Neuronal nicotinic acetylcholine receptors of chromaffin cells in the adrenal medulla are physiologically activated by acetylcholine to mediate catecholamine release into the bloodstream. The present study examined the subunit composition and functional properties of rat chromaffin cell neuronal nicotinic acetylcholine receptors using molecular biology, immunocytochemistry and whole-cell patch-clamp. Reverse transcription-polymerase chain reaction analysis indicated the presence of alpha2, alpha3, alpha4, alpha5, alpha7, beta2 and beta4 transcripts (alpha6 and beta3 could not be detected). Immunocytochemistry revealed most cells positive for alpha3, beta2, beta4 and alpha5 proteins. Few cells were immunoreactive for alpha2 and alpha4, while none was for alpha7. At single-cell level, colocalization could be demonstrated for alpha3alpha5 and alpha4beta2. Western blot analysis confirmed antibody specificity for alpha3, alpha4, alpha5, beta2 and beta4 subunits. Inward currents elicited by nicotine pulses were insensitive to alpha-bungarotoxin and low doses of methyllycaconitine, demonstrating lack of functional alpha7 receptors. Partial block of nicotine currents was observed with either AuIB alpha-conotoxin (selective against alpha3beta4 receptors) or MII alpha-conotoxin (selective against alpha3beta2 receptors). With high concentrations of co-applied toxins, antagonism occlusion developed, suggesting loss of subunit selectivity. Antagonism by dihydro-beta-erythroidine summated nonlinearly with AuIB and MII inhibition, confirming heterogeneity of neuronal nicotinic acetylcholine receptor block. The present results suggest that the most frequently encountered receptors of rat chromaffin cells should comprise alpha3beta4, alpha3beta2 with the addition of alpha5 subunits. Because of the prevailing subunit composition, rat chromaffin cell neuronal nicotinic acetylcholine receptors are suitable models, particularly for the alpha3beta4 subclasses of mammalian brain receptors recently demonstrated in discrete cerebral areas.
1 Neuronal nicotinic ACh receptors (nAChRs) readily desensitize in the presence of an agonist. However, when the agonist is applied for minutes, hours or days, it is unclear how extensive desensitization is, how long it persists after agonist removal and whether nAChRs consequently change their pharmacological properties. 2 These issues were explored with electrophysiological studies of native receptors of voltage-clamped human neuroblastoma SH-SY5Y cells. Puffer pulses of nicotine (1 mM)-evoked inward currents partly antagonized by methyllycaconitine (MLA; 10 nM) or a-conotoxin MII (MII; 10 nM), suggesting contribution by a7 and a3 subunit containing receptors, respectively. Nicotine-evoked currents desensitized with 150 ms time constant and fully recovered after a few s washout. 3 Although the current induced by 10 min application of nicotine (10 mM) decayed to baseline indicating complete desensitization, puffer applications of maximally effective doses of nicotine still generated small responses (22% of control). Similar responses to puffer-applied nicotine were observed when nicotine was chronically incubated for 8 or 48 h. On nicotine washout, cells recovered their response amplitude within 5 min and then increased it (about 50% of untreated controls) after 30 min without altering response kinetics or sensitivity to MLA and MII. 4 The present results suggest that native nAChRs of SH-SY5Y cells preserved a degree of responsiveness during chronic application of nicotine, and that they rapidly recovered on washout to generate larger responses without changes in kinetics or pharmacology. These data indicate strong compensatory mechanisms to retain nicotinic receptor function during long-term exposure to nicotine.
1 Nicotinic drug treatment can affect the expression of neuronal nicotinic acetylcholine receptors (nAChR) both in vivo and in vitro through molecular mechanisms not fully understood. The present study investigated the effect of the novel cytisine dimer 1,2-bisN-cytisinylethane (CC4) on nAChR natively expressed by SH-SY5Y neuroblastoma cells in culture. 2 CC4 lacked the agonist properties of cytisine and was a potent antagonist (IC 50 ¼ 220 nM) on nAChRs. Chronic treatment of SH-SY5Y cells with 1 mM CC4 for 48 h increased the expression of 3 H-epibatidine ( 3 H-Epi; 3-4-fold) or 125 I-a-bungarotoxin ( 125 I-aBgtx; 1.2-fold) sensitive receptors present on the cell membrane and in the intracellular pool. Comparable data were obtained with nicotine or cytisine, but not with carbamylcholine, d-tubocurarine, di-hydro-b-erythroidine or hexametonium. 3 Immunoprecipitation and immunopurification studies showed that the increase in 3 H-Epi-binding receptors was due to the enhanced expression of a3b2 and a3b2b4 subtypes without changes in subunit mRNA transcription or receptor half-life. The upregulation was not dependent on agonist/antagonist properties of the drugs, and did not concern muscarinic or serotonin receptors. 4 Whole-cell patch clamp analysis of CC4-treated cells demonstrated larger nicotine-evoked inward currents with augmented sensitivity to the blockers a-conotoxin MII or methyllycaconitine. 5 In conclusion, chronic treatment with CC4 increased the number of nAChRs containing b2 and a7 subunits on the plasma membrane, where they were functionally active. In the case of b2-containing receptors, we propose that CC4, by binding to intracellular receptors, triggered a conformational reorganisation of intracellular subunits that stimulated preferential assembly and membrane-directed trafficking of b2-containing receptor subtypes.
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