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Neurotensin receptor binding, regional and subcellular distributions favor transmitter role
European Journal of Pharmacology, 41 (1977) 89--91
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© Elsevier/North-Holland Biomedical Press, Amsterdam -- Printed in The Netherlands Rapid c o m m u n i c a t i o n N E U R O T E N S I N R E C E P T O R BINDING, R E G I O N A L AND S U B C E L L U L A R DISTRIBUTIONS F A V O R T R A N S M I T T E R ROLE
GEORGE R. UHL and SOLOMON H. SNYDER Departments of Pharmacology and Experimental Therapeutics and Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, U.S.A.
Received 11 November 1976, accepted 15 November 1976
Neurotensin, a biologically active tridecapeptide ~ Glu-Leu-Tyr-Glu-Asn-Lys-Pro-ArgArg-Pro-Tyr-Ile-Leu-OH, was elegantly isolated from bovine hypothalamic extracts and characterized by Carraway and Leeman (1973, 1975). These authors have reported development of a radioimmunoassay allowing detection of neurotensin immunoreactivity in hypothalami of several different species (Carraway and Leeman, 1974). ~2SI-Neurotensin binds to brain membranes with high affinity and substrate specificity suggesting interactions with physiological neurotensin receptors (Uhl et al., 1977). This receptor binding, as well as a preferential localization of brain neurotensin immunoreactivity to synaptosomal fractions and to specific grey matter areas (Uhl and Snyder, 1976; S.E. Leeman, personal communication) is consistent with a neurotransmitter candidate role for neurotensin in the brain. Synthetic neurotensin (Beckman) was iodinated by a Chloramine T m e t h o d and purified by adsorption to talc; specific activity calculated on the basis of membrane binding was 169 Ci/mmole (Uhl et al., 1977). Membranes were prepared by homogenizing brain regions with a Polytrone ®, centrifuging (50,000 X g, 8 min), and washing twice with resuspension and recentrifugation. Washed membrane suspensions were incubated for 30 min at 4°C in 20 mM Tris buffer pH 7.5
with 0.5% bovine serum albumin. Pellets resulting from 10 min 17,500 × g centrifugation were superficially rinsed and counted. Specific 12SI-neurotensin binding was calculated by subtracting from total b o u n d radioactivity the non-specific binding in the presence of 1 pM unlabeled neurotensin. After 4°C 30 rain incubation with brain membranes, 12SI-neurotensin is not appreciably degraded as demonstrated by thin layer chromatography, high voltage electrophoresis, rebinding to membranes, and antiserum binding, though degradation can be demonstrated after 37 ° C incubations (Uhl et al., 1977). Unlabeled neurotensin competes with high affinity for the binding of l:s Ineurotensin, with 50% displacement at a b o u t 3 nM and a Hill coefficient at 1.2, indicating the absence of cooperativity. Saturation of specific binding is evident with increasing levels of 125I-neurotensin. One binding site is detected by Scatchard analysis with maximal binding capacity of 3 pmoles/g rat cerebral cortex. The association of l:sIneurotensin at 4°C to specific membrane binding sites resembles a bimolecular interaction, with an association constant ( k , ) of 4 × 10 s/M/sec while dissociation is exponential with a rate constant (k ~) of 1.5 × 10-3/sec. The K D determined as the ratio of the rate constant for dissociation to the rate constant for association is 3.7 nM, closely resembling values determined in equilibrium studies.
