ANGIOTENSIN-II RECEPTOR SUBTYPES AND COUPLING TO SIGNALING SYSTEMS IN CULTURED FETAL FIBROBLASTS

Angiotensin-II (AII) receptors and their coupling to signaling transduction systems were studied in fetal skin fibroblasts using the receptor subtype-specific antagonists Dup753 (type 1) and PD123177 (type 2). In primary cultures, total binding per cell remained constant from days 2-6 of culture, but after subculture levels were initially decreased, then increased until the cells reached confluence. In freshly isolated cells more than 90% of the receptors were type 2, and only a minor fraction were type 1, findings that are consistent with autoradiographic analyses and membrane binding assays. The proportions tended to reverse during culture, with type 1 receptors accounting for 60-80% and type 2 for 20-40% by day 6 of culture. In secondary culture, subtype 1 was about 50% on day 1 and increased to 70% by day 6. At subsequent passages, the proportion of receptor subtypes remained constant up to 6 days, with 90% being of the type 1 variety and 10% of type 2. Scatchard analysis of binding in the presence of the selective antagonists showed similar binding affinities for both subtypes, and the changes in receptor subtype during culture were due primarily to changes in receptor number. Treatment of primary cultures with actinomycin-D for 24 h prevented the transition from AII receptor type 2 to type 1 by increasing the absolute number of type 2 receptors, suggesting that receptor synthesis is regulated at the posttranscriptional level. Stimulation of the cells with 100 nM AII resulted in increases in inositol phosphate accumulation, an effect that was prevented by the type 1 antagonist, but not by the type 2 antagonist. In contrast with other systems in which AII inhibits adenylate cyclase, in fetal fibroblasts AII stimulated cAMP production. The increases in cAMP were more pronounced in secondary cultures in which type 1 receptor content is higher, and the effect was prevented by the type 1, but not the type 2, antagonists. These findings demonstrate the presence of both AII receptor subtypes in fetal fibroblasts, but only the type 1 receptors are coupled to a known intracellular signalling system. The changes in AII receptor subtypes may be the consequence of alterations in intercellular communication, endocrine, or paracrine factors during culture.