Brain prostaglandins mediate the bombesin-induced increase in plasma levels of catecholamines

Life Sciences, Vol. 59, NO. IS, pp. 1217-1225, 1996 Copyright 0 1996 Elsevier Science Inc. Printed in the USA. All rights reserved 0024-3205/% $15.00 + .oo

PI1 SOO243205(96)00445-6

ELSEVIER

BRAIN

PROSTAGLANDINS IN PLASMA

MEDIATE LEVELS

THE BOMBESIN-INDUCED OF CATECHOLAMINES

INCREASE

Yasunobu Okuma, Kunihiko Yokotani and Yoshitsugu Osumi Department of Pharmacology, Kochi Medical School, Nankoku, Kochi 783, Japan (Received

in final form August 5, 19%)

Intracerebroventricular (i.c.v.) administration of bombesin (0.1, 1.O, 10.0 nmol/animal) in urethane-anesthetized rats induced long-lasting and dose dependent increases in plasma levels of adrenaline and noradrenaline. These effects of bombesin were inhibited by i.c.v. pretreatment with indomethacin (50-500 pg/animal), while the same dose of indomethacin by the intravenous route was without effect. The bombesin-induced increases in plasma levels of catecholamines were also inhibited by i.c.v. pretreatment with diclofenac (100-500 pg/animal), a cyclooxygenase inhibitor other than indomethacin. I.c.v. administration of thyrotropin releasing hormone (TRH) (10 nmol/animal) also induced increases in plasma levels of adrenaline and noradrenaline, however, these increases were not modified by i.c.v. pretreatment with indomethacin. The present results suggest that the bombesin-induced increases in plasma levels of catecholamines are probably due to prostaglandins synthetized in the brain with this neuropeptide. Furthermore, it is likely that prostaglandins are not always involved in central activation of sympathoadrenomedullary system by other brain neuropeptides. Key Words-: thyrotropin

indomethacin, hormone

bombesiu,

releasing

diclofenac,

plasma

adrenaline,

plasma

noradrenaline,

prostaglamlin,

Centrally applied bombesin increases plasma levels of catecholamines, especially of adrenaline (l-3). Recently, we have demonstrated a direct electrophysiological evidence that centrally applied bombesin elevates nerve activities of both sympathetic and adrenal branches of splanchnic nerves (4). However, brain mechanisms of bombesin to activate the sympathoadrenomedullary functions have not been elucidated. Several independent lines of evidence indicate that prostaglandins are involved in the effects of brain neuropeptide on various physiological functions. Indomethacin, an inhibitor of cyclooxygenase, given i.c.v. reduces hypertension and behavioral excitation caused by bradykinin (5, 6). Central administration of calcitonin induces anorexia and gastrointestinal motor effect, which are blocked by i.c.v. pretreatment with indomethacin (7). I.c.v. administration of neurotensin and calcitonin to fed rats restore the migrating myoelectric complex. These effects are blocked by i.c.v. pretreatment with indomethacin (8). Central effect of bombesin to inhibit gastric acid secretion might be mediated by central prostaglandins (9). Prostaglandins in the brain are known to affect various physiological functions, including body temperature (lo), cardiovascular functions (11, 12) and gastrointestinal functions (8, 13, 14). With regard to sympatho-adrenomedullary functions, centrally applied prostaglandins increases plasma levels of catecholamines (15-19). In the present study, therefore, the effect of indomethacin Correspondence to: Yasunobu Okuma, Department of Pharmacology, Nankoku, Kochi 783, Japan, Phone: (888)80-2327, FAX: (888)80-2328

Kochi Medical

School,

Vol. 59, No. 15, 1996

Brain Bombesin and Plasma Catecholamines

1218

on the bombesin-induced increases anesthetized with urethane.

