Peptide-containing nerve fibres in guinea-pig coronary arteries: Immunohistochemistry, ultrastructure and vasomotility

Journal of the Autonomic Nervous System, 31 (1990) 153-168

153

Elsevier

JANS 01096

Peptide-containing nerve fibres in guinea-pig coronary arteries: immunohistochemistry, ultrastructure and vasomotility Sergio Gulbenkian 1, Lars Edvinsson 2, Ole Saetrum Opgaard 2, John Wharton 3, Julia M. Polak 3 and Jos6 F. David-Ferreira 1 i Department of Cell Biology, Gulbenkian Institute of Science, Oeiras, Portugal, : Department of Internal Medicine, Unwersity Hospital of Lund, Lund, Sweden and 3 Department of Histochemistry, Royal Postgraduate Medical School, London, U.K. (Received 12 February 1990) (Revision received and accepted 11 July 1990)

Key words: Neuropeptide Y; Vasoactive intestinal polypeptide; Calcitonin gene-related peptide; Tachykinins; Coronary innervation; Immunohistochemistry; In vitro pharmacology Abstract The peptidergic innervation of guinea-pig coronary arteries was investigated by means of immunohistochemical, ultrastructural and in vitro pharmacological techniques. A network of nerves was demonstrated in all major epicardial arteries by means of an antiserum to the neuronal marker protein gene product 9.5. The majority of nerve fibres possessed neuropeptide Y (NPY) and tyrosine hydroxylase (TH) immunoreactivity, the number and distribution of nerves immunoreactive for N P Y being similar to that of nerves containing T H immunoreactivity. N u m e r o u s nerve fibres displaying immunoreactivity for substance P, neuropeptide K and calcitonin gene-related peptide (CGRP) were also found. In double-stained preparations substance P immunoreactivity was co-localized with C G R P and with neuropeptide K immunoreactivities in the same varicose nerve fibres. Ultrastructural studies revealed the presence of numerous axon varicosities at the adventitial-medial border. N P Y immunoreactivity was localized in large granular vesicles in nerve varicosities which also contained numerous small granular vesicles. Large granular vesicle-containing nerves also displayed immunoreactivity for dopamine-fl-hydroxylase. With an in vitro method, the vasomotor responses to perivascular peptides were characterized in epicardial and intramyocardial arteries. In epicardial arteries neither noradrenaline nor N P ¥ elicited a contractile response. Only in some intramyocardial arteries was an NPY-mediated contraction demonstrated. N o potentiating effect of noradrenaline and N P Y was observed in either epicardial or intramyocardial arterial segments. In contrast, C G R P , substance P and vasoactive intestinal peptide (VIP) all produced a concentration-dependent relaxation of both epicardial and intramyocardial arteries. These results suggest that peptide-containing nerves associated with guinea-pig coronary arteries may predominantly be involved in mediating vasodilation.

Introduction

With the development of histochemical and immunohistochemical techniques it has been

Correspondence: S. Gulbenkian, Laboratrrio de Biologia Celular, Instituto Gulbenkian de CiSncia, Apartado 14, 2781 Oeiras Codex, Portugal.

established that in addition to the "classical" transmitters, noradrenaline and acetylcholine, cardiovascular nerves contain a number of neuropeptides (for review see ref. [68]). These peptides have marked effects on cardiovascular tissues, acting both directly via specific peptide receptors and indirectly by modulating the action of other neurotransmitters [13,51]. The most widely distributed of the neuropeptides identified in mam-

0165-1838/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

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malian cardiovascular tissues are neuropeptide tyrosine (NPY), calcitonin gene-related peptide (CGRP), tachykinins such as substance P and vasoactive intestinal polypeptide (VIP). Nerves containing these neuropeptides supply most vascular beds, but exhibit both regional and species variations in their distribution pattern [17,19,27, 62,67,68]. Neural and humoral factors are involved in the control of both epicardial and intramyocardial arteries [28,29,71,72]. Neuropeptides, such as NPY, CGRP and substance P are known to affect coronary blood flow and epicardial vessel size [2,8,9,23,56], but the peptidecontaining innervation of mammalian epicardial arteries has received relatively little attention. In the present study, we have employed immunohistochemical, ultrastructural and pharmacological techniques to examine the peptide-containing innervation of guinea-pig coronary arteries.

