Vol. 165, No. 3, 1989 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

December 29, 1989 Pages 1399-1405

INCREASED IMMUNOREACTIVE NEUROPEPTIDE Y IN PLATELETS OF SPONTANEOUSLY HYPERTENSIVE RATS (SHR)

Tsuneo Ogawa, Kazuo Kitamura, Mari Kawamoto, Tanenao Eto and Kenjiro Tanaka

First Department of Internal Medicine, Miyazaki Medical College, Kiyotake, Miyazaki 889-16, Japan

Received November 21, 1989

Summary: A high concentration of immunoreactive neuropeptide Y was observed in rat platelets using a specific and sensitive radioimmunoassay for

neuropeptide Y. Three kinds of high performance liquid chromatography

combined with radioimmunoassay for neuropeptide Y showed that immunoreactive neuropeptide Y in rat platelets is identical to rat

authentic neuropeptide Y. To investigate the pathological role of platelet neuropeptide Y in genetic hypertensive rats, the platelet content and plasma concentration of neuropeptide Y were measured by a sensitive

radioimmunoassay for rat neuropeptide Y in 5-, lo- and 15-wk old spontaneously hypertensive rat and age-matched Wistar Kyoto rat. Platelet content of immunoreactive neuropeptide Y in 5-, lo- and 15-wk old

spontaneously hypertensive rat was higher than that in Wistar Kyoto rat at each age. No difference was observed in plasma concentration of immunoreactive neuropeptide Y between spontaneously hypertensive rat and

Wistar Kyoto rat at each age. ir7 1989 Academic PRSS, Inc.

Neuropeptide Y (NPY) is a tyrosine rich 36 amino acid peptide

originally isolated from porcine brain, having considerable sequence

homology with pancreatic polypeptide and peptide YY (1). NPY is widely

spread throughout the brain, and various central regulatory effects of NPY

have been postulated (2). NPY is also observed in nerve endings in a

variety of tissue, including heart, spleen, intestine, reproductive organs,

and large cerebral blood vessels (3). It has been reported that NPY is a

potent vasoconstrictor increasing mean arterial pressure and total

peripheral resistance (4), suggesting that NPY may be important in

controlling systemic blood pressure as well as local blood flow.

Immunohistochemical studies of rat bone marrow demonstrated de novo

synthesis of this peptide in megakaryocytes (5). Very recently, it has been

reported that immunoreactive NPY is stored in rat platelets and released

ABBREVIATIONS: NPY, neuropeptide Y; SHR, spontaneously hypertensive rats; ir-, immunoreactive; WKY, Wistar Kyoto rats; RIA, radioimmunoassay; HPLC, high performance liquid chromatography: PRP, platelet rich plasma; PPP, platelet poor plasma; TFA, trifluoroacetic acid: VIP, vasoactive intestinal polypeptide.

0006-291x/89 $1.50

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Vol. 165, No. 3, 1989 BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS

during platelet aggregation (6). However the molecular form and biological

function of immunoreactive NPY in rat platelets have not been elucidated.

In the present study, high content of immunoreactive (ir)TNeY was

observed in rat platelets, and the molecular form of ir-NPY was

chromatographically characterized. We found that the content of ir-NPY in

platelets is high in spontaneously hypertensive rats (SHR) compared with

that in normotensive control Wistar Kyoto rats (WKY).

Materials and Methods

Materials: Rat NPY and antiserum for ----_--__ NPY were purchased from Peptide Institute, Inc., (Osaka, Japan). Bovine serum albumin (BSA, Fraction V) for

radioimmunoassay (RIA) was purchased from Sigma Chemical Co., (St. Louis, MO) and was twice recrystallized in our laboratory,

