Angiotensin (at1a) receptor mediated increases in transcellular sodium transport in proximal tubule cells.. Thekkumkara, Thomas J., Rochelle Cookson, and Stuart L. Linas. Department of Medicine, University of Colorado Health Sciences Center, Denver Health Medical Center, 4200 East Ninth Ave, Denver, CO 80262
APStracts 5:0026F, 1998.
Angiotensin II (AngII) acting through Angiotensin type 1A receptors (AT1A) is important in regulating proximal tubule salt and water balance. AT1A are present on apical (Ap) and basolateral (Bl) surfaces of proximal tubule epithelial cells (PTEC). The molecular mechanism of AT1A function in epithelial tissue is not well understood because specific binding of AngII to intact PTEC has not been found and a number of isoforms of AT receptors are present in vivo. To overcome this problem, we developed a cell line from opossum kidney (OK) proximal tubule cells which stably express AT1A (Kd = 5.27 nM, Bmax = 6.02 pmol/mg protein). Characterization of non -transfected OK cells revealed no evidence of AT1A mRNA (Reverse transcriptase-polymerase chain reaction analysis) or protein ([125I] AngII binding studies) expression. In cells stably expressing AT1A, AngII binding was saturable, reversible and regulated by G proteins. Transfected receptors were coupled to increases in intracellular calcium and inhibition of cAMP. To determine the polarity of AT1A expression and function in proximal tubules, transfected cells were grown to confluence on membrane inserts under conditions which allowed selective access to Ap or Bl surfaces. AT1A were expressed on both Ap (Kd = 8.7 nM, Bmax = 3.33 pmol/mg protein) and Bl surfaces (Kd = 10.1 nM, Bmax = 5.50 pmol/mg protein). Both Ap and Bl AT1A receptors underwent agonist-dependent endocytosis (Ap receptor; t1/2 = 7.9 min, Ymax = 78.5% and Bl receptor; t1/2 = 2.1 min, Ymax = 86.3%). In cells transfected with AT1A, AngII caused time and concentration dependent increases in transepithelial 22Na transport (two fold over control at 20 min) by increasing Na/H exchange. In conclusion, we have established a stable proximal tubule cell line which express AT1A on both Ap and Bl surfaces, undergo agonist -dependent receptor endocytosis and are functional as evidenced by inhibition of cAMP and increases in cytosolic calcium mobilization and transepithelial sodium movement. This cell line should prove useful for understanding the molecular and biochemical regulation of AT1A expression and function in PTEC. 

Received 11 August 1997; accepted in final form 20 January 1998.
APS Manuscript Number F267-7.
Article publication pending Am. J. Physiol. (Renal Physiology).
ISSN 1080-4757 Copyright 1998 The American Physiological Society.
Published in APStracts on 28 January 1998