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.
It is becoming clear that stress proteins play a role in various aspects of postischemic myocardial recovery and that the cytoskeleton of cardiac myocytes is an important determinant for cellular survival during ischemia and energy depletion. In the present study, we addressed the question of whether the cytoskeleton-binding stress protein αB-crystallin may be involved in early cellular responses of rat and porcine myocardium to ischemia. Immunostaining and subcellular fractionation revealed a rapid ischemia-induced redistribution of αB-crystallin from a cytosolic pool to intercalated disks and Z lines of the myofibrils. This striking translocation of αB-crystallin from the cytosol to sites of the myofibrillar system that are known to be sensitive to ischemiareperfusion injury was accompanied by a rapid shift of a fraction of αB-crystallin to a more acidic isoelectric point. This shift is caused by αB-crystallin phosphorylation, as identified by its augmentation in the presence of phosphatase inhibitors (vanadate, fluoride) and comigration of the acidic αB-crystallin form with the phosphorylated B1 form of lenticular αB-crystallin. In view of the chaperone-like function of αB-crystallin in conjunction with its high level of constitutive expression in the myocardium (1–2% of soluble protein content), we consider αB-crystallin an excellent candidate to play a role in early aspects of the protection of the myocardial contractile apparatus against ischemia-reperfusion injury.
Poly- and monoclonal antibodies have been prepared against the cytoplasmic domain (43 kDa) and the 17-, 20-, and 35-kDa fragments of the membrane-spanning domain of the human erythrocyte anion exchanger, band 3. The antibodies were used to localize and further characterize analogues of band 3 in the human kidney. We report here that the basolateral membrane of intercalated cells of the connecting tubules and collecting ducts contains an analogue of band 3 that appears to be highly homologous to the erythrocyte anion exchanger. This band 3-like protein is probably important for reabsorption of bicarbonate in the collecting duct system and thus for acidification of the forming urine. The band 3-like protein of the intercalated cells contain immunoreactive sites of both the cytoplasmic domain and the three major fragments of the membrane-spanning domain of erythrocyte band 3. Although no immunological differences were detected between the membrane-spanning domains of band 3 in erythrocytes and intercalated cells, there are at least three sites along the cytoplasmic domain of kidney band 3 that differ from erythrocyte band 3 in either amino acid composition or posttranslational modifications. The main kidney analogue of band 3 that contains epitopes of the cytoplasmic domain as well as the 17- and 35-kDa membrane-spanning domain of erythroid band 3 is a polypeptide with an apparent molecular mass of 100-110 kDa. Further immunoreactive polypeptides at approximately 180, approximately 140, approximately 38, approximately 25-30 kDa that were detected at lower stringency and higher sensitivity of the immunoblotting procedure may be members of a multigene family that encodes a series of related proteins.
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