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Increased α2- and β2-adrenergic receptors in cerebral microvessels in Alzheimer disease
Neuroscience Letters, 106 (1989) 233-238 Elsevier Scientific Publishers Ireland Ltd.
233
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Increased 2- and fl2-adrenergic receptors in cerebral microvessels in Alzheimer disease R a j e s h N . K a l a r i a a n d S a m i I. H a r i k Department of Neurology, University Hospitals of Cleveland and Case Western Reserve University School of Medicine Cleveland, OH 44106 (U.S.A.)
(Received 18 May 1989; Revised version received 10 July 1989; Accepted 13 July 1989) Key words: Adrenergic receptor; Alzheimer disease; Aging; Cerebral microvessel; Cerebral cortex; Blood-brain barrier
Adrenergic receptors exist in brain microvessels which are innervated by noradrenergic locus ceruleus neurons. Biochemical and pathological studies indicate locus ceruleus degeneration in Ahheimer disease (AD), which can cause adrenergic receptor alterations in brain microvessels. To assess this, we studied adrenergic receptors in human brain microvessels from AD subjects and age-matched controls by ligand binding methods. Total fl receptors of cerebral microvessels and f12receptors, the type which predominates in microvessels, were significantly increased in AD. Compared to the cerebral cortex, there was a paucity of ~q-adrenergic receptors in cerebral microvessels, and they did not change in AD. Binding to ct2receptors in cerebral microvessels was ~ 50 % of that in the cortex, and these receptors increased by ~ 60 % in cerebral microvessels of AD subjects. These findings suggest adrenergic receptor 'upregulation' in response to noradrenergic deafferentation in AD, which may have functional consequences at the blood-brain barrier. A l t h o u g h several studies describe a b n o r m a l i t i e s in the b r a i n ' s n o r a d r e n e r g i c systems in A h h e i m e r disease ( A D ) [1, 2, 10, 15], there is conflicting evidence as to w h e t h e r b r a i n ~t- a n d fl-adrenergic receptors are affected [4, 5, 20, 25, 26]. It is n o w well established t h a t a d r e n e r g i c receptors, in p a r t i c u l a r fl receptors, exist in b r a i n microvessels [7, 13, 18, 20, 22]. These small i n t r a p a r e n c h y m a l vessels are p r o b a b l y i n n e r v a t e d , at least in part, by locus ceruleus n o r a d r e n e r g i c n e u r o n s [6, 14, 18, 27] but, thus far, there are no studies o f a d r e n o c e p t o r a l t e r a t i o n s in b r a i n microvessels in A D . Here, we investigated b y specific ligand b i n d i n g m e t h o d s the status o f ct- a n d fl-adrenergic r e c e p t o r s u b t y p e s in cerebral microvessels o f subjects with A D a n d in a g e - m a t c h e d controls. H u m a n b r a i n s a m p l e s were o b t a i n e d at a u t o p s y f r o m 14 n o r m a l c o n t r o l s ( 7 M / 7 F ) aged 55-78 years ( 6 9 + 2 , m e a n _ S . E . M . ) a n d 15 A D subjects ( 9 M / 6 F ) aged 61-79 years (72 __+2). The interval between d e a t h a n d b r a i n s a m p l i n g was 1 1 . 4 _ 1.8 h in controis a n d 7.7 + 1.3 h in A D subjects. Microvessels were harvested f r o m 50-100 g samCorrespondence: R.N. Kalaria, Department of Neurology, University Hospitals of Cleveland, 2074 Abington Road, Cleveland, OH 44106, U.S.A.
0304-3940/89/$ 03.50 © 1989 Elsevier Scientific Publishers Ireland Ltd.
