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Neuroscienee Letters, 67 (1986) 275-278 275 Elsevier Scientific Publishers Ireland Ltd. NSL 03976 INCREASED NEUROPEPTIDE Y (NPY) RECEPTOR BINDING IN HIPPOCAMPUS AND CORTEX OF SPONTANEOUS HYPERTENSIVE (SH) RATS COMPARED TO NORMOTENSIVE (WKY) RATS RAYMOND S.L. CHANG*, VICTOR J. LOTTI and TSING-BAU CHEN Merck Sharp and Dohme Research Laboratories, Department of Microbial Pharmacometries, West Point, PA 19486 (U.S.A.) (Received January 28th, 1986; Revised version received and accepted March 7th, 1986) Key words: neuropeptideY NPY receptor brain SH rat-WKY rat Specific ~:51-NPY binding in various brain regions of spontaneous hypertensive (SH) rats and age- matched normotensive (WKY) rats was compared. SH rats exhibited significantly greater ~2~I-NPY bind- ing than WKY rats in the hippocampus (43%) and cortex (18%), but not hypothalamus, midbrain, striatum or pons-medulla. Scatchard analysis indicated that the increased t2SI-NPY binding in the hippocampus of SH rats represents a greater number of NPY binding sites. Neuropeptide Y (NPY), a member of the pancreatic polypeptide family, has been isolated from mammalian central nervous system [10]. Several lines of evidence sug- gest that NPY may be involved in the central control of blood pressure. NPY coexists with epinephrine and norepinephrine in some central adrenergic neurons [8] which are associated with blood pressure regulation [3]. Intracisternal administration of NPY in rats also causes a marked hypotensive response [7]. Recently, NPY has been found in-vitro to increase the number of ct2-adrenergic binding sites in the medulla oblongata of normotensive, but not spontaneous hyper- tensive (SH) rats [1, 2]. Differences in the regional brain concentrations of NPY of SH and normotensive (WKY) rats have also been reported [9]. The present studies were undertaken to determine whether such differences in NPY between these rat strains might be associated with differences in the number or distribution of NPY binding sites. 125I-NPY binding in several brain regions was compared in SH and normotensive (WKY) rats. Male SH rats and age-matched (3- and 6-month-old) WKY rats were obtained from Charles River. 125I-Bolton-Hunter NPY binding was performed as described pre- viously [4]. In concurrent studies, SH and WKY rats were sacrificed by decapitation, the brains removed and various brain regions dissected. The brain tissues were homoge- *Author for correspondence.

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Page 1: Increased neuropeptide Y (NPY) receptor binding in hippocampus and cortex of spontaneous hypertensive (SH) rats compared to normotensive (WKY) rats

Neuroscienee Letters, 67 (1986) 275-278 275 Elsevier Scientific Publishers Ireland Ltd.

NSL 03976

INCREASED NEUROPEPTIDE Y (NPY) RECEPTOR BINDING IN HIPPOCAMPUS AND CORTEX OF SPONTANEOUS HYPERTENSIVE (SH) RATS COMPARED TO NORMOTENSIVE (WKY) RATS

RAYMOND S.L. CHANG*, VICTOR J. LOTTI and TSING-BAU CHEN

Merck Sharp and Dohme Research Laboratories, Department of Microbial Pharmacometries, West Point, PA 19486 (U.S.A.)

(Received January 28th, 1986; Revised version received and accepted March 7th, 1986)

Key words: neuropeptideY NPY receptor brain SH r a t - W K Y rat

Specific ~:51-NPY binding in various brain regions of spontaneous hypertensive (SH) rats and age- matched normotensive (WKY) rats was compared. SH rats exhibited significantly greater ~2~I-NPY bind- ing than WKY rats in the hippocampus (43%) and cortex (18%), but not hypothalamus, midbrain, striatum or pons-medulla. Scatchard analysis indicated that the increased t2SI-NPY binding in the hippocampus of SH rats represents a greater number of NPY binding sites.

Neuropeptide Y (NPY), a member of the pancreatic polypeptide family, has been isolated from mammalian central nervous system [10]. Several lines of evidence sug- gest that NPY may be involved in the central control of blood pressure. NPY coexists with epinephrine and norepinephrine in some central adrenergic neurons [8] which are associated with blood pressure regulation [3]. Intracisternal administration of NPY in rats also causes a marked hypotensive response [7].

Recently, NPY has been found in-vitro to increase the number of ct2-adrenergic binding sites in the medulla oblongata of normotensive, but not spontaneous hyper- tensive (SH) rats [1, 2]. Differences in the regional brain concentrations of NPY of SH and normotensive (WKY) rats have also been reported [9]. The present studies were undertaken to determine whether such differences in NPY between these rat strains might be associated with differences in the number or distribution of NPY binding sites. 125I-NPY binding in several brain regions was compared in SH and normotensive (WKY) rats.

