Sensory-motor neuromodulation of sympathetic vasoconstriction in the rabbit central ear artery

European Journal of Pharmacology, 187 (1990) 171-182

171

Elsevier EJP 51473

Sensory-motor neuromodulation of sympathetic vasoconstriction in the rabbit central ear artery K e n n e t h I. M a y n a r d , Valerie L. Saville 1 a n d G e o f f r e y B u r n s t o c k Department of Anatomy and DevelopmentalBiology and Centrefor Neuroscience, University CollegeLondon, GowerStreet, London WCIE 6BT, U.K.

Received 15 May 1990, accepted 26 June 1990

Histochemical and pharmacological studies were performed on the rabbit central ear artery. In perivascular nerves, positive immunoreactivity for calcitonin gene-related peptide and substance P was demonstrated. Calcitonin gene-related peptide-like immunoreactivity was also found to be colocalised with substance P-like immunoreactivity in a subpopulation of perivascular nerves. In vitro incubation with 6-hydroxydopamine did not alter the intensity and/or density of either the calcitonin gene-related peptide- or substance P-like immunoreactive fibres, whereas incubation with capsaicin significantly reduced both. In pharmacological studies, calcitonin gene-related peptide reduced the vasoconstrictor responses to exogenous noradrenaline and a,fl-methylene ATP and to electrical field stimulation in a concentration-dependent manner. In segments of the central ear artery preconstricted with noradrenaline, relaxation mediated by calcitonin gene-related peptide was endothelium-independent. These results shed new light on the innervation and nervous control of the rabbit central ear artery previously thought to be exclusively under sympathetic (adrenergic and purinergic) control. Further, the results suggest that calcitonin gene-related peptide localised in sensory nerves in the rabbit central ear artery may act as an inhibitory modulator of excitatory sympathetic vascular neurotransmission. Calcitonin gene-related peptide (CGRP); Noradrenaline; a,fl-Methylene ATP; Sympathetic nerve stimulation; Ear artery (rabbit, central)

1. Introduction

A large number of peptides has now been identified and localised in the peripheral, vascular nervous system (for reviews see Wharton and Gulbenkian, 1987; Dhital and Burnstock, 1989). To date, the only peptide localised immunohistochemically in the rabbit central ear artery (REA)

i Present address: Department of Biology, Roche Products Ltd., P.O. Box 8, Welwyn Garden City, Hertfordshire AL7 38Y, U.K. Correspondence to: G. Burnstock, Department of Anatomy and DevelopmentalBiology and Centre for Neuroscience, University College London, Gower Street, London WC1E 6BT, U.K.

is substance P (SP) (Morris and Bevan, 1986). Other peptides, however, such as neuropeptide Y (Edvinsson et al., 1984; Glover, 1985; Daly and Hieble, 1987; Juan et al., 1988; Wong-Dusting and Rand, 1988; Budai et al., 1989), calcitonin gene-related peptide (CGRP) (Hanko et al., 1985; Dogramatzis et al., 1987; Maynard and Burnstock, 1989; Moritoki et al., 1990), [Met]- and [Leu]enkephalin (IUes et al., 1983; Ganten et al., 1984) and somatostatin (Cohen et al., 1978) are known to have pharmacological actions on the REA. The aim of this study was to determine whether C G R P could be localised in the REA, and if so, to see if it was colocalised with SP as has been reported for other perivascular, sensory nerves (Wharton and Gulbenkian, 1987; Kawasaki et al.,

0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

172

1988; Dhital and Burnstock, 1989; Saito et al., 1989). In addition, the pharmacological actions of C G R P on raised tone preparations, on responses to exogenous noradrenaline (NA), a, fl-methylene ATP (a,fl-mATP) and on electrical field stimulation of the perivascular nerves were investigated. Responses to exogenous NA and a, fl-mATP were studied because it has been shown that there are adrenergic and purinergic components to the sympathetic transmission in the REA (Kennedy et al., 1986; Saville and Burnstock, 1988). By comparing the effects of C G R P on postjunctional actions of exogenous NA and a,fl-mATP with its effects on responses to electrical field stimulation, we hope to determine whether C G R P has a pre- a n d / o r postjunctional action on sympathetic transmission in this blood vessel.

2. Materials and methods

2.1. General Male New Zealand white rabbits (2.5-3.5 kg) were killed by a blow to the back of the head or an overdose of sodium pentobarbitone (Sagatal), followed by exsanguination. The REA was dissected out and divided into specific portions for the various investigations to be carried out. The proximal 20 mm of the artery was used for pharmacological investigations and segments from along the entire artery for immunohistochemical studies. A modified Krebs solution of the following composition (mM): NaC1 120.0, KC1 5.0, K H 2 P O 4 1.20, N a H C O 3 25.0, MgSO 4 1.2, glucose 11.0, and CaC12 2.5 was used in all experiments. 2.2. Histochemistry 2.2.1. Immunohistochemical whole-mount preparations The localisation of C G R P and SP in the REA was performed on whole-mount (stretch) preparations. Segments of the artery were dissected out and placed in gassed (95% 02-5% CO2) Krebs solution. Following the removal of adipose and connective tissue, the segments were cut open lengthwise and stretched, adventitial side upper-