234 pies of prefrontal cortex (Brodmann area 10) by the bulk isolation method of Goldstein et al. [9], as modified for human autopsy material in our laboratory [16, 17]. Microvessels were checked for their purity by microscopy and by biochemical assays of marker enzymes: ),-glutamyl transpeptidase, alkaline phosphatase and angiotensin-converting enzyme. Microvessels from AD subjects and controls showed no significant differences in these endothelial cell marker enzymes [16, 17]. Particulate membrane fractions of both cerebral microvessels and the prefrontal cortex from which the microvessels were obtained, were prepared by homogenization in a Brinkmann polytron and extensively washed in dilute buffer by centrifugation and resuspension, and used for binding studies. Adrenergic receptors were assessed by established ligand binding methods as follows: fl receptors and their subtypes were assayed using (-)-[125I]iodopindolol (spec. act. 2200 Ci/mmol, NEN), as described before [19, 23]. Tissues were incubated in 20 mM Tris-HC1 buffer, pH 7.5 containing 150 mM NaC1, 2.5 mM MgCI2, 0.5 mM ascorbate and varying concentrations of [125I]iodopindolol for 30 min at 37°C. fll and [~ receptors were determined using 60 nM ICI-118,551 and 80 nM ICI-89,406, antagonists at f12 and/31 receptors, respectively [23]. We have confirmed in earlier experiments that at these concentrations, the antagonists selectively recognize specific fl-receptor subtypes (Fig. 1 of ref. 19). Non-specific binding was determined using 100 I~M (-)-isoproterenol. 21 Receptors were assayed using (_+)-fl-([125I]iodo-4-hydroxyphenyl)-ethyl-aminomethyl-tetralone (HEAT) (spec. act. 2200 Ci/mmol, NEN). The ligand (0.2 mM) was incubated with 30-50/tg of tissue protein in 50 mM Na+-K + phosphate buffer, pH 7.4 for 1 h at 22°C [8]. Non-specific binding was determined in the presence of 10 ItM prazosin. [3H]p-aminoclonidine (PAC) (spec. act. 62.5 Ci/ mmol, NEN) at 1 nM was used to assess 22-adrenergic receptors [3]. Tissues were incubated in 20 mM Na+-HEPES buffer, pH 7.4, with 0.1 mM ascorbate for 1 h at 22'C. Non-specific binding was determined in the presence of 1/~M clonidine. In all cases equilibrium binding was terminated by rapid filtration under reduced pressure and washing 3 times with 4 ml of cold buffer. In experiments using 3H-ligands, the filters were counted in a fl scintillation counter at ~45% efficiency, while in experiments using ~25I-labeled ligands, the filters were counted in a gamma counter at ~ 70% efficiency. Specific binding was calculated by subtracting nonspecific binding from total binding. Maximal binding (Bmax) and (Kd) were calculated for fl receptors according to Scatchard [24]. Specific binding of [125I]iodopindolol, [125I]HEAT or [3H]PAC to cerebral microvessels correlated neither with the post-mortem interval nor with time of tissue storage. There were no differences between men and women in the binding of any ligands. /3 Receptor binding was performed primarily at a single [125I]iodopindolol concentration because of insufficient material to allow for Scatchard plots in microvessels obtained from any one subject. Fig. 1 shows that total fl receptor binding was significantly higher in cerebral microvessels obtained from AD subjects than in those from age-matched controls (P < 0.05). Analysis of total fl receptors into fll and f12 subtypes showed that f12 adrenoceptors predominate in human cerebral microvessels (Fig. 1), a finding consistent with our previous results in pig and rat brain microvessels [13,
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Fig. 1. Histograms show specific [~25I]pindololbinding to cerebral microvessels in AD subjects (crosshatched columns) and age-matched controls (open columns). The columns represent means and the bars represent S.E.M. for 12 individual AD subjects and 11 controls. Binding was performed at a single concentration of 150 pM [~25I]pindolol. Statistical significance was assessed by the 2-sample Student's t-test (2-tailed). *Different from control at P< 0.05. **Different from control at P<0.01. Fig. 2. Histograms show specific [125I]HEATbinding to ~ receptors and specific [3H]PAC binding to ~2 receptors in cerebral microvessels and prefrontal cortex in AD subjects (cross-hatched columns) and agematched controls (open columns). The columns represent means and the bars represent S.E.M. for 15 AD subjects and 14 controls. Binding was performed at a single concentration of 150 pM for [tzSI]HEATand 1 nM for [3H]PAC.Binding results for cortex (unpublished observations) are shown here for comparison to microvessels. **Different from controls at P < 0.01. ***Different from controls at P < 0.001.
