European Journal of Pharmacology, 60 (1979) 79--89
79
© Elsevier/North-Holland Biomedical Press
PRESYNAPTIC NORADRENERGIC a-RECEPTORS AND MODULATION OF 3H-NORADRENALINE RELEASE FROM RAT BRAIN SYNAPTOSOMES CEES D.J. DE LANGEN, FRANCOIS HOGENBOOM and ARIE H. MULDER *
Department of Pharmacology, Free University Medical Faculty, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands Received 19 February 1979, revised MS received 25 June 1979, accepted 23 August 1979
C.D.J. DE LANGEN, F. HOGENBOOM and A.H. MULDER, Presynaptic noradrenergic s-receptors and modulation of 3H-noradrenaline release from rat brain synaptosomes, European J. Pharmacol. 60 (1979) 79--89. The depolarization (15 mM K*)-induced release of 3H-NA from superfused rat brain synaptosomes and the effects of ~-noradrenergic drugs thereon were studied. Noradrenaline (NA; in the presence of the uptake inhibitor desipramine) reduced synaptosomal 3H-NA release. Reduction of the concentration of calcium ions in the medium during K ÷ stimulation greatly enhanced the sensitivity of 3H-NA release to s-receptor-mediated inhibition. Under these conditions NA dose-dependently inhibited 3H-NA release from synaptosomes obtained from cortex or hypothalamus, but did not affect 3H-NA release from striatal (i.e. dopaminergic) synaptosomes. Adrenaline, clonidine and oxymetazoline potently inhibited 3H-NA release from cortex synaptosomes at concentrations in the nanomolar range. Phentolamine by itself did not affect synaptosomal 3H-NA release, but antagonized the inhibitory effects of both noradrenaline and adrenaline. The data obtained further substantiate the hypothesis that the s-receptors mediating a local negative feedback control of NA release are localized on the varicosities of central noradrenergic neurons. Furthermore, noradrenergic nerve terminals in the hypothalamus appear to be less sensitive to s-receptor-mediated presynaptic inhibition than those in the cortex. Presynaptic s-receptors Noradrenaline release
Rat brain
Synaptosomes
1. Introduction In recent years much evidence has accumulated indicating that the amount of noradrenaline (NA) released upon firing of noradrenergic neurons may be under the control of a variety of local regulatory mechanisms, i.e. not primarily operating via alterations in firing rate. For instance, various types of presynaptic receptors, such as a-noradrenergic, cholinergic and opiate receptors are thought to be present on varicosities in the central nervous system (for reviews see Langer, 1977; Starke et al., 1977). Activation of presynaptic a-receptors has
* To whom reprint requests should be addressed.
Calcium ions
been shown to inhibit the release of NA and this would provide a mechanism for local negative feedback control o f transmitter release from noradrenergic neurons. In the CNS, inhibition of depolarization-induced NA release by a-receptor agonists has been demonstrated using brain slices labeled with the radioactive catecholamine (Farnebo and Hamberger, 1971; Starke and Montel, 1973; Dismukes and Mulder, 1976). In a previous paper (Dismukes et al., 1977) we presented some evidence suggesting that lowering the Ca2+-concentration in the medium enhanced the susceptibility of depolarization-induced NA release from cortex slices to inhibition by a-receptor agonists. Furthermore, in a recent preliminary report (Mulder et al., 1978) we demonstrated that
80
K*-induced release of 3H-NA from cortex synaptosomes was inhibited by a-receptor agonists. Therefore, in the present study we used superfused synaptosomes prepared from a number of brain regions to further substantiate the hypothesis that release-modulating a-receptors are localized on noradrenergic nerve terminals in the brain. In addition, the data obtained show that the extent of a-receptor-mediated inhibition of 3H-NA release from synaptosomes is strongly dependent on the Ca2+-concentration in the medium.
2. Materials and methods
C.D.J. DE LANGEN ET AL.