in plasma

levels of catecholamines

was examined

in rats

Methods Male Wistar rats weighing 400-4508 were maintained in a room at 22-24°C under a constant day-night rhythm for 7-10 days and given food (laboratory chow, CE-2, CLEA Co., Tokyo) and tap water, ad libitum. Experiments were performed under urethane anesthesia (1.2 g/kg, i.p.). The femoral artery and the femoral vein were cannulated and physiological saline was infused i.v. at a rate of 1.6 ml/h. Animals were placed in a stereotaxic apparatus. Drugs dissolved in saline was given into the lateral cerebral ventricle (AP: 7.5, L:1.2, H: 3.8 below the surface of the brain) in a volume of 10 yl through a stainless steel micropipette (0.35 mm outer diameter). Blood samples (400 p-1) were collected through the arterial catheter for measurement of catecholamines. Catecholamines in the plasma were extracted by the method of Anton and Sayre (20) with slight modifications and were assayed electrochemically by high performance liquid chromatography (HPLC). The modifications were as follows: plasma (180 ~1) was transferred to a centrifuge tube containing 30 mg of activated aluminum oxide, 2 ml of twice deionized water and 1 ml of 1.5 M Tris Buffer (pH 8.6) containing 0.1 M disodium EDTA, and then the preparation was

3000 ,

2500 c‘ 5E

Bombeein or vehicle i.c.v.

_*

2000

4 s

1500 -

2 4 P

1000 -

500 -

O-

NA =‘

1500-

g 1

lOOO-

fz

500-

o. 0’

I

0

20

40

60

Time (min)

Fig. 1 The effect of bombesin applied i.c.v. on plasma levels of catecholamines. AD: 0 : Vehicle (n=lO), A : Bombesin 0.1 nmol (n=3), adrenaline, NA: noradrenaline, 0 : Bombesin 1 nmol (n=5), n : Bombesin 10 nmol (n=4). *: PcO.05 (statistically significant from the respective control values with vehicle. Statistical analysis was made using Dunnett’s test.).

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immediately shaken for 5 min. After several washings with 4-ml aliquots of ice-cold twice deionized water, catecholamines adsorbed onto aluminum oxide were eluted with 300 pl of 4% acetic acid containing 0.1 mM disodium EDTA. A pump (880 PU; Japan Spectroscopic Co., Ltd., Tokyo, Japan) and an electrochemical detector (ECD-100, Eicom Co., Ltd., Kyoto, Japan) were used with HPLC. Analytical conditions were as follows: detector, 0.7 volt potential; column, Cosmosilpacked column (ODS, 4.6 x 150 mm, Nacalai Tesque, Inc. Kyoto, Japan); mobile phase, 0.1 M phosphate buffer pH 3.5, 20 mM EDTA, 4 mM l-octane sulfate sodium (Nacalai Tesque, Inc.) containing 16% methanol. The amount of catecholamines in each sample was calculated by using the peak height ratio relative to 3,4-dihydroxy benzylamine, an internal standard. By this assay, coefficient of variation (CV) value was 2% and 1 pg of noradrenaline and adrenaline could be determined accurately.

1600

AD

1

600 -

NA

1200 = E 5,P

lOOO800 -

s

600 -

E jj P

400200 0’

I

-40

-20

0 Time

20

40

60

(min)

Fig. 2 Effect of indomethacin (i.c.v.) on the bombesin-induced increases in plasma catecholamines. I.c.v. pretreatment with indomethacin was performed 40 min before 0-O: Vehicle + Bombesin 1 nmol (n=5), A----A: the administration of bombesin. Indomethacin 50 pg/animal + Bombesin 1 nmol (n=4), m----m: Indomethacin 500 l.tg/animal + Bombesin 1 nmol (n=8). *: PcO.05 (statistically significant from the respective controls received bombesin without indomethacin. Statistical analysis was made using Dunnett’s test.).

Brain Bombesin

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Drugs used were: bombesin (Peptide Institute, Inc., Osaka, Japan); water-soluble indomethacin sodium trihydrate kindly provided by Merk and Co. (USA); diclofenac sodium (Wako Pure Chemical Industries LTD, Osaka, Japan). Statistics; Results were expressed as the mean Y!Z S.E. Statistical analysis was performed with Dunnett’s test for multiple comparison after one-way analysis of variance (ANOVA). Paired Student’s t-test was also used for comparison between two groups. (Fig.4). Significant values are those with P
AD

1600 1400 1 =

1200.