RIA for NPY: --- --- --- lz51-NPY was prepared by the lactoperoxidase method and purified by reverse phase high performance liquid chromatography (HPLC) as

described (7). The incubation buffer for RIA was 0.05M sodium phosphate

buffer (pH 7.4) containing 1% BSA, 0.1% Triton X-100, 0.08M NaCl, 0.025M EDTA-2Na, 0.05% NaN3 and aprotinin 500 KIU/ml. All procedures were performed at 4°C. Standard NPY or the unknown sample (100 ~1) was incubated

with 50 ~1 of anti-NPY antiserum at a final concentration of l:lO,OOO. After standing for 15 hr, the tracer solution (18,000~20,000 cpm in 50 ~1) was added. After incubation for 24 hr, anti-rabbit IgG goat serum diluent

was added. After standing for 40 hr, the tubes were centrifuged at 2000 g x for 30 min, and radioactivity of the precipitate was measured by gamma counter (Aloka ARC-600, Tokyo, Japan).

SaQle preparation: SHR and WKY were purchased from Charles River Co. TAts&i

------- ,-Japan). Systolic blood pressure was measured using a

plethysmograph by the tail-cuff method. Platelet rich plasma (PRP) and platelet poor plasma (PPP) were prepared by the described methods (8).

After intraperitoneal injection of pentobarbital sodium (50mg/kg) and 0.5 ml of 3.8% sodium citrate, blood was collected from the inferior vena cava

in polypropyrene tubes containing 0.38% sodium citrate. The blood was centrifuged at 700 g x for 7 min. After counting the platelet number with a

Coulter counter (Coulter Electronics Inc., Hialeah, FL), the supernatant was centrifuged again at 1600 g x for 10 min. The supernatant was used for

the PPP sample and the sediment was used for the platelet sample. The platelet sample prepared as above was homogenized with a polytoron

homogenizer in 2 ml of IN acetic acid and boiled for 10 min to inactive intrinsic proteases. After chilling, the homogenate was centrifuged at 4OC

for 30 min at 24,000 g x. The supernatant was lyophilized and the residual materials were reconstituted with RIA buffer and subjected to the RIA for NPY. Two ml of PPP sample was applied to a Sep-Pak Cl8 cartridge (Waters Association, Milford, MA), which was previously equilibrated with 0.5N acetic acid, and the absorbed material was eluted with 2 ml of 60% acetonitrile in 0.1% trifluoroacetic acid (TFA). The eluate was

lyophilized, the residual materials were dissolved with RIA buffer, and the clear solution was submitted to the RIA for NPY.

Characterization of ir-NPY in platelets: --- The platelet sample from 3 ml of

PRP was loaded onto a Sep-Pak Cl8 cartridge. The cartridge was washed with

0.5N acetic acid and eluted with 3 ml of 60% CH CN and 0.1% TFA. The ir- NPY in this sample was analyzed by three kinds o + HPLC. One-third of the sample was lyophilized and dissolved in 100 ~1 of 300mM NaCl containing 40%

CH CN and 0.1% TFA. This sample was applied to gel filtration HPLC on a T&GEL G2OOOSW (7.5 x 600 mm Toyosoda, Tokyo, Japan) equilibrated with

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300mM NaCl containing 40% CH3CN and 0.1% TFA. Another one-third of the

sample was lyophilized and dissolved in 500 ~1 of 10mM HCOONH4 (pH 6.5) containing 10% CH CN and the sample was applied to cation exchange HPLC on

a TSK-GEL CM-2SW ? 4.6 x 250 mm Toyosoda, Tokyo, Japan) column with a linear gradient elution of HCOONH4 (pH 6.5) (IOmM-500mM) in 10% CH CN. The rest

of the sample was lyophilized and dissolved in 1 ml of H20:$H3CN:lO%TFA = 9O:lO:l. This sample was analyzed by reverse phase HPLC on Cosmos11 ODS-300 column (4.6 x 250 mm, Nakarai Chemicals LTD., Kyoto, Japan) with a linear

gradient of CH CN (IO%-60%) in 0.1% TFA. Each fraction was monitored with the RIA for NP 3 .

