`
`Br. J. Pharmacot. (1993), 110, 772 776
`
`'PI Macmillan Press Ltd, 1993
`
`Macmillan Press Ltd, 1993
`
`Effect of a calcitonin gene-related peptide antagonist
`(CGRP8-37) on skin vasodilatation and oedema induced by
`stimulation of the rat saphenous nerve
`K. Jane Escott & 'Susan D. Brain
`
`Pharmacology Group, Division of Biomedical Sciences, King's College, Manresa Road, London SW3 6LX
`
`1 The effect of the calcitonin gene-related peptide antagonist (CGRP837, 400 nmol kg-', i.v.) on the
`increased blood flow induced by calcitonin gene related peptide (CGRP), vasodilator prostaglandins,
`and topical capsaicin was measured with a laser Doppler blood flow meter in rat abdominal skin.
`2 The saphenous nerve was electrically stimulated and the effect of CGRP8-37 (400 nmol kg-', i.v.) on
`the increased blood flow (measured by laser Doppler flowmetry) and oedema formation (measured by
`the extravascular accumulation of ['25f]-albumin) was investigated in the rat hind paw.
`CGRP8-37 (400 nmol kg-', i.v.) had no effect on basal cutaneous blood flow at uninjected sites and
`3
`sites injected with Tyrode buffer, but acted selectively to inhibit the increased blood flow induced by
`intradermal CGRP (10 pmol/site, P< 0.05), but not that induced by prostaglandin E2 (PGE2, 300 pmol/
`site) or carba-prostacyclin (cPGI2, 100 pmol/site).
`Capsaicin (0.1-33 mM), applied topically, acted in a dose-related manner to increase blood flow.
`4
`CGRP837 (400 nmol kg-', i.v.) almost totally inhibited blood flow induced by capsaicin (10 mM;
`P <0.05) but did not significantly inhibit blood flow induced by a higher dose of capsaicin (33 mM).
`5 The increased blood flow induced by short stimulation of the saphenous nerve (10 V, 1 ms, 2 Hz for
`30 s) was inhibited by 76%, 5 min after i.v. CGRP837 (400 nmol kg-', i.v., P<0.05).
`6 A longer (5 min) electrical stimulation of the saphenous nerve caused oedema formation, in addition
`to increased blood flow. The oedema formation was significantly inhibited by CGRP8-37 (400 nmol kg-',
`i.v., P<0.05).
`7 The results suggest that the potent microvascular vasodilator neuropeptide, CGRP, is responsible for
`the increased blood flow observed after short stimulation of the saphenous nerve and that endogenous
`CGRP contributes in a pro-inflammatory manner to neurogenic oedema formation in the rat hind paw.
`Keywords: Calcitonin gene-related peptide (CGRP); substance P; neurokinin-1 receptors; saphenous nerve; skin blood flow;
`laser Doppler flowmetry; neurogenic inflammation
`
`Introduction
`
`Our group has previously studied the release and activity of
`the 37 amino acid vasodilator sensory neuropeptide cal-
`citonin gene-related peptide (CGRP) in the rabbit cutaneous
`microvasculature. The studies show that the increased blood
`flow induced in rabbit skin by intradermal injection of cap-
`saicin and analogues (e.g. olvanil), can be inhibited by the
`co-injection of the 8-37 amino acid C-terminal peptide of
`CGRP (CGRP8-37), a selective competitive antagonist of
`CGRP (Chiba et.al., 1989; Gardiner et al., 1990; Han et al.,
`1990). Early experiments suggested that CGRP8-37 could par-
`tially
`inhibit capsaicin-induced vasodilatation (Hughes &
`Brain, 1991), whilst more recent experiments using CGRP8-37
`synthesized by improved techniques have shown that a 60%
`increase in blood flow in response to capsaicin (100 nmol/
`site) is totally inhibited by CGRP8-37 (Hughes et al., 1992).
