Life Sciences, Vol. 34, pp. 1087-1093 Pergamon Press Printed in the U.S.A.

ADRENALINE INVOLVEMENT IN THE PRESYNAPTIC B-ADRENOCEPTOR-MEDIATED MECHANISM OF DOPAMINE RELEASE FROM SLICES OF THE RAT HYPOTHALAMUS

Hiroshi Ueda I, Yoshio Goshima, Takao Kubo and Yoshimi Misu 2

Department of Pharmacology, Yokohama City University School of Medicine, Yokohama 232, Japan

(Received in final form January 4, 1984)

Summary

Slices of rat hypothalamus were superfused and endogenous release of dopamine (DA) was measured by high performance liquid+ chromatography combined with electrochemical detection. The K (20 mM)-evoked release in the presence of tetrodotoxin was Ca 2+- dependent. The evoked release was facilitated by a B-a~onist, iso- proterenol and this effect was completely abolished by a B-antago- nist, l-propranolol. Isoproterenol also concentration-dependently facilitated the electrically (at 5Hz) evoked release of DA. The pretreatment with l-Dropranolol, B1-antagonist, atenolol and B2- antagonist, butoxamine shifted the concentration-effect curve of isoproterenol to the right. On the other hand, B1-agonist,tazolol, ~2-agonist, salbutamol and low concentration (10-~M) of adrenaline also facilitated the release, l-Propranolol alone reduced the elec- trically (at 2 Hz) evoked release, and this effect was completely abolished when the adrenaline content in the brain was drastically reduced by use of a potent PNMT inhibitor, DCMB. These findings suggest that in the rat hypothalamus adrenaline released from adrenaline-containing nerve terminals probably modulates DA release via presynaptic BI- and B2-adrenoceptors on DA nerve terminals.

It is well established that dopamine (DA) regulates its own release via presynaptic DA-receptor-mediated feedback mechanism (i). Most recently we dem- onstrated that DA release is also modulated via presynaptic ~- and B-adreno- ceptor-mediated mechanisms (2,3). However, little is known of the physiological roles of these "heteroreceptor"-mediated mechanisms of DA release.

We report here findings that adrenaline may play an important role in the presynaptic B-adrenoceptor-mediated facilitation of DA release from rat hypo- thalamic slices.

Methods

Male, 8-week old Sprague-Dawley rats were given food pellets and water ad libitum and were kept on a day and night schedule (lights on at a.m. 5 and off at 7 p.m.). These rats were decapitated and the brains removed and placed on ice. The hypothalamus was dissected out using the method of Glowinski and Iversen (4), and was cut sagittally into 6 pieces. Superfusion technique of

i Present address: Department of Pharmacology, Faculty of Pharmaceutical Sciences, Kyoto University, Kyoto 606, Japan.

2 To whom reprint requests should be addressed.

0024-3205/84 $3.00 + .00 Copyright (c) 1984 Pergamon Press Ltd.

1088 Adrenaline and Presynaptic B-receptors Vol. 34, No. ii, 1984

the slices were as described previously (2,3). The DA-containing superfusates were collected subsequently every 3 min, partially purified by the use of alumina and measured by high performance liquid chromatography with an electro- chemical detector. Details in the chromatographical data were as described pre- viously (2,3).

In some sets of experiments, high potassium (20 mM KCI,5 min) stimulations of the slices were performed twice, at 60 ($I) and 120 ($2) min after the onset of superfusion,in the presence of 2 x 10-SM cocaine and 3 x 10-TM tetrodotoxin, which might exclude the influence of neuronal loops (3,5). Evoked release S1 was estimated by the amounts of total minus basal release for 12 min, as des- cribed by Kamal et al. (6). Basal release was estimated by interpolation bet- ween the amounts during 57-60 min and those during 72-75 min after the onset of superfusion. Evoked release $2 was as the case in $I. Spontaneous release (Spl or Sp2) was estimated by the amounts for 3 min immediately before the stimula- tion. In the others, electrical field stimulations were performed 60($I) and 90 ($2) min after the onset of superfusion by the method as described previously (2). Briefly, cocaine 2x 10-SM was contained in the superfusion medium through- out the experiments when the electrical stimulations at 5Hz for 3 min were used, on the other hand, cocaine 5 x 10-SM was contained when at 2 Hz. Evoked release $I or $2 was estimated by the amounts of total minus basal release for 9 min. Other details were essentially as above-mentioned.

