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 (σ∆π) opposing filtration is greater than that measured between lumen and interstitial fluid. To test this we measured σ∆π 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 σ∆π 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 µm 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. The classic Starling equation describing the balance of hydrostatic and colloid osmotic (oncotic) forces which determine filtration and reabsorption across the capillary wall includes four forces:where P c and P t are the hydrostatic pressures in the capillary lumen and tissue, respectively, and π c and π t are the corresponding lumen and tissue oncotic pressures. J v /A is the filtration rate per unit area, σ is the reflection coefficient to the plasma proteins and L p is the hydraulic conductivity of the vessel wall. This relation has been tested by changing P c and π c (only rarely by changing P t or π t ) and confirmed many times in both whole organ and isolated microvessels,

. PAF-and bradykinin-induced hyperpermeability of rat venules is independent of actin-myosin contraction. Am J Physiol Heart Circ Physiol 285: H406-H417, 2003. First published March 20, 2003 10.1152/ajpheart. 00021.2003.-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 (L p) was measured to monitor hyperpermeability response to the platelet-activating factor (PAF) 1-O-hexadecyl-2-acetylsn-glycero-3-phosphocholine or bradykinin. Perfusion withϪ7 cm/(s ⅐ cmH2O)] that peaked in 8.9 Ϯ 0.5 min and then returned toward control L p [1.6 Ϯ 0.1 ϫ 10 Ϫ7 cm/(s ⅐ 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 M 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML-7) failed to block the PAF L p 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 Ϯ 0.7 to 3.2 Ϯ 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 endothelialendothelial cell adhesion structures in the absence of contraction by the actin-myosin network.bradykinin; platelet-activating factor; inflammatory gaps; vascular endothelium; ML-7; myosin light chain kinase

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

Haraldsson and Rippe suggested that the circulating glycoprotein orosomucoid (alpha 1-acid glycoprotein) contributes to the net charge on microvessel walls (Acta Physiol. Scand. 129: 127-135, 1987). We tested their hypothesis in individually perfused microvessels of frog mesentery by measuring solute permeability coefficients of two globular proteins (alpha-lactalbumin and ribonuclease) having approximately the same size (Stokes radius, 2 nm) but different charge (-11 and +3, respectively). In vessels perfused with orosomucoid (0.1 and 1 mg/ml) in a Ringer-albumin perfusate, the solute permeability coefficient of alpha-lactalbumin decreased to one-half [0.47 +/- 0.25 (SD)] the value in the absence of orosomucoid, and the solute permeability coefficient of ribonuclease was close to six times as large as alpha-lactalbumin permeability. Both results may be accounted for if orosomucoid increases the net negative charge on microvessel walls in frog mesentery from 11.2 to 28 meq/l. A similar change in microvessel charge would be more than sufficient to account for the decrease in albumin clearance in the presence of orosomucoid reported by Haraldsson and Rippe in rat muscle microvessels.

Therefore, the loss of sodium fluorescein through the upper mesothelium does not significantly alter permeability under these conditions. These techniques enable detailed analysis of solute concentration gradients surrounding microvessels.