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First published online January 16, 2009
Journal of Experimental Biology 212, 329-340 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.024646
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Signaling to the apical membrane and to the paracellular pathway: changes in the cytosolic proteome of Aedes Malpighian tubules

Klaus W. Beyenbach1,*, Sabine Baumgart2, Kenneth Lau1, Peter M. Piermarini1 and Sheng Zhang2

1 Department of Biomedical Sciences, VRT 8004, Cornell University, Ithaca, NY 14853, USA
2 Proteomics and Mass Spectrometry Core Facility, 143 Biotechnology Building, Cornell University, Ithaca, NY 14853, USA


Figure 1
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  		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 (Vt) and resistance (Rt) and on the basolateral and apical membrane voltages (Vbl, Va) 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, 2003Go; Hayes et al., 1989Go; Pannabecker et al., 1993Go; Yu and Beyenbach, 2004Go).

 

Figure 2
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  		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).

 

Figure 3
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  		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.

 

Figure 4
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  		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.

 

Figure 5
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  		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., 2005Go). In the present study, AnogaDH31 served as a positive control for the bioassay. Data are means ± s.e.; ***, P<0.001.

 

Figure 6
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  		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.

 

Figure 7
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  		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.

 

Figure 8
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  		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; {alpha},β,{gamma}, 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.

 

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