Ang-1: Tie-ing up endothelial adhesion?
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Angiopoietin-1↗
Biomedical subjects
F E Curry
Publications and source records attributed to F E Curry.
Cytosolic Ca2+ concentration and rate of increase of the cytosolic Ca2+ concentration in the regulation of vascular permeability in Rana in vivo.
Vascular permeability is assumed to be regulated by the cytosolic Ca(2+) concentration ([Ca(2+)](c)) of the endothelial cells. When permeability is increased, however, the maximum [Ca(2+)](c) appears to occur after the maximum permeability increase, suggesting that Ca(2+)-dependent mechanisms other than the absolute Ca(2+) concentration may regulate permeability. Here we investigate whether the rate of increase of the [Ca(2+)](c) (d[Ca(2+)](c)/dt) may more closely approximate the time course of the permeability increase. Hydraulic conductivity (L(p)) and endothelial [Ca(2+)](c) were measured in single perfused frog mesenteric microvessels in vivo. The relationships between the time courses of the increased L(p), [Ca(2+)](c) and d[Ca(2+)](c)/dt were examined. L(p) peaked significantly earlier than [Ca(2+)](c) in all drug treatments examined (Ca(2+) store release, store-mediated Ca(2+) influx, and store-independent Ca(2+) influx). When L(p) was increased in a store-dependent manner the time taken for L(p) to peak (3.6 +/- 0.9 min during store release, 1.2 +/- 0.3 min during store-mediated Ca(2+) influx) was significantly less than the time taken for [Ca(2+)](c) to peak (9.2 +/- 2.8 min during store release, 2.1 +/- 0.7 min during store-mediated influx), but very similar to that for the peak d[Ca(2+)](c)/dt to occur (4.3 +/- 2.0 min during store release, 1.1 +/- 0.5 min during Ca(2+) influx). Additionally, when the increase was independent of intracellular Ca(2+) stores, L(p) (0.38 +/- 0.03 min) and d[Ca(2+)](c)/dt (0.30 +/- 0.1 min) both peaked significantly before the [Ca(2+)](c) (1.05 +/- 0.31 min). These data suggest that the regulation of vascular permeability by endothelial cell Ca(2+) may be regulated by the rate of change of the [Ca(2+)](c) rather than the global [Ca(2+)].
Animals↗
Clostridium perfringens epsilon-toxin increases permeability of single perfused microvessels of rat mesentery.
Epsilon-toxin, the primary virulence factor of Clostridium perfringens type D, causes mortality in livestock, particularly sheep and goats, in which it induces an often-fatal enterotoxemia. It is believed to compromise the intestinal barrier and then enter the gut vasculature, from which it is carried systemically, causing widespread vascular endothelial damage and edema. Here we used single perfused venular microvessels in rat mesentery, which enabled direct observation of permeability properties of the in situ vascular wall during exposure to toxin. We determined the hydraulic conductivity (L(p)) of microvessels as a measure of the response to epsilon-toxin. We found that microvessels were highly sensitive to toxin. At 10 microg ml(-1) the L(p) increased irreversibly to more than 15 times the control value by 10 min. At 0.3 microg ml(-1) no increase in L(p) was observed for up to 90 min. The toxin-induced increase in L(p) was consistent with changes in ultrastructure of microvessels exposed to the toxin. Those microvessels exhibited gaps either between or through endothelial cells where perfusate had direct access to the basement membrane. Many endothelial cells appeared necrotic, highly attenuated, and with dense cytoplasm. We showed that epsilon-toxin, in a time- and dose-dependent manner, rapidly and irreversibly compromised the barrier function of venular microvessel endothelium. The results conformed to the hypothesis that epsilon-toxin interacts with vascular endothelial cells and increases the vessel wall permeability by direct damage of the endothelium.
Animals↗
Regulation of actin dynamics is critical for endothelial barrier functions.