18]. These f12 receptors were significantly increased in microvessels o f A D subjects (Fig. 1, P < 0 . 0 1 ) . To ascertain that the increased binding to total fl and fl2-adrenoceptors was due to increased binding sites rather than higher affinity o f binding in microvessels from A D subjects, we performed Scatchard plots on cerebral microvessels that were pooled from several subjects within each group. The Bmax for [125I]iodopindolol binding to total fl receptors was 32 + 0.7 fmol/mg protein in microvessels o f A D subjects and 2 6 + 0 . 9 fmol/mg protein in microvessels o f controls (means + S.E.M., n = 4 , P < 0.01, Student's t-test, 2-tailed). The Kd o f binding was 142 + 30 p M in A D subjects and 106 + 20 p M in controls (P > 0.05). The density and affinity o f f l adrenoceptors in cerebral microvessels in h u m a n control subjects is similar to the density and affinity o f binding in the cerebral cortex [19]. Similar findings were previously noted in rats and pigs [13]. Also, f12 adrenoceptors were increased in the cerebral cortex o f A D subjects when c o m p a r e d to controls, suggesting fl receptor 'upregulation' [19]. There were no significant changes in ~1 receptors in cerebral microvessels in A D (Fig. 2), although those receptors were significantly decreased in the cerebral cortex o f A D subjects. Specific [125I]HEAT binding in brain microvessels was a b o u t one third o f that in the cortex (Fig. 2). A l t h o u g h this result is consistent with a recent study [7] in which [3H]prazosin was used as the ligand, it is at variance with the study in pig and rat cerebral microvessels where [3H]WB-4101 was used [13]. The inconsistency is p r o b a b l y due to differences in species and receptor ligands.
236 The binding of [3H]PAC to ~2 receptors in cerebral microvessels was ~ 50% that of the cerebral cortex of control subjects (Fig. 2). This observation is consistent with earlier reports in humans [7] and other mammalians [13, 22]. However, in contrast to [3H]PAC binding in the cerebral cortex which was decreased by ~ 50% in AD subjects (P < 0.001), [3H]PAC binding in cerebral microvessels was increased by ~ 60% in AD ( P < 0.01, Fig. 2). The increased [3H]PAC binding in cerebral microvessels was due to a change in the Bmax rather than the Kd. Thus, our results show increased f12 and ~2 adrenoceptors in cerebral microvessels from AD subjects but no significant alterations in ~1 receptors. The increased f12 receptors in microvessels may be the result of noradrenergic deafferentation of cerebral microvessels in AD. These observations are similar to the 'denervation supersensitivity' of microvessel [32 receptors seen in rats with lesions of locus ceruleus [18], although the pathological processes in AD are much more complex. It is not clear why upregulation of fl~ adrenoceptors does not occur in the cerebral cortex of AD subjects where this receptor subtype predominates, as it does in rats after locus ceruleus lesion [18]. However, it is possible that in AD, the cerebral cortex is not capable of reacting to noradrenergic denervation while cerebral microvessels retain this capability. Cerebral microvessel 22 adrenoceptors also increase in A D but decrease in the cerebral cortex (Fig. 2). A similar mechanism as that invoked above for • receptors may be the explanation. Alternatively, these opposite changes in 22 receptors may be due to their different location. It is possible that the 22 receptors recognized by [3H]PAC are predominantly autoreceptors on presynaptic terminals in the human cerebral cortex [28], but are located postsynaptically on capillary endothelial cells [7]. The loss of ~2 receptors in the cortex is then the result of impaired synaptic connectivity in AD. Whatever the underlying cause of the adrenoceptor alterations in cerebral microvessels of A D subjects, these changes may have important functional consequences at the blood brain barrier. We have previously demonstrated that lesion of the nucleus locus ceruleus and norepinephrine depletion in the ipsilateral cerebral cortex is associated with decreased N a + , K + - A T P a s e in brain microvessels and increased permeability of the blood brain barrier to macromolecules, especially under certain pathophysiological conditions such as seizures and hypertension [i 1, 12]. Recent evidence also invokes abnormalities of the blood-brain barrier in explaining some of the brain pathology in AD [21]. We thank the Neuropathology Division, Institute of Pathology, C W R U for help in obtaining human postmortem tissue and Jeanette Barnhart for manuscript preparation. This work was supported in part by a grant to R.N.K. from the A D R D A , by USPHS Grants MH-43444 and HL~35617 and by the David S. Ingalls Fund. I Arai, H., Kosaka, K. and lizuka, R., Changes of biogenic amines and their metabolites in postmortem brains from patients with Alzheimer-typedementia, J. Neurochem., 43 (1984) 388 393. 2 Bondareff,W., Mountjoy, C.Q. and Roth, M., Loss of neurons of origin of the adrenergic projection to cerebral cortex (nucleus locus ceruleus) in senile dementia, Neurology, 32 (1982) 164 168.
