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Presynaptic mediation by α2-, β1- and β2-adrenoceptors of endogenous noradrenaline and dopamine release from slices of rat hypothalamus
Pergamon Press
Life Sciences, Vol. 33, pp. 371-376 Printed in the U.S.A.
PRESYNAPTIC MEDIATION BY a2-' B1 - AND B2-ADRENOCEPTORS OF ENDOGENOUS NORADRENALINE AND DOPAMINE RELEASE FROM SLICES OF RAT HYPOTHALAMUS Hiroshi Ueda, Yoshio Goshima and Yoshimi Misu Department of Pharmacology, Yokohama City University School of Medicine, Yokohama, 232, Japan (Received in final form May 10, 1983) Summary Using high performance liquid chromatography with an electrochemical detector, we studied effects of different compounds on the impulse-evoked release of endogenous noradrenaline (NA) and dopamine (DA) release from slices of the rat hypothalamus. Adrenaline (10-7 M), with a potent a - a goni s t i c action decreased both NA and DA release, and these effects were blocked by pretreatment with yohimbine (10-7M) . The a 2- a n t a go ni s t , yohimbine alone (10-s-10-6 M) concentration-dependently increased these releases, while a i - a nt a goni s t , prazosin showed weak increase on NA but not DA release at 10-6M• Isoproterenol (lO-IO_lO-sM) concentration-dependently increased these releases and the effects were antagonized by pretreatment with a non-selective Bantagonist, i-propranolol, a BI- an t a gon i s t , atenolol or a B2-antagonist, butoxamine. i-Propranolol (3xlO- 7M) alone, but not the d-isomer inhibi ted the releases. Thus, in the rat hypothalamus', the release of NA and DA may be mediated via presynaptic a2 - , Bl- and B2-adrenoceptors . The role of presynaptic a - a dr eno cep t o r s in the regulation of the release of the transmitter noradrenaline (NA) through a negative feedback mechanism in the sympathetic nervous systems has been well established (for review see I, 2), and it has been proposed that there is additional posi ti ve feedback mechanism through presynaptic B-adrenoceptors (1-4). Little is known of presynaptic a- and Badrenoceptors on the catecholaminergic neurons in the central nervous system (1,5) • We used high performance liquid chromatography with an electrochemical detector and studied presynaptic regulation mechanism via a - and B-adrenoceptors on the impUlse-evoked release of e ndo ge nous noradrenaline and dopamine from slices of the rat hypothalamus. Part of the results has been previously presented in the Japan Neuroscience Society (6). Methods Male, 8-week old Sprague-Dawley rats were given food pellets and water ad l ibitum and were kept on a regular day and night schedule (lights on at 5 a.m. and off 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 (7), and was cut sagittally into 6 pieces. The slices were transferred to a glass chamber (10 rnm~ x 5 rnm) and superfused in an overflow manner at the rate of 0.45 ml/rnin at 37°C with Krebs' medium containing NaCI 113 (mM), KCl
0024-3205/83 $3.00 + .00 Copyright (c) 1983 Pergamon Press Ltd.