Materials and Methods

Immunofluorescence staining Tissues for immunostaining were obtained from male guinea-pigs (300-400 g) which had been perfused with 200 ml of phosphate buffered saline (PBS) followed by 300-400 ml of Zamboni's fixative [58] at 4 ° C. Following the perfusion fixation, the heart was removed and immersed in the same

fixative for a further 16 h at 4 ° C and then washed in PBS containing 15% sucrose (w/v) and 0.01% (w/v) sodium azide. The major coronary arteries (left coronary artery, left circumflex coronary artery, left anterior descending artery, and the right coronary artery) were then dissected to obtain both epicardial and intramyocardial regions. A modified indirect immunofluorescence method was performed on whole mount preparations of the arteries as previously described [35]. Briefly, after pretreatment with a solution containing 0.2% Triton X-100 in PBS for 2 h at room temperature and impregnation with the dye Pontamine sky blue [16] for 30 min, blood vessels were incubated in diluted primary antisera (Table I) overnight at room temperature. The preparations were then washed in PBS and incubated with fluorescein isothiocyanate conjugated goat antirabbit IgG (1:100 dilution; Sigma) for 1 h at room temperature. For the simultaneous localization of two antigens, preparations were first exposed to a primary antiserum raised in rabbit which was visualized by a rhodamine-labelled goat anti-rabbit IgG ( 1 : 6 0 dilution; Sigma) and then to a second primary antiserum raised in rat which was visualized by a fluorescein isothiocyanatelabelled goat anti-rat IgG (1 : 100 dilution; Sigma). The preparations were finally examined using an Olympus BH-2 microscope equipped for epi-illumination with filters selective for fluorescein (BP-490) and rhodamine (BP-545) fluorescence.

TABLE I

Source and characterization of the primary antisera Antigen

D o n o r species

Code no.

Dilutions LM

P G P 9.5 ( H u m a n ) T H (Rat) D B H (Bovine) N P Y (Porcine) C-PON (Human) C G R P (Rat) Substance P (Mammalian) Substance P (Mammalian) Neuropeptide K (Mammalian) VIP (Porcine)

Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rat Rabbit Rabbit

LM, light microscopy; EM, electron microscopy.

RA 95103 AS2 AB 145 1086 1411 1208 910 MAS 035b 15-36 R2 652

I : 1600 1 : 300 1 : 200 1 : 400 1 : 600 1 : 200 1 : 500 l : 100 1 : 600 1 : 2000

References EM

1 : 800 1 : 2000 I : 2500

Utraclone, U.K. [35] [35,60] Chemicon, U.S.A. [33,35] [33] [35,67] [35,67] Sera Lab, U.K. [35,67] [47,64] [69]

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Conoentional electron microscopy and post-embedding immunogold staining Guinea-pigs were perfused with 2.5% glutaraldehyde (v/v) in 0.1 M phosphate buffer (pH 7.2). After removing the perfused fixed heart, the coronary arteries were dissected and immersed in the same fixative for a further 2 h at 4 ° C. Arteries were then washed in buffer containing 0.1 M sucrose, post-fixed in 1% osmium tetroxide in 0.1 M phosphate buffer for 1 h at 4 ° C, rinsed in buffer containing 0.1 M sucrose, dehydrated in a graded series of ethanol concentrations, cleared in propylene oxide and infiltrated with Epon resin. Ultrathin sections of silver interference colour (70-90 nm) were collected onto 300-mesh formvar coated grids. For the localization of peptide-immunoreactive sites, grid-mounted ultrathin sections were treated for 20 rain with a solution containing 5% ( w / v ) sodium metaperiodate in distilled water prior to immunostaining. Single immunogold staining using a gold-labelled (15 nm gold particles) IgG ( 1 : 1 5 dilution; Janssen Pharmaceutica, Belgium) was carried out as previously described [65]. All sections were finally counterstained with uranyl acetate and lead citrate and examined using a Jeol 100 CX electron microscope operating at 60 kV.

