Substructurel features of LLC-PK1porcine kidney cells grown on Anocell filter supports

Abstract

LLC-PK1cells, derived from porcine kidney cortex, have been used for a variety of studies including sodium-coupled hexose transport, vasopressin/calcitonin sensitive adenylate cyclase(1) and vasopressin V1and V2receptor-mediated endocytosis involving clathrin coated pits and vesicles(2,3,4,5). Recently, we reported on the morphological profile of these cells using the techniques of scanning electron microscopy (SEM). SEM studies of confluent LLC-PK1cells exhibited surface morphology similar to intact tissue. These cells also responded to ADH (antidiuretic hormone, vasopressin) or MZ (mezerein), a non-phorbol activator of protein kinase C (PKC) with a propagation of numerous microvilli over the apical plasma membrane typical of ADH responsive tissue. The current study evaluates the cytomorphological features of cultured LLC-PK1cells following hormone addition.LLC-PK1cells were grown on Anocell filter supports using DMEM media in a 5% CO2/air environment in an incubator at 37°C. Upon confluency, cultured cells were washed in buffer and then treated with 100 mU/ml ADH or MZ10-6M for 15 min. Control and stimulated tissues were fixed in 2% glutaraldehyde in PIPES with a postfixation in 1% osmium tetroxide for 1 hr for either SEM or TEM.