Patch clamp study on primary culture of isolated proximal convoluted tubules

Abstract

Primary cultures were obtained from microdissected rabbit proximal tubules (S1 segments). The growing epithelia were maintained in culture for up to 30 days. Electron microscopy study revealed that the cells formed a monolayer and showed a morphological polarity with apical microvilli and tight junctions. An immunofluorescence technique using two monoclonal antibodies raised against two apical brush border enzymes of the proximal tubule (LAP, DPP IV) revealed that these enzymes were expressed in the cultured cells. Membrane associated and cytosolic enzyme activities were measured on 12, 20 and 30-day-old cultures. Cultured epithelia exhibited leucine aminopeptidase, gamma glutamyl transferase and fructose 1-6 biphosphatase activities that remained constant for up to 30 days, whereas alkaline phosphatase activity decreased in the oldest cultures. Hexokinase activity on the other hand, increased after 12 days of culture. Cyclic AMP synthesis was stimulated by parathyroid hormone at 12, 20 and 30 days of culture and was insensitive to arginine vasopressin. After 20 days of culture the epithelia grown on permeable supports developed a transepithelial potential of -0.13 mV (apical negative) and a transepithelial resistance of 37 omega cm2 that increased to -1.13 mV and 60 omega cm2 respectively in 30-day-old cultures. The patch clamp technique was applied to the apical membrane of 12-15-day-old cultures. In the whole cell recording configuration, a cellular potential of -61.5 mV was measured, which was mainly due to K+ diffusion. A non-selective cationic channel was present in the apical membrane of the cultured cells. In cell-attached patches the channel carried an inward current and had a conductance of 13 pS. On excised patches the channel discriminated poorly between Na+ and K+ and was impermeant to Cl- and its conductance ranged between 20 and 28 pS. The channel activity was not voltage dependent but required a high calcium concentration (1 mM Ca2+) on the cytoplasmic face.