most, onto strips of Sylgard silicone resin. The tissues were then fixed for 1-1.5 h in 4% paraformaldehyde and subsequently processed for immunofluorescent localisation of neuropeptides according to the method of Costa et al. (1980). Briefly, REA segments were dehydrated, cleared, rehydrated and then washed in phosphate-buffered saline ( P B S ) / T r i t o n X-100 solution before being incubated (12-18 h) with C G R P antiserum or SP antiserum in a humid chamber at room temperature. After three washes in PBS/Triton X-100 solution, the segments were incubated for 1 h with an appropriate (see later) fluorescein isothiocyanate (FITC)-conjugated secondary antiserum. Segments were finally washed in PBS containing pontamine sky blue (PSB 0.05-0.1%) and dimethylsulphoxide (DMSO 1%) and mounted in Citifluor. Various dilutions ranging from 1 : 500 to 1 : 30 were used in testing for peptide-like immunoreactivity, but C G R P antiserum had an optimal dilution factor of 1:200, and 1:30 was used for SP antiserum (Morris and Bevan, 1986). The second fluorescent-conjugated antibody used for the C G R P primary antiserum was FITC-conjugated goat anti-rabbit with a dilution factor of 1:50. Rabbit anti-rat FITC (1:20) (Morris and Bevan, 1986) was used for the monoclonal SP primary antiserum. Antiserum-specific controls were performed on both antisera. The antibodies were preadsorbed with their respective peptides using a series of concentrations (0.1 nM-10 /LM). The preadsorbed antisera to C G R P and SP did not produce CGRPlike or SP-like immunoreactivity (CGRP-LI or SP-LI, respectively) when incubated with segments of the artery at the following concentrations: C G R P 0.1 t~M and SP 10 /~M. This confirms that the antisera were specific. 2.2.2. Direct double staining This procedure (Legay et al., 1984) was carried out on mid-segment REA samples using a modified version of the immunostaining method previously described. Initially the segments were incubated in SP primary antiserum followed by rabbit anti-rat FITC, preadsorbed with rabbit immunoglobulin G (IgG). The segments were then

173 washed six to eight times in PBS/Triton X-100. This was followed by a further incubation with CGRP primary antiserum and goat anti-rabbit secondary antiserum-conjugated tetrarhodamine isothiocyanate (TRITC), preadsorbed with rabbit IgG. After rinsing in PBS containing PSB and DMSO, the segments were mounted in Citifluor. Control experiments were performed to check that there was no cross reactivity i.e. binding of secondary antibodies to inappropriate primary antibodies. The tissue was prepared as previously described. Segments incubated with SP primary antiserum were then incubated with preadsorbed goat anti-rabbit TRITC. These were viewed under the microscope and showed no fluorescence. Additional segments incubated with CGRP primary antiserum were subsequently incubated with preadsorbed rabbit anti-rat FITC and also showed no fluorescence. These controls showed that there was no cross reactivity between CGRP and SP antisera.

2.2.3. In vitro incubation with neurotoxins Segments of the REA were incubated in vitro at 37 °C and gassed with Krebs solution containing either 6-hydroxydopamine (6-OHDA, 0.4 mM) for 4 h (Warland and Burnstock, 1987) or capsaicin (CAPS, 1/~M) for 30 min (Saito at al., 1989). Controls consisted of segments gassed in normal Krebs solution and also Krebs solution containing 0.05% Tween 80 and 1% alcohol, which was the vehicle used to dissolve capsaicin. After incubation, tissues were rinsed in Krebs solution and processed for fluorescence histochemistry. 2.2.4. Catecholamine fiuorescence In addition to immunohistochemistry, fluorescence histochemical localisation of catecholaminecontaining nerves was also conducted on wholemount preparations using the glyoxylic acid fluorescence method detailed by Lindvall and BjiSrklund (Lindvall and BjiSrklund, 1974). Briefly, segments of REA were incubated at room temperature for 1.5 h in 2% glyoxylic acid in 0.1 M phosphate buffer at pH 7.2. Tissue segments were then stretched on glass slides (adventitial side uppermost) and air dried until translucent. Stretched vessels were then incubated at 100 °C

for 4 rain, mounted in liquid paraffin and viewed using a Zeiss photomicroscope.