E 3l p

lOOO-

a

600

-

600

-

ii f P

400 200 1

0’

NA

y-1

,p-----------

1200-

I

600-

&_____----___-__6”

0’

1

-40

-20

0

20

40

60

Time (min)

Fig. 3 Effect of indomethacin (i.v.) on the bombesin-induced increases in plasma catecholamines. Intravenous pretreatment with indomethacin was performed 40 min before the administration of bombesin. 0-O: Bombesin 1 nmol (n=5) without indomethacin, cited from fig. 1, U---U: Indomethacin 500 pg/animal + Bombesin 1 nmol (n=3). *: PcO.05 (statistically significant from the respective controls received bombesin without indomethacin. Statistical analysis was made using Dunnett’s test.).

Brain Bombesin and Plasma Catecholamines

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1221

Basal plasma levels of adrenaline and noradrenaline in rats under urethane anesthesia were 222f16 pg/ml and 419k24 pg/ml, n=22, respectively. Blood sampling for 5 times over a lOO-min period did not affect plasma levels of both adrenaline and noradrenaline as observed in the controls. I.c.v. administration of bombesin (0.1-10.0 nmol/animal) induced significant and dose-dependent elevations of plasma catecholamines, especially of adrenaline. Plasma levels of adrenaline and noradrenaline 30 min after the administration of bombesin 10 nmol reached to 2329+124 and 1313+179 pg/ml, n=4, respectively (Fig. 1). Pretreatment with indomethacin (50-500 l.&tnimal) by the i.c.v. route dose-dependently inhibited the bombesin (1 nmol)-induced increases in plasma levels of both adrenaline and noradrenaline at 30 and 60 min after the administration of this peptide (Fig. 2). In the animals and pretreated with indomethacin 500 yg/animal i.c.v., plasma levels of both adrenaline noradrenaline at 30 min collection period after the administration of bombesin were 508+56 and 383tiO pg/ml, n=8; these values were significantly different from the values in control animals

ABP 30

B

6 2 z 0 h

Bombesin 1 nmol i.c.v.

1

____&__------A I ‘I

i

ZO-

____i_-________[

lo-

-+_______J *

O-

B 9 P E $ w

T

I *

-lO-

*

-20 I

0

I

10

20

30

40

50

60

Time (min)

Fig. 4 Effects of the i.c.v. and intravenous pretreatments with indomethacin on the ABP: Values were expressed as changes in mean hypertensive action of bombesin. blood pressure after the administration of bombesin. l d: Vehicle (i.c.v.) + Bombesin 1 nmol (n=5), A----A: Indomethacin 50 &animal (i.c.v.) + Bombesin 1 nmol (n=4), W---W: Indomethacin 500 l&animal (i.c.v.) + Bombesin 1 nmol (n=8). O----O: Indomethacin 500 pg/animal (i.v.) + Bombesin 1 nmol (n=3). The values of mean blood pressure just before the administration of bombesin in each group were 96.8k3.6 mmHg (Vehicle, i.c.v.), lOO.Of3.2 mmHg (Indomethacin 50 pg/animal, i.c.v.), 99.8f1.7 mmHg (Indomethacin 500 yg/animal, i.c.v), 102.M0.7 mmHg (Indomethacin 500 pg/animal, i.v.), respectively. *: PcO.05 (statistically significant from the respective controls received bombesin without indomethacin. Statistical analysis was made using Dunnett’s test.).

