Leukocyte adhesion and microvessel permeability

To investigate the direct effect of leukocyte adherence to microvessel walls on microvessel permeability, we developed a method to measure changes in hydraulic conductivity (L(p)) before and after leukocyte adhesion in individually perfused venular microvessels in frog mesentery. In 19 microvessels that were initially free of leukocyte sticking or rolling along the vessel wall, control L(p) was measured first with Ringer-albumin perfusate. Blood flow was then restored in each vessel with a reduced flow rate in the range of 30-116 microm/s to facilitate leukocyte adhesion. Each vessel was recannulated in 45 min. The mean number of leukocytes adhering to the vessel wall was 237 +/- 22 leukocytes/mm(2). At the same time, L(p) increased to 4.7 +/- 0.5 times the control value. Superfusion of isoproterenol (10 microM) after leukocyte adhesion brought the increased L(p) back to 1.1 +/- 0.2 times the control in 5-10 min (n = 9). Superfusing isoproterenol before leukocyte adhesion prevented the increase in L(p) (n = 6). However, the number of leukocytes adhering to the vessel wall was not significantly affected. These results demonstrated that leukocyte adhesion caused an increase in microvessel permeability that could be prevented or restored by increasing cAMP levels in endothelial cells using isoproterenol. Thus cAMP-dependent mechanisms that regulate inflammatory agent-induced increases in permeability also modulate leukocyte adhesion-induced increases in permeability but act independently of mechanisms that regulate leukocyte adhesion to the microvessel wall. Application of ketotifen, a mast cell stabilizer, and desferrioxamine mesylate, an iron-chelating reagent, attenuated the increase in L(p) induced by leukocyte adhesion, suggesting the involvement of oxidants and the activation of mast cells in leukocyte adhesion-induced permeability increase. Furthermore, with the use of an in vivo silver stain technique, the locations of the adherent leukocytes on the microvessel wall were identified quantitatively in intact microvessels.     

Tumor necrosis factor-alpha-induced leukocyte adhesion and microvessel permeability

The objective of this study was to investigate whether leukocyte adhesion and/or emigration are critical steps in increased microvessel permeability during acute inflammation. To conduct this study, we combined autologous blood perfusion with a single microvessel perfusion technique, which allows microvessel permeability to be measured precisely after the endothelium has interacted with blood-borne stimuli. Experiments were carried out in intact venular microvessels in rat mesenteries. Firm attachment of leukocytes to endothelial cells was induced by intravenous injection of TNF-alpha (3.5 microg/kg) and resuming autoperfusion in a precannulated microvessel. Leukocyte emigration was facilitated by superfusion of formyl-Met-Leu-Phe-OH. Microvessel permeability was measured as hydraulic conductivity (L(p)) or the solute permeability coefficient to tetramethylrhodamine isothiocyanate-labeled alpha-lactalbumin before and after leukocyte adhesion and emigration in individually perfused microvessels. We found that perfusion of a microvessel with TNF-alpha did not affect basal microvessel permeability, but intravenous injection of TNF-alpha caused significant leukocyte adhesion. However, the significant leukocyte adhesion and emigration did not cause corresponding increases in either L(p) or solute permeability. Thus our results suggest that leukocyte adhesion and emigration do not necessarily increase microvessel permeability and the mechanisms that regulate the adhesion process act independently from mechanisms that regulate permeability. In addition, silver staining of endothelial boundaries demonstrated that leukocytes preferentially adhere at the junctions of endothelial cells. The appearance of the silver lines indicates that the TNF-alpha-induced firm adhesion of leukocyte to microvessel walls did not involve apparent changes in the junctional structure of endothelial cells, which is consistent with the results of permeability measurements.     

