The Researches Of α₇-Subtype Nicotinic Acetylcholine Receptors In Brain

α₇-subtype nicotinic acetylcholine receptors (nAChRs) belong to a family of ligand-gated ion channels, which are one of the two most abundant nicotinic receptor subtypes expressed in brain. With the help of some advanced technologies including molecular clone, we have found some differences between α₇ nAChRs and other nAChRs subtypes, such as α₄β₂ receptors, in molecular structure, biological characteristics, and so on. These differences make α₇ nAChRs have peculiar physiological functions and play an important role in the occurence, development and treatment of some diseases. We take α₇ nAChRs as subject in the paper, and firstly delineate the characteristics and mechanisms of homologous upregulation of them; prove the antinociceptive effects of α₇ nAChRs in acute thermal pain; find the similarities and differences of α₇ nAChRs between brain and endothelial cell and screen some compounds which have high affinity with α7 nAChRs in difference tissues as the subject of further research. Abstract as follow:â… . Characteristics and Mechanisms of Homologous Up-regulation of α₇ nAChRsWhen we use drugs that target α₇ nAChRs to treat an illness, α₇ nAChRs have to be exposed to their ligands for a long time. According to thetraditional views, chronic exposure to an agonist generally down-regulates the receptors, while chronic exposure to antagonist usually up-regulates the receptors, such as muscarinic acetylcholine receptors. But there is an exception to 014P2 nAChRs, whose number up-regulate after exposed to both agonists and antagonists for a long time, and the actions of them will decrease on the contrary. Whether the same phenomena as CX4P2 nAChRs will happen on 07 nAChRs is still unknown.To delineate the questions above, hippocampus neurons of newborn rats were primarily cultured. After 3 days, cells were exposed to selective agonist choline, selective antagonist methyllycaconitine (MLA) and nicotine, agonist to nAChRs respectively in different doses for 7 days. Using these cells, we did two kinds of experiments, first is radioligand binding assays, which with the help of a-Bungarotoxin (a-Bgt) to estimate the changes of (X7 nAChRs; second is calcium fluorimetry, which with the help of fluo-3-AM to monitor fluorescence changes of intracellular Ca2+ after choline administration and speculate the actions of 017 nAChRs. We found these results:(1) After exposed to choline (10"9¹0"6M), or MLA (10'5¹0'4M) or nicotine (10'5¹0'4M), the maximal binding of [125I] a-Bgt to a7 nAChRs that existed in the surface of hippocampus cells' membranes increased (P<0.05), while the affinity between them did not change in the duration (P>0.05).(2) Choline (lmM) generated a rapid elevation in intracellular Ca2+ fluorescence in hippocampus cells, which effects could be inhibited by a? nAChRs selectively antagonists, MLA. After immediately got rid of choline (10"7M), or MLA (10'5M) or nicotine (10"5M) who coexisted with cells for a long time (7d), choline (lmM) could not generate a different changes to intracellular Ca2+ in contrast with controls (P<0.05), but at the same time, the maximal binding of aj nAChRs for [125I]a-Bgt increased significantly (P>0.05).These results indicate:(1) After expose to agonists or antagonists for a long time, the numbers ofa? nAChRs existing on cells membrane increase, which is as same as 014P2 nAChRs.(2) Only treated with the selective agonist of a? nAChRs, choline, in a low dose, the numbers of a? nAChRs will increase. When the dose of choline is higher than 10"6M, there will be little effect on the numbers of 017. These characteristics are different from MLA and nicotine, which are dose-depended.(3) The effect of choline on intracellular Ca is produced through 017 nAChRs.(4) Although the numbers of a? nAChRs increase, the total reactions of (X7 nAChRs in one cell do not change, which may be caused by decreasing the function of single a? nAChR.In other transmitter systems, chronic exposure to ligands will change the numbers of receptors, and this is correlated with a variety of mRNA expression. However, according the data from our labs and other references, the up-regulation of nAChRs is unrelated with the mRNA level. These facts suggest that the up-regulation is caused by a post-transcriptional event. Generally, changes in receptor numbers depend on the relative kinetics of receptor degradation on the one hand, and new receptors synthesized on the other. Who on earth is the key to a-i nAChRs up-regulation is still unknown. So, Cycloheximide, a protein synthesize inhibitor that can inhibit new a? synthesize, was used with effective doses agonists or antagonists on hippocampus cells at the same time. After seven days, radioligand binding assays were do with the help of lOnM [125I]a-Bgt to resolve the question above. Other studies indicated the activation of protein kinase A and related signal pathway appear to be involved in the up-regulation of (X4P2 nAChRs. The intracellular domain of the a? subunit, containing a conserved consensus sequence of protein kinase A around ser-342, is specifically phosphorylated by cAMP-dependent protein kinase. Whether the up-regulation of 07 nAChRs is related with PKA pathway have not been reported. So, a serial doses of Forskolin, which can activate the PKA pathway by increasing theconcentration of intracellular cAMP, were used to decide the hypothesis. Results as follow:(1) Exposed with 0.4u.M Cycloheximide alone for seven days, levels of [i25I]a-Bgt binding to the cells decreased significantlyC/^O.OS), but the number of live cells were not affected.