Nicotinic acetylcholine receptors are key molecules in cholinergic transmission in the nervous system. Because of their structural complexity, only a limited number of subtype-specific agonists and antagonists are available to study nicotinic receptor functions. To overcome this limitation, we used voltageclamp recordings to examine the effects of several frog skin alkaloids on acetylcholine-elicited currents in Xenopus laevis oocytes expressing major types of neuronal nicotinic receptors (␣4␤2, ␣7, ␣3␤2, ␣3␤4, and ␣4␤4). We found that the 5,8-disubstituted indolizidine (Ϫ)-235B acted as a potent noncompetitive blocker of ␣4␤2 nicotinic receptors (IC 50 ϭ 74 nM). This effect was highly selective for ␣4␤2 receptors compared with ␣3␤2, ␣3␤4, and ␣4␤4 receptors. The inhibition of ␣4␤2 currents by (Ϫ)-235B was more pronounced as the acetylcholine concentration increased (from 10 nM to 100 M). Moreover, the blockade of ␣4␤2 currents by (Ϫ)-235B was voltage-dependent (more pronounced at hyperpolarized potentials) and usedependent, indicating that (Ϫ)-235B behaves as an openchannel blocker of this receptor. Several other 5,8-disubstituted indolizidines (5-n-propyl-8-n-butylindolizidines), two 5,6,8-trisubstituted indolizidines ((Ϫ)-223A and (ϩ)-6-epi-223A), and a 1,4-disubstituted quinolizidine ((ϩ)-207I) were less potent than (Ϫ)-235B, and none showed selectivity for ␣4␤2 receptors. The quinolizidine (Ϫ)-1-epi-207I and the tricyclic (ϩ)-205B had 8.7-and 5.4-fold higher sensitivity, respectively, for inhibition of the ␣7 nicotinic receptor than for inhibition of the ␣4␤2 receptor. These results show that frog alkaloids alter the function of nicotinic receptors in a subtype-selective manner, suggesting that an analysis of these alkaloids may aid in the development of selective drugs to alter nicotinic cholinergic functions.

Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that mediate and/or modulate synaptic transmission in the nervous system. [1][2][3] In mammalian brain, neuronal nAChR subunits are identified as a2-a7, a9, a10, and b 2-b 4. 4,5) Ligand-binding studies indicate that the most abundant native nAChRs in the central nervous system are a4b 2 heteromeric receptors and a7 homomeric receptors.6) Although nAChRs have been implicated in several neurological disorders including nicotinic addiction, epilepsy, Parkinson's disease and Alzheimer's disease, 7-9) nicotinic ligands and nicotinic-based therapies have only slowly and minimally become available. [10][11][12][13] A variety of alkaloids were isolated from amphibian skins, including indolizidines, quinolizidines, decahydroquinolines and piperidines, some of which have been identified as noncompetitive blockers of nAChRs at neuromuscular junction and in ganglionic PC12 cells 14) and recombinant neuronal nAChRs expressed in Xenopus oocytes.15) To date, a wide range of toxins, including different types of decahydroquinolines and quinolizidines, have been isolated from ascidians (tunicates, subphylum Urochordata, class Ascidiacea) commonly known as sea squirts.16) Nevertheless, none of the ascidian alkaloids has been tested in pharmacological research to develop therapeutically useful nicotinic drugs. Previously, we have redefined the absolute configuration of two ascidian alkaloids by enantioselective total synthesis: (Ϫ)-pictamine, a quinolizidine alkaloid from Clavelina picta, and (Ϫ)-lepadin B, a decahydroquinoline alkaloid from Clavelina lepadiformis. In the present study, we investigated the effects of (Ϫ)-pictamine and (Ϫ)-lepadin B on the major types of nicotinic receptors (a4b 2 and a7) expressed in Xenopus oocytes. MATERIALS AND METHODS MaterialsAll alkaloids used in this study were synthesized and are shown in Fig. 1. The synthesis of (Ϫ)-pictamine and (Ϫ)-lepadin B was performed as reported previously.17,18) The 3-piperidinol alkaloid (+)-prosafrinine, originally isolated from the leaves of Prosopis africana, has also been synthesized as described previously.19) The structures of these alkaloids are identical to those of the natural products. The other reagents were purchased from Sigma (St. Louis, MO, U.S.A.), unless indicated otherwise.Expression in Xenopus Oocytes To assess the nAChR activity, we measured current through the receptor-channel following the techniques described in detail by Tsuneki et al. 15) In brief, oocytes were isolated from Xenopus laevis frogs and were microinjected with 20 ng cDNAs in the nucleus. The mixture of cDNAs of mouse a4 and mouse b 2 subunits was injected in a ratio of 1 : 1, whereas mouse a7 cDNA was injected alone. Oocytes were used in electrophysiological recordings 3 to 8 d after the injection. All experiments were carried out in accordance with guidelines approved by the Toyama Medical and Pharmaceutical Univer- Ascidians (sea squirts) contain a wealth of alkaloids, but their influence o...

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The human aldo-keto reductase (AKR) 1C3, also known as type-5 17β-hydroxysteroid dehydrogenase and prostaglandin F synthase, has been suggested as a therapeutic target in the treatment of prostate and breast cancers. In this study, AKR1C3 inhibition was examined by Brazilian propolis-derived cinnamic acid derivatives that show potential antitumor activity, and it was found that baccharin (1) is a potent competitive inhibitor (K(i) 56 nM) with high selectivity, showing no significant inhibition toward other AKR1C isoforms (AKR1C1, AKR1C2, and AKR1C4). Molecular docking and site-directed mutagenesis studies suggested that the nonconserved residues Ser118, Met120, and Phe311 in AKR1C3 are important for determining the inhibitory potency and selectivity of 1. The AKR1C3-mediated metabolism of 17-ketosteroid and farnesal in cancer cells was inhibited by 1, which was effective from 0.2 μM with an IC(50) value of about 30 μM. Additionally, 1 suppressed the proliferation of PC3 prostatic cancer cells stimulated by AKR1C3 overexpression. This study is the first demonstration that 1 is a highly selective inhibitor of AKR1C3.