Showing papers by "Peter W. Reeh published in 2001"

Sustained sensitization and recruitment of rat cutaneous nociceptors by bradykinin and a novel theory of its excitatory action

Abstract: Among the endogenous algogens and inflammatory mediators, bradykinin (BK), serotonin, prostaglandins and histamine, BK has been found to be the most effective agent acting on cutaneous nociceptors in the rat (Lang et al. 1990; Handwerker & Reeh, 1991; Reeh 1994). These studies, however, have shown that BK alone activates less than half of the C-fibres in the rat skin. This prevalence is much lower than that in deep tissues where it has been reported to be about 90 % (Baker et al. 1980; Kumazawa & Mizumura, 1980; Haupt et al. 1983; Kanaka et al. 1985). In addition, the excitatory effect of BK on nociceptive nerve fibres appears to be transient and prone to tachyphylaxis (Kumazawa et al. 1987; Lang et al. 1990; Koltzenburg et al. 1992). Moreover, the BK concentrations needed to excite nerve endings (≈10−6m) are clearly higher than those determined in exudates of painful inflammations (Handwerker & Reeh, 1991). Together, these arguments seem to indicate that the excitatory effect of BK is less important in the overall induction of pain. On the other hand, BK-induced sensitization of primary afferent nociceptors to heat (Kumazawa et al. 1991; Khan et al. 1992; Koltzenburg et al. 1992; Schuligoi et al. 1994), mechanical (Levine et al. 1986; Steranka et al. 1988; Neugebauer et al. 1989) and endogenous chemical (Kessler et al. 1992; Koppert et al. 1993; Vyklickýet al. 1998) stimulation has been well established. The sensitizing effects of BK have been reported to be more sustained than the excitatory ones (Neugebauer et al. 1989; Manning et al. 1991), and they can occur independently of BK inducing discharge (Beck & Handwerker, 1974; Khan et al. 1992). In the monkey skin, it has been reported that all mechano-heat-sensitive fibres are sensitized to heat stimulation by BK, but only some of them show an excitatory response to the chemical stimulus itself (Khan et al. 1992). Therefore, the sensitizing rather than excitatory effect of BK seems to be the more important one, inducing and eventually maintaining hyperalgesia and pain from harmless stimuli. In rat and monkey skin it has been shown that BK sensitizes the afferent units to heat but not to mechanical stimulation (Lang et al. 1990; Koltzenburg et al. 1992; Khan et al. 1992). Vice versa, previous heat stimulation was found to leave the afferents with prolonged sensitization to BK stimulation and could even uncover a latent BK responsiveness in units which were previously not excited by BK (Beck & Handwerker, 1974; Lang et al. 1990; Mizumura et al. 1992). These observations suggest a particular functional interrelationship in the transduction of heat and BK stimuli. It is conceivable that such a mutual sensitization may play an important role in inflammatory pain and hyperalgesia since both local temperature and BK concentration are increased and maintained at high levels in inflamed tissue (Hargreaves et al. 1988; Damas et al. 1990). However, little is known about the persistence or possible tachyphylaxis of the sensitizing effect and to what extent BK can induce heat sensitization in the majority of nociceptive fibres which are not excited by BK or insensitive to heat. To answer these questions we have performed the present study using isolated rat skin and recordings from unmyelinated nociceptive nerve fibres. A short account of the present work has previously been published in abstract form (Haake et al. 1996).

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.

Bradykinin-induced nociceptor sensitization to heat is mediated by cyclooxygenase products in isolated rat skin.