Pre-embedding peroxidase anti-peroxidase (PAP) staining Guinea-pigs were perfused with 4% paraformaldehyde (w/v) in 0.1 M phosphate buffer and subsequently the dissected coronary arteries were immersed in the same fixative for a further 2 h at 4 ° C. The tissues were then washed in buffer and immunostained using a method modified from that previously described [53]. Briefly, dissected coronary arteries were incubated in a solution of 0.2% Triton X-100 in PBS for 15 rain and then in normal goat serum (diluted 1 : 20 in buffer) for 30 min. After washing in buffer, the preparations were incubated in primary antisera for 16 h at 4 ° C , goat anti-rabbit IgG ( 1 : 1 0 dilution; Miles) for 2 h and with rabbit PAP complex (1 : 50 dilution; Miles) for 2 h. The immunoreaction was then visualized by immersing the preparations in 0.025 % 3.3 diaminobenzidine in 0.05 M Tris-HC1 buffer

containing 0.03% hydrogen peroxide for 5 min. Tissues were then post-fixed for 1 h in 1% osmium tetroxide in buffer, rinsed in buffer containing 0.1 M sucrose, dehydrated and embedded in Epon resin.

A n tisera The antisera used in this study are listed in Table I. The polyclonal (code 910) and monoclonal (code MAS 035b) antisera raised against substance P showed partial cross-reactivity with neurokinin A and neurokinin B; the antiserum raised against neuropeptide K (code 15-36 R2) cross-reacted with neurokinin A and B, but did not cross-react with substance P. Since the peptide sequence of NPY and the C-flanking peptide of NPY (C-PON) occur together in the same precursor molecule and have an identical distribution pattern [33], immunostaining detected using the antisera raised against either NPY (code 1086) or C-PON (code 1411) will be referred to as N P Y / C PON immunoreactivity for the sake of brevity.

Immunohistochemical controls In control experiments no immunostaining was observed when primary antisera (Table I) were omitted, replaced with non-immune serum or preabsorbed with their c o r r e s p o n d i n g a n t i g e n s (10-5-10 -6 M) for 24 h at 4 ° C . Labelled secondary antisera also exhibited no cross-reactivity with IgG from inappropriate species.

In vitro pharmacology Circular epicardial and intramyocardial arterial segments, 1 - 2 nun long, were excised and mounted in a temperature-controlled tissue bath (37°C) containing a buffer solution of the following composition: 119 m M NaC1; 15 m M NaHCO3; 4.6 m M KC1; 1.5 mM CaC12; 1.2 mM NaHzPO4; 1.2 m M MgCI2; and 11 mM glucose. The buffer solution was bubbled continuously with a mixture of 5% CO 2 and 95% O 2, giving a pH of approximately 7.4; for further details see ref. [38]. Each vessel segment was suspended between two L-shaped metal holders (0.05 to 0.1 mm in diameter) one of which was attached to a Grass FT-03 transducer for continuous recording of iso-

156

metric tension on a Grass polygraph. The position of the other holder could be changed by means of a movable unit allowing fine adjustment of the vascular tension by varying the distance between the metal prongs. A tension of 1-2 mN was applied, this resulted in spontaneous relaxation, which was compensated for by frequent stretches in order to maintain a resting tension of 1-2 mN (depending on vessel size in situ). After approximately 1 h, when the tension had stabilized at the desired level, the vessel was exposed to a buffer solution containing 62 mM KC1, obtained by substituting equimolar concentrations of NaC1 for KC1 in the above buffer solution. After two reproducible contractions (variation less than 10%) had been evoked by the K+-rich solution, agonists were added in a cumulative fashion and concentration-response curves were constructed. The contraction evoked by the K+-rich solution was set at 100% and used as an internal standard because the length of the vascular segments and the thickness of the arteries could vary slightly. By setting the K+-induced contraction at 100% it was possible to compare the specimens. Since vessels during these conditions are almost completely relaxed, precontraction was regularly induced by 62 mM K + to study relaxant effects. Although histamine is a powerful vasoconstrictor it does not maintain tone and was only used occasionally. Thus, when tone of the vessels was induced by KC1, this resulted in a tension of 1.5 +_ 0.3 mN (n = 50) in the proximal epicardial artery and 1.1 _+ 0.2 mN in the intramyocardial artery (n = 40), and lasted for at least 20 min without a significant fall in tone.