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Marked regional variations exist in neurotensin binding. Highest levels are found in the dorsomedial thalamus, parahippocampal cortex, and hypothalamus, lowest levels in the cerebellum and brainstem, and intermediate values in the basal ganglia (table 1). The relative abilities of five partial se-
quence fragments to compete for 12SI-neurotensin binding in general resembles their relative pharmacological potencies in a number of peripheral systems (S.E. Leeman, personal communication) (table 1). The 2--13 and 4--13 fragments have about the same potency as neurotensin in the displace-
TABLE 1 Characteristics of neurotensin membrane binding and radioimmunoassay. Brain membrane binding
Radioimmunoassay
Dissociation constant (KD) 3 nM
50% displacement 1 nM (200 fmoles)
Rate constant, association (k 1 ) 4.1 X 10S/M/sec Rate constant, dissociation ( k 1 ) 1.5 X 10-3/sec Binding site number (Bmax)3.1 pmoles/g rat cerebral cortex
Sensitivity less than 75 fmoles
Specificity (ICs0 values, nM) (2--13) 2.0 (4--13) 2.7 (6--13) 5.8 (8--13) 48 (9--13) 830 ICs0 values greater than 1 ]2M: angiotensin, bacitracin, enkephalins, glucagon, substance P, prolactin,
Recovery /> 90% Specificity less than 0.1% cross reactivity: (2--13), (4--13), (6--13), (8--13) and (9--13) neurotensin sequence fragments, enkephalins, endorphins, GnRH, TRH, angiotensins, bradykinin, substance P, glucagon Subcellular distribution (rat hypothalamus) Fraction immunoreactive neurotensin (pmoles/g)
Calf brain regional distribution High regions (2.5--1.2 X frontal pole): thalamus (dorsomedial, ventral, anterior) hypothalamus (anterior, medial, mamillary body) cerebral cortex (parahippocampal, cingulate, occipital) Intermediate regions (1.2-0.7 X frontal pole) : cerebral cortex (precentral, postcentral, frontal, insular, colliculi, pulvinar of thalamus, caudate, putamen, globus pallidus, hippocampus, amygdala Low regions (less than 0.7 X frontal pole) : pons, cerebellar cortex, cervical spinal cord, medulla oblongata, cerebral white
Whole P1 (10,000 × g, 10 min) P2 ( 7,500 X g, 20 rain) S2
30 4 15 21
P2 subfractionation: A (0.32--0.8 M sucrose) B (0.8--1.2 M sucrose) C (1.2 M sucrose and pellet) Calf brain regional distribution High regions (>/8 pmoles/g wet): hypothalamus (anterior, medial, mamillary body) caudate, globus pallidus Intermediate regions (2.5--8 pmoles/g wet): cortex (parahippocampal, cingulate, occipital) thalamus (anterior), hippocampus, colliculi Low regions (1--2.5 pmoles/g wet): cortex (frontal, precentral, parietal), amygdala, pons, medulla oblongata, cervical spinal cord Very low regions (less than 1 pmole/g wet): cerebral white, cerebellar cortex
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ment of 12SI-neurotensin binding, while the 6--13 fragment is 0.5%, the 8--13 fragment is 0.1%, and the 9--13 fragment is 0.5% as potent as neurotensin itself in this system. The existence of binding in fragments with deletions of up to seven amino acids from the Nterminus parallels retention of pharmacological activity in these fragments; both binding and pharmacological activities are lost in conversion of the 8--13 to the 9--13 fragments. However, the 2--13 and 4--13 fragments are relatively more potent in displacing bound 12SI-neurotensin than in peripheral pharmacological tests. Numerous other peptides and nonpeptides have negligible affinity for neurotensin binding sites (table 1). The high affinity, reversible, selective binding properties of neurotensin and the general parallel with pharmacological activity of fragments suggests that the binding sites labeled here represent physiologically relevant neurotensin receptors in the brain, A sensitive, selective radioimmunoassay for neurotensin with characteristics shown in table 1 has facilitated comparison of regional distributions of immunoreactive neurotensin and of receptor binding (Uhl and Snyder, 1976) (table 1). The calf hypothalamus is rich in neurotensin immunoreactivity and in binding sites, while white matter, brainstem and cerebellar cortical regions are low in both. Discrepancies between regional binding and immunoreactivity data may also be noted; thalamocortical regions rank relatively higher in the former, basal ganglia rank higher in the latter. The regional distribution of neurotensin immunoreactivity in the rat parallels findings in the calf (S.E. Leeman, personal communication; G.R. Uhl, and S.H. Snyder, in preparation).
Neurotensin immunoreactivity is preferentially localized in crude mitochondrial (P2) and microsomal (P3) subcellular fractions (Uhl and Snyder, 1976) (table 1). When P2 is further fractionated on discontinuous sucrose gradients, neurotensin immunoreactivity is enriched in the synaptosome containing fraction. Although this fraction contains particles other than synaptosomes, preferential localization of neurotensin immunoreactivity to subcellular synaptosomal fractions, along with the existence of binding with receptor-like properties, is consistent with a neurotransmitter candidate role for neurotensin.
Acknowledgements Supported by USPHS grants DA-00266 and MH33128 (to S.H.S.) and 5 TO 1 GM01183-13 (to G.R.U.).
References Carraway, R. and S.E. Leeman, 1973, The isolation of a new hypotensive peptide, neurotensin, from bovine hypothalami, J. Biol. Chem. 248, 6854. Carraway, R. and S.E. Leeman, 1974, The amino acid sequence, chemical synthesis, and radioimmunoassay of neurotensin, Federation Proc. 33, 548. Carraway, R. and S.E. Leeman, 1975, The amino acid sequence of a hypothalamic peptide, neurotensin, J. Biol. Chem. 250, 1907. Uhl, G.R. and S.H. Snyder, 1976, Regional and subcellular distributions of brain neurotensin, Life Sci. (in press). Uhl, G.R., J.P. Bennett, Jr. and S.H. Snyder, 1977, Neurotensin, a central nervous system peptide: apparent receptor binding in brain membranes, Brain Res. (in press).