Results and Discussion

Radioimmunoassay for NPY: The results of RIA shown in Fig. 1 indicate that --

half-maximum inhibition of the binding of radioiodinated NPY by synthetic

NPY was 80 pg/tube. The RIA used here has less than 0.1% crossactivity

with pancreatic polypeptide and peptide YY and has no crossactivity against

other neuropeptides and atria1 natriuretic polypeptide. Fig. 1 also shows

that the dilution of the RIA sample from rat platelets and plasma were

roughly parallel with the standard curves of NPY. Several RIAs for NPY have

been reported (9,10,11), but the RIA presented here is one of the most

sensitive among those thus far reported, because the 1251-NPY used here is

completely separated from non-labelled NPY using reverse phase HPLC.

Characterization of ir-NPY in rat platelets: The ir-NPY in WKY rat _---_--__------- -- ------ -- --- -----_--

platelets was characterized by gel filtration HPLC, cation exchange HPLC

and reverse phase HPLC coupled with the RIA for NPY. As shown in Fig. ZA,

on gel filtration HPLC using a TSK-GEL GZOOOSW column (7.5 x 600 mm,

Toyosoda, Tokyo, Japan) equilibrated with 300mM NaCl, 40% CH3CN and 0.1%

TFA, the major ir-NPY emerged at a position identical to that of authentic

NPY. Fig. 2B shows that one major ir-NPY in rat platelets emerged at the

I

100

80

PRP sample (pi/tube

;uf

1 10 102 103

NPY (pg/tube)

Fig.l Standard curve of radioimmunoassay for NPY. Inhibition of 1251-NPY binding to the antiserum by serial dilution of rat NPY(+-+). The dilution curves of PRP (-0-0) and PPP (-Q-C-) 10 wk WKY samples are roughly parallel to the dilution curve of standard NPY.

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0 10 20 30 40 50 60 Time (min)

Fiq.2A Gel filtration HPLC of 10 wk WKY rat platelet extract (PRP 1 ml)

Column: TSK-GEL G2000SW (7.5 x 600 mm, Toyosoda, Tokyo, Japan). Eluent: 300mM NaCl, 40% CH3CN and 0.1% TFA. Flow rate: 0.5 ml/min. Aliquots of all fractions were submitted to RIAs for NPY. Arrows indicate elution positions of (a)void volume, (b)trasylol, (c)authentic NPY, (d) bacitracin and (e)NaCl, respectively. Fiq.28 Cation exchange HPLC of 10 wk WKY rat platelet extract (PRP 1 ml) Column: TSK-GEL CM-PSW, (4.6 x 250 mm, Toyosoda, Tokyo Japan).

Flow rate: 1.0 ml/min. Solvent system: (a) 1OmM HCOONH4(pH6.5):CH3CN = 9O:lO(v/v)

(b) l.OM HCOONH4(pH6.5):CH CN = 9O:lO(v/v)

Linear gradient elution from (a):(b)=lOO:d to (a):(b)=50:50 (80 min), and

(a):(b)=50:50 to (a):(b)=O:lOO (IO min). Arrow indicates elution position of authentic NPY. Fiq.2C Reverse phase HPLC of 10 wk WKY rat platelet extract (PRP 1 ml) Column: Cosmosil ODS-300 (4.6 x 250 mm, Nakarai Chemicals LTD., Kyoto,

Japan). Flow rate: 1.0 ml/min.

Solvent system: H20:CH3CN:10%TFA=(a)90:10:1, (b)40:60:1 (v/v). Linear gradient from (a) to (b) for 60 min. Arrow indicates elution position of authentic NPY.

same position as authentic NPY on cation exchange HPLC using a TSK-GEL CM-

2SW column (4.6 x 250 mm, Toyosoda, Tokyo, Japan), which was eluted by a

linear gradient of NH4COOH (IO-500mM) in 10% CH3CN. As shown in Fig. 2C,

ir-NPY in rat platelets eluted at the same position as authentic NPY on

reverse phase HPLC using a Cosmosil OOS 300 column (4.6 x 250 mm, Nakarai

Chemicals LTD. Kyoto, Japan). The data presented here indicate that ir-NPY

in rat platelets is identical to authentic NPY. Recently it has been

reported that blood cells contain an abundance of immunoreactive vasoactive

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intestine

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3, 1989 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

polypeptide (VIP) (12). VIP was originally discovered in porcine

(12) and its carboxy terminal is amidated. But Edward J. Goetzl

hat ir-VIP in blood cells is VIPlO-28 and its carboxy terminal is

not amidated but is COOH (12). However, ir-NPY in rat platelets is

identical to authentic NPY whose carboxy terminal is amidated.

