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Innervation of the human middle meningeal artery: immunohistochemistry, ultrastructure, and role of endothelium for vasomotility
Peptides, Vol. 19, No. 7, 1998, pp. 1213–1225 Copyright © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0196-9781/98 $19.00 1 .00
PII S0196-9781(98)00066-7
Innervation of the Human Middle Meningeal Artery: Immunohistochemistry, Ultrastructure, and Role of Endothelium for Vasomotility LARS EDVINSSON,*1 SERGIO GULBENKIAN,† CARLA P. BARROSO,† MANUEL CUNHA E S´A,†‡ JULIA M. POLAK,§ ANDERS MORTENSEN,¶ LINDA JØRGENSENi AND INGER JANSEN-OLESEN# *Department of Internal Medicine, University Hospital of Lund, S-221 85 Lund, Sweden †Unit of Cell Morphology and Image Processing, Gulbenkian Institute of Science, 2781 Oeiras, Portugal ‡Department of Neurosurgery, Hospital Garcia de Orta, Almando, Portugal §Department of Histochemistry, Royal Postgraduate Medical School, Hammersmith Hospital, London W12 ONN, United Kingdom ¶Department of Neurosurgery, Copenhagen University, Glostrup Hospital, 2600 Copenhagen, Denmark iDepartment of Neurosurgery, Copenhagen University, Rigshospitalet, 2100 Copenhagen Ø, Denmark, Department of Biological Sciences, The Royal Danish School of Pharmacy, Universitetsparken 2, 2100 Copenhagen Ø, Denmark Received 30 December 1997; Accepted 13 April 1998 EDVINSSON, L., S. GULBENKIAN, C. P. BARROSO, M. CUNHA E SA´ , J. M. POLAK, A. MORTENSEN, L. JØRGENSEN AND I. JANSEN-OLESEN. Innervation of the human middle meningeal artery: Immunohistochemistry, ultrastructure and role of endothelium for vasomotility. PEPTIDES 19(7) 1213–1225, 1998.—The majority of nerve fibers in the middle meningeal artery and branching arterioles are sympathetic, storing norepinephrine and neuropeptide Y (NPY). A sparse supply of fibers contain acetylcholinesterase activity and immunoreactivity toward vasoactive intestinal peptide (VIP), peptidine histidine methionine (PHM), and calcitonin gene-related peptide (CGRP). Only few substance P and neuropeptide K immunoreactive fibers are noted. Electronmicroscopy shows axons and terminals at the adventitial medial border of the human middle meningeal artery, with a fairly large distance to the smooth muscle cells (.500 nM). Several axon profiles contain vesicles of different types, including putative sensory profiles. The perivascularly stored signal substances, norepinephrine and NPY induced vasoconstrictor. Relaxations were induced by acetylcholine and substance P, and these were significantly reduced in arteries without endothelium, while the responses to norepinephrine, NPY, VIP, PHM, and CGRP were not changed by endothelium removal. Blockade experiments showed that the vasomotor responses to norepinephrine were blocked by prazosin, to NPY by BIBP 3226, acetylcholine by atropin, substance P by RP 67580, and the human a-CGRP response by human a-CGRP8 –37. © 1998 Elsevier Science Inc. Neuropeptide Y Vasoactive intestinal peptide Calcitonin gene-related peptide Tachykinins Acetylcholinesterase Middle meningeal artery Immunohistochemistry Endothelium in vitro pharmacology
WOLFF and colleagues demonstrated more than fifty years ago that stimulation of vessels in the dura mater, such as the middle meningeal artery and the superior sagittal sinus (SSS), caused aching, throbbing ,and penetrating headaches as well as typical referred pain (42). This has served as one
important element in the current discussion of migraine pathogenesis with dural vessels currently held as one possible origin of the migraine pain. Stimulation of the SSS may produce activation of sensory neurons in the trigeminal nucleus caudalis and release of CGRP and VIP (15,49).
1 Requests for reprints should be addressed to Lars Edvinsson M.D., Ph.D., Department of lnternal Medicine, University Hospital of Lund S-221 85 Lund, Sweden. E-mail: [email protected]
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EDVINSSON ET AL. TABLE 1 SOURCE AND CHARACTERIZATION OF THE PRIMARY ANTISERA Antigen
Species
Code No.
Dilutions
Source
PGP 9.5 TH NPY VIP PHM SP SP NPK CGRP NOS endot NOS neuro
Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rat Rabbit Rabbit Rabbit Rabbit
Ra95103 Tel01 1086 652 1655 910 MASO35b 15-36R2 1208 2431 2393
1:9600 1:1600 1:1200 1:4000 1:4000 1:1600 1:100 1:3000 1:1600 1:1000 1:1600
Ultraclone; UK Eugene Tech, USA Hammersmith Hosp. Hammersmith Hosp. Hammersmith Hosp. Hammersmith Hosp. Sera Lab, UK Dr. Valentino, USA Hammersmith Hosp. Hammersmith Hosp. Hammersmith Hosp.
PGP 9.5, protein gene product 9.5; TH, tyrosine hydroxylase; NPY, neuropeptide tyrosine; VIP, vasoactive intestinal polypeptide; PHM, peptide histidine methionine; SP, substance P; NPK, neuropeptide K; CGRP, calcitonin gene-related peptide; NOS endot, nitric oxide synthase (endothelial form); NOS neuro, nitric oxide synthase (neuronal form). Characterization of the antisera: (17,18,29,48).
Stimulation of the trigeminal ganglion in rodents elicits a neurogenic inflammation, which is a complex process characterized by vasodilatation, plasma extravasation, endothelium activation, and mast cell degranulation in the dura mater but not in the central nervous system (CNS; References 6,33,34). This process can be inhibited by antimigraine drugs. The mechanisms involved are not clear in detail but putatively pre- and post-synaptic receptor mechanisms are involved, mediating both presynaptic inhibition of neuropeptide release (4,15) and cranial vasoconstriction (23). Neuroanatomical tracing studies from the meningeal in rat (47) and in cat (35) have revealed projections of sympathetic fibers from the superior cervical ganglion, parasympathetic fibers from the sphenopalatine and otic ganglia, and sensory fibers from the first division of the trigeminal ganglion. Histochemical studies in laboratory animals early revealed a moderate supply of perivascular fibers in the dura mater (10), whereas the detailed innervation of the dura mater in man remains to be characterized (30). The present study aims at examining in detail the perivascular nerve supply of the human middle meningeal artery, removed during neurosurgical operations, using immunocytochemistry and electron microscopy. In addition, the vasomotor responses of ring segments of the human middle meningeal artery to stored neurotransmitters and the role of the endothelium for the responses were studied using a sensitive in vitro system. METHOD
16 –24 h at 4°C in a solution containing 85 ml of 2% (w/v) paraformaldehyde in 0.1 M phosphate buffer (pH 7.2) and 15 ml of saturated picric acid per 100 ml of fixative. The tissue was rinsed in several changes of phosphate-buffered saline (PBS; 0.01 M, pH 7.2) containing 15% (w/v) sucrose and 0. l% (w/v) sodium azide, then processed for cryostat sectioning or whole-mount preparations. Cryostat section (14-mm thick) were immunostained using an biotin–streptavidin–fluorescence method. A modified biotin—streptavidin–fluorescence method was performed on whole-mount preparations of the arteries. 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 (BDH, Poole, UK) for 30 min to reduce background fluorescence, the whole-mount preparations were incubated in diluted primary antisera (Table 1) overnight at room temperature. The preparations were then washed in PBS, incubated in biotinylated goat anti-rabbit IgG (1:200 dilution; Sigma Chemical Co., St Louis, MO) for l h at room temperature rinsed in PBS, and incubated with fluorescein isothiocyanate-labeled streptavidin (l:100 dilution; Sigma) for l h at room temperature. For the simultaneous localization of two antigens, whole-mount preparations were first exposed to a primary antiserum raised in rabbit that was visualized by a rhodamine-labeled goat anti-rabbit IgG (1:100 dilution; Sigma) and then to a second primary antiserum raised in rat that was visualized by a fluorescein isothiocyanate labeled goat anti rat IgG (l:100 dilution; Sigma). The preparations were finally examined using an Olympus BH-2 microscope equipped for epi-illumination with filters selective for fluorescein and rhodamine fluorescence.