We tested the hypothesis that the equilibrium between F- and G-actin in endothelial cells modulates the integrity of the actin cytoskeleton and is important for the maintenance of endothelial barrier functions in vivo and in vitro. We used the actin-depolymerizing agent cytochalasin D and jasplakinolide, an actin filament (F-actin) stabilizing and promoting substance, to modulate the actin cytoskeleton. Low doses of jasplakinolide (0.1 microM), which we have previously shown to reduce the permeability-increasing effect of cytochalasin D, had no influence on resting permeability of single-perfused mesenteric microvessels in vivo as well as on monolayer integrity. The F-actin content of cultured endothelial cells remained unchanged. In contrast, higher doses (10 microM) of jasplakinolide increased permeability (hydraulic conductivity) to the same extent as cytochalasin D and induced formation of intercellular gaps in cultured myocardial endothelial (MyEnd) cell monolayers. This was accompanied by a 34% increase of F-actin and pronounced disorganization of the actin cytoskeleton in MyEnd cells. Furthermore, we tested whether an increase of cAMP by forskolin and rolipram would prevent the cytochalasin D-induced barrier breakdown. Conditions that increase intracellular cAMP failed to block the cytochalasin D-induced permeability increase in vivo and the reduction of vascular endothelial cadherin-mediated adhesion in vitro. Taken together, these data support the hypothesis that the state of polymerization of the actin cytoskeleton is critical for maintenance of endothelial barrier functions and that both depolymerization by cytochalasin D and hyperpolymerization of actin by jasplakinolide resulted in an increase of microvessel permeability in vivo. However, cAMP, which is known to support endothelial barrier functions, seems to work by mechanisms other than stabilizing F-actin.
Actin Cytoskeleton↗
cAMP protects endothelial barrier functions by preventing Rac-1 inhibition.
cAMP enhances endothelial barrier properties and is protective against various inflammatory mediators both in vivo and in vitro. However, the mechanisms whereby cAMP stabilizes the endothelial barrier are largely unknown. Recently we demonstrated that the Rho family GTPase Rac-1 is required for maintenance of endothelial barrier functions in vivo and in vitro. Therefore, in the present study we investigated the effect of forskolin (5 microM)- and rolipram (10 microM)-induced cAMP increase on reduction of barrier functions in response to Rac-1 inhibition by Clostridium sordellii lethal toxin (LT). Forskolin and rolipram treatment blocked LT (200 ng/ml)-induced hydraulic conductivity (Lp) increase in mesenteric microvessels in vivo. Likewise, LT-induced intercellular gap formation in monolayers of cultured microvascular myocardial endothelial (MyEnd) cells and LT-induced loss of adhesion of vascular endothelial cadherin-coated microbeads were abolished. Inhibition of PKA by myristoylated inhibitor peptide (14-22) of PKA (100 microM) reduced the protective effect of cAMP on LT-induced Lp increase in vivo and gap formation in vitro, indicating that the effect of cAMP on Rac-1 inhibition was PKA dependent. Glucosylation assays demonstrated that cAMP prevents inhibitory Rac-1 glucosylation by LT, indicating that one way that cAMP enhances endothelial barrier functions may be by regulating Rac-1 signaling. Our study suggests that cAMP may provide its well-established protective effects at least in part by regulation of Rho proteins.
Animals↗
Oncotic pressures opposing filtration across non-fenestrated rat microvessels.
We hypothesized that ultrafiltrate crossing the luminal endothelial glycocalyx through infrequent discontinuities (gaps) in the tight junction (TJ) strand of endothelial clefts reduces albumin diffusive flux from tissue into the 'protected region' of the cleft on the luminal side of the TJ. Thus, the effective oncotic pressure difference (sigma black triangle down pi) opposing filtration is greater than that measured between lumen and interstitial fluid. To test this we measured sigma black triangle down pi across rat mesenteric microvessels perfused with albumin (50 mg ml(-1)) with and without interstitial albumin at the same concentration within a few micrometres of the endothelium as demonstrated by confocal microscopy. We found sigma black triangle down pi was near 70% of luminal oncotic pressure when the tissue concentration equalled that in the lumen. We determined size and frequency of TJ strand gaps in endothelial clefts using serial section electron microscopy. We found nine gaps in the reconstructed clefts having mean spacing of 3.59 microm and mean length of 315 nm. The mean depth of the TJ strand near gaps was 67 nm and the mean cleft path length from lumen to interstitium was 411 nm. With these parameters our three-dimensional hydrodynamic model confirmed that fluid velocity was high at gaps in the TJ strand so that even at relatively low hydraulic pressures the albumin concentration on the tissue side of the glycocalyx was significantly lower than in the interstitium. The results conform to the hypothesis that colloid osmotic forces opposing filtration across non-fenestrated continuous capillaries are developed across the endothelial glycocalyx and that the oncotic pressure of interstitial fluid does not directly determine fluid balance across microvascular endothelium.