Vol. 33, No.4, 1983
372
4.05, NaHC03 25, KH 2P0 4 1.18, MgS04 1.19, caC1 2 2.52, glucose 11.1, EDTA~2Na 0.029 and ascorbic acid 0.29 in the presence of cocaine 2 or 5 x 10-sM. Electrical field stimulation was performed by 30 rnA rectangular pulses of 2 msec duration at a frequency of 2 or 5 Hz for 3 min at 30 min intervals through platinum spiral electrodes set up at the two ends of the chamber, using an electrical stimulator with an isolator (SEN-320l and SS-102J, Nihon Kohden, Tokyo). Stimulation was applied only to "condition" the slices 30 min after the start of superfusion and data on the evoked releases were discarded. After a further equilibration period of 30 min, field stimulations were performed twice 60 (51) and 90 (S2) min after the beginning of superfusion and 3 min samples of the superfusates were successively collected throughout the rest of the experiment. Catecholamine release evoked by electrical stimulation was calculated by subtracting the estimated basal release from the total release . The effects of drugs on catecholamine release were evaluated by means of S 2/S1 ratio of evoked amounts. Adrenergic agonists or antagonists alone were always added 15 min before the S2 period of 's t i mul at i on . Pretreatments with a- or B-antagonists were initiated at the start of superfusion and were continued throughout the experiments. Measurements of catecholamines were made as described by Keller et al . (8). Catecholamine containing samples (1.35 ml) of superfusates were transferred to polypropylene tubes (1.7 ml) co ntaining activated alumina (10 mg), EDTAo 2Na (10 mg) and dihydroxybenzylamine (10 pmol), as an internal standard. To the tubes was added 0.2 ml of 1.5 M Tris - HCl buffer (pH 8.6), and t he p r ep a r a t i on s were placed in a mixer for 15 min. The supernatant was discarded and the alumina washed three times with 1 ml of water containing 5 roM Tris-HCl buffer (pH 8.6) and 1 roM NaHS03. Adsorbed catecholamines were eluted from the alumina with 100 ~l of 0.1 N HCl. To minimize the contaminants, catecholamines were readsorbed to the alumina by adding 1 ml of 0.5 M Tris-HCl buffer containing 1 roM NaHS03 and the preparations returned to the mixer for another 15 min. Washing and elution were repeated as above-mentioned. Eluted catecholamines were measured by high performance liquid chromatography with an electrochemical detector (Yanaco Co. Ltd.). Details on the chromatographical data are as follows; column: Cosmosil ~l e (4.6 mm ¢ x 15 0 mm), mobile phase: 0.1 M phos ph a t e potassium buffer (pH 5.8) containing 7 % of methanol, 0 . 02 % of l-heptanesulfonate sodium and 1 roM EDTAo2 Na, applied potential: 700 mV vs Ag/AgCl, flow rate: 1.5 ml/min. The lower limitation of sensitivity of noradrenaline (NA), dopamine (DA) and adrenaline (Ad) was 0 .1 pmol . Drugs used were i-adrenaline bitartrate and yohimbine hydrochloride (Nakarai Chern., Kyoto), i-isoproterenol hydrochloride (Sigma Chern. Co., U.S.A.), 1- and d-propranolol hydrochloride and atenolol hydrochloride (Imperial Chern. Ind., Wilmslow, Cheshire, England), and butoxamine hydrochloride (BurroughsWellcome Research, Triangle Park, N. Carol.). The statistical significance of differences was calculated using Student's t-test. Results NA and DA, but not Ad in the samples of the superfusates were consistently detectable with high performance liquid chromatography with an electrochemical detector. Experiments were performed in the presence of cocaine 2 x 10-sM with the
Vol. 33, No.4, 1983
373
electrical stimulation at 5 Hz, unless otherwise atated. Evoked release 52 of NA and DA by the stimulation at 60. min after the start of the superfusion was 4.19 ~ 0.29 and 5.75 ± 0.28 pmol, respectively (n=14). The ratio of 52 to 51 in the absence of drugs was 85.6 ± 4.6 % for NA and 90.0 ~ ~.8 % for DA (n=14). This ratio was drastically inhibited when extracellular Ca 2 was omitted from the Krebs' medi~ (NA: 25.3 ± 9.4 %, n=4, DA: 16.6 ± 11.0 %, n=4). Addition of 2 mM EGTA to Ca 2 free medium completely blocked the release of both amines (Data not shown). Tetrodotoxin at 3 x 10-7 M completely blocked these releases (NA: 8 .8 ~ 6.2 %, n=4, DA: 1.2 ± 0.4 %, n=4). Pretre.t-.nt
0
1.0
0
50/5 ,
1.0
50/5,
NA
DA
I.
Yohiabine 10-'M
YohilObine 10-'M
.I
.
Adrenaline 10-'M
I --.J
Adrenaline 10-'M
I
---.J
Fig. 1 Inhibitory effects of adrenaline on the evoked release of NA and DA from rat hypothalamic slices. The pretreatment with antagonist, yohimbine was performed at the start of superfusion. The drug, adrenaline was applied 15 min before 5 period of stimulation. Each value represents the mean ± 5EM of at least 5 experiments. *: p<0.05, compared to control (no drug). Adrenaline significantly inhibited both NA and DA release, and these effects were antagonized by the pretreatment with yohimbine (Fig. 1). Yohimbine alone increased NA release concentration-dependently, while DA release was increased only with a relatively high concentration (Fig .2) . A high concentration of prazosin increased NA but not DA release. Prazosin is less potent in the facilitation of NA release than yohimbine by a factor of approximately 2 orders.