237 3 Bylund, D.B. and U'Prichard, D.C., Characterization of alphar and alphaz-adrenergic receptors, Int. Rev. Neurobiol., 24 (1983) 343~431. 4 Cross, A.J., Crow, T.J., Johnson, J.A., Perry, E.K., Perry, R.H., Blessed, G. and Tomlinson, B.E., Studies in neurotransmitter receptor systems in neocortex and hippocampus in senile dementia of the Alzheimer-type, J. Neurol. Sci., 64 (1984) 109-117. 5 D'Amato, R.J., Zweig, R.M., Whitehouse, P.J., Wenk, G.L., Singer, H.S., Mayeux, R., Price, D.L. and Snyder, S.H., Aminergic systems in Ahheimer's disease and Parkinson's disease, Ann. Neurol., 22 (1987) 229-236. 6 Edvinsson, L., Lindvall, N., Nielsen, K.C. and Owman, C., Are brain vessels innervated also by central (non-sympathetic) adrenergic neurons?, Brain Res., 63 (1973) 496~499. 7 Ferrari-DiLeo, G. and Potter, L.T., Alpha-adrenoceptors and muscarine receptors in human pial arteries and microvessels: Receptor binding study, J. Cereb. Blood Flow Metab., 5 (1985) 458~,64. 8 Glossman, H., Lubbecke, F. and Belleman, P., ['25I]HEAT, A selective, high-affinity, high specific activity ligand for ~radrenoceptors, Naunyn-Schmiedeberg's Arch. Pharmacol., 318 (1981) 1-9. 9 Goldstein, G.W., Wolinsky, J,S., Csejtey, J. and Diamond, I., Isolation of metabolically active capillaries from rat brain, J. Neurochem., 25 (1975) 715 717. 10 Gottfries, C.G., Adolfsson, R., Aquilonius, S.M., Carlsson, A., Eckernas, S.K.A., Nordberg, A., Oreland, L., Svennerholm, L., Wiberg, A. and Winblad, B., Biochemical changes in the dementia disorders of Alzheimer type (AD/SDAT), Neurobiol. Aging, 4 (1984) 261-271. 11 Harik, S.I., Blood-brain barrier sodium-potassium pump: Modulation by central noradrenergic innervation, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 4067~4070. 12 Harik, S.I. and McGunigal, T,, The protective influence of the locus ceruleus on the blood-brain barrier, Ann. Neurol., 15 (1984) 568 574. 13 Harik, S.I., Sharma, V.K., Wetherbee, J.R., Warren, R.H. and Banerjee, S.P., Adrenergic and cholinergic receptors of cerebral microvessels, J. Cereb. Blood Flow Metab., 1 (1981) 329 338. 14 Hartman, B.K., Zide, D. and Udenfriend, S., The use of dopamine beta-hydroxylase as a marker for the central noradrenergic neuro system in rat brain, Proc. Natl. Acad. Sci. U.S.A., 69 (1972) 2722 2726. 15 Iversen, L.L., Rossor, M.N., Reynolds, G,P., Hills, R., Roth, M,, Mountjoy, C.Q., Foote, S.L., Morrison, J.H. and Bloom, F.E., Loss of pigmented dopamine beta hydroxylase positive cells from locus ceruleus in senile dementia of Alzheimer's type, Neurosci. Lett., 39 (1983) 95 100. 