2.2.5. Fluorescence photomicroscopy The immunohistochemical preparations were viewed and photographed on a Carl Zeiss photomicroscope equipped with a 3RS epi-illumination system for viewing catecholamine, FITC and TRITC fluorescence. Selected areas were photographed on Kodak (Tri-X-Pan and TMAX P3200) film. 2.3. Pharmacology Proximal segments of the artery were dissected and mounted as described by Kennedy and Burnstock, the only modification being that the Krebs solution contained bovine albumin serum (50 mg/1) and bacitracin (30 mg/l) to prevent the peptides sticking to the glassware or being degraded by non-specific peptidases respectively (Kennedy and Burnstock, 1985). Changes in isometric tension were measured in each segment of the artery after they had been equilibrated for 1 h under a resting tension of 0.75 g. The effects of CGRP (0.263/~M) were investigated on the responses to exogenous NA (0.5-10 #M) and a,fl-mATP (0.5-10 #M). CGRP (2.63263 nM) was also examined on the responses to electrical field stimulation (platinum wire electrodes were placed on each side of the preparation and electrical stimulation was applied at supramaximal voltage, 0.1 ms pulse width for 1 s at 16 and 64 Hz). NA and a, fl-mATP were administered and left in contact with the tissue until the responses reached a peak (2-4 rain for NA; approximately 1 latin for a, fl-mATP). NA was applied every 20 min, whereas 40 min cycles were used for a,fl-mATP as the latter desensitises its own receptor when applied exogenously. After constant responses had been obtained, CGRP was added to the organ bath and left for 2 min before retesting the responses to NA and a,fl-mATP. Responses to electrical field stimulation were elicited and recorded every 2 min. Following stabilisation of these control responses, CGRP was added during a 2 rain interval. Periods of stimulation were continued for 8 min after the

174

addition of CGRP. An earlier study using intervals of 30 s enabled us to follow the time course of the maximum inhibitory action of C G R P on contractions elicited by electrical field stimulation. Thus, the 2-3 min incubation period used during these experiments was calculated based upon the results of earlier experiments. The action of C G R P was tested on raised-tone preparations with and without endothelium. The tone was raised by the addition and maintained presence of 1 # M NA. Once a steady raised tone had been established, the peptide was added. In some preparations the endothelium was removed by passing cotton through the lumen of the vessel followed by cold distilled water. To confirm that the endothelium had been removed, acetylcholine (ACh 1 /~M) was added to raised-tone preparations. If no relaxation was observed it confirmed that the endothelium had been removed, since ACh acts via an endothelium-dependent mechanism to cause relaxation of raised-tone blood vessel preparations (Furchgott and Zawadzki, 1980; Kennedy and Burnstock, 1985). Responses to drugs and electrical field stimulation were measured using a Grass FT03 transducer and recorded on a Grass 79D polygragh. Electrical stimulation was provided by a Grass $44 stimulator. Responses are expressed in (mg) tension, a n d / o r as a percentage of the control maximal response to NA, or as a percentage inhibition of the control response. The effects of C G R P were measured at the point of maximal inhibition. The values on the graphs represent means _+ S.E.M. Statistical significance was calculated using Student's paired t-test and a level of 0.05 was considered significant.

antiserum (rabbit) were obtained from Cambridge Research Biochemicals. Monoclonal SP (rat) antiserum was obtained from Sera-lab Ltd., and Citifluor from Citifluor Ltd., London.

3. Results

3.1. Immunohistochemical localisation of neuropeptides in the REA 3.1.1. General Positive immunoreactivity in perivascular nerves was obtained with antisera against C G R P and SP. SP-LI was also found colocalised with CGRP-LI. 3.1.2. S P All regions of the artery tested showed positive SP-LI (n = 19). The distribution of these fibres was sparse with as little as one fibre supplying the distal region of the artery and two or three branches from a main fibre supplying the proximal region. The nerves ran along the length of the vessel and were mainly single varicose fibres (fig. la). 3.1.3. CGRP Many CGRP-LI nerve fibres were seen in the vessel segments of all animals examined (n = 19). The nerve fibres were mostly single or in very small bundles and ran parallel to the longitudinal axis of the artery. The fluorescence in these fibres was bright, sharp and showed both varicose and non-varicose fibres (fig. lb). The nerve fibres were evenly distributed in the proximal, middle and distal regions of the artery.

2.4. Drugs and compounds used a,/3-mATP (lithium salt), L-ascorbic acid, Larterenol bitartrate, bacitracin, bovine serum albumin, 8-methyl-N-vanillyl-6-noneamide (capsaicin), glyoxylic acid monohydrate, 6-hydroxydopamine hydrobromide, and polyoxyethylenesorbitan monooleate (Tween 80) were all obtained from the Sigma Chemical Company. Sodium pentobarbitone (Sagatal) was obtained from RMB Animal Health Ltd. Both a C G R P (rat) and C G R P

3.1.4. Effects of in vitro incubation with neurotoxins Segments incubated in 6-OHDA (0.4 mM) showed no change in SP- (fig. lc) or CGRP-LI (fig. ld) in comparison with controls (fig. la,b). Treated segments, however, showed no catecholamine fluorescence in comparison with the control segments which showed dense catecholaminergic, perivascular, sympathetic fibres. Segments incubated in CAPS (1/~M), however, showed a substantial reduction in the intensity

