- Email: [email protected]
Effect of a neurokinin-1 (NK1) receptor antagonist on oedema formation induced by tachykinins, carrageenin and an allergic response in guinea-pig skin
Neuropeptides (1994) 26.4054
qf Longman
11
Group Ltd 1994
Effect of a Neurokinin-1 (NK,) Receptor Antagonist on Oedema Formation Induced by Tachykinins, Carrageenin and an Allergic Response in Guinea-pig Skin P. WILSONCROFT,
H. EUZGER, and S. D. BRAIN
Pharmacology Group and Vascular Biology Research Centre, Division of Biomedical King’s College, Manresa Road, London SW3 SLX, UK (Reprint requests to SDB)
Sciences,
Abstract-The effect of the neurokinin-1 (NK,) receptor antagonist RP67580 in modulating inflammatory oedema formation has been investigated in guinea-pig skin. Oedema formation was measured over 30 min by the extravascular accumulation of intravenously-injected 1251albumin in the anaesthetised guinea-pig. RP67580 was injected intradermally with the agents under test. lntradermal RP67580 (IO nmol/site) inhibits oedema formation induced by substance P (30 pmol) and neurokinin A (100 pmol), but not that induced by bradykinin (10-1000 pmol) or histamine (10 nmol). Substance P-induced oedema formation is similar in control (saline) and mepyramine (histamine H, receptor antagonist) pretreated guinea-pigs suggesting a minimal involvement of histamine in substance P induced oedema formation in guinea-pig skin. Oedema formation induced by intradermal carrageenin (0.2%) was not inhibited by RP67580 (l-10 nmol). A significant but partial inhibition of oedema formation induced in a passive cutaneous anaphylaxis (PCA) response was observed. The oedema formation in the PCA was inhibited 50% by mepyramine pretreatment but in the presence of mepyramine no further inhibition of the PCA response by RP67580 was observed.
Introduction
The tachykinins are a group of structurally related peptides which share a common C-terminal sequence and include substance P (SP) and neurokinin A (NKA). These peptides are released from sensory nerves in skin and are well established as .____ Date received Date accepted
19 October IO January
1993 1994
potent mediators of increased microvascular permeability in skin.’ The potent vasodilator calcitonin gene-related peptide (CGRP), is also released from skin, but this peptide does not act directly to increase microvascular permeability, although it can potentiate oedema formation induced by tachykinins.2.3 The tachykinins act via at least 2 mechanisms to induce oedema formation. Tachykinin receptor-mediated plasma extravasation is now considered to be mediated by neurokinin NK,
405
406
NEUROPEPTIDES
receptors, in tissues that include the guinea-pig skin.4,5In addition SP is a potent mediator of mast cell degranulation in tissues that include human and rat skin6 thus it can also act to increase microvascular permeability via mast cell-derived histamine. The relative contribution of the latter mechanism to SP-induced oedema formation is debatable. Recently non-peptide NK, receptor antagonists have been developed. These have included RP67580, a perhydroisoindolone derivative, which has been shown to be a selective antagonist of NK, receptor-mediated increased microvascular permeability in the rat.‘.* There is a species selectivity of the affinities of known selective NK, receptor antagonists. RP67580 has a lO-30-fold higher affinity for mouse and rat NK1 receptors when compared with other species including guinea-pigs and man.’ However in an initial study we observed that intradermal RP67580 inhibited intradermal SP induced oedema in the guinea-pig. This suggested to us that we could use this antagonist as a local treatment in guinea-pig skin where adjacent sites in the same animal which received SP alone could be used as internal controls. Thus in the present study we have examined the effect of intradermal RP67580 on the increased microvascular permeability induced by a range of agents in guineapig skin. The aim of this study is to determine whether endogenous neuropeptides act via NK, receptors to mediate increased microvascular permeability induced by bradykinin, carrageenin and an allergic reaction in the guinea-pig.