"
IIA
1.11
u
1.0
! 0
~: /10"
10"
"
Dol
1.11
U
'.0
10" 1111
! 0
~.
~-
~
10"
10" CIII
Fig. 2 Facilitatory effects of a-antagonists on evoked release of NA and DA from rat hypothalamic slices. Drugs were applied 15 min before 52 period of stimulation. Each points represents the mean ± SEM of at least 4 experiments. *: p<0.05, compared to control (a closed circle at 0 M).
Vol. 33, No.4, 1983
374
TABLE I Effects of several types of B-antagonists on isoproterenolinduced facil itation of evoked release of NA and DA from rat hypothalamic slices. Pretreatment
Isoproterenol
(M)
(M)
Propranolol 10- 8 10- 6 Atenolol Butoxamine
10- 8
S 2/ S 1 ratio (%) NA
DA
(14)
85.6 ±
4.6
90.0 ± 6.8
10 -10
(7)
98.0 ±
8.8
95.4 ± 6 .8
10 -9
(9)
10 -
(14) 115.0 ± 6.9*
8
10 -8
105.4 ± 6.0
109.0 ± 4.9*
115 .1 ± 6.0*
(10)
89.9 ± 5.3t
86.4 ± 6.4t
10 -8
(6 )
86.7 ± 11. 2t
85 .1 ± 6. 7 t
10 -
(7)
89. 1 ± 3.8 t
87.8 ± 6.4t
8
() : number of experiments, *: p
NA Control
(5) 10-7 I-Propranolol 3 x M (9) a-Propranolol 3 x 10-7M (5)
DA
97.1 ± 6.5
99.0 ± 3.9
63.3 ± 5.6*
77.2 ± 6.9*
99.3 ± 4.1
101.4 ± 7.0
Experiments were performed in the presence of cocaine 5 x 10-sM with the electrical stimulation at 2 Hz. Evoked release SI of NA and DA were 3.52 ± 0.26 and 6.66 ± 0.49 pmol, respectively (n=19). * : p <0.05,compared to control . Other details are as in TABLE I.
Vol. 33, No.4, 1983
375
Discussion Impulse-evoked release of endogenous NA and DA from slices of the rat hypothalamus wa~ measured using high performance liquid chromatography. This release was Ca2 -dependent and tetrodotoxin-sensitive. Endogenous NA and DA releases seem to be from the intrinsic neurons. Although it cannot be excluded that DA is released as a precursor from NA and/or Ad nerve endings, most of the released DA may originate from DA nerve endings, since yohimbine alone showed more potent facilitatory effects on NA than DA release (Fig. 2). In the central nervous system, the possibility exists that interneurons, axons converging near NA and DA nerve endings may have a role in the regulation of NA and DA release. Most recently we demonstrated the inhibition by adrenaline 10-7M and fnhancementby isoproterenol 10-sM of endogenous NA and DA release by 20 roM K in the presence of tetrodotoxin 3 x 10-7M (9), suggesting that interneurons are not mediating the effects of these drugs and that these presynaptic a- and 8-receptors preferentially exist on the nerve endings but not on soma/dendrites. Adrenaline with a potent a-agonistic action inhibited the release of both NA and OA, and this inhibition was completely antagonized by yohimbine, thereby suggesting the presence of presynaptic a-receptors probably on both NA and DA neurons. Our findings regarding the hypothalamus are consistent with data obtained by Galzin et al. (10). Furthermore, increase in NA and DA releases elicited by yohimbine alone suggests that endogenously released NA may act physiologically on the presynaptic a-receptors. An important finding is that prazosin, an