Selective activation of nociceptors by P2X receptor agonists in normal and inflamed rat skin

Abstract

1. ATP can elicit pain in humans and, together with other P2X channel agonists, can produce nocifensive responses in rodents. We used the rat in vitro skin-nerve preparation to quantify primary afferent responses to ATP and its stable analogue alpha,beta-methylene ATP in normal and carrageenan-inflamed skin. 2. Both ATP and alpha,beta-methylene ATP were found to specifically activate the peripheral terminals of Adelta and C-fibre nociceptors in the skin. Thirty-nine per cent of the nociceptors tested responded to the maximal dose of alpha,beta-methylene ATP (5 mM). In contrast, non-nociceptive, low-threshold mechano-sensitive fibres were never activated by the same agonist concentrations. 3. Amongst the nociceptor population, C-mechanoheat fibres (C-MH or polymodal nociceptors) were markedly more responsive to P2X agonists than mechanonociceptors (C-M nociceptors) with Adelta- or C-fibre axons. Both C-mechanoheat and C-mechanonociceptors were activated by alpha,beta-methylene ATP doses as low as 50 microM. 4. In skin inflamed with carrageenan 3-4 h before recording both the number of responsive C-fibre nociceptors and their response magnitude increased. The increased neural response under inflammatory conditions was largely observed in C-mechanoheat or polymodal nociceptors. After low doses of P2X agonists C-MH fibres but not C-M fibres developed elevated ongoing activity and this effect was only seen after carrageenan inflammation. The time course of alpha,beta-methylene ATP-evoked discharges in nociceptors was found to correlate well with the time course of behavioural nocifensive responses in rats to the same agonist described in a previous study (Hamilton et al. 1999). 5. We conclude that the rapid increase in the number of alpha,beta-methylene ATP responsive nociceptors and the increased magnitude of the neural response following carrageenan inflammation explains why very low concentrations of such agonists can cause pain in inflammatory states.

Figure 1. Examples of responses to α,β-methylene ATP

A, response of a single C-MH fibre to α,β-methylene ATP (5 mm, 5 min). B, example of an Aδ-nociceptor responding to a 2 min application of α,β-methylene ATP (5 mm) (left panel). None of the Aβ-fibres tested responded to application of α,β-methylene ATP (5 mm); an example is shown in the right panel. At the end of the wash-out period the A-fibre was tested with a mechanical stimulus (*) to confirm that the spike shape was unchanged. In C the average C-MH fibre response (n = 12 different fibres) to a 5 min application of α,β-methylene ATP (5 mm) is depicted as a spike frequency histogram. Note that the mean response desensitises in the continued presence of the agonist.

Figure 2. Dose-response relationships of C-fibre nociceptors to α,β-methylene ATP

A and B show the responses of C-MH fibres. In A the dose-related increase in the magnitude of the response of C-MH fibres in normal (^) and inflammatory (□) conditions is shown (two-way ANOVA, P < 0.05, n = 6–16). The proportion of C-MH units with a response to the agonist is shown in B. Note that in inflamed skin a larger proportion of C-MH fibres respond to the agonist compared with normal skin at low concentrations of α,β-methylene ATP (50 μm to 0.5 mm). At the highest concentration tested (5 mm) there is no significant difference in the percentage of responsive C-MH units in inflamed and in normal skin. In C and D equivalent data are plotted for C-M fibres. In C the response to equivalent concentrations of agonist is shown for C-M fibres recorded in normal (^) and inflamed (□) skin. Note that C-M fibres do not respond so well as C-MH fibres to α,β-methylene ATP and that the response is not dose dependent in normal skin. In inflamed skin the response of C-M fibres to the agonist was only increased at the highest dose of α,β-methylene ATP. In D the proportion of C-M fibres that respond to the agonist is shown at all doses. The proportion of C-M fibres did not increase in a dose-related manner in normal or inflamed skin. However, the proportion of fibres responding to α,β-methylene ATP was almost doubled at all doses in inflamed compared with normal skin. Overall, the proportion of responsive C fibres (C-MH and C-M) was significantly greater in inflamed skin than in normal skin when tested with 50 μmα,β-methylene ATP (χ² test, P < 0.05, n = 30–44).

Figure 3. C-fibres exhibit tachyphylaxis to α,β-methylene ATP

The time course of the response to the same chemical stimulus of a naive population of C-MH units (□) and the response of a population of C-MH units previously exposed to lower doses of α,β-methylene ATP (50 μm and 0.5 mm, ^) are shown (n = 10–12). Note that after previous exposure to the agonist the mean response magnitude is reduced throughout the agonist application.

Figure 4. Dose-dependent activation of C-MH and C-M fibres by α,β-methylene ATP in normal and inflamed skin

In A the time course of C-MH fibre activation by α,β-methylene ATP (n = 16–21) is shown; spontaneous activity was not subtracted in this case. For clarity the activity between agonist doses is not shown (note the breaks in the abscissa). Note that in inflamed skin (□) low doses of α,β-methylene ATP induce a long-lasting increase in on-going activity in C-MH fibres that was not observed in normal skin (^). Thus the mean activity is already high in these fibres shortly before application of the second and third doses of α,β-methylene ATP. In B the equivalent data from C-M fibres are plotted (n = 5-15). Note that these fibres are much less affected by carrageenan inflammation and no significant effect was observed of low doses of agonist on on-going activity.

Figure 5. Comparison of primary afferent response and nocifensive behaviour

Data obtained from the present study are compared with already published data on the time course of nocifensive responses in behaving rats and humans (see text for details). The time course of the response to ATP analogues is shown in three different models: rat nocifensive behaviour following intraplantar injection of α,β-methylene ATP (▵, dashed line) (Hamilton et al. 1999), transcutaneous iontophoresis of ATP in humans (□, continuous line) (Hamilton et al. 2000) and single C-fibre unit recordings from the in vitro skin-nerve preparation (• with error bars, continuous line). The method of quantifying the response to ATP analogues is indicated in the y-axis: paw lifting in 2 min bins for the rat behavioural model; the average number of spikes in 10 s bins for the single unit recordings; and the average visual analog score (VAS rating) every 20 s for human subjects.