2.5% w/v in saline) was injected intravenously using
a 27 gauge needle into a peripheral vein in either the ear or the hind paw. Agents (in 0.1 ml Tyrode solution) were then intradermally injected and an accumulation period of 30-60 min, depending on protocol, timed from midway through the injections. A blood sample was then taken via cardiac puncture, the blood centrifuged and 2 plasma samples of known volume were prepared. The animal was killed by anaesthetic overdose and the dorsal skin was removed. The injected skin sites were punched out using a 16 mm diameter punch. Skin sites and plasma samples were counted in an automatic gamma counter. Leakage at each skin site was expressed as ~1 of plasma by comparing counts in skin sites with those in 1 ml of plasma. For all protocols RP67580 was injected intradermally, in the same solution, with the agent used to induce oedema formation. In experiments where mepyramine (10 mg/kg) was used, the antagonist or saline (for control) were injected intraperitoneally 15 min before intradermal injections. Intradermal test agents were injected as described above, with the exception of passive cutaneous anaphylaxis (PCA) responses where a pretreatment was necessary as follows. After anaesthetising and shaving the dorsal skin of the guinea-pig, purified IgG, anti-ovalbumin was injected i.d. in 50 ~1 volumes into the dorsal skin, with 50 ~1 Tyrode given at appropriate control sites. The guinea-pig was used in an oedema assay after a 24 h period which allowed fixation of the antibody to appropiate cells. Results
Materials and methods Measurement
of‘oedema formation
in guinea-pig skin
Male Dunkin-Hartley guinea-pigs (300-500 g) were anaesthetised with sodium pentobarbitone (Sagatal) 30-50 mg/kg injected intraperitoneally. The dorsal skin was then shaved and sites for intradermal injections marked to enable a balanced site injection plan where each agent was injected in duplicate, one injection on each flank of the dorsal skin. Up to 12 test agents were injected in each experiment and allocation of test agents to sites was randomised. “‘I-human serum albumin (‘*‘I-HSA; 0.18 MBq/kg) mixed with Evans Blue (0.3 ml of
RP67580 was tested at a series of doses for its ability to inhibit neurokinin-induced oedema. Figures 1 and 2 show that RP67.580 (10 nmol/site) significantly inhibited oedema formation induced by SP (30 pmol) and NKA (100 pmol), whilst not affecting that induced by bradykinin (100 pmol) and histamine (10 nmol). Thus this dose of antagonist was used in further experiments. Indeed RP67580 had no effect on oedema formation induced by bradykinin at varying doses (Fig. 2). In some species (e.g. the rat, see Brain and Williams, 1989) SP induces oedema formation that is partially dependent on the release of mast cell amine. Thus the effect of SP on oedema in guinea-
NK, RECEPTORS AND OEDEMA IN GUINEA-PIG
407
SKIN
80 70 60
6 z
1 5o E 0” g I
RP67580 (IOnmoVsite)
403020 -
NKA
Tyrode
BK
(1OOpmol)
(100pmoI)
Fig. I The effect of RP67580 (10 nmol) on SP (30 pmol), NKA (100 pmol) and bradykinin (100 pmol) induced oedema. The dotted line represents Tyrode injected sites alone. The results are expressed as the mean with bars representing *SEM. n = 5 guinea-pigs, where each result is the average of 2 injections per animal. *p > 0.05 indicates a significant difference compared with sites which received mediator alone.
pigs treated with the histamine HI receptor antagonist mepyramine was investigated. Mepyramine totally inhibited oedema formation induced by intradermal histamine (10 nmol) but had little effect
on oedema formation induced by SP in the presence or absence of RP67580 as shown in Figure 3. RP67580 was tested in two inflammatory models. The effect of RP67580 was tested in a PCA model
l-
80
T
+ RP67580 1 Onmol RP67580 (10nmoUsite)
1 Onmol HA
10 100 1000 BK pmol
Tyrode
Fig. 2 The effect of RP67580 (10 nmol) on SP (30 pmol), histamine (10 nmol) and bradykinin (10-1000 pmo)) induced oedema. The shaded symbols denote sites which received RP67580. The dotted line represents Tyrode injected sites alone. The results are expressed as the mean with bars representing +SEM. n = 3-7 guinea-pigs. where each result is the average of 2 injections per animal.
