European Journal of Pharmacology, 192 (1991) 123-132 © 1991 Elsevier Science Publishers B.V. (Biomedical Division) 0014-2999/91/$03.50 ADONIS 001429999100077L
123
EJP 51635
Autoradiographic localization of dopamine receptors in rat cerebral blood vessels F. A m e n t a a,2, A. Ricci 2 a n d J.A. V e g a 3 1 Dipartimento di Sanitdt Pubblica e Biologia Cellulare, Universit& "Tor Vergata', Rome, Italy, e Dipartimento di Scienze Cardooascolari, 'La Sapienza; Rome, Italy and 3 Departamento de Morfologia y Biologia Celular, Universidad de Ooiedo, Oviedo, Spain Received 31 August 1990, accepted 25 September 1990
Combined in vitro radioreceptor binding and autoradiographic techniques were used to analyze the pharmacological profile and the anatomical localization of dopamine (DA) DA 1 and DA 2 receptor sites in the arteries and arterioles of the pial-arachnoid membrane in the rat. [3H]SCH 23390, used as a ligand of DA1 receptors, was bound by sections of rat cerebral vessels in a manner consistent with the labehng of DA1 receptors, with a value of 2.2 nM for the dissociation constant (Kd) and of 68.4 fmol/mg protein for the density of binding sites (Bmax). The ligand was bound primarily by the medial layer of cerebral arteries. The density of silver grains developed after exposure of cerebral vessel sections to [3H]SCH 23390 was unchanged after chemical sympathectomy with 6-hydroxydopamine (6-OHDA) and was not significantly different in either the circle of Willis arteries or in the pial-arachnoid arteries and arterioles. [3H]Spiroperidol was used as ligand of DA2 receptors in the presence of ketanserin to block the possible binding to 5-HT2 receptors. [3H]Spiroperidol was bound by sections of rat cerebral vessels in a manner consistent with labehng of DA 2 receptors, with K d and Bm~ values of 1.9 nM and 39.8 fmol/mg protein, respectively. The ligand was bound primarily by the adventitia and the adventitial-medial border as well as by the intimal layer of cerebral arteries. The accumulation of adventitial and adventitial-medial silver grains developed after exposure of cerebral vessels sections to [3H]spiroperidol did not occur in 6-OHDA-treated animals. In contrast, chemical sympathectomy was without effect on the density of intimal silver grains. The density of adventitial silver grains was higher in the anterior than in the posterior circle of Willis and pial-arachnoid arteries and arterioles, as well as in the pial-arachnoid arteries and arterioles than in circle of Willis arteries. These findings indicate the existence of both D A 1 and DA 2 receptor sites in the rat cerebrovascular tree. Dopamine DA 1 receptors; Dopamine DA 2 receptors; Circle of Wilhs arteries; Pial-arachnoid arteries; Radioreceptor binding; Autoradiography
I. Introduction A possible role of dopamine (DA) in the control of cerebrovascular circulation was suggested by several physiological and pharmacological observations. In vitro experiments have shown that DA, after a- and fl-adrenoceptor blockade, mediates relaxation of smooth muscle of major cerebral arteries (e.g. internal carotid, middle cerebral artery and basilar artery). These effects are mimicked by dopaminergic agonists such as apomorphine, 6,7-ADTN, epinine and S K & F 38393 and antagonized by bulbocapnine, (+)-butaclamol, cis-aflupentixol, fluphenazine and haloperidol, suggesting that they are due to the interaction with specific D A receptors (Toda et al., 1976; Boullin et ai., 1977; 1978; Oudart et al., 1981; Edvinsson et al., 1982; Forster et al,, 1983). In vivo experiments demonstrated cerebral vasodilation elicited by D A and antagonized by the D A
Correspondence to: F. Amenta, Dipartimento di Sanith Pubblica, Via A. Borelli 50, I 00161 Rome, Italy.
receptor antagonist, pimozide (Von Essen, 1974; Von Essen and Roos, 1974). Moreover, in vivo activation of D A receptors in cerebral tissue results in increased local cerebral glucose utilization (McCulloch et al., 1979) and in cerebrovascular vasodilation (McCulloch et al., 1982). DA-induced cerebral vasodilation has also been described after in vivo microapplication of DA to brain vessels (Edvinsson et al., 1978), perivascular microinjections of D A to pial arterioles (Altura et al., 1980) and subarachnoid perivascular microinjections of DA and of putative D A receptor agonists (Edvinsson et al., 1985). The existence of D A receptors in the cerebrovascular tree has been demonstrated biochemically by studies describing a DA-sensitive c A M P generated system linked to D A receptors in m e m b r a n e particles of cerebral microvessels (Baca et al., 1978; Palmer, 1981), of circle of Willis and pial-arachnoid arteries (Amenta et al., 1984) and of internal carotid and middle cerebral artery (Amenta et al., 1988). Moreover, the use of radioreceptor binding techniques has shown the existence of D A 1 but not DA 2 receptors in membrane preparations of bovine cerebral blood vessels (De Keyser et al., 1988).
124 Since little information is available concerning the localization of DA receptors in the cerebrovascular tree, we now analyzed the pharmacological characteristics and the anatomical distribution of DA receptors in the rat circle of Willis and pial-arachnoid arteries by means of combined radioreceptor binding and autoradiographic techniques.