`We have concluded from these results that CGRP is an
`important vasodilator neuropeptide released from capsaicin
`sensitive nerves in rabbit skin. It is generally assumed that
`substance P, which is often co-localised with CGRP also has
`an important vasoactive effect. However, substance P is an
`extremely weak vasodilator in rabbit skin (Brain et al., 1985)
`and a neurokinin-I (NK,) receptor selective antagonist had
`little effect on capsaicin-induced blood flow in this species
`(Hughes et al., 1992).
`In this study we have investigated the release and activity
`of CGRP in rat skin, where neurokinins (especially substance
`P) have been established as potent vasoactive mediators
`(Lembeck & Holzer 1979; Gamse & Saria 1985; Brain &
`
`'Author for correspondence.
`
`Williams 1989). We know, from a previous study that
`CGRP8-37 cannot be administered intradermally in the rat due
`to a pro-inflammatory activity, possibly related to basic
`residues in the N-terminus region (Brain et al., 1992). Thus
`we have administered CGRP8-37 intravenously (i.v.) which has
`been shown to be effective in inhibiting the hypotensive
`effects of i.v. CGRP in several studies (Donoso et al., 1990;
`1990). We have examined the ability of
`Gardiner et al.,
`influence the sensory nerve-dependent pro-
`CGRP8-37 to
`inflammatory responses in the rat in response to capsaicin
`administration and electrical stimulation of the saphenous
`nerve.
`
`Methods
`
`Blood flow and oedema formation were measured in the skin
`of male Wistar rats (200-300 g), anaesthetized with sodium
`pentobarbitone (Sagatal, May and Baker, initial i.p. injection
`50-80 mg kg-', maintained by additional 20 mg kg- h-',
`given i.v.). Body temperature was maintained at 36-38°C by
`automatic control of a heating pad. Rats were pretreated
`with guanethidine (20mgkg-', s.c.) 24h before saphenous
`nerve stimulation, to prevent vasoconstriction induced by
`concomitant stimulation of the sympathetic fibres of the
`saphenous nerve (Gamse & Saria, 1987).
`
`Measurement of bloodflow in abdominal skin
`The animal was anaesthetized, and the abdominal skin
`
`1
`
`EX2007
`Eli Lilly & Co. v. Teva Pharms. Int'l GMBH
`IPR2018-01427
`
`
`
`CGRP RELEASE FROM SAPHENOUS NERVE
`
`773
`
`Materials
`The following drugs were used: prostglandin El (PGEI) from
`Sigma Chemical Company, U.K., human a-calcitonin gene-
`related peptide (CGRP) a gift from Dr U. Ney, Celltech,
`U.K., human CGRP8-37 from Bachem, U.K.,
`synthetic
`vanillylamide
`from
`capsaicin-pelargonic
`acid
`Fluka
`Chemicals Ltd., U.K., carba-prostacyclin (cPGI2) from Cas-
`cade Biochem. Ltd., U.K., and guanethidine from Ciba-
`Geigy, U.K. CGRP, PGE, and PGI2 were stored in stock
`solutions at - 20°C and then diluted with Tyrode solution
`immediately prior to use. CGRP837 was prepared in 0.1%
`bovine serum albumin (BSA) in saline. Capsaicin was dis-
`solved in ethanol at 100 mg ml-' and diluted in a solution of
`alcohol:Tween 80:saline, at a ratio of 20:10:70. '25I-labelled
`human serum albumin was obtained from Amersham Inter-
`national, U.K. Immac (Reckitt & Colman, U.K.) was used to
`remove remaining hair stubble. The composition of the
`Tyrode solution was a follows (mM): NaCl 136.89, KCI 2.68,
`NaH2PO4 0.42, NaHCO3 11.9, MgCl2 1.05 and glucose 5.55.
`
`Results
`
`The intradermal injection of CGRP, PGE, and carba-
`prostacyclin (cPGI2) led to an increased blood flow as
`measured by laser Doppler flowmetry, in keeping with
`previous observations (Brain & Williams, 1989). CGRP837
`(400 nmol kg-', i.v.) had no significant effect on basal blood
`flow as shown in Figure 1. However, this figure shows that
`CGRP8-37 significantly inhibited the increased blood flow
`induced by CGRP but not that induced by the prostaglan-
`dins. These results indicate that the administration of int-
`ravenous CGRP8-37 can be used to investigate the release of
`vasoactive amounts of endogenous CGRP.