175

Fig. 1. (a) SP-like immunoreactive (SP-LI) nerve fibres in the rabbit central ear artery. Note the presence of sparse, single, varicose fibres. Calibration bar = 30 /~m. (b) CGRP-like immunoreactivity (CGRP-LI) in predominantly single varicose, perivascular nerve fibres in the rabbit central ear artery. Note the higher density of innervation in comparison with SP-LI. Calibration bar = 30/,tm. (c) SP-LI in the rabbit central ear artery after in vitro incubation with 6-hydroxydopamine (0.4 mM). The fluorescent, perivascular fibres appear unchanged in intensity and density, in comparison with the control preparations (a). Calibration bar = 30/~m. (d) CGRP-LI after in vitro incubation with 6-hydroxydopamine (0.4 raM). There appears to be no change in density and intensity, in comparison with control rabbit central ear artery preparations (b). Calibration bar = 30 ~m. (e) Marked reduction of SP-LI density and intensity in the fluorescent perivascular fibres in the rabbit central ear artery after in vitro incubation with capsaicin (1 /.tM). Calibration bar = 30 /~m. (f) Significant reduction in the density and intensity of CGRP-LI perivascular nerve fibres in the rabbit central ear artery after in vitro incubation with capsaicin (1/~M). The arrows indicate reduced fluorescent varicosities visible using long exposure times (hence the comparibly light background in comparison with figs. b, d) to highlight them. Calibration bar = 30 ttm.

and often the density of SP- (fig. le) and CGRP-LI nerve fibres (fig. lf), whereas segments incubated with vehicle-containing Krebs solution sometimes

showed a slightly reduced intensity of SP- and CGRP-LI in comparison with normal control preparations (fig. la,b). Catecholarnine fluorescence

176 TABLE 1

TABLE 2

The effect of CGRP (0.263/~M) on the contractile responses to noradrenaline in the rabbit central ear artery, n = number of observations. The level of significance was calculated using Student's paired t-test.

The effect of CGRP (0.263/tM) on the contractile responses to a,fl-methylene ATP in the rabbit central ear artery, n = number of observations. The level of significance was calculated using Student's paired t-test.

Noradrenaline (~M) 0.5 1.0 10.0

n Tension (mg) Control 5 793_+119 5 915+ 72 5 1880_+159

+ CGRP

% reduc-

Level of

a,/3-

tion

significance

Methylene ATP (/~M)

480-+161 46-+12 595-+ 68 35_+ 5 1700_+157 11_+ 2

P < 0.01 P<0.005 P<0.05

0.5 1.0 10.0

n

9 9 9

Tension (mg)

% reduc-

Level of

Control

+ CGRP

tion

significance

230_+ 42 805+_107 1013-+149

9 7 _ + 17 459+140 645-+132

51_+ 9 47_+11 38+ 5

P<0.05 P<0.05 P<0.005

was unaltered in control, vehicle control and capsaicinised preparations. 3.1.5. Coexistence of SP and C G R P Vessel segments from six animals were examined for the coexistence of C G R P - L I and SP-LI using the direct double-staining technique. Although all SP-LI fibres observed also showed C G R P - L I (fig. 2a,b), many C G R P - L I fibres did not show SP-LI. Hence, there were many more C G R P - L I than SP-LI perivascular nerve fibres.

3.2, The pharmacological actions of C G R P in the REA 3.2.1. Effect of C G R P on the responses to exogenous NA The administration of exogenous N A produced a monophasic and well-maintained contractile response at each concentration examined. These NA-induced contractions were significantly at-

Fig. 2. Immunofluorescent micrographs of the rabbit central ear artery showing (a) CGRP and (b) SP colocalisation. Note that the arrows indicate varicosities which are identical in each of the micrographs. Calibration bars = 30 ttm.

177 80-

a. Noradrenaline 1 IJM

"

to

\~w

60-

4mln

O ¢... Z

CGRP (0.263 IJM)

2 40rr

NA (IlJM)

X <

b. 0c,~-methylene ATP 1 IIM

20-

0-

L

4mln

CGRP (0.263 gM) -- a , I ~ - m A T P (IlJM) Fig. 3. Traces showing the effect of CGRP (0.263/xM) on the contractile responses to (a) exogenous NA (1 /xM) and (b) ct,fl-mATP (1 /~M) in the rabbit central ear artery. Horizontal lines indicate the time for which the drug a n d / o r peptide was present.

tenuated in a concentration-dependent manner in the presence of C G R P (table 1; figs. 3a, 4).

I I 0.5 1.0 CA31~ENIRATION pM

I I 10.0

Fig. 4. Effect of CGRP (0.263/~M) on exogenous noradrenaline-induced (circles) and a,fl-methylene ATP-induced (triangles) contractions of the rabbit central ear artery. Control responses are represented as closed symbols and CGRP-treated and inhibited responses by open symbols. Symbols represent the mean and vertical lines show the S.E.M. The results were calculated using Student's paired t-test. * indicates P < 0.05; • *, P < 0.01 and * * *, P < 0.005. In each case n >/4. Exact n values are listed in tables 1 and 2.

these responses at each concentration examined (table 2; figs. 3b, 4).

