Interactions of inflammatory mediators stimulating release of calcitonin gene-related peptide, substance P and prostaglandin E2 from isolated rat skin

doi:10.1016/S0028-3908(00)00171-4

Neuropharmacology

Volume 40, Issue 3, March 2001, Pages 416-423

https://doi.org/10.1016/S0028-3908(00)00171-4 Get rights and content

Abstract

Inflammatory mediators acting directly on nociceptive primary afferents induce neuropeptide release. In this study we investigated interactions between bradykinin, serotonin, histamine, prostaglandin and acid pH in stimulating the release of substance P (SP), calcitonin gene-related peptide (CGRP) and prostaglandin E₂ (PGE₂) from isolated flaps of rat back skin using enzyme immunoassays. Stimulation with bradykinin (10⁻⁵ M) augmented the release of SP, CGRP and PGE₂ significantly. Serotonin, histamine and PGE₂ individually tested (10⁻⁵ M) had no effect on neuropeptide release but they facilitated the bradykinin-evoked neuropeptide release. When bradykinin was combined with both serotonin and histamine, neither additional PGE₂ nor acid pH showed any further effect, suggesting that the facilitation had reached a maximum. Exposure of the skin to acid pH (6.1 or 5.2) significantly increased CGRP release. SP release was only slightly enhanced and PGE₂ release, in contrast, was suppressed by low pH stimulation, probably due to pH-dependent inhibition of phospholipase A₂. Treatment of the rats with flurbiprofen (25 mg/kg i.p.) one hour before dissection reduced PGE₂ to detection level and inhibited the CGRP secretion evoked by the combination of bradykinin, serotonin and histamine (all 10⁻⁶ M). As this suppression could not be overcome by substitution of PGE₂ (10⁻⁶ M), it is likely that exogenously applied PGE₂ differs in effect from endogenous, intracellularly synthesized prostaglandins that are accompanied by active intermediates and byproducts.

Introduction

The classical inflammatory mediators bradykinin (BK), serotonin (5-HT), histamine and prostaglandins, as well as acid pH, show direct excitatory and/or sensitising effects on nociceptive primary afferents (reviewed by Kress and Reeh, 1996). By acting directly on nociceptors, the mediators induce neuropeptide secretion, and they stimulate prostaglandin formation in the surrounding tissue matrix. This has been demonstrated in several preparations (Juan, 1977, Griesbacher and Lembeck, 1987, Franco-Cereceda et al., 1989, Geppetti et al., 1991, Hua and Yaksh, 1993, Sauer et al., 1998). Since rat skin has been well characterised in terms of nociceptor responsiveness to chemical stimuli, it is now of interest to determine the correlation of stimulated neuropeptide release with previous findings. Therefore, this study was restricted to those inflammatory mediators and concentrations that showed excitatory and/or sensitising effects in the rat skin–nerve preparation in previous studies, and their interactions in stimulating release of calcitonin gene-related peptide (CGRP), substance P (SP) and PGE₂ were investigated in the same isolated preparation. The chosen inflammatory mediators 5-HT, histamine and PGE₂ have been shown not to stimulate neuropeptide release when tested individually in previous studies (Hua and Yaksh, 1993, Franco-Cereceda et al., 1989, Kress et al., 1999); therefore, the investigations focused on the facilitatory effects of these inflammatory mediators on the BK-evoked neuropeptide and PGE₂ release.

The inflammatory mediators BK, 5-HT and histamine are well known to stimulate phospholipase A₂ and prostaglandin synthesis in consequence (Dennis, 1994). Thus, endogenous PGE₂ could confound the interactions studied. We have investigated this by cyclooxygenase blockade using a non-steroidal anti-inflammatory drug, flurbiprofen. We also tested whether its effects were reversible upon substitution of PGE₂.

Short accounts of the present work have been published in abstract form (Averbeck and Reeh, 1997).

Section snippets

Methods

All agents under test were obtained from Sigma (Deisenhofen, Germany). The release experiment was carried out in synthetic interstitial fluid, a buffer that consisted of (in mM) NaCl 108, KCl 3.5, CaCl₂ 1.5, MgSO₄ 0.7, NaHCO₃ 26, NaH₂PO₄ 1.7, sodium gluconate 9.6, glucose 5.5 and sucrose 7.6 (Bretag, 1969). Before use, the buffer was gassed with carbogen to adjust the pH to 7.4. Solutions of low pH were prepared by gassing the buffer with pure CO₂ instead of carbogen to reach pH 6.1 or by

Effects of low pH on SP, CGRP and PGE₂ release

Stimulation of rat skin with low pH of 6.1 and 5.2 increased CGRP release, decreased PGE₂ release but had no significant effect on SP release (Table 1 and Fig. 1a). The suppression of PGE₂ release at pH 6.1 could be counteracted in a dose-dependent manner by the addition of the triple combination of inflammatory mediators BK, 5-HT and histamine at 10⁻⁶ M and 10⁻⁵ M (Table 1, Table 2). This test was not attempted using the stimulation with pH 5.2.

SP, CGRP and PGE₂ release induced by inflammatory mediators at 10⁻⁵ M

The incubation of rat skin with inflammatory

Discussion

Several endogenous mediators and algogens are liberated in inflamed tissue. They stimulate prostaglandin secretion from cells of the tissue matrix (Juan, 1977, Griesbacher and Lembeck, 1987, Sauer et al., 1998) and, by acting directly on nociceptors, they induce neuropeptide release from the peripheral nerve endings (Franco-Cereceda et al., 1989, Geppetti et al., 1991, Hua and Yaksh, 1992). Elevated intracellular Ca²⁺ levels are necessary and sufficient to release neuropeptides from vesicles in

Acknowledgements

The authors thank A. Kuhn and I. Izydorczyk for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 353 B12).

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Interactions of inflammatory mediators stimulating release of calcitonin gene-related peptide, substance P and prostaglandin E2 from isolated rat skin

Abstract

Introduction

Section snippets

Methods

Effects of low pH on SP, CGRP and PGE2 release

SP, CGRP and PGE2 release induced by inflammatory mediators at 10−5 M

Discussion

Acknowledgements

Neuroscience

Life Sciences

FEBS Letters

Neuroscience

European Journal of Pharmacology

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Journal of Biological Chemistry

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British Journal of Pharmacology

Neuron

Neuroscience Letters

Neuroscience Letters

Neuroscience

Brain Research

General Pharmacology

Neuroscience Letters

Neuroscience Letters

Life Sciences

Neuroscience Letters

Pain

Biochemical and Biophysical Research Communications

Neuroscience Letters

Interactions of inflammatory mediators in releasing CGRP, SP and PGE2 from rat skin, in vitro

European Journal of Physiology—Pfluegers Archiv

Bradykinin and serotonin effects on various types of cutaneous nerve fibers

European Journal of Physiology—Pfluegers Archiv

Interactions of inflammatory mediators stimulating release of calcitonin gene-related peptide, substance P and prostaglandin E₂ from isolated rat skin

Effects of low pH on SP, CGRP and PGE₂ release

SP, CGRP and PGE₂ release induced by inflammatory mediators at 10⁻⁵ M

Interactions of inflammatory mediators in releasing CGRP, SP and PGE₂ from rat skin, in vitro