Excitatory nicotinic and desensitizing muscarinic (M2) effects on C-nociceptors in isolated rat skin

Abstract

The actions of different cholinergic agonists and antagonists were investigated on nociceptive afferents using the rat skin-saphenous nerve preparation, in vitro. Nicotine was able to weakly excite C-nociceptors and to induce a mild sensitization to heat stimulation (in 77% of tested fibers) in a dose-dependent manner (10(-)6 to 10(-)5 m), but it caused no alteration in mechanical responsiveness tested with von Frey hairs. Muscarine did not induce a significant nociceptor excitation, but almost all fibers exhibited a marked desensitization to mechanical and heat stimuli in a dose-dependent manner (from 10(-)6 to 10(-)4 m). The muscarinic effects could be prevented by the general muscarinic antagonist scopolamine (10(-)5 m), by the M3 antagonist 1,1-dimethyl-4-diphenylacetoxypiperidium oxide (10(-)6 m) co-applied with the M2 antagonist gallamine (10(-)5 m), and by gallamine alone. As positive control we used the relatively M2-selective agonist arecaidine (10(-)6 to 10(-)5 m), obtaining a similar desensitizing effect as with muscarine. Finally, we performed an immunocytochemical study that demonstrated the presence of M2 but not M3 receptors in thin epidermal nerve fibers of the rat hairy skin. Altogether, these data demonstrate opposite effects of nicotinic and muscarinic receptor stimulation on cutaneous nociceptors. M2 receptor-mediated depression of nociceptive responsiveness may convey a therapeutic, i.e., analgesic or antinociceptive, potential.

Fig. 1.

Original recording from two C-MH fibers of the saphenous nerve with receptive fields in isolated rat hairy skin, illustrating also the typical experimental protocol. Instantaneous discharge rates are plotted over a time scale of two different resolutions showing heat responses (32–46°C in 20 sec; gray columns) and responses to chemical stimuli (of 5 min duration); von Frey thresholds are indicated by open triangles.A shows nicotine responses and a dose-dependently induced sensitization to heat. B shows lack of muscarine-induced excitation and dose-dependent desensitization to heat and mechanical (von Frey) stimulation.

Fig. 2.

Concentration–response curves for induced C-fiber excitation. Nicotine-induced discharge was of a significantly higher rate than spontaneous activity (spa; recorded during 5 min before chemical stimulation), and 10⁻⁵m nicotine was significantly more effective than 10⁻⁶m(*p < 0.05, t test). Both concentrations of nicotine induced significantly more discharge than muscarine (*p < 0.05, ttest).

Fig. 3.

Heat thresholds of C-MH fibers before and after administration of cholinergic agonists and antagonists.Arrowheads in C and Dindicate fibers no longer excited up to 46°C.

Fig. 4.

Averaged per stimulus-time histograms of heat responses of C-MH units. Nicotine (A) induced a dose-dependent sensitization, lowering the threshold and increasing the mean response. Muscarine (B) and the M2-agonist arecaidine (C) induced a dose-dependent desensitization with increased threshold and reduced response.D illustrates the heat stimulus. In A, a few white columns are masked by grayones, and in C three black columns vanish behind gray ones, but in all cases the differences were negligible (see Results for statistics).

Fig. 5.

Mechanical thresholds tested with von Frey hairs before and after administration of cholinergic agonists and antagonists. Arrowheads in C andD indicate fibers responding only to a glass rod pressure (∼1000 mN).

Fig. 6.

Immunocytochemical detection of M2 but not M3 ACh receptors in rat cutaneous nerve fibers. Immunoreactivity for M2R, M3R, and PGP 9.5 at the epidermis–dermis border of the rat skin. Some nerve fibers showed immunoreactivity for M2R and PGP 9.5 (arrowheads in a, b), whereas M3R immunoreactivity could not be observed in PGP 9.5 nerve fibers at the epidermis–dermis border (arrows inc, d). Note the strong M2R-IR of the upper epidermal and the strong M3R-immunoreactivity of the basal epidermal layer. Scale bar, 40 μm.