Albumins↗
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.
Actins↗
Requirement of Rac activity for maintenance of capillary endothelial barrier properties.
Our previous experiments indicated that GTPases, other than RhoA, are important for the maintenance of endothelial barrier integrity in both intact microvessels of rats and mice and cultured mouse myocardial endothelial (MyEnd) cell monolayers. In the present study, we inhibited the endothelial GTPase Rac by Clostridium sordellii lethal toxin (LT) and investigated the relation between the degree of inhibition of Rac by glucosylation and increased endothelial barrier permeability. In rat venular microvessels, LT (200 ng/ml) increased hydraulic conductivity from a control value of 2.5 +/- 0.6 to 100.8 +/- 18.7 x 10-7 cm x s(-1) x cm H2O(-1) after 80 min. In cultured MyEnd cells exposed to LT (200 ng/ml), up to 60% of cellular Rac was glucosylated after 90 min, resulting in depolymerization of F-actin and interruptions of junctional distribution of vascular endothelial cadherin (VE-cadherin) and beta-catenin as well as the formation of intercellular gaps. To understand the mechanism by which inhibition of Rac caused disassembly of adherens junctions, we used laser tweezers to quantify VE-cadherin-mediated adhesion. LT and cytochalasin D, an actin depolymerizing agent, both reduced adhesion of VE-cadherin-coated microbeads to the endothelial cell surface, whereas the inhibitor of Rho kinase Y-27632 did not. Stabilization of actin filaments by jasplakinolide completely blocked the effect of cytochalasin D but not of LT on bead adhesion. We conclude that Rac regulates endothelial barrier properties in vivo and in vitro by 1) modulation of actin filament polymerization and 2) acting directly on the tether between VE-cadherin and the cytoskeleton.
Actins↗
Thrombin increases permeability only in venules exposed to inflammatory conditions.
Thrombin is widely used to stimulate a variety of responses in cultured endothelial cell monolayers as a model of acute vascular endothelial response to inflammatory mediators. However, preliminary results indicated that rat mesenteric venules did not respond acutely to thrombin. We tested the hypothesis that rat venules would respond to thrombin 24 h after prior injury by microperfusion. Vessel responsiveness was measured as hydraulic conductivity (Lp). When venules were exposed to rat thrombin (10 U/ml) within 2 h of initial perfusion with vehicle control, there was no increase in Lp of any vessel from a mean baseline of 1.2 +/- 0.2 x 10(-7) cm.s-1.cmH2O-1. In contrast, when perfused with thrombin at 25-27 h after initial perfusion, every venule responded to thrombin with a transient increase in Lp. The mean peak Lp on day 2 in response to thrombin was 24 +/- 4.2 x 10(-7) cm.s-1.cmH2O-1. Our results suggest that prior endothelial injury modifies the endothelial cell phenotype and alters the response of endothelial cells to thrombin after 24 h. Phenotypic plasticity of endothelial cells may play a key role in the regulation of permeability of some endothelial cells in culture and in intact venules, where localized leaky sites may form where there had been a previous inflammatory response.
Animals↗
PAF- and bradykinin-induced hyperpermeability of rat venules is independent of actin-myosin contraction.
We tested the hypothesis that acutely induced hyperpermeability is dependent on actin-myosin contractility by using individually perfused mesentery venules of pentobarbital-anesthetized rats. Venule hydraulic conductivity (Lp) was measured to monitor hyperpermeability response to the platelet-activating factor (PAF) 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine or bradykinin. Perfusion with PAF (10 nM) induced a robust transient high Lp [24.3 +/- 1.7 x 10-7 cm/(s.cmH2O)] that peaked in 8.9 +/- 0.5 min and then returned toward control Lp [1.6 +/- 0.1 x 10-7 cm/(s.cmH2O)]. Reconstruction of venular segments with the use of transmission electron microscopy of serial sections confirmed that PAF induces paracellular inflammatory gaps. Specific inhibition of myosin light chain kinase (MLCK) with 1-10 microM 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML-7) failed to block the PAF Lp response or change the time-to-peak Lp. ML-7 reduced baseline Lp 50% at 40 min of pretreatment. ML-7 also increased the rate of recovery from PAF hyperpermeability measured as the decrease of half-time of recovery from 4.8 +/- 0.7 to 3.2 +/- 0.3 min. Inhibition of myosin ATPase with 5-20 mM 2,3-butanedione 2-monoxime also failed to alter the hyperpermeability response to PAF. Similar results were found using ML-7 to modulate responses. These experiments indicate that an actin-myosin contractile mechanism modulated by MLCK does not contribute significantly to the robust initial increase in permeability of rat venular microvessels exposed to two common inflammatory mediators. The results are consistent with paracellular gap formation by local release of endothelial-endothelial cell adhesion structures in the absence of contraction by the actin-myosin network.
