214
Brain Reaearch, 113 (1976) 214 218 :~:- Elsevier Scientific Publishing Company, Amsterdam
Printed in The Netherlands
fl-Adrenergic receptor binding: synaptic localization in rat brain
JAMES N. DAVIS and ROBERT J. LEFKOWITZ
Neurology Section, Durham V. A. Hospital, and Departments of Medicine and Biochemistry, Duke University, Durham, N.C. 27705 (U.S.A.) (Accepted May 14th, 1976)
Despite extensive studies of the anatomy, pharmacology and electrophysiology of noradrenergic neurons in the central nervous system, little is known about adrenergic receptors in the brain. In the peripheral sympathetic nervous system the classification of a and fl receptors is based on the potency of agonists, such as epinephrine, norepinephrine and isoproterenol, in eliciting receptor-coupled responses and on the specificity of antagonists, such as propranolol and phentolamine, which preferentially block one type of receptor, fl-adrenergic receptors are coupled to the membrane enzyme, adenylate cyclase, in peripheral organs allowing the study of receptor responses in tissue homogenates by measurements of adenylate cyclase activation. Brain homogenization appears to uncouple the fl-adrenergic receptor from adenylate cyclase, although some recent reports suggest that some adrenergic activation of adenylate cyclase can be obtained3, 5,11. The study of adrenergic receptors in brain slices has been complicated since cyclic-AMP accumulation occurs in response to both a- and fl-adrenergic agents and varies among brain regions and speciesL The measurement of fl-adrenergic receptors by radioligand binding assays is now possible. We recently described the binding of (--)-[SH]dihydroalprenolol (DHA), a potent fl-adrenergic receptor antagonist, to rat cerebral cortex membranes 1. We concluded that DHA was binding to a site with the properties of a fll-adrenergic receptor based on the potency of inhibition of DHA binding by a series of agonists and antagonists. In the present study we report the distribution of DHA binding in subcellular fractions of rat cortical homogenates and a striking localization of binding to synaptosomes in the crude mitochondrial fraction. Rat cerebral cortex was homogenized in 0.32 M sucrose, pH 7.4, in a teflon-glass homogenizer with a 250/~1 clearance using 10 slow up-and-down strokes at a pestle speed of 300 rev/min. The homogenate was centrifuged at 800 × g for 15 min, the supernatant decanted, and the pellet rehomogenized with two up-and-down strokes by hand. A second 800 × g centrifugation for 15 min was performed and this pellet taken as the nuclear fraction. The combined supernatants were centrifuged at 11,000 × g for 20 min and the resultant pellet resuspended in sucrose and recentrifuged at I 1,000 × g to yield the crude synaptosomal fraction. In some experiments this synaptosomal fraction was incubated at 23 °C with (--)-[3H]norepinephrine (10 -8 M) in a mod-
215 ified Krebs-Ringer solution 4. The combined supernatants from the 11,000 centrifugations were centrifuged at 100,000 × g for 60 min to yield a microsomal pellet. The crude synaptosomal pellet after exposure to (--)-[aH]norepinephrine was centrifuged, resuspended in 0.32 M sucrose, and layered on a 0.8-1.5 M linear sucrose density gradient. These gradients were centrifuged for 60 rain in a SW 41 rotor at 100,000 × g; 0.5 ml fractions were collected from the gradients by puncturing the bottom of the tubes. An aliquot of each fraction was diluted to 0.4 M sucrose with additional 0.32 M sucrose and centrifuged at 14,000 x g for 20 min. The resultant pellets were suspended in 0.1 N HC1 and the [aH]norepinephrine was counted directly in a Triton-based scintillation fluid with a Packard liquid scintillation counter. A second aliquot of each fraction was used to determine the sucrose concentration by refractive index and the protein by the method of Lowry 10. A final aliquot of the fractions from the sucrose gradients was washed repeatedly in large volumes of buffer (75 mM TrisHC1, pH 7.4; 25 mM MgCI2). The membranes resulting from this wash were assayed for adenylate cyclase