408
NEUROPEPTlDES
Saline ip -15min
Mepyramine
lOmg/kg.
ip -15min
60
substance
P substance P (pmol) (pmol) Fig. 3 The effect of RP67580 (10 nmol) on SP (3-30 pmol) induced oedema in control guinea-pigs pretreated with saline (i.p.) and guinea-pigs pretreated with mepyramine (10 mgkg-’ i.p. - 15 min). The graph also demonstrates the effect of mepyramine pretreatment on histamine (10 nmol) induced oedema. The dotted line represents Tyrode injected sites alone. The results are expressed as the mean with bars representing k SEM, n = 6 guinea-pigs per group, where each result is the average of 2 injections per animal.
where a partial but significant inhibitory response of oedema formation was observed as shown in Figure 4. Mepyramine inhibited the oedema formation by 50% and interestingly no further inhibition of oedema formation was observed in mepyramine-treated sites when RP67580 was administered. In the second model RP67580 was tested against carrageenin-induced oedema as shown in Figure 5, where no inhibitory effect was observed. Discussion
The results show that RP67580 did not have any effect on oedema formation when injected intradermally alone at doses up to 10 nmol. However, RP67580 inhibited SP and NKA-induced oedema formation whilst having little inhibitory effect on bradykinin and histamine-induced oedema formation. This suggests that RP67580 is a selective NK, receptor antagonist when injected intradermally. Further, the results suggest that bradykinin and
histamine do not act in part via a NK, mediated neurogenic component to mediate their potent effects on increased microvascular permeability in guinea-pig skin. Results from a previous study in guinea-pig skin led to the suggestion that whilst NK, receptors play a role in mediating tachykinininduced increased microvascular permeability in guinea-pig, NK2 and NK, receptors also play a role.’ The present studies, demonstrate a clear involvement of NKI receptors, but do not elucidate the possible involvement of other neurokinin receptors in mediating neurogenic oedema formation in guinea-pig skin. SP induced a similar oedema formation when injected intradermally into rats treated systemically with the histamine H, receptor antagonist mepyramine, in experiments where oedema formation induced by histamine was totally abolished. This suggests that SP, when injected intradermally at least, does not act to stimulate degranulation of guinea-pig mast cells. This finding is in contrast to results obtained in rat skin where intradermal SP
NK, RECEPTORS AND OEDEMA IN GUINEA-PIG
409
SKIN
70 1
60
Tyrode
PCA
PCA + Mepyramine (1 vsls~e)
Fig. 4 The effect of RP67580 (10 nmol) on PCA in the absence and presence of mepyramine (1 pglsite). The dotted line represents Tyrode injected sites alone. The results are expressed as the mean with bars representing f SEM, n = 5-7 guinea-pigs. where each result is the average of 2 injections per animal. *p i 0.05 indicates a significant difference compared with PCA alone.
induces oedema formation that is partially dependent on mast cell amines,” although evidence suggests that endogenous tdchykinins released from the saphenous nerve act independently of mast cells in the rat paw.” Moskowitz and co-workers have presented evidence which suggests that neurogenic inflammation might be involved in vascular responses in the cranial tissues, relevant to the pathological processes involved in migraine.” This has led to studies where NK, receptor mediated oedema formation has been studied in guinea-pig cranial tissues. O’Shaughnessy and Connor have demonstrated that the NK, receptor agonist GR73632 mediated plasma protein extravasation in guinea-pig dura and conjunctiva
when injected intravenously and that the NK, receptor antagonist GR82334 blocked this response in both tissuesI By comparison NK2 and NK3 receptor-selective agonists had no significant effect on plasma protein extravasation, although interestingly a trend towards an increase in plasma protein extravasation in the dura was observed with the NK,-selective agonist. Moussaoui and co-workers have used RP67580 to investigate the inhibition of intravenous SP-induced oedema formation in tissues from guinea-pig and rat.8 RP67580 significantly inhibited oedema formation in the dura of rat and guinea-pig and in rat skin. In these studies the effect of RP67580 in guinea-pig skin was not determined. RP67580 has also been shown to
NEUROPEPTIDES
Tyrode
SP
30pmol CGRP
sp + CGRP RP 1Onmol
1Opmol
0.2% Canageenin
0.2% Carrageenin + RP
Fig. 5 The effect of RP67580 (10 nmol) on carrageenin (0.2%) induced oedema. The dotted line represents the sites injected with Tyrode alone. The results are expressed as the mean with bars representing &SEM, n = 4 guinea-pigs, where each result is the average of 2 injections per animal.
