Purnyn' O. Pharmacological Properties and alpha-Subunit Composition of the Nicotinic Acetylcholine Receptors of the Autonomic Ganglia Neurons of the Rat

The dissertation is devoted to investigation of subunit composition and pharmacological properties of nicotinic acetylcholine receptors (nAChRs) of superior cervical ganglion (SCG), submandibular ganglion (SMG) and intracardiac ganglion (ICG) neurons of the rat. We carried out experiments by the patch-clamp method in the "whole cell" configuration and by the method of microelectrode intracellular recording of excitatory postsynaptic potentials (EPSPs) or acetylcholine-induced potentials (ACh-potentials) on neurons of three-week age rats. One of the main aims of our work was to clarify whether the blocking effects produced by different subunit-specific antibodies (Abs) are additive. Affinity purified rabbit Abs to synthetic fragments of alpha4(181-192) and falpha7(179-190), as well as mouse monoclonal Abs to alpha(181-192) and alpha5(180-191), were added to perfusion solution. We show that on the six neurons of SCG the blocking effects were additive after sequential addition of alpha7- and alpha3-specific Abs. The alpha-subunit composition of nAChRs expressed in the neurons of rat ICG was investigated using specific Abs againstalphaб3, alpha4, alpha5 and alpha7 nAChR subunits. Our data suggested that rat ICG neurons expressed nAChRs containing each of the above subunits. To clarify whether the same neuron possessed nAChRs with different alpha-subunits, we studied the blocking effects of two or more different antibodies applied to the same neuron in succession. Each nAChR subtype was present only in a part of the neurons. The neurons were heterogeneous as to the nAChR subtypes expressed. The effects of application of alpha-Abs on ACh-potentials and EPSP were studied in neurons of the rat SMG. A block of ACh-potentials evidenced that about 75% of neurons in SMG contained alpha3 or alpha4, or alpha5 subunits, while only about 30% of them contained alpha7-subunit. The EPSPs’ depression by the same Abs showed that the part of alpha3- and alpha4-containing nAChRs in each cell of SMG appeared more prominent thanthat of alpha7- and alpha5-containing nAChRs. alpha3-Subunits were more numerous in the neuronal somata (as evidenced by ACh-potentials) than in post-synaptic membranes (as evidenced by EPSPs). Our data indicate that both the somata and post-synaptic membranes of SMG neurons contain nAChRs composed of more than one alpha-subunit. It was shown that each neuron of SCG, SMG and ICG can expressed various subtypes of nAChRs. Spectra of the submitted subtypes of nAChRs of different ganglion neurons are different. Key words:nAChR, subunit composition, specific antibodies, SCG, SMG, ICG.