POLARITY OF CONSTITUTIVE AND REGULATED VON-WILLEBRAND-FACTOR SECRETION BY TRANSFECTED MDCK-II CELLS

Von Willebrand factor (vMF), synthesized by endothelial cells, is both rapidly secreted by the constitutive pathway and stored in Weibel-Pal ade bodies. Secretion from these organelles occurs upon activation of the protein kinase C signal transduction pathway and yields highly mul timerized vWF. Highly multimerized vWF acts as a more effective adhesi ve ligand than the lower molecular weight forms that are constitutivel y secreted. We employed the extensively characterized polar Madin-Darb y Canine Kidney II (MDCK-II) epithelial cell line, stably transfected with full-length VWF cDNA or deletion mutants thereof, to gain insight in the polarity of vWF secretion by either one of the two pathways. I mmunofluorescence analysis and metabolic labeling experiments revealed that multimeric ''wild-type'' vWF is stored in MDCK-II cells and rele ased upon stimulation with phorbol esters. Furthermore, we show that 6 2.0 +/- 3.8% of constitutively secreted and 83.2 +/- 6.6% of the regul ated secreted wild-type vWF is encountered at the apical side of the c ell. The polarity of the constitutive secretion of deletion mutant vWF delD'D3 is similar to that of constitutively secreted wild-type vWF, w hereas deletion mutant vWFdelD1D2 displays no polar secretion (50.1 +/ - 5.7% apical). (C) 1997 Academic Press.