in the presence of 10 mM NaF 9, monoamine oxidase activity 14, and (--)-[3H]dihydroalprenolol binding la. Electron microscopy was performed on appropriate fractions from the sucrose gradient after centrifugation, exposure to buffered glutaraldehyde and staining with osmium tetroxide. fl-Adrenergic receptor binding activity was present in the nuclear, synaptosomalmitochondrial and microsomal pellets from rat cerebral cortex. The nuclear pellet contained 0.13 pmole/mg protein of DHA binding sites representing 4 ~ of the total DHA binding in the homogenate. Binding in the crude synaptosomal pellet (0.26 pmole/mg protein) and the microsomal pellet (0.25 pmole/mg protein) represented 55 ~ and 41 ~ of the total DHA binding respectively. The crude synaptosomal fraction was characterized by density gradient centrifugation. The uptake of [3H]norepinephrine was maximal in particles sedimenting between 1.0 and 1.2 M sucrose (Fig. 1B) and marked the distribution of synaptosomes. A close association of (--)-[SH]norepinephrine uptake and fl-adrenergic receptor binding was present in the 6 gradients studied (Fig. 1). In contrast both basal and fluoride-stimulated adenylate cyclase activities were found throughout the gradient and were most concentrated in membranes from lighter fractions than the fl-adrenergic receptor binding. Catecholaminestimulated adenylate cyclase was not detected. Monoamine oxidase activity was also present throughout the gradient but was predominantly in membranes from the heavier fractions. Fractions from the peak of monoamine oxidase activity on these gradients (Fig. IA) contained many mitochondria, a rare synaptosome and a moderate number of loose membranes when examined with the electron microscope (Fig. 2). Fractions taken at the peak of fl-adrenergic receptor binding and (--)-[3H]norepinephrine uptake (Fig. IB) contained many synaptosomes; often synaptosomes had attached postsynaptic membranes (Fig. 2). Fraction C from the top of the gradient contained loose membranes and myelin. It has been suggested that brain adenylate cyclase is associated with synaptosomal membranes 6,12. However these previous studies have reported the distribution of
216 x
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B-ADRENERGIC ~ RECEPTORS ,f~ ~
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Fig. 1. Linear sucrose gradient of the mitochondrial-synaptosomal fraction of rat cerebral cortex. A, B, and C denote the fractions taken for electron microscopy and correspond to A, B, and C in Fig. 2. IAA, indoteacetic acid; C-AMP, cyclic adenosine 3',5'-monophosphate.
adenylate cyclase on szmple discontinuous sucrose gradients. The present study is the first report of the distribution of adenylate cyclase from the crude mitochondrial pellet on a linear sucrose density gradient. Fluoride-stimulated adenylate cyclase activity was widely distributed on these gradients and was associated with both synaptosomes and loose membranes. Brain is a unique tissue for the study of membrane-bound enzymes since synaptosomes form when homogenization is carried out in sucrose. Thus a crude separation of membrane components is possible on linear sucrose density gradients of the synaptosomal-mitochondrialfraction. In the present study ~-adrenergic receptor binding was separated from the bulk of adenylate-cyclase containing membranes. The striking feature of these studies is the association of fl-adrenergic receptor binding with synaptosomes. Glial cells in culture demonstrate fl-adrenergic receptorcoupled adenylate cyclase activity7 and thus the possibility that DHA may be binding to non-neuronal fl-adrenergic receptors in this study cannot be excluded. An argument has recently been made that 'gliosomes', glial membrane fragments, might form during homogenization in sucrose and sediment with synaptosomess. However, this hypoth-
Fig. 2. Electron microscopy of fractions from the linear sucrose gradient in Fig. 1. A, B, and C correspond to fractions marked A, B, and C in Fig. 1. A synaptosome with attached postsynaptic membrane is indicated by the arrow in B.
