Neurotensin, a central nervous system peptide: apparent receptor binding in brain membranes

Brain Research, 130 (1977) 299-313 ,~',) Elsevier/North-Holland Biomedical Press

299

N E U R O T E N S I N , A C E N T R A L NERVOUS SYSTEM PEPTIDE: A P P A R E N T R E C E P T O R B I N D I N G IN BRAIN M E M B R A N E S

GEORGE R. UHL, JAMES P. BENNETT, JR. and SOLOMON H. SNYDER* Departments of Pharmacology and Experimental Therapeutics and Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, Md. 21205 (U.S.A.)

(Accepted November 3rd, 1976)

SUMMARY Neurotensin, a tridecapeptide recently isolated from bovine hypothalami, has potent pharmacologic effects in several peripheral systems and a regional distribution in rat brain suggestive of a specialized function. 125I-neurotensin binds to membrane preparations from rat brain saturably, reversibly, and with high affinity (apparent KD :~ 3 nM) under conditions that minimize degradation of the polypeptide. This binding is displaced by neurotensin sequence fragments with relative potencies generally paralleling their potencies in peripheral systems. 125I-neurotensin binding is highest in specific thalamic, cerebral cortical, and hypothalamic areas of rat and calf brain. White matter, brain stem and cerebellar regions have substantially lower amounts of binding. Characteristics of this binding suggest an association with a physiologically relevant neurotensin receptor.

INTRODUCTION Several peptides, such as angiotensin, substance P and enkephalin, have prominent central nervous system influences suggestive of synaptic roles for these substances in the brainll,lL Recently, Carraway and Leeman isolated from bovine hypothalamic extracts a tridecapeptide, neurotensin, whose sequence is < Glu-Leu-TyrGlu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu-OH~-5. Neurotensin has potent pharmacological effects both in vivo and in vitro. Peripherally administered neurotensin elicits hypotension, increased vascular permeability, pain sensation, increased hematocrit, cyanosis, morphine-inhibitible stimulation of ACTH secretion, increased LH secretion, increased FSH secretion, and hyperglycemia G. In vitro smooth muscle effects of neurotensin include contraction of the estrous rat uterus and guinea pig ileum, and * To whom correspondence should be sent.

300 relaxation of the rat duodenum 6. Neurotensin injected intraventricularly ~ rats enhances barbiturate effects1°. Brain neurotensin levels determined by radioimmunoassay show striking regional variations. Levels in the hypothalamus are almost 30 times higher than cerebral cortical levels and about 200 times higher than cerebetlar concentrations 9 (G, Uhl and S.H. Snyder, in preparation). in the present study, we describe high affinity binding of ~al-neurotensin to membrane preparations from rat and calf brain that has characteristics resembling those anticipated of binding to physiological neurotensin receptors. METHODS

Radiolabeling of neurotensin Synthetic neurotensin was iodinated by a modification of the procedure of Cuatrecasas v. Ten #g of neurotensin dissolved in 64 #1 0.01 M acetic acid were added to a polyethylene centrifuge tube containing 100 #1 0.25 M sodium phosphate buffer (pH 7.4) and 2 mCi of NaV)SI. Iodination at 22 ~C was begun by adding 20,ul of chloramine T solution (5 mg/ml H20). The reaction was stopped after 30 sez by rapidly adding 20 #1 sodium metabisulfite (10 mg/ml H20) and 8 ml ice-cold 0.25 M sodium phosphate buffer (pH 7.4) containing 0.1 °/o bovine serum albumin (BSA). The peptide product was adsorbed to two 25 mg talc tablets which were vigorously suspended in the reaction mixture. The talc pellet produced by centrifugation at 50,000 ;z g for 10 rain (4 °C) was washed 5 times by resuspension in 10 ml sodium phosphate buffer (pH 7.4) and recentrifugation, lodinated peptide was eluted from the pellet by resuspension twice in 3 ml of 0.35 M HCt containing 5 }o BSA and centrifugation at 770 ~, g for 30 min (4 °C). The combined eluates were centrifuged twice (50,000 > g, 10 rain) to remove talc remnants, neutralized to pH 6 with NaOH, and rapidly frozen in aliquots of dry ice-acetone. l~Sl-neurotensin prepared by this method migrated as a single peak on cellulose thin-layer chromatography (n-butanol-acetic acid-H20, 4: I:1) and on paper electrophoresis (30 V/cm, 2 h, formic acid-acetic acid-H20, 8 : 2: 90). There was no detectable peak corresponding to free 125I. Biological activity of the iodinated neurotensin was not tested directly. However, 1251-neurotensin prepared in the same fashion binds with high affinity to a specific antiserum (see below). Binding experiments reported in detail here were performed using a single pool of lZSl-neurotensin.

