The Journal of Neuroscience, May 1993, 73(5): 1947-l 953

Pharmacology of the Effects of , , and on the Release of Gene-related from C-Fiber Terminals in the Rat Trachea

Xiao-Ying Hua and Tony L. Yaksh Department of Anesthesiology, University of California at San Diego, La Jolla, California 92093-0818

The effects of inflammatory substances, bradykinin (BK), matory cell function (see Szolcsanyi, 1984). The effects are 5-HT, and histamine (HIS), on the release of calcitonin gene- thought to be mediated by the local releaseof mediators such related peptide (CGRP) from the peripheral terminals of sen- as the tachykinins and calcitonin gene-related peptide (CGRP) sory afferents in the rat trachea were examined ex viva. With (seeHolzer, 1988). The mammalian respiratory tract, including intralumenal perfusion, the isolated rat trachea displays low the trachea, is innervated by unmyelinated afferents containing but measurable secretion of CGRP (32 + 4.6 fmol/lO min CGRP and, to a lesserextent, substanceP and fraction). The addition of BK (1O-B to lo-’ M) to the super- (Lundberg et al., 1983; Cadieux et al., 1986). Thus, manipula- fusate resulted in an immediate, concentration-dependent tions that evoke neurogenic inflammatory responsesobserved increase in the level of CGRP (5-30-fold increase above in the trachea, for example, an increasein vascular permeability baseline) in the perfusates, and this effect showed a con- by local application of (CAP) (Lundberg and Saria, centration-dependent tachyphylaxis. [Des-Arg’O]-, a 1983) or an increase in tracheal blood flow by antidromically B, , at concentrations of up to lo-’ M did electrical stimulation of vagal nerve (seeMartling, 1987), may not induce any significant increase in CGRP outflow from do so by the local releaseof thesepeptides. the rat trachea. HIS at 1 O-4 M caused a modest but progres- In previous work, using the intralumenally perfusedisolated sive augmentation in the release of CGRP. 5-HT at lOma M trachea, wehave shown that CAP, excesspotassium, or electrical had no effect upon the resting efflux of CGRP, but at a con- field stimulation will yield an acute stimulus-dependentincrease centration of 10 m(LM significantly enhanced the release of in the extracellular levels of CGRP (Hua and Yaksh, 1992).This CGRP evoked by capsaicin (lOme M). Similar conditioning releaseis dependent and can be induced by the local studies carried out with HIS and BK showed no augmenta- excitation of the TTX-treated axon (Hua and Yaksh, 1991), tion. BK-evoked CGRP efflux was significantly inhibited by emphasizingthat CGRP and tachykinin activity is releasedfrom [o-Argo, Hyp5, This~a, o-Phe+BK (B, antagonist) and indo- thesetracheal afferents by local terminal depolarization. methacin. While [Des-Args, Leu*]-BK (B, antagonist) also It has been shown that local application of a variety of ma- caused a reduction of BK-induced release, its effect did not terials typically found in the tissue following local injury can reach statistical significance. The facilitatory effect of 5-HT evoke activity in C-fibers. Thus, degranulation leads on capsaicin-evoked release was not markedly affected by to the release of histamine (HIS) and 5-HT as well as other 5-HT, or 5-HT, antagonists, (s)-(-)propranolol or methyser- inflammatory mediators (Fewtrell et al., 1982; Schleimer et al., gide, but was totally abolished by the 5-HT, receptor antag- 1986). Activation of the Hageman factor and the initiation of onist ICS 205-930 and also by indomethacin. These data certain Hageman-dependentpathways lead to the formation of suggest that BK, acting through a B, receptor, activates CGRP BK (Miller and Margolius, 1988).These agents have been shown release from the peripheral terminals of capsaicin-sensitive to evoke sensoryC-fiber excitation (Kaufman et al., 1980; Lang sensory afferents. The sensitizing effect of 5-HT on the cap- et al., 1990). Electrophysiological study of the rat skin has re- saicin-evoked release of CGRP may be mediated via a 5-HT, vealed that 5-HT excited 43% of the polymodal C-fibers that receptor. Both actions appear to require the formation of are also driven by BK and CAP. Furthermore, conditioning cyclooxygenase products for their manifestation. treatment with 5-HT causeda sensitization to subsequentBK [Key words: bradykinin, 5-HT, histamine, capsaicin, cal- stimulation (Lang et al., 1990). citonin gene-related peptide, release, trachea] Given the correlation outlined above between terminal ac- tivity and local release,it follows that pharmacologicalstimuli The peripheral terminals of unmyelinated sensory axons are known to activate C-fiber terminals may result in the local re- widely distributed. Activation of these terminals is known to leaseof terminal contents. The current thinking regarding the influence blood flow, capillary permeability, and local inflam- releaseof CGRP from the afferent terminal suggeststhat stimuli that lead to the activation of the C-fiber terminal will in fact evoke the exocytotic release of the vesicular contents of the Received June 10, 1992; revised Nov. 5, 1992; accepted Nov. 11, 1992. terminal. Thus, studiescarried out in guinea pig (Saria et This work was supported by funds provided by the Cigarette and Surtax Fund of the State of California through the Tobacco-Related Disease Research al., 1988) or heart (France-Cerecedaet al., 1989; Geppetti et Program of the University of California (X.-Y.H.). al., 1991) have shown that, consistent with the stimulatory ef- Correspondence should be addressed to Xiao-Ying Hua, Department of An- esthesiology 08 18, University of California at San Diego, 9500 Gilman Drive, La fect, local application of BK will evoke the subsequentrelease Jolla, CA 92093-08 18. ofthe peptidesfrom the peripheral terminals. This measurement Copyright 0 1993 Society for Neuroscience 0270-6474/93/l 3 1947-07$05.00/O of peripheral afferent releasethus provides a direct method for 1949 Hua and Yaksh * BK and 5-HT on CGRP Release