2.2. Superfusion procedure and addition of drugs After 25 min of superfusion 8 consecutive 5-min fractions were collected to determine the efflux of radioactivity before, during and after K+-stimulation. At t = 40 min depolarization of the synaptosomes was effected by superfusion with medium containing 15 mM K ÷ for 15 min {fig. 1). In the media containing an elevated K ÷ concentration the NaC1 concentration was reduced equivalently in order to maintain iso-osmolarity. In a number of experiments (figs. 2 and 3; table 1) 1.2 mM CaC12 was continuously present in all media used. However, in most
2.1. Preparation and labeling of synaptosomes Male Wistar rats (140-180 g b o d y weight) were sacrificed by decapitation and the brains were rapidly removed and dissected according to Glowinski and Iversen (1966). Tissue obtained from various brain regions (in most experiments neocortex) was homogenized in 9 volumes of 0.32 M sucrose at 4°C in a Potter-Elvehjem glass homogenizer with a rotating teflon pestle. The homogenate was centrifuged at 1000 × g for 10 min and 2 ml of the supernatant was added to 8 ml of oxygenated Krebs-Ringer-bicarbonate medium. The medium had the following composition (raM): NaC1 (121); KC1 (1.85); CaC12 (1.2); MgSO4 (1.15); KH2PO4 (1.15); NaHCO3 (25); glucose (11.1) pH 7.2-7.4; oxygenated with 95% O2/5%CO2. After 10 min of equilibration in a D u b n o f f metabolic shaker 1-3H-NA was added (10 pCi; final concentration in the medium a b o u t 0.1 tiM) and incubation was continued for 15 min. After sedimentation at 600 × g the labeled synaptosomes were carefully resuspended in 1 ml of Krebs-Ringer medium and 100 til of this suspension (containing the equivalent of a b o u t 20 mg of fresh tissue) was applied on t o p of a small layer (200 pl) of Sephadex G-15 in each of 8 chambers of a superfusion system (Mulder et al. 1975).
0.25-
0.20-
0.15"
0.10"
0,05" 15mM
K+ II
4'o Fig. 1. Calcium dependence of 3H-NA release. Brain cortex synaptosomes were superfused with medium from which CaCi2 had been eliminated. At t = 40 min, depolarization (indicated by a black bar on the abscissa) was effected by superfusion with medium containing 15 mM K ÷ and 1.2 mM CaC12 (control, solid line) or with medium containing 15 mM K ÷ without CaC12 (no Ca 2÷, broken line), EGTA (10 pM) was present in all media. The values represent the mean of 4 separate experiments (average S.E.M. 8% of the mean). Ordinate: tritium efflux (fractional rate per 5 rain). Abscissa: superfusion time (rain).
PRESYNAPTIC s-RECEPTORS ON SYNAPTOSOMES experiments CaC12 was eliminated from the incubation and superfusion media, and was only added in various concentrations (including zero) during depolarization with 15 mM K ÷. In these experiments all media contained 10 pM EGTA. The exact experimental conditions with respect to the Ca 2÷ concentrations in the media are mentioned in the legends to figures and tables. Desipramine or phentolamine, when used, were present in the superfusion media throughout the experiment from t = 20 min. The s-receptor agonists 1-noradrenaline, 1-adrenaline, clonidine or oxymetazoline were continuously present from t = 30 min. At the end of each experiment radioactivity remaining in the synaptosomes was e x t r a c t e d with 0.1 N hydrochloric acid. The radioactivity in the superfusion samples and the tissue extracts was assayed b y liquid scintillation counting and corrected for quenching b y the external standard channels ratio method.
2.3. Calculation of the data The efflux of radioactivity during each 5-min collection period was expressed as a fractional rate, i.e. the radioactivity in that fraction divided b y the total a m o u n t of radicfactivity present in the tissue at the onset of collection. In order to evaluate the effect of a drug on baseline (spontaneous) efflux, the fractional rate (in the presence of the drug) for 5 min before depolarization was expressed as % of the fractional rate of the control efflux (in most cases w i t h o u t drug) in parallel superfusion chambers. The K÷-induced 3Hoverflow was calculated as the area under the curve describing the fractional rate efflux vs. time (fig. 1) after subtraction of the baseline efflux, which was estimated b y averaging the fractional rates obtained for 5 min before and 15 after depolarization. The K*-induced 3Hoverflow was expressed as percent of total tritium present in the synaptosomes during the K÷-depolarization. The K÷-induced 3Hoverflow was almost completely dependent on the presence of Ca 2÷ ions in the medium
81 (fig. 1) and a b o u t 90% of it co-chromatographed with noradrenaline on ion-exchange columns according to the method of Kehr (1974). The effects of s-receptor agents on K*-induced 3H-NA release were evaluated in duplicate in individual experiments and expressed as percent of the 3H-NA release observed in duplicate parallel control chambers. The experiments were repeated at least three times. The statistical significance of differences between means was determined by Student's two-tailed t-test; n denotes the number of observations.