Binding assay Male Sprague-Dawley rats (t 50-250 g) were decapitated. Brains were removed rapidly, placed in ice-cold Krebs-Ringer-Tris buffer (pH 7.4 at 37 °C) and dissected on dental wax over ice into cerebral hemispheres (cerebral cortex with white matter included) or cerebral cortex (from which most white matter had been removed), thalamus, hypothalamus, corpus striatum, hippocampus, midbrain, pons/medullaoblongata, and cerebellum. Tissue was drained, weighed, and disrupted in t0 vols (w/v) ice-cold 50 mM Tris buffer (pH 7.4 at 37 °C) with a Brinkmann Polytron (setting 6.5, I min). The pellet produced by an 8 rain centrifugation (4 °C, 50,000 × g) was

301 washed twice by Polytron resuspension in 10 vols Tris buffer and 5 rain (4 °C, 50,000 g) centrifugation. Washed pellets were resuspended in 8.6 vols of incubation buffer (20 mM Tris, pH 7.5 at 4 °C with 0.5 ~, BSA), and 0.86 ml of this ice-cold suspension was dispensed with stirring into palypropylene incubation tubes. Unlabeled peptides or other drugs and lZSl-neurotensin were added so that each incubation mixture routinely contained washed membranes from 100 mg wet brain weight in a final volume of I ml. Incubation for 30 rain at 4 °C was terminated by rapidly adding 3 ml Krebs-Ringer-Tris buffer and centrifuging (4 °C, 17,500 x g) for 8 rain. After discarding the supernatant fluid, the insides of the tubes and the pellets were rinsed three times with 5.5 ml aliquots of ice-cold 50 mM Tris buffer. The outsides of the inverted tubes were rinsed with warm tap water, and the tubes and pellets were counted in a Searle gamma spectrometer at an lZal counting efficiency of 90 Y~Jo. 'Specific a2'~l-neurotensin binding' was calculated by subtracting from the 'total' bound radioactivity the 'non-specifically' bound radioactivity not displaceable by 1 # M non-radioactive neurotensin. Each data point represents the mean of triplicate determinations with standard errors of the mean less than 5 °/,i of the indicated value, unless noted otherwise.

Radioimmunoassay Neurotensin radioimmunoassay was performed as described in detail elsewhere (G. R. Uhl and S. H. Snyder, in preparation). Briefly, an antiserum raised in guinea pigs after a series of injections of a neurotensin-hemocyanin conjugate bound 50-65 0J,/; of 10 fmoles added 125I-neurotensin at 1:500-1:600 dilution. Free a251-neurotensin was adsorbed to a charcoal-dextran slurry, which was pelleted by centrifugation. 'Bound' supernatant and 'free' pellet fractions were counted, and the per cent bound calculated. Unlabeled neurotensin displaced about half of 1251-neurotensin binding to this antiserum at I nM, while the N-cleaved (2-13), (4-13), (6-13), (8-13) and (9-13) neurotensin sequence fragments were less than 0.1 ~ as potent. Other polypeptides showing less than 0.1 ~ of neurotensin's potency at displacing leaI-neurotensin from this antiserum included: met-enkephalin, leu-enkephalin, a-endorphin, /5'-endorphin, GnRH, TRH, angiotensin l, angiotensin II, angiotensin il hexapeptide, angiotensin I I pentapeptide, substance P, bradykinin, glucagon, prolylleucylglycinamide, and histidylleucinamide.

Materials Synthetic neurotensin, neurotensin sequence fragments, substance P, angiotensin, and angiotensin sequence fragments were purchased from Beckman Instruments, Spinco Division, Palo Alto, Calif. NalZSl (IMS 0.30) was purchased from Amersham-Searle, Chicago, Ill. Cellulose thin-layer chromatography plates and chloramine T were obtained from Eastman Kodak, Rochester, N.Y. BSA was Sigma, fraction V. Incubation tubes were Fa[con no. 2053. Other reagents were obtained from commercial suppliers. Calf brains were obtained from a local slaughterhouse, and kept in ice-cold saline until use.

302

J251 REMAINING 6O,OO(.IN SUPERNATANT AFTER q*30' INCUBATION 3,060

AUTHENTIC Z CO( IZSI. FIEUROTENSIN '

45,00C-

Z,O00

.....!]

!25I REMAPNrNG IN SUPERNATANT AFTER 37"30' INCUBAT~N

.....