Analysis of in vitro data All concentration-response curves were plotted graphically and /max (maximum effect obtained with an agonist) and pD 2 (negative logarithm of the concentration of agonist that elicited half maximum effect) were calculated arithmetically from each individual concentration-response curve. Values are given as mean + SEM. Student's t-test was used to determine statistical significance with respect to differences in pD 2 and /max values. For multiple group comparison Bonferroni correction was performed [66].

Drugs The following pharmacological agents were used: acetylcholine HC1 (ACh; Sigma, U.S.A.), cocaine HC1 (Sigma), rat cyclic calcitonin gene-related peptide (CGRP; Sigma), histamine HC1 (Sigma) neurokinin A (NKA; Sigma), neuropeptide Y (NPY; Sigma), papaverine HC1 (Sigma), peptide histidine isoleucine - 27 (PHI; Sigma) propranolol (Inderal; ICI, U.K.), prostaglandin F2 (PGF2; Amoglandin R, Astra, Sweden), vasoactive intestinal peptide (VIP; Sigma). All agents were dissolved and diluted in saline containing 10 -4 M ascorbic acid to minimize oxidation.

Results

Light microscopical immunohistochemistry Immunofluorescence staining with the antiserum to the general neuronal marker protein gene product (PGP) 9.5 (Fig. 1) demonstrated that guinea-pig epicardial coronary arteries possess a dense supply of nerves forming an extensive network in the adventitia. The distribution of this perivascular innervation was found to be similar in all the vessels examined and was arranged in two layers, consisting of an outer layer of nonvaricose nerve fibres and fascicles and an inner layer of varicose and non-varicose nerve fibres which mainly run around the vessel in a circular direction at the adventitial-medial border (Fig. 1). The majority of the nerve fibres displayed N P Y / C - P O N immunoreactivity (Figs. 2 and 4). The number and distribution of N P Y / C - P O N - i m munoreactive nerves was similar from one artery to another and paralleled that of nerves containing T H immunoreactivity (Fig. 3). In all coronary arteries analysed, an increase in the relative number of N P Y / C - P O N - and TH-immunoreactive nerves with declining vessel diameter was observed (Figs. 5 and 6). Proximal regions of the epicardial arteries possessed a moderate supply of CGRP-, substance P- and neuropeptide K-immunoreactive nerve fibres, forming a loose network with a predominant circular orientation around the long axis of the vessel (Figs. 7a,b). As the vessels were followed distally, however, CGRP(Figs. 8 and 9), substance P- (Fig. 10a) and neuro-

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Figs. 1-6. Whole mount preparations of guinea-pig left anterior descending (Figs. 1, 2, 3, 6), left circumflex (Fig. 4) and right coronary arteries (Fig. 5) immunostained for PGP 9.5 (Fig. 1), NPY (Figs. 2,6), C-PON (Fig. 4) and TH (Figs. 3, 5). The number and distribution of NPY-, C-PON- and TH-immunoreactive nerves is similar from one artery to another and increases with declining vessel size (Figs. 5, 6). Bar = 50 #m.

