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.     

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.     

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.     

Macromolecule permeability of in situ and excised rodent skeletal muscle arterioles and venules

In microvessels, acute inflammation is typified by an increase in leukocyte-endothelial cell interactions, culminating in leukocyte transmigration into the tissue, and increased permeability to water and solutes, resulting in tissue edema. The goal of this study was to establish a method to quantify solute permeability (P(s)) changes in microvessels in intact predominantly blood-perfused networks in which leukocyte transmigratory behavior could be precisely described using established paradigms. We used intravital confocal microscopy to measure solute (BSA) flux across microvessel walls, hence P(s). A quantitative fluorescence approach (Huxley VH, Curry FE, and Adamson RH. Am J Physiol Heart Circ Physiol 252: H188-H197, 1987) was adapted to the imaged confocal tissue slice in which the fluorescent source volume and source surface area of the microvessel were restricted to the region of vessel that was contained within the imaged confocal tissue section. P(s) measurements were made in intact cremaster muscle microvasculature of anesthetized mice and compared with measurements of P(s) made in isolated rat skeletal muscle microvessels. Mouse arteriolar P(s) was 9.9 +/- 1.1 x 10(-7) cm/s (n = 16), which was not different from 8.4 +/- 1.3 x 10(-7) cm/s (n = 6) in rat arterioles. Values in venules were significantly (P < 0.05) higher: 44.4 +/- 7.9 x 10(-7) cm/s (n = 14) in mice and 25.0 +/- 3.7 x 10(-7) cm/s in rats. Convective coupling was estimated to contribute <10% to the measured P(s) in both microvessel types and both animal models. We conclude that this approach provides an appropriate quantification of P(s) in the intact microvasculature and that arteriolar P(s), while lower than in venules, is nevertheless consistent with arterioles being a significant source of interstitial protein.     

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.     

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.     

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.     

A model for the modulation of microvessel permeability by junction strands

To investigate the effect of junction strands on microvessel permeability, we extend the previous analytical model developed by Fu et al. (1994, J. Biomech. Eng., 116, pp. 502-513), for the interendothelial cleft to include multiple junction strands in the cleft and an interface between the surface glycocalyx layer and the cleft entrance. Based on the electron microscopic observations by Adamson et al. (1998, Am. J. Physiol., 274(43), pp. H1885-H1894), that elevation of intracellular cAMP levels would increase number of tight junction strands, this two-junction-strand and two-pore model can successfully account for the experimental data for the decreased permeability to water, small and intermediate-sized solutes by cAMP.     

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.     

Determination of Solute Permeability in Chara Internodes by a Turgor Minimum Method : Effects of External pH

The analysis of Sha'afi et al. (Sha'afi, Rich, Mickulecky, Solomon 1970 J Gen Physiol 55: 427-450) for determining solute permeability in red blood cells has been modified and applied to turgid plant cells. Following the addition of permeating solute to the external medium, a biphasic response of cell turgor can be measured with the pressure probe in isolated internodes of Chara corallina. After an initial decrease in turgor due to water flow (water phase), turgor increases due to the uptake of the solute (solute phase) until the original turgor is reattained. From the pressure/time course in the neighborhood of the minimum turgor, the permeability of the osmotic solute can be determined. The data obtained by the minimum method for rapidly permeating (ethanol, methanol) and slowly permeating (formamide, dimethylformamide) solutes are similar to those calculated from the half-time of pressure changes during the solute phase and to data obtained by other workers using radioactive tracers. The methods employing the pressure probe were applied to examine the effect of high pH (up to pH 11) on the membrane permeability. There appeared to be no effect of high pH on the permeability coefficients, reflection coefficients, and hydraulic conductivity.     

A folding variant of human alpha-lactalbumin induces mitochondrial permeability transition in isolated mitochondria.