Abstract: Bradykinin can excite C-polymodal nociceptors and sensitize them to heat and it can also enhance prostaglandin synthesis, but it is unclear whether these effects are causally related. The role of cyclooxygenase products was investigated using two enantiomers of the cyclooxygenase inhibitor flurbiprofen of which S(+)- is more potent than R(-)-flurbiprofen. Single-unit activity was recorded from mechano-heat-sensitive, polymodal C-fibers in the isolated rat skin-saphenous nerve preparation. Bradykinin pretreatment (10 microM, 5 min) induced a 219 +/- 26% increase in the number of spikes evoked by noxious heat stimulation and a drop in the heat threshold by 5.2 +/- 0.6 degrees C in a fully reproducible manner. S(+)-flurbiprofen (1 microM) abolished the bradykinin-induced heat sensitization but did not alter the unconditioned heat response itself. Under R(-)-flurbiprofen (1 microM) bradykinin still induced a significant heat sensitization which was reduced by 33 +/- 21% (P = 0.11) of its previous extent; this effect may be due to the limited purity of the enantiomer preparation or to a cyclooxygenase-independent action of flurbiprofen. The heat sensitization suppressed by S(+)-flurbiprofen could be significantly restored (to 43 +/- 12%) by addition of PGE(2) plus PGI(2) (10 microM both) to bradykinin. Neither S(+)- nor R(-)-flurbiprofen had an influence on the magnitude of the excitatory effect of bradykinin. It is concluded that (i) cyclooxygenase products are the main mediators of nociceptor sensitization to heat following bradykinin treatment in the isolated rat skin; (ii) PGE(2)/I(2) are essential but perhaps not the only relevant cyclooxygenase products involved and (iii) neither S(+)- nor R(-)-flurbiprofen inhibit the unconditioned noxious heat response and the excitatory bradykinin response of the polymodal C-nociceptors.

Pro- and anti-inflammatory actions of ricinoleic acid: similarities and differences with capsaicin.

Abstract: We have investigated the pro- and anti-inflammatory effects of ricinoleic acid (RA), the main active principle of castor oil, in an experimental model of blepharitis induced by intradermal injection of carrageenan in the guinea-pig eyelid and its possible capsaicin-like mode of action on acutely dissociated rat dorsal root ganglia (DRG) neurons in vitro. Topical treatment with RA (10-100 mg/guinea-pig) or capsaicin (1-10 mg/guinea-pig) caused eyelid reddening and oedema. At lower doses (0.3-3 mg/guinea-pig and 0.009-0.09 mg/guinea-pig for RA and capsaicin, respectively) both drugs significantly potentiated the eyelid oedema induced by carrageenan. The tachykinin NK1 receptor antagonist FK 888 (0.59 mg/kg s.c.) abolished the potentiation of carrageenan-induced eyelid oedema induced by either RA or capsaicin. The neutral endopeptidase inhibitor, thiorphan (1.3 mg/kg i.v.) significantly enhanced the potentiation of carrageenan-induced eyelid oedema produced by RA. This potentiating effect was abolished by FK 888. Repeated (8 days) topical application of RA (0.9 mg/guinea-pig) or capsaicin (0.09 mg/guinea-pig) inhibited the carrageenan-induced eyelid oedema. This anti-inflammatory effect was accompanied by a reduction (75%-80% of SP and 46%-51% of NKA) in tachykinin content of the eyelids, as determined by radioimmunoassay. In dissociated rat DRG neurons, RA (0.1 mM for 5 min) significantly inhibited the inward currents induced by application of capsaicin (1 microM) and/or low pH (5.8), without inducing any currents by itself or changing voltage-dependent currents. Moreover, after 24-h incubation, RA (0.1 mM) significantly decreased the capsaicin (1 microM)-induced calcitonin gene-related peptide (CGRP) release from rat DRG neurons, whereas acute drug superfusion did not evoke CGRP release by itself. Summarizing, RA possesses capsaicin-like dual pro-inflammatory and anti-inflammatory properties which are observed upon acute and repeated application, respectively. However, unlike capsaicin, RA does not induce inward current in DRG neurons and it is devoid of algesic properties in vivo.

Noxious heat-induced CGRP release from rat sciatic nerve axons in vitro.