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Figs. 7-10. Whole mount preparations of guinea-pig left anterior descending (Figs. 7a,b, 10a,b), left circumflex (Fig. 8) and right coronary arteries (Fig. 9) immunostained for CGRP (Figs. ?a, 8, 9), substance P (Figs. 7b, 10a) and neuropeptide K (NK) (Fig. 10b). The co-localization of substance P with CGRP (Fig. 7a, b) as well as with NK (Fig. 10a,b) was determined by double immunostaining the same preparations. Varicose nerve fibres (arrows) displaying substance P-immunofluorescence staining also possess CGRP (Fig. 7a,b) and NK immunoreactivity (Fig. 10a,b). As the vessel calibre declines, CGRP-, substance P- and NK-immunoreactive nerves display an orientation which is mainly parallel to the long axis of the vessel (Figs. 8, 9, 10a,b). Bar = 50 ~m,

159

Figs. 11-14. Electron micrographs of nerve varicosities at the adventitial-medial border of the guinea-pig right (Fig. 11) and left anterior descending coronary arteries (Figs. 12-14). The cleft, between the varicosities and the muscle cell membrane (double-headed arrow) is generally greater than 300 nm wide (Fig. 11). Several axon profiles may be demonstrated containing numerous small granular vesicles (Fig. 11; arrows); small round (arrowheads) and flattened (arrows) agranular vesicles (Fig. 12); large granular and small agranular vesicles or mainly small granular vesicles (asterisk; Fig. 13). Other axon bundles contain varicosities possessing small mitochondria and dense bodies as well as varicosities possessing numerous small granular vesicles (asterisks) (Fig. 14). SM, smooth muscle; SC, Schwann cell. Bar = 400 nm.

160 p e p t i d e K- (Fig. lOb) i m m u n o r e a c t i v e fibres disp l a y e d an o r i e n t a t i o n which was m a i n l y p a r a l l e l to the long axis of the vessel. In all the c o r o n a r y arteries examined, the n u m b e r a n d d i s t r i b u t i o n of C G R P - i m m u n o r e a c t i v e fibres was similar to that of fibres c o n t a i n i n g substance P a n d n e u r o p e p t i d e

K i m m u n o r e a c t i v i t y . F u r t h e r m o r e , when d o u b l e i m m u n o s t a i n i n g for either C G R P a n d substance P (Fig. 7a,b) or s u b s t a n c e P a n d n e u r o p e p t i d e K (Fig. 10a,b) was p e r f o r m e d , the p e p t i d e imm u n o r e a c t i v i t i e s coexisted in the same varicose nerve fibres.

Figs. 15, 16. Electron micrographs demonstrating the ultrastructural localization of NPY (Fig. 15) and DBH (Fig. 16) immunoreactivity in nerve varicosities of the guinea-pig left anterior descending coronary artery. Fig. 15: NPY-immunogold labelling (15 nm gold particles, arrows) is localized over large granular vesicles in varicosities which also contain numerous unlabelled small granular vesicles (arrowheads). Fig. 16: DBH-PAP immunostaining is found in large granular vesicles (arrows)-containing nerves. SM, smooth muscle. Bar = 200 nm.

161 No nerves fibres containing VIP immunoreactivity were detected in association with coronary arteries.

Conventional electron microscopy Numerous unmyelinated axons ran in the adventitia. At the adventitial-medial border axon varicosities were usually separated from a muscle cell membrane by a cleft 300 to 800 nm wide (Figs. 11-14). Although varicosities represent a spectrum of profiles containing a heterogeneous vesicle population, four principal types were distinguishable. (1) Varicosities containing a large number of small granular vesicles (40-60 nm in diameter) and a few large granular vesicles (80-100 nm in diameter). In these varicosities a varying number of small vesicles with almost indistinct granular cores as well as agranular vesicles (40-60 nm in diameter) were usually found (Fig. 11). (2) Varicosities containing a heterogeneous population of small round (40-60 nm in diameter) and flattened agranular (20-40 nm in diameter and extending for up to 70 nm) vesicles, as well as few occasional large granular ones (Fig. 12). (3)Varicosities containing large granular vesicles (80-100 nm in diameter) together with varying number of small round and flattened agranular vesicles (Fig. 13). (4) Varicosities containing a large number of tightly packed mitochondria as well as some dense bodies (Fig. 14). Electron microscopical immunoh&tochemistry Post-embedding immunogold staining demonstrated that N P Y / C - P O N immunoreactivity consistently occurred i n the large granular vesicles (80-100 nm in diameter) in varicosities which also contained numerous small granular vesicles (40-60 nm in diameter) (Fig. 15). Pre-embedding PAP immunostaining also demonstrated that varicosities containing large granular vesicles were immunoreactive for the catecholamine synthesising enzyme dopamine-fl-hydroxylase (DBH) (Fig. 16). In vitro pharmacology Studies were performed with noradrenaline and NPY, administered in increasing concentrations. In the majority of the tests, cocaine (10 -6 M) and propranolol (10-7 M) were present, however some

