[Show abstract][Hide abstract] ABSTRACT: In the past, we have used the kinins of the cockroach Leucophaea (the leucokinins) to evaluate the mechanism of diuretic action of kinin peptides in Malpighian tubules of the yellow fever mosquito Aedes aegypti. Now using the kinins of Aedes (the aedeskinins), we have found that in isolated Aedes Malpighian tubules all three aedeskinins (1 microM) significantly 1) increased the rate of fluid secretion (V(S)), 2) hyperpolarized the basolateral membrane voltage (V(bl)), and 3) decreased the input resistance (R(in)) of principal cells, consistent with the known increase in the Cl(-) conductance of the paracellular pathway in Aedes Malpighian tubules. Aedeskinin-III, studied in further detail, significantly increased V(S) with an EC(50) of 1.5 x 10(-8) M. In parallel, the Na(+) concentration in secreted fluid significantly decreased, and the K(+) concentration significantly increased. The concentration of Cl(-) remained unchanged. While the three aedeskinins triggered effects on V(bl), R(in), and V(S), synthetic kinin analogs, which contain modifications of the COOH-terminal amide pentapeptide core sequence critical for biological activity, displayed variable effects. For example, kinin analog 1578 significantly stimulated V(S) but had no effect on V(bl) and R(in), whereas kinin analog 1708 had no effect on V(S) but significantly affected V(bl) and R(in). These observations suggest separate signaling pathways activated by kinins. One triggers the electrophysiological response, and the other triggers fluid secretion. It remains to be determined whether the two signaling pathways emanate from a single kinin receptor via agonist-directed signaling or from a differentially glycosylated receptor. Occasionally, Malpighian tubules did not exhibit a detectable response to natural and synthetic kinins. Hypothetically, the expression of the kinin receptor may depend on developmental, nutritional, and/or reproductive signals.
[Show abstract][Hide abstract] ABSTRACT: Transepithelial fluid secretion across the renal (Malpighian) tubule epithelium of the mosquito (Aedes aegypti) is energized by the vacuolar-type (V-type) H(+)-ATPase and not the Na(+)-K(+)-ATPase. Located at the apical membrane of principal cells, the V-type H(+)-ATPase translocates protons from the cytoplasm to the tubule lumen. Secreted protons are likely to derive from metabolic H(2)CO(3), which raises questions about the handling of HCO(3)(-) by principal cells. Accordingly, we tested the hypothesis that a Cl/HCO(3) anion exchanger (AE) related to the solute-linked carrier 4 (SLC4) superfamily mediates the extrusion of HCO(3)(-) across the basal membrane of principal cells. We began by cloning from Aedes Malpighian tubules a full-length cDNA encoding an SLC4-like AE, termed AeAE. When expressed heterologously in Xenopus oocytes, AeAE is both N- and O-glycosylated and mediates Na(+)-independent intracellular pH changes that are sensitive to extracellular Cl(-) concentration and to DIDS. In Aedes Malpighian tubules, AeAE is expressed as two distinct forms: one is O-glycosylated, and the other is N-glycosylated. Significantly, AeAE immunoreactivity localizes to the basal regions of stellate cells but not principal cells. Concentrations of DIDS that inhibit AeAE activity in Xenopus oocytes have no effects on the unstimulated rates of fluid secretion mediated by Malpighian tubules as measured by the Ramsay assay. However, in Malpighian tubules stimulated with kinin or calcitonin-like diuretic peptides, DIDS reduces the diuretic rates of fluid secretion to basal levels. In conclusion, Aedes Malpighian tubules express AeAE in the basal region of stellate cells, where this transporter may participate in producing diuretic rates of transepithelial fluid secretion.
[Show abstract][Hide abstract] 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.