Immunohistochemistry Middle meningeal arteries were obtained from six patients during neurosurgical tumor resections. Immediately after excision, vessel segments were fixed by immersion for
Acetylcholinesterase (AChE) staining The histochemical demonstration of acetylcholinesterase (AChE) activity in whole-mount preparations was per-
PERIVASCULAR NERVES AND ENDOTHELIUM IN MIDDLE MENINGEAL ARTERY
formed as previously described (17). Briefly, whole-mount preparations were immersed in incubation medium (1 mg acetylcholine iodide, l.263 ml of 0. l M sodium acetate, 0.04 ml of 0. l M acetic acid, 0.096 ml of 0. l M sodium citrate, 0.2 ml 30 of mM cupric sulfate, 0.2 ml 5 mM potassium ferricyanide, and 38.2 ml of distilled water) for 30 min at 37°C. After rinsing in distilled water, the AChE activity was visualized by immersing the preparations for 5 min in Tris-HCl buffer (50 mM, pH 7.6) containing 0.04% 3,39diaminobenzidine tetrahydrochloride and 0.3% nickel ammonium sulphate and then for a further 5–10 min with the addition of 0.003% hydrogen peroxide. After a brief wash in distilled water, whole-mount preparations were mounted on glass slides, dehydrated in an ascending series of acetone concentrations, cleared in xylene, and mounted in DPX. The preparations were finally observed and photographed with an Olympus BH-2 microscope using the transmitted bright field illumination. Conventional Electron Microscopy Vessel segments were immersed in 2.5% glutaraldehyde (v/v) in 0. l M phosphate buffer (pH 7.2) for 2 h at 4°C. Arteries were then washed in buffer containing 0. l M sucrose, postfixed in l % osmium tetroxide for l h at 40°C, rinsed in buffer containing 0. l M sucrose, dehydrated in a graded series of ethanol concentrations, cleared in propylene oxide, and infiltrated with Epon resin. Ultrathin sections of silver interference color (70 –90 nm) were collected onto 300-mesh formvar-coated grids. Grid-mounted sections were finally counterstained with uranyl acetate and lead citrate and were examined and photographed using a Jeol 100 CX electron microscope operating at 8OkV. Antisera The antisera used in this study are listed in Table l. The polyclonal (code 910) and monoclonal (code MAS 035b) antisera raised against SP 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 SP. Immunohistochemical and Histochemical Controls In control experiments, no immunostaining was observed when one primary antiserum (Table 1) was omitted, replaced with nonimmune serum, or reabsorbed with the corresponding antigens (1025 –1026 M) for 24 h at 4°C. In double-immunofluorescence staining experiments, labeled secondary antisera exhibited no cross-reactivity with IgG from inappropriate species. For the demonstration of AChE activity, the following control incubation media were used: 1) 1024 –1026 M tetraisopropyl-pyrophospharamine (isoOMPA; Sigma) was added to the incubation medium as an inhibitor for non-
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specific cholinesterases. (2) Preparations were incubated in a substrate-free medium omitting acetylthiocholine. Vasomotor Responses in vitro Middle meningeal arteries were obtained from 26 patients during neurosurgical tumor resections. Immediately after excision, vessel segments were immersed in aerated (5% CO2 in O2) buffer solution (4°C) and transported to the laboratory for studies of vasomotor reactivity. Two- to three-mm long ring segments of the artery were suspended between two L-shaped metal prongs (0.2 mm) in small tissue baths containing a buffer solution aerated with 5% CO2 and 95% O2. The vessels were given a passive load of 2– 4 mN depending on the vessel size and allowed to stabilize at this level of tension for an equilibration period of l.5 h before isometric circular contractions were recorded. The contractile capacity of each preparation was first tested by exposure to a buffer solution containing 60 mM potassium; this resulted in strong contractions of middle meningeal arteries; 23.0 6 2.0 mN (n 5 49). Only vessels showing reasonably strong (.1 mN) and reproducible (,10% variation between two tests) responses were used. Prostaglandin F2a (PGF2a; 3 3 1026 M) was used as a contractile agent and evoked a stable level of tension; (11.6 6 l.1 mN). During this precontraction relaxant responses to acetylcholine and the different peptides were examined. In all studies, controls were run in parallel. To remove the endothelium, arterial segments were perfused during 20 s with the above buffer solution also containing 0.1% Triton X-100. Successful removal of the endothelium was always demonstrated by the absence of relaxation to acetylcholine (19). The in vitro data are expressed as pEC50 or pIC50 values (2log concentration of agonist eliciting half maximum contraction or relaxation, respectively) and as Emax or Imax (maximum contraction or relaxation, respectively). Data are presented as the means 6 standard error of the mean (SEM) of the responses obtained in a given number (n) of vessel segments, one or two from each patient. Solutions and drugs. The buffer solution used was of the following composition (mM): NaCl, 119; NaHCO3, 15; KCl, 4.6; CaCl2, l.5; MgCl2, l.2; NaH2PO4, l.2; glucose, 5.5. The potassium buffer was obtained by an equimolar substitution of NaCl for KCl, resulting in a potassium concentration of 60 mM. Pharmacological agents. Acetylcholine hydrochloride, norepinephrine hydrochloride (Sigma, USA), and prostaglandin F2a (AmoglandinR; Astra, Sweden) were diluted in 0.9% saline. Human a-CGRP, NKA, PHM-27 (CRB, Cambridge, U.K.), and human NPY, SP, VIP (Sigma, USA) were dissolved in 0.9% saline containing 1% bovine serum albumin (BSA) and 0. l mM ascorbic acid. Prazosin HCl (Fermion, Orion Corp. Ltd., Finland) was dissolved in methanol and 1 mM HCl, giving it a final concentration of
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FIG. 1. Cryostat section (a) and whole-mount preparations (b-j) of human middle meningeal artery immunostained for PGP (a-b), TH (c), NPY (d), CGRP (e), SP (f), NPK (g), VIP (h), and PHM (i). In j, perivascular nerve fibers display ACHE activity. The majority of the perivascular nerves mainly run parallel to the long axis of the vessel at the adventitialmedial border. The relative density for the different nerve populations was TH . NPY . CGRP 5 SP 5 NPK . VIP 5 PHM 5 ACHE. LA indicates longitudinal axis of the vessel. a, adventitia; m, medial layer. Magnification 3250.
0.1 mM, and then further diluted in 0.9% saline. BIBP 3226 (gift of: Dr. Karl Thomae AG, Germany); L-NMMA (Sigma); Indometacin: (Sigma). Statistics. Statistical analysis of the results was performed by using Kruskal–Wallis test (two-tailed) followed by Mann–Whitney U-test for comparison between each individual group. A probability value of 0.05 was accepted as significant for differences between groups of data.
RESULTS Morphology Light microscopical immunohistochemistry and histochemistry. Immunofluorescence staining with the antiserum to the general neuronal marker protein gene product 9.5 (PGP 9.5) showed that the human middle meningeal artery possesses a dense supply of nerve fibers in the adventitia and at
PERIVASCULAR NERVES AND ENDOTHELIUM IN MIDDLE MENINGEAL ARTERY
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FIG. 2. Whole-mount preparations of human middle meningeal artery. The co-localization (arrows) of SP with CGRP (a and b) as well as with neuropeptide K (NPK; c and d) was determined by double immunostaining of the same preparations. Magnification 3413.
the adventitial–medial border (Fig. 1a and b). Perivascular nerve fibers displayed an orientation that was mainly parallel to the long axis of the vessel (Fig. 1b). The majority of the nerve fibers displayed tyrosine hydroxylase (TH) and NPY immunoreactivity (Fig. 1c and d). The relative number and distribution of NPY-immunore-
active nerves was similar to that of nerves containing immunoreactivity for the catecholamine synthesizing enzyme TH (Fig. 1c and d). The middle meningeal artery was found to be supplied with a moderate number of nerve fibers containing CGRP (Fig. 1e), substance P (SP; Fig. 1f) and neuropeptide K (Fig. 1g).
FIG. 3. Cryostat section of a human middle meningeal artery immunostained for nitric oxide synthase (NOS-endothelial form). Endothelial cells display a strong immunofluorescence staining (arrows). LEI, Lamina elastica interna. Magnification, 3825.
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FIG. 4. Electron micrographs of nerve varicosities at the adventitial-medial border of the human middle meningeal artery. The cleft, between the varicosities and the smooth muscle (sm) membrane (double-headed arrow) is generally greater than 500 nm wide (a). Several axon profiles may be demonstrated containing numerous small, round and flattened agranular vesicles (a and b, arrows), small granular vesicles (c, arrows). These axon profiles also contained a few large granular vesicles (b and c, arrow heads). Putative sensory axon profiles were also observed (d-f). These contained an unusual abundance of mitochondria (d), pleomorphic dense bodies (d and e), autophagic vacuoles (d and e, asterisks), and large amounts of glycogen-like granules (f, asterisks. m, mitochondria; sm, smooth muscle. Magnification 328,000).
The use of the double immunofluorescence staining technique revealed that SP immunoreactivity was co-localized with neuropeptide K and CGRP immunoreactivities in the same varicosed nerve fibers (Fig. 2). Only a few scattered nerve fibers displaying immunore-
activity for VIP (Fig. 1h), PHM (Fig. 1i), and AChE activity (Fig. 1j) were observed. In marked contrast to the distribution of the neuropeptides, TH and AChE, nitric oxide synthase (NOS) was only detected in the cytoplasm of endothelial cells of the middle meningeal artery (Fig. 3).
PERIVASCULAR NERVES AND ENDOTHELIUM IN MIDDLE MENINGEAL ARTERY
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Electron Microscopy Electron microscopical examination of the human middle meningeal artery revealed the presence of numerous unmyelinated axons in the adventitia. At the adventitial–medial border, axon varicosities were separated from adjacent smooth muscle cell membranes by a cleft which generally was greater than 500 nm wide (Fig. 4a). Although axon varicosities represent a spectrum of profiles containing a heterogeneous vesicle population, several principal types were distinguishable. Axon profiles containing numerous small, round agranular vesicles (40 – 60 nm in diameter) (Fig. 4a and b) or small granular vesicles (40 – 60 nm in diameter; Fig. 4c) were found. These two types of axon profiles contained a few large granular vesicles (80 –150 nm in diameter). Putative sensory axon profiles were also detected. These contained an unusual abundance of mitochondria (Fig. 4d), autophagic vacuoles (Fig. 4e) and large amounts of glycogen-like granules (Fig. 4f). Functional Analysis Contractile responses. Norepinephrine (1029 –3 3 1025 M) and NPY caused concentration-dependent contractions of meningeal arteries (Fig. 5). The maximum amount of contraction (Emax) relative to the response of the vessel to 60 mM K1 was for norepinephrine 100 6 6% (n 5 10) and for NPY 69 6 26% (n 5 7) and the pEC50 values were 6.48 6 0.13 and 6.77 6 0.11, respectively. Removal of the endothelium did not change significantly the Emax (89 6 11%, n 5 13; 125 6 20%, n 5 9) or the pEC50 values (6.73 6 0.15; 6.79 6 0.11) during to cumulative application of norepinephrine or NPY, respectively, or of the contractile responses to 60 mM K1 or to PGF2a (data not shown). The a1-adrenoceptor antagonist prazosin (1029–1027 M) caused a significant shift of the concentration–response curve to norepinephrine toward higher norepinephrine concentrations, resulting in a pA2 value of 9.47 and with a slope of – 0.95 in the Schild plot (Fig. 6a and b). The contraction induced by NPY was blocked by the NPY Y1 antagonist BIBP 3226 (Fig. 6c and d). Construction of a Schild plot revealed a line with a slope of – 0.13. Since this significantly differed from a slope of –1, we did not calculate the pA2 value. Relaxant Responses The parasympathetic transmitters ACh, VIP, and PHM (Table 2) and the sensory peptides SP and CGRP (Table 3) induced concentration-dependent relaxations in PGF2a precontracted arteries. Removal of the endothelium significantly reduced the maximum amount of relaxation (Imax) of ACh from 58 6 12% (n 5 7) in arteries with endothelium to 35 6 9% (n 5 11) in arteries without endothelium (Fig. 7a). The responses to VIP and PHM were not altered by
FIG. 5. Contractile responses, in % of contraction induced by 60 mM K1 to norepinephrine (a) and NPY (b) in human middle meningeal arteries with and without endothelium. Values are given as SEM. Number of experiments was 5–7 from three to five patients.