Platelet-activating factor increases endothelial [Ca2+]i and NO production in individually perfused intact microvessels

We have demonstrated that inhibition of NO synthase (NOS) in endothelial cells by either the NOS inhibitor N(omega)-monomethyl-l-arginine (l-NMMA) or the internalization of caveolin-1 scaffolding domain attenuated platelet-activating factor (PAF)-induced increases in microvessel permeability (Am J Physiol Heart Circ Physiol 286: H195-H201, 2004) indicating the involvement of an NO-dependent signaling pathway. To investigate whether an increase in endothelial cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) is the initiating event and Ca(2+)-dependent NO production is crucial for permeability increases, PAF (10 nM)-induced changes in endothelial [Ca(2+)](i) and NO production were measured in individually perfused rat mesenteric venular microvessels via fluorescence microscopy. When venular microvessels were exposed to PAF, endothelial [Ca(2+)](i) increased from 69 +/- 8 nM to a peak value of 374 +/- 26 nM within 3 min and then declined to a sustained level at 190 +/- 12 nM after 15 min. Inhibition of NOS did not modify PAF-induced increases in endothelial [Ca(2+)](i). PAF-induced NO production was visualized and quantified at cellular levels in individually perfused microvessels using 4,5-diaminofluorescein diacetate and fluorescence imaging. Increased fluorescence intensity (FI), which is an indication of increased NO production, occurred in 75 +/- 7% of endothelial cells in each vessel. The mean maximum FI increase was 140 +/- 7% of baseline value. This increased FI was abolished by pretreatment of the vessel with l-NMMA and attenuated in the absence of extracellular Ca(2+). These results provide direct evidence from intact microvessels that increased endothelial [Ca(2+)](i) is the initial signal that activates endothelial NOS, and the subsequent increased NO production contributes to PAF-induced increases in microvessel permeability.     

fMLP-stimulated release of reactive oxygen species from adherent leukocytes increases microvessel permeability

Our previous study (Am J Physiol Heart Circ Physiol 288: H1331-H1338, 2005) demonstrated that TNF-alpha induced significant leukocyte adhesion without causing increases in microvessel permeability, and that formyl-Met-Leu-Phe-OH (fMLP)-stimulated neutrophils in the absence of adhesion increased microvessel permeability via released reactive oxygen species (ROS). The objective of our present study is to investigate the mechanisms that regulate neutrophil respiratory burst and the roles of fMLP-stimulated ROS release from adherent leukocytes in microvessel permeability. A technique that combines single-microvessel perfusion with autologous blood perfusion was employed in venular microvessels of rat mesenteries. Leukocyte adhesion was induced by systemic application of TNF-alpha. Microvessel permeability was assessed by measuring hydraulic conductivity (L(p)). The 2-h autologous blood perfusion after TNF-alpha application increased leukocyte adhesion from 1.2 +/- 0.2 to 13.3 +/- 1.6 per 100 microm of vessel length without causing increases in L(p). When fMLP (10 microM) was applied to either perfusate (n = 5) or superfusate (n = 8) in the presence of adherent leukocytes, L(p) transiently increased to 4.9 +/- 0.9 and 4.4 +/- 0.3 times the control value, respectively. Application of superoxide dismutase or an iron chelator, deferoxamine mesylate, after fMLP application prevented or attenuated the L(p) increase. Chemiluminescence measurements in isolated neutrophils demonstrated that TNF-alpha alone did not induce ROS release but that preexposure of neutrophils to TNF-alpha in vivo or in vitro potentiated fMLP-stimulated ROS release. These results suggest a priming role of TNF-alpha in fMLP-stimulated neutrophil respiratory burst and indicate that the released ROS play a key role in leukocyte-mediated permeability increases during acute inflammation.     

Internalization of caveolin-1 scaffolding domain facilitated by Antennapedia homeodomain attenuates PAF-induced increase in microvessel permeability

