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.

We tested the hypothesis that increased endothelial cell adenosine 3′,5′-cyclic monophosphate (cAMP) decreases microvascular permeability in vivo. The effects of cAMP-specific phosphodiesterase type IV inhibition and adenylate cyclase activation on microvascular hydraulic conductivity ( L p) were investigated in intact individual capillaries and postcapillary venules in mesentery of pithed frogs ( Rana pipiens). Treatment with rolipram (10 μM) and forskolin (5 μM) for 25 min decreased L p to 37% of control. Rolipram alone also significantly decreased L p. Isoproterenol (10 μM) decreased L p to 27% of control within 20 min. A subgroup of eight vessels treated with rolipram and forskolin, in which mean L p fell to 25% of control, was examined with transmission electron microscopy. The mean number of tight junctions in the treated vessels was 2.2 per cleft (303 clefts), significantly higher than in a matched control group (192 clefts), which was 1.7 per cleft. The results indicate that microvascular L pcan be modulated by intracellular cAMP and that one of the structural end points of stimulated cAMP levels is an increase in the mean number of tight-junction strands between endothelial cells.

Porcine arterial endothelial cells (PAECs)Pulmonary arterial endothelial cells were obtained from freshly slaughtered pigs by exposure of the excised pulmonary artery to 0.1 % collagenase for 12-15 min. Cells were cultured in medium 199 containing 10 % fetal calf serum (GIBCO, Karlsruhe, Germany), passaged and characterized as described by Suttorp et al. (1988). Only cultures of passages 2-5 were used.Cell culture studies Cells were grown on coverslips, coated with gelatin cross-linked with glutaraldehyde (Schnittler et al. 1993). Toxin B was added to R. H. Adamson and others 296

To investigate the mechanism whereby nitric oxide (NO) signaling pathways regulate microvessel permeability in vivo, we measured changes in microvessel hydraulic conductivity (Lp) and endothelial cytoplasmic calcium concentration ([Ca2+]i) in response to calcium ionophore, ionomycin (5 microM), and ATP (10 microM) before and after the use of NO synthase (NOS) inhibitors in single perfused frog mesenteric venular microvessels. Ionomycin induced a transient increase in endothelial [Ca2+]i and an associated increase in Lp. The NOS inhibitors N omega-nitro-L-arginine methyl ester (10 and 300 microM) and N omega-monomethyl-L-arginine (L-NMMA; 10, 50, and 100 microM) significantly attenuated the peak increase in Lp induced by ionomycin. A similar inhibitory effect was also observed with the increase in Lp mediated by ATP. In contrast, D-NMMA, a biologically inactive isomer of L-NMMA, showed no effect on ionomycin-induced increase in Lp L-Arginine (3 mM) reversed the inhibitory effect of L-NMMA (10 microM) on Lp. However, the NOS inhibitors did not alter the magnitude and time course of the biphasic increase in endothelial [Ca2+]i induced by both ionomycin and ATP. These data suggest that 1) calcium-dependent NO release is a necessary step to increase microvessel permeability, and 2) the action of NOS inhibitors in attenuating the permeability increase in response to ionomycin and ATP occurs down-stream from calcium entry and does not involve modification of the initial increase in endothelial [Ca2+]i.

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