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Evidence for the Existence of Stereoselective Presynaptic β1-Adrenoceptors on Noradrenergic and Dopaminergic Neurons in the Rat Hypothalamus
Evidence
for the Existence
of Stereoselective
Presynaptic
~1-Adrenoceptors on Noradrenergic and Dopaminergic Neurons in the Rat Hypothalamus Chia Cheng CHANG*,Yoshio GOSHIMAand Yoshimi MISU** Department of Pharmacology, Yokohama CityUniversity Schoolof Medicine, Yokohama 232,Japan AcceptedAugust29,1986 Abstract-Using high performance liquid chromatography with an electrochemical detector, we examined antagonistic effects of / and d-acebutolol and atenolol against isoproterenol-induced facilitation of impulse-evoked release of endogenous norepinephrine and dopamine in rat hypothalamic slices. Isoproterenol (10 nM) increased the release of the both catecholamines, and this facilitation was similarly antagonized by 100 nM /-acebutolol and 100 nM atenolol, but was not antagonized by 100 nM d-acebutolol. In conclusion, there exist stereoselective presynaptic 81-adrenoceptors on noradrenergic and dopaminergic neurons in the rat hypo thalamus. It has been proposed that there is a facili tatory regulatory mechanism of the release of the transmitter norepinephrine through presyn aptic Q-adrenoceptors in peripheral sympa thetic nerve terminals (1-4). In the central nervous system, we demonstrated the coexistence of presynaptic 31 and 12 adrenoceptors on dopaminergic (5, 6), nor adrenergic (5, 7, 8) and adrenergic (9) neuron terminals in the rat hypothalamus. One of the requisites for clarifying whether presynaptic 8-adrenoceptors are present is to demonstrate a stereoselective nature in line with that of classical postsynaptic /9 adrenoceptors (4). We already demonstrated the stereoselective blocking actions of pro pranolol alone, a nonselective Q-antagonist, on presynaptic 8-adrenoceptors to facilitate tonically the release of 3 kinds of catechol amines in rat hypothalamic slices (5, 9). However, there have only been a few findings regarding the stereoselectivity of presynaptic 31-adrenoceptors (2, 4). Thus, in the present experiments, we attempted to * Present Surgery, Medicine, ** To whom
address: Yokohama
Department City
Yokohama 232, correspondence
of
Neurological
University Japan should
School be
addressed
of .
clarify the existence of stereoselective presynaptic i9 -adrenoceptors which par ticipate in facilitating the release of endogenous norepinephrine and dopamine in rat hypothalamic slices by means of / and d-acebutolol. Eight-week old male Sprague-Dawley rats were decapitated, and the hypothalamus was dissected out as described previously (5, 6). The slices were transferred to a glass chamber (10 mm x 5 mm) and superfused in an over flow manner at the rate of 0.45 ml/min at 37'C with Krebs' medium bubbled with 95% 02 and 5% CO2 in the presence of 20 ,eM cocaine. The composition of the medium was as follows (mM): NaCI, 113; NaHCO3, 25; KCI, 4.75; KH2PO4, 1.18; CaCl2, 2.52; MgSO4, 1.19; glucose, 11.1: disodium EDTA, 0.029, and ascorbic acid, 0.29. Electrical field stimulations were performed twice by 30 mA rectangular pulses of 2 msec duration at a frequency of 5 Hz for 3 min at 60 (S1) and 90 (S2) min after the start of superfusion through platinum spiral electrodes set up at the ends of the chamber, using an electrical stimulator with an isolator. Samples were collected every 3 min throughout the experi ments. Measurements of norepinephrine and dopamine were made using high-performance