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© Elsevier/North-Holland Biomedical Press, Amsterdam -- Printed in The Netherlands Rapid c o m m u n i c a t i o n N E U R O T E N S I N R E C E P T O R BINDING, R E G I O N A L AND S U B C E L L U L A R DISTRIBUTIONS F A V O R T R A N S M I T T E R ROLE
GEORGE R. UHL and SOLOMON H. SNYDER Departments of Pharmacology and Experimental Therapeutics and Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, U.S.A.
Received 11 November 1976, accepted 15 November 1976
Neurotensin, a biologically active tridecapeptide ~ Glu-Leu-Tyr-Glu-Asn-Lys-Pro-ArgArg-Pro-Tyr-Ile-Leu-OH, was elegantly isolated from bovine hypothalamic extracts and characterized by Carraway and Leeman (1973, 1975). These authors have reported development of a radioimmunoassay allowing detection of neurotensin immunoreactivity in hypothalami of several different species (Carraway and Leeman, 1974). ~2SI-Neurotensin binds to brain membranes with high affinity and substrate specificity suggesting interactions with physiological neurotensin receptors (Uhl et al., 1977). This receptor binding, as well as a preferential localization of brain neurotensin immunoreactivity to synaptosomal fractions and to specific grey matter areas (Uhl and Snyder, 1976; S.E. Leeman, personal communication) is consistent with a neurotransmitter candidate role for neurotensin in the brain. Synthetic neurotensin (Beckman) was iodinated by a Chloramine T m e t h o d and purified by adsorption to talc; specific activity calculated on the basis of membrane binding was 169 Ci/mmole (Uhl et al., 1977). Membranes were prepared by homogenizing brain regions with a Polytrone ®, centrifuging (50,000 X g, 8 min), and washing twice with resuspension and recentrifugation. Washed membrane suspensions were incubated for 30 min at 4°C in 20 mM Tris buffer pH 7.5
with 0.5% bovine serum albumin. Pellets resulting from 10 min 17,500 × g centrifugation were superficially rinsed and counted. Specific 12SI-neurotensin binding was calculated by subtracting from total b o u n d radioactivity the non-specific binding in the presence of 1 pM unlabeled neurotensin. After 4°C 30 rain incubation with brain membranes, 12SI-neurotensin is not appreciably degraded as demonstrated by thin layer chromatography, high voltage electrophoresis, rebinding to membranes, and antiserum binding, though degradation can be demonstrated after 37 ° C incubations (Uhl et al., 1977). Unlabeled neurotensin competes with high affinity for the binding of l:s Ineurotensin, with 50% displacement at a b o u t 3 nM and a Hill coefficient at 1.2, indicating the absence of cooperativity. Saturation of specific binding is evident with increasing levels of 125I-neurotensin. One binding site is detected by Scatchard analysis with maximal binding capacity of 3 pmoles/g rat cerebral cortex. The association of l:sIneurotensin at 4°C to specific membrane binding sites resembles a bimolecular interaction, with an association constant ( k , ) of 4 × 10 s/M/sec while dissociation is exponential with a rate constant (k ~) of 1.5 × 10-3/sec. The K D determined as the ratio of the rate constant for dissociation to the rate constant for association is 3.7 nM, closely resembling values determined in equilibrium studies.