* CAP 1 O-5M BK 1 O-5M

200 ihi BK 1 O-4M lo-4M f‘ 600 = 0 E 150 t z E k 400

1 2 3 4 5 6 FRACTION

Figure 2. The effect of CAP desensitization on BK-induced CGRP release from perfused rat trachea. CAP at 1O-5 M was applied atfractions 2, 3, and 4. BK 1O-5 M was administrated at fraction 5. CGRP level is expressed as femtomoles per 10 min fraction, and the data are given as mean + SEM of five experiments.

Materials and Methods Release experiment. Male Sprague-Dawley rats (300-350 gm body weight) were used in the present study. The animals were anesthetized with sodium pentobarbital (Anthony Prod; 50 mg/kg, i.p.) and decap- itated. The trachea (from larynx to carina) was carefully removed and placed in a perfusion bath (37°C). Intralumenal tracheal perfusion was carried out with oxygenated (95% O,, 5% CO,) Krebs’-bicarbonate solution, which contained (in mM) NaCl, 118.3; KCl, 4.7; CaCl,, 2.5; MgSO,, 1.2; NaHCO,, 25; KH,PO,, 1.2; and, , 11; 0.3% baci- tracin added to prevent possible peptide degradation; pH 7.35-7.45. The perfusion flow rate was 0.2 mUmin. After a 30 min equilibration period, perfusates (2 ml) were collected at 10 min intervals in test tubes containing acetic acid (final concentration of 2 M). To investigate the possible facilitatory effects on CGRP release, BK, 5-HT, and HIS (all given at a concentration of 1O-6 M) were added to the perfusate in the sample preceding and during the administration of CAP ( 1O-6 M). To study the antagonist pharmacology of the effects of two BK analogs, lo-6M lo-6M [D-A@, Hyp3, Thi5,8, o-Phe7]-BK(B,) (Dray and Perkins, 1988) or [Des- Arg9, Leu8]-BK (B,) (Regoli et al., 1990), (1O-6 or 5 x 1O-6 M) was applied in the sample 10 min before and during the application of BK. For 5-HT, the 5-HT antagonists (s)-(-)propranolol (5-HT,) (Hide et al., 1989), methysergide (5-HT,) (Fozard, 1984), and.ICS 265-930 (5- HT,) (Eschalier et al.. 1989) (1O-6 M) were similarlv aDDlied 10 min before-and during the’administration of 5-HT. To cdnsihkr the role of k loo cyclooxygenase products, indomethacin (1O-5 M) was applied 20 min before and during BK or 5-HT application. To examine the effects of 8 50 capsaicin desensitization, CAP ( 1O-5 M) was applied in samples 2-4 and BK was then applied in sample 5 (see Fig. 2). All fractions were then frozen and lyophilized before radioimmunoassay (RIA). Radioimmunoassay. CGRP determination was achieved with RIA 0 using CGRP antibody G987 (1:84,000) and 12+Ty?