15,o~o

-

Distance

F r o m Origin ( C M )

Fig. 1. Thin-layer chromatography of preincubated and non-preincubated 12~l-neurotensin. 1251neurotensin was preincubated with membranes prepared from 100 mg wet weight rat cerebral cortex for 30 min at 4 °C and for 30 min at 37 °C. Aliquots of supernatant obtained followingcentrifugation of the incubation mixtures were chromatographed alongside non-pretreated authentic lzSI-neurotensin on cellulose TLC plates in 1-butanol-acetic-acid H~O, 4:1 : 1. Plates were cut into 1 cm strips and counted. RESULTS

Identity of 125I-neurotensin following &cubation with membranes Because many peptides are extensively degraded by soluble and membrane bound enzymes, receptor binding experiments require incubation conditions which minimize such degradation. In preliminary incubations at 37 °C for 30 min, considerable degradation of 125I-neurotensin is apparent in thin-layer chromatographic analyses of incubation media (Fig. 1, right). When incubations are conducted at 4 °C for 30 min in the presence of 0.5 ~ bovine serum albumin, negligible degradation of l~5l-neurotensin can be detected by thin-layer chromatographic analyses in solvent systems including 1-butanol-acetic acid-H20 (4:1 : 1) (Fig. 1, left). With these incubation conditions, high voltage electrophoretic analysis of radioactivity bound to membranes or remaining in the supernatant after centrifugation indicates no apparent catabolism of 125I-neurotensin (Fig. 2). It is conceivable that subtle changes in a peptide, not detectable by thin-layer chromatography or high voltage electrophoresis. might alter the biological properties of the peptide. Accordingly, after pre-incubating lzSI-neurotensin with membranes we assessed the ability of radioactive material eluted from these membranes and of radioactivity remaining in the supernatant to bind to fresh membrane preparations and to a specific anitserum to neurotensin (Table I). The membrane and antiserum binding properties of lzSI-neurotensin subjected to routine incubation procedures resemble those of non-preincubated authentic lZZlneurotensin. Variations in metabolism from one rat brain region to another are not evident in these studies.

Displacement of 12aI-neurotensin binding by unlabeled neurotensin Unlabeled neurotensin competes with high affinity for binding of subsaturating concentrations of 125 l-neurotensin (Fig. 3). Negligible displacement of the 125I-labeled

303

AUTHENTIC ¢/125I~NEUROTEN¢.

40

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REMAINING

35

30

25

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.

+

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a~

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lesI ELUTEO

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CM From Origin Fig. 2. Electrophoresis of preincubated and non-preincubated 125]-neurotensin. l~[-neurotensin was preJncubated for 30 rain at 4 °C with membranes prepared from 100 mg wet weight rat cerebra]

hemispheres. The pellet was rinsed 3 times with Tris buffer, eluted with l ml 0.0l M acetic acid by vortexing, and further incubated for 15 rain at 4 °C. Centrifugation (17,500 × g, 10 min) separated the 'eluate' from the eluted pellet. Aliquots of supernatant and eluate were electrophoresed on paper alongside untreated 'authentic' 125I-NTat 30 V/cm for 2 h in formic acid-acetic acid-H~O (8:2:90). One cm strips were cut and counted. ligand is apparent at concentrations of unlabeled neurotensin below 1 nM. At higher concentrations, however, displacement increases with 50~/o reduction of 125I-neurotensin binding at 3-4 n M displacer and with maximal displacement at unlabeled neurotensin concentrations exceeding 0.1 #M. Log-probit analysis of these data yields an apparent ICs0 (concentration inhibiting binding by 50 ~ ) of 3.7 nM. To examine for the presence of cooperativity, the data were also plotted according to the Hill equation. The Hill coefficient of 1.2 indicates the possibility of only a small degree of positive cooperativity. Hill coefficients from 4 replicate experiments show a mean of 1.17 ± 0.07 S.E.M.

