Adenosine 5'-triphosphate and its relationship with other mediators that activate pelvic nerve afferent neurons in the rat colorectum

Abstract

Evidence of a role for purinergic signalling in visceral afferents involving P2X(2), P2X(3) and P2Y(1) receptors exists, which appears to be important during inflammation. This study aimed to evaluate the degree of interaction between adenosine 5'-triphosphate (ATP) and other mediators that activate sensory nerves in the colorectum. Recordings from pelvic nerve afferents were made during application of agents to the in-vitro colorectal preparation. Analysis allowed calculation of single unit activity. When applied individually, bradykinin (78%) and 5-hydoxytryptamine (77%) activated the greatest number of neurons, followed by substance P, protons, ATP and capsaicin. Prostaglandin E(2) stimulated the least number (54%) and had a longer latency. Seventy-seven percent of all units studied either responded to both ATP and capsaicin or to neither, giving the greatest degree of activity correlation. Five percent of units were activated by all seven agents and no units were activated by a single agent alone. 5-hydroxytryptamine, capsaicin and protons, when co-applied with ATP, increased pelvic nerve activity to a greater degree than the sum of the individual responses. It is concluded that ATP activates pelvic nerve afferents and acts synergistically with protons, capsaicin and 5-hydroxytryptamine. The pattern of neuronal activation suggests that visceral afferents are polymodal but the receptor expression on their terminals is variable.

Figure 1

Typical responses from pelvic nerve afferents during application of the various agents used in the study. The traces show the frequency of spike firing (Hz) during multifibre recording. As labelled, the responses to (a) ATP, (b) 5-HT, (c) protons, (d) capsaicin, (e) bradykinin, (f) prostaglandin E₂ and (g) substance P are shown. Arrows denote point of application.

Figure 2

The latencies of activation of the agents applied to the serosal surface of the colorectum were similar except for prostaglandin E₂ (a). The time taken to reach peak spike frequency is shown in (b), where capsaicin was fastest and prostaglandin substantially slower than the others (cap, capsaicin; H, protons). ^*P ≤ 0.05.

Figure 3

A typical response of a pelvic nerve multiunit recording to distension (50 mmHg).

Figure 4

Two continuous traces from the same experiment are shown. On the left, the pelvic nerve (multifibre) response to application of ATP 1 mM to the colorectum is demonstrated. On the right (after 30 min washout) the response to capsaicin (Cap) 1 µM is shown, followed shortly afterwards by a repeat application of ATP 1 mM. The ATP-sensitive fibres are still responsive after capsaicin, but multifibre activity is reduced compared to the previous application of ATP. Top trace: spike frequency (Hz), bottom trace: neurogram.

Figure 5

Pelvic nerve recordings are shown during the application of 5-HT (a), capsaicin (b) and protons (c). For each experiment, three traces are shown: the top trace shows the frequency of activity, the middle trace the neurogram and the bottom line shows the pressure, which remained at zero throughout. The figures show the application of low concentration agent (left hand traces), followed by ATP 1 mM (middle traces) and then the coapplication of both (right hand traces) showing greater activity than the sum of the two individual responses.

Figure 6

(a) Co-application of 5-HT (5 or 100 µM) and ATP (1 mM or 50 µM, respectively) resulted in greater nerve activity than the sum of the individual responses suggesting synergism (combined data for high and low concentrations is shown). This was also the case for low concentration data capsaicin (Cap) 100 nM and ATP 1 mM (b). Responses to ATP 1 mM are greater at pH 6.8 (c) and even more so at pH 6.5 (d). For each graph, the relative proportion of the sum of the individual responses is shown, so the first two bars add up to 1.0. The third bar (on the right) then demonstrates the degree of increased activity when the two agents are applied together. ^*P ≤ 0.05; ^***P < 0.0001.

Figure 7

An example of a single unit in the pelvic nerve that responds to both 5-HT and ATP individually. When the two mediators are applied together, the activity is greatly increased.