ai-selective antagonist, produced an approximately 100 times less potent action on the NA release than did yohimbine, an a2-selective antagonist in the rat hypothalamus. This finding indicates that presynaptic a-adrenoceptors on NA and DA nerve endings are predominantly a 2-subtypes in the rat hypothalamus, as well as in the peripheral sympathetic neurons (11). Isoproterenol at low concentrations (10- 10 -lO-sM) facilitated both NA and DA releases by 10 - 30 , and these facilitatory effects were abolished by the pretreatment with BI - and B2-antagonists. These findings provide evidence for the co-existence of presynaptic BI- and B2-receptors on NA and DA nerve endings in the rat hypothalamus. Furthermore, i-propranolol alone but not the d-isomer inhibited these releases, suggesting that these presynaptic B-receptors may play physiologically roles in the regulation of NA and DA release. Theses findings are in a good agreement with the report by Dietl et al (5), who demonstrated that tazolol, a 61-agonist, salbutamol, a 6 2-agonist and isoproterenol facilitated and i -propranolol inhibited the in vivo catecholamine release from the cat posterior hypothalamus. In the peripheral sympathetic neurons, most of presynaptic 6-adrenoceptors are characterized as B2-subtypes (2, 12) except for 61-adrenoceptors in the arterial vessels of cat hind limbs (3). Majewski et al. (13) emphasized that NA and Ad, as co-transmitters play roles in a 8-receptormediated pos itive feedback mechanism for NA release in the peripheral sympathetic neurons. It is noteworthy that presynaptic 61 - and 62-receptors co-exist in the hypothalamus, through which endogenously released NA and/or Ad probably mediate the release of these amines, via a positive feedback mechanism. We conclude that release of NA and DA in the rat hypothalamus may be mediated via presynaptic a2-, 61- and 62-adrenoceptors. Acknowledgement We thank M. Ohara (Kyuspu univ.) for comments on the manuscript.
Vol. 33, No.4, 1983
376
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
K. STARKE, Rev. Physiol. Biochem. Pharmacol., 77 1-124 (1977). T.C. WESTFALL, Physiol. Rev., 57 659-728 (1977~ C. DAHLOF, Acta Physiol. Scand~ ~pl. 500 1-147 (1981). Y. MISU, M. KAIHO, K. OGAWA and T. KUBO, J. Pharmacol. Exp. Ther., 218 242247 (1981). H. DIETL, J.N. SINHA and A. PHILIPPU, Brain Res., 208 213-218 (1981). H. UEDA, Y. GOSHIMA and Y. MISU, Neurosci. lett., in press. J. GLOWINSKI and L.L. IVERSEN, J. Neurochem., !l 655-669 (1966). R. KELLER, A. OKE, I. MEFFORD and R.N. ADAMS, Life Sci., 19 995-1004 (1976). H. UEDA, Y. GOSHIMA andY. MISU, Japan J. Pharmacol., in press. A.M. GALZIN, M.L. DUBOCOVICH and S.Z. LANGER, J. Pharmacol. Exp. Ther., 221 461-471 (1982). J.C. DOXEY, C.F.C. SMITH and J.M. WALKER, Br. J. Pharmacol., 60 91-96 (1977). Y. MISU, M. KAIHO and T. KUBO, Blood Vessels, 18 219 (1981). H. MAJEWSKI, M.J. RAND and L.-H. TUNG, Br. J. Pharmacol., 73 669-679 (1981).
Life Sciences, Vol. 33, pp. 371-376 Printed in the U.S.A.