removal of the endothelium (Fig. 7b and c). Removal of the endothelium significantly decreased the maximum amount of relaxation (Imax) to SP from 56 6 9% (n 5 5) in arteries with endothelium to 26 6 7% (n 5 6) in arteries without endothelium (Fig. 8a). The response to CGRP was not altered by endothelium removal (Fig. 8b). The muscarinic antagonist atropin (1028–1026 M) caused a significant shift of the concentration–response curve to ACh toward higher ACh concentrations, resulting in a pA2 value of 8.68 and with a slope in the Schild plot of –1.10 (Fig. 9a and b). The relaxant response to SP was blocked by the NK-1 antagonist RP 67580 (1027 to 1025 M). Construction of a Schild plot revealed a line with a slope of –1.28, and pA2-value of 7.26 (Fig. 10a and b).
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FIG. 6. Effect of prazosin 1029 –1027 M on norepinephrine-induced contraction (a) and BIBP 3226 1028 –1026 M on NPY induced contraction in human middle meningeal arteries (c). Schild plots are shown for the antagonistic effects of prazosin (b) and BIBP 3226 (d) on norepinephrine- and NPY-induced contractions, respectively. The slope of regression line was – 0.95 for prazosin of norepinephrine-induced contractions and – 0.13 for BIBP 3226 of NPY-induced contractions. The pA2 for prazosin was 9.47.
The relaxant response to CGRP was blocked by the CGRP1 receptor antagonist human a-CGRP8 –37 (1026 –3 3 10— 6 M). Construction of a Schild plot revealed a line with a slope of –1.07, and the pA2-value from this was 6.82 (Fig. 11a and b).
DISCUSSION Morphology The present study demonstrates that the human middle meningeal artery is richly innervated. Nerve varicosities present at the adventitial-medial border were found to lack pre- or post-synaptic membrane specializations, being sep-
TABLE 2 RELAXANT RESPONSES OF HUMAN MIDDLE MENINGEAL ARTERIES TO ACETYLCHOLINE, VASOACTIVE INTESTINAL PEPTIDE (VIP) AND PEPTIDE HISTIDINE METHIONINE (PHM-)
Acetylcholine VIP PHM
pIC50
Imax(%)
N
6.81 6 0.59 7.50 6 0.22 6.74 6 0.05
41 6 9 72 6 7 63 6 8
7 9 6
pIC50 5 the negative logarithm of agonist concentration (M) that elicits half maximum relaxation. Imax 5 maximum relaxant response. Data are presented as mean values 6 SEM. N 5 number of vessels examined.
TABLE 3 RELAXANT RESPONSES OF HUMAN MIDDLE MENINGEAL ARTERIES TO SUBSTANCE P AND CALCITONIN GENE-RELATED PEPTIDE (CGRP)
Substance P CGRP
pIC50
Imax(%)
N
9.61 6 0.25 7.42 6 0.33
56 6 9 102 6 11
5 5
pIC50 5 the negative logarithm of agonist concentration (M) that elicits half maximum relaxation. Imax 5 maximum relaxant response. Data are presented as mean values 6 SEM. N 5 number of vessels examined.
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FIG. 8. Relaxant responses, in % of precontraction induced by prostaglandin F2a, for substance P (a) and CGRP (b) in human middle meningeal arteries with and without endothelium. Values are given as SEM. Number of experiments was 5–7 from three to five patients.
FIG. 7. Relaxant responses, in % of precontraction induced by prostaglandin F2a, for acetylcholine (a), VIP (b), and PHM-27 (c) in human middle meningeal arteries with and without endothelium. Values are given as SEM. Number of experiments was 5–7 from three to five patients.
arated from the smooth muscle cells by a cleft which was usually several hundred nanometers wide. A spectrum of axon varicosity profiles was also identified at the ultrastructural level.
They included nerve profiles that contain, in addition to some large granular vesicles, populations of small round agranular or granular vesicles which are thought to correspond to cholinergic and noradrenergic terminals, respectively (3,25). Other axon profiles were interpreted as sensory because they displayed ultrastructural features similar to the ones described for presumed sensory or baroreceptor nerve terminals (18). These distinguishing features include an unusual abundance of mitochondria, autophagic vacuoles, pleomorphic dense bodies, and large amounts of glycogen-like granules. In the present study, it was observed that the human middle meningeal artery is supplied with numerous peptidecontaining nerve populations. The majority of the perivascular nerve fibers contain NPY and TH immunoreactivity and are thought to represent noradrenergic sympathetic neurones (11,32). The nerve supply of sensory fibers storing CGRP, SP, and neuropeptide K was found to be moderate.
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munoreactivity (neuronal form) were detected in the present study. The reason for the lack of NOS-immunoreactive fibers is not clear, but it may well reflect the limited distribution of NOS-containing fibers around the human middle meningeal artery and/or indicate possible qualitative differences in the antisera used. On the other hand, NOS immunoreactivity (endothelial form) was detected in the cytoplasm of the vascular endothelial cells. The present results correlate well with previous work done on cryostat sections of the same artery (30). However, the application of immunohistochemical techniques on whole-mount preparations of the middle meningeal artery has allowed us to analyse the relationship between different nerve subtypes in large tissue areas providing a more accurate picture of the overall vascular innervation than can be obtained with sections alone. The identity of the perivascular peptide-containing nerve fibers and quantitative levels in the human middle meningeal artery were verified in our previous study using radioimmunoassay and HPLC (30).
FIG. 9. Effect of atropine 1029 –1027 M on acetylcholine-induced relaxation in human middle meningeal arteries (a). In b, Schild plot for the antagonistic effects of atropine on acetylcholine-induced relaxations. The slope of regression line was –1.10, and the pA2 was 8.68.
It was also shown that SP and neuropeptide K co-exist with CGRP in the same nerve fibers. These observations are similar to results obtained in the human superficial temporal artery (29) and correlate well with the results of previous studies showing that tachykinins and CGRP coexist in capsaicin-sensitive neurones (22,48). VIP-, PHM-, and AChE-containing nerve fibers were comparatively sparse. Perivascular nerve fibers immunoreactive for VIP and PHM, two peptides derived from the same precursor molecule (24) are generally presumed to represent cholinergic neurones (17,20,43). It should be noted, however, that not all cholinergic nerve fibers may be positive for VIP immunoreactivity, or vice versa (37). NOS (known to label NO-containing neurones; References 41,46) has been reported to coexist with VIP in putative cholinergic nerve fibers supplying human cerebral vessels, however, no perivascular nerve fibers containing NOS im-
FIG. 10. Shows the effect of RP 67580 (1027 –1025 M) on substance P-induced relaxation in human middle meningeal arteries (a). In b, Schild plot for the antagonistic effects of RP 67580 on substance P-induced relaxations. The slope of regression line was –1.28, and the pA2 for was 7.26.
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activation of receptors located to the smooth muscle cells. The responses to CGRP and VIP have in previous studies been shown to act directly on smooth muscle cell receptors and cause activation of adenylyl cyclase (8). PHM is a neuropeptide with structural homology to VIP and is a putative vasodilatory transmitter. Only few studies have been performed investigating the effect and receptor actions of PHM in the cranial circulation (9). In the present study, PHM caused a concentration-dependent relaxation of the human meningeal artery. This has previously been observed shown by us in another study (30). The pD2 value obtained in the present study is close to that found previously. The relaxation induced by PHM was independent of the endothelium. PHM has also been shown to induce relaxation that is dependent on an intact endothelium in the human pulmonary artery (44). In some vascular preparations, CGRP is dependent on an intact endothelium to induce relaxation (2,16,31). However, in most other vascular areas the CGRP induced relaxation seems to be independent of an intact endothelium (8).
FIG. 11. Effect of human a-CGRP8 –37 (1026 –3 3 1026 M) on human a-CGRP-induced relaxation in human middle meningeal arteries (a). In b, Schild plot for the antagonistic effects of human a-CGRP8 –37 on CGRP-induced relaxations. The slope of regression line was –1.07, and the pA2 was 6.82.
