(53g) cAMP Stimulation Regulates Aquaporin-1 Mediated Transendothelial Water Transport In Bovine Aortic Endothelium Monolayers

Abstract — The pressure inside large arteries is typically ~100 mmHg higher than outside of them. This difference, ΔP, drives a flow of plasma, mainly water and advected/swept-along solutes, that would otherwise only transport by (much slower) diffusion [1], across the porous vessel wall. It is natural to ask whether this flow enters the wall across the endothelium solely through inter-endothelial cell (EC) junctions or whether a portion of the water traverses the ECs via membrane proteins from the ubiquitous aquaporin (AQP) family [2].  Data from our group show that bovine aortic ECs (BAECs) express AQP-1 in cultured monolayers and that rat aortic ECs (RAECs) express AQP-1 in culture and in excised vessels.  Chemically blocking AQP-1 or knocking down AQP-1 expression significantly reduces endothelial hydraulic conductivity (L_pe).  Upregulating AQP-1 expression on L_pe in vessels and monolayers might be a route towards washing  LDL out of the subendothelial intima (SI) before it can bind significantly, a process that can lead to lesions. 

Belkacemi et al. [3] showed a 4-fold protein expressional change of AQP1 in trophoblast cells after treatment with arginine vasopressin (AVP).  Additionally, expression of vasopressin type-2 receptors (V2R) has been confirmed in human lung microvascular endothelial cells [4] and rat aortic strips [5].  We sought to investigate the effect of V2R stimulation on L_pe and AQP-1 expression in BAEC monolayers for both short and long term treatments. 

We found that AVP increased L_pe  34% and AQP-1 expression 52% in BAEC monolayers as compared to untreated monolayers.

AVP treatment had no effect on albumin transport across BAEC monolayers under either convective or diffusive conditions, indicating  no apparent affect on junctional tightness or length.  Since albumin transports significantly by the paracellular route, this indicates that AVP treatment does not affect this pathway and the change  in L_pe is likely due to the transcellular AQP-1 route.

Short term forskolin (F) treatment is known to increase intercellular cAMP, which causes membrane shuttling of internal AQP-1 to the cell membrane. We found that short term F treatment increased L_pe  172% but did not significantly change AQP-1 expression, which is consistent with the shuttling hypothesis. Satavaptan (S), an inverse V2R agonist, treatment decreased L_pe -46% and AQP-1 expression -44%.  F/S treatment did not significantly change L_pe and decreased aquaporin-1 expression -29%.  A/S treatment decreased L_pe -38 and caused no significant change in AQP-1 expression. 

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[2]   Preston, G. M. and Agre P. (1991). "Isolation of the cDNA for erythrocyte integral membrane protein of 28 kilodaltons: member of an ancient channel family." Proc Natl Acad Sci U S A 88(24): 11110-4.

[3]   Belkacemi, L., Beall, M.H., Magee, T.R., Pourtemour, M., Ross, M.G. (2008). "AQP1 gene expression is upregulated by arginine vasopressin and cyclic AMP agonists in trophoblast cells." Life Sci. 82(25-26): 1272-80.

[4]   Kaufmann, J.E. and Vischer, U.M. (2003). “Cellular mechanisms of the hemostatic effects of desmopressin (DDAVP).“J Thrombosis and Haemostasis, 1: 682–689.

[5]   Méchaly I, Laurent F, Portet K, Serrano J, Cros G. (1999). "Vasopressin V2 (SR121463A) and V1a (SR49059) receptor antagonists both inhibit desmopressin vasorelaxing activity." Eur J Pharmacol. 383(3):287-90.