PRESYNAPTIC MEDIATION BY a2-' B1 - AND B2-ADRENOCEPTORS OF ENDOGENOUS NORADRENALINE AND DOPAMINE RELEASE FROM SLICES OF RAT HYPOTHALAMUS Hiroshi Ueda, Yoshio Goshima and Yoshimi Misu Department of Pharmacology, Yokohama City University School of Medicine, Yokohama, 232, Japan (Received in final form May 10, 1983) Summary Using high performance liquid chromatography with an electrochemical detector, we studied effects of different compounds on the impulse-evoked release of endogenous noradrenaline (NA) and dopamine (DA) release from slices of the rat hypothalamus. Adrenaline (10-7 M), with a potent a - a goni s t i c action decreased both NA and DA release, and these effects were blocked by pretreatment with yohimbine (10-7M) . The a 2- a n t a go ni s t , yohimbine alone (10-s-10-6 M) concentration-dependently increased these releases, while a i - a nt a goni s t , prazosin showed weak increase on NA but not DA release at 10-6M• Isoproterenol (lO-IO_lO-sM) concentration-dependently increased these releases and the effects were antagonized by pretreatment with a non-selective Bantagonist, i-propranolol, a BI- an t a gon i s t , atenolol or a B2-antagonist, butoxamine. i-Propranolol (3xlO- 7M) alone, but not the d-isomer inhibi ted the releases. Thus, in the rat hypothalamus', the release of NA and DA may be mediated via presynaptic a2 - , Bl- and B2-adrenoceptors . The role of presynaptic a - a dr eno cep t o r s in the regulation of the release of the transmitter noradrenaline (NA) through a negative feedback mechanism in the sympathetic nervous systems has been well established (for review see I, 2), and it has been proposed that there is additional posi ti ve feedback mechanism through presynaptic B-adrenoceptors (1-4). Little is known of presynaptic a- and Badrenoceptors on the catecholaminergic neurons in the central nervous system (1,5) • We used high performance liquid chromatography with an electrochemical detector and studied presynaptic regulation mechanism via a - and B-adrenoceptors on the impUlse-evoked release of e ndo ge nous noradrenaline and dopamine from slices of the rat hypothalamus. Part of the results has been previously presented in the Japan Neuroscience Society (6). Methods Male, 8-week old Sprague-Dawley rats were given food pellets and water ad l ibitum and were kept on a regular day and night schedule (lights on at 5 a.m. and off 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 (7), and was cut sagittally into 6 pieces. The slices were transferred to a glass chamber (10 rnm~ x 5 rnm) and superfused in an overflow manner at the rate of 0.45 ml/rnin at 37°C with Krebs' medium containing NaCI 113 (mM), KCl
0024-3205/83 $3.00 + .00 Copyright (c) 1983 Pergamon Press Ltd.
Vol. 33, No.4, 1983
372
4.05, NaHC03 25, KH 2P0 4 1.18, MgS04 1.19, caC1 2 2.52, glucose 11.1, EDTA~2Na 0.029 and ascorbic acid 0.29 in the presence of cocaine 2 or 5 x 10-sM. Electrical field stimulation was performed by 30 rnA rectangular pulses of 2 msec duration at a frequency of 2 or 5 Hz for 3 min at 30 min intervals through platinum spiral electrodes set up at the two ends of the chamber, using an electrical stimulator with an isolator (SEN-320l and SS-102J, Nihon Kohden, Tokyo). Stimulation was applied only to "condition" the slices 30 min after the start of superfusion and data on the evoked releases were discarded. After a further equilibration period of 30 min, field stimulations were performed twice 60 (51) and 90 (S2) min after the beginning of superfusion and 3 min samples of the superfusates were successively collected throughout the rest of the experiment. Catecholamine release evoked by electrical stimulation was calculated by subtracting the estimated basal release from the total release . The effects of drugs on catecholamine release were evaluated by means of S 2/S1 ratio of evoked amounts. Adrenergic agonists or antagonists alone were always added 15 min before the S2 period of 's t i mul at i on . Pretreatments with a- or B-antagonists were initiated at the start of superfusion and were continued throughout the experiments. Measurements of catecholamines were made as described by Keller et al . (8). Catecholamine containing samples (1.35 ml) of superfusates were transferred to polypropylene tubes (1.7 ml) co ntaining activated alumina (10 mg), EDTAo 2Na (10 mg) and dihydroxybenzylamine (10 pmol), as an internal standard. To the tubes was added 0.2 ml of 1.5 M Tris - HCl buffer (pH 8.6), and t he p r ep a r a t i on s were placed in a mixer for 15 min. The supernatant was discarded and the alumina washed three times with 1 ml of