ABSTRACT
Ascidians (sea squirts) contain a wealth of alkaloids, but their influence over neuronal nicotinic acetylcholine receptors (nAChRs) has not been evaluated. In this study, we examined the effects of two synthetic compounds, (-)-pictamine, a quinolizidine alkaloid from Clavelina picta, and (-)-lepadin B, a decahydroquinoline alkaloid from Clavelina lepadiformis, on major types of neuronal nicotinic receptors (alpha4beta2 and alpha7) expressed in Xenopus oocytes. We found that these alkaloids are potent blockers at these receptors: acetylcholine-elicited currents through alpha4beta2 and alpha7 receptors were blocked by (-)-pictamine with IC(50) values of 1.5 microM and 1.3 microM, respectively, and by (-)-lepadin B with IC(50) values of 0.9 microM and 0.7 microM, respectively. Interestingly, no recovery was observed after the removal of (-)-pictamine in oocytes expressing alpha4beta2 receptors, whereas the inhibited alpha7 currents quickly recovered after the removal of (-)-pictamine. Since there are few compounds that elicit irreversible blocks of alpha4beta2 receptors, (-)-pictamine will be a novel, valuable tool to remove the alpha4beta2-nAChR action from neuronal activities mediated by these two major types of nAChRs.
Subject(s)
Alkaloids/pharmacology , Heterocyclic Compounds, 2-Ring/pharmacology , Nicotinic Antagonists/pharmacology , Quinolines/pharmacology , Urochordata/chemistry , Alkaloids/chemistry , Animals , Dose-Response Relationship, Drug , Heterocyclic Compounds, 2-Ring/chemistry , Inhibitory Concentration 50 , Membrane Potentials/drug effects , Nicotinic Antagonists/chemistry , Oocytes/drug effects , Oocytes/metabolism , Quinolines/chemistry , Receptors, Nicotinic/metabolism , Xenopus laevisABSTRACT
Nicotinic acetylcholine receptors are found in microvascular endothelial cells. To reveal the functional role in cerebral angiogenic processes, we studied the nicotinic modulation of proliferation activity in cultured bovine and porcine cerebral microvascular endothelial cells. The proliferation activity was determined by an increase in the number of cells present in culture dishes. When the bovine cerebral endothelial cells at different passages were cultured in the presence of nicotine (10 nM), the proliferation activities were significantly increased in the cells at passage 1 and passage 3, but not at passage 4. Reverse transcriptase-polymerase chain reaction studies demonstrated that the expression of mRNAs coding for alpha3 nicotinic receptor subunit was significantly reduced in the bovine cerebral endothelial cells at passage 4, compared with that at passage 1. The proliferation of porcine cerebral endothelial cells (passage 1) was enhanced by acetylcholine (10 nM-100 microM) in the presence of atropine, a muscarinic antagonist, and this enhancing effect was inhibited by hexamethonium (100 microM, a nicotinic antagonist). The stimulation by acetylcholine (1 microM, with atropine) or nicotine (10 nM) induced the phosphorylation of a mitogen-activated protein (MAP) kinase (extracellular-signal regulated kinase: ERK) in the serum-starved endothelial cells. In the presence of PD98059 (2 microM, a MAP kinase kinase inhibitor) and atropine, acetylcholine (1 microM) failed to enhance the proliferation of porcine cerebral endothelial cells. These results demonstrate that nicotinic stimulation promotes the proliferation of bovine and porcine cerebral microvascular endothelial cells, at least in part, through the MAP kinase activation.
Subject(s)
Cell Proliferation/drug effects , Endothelium, Vascular/drug effects , Nicotine/pharmacology , Animals , Cattle , Dose-Response Relationship, Drug , Endothelium, Vascular/cytology , Endothelium, Vascular/metabolism , Microcirculation/cytology , Microcirculation/drug effects , Microcirculation/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , Receptors, Nicotinic/metabolism , SwineABSTRACT
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 (alpha4beta2, alpha7, alpha3beta2, alpha3beta4, and alpha4beta4). We found that the 5,8-disubstituted indolizidine (-)-235B' acted as a potent noncompetitive blocker of alpha4beta2 nicotinic receptors (IC50 = 74 nM). This effect was highly selective for alpha4beta2 receptors compared with alpha3beta2, alpha3beta4, and alpha4beta4 receptors. The inhibition of alpha4beta2 currents by (-)-235B' was more pronounced as the acetylcholine concentration increased (from 10 nM to 100 microM). Moreover, the blockade of alpha4beta2 currents by (-)-235B' was voltage-dependent (more pronounced at hyperpolarized potentials) and use-dependent, indicating that (-)-235B' behaves as an open-channel blocker of this receptor. Several other 5,8-disubstituted indolizidines (5-n-propyl-8-n-butylindolizidines), two 5,6,8-trisubstituted indolizidines ((-)-223A and (+)-6-epi-223A), and a 1,4-disubstituted quinolizidine ((+)-207I) were less potent than (-)-235B', and none showed selectivity for alpha4beta2 receptors. The quinolizidine (-)-1-epi-207I and the tricyclic (+)-205B had 8.7- and 5.4-fold higher sensitivity, respectively, for inhibition of the alpha7 nicotinic receptor than for inhibition of the alpha4beta2 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.