2.4. Chemicals and drugs 1-[7-3H]-Noradrenaline (10 Ci/mmole) was purchased from the Radiochemical Centre (Amersham). The following substances were obtained commercially: 1-noradrenaline, 1-adrenaline, phentolamine and EGTA. Desipramine was a generous gift from CibaGeigy, clonidine from Boehringer-Ingelheim and oxymetazoline from Merck-Pharma.
3. Results
3.1. Inhibition by desipramine of the uptake of exogenous NA during superfusion Ol synaptosomes Addition of 1 pM NA to the medium superfusing synaptosomes previously labeled with 3H-NA strongly enhanced the efflux of tritium (fig. 2), since unlabeled NA was taken up into nerve terminals and displaced, or exchanged with 3H-NA. Within 10 min following the addition o f exogenous NA the fractional rate of 3H-efflux was increased b y a b o u t 200% compared to the control baseline efflux. This effect of NA was completely blocked b y 3/~M desipramine, an inhibitor of catecholamine uptake into noradrenergic nerve terminals (Horn et al., 1971), when desipramine was added 10 min prior to NA (fig. 2). At this concentration desipramine did n o t
82
C.D.J, DE LANGEN ET AL.
0.20*T & X /
0.15-
I
X
I
. ~
J
T
x t
1/
100
I
T ~
i _ _ NA 0.10"
r-I
~
-
~
....
0 . 0 5 - ~ ~ i
OM,
L
I
20
'
_ I/NA+DMI , ----, - - ~ - CONTROL
75
HA ,
30
,
,
40
,
50
,
60
W
Fig. 2. Displacement of 3H-NA by exogenous noradrenaline and its inhibition by desipramine (DMI). Brain cortex synaptosomes were superfused with medium containing no drugs (control), noradrenaline (NA, 1 gM), or noradrenaline plus desipramine (NA, 1/~M, DMI, 3 pM) added at the times indicated on the abscissa. Ca 2+ (1.2 raM) was present in all media throughout the experiments. The 3H-efflux was expressed as a fractional rate per 5 rain. Values represent the mean of 4 experiments (S.E.M. averaged about 10% of the means and are not shown). Ordinate: 3H-efflux (fractional rate per 5rain). Abscissa: superfusion time (min).
affect the baseline 3H-efflux nor the K ÷induced 3H-NA release, clearly indicating that reuptake of released 3H-NA was largely, if not totally, prevented during continuous superfu-
0.3
1.0
3.0
Fig. 3. Reduction o f 3H-NA release f r o m superfused brain cortex synaptosomes by noradrenaline and antagonism by phentolamine in the presence of desipramine (3 pM). NA was added as indicated on the abscissa, in the absence ($ ; ) or presence (× . . . . . . ×) of phentolamine (1 pM). Ca 2+ (1.2 mM) was present in all media throughout the experiments. 3H-NA release (15 mM K*) was expressed as % of control (no NA) and averaged 14.8% (without phentolamine) and 14.2% (with phentolamine) of total 3H. Values represent means -+ S.E.M. (n = 6). * Significantly higher release than at the corresponding NA concentration without phentolamine ( P < 0.005) but not significantly different from control (no NA; phentolamine only). ** Significantly lower release than control (no NA, P < 0.05). Ordinate: 3H-NA release % of control. Abscissa: NA concentration (gM).