liquid chromatography with an electro chemical detector as described previously (5, 6). The evoked release of both catecholamines was calculated by subtracting the estimated basal release from the total release. Isopro terenol was added 15 min before S2, and the effect was evaluated by means of the ratio of amounts evoked by the S2 and St periods of stimulation (S2/S1). l9-Antagonists were applied before S1 and were present through out the experiments. Drugs used were / isoproterenol hydrochloride (Sigma), / and d-acebutolol hydrochloride (Rhone-Poulenc) and atenolol hydrochloride (ICI). Statistical significance of difference was calculated using Student's t-test. In control preparations, the spontaneous release of norepinephrine and dopamine immediately before St was 580±40 and 312±55 fmol/mg of protein, respectively, and the evoked release of norepinephrine and dopamine during S1 was 419±29 and 580± 30 fmol/mg of protein, respectively (n=14) (6, 7). Tissue contents of norepinephrine and dopamine in slices after these superfusion experiments were 56.5±1.9 and 92.1±10.0 pmol/mg of protein, respectively (n=12) (6, 7). The spontaneous release of norepine phrine and dopamine before S2 was slightly attenuated, and the ratio of spontaneous release S2/S1 was 0.80±0.04 and 0.71 ±0.04, respectively. The drugs studied produced no
modifications of the absolute amounts of spontaneous and evoked release at S1, S2/S1 ratios of spontaneous release and tissue contents of both catecholamines. Isopro terenol at 10 nM significantly facilitated impulse-evoked release of norepinephrine and dopamine (6, 7) (Table 1). Pretreatment with 10 nM /-acebutolol did not antagonize, but 100 nM antagonized significantly isopro terenol-induced facilitation of the release of norepinephrine and dopamine. /-Acebutolol at 100 nM, alone, applied before St produced no effect. Atenolol at 10 nM did not an tagonize, but 100 nM antagonized sig nificantly the isoproterenol-induced facili tation of norepinephrine (7) and produced a tendency to antagonize that of dopamine. A higher concentration of 1 /tM atenolol significantly antagonized the isoproterenol induced facilitation of dopamine (5). Fur thermore, a clear dissociation was seen between the antagonisms by 100 nM / and d-acebutolol against isoproterenol-induced facilitation of the release of norepinephrine and dopamine (Fig. 1). /-Acebutolol, a selective a1-antagonist, antagonized isoproterenol-induced facili tation of impulse-evoked release of endo genous norepinephrine and dopamine from rat hypothalamic slices. This is consistent with our previous findings that tazolol, a selective Q1-agonist, facilitated the release of
Table 1. Antagonism by /-acebutolol and atenolol against isoproterenol-induced facilitation of impulse evoked release of endogenous norepinephrine (NE) and dopamine (DA) from rat hypothalamic slices
Electrical field stimulation (5 Hz, 2 msec, 30 mA, 3 min) was performed twice in the presence of 20 I_iM cocaine at 60 (S,) and 90 (S2) min after the start of superfusion. Isoproterenol was applied 15 min before S2. j9-Antagonists were applied before S, and were present throughout the experiments. Data shown are ratios of NE and DA output released by S2 and S, stimulations (S2/S,). Values are means± S.E. and n is the number of experiments. 'P<0.05, compared to the control; tP<0.05, ttP<0.01, compared to isoproterenol alone.
of stereoselective presynaptic a, -adrenocep tors in noradrenergic and dopaminergic systems in rat hypothalamic slices and are consistent with our previous findings that propranolol alone stereoselectively blocked presynaptic Q-adrenoceptors on the nor adrenergic and dopaminergic nerve terminals in the same preparations (5, 7).
11.1.1
Fig. 1. Stereoselective antagonism by acebutolol against isoproterenol (ISOP)-induced facilitation of impulse-evoked release of endogenous norepine phrine (NE) and dopamine (DA). Ordinates show ratios (S2/S1) of NE (upper half) and DA (lower half) output released by S2 and S, stimulations. Vertical bars represent the mean±S.E., and numbers of estimations are indicated in parentheses. *P<0 .01, compared to the corresponding control (C). Other details are as in Table 1.