Saturation of ~251-neurotensin binding Addition of increasing amounts of 125[-neurotensin brane preparation results in saturation of specific binding plateaus at 2-4 × 106 disint./min/ml with half-maximal 1.6 x 106 disint./min/ml. In routine assays in which 0.2 ×

to fixed amounts of mem(Fig. 4A). Specific binding specific binding at about 106 disint./min/ml of 125I-

304 TABLE I Binding o f preincubated and non-preincubated ~eSl-neurotensin to rat cerebral cortical membranes attd to neurotensin antibodies ~2H-neurotensin (~eal-NT) was preincubated for 30 min at 4 C with membranes prepared from 100 mg rat cerebral hemispheres (membrane binding) or from cerebral hemispheres, hipp0campus, or cerebellum (antiserum binding). The triply rinsed pellet was eluted with l ml 0.01 M acetic acid by vortexing and allowing dissociation for 15 rain at 4 'C. Centrifugation (17,500 ~ g, 10 min) separated the 'eluate' from the eluted pellet. Aliquots of the incubation medium, of NaOH-neutralized eluate, and of authentic neurotensin were added to both fresh membrane preparations and to a 1 : 500 dilution of guinea pig antineurotensin serum. Binding was determined as described in Methods. This experiment was replicated twice. Ligand

% added r~tdioactivity specifically bound to [resh rat cerebral hemisphere membrane preparations

% added radioactivity bound to specific antiserum and displaeible by I ItM unlabeled neurotensin

'Authentic' non-preincubated 12'~I-NT Supernatant v'~I-NT preincubated with cerebral hemisphere membranes Eluted 125I-NT preincubated with cerebral hemisphere membranes Supernatant ~25I-NT preincubated with hippocampat membranes Eluted 125I-NT preincubated with hippocampal membranes Supernatant 12~I-NT preincubated with cerebellar membranes Eluted ~5I-NT preincubated with cerebellar membranes

6. I I

41.0

5.43

39.9

5.20

40.4

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39.4 38.9

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38.2

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39.0

Rat Cerebral Cortex

4oc

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g

%

"D~2C ,o' °

~;~

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,b" [Neurotensin] (M)

,b-"

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Fig. 3. Displacement of 12~I-neurotensin binding by unlabeled neurotensin. Membrane preparations from 100 mg original wet weight of rat cerebral cortex were incubated with 0.5 n M ~2~l-neurotensin and varying concentrations of unlabeled neurotensin for 30 rain at 4 °C. Specific 12~l,neurotensin binding was assayed as described in Methods. Each point represents the mean of quadruplicate determinations with standard errors of the mean less than 2.5 ~ , This experiment has been replicated three times.

305 24-

RotCerebral

Hemispheres

.05

20-

~

KD=ICerebral 6ROtHemispheres •

18,0 16-

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NeurotensiAdded n (DPMx

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Fig. 4. Saturation of leSl-neurotensin binding. Increasing amounts of r'aI-neurotensin were incubated with membranes prepared from 100 mg wet weight rat cerebral hemispheres for 30 rain at 4 "C in the presence and absence of l i,M unlabeled neurotensin. Total, specific and nonspecific binding were determined as described in Methods (A). Scatchard analysis of the same data is shown in B. Linear regression of this data yields a slope corresponding to a KD of 1.6 x l0 n disint./min and a Bma× for cerebral hemisphere tissue of 8.83 × 104 disint./min. Replication of this experiment with cerebral cortex yielded a KD of 1.6 X 106 disint./min and a Bma~of 1.35 x 105 disint./min by linear regression. Since the cerebral white stripped from 'cerebral hemispheres' shows little binding, and since it constitutes one-quarter to one-third of the wet weight of the hemisphere, these two experiments show good agreement. This experiment has also been repeated three times with other ~e'~l-neurotensin preparations.

neurotensin are added, total binding is 2 4 times non-specific binding d e t e r m i n e d in the presence o f 1 # M unlabeled neurotensin. The ratio o f total to non-specific binding declines progressively at higher m e d i u m concentrations o f 12q-neurotensin. Scatchard analysis o f the same d a t a indicates the presence o f a single p o p u l a t i o n o f binding sites with an a p p a r e n t dissociation constant, (l/slope), o f 1.6 × 10 ~ disint./min/ml (Fig. 4B). If iodinated neurotensin has the same affinity for binding sites as native neurotensin, the a p p a r e n t dissociation constant for lZal-neurotensin binding o b t a i n e d from s a t u r a t i o n experiments should a g p r o x i m a t e the c o n c e n t r a t i o n o f unlabeled neurotensin which elicits h a l f - m a x i m a l displacement o f specific binding o f s u b s a t u r a t i n g concentrations o f 1251-neurotensin. This i n f o r m a t i o n permits an estimation o f the specific activity o f the 12q-neurotensin used in theae experiments as 169 Ci/mmole. Correspondingly, a maximal binding capacity (Bmax) o f 3.1 pmoles/g wet weight may be estimated for cerebral cortex.