3.2.2. Effect of C G R P on the responses to exogenous a, fl-mA TP The application of a, fl-mATP (0.5-10 /~M)

3.2.3. Effect of C G R P on the responses to perivascular nerve stimulation

elicited a rapid contractile response that was not well maintained. CGRP significantly attentuated

Electrical field stimulation of the perivascular nerves in the REA (supramaximal voltage, 0.1 ms

TABLE 3 The effect of CGRP (2.63-263 nM) on the contractile responses elicited by electrical field stimulation (supramaximal voltage, 0.1 ms pulse width, 1 s train duration) at 16 and 64 Hz in the rabbit central ear artery, n = number of observations. NS means not significant. The level of significance was calculated using Student's paired t-test. Frequency

CGRP

(Hz)

(nM)

16 64 16 64 16 64

2.63 2.63 26.3 26.3 263 263

n

7 6 7 6 4 4

Tension (mg)

% reduction

Control

+ CGRP

877 + 101 1546+201 804+124 1683+171 888 4- 88 1694 + 149

763 + 78 1400+213 257+ 80 938+141 288 4- 97 918 4-172

Level of significance

11 + 3 11+ 3 72+ 7 37+ 7 69 + 10 46 4-10

P< NS P< P< P< P<

0.05 0.001 0.05 0.001 0.05

178 80-

#M) relaxed the vessel by 43.0 _+ 9.2% (n = 3) (fig. 7aii). When the endothelial cell layer was mechanically removed, the addition of ACh (1/~M) had no effect (fig. 7bi) or produced a small contraction, while the addition of C G R P (0.263/~M) still produced a relaxation and reduced the tone of the preparation by 43.3 _+ 9.2% (n = 3) (fig. 7bii). Thus the relaxant responses to C G R P were not significantly different between endothelium-denuded and endothehum-intact preparations.

/

60-

Z

2

rn -r 7

40--

/

./,

20--

4. Discussion

I I I 2.63 26.3 263 CGRP CONGENqqqATC3N

I

nM Fig. 5. Concentration-dependent, inhibitory effect of CGRP on contractions of the rabbit central ear artery, induced by electrical field stimulation (supramaximal voltage, 0.1 ms pulse width, 1 s train duration every 2 rain) at 16 Hz (open circles) and 64 Hz (closed circles). On the ordinate, responses are represented as % inhibition (i.e. percentages of the control contraction). The results were calculated using Student's paired t-test. * indicates P < 0.05 and * * * P < 0.001. These P values represent the significant differences between control and CGRPtreated responses. Exact n values are listed in table 3, n >/4 in each case.

The results reported in this study demonstrate for the first time the presence of CGRP-LI in the REA. In addition, they confirm the results of Morris and Bevan (1986) by demonstrating the presence of capsaicin-sensitive SP-LI. Moreover, SP- and C G R P - L I were shown to coexist in the same varicosities of single nerve fibres, although a.

Electrical Stimulation

16 Hz

4min

pulse width, 16 and 64 Hz for 1 s) produced rapid frequency-dependent contractions which were abolished by tetrodotoxin (1/~M). There were significant concentration-dependent reductions ( C G R P 2.63-263 nM) in the responses to perivascular nerve stimulation at both frequencies of stimulation (16 and 64 Hz) following incubation with C G R P (table 3; fig. 5). The maximal effect of C G R P was seen after approximately 2 min following which the responses began to return to control levels (fig. 6).

CGRP (0.263 pM)

b.

Electrical Stimulation

////

6 4 Hz

, //

3.2.4. Relaxation to A C h and C G R P - role o f the endothefium

The addition and maintained presence of NA (1/~M) produced a steady raised tone in the REA. In preparations in which the endothelial cell layer was intact ACh (1 /~M) produced a relaxation of 53.1 _+ 8.2% (n = 11) (fig. 7ai) while C G R P (0.263

CGRP (0.263

pM)

Fig. 6. Traces showing the effect of CGRP (0.263 /~M) on responses to electrical field stimulation in the rabbit central ear artery (supramaximal voltage, 0.1 ms pulse duration (a) 16 or (b) 64 Hz for 1 s). Responses were elicited every 2 min and continued for 8 min after addition of the peptide.

179

0.25 gl a.

W i t h Endothellum

I. NA and Ach

- -

-

b.

Ach(llJM) NA(1pM)

4

rain

li. NA and CGRP

--

CGRP(O.2631~M) NA(IlJM)

Without Endothelium

I. NA and Ach

--

Ach(llJM) NA ( I IJM)

ii. NA and CGRP

--CGRP (O. 2 6 3 p M ) NA(1pM)

Fig. 7. The effect of (i) ACh (1 ~tM) and (ii) CGRP (0.263/tM) on rabbit central ear artery segments preconstricted with N A (1 /tM), (a) in the presence of endothelium and (b) in the absence of endothelium. Horizontal lines indicate the time for which the drug and/or peptide was present.

m a n y fibres showed only C G R P - L I . This was expected as there were m a n y more C G R P - L I than SP-LI nerves. C G R P - L I nerves were found to have a significant density of innervation that was uniform throughout the length of the REA, whereas SP-LI nerves were sparse and unevenly distributed. The results from the use of the neurotoxins 6 - O H D A and CAPS suggest that these C G R P and SP-LI nerves are sensory in origin. Firstly, there was no change in the pattern or intensity of C G R P - and SP-LI after in vitro incubation with 6 - O H D A , although there was no catecholaminergic fluorescence, indicating that the sympathetic nerves had been damaged (Thoenen and Tranzer, 1968; Bennett et al., 1970; Jonsson and Sachs, 1972). Secondly, after in vitro incubation