inhibit neurogenic oedema formation induced by stimulation of the trigeminal nerve in the rat.15 The results do not suggest a major role of neuropeptides in carrageenin-induced oedema formation in guinea-pig skin. However, the inhibition of oedema formation in the passive cutaneous anaphylaxis response is of interest. This suggests that a neurokinin is involved in mediating allergic oedema formation. The PCA is a mast cell-dependent phenomenon and it is well established that histamine plays a major, but not total, role in the early oedematous phase of the response. It has also been suggested that other mediators of increased microvascular permeability are also involved as an inhibition of oedema formation is observed when sites are pretreated with a mixture of histamine, leukotriene and PAF antagonists.16 To our knowledge our data is the first to suggest that a NK, receptor antagonist inhibits PCA-induced oedema formation in guinea-pig skin. However Bertrand and
co-workers have recently shown that the non-peptide NK, antagonist CP96,345 inhibits oedema formation induced over a 5-10 min period after an antigen induced response in the trachea in actively sensitized guinea-pigs.” The latter finding suggests that the NK,-mediated effects only occur after an early phase of extravasation has taken place. Interestingly no further inhibition of the PCA by RP67580 was observed when tested in the presence of mepyramine. This might suggest that histamine has some role, possibly in conjunction with, as yet, unknown mechanisms to activate the efferent function of sensory nerves. It is concluded that endogenous neurokinins such as SP stimulated increased microvascular permeability in guinea-pig skin via NK, receptors, with a minimal involvement of mast cell-derived histamine. The results suggest that endogenous neurokinins play a role in the oedema formation induced in acute allergic responses. It will be of
NK, RECEPTORS
AND OEDEMA
IN GUINEA-PIG
interest to learn more about conditions where endogenous neurokinins may play a pro-inflammatory role.
8.
9.
Acknowledgement This study is supported
411
SKIN
by the Wellcome
Trust.
References Gardiner. S. M., Comptom, A. M., Kemp, P. A., Bennett, T.. Foulkes. R. and Hughes, B. Haemodynamic effects of human acalcitonin gene-related peptide following administration of endothelin-1 or No-nitro-L-arginine methyl ester in conscious rats. Br. J. Pharmacol. 1991; 105: 1256-1262. Brain, S. D. and Williams. T. J. Inflammatory oedema induced by synergism between calcitonin gene-related peptide (CGRP) and mediators of increased vascular permeability. Br. J. Pharmacol. 1985: 86: 855-860. Gamse. R. and Saria, A. Potentiation of tachykinin-induced plasma protein extravasation by calcitonin gene-related peptide. Eur. J. Pharmacol. 1985; 114: 61-66. Doutremepuich, J. D., Barbier, A., Vilain, P. and Lacheretz, F. Effect of Substance P and Sar9Met(0*)“-Substance P on cutaneous capillary permeability in guinea pig skin. Skin Pharmacol. 1992; 5: 171-176. Iwatoto. I. and Nadel, J. A. Tachykinin receptor subtype that mediates the increase in vascular permeability in guinea pig skin. Life Sci. 1989; 44: 1089-1095. Foreman, J. C., Jordan, C. C., Oehme, P. and Renner, H. Structure-activity relationships for some substance P-related peptides that cause wheal and flare reactions in human skin. J. Physiol. 1983; 335: 449465. Garret, C., Carruette, A., Fardin, V. et al. Pharmacological properties of a potent and selective non-peptide substance
IO.
11.
12.
13.
14.
15.
16.
17.