-CGRP tracer. CGRP 1 2 3 4 5 6 7 8 91011 antibody G987 is a gift from Dr. H. Heath (Mayo Clinic, MN), and FRACTION details of the antibody have been reported (Cater et al., 1991). The absolute sensitivity of G987 is 4.0 fmol/assay tube, CGRP antibody Figure I. CGRP level (femtomoles per 10 min sample) in the per& G987 is 100% cross-reactive to rat CGRP, human CGRP-LU, and human ates from intralumenally perfused rat trachea. BK (10m6 to 1O-4 M) was CGRP-& but does not cross-react to calcitonin, CGRP,,,,, CGRP,,,,, applied in fractions 4 and 9. Eachvalue representsthe mean+ SEM or SP (< 1% at 1 pmol/tube). In a previous study, we have demonstrated of four or five experiments.*, P < 0.05as comparedfraction 3 versus that the immunoreactivity released from the rat trachea detected by 4, 8 versus9, and4 versus9, by usingone-way ANOVA for repeated antibody G987 was eluted as a single peak at the same fraction as measurementsfollowed by Dunnett’stest. svnthetic rat CGRP-B fHua and Yaksh. 1992). CGRP trace was nurified by elution from a reverse-phase column usingHPLC with an aceionitrile gradient. The assay was carried out in duplicate, and nonspecific binding assessingthe local pharmacology of the peripheral terminal. and blanks were assessed. The peptide levels are expressed as femto- Given the well-defined releaseof CGRP from C-fibers in the moles per 10 min fraction. The background blanks were examined in trachea and the known stimulatory effectsof several autocoids normal Krebs’ buffer or buffer containing the substances examined, on vagal afferents,we sought, in the present study, to examine neither of them interfered with CGRP RIA. Drugs. We used bradykinin acetate salt (Sigma), 5-hydroxytryptamine the effectsof three inflammatory substances,BK, HIS, and 5-HT, hydrochloride (5-HT, Sigma), histamine (Sigma), indomethacin (Sig- on tracheal afferent CGRP releaseand to define the receptor ma), (s)-( -)propranolol (Sigma), methysergide maleate (Sandoz), ICS pharmacology of their actions. 205-930 (Sandoz), [Des-ArglO]-kallidin (a gift from Dr. Eric Whalley, The Journal of Neuroscience, May 1993, 13(5) 1949

Camaicin His His * lo-4M lo-4M 80 n CONTROL C

s’ 60

n u 200

8 100

0 1 2 3 4 5 &ION 1001 5-HT 5-HT Figure 4. CGRP level (femtomoles per 10 min fraction) from the lo-4M lo-4M isolated rat trachea during the administration of CAP ( 10m6 M) alone 80 - ” (CONTROL) in fraction 4 or CAP at the sameconcentration with con- ditioning application of BK, HIS, or 5-HT, all at 10d6 M in fractions 3 E 60- and 4. The valuesrepresent the mean? SEM of 7-12 experiments.*, % 4 P < 0.05 as compared with 5-HT group versus control group using one- way ANOVA for independentgroups followed by Dunnett’stest.