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which received vehicle alone (121&l 19 and 994f102 pg/ml, n=5, respectively). However, 10 min after administration of bombesin in these indomethacin-pretreated animals, levels of both adrenaline and noradrenaline were not lower but rather higher than those in animals treated bombesin alone. On the other hand, intravenous administration of indomethacin 500 pg/animal did not significantly inhibit the bombesin-induced increases in plasma levels of catecholamines (Fig. 3). In general, the increase in mean arterial blood pressure of the bombesin-treated animals corresponded well with that in the plasma levels of catecholamines (Fig. 4). The mean increment at 30 min after the administration of this peptide (1 nmol) was 25.6i1.5 mmHg, n=5; these values were significantly different from the basal level (Statistical analysis was made using paired Student’s t-test.). In the animals pretreated with indomethacin 50 and 500 pLg/animal i.c.v., the bombesin (1 nmol)-induced elevation of arterial blood pressure at 30 min was reduced to +l l.0k2.4 mmHg, n=4 and +0.3+3.5 mmHg, n=8, respectively. However, in the animals pretreated with indomethacin (500 pg/animal i.c.v), a transient decrease rather than increase in arterial blood pressure was observed immediately after the administration of this peptide. The maximal decrease was observed at 2 min (14.5f2.8 mmHg, n=8, statistically significant from the basal level.), and arterial blood pressure returned toward the basal level within 5 to 15 min. I.c.v. pretreatment with diclofenac, a cyclooxygenase inhibitor other than indomethacin also dose-dependently (100-500 pg/animal) inhibited the bombesin (1 nmol)-induced increases in plasma levels of both adrenaline and noradrenaline (Table I). In these animals pretreated with diclofenac 100 and 500 pg/animal i.c.v., the bombesin (1 nmol)-induced elevation of arterial blood pressure at 30 min was reduced to +15.2+6.1 mmHg, n=5 and +8.2f3.0 mmHg, n=6, respectively.

TABLE I Effects of the I.c.v. Pretreatments with Diclofenac and Indomethacin Increases in Plasma Catecholamines Pretreatment

(i.c.v.)

Vehicle Diclofenac 100 j.tg Diclofenac 500 c(g Vehiclea

n __~~~ 9 5 5

lndomethacin

50 pga

5 4

lndomethacin

500 pga

8

I.c.v. pretreatment with diclofenac, before the administration of bombesin administration of bombesin. a: cited from the respective controls received using Dunnett’s test.).

on the Bombesin-Induced

Adrenaline (pg/ml)

Noradrenaline

1470f 75 1062 + 300

1041 f 61 704 f 123*

706 + 168* 1210+ 119

612*119* 994 + 102

675 + 167*

517f

41*

508 f

383f

60*

56*

(pg/ml)

indomethacin or vehicle was performed 40 min 1 nmol. Values were obtained at 30 min after the from fig. 2. *: PcO.05 (statistically significant bombesin alone. Statistical analysis was made

In the next series, additional experiments with TRH were performed as a reference for studies with bombesin. I.c.v. administration of TRH 10 nmol induced rapid and transient increases in plasma levels of both adrenaline and noradrenaline with a concomitant increase in arterial blood pressure. Plasma levels of adrenaline and noradrenaline 10 min after the administration of TRH 10 nmol reached to 612+132 and 741+87 pg/ml, n=4, respectively. These increases in plasma levels of catecholamines with TRH were much smaller and transient than those with bombesin. The mean

Brain Bombesin

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1223

and Plasma Catecholamines

increment in arterial blood pressure at 10 min after the administration of TRH was 2O.Ok4.5 mmHg, n=4. These effects of TRH were not modified by i.c.v. pretreatment with indomethacin (500 lq_&nimal) (Fig. 5). Furthermore, in the animals pretreated with indomethacin, TRH did not induce the rapid and transient decrease in blood pressure as observed by the administration of bombesin.

AD

TRH 10 nmol icv 800

600 lndomethacin

or vehicle

I 400’

200-

O800 -

600 -

400 -

;_________

200-

O-

ABP

T 20-

I’

‘\ ‘\

10-

*-. --_

-------

if%

O-

I

-40

-20

0

20

40

60

Time (min)

Fig. 5 Effect of indomethacin (i.c.v.) on the TRH (i.c.v.)-induced increases in plasma catecholamines and changes in arterial blood pressure. I.c.v. pretreatment with indomethacin was performed 40 min before the administration of TRH. 0-O: Vehicle + TRH 10 nmol (n=4), W---M: Indomethacin 500 pg/animal + TRH 10 nmol (n=3). The values of mean blood pressure just before the administration of TRH in each group were 95.5k1.7 mmHg (Vehicle), 100.7-t5.8 mmHg (Indomethacin 500 l.@inimal), respectively.