We demonstrated previously that inhibition of endothelial nitric oxide synthase (NOS), using pharmacological inhibitors, attenuated the ionomycin- and ATP-induced increases in microvessel permeability (Am J Physiol Heart Circ Physiol 272: H176-H185, 1997). Recently, the scaffolding domain of caveolin-1 (CAV) has been implicated as a negative regulator of endothelial NOS (eNOS). To examine the role of CAV-eNOS interaction in regulation of permeability in intact microvessels, the effect of internalized CAV on the platelet-activating factor (PAF)-induced permeability increase was investigated in rat mesenteric venular microvessels. Internalization of CAV was achieved by perfusion of individual vessels using a fusion peptide of CAV with Antennapedia homeodomain (AP-CAV) and visualized by fluorescence imaging and electron microscopy. Changes in microvessel permeability were evaluated by measuring hydraulic conductivity (Lp) in individually perfused microvessels. We found that the PAF (10 nM)-induced Lp increase was significantly attenuated from 6.0 +/- 0.9 (n = 7) to 2.0 +/- 0.3 (n = 5) times control after microvessels were perfused with 10 microM AP-CAV for 2 h. The magnitude of this reduction is comparable with that of the inhibitory effect of Nomega-monomethyl-l-arginine on the PAF-induced Lp increase. In contrast, perfusion with 10 microM AP alone for 2 h modified neither basal Lp nor the vessel response to PAF. These results indicate that CAV plays an important role in regulation of microvessel permeability. The inhibitory action of CAV on permeability increase might be attributed to its direct inactivation of eNOS. In addition, this study established a method for studying protein-protein interaction-induced functional changes in intact microvessels and demonstrated AP as an efficient vector for translocation of peptide across the cell membrane in vivo.     

Epac/Rap1 pathway regulates microvascular hyperpermeability induced by PAF in rat mesentery

Experiments in cultured endothelial cell monolayers demonstrate that increased intracellular cAMP strongly inhibits the acute permeability responses by both protein kinase A (PKA)-dependent and -independent pathways. The contribution of the PKA-independent pathways to the anti-inflammatory mechanisms of cAMP in intact mammalian microvessels has not been systematically investigated. We evaluated the role of the cAMP-dependent activation of the exchange protein activated by cAMP (Epac), a guanine nucleotide exchange factor for the small GTPase Rap1, in rat venular microvessels exposed to the platelet-activating factor (PAF). The cAMP analog 8-pCPT-2'-O-methyl-cAMP (O-Me-cAMP), which stimulates the Epac/Rap1 pathway but has no effect on PKA, significantly attenuated the PAF increase in microvessel permeability as measured by hydraulic conductivity (Lp). We also demonstrated that PAF induced a rearrangement of vascular endothelial (VE)-cadherin seen as numerous lateral spikes and frequent short breaks in the otherwise continuous peripheral immunofluorescent label. Pretreatment with O-Me-cAMP completely prevented the PAF-induced rearrangement of VE-cadherin. We conclude that the action of the Epac/Rap1 pathway to stabilize cell-cell adhesion is a significant component of the activity of cAMP to attenuate an acute increase in vascular permeability. Our results indicate that increased permeability in intact microvessels by acute inflammatory agents such as PAF is the result of the decreased effectiveness of the Epac/Rap1 pathway modulation of cell-cell adhesion.     

Leukocyte-platelet aggregate adhesion and vascular permeability in intact microvessels: role of activated endothelial cells

Leukocyte-platelet aggregation and aggregate adhesion have been indicated as biomarkers of the severity of tissue injury during inflammation or ischemic reperfusion. The objective of this study is to investigate the mechanisms of the aggregate adhesion and quantitatively evaluate its relationship with microvessel permeability. A combined autologous blood perfusion with single microvessel perfusion technique was employed in rat mesenteric venular microvessels. The aggregate adhesion was induced by systemic application of TNF-alpha plus local application of platelet-activating factor (PAF). Changes in permeability were determined by measurements of hydraulic conductivity (Lp) before and after aggregate adhesion in the same individually perfused microvessels. The compositions of the adherent aggregates were identified with fluorescent labeling and confocal imaging. In contrast to leukocyte adhesion as single cells resulting in no increase in microvessel permeability, aggregate adhesion induced prolonged increases in microvessel Lp (6.1 +/- 0.9 times the control, n = 9) indicated by the initial Lp measurements after 3 h of blood perfusion, which is distinct from the transient Lp increase caused by PAF-induced endothelial activation in the absence of blood. Isoproteronol (Iso) attenuated aggregate adhesion-mediated Lp increases if applied after autologous blood perfusion and prevented the aggregate adhesion if the initial endothelial activation is inhibited by applying Iso before PAF administration but showed less effect on single leukocyte adhesion. This study demonstrated that leukocyte-platelet aggregate adhesion via a mechanism different from that of single leukocyte adhesion caused a prolonged increase in microvessel permeability. Our results also indicate that the initial activation of endothelial cells by PAF plays a crucial role in the initiation of leukocyte-platelet aggregate adhesion.     