2. Material and methods
2.1. Animals Male Wistar rats (Charles River, Calco, Italy) weighing 200-300 g were used. Six animals received a total dose of 100 mg/kg (i.v.) of 6-hydroxydopamine (6OHDA) 48 and 24 h before killing to cause sympathectomy (Jonsson, 1980). Another 10 rats were used as controls and were injected with vehicle alone. The effectiveness of 6-OHDA treatment was assessed by applying a method for histochemical detection of tissue stores of catecholamines (for technical details see Amenta et al., 1987) to whole mounts of irides from sympathectomized and control rats. The animals were killed by decapitation under diethylether anaesthesia. The cranium was opened and, with the aid of a stereomicroscope, the circle of Willis arteries and pial-arachnoid membrane from the lateral brain surface were dissected free and removed according to the procedure detailed earlier (Napoleone et al., 1987). The arteries of the circle of Willis and separate portions of the pial-arachnoid membrane (frontoparietal and occipital) were embedded in a cryoprotectant medium (OCT, Ames, U.S.A.) and oriented to obtain transverse sections perpendicular to the major axis of the vessels. OCT blocks were frozen in isopentane cooled with liquid nitrogen, cut serially with a microtome cryostat at - 2 0 ° C , mounted on gelatincoated microscope slides and air-dried. Thicker sections (10-12/~m) were used for radioreceptor binding; thinner sections (6-8/~m) were used for autoradiography. Both radioreceptor binding and autoradiography experiments were performed with tissues of control and 6-OHDAtreated animals.
2.2. Radioreceptor binding For the labeling of DA 1 receptors, sections of the circle of Willis arteries and of the pial-arachnoid membrane were incubated for 60 rain at room temperature in 170 nM Tris-HC1 buffer containing NaC1 (120 mM), KC1 (5 mM), CaCl 2 (1.5 raM), MgCl 2 (4 raM), EDTA (1 mM) (final pH 7.4), in the presence of increasing concentrations (0.1 to 10 nM) of [3H]SCH 23390. Nonspecific binding was defined by the presence in the
incubation medium of [3H]SCH 23390 plus 1 t~M 23390. For the labeling of DA 2 receptors, sections of the circle of Willis arteries and the pial-arachnoid membrane were incubated for 60 min at room temperature in 170 nM Tris-HC1 buffer containing NaC1 (120 mM), KC1 (5 mM), CaCI 2 (2 mM), MgC12 (1 mM) and ascorbic acid (0.001%) (final pH 7.4), in the presence of increasing concentrations (0.1 to 10 nM) of [3H]spiroperidol. Possible binding of the ligand to 5-HT2 receptors (Wamsley and Palacios, 1983) was prevented by adding the 5-HT2 receptor antagonist, ketanserin (40 nM), to the incubation medium. Non-specific binding was defined by the presence in the incubation medium of [3H]spiroperidol plus ketanserin and 0.1 #M spiroperidol. No significant differences in the amounts of [3H]spiroperidol specifically bound to sections of rat cerebral arteries were noticeable when 0.1 or 1 /~M spiroperidol was used to generate non-specific binding (data not shown). The optimal incubation and washing conditions were determined in a series of preliminary experiments (data not shown). It was found from these experiments that the selective DA 2 receptor antagonist, (-)-sulpiride (1 #M as used in binding experiment to generate nonspecific binding, for ref. see Amenta 1990), inhibited specific "[3H]spiroperidol binding by about 60%. Ketanserin, 40 nM, caused about 40% inhibition of specific [3H]spiroperidol binding. The addition of ( - ) sulpiride (1 #M) and ketanserin (40 nM) to the incubation medium reduced [3H]spiroperidol binding to sections of cerebral vessels to the non-specific value (data not shown). At the end of incubation, the slides were washed in ice-cold incubation buffer (2 × 5 min) and quickly rinsed in distilled water. The specificity of [3H]SCH 23390 and [3H]spiroperidol binding to DA 1 and to DA 2 receptors, respectively, was assessed by incubating sections containing the radioligands in the presence of various concentrations of dopaminergic, adrenergic and serotoninergic receptor agonists and antagonists. Sections used for binding experiments were wiped onto Whatman GF-B glass fibre filters and counted in a Beckman liquid scintillation spectrometer at an efficiency of 40%. Representative sections mounted on acid-washed slides (non-gelatin-coated) were sonicated and the protein content was determined according to Lowry et al. (1951) against a standard of bovine serum albumin.
2.3. A utoradiography For the autoradiographic demonstration of DA a receptor sites sections of the rat circle of Willis arteries and of the pial-arachnoid membrane were incubated for 60 min at room temperature in the above buffer containing 5 nM [3H]SCH 23390 in the presence or in the
125 absence of 1 #M SCH 23390 to generate non-specific binding. For the autoradiographic demonstration of DA2 receptor sites, sections of the rat circle of Willis arteries and of the pial-arachnoid membrane were incubated for 60 rain at room temperature in the above buffer containing 4 nM [3H]spiroperidol plus ketanserin (40 nM) in the presence or in the absence of 0.1 #M spiroperidol to generate non-specific binding. Sections were washed in ice-cold incubation buffer (2 × 5 min) to remove unbound radioligands, quickly rinsed in distilled water and air-dried. Sections were then processed for the autoradiographic demonstration of reversible and diffusible radioligands according to the technique proposed by Young and Kuhar (1979). Briefly, acid-washed, gelatin-coated coverslips were dipped in Ilford L4 nuclear emulsion (diluted 1:1 in distilled water) at 40 °C and were dried at room temperature for about 5 h in a lightproof box containing P205. Emulsion-coated coverslips were attached to the slides carrying radiolabeled sections by means of cyanoacrylate glue to one end and a binder clip at the other. Nuclear emulsion was exposed for 4-10 weeks, the binder clips were removed and the coverslips gently lifted from microscope slides at one end. The emulsions were then developed with Kodak D19, fixed with Agefix and washed in distilled water. Cerebral vessels sections were stained with toluidine blue and the coverslips were sealed permanently to the slides. The tissue and overlying silver grains in the emulsion layer were viewed and photographed with a bright- and a dark-field-equipped Zeiss II photomicroscope. The density of silver grains developed within the wall of the circle of Willis arteries and the pial-arachnoid arteries (diameter greater than 70 ttm) and arterioles (diameter less than 70 #m) (see Rodhin, 1967) was evaluated by reflectance photometry in dark-field sections according to the technique described elsewhere (Amenta et al., 1986; Napoleone et al., 1987). In brief, a 'Fluoval Photometrie' microphotometric unit was calibrated taking the background of control sections incubated without radioligands as 'zero'. All the reflectance measurements to be reported were made in a circular area of 10 /~m in diameter delineated by a measuring diaphragm. Measurements were made on the medial layer of blood vessels exposed to [3H]SCH 23390 and on the adventitial layer (including the adventitialmedial border) and on the intimal layer of blood vessels exposed to [3H]spiroperidol. The photometer readings were recorded in arbitrary units proportional to the grain density. The silver grain concentration of sections incubated with [3H]SCH 23390 plus 1 /~M SCH 23390 or with [3H]spiroperidol (in presence of ketanserin) plus 0.1/~M spiroperidol were considered due to non-specific retention of the radioligands and subtracted from the density of silver grains developed in sections incubated without excess of displacers. Vessel diameters were de-
termined from measurements performed directly under the microscope by means of an eyepiece micrometer.