experiments were also performed in their absence. Noradrenaline in concentrations of 1 0 - 9 - 1 0 - 4 M did not induce a contractile response in either epicardial (n = 6) or intramyocardial (n = 6) arterial segments. NPY in concentrations of 10 1°-10-6 M also did not induce the contraction of epicardial arteries (n = 6). In two out of six preparations of intramyocardial arteries, however, a concentration-dependent contraction was induced by NPY (starting at 10 -9 M and reaching a maximum at 10 -7 M). The same maximum effect was seen at 3 x 10 -7 M, amounting to 10% of that produced by the potassium rich buffer. To test any additive or potentiating effect of noradrenaline and NPY, noradrenaline was added in a cumulative manner (10-8-10 -4 M) in vessels pretreated with 10 -6 M NPY. No contractile response was seen in either epicardial or intramyocardial arterial segments. Similarly, the addition of increasing concentrations of NPY to epicardial arteries (n = 3) p r e t r e a t e d with 10 -4 M noradrenaline did not elicit a contractile response. As noted in the materials and methods section, studies of vasodilatation require the presence of an induced tone. Since histamine failed to maintain tone, a potassium rich buffer (62 mM) was used to produce a steady level of contraction during which various peptides were tested. CGRP, substance P and VIP all produced a concentration-dependent relaxation of both epicardial and intramyocardial arteries. The rank order of potency and maximum responses are given in Table II. In general, intramyocardial segments exhibited stronger relaxant responses to the peptides than did epicardial regions of the arteries (Table II). Substance P was the most potent peptide, followed by VIP and CGRP. The same rank order of potency was observed in both epicardial and intramyocardial regions (Fig. 17). To test whether there was any additive or potentiating effect of substance P on vessels already partially relaxed by VIP, cumulative additions of s u b s t a n c e P were given to vessels precontracted with the potassium buffer and relaxed by 3 x 10 _7 M VIP. This induced an additional c o n c e n t r a t i o n - d e p e n d e n t r e l a x a t i o n (which amounted to 32.9 + 5.7% in intramyocardial and 22.5 _+ 3.5% in epicardial vessels), but no potentia-

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Fig. 17. Concentration-dependent relaxation of (A) epicardial and (B) intramyocardial guinea-pig artery segments after the administration of calcitonin gene-related peptide (CGRP), substance P (SP) and vasoactive intestinal peptide (VIP) to vessels precontracted by a potassium rich buffer solution (62 raM). Values represent mean + SEM of 6 experiments in each point.

tion (the pD 2 value for substance P was 8.70 + 0.06 and 8.67 + 0.07 for the epicardial and intramyocardial vessels, respectively). The relaxant responses represent the sum of relaxation induced by VIP and substance P alone.

Discussion The present study indicates that the major epicardial coronary arteries of the guinea-pig are TABLE II

Relaxant response of precontracted segments of guinea-pig epicardial and intramyocardial arteries to the addition of increasing concentrations of substance P (SP), calcitonin gene-related peptide (CGRP) and vasoactive intestinal peptide (VIP) Values given represent m e a n s + S E M for 6 vessels tested in each group. Differences between mean values were performed using the Student's t-test with a critical P value < 0.05. For

multiple group comparison Bonferroni correction was performed [661. Peptide Epicardial artery

PD2 CGRP SP VIP

lmax%

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pD2

Imax%

8.36_+0.23 a 12.6+_1.0 b 8.20_+0.10 a'b 18.0-t-1.3 ~ 9.06 _+0.15 a 17.3+_3.2 9.14_+0.t3 ~ 31.7-+5.2 ..... 8.90_+0.11 16.9_+1.4 h 8.88+0.07 h 17.5+2.0 "