P antagonist. Proc. Nat]. Acad. Sci. USA 1991: 88: 102088 10212. Moussaoui, S. M., Philippe, L., Le Prado. N. and Garret, C. Inhibition of neurogenic inflammation in the meninges by a non-peptide NK, receptor antagonist. RP67580. Eur. J. Pharmacol. 1993; 238: 421424. Watling. K. J. and Krause, J. E. The rising sun shines on substance P and related peptides. TIPS 1993; 14: 81-84. Brain, S. D. and Williams, T. J. Interactions between the tachykinins and calcitonin gene-related peptide lead to the modulation of oedema formation and blood flow in rat skin. Br. J. Pharmacol. 1989; 97: 77-82. Kowalski, M. L.. Sliwinska-Kowalska, M. and Kaliner, M. A. Neurogenic inflammation, vascular permeability. and mast cells. 2. Additional evidence indicating that mast cells are not involved in neurogenic inflammation. J. Immunol. 1990: 145: 1214-1221. Moskowitz. M. A. Neurogenic versus vascular mechamsms of sumatriptan and ergot alkaloids in migraine. TIPS 1992; 13: 307-311. Buzzi. M. G. and Moskowitz, M. A. The antimigraine drug, sumatriptan, (GR43175), selectively blocks neurogenic plasma extravasation from blood vessels in dura mater. Br. J. Pharmacol. 1990; 99: 202-206. O’Shaughnessy. C. T. and Connor. H. E. Neurokinin NK, receptors mediate plasma protein extravasation in guineapig dura. Eur. J. Pharmacol. 1993; 236: 319-321. Shepheard. S. L.. Williamson, D. J.. Hill, R. G. and Hargreaves. R. J. The non-peptide neurokinin-1 receptor antagonist. RP 67580. blocks neuroeenic nlasma extravasation in the dura mater of rats. Br. J. Pharmacol. 1993: 108: 1 l-12. Weg, V. B.. Watson, M. L., Cordeiro. R. S. B. and Williams T. J. Histamine, IeukotrieneD, and platelet-activating factor in guinea pig passive cutaneous anaphylaxis. Eur. J. Pharmacol. 1991; 204: 1577163. Bertrand. C.. Geppetti. P., Baker, J., Yamawaki. I. and Nadel, J. A. Role of neurogenic inflammation in antigeninduced extravasation in guinea pig trachea. J. Immunol. 1993: 150: 1479-1485.
qf Longman
11
Group Ltd 1994
Effect of a Neurokinin-1 (NK,) Receptor Antagonist on Oedema Formation Induced by Tachykinins, Carrageenin and an Allergic Response in Guinea-pig Skin P. WILSONCROFT,
H. EUZGER, and S. D. BRAIN
Pharmacology Group and Vascular Biology Research Centre, Division of Biomedical King’s College, Manresa Road, London SW3 SLX, UK (Reprint requests to SDB)
Sciences,
Abstract-The effect of the neurokinin-1 (NK,) receptor antagonist RP67580 in modulating inflammatory oedema formation has been investigated in guinea-pig skin. Oedema formation was measured over 30 min by the extravascular accumulation of intravenously-injected 1251albumin in the anaesthetised guinea-pig. RP67580 was injected intradermally with the agents under test. lntradermal RP67580 (IO nmol/site) inhibits oedema formation induced by substance P (30 pmol) and neurokinin A (100 pmol), but not that induced by bradykinin (10-1000 pmol) or histamine (10 nmol). Substance P-induced oedema formation is similar in control (saline) and mepyramine (histamine H, receptor antagonist) pretreated guinea-pigs suggesting a minimal involvement of histamine in substance P induced oedema formation in guinea-pig skin. Oedema formation induced by intradermal carrageenin (0.2%) was not inhibited by RP67580 (l-10 nmol). A significant but partial inhibition of oedema formation induced in a passive cutaneous anaphylaxis (PCA) response was observed. The oedema formation in the PCA was inhibited 50% by mepyramine pretreatment but in the presence of mepyramine no further inhibition of the PCA response by RP67580 was observed.