`The effect of the application of topical capsaicin on rat
`cutaneous blood flow is shown in Figure 2. Capsaicin in-
`creased blood flow in a dose-related manner, whilst the vehi-
`cle had no effect on blood flow when tested at the highest
`concentration used. The graph is similar to that obtained by
`Lynn and co-workers (1992). CGRP8-37 caused an almost
`
`T
`
`200 -
`
`150-
`
`0 -
`
`ooo100
`50
`
`.0
`
`CGRP
`(10 pmol)
`
`PGE,
`(300 pmol)
`
`cPG12
`(100 pmoI)
`
`-50L
`Figure I The effect of calcitonin gene-related peptide (CGRP) and
`vasodilator prostaglandins on blood flow in rat abdominal skin.
`Results are expressed as percentage change in local skin blood flow
`at each injected site compared with blood flow at the site before
`injection. The results from control rats (0.1% BSA, i.v.) are shown
`by the open columns and from rats
`treated with CGRP8.37
`(400 nmol kgT efi.v.) are shown by the solid columns. The results are
`expressed as the meanrs.e.mean of 9 rats in each group. A
`significant effect of CGRP8-37 on the vasodilator activity of CGRP is
`shown by *P<0.05.
`
`shaved and depilated. Thirty minutes later CGRP8-37 or vehi-
`cle were given i.v. and blood flow was measured at several
`sites with a laser Doppler flowmeter. Prostaglandins, CGRP
`or Tyrode were injected intradermally (50,l) and capsaicin
`or vehicle (20% alcohol: 10% Tween 80: 70% saline) were
`(5 tLI). Blood flow was measured again
`applied topically
`between 5-O min after application of test agents. Blood
`flow changes were measured with a Moor dual probe laser
`Doppler (MBF3D) and measured as flux, as previously dis-
`cussed in detail (Lawrence & Brain, 1992). The results are
`expressed as percentage change in blood flow compared with
`pre-injected sites in Figures 1 and 2. In some experiments the
`protocols were designed to measure responses to vasoactive
`agents and to saphenous nerve stimulation in the same
`animals.
`
`Measurement of bloodflow after saphenous nerve
`stimulation
`After induction of anaesthesia the hind limbs of the rat were
`shaved and depilated with a commercial cream. The jugular
`vein was cannulated for i.v. administration of anaesthetic and
`test agents. The saphenous nerves were carefully dissected in
`both legs (stimulated or sham legs) tied centrally, placed on
`bipolar platinum electrodes and immersed in mineral oil. In
`order to study antidromic vasodilatation one laser Doppler
`probe was positioned and secured over the hind paw skin in
`a region innervated by the saphenous nerve, i.e. medio-dorsal
`side of the skin of the hind paw, as confirmed by the
`observation of Evans Blue dye extravasation after saphenous
`nerve stimulation. The other probe was positioned in a cor-
`responding site on the sham treated hind paw to monitor
`basal blood flow throughout the experiment. Not earlier than
`preparation was finished,
`30 min after
`the
`antidromic
`stimulation was performed using the following parameters:
`10 V, 1 ms, 2 Hz for 30 s. Three stimulations were given at
`20-30 min intervals. To investigate the effect of CGRP8-37 on
`blood flow changes induced by saphenous nerve stimulation,
`CGRP837 (400 nmol kg-'), or vehicle (0.1% BSA in saline)
`were administered i.v. 5 min prior to the second stimulation.
`Results in Figure 3 show the flux in 3a and 3b; whilst Figure
`3c shows percentage change in the vasodilator response to
`saphenous nerve stimulation, after i.v. treatments.