10m4M, however, was significantly diminished as compared to the first exposure [F(4,50) = 5.12, p < 0.011 (Fig. 1). [Des- ArglO]-kallidin, a B, agonist (Regoli et al., 1990) was without 12 3 4 5 6 7 8 91011 effect at 1O-6 M and 10m5M (data not shown). A modest nu- FRACTION merical increasein CGRP was seenat 1O-4M (basal, 33 + 5.7 fmol; [Des-ArglO]-kallidin, 45 -t 9.9 fmol per 10 min fraction; Figure 3. CGRP release (femtomoles per 10 min fraction)from the n = 6) but this changedid not achieve statistical significance. perfusedrat tracheain the presenceof HIS at 1O-4M and 5-HT at 10m4 M, which wereapplied to the tracheain samples4 and 9. The data are Continued application of CAP at 10m5M for 30 min evoked an given as meank SEM and representobservations from four experi- acute releasefollowed by a progressivedecline in the subsequent ments.*, P < 0.05, one-way ANOVA for repeated measurements fol- samplesto baseline.The subsequentapplication of BK at 1O-5 lowed by Dunnett’s test. M was then without effect (Fig. 2). 5-HT at 1O-4 M (Fig. 3) and HIS at 1O-5M (data not shown) Cortech,Inc., Denver, CO), [D-Argo,Hyp), Thi5.8,D-Phe7]-BK (Pen- did not causeany changein the efflux of CGRP. HIS at 1O-4M insula),[Des-Arg9, LeuS]-BK (Peninsula), rat CGRP-@(Peninsula), and induced a modest,gradual increasein CGRP outflow. Following capsaicin (Sigma; purity of 98%). Capsaicin and indomethacin were the secondapplication of HIS, there was a pronounced increase initially dissolvedin ethanoland subsequentlydiluted in Krebs’buffer with a final concentrationof 0.6-1.0%ethanol. In previouswork, we in release[F(3,32) = 6.5, p < O.OOl] at the sameconcentration haveshown that this vehicledid not influencethe basalrelease of CGRP (Fig. 3). (Hua and Yaksh, 1992).The other agentswere all dissolvedin Krebs’ The addition of a CAP-containing buffer (1 Om6M) induced a buffer. All agentswere reagent grade. remarkable efflux of CGRP from the perfused rat trachea Data analysis. Statisticalsignificance was calculatedusing one-way (ACGRP = 204 f 48 fmol, basallevel of the releasesubtracted) analysisof variance(ANOVA), with multiple comparisonsfor inde- pendentor repeatedmeasurements followed by Dunnett’stest. Critical (Fig. 4). Conditioning application of 5-HT, at a concentration valuesreaching a P < 0.05 level wereconsidered significant. (1Om6 M) that had no effect upon basalrelease, served to augment prominently the releaseevoked in the presenceof CAP (ACGRP Results = 405 f 53 fmol) [F(3,29) = 2.88, p < 0.051 (Fig. 4). CAP- Basal releaseof CGRP induced peptide releasewas not altered in the presenceof BK In the perfusate from the isolated rat trachea, basal outflow of or HIS (Fig. 4). CGRP was 32 + 5 fmol per 10 min fraction in untreated prep- aration (n = 30) (Figs. 14). In the absenceof pharmacological Bradykinin antagonists manipulation, this level remained stable for periods in excess [Des-Arg9,Leu*]-BK and [D-Argo, Hypl, Thi5.*, D-Phe’]-BK, both of 1.5 hr. at 1O-6 M, had no effect upon the releaseevoked by BK (5 x 1O-6M) (Fig. 5). [D-Argo, Hyp3, Thi5.8, D-Phe’]-BK at a concen- Evoked releaseof CGRP tration of 5 x 10m6M caused a significant reduction in the Exposure of the rat trachea to BK-containing ( 1O-6 to 10m4M) increasedCGRP releaseinduced by BK [F(5,35) = 3.16, p < buffer resultedin a concentration-dependentincrease of CGRP 0.051 (Fig. 5). [D-Argo, Hyp3, Thi5.*, D-Phe7]-BK at 5 x 10m6M level in the perfusates (Fig. 1). Forty minutes after the first served to enhanceCGRP releasein two out of six experiments. application with BK at 1O-5M, a secondexposure to BK at the [Des-Arg9, Leu*]-BK at a concentration of 5 x 10m6M also sameconcentration causeda similar amount of releaseof CGRP caused a reduction of BK-induced CGRP release;it did not, (Fig. 1). The releaseevoked by a second application of BK at however, reach a statistical significance.Indomethacin at 1O-5 1950 Hua and Yaksh l BK and 5-HT on CGRP Release