TABLE 1 Effects of desipramine and phentolamine on baseline tritium efflux and on depolarization-induced 3H-NA release. After labeling with 3H-NA, brain cortex synaptosomes were superfused and depolarized either in the absence (control) or presence of drugs, added at t = 20 min. Ca 2+ (1.2 mM) was present in all media throughout the " " 3 experiments. Depolarization was effected with 15 mM K + for 5 mm. Basehne H-efflux, 5 min before depolarization, was calculated as a fractional rate and expressed as % of control. 3H-NA release was calculated as % of total tissue tritium and expressed as % of control. This control release averaged 12% (desipramine) or 13% (phentolamine) of total tissue tritium. Values represent means -+ S.E.M. (n = 6-8). Drug
Desipramine
Phentolamine
Concentration (pM)
Baseline 3H-efflux (% of control)
Stimulated 3H-NA release (% of control)
1
9 7 -+ 2
3
98_~2
100 +- 13 96 + 7
1 3 10
101 -+ 2 98~4 105-+3
114 -+ 4 113 + 4 106-+ 5
PRESYNAPTIC c~-RECEPTORS O N S Y N A P T O S O M E S
83
sion of synaptosomes. Therefore, in further experiments 3 pM desipramine was routinely present in the superfusion medium; at this concentration desipramine had no effect on baseline 3H~efflux, nor on K÷-induced 3H-NA release (table 1 ).
3.3. Calcium-dependence of ~-receptor-medi. ated inhibition of 3H-NA release Table 2 shows the dependency of 3H-NA release on the Ca 2÷ concentration in the medium. In the absence of Ca 2÷ ions 2.2% of total tissue tritium was still released by 15 mM K ÷ stimulation. Table 2 also reveals the relative nature of the calcium dependence of 3H-NA release. Thus, when cortical synaptosomes were exposed to 15 mM K ÷ in the presence of 1.2 mM Ca 2÷ 22% of total tissue tritium was released, i.e. 90% of this release was dependent on the presence of Ca 2÷. However, when only 0.075 mM Ca 2÷ was present during depolarization 10% of total tritium was released and, consequently, 78% of this release was calcium dependent (table 2). The extent of inhibition by NA of 3H-NA release from cortical synaptosomes was compared under three different experimental conditions (1) 1.2 mM Ca 2÷ present throughout the experiment, (2) Ca2÷-free incubation and superfusion media, except during K*
79
© Elsevier/North-Holland Biomedical Press
PRESYNAPTIC NORADRENERGIC a-RECEPTORS AND MODULATION OF 3H-NORADRENALINE RELEASE FROM RAT BRAIN SYNAPTOSOMES CEES D.J. DE LANGEN, FRANCOIS HOGENBOOM and ARIE H. MULDER *
Department of Pharmacology, Free University Medical Faculty, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands Received 19 February 1979, revised MS received 25 June 1979, accepted 23 August 1979
C.D.J. DE LANGEN, F. HOGENBOOM and A.H. MULDER, Presynaptic noradrenergic s-receptors and modulation of 3H-noradrenaline release from rat brain synaptosomes, European J. Pharmacol. 60 (1979) 79--89. The depolarization (15 mM K*)-induced release of 3H-NA from superfused rat brain synaptosomes and the effects of ~-noradrenergic drugs thereon were studied. Noradrenaline (NA; in the presence of the uptake inhibitor desipramine) reduced synaptosomal 3H-NA release. Reduction of the concentration of calcium ions in the medium during K ÷ stimulation greatly enhanced the sensitivity of 3H-NA release to s-receptor-mediated inhibition. Under these conditions NA dose-dependently inhibited 3H-NA release from synaptosomes obtained from cortex or hypothalamus, but did not affect 3H-NA release from striatal (i.e. dopaminergic) synaptosomes. Adrenaline, clonidine and oxymetazoline potently inhibited 3H-NA release from cortex synaptosomes at concentrations in the nanomolar range. Phentolamine by itself did not affect synaptosomal 3H-NA release, but antagonized the inhibitory effects of both noradrenaline and adrenaline. The data obtained further substantiate the hypothesis that the s-receptors mediating a local negative feedback control of NA release are localized on the varicosities of central noradrenergic neurons. Furthermore, noradrenergic nerve terminals in the hypothalamus appear to be less sensitive to s-receptor-mediated presynaptic inhibition than those in the cortex. Presynaptic s-receptors Noradrenaline release
Rat brain
Synaptosomes
1. Introduction In recent years much evidence has accumulated indicating that the amount of noradrenaline (NA) released upon firing of noradrenergic neurons may be under the control of a variety of local regulatory mechanisms, i.e. not primarily operating via alterations in firing rate. For instance, various types of presynaptic receptors, such as a-noradrenergic, cholinergic and opiate receptors are thought to be present on varicosities in the central nervous system (for reviews see Langer, 1977; Starke et al., 1977). Activation of presynaptic a-receptors has
* To whom reprint requests should be addressed.