Vasomotor Responses Endothelium removal. The present study shows that norepinephrine and NPY induced contraction of the middle meningeal artery is not dependent on an intact endothelium as there was no difference in their responses in arteries with and without endothelium. These data are in accordance to previous studies in other human arteries that have shown that norepinephrine- and NPY-induced contractions are not dependent on an intact endothelium (14,29,36). The relaxant responses to ACh and substance P were significantly reduced in arteries in which the endothelium had been removed beforehand. Thus, they seem to act on receptors located to the endothelium (19). This has previously been shown in cerebral arteries from monkey, cat, and guinea pig (8,27,45). Relaxations induced by VIP, PHM and CGRP were not changed in arteries without endothelium. Thus, they act via
Antagonist Experiments The contractions induced by norepinephrine were in the human middle meningeal artery blocked by the a1-adrenoceptor antagonist prazosin. The pA2 value (9.47) found for prazosin in the present study is comparable to pA2 values for the interaction between prazosin and a1 receptors in several tissues (1,21,38) which suggests the occurrence of postjunctional a1 receptors in human middle meningeal arteries. The NPY Y1 receptor antagonist BIBP 3226 has in previous studies been shown to cause a rightward parallel shift of the NPY concentration–response curve in human cerebral and subcutaneous arteries (39,40). Also in the human middle meningeal artery did BIBP 3226 cause a rightward parallel shift of the NPY concentration-response curve. However, construction of a Schild plot showed a slope of – 0.13, that was significantly different from 1. An explanation for this could be, that we at the highest BIBP 3226 concentration (1026 M) could not add NPY in concentrations that were high enough to induce a response equal to Emax in the control segments and that the pEC50 value obtained for NPY in the presence of this concentration of BIBP 3226; thus, is too low. RP 67580 is a selective antagonist of NK-1 receptors (13). In the human middle meningeal artery it induced a parallel rightward shift of the SP-induced relaxations with a pA2-value of 7.26. This pA2 value is very close to that found in other tissues (5,12,13), which suggests the occurrence of postjunctional NK-1 receptors in human middle meningeal arteries. The CGRP1 receptor antagonist induced a parallel rightward shift of the human a-CGRP induced relaxation of the human middle meningeal artery. The pA2 value of 6.84 is
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EDVINSSON ET AL.
similar to that found in the guinea pig basilar artery (26). However, at present few pharmacological tools are available for the characterization of CGRP receptors, and the potency of CGRP8 –37 is used in order to discriminate between CGRP1 and CGRP2 receptors. If the pA2-value is 7.0 or higher, the receptor is classified as a CGRP1 receptor, whereas it is regarded as a CGRP2 receptor (or a nonCGRP1 receptor) if the pA2 value is below 7.0. Still unpublished studies from our group have shown that the CGRP2 agonist Cys(ACM2,7)–CGRP only has a dilatory effect on the human middle meningeal artery in very high concentrations; thus, the CGRP receptor present in this tissue is not a CGRP2 receptor. The pA2 value 6.84 is not significantly different from 7.0, and the presence of a CGRP1 receptor can therefore not be excluded. Further evidence for the presence of a CGRP1 receptor in human cranial arteries was
recently published: The present studies provide the basic morphological and functional background for understanding of intracranial processes involving the dura mater. This is of particular importance for primary headache patophysiology and its treatment. Expression of CGRP1 receptor mRNA was seen in trigeminal ganglion and human cerebral arteries (7) as well as in human temporal and middle meningeal arteries (28). ACKNOWLEDGEMENTS This study was supported by the Swedish Medical Research Council (grant nos. 05958 and 11238), the Swedish Society of Medicine, the Novo Nordisk Foundation, the Danish Pharmacist Foundation of 1991, the Lundbeck Foundation, and the Danish Medical Research Council. We thank Mrs. M.R. Alpiarca and A. Homem for the excellent technical assistance. C.P. Barroso was supported by a fellowship from PRAXIS XXI (BD/3575/ 94).
REFERENCES 1. Andersson, K. E.; Larsson, B.; Sjo¨gren, C. Characterization of the alpha-adrenoceptors in the female rabbit urethra. Br. J. Pharmacol. 81:293–300; 1984. 2. Brain, S. D.; Williams, T. J.; Trippins, J. R.; Morris, H. R.; MacIntyre, I. Calcitonin gene-related peptide is a potent vasodilator. Nature (London). 313:54 –56; 1985. 3. Burnstock, G.; Chamley, J. H.; Campbell, G. R. The innervation of arteries. In: Schwartz, J.; Werhessen, N. T.; Wolf, S.; eds. Structure and function of the circulation, Vol I. New York: Plenum Publishing Corp.; 1980:729 –767. 4. Buzzi, M. G.; Moskowitz, M. A.; Peroutka, S. J.; Byun, B. Further characterization of the 5-HT, receptor mediating blockade of neurogenic plasma extravasation within dura mater. Br. J. Pharmacol. 103:1421–1428; 1991. 5. Carruette, A.; Moussaoui, S. M.; Champion, A.; Cottez, D.; Goniot, P.; Garret, C. Comparison in different tissue preparation of the in vitro pharmacological profile of RP 67580, a new non-peptide substance P antagonist. Neuropeptides. 23:245– 250; 1992. 6. Dimitriadou, V.; Buzzi, M. G.; Theoharides, T. C.; Moskowitz, M. A. Ultrastructural evidence for neurogenically mediated changes in blood vessels of the rat dura mater and tongue following antidromic trigeminal stimulation. Neuroscience. 48:187–203; 1992. 7. Edvinsson, L.; Cantera, L.; Jansen-Olesen, I.; Uddman, R. Expression of calcitonin gene-related peptide-1 receptor mRNA in human trigeminal ganglia and cerebral arteries. Neurosci. Lett. 229:209 –211; 1997. 8. Edvinsson, L.; Fredholm, B. B.; Hamel, E.; Jansen, I.; Verrecchia, C. Perivascular peptides relax cerebral arteries concomitant with stimulation of cyclic adenosine monophosphate accumulation or release of an endothelium-derived relaxing factor in the cat. Neurosci. Lett. 58:213–217; 1985. 9. Edvinsson, L.; McCulloch, J. Distribution and vasomotor effects of peptide HI (PHI) in feline cerebral blood vessels in vitro and in situ. Regul. Pept. 10:345–356; 1985. 10. Edvinsson, L.; Uddman, R. Adrenergic, choloinergic and peptidergic nerve fibres in dura mater of guinea pig. Cephalalgia. 1:175–179; 1981. 11. Ekblad, E.; Edvinsson, L.; Wahlestedt, C.; Uddman, R.; Håkanson, R; Sundler, F. Neuropeptide Y co-exists and cooperates with noradrenaline in perivascular nerve fibres. Regul. Pept. 8:225–235; 1984.
12. Floch, A.; Fardin, V.; Cavero, I. Characterization of NK1 and NK2 tachykinin receptors in guinea-pig and rat bronchopulmonary and vascular systems. Br. J. Pharmacol. 111–759-768; 1994. 13. Garrett, C; Carruette, A.; Fardin, V.; Moussaoui, S.; Peyronel, J. F.; Blanchard, J. C.; Laduron, P. M. Pharmacological properties of a potent and selective nonpeptide substance P antagonist. Proc. Natl. Acad. Sci. USA. 88:10208 –10212; 1991. 14. Gaw, A. J.; Wadsworth, R. M. Pharmacological characterization of postjunctional alpha-adrenoceptors in cerebral arteries from the sheep. Br. J. Pharmacol. 98:741–746; 1989. 15. Goadsby, P. J.; Edvinsson, L. Peripheral and central trigeminovascular activation in cat in blocked by the serotonin (5HT)1D receptor agonist 311C90. Headache. 34:394 –399; 1994. 16. Grace, G. C.; Dusting, G. J.; Kemp, B. E.; Martin, T. J. Endothelium and the vasodilator action of rat calcitonin generelated peptide (CGRP). Br. J. Pharmacol. 91:729 –733; 1987. 17. Gulbenkian, S.; Valenqa, A.; Wharton, J.; Polak, J. M.; DavidFerreira, J. F. Simultaneous visualization of neuropeptide and acetylcholinesterase nerve subpopulations in the perivascular nerve plexus. Histochem. J. 23:553–558; 1991. 18. Gulbenkian, S; Wharton, J. Pattern of innervation of sensory afferent systems in the circulation: immunocytochemistry and ultrastructure. In: Edvinsson, L.; Uddman, R.; eds. Vascular innervation and receptor mechanisms, Chapter 12. San Diego: Academic Press; 1993:247–262. 19. Hamel, E.; Assumel Lurdin, C.; Edvinsson, L.; Fage, D.; MacKenzie, E. T. Neuronal versus endothelial origin of vasoactive acetylcholine in pial vessels. Brain Res. 420:391–396; 1987. 20. Hara, H.; Hamill, G. S.; Jacobowitz, D. M. Origin of cholinergic nerves to the rat major cerebral arteries: coexistence with vasoactive intestinal polypeptide. Brain Res. 14:179 –188; 1985. 21. Ho¨gesta¨tt, E. D.; Andersson, K. E. On the postjunctional alpha-adrenoreceptors in rat cerebral and mesenteric arteries. J. Auton. Pharmacol. 4:161–173; 1984. 22. Hua, X.-Y.; Theodorsson-Norheim, E; Brodin, E.; Lundberg, J.; Ho¨kfelt, T. Multiple tachykinins (neurokinin A, neuropeptide K and substance P) in capsaicin sensitive neurones in the guinea pig. Regul. Pept. 13:1–19; 1985. 23. Humphrey, P. P. A.; Feniuk, W.; Perren, M. J.; Beresford,