The effects on DA release were evaluated by means of $2/$I ratio of evoked amounts as described by Kamal et al.(6). B-Agonists and antagonists alone were always added 15 min before the $2 period of stimulation. Pretreatments with B- antagonists were initiated at the start of superfusion and were contained throughout the experiments.

Adrenaline (Ad) contents were lowered by the pretreatment with phenyl- ethanolamine N-methyltransferase (PNMT, EC 2.1.1.28) inhibitor, 2,3-dichloro-~- methylbenzylamine (DCMB), 80 mg/kg i.p. 2 hr prior to decapitation.

Drugs used were 1-adrenaline bitartrate (Nakarai Chem., Kyoto), l-isopro- terenol hydrochloride-(Sigma Chem. Co., U.S.A.), !- and d-propranolol hydro- chloride and atenolol hydrochloride (Imperial Chem. Ind., Wilmslow, Cheshire, England), butoxamine hydrochloride (Burroughs-Wellcome Research, Triangle Park, N. Carol.), tazolol hydrochloride (Syntex Corp., Palo Alto, Calif.) and salbutamol sulfate (Sankyo Co. Ltd., Tokyo).

Statistical significance of difference was calculated using Student's t-test.

Results

Dopamine in the samples of superfusates was consistently detectable with high performance liquid chromatography with an electrochemical detector. Spontaneous DA release for 3 min before the time evoked release was almost stabi- lized. Spontaneous release, Spl 3 min immediately before $I was described in the legend of each figure. In the absence o~ drugs, Sp2/Spl ratio was 61.2 ± 3.1% (n=ll) with the experiments using hiRh K ~ ~ stimulations and 2 x 10-SM cocaine and 3x 10-?M tetrodotoxin, 71.1 ± 3.9% (n=14) with electrical stimulations (at 5Hz) and 2 x 10-SM cocaine, and 81.6 +- 3.8 % (n=5) with electrical.stimulations (at 2 Hz) and 5 x 10-SM cocaine. In both experiments using high K - and electrical stimulations, all B-agonists, antagonists and the pretreatment withDCMBdid not affect Sp2/Spl ratios and $issue contents of DA after the experiments. Evoked release SI of ~A by high K was 1.28 ± 0.12 (n=ll) pmol/mg protein. As shown in Fig. i, high K -evoked DA release was only slightly attenuated at the second

Vol. 34, No. II, 1984 Adrenaline and Presynaptic B-receptors 1089

stimulation ($2/$I, 0.88 ± 0.05) in the presence of tetrodotoxin 3 x 10-?M, which completely abolished the release of DA evoked by elecSrical field stimula- tion at 5 Hz. This ratio was drastically reduced when Ca 2 was omitted fro~ and 2 mM EGTA added to the superfusion medium ($2/$I, 0.15 ± 0.03). High K evoked DA release was potentiated by isoproterenol lO-°M and this effect was abolished by the pretreatment with ~-propranolol 10-SM (Fig. i).

As previously reported (2), electrical stimulation-induced DA release was also facilitated concentration-dependently by a B-agonist, isoproterenol at the range of 10 -I° to 10-SM, and in the present study the pretreatment with l- propranolol 10-SM shifted the concentration-effect curve of isoproterenolto the right (~ig. 2). The pretreatment with B1-antagonist, atenolol or Bz-antagonist, butoxamine also shifted the curve to the right (Data not shown). The pA2 value of the B-antagonists against isoproterenol-induced effects was 10.6 (~-proprano- ioi), 10.4 (butoxamine) or 8.4 (atenolol). On the other hand, B1-agonist, tazolol or B2-agonist, salbutamol facilitated electrically evoked DA release (TABLE I).

As shown in Fig. 3, adrenaline 10 -9 to 10-TM produced biphasic effects, enhancement at a low concentration (10-gM) and inhibition at a relatively high concentration (10-?M) on DA release evoked by electrical stimulation. Inhibi- tory effect by adrenaline was abolished by an ~2-antagonist, yohimbinel0-TM (2). On the other hand, adrenaline-induced enhancement was significantly abolished by the pretreatment with ~-propranolol 10-SM ($2/$I : 0.93 + 0.07, p<0.05).