Modulation of venular microvessel permeability by calcium influx into endothelial cells.

It has been proposed that calcium ion influx into endothelial cells modulates the permeability of venular microvessels via a calcium-dependent contractile process. The results of recent investigations using permeabilized endothelial cell monolayers conform to this hypothesis by demonstrating a calcium-dependent interaction of endothelial actin and myosin during the retraction of adjacent endothelial cells exposed to inflammatory agents. Little is known about the pathway for calcium influx into endothelial cells after exposure to mediators of inflammation, but evidence suggests that the properties of the calcium entry pathways are similar to the calcium entry pathways that regulate the release of endothelium-derived relaxing factor (EDRF). Substances that stimulate EDRF release from arterial endothelium also increase venular microvessel permeability. Recently developed methods to measure cytoplasmic calcium concentration in the endothelial cells forming the walls of individually perfused microvessels enable a direct investigation of the modulation of the permeability of venular microvessels by calcium influx. These experiments demonstrate that the magnitude of the initial increase in the permeability of microvessels after exposure to an agent that increases permeability, such as a calcium ionophore, is determined by the magnitude of calcium ion influx into the endothelial cells. Furthermore, the magnitude of the calcium influx into endothelial cells is modulated by the membrane potential of the endothelial cells. Depolarization of the endothelial cell membrane reduces calcium influx and attenuates increases in permeability whereas hyperpolarization of the endothelial membrane increases calcium influx and potentiates increases in permeability. These data conform to the hypothesis that a passive conductance channel for calcium is a major pathway for calcium ion flux responsible to eliciting an increase in the permeability of the endothelial barrier in microvessels.     

Structural mechanisms in the abolishment of VEGF-induced microvascular hyperpermeability by cAMP

To investigate the structural mechanisms by which elevation of the intraendothelial cAMP levels abolishes or attenuates the transient increase in microvascular permeability by vascular endothelial growth factor (VEGF), we examined cAMP effect on VEGF-induced hyperpermeability to small solute sodium fluorescein (Stokes radius = 0.45 nm) P(sodium fluorescein), intermediate-sized solute alpha-lactalbumin (Stokes radius = 2.01 nm) P(alpha-lactalbumin), and large solute albumin (BSA, Stokes radius = 3.5 nm) P(BSA) on individually perfused microvessels of frog mesenteries. After 20 min pretreatment of 2 mM cAMP analog, 8-bromo-cAMP, the initial increase by 1 nM VEGF was completely abolished in P(sodium fluorescein) (from a peak increase of 2.6+/-0.37 times control with VEGF alone to 0.96+/-0.07 times control with VEGF and cAMP), in P(alpha-lactalbumin) (from a peak increase of 2.7+/-0.33 times control with VEGF alone to 0.76+/-0.07 times control with VEGF and cAMP), and in P(BSA) (from a peak increase of 6.5+/-1.0 times control with VEGF alone to 0.97+/-0.08 times control with VEGF and cAMP). Based on these measured data, the prediction from our mathematical models suggested that the increase in the number of tight junction strands in the cleft between endothelial cells forming the microvessel wall is one of the mechanisms for the abolishment of VEGF-induced hyperpermeability by cAMP.     