The concentration of NPY in rat platelets is estimated to be 3.05

ng/mg of wet tissue according to the calculation method described (13).

This concentration is estimated to be comparable with the concentration of

insulin in the pancreatic tail (14) or the concentration of atria1

natriuretic polypeptide in the atrium (15).

Comparison of ir-NPY in serum and Platelet from WKY and SHR: Table 1 ___-__ -- _----- -- ----- -- ------ ---- --- -- ---

summarizes body weight, systolic blood pressure, the plasma concentration

of ir-NPY, and the content of ir-NPY in platelets in each group of WKY and

SHR at 5, 10 and 15 wk of age. The ir-NPY content in platelets from male

SHR at 10 wk (56.3i3.92, mean?SE, pg/106 platelets) is about 1.9 times

higher than that from male WKY at the same age (30.752.81 pg/106

platelets). However, the ir-NPY concentration in serum from the 10 wk male

SHR (178.6t5.63 pg/ml) is almost the same as that from male WKY at 10 wk

(179.2+10.7 pg/ml).

To investigate more precisely the relationship between platelet NPY

and the development of SHR hypertension, we measured platelet NPY in SHR

and WKY at 5 wk and 15 wk of age. In 5 wk male rat, the blood pressure of

SHR has not yet been elevated, but the content of ir-NPY in SHR platelets

is already high compared with that of WKY. At 15 wk , hypertension has been

well established in SHR, but there is no increase in the already

comparatively high ir-NPY platelet content. These data indicate that the

increase of ir-NPY in SHR platelets is not the result of hypertension but

is a genetically determined characteristic in SHR.

Table 1. Comparison of WKY and SHR

Body Weight Blood Pressure Plasma NPY Plate et NPY (9) (mm& 1 (Pg/lOOul) b (pg/lO platelet)

SW:WKY(n=7) 116.3+1.30 118.0+3.78 164.1+12.4 SHR(n=7) 105.4z3.67 125.6z3.56 169.325.91

36.3+2.88, 58.5z7.84

lOW:WKY(n=7) 260.3+6.68 130.0+2.59* 179.2+10.7 30.7+2.81 SHR(n=7) 248.6zl1.5 211.9+9.1ET 178.6s5.63 56.3%3.92*

15W:WKY(n=7) SHR(n=7)

343.3+1.22, 294.317.23

146.754.15, 158.8+8.71 28.9+3.11 230.5k4.67 161.4+5.20 51.9z3.72*

Mean+SE; * p<o.o01.

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The pathophysiological significance of the higher content of ir-NPY in

SHR platelets remains obscure. However, since NPY functions as a potent

vasoconstrictor as does angiotensin II, NPY in rat platelets may play an

important role in the pathophysiology of hypertension. It has been reported

that platelet aggregability is elevated in SHR compared with WKY (16). and

that ir-NPY in rat platelets is released during aggregation (6). Therefore,

when endothelial cells are injured and platelets aggregate, a larger

amount of NPY should be released from SHR platelets than from WKY

platelets. The released NPY should act on vascular smooth muscle to

increase its tension and tonus.

Concerning the concentration of ir-NPY in plasma, we found no

difference between SHR and WKY plasma. These results are in agreement with

reports that there is no difference in the plasma concentration of ir-NPY

between healthy persons and patients with essential hypertension (17). On

the other hand, it has been found that NPY coexists with noradrenalin in

the same sympathetic nerve endings (18) and NPY is also reported to be

released together with noradrenalin during sympathetic activation (19). The

plasma concentration of noradrenalin is increased in SHR (20), whereas

plasma NPY levels are not increased in SHR. This discrepancy may be

explained by the possibility that there are some differential plasma

noradrenalin and NPY responses to stress between rat and human (19,21,22).