Abstract: Noxious heat may act as an endogenous activator of the ionotropic capsaicin receptor (VR1) and of its recently found homologue VRL1, expressed in rat dorsal root ganglion cells and present along their nerve fibres. We have previously reported that capsaicin induces receptor-mediated and Ca++-dependent calcitonin gene-related peptide (CGRP) release from axons of the isolated rat sciatic nerve. Here we extended the investigation to noxious heat stimulation and the transduction mechanisms involved. Heat stimulation augmented the CGRP release from desheathed sciatic nerves in a log-linear manner with a Q10 of approximately 15 and a threshold between 40 and 42 degrees C. The increases were 1.75-fold at 42 degrees C, 3.8-fold at 45 degrees C and 29.1-fold at 52 degrees C; in Ca++-free solution these heat responses were abolished or reduced by 71 and 92%, respectively. Capsazepine (10 microm) and Ruthenium Red (1 microm) used as capsaicin receptor/channel antagonists did not significantly inhibit the heat-induced release. Pretreatment of the nerves with capsaicin (100 microm for 30 min) caused complete desensitization to 1 microm capsaicin, but a significant heat response remained, indicating that heat sensitivity is not restricted to capsaicin-sensitive fibres. The sciatic nerve axons responded to heat, potassium and capsaicin stimulation with a Ca++-dependent CGRP release. Blockade of the capsaicin receptor/channels had little effect on the heat-induced neuropeptide release. We conclude therefore that other heat-activated ion channels than VR1 and VRL1 in capsaicin-sensitive and -insensitive nerve fibres may cause excitation, axonal Ca++ influx and subsequent CGRP release.

Role of nitric oxide in zymosan induced paw inflammation and thermal hyperalgesia.

Abstract: Objective: To assess the involvement of spinal inducible nitric oxide synthase (iNOS) in inflammation and nociception.¶Materials and Methods: The time course of iNOS mRNA expression in rat spinal cord and inflamed paw was assessed by means of quantitative real time RT-PCR. In addition, the effects of the iNOS inhibitor L-NIL on inflammatory paw edema and thermal hyperalgesia were studied in comparison to those of the NO-donor RE-2047. L-NIL (3, 9, 27 and 81 mg/kg) and RE-2047 (3, 9 and 27 mg/kg) or vehicle were administered orally 15 min prior to the intraplantar injection of 0.625 mg zymosan.¶Results: Following zymosan injection, mRNA expression of iNOS increased in the inflamed paw and spinal cord with a maximum at 2.5 and 4 h, respectively. In the spinal cord iNOS mRNA started to decline at 10 h whereas it remained at maximum in the inflamed paw up to the end of the observation period of 24 h. As expected, RE-2047 had significant pronociceptive and proinflammatory effects. L-NIL significantly reduced paw inflammation at 27 and 81 mg/kg but failed to reduce hyperalgesia at the doses tested.¶Conclusions: The results show that iNOS is upregulated in the inflamed tissue and spinal cord with a similar time course. The effects obtained with L-NIL suggest that iNOS differently contributes to the inflammatory and nociceptive response induced by zymosan.¶

Muscarinic M2 receptors inhibit heat-induced CGRP release from isolated rat skin.

Abstract: The action of cholinergic agonists on modulating basal and heat-induced CGRP release was investigated in isolated rat skin. Nicotine (10(-6), 10(-5) and 10(-4) M) induced a bimodal increase of CGRP release, that was significant for the two larger concentrations (by 113 and 36%, respectively). On the contrary, muscarine (10(-4) M) and arecaidine (10(-5) M) significantly decreased the basal CGRP release (by 16 and 23%, respectively). The substantial increase of CGRP release evoked by noxious heat (47 degrees C) remained unaltered upon co-application of nicotine, but was diminished by 35% upon muscarine. Arecaidine was more effective in this respect causing significant dose-dependent depressions by 30% (at 10(-6) M) and by 60% (at 10(-5) M). These data support a role of muscarinic M2 receptors in nociceptor desensitization.

Modulation of CGRP and PGE2 release from isolated rat skin by alpha-adrenoceptors and kappa-opioid-receptors.

Abstract: Norepinephrine (NE) reduces the release of neuropeptides from central terminals of primary afferent neurones by presynaptic inhibition. We investigated whether NE also affects stimulus-induced intracutaneous calcitonin gene-related peptide (CGRP) and secondary prostaglandin E2 (PGE2) release. For comparison, kappa-opioid effects were examined. Antidromic electrical nerve stimulation resulted in significant increases in the release of CGRP and PGE2. The PGE2 release was prevented by selective activation of alpha2-adrenoceptors whereas the CGRP release was not changed. In contrast, selective kappa-opioid receptor activation diminished electrically evoked release of both CGRP and PGE2. We conclude that NE affected stimulated PGE2 release via alpha2-adrenoceptors on cells other than cutaneous afferents while kappa-opioid receptors are expressed in peripheral terminals of cutaneous afferents and their activation reduced CGRP release and secondary PGE2 formation.