Publications (6)20.66 Total impact
-
Article: Methadone increases intracellular calcium in SH-SY5Y and SH-EP1-halpha7 cells by activating neuronal nicotinic acetylcholine receptors.
[show abstract] [hide abstract]
ABSTRACT: (-)-Methadone acts as an agonist at opioid receptors. Both (+)- and (-)-enantiomers of methadone have been suggested to be potent non-competitive antagonists of alpha3beta4 neuronal nicotinic acetylcholine receptors (nAChRs). In the present study, we have examined interactions of methadone with nAChRs by using receptor binding assays, patch-clamp recording and calcium fluorometry imaging with SH-SY5Y cells naturally expressing alpha7 and alpha3* nAChR subtypes and SH-EP1-halpha7 cells heterologously expressing human alpha7 nAChRs. Methadone potently inhibited binding of [3H]methyllycaconitine to alpha7 nAChRs and that of [3H]epibatidine to alpha3* nAChRs. Methadone pretreatment induced up-regulation of epibatidine binding sites in SH-SY5Y cells. Using whole-cell patch-clamp recording, both isomers of methadone activated cation currents via mecamylamine-sensitive nAChRs in SH-SY5Y cells. Nicotine and both (+)- and (-)-methadone evoked increases in [Ca2+]i in both fluo-3AM loaded cell lines, and these effects were blocked by mecamylamine and by the alpha7 selective antagonist methyllycaconitine, suggesting effects of methadone as alpha7-nAChR agonist. Sensitivity of sustained nicotine and methadone effects to blockade by CdCl2, ryanodine and xestospongin-c implicates voltage-operated Ca2+ channels and intracellular Ca2+ stores as downstream modulators of elevated [Ca2+]i. Collectively, our results suggest that methadone engages in complex and potentially pharmacologically significant interactions with nAChRs.Journal of Neurochemistry 10/2005; 94(5):1329-41. · 4.06 Impact Factor -
Article: High-affinity epibatidine binding of functional, human alpha7-nicotinic acetylcholine receptors stably and heterologously expressed de novo in human SH-EP1 cells.
[show abstract] [hide abstract]
ABSTRACT: Human nicotinic acetylcholine receptor (nAChR) alpha7 subunits were stably and heterologously expressed in native nAChR-null SH-EP1 human epithelial cells. Immunofluorescence staining shows alpha7 subunit protein expression in virtually every transfected cell. Microautoradiographic analysis identifies 125I-labeled alpha-bungarotoxin (I-Bgt) binding sites corresponding to human alpha7 (halpha7)-nAChRs on the surface of most cells. I-Bgt binds to halpha7-nAChRs in membrane fractions with a typical apparent K(D) value of approximately 5 nM and B(max) value of approximately 1 pmol/mg membrane protein, and 62% of these sites are expressed on the cell surface. Function of heterologously expressed halpha7-nAChRs is evident as rapid, transient inward current responses to (-)-nicotine. Nicotine treatment of transfected cells produces dose- and time-dependent increases (up to approximately 100%) in numbers of I-Bgt binding sites. Epibatidine is a useful ligand for studies of nAChRs containing alpha3 or alpha4 subunits (K(D) values of about 100 or 10 pM, respectively). halpha7-nAChRs expressed in transfected SH-EP1 cells also exhibit picomolar affinity binding of 3H-labeled epibatidine (K(D) value of approximately 0.6 nM). Studies of several forms of native or heterologously expressed rat or human alpha7-nAChRs confirm high-affinity and mutually exclusive interaction with both epibatidine and alpha-bungarotoxin. Rank order potencies for drugs acting to compete for binding of either radioligand are similar (methyllycaconitine > dimethylphenyl-piperazinium > nicotine approximately cytisine > carbamylcholine approximately D-tubocurarine). These results demonstrate that transfected SH-EP1 cells are excellent models for studies of heterologously expressed, human alpha7-nAChRs that exhibit ligand binding and functional properties like native alpha7-nAChRs and that epibatdine is useful as a probe for human alpha7-nAChRs.Journal of Pharmacology and Experimental Therapeutics 04/2005; 313(1):24-35. · 3.83 Impact Factor -
Article: Characterization of human alpha 4 beta 2-nicotinic acetylcholine receptors stably and heterologously expressed in native nicotinic receptor-null SH-EP1 human epithelial cells.
