Distribution and functional correlation of PKC isoforms in A6 epithelia

AIM: A6 cells are used to study the regulation of Na⁺-absorption by electrically-tight hormonally-responsive epithelia. Despite the presence of the cloned ENaC (Epithelial Na⁺-Channel) in these cells, their baseline electrophysiological properties and, moreover, their hormonal regulation by agents such as vasopressin and aldosterone, are variable. This variability may be attributed to differences in the activity or distribution of second messenger protein kinases. Protein kinase C (PKC) is a ubiquitous enzyme implicated in the regulation of many cellular functions. Multiple PKC isoforms exist in epithelial and non-epithelial cells and are thought to be responsible for the diversity of actions of this kinase. To determine whether the reported variability in the properties and regulation of A6 cells are attributed to PKC, we examined the effects of cell polarization and/or stimulation by aldosterone on the distribution and induction of PKC isoforms. METHODS: A6 epithelia were grown to confluency in flasks and on permeable supports. Cell polarity was assessed from the measured open circuit voltage (V_oc) and membrane resistance (R_T). Cells were treated with 1 μM aldosterone or vehicle overnight before harvesting. Western blot analysis of cytoplasmic and membrane protein fractions were carried out using monoclonal antibodies. RESULTS: Cells grown on permeable supports exhibited unidirectional Na⁺ transport as evident from an amiloride-sensitive V_oc of -49.4 ± 0.5 mV and an R_T of 12.6 ± 0.13 kΩ cm². Western blot analysis of both polarized and non-polarized cells revealed the presence of 2 members in each of the conventional (α and γ), novel (δ and ε), and atypical (ι and λ) PKC isoform groups. Where applicable these isoforms were shifted to the membrane fraction by the phorbol ester PMA, providing evidence for antibody specificity. Preliminary results indicate that cell polarization caused a shift in α-like immunoreactivity from cytosolic to membrane fractions. This was accompanied by an increase of cytoplasmic λ-like staining in the absence of detectable changes of membrane staining intensity. Aldosterone treatment of polarized cells (V_oc -99.3± 0.7 mV, and R_T 6.0 + 0.1 KΩ cm²) altered the distribution of λ-like immunoreactivity to increase its membrane versus cytoplasmic intensity. This potential activation of λ-PKC by aldosterone was not observed in non-polarized cells. CONCLUSIONS: A6 epithelia contain multiple isoforms in all 3 PKC categories. Cell polarization may affect the properties of the Na⁺-channel(s) in A6 epithelia by potentially stimulating α-PKC and inducing λ-PKC. Aldosterone may exert its actions on this epithelium, at least in part, by activating λ-PKC.