Kinetics of 1251-neurotensin binding Specific l~51-neurotensin binding at 4 °C increases with time in a p a t t e r n re-

306 1001

Rat CerebralCortex

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15

20

25

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55

50

iNCUBATION TIME (minu~es)

Fig. 5. Association of a25I-neurotensin to rat cerebral cortical membranes. Incubations at 4 °C for varying durations of membranes prepared from 100 mg original wet weight of rat cerebral cortex with 0.5 nM 12~I-neurotensin were terminated by rapidly adding 3 ml ice-cold Krebs-Ringer-Tris buffer and centrifuging. Specific and non-specific binding were determined as described in Methods. Assuming pseudo first-order kinetics for the initial portion (0.5-3 rain) of this curve allows calculation of k, -- 4.1 × 10S/mole/sec. The figure represents three experiments, each involving triplicate determinations with standard errors of the mean less than 5 ~. This experiment was repeated three times with other preparations of X~5l-neurotensin.

sembling that seen in bimolecular reactions (Fig. 5). Specific binding plateaus by 30 rain with half-maximal binding apparent at about 5-10 min. By contrast, non-specific binding occurs very rapidly; at the earliest time intervals measured non-specific binding has almost reached maximal values. Assuming pseudo first-order kinetics for early time points, the rate constant for association, kl, is equal to (d[R--L])/(dt)/[R] [L] with [R], the m a x i m u m specific binding site concentration estimated from Scatchard analysis of 125I-neurotensin binding (Fig. 4B, 3.1 pmole/g wet weight); [L], the ligand concentration (0.2 × l0 s disint., min/ml, estimated to be 0.5 nM), and (d[R--L])/(dt), the initial rate of specific binding (28.51 disint./min/sec, estimated to be 7 × 10-17 molesjsec). The calculated ka is 4.1 × l0 n/mole~sec. As might be anticipated, association of 12~l-neurotensin at 37 °C is considerably more rapid; plateau binding values reached in 3-5 rain at 37 °C are similar to values attained after 30 min at 4 °C. The dissociation of bound 12~I-neurotensin was examined by labeling rat cerebral cortical membranes with t25I-neurotensin in the standard 30 rain incubation at 4 °C. Non-radioactive neurotensin (1 ,aM) was then added, and residual binding assayed at various time intervals thereafter (Fig. 6). Dissociation of 1251-neurotensin is linear when plotted semi-logarithmically; the apparent half-life for dissociation at 4 °C is 7.5 rain. The rate constant for dissociation determined by the equation k2 --=-(0.693)/(t1/D is 1.54 × 10-a/sec. The KD for 12~I-neurotensin binding to cerebral cortex membrane preparations may be calculated as the ratio of the rate constant for dissociation to the rate constant for association. This value is 3.7 riM. Thus, the dissociation constant determined b y kinetic experiments resembles values obtained in equilibrium studies.

307

Rat Cerebral Cortex

t~5Minutes z TIME(minutes)

Fig. 6. Dissociation of 12~I-neurotensin from rat cerebral cortical membranes. Membranes prepared from 100 mg original wet weight of rat cerebral cortex were incubated for 30 min with 0.5 nM 12~Ineurotensin at 4 cC. Unlabeled neurotensin (1/~M) was then added and further 4 °C incubations of varying durations were terminated by the rapid addition of 3 ml ice-cold Krebs-Ringer-Tris buffer and centrifugation. Semi-logarithmic plot of 12~I-neurotensin remaining bound vs. dissociation interval is linear with an apparent tx/2 of 7.5 min. Each point represents the mean of triplicate determinations, whose standard errors were less than 5 ~. This experiment was replicated three times.

Other properties of 125I-neurotensin binding Under standard 1 ml incubation conditions, specific 125I-neurotensin binding is linear with membrane concentration in preparations from between 12.5 mg and 200 nag original wet weight of rat cerebral cortex. Specific 125I-neurotensin binding to rat cerebral cortical membranes displays a broad pH optimum between 7.0 and 8.0. Binding declines to roughly one-half of peak values at pH 6.5 and at pH 8.5. Specific 125I-neurotensin binding measured at 4 °C declines markedly with the addition of increasing concentrations of several ions (Table lI). Binding is reduced to 30-40 ~ of control levels in the presence of 150 m M NaCI, LiC1, KCI, NH4CI, NH4Br or NH4NO3. Binding measured after 37 °C 4 rain incubations is, however, less susceptible to reduction by the addition of ions. Addition of 320 m M sucrose, an osmotically but not ionically active substance, has no effect on binding.