[show abstract] [hide abstract]
ABSTRACT: Naturally expressed nicotinic acetylcholine receptors composed of alpha4 and beta2 subunits (alpha4beta2-nAChR) are the predominant form of high affinity nicotine binding site in the brain implicated in nicotine reward, mediation of nicotinic cholinergic transmission, modulation of signaling through other chemical messages, and a number of neuropsychiatric disorders. To develop a model system for studies of human alpha4beta2-nAChR allowing protein chemical, functional, pharmacological, and regulation of expression studies, human alpha4 and beta2 subunits were stably introduced into the native nAChR-null human epithelial cell line SHEP1. Heterologously expressed alpha4beta2-nAChR engage in high-affinity, specific binding of 3H-labeled epibatidine (H-EBDN; macroscopic KD = 10 pM; kon = 0.74/min/nM, koff = 0.013/min). Immunofluorescence studies show alpha4 and beta2 subunit protein expression in virtually every transfected cell, and microautoradiographic studies show expression of 125I-labeled iodo-deschloroepibatidine binding sites in most cells. H-EBDN binding competition studies reveal high affinity for nicotinic agonists and lower affinity for nicotinic antagonists. Heterologously expressed alpha4beta2-nAChR functional studies using 86Rb+ efflux assays indicate full efficacy of epibatidine, nicotine, and acetylcholine; partial efficacy for 1,1-dimethyl-4-phenyl-piperazinium, cytisine, and suberyldicholine; competitive antagonism by dihydro-beta-erythroidine, decamethonium, and methyllycaconitine; noncompetitive antagonism by mecamylamine and eserine; and mixed antagonism by pancuronium, hexamethonium, and d-tubocurarine. These results demonstrate utility of transfected SH-EP1 cells as models for studies of human alpha4beta2-nAChR, and they also reveal complex relationships between apparent affinities of drugs for radioligand binding and functional sites on human alpha4beta2-nAChR.Molecular Pharmacology 01/2004; 64(6):1283-94. · 4.88 Impact Factor -
Article: Functional properties of homomeric, human alpha 7-nicotinic acetylcholine receptors heterologously expressed in the SH-EP1 human epithelial cell line.
[show abstract] [hide abstract]
ABSTRACT: alpha 7-Nicotinic acetylcholine receptors (alpha 7-nAChRs) are broadly distributed in the central nervous system, where they play important roles in chemical and electrical signaling, and perhaps in neurite outgrowth, synaptic plasticity, and neuronal death/survival. To help elucidate their normal and pathophysiological roles, we have heterologously expressed human alpha 7-nAChR in transfected SH-EP1 human epithelial cells. Reverse transcription-polymerase chain reaction and mRNA fluorescence in situ hybridization analyses demonstrate expression of human alpha 7 subunits as messenger RNA. Patch-clamp recordings exploiting a novel strategy to prevent functional rundown of whole-cell peak current responses to repeated acute challenges with nicotinic agonists show successful expression of functional alpha 7-nAChR that mediate inward currents characterized by rapid phases of activation and inactivation. Concentration-response curves show that nicotine, acetylcholine, and choline are efficacious agonists at human alpha 7-nAChRs. Current-voltage relationships show inward rectification for agonist-induced currents. Human alpha 7-nAChRs exhibit some sensitivity to alpha 7-nAChR antagonists alpha-bungarotoxin (Bgt) or methyllycaconitine (MLA) when applied coincidentally with agonist, but much higher affinity block occurs when cells and alpha 7-nAChRs are pre-exposed to antagonists for 2 min before challenge with agonist. Both Bgt and MLA are competitive inhibitors of alpha 7-nAChR function. Whole-cell current peak amplitudes and half-times for inactivation of alpha 7-nAChR functional responses to nicotine are dramatically reduced in the absence of extracellular Ca2+, suggestive of high Ca2+ permeability of the alpha 7-nAChR channel. Thus, heterologously expressed human alpha 7-nAChR in mammalian cells have properties of native alpha 7-nAChR or of alpha 7-nAChR heterologously expressed in other systems and serve as excellent models for studies of molecular bases of alpha 7-nAChR function.Journal of Pharmacology and Experimental Therapeutics 07/2003; 305(3):1132-41. · 3.83 Impact Factor -