both catecholamines, whereas atenolol, a selective Q1-antagonist, antagonized iso proterenol-induced increases in the release of these catecholamines (5-7). The present finding further supports the existence of presynaptic 81 -adrenoceptors in the rat hypothalamus. The degree of the antagonism induced by /-acebutolol was qualitatively similar to that by atenolol. This is consistent with the findings that atenolol and acebutolol produce antagonisms of a similar degree against isoproterenol-induced activation of postsynaptic cardiac 81 -adrenoceptors, the positive inotropism in guinea-pig and human atrial appendages (10). On the other hand, d-acebutolol produced no antagonism in the present experiments. These facts clearly demonstrate the evidence for the existence
References 1 Langer, S.Z.: Presynaptic receptors and their role in the regulation of transmitter release. Br. J. Pharmacol. 60, 481-497 (1977) 2 Dahlof, C.: Studies on jS-adrenoceptor mediated facilitation of sympathetic neurotransmission. Acta Physiol. Scand. Supp. 500, 1-147 (1981) 3 Majewski, H.: Modulation of noradrenaline release through activation of presynaptic 1S adrenoceptors. J. Auton. Pharmacol. 3, 47-60 (1983) 4 Misu, Y. and Kubo, T.: Presynaptic Q adrenoceptors. Med. Res. Rev. 6, 197-225 (1986) 5 Ueda, H., Goshima, Y. and Misu, Y.: Presynaptic mediation by a2-, Q, and ~2-adrenoceptors of endogenous noradrenaline and dopamine release from slices of rat hypothalamus. Life Sci. 33, 371-376 (1983) 6 Ueda, H., Goshima, Y., Kubo, T. and Misu, Y.: Adrenaline involvement in the presynaptic fi adrenoceptor-mediated mechanisms of dopa mine release from slices of the rat hypothalamus. Life Sci. 34, 1087-1093 (1984) 7 Ueda, H., Goshima, Y., Kubo, T. and Misu, Y.: Involvement of epinephrine in the presynaptic beta adrenoceptor mechanism of norepinephrine release from rat hypothalamic slices. J. Phar macol. Exp. Ther. 232, 507-512 (1985) 8 Misu, Y., Goshima, Y., Ueda, H. and Kubo, T.: Presynaptic inhibitory dopamine receptors on noradrenergic nerve terminals: Analysis of biphasic actions of dopamine and apomorphine on the release of endogenous norepinephrine in rat hypothalamic slices. J. Pharmacol. Exp. Ther. 235, 771-777 (1985) 9 Goshima, Y., Kubo, T. and Misu, Y.: Autoregu lation of endogenous epinephrine release via presynaptic adrenoceptors in the rat hypo thalamic slice. J. Pharmacol. Exp. Ther. 235, 248-253 (1985) 10 Harms, H.H.: Cardioselectivity of beta-adre noceptor blocking agents; In vitro studies with human and guinea-pig preparations. In Beta Adrenoceptor Blocking Agents, Edited by Saxena, P.R. and Forsyth, R.P., p. 311-315, North-Holland Pub. Co., Amsterdam (1976)
for the Existence
of Stereoselective
Presynaptic
~1-Adrenoceptors on Noradrenergic and Dopaminergic Neurons in the Rat Hypothalamus Chia Cheng CHANG*,Yoshio GOSHIMAand Yoshimi MISU** Department of Pharmacology, Yokohama CityUniversity Schoolof Medicine, Yokohama 232,Japan AcceptedAugust29,1986 Abstract-Using high performance liquid chromatography with an electrochemical detector, we examined antagonistic effects of / and d-acebutolol and atenolol against isoproterenol-induced facilitation of impulse-evoked release of endogenous norepinephrine and dopamine in rat hypothalamic slices. Isoproterenol (10 nM) increased the release of the both catecholamines, and this facilitation was similarly antagonized by 100 nM /-acebutolol and 100 nM atenolol, but was not antagonized by 100 nM d-acebutolol. In conclusion, there exist stereoselective presynaptic 81-adrenoceptors on noradrenergic and dopaminergic neurons in the rat hypo thalamus. It has been proposed that there is a facili tatory regulatory mechanism of the release of the transmitter norepinephrine through presyn aptic Q-adrenoceptors in peripheral sympa thetic nerve terminals (1-4). In the central nervous system, we demonstrated the coexistence of presynaptic 31 and 12 adrenoceptors on dopaminergic (5, 6), nor adrenergic (5, 7, 8) and adrenergic (9) neuron terminals in the rat hypothalamus. One of the requisites for clarifying whether presynaptic 8-adrenoceptors are present is to demonstrate a stereoselective nature in line with that of classical postsynaptic /9 adrenoceptors (4). We already demonstrated the stereoselective blocking actions of pro pranolol alone, a nonselective Q-antagonist, on presynaptic 8-adrenoceptors to facilitate tonically the release of 3 kinds of catechol amines in rat hypothalamic slices (5, 9). However, there have only been a few findings regarding the stereoselectivity of presynaptic 31-adrenoceptors (2, 4). Thus, in the present experiments, we attempted to * Present Surgery, Medicine, ** To whom
address: Yokohama
Department City
Yokohama 232, correspondence
of
Neurological
University Japan should
School be
addressed
of .