Actins↗
Flow-dependent changes in microvascular permeability -- an important adaptive phenomenon.
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Adaptation, Physiological↗
Rho and rho kinase modulation of barrier properties: cultured endothelial cells and intact microvessels of rats and mice.
Previous experiments using cultured endothelial monolayers indicate that Rho-family small GTPases are involved in modulation of endothelial monolayer permeability by regulating assembly of the cellular actin filament scaffold, activity of myosin-based contractility and junctional distribution of the Ca2+-dependent endothelial cell adhesion molecule, VE-cadherin. We investigated these mechanisms using both cultured endothelial cells (from porcine pulmonary artery and mouse heart) and vascular endothelium in situ (mouse aorta, and individually perfused venular microvessels of mouse and rat mesentery). Exposure to Clostridium difficile toxin B (100 ng x ml(-1)) inactivated 50-90% of all endothelial Rho proteins within 60-90 min. This was accompanied by considerable reduction of actin filament stress fibres and junctional F-actin in cultured endothelial monolayers and in mouse aortic endothelium in situ. Also, VE-cadherin became discontinuous along endothelial junctions. Inhibition of Rho kinase with Y-27632 (30 microM) for 90-120 min induced F-actin reduction both in vitro and in situ but did not cause redistribution or reduction of VE-cadherin staining. Perfusion of microvessels with toxin B increased basal hydraulic permeability (L(p)) but did not attenuate the transient increase in L(p) of microvessels exposed to bradykinin. Perfusion of microvessels with Y-27632 (30 microM) for up to 100 min reduced basal L(p) but did not attenuate the permeability increase induced by platelet activating factor (PAF) or bradykinin. These results show that toxin B-mediated reduction of endothelial barrier properties is due to inactivation of small GTPases other than RhoA. Rho proteins as well as RhoA-mediated contractile mechanisms are not involved in bradykinin- or PAF-induced hyperpermeability of intact microvessels.
Acute-Phase Proteins↗
Mechanisms of heterogeneous endothelial cytoplasmic calcium increases in venular microvessels.
OBJECTIVE: Localized inflammatory leaky sites form at regions of the microvessel wall with the largest increase in endothelial cell cytoplasmic calcium concentration, [Ca(2+)](i). We investigated the mechanisms that modulate localized increases in [Ca(2+)](i) in individual endothelial cells of microvessels after exposure to ATP. METHODS: [Ca(2+)](i) was measured by using digital fluorescence microscopy and fura-2 in the endothelial cells forming the walls of individually perfused frog mesenteric microvessels. The spread of [Ca(2+)](i) from a localized mechanical stimulus was also measured. RESULTS: The peak [Ca(2+)](i) after ATP showed marked heterogeneity, ranging from 227 to 1469 nM from resting values of 69 +/- 5 nM. After depolarization with high-potassium solutions, the endothelial cells with the largest peak increase in [Ca(2+)](i) had the largest fractional reduction. Localized increases in [Ca(2+)](i) due to mechanical stimulus did not spread. CONCLUSION: The key mechanism regulating the heterogeneity in initial peak increase in [Ca(2+)](i) is a calcium-dependent process regulated by the calcium influx itself. One such mechanism, the calcium-dependent opening of additional potassium channels leading to membrane hyperpolarization and increased driving force for calcium entry through passive conductance pathways, accounts for a significant amount of the heterogeneity of [Ca(2+)](i) in our experiments. Further investigations of both localized calcium influx and membrane potentials in the endothelial cells of intact microvessels in both frog and mammals using the imaging methods developed for these investigations are needed to understand the formation of localized leaky sites in inflamed microvessels.