A human milk fraction containing multimeric alpha-lactalbumin (MAL) is able to kill cells via apoptosis. MAL is a protein complex of a folding variant of alpha-lactalbumin and lipids. Previous results have shown that upon treatment of transformed cells, MAL localizes to the mitochondria and cytochrome c is released into the cytosol. This is followed by activation of the caspase cascade. In this study, we further investigated the involvement of mitochondria in apoptosis induced by the folding variant of alpha-lactalbumin. Addition of MAL to isolated rat liver mitochondria induced a loss of the mitochondrial membrane potential (Delta Psi(m)), mitochondrial swelling and the release of cytochrome c. These changes were Ca(2+)-dependent and were prevented by cyclosporin A, an inhibitor of mitochondrial permeability transition. MAL also increased the rate of state 4 respiration in isolated mitochondria by exerting an uncoupling effect. This effect was due to the presence of fatty acids in the MAL complex because it was abolished completely by BSA. BSA delayed, but failed to prevent, mitochondrial swelling as well as dissipation of Delta Psi(m), indicating that the fatty acid content of MAL facilitated, rather than caused, these effects. Similar results were obtained with HAMLET (human alpha-lactalbumin made lethal to tumour cells), which is native alpha-lactalbumin converted in vitro to the apoptosis-inducing folding variant of the protein in complex with oleic acid. Our findings demonstrate that a folding variant of alpha-lactalbumin induces mitochondrial permeability transition with subsequent cytochrome c release, which in transformed cells may lead to activation of the caspase cascade and apoptotic death.     

Increased permeability of primary cultured brain microvessel endothelial cell monolayers following TNF-alpha exposure.

TNF-alpha is a cytokine that produces increased permeability in the peripheral vasculature; however, little is known about the effects of TNF-alpha on the blood-brain barrier (BBB). Using primary cultured bovine brain microvessel endothelial cells (BBMEC) as an in vitro model of the BBB, this study shows that TNF-alpha produces a reversible increase in the permeability of the brain microvessel endothelial cells. The BBMEC monolayers were pre-treated with 100 ng/ml of TNF-alpha for periods ranging from 2 to 12 hours. Permeability was assessed using three molecular weight markers, fluorescein (376 MW), fluorescein-dextran (FDX-4400; 4400 MW), and FDX-70000 (MW 70000). The permeability of BBMEC monolayers to all three fluorescent markers was increased two-fold or greater in the TNF-alpha treatment group compared to control monolayers receiving no TNF-alpha. Significant changes in permeability were also observed with TNF-alpha concentrations as low as 1 ng/ml. These results suggest that TNF-alpha acts directly on the brain microvessel endothelial cells in a dynamic manner to produce a reversible increase in permeability. Exposure of either the lumenal or ablumenal side of BBMEC monolayers to TNF-alpha resulted in similar increases in permeability to small macromolecules, e.g. fluorescein. However, when a higher molecular weight marker was used (e.g. FDX-3000), there was a greater response following lumenal exposure to TNF-alpha. Together, these studies demonstrate a reversible and time dependent increase in brain microvessel endothelial cell permeability following exposure to TNF-alpha. Such results appear to be due to TNF's direct interaction with the brain microvessel endothelial cell.     

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.     

Compactness of the kinetic molten globule of bovine alpha-lactalbumin: a dynamic light scattering study.

During folding of globular proteins, the molten globule state was observed as an equilibrium intermediate under mildly denaturing conditions as well as a transient intermediate in kinetic refolding experiments. While the high compactness of the equilibrium intermediate of alpha-lactalbumin has been verified, direct measurements of the compactness of the kinetic intermediate have not been reported until now. Our dynamic light scattering measurements provide a complete set of the hydrodynamic dimensions of bovine alpha-lactalbumin in different conformational states, particularly in the kinetic molten globule state. The Stokes radii for the native, kinetic molten globule, equilibrium molten globule, and unfolded states are 1.91, 1.99, 2.08, and 2.46 nm, respectively. Therefore, the kinetic intermediate appears to be even more compact than its equilibrium counterpart. Remarkable differences in the concentration dependence of the Stokes radius exist revealing strong attractive but repulsive intermolecular interactions in the kinetic and equilibrium molten globule states, respectively. This underlines the importance of extrapolation to zero protein concentration in measurements of the molecular compactness.     