~' Comparison between CGRP and SP; bcomparison between CGRP and VIP; ~ comparison between SP and VIP data.

supplied by an extensive network of perivascular nerve fibres. Nerve varicosities present at the adventitial-medial border were found to lack pre- or post-synaptic membrane specializations, being separated from the smooth muscle cells by a cleft which was usually several hundred nanometres wide. This structural arrangement is in agreement with the general model of the autonomic neuromuscular junction proposed by Burnstock [10]. Four principal types of varicosities were observed. These varicosities were similar to nerve profiles previously identified in intramyocardial resistance vessels [43,70] and in other vascular beds [7,12,42]. They include nerve profiles that contain a predominance of small granular vesicles and which are thought to correspond to sympathetic nerve varicosities. Other varicosities may be distinguished on the basis of the numerous large electron-dense vesicles they contain and have been collectively referred to as p-type (peptidecontaining) vesicles [3]. A further nerve profile contained a large number of tightly packed mitochondria and some dense bodies and is thought to represent sensory or baroreceptor nerve endings [11,14,54]. All major coronary arteries received a rich supply of N P Y / C - P O N - i m m u n o r e a c t i v e nerves. As in other vascular beds, NPY-C-PON-immunoreactive nerves were one of the major peptide-containing nerve populations and ex-

163 hibited a similar distribution to that of nerves containing the catecholamine synthesizing enzyme TH. These observations are consistent with the general assumption that NPY/C-PON-containing cardiovascular nerves represent postganglionic, sympathetic neurons [20,32,33,44]. On the other hand, we cannot rule out the possibility that some of the nerves supplying the guinea-pig coronary arteries may also originate from intrinsic cardiac ganglia [32,37]. The use of immunogold staining techniques revealed that in presumptive sympathetic varicosities, N P Y / C - P O N immunoreactivity was found in the large, but not in the small, dense-cored vesicles. This location confirms our earlier observations in human atrial myocardium [68,69] and is in agreement with subcellular fractionation studies in the spleen [24,26,46] and vas deferens [18,25] suggesting that noradrenaline occurs mainly in the small dense-cored vesicles, whereas NPY is found together with some noradrenaline in the large dense-cored vesicle population. The localization of D B H immunoreactivity in nerve fibres containing large dense-cored vesicles also correlates well with the results of biochemical studies, indicating that the large dense-cored vesicles in sympathetic nerves contain this catecholamine synthesizing enzyme [50]. It has recently been suggested that the storage of NPY and noradrenaline in two distinct subcellular compartments could facilitate the differential release of noradrenaline or NPY, the neuropeptide being preferentially released at high stimulation frequencies [45,46] and in the presence of calcium [36]. Despite the rich supply of NPY and TH-immunoreactive fibres, neither NPY nor noradrenaline induced a contractile response in preparations of epicardial arteries. In studies using human, monkey and dog epicardial arteries it has been reported that contractile responses were only seen after fl-blockade [6,61]. Little work has been performed on rodent coronary arteries, but in the rat we have observed a weak contraction by noradrenaline in concentrations above 10 -5 M which is enhanced by the presence of propranolol [57]. During precontraction a potent fl-adrenoceptor has been characterized in intramural rat coronary arteries [52]. Similarly Moreland and