Adenosine Triphosphate↗
Vascular endothelial growth factor increases Rana vascular permeability and compliance by different signalling pathways.
Vascular endothelial growth factor (VEGF) chronically increases microvascular permeability, compliance and vessel diameter. To determine the signalling pathways by which VEGF exerts these effects, we investigated the role of Ca2+ influx and mitogen-activated protein kinase (MAPK) phosphorylation on the increase in hydraulic conductivity (Lp), diameter and compliance in mesenteric microvessels in the anaesthetised frog (Rana species). The VEGF-mediated chronically increased permeability was attenuated by co-perfusion of VEGF with 5 mM NiCl2, previously shown to inhibit Ca2+ influx. MAPK phosphorylation inhibition by PD98059 did not affect the chronic increase in Lp. To determine whether other agonists which increased Ca2+ influx also chronically increased Lp, the effect of ATP perfusion on chronic Lp was measured. ATP perfusion also chronically increased Lp. The chronic increase in Lp was therefore dependent on an initial transient Ca2+ influx, and not MAPK activation, and was not unique to VEGF stimulation. Inhibition of Ca2+ influx did not inhibit the increase in microvascular diameter or compliance brought about by VEGF. Both these increases were inhibited by PD98059. The VEGF-mediated increase in compliance and diameter was therefore dependent on MAPK activation, not on Ca2+ influx. The chronic increase in Lp stimulated by VEGF perfusion 24 h previously was reduced when the vessel was perfused with 5 mM NiCl2. The sustained, high Lp was therefore dependent on Ca2+ influx. The endothelial cell calcium concentration ([Ca2+]i) of vessels previously perfused with VEGF or ATP, and with a chronically increased Lp, was not significantly increased compared to [Ca2+]i of endothelial cells in vessels before agonist perfusion These experiments show that VEGF acts through different pathways to stimulate increased permeability and compliance. The data are consistent with the hypothesis that VEGF chronically increases Lp through an acute stimulation of Ca2+ influx, but increases compliance and diameter by acute stimulation of the MAPK signalling pathway. They also suggest that the increase in Lp is dependent on a sustained Ca2+ influx, even though the endothelial [Ca2+]i is not raised.
Adenosine Triphosphate↗
Dominant role of cAMP in regulation of microvessel permeability.
We reported previously that increasing cAMP levels in endothelial cells attenuated ATP-induced increases in hydraulic conductivity (L(p)), and that the activation of cGMP-dependent pathways was a necessary step to increase L(p) in response to inflammatory mediators. The aim of the present study was to evaluate the role of basal levels of cAMP in microvessel permeability under resting conditions and to evaluate the cross talk between cAMP- and cGMP-dependent signaling mechanisms in regulation of microvessel permeability under stimulated conditions, using individually perfused microvessels from frog and rat mesenteries. We found that reducing cAMP levels by inhibition of adenylate cyclase or inhibiting cAMP-dependent protein kinase through the use of H-89 increased basal L(p) in both frog and rat mesenteric venular microvessels. We also found that 8-bromocAMP (8-BrcAMP, 0.2 and 2 mM) was sufficient to attenuate or abolish the increases in L(p) due to exposure of frog mesenteric venular microvessels to 8-BrcGMP (2 mM) and ATP (10 microM). Similarly, in rat mesenteric venular microvessels, application of 8-BrcAMP (2 mM) abolished the increases in L(p) due to exposure to 8-BrcGMP alone (2 mM) or with the combination of bradykinin (1 nM). In addition, application of erythro-9-(2-hydroxy-3-nonyl)adenine, an inhibitor of cGMP-stimulated phosphodiesterase, significantly attenuated both 8-BrcGMP- and bradykinin-induced increases in L(p). These results demonstrate that basal levels of cAMP are critical to maintaining normal permeability under resting conditions, and that increased levels of cAMP are capable of overcoming the activation of cGMP-dependent pathways, therefore preventing increases in microvessel permeability. The balance between endothelial concentrations of these two opposing cyclic nucleotides controls microvessel permeability, and cAMP levels play a dominant role.
8-Bromo Cyclic Adenosine Monophosphate↗
VEGF and ATP act by different mechanisms to increase microvascular permeability and endothelial [Ca(2+)](i).