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Brain Bomb&n

and Plasma Catecholamines

Vol. 59,No. 15,1996

Discussion The brain mechanisms in mediating the central actions of bombesin are poorly understood. Corticotropin releasing hormone (CRH) receptor antagonist did not alter the increased plasma concentrations of adrenaline or noradrenaline following i.c.v. administration of bombesin (21). Bombesin-induced anorexia is independent of interaction with brain catecholaminergic system (22). Central action of bombesin to inhibit gastric acid secretion is independent of brain dopamine, catecholamine, serotonin and opioid pathways (23, 24). We have reported that the inhibition of gastric acid secretion induced by i.c.v. applied bombesin was abolished by bilateral adrenalectomy plus chemical sympathectomy with 6-hydroxydopamine as well as by bilateral cutting of the greater splanchnic nerves. From these results, we suggested that centrally applied bombesin probably excites the sympatho-adrenomedullary system and thus induces inhibition of gastric acid secretion (25). Bombesin increases plasma levels of catecholamines, especially of adrenaline (l-3). In the present study, bombesin-induced increases in plasma levels of catecholamines and a rise in blood pressure were inhibited by i.c.v. pretreatment with indomethacin, while the same dose of this reagent by the intravenous route was without effects. The bombesin-induced increases in plasma levels of catecholamines were also inhibited by i.c.v. pretreatment with diclofenac, a cyclooxygenase inhibitor other than indomethacin. These results suggest that centrally administered bombesin-induced increases in plasma levels of catecholamines and a rise in blood pressure are probably mediated by prostaglandins synthetized in the brain. Both prostaglandin E2 and F2a applied into the brain have been shown to increase plasma levels of catecholamines (15-18). We have also demonstrated that EP3-receptors in the brain are involved in the prostaglandin E2-induced elevation of plasma level of noradrenaline ( 19). We have to discuss here why i.c.v. pretreatment with indomethacin (500 @animal) did not prevent but rather potentiated the bombesin-induced increases in plasma levels of catecholamines at 10 min after the administration of bombesin. In these animals, a rapid and transient decrease rather than increase in arterial blood pressure was observed immediately after the administration of this peptide. It is well known that a rapid fall in blood pressure activates sympatho-adrenomedullary system via baroreflex. We also confirmed this evidence in our previous study (4). It is therefore likely that the initial fall in blood pressure activates the sympatho-adrenomedullary system via baroreflex and resultant increase in plasma levels of catecholamines occurs in the present study (at 10 min collection period in Fig. 2). The mechanism of this rapid and transient decrease in blood pressure after the administration of bombesin in the animals pretreated with relatively large dose of indomethacin (500 l.@rtimal, i.c.v.) was, however, not elucidated in the present study. A variety of brain neuropeptides have been shown to increase plasma levels of catecholamines. I.c.v. administration of TRH increases plasma adrenaline levels more than plasma noradrenaline levels, while i.c.v. administration of CRH increases plasma noradrenaline levels more than plasma adrenaline levels (2). I.c.v. administration of calcitonin gene-related peptide selectively increases plasma noradrenaline levels (26, 27). Thus, the effect of TRH on the plasma levels of adrenaline and noradrenaline seems to be similar to that of bombesin. Then, in the next series, additional experiments with TRH were performed as a reference for studies with bombesin. Administration of TRH (i.c.v.) elevated plasma levels of both adrenaline and noradrenaline. These increases in plasma levels of catecholamines with TRH were much smaller and transient than those with bombesin. In contrast to bombesin, the TRH-induced increases in plasma levels of catecholamines and a rise in blood pressure were not modified by the i.c.v. pretreatment with Prostaglandins are, therefore, not always involved as signal mediators in central indomethacin. activation of sympatho-adrenomedullary system by brain neuropeptides.

AcknowledemenQ This work was supported in part by a Grant-in-Aid for Scientific Research No. 06670115 from the Ministry of Education, Science and Culture, Japan and a Grant from the Smoking Research Foundation, Japan.

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Brain Bombesin

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Beferences :. 3: 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. ;:: ;:: 27.

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