RESUMO
HEK293 cells were stably transfected with the cDNAs encoding full-length human neuronal nicotinic acetylcholine receptor (nAChR) subunit combinations alpha3beta2 or alpha4beta2. [(3)H]-(+/-)Epibatidine ([(3)H]-(+/-)EPI) bound to membranes from A3B2 (alpha3beta2) and A4B2.2 (alpha4beta2) cells with K(d) values of 7.5 and 33.4 pM and B(max) values of 497 and 1564 fmol/mg protein, respectively. Concentration-dependent increases in intracellular free Ca(2+) concentration were elicited by nAChR agonists with a rank order of potency of EPI>1,1-dimethyl-4-phenylpiperazinium (DMPP)>nicotine (NIC)=suberyldicholine (SUB)>cytisine (CYT)=acetylcholine (ACh) for A3B2 cells and EPI>CYT=SUB=NIC=DMPP>ACh for A4B2.2 cells. Antagonists of nAChRs blocked NIC-induced responses with a rank order of potency of d-tubocurarine (d-Tubo)=mecamylamine (MEC)>dihydro-beta-erythroidine (DHbetaE) in A3B2 cells and MEC=DHbetaE>d-Tubo in A4B2.2 cells. Whole-cell patch clamp recordings indicate that the decay rate of macroscopic ACh-induced currents is faster in A3B2 than in A4B2.2 cells and that A3B2 cells are less sensitive to ACh than A4B2.2 cells. ACh currents elicited in alpha3beta2 and alpha4beta2 human nAChRs are maximally potentiated at 20 and 2 mM external Ca(2+), respectively. Our results indicate that stably expressed alpha3beta2 and alpha4beta2 human nAChRs are pharmacologically and functionally distinct.
Assuntos
Receptores Nicotínicos/metabolismo , Northern Blotting , Western Blotting , Cálcio/metabolismo , Linhagem Celular , Estimulação Elétrica , Eletrofisiologia , Humanos , Rim/metabolismo , Ligantes , Membranas/efeitos dos fármacos , Membranas/metabolismo , Agonistas Nicotínicos/farmacologia , Técnicas de Patch-Clamp , RNA/biossíntese , RNA/isolamento & purificação , Ensaio Radioligante , Receptores Nicotínicos/efeitos dos fármacos , Receptores Nicotínicos/genética , Proteínas Recombinantes/químicaRESUMO
Nicotinic acetylcholine receptor (nAChR) subunit mRNA expression in the rat substantia nigra (SN) was assayed by semiquantitative RT-PCR following 6-hydroxydopamine (6-OHDA) lesion of nigrostriatal dopaminergic neurons. Six months after unilateral injection of 6-OHDA or saline into the SN, total RNA was isolated from ipsilateral and contralateral tissue samples. RT-PCR amplifications were performed with template titration using primers specific for sequences encoding 1. nAChR alpha 2-alpha 7 and beta 2-beta 4 subunits 2. Glutamic acid decarboxylase 3. Glyceraldehyde 3-phosphate dehydrogenase for normalization of template mass. PCR products specific for alpha 3, alpha 4, alpha 5, alpha 6, alpha 7, beta 2, beta 3, and glutamic acid decarboxylase were detected in the reactions containing SN RNA. This is the first evidence that alpha 7 may be expressed in the SN. alpha 2 and beta 4 PCR products were not detected in SN reactions, although they were observed in hippocampus and thalamus control reactions. A comparison of ipsilateral and contralateral SN RT-PCR reaction products showed substantial decreases in alpha 5, alpha 6, and beta 3 product yields following 6-OHDA, but not sham treatment. Neither the SN of sham-lesioned rats nor the thalamus of 6-OHDA-lesioned rats yielded similar results, indicating that the effects observed in 6-OHDA-treated SN were not caused by local mechanical damage or a nonspecific response, respectively. Effects of 6-OHDA treatment on alpha 3, alpha 4, alpha 7, beta 2, or glutamic acid decarboxylase product yields from SN samples were small or undetectable. The results suggest that alpha 5, alpha 6, and beta 3 subunit-encoding mRNAs are expressed at substantially higher levels in dopaminergic than in nondopaminergic cell bodies in the SN.
Assuntos
Dopamina/metabolismo , Neostriado/metabolismo , Neurônios/metabolismo , Oxidopamina/farmacologia , Receptores Nicotínicos/genética , Substância Negra/metabolismo , Animais , Glutamato Descarboxilase/genética , Gliceraldeído-3-Fosfato Desidrogenases/genética , Masculino , Neostriado/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Oligonucleotídeos , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Substância Negra/efeitos dos fármacos , Tálamo/metabolismoRESUMO
Human embryonic kidney (HEK293) cells were transfected with cDNA encoding the human beta4 neuronal nicotinic acetylcholine (ACh) receptor subunit in pairwise combination with human alpha2, alpha3 or alpha4 subunits. Cell lines A2B4, A3B4.2 and A4B4 were identified that stably express mRNA and protein corresponding to alpha2 and beta4, to alpha3 and beta4 and to alpha4 and beta4 subunits, respectively. Specific binding of [3H]epibatidine was detected in A2B4, A3B4.2 and A4B4 cells with Kd (mean +/- S.D. in pM) values of 42 +/- 10, 230 +/- 12 and 187 +/- 29 and with Bmax (fmol/mg protein) values of 1104 +/- 338, 2010 +/- 184 and 3683 +/- 1450, respectively. Whole-cell patch-clamp recordings in each cell line demonstrated that (-)nicotine (Nic), ACh, cytisine (Cyt) and 1, 1-dimethyl-4-phenylpiperazinium iodide (DMPP) elicit transient inward currents. The current-voltage (I-V) relation of these currents showed strong inward rectification. Pharmacological characterization of agonist-induced elevations of intracellular free Ca++ concentration revealed a distinct rank order of agonist potency for each subunit combination as follows: alpha2beta4, (+)epibatidine (Epi) > Cyt > suberyldicholine (Sub) = Nic = DMPP; alpha3beta4, Epi > DMPP = Cyt = Nic = Sub; alpha4beta4, Epi > Cyt = Sub > Nic > DMPP. The noncompetitive antagonists mecamylamine and d-tubocurarine did not display subtype selectivity. In contrast, the Kb value for the competitive antagonist dihydro-beta-erythroidine (DHbetaE) was highest at alpha3beta4 compared with alpha2beta4 or alpha4beta4 receptors. These data illustrate that the A2B4, A3B4.2 and A4B4 stable cell lines are powerful tools for examining the functional and pharmacological properties of human alpha2beta4, alpha3beta4 and alpha4beta4 neuronal nicotinic receptors.
