No abstract

The AT4 receptor specifically binds Angiotensin (Ang) IV and is distinct from the AT1 and AT2 receptors which bind Ang II and Ang III. The AT4 receptor in bovine adrenal cortex has a Kd of 0.74 +/- 0.14 nM and a Bmax of 3.82 +/- 1.12 pmol/mg prot. Competition curves demonstrated the following rank order of affinity: Ang IV >> Ang III >> d-Arg - Ang II >> Sar1,Ile8 - Ang II = Ang II = Ang II (1-7) >> DuP753 = CGP42112A = PD 123177. AT4 receptors were present in many tissues from several mammalian species including human and monkey. AT4 and AT1/AT2 receptors revealed a differential distribution in the rat kidney.

Rat brain angiotensin II (Ang II) receptors were solubilized with a yield of 30-40% using the synthetic detergent 3[(3-cholamidopropyl)dimethylammonio)]-1-propanesulfonate. Kinetic analysis employing the high-affinity antagonist 125I-Sar1,Ile8-Ang II indicated that the solubilized receptors exhibited the same properties as receptors present within intact brain membranes. Furthermore, there was a positive correlation (r = 0.99) between the respective pIC50 values of a series of agonist and antagonists competing for 125I-Sar1,Ile8-Ang II labeled binding sites in either solubilized or intact membranes. Moreover, covalent labeling of 125I-Ang II to solubilized receptors with the homo-bifunctional cross-linker disuccinimidyl suberate, followed by gel filtration, revealed one major and one minor binding peak with apparent molecular weights of 64,000 and 115,000, respectively. Two binding proteins of comparable molecular weights (i.e., 112,000 and 60,000) were also identified by covalent cross-linking of 125I-Ang II to solubilized brain membranes followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. In contrast, only the smaller molecular mass binding protein was observed when solubilized membranes were labeled with the antagonist 125I-Sar1,Ile8-Ang II prior to gel filtration, and chromatofocusing of antagonist labeled sites revealed only one peak with an isoelectric point of 6.2. The successful solubilization of these binding sites should facilitate continued investigation of Ang II receptors in the brain.

Although pituitary prolactin (PRL) exhibits size and charge variability, questions concerning the structural heterogeneity and biological potency of hormonal forms secreted in vivo remain. In the present studies, monomeric PRL in male rat pituitaries and plasma was fractionated by Sephacryl S-100 size exclusion chromatography and aqueous chromatofocusing to resolve size and charge forms under conditions compatible with optimum preservation of biological activity. Individual hormonal variants were subsequently evaluated for their ability to stimulate the growth of PRL receptor-bearing rat lymphoma cells in vitro. Pituitary elution profiles contained several cross-reactive size variants ranging from 30.4 to 21.5 kD in M_r; major hormonal peaks were eluted at 25.6, 24.3, and 23.6 kD. Multiple size forms of PRL were also detected in plasma profiles, with predominant peaks eluting between 26.5 and 21.5 kD M_Γ in size. Mean B/I ratios, established as an index of relative biopotency, varied significantly between size variants obtained from pituitary and plasma. Pituitary PRL size variants of 27.1 24.3, and 21.5 kD exhibited greatest potency in the in vitro bioassay, whereas the 25.6- and 23.6-kD forms were least potent under these conditions. Of the PRL size variants detected in peripheral plasma, those of 24.3 and 21.5 kD size were characterized by highest mean B/I ratios. Pituitary 24.3-kD PRL was chromatofocused as five charge variants of pI 5.34, 5.31, 5.26, 5.20, and 5.14; only some of these isomers are apparently secreted in vivo, since pI values for plasma charge isomers ranged from 5.26 to 5.14. Charge isomers of pituitary 24.3-kD PRL exerted variable mitogenicity in the Nb₂ in vitro bioassay. The highest mean B/I ratio was associated with the relatively basic 24.3-kD isomer of pI 5.31; the relative biopotency of more acidic charge isomers was progressively diminished with increasing acidic charge. In summary, the present findings show that both size and charge variants of male rat pituitary PRL exhibit differential biopotency in vitro, indicative of their functional heterogeneity. The demonstration of multiple hormonal forms in peripheral plasma suggests that the net biopotency of circulating PRL reflects the sum of activity of structurally and functionally diverse molecules.