16 Kalaria, R.N. and Harik, S.I., Adrenergic receptors and the nucleoside transporter in human brain vasculature, J. Cereb. Blood Flow Metab.. 8 (1988) 32 39. 17 Kalaria, R.N. and Harik, S.I., Reduced glucose transporter at the blood-brain barrier and in cerebral cortex in Alzheimer disease, J. Neurochem., 53 (1989) 1083 1088. 18 Kalaria, R.N., Stockmeier, C.A. and Harik, S.I., Brain microvessels are innervated by locus ceruleus noradrenergic neurons, Neurosci. Lett., 97 (1989) 203-208. 19 Kalaria, R.N., Andorn, A.C., Tabaton, M., Whitehouse, P.J., Harik, S.I. and Unnerstall, J.R., Adrenergic receptors in aging and Alzheimer's disease: increased fl2-receptors in prefrontal cortex and hippocampus, J. Neurochem., 53 (1989) in press. 29 Kobayashi, H., Frattola, L., Ferrarese, C., Spano, P. and Trabucchi, M., Characterization of betaadrenergic receptors on human cerebral microvessels, Neurology, 32 (1982) 1384-1387. 21 Mooradian, A.D., Effect of aging on the blood-brain barrier, Neurobiol. Aging 9 (1988) 31 39. 22 Peroutka. S.J., Moskowitz, M.A., Reinhard, J.F. and Snyder, S.H., Neurotransmitter receptor binding in bovine cerebral microvessels, Science, 208 (1980) 610-612. 23 Rainbow, T.C., Parsons, B. and Wolfe, B.D., Quantitative autoradiography of fl- and fl2-adrenergic receptors in rat brain, Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 1585 1589. 24 Scatchard, G., The attractions of proteins for small molecules and ions, Ann. N.Y. Acad. Sci., 51 (1949) 660 672. 25 Shimohama, S., Taniguchi, T., Fujiwara, M. and Kameyama, M., Changes in fl-adrenergic receptor subtypes in Alzheimer-type dementia, J. Neurochem., 48 (1983) 1215-1221.
238 26 Shimohama, S., Taniguchi, T., Fujiwara, M. and Kameyama, M., Biochemical characterization of ~-adrenergic receptors in human brain and changes in Alzheimer-type dementia, J. Neurochem., 47 (1986) 1294- t351. 27 Swanson, L.W., Connelly, M.A. and Hartman, B.K.. Ultrastructural evidence for central monoaminergic innervation of blood vessels in paraventricular nucleus of the hypothalamus, Brain Res., 136 (1977) 166 173. 28 Unnerstall, J.R., Kopajtic, T.A. and Kuhar, M.J., Distribution of alpha2 agonists binding sites in the rat and human central nervous system: analysis of some functional, anatomical correlates of the pharmacologic effects of clonidine and related adrenergic agents, Brain Res. Rev., 7 (1984) 69 101.
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Increased 2- and fl2-adrenergic receptors in cerebral microvessels in Alzheimer disease R a j e s h N . K a l a r i a a n d S a m i I. H a r i k Department of Neurology, University Hospitals of Cleveland and Case Western Reserve University School of Medicine Cleveland, OH 44106 (U.S.A.)