200

” T 400

300

200

100 CONT BK2 ant BK2 ant BKI ant BKI ant INDO IO-6M 5x1 O-6M 1 O-6M 5x1 o-6M 1 O-5M 0 7 Figure 5. The evoked CGRP release (ACGRP,femtomoles per 10min PROP METHY ICS INDO fraction, the basal level of CGRP subtracted) by BK (5 x 1O-6 M) alone CONT (CONT, control) or by pre-administrationof [D-Argo, Hyp3, This,*, Figure 6. The effectsof 5-HT antagonists(s)-(-)propranolol (PROP; D-Phe71-BK(B2 ant). IDes-A&. Leu*l-BK(BB1 ant), or indomethacin 1O-6M), methysergide(METHY; 1O-6M), and ICS205-930 (ZCS; 1O-6 (INDOj. *, P < 0.05 as comp%edwith control groupversus [D-Argo, M), and indomethacin(ZNDO; 1O-5M) on the enhancementof CAP- Hyp’, This,*,D-Phe7]-BK (B2 ant, 5 x 1O-6M); **, P < 0.01, control evoked(1O-6 M) CGRP releaseby 5-HT (1O-6M) (CONT, control). groupversus indomethacin group; one-way ANOVA for independent ACGRP:the evokedrelease in femtomolesper 10 min collection,with measurementsfollowed by Dunnett’stest (N = 4-8). the baselineof the releasesubtracted. **, P < 0.01,control group versus ICS 205-930;*, P < 0.05, control groupversus indomethacin group; one-wayANOVA for independentgroups followed by Dunnett’stest. M almost completely abolishedthe increasein CGRP outflow The data are givenas mean+ SEM and representobservations from by BK [F(5,35) = 3.84, p < 0.011 (Fig. 5). 7-l 2 experiments. 5-HT antagonists The basal level of CGRP efflux from the rat trachea was not dependentrelease of CGRP, which wasindomethacin sensitive. influenced in the presenceof 1O-6 M (s)-(-)propranolol, meth- This result is consistentwith previous work indicating that many ysergide, or ICS 205-930, or 1O-5 M indomethacin (data not of the actions of BK on and in activating the shown). (.s)-(-)Propranolol and methysergidecaused a reduc- peripheral terminal of primary sensoryneurons are mediated tion of 5-HT-induced (1 O+ M) enhancementof CGRP release by the releaseof prostaglandins(PGs) (seeRegoli et al., 1990). by CAP (1O-6 M) to someextent, but thesedifferences are not Importantly, BK will in fact stimulate PGE, releasefrom human statistically significant.In contrast, ICS 205-930( 1O-6M) [F(4,3 1) (Churchill et al., 1989) and canine tracheal epithelial cells (Lei- = 4.16, p < 0.011, aswell as indomethacin ( 1O-5M) [F(4,3 1) = kauf et al., 1985), PGI, from the guinea pig heart (Geppetti et 3.25, p < 0.051, induced a pronounced inhibition of the sen- al., 1991), and cultured endothelial cells from the bovine aorta sitization effect of 5-HT effect on the CGRP releaseevoked by (Hock et al., 1991). In the airway, PGE,, PGE,, and PGF,, have BK (Fig. 6). been found to activate afferent C-fibers (Coleridgeet al., 1976) and to facilitate vascular permeability induced by CAP (Saria Discussion and Lundberg, 1984). In other work, we have found that PGE,, In the presentstudy, CGRP levels in the perfusatesfrom isolated PGE,, PGF,,, and PGI, are all able to induce CGRP release rat trachea were elevated by BK and the CAP-evoked release from the CAP-sensitive afferent in the rat trachea, and this was augmented by 5-HT. Previous studies have shown that releaseis not altered by pretreatment with indomethacin (X.- CGRP is contained in vagal afferent C-fibers and thesestores Y. Hua et al., unpublishedobservations). have been shown to be CAP sensitive (Cadieux et al., 1986). The observation that repeated application of BK resulted in Immunohistochemical studieshave demonstratedthat, in ad- a subsequentdiminution of the amount of CGRP released,with dition to primary sensorynerve fibers, modestamounts of CGRP the degreeof reduction proportional to the first exposure con- immunoreactivity exist in endocrine-like cells in the epithelium centration, is consistent with other studieswith this peptide. of the rat trachea (Shimosegawaand Said, 1991). We do not Thus, in rat saphenousnerve preparation, half of the BK-sen- believe thesecells contribute to the present evoked releasebe- sitive C-fibers did not respondto a secondBK stimulus. This cause(1) thesecells appearto be CAP resistant (Shimosegawa desensitizationcould not be reversedby the application of lOO- and Said, 199l), and (2) the preliminary result has shown that fold higher BK concentration or by a 1.5-hr washout period tracheal CGRP releasestill exists after stripping of the epithe- (Lang et al., 1990). The mechanismof this inactivation is not lium (M. Hogman and X.