Chick heat-shock protein of Mr = 90,000, free or released from progesterone receptor, is in a dimeric form

A monoclonal antibody (BF4) has been used to characterize and purify the heat-shock protein of Mr approximately 90,000 (hsp 90) present in the chick oviduct. In low salt cytosol, the sedimentation coefficient of hsp 90 is approximately 6.8 S, the Stokes radius approximately 7.1 nm, and the calculated Mr approximately 204,000, thus suggesting a dimeric structure. In 0.4 M KCl cytosol, only slightly smaller values were determined (approximately 6.5 S, approximately 6.8 nm, and approximately 187,000). Following purification by ion exchange and immunoaffinity chromatography, hsp 90 migrated as a single silver-stained band at Mr approximately 90,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while the sedimentation coefficient 6.2 S, the Stokes radius approximately 6.8 nm, and the Mr approximately 178,000 confirmed the dimeric structure. However, in both antigen or antibody excess conditions, only one molecule of monoclonal antibody could be bound to the hsp 90 dimer. Whether steric hindrance in a homodimer or the presence of two different 90-kDa proteins in a heterodimer explains this result cannot yet be decided. The dimer is not dissociated by high salt (1 M KCl) or the chaotropic agent (0.5 M NaSCN), but is disrupted by 4 M urea, suggesting a stabilization of the structure by hydrogen bonds. The molybdate-stabilized progesterone receptor hetero-oligomer form of approximately 8 S sedimentation coefficient was purified, and its hsp 90 component was then released by salt treatment. It was found to sediment at approximately 5.8 S and have a Stokes radius approximately 7.1 nm, giving Mr approximately 174,000. This observation is consistent with a previous report suggesting from specific activity determination, scanning of polyacrylamide gels, and cross-linking experiments that each purified nontransformed progesterone receptor molecule includes one progesterone binding unit per two 90-kDa protein molecules (Renoir, J. M., Buchou, T., Mester, J., Radanyi, C., and Baulieu, E. E. (1984) Biochemistry 23, 6016-6023). This work brings direct evidence that both free hsp 90 and the non-hormone binding hsp 90 component released from the nontransformed steroid receptor in the cytosol are in a dimeric form.     

An electrodiffusion model for the blood-brain barrier permeability to charged molecules

The endothelial surface glycocalyx layer (SGL) and the basement membrane (BM) are two important components of the blood-brain barrier (BBB). They provide large resistance to solute transport across the BBB in addition to the tight junctions in the cleft between adjacent endothelial cells. Due to their glycosaminoglycan compositions, they carry negative charge under physiological conditions. To investigate the charge effect of the SGL and BM on the BBB permeability to charged solutes, we developed an electrodiffusion model for the transport of charged molecules across the BBB. In this model, constant charge densities were assumed in the SGL and in the BM. Both electrostatic and steric interaction and exclusion to charged molecules were considered within the SGL and the BM and at their interfaces with noncharged regions of the BBB. On the basis of permeability data for the positively charged ribonuclease (+4,radius=2.01?nm) and negatively charged α-lactalbumin (-10,radius=2.08?nm) measured in intact rat mesenteric and pial microvessels, our model predicted that the charge density in both SGL and BM would be ～30?mEq/L, which is comparable to that in the SGL of mesenteric microvessels. Interestingly, our model also revealed that due to the largest concentration drop in the BM, there is a region with a higher concentration of negatively charged α-lactalbumin in the uncharged inter-endothelial cleft, although the concentration of α-lactalbumin is always lower than that of positively charged ribonuclease and that of a neutral solute in the charged SGL and BM.     