(Received 18 May 1989; Revised version received 10 July 1989; Accepted 13 July 1989) Key words: Adrenergic receptor; Alzheimer disease; Aging; Cerebral microvessel; Cerebral cortex; Blood-brain barrier
Adrenergic receptors exist in brain microvessels which are innervated by noradrenergic locus ceruleus neurons. Biochemical and pathological studies indicate locus ceruleus degeneration in Ahheimer disease (AD), which can cause adrenergic receptor alterations in brain microvessels. To assess this, we studied adrenergic receptors in human brain microvessels from AD subjects and age-matched controls by ligand binding methods. Total fl receptors of cerebral microvessels and f12receptors, the type which predominates in microvessels, were significantly increased in AD. Compared to the cerebral cortex, there was a paucity of ~q-adrenergic receptors in cerebral microvessels, and they did not change in AD. Binding to ct2receptors in cerebral microvessels was ~ 50 % of that in the cortex, and these receptors increased by ~ 60 % in cerebral microvessels of AD subjects. These findings suggest adrenergic receptor 'upregulation' in response to noradrenergic deafferentation in AD, which may have functional consequences at the blood-brain barrier. A l t h o u g h several studies describe a b n o r m a l i t i e s in the b r a i n ' s n o r a d r e n e r g i c systems in A h h e i m e r disease ( A D ) [1, 2, 10, 15], there is conflicting evidence as to w h e t h e r b r a i n ~t- a n d fl-adrenergic receptors are affected [4, 5, 20, 25, 26]. It is n o w well established t h a t a d r e n e r g i c receptors, in p a r t i c u l a r fl receptors, exist in b r a i n microvessels [7, 13, 18, 20, 22]. These small i n t r a p a r e n c h y m a l vessels are p r o b a b l y i n n e r v a t e d , at least in part, by locus ceruleus n o r a d r e n e r g i c n e u r o n s [6, 14, 18, 27] but, thus far, there are no studies o f a d r e n o c e p t o r a l t e r a t i o n s in b r a i n microvessels in A D . Here, we investigated b y specific ligand b i n d i n g m e t h o d s the status o f ct- a n d fl-adrenergic r e c e p t o r s u b t y p e s in cerebral microvessels o f subjects with A D a n d in a g e - m a t c h e d controls. H u m a n b r a i n s a m p l e s were o b t a i n e d at a u t o p s y f r o m 14 n o r m a l c o n t r o l s ( 7 M / 7 F ) aged 55-78 years ( 6 9 + 2 , m e a n _ S . E . M . ) a n d 15 A D subjects ( 9 M / 6 F ) aged 61-79 years (72 __+2). The interval between d e a t h a n d b r a i n s a m p l i n g was 1 1 . 4 _ 1.8 h in controis a n d 7.7 + 1.3 h in A D subjects. Microvessels were harvested f r o m 50-100 g samCorrespondence: R.N. Kalaria, Department of Neurology, University Hospitals of Cleveland, 2074 Abington Road, Cleveland, OH 44106, U.S.A.
0304-3940/89/$ 03.50 © 1989 Elsevier Scientific Publishers Ireland Ltd.
234 pies of prefrontal cortex (Brodmann area 10) by the bulk isolation method of Goldstein et al. [9], as modified for human autopsy material in our laboratory [16, 17]. Microvessels were checked for their purity by microscopy and by biochemical assays of marker enzymes: ),-glutamyl transpeptidase, alkaline phosphatase and angiotensin-converting enzyme. Microvessels from AD subjects and controls showed no significant differences in these endothelial cell marker enzymes [16, 17]. Particulate membrane fractions of both cerebral microvessels and the prefrontal cortex from which the microvessels were obtained, were prepared by homogenization in a Brinkmann polytron and extensively washed in dilute buffer by centrifugation and resuspension, and used for binding studies. Adrenergic receptors were assessed by established ligand binding methods as follows: fl receptors and their subtypes were assayed using (-)-[125I]iodopindolol (spec. act. 2200 Ci/mmol, NEN), as described before [19, 23]. Tissues were incubated in 20 mM Tris-HC1 buffer, pH 7.5 containing 150 mM NaC1, 2.5 mM MgCI2, 0.5 mM ascorbate and varying concentrations of [125I]iodopindolol for 30 min at 37°C. fll and [~ receptors were determined using 60 nM ICI-118,551 and 80 nM ICI-89,406, antagonists at f12 and/31 receptors, respectively [23]. We have confirmed in earlier experiments that at these concentrations, the antagonists selectively recognize specific fl-receptor subtypes (Fig. 1 of ref. 19). Non-specific binding was determined using 100 I~M (-)-isoproterenol. 21 Receptors were assayed using (_+)-fl-([125I]iodo-4-hydroxyphenyl)-ethyl-aminomethyl-tetralone (HEAT) (spec. act. 2200 Ci/mmol, NEN). The ligand (0.2 mM) was incubated with 30-50/tg of tissue protein in 50 mM Na+-K + phosphate buffer, pH 7.4 for 1 h at 22°C [8]. Non-specific binding was determined in the presence of 10 ItM prazosin. [3H]p-aminoclonidine (PAC) (spec. act. 62.5 Ci/ mmol, NEN) at 1 nM was used to assess 22-adrenergic receptors [3]. Tissues were incubated in 20 mM Na+-HEPES buffer, pH 7.4, with 0.1 mM ascorbate for 1 h at 22'C. Non-specific binding was determined in the presence of 1/~M clonidine. In all cases equilibrium binding was terminated by rapid filtration under reduced pressure and washing 3 times with 4 ml of cold buffer. In experiments using 3H-ligands, the filters were counted in a fl scintillation counter at ~45% efficiency, while in experiments using ~25I-labeled ligands, the filters were counted in a gamma counter at ~ 70% efficiency. Specific binding was calculated by subtracting nonspecific binding from total binding. Maximal binding (Bmax) and (Kd) were calculated for fl receptors according to Scatchard [24]. Specific binding of [125I]iodopindolol, [125I]HEAT or [3H]PAC to cerebral microvessels correlated neither with the post-mortem interval nor with time of tissue storage. There were no differences between men and women in the binding of any ligands. /3 Receptor binding was performed primarily at a single [125I]iodopindolol concentration because of insufficient material to allow for Scatchard plots in microvessels obtained from any one subject. Fig. 1 shows that total fl receptor binding was significantly higher in cerebral microvessels obtained from AD subjects than in those from age-matched controls (P < 0.05). Analysis of total fl receptors into fll and f12 subtypes showed that f12 adrenoceptors predominate in human cerebral microvessels (Fig. 1), a finding consistent with our previous results in pig and rat brain microvessels [13,
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Fig. 1. Histograms show specific [~25I]pindololbinding to cerebral microvessels in AD subjects (crosshatched columns) and age-matched controls (open columns). The columns represent means and the bars represent S.E.M. for 12 individual AD subjects and 11 controls. Binding was performed at a single concentration of 150 pM [~25I]pindolol. Statistical significance was assessed by the 2-sample Student's t-test (2-tailed). *Different from control at P< 0.05. **Different from control at P<0.01. Fig. 2. Histograms show specific [125I]HEATbinding to ~ receptors and specific [3H]PAC binding to ~2 receptors in cerebral microvessels and prefrontal cortex in AD subjects (cross-hatched columns) and agematched controls (open columns). The columns represent means and the bars represent S.E.M. for 15 AD subjects and 14 controls. Binding was performed at a single concentration of 150 pM for [tzSI]HEATand 1 nM for [3H]PAC.Binding results for cortex (unpublished observations) are shown here for comparison to microvessels. **Different from controls at P < 0.01. ***Different from controls at P < 0.001.
