Substrate Adhesion of Rat Hepatocytes : A Comparison of Laminin and Fibronectin as Attachment Proteins

In previous studies rat hepatocytes have been shown to adhere to substrates composed of collagen or fibronectin . In the present communication, the basement membrane protein laminin is reported to mediate the attachment and spreading of hepatocytes. The cell attachment-mediating activity of laminin was compared with that of fibronectin . The activity of fibronectin was heat sensitive, whereas laminin retained its activity after boiling. On the other hand, reduction and alkylation or periodate oxidation of the proteins affected only the cell attachment activity of laminin. Preincubation of cells with soluble fibronectin inhibited initial cell attachment to fibronectin but not to laminin substrates, and, reversely, soluble laminin selectively inhibited cell attachment to laminin. These results suggest that attachment of cells to substrates of the two proteins involves different cellular receptors recognizing distinct and nonidentical structures in the proteins . were purchased from Sigma Chemical Co ., St. Louis, Mo., and F-10 medium (13) from Flow Laboratories, Inc ., Rockville, Md. Preparation of Cell Attachment Substrates Bacteriological plastic petri dishes, 35 mm in diameter (cat . no. 1008 ; Falcon Plastics, Div. Becton, Dickinson & Co ., Oxnard, Calif.), were coated by adding indicated amounts of protein in 2 ml of water. The dishes were then air-dried at 22°C and washed with water . Cell Attachment Assay Hepatocytes were isolated from male Sprague-Dawley rats by perfusion of the livers in situ with a solution of collagenase as previously described (14). The cells were seeded in 35-mm dishes in 2 ml of buffer 3 (see reference 14 ; 8.0 g NaCl, 0 .35 g KCI, 0.16 g MgS0,.7H20, 0.18 g CaC12-2H20, 2 .4 g HEPES, H2O to I liter, pH 7.4) and incubated at 37°C in humidified air . After incubation for various times the dishes were washed with 2 x 1 ml of buffer 3 . The number of cells attached to the substrate was determined as described previously (14). In short, the cells are lysed in a solution of Triton X-100, and the activity in the lysate of the enzyme, lactate dehydrogenase, which has been shown to be proportional to the cell number, is determined . When 2 x 108 cells were seeded on a substrate made from 10 f+g of fibronectin, -90% of the cells attached to the dish in a confluent layer (12). However, small variations in the number of attached cells were observed from one cell batch to another . Therefore, in each experiment, control cells were incubated for 60 min on a substrate made from 10 1% of fibronectin . The number of cells that attached under these conditions was