2.4. Data analysis All data are expressed as means + S.D. unless otherwise specified. The receptor densities (Bmax) and radioligand equilibrium dissociation constants (Kd) were calculated by linear regression analysis of Scatchard plots of the saturation binding data. Competition dissociation constant values (Ki) were determined according to the method of Cheng and Prusoff (1973). The statistical significance of the density of radioligands binding sites in the different circle of Willis arteries and in the different sized pial-arachnoid vessels was determined by analysis of variance (ANOVA).
2.5. Chemicals [3H]SCH 23390 (specific activity 85 Ci/mmol) and [3H]spiroperidol (specific activity 70 Ci/mmol) were purchased from Amersham Radiochemical Centre (U.K.). Isomers of butaclamol, ADTN, spiroperidol and SK&F 38393 were obtained from Research Biochemicals, Inc. (U.S.A.). Domperidone, haloperidol and ketanserin were purchased from Janssen (Belgium). Cisa n d trans-flupentixol were obtained from Lundbeck AS (Denmark). SCH 23390 and (-)-sulpiride were products of Schering Plough (U.S.A.) and Ravizza (Italy) respectively. Bromocriptine, phentolamine and quinpirole (LY 171555) were obtained from Sandoz AG (Switzerland), Ciba-Geigy (Switzerland) and Eli Lilly, Inc. (U.S.A.) respectively. Other chemicals were purchased from Sigma Chemical Co. (U.S.A.).
3. Results
3.1. Binding experiments [3H]SCH 23390 and [3H]spiroperidol were specifically bound by sections of circle of Willis arteries and of pial-arachnoid membrane. The binding of both ligands was saturable (fig. 1), reversible, time- and temperature-dependent (data not shown). Where a concentration of 5 nM was used approximately 41% of [3H]SCH 23390 was bound specifically (fig. 1A). Scatchard analysis of the binding isotherms showed K a and Bmax values of 2.2 + 0.3 nM and 68.4 + 4.7 fmol/mg protein, respectively (fig. 1A). Using a concentration of 4 nM, approximately 38% of [3H]spiroperidol was bound specifically (fig. 1B). Scatchard analysis of the binding isotherms showed a K d and a Bm~, of 1.9 + 0.4 nM and 39.8 + 2.6 fmol/mg protein, respectively (fig. 1B). At least in the range of concentrations of the two ligands used, both [3H]SCH 23390 and [3H]spiroperidol were
126
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TABLE 1
20 40 §0 8~) (3H) S C H 2 3 3 1 0 bound (f mol/m I proteinl
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Compound
90.
6O
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1
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3
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5
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(nM)
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100.
Pharmacological specificity of [3H]SCH 23390 and of [3H]spiroperidol binding to sections of rat cerebral vessels. [3H]SCH 23390 or [3H]spiroperidol binding to sections of rat circle of Willis arteries and of pial-arachnoid membrane was assayed as described in the text. The values are expressed in nM and represent the competitor dissociation constant (K i) determined according to the method of Cheng and Prusoff (1973). The data are means of three to five experiments carried out in triplicate. The standard error was less than 10%.
°1..\
10 20 3b 4b (3HI Spiroperidol bound
Z
J'
Ki
cis-Flupentixol trans-Flupentixol SCH 23390 SK&F 38393 ADTN Apomorphine ( + )-Butaclamol ( - )-Butaclamol Dopamine Bromocriptine Spiroperidol Domperidone Quinpirole ( - )-Sulpiride Ketanserin Phentolamine ( - )-Propranolol Serotonin
[3H]SCH 23390
[3H]Spiroperidol
6.3 850 6.1 24 6 050 93 9.7 9 700 350 > 5 000 > 10 000 > 10000 > 5 000 > 10 000 500 > 5 000 > 10 000 > 5 000
10.8 480 1000 > 10000 1530 104 10.9 7100 620 83 9.2 10.7 729 31 680 1250 > 10 000 2 250
80. n
._ h,.
DA 2 activity ((+)-butaclamol, transflupentixol, haloperidol, ADTN, apomorphine, DA) (see Seeman and Grigoriadis, 1987) compete effectively with [3H]SCH 23390 binding. In contrast, compounds known DA 1 and
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I
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2o. B v
1
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4
5
6
7
8
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(3H) Spiroperidol
(3H) Spiroperidol ( nM ) Fig. 1. Saturation curve of [3H]SCH 23390 (panel A) and of [3H]spiroperidol (panel B) binding to sections of rat circle of Willis arteries and of pial-arachnoid membrane. The points are the means 5: S.E.M. of triplicate determinations. Non-specific binding was generated by incubating sections with radioligands in the presence of 1 #M SCH 23390 (panel A) or of 0.1 /~M spiroperidol (panel B). Inset: Scatchard analysis of [3H]SCH 23390 binding (panel A) or of [3H]spiroperidol binding (panel B) to sections of rat circle of Willis arteries and of pial-arachnoid membrane. In panel A the Bmax value was 68.4 + 4,7 fmol/mg protein; in panel B it was 39.8 5:2.6 fmol/mg protein. • Total binding; • non-specific binding.
c
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bound to a single class of high-affinity binding sites (fig. 1). The pharmacological specificity of [3H]SCH 23390 binding to sections of rat cerebral arteries was cons i s t e n t w i t h l a b e l i n g o f D A z r e c e p t o r sites. A s s h o w n i n t a b l e 1, c o m p o u n d s k n o w n t o e x h i b i t D A 1 a c t i v i t y ( S C H 23390, S K & F 38393, c i s - f l u p e n t i x o l ) o r m i x e d
Fig. 2. Comparison of [3H]SCH 23390 and [3H]spiroperidol specifically bound to sections of rat circle of Willis arteries and pialarachnoid membrane of normal control (panel A, n =10) and 6OHDA-sympathectomized (panel B, n = 6) rats.
