N-ethylmaleimide (NEM) Causes Aquaporin-2 Trafficking in the Renal Inner Medullary Collecting Duct by Direct Activation of Protein Kinase A

Title: N-ethylmaleimide (NEM) Causes Aquaporin-2 Trafficking in the Renal Inner Medullary Collecting Duct by Direct Activation of Protein Kinase A
Authors: Shaw, Stephen; Marples, David
Publisher: American Journal of Physiology: Renal Physiology
Date Published: November 09, 2004
Reference Number: 677
The antidiuretic hormone arginine vasopressin (AVP) increases the osmotic water permeability of the renal collecting ducts by inducing the shuttling of aquaporin-2 (AQP2) water channels from intracellular vesicles to the apical plasma membrane of the principal cells. This process has been demonstrated to be dependent upon the cytoskeleton and protein kinase A (PKA). Previous studies in the toad urinary bladder, a functional homologue of the renal collecting duct, have demonstrated that the sulphydryl reagent N-ethylmaleimide (NEM) is also able to activate the vasopressin-sensitive water permeability pathway in this tissue. The aim of the present study was to investigate the effects of NEM on AQP2 trafficking in a mammalian system. We show that NEM causes translocation of AQP2 from the cytosol to the plasma membrane in rat inner medullary collecting ducts; like the response to AVP, this action was also dependent upon an intact cytoskeleton and PKA. This effect is not mediated by cAMP, but results from direct activation of PKA by NEM.
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This translation by the NDI Foundation is to assist the lay reader. To provide a clear, accessible interpretation of the original article, we eliminated or simplified some technical detail and complicated scientific language. We concentrated our translation on those aspects of the article dealing directly with NDI. The NDI Foundation thanks the researchers for their work toward understanding and more effectively treating this disorder.
© Copyright NDI Foundation 2007 (JC)

Aquaporin 2 (AQP2) is a protein that allows water to pass through the membrane of the principal cells in the kidney collecting duct. In order for AQP2 to perform this function, it must first travel to the apical section of the cell membrane from the cell interior. The molecular sequence that accomplishes this begins when the hormone, arginine vasopressin (AVP) binds with the vasopressin 2 receptor (V2R). This activates adenylate cyclase and results in an increase in cyclic adenosine monophosphate (cAMP), which in turn activates protein kinase A (PKA). This catalyzes the addition of a phosphate group on a specific point on the AQP2 protein. The AQP2 then is shuttled in tiny sacs called intracellular vesicles along the cell’s internal reinforcement system (the cytoskeleton) to the cell membrane.

Research using toad urinary bladders indicated another substance, N-ethylmaleimide (NEM) could affect transfer of AQP2 to the membrane. Shaw and Marples investigated the effects of NEM on AQP2 movement in mammalian cells by experimenting with laboratory rat cell cultures. They found that NEM is able to induce the movement of AQP2 from the rat cell interior to the cell membrane. Its ability to do this is dependent on the amount of NEM used in the laboratory cell culture, the most effective amount being 0.1 millimolar (mM). Just as with AVP, successful movement of AQP2 to the cell membrane initiated by NEM requires an intact cytoskeleton and the presence of PKA. The researchers reason that NEM stimulates AQP2 movement primarily by activating PKA directly, thus bypassing AVP receptors and adenylate cyclase.

The research team discovered that when both NEM and a maximal dose of AVP are applied to the cell culture, the amount of AQP2 moving to the membrane is increased. This suggests there are at least two molecular signaling paths involved in the efficient movement of AQP2 to the cell membrane. Shaw and Marples conclude that NEM’s ability to initiate AQP2 movement to the membrane via activation of PKA could be of value to researchers studying the cellular response of the kidney collecting duct to AVP. Potentially, such an approach might also be useful in patients with NDI.