The V-type H⁺-ATPase is targeted in anti-diuretic hormone control of the Malpighian ‘renal’ tubules

Abstract

Like other insects, secretion by mosquito MTs is driven by the V-type H+-ATPase (VA), which is densely packed in the apical membrane of principal cells. The anti-diuretic neurohormone CAPA inhibits tubule secretion stimulated by select diuretic hormones; however, the cellular effectors of this inhibitory signaling cascade remain unclear. Herein, we show that the VA inhibitor bafilomycin selectively inhibits serotonin (5HT)- and calcitonin-related diuretic hormone (DH31)-stimulated secretion. VA activity increases in DH31-treated MTs, whereas CAPA abolishes this increase. A critical feature of VA activation involves the reversible association of the cytosolic (V1) and membrane (Vo) complexes and results revealed higher V1 protein abundance in membrane fractions of DH31-treated MTs whereas CAPA significantly decreased V1 protein abundance in membrane fractions while increasing it in cytosolic fractions. Immunolocalization of V1 was observed strictly in the apical membrane of MTs treated with DH31 alone whereas immunoreactivity was dispersed following CAPA treatment. Elevated levels of cGMP in CAPA-treated MTs suggests activation of a phosphodiesterase (PDE), resulting in decreased levels of DH31-stimulated cAMP. Therefore, CAPA inhibition of MTs involves reducing VA activity and promotes complex dissociation hindering secretion. Collectively, these findings reveal a key target necessary for hormone-mediated inhibition of MTs required in countering diuresis providing a deeper understanding of this critical physiological process.Significance StatementThe V-type H+-ATPase (VA) proton pump, provides the driving force for transepithelial ion and fluid secretion in insect Malpighian tubules (MTs). While studies have shown that diuretic stimulation activates various signaling pathways that culminate with increased VA activity, our understanding of anti-diuretic signaling and its potential regulation involving the VA remains rudimentary. Here we show that a CAPA neuropeptide acts through the NOS/cGMP/PKG pathway that inhibits VA activity, supporting the notion that anti-diuretic regulation is mediated through reversible dissociation of the VA complexes. Our results demonstrate a critical role of VA inhibition and trafficking necessary for anti-diuretic signaling, advancing our understanding of the complex neuroendocrine regulatory mechanisms of the MTs in an important human disease vector mosquito.

Anti-diuretic hormone ITP signals via a guanylate cyclase receptor to modulate systemic homeostasis inDrosophila

“…For instance, in the locust Schistocerca gregaria , ITPa but not ITPL promotes ion and water reabsorption across the hindgut (Audsley et al ., 1992a, Audsley et al ., 1992b, King et al ., 1999, Wang et al ., 2000), thereby promoting anti-diuresis. Previous experiments have shown that the MTs are also targeted by anti-diuretic hormones: CAPA neuropeptides inhibit diuresis in some insects including Drosophila via direct hormonal actions on the renal tubules (Paluzzi et al ., 2008, MacMillan et al ., 2018, Sajadi et al ., 2020, Sajadi et al ., 2023). Given the expression of Gyc76C, a putative ITP receptor, in both the hindgut and renal tubules, ITP could modulate osmotic and/or ionic homeostasis by targeting these two excretory organs.…”

Molecular characterization, localization, and physiological roles of ITP and ITP-L in the mosquito,Aedes aegypti

“…The MTs, which are functional analogs of vertebrate kidneys, are responsible for the formation of primary urine (Coast, 2007), driven by the V-type H + -ATPase (Wieczorek, 1992) that permits transport of Na + and K + cations across the membrane (Beyenbach, 2003) via a putative H + /cation exchanger (Wieczorek et al, 2009). The MTs are regulated by various diuretic and anti-diuretic hormones, which in A. aegypti includes the biogenic amine 5-hydroxytryptamine (5HT) (Clark and Bradley, 1998; Veenstra, 1988), DH 31 (Coast et al, 2005), DH 44 (Clark et al, 1998; Clark et al, 1998), kinin-like peptides (Lu et al, 2011; Pietrantonio et al, 2005) and CAPA that inhibits the activity of select diuretic hormones (Sajadi et al, 2018; Sajadi et al, 2020; Sajadi et al, 2023). The primary urine then enters the hindgut, where it is further modified through secretory and reabsorptive processes.…”

The V-type H + -ATPase is targeted in antidiuretic hormone control of the Malpighian “renal” tubules

“…All study data are openly available at https://doi.org/10.5683/SP3/PADHU5 ( 83 ). All other data is included in the article and/or SI Appendix .…”