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular mechanisms regulating vascular permeability in cultured endothelial cell monolayers and the functional exchange properties of whole microvascular beds. A method to cannulate and perfuse venular microvessels of rat mesentery and measure the hydraulic conductivity of the microvessel wall is described. The main equipment needed includes an intravital microscope with a large modified stage that supports micromanipulators to position three different microtools: (1) a beveled glass micropipette to cannulate and perfuse the microvessel; (2) a glass micro-occluder to transiently block perfusion and enable measurement of transvascular water flow movement at a measured hydrostatic pressure, and (3) a blunt glass rod to stabilize the mesenteric tissue at the site of cannulation. The modified Landis micro-occlusion technique uses red cells suspended in the artificial perfusate as markers of transvascular fluid movement, and also enables repeated measurements of these flows as experimental conditions are changed and hydrostatic and colloid osmotic pressure difference across the microvessels are carefully controlled. Measurements of hydraulic conductivity first using a control perfusate, then after re-cannulation of the same microvessel with the test perfusates enable paired comparisons of the microvessel response under these well-controlled conditions. Attempts to extend the method to microvessels in the mesentery of mice with genetic modifications expected to modify vascular permeability were severely limited because of the absence of long straight and unbranched microvessels in the mouse mesentery, but the recent availability of the rats with similar genetic modifications using the CRISPR/Cas9 technology is expected to open new areas of investigation where the methods described herein can be applied.     

NO increases permeability of cultured human cervical epithelia by cGMP-mediated increase in G-actin

Human cervicalepithelial cells express mRNA for the nitric oxide (NO) synthase (NOS)isoforms ecNOS, bNOS, and iNOS and release NO into the extracellularmedium. N^G-nitro-L-arginine methylester (L-NAME), an NOS inhibitor, and Hb, an NO scavenger,decreased paracellular permeability; in contrast, the NO donors sodiumnitroprusside (SNP) andN-(ethoxycarbonyl)-3-(4-morpholinyl)sydnonimine increasedparacellular permeability across cultured human cervical epithelia onfilters, suggesting that NO increases cervical paracellular permeability. The objective of the study was to understand the mechanisms of NO action on cervical paracellular permeability. 8-Bromo-cGMP (8-BrcGMP) also increased permeability, and the effect wasblocked by KT-5823 (a blocker of cGMP-dependent protein kinase), butnot by LY-83583 (a blocker of guanylate cyclase). In contrast, LY-83583and KT-5823 blocked the SNP-induced increase in permeability. Treatmentwith SNP increased cellular cGMP, and the effect was blocked by Hb andLY-83583, but not by KT-5823. Neither SNP nor 8-BrcGMP had modulatedcervical cation selectivity. In contrast, both agents increasedfluorescence from fura 2-loaded cells in theCa²⁺-insensitive wavelengths, indicating that SNP and8-BrcGMP stimulate a decrease in cell size and in the resistance of thelateral intercellular space. Neither SNP nor 8-BrcGMP had an effect ontotal cellular actin, but both agents increased the fraction ofG-actin. Hb blocked the SNP-induced increase in G-actin, and KT-5823blocked the 8-BrcGMP-induced increase in G-actin. On the basis of theseresults, it is suggested that NO acts on guanylate cyclase andstimulates an increase in cGMP; cGMP, acting via cGMP-dependent proteinkinase, shifts actin steady-state toward G-actin; this fragments thecytoskeleton and renders cells more sensitive to decreases in cell sizeand resistance of the lateral intercellular space and, hence, toincreases in permeability. These results may be important forunderstanding NO regulation of transcervical paracellular permeabilityand secretion of cervical mucus in the woman.

     

fMLP-stimulated neutrophils increase endothelial [Ca2+]i and microvessel permeability in the absence of adhesion: role of reactive oxygen species

