Neuronal nicotin~c receptors (nAChRs) are important membrane proteins to signal intercellular communication in health and disease. Against a large body of data related to brain or muscle nAChRs, there is comparatively less information concerning such receptors on autonomic sensory neurons. The present investigation was focused on characterizing the expression and function of nAChRs in chromaffin cells of the rat adrenal medulla, on exploring how novel synthetic compounds could modulate acutely or clu·onically nAChRs by using SH-SY5Y cells which are tumorderived cells of chromaffin cell lineage, and how long-term application of nicotinic agents could alter the function of such receptors. Reverse transcription-polymerase chain reaction analysis indicated the presence of a2, a3, a4, a5, a7, ~2 and P4 transcripts (a6 and P3 could not be detected) in rat chromaffin cells. Immunocytochernistry and western blot analysis did not confirm the expression of the a7 subunit. Inward currents elicited by nicotine pulses were insensitive to a-bungarotoxin and low doses of methyllycaconitine, demonstrating lack of functional a7 receptors. Partial block of nicotine currents was observed with either AuIB a-conotoxin (selective against a3P4 receptors) or MII a-conotoxin (selective against a3P2 receptors). Antagonism by dihydro-p-erythroidine (selective at low doses against a4P2 receptors) summated nonlinearly with AuIB and MU inhibition, confirming heterogeneity of neuronal nicotinic acetylcholine receptor populations. These results suggest that the most frequently encountered receptors of rat chromaffin cells should comprise a3p'4, a3P2 with the addition of a5 subunits, and much less commonly a2p4, without excluding other subunit combinations. Using SH-SY5Y cells, more stable in culture and therefore suitable for chronic treatment, we investigated the effect of the novel cytisine dimer 1,2-bisNcytisinylethane (CC4). On nAChRs CC4 lacked the agonist properties of cytisine and was actually a potent antagonist (IC50 = 220 nM). Chronic treatment of SH-SYSY cells with 1 mM CC4 for 48 h significantly increased nicotine-evoked currents with augmented sensitivity to the blockers a-conotoxin MII or methyllycaconitine, indicating a relative increase of functional nicotinic receptors comprising Beta2 and alpha7 subunits on the cell membrane. Chronically treating SH-SYSY cells with nicotine showed that, despite desensitization, they preserved a degree of responsiveness to nicotine pulses, and that they rapidly recovered on washout to generate larger responses without changes in kinetics or pharmacology. In summary, these data have demonstrated the subunit expression and function of nAChRs on peripheral autonomic cells, and have identified new properties like their sustained ability to preserve function even in the presence of chronic agonist application and their compensatory up-regulation. The present study, thus, sheds new light on the plasticity of nAChRs and outlines new strategies for their pharmacological modulation. Because of the role of nAChRs in the control of chromaffin cell function in regulating blood pressure, it is suggested that changes in nAChR activity might influence the mechanisms responsible for changes in blood pressure in health and disease.
|Titolo:||Studies of neuronal nicotinic receptors of autonomic neurons: expression and function|
|Data di pubblicazione:||16-mag-2007|
|Appare nelle tipologie:||8.1 PhD thesis|