Calcium ions
been shown to inhibit the release of NA and this would provide a mechanism for local negative feedback control o f transmitter release from noradrenergic neurons. In the CNS, inhibition of depolarization-induced NA release by a-receptor agonists has been demonstrated using brain slices labeled with the radioactive catecholamine (Farnebo and Hamberger, 1971; Starke and Montel, 1973; Dismukes and Mulder, 1976). In a previous paper (Dismukes et al., 1977) we presented some evidence suggesting that lowering the Ca2+-concentration in the medium enhanced the susceptibility of depolarization-induced NA release from cortex slices to inhibition by a-receptor agonists. Furthermore, in a recent preliminary report (Mulder et al., 1978) we demonstrated that
80
K*-induced release of 3H-NA from cortex synaptosomes was inhibited by a-receptor agonists. Therefore, in the present study we used superfused synaptosomes prepared from a number of brain regions to further substantiate the hypothesis that release-modulating a-receptors are localized on noradrenergic nerve terminals in the brain. In addition, the data obtained show that the extent of a-receptor-mediated inhibition of 3H-NA release from synaptosomes is strongly dependent on the Ca2+-concentration in the medium.
2. Materials and methods
C.D.J. DE LANGEN ET AL.
2.2. Superfusion procedure and addition of drugs After 25 min of superfusion 8 consecutive 5-min fractions were collected to determine the efflux of radioactivity before, during and after K+-stimulation. At t = 40 min depolarization of the synaptosomes was effected by superfusion with medium containing 15 mM K ÷ for 15 min {fig. 1). In the media containing an elevated K ÷ concentration the NaC1 concentration was reduced equivalently in order to maintain iso-osmolarity. In a number of experiments (figs. 2 and 3; table 1) 1.2 mM CaC12 was continuously present in all media used. However, in most
2.1. Preparation and labeling of synaptosomes Male Wistar rats (140-180 g b o d y weight) were sacrificed by decapitation and the brains were rapidly removed and dissected according to Glowinski and Iversen (1966). Tissue obtained from various brain regions (in most experiments neocortex) was homogenized in 9 volumes of 0.32 M sucrose at 4°C in a Potter-Elvehjem glass homogenizer with a rotating teflon pestle. The homogenate was centrifuged at 1000 × g for 10 min and 2 ml of the supernatant was added to 8 ml of oxygenated Krebs-Ringer-bicarbonate medium. The medium had the following composition (raM): NaC1 (121); KC1 (1.85); CaC12 (1.2); MgSO4 (1.15); KH2PO4 (1.15); NaHCO3 (25); glucose (11.1) pH 7.2-7.4; oxygenated with 95% O2/5%CO2. After 10 min of equilibration in a D u b n o f f metabolic shaker 1-3H-NA was added (10 pCi; final concentration in the medium a b o u t 0.1 tiM) and incubation was continued for 15 min. After sedimentation at 600 × g the labeled synaptosomes were carefully resuspended in 1 ml of Krebs-Ringer medium and 100 til of this suspension (containing the equivalent of a b o u t 20 mg of fresh tissue) was applied on t o p of a small layer (200 pl) of Sephadex G-15 in each of 8 chambers of a superfusion system (Mulder et al. 1975).
0.25-
0.20-
0.15"
0.10"
0,05" 15mM
K+ II
4'o Fig. 1. Calcium dependence of 3H-NA release. Brain cortex synaptosomes were superfused with medium from which CaCi2 had been eliminated. At t = 40 min, depolarization (indicated by a black bar on the abscissa) was effected by superfusion with medium containing 15 mM K ÷ and 1.2 mM CaC12 (control, solid line) or with medium containing 15 mM K ÷ without CaC12 (no Ca 2÷, broken line), EGTA (10 pM) was present in all media. The values represent the mean of 4 separate experiments (average S.E.M. 8% of the mean). Ordinate: tritium efflux (fractional rate per 5 rain). Abscissa: superfusion time (rain).
