ABSTRACTa2-Adrenergic receptors comprise a heterogeneous population based on pharmacologic and molecular evidence. We have isolated a cDNA clone (pRNGa2) encoding a rat a2-adrenergic receptor. A rat kidney cDNA library was screened with an oligonucleotide complementary to a highly conserved region found in all biogenic amine receptors described to date. The deduced amino acid sequence displays many features of guanyl nucleotide-binding protein-coupled receptors except it does not have a consensus N-linked glycosylation site near the amino terminus. Membranes prepared from COS cells transfected with pRNGa2 DNA display high affinity and saturable binding to [3H]rauwolscine (Kd = 2 nM).Competition curve data analysis shows that RNGa2 protein binds to a variety of adrenergic drugs with the following rank order of potency: yohimbine 2 chlorpromazine > prazosin 2 clonidine > norepinephrine 2 oxymetazoline. RNGa2 RNA accumulates in both rat kidney and neonatal rat lung (predominant species is 4000 nucleotides). When a cysteine residue (a2-C10), isolated from a human genomic library, encodes an a2A subtype (3), whereas a second clone (a2-C4), isolated from a human kidney cDNA library, encodes a subtype with some of the properties expected of Bylund's a2B subtype (4). For example, the a2-C4 protein has a greater affinity for prazosin than oxymetazoline but is glycosylated following expression of the cDNA in COS cells. Venter and colleagues (5) have further shown that the a2A-adrenergic clone is functional in that Chinese hamster ovary cells transfected with this DNA respond to a2-adrenergic agonists by inhibiting forskolin-stimulated increases in cAMP. Southern blots of human genomic DNA hybridized to a2A-adrenergic receptor DNA showed the existence of three sets of bands, and only two of these were accounted for by known (i.e., a2-C10 and a2-C4) adrenergic receptor genes (3, 4). Thus it appears that there exists a third, closely related gene in the a2-adrenergic subfamily.To obtain the full set of a2-adrenergic clones for our studies on the role of a2-adrenergic receptors in blood pressure control, we screened a rat kidney cDNA library with an oligonucleotide derived from a consensus nucleotide sequence of known biogenic amine receptors. In this report we describe a cDNA encoding a molecule with the ligand binding, tissue distribution, and structural properties expected of an a2B-adrenergic receptor. § METHODS Cloning and Sequence Analysis. Two degenerate oligonucleotides (5'-CTNGAYGTGCTGTKCTGCACSKCSTC-CATCPTGMACCTGTGCG-3' and 5'-CAGSSYGATGPCG-CACAGGT-3'; N = G, A, T, or C; Y = T or C; K = G or T; S = G or C; P = A or G; and M = A or C) were synthesized on a Biosearch 8600 syfnthesizer and purified. The 3' terminal nonamers of these oligonucleotides are complementary; labeled, double-stranded DNA was prepared by the action ofthe Klenow fragment of DNA polymerase I on the annealed oligonucleotide templates in the presence of radiolabeled (32p) deoxynucleoside triphosphates. The resulting 54-residue, 8192-fold degenerat...

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In two multicenter phase III efficacy studies, blood samples were obtained to evaluate the serum concentrations of 17beta-estradiol (E2) and unconjugated estrone (E1) after administration of a percutaneous gel or transdermal patch containing estradiol. In postmenopausal women, normal laboratory E2 and E1 serum concentrations range from 10-30 pg/mL and 20-40 pg/mL, respectively. Study subjects were healthy postmenopausal women with moderate to severe hot flushes occurring at least seven times daily or 60 times per week. Study 1 was a randomized, double-blind, multicenter study of percutaneous E2 gel 1.25 or 2.5 g (0.75 and 1.5 mg E2, respectively) versus placebo gel. Study 2 was a double-blind (blinded to E2 gel dose), randomized, active-controlled, multicenter, 12-week phase 3 study of E2 gel 0.625, 1.25, or 2.5 g (0.375, 0.75, or 1.5 mg E2, respectively) versus a transdermal E2 patch delivering 0.05 mg E2 per day. Serum E2 and E1 concentrations were evaluated at baseline and at week 12 for study 1 and at baseline and weeks 4, 8, and 12 for study 2 using radioimmunoassay. Median serum concentrations of E2 after 1.25- and 2.5-g gel administration appeared to be dose-proportional throughout both studies. In study 1, the median serum concentrations of E2 at week 12 were 33.5 and 65.0 pg/mL for 1.25- and 2.5-g gel dose, respectively. The corresponding E1 values were 49.0 and 58.0 pg/mL. In study 2, both E2 and E1 concentrations were relatively stable at weeks 4, 8, and 12. E2 values at week 12 for 0.625-, 1.25-, and 2.5-g gel doses and E2 patch were 25.0, 32.0, 60.0, and 38.5 pg/mL, respectively. The corresponding E1 values were 39.0, 41.0, 62.5, and 40.0 pg/mL. Application of the 1.25-g gel dose and a transdermal patch delivering 50 microg per day of E2 resulted in comparable median E2 and E1 concentrations. However, the 0.625-g gel dose did not produce E2 levels in a range expected to be consistently therapeutic in most postmenopausal women.