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.