1.0-

0.5-

DA

c ~ t ,,op : .~ Isop

Fi~. I

Isoproterenol-induced facilitation on DA release evoked by high K + in the presence of tetrodotoxin. In superfssion, Krebs' medium con-

. ° -5 talnlng 2 x i0 M cocaine was used. High K stimulations were per- formed by increasing KCI concentration to 20 mM for 5 min. Each value represents the mean ± SEM of at least 7 experiments. Abbre- viation: Cont; control (no drug), Isop; isoproterenol 10-SM, 1-Prop; l-propranolol IO-SM was pretreated from the start of superfusion and continued throughout the experiments. Spontaneous release Spl of DA was 355 + 58 fmol/mg protein (n=ll). Evoked release $I of DA was 1.28 + 0.12 pmol/mg protein (n=ll). *: p<O.O5.

1090 Adrenaline and Presynaptic B-receptors Vol. 34, No. ii, 1984

%

1.2

1.0

0.8

DA

t f /

10-1o 10-9 10-e

I$oproterenol

10 -7 (M)

Fig. 2

Isoproterenol-induced facilitation on DA release evoked by electrical field stimulation. In superfusion, Krebs' medium containing 2 x 10 -5 M cocaine was used. Electrical field stimulations were performed at the frequency of 5 Hz. Each value represents the mean ± SEM of at least 4 experiments. Propranolol; !-propranolol 10-SM was pretreated from the start of superfusion and continued throughout the experi- ments. Spontaneous release Spz of DA was 312 ± 55 fmol/mg protein (n=14). Evoked release SI of DA was 580 ± 30 fmol/mg protein (n=14). *: p<0.05, compared to control (the closed circle at 0 M).

TABLE I

Effects of several B-agonists on DA release evoked by electrical stimulations

B-Agonists (n) S2/Sz ratio

Control (14) 0.91 ± 0.07

Isoproterenol 10-SM (81+82) (14) 1.15 ± 0.06 *

Tazolol IO-TM (81) (7) i. Ii ± 0.09 *

Salbutamol 10-SM (82) (i0) 1.12 ± 0.07 *

Details are as Fig. 2 legend. (): number of experiments. *: p<0.05, compared to control.

Vol. 34, No. ii, 1984 Adrenaline and Presynaptic B-receptors 1091

$~/$I

1.5

1.0

0.5

DA

/ / ' ~-8 ~-7 M 0 10 -°

Adrenaline

Fig. 3

Biphasic effects of adrenaline on DA release evoked by electrical stimulation. Details are as in the legend of'Fig. 2.

$2/$I

1.0-

0 . 5 -

DA

Cont l-Pr© l-ProD 10 .7 S x l O -7

OCMB +

I-Prop

3 x i 0 "7

1 DCMB

(M)

Fig. 4

l-Propranolol-induced effects and the blockade by the pretreatment with DCMB on DA release by electrical stimulation. In superfusion, Krebs' medium containing 5 x 10-SM cocaine was used. Electrical field stimulations were performed at the frequency of 2 Hz. Each value re- presents the mean ± SEM of at least 4 experiments. Without DCMB pre- treatment, spontaneous release Spl of DA was 538 ± 30 fmol/mg protein and evoked release $I was 718 ± 55 fmol/mg protein (n=27). With DCMB pretreatment, Spl was 636 ± 94 fmol/mg protein and $I was 775 ± 86 fmol/mg protein (n=10). l-Propranolol was applied to the medium 15 min before $2 stimulation. DCMB 80 mg/kg was given i.p. 2 hr prior to decapitation. *: p<0.05.

1092 Adrenaline and Presynaptic B-receptors Vol. 34, No. ii, 1984

l-Propranolol alone produced no alteration in the DA release evoked by electrical stimulation at 5Hz (Data not shown), but showed significant decrease in the release at 2 Hz (Fig. 4). When rats were pretreated with DCMB, !-pro- pranolol-induced inhibitory effects were abolished (Fig. 4). The tissue contents of adrenaline in the slices after the superfusion experiments, under conditions of pretreatment with DCMB, were drastically reduced, 0.54 ± 0.15 pmol/mg protein (n=ll), compared to the untreated, 1.42 ± 0.15 pmol/mg protein (n=12). On the other hand, DA contents were unchanged in these experiments (untreated: 92.1 ± i0.0 pmol/mg protein, n=12, vs pretreated: 96.4 ± ii.0 pmol/mg protein, (n=ll).