(2) After treated 0.4u.M cycloheximide with 10"7M choline or lO'^M nicotine or 10⁵M MLA on hippocampus cells for seven days, levels of [125I]a-Bgt binding to the cells decreased significantly in contrast with those treated with ligands alone (P<0.05); but levels of [l25I]a-Bgt binding to the cells increased significantly in contrast with those treated with cycloheximide alone (P<0.05).(3) There were no changes happened on [l2:T]a-Bgt binding both after exposed hippocampus cells with forskolin (l²40u.M) (P>0.05) and co-used with 0.4uM cycloheximide (P>0.05) for seven days.These results indicate that the up-regulation of 07 nAChR is caused by both the increasement of synthesize of new receptors and the decreasement of disassemble of original receptors in membrane, and there are no relations with PKA signal pathway. II. The Antinociceptive Effects of a-j nAChRs in BrainRecently, some researches indicate that agonists who target nAChRs in brain exert strong antinociceptive actions in a variety of pain tests. These characteristics lead to a new way for the study of novel target and selective compounds about antinociception. However, it is difficult to decide which subtype of nAChRs participates in the effect of agonist, because of the diversities of nAChRs. Some studies have proved that 04^2 nAChRs, one of the two most abundant nAChRs expressed in brain, take part in the antinociception of agonists. But there are some inconsistencies on the effect of 07 nAChRs up to the present. To prove the antinociceptive effects of 07 nAChRs, we used hot plate test for mice to measure the effects of choline, selective agonist of a-j nAChRs, on the behavior of mice after i.c.v. or i.v.administration. Furthermore, some kinds of antagonists were used before choline administration to prove the relationship between a7 nAChRs and pain. Results as follows:(1) In the hot plate test, the maximal antinociceptive effect of choline, 90ug/animal, i.c.v., appeared 15 min after injection (P<0.05), but not in 30, 45, 60 and 120 min(P>0.05).(2) In the hot plate test, there were no changes on response latency after i.c.v. administration of 30 or 60 |ig/animal choline compared with controls (P>0.05). Mice treated with choline at 90 or 120 ug/animal showed a dose-depended increase in latency time (P<0.05).(3) The doses of choline from 2 mg/kg to 64 mg/kg had no effect on the response latency 15 min after injection through the tail veins (P>0.05).(4) MLA (50 ug/animal, i.c.v.) or a-Bgt (2 ug/animal, i.c.v.) or atropine (0.1 jag/animal, i.c.v) could block the antinociceptive effects of choline (90 |ig/animal, i.c.v.) in hot plate test (P<0.05), but not mecamylamine (up to 2 ug/animal, i.c.v.)and naloxone (2 ug/animal, i.c.v.) (P>0.05).The results above prove the fact that 017 nAChRs are involved in the antinociceptive effects of choline.III. Pharmacological Characteristics of 017 nAChRs on the Membranes of Brain and Endothelial Cells and Compounds Sreened017 nAChRs are expressed not only in the nervous system but also in non-nervous systems or tissues, such as endothelium existing in blood vessels. So, it is difficult for new drugs studies on a7 nAChRs target, because the drugs target receptors in the brain may coupled with those existing in endothelium and produce severe vascular side effects. In order to gain maximal therapeutical effects, limit the happening of side effects and delineate whether had the possibility to develop a drug targeted ay nAChRs in selective tissue, we used brains and human umbilical vein endothelium cells as the subjects of our experiments, [125I]a-Bgt were also used to mark 017 nAChRs so that we could find the similarities and differences of pharmacological characteristic ofa7 nAChRs in different tissues with radioligand binding assays. Furthermore, we could design and optimize compounds according these results. In this part, we also screened almost 20 new synthesized compounds on brain or endothelium cells, so that we could get compounds that had high affinity with ay nAChRs, on the basis of which, we could investigate the characteristics of compounds on one hand, and on the other hand it would be helpful to the ligands design for 07 nAChRs. Results as follows:(1) In the radioligand saturation assays, the Kd values of [' 3I]a-Bgt for 017 nAChRs in brain is 3.49 + 1.23 nM, which lower than that of endothelium cells, 4.74±0.96nM (P<0.05); however, the maximal binding value of [l25IJa-Bgt for 07 nAChRs in brain is 52.25 + 6.8 fmol/mg protein, which higher than that of endothelium cells, 3.51 ±0.89 fmol/mg protein (PO.01).(2) In the radioligand competition binding assays, the Ki values of choline for 0L7 nAChRs in brain is 2.8+1.2mM, which has no difference with that in endothelium cells, 2.5 + 0.9mM (P>0.05); the Ki values of nicotine for 07 nAChRs in brain is 6.3 + 0.2uM, which is lower than that in endothelium cells, 13.1±0.6uM (P<0.01); the Ki values of MLA for a7 nAChRs in brain is 4.1 + 0.4nM, which is lower than that in endothelium cells, 12±1.5nM (P<0.01); the Ki values of ot-Bgt for ay nAChRs in brain is l.l+0.3nM, which is lower than that in endothelium cells, 2.8 + 0.7nM (PO.05).(3) We screened out two kinds of compounds that has high affinity with 07 nAChRs. The Ki values of compound 398 for 017 nAChRs in brain was 895 + 126u.M, which was lower than that in endothelium cells, 1860 ± 212u.M (P<0.01). INA-F only had high affinity with ay nAChRs in endothelium cells but not in brain, the Ki value of it was 2603 + 1005u.M.These results indicate there are differences between 07 nAChRs in brain and those in endothelium cells. The binding affinity of ligands for 017 nAChRs in endothelium are not higher than that in brain. It is hopeful to design compounds that can bind specific with 07 nAChRs in the tissue we want.In conclusion, our researches offer some referenced and theoretic proof for the clinic use, screen, design and optimization of drugs which target 017 nAChRs.