U.- Purdue U. at Indianapolis

It has been hypothesized that the hyperinsulinemic state may contribute to the development of hypertension by activating the amiloride-sensitive epithelial sodium channel (ENaC) thereby stimulating an increase in sodium reabsorption in the distal nephron. The phosphoinositide (PI) signaling cascade has been shown to initiate the sodium reabsorptive process, but it is evident there are many unidentified parts in this complex, branching pathway. We are using proteomics to identify these unknown effectors in a mouse cortical collecting duct cell line (mpkCCDcl4). Proteins are separated using isoelectric focusing followed by large format 2D-PAGE. Sequential protein and phosphoprotein staining followed by MALDI-tof mass spectrometry are used to identify proteins whose phosphorylation state is altered. We have identified 22 proteins modified by insulin within ten minutes. Of the 22 proteins, 21 have been previously shown to be stimulated by insulin in other cell types or are associated with the (PI) pathway. These identifications have allowed us to postulate a new hypothetical branch of the sodium absorptive pathway involving the microtubular network. This hypothesis will be tested in intact cellular monlayers using electrophysiological techniques. We thank Dr. A. Vandewalle (INSERM U478, Paris, France) for providing the mpkCCDcl4 cell line.