Predicted permeability parameters of human ovarian tissue cells to various cryoprotectants and water

This study presents a generic numerical model to simulate the coupled solute and solvent transport in human ovarian tissue sections during addition and removal of chemical additives or cryoprotective agents (CPA). The model accounts for the axial and radial diffusion of the solute (CPA) as well as axial convection of the CPA, and a variable vascular surface area (A) during the transport process. In addition, the model also accounts for the radial movement of the solvent (water) into and out of the vascular spaces. Osmotic responses of various cells within an human ovarian tissue section are predicted by the numerical model with three model parameters: permeability of the tissue cell membrane to water (L(p)), permeability of the tissue cell membrane to the solute or CPA (omega) and the diffusion coefficient of the solute or CPA in the vascular space (D). By fitting the model results with published experimental data on solute/water concentrations within an human ovarian tissue section, I was able to determine the permeability parameters of ovarian tissue cells in the presence of 1.5M solutions of each of the following: dimethyl sulphoxide (DMSO), propylene glycol (PROH), ethylene glycol (EG), and glycerol (GLY), at two temperatures (4 degrees C and 27 degrees C). Modeling Approach 1: Assuming a constant value of solute diffusivity (D = 1.0 x 10(-9) m(2)/sec), the best fit values of L(p) ranged from 0.35 x 10(-14) to 1.43 x 10(-14) m(3)/N-sec while omega ranged from 2.57 x 10(-14) to 70.5 x 10(-14) mol/N-sec. Based on these values of L(p) and omega, the solute reflection coefficient, sigma defined as sigma = 1-omega v(CPA)/L(P) ranged from 0.9961 to 0.9996. Modeling Approach 2: The relative values of omega and sigma from our initial modeling suggest that the embedded ovarian tissue cells are relatively impermeable to all the CPAs investigated (or omega approximately 0 and sigma approximately 1.0). Consequently the model was modified and used to predict the values of L(p) and D assuming omega = 0 and sigma = 1.0. The best fit values of L(p) ranged from 0.44 x 10(-14) to 1.2 x 10(-14) m(3)/N-sec while D ranged from 0.85 x 10(-9) to 2.08 x 10(-9) m(2)/sec. Modeling Approach 3: Finally, the best fit values of D from modeling approach 2 were incorporated into model 1 to re-predict the values of L(p) and omega. It is hoped that the ovarian tissue cell parameters reported here will help to optimize chemical loading and unloading procedures for whole ovarian tissue sections and consequently, tissue cryopreservation procedures.     

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.     

Sieve tube unloading and post-phloem transport of fluorescent tracers and proteins injected into sieve tubes via severed aphid stylets

A variety of fluorescent tracers and proteins were injected via severed aphid stylets into the sieve tubes of wheat (Triticum aestivum L.) grains to evaluate the dimensions of plasmodesmal channels involved in sieve element/companion cell (SE/CC) unloading and post-phloem transport. In the post-phloem pathway, where diffusion is the predominant mode of transport, the largest molecule to show mobility was 16-kD dextran, with a Stokes radius of 2.6 nm. This suggests that the aqueous channels for cell-to-cell transport must be about 8 nm in diameter. Even the largest tracer injected into the sieve tubes, 400-kD fluorescein-labeled Ficoll with a Stokes radius of about 11 nm, was unloaded from the SE/CC complex. However, in contrast to smaller tracers (< or =3 kD, with a Stokes radius < or = 1.2 nm), the unloading of fluorescein-labeled Ficoll and other large molecules from the SE/CC complex showed an irregular, patchy distribution, with no further movement along the post-phloem pathway. Either the plasmodesmal channels involved in SE/CC unloading are exceptionally large (perhaps as much as 42 nm in diameter), with only a very small fraction of plasmodesmata being conductive, or the larger tracers damage the plasmodesmata in some way, enlarging smaller channels.     

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.