Our previous study demonstrated that firm attachment of leukocytes to microvessel walls does not necessarily increase microvessel permeability (Am J Physiol Heart Circ Physiol 283: H2420-H2430, 2002). To further understand the mechanisms of the permeability increase associated with leukocyte accumulation during acute inflammation, we investigated the direct relation of reactive oxygen species (ROS) release during neutrophil respiratory burst to changes in microvessel permeability and endothelial intracellular Ca(2+) concentration ([Ca(2+)](i)) in intact microvessels. ROS release from activated neutrophils was quantified by measuring changes in chemiluminescence. When isolated rat neutrophils (2 x 10(6)/ml) were exposed to formyl-Met-Leu-Phe-OH (fMLP, 10 microM), chemiluminescence transiently increased from 1.2 +/- 0.2 x 10(4) to a peak value of 6.7 +/- 1.0 x 10(4) cpm/min (n = 12). Correlatively, perfusing individual microvessels with fMLP-stimulated neutrophils in suspension (2 x 10(7)/ml) increased hydraulic conductivity (L(p)) to 3.7 +/- 0.4 times the control value (n = 5) and increased endothelial [Ca(2+)](i) from 84 +/- 7 nM to a mean peak value of 170 +/- 7 nM. In contrast, perfusing vessels with fMLP alone did not affect basal L(p). Application of antioxidant agents, superoxide dismutase, vitamin C, or an iron chelator, deferoxamine mesylate, attenuated ROS release in fMLP-stimulated neutrophils and abolished increases in L(p). These results indicate that release of ROS from fMLP-stimulated neutrophils increases microvessel permeability and endothelial [Ca(2+)](i) independently from leukocyte adhesion and the migration process.     

Role of adhesion and contraction in Rac 1-regulated endothelial barrier function in vivo and in vitro

We demonstrated previously that inhibition of the small GTPase Rac-1 by Clostridium sordellii lethal toxin (LT) increased the hydraulic conductivity (L(p)) of rat venular microvessels and induced gap formation in cultured myocardial endothelial cells (MyEnd). In MyEnd cells, we also demonstrated that both LT and cytochalasin D reduced cellular adhesion of vascular endothelial (VE)-cadherin-coated beads. Here we further evaluate the contribution of actin depolymerization, myosin-based contraction, and VE-cadherin linkage to the actin cytoskeleton to LT-induced permeability. The actin-depolymerizing agent cytochalasin D increased L(p) in single rat mesenteric microvessels to the same extent as LT over 80 min. However, whereas the actin-stabilizing agent jasplakinolide blunted the L(p) increase due to cytochalasin D by 78%, it had no effect on the LT response. This conforms to the hypothesis that the predominant mechanism whereby Rac-1 stabilizes the endothelial barrier in intact microvessels is separate from actin polymerization and likely at the level of the VE-cadherin linkage to the actin cytoskeleton. In intact vessels, neither inhibition of contraction (butanedione monoxime, an inhibitor of myosin ATPase) nor inhibition of Rho kinase (Y-27632) modified the response to LT, even though both inhibitors lowered resting L(p). In contrast butanedione monoxime and inhibition of myosin light chain kinase completely inhibited LT-induced intercellular gap formation and largely reduced the LT-induced permeability increase in MyEnd monolayers. These results support the hypothesis that the contractile mechanisms that contribute to the formation of large gaps between cultured endothelial cells exposed to inflammatory conditions do not significantly contribute to increased permeability in intact microvessels.     

An electrodiffusion-filtration model for effects of endothelial surface glycocalyx on microvessel permeability to macromolecules

Endothelial surface glycocalyx plays an important role in the regulation of microvessel permeability by possibly changing its charge and configuration. To investigate the mechanisms by which surface properties of the endothelial cells control the changes in microvessel permeability, we extended the electrodiffusion model developed by Fu et al. [Am. J. Physiol. 284, H1240-1250 (2003)], which is for the interendothelial cleft with a negatively charged surface glycocalyx layer, to include the filtration due to hydrostatic and oncotic pressures across the microvessel wall as well as the electrical potential across the glycocalyx layer On the basis of the hypotheses proposed by Curry [Microcirculation 1(1): 11-26 (1994)], the predictions from this electrodiffusion-filtration model provide a good agreement with experimental data for permeability of negatively charged a-lactalbumin summarized in Curry [Microcirculation 1(1), 11-26 (1994)] under various conditions. In addition, we applied this new model to describe the transport of negatively charged macromolecules, bovine serum albumin (BSA), across venular microvessels in frog mesentery. According to the model, the convective component of the albumin transport is greatly diminished by the presence of a negatively charged glycocalyx under both normal and increased permeability conditions.     