`
`Measurement of oedema in the hind paw after saphenous
`nerve stimulation
`The saphenous nerves were prepared as described above and
`left for 5 min. Then Evans Blue (25 mg kg-') and '25I-labelled
`human serum albumin (50 kBq) were injected into the rat via
`the tail vein. CGRP8-37 (400 nmol kg-') or vehicle (0.1%
`BSA) were administered via the jugular vein, at least 5 min
`prior to stimulation. Then the nerve was stimulated: 1O V,
`1 ms, 2 Hz for 5 min. After stimulation, a blood sample was
`obtained by cardiac puncture and then the animal killed by
`anaesthetic overdose. The oedematous area of skin of the
`hind paw (as observed by Evans blue dye extravasation) was
`removed and weighed. An approximately equal amount of
`skin was also removed from the sham hind paw and weighed.
`Radioactivity was counted in 100 lAl of plasma and hind paw
`skin samples. Plasma extravasation was expressed as IL1 of
`plasma in 100 mg of skin and a ratio calculated: sham versus
`stimulated hind paw oedema for each rat.
`
`Statistical analysis
`Results are expressed as mean ± s.e.mean. Abdominal, dorsal
`skin and paw blood flow data were analysed by Bonferroni's
`modified t test, the standard error estimate for the analysis of
`variance was used to allow comparison of multiple sites. The
`significance of the hind paw skin oedema after saphenous
`nerve stimulation was tested by Student's unpaired t test.
`
`2
`
`
`
`10
`
`40
`30
`20
`Time (min)
`
`50
`
`60
`
`0
`
`b
`
`0
`
`10
`
`20 30 40 50 60
`Time (min)
`
`70
`
`r-
`
`M o1
`
`0, a
`
`40
`
`2
`
`3
`
`240
`
`200
`
`160
`
`120
`
`80
`
`40
`
`0
`
`240
`
`200
`
`160
`
`120
`
`80
`
`40
`
`0
`
`-40
`
`-80
`
`-1.
`
`x
`
`xL2
`
`0 V00 0
`
`)._
`
`Ca
`
`)
`o)
`
`Effect of CGRP8-37 on blood flow in the skin of rat hind
`Figure 3
`paw after saphenous nerve stimulation (10 V, 1 ms, 2 Hz, for 30 s).
`(a) Effect of repeated stimulations of the saphenous nerve on blood
`flow in the skin of a rat hind paw. The rat received vehicle (0.1%
`BSA in saline, i.v.) 5 min prior to the second stimulation. (b) Effect
`of CGRP8.37 (400 nmol kg-', i.v., 5 min before the second stimula-
`tion) on saphenous-induced blood flow in the rat hind paw skin. The
`results for (a) and (b) are expressed as Flux (arbitrary unit) and the
`dotted line indicates flux recorded on the contralateral paw. Stimula-
`tion of the saphenous nerve is represented by the upward arrows. (c)
`Collected results to show the effect of CGRP8-37 on blood flow
`induced by saphenous nerve stimulation. Results are expressed as
`percentage change in blood flow compared to the first stimulation
`and are the mean ± s.e.mean of 4 rats in each group. In each group
`CGRP8.37 or vehicle were administered i.v. at least 5 min prior to the
`second stimulation. CGRP8-37 inhibited blood flow after the second
`stimulation (*P< 0.05) and the third stimulation.
`
`plasma extravasation. In those experiments, blood flow was
`femoral
`measured
`by
`outflow.
`vein
`the
`In
`present
`experiments blood flow
`is measured by laser Doppler
`flowmetry. The use of the laser Doppler blood flow meter to
`measure saphenous nerve-stimulated increased blood flow
`was first used by Gamse & Saria
`(1987).
`It became
`immediately apparent that an increase in blood flow could be
`induced by a mild and short electrical stimulation when
`compared with the electrical stimulation necessary for the
`
`774
`
`K.J. ESCOTT & S.D. BRAIN
`
`Vehicle
`
`0.1
`
`10
`1
`Capsaicin (mM)
`
`33
`
`250
`
`200
`
`150
`
`100
`
`50
`
`0
`
`0 o0 a
`
`)
`a)a)
`c._,
`
`Figure 2 The effect of topical capsaicin on blood flow in rat
`abdominal skin. Results are expressed as percentage increase in local
`blood flow at each injected site compared with blood flow at the site
`before injection. The open columns represent control rats (0.1%
`BSA, i.v.) and the solid columns are animals treated with CGRP8-37
`(400 nmol kg-', i.v.). Each column is the mean ± s.e.mean of 9 rats.