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I. J. M.; Skingle, M. Serotonin and migraine. Ann. N.Y. Acad. Aci., 600:587–598; 1990. Itoh, N.; Obata, K.-I; Yanaihara, N.; Okamoto, H. Human prepro-vasoactive intestinal polypeptide contains novel PHI27-like peptide, PHM-27. Nature (London). 304:547–549; 1983. Iwayama, T.; Furness, J. B; Burnstock, G. Dual adrenergic and cholinergic innervation of the cerebral artery of the rat. An ultrastructural study. Circ. Res. 26:646 – 653; 1970. Jansen, I. Characterization of calcitonin gene-related peptide (CGRP) receptors in guinea pig basilar artery. Neuropeptides. 21:73–79; 1992. Jansen, I.; Alafaci, C.; McCulloch, J.; Uddman, R.; Edvinsson, L. Tachykinins (substance P, neurokinin A, neuropeptide K, and neurokinin B) in the cerebral circulation: vasomotor responses in vitro and in situ. J. Cereb. Blood Flow Metab. 11:567–575; 1991. Jansen-Olesen, I.; Gulbenkian, S.; Cantera, L.; Edvinsson, L. Evidence for calcitonin gene-related peptide-1 receptors in human cranial arteries. In: Olesen, J.; Edvinsson, L.; eds. Headache pathogenesis: Monoamines, neuropeptides, purines and nitric oxide. Philadelphia: Lippincott-Raven Publishers; 1997:229 –234. Jansen-Olesen, I.; Gulbenkian, S.; Valenca, A.; Antunes, J. L.; Wharton, J.; Polak, J. M.; Edvinsson, L. The peptidergic innervation of the human superficial temporal artery: immunohistochemistry, ultrastructure, and vasomotility. Peptides. 16:275–287; 1995. Jansen, I; Uddman, R.; Ekman, R.; Olesen, J.; Ottosson, A.; Edvinsson, L. Distribution and effects of neuropeptide Y, vasoactive intestinal peptide, substance P and calcitonin generelated peptide in human middle meningeal arteries: comparison with cerebral and temporal arteries. Peptides. 13:527– 536; 1992. Kubota, M.; Moseley, J. M.; Butera, L.; Dusting, G. J.; MacDonald, P. S., Martin, T. J. Calcitonin gene-related peptide stimulates cyclic AMP formation in rat aortic smooth muscle cells. Biochem. Biophys. Res. Commun. 132:88 –94; 1985. Lundberg, J. M.; Terenius, L.; Ho¨kfelt, T.; Martling, C. R.; Tatemoto, K.; Mutt, V.; Polak, J. M.; Bloom, S. R; Goldstein, M. Neuropeptide Y (NPY)-like immunoreactivity in peripheral noradrenergic neurones and effects of NPY on sympathetic function. Acta Physiol. Scand. 116:477– 480; 1982. Markowitz, S.; Saito, K.; Moskowitz, M. A. Neurogenically mediated leakage of plasma protein occurs from blood vessels in dura mater but not in brain. J. Neurosci. 7:4129 – 4136; 1987. Markowitz, S.; Saito, K.; Moskowitz, M. A. Neurogenically mediated plastma extravasation in dura mater: effect of ergot alkaloids. A possible mechanism of action in vascular headache. Cephalalgia. 8:83–91; 1988. Mayberg, M. R.; Zervas, N. T.; Moskowitz, M. A. Trigeminal projections to supratentorial pial and dural blood vessels in cat demonstrated by horseradish peroxidase histochemistry. J. Comp. Neurol. 223:46 –56; 1984.
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36. Mejia, J. A.; Pernow, J.; von Holst, H.; Rudehill, A.; Lundberg, J. M. Effects of neuropeptide Y, calcitonin gene-related peptide, substance P, and capsaicin on cerebral arteries in man and animals. J. Neurosurg. 69:913–918; 1988. 37. Miao, F. J.-P; Lee, T. J.-F. Cholinergic and VIPergic innervation in cerebral arteries: a sequential double-labeling immunohistochemical study. J. Cereb. Blood Flow Metab. 10:32– 37; 1990. 38. Minneman, K. P.; Fox, A. W.; Abel, P. W. Occupancy of alpha 1-adrenergic receptors and contraction of rat vas deferens. Mol. Pharmacol. 23:359 –368; 1983. 39. Nilsson, T.; Cantera, L.; Edvinsson, L. Presence of neuropeptide Y Y1 receptor mediating vasoconstriction in human cerebral arteries. Neurosci. Lett. 204:145–148; 1996a. 40. Nilsson, T.; Erlinge, D.; Cantera, L.; Edvinsson, L. Contractile effects of neuropeptide Y in human subcutaneous resistance arteries are mediated by Y1 receptors. J. Cardiovasc. Pharmacol. 28:764 –768; 1996b. 41. Nozaki, K.; Moskowitz, M. A.; Maynard, K.; Koketzu, N; Dawson, T. M.; Bredt, D. S.; Snyder, S. H. Possible origin and distribution of immunoreactive nitric oxide synthase-containing nerve fibers in cerebral arteries. J. Cereb. Blood Flow Metab. 13: 70 –79; 1993. 42. Ray, B. S; Wolff, H. G. Experimental studies on headaches; pain sensitive structures of the head and their significance in headaches. Arch. Surg. 41:813– 856; 1940. 43. Suzuki, N; Hardebo, J. E.; Owman, C. Origins and pathways of cholineacetyltransferase-positive parasympathetic nerve fibers to cerebral blood vessels in rat. J. Cereb. Blood Flow Metab. 10:399 – 408; 1990. 44. Thom, S.; Hughes, A.; Martin, G.; Sever, P. S. Endotheliumdependent relaxation in isolated human arteries and veins. Clin. Sci. 73:547–552; 1987. 45. Toda, N.; Kawakami, M.; Yamazaki, M.; Okamura, T. Comparison of endothelium-dependent responses of monkey cerebral and temporal arteries. Br. J. Pharmacol. 102:805– 810; 1991. 46. Tomimoto, H.; Ishimura, M.; Suenaga, T.; Nakamura, S.; Aldguchi, 1.; Waldta, H.; Kimura, J.; Mayer, B. Distribution of nitric oxide synthase in the human cerebral blood vessels and brain tissues. J. Cereb. Blood Flow Metab. 14:930 –938; 1994. 47. Uddman, R.; Edvinsson, L.; Hara, H. Axonal tracing of autonomic nerve fibers to the superficial temporal artery in the rat. Cell Tissue Res. 256:559 –565; 1989. 48. Wharton, J.; Gulbenkian, S.; Mulderry, P. K.; MacGregor, G. P.; Bloom, S. R; Polak, J. M. Capsaicin induces a depletion of calcitonin gene-related peptide (CGRP)-immunoreactive nerves in the cardiovascular system of the guinea pig and rat. J. Auton. Nerv. Syst. 16:535–542; 1986. 49. Zagami, Z.; Goadsby, P. J.; Edvinsson, L. Stimulation of the superior sagittal sinus in the cat causes release to vasoactive peptides. Neuropeptides. 16:69 –75; 1990.
PII S0196-9781(98)00066-7
Innervation of the Human Middle Meningeal Artery: Immunohistochemistry, Ultrastructure, and Role of Endothelium for Vasomotility LARS EDVINSSON,*1 SERGIO GULBENKIAN,† CARLA P. BARROSO,† MANUEL CUNHA E S´A,†‡ JULIA M. POLAK,§ ANDERS MORTENSEN,¶ LINDA JØRGENSENi AND INGER JANSEN-OLESEN# *Department of Internal Medicine, University Hospital of Lund, S-221 85 Lund, Sweden †Unit of Cell Morphology and Image Processing, Gulbenkian Institute of Science, 2781 Oeiras, Portugal ‡Department of Neurosurgery, Hospital Garcia de Orta, Almando, Portugal §Department of Histochemistry, Royal Postgraduate Medical School, Hammersmith Hospital, London W12 ONN, United Kingdom ¶Department of Neurosurgery, Copenhagen University, Glostrup Hospital, 2600 Copenhagen, Denmark iDepartment of Neurosurgery, Copenhagen University, Rigshospitalet, 2100 Copenhagen Ø, Denmark, Department of Biological Sciences, The Royal Danish School of Pharmacy, Universitetsparken 2, 2100 Copenhagen Ø, Denmark Received 30 December 1997; Accepted 13 April 1998 EDVINSSON, L., S. GULBENKIAN, C. P. BARROSO, M. CUNHA E SA´ , J. M. POLAK, A. MORTENSEN, L. JØRGENSEN AND I. JANSEN-OLESEN. Innervation of the human middle meningeal artery: Immunohistochemistry, ultrastructure and role of endothelium for vasomotility. PEPTIDES 19(7) 1213–1225, 1998.—The majority of nerve fibers in the middle meningeal artery and branching arterioles are sympathetic, storing norepinephrine and neuropeptide Y (NPY). A sparse supply of fibers contain acetylcholinesterase activity and immunoreactivity toward vasoactive intestinal peptide (VIP), peptidine histidine methionine (PHM), and calcitonin gene-related peptide (CGRP). Only few substance P and neuropeptide K immunoreactive fibers are noted. Electronmicroscopy shows axons and terminals at the adventitial medial border of the human middle meningeal artery, with a fairly large distance to the smooth muscle cells (.500 nM). Several axon profiles contain vesicles of different types, including putative sensory profiles. The perivascularly stored signal substances, norepinephrine and NPY induced vasoconstrictor. Relaxations were induced by acetylcholine and substance P, and these were significantly reduced in arteries without endothelium, while the responses to norepinephrine, NPY, VIP, PHM, and CGRP were not changed by endothelium removal. Blockade experiments showed that the vasomotor responses to norepinephrine were blocked by prazosin, to NPY by BIBP 3226, acetylcholine by atropin, substance P by RP 67580, and the human a-CGRP response by human a-CGRP8 –37. © 1998 Elsevier Science Inc. Neuropeptide Y Vasoactive intestinal peptide Calcitonin gene-related peptide Tachykinins Acetylcholinesterase Middle meningeal artery Immunohistochemistry Endothelium in vitro pharmacology
WOLFF and colleagues demonstrated more than fifty years ago that stimulation of vessels in the dura mater, such as the middle meningeal artery and the superior sagittal sinus (SSS), caused aching, throbbing ,and penetrating headaches as well as typical referred pain (42). This has served as one
important element in the current discussion of migraine pathogenesis with dural vessels currently held as one possible origin of the migraine pain. Stimulation of the SSS may produce activation of sensory neurons in the trigeminal nucleus caudalis and release of CGRP and VIP (15,49).