218 esis was based on observations o f glial m e m b r a n e s f r o m cultured glial cells on disc o n t i n u o u s sucrose gradients. In the present study loose m e m b r a n e s a n d small vesicles are present in all o f the fractions from the linear sucrose gradient. H o w e v e r the close association o f D H A binding with [3H]norepinephrine u p t a k e a n d the lack o f binding to m e m b r a n e s from n e a r b y fractions with a d e n y l a t e cyclase activity strongly suggest t h a t D H A binding is synaptic. It s h o u l d be noted t h a t the D H A binding in the nuclear a n d m i c r o s o m a l pellet c o u l d be n o n - n e u r o n a l and further that a differentation between pre- a n d p o s t s y n a p t i c receptors is n o t possible f r o m these studies. P e r h a p s the most significant implication o f these findings is the possibility o f using D H A binding as a biochemical marker. A t t e m p t s to isolate and purify synaptic m e m b r a n e proteins have been hindered by the lack o f a specific biochemical m a r k e r for this region 2. These studies suggest that D H A binding m a y be a useful tool for c h a r a c t e r i z i n g the synaptic region in rat brain.
1 Alexander, R. W., Davis, J. N. and Lefkowitz, R. J., Direct identification and characterization of fl-adrenergic receptors in the rat brain, Nature (Lond.), 258 (1975) 437-440. 2 Barondes, S. H., Synaptic macromolecules: identification and metabolism, Ann. Rev. Biochem., 43 (1974) 147-168. 3 Chasin, M., Mamrack, F. and Samaniego, S. G., Preparation and properties of a cell-free, hormonally responsive adenylate cyclase from guinea pig brain, J. Neurochem., 22 (1974) 1031-1038. 4 Coyle, J. T. and Snyder, S. H., Catecholamine uptake by synaptosomes in homogenates of rat brain; stereospecificity in different areas, J. Pharmacol., 170 (1969) 221-231. 5 Daly, J. W., Cyclic adenosine 3',5'-monophosphate role in the physiology and pharmacology of the central nervous system, Bioehem. PharmacoL, 24 (1975) 159-164. 6 De Robertis, E., De Lores Arnaiz, G. R., Alberici, M., Butcher, R. W. and Sutherland, E. W., Subcellular distribution of adenyl cyclase and cyclic phosphodiesterase in rat brain cortex, J. biol. Chem., 242 (1967) 3487-3493. 7 Gilman, A. G. and Nirenberg, M., Effect of catecholamines on the adenosine 3':5'-cyclic monophosphate concentrations of clonal satellite cells of neurons, Proc. nat. Acad. ScL (Wash.), 68 (1971) 2165-2169. 8 Henn, F. A., Anderson, D. J. and Rustael, D. G., Glial contamination of synaptosomal fractions, Brain Research, 101 (1976) 341-344. 9 Letkowitz, R. J., Stimulation of catecholamine-sensitive adenylate cyclase by 5'-guanylyl-imidodiphosphate, J. biol. Chem., 249 (1974) 6119-6124. 10 Lowry, O. H., Rosebrough, N. J., Farr, L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 11 Von Hungen, K. and Roberts, S., Adenylate-cyclasereceptors for noradrenergic neurotransmitters in rat cerebral cortex, Europ. J. Biochem., 36 (1973) 391-401. 12 Weiss, B. and Costa, E., Regional and subcellular distribution of adenylate cyclase and 3',5'-cyclic nucleotide phosphodiesterase in brain and pineal gland, Biochem. Pharmaeol., 17 (1968) 2107-2116. 13 Williams, L. T., Jarrett, L. and Lefkowitz, R. J., Adipocyte fl-adrenergic receptors. Identification and subcellular localization by (--)-[3H]dihydroalprenolol binding, J. biol. Chem., (1976) in press. 14 Wurtman, R. J. and Axelrod, J., A sensitive and specific assay for the estimation of monoamine oxidase, Biochem. PharmacoL, 12 (1963) 1439-1441.