`CGRP8-37 significantly inhibited the vasodilator activity of capsaicin
`at a dose of 10 nm, as shown by *P<0.05.
`
`total inhibition of blood flow induced by capsaicin (10 mM).
`However, little inhibitory effect of a higher dose of capsaicin,
`33 mM was observed.
`A short (30 s) electrical stimulation of the saphenous nerve
`caused an increased blood flow which returned to baseline
`levels within 5-O min after stimulation. This response was
`repeatable at least three times, as shown in Figure 3a.
`CGRP8-37 caused an inhibition of the peak response as
`shown in Figure 3b. The group data, with an inhibition of
`stimulated blood flow at both time points after saphenous
`nerve stimulation are shown in Figure 3c. No evidence of
`oedema formation was observed within this short stimulation
`period. By comparison increasing the stimulation period to
`5 min led to the well established neurogenic oedema forma-
`tion (shown in Figure 4) as first described by Jancso et al.
`(1967). The oedema was significantly attenuated by CGRP8-37.
`The results suggest that CGRP8-37 acted to inhibit approx-
`imately 50% of the oedema formation induced by electrical
`stimulation of the saphenous nerve.
`
`Discussion
`
`The results show that CGRP8-37 acts in a selective manner to
`antagonize the actions of exogenous CGRP in increasing
`blood flow. CGRP8.37 also significantly inhibited the in-
`creased blood flow induced by topical capsaicin (10 mM) and
`saphenous nerve stimulation, as well as oedema formation
`induced by saphenous nerve stimulation. CGRP8-37 failed to
`inhibit the increased blood flow induced by the highest dose
`of capsaicin (33 mM). The reason for this is unclear; possibly
`a maximal response combined with the release of increased
`levels of CGRP resulted in an inability of CGRP8-37 to inhibit
`the increased blood flow. Alternatively other vasodilator
`mediators might be involved in mediating the increased blood
`flow induced by this dose of capsaicin.
`The stimulation of the saphenous nerve in rats previously
`treated with guanethidine, to deplete a sympathetic compo-
`nent, has become an established small animal model of
`neurogenic inflammation, since Lembeck & Holzer (1979)
`provided evidence that substance P acts as a neurogenic
`of antidromic
`mediator
`neurogenic
`vasodilatation
`and
`
`3
`
`
`
`CGRP RELEASE FROM SAPHENOUS NERVE
`
`775
`
`1992). It is unlikely that substance P acts directly via NK,
`receptors to increase blood flow as the selective NK, receptor
`agonist, GR73632, does not increase blood flow in rat skin,
`although it has potent oedema inducing effects which are
`potentiated by CGRP (Birch et al., 1992; Richards et al.,
`is known that the selective NK, receptor
`1993). Further it
`antagonist RP67580 does not affect the increased blood flow
`induced by stimulation of the saphenous nerve at levels
`al.,
`to induce oedema formation (Shepheard et
`sufficient
`1992). Thus a vasodilator component is likely to be involved
`in the resulting oedema formation. There are several pos-
`sibilities as to the identity of this vasodilator. Firstly the
`increased blood flow could be totally induced by CGRP at
`concentrations that were not possible to antagonize in the
`present experiments when oedema formation occurs in res-
`ponse to a 5 min electrical stimulation period. It should be
`remembered that CGRP837 is a peptide antagonist which is
`poorly characterized whilst CP96345 and RP67580 are non-
`peptide structures, developed on the basis of their affinity and
`selectivity as NK, receptor antagonists. Alternatively it
`is
`possible that substance P is released from the saphenous
`nerve in sufficient quantities to stimulate mast cell degranula-
`tion and thus the release of the vasoactive amines histamine
`and 5-hydroxytryptamine (5-HT). This has been suggested by
`previous studies where evidence for the involvement of his-
`tamine and 5-HT has been obtained (Lembeck & Holzer,
`1979; Morton & Chahl, 1980). However this would appear
`unlikely as the amines do not participate in the oedema
`response which, as discussed above, is completely inhibited
`by NK, receptor antagonists. It is also possible that another
`distinct, perhaps novel vasodilator, in addition to CGRP, is
`released from sensory nerves upon saphenous nerve stimula-
`tion. This is the most exciting possibility but we have no
`evidence to date that this is so.