1 Requests for reprints should be addressed to Lars Edvinsson M.D., Ph.D., Department of lnternal Medicine, University Hospital of Lund S-221 85 Lund, Sweden. E-mail: [email protected]
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EDVINSSON ET AL. TABLE 1 SOURCE AND CHARACTERIZATION OF THE PRIMARY ANTISERA Antigen
Species
Code No.
Dilutions
Source
PGP 9.5 TH NPY VIP PHM SP SP NPK CGRP NOS endot NOS neuro
Rabbit Rabbit Rabbit Rabbit Rabbit Rabbit Rat Rabbit Rabbit Rabbit Rabbit
Ra95103 Tel01 1086 652 1655 910 MASO35b 15-36R2 1208 2431 2393
1:9600 1:1600 1:1200 1:4000 1:4000 1:1600 1:100 1:3000 1:1600 1:1000 1:1600
Ultraclone; UK Eugene Tech, USA Hammersmith Hosp. Hammersmith Hosp. Hammersmith Hosp. Hammersmith Hosp. Sera Lab, UK Dr. Valentino, USA Hammersmith Hosp. Hammersmith Hosp. Hammersmith Hosp.
PGP 9.5, protein gene product 9.5; TH, tyrosine hydroxylase; NPY, neuropeptide tyrosine; VIP, vasoactive intestinal polypeptide; PHM, peptide histidine methionine; SP, substance P; NPK, neuropeptide K; CGRP, calcitonin gene-related peptide; NOS endot, nitric oxide synthase (endothelial form); NOS neuro, nitric oxide synthase (neuronal form). Characterization of the antisera: (17,18,29,48).
Stimulation of the trigeminal ganglion in rodents elicits a neurogenic inflammation, which is a complex process characterized by vasodilatation, plasma extravasation, endothelium activation, and mast cell degranulation in the dura mater but not in the central nervous system (CNS; References 6,33,34). This process can be inhibited by antimigraine drugs. The mechanisms involved are not clear in detail but putatively pre- and post-synaptic receptor mechanisms are involved, mediating both presynaptic inhibition of neuropeptide release (4,15) and cranial vasoconstriction (23). Neuroanatomical tracing studies from the meningeal in rat (47) and in cat (35) have revealed projections of sympathetic fibers from the superior cervical ganglion, parasympathetic fibers from the sphenopalatine and otic ganglia, and sensory fibers from the first division of the trigeminal ganglion. Histochemical studies in laboratory animals early revealed a moderate supply of perivascular fibers in the dura mater (10), whereas the detailed innervation of the dura mater in man remains to be characterized (30). The present study aims at examining in detail the perivascular nerve supply of the human middle meningeal artery, removed during neurosurgical operations, using immunocytochemistry and electron microscopy. In addition, the vasomotor responses of ring segments of the human middle meningeal artery to stored neurotransmitters and the role of the endothelium for the responses were studied using a sensitive in vitro system. METHOD
16 –24 h at 4°C in a solution containing 85 ml of 2% (w/v) paraformaldehyde in 0.1 M phosphate buffer (pH 7.2) and 15 ml of saturated picric acid per 100 ml of fixative. The tissue was rinsed in several changes of phosphate-buffered saline (PBS; 0.01 M, pH 7.2) containing 15% (w/v) sucrose and 0. l% (w/v) sodium azide, then processed for cryostat sectioning or whole-mount preparations. Cryostat section (14-mm thick) were immunostained using an biotin–streptavidin–fluorescence method. A modified biotin—streptavidin–fluorescence method was performed on whole-mount preparations of the arteries. 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 (BDH, Poole, UK) for 30 min to reduce background fluorescence, the whole-mount preparations were incubated in diluted primary antisera (Table 1) overnight at room temperature. The preparations were then washed in PBS, incubated in biotinylated goat anti-rabbit IgG (1:200 dilution; Sigma Chemical Co., St Louis, MO) for l h at room temperature rinsed in PBS, and incubated with fluorescein isothiocyanate-labeled streptavidin (l:100 dilution; Sigma) for l h at room temperature. For the simultaneous localization of two antigens, whole-mount preparations were first exposed to a primary antiserum raised in rabbit that was visualized by a rhodamine-labeled goat anti-rabbit IgG (1:100 dilution; Sigma) and then to a second primary antiserum raised in rat that was visualized by a fluorescein isothiocyanate labeled goat anti rat IgG (l:100 dilution; Sigma). The preparations were finally examined using an Olympus BH-2 microscope equipped for epi-illumination with filters selective for fluorescein and rhodamine fluorescence.
Immunohistochemistry Middle meningeal arteries were obtained from six patients during neurosurgical tumor resections. Immediately after excision, vessel segments were fixed by immersion for
Acetylcholinesterase (AChE) staining The histochemical demonstration of acetylcholinesterase (AChE) activity in whole-mount preparations was per-
PERIVASCULAR NERVES AND ENDOTHELIUM IN MIDDLE MENINGEAL ARTERY
formed as previously described (17). Briefly, whole-mount preparations were immersed in incubation medium (1 mg acetylcholine iodide, l.263 ml of 0. l M sodium acetate, 0.04 ml of 0. l M acetic acid, 0.096 ml of 0. l M sodium citrate, 0.2 ml 30 of mM cupric sulfate, 0.2 ml 5 mM potassium ferricyanide, and 38.2 ml of distilled water) for 30 min at 37°C. After rinsing in distilled water, the AChE activity was visualized by immersing the preparations for 5 min in Tris-HCl buffer (50 mM, pH 7.6) containing 0.04% 3,39diaminobenzidine tetrahydrochloride and 0.3% nickel ammonium sulphate and then for a further 5–10 min with the addition of 0.003% hydrogen peroxide. After a brief wash in distilled water, whole-mount preparations were mounted on glass slides, dehydrated in an ascending series of acetone concentrations, cleared in xylene, and mounted in DPX. The preparations were finally observed and photographed with an Olympus BH-2 microscope using the transmitted bright field illumination. Conventional Electron Microscopy Vessel segments were immersed in 2.5% glutaraldehyde (v/v) in 0. l M phosphate buffer (pH 7.2) for 2 h at 4°C. Arteries were then washed in buffer containing 0. l M sucrose, postfixed in l % osmium tetroxide for l h at 40°C, rinsed in buffer containing 0. l M sucrose, dehydrated in a graded series of ethanol concentrations, cleared in propylene oxide, and infiltrated with Epon resin. Ultrathin sections of silver interference color (70 –90 nm) were collected onto 300-mesh formvar-coated grids. Grid-mounted sections were finally counterstained with uranyl acetate and lead citrate and were examined and photographed using a Jeol 100 CX electron microscope operating at 8OkV. Antisera The antisera used in this study are listed in Table l. The polyclonal (code 910) and monoclonal (code MAS 035b) antisera raised against SP 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 SP. Immunohistochemical and Histochemical Controls In control experiments, no immunostaining was observed when one primary antiserum (Table 1) was omitted, replaced with nonimmune serum, or reabsorbed with the corresponding antigens (1025 –1026 M) for 24 h at 4°C. In double-immunofluorescence staining experiments, labeled secondary antisera exhibited no cross-reactivity with IgG from inappropriate species. For the demonstration of AChE activity, the following control incubation media were used: 1) 1024 –1026 M tetraisopropyl-pyrophospharamine (isoOMPA; Sigma) was added to the incubation medium as an inhibitor for non-
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specific cholinesterases. (2) Preparations were incubated in a substrate-free medium omitting acetylthiocholine. Vasomotor Responses in vitro Middle meningeal arteries were obtained from 26 patients during neurosurgical tumor resections. Immediately after excision, vessel segments were immersed in aerated (5% CO2 in O2) buffer solution (4°C) and transported to the laboratory for studies of vasomotor reactivity. Two- to three-mm long ring segments of the artery were suspended between two L-shaped metal prongs (0.2 mm) in small tissue baths containing a buffer solution aerated with 5% CO2 and 95% O2. The vessels were given a passive load of 2– 4 mN depending on the vessel size and allowed to stabilize at this level of tension for an equilibration period of l.5 h before isometric circular contractions were recorded. The contractile capacity of each preparation was first tested by exposure to a buffer solution containing 60 mM potassium; this resulted in strong contractions of middle meningeal arteries; 23.0 6 2.0 mN (n 5 49). Only vessels showing reasonably strong (.1 mN) and reproducible (,10% variation between two tests) responses were used. Prostaglandin F2a (PGF2a; 3 3 1026 M) was used as a contractile agent and evoked a stable level of tension; (11.6 6 l.1 mN). During this precontraction relaxant responses to acetylcholine and the different peptides were examined. In all studies, controls were run in parallel. To remove the endothelium, arterial segments were perfused during 20 s with the above buffer solution also containing 0.1% Triton X-100. Successful removal of the endothelium was always demonstrated by the absence of relaxation to acetylcholine (19). The in vitro data are expressed as pEC50 or pIC50 values (2log concentration of agonist eliciting half maximum contraction or relaxation, respectively) and as Emax or Imax (maximum contraction or relaxation, respectively). Data are presented as the means 6 standard error of the mean (SEM) of the responses obtained in a given number (n) of vessel segments, one or two from each patient. Solutions and drugs. The buffer solution used was of the following composition (mM): NaCl, 119; NaHCO3, 15; KCl, 4.6; CaCl2, l.5; MgCl2, l.2; NaH2PO4, l.2; glucose, 5.5. The potassium buffer was obtained by an equimolar substitution of NaCl for KCl, resulting in a potassium concentration of 60 mM. Pharmacological agents. Acetylcholine hydrochloride, norepinephrine hydrochloride (Sigma, USA), and prostaglandin F2a (AmoglandinR; Astra, Sweden) were diluted in 0.9% saline. Human a-CGRP, NKA, PHM-27 (CRB, Cambridge, U.K.), and human NPY, SP, VIP (Sigma, USA) were dissolved in 0.9% saline containing 1% bovine serum albumin (BSA) and 0. l mM ascorbic acid. Prazosin HCl (Fermion, Orion Corp. Ltd., Finland) was dissolved in methanol and 1 mM HCl, giving it a final concentration of
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FIG. 1. Cryostat section (a) and whole-mount preparations (b-j) of human middle meningeal artery immunostained for PGP (a-b), TH (c), NPY (d), CGRP (e), SP (f), NPK (g), VIP (h), and PHM (i). In j, perivascular nerve fibers display ACHE activity. The majority of the perivascular nerves mainly run parallel to the long axis of the vessel at the adventitialmedial border. The relative density for the different nerve populations was TH . NPY . CGRP 5 SP 5 NPK . VIP 5 PHM 5 ACHE. LA indicates longitudinal axis of the vessel. a, adventitia; m, medial layer. Magnification 3250.
