Application of acetylcholine to peripheral nerve terminals in the skin is a widely used test in studies of human small-fiber functions. However, a detailed pharmacological profile and the subunit composition of nicotinic acetylcholine receptors in human C-fiber axons are not known. In the present study, we recorded acetylcholine-induced changes of the excitability and of the intracellular Ca2+ concentration in C-fiber axons of isolated human nerve segments. In addition, using immunohistochemistry, an antibody of a subtype of nicotinic acetylcholine receptor was tested. Acetylcholine and agonists reduced the current necessary for the generation of action potentials in C fibers by > 5-Iodo-A-85380 > 1,1-dimethyl-4-phenylpiperazinium iodide > nicotine > cytisine > acetylcholine; choline had no effect. The epibatidine-induced increase in axonal excitability was blocked by mecamylamine and, less efficiently, by methyllycacontine and dihydro-beta-erythroidine. Many C-fiber axons were labeled by an antibody that recognizes the alpha5 subunit of nicotinic acetylcholine receptors. In summary, electrophysiological and immunohistochemical data indicate the functional expression of nicotinic acetylcholine receptors composed of alpha3, alpha5, and beta4 but not of alpha4/beta2 or of alpha7 subunits in the axonal membrane of unmyelinated human C fibers. In addition, the observations suggest that the axonal membrane of C fibers in isolated segments of human sural nerve can be used as a model for presumed cholinergic chemosensitivity of axonal terminals.

The novel alpha-conotoxin Vc1.1 is a potential analgesic for the treatment of painful neuropathic conditions. In the present study, the effects of Vc1.1 were tested on the nicotine-induced increase in excitability of unmyelinated C-fiber axons in isolated segments of peripheral human nerves. Vc1.1 in concentrations above 0.1 microM antagonized the increase in axonal excitability produced by nicotine; the maximal inhibition was observed with 10 microM. We also demonstrate immunoreactivity for alpha 3 and alpha 5 subunits of neuronal nicotinic receptors on unmyelinated peripheral human axons. Blockade of nicotinic receptors on unmyelinated peripheral nerve fibers may be helpful in painful neuropathies affecting unmyelinated sympathetic and/or sensory axons.

Receptors for ATP in the peripheral nervous system may contribute to the transduction of sensory, including nociceptive, stimuli and are candidates in the pathogenesis of neuropathic pain. In a complex neural tissue, such as the human peripheral nerve trunk, ATP may activate P2X, P2Y, and adenosine receptors present on various cell types. Experiments were performed on segments of isolated human sural nerves. The experimental set-up enabled simultaneous recording of C fiber excitability, intracellular Ca(2+) ([Ca(2+)](i)) and extracellular K(+) activity (aK(e)). The increase in excitability of unmyelinated fibers seen during bath application of both ATP and adenosine was reversed to a reduction in axonal excitability in the presence of 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolol[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385), an antagonist of adenosine A2 receptors. The pharmacological profile of the axonal subexcitability indicates the presence and activation of adenosine A1 receptors. Intracellular Ca(2+) transients were observed during bath application of ATP but not of adenosine and were blocked by 2'-deoxy- N(6)-methyladenosine 3',5'-bisphosphate (MRS 2179), an antagonist at P2Y(1) receptors. K(+)-sensitive microelectrodes were used to search for a possible activation of P2X receptors by ATP. In isolated rat vagus nerve, activation of P2X receptors by alpha,beta-methylene-adenosine 5'-triphosphate (alpha,beta-meATP) and by diadenosine pentaphosphate (Ap5A) resulted in a rapid, transient rise in the extracellular K(+) activity. In contrast, in human nerve, application of P2X receptor agonists did not result in a detectable elevation of aK(e). The data suggest that ATP-induced changes in axonal excitability and of [Ca(2+)](i) result from activation of adenosine A2, A1 and P2Y nucleotide receptors in human nerve; a contribution of P2X receptors was not found with the methods used. It is suggested that antagonists of A2 receptors might suppress enhanced activity in human nociceptive afferent nerve fibers under conditions in which ATP and/or adenosine is released into the trunk of a human peripheral nerve.

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