Vascular endothelial growth factor (VEGF) increases hydraulic conductivity (L(p)) by stimulating Ca(2+) influx into endothelial cells. To determine whether VEGF-mediated Ca(2+) influx is stimulated by release of Ca(2+) from intracellular stores, we measured the effect of Ca(2+) store depletion on VEGF-mediated increased L(p) and endothelial intracellular Ca(2+) concentration ([Ca(2+)](i)) of frog mesenteric microvessels. Inhibition of Ca(2+) influx by perfusion with NiCl(2) significantly attenuated VEGF-mediated increased [Ca(2+)](i). Depletion of Ca(2+) stores by perfusion of vessels with thapsigargin did not affect the VEGF-mediated increased [Ca(2+)](i) or the increase in L(p). In contrast, ATP-mediated increases in both [Ca(2+)](i) and L(p) were inhibited by thapsigargin perfusion, demonstrating that ATP stimulated store-mediated Ca(2+) influx. VEGF also increased Mn(2+) influx after perfusion with thapsigargin, whereas ATP did not. These data showed that VEGF increased [Ca(2+)](i) and L(p) even when Ca(2+) stores were depleted and under conditions that prevented ATP-mediated increases in [Ca(2+)](i) and L(p). This suggests that VEGF acts through a Ca(2+) store-independent mechanism, whereas ATP acts through Ca(2+) store-mediated Ca(2+) influx.
Adenosine Triphosphate↗
Starling forces that oppose filtration after tissue oncotic pressure is increased.
We tested the hypothesis that the effective oncotic force that opposes fluid filtration across the microvessel wall is the local oncotic pressure difference across the endothelial surface glycocalyx and not the global difference between the plasma and tissue. In single frog mesenteric microvessels perfused and superfused with solutions containing 50 mg/ml albumin, the effective oncotic pressure exerted across the microvessel wall was not significantly different from that measured when the perfusate alone contained albumin at 50 mg/ml. Measurements were made during transient and steady-state filtration at capillary pressures between 10 and 35 cmH(2)O. A cellular-level model of coupled water and solute flows in the interendothelial cleft showed water flux through small breaks in the junctional strand limited back diffusion of albumin into the protected space on the tissue side of the glycocalyx. Thus oncotic forces opposing filtration are larger than those estimated from blood-to-tissue protein concentration differences, and transcapillary fluid flux is smaller than estimated from global differences in oncotic and hydrostatic pressures.
Animals↗
Endothelial cell shrinkage increases permeability through a Ca2+-dependent pathway in single frog mesenteric microvessels.
1. We tested whether calcium (Ca2+)-dependent mechanisms were essential for our previous observation that a change in the endothelial cell (EC)-extracellular matrix (ECM) attachment caused an increase in microvessel hydraulic permeability (Lp) after exposure to hypertonic solutions in single perfused mesenteric microvessels in pithed frogs (Rana pipiens). 2. In microvessels where integrin-dependent EC-ECM attachments were disrupted by pretreatment with the peptide Gly-Arg-Gly-Asp-Thr-Pro (GRGDTP; 0.3 mmol l-1), we measured microvessel Lp after exposure to hypertonic solutions under experimental conditions that reduced Ca2+ influx into endothelial cells. 3. High K+ solutions (59.7 and 100 mmol l-1 K+) were used to depolarize the endothelial membrane and therefore to reduce the electrochemical driving force for Ca2+ influx through conductive Ca2+ channels. These solutions abolished the increase in Lp caused by hypertonic solutions in the microvessels pretreated with GRGDTP. 4. We previously suggested that the removal of albumin from the perfusate may reduce EC-ECM attachment because hypertonic solutions increased the Lp of microvessels above that due to removal of albumin alone. This additional increase in Lp was attenuated by the 59.7 mmol l-1 K+ solution and was completely abolished by the 100 mmol l-1 K+ solution. 5. Bumetanide, an inhibitor of the Na+-K+-2Cl- co-transporter and one of the mechanisms of regulatory volume increase after exposure to hypertonic solutions in endothelial cells, did not change the response of microvessels to high K+ solutions. 6. Our findings indicate that Ca2+ entry into endothelial cells via passive conductance channels is necessary to increase microvessel Lp after exposure to hypertonic solutions in microvessels where EC-ECM attachments are disrupted.
Albumins↗