Signaling to the apical membrane and to the paracellular pathway: changes in the cytosolic proteome of Aedes Malpighian 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.

Fig. 1.

Leucokinin increases the paracellular Cl^– conductance in Malpighian tubules of Aedes aegypti. (A,B) Leucokinin-VIII increases the transepithelial secretion of both NaCl and KCl. Numbers in red indicate a statistical significant difference (P<0.05) from controls; (C) electrophysiological effects of LK-VIII on the transepithelial voltage (V_t) and resistance (R_t) and on the basolateral and apical membrane voltages (V_bl, V_a) indicate a transepithelial short-circuit brought about by the sudden increase in paracellular Cl^– conductance; (D) model of transepithelial electrolyte secretion in Aedes Malpighian tubules. The transepithelial transport of Na⁺ and K⁺ is active and mediated by principal cells; the transepithelial transport of Cl^– is passive and mediated by the paracellular pathway and stellate cells. However, under conditions of diuresis triggered by aedeskinin or kinin isoforms, the transcellular and paracellular pathways are electrically so well coupled that the rates of transcellular cation secretion and paracellular anion secretion are equivalent (Beyenbach, 2003; Hayes et al., 1989; Pannabecker et al., 1993; Yu and Beyenbach, 2004).

Fig. 2.

Two-dimensional electrophoresis of cytosolic proteins before (control, C) and after treating (T) Malpighian tubules with aedeskinin-III (10^–7 mol l^–1) for 1 min. Portions of the whole gel are shown. The SYPRO Ruby stain recognizes proteins, and the Pro-Q Diamond stain recognizes phosphoproteins. Circles identify some of the spots of interest in the present study: 352, endoplasmin; 565, subunit A of the V-type H⁺ ATPase; 710, subunit B of the V-type H⁺ ATPase; 782, subunit B of the V-type H⁺ ATPase and calreticulin; 913, adducin; 971, rab dissociation inhibitor; 1062, regulatory subunit type II of protein kinase A; 1079, actin (see also Table 1).

Fig. 3.

Effect of aedeskinin-III (10^–7 mol l^–1) on subunit A (spot 565) of the V-type H⁺ ATPase in the cytosol of Aedes Malpighian tubules. Negative SYPRO Ruby and Pro-Q Diamond C/T ratios indicate reductions in protein concentration and phosphorylation. Spot 565 was selected for analysis because the phosphorylation C/T ratio exceeded the criterion of ±1.5.

Fig. 4.

The PKA inhibitor H89 reverses the diuretic effect of aedeskinin-III in isolated Malpighian tubules of Aedes aegypti. Each of 10 Malpighian tubules was used as its own control. The concentrations of aedeskinin-III and H89 were 1 μmol l^–1 and 20 μmol l^–1, respectively. Data are means ± s.e.; ^***, P<0.002.

Fig. 5.

The lack of effect of aedeskinins (1 μmol l^–1) on intracellular cAMP levels in Malpighian tubules of Aedes aegypti. AnogaDH₃₁ (1 μmol l^–1) is known to increase intracellular [cAMP] in Aedes and Anopheles Malpighian tubules (Coast et al., 2005). In the present study, AnogaDH₃₁ served as a positive control for the bioassay. Data are means ± s.e.; ^***, P<0.001.

Fig. 6.

The paracellular pathway in Malpighian tubules of Aedes aegypti. (A) The paracellular pathway between two principal cells and part of a stellate cell (near the apical brush border). Mitochondria in microvilli of the brush border are unique to principal cells of the tubule. Microvilli of the brush border of stellate cells are devoid of mitochondria. (B,C) The paracellular pathway between a stellate cell and principal cell is occupied largely by a septate junction. Septa give rise to the ladder-like structure of the junction. Note that the septate junction can extend from apical to basal poles of epithelial cells. Abbreviations: bb, brush border; bmi, basolateral membrane infoldings; mt, microtubule; pc, principal cell; sc, stellate cell. White arrows point to the paracellular pathway in B and to septa in C.

Fig. 7.

Molecular, mechanical and regulatory models of the V-type H⁺ ATPase. (A) Molecular model. Subunits of catalytic complex V₁ bear capital letters; subunits of the V₀ complex in the apical membrane bear small letters. (B) Mechanical model of the proton pump consisting of a stator and rotor. (C) Regulatory model. The phosphorylation of subunit C is thought to be instrumental in the assembly of the two complexes to form the active proton pump. Candidate kinases (PK) that might phosphorylate subunit C are protein kinase A and/or protein kinase C.

Fig. 8.

Hypothetical model of aedeskinin-III signaling to the paracellular pathway in Malpighian tubules of the yellow-fever mosquito. The paracellular protein complex consists of cytoplasmic elements such as the cytoskeleton, scaffolding and regulatory proteins, and integral membrane proteins reaching into the paracellular space. The proteins defining the paracellular barrier/permeability properties are unknown in insect epithelia. ADF, actin depolymerizing factor; GPCR, G-protein-coupled receptor; α,β,γ, subunits of G protein; GTP, guanosine triphosphate; PLC, phospholipase C; PIP₂, phosphatidylinositol (4,5)-bisphosphate; DAG, diacylglycerol; PKC, protein kinase C; SERCA, sarcoplasmic and endoplasmic reticulum calcium ATPase.