Bohr [48] concluded from their functional studies that a-adrenoceptors were present in large coronary arteries whereas small coronary arteries were supplied almost exclusively with dilatory fladrenoceptors. The results of our study indicate that noradrenaline has virtually no contractile effect, even in the presence of propranolol, and thus suggest that t~-adrenoceptors are lacking in guinea pig coronary arteries. As pointed out by Moreland and Bohr [48], the main function of noradrenaline may be to mediate primary dilatation of the coronary circulation. These findings are also in agreement with pharmacological studies using isolated pig epicardial coronary arteries [28] which showed that noradrenaline had no effect on these vessels. In the present study we have demonstrated, however, that an NPY induced-contraction occurred in some intramyocardial arteries. Rioux and colleagues [55] showed that NPY increases myocardial perfusion pressure in guineapig isolated hearts. Although in their study the authors did not mention which parts of the coronary vasculature contracted, it seems reasonable to assume that the intramyocardial resistance vessels are the main vascular elements responsible for the variations in the perfusion pressure. Furthermore, the infusion of NPY into a coronary artery of patients with typical angina pectoris revealed a transient myocardial ischemia which appeared to be induced by the constriction of small resistance vessels rather than of epicardial arteries [15]. It is also possible that NPY may down-regulate the action of vasodilator agents such as substance P and acetylcholine [1,21]. Guinea-pig epicardial arteries were supplied by numerous perivascular nerve fibres containing substance P, neuropeptide K and C G R P immunoreactivity. The present findings also show that substance P, neuropeptide K and C G R P immunoreactivities invariably occurred in the same varicose nerve fibres. These observations are in agreement with previous reports demonstrating that tachykinins and C G R P immunoreactivities coexist in guinea-pig capsaicin-sensitive sensory neurons [30,40,67]. The findings also correlate well with the results of immunoelectron microscopical studies showing that C G R P and substance P immunoreactivities are co-localized in the same

164 secretory vesicles in both varicosities of perivascular nerve fibres and sensory ganglion cells in the guinea-pig [34]. Furthermore, the demonstration that substance P and neurokinin A are derived from a common precursor molecule (/~-preprotachykinin) [49,59], suggests that several peptides of the tachykinin family may be co-stored with C G R P in the same secretory vesicles. Some workers have tested the effects of C G R P and substance P on human and porcine coronary arteries [4,5,6,31] and reported that both peptides caused relaxation, but by different modes of action. The substance P response required the presence of an intact endothelium, whereas the one elicited by C G R P did not. We found that epicardial as well as intramyocardial coronary arteries relaxed upon exposure to both C G R P and substance P. Although both regions of the vessel showed a similar degree of sensitivity, the responses seemed to be more marked in the intramyocardial arteries. Furthermore, whereas the C G R P - i n d u c e d relaxation occurred without changes in the smooth muscle membrane potential, substance P relaxed and hyperpolarized the coronary arteries [4]. VIP-immunoreactive fibres have been reported to be associated with guinea-pig coronary arteries [17,22], although no perivascular nerves containing VIP immunoreactivity were detected in the present study. The reason for this apparent discrepancy is not clear, but it may well reflect the limited distribution of VIP-positive nerve fibres around guinea-pig coronary arteries and indicate possible qualitative differences in the antisera used. Data on the effects of VIP on the epicardial and intramyocardial circulation seems to be sparse. Brny and colleagues [4] showed that VIP causes dilatation of pig coronary arteries independently of the presence of endothelium. Furthermore, the VIP relaxation did not change the resting membrane potential. A substantial reduction in coronary perfusion pressure, as well as an increase in coronary blood flow has been reported for the guinea-pig and dog [39,63]. Furthermore, the presence of high affinity VIP binding sites in bovine coronary arteries has been reported [41]. Our results showing that VIP relaxed both epicardial and intramyocardial arteries complement the findings

of these studies. Furthermore, there was no difference in either the potency or maximum responses between the epicardial and the intramyocardial arteries. In view of our morphological observations showing a lack of VIP-immunoreactive fibres, the functional role of VIP in the guinea-pig coronary vasculature is still uncertain. In conclusion, the results indicate that the predominant neuropeptide mediated response in guinea-pig coronary arteries appears to be vasodilation.

Acknowledgements This work was funded in part by a grant from the Anglo-Portuguese Joint Research Programme (Treaty of Windsor). Antisera raised to regulatory peptides at the Hammersmith Hospital were produced in conjunction with Prof. S.R. Bloom. The authors are grateful to Dr. K. Valentino and Dr. J. Thibault for providing antisera to neuropeptide K and tyrosine hydroxylase respectively. We thank Mrs. M.R. Alpiar~a for excellent technical assistance.

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