18]. These f12 receptors were significantly increased in microvessels o f A D subjects (Fig. 1, P < 0 . 0 1 ) . To ascertain that the increased binding to total fl and fl2-adrenoceptors was due to increased binding sites rather than higher affinity o f binding in microvessels from A D subjects, we performed Scatchard plots on cerebral microvessels that were pooled from several subjects within each group. The Bmax for [125I]iodopindolol binding to total fl receptors was 32 + 0.7 fmol/mg protein in microvessels o f A D subjects and 2 6 + 0 . 9 fmol/mg protein in microvessels o f controls (means + S.E.M., n = 4 , P < 0.01, Student's t-test, 2-tailed). The Kd o f binding was 142 + 30 p M in A D subjects and 106 + 20 p M in controls (P > 0.05). The density and affinity o f f l adrenoceptors in cerebral microvessels in h u m a n control subjects is similar to the density and affinity o f binding in the cerebral cortex [19]. Similar findings were previously noted in rats and pigs [13]. Also, f12 adrenoceptors were increased in the cerebral cortex o f A D subjects when c o m p a r e d to controls, suggesting fl receptor 'upregulation' [19]. There were no significant changes in ~1 receptors in cerebral microvessels in A D (Fig. 2), although those receptors were significantly decreased in the cerebral cortex o f A D subjects. Specific [125I]HEAT binding in brain microvessels was a b o u t one third o f that in the cortex (Fig. 2). A l t h o u g h this result is consistent with a recent study [7] in which [3H]prazosin was used as the ligand, it is at variance with the study in pig and rat cerebral microvessels where [3H]WB-4101 was used [13]. The inconsistency is p r o b a b l y due to differences in species and receptor ligands.
236 The binding of [3H]PAC to ~2 receptors in cerebral microvessels was ~ 50% that of the cerebral cortex of control subjects (Fig. 2). This observation is consistent with earlier reports in humans [7] and other mammalians [13, 22]. However, in contrast to [3H]PAC binding in the cerebral cortex which was decreased by ~ 50% in AD subjects (P < 0.001), [3H]PAC binding in cerebral microvessels was increased by ~ 60% in AD ( P < 0.01, Fig. 2). The increased [3H]PAC binding in cerebral microvessels was due to a change in the Bmax rather than the Kd. Thus, our results show increased f12 and ~2 adrenoceptors in cerebral microvessels from AD subjects but no significant alterations in ~1 receptors. The increased f12 receptors in microvessels may be the result of noradrenergic deafferentation of cerebral microvessels in AD. These observations are similar to the 'denervation supersensitivity' of microvessel [32 receptors seen in rats with lesions of locus ceruleus [18], although the pathological processes in AD are much more complex. It is not clear why upregulation of fl~ adrenoceptors does not occur in the cerebral cortex of AD subjects where this receptor subtype predominates, as it does in rats after locus ceruleus lesion [18]. However, it is possible that in AD, the cerebral cortex is not capable of reacting to noradrenergic denervation while cerebral microvessels retain this capability. Cerebral microvessel 22 adrenoceptors also increase in A D but decrease in the cerebral cortex (Fig. 2). A similar mechanism as that invoked above for • receptors may be the explanation. Alternatively, these opposite changes in 22 receptors may be due to their different location. It is possible that the 22 receptors recognized by [3H]PAC are predominantly autoreceptors on presynaptic terminals in the human cerebral cortex [28], but are located postsynaptically on capillary endothelial cells [7]. The loss of ~2 receptors in the cortex is then the result of impaired synaptic connectivity in AD. Whatever the underlying cause of the adrenoceptor alterations in cerebral microvessels of A D subjects, these changes may have important functional consequences at the blood brain barrier. We have previously demonstrated that lesion of the nucleus locus ceruleus and norepinephrine depletion in the ipsilateral cerebral cortex is associated with decreased N a + , K + - A T P a s e in brain microvessels and increased permeability of the blood brain barrier to macromolecules, especially under certain pathophysiological conditions such as seizures and hypertension [i 1, 12]. Recent evidence also invokes abnormalities of the blood-brain barrier in explaining some of the brain pathology in AD [21]. We thank the Neuropathology Division, Institute of Pathology, C W R U for help in obtaining human postmortem tissue and Jeanette Barnhart for manuscript preparation. This work was supported in part by a grant to R.N.K. from the A D R D A , by USPHS Grants MH-43444 and HL~35617 and by the David S. Ingalls Fund. I Arai, H., Kosaka, K. and lizuka, R., Changes of biogenic amines and their metabolites in postmortem brains from patients with Alzheimer-typedementia, J. Neurochem., 43 (1984) 388 393. 2 Bondareff,W., Mountjoy, C.Q. and Roth, M., Loss of neurons of origin of the adrenergic projection to cerebral cortex (nucleus locus ceruleus) in senile dementia, Neurology, 32 (1982) 164 168.
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