0.1 mM, and then further diluted in 0.9% saline. BIBP 3226 (gift of: Dr. Karl Thomae AG, Germany); L-NMMA (Sigma); Indometacin: (Sigma). Statistics. Statistical analysis of the results was performed by using Kruskal–Wallis test (two-tailed) followed by Mann–Whitney U-test for comparison between each individual group. A probability value of 0.05 was accepted as significant for differences between groups of data.
RESULTS Morphology Light microscopical immunohistochemistry and histochemistry. Immunofluorescence staining with the antiserum to the general neuronal marker protein gene product 9.5 (PGP 9.5) showed that the human middle meningeal artery possesses a dense supply of nerve fibers in the adventitia and at
PERIVASCULAR NERVES AND ENDOTHELIUM IN MIDDLE MENINGEAL ARTERY
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FIG. 2. Whole-mount preparations of human middle meningeal artery. The co-localization (arrows) of SP with CGRP (a and b) as well as with neuropeptide K (NPK; c and d) was determined by double immunostaining of the same preparations. Magnification 3413.
the adventitial–medial border (Fig. 1a and b). Perivascular nerve fibers displayed an orientation that was mainly parallel to the long axis of the vessel (Fig. 1b). The majority of the nerve fibers displayed tyrosine hydroxylase (TH) and NPY immunoreactivity (Fig. 1c and d). The relative number and distribution of NPY-immunore-
active nerves was similar to that of nerves containing immunoreactivity for the catecholamine synthesizing enzyme TH (Fig. 1c and d). The middle meningeal artery was found to be supplied with a moderate number of nerve fibers containing CGRP (Fig. 1e), substance P (SP; Fig. 1f) and neuropeptide K (Fig. 1g).
FIG. 3. Cryostat section of a human middle meningeal artery immunostained for nitric oxide synthase (NOS-endothelial form). Endothelial cells display a strong immunofluorescence staining (arrows). LEI, Lamina elastica interna. Magnification, 3825.
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EDVINSSON ET AL.
FIG. 4. Electron micrographs of nerve varicosities at the adventitial-medial border of the human middle meningeal artery. The cleft, between the varicosities and the smooth muscle (sm) membrane (double-headed arrow) is generally greater than 500 nm wide (a). Several axon profiles may be demonstrated containing numerous small, round and flattened agranular vesicles (a and b, arrows), small granular vesicles (c, arrows). These axon profiles also contained a few large granular vesicles (b and c, arrow heads). Putative sensory axon profiles were also observed (d-f). These contained an unusual abundance of mitochondria (d), pleomorphic dense bodies (d and e), autophagic vacuoles (d and e, asterisks), and large amounts of glycogen-like granules (f, asterisks. m, mitochondria; sm, smooth muscle. Magnification 328,000).
The use of the double immunofluorescence staining technique revealed that SP immunoreactivity was co-localized with neuropeptide K and CGRP immunoreactivities in the same varicosed nerve fibers (Fig. 2). Only a few scattered nerve fibers displaying immunore-
activity for VIP (Fig. 1h), PHM (Fig. 1i), and AChE activity (Fig. 1j) were observed. In marked contrast to the distribution of the neuropeptides, TH and AChE, nitric oxide synthase (NOS) was only detected in the cytoplasm of endothelial cells of the middle meningeal artery (Fig. 3).
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Electron Microscopy Electron microscopical examination of the human middle meningeal artery revealed the presence of numerous unmyelinated axons in the adventitia. At the adventitial–medial border, axon varicosities were separated from adjacent smooth muscle cell membranes by a cleft which generally was greater than 500 nm wide (Fig. 4a). Although axon varicosities represent a spectrum of profiles containing a heterogeneous vesicle population, several principal types were distinguishable. Axon profiles containing numerous small, round agranular vesicles (40 – 60 nm in diameter) (Fig. 4a and b) or small granular vesicles (40 – 60 nm in diameter; Fig. 4c) were found. These two types of axon profiles contained a few large granular vesicles (80 –150 nm in diameter). Putative sensory axon profiles were also detected. These contained an unusual abundance of mitochondria (Fig. 4d), autophagic vacuoles (Fig. 4e) and large amounts of glycogen-like granules (Fig. 4f). Functional Analysis Contractile responses. Norepinephrine (1029 –3 3 1025 M) and NPY caused concentration-dependent contractions of meningeal arteries (Fig. 5). The maximum amount of contraction (Emax) relative to the response of the vessel to 60 mM K1 was for norepinephrine 100 6 6% (n 5 10) and for NPY 69 6 26% (n 5 7) and the pEC50 values were 6.48 6 0.13 and 6.77 6 0.11, respectively. Removal of the endothelium did not change significantly the Emax (89 6 11%, n 5 13; 125 6 20%, n 5 9) or the pEC50 values (6.73 6 0.15; 6.79 6 0.11) during to cumulative application of norepinephrine or NPY, respectively, or of the contractile responses to 60 mM K1 or to PGF2a (data not shown). The a1-adrenoceptor antagonist prazosin (1029–1027 M) caused a significant shift of the concentration–response curve to norepinephrine toward higher norepinephrine concentrations, resulting in a pA2 value of 9.47 and with a slope of – 0.95 in the Schild plot (Fig. 6a and b). The contraction induced by NPY was blocked by the NPY Y1 antagonist BIBP 3226 (Fig. 6c and d). Construction of a Schild plot revealed a line with a slope of – 0.13. Since this significantly differed from a slope of –1, we did not calculate the pA2 value. Relaxant Responses The parasympathetic transmitters ACh, VIP, and PHM (Table 2) and the sensory peptides SP and CGRP (Table 3) induced concentration-dependent relaxations in PGF2a precontracted arteries. Removal of the endothelium significantly reduced the maximum amount of relaxation (Imax) of ACh from 58 6 12% (n 5 7) in arteries with endothelium to 35 6 9% (n 5 11) in arteries without endothelium (Fig. 7a). The responses to VIP and PHM were not altered by
FIG. 5. Contractile responses, in % of contraction induced by 60 mM K1 to norepinephrine (a) and NPY (b) in human middle meningeal arteries with and without endothelium. Values are given as SEM. Number of experiments was 5–7 from three to five patients.
removal of the endothelium (Fig. 7b and c). Removal of the endothelium significantly decreased the maximum amount of relaxation (Imax) to SP from 56 6 9% (n 5 5) in arteries with endothelium to 26 6 7% (n 5 6) in arteries without endothelium (Fig. 8a). The response to CGRP was not altered by endothelium removal (Fig. 8b). The muscarinic antagonist atropin (1028–1026 M) caused a significant shift of the concentration–response curve to ACh toward higher ACh concentrations, resulting in a pA2 value of 8.68 and with a slope in the Schild plot of –1.10 (Fig. 9a and b). The relaxant response to SP was blocked by the NK-1 antagonist RP 67580 (1027 to 1025 M). Construction of a Schild plot revealed a line with a slope of –1.28, and pA2-value of 7.26 (Fig. 10a and b).
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FIG. 6. Effect of prazosin 1029 –1027 M on norepinephrine-induced contraction (a) and BIBP 3226 1028 –1026 M on NPY induced contraction in human middle meningeal arteries (c). Schild plots are shown for the antagonistic effects of prazosin (b) and BIBP 3226 (d) on norepinephrine- and NPY-induced contractions, respectively. The slope of regression line was – 0.95 for prazosin of norepinephrine-induced contractions and – 0.13 for BIBP 3226 of NPY-induced contractions. The pA2 for prazosin was 9.47.
The relaxant response to CGRP was blocked by the CGRP1 receptor antagonist human a-CGRP8 –37 (1026 –3 3 10— 6 M). Construction of a Schild plot revealed a line with a slope of –1.07, and the pA2-value from this was 6.82 (Fig. 11a and b).
DISCUSSION Morphology The present study demonstrates that the human middle meningeal artery is richly innervated. Nerve varicosities present at the adventitial-medial border were found to lack pre- or post-synaptic membrane specializations, being sep-
TABLE 2 RELAXANT RESPONSES OF HUMAN MIDDLE MENINGEAL ARTERIES TO ACETYLCHOLINE, VASOACTIVE INTESTINAL PEPTIDE (VIP) AND PEPTIDE HISTIDINE METHIONINE (PHM-)
Acetylcholine VIP PHM
pIC50
Imax(%)
N
6.81 6 0.59 7.50 6 0.22 6.74 6 0.05
41 6 9 72 6 7 63 6 8
7 9 6
pIC50 5 the negative logarithm of agonist concentration (M) that elicits half maximum relaxation. Imax 5 maximum relaxant response. Data are presented as mean values 6 SEM. N 5 number of vessels examined.
