The effect of the corticotropin releasing factor-like diuretic hormone, Manse-DH, on the molecular mechanism of activation of vacuolar-ATPase in Manduca sexta Malpighian tubules

The control of water balance is crucial to the survival of insects. Two types of hormones, diuretic and antidiuretic, have been implicated in maintaining the insect's ability to maintain its water balance. The first hormone to be identified is a corticotropin releasing factor-like diuretic hormone; it was discovered in Manduca sexta and has been shown to be active on the cryptonephric tubules of the larval stage. This diuretic hormone, Manse-DH, has been shown to elevate cAMP which then activates the Na+-K+-2Cl- cotransporter causing an increase in the transportation of ions across the cell to the lumen of the Malpighian tubule were they are taken to the rectum for reabsorption or excretion. Water follows ion movement through osmosis and is reabsorbed in the rectum.;It is postulated that the Vacuolar – ATPase (V-ATPase) is responsible for providing the power for specific ion transport proteins, i.e., Na +/H+ and K+/H+ antiporters, by hydrolyzing ATP. It had been unknown if Manse-DH had a direct effect on the activity of V-ATPase, although it had been shown that Manse-DH increases the excretion of Na+ and K+ ions. To address the effect of Manse-DH on the activity of the V-ATPase and to further identify key proteins involved in the activation, a technique to isolate the two distinct membranes of the principal cell was devised.;The isolation of the basolateral membrane, the location of the Manse-DH receptor and the Na+-K+-2Cl- cotransporter, was easily accomplished with differential centrifugation. The resulting method gave high signal intensity for the Na+-K +-2Cl- cotransporter antibody staining with only a 1% recovery of the mitochondrial membrane marker enzyme, succinate dehydrogenase, and a 1% recovery of the apical membrane marker enzyme, alkaline phosphatase.;The isolation of the apical membrane, the location of the V-ATPase and specific ion transport proteins, proved to be more difficult. The apical membrane is expected to be in close proximity to mitochondria to provide ATP for the V-ATPase and subsequently ion transport. The method developed here was only able to partially purify the apical membrane from contaminating mitochondrial membrane.;The partially purified apical membrane was used to determine ATP hydrolysis activity of the V-ATPase. An assay that measures ATP hydrolysis directly was created. The method measures the amount of liberated 33P from gamma labeled ATP which is monitored by liquid scintillation analysis.;With the devised ATPase assay the direct effect of Manse-DH on V-ATPase activity was demonstrated. A 2-fold increase in V-ATPase hydrolysis activity was observed in tissue treated with 10 nM Manse-DH as compared to non-treated V-ATPase. It also was observed that the addition of 10 μM H-89, a specific protein kinase A inhibitor, blocked the activation caused by Manse-DH.;Proteomic analysis of the changes in cytosolic protein suggested a change in the redox state of the cell as 6 different spots show changes in amount of glutathione S-transferase with 10 nM Manse-DH treatment vs. non-treated. Further analysis of the GST activity of the cell proved to be inconclusive as three different classes of GST were shown to be either higher in the Manse-DH treatment or in the non-treated, demonstrating the cells tight regulation of its overall redox states to maintain homeostasis.;In addition to the changes in the GST protein spots, a spot identified as 14-3-3, a protein known to interact with phosphorylation sites of other proteins changing their activity, was observed only in non-treated tissue. We attempted to observe if the lack of presence of 14-3-3 in the Manse-DH treated tissue was due to its binding to the A-subunit of the V-ATPase, an interaction observed in the barley V-ATPase after activation with blue light. This also proved to be incondusive, but the presence of 14-3-3 in the non-treated tissue and not in the Manse-DH treatment suggests its involvement in the increase of ion excretion of Malpighian tubules.