127
to exhibit DA 2 activity (domperidone, (-)-sulpiride, spiroperidol, bromocriptine and quinpirole) (see Seeman and Grigoriadis, 1987) did not compete with [3H]SCH 23390. An opposite rank order of potencies was obtained with [3H]spiroperidol binding (table 1), suggesting that the ligand labeled specifically DA 2 receptor sites. The binding of [3H]SCH 23390 and [3H]spiroperidol to sections of cerebral vessels was stereospecific: (-)-butaclamol competed significantly less potently than (+)-butaclamol with the two ligands (table 1). Among the non-dopaminergic agents tested, ketanserin competed the most effectively with [3H]spiroperidol binding (table 1). Chemical sympathectomy did not significantly change the K d value of [3H]SCH 23390 or of [3H]spiroperidol binding (data not shown) nor DA 1 receptor density in rat cerebral vessels (fig. 2). The Bmax value of [3H]spiroperidol was reduced by about 70% after 6-OHDA treatment (fig. 2). 3.2. A utoradiography Silver grains representing specific [3H]SCH 23390 or [3H]spiroperidol binding sites were observed within sections of rat cerebral vessels (figs. 3-5). The density of silver grains determined by the simultaneous presence in the incubation medium of [3H]SCH 23390 and 1/~M SCH 23390 or of [3H]spiroperidol (plus ketanserin) and 0.1/xM spiroperidol (non-specific binding) was substantially lower than that obtained by incubation in the presence of [3H]SCH 23390 or of [3H]spiroperidol (plus ketanserin) alone (figs. 3-4).
Sections incubated with 5 nM [3H]SCH 23390 (the concentration producing the highest specific binding, see fig. 1A) to label DA 1 receptors developed silver grains primarily over the medial layer of the different circle of Willis arteries and pial-arachnoid arteries (fig. 3) and arterioles. The silver grains were rather homogeneously distributed within the entire medial layer of the different circle of Willis arteries or pial arteries and arterioles without significant differences in the density of silver grains between circle of Willis arteries, frontoparietal or occipital pial arteries and arterioles or blood vessels of different size (table 2). Chemical sympathectomy was without effect on the density and pattern of DA receptor sites within circle of Willis arteries or pial arteries and arterioles (table 2). Exposure of cerebral vessel sections to 4 nM [3H]spiroperidol in the presence of ketanserin (the concentration producing the highest specific binding, see fig. 1B) to label DA 2 receptors caused the accumulation of silver grains primarily within the adventitia, advential-medial border and the intimal layer of circle of Willis arteries (fig. 4) and of pial arteries (fig. 5) and arterioles. The density of silver grains within the adventitia and the adventitial-medial border of circle of Willis arteries was higher in the anterior circle of Willis arteries than in the posterior ones (fig. 4 and table 3). The same was true for pial arteries and arterioles taken from the fronto-parietal pial-arachnoid in comparison with those obtained from the occipital pial-arachnoid (table 3). Moreover, the density of adventitial and adventitial-medial silver grains was higher in pial-arachnoid arteries and arterioles than in the circle of Willis arteries (table
Fig. 3. Autoradiographic localization of D A 1 receptors in sections of rat middle cerebral artery. Sections were incubated with 5 n M [3H] SCH 23390 alone (a) or plus 1 # M SCH 23390 to cause non-specific binding (c). Pictures a and c are dark-field images. Picture b is a bright-field image of picture a stained with toluidine blue to verify microanatomical details A = adventitia; M = media; L = lumen. The arrow shows the intima. Note that the accumulation of silver grains occurs primarily in the media ( × 140).
128
3). The density of silver grains developed within the intimal layer of cerebral vessels was not significantly changed in the different sized arteries examined (table
3).
Chemical sympathectomy caused the loss of adventitial and adventitial-medial silver grains developed after exposure of sections of cerebral vessels to [3H]spiroperidol, but was without effect on the density of grains in intimal binding sites (fig. 5 and table 3).
4. Discussion
Although a great deal of evidence has accumulated regarding the localization of central DA receptor sites
(for ref. see Wamsley and Palacios, 1983), little information is so far available as to the anatomical localization of peripheral DA receptor sites (see Amenta et al., 1988). This lack of information may be due partly to the fact that less interest has been devoted to peripheral dopaminergic mechanisms than to central dopaminergic mechanisms but there are also technical reasons. The lower density of peripheral DA receptors in comparison with that of central ones and the heterogeneity of peripheral tissues in comparison with central nervous system, may cause significant problems with the radioreceptor analysis and the autoradiographic localization of DA receptors in the periphery. The above data provide evidence that [3H]SCH 23390, the first selective radioligand for both central (D1) and peripheral (DA1) DA
/@
/
!, 0 Fig. 4. Autoradiographic localization of D A 2 receptors in sections of rat internal carotid (pictures a-c) and basilar artery (d-f). Sections were incubated with 4 n M [3H]spiroperidol plus 40 n M ketanserin in the absence (total binding, a and d) or in the presence of 0.1 /~M spiroperidol (non-specific binding c and f). Pictures a, c, d and f represent dark-field micrographs; pictures b and e are bright-field pictures of (a) and (d) respectively stained with toluidine blue to verify microanatomical details. A = adventitia; M = media; L = lumen; arrows indicate the intimal layer. The density of adventitial silver grains is higher in the internal carotid than in the basilar artery ( X 140).