Brain Reaearch, 113 (1976) 214 218 :~:- Elsevier Scientific Publishing Company, Amsterdam
Printed in The Netherlands
fl-Adrenergic receptor binding: synaptic localization in rat brain
JAMES N. DAVIS and ROBERT J. LEFKOWITZ
Neurology Section, Durham V. A. Hospital, and Departments of Medicine and Biochemistry, Duke University, Durham, N.C. 27705 (U.S.A.) (Accepted May 14th, 1976)
Despite extensive studies of the anatomy, pharmacology and electrophysiology of noradrenergic neurons in the central nervous system, little is known about adrenergic receptors in the brain. In the peripheral sympathetic nervous system the classification of a and fl receptors is based on the potency of agonists, such as epinephrine, norepinephrine and isoproterenol, in eliciting receptor-coupled responses and on the specificity of antagonists, such as propranolol and phentolamine, which preferentially block one type of receptor, fl-adrenergic receptors are coupled to the membrane enzyme, adenylate cyclase, in peripheral organs allowing the study of receptor responses in tissue homogenates by measurements of adenylate cyclase activation. Brain homogenization appears to uncouple the fl-adrenergic receptor from adenylate cyclase, although some recent reports suggest that some adrenergic activation of adenylate cyclase can be obtained3, 5,11. The study of adrenergic receptors in brain slices has been complicated since cyclic-AMP accumulation occurs in response to both a- and fl-adrenergic agents and varies among brain regions and speciesL The measurement of fl-adrenergic receptors by radioligand binding assays is now possible. We recently described the binding of (--)-[SH]dihydroalprenolol (DHA), a potent fl-adrenergic receptor antagonist, to rat cerebral cortex membranes 1. We concluded that DHA was binding to a site with the properties of a fll-adrenergic receptor based on the potency of inhibition of DHA binding by a series of agonists and antagonists. In the present study we report the distribution of DHA binding in subcellular fractions of rat cortical homogenates and a striking localization of binding to synaptosomes in the crude mitochondrial fraction. Rat cerebral cortex was homogenized in 0.32 M sucrose, pH 7.4, in a teflon-glass homogenizer with a 250/~1 clearance using 10 slow up-and-down strokes at a pestle speed of 300 rev/min. The homogenate was centrifuged at 800 × g for 15 min, the supernatant decanted, and the pellet rehomogenized with two up-and-down strokes by hand. A second 800 × g centrifugation for 15 min was performed and this pellet taken as the nuclear fraction. The combined supernatants were centrifuged at 11,000 × g for 20 min and the resultant pellet resuspended in sucrose and recentrifuged at I 1,000 × g to yield the crude synaptosomal fraction. In some experiments this synaptosomal fraction was incubated at 23 °C with (--)-[3H]norepinephrine (10 -8 M) in a mod-
215 ified Krebs-Ringer solution 4. The combined supernatants from the 11,000 centrifugations were centrifuged at 100,000 × g for 60 min to yield a microsomal pellet. The crude synaptosomal pellet after exposure to (--)-[aH]norepinephrine was centrifuged, resuspended in 0.32 M sucrose, and layered on a 0.8-1.5 M linear sucrose density gradient. These gradients were centrifuged for 60 rain in a SW 41 rotor at 100,000 × g; 0.5 ml fractions were collected from the gradients by puncturing the bottom of the tubes. An aliquot of each fraction was diluted to 0.4 M sucrose with additional 0.32 M sucrose and centrifuged at 14,000 x g for 20 min. The resultant pellets were suspended in 0.1 N HC1 and the [aH]norepinephrine was counted directly in a Triton-based scintillation fluid with a Packard liquid scintillation counter. A second aliquot of each fraction was used to determine the sucrose concentration by refractive index and the protein by the method of Lowry 10. A final aliquot of the fractions from the sucrose gradients was washed repeatedly in large volumes of buffer (75 mM TrisHC1, pH 7.4; 25 mM MgCI2). The membranes resulting from this wash were assayed for adenylate cyclase in the presence of 10 mM NaF 9, monoamine oxidase activity 14, and (--)-[3H]dihydroalprenolol