with CAPS, a sensory neurotoxin known to deplete sensory nerves (Kawasaki et al., 1988; Maggi and Meli, 1988), there was a marked reduction in intensity and density of both C G R P - and SP-LI fibres. The sympathetic, catecholamine-containing nerves however, were intact. The slight reduction of immunofluorescence observed in the vehiclecontrol preparations was probably caused by Tween 80, since the incubation was in vitro and not perfused (Kawasaki et al., 1988). Since both C G R P - and SP-LI nerves were sensitive to CAPS (a sensory neurotoxin), but not 6 - O H D A (a sympathetic neurotoxin), these perivascular nerves are probably sensory in origin. C G R P has been shown to have pharmacological activity by modulating the vasoconstrictor responses to exogenous NA, a, fl-mATP and field stimulation in a concentration-dependent manner. In addition, C G R P relaxed REA segments preconstricted with N A in an endothelium-independent manner. This confirms the results of Dogramatzis et al. (Dogramatzis et al., 1987) and comparable findings which were obtained using bovine coronary rings (Greenberg et al., 1986) and cat cerebral and h u m a n pial arteries (Hanko et al., 1985). This contrasts, however, with the results on aortic strips from both rat and rabbit, and rat mesenteric artery, where endothelium-dependent relaxation has been demonstrated (Gibson et al., 1984; Brain et al., 1985; Kubota et al., 1985; Grace et al., 1987). The demonstration that C G R P is an endothelium-independent vasodilator in the REA suggests that the postjunctional action of the peptide on sympathetic transmission is due to a direct action on the smooth muscle of the blood vessel. Although our results concerning the inhibitory actions Of C G R P on the vasoconstriction mediated by electrical field stimulation are consistent with those of H a n k o et al. (1985) and Moritoki et al. (1990) on the REA, our finding of a reduction of contractile responses due to the effect of C G R P on exogenous N A supports those of Moritoki et al. (1990), but contrasts with the results of H a n k o et al. (1985) on the same preparation. A probable explanation for the discrepancy is that H a n k o et al. (1985) added C G R P 5-10 min before exogenous N A application. Both this study and that of

180

Moritoki et al. (1990) have shown that the inhibitory response elicited by C G R P recovered with time. It is therefore possible that the 5-10 min incubation period used by Hanko et al. (1985) was too long. Moritoki et al. (1990) stated that CAPS acted prejunctionally in the rabbit central ear artery, and may inhibit the release of NA. They further suggested that C G R P may be the peptide involved in mediating the actions of CAPS. Although our present results support those of Moritoki et al. (1990) regarding the actions of C G R P on electrical field stimulation and exogenous NA, we previously showed that C G R P did not inhibit the release of N A in this preparation (Maynard and Burnstock, 1989). It is known however, that the sympathetic transmission of the rabbit central ear artery has a purinergic as well as a noradrenergic component (Kennedy et al., 1985; Saville and Burnstock, 1988). It seems possible, therefore, that as is the case in the guinea-pig vas deferens, C G R P may prejunctionally inhibit the release of ATP, but not N A (Ellis and Burnstock, 1989). Further experiments are required to examine this possibility. Various authors (Burnstock et al., 1984; Owman et al., 1986; Uddman et al., 1986; Benjamin et al., 1987; Wallengren and H~tkanson, 1987; McEwan et al., 1988; Maggi and Meli, 1988) have suggested a role for S P / C G R P nerve fibres involving a sensory-motor function. Morris and Bevan (1986) have shown (and this study has confirmed), that SP-LI in rabbit ear vessels is capsaicin sensitive. In addition, capsaicin has been shown to activate polymodal (mechanical and thermal) C-fibres in the rabbit ear (Szolcsfinyi, 1983). C G R P is often found colocalised with SP in C-fibre nerves (Brain and Williams, 1988) and C G R P has also been shown to be capsaicin sensitive (Saito et al., 1987; Maggi and Meli, 1988; this study). C G R P is probably more effective than SP as a peptidergic modulator in this blood vessel, because (i) CGRPLI perivascular nerve fibres are more abundant than SP-LI fibres as well as being colocalised with SP-LI (this study); (ii) C G R P has a potent pharmacological effect (this study; Hanko et al., 1985; Moritoki et al., 1990) on the sympathetic transmission; (iii) SP has no effect on neurogenic

contractions or NA release in the REA (Illes and Van Falkenhausen, 1986) and (iv) C G R P appears to be a selective arterial dilator, whereas SP is a more potent venodilator (Benjamin et al., 1987). The role of SP in the REA could be to regulate the actions of CGRP. It has been reported that when SP and C G R P are released from sensory nerves during antidromic nerve impulses, SP can attenuate the vasodilatory response of C G R P in vivo (Brain and Williams, 1988). In summary, this study has localised and colocalised CGRP-LI with SP-LI, (both CAPS-sensitive, but not affected by 6-OHDA), suggesting a sensory origin for these perivascular nerves in the REA. It has shown that C G R P has a direct, postjunctional action modulating contractile responses to exogenous NA, a,fl-mATP and electrical field stimulation. Considering the results of the present study in conjunction with previous reports, these CGRP- and C G R P / S P - L I perivascular nerves may be involved in a sensory-motor neuroregulatory mechanism controlling local blood flow in the rabbit central ear artery.