clarify the existence of stereoselective presynaptic i9 -adrenoceptors which par ticipate in facilitating the release of endogenous norepinephrine and dopamine in rat hypothalamic slices by means of / and d-acebutolol. Eight-week old male Sprague-Dawley rats were decapitated, and the hypothalamus was dissected out as described previously (5, 6). The slices were transferred to a glass chamber (10 mm x 5 mm) and superfused in an over flow manner at the rate of 0.45 ml/min at 37'C with Krebs' medium bubbled with 95% 02 and 5% CO2 in the presence of 20 ,eM cocaine. The composition of the medium was as follows (mM): NaCI, 113; NaHCO3, 25; KCI, 4.75; KH2PO4, 1.18; CaCl2, 2.52; MgSO4, 1.19; glucose, 11.1: disodium EDTA, 0.029, and ascorbic acid, 0.29. Electrical field stimulations were performed twice by 30 mA rectangular pulses of 2 msec duration at a frequency of 5 Hz for 3 min at 60 (S1) and 90 (S2) min after the start of superfusion through platinum spiral electrodes set up at the ends of the chamber, using an electrical stimulator with an isolator. Samples were collected every 3 min throughout the experi ments. Measurements of norepinephrine and dopamine were made using high-performance
liquid chromatography with an electro chemical detector as described previously (5, 6). The evoked release of both catecholamines was calculated by subtracting the estimated basal release from the total release. Isopro terenol was added 15 min before S2, and the effect was evaluated by means of the ratio of amounts evoked by the S2 and St periods of stimulation (S2/S1). l9-Antagonists were applied before S1 and were present through out the experiments. Drugs used were / isoproterenol hydrochloride (Sigma), / and d-acebutolol hydrochloride (Rhone-Poulenc) and atenolol hydrochloride (ICI). Statistical significance of difference was calculated using Student's t-test. In control preparations, the spontaneous release of norepinephrine and dopamine immediately before St was 580±40 and 312±55 fmol/mg of protein, respectively, and the evoked release of norepinephrine and dopamine during S1 was 419±29 and 580± 30 fmol/mg of protein, respectively (n=14) (6, 7). Tissue contents of norepinephrine and dopamine in slices after these superfusion experiments were 56.5±1.9 and 92.1±10.0 pmol/mg of protein, respectively (n=12) (6, 7). The spontaneous release of norepine phrine and dopamine before S2 was slightly attenuated, and the ratio of spontaneous release S2/S1 was 0.80±0.04 and 0.71 ±0.04, respectively. The drugs studied produced no
modifications of the absolute amounts of spontaneous and evoked release at S1, S2/S1 ratios of spontaneous release and tissue contents of both catecholamines. Isopro terenol at 10 nM significantly facilitated impulse-evoked release of norepinephrine and dopamine (6, 7) (Table 1). Pretreatment with 10 nM /-acebutolol did not antagonize, but 100 nM antagonized significantly isopro terenol-induced facilitation of the release of norepinephrine and dopamine. /-Acebutolol at 100 nM, alone, applied before St produced no effect. Atenolol at 10 nM did not an tagonize, but 100 nM antagonized sig nificantly the isoproterenol-induced facili tation of norepinephrine (7) and produced a tendency to antagonize that of dopamine. A higher concentration of 1 /tM atenolol significantly antagonized the isoproterenol induced facilitation of dopamine (5). Fur thermore, a clear dissociation was seen between the antagonisms by 100 nM / and d-acebutolol against isoproterenol-induced facilitation of the release of norepinephrine and dopamine (Fig. 1). /-Acebutolol, a selective a1-antagonist, antagonized isoproterenol-induced facili tation of impulse-evoked release of endo genous norepinephrine and dopamine from rat hypothalamic slices. This is consistent with our previous findings that tazolol, a selective Q1-agonist, facilitated the release of
Table 1. Antagonism by /-acebutolol and atenolol against isoproterenol-induced facilitation of impulse evoked release of endogenous norepinephrine (NE) and dopamine (DA) from rat hypothalamic slices
Electrical field stimulation (5 Hz, 2 msec, 30 mA, 3 min) was performed twice in the presence of 20 I_iM cocaine at 60 (S,) and 90 (S2) min after the start of superfusion. Isoproterenol was applied 15 min before S2. j9-Antagonists were applied before S, and were present throughout the experiments. Data shown are ratios of NE and DA output released by S2 and S, stimulations (S2/S,). Values are means± S.E. and n is the number of experiments. 'P<0.05, compared to the control; tP<0.05, ttP<0.01, compared to isoproterenol alone.