`We consider that the involvement of CGRP in neurogenic
`oedema formation is of interest. It has recently been demon-
`strated that the NK, receptor antagonist, RP67580, inhibits
`oedema formation in the rat dura induced by trigeminal
`nerve stimulation (Shepheard et
`al.,
`1993);
`a neurogenic
`model that is considered to be relevant to migraine in man.
`Also increased CGRP levels have been measured in venous
`blood samples taken from patients with migraine (Goadsby
`et al., 1990). As CGRP is colocalized with substance P it
`could be released in the rat dura in vasodilator amounts. It is
`not known whether the endogenous release of CGRP is
`pro-inflammatory in terms of vasodilatation or oedema for-
`mation in the rat dura; but our results obtained in this study
`would suggest that such a possibility may exist. However, it
`should also be pointed out that CGRP, at vasodilator doses,
`is anti-inflammatory in the hamster cheek pouch causing a
`clear
`inhibition of mediator induced oedema formation
`al.,
`(Raud et
`1991). Our studies suggest that this anti-
`inflammatory mechanism does not operate in rat skin or paw
`(Newbold & Brain, 1993); but the collected findings highlight
`the possibility that CGRP could well have multiple effects on
`the inflammatory response.
`In conclusion we have shown that CGRP8-37 inhibits
`vasodilatation induced by topical capsaicin and saphenous
`nerve stimulation in the rat and thus provide evidence that
`endogenous CGRP is the major vasodilator mediator in these
`models of neurogenic inflammation. CGRP837 also signifi-
`cantly inhibited neurogenic oedema formation induced by
`stimulation of the saphenous nerve. We consider this is good
`evidence to suggest that CGRP potentiates oedema forma-
`tion when co-released from sensory nerves with substance P.
`
`K.J.E. is the recipient of an MRC/Glaxo collaborative studentship.
`We thank Dr H. Connors and Dr D. Beattie from Glaxo Group
`research, U.K. for their helpful discussion and Dr U. Ney from
`Celltech, U.K. for the gift of the human a-CGRP.
`
`*
`
`4.0 r
`
`3.5 1-
`
`3.0
`
`2.5
`
`2.0
`
`1.5 p
`
`.In.
`
`E 0 C
`
`u
`
`I C
`
`ontrol
`
`1.0
`
`F- -
`
`0.5p-
`
`0.0o
`
`Effect of CGRP8-37 (400 nmol kg-', i.v.) on oedema forma-
`Figure 4
`tion after electrical stimulation of the saphenous nerve (10 V, I ms,
`2 Hz for 5 min). Results are expressed as the ratio of plasma ext-
`ravasation in the stimulated compared to the sham-treated hind paw
`skin. The dashed line at I indicates the basal level of plasma extra-
`vasation in unstimulated paws. Results are expressed as mean ± s.e.
`mean of 10 rats in each group. CGRP8-37 (solid column) significantly
`reduced plasma extravasation as indicated by *P <0.05, in com-
`parison to the control rats (0.1% BSA, i.v., open column).
`
`observation of oedema formation. The reason for this is not
`clear. It is probably related to the potency of endogenous
`CGRP as a vasodilator in skin. It is also possible that CGRP
`is released from A-delta fibres in addition to C-fibre nerves in
`initial saphenous nerve stimulation (Janig & Lisney, 1989).
`The present results are compatible with the suggestion that
`CGRP is the principle vasodilator released after mild and
`short stimulation of sensory nerves and this is in keeping
`with the findings of Delay-Goyet and co-workers (1992) who
`published a brief communication with similar findings whilst
`this study was in progress. They also investigated the effect of
`a neurokinin-l antagonist (RP67580) and showed that this
`had no effect on saphenous nerve stimulation-mediated
`changes in blood flow.