129
q
123
EJP 51635
Autoradiographic localization of dopamine receptors in rat cerebral blood vessels F. A m e n t a a,2, A. Ricci 2 a n d J.A. V e g a 3 1 Dipartimento di Sanitdt Pubblica e Biologia Cellulare, Universit& "Tor Vergata', Rome, Italy, e Dipartimento di Scienze Cardooascolari, 'La Sapienza; Rome, Italy and 3 Departamento de Morfologia y Biologia Celular, Universidad de Ooiedo, Oviedo, Spain Received 31 August 1990, accepted 25 September 1990
Combined in vitro radioreceptor binding and autoradiographic techniques were used to analyze the pharmacological profile and the anatomical localization of dopamine (DA) DA 1 and DA 2 receptor sites in the arteries and arterioles of the pial-arachnoid membrane in the rat. [3H]SCH 23390, used as a ligand of DA1 receptors, was bound by sections of rat cerebral vessels in a manner consistent with the labehng of DA1 receptors, with a value of 2.2 nM for the dissociation constant (Kd) and of 68.4 fmol/mg protein for the density of binding sites (Bmax). The ligand was bound primarily by the medial layer of cerebral arteries. The density of silver grains developed after exposure of cerebral vessel sections to [3H]SCH 23390 was unchanged after chemical sympathectomy with 6-hydroxydopamine (6-OHDA) and was not significantly different in either the circle of Willis arteries or in the pial-arachnoid arteries and arterioles. [3H]Spiroperidol was used as ligand of DA2 receptors in the presence of ketanserin to block the possible binding to 5-HT2 receptors. [3H]Spiroperidol was bound by sections of rat cerebral vessels in a manner consistent with labehng of DA 2 receptors, with K d and Bm~ values of 1.9 nM and 39.8 fmol/mg protein, respectively. The ligand was bound primarily by the adventitia and the adventitial-medial border as well as by the intimal layer of cerebral arteries. The accumulation of adventitial and adventitial-medial silver grains developed after exposure of cerebral vessels sections to [3H]spiroperidol did not occur in 6-OHDA-treated animals. In contrast, chemical sympathectomy was without effect on the density of intimal silver grains. The density of adventitial silver grains was higher in the anterior than in the posterior circle of Willis and pial-arachnoid arteries and arterioles, as well as in the pial-arachnoid arteries and arterioles than in circle of Willis arteries. These findings indicate the existence of both D A 1 and DA 2 receptor sites in the rat cerebrovascular tree. Dopamine DA 1 receptors; Dopamine DA 2 receptors; Circle of Wilhs arteries; Pial-arachnoid arteries; Radioreceptor binding; Autoradiography
I. Introduction A possible role of dopamine (DA) in the control of cerebrovascular circulation was suggested by several physiological and pharmacological observations. In vitro experiments have shown that DA, after a- and fl-adrenoceptor blockade, mediates relaxation of smooth muscle of major cerebral arteries (e.g. internal carotid, middle cerebral artery and basilar artery). These effects are mimicked by dopaminergic agonists such as apomorphine, 6,7-ADTN, epinine and S K & F 38393 and antagonized by bulbocapnine, (+)-butaclamol, cis-aflupentixol, fluphenazine and haloperidol, suggesting that they are due to the interaction with specific D A receptors (Toda et al., 1976; Boullin et ai., 1977; 1978; Oudart et al., 1981; Edvinsson et al., 1982; Forster et al,, 1983). In vivo experiments demonstrated cerebral vasodilation elicited by D A and antagonized by the D A
Correspondence to: F. Amenta, Dipartimento di Sanith Pubblica, Via A. Borelli 50, I 00161 Rome, Italy.
receptor antagonist, pimozide (Von Essen, 1974; Von Essen and Roos, 1974). Moreover, in vivo activation of D A receptors in cerebral tissue results in increased local cerebral glucose utilization (McCulloch et al., 1979) and in cerebrovascular vasodilation (McCulloch et al., 1982). DA-induced cerebral vasodilation has also been described after in vivo microapplication of DA to brain vessels (Edvinsson et al., 1978), perivascular microinjections of D A to pial arterioles (Altura et al., 1980) and subarachnoid perivascular microinjections of DA and of putative D A receptor agonists (Edvinsson et al., 1985). The existence of D A receptors in the cerebrovascular tree has been demonstrated biochemically by studies describing a DA-sensitive c A M P generated system linked to D A receptors in m e m b r a n e particles of cerebral microvessels (Baca et al., 1978; Palmer, 1981), of circle of Willis and pial-arachnoid arteries (Amenta et al., 1984) and of internal carotid and middle cerebral artery (Amenta et al., 1988). Moreover, the use of radioreceptor binding techniques has shown the existence of D A 1 but not DA 2 receptors in membrane preparations of bovine cerebral blood vessels (De Keyser et al., 1988).
124 Since little information is available concerning the localization of DA receptors in the cerebrovascular tree, we now analyzed the pharmacological characteristics and the anatomical distribution of DA receptors in the rat circle of Willis and pial-arachnoid arteries by means of combined radioreceptor binding and autoradiographic techniques.
2. Material and methods
2.1. Animals Male Wistar rats (Charles River, Calco, Italy) weighing 200-300 g were used. Six animals received a total dose of 100 mg/kg (i.v.) of 6-hydroxydopamine (6OHDA) 48 and 24 h before killing to cause sympathectomy (Jonsson, 1980). Another 10 rats were used as controls and were injected with vehicle alone. The effectiveness of 6-OHDA treatment was assessed by applying a method for histochemical detection of tissue stores of catecholamines (for technical details see Amenta et al., 1987) to whole mounts of irides from sympathectomized and control rats. The animals were killed by decapitation under diethylether anaesthesia. The cranium was opened and, with the aid of a stereomicroscope, the circle of Willis arteries and pial-arachnoid membrane from the lateral brain surface were dissected free and removed according to the procedure detailed earlier (Napoleone et al., 1987). The arteries of the circle of Willis and separate portions of the pial-arachnoid membrane (frontoparietal and occipital) were embedded in a cryoprotectant medium (OCT, Ames, U.S.A.) and oriented to obtain transverse sections perpendicular to the major axis of the vessels. OCT blocks were frozen in isopentane cooled with liquid nitrogen, cut serially with a microtome cryostat at - 2 0 ° C , mounted on gelatincoated microscope slides and air-dried. Thicker sections (10-12/~m) were used for radioreceptor binding; thinner sections (6-8/~m) were used for autoradiography. Both radioreceptor binding and autoradiography experiments were performed with tissues of control and 6-OHDAtreated animals.