Binding properties of partial sequences of neurotensin and of other substances Relative physiological and binding potencies of a series of related and unrelated compounds provide important evidence of selectivity in assessing whether binding of a neurotransmitter or hormone involves the physiologically relevant receptor 13. Carraway and Leeman have assessed the abilities of several partial sequence fragments of neurotensin to mimic neurotensin's action in producing hypotension in the anesthetized

308 TABLE !1 Effects 0]' added substances on l~51-neurotensin binding

Membrane preparations from 100 mg original wet weight of rat cerebral cortex were incubated at 4 C for 30 rain in the standard 20 mM Tris (pH 7.5 at 4 '~C)0.5~ BSA incubation buffer to which various ions were added. Specific neurotensin binding was assayed as described in Methods. This experiment has been replicated twice. Ion

Concentration (raM)

NaCI

10 5O 100

10 2 1

84 57 43 4O 81 57 43 40 38 96 40 74 84 95

150 150 150 150

45 100 30 28

150

NH4CI

10 50 100

LiCI KCI

150 5

150

150

CaCI~ KH4PO4 MgSO4 Choline chloride Sucrose NH4Br NHaNOa

% Control specific binding

rat, hyperglycemia in rats, permeability enhancement in guinea pig peripheral vasculature, and contraction of the guinea pig ileum ~. Fragment potencies in their systems decline as shorter fragments are tested, with the sharpest decrement occurring between the 8-13 and the 9-13 fragments. We compared the abilities of 5 partial sequence fragments of neurotensin with neurotensin itself to compete for lZSl-neurotensin binding (Table Iit, Fig. 7). The 2-13 and 4-13 fragments have about the same potency as neurotensin itself in the binding assay, while the 6-13 fragment is about one-half as active as native neurotensin, The 8-13 fragment is about one-tenth as potent as neurotensin, while the 9-13 fragment has only about 0.5% of the affinity for 125I-neurotensin binding sites. The existence of substantial binding potency in neurotensin fragments with deletions of up to 7 amino acids from the N-terminus parallel the retention of pharmacologic activity in the 2-13, 4-13, 6-13 and 8-13 fragments found by Carraway and Leeman 6. The loss of an additional arginine in conversion of the 8-13 to the 9-13 fragment results in a pronounced decline in binding potency and almost complete abolition of physiologic responses. There are, however, some discrepancies between binding potencies i n the brain and peripheral pharmacologic activity. The 4-13 fragment possesses onty25 % of the pharmacologic activity of native neurotensin, although the binding affinity of this fragment is about the same as that of neurotensin. In elicitating hypotension and

309 TABLE Ill

Di,~placement o/'le~l-neurotensin binding by neurotensin sequence.fragments IC50 values for displacement of ~2~I-neurotensin binding were calculated for each sequence fragment by log-probit analysis of the data displayed in Fig. 7. Substances with 1Cs0 values in excess of I /tM include: angiotensin If, angiotensin II pentapeptide, angiotensin II hexapeptide, L-aspartate, bacitracin, dopamine, leucine-enkephalin, methionine-enkephalin, glucagon, L-glutamate, glutathione, HipHis-Leu, His-Leu, norepinephrine, Pro-Leu-G[y, prolactin, serotonin, substance P, tyramine, octopamine, phenylethanolamine, imipramine, ergotamine, haloperidol, chlorpromazine, LSD, diazepam, strychnine, atropine, burimamide and hydralazine.

Compound

ICm) (riM)

Neurotensin < Gfu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-lle-Leu-OH (2 13) fragment H-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-ife-Leu-OH (4 13) fragment H-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-lle-Leu-OH (6 13) fragment H-Lys-Pro-Arg-Arg-Pro-Tyr-lle-Leu-OH (8-13) fragment H-Arg-Arg-Pro-Tyr-lle-Leu-OH (9 13) fragment H-Arg-Pro-Tyr-lle- Leu-OH

~ IOC

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3.7 2.0 2.7 5.8 48 830

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m

10"9

10-6

[Displocer] (M) Fig. 7. J2~l-neurotensin binding displacement by unlabeled neurotensin and neurotensin sequence fragments. Membrane preparations from 100 mg original wet weight of rat cerebral cortex were incubated with 0.5 nM 12~l-neurotensin and varying concentrations of unlabeled neurotensin ( A - A ) and its 2-13 (0-0), 4 13 (rag-[]), 6-13 (/'~-L), 8-13 (O-O), and 9 13 ( ~ - [ ] ) sequence fragments. Specific teaI-neurotensin binding was assayed as described in Methods. The figure represents data obtained in three experiments, each experiment being comprised of triplicate (fragments) or quadruplicate (neurotensin) determinations with standard errors of the mean less than 5 o,/