water containing 5 roM Tris-HCl buffer (pH 8.6) and 1 roM NaHS03. Adsorbed catecholamines were eluted from the alumina with 100 ~l of 0.1 N HCl. To minimize the contaminants, catecholamines were readsorbed to the alumina by adding 1 ml of 0.5 M Tris-HCl buffer containing 1 roM NaHS03 and the preparations returned to the mixer for another 15 min. Washing and elution were repeated as above-mentioned. Eluted catecholamines were measured by high performance liquid chromatography with an electrochemical detector (Yanaco Co. Ltd.). Details on the chromatographical data are as follows; column: Cosmosil ~l e (4.6 mm ¢ x 15 0 mm), mobile phase: 0.1 M phos ph a t e potassium buffer (pH 5.8) containing 7 % of methanol, 0 . 02 % of l-heptanesulfonate sodium and 1 roM EDTAo2 Na, applied potential: 700 mV vs Ag/AgCl, flow rate: 1.5 ml/min. The lower limitation of sensitivity of noradrenaline (NA), dopamine (DA) and adrenaline (Ad) was 0 .1 pmol . Drugs used were i-adrenaline bitartrate and yohimbine hydrochloride (Nakarai Chern., Kyoto), i-isoproterenol hydrochloride (Sigma Chern. Co., U.S.A.), 1- and d-propranolol hydrochloride and atenolol hydrochloride (Imperial Chern. Ind., Wilmslow, Cheshire, England), and butoxamine hydrochloride (BurroughsWellcome Research, Triangle Park, N. Carol.). The statistical significance of differences was calculated using Student's t-test. Results NA and DA, but not Ad in the samples of the superfusates were consistently detectable with high performance liquid chromatography with an electrochemical detector. Experiments were performed in the presence of cocaine 2 x 10-sM with the
Vol. 33, No.4, 1983
373
electrical stimulation at 5 Hz, unless otherwise atated. Evoked release 52 of NA and DA by the stimulation at 60. min after the start of the superfusion was 4.19 ~ 0.29 and 5.75 ± 0.28 pmol, respectively (n=14). The ratio of 52 to 51 in the absence of drugs was 85.6 ± 4.6 % for NA and 90.0 ~ ~.8 % for DA (n=14). This ratio was drastically inhibited when extracellular Ca 2 was omitted from the Krebs' medi~ (NA: 25.3 ± 9.4 %, n=4, DA: 16.6 ± 11.0 %, n=4). Addition of 2 mM EGTA to Ca 2 free medium completely blocked the release of both amines (Data not shown). Tetrodotoxin at 3 x 10-7 M completely blocked these releases (NA: 8 .8 ~ 6.2 %, n=4, DA: 1.2 ± 0.4 %, n=4). Pretre.t-.nt
0
1.0
0
50/5 ,
1.0
50/5,
NA
DA
I.
Yohiabine 10-'M
YohilObine 10-'M
.I
.
Adrenaline 10-'M
I --.J
Adrenaline 10-'M
I
---.J
Fig. 1 Inhibitory effects of adrenaline on the evoked release of NA and DA from rat hypothalamic slices. The pretreatment with antagonist, yohimbine was performed at the start of superfusion. The drug, adrenaline was applied 15 min before 5 period of stimulation. Each value represents the mean ± 5EM of at least 5 experiments. *: p<0.05, compared to control (no drug). Adrenaline significantly inhibited both NA and DA release, and these effects were antagonized by the pretreatment with yohimbine (Fig. 1). Yohimbine alone increased NA release concentration-dependently, while DA release was increased only with a relatively high concentration (Fig .2) . A high concentration of prazosin increased NA but not DA release. Prazosin is less potent in the facilitation of NA release than yohimbine by a factor of approximately 2 orders.
"
IIA
1.11
u
1.0
! 0
~: /10"
10"
"
Dol
1.11
U
'.0
10" 1111
! 0
~.
~-
~
10"
10" CIII
Fig. 2 Facilitatory effects of a-antagonists on evoked release of NA and DA from rat hypothalamic slices. Drugs were applied 15 min before 52 period of stimulation. Each points represents the mean ± SEM of at least 4 experiments. *: p<0.05, compared to control (a closed circle at 0 M).
Vol. 33, No.4, 1983
374
TABLE I Effects of several types of B-antagonists on isoproterenolinduced facil itation of evoked release of NA and DA from rat hypothalamic slices. Pretreatment
Isoproterenol
(M)
(M)
Propranolol 10- 8 10- 6 Atenolol Butoxamine
10- 8
S 2/ S 1 ratio (%) NA
DA
(14)
85.6 ±
4.6
90.0 ± 6.8
10 -10
(7)
98.0 ±
8.8
95.4 ± 6 .8
10 -9
(9)
10 -
(14) 115.0 ± 6.9*
8
10 -8
105.4 ± 6.0
109.0 ± 4.9*
115 .1 ± 6.0*
(10)
89.9 ± 5.3t
86.4 ± 6.4t
10 -8
(6 )
86.7 ± 11. 2t
85 .1 ± 6. 7 t
10 -
(7)
89. 1 ± 3.8 t
87.8 ± 6.4t
8
() : number of experiments, *: p
NA Control
(5) 10-7 I-Propranolol 3 x M (9) a-Propranolol 3 x 10-7M (5)
DA
97.1 ± 6.5
99.0 ± 3.9
63.3 ± 5.6*
77.2 ± 6.9*
99.3 ± 4.1
101.4 ± 7.0
Experiments were performed in the presence of cocaine 5 x 10-sM with the electrical stimulation at 2 Hz. Evoked release SI of NA and DA were 3.52 ± 0.26 and 6.66 ± 0.49 pmol, respectively (n=19). * : p <0.05,compared to control . Other details are as in TABLE I.