2.2. Radioreceptor binding For the labeling of DA 1 receptors, sections of the circle of Willis arteries and of the pial-arachnoid membrane were incubated for 60 rain at room temperature in 170 nM Tris-HC1 buffer containing NaC1 (120 mM), KC1 (5 mM), CaCl 2 (1.5 raM), MgCl 2 (4 raM), EDTA (1 mM) (final pH 7.4), in the presence of increasing concentrations (0.1 to 10 nM) of [3H]SCH 23390. Nonspecific binding was defined by the presence in the
incubation medium of [3H]SCH 23390 plus 1 t~M 23390. For the labeling of DA 2 receptors, sections of the circle of Willis arteries and the pial-arachnoid membrane were incubated for 60 min at room temperature in 170 nM Tris-HC1 buffer containing NaC1 (120 mM), KC1 (5 mM), CaCI 2 (2 mM), MgC12 (1 mM) and ascorbic acid (0.001%) (final pH 7.4), in the presence of increasing concentrations (0.1 to 10 nM) of [3H]spiroperidol. Possible binding of the ligand to 5-HT2 receptors (Wamsley and Palacios, 1983) was prevented by adding the 5-HT2 receptor antagonist, ketanserin (40 nM), to the incubation medium. Non-specific binding was defined by the presence in the incubation medium of [3H]spiroperidol plus ketanserin and 0.1 #M spiroperidol. No significant differences in the amounts of [3H]spiroperidol specifically bound to sections of rat cerebral arteries were noticeable when 0.1 or 1 /~M spiroperidol was used to generate non-specific binding (data not shown). The optimal incubation and washing conditions were determined in a series of preliminary experiments (data not shown). It was found from these experiments that the selective DA 2 receptor antagonist, (-)-sulpiride (1 #M as used in binding experiment to generate nonspecific binding, for ref. see Amenta 1990), inhibited specific "[3H]spiroperidol binding by about 60%. Ketanserin, 40 nM, caused about 40% inhibition of specific [3H]spiroperidol binding. The addition of ( - ) sulpiride (1 #M) and ketanserin (40 nM) to the incubation medium reduced [3H]spiroperidol binding to sections of cerebral vessels to the non-specific value (data not shown). At the end of incubation, the slides were washed in ice-cold incubation buffer (2 × 5 min) and quickly rinsed in distilled water. The specificity of [3H]SCH 23390 and [3H]spiroperidol binding to DA 1 and to DA 2 receptors, respectively, was assessed by incubating sections containing the radioligands in the presence of various concentrations of dopaminergic, adrenergic and serotoninergic receptor agonists and antagonists. Sections used for binding experiments were wiped onto Whatman GF-B glass fibre filters and counted in a Beckman liquid scintillation spectrometer at an efficiency of 40%. Representative sections mounted on acid-washed slides (non-gelatin-coated) were sonicated and the protein content was determined according to Lowry et al. (1951) against a standard of bovine serum albumin.
2.3. A utoradiography For the autoradiographic demonstration of DA a receptor sites sections of the rat circle of Willis arteries and of the pial-arachnoid membrane were incubated for 60 min at room temperature in the above buffer containing 5 nM [3H]SCH 23390 in the presence or in the
125 absence of 1 #M SCH 23390 to generate non-specific binding. For the autoradiographic demonstration of DA2 receptor sites, sections of the rat circle of Willis arteries and of the pial-arachnoid membrane were incubated for 60 rain at room temperature in the above buffer containing 4 nM [3H]spiroperidol plus ketanserin (40 nM) in the presence or in the absence of 0.1 #M spiroperidol to generate non-specific binding. Sections were washed in ice-cold incubation buffer (2 × 5 min) to remove unbound radioligands, quickly rinsed in distilled water and air-dried. Sections were then processed for the autoradiographic demonstration of reversible and diffusible radioligands according to the technique proposed by Young and Kuhar (1979). Briefly, acid-washed, gelatin-coated coverslips were dipped in Ilford L4 nuclear emulsion (diluted 1:1 in distilled water) at 40 °C and were dried at room temperature for about 5 h in a lightproof box containing P205. Emulsion-coated coverslips were attached to the slides carrying radiolabeled sections by means of cyanoacrylate glue to one end and a binder clip at the other. Nuclear emulsion was exposed for 4-10 weeks, the binder clips were removed and the coverslips gently lifted from microscope slides at one end. The emulsions were then developed with Kodak D19, fixed with Agefix and washed in distilled water. Cerebral vessels sections were stained with toluidine blue and the coverslips were sealed permanently to the slides. The tissue and overlying silver grains in the emulsion layer were viewed and photographed with a bright- and a dark-field-equipped Zeiss II photomicroscope. The density of silver grains developed within the wall of the circle of Willis arteries and the pial-arachnoid arteries (diameter greater than 70 ttm) and arterioles (diameter less than 70 #m) (see Rodhin, 1967) was evaluated by reflectance photometry in dark-field sections according to the technique described elsewhere (Amenta et al., 1986; Napoleone et al., 1987). In brief, a 'Fluoval Photometrie' microphotometric unit was calibrated taking the background of control sections incubated without radioligands as 'zero'. All the reflectance measurements to be reported were made in a circular area of 10 /~m in diameter delineated by a measuring diaphragm. Measurements were made on the medial layer of blood vessels exposed to [3H]SCH 23390 and on the adventitial layer (including the adventitialmedial border) and on the intimal layer of blood vessels exposed to [3H]spiroperidol. The photometer readings were recorded in arbitrary units proportional to the grain density. The silver grain concentration of sections incubated with [3H]SCH 23390 plus 1 /~M SCH 23390 or with [3H]spiroperidol (in presence of ketanserin) plus 0.1/~M spiroperidol were considered due to non-specific retention of the radioligands and subtracted from the density of silver grains developed in sections incubated without excess of displacers. Vessel diameters were de-
termined from measurements performed directly under the microscope by means of an eyepiece micrometer.