310 TABLE IV

Regional chLs'tribution of specific v'-al-neurotensin binding in calf brain Regions from fresh calf brains were dissected on ice. Cerebral cortical samples were taken from areas named by analogy to human cerebral cortical subdivision, as follows: 'frontal' and 'occipital' poles were sampled from the most anterior and posterior cerebral cortical gyri respectively, 'Anterior', 'mid' and 'posterior cingulate gyri were sampled from the cerebral gyri dorsal to the genu, body, and splenium of the corpus callosum, respectively. Fragments of 'parahippocampal' gyri were dissected from cortex immediately overlying the body of the hippocampus in the ventromedial aspect of the cerebral hemispheres. Cortical gyri anterior and posterior to the central sulcus were sampled as the 'precentral' and 'postcentral' gyri respectively. The 'frontal' and 'parietal' opercula were taken from the most lateral gyri in their respective lobes. Finally, cortex at the junction of the parietal, occipital, and temporal lobes was sampled as 'insular' cortex. Subcortical regions were dissected from their more standardized anatomic loci. Each regional dissection involved membranes prepared from one calf except for hypothalamic subdivisions, where tissues from 4 calves were pooled. Binding assays on membranes prepared from each dissection were performed in triplicate. Specific binding values obo.' tained from each region for each dissection were normalized, with frontal pole set as !00/0 specific binding. Frontal pole specifically bound about 13 fmoles per gram original wet weight following a rather lengthy tissue preparation. Reported values for each brain region are the mean : S.E.M. for 3 regional dissections.

Region

% specific binding C/i'ontal pole 100 "o)

Dorsomedial thalamus Superior parahippocampal gyrus Inferior parahippocampal gyrus Medial hypothalamus Anterior hypothalamus Ventral tier of thalamus Anterior cingulate gyrus Mammillary body Occipital pole Mid-cingulate gyrus Anterior thalamus Posterior cingulate gyrus Caudate nucleus Frontal pole Pulvinar of thalamus Amygdala Precentral gyrus Frontal operculum Insular cortex Parietal operculum Putamen Postcentral gyrus Superior colliculus Hippocampus Inferior colliculus Globus pallidus Dorsal pons Ventral ports Cerebellar cortex MeduUa-oblongata Cerebral white Cervical spinal cord

249 188 173 165 144 136 132 132 126 125

: 3.8 ~ 22 j: 8.0 j 22 i 21 4:38 13 16 -i 5 ~ 12 112 : 8.0 106 ~ 7.8 100 :! 13 100 99.0:1 12 98.0 ! 4.3 96.5 :! 13 96.5 :~ 25 95.2 - 9.5 92.4 ::i 11 90.6 12 88.6 i 9.3 88.2 [ 7.9 87.9 :~ 21 85.6 :k I 1 83.7 k 20 68.3 =~ 3.8 50.4 =~ 7.6 30.5 ~ 2.9 27.5 3:15 26.6 " 7 13.0 ~ 5.4

311 TABLE V Regional distribution oJ'specific 1251-neurotensin binding in rat brain

Regions from fresh rat brains were dissected on ice. Each regional dissection involved membranes prepared from 8 rats. Binding assays on membranes prepared from each dissection were performed in triplicate. Specific binding values obtained from each region for each dissection were normalized, with the pons/medulla-oblongata value for that dissection set as 100~ specific binding. Medulla-oblongata bound about 17 fmoles/g wet weight after a relatively brief period of tissue preparation. Reported values for each region are the mean ± S.E.M. for 5 rat dissections. Region

% Specific binding (medulla~ports = 100 %)

Cerebral cortex Hypothalamus Thalamus Midbrain Hippocampus Corpus striatum Cerebellum Medulla oblongata/pons