Introduction
The tachykinins are a group of structurally related peptides which share a common C-terminal sequence and include substance P (SP) and neurokinin A (NKA). These peptides are released from sensory nerves in skin and are well established as .____ Date received Date accepted
19 October IO January
1993 1994
potent mediators of increased microvascular permeability in skin.’ The potent vasodilator calcitonin gene-related peptide (CGRP), is also released from skin, but this peptide does not act directly to increase microvascular permeability, although it can potentiate oedema formation induced by tachykinins.2.3 The tachykinins act via at least 2 mechanisms to induce oedema formation. Tachykinin receptor-mediated plasma extravasation is now considered to be mediated by neurokinin NK,
405
406
NEUROPEPTIDES
receptors, in tissues that include the guinea-pig skin.4,5In addition SP is a potent mediator of mast cell degranulation in tissues that include human and rat skin6 thus it can also act to increase microvascular permeability via mast cell-derived histamine. The relative contribution of the latter mechanism to SP-induced oedema formation is debatable. Recently non-peptide NK, receptor antagonists have been developed. These have included RP67580, a perhydroisoindolone derivative, which has been shown to be a selective antagonist of NK, receptor-mediated increased microvascular permeability in the rat.‘.* There is a species selectivity of the affinities of known selective NK, receptor antagonists. RP67580 has a lO-30-fold higher affinity for mouse and rat NK1 receptors when compared with other species including guinea-pigs and man.’ However in an initial study we observed that intradermal RP67580 inhibited intradermal SP induced oedema in the guinea-pig. This suggested to us that we could use this antagonist as a local treatment in guinea-pig skin where adjacent sites in the same animal which received SP alone could be used as internal controls. Thus in the present study we have examined the effect of intradermal RP67580 on the increased microvascular permeability induced by a range of agents in guineapig skin. The aim of this study is to determine whether endogenous neuropeptides act via NK, receptors to mediate increased microvascular permeability induced by bradykinin, carrageenin and an allergic reaction in the guinea-pig.
2.5% w/v in saline) was injected intravenously using
a 27 gauge needle into a peripheral vein in either the ear or the hind paw. Agents (in 0.1 ml Tyrode solution) were then intradermally injected and an accumulation period of 30-60 min, depending on protocol, timed from midway through the injections. A blood sample was then taken via cardiac puncture, the blood centrifuged and 2 plasma samples of known volume were prepared. The animal was killed by anaesthetic overdose and the dorsal skin was removed. The injected skin sites were punched out using a 16 mm diameter punch. Skin sites and plasma samples were counted in an automatic gamma counter. Leakage at each skin site was expressed as ~1 of plasma by comparing counts in skin sites with those in 1 ml of plasma. For all protocols RP67580 was injected intradermally, in the same solution, with the agent used to induce oedema formation. In experiments where mepyramine (10 mg/kg) was used, the antagonist or saline (for control) were injected intraperitoneally 15 min before intradermal injections. Intradermal test agents were injected as described above, with the exception of passive cutaneous anaphylaxis (PCA) responses where a pretreatment was necessary as follows. After anaesthetising and shaving the dorsal skin of the guinea-pig, purified IgG, anti-ovalbumin was injected i.d. in 50 ~1 volumes into the dorsal skin, with 50 ~1 Tyrode given at appropriate control sites. The guinea-pig was used in an oedema assay after a 24 h period which allowed fixation of the antibody to appropiate cells. Results
Materials and methods Measurement
of‘oedema formation
in guinea-pig skin
Male Dunkin-Hartley guinea-pigs (300-500 g) were anaesthetised with sodium pentobarbitone (Sagatal) 30-50 mg/kg injected intraperitoneally. The dorsal skin was then shaved and sites for intradermal injections marked to enable a balanced site injection plan where each agent was injected in duplicate, one injection on each flank of the dorsal skin. Up to 12 test agents were injected in each experiment and allocation of test agents to sites was randomised. “‘I-human serum albumin (‘*‘I-HSA; 0.18 MBq/kg) mixed with Evans Blue (0.3 ml of
RP67580 was tested at a series of doses for its ability to inhibit neurokinin-induced oedema. Figures 1 and 2 show that RP67.580 (10 nmol/site) significantly inhibited oedema formation induced by SP (30 pmol) and NKA (100 pmol), whilst not affecting that induced by bradykinin (100 pmol) and histamine (10 nmol). Thus this dose of antagonist was used in further experiments. Indeed RP67580 had no effect on oedema formation induced by bradykinin at varying doses (Fig. 2). In some species (e.g. the rat, see Brain and Williams, 1989) SP induces oedema formation that is partially dependent on the release of mast cell amine. Thus the effect of SP on oedema in guinea-
NK, RECEPTORS AND OEDEMA IN GUINEA-PIG
407
SKIN
80 70 60
6 z
1 5o E 0” g I
RP67580 (IOnmoVsite)
403020 -
NKA
Tyrode
BK
(1OOpmol)
(100pmoI)
Fig. I The effect of RP67580 (10 nmol) on SP (30 pmol), NKA (100 pmol) and bradykinin (100 pmol) induced oedema. The dotted line represents Tyrode injected sites alone. The results are expressed as the mean with bars representing *SEM. n = 5 guinea-pigs, where each result is the average of 2 injections per animal. *p > 0.05 indicates a significant difference compared with sites which received mediator alone.