Article: Activity of cytisine and its brominated isosteres on recombinant human α7, α4β2 and α4β4 nicotinic acetylcholine receptors
[show abstract] [hide abstract]
ABSTRACT: Effects of cytisine (cy), 3-bromocytisine (3-Br-cy), 5-bromocytisine (5-Br-cy) and 3,5-dibromocytisine (3,5-diBr-cy) on human (h) α7-, α4β2- and α4β4 nicotinic acetylcholine (nACh) receptors, expressed in Xenopus oocytes and cell lines, have been investigated. Cy and its bromo-isosteres fully inhibited binding of both [α-125I]bungarotoxin ([α-125I]BgTx) to hα7- and [3H]cy to hα4β2- or hα4β4-nACh receptors. 3-Br-cy was the most potent inhibitor of both [α-125I]BgTx and [3H]cy binding. Cy was less potent than 3-Br-cy, but 5-Br-cy and 3,5-diBr-cy were the least potent inhibitors. Cy and 3-Br-cy were potent full agonists at hα7-nACh receptors but behaved as partial agonists at hα4β2- and hα4β4-nACh receptors. 5-Br-cy and 3,5-diBr-cy had low potency and were partial agonists at hα7- and hα4β4-nACh receptors, but they elicited no responses on hα4β2-nACh receptors. Cy and 3-Br-cy produced dual dose–response curves (DRC) at both hα4β2- and hα4β4-nACh receptors, but ACh produced dual DRC only at hα4β2-nACh receptors. Low concentrations of cy, 3-Br-cy and 5-Br-cy enhanced ACh responses of oocytes expressing hα4β2-nACh receptors, but at high concentrations they inhibited the responses. In contrast, 3,5-diBr-cy only inhibited, in a competitive manner, ACh responses of hα4β2-nACh receptors. It is concluded that bromination of the pyridone ring of cy produces marked changes in effects of cy that are manifest as nACh receptor subtype-specific differences in binding affinities and in functional potencies and efficacies.Journal of Neurochemistry 08/2001; 78(5):1029 - 1043. · 4.06 Impact Factor -
Article: Inducible, heterologous expression of human α7-nicotinic acetylcholine receptors in a native nicotinic receptor-null human clonal line
[show abstract] [hide abstract]
ABSTRACT: Tetracycline-regulated expression of recombinant nicotinic acetylcholine receptors (nAChR) composed of human α7 subunits is achieved in native nAChR-null SH-EP1 human epithelial cells. α7 subunits are heterologously expressed as messenger RNA and as components of -labeled α-bungarotoxin (I-Bgt)-binding nAChR (∼10 pmol per milligram of membrane protein) at levels sensitive to the amount of tetracycline in cell growth medium. I-Bgt-binding α7-nAChR appear on the cell surface pool and in intracellular pools. The pharmacological profile for drug competition toward I-Bgt binding to these recombinant α7-nAChR matches that of human native α7-nAChR naturally expressed in SH-SY5Y human neuroblastoma cells (rank order potency methyllycaconitine>1,1-dimethyl-4-phenylpiperazinium>(−)nicotine>cytisine>carbamylcholine⩾d-tubocurarine). Chronic exposure to nicotine induces up-regulation of human recombinant α7-nAChR (80% up-regulation at 10 μM nicotine) just as it does native α7-nAChR in other human cell lines. These studies confirm expression of nAChR as homooligomers of human α7 subunits from transgenes, establish a native nAChR-null background for such expression, and demonstrate that this expression can be regulated to facilitate studies of human α7-nAChR.Brain Research.
Top Journals
Institutions
-
2001–2005
-
Barrow Neurological Institute
- Division of Neurobiology
Phoenix, AZ, USA
-