90
Marked regional variations exist in neurotensin binding. Highest levels are found in the dorsomedial thalamus, parahippocampal cortex, and hypothalamus, lowest levels in the cerebellum and brainstem, and intermediate values in the basal ganglia (table 1). The relative abilities of five partial se-
quence fragments to compete for 12SI-neurotensin binding in general resembles their relative pharmacological potencies in a number of peripheral systems (S.E. Leeman, personal communication) (table 1). The 2--13 and 4--13 fragments have about the same potency as neurotensin in the displace-
TABLE 1 Characteristics of neurotensin membrane binding and radioimmunoassay. Brain membrane binding
Radioimmunoassay
Dissociation constant (KD) 3 nM
50% displacement 1 nM (200 fmoles)
Rate constant, association (k 1 ) 4.1 X 10S/M/sec Rate constant, dissociation ( k 1 ) 1.5 X 10-3/sec Binding site number (Bmax)3.1 pmoles/g rat cerebral cortex
Sensitivity less than 75 fmoles
Specificity (ICs0 values, nM) (2--13) 2.0 (4--13) 2.7 (6--13) 5.8 (8--13) 48 (9--13) 830 ICs0 values greater than 1 ]2M: angiotensin, bacitracin, enkephalins, glucagon, substance P, prolactin,
Recovery /> 90% Specificity less than 0.1% cross reactivity: (2--13), (4--13), (6--13), (8--13) and (9--13) neurotensin sequence fragments, enkephalins, endorphins, GnRH, TRH, angiotensins, bradykinin, substance P, glucagon Subcellular distribution (rat hypothalamus) Fraction immunoreactive neurotensin (pmoles/g)
Calf brain regional distribution High regions (2.5--1.2 X frontal pole): thalamus (dorsomedial, ventral, anterior) hypothalamus (anterior, medial, mamillary body) cerebral cortex (parahippocampal, cingulate, occipital) Intermediate regions (1.2-0.7 X frontal pole) : cerebral cortex (precentral, postcentral, frontal, insular, colliculi, pulvinar of thalamus, caudate, putamen, globus pallidus, hippocampus, amygdala Low regions (less than 0.7 X frontal pole) : pons, cerebellar cortex, cervical spinal cord, medulla oblongata, cerebral white
Whole P1 (10,000 × g, 10 min) P2 ( 7,500 X g, 20 rain) S2
30 4 15 21
P2 subfractionation: A (0.32--0.8 M sucrose) B (0.8--1.2 M sucrose) C (1.2 M sucrose and pellet) Calf brain regional distribution High regions (>/8 pmoles/g wet): hypothalamus (anterior, medial, mamillary body) caudate, globus pallidus Intermediate regions (2.5--8 pmoles/g wet): cortex (parahippocampal, cingulate, occipital) thalamus (anterior), hippocampus, colliculi Low regions (1--2.5 pmoles/g wet): cortex (frontal, precentral, parietal), amygdala, pons, medulla oblongata, cervical spinal cord Very low regions (less than 1 pmole/g wet): cerebral white, cerebellar cortex
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ment of 12SI-neurotensin binding, while the 6--13 fragment is 0.5%, the 8--13 fragment is 0.1%, and the 9--13 fragment is 0.5% as potent as neurotensin itself in this system. The existence of binding in fragments with deletions of up to seven amino acids from the Nterminus parallels retention of pharmacological activity in these fragments; both binding and pharmacological activities are lost in conversion of the 8--13 to the 9--13 fragments. However, the 2--13 and 4--13 fragments are relatively more potent in displacing bound 12SI-neurotensin than in peripheral pharmacological tests. Numerous other peptides and nonpeptides have negligible affinity for neurotensin binding sites (table 1). The high affinity, reversible, selective binding properties of neurotensin and the general parallel with pharmacological activity of fragments suggests that the binding sites labeled here represent physiologically relevant neurotensin receptors in the brain, A sensitive, selective radioimmunoassay for neurotensin with characteristics shown in table 1 has facilitated comparison of regional distributions of immunoreactive neurotensin and of receptor binding (Uhl and Snyder, 1976) (table 1). The calf hypothalamus is rich in neurotensin immunoreactivity and in binding sites, while white matter, brainstem and cerebellar cortical regions are low in both. Discrepancies between regional binding and immunoreactivity data may also be noted; thalamocortical regions rank relatively higher in the former, basal ganglia rank higher in the latter. The regional distribution of neurotensin immunoreactivity in the rat parallels findings in the calf (S.E. Leeman, personal communication; G.R. Uhl, and S.H. Snyder, in preparation).
Neurotensin immunoreactivity is preferentially localized in crude mitochondrial (P2) and microsomal (P3) subcellular fractions (Uhl and Snyder, 1976) (table 1). When P2 is further fractionated on discontinuous sucrose gradients, neurotensin immunoreactivity is enriched in the synaptosome containing fraction. Although this fraction contains particles other than synaptosomes, preferential localization of neurotensin immunoreactivity to subcellular synaptosomal fractions, along with the existence of binding with receptor-like properties, is consistent with a neurotransmitter candidate role for neurotensin.
Acknowledgements Supported by USPHS grants DA-00266 and MH33128 (to S.H.S.) and 5 TO 1 GM01183-13 (to G.R.U.).
References Carraway, R. and S.E. Leeman, 1973, The isolation of a new hypotensive peptide, neurotensin, from bovine hypothalami, J. Biol. Chem. 248, 6854. Carraway, R. and S.E. Leeman, 1974, The amino acid sequence, chemical synthesis, and radioimmunoassay of neurotensin, Federation Proc. 33, 548. Carraway, R. and S.E. Leeman, 1975, The amino acid sequence of a hypothalamic peptide, neurotensin, J. Biol. Chem. 250, 1907. Uhl, G.R. and S.H. Snyder, 1976, Regional and subcellular distributions of brain neurotensin, Life Sci. (in press). Uhl, G.R., J.P. Bennett, Jr. and S.H. Snyder, 1977, Neurotensin, a central nervous system peptide: apparent receptor binding in brain membranes, Brain Res. (in press).