Three-dimensional localization and quantification of PAF-induced gap formation in intact venular microvessels

Combining single-vessel perfusion technique with confocal microscopy, this study presents a new approach that allows three-dimensional visualization and quantification of endothelial gaps under experimental conditions identical to those used to measure permeability coefficients, endothelial calcium concentration, and nitric oxide production in individually perfused intact microvessels. This approach provides an efficient means for defining the transport pathways and cellular mechanisms of increased microvascular permeability during inflammation. Platelet-activating factor (PAF) was used to increase the permeability of individually perfused rat mesenteric venules. Fluorescent microspheres (FMs, 100 nm) were used as leakage markers, and confocal images were acquired at successive focal planes through the perfused microvessel. Perfusion of FMs under control conditions produced a thin, uniform layer of FMs in the vessel lumen, but in PAF-stimulated microvessels significant amounts of FMs accumulated at endothelial junctions. Reconstructed confocal images three-dimensionally delineated the temporal and spatial development of endothelial gaps in PAF-stimulated microvessels. The FM accumulation, quantified as the total fluorescence intensity per square micrometer of vessel wall, was 8.4 +/- 1.8 times the control value within 10 min of PAF perfusion and declined to 5.0 +/- 0.6 and 1.4 +/- 0.2 times the control value when FMs were applied 30 and 60 min after PAF perfusion. The changes in the magnitude of FM accumulation closely correlated with the time course of PAF-induced increases in hydraulic conductivity (L(p)), indicating that the opening and closing of endothelial gaps contributed to the transient increase in L(p) in PAF-stimulated microvessels. Electron microscopic evaluations confirmed PAF-induced gap formation and FM accumulation at endothelial clefts.     

Atrial natriuretic factor stimulates the frog urinary bladder osmotic permeability in presence of a cyclic nucleotide phosphodiesterase inhibitor

The role of atrial natriuretic factor (ANF) in regulation of osmotic water permeability was studied in isolated frog Rana temporaria L. urinary bladder. It was found that ANF (rANF, 1-28) added to the serosal solution at concentrations 5 x 10(-8) M and higher dosedependently stimulated the arginine-vasotocin (AVT)-induced increase of osmotic water permeability. The effect of ANF was revealed only in presence of 3-isobuthyl-1-methylxantine (180 microM) and was accompanied by significant elevation of cGMP level in urinary bladder homogenate and isolated mucosal epithelial cells. C-ANF (des[Gln18, Ser19, Gly20, Leu21, Gly22]-ANF-(4-23)-NH2), a specific agonist of NPR-C receptor, exerted no effect on osmotic water permeability. ANF induced a significant increase of cAMP in urinary bladder homogenates (AVT, 5 x 10(-11) M: 52.3 +/- 10.6; AVT + ANF, 10(-7) M: 114.2 +/- 26.9 pmol/mg protein, n = 5, p < 0.05). The activity of adenylate cyclase in crude plasmatic membrane fraction was not changed. Milrinone, a specific inhibitor of phosphodiesterase 3, at concentrations from 25 to 80 microM, enhanced both the hydroosmotic response to AVT and AVT-stimulated cAMP production. Altogether these data demonstrate that, in the frog urinary bladder, ANF stimulates the AVT-induced increase of osmotic water permeability acting probably through NPR-A receptor-coupled mobilization of cGMP and cGMP-dependent inhibition of phosphodiesterase 3.     

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.     