Male SH rats and age-matched (3- and 6-month-old) WKY rats were obtained from Charles River. 125I-Bolton-Hunter NPY binding was performed as described pre-

viously [4]. In concurrent studies, SH and WKY rats were sacrificed by decapitation, the

brains removed and various brain regions dissected. The brain tissues were homoge-

*Author for correspondence.

Page 2: Increased neuropeptide Y (NPY) receptor binding in hippocampus and cortex of spontaneous hypertensive (SH) rats compared to normotensive (WKY) rats

276

TABLE 1

SPECIFIC 125I-NPY B I N D I N G IN VARIOUS BRAIN REGIONS OF SH A N D W K Y RATS

Values are the mean _+ S.E.M. from 5 rats in each group. Values for SH and W K Y rats in the matched

brain regions were determined in the same experiments. *P < 0.005; (unpaired t-test); **P < 0.05 (unpaired

t-test).

Brain regions I251-NPY binding (fmol/g tissue)

6-month-old 3-month-old

W K Y SH W K Y SH

Hippocampus 590 + 19 844 + 54* 466 + 22 706 +_ 46* Hypotha lamus 289+ 12 331 + 12 206__ 14 230___20

Cortex 288 + 15 341 _+ 17** 172 __+ 13 266 ___ 22**

Midbrain 197 _+ 12 215 _+ 19 157 _+ 26 195 + 25 Striatum 194 + 12 167 + 22 145 _ 22 157 + 15

Pons-Medul la 90 + 15 107 _+ 26 75 _+ 14 73 __. 16

nized in 50 vols. of ice-cold 50 mM Tris-HCI (pH 7.4 at 37°C) with a polytron (Brink- mann PT 10, setting at 6 for 30 s) and the homogenates centrifuged at 50,000 g for 10 min. The pellets were washed by resuspending in 50 vols. buffer and incubated at 37°C for 40 min. The membranes were then centrifuged and washed two additional times by centrifugation and resuspension. The resulting membranes were resus- pended in 125 vols. of the above Tris-HCi buffer containing 5 mM MgCI2, 0.1 mg/ml soybean trypsin inhibitor, 0.1% bovine serum albumin and 0.25 mg/ml bacitracin unless other,vise indicated. For binding studies, 230 Ixl of brain membranes were added to 10 ~tl of buffer (for total binding) or 0.3 ~tM (final concentration) unlabeled NPY (for non-specific binding) containing 10 lal (final concentration) unlabeled NPY (for non-specific binding) containing 10 ~tl 125I-BH-NPY (25 pM). After incubation at 37°C for 90 rain, the incubation was terminated by adding 1 ml of ice-cold buffer and immediately centrifuged in a Brinkmann microfuge for 4 min. The supernatant was aspirated and the pellets were counted in a Beckman gamma-counter.

Specific 125I-NPY binding was significantly higher in the cortex (+ 18%) and parti- cularly the hippocampus (+44%) of 6-month~old SH rats compared to WKY rats (Table I). Other brain regions including the hypothalamus, midbrain striatum and pons-medulla did not show significant differences in specific 125I-NPY binding between these two strains of rats (Table I). Similar results were obtained using anoth- er group of younger (3-month-old) SH and WKY rats (Table I).

Scatchard analysis of 125I-NPY binding in hippocampal membranes of SH and WKY rats were performed to determine whether the observed differences in binding could be correlated with differences in the affinity of NPY for its binding site (Kd) or the estimated maximal number of NPY binding sites (Bmax). As shown in Fig. 1, the Bma x values obtained in SH rats (2.8 + 0.4 pmol/g) was significantly greater than WKY rats (1.8 __+ 0.3 pmol/g); the respective Kd values (0.08 + 0.02 nM vs 0.09 + 0.03

Page 3: Increased neuropeptide Y (NPY) receptor binding in hippocampus and cortex of spontaneous hypertensive (SH) rats compared to normotensive (WKY) rats

277

28

24

I

" 20 T

E 16

~ 12 kl_

c

o 8 m

4

C 0

0

o

0.4 0.8 1.2 1.6 2.0 2.4

Bound (pmole g-l)

Fig. 1. Scatchard analysis of the displacement of t2SI-NPY binding by NPY in WKY (0 ) and SH (O) rat hippocampal membranes. Each point represents a single experiment performed in triplicate. Thesc experiments were replicated 5 times and the mean + S.E.M. of the Ka and Bm~ values are given in the text.

nM) were not significantly different. The present finding of regional differences in brain 12SI-NPY binding in SH rats

compared to WKY rats do not appear to correlate anatomically with the recently reported differences in regional NPY brain concentrations in these rat strains [9]. NPY concentrations in the cortex of SH rats are appreciably reduced compared to WKY rats whereas, hippocampal NPY concentrations do not differ significantly [9]. In contrast, J25I-NPY binding is increased in the cortex and hippocampus of SH com- pared to WKY rats.

