RECEPTOR-MEDIATED ENDOCYTOSIS OF ANGIOTENSIN-II IN RAT MYOMETRIAL CELLS

The events involved in the processing of the angiotensin II (Ang II)-r eceptor complex were studied in primary cultures of rat myometrial cel ls. Ang II bound to rat myometrial cells in aspecific, time-and temper ature-dependent fashion. Pretreatment with cycloheximide did not inter fere with binding up to 3 ha, but inhibited increases in binding obser ved over longer periods. The [H-3]Ang II binding to intact cells was i nhibited by dithiothreitol (DTT), and the rank order of potency of Ang II and nonpeptide antagonists to inhibit the [H-3]Ang II binding was Ang II > Losartan much greater than PD 123319 or CGP 42112B, indicatin g the presence of the AT(1) receptor type. Whereas most of the [H-3]An g II binding at 4 degrees was susceptible to acid or pronase treatment , binding at 35 degrees was resistant to both treatments, suggesting a n internalization of the Ang II-receptor complex. Phenylarsine oxide ( PAO) and N ethylmaleimide (NEM) caused a concentration-dependent inhib ition when the binding assay was performed at 35 degrees, but no effec t was observed at 4 degrees, indicating that these agents did not alte r cell-surface binding but actually prevented the internalization proc ess. Simultaneous treatment with 1 mM DTT or beta-mercaptoethanol prev ented the inhibitory effect of NEM, but only DTT could prevent the inh ibition caused by PAO, indicating that two closely located sulfhydryl groups must be involved in the internalization process. Chloroquine (1 00 mu M) inhibited the [H-3]Ang II dissociation from cells, and monens in (25 mu M) induced a 30% inhibition of [H-3]Ang II binding (35 degre es, 3 hr), suggesting endosomal processing of the Ang II-receptor comp lex with receptor recycling to the cell surface. These results indicat e that Ang II binding to AT(1) receptors in rat myometrial cells is fo llowed by internalization of the Ang II-receptor complex and recycling of the receptor to the cell surface. (C) 1997 Elsevier Science Inc.