PRESYNAPTIC s-RECEPTORS ON SYNAPTOSOMES experiments CaC12 was eliminated from the incubation and superfusion media, and was only added in various concentrations (including zero) during depolarization with 15 mM K ÷. In these experiments all media contained 10 pM EGTA. The exact experimental conditions with respect to the Ca 2÷ concentrations in the media are mentioned in the legends to figures and tables. Desipramine or phentolamine, when used, were present in the superfusion media throughout the experiment from t = 20 min. The s-receptor agonists 1-noradrenaline, 1-adrenaline, clonidine or oxymetazoline were continuously present from t = 30 min. At the end of each experiment radioactivity remaining in the synaptosomes was e x t r a c t e d with 0.1 N hydrochloric acid. The radioactivity in the superfusion samples and the tissue extracts was assayed b y liquid scintillation counting and corrected for quenching b y the external standard channels ratio method.
2.3. Calculation of the data The efflux of radioactivity during each 5-min collection period was expressed as a fractional rate, i.e. the radioactivity in that fraction divided b y the total a m o u n t of radicfactivity present in the tissue at the onset of collection. In order to evaluate the effect of a drug on baseline (spontaneous) efflux, the fractional rate (in the presence of the drug) for 5 min before depolarization was expressed as % of the fractional rate of the control efflux (in most cases w i t h o u t drug) in parallel superfusion chambers. The K÷-induced 3Hoverflow was calculated as the area under the curve describing the fractional rate efflux vs. time (fig. 1) after subtraction of the baseline efflux, which was estimated b y averaging the fractional rates obtained for 5 min before and 15 after depolarization. The K*-induced 3Hoverflow was expressed as percent of total tritium present in the synaptosomes during the K÷-depolarization. The K÷-induced 3Hoverflow was almost completely dependent on the presence of Ca 2÷ ions in the medium
81 (fig. 1) and a b o u t 90% of it co-chromatographed with noradrenaline on ion-exchange columns according to the method of Kehr (1974). The effects of s-receptor agents on K*-induced 3H-NA release were evaluated in duplicate in individual experiments and expressed as percent of the 3H-NA release observed in duplicate parallel control chambers. The experiments were repeated at least three times. The statistical significance of differences between means was determined by Student's two-tailed t-test; n denotes the number of observations.
2.4. Chemicals and drugs 1-[7-3H]-Noradrenaline (10 Ci/mmole) was purchased from the Radiochemical Centre (Amersham). The following substances were obtained commercially: 1-noradrenaline, 1-adrenaline, phentolamine and EGTA. Desipramine was a generous gift from CibaGeigy, clonidine from Boehringer-Ingelheim and oxymetazoline from Merck-Pharma.
3. Results
3.1. Inhibition by desipramine of the uptake of exogenous NA during superfusion Ol synaptosomes Addition of 1 pM NA to the medium superfusing synaptosomes previously labeled with 3H-NA strongly enhanced the efflux of tritium (fig. 2), since unlabeled NA was taken up into nerve terminals and displaced, or exchanged with 3H-NA. Within 10 min following the addition o f exogenous NA the fractional rate of 3H-efflux was increased b y a b o u t 200% compared to the control baseline efflux. This effect of NA was completely blocked b y 3/~M desipramine, an inhibitor of catecholamine uptake into noradrenergic nerve terminals (Horn et al., 1971), when desipramine was added 10 min prior to NA (fig. 2). At this concentration desipramine did n o t
82
C.D.J, DE LANGEN ET AL.
0.20*T & X /
0.15-
I
X
I
. ~
J
T
x t
1/
100
I
T ~
i _ _ NA 0.10"
r-I
~
-
~
....