TABLE 3 RELAXANT RESPONSES OF HUMAN MIDDLE MENINGEAL ARTERIES TO SUBSTANCE P AND CALCITONIN GENE-RELATED PEPTIDE (CGRP)
Substance P CGRP
pIC50
Imax(%)
N
9.61 6 0.25 7.42 6 0.33
56 6 9 102 6 11
5 5
pIC50 5 the negative logarithm of agonist concentration (M) that elicits half maximum relaxation. Imax 5 maximum relaxant response. Data are presented as mean values 6 SEM. N 5 number of vessels examined.
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FIG. 8. Relaxant responses, in % of precontraction induced by prostaglandin F2a, for substance P (a) and CGRP (b) in human middle meningeal arteries with and without endothelium. Values are given as SEM. Number of experiments was 5–7 from three to five patients.
FIG. 7. Relaxant responses, in % of precontraction induced by prostaglandin F2a, for acetylcholine (a), VIP (b), and PHM-27 (c) in human middle meningeal arteries with and without endothelium. Values are given as SEM. Number of experiments was 5–7 from three to five patients.
arated from the smooth muscle cells by a cleft which was usually several hundred nanometers wide. A spectrum of axon varicosity profiles was also identified at the ultrastructural level.
They included nerve profiles that contain, in addition to some large granular vesicles, populations of small round agranular or granular vesicles which are thought to correspond to cholinergic and noradrenergic terminals, respectively (3,25). Other axon profiles were interpreted as sensory because they displayed ultrastructural features similar to the ones described for presumed sensory or baroreceptor nerve terminals (18). These distinguishing features include an unusual abundance of mitochondria, autophagic vacuoles, pleomorphic dense bodies, and large amounts of glycogen-like granules. In the present study, it was observed that the human middle meningeal artery is supplied with numerous peptidecontaining nerve populations. The majority of the perivascular nerve fibers contain NPY and TH immunoreactivity and are thought to represent noradrenergic sympathetic neurones (11,32). The nerve supply of sensory fibers storing CGRP, SP, and neuropeptide K was found to be moderate.
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munoreactivity (neuronal form) were detected in the present study. The reason for the lack of NOS-immunoreactive fibers is not clear, but it may well reflect the limited distribution of NOS-containing fibers around the human middle meningeal artery and/or indicate possible qualitative differences in the antisera used. On the other hand, NOS immunoreactivity (endothelial form) was detected in the cytoplasm of the vascular endothelial cells. The present results correlate well with previous work done on cryostat sections of the same artery (30). However, the application of immunohistochemical techniques on whole-mount preparations of the middle meningeal artery has allowed us to analyse the relationship between different nerve subtypes in large tissue areas providing a more accurate picture of the overall vascular innervation than can be obtained with sections alone. The identity of the perivascular peptide-containing nerve fibers and quantitative levels in the human middle meningeal artery were verified in our previous study using radioimmunoassay and HPLC (30).
FIG. 9. Effect of atropine 1029 –1027 M on acetylcholine-induced relaxation in human middle meningeal arteries (a). In b, Schild plot for the antagonistic effects of atropine on acetylcholine-induced relaxations. The slope of regression line was –1.10, and the pA2 was 8.68.
It was also shown that SP and neuropeptide K co-exist with CGRP in the same nerve fibers. These observations are similar to results obtained in the human superficial temporal artery (29) and correlate well with the results of previous studies showing that tachykinins and CGRP coexist in capsaicin-sensitive neurones (22,48). VIP-, PHM-, and AChE-containing nerve fibers were comparatively sparse. Perivascular nerve fibers immunoreactive for VIP and PHM, two peptides derived from the same precursor molecule (24) are generally presumed to represent cholinergic neurones (17,20,43). It should be noted, however, that not all cholinergic nerve fibers may be positive for VIP immunoreactivity, or vice versa (37). NOS (known to label NO-containing neurones; References 41,46) has been reported to coexist with VIP in putative cholinergic nerve fibers supplying human cerebral vessels, however, no perivascular nerve fibers containing NOS im-
FIG. 10. Shows the effect of RP 67580 (1027 –1025 M) on substance P-induced relaxation in human middle meningeal arteries (a). In b, Schild plot for the antagonistic effects of RP 67580 on substance P-induced relaxations. The slope of regression line was –1.28, and the pA2 for was 7.26.
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activation of receptors located to the smooth muscle cells. The responses to CGRP and VIP have in previous studies been shown to act directly on smooth muscle cell receptors and cause activation of adenylyl cyclase (8). PHM is a neuropeptide with structural homology to VIP and is a putative vasodilatory transmitter. Only few studies have been performed investigating the effect and receptor actions of PHM in the cranial circulation (9). In the present study, PHM caused a concentration-dependent relaxation of the human meningeal artery. This has previously been observed shown by us in another study (30). The pD2 value obtained in the present study is close to that found previously. The relaxation induced by PHM was independent of the endothelium. PHM has also been shown to induce relaxation that is dependent on an intact endothelium in the human pulmonary artery (44). In some vascular preparations, CGRP is dependent on an intact endothelium to induce relaxation (2,16,31). However, in most other vascular areas the CGRP induced relaxation seems to be independent of an intact endothelium (8).
FIG. 11. Effect of human a-CGRP8 –37 (1026 –3 3 1026 M) on human a-CGRP-induced relaxation in human middle meningeal arteries (a). In b, Schild plot for the antagonistic effects of human a-CGRP8 –37 on CGRP-induced relaxations. The slope of regression line was –1.07, and the pA2 was 6.82.
Vasomotor Responses Endothelium removal. The present study shows that norepinephrine and NPY induced contraction of the middle meningeal artery is not dependent on an intact endothelium as there was no difference in their responses in arteries with and without endothelium. These data are in accordance to previous studies in other human arteries that have shown that norepinephrine- and NPY-induced contractions are not dependent on an intact endothelium (14,29,36). The relaxant responses to ACh and substance P were significantly reduced in arteries in which the endothelium had been removed beforehand. Thus, they seem to act on receptors located to the endothelium (19). This has previously been shown in cerebral arteries from monkey, cat, and guinea pig (8,27,45). Relaxations induced by VIP, PHM and CGRP were not changed in arteries without endothelium. Thus, they act via
Antagonist Experiments The contractions induced by norepinephrine were in the human middle meningeal artery blocked by the a1-adrenoceptor antagonist prazosin. The pA2 value (9.47) found for prazosin in the present study is comparable to pA2 values for the interaction between prazosin and a1 receptors in several tissues (1,21,38) which suggests the occurrence of postjunctional a1 receptors in human middle meningeal arteries. The NPY Y1 receptor antagonist BIBP 3226 has in previous studies been shown to cause a rightward parallel shift of the NPY concentration–response curve in human cerebral and subcutaneous arteries (39,40). Also in the human middle meningeal artery did BIBP 3226 cause a rightward parallel shift of the NPY concentration-response curve. However, construction of a Schild plot showed a slope of – 0.13, that was significantly different from 1. An explanation for this could be, that we at the highest BIBP 3226 concentration (1026 M) could not add NPY in concentrations that were high enough to induce a response equal to Emax in the control segments and that the pEC50 value obtained for NPY in the presence of this concentration of BIBP 3226; thus, is too low. RP 67580 is a selective antagonist of NK-1 receptors (13). In the human middle meningeal artery it induced a parallel rightward shift of the SP-induced relaxations with a pA2-value of 7.26. This pA2 value is very close to that found in other tissues (5,12,13), which suggests the occurrence of postjunctional NK-1 receptors in human middle meningeal arteries. The CGRP1 receptor antagonist induced a parallel rightward shift of the human a-CGRP induced relaxation of the human middle meningeal artery. The pA2 value of 6.84 is
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similar to that found in the guinea pig basilar artery (26). However, at present few pharmacological tools are available for the characterization of CGRP receptors, and the potency of CGRP8 –37 is used in order to discriminate between CGRP1 and CGRP2 receptors. If the pA2-value is 7.0 or higher, the receptor is classified as a CGRP1 receptor, whereas it is regarded as a CGRP2 receptor (or a nonCGRP1 receptor) if the pA2 value is below 7.0. Still unpublished studies from our group have shown that the CGRP2 agonist Cys(ACM2,7)–CGRP only has a dilatory effect on the human middle meningeal artery in very high concentrations; thus, the CGRP receptor present in this tissue is not a CGRP2 receptor. The pA2 value 6.84 is not significantly different from 7.0, and the presence of a CGRP1 receptor can therefore not be excluded. Further evidence for the presence of a CGRP1 receptor in human cranial arteries was
recently published: The present studies provide the basic morphological and functional background for understanding of intracranial processes involving the dura mater. This is of particular importance for primary headache patophysiology and its treatment. Expression of CGRP1 receptor mRNA was seen in trigeminal ganglion and human cerebral arteries (7) as well as in human temporal and middle meningeal arteries (28). ACKNOWLEDGEMENTS This study was supported by the Swedish Medical Research Council (grant nos. 05958 and 11238), the Swedish Society of Medicine, the Novo Nordisk Foundation, the Danish Pharmacist Foundation of 1991, the Lundbeck Foundation, and the Danish Medical Research Council. We thank Mrs. M.R. Alpiarca and A. Homem for the excellent technical assistance. C.P. Barroso was supported by a fellowship from PRAXIS XXI (BD/3575/ 94).
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