binding la. Electron microscopy was performed on appropriate fractions from the sucrose gradient after centrifugation, exposure to buffered glutaraldehyde and staining with osmium tetroxide. fl-Adrenergic receptor binding activity was present in the nuclear, synaptosomalmitochondrial and microsomal pellets from rat cerebral cortex. The nuclear pellet contained 0.13 pmole/mg protein of DHA binding sites representing 4 ~ of the total DHA binding in the homogenate. Binding in the crude synaptosomal pellet (0.26 pmole/mg protein) and the microsomal pellet (0.25 pmole/mg protein) represented 55 ~ and 41 ~ of the total DHA binding respectively. The crude synaptosomal fraction was characterized by density gradient centrifugation. The uptake of [3H]norepinephrine was maximal in particles sedimenting between 1.0 and 1.2 M sucrose (Fig. 1B) and marked the distribution of synaptosomes. A close association of (--)-[SH]norepinephrine uptake and fl-adrenergic receptor binding was present in the 6 gradients studied (Fig. 1). In contrast both basal and fluoride-stimulated adenylate cyclase activities were found throughout the gradient and were most concentrated in membranes from lighter fractions than the fl-adrenergic receptor binding. Catecholaminestimulated adenylate cyclase was not detected. Monoamine oxidase activity was also present throughout the gradient but was predominantly in membranes from the heavier fractions. Fractions from the peak of monoamine oxidase activity on these gradients (Fig. IA) contained many mitochondria, a rare synaptosome and a moderate number of loose membranes when examined with the electron microscope (Fig. 2). Fractions taken at the peak of fl-adrenergic receptor binding and (--)-[3H]norepinephrine uptake (Fig. IB) contained many synaptosomes; often synaptosomes had attached postsynaptic membranes (Fig. 2). Fraction C from the top of the gradient contained loose membranes and myelin. It has been suggested that brain adenylate cyclase is associated with synaptosomal membranes 6,12. However these previous studies have reported the distribution of
216 x
7.
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20
B-ADRENERGIC ~ RECEPTORS ,f~ ~
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,~,o"
B
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400 N
200
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',
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~._
0
Fig. 1. Linear sucrose gradient of the mitochondrial-synaptosomal fraction of rat cerebral cortex. A, B, and C denote the fractions taken for electron microscopy and correspond to A, B, and C in Fig. 2. IAA, indoteacetic acid; C-AMP, cyclic adenosine 3',5'-monophosphate.
adenylate cyclase on szmple discontinuous sucrose gradients. The present study is the first report of the distribution of adenylate cyclase from the crude mitochondrial pellet on a linear sucrose density gradient. Fluoride-stimulated adenylate cyclase activity was widely distributed on these gradients and was associated with both synaptosomes and loose membranes. Brain is a unique tissue for the study of membrane-bound enzymes since synaptosomes form when homogenization is carried out in sucrose. Thus a crude separation of membrane components is possible on linear sucrose density gradients of the synaptosomal-mitochondrialfraction. In the present study ~-adrenergic receptor binding was separated from the bulk of adenylate-cyclase containing membranes. The striking feature of these studies is the association of fl-adrenergic receptor binding with synaptosomes. Glial cells in culture demonstrate fl-adrenergic receptorcoupled adenylate cyclase activity7 and thus the possibility that DHA may be binding to non-neuronal fl-adrenergic receptors in this study cannot be excluded. An argument has recently been made that 'gliosomes', glial membrane fragments, might form during homogenization in sucrose and sediment with synaptosomess. However, this hypoth-
Fig. 2. Electron microscopy of fractions from the linear sucrose gradient in Fig. 1. A, B, and C correspond to fractions marked A, B, and C in Fig. 1. A synaptosome with attached postsynaptic membrane is indicated by the arrow in B.
