Bacterial exotoxins and endothelial permeability for water and albumin in vitro

Abstract

Effects of Staphylococcus aureus alpha-toxin and Pseudomonas aeruginosa cytotoxin on the permeability of an endothelial monolayer were studied. Porcine pulmonary artery endothelial cells were grown on a polycarbonate membrane, mounted in a chamber, and exposed to a continuous hydrostatic pressure of 10 cmH2O. On application of this trans-endothelial pressure, endothelial monolayer became "sealed," i.e., the filtration rate for water decreased and the reflection coefficient for albumin increased, reaching a plateau after 1-2 h. Sealed monolayer had a hydraulic conductivity of 2.1 X 10(-6) cm.s-1.cmH2O and an albumin reflection coefficient of 0.73. Permeability of the monolayer was increased on addition of an excess of EDTA and reversed on readdition of calcium. Within 60-90 min after addition of 1 microgram/ml alpha-toxin, the filtration rate increased 75-fold, and the albumin reflection coefficient dropped to 0.20. These changes in permeability were accompanied by cell retraction and formation of large intercellular gaps between endothelial cells. Effects of alpha-toxin were abolished by preincubation with neutralizing antibodies and by inhibitors of calmodulin function. Pseudomonas aeruginosa cytotoxin (25 and 50 micrograms/ml) also increased the permeability of the endothelial monolayer, but it was only about one-third as effective as alpha-toxin.

Role of actin and myosin in the control of paracellular permeability in pig, rat and human vascular endothelium.

“…During this period the filtration rate per cm2 of monolayer typically dropped from initial values of 15-20 /sl/min to less than 5 ,ul/min. Concomitantly, the reflection coefficient for albumin showed a rise from about 0-15 to about 0-75 (Suttorp et al 1988).…”

“…The calmodulin antagonists TFP and W7 inhibit ionophore/Ca2+-induced permeability of the monolayer (for further documentation of experiments with PAEC monolayers see Suttorp et al 1989). with myosin-S1-NEM no gap formation occurred after addition of Ca2+/ATP (Fig.…”

“…Endothelial cells were isolated from the human umbilical cord (HUVEC) and from pulmonary arteries of freshly slaughtered pigs (PAEC) and cultured under conditions as described in detail elsewhere (Franke, Grafe, Schnittler, Seiffge, Mittermayer & Drenckhahn, 1984;Suttorp et al 1988). Briefly, cells of the second to fourth passage were plated on gelatin-coated polycarbonate filters or expanded polytetrafluoroethylene (ePTFE) membranes and cultured until confluency.…”

“…Filters covered with confluent monolayers were placed into a chamber in which the upper side of the monolayer was continuously exposed to a hydrostatic pressure of 10 cmH20. The permeability chamber used for polyearbonate filters has been described recently (Suttorp et al 1988). The chamber constructed for measurement of permeability of monolayers grown on ePTFE membranes is shown in Fig.…”

“…Exposure to A23187 in the presence of Ca2+ resulted in a 10-to 20-fold increase in the filtration rate across the HUVEC and PAEC monolayer (Fig. 8B) (for further details on PAEC, see Suttorp et al 1989). This was accompanied by formation of intercellular gaps that were visualized by phase-contrast microscopy (Fig.…”