Moreover the present finding of similar 125I-NPY binding in the pons-medulla of SH and WKY rats would suggest that differences in NPY binding does not account for the reported ability of NPY to increase cL2-adrenergic binding in the medulla of normotensive, but not SH, rats [1, 2].

The significance of enhanced 125I-NPY binding in the hippocampus and cortex of SH rats is unknown. However, the hippocampus has been proposed as an anatomical substrate for the central cardiovascular actions of vasopressin, and limbic and corti- cal structures are recognized as being important in the control of behavior [61. The possibility that increased NPY binding in the hippocampus and cortex of SH rats may be related to their cardiovascular or behavior differences from normotensive rats [5] remains to be determined.

Page 4: Increased neuropeptide Y (NPY) receptor binding in hippocampus and cortex of spontaneous hypertensive (SH) rats compared to normotensive (WKY) rats

278

1 Agnati, L.F., Fuxe, K., Benfenati, F., Battistini, N., Harfstrand, A., Tatemoto, D., H6kfelt, T. and Mutt, V., Neuropeptide Y in vitro selectively increases the number of c~2-adrenergic binding sites H1 membranes of the medulla oblongata of the rat, Acta Physiol. Scand., 118 (1980) 293-295.

2 Agnati, L.F,, Fuxe, K., Benfenati, F., Battistini, N., Harstrand, A., Hokfelt, T., Cavicchioli, L., Taeto~ moto, K. and Mutt, V., Failure of neuropeptide Y in vitro to increase the number of ~t2-adrenergic binding sites in membranes of medulla oblongata of the spontaneous hypertensive rat, Acta Physiol. Scand., 119 (1983) 309-312.

3 Borkowski, K.R. and Finch, L., The hypotensive actions of centrally administered adrenaline. In K. Fuxe, M. Goldstein, B. H6kfelt and T. H6kfelt (Eds.), Central Adrenaline Neurons: Basic Aspects and Their Role in Cardiovascular Functions, Pergamon, Oxford, 1979, pp. 469-480.

4 Chang, R.S.L., Lotti, V.J., Chen, T.B., Cerino, D.J. and Kling, P.J., Neuropeptide Y (NPY) binding sites in rat brain labeled with ~2Sl-Bolton-Hunter NPY: comparative potencies of various polypeptides on brain NPY binding and biological responses in the rat vas deferens, Life Sci., 37 (I 985) 2111--2122.

5 Danysz, W., Plaznik, A., Pucilowski, O., Plewako, M., Obersztyn and Kostowksi, W., Behavioral stud- ies in spontaneously hypertensive rats, Behav. Neural Biol., 39 (1983) 22-29.

6 De Jong, W.. Versteeg, C.A. and Bohus, B., Inhibition ofpressor responses induced by electrical stimu- lation of the meseneephalon by vasopressin and oxytocin, Clin. Exp. Hypertens., A6 (1984) 139-147.

7 Fuxe, K., Agnati, L.F., Harfstrand, A., Zini, I., Tatemoto, K., Pich, E.M., H6kfelt, T., Mutt, V. and Terenius, L., Central administration of neuropeptide Y induces hypotension, bradypnea and EEG syn- chronization in the rat, Acta Physiol. Scand., 118 (1983) 189-192.

8 H6kfelt, T., Lundberg, J.M., Tatemoto, K., Mutt, V., Terenius, L., Polak, J., Elde, R. and Goldstein. M., Neuropeptide Y (NPY) and FMRF-amide neuropeptide-like immuno-reactivities in catechola- mine neurons of the rat medulla oblongata, Acta Physiol. Scand., 117 (1983) 315-318.

9 Maccarrone, C. and Jarrott, B., Differences in regional brain concentration of neuropeptide Y in spon- taneously hypertensive (SH) and Wistar-Kyoto (WKY) rats, Brain Res., 345 (1985) 165-169.

10 Tatemoto, K., Calquist, M. and Mutt, V., Neuropeptide Y - a novel brain peptide with structural similarities to peptide YY and pancreatic polypeptide, Nature (London), 269 (1982) 659-660.