pigs treated with the histamine HI receptor antagonist mepyramine was investigated. Mepyramine totally inhibited oedema formation induced by intradermal histamine (10 nmol) but had little effect
on oedema formation induced by SP in the presence or absence of RP67580 as shown in Figure 3. RP67580 was tested in two inflammatory models. The effect of RP67580 was tested in a PCA model
l-
80
T
+ RP67580 1 Onmol RP67580 (10nmoUsite)
1 Onmol HA
10 100 1000 BK pmol
Tyrode
Fig. 2 The effect of RP67580 (10 nmol) on SP (30 pmol), histamine (10 nmol) and bradykinin (10-1000 pmo)) induced oedema. The shaded symbols denote sites which received RP67580. The dotted line represents Tyrode injected sites alone. The results are expressed as the mean with bars representing +SEM. n = 3-7 guinea-pigs. where each result is the average of 2 injections per animal.
408
NEUROPEPTlDES
Saline ip -15min
Mepyramine
lOmg/kg.
ip -15min
60
substance
P substance P (pmol) (pmol) Fig. 3 The effect of RP67580 (10 nmol) on SP (3-30 pmol) induced oedema in control guinea-pigs pretreated with saline (i.p.) and guinea-pigs pretreated with mepyramine (10 mgkg-’ i.p. - 15 min). The graph also demonstrates the effect of mepyramine pretreatment on histamine (10 nmol) induced oedema. The dotted line represents Tyrode injected sites alone. The results are expressed as the mean with bars representing k SEM, n = 6 guinea-pigs per group, where each result is the average of 2 injections per animal.
where a partial but significant inhibitory response of oedema formation was observed as shown in Figure 4. Mepyramine inhibited the oedema formation by 50% and interestingly no further inhibition of oedema formation was observed in mepyramine-treated sites when RP67580 was administered. In the second model RP67580 was tested against carrageenin-induced oedema as shown in Figure 5, where no inhibitory effect was observed. Discussion
The results show that RP67580 did not have any effect on oedema formation when injected intradermally alone at doses up to 10 nmol. However, RP67580 inhibited SP and NKA-induced oedema formation whilst having little inhibitory effect on bradykinin and histamine-induced oedema formation. This suggests that RP67580 is a selective NK, receptor antagonist when injected intradermally. Further, the results suggest that bradykinin and
histamine do not act in part via a NK, mediated neurogenic component to mediate their potent effects on increased microvascular permeability in guinea-pig skin. Results from a previous study in guinea-pig skin led to the suggestion that whilst NK, receptors play a role in mediating tachykinininduced increased microvascular permeability in guinea-pig, NK2 and NK, receptors also play a role.’ The present studies, demonstrate a clear involvement of NKI receptors, but do not elucidate the possible involvement of other neurokinin receptors in mediating neurogenic oedema formation in guinea-pig skin. SP induced a similar oedema formation when injected intradermally into rats treated systemically with the histamine H, receptor antagonist mepyramine, in experiments where oedema formation induced by histamine was totally abolished. This suggests that SP, when injected intradermally at least, does not act to stimulate degranulation of guinea-pig mast cells. This finding is in contrast to results obtained in rat skin where intradermal SP
NK, RECEPTORS AND OEDEMA IN GUINEA-PIG
409
SKIN
70 1
60
Tyrode
PCA
PCA + Mepyramine (1 vsls~e)
Fig. 4 The effect of RP67580 (10 nmol) on PCA in the absence and presence of mepyramine (1 pglsite). The dotted line represents Tyrode injected sites alone. The results are expressed as the mean with bars representing f SEM, n = 5-7 guinea-pigs. where each result is the average of 2 injections per animal. *p i 0.05 indicates a significant difference compared with PCA alone.