Acknowledgements This work was supported by the British Heart Foundation (Grant No. RG12). K.I. Maynard is an Overseas Research Student Award holder.

References Benjamin, N., C.T. Dollery, R.W. Fuller, S. Larkin and J. McEwan, 1987, The effects of calcitonin gene-related peptide and substance P on resistance and capacitance vessels, Br. J. Pharmacol. 90, 39P. Bennett, T., G. Burnstock, J.L.S. Cobb and T. Malmfors, 1970, An ultrastructural and histochemical study of the short-term effect of 6-hydroxydopamine on adrenergic nerves in the domestic fowl, Br. J. Pharmacol. 38, 802. Brain, S.D. and T.J. Wilharns, 1988, Substance P regulates the vasodilator activity of calcitonin gene-related peptide, Nature 335, 73. Brain, S.D., T.J. Williams, J.R. Tippins, H.R. Morris and I. Maclntyre, 1985, Calcitonin gene-related peptide is a potent vasodilator, Nature 313, 54. Budai, D., H.Q. Vu and S. Piper Duckies, 1989, Endothelium removal does not affect potentiation by neuropeptide Y in the rabbit ear artery, European J. Pharmacol. 168, 97.

181

Burnstock, G., S.G. Griffith and P. Sneddon, 1984, Autonomic nerves in the precapillary vessel wall, J. Cardiovasc. Pharmacol. 6, $344. Cohen, M.L., E. Rosing, K.S. Wiley and I.H. Slater, 1978, Somatostatin inhibits adrenergic and cholinergic neurotransmission in smooth muscle, Life Sci. 23, 1659. Costa, M., R. Buffa, J.B. Furness and E. Solcia, 1980, Immunohistochemical localization of polypeptides in peripheral autonomic nerves using whole mount preparations, Histochemistry 65, 157. Daly, R.H. and J.P. Hieble, 1987, Neuropeptide Y modulates adrenergic neurotransmission by an endothelium dependent mechanism, European J. Pharmacol. 138, 445. Dhital, K.K and G. Burnstock, 1989, Adrenergic and nonadrenergic neural control of the arterial wall, in: Diseases of the Arterial Wall, eds. J.-P. Camilleri, C.L. Berry, J.-N. Fiessinger and J. Barirty (Springer, London) p. 97. Dogramatzis, D., O.S. Steinsland, G.A. Nickols and C.W. Cooper, 1987, Mechanisms of the vasodilatory action of calcitonin gene-related peptide, Abstr. X Int. Conf. Pharmacol., p. 931. Edvinsson, L., E. Ekblad, R. HS_kanson and C. Wahlestedt, 1984, Neuropeptide Y potentiates the various vasoconstrictor agents on rabbit blood vessels, Br. J. Pharmacol. 83, 519. Ellis, J.L. and G. Burnstock, 1989, Modulation of neurotransmission in the guinea-pig vas deferens by capsaicin: involvement of calcitonin gene-related peptide and substance P, Br. J. Pharmacol. 98, 707. Furchgott, R.F. and J.V. Zawadzki, 1980, The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine, Nature 288, 373. Ganten, D., R.E. Lang, J. Archelos and T. Unger, 1984, Peptidergic systems: effects on blood vessels, J. Cardiovasc. Pharmacol. 6, $598. Gibson, S.J., J.M. Polak, S.R. Bloom, I.M. Sabate, P.M. Mulderry, M.A. Ghatei, G.P. McGregor, J.F.B. Morrison, J.S. Kelly, R.M. Evans and M.G. Rosenfeld, 1984, Calcitonin gene-related peptide immunoreactivity in the spinal cord of man and eight other species, J. Neurosci. 4, 3101. Glover, W.E., 1985, Increased sensitivity of the rabbit ear artery to noradrenaline following perivascular nerve stimulation may be a response to neuropeptide Y released as a cotransmitter, Clin. Exp. Pharmacol. Physiol. 12, 227. Grace, G.C., G.J. Dusting, B.E. Kemp and T.J. Martin, 1987, Endothelium and the vasodilator action of rat calcitonin gene-related peptide (CGRP), Br. J. Pharmacol. 91, 709. Greenberg, B., K. Rhoden and P. Barnes, 1986, Coronary artery relaxant effects of calcitonin gene-related peptide and vasoactive intestinal polypeptide, Fed. Proc. 45, 599. Hanko, J., J.E. Hardebo, J. KMlrstr~Sm, C. Owman and F. Sundler, 1985, Calcitonin gene-related peptide is present in mammalian cerebrovascular nerve fibres and dilates pial and peripheral arteries, Neurosci. Lett. 57, 91. Illes, P., N. Pfeiffer, N. Limberger and K. Starke, 1983, Presynaptic opioid receptors in the rabbit ear artery, Life Sci. 33, 307.