In conclusion, our results showed that ir-NPY in rat platelets is

authentic NPY and that the content of ir-NPY in SHR platelets is elevated.

It is well accepted that platelets play an important role in formation of

atherosclerosis and thrombosis in hypertension, so we surmise that NPY in

platelets may play a profound role in the pathophysiology of hypertension

and its complications.

Acknowledgments

We wish to thank Dr. Robert J. Adams for reading the manuscript. This

work was supported, in part, by a research grant from the Ministry of

Education, Science and Culture of Japan (No. 01641534).

1. 2.

3.

4.

5.

References

Tatemoto, K., Carlquist, M., and Mutt, V. (1982) Nature 296, 659-660.

Hilig, M., Wahlestedt, C., Ekman, R. and Widerlov, E. (1988) Eur. J. Pharmacol. 147, 465-467. Lundberg, J.M., Terenius, L., Hokfelt. T. and Goldstein, M. (1983)

Neurosci. Lett. 42, 167-172.

Zukowska-Grojec, Z., Marks, E.S., and Haass, M. (1987) Am. J. Physiol. 253, H1234-H1239. Ericsson, A., Schalling, M., Mclntyre, K.R., Lundberg, J.M., Larhammar, D Seroogy, K., Hokfelt, T., and Persson, H. (1987) Proc. Natl. Acad.

SC:. 84, 55855589.

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6. Myers, A.K., Farhat, M.Y., Vaz, C.A., Keiser, H.R. and Zukowska-Grojec,

Z. (1988) Biochem. Biophys. Res. Commun. 155, 118-722.

7. Kitamura, K., Tanaka, T., Kato, J., Eto, T. and Tanaka, K. (1989)

Biochem. Biophys. Res. Commun, 161, 348-352. 8. Duarte, A.P.T., Ramwell, P., and Myers, A. (1986) Thromb. Res. 43, 33-

39. 9. Theodorsson-Norheim, E., Hemsen, A. and Lundberg, J.M. (1985) Stand. J.

Clin. Lab. Invest. 45, 355-365. IO. Emson, P.C., Corder, R. and Lowry, P.J. (1984) Regul. Pept. 8,88-94. 11. Higuchi, H., Yang, H.T. and Costa, E. (1988) J.Neurochem. 50.1879-1886. 12. Goetzl, E.J., Sreedharan, S.P. and Christoph, W.T. (1988) J. Bio. Chem.

263, 9083-9086. 13. Simon, K. (1978) Blood. 51, 307-316.

14. Ishida, H., Kiyono, Y. and Takou, A. (1986) Gastrointestinal Hormones VI. 457-463. Igakutosyo Syuppan. Tokyo Japan.

15. Kato, J., Kida, O., Nakamura, S., Sasaki, A., Takiguchi, K. and Tanaka,

K. (1986) Life Sci. 39, 2623-2627. 16. Metha,J. and Metha,P. (1981) Am. J. Cardiol. 47.331-334. 17. Takahashi, K., Mouri, T., Itoi, K., Sone, M., Ohneda, M., Murakami, O.,

Nozuki, M., Tachibana, Y. and Yoshinaga, K. (1987) J. Hypertension. 5,

749-753. 18. Fried, G., Terenius, L., Hokfelt, T. and Goldstein, M. (1985) J.

Neurosci. 5, 450-456. 19. Morris, M.J., Russell, A.E., Kapoor,V., Cain, M.D., Elliott, J.M. West,

M.J., Wing, L.M.H. and Chalmers, J.P. (1986) J. autonomic Nervous System. 17, 143-149.

20. Nagaoka, A. and Lovenberg, W. (1976) Life Sci. 19, 29-34. 21. Pernow, J., Lundberg, J. M., Kaijser, L., Hjemdahl, P., Theodorsson,

E ., Martinsson, A. and Brnow, B. (1988) Clin. Physiol. 6, 561-578. 22. Zukowska-Grojec, Z., Konarska, M. and McCarty, R. (1988) Life Sci. 42,

1615-1624.

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