The role of prostaglandins in the bradykinin-induced activation of serosal afferents of the rat jejunum in vitro

Abstract

1. This study was performed to elucidate the role of prostaglandins in the action of bradykinin on serosal afferent neurones supplying the rat jejunum. Extracellular recordings of multi-unit activity were made from serosal afferents in isolation, using a novel in vitro preparation. The discharge of single afferents within the multi-unit recording was monitored using waveform discrimination software. 2. All afferents tested were both mechano- and capsaicin sensitive. Application of bradykinin elicited increases in whole nerve discharge in a concentration-dependent manner. The agonist potency estimate (EC50) was 0.62 +/- 0.12 microM and is consistent with an interaction at the B2 receptor subtype. 3. The stimulatory effect of bradykinin on serosal afferents was antagonized by a specific antagonist of the B2 receptor, HOE140. In contrast, a selective B1 receptor antagonist, [des-Arg10]HOE140, had no effect. The IC50 estimate obtained for HOE140 was 1.6 nM and again consistent with an interaction at B2 receptors. 4. The response to a submaximal concentration of bradykinin (1 microM) was significantly reduced to 24.4 +/- 54.9 % of control following blockade of cyclo-oxygenase activity with naproxen (10 microM). The addition of 1 microM prostaglandin E2 (PGE2), in the presence of naproxen, had no direct effect on afferent activity, but fully restored the response to bradykinin in 15 single afferents. 5. In summary, bradykinin stimulates serosal afferents by a direct action on kinin B2 receptors that are present on serosal afferent terminals. The response to bradykinin is dependent on the presence of prostaglandins, particularly PGE2. We suggest that bradykinin has a self-sensitizing action, whereby it stimulates the release of PGE2, which in turn sensitizes the endings of serosal afferent neurones responsive to bradykinin.

Figure 1. Bradykinin stimulates whole nerve mesenteric afferent discharge

Typical mesenteric afferent response to the application of bradykinin (1 μM, 2 min). Above the raw nerve trace is a rate histogram of the whole nerve discharge which clearly shows a large increase in nerve discharge in the presence of bradykinin. The histogram shows the number of action potentials in consecutive 5 s bins. Below is a segment of the nerve recording which has been expanded to illustrate the multi-unit nature of these recordings. However, action potentials of different amplitude can be clearly identified which, in some instances, enable single units to be discriminated using waveform analysis.

Figure 2. Sequential concentration-response curve for bradykinin (0.03-10 μM)

A is a rate histogram of the whole nerve discharge from a typical recording showing the total number of action potentials in consecutive 20 s bins; B is the mean CRC from 5 experiments in which nerve discharge from individual recordings has been normalized by expressing data as a percentage of the maximum response to bradykinin.

Figure 3. Single unit responses to bradykinin

Sequential rate histograms of 2 single afferent units discriminated from the same whole nerve recording using waveform analysis. The upper unit displays sensitivity to higher concentrations of bradykinin with a threshold around 0.1 μM and a maximum response at 3 μM. The lower unit already has a prominent response at 0.1 μM and peaks at around 1 μM. The histograms shows the number of action potentials in consecutive 20 s bins.

Figure 4. Summary of the effects of bradykinin receptor antagonists

A is a rate histogram showing the total number of action potentials in consecutive 1 s bins in response to repeated 2 min exposure to 1 μM bradykinin (shown by the bar). The area under the response curve for the second response is expressed as a percentage of the first response in the histograms below. Note that there is no desensitization to bradykinin in this time control experiment. B, the response to bradykinin (1 μM, 2 min, □) in time control experiments, and following 30 min incubation with the B₂ receptor antagonist HOE140 (1, 3 and 10 nM,) and the B₁ receptor antagonist [des-Arg¹⁰]HOE140 (100 nM, ▪).

Figure 5. Summary of the effect of the cyclo-oxygenase inhibitor naproxen on the afferent response to bradykinin

The mean percentage of the control response to bradykinin (1 μM, 2 min) remaining in the presence of naproxen (10 μM) is compared with the time matched control.

Figure 6. Mean CRCs for bradykinin after incubation with naproxen (10 μM) in the presence and absence of PGE₂ (1 μM)

Data are expressed as the percentage of the maximum response to bradykinin prior to treatment with naproxen. The control curve obtained by non-linear regression analysis of the bradykinin response data obtained prior to naproxen using a one-binding site equation (R² = 0.91) is shown for comparison.

Figure 7. Summary of the effect of prostaglandin E₂ (PGE₂) in the presence of naproxen on the response to bradykinin in 15 single units

The control response to bradykinin (1 μM, 2 min, ▪), the response in the presence of naproxen (10 μM, ) and the response following the further addition of PGE₂ (1 μM, □). The effect on spontaneous discharge frequency (A), mean discharge frequency in the presence of bradykinin (B), overall afferent discharge to bradykinin (C) and latency of the bradykinin response (D) are shown.