of stereoselective presynaptic a, -adrenocep tors in noradrenergic and dopaminergic systems in rat hypothalamic slices and are consistent with our previous findings that propranolol alone stereoselectively blocked presynaptic Q-adrenoceptors on the nor adrenergic and dopaminergic nerve terminals in the same preparations (5, 7).
11.1.1
Fig. 1. Stereoselective antagonism by acebutolol against isoproterenol (ISOP)-induced facilitation of impulse-evoked release of endogenous norepine phrine (NE) and dopamine (DA). Ordinates show ratios (S2/S1) of NE (upper half) and DA (lower half) output released by S2 and S, stimulations. Vertical bars represent the mean±S.E., and numbers of estimations are indicated in parentheses. *P<0 .01, compared to the corresponding control (C). Other details are as in Table 1.
both catecholamines, whereas atenolol, a selective Q1-antagonist, antagonized iso proterenol-induced increases in the release of these catecholamines (5-7). The present finding further supports the existence of presynaptic 81 -adrenoceptors in the rat hypothalamus. The degree of the antagonism induced by /-acebutolol was qualitatively similar to that by atenolol. This is consistent with the findings that atenolol and acebutolol produce antagonisms of a similar degree against isoproterenol-induced activation of postsynaptic cardiac 81 -adrenoceptors, the positive inotropism in guinea-pig and human atrial appendages (10). On the other hand, d-acebutolol produced no antagonism in the present experiments. These facts clearly demonstrate the evidence for the existence
References 1 Langer, S.Z.: Presynaptic receptors and their role in the regulation of transmitter release. Br. J. Pharmacol. 60, 481-497 (1977) 2 Dahlof, C.: Studies on jS-adrenoceptor mediated facilitation of sympathetic neurotransmission. Acta Physiol. Scand. Supp. 500, 1-147 (1981) 3 Majewski, H.: Modulation of noradrenaline release through activation of presynaptic 1S adrenoceptors. J. Auton. Pharmacol. 3, 47-60 (1983) 4 Misu, Y. and Kubo, T.: Presynaptic Q adrenoceptors. Med. Res. Rev. 6, 197-225 (1986) 5 Ueda, H., Goshima, Y. and Misu, Y.: Presynaptic mediation by a2-, Q, and ~2-adrenoceptors of endogenous noradrenaline and dopamine release from slices of rat hypothalamus. Life Sci. 33, 371-376 (1983) 6 Ueda, H., Goshima, Y., Kubo, T. and Misu, Y.: Adrenaline involvement in the presynaptic fi adrenoceptor-mediated mechanisms of dopa mine release from slices of the rat hypothalamus. Life Sci. 34, 1087-1093 (1984) 7 Ueda, H., Goshima, Y., Kubo, T. and Misu, Y.: Involvement of epinephrine in the presynaptic beta adrenoceptor mechanism of norepinephrine release from rat hypothalamic slices. J. Phar macol. Exp. Ther. 232, 507-512 (1985) 8 Misu, Y., Goshima, Y., Ueda, H. and Kubo, T.: Presynaptic inhibitory dopamine receptors on noradrenergic nerve terminals: Analysis of biphasic actions of dopamine and apomorphine on the release of endogenous norepinephrine in rat hypothalamic slices. J. Pharmacol. Exp. Ther. 235, 771-777 (1985) 9 Goshima, Y., Kubo, T. and Misu, Y.: Autoregu lation of endogenous epinephrine release via presynaptic adrenoceptors in the rat hypo thalamic slice. J. Pharmacol. Exp. Ther. 235, 248-253 (1985) 10 Harms, H.H.: Cardioselectivity of beta-adre noceptor blocking agents; In vitro studies with human and guinea-pig preparations. In Beta Adrenoceptor Blocking Agents, Edited by Saxena, P.R. and Forsyth, R.P., p. 311-315, North-Holland Pub. Co., Amsterdam (1976)