Endothelin-1 reduces mesenteric microvascular hydraulic permeability via cyclic AMP and protein kinase A signal transduction

We have previously shown that endothelin-1 (ET-1) decreases microvascular hydraulic permeability. In this study, we tested the hypothesis that ET-1 exerts its permeability-decreasing effect through cAMP, cGMP, and protein kinase A (PKA) by determining the effect of ET-1 on venular fluid leak during inhibition of cAMP synthesis, inhibition of cGMP degredation, and inhibition of PKA. Rat mesenteric venules were cannulated to measure hydraulic permeability, L(p) (units x 10(-7)cm/(s cmH(2)O)). L(p) was measured during continuous perfusion of 80 pM ET-1 and either (1) an inhibitor of cAMP synthesis (10 microM 2',5'ddA), (2) an inhibitor of cGMP degradation (100 microM Zaprinast), or (3) an inhibitor of PKA (10 microM H-89). Inhibition of cAMP synthesis blocked the permeability decreasing effects of ET-1. The peak L(p) of the cAMP inhibitor alone and with ET-1 was 4.11+/-0.53 and 3.86+/-0.19, respectively (p=0.36, n=6). Inhibition of cGMP degradation did not block the permeability decreasing effects of ET-1. The peak L(p) during inhibition of cGMP degradation alone and with ET-1 was 2.26+/-0.15 and 1.44+/-0.09, respectively (p<0.001, n=6). Inhibition of PKA activation blocked the permeability decreasing effects of ET-1. The peak L(p) of the PKA inhibitor alone and with ET-1 was 2.70+/-0.15 and 2.59+/-0.15, respectively (p=0.38, n=6). The data support the notion that the signal transduction mechanism of ET-1 with regard to decreasing microvascular fluid leak involves cAMP production and PKA activation, but not cGMP degradation. Further understanding of intracellular mechanisms that control microvascular fluid leak could lead to the development of a pharmacologic therapy to control third space fluid loss in severely injured or septic patients.     

Determination of microvessel permeability and tissue diffusion coefficient of solutes by laser scanning confocal microscopy

Interstitium contains a matrix of fibrous molecules that creates considerable resistance to water and solutes in series with the microvessel wall. On the basis of our preliminary studies, by using laser-scanning confocal microscopy and a theoretical model for interstitial transport, we determined both microvessel solute permeability (P) and solute tissue diffusion coefficient (D) of alpha-lactalbumin (Stokes radius 2.01 nm) from the rate of tissue solute accumulation and the radial concentration gradient around individually perfused microvessel in frog mesentery. P(alpha-lactalbumin) is 1.7 +/- 0.7(SD) x 10(-6) cm/s (n = 6). D(t)/D(free) for alpha-lactalbumin is 27% +/- 5% (SD) (n = 6). This value of D(t)/D(free) is comparable to that for small solute sodium fluorescein (Stokes radius 0.45 nm), while p(alpha-lactalbumin) is only 3.4% of p(sodium fluorescein). Our results suggest that frog mesenteric tissue is much less selective to solutes than the microvessel wall.     

Involvement of cGMP-dependent protein kinase in the relaxation of ovine pulmonary arteries to cGMP and cAMP.

Agonist-induced smooth muscle relaxation occurs following an increase in intracellular concentrations of cGMP or cAMP. However, the role of protein kinase G (PKG) and/or protein kinase A (PKA) in cGMP- or cAMP-mediated pulmonary vasodilation is not clearly elucidated. In this study, we examined the relaxation responses of isolated pulmonary arteries of lambs (age = 10 +/- 1 days), preconstricted with endothelin-1, to increasing concentrations of 8-bromo-cGMP (8-BrcGMP) or 8-BrcAMP (cell-permeable analogs), in the presence or absence of Rp-8-beta-phenyl-1,N(2)-etheno-bromoguanosine cyclic monosphordthioate (Rp-8-PET-BrcGMPS) or KT-5720, selective inhibitors of PKG and PKA, respectively. When examined for specificity, Rp-8-Br-PET-cGMPS abolished PKG, but not PKA, activity in pulmonary arterial extracts, whereas KT-5720 inhibited PKA activity only. 8-BrcGMP-induced relaxation was inhibited by the PKG inhibitor only, whereas 8-BrcAMP-induced relaxation was inhibited by both inhibitors. A nearly fourfold higher concentration of cAMP than cGMP was required to relax arteries by 50% and to activate PKG by 50%. Our results demonstrate that relaxation of pulmonary arteries is more sensitive to cGMP than cAMP and that PKG plays an important role in both cGMP- and cAMP-mediated relaxation.