induces oedema formation that is partially dependent on mast cell amines,” although evidence suggests that endogenous tdchykinins released from the saphenous nerve act independently of mast cells in the rat paw.” Moskowitz and co-workers have presented evidence which suggests that neurogenic inflammation might be involved in vascular responses in the cranial tissues, relevant to the pathological processes involved in migraine.” This has led to studies where NK, receptor mediated oedema formation has been studied in guinea-pig cranial tissues. O’Shaughnessy and Connor have demonstrated that the NK, receptor agonist GR73632 mediated plasma protein extravasation in guinea-pig dura and conjunctiva
when injected intravenously and that the NK, receptor antagonist GR82334 blocked this response in both tissuesI By comparison NK2 and NK3 receptor-selective agonists had no significant effect on plasma protein extravasation, although interestingly a trend towards an increase in plasma protein extravasation in the dura was observed with the NK,-selective agonist. Moussaoui and co-workers have used RP67580 to investigate the inhibition of intravenous SP-induced oedema formation in tissues from guinea-pig and rat.8 RP67580 significantly inhibited oedema formation in the dura of rat and guinea-pig and in rat skin. In these studies the effect of RP67580 in guinea-pig skin was not determined. RP67580 has also been shown to
NEUROPEPTIDES
Tyrode
SP
30pmol CGRP
sp + CGRP RP 1Onmol
1Opmol
0.2% Canageenin
0.2% Carrageenin + RP
Fig. 5 The effect of RP67580 (10 nmol) on carrageenin (0.2%) induced oedema. The dotted line represents the sites injected with Tyrode alone. The results are expressed as the mean with bars representing &SEM, n = 4 guinea-pigs, where each result is the average of 2 injections per animal.
inhibit neurogenic oedema formation induced by stimulation of the trigeminal nerve in the rat.15 The results do not suggest a major role of neuropeptides in carrageenin-induced oedema formation in guinea-pig skin. However, the inhibition of oedema formation in the passive cutaneous anaphylaxis response is of interest. This suggests that a neurokinin is involved in mediating allergic oedema formation. The PCA is a mast cell-dependent phenomenon and it is well established that histamine plays a major, but not total, role in the early oedematous phase of the response. It has also been suggested that other mediators of increased microvascular permeability are also involved as an inhibition of oedema formation is observed when sites are pretreated with a mixture of histamine, leukotriene and PAF antagonists.16 To our knowledge our data is the first to suggest that a NK, receptor antagonist inhibits PCA-induced oedema formation in guinea-pig skin. However Bertrand and
co-workers have recently shown that the non-peptide NK, antagonist CP96,345 inhibits oedema formation induced over a 5-10 min period after an antigen induced response in the trachea in actively sensitized guinea-pigs.” The latter finding suggests that the NK,-mediated effects only occur after an early phase of extravasation has taken place. Interestingly no further inhibition of the PCA by RP67580 was observed when tested in the presence of mepyramine. This might suggest that histamine has some role, possibly in conjunction with, as yet, unknown mechanisms to activate the efferent function of sensory nerves. It is concluded that endogenous neurokinins such as SP stimulated increased microvascular permeability in guinea-pig skin via NK, receptors, with a minimal involvement of mast cell-derived histamine. The results suggest that endogenous neurokinins play a role in the oedema formation induced in acute allergic responses. It will be of
NK, RECEPTORS
AND OEDEMA
IN GUINEA-PIG
interest to learn more about conditions where endogenous neurokinins may play a pro-inflammatory role.
8.
9.
Acknowledgement This study is supported
411
SKIN
by the Wellcome
Trust.
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