218 esis was based on observations o f glial m e m b r a n e s f r o m cultured glial cells on disc o n t i n u o u s sucrose gradients. In the present study loose m e m b r a n e s a n d small vesicles are present in all o f the fractions from the linear sucrose gradient. H o w e v e r the close association o f D H A binding with [3H]norepinephrine u p t a k e a n d the lack o f binding to m e m b r a n e s from n e a r b y fractions with a d e n y l a t e cyclase activity strongly suggest t h a t D H A binding is synaptic. It s h o u l d be noted t h a t the D H A binding in the nuclear a n d m i c r o s o m a l pellet c o u l d be n o n - n e u r o n a l and further that a differentation between pre- a n d p o s t s y n a p t i c receptors is n o t possible f r o m these studies. P e r h a p s the most significant implication o f these findings is the possibility o f using D H A binding as a biochemical marker. A t t e m p t s to isolate and purify synaptic m e m b r a n e proteins have been hindered by the lack o f a specific biochemical m a r k e r for this region 2. These studies suggest that D H A binding m a y be a useful tool for c h a r a c t e r i z i n g the synaptic region in rat brain.
1 Alexander, R. W., Davis, J. N. and Lefkowitz, R. J., Direct identification and characterization of fl-adrenergic receptors in the rat brain, Nature (Lond.), 258 (1975) 437-440. 2 Barondes, S. H., Synaptic macromolecules: identification and metabolism, Ann. Rev. Biochem., 43 (1974) 147-168. 3 Chasin, M., Mamrack, F. and Samaniego, S. G., Preparation and properties of a cell-free, hormonally responsive adenylate cyclase from guinea pig brain, J. Neurochem., 22 (1974) 1031-1038. 4 Coyle, J. T. and Snyder, S. H., Catecholamine uptake by synaptosomes in homogenates of rat brain; stereospecificity in different areas, J. Pharmacol., 170 (1969) 221-231. 5 Daly, J. W., Cyclic adenosine 3',5'-monophosphate role in the physiology and pharmacology of the central nervous system, Bioehem. PharmacoL, 24 (1975) 159-164. 6 De Robertis, E., De Lores Arnaiz, G. R., Alberici, M., Butcher, R. W. and Sutherland, E. W., Subcellular distribution of adenyl cyclase and cyclic phosphodiesterase in rat brain cortex, J. biol. Chem., 242 (1967) 3487-3493. 7 Gilman, A. G. and Nirenberg, M., Effect of catecholamines on the adenosine 3':5'-cyclic monophosphate concentrations of clonal satellite cells of neurons, Proc. nat. Acad. ScL (Wash.), 68 (1971) 2165-2169. 8 Henn, F. A., Anderson, D. J. and Rustael, D. G., Glial contamination of synaptosomal fractions, Brain Research, 101 (1976) 341-344. 9 Letkowitz, R. J., Stimulation of catecholamine-sensitive adenylate cyclase by 5'-guanylyl-imidodiphosphate, J. biol. Chem., 249 (1974) 6119-6124. 10 Lowry, O. H., Rosebrough, N. J., Farr, L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 11 Von Hungen, K. and Roberts, S., Adenylate-cyclasereceptors for noradrenergic neurotransmitters in rat cerebral cortex, Europ. J. Biochem., 36 (1973) 391-401. 12 Weiss, B. and Costa, E., Regional and subcellular distribution of adenylate cyclase and 3',5'-cyclic nucleotide phosphodiesterase in brain and pineal gland, Biochem. Pharmaeol., 17 (1968) 2107-2116. 13 Williams, L. T., Jarrett, L. and Lefkowitz, R. J., Adipocyte fl-adrenergic receptors. Identification and subcellular localization by (--)-[3H]dihydroalprenolol binding, J. biol. Chem., (1976) in press. 14 Wurtman, R. J. and Axelrod, J., A sensitive and specific assay for the estimation of monoamine oxidase, Biochem. PharmacoL, 12 (1963) 1439-1441.