0 . 0 5 - ~ ~ i
OM,
L
I
20
'
_ I/NA+DMI , ----, - - ~ - CONTROL
75
HA ,
30
,
,
40
,
50
,
60
W
Fig. 2. Displacement of 3H-NA by exogenous noradrenaline and its inhibition by desipramine (DMI). Brain cortex synaptosomes were superfused with medium containing no drugs (control), noradrenaline (NA, 1 gM), or noradrenaline plus desipramine (NA, 1/~M, DMI, 3 pM) added at the times indicated on the abscissa. Ca 2+ (1.2 raM) was present in all media throughout the experiments. The 3H-efflux was expressed as a fractional rate per 5 rain. Values represent the mean of 4 experiments (S.E.M. averaged about 10% of the means and are not shown). Ordinate: 3H-efflux (fractional rate per 5rain). Abscissa: superfusion time (min).
affect the baseline 3H-efflux nor the K ÷induced 3H-NA release, clearly indicating that reuptake of released 3H-NA was largely, if not totally, prevented during continuous superfu-
0.3
1.0
3.0
Fig. 3. Reduction o f 3H-NA release f r o m superfused brain cortex synaptosomes by noradrenaline and antagonism by phentolamine in the presence of desipramine (3 pM). NA was added as indicated on the abscissa, in the absence ($ ; ) or presence (× . . . . . . ×) of phentolamine (1 pM). Ca 2+ (1.2 mM) was present in all media throughout the experiments. 3H-NA release (15 mM K*) was expressed as % of control (no NA) and averaged 14.8% (without phentolamine) and 14.2% (with phentolamine) of total 3H. Values represent means -+ S.E.M. (n = 6). * Significantly higher release than at the corresponding NA concentration without phentolamine ( P < 0.005) but not significantly different from control (no NA; phentolamine only). ** Significantly lower release than control (no NA, P < 0.05). Ordinate: 3H-NA release % of control. Abscissa: NA concentration (gM).
TABLE 1 Effects of desipramine and phentolamine on baseline tritium efflux and on depolarization-induced 3H-NA release. After labeling with 3H-NA, brain cortex synaptosomes were superfused and depolarized either in the absence (control) or presence of drugs, added at t = 20 min. Ca 2+ (1.2 mM) was present in all media throughout the " " 3 experiments. Depolarization was effected with 15 mM K + for 5 mm. Basehne H-efflux, 5 min before depolarization, was calculated as a fractional rate and expressed as % of control. 3H-NA release was calculated as % of total tissue tritium and expressed as % of control. This control release averaged 12% (desipramine) or 13% (phentolamine) of total tissue tritium. Values represent means -+ S.E.M. (n = 6-8). Drug
Desipramine
Phentolamine
Concentration (pM)
Baseline 3H-efflux (% of control)
Stimulated 3H-NA release (% of control)
1
9 7 -+ 2
3
98_~2
100 +- 13 96 + 7
1 3 10
101 -+ 2 98~4 105-+3
114 -+ 4 113 + 4 106-+ 5
PRESYNAPTIC c~-RECEPTORS O N S Y N A P T O S O M E S
83
sion of synaptosomes. Therefore, in further experiments 3 pM desipramine was routinely present in the superfusion medium; at this concentration desipramine had no effect on baseline 3H~efflux, nor on K÷-induced 3H-NA release (table 1 ).
3.3. Calcium-dependence of ~-receptor-medi. ated inhibition of 3H-NA release Table 2 shows the dependency of 3H-NA release on the Ca 2÷ concentration in the medium. In the absence of Ca 2÷ ions 2.2% of total tissue tritium was still released by 15 mM K ÷ stimulation. Table 2 also reveals the relative nature of the calcium dependence of 3H-NA release. Thus, when cortical synaptosomes were exposed to 15 mM K ÷ in the presence of 1.2 mM Ca 2÷ 22% of total tissue tritium was released, i.e. 90% of this release was dependent on the presence of Ca 2÷. However, when only 0.075 mM Ca 2÷ was present during depolarization 10% of total tritium was released and, consequently, 78% of this release was calcium dependent (table 2). The extent of inhibition by NA of 3H-NA release from cortical synaptosomes was compared under three different experimental conditions (1) 1.2 mM Ca 2÷ present throughout the experiment, (2) Ca2÷-free incubation and superfusion media, except during K*