-Y. Hua, unpublishedobservations). clear. However, McGehee and colleaguesin their recent study These observations and the observation from the present study (McGehee et al., 1992) have provided evidence suggestingthat that BK did not induce any releasein the CAP-desensitized the -cyclic GMP pathway is involved in BK desen- trachea thus suggestthat the peptide responseto theseinflam- sitization in rat dorsal root ganglion cells. matory agentsreflects an action on the afferent terminal, which With regard to the pharmacology of the BK effect, BK and is ultimately reflected in the releaseof CGRP. The issuesrele- related exert their biological effectsby activating at least vant to the several autocoids will be consideredbelow. two different receptor types: B, and B, (seeRegoli et al., 1990). The BK receptor mediating the responseto BK is Bradykinin believed to have the pharmacologicalcharacteristics of the B, In the present study, we observed that local application of BK subtype (Dray and Perkins, 1988; Mizumura et al., 1990). Fur- to the inner lumen of the rat trachea induced a concentration- thermore, electrophysiologicalstudies have provided evidence The Journal of Neuroscience, May 1993, 13(5) 1951 that a B,-preferring agonist was unable to evoke activity in receptor (Higashi and Nishi, 1982; Round and Wallis, 1985; (Mizumura et al., 1990). Moreover, the B,, but not Eschalier et al., 1989). For example, 5-HT evokes depolarization B,, antagonist displayed an inhibitory effect on nociceptive re- of primary afferents in rabbit nodose ganglion that is blocked sponses to BK (Dray and Perkins, 1988). In the present study, in a competitive fashion by ICS 205-930 (Round and Wallis, two specific observations were made. First, only the BK, and 1985) but not by methysergide (Higashi and Nishi, 1982). Both not the B, agonist [Des-ArglO]-kallidin, could evoke CGRP re- 5-HT and 5-HT, agonist 2-methyl-5-HT caused a painful effect lease. Second, this BK-evoked peptide release was significantly when applied to a blister base of human forearm. ICS 205-930 suppressed by the B, antagonist [o-Argo, Hyp3, This,*, o-Phe7]- in the nanomolar range blocked both 5-HT-evoked and 5-HT- BK, but not by the B, antagonist [Des-Arg9, Leus]-BK. These potentiated nociceptive effects by BK in human blister (Rich- results jointly support the hypothesis that BK activates CGRP ardson et al., 1985). ICS 205-930 also blocked carrageenan- release from the peripheral terminals of CAP-sensitive sensory induced hyperalgesia in rats (Eschalier et al., 1989), which also afferent through a B, receptor. Significantly, [D-Argo, Hyp’, This,s, suggests the endogenous release of 5-HT as well as its involve- D-Phe’]-BK induced approximately an 80% reduction of CGRP ment in the sensitization of peripheral nociceptors and the role release at a concentration of 5 x 1Oe6 M. This value is essentially of the 5-HT, receptor. Comparable results were observed in the identical to the pA, value obtained in guinea pig taenia caeci present study. Thus, among the 5-HT antagonists examined, (PA, is about 5.5) (Field et al., 1988) and in guinea pig ileum only ICS 205-930 (5-HT, antagonist), and not (s)-(-)propran- (PA, = 6.0) (Steranka et al., 1989). This agent does, however, 0101or methysergide (5-HT, and/or 5-HT, antagonists), blocked appear to be more potent in the rat uterus (PA, = 6.9) (Steranka the facilitatory effect of 5-HT on the CAP-evoked release of et al., 1989). [Des-Arg9, L&l-BK, which is known to have a CGRP from the trachea. A putative 5-HT, receptor has been relatively low affinity for the B, receptor (pD, < 5.0), displayed recently proposed (Dumuis et al., 1988; Clarke et al., 1989), on a potent antagonistic effect on the rabbit aorta: a pure B, receptor which ICS 205-930 in the micromolar range is reported to func- system (PA, = 7.27) (see Regoli et al., 1990). It has been reported tion as an antagonist (Villalon et al., 1990, 1991). Thus, we that prostanoid synthesis activated by BK may also be mediated should not exclude the possibility of either or both a 5-HT, and via a B, receptor (Hock et al., 1991; Neppl et al., 