`Thus our findings indicate that CGRP mediates the in-
`crease in cutaneous blood flow observed with saphenous
`nerve stimulation. Results of previous studies in our group
`show that CGRP, does not act by itself to increase microvas-
`cular permeability, but as a consequence of its vasodilator
`activity potentiates oedema formation induced by mediators
`of increased microvascular permeability such as substance P
`and neurokinin A, in species that include the rat (Brain &
`1989). We therefore investigated whether
`Williams, 1985;
`affect oedema formation
`induced
`by
`CGRP8-37 could
`saphenous nerve stimulation. The results show an approx-
`imately 50% inhibition. Similar experimental protocols have
`been used to investigate the effect of recently developed
`non-peptide NK, receptor antagonists on oedema formation
`induced by saphenous nerve stimulation. CP96345 (Lembeck
`et al., 1992; Xu et al., 1992) and RP67580 (Garret et al.,
`1991) almost completely abolished oedema formation, whilst
`a NK2 receptor antagonist, MenlO207, had no inhibitory
`effect (Xu et al., 1992). These results are in keeping with the
`previous suggestion that the NK, receptor mediates increased
`microvascular permeability (Andrews et al., 1989). The par-
`tial inhibitory activity of CGRP837, on oedema formation
`induced by saphenous nerve stimulation,
`indicates
`that
`in neurogenic
`involved
`endogenously-released CGRP is
`oedema formation. An increase in blood flow is observed and
`this is concomitant with oedema formation (Shepheard et al.,
`
`4
`
`
`
`776
`
`K.J. ESCOTT & S.D. BRAIN
`
`References
`
`ANDREWS, P.V., HELME, R.D. & THOMAS, K.L. (1989). NK1 receptor
`mediation of neurogenic plasma extravasation in rat skin. Br. J.
`Pharmacol., 97, 1232-1238.
`BIRCH, P.J., HARRISON, S.M., HAYES, A.G., ROGERS, H. & TYERS,
`M.B. (1992). The non-peptide NK, receptor antagonist (± )-CP-
`96,345, produces antinociceptive and anti-oedema effects in the
`rat. Br. J. Pharmacol., 105, 508-510.
`BRAIN, S.D., CAMBRIDGE, H., HUGHES, S.R. & WILSONCROFT, P.
`(1992). Evidence that calcitonin gene-related peptide contributes
`to inflammation in the skin and joint. Proc. Natl. Acad. Sci.
`U.S.A., 657, 412-419.
`BRAIN, S.D. & WILLIAMS, T.J. (1985). Inflammatory oedema induced
`by synergism between calcitonin gene-related peptide (CGRP)
`and mediators of increased vascular permeability. Br. J. Phar-
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`
`(Received March 30, 1993
`Revised May 10, 1993
`Accepted May 18, 1993)
`
`5
`
`
`
`
`
`BIOCHEMISTRY AND MOLECULAR BIOLOGY OF NITRIC OXIDE
`
`
`
`
`
`SYMPOSIUM
`New and Emerging Mechanisms of Biosyntbesis, Metabolism, and Biological Actions of
`Nitric Oxide in Health and Disease
`
`July 16 - 21, 1994
`
`UCLA Sunset Village - Los Angeles, California
`Symposium Chairpersons — Louis J. Ignarro 0 Michael A. Marlena o Ferid Murad
`Symposium Advisors — Salvador Moncada . Solomon H. Snyder . John R. Vane
`
`Symposium Format
`° Continuous Plenary Sessions - invited speakers Iii/mm on tbe ltttic‘ltt’ttlt.\'l7_‘l’
`and molecular biology ofm‘tne oxide,
`' Poster Presentations - submitted abstracts in all fields (f basic
`and clinical research on nitric oxide
`- Special Plenary Session on Inbalational therapy will) Nitric Oxide
`
`Registration Fee $580
`Application pending for continuing medical education credit
`
`Forfmtber information please contact
`
`KREBS Convention Management Services 0 553 helium Street. Suite .200 0 Sztn Francisco, (LA 91' 10743548
`
`litx NH) 25"» 8100
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