Signaling to the apical membrane and to the paracellular pathway: changes in the cytosolic proteome ofAedesMalpighian tubules

Abstract

Using a proteomics approach, we examined the post-translational changes in cytosolic proteins when isolated Malpighian tubules of Aedes aegypti were stimulated for 1 min with the diuretic peptide aedeskinin-III (AK-III, 10(-7) mol l(-1)). The cytosols of control (C) and aedeskinin-treated (T) tubules were extracted from several thousand Malpighian tubules, subjected to 2-D electrophoresis and stained for total proteins and phosphoproteins. The comparison of C and T gels was performed by gel image analysis for the change of normalized spot volumes. Spots with volumes equal to or exceeding C/T ratios of +/-1.5 were robotically picked for in-gel digestion with trypsin and submitted for protein identification by nanoLC/MS/MS analysis. Identified proteins covered a wide range of biological activity. As kinin peptides are known to rapidly stimulate transepithelial secretion of electrolytes and water by Malpighian tubules, we focused on those proteins that might mediate the increase in transepithelial secretion. We found that AK-III reduces the cytosolic presence of subunits A and B of the V-type H(+) ATPase, endoplasmin, calreticulin, annexin, type II regulatory subunit of protein kinase A (PKA) and rab GDP dissociation inhibitor and increases the cytosolic presence of adducin, actin, Ca(2+)-binding protein regucalcin/SMP30 and actin-depolymerizing factor. Supporting the putative role of PKA in the AK-III-induced activation of the V-type H(+) ATPase is the effect of H89, an inhibitor of PKA, on fluid secretion. H89 reverses the stimulatory effect of AK-III on transepithelial fluid secretion in isolated Malpighian tubules. However, AK-III does not raise intracellular levels of cAMP, the usual activator of PKA, suggesting a cAMP-independent activation of PKA that removes subunits A and B from the cytoplasm in the assembly and activation of the V-type H(+) ATPase. Alternatively, protein kinase C could also mediate the activation of the proton pump. Ca(2+) remains the primary intracellular messenger of the aedeskinins that signals the remodeling of the paracellular complex apparently through protein kinase C, thereby increasing transepithelial anion secretion. The effects of AK-III on active transcellular and passive paracellular transport are additive, if not synergistic, to bring about the rapid diuresis.