1991). Based a 5-HT, receptor in the trachea. on antagonist analysis, in the guinea pig trachea, BK-induced, PG-dependent relaxation appeared not to involve activation of Aferent terminal sensitization either a B, or B, site (Rhaleb et al., 1988). A possible species Both BK and 5-HT are also well known for their ability to difference, consistent with other BK receptor types, must be sensitize the nociceptor (Lang et al., 1990; see Basbaum, 199 1). considered (Regoli et al., 1990). This facilitation of BK, however, has been proposed by an action Autoradiographic studies have revealed BK binding sites to on an intermediate system, such as the sympathetic terminal, be found in small dorsal root ganglion cells, and in high con- to release some substances which in turn results in facilitation centrations in regions of the dorsal horn to which primary af- (Levine et al., 1986; Taiwo and Levine, 1988). Our present ferents project and in the periphery (Steranka et al., 1988). While observations reveal that only 5-HT, and not BK or HIS, en- these binding data suggest that the BK sites may be on the central hanced CGRP release from the rat trachea induced by CAP. and peripheral terminals of CAP-sensitive primary afferents, it These observations are in accord with other studies (see Hand- is not clear that CGRP released by BK is via a receptor located werker, 1990; Lang et al., 1990) that indicate that 5-HT shows on the terminal. The certainty of a direct effect is called into a more reliable sensitizing effect than other mediators, such as question by observations that (1) the BK effect is mediated or PGE, . through the release of PGs (see above), and (2) sympathetic neurons may be involved in BK-induced hyperalgesia and in- The possible role of prostanoids flammation (Levine et al., 1986; Taiwo and Levine, 1988; Lee The fact that both the effects of BK stimulation and 5-HT en- et al., 1991). hancement of CGRP release were blocked by indomethacin suggests that prostanoid production is involved in both the stim- Histamine and S-HT ulatory and facilitatory actions on the nerve terminal. Two is- sues are emphasized. First, because each agent behaves differ- HIS and 5-HT have been reported to be able to excite certain entially, that is, BK evokes release but does not sensitize while limited populations of polymodal C-fiber afferents (see Hand- 5-HT sensitizes but does not evoke, it seems probable that werker, 1990). 5-HT- and HIS-evoked increases in vascular different elements of the cyclooxygenase cascade must be in- permeability in the airway are absent in the animal pretreated with CAP (Lundberg and Saria, 1983) suggesting a possible volved in the actions of the respective agents. Second, at present it is not clear from what source the cyclooxygenase products are stimulatory effect upon the C-fiber terminal. In the present study, however, 5-HT did not exert any direct excitatory effect on derived. PGs sensitize a majority of the C-polymodal afferents (Martin et al., 1987) and potentiate the vascular permeability CGRP release, and HIS displayed some modest activity at high increase induced by CAP (Saria and Lundberg, 1984). In the concentrations. The modest action might emphasize that HIS preliminary study, however, no PGs examined (PGE,, PGE,, and 5-HT are rapidly taken up and metabolized or that they PGF,,, PGI,) showed sensitizing activity on CAP-evoked pep- interact with axons that do not secrete CGRP. As will be noted tide release from the rat trachea, although all displayed direct below for 5-HT, this does not appear to be the case. excitatory effects in the same model (Hua et al., unpublished observations). Instability of thromboxane A, prevents the in- 5-HT receptor vestigation of the biological activity of this agent. Interestingly, Previous studies have emphasized that the action of 5-HT on a stable PGI, analog, carba-PGI,, has been reported to enhance the peripheral terminal of primary sensory afferents is mediated CAP-evoked release of and CGRP from rat sensory by a receptor having characteristics resembling those of a 5-HT, neuron (Hingtgen and Vasko, 1992). Further work employing 1952 Hua and Yaksh l BK and 5-HT on CGRP Release

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