Angiotensin binding sites in rabbit anterior uvea and human ciliary epithelial cells

Angiotensin binding sites in membrane homogenates of rabbit iris/ciliary body and human nonpigmented ciliary epithelial (NPE) cells grown in culture were characterized using radioligand binding assays with (125I)-sarcosine1-isoleucine8-angiotensin II [(125I)-SARILE]. Scatchard analysis of the binding of (125I)-SARILE yielded linear plots with a Kd value of 0.55 +/- 0.1 nM and a Bmax of 98 +/- 23 fmol/mg protein in rabbit iris/ciliary body, and a Kd value of 0.63 +/- 0.1 nM, and a Bmax of 36.2 +/- 24 fmol/mg protein in NPE cells. Studies of the inhibition of the binding of (125I)-SARILE in rabbit iris/ciliary body were performed with a series of competing ligands, including the angiotensin receptor antagonist SARILE and the agonists angiotensin I, angiotensin II and angiotensin III. Inhibition curves for the antagonist resulted in Hill coefficients of approximately 1, consistent with the presence of a single class of binding sites with high affinity for (125I)-SARILE. Competition for the binding of (125I)-SARILE to binding sites with each of the agonists resulted in inhibition curves with Hill coefficients significantly less than 1 in the absence of GTP. However, in the presence of 100 microM GTP the Hill coefficients increased to approximately 1. The order of potencies of these agents was consistent with the pharmacologic profile of angiotensin II receptors. Thus rabbit iris/ciliary body homogenates, which include vascular tissue, contain a homogeneous population of angiotensin binding sites coupled to a guanine nucleotide binding protein. The presence of binding sites in cultured NPE cells indicates that at least some are located on the cells thought to be responsible for aqueous humor secretion.