STABLE EXPRESSION OF A FUNCTIONAL-RAT ANGIOTENSIN-II (AT(1A)) RECEPTOR IN CHO-K1 CELLS - RAPID DESENSITIZATION BY ANGIOTENSIN-II

The octapeptide angiotensin II mediates the physiological actions of t he renin-angiotensin system through activation of several angiotensin II receptor subtypes; in particular the AT(1). In many tissues, the pr esence of multiple angiotensin II receptor subtypes, together with a l ow number of receptors, makes it difficult to study biological respons es to physiological concentrations (10(-11)-10(-9) M) of angiotensin I I. Also, cultured cells show diminished angiotensin II receptor bindin g with respect to time in culture and passage number. To address these problems, we expressed the recombinant AT(1A) receptor in CHO-K1 cell s. The stably transfected receptor was characterized using radioligand binding studies and functional coupling to cytosolic free calcium. Ra dioligand binding of [I-125] angiotensin II to the angiotensin II rece ptor was specific, saturable, reversible and modulated by guanine nucl eotides. Like the endogenous AT(1A) receptor, reported in a variety of tissues, the specific, noncompetitive, nonpeptide An receptor antagon ist, EXP3174, blocked binding of [I-125] angiotensin II to the transfe cted receptor. Scatchard analysis demonstrated that the transfected re ceptor had a dissociation constant of 1.9 nM with a density of 3.4 pmo l/mg protein. An important feature of many of the responses to angiote nsin II is the rapid desensitization that occurs following agonist occ upancy and the development of tachyphylaxis. In AT(1A) receptor transf ected CHO-K1 cells, angiotensin II (10(-9) M) stimulated a rapid incre ase in cytosolic free calcium that was completely desensitized within 50 sec following receptor occupancy. Agonist induced desensitization w as unaffected when receptor internalization was blocked by pretreatmen t with concanavalin A or incubation at 4 degrees C, and no changes in AT(1A) receptor affinity or number were observed, Receptor desensitiza tion was also unaffected by inhibition or activation of protein kinase C. Thus, we have established a permanent, high-level transfectant of the AT(1A) receptor in CHO-K1 cells and have shown that these receptor s rapidly desensitize following exposure to physiological concentratio ns of agonist. The mechanism of rapid desensitization is not related t o receptor sequestration, internalization or controlled by PKC phospho rylation. This provides an excellent model for studying AII actions me diated through a specific receptor subtype, at subnanomolar concentrat ions.