llles, P. and S. Van Falkenhausen, 1986, Functional characterization of substance P receptors in the rabbit ear artery, Naunyn-Schmiedeb. Arch. Pharmacol. 333, 52. Jonsson, G. and C. Sachs, 1972, Degenerative and nondegenerative effects of 6-hydroxydopamine on adrenergic nerves, J. Pharmacol. Exp. Ther. 180, 625. Juan, H., W. Sametz, A. Saria and G. Prch, 1988, Eicosapentaenoic acid inhibits vasoconstrictor- and noradrenalinepotentiating effects of neuropeptide Y in the rabbit isolated ear, J. Auton. Nerv. Syst. 22, 237. Kawasaki, H., K. Takasaki, A. Saito and K. Goto, 1988, Calcitonin gene-related peptide acts as a novel vasodilator neurotransmitter in mesenteric resistance vessels of the rat, Nature 335, 164. Kennedy, C. and G. Burnstock, 1985, ATP produces vasodilation via Pl-purinoceptors and vasoconstriction via P2purinoceptors in the isolated rabbit central ear artery, Blood Vessels 22, 145. Kennedy, C., V.L. Saville and G. Burnstock, 1986, The contributions of noradrenaline and ATP to the responses of the rabbit central ear artery to sympathetic nerve stimulation depend on the parameters of stimulation, European J. PharmacoL 122, 291. Kubota, M., J.M. Moseley, L. Butera, G.J. Dusting, P.S. MacDonald and T.J. Martin, 1985, Calcitonin gene-related peptide stimulates cyclic AMP formation in rat aortic smooth muscle cells, Biochem. Biophys. Res. Commun. 132, 88. Legay, C., M.J. Saffrey and G. Burnstock, 1984, Coexistence of immunoreactive substance P and serotonin in neurones of the gut, Brain Res. 302, 379. Lindvall, O. and A. Bj~Srklund, 1974, The glyoxylic acid fluorescence histochemical method: a detailed account of the methodology for the visualisation of central catecholamine neurons, Histochemistry 39, 97. Maggi, C.A. and A. Meli, 1988, The sensory-efferent function of capsaicin-sensitive sensory neurons, Gen. Pharmacol. 19, 1. Maynard, K.I. and G. Burnstock, 1989, Effect of CGRP on 3H-NA release in the rabbit ear artery, Reg. Pept. 26, 82. McEwan, J.R., N. Benjamin, S. Larkin, R.W. Fuller, C.T. Dollery and I. Maclntyre, 1988, Vasodilatation by calcitonin gene-related peptide and by substance P: a comparison of their effects on resistance and capacitance vessels of human forearms, Circulation 77, 1072. Moritoki, H., H. Takase and A. Tanioka, 1990, Dual effects of capsaicin on the responses of the rabbit ear artery to field stimulation, Br. J. Pharmacol. 99, 152. Morris, J.L. and R.D. Bevan, 1986, Proliferation of arteriovenous anastomoses in the developing rabbit ear artery is enhanced after denervation, Am. J. Anat. 176, 497. Owman, Ch., J.E. Hardebo, J. Kilhrstr~Sm and F. Sundler, 1986, Co-existence and interaction between substance P and calcitonin gene-related peptide in central and peripheral vasodilatation, Blood Vessels, Abstr. Mechanism of Vasodilatation p. 93. Saito, A., T. Masaki, Y. Uchiyama, T. J.-F. Lee and K. Goto, 1989, Calcitonin gene-related peptide and vasodilator nerves

182 in large cerebral arteries of the cat, J. Pharmacol. Exp. Ther. 248, 455. Saito, A., Y. Tomobe and K. Goto, 1987, Effects of capsaicin on smooth muscles of the rat vas deferens: Involvement of calcitonin gene-related peptide?, J. Pharmacol. Exp. Ther. 242, 666. Saville, V. and G. Burnstock, 1988, Use of reserpine and 6-hydroxydopamine support evidence for purinergic cotransmission in the rabbit ear artery, European J. Pharmacol. 155, 271. Szolcsfi,nyi, J., 1983, Disturbances of thermoregulation induced by capsaicin, J. Therm. Biol. 8, 207. Thoenen, H. and J.P. Tranzer, 1968, Chemical sympathectomy by selective destruction of adrenergic nerve endings with 6-hydroxydopamine, Naunyn-Schmiedeb. Arch. Pharmacol. 261,271. Uddman, R., L. Edvinsson, E. Ekblad, R. Hfi.kanson and F.

Sundler, 1986, Calcitonin gene-related peptide (CGRP): perivascular distribution and vasaodilatory effects, Reg. Pept. 15, 1. Wallengren, J. and R. Hfi~kanson, 1987, Effects of substance P, neurokinin A and calcitonin gene-related peptide in human skin and their involvment in the sensory nerve-mediated responses, European J. Pharmacol. 143, 267. Warland, J.J.I. and G. Burnstock, 1987, Effects of reserpine and 6-hydroxydopamine on the adrenergic and purinergic components of sympathetic nerve responses of the rabbit saphenous artery, Br. J. Pharmacol. 92, 871. Wharton, J. and S. Gulbenkian, 1987, Peptides in the mammalian cardiovascular system, Experientia 43, 821. Wong-Dusting, H.K. and M.J. Rand, 1988, Pre- and postjunctional effects of neuropeptide Y on the rabbit isolated ear artery, Clin. Exp. Pharmacol. Physiol. 15, 411.