shear stress (24.7%) and was further inhibited at time periods > 8 hours. In vivo elevations in shear stress were created by placing a stainless steel clip over a 12-mm region of the rat abdominal aorta. Restriction of vessel diameter increased blood flow velocity and caused reduction in vascular ACE activity by 40%. These studies suggest that elevations in the level of shear stress alter endothelial cell function by suppressing ACE gene and protein expression in vitro and in vivo.

Inhibition of ecto-ATPase by PPADS, suramin and reactive blue in endothelial cells, C6 glioma cells and RAW 264.7 macrophages.

1. Previous studies have shown that bovine pulmonary artery endothelium (CPAE) has P2Y and P2U purinoceptors, rat C6 glioma cells have P2U purinoceptors and mouse RAW 264.7 cells have pyrimidinoceptors, all of which are coupled to phosphoinositide-specific phospholipase C (PI-PLC). The dual actions of PPADS, suramin and reactive blue as antagonists of receptor subtypes and ecto-ATPase inhibitors were studied in these three cell types. 2. In CPAE, suramin, at 3-100 microM, competitively inhibited the PI responses induced by 2MeSATP and UTP, with pA2 values of 5.5 +/- 0.3 and 4.4 +/- 0.4, respectively. Reactive blue, at 1-3 microM, produced shifts to the right of the 2MeSATP and UTP curves, but no further right shift at 10 microM. PPADS, at 10 microM, caused a 3 fold right shift of the 2MeSATP curve, but no further shift at concentrations up to 100 microM. In contrast, a dose-dependent shift to the left of the UTP curve and a weak inhibition of the ATP response were seen with PPADS. 3. In RAW 264.7 cells, suramin and reactive blue, but not PPADS, competitively inhibited the UTP response, with pA2 values of 4.8 +/- 0.5 and 5.8 +/- 0.7, respectively. 4. In C6 glioma cells, although suramin and reactive blue inhibited the ATP response, a potentiation effect on ATP and UTP responses was seen with PPADS. 5. The ecto-ATPase inhibitory activity of these three receptor antagonists were determined. All three inhibited ecto-ATPase present in CPAE, C6 and RAW 264.7 cells, with IC50 values of 4, 4.8 and 4.7 for PPADS, 4, 4.4 and > > 4 for suramin, and 4.5, 4.7 and 4.7 for reactive blue. 6. This study indicates that PPADS, suramin and reactive blue ar ecto-ATPase inhibitors. This property, combined with their antagonistic selectivity for receptor subtypes, can result in inhibition of, potentiation of, or lack of effect on agonist-mediated PI responses. Reactive blue is a more potent antagonist than suramin on P2Y, P2U and pyrimidinoceptors, and PPADS is a weak antagonist for P2Y receptors.

Dose-Response Effects of Radiation on the Permeability of Endothelial Cells in Culture

Increased permeability is an early and universal response of the vasculature to radiation injury, yet the biological basis of this reaction is poorly understood. The present study determined the time course and the dose-response relationship of radiation-induced hyperpermeability in cultured bovine pulmonary artery endothelial (BPAE) cells. BPAE cells were grown to a confluent monolayer on microcarrier beads, and column chromatography methods were used to evaluate permeability to two low molecular weight compounds: sodium fluorescein (NaFlsc, mol. wt. = 342) and cyanocobalamin (B12, mol. wt. = 1355). This is a novel in vitro model to study mechanisms and modifiers of radiation-induced permeability of endothelial cells under flow conditions using nonradioactive tracers. Cell-covered beads were exposed to a single dose of 10 Gy Of 137Cs gamma rays and placed in the column, and permeability was measured every 30 min for 3 h. There was a time-dependent increase in permeability to both tracers, reaching significance by 2 h. Increased permeability was accompanied by perturbations in F-actin distribution in the BPAE cells as determined by rhodamine-phalloidin fluorescence microscopy. Neither catalase nor captopril ameliorated this hyperpermeability, but dibutyryl cAMP partially prevented it. At 3 h after 0, 1, 2, 5 and 10 Gy irradiation, permeability values of 11.8 +/- 2.1, 13.9 +/- 2.2, 20.9 +/- 3.6, 24.8 +/- 2.8 and 27.2 +/- 3.3 (10(-5) cm/s, +/- SEM), respectively, were observed using NaFlsc. The increase was significant (P < 0.05) at 2 Gy or higher. Permeability to B12 was significantly elevated after 5 or 10 Gy. These results suggest that permeability of endothelial cells to low molecular weight solutes increases within 3 h after therapeutic doses of radiation, and that cAMP ameliorates this response.