Vol. 33, No.4, 1983
375
Discussion Impulse-evoked release of endogenous NA and DA from slices of the rat hypothalamus wa~ measured using high performance liquid chromatography. This release was Ca2 -dependent and tetrodotoxin-sensitive. Endogenous NA and DA releases seem to be from the intrinsic neurons. Although it cannot be excluded that DA is released as a precursor from NA and/or Ad nerve endings, most of the released DA may originate from DA nerve endings, since yohimbine alone showed more potent facilitatory effects on NA than DA release (Fig. 2). In the central nervous system, the possibility exists that interneurons, axons converging near NA and DA nerve endings may have a role in the regulation of NA and DA release. Most recently we demonstrated the inhibition by adrenaline 10-7M and fnhancementby isoproterenol 10-sM of endogenous NA and DA release by 20 roM K in the presence of tetrodotoxin 3 x 10-7M (9), suggesting that interneurons are not mediating the effects of these drugs and that these presynaptic a- and 8-receptors preferentially exist on the nerve endings but not on soma/dendrites. Adrenaline with a potent a-agonistic action inhibited the release of both NA and OA, and this inhibition was completely antagonized by yohimbine, thereby suggesting the presence of presynaptic a-receptors probably on both NA and DA neurons. Our findings regarding the hypothalamus are consistent with data obtained by Galzin et al. (10). Furthermore, increase in NA and DA releases elicited by yohimbine alone suggests that endogenously released NA may act physiologically on the presynaptic a-receptors. An important finding is that prazosin, an ai-selective antagonist, produced an approximately 100 times less potent action on the NA release than did yohimbine, an a2-selective antagonist in the rat hypothalamus. This finding indicates that presynaptic a-adrenoceptors on NA and DA nerve endings are predominantly a 2-subtypes in the rat hypothalamus, as well as in the peripheral sympathetic neurons (11). Isoproterenol at low concentrations (10- 10 -lO-sM) facilitated both NA and DA releases by 10 - 30 , and these facilitatory effects were abolished by the pretreatment with BI - and B2-antagonists. These findings provide evidence for the co-existence of presynaptic BI- and B2-receptors on NA and DA nerve endings in the rat hypothalamus. Furthermore, i-propranolol alone but not the d-isomer inhibited these releases, suggesting that these presynaptic B-receptors may play physiologically roles in the regulation of NA and DA release. Theses findings are in a good agreement with the report by Dietl et al (5), who demonstrated that tazolol, a 61-agonist, salbutamol, a 6 2-agonist and isoproterenol facilitated and i -propranolol inhibited the in vivo catecholamine release from the cat posterior hypothalamus. In the peripheral sympathetic neurons, most of presynaptic 6-adrenoceptors are characterized as B2-subtypes (2, 12) except for 61-adrenoceptors in the arterial vessels of cat hind limbs (3). Majewski et al. (13) emphasized that NA and Ad, as co-transmitters play roles in a 8-receptormediated pos itive feedback mechanism for NA release in the peripheral sympathetic neurons. It is noteworthy that presynaptic 61 - and 62-receptors co-exist in the hypothalamus, through which endogenously released NA and/or Ad probably mediate the release of these amines, via a positive feedback mechanism. We conclude that release of NA and DA in the rat hypothalamus may be mediated via presynaptic a2-, 61- and 62-adrenoceptors. Acknowledgement We thank M. Ohara (Kyuspu univ.) for comments on the manuscript.
Vol. 33, No.4, 1983
376
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
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