Signaling and growth responses of LLC-PK1/Cl4 cells transfected with the rabbit AT1 ANG II receptor

Abstract

Angiotensin II (ANG II) receptors of the AT1 subtype are present on the apical and basolateral membranes of renal proximal tubule cells. Cells of the proximal tubulelike cell line, LLC-PK1/Cl4, were transfected with an expression plasmid containing cDNA encoding the rabbit AT1 ANG II receptor. In transfected cells, specific binding of 125I-ANG II was detected on both apical and basolateral membranes; wild-type LLC-PK1/Cl4 cells did not express ANG II receptors. In transfected cells, apical or basolateral ANG II increased both S6 kinase activity and incorporation of [3H]leucine. In cells pretreated with pertussis toxin, the stimulatory effect of apical or basolateral ANG II on [3H]leucine incorporation was abolished. In contrast, ANG II did not affect mitogenesis, determined by [3H]thymidine incorporation. Apical or basolateral ANG II (10(-6) M) stimulated phosphoinositide turnover by 13.4 +/- 4.4% (n = 8) and 16.3 +/- 4.2% (n = 9), respectively. The activity of protein kinase C, determined by phosphorylation of a specific protein kinase C peptide substrate, was also stimulated by ANG II in transfected cells. Apical or basolateral ANG II had no significant effect on cellular adenosine 3',5'-cyclic monophosphate levels. In permeabilized transfected cells, apical ANG II (10(-6) M) inhibited the phosphorylation of a specific peptide substrate of protein kinase A; lower apical concentrations or basolateral ANG II were without significant effect. These results indicate that AT1 ANG II receptors sort to both apical and basolateral membranes in renal epithelial cells and are coupled to activation of phospholipase C. ANG II stimulates protein synthesis by binding to either apical or basolateral receptors; this effect requires coupling to G proteins and may be mediated by activation of S6 kinase. Because high concentrations of ANG II exist in proximal tubule, binding to apical and basolateral receptors may regulate proximal tubule cell growth under physiological conditions.

Role of EGF Receptor Activation in Angiotensin II–Induced Renal Epithelial Cell Hypertrophy

“…Growth factors such as Ang II have been implicated in development of renal cell hypertrophy through activation of AT1R (21,33). We previously reported that AT1R-transfected LLCPKcl4 cells do not increase DNA synthesis but increase protein synthesis in response to Ang II, as indicated by increased [ 3 H]leucine incorporation without increases in [ 3 H]thymidine incorporation (21).…”

“…We previously reported that AT1R-transfected LLCPKcl4 cells do not increase DNA synthesis but increase protein synthesis in response to Ang II, as indicated by increased [ 3 H]leucine incorporation without increases in [ 3 H]thymidine incorporation (21). In rabbit proximal tubular cells and the rat kidney epithelial cell line NRK-52E, TGF-␤ converts EGF-induced hyperplasia into hypertrophy by inhibiting mitogen-induced hyperphosphorylation of pRB (the product of the retinoblastoma gene) (34).…”

“…This cDNA then was cloned into the NotI and HindIII sites of the mammalian expression vector pRc/CMV (Invitrogen, San Diego, CA) (21). A total of 1 g/ml of this AT1R cDNA construct or empty pRc/CMV vector alone was used for transfection into LLCPKcl4 cells using LipofectAMINE (Life Technologies, Inc.), and G418-resistant individual clones were isolated as described (19 …”

The Intrarenal Renin-Angiotensin System: From Physiology to the Pathobiology of Hypertension and Kidney Disease

“…Several studies have demonstrated that, despite normal or suppressed plasma renin activity, the intrarenal content of renin is increased in experimental diabetes models (Correa-Rotter et al, 1992;Jaffa et al, 1992;Burns and Harris, 1995;Carey and Siragy, 2003). Studies using Otsuka Long-Evans Tokushima Fatty rats, a model of type 2 diabetic nephropathy, showed increased activity of ACE in the kidney, although serum ACE activity was lower than that in control animals (Taniguchi et al, 2002).…”

Central role for Rho in TGF-β1-induced α-smooth muscle actin expression during epithelial-mesenchymal transition

“…LLC-PK 1 cells (Cl4) stably expressing the rabbit AT1 receptor were a kind gift from Dr. R. Harris (12). Cells were kept in DMEM containing 10% FBS, 100 U/ml penicillin, and 100 g/ml streptomycin in a humidified incubator at 37°C under 5% CO 2.…”