265 ~ 10 229 :~:: 9 216 ~= 10 195 ~ 11 162 ~ 15 136 i 14 105 ~ 7 100

hyperglycemia, the 8-13 fragment is more potent than the 6-13 fragment, although in the vascular permeability and receptor binding systems the 8-13 fragment is no more potent than the 6-13 fragment 6. The specificity of displacement of 1251-neurotensin binding is supported by the failure of several peptides including angiotensin II, angiotensin sequence fragments, substance P, the enkephalins, glucagon, prolactin, prolyleucylglycinamide, and bacitracin to displace more than 50 ~ of bound 1251-neurotensin at 1/~M concentration. Other non-peptide neurotransmitter candidates and drugs tested are similarly ineffective. Regional distribution o f lZSl-neurotensin binding Marked variations in specific 12~l-neurotensin binding to membranes prepared from various regions of rat and calf brain are detectable with our assay (Tables IV and V). Highest specific binding in the calf brain is detected in the area of the dorsomedial thalamus. The pulvinar, anterior thalamus and ventral tier thalamus have roughly half the specific binding of the dorsomedial nucleus. Significant variations in specific binding are also seen from region to region of the cerebral cortex. The 'parahippocampal' gyri display 70 ~ of the binding seen in the dorsomedial thalamus. By contrast, the postcentral gyrus, precentral gyrus, frontal operculum, insular cortex, and frontal pole have only about one-third of the binding seen in the dorsomedial thalamus. Binding values obtained from the cingulate gyri and occipital pole are intermediate between values obtained from the parahippocampal gyri and those detected in other cerebral cortical regions. Levels of binding in the hypothalamus are similar to the high values in the parahippocampal gyri. Little neurotensin is specifically bound to the medulla-oblongata, cerebellar cortex, cervical spinal cord, or cerebral white

312 matter. Binding to the cervical spinal cord is one-twentieth of binding to the dorsomedial thalamus. Similar regional differences are apparent in rat brain, where the lowest amounts of specific lZSl-neurotensin binding are seen in the pons/medulla-oblongata and the cerebellum. Highest levels of binding occur in the cerebral cortex and the hypothalamus. High levels of binding are also observed in the thalamus and midbrain, while the hippocampus and the corpus striatum display values intermediate between the low values of the cerebellum and pons/medulla-obtongata and the highest levels of the hypothalamus and cerebral cortex. DISCUSSION The major finding of this study is that ~eSI-neurotensin binds saturably, reversibly, with marked regional variation, and with high affinity to brain membranes in a fashion suggesting an association with a physiologically relevant neurotensin receptor. The relative potencies of various fragments of neurotensin in competing for binding to brain membranes parallel in a general way their relative pharmacologic activities on peripheral tissues. It is unclear whether the sites that mediate pharmacologic effects of neurotensin in the periphery are identical with binding sites in the central nervous system. The relative potencies of neurotensin fragments in various pharmacologic tests can differ substantially. For instance, the 8-13 fragment retains a great deal of activity in hypotensive actions in the anesthetized rat, but is much weaker than neurotensin in influencing vascular permeability~. Also, while the 2-13 fragment has the same potency as neurotensin in eliciting hyperglycemia and vascular permeability increases, it has only half of the activity of neurotensin in contracting the guinea pig ileum 6. In preliminary experiments we have detected binding of 12~l-neurotensin to membrane preparations from rat uterus. Displacement of uterine membrane binding by partial sequence fragments of neurotensin resembles the relative affinities of the fragments for binding sites in the brain. In comparing biological and binding activities of neurotensin fragments, it is important to consider possible metabolism. Our binding conditions have been designed to protect neurotensin and its partial sequences from degradation; we are unable to detect significant degradation of neurotensin incubated under these conditions with electrophoretic, chromatographic, membrane binding, or antiserum binding systems. It is possible that fragments of neurotensin are differentially degraded in various peripheral pharmacologic tests. Neurotensin binding appears to vary from region to region in the rat and calf brains. It seems unlikely that uneven regional distribution of neurotensin degradative activity accounts for these differences, l~5I-neurotensin samples previously incubated with membrane preparations from rat brain regions showing high, intermediate, and low levels of specific binding resemble authentic 12aI-neurotensin in binding to a specific antiserum. The regional binding variations show similarities to variations in endogenous levels of neurotensin detected by radioimmunoassay9, (G. R, Uhl and S. H. Snyder, in preparation). Thus the hypothalamus is enriched both in neurotensin binding and endogenous neurotensin, while the cerebellar cortex has the lowest levels

313 of binding and e n d o g e n o u s neurotensin. The disparity between the substantial neurotensin b i n d i n g and the low e n d o g e n o u s n e u r o t e n s i n levels in the cerebral cortex resembles disparities between the high levels of receptor b i n d i n g for serotonin, a-adrenergic, and fl-noradrenergic receptor b i n d i n g in the cerebral cortex a n d the low endogenous levels of these amines in this region1,9, s. ACKNOWLEDGEMENTS This research was supported by U S P H S Research G r a n t DA-00266, a Research Scientist Development Award, MH-33128 (S.H.S.) and a U S P H S T r a i n i n g G r a n t I-T01-GM-02191 (G.R.U.). We wish to t h a n k Dr. Morley Hollenberg for bringing n e u r o t e n s i n to our attention.

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