Discussion

High potassium and electrically evoked release of endogenous DA from slices of the rat hypothalamus were measured, using high performance liquid chromato- graphy with an electrochemical detector. Both types of the evoked release were Ca2~-dependent and the latter was tetrodotoxin-sensitive (2). Previously we have demonstrated the presence of presynaptic b-receptor-mediated mechanism of DA+re- lease by electrical stimulation (2). Presently we demonstrafed that high K - evoked release of DA was also enhanced by isoproterenol in the presence of the enhancement of DA release, but not to the blockade of DA uptake or activation of DA synthesis, since B-agonists such as isoproterenol did not show any increase of spontaneous DA release and tissue contents of DA after the experiments.

As tetrodotoxin might abolish action potential, it is suggested that tetro- dotoxin-sensitive interneurons are not mediating the effect of isoproterenol and that the presynaptic b-receptors preferentially exist on nerve terminals but not on soma/dendrites of DA neurons.

l-Propranolol alone (10 -7 to3xl0-TM) concentration-dependently decreased the release evoked by electrical stimulation at 2 Hz, whereas d-propranolol alone 3 x 10-7M failed to show any decrease (2). These findings indicate that facilitation of DA release via presynaptic B-adrenoceptors in the rat hypo- thalamus is selective at a low frequency and stereospecific, as is the case in the sympathetic noradrenaline neurons, and that electrically released endogenous B-agonist such as noradrenaline and adrenaline may act on the B-adrenoceptors on DA nerve terminals.

The physiological significance of presynaptic B-adrenoceptors in the sym- pathetic noradrenaline neurons has been well discussed (7-9). Majewski et al. (i0) emphasized that adrenaline once incorporated into the sympathetic noradre- naline terminals from the blood stream is released as a co-transmitter with noradrenaline, and plays roles in a B-adrenoceptor-mediated positive feedback mechanism for noradrenaline release. It is generally accepted that in the rat hypothalamus, adrenaline plays a neurotransmitter role, since the adrenaline- forming enzyme PNMT is concomitantly present (ii) and adrenalin$ is Ca2~-depen - dently released, in vitro, by depolarizing agents such as high K orveratridine (12). Although the amounts of adrenaline are 40 times less than those of nor- adrenaline in the rat hypohtalamus (12), it is noteworthy that a low concentra- tion of adrenaline facilitated the evoked DA release, via presynaptic B-adreno- ceptors.

Fuller and Perry (13) demonstrated that pretreatment with DCMB, PNMT inhibi- tor lowers adrenaline contents in the brain. When rats were pretreatedwithDCMB l-propranolol-induced inhibitory effects were abolished. The abolishment with DCMB may not be related to its B-antagonistic action, since in the guinea pig pulmonary arteries (14), the pretreatment did not show any antagonistic action

Vol. 34, No. ii, 1984 Adrenaline and Presynaptic B-receptors 1093

against isoproterenol-induced facilitation of 3H-noradrenaline release (Data not shown). These findings indicate that adrenaline is closely related to !-pro- pranolol-induced inhibition of the evoked release of DA via presynaptic 8- adrenoceptors on the DA nerve terminals.

In the sympathetic neurons, most presynaptic 8-adrenoceptors are character- ized as 82-subtypes (15,16) except for 81-adrenoceptors in the arterial vessels of the cat hind limbs (9). In the present study we found both 81- and 8z-sub- types of adrenoceptors co-exist on the DA nerve terminals in the rat hypothala- mus, suggesting that noradrenaline, which has only weak 82- but potent 81- agonistic activities comparable to adrenaline, may also play a physiological role in the presynaptie 8-adrenoeeptor-mediated mechanism in the rat hypothala- mus. Howevere as l-propranolol-induced inhibition was almost completely abol- ished in the slices in which adrenaline was drastically lowered using DCMB, it is plausible that adrenaline rather than noradrenaline is predominantly involved in this mechanism. Direct evidence that adrenaline-containing neurons form axo- axonic linkage with DA nerve terminals in the rat hypothalamus must await elec- tron microscopical observation as axo-axonie contacts between noradrenaline and non-noradrenaline terminals have been demonstrated in the nucleus tractus solitarii of rats (17).

In conclusion, adrenaline from adrenaline-containing nerve terminals pro- bably modulates DA release via presynaptic B-adrenoceptors on DA nerve terminals.

Acknowledgements

This work was partly supported by a grant No. 57570080 and 58770171, from the Ministry of Education, Science and Culture, Japan. We thank Dr.R.W. Fuller (Eli Lilly and Co. Ltd.) for the gift of DCMB and M. Ohara (Kyushu Univ.) for reading the manuscript.

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