2.4. Data analysis All data are expressed as means + S.D. unless otherwise specified. The receptor densities (Bmax) and radioligand equilibrium dissociation constants (Kd) were calculated by linear regression analysis of Scatchard plots of the saturation binding data. Competition dissociation constant values (Ki) were determined according to the method of Cheng and Prusoff (1973). The statistical significance of the density of radioligands binding sites in the different circle of Willis arteries and in the different sized pial-arachnoid vessels was determined by analysis of variance (ANOVA).
2.5. Chemicals [3H]SCH 23390 (specific activity 85 Ci/mmol) and [3H]spiroperidol (specific activity 70 Ci/mmol) were purchased from Amersham Radiochemical Centre (U.K.). Isomers of butaclamol, ADTN, spiroperidol and SK&F 38393 were obtained from Research Biochemicals, Inc. (U.S.A.). Domperidone, haloperidol and ketanserin were purchased from Janssen (Belgium). Cisa n d trans-flupentixol were obtained from Lundbeck AS (Denmark). SCH 23390 and (-)-sulpiride were products of Schering Plough (U.S.A.) and Ravizza (Italy) respectively. Bromocriptine, phentolamine and quinpirole (LY 171555) were obtained from Sandoz AG (Switzerland), Ciba-Geigy (Switzerland) and Eli Lilly, Inc. (U.S.A.) respectively. Other chemicals were purchased from Sigma Chemical Co. (U.S.A.).
3. Results
3.1. Binding experiments [3H]SCH 23390 and [3H]spiroperidol were specifically bound by sections of circle of Willis arteries and of pial-arachnoid membrane. The binding of both ligands was saturable (fig. 1), reversible, time- and temperature-dependent (data not shown). Where a concentration of 5 nM was used approximately 41% of [3H]SCH 23390 was bound specifically (fig. 1A). Scatchard analysis of the binding isotherms showed K a and Bmax values of 2.2 + 0.3 nM and 68.4 + 4.7 fmol/mg protein, respectively (fig. 1A). Using a concentration of 4 nM, approximately 38% of [3H]spiroperidol was bound specifically (fig. 1B). Scatchard analysis of the binding isotherms showed a K d and a Bm~, of 1.9 + 0.4 nM and 39.8 + 2.6 fmol/mg protein, respectively (fig. 1B). At least in the range of concentrations of the two ligands used, both [3H]SCH 23390 and [3H]spiroperidol were
126
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150.
E
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TABLE 1
20 40 §0 8~) (3H) S C H 2 3 3 1 0 bound (f mol/m I proteinl
i~,,,I '~P
j.
120.
Compound
90.
6O
"r
3O
1
2
3
4
5
6
( 3 H ) SCH 2 3 3 9 0 *"
7
B
9
10
(nM)
16
"~ 14o. ~2 g" "~
-
~ 4
100.
Pharmacological specificity of [3H]SCH 23390 and of [3H]spiroperidol binding to sections of rat cerebral vessels. [3H]SCH 23390 or [3H]spiroperidol binding to sections of rat circle of Willis arteries and of pial-arachnoid membrane was assayed as described in the text. The values are expressed in nM and represent the competitor dissociation constant (K i) determined according to the method of Cheng and Prusoff (1973). The data are means of three to five experiments carried out in triplicate. The standard error was less than 10%.
°1..\
10 20 3b 4b (3HI Spiroperidol bound
Z
J'
Ki
cis-Flupentixol trans-Flupentixol SCH 23390 SK&F 38393 ADTN Apomorphine ( + )-Butaclamol ( - )-Butaclamol Dopamine Bromocriptine Spiroperidol Domperidone Quinpirole ( - )-Sulpiride Ketanserin Phentolamine ( - )-Propranolol Serotonin
[3H]SCH 23390
[3H]Spiroperidol
6.3 850 6.1 24 6 050 93 9.7 9 700 350 > 5 000 > 10 000 > 10000 > 5 000 > 10 000 500 > 5 000 > 10 000 > 5 000
10.8 480 1000 > 10000 1530 104 10.9 7100 620 83 9.2 10.7 729 31 680 1250 > 10 000 2 250
80. n
._ h,.
DA 2 activity ((+)-butaclamol, transflupentixol, haloperidol, ADTN, apomorphine, DA) (see Seeman and Grigoriadis, 1987) compete effectively with [3H]SCH 23390 binding. In contrast, compounds known DA 1 and
60,
2
.5. 4o. ffl
I
~
2o. B v
1
i
;3
4
5
6
7
8
9
(3H)
10
SCH 23310
g-"-I
(3H) Spiroperidol
(3H) Spiroperidol ( nM ) Fig. 1. Saturation curve of [3H]SCH 23390 (panel A) and of [3H]spiroperidol (panel B) binding to sections of rat circle of Willis arteries and of pial-arachnoid membrane. The points are the means 5: S.E.M. of triplicate determinations. Non-specific binding was generated by incubating sections with radioligands in the presence of 1 #M SCH 23390 (panel A) or of 0.1 /~M spiroperidol (panel B). Inset: Scatchard analysis of [3H]SCH 23390 binding (panel A) or of [3H]spiroperidol binding (panel B) to sections of rat circle of Willis arteries and of pial-arachnoid membrane. In panel A the Bmax value was 68.4 + 4,7 fmol/mg protein; in panel B it was 39.8 5:2.6 fmol/mg protein. • Total binding; • non-specific binding.
c
2(2. 01
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C~ Z "-t
om Gl z ,,~
20.
bound to a single class of high-affinity binding sites (fig. 1). The pharmacological specificity of [3H]SCH 23390 binding to sections of rat cerebral arteries was cons i s t e n t w i t h l a b e l i n g o f D A z r e c e p t o r sites. A s s h o w n i n t a b l e 1, c o m p o u n d s k n o w n t o e x h i b i t D A 1 a c t i v i t y ( S C H 23390, S K & F 38393, c i s - f l u p e n t i x o l ) o r m i x e d
Fig. 2. Comparison of [3H]SCH 23390 and [3H]spiroperidol specifically bound to sections of rat circle of Willis arteries and pialarachnoid membrane of normal control (panel A, n =10) and 6OHDA-sympathectomized (panel B, n = 6) rats.
127
to exhibit DA 2 activity (domperidone, (-)-sulpiride, spiroperidol, bromocriptine and quinpirole) (see Seeman and Grigoriadis, 1987) did not compete with [3H]SCH 23390. An opposite rank order of potencies was obtained with [3H]spiroperidol binding (table 1), suggesting that the ligand labeled specifically DA 2 receptor sites. The binding of [3H]SCH 23390 and [3H]spiroperidol to sections of cerebral vessels was stereospecific: (-)-butaclamol competed significantly less potently than (+)-butaclamol with the two ligands (table 1). Among the non-dopaminergic agents tested, ketanserin competed the most effectively with [3H]spiroperidol binding (table 1). Chemical sympathectomy did not significantly change the K d value of [3H]SCH 23390 or of [3H]spiroperidol binding (data not shown) nor DA 1 receptor density in rat cerebral vessels (fig. 2). The Bmax value of [3H]spiroperidol was reduced by about 70% after 6-OHDA treatment (fig. 2). 3.2. A utoradiography Silver grains representing specific [3H]SCH 23390 or [3H]spiroperidol binding sites were observed within sections of rat cerebral vessels (figs. 3-5). The density of silver grains determined by the simultaneous presence in the incubation medium of [3H]SCH 23390 and 1/~M SCH 23390 or of [3H]spiroperidol (plus ketanserin) and 0.1/xM spiroperidol (non-specific binding) was substantially lower than that obtained by incubation in the presence of [3H]SCH 23390 or of [3H]spiroperidol (plus ketanserin) alone (figs. 3-4).
Sections incubated with 5 nM [3H]SCH 23390 (the concentration producing the highest specific binding, see fig. 1A) to label DA 1 receptors developed silver grains primarily over the medial layer of the different circle of Willis arteries and pial-arachnoid arteries (fig. 3) and arterioles. The silver grains were rather homogeneously distributed within the entire medial layer of the different circle of Willis arteries or pial arteries and arterioles without significant differences in the density of silver grains between circle of Willis arteries, frontoparietal or occipital pial arteries and arterioles or blood vessels of different size (table 2). Chemical sympathectomy was without effect on the density and pattern of DA receptor sites within circle of Willis arteries or pial arteries and arterioles (table 2). Exposure of cerebral vessel sections to 4 nM [3H]spiroperidol in the presence of ketanserin (the concentration producing the highest specific binding, see fig. 1B) to label DA 2 receptors caused the accumulation of silver grains primarily within the adventitia, advential-medial border and the intimal layer of circle of Willis arteries (fig. 4) and of pial arteries (fig. 5) and arterioles. The density of silver grains within the adventitia and the adventitial-medial border of circle of Willis arteries was higher in the anterior circle of Willis arteries than in the posterior ones (fig. 4 and table 3). The same was true for pial arteries and arterioles taken from the fronto-parietal pial-arachnoid in comparison with those obtained from the occipital pial-arachnoid (table 3). Moreover, the density of adventitial and adventitial-medial silver grains was higher in pial-arachnoid arteries and arterioles than in the circle of Willis arteries (table
Fig. 3. Autoradiographic localization of D A 1 receptors in sections of rat middle cerebral artery. Sections were incubated with 5 n M [3H] SCH 23390 alone (a) or plus 1 # M SCH 23390 to cause non-specific binding (c). Pictures a and c are dark-field images. Picture b is a bright-field image of picture a stained with toluidine blue to verify microanatomical details A = adventitia; M = media; L = lumen. The arrow shows the intima. Note that the accumulation of silver grains occurs primarily in the media ( × 140).
128
3). The density of silver grains developed within the intimal layer of cerebral vessels was not significantly changed in the different sized arteries examined (table
3).
Chemical sympathectomy caused the loss of adventitial and adventitial-medial silver grains developed after exposure of sections of cerebral vessels to [3H]spiroperidol, but was without effect on the density of grains in intimal binding sites (fig. 5 and table 3).
4. Discussion
Although a great deal of evidence has accumulated regarding the localization of central DA receptor sites
(for ref. see Wamsley and Palacios, 1983), little information is so far available as to the anatomical localization of peripheral DA receptor sites (see Amenta et al., 1988). This lack of information may be due partly to the fact that less interest has been devoted to peripheral dopaminergic mechanisms than to central dopaminergic mechanisms but there are also technical reasons. The lower density of peripheral DA receptors in comparison with that of central ones and the heterogeneity of peripheral tissues in comparison with central nervous system, may cause significant problems with the radioreceptor analysis and the autoradiographic localization of DA receptors in the periphery. The above data provide evidence that [3H]SCH 23390, the first selective radioligand for both central (D1) and peripheral (DA1) DA
/@
/
!, 0 Fig. 4. Autoradiographic localization of D A 2 receptors in sections of rat internal carotid (pictures a-c) and basilar artery (d-f). Sections were incubated with 4 n M [3H]spiroperidol plus 40 n M ketanserin in the absence (total binding, a and d) or in the presence of 0.1 /~M spiroperidol (non-specific binding c and f). Pictures a, c, d and f represent dark-field micrographs; pictures b and e are bright-field pictures of (a) and (d) respectively stained with toluidine blue to verify microanatomical details. A = adventitia; M = media; L = lumen; arrows indicate the intimal layer. The density of adventitial silver grains is higher in the internal carotid than in the basilar artery ( X 140).
129
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